SHORT TERM SCIENTIFIC MISSIONS (STSMs) Reference: Short Term Scientific Mission, COST Action FA1003 Beneficiary: Bocharova Valeriia, National Scientific Center Institute of viticulture and winemaking named after V.Ye. Tairov Host: Regner Ferdinand, HBLAuBA Klosterneuburg Period: from 14.11.011 to 7.11.011 Place: Langenzersdorf (Austria) Reference code: COST-STSM-ECOST-STSM-FA1003-141111-01131 Amount: EUR 1500 I. Purpose of the STSM: STSM Scientific Report The main aim of a short-term training in a HBLAuBA Klosterneuburg laboratory is a possibility to possess the new methods of molecular genetic analysis, get to know about new equipment and research strategies for studying grape genome which was selected on the base of NSC Tairov institute of viticulture and wine-making. In addition, establishing the missing scientific and educational relations between NSC Tairov institute of viticulture and winemaking and Department of Grapevine Breeding with possibility of cooperation in future. Besides, increasing the knowledge about: SSR fingerprinting and the other methods for genetic characterization, phenotyping and mapping, to become proficient in chloroplast typing and other PCR markers for characterization. Learning to use a new sequencing machine Li- Cor 4300 DNA Analyser. The knowledge will give the possibility to provide a research of grapevine genome rate on sufficiently high level. II. Description of the work carried out during the STSM: 14.11.011 The first day of DNA extraction from cane samples of cultivars and clones of grapevine (10 samples); Samples list:
1 3 4 5 6 7 8 9 10 11 Aligote 101 clone Cabernet Sauvignon 143141 clone Sukcholimansky beliy Odessky cherniy Cabernet Franc Smena Tairian Zagrey Jantar Aromatniy Muskat odesskiy Clones breeding by NSC Tairov institute of viticulture and wine-making Varieties breeding NSC Tairov institute of viticulture and wine-making propagate in Ukraine Varieties propagate in Ukraine New promising varieties breeding NSC Tairov institute of viticulture and winemaking The eleventh sample was brought as extracted DNA from NSC Tairov institute of viticulture and wine-making department of Molecular-genetic and Phytopathology (Muskat odesskiy). 15.11.011 The second day of DNA extraction of cultivars and clones of grapevine; Checking the concentration of DNA in agarose gel; Dilution of DNA samples to the desired concentration; The first PCR by SSR-locus VVS (control PCR); 16.11.011 Preparation of sequencing gel; Preparation samples for sequencing, pre-run; Sequencing by LI-COR Biosciences 4300 DNA Analyzer (SSR-locus VVS); The second PCR by SSR-loci VVMD7,VVMD7, VVMD5, VRZAG6, VRZAG79; 17.11.011 Sequencing by LI-COR Biosciences 4300 DNA Analyzer (SSR-loci VVMD7,VVMD7, VVMD5, VRZAG6, VRZAG79); Learning to work in the program SAGA Generation ; The third PCR by SSR-loci VVMD5,VVMD8, VVMD3; 18.11.011 Sequencing by LI-COR Biosciences 4300 DNA Analyzer (SSR-loci VVMD5,VVMD8, VVMD3 ); Data analysis; 1.11.011
Sequencing by LI-COR Biosciences 4300 DNA Analyzer; The PCR by chloroplast marker ccmp10; Preparation of gel for silver staining; Data analysis, statistical analysis;.11.011 Sequencing by LI-COR Biosciences 4300 DNA Analyzer (final results); Preparation of gel for silver staining; Data analysis; 3.11.011 PAGE electrophoresis by chloroplast DNA-marker ccmp10 of 10 samples; Silver staining; Data analysis; 4.11.011 Beginning of cloning PCR products with pgem-t and pgem-t easy vectors; The extraction of nucleic acid from agarose gels; 5.11.011 Ligation Using the purified PCR fragments; Data analysis; III. Description of the main results obtained: A molecular-genetic analysis of polymorphism grapevine cultivars from collection NSC Tairov institute of viticulture and wine-making using 9 microsatellite loci was conducted. The allele size of SSR-loci was obtained (table 1). Near the allele size is shown comparison the results of SSR-analysis was got in laboratory by Tairov Institute of viticulture and winemaking. Table 1 DNA profiles of Ukrainian cultivars analysed by 9 microsatellite loci VVS VVMD5 VVMD7 VVMD7 No. Sample Allele1 Allele Comp. Allele1 Allele Allele1 Allele Comp. Allele1 Allele Comp. 1 Aligote 101 clone 13 136 6 38 37-178 188 Cabernet Franc 138 146 141:149 4 38 37 61 40:64 180 188 179:185 3 Sukcholimansky beliy 13 150 6 30 37 55 174 188 4 Smena 134 14 34 36 39 47 180 188 5 Tairjan 134-34 38 37 47 178 184 6 Cabernet Sauvignon 143141 clone 138 150 141:153 30 38 37-40 174 188 175:185 7 Zagrey 14 136 6 34 37 45 178 18 8 Jantar 134 148 4 36 45 47 184 19 9 Aromatniy 13 14 4 30 45 47 180 188 10 Odessky cherniy 13 150 34 44 37 41 188 19 11 Muskat odesskiy 13 14 34-45 47 184 188
VRZAG6 VRZAG79 VVDM5 VVMD8 VVMD3 No. Allele1 Allele Comp. Allele1 Allele Comp. Allele1 Allele Allele1 Allele Allele1 Allele 1 19 194 40 4 39-7 35 38-19 0 195:06 44 56 50:60 39 55 7 35 38 56 3 186 19 4 44 41 49 33 35 38 54 4 18 186 56 58 55-33 43 54 56 5 186 19 36 48 39 49 33 57 38-6 186 19 189:195 44-50:50 55-33 35 38-7 19 0 40 5 39 49 6 43 34-8 184 194 5 54 49 55 43-38 54 9 0-40 58 41 55 33 47 38 7 10 186-44 54 41 49 35 43 38-11 50 60 33 67 36 7 The previous results was obtained by silver staining and shows difference by near 3-4 bp. Statistical assay by allele frequency, quantity of homo- and heterozygous, and homozygosity was obtained. The statistical analysis of studied SSR-loci showed the high level of polymorphism (Table ). Table Indicators of informativeness DNA-marker system studied grape varieties
VVS 14 0,045 0,0005 13 0,7 0,05159 134 0,18 0,03314 136 0,09 0,0081 138 0,09 0,0081 14 0,136 0,018496 9 1 10 0,86 146 0,045 0,0005 148 0,045 0,0005 150 0,136 0,018496 0,996 0,1439 VVMD5 4 0,136 0,018496 6 0,136 0,018496 30 0,136 0,018496 34 0,73 0,07459 36 0,09 0,0081 7 1 10 0,83 38 0,18 0,03314 44 0,045 0,0005 0,998 0,17366 VVMD7 37 0,409 0,16781 39 0,045 0,0005 41 0,045 0,0005 45 0,18 0,03314 47 0,7 0,05159 7 9 0,74 55 0,045 0,0005 61 0,045 0,0005 0,998 0,60034 VVMD7 174 0,09 0,0081 178 0,136 0,018496 180 0,136 0,018496 18 0,045 0,0005 184 0,136 0,018496 7 0 11 0,79 188 0,364 0,13496 19 0,09 0,0081 0,997 0,0609 Table
VRZAG6 18 0,05 0,005 184 0,05 0,005 186 0,3 0,09 19 0, 0,04 5 8 0,79 194 0, 0,04 0 0, 0,04 1 0,15 VRZAG79 36 0,045 0,0005 40 0,136 0,018496 4 0,09 0,0081 44 0,7 0,05159 48 0,045 0,0005 50 0,045 0,0005 5 0,09 0,0081 11 1 10 0,88 54 0,09 0,0081 56 0,09 0,0081 58 0,09 0,0081 60 0,045 0,0005 0,993 0,11865 VVDM5 39 0,5 0,065 41 0,15 0,05 49 0,5 0,065 4 8 0,73 55 0,35 0,15 1 0,7 VVMD8 6 0,045 0,0005 7 0,09 0,0081 33 0,73 0,07459 35 0,7 0,05159 43 0,7 0,05159 8 1 10 0,81 47 0,045 0,0005 57 0,045 0,0005 67 0,045 0,0005 0,997 0,193787 VVMD3 34 0,09 0,0081 36 0,045 0,0005 38 0,54 0,916 54 0,136 0,018496 6 5 6 0,66 56 0,09 0,0081 7 0,09 0,0081 0,991 0,33641 Common results - - 7,11 1,67 8 0,79
The average allele number of the locus was 7,11. Average heterozygosity or polymorphism level (Не) of nine studied loci of analyzed samples was 0.79. The next step of analysis was transforming the data from allele size into the n-code system (table 3). Transformed data of SSR-analysis to n-code Table 3 Sample VS VVMD 5 VVMD 7 VVMD5 VVMD8 VVMD3 VVMD 7 VRZag6 VrZag79 Aligote 101 clone N+10 N+14 N+4 N+16 N+6 N N+10 N+18 N+ N+ N+1 N+16 N+18 N N+ Cabernet Franc N+16 N+4 N+ N+16 N+6 N+30 N N+16 N+10 N+18 N+ N+0 N+4 N+1 N+16 N+6 N+4 N+16 Sukcholimansky beliy N+10 N+8 N+4 N+8 N+6 N+4 N+ N+10 N+16 N+18 N+ N+18 N- N+1 N+10 N+16 N N+4 Smena N+1 N+0 N+1 N+14 N+8 N+16 N+16 N+16 N+6 N+18 N+0 N+4 N+1 N+6 N+10 N+16 N+18 Tairian N+1 N+1 N+16 N+6 N+16 N N+10 N+16 N+40 N+ N+ N+8 N+10 N+16 N-4 N+8 Cabernet Sauvignon 14314 N+16 N+8 N+8 N+16 N+6 N+16 N+16 N+18 N+ N- N+1 N+10 N+16 N+4 Zagrey N+ N+14 N+4 N+1 N+6 N+14 N N+10 N+8 N+6 N- N+ N+10 N+16 N+6 N N+1 Jantar N+1 N+6 N+ N+14 N+14 N+16 N+10 N+16 N+6 N+ N+18 N+8 N+16 N+8 N+18 N+1 N+14 Aromatniy N+10 N+0 N+ N+8 N+14 N+16 N+ N+16 N+16 N+30 N+ N+36 N+4 N+1 N+6 N N+18 Odessky cherniy N+10 N+8 N+1 N+ N+6 N+0 N+ N+10 N+18 N+6 N+ N+1 N+16 N+10 N+4 N+14 Muskat odesskiy N+10 N+0 N+1 N+14 N+16 N+16 N+50 N N+36 N+8 N+1 N+10 N+0 (Fig. 1). The data was coded and analysed using MEGA 4 program for building genetic tree Fig. 1. Distribution tree studied grape varieties by genetic distances, calculated according to the SSR-analysis using UPGMA method (figures near the bifurcation branches - the authenticity of the selection of the cluster)
To improve the reliability of the results of cluster analysis bootstrap-test was conducted (Fig. ). Bootstrap method is loaded into the program MEGA 4, is most often used to test probability of formation branches on phylogenetic trees. Fig.. Tree obtained according bootstrap-test As shown in the tree, studied varieties are divided into two clusters. The first cluster is formed mainly technical cultivars, except table cultivar Tairian. And the second cluster is formed mainly table cultivars, except technical cultivars Muskat odesskiy and Aromatniy. The first cluster is formed by cultivars with mainly close genoms. The variety Sukcholimansky beliy is the result of a cross between the Chardonnay and Plavay (a Moldovan variety of white grape). Variety Aligote was obtained from a cross of Pino Noir and Gouais Blanc. Even the variety Chardonnay derived from a cross between the Pinot and Gouais Blanc (Heunisch) too. So the genetic distance between Sukcholimansky beliy and Aligote 101 is low (0,1801). The variety Odessky cherniy (Synonyms - Alibernet) - crossing of Alicante Bouschet x Cabernet Sauvignon was bred in 1950 in the NSC Tairov institute of viticulture and winemaking in Odessa. It is planted in Slovakia, Hungary and the Czech Republic. That's way genetic distance between variety Odessky cherniy and Cabernet Sauvignon 143141 is very low (0,1313). The cultivar Cabernet Sauvignon was a result of cross between Cabernet Franc x
Sauvignon blanc, and in this case Cabernet Sauvignon 143141 is close to Cabernet Franc on the tree (genetic distance 0,1801). Polymorphism of chloroplasts DNA As a result of chloroplast DNA analysis using ccmp10 chloroplast-marker polymorphism of grapevine varieties was derived (Fig. 3). Fig. 3. The results of electrophoresis by chloroplast DNA-marker ccmp 10 (M size standard marker 50 bp Ledder, Promega, 1 - Aligote 101 clone, - Cabernet Franc, 3 Sukcholimansky beliy, 4 - Smena, 5 - Tairian, 6 - Cabernet Sauvignon 143141 clone, 7 Zagrey, 8 - Jantar, 9 - Aromatniy, 10 - Odessky cherniy, 11 - Muskat odesskiy) Three polymorphic chlorotypes were observed: 1) 113 bp sample 1 (Aligote 101 clone); ) 114 bp 3, 5, 6, 7, 8, 9, 10 (Sukcholimansky beliy, Tairian, Cabernet Sauvignon 143141 clone, Zagrey, Jantar, Aromatniy, Odessky cherniy); 3) 115 bp, 4, 11 (Cabernet Franc, Smena, Muskat odesskiy). Interesting that the clone Cabernet Sauvignon 143141 doesn't have similar chlorotyp with parent cultivar Cabernet Franc. Because Cabernet Sauvignon inherited chloroplasts DNA from the mother's cultivar Sauvignon blanc. IV. Future collaboration with host institution (if applicable): The cultivars exchange between collections; Introduction of new clones and researches of resistance; Training of young scientist;
Publications; Development of genotyping and identification of cultivars using SSR-analysis; Somatic chimerism, genetic inheritance, and mapping of mutation in grapevine; Genetic diversity and geographical dispersal of Austrian and Ukraine cultivars using by microsatellite markers; V. Foreseen publications/articles resulting or to result from the STSM (if applicable): It is foreseen that after deepest data analysis publication of results from STSM will be performed in the Mitteilungen Klosterneuburg. VI. Confirmation by the host institution of the successful execution of the STSM: Valery Bocharova used her stay in our laboratories to get familiar with several methods for genotyping grapevine material. As she have done all steps of the protocols she is ready to reestablish her procedures at the lab at home institute. For several of her probes the complete way from isolation to the analysing of results was done. On the other side she had also the opportunity to genotype with chloroplast markers and cloning fragments for sequencing. During her stay she could give an evidence of her perfect manual and intellectual capacities as a researcher. Valery Bocharova is a hard working scientist with high demands for precision and quality for her research. On the other side she is skilled with high social and emotional competence that each chief would appreciate to have her in his scientific team. We wish her all the best for future scientific career.