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.     

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%).     

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.     

Genetic diversity of ash gourd [Benincasa hispida (Thunb.) Cogn.] inbred lines based on RAPD and ISSR markers and their hybrid performance

Ten inbred lines of ash gourd [Benincasa hispida (Thunb.) Cogn.] were crossed to produce 45 F₁ hybrids (without reciprocal) which were evaluated along with the parents for 20 growth- and yield-related traits, in a replicated field trial. High level of heterosis was observed among the hybrids for most of the traits examined, including yield. These inbred lines were analysed by using 42 RAPD primers those produced 282 DNA marker bands. A total of 130 RAPD markers were obtained with a mean of 3.1 per primer, which in combination discriminated all the inbreds from each other. Pair-wise genetic distance measurements ranged from 0.07 to 0.31, suggesting a wide genetic diversity for these inbreds. These inberds were also analysed with five ISSR primers of which four were informative. Twenty-six ISSR marker bands were generated of which 11 were polymorphic with an average of 2.80 per primers. The percentage of polymorphic bands produced were higher in ISSR markers (>80%) than generated through RAPD markers (46%). Although the results indicated significant positive correlations of genetic distance with hybrid performance and heterosis, the RAPD based genetic distance measures and use of limited ISSR markers in this present study could not effectively predict hybrid performance in this crop. The genetic variation among ash gourd inbred lines examined, herein, defined a marker array (combined ISSR and RAPD) for the development of a standard reference for further genetic analyses, and the selection of potential parents for predicting hybrid performance and heterosis.     

Genetic diversity of Dianthus accessions as assessed using two molecular marker systems (SRAPs and ISSRs) and morphological traits

Dianthus chinensis, Dianthus barbatus and Dianthus superbus are members of the Caryophyllaceae and are grown widely as ornamental plants. Information about relative genetic relationships can facilitate breeding programs. Here, we have compared two polymerase chain reaction (PCR)-based systems (sequence-related amplified polymorphisms (SRAPs) and inter-simple sequence repeats (ISSRs)) and morpological trait measurements for their relative effectiveness in estimating the genetic diversity found between 22 Chinese pink (D. chinensis) inbred-lines, one accession of D. barbatus and one accession of D. superbus. Interspecific differences were readily detected but the markers were less reliable in distinguishing the accessions according to their region of origin or in separating the wild species from the cultivars. Morphological traits were found to be the least effective genetic markers. The relative effectiveness of the three systems as markers for genetic diversity was concluded to be SRAP > ISSR > morphological traits, but the combined data from ISSR + SRAP analyses was superior to all three. The information generated by the SRAP marker system correlated more closely with morphological variability than did the ISSR marker system. The morphological markers of plant height/crown size ratio, lower leaf length, ovary shape index and calyx length showed strong correlations with the genetic diversity index (GD_ij, PPB(II) and PSB) as generated by the percentage of polymorphic bands and percentage of special bands of the PCR-based markers.     

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.     

丝瓜种质资源遗传多样性的形态和RAPD标记分析

应用形态标记和RAPD标记对26份丝瓜种质材料进行遗传多样性和亲缘关系分析。所观察的44个形态性状变异系数为5.27 %～107.00 %,平均变异系数为32.75 %。从200个随机引物中筛选出16个引物,共扩增出145条带,多态性带有125条,多态性比率为86.21 %,平均Shannon多样性信息指数为 0.325。基于形态标记的聚类分析将26份丝瓜种质分为普通丝瓜和有棱丝瓜两大类。基于RAPD标记的聚类分析将26份丝瓜种质也分为两大类,但有2份有棱丝瓜种质和普通丝瓜聚为了一类,与形态标记聚类结果不一致。     

Genetic variability in wild vs. cultivated Eruca vesicaria populations as assessed by morphological,agronomical and molecular analyses

In this study, the genetic diversity of 50 individuals of rocket, Eruca vesicaria, from five accessions, four of them wild type collected from different parts of Spain and one commercial, were evaluated using morphological, agronomical and inter simple sequence repeat DNA (ISSR) data. Molecular analysis was carried out using the ISSR technique with 20 primers. Out of these 20 primers, nine were polymorphic, producing a total of 395 DNA bands, 247 of which were polymorphic among the accessions. A dendrogram drawn on the basis of a similarity matrix using the UPGMA algorithm revealed that the 50 samples of rocket plants could be classified into three major clusters at a Nei's genetic distance of 0.36. The experiment shows that molecular markers such as ISSR are a good instrument for distinguishing and selecting rocket accessions to group different wild populations. In general, a high variation was observed for most of the 16 morphological and 6 agronomical traits showing significant differences. Some morphological traits such as leaf length, petiole length and lamina width explained 69.1% of the whole variation observed in the populations, and some agronomical traits such as leaf area, nitrate and chlorophyll contents accounted for 65.7%, but the clusters generated by means of agronomical and morphological variables were less evident than when ISSR markers used. Some accessions showed good qualities, such as small leaves, high chlorophyll content, late-flowering or low nitrate content. All these parameters, together with the high degree of genetic homogeneity found, could make the local accessions good candidates for a future breeding programme.     

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.     

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。     

Variety identification and comparative analysis of genetic diversity in yardlong bean (Vigna unguiculata spp. sesquipedalis) using morphological characters,SSR and ISSR analysis

Genetic diversity and relatedness of 23 yardlong bean (Vigna unguiculata spp. sesquipedalis) accessions and 7 accessions of a hybrid between cowpea (V. unguiculata spp. unguiculata) and yardlong bean (dwarf yardlong bean) in Thailand were estimated using morphological characters, simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers. In addition, two mungbean (Vignaradiata (L.) Wilczek) and two blackgram (Vigna mungo (L.) Hepper) accessions were also used as outgroup species for molecular analysis. Five morphological characters were diverse among most accessions. However, five groups of 2–3 accessions could not be distinguished from one another based on these morphological characters alone. Unweighted pair-group arithmetic average (UPGMA) analysis of these characters separated these 30 accessions into 2 major groups; the yardlong bean group and the dwarf yardlong bean group. Eleven of the sixteen SSR primers yielded clear SSRs, ten of which were polymorphic (90.91% polymorphism), detecting a total of 54 alleles with an average of 4.91 alleles per locus. These 10 polymorphic SSR markers successfully distinguished 28 yardlong bean and dwarf yardlong bean accessions. The polymorphic information content (PIC) among genotypes varied from 0.251 to 0.752 with an average of 0.597. Among the 16 ISSR primers used, a total of 312 ISSR fragments were amplified for these three Vigna species, revealing the polymorphism percentage of 91.03%. The average ISSR PIC value (0.197) with the range of 0.137–0.276 was lower than that of SSR. Nevertheless, the average marker index of this multilocus marker was 3.495, which was higher than that of SSR (0.669), owing to the differences in the effective multiplex ratio. In addition, Mantel test cophenetic correlation coefficient was higher for ISSR (0.566) than that of SSR (0.198). These results indicated higher efficiency of ISSR for estimating the levels of genetic diversity and relationships among yardlong beans and dwarf yardlong beans in this study. Pair-wise coefficients of SSR- and ISSR-based genetic similarity among all yardlong bean and dwarf yardlong bean accessions averaged 0.87 and 0.91, respectively, suggesting a narrow genetic base that emphasizes the need to broaden genetic diversity to ensure continued breeding success. Clustering of genotypes within groups was not similar when SSR and ISSR derived dendrograms from UPGMA analysis were compared. It appeared that ISSR was the most effective marker system in determining the genetic variability and relationships among yardlong bean and dwarf yardlong bean accessions and differentiating three Vigna species. In addition, ISSR was also most useful for variety identification since all 30 yardlong beans and dwarf yardlong bean accessions can be effectively distinguished by only four ISSR primers with the highest PIC values.     

Evaluation of genetic diversity among some Iranian wild asparagus populations using morphological characteristics and RAPD markers

An evaluation of genetic diversity in 39 wild asparagus populations was carried out using morphological and RAPD markers. A combination of morphological traits and random RAPD primers was used to examine the level of genetic variation and polymorphisms among the populations. A factor analysis using Ward's method on mean values of morphological characteristics indicated seven main factors resulting in four groups. Analysis of polymorphic bands using Jaccard's similarity coefficient indicated that genetic similarity ranged between 0.71 and 0.29. At a similarity level of 0.64, the populations were divided in three sub-clusters, containing 34, four and one populations, respectively. Significant regression associations were found between 21 morphological characteristics and 18 RAPD markers, revealing some informative markers associated with some traits. The highest R² was related to 18 RAPD markers associated with gender (53.5%) that among them BA-04₂₀₀₀ had a maximum R². The results showed that Iranian wild asparagus with its high levels of genetic variation could be considered as a valuable gene pool for future asparagus breeding programs. Furthermore, it could be inferred that morphological characteristics and RAPD markers are suitable tools to discriminate asparagus populations for the evaluation of genetic diversity.     

十一份不同地理居群野菊的ISSR分析

对11份野菊材料分别用ISSR分子标记和形态学特征进行了遗传分析。用13个引物共得到176个位点,其中166个为多态性位点,多态性比率(PPB)高达94.32%;结果表明:2种分类方法得到的结果不甚一致。说明了野菊是一个多型性的种,在形态上表现出体态、叶型、叶序、伞房花序式样以及茎叶毛被性等诸特征上的极大的多样性。从ISSR标记分析,湖北神农架野菊和安徽天堂寨野菊之间的遗传系数最大,为0.727;安徽天柱山野菊和南京野菊之间的遗传系数最小,为0.591。     

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.     

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.     

Association analysis of fruit traits in mulberry species (Morus L.)

Improvement of fruit traits is an important objective in current mulberry breeding programs. In this study, 93 mulberry accessions of diverse origin were genotyped using 15 ISSR markers to identify marker–trait associations with fruit traits. Fifteen ISSR primers generated a total of 104 amplification products, of which 94 were polymorphic, revealing 90.38% polymorphism; the mean PIC value was 0.2698. UPGMA cluster analysis showed clear genetic relationships between the 93 mulberry cultivars, and the major clusters were related to known pedigree relationships and their ecotype. The mean r² value for all intra-chromosomal loci pairs was 0.0210. Marker–trait associations were investigated using the unified mixed-model approach, considering both population structure (Q) and kinship (K). In total, 24 marker–trait associations (p < 0.01) were identified using different ISSR markers. The results suggest that association mapping in mulberry is a viable alternative to quantitative trait loci mapping, and detection of associations between markers and mulberry fruit traits will also provide important information for marker-assisted breeding.     

A preliminary genetic linkage map of chrysanthemum (Chrysanthemum morifolium) cultivars using RAPD,ISSR and AFLP markers

A genetic linkage map of chrysanthemum (Chrysanthemum morifolium) was constructed by genotyping 142 F₁ progeny of the bi-parental cross ‘Yuhualuoying’ × ‘Aoyunhanxiao’ with a combination of RAPD, ISSR and AFLP markers in a double pseudo-testcross mapping strategy. A total of 567 polymorphic markers, including 153 RAPDs, 61 ISSRs and 353 AFLPs, were used in linkage mapping. 336 of 567 (60%) markers were grouped on the two parental maps, leaving 231 (40%) markers unlinked. In the ‘Yuhualuoying’ linkage map, 210 markers including 116 testcross and 94 intercross markers were placed in 12 major and 32 minor (8 triplets and 24 doublets) linkage groups, covering 1034 cM with an average map distance of 6.2 cM between adjacent markers. In ‘Aoyunhanxiao’ linkage map, 190 markers consisting of 113 testcross and 77 intercross markers were resolved into 9 major and 24 minor linkage groups, with genome coverage of 1095 cM and a mean inter-marker separation of 6.9 cM between adjacent markers. Six pairs of homologous linkage groups were established on the basis of 64 intercross markers shared by the two parental maps. The maps lay a foundation for further quantitative traits loci (QTL) mapping and marker-assisted breeding of chrysanthemum.     

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.     

Use of ISSR markers to assess genetic diversity of African edible seeded Citrullus lanatus landraces

We used the twenty primers to evaluate the genetic variability of 80 individuals belonging to four accessions of edible seeded Citrullus lanatus originated from Côte d’Ivoire. Edible seeded C. lanatus, named “egusi” or “pistachio”, had a great importance in nutrition in West Africa. Nevertheless, due to its neglected status no study to our knowledge has been devoted to its genetic variability using DNA markers. The twenty ISSR primers generated 258 bands among which 252 were polymorphic (97.67%). On the whole, the bands generated revealed three types of profile sharply distinct from each other with minor differences within each type. One profile (P1) was most frequent with 65 individuals. Three accessions (NI084, NI127 and NI145) generated the three types of profile and had medium values of genetic diversity (GD = 0.246–0.275, respectively). On the opposite, the accession NI076 only contained individuals of the most represented type of profile (P1) and had the lowest genetic diversity (GD = 0.055 ± 0.017). The pairwise genetic distance between the 80 individuals varied from 0 to 0.61. The Factorial Component Analysis and the dendrogram clearly separated the 80 individuals into three clusters corresponding to the three types of profile. The results showed that clusters were well separated from each other whereas accessions were not. Our results suggest that high number of individuals should be taken into account for sampling missions and conservation strategies because accessions were not well differentiated from each other. Local agricultural practices consisting of frequent seeds exchanges between farmers and the conservation of harvested seeds for next year culture could be one explanation.