基于KASP技术的水牛SNP标记开发及其分子身份证构建  

Development of SNP markers and establishment of molecular identity cards for water buffalo based on KASP technology

作　　者：庞春英 梁莎莎 文崇利 钟华配 韦科龙 梁淦 梁宇辰 陈笑寒 黄斌 冯玲 梁贤威 PANG Chunying;LIANG Shasha;WEN Chongli;ZHONG Huapei;WEI Kelong;LIANG Gan;LIANG Yuchen;CHEN Xiaohan;HUANG Bin;FENG Ling;LIANG Xianwei(Guangxi Buffalo Research Institute/Guangxi Key Laboratory of Buffalo Genetics,Breeding and Reproduction Technology,Ministry of Agriculture and Rural Affairs,Nanning 530001,China)

机构地区：[1]广西水牛研究所/农业农村部(广西)水牛遗传繁育重点实验室,广西南宁530001

出　　处：《畜牧与兽医》2025年第3期1-10,共10页Animal Husbandry & Veterinary Medicine

基　　金：农业农村部畜禽种质资源精准鉴定项目(202103);广西科技重大专项(桂科AA16450002)。

摘　　要：旨在利用竞争性等位基因特异性聚合酶链式反应(competitive allele specific PCR,KASP)技术,探讨水牛分子身份证构建的方法,为今后水牛品种精准鉴定及其品种资源的保护等提供技术支撑。基于水牛基因组重测序获得单核苷酸多态性(SNP)位点信息,筛选合适SNP位点,通过转化标记获得可用于分子身份证构建的SNP位点,进行遗传多样性、群体结构分析,并分别使用二进制和十进制构建14个水牛品种的DNA分子身份证。结果:基于前期研究的10头不同水牛品种基因组重测序数据,筛选获得284个在不同种间有差异且品种内一致的SNPs位点,在40个水牛样本中测序并结合桑格(Sanger)测序验证获得的60个SNPs位点,6个位点未能正常分型,转化成功率为90%,有效等位基因(Ne)平均值为1.522,香农信息指数(SHA)平均值为0.503,观察杂合度(Ho)和期望杂合度(He)平均值分别为0.182和0.328,多态性信息含量值(PIC)平均值为0.269;除去20个在部分水牛品种中位点缺失的SNPs位点,应用34个SNPs位点进行群体遗传结构分析表明14个水牛品种分为2个类群,其中槟榔江水牛、尼里/拉菲水牛和摩拉水牛的遗传距离与其他11种水牛的较远,同为1个类群;最终根据34个SNPs位点构建了14个水牛品种的分子身份证。综上,应用KASP技术并结合遗传多样性和群体结构分析,成功开发出能精准区分全部供试水牛品种的34个SNPs位点,并构建了14个水牛品种具有唯一性的二进制和十进制分子身份证,说明此方法可有效构建水牛分子身份证。The competitive allele specific PCR(KASP)technology was used in this study to establish a method of constructing molecular identity cards for water buffaloes,in order to offer technical support for precise identification of water buffalo breeds and protection of breed resources in the future.Based on water buffalo genome resequencing,SNP site information was obtained,suitable SNP sites were screened,and SNP sites that can be used for molecular identity card construction were obtained through transformation markers.Genetic diversity and population structure analysis were conducted,and DNA molecular identity cards for 14 water buffalo breeds were constructed using binary and decimal systems,respectively.Based on genome resequencing data from 10 different breeds of water buffalo,284 SNPs with differences be⁃tween different breeds and consistency within the breed were screened.60 SNPs were obtained by sequencing 40 water buffalo samples and by combining with Sanger sequencing verification.Six SNPs failed to be classified normally,resulting in a conversion power of 90%.The average effective allele(Ne)value was 1522,and the average Shannon information index(SHA)value was 0503The average values of observed heterozygosity(Ho)and expected heterozygosity(He)were 0182 and 0328,respectively,and the polymorphism information content(PIC)was 0269Excluding 20 SNPs that were missing in some water buffalo breeds,a population genetic structure analysis using 34 SNPs showed that 14 water buffalo breeds were divided into 2 groups.Among them,the genetic distance of the Binglangjiang buffalo,Nili/Lafite buffalo and Mora buffalo was far from that of the other 11 water buffalo breeds,and they belong to the same group.Finally,molecular identi⁃fication cards for 14 water buffalo breeds were constructed based on 34 SNPs.By applying the KASP technology combined with genetic diver⁃sity and population structure analysis,34 SNPs that can accurately distinguish all 14 tested water buffalo breeds were successfully developed,and 14 unique bi

关 键 词：水牛 竞争性等位基因特异性技术 SNP标记 分子身份证 

分 类 号：S823[农业科学—畜牧学]