4.4 Linking the LDH enzymes to genes
Contrasting homozygosity for the allozyme locus LDH-1 detected
the sympatric populations in Lake Bunnersjöarna
(Allendorf et al., 1976;
Ryman et al., 1979), and we tried to
identify the LDH-1 gene in the genome and find the sequence
differences that result in the allozyme divergence. We were not fully
successful in this, however. We located two LDH-A loci as
expected from the genome duplication event. One locus was located on
chromosome 7 and the other on chromosome 17. The amino acid composition
of the protein products from these loci suggested the LDH locus on
chromosome 7 to be LDH-1 due to a slightly more positive charge
of the product at this locus as compared to the one on chromosome 17, as
expected from allozyme patterns (Allendorf
et al., 1984). However, our SNP- and gene-wise analyses revealed that
neither copy of LDH-A that we identified in the reference genome
shows a fixed or even strong differentiation between the demes.
When investigating the Pool-seq reads mapping to the two gene copies
visually in the Integrative Genomics Viewer (IGV), we identified a
region in the 3’ UTR of the LDH-A on chromosome 17 that showed
strong divergence, albeit not fixed differences, between the two demes,
which was confirmed by allele frequency calculations (Figures S7, S9).LDH-A is highly regulated at the transcriptional and
posttranscriptional levels (Jungmann,
Huang, & Tian, 1998), and the 3’ UTR region is known to be a critical
determinant for the mRNA stabilizing activity for this gene
(Tian, Huang, Short, Short, & Jungmann,
1998). We hypothesized that the divergence in this region could result
in the null allele for Deme II observed in allozyme data, but this would
imply that LDH-1 is located on chromosome 17 (and not on 7 as
indicated by the amino acid charges). A previous study suggests that
regulatory regions may primarily be involved in divergence between the
two sympatric forms of whitefish – dwarf and normal – in Cliff Lake in
St John River drainage in Maine, USA, since outliers were predominantly
found in non-coding regions within genes
(Hebert, Renaut, & Bernatchez, 2013).
When we examined the 3´ UTR of the LDH-A on chromosome 17 in the
whole-genome sequences from a total of 16 individuals (from 7 lakes,
including 2 individuals from each of the demes) scored for allozyme
genotype in LDH-1, we found that the majority of the individuals
(n =11) showed similar patterns as Deme I in Pool-seq data.
However, one individual from Lake Saxvattnet, and all four individuals
from Lakes Bunnersjöarna (both demes) showed the Deme II Pool-seq
genotype at the 3’ UTR region of chromosome 17 (Figure S9). These
results indicate that LDH-1 expression is a complex feature, and
we were not able to resolve the genomic background to the contrasting
allozyme expressions in the demes. Rather, further studies need to
identify the exact genetic control behind the LDH-1 enzyme in brown
trout. Our results suggest that contrasting homozygosity of an allozyme
(F ST=1) may not result directly from contrasting
homozygosity in a DNA sequence. Identifying the genes and the regulatory
mechanisms underlying these enzymes is particularly challenging in
duplicated genomes.