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The short and similar sized chromosomes of Acropora pose a challenge for karyotyping. Conventional methods, such as staining of heterochromatic regions, provide unclear banding patterns that hamper identification of such chromosomes. In this study, we used short single-sequence probes from tandemly repetitive 5S ribosomal RNA (rRNA) and core histone coding sequences to identify specific chromosomes of Acropora pruinosa. Both the probes produced intense signals in fluorescence in situ hybridization, which distinguished chromosome pairs. The locus of the 5S rDNA probe was on chromosome 5, whereas that of core histone probe was on chromosome 8. The sequence of the 5S rDNA probe was composed largely of U1 and U2 spliceosomal small nuclear RNA (snRNA) genes and their interspacers, flanked by short sequences of the 5S rDNA. This is the first report of a tandemly repetitive linkage of snRNA and 5S rDNA sequences in Cnidaria. Based on the constructed tentative karyogram and whole genome hybridization, the longest chromosome pair (chromosome 1) was heteromorphic. The probes also hybridized effectively with chromosomes of other Acropora species and population, revealing an additional core histone gene locus. We demonstrated the applicability of short-sequence probes as chromosomal markers with potential for use across populations and species of Acropora.

We performed conventional and molecular cytogenetic studies on the Favites pentagona Esper, 1795, a scleractinian coral mostly found along the west coast of Japan. Karyotype analysis of F. pentagona by G-banding revealed a karyogram containing a homogenously staining region (HSR) on chromosome 10 in more than 50% of the examined metaphase spreads. This HSR consisted of sequences from 18S ribosomal RNA (rRNA) genes, as demonstrated by fluorescence in situ hybridization (FISH) and DNA sequencing. We highlighted the development of four chromosomal FISH markers from repetitive genes such as U2 small nuclear RNA linked to 5S rRNA sequence (U2 snRNA-5S), 18S rRNA, histone H3, and uncharacterized gene FP-9X. The chromosomal locations of the U2 snRNA-5S and 18S RNA were on the terminal end of long arm of chromosomes 2 and 10, respectively, while the histone H3 and the uncharacterized gene were located near the centromeres of chromosomes 1 and 9, respectively. These FISH markers will improve the karyotyping of F. pentagona from mitotic preparations which helps in widening our understanding of coral genetic structure and chromosome organization. In addition, these improvements in karyotyping will provide the basis in constructing of chromosome-level genome assembly for F. pentagona.