Detection and mapping of homologous and homoeologous segments in homoeologous groups of allotetraploid cotton by BAC-FISH

Abstract

Background: Cotton, as an allopolyploid species, contains homoeologous A and D subgenomes. The study of the homoeologous (duplicated) segments or chromosomes can facilitate insight into the evolutionary process of polyploidy and the development of genomic resources. Fluorescence in situ hybridization (FISH) using bacterial artificial chromosome (BAC) clones as probes has commonly been used to provide a reliable cytological technique for chromosome identification. In polyploids, it also presents a useful approach for identification and localization of duplicated segments. Here, two types of BACs that contained the duplicated segments were isolated and analyzed in tetraploid cotton by FISH.

Results: Homologous and homoeologous BACs were isolated by way of SSR marker-based selection and then used to develop BAC-FISH probes. Duplicated segments in homoeologous chromosomes were detected by FISH. The FISH and related linkage map results followed known reinforced the relationships of homoeologous chromosomes in allotetraploid cotton, and presented a useful approach for isolation of homoeologous loci or segments and for mapping of monomorphic loci. It is very important to find that the large duplicated segments (homologous BACs) do exist between homoeologous chromosomes, so the shot-gun approach for genome sequencing was unavailable for tetraploid cotton. However, without doubt, it will contain more information and promote the research for duplicated segments as well as the genome evolution in cotton.

Conclusion: These findings and the analysis method by BAC-FISH demonstrated the powerful nature and wide use for the genome and genome evolutionary researches in cotton and other polyploidy species.

Figure 1

Distribution analysis of homoeologous segments in tetraploid cotton by FISH. (A) Figure showed that the identification of the BAC clone 75F07 (lane 5) containing the polymorphic locus of NAU837_-205, and 68D15 (lane 6) containing the monomorphic locus NAU837_-195 between TM-1 and Hai7124 by SSR marker NAU837. Lanes 1–4 were Hai7124, F₁(TM-1 × Hai7124), TM-1 and restorer line 0-613-2R, respectively. (B) FISH image showed that the signals of the polymorphic allele BAC 75F07 (green signals, arrows) and chromosome A6-specific BAC 47N15 (red signals, arrowheads) were located on the same chromosome. It indicated that the polymorphic allele BAC 75F07 derived from chromosome A6. (C) FISH image showed that the BAC clone 68D15 (red signals, arrowheads) located on the same chromosome with chromosome-specific BAC clone 24K19 (green signals, arrows) of chromosome D6.

Figure 2

The linkage mapping of monomorphic locus-derived markers. The monomorphic BAC-derived markers Y2446, Y2482 and Y2478 were indicated in bold italics, and linked with the corresponding polymorphic loci which were underlined. Other duplicated loci were also connected by solid bar between the homoeologous chromosomes.

Figure 3

Distribution analysis of homologous segments in tetraploid cotton by FISH. (A) BAC clone 09D09 produced two pairs of bright signals (arrows) in tetraploid cotton mitotic cell. (B) repeated-FISH showed the locations of two chromosome-specific probes 87P01 (red signal, arrows) of chromosome A5 and 50D03 (green signal, arrowheads) of chromosome D5. The results showed that the signals produced by clone 09D09 were located on the homoeologous chromosomes A5 and D5.

Figure 4

FISH photomicrographs of G. hirsutum duplicated segments hybridized to metaphase chromosomes of G. arboreum and G. raimondii. BAC clone 09D09 produced one pair of signals (arrows) on one pair of mitotic chromosomes of G. arboreum (A), but no signals on the chromosomes of G. raimondii (B).