Use of ISSR,SRAP, and RAPD markers to assess genetic diversity in Turkish melons

The genetic relationships among 63 melon (Cucumis melo L.) genotypes collected from various regions of Turkey were determined by comparing their molecular ISSR, SRAP, and RAPD markers with those of 19 foreign melon genotypes to investigate the taxonomic relationships and genetic variation of Turkish melon germplasm. Total 162 polymorphic markers (69, 18, and 75 obtained from ISSR, SRAP, and RAPD primers, respectively) were used to define the genetic similarity among the melon genotypes by dendrogram or two and three dimensional scalings. The average similarity (SM coefficient) between any two pairs of accessions examined as estimated by molecular variation was 0.73 ± 0.48. Within-group genetic similarities ranged between 0.46 and 0.96. Related genotypes or genotypes collected from similar regions were partitioned to similar clusters. Southeastern Anatolian genotypes were distinctly apart from group inodorus and group cantalupensis (sweet) genotypes. This reinforced the position of Turkey in the secondary genetic diversity center of melon. The genetic diversity among Turkish genotypes (H = 0.28 and I = 0.42) was only a little less than that of the world accessions (H = 0.30 and I = 0.45). On the other hand, the percentage of polymorphic loci among Turkish melon genotypes (90.7%) was even higher than that of the world accessions (87.6%).     

A comparative analysis of the genetic diversity between inbred lines of Zinnia elegans using morphological traits and RAPD and ISSR markers

Genetic diversity within Zinnia elegans is key to the genetic improvement of this important ornamental species. Here, morphological traits and RAPD and ISSR molecular markers were used to assess levels of polymorphism across 20 inbred lines. Thirty-four morphological traits were scored and also 147 RAPD marker-fragments, as amplified by 12 arbitrary primers, and 128 ISSR marker-fragments as generated by 9 primers. The number of polymorphic loci, the percentage of polymorphic loci, Shannon's Information index (I) and the effective number of alleles (Ne) were calculated from the RAPD data as 100, 68.03%, 0.3559 and 1.4169, respectively. From the ISSR data, these respective statistics were calculated as 97, 76.38%, 0.4013 and 1.4728. Thus, ISSR markers were considered slightly superior to RAPD markers for assessing genetic diversity between the accessions; however, Mantel's test indicated significant correlation (R = 0.733) of the RAPD and ISSR results. By contrast, the morphological matrix showed low correlation with both RAPD and ISSR data matrices (R = 0.3814 and 0.3765, respectively). Cluster analysis showed that groupings of the accessions according to all three methods correlated well with their geographic region of origin, but flower color was not strongly associated with the genetic classification of these inbred lines of Z. elegans.     

Analysis of genetic diversity among Indian short-day onion (Allium cepa L.) cultivars using RAPD markers.

Summary

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate genetic diversity among 24 cultivars of short-day onions. Total genomic DNA was extracted and subjected to RAPD analysis using 90 arbitrary 10-mer primers. Of these, 15 primers were selected which yielded 137 bands, 91.24% of which were polymorphic. None of the primers produced a unique banding pattern for each cultivar. RAPD data were used to calculate a Squared-Euclidian Distance matrix which revealed a minimum genetic distance between cultivars ‘AFLR-722’ and ‘PBR-140’, and a maximum genetic distance between cultivars ‘PBR-139’ and ‘A.Kalyan’, and ‘MS-48’ and ‘A.Kalyan’. Based on the distance matrix, cluster analysis was done using a minimum variance algorithm.The dendrogram thus generated, based on Ward’s method, grouped the 24 onion cultivars into two major clusters. The first cluster consisted of cultivars from the northern region, and the second of cultivars from the southern region of India. The present study shows that there is high diversity among the onion cultivars selected and indicates the potential of RAPD markers for identification and maintenance of onion germplasm for crop improvement purposes.     

Genetic structure of Buxus sinica var. parvifolia, a rare and endangered plant

Buxus sinica var. parvifolia, a rare and endangered tree species in some semitropics alpine areas of China, plays an important role in the maintenance of the landscape and ecosystem. In this study, RAPD and ISSR markers were used to investigate the genetic diversity and structure of five natural populations and one tamed population of B. sinica var. parvifolia. 21 RAPD primers amplified 209 bands with 167 (79.90%) polymorphic and 21 ISSR primers amplified 518 bands with 467 (90.15%) polymorphic. The genetic diversity, estimated by Shannon’ index, was 0.4343 (by RAPDs) and 0.3661 (by ISSRs). Both RAPD and ISSR analyses revealed a high level of genetic diversity in natural populations of B. sinica var. parvifolia. Furthermore, analysis of molecular variance (AMOVA) was used to apportion the variation within and between populations. The proportion of variation attributable to within-population differences was very high (69.2% by RAPDs; 84.51% by ISSRs). Moderate differentiation was detected among populations using RAPDs (30.80%), while only a small amount of variation (15.49%) was detected among populations using ISSRs. We suggest that the present genetic structure is due to high levels of environmental variability and gene flow, which still need further study to confirm. Conservation measures are suggested, including in situ and ex situ strategies, based on the observed population genetic information.     

RAPD markers reveal polymorphism among some Iranian pomegranate (Punica granatum L.) genotypes

In this study RAPD markers were used to determine the diversity level among 24 Iranian pomegranate genotypes. One hundred decamer random primers were used for PCR reactions, among which 16 showed reliable polymorphic patterns. These primers produced 178 bands, of which 102 were polymorphic. Cluster analysis of the genotypes was performed based on data from polymorphic RAPD bands, using Jaccard's similarity coefficient and UPGMA clustering method. The highest and lowest similarities detected between genotypes were 0.89 and 0.29, respectively. At a similarity of 60%, the genotypes were divided into four sub-clusters. Cophenetic correlation coefficient between similarity matrix and cophenetic matrix of dendrogram was relatively high (r = 0.9) showing the goodness of fit of the dendrogram. RAPD markers showed to be a useful tool for studying the genetic diversity of pomegranate.     

Genetic characterization of different demes in Prunus persica revealed by RAPD markers

DNAs of 180 accessions in 10 demes in Prunus persica were amplified with twenty-two, 10-base primers selected from 200 arbitrary primers using Randomly Amplified Polymorphic DNA (RAPD) technology. One hundred and eighty loci were observed and recorded. With statistical analyses of the data from the study, genetic diversity of the demes was expressed as follow: yellow peach group > honey peach group > flat peach group > red leaf peach group > crisp peach group > bitao group and juicy peach group > nectarine group > shouxingtao group > weeping peach group. Genetic variations among and within groups by AMOVA analyses were 11.9, 88.1%, respectively. Demes clustered by UPGMA modified from NEIGHBOR procedure of PHYLIP Version 3.5, the edible peaches of which were combined as a section, while the ornamental species were classified into separate sections. Through analyses of genetic diversity and genetic structure, the results could provide molecular biological evidence for conservation and utilization of P. persica germplasm.     

Genetic diversity evaluation of wild Persian shallot (Allium hirtifolium Boiss.) using morphological and RAPD markers

Persian shallot, a bulb producing plant from Alliaceae, is a wildly growing plant collected for its bulbs. Bulbs of Persian shallot, called “Mooseer” in Farsi, are oval, white skinned, usually of one and rarely of two main bulbs and are completely different from common shallot (Allium ascalonicum). There is no information about genetic diversity of this species; therefore, the diversity of 17 wild Persian shallot populations collected from different parts of Iran across the Zagross Mountains were evaluated by morphological and RAPD markers. Fifteen random decamer primers could amplify DNA well and produced polymorphic bands among populations. The most fragment number amplified by one primer was 27 out of which 15 fragments were polymorphic and the least was 11 with 6 polymorphic bands. At the similarity of 0.60 on dendrogram constructed on the base of RAPD data, the samples were divided into eight sub-clusters. The highest and lowest detected similarities were 0.73 and 0.49, respectively. Clustering based on morphological traits divided the populations into three groups. Result of cluster analysis based on RAPD data did not show any correlation with morphological characters based on Mantel’s test (r = 0.03). The mineral and fatty acid analysis for defining the nutrient composition of this plant also revealed some differences among populations.     

Characterization of white sapote (Casimiroa edulis Llave & Lex.) germplasm using floral morphology,RAPD and AFLP markers

Floral morphology, random amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) were used to characterize and verify genetic diversity within a white sapote cultivar collection and to develop molecular markers for germplasm identification. On the basis of floral morphology, the cultivars were classified into three types: type I included 23 cultivars with large ovaries and small anthers; type II included 13 cultivars with small ovaries and large anthers; and type III included one cultivar, named ‘Maltby’, with a large ovary and large anthers. DNA was isolated from 39 cultivars of white sapote and subjected to RAPD and AFLP analysis using 24 and 7 primers, respectively. One hundred and sixty-eight RAPD and 286 AFLP bands were used to assess genetic characterization among white sapote. Sixty percent of the RAPD and 77% of the AFLP amplification products were polymorphic among accessions. RAPD or AFLP markers differentiated all white sapote cultivars effectively. Moreover, each flower type was characterized as specially associated with two RAPD bands. UPGMA dendrograms based on RAPD and AFLP data, showed the majority of the cultivars from flower type I and flower type II clustering together. Finally 101 RAPD markers and 220 AFLP markers were used to construct a neighbor-joining dendrogram. This showed that the 37 cultivars could be classified into six distinct clusters, between which the similarity coefficient was as low as 0.00–0.55, even though the cultivars were morphologically very similar. The remaining two cultivars namely ‘Smathers’ and ‘Maltby’ were found genetically very distant from the other cultivars in RAPD, AFLP or combined RAPD and AFLP based dendrograms. The results suggested that the level of genetic variation among white sapote cultivars is diverse and the morphological and molecular data may lead to representation of the cultivar relationships as well as flower type discrimination.     

Comparative analysis of genetic diversity in Tunisian apricot germplasm using AFLP and SSR markers

Eighty-one accessions representing apricot germplasm in Tunisia were collected from different areas of cultivation and fingerprinted using amplified fragment length polymorphism (AFLP) and microsatellites (SSR) markers. A total of 339 polymorphic markers were revealed using 5 AFLP primers combinations and 24 SSR loci. AFLP and SSR markers expressed a high level of polymorphism allowing the distinction of the accessions with an efficiency coefficient of discrimination of 100% for AFLP and 97% for SSR markers. Genetic diversity structure was assessed with AFLPs and SSRs markers separately then with combined matrix data by the help of hierarchical clustering elaborated using Wards method based on Nei and Li (1979) distances. Comparison of the obtained dendrograms revealed a phylogeographic structure into two major groups with significant conservation between the observed subgroups in relation with the geographic origin of the accessions. The relative efficiency of the markers in determining the genetic relationships among apricot accessions has been assessed and a combination of AFLPs and SSRs markers was the most effective. In addition, Mantel test based on genetic distances indicated highly significant correlation between AFLP-SSR data and each of the AFLP and SSR ones, with Pearson correlation values of r = 0.873 and r = 0.692, respectively, revealing the higher efficiency of the combination of both molecular techniques (AFLP and SSR) to estimate the levels of genetic variability among apricot germplasm.     

Study of genetic diversity in Chamomile (Matricaria chamomilla) based on morphological traits and molecular markers

Chamomile is one of the most important medicinal plants in the world trade that has many applications in drug and sanitary industrials. In order to evaluate the genetic diversity of different chamomile landraces based on morphological and molecular markers, 20 landraces were collected from different area of Iran. In addition to that, five populations imported from European were examined. The augmented design with four blocks and five controls were used to assess morphological traits. The RAPD method was utilized for evaluating the genetic diversity. Results showed that the economical yield, the number of flowers in plant, and the essential oil content had maximum variance coefficient. The flower's diameter and height had minimum variance coefficient. According to the cluster analysis on both morphological and molecular markers, 25 populations were classified into 5 clusters, but the population intra-groups were different. From 29 reliable primers that were used, 369 bands were detected and from which 314 (85.44%) bands were polymorphic. Genetic Jaccard's similarity coefficient was estimated in the range of 0.15–0.63, and with a mean of 0.35. Results showed that the genetic diversity was not according to the geographical diversity.     

Assessment of genetic diversity in cashew germplasm using RAPD and ISSR markers

Diversity and genetic relationship in 100 cashew germplasm accessions were analyzed by using RAPD and ISSR markers. Using 10 selected RAPD primers 60 bands were generated, of which 51 bands were polymorphic (85%), and with 10 selected ISSR primers 67 amplified bands were observed with 58 polymorphic bands (86.6%). Though both kinds of markers discriminated the accessions effectively, analysis of combined data of markers (RAPD + ISSR) resulted in better distinction of accessions. By combining markers, a total of 127 bands were detected, of which 109 bands (85.8%) were polymorphic and produced on an average of 5.45 polymorphic bands per primer. Primers with high polymorphic information content and marker index were identified for discriminating accessions. High percentage of polymorphism (>85%) observed with different markers indicated high level of genetic variation existing among the accessions. Genetic relationship estimated using similarity co-efficient (Jaccard’s) values between different pair of accessions varied from 0.43 to 0.94 in RAPD, 0.38 to 0.89 in ISSR and 0.43 to 0.87 with combined markers suggested a diversity (dissimilarity) ranging from 6 to 57%, 11 to 62% and 13 to 57% respectively and the diversity skewed around 50% indicated moderate diversity. The cluster analysis with UPGMA method separated the accessions broadly into 13 clusters and in that three into smaller clusters. Some correspondence between the molecular groupings and the morphological clusters were observed. Among the accessions, NRC-142 and NRC-12 were highly divergent and NRC-231 and NRC-232 were genetically similar.     

Molecular polymorphism of cytoplasmic DNA in Ficus carica L.: Insights from non-coding regions of chloroplast DNA

The trnL (UAA) intron and the intergenic spacer between the 3′ exon of trnL (UAA) and trnF (GAA) sequences were used as genetic markers for differentiating Ficus carica cultivars and establishing refined genetic relationships. The study was based on 20 fig cultivars, collected from south and centre of Tunisia. Since, the intron was thought to be more variable among close relatives than is the chloroplast spacer. The size of these non-coding regions varied from 554 to 589 and from 989 to 1022 bases pairs for the intron and the combined sequences correspondingly. The average of GC content was 33.9% and 34.6% in the intron and the combined intron and spacer respectively. High values of A + T contents were detected in both data sets and may explain the high proportions of transversions founded. The observed variation pattern of plastid DNA provides evidence of an important genetic diversity. The overall transition/transversion bias (R) was 0.202 in the intron and 0.27 in the combined regions. The RI index of 0.592 indicates that these combined sequences have clearly more homoplasy then the intron (RI = 0.705) and spacer (RI = 0.777) sequences separately. Phylogenetic trees were generated based on maximum parsimony (MP) and neighbor-joining (NJ) analysis of the chloroplast sequences data. Results proved that a typically continuous genetic diversity characterizes the local fig germplasm. In fact, relationships inferred from the cpDNA analysis suggest several clades, which do not show geographical or tree sex correspondence. Although the level of apparent diversity is considerable, we may conclude that non-coding regions of chloroplast genome provide a new and practical opportunity to evaluate genetic diversity and to discriminate fig cultivars. Revealed cytoplasmic DNA markers are reliable to elaborate a molecular data base to conduct management and breeding programs on local fig germplasm.     

Utility of RAPD,ISSR, IRAP and REMAP markers for the genetic analysis of Citrus spp.

The application and informativeness of RAPD, ISSR, IRAP and REMAP markers to study the genetic diversity and relationship among the Citrus and its relative genotypes were investigated. High levels of polymorphism were observed for all four marker systems. The RAPD technique generated the highest number of polymorphic bands and average number of polymorphic band per assay unit. Average limit of the discriminating power was very close to its actual discriminating power of each marker. The highest and the lowest values of effective number of patterns were obtained from the marker REMAP (5.94) and RAPD (4.48), respectively. Correlation between the genetic similarities matrices were estimated from all four markers using the Mantel matrix correspondence test, and results showed significant correlations among the RAPD, ISSR, IRAP and REMAP similarity matrices. The highest correlations were found comparing RAPD and ISSR markers, whereas RAPD and REMAP (r = 0.143) markers were poorly correlated. To assess the usefulness of the overall information provided by these marker data for establishing phylogenetic relationships and Citrus germplasm classification, cluster analysis was performed. All four techniques, solely or in combination, discriminated the genotypes very efficiently and generated a high similarity in dendrogram topologies, although some differences were observed. The linkage analysis was conducted based on the segregation of 38 RAPD, 13 ISSR, 19 IRAP and 9 REMAP loci in 96 progeny of an intergeneric cross Citrus sinensis and Poncirus trifoliata. Among the 81 studied loci 65 loci distributed on five linkage groups. Comparing the result obtained with RAPD, ISSR, IRAP and REMAP markers in this study, IRAP and REMAP proved to be as a reliable molecular marker for fingerprinting, mapping and diversity study of Citrus and its relatives.     

Wide genetic diversity of Rosa damascena Mill. germplasm in Iran as revealed by RAPD analysis

The genetic relationships among 41 Rosa damascena accessions from various cultivation areas of Iran and one accession from Bulgaria were analyzed using 31 RAPD (Randomly Amplified Polymorphic DNA) primers. Each primer exhibited 3–12 banding patterns for a total of 343 scorable and 184 polymorphic bands. The combination of 11 primers was found optimal for discrimination of 42 accessions with very low values of cumulative confusion probability (9.7 × 10⁻⁵); indicating that only one pair from over 10,000 distinct pairs of accessions would be indistinguishable. Unweighted pair group method cluster analysis based on similarity values revealed 10 groups at the distance of 0.85. The Bulgarian genotype grouped with the majority of the Iranian genotypes in a main cluster. Results of molecular variance analysis (AMOVA) indicated that the major proportion (65.7%) of the total genetic variation was within collecting provinces rather than between them. The wide genetic variation seen for R. damascena in Iran indicates that Iran is a center of genetic diversity for this species and that there is a promising future for the breeding.     

杨桃遗传多样性的形态特征与RAPD标记的相关性分析

【目的】为指导杨桃种质资源的引进以及良种选育提供科学依据,【方法】采用形态标记数量聚类分析和RAPD分子标记相结合,对广东地区10份杨桃品种资源进行遗传多样性分析,并将形态学聚类和RAPD分子标记聚类结果加以对比。【结果】在所观察的12个形态性状中,变异系数为14.08%~49.71%,平均变异系数为25.38%。从100条RAPD随机引物中筛选出10个引物,共扩增出58条带,其中53条为多态性带,多态率达93.02%。聚类分析结果表明,形态标记和RAPD标记都可依果实风味将供试材料分组,即甜味和酸味。2种标记方法的相关系数为r0.01=0.685 3,达到显著水平。【结论】杨桃具有丰富的遗传多样性,2种方法聚类结果相似,且相关性高,具有较高的可靠性。     

Genetic diversity and population's structure in Tunisian strawberry tree (Arbutus unedo L.)

A growing interest in the use of the Strawberry tree (Arbutus unedo L., Ericaceae) has been recently reported for industrial, pharmaceutical and chemical fields. However, the bulk material comes from natural populations because of the lack of selection of interesting cultivars. In Tunisia, A. unedo populations are severely destroyed due to deforestation and over-collecting. The species occurs in small scattered populations decreasing progressively in size. Yet, no conservation or improvement programs are attempted to preserve and promote the potential value of this resource. In this work, we assessed the genetic diversity of nine Tunisian populations of A. unedo L. from different bioclimates, using 65 polymorphic RAPD loci. The analysis of the genetic variation within and among populations is primordial to elaborate conservation and improvement programs. A low genetic diversity within a population estimated by both Nei's (He) and Shannon's diversity (H′) indices (0.155 < He < 0.248; 0.229 < H′ < 0.364) was observed due to genetic drift and selfing. At the species level, the amount of the within population variation estimated by Shannon's index (H_POP/H_SP = 0.686) and the molecular variance (80.67%) was higher than that among populations. A moderate genetic differentiation among populations (Φ_ST = 0.193 and G_ST = 0.314) which could be attributed to the long seed distance dispersal was detected. The UPGMA dendrogram based on Φ_ST values showed three clusters each including populations without relationship to bioclimatic or geographical origin indicating that differentiation occurs at a local space scale. The in situ protection measures should be made appropriately according to a population within bioclimates. The ex situ conservation and the selection of genotypes should involve extensive collection of seeds or cuttings from the within populations rather than among them.     

Comparative analysis of genetic diversity in Indian bitter gourd (Momordica charantia L.) using RAPD and ISSR markers for developing crop improvement strategies

A genetic analysis of 38 diverse Indian bitter gourd (Momordicacharantia var. charantia, and var. muricata) accessions was performed using 29 RAPD and 15 ISSR markers. RAPD primers yielded 208 amplicons of which 76 (36.5%) were polymorphic providing an average of 2.6 amplicons per primer. RAPD amplicons per primer ranged from 3 (OPE-19, OPW-09) to 15 (OPW-05), and varied in size from 200 bp to 3000 bp. Fifteen ISSR primers provided a total of 125 bands of which 94 (74.7%) were polymorphic. Polymorphic ISSR markers ranged from 0 (UBC-841) to 12 (UBC-890) providing a mean of 6.3 amplicons per primer that ranged in size from 150 bp to 2700 bp. Nevertheless, the concordance among bitter gourd accession groupings after cluster analysis was relatively high (r = 0.77), indicating that RAPD- and ISSR-based diversity assessments in this germplasm array were generally consistent. The M.charantia var. charantia (domesticated) and var. muricata (wild, free-living) accessions examined were genetically distinct, and these differences provided for the development of strategies for genetic analyses and crop improvement in this species.     

Detection of genetic diversity among populations of sweet cherry (Prunus avium L.) by AFLPs

Summary

The high degree of polymorphism of AFLPs provides an efficient system for identification and genome analysis of sweet cherry (Prunus avium) cultivars and selections. The cultivars of sweet cherry have usually been characterized by assessment of phenotypic and pomological traits. AFLP markers were employed to identify 38 sweet cherry accessions and estimate the genetic diversity among this material. Ten of 18 tested primer combinations were informative with up to 80 bands per primer combination. Seven to 33% of the amplfied bands were polymorphic depending upon primer combination. Allcultivars and selections tested could be clearly identified. The objective of this work was to demonstrate the usefulness of molecular markers in revealing the genetic diversity among different sweet cherry genotypes.     

Molecular characterization and genetic relationship among almond cultivars assessed by RAPD and SSR markers

RAPDs and SSRs were used to study the genetic diversity of Iranian almond cultivars and their relationship to important foreign cultivars and three related species. Eight unidentified almond Shahrodi cultivars and three wild almonds (Prunus communis, Prunus orientalis and Prunus scoparia) were also included. Of the primers tested, 42 (out of 80) RAPD and 18 (out of 26) SSR primers were selected for their reproducibility and high polymorphism. A total of 664 polymorphic RAPD bands were detected out of 729 bands. The number of presumed alleles revealed by the SSR analysis ranged from 3 to 10 alleles per locus with a mean value of 6.64 alleles per locus. Both techniques discriminated the genotypes very effectively, but only RAPDs were able to discriminate the cultivars Monagha and Sefied. Results demonstrated an extensive genetic variability within the tested cultivars as well as the value of SSR markers developed in peach for characterization of almond and related species of Prunus. Dice similarity coefficient was calculated for all pair wise comparisons and was used to construct a UPGMA dendrogram. For both markers a high similarity in dendrogram topologies was obtained although some differences were observed. All dendrograms, including that obtained by the combined use of both the marker data, depicted the phenetic relationships among the cultivars and species, depending upon their geographic region and/or pedigree information. Almond cultivars clustered with accession of P. communis showing their close relationship. P. orientalis and P. scoparia were clustered out of the rest of P. dulcis.