[The Development of SpCas9 Variants with High Specificity and Efficiency Based on the HH Theory].

Streptococcus pyogenes Cas9 (SpCas9) is the most popular tool in gene editing; however, off-target mutagenesis is one of the biggest impediments in its application. In our previous study, we proposed the HH theory, which states that sgRNA/DNA hybrid (hybrid) extrusion-induced enhancement of hydrophobic interactions between the hybrid and REC3/HNH is a key factor in cleavage initiation. Based on the HH theory, we analyzed the interactions between the REC3 domain and hybrid and obtained 8 mutant sites. We designed 8 SpCas9 variants (V1-V8), used digital droplet PCR to assess SpCas9-induced DNA indels in human cells, and developed high-fidelity variants. Thus, the HH theory may be employed to further optimize SpCas9-mediated genome editing systems, and the resultant V3, V6, V7, and V8 SpCas9 variants may be valuable for applications requiring high-precision genome editing.

Regulatory role of phosphoproteins in the development of bovine small intestine during early life.

The small intestine is the primary site of nutrient digestion and absorption, which plays a key role in the survival of neonatal calves. A comprehensive assessment of the phosphoproteomic changes in the small intestine of neonatal calves is unavailable; therefore, we used phosphopeptide enrichment coupled with liquid chromatography-tandem mass spectrometry to investigate the changes in the phosphoproteome profile in the bovine small intestine during the first 36 h of life. Twelve neonatal male calves were assigned to one of the following groups: (1) calves not fed colostrum and slaughtered approximately 2 h postpartum (n = 3), (2) calves fed colostrum at 1 to 2 h and slaughtered 8 h postpartum (n = 3), (3) calves fed 2 colostrum meals (at 1-2 and 10-12 h) and slaughtered 24 h postpartum (n = 3), (4) calves fed 3 colostrum meals (at 1-2, 10-12, and 22-24 h) and slaughtered 36 h postpartum (n = 3). Mid-duodenal, jejunal, and ileal samples of the calves were collected after slaughter. We identified 1,678 phosphoproteins with approximately 3,080 phosphosites, which were mainly Ser (89.9%), Thr (9.8%), and Tyr (0.3%) residues; they belonged to the prodirected (52.9%), basic (20.4%), acidic (16.6%), and Tyr-directed (1.7%) motif categories. The regional differentially expressed phosphoproteins included zonula occludens 2, sorting nexin 12, and protein kinase C, which are mainly associated with developmental processes, intracellular transport, vesicle-mediated transport, and immune system process. They are enriched in the endocytosis, tight junction, insulin signaling, and focal adhesion pathways. The temporal differentially expressed phosphoproteins included occludin, epsin 1, and bridging integrator 1, which were mainly associated with macromolecule metabolic process, cell adhesion, and growth. They were enriched in the spliceosomes, adherens junctions, and tight junctions. The observed changes in the phosphoproteins in the tissues of small intestine suggest the protein phosphorylation plays an important role in nutrient transport and immune response of calves during early life, which needs to be confirmed in a larger study.

Evaluation of colostrum bioactive protein transfer and blood metabolic traits in neonatal lambs in the first 24 hours of life.

Colostrum is a unique resource that contributes to the passive transfer of immunity and plays a central role in the health status of neonatal ruminants. However, digestion and absorption of colostral proteins in the gut remain incompletely understood. Therefore, this study aimed to investigate the effect of bovine colostrum feeding on blood metabolic traits and to quantify colostral bioactive proteins in the gastrointestinal digesta and blood to evaluate intestinal transfer in neonatal lambs in the first 24 h of life. Fifty-four newborn lambs were used in this study, including 27 lambs fed pooled bovine colostrum and slaughtered at 6 (C6h), 12 (C12h), or 24 h (C24h) after birth; 18 lambs not fed any colostrum or milk and slaughtered at birth (N0h) or 24 h (N24h) after birth; and 9 milk-fed lambs slaughtered at 24 h (M24h) after birth. Lambs receiving colostrum or milk were bottle-fed within the first 2 h to obtain intakes of 8% of body weight at birth. Samples of blood and digesta from the abomasum, jejunum, and ileum were collected after slaughter. Serum concentrations of glucose, insulin, total protein, and aspartate aminotransferase were higher in colostrum-fed lambs than in N0h lambs. Serum concentrations of insulin, total protein, insulin-like growth factor 1, and γ-glutamyl transpeptidase were higher in C24h lambs than in N24h or M24h lambs. Apparent efficiencies of IgG absorption in C6h, C12h, and C24h lambs were 14.4, 26.8, and 17.2%, respectively, whereas apparent efficiencies of lactoferrin (LF), α-lactalbumin (α-LA), and ß-lactoglobulin (ß-LG) absorption were very low in colostrum-fed lambs, with mean values of 0.06, 0.002, and 0.003%, respectively. Concentrations of IgG, LF, α-LA, and ß-LG in the digesta of the abomasum, jejunum, and ileum rapidly decreased from C6h to C24h lambs, and the disappearance rates of IgG, LF, α-LA, and ß-LG were higher in lambs from C6h to C12h (62.1, 75.7, 91.3, and 95.0% for IgG, LF, α-LA, and ß-LG, respectively) than from C12h to C24h (34.6, 22.5, 7.5, and 2.2% for IgG, LF, α-LA, and ß-LG, respectively). These results indicated that bovine colostrum feeding improved the metabolic and immunological status of lambs, and that ingested colostral IgG was prone to intact uptake into the blood, whereas almost all ingested LF, α-LA, and ß-LG disappeared in the lumen of the gastrointestinal tract in a time-dependent manner. The findings provide novel information for exploring selective absorption of colostral compounds in the small intestine of lambs.

Quantitative comparative phosphoproteomic analysis of the effects of colostrum and milk feeding on liver tissue of neonatal calves.

Posttranslational modifications, mostly phosphorylation, are critical for protein structure and function. However, the association between liver phosphoproteins in neonatal calves and colostrum intake is not well understood. In this study, we examined the liver phosphoproteome profile in neonatal calves after receiving colostrum or milk. Liver tissue samples were collected from control calves (CON, n = 3) 2 h after birth and from calves that received colostrum (CG, n = 3) or milk (MG, n = 3) 24 h after birth. Hepatic phosphoprotein expression profiles were analyzed using quantitative proteomics based on the liquid chromatography-tandem mass spectrometry method. In total, 1,587 phosphorylated sites were identified in 1,011 liver proteins. The most abundant phosphorylation site AA was serine (87.5%), followed by threonine (11.9%) and tyrosine (0.5%). Among the 1,011 phosphoproteins, 219, 453, and 26 displayed differential expression in the CG versus MG, CG versus CON, and MG versus CON comparisons, respectively. Differentially expressed phosphoproteins in the CG-MG comparison included 3-phosphoinositide-dependent protein kinase 1, glucose transporter member 4, protein kinase N2, and vinculin, which were mainly involved in the glycogen metabolic process, transport, growth and development, and cell adhesion process, according to Gene Ontology analysis. Pathway analysis indicated their enrichment in the insulin signaling pathway, spliceosome, and adherens junction. The CG-CON comparison identified differentially expressed phosphoproteins and their target genes that were largely involved in the cellular process, macromolecule metabolic process, developmental process, and transport. Pathway analysis indicated their association with endocytosis, mechanistic target of rapamycin, AMP-activated protein kinase, and insulin signaling pathways. These data demonstrate that changes in the phosphoproteins of liver tissues may play an important role in energy metabolism and immune response in the calves that received colostrum. These results provide novel insights into the crucial roles of protein phosphorylation during the early life of newborn calves.

Changes in bacterial community and expression of genes involved in intestinal innate immunity in the jejunum of newborn lambs during the first 24 hours of life.

The newborn gut undergoes rapid colonization by commensal microorganisms and possible exposure to pathogens. The contribution of colostrum intake to host protection is well known; however, limited research exists on the intestinal innate immunity corresponding to colostrum intake during the passive immune transfer period in newborn ruminants. The aim of this study was to investigate the changes in bacterial community and expression of genes encoding toll-like receptors (TLR), mucins (MUC), antimicrobial peptides, and tight junctions in the jejunum of lambs that were fed colostrum during the first 24 h of life. Twenty-seven newborn lambs were used in this study, of which 18 lambs were bottle-fed pooled bovine colostrum within the first 2 h after birth to obtain an intake of approximately 8% of body weight. Lambs were slaughtered at 12 (n = 9) and 24 h (n = 9) after birth. The remaining 9 lambs without any feeding were slaughtered at 30 min after birth (0 h). Tissue and ligated segment samples from the jejunum were collected immediately after the lambs were slaughtered. The bacterial profile in the ligated jejunum segment was assessed using amplicon sequencing. The gene expression in the jejunum tissue was determined using quantitative real-time PCR. The relative abundances of Escherichia-Shigella, Lactobacillus, Lactococcus, and Streptococcus increased, whereas those of Sphingomonas, Phyllobacterium, Bradyrhizobium, and Rudaea decreased during the first 24 h of life. Expression of TLR2 and ß-defensin 109-like was upregulated at 12 h after birth, but a recovery was detected at 24 h; TLR3, TLR5, LYZ, MUC1, MUC13, MUC20, and CLDN7 showed a higher expression level in samples taken at 24 h than in those taken at 0 h. In addition, expression level of CLDN1, CLDN4, and the junctional adhesion molecule-1 tended to be higher at 24 h than at 0 h after birth. Correlation analysis indicated that TLR2 expression was negatively correlated with the relative abundance of Lactobacillus and Bradyrhizobium, whereas TLR5 expression was positively correlated with the relative abundance of Escherichia-Shigella and Pelagibacterium. These results suggest that TLR, MUC, antimicrobial peptides, and CLDN act together and play an important role in intestinal defense during the passive immune transfer period. They are potentially associated with microbial colonization. The findings from this study provide novel information to elucidate the role of colostrum components in regulating the development of the intestinal mucosal immune barrier in newborn lambs during the passive immune transfer period.

Label-free quantitative proteomics analysis reveals the fate of colostrum proteins in the intestine of neonatal calves.

The contribution of intestinally absorbed colostral immunoglobulins to the transmission of passive immunity is widely reported in neonatal calves. However, changes in the colostral proteome in the gastrointestinal digesta remain unclear. Therefore, this study aimed to investigate changes in colostral proteome affected by gastrointestinal proteases in neonatal calves. Twenty-one neonatal Holstein calves were used in this study, including 18 colostrum-fed calves slaughtered at 8 (CI, n = 6), 24 (CII, n = 6), and 36 h (CIII, n = 6) postpartum and 3 milk-fed calves slaughtered 24 h postpartum (MI, n = 3). The ingested colostrum and milk samples were collected from the mid-jejunum segment, following the sacrifice. The undigested colostrum or milk along with their ingested colostrum or milk samples were investigated using a label-free proteomics approach. Hierarchical clustering and principal component analysis of the quantified proteins revealed that the ingested colostrum from the CII and CIII groups and the ingested mature milk from the MI group appeared to share similar patterns. Analysis of the intestinal digesta revealed a time-dependent decrease in caseins, lactoferrin, and osteopontin protein levels, and an increase in cationic trypsin, chymotrypsin, and carboxypeptidase. Several protease inhibitors, such as α-1-antiproteinase, α-2-antiplasmin, and early lactation protein, were identified in the colostrum and intestinal digesta. In addition, we detected identical levels in the intestinal digesta and colostrum for albumin, α-1-acid glycoprotein, and plasminogen. Pathway analysis indicated that proteins increased in the intestinal digesta belonged to the following categories: biosynthesis of antibiotics, carbon metabolism, and biosynthesis of amino acids. These results indicated that selected colostral proteins were digested by gastrointestinal proteases, contributing to their intestinal absorption in calves. These findings provide new insights into the fate of the colostral proteome in the gastrointestinal tract and may aid in the identification of factors contributing to health management in neonatal calves.

Changes in expression of antimicrobial peptides and Fc receptors in the small intestines of neonatal calves during the passive immunity period.

The contribution of colostrum to passive immunity transfer and intestinal protection is well known; however, the effects of colostrum intake on the expression of antimicrobial peptides (AP) and Fc receptors in the intestine of neonatal calves are unclear. Our aim was to investigate changes in the expression of AP and Fc receptor in the small intestine of calves in the first 36 h postpartum. Twenty-four Holstein bull calves were used in this study, of which 18 calves were administered 3.2 L of pooled colostrum for each calf per meal via an esophageal tube. Calves were slaughtered at 8 h (1 meal at 1-2 h), 24 h (2 meals at 1-2 h and 10-12 h), and 36 h (3 meals at 1-2 h, 10-12 h, and 22-24 h) postpartum. The remaining 6 calves without any milk administration were slaughtered at 2 h postpartum. Samples of blood and jejunum digesta were collected to determine immunoglobulin concentration using ELISA. Samples of the duodenum, jejunum, and ileum tissues after slaughter were collected to determine AP and Fc receptor expression using quantitative real-time PCR. In calves administered colostrum, IgG concentration in jejunum digesta rapidly decreased in an age-dependent manner (33.41, 9.47, and 0.34 mg/mL at 8, 24, and 36 h, respectively), whereas serum IgG concentration increased significantly, from 0.25 µg/mL at 2 h to 21.72 mg/mL at 24 h. Cathelicidin-4, ß-defensin (DEFB)-7, and enteric ß-defensin expression was upregulated at 8 h postpartum in the duodenum and jejunum compared with that at 2 h, but progressive recovery was detected from 24 h onward. Higher expression of cathelicidin-4, regenerating family member 3γ, lysozyme (LYZ), LYZ1, and LYZ2 and lower expression of DEFB, DEFB1, DEFB7, DEFB10, and enteric ß-defensin were observed in the duodenum and jejunum compared with the ileum. Differences in AP expression between intestinal regions suggested that the innate immune defense mechanism varied significantly among the duodenum, jejunum, and ileum. No difference in the expression of Fc fragment of the IgG receptor was observed either among ages or small intestinal regions. The Fcγ receptor (FcγR)Ia and FcγRIIb expression was the highest at 8 h compared with that at 2, 24, and 36 h, and expression of FcγRIa, FcγRIIb, and FcγRIIIa was higher in the duodenum and jejunum than in the ileum. These results indicated that AP and Fcγ receptors might play important roles in intestinal defense during the passive immunity period.

Alterations of the circular RNA profile in the jejunum of neonatal calves in response to colostrum and milk feeding.

Circular RNA (circRNA) have been suggested to contribute to regulating gene expression in various tissues and cells of eukaryotes. However, little is known regarding the expression pattern of circRNA and their potential function in the small intestine of neonatal calves that receive colostrum. In the current study, jejunum tissue samples were collected from control calves (2 h after birth; CT; n = 3) and neonatal calves that ingested colostrum (24 h after birth; CO; n = 3) or milk (24 h after birth; MK; n = 3) to compare the circRNA expression patterns using a high-throughput RNA sequencing approach. A total of 21,213, 17,861, and 21,737 circRNA were identified in the CT, CO, and MK groups, respectively. Only 13,254 of these circRNA were common to the 3 groups, suggesting high specificity of circRNA expression depending on nutrient type. In total, 243, 249, and 283 circRNA were differentially expressed in the CO versus CT, CO versus MK, and MK versus CT comparisons, respectively. Gene ontology analysis showed that the differentially expressed circRNA and their predicted or known target genes from the CO and MK groups were mainly involved in macromolecule metabolic process, response to stress, and vesicle-mediated transport. Moreover, pathway analysis showed that the Rap1 signaling pathway, focal adhesion, ubiquitin-mediated proteolysis, and extracellular matrix-receptor interaction were the most significantly enriched pathways. These data collectively indicate that circRNA are abundant and dynamically expressed when calves receive colostrum and act as microRNA sponges to regulate their target genes for jejunum function during the early development of newborn calves.

Changes in serum metabolites in response to ingested colostrum and milk in neonatal calves, measured by nuclear magnetic resonance-based metabolomics analysis.

Uptake of colostrum is of central importance for establishing a passive immunity transfer in neonatal calves. Studies of absorption and transmission of colostral immunoglobulins have been widely reported; however, changes in the serum in response to the absorption of colostral components in neonatal calves have not been completely characterized. Here, a nuclear magnetic resonance-based metabolomics approach was used to investigate the changes in metabolites in ingested colostrum, milk, and serum after neonatal calves were fed colostrum or milk. Twenty-seven neonatal male Holstein calves were assigned to 1 of the following groups: (1) calves not fed colostrum or milk and slaughtered approximately 2 h after birth (control group, n = 6), (2) calves fed colostrum at 1 to 2 h after birth and slaughtered 8 h after birth (n = 6), (3) calves fed 2 colostrum meals (at 1-2 and 10-12 h after birth) and slaughtered 24 h after birth (n = 6), (4) calves fed 3 colostrum meals (at 1-2, 10-12, and 22-24 h after birth) and slaughtered 36 h after birth (n = 6), or (5) calves fed 2 milk meals (1-2 and 10-12 h after birth) and slaughtered 24 h after birth (n = 3). Concentrations of valine, leucine, lactate, lysine, and isoleucine were higher and concentrations of lactose were lower in the groups fed colostrum and milk compared with groups not fed colostrum and milk, respectively. Metabolite changes between groups fed or not fed colostrum and milk were similar and may reflect the primary metabolic requirements of ingestion by the small intestine of neonatal calves. Concentrations of serum metabolites choline, valine, leucine, and glutamate were higher in the serum of calves that received colostrum compared with control calves. Furthermore, concentrations of serum phenylalanine, valine, and glutamate were significantly higher, whereas serum concentrations of citrate and very low density lipoproteins were lower in calves that received colostrum compared with calves fed milk. Our results indicate that concentrations of leucine, valine, and glutamate, which were higher in the calves that ingested colostrum, may transfer into the bloodstream, and that these metabolites are associated with health benefits in the neonatal calves that received colostrum. These findings provide novel information to help us understand the mechanism by which colostrum components are metabolized and absorbed in the small intestine and then transferred into bloodstream of neonatal calves.

The transformer genes in the fig wasp Ceratosolen solmsi provide new evidence for duplications independent of complementary sex determination.

Transformer (tra) is the key gene that turns on the sex-determination cascade in Drosophila melanogaster and in some other insects. The honeybee Apis mellifera has two duplicates of tra, one of which (complementary sex determiner, csd) is the primary signal for complementary sex-determination (CSD), regulating the other duplicate (feminizer). Two tra duplicates have been found in some other hymenopteran species, resulting in the assumption that a single ancestral duplication of tra took place in the Hymenoptera. Here, we searched for tra homologues and pseudogenes in the Hymenoptera, focusing on five newly published hymenopteran genomes. We found three tra copies in the fig wasp Ceratosolen solmsi. Further evolutionary and expression analyses also showed that the two duplicates (Csoltra-B and Csoltra-C) are under positive selection, and have female-specific expression, suggesting possible sex-related functions. Moreover, Aculeata species exhibit many pseudogenes generated by lineage-specific duplications. We conclude that phylogenetic reconstruction and pseudogene screening provide novel evidence supporting the hypothesis of independent duplications rather an ancestral origin of multiple tra paralogues in the Hymenoptera. The case of C. solmsi is the first example of a non-CSD species with duplicated tra, contrary to the previous assumption that derived tra paralogues function as the CSD locus.

Diversity and evolution of Ty1-copia retroelements within Chalcidoidea by reverse transcriptase domain analysis.

Ty1-copia retrotransposons are widespread and diverse in insects. Some features of their hosts, such as mating and genetic systems, are predicted to influence the spread of selfish genetic elements like Ty1-copia. Using part of the reverse transcriptase gene as a reference, we experimentally surveyed Ty1-copia elements in eight species of fig wasps (Hymenoptera: Chalcidoidea), and performed an in silico analysis of six available genomes of chalcid wasps. Contrary to initial expectations that selfish elements such as Ty1-copia would be purged from the genomes of these species because of inbreeding and haplodiploidy, almost all of these wasps harbour an abundance of diverse Ty1-copia elements. Phylogenetic analyses suggest that the families of Ty1-copia elements found in these species have had a long association with their chalcid hosts. These results suggest an evolutionary scenario in which there was ancestral polymorphism followed by some taxa-specific events including stochastic loss and further diversification. Furthermore, estimating natural selection within the internal and terminal portions of the Ty1-copia phylogenies demonstrated that the elements are under strong evolutionary constraints for their long-term survival, but evolve like pseudogenes in the short term, accompanied by the rise and fall of parasitic elements in the history of wasp lineage.

Subtraction suppressive hybridisation analysis of differentially expressed genes associated with puberty in the goat hypothalamus.

The cost of developing replacement nanny goats could be reduced by decreasing the age at puberty because this way nanny goats could be brought into production at an earlier age. The aim of the present study was to screen genes related to puberty to investigate the molecular mechanisms of puberty. Subtracted cDNA libraries were constructed for hypothalami from juvenile (Group A), pubertal (Group B) and age-matched control pubertal (Group E) Jining grey (JG) and Liaoning cashmere (LC) goats using suppression subtractive hybridisation (SSH). Differentially expressed genes were analysed by bioinformatics methods. There were 203 expressed sequence tags (ESTs) in the subtracted cDNA libraries that were differentially expressed between JG and LC goats at the juvenile stage, 226 that were differentially expressed at puberty and 183 that were differentially expressed in the age-matched control group. The differentially expressed ESTs in each subtracted cDNA library were classified as known gene, known EST and unknown EST according to sequence homology in the GenBank non-redundant (NR) and EST database. According to gene function analysis in the COG (Cluster of Orthologous Groups) database, the known genes were grouped into 10 subdivisions in Group A, into seven subdivisions in Group E and into nine subdivisions in Group B under three categories: cellular processes and signalling, information storage and processing, and metabolism. Pathway analysis in the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database of known genes revealed that the three pathways that most differentially expressed genes were involved in were metabolic pathways, Parkinson's disease and oxidative phosphorylation. Protein interaction analysis of the high homology genes revealed the most dominant network to be structure of ribosome/protein translation, oxidative phosphorylation and carbohydrate metabolism. The results reveal that the onset of puberty is a complex event involving multiple genes in multiple biological processes. The differentially expressed genes include genes related to both neuroendocrine and energy metabolism.

Odorant-binding protein (OBP) genes affect host specificity in a fig-pollinator mutualistic system.

The interaction between figs and their pollinating wasps is regarded as a model system for studying specialized co-evolved mutualism. Chemoreception of fig wasps plays an important role in this interaction, and odorant-binding proteins (OBP) function in the first step of odorant detection. The OBP repertoire of the fig wasp Ceratosolen solmsi is reported to be one of the smallest among insects; however, it is unknown how these OBPs are related to the complicated mating process occurring within the fig cavity and the extreme host specificity of the species. In the present study, we combined a structural analysis of the conserved cysteine pattern and motif order, a phylogenetic analysis, and previous studies on ligand-binding assays to deduce the function of OBPs. We also quantified the expression of OBP genes in different life stages of female and male fig wasps by using real-time quantitative PCR, which can help to predict the function of these genes. The results indicated that CsolOBP1 and CsolOBP2 (or CsolOBP5) in males may bind to pheromones and play important roles in mate choice, whereas CsolOBP4 and CsolOBP5 may primarily function in host localization by females through binding of volatile compounds emitted by receptive figs.

Multiple ancient horizontal gene transfers and duplications in lepidopteran species.

Eukaryotic horizontal gene transfer (HGT) events are increasingly being discovered yet few reports have summarized multiple occurrences in a wide range of species. We systematically investigated HGT events in the order Lepidoptera by employing a series of filters. Bombyx mori, Danaus plexippus and Heliconius melpomene had 13, 12 and 12 HGTs, respectively, from bacteria and fungi. These HGTs contributed a total of 64 predicted genes: 22 to B. mori, 22 to D. plexippus and 20 to H. melpomene. Several new genes were generated by post-transfer duplications. Post-transfer duplication of a suite of functional HGTs has rarely been reported in higher organisms. The distributional patterns of paralogues for certain genes differed in the three species, indicating potential independent duplication or loss events. All of these HGTs had homologues expressed in some other lepidopterans, indicating ancient transfer events. Most HGTs were involved in the metabolism of sugar and amino acids. These HGTs appeared to have experienced amelioration, purifying selection and accelerated evolution to adapt to the background genome of the recipient. The discovery of ancient, massive HGTs and duplications in lepidopterans and their adaptive evolution provides further insights into the evolutionary significance of the events from donors to multicellular host recipients.

Analysis on DNA sequence of TSHB gene and its association with reproductive seasonality in goats.

Thyroid stimulating hormone beta chain (TSHB) is mainly expressed in pituitary and its expression is closely related to photoperiodic control of seasonal reproduction in animals. In the present study, ten primer pairs have been used to clone the DNA sequence and to detect genetic mutations of goat TSHB gene. Two DNA fragments of goat TSHB gene were obtained, which were 2,614 and 1,031 bp in length, respectively. They comprised about 2.5 kb 5' regulatory region, all of the three exons and two introns. Goat TSHB gene has a coding region of 417 bp, encoding 138 amino acids which was predicted to be a secretory protein with a signal peptide of 16 amino acids. The sequence of TSHB gene is highly conserved among mammals. In addition, five mutations (C53A, 3 bp Indel at the 287-289 locus, 34 bp Indel at the 584-617 locus, A1819C and E2_72TA) were found in goat TSHB gene and they were shown to be in strong linkage disequilibrium. Interestingly, the genotype distributions for both single locus and haplotype have shown to be significant different between seasonal and nonseasonal goat breeds. And haplotype H2 and diplotype H2/H4 may be related to year-round estrus. We preliminarily presumed that the five closely linked mutations of goat TSHB gene may be part of the causal sources for the diversities of reproductive seasonality in goats. Our study may provide a possible efficient genetic way to decrease seasonality in goats.

Analysis on DNA sequence of goat RFRP gene and its possible association with average daily sunshine duration.

Goat RFRP gene was cloned and its mutations were detected in thirteen goat breeds whose reproductive seasonality and litter size were different. Then sequence characteristics were analyzed and association analyses were performed to reveal the relationships between mutations of RFRP gene and average daily sunshine duration, reproductive seasonality as well as litter size in goats. A 4,862 bp DNA fragment of goat RFRP gene was obtained and the complete CDS of 591 bp encodes 196 amino acids, having high homology with that of other mammals. The protein was predicted to be a secreted protein with a signal peptide of 21 amino acids. Moreover, two mutations (A712G, T1493C) in 5' regulatory region and one mutation (A3438T) in exon 2 were detected. The test of genotype distribution in six selective goat breeds showed that there was no uniform significant association between the three polymorphisms and seasonal reproduction. The association just existed in some goat breeds for each locus. Interestingly, however, there was a strong positive correlation (r = 0.830, P = 0.003) between the G allele frequency of the A712G locus and average daily sunshine duration in ten local goat breeds, suggesting that RFRP gene has undergone a selective pressure in sunshine duration and may have indirect relationship with reproductive seasonality in goats. Additionally, no significant difference was found in litter size between genotypes in prolific Jining Grey goats.

Polymorphism of 5' regulatory region of ovine FSHR gene and its association with litter size in Small Tail Han sheep.

Single nucleotide polymorphisms of 5' regulatory region of follicle-stimulating hormone receptor (FSHR) gene were detected in two high prolificacy sheep breeds (Small Tail Han and Hu sheep) and two low prolificacy sheep breeds (Corriedale and Chinese Merino sheep) by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The results indicated that there were three genotypes (AA, AB and BB) detected by primer 1 in Hu sheep while only one genotype (AA) in other three sheep breeds, and frequencies of AA, AB and BB genotypes in Hu sheep were 0.700, 0.225 and 0.075, respectively. There were three genotypes (EE, EF and EG) detected by primer 3 in Small Tail Han sheep while only EE genotype occurred in other three sheep breeds, and frequencies of EE, EF and EG genotypes in Small Tail Han sheep were 0.775, 0.200 and 0.025, respectively. No polymorphism was detected in four sheep breeds by primer 2 and primer 4. The sequencing results showed that there were two nucleotide mutations (g. -681T>C and g. -629C>T) in genotype BB compared with AA for primer 1. As for primer 3, two mutations (g. -197G>A and g. -98T>C) in genotype EF compared with EE and two mutations (g. -200G>A and g. -197G>A) in genotype EG compared with EE. The heterozygous ewes with EG or EF had 0.89 (P < 0.05) or 0.42 (P < 0.05) lambs more than homozygous ewes (EE genotype) in Small Tail Han sheep, respectively, while there was no significant difference on litter size between EG and EF ewes.

Polymorphism of exon 2 of BoLA-DRB3 gene and its relationship with somatic cell score in Beijing Holstein cows.

In the present study, the exon 2 and 3' end sequence of intron 1 of BoLA-DRB3 gene of 211 Beijing Holstein cows was amplified and a uniform fragment of 284 bp was obtained. The genetic polymorphism was investigated by PCR-RFLP using two restriction endonucleases HaeIII and BstYI. Seven genotypes were detected by digesting the PCR products with HaeIII. The frequency of AA, AB, AC, AD, BB, BC and BF genotypes was 0.4638, 0.0193, 0.0193, 0.3478, 0.0290, 0.0386 and 0.0822, respectively. Three genotypes were found by digesting the PCR products with BstYI. The frequency of AA, AB and BB genotypes was 0.0569, 0.2844 and 0.6587, respectively. The relationship between the polymorphisms in exon 2 of BoLA-DRB3 gene and somatic cell score (SCS) in Beijing Holstein cows was analyzed by least squares linear model. No significant difference was detected among least squares means of SCS for seven HaeIII-RFLP genotypes (P > 0.05). As for BstYI-RFLP analysis, least squares mean of SCS for AA was significantly lower than that for AB (P < 0.05) or BB (P < 0.05), and no significant difference was detected between AB and BB genotypes (P > 0.05). BstYI AA was the most favorable genotype and BstYI BB was the most unfavorable genotype for mastitis resistance. The information found in the present study is very important for improving mastitis resistance in dairy cattle by marker assisted selection.

Polymorphism of insulin-like growth factor 1 gene and its association with litter size in Small Tail Han sheep.

The insulin-like growth factor 1 (IGF1) gene was studied as a candidate gene for high prolificacy in sheep. Polymorphisms of 5' regulatory region and all four exons of IGF1 gene were detected in Small Tail Han (n = 277), Hu (n = 58), Texel (n = 48) and Dorset (n = 46) sheep by PCR-RFLP and PCR-SSCP analysis. A microsatellite polymorphic site and a restriction fragment length polymorphism were shown in the 5' regulatory region of IGF1 gene. The ewes with genotype 123/123 bp had 0.81 (P < 0.05) or 1.03 (P < 0.01) lambs more than those with genotype 125/125 bp or 125/127 bp, the ewes with genotype 123/125 bp had 0.46 (P < 0.05) or 0.68 (P < 0.01) lambs more than those with genotype 125/125 bp or 125/127 bp. In addition, there were two mutations (C1511G and A1513G) in 5' regulatory region of IGF1 gene. The ewes with genotype BB or AB had 0.96 (P < 0.05) or 0.38 (P < 0.05) lambs more than those with genotype AA, but there were no significant differences between BB and AB genotypes (P > 0.05) in Small Tail Han sheep. These results preliminarily indicated that these polymorphisms of IGF1 gene could be used in molecular marker-assisted selection for sheep breeding programs.