Establishment And Assessment Of SSAP For Waxy Corn

Waxy corn originated in China, it is one subspecies of corn. After several centuries'variation, Waxy corns have large differences in height, color grain, yield, resistance, quality and other aspects. They are our country's precious maize resources. Waxy corn possesses characters of sweet taste, wax and fragrance beyond of normal maize. Its soluble sugar higher than ordinary corn and lower than sweet corn, more suitable for developed countries'tasting. The products made of waxy corn will join in recipes with the distinctive taste. So, it is important to do further research in genetic diversity of waxy corns. The research based on sequence-specific amplification polymorphisms(SSAP)created by R.Waugh in 1997,and our research made a foundation of germplasm resources and genetic diversity for waxy corn.the results as follows :1. Combine with existing DNA extraction methods and the nature of the waxy corn, waxy corn genome DNA extraction was improved: high salt (1.4mol/lNaCl) in 2% CTAB to break cells;2% of the soluble PVP and 2%β-Mercaptoethanol to remove phenols;first phenol: chloroform: isoamyl alcohol and then chloroform: isoamyl alcohol to remove proteins; at last digest RNA with RNase,the high quality DNA will be get.The value of the DNA in A260 / A280 between 1.8 and 2.0, A260 / A230 greater than 2.0 and genome DNA can be restriction by MseⅠc ompletely, and the DNA can meet the follow-up test. The DNA extraction method is rapid, efficient, economic, the quality of DNA is high, especially suitable for the large number of samples extraction.2. Base on the single enzyme (MseI) and dual-enzyme (EcoRI, MseI) comparative experiments, we confirmed that a single frequency enzyme restriction is not suitable for SSAP analysis of waxy corn; In experiment of EcoR I and Mse I restrction, we found that EcoR I primers combine with specific primers can got more bands and polymorphisms are also high. At the same time we found that many people now use the high frequency enzyme primers do not apply to waxy corn SSAP analysis.3. Initially established of waxy corn SSAP analysis method as follows:(1)Restriction: EcoRI and MseI. First EcoRI restrction at 37℃for 3h(Buffer:3μl,EcoRI:0.5μl,template DNA: 500ng,ddH2O up to 15μl).Then MseI restrction at 65℃for 1h(Buffer:1μl,MseI:0.2μl,ddH2O: 3.8μl).10μl for next step. (2)ligation: EcoRⅠand MseⅠadaptor ligation at 22℃for 2h(Buffer:2.0μl Ead: 0.5μl, Mad:0.5μl, T4 Ligase:0.15μl,ddH2O up to 10μl).(3)Pre-amplified:10XBuffer 2.5μl, ligation DNA 2.4μl, dNTPs(2.5mM) 1.6μl, EcoRI primer (25ng/μl) 4.0μl, MseⅠprimer (25ng/μl) 4.0μl,Taq DNA polymerase 1U,ddH2O: up to 25μl at 94℃:5min, 94℃:30s, 56℃: 1min,72℃:1min, 30cycles, 72℃:5min. then,diluted 4 times by TE.(4)Selective amplification:10XBuffer:1.0μl,dNTPs(2.5mM):0.8μl, pre-amplified DNA: 0.75μl, EcoRI primer(25ng/μl): 0.6μl, specific primer (25ng/μl):1.2μl, Taq DNA polymerase: 0.5U, ddH2O:up to 10μl at 94℃: 5min, 94℃:30s, 65℃:30s(touchdown, -0.7℃for every step), 72℃1min, 13 cycles, then at 94℃:30s, 56℃:30s, 72℃: 1min for 26cycles, 72℃for 7min.4. SSAP is a new molecular marker technology based on retrotransposons. We need to known a specific sequence which is on conservation region of retrotransposons. At the same time we found that it is serious difference in number of bands and polymorphisms when different specific primers combine with same restriction enzyme primer. So we require sufficient specific primers to choose. Because of different GC content in different specific primers, we need to explore suitable annealing temperature respectively. SSAP seems to be lower repetition than AFLP, but more bands, higher polymorphisms and relatively less number of primers, no doubt we can to save a lot of time. At this point, we can confirm it and continue to do more study.