基于转录组测序的葛根SSR标记研究与利用（英文）
|
摘要
该研究以‘桂粉葛1号’为材料,通过转录组测序的方法测得8.9Gb clean reads,组装成137 629个转录本,最终得到83 811个Unigene序列。进化树分析表明,葛根和苜蓿、花生聚为一支。用MISA软件在83 811个序列中检测到25 452个简单重复序列(SSR)位点,三核苷酸重复的SSR数量最多,其次是二核苷酸重复。三核苷酸重复中,(AAG)n是最普遍的重复单元(27.87%)。共设计了229对SSR引物,其中28对引物可以产生清晰的条带和丰富的多态性,被用于检测44份葛根资源的遗传多样性。在44个葛根资源的基因组DNA中共扩增出90个片段,其中89个条带有多态性,平均等位基因数为3.178 6。多态性信息量范围为0.083 0～0.774 2(平均数为0.4557)。聚类分析显示遗传相似性系数范围为0.266 7～1.000 0。这些结果提示所检测的葛根资源在DNA分子水平存在着丰富的遗传多样性。当阈值为0.58时,44个资源可以划分为2个类群,且44份资源的类群划分与地理来源之间没有直接关系,但这些标记将是葛根遗传多样性研究可用的基因组资源。
In this study,137 629 transcripts were assembled from 8.9 Gb of clean Illumina DNA sequencing read data,yielding 83 811 unigene sequences from Pueraria thomsonii ‘No.1'.A phylogenetic analysis indicated that Pueraria lobata and Medicago sativa clustered together with Arachis hypogaea(peanut).We detected 25 452 SSR loci in the 83 811 assembled unigenes using MISA software.Tri-nucleotide repeats were the most abundant followed by di-nucleotide repeats.Among the tri-nucleotide motifs,(AAG)n(27.87%),was the most common repeat unit.A total of 229 SSR primer pairs were designed,and 28 markers that gave clear,polymorphic amplification products were used to analyze the genetic diversity within a panel of 44 Puerariaaccessions.Ninety SSR fragments,consisting of 89 alleles,were amplified from genomic DNA of the 44 accessions.The average allele number is 3.178 6.Polymorphic information content(PIC)values ranged between 0.083 0 and 0.774 2(mean = 0.455 7).Cluster analysis showed that the genetic similarity coefficients among the accessions ranged from 0.266 7 to 1.000 0.These resultssuggest that the detected P.lobataresources have abundant genetic diversity at DNA molecular level.The in-group similarity coefficient(0.58)was observed in the 44 germplasm accessions,and all accessions could be clearly divided into two groups.The clustering results of tested P.lobataresource did not show clear correlation to their geographic origin.These markers are reliable genomic resource for genetic diversity analysis in Pueraria.
In this study,137 629 transcripts were assembled from 8.9 Gb of clean Illumina DNA sequencing read data,yielding 83 811 unigene sequences from Pueraria thomsonii ‘No.1'.A phylogenetic analysis indicated that Pueraria lobata and Medicago sativa clustered together with Arachis hypogaea(peanut).We detected 25 452 SSR loci in the 83 811 assembled unigenes using MISA software.Tri-nucleotide repeats were the most abundant followed by di-nucleotide repeats.Among the tri-nucleotide motifs,(AAG)n(27.87%),was the most common repeat unit.A total of 229 SSR primer pairs were designed,and 28 markers that gave clear,polymorphic amplification products were used to analyze the genetic diversity within a panel of 44 Puerariaaccessions.Ninety SSR fragments,consisting of 89 alleles,were amplified from genomic DNA of the 44 accessions.The average allele number is 3.178 6.Polymorphic information content(PIC)values ranged between 0.083 0 and 0.774 2(mean = 0.455 7).Cluster analysis showed that the genetic similarity coefficients among the accessions ranged from 0.266 7 to 1.000 0.These resultssuggest that the detected P.lobataresources have abundant genetic diversity at DNA molecular level.The in-group similarity coefficient(0.58)was observed in the 44 germplasm accessions,and all accessions could be clearly divided into two groups.The clustering results of tested P.lobataresource did not show clear correlation to their geographic origin.These markers are reliable genomic resource for genetic diversity analysis in Pueraria.
引文
[1]HIEN T T,KIM H G,HAN E H,et al.Molecular mechanism of suppression of MDR1by puerarin fromPueraria lobata via NF-kappaB pathway and cAMP-responsive element transcriptional activity-dependent up-regulation of AMP-activated protein kinase in breast cancer MCF-7/adr cells[J].Molecular Nutrition&Food Research,2010,54(7):918-928.
[2]LIU B,WU Z Y,LI Y P,et al.Puerarin prevents cardiac hypertrophy induced by pressure overload through activation of autophagy[J].Biochemical and Biophysical Research Communications,2015,464(3):908-915.
[3]YEUNG D K,LEUNG S W,XU Y C,et al.Puerarin,an isoflavonoid derived from Radix puerariae,potentiates endothelium-independent relaxation via the cyclic AMP pathway in porcine coronary artery[J].European Journal of Pharmacology,2006,552(1-3):105-111.
[4]WANG J B.ISSR markers and their applications in plant genetics[J].Hereditas,2002,24(5):613-616.
[5]JING X,XU L,CHEN J Y,et al.Genetic diversity of arrowroot(Pueraria L.)varieties revealed by RAPD analysis in Chongqing area[J].Chinese Agricultural Science Bulletin,2010,26(24):80-82.
[6]CHEN D X,PENG R,LI L Y,et al.Analysis of genetic relationships of Pueraria thomsonii based on SRAP markers[J].China Journal of Chinese Material Medica,2011,36(5):538-541.
[7]GUO Y Y,CHEN C Y,HUANG J L,et al.ISSR analysis of genetic relationships in RadixPuerariae from different original place[J].Popular Science&Technology,2013,15(4):134-136.
[8]ZHOU J H,JIE Y C,DU X H,et al.RAPD analysis of arrowroot(Pueraria L.)germplasm genetic relationship[J].Crop Research,2013,27(4):347-350.
[9]YUAN C,ZHONG WJ,GONG YY,et al.Genetic diversity and trait association analysis of Pueraria lobataresources[J].Journal of Plant Genetic Resources,2017,18(2):233-241.
[10]POWELL W,MACHRAY G C,PROVAN J.Polymorphism revealed by simple sequence repeats[J].Trends in Plant Science,1996,1(7):215-222.
[11]ZHANG L W,WAN X B,XU J T,et al.De novo assembly of kenaf(Hibiscus cannabinus)transcriptome using Illumina sequencing for gene discovery and marker identification[J].Molecular Breeding,2015,35(10):192.
[12]GRABHERR M G,HAAS B J,YASSOUR M,et al.Fulllength transcriptome assembly from RNA-Seq data without a reference genome[J].Nature Biotechnology,2011,29(7):644-652.
[13]DAVIDSON N M,OSHLACK A.Corset:enabling differential gene expression analysis for de novo assembled transcriptomes[J].Genome Biology,2014,15(7):410.
[14]EDGAR R C.MUSCLE:multiple sequence alignment with high accuracy and high throughput[J].Nucleic Acids Research,2004,32(5):1 792-1 797.
[15]LI L,STOECKERT C J Jr,ROOS D S.OrthoMCL:identification of ortholog groups for eukaryotic genomes[J].Genome Research,2003,13(9):2 178-2 189.
[16]KUMAR S,STECHER G,TAMURA K.MEGA7:molecular evolutionary genetics analysis version 7.0for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1 870-1 874.
[17]MURRAY M G,THOMPSON W F.Rapid isolation of high molecular weight plant DNA[J].Nucleic Acids Research,1980,8(19):4 321-4 325.
[18]ZHANG L W,LI A Q,WANG X F,et al.Genetic diversity of kenaf(Hibiscus cannabinus)evaluated by inter-simple sequence repeat(ISSR)[J].Biochemical Genetics,2013,51(9-10):800-810.
[19]NEI M.Molecular Evolutionary Genetics[M].New York:Columbia University Press,1987:145-163.
[20]MACDONALD M.A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntingtons disease chromosomes[J].Cell,1993,72(6):971-983.
[21]AYERS N M,MCCLUNG A M,LARKIN P D,et al.Microsatellites and a single nucleotide polymorphism differentiate apparent amylose classes in an extended pedigree of US rice germplasm[J].Theoretical&Applied Genetics,1997,94(6-7):773-781.
[22]ZHANG X W,YE Z W,WANG T K,et al.Characterization of the global transcriptome for cotton(Gossypium hirsutum L.)anther and development of SSR marker[J].Gene,2014,551(2):206-213.
[23]LIANG M,YANG X M,LI H,et al.De novotranscriptome assembly of pummelo and molecular marker development[J].PLoS One,2015,10(3):e0120615.
[24]WEI M M,CHEN Y H,LIU F Z,et al.Development of SSR Markers for eggplant with transcriptome sequencing data[J].Journal of Plant Genetic Resources,2016,7(6):1 082-1 091.
[25]FENG S J,ZHAO L L,LIU Z S,et al.De novo transcriptome assembly of Zanthoxylum bungeanumusing Illumina sequencing for evolutionary analysis and simple sequence repeat marker development[J].Scientific Reports,2017,7(1):16 754.
[26]RUNGIS D,BRUBY,ZHANG J,et al.Robust simple sequence repeat markers for spruce(Picea spp.)from expressed sequence tags[J].Theoretical and Applied Genetics,2004,109(6):1 283-1 294.
[27]DUTTA S,KUMAWAT G,SINGH B P,et al.Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea[Cajanu scajan(L.)Millspaugh][J].BMCPlant Biology,2011,11(1):17.
[28]DENG S Y,WANG X R,ZHU P L,et al.Development of polymorphic microsatellite markers in the medicinal plant Gardenia jasminoides(Rubiaceae)[J].Biochemical Systematics and Ecology,2015,58:149-155.
[29]LIU F,WANG Y S,TIAN X L,et al.SSR mining in pepper(Capsicum annuum L.)transcriptome and the polymorphism analysis[J].Acta Horticulturae Sinica,2012,39(1):168-174.
[30]AGGARWAL R K,HENDRE P S,VARSHNEY R K,et al.Identification,characterization and utilization of EST-derived genic microsatellite markers for genome analyses of coffee and related species[J].Theoretical and Applied Genetics,2007,114(2):359-372.
[2]LIU B,WU Z Y,LI Y P,et al.Puerarin prevents cardiac hypertrophy induced by pressure overload through activation of autophagy[J].Biochemical and Biophysical Research Communications,2015,464(3):908-915.
[3]YEUNG D K,LEUNG S W,XU Y C,et al.Puerarin,an isoflavonoid derived from Radix puerariae,potentiates endothelium-independent relaxation via the cyclic AMP pathway in porcine coronary artery[J].European Journal of Pharmacology,2006,552(1-3):105-111.
[4]WANG J B.ISSR markers and their applications in plant genetics[J].Hereditas,2002,24(5):613-616.
[5]JING X,XU L,CHEN J Y,et al.Genetic diversity of arrowroot(Pueraria L.)varieties revealed by RAPD analysis in Chongqing area[J].Chinese Agricultural Science Bulletin,2010,26(24):80-82.
[6]CHEN D X,PENG R,LI L Y,et al.Analysis of genetic relationships of Pueraria thomsonii based on SRAP markers[J].China Journal of Chinese Material Medica,2011,36(5):538-541.
[7]GUO Y Y,CHEN C Y,HUANG J L,et al.ISSR analysis of genetic relationships in RadixPuerariae from different original place[J].Popular Science&Technology,2013,15(4):134-136.
[8]ZHOU J H,JIE Y C,DU X H,et al.RAPD analysis of arrowroot(Pueraria L.)germplasm genetic relationship[J].Crop Research,2013,27(4):347-350.
[9]YUAN C,ZHONG WJ,GONG YY,et al.Genetic diversity and trait association analysis of Pueraria lobataresources[J].Journal of Plant Genetic Resources,2017,18(2):233-241.
[10]POWELL W,MACHRAY G C,PROVAN J.Polymorphism revealed by simple sequence repeats[J].Trends in Plant Science,1996,1(7):215-222.
[11]ZHANG L W,WAN X B,XU J T,et al.De novo assembly of kenaf(Hibiscus cannabinus)transcriptome using Illumina sequencing for gene discovery and marker identification[J].Molecular Breeding,2015,35(10):192.
[12]GRABHERR M G,HAAS B J,YASSOUR M,et al.Fulllength transcriptome assembly from RNA-Seq data without a reference genome[J].Nature Biotechnology,2011,29(7):644-652.
[13]DAVIDSON N M,OSHLACK A.Corset:enabling differential gene expression analysis for de novo assembled transcriptomes[J].Genome Biology,2014,15(7):410.
[14]EDGAR R C.MUSCLE:multiple sequence alignment with high accuracy and high throughput[J].Nucleic Acids Research,2004,32(5):1 792-1 797.
[15]LI L,STOECKERT C J Jr,ROOS D S.OrthoMCL:identification of ortholog groups for eukaryotic genomes[J].Genome Research,2003,13(9):2 178-2 189.
[16]KUMAR S,STECHER G,TAMURA K.MEGA7:molecular evolutionary genetics analysis version 7.0for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1 870-1 874.
[17]MURRAY M G,THOMPSON W F.Rapid isolation of high molecular weight plant DNA[J].Nucleic Acids Research,1980,8(19):4 321-4 325.
[18]ZHANG L W,LI A Q,WANG X F,et al.Genetic diversity of kenaf(Hibiscus cannabinus)evaluated by inter-simple sequence repeat(ISSR)[J].Biochemical Genetics,2013,51(9-10):800-810.
[19]NEI M.Molecular Evolutionary Genetics[M].New York:Columbia University Press,1987:145-163.
[20]MACDONALD M.A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntingtons disease chromosomes[J].Cell,1993,72(6):971-983.
[21]AYERS N M,MCCLUNG A M,LARKIN P D,et al.Microsatellites and a single nucleotide polymorphism differentiate apparent amylose classes in an extended pedigree of US rice germplasm[J].Theoretical&Applied Genetics,1997,94(6-7):773-781.
[22]ZHANG X W,YE Z W,WANG T K,et al.Characterization of the global transcriptome for cotton(Gossypium hirsutum L.)anther and development of SSR marker[J].Gene,2014,551(2):206-213.
[23]LIANG M,YANG X M,LI H,et al.De novotranscriptome assembly of pummelo and molecular marker development[J].PLoS One,2015,10(3):e0120615.
[24]WEI M M,CHEN Y H,LIU F Z,et al.Development of SSR Markers for eggplant with transcriptome sequencing data[J].Journal of Plant Genetic Resources,2016,7(6):1 082-1 091.
[25]FENG S J,ZHAO L L,LIU Z S,et al.De novo transcriptome assembly of Zanthoxylum bungeanumusing Illumina sequencing for evolutionary analysis and simple sequence repeat marker development[J].Scientific Reports,2017,7(1):16 754.
[26]RUNGIS D,BRUBY,ZHANG J,et al.Robust simple sequence repeat markers for spruce(Picea spp.)from expressed sequence tags[J].Theoretical and Applied Genetics,2004,109(6):1 283-1 294.
[27]DUTTA S,KUMAWAT G,SINGH B P,et al.Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea[Cajanu scajan(L.)Millspaugh][J].BMCPlant Biology,2011,11(1):17.
[28]DENG S Y,WANG X R,ZHU P L,et al.Development of polymorphic microsatellite markers in the medicinal plant Gardenia jasminoides(Rubiaceae)[J].Biochemical Systematics and Ecology,2015,58:149-155.
[29]LIU F,WANG Y S,TIAN X L,et al.SSR mining in pepper(Capsicum annuum L.)transcriptome and the polymorphism analysis[J].Acta Horticulturae Sinica,2012,39(1):168-174.
[30]AGGARWAL R K,HENDRE P S,VARSHNEY R K,et al.Identification,characterization and utilization of EST-derived genic microsatellite markers for genome analyses of coffee and related species[J].Theoretical and Applied Genetics,2007,114(2):359-372.