The Construction Of Physical Mapping Of 12A And 12D Homoeologous Chromosomes Based On Pachytene-fluresence In Situ Hybridization Technique In Cotton

Fluorescence in situ hybridization (FISH) has been widely applied for cytogenetic development up to now. We mainly presented development of FISH based on pachytene chromosomes in cotton. Based on it, the 12A and 12D homoeologous chromosomes of tetraploid cotton were analyzed.The main results were as follows:1. The construction of FISH based on pachytene chromosomes:Young buds with suitable size were fixed in ethanol-acetic acid (3:1) fixative at room temperature. Then the modifications of prolonged time of digestion with higher-concentration pectolytic enzymes (2% cellulase and 2% pectolyase,5-6 h) were applied in the anthers stayed in suitable phase. After that, the anthers would be squashed on a hot plate. The protocol of pachytene chromosome preparation for tetraploid cotton was constructed through these modifications. Based on it, two important parameters, axial-resolution and sensitivity, of FISH on mitotic metaphase and pachytene chromosomes were systematically analyzed, and it is reported for the first time in the world. The results demonstrated that DNA targets separated by 0.6 cM and low-copy targets as small as 3-kb were resolved and detected, respectively, in pachytene FISH. Based on it, The FISH protocol of pachytene chromosome for tetraploid cotton was successfully constructed.2. The application of FISH based on pachytene chromosomes:based on the pachytene FISH, an integrated high axial-resolution cytogenetic map was constructed in cotton.in this study, specific SSR markers were selected along the entire linkage maps of 12A and 12D, and used to screen two genomic BAC libraries derived from TM-1 and a restorer line, 0-613-2R (G. hirsutum), for cytoplasmic male sterile line of G. harknessii for the corresponding BACs. After validated in FISH, the BACs which consistently produced unambiguous bright FISH signals on chromosome 12A or 12D were used in the construction of cytogenetic maps on pachytene chromosomes. The integration of genetic loci into physical localizations showed that the orders and positions of the BACs on chromosomes 12A and 12D were concordant with marker positions in the corresponding linkage group, and the results have also been shown that suppression of genetic recombination in the pericentromeric regions of the two homoeologous chromosomes led to the discrepancies between genetic and physical distances. To sum up, the construction of high axial-resolution and sensitivity cytogenetic maps on pachytene chromosomes has a specific advantage in the research of the cotton genome and it is very meaningful for studying the polyploidy homoeologous chromosomes and sequencing of complex genomes in plant species.