Large-scale variation in single nucleotide polymorphism density within the laboratory axolotl (Ambystoma mexicanum)

Abstract

Background: Recent efforts to assemble and analyze the Ambystoma mexicanum genome have dramatically improved the potential to develop molecular tools and pursue genome-wide analyses of genetic variation.

Results: To better resolve the distribution and origins of genetic variation with A mexicanum, we compared DNA sequence data for two laboratory A mexicanum and one A tigrinum to identify 702 million high confidence polymorphisms distributed across the 32 Gb genome. While the wild-caught A tigrinum was generally more polymorphic in a genome-wide sense, several multi-megabase regions were identified from A mexicanum genomes that were actually more polymorphic than A tigrinum. Analysis of polymorphism and repeat content reveals that these regions likely originated from the intentional hybridization of A mexicanum and A tigrinum that was used to introduce the albino mutation into laboratory stocks.

Conclusions: Our findings show that axolotl genomes are variable with respect to introgressed DNA from a highly polymorphic species. It seems likely that other divergent regions will be discovered with additional sequencing of A mexicanum. This has practical implications for designing molecular probes and suggests a need to study A mexicanum phenotypic variation and genome evolution across the tiger salamander clade.

Keywords: SNPs; axolotl; genome; hybrid; salamander.

FIGURE 1

Examining variation in the density of polymorphisms identifies regions with an excess or dearth of polymorphisms. A, Homozygous polymorphisms between two sequenced A mexicanum individuals: a d/d strain male and a wildtype AGSC female. Note a region of excess polymorphism on chromosome 1. B, Heterozygous polymorphisms identified from low-coverage sequencing of the d/d strain male. Note regions of excess polymorphism on chromosomes 4, 5, and 9, and several stretches with decreased polymorphism on all chromosomes presumably due to recent history of inbreeding. C, Heterozygous polymorphisms identified for the wildtype AGSC female. Note regions of excess polymorphism on chromosomes 4, and 7, as well as a few small stretches with decreased polymorphism (eg, chr8). Scalebar: 1 Gb

FIGURE 2

Histogram of SNP densities within sequenced individuals. Plots show densities of homozygous (red) and heterozygous (blue) SNPs identified in A, the d/d strain male, B, the AGSC female, C, the sampled A tigrinum, and D, Three regions of high differentiation that were characterized in this study

FIGURE 3

Patterns of polymorphism in a wild caught Ambystoma tigrinum provide perspective on those observed in laboratory axolotls. A, Fixed homozygous polymorphisms are higher on average between A tigrinum and A mexicanum than between the two sampled A mexicanum). B, On average this individual has higher levels of heterozygosity than either of the sampled A mexicanum. Notably, both homozygous and heterozygous SNPs show decreases in frequency overlapping regions of excess heterozygosity in the d/d A mexicanum at chr 4, 5, and 9. Careful examination of signal on chromosome 3 indicates that decreased polymorphism rate is due to the presence of several large gaps in this region

FIGURE 4

Differences in polymorphism frequency and copy number over a 150 Mb interval on chromosome 1. The frequency of fixed homozygous polymorphisms between the d/d strain male and wildtype AGSC female is higher over this region in comparison to the rest of the genome. Heterozygous sites detected from the d/d strain male are more likely to match alleles found in A tigrinum. Notably, depth of sequence coverage analyses reveal that A tigrinum has substantially increased read depth over this region (due to the presence of additional repetitive element copies elsewhere in the A tigrinum genome). Increased copy number is also observed in pooled sequence data from 48 backcross hybrids between A mexicanum and A tigrinum. The top half of the chromosome (roughly the P arm) is shown here - larger chromosomes were split in half to circumvent length limitations when originally submitted to NCBI

FIGURE 5

Identification of alternate haplotypes by PCR. A, Example of CIRBP amplicons showing CIRBP^AGSC homozygotes (single band) and CIRBP^AGSC/CIRBP^dd heterozygotes (two bands). B, Genotype and allele frequencies within a sample of 162 individuals from the Ambystoma Genetic Stock Center

FIGURE 6

Evidence for widespread presence of the colony-like variant of the chromosome 1 polymorphic region. Analysis of polymorphism frequencies from published transcriptome datasets identifies six published studies that contain only animals with the colony-like variant, including one (BioProject: PRJNA354434) that sampled animals from their native population. Three other studies contain animals that are homozygous for the colony-like variant and other animals that are either heterozygous or homozygous for the d/d-like variant. Samples that are homozygous for the colony-like genotypes or possessing d/d-like genotypes are separated into upper and lower panels (respectively) for these three projects. Notably, a second potential variant was observed in two of these studies (PRJNA312389 and PRJNA300706): marked by an asterisk

FIGURE 7

Estimated copy number of repetitive elements in A mexicanum and A tigrinum. Counts of repetitive elements are estimated across the length of each of three consensus repeats that were identified in the chr1P polymorphic region

FIGURE 8

Young repeat families found in the chr1P polymorphic region and other polymorphic regions. A-C, Counts of full-length elements extracted from the A mexicanum assembly, with varying sequence identity with the chr1 consensus A, Harbinger, B, Epsilon, and C, Gypsy. D-F, Phylogenetic trees of D, 500 Harbinger elements, E, 1000 Epsilon elements and F, 400 Gypsy elements. Trees include all elements in polymorphic regions and additional randomly selected members from age classes that are closest to the chr1P consensus (rightmost peaks). Clades with members in the chr1P polymorphic region are highlighted in red

FIGURE 9

Differences in polymorphism frequencies over a 243 megabase interval on chromosome 7. The frequency of heterozygous polymorphisms between the d/d strain male and AGSC individuals is higher over this region in comparison to the rest of the genome

FIGURE 10

Patterns of recombination across two chromosomes. A, chromosome 7 and B, chromosome 1. The relationship between map distance (centiMorgans) and assembly length is shown. The albino introgression region is highlighted in red in panel A and the chr1P polymorphic region is highlighted in red in panel B

FIGURE 11

Differences in polymorphism frequency over a 120 Mb interval on chromosome 4 and a smaller 51 Mb interval on the same chromosome. The frequency of heterozygous polymorphisms between the d/d strain male and AGSC individual is higher over a 120 Mb region (yellow highlight) in comparison to the rest of the genome. By contrast, there are fewer differences between the d/d strain male and A tigrinum over a second interval (pink). The top half of the chromosome (roughly the P arm) is shown here

FIGURE 12

Other regions with an excess of polymorphism. A-B, Regions on A, chr5 and B, chr9 have increased rates of heterozygosity in the d/d strain male. The bottom half of chr5 (roughly the Q arm) is shown here. C, A small region of chr2 with an increased rate of homozygous polymorphism between the d/d strain male and AGSC wildtype female. The bottom half of chr2 (roughly the Q arm) is shown here

FIGURE 13

Assessment of potentially biologically relevant polymorphisms. A-C, Proportion of substitutions resulting in nonsynonymous amino acid changes across chromosomes/chromosome arms and within candidate introgression intervals. D-F, Proportion of substitutions resulting in changes within 5kp upstream of annotated genes. Values are normalized by the density of coding bases, A-C or genes, D-F, annotated to each chromosome or region and are plotted separately for polymorphism classes: A,D, the chr1P region with an excess of homozygous differences; B,E, chr 4P and 7 regions with an excess of heterozygous polymorphism in the AGSC female; C,F, chr 4P, 5Q and 9 regions with an excess of heterozygous polymorphism in the d/d male

FIGURE 14

Evidence for expression of repetitive elements from regeneration and gonadal RNAseq studies. Four genomic intervals are shown for each repeat family, Mapped RNAseq reads are color coded by study: orange, green, and blue