摘要
【目的】鉴定花生RNA-seq数据中的SSR位点,明确转录组中SSR位点的分布和结构特点,开发与花生基因相关联的SSR标记,为花生重要功能基因的挖掘、等位变异研究和分子标记辅助育种奠定基础。【方法】根据栽培种花生全生育期中22种不同类型的组织RNA-Seq数据,使用MISA软件分析SSR位点分布及特征,采用Primer 3设计基因关联的SSR引物,并利用电子PCR软件对引物的质量进行检测,随机合成38对引物,进行多态性检测。【结果】从52280条转录本中共鉴定19143个SSR位点,分布于14084条转录本,发生频率为26.94%。重复单元类型为单核苷酸—五核苷酸,以单核苷酸和三核苷酸为重复单元的SSR位点数最多,分别占位点总数的39.24%和38.40%。各重复单元优势基序类型分别为A/T、AG/CT、AAG/CTT、AAAG/CTTT和AACAC/GTGTT,占所在重复单元中的比例分别为97.62%、72.01%、30.96%、24.59%和16.67%。重复单元的重复次数为5—47次,单个SSR位点的长度的分布范围为10—47 bp,基序长度主要集中在10—14 bp;复合SSR位点的长度范围为21—249 bp,以31—40 bp为主。鉴定的SSR位点中共有13477个SSR位点可以进行引物设计,其中5020条转录本序列对应到特定的基因,共包含5859个可进行引物设计的SSR位点,这些SSR位点在A基因组和B基因组共20条染色体上不均匀分布,其中B03染色体上SSR位点最多,为484个。对特定基因SSR引物进行电子PCR检测,在A.duranensis、A.ipaensis、A.hypogaea基因组中有效扩增位点分别为4468、4929和10188个,有效引物数分别为3968(67.74%)、4232(72.25%)和5174(88.33%)对,在A.hypogaea基因组中,SSR引物扩增位点主要以2个位点为主,其中,有1477对引物单位点扩增。根据SSR引物扩增位点在栽培种花生基因组中的位置信息绘制了SSR位点的物理图谱。在38对SSR引物中,共有35对(92.1%)SSR引物可以扩增出清晰的条带,其中,有11对(28.9%)SSR引物在2个品种间扩增�
【Objective】This study aimed to identify SSRs in peanut RNA-seq data,clarify their distribution and structural characteristics,and develop gene-associated SSR markers.The study may lay the foundation for the excavation of important functional genes of peanut,the study of isometric variation and molecular markers assisted breeding.【Method】From 22 different cultivated peanut tissue types and ontogenies that represent its full development,the reported RNA-Seq data,were used to analyze the distribution and characteristics of SSR using MISA software.Gene-associated SSR primers were designed by Primer 3.0 and its quality were detected by e-PCR 2.3.9.38 pairs of primers were randomly synthesized for polymorphism testing.【Result】A total of 19143 SSRs were identified from 52280 transcripts,distributed in 14084 transcripts,with a frequency of 26.94%.The dominant SSR repeat unit types were mononucleotide and trinucleotide in mononucleotide to pentanucleotide,accounting for 39.24%and 38.40%of the total SSR locus.Dominant motif types of each repeat unit were A/T,AG/CT,AAG/CTT,AAAG/CTTT,AACAC/GTGTT,accounting for 97.62%,72.01%,30.96%,24.59%,and 16.67%in the corresponding repeat units,respectively.The repetition of repeat units was 5-47 times,and the length distribution range of single SSR site was 10-47bp,mainly concentrated at 10-14 bp.The length range of compound SSR locus was 21-249 bp,mainly concentrated at 31-40 bp.Among all the SSR,13477 SSR could be used to develop SSR markers,of which 5020 transcript sequences were annotated to specific genes,containing 5859 SSR markers locus.These SSRs were unevenly distributed on the 20 chromosomes of A and B genomes,and chromosomes B03 had the most SSR locus of 484.Using electronic PCR,4468,4929 and 10188 effective loci were amplified in the genome of A.duranensis,A.ipaensis,A.hypogaea,with 3968(67.74%),4232(72.25%)and 5174(88.33%)effective markers,respectively.In the genome of A.hypogaea,SSR primers amplified mainly with 2 loci,while 1477 pairs of SSR primers were single
作者
徐志军
赵胜
徐磊
胡小文
安东升
刘洋
XU ZhiJun;ZHAO Sheng;XU Lei;HU XiaoWen;AN DongSheng;LIU Yang(Zhanjiang Experiment Station,Chinese Academy of Tropical Agricultural Sciences/Guangdong Engineering Technology Research Center for Dryland Water-saving Agriculture,Zhanjiang 524013,Guangdong;Shenzhen Agricultural Genome Research Institute,Chinese Academy of Agricultural Sciences,Shenzhen 518120,Guangdong)
出处
《中国农业科学》
CAS
CSCD
北大核心
2020年第4期695-706,共12页
Scientia Agricultura Sinica
基金
中国热带农业科学院科技创新团队专项资金（1630102017002）
