Genetic Diversity Of Different Flesh Color In Actinidia And It's Germ Plasm Identification

Twenty-one Actinidia cultivars such as 'Chuanmi 1', 'Hongyang' and 'Hayward' were used as materials to ravel their genetic relationship and diversity by RAPD. The aim of the study is to give some theory bases of making use of the germplasm and selecting the parents of hybridization. The results were as follows:First, to find a rapid, simple and effective total DNA extraction method for Actinidia, three methods, SDS, improved CTAB and CTAB regular method, were compared. The total DNA was detected by spectrophotometer, agarose gel electrophoresis and RAPD amplification. The result showed that improved CTAB method was hish-yield, high-purity, low levels of impurity and DNA fragments. RAPD fragments amplified with DNA template extracted by improved CTAB method showed clear bands, and good polymorphic, stability and repetition. The improved CTAB method was optimal for total DNA extraction in kiwifruit, which was suitable for PCR amplification and other molecular biology researches.Second, Several main factors infiuencing the RAPD-PCR reaction were analyzed in order to establish the stable system in Actinidia. The optimal system was as follows (in 25μl reaction system): template DNA 30ng, 10×buffer2.5μl, Mg²⁺2.0mmol/L, primer 0.6μmol/L, dNTPs0.20mmol/L, TaqDNA polymerase1.6U. Amplified procedure was programmed for 5min at 94℃; 1min at 94℃, 1min at annealing temperature 36℃, extension at 72℃for 2min, 45 cycles; then a final extension at 72℃for 10min and then keep at 4℃.Third, of the 90 random primers screened, 21 primers exhibited sufficient polymorphic band patterns. A total of 267 RAPD bands were generated by 21 selected arbitrary primers, among which 228 (83.7%) bands were polymorphic. Genetic distance among the 21 Actinidia cultivars ranged from 0.12 to 0.54 with an average of 0.36. The results showed that the genetic relationship and diversity between all cultivars were very complex, there was a little high correlation the two facts of genetic relationship and geographic distribution, but have nothing to the flesh color.Fourth, we found two variety materials of which flesh of both inner and outer layer was red in Actinidia. Through RAPD analysis, they had closer genetic relationship with other cultivars, which was much different in morphology. It indicated that the function of eanthocyanin content of flesh was very complex in genomic DNA of Actinidia.Fifth, A total of 267 RAPD bands were generated by 21 selected arbitrary primers, of which 228 (83.7%) bands were polymorphic. It proved that the vegetative variation occurred in a wide range of genomic DNA, while no one characteristic bands among the 21 ture and fake Actinidia through comparing, and following results were obtained: 'hongyang' which came from Cangxi and Yaan were the same materials, while not to 'Hayward'; In the result of amplified bands analysis, the unknowing materials could be distinguished but couldn't know the cultivar name.