Variation in ovine KRTAP8-1 affects mean staple length and opacity of wool fiber.

In this study, keratin-associated proteins gene (KRTAP8-1) from five different sheep breeds and breed-crosses (n = 310) was genotyped using a Polymerase Chain Reaction-Single Strand confirmation Polymorphism (PCR-SSCP). Six unique genotypes were observed: AA, AC, AD, AE, DD and EE, with AA being the most common in the different breeds and crosses. Twelve wool characteristics: yield, mean staple length (MSL), bulk, mean fiber diameter (MFD), fiber diameter standard deviation (FDSD), coefficient of variation of fiber diameter (CVFD), medullation, standard deviation of medullation (MeSD), coefficient of variation of medullation (CVMed), opacity, standard deviation of opacity (OpSD), and coefficient of variation of opacity (CVOp) were measured on wool derived from the sheep. Variation in KRTAP8-1 was found to have strong association with MSL, OpSD and CVOp (p ≤ 0.027). The MSL of sheep of genotype AE was greater (p = 0.027) than for sheep of genotype AA. The OpSD of sheep of genotype AA was less (p = 0.017) than sheep with the AE genotype, and the CVOp of sheep with genotype AA was less (p = 0.018) than sheep with genotype AE. Further studies are required to confirm the role of variation in KRTAP8-1 in improving quality wool production.

Identification of sequence variation in the oocyte-derived bone morphogenetic protein 15 (BMP15) gene (BMP15) associated with litter size in New Zealand sheep (Ovis aries) breeds.

Advances in the study of reproductive traits indicate that functional variation in fertility genes may be useful for improving sheep fertility. The aim of this study was to search for variation in the bone morphogenetic protein 15 gene (BMP15) and ascertain any association with litter size in purebred Finnish Landrace sheep (n = 148), Finnish Landrace × Texel-cross sheep (n = 45), and composite sheep (of varying breed background; n = 58) from New Zealand (NZ). A 482 bp and 312 bp fragment of exon 1 and 2, respectively, of BMP15 were analysed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The additive and dominance effect of BMP15 variation on litter size were estimated using animal and sire models. Two variants (A and B) were detected in exon 1; no sequence variation was detected in exon 2. Variant A had the nucleotide sequence CTT between positions c.31 and c.33, while variant B had a deletion (c.31_33del). The observed frequency for variant A in the Finnish Landrace sheep, Finnish Landrace × Texel-cross sheep and the composite sheep, was 0.77, 0.92, and 0.68, respectively while the frequency of variant B (c.31_33del) was 0.23, 0.08, and 0.32, respectively. An association between litter size and c.31_33del (P < 0.001) was observed in composite sheep. Analysis of more sheep will be required to confirm these results. Litter size did not differ significantly between sheep breeds regardless of the presence/absence of c.31_33del. Results suggested that c.31_33del might be a genetic marker for improving fecundity in some NZ sheep.

Identification and characterization of circular RNAs in mammary gland tissue from sheep at peak lactation and during the nonlactating period.

Circular RNAs are a class of noncoding RNA with a widespread occurrence in eukaryote tissues, and with some having been demonstrated to have clear biological function. In sheep, little is known about the role of circular RNAs in mammary gland tissue, and therefore an RNA sequencing approach was used to compare mammary gland tissue expression of circular RNAs in 9 Small Tail Han sheep at peak lactation, and subsequently when they were not lactating. These 9 sheep had their RNA pooled for analysis into 3 libraries from peak lactation and 3 from the nonlactating period. A total of 3,278 and 1,756 circular RNAs were identified in the peak lactation and nonlactating mammary gland tissues, respectively, and the expression and identity of 9 of them was confirmed using reverse transcriptase-polymerase chain reaction analysis and DNA sequencing. The type, chromosomal location and length of the circular RNAs identified were ascertained. Forty upregulated and one downregulated circular RNAs were characterized in the mammary gland tissue at peak lactation compared with the nonlactating mammary gland tissue. Gene ontology enrichment analysis revealed that the parental genes of these differentially expressed circular RNAs were related to molecular function, binding, protein binding, ATP binding, and ion binding. Five differentially expression circular RNAs were selected for further analysis to predict their target microRNAs, and some microRNAs reportedly associated with the development of the mammary gland were found in the constructed circular RNA-microRNA network. This study reveals the expression profiles and characterization of circular RNAs at 2 key stages of mammary gland activity, thereby providing an improved understanding of the roles of circular RNAs in the mammary gland of sheep.

Identification and characterization of circular RNA in lactating mammary glands from two breeds of sheep with different milk production profiles using RNA-Seq.

CircRNA is a specific type of non-coding RNA that has been shown to have an important role in mammary gland (MG) activity, but no study of MG circRNA activity in sheep so far. In this study, the expression profile of circRNAs was investigated using RNA-Seq in MG parenchyma at peak lactation from Small-Tailed Han sheep and Gansu Alpine Merino sheep with phenotypic differences in milk yield and components. A total of 4, 906 circRNAs were found and 33 of these were differentially expressed between breeds. GO and KEGG results showed that the parental genes of differentially expressed circRNAs were mainly enriched in heterocyclic compound binding, kinase activity, adherens junction, the TGF-ß signaling pathway, and the MAPK signaling pathway. This study provides an overview of circRNA expression in the ovine MG and the interaction between some key circRNAs and their target miRNAs. It improves our knowledge of the role of circRNA in sheep milk synthesis.

Identification of polymorphisms in the oocyte-derived growth differentiation growth factor 9 (GDF9) gene associated with litter size in New Zealand sheep (Ovis aries) breeds.

Having the ability to control litter size is important for sheep farmers and breeders worldwide. However, making genetic gain in key livestock traits like reproductive performance needs typically a lot of time, and both the fecundity and fertility traits have a great economic importance. Attention has therefore turned to better understanding the genes that control reproductive performance. Of these genes, research has focussed on the growth differentiation growth factor 9 (GDF9) gene (GDF9). In this study, a PCR-single strand conformation polymorphism (PCR-SSCP) approach was used to investigate variation in this gene in separate groups of purebred Finnish Landrace sheep, Finnish Landrace × Texel-cross sheep and composite sheep of undefined breed background, but based on New Zealand Romney-type genetics. Three GDF9 variants (named A, B and C) were found, and upon DNA sequencing, the nucleotide substitutions c.978A>G, c.994G>A and c.1111G>A were revealed. The frequency of variant A (containing nucleotides c.978A, c.994G and c.1111G) in the Finnish Landrace, Finnish Landrace × Texel-cross and composite sheep was 0.86, 0.78 and 0.76, respectively. In these three sheep groups, the frequency of B (defined by the presence of nucleotides c.978G and c.994A) was 0.01, 0.03 and 0.23 and for C (containing c.1111A) was 0.13, 0.18 and 0.01, respectively. An animal model was used to estimate the additive effect of fertility data for Finnish Landrace × Texel-cross sheep and revealed an association between litter size and the c.1111G>A variation (p = .036), but this was not observed for the Finnish Landrace sheep (p = .27) or the composite sheep (p = .17). When all the sheep were analysed together, the presence of c.1111A was associated (p < .05) with increased litter size, when compared to ewes that had c.1111G. Litter size did not differ between sheep with and without c.994A in all three groups of sheep investigated. This study suggests that c.1111A could be a useful genetic marker for improving fecundity in New Zealand sheep breeds and that it could be introgressed into other breeds, but analysis of more sheep will be required to confirm the associations that have been observed here.

Variation in the yak lipin-1 gene and its association with milk traits.

The aim of this research was to identify variation in the yak lipin-1 gene (LPIN1) and determine whether this variation affects milk traits. PCR-single stranded conformational polymorphism (PCR-SSCP) analysis was used to detect variation in the 5' untranslated region of LPIN1 in 500 yaks from four populations: Tianzhu white yaks, Qinghai yaks, wild × domestic-cross yaks and Gannan yaks. Four unique PCR-SSCP patterns, representing four different DNA sequence variants (named A, B, C and D), were observed. These contained six single nucleotide polymorphisms. Female Gannan yaks with BC genotype produced milk with a higher fat content (P < 0.001) and total milk solids (P < 0.001), than those with the AA, AB and BB genotypes. These results would suggest that LPIN1 is having an effect on yak milk fat synthesis.

Variation in the KRTAP6-3 gene and its association with wool characteristics in Pakistani sheep breeds and breed-crosses.

This study investigated variation in the keratin-associated proteins gene, KRTAP6-3, in 5 Pakistani sheep breeds/crosses using polymerase chain reaction-single strand confirmation polymorphism (PCR-SSCP) analysis. Different banding patterns were revealed, including previously described patterns and a novel pattern (named variant H). The amplified PCR product of the novel banding pattern was directly sequenced, and a synonymous nucleotide variation c.51T>C was revealed. Among the wool traits assessed, a strong correlation (r = 0.929; P < 0.001) was observed between fibre diameter standard deviation (FDSD) and coefficient of variation of fibre diameter (CVFD), between FDSD and medullation (r = 0.720; P < 0.001), between FDSD and medullation standard deviation (MeSD) (r = 0.734; P < 0.001), between MeSD and coefficient of variance of medullation (CVMed), (r = 0.903, P < 0.001), and between CVFD and medullation (r = 0.660), CVFD and MeSD (r = 0.786; P < 0.001), CVFD and CVMed (r = 0.701; P < 0.001) and medullation and MeSD (r = 0.771; P < 0.001). Variant B was found to be associated (P = 0.018) with CVFD; the presence of B being associated with a higher CVFD, than in its absence (41.08 ± 3.98 versus 36.34 ± 3.08). Variant C was associated with CVMed (P = 0.040), where sheep with C had a lower CVMed than sheep where it was absent. Variation in KRTAP6-3 was found to affect fibre diameter related traits of wool.

Variation in the Toll-like Receptor 4 (TLR4) gene affects milk traits in dairy cows.

The objective of this Research Communication was to use polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis to investigate a region of the bovine TLR4 gene (TLR4) in pasture-fed New Zealand (NZ) Holstein-Friesian × Jersey (HF × J) cross dairy cows and to determine whether gene variation was associated with milk production traits. Genetic variation was observed, with two variants (A and B) containing a single nucleotide polymorphism (SNP) (c.2021C/T) that was non-synonymous and putatively results in a p.Thr674Ile substitution in the transmembrane/cytoplasmic domain of TLR4. Variant A was associated with higher milk yields, but lower milk fat percentages, whereas B was associated with lower milk yields, but higher fat and protein percentages. Cows of genotype AA produced more milk than AB or BB cows, but the milk produced by AA cows contained less fat than AB or BB cows.

Haplotyping using a combination of polymerase chain reaction-single-strand conformational polymorphism analysis and haplotype-specific PCR amplification.

A single nucleotide polymorphism (SNP) may have an impact on phenotype, but it may also be influenced by multiple SNPs within a gene; hence, the haplotype or phase of multiple SNPs needs to be known. Various methods for haplotyping SNPs have been proposed, but a simple and cost-effective method is currently unavailable. Here we describe a haplotyping approach using two simple techniques: polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and haplotype-specific PCR. In this approach, individual regions of a gene are analyzed by PCR-SSCP to identify variation that defines sub-haplotypes, and then extended haplotypes are assembled from the sub-haplotypes either directly or with the additional use of haplotype-specific PCR amplification. We demonstrate the utility of this approach by haplotyping ovine FABP4 across two variable regions that contain seven SNPs and one indel. The simplicity of this approach makes it suitable for large-scale studies and/or diagnostic screening.

Exploring Variation in Ovine KRTAP19-5 and Its Effect on Fine Wool Fibre Curvature in Chinese Tan Sheep.

Sheep's wool is known to have unique biological, physical and chemical properties. The fibre primarily consists of proteins, but these have amino acid sequence variation, and at the phenotypic level wool fibre varies considerably. This can affect its utility and value. Unravelling the genetic factors that underpin the protein and phenotypic variability is crucial if we are to contemplate improving wool quality. Accordingly, this study investigates the high glycine and tyrosine content keratin-associated protein 19-5 gene (KRTAP19-5) in sheep. PCR-single strand confirmation polymorphism analysis, coupled with DNA sequencing of a region spanning whole coding sequence, revealed six sequence variants containing seven single nucleotide polymorphisms (SNPs). Five of the SNPs were located within the coding region, with four leading to amino acid changes if expressed. In 247 Chinese Tan sheep derived from 10 sire-lines, and renowned for their distinct 'spring-like' crimped wool at up to approximately 35 days after birth, one of the variants was found to be associated with decreased curvature of the fine wool fibres in the fleece. No associations were detected with other fibre traits or with variation in the heterotypic hair fibres of the Tan sheep. While these findings may be useful for developing gene markers to alter mean wool fibre curvature and improve sheep breeding, many other genes and environmental factors are known to contribute to variation in fibre traits.

Variation in the Exon 3-4 Region of Ovine KRT85 and Its Effect on Wool Traits.

α-keratins are structural proteins in the cortex of wool fibres and assemble in an organized fashion into keratin intermediate filaments. Variation in these keratin proteins affects the structure and characteristics of wool fibre, making keratin genes ideal candidates for the development of gene markers that describe variations in wool traits. A region of KRT85 spanning exon 3-4 (including the entire exon 3, intron 3, exon 4 and part of intron 4) was investigated. Two banding patterns defining two variant sequences (A and B) were observed in this region, and these were characterised by the presence of two single nucleotide polymorphisms. The effect of this variation in the exon 3-4 region of KRT85 on wool traits was investigated in 463 Merino × Southdown-cross lambs. The frequencies of these two variants in these sheep were 55.6% and 44.4%, respectively. Three different genotypes were observed with frequencies of 32.6%, 46.1% and 21.3% for AA, AB and BB, respectively. The presence of A was associated with an increase in greasy fleece weight and clean fleece weight, while the presence of B was associated with an increased wool prickle factor. These findings should be replicated in a broader range of sheep breeds to determine whether the associations are robust and to clarify whether the observed effects are attributable to breed differences or to gene effects themselves.

Effects of KRTAP20-1 Gene Variation on Wool Traits in Chinese Tan Sheep.

Chinese Tan sheep lambs are recognised for having tight 'spring-like' curly wool when young, but this phenotype disappears with age. This wool consists of shorter, fine wool fibres (which are usually unmedullated) and heterotypic hair fibres (which are frequently medullated), which are referred to as 'halo hair'. Both the wool and hair fibres consist of α-keratin proteins embedded in a keratin-associated protein (KAP) matrix. Of these KAPs, the KAP20-1 gene (designated KRTAP20-1) and its effect on four fibre traits (mean fibre curvature, mean fibre diameter, fibre diameter standard deviation, and coefficient of variation of fibre diameter) of Tan lambs was studied. Seven previously identified KRTAP20-1 variants (A, B, D, E, F, G, and H) of KRTAP20-1 were revealed, but the previously identified variant C was not present. Of the seven variants detected, only two (A and G) were common and present at frequencies greater than 5%, and the effect of these on the fibre traits of the finer wool fibres was assessed. It was found that variant G was associated with an increased mean fibre curvature in these wool fibres. This suggests that KRTAP20-1 might possibly be expressed differentially in the two fibre types, which may be of future value in breeding.

Ovine KRT81 Variants and Their Influence on Selected Wool Traits of Commercial Value.

Keratins are the main structural protein components of wool fibres, and variation in them and their genes (KRTs) is thought to influence wool structure and characteristics. The PCR-single strand conformation polymorphism technique has been used previously to investigate genetic variation in selected coding and intron regions of the type II sheep keratin gene KRT81, but no variation was identified. In this study, we used the same technique to explore the 5' untranslated region of KRT81 and detected three sequence variants (A, B and C) that contain four single nucleotide polymorphisms. Among the 389 Merino × Southdown cross sheep investigated, variant B was linked to a reduction in clean fleece weight, while C was associated with an increase in both greasy fleece weight and clean fleece weight. No discernible effects on staple length or mean-fibre-diameter-related traits were observed. These findings suggest that variation in ovine KRT81 might influence wool growth by changing the density of wool follicles in the skin, the density of individual fibres, or the area of the skin producing fibre, as opposed to changing the rate of extrusion of fibres or their diameter.

Screening and identification of lncRNAs in preadipocyte differentiation in sheep.

Studies of preadipocyte differentiation and fat deposition in sheep have mainly focused on functional genes, and with no emphasis placed on the role that long non-coding RNAs (lncRNAs) may have on the activity of those genes. Here, the expression profile of lncRNAs in ovine preadipocyte differentiation was investigated and the differentially expressed lncRNAs were screened on day 0 (D0), day 2(D2) and day 8(D8) of ovine preadipocyte differentiation, with their target genes being predicted. The competing endogenous RNA (ceRNA) regulatory network was constructed by GO and KEGG enrichment analysis for functional annotation, and some differentially expressed lncRNAs were randomly selected to verify the RNA-Seq results by RT-qPCR. In the study, a total of 2517 novel lncRNAs and 3943 known lncRNAs were identified from ovine preadipocytes at the three stages of differentiation, with the highest proportion being intergenic lncRNAs. A total of 3455 lncRNAs were expressed at all three stages of preadipocyte differentiation, while 214, 226 and 228 lncRNAs were uniquely expressed at day 0, day 2 and day 8, respectively. By comparing the expression of the lncRNAs between the three stages of differentiation stages, a total of 405, 272 and 359 differentially expressed lncRNAs were found in D0-vs-D2, D0-vs-D8, and D2-vs-D8, respectively. Functional analysis revealed that the differentially expressed lncRNAs were enriched in signaling pathways related to ovine preadipocyte differentiation, such as mitogen-activated protein kinase (MAPK) pathway, the phosphoinositide 3-kinase protein kinase B (PI3K-Akt) pathway, and the transforming growth factor beta (TGF-ß) pathway. In summary, lncRNAs from preadipocytes at different stages of differentiation in sheep were identified and screened using RNA-Seq technology, and the regulatory mechanisms of lncRNAs in preadipocyte differentiation and lipid deposition were explored. This study provides a theoretical reference for revealing the roles of lncRNAs in ovine preadipocyte differentiation and also offers a theoretical basis for further understanding the regulatory mechanisms of ovine preadipocyte differentiation.

Spatiotemporal Expression and Haplotypes Identification of KRT84 Gene and Their Association with Wool Traits in Gansu Alpine Fine-Wool Sheep.

Keratin (K) is a major protein component of hair and is involved in hair growth and development. In this study, we analysed the expression, localization, and polymorphism of the K84 gene (KRT84) in Gansu Alpine Fine-wool sheep using immunofluorescence, RT-qPCR, and PARMS (penta-primer amplification refractory mutation system). Haplotypes of KRT84 were also constructed and their relationship with wool traits analysed. It was revealed that KRT84 was highly expressed in hair follicles, including the inner root sheath, outer root sheath, and hair medulla and at all six lamb ages investigated from 1 to 270 days of age. Three SNPs were detected in KRT84 exon 1, and they formed three haplotypes (named H1, H2, and H3) and six genotypes. Analyses revealed an association between haplotype combinations (diplotypes) and the mean fibre curvature, mean staple length, mean staple strength, mean fibre diameter, the coefficient of variation of fibre diameter, and comfort factor for these sheep. These results suggest that KRT84 is of importance in determining several key traits in Gansu Alpine Fine-wool sheep and that the gene could possibly be used as a genetic marker for wool trait selection in these sheep.

Ovine forkhead box class O 3 (FOXO3) gene variation and its association with lifespan.

FOXO3 is a member of the FOXO (forkhead box class O) transcription factor family and has roles in cell cycle control, apoptosis, neural and hematopoietic cell differentiation and DNA repair among other functions. Several human studies provide evidence for an association between aging, longevity and variation in FOXO3. Recently variation has been identified in exon 2 of ovine FOXO3. This exon encodes the C-terminus of the DNA-binding domain and a transcription activation domain that is the key regulator of transcriptional activity in target genes. The association of genetic variation in exon 2 with lifespan was investigated in 1,732 New Zealand (NZ) sheep. Of the seven haplotypes detected, the presence of the D haplotype, which codes for an amino acid substitution (a conserved methionine residue is replaced by a valine) at residue 407, is associated with a decrease of 0.39 years in mean age (P = 0.034). Significant differences in mean age were also detected between genotypes containing D (AD: 4.7 ± 0.21; BD: 4.7 ± 0.25) and genotypes that did not contain D (AA: 5.1 ± 0.14; AB: 4.9 ± 0.18; BB: 5.3 ± 0.25). Other genotypes were rare in the sheep investigated and were not analyzed. This suggests that genetic variation in ovine FOXO3 influences the lifespan of sheep either directly or indirectly by impacting on factors that lead to reduced productivity and increased likelihood of culling.

Halplotypes of the ovine ADRB3 gene (ADRB3) and their association with post-weaning growth in New Zealand Suffolk sheep.

The ß3-adrenergic receptor (ADRB3) regulates thermogenesis and lipolysis in brown and white adipose tissue. Previously, sixteen ovine ADRB3 haplotypes have been defined. In this study, the relationship between these ADRB3 haplotypes and variation in post-weaning growth was investigated in 797 New Zealand Suffolk lambs from 38 sire lines and eight studs, using PCR-SSCP and General Linear Mixed-effects Models. Seven haplotypes were found in these sheep and they comprised five previously reported intron sequences and four previously reported 3'UT sequences. The frequencies of the various diplotypes ranged from 0.1 to 17.6% and individual haplotypes from 0.8 to 32.5%. The presence of haplotype A-b was associated with a decreased weaning-weight (P=0.001). Sheep with the B-c/F-e diplotype had a higher mean weaning-weight than those with A-b/B-c or A-b/E-e (P<0.05). The presence of C-a was found to be associated with increased post-weaning growth (P=0.008), while the presence of B-c was associated with decreased post-weaning growth (P=0.005). Sheep with A-b/C-a had higher mean post-weaning growth than those with A-b/A-b, A-b/B-c, B-c/B-c, B-c/E-e or B-c/F-e (P<0.05). Sheep with B-c/B-c had lower mean post-weaning growth than those with A-b/C-a or B-c/C-a (P<0.05). Additive effects for the different forms of the B-c haplotypes on post-weaning growth were identified. The effects of the ovine ADRB3 haplotypes on weaning-weight and post-weaning growth confirm that they could be used as a candidate gene-marker for improving sheep growth.

Allelic variation in ovine fatty acid-binding protein (FABP4) gene.

The fatty acid-binding protein 4 (FABP4) plays a role in lipid metabolism and has been implicated in intra-cellular lipid transport. While FABP4 variation has been reported in some species, variation in the coding sequence has not been reported in sheep. In this study two regions of ovine FABP4 were analysed using PCR-SSCP and sequencing. Five different PCR-SSCP patterns, representing five specific sequences (A ( 1 )-E ( 1 )) were detected in region 1 (exon 2-intron 2) with sequence analysis revealing three nucleotide substitutions and one deletion in the intron. In region 2 (exon 3-intron 3), four different PCR-SSCP patterns (A ( 2 )-D ( 2 )) were observed and four nucleotide substitutions were revealed. In total, fourteen haplotypes through both regions were defined. There was a difference (P < 0.001) in allele frequencies between two selection lines of Coopworth sheep that have been bred over many generations to be lean or fat. In region 1, A ( 1 ) and B ( 1 ) were most common (at a frequency of 50 and 30 % respectively) in the fat line, whereas these two variants were absent or rare in the lean line in which C ( 1 ) predominated (89 %). In region 2, C ( 2 ) was the most common variant (59 %) in the lean line but was absent in the fat line, whereas B ( 2 ) was predominant (83 %) in the fat line but was rare (3 %) in the lean line. These results indicate that ovine FABP4 is polymorphic and suggest further analysis is required to see if the variation detected affects fat deposition in sheep.