Delimiting the Origin of a B Chromosome by FISH Mapping, Chromosome Painting and DNA Sequence Analysis in Astyanax paranae (Teleostei, Characiformes)

Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.This research was funded by grants from the State of São Paulo Research Foundation (FAPESP) to DMZAS (2011/16825-3) and CO (2010/17009-2), grants from National Council for Research and Development (CNPq) to FF and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Nucleotide diversity (π) for the DNA sequences analysed, and Student (t) tests comparing A chromosome (0B-gDNA) and microdissected B chromosome (µB) sequences.

<p>Where n =  number of sequences, S =  number of segregating sites, Hap =  number of haplotypes, SE =  standard error, df =  degrees of freedom, P =  probability.</p

Metaphases of <i>Astyanax paranae</i> from the Capivara River after double-FISH with 5S rDNA and H1 histone (A), H1 and H3 histone (B), H1 and H4 histone (C), <i>Rex1</i> and 18S rDNA (D), <i>Rex3</i> and 18S rDNA (E) and after chromosome painting with µBm-probe (F).

<p>Bar = 10 µm.</p

Number of synonymous and non-synonymous substitutions per synonymous (dS) and non-synonymous (dN) site, respectively, observed in the DNA sequences of the H1 and H3 histone genes obtained from 0B genomic DNA (0B-gDNA) and B microdissected DNA (µB).

<p>Number of synonymous and non-synonymous substitutions per synonymous (dS) and non-synonymous (dN) site, respectively, observed in the DNA sequences of the H1 and H3 histone genes obtained from 0B genomic DNA (0B-gDNA) and B microdissected DNA (µB).</p

Maximum likelihood tree built with the concatenated ITS rDNA sequences.

<p>The sequences were obtained from the microdissected B chromosomes, gDNA from 0B <i>A. paranae</i> individuals, and gDNA from <i>A. bockmanni, A. fasciatus</i> and <i>A. altiparanae</i> (Aalti) specimens. Note the high similarity of the B chromosome sequences (uBpar) and the sequences obtained from 0B-gDNA in <i>A. paranae</i> (Apar) and <i>A. bockmanni</i> (Abock), and the lower similarity to those from <i>A. fasciatus</i> (Afasc).</p

Species tree built with the histone genes and ITS regions by BEAST.

<p>Note that the DNA sequences of <i>A. paranae</i> obtained from the microdissected B chromosomes (µB-DNA) are most similar to those in the host genome (0B-gDNA).</p

Ideogram of <i>Astyanax paranae</i> from the Capivara River showing the main cytogenetic markers localisation used in this paper.

<p>In the box, a summary of the FISH markers and chromosome painting associated with B chromosome in chromosomes 2, 23, Bm and Bsm.</p