Assessing Genetic Diversity in <Emphasis Type="Italic">Olea europaea</Emphasis> L. Using ISSR and SSR Markers

Olea europaea L. is one of the most economically important crops in the Mediterranean area, and known for having large genetic variability. In order to assess the genetic diversity, DNA from 41 olive cultivars, present in the protected denomination of origin (PDO) region of Trás-os-Montes, was screened using inter simple sequence repeat (ISSR) and microsatellite (SSR) markers. Eleven ISSR primers amplified 135 reproducible bands of which 108 were polymorphic. The percentage of polymorphic bands detected by ISSR was 79%. The highest number of polymorphic bands was obtained by the use of primers UBC807 (15) and UBC809 (16). A total of 67 alleles were detected by six SSR primers, with an average of 11 alleles per primer. The number of alleles per locus ranged from five (ssrOeUaDCA05) to 15 (ssrOeUaDCA03). The observed heterozygosity ranged from 0.219 (ssrOeUaDCA05) to 0.900 (ssrOeUaDCA04), while the expected heterozygosity varied between 0.426 (ssrOeUaDCA05) and 0.887 (ssrOeUaDCA03). The polymorphism information content (PIC) values ranged from 0.392 (ssrOeUaDCA05) to 0.863 (ssrOeUaDCA03). The collection of primers selected gave a reasonable number of amplification products for the genetic diversity analysis. Based on the results, the genetic diversity among 41 olive cultivars is discussed. This study reveals the great importance of guaranteeing the differentiation of olive cultivars and their application for certification purposes.     

Genetic diversity revealed by EST-SSR markers in carob tree (Ceratonia siliqua L.)

Carob is a caesalpinoid tree species widespread in the Mediterranean. In this paper, carob EST-SSR (Expressed Sequence Tags-Simple Sequence Repeat) markers were used to assess the level of genetic diversity among 71 cultivars and accessions collected in three Mediterranean countries (Italy, Malta and Spain). Starting from 20 microsatellite loci, we identified a set of 9 polymorphic EST-SSRs, with a number of alleles per locus ranging from 2 to 7, and a number of genotypes from 3 to 13. The nine polymorphic EST-SSRs discriminated most of the analyzed genotypes with the exceptions of some cultivated clones having similar phenotypic features and probably belonging to cultivar-populations, and clarified some cases of homonymy.A neighbor joining dendrogram generated three main clusters which did not show a correlation with the distribution areas of the analyzed genotypes. A Bayesian analysis evidenced a high degree of admixture among the gene pools from the three countries. The low level of diversification among geographical areas reveals that the asexual propagation of selected clones played a major role in the diffusion of the species in the western Mediterranean. The work described in this paper represents the first report of carob genotyping achieved through microsatellite markers.     

Genetic diversity analysis of common beans based on molecular markers

A core collection of the common bean (Phaseolus vulgaris L.), representing genetic diversity in the entire Mexican holding, is kept at the INIFAP (Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias, Mexico) Germplasm Bank. After evaluation, the genetic structure of this collection (200 accessions) was compared with that of landraces from the states of Oaxaca, Chiapas and Veracruz (10 genotypes from each), as well as a further 10 cultivars, by means of four amplified fragment length polymorphisms (AFLP) +3/+3 primer combinations and seven simple sequence repeats (SSR) loci, in order to define genetic diversity, variability and mutual relationships. Data underwent cluster (UPGMA) and molecular variance (AMOVA) analyses. AFLP analysis produced 530 bands (88.5% polymorphic) while SSR primers amplified 174 alleles, all polymorphic (8.2 alleles per locus). AFLP indicated that the highest genetic diversity was to be found in ten commercial-seed classes from two major groups of accessions from Central Mexico and Chiapas, which seems to be an important center of diversity in the south. A third group included genotypes from Nueva Granada, Mesoamerica, Jalisco and Durango races. Here, SSR analysis indicated a reduced number of shared haplotypes among accessions, whereas the highest genetic components of AMOVA variation were found within accessions. Genetic diversity observed in the common-bean core collection represents an important sample of the total Phaseolus genetic variability at the main Germplasm Bank of INIFAP. Molecular marker strategies could contribute to a better understanding of the genetic structure of the core collection as well as to its improvement and validation.     

利用SRAP标记分析河南小麦栽培品种的遗传多样性

利用小麦SRAP标记对22个河南省小麦品种进行了遗传多样性分析,10对引物组合扩增获得169个条带,其中70个条带具有多态性,多态条带百分率为41.42％,每对引物平均产生7个多态性条带。22个供试材料的带型按照条带的有,无分别记录为1,0后,采用Nei 72方法计算不同品种的遗传距离,利用NTSYS软件进行非加权成组法（UPGMA）进行了聚类分析。结果表明SRAP标记技术能较真实地反映小麦品种间的亲缘关系,可以用于小麦品种遗传多样性的研究。     

利用SRAP标记分析河南小麦栽培品种的遗传多样性

利用小麦SRAP标记对22个河南省小麦品种进行了遗传多样性分析,10对引物组合扩增获得169个条带,其中70个条带具有多态性,多态条带百分率为41．42％,每对引物平均产生7个多态性条带。22个供试材料的带型按照条带的有、无分别记录为1、0后,采用Nei72方法计算不同品种的遗传距离,利用NTSYS软件进行非加权组法（UPGMA）聚类分析。结果表明SRAP标记技术能较真实地反映小麦品种间的亲缘关系,可以用于小麦品种遗传多样性研究。     

Comparative study of the discriminating capacity of RAPD,AFLP and SSR markers and of their effectiveness in establishing genetic relationships in olive

RAPDs, AFLPs and SSRs were compared in terms of their informativeness and efficiency in a study of genetic diversity and relationships among 32 olive cultivars cultivated in Italy and Spain. SSRs presented a higher level of polymorphism and a greater information content, as assessed by the expected heterozygosity, than AFLPs and RAPDs. The lowest values of expected heterozygosity were obtained for AFLPs, which, nevertheless were the most efficient marker system due to their capacity to reveal the highest number of bands per reaction and because of the high values achieved for a considerable number of indexes. All three techniques discriminated the genotypes very effectively, but only SSRs were able to discriminate the cultivars Frantoio and Cellina. The correlation coefficients of similarity were statistically significant for all three marker systems used but were lower for the SSR data than for RAPDs and AFLPs. For all markers a high similarity in dendrogram topologies was obtained although some differences were observed. All the dendrograms, including that obtained by the combined use of all the marker data, reflect some relationships for most of the cultivars according to their geographic diffusion. AMOVA analysis detected greater genetic differentiation among cultivars within each country than it did between the two countries.Communicated by H.F. Linskens     

红掌品种亲缘关系SRAP分析

利用相关序列扩增多态性（SRAP）分子标记,从100对引物组合中筛选出 26对多态性高、条带清晰的SRAP引物,对33个红掌品种进行遗传多样性和亲缘关系分析。结果如下：（1）26对引物共扩增出366条条带,其中有314条多态性条带,多态性比率为85.79%。引物组合产生的条带数在9～23之间,平均每对引物组合扩增出14.1条和12.1条多态性条带。（2）根据SRAP扩增结果,利用UPGMA法进行聚类分析,33份材料的遗传相似系数在0.55～0.94之间,在遗传相似系数0.786处可将33个红掌品种分为5个类群。结果表明,供试品种遗传多样性丰富,本研究为品种鉴定和杂交育种提供了参考信息。     

Genetic diversity in colonial bentgrass (Agrostis capillaris L.) revealed by EcoRI-MseI and PstI-MseI AFLP markers.

Colonial bentgrass (Agrostis capillaris L.) is a potential source for genetic improvement of resistance to environmental stress and disease for other bentgrass species (Agrostis spp.). To conserve and study the existing genetic resources of colonial bentgrass for use in breeding, genetic diversity was investigated using amplified fragment length polymorphism (AFLP) markers. Included in this study were 22 accessions from US Department of Agriculture germplasm collected from 11 countries, in conjunction with 14 accessions from northern Spain and 3 commercial cultivars. Ten EcoRI-MseI and 6 PstI-MseI AFLP primer combinations produced 181 and 128 informative polymorphic bands, respectively. Cluster analysis of genetic similarity estimates revealed a high level of diversity in colonial bentgrass species with averages of 0.51 (EcoRI-MseI) and 0.63 (PstI-MseI). Greater genetic diversity was detected by the EcoRI-MseI AFLP primer combinations. A low but significant positive correlation (r = 0.44, p = 0.0099) between the 2 Jaccard similarity matrices was obtained by the Mantel test. Commercial cultivars of bentgrass showed a narrow genetic background. The assessment of genetic diversity among colonial bentgrass accessions suggested the potential value of the colonial bentgrass germplasm in turfgrass cultivar improvement.     

AFLP reveals structural details of genetic diversity within cultivated olive germplasm from the Eastern Mediterranean

Amplified fragment length polymorphism (AFLP) analysis was used to assess genetic inter-relationships among olive varieties cultivated in the Eastern Mediterranean Basin. The genotypes sampled included most of the important cultivars from Turkey, Greece and the Middle East and selected genotypes from the Western Mediterranean area. A total of 119 polymorphic markers were generated from five selective primer-pair combinations. The combined data sets generated by just two primer-pairs were adequate to discriminate between all 65 genotypes, while each primer-pair could individually identify up to 64 genotypes. A factorial correspondence analysis (FCA) plot indicated that the cultivars clustered into two relatively modestly defined groups. The first broad group was dominated by cultivars from Turkey but also included genotypes originating from the Middle East (Syria and Lebanon) that collectively formed a tight subcluster. The second group comprised Greek cultivars and those originating from the Western Mediterranean. A significant genetic distance value between Greek and Turkish cultivars was provided by an analysis of molecular variance (amova). There was also evidence of substructure here, with an apparent separation of most Spanish and Italian clones. These findings are in general accordance to previous suggestions of an East-West divergence of olive cultivars, although the dichotomy is less extensive than reported previously and complicated by regional variation within each group.     

Analysis of the Genetic Structure of Sclerotinia sclerotiorum (Lib.) de Bary Populations from Different Regions and Host Plants by Random Amplified Polymorphic DNA Markers

The genetic diversity and genetic structure of a population of isolates of Sclerotinia sclerotiorum (Lib.) de Bary from different regions and host plants were investigated using the random amplified polymorphic DNA (RAPD) method with 20 random decamer primer pairs in order to provide some information on the phylogenetic taxa and breeding for resistance to sclerotinia stem rot. A minimum of three and a maximum of 15 unambiguously amplified bands were generated, furnishing a total of 170 bands ranging in size from 100to 3 200 bp, corresponding to an average of 8.5 bands per primer pair. One hundred and four of these 170bands (61.2%) were polymorphic, the percentage of polymorphic bands for each primer pair ranging from 0.0% to 86.7%. The genetic relationships among the isolates, based on the results of RAPD analysis, were examined. The genetic similarity of all selected isolates was quite high. At the species level, the genetic diversity estimated by Nei's gene diversity (h) was 0.197 and S hannon's index of diversity (I) was 0.300. The unweighted pair-group mean analysis (UPGMA) cluster analysis showed that most isolates from the same regions were grouped in the same cluster or a close cluster. The population of isolates from Hefei (Anhui Province, China) was more uniform and relatively distant to other populations. The Canadian population collected from carrot (Daucus carota var. sativa DC.) was relatively close to the Polish population collected from oilseed rape (Brassica napus L.) plants. There was no relationship between isolates from the same host plants. An analysis of molecular variance (AMOVA) revealed that the percentage of variance attributable to variation among and within populations was 50.62% and 49.38%, respectively. When accessions from China, Europe, and Canada were treated as three separate groups, the variance components among groups,among populations within groups, and within populations were -0.96%, 51.48%, and 49.47%, respectively.The genetic differentiations among and within populations were highly significant (P ＜ 0.001). Similarly, the coefficient of gene differentiation (Gst) in total populations calculated by population genetic analysis was 0.229 4, which indicated that the genetic variation among populations was 22.94%. The gene flow (Nm)was 1.68, which indicated that the gene permutation and interaction among populations was relatively high.     

Microsatellite marker-based identification and genetic relationships of olive cultivars from the Extremadura region of Spain

Seventy-seven olive accessions corresponding to 25 cultivars from the Extremadura region of Spain were studied using four microsatellite or SSR markers in order to fingerprint them, and evaluate genetic similarity and relationships between local and introduced olive cultivars. The number of alleles per locus ranged from 4 to 8, with a mean of 6.25 alleles per primer pair (a total of 25 alleles). The observed heterozygosity ranged from 0.58 to 0.95, while the expected heterozygosity varied between 0.68 and 0.83. The polymorphism information content values ranged from 0.63 to 0.79. The mean polymorphism information content value of 0.70 for the SSR loci provided sufficient discriminating ability to evaluate the genetic diversity among the cultivars. The SSR data allowed unequivocal identification of all the cultivars; a combination of three SSR markers was sufficient to discriminate all 25 olive cultivars. A dendrogram was prepared, using the unweighted pair-group method with arithmetic mean clustering algorithm; it depicted the pattern of relationships between the cultivars. Most of the local cultivars grouped according to their geographic origin. No clear clustering trends were observed when the morphological traits of fruit endocarps or fruit use of cultivars were employed as analysis criteria. We conclude that there is a high level of variability among local olive cultivars from the Extremadura region at both the morphological and molecular levels; these data should be useful for identifying and distinguishing local germplasm.     

An analysis of genetic variation in natural populations of Sticherus flabellatus [R. Br. (St John)] using amplified fragment length polymorphism (AFLP) markers

Amplified fragment length polymorphisms (AFLPs) were used to characterize the genetic diversity within and among natural populations of Sticherus flabellatus. Eight populations within the Sydney region of New South Wales, Australia were surveyed using 11 primer combinations. A total of 1108 reproducible bands were detected of which 469 (42%) were polymorphic. FST estimates averaged over all polymorphic loci indicated that significant genomic differentiation occurs among populations (average = 0.783). Genetic diversity within populations was assessed according to average heterozygosity (H) and percentage polymorphic loci (P) per population. Within-population diversity ranged from H = 0.12 and P = 33.69 to H = 0.04 and P = 15.99. Analysis of genetic similarity among populations suggested that the eight populations studied fall into two groups of four populations, based on population size and the condition of the habitat. Phenetic analysis (AMOVA) indicated that genetic variation is greater among populations (74.34%) than within populations (25.66%). These findings suggest that the breeding system of S. flabellatus is predominantly inbreeding, with genetic diversity maintained by occasional outcrossing in larger populations. The results presented in this study could provide evidence to support the proposal to protect natural stands of S. flabellatus, which has implications for the Australian horticulture industry.     

Genetic Diversity and Genetic Structure of an Endangered Species, Trillium tschonoskii

The genetic diversity and genetic structure of Trillium tschonoskii (Maxim) were investigated using amplified fragment length polymorphism markers. Eight primer combinations were carried out on 105 different individuals sampled from seven populations. Of the 619 discernible DNA fragments generated, 169 (27.3%) were polymorphic. The percentage of polymorphic bands within populations ranged from 4.52 to 10.50. Genetic diversity (H_E) within populations ranged from 0.0130 to 0.0379, averaging 0.0536 at the species level. Genetic differentiation among populations was detected based on Nei's genetic diversity analysis (53.03%) and analysis of molecular variance (AMOVA) (52.43%). AMOVA indicated significant genetic differentiation among populations (52.43% of the variance) and within populations (47.57% of the variance) (p < 0.0002). Gene flow was low (0.4429) among populations. Species breeding system and limited gene flow among populations are plausible reasons for the high genetic differentiation observed for this species. We propose an appropriate strategy for conserving the genetic resources of T. tschonoskii in China.     

Temporal changes in SSR allelic diversity of major rice cultivars in China

Forty simple sequence repeats （SSRs） were used to assess the changes of diversity in 310 major Chinese rice cultivars grown during the 1950s-1990s. Of the 40 SSR loci, 39 were polymorphic. A total of 221 alleles were detected with an average of 5.7 alleles per locus （Na）. The Nei＇s genetic diversity index （He） varied drastically among the loci （0.207 to 0.874, mean 0.625）. Comparing the temporal changes in Na and He, the cultivars from the 1950s had more alleles and higher He scores than the cultivars from the other four decades. Analysis of molecular variance （AMOVA） indicated that the genetic differentiation among the five decades was not significant in the whole set, but significant within indica and japonica. More changes among the decades were revealed in indica cultivars than in japonica cultivars. Some alleles had been lost in current rice cultivars in the 1990s, occurring more frequently in indica. These results suggest that more elite alien genetic resources should be explored to widen the genetic backgrounds of rice cultivars currently grown in China.     

Multiple origins for Mediterranean olive (Olea europaea L. ssp. europaea) based upon mitochondrial DNA polymorphisms

A study of nuclear and cytoplasmic genetic diversity of cultivated olive, oleaster and other taxa belonging to the complex O. europaea was performed. Nuclear DNA polymorphism (RAPDs) in oleaster displays a gradient between the east and west of the Mediterranean Basin. In cultivars, the gradient is less visible owing to their diffusion and selection. Furthermore, three mitotypes (ME1, MOM and MCK) were detected in both cultivated olive and oleaster. A fourth mitotype, ME2, was unique to some cultivars. The preponderant mitotype, ME1, marks the Near Eastern origin of olive in oleaster. In the west of the Mediterranean, another mitotype, MOM, was found in most oleaster. and a few cultivars. The third, MCK, was found in a few oleaster from the west and in cultivars originating in Kabylie and Languedoc. We argue that MCK marks an ancestral Mediterranean population. The mitotypes mark independent cultivated olive origins which were not detected with DNA nuclear diversity.     

Genetic structure and core collection of the World Olive Germplasm Bank of Marrakech: towards the optimised management and use of Mediterranean olive genetic resources

The conservation of cultivated plants in ex-situ collections is essential for the optimal management and use of their genetic resources. For the olive tree, two world germplasm banks (OWGB) are presently established, in Córdoba (Spain) and Marrakech (Morocco). This latter was recently founded and includes 561 accessions from 14 Mediterranean countries. Using 12 nuclear microsatellites (SSRs) and three chloroplast DNA markers, this collection was characterised to examine the structure of the genetic diversity and propose a set of olive accessions encompassing the whole Mediterranean allelic diversity range. We identified 505 SSR profiles based on a total of 210 alleles. Based on these markers, the genetic diversity was similar to that of cultivars and wild olives which were previously characterised in another study indicating that OWGB Marrakech is representative of Mediterranean olive germplasm. Using a model-based Bayesian clustering method and principal components analysis, this OWGB was structured into three main gene pools corresponding to eastern, central and western parts of the Mediterranean Basin. We proposed 10 cores of 67 accessions capturing all detected alleles and 10 cores of 58 accessions capturing the 186 alleles observed more than once. In each of the 10 cores, a set of 40 accessions was identical, whereas the remaining accessions were different, indicating the need to include complementary criteria such as phenotypic adaptive and agronomic traits. Our study generated a molecular database for the entire OWGB Marrakech that may be used to optimise a strategy for the management of olive genetic resources and their use for subsequent genetic and genomic olive breeding.     

Genetic diversity within and among populations of a threatened species: Erodium paularense Fern. Gonz. & Izco

Erodium paularense Fern. Gonz. & Izco (Geraniaceae) is a perennial, endemic species of central Spain. The restricted distribution of its three populations has made it necessary to consider measures for conservation. Random amplified polymorphic DNA markers were used to determine genetic variation among and within populations. Sixty-three per cent of the bands were polymorphic. No population-specific marker was identified. Cluster analysis based on Jaccard's coefficient of all the individuals grouped most of them according to the present populations. Within-population variability was of a similar order in the three populations. Highly significant genetic differences between populations were detected through AMOVA . Within population diversity contributed 80–85% to the species diversity, a value typical of an outbreeding plant.     

用SSR和AFLP技术分析花生抗青枯病种质遗传多样性的比较

由Ralstonia solanacearum E.F.Smith引起的青枯病是若干亚洲和非洲国家花生生产的重要限制因子,利用抗病品种是防治这一病害最好的措施。虽然一大批抗青枯病花生种质资源材料已被鉴定出来,但对其遗传多样性没有足够的研究,限制了在育种中的有效利用。本研究以31份对青枯病具有不同抗性的栽培种花生种质为材料,通过简单序列重复(SSR)和扩增片段长度多态性(AFLP)技术分析了它们的遗传多样性。通过78对SSR引物和126对AFLP引物的鉴定,筛选出能显示抗青枯病种质多态性的SSR引物29对和AFLP引物32对。所选用的29对多态性SSR引物共扩增91条多态性带,平均每对引物扩增3.14条多态性带;32对多态性AFLP引物共扩增72条多态性带,平均扩增2.25条多态性带。在所筛选引物中,4对SSR引物(14H06,7G02,3A8,16C6)和1对AFLP引物(P1M62)检测花生多态性的效果优于其他引物。SSR分析获得的31个花生种质的遗传距离为0.12-0.94,平均为0.53,而AFLP分析获得的遗传距离为0.06～0.57,平均为0.25,基于SSR分析的遗传距离大于基于AFLP分析的遗传距离,疏枝亚种组的遗传分化相对大于密枝亚种组。基于两种分析方法所获得的聚类结果基本一致,但SSR数据聚类结果与栽培种花生的形态分类系统更为吻合。根据分析结果,对构建青枯病抗性遗传图谱群体的核心亲本和抗性育种策略提出了建议。     

Simple Sequence Repeat Analysis of Genetic Diversity in Primary Core Collection of Peach （Prunus persica）

In this study, the genetic diversity of 51 cultivars in the primary core collection of peach （Prunus persica （L.） Batsch） was evaluated by using simple sequence repeats （SSRs）. The phylogenetic relationships and the evolutionary history among different cultivars were determined on the basis of SSR data. Twenty-two polymorphic SSR primer pairs were selected, and a total of 111 alleles were identified in the 51 cultivars, with an average of 5 alleles per locus. According to traditional Chinese classification of peach cultivars, the 51 cultivars in the peach primary core collection belong to six variety groups. The SSR analysis revealed that the levels of the genetic diversity within each variety group were ranked as Sweet peach 〉 Crisp peach 〉 Flat peach 〉 Nectarine 〉 Honey Peach 〉 Yellow fleshed peach. The genetic diversity among the Chinese cultivars was higher than that among the introduced cultivars. Cluster analysis by the unweighted pair group method with arithmetic averaging （UPGMA） placed the 51 cultivars into five linkage clusters. Cultivar members from the same variety group were distributed in different UPGMA clusters and some members from different variety groups were placed under the same cluster. Different variety groups could not be differentiated in accordance with SSR markers. The SSR analysis revealed rich genetic diversity in the peach primary core collection, representative of genetic resources of peach.     

Morphological traits and RAPD markers for characterization and identification of minor Spanish olive cultivars from the Extremadura region

A total of 91 wild olive accessions and 31 olive cultivars growing in the Extremadura region of central-western Spain were analyzed using morphological traits and RAPD markers. We focused on three main and 16 minor Spanish olive cultivars that are recognized as native or local to the Extremadura region. The five arbitrary 10-mer primers tested on the olive cultivars gave 67 polymorphic bands, representing 91% of the total amplification products. The number of bands per primer ranged from 9 to 18, whereas the number of polymorphic bands ranged from 8 to 17. All the cultivars could be identified by a combination of three primers (OPF-6, OPA-8, and OPK-16); four cultivar-specific markers were detected. The minor local "Jariego" and "Tempranillo" cultivars showed the most distal similarities. The resulting dendrogram, using the unweighted pair-group method with arithmetic mean clustering algorithm, depicted the pattern of relationships between the local Extremadura cultivars and the cultivars from geographically connected regions. This analysis showed a correlation between most of the minor local cultivars and the geographical origin; there was no apparent clustering according to morphological traits or fruit use of olive cultivars when these parameters were used as analysis criteria.