基于荧光SSR的宁夏糜子DNA分子身份证的构建  

Construction of DNA molecular identity card of proso millet in Ningxia based on fluorescent SSR

作　　者：曹越 张立媛[3] 辛旭霞 冯智尊 郭娟 王晓丹 曹晓宁[2] SANTRA Dipak K 陈凌 乔治军[2] 王瑞云 CAO Yue;Zhang Li-Yuan;XIN Xu-Xia;FENG Zhi-Zun;GUO Juan;WANG Xiao-Dan;CAO Xiao-Ning;SANTRA Dipak K;CHEN Ling;QIAO Zhi-Jun;WANG Rui-Yun(Agronomy College,Shanxi Agricultural University,Taigu 030801,Shanxi,China;Center for Agricultural Genetic Resources Research,Shanxi Agricultural University/Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess Plateau,Ministry of Agriculture and Rural Affairs/Shanxi Key Laboratory of Genetic Resources and Genetic Improvement of Minor Crops,Taiyuan 030031,Shanxi,China;Chifeng Institute of Agriculture and Animal Husbandry Science,Chifeng 024031,Inner Mongolia,China;Panhandle Research&Extension Center,Department of Agronomy and Horticulture,University of Nebraska-Lincoln,Scottsbluff 69361,Nebraska,USA)

机构地区：[1]山西农业大学农学院,山西太谷030801 [2]山西农业大学农业基因资源研究中心/农业农村部黄土高原作物基因资源与种质创制重点实验室/杂粮种质资源发掘与遗传改良山西省重点实验室,山西太原030031 [3]赤峰市农牧科学研究所,内蒙古赤峰024031 [4]内布拉斯加大学林肯分校农艺系小宗粮豆研究与推广中心,美国内布拉斯加州斯克茨布拉夫69361

出　　处：《作物学报》2024年第11期2699-2711,共13页Acta Agronomica Sinica

基　　金：财政部和农业农村部国家农作物种质资源平台项目(NCGRC-2024-026);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-06-14.5-A16);山西省现代农业产业技术体系建设专项资金项目(2024CYJSTX03-12);山西省重点研发项目(2022ZDYF110);山西农业大学农学院研究生教育改革与质量提升工程项目(2023YCX33,2023YCX48,2023YCX49,2023YDT05)资助。

摘　　要：糜子(Panicum miliaceum L.)种质资源丰富,在干旱环境中具生产优势,基于荧光SSR标记构建其DNA分子身份证可为资源的数字化管理提供理论依据和分子检测工具。本文以274份宁夏糜子核心种质为试验材料,对山西农业大学前期开发的糜子特异性SSR标记进行多次PCR筛选和优化后获取核心引物。基于糜子参考基因组信息,经过BLAST序列比对后将核心标记进行染色体定位。在SSR引物的5′端标注荧光(FAM/HEX),利用毛细管电泳给出材料的基因型,采用“0,1”二进制编码方式记录扩增条带的有无,使用IDAnalysis 4.0检测材料的区分程度。采用十进制(0~9)统计扩增片段大小以获得材料的字符串分子身份证。使用Popgene、Powermarker、MEGA、NTSYS进行遗传多样性、遗传聚类和主成分分析。利用二维码在线软件(https://cli.im/)给出材料的二维码DNA分子身份证。PCR扩增结果发现,10个荧光SSR(RYW6、RYW125、RYW43、RYW3、RYW40、RYW37、RYW42、RYW8、RYW28和RYW124)组合在一起可以将274份材料全部区分开。BLAST结果表明,RYW124分布在12号染色体上,位于7.8 cM处;RYW40分布在4号染色体上,位于42.64 cM处;RYW42分布在13号染色体上,位于34.63 cM处,RYW28分布在16号染色体上,位于2.34 cM处,RYW8分布在3号染色体上,位于9.90 cM处。274份材料在10个位点共检出125个等位变异,平均每个位点为12.5个,变幅为5.0000(RYW3)~25.0000(RYW6);检出的Shannon多样性指数(I)为1.2458(RYW3)~2.6568(RYW6),平均为1.8532;观测杂合度(Ho)为0.5185(RYW40)~0.9964(RYW124),平均为0.8674;期望观测杂合度(He)为0.5724(RYW40)~0.9108(RYW42),平均为0.7784;Nei’s基因多样性指数(Nei)为0.5711(RYW40)~0.9091(RYW42),平均为0.7767;多态性信息含量(PIC)为0.6563(RYW3)~0.9602(RYW42),平均为0.8399。聚类分析和主成分分析均将材料划归4个类群。将电泳条带进行数字编码,利用10个标记组合,构建了全部材料的字符串和二维码DNA分子�Objective proso millet(Panicum miliaceum L.)possesses a rich in germplasm and demonstrates advantages in arid environments..Developing a DNA identification system for proso millet using fluorescent SSR markers can establish a theoretical foundation and provide a molecular detection tool for digital resource management.In this study,274 core germplasm samples of Ningxia proso millet were selected as an experimental materials,Proso millet specific SSR markers developed by Shanxi Agri-cultural University were subjected to multiple rounds of PCR screening and optimization.The core markers were then mapped using BLAST sequence comparison based on the reference genome information of proso millet.The SSR primers were labeled with FAM/HEX at the 5'end,and the genotypes of the materials were obtained through capillary electrophoresis..The presence of amplified bands was recorded using“0,1”binary coding,while the differentiation degree of the materials was assessed using IDAnalysis4.0.The size of the amplified fragments was represented in decimal form(0-9)to generate a unique molecular ID for each material.Genetic diversity,genetic clustering and principal component analysis.were conducted using software such as Popgene,Powermarker,MEGA,and NTSYS.The molecular IDs of the materials were obtained by generating QR codes using an online QR code software(https://cli.im/).PCR amplification results showed that a combination of 10 fluorescent SSR markers(RYW6,RYW125,RYW43,RYW3,RYW40,RYW37,RYW42,RYW8,RYW28,and RYW124)could distinguish all 274 mate-rials.BLAST analysis indicated that RYW124 was located at 7.8 cM on chromosome 12,RYW40 was located at 42.64 cM on chromosome 4,RYW42 was positioned at 34.63 cM on chromosome 13,RYW28 was at 2.34 cM on chromosome 16,and RYW8 was found at 9.90 cM on chromosome 3.Across the 10 loci,a total of 125 alleles were detected at 10 loci in the 274 samples,with an average of 12.5 alleles per locus and a range of variation from 5.0000(RYW3)to 25.0000(RYW6).The Shannon diversity index(I)ranged fro

关 键 词：糜子 宁夏 毛细管电泳 荧光SSR DNA分子身份证 染色体定位 

分 类 号：S51[农业科学—作物学]