Genome-wide association study to identify genetic loci associated with gastrointestinal nematode resistance in Katahdin sheep

Resistance to gastrointestinal nematodes has previously been shown to be a moderately heritable trait in some breeds of sheep, but the mechanisms of resistance are not well understood. Selection for resistance currently relies upon faecal egg counts (FEC), blood packed cell volumes and FAMACHA visual indicator scores of anaemia. Identifying genomic markers associated with disease resistance would potentially improve the selection process and provide a more reliable means of classifying and understanding the biology behind resistant and susceptible sheep. A GWAS was conducted to identify possible genetic loci associated with resistance to Haemonchus contortus in Katahdin sheep. Forty animals were selected from the top and bottom 10% of estimated breeding values for FEC from a total pool of 641 sires and ram lambs. Samples were genotyped using Applied Biosystems™ Axiom™ Ovine Genotyping Array (50K) consisting of 51 572 SNPs. Following quality control, 46 268 SNPs were included in subsequent analyses. Analyses were conducted using a linear regression model in plink v1.90 and a single-locus mixed model in snp and variation suite . Genome-wide significance was determined by a Bonferroni correction for multiple testing. Using linear regression, loci on chromosomes 2, 3, 16, 23 and 24 were significantly associated at the genome level with FEC estimated breeding values, and we identified a region on chromosome 2 that was significant using both statistical analyses. We suggest a potential role for the gene DIS3L2 for gastrointestinal nematode resistance in Katahdin sheep, although further research is needed to validate these findings.     

Genome‐wide association study reveals novel genes for the ear size in sheep (Ovis aries)

Variations in ear size can be observed in livestock such as sheep; however, the genetic basis of variable ear size in sheep is still poorly understood. To investigate causative genes associated with ear size in sheep, a genome‐wide association study was performed in 115 adult Duolang sheep with different‐sized floppy ears using the Ovine Infinium HD BeadChip. We found 38 significant SNPs at the genome‐wide or chromosome‐wise 5% significance level after Bonferroni correction. The most significant association (P = 1.61 × 10^?6) was found at SNP rs402740419, located in the DCC gene, which plays a critical role in ear development. Also, we observed two additional significant SNPs, rs407891215 in PTPRD and rs407769095 in SOX5, both of which are functionally associated with ear developmental processes. Our results are useful for future sheep breeding and provide insights into the genetic basis of ear size development in sheep and other livestock.     

Sex without sex chromosomes: genetic architecture of multiple loci independently segregating to determine sex ratios in the copepod Tigriopus californicus

Sex‐determining systems are remarkably diverse and may evolve rapidly. Polygenic sex‐determination systems are predicted to be transient and evolutionarily unstable, yet examples have been reported across a range of taxa. Here, we provide the first direct evidence of polygenic sex determination in Tigriopus californicus, a harpacticoid copepod with no heteromorphic sex chromosomes. Using genetically distinct inbred lines selected for male‐ and female‐biased clutches, we generated a genetic map with 39 SNPs across 12 chromosomes. Quantitative trait locus mapping of sex ratio phenotype (the proportion of male offspring produced by an F2 female) in four F2 families revealed six independently segregating quantitative trait loci on five separate chromosomes, explaining 19% of the variation in sex ratios. The sex ratio phenotype varied among loci across chromosomes in both direction and magnitude, with the strongest phenotypic effects on chromosome 10 moderated to some degree by loci on four other chromosomes. For a given locus, sex ratio phenotype varied in magnitude for individuals derived from different dam lines. These data, together with the environmental factors known to contribute to sex determination, characterize the underlying complexity and potential lability of sex determination, and confirm the polygenic architecture of sex determination in T. californicus.     

Reconstructing the plant mitochondrial genome for marker discovery: a case study using Pinus

Whole‐genome‐shotgun (WGS) sequencing of total genomic DNA was used to recover ~1 Mbp of novel mitochondrial (mtDNA) sequence from Pinus sylvestris (L.) and three members of the closely related Pinus mugo species complex. DNA was extracted from megagametophyte tissue from six mother trees from locations across Europe, and 100‐bp paired‐end sequencing was performed on the Illumina HiSeq platform. Candidate mtDNA sequences were identified by their size and coverage characteristics, and by comparison with published plant mitochondrial genomes. Novel variants were identified, and primers targeting these loci were trialled on a set of 28 individuals from across Europe. In total, 31 SNP loci were successfully resequenced, characterizing 15 unique haplotypes. This approach offers a cost‐effective means of developing marker resources for mitochondrial genomes in other plant species where reference sequences are unavailable.     

Confirmation and fine‐mapping of clinical mastitis and somatic cell score QTL in Nordic Holstein cattle

A genome‐wide association study of 2098 progeny‐tested Nordic Holstein bulls genotyped for 36 387 SNPs on 29 autosomes was conducted to confirm and fine‐map quantitative trait loci (QTL) for mastitis traits identified earlier using linkage analysis with sparse microsatellite markers in the same population. We used linear mixed model analysis where a polygenic genetic effect was fitted as a random effect and single SNPs were successively included as fixed effects in the model. We detected 143 SNP‐by‐trait significant associations (P < 0.0001) on 20 chromosomes affecting mastitis‐related traits. Among them, 21 SNP‐by‐trait combinations exceeded the genome‐wide significant threshold. For 12 chromosomes, both the present association study and the previous linkage study detected QTL, and of these, six were in the same chromosomal locations. Strong associations of SNPs with mastitis traits were observed on bovine autosomes 6, 13, 14 and 20. Possible candidate genes for these QTL were identified. Identification of SNPs in linkage disequilibrium with QTL will enable marker‐based selection for mastitis resistance. The candidate genes identified should be further studied to detect candidate polymorphisms underlying these QTL.     

Trait sorting in Daphnia colonising man‐made lakes

1. We used a zooplankton metacommunity to ask how dispersal, genetic drift and selection act to determine the local and regional distributions of trait variation. Since the formation of the lakes 80 years ago, cladoceran species have sorted into local assemblages that cluster by lake depth. Given this species sorting, we ask whether intraspecific variation in an ecologically important phenotypic trait – body size – has sorted as well. 2. We quantified changes in body size through time by measuring ephippia from D. pulicaria, D. dentifera and D. ambigua recovered from sediment cores from two lakes. We then estimated mean body size of contemporary populations of two competing species, Daphnia pulicaria and D. dentifera, in a laboratory common garden experiment. Finally, we used microsatellite loci to characterise genetic diversity and gene flow among local sites in the metacommunity. 3. Body size was variable both within and among years for the three species of Daphnia examined using sediment cores. For two lakes where we examined body size distributions through time, we observed a significant shift in body size of the first species to arrive after colonisation by other Daphnia species, which suggests selection has occurred historically. 4. Despite heritable variation in body size in the laboratory, evidence for trait sorting was only found for D. pulicaria, which was larger in deeper lakes. Mean body size varied among lakes, but did not sort relative to depth for D. dentifera. 5. Microsatellite data indicated that neutral genetic diversity was low in the region; only 27% of the individuals assayed were unique multi‐locus genotypes. We also found significant patterns of isolation by distance for both species. However, population structure was stronger in D. dentifera than in D. pulicaria. Hence, we conclude that a limited number of colonists have successfully invaded this metacommunity, and those genotypes arriving in this new region have experienced significant dispersal limitation among local sites. 6. Overall, while dispersal and selection have clearly led to the development of predictable community assemblages related to depth in this metacommunity, the distribution of phenotypic traits within species can differ substantially even between two trophically similar species. Our results highlight the complex roles of colonisation history, dispersal, selection and stochasticity in determining inter‐ and intra‐specific patterns in metacommunities.     

Genome‐wide association study to identify genomic regions affecting prolificacy in Lori‐Bakhtiari sheep

Several causative mutations in candidate genes affecting prolificacy have been detected in various sheep breeds. A genome‐wide association study was performed on estimated breeding values for litter size in Lori‐Bakhtiari sheep. Prolific ewes with twinning records and others with only singleton records were genotyped using the medium‐density Illumina Ovine SNP50 array. Four single nucleotide polymorphisms (SNPs) associated with litter size were identified on chromosomes 3, 6 and 22. The region on sheep chromosome 3 between 75 739 167 and 75 745 152 bp included two significant SNPs (s52383.1 and OAR3_80038014_X.1) in high linkage disequilibrium with each other. The region that surrounds these SNPs contains a novel putative candidate gene: luteinizing hormone/choriogonadotropin receptor (LHCGR), known to be involved in ovarian steroidogenesis and organism‐specific biosystem pathways in sheep. Known prolificacy genes BMPR1B, BMP15 and GDF9 were not associated with litter size in Lori‐Bakhtiari sheep, suggesting that other biological mechanisms could be responsible for the trait's variation in this breed.     

Genome mapping of kernel characteristics in hard red spring wheat breeding lines

Kernel characteristics, particularly kernel weight, kernel size, and grain protein content, are important components of grain yield and quality in wheat. Development of high performing wheat cultivars, with high grain yield and quality, is a major focus in wheat breeding programs worldwide. Here, we report chromosome regions harboring genes that influence kernel weight, kernel diameter, kernel size distribution, grain protein content, and grain yield in hard red spring wheat breeding lines adapted to the Upper Midwest region of the United States. A genetic linkage map composed of 531 SSR and DArT marker loci spanned a distance of 2,505 cM, covering all 21 chromosomes of wheat. Stable QTL clusters influencing kernel weight, kernel diameter, and kernel size distribution were identified on chromosomes 2A, 5B, and 7A. Phenotypic variation explained by individual QTL at these clusters varied from 5 to 20% depending on the trait. A QTL region on chromosome 2B confers an undesirable pleiotropic effect or a repulsion linkage between grain yield (LOD = 6.7; R ² = 18%) and grain protein content (LOD = 6.2; R ² = 13.3%). However, several grain protein and grain yield QTL independent of each other were also identified. Because some of the QTL identified in this study were consistent across environments, DNA markers will provide an opportunity for increasing the frequency of desirable alleles through marker-assisted selection.     

Sixteen microsatellite loci for the Bufo boreas group

The four species that comprise the Bufo boreas group of toads are critically imperiled in all or portions of their geographical ranges. We present data from 16 microsatellite loci isolated from B. boreas that cross‐amplify in these four species. These markers have proven useful in the analyses of population structure and conservation genetics, and provide a powerful tool for future researchers who seek to understand the conservation genetics of these rare toad species. Polymorphism was assessed for 339 individuals from seven populations representing the four species. All loci were polymorphic (X? = 8, range of four to 19 alleles). Three loci were not in Hardy–Weinberg equilibrium (HWE) in one population, and one of these loci was out of HWE in a second population (P < 0.003 after Bonferroni correction for multiple tests). However, there were no systematic deviations from HWE across all study populations. Small populations in fragmented habitat appear to explain the existing HWE deviations.     

Meta‐analysis reveals lower genetic diversity in overfished populations

While population declines can drive the loss of genetic diversity under some circumstances, it has been unclear whether this loss is a general consequence of overharvest in highly abundant marine fishes. We compiled data from 11 049 loci across 140 species and found that allelic richness was lower in overfished populations within 9 of 12 genera and families. A multiple linear regression showed that allelic richness was on average 12% lower (P < 0.0001) in overharvested populations after accounting for the effects of body size, latitude and other factors. Heterozygosity was on average 2% lower (P = 0.030). Simulations confirmed that these patterns are consistent with a recent bottleneck in abundant species and also showed that our analysis likely underestimates the loss of rare alleles by a factor of two or three. This evidence suggests that overharvest drives the decay of genetic diversity across a wide range of marine fishes. Such reductions of genetic diversity in some of the world's most abundant species may lead to a long‐term impact of fishing on their evolutionary potential, particularly if abundance remains low and diversity continues to decay.     

Genome sequence of M6, a diploid inbred clone of the high‐glycoalkaloid‐producing tuber‐bearing potato species Solanum chacoense,reveals residual heterozygosity

Cultivated potato (Solanum tuberosum L.) is a highly heterozygous autotetraploid that presents challenges in genome analyses and breeding. Wild potato species serve as a resource for the introgression of important agronomic traits into cultivated potato. One key species is Solanum chacoense and the diploid, inbred clone M6, which is self‐compatible and has desirable tuber market quality and disease resistance traits. Sequencing and assembly of the genome of the M6 clone of S. chacoense generated an assembly of 825 767 562 bp in 8260 scaffolds with an N50 scaffold size of 713 602 bp. Pseudomolecule construction anchored 508 Mb of the genome assembly into 12 chromosomes. Genome annotation yielded 49 124 high‐confidence gene models representing 37 740 genes. Comparative analyses of the M6 genome with six other Solanaceae species revealed a core set of 158 367 Solanaceae genes and 1897 genes unique to three potato species. Analysis of single nucleotide polymorphisms across the M6 genome revealed enhanced residual heterozygosity on chromosomes 4, 8 and 9 relative to the other chromosomes. Access to the M6 genome provides a resource for identification of key genes for important agronomic traits and aids in genome‐enabled development of inbred diploid potatoes with the potential to accelerate potato breeding.     

Hybridization and divergence in multi‐species oak (Quercus) communities

Oaks (Quercus: Fagaceae) commonly interbreed yet retain their morphological, genetic and ecological distinctiveness. Post‐zygotic isolation mechanisms, such as ecologically dependent selection on adaptive loci, may therefore limit introgression. To test this hypothesis, we quantified hybridization and genetic divergence across the contact zone of four red oaks (Quercus section Lobatae) in the Great Lakes region of North America using a suite of 259 amplified fragment length polymorphisms and 27 genic and genomic microsatellite markers. First, we identified hybrids using genetic structure analysis and confirmed the reliability of our assignments via simulations. Then, we identified candidate loci for species maintenance with three complementary tests for selection and obtained partial gene sequences linked to an outlier locus and three other loci. We detected evidence of recent hybridization among all species and considerable gene flow between Q. ellipsoidalis and Q. velutina. Overall, c. 20% of Q. velutina had recent ancestry from Q. ellipsoidalis, whereas nearly 30% of Q. ellipsoidalis had a Q. velutina ancestor. Most loci were negligibly to weakly differentiated among species, but two gene‐linked microsatellites deviated significantly from neutral expectations in multiple, complementary outlier tests. Both outlier loci were located in the same 15‐cM bin on an existing Q. robur linkage map, a region under divergent selection in other oak species. Adaptive loci in this highly differentiated genomic region may contribute to ecological divergence among species and limit introgression.     

Genetic differences between wild and hatchery‐bred brown trout (Salmo trutta L.) in single nucleotide polymorphisms linked to selective traits

To study effects from natural selection acting on brown trout in a natural stream habitat compared with a hatchery environment, 3,781 single nucleotide polymorphism (SNP) markers were analyzed in three closely related groups of brown trout (Salmo trutta L.). Autumn (W/0+, n = 48) and consecutive spring (W/1+, n = 47) samples of brown trout individuals belonging to the same cohort and stream were retrieved using electrofishing. A third group (H/1+, n = 48) comprised hatchery‐reared individuals, bred from a mixture of wild parents of the strain of the two former groups and from a neighboring stream. Pairwise analysis of F_ST outliers and analysis under a hierarchical model by means of ARLEQUIN software detected 421 (10.8%) candidates of selection, before multitest correction. BAYESCAN software detected 10 candidate loci, all of which were included among the ARLEQUIN candidate loci. Body length was significantly different across genotypes at 10 candidate loci in the W/0+, at 34 candidate loci in the W/1+ and at 21 candidate loci in the H/1+ group. The W/1+ sample was tested for genotype‐specific body length at all loci, and significant differences were found in 10.6% of all loci, and of these, 14.2% had higher frequency of the largest genotype in the W/1+ sample than in W/0+. The corresponding proportion among the candidate loci of W/1+ was 22.7% with genotype‐specific body length, and 88.2% of these had increased frequency of the largest genotype from W/0+ to W/1+, indicating a linkage between these loci and traits affecting growth and survival under this stream's environmental conditions. Bayesian structuring of all loci, and of the noncandidate loci suggested two (K = 2), alternatively four clusters (K = 4). This differed from the candidate SNPs, which suggested only two clusters. In both cases, the hatchery fish dominated one cluster, and body length of W/1+ fish was positively correlated with membership of one cluster both from the K = 2 and the K = 4 structure. Our analysis demonstrates profound genetic differentiation that can be linked to differential selection on a fitness‐related trait (individual growth) in brown trout living under natural vs. hatchery conditions. Candidate SNP loci linked to genes affecting individual growth were identified and provide important inputs into future mapping of the genetic basis of brown trout body size selection.     

Genome‐wide association analysis identifies quantitative trait loci for growth in a Landrace purebred population

Growth‐related traits are complex and economically important in the livestock industry. The aim of this study was to identify quantitative trait loci (QTL) and the associated positional candidate genes affecting growth in pigs. A genome‐wide association study (GWAS) was performed using the porcine single‐nucleotide polymorphism (SNP) 60K bead chip. A mixed‐effects model and linear regression approach were used for the GWAS. The data used in the study included 490 purebred Landrace pigs. All experimental animals were genotyped with 39 438 SNPs located throughout the pig autosomes. We identified a strong association between a SNP marker on chromosome 16 and body weight at 71 days of age (ALGA0092396, P = 5.35 × 10^?9, Bonferroni adjusted P < 0.05). The SNP marker was located near the genomic region containing IRX4, which encodes iroquois homeobox 4. This SNP marker could be useful in the selective breeding program after validating its effect on other populations.     

Polymorphic microsatellites in largemouth bronze gudgeon (Coreius guichenoti) developed from repeat‐enriched libraries and cross‐species amplifications

Largemouth bronze gudgeon (Coreius guichenoti) is a medium‐sized fish endemic from the upper Yangtze River of China and its survival is threatened by the construction of the Three Gorges Dam. This study reports 20 new polymorphic microsatellites from a repeat‐enriched genomic library with a mean number allele of 5.2, and observed and expected heterozygosities ranging from 0.035 to 1, and from 0.13 to 0.917, respectively. In a cross‐species amplification test, nine of the 37 tested loci were found to be also polymorphic in a congeneric species, brass gudgeon (C. heterodon). In addition, other four loci from common carp (Cyprinus carpio) were also polymorphic in C. guichenoti. Out of these 24 polymorphic microsatellites, only three loci significantly deviated from Hardy–Weinberg equilibrium in the sampled population (P < 0.0025), and all pairwise tests for linkage disequilibrium among loci were nonsignificant after applying sequential Bonferroni correction (P > 0.0026). These novel microsatellites provide sufficient levels of polymorphism for studies on population genetics and conservation in C. guichenoti and its related species.     

Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB‐LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations

RenSeq is a NB‐LRR (nucleotide binding‐site leucine‐rich repeat) gene‐targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB‐LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB‐LRRs and can be accessed through a genome browser that we provide. We compared these NB‐LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ~80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum ‘Heinz 1706’ extended the NB‐LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co‐segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi‐ber2) and S. ruiz‐ceballosii (Rpi‐rzc1), we were able to apply RenSeq successfully to identify markers that co‐segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy‐to‐adapt Galaxy pipelines.     

Identification of polymorphic microsatellite loci in the mud crab Scylla serrata (Brachyura: Portunidae)

Five microsatellite loci are identified and characterized from the genome of Scylla serrata, a widespread and commercially important species of coastal marine crab. The loci were detected by randomly screening for di‐ and tri‐nucleotide repeat units within a partial genomic library developed for the species. The five loci consist of dinucleotide repeats and are both co‐dominant and polymorphic within the species. A sample (N = 36) of S. serrata from one Australian population has an average observed heterozygosity of 0.875 and provides no evidence of either linkage among loci or significant deviation from random mating expectations across loci. PCR products for each of the five loci were also observed from a small sample of three other species within the Scylla genus. These markers may provide genetic information that will be useful for both aquaculture and studies of natural populations of the genus.     

Genetic mapping shows intraspecific variation and transgressive segregation for caterpillar‐induced aphid resistance in maize

Plants in nature have inducible defences that sometimes lead to targeted resistance against particular herbivores, but susceptibility to others. The metabolic diversity and genetic resources available for maize (Zea mays) make this a suitable system for a mechanistic study of within‐species variation in such plant‐mediated interactions between herbivores. Beet armyworms (Spodoptera exigua) and corn leaf aphids (Rhopalosiphum maidis) are two naturally occurring maize herbivores with different feeding habits. Whereas chewing herbivore‐induced methylation of 2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐3‐one glucoside (DIMBOA‐Glc) to form 2‐hydroxy‐4,7‐dimethoxy‐1,4‐benzoxazin‐3‐one glucoside (HDMBOA‐Glc) promotes caterpillar resistance, lower DIMBOA‐Glc levels favour aphid reproduction. Thus, caterpillar‐induced DIMBOA‐Glc methyltransferase activity in maize is predicted to promote aphid growth. To test this hypothesis, the impact of S. exigua feeding on R. maidis progeny production was assessed using seventeen genetically diverse maize inbred lines. Whereas aphid progeny production was increased by prior caterpillar feeding on lines B73, Ki11, Ki3 and Tx303, it decreased on lines Ky21, CML103, Mo18W and W22. Genetic mapping of this trait in a population of B73 × Ky21 recombinant inbred lines identified significant quantitative trait loci on maize chromosomes 1, 7 and 10. There is a transgressive segregation for aphid resistance, with the Ky21 alleles on chromosomes 1 and 7 and the B73 allele on chromosome 10 increasing aphid progeny production. The chromosome 1 QTL coincides with a cluster of three maize genes encoding benzoxazinoid O‐methyltransferases that convert DIMBOA‐Glc to HDMBOA‐Glc. Gene expression studies and benzoxazinoid measurements indicate that S. exigua ‐induced responses in this pathway differentially affect R. maidis resistance in B73 and Ky21.     

Mapping the four‐horned locus and testing the polled locus in three Chinese sheep breeds

Four‐horned sheep are an ideal animal model for illuminating the genetic basis of horn development. The objective of this study was to locate the genetic region responsible for the four‐horned phenotype and to verify a previously reported polled locus in three Chinese breeds. A genome‐wide association study (GWAS) was performed using 34 two‐horned and 32 four‐horned sheep from three Chinese indigenous breeds: Altay, Mongolian and Sishui Fur sheep. The top two significant single nucleotide polymorphisms (SNPs) associated with the four‐horned phenotype were both located in a region spanning positions 132.6 to 132.7 Mb on sheep chromosome 2. Similar locations for the four‐horned trait were previously identified in Jacob, Navajo‐Churro, Damara and Sishui Fur sheep, suggesting a common genetic component underlying the four‐horned phenotype. The two identified SNPs were both downstream of the metaxin 2 (MTX2) gene and the HOXD gene cluster. For the top SNP—OAR2:g.132619300G>A—the strong associations of the AA and AG genotypes with the four‐horned phenotype and the GG genotype with the two‐horned phenotype indicated the dominant inheritance of the four‐horned trait. No significant SNPs for the polled phenotype were identified in the GWAS analysis, and a PCR analysis for the detection of the 1.8‐kb insertion associated with polled sheep in other breeds failed to verify the association with polledness in the three Chinese breeds. This study supports the hypothesis that two different loci are responsible for horn existence and number. This study contributes to the understanding of the molecular regulation of horn development and enriches the knowledge of qualitative traits in domestic animals.     

Ear type in sheep is associated with the MSRB3 locus

Ear morphology is an important determinant of sheep breeds. It includes different variable traits such as ear size and erectness, suggesting a polygenic architecture. Here, we performed a comprehensive genome-wide analysis to identify regions under selection for ear morphology in 515 sheep from 17 breeds fixed for diverse ear phenotypes using 34k SNP genotyping data. GWASs for two ear type traits, size and erectness, revealed a single genome-wide significant association on ovine chromosome 3. The derived marker alleles were enriched in sheep with large and/or floppy ears. The GWAS signal harboured the MSRB3 gene encoding methionine sulphoxide reductase B3, which has already been found to be associated with different ear types in other species. We attempted whole-genome resequencing to identify causal variant(s) within a 1 Mb interval around MSRB3. This experiment excluded major copy number variants in the interval, but failed to identify a compelling candidate causal variant. Fine-mapping suggested that the causal variant for large floppy ears most likely resides in a 175 kb interval downstream of the MSRB3 coding region.