| Grape is an important economic fruit crop. Great progress has been made in conventionalcross breeding, but grape lags behind other economic crops in molecular genetic mapconstruction and marker assisted breeding in Chian. In this study,94F1progenies derivedfrom the cross ‘Red Globe’（Vitis vinifera L.）בShuangyou’（Vitis amurensis Rupr.） and94F2progenies derived from ‘Beibinghong’（Vitis vinifera L.×Vitis amurensis Rupr.） selfingwere used as two mapping populations, and molecular genetic linkage maps based on SSRand SRAP markers were constructed for ‘Red Globe’,‘Shuangyou’ and ‘Beibinghong’respectively. Grape cold hardiness was measured, and QTL mapping for cold hardiness wasstudied by interval mapping. High density molecular genetic maps of grape and QTL mappingfor grape cold hardiness provide reliable theoretical basis, methodology and materials forgrape cold hardiness gene isolation, gene clone and molecular assisted breeding. This study isof great importance in grape cold hardiness breeding. The main results are as follows:1. SRAP-PCR reaction system was optimized for Vitis amurensis Rupr. and its progenies.L16（45） orthogonal design results showed that the optimized20μl SRAP-PCR reaction systemcontains10ng template DNA,0.8μmol·L-1Primer,0.2mmol·L-1dNTPs,3.2mmol·L-1Mg2+,Taq DNA polymerase2.0U as well as2μl10×buffer （Mg2+free）.24progenies derived fromthe cross ‘Red Globe’בShuangyou’ was amplified by this optimized system, resulting inclear bands with good polymorphism, which implies that the system can be used in geneticlinkage map construction and genetic diversity analysis.2. Optimal annealing temperatures of447grape SSR primer pairs were selected. Testedannealing temperatures were69℃,68.2℃,66.7℃,63.9℃,60.5℃,57.8℃,55.9℃and55℃.Clear and stable bands can be amplified by the primer pairs under their optimal annealingtemperatures.3. Seedlings derived from the cross ‘Red Globe’בShuangyou’ was successfullyidentified for their authenticity using the optimized SRAP-PCR reaction system. Along withmorphological characters in the field,92.6%of94seedlings were identified true hybrid withonly5SRAP primer pairs carrying male parent specific band.4. Population derived from the cross ‘Red Globe’בShuangyou’ was amplified using68SRAP primer pairs and186SSR primer pairs, creating totally347SRAP segregation loci and186SSR segregation loci, among which132SRAP loci were female parent specific,48SSRloci only segregated in female parent,155SRAP loci were male parent specific,54SSR loci only segregated in male parent,60SRAP loci were in common for both parents and84SSRloci segregated in both parents. Population derived form ‘Beibinghong’ selfing was amplifiedusing57SRAP primer pairs and174SSR primer pairs, producing totally279SRAPsegregating loci and174SSR segregation loci.5. Molecular genetic map for ‘Red Globe’,‘Shuangyou’ and ‘Beibinghong’ wassuccessfully constructed.‘Red Globe’ molecular genetic map contains266loci and19linkagegroups, and covers a total length of1370cM with an average distance between markers of4.8cM.‘Shuangyou’ molecular genetic map contains233loci and20linkage groups, andcovers a total length of1254cM with an average distance between markers of5.0cM.‘Beibinghong’ molecular genetic map contains228loci and21linkage groups, and covers atotal length of1123cM with an average distance between markers of4.5cM.6. Cold hardiness for ‘Red Globe’,‘Shuangyou’ and their82F1progenies as well as‘Beibinghong’ and its92F2progenies was phynotyped. Annual canes of all materials weretreated under gradient low temperatures as follows:-15℃,-20℃,-25℃,-30℃,-35℃,-40℃and-45℃. Relative conductivity was measured. Fitting Logistic equation, half lethaltemperature for all materials was calculated to evaluate cold hardiness. Based on this, QTLsfor grape cold hardiness were detected with software MapQTL5.0following IntervalMapping model.4QTLs were detected on ‘Red Globe’ genetic map, and1on ‘Shuangyou’and ‘Beibinghong’ genetic map each. |
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