Investigation of Astyanax mexicanus (Characiformes, Characidae) chromosome 1 structure reveals unmapped sequences and suggests conserved evolution

Abstract

Natural selection in the cave habitat has resulted in unique phenotypic traits (including pigmentation loss and ocular degeneration) in the Mexican tetra Astyanax mexicanus, considered a model species for evolutionary research. A. mexicanus has a karyotype of 2n = 50 chromosomes, and long-read sequencing and quantitative trait linkage maps (QTLs) have completely reconstructed the reference genome at the chromosomal level. In the current work, we performed whole chromosome isolation by microdissection and total amplification using DOP-PCR and Whole Chromosome Painting (WCP), followed by sequencing on the Illumina NextSeq platform, to investigate the microstructure of the large and conserved metacentric chromosome 1 of A. mexicanus. The sequences aligned to linkage block 3 of the reference genome, as determined by processing the reads with the DOPseq pipeline and characterizing the satellites with the TAREAN program. In addition, part of the sequences was anchored in linkage blocks that have not yet been assigned to the chromosomes. Furthermore, fluorescence in situ hybridization using WCP 1 carried out in other nearby species revealed a high degree of chromosome conservation, which allows us to hypothesize a common origin of this element. The physical mapping of the repetitive marker sequences provided a micro- and macrostructural overview and confirmed their position in chromosome pair 1. These sequences can serve as comparative tools for understanding the evolution and organization of this chromosome in other species of the family in future studies.

Fig 1. Karyotype, mapping of the probe and anchored reads of chromosome 1 of Astyanax mexicanus.

A) Conventional Giemsa staining revealed a karyotype for A. mexicanus as 2n = 50. B) FISH-mapping of the probe isolated by microdissection of chromosome 1 in A. mexicanus, in A. altiparanae (C); in Psalidodon bifasciatus (D); and P. scabripinnis (E). In B and E the probe is marked with digoxigenin—rhodamine (red), the chromosomes are counter-stained by DAPI (blue), and in C and D the probe is marked with Spectrum Orange-dUTP and counter-stained with propidium iodide. The arrows indicate the chromosome 1 pair. Bar: 10 μm.; F) Reads anchored in the first three chromosomes of the karyotype, represented in the reference genome.

Fig 2. Mapping of satellite DNAs markers for chromosome 1 of Astyanax mexicanus.

A) SatA_mex (574bp), B) SatB_mex (584 bp), C) SatC_mex (178bp) and D) SatD_mex (54bp). The satellites SatE_mex (229 bp) and SatF_mex (152bp), revealed signals outside of chromosome 1, used as a negative control. The sequences were marked with Streptavidin (green) or Digoxigenin—rhodamine (red). The arrows indicate chromosome 1 pair. Bar: 10 μm. F) Chromosome 1 and its satellite DNA distribution. G) Idiogram of all satellite DNAs mapped on chromosome 1 (left homologue) and with the satellite DNAs and the exclusive monogenic amplified sequences mapped (right homologue).

Fig 3. Signals of satellite DNAs probes for chromosome 1 of A. mexicanus in Psalidodon scabripinnis.

The sequences were marked with Streptavidin (green) or Digoxigenin—rhodamine (red). The arrows indicate the chromosome pair 1. Bar: 10 μm.