| Strawberry (Fragaria ananassa Duch.), belongs to Rosaceae Fragaria, is widely cultivated with high nutritional and economic value in our country. Low temperature is one of the main factors affecting the yield and quality of strawberry in actual cultivation, especially the cold season and the comparatively simple facilities in the north. In this experiment,10 Hefei local cultivars of strawberry leaves were chosed as materials, and treated with 5 temperature gradient stress,0℃,-2℃,-4℃,-6℃,-8℃ respectively, calculating the ten strawberry material LT50, then made analysis of correlation with the chilling injury index, initially identified the cold-resistence ability of strawberry varieties. After that, single factor experiment design methods were adoted, Mg2+, dNTPs, Taq DNA polymerase, primer concentration, template DNA concentration and annealing temperature and other factors, which affected strawberry SRAP reaction system, to be optimized, so as to establish SRAP-PCR suitable system.55 pairs of polymorphic which were good, clear and stable bands primer pairs were screened from 110 pairs of primers, and statistical analysis were made of 55 pairs of these polymorphic primers, and built the clustering tree diagram. Preliminary studies were made with strawberries on cold-resistence SRAP marked, which laid the foundation for strawberry breeding cold-resistence varieties. Main results obtained in this study are as follows:(1) Under different temperature stress, electrolyte penetration was increased as the temperature reduced, and there was a negative correlation between the processing temperature and electrolyte penetration. Depending on the size of semi-lethal temperature hardiness, ten strawberry cultivars sort results are as follows:Toyonoka> Sweet berries> Xue Mei> Er Ma Wo> Saga fragrance> Ning Feng> Flange>Li Yi Nv> Jing Cheng Xiang > Zhang ji,and the semi-lethal temperature of each cultivar is-5.32℃、-4.74℃、-4.24℃、-3.89℃、-3.88℃、-3.09℃、-2.97℃、-2.43℃、-2.19℃ and-1.70℃.(2) The statistics obtained that,10 varieties of chilling injury index is between 0.5 to 0.9, and made correlation analysis with the semi-lethal temperature, the correlation coefficient was 0.735, reached significant levels. Using semi-lethal temperature to identify the cold-resistance of strawberry was reliable.(3) The modified CTAB method was adopted to successfully extract high-quality strawberry genomic DNA, which OD260/OD280 basic values were between 1.7-2.0, and agarose gel electrophoresis results also show that the extracted DNA purity is good, no protein contamination.(4) After optimizing the strawberry SRAP-PCR reaction system, and then suitable SRAP-PCR reaction system was established:20μL reaction system containing 10×PCR buffer 2μL, Mg2+ 2.0mmol/L, dNTPs 0.3mmol/L, both forward and reverse each primer to 0.6μmol/L, Taq DNA polymerase 1.0U, template DNA was 100ng.(5) 55 primer pairs with clear and rich bands, better polymorphism were screened from 110 pairs of SRAP primers, accounting for 50% of primer pairs.311 were amplified polymorphic bands, with an average rate of 73.43% polymorphism. Polymorphic information content (PIC value) was between 0.9001-0.2688, with an average PIC value of 0.6993. Cluster analysis showed that 10 strawberry varieties can be divided into 3 categories, which weakest " Zhang Ji " into a single category, however, a stronger cold resistance "Feng Xiang", "Xue Mei" in the first sub-category of third category.It can explain that the use of SRAP molecular markers can provide some reference for the study of cold resistance strawberry. |
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