Analysed The Genetic Diversity In Squash By SRAP And ISSR Markers

Breeders think that cultivating high-quality squash varieties is more and more important, because of the Squash contains rich nutritional value, function of health care and processing value. Analysing the genetic diversity and genetic relationship by using the molecular biological techniques for the existing squash varieties is very important. In my study, analysed the genetic diversity and established genetic clustering figure and identified the genetic relationship of 85 Squashes by SRAP and ISSR. Meanwhile combined with field observation to provide the theory basis of identification of germplasm resources, classification and the cross breeding work. Main results were as follows.1. Establishment of SRAP amplification system of SquashWe took an optimization experiment of SRAP amplification system with single factor design which focused on the concentrated of Mg2+, dNTP, Taq DNA polymerase, primer, template DNA and the amplification procedures of annealing temperature and cycle by using the Squash ’mien’as the material of filter system. Experimental results showed the optimal 20μL SRAP amplification of Squash which contained 0.20mmol/L dNTP,1.50U Taq DNA polymerase,80ng template DNA, 0.16μmol/L single primer,1.50mmol/L Mg2+,2μL 10×Buffer. The best amplification procedures was pre-denaturation at 94℃ for 5min; Next enter 5 cycles, inculding 94℃ for 1min,35℃ for 1min,72℃ for lmin; Then enter 35 cycles, inculding 94℃ for 1min,52℃ for 1min and 72℃ for 1min; Finally, extension at 72℃ for 10min.2. Analysed the genetic diversity in Squash by SRAPThe results showed:17 pairs of primers were screened out of 100 pairs of SRAP primers,which produced 200 DNA bands, polymorphic bands were 187,13 bands were shared with all tested primers, the proportion of polymorphic was 93.50%. The genetic similarity coefficient of 85 accessions in squash was between 0.4500-0.9850 which was calculated by Ntsys2.10e. Through cluster analysis, Squashes were divided into three groups:Cucurbita pepo L, Cucurbita moschata, Cucurbita maxima. The relationship of Cucurbita moschata and Cucurbita pepo L were close, whereas their kinship with Cucurbita maxima were far.3. Establishment of ISSR amplification system of Squashwe took an optimization experiment of ISSR amplification system with single factor design which focused on the concentrated of Mg2+, dNTP, Taq DNA polymerase, primer, template DNA by using the Squash’miben’as the material of filter system. The greatest 25μL ISSR reaction system which contained with 2.50μL 10×Buffer,2.00mmol/L Mg2+,0.30mmol/L dNTP,1.00U Taq DNA polymerase, 0.48μmol/L primer,75ng template DNA. On this basis, optimized annealing temperature of primers which had been screened, and ultimately determined the optimal annealing temperature of 12 primers.4. Analysed the genetic diversity in Squash by ISSRThe results showed:12 polymorphic primers were screened out of 100 polymorphic primers, which generated 125 DNA bands,115 bands were polymorphic, 10 bands were shared with all tested primers. The ratio of polymorphic bands was 92.00%, And the genetic similarity coefficient of 85 accessions in squash ranged between 0.3840 to 1.0000, which was calculated by Ntsys2.10e. The cluster analysis showed that squashes could be divided into three groups:Cucurbita pepo L, Cucurbita moschata, and Cucurbita maxima. The relationship of Cucurbita moschata and Cucurbita pepo L was close, whereas their kinship with the Cucurbita maxima was far.5. The survey result of filed characters in squashThe survey result of filed characters were as follows, cotyledon length was 3.40cm-7.30cm; cotyledon width was 2.15cm-4.38cm; aspect ratio of cotyledon was 1.30-2.30; squashes’ weight was 0.41kg-8.60kg; outer diameter of squash was 8.60cm-26.40cm; inside diameter of squash was 4.70cm-19.80cm; thickness of pulp was 1.85cm-3.48cm; longitudinal diameter of squash was 5.20cm-51.20cm; growth had strong and moderate; shape of main vine had circular and five prismatic; white spot of leaf had large, medium and small; shape of blade had heart shaped, heart shaped with five angle, subtriangular, suborbicular, palmate with five angle and palmate; growth potential had low-growing and trailing; skin color of young squash had light-green, dark-green, light-yellow, green, orange and yellow; skin color of old squash had orange, yellow, pale-brown, orange-red, dark-green, blackish-green and yellowish-brown; pulp color of old squash had orange, golden-yellow, light-yellow, yellow and white; characteristics of squash had smooth, ditch, tumor, shrivel and multi edge; shape of squash had long neck with cylinder, oval, pear-shaped, oblate, suborbicular and long tube; surface powder of squash had large, small and nothing.