Study On The Purity Identification Of Cultivated Watermelon By SRAP Molecular Marker

Purity identification of five shares watermelon hybrids were studied by the SRAP molecular marker through using the polymorphic primer pairs selected from 64 primer pairs,which were checked by 3 watermelon hybrid combinations and their parents.The related technology of SRAP marker on purity identification were optimized.The results of this study were as follows:(1) The optimized SRAP marker suitable for watermelon DNA analysis was established.1) The meathed of DNA extraction suitable for SRAP analysis were established. CTAB extraction to the watermelon's euphylla was best when EDTA density is 20 mmol·L^-1.2) Taking watermelon genome DNA as template,the major components of SRAP, such as concentrations of Mg²⁺,dNTPs,primer,DNA,Taq polymerase,were optimized by design of seven levels of five factors,respectively.The results showed that the optimum SRAP reaction system includes Mg²⁺ 2.0 mmol·L^-1,dNTPs 0.2 mmol·L^-1, prmier 60 ng,DNA 100 ng and Taq polymerase 0.5 U in the 15μL volume reaction. The optimum SRAP reaction program was as follows:pre-denaturation at 94℃for 5 min followed by 5 cycles of denaturation at 94℃for 1 min,anneal at 35℃for 1 min and extension at 72℃for 1 min;then 35 cycles of 94℃for 1 min,50℃for 1 min and 72℃for 1 min,and a final extension at 72℃for 5 min;kept at 4℃.The program and system could meet the demands for genome SRAP marker in watermelon.3) The examination technology of amplification reaction production was optimized. The gradient establishment experiment showed that the results of the silver dyeing was best when the fixed time was 15 rain,the dyeing liquor concentration was 0.133%, stationary liquor concentration was 1.2%.Moreover,the slab rubber stabilized image after stationary were favorable for the preservation and the later research analysis because of avoiding the phenomenon of dry shrink and fall.(2) Parents purity identification and polymorphic primer pairs seleted.The purity identification of Parents by 3 primer pairs seleted showed the parent seed quite has high purity.Seletion was carried among the 64 primer pairs by HR-F,HR-M,HR-H-1, DF-12-F,DF-12-M,DF-12-H,my-1-F,my-1-M and my-1-H using the SRAP-PCR reaction program and system optimized.The results showed that there were 32 primer pairs displaying polymorphism.Each primer pairs has 37～129 clear amplified fragments and the total was 2 184,average was 68.25;Each primer pairs has 1～14 polymorphic fragments and the total was 132,average was 4.13.The percentage of polymorphic fragments was 1.37%～18.18%,average was 6.04%.(3) SRAP marker could be applied in the purity identification of watermelon hybrids.Seed purities of five shares watermelon cultivars were tested by the SRAP-PCR reaction program and system optimized.The results showed that the purity of five shares watermelon cultivars was 94.92%,98.18%,94.92%,94.92%and 94.64%, respectively,which were lower than field-planting identification's.The accordant rate between SRAP technology and field-planting identification was 95.87%,99.17%, 97.85%,97.85%and 98.59%,respectively.It showed that SRAP had preferably ability to identify purity than field-planting identification.Moreover,the accordant rate and correlative rate beteen SRAP identification and field-planting identification have good consistency.Therefore,SRAP technology could replaces field-planting identification, overcoming the shortcomings such as long appraisal cycle and easy government by the environmental condition of field-planting identification.