Identificação de vírus em amendoim forrageiro e pimenta por sequenciamento de nova geração
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Data
2020-03-20
Autores
Pantoja, Késsia de Fátima da Cunha [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A espécie Arachis pintoi, conhecida como amendoim forrageiro, é uma leguminosa nativa do Brasil. Muitas são as utilidades atribuídas ao amendoim forrageiro, sendo seu uso mais comum como espécie forrageira, fornecendo alimento em grande quantidade e qualidade aos animais, em plantios puros ou consórcio com gramíneas. A ocorrência de sintomas de viroses em genótipos e cultivos de amendoim forrageiro tem sido observada por pesquisadores em diferentes estados brasileiros. No Brasil, apenas duas espécies virais já foram relatadas: o Peanut mottle virus- PeMoV e o Cowpea mild mottle virus - CpMMV. O objetivo deste trabalho foi estudar os vírus de plantas de amendoim forrageiro do Banco de Germoplasma da Embrapa Acre e detectar viroses em plantas de pimenta Cumari-do-Pará. Para detectar possíveis vírus nesses genótipos, uma análise de sequenciamento de nova geração foi realizada. A extração total de RNA dos 22 acessos foi realizada com o kit RNA Viral PureLink (Invitrogen) seguida de preparação da biblioteca e sequenciamento do transcriptoma utilizando a plataforma Illumina HiSeq2500. A montagem de novo das leituras de 24.659.442 foi realizada usando o software CLC Genomics Workbench v7.0.3. Os 9.709 contigs obtidos foram submetidos a uma pesquisa BLASTn usando o software Geneious v.9.1.5. A análise metagenômica permitiu a identificação de sete espécies de vírus: Peanut mottle virus - PeMoV (Potyvirus), Cucumber mosaic virus sub-grupo IB (Cucumovirus), Cowpea chlorotic mottle virus - CCMV (Bromovirus) e um provável novo mebro da família Potyviridae, e também novas espécies dos gêneros Emaravirus, Polerovirus e Ampelovirus em amendoim forrageiro. A análise de NGS permitiu obter a sequência completa do genoma do Peanut mottle virus (PeMoV) com 9.680pb (GenBank MK396065), que foi confirmada em dois genótipos de A. pintoi (BRA cv. BRS Mandobi, BRA042269) por RT-PCR, seguido de sequenciamento de Sanger, usando os primers específicos desenhados para PeMoV. O CCMV foi identificado baseado na sequência dos três componentes do genoma viral, tratando-se da primeira identificação deste vírus em amendoim forrageiro. A sequência nucleotídica completa de um novo membro da família Potyviridae foi relatada em plantas de amendoim forrageiro (Arachis pintoi) no Brasil exibindo sintomas típicos de vírus, que propomos nomear como Arachis pintoi mottle virus (ArPMV). O genoma do RNA de sentido positivo ArPMV possui 9.213 nucleotídeos. A ORF única completa do ArPMV compartilha 47% e 34% de identidade de nucleotídeo e aminoácido, respectivamente, com o isolado mais próximo relacionado, o soybean yellow shoot virus. As análises de sequenciamento de nova geração plataforma Illumina HiSeq2500 também foram realizadas para uma amostra de pimenta cv. Cumari-do-Pará (Capsicum chinense) coletada em 2017 em uma fazenda próxima ao município de Altamira, no estado do Pará, mostrando sintomas de amarelecimento internerval, redução de lâmina foliar e encurtamento dos entrenós. Embora seja cultivada em todos os estados brasileiros, a produção é fortemente focada nos estados do norte do país. Os contigs foram submetidos à analise por BLASTX com o software Geneious v9.1.5. no banco de dados viral do NCBI, que revelou identidades de nucleotídeos superiores a 90% com a espécie Pepper vein yellows virus- PeVYV (gênero Polerovirus, família Luteoviridae) e também sequências relacionadas aos gêneros Ampelovirus e Cucumovirus. Posteriormente, através de montagem “de novo”, foi possível obter o genoma completo de uma possível nova espécie pertencente ao complexo de Polerovirus da pimenta, cujo nome proposto foi Pepper vein yellows virus 8 (PeVYV-8). Este é o primeiro relato de um polerovírus infectando a pimenta Cumari-do-Pará no Brasil e a distribuição do vírus em outras regiões do país precisa ser melhor avaliada.
The species Arachis pintoi, known as forage peanuts is a native legume from Brazil. There are many uses attributed to forage peanut, being its most common use as a forage species, providing food in large quantity and quality to the animals, in single crops or consortium with grasses. The occurrence of virus symptoms in forage peanut genotypes and crops has been observed by researchers in different Brazilian states. In Brazil, only two viral species have been reported: Peanut mottle virus-PeMoV and Cowpea mild mottle virus-CpMMV. The objective of this work was to study the forage peanut plant viruses of the Embrapa Acre Germplasm Bank and to detect viruses in Cumari-do-Pará pepper plants. To detect possible viruses in these genotypes, a new generation sequencing analysis was performed. Total RNA extraction from the twenty-two accessions was performed with the PureLink Viral RNA Kit (Invitrogen) followed by library preparation and transcriptome sequencing using the Illumina HiSeq2500 platform. Re-assembly of the 24,659,442 readings was performed using the CLC Genomics Workbench v7.0.3 software. The 9,709 contigs obtained were subjected to a BLASTn search using the Geneious v.9.1.5 software. Metagenomic analysis allowed the identification of seven virus species: Peanut mottle virus - PeMoV (Potyvirus), Cucumber mosaic virus subgroup IB (Cucumovirus), Cowpea chlorotic mottle virus - CCMV (Bromovirus) and a probable new member of the Potyviridae family, and also new species of the genera Emaravirus, Polerovirus and Ampelovirus in forage peanuts. NGS analysis yielded the complete 9,680 bp Peanut mottle virus (PeMoV) genome sequence (GenBank MK396065), which was confirmed in two A. pintoi (BRA cv. BRS Mandobi, BRA042269) genotypes by RT–PCR, followed by Sanger, sequencing using PeMoV-specific primers. The CCMV was identified based on the sequence of the three components of the viral genome, being the first identification of this virus in forage peanut. The complete nucleotide sequence of a new member of the family Potyviridae was reported in forage peanut plants (Arachis pintoi) in Brazil exhibiting typical virus symptoms, which we propose to name as Arachis pintoi mottle virus (ArPMV). The ArPMV positive-sense RNA genome is 9,213 nucleotides. The complete ArPMV single ORF shares 47% and 34% nucleotide and amino acid identity, respectively, with the closest related isolate, soybean yellow shot virus. Sequencing analysis of the new generation Illumina HiSeq2500 platform were also performed for a cv pepper sample. Cumari-do-Pará (Capsicum chinense) collected in 2017 on a farm near the municipality of Altamira, state of Pará, showing symptoms of internerval yellowing, leaf blade reduction and internode shortening. The contigs were subjected to BLASTX analysis with Geneious v9.1.5 software. In the NCBI viral database, which revealed nucleotide identities greater than 90% with the Pepper vein yellows virus-PeVYV species (genus Polerovirus, family Luteoviridae) and also sequences related to the genera Ampelovirus and Cucumovirus. Subsequently, by "de novo" assembly it was possible to obtain the complete genome of a possible new species belonging to the pepper polerovirus complex, whose proposed name was Pepper vein yellows virus 8 (PeVYV-8). This is the first report of a polerovirus infecting Cumari-do-Pará pepper in Brazil and the distribution of the virus in other regions of the country needs to be better evaluated.
The species Arachis pintoi, known as forage peanuts is a native legume from Brazil. There are many uses attributed to forage peanut, being its most common use as a forage species, providing food in large quantity and quality to the animals, in single crops or consortium with grasses. The occurrence of virus symptoms in forage peanut genotypes and crops has been observed by researchers in different Brazilian states. In Brazil, only two viral species have been reported: Peanut mottle virus-PeMoV and Cowpea mild mottle virus-CpMMV. The objective of this work was to study the forage peanut plant viruses of the Embrapa Acre Germplasm Bank and to detect viruses in Cumari-do-Pará pepper plants. To detect possible viruses in these genotypes, a new generation sequencing analysis was performed. Total RNA extraction from the twenty-two accessions was performed with the PureLink Viral RNA Kit (Invitrogen) followed by library preparation and transcriptome sequencing using the Illumina HiSeq2500 platform. Re-assembly of the 24,659,442 readings was performed using the CLC Genomics Workbench v7.0.3 software. The 9,709 contigs obtained were subjected to a BLASTn search using the Geneious v.9.1.5 software. Metagenomic analysis allowed the identification of seven virus species: Peanut mottle virus - PeMoV (Potyvirus), Cucumber mosaic virus subgroup IB (Cucumovirus), Cowpea chlorotic mottle virus - CCMV (Bromovirus) and a probable new member of the Potyviridae family, and also new species of the genera Emaravirus, Polerovirus and Ampelovirus in forage peanuts. NGS analysis yielded the complete 9,680 bp Peanut mottle virus (PeMoV) genome sequence (GenBank MK396065), which was confirmed in two A. pintoi (BRA cv. BRS Mandobi, BRA042269) genotypes by RT–PCR, followed by Sanger, sequencing using PeMoV-specific primers. The CCMV was identified based on the sequence of the three components of the viral genome, being the first identification of this virus in forage peanut. The complete nucleotide sequence of a new member of the family Potyviridae was reported in forage peanut plants (Arachis pintoi) in Brazil exhibiting typical virus symptoms, which we propose to name as Arachis pintoi mottle virus (ArPMV). The ArPMV positive-sense RNA genome is 9,213 nucleotides. The complete ArPMV single ORF shares 47% and 34% nucleotide and amino acid identity, respectively, with the closest related isolate, soybean yellow shot virus. Sequencing analysis of the new generation Illumina HiSeq2500 platform were also performed for a cv pepper sample. Cumari-do-Pará (Capsicum chinense) collected in 2017 on a farm near the municipality of Altamira, state of Pará, showing symptoms of internerval yellowing, leaf blade reduction and internode shortening. The contigs were subjected to BLASTX analysis with Geneious v9.1.5 software. In the NCBI viral database, which revealed nucleotide identities greater than 90% with the Pepper vein yellows virus-PeVYV species (genus Polerovirus, family Luteoviridae) and also sequences related to the genera Ampelovirus and Cucumovirus. Subsequently, by "de novo" assembly it was possible to obtain the complete genome of a possible new species belonging to the pepper polerovirus complex, whose proposed name was Pepper vein yellows virus 8 (PeVYV-8). This is the first report of a polerovirus infecting Cumari-do-Pará pepper in Brazil and the distribution of the virus in other regions of the country needs to be better evaluated.
Descrição
Palavras-chave
Arachis pintoi, Capsicum, NGS, Polerovirus, Virus