黄瓜单性结实和种质资源遗传多样性的ISSR分析
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摘要
黄瓜是一种重要的果菜类蔬菜作物,品种资源较为丰富,利用分子标记进行黄瓜种质资源鉴定和分类对黄瓜种质创新和选育新品种具有重要意义。目前尚未见有利用ISSR分子标记进行黄瓜资源鉴定和遗传多样性分析等的研究报道。本研究利用正交试验法成功设计并优化了黄瓜ISSR技术体系。黄瓜单性结实特性是其重要的经济性状,选育单性结实品种是现代黄瓜育种中重要的目标性状之一。本文基于黄瓜单性结实品系(EP-6)与非单性结实品系(ZR-2)构建了包含135个单株的F_2分离群体,对单性结实和非单性结实的分子特征进行了分析。利用ISSR分子标记技术对46份黄瓜品种资源进行了多态性分析,8个ISSR引物在46份黄瓜材料中获得了42条扩增带,采用Jaccard方法计算出遗传相似系数矩阵,以UPGMA法对46份黄瓜品种进行了聚类分析。利用ISSR技术对3个黄瓜组合的双亲和F_1进行了引物筛选,初步建立了应用于鉴定黄瓜亲本及其F_1种子的ISSR指纹图谱。研究的主要结果如下:
1.利用正交试验设计方法,从Taq酶、dNTPs、引物、Mg~(2+) 4种因素各3个水平来优化黄瓜ISSR反应体系。通过实验确定的黄瓜ISSR最佳反应体系为:在25μl反应体系中各反应成分为:1×PCR buffer,1.5mmol/L Mg~(2+),25ng黄瓜模板DNA,1U Taq酶,200μmol/L dNTPs,0.75μmol/L引物,ddH2O。这一体系的建立为利用ISSR技术进行黄瓜种质资源鉴定、遗传图谱构建和基因定位奠定了初步的技术基础。
2.以黄瓜单性结实品系EP-6和非单性结实品系ZR-2为亲本构建了分离群体,采用分离集团混合分析法(BSA)法筛选与黄瓜单性结实基因连锁的ISSR分子标记。引物N92在亲本和DNA池之间扩增出一条大小为850bp多态性片段,经F_2代群体135个单株验证,该片段稳定出现,在单性结实植株中表现为无带,在非单性结实植株中表现为有带,可以作为鉴别单性结实与非单性结实的标记引物。这一结果为今后黄瓜单性结实分子标记辅助育种和基因定位奠定了初步的基础。
3. 8个ISSR引物在46份黄瓜品种中扩增出42条条带,其中多态性条带为36条,多态性比率为85.71 %,表明ISSR标记在黄瓜上具有较高的多态性。46份材料间遗传相似系数的变化范围是0.3030~0.8750,其中23(ZN1)和42(四川寸金子)的遗传系数最小(0.3030),其遗传距离最远;34(Edipse)与43(杨泾黄瓜)之间的遗传相似系数最高(0.8750),其遗传距离最近。聚类分析结果表明,若以相似性系数0.52为阈值时,可将46份黄瓜品种聚为两类,一类以较强的雌性表现为特征,另一类则多表现为普通性型。若以0.54的相似系数为阈值,46份黄瓜材料可以被分为四大类,虽然不能完全按照生态型将本研究中的黄瓜资源聚类,但总体而言,来自于同一生态型的多数资源可以聚在同一类或亚类中。各个类中均分布有具有单性结实性能的品种,前三类包括了较多的单性结实种质资源,占该类资源的46.10%。这既说明黄瓜单性结实性在品种间的普遍性,同时更说明不同品种间单性结实性又存在差异。
4.利用ISSR技术对由双亲及其F_1组成的3个黄瓜组合进行了多态性分析, 65个引物中筛选出5个具有多态性,其中4个引物(J1、UBC818、N86和I-10)在组合1中均表现多态,F_1中双亲的特异带均能被扩增出来,表明四个引物均能将组合1的双亲及其F_1进行有效鉴别。引物N86和I-10对组合2和组合3的扩增结果表明,F_1中双亲的特异带均能被扩增出来,因此,引物N86和I-10均能将组合2和组合3的双亲及其F_1进行有效鉴别。据此初步建立了应用于鉴定黄瓜三个组合亲本及其杂种的ISSR指纹图谱。为黄瓜种子纯度的鉴定提供了新的技术途径,为实施黄瓜新品种知识产权保护提供了分子基础。
Cucumber(Cucumis sativus L.) is a very important vegetable crop with an adundant germplasms. Researches on evaluation and classification of cucumber germplasms with molecular markers play an important role in innovation of germplasms and breeding for new varieties. It hasn’t been reported that ISSR is used to indentify cucumber germplasms and genetic diversity . Orthogonal design was used to optimize the cucumber ISSR reaction system in the present research. Parthenocarpy is one of important economic characters and has been an important aim character in the field of modern cucumber breeding. Based on the separate progeny from the cross between parthenocarpic line EP-6 and non-parthenocarpic line ZR-2, 135 individuals were tested in molecule characters linked with parthenocarpy. 46 cultivated accessions were used in this study to analyze their polymorphisms with ISSR primers. 42 polymorphic ISSR bands were amplified with 8 ISSR primers. Genetic similarity coefficients were calculated by Jaccard method and culstering was conducted for 46 cultivars by UPGMA.The DNA fingerprinting of hybridity with ISSR markers were contructed preliminarly in three cucumber crosses . The main results were as follows:
1 In this study, Orthogonal design with three levels of four factors(Taq DNA polymerase、dNTPs、primer、Mg~(2+)) was used to optimize the cucumber ISSR reaction system. The results showed that a better amplification of ISSR was obtained with the reaction system containing 1×PCR buffer,1.5mmol/L Mg~(2+),25ng DNA,ddH2O,1UTaq DNA polymerase,200μmol/L dNTPs,0.75μmol/L. It provided the basis for the analysis of diversity, map construction and gene localization of important traits in cucumber with ISSRmarkers.
2 The separate progeny from the cross between parthenocarpic line EP-6 and non-parthenocarpic line ZR-2 was employed to screen the molecular markers linked to the parthenocarpy of cucumber with the method of BSA(bulked segregation anlysis). A band of 850bp was tested not only in two parents but also in two DNA pools with the primer N92. The product of electrophoresis with the primer N92 showed that non-parthenocarpic line had the band but parthenocarpy line none. Therefore the primer N92 could be used to identify cucumber parthenocarpy. It provided the basis for marker assistance selection in breeding and gene localization of cucumber parthenocarpy.
3 With 8 ISSR primers, 42 bands were amplified in 46 cultivated accessions of cucumber, of 36 bands (85.71 %) were polymorphic. The genetic similarity coefficients of 46 cultivated accessions were ranged from 0.3030 to 0.8750, the coefficient(0.3030) between 23(ZN1)and 42(SichuanCunjinzi)was the lowest, meaning the longest genetic distance between them and the highest coefficient was found between 34(Edipse)and 43(Yangjing),meaning the shortest genetic distance between them. The culstering of 46 cultivated accessions showed that the cultivars are divided into 2 groups at the threshold of 0.52 and was realted to the sex phenotypes of cucumber. One group was charactered with more gynoecious phyenotypes ,and the other for general sex phenotypes. 46 cultivated accession were culstered four groups at the threshold of 0.54. Most of the same ecotypes could be grouped into same group and subgroup. Cultivar charactered by the specific band for parthenocarpy was found in different groups and the parthenocarpy accession of the first three groups accounts for 46.10%. It suggested that parthenocarpy of cucumber exist widely, but differences in this character be in various accessions.
4 65 ISSR primers were used in three cucumber crosses, and of 5 primers showed polymorphisms in parents and their F_1. Primer J1、UBC818、N86 and I-10 have polymorphic bands in the cross1, and the specific bands of parents were found in their F_1. Polymorphisms could be found in the cross2 and cross3 with two primers(N86 and I-10),and the specific bands of parents were found in their F_1.The DNA fingerprinting of hybridity with ISSR markers were contructed preliminarly. It provided a new technique for distingishing uniform and a molecular basis for protecting intellectual property rights of new cucumber varieties.
1.利用正交试验设计方法,从Taq酶、dNTPs、引物、Mg~(2+) 4种因素各3个水平来优化黄瓜ISSR反应体系。通过实验确定的黄瓜ISSR最佳反应体系为:在25μl反应体系中各反应成分为:1×PCR buffer,1.5mmol/L Mg~(2+),25ng黄瓜模板DNA,1U Taq酶,200μmol/L dNTPs,0.75μmol/L引物,ddH2O。这一体系的建立为利用ISSR技术进行黄瓜种质资源鉴定、遗传图谱构建和基因定位奠定了初步的技术基础。
2.以黄瓜单性结实品系EP-6和非单性结实品系ZR-2为亲本构建了分离群体,采用分离集团混合分析法(BSA)法筛选与黄瓜单性结实基因连锁的ISSR分子标记。引物N92在亲本和DNA池之间扩增出一条大小为850bp多态性片段,经F_2代群体135个单株验证,该片段稳定出现,在单性结实植株中表现为无带,在非单性结实植株中表现为有带,可以作为鉴别单性结实与非单性结实的标记引物。这一结果为今后黄瓜单性结实分子标记辅助育种和基因定位奠定了初步的基础。
3. 8个ISSR引物在46份黄瓜品种中扩增出42条条带,其中多态性条带为36条,多态性比率为85.71 %,表明ISSR标记在黄瓜上具有较高的多态性。46份材料间遗传相似系数的变化范围是0.3030~0.8750,其中23(ZN1)和42(四川寸金子)的遗传系数最小(0.3030),其遗传距离最远;34(Edipse)与43(杨泾黄瓜)之间的遗传相似系数最高(0.8750),其遗传距离最近。聚类分析结果表明,若以相似性系数0.52为阈值时,可将46份黄瓜品种聚为两类,一类以较强的雌性表现为特征,另一类则多表现为普通性型。若以0.54的相似系数为阈值,46份黄瓜材料可以被分为四大类,虽然不能完全按照生态型将本研究中的黄瓜资源聚类,但总体而言,来自于同一生态型的多数资源可以聚在同一类或亚类中。各个类中均分布有具有单性结实性能的品种,前三类包括了较多的单性结实种质资源,占该类资源的46.10%。这既说明黄瓜单性结实性在品种间的普遍性,同时更说明不同品种间单性结实性又存在差异。
4.利用ISSR技术对由双亲及其F_1组成的3个黄瓜组合进行了多态性分析, 65个引物中筛选出5个具有多态性,其中4个引物(J1、UBC818、N86和I-10)在组合1中均表现多态,F_1中双亲的特异带均能被扩增出来,表明四个引物均能将组合1的双亲及其F_1进行有效鉴别。引物N86和I-10对组合2和组合3的扩增结果表明,F_1中双亲的特异带均能被扩增出来,因此,引物N86和I-10均能将组合2和组合3的双亲及其F_1进行有效鉴别。据此初步建立了应用于鉴定黄瓜三个组合亲本及其杂种的ISSR指纹图谱。为黄瓜种子纯度的鉴定提供了新的技术途径,为实施黄瓜新品种知识产权保护提供了分子基础。
Cucumber(Cucumis sativus L.) is a very important vegetable crop with an adundant germplasms. Researches on evaluation and classification of cucumber germplasms with molecular markers play an important role in innovation of germplasms and breeding for new varieties. It hasn’t been reported that ISSR is used to indentify cucumber germplasms and genetic diversity . Orthogonal design was used to optimize the cucumber ISSR reaction system in the present research. Parthenocarpy is one of important economic characters and has been an important aim character in the field of modern cucumber breeding. Based on the separate progeny from the cross between parthenocarpic line EP-6 and non-parthenocarpic line ZR-2, 135 individuals were tested in molecule characters linked with parthenocarpy. 46 cultivated accessions were used in this study to analyze their polymorphisms with ISSR primers. 42 polymorphic ISSR bands were amplified with 8 ISSR primers. Genetic similarity coefficients were calculated by Jaccard method and culstering was conducted for 46 cultivars by UPGMA.The DNA fingerprinting of hybridity with ISSR markers were contructed preliminarly in three cucumber crosses . The main results were as follows:
1 In this study, Orthogonal design with three levels of four factors(Taq DNA polymerase、dNTPs、primer、Mg~(2+)) was used to optimize the cucumber ISSR reaction system. The results showed that a better amplification of ISSR was obtained with the reaction system containing 1×PCR buffer,1.5mmol/L Mg~(2+),25ng DNA,ddH2O,1UTaq DNA polymerase,200μmol/L dNTPs,0.75μmol/L. It provided the basis for the analysis of diversity, map construction and gene localization of important traits in cucumber with ISSRmarkers.
2 The separate progeny from the cross between parthenocarpic line EP-6 and non-parthenocarpic line ZR-2 was employed to screen the molecular markers linked to the parthenocarpy of cucumber with the method of BSA(bulked segregation anlysis). A band of 850bp was tested not only in two parents but also in two DNA pools with the primer N92. The product of electrophoresis with the primer N92 showed that non-parthenocarpic line had the band but parthenocarpy line none. Therefore the primer N92 could be used to identify cucumber parthenocarpy. It provided the basis for marker assistance selection in breeding and gene localization of cucumber parthenocarpy.
3 With 8 ISSR primers, 42 bands were amplified in 46 cultivated accessions of cucumber, of 36 bands (85.71 %) were polymorphic. The genetic similarity coefficients of 46 cultivated accessions were ranged from 0.3030 to 0.8750, the coefficient(0.3030) between 23(ZN1)and 42(SichuanCunjinzi)was the lowest, meaning the longest genetic distance between them and the highest coefficient was found between 34(Edipse)and 43(Yangjing),meaning the shortest genetic distance between them. The culstering of 46 cultivated accessions showed that the cultivars are divided into 2 groups at the threshold of 0.52 and was realted to the sex phenotypes of cucumber. One group was charactered with more gynoecious phyenotypes ,and the other for general sex phenotypes. 46 cultivated accession were culstered four groups at the threshold of 0.54. Most of the same ecotypes could be grouped into same group and subgroup. Cultivar charactered by the specific band for parthenocarpy was found in different groups and the parthenocarpy accession of the first three groups accounts for 46.10%. It suggested that parthenocarpy of cucumber exist widely, but differences in this character be in various accessions.
4 65 ISSR primers were used in three cucumber crosses, and of 5 primers showed polymorphisms in parents and their F_1. Primer J1、UBC818、N86 and I-10 have polymorphic bands in the cross1, and the specific bands of parents were found in their F_1. Polymorphisms could be found in the cross2 and cross3 with two primers(N86 and I-10),and the specific bands of parents were found in their F_1.The DNA fingerprinting of hybridity with ISSR markers were contructed preliminarly. It provided a new technique for distingishing uniform and a molecular basis for protecting intellectual property rights of new cucumber varieties.
引文
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