The Individual Chromosome Identification And RDNA Location In Tetraploids Of Gossypium And Their Progenitorial Genomes

Cotton (Gossypium) is an important fiber economic crop, also is a model plant for the researches in cytogenetics, genomics and evolutionary biology. It is controversy about donors of the tetraploid cottons so far. The research on the donors will contribute to understand diversity and genomic sequencing of cottons. It is also helpful to establish a theoretical basis of using the wild gene bank to exploit more genes on purpose for cotton breeding. The chromosome recognition is the foundation of research on cotton genomics and will provide powerful evidences both for the evolution of Gossypium and the determination of the donors of tetraploids. In this study, the experiments on individual chromosome assignments and physical locations of ribosomal DNA (rDNA) for tetraploid and their donor genomes (A and D) were conducted using a multi-probe fluorescence in situ hybridization (FISH) system. The FISH probes contained two sets of bacterial artificial chromosome (BAC) clones specific to 26 individual chromosomes from A and D subgenomes of G. hirsutum and D genome centromere-specific BAC clone 150D24, 45S and 5S rDNA clones. The genetic map and physical map were also integrated.The main results were as follows:1. The chromosome-specific BAC clones were screened using chromosome-specific SSR markers derived from A and D subgenomes, respectively. Thirteen SSR markers from A subgenome were examined to be all-existent in A subgenome of G. darwinii, A subgenome of G. barbadense, G. herbaceum and G. herbaceum var. africanum using PCR. In addition, thirteen pairs of SSR markers from D subgenome were examined to be all-existent in D subgenome of G. barbadense, D subgenome of G. darwinii, G. thurberi, G. trilobum, G. klotzschianum, G. davidsonii, G. armourianum, G. aridum and G. raimondii, using PCR. Although the size of some products of the amplifications was not accordant to the expecting size, most of the SSR markers were amplified with expecting size. Therefore, two sets of BAC clone (Ah01-Ah13 and Dh01-Dh13) from the A and D subgenomes of G. hirsutum can be used as FISH markers to identify the individual chromosomes for G. barbadense, G. darwinii, and their donor genomes (A and D).2. All the individual metaphase chromosomes of mitosis were identified by BAC-FISH (FISH probed with BAC clone) for 11 species, which were G. barbadense, G. darwinii, G. thurberi, G. trilobum, G. klotzschianum, G. davidsonii, G. armourianum, G. aridum, G. raimondii, G. herbaceum and G. herbaceum var. africanum. The results showed all 13 Ah chromosome-specific BAC clones were located to the corresponding chromosomes of A genomes and A subgenome of tetrapoid cottons. All 13 Dh chromosome-specific BAC clones were also located to the corresponding chromosomes and chromosomal arms of D genomes and D subgenome of tetrapoid cottons. All the BAC clones had chromosome-specific signals for all cottons used but some BAC clones without chromosome-specific signals in G. raimondii. Therefore, it is reliable to identify the individual chromosome for the species, which mentioned (three chromosomes of G. raimondii are exceptions) with two sets of chromosome-specific BAC clones as markers. According to the homology within D subgenomes, within A subgenomes, between D genome and D subgenome, between A genome and A subgenome, the systematic nomenclature of chromosomes for all species studied were established. The chromosomes of A/D subgenome (At/Dt) in tetraploid cottons, A genomes (Ag) and D genomes (Dg) were named At01-At13 (Dt01-Dt13), Ag01-Ag13 and Dg01-Dg13, respectively. To distinguish the names of subgenomes in different tetraploid cottons, the chromosomes of A (D) subgenomes in G. hirsutum, G. barbadense and G. darwinii were named Ah01-Ah13 (Dh01-Dh13), Ab01-Ab13 (Db01-Db13), Ad01-Ad13 (Dd01-Dd13), respectively. The chromosomes of G. herbaceum and G. herbaceum var. africanum were named A101-A113 and A1a01-A1a13, respectively. The chromosomes of G. thurberi (D1), G. trilobum (D8), G. klotzschianum (D3k), G. davidsonii (D3d), G. armourianum (D2-1), G. aridum (D4) and G. raimondii (D5) were named D101-D113, D801-D813, D3k01-D3k13, D3d01-D3d13, D2-101-D2-113, D401-D413 and D501-D513, respectively.3. The BAC clones of chromosomes Ah07, Ah09, Dh07 and Dh09 were used to identify four corresponding chromosomes in G. tomentosum, and produced chromosome-specific signals. The BAC clones of chromosomes Ah05, Ah07, Ah09, Dh03, Dh07, Dh09 and Dh12 were used to identify seven corresponding chromosomes in G. mustelinum, and produced chromosome-specific signals. The BAC clones of chromosomes Ah05, Ah07 and Ah09 were used to identify the three corresponding chromosomes in G. arboreum, and produced chromosome-specific signals. The BAC clones of chromosomes Dh05, Dh07, Dh09 and Dh12 were used to identify the four corresponding chromosomes in G. laxum, G. schwendimanii and G. gossypioides, and produced chromosome-specific signals, respectively. Similarly, seven chromosomes of G. mustelinum named as chromosomes Am05, Am07, Am09, Dm03, Dm07, Dm09 and Dm12, respectively. Four chromosomes of G. tomentosum were named chromosomes At07, At09, Dt07 and Dt09, respectively. Three chromosomes of G. arboreum were named A205, A207 and A209, respectively. Four chromosomes of G. laxum were named D905, D907, D909 and D912, respectively. Four chromosomes of G. schwendimanii were named D1105, D1107, D1109 and D1112, respectively. Four chromosomes of G. gossypioides were named D605, D607, D609 and D612, respectively.4. Based on the successful identification of individual chromosomes,45S and 5S rDNA were located to chromosomes and chromosome arms for almost all tetraploid cottons, A genomes and D genomes. Generally,45S and 5S rDNA were revealed both consistency and polymorphism in the number of rDNA loci, in the number of rDNA repeats, in their chromosomal positions and the synteny relationship among the species studied. Three 45S rDNA loci were revealed on the chromosomes Am07 and Am09 of G. mustelinum and At09, Dt07 and Dt09 of other four tetraploid cottons. Three 45S rDNA loci on the chromosomes Ag05, Ag07 and Ag09 were revealed in all A genomes of diploid cottons. In the D genomes of diploid cottons, only two 45S rDNA loci was located to the chromosomes D607 and D609 of G. gossypioides, whereas three or four loci mostly marked on the chromosomes Dg05, Dg07, Dg09 and Dg12 were found in other D genomes studied. The copy number of 45S rDNA loci was discrepant in the different chromosomes of the same species or the corresponding chromosomes of different species. Besides the discrepancy that D genome of diploid cotton had much more than D subgenome of tetraploid cottons, the copy number of 5S rDNA was highly conserved in the number of rDNA loci, in the number of rDNA repeats and in their chromosomal positions. Only one 5S rDNA locus was found on the 9th chromosome (At09/Dt09 and Ag09/Dg09).45S and 5S rDNA showed synteny in A genome and D genome of diploid cottons, A subgenome of tetraploid cotton and D subgenome of tetraploid cotton except from D subgenome of G. mustelinum which was non-synteny (No 45S rDNA was detected in Dm).5. Based on the physical location of rDNA in 18 species, the results support polyphyletic origination theory. It was potentially different that the possible donor of Dm for G. mustelinum and the possible donor(s) of Dt for the other four tetraploid cottons, which there was more than one donor for the latter. It was possible that the donor of Dm became extinct or was not found. The possible donor(s) of Dt for the other tetraploid cottons were G. gossypioides, G. davidsonii, G. klotzschianum, G. armourianum, G. aridum, G. laxum and G. schwendimanii rather than G. raimondii, G. thurberi and G. trilobum. Moreover, the possible donor of A subgenome in tetraploid cottons was G. herbaceum var. africanum. 6. There were chromosomal collinearity about the BAC clones among different species by the comparison of the BACs positions suggesting that the majority of chromosome segment homology between D genome and D subgenome, as well as between A genome and A subgenome. The genetic map and physical map were integrated, and the orientations of genetic maps for D subgenome of tetraploid cotton and D genome of diploid cotton were established. Some orientations of chromosomes in genetic maps (Dt03, 04,06,09,10 and12) were switched. The SSR marker in the middle of linkage group 04 was located near the end of chromosome 04 by FISH.7. Three Dh chromosome-specific BAC clones were hybridized to more than one chromosome rather than one expected and specific chromosome. The nonspecific BAC signals in chromosomes of G. raimondii suggested that there were more homoeologous segments among these chromosomes. The different positions of chromosomes D507 and D511 of G. raimondii from other D genome species and D subgenome of tetraploid cottons indicated that chromosomal rearrangements might occur in both of the chromosomes. Therefore, these results indicated that G. raimondii was a very specific species in D genome.