同工酶及AFLP分子标记在秦巴山区天麻种质资源研究中的应用
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摘要
天麻(Gastrodia elata B1)是兰科(Orchidaceae)多年生共生寄生草本植物,是中国传统的名贵中药,秦巴山区是天麻道地产区之一。本实验运用同工酶标记和AFLP分子标记对天麻不同种质进行研究,对秦巴山区天麻遗传多样性进行了综合评价,为天麻种质资源合理保护利用及优良种质资源的选育奠定基础。主要结果如下:
1、天麻同工酶分析
POD、EST同工酶标记分析结果显示:天麻同工酶具有丰富的遗传多样性;同工酶谱条带最少的卵果天麻较原始,条带最多的红杆天麻分化程度较大,是3种种质中最为进化种质;红杆天麻与绿杆天麻亲缘关系比较近,红杆天麻与卵果天麻亲缘关系相对较远。
2、适合AFLP分子标记的天麻基因组DNA提纯方法的优化
以新鲜天麻花茎为实验材料,采用改良CTAB结合苯酚/氯仿(1:1)法提取方法的改良:即将温浴提取时间从1 h延长至3 h,改苯酚/氯仿/异戊醇(25:24:1)为苯酚/氯仿(1:1)抽提蛋白及酚类等杂质,改无水乙醇为异丙醇沉淀DNA,改离心获取DNA为挑取絮状DNA。经检测:DNA浓度为1.458μg/mL,A260/A280为1.93,OD值更接近于1.8,单位质量实验材料提取DNA平均得率提高180%,说明用改良CTAB法从新鲜天麻花茎中提取的天麻基因组DNA质量提高,得率增加,且DNA无降解,无杂质污染,适于天麻分子生物学研究。
3、天麻AFLP最适反应体系构建
在利用AFLP技术研究天麻遗传多样性过程中,从引物筛选、酶切连接、扩增条件、检测方法等几个方面进行优化,建立天麻AFLP最适反应体系为:500ng/μL DNA用3U PstI和3U MseI在37℃4h温浴双酶切;16℃下连接9h;预扩增时取连接产物2μL,50-58ng/μL预扩引物各0.6μL,Taq酶0.4μL ,(5U/μL)10×buffer2μL,dNTP(10mM) 0.4μL,MgC12(25mM)1.2μL ,ddH2O补至20μL;取5μL稀释20倍后的预扩增产物,50ng/μL PstI、MseI选扩引物各1μL,总体系20μL进行选择性扩增。在此优化体系下,可以得到重复性好、稳定且多态性高的天麻AFLP条带。
4、利用AFLP分子标记研究秦巴山区天麻遗传多样性
AFLP结果显示:12对PstI/MseI引物组合共获得605条带,每个引物组合在个体间扩增条带的数目在34-64条之间,平均每条引物能扩增条带50.4条;检测位点323个,其中多态性位点193个,平均多态性检出率为43.03%;每个引物组合在个体间扩增条带平均PIC为0.33,变化范围为0.21-0.39;天麻群体内的相似系数位在0.79-0.86之间;红杆天麻与绿杆天麻之间的遗传距离最小,卵果天麻与红杆天麻之间遗传距离最大。AFLP分子标记结果与同工酶分析结果吻合。
5、天麻AFLP指纹图谱构建
在利用AFLP分子标记研究天麻遗传多样性基础上,筛选出2、5、8、10四对可用于构建秦巴山区天麻不同种质的AFLP特异引物,这4对引物组合扩增条带丰富(232条),检测特异位点多(83),PIC值均达到或超过0.35。不同天麻种质都存在多个特异性带(15-20个)。结果表明AFLP分子标记技术是种质鉴定的一种高效方法,建议在天麻遗传多样性研究中应优先采用这些引物组合。
Gastrodia elata Bl(Tian Ma in China),belonging to orchid family,is a very important Chinese herb medicine, of which Qinba Mountainous Area is one of the genuine producing regions. In this Study of Gastrodia elata B1 Resources, the genetic diversity was investigated by Isozyme and AFLP markers, in order to make further exploitation on Gastrodia elata Bl and provide basis for Gastrodia elata B1 genetics and breeding research .The main results showed as follows:
1. The POD and EST Isozyme analysis results shows that: There were firmly high Polymorphisms in Gastrodia elata B1 Resources from Qinba Mountainous Area. Gastrodia elata Bl.var. obovata sp nov., which has the least belts, is the most original germplasm. Gastrodia elata Bl.f.elata chich has the most belts, is the most developed germplasm in the three. the relative between Gastrodia elata Bl.f.elata and Gastrodia elata Bl.f.viridis Makino is nearest, and Gastrodia elata Bl.f.elata is the most far away from Gastrodia elata Bl.var. obovata sp nov.
2. Optimization of DNA extraction and purification methods from Gastrodia elata for AFLP analysis
The results shows that the method (tissues of stem, optimized CTAB solution, extraction for 3h, extract protein by phenol/chloroform (1:1), precipitate DNA by isopropanol, DNA isolating method) was optimal. This method has the advantages that not only polysaccharide and proteins, but also polyphenol and quinonoid compounds are eliminated. As a result, DNA with high quantity and purity was obtained. The results of UV-spectrophotometric method show A260/A280 is 1.93, and the content of DNA is 1.458μg/ml. Agarose Gelelectrophoresis analysis shows that DNA belts are clear and are suitable for AFLP-PCR.
3. The factors affecting AFLP analysis including primer selection, digestion and ligation of DNA, amplified conditions and detection ways were optimized. An optimization system of AFLP markers was established: 500ng/μL DNA was digested with 3U PstI and 3U MseI at 37℃f or 4h , ligation mixture was kept at 16℃f or 9h, pre-selective amplification was set up with 2μL restriction-ligation sample DNA, 0.6μL PstI and MseI primer(50-58ng/μL), 0.4μL Taq DNA polymerase, 2μL10×buffer(5U/μL) , 0.4μL dNTP(10mM) , 1.2μL MgC12(25mM), ddH2O was added up to 20μL. In 20μL selective amplification, 5μL diluted(20×)DNA pre-selective amplification product, 1μL 50ng/μL PstI and MseI selective primers, Under this optimization system, clear, steady and higher polymorphic Gastrodia elata Bl AFLP bands were obtained.
4. In the experiment, All of the 12 primers can amplify 605 bands, each primers can amplify bands between 34 and 64. 193 of 323 amplified bands were polymorphic bands, which acccout for 43.03%. the average PIC is 0.33, varied from .021 to 0.39, with 0.14-0.21 genetic distance. the relative between Gastrodia elata Bl.f.elata and Gastrodia elata Bl.f.viridis Makino is nearest, and Gastrodia elata Bl.f.elata is the most far away from Gastrodia elata Bl.var. obovata sp nov. the results of AFLP analysis was the same as Isozyme analysis.
5. Based on the genetic diversity analysis using AFLP marker, four primer pairs were selected from 12 primer pairs for AFLP analysis, which amplified 232 bands, detected 83 polymorphic sites(15-20bands for each primer pair), and PIC overrun 0.35. the results showed that the AFLP marker is a very important method for Gastrodia elata Bl rermplasm identification, and the four primer pairs should be used first during analysising the genetic diversity.
1、天麻同工酶分析
POD、EST同工酶标记分析结果显示:天麻同工酶具有丰富的遗传多样性;同工酶谱条带最少的卵果天麻较原始,条带最多的红杆天麻分化程度较大,是3种种质中最为进化种质;红杆天麻与绿杆天麻亲缘关系比较近,红杆天麻与卵果天麻亲缘关系相对较远。
2、适合AFLP分子标记的天麻基因组DNA提纯方法的优化
以新鲜天麻花茎为实验材料,采用改良CTAB结合苯酚/氯仿(1:1)法提取方法的改良:即将温浴提取时间从1 h延长至3 h,改苯酚/氯仿/异戊醇(25:24:1)为苯酚/氯仿(1:1)抽提蛋白及酚类等杂质,改无水乙醇为异丙醇沉淀DNA,改离心获取DNA为挑取絮状DNA。经检测:DNA浓度为1.458μg/mL,A260/A280为1.93,OD值更接近于1.8,单位质量实验材料提取DNA平均得率提高180%,说明用改良CTAB法从新鲜天麻花茎中提取的天麻基因组DNA质量提高,得率增加,且DNA无降解,无杂质污染,适于天麻分子生物学研究。
3、天麻AFLP最适反应体系构建
在利用AFLP技术研究天麻遗传多样性过程中,从引物筛选、酶切连接、扩增条件、检测方法等几个方面进行优化,建立天麻AFLP最适反应体系为:500ng/μL DNA用3U PstI和3U MseI在37℃4h温浴双酶切;16℃下连接9h;预扩增时取连接产物2μL,50-58ng/μL预扩引物各0.6μL,Taq酶0.4μL ,(5U/μL)10×buffer2μL,dNTP(10mM) 0.4μL,MgC12(25mM)1.2μL ,ddH2O补至20μL;取5μL稀释20倍后的预扩增产物,50ng/μL PstI、MseI选扩引物各1μL,总体系20μL进行选择性扩增。在此优化体系下,可以得到重复性好、稳定且多态性高的天麻AFLP条带。
4、利用AFLP分子标记研究秦巴山区天麻遗传多样性
AFLP结果显示:12对PstI/MseI引物组合共获得605条带,每个引物组合在个体间扩增条带的数目在34-64条之间,平均每条引物能扩增条带50.4条;检测位点323个,其中多态性位点193个,平均多态性检出率为43.03%;每个引物组合在个体间扩增条带平均PIC为0.33,变化范围为0.21-0.39;天麻群体内的相似系数位在0.79-0.86之间;红杆天麻与绿杆天麻之间的遗传距离最小,卵果天麻与红杆天麻之间遗传距离最大。AFLP分子标记结果与同工酶分析结果吻合。
5、天麻AFLP指纹图谱构建
在利用AFLP分子标记研究天麻遗传多样性基础上,筛选出2、5、8、10四对可用于构建秦巴山区天麻不同种质的AFLP特异引物,这4对引物组合扩增条带丰富(232条),检测特异位点多(83),PIC值均达到或超过0.35。不同天麻种质都存在多个特异性带(15-20个)。结果表明AFLP分子标记技术是种质鉴定的一种高效方法,建议在天麻遗传多样性研究中应优先采用这些引物组合。
Gastrodia elata Bl(Tian Ma in China),belonging to orchid family,is a very important Chinese herb medicine, of which Qinba Mountainous Area is one of the genuine producing regions. In this Study of Gastrodia elata B1 Resources, the genetic diversity was investigated by Isozyme and AFLP markers, in order to make further exploitation on Gastrodia elata Bl and provide basis for Gastrodia elata B1 genetics and breeding research .The main results showed as follows:
1. The POD and EST Isozyme analysis results shows that: There were firmly high Polymorphisms in Gastrodia elata B1 Resources from Qinba Mountainous Area. Gastrodia elata Bl.var. obovata sp nov., which has the least belts, is the most original germplasm. Gastrodia elata Bl.f.elata chich has the most belts, is the most developed germplasm in the three. the relative between Gastrodia elata Bl.f.elata and Gastrodia elata Bl.f.viridis Makino is nearest, and Gastrodia elata Bl.f.elata is the most far away from Gastrodia elata Bl.var. obovata sp nov.
2. Optimization of DNA extraction and purification methods from Gastrodia elata for AFLP analysis
The results shows that the method (tissues of stem, optimized CTAB solution, extraction for 3h, extract protein by phenol/chloroform (1:1), precipitate DNA by isopropanol, DNA isolating method) was optimal. This method has the advantages that not only polysaccharide and proteins, but also polyphenol and quinonoid compounds are eliminated. As a result, DNA with high quantity and purity was obtained. The results of UV-spectrophotometric method show A260/A280 is 1.93, and the content of DNA is 1.458μg/ml. Agarose Gelelectrophoresis analysis shows that DNA belts are clear and are suitable for AFLP-PCR.
3. The factors affecting AFLP analysis including primer selection, digestion and ligation of DNA, amplified conditions and detection ways were optimized. An optimization system of AFLP markers was established: 500ng/μL DNA was digested with 3U PstI and 3U MseI at 37℃f or 4h , ligation mixture was kept at 16℃f or 9h, pre-selective amplification was set up with 2μL restriction-ligation sample DNA, 0.6μL PstI and MseI primer(50-58ng/μL), 0.4μL Taq DNA polymerase, 2μL10×buffer(5U/μL) , 0.4μL dNTP(10mM) , 1.2μL MgC12(25mM), ddH2O was added up to 20μL. In 20μL selective amplification, 5μL diluted(20×)DNA pre-selective amplification product, 1μL 50ng/μL PstI and MseI selective primers, Under this optimization system, clear, steady and higher polymorphic Gastrodia elata Bl AFLP bands were obtained.
4. In the experiment, All of the 12 primers can amplify 605 bands, each primers can amplify bands between 34 and 64. 193 of 323 amplified bands were polymorphic bands, which acccout for 43.03%. the average PIC is 0.33, varied from .021 to 0.39, with 0.14-0.21 genetic distance. the relative between Gastrodia elata Bl.f.elata and Gastrodia elata Bl.f.viridis Makino is nearest, and Gastrodia elata Bl.f.elata is the most far away from Gastrodia elata Bl.var. obovata sp nov. the results of AFLP analysis was the same as Isozyme analysis.
5. Based on the genetic diversity analysis using AFLP marker, four primer pairs were selected from 12 primer pairs for AFLP analysis, which amplified 232 bands, detected 83 polymorphic sites(15-20bands for each primer pair), and PIC overrun 0.35. the results showed that the AFLP marker is a very important method for Gastrodia elata Bl rermplasm identification, and the four primer pairs should be used first during analysising the genetic diversity.
引文
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