Genetic relationships of 34 grapevine varieties and construction of molecular fingerprints by SSR markers

ABSTRACT In order to protect and promote the effective differentiation and rational utilization of grape germplasm resources, 34 grapevine varieties were selected from the National Germplasm Resources, Taigu Grape Nursery in China: 12 wine grapes (Group I), 14 table grapes (Group II) and 12 seedless grapes (Group III), 4 of which were in Group II, too. For the purpose of genetic characterization, 15 pairs of SSR (simple sequence repeat) primers were used. Sixty-six alleles were generated among Group I, including 64 polymorphic bands, with 1–8 alleles per locus. In Group II, a total of 54 alleles, from 1 to 8 (3.6 on average) alleles per locus, were identified. In Group III, 54 alleles with an average of 3.6 alleles per locus were identified and the percentage of polymorphic alleles was 94%. The 34 varieties clustered into two major clades in the dendrogram: V. vinifera with hybrid of V. vinifera and V. labrusca or hybrid of V. amurensisi and V. vinifera were clearly differentiated. After processing and filtering the raw data, we produced the molecular fingerprint code of each variety. These results showed that the SSR markers are useful for discrimination and analysis of genetic diversity of grapevine varieties. The SSR markers could be used to examine and distinguish the genetic resources among closely related varieties. This is also an effective tool for construction of a grapevine molecular fingerprinting system. The obtained data will be useful in grape breeding in the future.


Introduction
Grapevine (Vitis vinifera L.) is economically important around the world, as grape can be consumed directly or processed into wine, juice and others. There are more than 70 species in the Vitis genus worldwide, mainly distributed in the temperate regions of the Northern Hemisphere. With the grape industry growing, authorized varieties increase rapidly; consequently, a lot of different grape varieties use the same name (homonyms) or varieties get more than one name (synonyms). The growing seedling production, the protection of varieties and intellectual property rights, management of germplasm resources and demand of grape breeding put forward the requirements for grape variety identification. The traditional field evaluation method had been considered an important approach for the grape variety identification, however, it is time-consuming, laborious and error-prone due to environmental factors, which poses great challenges [1]. As an alternative, SSR (simple sequence repeat) markers, also known as short tandem repeats, are now widely used and have become a rewarding tool in the construction of genetic maps, description of distinctive individuals and assessment of genetic relatedness of grapevine varieties, for their reproducibility, high polymorphism, openness and co-dominance [2][3][4]. Since 1993, when Thomas et al. [5] first used SSR markers to identify multiple Eurasian grape varieties and several other representatives of genus Vitis, SSR markers became more and more widely used in the research of different species [6,7], cultigens and rootstock varieties [8,9], and in distinguishing and identification of homonym and synonym varieties [3,[10][11][12][13]. This et al. [14] demonstrated the usefulness of SSR markers in the identification of grape varieties, and Aradhya et al. [15] reported that SSR genetic analysis with 244 grape varieties matched the classical eco-geographic grouping of grape cultivars: occidentalis, pontica and orientalis. Sefc et al. [16] showed that 162 grape cultivars from seven European vine-growing regions had significantly different allele frequencies among the regions, speculating that these cultivars could possibly be assigned to their regions of origin according to their genotypes. Compared with the phenotypic characterization methods, which may cause incorrect judgment, SSR markers technology could directly reflect the existing differences, producing more trustworthy results which are not affected by the environmental factors [17]. Therefore, this technology gradually became a very useful tool in the study of the origin of species, as well as in the investigation of the evolution and genetic diversity of grape varieties [18].
Some researchers began to build the molecular fingerprinting maps of grape varieties by SSR markers. In 2004, ten different laboratories from France, Germany, Italy and four other countries chose six high polymorphism SSR primers and analyzed 46 grape cultivars for the standardization of reference allele coding [14].
In this study, we selected 34 grape varieties: 12 wine grapes (Group I), 14 table grapes (Group II) and 12 seedless table grapes (Group III), four of which were in Group II, from the National Germplasm Resources, Taigu grape Nursery in China. Fifteen SSR markers were used to investigate the relationship and genetic diversity among three groups of different types grape varieties. The molecular fingerprint code of each variety was also produced. The obtained results would be a useful reference for the more efficient utilization of grape germplasm and would be helpful in the selection of breeding parents and research in the future.

Plant materials
The leaf samples of 34 grapevine varieties used in this study were gathered from the national fruit germplasm resources, Taigu grape nursery of Pomology Institute, Shanxi Academy of Agricultural Sciences (37 23ˊN, 112 32ˊE). Fresh samples were frozen and kept at ¡80 C for genomic DNA extraction. The accession names, species, genetic relationship and their geographic origins are listed in Table 1.

Genomic DNA extraction
Genomic DNA was extracted from young leaf tissues by the method of Thomas and Scott [4] with minor modifications. DNA integrity was confirmed using 1% (w/v) agarose gel electrophoresis, and DNA concentration and purity were determined by Bio-Photometer (Eppendorf BioSpectrometer Germany). Polymerase chain reaction (PCR) was performed using the diluted DNA solution in 20 ng/mL that was stored in ¡20 C. We chose a total of 44 internationally well-known grape SSR primers derived from the NCBI public database and related references [19][20][21], 15 of which gave clear bands and had rich polymorphism ( Table 2). The primers were synthesized by Bio-engineering (Shanghai) co., LTD.
The PCR was performed using 10 mL reaction mixtures containing 20 ng genomic DNA, 5 mL PCR Mix, 5 pmol of each primer, 1 U Taq DNA Polymerase (Promega) and 3 mL ddH 2 O. Reactions without DNA were used as negative controls. The PCR was carried out in a Biome-tra® PCR System with the following program: an initial denaturation step of 5 min at 94 C, followed by 35 cycles of 30 s at 94 C, 30 s at 52»60 C and 90 s at 72 C, with a final extension at 72 C for 10 min, 16 C 1. The amplification products were visualized using capillary electrophoresis method to estimate their sizes.

Genetic analysis
SSR data were scored as presence (1) or absence (0) of bands and each band was regarded as a locus, according to the statistics of 0/1 data matrix. The genetic similarity coefficients between different samples were calculated using the software Numerical Taxonomy and Multiware Analysis System, version 2.0 (NTSYS-pc2.10) [22]. Cluster analysis was done using the unweighted pair-group method with arithmetic means (UPGMA) [23].
Through polymorphism band order code conversion, a molecular identification system was built. The standard for the assignment was as follows: 1) 15 decimal numbers (0-9), corresponding to 15 pairs of SSR primers and the order listed in Table 2; 2) the amplification bands of each material and each primer are arranged in order of size; the fingerprint code indicates the order of all bands of this primer; 3) if a primer has more than 10 bands, the banding pattern is defined as 0; 4) if there are multiple bands corresponding to a primer, the smallest band order is taken as the fingerprint code of the primer.

Polymorphism analysis of 34 grape varieties
All the 34 samples were successfully amplified using the15 SSR primers. The SSR analysis results of 12 wine grape varieties in Group I illustrated that a total number of 66 alleles, with 1-8 alleles per locus, average value of 4.4 alleles per locus, were genetically identified (Table 3). In Group II, 14 table grape varieties were analyzed. A total number of 54 alleles were genetically identified, also with 1-8 alleles per locus; and an average value of 3.6 alleles per locus (Table 4). In Group III, there were 12 seedless-table grape varieties. A total number of 54 alleles were genetically identified, with 1-7 alleles per Table 2. Nucleotide sequence of 15 microsatellite primers used for construction of a molecular fingerprinting system of 34 grapevine varieties.
In Group I and II, all primers had polymorphic bands, except the VVMD19 primer. There were three primers, SCU04VV, SCU15VV and VVMD19, that had no polymorphic bands in Group III. In Group I, the UDV134 locus had the highest effective number of eight alleles, whereas in Group II and III, the VVMD5 locus had the highest effective number of eight alleles among the 15 loci.
Genetic similarity coefficient analysis of 34 grape varieties  (Tables 6-8 and Figures 1-3). All these results indicated that the lowest genetic similarity coefficients were those from different species; the parents-offsprings varieties or sisters varieties had the highest genetic similarity in each group, which is in agreement with precious reports [14].

Phylogenetic analysis of 34 grape varieties
To reveal the genetic relationship of the grape varieties in three groups, we performed UPGMA cluster analysis to construct a dendrogram from the 15 SSR loci (Figures 1-3). In Group I, two hybrids of V. amurensisi and V. vinifera, Gongniang 1 and Gongniang 2, and one V. amurensisi Zuoshan were clustered in one group and separated from the other group, which was all V. vinifera varieties. The second V. vinifera varieties group contained nine varieties and this group was further divided into three subgroups. Three varieties (Cabernet sauvignon, Merlot181 and Merlot343) were grouped in one subgroup; two varieties (Muscat blanc and Muscat hamburg) with muscat flavour were included in another subgroup; and the third subgroup contained the remaining four varieties (Petit manseng, Petit verdot, Chinin blanc and Blue french) (Figure 1).
In Group II, three hybrids of V. vinifera and V. labrusca, Kyoho, Black Olympia and Jingya, and Meixiangbao, which is an offspring of Kyoho, were clustered together in one group and separated from the remaining V. vinifera varieties. The V. vinifera varieties group contained 10 varieties and was further divided into three subgroups. Centennial seedless with its three offsprings,   Figure 2). In Group III, two hybrids of V. vinifera and V. Labrusca, Venus seedless and Mars seedless, were grouped together in one cluster, and separated from the remaining varieties. Another cluster contained 10 varieties and was further divided into two subgroups. Otilia seedless and Dawn seedless were included in the first subgroup. The second subgroup contained the   The results of clustering indicated that the grape varieties of different species could be distinguished clearly, which agreed with the classical eco-geographic grouping theory [15]. Although most Chinese local cultivars are genetically separated from foreign cultivars to form a group, it also illustrated that the origins of some Chinese local cultivars might be related to foreign cultivars. The separation might be the result of long-term

Construction of molecular fingerprints of 34 grape varieties
According to the amplification results of each primer and the assignment standard of allele, each of 34 varieties received a specific SSR molecular fingerprint code (Table 9). The 15 SSR molecular markers can make a clear distinction between each of the varieties tested (except the two clones Merlot181 and Merlot 343), as well as between the relatives of the same parents and sistervarieties (Gongniang 1, Gongniang 2 and Zaokangbao, Lihongbao, Jinghongbao). It can also be applied to related appraisal, including the materials from different parents, different species or different subgenus.
In this study, we established the specific molecular identification of 34 grape germplasms. The sister varieties with the same parents, Lihongbao, Jinghongbao, Zaokangbao, and Gongniang 1, Gongniang 2, Jingya, the seedling of Black olympia, all could be effectively distinguished, but the two 'Merlot' lines 'Merlot181' and 'Mer-lot343' could not. Further work needs to address the molecular fingerprinting of these varieties using SSR molecules. The development of core molecular markers is one direction of our future research.
Using SSR markers as a tool for genetic mapping, cultivars identification, genetic diversity investigations, parentage analysis, as well as molecular fingerprint construction are highly accepted throughout the world [31]. The SSR analysis data reported here might provide worthy information for further grape protection, exploitation and utility, grape selection and breeding research in the whole world. Moreover, the established SSR data could enable researchers to preserve the valuable genetic cultivars for variety improvement, offer reference for the future studies on grape cultivars.
In this work, we used 15 pairs SSR markers selected from 44 pairs, but still did not cover the different linkage groups of the whole grape genome [32]. Complete and  irrefutable identification needs a larger number of loci [4]. The main purpose of our research was to perform a preliminary study of the genetic diversity and explore the molecular fingerprinting system of grape varieties. Thus, our work will be of benefit for future research in the grape research community.

Conclusions
Fifteen SSR primer pairs were selected to identify 34 grape varieties including 14 wine grape varieties, 22 table grape varieties of which 12 were seedless, six newly-developed table grape germplasms and their parents in China. The polymorphic band patterns among the 34 grapevine varieties were used to perform cluster analysis using UPGMA methods based on genetic similarity. All the three groups of grape varieties were clustered into two main groups, one is the V. vinifera and the other is the hybrid of V. vinifera and V. Labrusca or hybrid of V. amurensisi and V. vinifera. The SSR molecular fingerprint coding of each variety was established, even when there are sister varieties, or one is another variety's seedling. This research indicated that the SSR markers are useful for identification, analysis of genetic diversity of grape, molecular fingerprint database construction and also for grape breeding in the future.