Fish Study Of Four Tetraploid Species Of Gossypium

It has been proposed previously that extant A- and D-genome species are the diploid progenitors of the tetraploid cotton(AD genome),Yet few data address the question of the actual diploid ancestor of tetraploid A- or D-subgenome, and the germplasm disproportionation in the process of origination, and the relationship between the diploid genomes and tetraploid genomes, and some diploids'homologous relationship, et al. Cotton is the most important fiber crops in China even in the world. Clarifying actual diploid ancient donor species of tetraploid cotton, can not only resolve the long-term debate of theoretical issues, but also help to select the preferred cotton species for genome sequencing. Meanwhile, clarifying the genetic evolution and relationships among the cotton species has a very important theoretical and practical significance to the cotton genetic improvement. Combining with planning of the overall experimental design, in this study we used four tetraploid cottons containing G.hirsutum[(AD)1],G.barbadense[(AD)2], G.tomentosum[(AD)3], and Gdarwinii[(AD)5] mitotic metaphase chromosomes as the target materials which were analyzed by FISH, using all of the available kinds of diploid A, D genomes gDNA, the representative specie of diploid B, C, E, F, G genome species gDNA and 45 S rDNA as probes, in order to investigate the donor of tetraploid cotton species,45S rDNA evolution law and the relationship between diploid species of cotton. The main results were as follows:1. Six NOR signals were all observed by FISH to (AD)1, (AD)2, (AD)3, and (AD)5 mitotic metaphase chromosomes with 45S rDNA as probe, using salmon sperm DNA as block DNA,and we found that except the (AD)5 chromosomes generated 4 large 2 small signals, the other tetraploid cotton all generated six large signals.We also found all the NOR signals were located in the ends of chromosome which adjacent to satellite sites.2. GISH to (AD)1, (AD)2, and (AD)3 mitotic metaphase chromosomes with G.herbaceum var.arfrium(A1-a) as probe, salmon sperm DNA as blocking, besides the fluorescent signals distributed on 26 A-subgenome chromosomes, we found six GISH-NOR signals, of which two in the A sub-genome chromosomes, and four in the D sub-genome chromosomes. But we didn't detect the GISH-NOR signals when using G. herbaceum var. hongxing(A1) and G. arboreum var. shixiya 1(A2) as probes. 45S rDNA IGS sequences of Gherbaceum var.africanum maybe has some homology with diploid D genome, while the A genome Gherbaceum hongxincaomian and Garboreum shixiya 1 have the low homology. Gherbaceum var.africanum(A1-a) is the the diplod progenitor of A-subgenome of all tested tetraploid cottons.3. GISH with all D genome species gDNA as probes, besides the red fluorescent signals were observed on D sub-genome chromosomes, and we also found six GISH-NOR signals, whether or not blocking with A genome, and two in the A sub-genome chromosomes, four in the D sub-genome chromosomes.The 45S rDNA IGS sequences of all diploid D genomes are similar with the tetraploid cottons. Gdavisonii(D3-d) is the the diplod progenitor of D-subgenome of G.hirsutum[(AD)1] and G.darwinii[(AD)5], and is also possible the diplod progenitor of D-subgenome of Gtomentosum [(AD)3].Graimondii(D5) is the diplod progenitor of D subgenome of the Gbarbadense[(AD)2].This results supported that teroploid cotton is polyphyletic.4. GISH to (AD)1 and (AD)2 mitotic metaphase chromosomes with D genome specie Ggossypiodies(D6) gDNA as probe, blocking with ssDNA, the fluorescence signals were distributed both on A- and D-subgenome chromosomes, we couldn't separate A- and D-subgenome.This showed that D6 contains some A genomic compositions or some of the genomic components of other African diploid cottons, it is very special in diploid D genome species.And further confirmed the results of previous studies.5. The Dual-color FISH with genomic DNA and 45S rDNA as probes further verified the unity of GISH-NORs and NORs signals, that's the number, position, and size were identified as similar or identical.6. FISH-based karyotype analysis of (AD)2 showed that the longest chromosome of D-subgenome was longer than the shortest two pairs of chromosomes of A- subgenome. This showed that only depend on the relative length of chromosomes from long to short order to separate A- and D-subgenome in teroploid cotton of Gossypium was not enough reliable. This is the problem that traditional method of karyotype analysis can not avoid. And this indicated FISH-based karyotype analysis was more accurate and reliable.7. FISH study showed that 45S rDNA of (AD)1, (AD)2, (AD)3, and (AD)5 had all concerted evolution, accompaning a large number of rDNA loci deleted.8. GISH to (AD)1, (AD)2, and (AD)3 mitotic metaphase chromosomes with B genome specie G.anomalum (B1), E genome specie G.somalense (E2), and F genome specie G.longicalyx (F1) as probes, blocking with ssDNA, the fluorescence signals were all mainly distributed on the longer A-subgenome chromosomes, and all generated six GISH-NORs, the signal strength of which is similar with those when used D genome specie as probes. The sequence of diplid B genome Ganomalum, E genome Gsomalense and F Genome Glongicalyx has close relationship with diploid A genome while their repetitive sequence of 45S rDNA IGS is similar with diploid D genome.9. GISH with C genome specie Gsturtianum (C1) and G genome specie Gbickii (G1) as probe, blocking with ssDNA, the fluorescence signals were distributed on both A- and D-subgenome chromosomes, accompaning six GISH-NOR signals produced, which are similar with that of D genome specie G.gossypiodies(D6) probe. This showed C genome Gsturtianum and G genome G.bickii contain both diploid A,D genomic component, while their 45S rDNA IGS sequences are similar with diploid D Genome.