沙打旺EST-SSR分子标记开发及其遗传多样性分析

doi:10.11686/cyxb2018802

摘要/Abstract

摘要： 沙打旺是一种高产优质、抗逆性强的多年生异花授粉豆科牧草,但分子标记的缺乏限制了其在遗传育种等方面的研究和利用。本研究旨在开发大量沙打旺EST-SSR分子标记,为沙打旺种质改良和遗传多样性分析提供参考资源。首先利用De novo转录组测序技术对两个沙打旺种质(CF019650, CF020070)进行RNA-seq测序,并对测序数据进行拼接获得总长度为190587631 bp的151516个unigenes。进一步在其中的30262个unigenes中检测到39163个EST-SSR位点,SSRs分布频率为25.85%。其中6635 (21.93%)条unigenes含有两个及以上SSR位点,复合SSRs有3514个(11.61%)。对所有EST-SSR位点进行引物设计,共得到22367对特异性引物。利用两个沙打旺种质(CF019650, CF020070)对随机合成的100对引物进行初步筛选,其中90对可扩增出目的特异性条带。随机选择其中51对引物对27个沙打旺种质的遗传多样性进行评估,结果表明:51对引物的平均等位基因数、平均多态性信息含量(PIC)、平均期望杂合度(He)和平均观测杂合度(Ho)分别为8.750、0.682、0.719和0.730。主成分及聚类分析结果揭示不同生态型(匍匐或直立)沙打旺种质的遗传分布具有明显的种质特异性,且聚类结果与其地理来源之间具有较高的相关性。新开发的EST-SSR分子标记可促进沙打旺遗传改良和基因组学研究,有助于沙打旺分子标记辅助育种、QTL定位和遗传变异分析。

关键词: 沙打旺, EST-SSR, 转录组, 遗传多样性

Abstract: Erect milk vetch (Astragalus adsurgens) is a perennial cross-pollinated legume forage with superior yield, high quality, and strong stress resistance. The lack of molecular markers has limited research on, and genetic breeding of, this species. The aim of this study was to develop a large set of expressed sequence tag-simple sequence repeat (EST-SSR) molecular markers to provide reference resources for the improvement and genetic diversity analysis of erect milk vetch accessions. First, RNA-seq sequencing of two erect milk vetch accessions (CF019650, CF020070) was performed by de novo transcriptome sequencing technology, and 151516 unigenes with a total length of 190587631 bp were obtained by splicing the sequencing data. A total of 39163 EST-SSR loci were detected from 30262 unigene sequences at a frequency of 25.85%, of which 6635 (21.93%) contained two or more SSRs, and 3514 (11.61%) were compound SSRs. Primer pairs (PPs) were designed for all EST-SSR loci (in total, 22367 EST-SSR PPs). In addition, 100 PPs were synthesized randomly and preliminarily screened in two accessions (CF019650, CF020070), and 90 of them were determined to be clear and stable EST-SSR markers. Fifty-one PPs were randomly selected to assess the genetic diversity of 27 erect milk vetch accessions. The average allele number, average polymorphism information content, average expected heterozygosity, and average observed heterozygosity values obtained using the 51 PPs were 8.750, 0.682, 0.719, and 0.730, respectively. Principal coordinate and cluster analyses revealed clear germplasm specificity in the genetic distribution of accessions with creeping and erect ecotypes, as well as a relatively high correlation between clusters and geographic origin. These newly developed EST-SSR molecular markers will be useful for the genetic improvement of, and genomic research on, erect milk vetch, and can be used in molecular marker-assisted breeding, quantitative trait loci mapping, and genetic variation analyses.

Key words: erect milk vetch, EST-SSR, transcriptome, genetic diversity

宫文龙, 王赞, 赵桂琴, 马琳, 韦宝, 龚攀, 刘希强. 沙打旺EST-SSR分子标记开发及其遗传多样性分析[J]. 草业学报, 2019, 28(11): 147-158.

GONG Wen-long, WANG Zan, ZHAO Gui-qin, MA Lin, WEI Bao, GONG Pan, LIU Xi-qiang. Development of EST-SSR molecular markers and analysis of genetic diversity of erect milk vetch (Astragalus adsurgens)[J]. Acta Prataculturae Sinica, 2019, 28(11): 147-158.

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