Uncovering the Ancestry of B Chromosomes in Moenkhausia sanctaefilomenae (Teleostei, Characidae).

B chromosomes constitute a heterogeneous mixture of genomic parasites that are sometimes derived intraspecifically from the standard genome of the host species, but result from interspecific hybridization in other cases. The mode of origin determines the DNA content, with the B chromosomes showing high similarity with the A genome in the first case, but presenting higher similarity with a different species in the second. The characid fish Moenkhausia sanctaefilomenae harbours highly invasive B chromosomes, which are present in all populations analyzed to date in the Parana and Tietê rivers. To investigate the origin of these B chromosomes, we analyzed two natural populations: one carrying B chromosomes and the other lacking them, using a combination of molecular cytogenetic techniques, nucleotide sequence analysis and high-throughput sequencing (Illumina HiSeq2000). Our results showed that i) B chromosomes have not yet reached the Paranapanema River basin; ii) B chromosomes are mitotically unstable; iii) there are two types of B chromosomes, the most frequent of which is lightly C-banded (similar to euchromatin in A chromosomes) (B1), while the other is darkly C-banded (heterochromatin-like) (B2); iv) the two B types contain the same tandem repeat DNA sequences (18S ribosomal DNA, H3 histone genes, MS3 and MS7 satellite DNA), with a higher content of 18S rDNA in the heterochromatic variant; v) all of these repetitive DNAs are present together only in the paracentromeric region of autosome pair no. 6, suggesting that the B chromosomes are derived from this A chromosome; vi) the two B chromosome variants show MS3 sequences that are highly divergent from each other and from the 0B genome, although the B2-derived sequences exhibit higher similarity with the 0B genome (this suggests an independent origin of the two B variants, with the less frequent, B2 type presumably being younger); and vii) the dN/dS ratio for the H3.2 histone gene is almost 4-6 times higher for B chromosomes than for A chromosome sequences, suggesting that purifying selection is relaxed for the DNA sequences located on the B chromosomes, presumably because they are mostly inactive

Metaphase plates of <i>M</i>. <i>sanctaefilomenae</i> after whole-chromosome painting with B₁ and B₂ probes and their respective sequential C-banding patterns.

<p>(a-d) represent <i>M</i>. <i>sanctaefilomenae</i> from the Batalha River. (e-f) represent <i>M</i>. <i>sanctaefilomenae</i> from the Novo River.</p

Metaphase plates of <i>M</i>. <i>sanctaefilomenae</i> from the Batalha River after FISH with different repetitive probes and sequential C-banding to show the clustering of 18S rDNA, H3 histones and satDNAs on different B chromosomes.

<p>Note that B₁ is euchromatic showing small blocks of 18S rDNA, and B₂ is heterochromatic showing larger amounts of 18S rDNA.</p

<i>M</i>. <i>sanctaefilomenae</i> karyotypes constructed from mitotic metaphase cells subjected to FISH with different repetitive DNA probes.

<p>a) Novo River population. b) Batalha River population. Note the differential distribution of H3 histone genes and 5S and 18S rDNA clusters on the A chromosomes in both populations.</p

Comparison of the mean number of B chromosomes and mitotic instability index (MI) per individual between the present data and those previously reported for the Tiete River by Foresti et al. (1998) and Dantas et al. (2007).

<p>Comparison of the mean number of B chromosomes and mitotic instability index (MI) per individual between the present data and those previously reported for the Tiete River by Foresti et al. (1998) and Dantas et al. (2007).</p

Genetic variation observed in MS1, MS2, H3.2 and H3.3 sequences obtained through microdissection and PCR amplification and directly from reads.

<p>Genetic variation observed in MS1, MS2, H3.2 and H3.3 sequences obtained through microdissection and PCR amplification and directly from reads.</p

Number of synonymous and non-synonymous substitutions per synonymous (dS) and non-synonymous (dN) site, respectively, observed in the DNA sequences of H3.2 histone genes

<p>Number of synonymous and non-synonymous substitutions per synonymous (dS) and non-synonymous (dN) site, respectively, observed in the DNA sequences of H3.2 histone genes</p

Minimum spanning trees (MST) showing the relationships between the different haplotypes of MS3 and MS7 satDNAs obtained from distinct libraries.

<p>Haplotypes retrieved directly from Illumina reads are represented by light/dark grey circles, and the diameter of the circles is proportional to their abundance, whereas PCR-amplified haplotypes are represented by coloured rectangles, with the percentage of clones corresponding to each haplotype. Each black dot represents a mutational step.</p