DOI: 10.5061/dryad.n8pk0p2xs
title: Genome-wide sequencing identifies a thermal tolerance related synonymous mutation in the mussel Mytilisepta virgata

description: The roles of ‘silent’ synonymous mutations for organisms adapting to
 stressfully thermal environments are of fundamental biological and
 ecological interests but poorly understood. To study whether synonymous
 mutations influence the thermal adaptation of animals to heat stress at
 specific microhabitats, a genome-wide genotype-phenotype association
 analysis was carried out in the black mussels Mytilisepta virgata
 inhabiting different microhabitats. A synonymous mutation of
 Ubiquitin-specific Peptidase 15 (MvUSP15) was significantly associated
 with the physiological upper thermal limit of the mussel. The individuals
 carrying GG genotype (the G-type) at the mutant locus owned significantly
 lower heat tolerance compared to the individuals carrying GA and AA
 genotype (the A-type). Furthermore, when heated to sublethal temperature,
 the G-type exhibited higher inter-individual variations in the MvUSP15
 expression, especially for the mussels on the sun-exposed microhabitats.
 Taken together, a synonymous mutation in MvUSP15 can affect the gene
 expression profile and interact with microhabitat heterogeneity to
 influence thermal resistance. This integrative study sheds light on the
 ecological importance of adaptive synonymous mutations as an
 underappreciated genetic buffer against heat stress and emphasizes the
 importance of integrative studies with the consideration of genetic,
 physiological, and environmental heterogeneity at a microhabitat scale for
 evaluating and predicting the impacts of climate change.
description: <strong>(a)
 Microhabitat-scale
 temperature and mortality statistics survey of M. virgata in the
 field</strong> The operative temperatures of <em>M.
 virgata</em> under filed conditions in the Dongshan Swire Marine
 Station (D-SMART), China (23.65°N, 117.49°E) were continuously recorded
 from July to August 2020 using biomimetic thermal loggers (electronic
 supplementary material). Two loggers were deployed separately in one
 sun-exposed and one shaded microhabitat, where both <em>M.
 virgata</em> had an abundance. In two types of microhabitats, the
 99th percentile of all temperatures recorded (<em>T</em>99)
 and the average daily maximum temperature (ADM) were calculated. The
 former was described as ‘acute’ thermal stress, while the latter as a
 measure of ‘chronic’ high-temperature exposure [29]. Mortality of the mussels inhabiting at
 sun-exposed and shaded microhabitat in summer was also investigated using
 quadrat method (electronic supplementary material).
 <strong>(b)
 Genome-wide association
 mapping analysis</strong> ddRADseq data from our previous study
 (BioProject ID: PRJNA517974; BioSample accessions: SAMN10849586 -
 SAMN10849649) and our unpublished <em>M. virgata</em> genome
 were used for identified SNPs in 64 samples (21 collected in April, 23 in
 August, and 20 in December) at genome-wide level. The reads were first
 cleaned using fastp [30], aligned to reference with BWA-MEM algorithm
 [31], and sorted using SAMtools
 [32]. The BAM outputs were then processed via
 gstacks script of package Stacks [33] to create loci and identify SNPs with unpaired
 reads discarded (--rm-unpaired-reads). Candidate loci were then filtered
 and converted to VCF file using populations scripts of package Stacks. A
 locus was retained using a minimum number of populations of three (−p 3),
 a maximum number of missing samples per locus of 20% (−r 0.8) and a minor
 allele frequency of 0.05 (−min_maf 0.05). Filtered loci were functionally
 annotated with the ANNOVA [34]. Finally, the genotype-phenotype association
 analysis using the 48 out of 64 sample’s ABT was used as thermal-tolerant
 phenotypic traits was performed with PLINK [35]. Those loci with missing frequencies greater
 than 0.1 (--geno 0.1) and Hardy-Weinberg equilibrium exact test p-values
 below 1e-6 (--hwe 1e -6) were excluded, and the retained loci were used in
 association analysis. 
 <strong>(c) Sequence
 Analysis and Phylogenetic Tree Construction of M. virgata USP15
 gene</strong> The coding domain sequence (CDS) of <em>M.
 virgata</em> <em>USP15 gene</em>
 (<em>MvUSP15</em>) was verified through molecular experiments
 (electronic supplementary material) and used for subsequent analysis. The
 codon usage bias in MvUSP15 was analyzed using DnaSP [36]. The prediction of <em>MvUSP15
 </em>protein domain architectures based on the deduced amino
 sequence from verified CDS protein was referred to methods described in
 the previous study [37]. The <em>MvUSP15 </em>protein
 structure was predicted using homology modelling, which was performed on
 the SWISS-MODEL website [38]. The <em>MvUSP15</em> protein
 sequence and homologous sequences obtained from GenBank (table S2) were
 aligned using MEGA Ⅹ [39]. Evolutionary analysis and Bayesian phylogenetic
 tree construction were further performed using MCMC methods on BEAST [40].
 Motif structure analysis for every protein sequence was conducted on the
 MEME website [41]. <strong>(d) Expression patterns of MvUSP15
 gene</strong> A total of 187 mussels were collected in the
 sun-exposed and shaded habitats along a ∼500 m shoreline in D-SMART on July 31st, 2021
 and acclimated for over two months as previously described
 [24]. After acclimation, the mussels were randomly
 allocated into heat-treated group or control group. The mussels in the
 heat-treated group were heated at a rate of 6°C h<sup>-1</sup>
 in the air from the acclimation temperature (22°C) to 42°C, the sub-lethal
 temperature for <em>M. virgata</em> [24]; while the control group was kept at 22°C.
 After the heating process, both the heat-treated (n = 135) and control
 group (n = 36) were stored in liquid nitrogen. The total DNA of these
 mussels were extracted and used for genotyping at the focused loci (see
 electronic supplementary material for further details). Then qRT-PCR
 experiment was performed to identify the relative expression level of
 <em>MvUSP15</em>.