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Molecular Cytogenetic Differentiation of Paralogs of Hox Paralogs in Duplicated and Re-Diploidized Genome of the North American Paddlefish (Polyodon Spathula)


Molecular Cytogenetic Differentiation of Paralogs of Hox Paralogs in Duplicated and Re-Diploidized Genome of the North American Paddlefish (Polyodon Spathula)

Radka Symonová et al. BMC Genet.


Background: Acipenseriformes is a basal lineage of ray-finned fishes and comprise 27 extant species of sturgeons and paddlefishes. They are characterized by several specific genomic features as broad ploidy variation, high chromosome numbers, presence of numerous microchromosomes and propensity to interspecific hybridization. The presumed palaeotetraploidy of the American paddlefish was recently validated by molecular phylogeny and Hox genes analyses. A whole genome duplication in the paddlefish lineage was estimated at approximately 42 Mya and was found to be independent from several genome duplications evidenced in its sister lineage, i.e. sturgeons. We tested the ploidy status of available chromosomal markers after the expected rediploidization. Further we tested, whether paralogs of Hox gene clusters originated from this paddlefish specific genome duplication are cytogenetically distinguishable.

Results: We found that both paralogs HoxA alpha and beta were distinguishable without any overlapping of the hybridization signal - each on one pair of large metacentric chromosomes. Of the HoxD, only the beta paralog was unequivocally identified, whereas the alpha paralog did not work and yielded only an inconclusive diffuse signal. Chromosomal markers on three diverse ploidy levels reflecting different stages of rediploidization were identified: quadruplets retaining their ancestral tetraploid condition, semi-quadruplets still reflecting the ancestral tetraploidy with clear signs of advanced rediploidization, doublets were diploidized with ancestral tetraploidy already blurred. Also some of the available microsatellite data exhibited diploid allelic band patterns at their loci whereas another locus showed more than two alleles.

Conclusions: Our exhaustive staining of paddlefish chromosomes combined with cytogenetic mapping of ribosomal genes and Hox paralogs and with microsatellite data, brings a closer look at results of the process of rediploidization in the course of paddlefish genome evolution. We show a partial rediploidization represented by a complex mosaic structure comparable with segmental paleotetraploidy revealed in sturgeons (Acipenseridae). Sturgeons and paddlefishes with their high propensity for whole genome duplication thus offer suitable animal model systems to further explore evolutionary processes that were shaping the early evolution of all vertebrates.

Keywords: Ancient fish genome; HoxA/D paralogs mapping; Rediploidization; Sturgeon whole genome duplication.


Fig. 1
Fig. 1
Summary of karyological data. DAPI stained metaphase reversed to black and white (a), Chromomycin A3 positive small submetacentric chromosomes, reversed to black and white (b), DAPI positive small submetacentric chromosomes (c), DAPI positive middle-sized subtelocentric chromosomes (d), AgNOR positive chromosomes (e), acipenseriform marker chromosomes - the largest acrocentric chromosomes, the first two of them with two distinct DAPI positive bands (f). Karyotype of the DAPI stained metaphase with macrochromosomes arranged into quadruplets, semi-quadruplet and duplets based on the DAPI signal (g). Karyotype of Giemsa stained chromosomes with macrochromosomes arranged only into quadruplets (h). Scale bars equal 10 μm
Fig. 2
Fig. 2
FISH with rDNAs and Hox clusters. FISH with 28S rDNA (a, red), 5S rDNA (b, red) and FISH with BACs of the paralogs Hox-A alpha (c, red) and Hox-A beta (c, green) and of the paralogs Hox-D beta (d, red) and Hox-D alpha (d, green – the diffuse character of this signal causes a greenish coloration of almost all chromosomes). Scale bars equal 10 μm
Fig. 3
Fig. 3
Ideogram summarizing all chromosomal markers investigated in this study
Fig. 4
Fig. 4
Microsatellite analysis of ploidy level. Microsatellite data (number of alleles) in Polyodon spathula and comparison with sturgeons related to chromosomes numbers and ploidy levels provide evidence of partial genome rediploidization in P. spathula (formula image) and sturgeons (●). The figure is based on data from this study and previous studies [18, 28]
Fig. 5
Fig. 5
Suggested main events in evolution of Acipenseriformes. S1 = split of Polyodontidae and Acipenseridae (~170 Mya); S2 = Split of Atlantic and Pacific lineage in Acipenseridae (~121 Mya); S3 = split of 2n and 4n species within Atlantic lineage (~80 Mya); S4 = Split of Polyodon spathula and Psephurus gladius (~68Mya); S5 = Split of Acipenser brevirostrum (~36 Mya). Whole genome duplication (WGD) events are: 1 = WGD specific to Polyodon spathula (60 → 120 chromosomes;~ 42 Mya [12]); 2 = WGD in Atlantic lineage (120 → 250 chromosomes; ~ 53 Mya); 3 = WGD in Pacific lineage (120 → 250 chromosomes; ~ 70 Mya); 4 = WGD specific to Acipenser brevirostrum (250 → 360 chromosomes ~ 35 Mya); D1 = the first WGD in Acipenseridae (60 → 120 chromosomes) had to take place between ~ 170 Mya and ~ 121 Mya; D2 = probable WGD (60 → 120 chromosomes) specific to Psephurus gladius. The data are based on study by Peng et al. [2] and Crow et al. [12]

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