Karyotype Analysis By Telomere-FISH And Primary Development Of High-resolution Cytological Map In Cotton

Cotton is the most important fiber cash crop in the world. Due to its global economic importance, the project of sequencing the cotton genome is on the agenda. Study on the interspecies relationship in cotton and the development of cytology map by fluorescence in situ hybridization (FISH) are important work in sequencing genome. In this research, we firstly investigated on the telomere in cotton and developed the technology about telomere-FISH and pachytene-FISH, by which we studied the variation and evolution of karyotype in diploid cotton and developed the high-resolution cytology map of diplod A genome of cotton. The main results are as follows:(1) By using the primer designed by BAC (F23H6) of 3# chromosome of Arabidopsis, the Arabidopsis-type telomere sequence was obtained and cloned into plasmid pAT1. In situ hybridizations with pAT1 probe indicated that the signals were located at all chromosome ends of cotton. To identify the signals of FISH, Bal31 digestion kinetics was introduced in this study. The result of Bal31 digestion indicated that the hybrideated signals represents the outermost DNA sequence of each cotton chromosome. So we first proved that the telomeric repeats of cotton cross-hybridizes with that of Arabidopsis. To measure the length of telomere in the genome of cultivated and wild cotton, we used the terminal restriction-fragment (TRF) asssy, which used a genomic southern blot with terminal restriction fragments of genomic DNA from 9 cotton species (of genome A, B, C, D, E, F, G and AD) producted by Haeâ…¢, Sau3Aâ… , Hhaâ… , Hapâ…¡and were hybridized with the telomere probe. All samples had smeared hybridization patterns, and most of them were 6-20 kb in length. The telomere length of cultivated cotton was about 20 kb and was larger than that of wild cotton which was ranging from 6 kb to 20 kb.(2) In order to determine the relationships within diploid species, the karyotypes of 7 diploid species representing 7 basic genome groups in cotton were first analysised by telomere-FISH in this paper. According to the length of chromosome, the ration of longest chromosome to shortest chromosome, arm ratio, chromosome type and the number of satellites of above cotton species, we inferred that G.anomalum (B genome) is the most primitive type. G.raimondii(D genome), G.nelsonii(C genome) and G.somalense(E genome) have primitive karyotypes, but their karyotypes are more evolutionary than that of G.anomalum. The karyotypes of G.herbaceum (A genome), G.longicalyx (F genome) and G.bickii(G genome), which have respective characteristics, are more evolutionary than other diploid species. A satellite of G.herbaceum was located on the relative larger chromosome which was different from other diploid species. The range of relative length of chromosome in G.longicalyx was wider than that of other species and there were no satellites can be detected in the end of short arms. The karyotype of G.bickii, which had the largest number of sm-type chromosome in diploid species, was relative evolutionary karyotype. According to the result of this experiment and others, we proposed that B genome is the most primitive type in cotton. In the process of cotton evolution, with the differentiation and migration of the B genome as a radiating center, deriving from it are the diploid species of C, D, E genome in earlier time and of A genome in later time. The diploid species of F genome are derived from E genome and that of G genome are derived from C genome. Eventually, it forms the specific distribution at present time.(3) We developed the technique of pachytene-FISH in this study, which was capable of obtain well-diffrentiated chromosomes suitable for FISH with less cell damage and clear backgroud. The late pachytene chromosome preparations of cotton were subjected to hybridization with cotton telomere probes and the results shown that the telomere signal can be detected at the end of all 13 extended bivalents. Based on the fluorescein in situ hybridization of pachytene chromosome of G.arboreum and G.herbaceum, with the probes of telomere and 45S rDNA, the FISH results revealed that there were 3 linear hybridized 45S rDNA signals, two strong and one weak, on chromosomes of G.arboreum, near to nuclear, which were located in chromosome number 3, 6, 8, respectively. And there were also 3 hybridized 45S rDNA signals, two strong and one weak, on chromosomes of G.herbaceum, near to nuclear, which were located in chromosome number 5, 7, 9, respectively. Coupled with the hybridized signals about 45S rDNA and telomere, and the extended pachytene chromosome of G.arboreum and G.herbaceum, each of which exhibited a well-differentiated pattern of eu- and heterochromatin, allowing to permits the identification of all 13 bivalents respectively, we preliminary developed the high resolution cytological map of A genomic groups of Gossypium.