| Cotton (Gossypium hirsutum L.) is one of the important economic crops in China and has an important position in national economy. Because of its obvious heterosis, using the cytoplasmic male sterile (CMS) to achieve the "three lines" system is the goal to the cotton breeders for a long time. Revealing the mechanism of plant cytoplasmic male sterility is a significant research direction at home and abroad, it can help researchers to creat new CMS germplasm resources, and provide theoretical basis for effectively regulate the expression of the CMS.A kind of new transgenic cotton was used in this study, which was based on transfering the kenaf HcPDIL5-2a gene into the gossypium barbadense to creat a new CMS line H276A. At the same time, used its non-transgenic maintainer H276B (H276A wild type) for comparison. In addition, one of the molecular marker technologies AFLP, which has rich polymorphism, strong stability and high sensitivity, was chosen to analyse the genomic DNA by using 256 pairs selective amplification primers. The main results obtained of the dissertation were as follows:1 AFLP system optimizationFirst of all, this study has built a high-purity and high-quality DNA extraction method that can be applied to cotton spire. This method was on the basis of the traditional CTAB and combined purification pillar extraction. The quality of the DNA can meet the AFLP requirements and completely suitable for subsequent analysis. Subsquently, In order to set up the most suitable AFLP technology system and reaction condition for the experimental research, the template dosage, enzyme digestion time, the dilution ratio of connected products and preamplification were optimized. At last, the denaturing polyacrylamide gel silver stain has been modified and optimized to make the enhancement effect better.2 H276A/H276B AFLP analysisUsed AFLP (include 256 pairs selective amplification primers) to analyse H276A and H276B from the level of genome DNA, there were 17 primers can stably amplified different bands, finally received 24 pieces of different fragments. Among the different fragments, H276A had 13 pieces, polymorphism proportion (2.63%); H276B had 11 pieces, polymorphism proportion (2.23%). Through recycling these different segments, I successfully recovered 19 pieces, mainly concentrated in 100-700bp.3 Specific fragments cloning and sequencingThe purification recovery fragments were linked to T-vector, subsquently transformed and detected by bacterial PCR. In the end,13 target segments were successfully detected and carried out for sequencing. Ultimately NCBI blast analysis for further comparison. As a result,6 genes were the cytoplasm genes homologous sequences in Gossypium hirsutum. Respectively 2 chloroplast homologous sequences, all were parts of the coding sequences of ndhH; and 4 mitochondria homologous sequences, two genes were located in ccmC upstream non-coding region, one gene were located in another non-coding region between nad6 and sdh3, the fourth gene were part of rps3 coding sequences. These differences between the two materials were cloned again and the sequences were listed in the GenBank for sequences alignment. At last 6 bases substitution and 5 bases deletion mutation were found.4 The development of molecular markersBased on the one-base substitution difference located in the mitochondrial gene ccmC upstream conservative region 91 bp between H276A and H276B, two specific molecular markers distinctively to male sterile cytoplasm had been developed. In order to screen male sterile cytoplasm from the germplasm using the two molecular markers, other 5 couples varieties, which had the same cytoplasm of H276A/H276B, were used for further verify. Ultimately, according to the presence of the target bands we can clearly distinguish the sterile cytoplasm and fertile cytoplasm. |
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