常绿木质藤本植物黑老虎基因组SSR特征分析及引物开发  

Characters of genomic SSRs and development of 28 SSR markers for Kadsura coccinea,an evergreen woody vine

作　　者：邹建文[1] 罗先权[1] 饶红欣[1] 李小飞 颜立红[1] 何润华 陈灵[1] ZOU Jianwen;LUO Xianquan;RAO Hongxin;LI Xiaofei;YAN Lihong;HE Runhua;CHEN Ling(Hunan Forest Botanical Garden,Changsha 410116,Hunan,China)

机构地区：[1]湖南省森林植物园,湖南长沙410116

出　　处：《中南林业科技大学学报》2021年第4期130-138,共9页Journal of Central South University of Forestry & Technology

基　　金：湖南创新型省份建设专项(2020NK2052);湖南省林业科技创新项目(XLK201815)。

摘　　要：【目的】开发黑老虎SSR标记,为其群体遗传学研究以及种质资源收集、保存、评价以及良种选育提供依据。【方法】基于高通量测序获取候选SSR位点,设计引物进行PCR扩增及其产物多态性检测;以湖南会同、城步和桂东3个天然群体66个黑老虎植株为供试材料,选取多态位点开展遗传多样性分析,并应用5个近缘种25个植株探讨SSR标记的通用性。【结果】通过高通量测序检索到152207个候选SSR位点,以二核苷酸重复比例(57.69%)最大,其次为单核苷酸(17.30%)和四核苷酸重复(10.68%);单、二、三、四、五和六核苷酸重复单元分别以A/T(98.06%)、AC/GT(57.30%)、AAG/CTT(41.91%)、AAAT/ATTT(29.55%)、AAAAT/ATTTT(29.18%)和ACACAT/ATGTGT(40.31%)最为普遍。从160对SSR引物中筛选出28对,能成功进行PCR扩增且获得多态位点;3个天然群体的遗传多样性分析结果表明,28个位点共有168个等位基因,每位点等位基因数为2~14,实际杂合度和期望杂合度分别为0.000~0.900和0.000~0.860;会同、城步和桂东群体分别有9、13和7个位点显著偏离Hardy-Weinberg平衡(P<0.05),未发现位点间连锁不平衡现象(P≥0.05)。28个位点中,有24和14个可应用于南五味子和异形南五味子,而对于翼梗五味子、华中五味子和狭叶五味子,则通用性偏低,仅6~9个位点得以成功扩增。【结论】开发出的28个SSR标记,可应用于黑老虎及其近缘种的遗传多样性分析、种质资源评价、种质鉴定以及分子标记辅助育种等。【Objective】Kadsura coccinea is an ever-green woody vine species indigenous to southeast Asia and south China.It has broad prospects in medicine,fruit,and ornamental gardening.It is essential to develop its SSR markers for studies on its population genetics,collection,conservation and evaluation of its germplasm resources,and selection breeding.【Method】Candidate SSR loci were searched out based on high-throughput sequencing,their primers were then designed and used to PCR,and amplification products were detected to determine polymorphism of these loci.Polymorphic loci were further applied to analyze genetic diversity of three natural populations containing 66 individuals from Huitong,Chenbu,and Guidong counties,Hunan province,and to assess their cross-species transferability with 25 individuals of five relatives of this species.【Result】152207 candidate SSR loci were obtained through high-throughput sequencing,among which the ratio of di-nucleotide was the highest(57.69%),followed by mono-nucleotide(17.30%)and tetra-nucleotide(10.68%).A/T(98.06%),AC/GT(57.30%),AAG/CTT(41.91%),AAAT/ATTT(29.55%),AAAAT/ATTTT(29.18%),and ACACAT/ATGTGT(40.31%)were the most common repeat motifs of mono-,di-,tri-,tetra-,penta-,and hexanucleotides,respectively.28 primer pairs were screen out from 160,which could generate polymorphic SSR markers through PCR amplifications.It was shown from diversity analysis on three natural populations that 168 alleles were obtained totally at 28 loci,the number of alleles per locus ranged from 2 to 14,the observed and expected heterozygosity in the three natural populations ranged from 0.000 to 0.900 and from 0.000 to 0.860,respectively.9,13,and 7 loci remarkedly deviated from Hardy-Weinberg equilibrium in three natural populations of Huitong,Chenbu and Guidong,respectively(P<0.05),and significant linkage disequilibrium was absent for any pairs of loci(P≥0.05).Among 28 polymorphic loci,24 and 14 loci could be successfully amplified for K.longipedunculata and K.heteroclita,and 6 to 9 for Schi

关 键 词：黑老虎 SSR标记 种间通用性 高通量测序 遗传多样性 

分 类 号：S718.3[农业科学—林学]