Isolation Of Single Chromosomes And Establishment Of AFLP Molecular Markers In Pomelo

This item micro-isolated a lot of single chromosomes of the pomelo in advance, then identified and classified them by LA-PCR and AFLP technology. This will make it possible to set up a direct and simply manipulative system for the single chromosomes' molecular markers. The major results obtained from the studies were summarized as follows:1 Isolation of single chromosomes in pomeloThe studies used 100 d young fruit of Citrus grandis cv. Guanximiyou as the material to micro-isolate the single chromosomes. In the course of making chromosome slides, it was found that different results could be obtained by using different treatment time of enzymatic digestion. There were high-quality chromosome slides when we used the mixture of 2% cellulose enzyme and 0.5% pectic enzyme to deal 40min～1h with the cells of young embryos, the background of which were clear and different chromosomes were adequately spread around. After post-seeping, 20～30 min treatment was appropriate by using ice water to treat the cells of young embryos. 32 single chromosomes were randomly micro-isolated in the liquid by using suitable glass needles made by myself.2 Extracting of genomic DNA and LA-PCR amplification of single chromosomes1) Genomic DNA was extracted by the improved method, with better quality, higher purity and no contamination of RNA and protein. Its producetion of Sau3A I digestion showed a well-proportioned, consecutive strap ranging from 0.3～2 kb.2) 32 single chromosomes were amplified by two rounds of LA-PCR amplification. Different single chromosomes showed different results: some single chromosomes' distributing range of fragments was about 0.6～2 kb; and others' was about 0.4～0.8 kb.3 Establishment of AFLP technical system and analysis of fingerprinting in single chromosomes20 pairs primers were filtrated in this experiment, 9 pairs primers of which were better than others. The primers A/D, H/L, X/S, C/G and B/N. were the best 5 pairs primers in the 9 pairs. Their products were clear and identifiable and steady-going in the repeated experiment. So finally the 5 pairs primers were used to amplify the 32 single chromosomes.It was found that the best producetions could be amplified by restructuring its PCR program, which including continuously progessing 4 times and then amplifying at low anneal temperature.32 single chromosomes showed different polymorphism bands in AFLP. And the first group was also amplified by 7 single primers.Using general analysis of the amplified producetions, the 32 single chromosomes were classified as 21 kinds chromosomes, but not 9 kinds ones. Presented this complexion, the reasons could be as follows: 1) the single chromosomes micro-isolated could not avoid to lose some partial sections in the micro-isolating course. Some equirotal bands didn't be showed in the agar-gel electrophoresis, so they could not classify as a kind of chromosome. 2) the single chromosomes micro-isolated might take place the structural variance, so it increased difficulty of identifying and classifying work. 3) the localization of experimental manipulation process of molecular biology could lead to some errores.