Bisexual Attract-and-Kill: A Novel Component of Resistance Management for Transgenic Cotton in Australia.

In Australia, destruction of overwintering pupae of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) has been a key component of mandatory resistance management schemes to constrain development of resistance to Bt toxins in transgenic cotton. This has been accomplished by tillage ('pupae busting'), but it is expensive and can interfere with farming operations. Bisexual attract-and-kill technology based on plant volatile formulations offers a potential alternative in some circumstances. We discuss strategies for using such products and describe two trials in which three applications of an attract-and-kill formulation substantially reduced the numbers of Helicoverpa spp. moths and the numbers of potentially overwintering eggs they laid. One trial tested a curative strategy in which the last generation of moths emerging from transgenic cotton was targeted. The other tested a preventive strategy which aimed to reduce the numbers of eggs in the last generation. The preventive strategy reduced egg numbers by about 90% and is now included as an optional alternative to pupae busting in resistance management strategies for Australian cotton. It is limited to fields which have not been defoliated prior to 31 March and was developed to be used primarily in southern New South Wales. In the 2020-2021 cotton season, it was adopted on approximately 60% of the eligible cotton area. We describe the process whereby the strategy was developed in collaboration with the transgenic technology provider, supported by the cotton industry, and approved by the regulatory authority.

Strip spraying delays pyrethroid resistance in the redlegged earth mite, Halotydeus destructor: a novel refuge strategy.

BACKGROUND: Pesticide resistance has seen control options for the redlegged earth mite (RLEM), Halotydeus destructor, dwindle for Australian grain farmers. The recent discovery of high recessiveness for pyrethroid resistance in RLEM provided an opportunity to examine the feasibility of a refuge strategy to slow the evolution of resistance. Unlike lepidopterous pests in Bt crops, where refuge strategies are routinely practiced, RLEM is a slow-moving pest, which will impact the design of susceptible refuges. RESULTS: Firstly, we confirmed the pyrethroid resistant allele is recessive to the susceptible (wildtype) allele (in terms of resistance) across spatially separated Australian populations. Secondly, we demonstrated that a small, localized resistant mite population can revert to susceptibility at field relevant scales and conditions. Next, we used a simulation modelling approach to design a practical refuge strategy to maintain susceptibility to pyrethroids in populations with a low incidence of resistance. Certain configurations (e.g. a pesticide strip width of 50 m and refuge spacing of 10 m) maintained low levels of resistance across a 10-year time horizon, with lower mite abundance and minimal yield loss. A larger refuge proportion did not always delay resistance, and, under certain conditions, increased resistance frequency. CONCLUSION: Strip spraying to maintain refuges can be readily incorporated into RLEM management programs where sprayer widths in commercial cropping contexts are typically between 20-40 m. A refuge approach to RLEM management that uses strip spraying may enhance long term control options in the absence of new chemical registrations but will now require field validation. © 2021 Society of Chemical Industry.

Lifecycle of the invasive omnivore, Forficula auricularia, in Australian grain growing environments.

BACKGROUND: The European earwig, Forficula auricularia (L.) (Dermaptera: Forficulidae), is regarded as an important beneficial in many orchard environments but has the potential to be a plant pest in other systems, including to grain crops. Due to its agricultural importance, the lifecycle of F. auricularia has been widely studied in North America and Europe. However, much less is known in the southern hemisphere, including Australia where F. auricularia has been present for over 170 years. RESULTS: To elucidate the lifecycle of F. auricularia, we monitored five sites in grain-growing areas of southern Australia using two different trap types. Adults were found year-round, but most prevalent from late-spring to mid-winter. First instars were typically found from mid to late winter. Second, third and fourth instars occurred from winter through to late spring. The seasonal development of F. auricularia in Australia may be much earlier than observed in comparable North American studies. Degree day modelling highlights variation in development times across the active season for F. auricularia across our sites. CONCLUSION: Forficula auricularia is well adapted to the Australian grain growing environments. The timing of egg hatching aligns closely with crop emergence, juveniles then develop alongside the crop, and adult development occurs by harvest time in late spring. These findings are important given that many of these crops (canola, lucerne, pulses) are vulnerable to attack by F. auricularia during emergence and development. They also suggest a phenotypic capacity of this species to adapt different phenology after introduction into a novel environment. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Morphological and Molecular Analysis of Australian Earwigs (Dermaptera) Points to Unique Species and Regional Endemism in the Anisolabididae Family.

Dermaptera (earwigs) from the Anisolabididae family may be important for pest control but their taxonomy and status in Australia is poorly studied. Here we used taxonomic information to assess the diversity of southern Australian Anisolabididae and then applied cox1 barcodes as well as additional gene fragments (mitochondrial and nuclear) to corroborate classification and assess the monophyly of the putative genera. Anisolabididae morphospecies fell into two genera, Anisolabis Fieber and Gonolabis Burr, based on paramere morphology. Combinations of paramere and forceps morphology distinguished seven morphospecies, which were further supported by morphometric analyses. The morphospecies were corroborated by barcode data; all showed within-species genetic distance < 4% and between-species genetic distance > 10%. Molecular phylogenies did not support monophyly of putative genera nor clades based on paramere shape, instead pointing to regional clades distinguishable by forceps morphology. This apparent endemism needs to be further tested by sampling of earwig diversity outside of agricultural production regions but points to a unique regional insect fauna potentially important in pest control.

Climate, human influence and the distribution limits of the invasive European earwig, Forficula auricularia, in Australia.

BACKGROUND: By modelling species-environment relationships of pest species, it is possible to understand potential limits to their distributions when they invade new regions, and their likely continued spread. The European earwig, Forficula auricularia, is a non-native invasive species in Australia that has been in the country for over 170 years. However, in the last few decades it has invaded new areas. Unlike in other countries, F. auricularia is a pest species of grain production in Australia. In this study we detail the Australian distribution of this species, adding new samples focused around grain-growing regions. Using this information, we build global species distribution models for F. auricularia to better understand species-environment relationships. RESULTS: Our models indicate that the distribution of F. auricularia is strongly associated with temperate through to semi-arid environments, a high winter rainfall and pronounced temperature seasonality. We identified regions that hold suitable, but as yet vacant, niche space for Australian populations, suggesting further potential for range expansion. Beyond climate, an index describing human influence on the landscape was important to understand the distribution limits of this pest. We identified regions where there was suitable climate space, but which F. auricularia has not occupied, probably due to low levels of human impact. CONCLUSION: Modelling the global distribution of a non-native pest species aided understanding of the regional distribution limits within Australia and highlighted the usefulness of human impact measures for modelling globally invasive insect species. © 2018 Society of Chemical Industry.

Polymorphism at expressed DQ and DR loci in five common equine MHC haplotypes.

The polymorphism of major histocompatibility complex (MHC) class II DQ and DR genes in five common equine leukocyte antigen (ELA) haplotypes was determined through sequencing of mRNA transcripts isolated from lymphocytes of eight ELA homozygous horses. Ten expressed MHC class II genes were detected in horses of the ELA-A3 haplotype carried by the donor horses of the equine bacterial artificial chromosome (BAC) library and the reference genome sequence: four DR genes and six DQ genes. The other four ELA haplotypes contained at least eight expressed polymorphic MHC class II loci. Next generation sequencing (NGS) of genomic DNA of these four MHC haplotypes revealed stop codons in the DQA3 gene in the ELA-A2, ELA-A5, and ELA-A9 haplotypes. Few NGS reads were obtained for the other MHC class II genes that were not amplified in these horses. The amino acid sequences across haplotypes contained locus-specific residues, and the locus clusters produced by phylogenetic analysis were well supported. The MHC class II alleles within the five tested haplotypes were largely non-overlapping between haplotypes. The complement of equine MHC class II DQ and DR genes appears to be well conserved between haplotypes, in contrast to the recently described variation in class I gene loci between equine MHC haplotypes. The identification of allelic series of equine MHC class II loci will aid comparative studies of mammalian MHC conservation and evolution and may also help to interpret associations between the equine MHC class II region and diseases of the horse.

Heritability of Recurrent Exertional Rhabdomyolysis in Standardbred and Thoroughbred Racehorses Derived From SNP Genotyping Data.

Recurrent exertional rhabdomyolysis (RER) in Thoroughbred and Standardbred racehorses is characterized by episodes of muscle rigidity and cell damage that often recur upon strenuous exercise. The objective was to evaluate the importance of genetic factors in RER by obtaining an unbiased estimate of heritability in cohorts of unrelated Thoroughbred and Standardbred racehorses. Four hundred ninety-one Thoroughbred and 196 Standardbred racehorses were genotyped with the 54K or 74K SNP genotyping arrays. Heritability was calculated from genome-wide SNP data with a mixed linear and Bayesian model, utilizing the standard genetic relationship matrix (GRM). Both the mixed linear and Bayesian models estimated heritability of RER in Thoroughbreds to be approximately 0.34 and in Standardbred racehorses to be approximately 0.45 after adjusting for disease prevalence and sex. To account for potential differences in the genetic architecture of the underlying causal variants, heritability estimates were adjusted based on linkage disequilibrium weighted kinship matrix, minor allele frequency and variant effect size, yielding heritability estimates that ranged between 0.41-0.46 (Thoroughbreds) and 0.39-0.49 (Standardbreds). In conclusion, between 34-46% and 39-49% of the variance in RER susceptibility in Thoroughbred and Standardbred racehorses, respectively, can be explained by the SNPs present on these 2 genotyping arrays, indicating that RER is moderately heritable. These data provide further rationale for the investigation of genetic mutations associated with RER susceptibility.

Developing Bisexual Attract-and-Kill for Polyphagous Insects: Ecological Rationale versus Pragmatics.

We discuss the principles of bisexual attract-and-kill, in which females as well as males are targeted with an attractant, such as a blend of plant volatiles, combined with a toxicant. While the advantages of this strategy have been apparent for over a century, there are few products available to farmers for inclusion in integrated pest management schemes. We describe the development, registration, and commercialization of one such product, Magnet(®), which was targeted against Helicoverpa armigera and H. punctigera in Australian cotton. We advocate an empirical rather than theoretical approach to selecting and blending plant volatiles for such products, and emphasise the importance of field studies on ecologically realistic scales of time and space. The properties required of insecticide partners also are discussed. We describe the studies that were necessary to provide data for registration of the Magnet(®) product. These included evidence of efficacy, including local and area-wide impacts on the target pest, non-target impacts, and safety for consumers and applicators. In the decade required for commercial development, the target market for Magnet(®) has been greatly reduced by the widespread adoption of transgenic insect-resistant cotton in Australia. We discuss potential applications in resistance management for transgenic cotton, and for other pests in cotton and other crops.

Non-Target Impacts of an Attract-and-Kill Formulation Based on Plant Volatiles: Responses of some Generalist Predators.

Responses of non-target insects to a blend of plant volatiles used as components in an attract-and-kill formulation for Helicoverpa spp. (Lepidoptera: Noctuidae) were studied in an Australian cotton field. Two experiments, one involving suction sampling during the day and the other at night, were conducted. Rows that had been treated with the volatile blend, with no added insecticide, were sampled with a large suction sampler 18, 42, and 85 h (day experiment) and 6, 30, and 78 h (night experiment) after treatment. Rows located 5, 10, 20, and 300 m away from the treated row were similarly sampled. Of seven generalist predators, only one accumulated on the treated rows compared to the untreated rows. Of the other six, five were found in lower numbers on the treated rows, and for one no significant effects were detected. Compared to pre-spray baseline levels, numbers of several taxa increased across the whole field after spraying, suggesting area-wide attraction, but localized responses to the treated rows were weak, and apparent repellence was more common than attraction. We suggest that attract-and-kill with plant volatiles should have minimal effects on populations of these predators, and is likely to be compatible with integrated pest management.

Morphological traits: predictable responses to macrohabitats across a 300 km scale.

Species traits may provide a short-cut to predicting generalities in species turnover in response to environmental change, particularly for poorly known taxa. We ask if morphological traits of assemblages respond predictably to macrohabitats across a large scale. Ant assemblages were collected at nine paired pasture and remnant sites from within three areas along a 300 km distance. We measured ten functional morphological traits for replicate individuals of each species. We used a fourth corner model to test associations between microhabitat variables, macrohabitats (pastures and remnants) and traits. In addition, we tested the phylogenetic independence of traits, to determine if responses were likely to be due to filtering by morphology or phylogeny. Nine of ten traits were predicted by macrohabitat and the majority of these traits were independent of phylogeny. Surprisingly, microhabitat variables were not associated with morphological traits. Traits which were associated with macrohabitats were involved in locomotion, feeding behaviour and sensory ability. Ants in remnants had more maxillary palp segments, longer scapes and wider eyes, while having shorter femurs, smaller apical mandibular teeth and shorter Weber's lengths. A clear relationship between traits and macrohabitats across a large scale suggests that species are filtered by coarse environmental differences. In contrast to the findings of previous studies, fine-scale filtering of morphological traits was not apparent. If such generalities in morphological trait responses to habitat hold across even larger scales, traits may prove critical in predicting the response of species assemblages to global change.

Accumulating mutations in series of haplotypes at the KIT and MITF loci are major determinants of white markings in Franches-Montagnes horses.

Coat color and pattern variations in domestic animals are frequently inherited as simple monogenic traits, but a number are known to have a complex genetic basis. While the analysis of complex trait data remains a challenge in all species, we can use the reduced haplotypic diversity in domestic animal populations to gain insight into the genomic interactions underlying complex phenotypes. White face and leg markings are examples of complex traits in horses where little is known of the underlying genetics. In this study, Franches-Montagnes (FM) horses were scored for the occurrence of white facial and leg markings using a standardized scoring system. A genome-wide association study (GWAS) was performed for several white patterning traits in 1,077 FM horses. Seven quantitative trait loci (QTL) affecting the white marking score with p-values p≤10(-4) were identified. Three loci, MC1R and the known white spotting genes, KIT and MITF, were identified as the major loci underlying the extent of white patterning in this breed. Together, the seven loci explain 54% of the genetic variance in total white marking score, while MITF and KIT alone account for 26%. Although MITF and KIT are the major loci controlling white patterning, their influence varies according to the basic coat color of the horse and the specific body location of the white patterning. Fine mapping across the MITF and KIT loci was used to characterize haplotypes present. Phylogenetic relationships among haplotypes were calculated to assess their selective and evolutionary influences on the extent of white patterning. This novel approach shows that KIT and MITF act in an additive manner and that accumulating mutations at these loci progressively increase the extent of white markings.

Assessing insect responses to climate change: What are we testing for? Where should we be heading?

To understand how researchers are tackling globally important issues, it is crucial to identify whether current research is comprehensive enough to make substantive predictions about general responses. We examined how research on climate change affecting insects is being assessed, what factors are being tested and the localities of studies, from 1703 papers published between 1985 and August 2012. Most published research (64%) is generated from Europe and North America and being dedicated to core data analysis, with 29% of the studies analysed dedicated to Lepidoptera and 22% Diptera: which are well above their contribution to the currently identified insect species richness (estimated at 13% and 17% respectively). Research publications on Coleoptera fall well short of their proportional contribution (19% of publications but 39% of insect species identified), and to a lesser extent so do Hemiptera, and Hymenoptera. Species specific responses to changes in temperature by assessing distribution/range shifts or changes in abundance were the most commonly used methods of assessing the impact of climate change on insects. Research on insects and climate change to date is dominated by manuscripts assessing butterflies in Europe, insects of economic and/or environmental concern in forestry, agriculture, and model organisms. The research on understanding how insects will respond to a rapidly changing climate is still in its infancy, but the current trends of publications give a good basis for how we are attempting to assess insect responses. In particular, there is a crucial need for broader studies of ecological, behavioural, physiological and life history responses to be addressed across a greater range of geographic locations, particularly Asia, Africa and Australasia, and in areas of high human population growth and habitat modification. It is still too early in our understanding of taxa responses to climate change to know if charismatic taxa, such as butterflies, or disease vectors, including Diptera, can be used as keystone taxa to generalise other insect responses to climate change. This is critical as the basic biology of most species is still poorly known, and dominant, well studied taxa may show variable responses to climate change across their distribution due to regional biotic and abiotic influences. Indeed identifying if insect responses to climate change can be generalised using phylogeny, functional traits, or functional groups, or will populations and species exhibit idiosyncratic responses, should be a key priority for future research.

Genetic diversity in the modern horse illustrated from genome-wide SNP data.

Horses were domesticated from the Eurasian steppes 5,000-6,000 years ago. Since then, the use of horses for transportation, warfare, and agriculture, as well as selection for desired traits and fitness, has resulted in diverse populations distributed across the world, many of which have become or are in the process of becoming formally organized into closed, breeding populations (breeds). This report describes the use of a genome-wide set of autosomal SNPs and 814 horses from 36 breeds to provide the first detailed description of equine breed diversity. F(ST) calculations, parsimony, and distance analysis demonstrated relationships among the breeds that largely reflect geographic origins and known breed histories. Low levels of population divergence were observed between breeds that are relatively early on in the process of breed development, and between those with high levels of within-breed diversity, whether due to large population size, ongoing outcrossing, or large within-breed phenotypic diversity. Populations with low within-breed diversity included those which have experienced population bottlenecks, have been under intense selective pressure, or are closed populations with long breed histories. These results provide new insights into the relationships among and the diversity within breeds of horses. In addition these results will facilitate future genome-wide association studies and investigations into genomic targets of selection.

Genome-wide analysis reveals selection for important traits in domestic horse breeds.

Intense selective pressures applied over short evolutionary time have resulted in homogeneity within, but substantial variation among, horse breeds. Utilizing this population structure, 744 individuals from 33 breeds, and a 54,000 SNP genotyping array, breed-specific targets of selection were identified using an F(ST)-based statistic calculated in 500-kb windows across the genome. A 5.5-Mb region of ECA18, in which the myostatin (MSTN) gene was centered, contained the highest signature of selection in both the Paint and Quarter Horse. Gene sequencing and histological analysis of gluteal muscle biopsies showed a promoter variant and intronic SNP of MSTN were each significantly associated with higher Type 2B and lower Type 1 muscle fiber proportions in the Quarter Horse, demonstrating a functional consequence of selection at this locus. Signatures of selection on ECA23 in all gaited breeds in the sample led to the identification of a shared, 186-kb haplotype including two doublesex related mab transcription factor genes (DMRT2 and 3). The recent identification of a DMRT3 mutation within this haplotype, which appears necessary for the ability to perform alternative gaits, provides further evidence for selection at this locus. Finally, putative loci for the determination of size were identified in the draft breeds and the Miniature horse on ECA11, as well as when signatures of selection surrounding candidate genes at other loci were examined. This work provides further evidence of the importance of MSTN in racing breeds, provides strong evidence for selection upon gait and size, and illustrates the potential for population-based techniques to find genomic regions driving important phenotypes in the modern horse.

A high density SNP array for the domestic horse and extant Perissodactyla: utility for association mapping, genetic diversity, and phylogeny studies.

An equine SNP genotyping array was developed and evaluated on a panel of samples representing 14 domestic horse breeds and 18 evolutionarily related species. More than 54,000 polymorphic SNPs provided an average inter-SNP spacing of ∼43 kb. The mean minor allele frequency across domestic horse breeds was 0.23, and the number of polymorphic SNPs within breeds ranged from 43,287 to 52,085. Genome-wide linkage disequilibrium (LD) in most breeds declined rapidly over the first 50-100 kb and reached background levels within 1-2 Mb. The extent of LD and the level of inbreeding were highest in the Thoroughbred and lowest in the Mongolian and Quarter Horse. Multidimensional scaling (MDS) analyses demonstrated the tight grouping of individuals within most breeds, close proximity of related breeds, and less tight grouping in admixed breeds. The close relationship between the Przewalski's Horse and the domestic horse was demonstrated by pair-wise genetic distance and MDS. Genotyping of other Perissodactyla (zebras, asses, tapirs, and rhinoceros) was variably successful, with call rates and the number of polymorphic loci varying across taxa. Parsimony analysis placed the modern horse as sister taxa to Equus przewalski. The utility of the SNP array in genome-wide association was confirmed by mapping the known recessive chestnut coat color locus (MC1R) and defining a conserved haplotype of ∼750 kb across all breeds. These results demonstrate the high quality of this SNP genotyping resource, its usefulness in diverse genome analyses of the horse, and potential use in related species.

Accurate determination of phenotypic information from historic thoroughbred horses by single base extension.

Historic DNA data have the potential to identify phenotypic information otherwise invisible in the historical, archaeological and palaeontological record. In order to determine whether a single nucleotide polymorphism typing protocol based on single based extension (SNaPshot™) could produce reliable phenotypic data from historic samples, we genotyped three coat colour markers for a sample of historic Thoroughbred horses for which both phenotypic and correct genotypic information were known from pedigree information in the General Stud Book. Experimental results were consistent with the pedigrees in all cases. Thus we demonstrate that historic DNA techniques can produce reliable phenotypic information from museum specimens.

A cis-acting regulatory mutation causes premature hair graying and susceptibility to melanoma in the horse.

In horses, graying with age is an autosomal dominant trait associated with a high incidence of melanoma and vitiligo-like depigmentation. Here we show that the Gray phenotype is caused by a 4.6-kb duplication in intron 6 of STX17 (syntaxin-17) that constitutes a cis-acting regulatory mutation. Both STX17 and the neighboring NR4A3 gene are overexpressed in melanomas from Gray horses. Gray horses carrying a loss-of-function mutation in ASIP (agouti signaling protein) had a higher incidence of melanoma, implying that increased melanocortin-1 receptor signaling promotes melanoma development in Gray horses. The Gray horse provides a notable example of how humans have cherry-picked mutations with favorable phenotypic effects in domestic animals.

L-2-hydroxyglutaric aciduria: characterisation of the molecular defect in a spontaneous canine model.

l-2-hydroxyglutaric aciduria (l-2-HGA) is a neurometabolic disorder that produces a variety of clinical neurological deficits, including psychomotor retardation, seizures and ataxia. The biochemical hallmark of l-2-HGA is the accumulation of l-2-hydroxyglutaric acid (l-2-HG) in cerebrospinal fluid, plasma and urine. Mutations within the gene L2HGDH (Entrez Gene ID 79944) on chromosome 14q22 encoding L-2-hydroxyglutaric acid dehydrogenase have recently been shown to cause l-2-HGA in humans. Using a candidate gene approach in an outbred pet dog population segregating l-2-HGA, the causal molecular defect was identified in the canine homologue of L2HGDH and characterised. DNA sequencing and pedigree analysis indicate a common founder effect in the canine model. The canine model shares many of the clinical and MRI features of the disease in humans and represents a valuable resource as a spontaneous model of l-2-HGA.

Characterisation of the genomic canine Fgf8 locus and screen for genetic variants in 4 dogs with different face types.

We are investigating the genetic basis of morphological differences in skull shape between domestic dogs of different breeds using a candidate gene approach to identify genes involved in the genetic regulation. One such candidate is Fgf8. Fgf8 is a signalling molecule important in the embryonic development and patterning of the craniofacial region. Mice conditional null for the expression of Fgf8 after E9.5 have a short foreface and a wide skull (Trumpp et al. 1999). Using a combination of bioinformatics and PCR cloning, we have characterised the genomic loci of the canine Fgf8 gene. Like the mouse homologue, it is composed of six exons and we also predict that like the mouse, there are eight alternative isoforms that are generated by alternative splicing events. We have identified a short 200 bp sequence upstream of the Fgf8 gene that is highly conserved between species and have predicted putative transcription factor binding sites using the Transfac database. Genetic analysis of 4 dogs with different skull types identified genetic variation. None of the variants however, were predicted to have any functional significance.

Exclusion of linkage of the RYR1, CACNA1S, and ATP2A1 genes to recurrent exertional rhabdomyolysis in Thoroughbreds.

OBJECTIVE: To determine whether there was genetic linkage between the recurrent exertional rhabdomyolysis (RER) trait in Thoroughbred horse pedigrees and DNA markers in genes (the sarcoplasmic reticulum calcium release channel [RYR1] gene, the sarcoplasmic reticulum calcium ATPase [ATP2A1] gene, and the transverse tubule dihydropyridine receptor-voltage sensor [CACNA1S] gene) that are important in myoplasmic calcium regulation. ANIMALS: 34 horses in the University of Minnesota RER resource herd and 62 Thoroughbreds from 3 families of Thoroughbreds outside of the university in which RER-affected status was assigned after 2 or more episodes of ER had been observed. PROCEDURES: Microsatellite DNA markers from the RYR1, ATP2A1, and CACNA1S gene loci on equine chromosomes 10, 13, and 30 were identified. Genotypes were obtained for all horses in the 4 families affected by RER, and data were used to test for linkage of these 3 loci to the RER phenotype. RESULTS: Analysis of the RYR1, CACNA1S, and ATP2A1 microsatellites excluded a link between those markers and the RER trait. CONCLUSIONS AND CLINICAL RELEVANCE: It is likely that the heritable alterations in muscle contractility that are characteristic of RER are caused by a gene that is not yet known to cause related muscle disease in other species.