In-Frame Indel Mutations in the Genome of the Blind Mexican Cavefish, Astyanax mexicanus

Abstract

Organisms living in the subterranean biome evolve extreme characteristics including vision loss and sensory expansion. Despite prior work linking certain genes to Mendelian traits, the genetic basis for complex cave-associated traits remains unknown. Moreover, it is unclear if certain forms of genetic variation (e.g., indels, copy number variants) are more common in regressive evolution. Progress in this area has been limited by a lack of suitable natural model systems and genomic resources. In recent years, the Mexican tetra, Astyanax mexicanus, has advanced as a model for cave biology and regressive evolution. Here, we present the results of a genome-wide screen for in-frame indels using alignments of RNA-sequencing reads to the draft cavefish genome. Mutations were discovered in three genes associated with blood physiology (mlf1, plg, and wdr1), two genes associated with growth factor signaling (ghrb, rnf126), one gene linked to collagen defects (mia3), and one gene which may have a global epigenetic impact on gene expression (mki67). With one exception, polymorphisms were shared between Pachón and Tinaja cavefish lineages, and different from the surface-dwelling lineage. We confirmed the presence of mutations using direct Sanger sequencing and discovered remarkably similar developmental expression in both morphs despite substantial coding sequence alterations. Further, three mutated genes mapped near previously established quantitative trait loci associated with jaw size, condition factor, lens size, and neuromast variation. This work reveals previously unappreciated traits evolving in this species under environmental pressures (e.g., blood physiology) and provides insight to genetic changes underlying convergence of organisms evolving in complete darkness.

Keywords: Astyanax; RNA-sequencing; genetic lesion; troglomorphy.

Fig. 1.

—RNA-sequencing alignments to the Astyanax genome uncovered indels in the expressed genes ghrb, mia3, mki67, and mlf1. The gene ghrb has a genomic length of ∼10.21 kb. Pachón and Tinaja cavefish harbor a 3-bp insertion in the seventh (last) exon of this gene, which was not present in surface fish (A). Similarly, the gene mia3 (∼17.69 kb) demonstrates a 6-bp insertion (exon 4), which is only present in Pachón and Tinaja cavefish (B). The gene mki67 (13.54 kb) has two sequential lesions in the ninth exon: a 36-bp insertion followed by a 3-bp deletion (C). Both mutations were observed in the cavefish lineages and were absent from surface fish. The gene mlf1 (∼14.95 kb) demonstrates a 12-bp insertion in the third exon of Pachón and Tinaja cavefish, which was not present in surface fish (D). RNA-seq alignments are presented in (A), (B), and (C); detected lesions are highlighted in yellow. Validated lesions based on direct PCR sequencing are presented in (A)–(D) (Pachón top, Tinaja middle, and Surface bottom).

Fig. 2.

—RNA-sequencing alignments to the Astyanax genome uncovered indels in the expressed genes plg, rnf126, and wdr1. The gene plg has a genomic length of ∼12.17 kb. Cavefish from both localities harbor a 1-bp insertion followed by a subsequent 1-bp deletion in the fourth exon of this gene, neither of which is present in surface fish (A). The gene rnf126 (∼14.43 kb) harbors a 5-bp deletion in the tenth exon, which is only present in the cavefish localities (B). The gene wdr1 demonstrates a 6-bp insertion in Tinaja cavefish, which is absent from Pachón and surface-dwelling fish (C). RNA-seq alignments are presented in (A)–(C); detected lesions are highlighted in yellow. Validated lesions based on direct PCR sequencing are presented in (A)–(C) (Pachón top, Tinaja middle, and Surface bottom).

Fig. 3.

—Expression analyses reveal largely patterns between cave and surface fish embryos. Highly conserved patterns of gene expression based on whole-mount in situ hybridization were observed in 72 hpf embryos for the genes ghrb (A, B), mia3 (C, D), mki67 (E, F), mlf1 (G, H), plg (I, J), rnf126 (K, L), and wdr1 (M, N). A no-probe control (O, P) is provided for comparison. RNA-sequencing expression data reveal largely conserved expression for genes across four stages of development (10, 24, 36, and 72 hpf), including ghrb (Q), mia3 (R), and mki67 (S). The gene mlf1 (T) demonstrates differences in expression at the first two developmental stages, whereas plg (U) appears to be more conserved early in development, becoming polarized at the 72-hpf stage. Both rnf126 (V) and wdr1 (W) are highly similar in expression between morphs at all stages of development. For comparison, we present stable and highly similar expression patterns of rps18 (X), a housekeeping gene used in prior transcriptomic studies. All whole-mount images were taken at 100×, except (I), (J), (O), and (P) which were taken at 45×. Scale bars = 500 µM.

Fig. 4.

—A Circos representation of the relative positions of six genes with indel mutations and QTL associated with diverse traits in Astyanax cavefish. QTL positions are depicted relative to the draft Astyanax genome, alongside six candidate genes. On chromosome 2, the gene wdr1 maps ∼29.2 Mb from microsatellite marker 229a (peak marker associated with anal fin ray morphology). The gene mia3 resides on chromosome 3, near markers 203f (associated with lens size and superficial neuromasts) and 218d (associated with fin placement). The gene ghrb maps to chromosome 14, close to marker 129b (associated with jaw bone size), and more distantly to 55a (associated with eye size variation). The genes mlf1 and rnf126 both map to chromosome 17, near markers 208a (associated with condition factor), and 222e (associated with eye size, craniofacial bone size, and sex), respectively. Although mki67 maps to chromosome 4, it is not located near any known QTL. In the current draft genome, plg resides on an unlocated chromosome.