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%).     

Characterization of progenies derived from pollination of pomegranate cv. Malase-Tourshe-Saveh using fruit traits and RAPD molecular marker

Fruit traits and molecular markers (RAPD) were used to survey genetic similarities and inheritance pattern of offspring, derived from self- and open pollination of pomegranate cv. Malase-Tourshe-Saveh (MTS) as well as progenies derived from cross between ‘MTS’ and ‘Narm-Daneh’ (ND) a soft-seed cultivar in the present study. Clustering based on fruit traits, divided the accessions firstly into two major groups, sweets and sour or sweet–sours. Members of sub-clusters were different in fruit characters, and seed softness also was affecting the grouping of individuals. Offspring of ‘MTS’ × ’ND’ showed somehow seed softness with varying degrees compared to other individuals. In RAPD analysis, by using 26 polymorphic primers, 325 fragments were produced from 39 individuals, among which 70 reproducible, polymorphic bands were scored for data analysis. NTsys and Bootstrap software were used for RAPD data analysis and respective dendrogram depicted by Jaccard's similarity coefficients using UPGMA method. The similarity coefficients were ranging from 0.2 to 0.94 and the lowest was obtained between pollen parent (ND cv.) and a genotype from open pollination of MTS cultivar. The highest similarity coefficient (0.94) was observed between two offspring both from self-pollination of MTS cultivar. In corresponding dendrogram, pollen parent was separated from other genotypes and laid individually in a sub-cluster, while the mother plant clustered with its progenies. In addition, some marker–trait associations were observed, especially for seed softness.     

Evaluation of genetic diversity among Iranian soft-seed pomegranate accessions by fruit characteristics and RAPD markers

Soft-seedness in pomegranate is a desirable trait for fresh consumption of this valuable fruit. At the main Iran pomegranate collection, 21 pomegranate accessions gathered from different parts of Iran are registered as soft-seed genotypes. The aim of this research was to study these soft-seed pomegranate accessions using fruit morphopomological traits and DNA markers to reveal their relatedness. Thirty-six fruit characteristics were measured in these accessions together with applying 29 random decamer primers already reported to be polymorphic on pomegranate. Factor analysis on mean values of fruit characteristics determined 10 main factors and applied for grouping of the accessions using Ward's method. Also 14 of the random primers showed good amplification and polymorphism on these samples, and a total of 43 RAPD markers were produced. Estimates of genetic similarity, using Jaccard's similarity coefficient, ranged from 0.13 to 1.0 using the RAPD data. Grouping based on the fruit traits compared with that based on RAPD data did not produce a significant correlation (r = −0.36). Morphometric measurements and sensory evaluation confirmed that some accessions are hard or semi-hard seeded. This study showed that information based on fruit characteristics and RAPD markers are complementary for genetic discrimination in soft-seed pomegranate accessions. This might be due to the high level of similarity between soft-seed pomegranate accessions.     

Inter- and intra-specific genetic diversity among cyclamen accessions investigated by RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to characterize genetic diversity of 26 Cyclamen persicum and Cyclamen com accessions. Eighty-four arbitrary primers tested, among which nine primers showed reliable polymorphic banding patterns and yielded 104 polymorphic markers. Jaccard's similarity coefficient among accessions ranged from 0.99 to 0.08. At a similarity of 68%, accessions were divided into three clusters. The cophenetic correlation coefficient between the similarity matrix and the dendrogram was relatively high (r = 0.9) showing the goodness of fit of the dendrogram. The RAPD analysis offered a rapid and reliable tool for the estimation of inter- and intra-specific variability in cyclamens. The wide genetic variation observed for cyclamens within Iran guarantees a promising future of breeding.     

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.     

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.     

中国北方引种的部分越橘品种的RAPD分析

利用RAPD技术对我国北方引种的15份越橘(Vaccinium spp.)栽培品种进行了遗传多样性分析。从70条随机引物中筛选出15条引物,共扩增出94个位点,其中多态性位点83个(88.3%)。材料间遗传相似系数变化为0.5426～0.8936,表明各品种间具有一定的遗传差异。UPGMA聚类分析结果可将供试材料分为2大类、5个亚类。聚类结果与各品种的杂交系谱相对应,从分子水平揭示了品种间的亲缘关系。     

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.     

Genetic Relationship of Iranian Pear Genotypes with European and Asian Pears as Revealed by Random Amplified Polymorphic DNA Markers

Genetic variation within specific fruit tree germplasms is an important tool in fruit tree breeding programs. In the present work, the genetic relationship of 31 European and Iranian (Pyrus communis L.) and Asian (Pyrus serotina Rehd) genotypes of pear were studied using 19 randomly amplified polymorphic DNA (RAPD) primers. Fifteen out of the 19 primers used in this study amplified 3373 clear and reproducible bands associated with 150 loci and many of them were polymorphic. The dendrogram resulting from the unweighted pair-group method of arithmetic cluster analysis separated the cultivars into eight groups. The correlation coefficient between the cophenetic matrix and the similarity matrix was 0.82 (r = 0.82). There was a significant difference between populations and most studied genotypes clustered closely together based on their geographic origin and Iranian pears placed between two groups of pears. Results showed the suitability of RAPD analysis in genetic diversity study of pear.     

山楂（Crataegus pinnatifida Bge.)遗传多样性的RAPD和ISSR标记分析

 利用RAPD和ISSR标记对35份山楂（Crataegus pinnatifida Bge.）资源进行了DNA多态性分析。12个RAPD引物共扩增出110条清晰的谱带,其中89条显示多态性,平均每个引物扩增出7.4条多态性谱带。13个ISSR引物共扩增出110条清晰的谱带,其中94条显示多态性,平均每个引物扩增出7.2条多态性谱带。基于RAPD和ISSR标记,利用UPGMA分别构建了35份山楂资源的聚类树状图。距离系数分别为0～0.62（RAPD）和0～0.64（ISSR）,表明山楂具有较高的遗传多样性。     

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.     

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.     

Genetic relationships of gladiolus cultivars inferred from fluorescence based AFLP markers

Gladiolus is one of the important commercial flowers with a large number of cultivars. However, genetic relationships among its genotypes have not been reported. This study analyzed genetic relatedness of 54 gladiolus cultivars using amplified fragment length polymorphism (AFLP) markers. A total of 24 AFLP primer pairs with three samples were initially screened, from which 9 primer sets that showed clear scorable and highly polymorphic bands were selected for AFLP reactions. Fluorescence-labeled amplification products were subjected to electrophoresis and then analyzed using an automated sequencer. A dendrogram was constructed by the unweighted pair group method using the arithmetic average (UPGMA). The number of AFLP fragments generated per primer set ranged from 10 to 151 with fragment sizes varying from 50 to 450 bp. A total of 660 AFLP fragments were detected, of which 658 (99.70%) were polymorphic. All the primers except E-AGG/M-CTA displayed 100% polymorphism. All cultivars were clearly differentiated by their AFLP profiles. The AFLP data were compared with previously obtained RAPD data and combined to generate a common dendrogram. The first cluster was dominated with indigenously bred cultivars while the second was dominated with exotic cultivars. This shows that most of the exotic cultivars as well as indigenous cultivars are closely related with each other. However, two indigenous cultivars viz., Pusa Suhagin and Pusa Archana share genetic similarity with exotic cultivars. Among the genotypes selected for the investigation, Pusa Gunjan was identified as the most distinct genotype. The AFLP markers developed will help future Gladiolus cultivar identification, germplasm conservation and new cultivar development. The assessed genetic relationships among gladiolus cultivars may enhance the efficiency of breeding program by selecting desirable parents with reduced breeding cycle.     

Genetic diversity analysis in date palm (Phoenix dactylifera L.): a comparative assessment using ISSR and RAPD marker assays

SUMMARY

A comparative study was conducted to evaluate genetic diversity in 45 genotypes of date palm (Phoenix dactylifera L.), including both male and female plants, employing RAPD and ISSR marker systems. The data were analysed to calculate the total number of bands, the number of polymorphic bands, the percentage polymorphism, the average number of bands per primer, the effective multiplex ratio (EMR), the polymorphic information content (PIC), the marker index (MI), and genetic similarity coefficients. The 37 RAPD and 53 ISSR primers used generated 363 and 608 scorable amplified products, respectively, of which 95.0% and 90.9% were polymorphic. The ISSR markers produced more information than the RAPD markers due to their higher EMR and MI values. Jaccard similarity values among male plants, female plants, and between all male and all female plants varied between 0.72 – 0.80. The results indicate the effectiveness of these two marker systems for demonstrating genetic relationships among date palm genotypes.     

杧果种质遗传多样性的RAPD分析

对杧果不同生态型、不同胚性、不同果形与果色的38份品种和1个近缘种—扁桃进行了RAPD分析,19个引物在39份种质中共扩增出223条带,其中多态性带为196条,多态性带的百分率为87.89%,表明品种间存在着广泛的遗传基础。利用UPGMA进行聚类分析,在相似性系数0.755的水平上将38个品种分成3组,该结果与传统上以胚型为依据进行品种类群划分比较吻合。发现了多个与胚性密切相关和1个与果皮颜色密切相关的RAPD标记,并就部分品种的系谱关系、胚性和果皮颜色的遗传进行了探讨。     

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.     

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.     

雷州半岛菠萝蜜种质遗传多样性的RAPD分析

 用RAPD标记方法对我国雷州半岛的65份菠萝蜜(Artocarpus heterophyllus Lam. ) 实生种质资源DNA的遗传多样性进行了检测, 16个引物共检测到78条带, 其中69条具多态性(占88.4% ) 。聚类分析表明, 65份菠萝蜜材料在遗传距离0.26处可分为3大类。供试种质虽具有丰富的形态性状变异, 但在DNA水平上平均相似性系数为0.7341。干、湿胞类型及引自马来西亚的种质均不能独立聚类。对菠萝蜜品种的干、湿胞分类法及菠萝蜜品种的引种区域进行了讨论。     

31份野生树莓资源的RAPD分析

用RAPD技术分析了31份野生树莓资源的亲缘关系,从100个A系列随机引物中选出了15个多态性高、分辨能力强的引物。将这15个引物应用于31份野生树莓材料,共扩增出182条谱带,其中170条为多态性标记(占93.41%),平均每个引物扩增多态性谱带12.13条,表明不同树莓资源基因型间存在着极为丰富的遗传多样性。基于RAPD的扩增结果建立UPGMA聚类分析树状图,31份野生树莓资源在遗传距离0.5100处被划分为4个类群。从分子水平研究了树莓属资源的亲缘关系,为树莓种质资源的保存和利用及其杂交亲本的选配提供了依据。