江西铅山红芽芋低温疗法脱毒苗遗传稳定性的SSR检测
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摘要
为了检测江西铅山红芽芋低温疗法脱毒苗是否存在遗传变异,本研究采用SSR分子标记技术与毛细管电泳技术相结合的方法来检测江西铅山红芽芋低温疗法脱毒苗的遗传稳定性。结果表明,根据PAGE检测从38对引物中选出10对引物(P10,P11,P18,P23,P24,XP3,XP5,XP7,XP8,XP10);10对引物的30个样本(包括低温疗法脱毒苗和大田苗)分型基本相同,且其多样性指数较低,观测纯合度和期望纯合度也均高于观测杂合度和期望杂合度;30个样本的遗传距离大部分均为0,少量为0.032 3、0.033 9或0.198 6,遗传相似系数大部分均为1,少量为0.968 2,表明遗传分化程度极低;按照UPGMA法进行聚类分析,30个样本聚为一类。以上研究表明,江西铅山红芽芋低温疗法脱毒苗无遗传变异,低温疗法脱毒可保证其遗传稳定性。本研究可为江西铅山红芽芋低温疗法脱毒提供遗传稳定性方面的依据。
In order to detect whether there was genetic variation in the genetic stability in Jiangxi Yanshan red bud taro virus-free plantlets bywith Ccryotherapy, the method of SSR molecular markers combined with capillary electrophoresis was applied in this study to detect the genetic stability in Jiangxi Yanshan red bud taro virus-free plantlets with cryotherapy. The results showed that 10 pairs of primers(P10, P11, P18, P23, P24, XP3, XP5, XP7,XP8, and XP10) were selected from 38 pairs of primers according to the PAGE test. The typing results of 30 samples(i Including virus-free plantlets by cryotherapy and field seedlings) based on 10 pairs of primers were basically the same., Ttheir diversity index was low, and the observed heterozygosity and expected heterozygosity were also higher than the observed heterozygosity and expected heterozygosity. The genetic distances of 30 samples were mostly 0, and a few were 0.032 3, 0.033 9 or 0.198 6. The majority of genetic similarity coefficients were 1 and a small amount were 0.968 2, indicating that the degree of genetic differentiation was very low. Cluster analysis was conducted according to UPGMA method, and 30 samples were clustered into only one category. The above results showindicated that Jiangxi Yanshan red bud taro virus-free plantlets by cryotherapy had no genetic variation, and virus-free by cryotherapy could ensure its genetic stability. This study couldwhich can provide a basisreference for genetic stability of Jiangxi red bud taro virus-free by cryotherapy.
In order to detect whether there was genetic variation in the genetic stability in Jiangxi Yanshan red bud taro virus-free plantlets bywith Ccryotherapy, the method of SSR molecular markers combined with capillary electrophoresis was applied in this study to detect the genetic stability in Jiangxi Yanshan red bud taro virus-free plantlets with cryotherapy. The results showed that 10 pairs of primers(P10, P11, P18, P23, P24, XP3, XP5, XP7,XP8, and XP10) were selected from 38 pairs of primers according to the PAGE test. The typing results of 30 samples(i Including virus-free plantlets by cryotherapy and field seedlings) based on 10 pairs of primers were basically the same., Ttheir diversity index was low, and the observed heterozygosity and expected heterozygosity were also higher than the observed heterozygosity and expected heterozygosity. The genetic distances of 30 samples were mostly 0, and a few were 0.032 3, 0.033 9 or 0.198 6. The majority of genetic similarity coefficients were 1 and a small amount were 0.968 2, indicating that the degree of genetic differentiation was very low. Cluster analysis was conducted according to UPGMA method, and 30 samples were clustered into only one category. The above results showindicated that Jiangxi Yanshan red bud taro virus-free plantlets by cryotherapy had no genetic variation, and virus-free by cryotherapy could ensure its genetic stability. This study couldwhich can provide a basisreference for genetic stability of Jiangxi red bud taro virus-free by cryotherapy.
引文
Ai P.F.,and Luo Z.R.,2004,Cryopreservation of in vitro shoottips of Persimmon and Date Plum by vitrification and genetic stability of regenerated plantlets,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),37(12):2023-2027(艾鹏飞,罗正荣,2004,柿和君迁子试管苗茎尖玻璃化法超低温保存及再生植株遗传稳定性研究,中国农业科学,37(12)2023-2027)
Bai J.M.,Chen X.L.,Lu X.X.,Guo H.C.,Xin X.,Zhang Z.E.and Xin P.P.,2010,Cryopreservation of in vitro shoot tips of potato by droplet vitrification and genetic stability of regenerated plantlets,Yuanyi Xuebao(Acta Horticulturae Sinica)37(9):1431-1438(白建明,陈晓玲,卢新雄,郭华春,辛霞,张志娥,辛萍萍,2010,马铃薯茎尖小滴玻璃化法超低温保存及其再生植株的遗传稳定性,园艺学报,37(9):1431-1438)
Chen F.,Wang Z.C.,He Y.X.,and Qu X.,2009,Analysis of genetic variation of wheat seeds and seedlings caused by ultra-low-temprature preservation,Henongxue Bao(Journal of Nuclear Agricultural Sciences),23(4):548-554(陈芳,王子成,何艳霞,曲先,2009,超低温保存小麦种子和幼苗的遗传变异分析,核农学报,23(4):548-554)
Feng C.H.,2014,Cryogenic techniques for germplasm conservation and virus eradication in apple(Malus),Dissertation for Ph.D.,Northwest Agriculture and Forestry University,Supervisor:Wang Q.C.,pp.55-57(冯超红,2014,苹果茎尖超低温保存技术及脱毒效率的研究,博士学位论文,西北农林科技大学园艺学院,导师:王乔春,pp.55-57)
He L.,and Wang Q.,2010,Review on the PCR-Based SSR markers isolation methods,Jiyin Zuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),29(4):775-782(何琳,王群,2010,基于PCR的SSR标记分离方法综述,基因组学与应用生物学,29(4):775-782)
Jiang Q.,Liao H.J.,Dong L.,Chen J.X.,Ning Z.Y.,Li W.W.,and Xu X.T.,2015,Induction of multiple shoot and establishment of regeneration system in Clocasia escalenta Schott,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(3):675-679(江芹,廖华俊,董玲,陈静娴,宁志怨,李卫文,许晓婷,2015,红芽芋的丛生芽诱导和再生体系建立,分子植物育种,13(3):675-679)
Jiang S.T.,Chen Y.Z.,Zheng Z.,and Pan L.J.,2012,Analysis and evaluation of nutritional components of red bud taro(Colocasia esulenla L.Schott),Shipin Kexue(Food Science),33(11):269-272(姜绍通,程元珍,郑志,潘丽军,2012,红芽芋营养成分分析及评价,食品科学,33(11):269-272)
Jin H.J.,2009,Study on oriental lily virus detection and shoot tip virus elimination by cryopreservation,Thesis for M.S.,Fujian Agriculture And Forestry University,Supervisor:Wang J.F.,pp.74-84(靳慧洁,2009,东方百合病毒检测与茎尖超低温脱毒研究,硕士学位论文,福建农林大学园艺学院,导师:王家福,pp.74-84)
Luo R.,Wu W.L.,Zhang Y.,and Li Y.H.,2010,SSR Marker and its application to crop genetics and breeding,Jiyin Zuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),29(1):137-143(罗冉,吴委林,张旸,李玉花,2010,SSR分子标记在作物遗传育种中的应用,基因组学与应用生物学,29(1):137-143)
Vieira R.L.,Silva A.L.D.,Zaffari G.R.,Steinmacher D.A.,de Freitas Fraga H.P.,and Guerra M.P.,2015,Efficient elimination of virus complex from garlic(Allium sativum L.)by cryotherapy of shoot tips,Acta Physiol.Plant,37(1):1733
Wang B.,2014,Cryopreservation,cryo-injury analysis and application to virus eradication in shoot tips of potato(Solanum tuberosum),Dissertation for Ph.D.,Northwest Agriculture and Forestry University,Supervisor:Wang Q.C.,pp.67-71(王彪,2014,马铃薯茎尖超低温保存与伤害解析及其脱毒效应研究,博士学位论文,西北农林科技大学园艺学院,导师:王乔春,pp.67-71)
Wang Q.C.,and Valkonen J.P.T.,2008a,Elimination of two viruses which interact synergistically from sweetpotato by shoot tip culture and cryotherapy,J.Virol.Methods,154(1)135-145
Wang Q.C.,and Valkonen J.P.T.,2008b,Efficient elimination of sweetpotato little leaf phytoplasma from sweetpotato by cryotherapy of shoot tips,Plant Pathol.,57(2):338-347
Wang Q.C.,Mawassi M.,Li P.,Gafny R.,Sela I.,and Tanne E.2003,Elimination of grapevine virus A(GVA)by cryopreservation of in vitro-grown shoot tips of Vitis vinifera L.Plant Sci.,165(2):321-327
Wang R.R.,2014,Cryopreservation for germplasm conservation and pathogen eradication in Chrysanthemum,Chinese jujube and potato,Dissertation for Ph.D.,Northwest Agriculture and Forestry University,Supervisor:Wang Q.C.,pp.39-41(王仁睿,2014,菊花,枣和马铃薯种质资源超低温保存技术与脱毒的研究,博士学位论文,西北农林科技大学园艺学院,导师:王乔春,pp.39-41)
Zhang J.M.,Zhang X.N.,Lu X.X.,Xin X.,Yin G.K.,He J.J.,and Chen X.L.,2014,Optimization of droplet-vitrification protocol for carnation genotypes and ultrastructural studies on shoot tips during cryopreservation,Acta Physiol.Plant,36(12):3189-3198
Zhu W.T.,Zhou H.C.,and Wang Z.C.,2013,Cryopreservation of Fragaria pentapylla and analysis of genetic stability,Guoshu Xuebao(Journal of Fruit Science),30(1):55-61(朱文涛,周厚成,王子成,2013,五叶草莓超低温保存及遗传稳定性分析,果树学报,30(1):55-61)
Bai J.M.,Chen X.L.,Lu X.X.,Guo H.C.,Xin X.,Zhang Z.E.and Xin P.P.,2010,Cryopreservation of in vitro shoot tips of potato by droplet vitrification and genetic stability of regenerated plantlets,Yuanyi Xuebao(Acta Horticulturae Sinica)37(9):1431-1438(白建明,陈晓玲,卢新雄,郭华春,辛霞,张志娥,辛萍萍,2010,马铃薯茎尖小滴玻璃化法超低温保存及其再生植株的遗传稳定性,园艺学报,37(9):1431-1438)
Chen F.,Wang Z.C.,He Y.X.,and Qu X.,2009,Analysis of genetic variation of wheat seeds and seedlings caused by ultra-low-temprature preservation,Henongxue Bao(Journal of Nuclear Agricultural Sciences),23(4):548-554(陈芳,王子成,何艳霞,曲先,2009,超低温保存小麦种子和幼苗的遗传变异分析,核农学报,23(4):548-554)
Feng C.H.,2014,Cryogenic techniques for germplasm conservation and virus eradication in apple(Malus),Dissertation for Ph.D.,Northwest Agriculture and Forestry University,Supervisor:Wang Q.C.,pp.55-57(冯超红,2014,苹果茎尖超低温保存技术及脱毒效率的研究,博士学位论文,西北农林科技大学园艺学院,导师:王乔春,pp.55-57)
He L.,and Wang Q.,2010,Review on the PCR-Based SSR markers isolation methods,Jiyin Zuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),29(4):775-782(何琳,王群,2010,基于PCR的SSR标记分离方法综述,基因组学与应用生物学,29(4):775-782)
Jiang Q.,Liao H.J.,Dong L.,Chen J.X.,Ning Z.Y.,Li W.W.,and Xu X.T.,2015,Induction of multiple shoot and establishment of regeneration system in Clocasia escalenta Schott,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(3):675-679(江芹,廖华俊,董玲,陈静娴,宁志怨,李卫文,许晓婷,2015,红芽芋的丛生芽诱导和再生体系建立,分子植物育种,13(3):675-679)
Jiang S.T.,Chen Y.Z.,Zheng Z.,and Pan L.J.,2012,Analysis and evaluation of nutritional components of red bud taro(Colocasia esulenla L.Schott),Shipin Kexue(Food Science),33(11):269-272(姜绍通,程元珍,郑志,潘丽军,2012,红芽芋营养成分分析及评价,食品科学,33(11):269-272)
Jin H.J.,2009,Study on oriental lily virus detection and shoot tip virus elimination by cryopreservation,Thesis for M.S.,Fujian Agriculture And Forestry University,Supervisor:Wang J.F.,pp.74-84(靳慧洁,2009,东方百合病毒检测与茎尖超低温脱毒研究,硕士学位论文,福建农林大学园艺学院,导师:王家福,pp.74-84)
Luo R.,Wu W.L.,Zhang Y.,and Li Y.H.,2010,SSR Marker and its application to crop genetics and breeding,Jiyin Zuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),29(1):137-143(罗冉,吴委林,张旸,李玉花,2010,SSR分子标记在作物遗传育种中的应用,基因组学与应用生物学,29(1):137-143)
Vieira R.L.,Silva A.L.D.,Zaffari G.R.,Steinmacher D.A.,de Freitas Fraga H.P.,and Guerra M.P.,2015,Efficient elimination of virus complex from garlic(Allium sativum L.)by cryotherapy of shoot tips,Acta Physiol.Plant,37(1):1733
Wang B.,2014,Cryopreservation,cryo-injury analysis and application to virus eradication in shoot tips of potato(Solanum tuberosum),Dissertation for Ph.D.,Northwest Agriculture and Forestry University,Supervisor:Wang Q.C.,pp.67-71(王彪,2014,马铃薯茎尖超低温保存与伤害解析及其脱毒效应研究,博士学位论文,西北农林科技大学园艺学院,导师:王乔春,pp.67-71)
Wang Q.C.,and Valkonen J.P.T.,2008a,Elimination of two viruses which interact synergistically from sweetpotato by shoot tip culture and cryotherapy,J.Virol.Methods,154(1)135-145
Wang Q.C.,and Valkonen J.P.T.,2008b,Efficient elimination of sweetpotato little leaf phytoplasma from sweetpotato by cryotherapy of shoot tips,Plant Pathol.,57(2):338-347
Wang Q.C.,Mawassi M.,Li P.,Gafny R.,Sela I.,and Tanne E.2003,Elimination of grapevine virus A(GVA)by cryopreservation of in vitro-grown shoot tips of Vitis vinifera L.Plant Sci.,165(2):321-327
Wang R.R.,2014,Cryopreservation for germplasm conservation and pathogen eradication in Chrysanthemum,Chinese jujube and potato,Dissertation for Ph.D.,Northwest Agriculture and Forestry University,Supervisor:Wang Q.C.,pp.39-41(王仁睿,2014,菊花,枣和马铃薯种质资源超低温保存技术与脱毒的研究,博士学位论文,西北农林科技大学园艺学院,导师:王乔春,pp.39-41)
Zhang J.M.,Zhang X.N.,Lu X.X.,Xin X.,Yin G.K.,He J.J.,and Chen X.L.,2014,Optimization of droplet-vitrification protocol for carnation genotypes and ultrastructural studies on shoot tips during cryopreservation,Acta Physiol.Plant,36(12):3189-3198
Zhu W.T.,Zhou H.C.,and Wang Z.C.,2013,Cryopreservation of Fragaria pentapylla and analysis of genetic stability,Guoshu Xuebao(Journal of Fruit Science),30(1):55-61(朱文涛,周厚成,王子成,2013,五叶草莓超低温保存及遗传稳定性分析,果树学报,30(1):55-61)