Koh Joshua C O, Barbulescu Denise M, Norton Sally, Redden Bob, Salisbury Phil A, Kaur Sukhjiwan, Cogan Noel, Slater Anthony T
Department of Economic Development, Jobs, Transport and Resources, Grains Innovation Park, 110 Natimuk Rd, Horsham, VIC 3401 Australia.
Department of Economic Development, Jobs, Transport and Resources, Australian Grains Genebank, Private Bag 260, Horsham, VIC 3401 Australia.
Plant Methods. 2017 Jun 15;13:49. doi: 10.1186/s13007-017-0200-8. eCollection 2017.
Within the Brassicaceae, six species from the genus are widely cultivated throughout the world as oilseed, condiment, fodder or vegetable crops. The genetic relationships among the six species are described by U's triangle model. Extensive shared traits and diverse morphotypes among 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 60K single nucleotide polymorphism (SNP) array has been used to identify species and assess genetic diversity of collections. However, these methods are technically challenging, expensive and time-consuming, making them unsuitable for routine or rapid screening of accessions for germplasm management. A cheaper, faster and simpler method for species identification is described here.
A multiplex polymerase chain reaction (MPCR) consisting of new and existing primers specific to the A, B and C genomes was able to reliably distinguish all six species in the triangle of U with 16 control samples of known species identity. Further validation against 120 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 60K SNP array. In addition, the MPCR was sensitive enough to detect seed contaminations in pooled seed samples of accessions.
A cheap and fast multiplex PCR assay for identification of 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 germplasm collections in genebanks.
在十字花科中，该属的六个物种作为油料作物、调味品、饲料或蔬菜作物在全球广泛种植。六个物种之间的遗传关系由U三角模型描述。物种间广泛的共同特征和多样的形态类型使得仅基于表型数据进行鉴定和分类具有挑战性且不可靠，尤其是在处理大量种质资源时。因此，基因库收集的一个主要问题是确保物种的正确鉴定。基于简单序列重复（SSR）标记测序或Illumina Infinium 60K单核苷酸多态性（SNP）阵列的分子基因分型已被用于鉴定物种和评估该属收集物的遗传多样性。然而，这些方法在技术上具有挑战性、成本高且耗时，使其不适用于种质管理中对该属种质的常规或快速筛选。本文描述了一种更便宜、更快且更简单的该属物种鉴定方法。
由针对该属A、B和C基因组的新引物和现有引物组成的多重聚合酶链反应（MPCR）能够通过16个已知物种身份的对照样本可靠地区分U三角中的所有六个物种。对先前进行基因分型的120份该属种质的进一步验证表明，MPCR高度准确，与SSR标记测序或Illumina Infinium 60K SNP阵列等更先进的技术相当。此外，MPCR足够灵敏，能够检测该属种质混合种子样本中的种子污染。
本研究开发并验证了一种用于鉴定U三角中该属物种的廉价且快速的多重PCR检测方法。MPCR检测方法可在任何基础分子实验室轻松实施，应证明对基因库中该属种质的管理有用。
