| Melon (Cucumis melo L.) is one of the most important horticultural crops in terms of its planting scale and economic value on the world. But the continuous cropping obstacles caused by the spread of the soil-borne diseases severely restricts the development of melon production in melon cultivation process, Grafting is an efficient method to improve the resistence of soil-borne diseases. Pumpkins, including the Cucurbita maxima, Cucurbita moschata, and their interspecific hybrids, are the most popular rootstocks of grafted melons. However, majority of the pumpkin cultivars were found to have negative effects on the fruit quality of grafted melon when they were used as rootstocks. As a result, evaluating and breeding high quality rootstock varieties specifically for melon is of great importance in commercial melon production. In addition, pumpkin used as the rootstock has its particularity. We can't know it germplasm homozygosity through the phenotypic traits observation in the whole growth period. So, using the transcriptome sequencing sequence to develop Cucurbita SSR makers is particularly important before the pumpkin genome-wide published. In this study, we evaluated and screened the pumpkin germplasm resources, evaluating the utility of pumpkin inbred lines as the rootstocks of grafted melon, creating new pumpkin cross combinations and evaluating the utility of it as the rootstocks of grafted melon, and developed Cucurbita SSR makers use the transcriptome sequencing. We aimed to select suitable rootstocks and provide the germplasm materials for grafted melon production, and quickly identify pumpkin germplasm resources and the hybrid purity of rootstock cross combinations. The main results are as follows:1. Investigating and evaluating the phenotypic traits of 89 long-term selfing purification pumpkin germplasm resources from the National Vegetable Germplasm Repository for mid-termin. The investigation index including 20 quantitative characters like lord tendril length/diameter, leaf length/wide, mature fruit transverse and longitudinal diameter, weight of single mature fruit, seed length/wide, thousand seed weight and 28 qualitative characters like Young fruit skin color, mature fruit skin color, fruit shape. The results showed that the coefficient of variation was between 0.11 and 1.28, and the average coefficient of variation of 48 phenotypic traits was 0.42, seed periphery had the largest coefficient of variation for 1.28 and fruit peduncle had the smallest coefficient of variation for 0.11. Comprehensive evaluated the phenotypic traits and growth condition in the field, we select 29 pumpkin inbred lines as the material for the rootstock evaluating of grafted melon.2. Using 29 screened pumpkin inbred line rootstock materials (including 27 C.moschata materials and 2 C.maxima materials) and two popular commercial rootstock varieties of melon were used as the controls. The non-grafted Elizabeth plants were also included to evaluate the grafting on melon performances. Significantly different effects on the growths and fruit qualities of grafted melon plants were observed when the cross combinations were used as rootstocks as compared to the non-grafted melon plants, we measured the grafting affinity, the yield and fruit quality of grafted melon plants, as well as the hypocotyl thickness and length of the cross combinations and their tolerances to root-rot. On the comprehensive analysis and evaluation, we selected 9 pumpkin inbred line rootstocks which were suitable for grafted melon, to create new rootstock cross combinations as parent materials.3. Using 9 screened pumpkin rootstock materials (including 7 C.moschata materials and 2 C.maxima materials). We created 25 pumpkin cross combinations, including 13 interspecific hybrids of C. maxima and C. moschata and 12 intraspecific hybrids of C. moschata. Under the temperature condition of 10?/5?, we did cold tolerance identification of the rootstock combinations seedling, the results showed that the pumpkin rootstock materials is generally resistance to low temperature, and the interspecific hybridization combinations has much better cold tolerance than the intraspecific hybridization combinations; The widely cultivated melon cultivar'Elizabeth'was used as scion in autumn experiment in Wuhan and 'Nanhaimi' was used as scion in spring experiment in Sanya to appraise the suitability of these pumpkin hybrids as rootstocks, taking the hypocotyl thickness and length, grafting affinity, melon plant growing after grafted, fruit characters and taste quality of the cross combinations into consideration. On the comprehensive analysis and evaluation of 25 pumpkin cross combinations, we selected two potential rootstock combinations.4. Analyzing the transcriptome sequencing data of rootstock pumpkin of our research group. We got 747 potential SSR makers by search software. We designed 211 pairs of primers and verified the polymorphism by polyacrylamide gel electrophoresis with 61 commercial pumpkin rootstocks and 4 pumpkin inbred lines. Totally we got 110 pairs of primers which had clear electrophoresis banding and high polymorphism. Analyzing the information of SSR loci and the characteristics of polymorphism primers, the result showed that the most loci were dinucleotide and trinucleotide repeats in the polymorphism markers loci, the percentage of them were 26.36% and 62.73% respectively. The polynucleotide repeats loci were less with total content of 10.9%. In addition, the average polymorphism information content (PIC) of 110 pairs of primers was 0.34, the primer which had the biggest PIC was LySSR102 for 0.5, and the smallest was LySSR157 for 0.09. Based on the amplificated results by 110 pairs of primers with 65 pumpkin rootstock materials, we did Clustering analysis. The result showed that in the similarity coefficient of 0.57,65 pumpkin materials could be divided into two groups, one group was C. maxima or the interspecific hybrids of C. maxima and C. moschata, the other group was C. moschata or the intraspecific hybrids of C. moschata. The screened polymorphism primers can also be applied to the construction of fingerprint for new combination and the identification of pumpkin rootstocks hybrid purity and so on.5. Based on the development of Cucurbita SSR markers, we selected a group which had 3 SSR makers, LySSR01, LySSR90 and LySSR131, which had high polymorphism, good banding pattern, also the differences of the amplification product size were around 50bp and the primers can't match each other to form the hairpins. We explored the multiple PCR system in molecular markers development and optimize it, and finally we found the most appropriate Cucurbita multiple PCR system. We used 4 groups of SSR makers, LySSR69, LySSR14, LySSR131; LySSR70, LySSR63, LySSR131; LySSR121, LySSR59, LySSR138; LySSR210, LySSR100, LySSR138 to verify the optimized Cucurbita multiple PCR system. The pumpkin multiple PCR system significantly improved the efficiency of the SSR marker analysis. It can be used for pumpkin cultivars identification, fingerprint build and hybrid purity identification and so on.6. Based on Cucurbita SSR markers LySSR121 and LySSR138 we had developed, we did qRT-PCR with 2 pumpkin parents and their hybrid F1. Analyzing the dissolution curve in the process of PCR and exploring the application of Cucurbita SSR-HRM in pumpkins germplasm genetic diversity, fingerprint build and hybrid purity identification. The results showed that compared with traditional electrophoresis detection method, using SSR-HRM could work more efficient and rapid. |
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