Structure and size variations between 12A and 12D homoeologous chromosomes based on high-resolution cytogenetic map in allotetraploid cotton.

Cotton is a model system for studying polyploidization, genomic organization, and genome-size variation because the allotetraploid was formed 1-2 million years ago, which is old enough for sequence divergence but relatively recent to maintain genome stability. In spite of characterizing random genomic sequences in many polyploidy plants, the cytogenetic and sequence data that decipher homoeologous chromosomes are very limited in allopolyploid species. Here, we reported comprehensive analyses of integrated cytogenetic and linkage maps of homoeologous chromosomes 12A and 12D in allotetraploid cotton using fluorescence in situ hybridization and a large number of bacterial artificial chromosomes that were anchored by simple sequence repeat markers in the corresponding linkage maps. Integration of genetic loci into physical localizations showed considerable variation of genome organization, structure, and size between 12A and 12D homoeologous chromosomes. The distal regions of the chromosomes displayed relatively lower levels of structural and size variation than other regions of the chromosomes. The highest level of variation was found in the pericentric regions in the long arms of the two homoeologous chromosomes. The genome-size difference between A and D sub-genomes in allotetraploid cotton was mainly associated with uneven expansion or contraction between different regions of homoeologous chromosomes. As an attempt for studying on the polyploidy homoeologous chromosomes, these results are of general interest to the understanding and future sequencing of complex genomes in plant species.

Higher axial-resolution and sensitivity pachytene fluorescence in situ hybridization protocol in tetraploid cotton.

Fluorescence in situ hybridization (FISH) based on pachytene chromosomes has become an important cytogenetic tool to construct high axial-resolution and sensitivity cytogenetic maps. However, the application of this technique in cotton has lagged behind due to difficulties in chromosome preparation. To date, successful FISH based on cotton pachytene chromosomes has not been reported. In this study, the first protocol developed for pachytene chromosome preparation in tetraploid cotton is presented. This protocol yielded chromosome spreads suitable for large and small DNA probe FISH labeling. Two important parameters, axial-resolution and sensitivity, of FISH on mitotic metaphase and pachytene chromosomes were systematically analyzed. 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. The application of our FISH protocol will continue to improve and provide a point of departure for constructing an integrated high axial-resolution cytogenetic map in cotton.