基于ISSR分子标记的野生桃儿七遗传多样性研究
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摘要
通过对甘肃南部青藏高原边缘区道地性药材桃儿七遗传多样性研究,为野生桃儿七资源的保护提供依据。采用ISSR分子标记技术,对13个野生桃儿七居群153份DNA进行PCR扩增,对其扩增条带进行遗传多样性分析,在所得遗传距离的基础上进行UPGMA聚类和Mantel检验地理距离,对野生桃儿七居群间遗传多样性差异进行分析。11条ISSR引物共检测到155个条带,每条引物10～17个,平均14.1个,共1320个多态性位点;居群平均多态性位点百分比(PPL)65.50%;Nei′s遗传多样性指数(H)为0.2101,Shannon′s信息指数(I)为0.3191;遗传距离变化范围0.0316～0.3769;Mantel检验P=0.4220。甘肃南部青藏高原边缘区13个野生桃儿七居群间具有较高的遗传多样性,种群内的基因流十分丰富,居群内的遗传分化明显比居群之间的分化要大,各居群间遗传距离与地理距离无相关关系。
In order to provide information for the domestication and protection of wild Sinopodophyllum hexandrum(Royle)T.S.Ying that serves as genuine medicinal material,we analyzed the genetic diversity by using ISSR markers in thirteen populations with 153 DNA samples,which were collected from the southern part of Gansu province.By cluster analysis using the un-weighted pair-group method with arithmetic average(UPGMA)and Mantel test with geographic distance,we constructed a dendrogram out based on the molecular data.In total,155 alleles and 1320 polymorphic loci were amplified using these 11 ISSR primers.Each marker produced 10-17 alleles,with an average of 14.1.The average percentage of polymorphic loci(PPL)was 65.50%.The Nei′s gene diversity(H)and Shannon′s information index(I)were 0.2101 and 0.3191,respectively.The genetic distance range from 0.0316 to 0.3769.The genetic diversity among 13 populations of Sinopodophyllum hexandrum(Royle)T.S.Ying was relatively abundant and the gene flow within the populations were frequently observed.The genetic differentiation within the population was significantly higher than that between populations,and no correlation was observed between the genetic diversity and the distances among populations.
In order to provide information for the domestication and protection of wild Sinopodophyllum hexandrum(Royle)T.S.Ying that serves as genuine medicinal material,we analyzed the genetic diversity by using ISSR markers in thirteen populations with 153 DNA samples,which were collected from the southern part of Gansu province.By cluster analysis using the un-weighted pair-group method with arithmetic average(UPGMA)and Mantel test with geographic distance,we constructed a dendrogram out based on the molecular data.In total,155 alleles and 1320 polymorphic loci were amplified using these 11 ISSR primers.Each marker produced 10-17 alleles,with an average of 14.1.The average percentage of polymorphic loci(PPL)was 65.50%.The Nei′s gene diversity(H)and Shannon′s information index(I)were 0.2101 and 0.3191,respectively.The genetic distance range from 0.0316 to 0.3769.The genetic diversity among 13 populations of Sinopodophyllum hexandrum(Royle)T.S.Ying was relatively abundant and the gene flow within the populations were frequently observed.The genetic differentiation within the population was significantly higher than that between populations,and no correlation was observed between the genetic diversity and the distances among populations.
引文
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[8]Kong Y,Xiao J J,Meng S C,Dong X M,Ge Y W,Wang R F,Shang M Y,Cai S Q. A new cytotoxic flavonoid from the fruit ofSinopodophyllumhexandrum.Fitoterapia,2010,81:367-370
[9]王笠,赵志礼,倪梁红,嘎务,米玛.基于ISSR标记的麻花艽遗传多样性分析.中草药,2017,48(15):3168-3174Wang L,Zhao Z L,Ni L H,Gaawe Dorje,Mi M. Assessment ofgeneticdiversityonGentianastramineabasedonISSR markers. Chinese Traditional and Herbal Drugs,2017,48(15):3168-3174
[10]Gupta M,Chyi Y S,Romero-Severson J,Owen J L. Amplification ofDNAmarkersfromevolutionarilydiversegenomesusing singleprimersofsimple-sequencerepeats.Theoreticaland Applied Genetics,1994,89(7-8):998-1006
[11]钱韦,葛颂,洪德元.采用RAPD和ISSR标记探讨中国疣粒野生稻的遗传多样性.植物学报,2000,42(7):741-750Qian W,Ge S,Hong D Y. Assessment of genetic variation of oryza granulata detected by RAPDs and ISSRs. Acta Botanica Sinica,2000,42(7):741-750
[12]McGregor C E,Lambert C A,Greyling M M.A comparative assessment of DNA fingerprinting techniques(RAPD,ISSR,AFLP and SSR)in tetraploid potato(Solanum tuberosumL.)germplasm. Euphytica,2000,113(2):135-144
[13]WolffK,ZietkiewiczE,HofstraH.Identificationof chrysanthemumcultivarsandstabilityofDNAfingerprint patterns. Theoretical and Applied Genetics,1995,91(3):439-447
[14]任梦云,陈彦君,张盾,杜乐山,刘方,关潇,张银东. ISSR标记技术在药用植物资源中的研究进展及应用.生物技术通报,2017,33(4):63-69Ren M Y,Chen Y J,Zhang D,Du L S,Liu F,Guan X,Zhang Y D. Application and research progress of inter-simple sequence repeat(ISSR)markerinmedicinalplants.Biotechnology Bulletin,2017,33(4):63-69
[15]张盾,任梦云,关潇,张银东.濒危藏药桃儿七ISSR-PCR反应体系的建立与优化.分子植物育种,2017,15(4):1408-1416ZhangD,RenM Y,GuanX,Zhang YD.Establishmentand optimization of ISSR-PCR reaction System for Sinopodophyllum hexandrum(Royle)ying. Molecular Plant Breeding,2017,15(4):1408-1416
[16]Ye H F C,Yang R C,Boyle T B J.Popgene:Microsoft windows?-Basedfreewareforpopulationgeneticanalysis,Version132.Canada:Universityof AlbertaandCentrefor International Forestry Research,1997
[17]Rohlf F J. NTSYS-pc:numerical taxonomy and multivariate analysis system. Applied Biostatistics,1992
[18]OksanenJ,KindtR,LegendreP. Theveganpackage.Community ecology package,2007,10:631-637
[19]Burns E L,Eldridge M D,Houlden B A.Microsatellite variation and population structure in a declining Australian Hylid Litoria aurea. Molecular Ecology,2004,13(7):1745-1757
[20]张惠源,赵润怀,袁昌齐,孙传奇,张志英.我国的中药资源种类.中国中药杂志,1995(7):387-390Zhang H Y,Zhao R H,Yuan C Q,Sun C Q,Zhang Z Y. Types ofChinesetraditionalmedicineresourcesinChina.China Journal of Chinese Materia Medica,1995(7):387-390
[21]董静洲,易自力,蒋建雄.我国药用植物种质资源研究现状.西部林业科学,2005,34(2):95–101Dong J Z,Yi Z L,Jiang J X. Current situation of the research on the germ plasm resources of medicinal plant in China. Ourna l of West China Forestry Science,2005,34(2):95–101
[22]时圣明,潘明佳,王洁,陈常青.分子鉴定技术在中药中的应用.中草药,2016,47(17):3121-3126Shi S M,Pan M J,Wang J,Chen C Q. Application of molecular identificationtechniquesinChinesemateriamedica.Chinese Traditional and Herbal Drugs,2016,47(17):3121-3126
[23]蒋志刚,马克平.中国生物多样性保护的国家意志,科学决策和公众参与:第一份省域物种红色名录研究.生物多样性,2017,25(7):794-795Jiang Z G,Ma K P. The state′s will,scientific decision and citizen participation:in memory of the first provincial species red list in China. Biodiversity Science,2017,25(7):794-795
[24]贺金生,马克平.保护生物学.杭州:浙江科学技术出版社,1997:20-33HeJS,MaKP.Conservationbiology.Hangzhou:Zhejiang Science and Technology Press,1997:20-33
[25]薛达元.《名古屋议定书》的主要内容及其潜在影响.生物多样性,2011,19(1):113-119Xue D Y. Analysis for the main elements and potential impacts of Nagoya Protocol. Biodiversity Science,2011,19(1):113-119
[26]Nag A,Ahuja P S,Sharma R K. Genetic diversity of highelevationpopulationsofanendangeredmedicinalplant. AoB Plants,2015:7
[27]任秀霞,张盈,薛璟祺,朱富勇,石丰瑞,王顺利,张秀新.滇牡丹天然居群的遗传多样性分析.植物遗传资源学报,2015,16(4):772-780RenXX,Zhang Y,XueJQ,ZhuF Y,ShiFR,WangSL,Zhang X X. Genetic Diversity analysis of natural populations in Paeonia delavayi. Journal of Plant Genetic Resources,2015,16(4):772-780
[28]刘强,尹翔,杨艳,陈景震,蒋丽娟.白檀自然居群遗传结构与遗传多样性研究.植物遗传资源学报,2015,16(4):751-758LiuQ,YinX,YangY,ChenJZ,JiangLJ. Analysisof genetic diversity and genetic structure in natural populations of Symplocos paniculata.Journal of Plant Genetic Resources,2015,16(4):751-758
[2]傅立国.中国植物红皮书—稀有濒危植物.北京:科学出版社,1991:184FuLG.Chineseplantreddatabook-Rareandendangered plants.Beijing:Science Press,1991:184
[3]陈大霞,赵纪峰,刘翔,王昌华,张植伟,秦松云,钟国跃.濒危药用植物桃儿七野生居群遗传多样性与遗传结构的SCOT分析.中国中药杂志,2013,38(2):278-283ChengDX,ZhaoJF,LiuX,WangCH,ZhangZ W,Qing S Y,ZhongG Y.Geneticdiversityandgeneticstructureof endangered wild Sinopodophyllum emodi by start codon targeted polymorphism.ChinaJournalofChineseMateriaMedica,2013,38(2):278-283
[4]应俊生.小檗科八角莲属和桃儿七属(新属)的研究.植物分类学报,1979,17(1):15-23YingJS.OnDysosma WoodsonandSinopodophyllumYing,gen.nov.oftheBerberidaceae.JournalofSystematicsand Evolution,1979,17(1):15-23
[5]马绍宾,胡志浩.小檗科鬼臼亚科的地理分布与系统发育.云南植物研究,1997,19(1):48-56Ma S B,Hu Z H.A contribution to the geographical distribution andphylogenyofpodophylloideae(berberidaceae). Acta Botanica Yunnanica,1997,19(1):48-56
[6]李经纬.中医大词典.北京:人民卫生出版社,2004:143LiJ W.Dictionaryoftraditionalchinesemedicine.Beijing:People′s Health Publishing House,2004:143
[7]陈毓亨.我国鬼臼类植物资源的研究.药学学报,1979,14(2):101-107Chen YH. AstudyontheresourcesofChinesepodophyllin plants. Acta Pharmaceutica Sinica,1979,14(2):101-107
[8]Kong Y,Xiao J J,Meng S C,Dong X M,Ge Y W,Wang R F,Shang M Y,Cai S Q. A new cytotoxic flavonoid from the fruit ofSinopodophyllumhexandrum.Fitoterapia,2010,81:367-370
[9]王笠,赵志礼,倪梁红,嘎务,米玛.基于ISSR标记的麻花艽遗传多样性分析.中草药,2017,48(15):3168-3174Wang L,Zhao Z L,Ni L H,Gaawe Dorje,Mi M. Assessment ofgeneticdiversityonGentianastramineabasedonISSR markers. Chinese Traditional and Herbal Drugs,2017,48(15):3168-3174
[10]Gupta M,Chyi Y S,Romero-Severson J,Owen J L. Amplification ofDNAmarkersfromevolutionarilydiversegenomesusing singleprimersofsimple-sequencerepeats.Theoreticaland Applied Genetics,1994,89(7-8):998-1006
[11]钱韦,葛颂,洪德元.采用RAPD和ISSR标记探讨中国疣粒野生稻的遗传多样性.植物学报,2000,42(7):741-750Qian W,Ge S,Hong D Y. Assessment of genetic variation of oryza granulata detected by RAPDs and ISSRs. Acta Botanica Sinica,2000,42(7):741-750
[12]McGregor C E,Lambert C A,Greyling M M.A comparative assessment of DNA fingerprinting techniques(RAPD,ISSR,AFLP and SSR)in tetraploid potato(Solanum tuberosumL.)germplasm. Euphytica,2000,113(2):135-144
[13]WolffK,ZietkiewiczE,HofstraH.Identificationof chrysanthemumcultivarsandstabilityofDNAfingerprint patterns. Theoretical and Applied Genetics,1995,91(3):439-447
[14]任梦云,陈彦君,张盾,杜乐山,刘方,关潇,张银东. ISSR标记技术在药用植物资源中的研究进展及应用.生物技术通报,2017,33(4):63-69Ren M Y,Chen Y J,Zhang D,Du L S,Liu F,Guan X,Zhang Y D. Application and research progress of inter-simple sequence repeat(ISSR)markerinmedicinalplants.Biotechnology Bulletin,2017,33(4):63-69
[15]张盾,任梦云,关潇,张银东.濒危藏药桃儿七ISSR-PCR反应体系的建立与优化.分子植物育种,2017,15(4):1408-1416ZhangD,RenM Y,GuanX,Zhang YD.Establishmentand optimization of ISSR-PCR reaction System for Sinopodophyllum hexandrum(Royle)ying. Molecular Plant Breeding,2017,15(4):1408-1416
[16]Ye H F C,Yang R C,Boyle T B J.Popgene:Microsoft windows?-Basedfreewareforpopulationgeneticanalysis,Version132.Canada:Universityof AlbertaandCentrefor International Forestry Research,1997
[17]Rohlf F J. NTSYS-pc:numerical taxonomy and multivariate analysis system. Applied Biostatistics,1992
[18]OksanenJ,KindtR,LegendreP. Theveganpackage.Community ecology package,2007,10:631-637
[19]Burns E L,Eldridge M D,Houlden B A.Microsatellite variation and population structure in a declining Australian Hylid Litoria aurea. Molecular Ecology,2004,13(7):1745-1757
[20]张惠源,赵润怀,袁昌齐,孙传奇,张志英.我国的中药资源种类.中国中药杂志,1995(7):387-390Zhang H Y,Zhao R H,Yuan C Q,Sun C Q,Zhang Z Y. Types ofChinesetraditionalmedicineresourcesinChina.China Journal of Chinese Materia Medica,1995(7):387-390
[21]董静洲,易自力,蒋建雄.我国药用植物种质资源研究现状.西部林业科学,2005,34(2):95–101Dong J Z,Yi Z L,Jiang J X. Current situation of the research on the germ plasm resources of medicinal plant in China. Ourna l of West China Forestry Science,2005,34(2):95–101
[22]时圣明,潘明佳,王洁,陈常青.分子鉴定技术在中药中的应用.中草药,2016,47(17):3121-3126Shi S M,Pan M J,Wang J,Chen C Q. Application of molecular identificationtechniquesinChinesemateriamedica.Chinese Traditional and Herbal Drugs,2016,47(17):3121-3126
[23]蒋志刚,马克平.中国生物多样性保护的国家意志,科学决策和公众参与:第一份省域物种红色名录研究.生物多样性,2017,25(7):794-795Jiang Z G,Ma K P. The state′s will,scientific decision and citizen participation:in memory of the first provincial species red list in China. Biodiversity Science,2017,25(7):794-795
[24]贺金生,马克平.保护生物学.杭州:浙江科学技术出版社,1997:20-33HeJS,MaKP.Conservationbiology.Hangzhou:Zhejiang Science and Technology Press,1997:20-33
[25]薛达元.《名古屋议定书》的主要内容及其潜在影响.生物多样性,2011,19(1):113-119Xue D Y. Analysis for the main elements and potential impacts of Nagoya Protocol. Biodiversity Science,2011,19(1):113-119
[26]Nag A,Ahuja P S,Sharma R K. Genetic diversity of highelevationpopulationsofanendangeredmedicinalplant. AoB Plants,2015:7
[27]任秀霞,张盈,薛璟祺,朱富勇,石丰瑞,王顺利,张秀新.滇牡丹天然居群的遗传多样性分析.植物遗传资源学报,2015,16(4):772-780RenXX,Zhang Y,XueJQ,ZhuF Y,ShiFR,WangSL,Zhang X X. Genetic Diversity analysis of natural populations in Paeonia delavayi. Journal of Plant Genetic Resources,2015,16(4):772-780
[28]刘强,尹翔,杨艳,陈景震,蒋丽娟.白檀自然居群遗传结构与遗传多样性研究.植物遗传资源学报,2015,16(4):751-758LiuQ,YinX,YangY,ChenJZ,JiangLJ. Analysisof genetic diversity and genetic structure in natural populations of Symplocos paniculata.Journal of Plant Genetic Resources,2015,16(4):751-758