Two single nucleotide polymorphisms in the promoter of the ovine myostatin gene (MSTN) and their effect on growth and carcass muscle traits in New Zealand Romney sheep.

Myostatin is a negative regulator of muscle growth and development in mammals, and variation in ovine myostatin gene (MSTN) has been demonstrated to be associated with variation in the muscularity of sheep. Polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) was used to look for single nucleotide polymorphisms (SNPs) in a 304-bp amplicon from the promoter region of ovine MSTN. Sequence analyses revealed two previously identified SNPs (c.-2449G/C and c. -2379T/C) that resulted in three haplotypes (H1 (c.[-2449G; -2379C]), H2 (c.[-2449C; -2379C]) and H3 (c.[-2449G; -2379T]). The effect of these SNPs on growth and carcass traits was investigated in 357 NZ Romney lambs. General linear mixed-effect models revealed that sheep with the genotype c.-2449GC had a higher loin meat yield (p = 0.032) and proportion loin yield (p = 0.028), than those with the genotype c.-2449GG. The genotype c.-2379CC was associated with an increase in three weight traits: birthweight (p = 0.003), tailing weight (p = 0.009) and weaning weight (p = 0.028), when compared with the genotype c.-2379TC, but it was not found to have an association with growth rate. This suggests that c.-2379T/C has an effect that originates at, or before birth. Haplotype H3 was associated with a decrease in birthweight (p = 0.002), tailing weight (p = 0.003) and weaning weight (p = 0.011). Haplotype H2 was associated with increased loin yield (p = 0.012) and proportion loin yield (p = 0.002). The SNPs may have value as genetic markers for improved Romney breeding.

A 57-bp deletion in the ovine KAP6-1 gene affects wool fibre diameter.

High glycine-tyrosine keratin-associated proteins (HGT-KAPs) are predominantly present in the orthocortex of wool fibres. They vary in abundance in different wools and have been implicated in regulating wool fibre properties, but little is known about the functional roles of these proteins in the fibre matrix. In this study, we used polymerase chain reaction--single-strand conformational polymorphism (PCR-SSCP) analysis to screen for variation in a gene encoding the ovine HGT-KAP6-1 protein. We identified three gene variants (A, B and C). Variants A and B were similar to each other, with only three nucleotide differences occurring downstream of the coding sequence. However, variant C had a 57-bp deletion that would notionally result in a loss of 19 amino acids in the protein. The presence of C was found to be associated with an increase in mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), coefficient of variation of fibre diameter (CVFD) and prickle factor (percentage of fibres over 30 microns; PF). Sheep of genotype BC produced wool of greater MFD, FDSD and PF than sheep of genotypes AA, AB and BB. The CVFD was greater in the BC sheep than the AB sheep. The results suggest that variation in ovine KRTAP6-1 affects wool fibre diameter-associated traits and that the 57-bp deletion in this gene would lead to coarser wool with greater FDSD, CVFD and PF.

Genetic variations in the myostatin gene (MSTN) in New Zealand sheep breeds.

Myostatin, which is also known as growth and differentiation factor 8 (GDF8), acts as a negative regulator of skeletal muscle growth. Variation in the myostatin gene (MSTN) has been associated with variation in muscularity in many animals including sheep. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis was used to investigate MSTN in a diverse range of sheep breeds including the New Zealand (NZ) Romney, Coopworth, Corriedale, Dorper, Perendale, Suffolk, Merino, Dorset Down, Poll Dorset, Texel and other NZ cross-bred sheep. A total of 28 nucleotide substitutions were identified from nucleotide c.-1199 in the promoter region to c.*1813 (based on NCBI GenBank accession number DQ530260) and including the well-described substitution c.*1232G>A (MSTN g+6223G>A). Of these 28 substitutions, 3 were located in the promoter region, 3 in the 5'UTR, 11 in intron 1, 5 in intron 2 and 5 in the 3'UTR. One substitution in exon 1 (c.101G>A) potentially results in an amino acid substitution of glutamic acid (Glu) with glycine (Gly) at codon 34. Ten of these substitutions have not been reported previously. The genetic variation revealed in this study suggests this gene is more variable than hitherto reported and provides a foundation for future research into how this variation affects muscle and growth traits.

A premature stop codon in the ADAMTS2 gene is likely to be responsible for dermatosparaxis in Dorper sheep.

We have used polymerase chain reaction-single-strand conformational polymorphism analysis to investigate variation in exon 2 of the ADAM metalloproteinase with thrombospondin type I motif, 2 (ADAMTS2) gene in 598 sheep, including three white Dorper lambs that had a pathology consistent with dermatosparaxis. Four sequence variants (A, B, C and D) were identified at this exon, with the lambs having the dermatosparaxis phenotype being uniquely B homozygous and their mothers being B-containing heterozygous for ADAMTS2. Analysis of the amplified exon 2 sequences revealed the B variant had a nucleotide substitution that creates a premature stop codon and would notionally abbreviate the ADAMTS2 peptide. The B variant was not found in any other breed aside from the white Dorper sheep that were studied.

Ovine FABP4 Variation and Its Association With Flystrike Susceptibility.

Flystrike is a major cost and a welfare issue for the New Zealand sheep industry. There are several factors that can predispose sheep to flystrike, such as having fleecerot, a urine-stained breech, and "dags" (an accumulation of fecal matter in the wool of the breech). The FABP4 gene (FABP4) has been associated with variation in ovine fleecerot resistance, with a strong genetic correlation existing between fleecerot and flystrike occurrence. In this study, blood samples were collected from sheep with and without flystrike for DNA typing. PCR-SSCP analyses were used to genotype two regions of ovine FABP4. Sheep with the A 1 variant of FABP4 were found to be less likely (odds ratio 0.689, P = 0.014) to have flystrike than those without A 1. The likelihood of flystrike occurrence decreased as copy number of A 1 increased (odds ratio 0.695, P = 0.006). This suggests that FABP4 might be a candidate gene for flystrike resilience in sheep, although further research is required to verify this association.

Polymorphisms in the ovine myostatin gene (MSTN) and their association with growth and carcass traits in New Zealand Romney sheep.

Myostatin is a regulator of myogenesis and has been implicated in the regulation of adiposity and in controlling the structure and function of tendons. Polymerase Chain Reaction Single-Stranded Conformational Polymorphism (PCR-SSCP) analysis of intron-1 was used to identify five variants (designated A-E) of the myostatin gene (MSTN). The effect of this genetic variation on growth and carcass traits was investigated in 517 Romney male lambs from 17 sire-lines, born on a South Island New Zealand farm. General linear mixed effect models revealed that the presence of allele A in a lamb's genotype was associated with decreased leg, loin and total yield of lean meat, whereas the presence of allele B was associated with increased loin yield and proportion loin yield (loin yield divided by total yield expressed as percentage). The effect of the number of allele copies present was investigated, and it was found that the absence of A, or the presence of two copies of B, was associated with increased mean leg yield, loin yield and total yield. Two copies of B were also associated with a decrease in proportion of shoulder yield, whereas two copies of A were associated with a decrease in proportion of loin yield. Associations with allele C were not detected. No associations of MSTN variation with birth weight, weaning weight, pre-weaning growth rate, draft age and hot carcass weight (H-W) were detected. These results suggest that variation in ovine MSTN is associated with meat production, but not birth weight or growth rate in New Zealand Romney sheep.

An effective method for silver-staining DNA in large numbers of polyacrylamide gels.

Silver-staining of nucleic acid has been used for various biological analyses, including polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. A variety of methods have been described, but these methods are not that effective for staining more than a few PCR-SSCP gels, especially rapidly and with high sensitivity, because they include a number of time-consuming or hazardous manual steps that are often time dependent. Here we report a silver-staining method that can efficiently stain up to 14 gels at one time and with a detection limit of approximately 10 pg of DNA/mm(2), which is comparable to other methods.

Haplotypic diversity within the ovine calpastatin (CAST) gene.

Calpastatin (CAST) is a protein inhibitor that acts specifically on calpains and plays a regulatory role in postmortem beef tenderization and muscle proteolysis. Polymorphisms in the bovine CAST gene have been associated with meat tenderness, but little is known about how the ovine CAST gene may affect sheep meat quality traits. In this study, we selected two parts of the ovine CAST gene that have been previously reported to be polymorphic (region 1-part of intron 5 and exon 6, and region 2-part of intron 12), to investigate haplotype diversity across an extended region of the ovine gene. First, we developed a simple and efficient polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) method for genotyping region 2, which allowed the detection of a novel allele as well as the three previously reported alleles. Next, we genotyped both regions 1 and 2 of the ovine CAST gene from a large number of sheep to determine the haplotypes present. Nine different haplotypes were found across this extended region of the ovine CAST gene and four haplotypes were identified that suggested historical recombination events within this gene. Haplotypes are typically more informative than single nucleotide polymorphisms (SNPs) for analyzing associations between genes and complex production traits, such as meat tenderness, but the potential for intragenic recombination within the ovine CAST may make finding associations challenging.

Association between variation in faecal egg count for a mixed field-challenge of nematode parasites and IGHA gene polymorphism.

Research has shown that variation in ovine immunoglobulin A (IgA) levels are associated with reduced faecal egg counts (FECs) in sheep hosting gastro-intestinal (GI) parasites. Variation in the constant region of the ovine IgA heavy alpha chain gene (IGHA) may result in structurally and functionally different IgA molecules and may consequently lead to variation in the IgA response to parasitisation. This study involved three sheep breeds (Merino, Polwarth and Corriedale) and a total of 2098 lambs from eight New Zealand farms that underwent a mixed field-challenge of nematode parasites. Faecal samples were taken at approximately 4 and 9 months of age and FECs for Nematodirus and Strongyle species determined along with total eggs per gram (EPG). Analysis of all eight farms collectively revealed no significant differences in FECs associated with the presence or absence of a particular IGHA allele. However, when the data was split into predominant challenge type groups, associations were detected. In 4-month-old lambs predominantly challenged by Nematodirus sp., the presence of the IGHA allele *01 was associated (P<0.05) with higher Strongyle FECs. In 9-month-old lambs predominantly challenged by Trichostrogylus sp., the presence of IGHA allele *02 was associated (P<0.006) with a higher mean total EPG at 9 months of age. These results suggest that IGHA gene variation will not be an effective gene-marker for reducing overall FEC but may be useful in defined or specific species challenges.

Polymorphism of the ovine beta3-adrenergic receptor gene (ADRB3) and its association with wool mean staple strength and yield.

We investigated the possibility that variation in ovine ADRB3 is associated with various wool traits, in particular mean staple strength (MSS). Polymerase chain reaction-single strand conformational polymorphism analysis of part of the ADRB3 intron was used to genotype 695 Merino lambs born on three farms in the South Island of New Zealand and which were shorn as 2-tooths. For each fleece, MSS, mean fibre diameter, mean staple length and yield were measured. The results from mixed-effects models and half-sib analyses suggest that ADRB3 alleles A and D have a negative impact on some wool traits, whereas ADRB3 alleles C and E appear to have a positive impact, with allele C potentially having a greater impact than allele E on MSS. This variation in the ADRB3 may assist in the genetic selection for increased MSS and yield in Merino sheep.

Clonal polymerase chain reaction-single-strand conformational polymorphism analysis: an effective approach for identifying cloned sequences.

The amplification of target sequences from genomic DNA can result in more than one amplicon sequence being produced even when highly specific primers are used. Here we present a clonal polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) approach for screening cloned amplicons and identifying particular clones prior to sequence determination. Comparison of the PCR-SSCP patterns of the cloned amplicons with the PCR-SSCP patterns observed for the DNA templates from which the clones were derived allows PCR artifacts, different alleles, and even different loci to be differentiated prior to sequencing. Using this approach, the number of clones required for reliable sequence determination is minimized, and complex "mixed" amplicons can be resolved easily, cost-effectively, and reliably.

Allelic polymorphism of the caprine calpastatin (CAST) gene identified by PCR-SSCP.

Calpastatin (CAST) specifically inhibits calpains and there is evidence that it plays a role in meat tenderization and myogenesis. Although the CAST gene has been extensively investigated in sheep and cattle, no studies have been reported in goats. In this study, a fragment of caprine CAST was analyzed using PCR-SSCP analysis. Seven novel SSCP patterns, representing seven different nucleotide sequences, were identified. All the sequences shared high homology with the published ovine and bovine CAST sequences. Sequence analysis revealed non-synonymous amino acid variation in exon 6, which would result in a Ser/Arg substitution in domain L of the protein. Considerable variation was detected in an intron region close to the acceptor splice site, with both sequence variation and length variation being observed in this region. Variation detected here might have an impact on both the function and expression of caprine CAST.

Short communication: Identification of allelic variation at the bovine DRA locus by polymerase chain reaction-single strand conformational polymorphism.

The major histocompatibility complex DRA locus is noteworthy among the major histocompatibility complex class II loci for the little or no variation reported in many species. In cattle, DRA has not been investigated in depth, and the extent of variation at the locus is not yet known. In this study, we used PCR-single strand conformational polymorphism (SSCP) analysis to screen for potential sequence variation in the second exon of bovine DRA, which encodes the antigen-presentation groove. Four unique SSCP patterns were detected among 384 cattle from New Zealand. Sequence analysis revealed that these SSCP patterns represent 4 DRA alleles; and 3 single nucleotide polymorphisms were detected in exon 2. However, all of the single nucleotide polymorphisms were synonymous, and no amino acid change was therefore expected in this region. The polymorphism detected may be linked to variation elsewhere in the gene that affects its structure or function.

Association between variation in faecal egg count for a natural mixed field-challenge of nematode parasites and TLR4 variation.

Research has shown that Toll-like receptor 4 (TLR4) is important in immune responses to some helminth parasites. In sheep, variation in the PAMP region of TLR4 may result in structurally and thus functionally different TLR4 molecules, and this may consequently lead to variation in the TLR4 response to parasite infections. This study involved three separate, but related sheep breeds (Merino, Polwarth and Corriedale sheep) and a total of 885 lambs from five New Zealand farms that underwent a mixed field-challenge from gastro-intestinal parasites. Faecal samples were collected at approximately 4 and 9 months of age and faecal egg counts (FECs) for Nematodirus spp. and Strongyle species determined, along with the total number of eggs per gram (EPG). Analysis of the five farms collectively revealed an association (P=0.023) between the presence of TLR4 variant *02 (mean 24 EPG) and the absence of the variant (mean 32 EPG) at 9 months of age. Conversely the presence of *03 had a significantly (P=0.047) higher mean Nematodirus spp. FEC (mean 42 EPG) compared to the absence (mean 28 EPG) at 9 months of age. More associations were revealed when the data were split according to the dominant faecal parasite species. With a predominantly Trichostrongylus spp. FEC group of lambs at 9 months of age, the presence of TLR4 variant *02 was found to have significantly (P=0.003) lower Nematodirus spp. FEC (mean 4 EPG), and also significantly (P=0.033) lower total FEC (mean 312 EPG) when compared to sheep without the variant (mean 15 EPG and 449 EPG, respectively). The presence of TLR4 variant *03 and *04 were associated or tended to be associated (P=0.010 and P=0.088, respectively) with higher Nematodirus spp. FEC (mean 25 EPG and 22 EPG, respectively) when compared to lambs without the variant (mean 10 EPG and 11 EPG, respectively). These results suggest that TLR4 variation may be affecting the immune response to gastro-intestinal parasites in sheep, although principally to Nematodirus spp. infections and not Strongyle species infections.

Variation in the bovine FABP4 gene affects milk yield and milk protein content in dairy cows.

Fatty acid binding proteins (FABPs) bind long-chain fatty acids and are involved in their intracellular transport. Of the known bovine FABP genes, FABP4 has been mapped to a region on chromosome 14 that contains quantitative trait loci for milk traits. This study investigated the association of FABP4 haplotypes with milk production traits in 719 Holstein-Friesian × Jersey cows. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of a variable region of the gene revealed three haplotypes (A, B and C). Five single nucleotide polymorphisms (SNPs) were identified: two in exon 3 and three in intron 3. A was associated (P=0.032) with increased milk protein percentage (present: 4.00 ± 0.02%; absent: 3.95 ± 0.02%) and B was associated (P=0.009) with increased milk yield (present: 23.81 ± 0.23 kg/d; absent: 23.06 ± 0.21 kg/d), but tended to be associated with a decrease in protein percentage and an increase in protein yield. Cows with genotypes AA, AB and AC produced less milk, but with a higher protein percentage than BC cows. This suggest that FABP4 affects milk yield and milk protein content, both economically important traits, and that further study of this gene is warranted.

Increased vibrissa growth in transgenic mice expressing insulin-like growth factor 1.

Insulin-like growth factor 1 (IGF-1) mediates many of the actions of growth hormone. Overexpression of IGF-1 has been reported to have endocrine and paracrine/autocrine effects on somatic growth in transgenic mice. To study the paracrine/autocrine effects of IGF-1 in hair follicles, transgenic mice were produced by pronuclear microinjection of a construct containing a mouse ultra-high sulfur keratin (UHS-KER) promoter linked to an ovine IGF-1 cDNA. This UHS-KER promoter has previously been shown to direct expression of a reporter gene to the hair follicles of transgenic mice. Four transgenic mouse lines were established as a result of microinjection of 435 embryos. Transgene expression was found in skin at day 8 and day 15 of age in three of the lines. Progeny tests were carried out by mating two of the transgenic expressing males to nontransgenic females. Mice from one line were all nonexpressors while four of the 12 mice from the other showed integration of the transgene and three expressed transgene IGF-1 mRNA in the skin. Vibrissa growth at 11-21 d of age was significantly greater in transgenic expressors than in their nontransgenic littermates. Specifically, the increase in vibrissa length for transgenics at days 11-16 (20.5%) is approximately 2-fold compared with days 16-21 (11.9%). These results demonstrate that local overexpression of IGF-1 in transgenic mice is capable of stimulating vibrissa growth during the first neonatal hair cycle.

Differential expression of a gene homologous to a G-alpha protein gene in neonatal mouse skin during development of hair follicles.

The development of mouse hair follicles depends on the proliferation, differentiation and migration of epithelial matrix cells in the follicle bulb. In particular, induction of the proliferation of epithelial cells is thought to be signalled by the dermal papilla at the base of the bulb. Neonatal mouse skin is useful for studying changes in gene expression during development of the follicles, as the mitotic activity of skin cells changes shortly after birth. Using RNA differential display, a 248-bp message has been identified, which is expressed in the skin, specifically on day 2 and day 3 but not on day 4 after birth. Confirmation of expression of this gene by ribonuclease protection assay showed that strong expression is seen on day 2 and day 3, but weak expression is also shown on day 1, day 4 and day 5. In situ hybridization data revealed that it is mainly localized in the dermal papilla. Analysis of its nucleotide sequence showed 99% identity between nucleotide 2 and 232 of the mouse uncoupled S49 cell mRNA for stimulatory GTP-binding protein (G(S)) alpha subunit, suggesting it is a segment of G(S)alpha. As the G(S)alpha subunit is involved in transducing extracellular signals across the cell, the finding of its expression in the papilla suggests it may be a molecular signal to the induction of epithelial proliferation in the follicle bulb. Evidence of strong expression on day 2, at the time when the mitotic activity of epithelial matrix cells starts to increase, also suggests that the G(S)alpha is a potential candidate for involvement in the initiation of follicle growth.

Extensive diversity in New Zealand Dichelobacter nodosus strains from infected sheep and goats.

Footrot is a contagious bacterial disease of ruminants spread by the Gram-negative, anaerobic organism, Dichelobacter nodosus. It is endemic in New Zealand and throughout sheep and goat farming regions of the world. Using the polymerase chain reaction (PCR) to amplify fragments of the fimbrial gene (fimA), D. nodosus was detected in 14 hoof scrapings, sampled from six farming regions within New Zealand. DNA sequencing revealed 15 strains covering eight serogroups on the New Zealand farms. The predominant serogroup was B which contained six strains, followed by serogroups F, H and G. No strains from serogroups D and I were detected in this investigation. Eleven out of the 15 D. nodosus strains had fimbriae sequences different to those previously reported and the presence of multiple strains on a single hoof was common (86% samples). Individual sheep from the same farm, or the same paddock, were often infected by a different range of strains, which suggests a host role in mediating footrot infection.

Dichelobacter nodosus serotype M fimbrial subunit gene: implications for serological classification.

Dichelobacter nodosus fimbrial subunit gene (fimA) from a serotype M strain (M-SPAHL) was investigated in this study. A primer set targeting the relatively conserved fimA regions and based on the published sequence from Nepalese serogroup M isolates (Nepalese M), failed to amplify the fimA of M-SPAHL. However, when the downstream primer was substituted with a primer that is specific for other serogroups of D. nodosus, the fimA was successfully amplified. Cloning followed by DNA sequencing, revealed that the M-SPAHL fimA was different to the Nepalese M fimA. The predicted amino acid sequence of the M-SPAHL fimA did not show homology to any known serogroups or serotypes. The most similar sequence was from serotype F1, and not Nepalese M. The consequences of serological relatedness and sequence dissimilarity are discussed.