摘要
【目的】开发一组能够区分中国主栽葡萄品种的竞争性等位基因特异性PCR(KASP)分子标记,为中国葡萄品种保护、品种登记和市场维权等提供技术支撑。【方法】基于前人筛选的60个葡萄高多态性SNP位点,转化为KASP标记。分别使用23份代表性品种和76份主要栽培品种对转化成功的KASP标记进行初筛、复筛、验证,获得一组高质量的KASP标记,并用于构建76份葡萄品种的DNA指纹图谱。【结果】60个SNP位点有3个不具备基因组特异性,6个无法设计KASP-PCR引物,最终51个SNP成功转化为KASP标记,转化率达到89.47%。利用LGC-SNPline平台对23份代表性品种进行基因分型,51个KASP标记全部成功分型。基于次要等位基因频率(MAF)大于0.25、多态性信息含量(PIC)大于0.35、缺失率小于0.2、杂合率小于0.6等参数,初步筛选出27个优质KASP标记。使用构建指纹图谱的76份品种复筛出22个高质量KASP标记。22个KASP标记的缺失率均小于0.12,PIC均大于0.3,杂合率在0.4-0.6的标记占77.27%,MAF大于0.3的标记占95.45%。此外,利用这22个标记对同一品种不同树体提取的23份代表性品种的DNA扩增检测,前后两次检测分型结果稳定一致,表明这22个标记具有较好的重复性和稳定性。进一步将基于22个标记获得的76份葡萄品种分型结果转化为二元编码数据,得到76份葡萄品种的指纹图谱。经邻接聚类分析和群体结果分析,可将76份葡萄品种分为3类,并能正确区分二倍体和多倍体。仅用10个标记(VIT_15_18567587、VIT_6_4258638、VIT_8_3320936、VIT_12_(2)2228357、VIT_11_19390306、VIT_16_17950801、VIT_18_11138668、VIT_12_739916、VIT_16_13454358、VIT_16_(2)1202286)就能区分70份品种,其中有54份品种达到品种鉴定的标准(差异位点数≥2)。【结论】从60个葡萄SNP中成功转化51个KASP标记,并筛选出22个高质量KASP标记,构建了76份葡萄品种的SNP指纹图谱,首次验证了KASP技术在我国�
【Objective】This study was aimed to develop a set of kompetitive allele specific PCR(KASP)markers that can be used to distinguish the main cultivated grape cultivars in China,so as to provide the technical support for domestic grape cultivar protection,cultivar registration and market right protection.【Method】60 highly polymorphic SNP loci from previous studies in grapes were selected to design KASP markers.23 representative grape cultivars and 76 main cultivated grape cultivars in China were used for preliminary screening,re-screening,and verification of the successfully transformed KASP markers.A set of high-quality KASP markers was further selected to establish the DNA fingerprint database for the 76 grape cultivars.【Result】Among the 60 SNP loci,3 were not genome-specific,6 could not be used to design KASP-PCR markers,and 51 could be successfully transformed to KASP markers with a conversion rate of 89.47%.Next,23 representative grape cultivars were successfully genotyped by using the 51 KASP markers on LGC-SNP line platform.Then,27 KASP markers of high quality were selected successfully based on the minor allele frequency(MAF)greater than 0.25,polymorphism information content(PIC)greater than 0.35,deletion rate less than 0.2,and heterozygosity rate less than 0.6.Finally,22 KASP markers were successfully re-screened in the 76 main cultivated grape cultivars.The missing rate of the 22 markers was less than 0.12,the PIC value of them was greater than 0.30,the heterozygosity rate of 0.40-0.60 was 77.27%,and the MAF of greater than 0.30 was 95.45%.In addition,the consistent genotype data were acquired with different trees from each of the 23 representative cultivars by using the 22 KASP markers,indicating that these 22 KASP markers had good reproducibility and stability for grape cultivar identification.The SNP genotyping results of 76 grape cultivars based on 22 markers were converted to binary coding data,and the fingerprints of 76 grape cultivars were obtained.Based on neighbor-joining cluster analys
作者
王富强
张建
温常龙
樊秀彩
张颖
孙磊
刘崇怀
姜建福
WANG FuQiang;ZHANG Jian;WEN ChangLong;FAN XiuCai;ZHANG Ying;SUN Lei;LIU ChongHuai;JIANG JianFu(Zhengzhou Fruit Research Institute,Chinese Academy of Agricultural Sciences,Zhengzhou 450009;Beijing Vegetable Research Center,Beijing Academy of Agricultural and Forestry Sciences,Beijing 100097)
出处
《中国农业科学》
CAS
CSCD
北大核心
2021年第13期2830-2842,I0001-I0004,共17页
Scientia Agricultura Sinica
基金
国家重点研发计划(2019YFD1001401)
国家现代农业产业技术体系(CARS-29-yc-1)
中国农业科学院科技创新工程(CAAS-ASTIP-2019-ZFRI)。
