基于SLAF-seq技术的山桐子SSR标记开发及应用  

Development and Application of SSR Markers in Idesia polycarpa Based on SLAF-seq Technology

作　　者：邱文明[1] 严莉 仝铸[1] 苏春莉 何秀娟[1] 肖翠 王泽琼 周明芹[2] 袁龙义[2] 孙中海[1] Qiu Wenming;Yan Li;Tong Zhu;Su Chunli;He Xiujuan;Xiao Cui;Wang Zeqiong;Zhou Mingqin;Yuan Longyi;Sun Zhonghai(Fruit and Tea Subcenter of Hubei Innovation Center of Agricultural Science and Technology,Institute of Fruit and Tea,Hubei Academy of Agricultural Sciences,Wuhan,430064;College of Horticulture and Gardening,Yangtze University,Jingzhou,434025)

机构地区：[1]湖北省农业科学院果树茶叶研究所,湖北省农业科技创新中心果树茶叶研究分中心,武汉430064 [2]长江大学园艺园林学院,荆州434025

出　　处：《分子植物育种》2024年第12期3929-3938,共10页Molecular Plant Breeding

基　　金：湖北省农业科技创新中心资助项目(2019-620-000-001-23);湖北省农业科学院青年拔尖人才计划项目(Q20-18028)共同资助。

摘　　要：为开发山桐子(Idesia polycarpa Maxim.)简单序列重复(simple sequence repeat,SSR)分子标记,本研究构建了山桐子特异性位点扩增片段测序(specific-locus amplified fragment sequencing,SLAF-seq)数据库。基于多态性SLAF标签序列,利用MISA程序识别到SSR位点30121个,SSR的发生频率为6.16%。SSR序列中包括3451种基元重复类型,其中单核苷酸、二核苷酸和三核苷酸为优势重复基元,SSR位点数分别为22191(73.67%)、3184(10.57%)和1338(4.44%)。单核苷酸中A/T类型比例最高,为22117(73.42%);二核苷酸和三核苷酸中以TA/AT(1743;5.79%)和AA~(A,G,C,T四种碱基均可)(233;0.77%)重复比例最高,78.52%的SSR长度主要在10~15 bp之间。为验证SSR的可靠性,随机设计合成100对SSR引物,经PCR分析,其中96对引物能扩增出预期条带,35对引物能扩增出多态性条带。选择6对多态性引物对30份山桐子样品进行遗传分析,聚类结果很好地反映出样品来源、地理分布和资源特征。以上结果表明本研究开发的SSR标记成本低、多态性高,未来可用于山桐子种质资源分析、遗传图谱构建和分子辅助育种等研究。To develop SSR(simple sequence repeat)markers in Idesia polycarpa Maxim.,we constructed a SLAF-seq(specific locus amplified fragment sequencing)database of I.polycarpa.Based on the polymorphic SLAF-tags,we identified 30121 SSR loci using MISA software,the occurrence frequency of SSR was 6.16%.And there were 3451 repeat motif types within the SSR sequences,among which mononucleotide,dinucleotide and trinucleotide were the dominant repeat motifs,the SSR number was 22191(73.67%),3184(10.57%)and 1338(4.44%),respectively.The proportion of A/T motif repeat in mononucleotide type was the highest,a total of 22117(73.42%)SSR loci were identified.TA/AT(1743;5.79%)and AA~(A,G,C,T)(233;0.77%)were the highest in dinucleotides and trinucleotides types,and 78.52%SSR had a length from 10 bp to 15 bp.To verify the feasibility of these SSR markers,100 pairs of primers were designed and synthesized randomly,the SSR-PCR results showed that 96 pairs of primers could amplify expected bands,and 35 pairs of primers could amplify polymorphic bands.Furthermore,6 pairs of primers were used to analyze the genetic relationship of 30 I.polycarpa samples,the clustering results well reflected the source,geographical distribution and resource characteristics of these samples.Above results confirmed that the SSR markers developed in this study had low cost and high polymorphism,which could be used for germplasm resources analysis,genetic map construction and molecular assisted breeding of I.polycarpa in the future.

关 键 词：山桐子 简单序列重复(SSR) 简化基因组测序(SLAF-seq) 

分 类 号：S794.9[农业科学—林木遗传育种]