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

doi:10.11686/cyxb2018802

Abstract

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

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.

References

[1] Liu J, Li Y, Creamer R.A re-examination of the taxonomic status of Embellisia astragali. Current Microbiology, 2016, 72: 404-409.
[2] Chen W, Sun L, Lu J, et al. Diverse nodule bacteria were associated with Astragalus species in arid region of northwestern China. Journal of Basic Microbiology, 2015, 55: 121-128.
[3] Wu H, Li X Y, Jiang Z, et al. Contrasting water use pattern of introduced and native plants in an alpine desert ecosystem, Northeast Qinghai-Tibet Plateau, China. Science of the Total Environment, 2016, 542: 182-191.
[4] Xu B C, Shan L, Li F M.Comparison of eco-physiological characteristics of seven plant species in semiarid loess hilly-gully region. Chinese Journal of Biotechnology, 2007, 18: 990-996.
徐炳成, 山仑, 李凤民. 半干旱黄土丘陵区七种植物的生理生态特征比较. 生物工程学报, 2007, 18: 990-996.
[5] Zhao M, Zhang H, Yan H, et al. Mobilization and role of starch, protein, and fat reserves during seed germination of six wild grassland species. Frontiers in Plant Science, 2018, 9: 234-244.
[6] He H, Dong Z, Pang J, et al. Phytoextraction of rhenium by lucerne (Medicago sativa) and erect milkvetch (Astragalus adsurgens) from alkaline soils amended with coal fly ash. Science of the Total Environment, 2018, 630: 570-577.
[7] Li Y, Wei W, Zhang J, et al. Structures and antipathogenic fungi activities of flavonoids from pathogen-infected Astragalus adsurgens. Natural Product Research, 2017, 20: 1-5.
[8] Xu B C, Shan L, Li F M.Responses of Medicago sativa and Astragalus adsurgens seedlings growth and water use to soil moisture regime. Chinese Journal of Applied Ecology, 2005, 16(12): 2328-2332.
徐炳成, 山仑, 李凤民. 苜蓿与沙打旺苗期生长和水分利用对土壤水分变化的反应. 应用生态学报, 2005, 16(12): 2328-2332.
[9] Luo J P, Jia J F, Gu Y H, et al. Improved protoplast-derived plants of Astragalus adsurgens through somatic embryogenesis. Chinese Journal of Biotechnology, 2000, 16(1): 17-21.
罗建平, 贾敬芬, 顾月华, 等. 沙打旺胚性原生质体培养优化及高频再生植株. 生物工程学报, 2000, 16(1): 17-21.
[10] Huang L K, Chen Z H, Zhang X Q, et al. A comparative analysis of molecular diversity of erect milkvetch (Astragalus adsurgens) germplasm from north China using RAPD and ISSR markers. Biochemical Genetics, 2009, 47: 92-99.
[11] Li R F, Li C, Su J K.Genetic diversity of Astragalus adsurgens Pall. germplasm by RAPD analysis. Acta Agrestia Sinica, 2001, 9(3): 171-175, 190.
李瑞芬, 李聪, 苏加楷. 沙打旺种质资源遗传多样性RAPD分析. 草地学报, 2001, 9(3): 171-175, 190.
[12] Pandey S, Ansari W A, Pandey M, et al. Genetic diversity of cucumber estimated by morpho-physiological and EST-SSR markers. Physiology and Molecular Biology of Plants, 2018, 24: 135-146.
[13] Ban Q, Xie C Y, Fan G H, et al. DNA finger printing of Ixeris polycephalya varieties based on EST-SSR and SRAP markers. Acta Prataculturae Sinica, 2018, 27(4): 111-122.
班骞, 谢彩云, 范国华, 等. 基于EST-SSR及SRAP标记构建苦荬菜品种(系)DNA指纹图谱. 草业学报, 2018, 27(4): 111-122.
[14] Liu H, Zhang X Q, Ma X, et al. Construction of EST-SSR fingerprinting based on fluorescence detection technology for Italian ryegrass. Scientia Agricultura Sinica, 2017, 50(3): 437-450.
刘欢, 张新全, 马啸, 等. 基于荧光检测技术的多花黑麦草EST-SSR指纹图谱的构建. 中国农业科学, 2017, 50(3): 437-450.
[15] Yan Z Z, Ren Y, Wu F, et al. The development and screening of EST-SSR markers in Melilotus albus. Pratacultural Science, 2017, 34(9): 1802-1814.
剡转转, 任艳, 吴凡, 等. 白花草木樨EST-SSR标记的开发与筛选. 草业科学, 2017, 34(9): 1802-1814.
[16] Yuan S, Zeng G, Shi M, et al. Development of EST-SSR markers for Primula ovalifolia (Primulaceae) by transcriptome sequencing. Applications in Plant Sciences, 2017, 5(12): 1700100.
[17] Jia H, Yang H, Sun P, et al. De novo transcriptome assembly, development of EST-SSR markers and population genetic analyses for the desert biomass willow, Salix psammophila. Scientific Reports, 2016, 6(1): 39591.
[18] Chen C, Xu M, Wang C, et al. Characterization of the Lycium barbarum fruit transcriptome and development of EST-SSR markers. PLoS One, 2017, 12(11): e0187738.
[19] Guo Q, Wang J X, Su L Z, et al. Development and evaluation of a novel set of EST-SSR markers based on transcriptome sequences of black locust (Robinia pseudoacacia L.). Genes, 2017, 8: 177.
[20] Wang Y, Liu K, Bi D, et al. Characterization of the transcriptome and EST-SSR development in Boea clarkeana, a desiccation-tolerant plant endemic to China. Peer Journal, 2017, 5(2): e3422.
[21] Liu C, Dou Y, Guan X, et al. De novo transcriptomic analysis and development of EST-SSRs for Sorbus pohuashanensis (Hance) Hedl. PLoS One, 2017, 12(6): e0179219.
[22] Harismendy O, Ng P C, Strausberg R L, et al. Evaluation of next generation sequencing platforms for population targeted sequencing studies. Genome Biology, 2009, 10: R32.
[23] Liu K, Muse S V.Power Marker: An integrated analysis environment for genetic marker data. Bioinformatics, 2005, 21: 2128-2129.
[24] Hall B G.Building phylogenetic trees from molecular data with MEGA. Molecular Biology and Evolution, 2013, 30: 1229-1235.
[25] Peakall R O D, Smouse P E. GENALEX 6: Genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes, 2006, 6: 288-295.
[26] Botstein D, White R L, Skolnick M, et al. Construction of genetic linkage maps in man using restriction fragment length polymorphisms. American Journal of Human Genetics, 1980, 32: 314-331.
[27] Zhai L, Xu L, Wang Y, et al. Novel and useful genic-SSR markers from de novo transcriptome sequencing of radish (Raphanus sativus L.). Molecular Breeding, 2014, 33: 611-624.
[28] Zhu Y Q, Peng D D, Lin C W, et al. Development of SSR markers based on transcriptome sequence and analysis of genetic diversity in Sorghum sudanense. Acta Prataculturae Sinica, 2018, 27(5): 178-189.
朱永群, 彭丹丹, 林超文, 等. 苏丹草转录组SSR分子标记开发及遗传多样性评价. 草业学报, 2018, 27(5): 178-189.
[29] Wang Z, Yan H, Fu X, et al. Development of simple sequence repeat markers and diversity analysis in alfalfa (Medicago sativa L.). Molecular Biology Reports, 2013, 40: 3291-3298.
[30] Yan Z, Wu F, Luo K, et al. Cross-species transferability of EST-SSR markers developed from the transcriptome of Melilotus and their application to population genetics research. Scientific Reports, 2017, 7: 17959.
[31] Bérubé Y, Zhuang J, Rungis D, et al. Characterization of EST-SSRs in loblolly pine and spruce. Tree Genetics and Genomes, 2007, 3: 251-259.
[32] Tóth G, Gáspári Z, Jurka J.Microsatellites in different eukaryotic genomes: Survey and analysis. Genome Research, 2000, 10: 967-981.
[33] Chen J, Li R, Xia Y, et al. Development of EST-SSR markers in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) based on de novo transcriptomic assemblies. PLoS One, 2017, 12(9): e0184736.
[34] Long Z C, Gichira A W, Chen J M, et al. Development of EST-SSR markers in the relict tree Davidia involucrata (Davidiaceae) using transcriptome sequencing. Genetics and Molecular Research, 2016, 15(4): gmr15048539.
[35] Li D, Deng Z, Qin B, et al. De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.). BMC Genomics, 2012, 13: 192.
[36] Parra-González L B, Aravena-Abarzúa G A, Navarro-Navarro C S, et al. Yellow lupin (Lupinus luteus L.) transcriptome sequencing: Molecular marker development and comparative studies. BMC Genomics, 2012, 13: 425.
[37] Zhou Q, Luo D, Ma L, et al. Development and cross-species transferability of EST-SSR markers in siberian wildrye (Elymus sibiricus L.) using Illumina sequencing. Scientific Reports, 2016, 6: 20549.
[38] Long Y, Wang Y, Wu S, et al. De novo assembly of transcriptome sequencing in Caragana korshinskii Kom. and characterization of EST-SSR markers. PLoS One, 2015, 10(1): e0115805.
[39] Qiang H, Chen Z, Zhang Z, et al. Molecular diversity and population structure of a worldwide collection of cultivated tetraploid alfalfa (Medicago sativa subsp. sativa L.) germplasm as revealed by microsatellite markers. PLoS One, 2015, 10(4): e0124592.
[40] Muriira N G, Muchugi A, Yu A, et al. Genetic diversity analysis reveals genetic differentiation and strong population structure in Calotropis plants. Scientific Reports, 2018, 8: 7832.
[41] Barboza K, Beretta V, Kozub P C, et al. Microsatellite analysis and marker development in garlic: Distribution in EST sequence, genetic diversity analysis, and marker transferability across Alliaceae. Molecular Genetics and Genomics, 2018, 293(5): 1091-1106.
[42] Doucet A, Overall C M.Amino-terminal oriented mass spectrometry of substrates (ATOMS) N-terminal sequencing of proteins and proteolytic cleavage sites by quantitative mass spectrometry. Methods in Enzymology, 2011, 501: 275-293.