中国农业科学 ›› 2018, Vol. 51 ›› Issue (14): 2771-2787.doi: 10.3864/j.issn.0578-1752.2018.14.014

• 园艺 • 上一篇    下一篇

甘蓝SNP标记开发及主要品种的DNA指纹图谱构建

李志远,于海龙,方智远,杨丽梅,刘玉梅,庄木,吕红豪,张扬勇   

  1. 中国农业科学院蔬菜花卉研究所,北京 100081
  • 收稿日期:2018-01-11 出版日期:2018-07-16 发布日期:2018-07-16
  • 通讯作者: 张扬勇,E-mail:zhangyangyong@caas.cn
  • 作者简介:李志远,E-mail:lizy1992@126.com。于海龙,E-mail:yuhailongnet@163.com。李志远和于海龙为同等贡献作者。
  • 基金资助:
    国家重点研发计划(2016YFD0101702,2016YFD0100204)、国家现代农业产业技术体系建设专项资金(CARS-25)、农业部园艺作物生物学与种质创制重点实验室项目

Development of SNP Markers in Cabbage and Construction of DNA Fingerprinting of Main Varieties

LI ZhiYuan, YU HaiLong, FANG ZhiYuan, YANG LiMei, LIU YuMei, ZHUANG Mu, LÜ HongHao, ZHANG YangYong   

  1. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2018-01-11 Online:2018-07-16 Published:2018-07-16

摘要: 【目的】搜集生产上的主要甘蓝品种,构建基于SNP标记的品种指纹图谱,为鉴定甘蓝品种的特异性、真实性提供依据。【方法】首先,利用50个甘蓝高代自交系的重测序数据,与甘蓝参考基因组(02-12)进行比对筛选SNP位点。挑选多态性较好、在染色体上均匀分布的SNP位点,并将其转化为KASP标记,利用KASP平台对供试材料进行检测,得到59个甘蓝品种的基因分型数据。根据基因分型数据,筛选出多态性信息含量(PIC)高、无基因型数据缺失且在染色体上均匀分布的标记作为构建指纹图谱的核心标记。将核心标记的分型结果转化为二元编码数据,获得甘蓝品种SNP-DNA指纹图谱。在59个甘蓝品种中随机挑选15个品种,另外增加5个未推广的新组合用于构建人工虚拟混合群体,并利用此群体对甘蓝品种指纹鉴定技术进行验证,检测核心标记构建的SNP-DNA指纹图谱的准确性与可靠性。【结果】利用重测序数据与参考基因组(02-12)进行比对开发SNP标记,最终获得2.54×106个SNP标记。从中筛选出500个SNP位点用于KASP标记开发,平均每条染色体上55.6个。500个SNP位点中有442个成功转化为KASP标记,转化成功率为88.4%。KASP平台检测结果显示,在442个SNP标记中,有25个位点的未分型材料数>5,在后续分析中去除。剩余417个SNP位点的PIC值的变化范围为0.12—0.38,平均值为0.36,表现为中度多态性。在59个供试甘蓝品种中,全部417个SNP位点的杂合度大于30%的有57个,占所有品种的96.6%。其中,品种‘豫生早熟牛心’的杂合度最高,为67.8%。最终筛选出50个SNP位点作为核心标记,并利用这些标记构建甘蓝主要品种的指纹图谱。50个核心位点的PIC值的变化范围为0.35—0.38,平均值为0.36,其中位点Bol2-56的PIC值最高。基于核心SNP标记的聚类分析结果表明,59个品种的遗传相似系数为0.43—0.98。利用人工虚拟混合群体对核心SNP标记进行了验证,结果表明,利用核心标记可对甘蓝品种的特异性和真实性进行有效鉴定。【结论】基于甘蓝自交系重测序数据进行比对,共获得SNP位点2.54×106个;从中筛选获得50个核心SNP标记用于构建59个甘蓝品种的指纹图谱,并采用人工虚拟混合群体对核心SNP标记进行了验证。

关键词: 甘蓝, SNP标记, 主要品种, 指纹图谱

Abstract: 【Objective】The main cabbage varieties in production were collected and DNA fingerprinting of cabbage varieties were constructed with SNP markers to provide reference for variety distinctness and authenticity identification. 【Method】SNP loci were obtained by aligning resequencing data of 50 cabbage inbred lines to the reference genome of cabbage (02-12). The SNP loci were screened with two criteria: (i) high polymorphism information content (PIC), (ii) evenly distribution on each chromosome. And then KASP primers were designed based on these loci. The KASP platform was used to genotype the fifty-nine cabbage varieties. According to the result of genotyping, core primers were selected with high PIC value, no loci missing and even distribution on each chromosome. These core primers were used to establish SNP fingerprinting of fifty-nine cabbage varieties. Fifteen varieties randomly selected from the fifty-nine main varieties and five new unreleased combinations were mixed together to construct an artificial population. The artificially mixed population was used to validate the core primers in the variety distinctness and authenticity identification. 【Result】Two million five hundred and forty thousand SNP loci were obtained by aligning the resequencing data of fifty cabbage inbred lines to the reference genome of cabbage. Five hundred SNP markers were selected with high PIC value, low missing rates and even distribution on each chromosome, with 55.6 loci per chromosome. 442 of them were successfully transformed into KASP markers, occupying 88.4% of all the 500 SNP markers. The genotyping results showed that twenty-five pairs of KASP primers were unsuccessfully genotyped in more than five materials and were removed from further analysis. The PIC values among the 417 primers ranged from 0.12 to 0.38. The average PIC value was 0.36, showing moderate polymorphism. The number of varieties with heterozygous marker ratio greater than 30% was 57, occupying 88.4% of all the main varieties. The heterozygous marker ratio of ‘Yusheng Zaoshu Niuxin’ was the highest, which reaches 67.8%. Finally, fifty core markers were selected to construct DNA fingerprinting of 59 main varieties. PIC values of 50 core makers ranged from 0.35 to 0.38, and the average PIC was 0.36. The result of cluster analysis of core SNP markers indicated that the genetic similarity coefficient of 59 varieties varied from 0.43 to 0.98. The core SNP markers were validated by artificially mixed population and the results showed that SNP fingerprinting constructed with core markers could be used to identify the variety distinctness and authenticity effectively. 【Conclusion】Two million five hundred and forty thousand SNP loci were obtained by aligning resequencing data of cabbage inbred lines. Fifty pairs of core makers were selected and used to establish DNA fingerprint database of 59 cabbage varieties, and the core SNP markers were validated by artificially mixed population.

Key words:  cabbage, SNP marker, main varieties, fingerprinting