Comparative Structural Analysis Of 12A And 12D Homoeologous Chromosomes And Isolation And Identification Of Centromere-Associated Sequnces In Gossypium Hirsutum

Recently, the progress of cotton genome research has been extensive improved by the development of tons of molecular markers, the successful construction of high-density genetic maps and high-resolution cytogenetic maps, deep sequencing of transcriptome and the completion of whole genome sequencing of diploid Gossypium raimondii (DD,2n=26) and Gossypium arboreum (AA,2n=26). However, due to the complexity of cotton genomic structure and composition, there are still many problems to be resolved for ongoing genome research, such as the unknown internal dynamics of structural variation between homoeologous chromosomes and unraveled centromeric sequences and their genetic and physical position. Here, using molecular cytogenetic techniques combing with bioinformatic analysis, we have performed exploration from three aspects. Firstly, we developed successfully DNA fiber fluorescence in situ hybridization (DNA fiber-FISH) for cotton. Secondly, combined with high-resolution molecular cytogenetic map, we assayed the structural differences between 12A and 12D homoeologous chromosomes of Gossypium hirsutum (AADD,2n=52) at the DNA sequence level. Thirdly, we identified cotton centromere-associated retrotransposons and identified the position of cotton centromeres in the physical and genetic maps.The main findings are as follows:1. Development and application of cotton DNA fiber-FISH techniqueCotton cell is in rich of polyphenols, carbohydrates, lipids and other secondary materials which interfered the isolation of nuclei and preparation of DNA fibers. So conventional plant fiber-FISH technique can not be directly applied to cotton. Here we have developed a fiber-FISH protocol which is suitable for cotton. The systemic application by analysis of telomere and 5S rDNA proved that the technique is feasible and reliable in cotton.2. Comparative structural analysis of 12A and 12D homoeologous chromosomes of G. hirsutumStudies based on high-resolution cytogenetic map have shown that homoeologous chromosomes 12A and 12D presented inconsistent structural differences along the chromosomes. To investigate the internal impact factors of structural differences at DNA sequence level, BAC clones in the distal and centromeric regions were sequenced and analyzed. Sequence alignment analysis indicated that the sequences were highly conserved in the distal regions but that are highly variable in the centromeric regions on 12A and 12D homoeologous chromosomes. Further analysis showed that the extensive differences in centromeric regions mainly due to the uneven accumulation of Gorge3-like retrotransposons. The intron length variation and small indels played slight role in the structural differences. The conclusions facilitate the study of cotton genome structural difference.3. Isolation and identification of cotton centromere-associated sequencesWe identified a cotton centromere-specific BAC clone which was then sequenced and analyzed. Four LTR retrotransposons were identified and showed specifically locating in cotton centromeric regions by FISH assay. We name them as GhCR1, GhCR2, GhCR3 and GhCR4. According to fiber-FISH analysis, GhCRs showed clustered distribution and mingled with each other. By cotton whole sequence searching using the GhCRs, the centromeric regions were identified. By in silico PCR analysis, we also identified the centromeric markers and then marked the position of centromeric regions in the cotton genetic map.