New Germplasm Creation Of ZYMV Resistance Squash By Using Marker And Double Haploid Technique

Zucchini yellow mosaic virus (ZYMV), is the main disease of Cucurbita. There are no effective prevention measures to this virus. Breeding resistant varieties is effective way to ensure the squash (Cucurbita pepo L.) safety production, while there are less disease-resistant cultivars to ZYMV. Using molecular marker assisted selection breeding can speed the breeding process, improve the accuracy of choice. Molecular marker assisted breeding research for squash (Cucurbita pepo L.) started much later, available marks are too less. The study of genetic gene analysis of squash resistance to ZYMV showed that the disease resistant is control by dominant single gene. Therefore, we developed 216 pairs of EST-SSR primers, synthesized 48 pairs of primers randomly, together with other available 204 pairs of SSR primers, analyzed the preliminary location of Zucchini resistant gene to ZYMV. ZYMV resistance gene was located at two molecular markers, between CmTm227 and WMG28, genetic distance is 3.9cM and 13.8 cM respectively.In this study, 330, 331 and 332, three genotype squash materials, were used as test materials, unpollinated ovaries were cultured in vitro one day before flowering. The result showed that induction rate of embryoid was variable for different genotype. The highest rate of plants regenerative was 332, followed by 331, 330was lowest. The effect of different media to induction of embryoid was significant, MS medium was much higher than N6 medium, reduce concentration of sucrose also is helpful for embryoid induction. The optimal concentration of sucrose in MS medium was 20g/L, 30g/L in N6 medium. Increasing content of B vitamins and inositol could also benefit to embryoid induction. The optimal concentration of TDZ in both MS and N6 medium was 1.0 mg/L.We also researched the effect of different genotypes and irradiation dose (25-400Gy) to induction of haploid plants of squash. The results showed that both irradiation dose and genotypes can affect the rate of haploid induction significantly. It was suitable for squash haploid induction when irradiation dose rate was 2Gy/min, irradiation dose was 125-250Gy, it was helpful for haploid plant produce. Rate of haploid induction of genotype 330 was 5.08%, 331 was 7.40%, and no haploid plant was obtained from 332. Haploid plant was mainly developed from point and arrow embryos. We obtained maximum number of total embryos in 75Gy, most embryos were point embryos in 75Gy and 100Gy, in 125Gy and 150Gy, it was inclined to produce Globular embryos.Ploidy identify was analyzed using count chloroplast in guard cells and flow cytometry analysis, and analyzed the haploid of regeneration plant and natural doubled haploid using specific EST-SSR primers.