Fish Based Karyotype Analysis Of 21 Gossypium Species

It is the most critical technology link in the karyotype analysis to distinguish properly homologous chromosomes in mitotic cells and then obtain the accurate basic data about chromosomes. Non-FISH karyotype analysis is difficult to match accurately homologous chromosomes and often makes mistakes. FISH (fluorescent in situ hybridization) based karyotype analysis breaks through the bottleneck. Clarifying parental donors of polyploid cottons not only resolves the issues of long-term theoretical arguments in Gossypium and but also helps to select the prior cotton species for genome sequencing, which is very important in whole-genome sequencing era. Therefore, taking the advantage of materials in our institute, we carried out a comprehensive karyotype analysis of existing cotton species based on FISH. In this study we collected 21 Gossypium species including A, B, C, D, E, F, G genomes and AD genome except K genome. We used genomic DNA (gDNA) and rDNA as probes and obtained the FISH results. The results were as follows:GISHs of the tetraploid species showed clearly and directly the two subgenomes of chromosomes, and the chromosomes in one subgenome were generally bigger than those in another, which confirmed the theories that tetraploid cottons were disomic nature and chromosomes from A genome in Gossypium were bigger than the D genome chromosomes. We found that some chromosomes in smaller subgenome were bigger than some in larger one in all tetraploid GISHs, which was very difficult for non-FISH karyotype analysis method to properly match those chromosomes between the two subgenomes mainly according to the chromosome relative lengths from the longest to the shortest, which indicated FISH-based karyotype analysis was more accurate and reliable. Comparisons of the basic parameters of karyotype and cluster analysis of karyotype overlapping percentages showed that G. herbaceum var.africanum was rather different from G. arboreum and G. herbaceum. G. herbaceum var.africanum may have a unique evolution pathway and should be given a "species" status in genus Gossypium. G. australe and G. nelsonii were rather different from C genome species G. sturtianum, and also much different from G. bickii. Therefore the restults did not support the the suggestion that G. australe and G. nelsonii were classed into G. bickii involved G genome or included in the any other same genome with G. bickii.The specific relationships among the four tetraploid cottons tested here in this study showed that G. hirsutum was very closer to G. barbadense, whereas these two species including G. darwinii were very distinct from G. mustelinum. And G. mustelinum was very special as it should be an independent branch in the evolution of tetraploid cottons. Data from this study further supported the theory that the two subgenomes of the tetraploid cottons formed from A and D genome species. Probably, A–subgenome of the tetraploid cottons had a common donor, which was G. herbaceum var.africanum. And the detail donor species of D–subgenome for each tetraploid was not very clear, but it was certain that polyphyletic origination theory was supported. Also the possible donors were G. raimondii and G. trilobum. But G. raimondii may not be the donor species of G. hirsutum D-subgenome. The distinguishing ability of identification satellite on chromosome was significantly improved, after locating the 45S rDNAs on the diploid cotton chromosomes. The results showed that the distribution of the satellites and the 45S rDNA loci were characterized with the "cluster region". That is they focused on some or a certain number of chromosomes, such as the D genome species had 3 regions with chromosome 5, 9 and 13 as the region center. Moreover, the 5S rDNAs of 9 diploid cottons were located. The results showed that the loci of 5S and 45S rDNAs from seven species were in the same chromosome, speculating that 45S and 5S rDNAs may have a high degree of collinearity.The results about karyotype analysis showed the overall evolution level in Gossypium was low. G. arboreum and G. captis-viridis were the two typical species of the highest and lowest evolutional level among the diploid cottons, respectively. Moreover, the possible ways of origin and evolution of each genome were analyzed and discussed in the dissertation.