Relationships between an Italian Strawberry Ecotype and its Ancestor using RAPD Markers

Random-amplified polymorphic DNA (RAPD) markers were used to evaluate genetic variability among populations of an Italian strawberry ecotype, and to determinate genetic relationships between genotypes and their putative ancestor. A total of 65 selections and one cultivar ‘Madame Moutot’ (MM), were analysed to evaluate genetic variability present in Etna mountain area and to confirm as MM was one of the cultivars that originated the ecotype. A total of 222 RAPD markers was obtained using 16 decamer primers and 6 longer primers, 90.8% of the markers obtained by selected primers resulted polymorphic at least within analysed genotypes. RAPDs were used to calculate genetic similarity coefficients and to generate dendrograms representing genetic relationships among genotypes analysed. Cluster analysis displays as RAPD polymorphisms were able to characterize the genotype variability among closely related groups. The data show as MM could be considered the ancestral genotypes introduced in that area. The results obtained confirm that RAPD markers could be used as reliable markers to perform phylogenetic studies in Fragaria×ananassa Duch. ex Rozier. Giuseppe Bertino and Piero Spada - Coauthor involved in genotype selection and field management     

Genetic Characterization of Brazilian Annual <Emphasis Type="Italic">Arachis</Emphasis> Species from Sections <Emphasis Type="Italic">Arachis</Emphasis> and <Emphasis Type="Italic">Heteranthae</Emphasis> using RAPD Markers

The genus Arachis is divided into nine taxonomic sections. Section Arachis is composed of annual and perennial species, while section Heteranthae has only annual species. The objective of this study was to investigate the genetic relationships among 15 Brazilian annual accessions from Arachis and Heteranthae using RAPD markers. Twenty-seven primers were tested, of which nine produced unique fingerprintings for all the accessions studied. A total of 88 polymorphic fragments were scored and the number of fragments per primer varied from 6 to 17 with a mean of 9.8. Two specific markers were identified for species with 2n = 18 chromosomes. The phenogram derived from the RAPD data corroborated the morphological classification. The bootstrap analysis divided the genotypes into two significant clusters. The first cluster contained all the section Arachis species, and the accessions within it were grouped based upon the presence or absence of the ‘A’ pair and the number of chromosomes. The second cluster grouped all accessions belonging to section Heteranthae.     

Inter Simple Sequence Repeat (ISSR) Analysis of Diploid Coffee Species and Cultivated Coffea arabica L. from Tanzania

Inter simple sequence repeat (ISSR) markers were used to evaluate levels of genetic similarity among Coffea arabica L. accessions from Tanzania and to estimate levels of genetic similarities in C. arabica and diploid coffee species. The six ISSR primers used generated a total of 82 fragments and the dissimilarity values ranged from 0.21 to 1. Mean dissimilarity values between provenances (0.56–0.85) were higher than within provenances (0.37–0.68). Cluster analysis based on Nei’s genetic distances showed C. arabica provenances grouping based on geographical origin. Two major clusters were formed that constituted of provenances from Kilimanjaro and Arusha in one sub-cluster; Tanga and Morogoro in the other; the second cluster had Mbeya provenances and diploid species, respectively. The implication is that Mbeya provenances are different from the rest of Tanzanian C. arabica. A principal coordinate analysis (PCA), whose first three coordinates explained 43% of the variation, showed similar groupings as in the cluster analysis. A separate cluster analysis of diploid species showed a distinct separation of the three species used. ISSR data gave results similar to previous findings from random amplified polymorphic DNA(RAPD) analysis. The results also confirm the limited diversity present in cultivated C. arabica in Tanzania     

Comparative analysis of genetic diversity using molecular and morphometric markers in Andrographis paniculata (Burm. f.) Nees

Andrographis paniculata is a medicinal plant of immense therapeutic value. The present study was aimed to elucidate its genetic diversity based on morphochemical and RAPD markers from 53 accessions belonging to 5 eco-geographic regions. Analysis of variance and D ² statistics revealed significant differences in all the metric traits and sufficient inter-cluster distances indicating considerable diversity among the accessions. The complementary approach of RAPD was used to evaluate the genetic dissimilarities among all the accessions using 6 highly polymorphic primers. The average proportion of polymorphic loci across primers was 96.28%. The molecular genetic diversity based on Shannon index per primer averaged 5.585 with values ranging from 3.08 to 8.70 indicating towards wide genetic base. RAPD based UPGMA and D ² cluster analysis also revealed that various accessions available in different eco-geographic regions might have originated from native places of wild abundance. Similarity matrices were generated for molecular markers and morphometric data to determine the degree of congruence between the two. A highly significant but low correlation (r = 0.547, P < 0.001) was obtained thus implying the correspondence between the two. The species is hermaphroditic and a habitual inbreeder. The present study yielded a typical triangular congruence between its breeding system, morphometric traits and RAPD markers thus elucidating the usefulness of complementary approaches to make diversity analysis more explanatory and purposeful for optimum genetic amelioration and effective conservation of its genotypic variability.     

Discrimination among subterranean clover (<Emphasis Type="Italic">Trifolium subterraneum</Emphasis> L. complex) genotypes using RAPD markers

RAPD markers were applied to subterranean clover aiming at: (i) assessing the genetic relationships among the subspecies subterraneum L., brachycalycinum Katzn. et Morley, yanninicum Katzn. et Morley, as their taxonomic status is still debated; (ii) verifying the adoption of RAPDs to supplement the common morphological markers used for cultivar identification and protection; (iii) assessing the possible genetic diversity in relation to the geographic origin. Eight primers were selected for genetic analysis of 18 genotypes: 10 subsp. yanninicum (five from Greece and five from Sardinia), six subsp. subterraneum (forming three pairs, each one difficult to distinguish by morphological markers), and two subsp. brachycalycinum. Cluster analysis, performed on the Jaccards coefficients of association computed across the eight primers, formed three groups of genotypes, corresponding to the three subspecies. The results supported at the DNA level previous inferences, made at cytological, karyological, and isoenzymatic levels, on the ongoing speciation process within the subterranean clover complex, although not warranting yet the full species rank to the three forms. The genotypes of subsp. yanninicum were genetically closer to those of subsp. subterraneum than either group was to the subsp. brachycalycinum genotypes. Within the subsp. yanninicum cluster, the Sardinian genotypes appeared fairly distinct from those from Greece, suggesting a possible, independent evolution going on in different centres of diversity of this subspecies. In two pairs of subsp. subterraneum genotypes, the members could be unequivocally distinguished, thus supporting the role of RAPD fingerprinting in cultivar identification. In the third pair, the two genotypes appeared to be the same, inadvertently duplicated within the germplasm collection.     

Inter-simple sequence repeat (ISSR) and RAPD variation among wild barley (Hordeum. vulgare subsp. spontaneum) populations from west Turkey

Inter-Simple Sequence Repeat (ISSR) and Randomly Amplified Polymorphic DNA (RAPD) markers were used to analyze genetic distance among H. vulgare subsp. spontaneum populations from west Turkey. Fifty-five RAPD and 10 ISSR primers were used to detect variation among sample. A total of 55 polymorphic loci were found using 65 primers. Two distinct cluster groups were clearly established among populations. The minimum variation was detected between Pinarbasi and Bornova (GD = 0.192) populations and the maximum was found between Icmeler and Aydin populations (GD = 0.926). As two dominant markers, RAPD and ISSRs are effective and promising marker systems for detecting genetic variation.     

Diversity among landraces of Indian snapmelon (<Emphasis Type="Italic">Cucumis melo</Emphasis> var. <Emphasis Type="Italic">momordica</Emphasis>)

Diversity among 36 snapmelon landraces, collected from 2 agro-ecological regions of India (9 agro-climatic sub-regions), was assayed using RAPD primers, morphological traits of plant habit and fruit, 2 yield-associated traits, pest and disease resistance and biochemical composition (TSS, ascorbic acid, titrable acidity). Typical differences among accessions were observed in plant and fruit characteristics and snapmelon germplasm with high titrable acidity and possessing resistance to downy mildew, Cucumber mosaic virus, Zucchini yellow mosaic virus, Papaya ringspot virus, Aphis gossypii and Meloidogyne incognita was noticed in the collection. RAPD based grouping analysis revealed that Indian snapmelon was rich in genetic variation and region and sub-region approach should be followed across India for acquisition of additional melon landraces. Accessions of var. agrestis and var. momordica clustered together and there was a separate cluster of the accessions of var. reticulatus. Comparative analysis of the genetic variability among Indian snapmelons and an array of previously characterized reference accessions of melon from Spain, Israel, Korea, Japan, Maldives, Iraq, Pakistan and India using SSRs showed that Indian snapmelon germplasm contained a high degree of unique genetic variability which was needed to be preserved to broaden the genetic base of melon germplasm available with the scientific community. N. P. S. Dhillon and Ranjana contributed equally to this work and are considered the first authors.     

Assessment of genetic relationships among Pyrus species and cultivars using AFLP and RAPD markers

Twenty-five Pyrus communis L. cultivars including eight traditional Portuguese pears, and four commercial Pyrus pyrifolia (Burm.) Nak. (Japanese pear or `nashi') cultivars were analysed by RAPD and AFLP techniques focusing on their molecular discrimination and the assessment of their genetic relatedness. Twenty-five primers generated 324 RAPD markers, among which 271 (84%) were polymorphic. The AFLP technique, using seven primer combinations, revealed a similar level of molecular polymorphisms (87%), representing 418 polymorphic bands among a total of 478 scored in autoradiographs. The high reproducibility of RAPD and AFLP techniques was confirmed comparing DNA samples from different extractions and different digestions of DNA from the same plant. Three genetic similarity matrices and respective dendrograms were elaborated on using RAPD, AFLP or joint RAPD and AFLP data. Both molecular marker techniques proved their reliability to assess genetic relationships among pear cultivars. P. pyrifolia cultivars exhibit a closer genetic relatedness, clustering apart from P. communis cultivars. Within P. communis, `William's', as well as `Doyenne du Comice', cluster close to their hybrids. Most of the Portuguese cultivars tend to cluster together, indicating to constitute a relatively independent genetic pool, which can be of interest in pear breeding programs.     

Morphological and molecular diversity analysis among the Indian clones of <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis> L.

Sesuvium portulacastrum L. (seapurslane) is a halophyte used as pioneer species in sand dune fixation and stabilization of saline soil. Studies on the morphological and molecular diversity were carried out for the 14 clones of Sesuvium collected from the different coastal regions of India. Significant differences were observed for morphological traits viz., length, width, diameter and area of leaf, internodal distance and stem diameter for different clones when compared with the clone from Gujarat state (GJ1). A UPGMA dendrogram for morphological traits based on the Pearson’s similarity coefficient clustered the clones into three groups considering 80% polymorphism as criteria. Molecular diversity among the clones was studied using Randomly Amplified Polymorphic DNA (RAPD), Internal Transcribed Spacer (ITS) and markers specific to Ac homologous region. Of the total 749 RAPD loci amplified with 70 random primers, 294 were polymorphic with 39.25% diversity. A phylogenetic tree constructed with UPGMA and SHAN, grouped the clones into three major clades based on RAPD data. The molecular diversity studied with ITS and markers specific to Ac homologous region revealed 37.50% and 66.66% polymorphism and clustered the clones into three and four clades, respectively. The genetic diversity analysis revealed wide variations among the S. portulacastrum clones, reflecting a high level of diversity within the species which might be due to anthropogenic impact and geographic environmental conditions. Further, the various clones from the different eco-geographic coastal localities might have originated from native places of wild abundance. To the best of our knowledge, this is the first attempt to evaluate both morphological and genetic diversity among the Sesuvium clones collected from the distant habitats of the coastal regions of the India.     

Analysis of genetic diversity in Piper nigrum L. using RAPD markers

RAPD analysis was conducted in 22 cultivars of P. nigrum(black pepper) from South India and one accession each of P. longum and P. colubrinum. Twenty-four primers generated 372 RAPD markers of which 367 were polymorphic. Jaccard's similarity between pairs of accessions ranged between 0.11 and 0.66 with a mean of 0.38. Among P. nigrum cultivars, the similarity ranged between 0.20 and 0.66 and the mean was 0.42. The existence of wide genetic diversity as revealed in the present study is supported by earlier reports of extensive inter- and intrapopulation morphological variability in pepper cultivars from South India. UPGMA dendrogram and PCO plot revealed P. colubrinum to be most distant of the three species. Genetic proximity among P. nigrum cultivars could be related to their phenotypic similarities or geographical distribution. Greater divergence was observed among landraces than among advanced cultivars. Landraces grown in southern parts of coastal India and those grown in more northern parts were grouped in separate clusters of the dendrogram.     

Genetic Relationships and Variation among Biotypes of Dallisgrass (Paspalum dilatatum Poir.) and Related Species Using Random Amplified Polymorphic DNA Markers

The genus Paspalum L. consists of more than 400 species. Around twenty-five informal groups of species are recognized in Paspalum and the Dilatata group is of special interest because its members are excellent potential forage grasses. Seventy-five germplasm accessions, representing 15 taxa, were analyzed using randomly amplified polymorphic DNA (RAPD). Polymorphisms were observed with twenty-two primers in the Dilatata group and 16 of those were analyzed. Four hundred and four different RAPD fragments were generated, resulting in an average of 25.2 bands per primer. Among the 404 markers analyzed, 48 (11.88%) were exclusive for the P. dilatatum Poir. biotypes, 31 (7.67%) were exclusive to taxa belonging to other groups included in this study, 28 markers (6.93%) were diagnosed for other species of the Dilatata group and 16 (3.96%), for natural hybrids. Extensive RAPD variation was found among the species studied. Inter- and intra-taxonomic polymorphisms were detected. A dendrogram based on the RAPD data shows some clusters corresponding to the same taxa. However, the biotypes of P. dilatatum do not form a cluster. The present work confirms that the RAPD technique can be used to determine genetic relationships between the taxa belonging to the Dilatata group.     

Relationships among species of Hystrix Moench and Elymus L. assessed by RAPDs

To assess the generic delimitation and the interspecific relationships between Hystrix and Elymus, three Hystrix and 10 Elymus species were used for random amplified polymorphic DNA(RAPD) assay. Of the 54 primers tested, 26 (48%) produced polymorphic products. A total of 167 products amplified from 16 primers were selected for RAPD analysis, among which 156 (93.4%) amplified products were found to be polymorphic among the 13 species. The polymorphism produced by each primer ranged from 4 to 13, with an average of 9.8. Data were used to generate Jaccard's similarity coefficients and to construct a dendrogram using UPGMA in the NTSYS computer programs. It is concluded from this study that: (1) there were clear differences between Hystrix and Elymus, which possibly suggest that Hystrix is a valid genus; (2) great diversity existed among the species of Hystrix and Elymus; (3) the species similar to each other in morphological characters were grouped together; (4) the species from neighboring geographical regions were clustered; (5) the species with the same genomes and polyploidy level were clustered together; (6) RAPD results are comparable with those obtained from studies on morphology and cytology. It is a useful additional method for assessing the relationships among genera and species in Triticeae.     

Genetic diversity among <Emphasis Type="Italic">Jatropha</Emphasis> species as revealed by RAPD markers

The genus Jatropha is native of tropical America with more than 200 species that are widely distributed in tropics with a promise for use as an oil crop for biodiesel. This investigation was carried out to assess the genetic diversity of 12 Jatropha species based on random amplified polymorphic DNA markers. From 26 random primers used, 18 primers gave reproducible amplification banding patterns of 112 polymorphic bands out of 134 bands scored accounting for 80.2% polymorphism across the genotypes. Three primers viz., OPA 4, OPF 11, and OPD 14 generated 100% polymorphic patterns. The polymorphic information content was highest for the primer OPD 14 (0.50) followed by the primers OPF 11 and OPAD 11 (0.48). Jaccard’s coefficient of similarity varied from 0.00 to 0.85, indicative of high level of genetic variation among the genotypes studied. UPGMA cluster analysis indicated three distinct clusters, one comprising all accessions of J. curcas L., while second included six species viz., J. ramanadensis Ramam., J. gossypiifolia L., J. podagrica Hook., J. tanjorensis J. L. Ellis et Saroja J. villosa Wight and J. integerrima Jacq. J. glandulifera Roxb. remained distinct and formed third cluster indicating its higher genetic distinctness from other species. The overall grouping pattern of clustering corresponds well with principal component analysis confirming patterns of genetic diversity observed among the species. The result provides valid guidelines for collection, conservation and characterization of Jatropha genetic resources.     

Assessment of Genetic Relationships between Wild and Cultivated Mulberry (Morus) Species using PCR Based Markers

In order to formulate appropriate strategies for the conservation and utilization of the wild mulberry genetic resources available in India, a study was undertaken with 20 mulberry genotypes from the four different species. Seventeen intersimple sequence repeat primers were used to generate a total of 114 markers, of which 98 (85.96%) were polymorphic. Seven unique bands for Morus serrata Roxb. and one for both M. serrata Roxb. and Morus macroura Miq. were identified, of which one fragment has been sequenced and deposited in the EMBL-GeneBank (AJ-585512). The genetic dissimilarity coefficients varied from 0.078 to 0.530 among these genotypes and from 0.168 to 0.465 among the species. The dendrograms realized from these markers clustered the genotypes into three groups. The outermost group was M. serrata Roxb., which was followed by the group of M. macroura Miq. and the innermost group contained genotypes of Morus indica L. and Morus alba L. This intermixing of genotypes of M. indica and M. alba supports the view that M. indica is merely a synonym of M. alba. Distribution of the genotypes on a two-dimensional figure upon multidimensional scaling with ALSCAL program, further, confirmed the genetic divergence between the cultivated and wild mulberry groups. On the basis of the results a few potential wild mulberry genotypes were identified for its conservation and utilization in breeding programs to confer the stress tolerance to the cultivated varieties of mulberry.     

Analysis of genetic diversity in Indian taro [Colocasia esculenta (L.) Schott] using random amplified polymorphic DNA (RAPD) markers

Taro [Colocasia esculenta (L.) Schott] germplasm accessions collected from different parts of India were subjected to RAPD (Random Amplified Polymorphic DNA) analysis to assess the genetic diversity prevalent and also to test the genetic basis of morphotypic classification. Thirteen random decamer primers out of the 22 tested were used to analyse 32 taro accessions belonging to 28 morphotypes. Three out of these thirteen primers analysed showed 100 per cent polymorphism. Per cent polymorphism varied from 60 to 100 among the polymorphic primers. High genetic diversity was revealed as the similarity coefficient values ranged from 0.50 to 0.98. No two accessions analysed in the present study showed a similarity coefficient value of one thereby indicating their distinctness and presence of high genetic diversity in Indian taro germplasm. Dendrogram obtained from UPGMA analysis grouped 32 accessions in four clusters and three accessions were placed as outliers. Clustering pattern did not show any strict relationship with geographical distribution, morphotype classification and genotypic diversity. Further, accessions classified, as belonging to the same morphotypic group did not always cluster together. Presence of a very close genepool of the wild, weedy and cultivated forms with extreme levels of phenotypic and genotypic variation is suggested as the reason for high genetic diversity reported. Usefulness of DNA markers such as RAPD in characterising and assessing the genetic diversity in Indian taro germplasm is hereby demonstrated.     

Genetic diversity in bambara groundnut (Vigna subterranea (L.) Verdc) landraces assessed by Random Amplified Polymorphic DNA (RAPD) markers

Genetic diversity in 12 landraces of bambara groundnut (Vigna subterranea), an indigenous African legume, was evaluated using Random Amplified Polymorphic DNA (RAPD) markers. DNA from individuals of each landrace was also analysed to determine the level of heterogeneity within landraces. RAPDs revealed high levels of polymorphism among landraces. The percentage polymorphism ranged from 63.2% to 88.2% with an average of 73.1% for the 16 RAPD primers evaluated. The construction of genetic relationships using cluster analysis groups the 12 landraces in two clusters. RAPDs are useful for the genetic diversity studies in V. subterranea and can identify variation within landraces.     

Studying the extent of genetic diversity among Gossypium arboreum L. genotypes/cultivars using DNA fingerprinting

Genetic diversity is an area of concern for sustaining crop yield. Information on genetic relatedness/diversity among Gossypium arboreum L. cultivars/genotypes is scanty. We have used random amplified polymorphic DNA (RAPD) analysis to assess the genetic divergence/relationship among 30 genotypes/cultivars of G. arboreum. Of 45 primers surveyed, 63% were polymorphic. Out of the total number of loci amplified, 36% were polymorphic. The calculated genetic similarity between the cultivars/genotypes was in the range of 47.05–98.73%. Two genotypes, HK-244 and Entry-17, were the most distantly related. The average genetic relatedness among all the genotypes was 80.46%. However, most of the cultivated varieties showed a close genetic relationship, indicating a narrow genetic base in comparison to the non-cultivated germplasm. The calculated coefficients were used to construct a dendrogram using the unweighted pair group of arithmetic means (UPGMA) algorithm, which grouped the genotypes/cultivars into two major and three smaller clusters. The study is the first comprehensive analysis of the genetic diversity of G. arboreum germplasm and identifies cultivars that will be useful in extending the genetic diversity of cultivated varieties and future genome mapping projects.     

RAPD and seed coat morphology variation in annual and perennial species of the genus Cicer L.

Molecular and morphological variation of six perennial and five annual species including domesticated chickpea, C. arietinum, were inferred on the basis of RAPD and S.E.M. seed coat features using three outgroup taxa (Lens ervoides, Lathyrus japonica and Pisum sativum). Of the 66 decamer arbitrary primers tested, eight primers revealed 87 informative fragments. Neighbor-joining cluster analysis using Jaccard's coefficient of similarity on the basis of polymorphic fragments indicated a narrow variation in C. arietinum and recognized two major clusters in the genus Cicer. The first one included four species of sect. Monocicer: C. echinospermum, C. arietinum, C. reticulatum and Iranian material of C. bijugum. The second cluster contained annual and perennial species belonging to sections Chamaecicer, Polycicer and Acanthocicer. The character state of morphological and ecological traits on the RAPD phenogram indicated no monophyletic incision. Our results suggested that the high genetic difference between annual and perennial species might be regarded as a rapid speciation of section Monocicer. Reconsideration of traditional classification of sections Polycicer and Acanthocicer is necessary. The Desi and Kabuli types of C. arietinum could not be separately grouped at the DNA level, and the low level of genetic variation of C. arietinum may result from a bottleneck during the domestication process.     

Variation and Intraspecific Relationships in Indian Wild Musa balbisiana (BB) Population as Evidenced by Random Amplified Polymorphic DNA

Sixteen collections of the wild Musa species, Musa balbisiana Colla collected from different regions of India were studied for their intraspecific relationships using random amplified polymorphic DNA (RAPD) markers. Out of 80 primers screened, 34 primers produced reproducible bands and four primers among them showing polymorphic bands were used. In all, 43 DNA fragments were amplified averaging 10.75 per primer. Of these, 31 amplified fragments showed polymorphism (averaging of 7.75 per primer). The extent of polymorphism (74.6%) has indicated the existence of considerable variation at the DNA level within the species. The 16 accessions were clustered into four as against seven clusters obtained through morphotaxonomic characterization. The inter relationships based on geographical origin in comparison with molecular characterization have been discussed.     

Genetic variations and interrelationships in Vanilla planifolia and few related species as expressed by RAPD polymorphism

Vanilla is naturally distributed in Mexico and parts of Central America and the history of origin of cultivated vanilla suggests that the entire stock outside Mexico may be from a single genetic source. In the present study, RAPD polymorphism was used to estimate the level of genetic diversity and interrelationships among different collections of Vanilla planifolia Andr., and few related species, including both leafy and leafless types such as V. tahitensis J.W.Moore, V. andamanica, Rolfe, V. pilifera Holtt., and V. aphylla Blume. Studies revealed that there are very limited variation within collections of V. planifolia, indicative of its narrow genetic base, and of the related species we tested, V. tahitensis is nearest to V. planifolia. The species studied are diverse and have a similarity ranging from 1.2 to 57.3 %. Of the sampled taxa, V. andamanica is the most divergent and there is also reasonable variability within its collections, indicating the possibility of natural seed set. A total of 82 polymorphic bands expressed in the RAPD profiles were used to generate a genetic distance matrix, which was then used in cluster analysis. Specific groupings were revealed by the cluster analysis whereby the leafless forms (V. aphylla, V. pilifera and the new species) and V. andamanica formed separate clusters. This is the first report of species interrelationship studies, including both cultivated and wild vanilla species.