Establishment and application of an SNP molecular identification system for grape cultivars

doi:10.1016/S2095-3119(21)63654-7

摘要/Abstract

摘要：

基于简化基因组测序技术（SLAF-seq）对304份葡萄种质测序获得的4241729个SNP位点，依次按照SNP位点只在染色体上的DNA链上前后大于50bp序列保守；具有二态性；SNP位点在304份葡萄种质中的基因型缺失率（Missing rate）<0.05，MAF>0.3；304份葡萄种质的重测序单个SNP位点平均测序深度>=10×；具备特异性等条件，筛选一组高质量SNP位点，用于KASP标记的转化。使用Perl语言计算出一组能够有效区分304份测序种质的标记，并用23份葡萄代表性品种进行初筛。充分结合前人研究进展，将课题组已筛选出的22个KASP标记同初筛出的标记一起使用348份葡萄种质进行复筛。对复筛的结果进行群体结构分析、主成分分析、遗传进化聚类分析验证标记的可靠性和准确性，并确定出一组核心KASP标记，构建葡萄品种SNP分子鉴定体系。从基于简化基因组测序技术（SLAF-seq）对304份葡萄种质测序获得的4 241729个群体SNP位点中筛选517个高质量的SNP位点，并成功设计出442个KASP标记。使用程序计算出一组只用能完全区分304份葡萄种质的27个标记组合，并使用23个葡萄代表品种筛选出26个合格的标记。加上课题组前期筛选出的22个标记，共48个KASP标记基于348份葡萄种质复筛出46个优质KASP标记，其MAF值大于0.3的占96%，PIC值均大于0.4，均为高度多态性标记。群体结构分析、主成分分析、聚类分析均显示348份葡萄种质分为2大类最佳。另外，聚类分析更加细化的将其分为6个亚类，证明美洲种、刺葡萄、毛葡萄、山葡萄同欧亚种的遗传距离较远，而欧美杂种、欧山杂种介于两者之间。发现有11组基因型一致的种质，获得一组能够有效鉴别333份葡萄种质的25个KASP标记，鉴定效率达到了95.69%，并作为我国葡萄品种SNP分子鉴定体系的核心标记，其余21个标记作为扩展标记。从4 241 729个群体SNP中筛选517个高质量的SNP位点，并成功设计出442个KASP标记。先后使用23个葡萄代表品种和348份葡萄种质筛选出46个优质KASP标记，构建348份葡萄种质的SNP指纹图谱，并将其中能够有效鉴别333份葡萄种质的25个KASP标记作为我国葡萄品种SNP分子鉴定体系的核心标记，其余21个标记作为扩展标记。因此，基于KASP分型技术的SNP分子标记为葡萄品种的DNA指纹图谱绘制提供了一种新途径。该技术效率高，准确性高，成本低，比目前的其他鉴定方法更具竞争力，在葡萄的DUS测试以及在市场维权等方面具有极好的应用前景。

Abstract: We aimed to develop a set of single nucleotide polymorphism (SNP) markers that can be used to distinguish the main cultivated grape (Vitis L.) cultivars in China and provide technical support for domestic grape cultivar protection, cultivar registration, and market rights protection. A total of 517 high-quality loci were screened from 4 241 729 SNPs obtained by sequencing 304 grape accessions using specific locus amplified fragment sequencing, of which 442 were successfully designed as Kompetitive Allele Specific PCR (KASP) markers. A set of 27 markers that completely distinguishes 304 sequenced grape accessions was determined by using the program, and 26 effective markers were screened based on 23 representative grape cultivars. Finally, a total of 46 out of 48 KASP markers, including 22 markers selected by the research group in the early stage, were re-screened based on 348 grape accessions. Population structure, principal component, and cluster analyses all showed that the 348 grape accessions were best divided into two populations. In addition, cluster analysis subdivided them into six subpopulations. According to genetic distance, V. labrusca, V. davidii, V. heyneana, and V. amurensis were far from V. vinifera, while V. vinifera×V. labrusca and V. amurensis×V. vinifera were somewhere in between these two groups. Furthermore, a core set of 25 KASP markers could distinguish 95.69% of the 348 grape accessions, and the other 21 markers were used as extended markers. Therefore, SNP molecular markers based on KASP typing technology provide a new way for mapping DNA fingerprints in grape cultivars. With high efficiency and accuracy and low cost, this technology is more competitive than other current identification methods. It also has excellent application prospects in the grape distinctness, uniformity, and stability (DUS) test, as well as in promoting market rights protection in the near future.

Key words: grape , KASP marker , variety identification , fingerprint , genetic diversity analysis

. [J]. Journal of Integrative Agriculture, 2022, 21(4): 1044-1057.

WANG Fu-qiang, FAN Xiu-cai, ZHANG Ying, SUN Lei, LIU Chong-huai, JIANG Jian-fu. Establishment and application of an SNP molecular identification system for grape cultivars[J]. Journal of Integrative Agriculture, 2022, 21(4): 1044-1057.

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