Linking genotype to phenotype in a changing ocean: inferring the genomic architecture of a blue mussel stress response with genome‐wide association

A key component to understanding the evolutionary response to a changing climate is linking underlying genetic variation to phenotypic variation in stress response. Here, we use a genome‐wide association approach (GWAS) to understand the genetic architecture of calcification rates under simulated climate stress. We take advantage of the genomic gradient across the blue mussel hybrid zone (Mytilus edulis and Mytilus trossulus) in the Gulf of Maine (GOM) to link genetic variation with variance in calcification rates in response to simulated climate change. Falling calcium carbonate saturation states are predicted to negatively impact many marine organisms that build calcium carbonate shells – like blue mussels. We sampled wild mussels and measured net calcification phenotypes after exposing mussels to a ‘climate change’ common garden, where we raised temperature by 3°C, decreased pH by 0.2 units and limited food supply by filtering out planktonic particles >5 μm, compared to ambient GOM conditions in the summer. This climate change exposure greatly increased phenotypic variation in net calcification rates compared to ambient conditions. We then used regression models to link the phenotypic variation with over 170 000 single nucleotide polymorphism loci (SNPs) generated by genotype by sequencing to identify genomic locations associated with calcification phenotype, and estimate heritability and architecture of the trait. We identified at least one of potentially 2–10 genomic regions responsible for 30% of the phenotypic variation in calcification rates that are potential targets of natural selection by climate change. Our simulations suggest a power of 13.7% with our study's average effective sample size of 118 individuals and rare alleles, but a power of >90% when effective sample size is 900.     

The brown coat colour of Coppernecked goats is associated with a non‐synonymous variant at the TYRP1 locus on chromosome 8

The recent development of a goat SNP genotyping microarray enables genome‐wide association studies in this important livestock species. We investigated the genetic basis of the black and brown coat colour in Valais Blacknecked and Coppernecked goats. A genome‐wide association analysis using goat SNP50 BeadChip genotypes of 22 cases and 23 controls allowed us to map the locus for the brown coat colour to goat chromosome 8. The TYRP1 gene is located within the associated chromosomal region, and TYRP1 variants cause similar coat colour phenotypes in different species. We thus considered TYRP1 as a strong positional and functional candidate. We resequenced the caprine TYRP1 gene by Sanger and Illumina sequencing and identified two non‐synonymous variants, p.Ile478Thr and p.Gly496Asp, that might have a functional impact on the TYRP1 protein. However, based on the obtained pedigree and genotype data, the brown coat colour in these goats is not due to a single recessive loss‐of‐function allele. Surprisingly, the genotype distribution and the pedigree data suggest that the ⁴⁹⁶Asp allele might possibly act in a dominant manner. The ⁴⁹⁶Asp allele was present in 77 of 81 investigated Coppernecked goats and did not occur in black goats. This strongly suggests heterogeneity underlying the brown coat colour in Coppernecked goats. Functional experiments or targeted matings will be required to verify the unexpected preliminary findings.     

Host and symbiont genetic determinants of nutritional phenotype in a natural population of the pea aphid

A defining feature of the nutritional ecology of plant sap‐feeding insects is that the dietary deficit of essential amino acids (EAAs) in plant sap is supplemented by EAA‐provisioning microbial symbionts in the insect. Here, we demonstrated substantial variation in the nutritional phenotype of 208 genotypes of the pea aphid Acyrthosiphon pisum collected from a natural population. Specifically, the genotypes varied in performance (larval growth rates) on four test diets lacking the EAAs arginine, histidine and methionine or aromatic EAAs (phenylalanine and tryptophan), relative to the diet containing all EAAs. These data indicate that EAA supply from the symbiotic bacteria Buchnera can meet total aphid nutritional demand for only a subset of the EAA/aphid genotype combinations. We then correlated single nucleotide polymorphisms (SNPs) identified in the aphid and Buchnera genomes by reduced genome sequencing against aphid performance for each EAA deletion diet. This yielded significant associations between performance on the histidine‐free diet and Buchnera SNPs, including metabolism genes predicted to influence histidine biosynthesis. Aphid genetic correlates of performance were obtained for all four deletion diets, with associations on the arginine‐free diet and aromatic‐free diets dominated by genes functioning in the regulation of metabolic and cellular processes. The specific aphid genes associated with performance on different EAA deletion diets are largely nonoverlapping, indicating some independence in the regulatory circuits determining aphid phenotype for the different EAAs. This study demonstrates how variation in the phenotype of associations collected from natural populations can be applied to elucidate the genetic basis of ecologically important traits in systems intractable to traditional forward/reverse genetic techniques.     

A genome‐wide association study in Caucasian women suggests the involvement of HLA genes in the severity of facial solar lentigines

Solar lentigines are a common feature of sun‐induced skin ageing. Little is known, however, about the genetic factors contributing to their development. In this genome‐wide association study, we aimed to identify genetic loci associated with solar lentigines on the face in 502 middle‐aged French women. Nine SNPs, gathered in two independent blocks on chromosome 6, exhibited a false discovery rate below 25% when looking for associations with the facial lentigine score. The first block, in the 6p22 region, corresponded to intergenic SNPs and also exhibited a significant association with forehead lentigines (P = 1.37 × 10^?8). The second block, within the 6p21 HLA region, was associated with decreased HLA‐C expression according to several eQTL databases. Interestingly, these SNPs were also in high linkage disequilibrium with the HLA‐C*0701 allele (r² = 0.95). We replicated an association recently found by GWAS in the IRF4 gene. Finally, a complementary study on 44 selected candidate SNPs revealed novel associations in the MITF gene. Overall, our results point to several mechanisms involved in the severity of facial lentigines, including HLA/immunity and the melanogenesis pathway.     

Genome‐wide association study identifies variants in the CAPN9 gene associated with umbilical hernia in pigs

Pig umbilical hernia (UH) affects pig welfare and brings considerable economic loss to the pig industry. To date, the molecular mechanisms underlying pig UH are still poorly understood. To identify potential loci for susceptibility to this disease, we performed a genome‐wide association study in an Erhualian × Shaziling F₂ intercross population. A total of 45 animals were genotyped using Illumina Porcine SNP60 BeadChips. We observed a SNP (rs80993347) located in the calpain‐9 (CAPN9) gene on Sus scrofa chromosome 14 that was significantly associated with UH (P = 1.97 × 10^?10). Then, we identified a synonymous mutation rs321865883 (g.20164T>C) in exon 10 of the CAPN9 gene that distinguished two affected individuals (CC) from their normal full‐sibs (TC). Finally, quantitative polymerase chain reaction was explored to investigate the mRNA expression profile of the CAPN9 gene in 12 tissues in Yorkshire pigs at different developmental stages (3, 90 and 180 days). CAPN9 showed high expression levels in the gastrointestinal tract at these three growth stages. The results of this study indicate that the CAPN9 gene might be implicated in UH. Further studies are required to establish a role of CAPN9 in pig UH.     

Rainbow trout resistance to bacterial cold water disease: two new quantitative trait loci identified after a natural disease outbreak on a French farm

In rainbow trout farming, Flavobacterium psychrophilum, the causative agent of bacterial cold water disease, is responsible for important economic losses. Resistance to F. psychrophilum is heritable, and several quantitative trait loci (QTL) with moderate effects have been detected, opening up promising perspectives for the genetic improvement of resistance. In most studies however, resistance to F. psychrophilum was assessed in experimental infectious challenges using injection as the infection route, which is not representative of natural infection. Indeed, injection bypasses external barriers, such as mucus and skin, that likely play a protective role against the infection. In this study, we aimed at describing the genetic architecture of the resistance to F. psychrophilum after a natural disease outbreak. In a 2000‐fish cohort, reared on a French farm, 720 fish were sampled and genotyped using the medium‐throughput Axiom? Trout Genotyping Array. Overall mortality at the end of the outbreak was 25%. Genome‐wide association studies were performed under two different models for time to death measured on 706 fish with validated genotypes for 30 060 SNPs. This study confirms the polygenic inheritance of resistance to F. psychrophilum with a few QTL with moderate effects and a large polygenic background, the heritability of the trait being estimated at 0.34. Two new chromosome‐wide significant QTL and three suggestive QTL were detected, each of them explaining between 1% and 4% of genetic variance.     

Finding needles in a genomic haystack: targeted capture identifies clear signatures of selection in a nonmodel plant species

Teasing apart neutral and adaptive genomic processes and identifying loci that are targets of selection can be difficult, particularly for nonmodel species that lack a reference genome. However, identifying such loci and the factors driving selection have the potential to greatly assist conservation and restoration practices, especially for the management of species in the face of contemporary and future climate change. Here, we focus on assessing adaptive genomic variation within a nonmodel plant species, the narrow‐leaf hopbush (Dodonaea viscosa ssp. angustissima), commonly used for restoration in Australia. We used a hybrid‐capture target enrichment approach to selectively sequence 970 genes across 17 populations along a latitudinal gradient from 30°S to 36°S. We analysed 8462 single‐nucleotide polymorphisms (SNPs) for F_ST outliers as well as associations with environmental variables. Using three different methods, we found 55 SNPs with significant correlations to temperature and water availability, and 38 SNPs to elevation. Genes containing SNPs identified as under environmental selection were diverse, including aquaporin and abscisic acid genes, as well as genes with ontologies relating to responses to environmental stressors such as water deprivation and salt stress. Redundancy analysis demonstrated that only a small proportion of the total genetic variance was explained by environmental variables. We demonstrate that selection has led to clines in allele frequencies in a number of functional genes, including those linked to leaf shape and stomatal variation, which have been previously observed to vary along the sampled environmental cline. Using our approach, gene regions subject to environmental selection can be readily identified for nonmodel organisms.     

Red and blond Mangalitza pigs display a signature of divergent directional selection in the SLC45A2 gene

The Mangalitza lard‐type pig breed is well known for its fat appearance and curly hair, and it is mainly distributed in Eastern Europe. Four main lines were created in the nineteenth century by artificial selection: Blond Mangalitza, Black Mangalitza, Swallow‐Belly Mangalitza and Red Mangalitza. The Swallow‐Belly line has a black coat combined with yellow‐blond throat and underbelly. In the current work, we aimed to investigate if the colourations of Mangalitza pigs are genetically determined by one or a few loci whose frequencies have been modified by artificial selection. The results of selection scans, with Hap FLK and BayeScan , and of a GWAS for coat colour highlighted the existence of one region on SSC16 (18–20 Mb) with potential effects on hair pigmentation (Red vs. Blond contrast). The analysis of the gene content of this region allowed us to detect the solute carrier family 45 member 2 (SLC45A2) locus as a candidate gene for this trait. The polymorphism of the SLC45A2 locus has been associated with reduced levels or the absence of melanin in several mammalian species. The genotyping of four missense polymorphisms evidenced that rs341599992:G > A and rs693695020:G > A SNPs are strongly but not fully associated with the red and blond coat colours of Mangalitza pigs, a result that was confirmed by performing a haplotype association test. The near fixation of alternative SLC45A2 genotypes in Red and Blond Mangalitza pigs provides a compelling example of the consequences of a divergent directional selection for coat colour in a domestic species.     

The genetic basis of drought tolerance in the high oil crop Sesamum indicum

Unlike most of the important food crops, sesame can survive drought but severe and repeated drought episodes, especially occurring during the reproductive stage, significantly curtail the productivity of this high oil crop. Genome‐wide association study was conducted for traits related to drought tolerance using 400 diverse sesame accessions, including landraces and modern cultivars. Ten stable QTLs explaining more than 40% of the phenotypic variation and located on four linkage groups were significantly associated with drought tolerance related traits. Accessions from the tropical area harboured higher numbers of drought tolerance alleles at the peak loci and were found to be more tolerant than those from the northern area, indicating a long‐term genetic adaptation to drought‐prone environments. We found that sesame has already fixed important alleles conferring survival to drought which may explain its relative high drought tolerance. However, most of the alleles crucial for productivity and yield maintenance under drought conditions are far from been fixed. This study also revealed that pyramiding the favourable alleles observed at the peak loci is of high potential for enhancing drought tolerance in sesame. In addition, our results highlighted two important pleiotropic QTLs harbouring known and unreported drought tolerance genes such as SiABI4, SiTTM3, SiGOLS1, SiNIMIN1 and SiSAM. By integrating candidate gene association study, gene expression and transgenic experiments, we demonstrated that SiSAM confers drought tolerance by modulating polyamine levels and ROS homeostasis, and a missense mutation in the coding region partly contributes to the natural variation of drought tolerance in sesame.     

Development,validation and genetic analysis of a large soybean SNP genotyping array

Cultivated soybean (Glycine max) suffers from a narrow germplasm relative to other crop species, probably because of under‐use of wild soybean (Glycine soja) as a breeding resource. Use of a single nucleotide polymorphism (SNP) genotyping array is a promising method for dissecting cultivated and wild germplasms to identify important adaptive genes through high‐density genetic mapping and genome‐wide association studies. Here we describe a large soybean SNP array for use in diversity analyses, linkage mapping and genome‐wide association analyses. More than four million high‐quality SNPs identified from high‐depth genome re‐sequencing of 16 soybean accessions and low‐depth genome re‐sequencing of 31 soybean accessions were used to select 180 961 SNPs for creation of the Axiom^® SoyaSNP array. Validation analysis for a set of 222 diverse soybean lines showed that 170 223 markers were of good quality for genotyping. Phylogenetic and allele frequency analyses of the validation set data indicated that accessions showing an intermediate morphology between cultivated and wild soybeans collected in Korea were natural hybrids. More than 90 unanchored scaffolds in the current soybean reference sequence were assigned to chromosomes using this array. Finally, dense average spacing and preferential distribution of the SNPs in gene‐rich chromosomal regions suggest that this array may be suitable for genome‐wide association studies of soybean germplasm. Taken together, these results suggest that use of this array may be a powerful method for soybean genetic analyses relating to many aspects of soybean breeding.     

Metabolome‐genome‐wide association study dissects genetic architecture for generating natural variation in rice secondary metabolism

Plants produce structurally diverse secondary (specialized) metabolites to increase their fitness for survival under adverse environments. Several bioactive compounds for new drugs have been identified through screening of plant extracts. In this study, genome‐wide association studies (GWAS) were conducted to investigate the genetic architecture behind the natural variation of rice secondary metabolites. GWAS using the metabolome data of 175 rice accessions successfully identified 323 associations among 143 single nucleotide polymorphisms (SNPs) and 89 metabolites. The data analysis highlighted that levels of many metabolites are tightly associated with a small number of strong quantitative trait loci (QTLs). The tight association may be a mechanism generating strains with distinct metabolic composition through the crossing of two different strains. The results indicate that one plant species produces more diverse phytochemicals than previously expected, and plants still contain many useful compounds for human applications.     

Breaking RAD: an evaluation of the utility of restriction site‐associated DNA sequencing for genome scans of adaptation

Understanding how and why populations evolve is of fundamental importance to molecular ecology. Restriction site‐associated DNA sequencing (RADseq), a popular reduced representation method, has ushered in a new era of genome‐scale research for assessing population structure, hybridization, demographic history, phylogeography and migration. RADseq has also been widely used to conduct genome scans to detect loci involved in adaptive divergence among natural populations. Here, we examine the capacity of those RADseq‐based genome scan studies to detect loci involved in local adaptation. To understand what proportion of the genome is missed by RADseq studies, we developed a simple model using different numbers of RAD‐tags, genome sizes and extents of linkage disequilibrium (length of haplotype blocks). Under the best‐case modelling scenario, we found that RADseq using six‐ or eight‐base pair cutting restriction enzymes would fail to sample many regions of the genome, especially for species with short linkage disequilibrium. We then surveyed recent studies that have used RADseq for genome scans and found that the median density of markers across these studies was 4.08 RAD‐tag markers per megabase (one marker per 245 kb). The length of linkage disequilibrium for many species is one to three orders of magnitude less than density of the typical recent RADseq study. Thus, we conclude that genome scans based on RADseq data alone, while useful for studies of neutral genetic variation and genetic population structure, will likely miss many loci under selection in studies of local adaptation.     

Association between putative functional variants in the PSMB9 gene and risk of melanoma – re‐analysis of published melanoma genome‐wide association studies

To mine possibly hidden causal single‐nucleotide polymorphisms (SNPs) of melanoma, we investigated the association of SNPs in 76 M/G1 transition genes with melanoma risk using our published genome‐wide association study (GWAS) data set with 1804 melanoma cases and 1026 cancer‐free controls. We found multiple SNPs with P < 0.01 and performed validation studies for 18 putative functional SNPs in PSMB9 in two other GWAS data sets. Two SNPs (rs1351383 and rs2127675) were associated with melanoma risk in the GenoMEL data set (P = 0.013 and 0.004, respectively), but failed in validation using the Australian data set. Genotype–phenotype analysis revealed these two SNPs were significantly correlated with mRNA expression level of PSMB9. Further experiments revealed that SNP rs2071480, which is in high LD with rs1351383 and rs2127675, may have a weak effect on the promoter activity of PSMB9. Taken together, our data suggested that functional variants in PSMB9 may contribute to melanoma susceptibility.     

Genome‐wide analyses suggest parallel selection for universal traits may eclipse local environmental selection in a highly mobile carnivore

Ecological and environmental heterogeneity can produce genetic differentiation in highly mobile species. Accordingly, local adaptation may be expected across comparatively short distances in the presence of marked environmental gradients. Within the European continent, wolves (Canis lupus) exhibit distinct north–south population differentiation. We investigated more than 67‐K single nucleotide polymorphism (SNP) loci for signatures of local adaptation in 59 unrelated wolves from four previously identified population clusters (northcentral Europe n = 32, Carpathian Mountains n = 7, Dinaric‐Balkan n = 9, Ukrainian Steppe n = 11). Our analyses combined identification of outlier loci with findings from genome‐wide association study of individual genomic profiles and 12 environmental variables. We identified 353 candidate SNP loci. We examined the SNP position and neighboring megabase (1 Mb, one million bases) regions in the dog (C. lupus familiaris) genome for genes potentially under selection, including homologue genes in other vertebrates. These regions included functional genes for, for example, temperature regulation that may indicate local adaptation and genes controlling for functions universally important for wolves, including olfaction, hearing, vision, and cognitive functions. We also observed strong outliers not associated with any of the investigated variables, which could suggest selective pressures associated with other unmeasured environmental variables and/or demographic factors. These patterns are further supported by the examination of spatial distributions of the SNPs associated with universally important traits, which typically show marked differences in allele frequencies among population clusters. Accordingly, parallel selection for features important to all wolves may eclipse local environmental selection and implies long‐term separation among population clusters.     

Replicated analysis of the genetic architecture of quantitative traits in two wild great tit populations

Currently, there is much debate on the genetic architecture of quantitative traits in wild populations. Is trait variation influenced by many genes of small effect or by a few genes of major effect? Where is additive genetic variation located in the genome? Do the same loci cause similar phenotypic variation in different populations? Great tits (Parus major) have been studied extensively in long‐term studies across Europe and consequently are considered an ecological ‘model organism’. Recently, genomic resources have been developed for the great tit, including a custom SNP chip and genetic linkage map. In this study, we used a suite of approaches to investigate the genetic architecture of eight quantitative traits in two long‐term study populations of great tits—one in the Netherlands and the other in the United Kingdom. Overall, we found little evidence for the presence of genes of large effects in either population. Instead, traits appeared to be influenced by many genes of small effect, with conservative estimates of the number of contributing loci ranging from 31 to 310. Despite concordance between population‐specific heritabilities, we found no evidence for the presence of loci having similar effects in both populations. While population‐specific genetic architectures are possible, an undetected shared architecture cannot be rejected because of limited power to map loci of small and moderate effects. This study is one of few examples of genetic architecture analysis in replicated wild populations and highlights some of the challenges and limitations researchers will face when attempting similar molecular quantitative genetic studies in free‐living populations.