华仁杏遗传多样性的SSR和ISSR分析
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摘要
华仁杏(Armeniaca cathayana D. L. Fu et al.)属于蔷薇科(Rosaceae)李亚科(Subfam. Prunoideae)杏属(Armeniaca Scop.),其果核薄、杏仁大、含油率高、油质上乘,具有很高的经济价值和广阔的开发利用前景。同时,华仁杏又是抗旱“先锋”经济树种,丘陵、山区和沙区经济开发的“绿色银行”,具有显著的生态效益和社会效益。
目前关于华仁杏的分子遗传学方面的研究报道很少。本文采用SSR和ISSR两种分子标记技术对华仁杏种质资源48份材料(其中大扁杏36份)和24份杂交后代材料及其近缘种14份材料,包括杏(A. vulgaris Lam.)种质资源10份,山杏(A. sibirica (L.) Lam.)种质资源2份,长果杏(A. elongata D. L. Fu et al., sp. nov. ined.)1份和杏李杂交材料1份,率先开展了华仁杏种间亲缘关系、种内遗传多样性、杂交后代变异等相关研究,通过聚类分析、主成分分析、遗传变异系数分析等方法探讨了华仁杏的遗传多样性,为华仁杏新品种选育和遗传改良提供了重要理论依据。实验结果如下:
(1)在对杏属植物材料进行微卫星DNA显带实验时,通过科学实验,提出了一种新的微卫星DNA的PAGE显带技术──荧光显带技术。该法与目前微卫星DNA的主要显带技术PAGE银染法相比,具有分辨率高、快速高效、简单易行、背景清晰等优点,是一种值得在实验室推广的DNA显带技术。
(2)采用SSR分子标记技术研究了华仁杏与其近缘种杏、山杏等之间的亲缘关系。结果表明:华仁杏与杏、山杏的亲缘关系较远,在DNA水平上支持了华仁杏种级分类地位的成立;大扁杏与华仁杏亲缘关系较近,而与杏、山杏亲缘关系较远,分子水平上支持大扁杏应属于华仁杏的变异类群,不属于杏、山杏的变异类群,也不是杏与山杏的杂交种。
(3)采用SSR和ISSR分子标记技术相结合的方法探讨了47个华仁杏种质资源的遗传多样性,结果表明:SSR引物共检测到65个等位基因(Na),多态性位点率(PPB)平均为100.00%,遗传相似系数在0.4769-0.9846之间;ISSR引物共检测到92个等位基因(Na),多态性位点率(PPB)平均为80.43%,遗传相似系数在0.5978-0.9891之间。两种分子标记技术均表明华仁杏具有丰富的遗传多样性。
(4)通过筛选具有双亲互补带型的SSR引物来甄别华仁杏6个杂交组合24份杂交后代的真假。试验中共筛选出具有双亲互补带型的SSR引物6对,除2个杂交后代无法鉴别外,其余杂交后代均可鉴别为真杂种。通过聚类分析、遗传距离分析和遗传变异分析,各个杂交组合后代表现出来较高的一致性,遗传多样性水平相差不大。
(5)采用ISSR分子标记技术对华仁杏3个杂交组合的杂交后代进行了杂种优势预测。研究结果显示:3个杂交后代同父母本之间的遗传相似系数基本上均低于亲本间的遗传相似系数,而有效等位基因数、期望杂合度、shannon多样性指数均高于两亲本,表明杂交后代的遗传多样性水平得到明显提高,极可能会产生潜在的杂种优势。作者认为利用分子标记技术进行杂种优势预测,可以作为一种辅助手段为杂交育种亲本选配等提供分子水平上的理论参考。
Armeniaca cathayana D. L. Fu et al., a species of Rosaceae Subfam. Prunoideae Armeniaca Scop., has important economic values, wide development and utilization prospect because of its thin endocarps, sweet kernels, and sources of high quality oil. A. cathayana has high ecological benefit and social benefit for "Pioneer" species in drought-resistance and "green bank" in hills,mountains and sand area.
The molecular genetics of A. cathayana has not seen so far. Based on 48 germplasm (36 kernel-apricot) and 24 hybrid experimental materials of A. cathayana, and 14 related experimental materials, 2 of A. sibirica, 10 of A. vulgaris, 1 of A. elongata, sp. nov. ined. and 1 of aprium, this paper using SSR and ISSR molecular markers takes the lead in investigating the genetic relationship, genetic diversity, hybrid offspring variation of A. cathayana. The genetic diversity of the species were analysed through cluster analysis, principal component analysis and genetic variation coefficient analysis, which can provide scientific basis for the breeding and genetic improvement of A. cathayana. The results showed that:
(1) Through the scientific experiments in DNA bands display of Armeniaca Scop., the paper proposed a new DNA bands display technology of Microsatellite DNA, which called Fluorescent Imaging Technology. Compared with Silver Stain Technology, the main technology of microsatellite DNA band display so far, the new technique has more advantages, such as high resolution, efficient, simple and clear, and clear background. Fluorescent Imaging Technology is a new DNA bands display technology worthy of popularizing in the laboratory.
(2) Using SSR molecular markers, this study investigated the genetic relationships among the species of A. cathayana, A. vulgaris and A. sibirica. Research results showed that A. cathayana had farther genetic relationship with A. vulgaris and A. sibirica, which provided evidence to the morphology classification of A. cathayana from DNA level. Dabianxing-apricot had closer genetic relationship with A. cathayana and farther genetic relationship with A. vulgaris and A. sibirica, so it should belong to variation groups of A. cathayana, not variation groups or hybrids of A. vulgaris and A. sibirica in molecular level.
(3) The genetic diversity of 47 A. cathayana germplasm experimental materials were studied using SSR and ISSR markers. The results showed that: Using SSR molecular markers, a total of 65 alleles(Na)were detected, the polymorphisms rate (PPB) was 100%, and the genetic similarity coefficient ranged from 0.4769 to 0.9846. Using ISSR molecular markers, a total of 92 alleles(Na)were detected, the polymorphisms rate (PPB) was 80.43% and the genetic similarity coefficient ranged from 0.5978 to 0.9891. These results suggested that there was rich genetic diversity of A. cathayana.
(4) Six primers with parents complementary belt type were selected by using SSR molecular marker technology, which can be used to discern true or false hybrids. Research results showed 22 of 24 hybrid maerials of six hybridized combinations of A. cathayana can be identified as true hybrids except two hybrids can not be identified. In addition, cluster analysis, genetic relationship and genetic variation analysis results showed that six combination hybrids displayed high consistency and not significant genetic diversity.
(5) Using ISSR molecular markers it was studied that the heterosis prediction of A. cathayana parents and hybrid materials in three hybrid combinations. The results showed that the genetic similarity coefficient between hybrids and their parents were lower than their parents, but the hybrids were higher than the parents in alleles, expected alleles, expected heterozygosity and shannon diversity index. These showed hybridization could improve genetic diversity level of A. cathayana obviously and produce some new cultivars with potential heterosis. The author think it is a means to predict heterosis using molecular markers, which can provide theory basis for parent selection in hybridization breeding.
目前关于华仁杏的分子遗传学方面的研究报道很少。本文采用SSR和ISSR两种分子标记技术对华仁杏种质资源48份材料(其中大扁杏36份)和24份杂交后代材料及其近缘种14份材料,包括杏(A. vulgaris Lam.)种质资源10份,山杏(A. sibirica (L.) Lam.)种质资源2份,长果杏(A. elongata D. L. Fu et al., sp. nov. ined.)1份和杏李杂交材料1份,率先开展了华仁杏种间亲缘关系、种内遗传多样性、杂交后代变异等相关研究,通过聚类分析、主成分分析、遗传变异系数分析等方法探讨了华仁杏的遗传多样性,为华仁杏新品种选育和遗传改良提供了重要理论依据。实验结果如下:
(1)在对杏属植物材料进行微卫星DNA显带实验时,通过科学实验,提出了一种新的微卫星DNA的PAGE显带技术──荧光显带技术。该法与目前微卫星DNA的主要显带技术PAGE银染法相比,具有分辨率高、快速高效、简单易行、背景清晰等优点,是一种值得在实验室推广的DNA显带技术。
(2)采用SSR分子标记技术研究了华仁杏与其近缘种杏、山杏等之间的亲缘关系。结果表明:华仁杏与杏、山杏的亲缘关系较远,在DNA水平上支持了华仁杏种级分类地位的成立;大扁杏与华仁杏亲缘关系较近,而与杏、山杏亲缘关系较远,分子水平上支持大扁杏应属于华仁杏的变异类群,不属于杏、山杏的变异类群,也不是杏与山杏的杂交种。
(3)采用SSR和ISSR分子标记技术相结合的方法探讨了47个华仁杏种质资源的遗传多样性,结果表明:SSR引物共检测到65个等位基因(Na),多态性位点率(PPB)平均为100.00%,遗传相似系数在0.4769-0.9846之间;ISSR引物共检测到92个等位基因(Na),多态性位点率(PPB)平均为80.43%,遗传相似系数在0.5978-0.9891之间。两种分子标记技术均表明华仁杏具有丰富的遗传多样性。
(4)通过筛选具有双亲互补带型的SSR引物来甄别华仁杏6个杂交组合24份杂交后代的真假。试验中共筛选出具有双亲互补带型的SSR引物6对,除2个杂交后代无法鉴别外,其余杂交后代均可鉴别为真杂种。通过聚类分析、遗传距离分析和遗传变异分析,各个杂交组合后代表现出来较高的一致性,遗传多样性水平相差不大。
(5)采用ISSR分子标记技术对华仁杏3个杂交组合的杂交后代进行了杂种优势预测。研究结果显示:3个杂交后代同父母本之间的遗传相似系数基本上均低于亲本间的遗传相似系数,而有效等位基因数、期望杂合度、shannon多样性指数均高于两亲本,表明杂交后代的遗传多样性水平得到明显提高,极可能会产生潜在的杂种优势。作者认为利用分子标记技术进行杂种优势预测,可以作为一种辅助手段为杂交育种亲本选配等提供分子水平上的理论参考。
Armeniaca cathayana D. L. Fu et al., a species of Rosaceae Subfam. Prunoideae Armeniaca Scop., has important economic values, wide development and utilization prospect because of its thin endocarps, sweet kernels, and sources of high quality oil. A. cathayana has high ecological benefit and social benefit for "Pioneer" species in drought-resistance and "green bank" in hills,mountains and sand area.
The molecular genetics of A. cathayana has not seen so far. Based on 48 germplasm (36 kernel-apricot) and 24 hybrid experimental materials of A. cathayana, and 14 related experimental materials, 2 of A. sibirica, 10 of A. vulgaris, 1 of A. elongata, sp. nov. ined. and 1 of aprium, this paper using SSR and ISSR molecular markers takes the lead in investigating the genetic relationship, genetic diversity, hybrid offspring variation of A. cathayana. The genetic diversity of the species were analysed through cluster analysis, principal component analysis and genetic variation coefficient analysis, which can provide scientific basis for the breeding and genetic improvement of A. cathayana. The results showed that:
(1) Through the scientific experiments in DNA bands display of Armeniaca Scop., the paper proposed a new DNA bands display technology of Microsatellite DNA, which called Fluorescent Imaging Technology. Compared with Silver Stain Technology, the main technology of microsatellite DNA band display so far, the new technique has more advantages, such as high resolution, efficient, simple and clear, and clear background. Fluorescent Imaging Technology is a new DNA bands display technology worthy of popularizing in the laboratory.
(2) Using SSR molecular markers, this study investigated the genetic relationships among the species of A. cathayana, A. vulgaris and A. sibirica. Research results showed that A. cathayana had farther genetic relationship with A. vulgaris and A. sibirica, which provided evidence to the morphology classification of A. cathayana from DNA level. Dabianxing-apricot had closer genetic relationship with A. cathayana and farther genetic relationship with A. vulgaris and A. sibirica, so it should belong to variation groups of A. cathayana, not variation groups or hybrids of A. vulgaris and A. sibirica in molecular level.
(3) The genetic diversity of 47 A. cathayana germplasm experimental materials were studied using SSR and ISSR markers. The results showed that: Using SSR molecular markers, a total of 65 alleles(Na)were detected, the polymorphisms rate (PPB) was 100%, and the genetic similarity coefficient ranged from 0.4769 to 0.9846. Using ISSR molecular markers, a total of 92 alleles(Na)were detected, the polymorphisms rate (PPB) was 80.43% and the genetic similarity coefficient ranged from 0.5978 to 0.9891. These results suggested that there was rich genetic diversity of A. cathayana.
(4) Six primers with parents complementary belt type were selected by using SSR molecular marker technology, which can be used to discern true or false hybrids. Research results showed 22 of 24 hybrid maerials of six hybridized combinations of A. cathayana can be identified as true hybrids except two hybrids can not be identified. In addition, cluster analysis, genetic relationship and genetic variation analysis results showed that six combination hybrids displayed high consistency and not significant genetic diversity.
(5) Using ISSR molecular markers it was studied that the heterosis prediction of A. cathayana parents and hybrid materials in three hybrid combinations. The results showed that the genetic similarity coefficient between hybrids and their parents were lower than their parents, but the hybrids were higher than the parents in alleles, expected alleles, expected heterozygosity and shannon diversity index. These showed hybridization could improve genetic diversity level of A. cathayana obviously and produce some new cultivars with potential heterosis. The author think it is a means to predict heterosis using molecular markers, which can provide theory basis for parent selection in hybridization breeding.
引文
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