Characterization of genetic variation and relationships among Choix germplasm accessions using RAPD markers

Choix, a plant in the tribe Maydeae of the grass family, has been cultivated in Asia for several thousand years. It is a potential gene resource for improvement of other cereal crops because of its nutritional value and tolerance to stress. Genetic variation and relationships among 21 Choix lachryma-jobi L. accessions were characterized by random amplified polymorphic DNA (RAPD) markers. A total of 205 DNA fragments across all materials were amplified with 31 random primers, averaging 6.61 per primer. Among amplified fragments, 115 showed polymorphism averaging 3.71 per primer. Of amplified markers, 56.1% were polymorphic, indicating considerable variation at the DNA level among these accessions. Some fragments were accession-specific. Pair-wise genetic similarity (GS) among 21 accessions ranged from 0.809 to 0.301. The 21 accessions clustered into two major groups. Three exotic Choix accessions clustered together. Three other Choix accessions, collected from Guangxi, China, clustered into a cohesive subgroup. Four wild types of Choix clustered into the same subgroup. These results indicated that the classification by RAPD data reflected the differences in geographic origins and evolution in Choix.     

Phenotypic variation in caryopsis dormancy and seedling salt tolerance in wild barley, Hordeum spontaneum, from different habitats in Israel

The phenotypic variation in caryopsis dormancy and seedling salt tolerance was investigated in 16 wild barley ecotypes in Israel. Depth of dormancy, as reflected by the time to maximum germination percentage, ranged from 15 to 103 days under dormancy-break treatment. Lower dormancy was characteristic of the mesic ecotypes, whereas deeper dormancy was characteristic of the xeric ecotypes. Dormancy-break patterns were revealed by growth curves: the xeric ecotype showed an S-shaped curve, whereas the mesic ecotype displayed a reverse L-shaped curve. Seedling salt tolerance was assessed by the ratio of root or coleoptile length in a seedling grown in 100 or 200 mM NaCl solution to that of a seedling grown in water. The root- and coleoptile-length ratios of mesic ecotypes were much higher than that of the xeric ecotypes, except that there was no observable difference in coleoptile-length ratio at 100 mM NaCl. The mesic ecotype was more tolerant to salt than the xeric ecotype at the young seedling stage, and seedling salt tolerance was negatively correlated with caryopsis dormancy depth. Thus evolutionary background environments have had a strong effect on the intensity of caryopsis dormancy in wild barley. Through natural selection, wild barley has adapted to dry and hot environments by increasing dormancy but not young seedling salt tolerance.     

Genetic diversity in the Olive tree (Olea europaea L. subsp. europaea) cultivated in Portugal revealed by RAPD and ISSR markers

To assess the genetic diversity of the most important olive cultivars used in Portugal, a base collection was established with two hundred and one accessions of eleven cultivars from the different agro-ecological-regions (AER) of olive oil production. Inter-cultivar diversity was evaluated using seven RAPD primers producing fifty-nine polymorphic markers that enable cultivar distinction. Discriminant analysis according to fruit use and AER revealed a genetic structure associated with local selection both for fruit exploitation and agro-ecological adaptation. Intra-cultivar diversity of the ancient cultivar Galega was also investigated. Three RAPD and five ISSR primers produced ninety-three polymorphic markers upon seventy-seven accessions from five AERs. Total accession discrimination was achieved. UPGMA clustering and discriminant analysis revealed that the genetic diversity was predominantly structured according to accessions origin. The within and among AER variation revealed by AMOVA supported this genetic structure and showed a high proportion of intra-AER variability. These evidences suggest that Galega is composed by a mixture of different genotypes adapted to local conditions, indicating that this cultivar is in an early stage of domestication and should be treated as a landrace instead of a uniform cultivar. The assessment of Galega genetic diversity within each of the five AERs indicated the highest significant level (H_g = 6.23 at p< 0.001) in Ribatejo-Santarém. This finding associated with the distinctiveness of Galega in relation to other Portuguese cultivars and with the recent insights of olive tree domestication allowed us to hypothesize that Ribatejo-Santarém was the ecological region of origin and dispersion of this ancient cultivar.     

Genetic variability evaluation in a Moroccan collection of barley, Hordeum vulgare L., by means of storage proteins and RAPDs

The genetic variation existing in a set of barley (Hordeum vulgare L.) landrace samples recently collected in Morocco was estimated. Two kinds of genetic markers, seed storage proteins (hordeins) and random amplified polymorphic DNA (RAPD), were used. Only six out of 31 landraces were subjected to RAPD analysis. Both kinds of markers, RAPD and storage proteins, yielded similar results, showing that the level of variation observed in Moroccan barley was high: all landraces showed variability; 808 different storage protein patterns (multilocus associations) were observed among 1897 individuals (2.32 seeds per association, on average) with an average of 43 multilocus associations per accession. In general, genetic variation within accessions was higher than between accessions. The 100 polymorphic RAPD bands generated by 21 effective primers were able to generate enough patterns to differentiate between uniform cultivars and even between individuals in variable accessions. One of the aims of this work was to compare the effectiveness of RAPD versus storage protein techniques in assessing the variability of genetic resource collections. On average hordeins were more polymorphic than RAPDs: they showed more alternatives per band on gels and a higher percentage of polymorphic bands, although RAPDs supply a higher number of bands. Although RAPD is an easy and standard technique, storage protein analysis is technically easier, cheaper and needs less sophisticated equipment. Thus, when resources are a limiting factor and considering the cost of consumables and work time, seed storage proteins must be the technique of choice for a first estimation of genetic variation in plant genetic resource collections.     

Genetic variation within and among species of five sections of the genus Arachis L. (Leguminosae) using RAPDs

Some Arachis species are widely used as commercial plants, e.g. the groundnut A. hypogaea, an important source of good quality protein and oil, and A. pintoi and A. glabrata, that are utilized as forage species. Germplasm of most Arachis species is available in germplasm banks. However, little it is known about the genetic attributes of this germplasm, and mainly about its genetic variability, which is very important for its maintenance. In the present study RAPDs were used to assay the genetic variation within and among 48 accessions of five sections of the genus Arachis and to establish the genetic relationships among these accessions. Ten of 34 primers tested were selected for DNA amplification reactions since they yielded the largest numbers of polymorphic loci. A dendrogram was constructed based on data from the 10 primers selected. Eighty RAPD polymorphic bands were analyzed among the accessions studied. The relationships among species based on RAPDs were similar to those previously reported based on morphological, cytological and crossability data; demonstrating that RAPDs can be used to determine the genetic relationships among species of the different sections of the genus Arachis. In general, wide variation was found among accessions and low variation was found within the accessions that had two or more plants analyzed. However, higher polymorphism was found in the section Trierectoides and in one accession of A. major, indicating that generalizations should be avoided and each species should be analyzed in order to establish collection and maintenance strategies.     

Variation of cultivated flax (Linum usitatissimum L. subsp.usitatissimum) and its wild progenitor pale flax (subsp.angustifolium (Huds.) Thell.)

The domestication of cultivated flax (Linum usitatissimum L. subsp.usitatissimum) is briefly discussed. Using data documented as a matter of routine in genebank work, 63 accessions of cultivated flax from the flax germplasm collection of the Gatersleben Genebank are compared with 73 accessions of its wild progenitor pale flax (subsp.angustifolium (Huds.) Thell.), which have been observed in systematic field trials. Range of variation, genetically based variation, heritability and correlation of several characters are considered, especially with respect to the influence of domestication. Cultivated flax shows higher variation in the characters of generative plant parts, while pale flax varies more in the vegetative parts of the plant. The character correlations are similar in both subspecies. Of the 29 characters studied in pale flax the parameters describing tillering, height of plant, weight of seeds, width of petals and width of leaves are especially suitable for distinguishing between different accessions. In spite of the high heritabilities of most of the characters, the influence of the environment is significant in most cases.     

Comparison between phenetic characterisation using RAPD and ISSR markers and phenotypic data of cultivated chestnut (Castanea sativa Mill.)

Patterns of phenotypic and phenetic variability in sixPortuguese cultivars of chestnut (Castanea sativa Mill.) are evaluated. Morphological characterisationwas based on the quantification of seventeen traits. Varianceanalysis showed significant differences among cultivars, and cultivar× year for all the traits studied, and trees within cultivarsshowed also some significant differences for some of themorphological variables. A significant correlation was obtainedbetween length of the leaf blade and the percentage of unisexual andandrogynic inflorescence with the effective thermal index,accumulated rainfall from April to October and from July to October,or the accumulated temperature below seven during the dormant period.Principal Component and cluster analysis were performed to group thecultivars, according to their similarity coefficients. For molecularcharacterisation, 125 RAPD and 157 ISSR polymorphic markers wereamplified using 28 and 7 primers respectively. High level ofcongruence among the two marker systems (r =90.5%) was obtained from comparison of pheneticsimilarities based on the percentage of shared fragments. ISSRmarkers revealed important advantages over RAPDs, due to a higheffective multiplex ratio (12.5 for ISSR compared with 2.2 forRAPD analysis) and reproducibility. Although morphological andmolecular results are comparable, slight differences are showed incluster analysis UPGMA dendrograms. Molecular analysis explainedhomonym situations among 'Martainha' and'Longal' cultivars in Portugal.     

RAPD Polymorphism of the White-Flowered Gourd (Lagenaria siceraria (Molina) Standl. Landraces and its Wild Relatives in Kenya

The present study was performed to investigate genetic diversity of Kenyan landraces of the white-flowered gourd (Lagenaria siceraria), which exhibits tremendous morphological variation. RAPD analyses were performed on 53 landraces of the cultivated species L. siceraria and 42 accessions of three wild species (40 L. sphaerica, 1 L. abyssinica, and 1 L. breviflora). A total of 432 polymorphic bands were detected using 54 primers. The four species were clearly differentiated from one another. Intra-specific variations were investigated with L. siceraria and its wild relative L. sphaerica. Landraces of the cultivated species collected from different ethnic communities or regions were differentiated. Morphological variations were not associated with RAPD variations. Bitter landraces collected in Maasai communities showed two specific RAPD bands. In the wild species, accessions collected from the eastern and western sides of the Great Rift Valley were genetically differentiated from each other. In both species, genetic and geographical distance matrices computed among all pairs of accessions were significantly correlated, implying that the observed geographical variation can be explained by the 'Isolation by distance model'. Progeny plants derived from a common mother in L. siceraria showed a low level of segregation in RAPD pattern, suggesting that collected landraces are cultivated, maintaining their inherent traits although they are monoecious and insect-pollinated, whereas the wild relative L. sphaerica showed a higher level of segregation. The morphological diversity observed among landraces of L. siceraria is the result of human selection and their genetic identities are maintained by inbreeding probably resulting from frequent self-pollination.     

Analysis of genetic diversity among European and Asian fig varieties (<Emphasis Type="Italic">Ficus carica</Emphasis> L.) using ISSR,RAPD, and SSR markers

Nineteen fig varieties and lines from Europe and Asia have been fingerprinted by ISSR, RAPD, and SSR markers, respectively, using 13, 19, and 13 primer combinations. All primers produced 258 loci, with the highest number of loci (119) generated by RAPD (R _p: 48.42). Clustering analysis was applied to the three marker datasets to elucidate the genetic structure and relationships among these varieties. Mean genetic similarities were 0.787, 0.717, and 0.749, respectively, as determined using ISSR, RAPD, and SSR. Each marker system produced incompletely separated clusters, although a weak binding group based on race type appeared in the combined dataset. Comparisons of coefficients revealed no correlation between different similarity matrices; congruence was observed between similarity matrices and co-phenetic matrices in all markers. Analysis of molecular variance (AMOVA) showed that most of the total polymorphism was attributable to within-group variance (ISSRs + RAPDs, 97.41%; SSRs, 90.18%). These results suggest that the genetic diversity of this fig population is low and that multiple marker utilization is critical to estimate the relatedness of figs at the variety level. Additionally, it was presumed that ‘Houraihi’, the oldest variety in Japan, was disseminated independently of other foreign varieties in the 17th century or before then.     

Levels and distribution of allozyme and RAPD variation in populations of Elymus fibrosus (Schrenk) Tzvel. (Poaceae)

To study the magnitude and nature of genetic variation in E. fibrosus, the levels and distribution of allozyme and RAPD variations were investigated in populations collected from Finland and Russia. The results obtained from the allozyme and RAPD studies were compared to each other in 10 of the populations. The allozyme analysis showed that 6 of 12 presumed loci (50%) were polymorphic within the species, while the mean number of polymorphic loci within populations was 4.8%. The mean number of allele per locus for the species was 1.5 and 1.05 across the populations. Genetic diversity at the species level was low (H _es = 0.025), and the mean population genetic diversity was even lower (H _ep = 0.007). Both these values were much lower than the average for other Elymus and self-fertilising species. The largest proportion of the total allozyme diversity was found among, rather than within the populations (G _ST = 0.70). The allozyme genetic distances between the populations did not reflect geographic distances. Cluster and principal coordinates analyses revealed the same allozyme relationship patterns among the populations. A comparison of allozyme and RAPD variation in 10 of the populations showed differences in the amount of genetic variation. The RAPD analysis revealed higher levels of variation (A _p = 1.19, P _p = 20.3 and H _ep = 0.09) than the allozyme one) A _p = 1.06, P _p = 5.8 and H _ep = 0.008). For both markers, the largest proportion of the total gene diversity was found among the populations studied (G _st = 0.63 for RAPDs and G _st = 0.65 for allozyme). In contrast to the allozyme analysis, the RAPD based genetic distances did reflect geographic distances. The cluster and principal coordinates analyses showed different grouping of populations for each data set. There was a positive, but not significant, correlation (r = 0.41) between the genetic distance matrices resulting from these markers. Regional comparison revealed that the Finnish populations had a higher diversity than the Russian ones. Generally, this study indicates that E. fibrosus contains low genetic variation in its populations. The results are discussed in the context of conservation of the species.     

Genetic Diversity among Wild Common Beans from Northwestern Argentina Based on Morpho-agronomic and RAPD Data

The genetic diversity among 10 wild populations of common bean Phaseolus vulgaris var. aborigineus was analyzed by means of RAPD markers and morpho-agronomic data. The study was performed on populations collected from different sites located in the provinces of Jujuy, Salta and Tucumán in northwestern Argentina. Ten quantitative traits and 33 random primers were scored. Clustering based on morpho-agronomic traits and RAPD markers generated similar phenograms that grouped bean populations based on their site of collection. The levels of diversity observed among populations were low suggesting they have a common ancestor. The levels of diversity shown by morpho-agronomic traits were higher compared to those of molecular markers, most probably due to the effect of the environment. Furthermore, a 480-bp DNA band identified a group of wild populations collected from similar sites. Breeding strategies need to exploit this diversity to broaden the genetic base of commercial beans to develop high yield cultivars.     

RAPD and ISSR molecular markers in <Emphasis Type="Italic">Olea europaea</Emphasis> L.: Genetic variability and molecular cultivar identification

Thirty Portuguese and eight foreign olive (Olea europaea L.) cultivars were screened using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) markers. Twenty RAPD primers amplified 301 reproducible bands of which 262 were polymorphic; and 17 ISSR primers amplified 204 bands of which 180 were polymorphic. The percentage of polymorphic bands detected by ISSR and RAPD was similar (88 and 87%, respectively). The genetic variability observed was similar in the Portuguese and foreign olive cultivars. Seven ISSR and 12 RAPD primers were able to distinguish individually all 38 olive cultivars. Twenty specific molecular markers are now available to be converted into Sequence Characterised Amplified Region (SCAR) markers. Relationships among Portuguese and foreign cultivars is discussed.     

Genetic diversity changes and relationships in spring barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) germplasm of Nordic and Baltic areas as shown by SSR markers

To examine changes in the level of and pattern in variability in 197 Nordic and Baltic spring barley cultivars over time we used 21 mapped barley simple sequence repeats (SSRs). A total number of 191 alleles were found from 22 SSR loci. The number of alleles per locus ranged from 2 to 23, with average of 8.63 107 alleles were rare (frequency <0.05) among the cultivars and only one allele was frequently observed (frequency >0.95). The gene diversity between loci in Nordic and Baltic material varied between 0.033 and 0.891. Average gene diversity was 0.623. The SSR data separated two-rowed and six-rowed cultivars. According to analysis of molecular variance (AMOVA) differentiation in two-rowed vs six-rowed accounted for 23.6% of the total variation. Overall no significant decrease of average gene diversity over time could be found. However, differences were observed when spring barleys from northern (north of ∼58°) and southern (south of ∼58°) parts of the Nordic and Baltic area were compared. For the southern ecogeographical region significant decrease of genetic diversity was observed in the middle of the 20th century, whereas no significant changes in the northern part were found. We found larger differentiation between modern and old cultivars in the South compared to the ones in North parts of the region. The magnitude of changes in genetic diversity differed also with the country of origin. Danish cultivars had a significant decrease in diversity in the middle of century, whereas changes in Finland, Norway and Sweden were not significant.     

Genetic diversity in wild Tunisian populations of Mentha pulegium L. (Lamiaceae)

The allozyme variation of 15 Tunisian wild populations of Mentha pulegium L. threatened by human activities (clearing, hard-grazing, ploughing, traditional uses) was surveyed by the analysis of 14 isozyme loci using horizontal gel starch electrophoresis. The species exhibited a high level of genetic variation within populations (the mean Ap = 2.20, P = 72%, Ho = 0.349 and He = 0.229), which indicates a predominately outcrossing mating system and the recruitment of new genotypes via dispersal seeds. The genetic structure analysis of the populations using F statistics indicates no inbreeding, and showed an excess of heterozygosity for few loci. The moderate differentiation of populations (F_ST = 0.110) and the low rate of gene flow between them (Nm = 2.02) might been caused by recent isolation of the populations through biotope disturbances. The value of Nei's genetic identity varied from 0.839 to 0.999 reflecting a relatively low genetic divergence between populations. Cluster analysis using UPGMA method and Nei's genetic identity values, showed that populations geographically close didn't always cluster together. However, populations within the same bioclimatic stage generally subclustered together indicating that differentiation between bioclimatic regions occurred.     

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.     

Genetic variation of Ethiopian and Eritrean sorghum (Sorghum bicolor (L.) Moench) germplasm assessed by random amplified polymorphic DNA (RAPD)

The extent and patterns of distribution of genetic variation among 80 sorghum (Sorghum bicolor (L.) Moench) germplasm accessions from Ethiopia and Eritrea were investigated using RAPD with 20 oligonucleotide primers. The primers generated a total of 147 polymorphic bands across the 80 accessions with a mean of 7.35 bands per primer. Estimation of the extent of variation by the Shannon-Weaver diversity index revealed an intermediate level of overall variation (H = 53), although the levels varied among regions of origin of the accessions. Partitioning of the total variation revealed considerable variation (77%) within the region of origin of the accessions and the remainder (23%) among regions of origin. Similarly, a large portion (94%) of the total variation was found within the adaptation zones compared to among the adaptation zones (6%). The results suggest a weak differentiation of the sorghum material both on regional and agro-ecological bases, which could be ascribed to the high rate of outcrossing in cultivated sorghum and its free natural hybridization with its wild and weedy relatives, as well as to seed movement by humans. The average genetic dissimilarity was found to be 36% among the 80 accessions and 13% among the 15 regions of origin. Cluster analysis failed to group accessions of the same region or the same adaptation zone, which further confirmed the weak differentiation of the material studied. The clustering pattern of the regions of origin was broadly concordant with previous clustering patterns obtained using morphological characters, in which regions with broad agro-climatic conditions were grouped together.     

Assessment of genetic diversity, and phylogenetic relationships based on ribosomal DNA repeat unit length variation and Internal Transcribed Spacer (ITS) sequences in chickpea (Cicer arietinum) cultivars and its wild species

Repeat unit length variation and internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA were used to assess genetic diversity, and phylogenetic relationships in chickpea (C. arietinum) cultivars, and its related wild species. Total genomic DNAs of 76 accessions of 10 Cicer species, belonging to three sections of the genus, were restricted with seven enzymes and the restriction fragments were hybridized to heterologous ribosomal clones of wheat pTa71 and Vicia faba probes Ver 6-5 and Ver18-6. A single repeat unit length class of 11.4 kb or 10.5 kb was recognized across Cicer accessions with pTa71. The intraspecific variation was negligible in those species where more than one accession was studied, except the four C. judaicum accessions, which were different from the rest. EcoRI and DraI digests gave two and one-two fragments, respectively. All the accessions produced three and three-five bands with BamHI and SacI, respectively. Both the accessions of C. yamashitae differed in their rDNA repeat unit length as well as restriction site variation. Maximum likelihood tree with rDNA RFLP recognized five clades which were more or less congruent with the previous data. Length of ITS-1 region was more variable (235–239 bp) than the ITS-2 region (212–213 bp). Cladistic analysis of ITS data revealed two major clades, clade I consisting of C. arietinum, C. reticulatum and C. echinospermum, and clade II comprised of C. judaicum, C. chorassanicum, C. bijugum and C. cuneatum. C. microphyllum grouped with the above four species. C. pinnatifidum was present as a separate branch. C. yamashitae emerged as the most distinct species.     

Assessing the Genetic Diversity in the International Cocoa Genebank,Trinidad (ICG,T) using Isozyme Electrophoresis and RAPD

Random amplified polymorphic DNA (RAPD) and isozyme electrophoresis (IE) techniques were used to estimate the level of genetic diversity in a sample of cacao germplasm existing at the International Cocoa Genebank, Trinidad. Twenty-six cocoa populations represented by 459 cocoa genotypes were analysed using IE and 22 populations represented by 353 cocoa genotypes were analysed using RAPD. Despite few differences in the classification of the populations, both techniques revealed three major groups: the indigenous trees, the cultivated Trinitario and the cultivated trees from Ecuador. Two-thirds of the partitioned diversity were found within populations and one-third between the populations, with both techniques.