摘要
【目的】开发黑果枸杞基因组SSR标记并验证其有效性,为黑果枸杞SSR分子开发应用提供依据。【方法】利用Illumina Hiseq 2500测序平台对2年生的黑果枸杞Lr-01无性系的基因组进行PE150测序,用MISA软件对获得的基因组序列进行检索与分析。在此基础上,以18份宁夏枸杞、1份中国枸杞、23份黑果枸杞和6份其他枸杞种质为供试材料,利用Primer 3.0设计SSR引物,进行SSR引物筛选和多样性验证。【结果】在黑果枸杞中共检索到2 326条Scaffolds,含有2 494个SSR位点,每5.29 kb就有1个SSR位点,优势重复基序为单核苷酸、二核苷酸和三核苷酸,分别占总SSR位点的59.18%,27.11%和11.75%。重复基元类型共21种,其中类型最多的为单核苷酸重复A/T,占总数的56.05%;其次为二核苷酸重复AT/AT,占总数的12.35%。从设计的SSR引物中,随机挑选合成150对引物进行PCR扩增,筛选出10对高多态性SSR引物,分析其在48份枸杞种质中遗传参数,结果共检测到186个等位基因,平均为19个;观察杂合度、期望杂合度和多态信息含量平均值分别为0.615,0.834和0.817。UPGMA聚类分析结果表明,48份枸杞种质被分为2个类群,其中类型Ⅰ包括23份种质,全部为黑果枸杞;类型Ⅱ包括25份种质,主要为宁夏枸杞。分析结果显示,48份枸杞种质群体结构和群体种质资源遗传结构均可分为2个类群,与聚类分析结果基本一致。【结论】通过高通量测序技术开发黑果枸杞SSR标记是一种简单而高效途径,这些SSR标记为黑果枸杞种质资源的遗传多样性分析及遗传图谱构建等研究提供了可靠的标记选择。
【Objective】The development and validation of genomic SSR markers was conducted to provide basis for development and application of SSR molecule of Lycium ruthenicum Murr..【Method】Illumina Hiseq 2500 sequencing platform was used to sequence the genome of L.ruthenicum by PE150 sequencing,and the obtained genome sequence was screened and analyzed by MISA.On the basis,18 L.barbarum,1 Lycium chinense,23 L.ruthenicum and 6 other L.barbarum germplasms were used as test materials.Primer 3.0 was used to design SSR primers for SSR primer screening and diversity verification.【Result】A total of 2326 SSR Scaffolds were obtained and there were 2494 SSR loci,with one SSR locus per 5.29 kb in black fruit wolfberry berries.The dominant repeat motifs were single nucleotide,dinucleotide and trinucleotide,accounting for 59.18%,27.11%and 11.75%of the total,respectively.There were 21 types of repeat motifs,the most of which were single nucleotide repeat A/T with contribution of 56.05%,followed by dinucleotide repeat AT/AT with contribution of 12.35%.SSR primers were designed by Primer 3.0,of which 150 pairs were randomly selected and synthesized for PCR amplification.The results revealed that 10 pairs of primers showed high polymorphism,and 186 alleles were detected in 48 germplasms,with an average of 19 alleles.The average values of heterozygosity,expected heterozygosity and polymorphic information content were 0.615,0.834 and 0.817,respectively.UPGMA cluster analysis showed that 48 germplasms were divided into two groups,of which typeⅠincluded 23 germplasms,all L.ruthenicum and typeⅡincluded 25 germplasms,mainly L.barbarum.Population structure analysis revealed that 48 accessions were also divided into two groups.The results were in line with UPGMA cluster analysis.【Conclusion】High throughput sequencing technology is a simple and efficient method to develop SSR markers of L.ruthenicum.These SSR markers could provide reliable marker selection for genetic diversity analysis and genetic map construction of L.ruthenicum.
作者
黄兴发
尹跃
赵建华
姚佳依
秦小雅
汪淑芬
曹有龙
战祥强
HUANG Xingfa;YIN Yue;ZHAO Jianhua;YAO Jiayi;QIN Xiaoya;WANG Shufen;CAO Youlong;ZHAN Xiangqiang(College of Horticulture,Northwest A&F University,Yangling,Shaanxi 712100,China;National Wolfberry Engineering Research Center,Ningxia Academy of Agriculture and Forestry Sciences,Yinchuan,Ningxia 750002,China)
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2021年第1期126-135,共10页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家自然科学基金项目(31760218)
第三批宁夏青年科技人才托举工程项目(TJGC2018022)
全产业链创新示范项目(YES-16-0405)
宁夏农业特色优势产业新品种选育专项(2013NYYZ0101)
陕西省自然科学基础研究计划项目(2017JM3019)。
