Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.     

Genetic fingerprinting of Australian cotton cultivars with RAPD markers.

RAPD (random amplified polymorphic DNA) markers generated by 30 random decamer primers were used to fingerprint 12 released cultivars and a breeding line of Gossypium hirsutum and 1 cultivar of G. barbadense presently under cultivation in Australia. Among a total of 453 developed markers, 69 (15.2%) were only present (unique) in the G. barbadense cultivar Pima S-7. Of the remaining markers, 128 (33.3%) were fixed in all 13 G. hirsutum cultivars. In pairwise comparisons of the degree of band sharing, nine closely-related cultivars showed 92.1-98.9% genetic similarity. Cluster analysis of genetic distance estimates between each of the cultivars revealed phylogenetic relationships in broad agreement with the known lineage of the cultivars. Ten of the G. hirsutum cultivars can be characterized individually based upon cultivar-specific RAPD markers, thus making it possible to differentiate closely related cultivars by molecular markers.     

Estimation of genetic diversity in varieties of Mucuna pruriens using RAPD

Genetic diversity was estimated in 13 accessions of the otherwise self pollinated Mucuna pruriens (L.) DC. (velvetbean) comprising varieties pruriens and utilis collected from tropical humid forest using 15 RAPD primers. Similarity index value of 0.68 based on Nei and Li's similarity coefficient indicated high degree of genetic variability. Analysis of various genetic diversity indices like total heterozygosity, Nei's gene diversity, percentage of polymorphic loci, expected and observed number of alleles and Shannon index strongly suggests that variety pruriens is genetically more diverse than variety utilis. Chemical analysis with respect to 3,4-dihydroxy-L-phenylalanine (L-DOPA) content showed uniform distribution. Cluster analysis showed grouping of accessions into two major clusters and tendency of accessions of variety pruriens to group according to their geographical locations. Bootstrap analysis confirmed the robustness of the phenogram. The putative hybrid MMP6 with relatively low similarity value index and low L-DOPA content was promising as food or fodder.     

Construction of phylogenetic tree forNicotiana species based on RAPD markers

To apply random amplified polymorphic DNA for analysis of phylogenetic relationships, we used 34 synthetic oligonucleotides as primers to examine interspecific and intraspecific variations among 18 genotypes, nine species ofNicotiana. The nine species used in this study belong to sectionsTomentosae andAlatae. In addition, we attempted to clarify the taxonomic position ofN. sylvestris. A total of 354 distinct DNA fragments were obtained by polymerase chain reaction. Pair-wise comparisons of unique and shared amplification products were used to generate Jaccard's similarity coefficients and Nei and Li's similarity coefficients with the computer software of numerical taxonomy and multivariate analysis system. On the basis of the dendrogram constructed with the similarity coefficients, the 18Nicotiana genotypes were divided into two clusters. The classification analyzed by RAPD markers is in accordance with the classification of Goodspeed thatN. sylvestris is a member of sectionAlatae.     

Assessment of genetic variation within Indian mustard (Brassica juncea) germplasm using random amplified polymorphic DNA markers

Genetic diversity among 45 Indian mustard (Brassica Juncea L.) genotypes comprising 37 germplasm collections, five advance breeding lines and three improved cultivars was investigated at the DNA level using the random amplified polymorphic DNA (RAPD) technique. Fifteen primers used generated a total of 92 RAPD fragments, of which 81 (88%) were polymorphic. Of these, 13 were unique to accession 'Pak85559'. Each primer produced four to nine amplified products with an average of 6.13 bands per primer. Based on pairwise comparisons of RAPD amplification products, Nei and Li's similarity coefficients were calculated to evaluate the relationships among the accessions. Pairwise similarity indices were higher among the oilseed accessions and cultivars showing narrow ranges of 0.77-0.99. An unweighted pair-group method with arithmetic averages cluster analysis based on these genetic similarities placed most of the collections and oilseed cultivars close to each other, showing a low level of polymorphism between the accessions used. However, the clusters formed by oilseed collections and cultivars were comparatively distinct from that of advanced breeding lines. Genetically, all of the accessions were classified into a few major groups and a number of individual accessions. Advanced breeding lines were relatively divergent from the rest of the accessions and formed independent clusters. Clustering of the accessions did not show any pattern of association between the RAPD markers and the collection sites. A low level of genetic variability of oilseed mustard was attributed to the selection for similar traits and horticultural uses. Perhaps close parentage of these accessions further contributed towards their little diversity. The study demonstrated that RAPD is a simple and fast technique to compare the genetic relationship and pattern of variation among the gene pool of this crop.     

Molecular Assessment of Diversity among Pathotypes of Colletotrichum falcatum Prevalent in Sub-Tropical Indian Sugarcane

Summary Six pathotypes of Colletotrichum falcatum, responsible for Red-rot in sugarcane, prevalent in subtropical India were examined for genetic relationships using RAPD markers. A high degree of polymorphism (78.6%) was observed using 40 RAPD markers. More than 50% genetic divergence was found among the pathotypes and UPGMA cluster analysis of genetic similarity indices grouped the six pathotypes into two clusters. Cluster I comprised pathotypes Cf01 and Cf09, while cluster II comprised the remaining four pathotypes. Cf02 and Cf08 were the most closely related among all the pathotypes. Pathotype-specific unique bands generated in RAPD profiling are being used for developing markers for pathotype identification in diseased cane samples.     

Genetic Analysis of Indian Lentil (Lens culinaris Medikus) Cultivars and Landraces Using RAPD and STMS Markers

Molecular analysis of 29 lentil (Lens culinaris) cultivars and landraces of Indian origin was carried out using twenty RAPD and ten cross-species STMS primers. A total of 97 markers (72 RAPD and 25 STMS) were amplified of which 42.3% were polymorphic. Genetic similarity among the cultivars and landraces was 89.7%. The observed results indicated low level of genetic diversity in the studied material. UPGMA cluster analysis for the combined data of RAPD and STMS revealed two broad clusters — Cluster I with three landraces and Cluster II containing all remaining landraces and cultivars except Precoz. Germplasm line Precoz was found to be the most distinct in individual as well as combined analyses. All cultivars and landraces except K-75 and L4076 could be discriminated from one another using combined data for the two techniques. Germplasm lines Precoz, L830 and cultivars L4147 and JL3 were quite distinct and could be potential germplasm resource.     

Genetic relationships among Mexican white pines (Pinus, Pinaceae) based on RAPD markers

Genetic relationships among Mexican white pines have not been completely resolved by DNA sequencing analyses. The use of random amplified polymorphic DNA (RAPD) markers for the study of interspecific relationships has been questioned because of the possible lack of homology of co-migrating bands between species. However, several RAPD based studies on pines have provided sufficient information to discriminate between closely related taxa. Genetic relationships among four species of Mexican white pines (Pinus ayacahuite, Pinus strobiformis, Pinus lambertiana and Pinus chiapensis) were estimated based on RAPD markers. Sixty-nine primers generated 247 bands in pooled DNA samples from ten populations. In addition, four selected primers generated 27 bands in 176 individual DNA samples. Unweighted Pair Group Method with Arithmetic Average (UPGMA) dendrograms based on Jaccard similarity indices were constructed. The results suggest that the closest pine species analyzed were P. ayacahuite and P. strobiformis, followed by P. lambertiana. The most genetically distant species was P. chiapensis. Cluster analyses did not support P. strobiformis as a distinct species from P. ayacahuite.     

Analysis of Genetic Diversity amongIxoraCultivars (Rubiaceae) using Random Amplified Polymorphic DNA

We have optimized the genomic DNA extraction method from freshand dry laminas, as well as fresh and dry corolla lobes ofIxoracultivars.Some woody tropical species such as these contain excessiveamounts of phenolic compounds that co-precipitate with DNA resultingin poor or no amplification during the polymerase chain reaction(PCR). Repeated precipitation with CsCl coupled with phenol:chloroformextraction yielded high quality DNA suitable for consistentPCR amplification. The DNA from fresh laminas of 22 cultivarsofIxorawas subjected to random amplified polymorphic DNA (RAPD)analysis. Individual taxa could be identified using specificDNA markers from the RAPD profiles. Cluster analysis of datafrom six primers grouped all 22 cultivars distinctly under twocultivar groups, viz.,IxoraCoccinea andI.Javanica. The percentagegenetic similarity was calculated for all the cultivars basedon the RAPD data. The two cultivar groups and the outgroup plantswere also clearly distinguishable with polar ordination usinga matrix of genetic dissimilarities (one minus similarity).Our data indicate that besides the use of RAPD markers for identificationof particularIxoracultivars within a germplasm collection, thephylogenetic relationships generated by RAPD analysis may beuseful for future breeding programmes. IxoraL. cultivars; Rubiaceae; RAPD fingerprinting; DNA extraction; woody tropical species     

Comparative genetic analysis of trichome-less and normal pod genotypes of Mucuna pruriens (Fabaceae)

Velvet bean (Mucuna pruriens) seeds contain the catecholic amino acid L-DoPA (L-3,4-dihydroxyphenylalanine), which is a neurotransmitter precursor and used for the treatment of Parkinson's disease and mental disorders. The great demand for L-DoPA is largely met by the pharmaceutical industry through extraction of the compound from wild populations of this plant; commercial exploitation of this compound is hampered because of its limited availability. The trichomes present on the pods can cause severe itching, blisters and dermatitis, discouraging cultivation. We screened genetic stocks of velvet bean for the trichome-less trait, along with high seed yield and L-DoPA content. The highest yielding trichome-less elite strain was selected and indentified on the basis of a PCR-based DNA fingerprinting method (RAPD), using deca-nucleotide primers. A genetic similarity index matrix was obtained through multivariant analysis using Nei and Li's coefficient. The similarity coefficients were used to generate a tree for cluster analysis using the UPGMA method. Analysis of amplification spectra of 408 bands obtained with 56 primers allowed us to distinguish a trichome-less elite strain of M. pruriens.     

Differentiation of <Emphasis Type="Italic">Vaccinium</Emphasis> Cultivars and Wild Clones Using RAPD Markers

The potential use of the random amplified polymorphic DNA (RAPD) technique for characterization and assessment ofgenetic relatedness was investigated in 13 wild cranberry (Vaccinium macrocarpon Ait) clones collected from Newfoundlandand Labrador, Canada. RAPD markers were also used to distinguish representatives of three different Vaccinium species.Twenty-two decamer arbitrary primers yielded informative, reproducible and polymorphic banding patterns in 13 cranberryclones and produced a total of 134 bands, of which 114 were polymorphic. The UPGMA cluster analysis separated the clonesinto two main groups: Cluster I of seven and Cluster II of six with 0.62 to 0.91 and 0.50 to 0.77 Nei and Li’s similarity range,respectively. In another experiment, a subset of eight primers was used and the RAPD markers discriminated the threeVaccinium species: cranberry, lowbush blueberry (V. angustifolium Ait) and lingonberry (V. vitis-idaea L). The RAPDpolymorphisms will be useful for Vaccinium genotype differentiation and the technology should be valuable for themaintenance of germplasm banks and the efficient choice of parents in the current Vaccinium germplasm improvementprogram.     

RAPD analysis of selected local Turkish grape cultivars (Vitis vinifera)

Turkey is very rich in local grape varieties. The solution to the problem of identifying local cultivars, which is considered an important deficiency for the region, will only be possible when they can be defined with molecular markers. Forty-nine local grapevine cultivars from ?anl?urfa (Turkey) were characterized with RAPD markers. Twenty-five decamer primers selected from 60 primers were used in this analysis. A total of 171 bands were obtained with the 25 primers, of which 112 were polymorphic; the level of DNA polymorphism was 65.49% in these local cultivars. Among the selected primers, OPA-18, OPO-07 and P-123 gave the maximum number of polymorphic bands (seven). Genetic relationships among these cultivars were determined with a similarity index and using a dendogram. Among the grape cultivars, the lowest similarity ratio (0.578) was observed among the Külahi-K?z?lbanki cultivars and the highest similarity ratio (0.908) was observed for the ?ilorut-D?külgen combination. The high similarity ratio among the grape cultivars of ?anl?urfa Province was also reflected in the dendogram. In general, no relationships were encountered between the genetic identification of the cultivars and their ampelographic properties.     

RAPD analysis of Fusarium Isolates Causing “Mango Malformation” Disease in Pakistan

Mango Malformation (MM) disease is a major constraint to mango production. A total of 20 Fusarium isolates from MM-affected mango plants were collected from 14 locations in Pakistan and assessed for genetic diversity using the random amplified polymorphic DNA (RAPD) technique. A total of 393 fragments were amplified after screening with 50 random primers. The amplifications with 45 primers identified scoreable polymorphisms among the isolates. A genetic similarity matrix based on Nei and Li’s index determined coefficients ranging from 46.46% to 92.51%. These coefficients were used to construct a dendrogram using the UPGMA algorithm. The isolates grouped into two main clusters, comprising 13 and 7 isolates respectively, at a genetic relatedness of 52%. Within the clusters, Fusarium isolates were not necessarily related either by geographic origin or by the mango cultivar from which they were isolated. RAPD proved a reproducible and tractable means of differentiating Fusarium isolates. These findings also suggest that some infections originate not from adjacent plants within an orchard but from geographically distant areas; indicating that most probably infection occurs in nurseries prior to plants being transported around the country for subsequent cultivation, and that improved plant hygiene could significantly curb MM infection and spread.     

利用RAPD研究桂林桂花品种间的亲缘关系

采用随机扩增多态性DNA(RAPD)技术,从100个随机引物中筛选出扩增效果较好的20个引物,分 析桂林市23个桂花品种的基因组多态性。20个随机引物共检测到193个位点,其中多态位点114个,占 59.1%。并进行了聚类分析,构建出树状聚类图,将这些品种划分为4个品种群,与传统分类学结果一致。结 果表明,以基因型而不是以表现型为基础,分析桂花品种间的区别是可能的。该技术为解决桂林市的桂花品 种分类问题提供了重要依据。     

RAPD polymorphisms in spring wheat cultivars and lines with different level of Fusarium resistance

Random amplified polymorphic DNA (RAPD) markers have been used to characterize the genetic diversity among 35 spring wheat cultivars and lines with different levels of Fusarium resistance. The objectives of this study were to determine RAPD-based genetic similarity between accessions and to derive associations between Fusarium head blight (FHB) and RAPD markers. Two bulked DNA from either highly resistant lines or susceptible lines were used to screen polymorphic primers. Out of 160 screened primers, 17 primers generated reproducible and polymorphic fragments. Genetic similarity calculated from the RAPD data ranged from 0.64 to 0.98. A dendrogram was prepared on the basis of a similarity matrix using the UPGMA algorithm, which corresponded well with the results of principal component analysis and separated the 35 genotypes into two groups. Association analysis between RAPD markers and the FHB index detected three RAPD markers, H19(1000), F2(500) and B1(2400), significantly associated with FHB-resistant genotypes. These results suggest that a collection of unrelated genotypes can be used to identify markers linked to an agronomically important quantitative trait like FHB. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning.     

Assessment of genetic diversity of pigeonpea cultivars using RAPD analysis

In our present study assessment of genetic diversity and identification of pigeonpea cultivars has been done by employing 76 random amplified polymorphic DNA (RAPD) primers. Out of 796 amplified products, 587 showed polymorphism (73.7 %) and an average of 10.47 bands were amplified per primer. Cluster analysis based on Jaccard’s similarity coefficient using UPGMA grouped all the cultivars into three clusters. The cluster I consists of 7 cultivars, cluster II of 11 cultivars in 4 sub-clusters and cluster III 4 cultivars. Two cultivars were not included in any cluster. The clustering was strongly supported by high bootstrap values. Furthermore, high values of the average heterozygosity (H_av) and marker index (MI) also indicated the efficiency of RAPD as a marker system.     

随机扩增多态DNA（RAPD）标记用于丁香品种遗传分析及品种分类

采用随机扩增多态ＤＮＡ（ＲＡＰＤ）标记分析了１５个丁香品种的ＤＮＡ扩增产物。研究选用了１６个随机引物,共扩增出９６条带,其中５５条带为可重复性条带,有价值条带大小多在５１７ｂｐ至１６３６ｂｐ之间。这些标记足以区分这些丁香品种。欧丁香（Ｓｙｒｉｎｇａｖｕｌｇａｒｉｓ）与Ｓ．×ｈｙａｃｉｎｔｈｉｆｌｏｒａ间的相似系数为６１．５％,欧丁香与Ｓ．×ｐｒｅｓｔｏｎｉａｅ间的相似系数为４７．２％,Ｓ．×ｈｙａｃｉｎｔｈｉｆｌｏｒａ与Ｓ．×ｐｒｅｓｔｏｎｉａｅ间的相似系数为４３．６％。结果表明,欧丁香与Ｓ．×ｈｙａｃｉｎｔｈｉｆｌｏｒａ亲缘关系最近。应用ＲＡＰＤ资料分析讨论了一些品种的起源。ＲＡＰＤ技术为丁香品种分类鉴定提供了可靠方法。     

Detection of DNA polymorphism among pea cultivars using RAPD technique

An influence of some Random Amplified Polymorphic DNA (RAPD) reaction factors on resulting banding pattern and the ability of RAPD technique to detect DNA polymorphism among six economically important pea cultivars was tested. Relatively high level of DNA polymorphism among peas was observed, using polyacrylamide/urea gels and silver staining. Altogether 13 arbitrarily designed primers produced 313 amplification products. In addition 59 polymorphisms were found. These polymorphisms can serve as potential genetic markers. RAPD data were processed using cluster analysis and plotted as dendrogram. Each tested cultivar was clearly distinguished from the others. Moreover,Pisum sativum andP. sativum subsp.arvense cultivars were separated into 2 different clusters, according to their systematic relationships.     

Analysis of genetic diversity and relationships in East African banana germplasm

The genetic diversity and phylogenetic relationships of 29 East African highland banana (Musa spp.) cultivars and two outgroup taxa, M. acuminata Calcutta 4 and Agbagba were surveyed by RAPD analysis. A genetic similarity matrix was established based on the presence or absence of polymorphic amplified fragments. Phylogenetic relationships were determined by UPGMA cluster analysis. RAPDs showed that the highland bananas are closely related with a narrow genetic base. Nevertheless, there were sufficient RAPD polymorphisms that were collectively useful in distinguishing the cultivars. The dendrogram was divisible into a major cluster composed of all the AAA highland banana cultivars and Agbagba (AAB) and a minor cluster consisting of Kisubi (AB), Kamaramasenge (AB) and Calcutta 4 (AA). Several subgroups are recognized within the major cluster. RAPD data did not separate beer and cooking banana cultivars. Our study showed that RAPD markers can readily dissect genetic differences between the closely related highland bananas and provide a basis for the selection of parents for improvement of this germplasm. Received: 28 June 2000 / Accepted: 1 August 2000     

Assessment of genetic diversity among 16 promising cultivars of ginger using cytological and molecular markers

Ginger (Zingiber officinale Roscoe) is an economically important plant, valued all over the world. The existing variation among 16 promising cultivars as observed through differential rhizome yield (181.9 to 477.3 g) was proved to have a genetic basis using different genetic markers such as karyotype, 4C nuclear DNA content and random amplified polymorphic DNA (RAPD). The karyotypic analysis revealed a differential distribution of A, B, C, D and E type of chromosomes among different cultivars as represented by different karyotype formulas. A significant variation of 4C DNA content was recorded in ginger at an intraspecific level with values ranging from 17.1 to 24.3 pg. RAPD analysis revealed a differential polymorphism of DNA showing a number of polymorphic bands ranging from 26 to 70 among 16 cultivars. The RAPD primers OPC02, OPA02, OPD20 and OPN06 showing strong resolving power were able to distinguish all 16 cultivars. The extent of genetic diversity among these cultivars was computed through parameters of gene diversity, sum of allele numbers per locus and Shannon's information indices. Cluster analysis, Nei's genetic similarity and genetic distances, distribution of cultivars into special distance classes and principal coordinate analysis and the analysis of molecular variance suggested a conspicuous genetic diversity among different cultivars studied. The genetic variation thus detected among promising cultivars of ginger has significance for ginger improvement programs.