Genetic distance detected with RAPD markers among selected Australian commercial varieties and boron-tolerant exotic germplasm of pea (Pisum sativum L.)

The optimisation of polymerase chain reaction (PCR) for random amplified polymorphic DNA (RAPD) analysis in pea was investigated and the results were applied to an analysis of five representative Australian varieties and five selected boron-tolerant accessions derived from different geographical regions. Genotypes were compared using 34 random primers (Operon Technologies, Alameda, CA) which generated 180 polymorphic bands. Genetic similarity among genotypes was estimated on the basis of the percentage of common bands between genotypes and a dendrogram was constructed by the unweighted pair grouping method. A pattern of RAPD reaction corresponding to two main groups was discerned. The genetic divergence between Australian varieties and the boron-tolerant accessions suggests an intensive back-crossing programme would be required to transfer boron tolerance to a locally adapted genetic background. Our results show RAPD to be useful for clarifying phylogenic relationships within a species and also to provide useful genetic markers for varietal identification in pea.     

RAPD markers for constructing intraspecific tomato genetic maps

The existing molecular genetic maps of the tomato, Lycopersicon spp, are constructed based on isozyme and RFLP polymorphisms between tomato species. These maps are useful for certain applications but have few markers that exhibit sufficient polymorphisms for intraspecific analysis and manipulations within the cultivated tomato. The purpose of this study was to investigate the relative potential of RAPD technology, as compared to isozymes and RFLPs, to generate polymorphic DNA markers within cultivated tomatoes. Sixteen isozymes and 25 RFLP clones that were known to detect polymorphism between L. esculentum and L. pennellii, and 313 random oligonucleotide primers were examined. None of the isozymes and only four of the RFLP clones (i.e., 16%) revealed polymorphism between the cultivated varieties whereas up to 63% of the RAPD primers detected one or more polymorphic DNA fragments between these varieties. All RAPD primers detected polymorphism between L. esculentum and L. pennellii genotypes. These results clearly indicate that RAPD technology can generate sufficient genetic markers exploiting sequence differences within cultivated tomatoes to facilitate construction of intraspecific genetic maps.Abbreviations RFLP restriction fragments length polymorphism - RAPD random amplified polymorphic DNA - PCR polymerase chain reaction - QTLs quantitative trait loci     

利用RAPD和ISSR标记分析烤烟品种间遗传关系

利用RAPD和ISSR标记对22份烤烟(Nicotiana tabacumL.)品种进行了遗传关系研究。在RAPD分析中筛选到13个引物,共扩增出167条带,其中多态性带50条,多态性比率为29.9%;在ISSR分析中筛选出7个引物,共扩增出96条带,其中多态性带44条,多态性比率为45.8%。两种标记相结合估算出的品种间遗传相似系数在0.881~0.979之间,平均为0.933。单独基于RAPD标记和ISSR标记的聚类结果有一定差异;两种标记结合起来的聚类分析结果与系谱信息吻合程度更高。定向选择可能对烤烟品种间遗传关系有较大影响;国外引进品种与国内育成品种并未完全分开,表明分子水平的遗传关系和地理来源间缺乏必然联系。     

Genetic relationships and discrimination of ten influential Upland cotton varieties using RAPD markers

Influential Upland cotton ( Gossypium hirsutum L.) varieties are those that have the higher genetic contributions to modern Upland cultivars than other germplasms. Our previous research has shown significant differences in general combining ability (GCA) effects for yield, yield components, and fiber properties among ten influential cotton varieties. In this study, we used random amplified polymorphic DNA (RAPD) data to evaluate DNA variation of these ten varieties. Of 86 random decamer primers screened for their capability of amplifying DNA via the polymerase chain reaction (PCR), 63 generated a total of 312 DNA fragments. Forty two bands were polymorphic, which showed a low percentage (13.5%) of DNA variation among these influential varieties. Genetic similarities among the ten varieties based on RAPD data were from 92.7% to 97.6%. All of the varieties were individually identified by variety specific markers in genetic fingerprinting. One primer, UBC-149, amplified a 1,430-bp DNA fragment that was absent in five varieties and present in the other five varieties. This RAPD marker had significant negative relationships with GCA-effect estimates for seed cotton yield, lint yield, number of bolls per plant and micronaire, and significant positive relationships with GCA effects for boll size and seed index. This finding, for the first time, identifies a DNA fragment in cotton that is a potential DNA marker linked to a yield gene(s) or a yield-related gene(s).     

A comparison of methods for the estimation of genetic diversity in strawberry cultivars

RAPD markers were used to examine the genetic relatedness of eight strawberry cultivars released from four breeding programmes around the world. Ten random primers successfully amplified DNA fragments from each cultivar and specific fingerprints were generated from the molecular marker data. The cultivars were traced back to founding clones and the relationships between the cultivars were examined from both the molecular and the pedigree data.     

Identification of cultivars and validation of genetic relationships in Mangifera indica L. using RAPD markers

Twenty-five accessions of mango were examined for random amplified polymorphic DNA (RAPD) genetic markers with 80 10-mer random primers. Of the 80 primers screened, 33 did not amplify, 19 were monomorphic, and 28 gave reproducible, polymorphic DNA amplification patterns. Eleven primers were selected from the 28 for the study. The number of bands generated was primer- and genotype-dependent, and ranged from 1 to 10. No primer gave unique banding patterns for each of the 25 accessions; however, ten different combinations of 2 primer banding patterns produced unique fingerprints for each accession. A maternal half-sib (MHS) family was included among the 25 accessions to see if genetic relationships could be detected. RAPD data were used to generate simple matching coefficients, which were analyzed phenetically and by means of principal coordinate analysis (PCA). The MHS clustered together in both the phenetic and the PCA while the randomly selected accessions were scattered with no apparent pattern. The uses of RAPD analysis for Mangifera germ plasm classification and clonal identification are discussed.     

Genetic segregation of random amplified polymorphic DNA in diploid cultivated alfalfa.

Polymerase chain reaction was used, with single 10-mer primers of arbitrary sequence, to amplify random regions of genomic DNA from a diploid cultivated alfalfa backcross population. Segregation of the random amplified polymorphic DNA (RAPD) fragments was analysed to determine if RAPD markers are suitable for use as genetic markers. Of the 19 primers tested, 13 amplified a total of 37 polymorphic fragments, of which 28 (76%) segregated as dominant Mendelian traits. RAPD markers appear useful for the rapid development of genetic information in species like alfalfa where little information currently exists or is difficult to obtain.     

Genetic variation in monoploids of diploid potatoes and detection of clone-specific random amplified polymorphic DNA markers

Randomly amplified polymorphic DNA (RAPD) markers have been used to study the genetic variation among androgenetic monoploids of diploid Solanum species. Cluster analysis of pairwise genetic distances was used to construct a genetic relationship among anther donor and anther-derived potato plants. The clustering based on Rogers' distances resembled classifications based on parental origins and hybrid combinations. Six of the 32 RAPD primers used resulted in the selective amplification of DNA fragments which were polymorphic between the two S. phureja parental clones, 1.22 and A95. It should be possible to construct a genetic linkage map, without making crosses, using monoploids derived from a single heterozygous diploid clone and RAPD markers.     

RAPD技术在芦荟属植物分类研究中的应用

采用CTAB法提取11个芦荟材料的基因组DNA为模板,以50个随机引物进行RAPD分析。结果表明：大部分引物可以在不同模板上扩增出条带,但仅有6个引物可以同时在11个芦荟材料的DNA上扩增出条带,对11个芦荟种变种的RAPD结果进行聚类分析,结果表明基本符合传统分类观点。对RAPD技术在芦荟属植物分类研究中的问题进行了讨论。     

Genetic relationships and variation among ecotypes of seashore paspalum (Paspalum vaginatum) determined by random amplified polymorphic DNA markers.

Random amplified polymorphic DNA (RAPD) markers were used to assess genetic relationships and variation among ecotypes of the turfgrass seashore paspalum (Paspalum vaginatum Swartz). Vegetative tissues or seeds of 46 seashore paspalum ecotypes were obtained from various locations in the United States, Argentina, and South Africa. Leaf DNA extracts were screened for RAPD markers using 34 10-mer random primers. A total of 195 reproducible RAPD fragments were observed, with an average of six fragments per primer. One hundred and sixty-nine fragments (87% of the total observed) were polymorphic, among which 27 fragments (16%) were present in three or less ecotypes, indicating the occurrence of a high level of genetic variation among the examined accessions of this species. Cluster analysis (UPGMA) and principal coordinates analysis were performed on the RAPD data set. The results illustrate genetic relationships among the 46 ecotypes, and between ecotypes and their geographical origins. Ecotypes from southern Africa could be differentiated from the U.S. and most of the Argentinean ecotypes. With a few exceptions, ecotypes collected from Argentina, Hawaii, Florida, and Texas were separated into distinct clusters.     

Analysis of genetic stability of in vitro propagated potato microtubers using DNA markers

The genetic stability of in vitro propagated potato microtubers was assessed using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. Microtubers were developed through in vitro from potato microplants using standardized protocols. The microtubers were conserved for 1 year under three different culture media and consequently microplants were regenerated for the DNA analyses. During the study, a total of 38 (10 RAPD, 11 ISSR, 12 SSR and 5 AFLP) primers produced a total of 407 (58 RAPD, 56 ISSR, 96 SSR and 197 AFLP) clear, distinct and reproducible amplicons. Cluster analysis revealed 100 % genetic similarity among the mother plant and its derivatives within the clusters by SSR, ISSR and RAPD analyses, whereas AFLP analysis revealed from 85 to 100 % genetic similarity. Dendrogram analysis based on the Jaccard’s coefficient classified the genotypes into five clusters (I–V), each cluster consisting of mother plant and its derivatives. Principal component analysis (PCA) also plotted mother plant and its genotypes of each cluster together. Based on our results, it is concluded that AFLP is the best method followed by SSR, ISSR and RAPD to detect genetic stability of in vitro conserved potato microtubers. The in vitro conservation medium (T2) is a safe method for conservation of potato microtubers to produce true-to-type plans.     

Pathogenic and molecular characterisations of pigeonpea wilt pathogen Fusarium udum

Thirty two pathogenic isolates of Fusarium udum from different pigeonpea growing areas in India were studied for pathogenic and molecular variability. Pathogenic variability was tested on 12 pigeonpea differential genotypes, which revealed prevalence of five variants in F. udum. The amount of genetic variation was evaluated by Polymerase Chain Reaction (PCR) amplification with 20 random amplified polymorphic DNA (RAPD) markers and nine microsatellite markers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 137 polymorphic fragments were scored for the RAPD markers and 16 alleles for the simple sequence repeat (SSR) markers. RAPD primers showed 86% polymorphism. Genetic similarity was calculated using Jaccard's similarity coefficient and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates could be grouped into three subpopulations based on molecular analysis. Results indicated that there is high genetic variability among a subpopulation of F. udum as identified by RAPD and SSR markers and pathogenicity on differential genotypes.     

RAPD Analysis of Genetic Diversity in Indian Tetraploid and Diploid Cotton (Gossypium spp)

Random Amplified Polymorphic DNA (RAPD) analysis was used to evaluate genetic diversity among commercial Indian cotton varieties. Fifteen varieties belonging to Gossypium hirsutum L and seven to G. arboreum L were analyzed with 50 random decamer primers using the polymerase chain reaction (PCR). Twenty six of the primers detected polymorphism in all 22 cotton varieties. A total of 371 bands were amplified, 87% of which were polymorphic. Cluster analysis by the unweighted pair group method of arithmetic means (UPGMA) showed that diploids and tetraploids can be divided in two groups at a similarity of 30%. Diploid variety C402W showed the least similarity to all the others in the group. Among tetraploids, closely related varieties Pusa 8-6, 4515 and RS 875 were distinctly different from the rest. The analysis revealed that the intervarietal genetic relationships of several varieties is related to their pedigree. The results also revealed that tetraploids show a much narrower genetic base (similarity range of 65–95%) than the diploids (similarity range 54–88%). The results obtained can be used for the selection of parents to generate a mapping population and begin a breeding programme.     

The use of ISSR and RAPD markers for detecting DNA polymorphism,genotype identification and genetic diversity among barley cultivars with known origin

The potential of bulk analyses of RAPD and ISSR-PCR markers for fingerprinting purposes was evaluated using ten RAPD and ten ISSR primers. The phylogenetic relationships of 16 barley cultivars from different countries, and all having a known pedigree, were analysed using 353 PCR markers (125 RAPDs and 228 ISSRs). The band profiles generated were reproducible in spite of the different DNA extractions, PCR techniques, electrophoretic methods and gel scorings used. The RAPD primer S10 and four ISSR primers (811, 820, 835 and 881) were both able to distinguish all cultivars. A strong and quite linear relationship was observed between Resolving Power (Rp) of a primer and its ability to distinguish genotypes. The dendrograms obtained using these two molecular markers are in agreement with their known origin, showing clusters that separate very well the spring/winter and six-rows/two-rows cultivars. Thus, bulk analyses of RAPD and ISSR PCR markers provides a quick, reliable and highly informative system for DNA fingerprinting and also permit to establish genetic relationships which agree with, by other means, known origin of the cultivars.     

Analysis of Genetic Diversity in Cicer arietinum L Using Random Amplified Polymorphic DNA Markers

PCR-based random amplified polymorphic DNA (RAPD) markers were employed to assess genetic diversity in 23 chickpea genotypes. Forty of the 100 random primers screened revealed polymorphism among the genotypes. Most of the primers revealed single polymorphic band, and only 14.1 2% of the products were polymorphic. Estimates of genetic similarity based on Jaccard’s coefficient ranged from 0.92 to 0.99, indicating narrow genetic variability among the genotypes based on RAPD markers.The 23 chickpea genotypes formed two major clusters in the dendrogram.The low RAPD polymorphism among chickpea genotypes suggests that more number of polymorphic primers need to be analysed to determine genetic relationships. It was observed that RAPD analysis employing 30 polymorphic primers could provide better estimates of genetic relationships in chickpea.     

Characterization of genetic identities and relationships of Brassica oleracea L. via a random amplified polymorphic DNA assay

Summary Effective conservation and the use of plant genetic resources are essential for future agricultural progress. Critical to this conservation effort is the development of genetic markers which not only distinguish individuals and accessions but also reflect the inherent variation and genetic relationships among collection holdings. We have examined the applicability of the random amplified polymorphic DNA (RAPD) assay for quick, cost-effective, and reliable use in addressing these needs in relation to collection organization and management. Twenty-five decamer oligonucleotide primers were screened individually with a test array composed of individuals representing a range of genetic relationships in Brassica oleracea L. (vegetable and forage cole crops). Over 140 reproducible, polymorphic fragments were generated for study. Each individual of the test array exhibited a unique molecular genotype and composites specific for accessions and botanical varieties could be established. An analysis of similarity based on amplified DNA fragments reflected the known genetic relationships among the selected entries. These results demonstrated that RAPD markers can be of great value in gene bank management for purposes of identification, measurement of variation, and establishment of genetic similarity at the intraspecific level.     

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.     

用EST-SSR标记检测我国甘蓝型油菜品种的遗传多样性

杂交育种依然是我国油菜育种的主要方法,杂种优势的利用仍然是提高产量的重要途径.为了解我国甘蓝型油菜的遗传变异,采用16个EST-SSR标记对近年来推广的91个品种的遗传多样性进行了分析.共扩增到100个条带,其中84个多态性带,多态性比率为84%.平均每对引物扩增的带数和多态性带数分别为6.25个和5.25个.多态性信息含量(PIC)变化在0.022-0.926之间,平均为0.677,所揭示的基因型数变化于2-24之间,平均为12.44个.供试材料间遗传距离变幅较大(0.0530-0.7223之间),说明它们具有广泛的遗传变异.其中,杂交种和2000年以后育成品种的遗传基础较宽,遗传多样性分别明显高于常规品种和2000年以前育成的品种.按非加权成对平均数法(UPGMA)进行的聚类分析显示,在遗传距离为0.313处,参试材料可以分为三大类,其中,包含87份材料的第一大类在遗传距离为0.233处又可进一步分为10个亚类.聚类结果与系谱来源基本一致,比较真实反映了所用材料的遗传变异情况.     

RAPD and pedigree-based genetic diversity estimates in cultivated diploid potato hybrids

In this study, RAPD and pedigree data were used to investigate the genetic relationships in a group of 45 diploid hybrid potato clones used in the breeding and genetics program of the Agriculture and Agri-Food Canada Potato Research Centre in Fredericton, New Brunswick, and used for the potato after-cooking darkness program at the Nova Scotia Agricultural College. These hybrids were derived from crossing primitive cultivated South American diploid species such as Solanum phureja or Solanum stenotomum and wild diploid species such as Solanum chacoense and other wild Argentine species with haploids of Solanum tuberosum. These hybrids have subsequently undergone up to 30 years of breeding and selection, for adaptation to local growing and storage conditions, processing traits and pest resistances. The objectives of this study were to estimate the level of genetic similarity (GS) among these sets of clones and to investigate the correlation between RAPD-based GS and f, based on pedigree information. Genetic similarity coefficients varied from 0.29 to 0.90 with a mean of 0.65 when based on the RAPD data, whereas the coefficient of parentage varied from zero to 0.75 with a mean of 0.11. The degree of relationship between the similarity matrices based on RAPD and pedigree was measured by comparing the similarity matrices with the normalized Mantel test. A low positive correlation (R = 0.104, p = 0.999) between the two matrices was observed. Cluster analysis using GS divided the clones into many subgroups that did not correspond well with the grouping based on pedigree. The level of genetic variation present in this set of potato clones is very high. Rigorous selection pressure aimed at different breeding purposes may result in the genetic differentiation of the clones from the same origin.     

Genetic diversity in important members of Cucurbitaceae using isozyme, RAPD and ISSR markers

Biochemical and molecular markers have been used on eleven species of Cucurbitaceae collected from lower Gangetic plains. Six enzyme systems were selected. Among 40 primers examined, 14 random amplified polymorphic DNA (RAPD) and 10 inter-simple sequence repeat (ISSR) primers were selected for the analysis. Generated RAPD (100) and ISSR (100) fragments showed high variations among the species. Jaccard similarity coefficients were used for the evaluation of pairwise genetic divergence; cluster analysis of the similarity matrices was performed to estimate interspecific diversity. Further, principal coordinate analysis was performed to evaluate the resolving power of the three marker systems to differenciate among the species.