芋种质资源遗传多样性RAPD分析
摘要
芋[Colocasia esculenta(L.)Schott]属天南星科芋属一年生或多年生草本植物，是许多国家人们的主食淀粉来源之一。在世界范围内，其消费量在蔬菜中居14位。作为一种以无性繁殖为主的作物，芋在世界各国的研究相对较少，特别是在品种资源、遗传进化方面。另一方面，由于芋种质资源种的划分，主要是根据花器官的雌花序、雄花序、中性花及附属器大小及性状来划分，而芋又极少开花，芋的分类甚为困难。目前对芋的分类大多基于形态分类，由于分类标准不统一，分类手段的局限性，导致分类结果不一致，部分品种来源不清，品种亲缘关系不明，同物异名及同名异物现象十分普遍，不利于芋种质资源的研究和品种的选育和推广。利用现代生物技术从DNA水平上进行芋遗传多样性和分类研究显得尤为重要。本试验利用RAPD技术，对47份芋属材料和2份海芋属材料进行了遗传多样性分析。主要试验结果如下：
1、用CTAB、SDS、高盐低pH三种方法提取芋总DNA，并分别对它们进行紫外吸收检测、电泳检测、RAPD扩增检测，结果表明：此三种方法提取的DNA完整性好，RAPD扩增谱带清晰，都可用于芋基因组DNA提取，其中尤以CTAB法提取的DNA纯度最好，因此本文选择CTAB法提取DNA。
2、对芋RAPD-PCR条件进行了优化，优化体系为：(1)反应体系：每一反应总体积为25ul，其中10×buffer(已含20mmol/L Mg~(2+))2.5ul，模板DNA(10ng/ul)3ul，引物(4.625umol/L)2ul，dNTP(10mmol/L)0.5ul，Taq DNA聚合酶(2U/ul)0.5ul，超纯水16.5ul。(2)扩增程序：94℃预变性3min，94℃变性1min，38℃退火1min，72℃延伸2min，循环38次，再72℃后延伸5min。
3、从150条10bp随机引物中筛选出了20条扩增好、多态性稳定的引物。将这些引物用于49份材料的扩增，扩增片断大小为300bp～2000bp。从20个引物产生的谱带中共获得150个位点，即DNA扩增片段，其中127个位点具有多态性，多态率高达84.7％。平均每个引物获得7.50个扩增位点，其中具有多态性的位点为6.35个。这说明本文所用的49份材料的遗传多样性很高。
4、根据遗传距离及以它为依据建立的UPGMA聚类分析图，49份材料明显分为
两大类:第一大类为海芋属的2份材料，第二大类为芋属的47份材料。其中，
芋属的47份材料又可分为6组:第一组为绿白芋类，第二组为紫芋类，第三
组为魁芋类，第四组为红芽芋类，第五组为叶用芋类，第六组为花茎用芋类，
其中包括云南红芋和湖北古夫芋的花茎用芋类属于芋类的高级进化类型。此
结果与据植物学性状初分类结果相符合。它们的亲缘关系为:花茎用芋类与
叶用芋类最近，然后与红芽芋类、魁芋类、紫芋类、绿白芋类的亲缘关系依
次变远。蔡克华(1995)和普迎冬(1999)认为可以将芋分为3个变种，即
叶用芋变种(var尹eriolarus Chang)、球茎用芋变种(var cormosus Chang)和
花苹用变种(va:力谬刀ore.tcen.v)，组成芋种以下三个变种的园艺学分类系统:本
文结果支持此观点，即云南红芋等一类主食花茎的芋属于花茎用芋变种。
Taro[Colocasia esculenta(L.) Schott],belonging to Colocasia of Araceae,is a perennial or annual training herb. It is one of the major starchy food plants in many countries and ranked 14th in consumption amount among the major vegetables in the world in 1997. Because taro is a kind of vegetatively propagated crop, few extensive studies on taro have been conducted, especially in genetic resources and phylogeny. On the other hand, taro classification is mainly based on flower characters such as female and male inflorescence, and size and character of neutral flower and its accessories, and it seldom flowers, so it is very difficult to classify taro. At present, the most taro classification characters are morphological. Taro classification is different, and sources of some accessions and theirs relation are unclear. As a result, it is not useful for the research of taro resources and variety breeding. In consequence, it is essential to conduct researches on genetic diversity and classification of taro at the DNA level using modern biological technique. Here, genetic diversity of 47 taro materials and 2 genus Alocasia (Schott) GDon materials had been conducted using RAPD, and the main results as follows:
1.Three methods-CTAB method, SDS method, and high salinity and low pH method were used to extract genomic DNA from leaves of taro. The DNA samples of the three methods were tested by spectrophotometer, agarose gel electrophoresis and RAPD. The result was that all of the three methods could be used for extracting genomic DNA from leaves of taro, and that the CTAB method was the best ones through which the production had the highest purity. So, we choose CTAB method to conduct following experiments.
2. Some essential factors that might affect the result of RAPD assay were tested and compared. An optional reaction system suitable for the assay and usage of RAPD in taro was established. The volume of amplified reaction was 25ul,containing 10 X buffer (Mg2+, 20mmol/L) 2.5ul, genomic DNA (10ng/ul) 3ul, primer (4.625umol/L) 2ul, dNTP (10mmol/L) 0.5ul, Taq DNA ployrase (2U/ul) 0.5ul, and
pure water 16.5ul. The reaction program was devised for 38 cycles, each with 94 C denaturation for 1min, 38C annealing for Imin, 72C extension for 2min, and 94C predenature for 3min in the former denature and 72C postextension for 5min in the final extension.
3. In the paper , forty-seven accessions of Colocasia escidenta, and two accessions of genus Aloca.sia were evaluated for genetic diversity using random amplified polymorphic UNA (RAPD) markers. Twenty primers were chosen for they showed strong , reproducible amplification and distinct polymorphisms from 150 10-base arbitrary primers. A total of 150 DNA fragments were amplified from 300bp~2000bp, among which 127 were polymorphics, and which accounted for 84.7%. The average number DNA bands produced by each primer was 7.5. Those above results showed that the genetic diversity of 49 accessions was high.
4.Through UPGMA cluster analysis of genetic linkage distance, the accessions were separated into 2 main groups with Colocasia esculenta divided into 6 sections, which were green, purple, mother, red-bud, petiole and inflorescence types of taro, respectively. It was found that the classification result through RAPD markers was accordant with that resulted from some botanic characters. It was clear from the morphology observation and RAPD analysis of taro that inflorescence types of taro such as Yunnan Hongyu and Hubei Gufuyu was closest to petiole taro in relationship than purple and green types of taro. This result suggested that the eating inflorescences taro such as Yunan Hongyu and Hubei Gufuyu could be classified as var. Inflorescens, which supported the opinions of Cai K H (1995) and Pu Y D (1999). They suggested that taro could be classified into three varieties, which are var. petiolatus Chang, var. cormosus Chang, and var. Inflorescens.
1、用CTAB、SDS、高盐低pH三种方法提取芋总DNA，并分别对它们进行紫外吸收检测、电泳检测、RAPD扩增检测，结果表明：此三种方法提取的DNA完整性好，RAPD扩增谱带清晰，都可用于芋基因组DNA提取，其中尤以CTAB法提取的DNA纯度最好，因此本文选择CTAB法提取DNA。
2、对芋RAPD-PCR条件进行了优化，优化体系为：(1)反应体系：每一反应总体积为25ul，其中10×buffer(已含20mmol/L Mg~(2+))2.5ul，模板DNA(10ng/ul)3ul，引物(4.625umol/L)2ul，dNTP(10mmol/L)0.5ul，Taq DNA聚合酶(2U/ul)0.5ul，超纯水16.5ul。(2)扩增程序：94℃预变性3min，94℃变性1min，38℃退火1min，72℃延伸2min，循环38次，再72℃后延伸5min。
3、从150条10bp随机引物中筛选出了20条扩增好、多态性稳定的引物。将这些引物用于49份材料的扩增，扩增片断大小为300bp～2000bp。从20个引物产生的谱带中共获得150个位点，即DNA扩增片段，其中127个位点具有多态性，多态率高达84.7％。平均每个引物获得7.50个扩增位点，其中具有多态性的位点为6.35个。这说明本文所用的49份材料的遗传多样性很高。
4、根据遗传距离及以它为依据建立的UPGMA聚类分析图，49份材料明显分为
两大类:第一大类为海芋属的2份材料，第二大类为芋属的47份材料。其中，
芋属的47份材料又可分为6组:第一组为绿白芋类，第二组为紫芋类，第三
组为魁芋类，第四组为红芽芋类，第五组为叶用芋类，第六组为花茎用芋类，
其中包括云南红芋和湖北古夫芋的花茎用芋类属于芋类的高级进化类型。此
结果与据植物学性状初分类结果相符合。它们的亲缘关系为:花茎用芋类与
叶用芋类最近，然后与红芽芋类、魁芋类、紫芋类、绿白芋类的亲缘关系依
次变远。蔡克华(1995)和普迎冬(1999)认为可以将芋分为3个变种，即
叶用芋变种(var尹eriolarus Chang)、球茎用芋变种(var cormosus Chang)和
花苹用变种(va:力谬刀ore.tcen.v)，组成芋种以下三个变种的园艺学分类系统:本
文结果支持此观点，即云南红芋等一类主食花茎的芋属于花茎用芋变种。
Taro[Colocasia esculenta(L.) Schott],belonging to Colocasia of Araceae,is a perennial or annual training herb. It is one of the major starchy food plants in many countries and ranked 14th in consumption amount among the major vegetables in the world in 1997. Because taro is a kind of vegetatively propagated crop, few extensive studies on taro have been conducted, especially in genetic resources and phylogeny. On the other hand, taro classification is mainly based on flower characters such as female and male inflorescence, and size and character of neutral flower and its accessories, and it seldom flowers, so it is very difficult to classify taro. At present, the most taro classification characters are morphological. Taro classification is different, and sources of some accessions and theirs relation are unclear. As a result, it is not useful for the research of taro resources and variety breeding. In consequence, it is essential to conduct researches on genetic diversity and classification of taro at the DNA level using modern biological technique. Here, genetic diversity of 47 taro materials and 2 genus Alocasia (Schott) GDon materials had been conducted using RAPD, and the main results as follows:
1.Three methods-CTAB method, SDS method, and high salinity and low pH method were used to extract genomic DNA from leaves of taro. The DNA samples of the three methods were tested by spectrophotometer, agarose gel electrophoresis and RAPD. The result was that all of the three methods could be used for extracting genomic DNA from leaves of taro, and that the CTAB method was the best ones through which the production had the highest purity. So, we choose CTAB method to conduct following experiments.
2. Some essential factors that might affect the result of RAPD assay were tested and compared. An optional reaction system suitable for the assay and usage of RAPD in taro was established. The volume of amplified reaction was 25ul,containing 10 X buffer (Mg2+, 20mmol/L) 2.5ul, genomic DNA (10ng/ul) 3ul, primer (4.625umol/L) 2ul, dNTP (10mmol/L) 0.5ul, Taq DNA ployrase (2U/ul) 0.5ul, and
pure water 16.5ul. The reaction program was devised for 38 cycles, each with 94 C denaturation for 1min, 38C annealing for Imin, 72C extension for 2min, and 94C predenature for 3min in the former denature and 72C postextension for 5min in the final extension.
3. In the paper , forty-seven accessions of Colocasia escidenta, and two accessions of genus Aloca.sia were evaluated for genetic diversity using random amplified polymorphic UNA (RAPD) markers. Twenty primers were chosen for they showed strong , reproducible amplification and distinct polymorphisms from 150 10-base arbitrary primers. A total of 150 DNA fragments were amplified from 300bp~2000bp, among which 127 were polymorphics, and which accounted for 84.7%. The average number DNA bands produced by each primer was 7.5. Those above results showed that the genetic diversity of 49 accessions was high.
4.Through UPGMA cluster analysis of genetic linkage distance, the accessions were separated into 2 main groups with Colocasia esculenta divided into 6 sections, which were green, purple, mother, red-bud, petiole and inflorescence types of taro, respectively. It was found that the classification result through RAPD markers was accordant with that resulted from some botanic characters. It was clear from the morphology observation and RAPD analysis of taro that inflorescence types of taro such as Yunnan Hongyu and Hubei Gufuyu was closest to petiole taro in relationship than purple and green types of taro. This result suggested that the eating inflorescences taro such as Yunan Hongyu and Hubei Gufuyu could be classified as var. Inflorescens, which supported the opinions of Cai K H (1995) and Pu Y D (1999). They suggested that taro could be classified into three varieties, which are var. petiolatus Chang, var. cormosus Chang, and var. Inflorescens.
引文
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