Comparative analysis of genetic diversity in Prunus L. as revealed by RAPD and SSR markers

RAPD and SSR markers were used for genetic diversity evaluations among 15 genotypes selected from the genus Prunus L. Altogether 40 RAPD primers and 21 primer pairs designated for microsatellite loci were applied on the whole group of genotypes.     

Assessment of genetic diversity among mango (Mangifera indica L.) genotypes using RAPD markers

Knowledge about the extent of genetic diversity/relatedness in mango germplasm is vital for developing coherent strategies for future gains in productivity. The genetic diversity/relatedness among mango cultivars/genotypes developed in Pakistan has not been investigated previously. We have assessed the genetic diversity among 25 mango genotypes/cultivars using randomly amplified polymorphic DNA (RAPD). Sixty random ten-mer primers were surveyed, out of which 45 yielded amplicons in all the genotypes. Genetic similarity between genotypes/cultivars was in the range of 64–89% with an average of 74%. Similarly, the genetic relatedness among all variants derived from a mango cultivar Chaunsa was in the range of 81.18–88.63%. These coefficients were utilized to construct a dendrogram using the unweighted pair group of arithmetic means (UPGMA). The genotypes were grouped into three (A, B, C) clusters. Generally, genotypes originating from Pakistan were grouped in cluster ‘A’ while cluster ‘B’ primarily composed of southern India as well as Florida cultivars. Kensington Pride was the most distantly related genotype which grouped with Maya and Yakta, forming a distinct cluster ‘C’.     

Genetic diversity of bitter gourd (Momordica charantia L.) genotypes revealed by RAPD markers and agronomic traits

Bitter gourd or bitter melon (Momordica charantia L.) is considered as minor cucurbitaceous vegetable in spite of having considerable nutritional and medicinal properties. Although some reports on genetic diversity based on morphological characterization are available, no work has been conducted to estimate genetic diversity using molecular markers in this crop. In the present study, 38 genotypes of M. charantia including few commercially cultivars collected from different parts of India based on agro-ecological zones were analysed for diversity study both at morphological and molecular levels. Genomic DNA was extracted from young healthy leaves following the procedure of Doyle and Doyle [Doyle, J.J., Doyle, J.L., 1990. A rapid DNA isolation procedure from small quantity of fresh leaf material. Phytochem. Bull. 119, 11–15]. Pair-wise comparison of genotypes was calculated as per the procedure of Jaccard [Jaccard, P., 1908. Nouvelles recherches sur la distribution florale. Bull. Soc. Vaud. Sci. Nat. 44, 223–270]. Dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA) and the computation for multivariate analysis was done using the computer programme NTSYS-pc Version 2.0 [Rohlf, F.J., 1998. NTSYS-pc Numerical Taxonomy and Multivariate Analysis System, Version 2.01. Exeter Software, Setauket, NY, USA]. Diversity based on yield related traits and molecular analysis was not in consonance with ecological distribution. Among 116 random decamer primers screened 29 were polymorphic and informative enough to analyse these genotypes. A total of 208 markers generated of which 76 (36.50%) were polymorphic and the number of bands per primer was 7.17 out of them 2.62 were polymorphic. Pair-wise genetic distance (GD) based on molecular analysis ranged from 0.07 to 0.50 suggesting a wide genetic base for the genotypes. The clustering pattern based on yield related traits and molecular variation was different.     

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.     

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.     

Molecular based assessment of genetic diversity within Barbary fig (Opuntia ficus indica (L.) Mill.) in Tunisia

In this work, we report for the first time on the analysis of genetic diversity within a set of 36 Tunisian Opuntia ficus indica (L.) Mill. ecotypes using RAPD markers.     

Using bulked AFLP analysis to assess genetic diversity in Echinacea species

Echinacea is an allogamous genus, thus its cultivars or populations are genetically heterogeneous. Using amplified fragment length polymorphism (AFLP) to estimate the genetic diversity of Echinacea is generally limited by the large number of individual plants and the higher cost that need to be processed. In the present study, effectiveness of several sizes of DNA bulking (10, 15, 20, 25 and 30 individuals) with 20, 36 and 55 primer pairs was compared using AFLP in determining the genetic diversity of Echinacea species. The results indicated that the use of bulked DNA-based AFLP analysis by using the selected eight primer pairs was capable of detecting genetic diversity between the tested Echinacea species, provided that the potential presence of low frequency variants was ignored and a possible bias in the estimates of genetic similarity was accepted. The assessments showed that a bulk of 15 individuals could detect sufficient AFLP variations at most genomic sites. Additionally, 20 primer pairs could generate sufficient polymorphic fragments to achieve high resolving power of AFLP for the tested Echinacea species.     

Genome composition and genetic diversity of Musa germplasm from China revealed by PCR-RFLP and SSR markers

Banana (Musa spp.) is one of the most important crops in the world. In this study, 216 banana accessions, 184 from the National Banana Germplasm Collection of China (NBGCC) and 32 from the International Network for the Improvement of Banana and Plantain (INIBAP), were used to determine the genome composition of banana plants in these collections and to estimate their genetic diversity. The genome composition was examined using PCR-RFLP markers. The molecular data for all but one accession (ITC 1231) from INIBAP were in agreement with the initial records based on phenotypic characteristics. Microsatellite (SSR) markers were used to investigate the genetic variability and relationships among these banana accessions. Ten of the 47 primer pairs tested consistently produced reproducible and discrete fragments. We identified a total of 92 alleles, ranging from 5 to 15 per locus. The genetic similarity between the accessions ranged between 0.1 and 1, when estimated using Jaccard's coefficient. The UPGMA method based on genetic similarities, grouped the NBGCC accessions according to those containing the ‘A’ and ‘B’ genomes. However, this analysis could not separate all the accessions, especially the somatic mutations, using the primers in this study. These data indicated that limited genetic variation exists within these accessions and the collections of NBGCC should include a much wider range of banana plant material.     

Assessment of genetic diversity of Highland bananas from the National Banana Germplasm Collection at Rubona,Rwanda using RAPD markers

The genetic similarities of 49 accessions of bananas from The National Banana Collection at Rubona were investigated using random amplified polymorphic DNA markers. A total of 120 primers were screened for their usefulness in amplifying DNA fragments of four cultivars belonging to the subgroup Mutika–Lujugira. Fifteen random primers were selected for more detailed analysis, on the basis of providing reproducible amplification and revealing a high level of variation between the four cultivars. The genetic similarity was estimated using a simple matching coefficient which showed the lowest value of 0.46 between ‘Ingumba’ and ‘Ishika’ and the highest value of 0.85 between ‘Kirayenda’ and ‘Inyabukuwe’. The data of matrix of coefficient of similarity was subjected to cluster analysis using the unweighted pair group method with arithmetic average (UPGMA). Each accession was clearly separated. The results of this study are important for the curation of the banana germplasm collection in Eastern Central Africa and for future breeding of this crop.     

Assessment of genetic diversity of Latvian and Swedish sweet cherry (Prunus avium L.) genetic resources collections by using SSR (microsatellite) markers

Three previously described highly polymorphic SSR (microsatellite) primer pairs were tested on 126 sweet cherry (Prunus avium L.) accessions to adapt a fast, reliable method for preliminary screening of sweet cherry germplasm collections and to compare two sweet cherry germplasm collections: at the Latvia State Institute of Fruit-Growing, Dobele (LIFG-Dobele) and at the Division of Horticultural Genetics and Plant Breeding at Balsgård, Department of Crop Sciences, Swedish University of Agricultural Sciences (SLU-Balsgård). The SSR loci were highly polymorphic with 4–10 different alleles and 5–18 genotypes. Heterozygosity values ranged from 0.431 to 0.809, gene diversity (PIC) values ranged from 0.400 to 0.753, and the discriminating power of each locus varied from 0.631 to 0.894. The combined discriminating power of all loci was highly effective (0.996). Sixteen identical accession groups with the same allele profile were discovered in both collections. This study demonstrated that SSR fingerprinting with the three primer pairs tested, can be used for preliminary characterization of sweet cherry germplasm collections.     

Evaluation of genetic relationships among Iranian pistachios using microsatellite markers developed from Pistacia khinjuk Stocks

To evaluate the genetic relationships among wild and cultivated Pistacia species grown in Iran and the analysis of genetic variation among Iranian pistachio genotypes, two DNA libraries enriched for dinucleotide (AG)_n and trinucleotide (ATG)_n microsatellite motifs were developed from Pistacia khinjuk genome. Following screening of clones by colony PCR technique, 44 clones were sequenced and 27 pairs of primers designed from flanking regions of the repeats. The examination of primer pairs, designed from P. khinjuk sequences, showed successful cross-species amplification within the genus Pistacia. A dendrogram constructed on the basis of the Minimum Evolution clustering algorithm revealed that Pistacia vera has closer relationships with P. khinjuk, than with Pistacia integerrima, Pistacia palaestina, Pistacia atlantica and Pistacia mutica. The dendrogram further distinguished the wild Sarakhs pistachio from the rest of P. vera genotypes suggesting that the domesticated genotypes of P. vera are evolved from P. vera var. Sarakhs and then this wild genotype likely develops to other local pistachios. Hence, it seems that the wild Sarakhs pistachio plays an important role in evolutionary trend of the edible pistachios in Iran. The results indicated that microsatellites developed in P. khinjuk are distributed in the genome of indigenous pistachio species including P. vera genotypes and therefore they will be useful in characterization of Iranian pistachio genotypes.     

RAPD analysis of genetic diversity among and within Portuguese landraces of common white bean (Phaseolus vulgaris L.)

Seventeen landraces of common beans (Phaseolus vulgaris L.) sampled in the North and Centre of Portugal were analyzed at population level for 689 RAPD loci amplified by using forty random primers. The two different parameters used to estimate the genetic variability in and between samples indicate that the inter-population component of the genetic variability is mainly responsible for the diversity found, since only about a 10% would be of an intra-population nature. In addition, the gametic disequilibrium was estimated and reached an average value of 40% for the different combinations of pairs in the 689 loci studied taking the 17 samples as a whole population. Self-pollination, genetic drift and adaptation would thus be favouring the formation of multilocus associations. In addition, the fingerprinting study suggests that each landrace produced unique amplification products allowing it to be distinguished from the other tested genotypes, but, nevertheless, the landraces show a considerable genetic similarity (56% and 70.5% using two different methods). The Neighbour-Joining dendrogram did not show any relation between the geographical distribution of landraces and genetic distance. The results suggest that these Portuguese landraces conserved an important genetic diversity that can be useful to widen the genetic base of currently cultivated beans.     

Genetic diversity in local Tunisian pears (Pyrus communis L.) studied with SSR markers

Few records are available about local Tunisian pear cultivars characterized by low chilling requirements and adaptation to dry conditions. In this work, seven SSRs derived from apple were successfully transferred to 25 local Tunisian pear genotypes and 6 common varieties of Pyrus communis cultivated in Europe. The 7 SSRs used amplified a total of 36 fragments. All the microsatellites except one seem to amplify more than one locus in some of the genotypes studied. Only 12 different fingerprinting patterns could be distinguished among the 25 Tunisian cultivars studied indicating a high number of synonymies. The mean expected and observed heterozygosities in the 25 Tunisian cultivars analyzed averaged 0.71 indicating a high level of genetic diversity among the local Tunisian pear germplasm. These markers will be useful to optimize the conservation of this highly threatened germplasm.     

Analysis of genetic diversity among wild pomegranates in Western Himalayas,using PCR methods

The genus Punica (Punicaceae) is distributed in Iran, Afghanistan, India and Mediterranean countries. Iran is considered to be its primary center of origin. In India Punica granatum is found in wild only in Western Himalayan regions comprising, Jammu and Kashmir, Himachal Pradesh and Uttarakhand states. However, there is little information available about the genetic variation present in pomegranates in the regions. In this paper we describe the use of DAMD and RAPD methods that generate the profiles, to study genetic diversity in wild genotypes of the P. granatum in India. Forty-nine accessions representing two regions of Western Himalaya were analyzed. Similarity coefficient value varied from 0.08 to 0.79 across different accessions. The results indicate that DAMD (97.08%) revealed more polymorphism in comparison to RAPD (93.72%). The results show that these methods are sufficiently informative to unravel the genetic variations in wild pomegranates in Western Himalayas.     

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 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.     

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 common bean (Phaseolus vulgaris L.) germplasm using amplified fragment length polymorphism (AFLP)

AFLP technique was applied to assess genetic diversity among 44 common bean accessions that included 6 exotic accessions, 15 Indian land races and 23 released varieties. Eight AFLP primer pairs were used that produced 820 products of which 698 were polymorphic (85.12%). Wide variations were observed among all the accessions for the number of amplification products, percent polymorphism and average polymorphism information content (PIC). The Jaccard's similarity indices (J) based on the AFLP profiles were subjected to UPGMA cluster analysis. The dendrogram generated revealed seven major groups. Seventeen out of 23 released varieties were restricted to clusters VI and VII. The value of r = 0.934 in Mantel's test for cophenetic corrlelation applied to the cluster analysis indicated the high fitness of the accessions to a group. The germplasm used in the present study had narrow genetic base, although moderate to high genetic diversity was observed. The details of diversity analysis and the potential use of Indian common bean accessions in common bean breeding programme are provided in the present study.     

Genetic diversity for mineral accumulation in the foliage of Chenopodium spp.

Chenopodium spp. is being used as a leafy vegetable and subsidiary grain crop in different parts of the world due to its rich nutritional quality and its ability to grow in stress conditions. A field experiment was conducted in Lucknow (India) to assess the genetic diversity in 40 accessions of Chenopodium spp. based on mineral composition of the leaves. Principal Component Analysis (PCA) showed that the first 4 PCs (Principal Component) accounted for 74.70% of the total variance among the accessions. The first PC (PC1) accounted for 41.96% of the total qualitative variation and had nickel, zinc and chromium with high positive and copper with high negative coefficients. The most important loadings for PC2 were calcium and potassium. Cluster analysis grouped the accessions into 4 major clusters. The first cluster, which showed maximum diversity, had 17 accessions, all of C. quinoa having high content of most of the heavy metals viz. zinc, chromium, nickel and cadmium. Cluster II was the largest consisting of 18 accessions which had low content of nickel, cadmium and chromium. Cluster III contained 3 accessions that had lowest amount of calcium, iron, magnesium and zinc, while accessions in cluster IV were characterized by high levels of calcium, sodium, magnesium, nickel, chromium and cadmium. Significant genotypic differences existed in the heavy metal uptake by plants. Mineral uptake and concentration in Chenopodium spp. corresponds to the taxonomic categorization in the genus and could also be utilized as a supplementary taxonomic tool for grouping together of closely related taxa. Conclusively the diversity can be exploited in plant breeding programs for increasing nutritional value and for bioremediation purposes.     

Genetic diversity and population structure of an endangered Orchid (Dendrobium loddigesii Rolfe) from China revealed by SRAP markers

Dendrobium loddigesii Rolfe is an endangered perennial herb with ornamental and medicinal value. Due to habitat deterioration and human over-exploitation, it has suffered a significant decline in abundance. Determining the level of genetic diversity and pattern of population genetic structure of this species would be helpful for its conservation and management. In this paper, sequence-related amplified polymorphism (SRAP) markers were applied to assess the level and pattern of genetic diversity in seven populations of D. loddigesii. Seventeen SRAP primer combinations generated a total of 231 clear amplification bands encompassing 187 (80.95%) polymorphic bands. A high level of genetic diversity was detected (PPB = 80.52%, H = 0.2743, I = 0.4113) at the species level. There was a moderate genetic differentiation (G_st = 0.304) among populations. Two main clusters were detected by cluster analysis using the unweighted pair-group method with arithmetic average (UPGMA). Mantel test revealed that no significant positive correlation was found between genetic distances and geographic distances (r = 0.2302; P > 0.05). Recommendations for conservation of the endangered species resources are proposed.