Construction Of AFLP Specific Molecular Marking System On Pomole (Citrus Grandis (L.) Osbeck) Single Chromosome

Pomole (Citrus grandis (L.) Osbeck) is one of the most important fruit in citrus. However, it is hard to construct accurate genetic map of pomole, because of the complex genetic background, long growth cycle. Which cause difficulty in the research of pomole genomic analysis, molecular marker assisted breeding, and the gene clone of some important horticultural traits. To obtain some specific molecular markers in polome single chromosome, establish a molecular marker system to identified single chromosome. Consequently, it can establish a single chromosome library, which can greatly raise the work efficiency in drawing polome genetic mapping. On this basis, AFLP method was used to identified pomole specific molecular markers, and acquire the following major results:1. The immature embryo of polome (Citrus grandis) cultivar"guanxi"was used in all experiments. We isolated 56 single chromosomes by glass needle, and then conducted enzyme digestion, ligation to adaptors, twice LA-PCR amplification. And we constructed single chromosome library of pomole. Agarose gel eletrophoresis analysis shows that DNA amplified fragments mainly distributed in 300～2000 bp, and there are some differences in DNA amplified fragments of different single chromosomes.2. We screened L/X and F/I in 42 primer pairs. Then we used AFLP to analyze the 56 single chromosomes, and obtained a clear AFLP fingerprints. UPGMA Cluster analysis showed that 56 single chromosomes can classify into 11 groups at the similarity coefficient of 0.76.3. AFLP analysis of genetic polymorphism showed that there are 361 differential fragments. We obtained 32 single chromosomes AFLP specific molecular markers of 129 different fragments by Southern blotting analysis. Then we discussed the distribution of AFLP specific molecular markers, and confirmed the 9 groups of 22 single chromosomes.4. Single chromosome specific molecular markers were studied by DNA sequencing directly, and we obtained 11 sequences of specific molecular markers. Comparsion analysis of DNA sequences using BLAST showed that eight specific molecular markers are high homology with those of other plants, including three Poncirus trifoliata citrus tristeza virus resistance gene locus, one LINE retrotransposon DNA.We establish the AFLP molecular marker system of polome single chromosome by AFLP and Southern blotting analysis, which may lay a foundation in constructing single chromosome library, gene location and drawing genetic mapping of pomole.