| Tomato (Lycopersicon esculentum) is used as a model plant, the researches conducting on physiology, biochemistry, genetics and molecular biology of tomato are widely and deeply. Our country is a main tomato cultivation country, in order to guarantee the authentic and purity of seeds, protect agriculture production and legitimate interests of farmers, it is essential to develop fast and accurate method of cultivars identification. Among the main sources of impurity in tomato F1 hybrid seed production, the most difficult to avoid is self-pollination of female parent. There are researches using RAPD markers to identify the purity of F1 hybrid, they use seedling leaf of tomato to analysis. Aimed at performing earlier determination of the purity of seeds, it will have application vista by using dry seed to achieve the analysis. In recent years, along with perfecting of tomato molecular genetic linkage map and marked or located many important agronomy or phenotype characters on the map day by day, established foundation for thoroughly understand of genetic mechanism in tomato. WO gene confers resistance to insect, it makes the whole plant woolly and has the effect of homozygous lethal. There is no molecular marker of WO gene so far; constructing RAPD markers toward WO gene would help us to find out the reason of homozygous lethal and the mechanism of trichome initiation or development in tomato. Tm-2 gene is an important gene of tomato, it confers resistance to TMV, Tm-2 and nv (netted virescent) character have tight linkage (no recombination). Many markers have been mapped close to Tm-2 locus, but the Tm-2 gene have not been isolated, keep searching on molecular marker toward this gene will contribute to understanding the structure of this gene. In this paper, we established DNA extraction method from dry tomato seed; optimized and established RAPD reaction system; established RAPD markers for purity control of three tomato F1 hybrids -'wool-pink 808', 'wool-pink 818'and 'qiangxuan 1'; identified purity of 'qiangxuan 1' using dry seed; established RAPD markers for identification of five tomato commercial cultivars-'wool-pink 808', 'wool-pink 818', 'qiangxuan 1', 'wool-pink 802'and 'west-pink 3' to two groups; conducted searching RAPD markers for WO gene; cloned and sequenced three RAPD markers, among these markers, S1072683 is the marker for purity control, S13161057 is the marker for cultivars identification, S1388952 is the marker of Tm-2(nv) locus; established two markers S178570 and S1388952 tightly linked to the Tm-2 (nv) locus of tomato. DNA extraction method from young leaf of plants was according to the method of Yang Chong-ling (with minor modification). The DNA sample used for screening RAPD markers was male parent (tm-2 NV wo/wo) and female parent (Tm-2 nv WO/wo) of 'wool-pink808'. The RAPD primers, which can produce paternal-specific polymorphic DNA fragments, can be used for purity control; the RAPD primers, which can produce maternal-specific polymorphic DNA fragments, can be the candidate of the markers of Tm-2 (nv) or WO genetic locus. 350 random 10-base primers were used for polymerase chain reaction amplifications and identification of polymorphic markers. Produced 3590 discrete PCR products in total (an average of 10.3 products per primer), their size ranged from 0.1-3.5kb. 14 reproducibly amplified products(0.39%)amplified by 12 primers(3.43%) exhibited polymorphisms among the 'wool-pink808' parents were identified. The 6 RAPD markers that can be used to identify F1 hybrid tomato cultivars- 'wool-pink 808', 'wool-pink 818'and 'qiangxuan 1' are: S1019400, S14221270, S16950, S1503750, S1072683 and S1388800. The 8 maternal RAPD markers are: S5051700, S1458650, S169570, S66780, S178570, S1388952, S66740 and S13161057. Among the 8 maternal RAPD markers, 2 RAPD markers S66740 and S13161057 can divided the 5 tomato cultivars to 2 groups, one group have 'wool-pink 808', 'wool-pink 818' and 'wool-pink 802'; another group have 'qiangxuan 1' and 'west-pink3'. Following RAPD assays, the p... |
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