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Acta Prataculturae Sinica ›› 2019, Vol. 28 ›› Issue (11): 147-158.DOI: 10.11686/cyxb2018802

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Development of EST-SSR molecular markers and analysis of genetic diversity of erect milk vetch (Astragalus adsurgens)

GONG Wen-long1, WANG Zan2, ZHAO Gui-qin1,*, MA Lin2, WEI Bao2, GONG Pan2, LIU Xi-qiang2   

  1. 1.College of Pratacultural Science, Gansu Agricultural University, Grassland Ecosystem Key Laboratory of Ministry of Education, Sino-U.S. Research Centers for Sustainable Grassland and Livestock Management, Lanzhou 730070, China;
    2.Institute of Animal Sciences, Chinese Academy of Agriculture Sciences, Beijing 100193, China
  • Received:2018-12-13 Online:2019-11-20 Published:2019-11-20
  • Contact: *. E-mail: zhaogq@gsau.edu.cn

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