Association analysis between Acetyl-Coenzyme A Acyltransferase-1 gene polymorphism and growth traits in Xiangsu pigs.

Introduction: Acetyl-Coenzyme A Acyltransferase-1 (ACAA1) is a peroxisomal acyltransferase involved in fatty acid metabolism. Current evidence does not precisely reveal the effect of the ACAA1 gene on pig growth performance. Methods: The present study assessed the mRNA expression levels of the ACAA1 gene in the heart, liver, spleen, lung, kidney of 6-month-old Xiangsu pigs and in the longissimus dorsi muscle at different growth stages (newborn, 6 months and 12 months of age) using RT-qPCR. The relationship between single-nucleotide polymorphisms (SNPs) of ACAA1 gene and growth traits in 6-month-old and 12-month-old Xiangsu pigs was investigated on 184 healthy Xiangsu pigs using Sanger sequencing. Results: The ACAA1 gene was expressed in heart, liver, spleen, lung, kidney, and longissimus dorsi muscle of 6-month-old pigs, with the highest level of expression in the liver. ACAA1 gene expression in the longissimus dorsi muscle decreased with age (p < 0.01). In addition, four SNPs were identified in the ACAA1 gene, including exon g.48810 A>G (rs343060194), intron g.51546 T>C (rs319197012), exon g.55035 T>C (rs333279910), and exon g.55088 C>T (rs322138947). Hardy-Weinberg equilibrium (p > 0.05) was found for the four SNPs, and linkage disequilibrium (LD) analysis revealed a strong LD between g.55035 T>C (rs333279910) and g.55088 C>T (rs322138947) (r 2 = 1.000). Association analysis showed that g.48810 A>G (rs343060194), g.51546 T>C (rs319197012), g.55035 T>C (rs333279910), and g.55088 C>T (rs322138947) varied in body weight, body length, body height, abdominal circumference, leg and hip circumference and living backfat thickness between 6-month-old and 12-month-old Xiangsu pigs. Conclusion: These findings strongly demonstrate that the ACAA1 gene can be exploited for marker-assisted selection to improve growth-related phenotypes in Xiangsu pigs and present new candidate genes for molecular pig breeding.

Association between polymorphisms in NOBOX and litter size traits in Xiangsu pigs.

The newborn ovary homeobox gene (NOBOX) regulates ovarian and early oocyte development, and thus plays an essential role in reproduction. In this study, the mRNA expression level and single nucleotide polymorphism (SNP) of NOBOX in various tissues of Xiangsu pigs were studied to explore the relationship between its polymorphism and litter size traits. Also, bioinformatics was used to evaluate the effects of missense substitutions on protein structure and function. The results revealed that NOBOX is preferentially expressed in the ovary. Six mutations were detected in the NOBOX sequence, including g.1624 T>C, g.1858 G>A, g.2770 G>A, g.2821 A>G, g.5659 A>G, and g.6025 T>A, of which g.1858 G>A was a missense mutation. However, only g.1858 G>A, g.5659 A>G, and g.6025 T>A were significantly associated with litter size traits (p < 0.05). Further prediction of the effect of the missense mutation g.1858 G>A on protein function revealed that p.V82M is a non-conservative mutation that significantly reduces protein stability and thus alters protein function. Overall, these findings suggest that NOBOX polymorphism is closely related to the litter size of Xiangsu pigs, which may provide new insights into pig breeding.

Genetic variation in the BLM gene and its expression in the ovaries is closely related to kidding number in goats.

Bloom (BLM) helicase plays an important role in DNA replication and the maintenance of genome integrity. BLM protein deficiency, which plays a vital role in the sperm-egg union and germ-cell development during reproduction, can lead to severe DNA damage in goats. However, the effect of BLM protein deficiency on goat litter size has not been reported. Herein, we studied the association between the genetic variation in the BLM gene and the number of kids per litter in Guizhou white goats. We explored differences in the expression of the BLM protein in the follicles of single and multi-kid nanny goats. We also analyzed the effects of dysregulated BLM gene expression on the proliferation and apoptosis of ovarian granulosa cells and the expression of genes related to follicle development in goats. Five single nucleotide polymorphism (SNP) loci, including the non-synonymous mutations g.38179 A > G, g.40626 G > C and g.89621 T > G; the intron synonymous mutation g.56961 G > A and the exon synonymous mutation g.65796 C > T were found in the BLM gene. All SNPs loci were in Hardy-Weinberg equilibrium, and correlation analysis showed that the g.65796 C > T and g.89621 T > G loci polymorphism was strongly associated with litter size in the first three litters (P < 0.05). The diplogenotype Hap 2/2 (AAGGAACCTT) showed no significant difference in litter size between different births, indicating that the diploid genotype is stable in different litter sizes. Bioinformatics analysis showed that three non-synonymous mutation loci (p.T488A, p.A662S, and p.S1373A) could affect BLM protein stability, and mutations in p.T488A and p.S1373A led to changes in amino acid polarity and associated interactions. qPCR results showed that the expression level of the BLM gene in the uterus and ovaries of TT genotype nanny goats was significantly higher than that of GG genotype nanny goats. Indirect immunofluorescence assay (IF) showed that the BLM protein was significantly overexpressed in both the primordial and growing follicles of nanny goats with multiple kids (P < 0.01). Disrupting BLM gene expression in the ovarian granulosa cells down-regulated the expression of the Cyp19A1 gene. It also significantly inhibited the proliferation of follicles and induces early apoptosis of the granulosa cells. These findings confirm that polymorphism in the BLM gene is closely related to the littering traits of Guizhou white goats, and it affects the reproductive performance of nanny goats by regulating the development of the oocytes and granulosa cells. This work provides new evidence on the regulatory effect of the BLM gene on the litter size of nanny goats.

A novel nonsynonymous SNP in the OLR1 gene associated with litter size in Guizhou white goats.

Oxidized low-density lipoprotein receptor-1 (OLR1) encodes a low-density lipoprotein receptor belonging to the C-type lectin superfamily, which is closely related to reproduction. OLR1 is associated with fecundity in Awassi sheep. However, its effect on litter size has not been investigated in goats. In this study, OLR1 sequences and their mRNA expression levels in the gonadal axis of Guizhou white goats were evaluated to investigate the relationship between gene polymorphisms and litter size. In addition, the potential effects of a nonsynonymous substitution were evaluated using a bioinformatics approach. The expression levels of OLR1 were highest in the uterus of mothers with multiple kids and highest in the ovaries of mothers with single kids. OLR1 mRNA expression levels in the ovaries of mothers with single kids were two times higher than in the ovaries of mothers with multiple kids. The sequencing results revealed five SNPs in OLR1; however, only g.294 T > A, g.2260 T > C, and g.2268 C > T were significantly associated with litter size (P < 0.05). Linkage disequilibrium was detected between g.2260 T > C and g.2268 C > T (r2 = 0.322, D' = 0.6). Additionally, goats with the Hap 1/1 diplotype had a greater litter size than others (P < 0.05). g.2260 T > C was a nonsynonymous mutation that resulted in the replacement of valine with alanine at the amino acid residue 54 of the OLR1 protein. Bioinformatic analyses revealed that the p.V54A locus was relatively conserved in cloven-hoofed species. Mutations at this locus could change the local conformation and reduce the stability of OLR1, affecting its half-life and the litter size of the nanny goat. These findings confirm that OLR1 affects goat kidding traits and provide a novel insight into the regulatory mechanism underlying the effect of OLR1 on litter size.

Association analysis of PLIN2 gene polymorphisms and lambing performance in Qianbei Ma goats.

Qianbei Ma goats are one of the three most valued local goat breeds in Guizhou, China; furthermore, it has lower litter size performance. The purpose of this study was to explore the correlation between SNP (single-nucleotide polymorphisms) of PLIN2 gene and lambing performance. The bioinformatics analysis, DNA sequencing, RT-qPCR and correlation analysis methods were used to analyse the evolutionary relationship of PLIN2 protein in 13 species, to detect the expression pattern of PLIN2 gene in the gonad axis of Qianbei sheep, to explore the dominant genotype of PLIN2 related to lambing traits and to screen molecular markers related to lambing performance to guide the breeding of Qianbei Ma goats. Results showed that the Qianbei Ma goat PLIN2 protein had the closest genetic relationship with sheep and the furthest from mice, there were significant or extremely significant differences in the expression levels of the PLIN2 gene in the gonadal axis of the mothers of single- and multi-lamb groups. Compared with the reference sequence, four SNPs were found, which were g.1006 C → A and g.1171 A → G in the first and second intron regions of the PLIN2 gene, g.8514 C → T in the exon 8 region and g.9122 A → T in the 3'UTR. The correlation analysis showed that g.1006 C → A, g.8514 C → T and g.9122 A → T had significant indigenous effects on the lambing performance of Qianbei Ma goats (p < .05). The number of third births for diploid H2H5 was significantly higher than that of diploid H1H2, and the number of first to third births for diploid H2H5 was large and stable. The results showed that PLIN2 gene could be used as a candidate gene related to lambing traits of Qianbei Ma goat.

Association Analysis of PRKAA2 and MSMB Polymorphisms and Growth Traits of Xiangsu Hybrid Pigs.

In this study, Xiangsu hybrid pig growth traits were evaluated via PRKAA2 and MSMB as candidate genes. Sanger sequencing revealed three mutation sites in PRKAA2, namely, g.42101G>T, g.60146A>T, and g.61455G>A, and all these sites were intronic mutations. Moreover, six mutation sites were identified in MSMB: intronic g.4374G>T, exonic g.4564T>C, exonic g.6378G>A, exonic g.6386C>T, intronic g.8643G>A, and intronic g.8857A>G. Association analysis revealed that g.42101G>T, g.60146A>T, g.61455G>A, g.4374G>T, g.4564T>C, g.6378G>A, g.6386C>T, g.8643G>A, and g.8857A>G showed different relationship patterns among body weight, body length, body height, chest circumference, abdominal circumference, tube circumference, and chest depth. Real-time polymerase chain reaction results revealed that the expression of PRKAA2 was highest in the longissimus dorsi muscle, followed by that in the heart, kidney, liver, lung, and spleen. The expression of MSMB was highest in the spleen, followed by that in the liver, kidney, lung, heart, and longissimus dorsi muscle. These results suggest that PRKAA2 and MSMB can be used in marker-assisted selection to improve growth related traits in Xiangsu hybrid pigs, providing new candidate genes for Pig molecular breeding.