Gosling Research Institute for Plant Preservation (GRIPP), University of Guelph, 50 Stone Rd E, Guelph, ON, N1G 2W1, Canada.
USDA-ARS, National Clonal Germplasm Repository, 33447 Peoria Rd, Corvallis, OR, 97333, USA.
Planta. 2019 Jun;249(6):1863-1874. doi: 10.1007/s00425-019-03131-4. Epub 2019 Mar 11.
This DNA fingerprinting test confirmed 195 unique Corylus sp. accessions that were used to build a reference database for identity verification of unknown hazelnut trees from three locations in Ontario. Hazelnut is one of the most profitable tree nuts worldwide. Development of a hazelnut industry in Ontario is urgently required, but economically important cultivars must be genetically verified first in order to meet industry standards. Traditional methods for cultivar identification are largely trait-based and unreliable. In this study, a multiplexed fingerprinting test was modified to allow for hazelnut cultivar discrimination at the DNA level. Fourteen highly polymorphic SSR markers covering the 11 linkage groups of Corylus genome were PCR amplified in multiplex using fluorescent-labelled primers. PCR conditions and primer physical properties were optimized to generate a clear signal for each locus. The 14 SSRs were used to fingerprint 195 unique Corylus accessions collected from the USDA-NCGR. Fragment sizes were subjected to a UPGMA clustering analysis which separated Corylus accessions based on species and geographic origin. For validation purposes, hazelnut leaves from three locations in Ontario were collected for identity verification using this DNA fingerprinting test. As a result, 33.3% of the unknown trees were duplicates of seven distinct genotypes and a small percentage (8.3%) of these were identical to reference Corylus hybrids. These results reflect common mislabelling issues and genotype duplications that can prevent a uniform plant propagation system. Implementation of this test together with the addition of more unique accessions to the reference database will help verification of trueness-to-type of economically important cultivars for the hazelnut industry.
该 DNA 指纹测试证实了 195 个独特的榛属植物品种，这些品种被用来建立一个参考数据库，用于验证安大略省三个地点的未知榛树的身份。榛子是全球最赚钱的坚果之一。安大略省迫切需要发展榛子产业，但为了达到行业标准，首先必须对经济上重要的品种进行基因验证。传统的品种鉴定方法主要基于性状，且不可靠。在这项研究中，修改了多重指纹测试，以允许在 DNA 水平上对榛子品种进行区分。使用荧光标记引物，在多重 PCR 中扩增了覆盖榛子基因组 11 个连锁群的 14 个高度多态性 SSR 标记。优化了 PCR 条件和引物物理性质，以产生每个基因座的清晰信号。使用这 14 个 SSR 对来自美国农业部- NCGR 的 195 个独特的榛属品种进行指纹图谱分析。片段大小进行了 UPGMA 聚类分析，根据物种和地理起源将榛属品种分开。为了验证目的，从安大略省的三个地点采集榛树叶片，使用该 DNA 指纹测试进行身份验证。结果，33.3%的未知树木是七个不同基因型的重复，其中一小部分（8.3%）与参考榛子杂种相同。这些结果反映了常见的标签错误和基因型重复问题，这些问题可能会阻止统一的植物繁殖系统。实施这项测试并将更多独特的品种添加到参考数据库中，将有助于榛子产业中经济上重要品种的真实性验证。
