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.     

Characterization of pea accessions by SRAP's markers

Sequence-related amplified polymorphism (SRAP) and morphological markers to estimate the genetic relations among forty pea varieties (Pisum sativum L.) were studied. Data on 15 morphological traits were collected and analyzed. A total of 162 polymorphic SRAP's bands were scored using seven combinations of primers. Cluster analysis and both principal component and principal coordinate analysis were carried out. The varieties were grouped in four clusters through procrustes generalized analysis. Relationships among varieties revealed by molecular markers were significantly correlated with those based on the agronomic traits, suggesting that the two systems give similar estimates of genetic relations among the varieties. Parents selection depend on the specific objectives in further breeding programs.     

Genetic divergence among gerbera accessions evaluated by RAPD

Genetic diversity was evaluated by RAPD markers and morpho-agronomic characters for a total of 42 accessions of Barberton daisy (Gerbera jamesonii) consisting of 29 commercial and 13 wild accessions. A total of 74 polymorphic bands were obtained employing a set of 12 primer pairs. The average genetic similarity coefficient for the 42 accessions, evaluated by Jaccard index was 0.55 ranging from 0.28 to 1.00. The genetic structure found among Barberton daisy accessions was evaluated by hierarchic classification analyses and UPGMA modeling, revealing six clusters of genotypes where two of them include the wild accessions and the remaining four including commercial material, except for wild genotype number 9. Shannon (H′) index was calculated using the molecular markers to investigate the genetic variation among the Gerbera accessions and showed higher values for the commercial cluster in comparison to the values obtained for the individuals from the non-commercial cluster, namely 0.34 versus 0.27, respectively. Therefore, both calculated indices (Jaccard and Shannon) indicated the presence of higher genetic variation among commercial accessions in comparison to the cluster representing non-commercial accessions, suggesting that genetic breeding programs may focus on commercial accessions to recombine interesting genotypes with commercially important and marketing-desired characteristics.     

RAPD and ITS-based variability revealed in Atriplex species introduced to semi-arid zones of Morocco

Variability related to RAPD markers and sequences of nrDNA ITS region has been studied in seven species of the genus Atriplex (A. amnicola, A. canescens, A. halimus from Morocco (MAR) and from USA, A. lentiformis, A. nummularia, A. semibaccata and A. undulate). As a whole, the results show a high variability among the species. The biggest diversity was obtained by RAPD data, followed by ITSs. According to RAPD markers, two major groups can be distinguished, one formed by A. semibaccata and A. undulata, the least similar to the rest of species. A. lentiformis was closer to A. halimus. Phylogenetic analysis confirmed the divergence of A. semibaccata from all species and the closeness of A. lentiformis to A. halimus. The intraspecies variability was also high, as 39 individual RAPD haplotypes were obtained by analyzing 40 plants. Genetic diversity was bigger among species (60.23%) than among individuals (39.77%). The amplification of ITS region leads to three well-defined clades. The heterogeneous and larger clade includes all A. amnicola individuals and some individuals from A. nummularia, A. lentiformis, A. halimus USA, A. halilmus MAR and A. undulata. Both RAPDs and ITS analyses revealed with some exceptions, that all individuals from one species grouped together. RAPDS turn out to be more appropriate than ITS to differentiate Atriplex species. The two markers gave rise to the same species relationships, but to a different structure of the Atriplex genetic diversity. AMOVA analysis estimated interspecies differences for 11.20% of the total ITS variation.     

Morphological and molecular analyses of Rosa damascena × R. bourboniana interspecific hybrids

Rosa damascena Mill is the most important scented rose species cultivated for rose oil production. Rosa bourboniana L. (Edward rose), a related species, is popular on account of its longer blooming period and ease of propagation. With an aim to combine the oil quality of R. damascena and recurrent flowering habit of R. bourboniana, two cultivars (Jwala and Himroz) of R. damascena were crossed with R. bourboniana. The F1 hybrids obtained were evaluated using morphological, random amplified polymorphic DNA (RAPD) and microsatellite (SSR) markers. Twenty-two selected RAPD and three SSR primer pairs were utilized for hybrid identification. According to presence or absence of bands RAPD and SSR markers were classified into seven types of markers. The bands specific for the pollen parent and occurring in the hybrids were good markers to confirm the hybridity. The non-parental bands expressing uniquely in hybrids were effective in distinguishing the hybrids from each other. Cluster analysis, based on Jaccard's similarity coefficient using unweighted pair group method based on arithmetic mean (UPGMA), reliably discriminated the hybrids into two main clusters. These results indicate the practical usefulness of RAPD and SSR markers in hybrid identification in scented roses. The approach is advantageous for its rapidity and simplicity, for identification of hybrids at the juvenile stage. One of the studied morphological traits – prickle density, can also complement in the identification of interspecific hybrids between R. damscena (♀) and R. bourboniana (♂).     

Genetic diversity and classification of Nelumbo germplasm of different origins by RAPD and ISSR analysis

In this study, RAPD and ISSR markers were used to investigate the genetic diversity and genetic relationships among different germplasm of Nelumbo including 70 Chinese ornamental cultivars, 7 wild Thai genotypes, 2 Nelumbo lutea genotypes and 8 hybrids of Nelumbo nucifera and N. lutea. High genetic diversities of 96.4% and 91.2% respectively were detected in the Nelumbo accessions using RAPD and ISSR markers. A dendrogram based on both RAPD and ISSR clustering data indicated that: (1) the genotypes of N. nucifera and N. lutea from different geographical origins were clustered into different groups. This indicated significant genetic differentiation attributed to extensive periods of geographical isolation and lack of gene exchange; (2) the Thai wild genotypes were separated from Chinese genotypes. This indicated genetic divergence between germplasm from Southeast Asia and that from China. Geographical location appears to have affected genetic diversity due to adaptation of the plants to the different environments. A new Southeastern Asia Lotus category is suggested as an addition to the current lotus cultivars classification system; (3) data on three morphological traits (namely: plant size, petal shape and flower color), showed that only the data on plant size was consistent with the dendrogram constructed from molecular data. This finding suggests that using data on genetic relationships in combination with morphological characteristics would serve to improve the classification system of lotus cultivars currently in use. The finding of previously unknown germplasm in this study indicated the potential of RAPD and ISSR techniques in identifying and managing lotus resources. Both marker techniques are potentially useful in improving the current strategies in breeding and germplasm conservation to enhance the ornamental and economic value of lotus.     

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.     

Comparison of the use of morphological,protein and DNA markers in the genetic characterization of Iranian wild Prunus species

In this study, the genetic diversity of four Iranian wild Prunus species including Prunus eleagnifolia, Prunus hauskenchtii, Prunus scoparia and Prunus lycioides were investigated using morphological, protein and DNA markers. DNA markers included nuclear and chloroplast SSRs and self-incompatibility (S) allele amplification. At the morphological level, leaf width showed significant differences between the four wild Prunus species. Concerning fruit and kernel characters, their values are relatively similar indicating the high degree of homoplasy described in Prunus. Nuclear SSR markers have been the most abundant markers with a higher polymorphism in comparison with morphological, protein and chloroplast SSR markers. Results also indicated the high variability present in the S locus. On the other hand, the correlation between the clustering based on DNA markers and protein were in general low. Dendogram performed using nuclear and chloroplast SSR indicated a more diffuse clustering between the wild almond species probably due to the natural introgression of genes observed in these wild almond species. Data from the analysis of the total protein seems to be more accurate to establish taxonomy relationships in these very close wild species.     

Genetic diversity and phylogenetic relationships among and within species of oriental cymbidiums based on RAPD analysis

The classifications of oriental cymbidiums that are native to Korea, China, Taiwan and Japan were examined by molecular analysis. A total of 21 cymbidiums, 15 species including three Cymbidium gyokuchin, 4 Cymbidium kanran and 2 Cymbidium goeringii cultivars, were analyzed by using randomly amplified polymorphic DNA (RAPD) to determine the interspecific and intraspecific relationships. Twenty-two primers were used in the RAPD analysis to distinguish, by comparing differences in DNA banding patterns, all species and cultivars. Similarity values ranged from 0.501 for Cymbidium aloifoilum and C. kanran to 0.935 for Cymbidium ensifolium and Cymbidium marginatum with analysis of total bands score. Phylogenetic tree analysis of the RAPD results from the 21 cymbidiums identified specific groupings. The cymbidiums could be divided into two clusters based upon ecological traits. One trait was temperate zone preference, with each cymbidium preferring either an Asian or subtropical temperate zone. The group that comprised the subtropical cymbidiums was C. aloifoilum, Cymbidium insigne and Cymbidium lowianum. Additionally, we found that Cymbidium lancifolium and Cymbidium aspidistrifolium could be separated based on different flowering physiology and unique leaf form. The groups identified by morphological, physiological and ecological characteristics were in full agreement with those determined by RAPD analysis. The phylogenetic tree derived from the RAPD results was similar to that of the traditional classification. The data acquired from this study could be used for identification and classification of other orchid genera and oriental cymbidium.     

Genetic relatedness (diversity) and cultivar identification by randomly amplified polymorphic DNA (RAPD) markers in teasle gourd (Momordica dioica Roxb.)

Genetic relationship and variation of 29 accessions of teasle gourd (Momordica dioica Roxb.) and 1 accession of Momordica cochinchinensis Spreng. (wild relatives of teasle gourd) were examined by RAPD analysis using 44 dodecamer oligonucleotide primers. A total of 496 fragments were produced by 44 primers of which 95% bands were polymorphic. Using presence or absence of specific RAPD markers or combination of primers, 23 out of 30 accessions were identified. The genetic relatedness or genetic distance based on Nei and Li's genetic similarity varied from 0.86 to 0.65 with an average of 0.74 among 29 M. dioica accessions (when M. cochinchinensis excluded). In the phenetic dendrogram developed from cluster analysis using UPGMA method, M. cochinchinensis was out grouped as single accession, while others showing relatively weak grouping formed four groups. Clustering pattern did not demonstrate any relationship between geographical origin and genetic diversity. A DNA extraction method has been standardized. This is the first report of using RAPD techniques in teasle gourd. It was concluded that RAPD analysis is a useful tool for genotypic identification and estimation of genetic similarity in teasle gourd.     

Analysis of Genetic Relationships Between Wild Roses (<Emphasis Type="Italic">Rosa</Emphasis> L. Spp.) Growing in Turkey

Rosa L. taxa are dominantly used for horticulture, food, medicinal, ornamental and aesthetic purposes. Rosa is represented with approximately 200 wild species in the world. The number of natural Rosa species in Turkey is reported as about 31. Rosa species are widely distributed in all regions in Turkey. In this study, RAPD and ISSR markers were applied to assess genetic diversity and genetic relationships among 27 species of Rosa. The UPGMA cluster was constructed using a combination of data from RAPD and ISSR markers. The dendrogram revealed two main clusters. Each cluster was divided into subgroups. This investigation showed that genetic distance was relatively significant among the species. As the results of the study close genetic relationships were found between evolutionary relations, morphplogical properties, and phytogeographical distributions of the Rosa species, The results also propose that RAPD and ISSR markers are useful tools for indicating genetic relationships among Rosa genotypes.     

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 in Portuguese Ceratonia siliqua L. cultivars using RAPD and AFLP markers

Although carob (Ceratonia siliqua L.) is of great economic importance little is still known about the pattern of genetic variation within this species. Morphological characteristics based on 31 fruit and seeds of continuous characters determinant for agro-industrial uses, were compared with RAPD and AFLP markers for assessing genetic distances in 68 accessions of carob trees, from different cultivars, varieties and eco-geographic regions of Algarve. Eighteen selected RAPD primers applied to the 68 accessions produced a total of 235 fragments ranging from 200 to 2000 bp, of which 93 (40%) were polymorphic. Four AFLP selective primer combinations generated a total of 346 amplification fragments of which 110 were polymorphic. The average level of polymorphism based on four primer combinations was 31.8%. The phenetic trees based on RAPD and AFLP analyses gave high co-phenetic correlation values, and were found to be consistent in general with the analysis of morphological data, carried out on the same accessions. A number of RAPD and AFLP markers were found to be diagnostic for ‘Canela’ cultivar and 13 wild ungrafted trees.     

Application of RAPD and SI-typing techniques to confirm genetic integrity of radish (Raphanus sativus L.) varieties

The random amplified polymorphic DNA (RAPD) and self-incompatibility (SI) typing techniques were applied to settle a lawsuit between a Seed Company and a farmer concerning seed impurity in radish (Raphanus sativus L.). After sowing “Halra Woldong” (HW) a winter radish variety, two morphologically different radish plants grew. Genomic DNA from each of the two types were prepared and analyzed using ninety-six RAPD markers and four variety-specific markers were found. The polymorphic RAPD band patterns were compared with those of the twenty Korean radish varieties. The genetic integrity of the two varieties in question was identified and further confirmed by SI-typing of S-locus glycoprotein (SLG) and S-locus receptor kinase (SRK) genes.     

Genetic diversity analysis of ginger (Zingiber officinale Rosc.) germplasm based on RAPD and ISSR markers

A global collection of ginger germplasm consisting of 46 accessions were characterized using two types of molecular markers, RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats). UPGMA dendrograms constructed based on three similarity coefficients, i.e., Jaccard's, Sorensen–Dice and Simple Matching using the combined RAPD and ISSR markers placed the accessions in four similar clusters in all the three dendrograms revealing the congruence of clustering patterns among the similarity coefficients and a rather less genetic distance among the accessions. Improved varieties/cultivars are grouped together with primitive types. Moreover, in the clustering pattern of the accessions, a geographical bias was also evident implying that germplasm collected from nearby locations especially with vernacular identity may not be genetically distinct. The clustering of the accessions was largely independent of its agronomic features.     

Genetic relationships among nine cultivated taxa of Calliandra Benth. (Leguminosae: Mimosoideae) using random amplified polymorphic DNA (RAPD) markers

Random amplified polymorphic DNA (RAPD) marker was used to assess genetic diversity and inter-specific relationships among nine taxa of Calliandra (Leguminosae: Mimosoideae) grown in Indian gardens. DNA from leaf sample was isolated and RAPD analysis was performed using 22 primers. The genetic similarities were analyzed from the dendrogram constructed by the RAPD data using a similarity index which supported the segregation of the nine taxa of the genus into two groups; the sect. Androcallis with seven taxa, viz. C. haematocephala, C. haematocephala var. alba, C. surinamensis, C. tweedii, C. tergemina var. emarginata and C. selloi and sect. Calliandra having two species namely, C. inermis and C. calothyrsus. The intra-generic classification and phylogeny inferred from molecular markers supported the traditional classification of the genus based on morphological characters at the level of sections and series except in case of C. selloi (C. brevipes) which did not show much genetic similarity with C. tweedii and C. surinamensis; all the three species being members of the sect. Androcallis series Androcallis.     

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.