Genetic diversity and geographical dispersal in grapevine clones revealed by microsatellite markers.

Intravarietal genetic diversification associated with geographical dispersal of a vegetatively propagated species was studied using grapevine Vitis vinifera L. 'Cabernet Sauvignon' as a model. Fifty-nine clonal samples obtained from 7 countries (France, Chile, Spain, Australia, Hungary, USA, and Italy) were analyzed using 84 microsatellite markers. Eighteen polymorphic microsatellite loci (21.4%) were detected, finding 22 different genotypes in the population analyzed with a genetic similarity of over 97%. The presence of chimeric clones was evidenced at locus VMC5g7 by means of a segregation analysis of descendants by self-pollination of a triallelic Chilean clone and by somatic embryogenesis analysis, showing a mutation in L2 cell layer. Only 2 clones (obtained from France and Australia) presented the ancestral genotype, and the most divergent genotype was exhibited by another French clone, which had accumulated 5 somatic mutations. The 2 largest populations considered (from France and Chile) showed a clear divergency in the polymorphisms detected. These antecedents enabled the tracing of geographical dispersal with a phylogenetic hypothesis supporting France as the center of origin of diversification of Cabernet Sauvignon. The results obtained could help to explain diversification processes in other grapevine cultivars. The possibility that this kind of genetic variability occurs in other vegetatively propagated species is discussed, focusing on possible fingerprinting applications.     

SSR-based assessment of genetic diversity in South American Vitis vinifera varieties

Six SSR loci, previously developed for grapevine, were analyzed to evaluate the genetic variability and cultivar relatedness in a collection of 25 autochthonous Vitis vinifera varieties from Perú and Argentina.

The number of alleles per locus ranged from 6 to 13, while the number of microsatellites genotypes varied between 9 and 16. The expected heterozygosity varied between 0.71 and 0.89 and the polymorphism information content ranged from 0.70 to 0.88 indicating that the SSRs were highly informative. It was possible to identify 76 different genotypes, with all accessions showing-at least one-specific combination of alleles. Triallelic loci were observed with some SSR. Sequence analysis revealed that variation in the number of repeats and insertion/deletions (InDels) accounted for the polymorphisms observed. Clustering analysis resulted in four separate groups of varieties sharing at least 75% of the markers. A few cases of synonymies were found within the Peruvian accessions. Varieties were clustered following a general pattern of shared morphological and enological traits, rather than geographical origin.     

Evidence of a secondary grapevine domestication centre detected by SSR analysis

The origin of the grapevine was investigated with archaeobotanical, cultural and historical data. A primary domestication centre was located in the Near East region but there is no agreement on the existence or role of secondary domestication centres. In this work, PCR-based microsatellite analysis has been applied to study the origin of some Italian cultivated grapevines from in situ direct domestication of the wild autoctonous grapevine. Three different Italian locations in Grosseto, Cosenza and Nuoro were identified for this study, and domesticated grapevine as well as wild local accessions growing in these location, were analysed by SSR markers. Cluster analysis performed on Cosenza and Grosseto samples showed a high value of genetic distance between domesticated and wild accessions. On the contrary two cultivars (Bovale Murru and Bovale Muristellu) recovered in Nuoro (in the Sardinia island) were very close to some wild varieties. This suggests that the latter two cultivars may have originated from wild grapevines and consequently that in this location a secondary grapevine domestication event occurred. Six Lambrusco varieties were also included in this analysis as ancient putative ancestors of the cultivated grapevines. The molecular analysis excluded this hypothesis and suggest Lambrusco as an independent Vitis taxon.The first two authors contributed equally to this research     

SSR分子标记检测出的花生类型内遗传变异

花生是我国重要的食用油和蛋白质来源作物,鉴定其DNA分子多态性对品种改良和资源评价具有重要的意义。从已公布的花生Genomic-SSR和EST-SSR引物中筛选出34对引物,用来分别鉴定花生4大类型各24份共96份品种资源的分子变异,其中龙生型资源全部来自广西,普通型资源中有11份从国外引进,有13份来自广西和国内其他省市,多粒型资源只有两份来自中国,其他22份分别来自印度、美国和非洲等地,珍珠豆型资源中有22份是来自中国各地的育成品种或农家品种,有2份来自国外。研究结果为:分别有10～16对SSR引物能在4大类型花生资源中扩增出多态性DNA片段;这些多态性SSR引物都具有多位点特性;首次为SSR分子标记设立了一个新的评价指标——区别指数,多态性SSR引物的区别指数最高达0.992;资源间的平均遗传距离,多粒型为0.59,普通型为0.48,珍珠豆型为0.38,龙生型为0.17。根据遗传距离采用最长距离法对4大类型花生资源分别进行了聚类分析,构建了资源间的遗传关系图,花生4大类型可进一步分成不同类群,资源间的亲缘关系与其来源相关。观察到PM15和PMc297的扩增产物具有类型特异性,PM15能在龙生型、普通型和多粒型花生资源中扩增出多态性条带,而在珍珠豆型花生中扩增条带完全相同,PMc297也有相似的扩增结果。由于在多粒型花生资源中检测出的遗传多样性最丰富,研究结果支持西班牙专家Krapovickas 1994年公布的花生栽培种分类系统。总之在花生4大类型内资源中能检测出丰富的SSR分子标记,开发出更多的SSR分子标记将能充分揭示花生分子水平的变异,从而使花生遗传图谱构建、分子标记辅助育种成为可能。     

Assessment of genetic variation and differentiation of hop genotypes by microsatellite and AFLP markers.

Microsatellites have many desirable marker properties and have been increasingly used in crop plants in genetic diversity studies. Here we report on the characterisation of microsatellite markers and on their use for the determination of genetic identities and the assessment of genetic variability among accessions from a germplasm collection of hop. Thirty-two polymorphic alleles were found in the 55 diploid genotypes, with an average number of eight alleles (3.4 effective alleles) for four microsatellite loci. Calculated polymorphic information content values classified three loci as informative markers and two loci as suitable for mapping. The average observed heterozygosity was 0.7 and the common probability of identical genotypes was 3.271 x 10(-4). An additional locus, amplified by one primer pair, was confirmed by segregation analysis of two crosses. The locus discovered was heterozygous, with a null allele in the segregating population. The same range of alleles was detected in nine triploid and five tetraploid hop genotypes. Cultivar heterozygosity varied among all 69 accessions, with only one cultivar being homozygous at four loci. Microsatellite allele polymorphisms distinguished 81% of all genotypes; the same allelic profile was found mainly in clonally selected cultivars. Cultivar-specific alleles were found in some genotypes, as well as a specific distribution of alleles in geographically distinct hop germplasms. The genetic relationship among 41 hop accessions was compared on the basis of microsatellite and AFLP polymorphisms. Genetic similarity dendrograms showed low correlation between the two marker systems. The microsatellite dendrogram grouped genetically related accessions reasonably well, while the AFLP dendrogram showed good clustering of closely related accessions and, additionally, separated two geographically distinct hop germplasms. The results of microsatellite and AFLP analysis are discussed from the point of view of the applicability of the two marker systems for different aspects of germplasm evaluation.     

Comparison of a retrotransposon-based marker with microsatellite markers for discriminating accessions of Vitis vinifera

Identification and knowledge concerning genetic diversity are fundamental for efficient management and use of grapevine germplasm. Recently, new types of molecular markers have been developed, such as retrotransposon-based markers. Because of their multilocus pattern, retrotransposon-based markers might be able to differentiate grapevine accessions with just one pair of primers. In order to evaluate the efficiency of this type of marker, we compared retrotransposon marker Tvv1 with seven microsatellite markers frequently used for genotyping of the genus Vitis (VVMD7, VVMD25, VVMD5, VVMD27, VVMD31, VVS2, and VZAG62). The reference population that we used consisted of 26 accessions of Vitis, including seven European varieties of Vitis vinifera, four North American varieties and hybrids of Vitis labrusca, and 15 rootstock hybrids obtained from crosses of several Vitis species. Individually, the Tvv1 and the group of seven SSR markers were capable of distinguishing all accessions except 'White Niagara' compared to 'Red Niagara'. Using the Structure software, the retrotransposon marker Tvv1 generated two clusters: one with V. vinifera plus North American varieties and the other comprising rootstocks. The seven SSR markers generated five clusters: V. vinifera, the North American varieties, and three groups of rootstock hybrids. The percentages of variation explained by the first two components in the principal coordinate analysis were 65.21 (Tvv1) and 50.42 (SSR markers) while the Mantel correlation between the distance matrixes generated by the two types of markers was 42.5%. We conclude that the Tvv1 marker is useful for DNA fingerprinting, but it lacks efficiency for discrimination of structured groups.     

Characterization of Spanish grapevine cultivar diversity using sequence-tagged microsatellite site markers.

A broad germplasm bank collection containing most of the autochthonous Spanish grapevine cultivars was analyzed using six sequence-tagged microsatellite site (STMS) loci: VVS2, VVMD5, VVMD7, ssrVrZAG47, ssrVrZAG62, and ssrVrZAG79. The number of alleles obtained ranged from 9 in ssrVrZAG47 to 13 in VVS2, and the observed genotypes per locus varied between 24 (ssrVrZAG47) and 41 (VVSS2). A total of 57 unique genotypes were obtained considering all 6 loci, and 40 varieties presented at least 1 of these specific genotypes. The genotypic combinations for the 6 loci have generated 163 different profiles in the 176 cultivars. Ten pairs of accessions and one group of four Garnacha's cultivars can not be differentiated. The observed heterozygosity varied between 75.6 (VVMD7) and 90.9% (VVMD5), without significant differences from the expected values for any loci. The VVMD5 locus was the most informative, and also showed the highest discrimination power. The cumulative discrimination power for all six loci was practically 1; however, in fact, these STMS loci have differentiated only about 93% of the accessions, probably owing to high relatedness of the plant material. Usefulness of this STMS set for characterization of a Spanish grapevine collection is emphasized, as well as the elaboration of databases with these molecular markers.     

Analysis of potential duplicates in barley gene bank collections using re-sampling of microsatellite data

Redundant duplication among putative Nordic spring barley material held at 12 gene banks worldwide was studied using 35 microsatellite primer pairs covering the entire barley genome. These microsatellite markers revealed an average of 7.1 alleles per locus, and a range of 1 to 17 different alleles per locus. Similarity of accession name was initially used to partition the 174 repatriated accessions into 36 potential duplicate groups, and one group containing 36 apparently unique or unrelated accessions. This partitioning was efficient to produce a distribution of mainly small average genetic distances within potential duplicate groups compared to distances from the group of unique accessions. However, comparisons within potential duplicate groups still contained large genetic distances of the same size as distances between unique accessions indicating classification errors. A bootstrap approach based on re-sampling of both microsatellite markers and alleles within marker loci was used to test for homogeneity within potential duplicate groups. The test was used in each group for sequential elimination of accessions with a significantly large average genetic distance to identify a homogeneous group. Such genetically homogeneous groups of two or more accessions were identified in 22 among the 36 potential duplicate groups studied. Results from the genetic analysis of some potential duplicate groups supported previous conclusions based on passport data through inclusion of the historically most-original accession in the genetically homogeneous group. In other potential duplicate groups the apparently most-original accession according to passport data was not included in the homogeneous set of accessions, indicating that this most-original accession does not have duplicate accessions in the group. During the present study the largest average genetic distance accepted in any homogeneous group was smaller than the smallest distance declared significant in any group, with a threshold average genetic distance of approximately 0.14. The results are discussed with respect to the identification of duplicate accessions within potential duplicate groups, as well as the elimination of genetic off types in such groups. Furthermore, large barley gene bank collections may be screened for potential duplicates with genetic distances below the suggested threshold of 0.14.     

Development of microsatellite markers and analysis of intraspecific genetic variability in chickpea (Cicer arietinum L.)

Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT) _n and (CT/GA) _n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08–0.91) and 0.74 (0.23–0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers developed in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.     

Combining chloroplast and nuclear microsatellites to investigate origin and dispersal of New World sweet potato landraces

We analysed a representative collection of New World sweet potato landraces (329 accessions from Mexico to Peru) with both chloroplast and nuclear microsatellite markers. Both kinds of markers supported the existence of two geographically restricted genepools, corresponding to accessions from the north-western part of South America and accessions from the Caribbean and Central America region. Our conservative cpSSRs markers revealed that the divergence between the two haplotype groups is associated with numerous mutation events concerning various markers, supporting the idea that this divergence may be ancient, predating domestication. For both kinds of markers, we found no significant difference in diversity between the two genepools and detected region-specific alleles in both groups. Previous studies have favoured the hypothesis of a single domestication of this crop. Our analysis suggests at least two independent domestications, in Central/Caribbean America and in the north-western part of South America. Sweet potato was then dispersed from these centres throughout tropical America. Comparison of nuclear and chloroplast data suggests that exchanges of clones and sexual reproduction were both important processes in landrace diversification in this clonally propagated crop. Our analysis provides useful tools for rationalizing the conservation and use of sweet potato germplasm collections.     

An extensive study of the genetic diversity within seven French wine grape variety collections

The process of vegetative propagation used to multiply grapevine varieties produces, in most cases, clones genetically identical to the parental plant. Nevertheless, spontaneous somatic mutations can occur in the regenerative cells that give rise to the clones, leading to consider varieties as populations of clones that conform to a panel of phenotypic traits. Using two sets of nuclear microsatellite markers, the present work aimed at evaluating and comparing the intravarietal genetic diversity within seven wine grape varieties: Cabernet franc, Cabernet Sauvignon, Chenin blanc, Grolleau, Pinot noir, Riesling, Savagnin, comprising a total number of 344 accessions of certified clones and introductions preserved in French repositories. Ten accessions resulted in being either self-progeny, possible offspring of the expected variety or misclassified varieties. Out of the 334 remaining accessions, 83 displayed genotypes different from the varietal reference, i.e., the microsatellite profile shared by the larger number of accessions. They showed a similarity value ranging from 0.923 to 0.992, and thus were considered as polymorphic monozygotic clones. The fraction of polymorphic clones ranged from 2 to 75% depending on the variety and the set of markers, the widest clonal diversity being observed within the Savagnin. Among the 83 polymorphic clones, 29 had unique genotype making them distinguishable; others were classified in 21 groups sharing the same genotype. All microsatellite markers were not equally efficient to show diversity within clone collections and a standard set of five microsatellite markers (VMC3a9, VMC5g7, VVS2, VVMD30, and VVMD 32) relevant to reveal clonal polymorphism is proposed.     

Development of an allele-mining set in rice using a heuristic algorithm and SSR genotype data with least redundancy for the post-genomic era

The allelic diversity of a collection of 4046 rice accessions was assessed using 15 neutral SSR markers distributed throughout the genome. A total of 482 alleles were detected; the average allelic richness was 32.1 alleles per locus. Using a heuristic approach, an allele-mining set was successfully developed on the basis of SSR marker data. 162 accessions of the allele-mining set, accounting for about 4.0% of the entire collection, captured all of the alleles (482) retained in the entire collection, which showed 100% coverage of alleles with minimum redundancy. As a result of validation of this heuristic approach using another 14 SSR markers associated with starch, 70% of the total alleles and 83% of the restricted alleles (allele frequency > 0.05%) were captured in this allele-mining set. The results showed that the heuristic approach meets the condition as an allele-mining set even when applied to another specific set of markers related to starch synthesis in the same entire and allele-mining set. The newly developed methodology for developing allele-mining sets can be used in other crop species. By retaining all alleles of the entire collection, this allele-mining set will be useful for future studies on introducing unused useful alleles into elite rice varieties by breeders in the post-genomic era.     

基于TP-M13-SSR指纹图谱的中国原产苹果属植物分子身份证的建立

利用TP-M13-SSR分子标记方法,构建27份中国原产苹果属植物在12个SSR位点的指纹图谱,运用条码技术生成其分子身份证。12对引物共获得251个等位基因,平均21个。引物多态性好,仅用引物CH05b06即可区分全部供试材料。27份苹果材料在12个SSR位点遗传多样性、多态性信息含量和位点杂合度的变化范围为0.6620～0.9455、0.6327～0.9211和0.6538～0.9319。基于CH05b06位点处获得的指纹谱图即可得到每份供试材料独有的分子身份证。TP-M13-SSR分子标记技术适用于苹果属植物种质资源的指纹图谱构建,利于分子基础数据库的积累。基于苹果种质资源TP-M13-SSR指纹图谱可获得每份苹果种质资源独有的分子身份证。     

Development,characterization and utilization of genomic microsatellite markers in turmeric (Curcuma longa L.)

Development of a robust set of 18 genomic microsatellite markers from turmeric (Curcuma longa L.) and its effective utilization in estimating the genetic diversity of 20 turmeric accessions are described. A total of 103 alleles were detected with an average of 5.7 alleles per locus. These markers displayed varied levels of polymorphism as evident from its discriminating power ranging from 0.19 to 0.70. The UPGMA cluster analysis of genetic distance values resolved the 20 turmeric accessions into five main groups. Three sets of genetically identical accessions were detected within the analyzed accessions, suggesting a revisit of the germplasm collection strategy based on vernacular identity. The entire grouping pattern of the entities was loose and independent of their geographical origins. These polymorphic SSR markers would be useful for the population genetic studies and germplasm management of turmeric.     

Assessing the genetic diversity of Panicum coloratum var. makarikariense using agro‐morphological traits and microsatellite‐based markers

Panicum coloratum var. makarikariense is a perennial C₄ grass native to South Africa with relatively good forage production under limited‐resource conditions. Genetic characterisation and breeding efforts have been scant, thus limiting its use in cattle raising systems. The goal of the present study was to assess the genetic diversity of a collection of P. coloratum var. makarikariense using agro‐morphological traits and molecular markers, in comparison with one accession of var. coloratum and one population of Panicum bergii. Agro‐morphological variability between and within accessions of var. makarikariense in a common garden setting was observed, showing that there is still opportunity for selection. Some accessions performed better than the commercialised material in relation to potential forage production. A total of 117 ISSR bands and 48 SSR alleles allowed the detection of genetic variability between and within accessions. The presence of accession‐specific bands suggested distinctness and limited gene flow. The genetic variability encountered in the commercialised material suggested that it is a stabilised population which has not undergone a strong selection process. Low correlation between agro‐morphologic and molecular variability was observed indicating that both approaches provide complementary information. Both morphological and molecular markers reveal genetic differentiation between varieties and species. This study provides a set of new SSR markers available for diversity assessment and valuable information that can be applied directly in collection management for breeding and conservation programmes.     

Microsatellite analysis of Aegilops tauschii germplasm

The highly polymorphic diploid grass Aegilops tauschii isthe D-genome donor to hexaploid wheat and represents a potential source for bread wheat improvement. In the present study microsatellite markers were used for germplasm analysis and estimation of the genetic relationship between 113 accessions of Ae. tauschii from the gene bank collection at IPK, Gatersleben. Eighteen microsatellite markers, developed from Triticum aestivum and Ae. tauschii sequences, were selected for the analysis. All microsatellite markers showed a high level of polymorphism. The number of alleles per microsatellite marker varied from 11 to 25 and a total of 338 alleles were detected. The number of alleles per locus in cultivated bread wheat germplasm had previously been found to be significantly lower. The highest levels of genetic diversity for microsatellite markers were found in accessions from the Caucasian countries (Georgia, Armenia and the Daghestan region of Russia) and the lowest in accessions from the Central Asian countries (Uzbekistan and Turkmenistan). Genetic dissimilarity values between accessions were used to produce a dendrogram of the relationships among the accessions. The result showed that all of the accessions could be distinguished and clustered into two large groups in accordance with their subspecies taxonomic classification. The pattern of clustering of the Ae. tauschii accessions is according to their geographic distribution. The data suggest that a relatively small number of microsatellites can be used to estimate genetic diversity in the germplasm of Ae. tauschii and confirm the good suitability of microsatellite markers for the analysis of germplasm collections. Received: 8 September 1999 / Accepted: 7 October 1999     

Genetic diversity of traditional South American Landraces of Cassava (Manihot esculenta Crantz): an analysis using microsatellites

The extent and structure of the genetic variability of traditional varieties of cassava (Manihot esculenta Crantz) have been little documented, despite considerable evidence for this crop? great varietal diversity in traditional agroecosystems. We used microsatellite markers to assess the genetic structure of traditional landraces of sweet and bitter cassava collected from five South American sites. As reference, we used a sample of 38 accessions from a world collection of cultivated cassava. For a total of 10 loci examined, we found 15 alleles that were not represented in this sample. Ten of these had been previously detected in wild Manihot species. The geographical structure of genetic variability was weak, but the genetic differentiation between bitter and sweet landraces was significant, suggesting that each form had evolved separately after domestication. Our results showed that traditional landraces form an important source of genetic diversity and merit more attention from managers of crop genetic resources.     

Microsatellite variability in apricots (<Emphasis Type="Italic">Prunus armeniaca</Emphasis> L.) reflects their geographic origin and breeding history

A collection of 133 apricot cultivars and three related species originating from different geographical regions were studied with 10 polymorphic microsatellite markers developed in apricot. Altogether, 133 alleles were identified in the set of accessions, with an average of 13.30 alleles per locus. Out of them, 32 alleles occurred only once in the investigated samples, especially in apricots originating from different eco-geographic groups or in different species. The observed heterozygosity for individual loci ranged from 0.8636 to 0.3182, with an average of 0.6281. An unweighted pair group method with arithmetic mean dendrogram based on Nei's genetic distance grouped the accessions according to their eco-geographical origin and/or their pedigree information. Central Asian cultivars have a distinct position on the dendrogram, which supports the assumption that most cultivars have an Asian ancestor. Most East European cultivars analysed cluster together, and the data even revealed a few synonyms. Results show that American cultivars have not only European germ plasm in their pedigree, but they have also been enriched with germ plasm of Asian origin. The implications of these data for the use of simple sequence repeat (SSR) markers as a tool for fingerprinting cultivars in breeders' rights protection and apricot breeding are discussed. In this paper, we demonstrate for the first time the variability of apricot SSRs in a large collection of apricot cultivars and closely related species.     

Assessment of genetic diversity within and among germplasm accessions in cultivated sorghum using microsatellite markers

Microsatellite markers are increasingly being used in crop plants to discriminate among genotypes and as tools in marker-assisted selection. Here we evaluated the use of microsatellite markers to quantify the genetic diversity within as well as among accessions sampled from the world germplasm collection of sorghum. Considerable variation was found at the five microsatellite loci analysed, with an average number of alleles per locus equal to 2.4 within accessions and 19.2 in the overall sample of 25 accessions. The collection of sorghum appeared highly structured genetically with about 70% of the total genetic diversity occurring among accessions. However, differentiation among morphologically defined races of sorghum, or among geographic origins, accounted for less than 15% of the total genetic diversity. Our results are in global agreement with those obtained previously with allozyme markers. We were also able to show that microsatellite data are useful in identifying individual accessions with a high relative contribution to the overall allelic diversity of the collection. Received: 10 August 1999 / Accepted: 27 August 1999