Cytological Observation Of Pepper CMS And MtDNA-AFLP Analysis

The materials of A16 and B16 were provided by the college of horticulture in Northwest A & F University. We observed and analyzed the microspore developments of CMS (cytoplasmic male sterility)line (A16) and its maintainer line(B16).AFLP was used to isolate and cloned the genes related to male sterility during CMS line and its maintainer line, and we also cloned the cytochrome P450 gene. The main results were as follows:1. CMS pepper varieties A16 showed different pistil length, almost no pollen, as a result the sterility of A16 is good.By cytology observation we discovered that, the microspore abortion of this test material happened on the formation of tetrad to microspore tetrad. The tetrad was Compressed to deformation, depredated and can not form a normal microspores.2. The mitochondrial DNA extraction system was optimized. In order to eliminate the final mtDNA pollution, we added DNase I to promote the degradation of the DNA released by nuclei and organelles through homogenate before the mitochondria cracked, Then we added RNase A to mtDNA to eliminate the small amount of RNA pollution and ensure the extraction quality. We compared the different amount of mitochondrial DNA extracted from different tissues of pepper (leaves, tender seeds and placenta) and discovered that tender seeds and placenta contains larger amount of mitochondrial DNA than leaves. Besides, it is easy to get high quality of mitochondrial DNA with small amount of tender seeds and placenta.3. We used the AFLP technique to study the difference in mitochondrial DNA in pepper male sterile material A16 and its maintenance line B16, and obtained 11 different gene fragments. Among which 7 fragments were obtained from male sterile material A16 and 4 were got from its maintenance line B16. the CanLE sequence got from the male sterile material showed great similarity to pepper cytochrome P450 protein, and the specific sequence CanLB which was got from the maintenance line showed 60 percent similarity to lysyl-tRNA synthetase of rice and castor. This means these two fragments have high homology.4. Using the mtDNA from the CMS A16 and maintainer lines B16 as the template, we cloned a cytochrome P450 gene, and proved that this gene was existed in the mitochondrial genome of the two-line. Then we compared the cytochrome P450 genes that amplified from these two lines and discovered that there were 5 bp mutations, in which three were silent mutations, while two led to amino acid change.