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.

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.

Sequence Variation in Caprine KRTAP6-2 Affects Cashmere Fiber Diameter.

Keratin-associated proteins (KAPs) are a structural component of cashmere fibers and in part determine fiber attributes. The gene encoding the high-glycine/tyrosine KAP6-2 (called KRTAP6-2) has been described in sheep, but it has not been identified goats. In this study, a 252-bp open reading frame with similarity to ovine KRTAP6-2 was found on goat chromosome 1, with its upstream and downstream flanking sequences are closely related with ovine KRTAP6-2 but are clearly distinct from other ovine KRTAP6-n sequences. Polymerase chain reaction amplification followed by single strand conformation polymorphism analysis of this region revealed five distinct banding patterns representing five different sequences (A to E) in 230 Longdong cashmere goats. Eleven diallelic single nucleotide polymorphisms (SNPs), a three-nucleotide sequence variation, and a 12-bp insertion/deletion were found among these five sequences, with most SNPs being either outside the coding region or synonymous. The presence of variant D was found to be associated with decreased mean fiber diameter (MFD; present: 13.26 ± 0.07 µm; absent: 13.55 ± 0.04 µm; p < 0.001), suggesting that variation in KRTAP6-2 may affect fiber diameter and have value as a molecular marker for improving the cashmere fiber diameter trait.

Identification of differentially expressed genes at different post-natal development stages of longissimus dorsi muscle in Tianzhu white yak.

The regulatory mechanisms controlling post-natal muscle development in the yak (Bos grunniens) are still largely unknown, yet the growth and development of muscle is a complex process that plays a crucial role in determining the yield and quality of an animal's meat. In this study, we performed a transcriptome analysis based on the RNA sequencing (RNA-Seq) of yak longissimus dorsi muscle tissue obtained from calves (6 months of age; 6 M), young adults (30 months of age; 30 M) and adult (54 months of age; 54 M) to identify which genes are differentially expressed and to investigate their temporal expression profiles. In total, 1788 differentially expressed genes (DEGs) (|log2FC| ≥ 1, P-adjusted < 0.05) were detected by pairwise comparisons between the different age groups. The expression levels of 10 of the DEGs were confirmed using reverse transcription-quantitative PCR (RT-qPCR), and the results were consistent with the transcriptome profile. A time-series expression profile analysis clustered the DEGs into four groups that could be divided into two classes (P < 0.05): class 1 profiles, which had up-regulated patterns of gene expression and class 2 profiles, which featured down-regulated patterns. Based on that cluster analysis, GO enrichment analysis revealed 1073, 127, and 184 terms as significantly enriched in biological process (BP), cellular component (CC), and molecular function (MF) categories in the class 1 profiles, while 714, 66, and 206 terms were significantly enriched in BP, CC, and MF in the class 2 profiles. A KEGG pathway analysis revealed that DEGs from the class 1 profiles were enriched in 62 pathways, with the most enriched being the phosphoinositide 3-kinase (PI3K) - protein kinase B (Akt)-signaling pathway. The DEGs from the class 2 profiles were enriched in 16 pathways, of which forkhead box protein O (FoxO) - signaling was the most enriched. Taken together, these results provide insight into the mechanisms of skeletal muscle development, as well suggesting some potential genes of importance for yak meat production.

Variation in caprine KRTAP1-3 and its association with cashmere fibre diameter.

Keratin-associated proteins (KAPs) are components of cashmere fibres. The gene encoding the KAP1-3 protein (KRTAP1-3) has been described in goats, but little is known about sequence variation in this gene and if it affects cashmere fibre traits. In this study, we used a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to screen for nucleotide sequence variation in caprine KRTAP1-3 in 327 Longdong cashmere goats, then analysed association between the genetic variation that was revealed and some cashmere fibre traits. Six PCR-SSCP patterns representing six different variant sequences of KRTAP1-3 (named A to F) were revealed. Among these variant sequences, seven single nucleotide polymorphisms (SNPs) were detected, with two of them being non-synonymous. Goats with genotype AC had higher mean fibre diameter (MFD) than those with genotype AB (P < 0.001), while goats with genotype AB had higher MFD than those with AA (P < 0.001). The presence of C (P < 0.001) and B (P = 0.006) in a genotype was associated with increased MFD, and together this suggests that variation in caprine KRTAP1-3 affects the key fibre trait of MFD.

MicroRNA-432 inhibits milk fat synthesis by targeting SCD and LPL in ovine mammary epithelial cells.

The microRNA (miR)-432 is differentially expressed in the mammary gland of two breeds of lactating sheep with different milk production traits, and between the non-lactating and peak-lactation periods, but there have been no reports describing the molecular mechanisms involved. In this study, the effect of miR-432 on the proliferation of ovine mammary epithelial cells (OMECs) and the target genes of miR-432 were investigated. The effects of miR-432 on the expression of the target genes and the content of triglycerides in the OMECs were also analyzed. Transfection with a miR-432 mimic was found using CCK8 and Edu assays, to inhibit the viability of OMECs and reduce the number of proliferated OMECs. In contrast, a miR-432 inhibitor had the opposite effect to the miR-432 mimic, and together these results suggest that miR-432 inhibits the proliferation of OMECs. A dual luciferase assay revealed that the genes for stearoyl-CoA desaturase (SCD) and lipoprotein lipase (LPL) are targeted by miR-432. The transfection of miR-432 mimic into OMECs resulted in decreases in the expression of SCD and LPL, and three other milk fat synthesis marker genes; FABP4, LPIN1 and ACACA. The mimic also decreased the content of triglycerides. The miR-432 inhibitor had the opposite effect to the mimic on the expression of these genes and the level of triglycerides. This is the first study to reveal the biological mechanisms by which miR-432 inhibits milk fat synthesis in sheep.

Variation in the Ovine Glycogen Synthase Kinase 3 Beta-Interaction Protein Gene (GSKIP) Affects Carcass and Growth Traits in Romney Sheep.

The glycogen synthase kinase 3 beta (GSK3ß)-interacting protein (encoded by the gene GSKIP) is a small A-kinase anchoring protein, which complexes with GSK3ßand protein kinase A (PKA) and acts synergistically with cAMP/PKA signaling to inhibit GSK3ß activity. The protein plays a role in regulating glycogen metabolism, protein synthesis, the cell cycle, and in regulating gene expression. In this study, PCR-single strand conformation polymorphism (PCR-SSCP) analyses were used to screen for variation in exon 1 and exon 2 of GSKIP in 840 New Zealand (NZ) Romney sheep. Two SSCP banding patterns representing two different nucleotide variants (A and B) were detected in an exon 1 region, whereas in an exon 2 region only one pattern was detected. Variants A and B of exon 1 had one non-synonymous nucleotide difference c.37A/G (p.Met13Val). The birthweight of sheep of genotype AA (5.9 ± 0.06 kg) was different (p = 0.023) to sheep of genotype AB (5.7 ± 0.06 kg) and BB (5.7 ± 0.06 kg). The hot carcass weight (HCW) of sheep of genotype AA (17.2 ± 0.22 kg) was different (p = 0.012) to sheep of genotype AB (17.6 ± 0.22 kg) and BB (18.0 ± 0.29 kg), and the fat depth at the 12th rib (V-GR) of sheep of genotype AA (7.7 ± 0.31 mm) was different (p = 0.016) to sheep of genotype AB (8.3 ± 0.30 mm) and BB (8.5 ± 0.39 mm). The results suggest that the c.37A/G substitution in ovine GSKIP may affect sheep growth and carcass traits.

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.

Variation in bovine leptin gene affects milk fatty acid composition in New Zealand Holstein Friesian × Jersey dairy cows.

Leptin is a protein hormone secreted from white adipose tissue. It regulates food/feed intake, body weight, immune function and reproduction. In our investigation, the polymerase chain reaction (PCR) amplification coupled with single-strand conformational polymorphism (SSCP) analysis was used to reveal variation in bovine leptin gene (LEP) in New Zealand (NZ) Holstein Friesian  × â€¯Jersey (HF  × â€¯J) dairy cows. Subsequent sequence analysis of a 430 bp amplicon spanning the entirety of exon 3 and part of the intron 2 region revealed three variant sequences ( A 3 , B 3 and C 3 ) containing a total of five nucleotide substitutions, all of which have been reported previously. Using general linear mixed-effect model analyses, the presence of variant A 3 (the most common variant) was associated with a decreased level of C15:1, C18:1 trans-11, C18:1 all trans, C18:2 trans-9, cis-12, C22:0 and C24:0 levels but increased levels of C12:1 and C13:0 iso ( p < 0.05 ). Variant B 3 was associated with reduced levels of C6:0, C8:0, C11:0, C13:0 and C20:0 but increased C17:0 iso and C24:0 levels ( p < 0.05 ). Variant C 3 was associated with decreased C17:0 iso levels but increased C20:0 ( p < 0.05 ) levels. In a genotype model, the A 3 B 3 genotype was associated with increased levels of C22:0 and C24:0 but decreased C8:0, C10:0, C11:0, C13:0, C15:0 and grouped medium-chain fatty acid (MCFA) levels ( p < 0.05 ). Genotype A 3 C 3 was found to be associated with decreased levels of C10:0, C11:0, C13:0 and grouped MCFA ( p < 0.05 ). This is the first report of findings of this kind in NZ HF  × â€¯J cows, and they suggest that variation in exon 3 of bovine leptin gene could be explored as a means of decreasing the concentration of saturated fatty acids in milk.

Nucleotide sequence variation of the major histocompatibility complex class II DQA1 gene in different cattle breeds from Nigeria and New Zealand.

The major histocompatibility complex (MHC) plays a role in immune response. Among other activities, the bovine MHC genes (BoLA) trigger immune responses, including the activation of antibody-producing B-cells. In this study, White Fulani (n = 24), Red Bororo (n = 5) and Holstein-White × Fulani-cross (n = 11) cattle from Nigeria, and New Zealand Holstein-Friesian × Jersey-cross (n = 40) cattle were used to investigate variability in exon 2 of BoLA-DQA1. Ten alleles were identified using a PCR-Single Strand Conformation Polymorphism (SSCP) approach and their nucleotide sequences confirmed by DNA sequencing. A total of 12.60 % of all nucleotide positions analysed were revealed to be variable and two novel BoLA-DQA1 alleles are reported here for the first time.

Variation in a Newly Identified Caprine KRTAP Gene Is Associated with Raw Cashmere Fiber Weight in Longdong Cashmere Goats.

Keratin-associated proteins (KAPs) and keratins determine the physical and chemical properties of cashmere fibers as they are the main components of the fibers. It has been reported that ovine KRTAP1-2 affects clean fleece weight, greasy fleece weight and yield in sheep, but the gene has not been described in goats and its effects on fiber traits are unknown. In this study, we identify the keratin-associated protein 1-2 gene (KRTAP1-2) in the goat genome and describe its effect on cashmere fiber traits in 359 Longdong cashmere goats. Six sequence variants (named CAPHI-KRTAP1-2*A to CAPHI-KRTAP1-2*F) were revealed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. These sequences have the highest homology with ovine KRTAP1-2 sequences. There were a 60-bp deletion, a 15-bp insertion and five single nucleotide polymorphisms (SNPs) including two non-synonymous SNPs in the coding sequence. The caprine KRTAP1-2 gene was expressed in the skin tissue, but a signal was not observed for the kidneys, liver, lungs, spleen, heart and longissimus dorsi muscle. Variation in caprine KRTAP1-2 was found to be associated with raw cashmere fiber weight, but not with fiber diameter and length.

Transcriptome Analysis Reveals Genes Involved in Thermogenesis in Two Cold-Exposed Sheep Breeds.

Thermogenesis plays an important role in the survival of sheep exposed to low temperatures; however, little is known about the genetic mechanisms underlying cold adaptation in sheep. We examined 6 Altay (A) and 6 Hu (H) six-month-old ewe lambs. Altay sheep are raised in northern China and are adapted to dry, cold climates, while Hu sheep are raised in southern China and are adapted to warm, humid climates. Each breed was divided into two groups: chronic cold sheep, exposed to -5 °C for 25 days (3 Ac; 3 Hc), and thermo-neutral sheep, maintained at 20 °C (3 Aw; 3 Hw). The transcriptome profiles of hypothalamus, tail-fat and perirenal fat tissues from these four groups were determined using paired-end sequencing for RNA expression analysis. There are differences in cold tolerance between Hu and Altay sheep. Under cold exposure of the lambs: (1) UCP1-dependent thermogenesis and calcium- and cAMP-signaling pathways were activated; and (2) different fat tissues were activated in Hu and Altay lambs. Several candidate genes involved in thermogenesis including UCP1, ADRB3, ADORA2A, ATP2A1, RYR1 and IP6K1 were identified. Molecular mechanisms of thermogenesis in the sheep are discussed and new avenues for research are suggested.

Differentially phosphorylated proteins in the crimped and straight wool of Chinese Tan sheep.

Proteins can be post-translationally modified and this can be important in the regulation of cellular processes and function. However, little is known about whether protein phosphorylation plays a role in regulating wool fibre properties. In this study, we used a chemical labelling method combined with a high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis to compare the phosphopeptides present in the wool of three Tan sheep with highly crimped wool and three Tan sheep with straighter wool. Thirty-six phosphopeptides that had differences in relative abundance between these two types of wool were identified. These peptides were derived from 28 to 33 different proteins, including two keratins (Ks) and 7 to 12 keratin-associated proteins (KAPs), with these proteins being common structural components of the wool fibre. The crimped wool had a higher relative abundance of phosphorylated K38, K72 and KAP13-x, whereas the straighter wool had a higher relative abundance of phosphorylated KAP2-1, KAP6-1, KAP4-x, KAP10-x and KAP13-y. These results confirm the phosphorylation of wool Ks and KAPs, and suggest that differential phosphorylation of Ks and KAPs may affect wool fibre crimping in Tan sheep. SIGNIFICANCE: Protein phosphorylation can alter the structural conformation and interaction of a protein, and hence affect the cellular processes that the protein undertakes. In this study, we compared the suite of phosphorylated proteins in crimped and straight wool from Chinese Tan sheep and found that some keratins and keratin-associated proteins were phosphorylated. Crimped wool had more keratin phosphorylation, while straight wool had more keratin-associated protein phosphorylation, with this suggesting that wool fibre crimping may be a regulated by phosphorylation of some wool proteins. This suggests that wool traits may be under epigenetic control and that post-translation modifications need to be considered in breeding for different wool types.

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.

Nucleotide Sequence Variation in the Insulin-Like Growth Factor 1 Gene Affects Growth and Carcass Traits in New Zealand Romney Sheep.

Insulin-like growth factor 1 (IGF1) is a mediator of the effects of growth hormone and polymorphism in the IGF1 gene (IGF1) is reported to affect fat deposition in some livestock species. In this study, nucleotide sequence variation in three regions of ovine IGF1 (part of the 5' flanking region, the exon 3 region, and the exon 4 region) was investigated in 848 New Zealand Romney lambs using PCR-single strand conformation polymorphism (SSCP) analyses to ascertain if single nucleotide polymorphisms (SNPs) existed. Six SNPs were identified across these three regions. The effect of the sequence variation in the exon 3 and exon 4 regions on growth and carcass traits were investigated. One of the PCR-SSCP sequence variants in the exon 3 region was associated with variation in hot carcass weight, carcass fat depth at the 12th rib measured using video imaging and the percentage proportion of leg lean meat, whereas the other was associated with variation in growth rate to weaning. No associations were detected for the other gene regions analyzed. The results suggest that polymorphism in exon 3 of ovine IGF1 has potential for use as a gene-marker for some carcass and growth traits.

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.

Identification of novel nucleotide sequence variations in an extended region of the bovine leptin gene (LEP) across a variety of cattle breeds from New Zealand and Nigeria.

Leptin is mainly secreted by white adipose tissue in animals. Leptin acts by stimulating or inhibiting the release of a neurotransmitter, which eventually results in a decrease in food/feed intake and an increase in energy expenditure. In this investigation, the polymerase chain reaction (PCR) coupled with single-strand conformation polymorphism (SSCP) analysis was used to reveal nucleotide sequence variations in bovine leptin gene (LEP) in 338 cattle of a variety of breeds farmed in New Zealand (NZ) and Nigeria. These included NZ Hereford, Angus, Shorthorn, and crossbred Holstein-Friesian  × â€¯Jersey cattle and the Nigerian Sokoto Gudali, Red Bororo, White Fulani, and crossbred Holstein-Friesian  × â€¯White Fulani cattle. Sequence analysis of three regions of bovine LEP that encompassed selected coding and non-coding regions, revealed a total of 12 nucleotide sequence variations (six in exons and six in introns). Of these, three are reported here for the first time, whereas nine have been previously described. Some of the variations identified were common in both the NZ and Nigerian cattle breeds, while others were peculiar to particular breeds from a specific region. The sharing of common variants across different breeds irrespective of geography may indicate an evolutionary relationship, just as the differences within a breed might be attributable to either selective pressure for specific traits or random genetic drift. The detection of both new and previously documented variations in bovine LEP suggests that the gene is highly variable.

Variation in the Caprine Keratin-Associated Protein 27-1 Gene is Associated with Cashmere Fiber Diameter.

Variation in some caprine keratin-associated protein (KAP) genes has been associated with cashmere fiber traits, but many KAP genes remain unidentified in goats. In this study, we confirm the identification of a KAP27-1 gene (KRTAP27-1) and describe its effect on cashmere traits in 248 Longdong cashmere goats. A polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis was used to screen for sequence variation in this gene, and three sequence variants (named A to C) were found. These sequences have the highest similarity (77% identity) to a human KRTAP27-1 sequence, while sharing some homology with a predicted caprine KRTAP27-1 sequence ENSCHIG00000023347 in the goat genome construct (ARS1:CM004562.1) at chromosome 1 position 3,966,193-3,973,677 in the forward strand. There were two single nucleotide polymorphisms (SNPs) detected in the coding sequence, including one nonsynonymous SNP (c.413C/T; p.Ala138Val) and one synonymous SNP (c.495C/T). The C variant differed from A and B at c.413C/T, having cytosine in its nucleotide sequence, while the B variant differed from A and C at c.495C/T, having thymine in its nucleotide sequence. Goats of the genotypes AB and BB produced cashmere fibers of higher mean fiber diameter (MFD) than goats of genotype AA, but no difference in MFD was detected between the AB and BB goats. These results suggest that B is associated with increased MFD. Expression of the caprine KRTAP27-1 sequence was predominantly detected in the skin tissue of goats but not or only weakly detected in other tissues, including longissimus dorsi muscle, heart, kidney, liver, lung and spleen.

Associations between the Bovine Myostatin Gene and Milk Fatty Acid Composition in New Zealand Holstein-Friesian × Jersey-Cross Cows.

The myostatin gene (MSTN), which encodes the protein myostatin, is pleiotropic, and its expression has been associated with both increased and decreased adipogenesis and increased skeletal muscle mass in animals. In this study, the polymerase chain reaction, coupled with single strand conformation polymorphism analysis, was utilized to reveal nucleotide sequence variation in bovine MSTN in 410 New Zealand (NZ) Holstein-Friesian × Jersey (HF × J)-cross cows. These cows ranged from 3 to 9 years of age and over the time studied, produced an average 22.53 ± 2.18 L of milk per day, with an average milk fat content of 4.94 ± 0.17% and average milk protein content of 4.03 ± 0.10%. Analysis of a 406-bp amplicon from the intron 1 region, revealed five nucleotide sequence variants (A-E) that contained seven nucleotide substitutions. Using general linear mixed-effect model analyses the AD genotype was associated with reduced C10:0, C12:0, and C12:1 levels when compared to levels in cows with the AA genotype. These associations in NZ HF × J cross cows are novel, and they suggest that this variation in bovine MSTN could be explored for increasing the amount of milk unsaturated fatty acid and decreasing the amount of saturated fatty acid.