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 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.     

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.     

Microsatellite markers and genetic diversity assessment in Lolium temulentum

Lolium and Festuca are two important genera of cool-season forage and turf grasses worldwide. Lolium temulentum L. (darnel ryegrass) has been proposed as a model species for genomics studies of cool-season forage and turf grasses. A study with 41 darnel ryegrass, three tall fescue (Festuca arundinacea Schreb.), two tetraploid fescue (F. glaucescens), and two meadow fescue (F. pratensis) genotypes was initiated to (i) identify a set of microsatellite (simple sequence repeats) markers useful for L. temulentum L., and (ii) to utilize such markers for assessing the genetic variability of L. temulentum accessions collected from different geographical regions of the world. A total of 40 tall fescue (TF) EST-SSRs and 60 Festuca–Lolium (F × L) genomic SSRs were screened on a subset of eight genotypes. The selected 30 tall fescue EST-SSRs and 32 F × L genomic SSRs were used for further analysis of genotypes. The TF-EST- and the F × L genomic-SSRs identified 10.3 and 9.3 alleles per marker, respectively with an average polymorphic information content (PIC) value of 0.66. The phenogram based on 319 EST-SSR and 296 genomic SSR fragments, grouped L. temulentum accessions into three major clusters except for accession ABY-BA 8892.78. Lolium temulentum accession ABY-BA 8892.78 did not cluster with any other accession. The Festuca clusters were distantly related with darnel ryegrass clusters with a similarity coefficient of 0.26. The selected set of tall fescue EST- and F × L genomic SSRs were useful in assessing L. temulentum genetic diversity and could benefit the genetic improvement of members of the Festuca–Lolium complex.     

Genetic relationships and diversity among Brazilian cultivars and landraces of common beans (Phaseolus vulgaris L.) revealed by AFLP markers

The genetic variation and relationships among 31 accessions of Phaseolus vulgaris L., and two representatives of Vigna unguiculata L., were evaluated by AFLP analysis. A total of 263 DNA fragments across all materials were scored using nine primer combinations, averaging 32 per primer. More than 95% of the amplification products showed polymorphism, indicating high variation at the DNA level among these accessions. Pair-wise genetic similarity (Jaccard's coefficient) ranged from 0.553 to 0.840, with a mean of 0.765. Twenty-three accessions (70%) clustered into three groups. A majority of the commercial cultivars (91%) clustered within a single group, whereas the landraces were distributed along all the variation. An apparent correlation with phaseolin types was detected. Results of this study suggest that Brazilian landraces truly represent the overall genetic variability of Phaseolus vulgaris, confirming the multiple origins of these materials, and their potential as a source of variation for breeding programs.     

Genetic diversity in Ethiopian hexaploid and tetraploid wheat germplasm assessed by microsatellite markers

The genetic diversity of a subset of the Ethiopian genebank collection maintained at the IPK Gatersleben was investigated applying 22 wheat microsatellites (WMS). The material consisted of 135 accessions belonging to the species T. aestivum L. (69 accessions), T. aethiopicum Jacubz. (54 accessions) and T. durum Desf. (12 accessions), obtained from different collection missions. In total 286 alleles were detected, ranging from 4 to 26 per WMS. For the three species T. aestivum, T. aethiopicum and T. durum on average 9.9, 7.9 and 7.9 alleles per locus, respectively, were observed. The average PIC values per locus were highly comparable for the three species analysed. Considering the genomes it was shown that the largest numbers of alleles per locus occurred in the B genome (18.4 alleles per locus) compared to A (10.1 alleles per locus) and D (8.2 alleles per locus) genomes. Genetic dissimilarity values between accessions were used to produce a dendrogram. All accessions could be distinguished, clustering in two large groups. Whereas T. aestivum formed a separate cluster, no clear discrimination between the two tetraploid species T. durum and T. aethiopicum was observed.     

Isozyme polymorphism and geographic differentiation in a collection of French perennial ryegrass populations

Summary A sample of 60 natural populations of perennial ryegrass from France has been studied for allelic variation at 7 polymorphic enzyme loci. Population genetic statistics are of the same magnitude than those previously reported for other outbreeding, short-lived perennial species (P = 64%, A = 2.75, H = 0.270).Genotype frequencies at most collection sites do not deviate significantly from Hardy-Weinberg expectations, with however a slight deficit of heterozygotes which may be accounted for by a Wahlund effect. Gene diversity is mainly explained by the within population component. The between population differentiation F_st averaged on 4 loci is only 0.054, which accounts for only 6% of the whole diversity.When mapped, most allele frequencies do not show any special structure. Only five alleles present a clinal trend from North to South. These 5 alleles are probably related to some climatic factors such as average temperature or potential evapotranspiration. The causal hypotheses about the low level of between-population differentiation and spatial structure are discussed with reference to the literature.The consequences of the found population structure for sampling and conservation strategies of natural populations for genetic resources are presented and discussed.     

Genetic diversity of introduced and local Spanish persimmon cultivars revealed by RAPD markers

Persimmon culture has become an alternative for fruit growers in Italy, Israel and Spain. However, there is some confusion concerning cultivar identification in non-Asian countries were the species was introduced. Authenticating the identity of germplasm resources of persimmon would be a great value for breeding. Due to the floral biology of the persimmon species the expected genetic variability is high. RAPD markers were chosen for this study of persimmon germplasm resources in Spain. Nineteen markers were obtained from 8 primers. The average of 2.4 markers/primer was higher than that obtained in other woody fruit species. The availability of data referenced from isozymes and pomological traits revealed complete agreement among groups identified by RAPDs, isozymes and pomological traits. Results suggest that RAPD technology is adequate for fingerprinting persimmon and the data is consistent with persimmon's hypothetical origins, adaptation history and previous classification by isozymes or pomological traits. Many synonyms have been identified, mostly in cultivars from local surveys, suggesting that a few new genotypes arose in Spain since the species was introduced to Europe. By means of RAPD markers, a better identification of the European persimmon germplasm is available.     

Influence of ecological factors on population differentiation in perennial ryegrass (Lolium perenne L.)

Summary Microgeographic differentiation was investigated at the phenotypic level for 10 agronomic traits between 24 pairs of adjacent populations of perennial ryegrass from Ireland. None of the univariate or multivariate statistical tests succeeded in finding any relationship between ecological characteristics of the contrasting sites and agronomic traits, which could have been interpreted as a result of directional selection.Complementary studies of isozyme loci on 12 populations indicated some genetic divergence for neutral genes in 3 out of 6 pairs. These genetically differentiated populations also differ for agronomic traits, and notably for heading date, which could lead to reproductive isolation.The possible influence of random drift, selection and reproductive isolation are discussed, as well as the consequences for collecting natural populations as genetic resources for breeding use.     

Inter-Simple Sequence Repeat fingerprints to assess genetic diversity in Tunisian fig (Ficus carica L.) germplasm

The genetic diversity of 18 Tunisian fig cultivars was investigated at the DNA level using the Inter Simple Sequence Repeat (ISSR) associated with the Polymerase Chain Reaction (PCR). Using a set of primers, the most informative ones were selected that were characterized by an important Resolving power value of 29.6. A total of 47 discernible fragments were scored from samples, with a mean of 11.7 fragments per primer. The 90.4% of sample that were polymorphic were scored as molecular markers to examine the Tunisian fig germplasm polymorphism at DNA level. A large genetic diversity as related to ISSR patterns was found within the local Tunisian fig germplasm. An UPGMA dendrogram exhibits the unstructured variability in this crop. Moreover, the principal component analysis shows that the observed diversity was typically continuous. Our data provide a large number of ISSR markers that are useful in the fingerprinting of Ficus carica L. cultivars, and in the understanding of the genetic relationships among these accessions.     

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.     

Analysis of genetic diversity in the endangered tropical tree species <Emphasis Type="Italic">Hagenia abyssinica</Emphasis> using ISSR markers

Genetic diversity within and among 12 populations of the dioecious tropical tree species Hagenia abyssinica (Bruce) J.F. Gmel. in Ethiopia was examined with eight inter simple sequence repeat (ISSR) primers. A total of 104 clearly scorable bands were generated, among which 84 (81%) were polymorphic. Jaccard similarity coefficient was calculated for pairwise comparisons among all 120 individuals and ranged from 0.30 to 0.88 while average within-population similarity ranged from 0.53 to 0.66. Within-population variability was estimated as percentage polymorphic loci (ranging from 52% to 87%), Shannon’s information index (0.30–0.50) and Nei’s genetic diversity (0.21–0.35). The highest variability values were obtained for one recently planted population and for one wild population growing in an undisturbed primary forest area. Significant overall differentiation among populations was detected by both Shannon’s information index (0.26) and G _ST (0.25). Relatedness among samples was estimated with a principal coordinate analysis, and relatedness among populations was estimated with a cluster analysis (UPGMA). A Mantel test indicated a significant association between genetic and geographic distances, and an autocorrelation analysis showed significant evidence of gene flow over distances up to 30 km. This study is the first of its kind for H. abyssinica, which has decreased recently in Ethiopia and now must be regarded as an endangered species. Both within-population and between-population diversity estimates are typical of outcrossing, longlived and late successional species, suggesting that recent anthropogenic disturbances have not yet had much impact on population genetic parameters. DNA marker data can, however, be used to identify the most suitable sites for in situ conservation and for collection of material for establishment of genebanks and plant improvement programs.     

Molecular diversity of Omani wheat revealed by microsatellites: I. Tetraploid landraces

Results of archaeological studies indicate a millennia-old cultivation history for wheat (Triticum spp.) in Oman. However, in spite of numerous collection surveys and efforts for phenotypic characterization of Omani wheat landraces, no attempts have been made using molecular tools to characterize this germplasm. To fill this gap, 29 microsatellite markers revealing 30 loci were used to study the genetic diversity of 38 tetraploid wheat landrace accessions comprising the species T. dicoccon, T. durum and T. aethiopicum. A total of 219 alleles were detected whereby the number of alleles per locus ranged from 2 to 16 with an average number of 7.1 alleles per locus. The highest number of alleles occurred in the B genome with on average 7.9 alleles per locus as compared to the A genome with 6.5 alleles per locus. Heterogeneity was detected for all microsatellites except for GWM 312, GWM 601 and GWM 192B with an average heterogeneity over all primers and lines of 14.4%. Approximately 10% of the accessions contained rare alleles with an average allele frequency <4%. Gene diversity across microsatellite loci ranged from 0.26 to 0.85. The pairwise comparison of genetic similarity ranged from 0.03 to 0.91 with an average of 0.2. Cluster analysis revealed a clear separation of the two species groups T. dicoccon versus T. durum and T. aethiopicum. Within the species clusters regional patterns of subclustering were observed. Overall, this study confirmed the existence of a surprisingly high amount of genetic diversity in Omani wheat landraces as already concluded from previous morphological analyses and showed that SSR markers can be used for landraces’ analysis and a more detailed diversity evaluation.     

47份水稻品种资源的ISSR遗传多样性分析

为研究广东省惠州市种植的常规水稻品种的遗传多样性,本实验利用ISSR标记对47份水稻品种资源进行遗传多样性检测。从49条引物中筛选出5条重复性好,条带清晰的引物进行PCR扩增,共扩增出53条带,每个引物可以扩增出9～13条带,平均为10．6条,其中47条具有多态性,比率为88．7％。不同水稻品种间遗传相似系数变幅为0．319～0．936,平均达0．691,说明ISSR标记能够揭示材料间较高的遗传多样性。通过聚类,从分子水平对水稻品种资源的遗传关系进行分析,并对47份水稻品种资源进行分类,ISSR标记能将47份水稻品种完全区分开,为水稻品种资源的研究利用提供参考。     

Genetic Diversity within and Between Populations of Lathyrus genus (Fabaceae) Revealed by ISSR Markers

In order to evaluate the genetic diversity in Lathyrus genus, the Inter Simple Sequence Repeats method (ISSR) was exploited in five populations. These consisted of two cultivated species belonging to section Lathyrus (L. sativus L. and L. cicera L.) and a wild one belonging to the section Clymenum (L. ochrus DC.). Two 3′anchored ISSR primers and two unanchored ones, generated a total of 60 useful polymorphic DNA bands. Our data provide evidence of high molecular polymorphism at the intra- and the inter-specific levels showing that both wild and cultivated forms constitute an important pool of diversity. Moreover, among the generated DNA bands, a 500 bp band, totally absent in the banding patterns of the section Clymenum, appears to be a molecular marker of section Lathyrus. Results provided for lineage and suggest recent origin of these species that might have evolved from a common ancestor producing both L. ochrus species and the two other species L. sativus and L. cicera. These relationships support previous studies based on morphological variation and molecular analysis.     

利用ISSR揭示不同类型月季遗传多样性

本研究应用正交设计法对ISSR反应体系中的各个主要影响因子进行了优化筛选,确立了适合月季ISSR-PCR反应的最佳体系。结果表明,25μL的ISSR反应体系中各组分的最适浓度分别为：1×PCR缓冲液、1U Taq DNA聚合酶、800pmol／L 引物、0.16mmol／L dNTPs、Mg^2＋ 1.5mmol／L。筛选了33个ISSR引物,共得到了11个多态性比较高的ISSR引物,占所筛引物的33.33％。利用筛选出的11条ISSR引物对3种月季类型的23份月季材料进行遗传多样性分析,共扩增出477条DNA带,其中多态性位点有14个,平均每条引物可以检测到4.5个多态性位点。用NTSYS软件对样品进行了UPGMA聚类分析,聚类结果表明丰花月季基本能聚为一类,切花月季与藤本月季交叉聚在一起。这表明月季种质的遗传差异与其应用分类的相关性不紧密。     

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.     

Genetic diversity and differentiation in Ethiopian populations of Phytolacca dodecandra as revealed by AFLP and RAPD analyses

Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analyses were performed on six populations (a total of 89 individuals) of Phytolacca dodecandra (endod) collected in Ethiopia. The populations were selected based on our previous investigation to represent two altitude groups: lowland/central-highland (1600–2500 m) and highland (2501–3000 m). A total of 197 AFLP and 68 RAPD markers were scored from 5 primer pairs and 12 random primers, respectively. The overall patterns obtained for AFLPs and RAPDs from diversity, cluster and principal component analyses were very comparable. However, the moderate correlation (r = 0.56) between AFLP and RAPD similarity matrices as well as the discrepancies in diversity estimates between the two techniques in some populations and in the lowland/central-highland plants could be due to differences in sensitivity of reaction conditions, bias in scoring of bands, number of markers used for analyses, and/or parts of the genome surveyed. For both AFLP and RAPD, the lowland/central-highland populations showed higher polymorphism and Shannon information measure (H) than the highlands. Cluster and principal component analyses performed for both marker types have also clearly demonstrated the differentiation of all the lowland/central highland plants from those of the highlands, in agreement with our previous conclusion. Markers scored from any of the five AFLP primer pairs were sufficient to clearly distinguish the two altitude groups; with RAPD, selection of about 8 informative markers produced by seven random primers was needed for the same purpose.     

Detection of genetic diversity in Libyan wheat genotypesusing wheat microsatellite markers

Twenty-four wheat microsatellites (WMS) wereused to estimate the extent of genetic diversity among 15 Libyanwheat genotypes. The WMS used determined 26 loci located on 20different chromosomes, and were capable of detecting 116 alleles withan average of 4.5 alleles per locus. Only two markers located on 2DSand 4DL, were monomorphic. The results indicated that the B genome(5.9 alleles per locus) was more variable than the A and Dgenomes (4.1 and 2.7 alleles per locus, respectively).Furthermore, the results obtained suggest that a relatively smallnumber of primers can be used to distinguish all genotypes used andto estimate their genetic diversity. Genetic dissimilarity valuesbetween genotypes, calculated by the WMS derived data, were used toproduce a dendrogram. The diversity within the analysed germplasm isdiscussed.     

Genetic diversity of <Emphasis Type="Italic">Nelumbo</Emphasis> accessions revealed by RAPD

Total 65 lotus accessions in genus Nelumbo mainly collected from China, were subjected to random amplified polymorphic DNA (RAPD) markers to estimate the genetic diversity and to test the genetic basis of the relationships between morphotypes and molecular markers. Seventeen primers generated a total of 195 highly reproducible and discernible loci, among which 173 were polymorphic. Percent polymorphism varied from 66.7 to 100 with an average of 88.72, and five primers out of them, OPC05, OPG10, OPN20, OPP09 and OPS17, showed 100% polymorphism. A relatively high genetic diversity was detected among all the samples with the similarity coefficient values ranging from 0.45 to 0.85, and Nei’s gene diversity (h) 0.30, and Shannon index (I) 0.46. The UPGMA dendrogram clustered 65 accessions in four clusters and the clustering pattern showed two groups, N. nucifera ssp. nucifera and those accessions related to the American lotus, and some special cultivars, landraces, hybrids and the American lotus. Principal Coordinate Analysis (PCA) further indicated that the genetic diversity of Nelumbo accessions was not evenly distributed, instead, was presented by a clustered distribution pattern. Similar to the results revealed by the dendrogram, two main groups representing the two subspecies of N. nucifera, as well as some special landraces, cultivars of Chinese lotus, the Japanese lotus and hybrids out of the two groups were obtained. Neither the UPGMA dendrogram nor the PCA analysis exhibited strict relationship with geographic distribution and morphotypes among the accessions.