Microsatellite fingerprinting of homonymous grapevine (Vitis vinifera L.) varieties in neighboring regions of South-East Turkey

Genotyping of Turkish grapevine (Vitis vinifera L.) germplasm was characterized by use of six highly polymorphic microsatellite loci (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, VrZAG79). In this study we aimed to clarify the relationships between homonymous varieties coming from different regions. Our results showed a large degree of genetic variability among most of the homonymous cultivars. The number of alleles per locus ranged from 10 to 21, and gene diversity (expected heterozygosity) values ranged from 0.85 to 0.93. Cultivars presenting the same names of Sergi karas? (sampled from ?anl?urfa and Gaziantep), Yediveren (sampled from ?anl?urfa, Gaziantep, and National Germplasm Repository Vineyard in Tekirda?) and Serpenek?ran (sampled from ?anl?urfa and Gaziantep) were clustered together, or very close to each other, in a phenogram. Moreover, the alleles at the six microsatellite loci analyzed were found to be similar in terms of base pairs within each of these three closely positioned varieties. However, all the other cultivars failed to show a suitable clustering pattern when comparing their DNA profiles and names. Similarly named cultivars were not generally grouped together in the phenogram. On the other hand, we detected a tendency for differently named homonymous grape cultivars to cluster together.     

Origin and genetic diversity of Tunisian grapes as revealed by microsatellite markers

Ten SSR loci, previously developed for grapevine, were analyzed to evaluate the genetic variability, cultivar relatedness, and parentage in a collection of 61 autochthonous Vitis vinifera cultivars from Tunisia.The number of alleles per locus ranged from 6 to 11, while the number of genotype patterns varied between 10 and 21. The expected heterozygosity varied between 0.621 and 0.855 and the observed heterozygosity was higher than 0.9 at 4 loci (VVMD28, VVMD5, VVIP31 and VVS2) indicating that the SSRs were highly informative.Cluster analysis using unweighted pair group method with arithmetic averaging (UPGMA) suggested 14 groups among studied cultivars and 53 grapevine denominations out of 61 were unequivocally distinguished, with all accessions showing at least one-specific combination of alleles.On the other hand, in order to overcome the existing confusion in Tunisian grapevine nomenclature, of the analyzed homonymous pairs of cultivars, only ‘Balta 2’ and ‘Balta 3’ have shown identical allelic profiles, consistent with their being the same genotype. Hence, nomenclature distinction is meaningless and only one denomination should be retained.Due to the high overall power of exclusion (Q) (greater than 99.99%) and to the absence of null alleles, the set of microsatellite loci used is appropriate to determine parentage in Tunisian grapevines beyond any reasonable doubt. The analysis of fingerprints indicated that the Tunisian grape vines have evolved through out crossing between five possible parents: Balta 1, Beldi Baddar, Beldi Rafraf, Beldi Local Rafraf and Khedhiri 3.     

SSR-based DNA fingerprints reveal the genetic diversity of Sicilian olive (Olea europaea L.) germplasm

Summary

Twelve published simple sequence repeat (SSR; microsatellite) markers, belonging to the ssrOeUA-DCA, GAPU and UDO series, were tested in a panel of 46 accessions of olive germplasm belonging to 30 unique cultivars collected in seven Provinces of Sicily. Four well-known reference olive cultivars were also added. The analysis was carried out on an automatic capillary sequencer using fluorescent dyes, and fragment sizes were determined using internal standards. The results allowed us to rank the SSRs assayed according to their information content and reproducibility. Up to 115 alleles were identified (119, if those unique to sport mutations were included), the frequency of which allowed genetic relationships among accessions to be investigated. The probability that two unrelated genotypes displayed the same SSR pattern at all loci examined was calculated to be as low as 1.18 10^–11. Sixteen accessions were identified as synonyms. Of these, eight matched perfectly with another accession at all SSR loci examined. The others showed one or two allelic differences from the reference accession. These were interpreted as mutations. Otherwise, all accessions were clearly separated from each other. Two likely parentages were also identified (‘Giarfara’ = ‘Nocellara del Belice’

‘Cacaridduni’; and ‘Pizzo di Corvo’ = ‘Nocellara Etnea’ ‘Tonda Iblea’). The genetic diversity of the pool represented by the unique accessions was very high, reflecting the richness of the olive germplasm accumulated in Sicily. A database of the accessions is available to the scientific community (http://www.unipa.it/germolive/ssr.html) to facilitate comparisons of data.     

Microsatellite characterization of grapevine (Vitis vinifera L.) genetic diversity in Asturias (Northern Spain)

The genetic heritage of the Asturian grapevine (Vitis vinifera L.) has been declining over the past century due to the phylloxera attack and the further abandonment of this culture. In addition, efforts in recent years to restore the Asturian vineyard with the pulling-up of old vineyards and replanting with cultivars endorsed by Cangas Quality Wine regulations are contributing even more to this genetic erosion. The aim of this study was the evaluation and identification of the phytogenetic resources of the Asturian grapevine. A total of 293 accessions were collected in old vineyards and analyzed through nine microsatellite markers. Forty-two different genotypes were obtained, including six profiles with allelic variations. Only 27 cultivars were identified when compared with national and international databases; some of them had not been found in this region before. Homonymies and synonymies have also been detected. These results provide an overview of the status of current grapevine phytogenetic resources in Asturias. Despite the substantial genetic erosion that the Asturian vineyard has suffered, a higher variability than expected has been detected. The finding of new grapevine genotypes is a fact of great importance. The genetic grapevine resources are being drastically reduced all over the world, so this new genetic material has to be included in germplasm banks for its conservation and further agronomical and enological evaluation.     

云南蔷薇属部分种质资源的SSR遗传多样性研究

利用简单重复序列SSR（Simple Sequence Repeat）标记技术对42份蔷薇属（Rosa L.）种质资源（包括13份野生种、变种、变型及29份栽培品种）的遗传多样性进行了研究。用筛选出的18对SSR引物对42份材料DNA进行PCR扩增,在18个位点共检测到148个等位基因,每一位点的等位基因变幅为6~14个,平均8.2个。材料间遗传相似系数变化范围为0.282~0.892,表明在分子水平上云南省蔷薇属植物具有丰富的遗传多样性。本研究发现,在相似系数为0.456时,基于SSR标记的聚类分析可以将 13个蔷薇野生种明显分为5个组,这与植物形态学分类结果大体一致。在遗传相似系数为0.43水平上,聚类分析将42份供试材料分为5大组群;同时初步探讨了野生种之间以及野生种与栽培品种之间的遗传亲缘关系。     

Assessment of genetic variability in Italian heritage peach resources from Emilia-Romagna using microsatellite markers

Summary

Heritage peach (Prunus persica L. Batsch) varieties in Emilia-Romagna, Italy’s leading peach-producing region, are largely marked by melting, juicy, aromatic white-fleshed fruit with a short shelf-life and susceptibility to bruising.While these varieties have rapidly been replaced since the 1950’s by yellow-fleshed peaches from US breeding programmes with improved handling resistance, the Fruit Research Unit of the Agricultural Research Council, at Forlì, has begun an effort to safeguard and characterise the heritage varieties of Emilia-Romagna. The programme has screened 26 local heritage accessions using a set of 16 highly polymorphic microsatellite (simple sequence repeat; SSR) markers to assess genetic diversity, to elucidate inter-relationships, and to resolve any cases of homonymy. Seven international peach cultivars have been included in order to standardise allele scoring. This study resulted in 19 unique molecular profiles among the heritage accessions, and a relatively high mean observed heterozygosity value of 0.49 for a germplasm pool from a restricted region, an indicator of potential genetic diversity. The main morphological features of the heritage peaches are reported, and potential benefits for future breeding programmes discussed.     

不同生态类型西瓜种质资源遗传多样性的SSR分析

采用SSR分子标记技术对96份不同生态型西瓜种质资源的遗传多样性进行研究。从398对引物中筛选出38对扩增条带清晰、多态性高的引物分析供试材料,共得到7043条清晰可辨条带,其中多态性条带826条,占11.7%。平均每对引物对96份材料扩增出185.34条带,其中21.74条具有多态性。96份材料间的相似系数为0.42～0.99。聚类分析结果表明,野生西瓜与栽培西瓜的亲缘关系较远。在栽培品种内华北生态型、华南生态型和西北生态型西瓜的亲缘关系较近,说明我国西瓜种质资源同源性较高,遗传基础狭窄,有必要引入更多种质资源以拓宽西瓜育种背景。     

Assessment of genetic relationships among 29 introduced and 49 local sweet cherry accessions in Turkey using AFLP and SSR markers

Summary

The characterisation of sweet cherry (Prunus avium L.) genetic resources in Turkey may help to increase their use in breeding programmes worldwide, as Turkey is the centre of origin of sweet cherry. Amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers were therefore used to analyse genetic diversity among a total of 78 local and introduced sweet cherry cultivars. Four AFLP primer combinations, and six SSR primer pairs for sweet cherry were used for genetic diversity analysis. A genetic similarity matrix was calculated using the combined data from AFLP and SSR analyses with simple matching coefficient. Genetic similarities among the sweet cherry genotypes studied were higher than 42%. No two accessions had an identical AFLP and SSR marker profile, indicating that all 78 genotypes were unique. An UPGMA dendrogram, based on the similarity matrix, revealed 18 separate Groups at or above the 70% similarity level. While some Groups consisted of both introduced and local genotypes, other Groups had only local genotypes. This result suggests that there was broad genetic diversity among the local Turkish sweet cherry genotypes, which was not present in the introduced sweet cherry accessions. The genetic variation present in local Turkish sweet cherry genotypes may be useful for future breeding programmes. We found that the use of both SSR and AFLP marker systems was effective for distinguishing between genetically-close sweet cherry genotypes. These marker systems can be used to complement pomological and morphological markers during the characterisation and identification of sweet cherry genotypes.     

Genetic diversity among olive varieties of Southern Italy and the traceability of olive oil using SSR markers

Summary

To evaluate germplasm variability and to identify DNA profiles useful for tracing the genetic identity of olive oil, we used six Simple Sequence Repeats (SSRs) in 47 cultivated olive varieties from Central and Southern Italy. A total of 80 polymorphic SSR markers were scored and both the observed heterozygosity and the polymorphic index content were, on average, high. Genetic similarities were investigated by agglomerative hierarchical clustering and principal component analysis. The results implied that most of the olive accessions from the Campania region were genetically different from those of other Italian regions. This finding was further supported by partitioning the genetic variability using analysis of molecular variance. Furthermore, we analysed the DNA isolated from the fruit and mono-varietal oils of three cultivars. Comparative analysis of the SSR profiles revealed that these were conserved in the agro-food chain, although our data also suggested that some issues, such as the sensitivity and performance of the assay in complex mixtures, may pose limitations. Our findings extend current knowledge of Italian olive germplasm and highlight the richness and specificity of the genetic resource of olives in some regions of Southern Italy. They also provide molecular information that can be exploited for the protection of genetic material and mono-varietal oils.     

SSR markers reveal the uniqueness of olive cultivars from the Italian region of Liguria

Twenty-three important Ligurian olive accessions corresponding to 16 cultivars were studied using 12 SSR markers and 40 Mediterranean cultivars were included in the study in order to investigate the relationships between Ligurian and Mediterranean germplasm. All SSRs produced polymorphic amplifications. One hundred and forty-nine alleles were found in the 63 accessions analysed. Twenty-two alleles were specific to germplasm from Liguria and of these 12 were unique to single cultivars. Heterozygosity and discriminating power calculated in this regional germplasm were high on average (0.70 and 0.74) and not so much lower than the values in the total sample that includes cultivars from different Mediterranean countries (0.77 and 0.88 respectively). No cases of genetic identities were found between Ligurian and Mediterranean accessions. Several cases of homonyms and synonyms within the Ligurian germplasm were explained. Cluster analysis generally revealed a clear discrimination of the profiles from Liguria and Italy with respect to the cultivars from other Mediterranean countries. Only one Ligurian cultivar, “Negrea”, appeared to have a different origin, grouping with the Mediterranean cultivars.     

二倍体马铃薯栽培品种遗传多样性的SSR标记分析

二倍体马铃薯基因组相对简单,借助二倍体进行育种可以加速马铃薯的育种进程,因此评价二倍体马铃薯种质的遗传多样性,挖掘和有效利用优良性状显得非常必要。为了筛选多态性的SSR标记,用55对SSR引物扩增39个遗传关系相对较远的二倍体马铃薯材料。选取分布在12条染色体上的12个具有高多态性的SSR标记评价192份二倍体马铃薯栽培品种的遗传多样性,共检测到98个等位位点,其中97个为多态性位点;每对SSR引物扩增出的等位位点为6~18个,平均8.2个。用非加权配对算术平均法(UPGMA)进行聚类,显示出所有供试材料的遗传关系:12对SSR引物可以将192份材料中的186份区分开;这192份材料被划分为11个组群,其中第一个组群包含了83.3%的材料。     

SSR-based identification key of cultivars of Olea europaea L. diffused in Southern-Italy

Olive (Olea europaea L.) is an important fruit species in Italy and Mediterranean basin constituted by a wide germplasm with a large number of cultivars present in all the Mediterranean area. SSRs are becoming the markers of choice for variability studies in olives as they are simple to perform, transferable, hypervariable, highly polymorphic and show a high information content.Olive genetic diversity was studied using 16 SSR markers on 30 cultivars diffused in Southern-Italy. Resolving Power (RP) and Power of Discrimination (PD) were calculated to evaluate the efficiency of the SSR markers investigated in studies of cultivars fingerprinting. Based on their high efficiency, two SSR markers, UDO43 and DCA16 were chosen to set up an identification key to distinguish the entire set of cultivars, confirming the high biodiversity of the Southern-Italian olive germplasm and the suitability of SSR markers in studies of cultivar diagnosis.     

Genetic Identification and Conservation of Local Turkish Grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) Genotypes on the Edge of Extinction

In the second half of the nineteenth century, intensive renovation of vineyards took place due to the losses caused by phylloxera and local varieties were mostly replaced by several worldwide cultivars. Shift in genotypic structure in favor of modern cultivars resulted in the decrease or even disappearance of regionally typical local varieties. A total of sixty five Turkish grape genotypes, including 5 references (four cultivars and one rootstock), were genotyped with 16 SSR and 15 SRAP markers. Sixteen SSR primers generated a total of 60 SSR amplicons in which 43 were polymorphic with 73.4% average polymorphism percentage. A total of 111 well-resolved clear DNA bands were obtained from 15 SRAP primers. Of these bands, 53 were highly polymorphic with an average of 47.74%. Cluster analysis based on pooled marker data generated a well resolved grouping pattern. The analyzed genotypes grouped basing on their geographical belongings. There were many cultivar pairs on the dendrogram most of which occurred between 0.75 and 0.90 levels. SSR and SRAP data revealed a wide genetic variability as well as certain synonyms among the historical grape varieties cultivated for decades in local vineyards lengthwise the mountainous regions of Konya, Karaman and Mersin provinces. All the genotypes have been maintained in a grapevine germplasm glasshouse. Preservation and use of these endangered genotypes will be helpful to avoid genetic erosion and diversity loss in this part of Turkey. Also, the molecular data generated in this study could be of great use in determining the optimal breeding strategies to allow continued progress in grapevine breeding.     

Varietal discrimination and genetic relationships of Vitis vinifera L. cultivars from two major Controlled Appellation (DOC) regions in Portugal

Thirty-nine grapevine cultivars widely grown in Portugal, especially in Vinhos Verdes and Douro regions, and two well known international cultivars as standards, were genotyped at 12 microsatellite loci. The number of alleles per locus ranged from 6 to 12, and the number of allelic combinations per locus from 13 to 26. The total number of unique genotypes in the 12 analysed loci was 120, having most of the cultivars (38 out of 41) at least one unique genotype in any of the loci. The microsatellite profiles were adequate to discriminate 41 cultivars. The level of observed heterozygosity at each locus varied from 70.7% to 95.1%. VVMD28 has been revealed as one of the most informative markers. Several synonymies between Spanish and Portuguese cultivars were confirmed, and some homonymies are discussed. The genetic profiles of all 41 cultivars were searched for possible parent-offspring groups. The data obtained revealed the possible descendence of Touriga Franca from Touriga Nacional and Marufo.     

Comparative analysis of genetic diversity in Tunisian apricot germplasm using AFLP and SSR markers

Eighty-one accessions representing apricot germplasm in Tunisia were collected from different areas of cultivation and fingerprinted using amplified fragment length polymorphism (AFLP) and microsatellites (SSR) markers. A total of 339 polymorphic markers were revealed using 5 AFLP primers combinations and 24 SSR loci. AFLP and SSR markers expressed a high level of polymorphism allowing the distinction of the accessions with an efficiency coefficient of discrimination of 100% for AFLP and 97% for SSR markers. Genetic diversity structure was assessed with AFLPs and SSRs markers separately then with combined matrix data by the help of hierarchical clustering elaborated using Wards method based on Nei and Li (1979) distances. Comparison of the obtained dendrograms revealed a phylogeographic structure into two major groups with significant conservation between the observed subgroups in relation with the geographic origin of the accessions. The relative efficiency of the markers in determining the genetic relationships among apricot accessions has been assessed and a combination of AFLPs and SSRs markers was the most effective. In addition, Mantel test based on genetic distances indicated highly significant correlation between AFLP-SSR data and each of the AFLP and SSR ones, with Pearson correlation values of r = 0.873 and r = 0.692, respectively, revealing the higher efficiency of the combination of both molecular techniques (AFLP and SSR) to estimate the levels of genetic variability among apricot germplasm.     

Isoenzyme and microsatellite analysis of Vitis vinifera L. varieties from the Hungarian grape germplasm

The aim of our work was to investigate the genetic diversity of grapevine with biochemical and molecular markers (isoenzyme and SSR). The isoenzyme patterns of 4 enzyme systems (catechol-oxidase, glutamate-oxalacetate-transaminase, acid phosphatase and peroxidase) and the microsatellite profile in 6 loci (VVS2, VVS16, VVMD7, VMC4A1, VMC4G6, VrZag79) of 48 grapevine varieties were analysed. The results with CO, GOT, AcP and PER enzymes were reproducible and the zymograms obtained from the woody stems were independent from the time of sampling during the dormant period of the grape. Based on the isoenzyme patterns of these 4 enzymes most of the investigated varieties (40/48) were identified. A correlation was found between the isoenzyme patterns and the classification to convarietas of the varieties. It was established, that while the varieties of the convarietas pontica differed from those of the convarietas orientalis and occidentalis, the two latter groups could have not been differentiated from each other. Based on the SSR (simple sequence repeat) analyses 46 of the 48 investigated varieties were identified. Even ‘Pinot blanc’ and ‘Pinot gris’ cultivars belonging to the same conculta (Pinot) could be differentiated in their VMC4A1 locus.     

Detection of genetic diversity among populations of sweet cherry (Prunus avium L.) by AFLPs

Summary

The high degree of polymorphism of AFLPs provides an efficient system for identification and genome analysis of sweet cherry (Prunus avium) cultivars and selections. The cultivars of sweet cherry have usually been characterized by assessment of phenotypic and pomological traits. AFLP markers were employed to identify 38 sweet cherry accessions and estimate the genetic diversity among this material. Ten of 18 tested primer combinations were informative with up to 80 bands per primer combination. Seven to 33% of the amplfied bands were polymorphic depending upon primer combination. Allcultivars and selections tested could be clearly identified. The objective of this work was to demonstrate the usefulness of molecular markers in revealing the genetic diversity among different sweet cherry genotypes.