Xiu Liu

Identification of the ovine keratin-associated protein 22-1 (KAP22-1) gene and its effect on wool traits

Keratin-associated proteins (KAPs) are structural components of wool and hair fibers. To date, eight high glycine/tyrosine KAP (HGT-KAP) families have been identified in humans, but only three have been identified in sheep. In this study, the putative ovine homolog of the human KAP22-1 gene (KRTAP22-1) was amplified using primers designed based on a human KRTAP22-1 sequence. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) was used to screen for variation in KRTAP22-1 in 390 Merino × Southdown-cross lambs and 75 New Zealand (NZ) Romney sheep. Three PCR-SSCP banding patterns were detected and DNA sequencing revealed that the banding patterns represented three different nucleotide sequences (A–C). Two single nucleotide polymorphisms (SNPs) were identified in these sequences. Variant B was most common with a frequency of 81.3% in NZ Romney sheep, while in the Merino × Southdown-cross lambs, A was more common with a frequency of 51.8%. The presence of B was found to be associated with increased wool yield and decreased mean fiber curvature (MFC). Sheep of genotype BB or AB had a higher wool yield than those of genotype AA. These results suggest that ovine KRTAP22-1 variation may be useful when developing breeding programs based on increasing wool yield, or decreasing wool curvature.

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.

Identification of the secondary follicle cycle of Hexi cashmere goat.

This experiment conducted to identify a periodic change of ultrastructures of secondary follicle characteristics during a whole year, reveal the molecule regulation of growth of cashmere. A total of 10 cashmere goats of 1‐year old were studied. The paraffin section and ultrathin slices of skin were made each month in a whole year, observed, photographed, and analyzed under light microscope and transmission electron microscope after stained. Following the development of down fiber, the ultrastructures of secondary follicle of Hexi cashmere goat showed a periodic change within a year. There were five different periods during a down fiber cycle. It was observed that the stage of telogen, proanagen, anagen, procatagen, and catagen was in January and February, March and April, May to August, September and October, and November and December, respectively. The key change observed in secondary follicle under transmission electron microscope was inner root sheath. This study illustrated the five different stage of secondary follicle of Hexi Cashmere goat within a whole growth cycle, and has provided more detailed information about the research field of Hexi cashmere goat. Choosing the suitable time to harvest the cashmere may get the profit maximization. Anat Rec, 2012.

Identification of the Caprine Keratin-Associated Protein 20-2 (KAP20-2) Gene and Its Effect on Cashmere Traits

The gene encoding the high glycine/tyrosine keratin-associated protein 20-2 (KAP20-2) gene has been described in humans, but has not been identified in any livestock species. A search for similar sequences in the caprine genome using the human KAP20-2 gene (KRTAP20-2) revealed a homologous sequence on chromosome 1. Three different banding patterns representing distinct sequences (A–C) in Longdong cashmere goats were identified using polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis. These sequences shared high sequence similarity with the human and mouse KRTAP20-2 sequences, suggesting that A–C are caprine variants of the human and mouse genes. Four single nucleotide polymorphisms (SNPs) were identified, and three of them were non-synonymous. KRTAP20-2 was found to be expressed in secondary hair follicles, but not in heart, liver, lung, kidney, spleen, or longissimus dorsi muscle. The presence of A was associated with increased cashmere fibre weight, while the presence of B was associated with a decrease in cashmere fibre weight and curly fibre length. Goats with genotype AA had a higher cashmere fibre weight and a higher curly fibre length than those with genotypes AB or BB. These results indicate that caprine KRTAP20-2 variation may have value as a genetic marker for improving cashmere fibre weight.

Effect of variation in ovine WFIKKN2 on growth traits appears to be gender-dependent

WFIKKN2 may play a role in the regulation of muscle growth and development, but to date there have been no reports on the effect of variation in WFIKKN2 on growth and carcass traits in livestock. In this study, the effect of variation in ovine WFIKKN2 was investigated in 800 New Zealand Romney lambs (395 male and 405 female), with five previously described variants (A to E) being identified. Variation in ovine WFIKKN2 was not found to affect various growth traits in the female lambs, but the presence of variant B was associated (P < 0.05) with decreased birth weight, tailing weight, weaning weight and pre-weaning growth rate; and increased post-weaning growth rate in male lambs. In male lambs, the presence of variant B was associated (P < 0.05) with an increased shoulder yield and proportion shoulder yield. No associations with growth or carcass traits were detected for the presence (or absence) of the other variants. These results suggest that variation in ovine WFIKKN2 may have a differential effect on growth in male and female lambs, and hence that the gene may be expressed in, or act in, a gender-specific fashion.

Variation in the ovine MYF5 gene and its effect on carcass lean meat yield in New Zealand Romney sheep

Myogenic factor 5 (MYF5) plays an important role in regulating skeletal muscle, but to date there have been no reports on whether the gene is variable and whether this variation is associated with meat yield in sheep. In this study, four variants (A to D) of ovine MYF5 containing two Single Nucleotide Polymorphisms (SNPs) and one basepair (bp) insertion/deletion were detected by Polymerase Chain Reaction - Single Stranded Conformational Polymorphism (PCR-SSCP) analysis. Breed differences in variant frequencies were observed. The effect of variation in ovine MYF5 on lean meat yield, predicted using VIAScan® technology, was investigated in 388 male NZ Romney lambs. Only genotypes AA and AB were found in these lambs. Lambs with genotype AA had a higher leg yield (P=0.044), loin yield (P=0.002) and total yield (P=0.012) than those with genotype AB. No association with shoulder yield was detected. These results suggest that ovine MYF5 may be a valuable genetic marker for improved lean meat yield.

Determination of Secondary Follicle Characteristics, Density, Activity, and Hoxc13 Expression Pattern of Hexi Cashmere Goats Breed.

This experiment was conducted to identify some aspects of secondary follicle (SF) characteristics of Hexi cashmere goats at five different growing stages in one year in order and discover the expression pattern of Hoxc13 in a SF cycling to provide morphological basis for studying the growth of cashmere. Ten cashmere goats of one‐year old (5 males, 5 females) were included in this study. The density and activity of SF were observed and measured by making paraffin sections, the ultra‐structural features of SF were studied under transmission electron microscopy (TEM) by making ultra‐thin sections, and the expression of Hoxc13 was investigated through the immunohistochemistry method. The average diameter of SF had the smallest value in the anagen stage, and significant difference (P < 0.05) was found between the anagen stage and other stages. The density of SF increased gradually through the different growing stages, and significant difference was found between the anagen and procatagen stages (P < 0.05). With an increase in time (months), the percentage of SF activity increased, and significant difference in the percentage of SF activity was found between the telogen and anagen stages (P < 0.05). At the telogen stage, the layers of connective tissue sheath (CTS) of SF were unclear, hemidesmosomes between the outer root sheath (ORS) and basement membrane disappeared, and dead cells were found at the top of the SF. The rudiments of new SF were found in the proanagen stage, CTS was thickened, and the cells of ORS were stretched out like fingers. At the anagen stage, the structure of SF was integral, and the inner root sheath (IRS) consisted of three concentric layers (Helen, Huxley, and Cuticle layers). The cells of Huxleys layer degenerated gradually, and pseudopodia were formed on the cells of ORS in the procatagen stage. At the catagen stage, the ORS was separated from the IRS, and IRS disappeared. Huxleys layer was absent in the inactive SF while, the ORS was present in the active SF. Hoxc 13 was expressed in the epidermis and sebaceous gland o, ORS, IRS, hair shaft of SF in the skin. Hoxc13 was expressed weakly during procatagen, catagen, and telogen stage, while with an increase in proanagen and anagen stage, significant difference was found between them. These findings demonstrated the ultra‐structural features of SF could provide the useful activity criteria, and Hoxc13 associated with the SF activity. Anat Rec, 298:1796–1803, 2015.

Molecular characterization of caprine KRTAP13-3 in Liaoning cashmere goat in China

Keratin-associated proteins are a main structural component of hair and wool fibers and may have effects on their characteristics. There are four KAP13 gene members that have been reported in humans, and the KAP13-3 gene has been identified in sheep and cattle, but it has not yet been identified in goats. This led us to search for the caprine KAP13-3 gene. In this study, the caprine KAP13-3 gene was polymerase chain reaction (PCR) amplified, and PCR single-stranded conformational polymorphism (PCR-SSCP) analysis was used to detect genetic variation in 214 Liaoning cashmere goats from China. PCR amplicons of 559 bp were obtained. Sequencing of the amplicons confirmed eight different sequences, and they were named alleles A–H. All of these sequences showed a high sequence homology to known KRTAP13-3 sequences from sheep and cattle. Nine nucleotide substitutions were identified within the KRTAP13-3 coding region, and all of these were nonsynonymous. All sequences showed an open reading frame of 471 nucleotides encoding 156 amino acids. This polypeptide contained high levels of serine (23.08–23.72 mol%), cysteine (10.26–11.54 mol%), and threonine (5.66 mol%). The polypeptide had high calculated pI values (9.02–9.59). The variation reported in the caprine KAP13-3 gene might affect gene expression, primary protein structure, and the character of the resultant cashmere fibers.

Y chromosomal haplotype characteristics of domestic sheep (Ovis aries) in China

Investigations on the variation present at the male-specific Y chromosome region provide strong information to understand the origin and evolution of domestic sheep. One SNP OY1 (g.88A>G) in the upstream region of SRY gene, and the microsatellite SRYM18 locus within ovine Y chromosome were analyzed in one hundred and forty five samples collected from eleven breeds in China. SNP OY1 was analyzed using PCR-SSCP method and sequencing. Two different PCR-SSCP patterns represented two specific sequences with sequence analysis revealing SNP-OY1 (g.88A>G) were observed, while SNP A-OY1 showed the most common frequency (82.8%). Sequencing of the SRYM18 region revealed one novel size fragment (A2) with different repetitive units. Seven haplotypes (H4, H5, H6, H7, H8, H9 and H12) and two novel haplotypes (Ha and Hb) were established using combined genotype analysis. H6 showed the highest frequency (43.4%) across all breeds, and H8 showed the second frequency (24.1%). Ha was only found in one breed (Tan), while Hb was present in three breeds (Gansu alpine, White Suffolk and Duolang). Our findings reveal one novel allele in SRYM18 region and two novel male haplotypes of domestic sheep in China.