Optimization Of SSR Reaction System And Research Of The No Vine Pumpkin Molecular Markers

Pumpkin(Cucurbitaceae) is one of the older crops in hunman history, in China has a long cultivation history, widely distributed, referred to as the vegetables in the world. Pumpkins have strong resistance, richful in varieties of resources, strong adaptability, use widely, resistant storage, high yield, fruit shape、size、color、quality is different, has the characteristics of significant biodiversity. Pumpkin has a high nutritional value and medicinal value. Pumpkin nutrition composition is a comprehensive and unique, especially the VA content in pumpkin pulp ranks first of all the vegetables. Study of pumpkin plants at home and abroad, lags far behind other crops, and number of molecular markers can be applied to the pumpkin is very limited. In this study to pumpkin SSR-PCR system, on the basis of optimization, to influence the production of pumpkin tendril traits were studied molecules, aimed at pumpkin and fine mapping and genetic control trailing characters of tag tendril gene will provide a scientific basis for gene choning.The main results were as follows:1. In order to obtain high quality pumpkin genomic DNA, improved by traditional CTAB method, CTAB method after the adjustment are obtained, every25ml CTAB extraction buffer to add0.25g PVP and1ul mercaptoethanol, with TE solution (containing10mg/ml of lul RNaseA) dissolving pumpkin genomic DNA. Effectively reduce the polyphenols in the leaves, polysaccharidc, protein and RNA, for subsequent PCR reaction with high quality genomic DNA.2. In order to obtain effective pumpkin SSR-PCR reaction system, to dwarf pumpkin inbred lines and sprawling inbred lines as test material, by adopting the orthogonal experiment method affecting the pumpkin SSR reaction system, the main conditions for research, and designed a gradient PCR annealing temperature on the pumpkin SSR reaction system was optimized, best of SSR-PCR reaction system is established. Reaction system for20ul, contain1.5mmol/L Mg2+,60ng DNA,0.20mmol/L dNTPs,0.25mmol/L primers and1.5U Taq polymerase, gradient PCR optimized annealing temperature, obtain the best annealing temperature is44.1℃.3. Using the selected36pairs of specific primers for24parent material amplify a total of2437bands, article2329of the polymorphism, accounting for95.57%of the total. Different primers amplified polymorphisms of total quantity of the product and get quantity difference is larger, product amplification bands ranging from2to8, an average of4. Polymorphism loci percentage up to100%, the lowest is35%.4. Using SSRmarkers to tag pumpkin tendril character length, homozygous group of no tendril at hybrid genetic distance was7.2693cM, homozygous no group with long tendril guroup genetic distance was10.1426cM, hybrid no group with long tendril group genetic distance was8.0847cM. Take the LOD values greater than3there are5markers can chain effectively. According to all the cluster analysis, confirm the pumpkin without stem traits are controlled by dominant gene.