基于NCBI数据库芦笋EST-SSR标记的开发
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摘要
为了明确芦笋EST序列中SSR的总体分布特点,开发芦笋EST-SSR标记,为利用EST-SSR分子标记进行芦笋遗传研究提供了理论依据。本研究从NCBI数据库上获得了8 615条EST,并利用在线软件ssrtool和websat检测所含SSR位点序列,并进行分析。共检索出483条SSR序列,检出率为5.61%。其中二核苷酸重复基元的EST-SSR占主导地位,占总SSR数目的 32.3%。利用在线引物设计软件Primer3.0开发了424对EST-SSR引物,随机挑选50对引物,以4份芦笋品种种质资源的DNA为模板,经8%非变性聚丙烯酰胺凝胶电泳,通过筛选验证获得了42对多态性引物,表明开发的EST-SSR具有较好的多态性。这些EST-SSR标记的开发不仅丰富了芦笋的分子标记数量,而且为其种质资源的遗传分析提供了理论依据。
In order to clarify the overall characteristics of SSR in EST sequences of Asparagus officinalis, the ESTSSR primers were developed to lay the theoretical foundation for genetic studies of Asparagus officinalis using EST-SSR primers. In this study, 8 615 ESTs were obtained from the NCBI database, and the SSR sequences contained in the databases were detected and analyzed by using online software ssrtool and websat. A total of 483 SSR sequences were retrieved and the detection rate was 5.61%. The dinucleotide repeat motif of EST-SSR dominated, accounting for 32.3% of the total SSR number. Then, 424 pairs of EST-SSR primers were developed using the Primer3.0 online primer design software. Among them, 50 pairs of primers were randomly selected and the DNA from 4 different germplasm resources of Asparagus officinalis were used as templates. The samples were screened by 8% non-denaturing polyacrylamide gel electrophoresis. Forty-two pairs of polymorphic primers were verified, indicating that the developed EST-SSR owned good polymorphism. The development of these EST-SSR markers not only enriched the number of molecular markers of Asparagus officinalis, but also would provide a basis for the genetic analysis of germplasm resources in Asparagus officinalis.
In order to clarify the overall characteristics of SSR in EST sequences of Asparagus officinalis, the ESTSSR primers were developed to lay the theoretical foundation for genetic studies of Asparagus officinalis using EST-SSR primers. In this study, 8 615 ESTs were obtained from the NCBI database, and the SSR sequences contained in the databases were detected and analyzed by using online software ssrtool and websat. A total of 483 SSR sequences were retrieved and the detection rate was 5.61%. The dinucleotide repeat motif of EST-SSR dominated, accounting for 32.3% of the total SSR number. Then, 424 pairs of EST-SSR primers were developed using the Primer3.0 online primer design software. Among them, 50 pairs of primers were randomly selected and the DNA from 4 different germplasm resources of Asparagus officinalis were used as templates. The samples were screened by 8% non-denaturing polyacrylamide gel electrophoresis. Forty-two pairs of polymorphic primers were verified, indicating that the developed EST-SSR owned good polymorphism. The development of these EST-SSR markers not only enriched the number of molecular markers of Asparagus officinalis, but also would provide a basis for the genetic analysis of germplasm resources in Asparagus officinalis.
引文
Chen H.L.,Zhang S.Q.,Gao J.M.,Zheng J.L.,Liu Q.L.,and Yi K.X.,2016,Morphological diversities of Asparagus officin alis L.germplasm resources,Redai Nongye Kexue(Ch nese Journal of Tropical Agriculture),36(5):55-63(陈河龙张世清,高建明,郑金龙,刘巧莲,易克贤,2016,芦笋种质资源形态多样性分析,热带农业科学,36(5):55-63)
Chen Y.,Zhang W.W.,Mo H.B.,Fu N.F.,and Tian D.K.,2017Construction of a genetic linkage map of lotus(Nelumb Adans.)using EST-SSR markers,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(6):2265-2273(陈岳,张微微,莫海波,付乃峰,田代科,2017,EST-SSR标记构建莲(Nelumbo Adans.)遗传连锁图谱,分子植物育种,15(6)2265-2273)
Deng C.L.,Gao J.,Cao Y.,Qin R.Y.,Gao W.J.,and Lu L.D.,2012Development and application of sex-specific EST-SSR marker in spinach(Spinacia oleracea L.),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),31(5)467-472(邓传良,高俊,曹莹,秦瑞云,高武军,卢龙斗2012,菠菜性别相关EST-SSR标记的开发及应用,基因组学与应用生物学,31(5):467-472)
Francesco M.,Paolo R.,Alex H.,Tea S.,Maria R.A.,Jim L.M.Agostino F.,and Francesco S.,2015,Single nucleotide polymorphism-based parentage analysis and population struc ture in garden asparagus,a worldwide genetic stock classi fication,Mol.Breeding,35:59
Han Y.Y.,Yang K.,Fan S.X.,and Zhang Z.X.,2007,AFLPmarkers linked with M gene in Asparagus officinalis L.Yuanyi Xuebao(Acta Horticulturae Sinica),34(3):779782(韩莹琰,杨凯,范双喜,张振贤,2007,与石刁柏性别基因M紧密连锁的AFLP新标记,园艺学报,34(3)779-782)
Li X.,Li S.H.,Yang L.,Yu J.Q.,Li B.H.,Zheng X.,and Bao YC.,2016,Genetic diversity analysis of asparagus based on SRAP markers,Nongxue Xuebao(Journal of Agriculture)6(3):40-45(李霞,李书华,杨林,于继庆,李保华,郑鑫,包艳存,2016,芦笋种质资源的SRAP遗传多样性分析,农学学报,6(3):40-45)
Lin Z.K.,Lin Y.H.,Cai K.X.,and Zheng T.,2017,Genetic rela tionships among 19 breeding parents of Asparagus by ISSRRedai Yaredai Zhiwu Xuebao(Journal of Tropical and Su btropical Botany),25(2):149-156(林宗铿,林艺华,蔡坤秀,郑涛,2017,基于ISSR方法的芦笋育种亲本间亲缘关系分析,热带亚热带植物学报,25(2):149-156)
Lu L.D.,Li R.L.,Gao W.J.,Deng C.L.,and Wang L.J.,2006Cloning and analyzing of the female-specific marker in the dioecious species Asparagus officinalis L.,Fenzi Xibao Shengwu Xuebao(Journal of Molecular Cell Biology),39(3):281-284(卢龙斗,李瑞丽,高武军,邓传良,王连军2006,芦笋雌性特异性片段的克隆和分析(简报),分子细胞生物学报,39(3):281-284)
Nicolai M.,Pisani C.,Bouchet J.P.,Vuylsteke M.,and Palloix A.2012,Discovery of a large set of SNP and SSR genetic markers by high-throughput sequencing of pepper(Capsicum annuum),Genet.Mol.Res.,11(3):2295-2300
Sun Q.M.,Li Y.Z.,Xiang X.,Chen D.M.,Yang X.Y.,Fang J.Wu X.B.,Zhou D.H.,Ma S.P.,and Ma W.C.,2013,A nov el litchi germplasm(Litchi chinensis Sonn.),Yujinqiu,iden tified by EST-SSR and SNP analysis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),11(3):403-414(孙清明,李永忠,向旭,陈道明,杨晓燕,方静,吴绪波,周东辉,马帅鹏马文朝,2013,利用SNP和EST-SSR分子标记鉴定荔枝新种质御金球,分子植物育种,11(3):403-414)
Wan X.B.,Li D.X.,Xu Y.,Xu J.T.,Zhang L.L.,Zhang L.M.,Lin L.H.,Qi J.M.,and Zhang L.W.,2017,Development and polymorphism evaluation of EST-SSR markers in kenaf,Zuo wu Xuebao(Acta Agronomica Sinica),43(8):1170-1180(万雪贝,李东旭,徐盈,徐建堂,张力岚,张列梅,林荔辉祁建民,张立武,2017,红麻EST-SSR标记的开发及其多态性评价,作物学报,43(8):1170-1180)
Wen Y.,Lu X.P.,Ren R.,Mi F.G.,Han P.A.,and Xue C.L.2013,Development of EST-SSR marker and genetic diver sity analysis in Sorghum bicolor×Sorghum sudanenes,Yichuan(Hereditas),35(2):225-232(温莹,逯晓萍,任锐,米福贵,韩平安,薛春雷,2013,高丹草EST-SSR标记的开发及其遗传多样性,遗传,35(2):225-232)
Chen Y.,Zhang W.W.,Mo H.B.,Fu N.F.,and Tian D.K.,2017Construction of a genetic linkage map of lotus(Nelumb Adans.)using EST-SSR markers,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(6):2265-2273(陈岳,张微微,莫海波,付乃峰,田代科,2017,EST-SSR标记构建莲(Nelumbo Adans.)遗传连锁图谱,分子植物育种,15(6)2265-2273)
Deng C.L.,Gao J.,Cao Y.,Qin R.Y.,Gao W.J.,and Lu L.D.,2012Development and application of sex-specific EST-SSR marker in spinach(Spinacia oleracea L.),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),31(5)467-472(邓传良,高俊,曹莹,秦瑞云,高武军,卢龙斗2012,菠菜性别相关EST-SSR标记的开发及应用,基因组学与应用生物学,31(5):467-472)
Francesco M.,Paolo R.,Alex H.,Tea S.,Maria R.A.,Jim L.M.Agostino F.,and Francesco S.,2015,Single nucleotide polymorphism-based parentage analysis and population struc ture in garden asparagus,a worldwide genetic stock classi fication,Mol.Breeding,35:59
Han Y.Y.,Yang K.,Fan S.X.,and Zhang Z.X.,2007,AFLPmarkers linked with M gene in Asparagus officinalis L.Yuanyi Xuebao(Acta Horticulturae Sinica),34(3):779782(韩莹琰,杨凯,范双喜,张振贤,2007,与石刁柏性别基因M紧密连锁的AFLP新标记,园艺学报,34(3)779-782)
Li X.,Li S.H.,Yang L.,Yu J.Q.,Li B.H.,Zheng X.,and Bao YC.,2016,Genetic diversity analysis of asparagus based on SRAP markers,Nongxue Xuebao(Journal of Agriculture)6(3):40-45(李霞,李书华,杨林,于继庆,李保华,郑鑫,包艳存,2016,芦笋种质资源的SRAP遗传多样性分析,农学学报,6(3):40-45)
Lin Z.K.,Lin Y.H.,Cai K.X.,and Zheng T.,2017,Genetic rela tionships among 19 breeding parents of Asparagus by ISSRRedai Yaredai Zhiwu Xuebao(Journal of Tropical and Su btropical Botany),25(2):149-156(林宗铿,林艺华,蔡坤秀,郑涛,2017,基于ISSR方法的芦笋育种亲本间亲缘关系分析,热带亚热带植物学报,25(2):149-156)
Lu L.D.,Li R.L.,Gao W.J.,Deng C.L.,and Wang L.J.,2006Cloning and analyzing of the female-specific marker in the dioecious species Asparagus officinalis L.,Fenzi Xibao Shengwu Xuebao(Journal of Molecular Cell Biology),39(3):281-284(卢龙斗,李瑞丽,高武军,邓传良,王连军2006,芦笋雌性特异性片段的克隆和分析(简报),分子细胞生物学报,39(3):281-284)
Nicolai M.,Pisani C.,Bouchet J.P.,Vuylsteke M.,and Palloix A.2012,Discovery of a large set of SNP and SSR genetic markers by high-throughput sequencing of pepper(Capsicum annuum),Genet.Mol.Res.,11(3):2295-2300
Sun Q.M.,Li Y.Z.,Xiang X.,Chen D.M.,Yang X.Y.,Fang J.Wu X.B.,Zhou D.H.,Ma S.P.,and Ma W.C.,2013,A nov el litchi germplasm(Litchi chinensis Sonn.),Yujinqiu,iden tified by EST-SSR and SNP analysis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),11(3):403-414(孙清明,李永忠,向旭,陈道明,杨晓燕,方静,吴绪波,周东辉,马帅鹏马文朝,2013,利用SNP和EST-SSR分子标记鉴定荔枝新种质御金球,分子植物育种,11(3):403-414)
Wan X.B.,Li D.X.,Xu Y.,Xu J.T.,Zhang L.L.,Zhang L.M.,Lin L.H.,Qi J.M.,and Zhang L.W.,2017,Development and polymorphism evaluation of EST-SSR markers in kenaf,Zuo wu Xuebao(Acta Agronomica Sinica),43(8):1170-1180(万雪贝,李东旭,徐盈,徐建堂,张力岚,张列梅,林荔辉祁建民,张立武,2017,红麻EST-SSR标记的开发及其多态性评价,作物学报,43(8):1170-1180)
Wen Y.,Lu X.P.,Ren R.,Mi F.G.,Han P.A.,and Xue C.L.2013,Development of EST-SSR marker and genetic diver sity analysis in Sorghum bicolor×Sorghum sudanenes,Yichuan(Hereditas),35(2):225-232(温莹,逯晓萍,任锐,米福贵,韩平安,薛春雷,2013,高丹草EST-SSR标记的开发及其遗传多样性,遗传,35(2):225-232)