Search

Chromosome painting is one of the key technologies in cytogenetic research, which can accurately identify chromosomes or chromosome regions. Oligonucleotide (oligo) probes designed based on genome sequences have both flexibility and specificity, which would be ideal probes for fluorescence in situ hybridization (FISH) analysis of genome structure. In this study, the bulked oligos of the two arms of chromosome seven of cotton were developed based on the genome sequence of Gossypium raimondii (DD, 2n = 2× = 26), and each arm contains 12,544 oligos. Chromosome seven was easily identified in both D genome and AD genome cotton species using the bulked chromosome-specific painting probes. Together with 45S ribosomal DNA (rDNA) probe, the chromosome-specific painting probe was also successfully used to correct the chromosomal localization of 45S rDNA in G. raimondii. The study reveals that bulked oligos specific to a chromosome is a useful tool for chromosome painting in cotton.

The activity of genome-specific repetitive sequences is the main cause of genome variation between Gossypium A and D genomes. Through comparative analysis of the two genomes, we retrieved a repetitive element termed ICRd motif, which appears frequently in the diploid Gossypium raimondii (D5) genome but rarely in the diploid Gossypium arboreum (A2Gossypium, Repetitive element, D genome, Fluorescence in situ hybridization (FISH), Genome-specific, Evolution ) genome. We further explored the existence of the ICRd motif in chromosomes of G. raimondii, G. arboreum, and two tetraploid (AADD) cotton species, Gossypium hirsutum and Gossypium barbadense, by fluorescence in situ hybridization (FISH), and observed that the ICRd motif exists in the D5 and D-subgenomes but not in the A2 and A-subgenomes. The ICRd motif comprises two components, a variable tandem repeat (TR) region and a conservative sequence (CS). The two constituents each have hundreds of repeats that evenly distribute across 13 chromosomes of the D5 genome. The ICRd motif (and its repeats) was revealed as the common conservative region harbored by ancient Long Terminal Repeat Retrotransposons. Identification and investigation of the ICRd motif promotes the study of A and D genome differences, facilitates research on Gossypium genome evolution, and provides assistance to subgenome identification and genome assembling. [ABSTRACT FROM AUTHOR]

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]

The study was conducted on the individual chromosome identification in Gossypium darwinii (AdDd), G. klotzschianum (D3k), G. davidsonii (D3d), G. armourianum (D2-1) and G. aridum (D4) using a multi-probe fluorescence of in situ hybridization (FISH) system. Comparative analysis on their genetic maps with that of physical maps was made as well. The FISH probes used contained two sets of bacterial artificial chromosome (BAC) clones [one is specific to 26 individual chromosomes from A and D subgenomes of G. hirsutum (Ah and Dh) while the other is a D genome centromere- specific BAC clone 150D24], 45S and 5S rDNA clones. The results showed that all Ad chromosomes were marked with the 13 Ah chromosome-specific BAC clones, whilst all Dd, D3k, D3d, D2-1 and D4 chromosomes and chromosomal arms were identified with the 13 Dh chromosome-specific BAC clones and the D genome centromere-specific BAC. According to the homology within D subgenomes which are between A (D) genome and A (D) subgenome, the systematic nomenclature for individual chromo- some in the five species was established. The chromosomes of A (D) subgenomes in G. darwinii were named as Ad01-Ad13 (Dd01-Dd13). The chromosomes in D3k, D3d, D2-1 and D4 were named as D3k01-D3k13, D3d01-D3d13, D2-101-D2-113 and D401-D413, respectively. Based on the successful identification for individual chromosomes, 45S and 5S rDNA were located to the special chromosomes and chromosomal arms for all five species. And there appeared chromosomal collinearity from the BAC clones among different species by comparing BACs positions, which suggested that the majority of chromosome segment homology may exist between D genomes and D subgenome. Moreover, as the genetic map and physical map were integrated, the orientations of genetic maps for Dd and D genomes of diploid cotton were established. The orientations of some of chromosomes in genetic maps (Dd03, Dd04, Dd06, Dd09, Dd10 and Dd12) were found switched. The SSR marker in the middle of linkage group 04 was corrected at nearby the end of chromosome 04 by FISH. The study will be helpful to establish a theoretical basis using the wild gene bank to exploit more genes aiming for cotton breeding and will provide powerful evidences both for the evolution of Gossypium and assembling the sequences of the obtained and as well the on-going whole genome sequencing projects of cotton. [ABSTRACT FROM AUTHOR]

Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established. [ABSTRACT FROM AUTHOR]

The experiment on individual chromosome assignments and chromosomal diversity was conducted using a multi-probe fluorescence in situ hybridization (FISH) system in D subgenome of tetraploid Gossypium barbadense (Db), G. thurberi (D1) and G. trilobum (D8), which the later two were the possible subgenome donors of tetraploid cottons. The FISH probes contained a set of bacterial artificial chromosome (BAC) clones specific to 13 individual chromosomes from D subgenome of G. hirsutum (Dh), a D genome centromere-specific BAC clone 150D24, 45S and 5S ribosomal DNA (rDNA) clones, respectively. All tested chromosome orientations were confirmed by the centromere-specific BAC probe. In D1 and D8, four 45S rDNA loci were found assigning at the end of the short arm of chromosomes 03, 07, 09 and 11, while one 5S rDNA locus was successfully marked at pericentromeric region of the short arm of chromosome 09. In Db, three 45S rDNA loci and two 5S rDNA loci were found out. Among them, two 45S rDNA loci were located at the terminal of the short arm of chromosomes Db07 and Db09, whilst one 5S rDNA locus was found situating near centromeric region of the short arm of chromosome Db09. The positions of the BAC clones specific to the 13 individual chromosomes from Dh were compared between D1, D8 and Db. The result showed the existence of chromosomal collinearity within D1 and D8, and as well between them and Db. The results will serve as a base for understanding chromosome structure of cotton and polyploidy evolution of cotton genome and will provide bio-information for assembling the sequences of finished and the on-going cotton whole genome sequencing projects. [ABSTRACT FROM AUTHOR]