高通量RNA-seq技术开发巴旦木目的基因SSR标记

Development of SSR Markers for Targeted Genes in Badam(Amygdalus communis L.)Based on High Throughput RNA-seq Technology

为了开发适合于木本油料巴旦木目的性状基因SSR分子标记,基于巴旦木叶片高通量RNA-seq数据,共组装得到41849个unigenes,其中22894个获得功能注释;利用MISA软件从4862个unigenes中挖掘到5260个SSR标记,它们的主要重复基序为单核苷酸(占总SSR位点数的14.30%)、二核苷酸(47.64%)和三核苷酸(31.35%)。二核苷酸重复基序中最多的重复类型是AG/CT(22.83%),三核苷酸重复基序中AAG/CTT重复类型最多(3.83%);根据unigenes功能注释和KEGG途径分析结果,筛选到13个含有SSR标记且与油脂合成、脂肪酸合成、种子大小与粒重等相关的目的基因,并设计48个目的基因SSR标记引物;以巴旦木叶片基因组DNA为模板验证SSR标记引物有效性,其中33对引物可以获得清晰的目的条带。这为巴旦木种质的遗传多样性分析、分子辅助育种和性状关联分析等提供了有效的目的基因SSR标记。

To develop SSR markers for targeted genes in Badam(Amygdalus communis L.),this study was based on high-throughput RNA-seq data of Badam leaves,and 41,849 unigenes were assembled,of which 22,894 unigenes were annotated.A total of 5,260 SSR sites were detected from 4,862 unigenes by using MISA.The dominant repeat motifs were single nucleotides(14.30%of the total SSR sites),followed by dinucleotides(47.64%)and trinucleotides(31.35%).The most frequent type of dinucleotide repeat motif is AG/CT(22.83%),and AAG/CTT repeat type in the trinucleotide repeat motif is the most(3.83%).According to the unigene annotation and KEGG pathway analysis,13 functional genes containing SSR markers associated with lipid synthesis,fatty acid synthesis,seed size and grain weight were discovered,48 pairs of targeted gene SSR primers were designed.The validity of these SSR primers were verified using genomic DNA of Badam leaf,and 33 pairs of primers amplified the clear bands.These SSRs will provide effective targeted gene markers for genetic diversity analysis,molecular-assisted breeding and association analysis of fruit quality traits of Badam germplasm.