Genetic diversity among elite Bulgarian barley varieties evaluated by RFLP and RAPD markers

Genetic variation among five elite winter barley cultivars (H. vulgare L.) currently grown in Bulgaria was assessed at the molecular level using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) markers. The present study sampled RFLPs in four well characterized multigene families in barley: the seed storage protein loci; the 18S, 5.8S and 26S ribosomal DNA loci; the loci coding for 5S ribosomal RNA and the loci coding subunit α of ATP-A complex in the mitochondrial genome. RFLPs were detected in three out of five investigated chromosomal loci in the barley cultivars studied. RAPD assay using arbitrary 10-base primers was applied to generate amplified length polymorphic markers in barley. Overall a total of 15 polymorphic phenotypes were found among the studied barley cultivars by using 11 out of 25 tested primers. All RAPDs were considered as dominant genetic markers except for two, where PCR and Southern blot analysis indicated the presence of codominant amplification products. Five RAPD polymorphisms in F₁ and F₂ progenies of the cross between Alpha and Obzor were inherited in Mendelian fashion. The determined values for the genetic variation proved a high genetic similarity among the tested cultivars. Genetic similarity (GS) calculated from RFLP and RAPD data ranged from 0.888 to 0.997 with a mean GS – 0.933. This revised version was published online in July 2006 with corrections to the Cover Date.     

DNA-RAPDs detect genetic variation and paternity in Malus

Summary DNA amplification fingerprinting using arbitrary primer(s) was applied to the identification of Malus species. Highly variable DNA fragment patterns were clearly detected by polyacrylamide gel electrophoresis of the amplified extension products, although three sports of Delicious exhibited the same fingerprint as the original cultivar. The fingerprinting of two triploid apple cultivars suggested that the parent contributing the 2n gamete was the female. We applied this fingerprinting to paternity analysis of an apple cultivar of which the pollen parent was unknown. By using 5 arbitrary primers and RFLP analysis of the amplified products, one cultivar was singled out for paternity among six putative candidates.     

Neutral DNA markers fail to detect genetic divergence in an ecologically important trait

The development of strategies for in situ, ex situ conservation and reforestation of the monkey puzzle tree (Araucaria araucana), a vulnerable tree endemic to southern South America, has led to an interest in the level and distribution of the genetic diversity of the species. Neutral DNA markers (RAPDs) and quantitative genetic techniques were used to characterise genetic heterogeneity within and among populations from throughout the natural range of the species. Both the level and pattern of genetic variation estimated using the different techniques were essentially uncorrelated. An important discrepancy was found with the neutral markers failing to detect an important quantitative genetic divergence across the Andean Range relating to drought tolerance. This study clearly demonstrates the potential problems associated with making recommendations for conserving the genetic resource of threatened species based solely on neutral marker studies. Alternative approaches are discussed, including a stronger focus on ecologically important traits and the potential use of surrogate measures of genetic variability.     

Genetic Structure of Wild and Domesticated Populations of Capsicum annuum (Solanaceae) from Northwestern Mexico Analyzed by RAPDs

Levels of genetic variation and genetic structure of 15 wild populations and three domesticated populations of Capsicum annuum were studied by RAPD markers. A total of 166 bands (all of them polymorphic) and 126 bands (125 of them polymorphic) were amplified in wild and domesticated populations, respectively. Mean percentage of polymorphism was 34.2% in wild populations and 34.7% in domesticated populations. Mean and total genetic diversity were 0.069 and 0.165 for wild populations and 0.081 and 0.131 for domesticated populations. Parameters of genetic diversity estimated from 54 bands with frequencies ≥1 − (3/n) (n = sample size) showed that 56.7% of the total variation was within and 43.3% among wild populations, whereas 67.8% of the variation was within and 32.2% among domesticated populations. AMOVA indicated that total genetic diversity was equally distributed within (48.9 and 50.0%) and among (50.0 and 51.1%) populations in both wild and domesticated samples. Wild and domesticated populations were clearly resolved in a UPGMA dendrogram constructed from Jaccard’s distances (average GD = 0.197), as well as by AMOVA (17.2% of variance among populations types, p = 0.001) and by multidimensional scaling analysis. Such differentiation can be associated with domestication as well as different origin of gene pools of the wild (Northwestern Mexico) and cultivated (more probably Central Mexico) samples analyzed. The considerable genetic distances among cultivars (average GD = 0.254) as well as the high number of diagnostic bands per cultivar (33 out of 126 bands), suggest that genetic changes associated with domestication could have resulted from artificial selection intervening in different directions, but the inclusion of more domesticated samples might clarify the nature of distinctions detected here.     

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.     

Genetic variation in wild and cultivated artichoke revealedby RAPD markers

Determination of intraspecific genetic diversity is an important first step for the utilization of genetic resources and canprovide useful information on crop evolution. A living collection of Italian and foreign artichoke varieties and ecotypes is maintained at the Germplasm Institute, Bari, Italy. A total of 32 accessions of cultivated (Cynara cardunculus L. var.scolymus (L.) Fiori), three of wild (Cynara cardunculus var.cardunculus L.) artichoke and two ofcultivated cardoon (Cynara cardunculus var.altilis L.) were analysed in order to studygenetic variation and relationships within the species, using RAPDmarkers. Cultivated accessions were selected according tomorphological variation and geographical distribution available inthe collection. Fifty arbitrary decamer primers were initially tested, 18 ofwhich showed from 3 to 10 unambiguously interpretable fragments. Anintra-accession analysis using 4 varieties and 8 polymorphicprimers revealed that no RAPD variation was detected amongindividuals. Jaccard's similarity index (JSI) was comprisedamong 1 and 0.693 when all accessions were considered, on the otherhand, within the cultivated artichoke, JSI ranged between 1 and0.817. A UPGMA dendrogram based on the similarity matrix showed thatwild artichokes were clearly separated from cultivated accessions.Moreover, within cultivated artichokes, several groups could bedistinguished.     

Genetic structure of natural Tunisian Hypericum humifusum L. (Hypericacae) populations as assessed by allozymes and RAPDs

The genetic diversity within and among seven Tunisian natural populations of Hypericum humifusum L., from different geographic regions and bioclimates, was assessed using 11 isozymic polymorphic loci, and 166 RAPD markers amplified by 8 primers. The genetic diversity within populations based on allozymes was higher (P = 72.46%, Ap = 2.01 and H_e = 0.29), than that revealed by RAPDs (29.52 < P < 39.16% and 0.150 < H < 0.200). Both markers yielded high estimates of genetic differentiation and low gene flow (Nm = 0.257 and 0.508 for RAPD and allozymes, respectively) among populations at all space scales. However, the level of differentiation revealed by RAPDs (Φ_ST = 0.494; G_ST = 0.561) was higher than that based on allozymes (F_ST = 0.117). No correlation (Mantel test) among genetic (F_ST and Φ_ST matrices) and geographical distance matrices was observed indicating no isolation by distance. Cluster analyses from allozyme and RAPD loci did not completely agree. The dendrogram based on allozymes yielded higher separation among most populations, while that from RAPDs separated populations into three distinct subclusters. Groupings of populations, in both dendrograms, did not reflect spatial geographic or bioclimatic patterns, indicating specific adaptation of populations to local environments. The correlation between matrices of allozyme and RAPD band frequencies was not significant (Mantel test). The dendrogram obtained from combined data yielded similar population groupings to that probed by RAPDs suggesting higher accuracy of these markers. Given the high differentiation among all populations even at a low geographic distance, ex situ conservation should involve extensive seed collection from all populations from all bioclimatic zones. The populations from the upper semi-arid bioclimate exhibiting relatively high level of genetic diversity should be first protected.     

Analysis of genetic variation in Cucurbita moschata by random amplified polymorphic DNA (RAPD) markers

Knowledge of genetic relationships among genotypes is essential for the effective utilisation of germplasm, especially for poorly characterised species. Random amplified polymorphic DNA (RAPD) analysis provides a quick and reliable method for resolving genetic relationships. Although Cucurbita moschata Duch, also known as tropical pumpkin, is one of the most important vegetable crops in Africa, being adapted to a wide range of climatic and soil conditions, it is a scientifically neglected species. The objectives of this study were to (1) analyse the amount of genetic diversity inC. moschata landraces grown in south-central Africa and (2) classify the landraces to assist in selection of parent genotypes for improvement of fruit characteristics. Cluster analysis, based on 39 polymorphic and 105 monomorphic DNA fragments amplified by 16 primers, was used to show relationships among 31 genotypes obtained from Zambia and Malawi. The analysis revealed four clusters, with genotypes from Malawi mainly clustering in three clusters while all genotypes from Zambia and three from Malawi clustered in one cluster. The pair-wise mean genetic distance was 0.32 ± 0.04 for samples from Malawi and 0.26 ± 0.04 for samples from Zambia. The possible application of the resulting classification in breeding of C. moschata is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Phylogeny of Vicia Subgenus Vicia (Fabaceae) Based on Analysis of RAPDs and RFLP of PCR-Amplified Chloroplast Genes

We report the results of two methods of DNA analysis to elucidate phylogenetic relationships among 29 Vicia subgen. Vicia species in comparison with two species of subgenus Vicilla sect. Vicilla. The methods employed were RAPD analysis of total genomic DNA and PCR-RFLP analysis of five chloroplast genes, rbcL, rpoB, 16S, psaA and trnK. The results of each method were similar and complementary, and support the current taxonomic systems of subsp. Vicia. According to RAPD and PCR-RFLP analysis the Narbonensis complex can be considered a well separated section, which may be related to section Vicia. Sections Vicia, Atossa and Wiggersia are separate, but closely related sections. Species of the section Hypechusa form a single monophyletic section, where V. lutea, V. anatolica and V. hyrcanica are quite remote from other species. Our results suggest that within the subgenus Vicia, V. faba is more closely related to V. bithynica and that these two species are most closely related to section Peregrinae. We found that PCR-RFLP of cp DNA provided more precise information concerning relationships between Vicia sections than RAPD analysis. However, RAPD analysis was more informative concerning diversity of closely related Vicia taxa, such as the variable groups, section Narbonensis and V. sativa aggregate.     

分子标记法和形态学特征对杏仁栽培种及野生种基因型的遗传关系构建的辨别

应用23个形态学特征,19个扩增片段长度多态性(AFLP)引物组合,80个随机扩增多态DNA(RAPD)引物和32个简单序列重复(SSR)引物对,比较三种分子标记法在29个杏仁栽培种和3个野生种遗传关系构建中的信息景和效率.根据预期杂合度的评价,与AFLPs和RAPDs相比,SSRs具有较高水平的多态性和较大的信息量.AFLPs预期朵合度值最低,但其辨别效率值最高,因为AFLPs能揭示每个反应中的大量条带,导致各种类型的多样性指数均较高.三种分子标记法对杏仁基因型的辨别效率均较高,只是SSRs无法辨别‘Monagha'和‘Sefied'杏仁基因型.三种分子标记法基因型相似性相关系数统计上显著,但SSR数据要低于RAPDs和AFLPs的值.尽管三种分子标记法树形图拓扑结构存在一些差异,但相似性水平均较高.SSRs、RAPDs和AFLPs的系统树图及其综合数据都能依据地理散布反映大多数栽培种的关系.AMOVA检测到每个地理组中栽培种和野生种的变异.辅助程序分析表明,实验所应用的标记物数量足以保证基因相似性估计的可靠性和标记法间的比较是有意义的.