| 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.
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