Ampelographic and genetic characterisation of ancestral grapevine (Vitis vinifera L.) accessions present in the Umbria Region (Central Italy)

Summary

During an ongoing effort to recover and preserve local germplasm, 14 accessions of indigenous minor grapevine (Vitis vinifera L.) cultivars from the Umbria Region, Central Italy, were chosen because they had been neglected and were threatened with extinction. Their phenotypic and genetic characteristics were evaluated through an ampelographic study of their shoots, mature leaves, bunches, and berries and by genomic analysis using an international set of nine microsatellite (simple sequence repeat; SSR) markers (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, VrZAG79, VVMD25, VVMD28, and VVMD32). Comparisons of the SSR profiles of all 14 accessions with grapevine accessions in several databases permitted the identification of unique genotypes, as well as possible synonyms. Information on these older, neglected cultivars will help to reduce the genetic erosion of grapevine germplasm, improve conservation and possible recovery, and assist in the future production of new, distinctive wines.     

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.     

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.     

Characterization of variability and genetic similarity of European pear using microsatellite loci developed in apple

Seven microsatellite loci (SSR) developed in apple were used for the identification of 63 European pear cultivars. A total of 46 fragments were amplified, with an average of 6.6 alleles per SSR. Only one microsatellite amplified more than one locus. The mean expected and observed heterozygosities over the six single-locus SSRs averaged 0.68 and 0.44, respectively, and the number of effective alleles per loci was 3.43. The amplified fragments produced 61 different fingerprinting patterns that allowed to unequivocally distinguish all the varieties analyzed. Only two varieties, which derive from mutations, could not be distinguished from the original variety. Cluster analysis of the estimated genetic similarity, grouped the varieties according to their pedigree and their geographic origin. The variability detected with the SSRs in European pear varieties was low when compared with the variability detected in other fruit crops in the Rosaceae.     

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.     

Microsatellite markers based assessment of genetic diversity in Iranian olive (Olea europaea L.) collections

To study the genetic variation in Iranian olive collections and some foreign olive cultivars, 47 accessions of 18 local cultivars from 6 olive collections of Iran (Roudbar, Zanjan, Ahvaz, Dezful, Kazeroon and Shiraz), were analyzed along with 30 imported cultivars using 16 microsatellite primer pairs. All the used microsatellite loci revealed polymorphism in the studied genotypes, except GAPU14 and GAPU113 markers. Fourteen microsatellite primers amplified 126 polymorphic alleles in the 87 selected olive accessions. The average number of alleles per locus was 9, ranging from 3 to 14. Polymorphic information content (PIC) was 0.85. The genetic similarity based on Jaccard coefficient ranged from 0.15 to 1. The genetic relationships among accessions were investigated using cluster analysis and principal component analysis (PCA). Most of the accessions with the same name were grouped together; some exceptions were also observed. As expected, close relationship was observed among accessions within same cultivar. Most of the Iranian olive accessions were clustered to a main distinct group. Two-dimensional scatter plot of principal component analysis revealed a clear separation of most of the Iranian olives from Syrian and other introduced cultivars. These suggest that Iranian cultivars have different origin related to West Mediterranean basin cultivars and have evolved independently from the others. Between and within Iranian and foreign cultivars (cultivars including three or more accessions) genetic diversity was analyzed using analysis of molecular variance (AMOVA). AMOVA revealed higher within cultivar genetic variation (62.76%) as compare to that between cultivar variations (37.24%). The intra- and inter-cultivar variance tested by permutation test showed significant genetic variation at both levels. The high level within cultivar genetic variance could be due to mislabeling and presence of homonyms in cultivars produced by vegetative propagation from original plants.     

Discrimination of the grapevine cultivars ‘Picolit’ and ‘Kéknyelű’ with molecular markers

The name of the grapevine cultivar ‘Kéknyel?’ has become inseparable from the name of the Badacsony vine region, whose fame is well known beyond our frontier as well. In the Vitis International Variety Catalogue (http://www.genres.de/idb/vitis/) ‘Kéknyel?’ is reported, as the synonym of the Italian grapevine cultivar ‘Picolit’. Vertical poliacrylamide-gel electrophoresis was used for the investigation isoenzymes of catechol-oxidase (CO) and acid phosphatase (AcP). Microsatellite analyses were carried out at 6 loci (VVS2, VVS16, VrZag79, VVMD7, VMC4A1, VMC4G6). The results of the isoenzymatic and microsatellite analyses confirmed, that this two cultivars are different.     

Comparative analysis of autochthonous almond (Prunus amigdalus Bastch) material and commercial varieties in the Castilla-La Mancha Region (Spain)

The Castilla-La Mancha Region is characterized by a large variety of microclimates, allowing the cultivation of species with different climate requirements. While the districts of “Sierra del Segura” and “Campos de Hellín” account for the largest almond growing area in the region. This comparative study is based on the existence of native almond material (local names De Santos and Daniel) which shows kernel morphology and nut quality quite similar to the two most well known commercial varieties of almonds: Marcona and Desmayo Largueta. The identification of this native almond material is of great importance due to its adaptation to local environmental conditions (low temperatures, drought, hot and dry summers) and its morphological similarities with these two almond commercial varieties. A set of 6 Prunus SSR markers were used to identify and differentiate a total of 27 almond samples taken in the “Sierra del Segura” district, including local plant material and material from commercial varieties as a reference. Ten different SSR profiles were discriminated. The number of alleles per locus ranged from seven to twelve with a total of 52 alleles for all loci and an average of 9 alleles per locus. The analysis showed that they share 58% of the studied alleles with one common allele size in both cases for all the SSRs studied. This is the first molecular characterization of native almond germplasm in the Castilla-La Mancha Region. The results provide useful information that could be included in the future almond germplasm bank.     

DNA fingerprinting of olive varieties in Istria (Croatia) by microsatellite markers

The Istria region, where olives have been cultivated for many centuries, is characterized by a considerable variety of microclimates. The study of varieties traditionally cultivated in Croatian Istria and their relationships with varieties in historically and geographically connected regions is very important in order to identify native olive germplasm, well adapted to local conditions, and to characterize the oil of regional origin. Twelve olive microsatellite markers were used for identification and differentiation of a set of 27 olive accessions grown in Istria (Croatia). Among the 27 accessions, 18 different SSR profiles were discriminated. All 12 microsatellite markers analysed were polymorphic, revealing a total of 81 alleles. The number of alleles per locus ranged from four to nine. This is the first molecular characterization of olive germplasm in Croatian Istria. The analysis clarified the genetic relationships of varieties native to Croatian Istria with introduced olive varieties, as well as with varieties in the neighbouring Slovene Istria region. Numerous varieties in neighbouring regions showed high similarity and a few cases of synonymy (‘Bilica’-‘Bjankera’; ‘Buga’-‘?rna’) and one Croatian-Slovenian homonymy (‘Bu?a’-‘Buga’) were observed. The results provide useful information for a native germplasm survey and can be used for the construction of a unique database comprising all olive varieties in the Istrian region of Croatia and Slovenia.     

Use of chloroplast microsatellite markers as a tool to elucidate polymorphism,classification and origin of Tunisian grapevines

Chloroplast microsatellite markers were used in this study to genotype 43 grapevines accessions grown in Tunisia. Size variation was observed for the three cpSSR loci, both in the sample of cultivars and in wild accessions. The seven alleles observed in the sample of cultivars for the three loci are present in wild accessions except that their distribution is different. Levels of genetic diversity obtained for the Tunisian grapevines either in wild or cultivated gene pools are high and comparable with values obtained with other studied samples of Vitis vinifera. The distribution of haplotypes within the two samples is differential. Indeed, the chlorotype A is most abundant in the wild sample, whereas the chlorotype C is majority in the sample of cultivars. Haplotypes frequencies for cultivated grapevine distinguish haplotypes B and C as the most frequent (28% and 44% respectively) and haplotypes A and D as the least frequent (16% and 12% respectively). For wild grapevines, the seven alleles combined in three haplotypes, A, C and D. The haplotype A is the most frequent (44%) in the analyzed sample of wild accessions while haplotypes C and D show a frequency of 28%. Chlorotype distribution in Tunisian cultivars is comparable with that of cultivars in the Eastern Region representing the primary centre of domestication of the species. These results agree with the higher relevance of table grape cultivars in Tunisian viticulture and support an oriental origin of a large part of autochthons cultivars. Our results agree with other studies based in nuclear and chloroplast microsatellite markers and suggest independent domestication events for V. vinifera L. species.     

Genetic relatedness of sweet cherry (Prunus avium L.) cultivars from Ukraine determined by microsatellite markers

Microsatellite (SSR) markers were used to characterise 23 sweet cherry cultivars of Ukrainian, and four cultivars of non-Ukrainian, origin. To assess their genetic diversity and relatedness, 11 pairs of primers were applied to microsatellite loci, resulting in amplification of 66 SSR alleles. The mean value of the number of different alleles, and the polymorphic index content, amount to 7.333 and 0.700, respectively, demonstrating a significant genetic diversity of the investigated sweet cherry cultivars. Four highly polymorphic SSR loci (EMPAS02, EMPAS06, PceGA34, UDP98-412), which belong to the list recommended by the European Cooperative Program for Plant Genetic Resources, can be used as a minimum genetic marker set for identification of the majority of the studied cultivars; however, for successful discrimination of the most similar cultivars, more markers, located on all chromosomes of sweet cherry, appear to be necessary. Application of unweighted variable-group method using averages clustering allowed elucidation of the relatedness among the sweet cherry varieties, and showed that the Ukrainian cultivars combine genetic material of local, western European, and probably Caucasian origin; however, the origin of several cultivars still remains unclear, and should be studied additionally.     

板栗主栽品种的遗传多样性及其亲缘关系分析

 采用9 个酶系统的15 个同工酶位点, 对89 个板栗品种进行了遗传多样性分析, 并以品种间的遗传距离构建UPGMA 聚类图, 鉴别板栗品种和评价它们之间的遗传关系。结果表明: ( 1) 我国板栗主要产区遗传多样性较高, 如浙江、山东、湖北和江苏省; ( 2) 在供试的89 个板栗品种中, 除5 个品种外, 其它均可用多位点同工酶对其作专一性鉴定; ( 3) 基于品种间等位酶遗传距离的UPGMA 聚类图将山东、湖北、江苏以及河南的大部分板栗品种分别聚在一起, 体现了在遗传构成上同地域的板栗品种具有遗传关系相近的特征。     

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。     

Intra-cultivar variability of three major olive cultivars grown in different areas of central-southern Italy and studied using microsatellite markers

A collection of 70 olive samples, originating from diverse areas in central-southern Italy (Abruzzo, Apulia, Calabria, and Umbria) and corresponding to 3 major cultivars denominations (‘Carolea’, ‘Coratina’ and ‘Frantoio’), was genotyped at 10 microsatellite loci. In total, 44 alleles with a mean number of 4.4 alleles per locus were detected. The molecular analysis, allowed the study to show a clear genetic diversity between the three cultivars ‘Carolea’, ‘Coratina’ and ‘Frantoio’ and to state that ‘Carolea’ is a polyclonal cultivar, while ‘Coratina’ and ‘Frantoio’, are probably monoclonal ones. The analysis of intra-varietal polymorphism, through the SSR analysis, proved to be very useful both for varietal identification and for intra-varietal ones. Our work shows that the current designations of olive cultivars fall short of describing the genetic variability among economically important plant material. A thorough investigation of the existing variability will prove of major importance for both management and economic production of olive trees.     

Assessment of genetic diversity of Latvian and Swedish sweet cherry (Prunus avium L.) genetic resources collections by using SSR (microsatellite) markers

Three previously described highly polymorphic SSR (microsatellite) primer pairs were tested on 126 sweet cherry (Prunus avium L.) accessions to adapt a fast, reliable method for preliminary screening of sweet cherry germplasm collections and to compare two sweet cherry germplasm collections: at the Latvia State Institute of Fruit-Growing, Dobele (LIFG-Dobele) and at the Division of Horticultural Genetics and Plant Breeding at Balsgård, Department of Crop Sciences, Swedish University of Agricultural Sciences (SLU-Balsgård). The SSR loci were highly polymorphic with 4–10 different alleles and 5–18 genotypes. Heterozygosity values ranged from 0.431 to 0.809, gene diversity (PIC) values ranged from 0.400 to 0.753, and the discriminating power of each locus varied from 0.631 to 0.894. The combined discriminating power of all loci was highly effective (0.996). Sixteen identical accession groups with the same allele profile were discovered in both collections. This study demonstrated that SSR fingerprinting with the three primer pairs tested, can be used for preliminary characterization of sweet cherry germplasm collections.     

Microsatellite marker-based identification of mother plants for the reliable propagation of olive (Olea europaea L.) cultivars in Australia

Summary

Olive production in Australia has continued to increase in recent years, however there remains a high degree of confusion on the genetic identities of the cultivars being grown. In the present study, seven microsatellite (simple sequence repeat; SSR) loci were used to identify a set of 53 olive tree samples from different sources. The microsatellite DNA profiles of all 53 tree samples, including seven unknown trees, were compared with the SSR profiles of 14 reference olive cultivars. A total of 60 fragments (alleles), averaging 8.57 alleles per microsatellite locus, were amplified. High average values were found for the observed heterozygosity, the expected heterozygosity, and the polymorphic information content (0.73, 0.74, and 0.72, respectively). While all seven microsatellite markers proved useful for characterisation and identification purposes, a combination of three SSR primer pairs (DCA9, DCA18, and EM030) was sufficient to distinguish all 53 olive samples. The microsatellite allelic profiles allowed the 53 tree samples to be grouped into 23 genotypes. The allelic profiles of 14 of these genotypes matched with their reference cultivars, while the genetic identities of the remaining nine genotypes could not be confirmed. Some of these unknown genotypes may have been derived from feral olive trees, or were due to mislabelling and/or planting errors among Australian olive cultivars. Our results confirm the usefulness of microsatellite markers as a tool for cultivar differentiation and identification, and indicate the need for reliable identification of mother plants for commercial propagation.     

Genetic diversity of apricot revealed by a set of SSR markers from linkage group G1

Eight polymorphic simple sequence repeat (SSR) markers located in the G1 linkage group of apricot (Prunus armeniaca L.) were previously developed and evaluated in a small set of cultivars. Those primers were used for studying variability in 77 apricot cultivars belonging to five different geographical groups, such as Chinese, Asian (Irano-Caucasian and Central Asian), North American, Mediterranean and Western European as well as Middle European cultivars. Six of the markers were polymorphic and revealed a total of 71 alleles ranging from 5 (aprigms11) to 20 (aprigms1) alleles per locus with a mean value of 11.83 alleles per locus. In conclusion, the SSR loci located in the G1 linkage group show a level of polymorphism which is similar to loci dispersed throughout the entire genome. The total number of alleles and the number of unique alleles were the highest in Chinese apricots and the lowest in Middle European cultivars. Heterozygosity also showed a decrease from Asia and China to Middle Europe. No association could have been observed between any SSR markers tested and plum pox virus (PPV) resistant phenotype of cultivars. PPV resistant cultivars did not form a separate clade on the dendrogram obtained by UPGMA cluster analysis. Middle European and Chinese cultivars formed separate clusters while other genotypes formed smaller multiple sub-groups or scattered among different clusters. Our results support previous hypotheses on the origin of PPV resistance in North American apricots. The allele data was also presented in a form that allowed the easy observation of allele frequencies in each geographical group at each locus. Using this data field, differences and similarities between cultivar groups can be easily assessed. The analysis demonstrated the links between the North American and Mediterranean apricot germplasm and confirmed that the Chinese and Eastern European cultivars are distantly related.     

Development of new microsatellite markers for molecular diversity analysis of Citrus species

Summary

Thirty microsatellite loci for further genetic analysis of Citrus species were developed by constructing a microsatellite-enriched library using capture with streptavidin-coated magnetic beads and used to assess genetic diversity in 40 Citrus accessions. In total, 150 alleles were detected, with an average of five alleles per locus. The average gene diversity and polymorphism information content values were 0.58 and 0.52, and ranged from 0.35 – 0.74 and from 0.32 – 0.70, respectively. Values for the observed and expected heterozygosities ranged from 0.13 – 1.00 and from 0.36 – 0.75, respectively. Fifteen loci deviated from the Hardy-Weinberg equilibrium (P < 0.05). The mean similarity coefficient among accessions was 0.5906. Based on the UPGMA algorithm, two main groups were successfully identified. These new microsatellite markers can be used to further investigate the genetics of, and phylogenic relationships in Citrus spp.