The Construction Of Cytogenetic Map Of Gossypium Herbaceum Chromosome 1 & The Discovery And Analysis Of CCICR Transposable Element Family

The principle of fluorescence in situ hybridization(FISH) technique is complementary base pairing. It can be used to locate specific DNA sequences accurately on chromosomes, interphase nuclei or DNA fibers. With continuous improvement and perfection of the technology, it has been widely used on researches such as the identification of chromosomes, the construction of cytogenetic maps, gene localization, and genome evolution. In this study the cytogenetic map of Gossypium herbaceum chromosome 1 was constructed by using fluorescence in situ hybridization. And in the process of constructing cytogenetic map, a peculiar retrotransposon family CCICR(Chinese Central Institute of Cotton Reserch) was discoveried and identified. CCICR family accounts for 36.2% of A1 genome, while almost complete absent in D5 genome. The results will be helpful on researches of Gossypium evolution. The main contents are as following:1. A total of 17 SSR molecular markers from 5 genetic linkage maps were selected, and all the SSR molecular markers were used to screen the Gossypium barbadense Pima 90-53 BAC library. Finally 47 positive BAC clones were obtained. The all positive BAC clones were used as fluorescent probes seperately, and G. herbaceum mitotic metaphase chromosomes were used as target DNA. The positons of clear fluorescent hybridization signals of BACs were obtained by measuring many cells with good chromosomes spread, thus the cytogenetic map of G.herbaceum chromosome 1 including 10 BAC clones was constructed.2. The cytogenetic map of G. herbaceum chromosome 1 was compared with five cotton genetic linkage maps of chromosome 1. We found that the orders of the most common molecular markers were nearly the same in the two kinds of maps, except three molecular markers which showed difference in different maps, indicating that small chromosome rearrangements may occurre in G. herbaceum chromosome 1 in its evolution history.3. The cytogenetic map of G. herbaceum chromosome 1 was compared with four cotton genetic linkage maps of chromosome 1 and the positions of common molecular markers were analyzed by using RMP(relative map positon). The results showed that the positons of most common molecular markers in different map showed some differences. The reason may be due to the different exchange rate on different chromosomal parts.4. All the BAC clones which localized in the cytogenetic map of G. herbaceum chromosome 1 were used as fluorescent probes trying to be localized on G. raimondii metaphase chromosomes. As a result, two of the BAC clones 305A19 and 216B15 showed clear fluorescent hybridization signals on G. raimondii chromosome 1. The corresponding molecular markers of the two BAC clones are NAU2015 and NAU4891, which were the first time to be positioned on chromosomes D501. The first location of the two SSR markers on chromosome D501 will facilitate mapping of genes for cotton improvement.5. The two BAC clones 305A19 and 216B15 were also successfully positioned to G. hirsutum Ah01 and Dh01 chromosomes. And the positions of the two BAC clones on different chromosomes were determined by measuring many cells. The positions of the two BAC clones in four chromosomes A101, D501, Ah01 and Dh01 were compared. We found that the positions of two BAC clones in D501 and Dh01 are almost the same, and the situation is same when comparing positions on chromosomes A101 and Ah01. In addition, the centromere positions of four chromosomes A101, D501, Ah01 and Dh01 were determined by measuring many cells with good chromosome spread. Centromere is an important part for a chromosome, and the obtainment of the position of centromere will help understanding the structure of centromere and chromosome.6. In the process of constructing cytogenetic map of G. herbaceum chromosome 1, a peculiar retrotransposon family CCICR was discoveried and identified. Through bioinformatics analysis, the retrotransposon family CCICR was found to be accounted for 36.2% of A1 genome, due to almost complete absence of this LTR element family in D5 genome. About 70% of the 800 Mb genome size gap between G. arboretum and G. raimondii(1.6G: 0.8G) can be explained by dynamics of this LTR retrotransposon.7. By FISH analysis, we found that the retrotransposon family CCICR was only distributed in A(sub) genome and B genome, and the repeat number was huge, while it was absent in other genomes(the materials of K genome species were scarce, not to be tested).Through bioinformatics analysis, we found that the amplification of the retrotransposon family started 3.5-4 million years ago, reached the peak 2-3 million years ago, became silence 1-1.5 million years ago. According to its distribution in different genomes, the evolution of Gossypium genus was speculated. The divergence of A genome and B genome should be after 2-3 million years ago, the divergence of A genome and E&F genome should be prior 3.5-4 million years ago. From the transposon’s analysis, A genome and B genome are most homologous. And the lack of the transposon families in D sub-genome may indicate that allotetraploid cotton was formed after the silence of the transposon family 1-1.5 million years ago.