| In the present study, two multiple-marker lines i.e. T582 and T586 of upland cotton were crossed to produce F1 and F2 plants, which were used as constituted mapping population of molecular marker linkage map. Two types of DNA molecular marker, simple sequence repeats (SSR) and amplified fragment length polymorphism (AFLP), were applied. The different molecular techniques like isolation of cotton genomic DNA, PCR reaction, detection of DNA polymorphism etc. were optimized through repeated experiment. A new framework of DNA molecular marker linkage map in upland cotton was constructed for the first time in China. At the same time, some genes in cotton were inserted into the marker linkage groups. The main results as follows: 1 Study on isolation of cotton genomic DNAThere are two basic methods of isolation of plant genomic DNA i.e. SDS method and CTAB method. If these methods are modified, they will be named modified SDS method or CTAB method. The four methods of isolating genomic DNA i.e. original SDS, modified SDS, original CTAB and modified CTAB methods using different tissues of cotton were compared in this study. There were three main of differences between modified SDS method and original SDS method, (1) In modified SDS method, we added chloroform and isoamyl alcohol (24:1) into top aqueous before depositing DNA by isopropyl alcohol, and (2) Picking out DNA pellets instead of centrifuging at high speed. (3) Adding 1-2% PVP into extraction buffer. Similarly, there were three main differences between modified CTAB method and original CTAB method, one of them was that in modified CTAB method, DNA isolation from CTAB and nucleic acid compound dissolved in the high salt solution (1.4mmol/L NaCl) relies on adding 0.6-1 volume isopropyl alcohol directly instead of on reducing salt concentration (0.7mmol/L NaCl), and others were the same as the modified SDS method. The results showed that both the modified SDS and CTAB methods are better than the original SDS and CTAB methods. Yield rate of genomic DNA was not only related to extraction methods, but also to cotton tissues. DNA yield rate of modified CTAB method was higher than modified SDS method. Among the material of cotton studied, the yield ofDNA was recorded highest from anther followed by petal, leave and fiber. The cotton genomic DNA isolated using modified methods could be digested by enzyme and also good to meet the demand of subsequent molecular operations such as SSR and AFLP analysis. Moreover, the two modified methods were also suitable for isolation of DNA of other plant, for e.g., the modified SDS method was suited especially for plant with high protein or glutinous amylose and the modified CTAB method for plant with poly-hydroxybenzene.2 Study on DNA molecular marking technique of cottonPCR reaction and detection system of DNA polymorphism are the two key techniques in the molecular biology research. PCR reaction and detection method of DNA polymorphism on microsatellite DNA was optimized in this study. The result showed that the best annealing temperature was 55 and the best annealing time was 45 seconds for the most among fifty pairs of SSR-primers. According to our results, for amplification products of SSR, polypropylene gel electrophoresis and silver staining was better than agarose gel electrophoresis and ethidium bromide staining. The concentration of polypropylene gel needs to be modified along with change of fragments size of DNA sequence. In general, 6% concentration of polypropylene gel was suitable. However, for the DNA fragments smaller than 100 bp, 8% concentration of polypropylene gel was found better than the 6%. Amount of 3 l amplified product added on gel wells was found enough. The best electrophoresis time was 1.5 hour and 2 hour at 6% and 8% concentration of polypropylene gel, respectively. Developing band time should be limited within 4 min. so as to make DNA intensive and clear if developing solution was in the cool condition.3 Analysis of DNA polymorphism of parents and hybrid F1Polymorphism analysis of...
|
PDF Full Text Request