| This paper aimed at the analysis on walnut resources which belong to the mountain and ravine region of southwest Sichuan by Using RAPD(Random Amplified Polymorphic DNA) method. The 109 selected plants come of seven counties which belong to two cantons and one city. Batang county, Xiangcheng county and Derong county belong to GanZi canton; Huili county, Meigu county and Muli county belong to Liangshan canton; Shimian county belong to Yaan city. The selection of amplified reaction program and system was carried out. 14 primers for RAPD analysis were obtained during the first and second selection from 120 primers, The 14 primers could be used for UMPGA (unweighted pair group method using arithmetic average).The results were as follows:1. Nucleus deposition method was adopted to isolate genomic DNA from walnut leaf. The results indicate that the nucleus deposition method was effective to remove most of polysaccharides in the cytoplast and to inhibit the formation of oxidized polyphenolic compounds in the leaves. This kind of high quality DNA was much idealer to the further research of molecular biology in Walnuts.2. Based on the genomic DNA extracted from Walnut, essential factors affecting the result of RAPD assay were tested and compared. The followed results approve that an optimal reaction system suitable for the assay and usage of RAPD in Walnut.PCR amplification program was denature at 94℃ in advance for 5 min, then followed 40cycles,every cycle: denature at 94℃ for 45s,anneal at 35℃ for 45s, extend at 72℃ for 90s. Then extend at 72℃ for 10min for one cycle and store at 4℃ .RAPD reaction system(25μL) optimum constitution: 20ng template, 10×PCR buffer 2.5μL, 0.04U/μL Taq DNA polymerase,0.36 μmol/L primer, 2.0 mmol/L Mg2+ ,0.2 mmol/L dNTPs and Anneal at 35℃.3. 120 Primers were firstly and secondly selected. At last ,14 primers were decided to apply to formal amplification.4. 224 bands were amplified by fourteen primers in walnut,210 bands were polymorphic and PPB was 93.75%. Primers amplified bands from 12 to 22 and polymorphic bands were... |
