Associations of CALM1 and DRD1 polymorphisms, and their expression levels, with Taihang chicken egg-production traits.

Egg production by hens is an important reproductive performance index in the poultry industry. To investigate the effects of the CALM1 and DRD1 genes on egg production in chicken, their mRNA expression and single nucleotide polymorphisms (SNP) levels were investigated, and bioinformatics and egg-production association analyses were performed. Three SNPs (g.44069941G > A and g.44069889A > G in CALM1 and g.10742639C > T in DRD1) were detected in the exons and introns of CALM1 and DRD1 in 400 Taihang chickens. Among them, g.44069941G > A was significantly associated with Taihang chicken egg production on the 500th day (p < 0.05), whereas g.10742639C > T was significantly associated with the 300th day (p < 0.05). The expression levels of CALM1 and DRD1 in ovarian tissues of a high-yielding Taihang group were greater than in a low-yielding group (p < 0.05). The bioinformatics analysis revealed that the mutations influenced the mRNA secondary structures of CALM1 and DRD1. This study provides new insights into the potential effects of CALM1 and DRD1 polymorphisms on chicken egg production. The two SNPs g.44069941G > A and g.10742639C > T are potential molecular markers for improving the reproductive traits of Taihang chicken.

CALM1 and DRD1 were two important genes for reproduction. In this study, the entire coding regions of both genes were sequenced and mutations were detected in Taihang chickens. The results showed that two single nucleotide polymorphisms (SNPs), g.44069941G > A in the CALM1 gene and g.10742639C > T in the DRD1 gene, were associated with egg-laying traits. g.10742639C > T is a synonymous mutation predicted to affect the secondary structure of mRNA. Therefore, these two mutations might be potential molecular markers for improving reproductive traits in Taihang chickens.

Identification and Characterization of lncRNAs Expression Profile Related to Goat Skeletal Muscle at Different Development Stages.

LncRNAs are essential for regulating skeletal muscle. However, the expression profile and function of lncRNAs in goat muscle remains unclear. Here, an average of ~14.58 Gb high-quality reads were obtained from longissimus dorsi tissues of 1-month-old (n = 3) and 9-month-old (n = 3) Wu'an black goats using RNA sequencing. Of a total of 3441 lncRNAs, 1281 were lincRNAs, 805 were antisense lncRNAs, and 1355 were sense_overlapping lncRNAs. These lncRNAs shared some properties with goats, such as fewer exons, shorter transcript, and open reading frames (ORFs) length. Among them, 36 differentially expressed lncRNAs (DE lncRNA) were identified, and then 10 random lncRNAs were validated by RT-qPCR. Furthermore, 30 DE lncRNAs were neighboring 71 mRNAs and several genes were functionally enriched in muscle development-related pathways, such as APC, IFRD1, NKX2-5, and others. Additionally, 36 DE lncRNAs and 2684 mRNAs were included in co-expression interactions. A lncRNA-miRNA-mRNA network containing 4 lncRNAs, 3 miRNAs, and 8 mRNAs was finally constructed, of which XR_001296113.2 might regulate PDLIM7 expression by interaction with chi-miR-1296 to affect skeletal muscle development. This study revealed the expression profile of goat lncRNAs for further investigative studies and provides a fuller understanding of skeletal muscle development.

Effect of polymorphisms in the 5'-flanking sequence of MC1R on feather color in Taihang chickens.

MC1R plays an important role in the regulation of the formation, transfer, and deposition of melanin in animals and is important for determining coat color. Many studies have reported on single nucleotide polymorphisms (SNPs) in the coding sequence of MC1R. However, few studies have investigated the polymorphisms in the 5'-flanking sequence of MC1R. In this study, we sequenced 2000 bp of the 5'-flanking sequence of MC1R in 300 Taihang chickens with brown feathers (MTH) and 300 Taihang chickens with black feathers (HTH). The sequencing results showed that 4 SNPs (MC1R g.18838722 G > C, g.18838624 T > C, g.18838694 G > A, and g.18838624 C > T) were located in the 5'-flanking sequence of MC1R between the MTH and HTH groups. Association analysis showed that there was a significant correlation between the 4 SNPs and feather color in Taihang chickens. The correlation between MC1R g.18838624 T >C and feather color of Taihang chicken was 100%, of which the CC (E1) genotype is MTH and the TT (E2) genotype is HTH. Furthermore, there was a significant correlation between MC1R g.18838624 T > C and egg production at 302 d. E1 (184.14 ± 0.674) was significantly higher than that in E2 (181.75 ± 0.577) (P < 0.05). Luciferase reporter assays were used to detect the transcriptional activity of MC1R with different SNP genotypes. The results showed that the luciferase activity of E2 was significantly higher than that of E1 (P < 0.05). In addition, transcription factor-binding site predictions showed that E2 creates a new binding site for ZEB1. RT‒qPCR results revealed that the expression of MC1R in E2 was significantly lower than that in E1 (P < 0.05), and the expression of ZEB1 in E2 was significantly higher than that in E1 (P < 0.05). Overexpression and shRNA experiments demonstrated that ZEB1 regulates the expression of MC1R in DF1 cells. ZEB1 has a negative regulatory effect on the transcriptional activity of MC1R; it inhibits the expression of MC1R and affects the feather color of Taihang chickens. This study provides new insight into the molecular mechanism of feather color formation and the transcriptional regulation of MC1R in Taihang chickens.

Association analysis and expression level of ACE polymorphisms with egg-laying trait in Taihang chicken.

The number of egg-laying is an important indicator of reproduction performance in poultry breeding. To investigate the relationship between the function of Angiotensin-converting enzyme (ACE) and egg-laying performance of Taihang chicken, the mRNA and protein expression and single nucleotide polymorphism (SNP) of ACE were detected. Analysis of ACE bioinformatics and association analysis of polymorphisms were then performed. The polymorphisms analysis of ACE showed that three SNP loci (g.5066812A>C, g.5080076G>A, and g.5072728A>G) were detected in 800 Taihang chickens with egg-laying records. Association analysis of egg-laying found that ACE g.5066812A>C mutation was significantly associated with the egg-laying performance of Taihang chickens (P < 0.05), and the individuals with the g.5066812A>C mutation showed significantly increasing egg-laying. The mRNA expression was significantly higher in individuals with the AA genotype mutation than those with the AC and CC genotypes (P < 0.01), and the expression of ACE protein levels was consistent with the mRNA expression. Bioinformatics analysis indicated that these mutations affected the secondary and tertiary structure of ACE. This study provides new insights into ACE affecting chicken egg production and some basis for improving the egg production rate of Taihang chickens.

Microbial Diversity and Community Composition of Duodenum Microbiota of High and Low Egg-Yielding Taihang Chickens Identified Using 16S rRNA Amplicon Sequencing.

The duodenum is an important digestive organ for poultry and houses a variety of microbes that help chickens to enhance nutrient absorption and improve production. To evaluate the characteristic of gut microbiome, duodenum content samples from 42-week-old native Taihang chickens with high (H) and low (L) egg-yielding were collected for 16S rRNA amplicon sequencing analysis. Consequently, 1,361,341 sequences were clustered into 2055 OTUs, with percentages of affiliation of 96.50 and 57.30% at phylum and genus levels. Firmicutes, Proteobacteria, Cyanobacteria and Bacteroidetes were the dominant phylum, with a lower ratio of Firmicutes/Bacteroidetes in H group than in L group (p < 0.05). At genus level, overrepresentation of Bacteroides, Faecalibacterim, and Enterococcus and underrepresentation of Romboutsia were found in H group. No significant difference in overall diversity of microbiota was observed between two groups. LEFSe analysis revealed Enterococcus was significantly enriched in H group. Importantly, Enterococcus and Lactobacillus were negatively correlated. Functional prediction analysis showed the proportion of microbiota involved in the metabolism process was the highest and enriched in H group. Differences in microbiota composition between the two groups, which may be related to intestinal function difference, also provide promising biomarkers for improving laying hen production.

Expression Profile Analysis to Identify Circular RNA Expression Signatures in Muscle Development of Wu'an Goat Longissimus Dorsi Tissues.

The growth and development of skeletal muscle is a physiological process regulated by a variety of genes and signaling pathways. As a posttranscriptional regulatory factor, circRNA plays a certain regulatory role in the development of animal skeletal muscle in the form of a miRNA sponge. However, the role of circRNAs in muscle development and growth in goats is still unclear. In our study, apparent differences in muscle fibers in Wu'an goats of different ages was firstly detected by hematoxylin-eosin (HE) staining, the circRNA expression profiles of longissimus dorsi muscles from 1-month-old (mon1) and 9-month-old (mon9) goats were screened by RNA-seq and verified by RT-qPCR. The host genes of differentially expressed (DE) circRNAs were predicted, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses (KEGG) of host genes with DE circRNAs were performed to explore the functions of circRNAs. The circRNA-miRNA-mRNA networks were then constructed using Cytoscape software. Ten significantly differentially expressed circRNAs were also verified in the mon1 and mon9 groups by RT-qPCR. Luciferase Reporter Assay was used to verify the binding site between circRNA and its targeted miRNA. The results showed that a total of 686 DE circRNAs were identified between the mon9 and mon1 groups, of which 357 were upregulated and 329 were downregulated. Subsequently, the 467 host genes of DE circRNAs were predicted using Find_circ and CIRI software. The circRNA-miRNA-mRNA network contained 201 circRNAs, 85 miRNAs, and 581 mRNAs; the host mRNAs were associated with "muscle fiber development" and "AMPK signaling pathway" and were enriched in the FoxO signaling pathway. Competing endogenous RNA (ceRNA) network analysis showed that novel_circ_0005314, novel_circ_0005319, novel_circ_0009256, novel_circ_0009845, novel_circ_0005934 and novel_circ_0000134 may play important roles in skeletal muscle growth and development between the mon9 and mon1 groups. Luciferase Reporter Assay confirmed the combination between novel_circ_0005319 and chi-miR-199a-5p, novel_circ_0005934 and chi-miR-450-3p and novel_circ_0000134 and chi-miR-655. Our results provide specific information related to goat muscle development and a reference for the goat circRNA profile.

Transcriptome Analysis Reveals miR-302a-3p Affects Granulosa Cell Proliferation by Targeting DRD1 in Chickens.

Egg production is an important economic trait in laying chickens as higher yields bring higher profits. Small yellow follicle (SYFL) development is a key determinant of chicken reproductive performance; however, the majority of SYFLs are not selected during the process of chicken reproduction and thus, atresia occurs. Although there have been numerous omic studies focused on egg production, the molecular mechanisms involved are still not well-understood. In this study, we used high-throughput technology to analyze the differences between the SYFL mRNA transcriptomes of high- (H) and low-egg-yielding (L) Taihang layer hens, with the aim of identifying the potential candidate genes involved in controlling the rate of egg production. We constructed six cDNA libraries, three from H and three from L Taihang hens and then performed high-throughput sequencing. Comparison of the H and L groups showed 415 differentially expressed genes (DEGs). In the high-yield group, 226 were upregulated and 189 were downregulated. Differentially enriched biological functions and processes were identified using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analysis. Ten of the candidate DEGs we identified (DRD1, MC5R, PCK1, CTSA, TGFBR3, AGO4, SLIT2, RGS1, SCNN1B, and ZP3) have been identified in previous studies as being involved in the development of small yellow follicles. DRD1 was significantly enriched in the gap junction pathway, which is an important pathway in chicken granulosa cells (GCs) to pass nutrition to an oocyte. Homology analysis showed that DRD1 was highly conserved in numerous species, indicating that it may be a productive target for improving egg production. Evidence from bioinformatics analysis revealed that gga-miR-302a-3p putatively targets the 3'UTR region of DRD1. We then identified the functions of gga-miR-302a-3p in follicular granulosa cell proliferation by targeting DRD1. RT-qPCR analysis showed that DRD1 and miR-302a-3p expression were inversely related in the SYLs of high and low egg-yielding chickens. Luciferase assays showed that miR-302a-3p targets the 3'UTR of DRD1, and overexpression of miR-302a-3p significantly inhibits the expression of DRD1 in chicken GCs (p < 0.01). Functional experiments revealed that by targeting DRD1, miR-302a-3p acts as an inhibitor of GC proliferation. Taken together, we concluded that miR-302a-3p affects chicken GC proliferation by targeting DRD1. Our data expanded the knowledge base of genes whose functions are important in egg production and the molecular mechanisms of high-yield egg production in chicken small yellow follicles.

Resveratrol Inhibits Human Visceral Preadipocyte Proliferation and Differentiation in vitro.

Visceral obesity is a high-risk factor for diabetes and metabolic syndrome. Resveratrol, a natural polyphenolic compound, has been reported to inhibit preadipocyte differentiation. However, the effect of resveratrol on human visceral preadipocyte (HPA-v) differentiation remains largely unknown. LIM domain only 3 (LMO3) promotes human preadipocyte differentiation by enhancing peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activity, which is the master regulator of adipogenesis. The purpose of our study was to determine the effect of resveratrol (0-50 µM) on HPA-v proliferation and differentiation, and the role of LMO3 in resveratrol-mediated regulation of HPA-v differentiation. Resveratrol inhibited HPA-v proliferation and differentiation in a dose-dependent manner, and significantly decreased the mRNA expression levels of PPARG, CCAAT/enhancer-binding protein α (CEBPA), fatty acid-binding protein 4 (FABP4), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) (p < 0.05) at 10, 20, and 50 µM. The mRNA and protein levels of LMO3 were significantly reduced by ≥20 µM resveratrol (p < 0.05), and overexpression of LMO3 partially attenuated resveratrol-induced reduction of HPA-v differentiation by enhancing the PPARG transcriptional activity. Together, our study suggested that resveratrol reduced HPA-v proliferation and differentiation, as well as LMO3, which was partially responsible for the reduction of resveratrol-mediated adipocyte differentiation.

Myostatin/SMAD4 signaling-mediated regulation of miR-124-3p represses glucocorticoid receptor expression and inhibits adipocyte differentiation.

The mechanism of adipocyte regulation specifically in muscle and the influence of muscle tissue on intramuscular fat deposition are unknown. Our previous studies have shown that myostatin, a myokine, is involved in inhibiting the differentiation of preadipocytes and may be a potential regulator that affects the deposition of intramuscular fat. Myostatin inhibited adipogenesis by downregulating the expression of glucocorticoid receptor (GR) in porcine preadipocytes. However, the mechanism of regulation is not yet clear. In this study, we demonstrate microRNA (miR-124-3p) mediates regulation of GR by myostatin. We found that miR-124-3p can target GR 3'-UTR and negatively regulate GR expression. We demonstrate that overexpression of miR-124-3p can reduce differentiation of 3T3-L1 cells by inhibiting GR, and vice versa. The expression of miR-124-3p was upregulated in 3T3-L1 cells treated with myostatin. Further study revealed that myostatin also promotes the expression of SMAD4 and its transfer and localization to the nucleus. The activated myostatin/SMAD4 signal promotes the expression of miR-124-3p by SMAD4 binding to the promoter region of miR-124-3p. When myostatin or SMAD4 activity is inhibited, the upregulation of miR-124-3p is also inhibited. All of these findings suggested that myostatin could inhibit adipogenic differentiation of 3T3-L1 cells by activating miR-124-3p to inhibit GR. These data may provide an explanation for how myostatin signaling affects intramuscular fat deposition in a tissue-specific manner.

A genome-wide landscape of mRNAs, lncRNAs, and circRNAs during subcutaneous adipogenesis in pigs.

BACKGROUND: Preadipocyte differentiation plays a critical role in subcutaneous fat deposition in pigs. However, the roles of different RNAs, such as messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) in the differentiation process of subcutaneous preadipocytes, are still largely unclear. In the present study, a transcriptome analysis, including the analysis of mRNAs, lncRNAs, and circRNAs, during different differentiation stages, namely, day 0 (D0), day 2 (D2), day 4 (D4), and day 8 (D8), of subcutaneous preadipocytes from Chinese Erhualian pigs was performed. RESULTS: A total of 1554, 470, 1344, 1777, and 676 differentially expressed (DE) mRNAs, 112, 58, 95, 136, and 93 DE lncRNAs, and 902, 787, 710, 932, and 850 DE circRNAs were identified between D2 and D0, between D4 and D2, between D8 and D4, between D4 and D0, and between D8 and D0, respectively. Furthermore, functional enrichment analysis showed that the common DE mRNAs during the entire differentiation process were mainly involved in lipid metabolic and cell differentiation processes. Additionally, co-expression network analysis identified the potential lncRNAs related to adipogenesis, e.g., MSTRG.131380 and MSTRG.62128. CONCLUSIONS: Our study provides new insights of the expression changes of RNAs during adipogenic differentiation, which might contribute to the phenotype of subcutaneous adipogenesis. These results greatly improve our understanding of the molecular mechanisms regulating subcutaneous fat deposition in pigs.

miR-130a regulates differential lipid accumulation between intramuscular and subcutaneous adipose tissues of pigs via suppressing PPARG expression.

To investigate the molecular mechanism underlying differential lipid deposition between intramuscular (IM) and subcutaneous (SC) fat tissues of pigs, peroxisome proliferator-activated receptor gamma (PPARG) expression levels were compared in these two types of adipose tissues. The results showed that both mRNA and protein levels of PPARG were significantly higher in SC fat than in IM fat. Correspondingly, the expression levels of miR-128 and miR-130a, which potentially targeting PPARG 3'-untranslated region (3'-UTR), in IM fat were 8.37 and 2.30-fold of those in SC fat respectively. Further dual luciferase activity assay showed that only miR-130a directly targeting PPARG. Moreover, over-expression of miR-130a in preadipocytes significantly inhibited its differentiation by suppressing PPARG expression. In conclusion, tissue variance of miR-130a levels results in the diverse of PPARG, and might be the reason for differential fat deposition between IM and SC fat tissues in pigs. Our study would provide the molecular foundation for IM fat deposition increase and therefore contribute to pork quality improvement.

miR-17-5p Regulates Differential Expression of NCOA3 in Pig Intramuscular and Subcutaneous Adipose Tissue.

Fat distribution affects economic value in pork production. Intramuscular adipose tissue (IMAT) improves meat quality, whereas subcutaneous adipose tissue (SCAT) is usually regarded as waste. In the present study, we analyzed IMAT/SCAT (I/S) ratios in each pig. Individuals selected from a population of 1200 Suhuai pigs were divided into two cohorts; those with high I/S ratios and those with low I/S ratios, and correlations between nuclear Receptor Co-activator 3 (NCOA3), a critical gene involved in regulating fat accumulation, and fat distribution were investigated. The ratio of IMAT NCOA3 to SCAT NCOA3 expression levels (NCOA3I/NCOA3S) was higher in the high I/S group compared with the low I/S group. The NCOA3 expression level in fat tissue was positively correlated with fat deposition. miR-17-5p was identified as a putative regulator of NCOA3 based on bioinformatics prediction analysis followed by gene expression analysis. The miR-17-5pI/miR-17-5pS ratio was negatively correlated with the NCOA3I/NCOA3S ratio. The predicted relationship between miR-17-5p and NCOA3 was further verified by dual luciferase activity assays, qPCR, and western blots. Overexpression of miR-17-5p in intramuscular preadipocytes inhibited NCOA3 expression and reduced preadipocyte differentiation. FABP4 and PPARG expression were also significantly decreased, as was triglyceride content. Meanwhile, knockdown of miR-17-5p significantly increased NCOA3 expression and promoted intramuscular preadipocyte differentiation. Based on these results, we propose that differential expression of NCOA3 in pig intramuscular and subcutaneous adipose tissue is regulated by miR-17-5p.

Muscle Conditional Medium Reduces Intramuscular Adipocyte Differentiation and Lipid Accumulation through Regulating Insulin Signaling.

Due to the paracrine effects of skeletal muscle, the lipid metabolism of porcine intramuscular (i.m.) preadipocytes was different from that of subcutaneous (s.c.) preadipocytes. To investigate the development of i.m. preadipocytes in vivo, the s.c. preadipocytes were cultured with muscle conditional cultured medium (MCM) for approximating extracellular micro-environment of the i.m. preadipocytes. Insulin signaling plays a fundamental role in porcine adipocyte differentiation. The expression levels of insulin receptor (INSR) and insulin-like growth factor 1 receptor (IGF-1R) in i.m. Preadipocytes were higher than that in s.c. preadipocytes. The effects of MCM on adipocyte differentiation, lipid metabolism and insulin signaling transdution were verified. MCM induced the apoptosis of s.c. preadipocytes but not of s.c. adipocytes. Moreover, MCM inhibited adipocyte differentiation at pre-differentiation and early stages of differentiation, while the expression levels of INSR and IGF-1R were increased. Furthermore, MCM treatment increased adipocyte lipolysis and fatty acid oxidation through induction of genes involved in lipolysis, thermogenesis, and fatty acid oxidation in mitochondria. Consistent with the above, treatment of s.c. adipocytes with MCM upregulated mitochondrial biogenesis. Taken together, MCM can approximate the muscle micro-environment and reduce intramuscular adipocyte differentiation and lipid accumulation via regulating insulin signaling.

Muscle-specific downregulation of GR levels inhibits adipogenesis in porcine intramuscular adipocyte tissue.

Intramuscular adipose is conducive to good pork quality, whereas subcutaneous adipose is considered as waste in pig production. So uncovering the regulation differences between these two adiposes is helpful to tissue-specific control of fat deposition. In this study, we found the sensitivity to glucocorticoids (GCs) was lower in intramuscular adipocytes (IMA) compared with subcutaneous adipocytes (SA). Comparison of glucocorticoid receptor (GR) revealed that IMA had lower GR level which contributed to its reduced GCs sensitivity. Higher methylation levels of GR promotor 1-C and 1-H were detected in IMA compared with SA. GR expression decrease was also found in adipocytes when treated with muscle conditioned medium (MCM) in vitro, which resulted in significant inhibition of adipocytes proliferation and differentiation. Since abundant myostatin (MSTN) was detected in MCM by ELISA assay, we further investigated the effect of this myokine on adipocytes. MSTN treatment suppressed adipocytes GR expression, cell proliferation and differentiation, which mimicked the effects of MCM. The methylation levels of GR promotor 1-C and 1-H were also elevated after MSTN treatment. Our study reveals the role of GR in muscle fiber inhibition on intramuscular adipocytes, and identifies myostatin as a muscle-derived modulator for adipose GR level.

C2C12 myotubes inhibit the proliferation and differentiation of 3T3-L1 preadipocytes by reducing the expression of glucocorticoid receptor gene.

Obesity is a well-established risk factor to health for its relationship with insulin resistance, diabetes and metabolic syndrome. Myocyte-adipocyte crosstalk model plays a significant role in studying the interaction of muscle and adipose development. Previous related studies mainly focus on the effects of adipocytes on the myocytes activity, however, the influence of myotubes on the preadipocytes development remains unclear. The present study was carried out to settle this issue. Firstly, the co-culture experiment showed that the proliferation, cell cycle, and differentiation of 3T3-L1 preadipocytes were arrested, and the apoptosis was induced, by differentiated C2C12 myotubes. Next, the sensitivity of 3T3-L1 preadipocytes to glucocorticoids (GCs), which was well known as cell proliferation, differentiation, apoptosis factor, was decreased after co-cultured with C2C12 myotubes. What's more, our results showed that C2C12 myotubes suppressed the mRNA and protein expression of glucocorticoid receptor (GR) in 3T3-L1 preadipocytes, indicating the potential mechanism of GCs sensitivity reduction. Taken together, we conclude that C2C12 myotubes inhibited 3T3-L1 preadipocytes proliferation and differentiation by reducing the expression of GR. These data suggest that decreasing GR by administration of myokines may be a promising therapy for treating patients with obesity or diabetes.

Genetic variants in ABCA1 promoter affect transcription activity and plasma HDL level in pigs.

Excess accumulation of cholesterol in plasma may result in coronary artery disease. Numerous studies have demonstrated that ATP-binding cassette protein A1 (ABCA1) mediates the efflux of cholesterol and phospholipids to apolipoproteins, a process necessary for plasma high density lipoprotein (HDL) formation. Higher plasma levels of HDL are associated with lower risk for cardiovascular disease. Studies of human disease and animal models had shown that an increased hepatic ABCA1 activity relates to an enhanced plasma HDL level. In this study, we hypothesized that functional mutations in the ABCA1 promoter in pigs may affect gene transcription activity, and consequently the HDL level in plasma. The promoter region of ABCA1 was comparatively scanned by direct sequencing with pool DNA of high- and low-HDL groups (n=30 for each group). Two polymorphisms, c. - 608A>G and c. - 418T>A, were revealed with reverse allele distribution in the two groups. The two polymorphisms were completely linked and formed only G-A or A-T haplotypes when genotyped in a larger population (n=526). Furthermore, we found that the G-A/G-A genotype was associated with higher HDL and ABCA1 mRNA level than A-T/A-T genotype. Luciferase assay also revealed that G-A haplotype promoter had higher activity than A-T haplotype. Single-nucleotide mutant assay showed that c.-418T>A was the causal mutation for ABCA1 transcription activity alteration. Conclusively, we identified two completely linked SNPs in porcine ABCA1 promoter region which have influence on the plasma HDL level by altering ABCA1 gene transcriptional activity.