Analysis of genetic diversity in Turkish sesame (Sesamum indicum L.) populations using RAPD markers⋆

The genetic diversity of 38 cultivated populations of Sesamum indicum L. from four different regions of Turkey was estimated at the DNA level with the random amplified polymorphic DNA (RAPD) technique. Sixty-one bands were obtained for all populations 78% of which were polymorphic. Analysis of molecular variance (AMOVA) was used to investigate the genetic diversity of the populations which yielded highly significant differences among populations within regions (91.9% of the total genetic diversity). According to AMOVA and Shannon's index that were performed separately for each region, the highest value of genetic variation was observed among Northwest region populations (CV = 7.7; H₀ = 0.304) and lowest in the Southeast regions' populations (CV = 2.6; H₀ = 0.068). Nei and Li's similarity index was calculated and phylogenetic tree was established using the neighbor-joining algorithm. This phenetic analysis grouped 35 of 38 accessions in six groups leaving three highly diverse accessions outside. Wagner phylogenetic method was used to assess the phylogenetic relationships among the populations. In the majority-rule consensus tree, only 7 of the 32 forks showed above 60% occurrence. Using Principal Coordinate Analysis (PCO) of the RAPD data set, the groups were clearly separated along the first three axis. These results indicate that RAPD technique is useful for sesame systematics, and should be valuable for the maintenance of germplasm banks and the efficient choice of parents in breeding programs.     

Interrelationships among Coconut (Cocos Nucifera L.) Accessions Using RAPD Technique

The genetic relationships among 33 coconut germplasm accessions were analyzed using RAPD markers. The germplasm accessions were collected from various coconut growing regions viz. South Asia (SA), South East Asia (SEA), South Pacific (SP), Atantic and America, and Africa. Forty-five random primers produced a total of 399 polymorphic markers. The Polymorphism Information Content (PIC) ranged from 0.031 to 0.392 and the Marker Index (MI) ranged from 6.28 to 0.031 among the primers. Based on the MI a set of 5, 10 and 15 informative and reproducible primers were identified. The mantel matrix correlation was calculated to compare the similarity matrices of a set of reproducible informative primers and global primers. There was significant correlation among the similarity matrices (r ≥ 0.50). The similarity matrix based on 399 polymorphic markers was used to construct the dendrogram to show the genetic relationship among the accessions. Similarity values ranged between 0.573 and 0.846. There was less genetic similarity (based on Jaccard's coefficient) among South Pacific and South East Asian accessions. The clustering pattern obtained in the present study was in agreement with the earlier reports based on RFLP, SSRs and AFLPs.     

Postharvest variation in apple (Malus x domestica Borkh.) Flavonoids following harvest, storage, and 1-MCP treatment

The impact of 1-methylcyclopropene (1-MCP) on the synthesis and retention of flavonoid compounds during storage and ripening of red Delicious (Malus x domestica Borkh.) apples was investigated. Numerous anthocyanins, flavonols, flavan-3-ols, and a hydroxycinnamic acid from three different fruit harvest maturities were monitored after a 120 day storage and 1 week shelf life period using high-performance liquid chromatography/diode array detector analysis. The total flavonoid concentration was 5% greater in fruit treated with 1-MCP, whereas chlorogenic acid levels were 24% lower. All compounds analyzed increased in concentration during fruit harvest; however, the anthocyanins generally declined after storage, while chlorogenic acid levels increased. 1-MCP treatment resulted in the retention of anthocyanins in the latter stages of storage but did not affect the flavonols and flavan-3-ols. Chlorogenic acid biosynthesis from early and optimal fruit harvest maturities was greatly inhibited by 1-MCP during storage and the 1 week shelf life period. However, 1-MCP did not affect chlorogenic acid concentrations in late-harvested fruit. Results suggest that 1-MCP may inhibit the activity of phenylalanine ammonia-lyase and subsequent biosynthesis of flavonoid compounds. However, because very little postharvest biosynthesis of flavonoids occurs in apples, 1-MCP treatment may be useful for maintaining some of the intrinsic flavonoid levels of red Delicious apples, if applied at the proper harvest maturity.     

Genetic diversity in cowpea [Vigna unguiculata (L.) Walp.] as revealed by RAPD markers

The present study, using RAPD analysis, was undertaken to characterize genetic variation in domesticated cowpea and its wild progenitor, as well as their relationships. The materials used consisted of 26 domesticated accessions, including accessions from each of the five cultivar-group, and 30 wild/weedy accessions, including accessions from West, East and southern Africa. A total of 28 primers generated 202 RAPD bands. One hundred and eight bands were polymorphic among the domesticated compared to 181 among wild/weedy cowpea accessions. Wild accessions were more diverse in East Africa, which is the likely area of origin of V. unguiculata var. spontanea. Var. spontanea is supposed to have spread westward and southward, with a loss of variability, loss counterbalanceed in southern Africa by introgressions with local perennial subspecies. Although the variabilty of domesticated cowpea was the highest ever recorded, cultivar-groups were poorly resolved, and several results obtained with isozyme data were not confirmed here. However primitive cultivars were more diverse than evolved cultivars, which still suggests two consecutive bottlenecks within domesticated cowpea evolution. As isozymes and AFLP markers, although with a larger number of markers, RAPD data confirmed the single domestication hypothesis, the gap between wild and domesticated cowpea, and the widespread introgression phenomena between wild and domesticated cowpea.     

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.     

Genetic diversity of Pakistan wheat germplasm as revealed by RAPD markers

Wheat breeding in Pakistan started in 1930s before partition in the United India and so far has released more than 68 cultivars, but no systematic analyses of the genetic diversity of Pakistan wheat have been made. Twenty Pakistan wheat cultivars released from 1933 to 2002 were examined for genetic diversity and relationships using random amplified polymorphic DNA (RAPD) markers. Forty-two RAPD primers were applied and 184 polymorphic bands were generated for each cultivar. Most of the cultivars were genetically interrelated, although six of them displayed some genetic distinctness. The RAPD variation observed among these cultivars was low. Only 40.7% of the total scorable bands were polymorphic, and 26.1% of the polymorphic bands were observed most frequently (f = 0.95) among the 20 cultivars. The proportions of polymorphic bands for each cultivar ranged from 0.67 in ‘Yecora’ to 0.84 in ‘C-250’ with an average of 0.76. About 1.4% of the RAPD variation might have been fixed over the 69 years of wheat breeding, but such fixation was not statistically significant. These results are significant for future improvement and conservation of Pakistan wheat.     

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.     

Genetic structure of quinoa (<Emphasis Type="Italic">Chenopodium quinoa</Emphasis> Willd.) from the Bolivian altiplano as revealed by RAPD markers

Quinoa (Chenopodium quinoa Willd.) is a pseudocereal originated from the Andes important for small farmers’ food security as well as for commercial production. Recently, it has been claimed that in Bolivia genetic erosion could result from the marginalization of the crop in the north and from its commercial standardization in the south. The aim of this study was to quantify the hierarchical structure of the genetic variation present in eight quinoa field populations, consisting of cultivated and weedy individuals, representative of the altiplano and interandean valleys of Bolivia. Randomly amplified polymorphic DNA markers show that quinoa has a strong population structure and a high intra-population variation. An effect of geographical structure of the populations was highlighted, due to population isolation, not simply linked to distance but more probably to climatic and orographic barriers present in the studied zone. The population structure is also reinforced by the limited seed exchanges among farmers as revealed by field interviews. This population structure appears related to three major biogeographic zones: the northern and central altiplano, the interandean valley, and the southern Salar. Intrapopulation genetic diversity was higher than that expected for a mainly autogamous species, and higher than that reported in anterior studies based on germplasm collections. These results are commented in view of current knowledge on phylogeny and reproductive biology of the species, and their implications regarding genetic resources management are discussed.     

The RAPD evidence for the phylogenetic relationship of the closely related species of cultivated apple

The phylogenetic relationship of cultivated apple and its closely related species is still not clear in the taxonomy of genus Malus. To try to find new evidence for the origin and evolution of the cultivated apple, random amplified polymorphic DNA (RAPD) markers of 14 taxa of Malus, among which a reference species (M. toringoides) and six presumably ancestral species of cultivated apple in the genus were investigated. The RAPD data obtained were used to construct both unrooted and rooted trees using TREECON software package. The result showed in our rooted tree that M. sieversii from the Xinjiang Autonomous Region of China is the species which is most closely related to the cultivated apple, M. domesticacv. `Golden Delicious'. The phylogenetic relationships among the species studied are discussed.     

Genetic diversity and relationships among cultivated and wild accessions of tartary buckwheat (Fagopyrum tataricum Gaertn.) as revealed by RAPD markers

The methodology of sampling and the selection of a proper marker systemfor the analysis of accessions are major concerns in the evaluation of gene bank material. In our study the RAPD analysis of bulked DNA samples and single seedsDNA was successfully employed to evaluate intra- and inter-population geneticvariability of cultivated and wild tartary buckwheat accessions. The bulkingapproach enabled the distinction of all 40 analysed accessions and theirseparation into geographically well defined clusters. Three wild populations,two from Sichuan and one from Qinghai, formed a group that was geneticallyrelatively distant from wild populations from Tibet and all cultivatedlandraces which, on the other hand, exhibited very close relationships. Thesingle seed study that was used after bulked DNA analysis provided detailedinformation of the genetic variation present within some accessions of specialinterest. A moderate level of genetic variability was detected betweenaccessions and the variability was partitioned into between- andwithin-population components. On average, most of the detected variation ispresent between F. tataricumpopulations. The genetic and geographic distribution of variability is furtherdiscussed. We demonstrated the usefulness of combining bulking and single seedstudy approaches for the effective evaluation of genetic variability inF. tataricum accessions that couldalso have wider applicability in the management of plant genetic resources andphylogenetic studies.     

Genetic variation analysis in the genus Lathyrus using RAPD markers

RAPD analysis was applied to five species belonging to the genusLathyrus (Fabaceae): L.sativus, L.cicera, L.ochrus, L.sylvestris and L.latifolius. All the species under study belong to thesection Lathyrus except L.ochrus which is in section Clymenum.Nine populations representing these species were used and ten random10-mer primers were sampled. A total of 129 amplification products,ranging in size from 0.3 to 3 Kb, were generated. Partitioning ofvariation was studied using the AMOVA technique. The genetic variation proved tobe nearly equally distributed among species and among populations withinspecies. A similar approach was carried out to distinguish populations, whichproved to be efficient. The between population dissimilarities were calculatedand a dendrogram of genetic relationships was drawn. The RAPDs obtained weresufficient to distinguish between the two accessions of a species and toseparate these accessions by clustering them according to species. A highgenetic similarity between the populations of L.sylvestris and L.latifolius was established. A similar result is also shownfor the populations of L. sativus andL. cicera.     

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.     

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.     

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.     

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.     

Genetic diversity analysis in Boerhavia diffusa L. of different geographic locations in India using RAPD markers

Boerhavia diffusa is extensively used in herbal medicines as well as in the Ayurvedic system, because it contains a set of clinically important compounds. In the present study, the genetic variability in Boerhavia diffusa between accessions of different geographical origin within the Indian territory is assessed through random amplified polymorphic DNA (RAPD) markers. Twenty-eight accessions of Boerhavia were screened with eighteen primers of which nine were found to be the most informative. The degree of polymorphism was found to be high in accessions collected from different places of Uttar Pradesh (Set II) in comparison to other states of India (Set I). A relatively lower level of polymorphism was recorded in accessions collected from diverse locations around Lucknow (Set III). Accessions from neighbouring geographical regions exhibited more similarity than those from distant regions (as revealed by the set I analysis). Certain diagnostic markers may be correlated with morphological character(s) such as plant type. BDL appeared most distinct and divergent from the rest of the accessions and the BDJ plant in set II also showed least similarity estimate. Fragments of 5.62 Kb and 4.47 Kb with primer GN59 was found to be unique for BDP and BD2 having ovate leaf character, whereas ovoid leaf genotype exhibited 0.79 Kb (GN34 primer) fragment. Similarly a unique band type (0.35 Kb) with primer GN83 was present in BDL and BD1 that share light pink flower. Jaccard's, and Nei and Li similarity coefficient values amongst the accessions were in the range of 0.22 to 0.89 and 0.33 to 0.93, respectively. Association of RAPD markers with the leaf characteristics, flower colour as well as with geographical locations has been made. This shows that RAPD markers are also useful for the study of genetic structure of Boerhavia populations.     

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.     

Heavy metals mobility,sources, and risk assessment in soils and uptake by apple (Malus domestica Borkh.) leaves in urban apple orchards

The aim of the study was determine potential mobility, sources, and environmental impacts of heavy metals in soil of urban apple orchards using a sequential extraction procedure and multivariate analysis. The soil and apple (Malus domestica Borkh.) leaf samples were collected from apple orchards of urban areas in Çanakkale, Turkey. A sequential extraction procedure was employed to determine the binding forms of Cd, Cr, Cu, Ni, Pb, and Zn in soil samples. Sequential extraction procedure revealed that mobile fractions (acid soluble, reducible, and oxidizable) of Cd (60.2%) and Pb (56.8%) were higher than their immobile fractions. Such higher mobile fractions indicate the anthropogenic sources, and multivariate analysis results also supported the existence of such sources. The relationship between pH and mobile fractions of the metals (sum of acid soluble, reducible, and oxidizable) indicates that Cd and Pb become more mobile under decreasing pH conditions. Considering all metals, except for Cd, a strong relationship was observed between metal concentrations of apple leaves and mobile fractions, and plants were primarily able to uptake the metals in these fractions. Contamination factor and risk assessment code results together also revealed that Cd was highly mobile; retained less and had high risk for the environment.     

RAPD markers efficiently distinguish heterogenous populations of wild potato (Solanum)

Genetic characterization of germplasm is important for setting objective guidelines for conservation. One common problem found in genebanks is determining the value of populations with insufficient or unreliable data regarding their geographic origin. In this study, a genetic analysis based on RAPD markers was conducted to characterize a `mystery' population of Solanum sucrense, a polysomic tetraploid potato (2n=4x=48), for which adequate documentation was lacking. The comparative analysis of genetic similarities between this mystery population and each one of 30 other S. sucrense populations in the genebank revealed that all populations within this species, including the mystery population, are significantly different from being duplicates, and are therefore worthy of separate conservation. RAPD markers also distinguished the mystery population from closely related tetraploid species S. oplocense, S. gourlayi and S. tuberosum ssp. andigena, suggesting that it is also not a duplicate of a population of these species. If RAPDs can clearly differentiate populations within highly heterogeneous tetraploids like S. sucrense, they should be generally useful for determining germplasm organization within potato species.     

Differential morphogenetic responses, ginsenoside metabolism and RAPD patterns of three Panax species

Genetic and metabolomic demarcations between two Indian and one American congeners of Genus Panax have been discerned. Genomic DNA was isolated from the root derived callus cultures of these species and amplified by AP-PCR. RAPD analyses of the DNA with six most responding arbitrary oligonucleotide decamers provided a total of 70 reproducible bands for computation of the similarity matrix amongst the Panax species. Only 18 of these were monomorphic giving an estimate of about 74% polymorphism among the test species examined. The similarity coefficient values based on the amplification pattern support an equidistant position of the three test species. The molecular demarcations between the species are also manifested in terms of their characteristic cultural requirements, in vitro growth kinetics, regeneration competence and ginsenoside complement of their calli. The Indian congeners i.e. P. sikkimensis and P. pseudoginseng were distinguishable by higher proportions of ginsenoside Rf and Ro (40% and 20%, respectively) in the crude saponin fractions. Furthermore P. quinquefolium calli mainly accumulated ginsenoside Rb₂ and Rg₁, whilst P. sikkimensis callus was rich in Rd fraction. P. quinquefolium showed high similarity with P. sikkimensis with respect to plasticity and totipotency for somatic embryogeny whereas P. pseudoginseng callus was highly recalcitrant and lacked regenerative capacity. The chemical and genetic fingerprints alongwith morphogenetic responses of the three species under controlled in vitro environment strongly advance the case of P. sikkimensis as an independent species, rather than a conglomerate of location specific variety or sub-species of P. pseudoginseng. The findings are also of relevance to formulations and evaluation of ginseng-based health foods.