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Background: Dissecting genome organization is indispensable for further functional and applied studies. As genome sequences data shown, cotton genomes contain more than 60% repetitive sequences, so study on repetitive sequences composition, structure, and distribution is the key step to dissect cotton genome. Results: In this study, a bacterial artificial chromosome (BAC) clone enriched in repetitive sequences, was discovered initiatively by fluorescence in situ hybridization (FISH). FISHing with allotetraploidy cotton as target DNA, dispersed signals on most regions of all A sub-genome chromosomes, and only middle regions of all D sub-genome chromosomes were detected. Further FISHing with other cotton species bearing A or D genome as target DNA, specific signals were viewed. After BAC sequencing and bioinformational analysis, 129 repeat elements, size about 57,172 bp were found, accounting for more than 62% of the BAC sequence (91,238 bp). Among them, a type of long terminal repeat-retrotransposon (LTR-RT), LTR/Gypsy was the key element causing the specific FISH results. Using the fragments of BAC matching with the identified Gypsy-like LTR as probes, the BAC-57I23-like FISH signals were reappeared. Running BLASTN, the fragments had good match with all chromosomes of G. arboreum (A2) genome and A sub-genome of G. hirsutum (AD1), and had relatively inferior match with all chromosomes of D sub-genome of AD1, but had little match with the chromosomes of G. raimondii (D5) genome, which was consistent with the FISH results. Conclusion: A repeats-enriched cytogenetic marker to identify A and D sub-genomes of Gossypium was discovered by FISH. Combined sequences analysis with FISH verification, the assembly quality of repetitive sequences in the allotetraploidy cotton draft genome was assessed, and better chromosome belonging was verified. We also found the genomic distribution of the identified Gypsy-LTR-RT was similar to the distribution of heterochromatin. The expansion of this type of Gypsy-LTR-RT in heterochromatic regions may be one of the major reasons for the size gap between A and D genome. The findings showed here will help to understand the composition, structure, and evolution of cotton genome, and contribute to the further perfection of the draft genomes of cotton. [ABSTRACT FROM AUTHOR]

Construction of single chromosomal DNA libraries by chromosome micromanipulation is a useful tool for pursuing genomic studies. Thus far, micromanipulation in cotton has not been reported yet, which may be due to difficulty in preparing chromosomes of similar sizes. In this study, single chromosome micromanipulation was successfully achieved in cotton. A single chromosome 5 of Gossypium arboreum (cultivar Shixiya-1) carrying a large satellite at mitotic metaphase was isolated by microdissection using the Cell Cut Plus Laser micromanipulation system. The chromosomal DNA was digested by Sau 3A and ligated to Sau 3A linker adaptors. After two rounds of linker adaptor PCR (LA-PCR) amplification, DNA fragments ranging from 300 to 2,500 bp were acquired. Southern hybridization revealed that the PCR products had homology with genomic DNA of the cultivar Shixiya-1, indicating that DNA of chromosome 5 has been successfully amplified. The second round LA-PCR products and 45S rDNA and chromosome-specific BAC clones were used as probes for fluorescence in situ hybridization analysis on metaphase chromosome. The results confirmed that the LA-PCR products were derived from the isolated target chromosome. Hybridization signals of the second round LA-PCR products were mainly detected along the entire chromosome 5; in addition, weak signals were also observed on other chromosomes, indicating that there were some homologous nucleotide sequences in other chromosomes. The second round LA-PCR products were cloned to generate a chromosome-specific DNA library which contains approximately 173,000 clones. Evaluation based on 136 randomly selected clones showed that the size of the inserts varied from 500 to 1,800 bp with an average of 750 bp. The no-load rate was less than 1 %, the titer of the library was 1.2 × 10 pfu mL, and the rate of the single and low copy sequences was over 47 %. This library will facilitate specific probe screening, molecular mapping, gene cloning, and DNA sequencing for this chromosome. [ABSTRACT FROM AUTHOR]

Two bacterial artificial chromosome (BAC) clones (350B21 and 299N22) of Pima 90-53 cotton [Gossypium barbadense Linnaeus, 1753 (2n=4x=52)] were screened from a BAC library using SSR markers. Strong hybridization signals were detected at terminal regions of all A genome (sub-genome) chromosomes, but were almost absent in D genome (sub-genome) chromosomes with BAC clone 350B21 as the probe. The results indicate that specific sequences, which only exist at the terminal parts of A genome (sub-genome) chromosomes with a huge repeat number, may be contained in BAC clone 350B21. When utilizing FISH with the BAC clone 299N22 as probe, a pair of obvious signals was detected on chromosome 13 of D genome (sub-genome), while strong dispersed signals were detected on all A genome (sub-genome) chromosomes. The results showed that peculiar repetitive sequence, which was distributed throughout all A genome (subgenome) chromosomes, may exist in BAC clone 299N22. The absence of the repetitive sequences, which exist in the two BAC clones, in D genome may account for the genome-size variation between A and D genomes. In addition, the microcolinearity analysis of the clone 299N22 and its homologous region on G. raimondii Ulbrich, 1932 chromosome 13 (D513) indicated that the clone 299N22 might come from A sub-genome of sea island cotton (G. barbadense), and a huge number of small deletions, illegitimate recombination, translocation and rearrangements may have occurred during the genus evolution. The two BAC clones studied here can be used as cytological markers but will be also be helpful to research in cotton genome evolution and comparative genomics. [ABSTRACT FROM AUTHOR]