Search

Telomeres are structures enriched in repetitive sequences at the end of chromosomes. In this study, using the telomere primer AA(CCCTAAA)3CCC for the single primer PCR, two DNA sequences were obtained from Gossypium hirsutum (Linnaeus, 1753) accession (acc.) TM-1. Sequence analysis showed that the two obtained sequences were all rich in A/T base, which was consistent with the characteristic of the telomere-associated sequence (TAS). They were designated as GhTAS1 and GhTAS2 respectively. GhTAS1 is 489 bp long, with 57.6% of A/T, and GhTAS2 is 539 bp long, with 63.9% of A/T. Fluorescence in situ hybridization results showed that both of the cloned TASs were located at the ends of the partial chromosomes of G. hirsutum, with the strong signals, which further confirmed that GhTAS1 and GhTAS2 were telomere-associated sequences including highly tandemly repetitive sequences. Results of blast against the assembled genome of G. hirsutum showed that GhTAS sequences may be missed on some assembled chromosomes. The results provide important evidence for the evaluation of the integrity of assembled chromosome end sequences, and will also contribute to the further perfection of the draft genomes of cotton.

Cytogenetic maps of Gossypium hirsutum (Linnaeus, 1753) homoeologous chromosomes Ah01 and Dh01 were constructed by fluorescence in situ hybridization (FISH), using eleven homoeologous-chromosomes-shared bacterial artificial chromosomes (BACs) clones and one chromosome-specific BAC clone respectively. We compared the cytogenetic maps with the genetic linkage and draft genome assembly maps based on a standardized map unit, relative map position (RMP), which allowed a global view of the relationship of genetic and physical distances along each chromosome, and assembly quality of the draft genome assembly map. By integration of cytogenetic maps with sequence maps of the two chromosomes (Ah01 and Dh01), we inferred the locations of two scaffolds and speculated that some homologous sequences belonging to homoeologous chromosomes were removed as repetitiveness during the sequence assembly. The result offers molecular tools for cotton genomics research and also provides valuable information for the improvement of the draft genome assembly.

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.

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 (sub-genome) 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.