利用SSR标记构建云南苦荞种质资源分子身份证
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摘要
苦荞是粮药兼备的重要作物,云南是苦荞的起源中心之一,也是遗传多样性和生态多样性中心,其种质资源的种类和数量很丰富。本研究为了将云南苦荞种质资源中的优良基因快速用于中国苦荞品种改良,利用SSR分子标记对48份云南苦荞种质资源进行分子身份证构建。结果表明,从分别在苦荞染色体上的100对SSR引物中,筛选出65对多态性引物,并用扩增48份云南苦荞种质资源,检测到161个等位基因,每对引物的等位基因数在2～5之间,平均为2.48个。Nei's基因多样性变化范围为0.101～0.693,平均值为0.508。引物位点的多态信息含量指数(PIC)变幅为0.041～0.500,平均为0.340。在多样性研究基础上,根据引物的等位基因数确定15对引物(Fes1326, BM06, BM28, BM38, BM43, BM53, BM55, BM56, BM62, BM66,BM68, BM69, BM71, BM74和BM76)组合,并用于构建48份种质资源的分子身份证,可有效区分各品种。本研究结果为云南苦荞种植资源鉴定和保护提供了依据。
Tartary buckwheat i s an important crop with both grain and medicine. Yunnan is one of the origin centers of Tartary buckwheat, is also the center of genetic diversity and ecological diversity and its germplasm resources are rich in variety and quantity. In order to rapidly apply the excellent genes of Yunnan Tartary buckwheat germplasm resources to the improvement of Chinese Tartary buckwheat varieties, molecular ID of 48 Yunnan Tartary buckwheat germplasm resources were constructed with SSR molecular marker. The results showed that 65 pairs of polymorphic primers were screened out from 100 pairs of SSR primers on Tartary buckwheat chromosomes and 161 alleles were detected by amplifying 48 Yunnan Tartary buckwheat germplasm resources. The alleles of each pair of primers ranged from 2 to 5, with an average of 2.48. The variation range of Nei's gene diversity was 0.101～0.693, with an average of 0.508. The polymorphism information content(PIC)index of the primer sites ranged from 0.041 to 0.500, with and average of 0.340. On the basis of diversity study, 15 pairs of primers(Fes1326, BM06, BM28, BM38, BM43, BM53, BM55, BM56, BM62, BM66, BM68, BM69,BM71, BM74 and BM76) were identified according to the number of alleles of the primers, and used to construct molecular ID of the 48 germplasm resources, which can effectively distinguish various varieties. The results would provide a basis for the identification and protection of Yunnan Tartary buckwheat germplasm resources.
Tartary buckwheat i s an important crop with both grain and medicine. Yunnan is one of the origin centers of Tartary buckwheat, is also the center of genetic diversity and ecological diversity and its germplasm resources are rich in variety and quantity. In order to rapidly apply the excellent genes of Yunnan Tartary buckwheat germplasm resources to the improvement of Chinese Tartary buckwheat varieties, molecular ID of 48 Yunnan Tartary buckwheat germplasm resources were constructed with SSR molecular marker. The results showed that 65 pairs of polymorphic primers were screened out from 100 pairs of SSR primers on Tartary buckwheat chromosomes and 161 alleles were detected by amplifying 48 Yunnan Tartary buckwheat germplasm resources. The alleles of each pair of primers ranged from 2 to 5, with an average of 2.48. The variation range of Nei's gene diversity was 0.101～0.693, with an average of 0.508. The polymorphism information content(PIC)index of the primer sites ranged from 0.041 to 0.500, with and average of 0.340. On the basis of diversity study, 15 pairs of primers(Fes1326, BM06, BM28, BM38, BM43, BM53, BM55, BM56, BM62, BM66, BM68, BM69,BM71, BM74 and BM76) were identified according to the number of alleles of the primers, and used to construct molecular ID of the 48 germplasm resources, which can effectively distinguish various varieties. The results would provide a basis for the identification and protection of Yunnan Tartary buckwheat germplasm resources.
引文
Ali M.L.,Rajewski J.F.,Baenziger P.S.,Gill K.S.,Eskridge K.M.,and Dweikat I.,2008,Assessment of genetic diversity and relationship among a collection of US sweet sorghum germplasm by SSR markers,Mol.Breeding,21(4):497-509
Chen C.W.,Cao K.,Wang L.R.,Zhu G.R.,and Fang W.C.,2011,Molecular ID establishment of main China peach varieties and peach related species,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),44(10):2081-2093(陈昌文,曹珂,王力荣,朱更瑞,方伟超,2011,中国桃主要品种资源及其野生近缘种的分子身份证构建,中国农业科学,44(10):2081-2093)
Gao Y.,Liu F.Z.,Wang K.,Wang D.J.,Gong X.,and Liu L.J.,2015,Establishment of molecular identity card for Malus Mill.originated from china based on the fingerprints of TP-M13-SSR,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plan Genetic Resources),16(6):1290-1297(高源,刘凤之,王昆,王大江,龚欣,刘立军,2015,基于TP-M13-SSR指纹图谱的中国原产苹果属植物分子身份证的建立,植物遗传资源学报,16(6):1290-1297)
Gao Y.L.,Zhu R.S.,Liu C.Y.,Li W.F.,Jiang H.W.,Li C.D.,Yao B.C.,Hu G.H.,and Chen Q.S.,2009,Establishment of molecular ID in soybean varieties in Heilongjiang,China,Zuowu Xuebao(Acta Agronomica Sinica),35(2):211-218(高运来,朱荣胜,刘春燕,李文福,蒋洪蔚,李灿东,姚丙晨,胡国华,陈庆山,2009,黑龙江部分大豆品种分子ID的构建,作物学报,35(2):211-218)
Hu Y.,Li Y.,Huang Y.Z.,and Liu P.W.,2016,Genetic diversity evaluation of sugar cane varieties using SSR and RAPDmarkers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):2494-2503(胡杨,李赟,黄有总,刘平武,2016,利用SSR与RAPD分子标记评估甘蔗品种的遗传多样性,基因组学与应用生物学,35(9):2494-2503)
Li C.H.,Yin G.F.,Wang Y.Q.,Sun D.W.,Lu W.J.,Chen R.K.,and Wang L.H.,2016,Analysis of genetic diversity of main characters of Tartary buckwheat germaplasm resources in Yunnan,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),17(6):993-999(李春花,尹桂芳,王艳青,孙道旺,卢文洁,陈蕤坤,王莉花,2016,云南苦荞种质资源主要性状的遗传多样性分析,植物遗传资源学报,17(6):993-999)
Li X.H.,Li X.H.,Gao W.W.,Tian Q.Z.,Li M.S.,Ma F.M.,and Zhang S.H.,2005,Establishment of DNA fingerprinting database of maize hybrids and its application in parentage identification,Zuowu Xuebao(Acta Agronomica Sinica),31(3):386-391(李晓辉,李新海,高文伟,田清震,李明顺,马凤鸣,张世煌,2005,玉米杂交种DNA指纹图谱及其在亲子鉴定中的应用,作物学报,31(3):386-391)
Liu K.J.,and Muse S.V.,2005,PowerMarker:an integrated analysis environment for genetic marker analysis,Bioinformatics,21(9):2128-2129
Liu P.W.,Zhou G.L.,Yang G.S.,and Fu T.D.,2005,Fingerprints construction of hybrid parents in Brassica napus and its utilization in hybrid purity test,Zuowu Xuebao(Acta Agronomica Sinica),31(5):640-646(刘平武,周国玲,杨光圣,傅廷栋,2005,双低甘蓝型油菜杂交种亲本指纹图谱构建和杂交种纯度鉴定,作物学报,31(5):640-646)
Liu W.L.,Zhang J.M.,Sheng W.M.,Sun Y.,Liu D.J.,Guo Y.F.,Yang S.P.,Zhang R.,and Zhang H.J.,2016,Genetic diversity analysis and molecular identities establishment of52 Russian potato varieties,Fengzi Zhiwu Yuzhong(Molecular Plant Breeding),14(1):251-258(刘文林,张举梅,盛万民,孙岩,刘东军,郭怡幡,杨淑萍,张睿,张宏纪,2016,52份俄罗斯引进马铃薯种质资源的遗传多样性与分子身份证构建,分子植物育种,14(1):251-258
Liu X.L.,Ma L.,Chen X.K.,Ying X.M.,Cai Q.,Liu J.Y.,and Wu C.W.,2010,Establishment of DNA fingerprint ID in sugarcane cultivars in Yunnan,China,Zuowu Xuebao(Acta Agronomica Sinica),36(2):202-210(刘新龙,马丽,陈学宽,应雄美,蔡青,刘家勇,吴才文,2010,云南甘蔗自育品种DNA指纹身份证构建,作物学报,36(2):202-210)
Morgante M.,and Olivieri A.M.,1993,PCR-amplified microsatellites as makers in plant genetics,The Plant Journal,3(1):175-182
Qiu Y.,Li X.X.,Li Q.X.,Chen Y.C.,Shen D.,Wang H.P.,and Song J.P.,2014,Establishment of the mo lecular identification for radish germplasm using SSR markers,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plan Genetic Resources),15(3):648-654(邱杨,李锡香,李清霞,陈亦辰,沈镝,王海平,宋江萍,2014,利用SSR标记构建萝卜种质资源分子身份证,植物遗传资源学报,15(3):648-654)
Russell J.R.,Fuller J.D.,Macaulau M.,Hatz B.G.,Jahoor A.,Powell W.,and Waugh R.,1997,Direct comparison of levels of genetic variation among barley accessions detected by RFLPs,AFLPs,SSRs and RAPDs,Theoretical and Applied Genetics,95(4):714-722
Shan F.,Li W.D.,Lin R.F.,and Jin Z.X.,2006,Effects of tartary extracts on the blood glucose of diabetic rat,Zhongguo Shiping Xuebao(Journal of Chinese Institute of Food Science and Technology),6(1):208-211(陕方,李文德,林汝法,金肇熙,2006,苦荞不同提取物对糖尿病模型大鼠血糖的影响,中国食品学报,6(1):208-211)
Song W.,and Xu J.R.,2000,Progress in application of germplasm identifieation and fingerprinting of fruit trees,Beijing Linye Daxue Xuebao(Journal of Beijing Forestry University),22(1):76-80(宋婉,续九如,2000,果树种质资源鉴定及DNA指纹图谱应用研究进展,北京林业大学学报,22(1):76-80)
Wang L.M.,Jiao S.J.,Jiang Y.X.,Yan H.D.,Su D.F.,and Sun G.Q.,2011,Establishment of molecular identity in 142 sweet sorghum varieties,Zuowu Xuebao(Acta Agronomica Sinica),37(11):1975-1983(王黎明,焦少杰,姜艳喜,严洪冬,苏德峰,孙广全,2011,142份甜高粱品种的分子身份证构建,作物学报,37(11):1975-1983)
Wang L.X.,Li Y.F.,Cheng L.F.,Huang L.,Li H.B.,Ge L.L.,Liu L.H.,Yao J.,and Zhao C.P.,2007,Method of ID constitution for Wheat cultivars,Zuowu Xuebao(Acta Agronomica Sinica),33(10):1738-1740(王立新,李云伏,常利芳,黄岚,李宏博,葛玲玲,刘丽华,姚骥,赵昌平,2007,建立小麦品种DNA指纹的方法研究,作物学报,33(10):1738-1740)
Wu W.,Deng Q.Y.,Zhou L.J.,Zhu X.Q.,Chen C.G.,Zhuang W.,Jin D.M.,and Wang B.,2008,Establishment of DNA fingerprinting with SSR for Y58S and some other elite parents of two line hybrid Rice,Zajiao Shuidao(Hybrid Rice),23(3):52-56(武文,邓启云,周丽洁,朱校奇,陈春光,庄文,金德敏,王斌,2008,利用SSR分子标记构建Y58S及部分重要两系杂交水稻亲本的DNA指纹图谱,杂交水稻,23(3):52-56)
Yang X.,Liu F.,Zhang Y.,Cheng Y.F.,Xue L.B.,and Chen X.H.,2016,Study on the genetic diversity of eggplant germplasm with SSR markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3450-3457(杨旭,刘飞,张宇,成玉富,薛林宝,陈学好,2016,利用SSR标记研究茄子种质资源遗传多样性,基因组学与应用生物学,35(12):3450-3457)
Zhao Z.C.,Zhou M.D.,Luo D.Z.,Hou X.,Wang Z.R.,Shen G.K.,Li F.L.,and Cao J.X.,2000,Genetic diversity of Fagoyrum tataricum cultivated in North Liangshan prefecture of Yi nationality Sichuan,China,Yichuan Xuebao(Acta Gentica Sinica),27(12):1084-1093,2000(赵佐成,周明德,罗定泽,侯鑫,王中任,沈国坤,李发良,曹吉祥,2000,四川省凉山州北部栽培苦荞麦的遗传多样性研究,遗传学报,27(12):1084-1093,2000)
Chen C.W.,Cao K.,Wang L.R.,Zhu G.R.,and Fang W.C.,2011,Molecular ID establishment of main China peach varieties and peach related species,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),44(10):2081-2093(陈昌文,曹珂,王力荣,朱更瑞,方伟超,2011,中国桃主要品种资源及其野生近缘种的分子身份证构建,中国农业科学,44(10):2081-2093)
Gao Y.,Liu F.Z.,Wang K.,Wang D.J.,Gong X.,and Liu L.J.,2015,Establishment of molecular identity card for Malus Mill.originated from china based on the fingerprints of TP-M13-SSR,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plan Genetic Resources),16(6):1290-1297(高源,刘凤之,王昆,王大江,龚欣,刘立军,2015,基于TP-M13-SSR指纹图谱的中国原产苹果属植物分子身份证的建立,植物遗传资源学报,16(6):1290-1297)
Gao Y.L.,Zhu R.S.,Liu C.Y.,Li W.F.,Jiang H.W.,Li C.D.,Yao B.C.,Hu G.H.,and Chen Q.S.,2009,Establishment of molecular ID in soybean varieties in Heilongjiang,China,Zuowu Xuebao(Acta Agronomica Sinica),35(2):211-218(高运来,朱荣胜,刘春燕,李文福,蒋洪蔚,李灿东,姚丙晨,胡国华,陈庆山,2009,黑龙江部分大豆品种分子ID的构建,作物学报,35(2):211-218)
Hu Y.,Li Y.,Huang Y.Z.,and Liu P.W.,2016,Genetic diversity evaluation of sugar cane varieties using SSR and RAPDmarkers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):2494-2503(胡杨,李赟,黄有总,刘平武,2016,利用SSR与RAPD分子标记评估甘蔗品种的遗传多样性,基因组学与应用生物学,35(9):2494-2503)
Li C.H.,Yin G.F.,Wang Y.Q.,Sun D.W.,Lu W.J.,Chen R.K.,and Wang L.H.,2016,Analysis of genetic diversity of main characters of Tartary buckwheat germaplasm resources in Yunnan,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),17(6):993-999(李春花,尹桂芳,王艳青,孙道旺,卢文洁,陈蕤坤,王莉花,2016,云南苦荞种质资源主要性状的遗传多样性分析,植物遗传资源学报,17(6):993-999)
Li X.H.,Li X.H.,Gao W.W.,Tian Q.Z.,Li M.S.,Ma F.M.,and Zhang S.H.,2005,Establishment of DNA fingerprinting database of maize hybrids and its application in parentage identification,Zuowu Xuebao(Acta Agronomica Sinica),31(3):386-391(李晓辉,李新海,高文伟,田清震,李明顺,马凤鸣,张世煌,2005,玉米杂交种DNA指纹图谱及其在亲子鉴定中的应用,作物学报,31(3):386-391)
Liu K.J.,and Muse S.V.,2005,PowerMarker:an integrated analysis environment for genetic marker analysis,Bioinformatics,21(9):2128-2129
Liu P.W.,Zhou G.L.,Yang G.S.,and Fu T.D.,2005,Fingerprints construction of hybrid parents in Brassica napus and its utilization in hybrid purity test,Zuowu Xuebao(Acta Agronomica Sinica),31(5):640-646(刘平武,周国玲,杨光圣,傅廷栋,2005,双低甘蓝型油菜杂交种亲本指纹图谱构建和杂交种纯度鉴定,作物学报,31(5):640-646)
Liu W.L.,Zhang J.M.,Sheng W.M.,Sun Y.,Liu D.J.,Guo Y.F.,Yang S.P.,Zhang R.,and Zhang H.J.,2016,Genetic diversity analysis and molecular identities establishment of52 Russian potato varieties,Fengzi Zhiwu Yuzhong(Molecular Plant Breeding),14(1):251-258(刘文林,张举梅,盛万民,孙岩,刘东军,郭怡幡,杨淑萍,张睿,张宏纪,2016,52份俄罗斯引进马铃薯种质资源的遗传多样性与分子身份证构建,分子植物育种,14(1):251-258
Liu X.L.,Ma L.,Chen X.K.,Ying X.M.,Cai Q.,Liu J.Y.,and Wu C.W.,2010,Establishment of DNA fingerprint ID in sugarcane cultivars in Yunnan,China,Zuowu Xuebao(Acta Agronomica Sinica),36(2):202-210(刘新龙,马丽,陈学宽,应雄美,蔡青,刘家勇,吴才文,2010,云南甘蔗自育品种DNA指纹身份证构建,作物学报,36(2):202-210)
Morgante M.,and Olivieri A.M.,1993,PCR-amplified microsatellites as makers in plant genetics,The Plant Journal,3(1):175-182
Qiu Y.,Li X.X.,Li Q.X.,Chen Y.C.,Shen D.,Wang H.P.,and Song J.P.,2014,Establishment of the mo lecular identification for radish germplasm using SSR markers,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plan Genetic Resources),15(3):648-654(邱杨,李锡香,李清霞,陈亦辰,沈镝,王海平,宋江萍,2014,利用SSR标记构建萝卜种质资源分子身份证,植物遗传资源学报,15(3):648-654)
Russell J.R.,Fuller J.D.,Macaulau M.,Hatz B.G.,Jahoor A.,Powell W.,and Waugh R.,1997,Direct comparison of levels of genetic variation among barley accessions detected by RFLPs,AFLPs,SSRs and RAPDs,Theoretical and Applied Genetics,95(4):714-722
Shan F.,Li W.D.,Lin R.F.,and Jin Z.X.,2006,Effects of tartary extracts on the blood glucose of diabetic rat,Zhongguo Shiping Xuebao(Journal of Chinese Institute of Food Science and Technology),6(1):208-211(陕方,李文德,林汝法,金肇熙,2006,苦荞不同提取物对糖尿病模型大鼠血糖的影响,中国食品学报,6(1):208-211)
Song W.,and Xu J.R.,2000,Progress in application of germplasm identifieation and fingerprinting of fruit trees,Beijing Linye Daxue Xuebao(Journal of Beijing Forestry University),22(1):76-80(宋婉,续九如,2000,果树种质资源鉴定及DNA指纹图谱应用研究进展,北京林业大学学报,22(1):76-80)
Wang L.M.,Jiao S.J.,Jiang Y.X.,Yan H.D.,Su D.F.,and Sun G.Q.,2011,Establishment of molecular identity in 142 sweet sorghum varieties,Zuowu Xuebao(Acta Agronomica Sinica),37(11):1975-1983(王黎明,焦少杰,姜艳喜,严洪冬,苏德峰,孙广全,2011,142份甜高粱品种的分子身份证构建,作物学报,37(11):1975-1983)
Wang L.X.,Li Y.F.,Cheng L.F.,Huang L.,Li H.B.,Ge L.L.,Liu L.H.,Yao J.,and Zhao C.P.,2007,Method of ID constitution for Wheat cultivars,Zuowu Xuebao(Acta Agronomica Sinica),33(10):1738-1740(王立新,李云伏,常利芳,黄岚,李宏博,葛玲玲,刘丽华,姚骥,赵昌平,2007,建立小麦品种DNA指纹的方法研究,作物学报,33(10):1738-1740)
Wu W.,Deng Q.Y.,Zhou L.J.,Zhu X.Q.,Chen C.G.,Zhuang W.,Jin D.M.,and Wang B.,2008,Establishment of DNA fingerprinting with SSR for Y58S and some other elite parents of two line hybrid Rice,Zajiao Shuidao(Hybrid Rice),23(3):52-56(武文,邓启云,周丽洁,朱校奇,陈春光,庄文,金德敏,王斌,2008,利用SSR分子标记构建Y58S及部分重要两系杂交水稻亲本的DNA指纹图谱,杂交水稻,23(3):52-56)
Yang X.,Liu F.,Zhang Y.,Cheng Y.F.,Xue L.B.,and Chen X.H.,2016,Study on the genetic diversity of eggplant germplasm with SSR markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3450-3457(杨旭,刘飞,张宇,成玉富,薛林宝,陈学好,2016,利用SSR标记研究茄子种质资源遗传多样性,基因组学与应用生物学,35(12):3450-3457)
Zhao Z.C.,Zhou M.D.,Luo D.Z.,Hou X.,Wang Z.R.,Shen G.K.,Li F.L.,and Cao J.X.,2000,Genetic diversity of Fagoyrum tataricum cultivated in North Liangshan prefecture of Yi nationality Sichuan,China,Yichuan Xuebao(Acta Gentica Sinica),27(12):1084-1093,2000(赵佐成,周明德,罗定泽,侯鑫,王中任,沈国坤,李发良,曹吉祥,2000,四川省凉山州北部栽培苦荞麦的遗传多样性研究,遗传学报,27(12):1084-1093,2000)