【作者】 熊发前；

【导师】 庄伟建； 唐荣华；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 栽培种花生是世界上第四大油料作物,它被广泛种植于热带和亚热带地区,中国是世界上最大的花生生产国和消费国。但是花生上的研究远远落后于其它作物,尤其在花生分子标记领域,由于栽培种花生狭窄的遗传基础,很少多态性分子标记被发现,因此,本论文的主要研究目的是:(1)应用最新的SCoT多态性分子标记技术来研究花生属资源的遗传多样性和亲缘关系,为利用野生种材料作为杂交亲本打下基础;(2)为充分理解栽培种花生的DNA多态性水平,多种随机和目标分子标记技术(SCoT,ISJAP,DAMD和URP)被用来检测栽培种花生的DNA多态性水平;(3)比较上述4种分子标记技术在检测栽培种花生多态性的效率程度;(4)开发了两种最新型目标分子标记技术,它们能在栽培种花生上检测到一定程度的DNA多态性,在其它物种上检测到丰富的DNA多态性。本论文研究的主要结果如下:1.本论文于国内外首次利用目标起始密码子多态性标记技术来研究花生属材料的遗传多样性和亲缘关系。结果表明:23条单引物共扩增出194条带,其中130条是多态性条带,聚类分析能准确地反映所研究材料间的亲缘关系。结果表明该技术能被用来研究花生属材料间的亲缘关系。2.为能在栽培种花生中获得大量的DNA多态性以及方便区别栽培种花生品种的基因型,本文首次利用目标起始密码子多态性标记技术来研究栽培种四大类型花生的遗传多样性和亲缘关系,一共筛选了36条引物,其中18条能产生清晰、可重复以及具有多态性的条带,18条引物产生了157条带,每条引物产生的条带从4到17条,平均8.72条,157条条带中有60条条带(38.22﹪)是多态性条带,每条引物能产生1到7条多态性条带,平均每条3.33条,多态比率从14.29﹪到66.67﹪,平均36.76﹪;结果揭示出,栽培种花生材料间具有非常高的遗传相似系数且并不是同一类型中的所有材料总是聚类在一起的;另外,在几种材料中发现了资源特异标记。所有这些结果显示:(1)目标起始密码子多态性标记技术能被用来检测栽培种花生的DNA多态性以及在分子水平上区别栽培种花生材料;(2)在研究栽培种花生的遗传多样性和亲缘关系上,该新型分子标记技术具有很大潜力。除栽培种花生外,该技术也能直接被用于其它植物物种上。3.栽培种花生是世界上重要的油料和经济作物之一。为了在栽培种花生中检测到大量的DNA多态性标记,三种分子标记技术(ISJAP、DAMD和URP)被用来研究栽培种花生的遗传多样性和亲缘关系并对最终结果作比较,判断这三种分子标记技术的效率程度。通过引物筛选,最终得到26条引物(14条ISJAP,,4条DAMD和8条URP)是多态性引物。14条ISJAP引物在16种栽培种花生中扩增,共扩增出121条条带,其中34条是多态性条带,每条引物在16种材料中扩增条带数从5条到12条,多态性比率从10.00%到62.50%,平均为27.74%;4条多态性DAMD引物在16种栽培种花生材料中共产生25条条带,其中16条具有多态性,每条引物在16种材料中扩增条带数从6条到7条,多态性比率从28.57﹪到50.00﹪,平均为36.31﹪;8条URP引物在16种栽培种花生中扩增,共扩增出50条条带,其中25条是多态性条带,每条引物在16种材料中扩增条带数从5条到8条,总条带数和多态性条带数平均为6.25和3.13,多态性比率从20.00﹪到80.00﹪,平均为49.53﹪。对这三种分子标记得到的结果进行比较,比较结果大概显示,URP标记相比ISJAP和DAMD标记更具效率,这三种标记的聚类结果或多或少地类似。4.根据SRAP、TRAP以及最近的CoRAP标记技术的原理,本文开发了一种基于PCR技术的新型分子标记技术,名叫相关序列扩增多态性基础上的内含子锚定扩增多态性(SRAP-IAAP),对SRAP-IAAP而言,一对引物被用来进行PCR,一条引物来自原始SRAP标记技术的正向或反向引物,另外一条引物是根据内含子拼接位点序列设计,PCR扩增条件和程序参考原始SRAP,仅做稍微改动,PCR产物通过标准的琼脂糖电泳来进行分离,本论文已将该技术应用于栽培种花生及其它2种物种的遗传多样性及亲缘关系分析上了且得到了好的结果。根据所用引物对组合的不同,该技术可以在栽培种花生、香蕉和龙眼上产生3到11条条带不等,在栽培种花生、香蕉和龙眼上产生的多态性条带所占比率分别是43.15%,80%,和79.25%。由于SRAP-IAAP标记技术所用引物的设计原理,这些引物组合可以被直接用于其它植物物种上的遗传学研究,该技术简单、方便、效率高以及无需所研究植物的基因组的序列信息,该目标分子标记技术可以作为SRAP、TRAP和最近CoRAP标记技术的有效补充,可以被应用于各个领域,比方目标数量性状位点定位、遗传多样性分析、遗传图谱构建。5.本文开发了另外一种产生分子标记的新方法,该方法使用长度从15bp到18bp的单引物进行PCR反应,单引物根据保守一致的BPS序列进行设计,PCR产物通过标准的琼脂糖电泳进行分离,本论文已将该技术应用于栽培种花生及其它3种物种的遗传多样性及亲缘关系分析上了且得到了好的结果,由于该技术所用单引物是根据基因内含子内部保守一致的短BPS序列设计的,所以这些单引物是通用的,可以被直接用于其它植物物种上的遗传学研究,该技术简单、方便、效率高以及无需所研究植物的基因组的序列信息,该技术可结合RAPD和ISSR标记技术来联合使用,可以被应用于各个领域。本论文研究结果:(1)为今后花生研究者在选择能在栽培种花生中检测到大量DNA多态性的分子标记技术提供了机会;(2)开发了两种新型分子标记技术。更多还原

【Abstract】 Peanut (Arachis hypogaea L.) is the world’s fourth largest oil crop which was grown worldwide in subtropical and tropical regions. China is the largest country of peanut production and comsumption in the world. But studies on peanut are far behind other crops. Especially in the field of molecular markers, less informative DNA polymorphic markers were found in cultivated peanut because of narrow genetic basis in cultivated peanut. So the main purposes of this study were: (1) to investigate the genetic diversity and relationships among accessions of genus Arachis for selecting the closer wild relatives which can be used to hybridize with cultivated peanut; (2) for comprehensive understanding the DNA polymorphisms in cultivated peanut, multiple random and gene-targeted molecular marker techniques (SCoT, ISJAP, DAMD and URP) were exploited for detecting DNA polymorphisms in cultivated peanut; (3) to compare the efficiency of these marker techniques for detecting DNA polymorphisms in cultivated peanut; and (4) to develop two novel and effective marker techniques for revealing a certain degree of DNA polymorphisms in cultivated peanut and revealing abundant DNA polymorphisms in other plant species. The main results of this investigation were obtained as follows:1 In the present study, SCoT polymorphism marker technique was firstly used to study the genetic diversity and relationships among sixteen accessions of genus Arachis. The results showed that 23 single primers amplified a total of 194 bands, of which 130 were polymorphic. Cluster analysis can reveal the genetic relationships between them correctly. This result demonstrated that start codon targeted polymorphism marker technique can be used to study genetic relationships among genus Arachis.2 For the purpose of obtaining considerable DNA polymorphisms and fingerprinting cultivated peanut genotypes conveniently, start codon targeted polymorphism technique was firstly used to study genetic diversity and relatedness among twenty accessions of four major botanical varieties of peanut in the present study. Of thirty six primers screened, eighteen primers could produce unambiguous and reproducible bands. All the 18 primers generated a total of 157 fragments with a mean of 8.72 ranging from 4 to 17 per primer. Of 157 bands, 60 (38.22﹪) bands were polymorphic. One to seven polymorphic bands were amplified per primer, with 3.33 polymorphic bands on average. The polymorphism per primer ranged from 14.29﹪to 66.67﹪with an average of 36.76﹪. The results also revealed that not all accessions of the same variety were grouped together and high genetic similarity was detected among the tested genotypes based on cluster analysis and genetic distance analysis, respectively. In addition, accession specific markers were found in several accessions. All these results demonstrated that: (1) Start codon targeted polymorphism technique can be utilized to identify DNA polymorphisms and fingerprint peanut cultivars in domesticated peanut and (2) It has great potential for studying genetic diversity and relationships among peanut accessions. In addition to cultivated peanut, this technique can be also applied to other plant species directly without further modifications.3 Cultivated peanut is one of the most important oil and economic crops in the world. For the purpose of detecting considerable DNA polymorphisms, three molecular marker techniques, intron-exon splice junction amplified polymorphism (ISJAP), directed amplification of minisatellite region DNA (DAMD) and universal rice primer (URP), were compared to study the genetic diversity and genetic relationships among tested 16 accessions of cultivated peanut. A total of 26 polymorphic primers (14 ISJAP, 4 DAMD and 8 URP) were used. Amplification of genomic DNA of 16 accessions, using ISJAP analysis, yielded 121 fragments, of which 34 were polymorphic. Number of amplified fragments with ISJAP primers ranged from five to twelve. Percentage polymorphism ranged from 10.00% to 62.50% with an average of 27.74%. The 4 DAMD primers produced 25 bands across 16 accessions, of which 16 were polymorphic. The number of amplified bands varied from six to seven. Percentage polymorphism ranged from 28.57﹪to 50.00﹪with an average of 36.31﹪. The 8 URP primers used in the study produced 50 bands across 16 accessions, of which 25 were polymorphic. The number of amplified bands varied from five to eight. The average numbers of bands per primer and polymorphic bands per primer were 6.25 and 3.13, respectively. Percentage polymorphism ranged from 20.00﹪to 80.00﹪with an average of 49.53﹪across all the genotypes. When these three marker techniques were compared, the results showed that URP markers were relatively more efficient than ISJAP and DAMD markers. Clustering of accessions remained more or less the same in ISJAP, DAMD and URP.4 On the basis of the principles of SRAP, TRAP and more recently CoRAP, a novel PCR-based molecular marker technique called sequence-related amplified polymorphism based on intron anchored amplified polymorphism (SRAP-IAAP) were developed in the present study. For SRAP-IAAP, a pair of primers is exploited to conduct PCR. One primer is derived from either forward or reverse primer of original SRAP technique. The other is designed from intron-exon splice junction sequence. PCR amplification is carried out according to the procedure of original SRAP technique with minor modifications. The PCR products are resolved through standard agarose gel electrophoresis in this study. We have applied this technique to study genetic diversity of cultivated peanut and to fingerprint other two plant species (banana and longan) and successful results were also achieved. Depending upon the primer pairs used, 3-11 bands were detected in peanut, banana, and longan. As high as 43.15%, 80%, and 79.25% of the polymorphic bands were detected in peanut, banana, and longan, respectively. Since the basis of primer pairs design, these primers would be universal across other plant species and SRAP-IAAP can be employed in other plant genotyping directly. This molecular marker technique is rapid, simple, and efficient and does not require preliminary sequence information of the plant genome of interest. Also this gene-targeted molecular marker technique could be complementary to SRAP, TRAP and even CoRAP to researchers for widely applications in the fields of targeted quantitative trait loci mapping, genetic diversity analyzing, genetic map constructing and important agronomic traits tagging. This technique provides a new way to develop molecular markers for plant genotyping.5 Another novel method for producing molecular markers in plants was also developed. This method uses single 15- to 18-mer primers designed from the short conserved consensus branch point signal sequences for PCR. The PCR products obtained with this kinds of primer are separated by means of standard agarose gel electrophoresis. This method was applied to fingerprint cultivated peanut and other several plant samples and good fingerprinting results were achieved. Since single primers were designed from relatively conserved branch point signal sequences within introns of genes, these primers would be universal across other plant species. It demonstrated this method is rapid, simple, efficient, and does not need sequence information of the plant genome of interest. This method could be used in conjunction with or as a substitute to conventional RAPD, ISSR techniques for further applications.The results obtained in the paper: (1) offered opportunities for peanut researchers to choose the better marker techniques for obtaining relatively abundant DNA polymorphisms in domesticated peanut; (2) offered two kinds of simple, novel, reliable, reproducible and effective marker techniques to conduct related study, such as genetic diversity analysis, phylogenetic relationships analysis, gene and/or QTL mapping, genetic linkage map construction.更多还原