Activity Report - INCT

Transcrição

Instituto Nacional de Ciência e Tecnologia de Genômica
para o Melhoramento de Citros
National Science and Technology Institute of Genomic for
Citrus Breeding
FAPESP 08/57909-2
CNPq 573848/08-4
First Annual Report
Coordinator: Marcos A. Machado
Members of the Program
Centro APTA Citros Sylvio Moreira, Instituto Agronômico
Embrapa Mandioca e Fruticultura Tropical
Esalq/USP
Cena/USP
Instituto Biológico
Universidade de Mogi das Cruzes
Universidade Federal de Campinas Grande
Universidade Estadual do Sudoeste da Bahia
Universidade de Santa Cruz (Ilhéus)
Unicamp
Estação Experimental de Citricultura de Bebedouro
Cordeirópolis – SP
- Abril, 2010 -
INSTITUTOS NACIONAIS DE CIÊNCIA E TECNOLOGIA – INCT
ACOMPANHAMENTO E AVALIAÇÃO
PERÍODO: de 14/5/2009 a 14/6/2010
IDENTIFICAÇÃO DO PROJETO
TÍTULO: INCT de Genômica para Melhoramento de Citros
PROCESSO Nº: 573848/2008-4
VIGÊNCIA: de 14/5/2009 a 13/5/2014
RECURSOS TOTAIS APROVAD0S: R$ 7.125.422,70
CUSTEIO – R$ 4.417.762,07
CAPITAL – R$ 1.852.609,91
BOLSAS – R$ 855.050,72
COORDENADOR: Marcos Antonio Machado
INSTITUIÇÃO SEDE: Instituto Agronômico de Campinas
INSTITUIÇÕES PARTICIPANTES DO PROJETO: (vide formulário de submissão)
EQUIPE DO PROJETO: (vide formulário de submissão)
PROJETO DE PESQUISA (Anexar Relatório Parcial)
HOUVE ALTERAÇÕES NOS OBJETIVOS E/OU METAS PROPOSTOS? ( x ) SIM ( ) NÃO
EM CASO POSITIVO REGISTRAR AS ALTERAÇÕES OCORRIDAS:
Plataforma de Informação Genômica
- Objetivos 1a (Ampliar a base de dados do CitEST) decidiu-se mudar a metodologia de
seqüenciamento de genoma expresso com a tecnologia Sanger original para a plataforma Illumina.
As razões para isso foram: menor custo de seqüenciamento terceirizado em empresas
especializadas, maior rapidez na obtenção de dados, maior eficiência na geração de resultados e
maior volume de informações sobre genoma expresso. Portanto, maiores ganhos para o programa.
- Objetivos 1b (Genoma completo de citros). É o projeto do Consórcio Internacional do Genoma
Citros. A participação do INCT Citros, inicialmente previsto com pagamento de cobertura 2x do
genoma com tecnologia Sanger, passou para 1x, em função da utilização de novas tecnologias de
re-seqüenciamento (particularmente com 454), uma das atividades previstas no programa original
(Objetivos 1c). Por outro lado, o serviço está sendo executado pelo AlphaHundson Institute of
Biotechnology da Universidade do Arkansas ao invés do Joint Genome Institute do Departamento
de Energia dos Estados Unidos.
O grande volume de informação em processamento (ESTs de citros da base CitEST e de outras
bases), assim como os dados a serem gerados com as novas plataformas de seqüenciamento,
exigem nova estrutura de computação, em aquisição no programa.
HOUVE ALTERAÇÕES NO CRONOGRAMA ORIGINAL? ( x ) SIM ( ) NÃO
EM CASO POSITIVO REGISTRAR AS ALTERAÇÕES OCORRIDAS:
Plataforma de Informação Genômica
A estruturação da base de dados de genoma expresso e genoma completo ainda está incompleta
em função das alterações apresentadas acima. Deve ser destacado que essas bases são
essenciais na montagem dos sistemas de expressão previstos na Plataforma de Aplicação
Genômica.
HOUVE PROBLEMAS E/OU DIFICULDADES NA EXECUÇÃO DO PROJETO?: ( ) SIM ( x ) NÃO
EM CASO POSITIVO DETALHAR:
EQUIPE
HOUVE ALTERAÇÃO NA COMPOSIÇÃO ORIGINAL DA EQUIPE? ( x ) SIM ( ) NÃO
EM CASO POSITIVO INDIQUE O NÚMERO DE INCLUSÕES E EXCLUSÕES:
Responsável p/ laboratório associado ( x ) Inclusão ( ) Exclusão
Justificar:
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Centro de Biologia Molecular Estrutural CeBiME) da Associação Brasileira de Tecnologia
de Luz Sincronton (ABTLus). Diretor: Kleber Gomes Franchini (ver ofício em anexo).
Laboratório referencia em biologia estrutural no Brasil.
Departamento de Ciências Biológicas, Faculdade de Ciências e Letras, Universidade
Estadual Paulista (UNESP). Grupo emergente na área de genética e biotecnologia vegetal
(ver ofício em anexo).
Pesquisador ( x ) Inclusão ( ) Exclusão
Justificar:
• O pesquisador Celso E. Benedetti do Centro de Biologia Molecular Estrutural (CeBiME) foi
convidado em função de seus trabalhos sobre genoma funcional de Xanthomonas
axonopodis pv citri e transformação genética de citros (ver ofício em anexo).
• O pesquisador Dario Abel Palmieri da Faculdade de Ciências e Letras e Departamento de
Ciências Biológicas foi convidado em função de seus trabalhos sobre marcadores
moleculares e mapeamento genético de citros (ver ofício em anexo).
Pós Doutorando ( x ) Inclusão ( ) Exclusão
Abaixo estão relacionados os Pós Doutorandos que atuam no programa com bolsas CNPq:
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Leonardo Pires Boava, Bolsa PDJ – Genoma funcional de citros sob estresse biótico
(infecção por Phytophthora parasítica). Centro de Citricultura Sylvio Moreira.
Supervisão: Mariângela Cristofani-Yaly
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Polyana Kelly Martins, Bolsa DTI (ex-PDJ) – Construção de vetores para transformação
genética de citros. Centro de Citricultura Sylvio Moreira.
Supervião: Juliana Freitas-Astúa
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Evandro Henrique Schinor, Bolsa DTI – Avaliação da rede experimental de citros. Centro
de Citricultura Sylvio Moreira.
Supervisão: Mariângela Cristofani-Yaly
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Maria Gerolina Silva Cardoso, Bolsa DTI – Avaliação de genótipos de porta-enxertos de
citros sobre estresse com alumínio. Embrapa Mandioca e Fruticultura Tropical.
Supervisão: Antonio da Silva Souza, Embrapa Mandioca e Fruticultura Tropical
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Luciano Takeshi Kishi, Bolsa DTI – Bioinformática do programa. Centro de Citricultura
Sylvio Moreira.
Supervisão: Marcos A. Machado
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Rosely Pereira da Silva, Bolsa PDJ – Produção de haplóides de citros. Embrapa Mandioca
e Fruticultura Tropical.
Supervisão: Walter dos Santos Soares Júnior
Estudante de Doutorado ( x ) Inclusão ( ) Exclusão
Justificar:
Os alunos relacionados abaixo foram aprovados em programas de pós graduação e estão
desenvolvendo suas Teses dentro do INCT Citros:
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Rosangela Naomi Inui, Curso Biologia Funcional e Molecular da Unicamp, com o tema
‘Prospeção e análise da atividade de peptídeos antimicrobianos no controle de patogenos
de citros’.
Orientação: Marcos A. Machado
Co-orientação: Juliana Freitas Astúa
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Danila Souza Oliveira Coqueiro, Curso Biologia Funcional e Molecular da Unicamp, com o
tema ‘Expressão gênica diferencial induzida por elicitores nos patossistemas
huanglongbing e CVC.
Orientação: Marcos A. Machado
Co-orientação: Alessandra Alves de Souza
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Tahise Magalhães de Oliveira, Curso de Genética e Biologia Molecular da Universidade
Estadual de Santa Cruz (Ilhéus), com o tema ‘Transformação genetica de citros para
tolerância a seca’.
Orientação: Marcio Costa
Co-orientação: Walter Soares Filho, Embrapa Mandioca e Fruticultura Tropical
Estudante de Mestrado ( ) Inclusão ( ) Exclusão
Justificar:
Os alunos relacionados abaixo foram aprovados em programas de pós graduação e estão
desenvolvendo suas Dissertações dentro do INCT Citros:
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Alexandre Dias Dutra, Curso de Ciências Agrárias da Universidade Federal do Recôncavo
Baiano. Bolsa CAPES.
Orientação: Abelmon da Silva Gesteira
Co-orientação: Walter Soares Filho, Embrapa Mandioca e Fruticultura Tropical
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Diana Matos Neves, Curso de Genética e Biologia Molecular da Universidade Estadual de
Santa Cruz (Ilhéus). Bolsa CAPES.
Orientação: Abelmon da Silva Gesteira
Co-Orientação: Márcio G.C. Costa
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Joadson Dutra de Souza, Curso de Genética, Biodiversidade e Conservação da
Universidade Estadual do Sudoeste da Bahia, Campus de Jequié. Bolsa CAPES.
Orientação: Antonio Carlos de Oliveira
Co-Orientação: Marcos A. Machado
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Luciana Faldoni, Curso de Agroecologia e Desenvolvimento Rural da UFSCar. Bolsa
CAPES.
OrientaçãoL Kátia Critina Kupper
Co-Orientação: Mariângela Cristofani-Yaly
Estudante de Graduação ( x ) Inclusão ( ) Exclusão
Justificar:
Os alunos relacionados abaixo estão desenvolvendo atividades de Iniciação Científica no
programa:
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Gabriela Marteloso Carrer (Bolsa IC) – Uniararas/SP.
Joice Fernanda Garbin (Bolsa IC) – UFCG/PB.
Jose Alberto Diogo (Bolsa IC) – Unicamp/SP.
Patrícia Herrmann Corrêa – (Bolsa IC) – Uniararas/SP.
Salete Rocha (Bolsa IC) – UFCG/PB.
Fernanda Nara Mauricio - (Bolsa ITI-A) – Uniararas.
Gessica Laize Berto Gomes (Bolsa II-A) – UFCG/PB.
Janaína Andréa Mendes – (Bolsa ITI-A) – Uniararas.
Mariana Cardoso Miguel - (Bolsa ITI-A) – UFSCar.
Mariela Thim Vitorino (Bolsa IC) – UFSCar/SP.
Outros (Bolsa de Capacitação ou Desenvolvimento Tecnológico) ( x ) Inclusão ( ) Exclusão
Justificar:
Os bolsistas relacionados abaixo estão envolvidos com as atividades do programa:
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Samanta Marengo, bolsa de Especialização na empresa Diversity Arrays Technology, em
Yarralumla, Austrália, para desenvolvimento de marcadores tipo DArt para citros.
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Adriano Malosso, bolsa DTI nas atividades de transformação genética de citros.
Orientação: Raquel Luciana Boscariol-Camargo.
Jacqueline Camargo Olivato, bolsa DTI nas atividades de genoma funcional de Xylella
fastidiosa. Orientação: Alessandra Alves de Souza
Silvia de Oliveira Dorta, bolsa DTI nas atividades de genoma funcional de plantas.
Orientação: Helvécio Della Coletta Filho
Orientação: Andrzej Kilian
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Responsável p/ laboratório associado ( x ) Inclusão ( ) Exclusão
Justificar:
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Ver acima.
Pesquisador ( x ) Inclusão ( ) Exclusão
Justificar:
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O pesquisador Abelmon da Silva Gesteira foi incluído no programa em função de sua
contratação pela Embrapa Mandioca e Fruticultura Tropical.
Pesquisador ( ) Inclusão ( x ) Exclusão
Justificar:
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O pesquisador Alexandre Morais do Amaral (Embrapa, porém sediado no Centro de
Citricultura Sylvio Moreira) assumiu o Labex da Inglaterra, afastando-se, portanto, das
atividades técnicas do programa.
DESCREVER OS MECANISMOS DE INTERAÇÃO UTILIZADOS ENTRE GRUPOS DE
PESQUISA PARTICIPANTES DO INCT
Alem da comunicação rotineira entre os membros do programa com o coordenador, foi
estabelecido um sistema de gestão de projetos com coordenadores definidos por área, sendo o
coordenador responsável por consolidação da informações e intercâmbios entre os grupos. Na
sede do INCT Citros foi feita uma reunião geral com todos os participantes do programa em 01 e 02
de outubro de 2009 para discussão geral (ver programa em anexo).
A gestão financeira é feita diretamente pelo Centro de Citricultura que concentra todas as compras
e prestações de contas. Todos os grupos participantes receberam planilhas financeiras com
valores aprovados pelo CNPq e FAPESP (grupos de São Paulo). A execução orçamentária é feita
com base na disponibilidade financeira do programa.
RELATAR EVENTUAIS DIFICULDADES ENCONTRADAS ENTRE OS GRUPOS DE PESQUISA
PARTICIPANTES DA REDE E POSSÍVEIS MECANISMOS UTILIZADOS PARA SUPERAR
ESTAS DIFICULDADES:
Embora sem afetar a condução do programa, merece ser destacado que nem sempre a equipe vê
no INCT Citros um programa conjunto. Existe a tendência de vê-lo como a reunião de projetos
individuais. A expectativa é que no segundo ano essa visão se altere.
HOUVE A INCLUSÃO OU EXCLUSÃO DE INSTITUIÇÕES E EMPRESAS? ( x ) SIM ( ) NÃO
EM CASO POSITIVO INDIQUE O NÚMERO:
Instituição de Ensino e/ou Pesquisa ( x ) Inclusão ( ) Exclusão
Justificar:
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Centro de Biologia Molecular Estrutural CeBiME) da Associação Brasileira de Tecnologia
de Luz Sincronton (ABTLus). O grupo tem atuado em genoma funcional e comparativo de
citros, alem de ter grande experiência em biologia molecular e estrutural, de acordo com as
linhas de atuação do INCT Citros.
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Departamento de Ciências Biológicas de Faculdade de Ciências e Letras da UNESP/Assis,
por ser um grupo novo e expansão, com experiência em mapeamento genético de citros. O
Dr. Dario Palmieri atuou no nosso grupo como Pós Doutorando durante o Instituto do
Milênio. Grupo emergente na área de genética e biotecnologia de plantas.
RESULTADOS OBTIDOS / METAS
ENUMERE E COMENTE OS RESULTADOS CIENTÍFICOS E/OU TECNOLÓGICOS OBTIDOS
ATÉ O MOMENTO PARA
A – PESQUISA
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Construído mapa genético com marcadores moleculares para Poncirus trifoliata e
Citrus sunki para avaliação de tolerância ao huanglongbing (greening).
A expressão de genes que codificam terpeno sintase (enzima chave na via de
síntese de óleos essenciais) é controlada ao nível de desenvolvimento nos frutos
de laranja doce.
Análises de rendimento e qualidade de óleos essenciais de tangerinas estão em
avaliação, bem como de expressão de genes que codificam terpeno sintases.
Clonados genes que codificam para limoneno hidroxilases, enzimas da via de
síntese de óleos essenciais, em vetores para expressão heteróloga.
Clonados promotores específicos de genes que se expressam no floema.
Comprovada a existência de enzima de restrição no genoma de Xylella fastidiosa,
agente causador da clorose variegada dos citros.
Mutante para formação de biofilme em Xylella fastidiosa comprova o envolvimento
do processo de formação de biofilme no desenvolvimento da clorose variegada
dos citros.
Obtidas plantas autotetraploides de variedades de porta-enxertos (limão Cravo,
tangerinas Cleopatra e Sunki, citrumelo Swingle, citrange Troyer e dois
citrandarins) e de variedades de copa (laranjas doces, tangor Murcott, tangerinas
Poncan e Clementina e Fortunella Meiwa).
Construção de bibliotecas genômicas de Candidatus. Liberibacter americanus,
agente do huanglongbing dos citros.
Seqüenciado parcialmente o genoma de Candidatus. Liberibacter americanus com
tecnologia 454 a partir de imunocaptura da bactéria em psilídeos.
Construção do primeiro chip de DNA de citros com a base de dados do genoma
seqüenciado no Centro de Citricultura Sylvio Moreira.
Análise através de chips de DNA dos genes de laranja doce durante o processo de
infecção por Ca. Liberibacter americanus.
Ampliada a base de dados de genoma expresso de citros com seqüências de
outros países. A base passou de 300 mil para 550 mil seqüências expressas.
Construído o primeiro mapa de proteoma do fungo Alternaria alternata, agente da
mancha marrom dos citros.
Construído o primeiro mapa de proteoma do ácaro Brevipalpus phoenicis, agente
da leprose dos citros.
Avaliados a expressão dos principais genes relacionados ao florescimento da
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laranja Valência através de chip de DNA (microarranjos).
Avaliados plantas transgênicas de laranja doce para tolerância ao huanglongbing e
ao cancro cítrico.
Produzidas plantas transgênicas de porta-enxertos com maior tolerância ao
estresse hídrico.
Seqüenciamento de genoma expresso do fungo Guignardia citricarpa, agente da
mancha preta dos citros.
Monitorado a flutuação populacional da bactéria Candidatus Liberibacter spp em
plantas de laranja doce em condições de campo.
Avaliado com chips de DNA os principais genes de Poncirus trifoliata e Citrus sunki
associados a infecção por Phytophthora parasítica.
Publicado o primeiro estudo sobre mapeamento de QTL (Quantitative Trait Loci)
para resistência à leprose dos citros.
Marcadores moleculares associados ao gene Ctv no mapa de Poncirus trifoliata
foram encontrados no mapa de citrumelo Swingle
Mapeados QTL (Quantitative Trait Loci) para resistência a gomose de
Phytophthora no mapa de ligação de citrumelo Swingle.
Foram encontrados marcadores moleculares microssatélites específicos para
algumas variedades de tangerinas e híbridos.
Seleção de 37 citrandarins (híbridos de Citrus sunki vs Poncirus trifoliata) como
potenciais porta-enxertos com resistência a tristeza, gomose e morte súbita do
citros, sendo que dentre eles alguns apresentam elevada tolerância ao estresse
hídrico.
Seleção de oito híbridos de limão Cravo vs citrumelo Swingle e onze híbridos de
limão Cravo vs Poncirus trifoliata resistentes à gomose de Phytophthora e à
tristeza dos citros.
Seleção de nove variedades de tangerinas resistentes à leprose.
Obtidas novas populações de híbridos de tangerina para seleção de plantas
resistentes a mancha marrom de alternaria.
Selecionados os porta-enxertos nanicantes: citrandarins Clementina x trifoliata,
Cleópatra x Swingle 715 e 1.614, Cleópatra x Rubidoux e Cleópatra x Cristhian.
Eles induziram a formação de laranjeiras Valencia com altura inferior a 3,5.
Comprovado que limão cravo Limeira, Ipanema e Pennivesiculata assim como o
limão Volkameriano Catania-2 e os limões rugosos FM, Schaub, do Cabo, 58329 e
58328 são tolerantes a morte súbita dos citros.
Demonstrado que os citrumelos Swingle, W-2, Swingle, F.80-7, F.80-5, F.80-8,
F.80-6, F.81-18, F.80-3 e 4481 são tolerantes a morte súbita dos citros e ao
declínio dos citros.
Comprovada a primeira incompatibilidade copa com porta-enxerto em laranja
Valência enxertada sobre trangpur (Limão Cravo x Citrange Carrizo) 1524.
Selecionados dez híbridos de tangor Murcott vs laranja Pêra e tangerina Cravo vs
laranja Pêra com resistência a mancha marrom de alternaria e com qualidade da
fruta com grande potencial para o mercado de fruta fresca.
Feito o levantamento de tolerância à mancha marrom de alternaria e ao cancro
cítrico no germoplasma de citros.
Comprovada a existência de resistência à mancha preta dos citros em laranja doce
de maturação tardia.
Concluída a caracterização nutracêutica e agronômica de frutos de laranjas
sanguíneas.
Produzido clones de Yuzu para a indústria de óleo essenciais.
Estabelecido o Sistema Protegido do Banco Ativo de Germoplasma de Citros com
recursos IAC/APTA/SAA.
Construído a primeira plataforma de micro arranjos de DNA para estudos de expressão
gênica global de laranja doce, tangerina e Poncirus trifoliata.
Duplicado o banco de genoma expresso de citros (ESts) em colaboração com grupos no
exterior.
Primeiros relatos da expressão diferencial de genes em laranja doce afetada por
Candidatus Liberibacter americanus, uma das bactérias causadoras do huanglongbing dos
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citros.
Identificação de híbridos de porta-enxertos de citros com tolerância à gomose de
Phytophthora e à tristeza (Programa Embrapa).
Identificação de híbridos de porta-enxertos com maior tolerância à seca (Programa
Embrapa).
Identificação de híbridos com potencial para plantas ornamentais.
B – FORMAÇÃO DE RECURSOS HUMANOS
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Incorporados 06 (seis) pós doutorando ao Programa.
Incorporados 03 (três) alunos de Doutorado e 04 (quatro) de Mestrado ao Programa.
Quatro bolsistas DTI (nível de graduação) estão atuando no Programa.
10 novos alunos de IC passaram a participar do Programa.
C – TRANSFERÊNCIA DE CONHECIMENTO E TECNOLOGIA
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Participação em eventos técnico-científicos.
Parceria com a empresa Fischer Agropecuária SA na montagem de experimentos para
avaliação de potenciais porta-enxertos e copa de citros em suas fazendas. As potenciais
variedades deverão ser utilizadas pela empresa, respeitando-se os direitos previstos na Lei
de Proteção de Cultivares.
Iniciada parceria com a empresa Cambuhy Agrícola (Matão) para a montagem de
experimentos de avaliação de variedades copa originadas do programa de melhoramento
do Centro de Citricultura.
D – EDUCAÇÃO E DIVULGAÇÃO DA CIÊNCIA
Nada a acrescentar.
ENUMERE OS IMPACTO(S) CAUSADO(S) PELAS AÇÕES E RESULTADOS DO PROJETO
PARA A AMPLIAÇÃO, MELHORIA E CONSOLIDAÇÃO DA COMPETÊNCIA TÉCNICOCIENTÍFICA NACIONAL PARA
A – PESQUISA
Embora a melhor maneira de medir impacto de pesquisa seja através de publicações
referenciadas. Essas estão relacionadas no relatório de atividades. No entanto, o INCT Citros tem
também o potencial de efetivamente reunir todos os pesquisadores brasileiros que trabalham com
citros, particularmente na área de melhoramento.
Com o avanço do huanglongbing (HLB, greening) o Centro iniciou a instalação do Sistema
Protegido do Banco Ativo de Germoplasma de Citros sob telado com proteção contra insetos
vetores de doenças. Ao BAG Citros foram incorporados mais de 500 novos híbridos resultantes do
programa de Melhoramento do Centro. Portanto, o Centro de Citricultura, como uma das unidades
do INCT Citros, continuará a ser um fornecedor de material básico com certificação genética e
fitossanitária.
O Centro continuou sendo referencia no fornecimento de material básico de propagação (sementes
de porta-enxertos e borbulhas de variedades copa), muito embora forneça essencialmente para
multiplicação de novas borbulheiras no setor privado. Nos últimos anos reduziu significativamente o
volume de borbulhas e sementes fornecidas, uma vez que o setor privado cada vez atende às suas
próprias necessidades.
- Métodos de diagnóstico de doenças de citros – ajustes os métodos para diagnóstico de Xylella
fastidiosa, Ca. Liberibacter spp e Xanthomonas axonopodis pv citri utilizando-se de PCR com
iniciadores fluorescentes, o que permite rapidez, precisão e economia nos testes. A metodologia
está sendo transferida para a Clínica Fitopatológica de Citros.
- Variedades livres de patogenos – Com a rotina de limpeza clonal por microenxertia, variedades de
clones novos e velhos do BAG Citros foram recuperadas e foram incluídas no programa de
proteção em ambiente protegido. Merece destaque a limpeza e liberação para testes de variedade
Yuzu, de interesse para a indústria de essenciais.
- Registro de variedades - Foram solicitados registros de 11 novos cultivares no RNC/MAPA
somando-se aos mais de 60 previamente registrados.
- Plantas geneticamente modificadas – Dentre vários eventos de transformação genética, algumas
plantas mostraram-se tolerantes ao cancro cítrico. Liberação controlada no ambiente está sendo
solicitada junto à CTNBio.
- Novos genes e promotores – A partir das informações sobre genoma foram identificados novos
genes e promotores dentro do grupo dos citros, permitindo a continuidade dos trabalhos de
obtenção de variedades geneticamente modificadas com genes do próprio grupo.
B – FORMAÇÃO DE RECURSOS HUMANOS
Praticamente todos os participantes de iniciação científica, treinamento técnico, pós graduação e
pós doutorado encontram-se inseridos em atividades do Programa e deverão ser capacitados em
áreas como biologia molecular, bioinformática, genética e melhoramento de citros. Todos são
potenciais pesquisadores que poderão estar envolvidos com pesquisa de citros no futuro próximo,
portanto, com efeito multiplicador.
C – TRANSFERÊNCIA DE CONHECIMENTO E TECNOLOGIA
Parte considerável de participam do INCT Citros, particularmente no Centro de Citricultura em
Cordeirópolis, estão envolvidos em eventos de transferência representado por eventos no Centro
destinados a produtores, extencionistas, viveiristas, etc. São eventos de caráter técnico destinados
a usuários diretos da informações tecnológicas (agronômicas). No período desse relatório foram
organizados e efetivados os seguintes eventos:
o
- 2 . Dia do Huanglongbing, no dia 13 de março de 2009, com sete palestras e mais de 300
participantes.
o
- 3 Dia do Huanglongbing, no dia 12 de março de 2010, com oito palestras e cerca de 350
participantes.
o
- 10 . Dia do Limão: no dia 26 de março de 2009, com três palestras e cerca de 60 participantes.
o
- 11 . Dia do Limão: no dia 25 de março de 2010, com debates sobre mercado e exportação e 55
participantes.
o
- 4 . Dia do Porta-Enxerto, em 30 de abril de 2009, com três palestras e 30 participantes.
o
- 5 . Dia do Porta-Enxerto, em 07 de maio de 2009, com três palestras e 25 participantes.
-XII Dia da Tangerina, concomitantemente com o 1º Encontro de Citricultura na Região Sudoeste
de São Paulo e o VIII Dia de Campo de Tangerina, em Capão Bonito (SP), no dia 18 de junho de
2009, com sete palestras e 80 participantes.
a
- 31 Semana da Citricultura, de 01 a 05 de junho de 2009, com apresentação de 41 palestras e
cerca de 6 mil participantes.
a
- 32 Semana da Citricultura, de 07 a 11 de junho de 2010, com apresentação de 43 palestras e
aproximadamente 8 mil participantes.
a
- 35 Expocitros, junto com a Semana da Citricultura, com participação de 54 empresas do setor
citrícola.
a
- 36 Expocitros, junto com a Semana da Citricultura, com participação de 45 empresas do setor
citrícola.
o
- 15 . Dia do Viveirista em 11 de agosto, com quatro palestras e 250 participantes.
o
- 9 . Dia da Laranja em 21 de outubro, em sistema de entrevista tipo Roda Viva com 150
participantes.
D – EDUCAÇÃO E DIVULGAÇÃO DA CIÊNCIA
O Centro de Citricultura, coordenador do INCT Citros, possui publicação anual da revista Laranja,
considerada técnico-científica, com grande penetração no setor de produção. A partir de 2010 a
revista passa a se chamar Citrus Research & Technology, com o objetivo de fomentar a melhoria
na qualidade de publicações e atender a sistemas de indexação exigidos pela Capes. (ver
citrusrt.centrodecitricultura.br).
O Centro ainda mantém um curso anual de citricultura destinado a alunos em final de curso e
recém formados, com abrangência em todas as áreas da citricultura. O curso tem carga de 80
horas. O Curso de Doenças de Citros e seu manejo, regularmente ministrado uma vez ao ano,
envolve aspectos aplicados no controle de doenças de citros e destina-se a todos os profissionais
do setor.
PARA FINS DE DIVULGAÇÃO, RELACIONAR RESULTADOS OBTIDOS QUE MEREÇAM
DESTAQUE PARA O DESENVOLVIMENTO CIENTÍFICO, TECNOLÓGICO E/OU SOCIAL:
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Mutante para formação de biofilme em Xylella fastidiosa comprova o envolvimento
do processo de formação de biofilme no desenvolvimento da clorose variegada
dos citros.
Seqüenciado parcialmente o genoma de Candidatus. Liberibacter americanus com
tecnologia 454 a partir de imunocaptura da bactéria em psilídeos.
Construção do primeiro chip de DNA de citros com a base de dados do genoma
seqüenciado no Centro de Citricultura Sylvio Moreira.
Ampliada a base de dados de genoma expresso de citros com seqüências de
outros países. A base passou de 300 mil para 550 mil seqüências expressas.
(Consórcio Internacional do Genoma Citros)
Construído o primeiro mapa de proteoma do fungo Alternaria alternata, agente da
mancha marrom dos citros.
Construído o primeiro mapa de proteoma do ácaro Brevipalpus phoenicis, agente
da leprose dos citros.
Avaliados plantas transgênicas de laranja doce para tolerância ao huanglongbing e
ao cancro cítrico.
Publicado o primeiro estudo sobre mapeamento de QTL (Quantitative Trait Loci)
para resistência à leprose dos citros.
Mapeados QTL (Quantitative Trait Loci) para resistência a gomose de
Phytophthora no mapa de ligação de citrumelo Swingle.
Seleção de 37 citrandarins (híbridos de Citrus sunki vs Poncirus trifoliata) como
potenciais porta-enxertos com resistência a tristeza, gomose e morte súbita do
citros, sendo que dentre eles alguns apresentam elevada tolerância ao estresse
hídrico.
Seleção de oito híbridos de limão Cravo vs citrumelo Swingle e onze híbridos de
limão Cravo vs Poncirus trifoliata resistentes à gomose de Phytophthora e à
tristeza dos citros.
Seleção de nove variedades de tangerinas resistentes à leprose.
Obtidas novas populações de híbridos de tangerina para seleção de plantas
resistentes a mancha marrom de alternaria.
Selecionados os porta-enxertos nanicantes: citrandarins Clementina x trifoliata,
Cleópatra x Swingle 715 e 1.614, Cleópatra x Rubidoux e Cleópatra x Cristhian.
Eles induziram a formação de laranjeiras Valencia com altura inferior a 3,5.
Comprovado que limão cravo Limeira, Ipanema e Pennivesiculata assim como o
limão Volkameriano Catania-2 e os limões rugosos FM, Schaub, do Cabo, 58329 e
58328 são tolerantes a morte súbita dos citros.
Demonstrado que os citrumelos Swingle, W-2, Swingle, F.80-7, F.80-5, F.80-8,
F.80-6, F.81-18, F.80-3 e 4481 são tolerantes a morte súbita dos citros e ao
declínio dos citros.
Selecionados dez híbridos de tangor Murcott vs laranja Pêra e tangerina Cravo vs
laranja Pêra com resistência a mancha marrom de alternaria e com qualidade da
fruta com grande potencial para o mercado de fruta fresca.
Comprovada a existência de resistência à mancha preta dos citros em laranja doce
de maturação tardia.
Estabelecido o Sistema Protegido do Banco Ativo de Germoplasma de Citros com
recursos IAC/APTA/SAA.
RESULTADOS EM NÚMEROS
A – INDICADORES DE PESQUISA
NÚMEROS DA PRODUÇÃO TÉCNICO-CIENTÍFICA E ARTÍSTICA NO PERÍODO
(anexar referências):
TIPO
LIVROS
QUANTIDADE
0
CAPÍTULOS DE LIVROS
ARTIGOS PUBLICADOS EM PERIÓDICOS NACIONAIS
ARTIGOS PUBLICADOS EM PERIÓDICOS INTERNACIONAIS
TRABALHOS
APRESENTADOS
EM
CONGRESSOS
NACIONAIS
TRABALHOS
APESENTADOS
EM
CONGRESSOS
INTERNACIONAIS
SOFTWARE
PATENTE
PRODUTOS
PROCESSOS
PRODUÇÃO ARTÍSTICA (ESPECIFICAR)
OUTROS (ESPECIFICAR):
0
09
08
12
07
0
0
0
0
0
B – INDICADORES DA FORMAÇÃO DE RECURSOS HUMANOS
NÚMEROS DA FORMAÇÃO DE RECURSOS HUMANOS NO PERÍODO
TIPO
QUANTIDADE
ENCERRADOS:
INICIAÇÃO CIENTÍFICA
MESTRE
DOUTOR
PÓS-DOUTOR
OUTROS (ESPECIFICAR):
5
1
0
0
0
EM ANDAMENTO:
INICIAÇÃO CIENTÍFICA
MESTRE
DOUTOR
PÓS-DOUTOR
OUTROS (ESPECIFICAR): BOLSAS DTI (APERFEIÇOAMENTO)
19
11
09
07
04
C – INDICADORES DE TRANSFERÊNCIA DE CONHECIMENTO E TECNOLOGIA
NÚMEROS DA PRODUÇÃO NO PERÍODO
(especificar e anexar referências):
TIPO
Dias Tematicos
Semana da Citricultura
QUANTIDADE
05
01
D – INDICADORES DE EDUCAÇÃO E DIVULGAÇÃO DA CIÊNCIA
NÚMEROS DA PRODUÇÃO NO PERÍODO
(especificar e anexar referências):
TIPO
QUANTIDADE
Revista Laranja
01
Curso de Citricultura
01
Curso de Doenças de Citros
01
INFORMAÇÕES ADICIONAIS
DESCREVER OUTRAS FORMAS DE DISPONIBILIZAÇÃO PÚBLICA DOS RESULTADOS DO
PROJETO:
Vídeo institucional do INCT Citros.
Página do Programa: http://www.centrodecitricultura.br/inct_citros.php
DESCREVER AS MELHORIAS IMPLANTADAS NAS INSTALAÇÕES FÍSICAS DA SEDE E DOS
LABORATÓRIOS
ASSOCIADOS
AO
INSTITUTO, COMO
ADPTAÇÕES
FÍSICAS,
EQUIPAMENTOS, ETC.
Embora o programa INCT Citros esteja contratado a pouco mais de um ano, foi somente a partir de
novembro de 2009 que o recurso do CNPq começou a ser utilizado. Houve dificuldades no uso do
Cartão de Pesquisa, posteriormente trocado por depósito em conta corrente. O único processo de
importação encaminhado ao CNPq em dezembro de 2009, até a presente data (junho de 2010)
ainda não se concretizou. Para complicar, o CNPq afirma que não mais irá importar nesses
projetos, repassando a função às instituições sedes do INCT. Um complicador quando a instituição
não tem capacitação para isso e o projeto está em nome do coordenador. Os grupos fora do
Estado de São Paulo dependem desse serviço para suas atividades no INCT.
De qualquer modo, foram adquiridos equipamentos com recursos da FAPESP para o Centro de
Citricultura (citometro, termociclador para RT-qPCR e servidor para análise de dados de
seqüenciamento). Para a Esalq foi adquirida estufa climatizada para manutenção de plantas
transgênicas.
HOUVE ATIVIDADES DE INTEGRAÇÃO COM OUTROS INCT’S: ( X ) SIM ( ) NÃO
EM CASO POSITIVO DETALHAR:
INCT de Controle Biorracional de Insetos pragas, da Universidade Federal de São Carlos, em
trabalhos sobre doenças de citros, como mancha marrom de alternaria, mancha preta (Guignardia
citricarpa) e clorose variegada dos citros (CVC), em parceria com a Profa. Dra. Maria de Fátima
G.F. da Silva.
CONSIDERAÇÕES FINAIS
COMENTAR OUTROS ASPECTOS RELEVANTES DO DESENVOLVIMENTO GERAL DO
PROJETO
De modo geral, o programa está sendo conduzido a contento, mas poderia ter avançado mais na
sua primeira parte (Plataforma de Informação Genômica) em função de desencontros dentro do
Consórcio Internacional e da saída do Join Genome Institute como centro de seqüenciamento do
genoma com tecnologia Sanger. Essa atividade está sendo conduzida no AlphaHudson Institute of
Biotechnology da Universidade do Alabama. Por outro lado também, houve um aumento
expressivo na base de dados de genoma expresso exigindo capacidade de processamento que
não havia no grupo, pois a estrutura usada no Instituto do Milênio (programa anterior ao INCT)
encontrava-se desatualizada e sem capacidade de processamento para aquele volume de dados.
QUAL O PAPEL DO INCT PARA A FORMAÇÃO DA REDE DE PESQUISA?
Como colocado acima o INCT tem sido uma oportunidade do grupo entender que o trabalho em
rede nem sempre é a soma de projetos individuais. Efetivamente existe o trabalho em rede em
alguns frentes do Programa, como na transgenia, onde o grupo tem boa afinidade. Nas atividades
de genoma esse trabalho fica restrito ao grupo do Centro de Citricultura, o único de tem
envolvimento maior com esses resultados. Nas atividades de melhoramento os grupos caminham
paralelos, muito provavelmente em função da natureza dessa atividade (experimentos de campo
de longa duração, áreas e focos distintos, material genético diverso). Como parte considerável
dessas atividades também estão no Centro de Citricultura, o INCT está se tornando o suporte
essencial ao programa de melhoramento.
AVALIE A INTERLOCUÇÃO DO INCT COM O CNPq E DEMAIS FINANCIADORES DO
PROGRAMA:
De modo geral, sempre muito boa. A equipe do CNPq responsável pelo INCT (Ana Lucia, Elpígio,
Alcebíades) sempre foram solícitos e colaboradores. A equipe da importação também. O impasse,
sobre se o CNPq continuará ou não a importar, persiste. Ofício encaminhado à Presidência ainda
não foi respondido.
Na FAPESP o contato principal é com a Diretoria Administrativa nos processos de compra e
prestação de contas.
Anexar relatório de resultados parciais, de no máximo 50 páginas, contendo:
1. Comitê Gestor – reuniões realizadas e decisões
Em anexo.
2. Atividades de cooperação entre os grupos de participantes do INCT.
Descritas no relatório técnico.
3. Atividades de cooperação entre INCT’s e com outras instituições (empresas, ongs,
instituições governamentais, etc).
4. Principais resultados técnico-científicos
Relatório técnico (em inglês)
5. Eventos nacionais e internacionais: apresentação de trabalhos, organização de cursos,
seminários; palestras; mesas redondas.
6. Atividades de formação e capacitação de recursos humanos
7. Perspectivas e futuros desdobramentos.
No relatório técnico.
LOCAL E DATA: Cordeirópolis, 07 de junho de 2010.
Marcos A. Machado
Coordenador
Instituto Nacional de Ciência e Tecnologia de
Genômica para o Melhoramento de Citros
National Science and Technology Institute of
Genomic for Citrus Breeding
FAPESP 08/57909-2
CNPq 573848/08-4
First Annual Report
Coordinator: Marcos A. Machado
Members of the Program
Centro APTA Citros Sylvio Moreira, Instituto Agronômico
Embrapa Mandioca e Fruticultura Tropical
Esalq/USP
Cena/USP
Instituto Biológico
Universidade de Mogi das Cruzes
Universidade Federal de Campinas Grande
Universidade Estadual do Sudoeste da Bahia
Universidade de Santa Cruz (Ilhéus)
Unicamp
Estação Experimental de Citricultura de Bebedouro
Cordeirópolis – SP
April 2010
Instituto Nacional de Ciência e Tecnologia
de Genômica para o Melhoramento de
Citros
National Science and Technology Institute
of Genomic for Citrus Breeding
FAPESP 08/57909-2
CNPq 573848/08-4
Program Summary
The citrus industry is one of the leading activities of the agribusiness in
Brazil, with the states of Sao Paulo, Sergipe and Bahia as the main citrus
producers. Brazil is by far the main producer and exporter of frozen
concentrated (FCOJ) and not from concentrated (NFC) orange juice in the
world. The low Brazilian yield (approximately 2 boxes 40.8 kg/tree/year) is
highly associated to the incidence of pests and diseases (resulting in a
substantial influence over the production costs), the production in non-irrigated
areas, and the narrow genetic basis that has been used.
It is estimated that more than 60 % of the costs for citrus production in
Brazil are associated with control of pests and diseases. Diseases like citrus
variegated chlorosis, leprosis, black and brown spot, sudden death, citrus
canker, gummosis, tristeza and, recently, huanglongbing (HLB, former
greening). Indeed, in recent years, the citrus industry has witnessed substantial
structural changes in its structures due to diseases like HLB. Most of the
models for the management of the disease vectors, including chemical control,
have failed due to their high economic and environmental costs, which enhance
even more the importance of studies on genetic breeding as a powerful and
durable strategy to control diseases. The Centro de Citricultura Sylvio Moreira
(CCSM) – IAC and Embrapa have been investigating citrus breeding for a long
time as well as producing and evaluating new genetic materials by using
biotechnological tools to accelerate genetic gains. As the leading member of the
Millennium Institute of CNPq /MCT (2002-2005), CCSM constructed an
extensive database on expressed citrus genome (CitEST), which comprises
tools for studying genetic breeding and comparative and functional genomics of
citrus and some of its pathogens. Besides producing the largest collection of
genomic data worldwide, the project enlarged profoundly the number of new
citrus hybrids under field evaluation, molecular markers for genetic mapping
and discovery of new genes discovery with potential association to disease
resistance.
This current program reflects the continuation of the program “Millennium
Institute”, not only bringing together the main groups that investigate citrus in
Brazil, but also focusing on subjects related to genetic breeding and functional
genomics of the citrus group, looking forward on the integration of three
platforms:
Platform of Genomic Information, which focuses on studies regarding
comparative and functional genomics to enlarge the basis of information,
including the complete genome associated with the International Consortium on
Citrus Genome and the database on expressed citrus genome, studies on gene
expression (microarrays) of citrus and some of its pathogens, support to
bioinformatics and genetic transformation to produce modified plants based on
data from the citrus genome.
Platform for Genomic Application, which the focus is the plant-pathogen
interaction in limiting pathosystems faced by the citrus industry and the plantenvironment interaction (water stress). Such studies will allow enlarging the
knowledge useful in other activities of the Program.
Platform of Apllied Genetics, which represents the advanced interface of
breeding from which previously generated data (markers, maps, genes,
promoters), as well as genetic material (hybrids of controlled crosses and
events of transformation already available) will be validated under field
conditions, to permit a reliable approach to the strategy of marker-assisted
breeding. From this platform, new materials will be released for the Brazilian
citrus industry.
The multi-disciplinar and multi-institutional network of the Program includes,
besides the CCSM as the coordinator member, Embrapa Mandioca e
Fruticultura and its network for citrus breeding, Embrapa Recursos Genéticos e
Biotecnologia, Escola Superior de Agricultura Luiz de Queiróz (ESALQ/USP)
and Centro de Energia Nuclear na Agricultura (Cena), Universidade Estadual
de Campinas (Unicamp), Instituto Biológico, Universidade Federal de Campina
Grande, Universidade Estadual do Sudoeste da Bahia (Vitória da Conquista),
Universidade de Mogi das Cruzes (UMC), and University of Florida.
Report Summary
The CitEST (Database of expressed citrus genome) was doubled and
shared with other citrus databases around the world. Thus the NSTI Citrus has
an expressive coverage of the expressed genome, which will support the
activities of annotation and anchorage of the complete citrus genome. The new
database needs to be processed and assembled as soon as the hardware
platforms are working. The current computer facilities in our laboratory are not
able to process this amount of data. Although originally planned to be conduced
in our facilities, the sequencing of new EST libraries with the objective to
increase the number of unigenes of other citrus species will be carried out
abroad at lower costs and higher efficiency. We also decided to use new
technologies for sequencing since such data will be useful for both EST
assembly and re-sequencing activities included in the Program. More important
than perform the sequencing activities inside the group is to acquire and to
process the generated data.
To accomplish this goal, we contacted Sequencing companies that
perform pyrosequencing, since it had been proposed as a third party service.
Using the Genome Sequencer FLX Instrument, powered by GS FLX Titanium
series reagents, we will be able to generate more than 1,000,000 individual
reads with improved Q20 read length of 400 bases per 10-hour instrument run.
Therefore, one run can generate 400 MB, which is more than the estimated
genome size. To get the job done we will need 4 runs per species and that is
the object of the discussion with the Sequencing companies.
The proposal to sequence the full genome of citrus within the
International Citrus Genome Consortium (ICGC) has faced problems of lack of
funds in US and Brazil, and lack of leadership especially in the European
groups. With the advance of new sequencing technologies, the ICGC decided
to include a set of such data and decrease the amount of sequences with
Sanger technology. Instead of the originally proposed 10x coverage, it was
decided to run 6x coverage with Sanger technology and 4x with 454 technology.
Thus Brazil, USA and Spain are in charge of 6x coverage (2x coverage each),
whereas France shall be responsible for 4x coverage, and Italy, 1x additional
coverage. Joint Genome Institute of DOE/US started to sequence 2x (American
part) but will not assume the sequencing of our part since they are moving to
the new sequencing technologies. AlphaHudson Institute of Biotechnology of
the University of Alabama will do the service for the NSTI Citrus.
Although the database did not reach the main program goals yet, it has
been used for several proposals in the NSTI Citrus, including search for new
genes, molecular markers inside the ESTs, and the built of microarray platforms
for studies on gene expression.
Slides were designed using UniGene selected from the CitEST database,
assembled from the ESTs submitted to NCBI. The sequences were used to
construct genome-wide 60-mer oligonucleotide cDNA microarrays by Roche
NimbleGen Systems using a multi-step approach to select probes with optimal
predicted hybridization characteristics. Slides were composed of 31,535
sequences of sweet orange (Citrus sinensis), plus probes for six additional
sequences, three probes per target, four copies on array, totalizing 378,420
probes of C. sinensis. Mixed slides (sweet orange, mandarin and Poncirus)
were also built. Additional arrays of mandarin and Poncirus trifoliata are also
planned as the database of unigenes are concluded. The DNA array technology
has been used as an approach for global gene expression of citrus and its
pathogens. Results on such approach are presented in the report, pointing out
the plant pathogen interaction in the pathosystems gummosis, leprosis, and
huanglongbing. Studies on interactions with Xylella fastidiosa and Xanthomonas
axonopodis pv. citri are underway.
Although the technology of DNA arrays has been the main proposed
approach for analysis of global gene expression, there is a tendency to replace
this transcriptome analysis using high throughput technology for sequencing.
Transcriptome by sequencing combines global gene expression with genome
studies and increase the efficiency in obtaining information in different citrus
pathosystems. However, for specific studies on gene expression the technology
of real time PCR has been more useful, and is well established in the routine of
several activities.
Expressive advance was done on the characterization of new candidate
genes for genetic transformation, all of them from the CitEST database. The
technology of transformation of juvenile tissue was well established with a
production of several potential events (transformed plants). The plants have
been challenged for biotic stresses to test for resistance or susceptibility to the
pathogens. Field experiments are planned after authorization of CTNBio.
In conclusion, both the platform of genomic information and of application
in the NSTI Citrus has extended the database of information on the genomes of
citrus and its pathogens, but has also pointed out emphasis in the use of new
approaches integrating with the breeding platform.
Next steps and priorities in these platforms will be:
•
to increase the database of expressed genome and its application
(annotation, molecular markers, new candidate genes);
•
to conclude the complete genome of citrus in the ICGC;
•
to conclude the experiments of global gene expression both by
transcriptome (microarrays and sequencing) or proteome, and
•
to produce new events of genetic transformation for disease
resistance.
The main challenge of the program is undoubtedly the integration of
information on genome to genetics at the breeding level. The proposed
approaches include the identification of molecular markers based on ESTs,
genetic mapping, genotyping of new population of hybrids, analysis of
expression QTLs, and production of genetically modified plants. An expressive
indicator of the program is the number o new hybrids under field evaluation both
as rootstock and scion potential varieties. Five populations of hybrids are being
used for genetic mapping with several classes of molecular markers including
DArts in a collaborative program with Australia.
With the increase of the citrus expressed genome database and the
availability of a complete genetic map with molecular markers, new approaches
could be launched. The possibility to map genes and to associate them in a
physical map of BAC libraries opens new perspectives in the associative
genetics. The Program should be prepared for this jump.
It should be pointed out that several field experiments have been
conduced with support of the industry, which maintain the orchards.
Next steps and priorities in these platforms will be:
•
to increase the number of molecular markers in the available
genetic maps;
•
to saturate the genetic maps with all classes of molecular markers;
•
to start a physical map using BAC libraries of three species;
•
to evaluate more hybrids of scion and rootstocks in the field;
•
to test genetically transformed varieties under field conditions.
All scientific and technical activities of the NSTI Citrus include
undergraduate and graduate students from several graduate programs.
Progress Report
1. Platform of Genomic Information
Platforms of genomic information are considered as the combined data both
from the citrus plant genome and its pathogens, which will play a role during the
program activities. The platforms have concentrated on studies regarding
comparative and functional genomics, the completion of the Citrus genome
sequencing (International Citrus Genomics Consortium) and the enrichment of
the expressed citrus genome databases, investigations on gene expression
(microarrays) of citrus plants as well as some of their pathogens, support to
genetic transformation based on data from the expressed citrus genome and
strengthening the bioinformatics to support the development of the tools that will
be needed during the development of the program.
Objective 1a. Increase CitEST database.
Goal 1a1: Construct and sequence cDNA libraries of sweet orange, mandarin,
trifoliate orange, Rangpur lime, and Sunki mandarin up to 30,000 unigenes per
species (approximately 120 thousand ESTs/species, in addition to the existing
CitEST database).
Indicators: Number of sequenced libraries
As described in the proposal, CitEST was composed of more than 280
thousands sequences, which resulted in more than 91 thousands unigenes.
From that, Citrus sinensis, C. reticulata and Poncirus trifoliata accounted for
most of the sequences, 32,121, 18,873, and 12,873 unigenes, respectively. For
increasing the CitEST database, we sequenced ESTs of 17 different new
libraries from different species as listed below.
Common name
Sweet orange
Mandarin
Poncirus
Species
Citrus sinensis
Citrus reticulata
Poncirus trifoliata
Library
name
CS
CR
PT
Number Number of
of library
ESTs
6
2
3
12,767
10,368
17,368
Sour orange
Tahiti lime
Mexican lime
Rangur lime
Sweet lime
Sunki mandarin
Swingle citrumelo
Citrus aurantium
Citrus aurantifolia
Citrus latifolia
Citrus limonia
Citrus limettiodes
Citrus sunki
Citrumelo swingle
CA
CG
LT
CL
CM
TS
CW
Total
1
1
1
2
1
1
1
19
960
960
960
5,278
480
1,536
10,552
3417,869
The goal of this part of the Program was to construct and sequence cDNA
libraries from sweet orange (C. sinensis), mandarin (C. reticulata), P. trifoliata,
C. sunki, and Rangpur lime (C. limonia). To these species we added others that
were interesting to our group but still we will accomplish the proposed goal for
each of the initially proposed species. However, due to the new technologies
that are fully available for DNA sequencing, we are planning to change the
method for increasing the CitEST database. Instead of the capillary Sanger
sequencing we are planning to use the Next-generation platform from Ilumina,
which is able to generate around 1.5 Gb per run. To make a rough comparison
with the Sanger technology, our reads are close to 750 bp maximum, which
means that it is proposed to sequence 90 Mb per species and, therefore, just
one run of the Genome Analyser II (Illumina) is enough to generate more bases
sequenced than the proposed for all the species together.
Because of that, we made contact with different companies that have this
service and chose Macrogen as service provider. We discussed the technical
conditions for sequencing our materials and we are now setting up the
bureaucratic aspects of the contract.
The material to be used for these experiments will be obtained from
experiments that were already set up for this project, like the evaluation of citrus
sudden death in the field, or material of plants infected with both bacteria of
huanglongbing.
The next step toward the accomplishment of this goal is to set the
agreement with Macrogen in relation to bureaucracy and prepare total RNA
from all the plant material to send for sequencing.
Goal 1a2: Process and analyze ESTs (approximately 310 thousand) database
and unigenes from all of the species of the Program and make available
annotation and search services.
Indicators: Number of assembled unigenes (singlets + high quality contigs).
We downloaded the data sequence from the University of California Riverside (214,224 ESTs) that comprises libraries prepared from different
laboratories in the world and includes different species. These sequences were
used for assembly with the CitEST set in our computational setup but we
completely failed in getting it done because our processing capacity was much
lower than the required for the task. On the other hand, small sets of ESTs were
successfully assembled, confirming the need for a more powerful setup to work
with large sets of sequences. The computational setup was included in the
original proposal.
Species
CitEST
Harvest
CitEST
+
Harvest
CS
112,420 89,182
201,602
CR
48,627
87,016
135,643
PT
34,920
20,981
5,5901
CA
6,797
960
7,757
CG
7,377
0
7,377
LT
6,380
0
6,380
CL
6,666
0
6,666
CM
2,077
0
2,077
TS
2,320
0
2,320
CW
6,908
8,758
15,666
TC
0
2,679
2,679
CF
0
1,728
1,728
CD
0
468
468
CY
0
1,635
1,635
ST
0
817
817
Total
234,492 2456,979 1682,1201
np: not clustered
New
Assembled
Unigenes
np
np
np
5,294
3,989
3,754
1,407
np
1,378
6,799
1,987
1,153
327
728
703
Because of that, we contacted several suppliers for increasing this
capacity by acquring new equipments. We talked to IBM, Apple and Silicon
Graphics, deciding for the former (see Goal 1.c.1 as well). A cluster was
acquired and is now in process of import. So the next step is to get the machine
working and do all the necessary assemblies.
Goal 1a3: Generate and release SSR markers based on ESTs.
Indicators: Number and validation of SSR markers (primers, amplification
conditions etc).
Coordinators: Mariângela Cristofani-Yaly, Marco Aurélio Takita and Luciano T.
Kishi
We implemented a service in our server to identify SSR markers in the
EST databases. For that we used Misa (pgrc.ipk-gatersleben.de/misa/) and
obtained the following results with the very first CitEST set of unigenes.
Number
Sequences
Identified
SSRs
SSR
containing
sequences
sequences
containing
more than
1 SSR
Number of
SSRs
present in
compound
formation
CS
32.121
26.588
CR
18.873
10.830
PT
12.873
10.794
CA
5.607
3.495
CG
4.327
2.779
LT
4.883
3.184
CL
5.945
8.642
CM
4.540
7.028
TS
2.652
4.768
14.416
6.866
6.066
2.253
1.785
2.178
4.026
2.861
1.842
5.988
2.254
2.377
750
573
659
1.962
1.759
1.107
7.544
2.389
2.994
699
565
591
3.216
3.023
2.083
The validation of some of these SSRs was done as mentioned in
Objective 3a. The main problem was the bioinformatics resource available for
the Program so far. As mentioned before, the server we have is not able to
assemble some sets of ESTs available in CitEST right now, mainly the most
comprehensive ones. As soon as the new machines arrive (the equipments are
expected to arrive within the next four months) we will do the assembly to
analyze the whole EST datasets.
Objective 1b. Participate in the effort of sequencing, assembling, and
annotating a model citrus genome with a local database available for the
Program in collaboration with the International Citrus Genomics Consortium
(ICGC).
Goal 1b1: Assemble bioinformatics database for the complete citrus genome;
Indicators: Percentage of partial and final coverage of the genome throughout
the sequencing and assembling processes.
Goal 1b2: Assemble tools that allow the comparison between complete versus
expressed genomes;
Indicators: Available structured database and tools in Brazil.
Coordinators: Marcos A. Machado, Juliana Freitas-Astúa, Marco Aurélio Takita
and Luciano T. Kishi
The below text reproduces the statements and agreements among the
members of the International Citrus Genomic Consortium (ICGC) in the meeting
at Joint Genome Institute/DOE, Walnut Creek, CA, 2007 and 2008.
‘The Steering Committee of the ICGC met to discuss plans and changes to our
previously described goals and strategies, as outlined in an earlier White Paper and in
the so-called Valencia Declaration of April 2003. The outcome conclusions following
two days of deliberations were accepted and approved unanimously by the members of
the Steering Committee. These conclusions represent the essence of the ICGC plan
for international collaboration in sequencing a citrus genome, and they are listed below.
1. The long-term goal of the international consortium is a freely available, high quality
reference genome sequence for Citrus.
Immediate short-term and mid-term goals
established by the ICGC are the selection of a target genome for sequencing by midsummer 2007, and the securing of funding commitments by late-fall 2007.’
Actually the sequencing started by mid-summer 2009 funded by Florida Citrus
Production Adviser Council (FCPRAC). A 2x cover was carried out by JGI using
cosmid libraries and Sanger quality. The Brazilian part (1x) will be sequenced
by HudsonAlpha Institute for Biotechnology at University of Alabama. The
contract between the Coordinator (Marcos A. Machado) and HudsonAlpha was
already signed, and payment will be done by FAPESP.
‘2. Based on various experiences from sequencing other heterozygous diploid plants
and the known allelic diversity of citrus, the highest quality reference sequence would
be provided by a haploid citrus genome.’
A haploid Clementine from Institute Valenciano de Investigaciones Agrícolas
(IVIA), Spain, was chosen as model species.
‘3. Requirements for the target sequencing genome:
•
Haploid (or di- or tri-haploid) tree that may be freely distributed internationally;
•
The candidate tree would preferably be free from infection with pathogens,
exhibit robust growth (as a partial guarantee against a defective genome), and
be relatively easy to maintain;
•
The candidate tree chromosome number should be verified.
•
The genome should be evaluated to assure that it is free large deletions or
other defects, as well as confirmed to be mono-allelic using molecular assays
including SSR marker assays, microarrays, and short-read sequencing
methods.’
Allmost all these activities were concluded inside the ICGC. The Brazilian group
was in charge to check the homozigozyty of the material using SSR markers.
‘5. Requirements for whole genome sequencing partnership:
•
Target of 8-10x total coverage (3 billion bp, US$3M estimated cost)
•
To begin ~fall 2007
•
Rapid deposition of data with free access to all partners
•
Sequencing partnership, pending funding approval, across Brazil, France,
Spain Italy and USA;
•
Use common libraries, participants contribute reads through exchange
•
Plan in place for the retention (multiple locations), maintenance and distribution
of clone resources (BACs and fosmids.)‘
With the possibility to use new technologies for sequencing the original proposal
was changed in the following way:
- 6x coverage with Sanger quality (2x for US, 2x for France, 1x for Brazil and 1x
for Italy) and 4x 454 quality (2x for Spain, 1x for Brazil and 1x for US).
- 1x Sanger quality of Brazilian group will be concluded by HudsonAlpha
Institute (University of Alabama), and will be paid by FAPESP (0.5x) and
Funarbe (0.5x) (Embrapa).
6.
Other components identified as essential components of genome sequencing
efforts:
•
A BAC library from the selected genome source, and BAC-end sequences
(greater than 7x, at least two enzymes)
•
Physical map of the selected genome, to aid genome sequence assembly
•
Probably no new large-scale EST sequencing is needed but complete
sequences from full length cDNAs would be desirable
•
Organized effort to characterize variation across Citrus and related Rutaceae
(species and varieties)
•
Integration of existing genetic maps to have highest density combined map
•
Informatics
o
Assembly to be performed at the various participating sequencing
centers, and released to the ICGC partners
o
Analysis of variation (e.g., sweet orange shotgun vs. genome)
o
“Structural annotation” (automated pipelines exist for gene prediction)
o
Distribution (various capabilities for “genome browsers”)
o
Curation’
BAC libraries of the haploid genome are available at Amplicon Express
(www.amplicon-express.com) and available to be purchased. We are ordering
not only libraries of haploid Clementine but also libraries of sweet orange
(Pineapple) and Poncirus trifoliata (Rubidoux).
Several activities above are not direct responsability of our group but can be
included in the Program as soon as they are concluded.
Objective 1c. Establish sequencing and assembling databanks of other citrus
species from the pyrosequencing effort.
Goal 1c1: 4x genome coverage (~380 Mb/species) of Ponkan mandarin,
Poncirus trifoliata and Rangpur lime with pyrosequencing sequences.
Indicators: Percentage of partial coverage of the genomes of these species.
To accomplish the goal of this part of the Program we made contact with
the Sequencing companies to verify the costs for pyrosequencing since it was
proposed as a third party service. For that, using the Genome Sequencer FLX
Instrument, powered by GS FLX Titanium series reagents, we will be able to
generate more than 1,000,000 individual reads with improved Q20 read length
of 400 bases per 10-hour instrument run. Therefore, one run can generate 400
MB, which is more than the estimated genome size. To get the job done we will
need 4 runs per species and that is the object of the discussion with the
Sequencing companies.
So far Macrogen was the one that offered us the best deal. We are now
discussing the technical conditions for sequencing our materials and setting up
the bureaucratic aspects of the contract. The material to be used for these
experiments will be obtained from plants grown in greenhouses.
We also evaluated the performance of two systems in order to have a
better idea about the computational resources needed for the task. We used
two different softwares, Mira (www.chevreux.org/projects_mira.html) and
Newbler (454 Life Sciences) to assemble two sets of 454 reads as listed below.
Reads
Base pairs
Assembling
time
Software
610178
183.994.141
7 hours
MIRA
1140276
388.321.828
> 30 days
MIRA
1140276
388.321.828
> 40 days
NEWBLER
OS
linux x
86_64
linux x
86_64
linux x
86_64
Server
8 core 48 GRAM
2 core 16 GRAM
2 core 16 GRAM
The results show that for 1x coverage a 2-core 16 GRAM machine is not
enough to process the information reasonably fast. An 8-core 48 GRAM
machine performed much better than the other machine, so we considered that
the minimum for running the project. We discussed with the IT companies what
we need and what we could get for the resources we have and decided to buy a
system from Silicon Graphics with one head node of 2 6-core processors, 48
GRAM, two compute nodes of 4 6-core processors, 48 GRAM, and 1 compute
node of 2 6-core processor, 144 GRAM. So each node will have at least 12 core
and 48 GRAM and, therefore, we expected a much better performance.
As mentioned before, the machines are already in process of import. As
soon as they arrive, we will rerun the samples as well as test some other
multiprocessor softwares. To do the assembly of genomes from other species it
is necessary to have the complete genome of Citrus clementina available to
guide the assembly of other species.
Goal 1c2: Assemble tools for analyzing pyrosequencing sequences.
Indicators: Assembled databanks and available tools.
Goal 1c3: Assemble tools that allow the comparison between complete
genome
(Sanger
quality)
versus
partial
genomes
obtained
from
pyrosequencing.
Indicators: Available tools.
The goals 1c2 and 1c3 depend on the results produced in 1b and 1c1.
Objective 1d. Assemble and analyze citrus high-density microarrays for gene
expression and gene expression QTLs studies;
Goal 1d1: Assemble and analyze sweet orange high-density microarrays (380
thousand spots) for large-scale expression.
Indicators: Number of assembled and analyzed slides (approximately 36 slides
for each bioassay) for gene expression studies on the plant-pathogen and plantwater stress interactions.
Slides were designed using UniGene selected from the CitEST database,
assembled from the ESTs submitted to NCBI (GenBank accession numbers
EY649559 to EY842485). The sequences were used to construct genome-wide
60-mer oligonucleotide cDNA microarrays by Roche NimbleGen Systems using
a multi-step approach to select probes with optimal predicted hybridization
characteristics. One of the slides was composed of 31,535 sequences of C.
sinensis, plus probes for six additional sequences three probes per target, four
copies on array, totalizing 378,420 probes of C. sinensis. According to the
experiment schedule (see goals 2a 2h) and upon request new sets of arrays
can be ordered.
Goal 1d2: Assemble and analyze high density microarrays (380 thousand
spots) for large scale gene expression studies in mandarin.
for each bioassay) for gene expression studies on the plant-pathogen and plantwater stress interactions (see Objectives 1a to 1f and 1h).
Slides of a total of 62,876 UniGenes (31,583 of C. sinensis, 18,712 of C.
reticulata and 12,581 of P. trifoliata) selected from the CitEST database,
assembled from the ESTs submitted to NCBI (GenBank accession numbers
EY649559 to EY842485) were used to construct genome-wide oligonucleotide
cDNA microarrays by Roche NimbleGen Systems using a multi-step approach
to select probes with optimal predicted hybridization characteristics. Three
probes were selected per UniGene, comprising a probe set, and each probe set
is represented on the final array by two replicates. All probes were designed as
perfect match oligonucleotides. The slide was composed of 377,256 probes.
spots) of Poncirus trifoliata for large scale expression.
for each bioassay) for gene expression studies on the plant-pathogen and plantwater stress interactions (see Objectives 1f, 1g and 1i).
The available number of unigenes of Poncirus trifoliata both of CitEST
and public is not enough to build such array yet. As soon as the number of
unigenes increases they will be used in the arrays.
spots) of sweet orange and Ponkan mandarin for expression QTL studies.
for each bioassay) for expression QTL studies (see Objectives 3f).
The available number of unigenes of Ponkan mandarin is also not
enough to build feasible arrays. As soon as the number of unigenes increases
they will be used in the arrays.
Goal 1d5: Develop proteome maps of sweet orange and mandarin during the
process of Alternaria infection.
Indicators: Number of maps consistently produced and number of identified
spots (see Objective 1g).
Two
dimensional
gel
electrophoresis
(2DE)
and
tandem
mass
spectrometry (MS/MS) were used to evaluate the interaction of citrus and
Alternaria alternata in a proteomic level. Young leaves (2-3 cm length) of
susceptible Murcott tangor and tolerant Pera sweet orange were inoculated with
a conidial suspension (106 spores/mL) of A. alternata. In order to normalize the
patterns of translated proteins before the inoculation, plants were maintained for
two weeks in a plant growth chamber at 27 °C, 12h photoperiod (light/dark), and
70% relative humidity. Healthy plants were used as experimental control.
Twelve hours after inoculation, leaves were collected, ground in liquid nitrogen,
and the proteins were extracted with phenol according to Hurkman and Tanaka
(1986). Isoelectric focusing was performed in polyacrylamide IPGstrips (pH 310NL) using 750g of proteins. For each genotype, spots differentially detected
between healthy and inoculated plants were excised and digested in-gel by
trypsin. MS/MS analysis are been conducted in a Waters-Micromass Q-TOFUltima API (ESI-MS/MS) coupled to a Waters CapLC system.
2DE gels were successfully obtained for healthy and inoculated plants of
each genotype (Figure 1). When treatments were compared, initial data
exhibited 191 and 76 differentially detected spots (p0.05) in Murcott tangor and
Pera sweet orange, respectively. All the spots were excised from 2DE gels for
further MS/MS analysis. In October 2009, the Mass spectrometer showed
technical problems and could not be used until March 2010. In order to improve
analysis and reduce the amount of peptides to be sequenced, only those spots
showing p0.01 were selected, corresponding to 56 spots from Murcott and 39
from Pera. During March 2010 9 digested proteins from Murcott and 21 from
Pera were sequenced (Table 1).
Amino acid sequences were automatically deduced using ProteinLynx
v.2.2.5 software (Waters) and Mascot server 2.3 (Matrix Science) coupled to
CitEST (http://biotecnologia.centrodecitricultura.br/), NCBI (http://www.ncbi.nlm.
nih.gov) and UniPROT/Swiss-Prot (http://www.expasy.ch/sprot) databanks. In
order to confirm automated deduction, sequences are been manually checked
(de
novo
sequencing)
using
PepSeq
(Waters)
and
mMass
v.3.0
(www.mmass.org) softwares. As soon as the mass spectrometer starts to work,
MS/MS analysis will be performed in order to conclude all the proteomic-related
experiments.
A2
A1
pH 3-10 NL
pH 3-10 NL
B2
B1
pH 3-10 NL
pH 3-10 NL
Figure 1. Coomassie Brilliant Blue G250 stained 2DE gels (pH 3-10NL) of healthy (A)
and 12 hours inoculated (B) plants of Murcott tangor (1) and Pera sweet orange (2).
Table 1. Citrus trypsin-digested proteins submitted to MS/MS analysis
Objective 1e. Assemble microarrays of the complete genome of citrus pathogens for
the functional genomics studies.
Goal 1e1: Microarray assay with the complete genome (~2,400 ORFs) of
Xylella fastidiosa.
for each bioassay) for the studies of biofilm formation during the process of
colonization of the xylem vessels.
DNA microarray chips were designed with thirteen 60-mer oligonucleotide
probes per gene, according to the available X. fastidiosa genomic sequences.
Probes were synthesized by Roche-NimbleGen, Inc. (Madison, WI, USA) in situ
by photolithography on glass slides using a computer-generated randomized
pattern on the array. All gene probe set were repeated five times in each chip
and we used three biological replicates per treatment. The raw data were
normalized, briefly, a Robust Multichip Average (RMA) convolution model was
applied for background correction, and the corrected probe intensities were then
normalized using a quantile-based normalization procedure. Differentially
expressed genes were identified, using the ArrayStar software, by Student's t
test (P.< 0.05) and the multiple test correction of raw p-values was performed
using the False Discovery Rate (FDR). We also used a cutoff of 2.0 fold change
in expression.
Goal 1e2: Microarray assay with the complete genome (of unkwnown size at
this point) of Candidatus Liberibacter americanus and Ca. L. asiaticus (if
bacterial DNA is available after cultivation).
for each bioassay) for the studies of infection in sweet orange.
The available database on genome of Ca. Liberibacter spp. does not
allow the construction of a robust array yet. As soon as more sequences are
submitted, allowing a minimal assembling of the genome, arrays will be
performed.
Objective 1f. Establish a routine analysis of large-scale gene expression using
RT-qPCR for gene validation.
Goal 1f1: Validate by RT-qPCR differentially expressed (up- or down-regulated)
genes in response to the interaction assays (see Objectives 1a to 1j, except for
1h).
Indicators: Number of genes validated for each assay.
To validate differentially expressed genes (up- or down-regulated) in
response to biotic stress [(Xylella fastidiosa, Phytophthora parasitica, and Citrus
leprosis virus C (CiLV-C)], relative quantification was made using the ABI
PRISM 7500 Sequence Detector System (Applied Biosystems). The primers
were designed using the ‘PrimerExpress’ (Applied Biosystems) program. All the
amplicons were sequenced using the automatic sequencer ABI 3730 to confirm
the amplification of the genes assessed. The primers amplification efficiency
were tested by serial dilution (at least 5) of cDNA samples and value of slope
assessed by E =10(-1/slope)-1. Selected primers were those with efficiency values
between 0.9 and 1. The reactions were done in triplicate using negative control
(without cDNA) to detect contaminants. Each reaction was composed by 2 μL of
cDNA, 1 μM by each primer (forward and reverse) and 12.5 μL of “SYBR green
PCR master mix” (Applied Biosystems); the final volume was adjusted for 25
μL. The reactions were incubated in 50ºC for 2 minutes, 10 minutes in 95ºC and
40 cycles of 15 seconds in 95ºC and 1 minute in 60ºC. The amplifications were
checked in ABI PRISM 7500. After amplifications, the reactions were submitted
to dissociation curve to identify contaminants in the test.
A short experiment to select the best normalizer genes was performed.
The -tubulin is frequently used for these types of studies without a robust
comparison with other candidate genes. For this experiment, the expression
levels of -tubulin, ubiquitin, ciclofilin, CEF2 and actin were tested with all the
samples of the experiments with CiLV-C inoculation. Their expression was
analyzed with the software GenEx, using the algorithm GeNorm. We concluded
that it is advisable to use two or three normalizers to provide a robust
comparison.
Gene expression of X.fastidiosa in biofilm condition - Some genes were
selected for validation by RT-qPCR, identified as probably related to persistent
cells. As endogenous control, genes with differential expressions in microarray
experiments are being tested.
Under tetracycline inhibitory conditions, 160 genes were differentially
expressed in DNA microarray experiments (114 genes repressed and 46
induced). From those, 10 were selected for validation by RT-qPCR. Under
copper inhibitory conditions, from the 868 differentially expressed genes in DNA
microarray experiments (407 repressed and 461 induced), 9 were selected for
validation by RT-qPCR.
The RT-qPCR assays are being conducted. The evaluation of possible
endogenous controls indicated that the genes XF1353 and XF0656 are good
candidates, since they were not differentially expressed from the control
(without antimicrobial compounds) in microarray experiments and RT-qPCR.
The expression of other genes, differentially expressed in microarray
experiments, is being tested.
Gene expression of Poncirus trifoliata, Citrus sunki and their hybrids during
infection of Phytophthora parasitica - In this study, genes differentially
expressed between hybrids of Poncirus trifoliata and Citrus sunki (resistant and
susceptible, respectively) may provide key candidates to identify transcripts
involved in disease resistance. We investigated gene expression in pools of four
resistant and four susceptible hybrids in comparison to their parents 48 hours
after P. parasitica inoculation using RT-qPCR. Our analysis searched
upregulated genes (fold > 2 and p-value 0.05) in the resistant genotypes
relative to the susceptible, found in previous microarray study. Nine genes were
selected due to the biological interest based on their function according to
CitEST database; among them, genes that encode enzymes participating in
defense-related
metabolic
pathways
such
as
the
biosynthesis
of
phenylpropanoids and antimicrobial compounds such as phytoalexins, flavonoid
biosynthesis and resistance genes such as CC-NBS-LRR and TIR-NBS-LRR.
Gene expression of Pera sweet orange, Murcott tangor and their hybrids during
infection of Xylella fastidiosa - A total of 163 unigenes in several functional
categories were detected as differentially expressed (Fold> 2) between the
pools in relation to resistant pools susceptible in microarray study. The
functional classification of induced transcripts revealed genes with defensive
function that candidates may be involved in resistance to CVC. Thirteen genes
were selected due to the biological interest based on their function according to
CitEST database, among them, genes that encode enzymes participating in
defense-related. The RT-qPCR assays are being tested for validation.
Gene expression of Pêra sweet orange and Murcott tangor in response to
infestation with Brevipalpus phoenicis non-viruliferous or viruliferous to CiLV This study compared the response of Pera sweet orange and Murcott tangor
(resistant and susceptible, respectively) to CiLV-C. Two-way ANOVA (p-value 0.1 and fold-change 2) analysis resulted in 80 differentially expressed genes,
68 of them upregulated and 12 downregulated. Among them, 11 genes were
chosen for validation by RT-qPCR based on their similarity with genes involved
in defense response to pathogens and fold-change. The RT-qPCR assays are
being tested for validation.
Gene expression in response to infection with Citrus leprosis virus C (CiLV-C) Hybrids of Pêra sweet orange and Murcott tangor were infested with
Brevipalpus phoenicis viruliferous for CiLV-C. These plants had been evaluated
for 6 years in prior field experiments order to find the most susceptible and the
most resistant individuals. A pool of seven resistant or seven susceptible plants
was collected in four biological repetitions for microarray analysis. The
comparison of the resistant pools to the susceptible ones showed 466 genes
differentially expressed. From these, 289 were upregulated and 177 were
downregulated. For validation, four genes upregulated and three downregulated
were chosen based on known genes for defense response to pathogens. In
addition to them, ten other genes without a clear function in the roll of plant
defense were chosen to try to identify genes not usually described.
Objective 1g. Increase the genetically modified citrus program.
Goal 1g1: Produce new sweet orange transformation events with constructions
derived from the Program database.
Indicators: Number of new constructions and events of transformation.
New constructions for candidate genes.
Defensins - Analysis of the citrus ESTs has shown the occurrence of at least
two genes encoding defensins similar to those described to other plant species
that present antifungal and/or antibacterial activities.
Primers were designed for the coding regions of these defensins that
resulted in the amplification of products of 815 bp (CsDef1) and 213 bp
(CsDef2) from Valencia sweet orange genomic DNA. The genes were cloned in
pGEM-T Easy vector (Promega) and submitted to DNA sequencing. CsDef1
has shown an intron interrupting the coding region while the same was not
observed for CsDef2. The defensin encoded by CsDef1 has 82 aa including 8
cysteins that are conserved among plant defensins and has shown 43.9%
sequence identity to defensin PDF1.4 from Arabidopsis thaliana. Amino acids
sequence analysis with the Psort program indicated the presence of a 26 aa
non-cleavable signal peptide and a putative localization in the endoplasmic
membrane. The defensin encoded by CsDef2 has 76 aa including the 8
conserved cysteins and has shown 59.7 % identity to A. thaliana PDF2.1. Psort
analysis indicated a 19 aa cleavable signal peptide and a high probability of
secretion to the outside of the cell.
The next step of vector construction is to isolate the cassettes
constructed and to transfer them to the binary vector pCambia2201
(www.cambia.org.au). After this, citrus plant transformation will be performed.
Citrus phloem protein 2 (PP2) promoter - The citrus phloem protein 2 (PP2)
promoter is capable to drive vascular tissue-specific expression of reporter
genes in citrus transgenic lines. The gene was characterized in the citrus
shotgun library and the upstream region was identified. Thus two regions (PP22 and PP2-3) of full promoter were isolated from leaves of Valencia sweet
orange (Citrus sinensis L. Osbeck) and cloned. These regions were digested
with Eco RI and Bgl II and cloned in the pCAMBIA 3301 vector. Citrus
transformation is in progress.
Npr1 from Citrus - Initially, using the Gene Projects environment of CitEST, we
selected 51 sequences either by npr1 keyword search or tBlast N. These
sequences were clusterized in 11 contigs and 6 singlets. The analysis of the
contigs allowed us to identify 12 clones carrying plasmids with sequences of
npr1 from Citrus. These clones used for plasmid extraction and DNA
sequencing. E. coli DH5 strain carrying plasmids from different CitEST
libraries were recovered from the -80oC freezers. The bacterial cells were grown
in 2 mL of LB medium, supplemented with ampicillin (100 μg/mL), at 37oC for 16
hours under rotation (250 rpm). Plasmidial DNA extraction was done using
alkaline lysis protocol.
The sequences were assembled in Seqman and 5 different forms of Npr1
were identified. To verify the absence of problems in the DNA sequence present
in each of the clone, new primers were designed using the contigs generated in
the assembly as templates. This was done because the fragment was large and
sequencing from the 5’ and 3’ ends was not enough to cover the entire
sequence. Twenty new primers were designed in Oligo Explorer 1.2
(http://www.genelink.com/tools/gl-oe.asp) and used for sequencing the DNAs.
P29 (putative coat protein) and P32 (movement protein) of Citrus leprosis virus
C (CiLV-C) - Genes coding for the viral proteins P29 and P32 were selected to
construct cassettes to induce silencing. This strategy is normally used to avoid
the virus multiplication in plants. The pKANNIBAL vector was used during the
cloning. Specific primers were designed for gene cloning in sense and
antisense using the program ‘Primer3’
After isolation of viral genes from the RT-PCR using cDNA of sweet
orange plants infected with CiLV-C, the genomic fragments (789 bp for P29 and
891 bp for P32) were purified, sub-cloned into vector pGEM-T (Promega) and
sequenced to confirm the inserts. The presence of the inserts was confirmed by
specific primers for the 35S promoter as well as primers for the genes of
interest.
As a next step, the constructions pKANNIBAL+sense gene (for the two
candidate genes) will be digested with specific enzymes for cloning of antisense
gene according to methods previously described.
Goal 1g2: Produce new transformation events of Rangpur lime, Sunki mandarin
and Swingle citrumelo (rootstocks) with constructions derived from the Program
database.
Indicators: Number of new constructions and events of transformation.
New constructions for candidate genes.
NAC4 and WRKY17 - The full-length cDNA sequences of the genes NAC4 and
WRKY17 were identified, cloned, and characterized from leaves of Citrus
reshni. These genes were excised from the pGEM-T vector, by means of
digestion with SalI and NotI, and subcloned in sense orientation at the same
restriction sites of the pUC118/CaMV35S plasmid. This plasmid contains the
CaMV
35S
promoter.
The
cassetes
have
been
now
excised
of
pUC118/CaMV35S, by digestion with PstI, and will be subcloned at the same
site in the pCAMBIA 3301 vector. This vector contains the chimeric genes bar
and gusA.
- Actions for the continuation and fulfillment of the goal in the next period.
In the next period, these constructions will be used to transform citrus
rootstocks.
P5CS - The coding region of p5cs gene (involved in the proline synthesis) from
Citrus sinensis was amplified by PCR, using Pfu DNA Polymerase with specific
primers. The gene amplified was cloned into pCR2.1 vector, using TOPO TA
Cloning kit (Invitrogen). After this, the coding region was isolated with restriction
enzymes BamHI and EcoRI and subcloned in p35SPT (pGreen vectors),
previously digested with the same enzymes. The expression cassette (P35Sp5cs-T35S) was isolated by digestion with EcoRV and cloned in pCambia
2305.1 in the SmaI restriction site. This vector was transferred to Agrobacterium
tumefaciens EHA105 and used for plant transformation.
Protocol for plant transformation - The vectors containing the desired
construction were introduced into EHA105 lines of A. tumefaciens, using the
freeze-thaw method. The verification of transformed colonies was done by PCR
using specific primers for the gene. Epicotyl segments of citrus (0.8 to 1 cm)
were obtained from seedlings germinated in vitro. Seeds were treated with
commercial bleaching solution diluted 1:3 with distillated water, for 20 minutes,
and placed for germination and elongation in the dark (4 weeks), at 27oC.
Epicotyl segment were inoculated with A. tumefaciens for 20 minutes. The
explants were incubated for 3 days at 24oC, and then transferred to EME
selection and regeneration medium, supplemented with BAP, cefotaxime and
kanamycin (100 mg L-1) or Finale® at 27oC, 16-h of light. Sprouts from
regenerated plants were analyzed by GUS hystochemical analysis and by PCR
with specific primers to detect the studied gene. Positive plants were
micrografted on selected rootstock for later acclimation in greenhouses.
Transformation of Rangpur lime and Sunki mandarin - Seeds of ‘Cravo Santa
Cruz’ Rangpur lime (Citrus limonia), ‘Sunki Maravilha’ mandarin (C. sunki), and
‘TSKxSW 314’ hybrid were collected in the active citrus germplasm bank
collection of Embrapa Cassava and Tropical Fruit (Cruz das Almas, BA). Seeds
were germinated in plastic dibble tubes (115 cm3 and 180 cm3) containing a mix
of Plantmax (Eucatex Agro.) substrate and coconut fiber, at 1:1 proportion,
under greenhouse conditions. Four months after germination, the plants were
transplanted to 15 L pots containing a mix of soil, sand, and Plantmax
substrate, at 2:1:1 proportion. After four months, plants of nucellar origin were
selected based on their uniformity and splitted in two groups: (i) a group of 4
plants of each genotype that were daily irrigated and (ii) a group of 11 plants of
each genotype that were drought stressed. All the pots were closed with
aluminum foil in order to allow the water loss only by transpiration. The water
content of the pots has been measured by time domain reflectometry (TDR)
probes, the stomatal resistance by a porometer, and the water potential by a
Scholander pump. Samples of leaf and root tissues have been collected for
further molecular analyses. At this point, three harvests of tissues were done
already. The RNA was extracted using the RNAqueous kit (Applied
Biosystems).
Objective 1h. Develop, improve, and implement bioinformatic tools in support
of the database.
Goal 1h1: Support the Program.
Indicators: Pipelines, services and interfaces in use.
Our citrus genome database, with 448,716 reads (CitEST, Harvest and
International Citrus Genome Consortium), requires constant updates to the
analysis of new sequences, including ESTs libraries on the following:
-
Submission System of electropherograms;
-
Pipeline to trim and assemble ESTs;
-
System Project Management (GP);
System for quantitative analysis of data from CitEST – in silico
hybridization;
Scripts to generate data in HTML format of the results of hybridization in
silico and to categorize and compare against a number of public databanks;
-
Support the work of expression by microarray, where it was necessary to
develop a database (MySQL) for storing microarray data and a web interface
(CGI-PERL) to make the results available, integrating with the Unipaper data
base;
-
Evaluation and development of scripts for new methodologies for
categorizing genes and systems for categorization via Gene Onthology;
-
Understanding
and
study
of
the
KEGG
metabolic
maps
for
implementation of unigenes in studies of metabolic pathways.
Difficulties found were related to low processing power and delay to
process the blasts and assembling programs with data of pyrosequencing.
2. Plataform of Genomic Application
Objective 2a. Transcriptome and gene expression profile to study citrusCandidatus Liberibacter spp. interactions
Goal 2a1: Characterize and compare at least two profiles (= bioassays) of
global gene expression of sweet orange infected with Candidatus L. spp.
Indicators: Number of differentially expressed genes putatively associated with
susceptibility to HLB.
To obtain healthy and infected plants, approximately 60 plants of sweet
orange (C. sinensis) originated from seeds and grafted onto Rangpur lime were
used. After reaching around 30 cm of vegetative growth, 25 plants were
inoculated with Ca. Liberibacter americanus (CLam), and other 25 with Ca.
Liberibacter asiaticus (CLas) by grafting of two buds infected from symptomatic
plants. All the plants were maintained under greenhouse conditions. Once
confirmed the presence of the bacteria in all of the inoculated plants, they were
pruned to induce sprouting and uniform vegetative growth.
The presence of the bacteria occurred approximately forty days after
pruning, with the appearance of the first shoots, having reached vegetative
growth of about 15 cm and tissues maturation of the first samples of infected
and healthy plants. The leaves and twigs tissues were collected from five citrus
plants positives for CLam; five plants positive for CLas, and three healthy
plants, with their developmental stage as uniform as possible. Plant tissues
were ground in liquid N2 and a small amount of different tissues of each plant
were evaluated to confirm the presence of the bacteria both by conventional
PCR and qPCR.
Once confirmed the presence of the bacteria in the tissues, RNAs from
these samples were extracted using the RNeasy Mini Kit (Qiagen) and sent to
NimbleGen for hybridization on microarrays containing about 32,121 speciesspecific unigenes of citrus (C. sinensis). CLas was first detected about 90 days
after inoculation (d.a.i) and the first symptoms appeared around 120-150 d.a.i in
approximately 95% of the infected plants. CLam was first detected 150 d.a.i and
the first symptoms appeared approximately 220 d.a.i in 30% of the inoculated
plants. Only three plants infected by CLas were confirmed by conventional
PCR. All other test plants were positive for CLam and CLas by qPCR. After 40
days of pruning it was possible to identify lack of uniformity in vegetative growth.
Moreover, plants infected with CLas exhibited leaf symptoms similar to nutrient
deficiency and, in plants infected with CLam, the leaves tended to curl down.
The RNA extracted from juvenile tissues (leaves and branches exemplified in
Fig.3) was of high quality and concentration (Fig. 4), varying from 790 to 4,496
ng/ul, ratio 260/280 varying from 1.53 to 2.12 and ratio 260/230 varying from
1.20 to 2.27. Preliminary results obtained by our group, using two biological
replicates, reveal 634 sweet orange genes differentially expressed after
inoculation with CLam compared to the healthy plants. From those, 419 were
induced and 215 repressed by CLam inoculation. Figure 5 A and B shows the
classification of the differentially expressed genes found according to MIPS
categorization.
Leaves
Branches
Figure 3. Sweet orange Plant 1 (Citrus
sinensis L. Osbeck) infected with CLam.
Leafs and branchs (juvenile tissues)
used in microarray analysis
Figure 4. RNA extracted of Sweet
orange Plant 1 (Citrus sinensis L.
Osbeck) infected with CLam. Leafs and
branchs tissues used in microarray
analysis.
Figure 5. Categorization of differentially expressed genes in sweet orange
(Citrus sinensis L. Osbeck) infected with Ca. Liberibacter americanus according
to MIPS database category of Arabdopsis thaliana.
Objective 2b. Transcriptome and gene expression profile to study citrus-Xylella
fastidiosa interactions.
Goal 2b1: Characterize and compare at least three profiles (= bioassays) of
global gene expression of sweet orange and mandarin infected with Xylella
fastidiosa.
susceptibility and resistance to CVC.
The experiments were conducted with plants of sweet orange and
mandarin grafted on Rangpur lime and kept in a protected environment. For
inoculation, 10l of X. fastidiosa suspension were deposited by needle.
Total genomic DNA (plant + bacteria) was extracted from inoculated
tissue after 1, 7, 14 and 21 days. These samples were used for the monitoring
of bacterial population through qPCR. The analyses were carried out with an
ABI PRISM 7500 Sequence Detector System. Each sample was tested in
triplicate with three biological replicates. The standard curve was produced
using known concentrations of genomic DNA of X. fastidiosa obtained from 10fold serial dilutions from 1x103 a 1x108 CFU per mL. This showed a linear range
with a correlation coefficient of 0.999. All the results reported from qPCR were
based on the number of cells of X. fastidiosa in each sample, which was
estimated from the standard curve. Negative (no template DNA) and positive
(DNA from X. fastidiosa) controls were included in all experiments to exclude or
detect any possible contamination.
It was found that sweet orange plants infected with X. fastidiosa showed
a gradual increase of population over time, which was expected due to the
susceptibility of this variety to CVC (Fig. 6). However, the population of bacteria
in tangerine remained stable at all time course evaluated (Fig. 6). This is
consistent with previous results where X. fastidiosa can stay for a certain period
of time in tolerant plants, but then the population decreased by a total absence.
Figure 6. Population mean of X. fastidiosa estimated by qPCR for analysis of
leaf samples taken from sweet orange and tangerine on various days after
inoculation. Bars represent errors of the averages of three biological replicates.
Originally the experiments were proposed to characterize and compare at
least three global gene expression profiles of sweet orange and tangerine
infected with X. fastidiosa. However, our group decided firstly investigate the
changes that occur in the early stage of response to infection that possible
involves resistance genes and signaling pathway. To determine which time
point could represent the time of expression of genes related with resistance
and signaling network, we did a pilot experiment with the four time points after
inoculation (1, 7, 14 and 21 days) in tangerine resistance plants through the
expression of some genes previously found in Citrus EST Project (CitEST).
Among the selected genes we chose those involved in early and delay stages
of plant response to pathogens (Table 2).
Table 2: ORFs from CitEST database possibly associated with resistance to X.
fastidiosa in mandarin.
ORF
Putative Function
NBS-LRR type disease
resistance protein
Resistance to plant pathogens
Apetala2/ethylene
responsive factor
Cell signaling
pad4
Involved in the synthesis of AS
and response to pathogens
npr1
Inductor of PR proteins
pr1
Inductor resistance and response
to pathogen
The same plant material collected from the experiment to follow up the
population of X. fastidiosa was used to do the pilot experiment. The same
material will also be used for microarray analysis. The total RNAs were
extracted with Trizol and treated with RNase-Free DNase Set. The samples
were done using a pool of three biological replicates for each time of evaluation
in plants infected or not (controls). For cDNA synthesis we used 500 ng/ l of
total RNA. The evaluations of gene expression were performed on the ABI
PRISM 7500 using relative quantification analysis. The detection of PCR
products was measured by monitoring the increase in fluorescence emitted by
SYBR green marker. The endogenous control (EC) used was -tubulin. For all
reactions the qPCR was performed in a dissociation curve to check for
nonspecific amplification resulting from contamination.
The results showed that all genes were induced after one day of
inoculation, but not apetala, which showed no expression at any time of
evaluation (Fig. 7). Based on these results we chose one day after inoculation
to evaluate the change of global gene expression by microarray. We expected
mainly identify the network of signalization genes in mandarin that might trigger
other genes and as a consequence reflecting in its resistance.
Figure 7. Relative quantification of the possible genes associated with
resistance in Poncan mandarin. cDNAs pool consisting of three biological
replicates for each time measured from plants challenged with X. fastidiosa, or
not. The samples were used for the quantification in the ABI PRISM 7500
Sequence Detector System (Applied Biosystems). The measures were
normalized using the threshold cycles (Cts) obtained for the amplifications of
the endogenous control run in the same plate. The values represent the fold
increase in gene expression compared with values obtained for cDNA from
plants without pathogen (calibrator). The results are averages of samples tested
in triplicate. Bars represent standard errors of the means.
Objective 2c. Transcriptome and gene expression profile to study citrusXanthomonas axonopodis pv citri interactions.
Goal 2c1: Characterize and compare at least two profiles (= bioassays) of
global gene expression of sweet orange and mandarin infected with
Xanthomonas axonopodis pv citri.
susceptibility and tolerance to citrus canker.
The experiments will be conduced in Spring 2011. Concentration of
bacteria, methods for inoculation into the tissue, and time of infections were
previously determinated.
Objective 2d. Transcriptome and gene expression profile to study citrus-Citrus
leprosis virus C (CiLV-C) interactions.
Goal 2d1: Characterize and compare at least two profiles (= bioassays) of
global gene expression of sweet orange, tangor, and lime infected with Citrus
leprosis virus C (CiLV-C).
susceptibility, resistance, and tolerance to leprosis.
Plants of the susceptible Pera sweet orange (C. sinensis) and the
tolerant Murcott tangor (Citrus sinensis x C. reticulata) were infested with
Brevipalpus phoenicis mite vector non-viruliferous or viruliferous for CiLV-C.
Samples were collected 48 hours post infestation for whole transcriptome
analysis. Total RNA from these samples were extracted using the RNeasy Mini
Kit (Qiagen) according to manufacturer´s instructions. The integrity, quality and
concentration of RNA was analyzed using denaturing gel and NanoDrop 8000
Spectrophotometer-Thermo Scientific equipment according to manufacturer’s
instructions and sent to Roche NimbleGen for hybridization on 385K density
microarrays containing 32,121, 18,873 and 12,873 unigenes from Pêra sweet
orange, Ponkan mandarin (C. reticulata) and Poncirus trifoliata, respectively.
These unigenes were obtained from the Citrus EST database (CitEST) of
Centro de Citricultura Sylvio Moreira/IAC.
Using a two-way ANOVA test (p-value 0.1 and fold-change 2), 80
differentially expressed genes were identified (Fig. 8). The majority of the upregulated genes were related to cellular communication/signal transduction
mechanism and transcription; the few genes related to cell rescue, defense and
virulence will be validated by RT-qPCR.
Upregulated
Figure 8. Categorization of differentially expressed upregulated genes in
Murcott tangor inoculated with CiLV-C according to MIPS database category of
Arabdopsis thaliana.
Because of the significant amount of work, as well as their economic
importance, we decided to compare the response of only two citrus genotypes
to leprosis: sweet orange (susceptible) and Murcott tangor (resistant/ tolerant).
On the other hand, another experiment was conducted aiming to find
differentially expressed genes in plants responding to Citrus leprosis virus C
(CiLV-C) infection. In this study, pools of seven highly tolerant and seven highly
susceptible hybrids from Murcott tangor and Pêra sweet orange were used for
comparison of response to the disease by microarray analysis. In order to
define susceptible and tolerant hybrids, they were infested by viruliferous mites
in 2002 and the symptoms were evaluated during six years. Symptomless
leaves of each hybrid were collected for RNA extraction. The experiment was
designed in ten experimental blocks, the leaves were collected from four blocks,
each being a biological repetition and each repetition containing seven
individuals. The same microarray chip containing 32,121; 18,873 and 12,873
unigenes from Pêra sweet orange, Ponkan mandarin and Poncirus trifoliata,
respectively, was used. The microarrays were performed by Roche Nimblegen
and Bayesian moderated T-test yielded 466 diferentially expressed genes (p.val
0.05 and fold-change 2). The differentially expressed genes and their
classification according to MIPS is showed in Figure 9. With exception of genes
encoding unknown proteins and with classification not clear yet, the majority
was related to metabolism, cell rescue, defense and virulence, and protein fate.
Up-regulated
Down-regulated
Figure 9. Categorization of differentially expressed genes in bulks of resistant
compared to susceptible hybrids according to MIPS database category of
Arabdopsis thaliana.
Goal 2d2: Characterize and compare at least two profiles (= bioassays) of
global gene expression of sweet orange, Murcott tangor, and lime infested with
the false spider mite Brevipalpus phoenicis (vector of CiLV-C).
response to the false spider mite.
The experiments will be conduced in Winter 2010. Number of mites, time
of virus transmission and incubation period were previously determined.
Objective 2e. Transcriptome and gene expression profile to study citrusCTV/CSD interactions.
Goal 2e1: Characterize and compare at least two profiles (= bioassays) of
global gene expression of Sunki mandarin and Poncirus trifoliata infected with
CTV.
susceptibility and resistance to tristeza.
The experiments will be conduced in Winter 2010. Rangpur lime will be
included in the evaluation.
Goal 2e2: Characterize and compare at least two profiles (= bioassays) of
global gene expression of Rangpur lime and Sunki mandarin infected with CTV
in plants exhibiting citrus sudden death (CSD) symptoms.
susceptibility and resistance to CSD.
Infected samples will be collected in the field in Spring 2011. Global gene
expression will be evaluated not only for Sunki mandarin, but also for Rangpur
lime and Swingle citrumelo, instead of Poncirus trifoliata.
Objective 2f. Transcriptome and gene expression profile to study citrusPhytophthora spp. interactions.
Goal 2f1: Characterize and compare at least two profiles (= bioassays) of
global gene expression of Sunki mandarin and Poncirus trifoliata infected with
Phytophthora parasitica.
susceptibility and tolerance to citrus gummosis.
A microarray containing 62,876 UniGene of P. trifoliata, Citrus sinensis
and C. reticulata selected from the CitEST database and prepared by
NimbleGen Systems was used for analyzing global gene expression 48 hours
after infection with P. parasitica. RNA was extracted from resistant and
susceptible hybrids (four each), and then the four samples were pooled into
resistant and susceptible samples.
A total of 8,522 UniGene transcripts distributed in several functional
categories (Figure 10) were detected as upregulated (fold change > 2) in P.
trifoliata relative to C. sunki, while 4,337 transcripts were detected as
upregulated in the resistant bulk samples relative to C. sunki, and 1,168
transcripts were upregulated in resistant hybrids relative to susceptible ones.
These analyses identified 102 upregulated (Figure 11) UniGene transcripts (pvalue 0.05) in all three comparative pair analyses, and nine of the top genes
with described functions retrieved from CitEST were selected for validation by
real-time RT-PCR (Figure 12). We encountered several previously reported
defense-related genes, such as one encoding CC-NBS-LRR and TIR-NBS-LRR
resistance protein, upregulated in the resistant genotypes compared to the
susceptible genotypes. NBS–LRR-mediated resistance has been identified
against numerous types of biotrophic or hemibiotrophic pathogens, including
fungi, oomycetes, viruses and bacteria, and these types of R genes have been
identified across a wide range of plants.
Our results showed that the libraries were useful for identifying genes
involved in the Phytophthora-citrus compatible interaction. On the other hand,
the use of a mixed-species DNA microarray was feasible because there was
enough complementarity among the libraries used to build the arrays. Pooled
samples can also be beneficial to find outgroup-specific expression profiles
despite large interindividual variation.
The genes that we have identified as upregulated across the resistant
genotypes will be valuable for ongoing work in eQTL mapping.
Figure 10. Functional categories according to MIPS classification scheme of
significantly upregulated UniGene transcripts (fold change > 2 and p-value 0.05) common to the three pairwise comparative analyses (Rub vs. Sunki, Pool
R vs. Sunki, and Pool R vs. Pool S) obtained using BLASTX searches against
the GenBank database.
Figure 11. Venn diagrams showing the differentially upregulated UniGene
transcripts common to the resistant genotypes based on three different
comparisons between resistant (Pool R) and susceptible (Pool S) F1 hybrids
and their parents, Citrus sunki (Sunki) and Poncirus trifoliata Rubidoux, (Rub),
respectively susceptible and resistant to P. parasitica, 48 hours after
inoculation. Summary of UniGene selection results by two methods: (A) Venn
diagram summarizing the number of UniGene transcripts identified using 2-fold
cut-offs without a p-value restriction; and (B) Venn diagram summarizing the
number of genes identified using 2-fold cut-off and p-value 0.05.
Figure 12. Validation of microarray data by quantitative real-time RT-PCR.
Real-time RT-PCR fold-changes are shown for nine genes upregulated in all
microarray pairwise comparisons (Rub vs. Sunki, Pool R vs. Sunki, and Pool R
vs. Pool S) 48 hours after P. parasitica inoculation, and wer compared with foldchanges obtained by microarray analysis. Real-time RT-PCR data were
normalized to the two most stable candidate endogenous control genes (UBQ
and CYP). Bars represent standard deviations of the means of three technical
replicates and three biological replicates. *, p-value 0.05; **, p-value 0.01.
Objective 2g. Transcriptome and gene expression profile to study citrusAlternaria alternata interactions.
Goal 2g1: Characterize ans compare at least two profiles of global gene
expression of sweet orange and mandarin during infection by Alternaria
alternata patotype tangerine.
susceptibility and tolerance to alternaria.
Instead
of
analyzing
gene
expression
using
microarrays,
the
transcriptome of the interaction between citrus and Alternaria will be carried out
by transcripts sequencing using the Illumina platform. The experiments are
planned for the next year.
Objective 2h. Citrus transcriptome and gene expression during water stress.
Goal 2h1: Characterize and compare at least two profiles (= bioassays) of
global gene expression of Sunki mandarin, Rangpur lime, and Poncirus trifoliata
under water stress conditions.
the response to hydric deficit.
Instead
of
analyzing
gene
expression
using
microarrays,
the
transcriptome of the interaction between the rootstostocks under water stress
conditions will be carried out by transcripts sequencing using the Illumina
platform. The experiments are planned for the next year.
Objective 2i. Xylella fastidiosa transcriptome and gene expression during
infection.
Goal 2j1: Characterize and compare conditions for the biofilm formation by
Xylella fastidiosa.
biofilm formation and pathogenicity.
Microarray analysis was peformed to identify genes associated with
programmed cell-death in Xylella fastidiosa biofilm. To accomplish that,
inhibitory concentration of copper and tetracycline were added when the biofilm
reached mature phase. The samples from three biological repetition where sent
to NimbleGen to microarray hybridization. Each oligonucleotide, which
represents one gene from X. fastidiosa genome, was spotted five times on the
chip. Each spot has 5 repetitions. The nomalization data was done using the
“quantile” methodology by NimbleScan software. The statistic analysis of the
normalized data was done using DNAStar software. The values of differentially
expressed genes were obtained using fold change more then 2 and T-Student
(95% of confidence) test.
3. Plataform of Applied Genetics
Objective 3a. Assess promising new citrus rootstock and scion hybrids under
biotic and abiotic stresses.
Goal 3a1: Assess rootstock trials for resistance to gummosis and tolerance to
CSD and tristeza.
Indicators: Number of assessed rootstocks; number of rootstocks with higher
or lower tolerance to CSD; number of rootstocks with higher or lower tolerance
or resistance to gummosis; number of rootstocks with higher susceptibility to
tristeza; number of rootstocks with potential to be recommended in new
experimental areas; number of rootstocks recommended as cultivars; number of
rootstocks registered in the “Serviço Nacional de Proteção de Cultivares” (or
National Plant Variety Protection Service).
Rootstock and scion breeding program of Instituto Agronômico
At this stage of the work, the agronomic characteristics of rootstock
hybrids under field and greenhouse conditions, with emphasis on disease
resistance, are being evaluated. In Table 3 there were described the
populations, traits and localities where the experiments were conducted.
Table 3. Description of the experiments in field and greenhouse, local,
pathosystem in evaluation and total number of plants evaluated.
Experiments in field
Hybrids
Sunki mandarin
x P. trifoliata
Sunki mandarin
x P. trifoliata
Pathosystem
Tristeza
Gummosis
Tristeza
Gummosis
Sudden death
Place
Cordeirópolis
Colômbia
Number
of
hybrids
Nr of
plants
Date
1104
2003
111 P/T
444
2004
111 V/T
165
V/T/LC
444
270
660
Experiments in greenhouse
Rangpur Lime x
Swingle
Tristeza
Gummosis
Cordeirópolis
120
480
2006
citrumelo
Rangpur Lime x
T. trifoliata
Sunki mandarin
vs P. trifoliata
Sudden death
Sudden death
Sudden death
Colina
Colina
Colina
120
65
198
720
2006
390
2006
594
2006
The results of the evaluation for resistance to CTV (Citrus tristeza virus),
CSD (Citrus Sudden Death) and Phytophthora gummosis provided hybrids with
level of resistance/tolerance similar to the resistant parental. Resistant/tolerant
hybrids to CTV and Phytophthora gummosis were obtained from the controlled
crosses: Citrus sunki x Poncirus trifoliata, C. limonia x P. trifoliata and C. limonia
x Swingle citrumelo. Some of them showed tolerance to hydric stress. These
hybrids have been included in breeding programs for citrus rootstocks.
The citrandarins (hybrids of Sunki mandarin x Poncirus trifoliata) were
evaluated for agronomic traits conferred by the rootstock to the scion variety
(Pera sweet orange) as water stress tolerance, plant height, incompatibility, fruit
quality and production. For tolerance to water stress, highly susceptible plants
and plants with tolerance comparable to Rangpur were found. In the present
study, only three hybrids displayed necrosis in the region of grafting, a typical
symptom of incompatibility between scion and rootstock. As for plant height, we
observed plants ranging from 0.99 to 2.37 m, indicating that rootstocks have
influence on their height. Thirty seven citrandarins were pre-selected with
potential for rootstocks varieties. Evaluations of the agronomic characteristics
and quality of the juice were performed in order to verify the importance of
rootstock on the development and ripening point of the scion variety, since
some hybrids induced early ripening of the scion (with the ratio above 16), late
ripening (with ratio below of 12) and hybrid plants with fruit ripening ideal for the
juice industry, with the ratio ranging between 14 and 16.
Evaluation of hybrids for scion varieties
The severity of ABS (Alternaria Brown Spot) was quantified by visual
assessment of the symptoms (scale of notes from 0 to 4). The hybrids were
evaluated in Bebedouro-SP, Botucatu-SP and Cordeirópolis-SP. The severity of
the CBS (Citrus Black Spot) was measured in 20 fruits of each treatment. We
used a scale consisting of six levels, 0 when there is no damage on the surface
of the fruit, and notes 1 to 6, corresponding respectively to the percentages of
areas damaged by 0.8%, 1.6%, 3.1%, 6.2%, 12.5% and 25%. The hybrids were
also evaluated in Cordeirópolis-SP.
For the evaluation of CVC, leaves of the hybrids were collected for total
DNA extraction and subsequent PCR diagnosis, quantifying the percentage of
infection from 0 to 100%. The hybrids were evaluated in Colina-SP.
A histogram of the distribution of mean severity of ABS for the hybrids in
each experiment is shown in Figure 13. It is observed that the highest disease
severity was detected in Botucatu.
Figure 13. Histogram distribution of alternaria brown spot severity in the
population of hybrids of tangor Murcott x Pera sweet orange (2009).
For CVC, during the evaluation performed in May 2009, 84 hybrids were
found PCR positive. The infection rate ranged from 25 to 100% among the
hybrids (Figure 14).
Figure 14: Histogram distribution of percentage of plants positive for CVC in the
Most hybrids showed notes of severity of CBS between 0 and 1 (0.8%
fruit area with lesions) and only five had notes between 1 and 3 (3.1%) (Figure
15).
Figure 15. Histogram distribution of citrus black spot mean severity in the
Rootstock breeding projects of Embrapa
Gummosis of Phytophthora - Nucelar seedlings from 25 genotypes were
cultivated in soil substrate containing Pinus spp. bark. After completing two
years of age, a portion of 5 mm in diameter of the bark was taken out by using a
cork borer at 15 cm from the bottom. Afterwards, a disk containing culture
media with Phytophthora parasitica var. nicotianae mycelium was inserted into
the lesion. Then, a section of the bark detached from the trunk was placed over
the mycelium disk. The inoculation spot was covered with wet cotton and
protected using tape. After the inoculation the seedlings were maintained at
25ºC and photoperiod of 12 hours of light. The experiment was installed in
complete random design with 25 genotypes and 10 replicates. Each
experimental plot was represented by one plant.
The genotypes Poncirus trifoliata, ‘Beneke’ selection, ‘Swingle’ citrumelo,
common ‘Sour Orange’, ‘Sunki’ mandarin, ‘Tropical’ selection, as well as the
hybrids: HTR - 010, HTR - 051, HTR - 069, HTR - 127, LRF x (TR x LCR) - 004,
LRF x (TR x LCR) - 005, LVK x LVA - 009, TSKC x CTSW - 018, TSKC x
CTSW - 019, TSKC x CTSW - 033, TSKC x CTSW - 041, TSKC x CTSW - 064,
TSKC x TRFD - 007 and TSKC x TRBK - 010 presented high tolerance to
Phytophthora foot rot. The genotypes, Rangpur’ lime, ‘Santa Cruz’ selection,
‘Sunki’ mandarin, ‘Maravilha CNMPF 02’ selection, as well as the hybrids LCR x
LRF - 034, TSKC x CTTR - 002, TSKFL x CTARG - 002 and TSKFL x CTARG 028 presented intermediate susceptibility. The common ‘Sunki’ mandarin
presented high susceptibility to the pathogen.
Citrus sudden death – Three experiments were installed from March 2007 to
July 2009 in areas with high incidence of CSD located in the county of
Colômbia, north of the State of São Paulo. The experimental design was in
random blocks with three replicates and variable number of plants per plot
(three to five). Disease evaluation, of possible viral etiology, will be carried out
mainly by observing the presence of yellowish color in the internal tissues of the
bark of rootstocks in the region of the functional phloem in contrast to the
tissues of the bark of the scions which are lighter.
Tristeza - 20 cm stems from 40 hybrid nucelar seedlings selected from the
CGBP were collected from the different quadrants of the scions and, after
removing the bark, evaluated regarding stem pitting symptoms using a grade
scale. The indirect ELISA test was carried out in order to detect the presence of
the virus in the tissues of the hybrids evaluated.
The TSKFL x CTARG - 029, TSKFL x CTSW - 004, TSKC x CTSW - 022,
TSKC x TRFD - 007, TCLN x TRDP - 015, HTR - 206 and TSKC x CTTR - 029
hybrids behaved as resistant; The TSKFL x CTARG - 023, TSKC x CTARG 015, TSKC x CTSW - 017, TSKC x CTSW - 058, TSKC x TRFD - 003, TSKC x
(TR x LCR) - 017, TSKC x CTQT1439 - 026, TSKFL x CTTR - 004, TSKFL x
CTTR - 021, TSKC x LHA - 001, HTR - 208 hybrids, behaved as very tolerant;
the TSKC x CTARG - 020, TSKC x CTARG - 069, TSKC x CTARG - 081,
TSKFL x CTSW - 009, TSKC x CTSW - 055, TSKC x CTSW - 060, TSKC x (TR
x LCR) - 018, TSKC x (TR x LCR) - 020, TSKC x CTQT1439 - 014, TSKC x
TRDP - 026, TCLC x CTSW - 005, TSKC x CTQT - 010, LVKC x CTSW - 009
and HTR - 207 hybrids behaved as tolerant; and the TSKC x CTARG - 079,
TSKC x CTSW - 025, TSKC x CTSW - 036, TSKC x (TR x LCR) - 032, TSKC x
CTQT - 003, TSKC x LHA - 007, TSKC x LHA - 004, TSKC x LVKCT2 - 001 and
TSKFL x CWEB - 004 hybrids behaved as non tolerant.
Goal 3a2: Assess rootstock trials in areas with hydric stress.
Indicators: Number of assessed rootstocks; number of rootstocks with higher
or lower tolerance to hydric stress; number of rootstocks with potential to be
recommended in new experimental areas; number of rootstocks recommended
as cultivars; number of rootstocks registered in the National Plant Variety
Protection Service.
During the period of intense water stress, evaluations based on the
presence or lack of leaf curling, characteristic of plants under water stress,
considering a grade criteria, were carried out.
The crosses which showed greater potential of drought tolerant hybrids
were: ‘Rangpur Santa Cruz’ lime (Citrus limonia) (LCRSTC) x common ‘Sour’
orange (C. aurantium) (LAZC), ‘Rangpur CNPMF-005’ lime (LCR CNPMF-05) x
‘Smooth Flat Seville Sour’ orange (LAZSFS), LCRSTC x LAZSFS, LCRSTC x
[common ‘Sunki’ (C. sunki) mandarin hybrid x P. trifoliata ‘English’ - 256) (TSKC
x TRENG - 256) and LCRSTC x (TSKC x TRENG - 264).
A competition assay with 50 rootstocks selected from the CGBP in
combination with ‘Valencia’ sweet orange scions was installed in the county of
Colômbia, SP. The experimental design was in random blocks with three
replicates with variable number of plants per plot (three to five). Visual
evaluations such as leaf curling characteristics and bud, flower and fruit
formation were carried out using the following grade criteria: 0 (presence) and 1
(absence).
The TSKC x (TR x LCR) - 001, TSKC x (TR x LCR) - 059, TSKC x (TR x
LCR) - 073, TSKC x CTSW - 033, TSKC x CTSW - 041, TSKC x CTQT1434 010, TSKC x CTQT1439 - 004, TSKC x CTQT1439 - 026, TSKC x CTTR - 002,
TSKC x CTARG - 001, TSKC x LHA - 006, TSKC x LHA - 011, LVK x LCR 010, TSKC x CTRK - 001, LCR x TR - 001, HTR - 053 and HTR - 116 hybrids,
as well as selections of the ‘Rangpur’ lime and ‘Tropical Sunki’ mandarin,
provided the least drought sensitivity to the scions of the ‘Valencia’ sweet
orange (no leaf curling occurred) and quick reaction to the presence of water
(previous rain at the time of the evaluations), according to the observations
regarding bud formation.
Objective 3b. Evaluate fruit quality from promising new hybrids between
oranges and mandarins or tangors as scion varieties;
Goal 3b1: Assess a number of new promising hybrids for scion varieties
regarding fruit quality in ongoing trials.
Indicators: Number of new potential varieties assessed; number of maturation
curves performed and evaluated; number of new scion varieties with potential to
be recommended in new experimental areas; number of new scion varieties
recommended as cultivars; number of scion varieties registered in the National
Plant Variety Protection Service.
Evaluations were carried out on physical-chemical characteristics of fruits
in September and October. For the analysis, 10 fruits were collected in the outer
portion of the canopy in the whole length of the perimeter of the plant. We
evaluated height and diameter of the fruit, color, total mass, juice yield, soluble
solids, acidity of the juice and soluble solids / acidity (ratio).
For the evaluation of fruit production, they were harvested and weighed
separately at each plant. We evaluated the total weight of fruit per plant, total
fruit number per plant and number of fruit per bag for collection of 27.2 kg,
which represents a measurement inversely proportional to the size of the fruit.
Some hybrids were selected showing a production of more than 1.52 boxes per
tree (average yield of Valencia / Natal oranges with 5 years without irrigation in
the region of evaluation) and juice percentage above 55%.
It was observed that some hybrids showed good characteristics for
consumption as fresh fruit and that they were more resistant to Alternaria brown
spot (Figure 16). The assessments of fruit quality will be continued in the
following years.
Figure 16: Plant and fruit of the hybrid between Murcott tangor x Pêra sweet
orange (TM x LP 281), resistant to Alternaria brown spot. A) detached leaves
subjected to in vitro inoculation of the fungus showing the absence of symptoms
and (B) the parental Murcott, highly susceptible to the fungus.
Objective 3c. Validate RGAs and SSR markers based on CitEST and map
them within the existing linkage maps;
Goal 3c1: Validate at least 500 new SSR markers and 30 new RGAs derived
from citrus ESTs.
Indicators: Number of SSR markers validated by PCR and inserted in the
following maps: Sunki mandarin (C. sunki) x Poncirus trifoliata cv Rubidoux,
Swingle citrumelo (C. paradisi x P. trifoliata) x Rangpur lime (C. limonia),
Murcott tangor (C. sinensis x C. reticulata) x Pêra sweet orange (C. sinensis),
Cravo mandarin (C. reticulata) x Pêra seet orange (C. sinensis).
Microsatellites were investigated in the unigene sequences from Citrus
spp. and Poncirus trifoliate (CitEST project). So far, 134 primer sequences
flanking SSR motifs were successfully designed and synthesized. In this stage
of work were assessed the SSR loci to build a consensus map through the
integration of four linkage maps: Pêra sweet orange (Citrus sinensis), Murcott
tangor (C. sinensis x C. reticulata), Cravo mandarin (C. reticulata) and
Pummelo (C. grandis).
The Isolation of DNA of 94 hybrids of each population: Pêra sweet
orange vs. Cravo mandarin (PE x CM), Pêra sweet orange vs. Murcott tangor
(PE x MU), and Pummelo vs Cravo mandarin (PU x CM) was performed as by
Murray and Thompson (1980). All hybrids and parents were genotyped with
SSR markers - 134 EST-SSRs and 171 Genomic-SSR.
From the total SSR markers (134 EST-SSRs and 171 Genomic-SSR),
35 pairs of primers SSR proceeding from sequences of ESTs were selected on
the basis of the polymorphism between the genitors and segregation in the
hybrid plants and 40 SSR pairs of primers were selected from the genomic
libraries.
From the 75 SSR pairs of primers, 25 pairs of primers produced
bands that segregated in an informative way in all the progenies, that is, both
parents were heterozygous and at least three different alellos were segregating,
so that four different genotypes could be identified in the population.
Goal 3c2: Map the molecular markers within the linkage maps already available
in the Program.
Indicators: Number of markers that can be inserted in the maps.
The total SSR evaluated 25 of them showed to be completely informative
(4 alleles) and they could be used as anchors to combine the different maps. In
a linkage analysis congregating the selected RAPD, SSR, AFLP and TRAPS
markers (Tab. 4), 336 out of 619 molecular markers grouped in ten linkage
groups totalizing 1,053 cM (Fig. 16). Almost 54% of the total number of the
markers could be integrated in one map. Only the linkage groups with markers
of at least two parents were considered to compose the consensus map.
Additionally to SSR markers, dominant markers as AFLP and RAPD that
segregated in a ratio of 3:1 functioned as anchors to combine the different
maps.
Table 4: Number of molecular markers of the Integrated Map from each
individual map
Species
Number of molecular markers
AFLP
RAPD
SSR
TRAPs
Pêra sweet orange
68
11
07
6
Murcott tangor
60
16
16
12
Cravo mandarin
22
47
09
-
Pummelo
Total
14
164
23
97
25
57
18
Total
92
104
78
62
336
New genetic maps were obtained for the populations of hybrids
(94
plants) Rangpur x Swingle Swingle and Rangpur lime x P. trifoliata (50 plants)
and location of regions associated with HLB, SDC, resistance to Phytophthora
and CTV.
Moreover, two populations of F1 hybrids derived from intraspecific
crosses between the Pera-de-Abril sweet orange (with monoembryonic seeds)
and Tobias sweet orange (plants with short juvenile cycle), obtained in 2006,
are being mapped using Traps and SSR molecular markers. Approximately
30% of the plants of both populations, with two years old, had already flowered
in the field. Screening for molecular data of 25 pair of Traps primers and 12 pair
of SSR primers have been generated with all plants of both populations and,
after that, the linkage map will be done.
0
1
2
3
6
7
8
9
11
12
15
18
19
27
29
33
38
41
45
51
54
58
60
61
65
68
69
70
71
73
77
83
85
86
90
98
103
105
115
117
121
153
A10-2015
AT7b
AV5-1626
AT5-484
AFLP488
AFLP383
G17-1101
B13-1832
AFLP393
AFLP391
AFLP392
N14-1386
AFLP390
E20-576
AFLP532 AFLP397
AFLP481
AFLP530 AFLP781
AFLP396 AFLP188
AFLP538
AFLP534
AFLP193
H15-335 AFLP537
AFLP540
P6-1339
AFLP665
G17-459
G18-1871
B2-1156
AFLP762 B11-2037
AFLP324 H16-705
B10-1986
H16-1349
AT13d
AFLP399
H1-1922
H19-1107
AFLP355
AFLP415 AV12-1987
AB8-1544
Pm99
AT7a
E17a
0
6
7
8
9
10
11
17
18
0
29
33
37
54
56
58
60
62
63
64
66
69
71
72
73
83
88
95
A4-717
U3-1107
B3-1698
AB5-1308
N15-597
G8-623 D15-1838
AB6-655
P9-1435 C6-652
E16-2267 I11-434
A8-341
C3-762
P11-1408
AFLP228
AB6-1268
A8-2011
B19-1571 B19-1730
H19-1673
G17-841
AT13-1432
129
Pc49
149
Pc55
160
Pc53
A19-2743
Q8-657
N15-1535
P20-635 N9-533
Q5-2961 B3-1778
AV12-1495 AT13-986
A7-643
A8-580 Q17-1822
Y11-1654
G19-1261
AB18-1357
0
8
11
19
20
0
1
2
8
12
14
16
17
19
20
21
26
27
31
32
33
34
38
39
41
42
43
44
46
48
49
50
52
53
54
57
58
64
68
R7-1565
AFLP1
AFLP78
N5-1249
C7-1779
B4-1650
AT7-620 Q6-738
AFLP584 AFLP155
AB14-767 AFLP411
B5-939
AT14-2115 AFLP637
AFLP351
AFLP341
AFLP743 AFLP79
AFLP350
AFLP720
AFLP222
AFLP332 AFLP220
AFLP589
AFLP719
AFLP403 AFLP55
AFLP631
AFLP270 AFLP223
AFLP714
AFLP119
AFLP218 AFLP152
AFLP156
AFLP335 AFLP154
AFLP376
AFLP221
AFLP319
AFLP145
AFLP227
AFLP352
AFLP414 AFLP118
AFLP501 AFLP225
A10-2015
AV5-1626
AT5-484
G17-1101
B13-1832
0
7
N14-1386
42
E20-576
71
74
77
84
85
86
88
96
99
106
114
121
Pc119
H15-335
P6-1339
G17-459
B2-1156
G18-1871 H16-705
B11-2037
B10-1986
H16-1349
AT13d H1-1922
H19-1107
AV12-1987 AFLP415
131
AB8-1544
138
AT7a
38
11
35
C6-1076
68
71
73
77
80
81
82
H19-1584
G17-1807
E16-805 N8-1362
M7-1426
M13-1189
M7-438
AB4-1226 M11-379
P1-1020 C6-866
A9-628 A8-497
AV8-822 P9-1692
R19-1027
P10-2261
G19-1203
B12-813
AV12-1840
83
84
86
88
89
90
AFLP52 AFLP805
AFLP46
AFLP37
AFLP468
AFLP51
AFLP50
AFLP482
AFLP48
AFLP513
AFLP356
AFLP475 AFLP53
AFLP581
AFLP236 AFLP44
AFLP791
AFLP731
AFLP474
AFLP789
AFLP134 AFLP473
AFLP653
AFLP5
AFLP457 AFLP1480
AFLP480
AFLP10
AFLP790
AFLP550
AFLP8 AFLP486
AFLP266
AFLP7
AFLP17
AFLP809
43
45
49
51
56
57
58
60
61
62
64
66
67
76
78
80
A8-769
H15-1014
U8-783 E16-1967
AT18-1053 N14-295
N15-612
9
33
0
8
10
17
24
25
26
27
29
31
33
1
14
16
20
22
24
25
27
30
32
35
36
39
40
42
44
46
49
50
52
54
57
61
67
71
AFLP207
AFLP168 AFLP160
AFLP67 AFLP833
AFLP831
AFLP167 AFLP303
AFLP162
AFLP296
AFLP272
AFLP306
AFLP248
AFLP147
AFLP244
AFLP243 AFLP295
AFLP273 AFLP302
AFLP240
AFLP242 AFLP291
AFLP241
AFLP290 AFLP238
AFLP234
AFLP285
AFLP276
AFLP237
AFLP301 AFLP279
AFLP281
AFLP280 AFLP283
AFLP286
AFLP275
0
Pt160
27
37
45
54
55
59
60
62
65
72
AFLP70
AFLP215
G10-1416
A9-1189
AFLP464
E19-1282
AFLP213
Q10-747
AFLP675
AFLP549
AFLP1549 AFLP211
AFLP214
AFLP504
AFLP785
AFLP512
AFLP545
AFLP522
AFLP309 AFLP636
AFLP515
AFLP514
AFLP524
AFLP5003
AFLP526 AFLP525
AFLP503
AFLP187 AFLP202
AFLP782
AFLP5001
74
76
78
81
83
84
86
89
92
95
98
102
103
104
116
135
0
A7-1261 A7-1201
62
65
68
G19-1261
A19-2743
Q8-657
N15-1535 N9-533
B3-1778 P20-635
Q5-2961
A8-580
A7-643 Y11-1654
Q17-1822
AT13-986
AV12-1495
AB18-1357
70
71
72
73
75
76
80
118
AFLP110
Figure 16. Consensus map of Citrus.
Goal 3c3: Genotype the molecular markers aiming the construction of
new linkage maps for the populations of intraspecific hybrids of sweet orange,
Rangpur lime x Poncirus trifoliata cv. Rubidoux and Clementine mandarin (C.
reticulata) x Murcott tangor.
Indicators: Number of markers that can be inserted in the maps.
Microsatellites were investigated in the unigene sequences from Citrus
spp. and Poncirus trifoliate (CitEST project). So far, 134 primer sequences
flanking SSR motifs were successfully designed and synthesized. In this stage
of work were assessed the SSR loci to build a consensus map through the
integration of four linkage maps: Pêra sweet orange (Citrus sinensis), Murcott
tangor (C. sinensis x C. reticulata), Cravo mandarin (C. reticulata) and
Pummelo (C. grandis).
The Isolation of DNA of 94 hybrids of each population: Pêra sweet
orange vs. Cravo mandarin (PE x CM), Pêra sweet orange vs. Murcott tangor
(PE x MU), and Pummelo vs Cravo mandarin (PU x CM) was performed as by
Murray and Thompson (1980). All hybrids and parents were genotyped with
SSR markers - 134 EST-SSRs and 171 Genomic-SSR.
The SSR markers were added to the preexisting maps built with RAPD,
AFLP and TRAPs markers. The genetic maps were obtained using the JoinMap
software v.3.0, through the simultaneous analysis of markers that had 1:1,
1:2:1, 3:1 and 1:1:1:1 Mendelian segregations.
Objective 3d. Include phenotypic data from assessments of promising new
hybrids within the respective linkage maps;
Goal 3d1: Validate the data on resistance or tolerance to CVC, leprosis, and
Alternaria brown spot on the assessed scion hybrids.
Indicators: Number of features inserted in the maps.
New evaluations for CVC, leprosis and ABS were conducted in field
experiments but the characteristis were not included in the map in this first
stage yet. Since several traits seem to be quantitative more evaluation should
be considered before to include them in the maps.
Goal 3d2: Validate the data on resistance or tolerance to tristeza, gummosis,
and water deficit on the assessed rootstock hybrids.
Indicators: Number of features inserted in the maps.
Detection and mapping of QTLs in the populations were analyzed with
the program MapQTL v. 4.0 (Van Ooijer et al., 2002), using parametric tests
Interval Mapping (IM) and "Multiple QTL Mapping (MQM).
QTL mapping for resistance to CTV in the linkage map of Swingle citrumelo
For the QTL location to CTV resistance, the phenotypic data measured
(absorbance in ELISA) was analyzed separately and the QTL was localized in
the linkage maps using the program MAPQTL v.4.0. The QTL was located
between SSR marker 109 and P1R1-420, the latter being a marker linked to
genes involved in the synthesis of lignin. This QTL may be considered of a large
effect since it explains 95.1% of the phenotypic variance.
CTV gene had been mapped in a previous work, in Poncirus trifoliate,
using a mapping population between P. trifoliata vs Sunki mandarin. Since
Swingle citrumelo is a hybrid of Citrus paradisi vs P. trifoliata, we used the
markers found in the linkage group of P. trifoliata gene associated with CTV
(Figure 18). Thus, one can see that the markers associated with the CTV gene in
the P. trifoliata map are also associated with the gene in map of C. Swingle. It
can be stated that the CTV resistance gene of P. trifoliata was inherited by C.
Swingle.
Figure 18. (A) Linkage map of Swingle citrumelo with markers near the CTV
gene of P. trifoliata, showing the location of a QTL for CTV resistance (B) a
group of P. trifoliata map of CTV with the gene (C) linkage group of Citrumelo
Swingle showing the location of a QTL for resistance to CTV, without the
markers found in P. trifoliata.
Mapping QTLs for resistance to Phytophthora in the linkage map of Swingle
citrumelo
The average length of lesions in hybrids and parents was between 5.6
and 25.23 mm. Parents Rangpur lime and Citrumelo Swingle had an average of
24.05 and 6.08 mm, respectively. In a linkage analysis with RAPD, SSR and
TRAP, 77 of 154 markers were grouped into eight linkage groups of Citrumelo
Swingle and 33 markers were grouped into 7 linkage groups of Rangpur lime.
Forty-four markers (26.78%) did not bind to any linkage group. The phenotypic
data were analyzed for localization of QTL through the program MAPQTL v.4.0.
The analysis by the method Multiple QTL Mapping (MQM) has detected the
presence of two QTLs for resistance to Phytophthora in linkage groups 3 and 6
(LG 3 and 6) of Swingle citrumelo. The permutation tests (1000 replicates)
detected the critical LOD scores critics that confirmed the presence of QTL (p
<0.05) that explained 50.8% and 37.7% of the phenotypic variation.
Figure 19. Swingle citrumelo linkage map with 86 markers (41 RAPD, 29 SSR
and 16 TRAP) in 8 linkage groups and QTL associated with resistance to
Phytophthora. Five RAPD and 4 SSR markers that were linked to CTV in the
Poncirus trifoliata map were incorporated into the linkage group 6.
0
F2P6_520
F2P3_320
0
5
10
A01_180
0
CTC/ACA_197
F4P4_400F2P2_4.300
22
AV03_750
2
AV02_2100
12
C06_450
R07_1610
AT14_600
CCSM111_180
2
3
3
4
P05_820
CAA/ACC_202
CAA/ACT_193
H04_1200
C05_750 M01_202
4
5
A18_3100
F4P1_380
13
21
26
28
29
30
34
37
38
46
51
57
1
N13_1400
1
N01_850
2
R14_800
2
M12_410
3
AV02_11
28
33
37
42
4
N14_100
48
5
M104_239
5
6
7
7
8
8
9
9
9
100
101
103
107
111
113
118
124
127
129
139
65
66
68
N01_1030
5
M02_1650
F4P5_680
M20_730
N08_310
AV05_1200
N14_400
81
A04_470
H04_110
N15_120
93
AT14_1550
N06_1500
N15_2000
Q05_500
105
C14_1500
AV08_1800
M06_1250
115
CAA/ACA_179
Q07_250
CAG/ACG_70
121
CAC/AAC_243
AT07_750
0
A10_200 CAT/ACA_105
0
AV11_13
A10_1200
M02_2110
1
CTC/ACT_30413
AV12_410
A10_300
14
CAT/ACA279
A10_800
17
AT07_3000
2
31
35
CTC/AAG_188
CTC/AAG_199
112
115
C05-1500
CCSM12_280
CAA/ACC_215
R20_1100
Z_680
AV11_2000
CAT/ACT_187
U05_740
AV02_1100
54
CCSM75_232
4
4
H04_1200
W13_1650
60
CCSM170_ 254
5
AV11_1300
AT07_3000
C06_1100
R01_1000
0
3
CCSM168_176
6
7
F1P5_710
7
95
M20_1030
93
R01_1100 F2P1_650
58
AV11_15
AT07_800
W13_580
23
26
30
34
39
41
55
60
85
A10_410
C14_1300
C14_650
N16_500
N16_510
C05_1410
A08_1622
H04_1400
C11_400
CAC/ACC_255
C15_110
C15_410
AV02_250
AV02_3050
M01_860
C10_170
F1P1_740 F2P1_850
C06_110
F4P4_150
N06_1510
AV08_1750
E15_1000
AT03_1000
F1P3_490
CTA/ACA316 F2P4_580
0
6
11
15
16
20
CTG/AAG_293
24
M05_420
CTC/ACA_270
28
F1P2_680
29
CAC/ACC_316
31
32
F2P3_510
33
F1P3_2100
36
38
M03_140
43
46
A04_780
51
57
CTG/AAC_194
65
11
15
16
48
6
6
59
70
82
83
2
5
11
12
22
28
30
35
37
38
39
41
43
47
49
50
51
55
57
58
66
67
75
N08_1300
CAC/ACG_144
C19_380
3
4
4
73
F2P6_800
Q07_430
CTC/ACT_294
A10_750 CCSM40_180
F4P1_200
F2P3_390
CCSM09_156
CAA/ACG_255
CTG/AAC_147 E07_100
AV12_330
CCSM156_310
CAC/ACC_344
0
AF3_153
AV05_250
CTG/ACA_121
A18_1800
F2P2_4900CAC/AAC_252
Q06_1020
E20_920 Q12_490
AV01_180
CCSM11_150
R01_710
C07_1400
M06_1100
U11_1550
UO3_1550
CCSM13_234
U05_750
CAC/ACT_407
CTC/AGC_199
F1P5_780
AV08_900
M16_300
CTG/ACA_347
N01_1550
U05_740
0
3
5
7
6
7
7
7
7
8
8
9
9
102
103
3
45
AV11_250
67
E07_800
N08_2500
AV11_15
AT07_400
E16_510
M05_310
N15_1050
CAC/AAC_294
F4P5_960
N12_1550
E20_510
N15_1610
Ct
W18_480
AV12_470
M01_140
A04_480
G02-540
CAT/ACA_107
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0
6
10
15
16
19
20
22
25
29
34
38
42
45
48
51
R09_710
CAC/ACT_215
R20_800
A04_100
M06_2300
M06_2000
Q06_1600
Q05_510
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M13_750
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F2P6_340
Q05_408
M12_1100
AT18_500
CAC/ACT_283
8
9
1
Figura 20. P. trifoliata linkage map with 202 markers (135 RAPD, 33 AFLP, 10
SSR and 24 TRAP) in 10 linkage groups; TRAP markers are highlighted in red
and SSR merkers in blue; QTLs are highlighted in yellow.
Objective 3e Assess expression QTLs (eQTLs) for disease resistance.
Goal 3e1: Validate through RT-qPCR at least 20 differentially expressed genes
in the parental lines and in pools or groups of 7 to 10 contrasting hybrids
regarding resistance to CVC, gummosis, CTV, Alternaria brown spot and
leprosis, and tolerance to water deficit, based on the microarray experiments;
Indicators: Number of differentially expressed genes validated.
Initially, the biological experiments of gene expression were established,
some genes were identified as induced in resistant hybrids and are being
validated by RT-qPCR. The step of quantification of transcripts in the hybrids
and mapping of eQTL will be held in the sequence of work.
Goal 3e2: Assess, through RT-qPCR, the level of expression of validated
genes (goal 3e1) in 94 hybrids from each map population (Sunki mandarin x
Poncirus trifoliata, Swingle citrumelo x Rangpur lime, Murcott tangor x Pêra
sweet orange, Cravo mandarin x Pêra sweet orange.
Indicators: Number of differentially expressed genes validated and mapped.
The activities depend on the results of the previous goal (3e1).
Goal 3e3: Map within the linkage groups of the eight genetic maps already
established, the genomic regions associated with disease resistance (CVC,
gummosis, CTV, Alternaria brown spot, and leprosis), and hydric stress
tolerance, based on phenotypic analyses (QTLs) (goals 3d1 and 3d2) and
expression QTLs (eQTLs) (goal 3e2).
Indicators: Number of mapped QTLs in each genetic map and correlation
between phenotypic analyses (QTLs) and expression (eQTLs)
The activities depend on the results of the previous goals (3d1, 3d2, 3e1
and 3e2).
Objective 3f. License genetically modified (GM) citrus plants previously
obtained in the Program for field trials.
Goal 3f1: Establish at least one experimental field area for evaluation of GM
plants (previously obtained in the Program) after challenging with each of the
pathogens studied (the causal agents of CVC, leprosis, HLB, and tristeza).
Indicators: Number of experimental areas and challenged plants.
A proposal for controlled experiments in the field is being prepared to
submit to CTNBio.
Objective 3g. Protect new cultivars (hybrids) after confirmation of their value of
culture and use.
Goal 3g1: Register and protect at least five new cultivars of scion and
rootstocks.
Indicators: Number of protected cultivars.
Among the new hybrids several individuals have been identified as
candidates to be new varieties both of rootstocks and scions. All of them have
been challenged under field conditions in four to seven years of experiments
(see Objetives 3a).
Objective 3h. Develop a database and web interface for managing field trials.
Goal 3h1: Establish a pipeline for management of the data obtained from the
field trials.
Indicators: Pipeline established.
This activity is planned for the next year.
Objective 3i: Prepare patent applications for processes and uses of products
developed based on the platforms of the Program.
Goal 3i1: Apply for patents on the use of novel genes and promotors.
Indicators: Number of patent applications and granted.
Several genes and promotors of the CitEST database are candidates to
be patented, but they need better characterization regarding vector construtions
and testing in heterologe systems.
Refereed Journal Articles
1. Baptista, JC, MA Machado, RA Homem, PS Torres, AA Vojnov & AM
Amaral 2010. Mutation in the xpsD gene of Xanthomonas axonopodis pv.
citri affects cellulose degradation and virulence. Genetics and Molecular
Biology, 33 (1): 46-153.
2. Basílio-Palmieri, AC, AM Amaral, RA Homem & MA Machado. 2009.
Differential expression of pathogenicity and virulence-related genes of
Xanthomonas axonopodis pv citri under copper stress. Genetics and
Molecular Biology
3. Bassanezi RB, LH Montesino, ES Stuchi. 2009. Effects of huanglongbing
on fruit quality of sweet orange cultivars in Brazil. European Journal of
Plant Pathology 125:565-572.
4. Bastianel M, VM Novelli, EK Kitajima, KS Kubo, RB Bassanezi, MA
Machado and J Freitas-Astúa. 2010. Citrus Leprosis: Centennial of an
Unusual Mite Virus Pathosystem. Plant Disease, v. 94, p. 284-292, 2010.
5. Bastianel, M, M Cristofani-Yaly, AC Oliveira, J Freitas-Astúa, AAF
Garcia, MDV Resende, V Rodrigues & MA Machado. 2009. Quantitative
trait loci analysis of citrus leprosis resistance in an interspecific
backcross family of (Citrus reticulata Blanco C. sinensis L. Osbeck) C. sinensis L. Osb. Euphytica. DOI:10.1007/s10681-009-9950-3.
6. Cantuarias-Avilés T, FAA Mourão Filho, ES Stuchi, SR Silva, E
Espinoza-Nuñez. 2010. Tree performance and fruit yield and quality of
`Okitsu’
Satsuma
mandarin
grafted
on
12
rootstocks.
Scientia
Horticulturae 123: 318-322.
7. Cavalcante IHL, ABG Martins, ES Stuchi, MCC Campos. 2009. Fruit
maturation as a parameter for selection of sweet orange cultivars in
Brazil.. International Journal of Food, Agriculture and Environment
(Online) 7:132-135.
8. Coletta-Filho, HD, EF Carlos, KCS Alves, MAR Pereira, LC Fender, LL
Lotto, RL Boscariol-Camargo,
AA Souza & MA Machado. 2009. In
planta multiplication and graft transmission of ‘Candidatus Liberibacter
asiaticus’ revealed by Real-Time PCR. European Journal of Plant
Pathology. DOI 10.1007/s10658-009-9523-2
9. Eiras M. SR Silva, ES Stuchi, MLPN Targon, SA Carvalho. 2009.
Viróides em citros. Tropical Plant Pathology 34:275-296.
10. Kubo, SK, J Freitas-Astúa, MA Machado & EW Kitajima. 2009. Orchid
fleck symptoms may be caused naturally by two different viruses
transmitted by Brevipalpus. J. Gen. Plant Pathol 75 (3): 250-255. DOI
10.1007/s10327-009-0167-z.
11. Kubo, SK, RM Stuart, J Freitas-Astúa, R Antonioli-Luizon, EC LocaliFrabis, HD Coletta Filho, MA Machado & EW Kitajima. 2009. Evaluation
of the genetic variability of orchid fleck vírus by single-strande
conformational polymorphism analysis and nucleotide sequencing of a
fragment from the nucleocapsid gene. Arch. Virology 154 (6): 1009-1014.
DOI 10.1007/s00705-009-0395-8.
12. Peroni, LA, M Lorencini, JRR Reis, MA Machado & DR Stach-Machado.
2009. Differential diagnosis of Brazilian strains of Citrus tristeza virus by
epitope mappingo of coat protein using monoclonal antibodies. Virus
Research. doi:10.1016/j.virusres.2009.05.014
13. Silva SR, JC Oliveira, ES Stuchi, ET Reiff. 2009. Qualidade e maturação
de tangerinas e seus híbridos em São Paulo. Revista Brasileira de
Fruticultura 31: 977-986.
14. Souza MC, ES Stuchi, A Góes. 2009. Evaluation of tangerine hybrid
resistance to Alternaria alternata. Scientia Horticulturae 123: 1-4.
15. Stuchi ES, ABG Martins, RR Lemo, T. Cantuarias-Avilés. 2009. Fruit
quality of 'Tahiti' lime (Citrus latifolia Tanaka) grafted on twelve
rootstocks. Revista Brasileira de Fruticultura. , v.31, p.454 - 460, 2009.
16. Tomasetto F, ES Stuchi, ABG Martins. 2009. Avaliação de cinco
seleções de laranjeira 'Valência' sobre dois porta-enxertos. Revista
Brasileira de Fruticultura 31: 480-486.
17. Weiler, RL, EC Brugnara, M Bastianel, MA Machado, MT SchifinoWittmann & SF Schwarz. 2009. Carcterização molecular de um progênie
de tangerineira Clementina Fina e Montenegrina. Scientia Agrária
(UFPR) 10(6): 429-435.
Abstracts
1. Cristofani-Yaly M, Bastianel M, Novelli VM, Machado MA. Agronomic
Characterization, Selection and Genetic Mapping of New Hybrids of
Citrus Scion and Rootstocks Varieties. 2nd International Citrus
Biotechnology Symposium, Catania, Italy, november 30 – december 2,
2009.
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8. Marengo, S, M Cristofani-Yaly, JA Diogo & MA Machado. 2009.
Mapeamento genético de tangerina Sunki e Poncirus trifoliata para
resistência ao Huanglongbing (Greening) dos citros. In: 55 Congresso
Brasileiro de Genética, 2009, Águas de Lindóia. Congresso Brasileiro de
Genética, 2009.
9. Conceição, JPS,
A Souza, KA Souza & W Soares Filho. 2009.
Obtenção e cultivo de calos nucelares de citros para isolamento de
protoplastos. In: Congresso Brasileiro de Floricultura e Plantas
Ornamentais e Congresso Brasileiro de Cultura de Tecicos de Plantas,
Aracaju. Anais... CD-ROM.
10. Pissinato, AGV, MA Coelho Filho, AS Gesteira, MGC Costa, W Soares
Filho. 2009. Protocolo experimental para estudos de déficit hídrico em
genótipos de citros passíveis de utilização como porta-enxertos. In:
Jornada Científica da Embrapa Mandioca e Fruticultura, 3., 2009, Cruz
das Almas. Anais... CD Rom.
11. Rocha, JS, W. Soares Filho. 2009. Desenvolvimento de variedades-copa
híbridas de citros: plantas ornamentais. In: Jornada Científica da
Embrapa Mandioca e Fruticultura, 3., 2009, Cruz das Almas. Anais... CD
Rom.
12. Santana, LG, W. Soares Filho, 2009. Programa de melhoramento
genético de citros da Embrapa Mandioca e Fruticultura Tropical:
obtenção de porta-enxertos híbridos. In: Jornada Científica da Embrapa
Mandioca e Fruticultura, 3., 2009, Cruz das Almas. Anais... CD Rom.
13. Soares Filho, W, OS Passos, FV Souza, J. Rocha, LG Santana, D. D.
S.; SANTOS, M. G. Recursos genéticos de citros: obtenção de plantas
híbridas ornamentais. In: Simpósio de Recursos Genéticos para América
Latina y Caribe, 7, Chile. Anais...v. 1, p. 483-484.
14. Soares Filho W, U Souza, CRC Oliveira, MG Santos, CAS Ledo, LGL
Santana, JS Rocha, AGV Pissinato, JSS Silva, AS Souza, OS Passos.
2009. Poliembrionia e potencial de obtenção de híbridos em citros. In: In:
3
Congresso
Brasileiro
de
Melhoramento
de
Plantas,
2009,
Guarapari/ES. Anais do 3 Congresso Brasileiro de Melhoramento de
Plantas, 2009.
15. Rodrigues AS, CJ Barbosa, EES Santos, W. Soares Filho, J.F. Astua.
2009. Tolerância de porta-enxertos híbridos de citros ao Citrus tristeza
virus e Bahia Bark Scaling disease. Tropical Plant Pathology 34.
Resumo 935.
16. Astua JF, ES Stuchi. 2009. Worldwide status of leprosis, its mite vector, and a
casa study: sampling, diagnostic and management of the disease in Brazil In:
International Workshop on Citrus Quarantine Pests, 2009, Villahermosa
Tabasco, México. (CD-ROM).
17. Stuchi ES, H Bremer Neto, E Spinoza-Nuñez, T Cantuarias-Aviles, FAA Mourão
Filho. 2009. Drought tolerance of rootstocks and clones of ‘Tahiti’ lime. In:
International Citrus Biotechnology Symposium, Abstracts p.89.
18. Stuchi ES, H Bremer Neto, E Spinoza-Nuñez, T Cantuarias-Aviles, FAA Mourão
Filho, D Milori. 2009. Integration of physiological methods to estimate water
deficit in scions, clones, rootstocks and interstocks of ‘ Tahiti’ acid lime. In:
International Citrus Biotechnology Symposium. Abstracts p.86.
19. Stuchi ES, T Cantuarias-Aviles,
FP Gonçalves. 2009. Current situation,
management and economic impact of citrus variegated chlorosis in Brazil In:
International Workshop on Citrus Quarantine Pests, 2009, Villahermosa
Tabasco, México. (CD-ROM).
Submitted manuscripts
1. Marengo S, M Cristofani-Yaly, HD Coletta-Filho, JA Diogo, MA
Machado. Genetic mapping of resistance of Poncirus trifoliata to
huanglongbing (HLB). Phytopathology.
2. Faria LM,
M Cristofani-Yaly, L Faldoni, B Bastianel, MA Machado.
Genetic mapping and detection of QTLs in Rangpur lime and Swingle
citrumelo. Euphytica.
3. Cristofani-Yaly M, VM Novelli, M Bastianel, MA Machado. Transferability
and level of heterozygosity of microsatellites markers in Citrus species".
Plant Molecular Biology Reporter.
4. Boava LP, M Cristofani-Yalym MS Mafra, K Kubo, LT Kishi, MA Takita, M
Ribeiro-Alves, MA Machado. Global gene expression of Poncirus
trifoliata, Citrus sunki and their hybrids under infection of Phytophthora
parasitica. 2010. BMC Genomics.
5. Santos J, MA Machado, DA Botrel, WH Pfenning. Efeito fitotóxico e
caracterização de Fusarium solani associados a plantas de citros com
sintomas de morte súbita. Tropical Plant Pathology.
6. Amaral AM, SA Carvalho, VA Silva, MA Machado. Host range reaction of
genotypes of citrus species and varieties to xanthomonas citri subsp. citri
under greenhouse conditions. Journal of Plant Pathology.
7. Santos, FA, AA Souza, HD Coletta Filho, MLPN Targon, LA Peroni, DR
Stach-Machado, SA Carvalho, MA Machado. Evidence of synergism
between subisolates of Citrus tristeza virus in Mexican lime. Archives of
Virology.
8. Winck, FV,
Novello, MA
AM Amaral, JC Baptista, D Martins, S Marangoni, JC
Machado. Protein profile in mutants of hrpF from
Xanthomonas axonopodis pv. Citri. Genetics and Molecular Biology.
Requested patents
Nothing to declare.
Book chapters
Nothing to declare.
Books
Nothing to declare.
Concluded Dissertations
Marengo, S. Mapeamento genético de tangerina Sunki e Poncirus trifoliata para
resistência
ao
Huanglongbing
(Greening)
dos
citros.
Dissertação
apresentada para obtenção do título de Mestre em Agricultura Tropical e
Subtropical Área de Concentração em Genética, Melhoramento Vegetal e
Biotecnologia. Campinas, 2009.
Concluded Thesis
Nothing to declare.
Cordeirópolis, 07 de junho de 2010.
Marcos A. Machado
Coordenador
1a. Reunião Anual
Data: 01 e 02/09/2009
Centro de Citricultura Sylvio Moreira
Agenda
Dia 01/10, Quinta-feira
09:00 – Recepção, apresentação da agenda e apresentação do grupo
09:15 - Apresentação geral do programa – Marcos
09:45 – Intervalo e Foto do Grupo
Apresentação dos grupos
10:00 – Genoma citros, Takita e Luciano Kishi
10:30 – Genoma funcional, Takita, Eliane, Juliana, Alexandre
11:00 - Transformação: Raquel, Polyana, Alexandre, Rodrigo
11:30 – Transformação na UESC: Márcio
12:00 – Almoço
13:30 – Construção de vetores e transformação no IB/ESALQ e CENA: Ricardo
14:00 – Mapeamento genético, Mariângela
14:30 - Melhoramento IAC, Marcos
15:00 - Melhoramento Embrapa, Walter
15:30 - Intervalo
16:00 – Discussão geral
17:00 – Encerramento
Dia 02/10, Sexta-feira
08:30 – Vários assuntos
- Constituição do Comitê Gestor
- Constituição do Comitê Externo de Avaliação
- Acordo de confidencialidade
- Revisão de metas anuais
- (Entrevistas para o vídeo)
10:30 – Intervalo
- Sistemática de compras; financeiro, recibos, notas, material permanente e
equipamentos, consumo, transporte, etc.
- Bolsas CNPq e CAPES: projetos, relatórios, implementação
- Relatórios dos grupos: cronograma e conteúdo
12:00 – Almoço
13:30 – (cont.)
- Cronograma de reuniões do Comitê Gestor: trimestral
- Cronograma de reuniões do INCT: semestral ou anual
- Cronograma de avaliações externas: anual
- Folder e vídeo
- (Entrevistas para o vídeo)
15:00 – Intervalo
Reuniões Coordenação e Grupos
16:30 – Encerramento
Ata da Reunião
Foi a primeira reunião com quase todos os participantes do INCT Citros.
O primeiro dia foi dedicado à apresentação dos vários subprojetos
componentes do programa, útil não só para normalizar as informações e
atividades, mas também para deixar bem definido a responsabilidade de cada
líder. A programação apresentada acima faz parte do programa aprovado no
edital do INCT. Foi feita uma reunião adicional com o grupo de transformação
genética e com o grupo de transformação para discussão de assuntos
específicos. No grupo de transformação acordou-se para a divisão de trabalho
na busca de genes, construção de vetores e protocolos de transformação. No
grupo de melhoramento (rede experimental de campo) foram apresentados os
experimentos em andamento, que resultarão em atividades do programa, mas
deixando definido a atuação institucional (Embrapa e Instituto Agronômico).
O segundo dia da reunião foi dedicado à discussão de questões
gerenciais do programa.
O Comitê Gestor foi nomeado, constituindo-se dos pesquisadores
Marcos A. Machado (Centro de Citricultura, coordenador), Alexandre Morais do
Amaral (Embrapa Cenargen), Juliana Freitas-Astúa (Embrapa Mandioca e
Fruticultura), Marco Aurélio Takita (Centro de Citricultura) e Francisco Mourão
Filho (Esalq/USP). Portanto, representante das principais unidades executoras
do programa. O grupo é de fácil capacidade de reunião e deverá se reunir na
solução de impasses. Sempre que necessário estarão resolvendo pendências
através de e-mail.
Cada grupo recebeu individualmente suas planilhas de orçamento,
definidas em função do que foi solicitado na elaboração do programa ao CNPq.
A execução orçamentária ficou evidentemente dependente da disponibilidade
financeira. Os procedimentos para compra foram estabelecidos e se concentram
no Centro de Citricultura, sendo vetada aos grupos a transferência direta de
recursos, uma vez que a modalidade de contratação junto ao CNPq foi a de
Auxílio Individual, em nome do Coordenador. Assim, os grupos devem enviar a
propostas de compras ao coordenador, obedecidos os critérios estabelecidos
pelo CNPq e FAPESP (para os grupos do Estado de São Paulo), que autorizará
o gasto de acordo com a disponibilidade financeira. O mesmo se aplica a
processos de importação que deverão ser executados pela FAPESP e pelo
CNPq.
Foi apresentada a sistemática de implementação de bolsas do CNPq e
da CAPES, sendo todas encaminhadas ao coordenador que as implementa no
sistema Carlos Chagas do CNPq ou encaminha documentação à CAPES. A
todos foi destacada a exigência de projeto vinculado ao programa, que deve ser
sempre enviada ao coordenador. Sem essa documentação nenhuma bolsa será
implementada.
Aos grupos foi também informado os prazos para relatório anual e a
necessidade de atendimento das metas estabelecidas. O primeiro relatório anual
ficou previsto para o final de abril (data da contratação do programa pela
FAPESP).
Discutiu-se também a constituição de um comitê externo de avaliação a
se reunir na reunião anual de 2010. Esse comitê tem a função de fazer uma
análise critica do programa e sugerir direcionamentos e integração, além de se
constituir de pesquisadores que podem contribuir com o grupo, por serem de
áreas afins. Os seguintes nomes foram sugeridos: Fred Gmitter, da Universidade
da Flórida, MIkeal Roose da Universidade da Califórnia (Riverside), Dario
Grattapaglia (Embrapa Cenargen). Ao coordenador cabe a função de fazer
contato e convidá-los a participar da reunião prevista para setembro de 2010.
Durante a reunião foi feito o vídeo institucional encaminhado ao CNPq e
FAPESP.
Cordeirópolis, 02 de Outubro de 2009.
Marcos A. Machado
Coordenador
Equipe
SECRETARIA DE
AGRICULTURA E ABASTECIMENTO

Activity Report - INCT

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