title: Development of methodologies for virus detection in soybean and wheat seeds.
abstract: Seeds that contain large amounts of oil, starch, fibers and phenols are the most difficult tissues for RNA extraction. Currently, there are some reports of virus detection in seeds using commercial kits for RNA extraction. However, individual seeds were used, which may not be always suitable for analyses that deal with large amounts of seeds. Sangha [1] described a simple, quick and efficient protocol for RNA extraction and downstream applications in a group of seeds of jatropha (Jatropha curcas), mustard (Brassica sp.) and rice (Oryza sativa). We tested this protocol for soybean (Glycine max), maize (Zea mays), wheat (Triticum aestivum) and triticale (×Triticosecale) seeds and further reverse transcription PCR (RT-PCR)/quantitative real-time PCR (qPCR) in order to have a faster and more practical method for virus detection from seeds than the traditional scheme of seed planting and subsequent Elisa/RT-PCR from leaves. The essential points in the method are:•Some modifications in the protocol [1] were done in order to increase performance: Wheat and triticale seeds are incubated with water prior to maceration. An amount of 1.2 g of dry soybean seeds is used to maceration.•RT-PCR is used for detection of Wheat streak mosaic virus from wheat seeds and RT-qPCR for detection of Soybean mosaic virus from soybean seeds.•The method may be tested for other viruses, however, pre-validation will be needed.
authors.affiliation: Embrapa Genetic Resources and Biotechnology, Brasilia, DF, Brazil.
authors.forename: Stephanie R A
authors.initials: SR
authors.lastname: Botelho
authors.affiliation: Embrapa Genetic Resources and Biotechnology, Brasilia, DF, Brazil.
authors.forename: Thais P
authors.initials: TP
authors.lastname: Martins
authors.affiliation: Embrapa Genetic Resources and Biotechnology, Brasilia, DF, Brazil.
authors.forename: Macária F
authors.initials: MF
authors.lastname: Duarte
authors.affiliation: Embrapa Genetic Resources and Biotechnology, Brasilia, DF, Brazil.
authors.forename: Andreza V
authors.initials: AV
authors.lastname: Barbosa
authors.affiliation: Embrapa Trigo, Passo Fundo, RS, Brazil.
authors.forename: Douglas
authors.initials: D
authors.lastname: Lau
authors.affiliation: Embrapa Plant Quarantine, Brasilia, Brazil.
authors.forename: Fernanda R
authors.initials: FR
authors.lastname: Fernandes
authors.affiliation: Embrapa Genetic Resources and Biotechnology, Brasilia, DF, Brazil.
authors.forename: Marcio M
authors.initials: MM
authors.lastname: Sanches
keywords: Quarantine; RT-PCR; SMV; Seed certification; WSMV; qPCR