Affected animals and carriers were all derived from the original Western Australian flock of OCPMD sheep . Disease status was scored based upon the following criteria: (1) noticeably stiffened gait in the hind limbs, leading to reduced ability to run effectively (in comparison to unaffected individuals), (2) skeletal muscle tissue wasting in affected muscle groups with adipose replacement, (3) presence of nemaline bodies in affected muscle tissues at biopsy, and (4) raised creatine kinase at rest, and particularly after exercise (in comparison to unaffected individuals). ‘Affected’ individuals could be descended from: (1) two affected individuals (100% chance), (2) an affected and a carrier, along with phenotypic presentation of disease pathology (50% chance), or (3) from two known carrier individuals, along with phenotypic presentation of disease pathology (25% chance). ‘Carrier’ individuals were descendants of either an affected and a carrier (50% chance), or from an affected and an unaffected individual (100% chance). ‘Wild-type’ individuals did not show any clinical signs of disease and were descended either from two animals outside the directed pedigree (close to 100% chance) or from two carrier individuals (25% chance). A full pedigree of the sheep flock analysed in the present study may be found in Additional File 1: Fig S1.
Animal husbandry and care
Sheep were housed outdoors in 0.5–1.0 acre paddocks exposed to ambient weather, with natural shelter (trees). Wild-type, carrier and affected sheep were all housed together in small sex-specific groups of 2–15 sheep. Sheep were maintained on a weighed ration of commercial ewe and lamb pellets (17.2% crude protein) along with oaten chaff and a loose sheep mineral mix. For pregnant and lactating ewes, mineral mix was supplemented with di-calcium phosphate. They also had access to a variety of grass and legume species in the paddock. Oaten hay was provided during times of low pasture availability, and Lucerne hay was provided to late pregnant ewes, at lambing, and until weaning time.
Sheep were maintained using industry best practice procedures. Lambs were vaccinated against Caseous Lymphadenitis and five clostridial diseases at 4 and 8 weeks of age and then annually (Glanvac 6, Zoetis Australia Pty Ltd); contagious ecthyma once as lambs (Scabigard, Zoetis Australia Pty Ltd); and Ovine Johne’s Disease once as lambs (Gudair, Zoetis Australia Pty Ltd). Individual faecal egg counts were performed at regular intervals throughout the year, and individual sheep were administered an anthelmintic (rotating drugs) if indicated by a high egg count. A preventative off-shears lice treatment (Spinosad, Extinosad Pour On, Elanco, Australia) was applied at shearing time. The sheep were weighed monthly and body condition scored on a weekly basis. Low stress sheep handling techniques were used and long-distance walks during hot weather were avoided.
Breeding was performed either by timed mating with natural sires, or by artificial insemination and embryo transfer (Genstock Pty Ltd, Kojonup, Western Australia). Oestrous cycle in ewes was synchronized using a controlled internal drug release device. Rams used for natural mating wore breeding harnesses in order to record mating dates. Affected rams had no apparent issues with natural mating, although breeding groups were housed in small yards to reduce the distance to ewes, and the ratio of ewes to rams was 4:1. Ewe lambs were not naturally mated until at least 12 months old. One month prior to expected lambing date, ewes were moved to a paddock with additional shelter. As lambs were born, the ewe and lamb were placed in individual temporary pens (lambing ‘jugs’) for 2–7 days to ensure good maternal behaviour and to allow close monitoring. Lambs were weighed and tagged 1–2 days after birth for identification purposes and released with the ewe into the paddock. Lambs had access to supplemental feed (alongside their mothers) from day one. Lambs were weaned at 3–4 months old, at which point they were weighed weekly for 1–2 months to ensure good growth.
SNP array and linkage analysis
Genomic DNA from 14 affected sheep and 6 carriers were analysed on an OvineSNP50 BeadChip (Illumina, USA). Typed SNPs that passed quality control standards were output in PLINK format (.ped) alongside a genomic map position of each SNP (.map). As existing homozygosity callers function off human datasets, homozygosity mapping was performed using a custom perl script. Homozygous region runs that were present only in carriers were discounted from further analyses. Runs shared between carriers and affected sheep were considered as potential candidates but were given less weight than runs present only in affected individuals. Association mapping was performed using PLINK , although the power of these tests was limited due to the small sample size, relatively sparse genome coverage and the inability to test relatedness of individuals. Linkage analysis was performed using the Merlin software package .
Whole genome sequencing
Illumina whole genome sequencing was performed on two affected sheep at 50× coverage by the service provider (Broad Institute). Reads were aligned to the Ovis aries (Texel sheep) reference genome, version 3.1 (Oar v3.1)  and variants called using the Genome Analysis Toolkit (GATK v3.5) . Homozygous variants were extracted from each variant call format (vcf) file, and hard filtering used to exclude low quality calls (quality of depth, QD > 20; depth, DP > 10). A subset of around 4 million variants common to both affected sheep was extracted and annotated using Ensembl Variant Effect Predictor (VEP, release 85).
Variants were subsequently filtered to include only those within exons or introns of known protein-coding genes. Low coverage (5×) Proton™ WGS data from an additional four affected and eight unaffected, unrelated Merino sheep (wild-type “controls”) were used to narrow down the list of potential disease-causing variants. As poor coverage limited accuracy and zygosity calling, these data were used only to support or refute the potential pathogenicity of high-quality homozygous variants identified in the two Illumina WGS datasets. A pipeline consisting of samtools , vt , and in-house python scripts was used to perform these analyses.
RT-PCR and DNA sequencing
To extract RNA, 20–30 µg cryopreserved sheep skeletal muscle tissue was homogenized in 300 µL Buffer RLT using a mini-bead beater (BioSpec Products, USA) at maximum oscillations per min in 30 s intervals. Total RNA was isolated using a RNeasy fibrous tissue mini kit with on-column DNase I digestion (QIAGEN, USA) and cDNA generated using the SuperScript III first-strand synthesis system (Thermo Fisher, USA). Standard RT-PCR reactions (25 µL) comprised: 1× GoTaq G2 master mix (Promega, USA), 0.5 µM forward and reverse primer, and 1 µL diluted cDNA.
Typical thermocycling conditions were as follows: 95 °C for 5 min; 15 cycles of touchdown PCR: 95 °C for 30 s, 65 °C for 30 s (− 0.5 °C/cycle), 72 °C for 2 min; 25 cycles of: 95 °C for 30 s, 57 °C for 30 s, 72 °C for 2 min, and a final extension at 72 °C for 5 min. Full-length TNNT1 cDNA was amplified using Platinum SuperFi PCR master mix (Thermo Fisher, USA). Cycling was as follows: 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 60 °C for 10 s and 72 °C for 2 min, and a final extension at 72 °C for 5 min. A full list of primers may be found in Additional file 2: Table S1. PCR products were analysed on a 1% agarose gel and fragments of interest purified using a QIAquick gel extraction kit (QIAGEN, USA). Purified products were sequenced by the Sanger method  and chromatograms aligned to reference transcripts in Benchling (benchling.com) using the MAFFT algorithm [16, 17].
TA cloning of RT-PCR products
As direct sequencing of some PCR products was unsuccessful or did not allow sequencing of the entire amplicon, PCR reactions were cloned into the pCR-2.1 vector using a TOPO TA cloning kit (Thermo Fisher, USA). Individual clones were picked and plasmid DNA isolated using a QIAprep spin miniprep kit (QIAGEN, USA). Plasmids were then screened for insert presence by digestion with EcoRI-HF (NEB, USA). Cloned PCR products were sequenced in both directions using M13 forward and M13 reverse primers (Additional file 2: Table S1) and aligned to the published Dorper sheep TNNT1 cDNA sequence (Accession #KT218690)  in Benchling (benchling.com) using the MAFFT algorithm [16, 17].
Protein multiple sequence alignments
TNNT1 amino acid sequences from sheep and six other mammalian species were aligned using Clustal Omega v1.2.4 . Protein sequences used for alignments were as follows: sheep (Ovis aries): AMR55385 (published AA sequence from K218690 CDS ), human (Homo sapiens): NP_0011196044 (NCBI RefSeq), cow (Bos taurus): NP_776899 (NCBI RefSeq), mouse (Mus musculus): NP_001264833 (NCBI RefSeq), rat (Rattus norvegicus): NP_001264191 (NCBI RefSeq), and dog (Canis lupus familiaris): XP_005616225 (NCBI predicted).
Western blot analysis
Total protein extracts were isolated from vastus intermedius muscle samples by homogenisation in lysis buffer (8 M urea, 125 mM Tris, 40% glycerol, 4% SDS; pH 8.8; and 15% protein inhibitor cocktail IV). Samples were heated at 95 °C, centrifuged for 5 min at 10,000 g and the supernatant collected. Total protein levels were measured using the bicinchoninic acid detection kit (Thermo Fisher, USA). Protein samples (10 µg) were briefly sonicated for 1 min before being resolved on 4–12% bis-tris midi gels (Thermo Fisher, USA), transferred to a polyvinylidene difluoride membrane (Thermo Fisher, USA) at 300 mA for 2 h at room temperature, and blocked for 1 h in 5% skim milk powder in PBS and 0.1% Tween 20. Membranes were incubated with a primary antibody against TNNT1 (1:5000; Sigma, Australia; HPA058448) at 4 °C overnight. Suitable horseradish peroxidase-conjugated secondary antibodies were used for detection with the Pierce™ ECL plus western blotting substrate kit (Thermo Fisher, USA). The gel was post-stained with Coomassie blue and the myosin band used as a loading control.
Histology and immunohistochemistry
Skeletal muscle samples were frozen in liquid nitrogen-cooled 2-methylbutane and stored at − 80 °C. Serial 10 µM sections were cut on a Leica CM3050S cryostat and used for standard histochemical techniques. For Gomori trichrome staining, sections were incubated in Gill’s haematoxylin (Sigma, Australia) for 7 min, washed with tap water, and then incubated with Gomori trichrome stain for 6 min. Slides were rinsed with 1% acetic acid, washed twice in 100% ethanol, washed twice in xylene substitute, and mounted in Entellan (ProSciTech, Australia).
Serial 10 µM muscle sections were blocked in phosphate buffered saline (PBS) with 10% fetal calf serum (FCS; Gibco, USA) and 1% bovine serum albumin (BSA; Sigma, Australia) for 1 h at room temperature. Rabbit polyclonal TNNT1 antibody (diluted 1:30; Sigma, Australia; HPA058448) or mouse monoclonal alpha-actinin antibody (diluted 1:20; Sigma, Australia; Clone EA-53; A7732) was applied and incubated at 4 °C overnight. Slides were washed in PBS, then incubated with phalloidin tetramethylrhodamine (diluted 1:1000; Sigma, Australia; P1951) and suitable secondary antibodies (diluted 1:500; Life Technologies, USA) for 1 h at room temperature. Lastly, slides were washed in PBS, counterstained in Hoechst (Sigma, Australia) and mounted in Hydromount (Electron Microscopy Sciences, USA).
All sections were imaged on a fluorescence microscope (model IX-71 or BX51, Olympus) equipped with a digital camera (model DP-74 or DP-80, Olympus).
Permeabilized myofibre mechanics
Skeletal muscle specimens were thawed and small strips were dissected from the muscle biopsies and permeabilized overnight as described previously [20, 21]. This procedure permeabilizes membranous structures in the myofibres, which enables activation of the myofilaments with exogenous calcium. Preparations were washed thoroughly with relaxing solution and stored in 50% glycerol/relaxing solution at − 20 °C. Single myofibres were dissected from the permeabilized strips and mounted using aluminium T-clips between a length motor (ASI 315C-I, Aurora Scientific Inc., Canada) and a force transducer element (ASI 403A, Aurora Scientific Inc., Canada) in a single myofibre apparatus (ASI 802D, Aurora Scientific Inc., Canada) mounted on the stage of an inverted microscope (Zeiss Axio Observer A1). Sarcomere length was determined using a high-speed VSL camera and ASI 900B software (Aurora Scientific Inc., Canada). Mechanical experiments were performed at a sarcomere length of 2.5 μm to ensure that the sarcomeres operate at an optimal length (middle of the plateau phase). Myofibre width and diameter were measured at three points along the fibre and the cross-sectional area was determined assuming an elliptical cross-section. The bathing solutions used during the experimental protocols were: a relaxing solution (100 mM BES, 6.97 mM EGTA, 6.48 mM MgCl2, 5.89 mM Na2-ATP, 40.76 mM K-propionate, 14.5 mM creatine phosphate), a pre-activating solution with low EGTA concentration (100 mM BES, 0.1 mM EGTA, 6.42 mM MgCl2, 5.87 mM Na2-ATP, 41.14 mM K-propionate, 14.5 mM creatine phosphate, 6.9 mM HDTA), and an activating solution (100 mM BES, 7.0 mM Ca-EGTA, 6.28 mM MgCl2, 5.97 mM Na2-ATP, 40.64 mM K-propionate, 14.5 mM creatine phosphate). The temperature of the bathing solutions was kept constant at 20 °C using a thermocouple thermometer/TEC controller (ASI 825A, Aurora Scientific Inc., Canada).
To investigate submaximal force generating capacities at the sarcomere level, force-pCa relations were established. To determine the force-pCa relation (pCa = − log of molar free Ca2+ concentration), permeabilized myofibres were sequentially bathed in solutions with pCa values ranging from 4.5 to 9.0 and the steady-state force was measured. Force values were normalized to the maximal force obtained at pCa 4.5. The obtained force-pCa data were fit to the Hill equation, providing the pCa10,20,50 and the Hill coefficient, nH, an index of myofilament cooperativity. Statistical differences between matched wild-type and affected samples were assessed using a two-tailed, unpaired t-test.
Myosin heavy chain isoform composition
As the contractile properties of myofibres are influenced by their myosin heavy chain composition, we used a specialized SDS-PAGE technique to analyse the myosin heavy chain isoform composition in the myofibres used in contractility experiments . In brief, skeletal muscle fibres were denatured by boiling for 2 min in SDS sample buffer. The stacking gel contained a 4% acrylamide concentration (pH 6.7), and the separating gel contained 7% acrylamide (pH 8.7) with 30% glycerol (v/v). The gels were run for 24 h at 15 °C and a constant voltage of 275 volts. Gels were silver-stained, scanned, and analysed with ImageQuant TL software (GE Healthcare, USA).
Graphs and statistics
Graphs and statistics were generated using GraphPad Prism V8. Sample comparisons were performed using a two-tailed, unpaired t-test. Statistical significance was assigned based on p < 0.05. Error bars represent mean ± SD.