蓝果忍冬（LONICERAL.SUBSECT.CAERULEAE）SRAP反应体系的建立及遗传多样性分析
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摘要
蓝果忍冬(Lonicera Caerulea L.)属忍冬科(caprifoliaceae)、忍冬属(Lonicera L.),多年生落叶小灌木,具有良好的环境适应性,主要分布在俄罗斯、日本、朝鲜和北美,中国的大小兴安岭、长白山等地也蕴含着丰富的野生资源;果实蓝紫色,由其加工成的果酱、饮料、罐头、果酒等无公害食品,既含有花青素、矿物质和维生素等生物活性物质,又具有很好的药用保健价值,深受世界各地人们的喜爱,是第三代果树之一,可与蓝莓相媲美,市场前景看好。
本实验于2009年～2010年在东北农业大学园艺学院进行,以我国第一个蓝果忍冬种质资源圃中的58份蓝果忍冬资源为试材,确立了蓝果忍冬叶片基因组DNA的最佳提取方法,讨论了SRAP-PCR反应体系中各因素对扩增结果的影响并对反应体系进行了优化,对58份蓝果忍冬的遗传多样性进行了分析,目的是为了解蓝果忍冬居群的遗传多样性水平和资源的保护与开发利用提供理论依据。主要结果如下:
1、DNA的分离提取是进行基因组多样性分析的基础,是分子生物学研究中重要的基本技术。不同的植物材料因其组织细胞中所含此生代谢产物的种类、含量不同,提取方法也不同。蓝果忍冬叶片富含多糖、多酚等次生代谢产物,这些物质会影响叶片基因组DNA提取的质量,本实验对常规CTAB法和改良CTAB法进行了比较,结果表明改良CTAB法提取出的DNA条带明亮,没有拖尾现象,适于蓝果忍冬基因组DNA的提取。改良CTAB法即在液氮研磨过程中加入PVP粉末,防止酚类物质氧化,用SET核分离缓冲液对样品进行预洗,将细胞核与多糖和多酚等次生物质分开,提取效果较好,OD_(260)/OD_(280)保持在1.7～1.9之间,能够满足SRAP扩增的要求。
2、PCR反应体系中各反应因素的用量对PCR反应的结果具有很大影响,本实验对反应体系中Tag DNA聚合酶、模板DNA、dNTPs、Mg~(2+)和引物进行单因素实验,建立了蓝果忍冬SRAP-PCR的最佳反应体系(20μl)为:2.0μl 10×PCR buffer、1.0U Tag DNA聚合酶、20ng模板DNA、0.20mmol/l dNTPs、2.0mmol/lMg~(2+)、0.3μmol/l引物。反应程序为:94℃预变性5min;94℃变性1min,35℃复性1min,72℃延伸90s,5个循环;94℃变性1min,50℃复性1min,72℃延伸90s,35个循环;72℃延伸8min。4℃保存。
3、在引物筛选过程中,以贝瑞尔、库页岛、大兴安岭野生资源为材料,从90个引物组合中筛选出20个多态性高、扩增条带清晰稳定的引物组合。利用所筛选的引物对58份材料进行扩增,共扩增出172个条带,其中多态性条带143个,占总条带的83.1%,其中每对引物扩增出5-12个条带。品种间遗传相似系数在0.53-0.96之间。
4、利用筛选出的引物和优化后的体系对参试品种进行PCR扩增,电泳条带按1/0形式进行数据转换,选择清晰、再现性强的谱带作为统计对象记为l,无扩增谱带记为0,UPGMA法聚类构建树状图并进行遗传多样性分析。58份资源被聚为7类,来自俄罗斯地区的资源聚为一类,伊春、尚志、勃利的资源聚在一起,新疆阿尔泰、大兴安岭野生和长白山野生资源分别单独聚类,各成一组,贝瑞尔、蓝鸟、蓝纺锤三个栽培品种和E3、E6、VIR聚在一起,杂交苗聚在一起成为一类。23号未知资源与海参崴资源聚在一起,说明23号可能是来自海参崴地区资源或是其杂交后代。其中来自东北地区的资源分成了四个组群,由此可知,东北地区的蓝果忍冬具有较高的遗传变异,同时也说明了东北地区是蓝果忍冬遗传多样性中心之一。
Lonicera Caerulea L. belongs to Caprifoliaceae family, was a perennial under shrub with strong environmental adaptability, it was widely distributed in Russia, Japan, Korea and North America. The Xingan and Changbai Mountains are rich in wildlife resources too. Fruits of Lonicera Caerulea L. are blue-purple, its processing into jams, beverages, canned food, wine and other pollution-free food both rich in nutrients such as anthocyanins, minerals, and vitamins and have medicinal value. Loved by people around the world, is one of the third generation fruit trees and can comparable with blueberries, has good market prospects.
The experiment was carried out in horticultural of Northeast Agricultural University from 2009 to 2010. 58 varieties of blue honeysuckle from China's first blue honeysuckle germplasm garden was used to establish the best genomic DNA extraction method, discussed the influence of amplification factors in amplification reaction system and optimization SRAP-PCR reaction system. Study the genetic diversity in 58 blue honeysuckles from different countries or region was assessed using SRAP markers. Purpose is to understand the blue honeysuckle populations’level of genetic diversity and the protection and utilization of resources to provide the theoretical basis. The main results are as follows:
1、DNA extraction is based on the analysis of genome diversity and an important basic skills in molecular biology study. Different tissues of plant material because of its metabolic products contained in this life, the type of content in different extraction methods are also different. Blue honeysuckle leaves are rich in polysaccharides and polyphenols and other secondary metabolites, these substances will affect the quality of genomic DNA extracted. In this study, the trial of conventional and modified CTAB method were compared and the results show that the modified CTAB method extraction out of the bright bands and no smearing, this method was efficient for blue honeysuckle. That is, in the process of adding liquid nitrogen grinding PVP powder, to prevent the oxidation of phenolic compounds. pre-wash the samples with SET, separated the nucleus and secondary compounds such as polysaccharides and polyphenols, DNA sample extracted by the methods had high purity, OD260/OD280 maintained at between 1.7 and 1.9, to meet the requirements of SRAP amplified.
2、The reaction factors have a significant impact in PCR reaction, in this study, we design for single-factor experiment to the reaction system, Tag DNA polymerase, template DNA, dNTPs, Mg~(2+) and primer. established 20μl reaction system of SRAP for blue honeysuckle included 2.0μl 10×PCR buffer, 1.0 U Taq DNA polymerase, 30 ng template DNA, 0.2 mmol/l dNTPs, 2.0mmol/l Mg~(2+) and 0.2μmol/l primer. The reaction procedure was as follows: denaturation at 94℃for 5 min ; denaturation at 94℃for 1 min ,anneal at 35℃for 1 min ,extension at 72℃for 90 s and in total 5 cycles ; denaturation at 94℃for 1 min ,anneal at 50℃for 1 min ,extension at 72℃for 90 s and in total 35cycles ;extension at 72℃for 8min ;preservation at 4℃.
3、During the primer screening, the L.caerulea subsp.altaicai×L.caerulea subsp.Kamtschatica, L.caerulea subsp.emphyllocalyx and daxinganling were used as experimental material, twenty out of 90 pairs of primer combinations were selected in order to amplify the genotypes, 172 bands were acquired by PCR, about 143 of them is diversity bands, so the rate of diversity is 83.1%, each primer pair produced 5-12 bands. The genetic similarity index among the 78 species varied from 0.53 to 0.96.
4、Using selected primers and optimized system to carry out PCR amplification of the tested varieties, electrophoretic bands by 1 / 0 form of data, choose clear, reproducible and strong bands for statistical object denoted 1and no amplified bands as 0, UPGMA method based on tree building and analysis of the genetic diversity. 58 materials were clustered into 7 categories, species from the Russian regions clustered in one group; Yichun, Shangzhi, Boli species together; Altay, Daxinganling and Changbaishan wild species cluster separately, each as a group; three cultivars L.caerulea subsp.altaicai×L.caerulea subsp.Kamtschatica, Blue bird and Blue spindle cluster with E3, E6 and VIR. The 23rd unknown species together with Vladivostok, it may come from Vladivostok region or its offspring. Resources from the northeast which is divided into four groups, it can be seen, blue honeysuckle in northeast have high genetic variation, also shows that the northeast is one of the genetic diversity centers of blue honeysuckle.
本实验于2009年～2010年在东北农业大学园艺学院进行,以我国第一个蓝果忍冬种质资源圃中的58份蓝果忍冬资源为试材,确立了蓝果忍冬叶片基因组DNA的最佳提取方法,讨论了SRAP-PCR反应体系中各因素对扩增结果的影响并对反应体系进行了优化,对58份蓝果忍冬的遗传多样性进行了分析,目的是为了解蓝果忍冬居群的遗传多样性水平和资源的保护与开发利用提供理论依据。主要结果如下:
1、DNA的分离提取是进行基因组多样性分析的基础,是分子生物学研究中重要的基本技术。不同的植物材料因其组织细胞中所含此生代谢产物的种类、含量不同,提取方法也不同。蓝果忍冬叶片富含多糖、多酚等次生代谢产物,这些物质会影响叶片基因组DNA提取的质量,本实验对常规CTAB法和改良CTAB法进行了比较,结果表明改良CTAB法提取出的DNA条带明亮,没有拖尾现象,适于蓝果忍冬基因组DNA的提取。改良CTAB法即在液氮研磨过程中加入PVP粉末,防止酚类物质氧化,用SET核分离缓冲液对样品进行预洗,将细胞核与多糖和多酚等次生物质分开,提取效果较好,OD_(260)/OD_(280)保持在1.7～1.9之间,能够满足SRAP扩增的要求。
2、PCR反应体系中各反应因素的用量对PCR反应的结果具有很大影响,本实验对反应体系中Tag DNA聚合酶、模板DNA、dNTPs、Mg~(2+)和引物进行单因素实验,建立了蓝果忍冬SRAP-PCR的最佳反应体系(20μl)为:2.0μl 10×PCR buffer、1.0U Tag DNA聚合酶、20ng模板DNA、0.20mmol/l dNTPs、2.0mmol/lMg~(2+)、0.3μmol/l引物。反应程序为:94℃预变性5min;94℃变性1min,35℃复性1min,72℃延伸90s,5个循环;94℃变性1min,50℃复性1min,72℃延伸90s,35个循环;72℃延伸8min。4℃保存。
3、在引物筛选过程中,以贝瑞尔、库页岛、大兴安岭野生资源为材料,从90个引物组合中筛选出20个多态性高、扩增条带清晰稳定的引物组合。利用所筛选的引物对58份材料进行扩增,共扩增出172个条带,其中多态性条带143个,占总条带的83.1%,其中每对引物扩增出5-12个条带。品种间遗传相似系数在0.53-0.96之间。
4、利用筛选出的引物和优化后的体系对参试品种进行PCR扩增,电泳条带按1/0形式进行数据转换,选择清晰、再现性强的谱带作为统计对象记为l,无扩增谱带记为0,UPGMA法聚类构建树状图并进行遗传多样性分析。58份资源被聚为7类,来自俄罗斯地区的资源聚为一类,伊春、尚志、勃利的资源聚在一起,新疆阿尔泰、大兴安岭野生和长白山野生资源分别单独聚类,各成一组,贝瑞尔、蓝鸟、蓝纺锤三个栽培品种和E3、E6、VIR聚在一起,杂交苗聚在一起成为一类。23号未知资源与海参崴资源聚在一起,说明23号可能是来自海参崴地区资源或是其杂交后代。其中来自东北地区的资源分成了四个组群,由此可知,东北地区的蓝果忍冬具有较高的遗传变异,同时也说明了东北地区是蓝果忍冬遗传多样性中心之一。
Lonicera Caerulea L. belongs to Caprifoliaceae family, was a perennial under shrub with strong environmental adaptability, it was widely distributed in Russia, Japan, Korea and North America. The Xingan and Changbai Mountains are rich in wildlife resources too. Fruits of Lonicera Caerulea L. are blue-purple, its processing into jams, beverages, canned food, wine and other pollution-free food both rich in nutrients such as anthocyanins, minerals, and vitamins and have medicinal value. Loved by people around the world, is one of the third generation fruit trees and can comparable with blueberries, has good market prospects.
The experiment was carried out in horticultural of Northeast Agricultural University from 2009 to 2010. 58 varieties of blue honeysuckle from China's first blue honeysuckle germplasm garden was used to establish the best genomic DNA extraction method, discussed the influence of amplification factors in amplification reaction system and optimization SRAP-PCR reaction system. Study the genetic diversity in 58 blue honeysuckles from different countries or region was assessed using SRAP markers. Purpose is to understand the blue honeysuckle populations’level of genetic diversity and the protection and utilization of resources to provide the theoretical basis. The main results are as follows:
1、DNA extraction is based on the analysis of genome diversity and an important basic skills in molecular biology study. Different tissues of plant material because of its metabolic products contained in this life, the type of content in different extraction methods are also different. Blue honeysuckle leaves are rich in polysaccharides and polyphenols and other secondary metabolites, these substances will affect the quality of genomic DNA extracted. In this study, the trial of conventional and modified CTAB method were compared and the results show that the modified CTAB method extraction out of the bright bands and no smearing, this method was efficient for blue honeysuckle. That is, in the process of adding liquid nitrogen grinding PVP powder, to prevent the oxidation of phenolic compounds. pre-wash the samples with SET, separated the nucleus and secondary compounds such as polysaccharides and polyphenols, DNA sample extracted by the methods had high purity, OD260/OD280 maintained at between 1.7 and 1.9, to meet the requirements of SRAP amplified.
2、The reaction factors have a significant impact in PCR reaction, in this study, we design for single-factor experiment to the reaction system, Tag DNA polymerase, template DNA, dNTPs, Mg~(2+) and primer. established 20μl reaction system of SRAP for blue honeysuckle included 2.0μl 10×PCR buffer, 1.0 U Taq DNA polymerase, 30 ng template DNA, 0.2 mmol/l dNTPs, 2.0mmol/l Mg~(2+) and 0.2μmol/l primer. The reaction procedure was as follows: denaturation at 94℃for 5 min ; denaturation at 94℃for 1 min ,anneal at 35℃for 1 min ,extension at 72℃for 90 s and in total 5 cycles ; denaturation at 94℃for 1 min ,anneal at 50℃for 1 min ,extension at 72℃for 90 s and in total 35cycles ;extension at 72℃for 8min ;preservation at 4℃.
3、During the primer screening, the L.caerulea subsp.altaicai×L.caerulea subsp.Kamtschatica, L.caerulea subsp.emphyllocalyx and daxinganling were used as experimental material, twenty out of 90 pairs of primer combinations were selected in order to amplify the genotypes, 172 bands were acquired by PCR, about 143 of them is diversity bands, so the rate of diversity is 83.1%, each primer pair produced 5-12 bands. The genetic similarity index among the 78 species varied from 0.53 to 0.96.
4、Using selected primers and optimized system to carry out PCR amplification of the tested varieties, electrophoretic bands by 1 / 0 form of data, choose clear, reproducible and strong bands for statistical object denoted 1and no amplified bands as 0, UPGMA method based on tree building and analysis of the genetic diversity. 58 materials were clustered into 7 categories, species from the Russian regions clustered in one group; Yichun, Shangzhi, Boli species together; Altay, Daxinganling and Changbaishan wild species cluster separately, each as a group; three cultivars L.caerulea subsp.altaicai×L.caerulea subsp.Kamtschatica, Blue bird and Blue spindle cluster with E3, E6 and VIR. The 23rd unknown species together with Vladivostok, it may come from Vladivostok region or its offspring. Resources from the northeast which is divided into four groups, it can be seen, blue honeysuckle in northeast have high genetic variation, also shows that the northeast is one of the genetic diversity centers of blue honeysuckle.
引文
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