A multiplex PCR for rapid identification of Brassica species in the triangle of U

Abstract

BackgroundWithin the Brassicaceae, six species from the genus Brassica are widely cultivated throughout the world as oilseed, condiment, fodder or vegetable crops. The genetic relationships among the six Brassica species are described by U’s triangle model. Extensive shared traits and diverse morphotypes among Brassica species make identification and classification based on phenotypic data alone challenging and unreliable, especially when dealing with large germplasm collections. Consequently, a major issue for genebank collections is ensuring the correct identification of species. Molecular genotyping based on simple sequence repeat (SSR) marker sequencing or the Illumina Infinium Brassica napus 60K single nucleotide polymorphism (SNP) array has been used to identify species and assess genetic diversity of Brassica collections. However, these methods are technically challenging, expensive and time-consuming, making them unsuitable for routine or rapid screening of Brassica accessions for germplasm management. A cheaper, faster and simpler method for Brassica species identification is described here.ResultsA multiplex polymerase chain reaction (MPCR) consisting of new and existing primers specific to the Brassica A, B and C genomes was able to reliably distinguish all six Brassica species in the triangle of U with 16 control samples of known species identity. Further validation against 120 Brassica accessions previously genotyped showed that the MPCR is highly accurate and comparable to more advanced techniques such as SSR marker sequencing or the Illumina Infinium B. napus 60K SNP array. In addition, the MPCR was sensitive enough to detect seed contaminations in pooled seed samples of Brassica accessions.ConclusionA cheap and fast multiplex PCR assay for identification of Brassica species in the triangle of U was developed and validated in this study. The MPCR assay can be readily implemented in any basic molecular laboratory and should prove useful for the management of Brassica germplasm collections in genebanks.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-017-0200-8) contains supplementary material, which is available to authorized users.

Development of a DNA Barcoding-Like Approach to Detect Mustard Allergens in Wheat Flours

“…The amplified regions were compared with NCBI GenBank. Results confirmed all primer pair specificities (i.e., B. juncea and B. napus for A6, B. juncea and B. nigra for B, and B. napus for C1), showing a 100% identity to the target species with BLASTn analysis [20,21].…”

“…In order to selectively distinguish each mustard species, three species-specific DNA regions were also considered (i.e., Bra019579 (Chromosome A6, 220 bp) specific for B. juncea and B. napus , pBNBH35 (Chromosome B, 280 bp) specific for B. juncea and B. nigra and Bo1g016520 (Chromosome C1, 130 bp) specific for B. napus ). These regions were previously identified by Koh et al [20] and Schelfhout et al [21]. All the primer pairs used in this study are reported in Table 1.…”

“…All the selected DNA barcoding markers and those found in Kho [20] and Schelfhout [21] were tested on all 12 plant samples tested. DNA extraction was successful for all 12 plant samples (see Table S1) with high DNA quality and good yield (i.e., 30–40 ng/μL).…”

“…The specificity of the amplification of the three loci identified by Kho and collaborators [20] and Schelfhout and collaborators [21] on the specific Brassicaceae samples was also tested. Concerning the amplification, A6, B, and C1 loci yielded a single band on electrophoresis and exhibited 100% amplification success with specific primer pairs (Table 2).…”

A Global Survey on Diseases and Pests in Oilseed Rape—Current Challenges and Innovative Strategies of Control

“…Oilseed rape (Brassica napus) is the second most important oilseed crop and belongs to the mustard family (Brassicaceae), which consists of 338 genera with more than 3,709 species cultivated worldwide, especially in temperate and mountainous regions (Al-Shehbaz et al, 2006;Warwick et al, 2010). B. napus is a relatively young crop species (AACC genome, 2n = 38) that derived from a spontaneous hybridization between B. rapa (AA genome, 2n = 20) and B. oleracea (CC genome, 2n = 18) (Koh et al, 2017). Europe, Canada, China, India, and Australia are the leading production zones with 25.5, 20.3, 13.3, 8.4, and 3.9 million metric tons of rapeseed production in 2018, respectively (FAO Database).…”