基于菊芋转录组的SSR分子标记开发及鉴定

Development and Identification of SSR Markers Based on Transcriptome of Helianthus tuberosus L.

利用菊芋(Helianthus tuberosus L.)转录组测序获得的63 089条Unigene(45.71 Mb)进行SSR查找分析,共检测出6 635个SSR位点,包含于5 452条Unigene中,出现频率为10.52%,SSR位点发生频率为8.64%,其中SSR位点的主要类型为二核苷酸重复,占总SSR的45.24%,其次为三核苷酸重复。SSR位点中共包含180种重复基序,其中出现频率较高的基序主要有AG/CT、ACC/GGT、ATC/ATG、AAG/CTT。设计36对多态性SSR引物组合进行扩增和多态性评价,其中,20对引物存在多态性差异,占55.56%。利用20对引物对18个菊芋资源样品进行多样性分析,分析表明,有效等位基因(Ne)在SSR位点上变化幅度较小;Shannon指数平均值为0.622;期望杂合度(He)和观察杂合度(Ho)平均值分别为0.434和0.447。系统进化树显示,菊芋资源可从块茎表皮颜色上分为白皮和红皮2大类。此外,从地域来源上进行聚类,能将18份菊芋种质资源聚为5类。以上分析表明,菊芋转录组测序产生的Unigene信息可作为开发SSR标记的有效来源,利用获得的SSR标记可为菊芋及其近缘种的遗传图谱构建、遗传多样性分析、基因组比较研究等提供丰富可靠的标记选择。

A total of 63 089 Unigene（45.71 Mb） were obtained by Helianthus tuberosus transcriptome sequencing,and 6 635 SSR loci were detected in 5 452 Unigenes,with the frequency of occurrence of 10.52%,and the frequency of SSR loci of 8.64%.The dominant type of SSR locus was dinucleotide repeat,accounting for 45.24% of total SSR,followed by trinucleotide repeat.There were 180 repeating motifs in the SSR locus,among which AG/CT,ACC/GGT,ATC/ATG and AAG/CTT were the most frequent motifs.36 pairs of polymorphic SSR primer combinations were designed to amplify and carry out polymorphism evaluation.Among them,20 pairs of primers were polymorphic,accounting for 55.56%.Diversity analysis of 18 Helianthus tuberosus was carried out by using 20 pairs of primers,and the results indicated effective allele（Ne） was less varied at the SSR locus.The average value of Shannon index was 0.622.The mean heterozygosity（He） and observed heterozygosity（Ho） was 0.434 and 0.447,respectively.The results of phylogenetic tree showed that Helianthus tuberosus resources could be divided into two types according to tuber color：white seed coat and red seed coat.In addition,18 species of Helianthus tuberosus germplasm resources could be clustered into 5 groups according to regional sources.The results indicated that the Unigene information generated by sequencing of Helianthus tuberosus could be used as an effective source for the development of SSR markers.The obtained SSR markers could provide more reliable markers for genetic diversity analysis and genetic map construction of Helianthus tuberosus and its related species selection.