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

Abstract

Background: Within 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.

Results: A 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.

Conclusion: A 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.

Keywords: Brassica; Brassicaceae; Genebank; Germplasm management; Species identification; Triangle of U.

Fig. 1

Brassica species within the triangle of U. Three diploid species (Brassica rapa, Brassica nigra and Brassica oleracea) which represent the AA, BB and CC genomes are shown. Also shown are three allotetraploid species (Brassica juncea, Brassica napus and Brassica carinata) which are hybrid combinations of the basic genomes. Diploid chromosome number (2n) is shown. Adapted from U (1935) [3]

Fig. 2

MPCR amplification of 16 control samples with known species identity. Primers specific to the Brassica A, B and C genomes were multiplexed in a PCR reaction. M: HyperLadder™ 50 bp DNA marker (Bioline, UK). Samples 1–2: B. rapa (AA) lines; 3–4: B. nigra (BB) lines; 5–6: B. oleracea (CC) lines; 7–9: B. juncea (AABB) lines; 10–12: B. napus (AACC) lines; 13–15: B. carinata (CCBB) lines; 16: R. sativus (Rs) line; 17: minus template (water) control. PCR products were resolved on 2% TAE agarose gel

Fig. 3

MPCR amplification of B. napus (AACC), B. juncea (AABB) and B. carinata (CCBB) lines with varying input DNA amounts. Genomic DNA amount in sample 1: 100 ng; 2: 10 ng; 3: 1 ng; 4: 0.1 ng; 5: 0.01 ng; 6: 0.001 ng; 7: minus template (water) control. PCR primers and their corresponding product sizes (bp) are indicated on left of figure

Fig. 4

MPCR amplification of genomic DNA extracted from Brassica accessions with mixed seeds. a China B (AGG ID 90542 was labelled as B. napus) and b ATR TW (AGG ID 94402 was labelled as B. napus). Sample 1: genomic DNA extracted from pooled seeds; samples 2–7: genomic DNA extracted from single seeds; sample 8: minus template (water) control. PCR primers and their corresponding product sizes (bp) are indicated on left of figure