Polymorphisms of Fatty Acid Synthase Gene and their Association with Milk Production Traits in Chinese Holstein Cows

Zuyang Zhou1, Xi He2, Yufang Liu1, Qiuling Li3, Pingqing Wang2, Yongfu An4, Ran Di1, Yuze Yang5 and Mingxing Chu1* 1Key Laboratory of Animal Genetics and Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China 2Bioengineering College, Chongqing University, Chongqing 400030, P.R. China 3College of Life Sciences, Langfang Teachers University, Langfang 065000, P. R. China 4Hebei Animal Science and Veterinary Medicine Institute, Baoding 071000, P.R. China 5Beijing General Station of Animal Husbandry, Beijing 100101, P.R. China Article Information Received 06 October 2021 Revised 05 November 2021 Accepted 14 November 2021 Available online 28 July 2023 (early access)


INTRODUCTION
N owadays, milk is one of the crucial nutrients in people's daily diet, in which fatty acid content and composition determine the nutritional value of milk. Fatty acid synthase (FASN) is a multi-enzyme complex whose main function is to catalyze the synthesis of fatty acids in the intercellular substance (Ye et al., 2021). The expression level of FASN was significantly increased in a variety of tumor cells and pre-tumor tissues, and its overexpression was closely related to tumor cell proliferation, metastasis, anti-apoptosis (Polonio-Alcalá et al., 2020). As a central regulator of lipid metabolism, FASN could rewire tumor cells to obtain greater energy flexibility to achieve their high energy requirements, therefore, FASN was also considered as a tumor marker (Fhu and Ali, 2020). As an important metabolic enzyme in fatty acid synthesis during the adult stage, FASN also played a central role during the embryo development in mammals (Chirala et al., 2003). FASN was required for mammary gland development and milk production during lactation (Suburu et al., 2014). The expressions of fatty acid synthase and its encoding gene (FASN) were studied at molecular biology level (Abe et al., 2009;Alim et al., 2014;Roy et al., 2005;Schennink et al., 2009;Li et al., 2016).
FASN gene had been cloned and sequenced in mouse, chicken, human, goose and bovine (Amy et al., 1990;Chirala et al., 1997;Jayakumar et al., 1995;Kameda et al., 1991;Roy et al., 2005). Bovine FASN is located on chromosome 19q22 (Roy et al., 2001) and its expression in brain, testis and adipose tissue is higher than that in liver and heart (Roy et al., 2005). Bovine FASN gene (AF285607) consists of 42 exons. TE domain in this gene is important in fatty acid synthesis as a substrate-binding site. TE domain determines the chain length of the fatty acids and effects on the fatty acid composition of bovine adipose tissue (Zhang et al., 2008). KR domain which was close to the TE domain can affect the function of TE domain by the changing the structure of the substrate-O n l i n e

F i r s t A r t i c l e
binding site (Abe et al., 2009). The objectives of this study were to analyze the polymorphism of TE domain of FASN gene by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and to examine the relationship between FASN gene and milk production traits in Chinese Holstein cows. The results could provide basic molecular data for marker assisted selection research in Chinese Holstein cows.

Blood sample collection and DNA preparation
Jugular blood samples (10 ml per cow) using acid citrate dextrose as an anticoagulant were collected from 407 Chinese Holstein cows calved in 2019 (first, second, or third parity) originated from four dairy farms (139 cows from the 1 st farm, 131 cows from the 2 nd farm, 69 cows from the 3 rd farm and 68 cows from the 4 th farm, respectively) in Hebei province, P.R. China.

Primer sequences and PCR amplification
Eleven pairs of primers (P1-P11) were designed to screen the SNP at KR and TE domain according to the DNA sequence of bovine FASN gene (GenBank Accession No. AF285607). Primers were synthesized by Shanghai Invitrogen Biotechnology Co. Ltd. (Shanghai, P.R. China). Primer sequence, product size, amplified region and annealing temperature were listed in Table I.

O n l i n e F i r s t A r t i c l e SSCP detection and genotyping
The 3 μL PCR product was mixed with 7 μL gel loading solution (98% formamide, 0.025% bromophenol blue, 0.025% xylene cyanol, 10 mmol/L EDTA (pH 8.0), 10% glycerol). The mixture was agitated and denatured at 98°C for 10 min, and quickly placed on ice for 7 min and loaded on 12% neutral polyacrylamide gels (acrylamide: bisacrylamide= 29:1). Electrophoresis was performed in 1 × Tris borate (pH 8.3)-EDTA buffer at 110 volts for 17 -22 h at 4 °C. After electrophoresis, the DNA fragments in the gels were visualized by silver nitrate staining, photographed and analyzed by using an AlphaImager TM 2200 and 1220 Documentation and Analysis Systems (Alpha Innotech Corporation, San Leandro, CA, USA). SSCP genotypes were identified by mobility shift due to conformational difference of the single-stranded DNAs of the amplified fragments by each primer, which was caused by nucleotide variation.

Cloning and sequencing
The PCR products with the variation detected by SSCP were cloned and sequenced according to the method of M.X. Chu (Chu et al., 2012).

Statistical analysis
Hardy-Weinberg equilibrium (HWE) and population parameters including gene heterozygosity (He), effective allele numbers (Ne) and polymorphism information content (PIC), were calculated using PopGene 32.
The association between each SNP and difference of five milk production traits (milk yield at 305 d, protein percentage, fat percentage, lactose percentage and dry matter percentage) were evaluated by the following linear model using general linear model procedure (GLM) of SAS (Ver 8.12) (SAS Institute Inc., Cary, NC, USA). y ijklmn = μ + B i + H j + P k + CS l + G m + e ijklmn Where y ijklmn is observation of milk production traits; μ is overall mean; B i is the fixed effect of the i th bull (i=1, 2, 3, 4, 5); H j is the fixed effect of the jth herd (j=1, 2, 3, 4); P k is the fixed effect of the k th parity (k=1, 2, 3); CS l is the fixed effect of the l th calving season (l=1, 2, 3, 4); G m is the fixed effect of the m th genotype (m=1, 2, 3); e ijklmn is the random error effect of each observation.

Bovine FASN gene
The KR and TE domains in FASN gene of Chinese Holstein cows were successfully PCR amplified (Fig. 1). The sizes of amplification fragments were consistent with the target ones and had a good specificity, which could be directly analyzed by SSCP. The results of SSCP analysis showed that only the PCR products amplified by P3 and P8 displayed polymorphisms. Three genotypes were detected by primer P3 (Fig. 2A, GG, GA and AA) and primer P8 (Fig. 2B, GG, GA and AA) respectively. For primer P3, sequencing result revealed that g.16024 G>A was existed between genotype GG and genotype AA. For primer P8, g.17924 G>A was existed between genotype GG and genotype AA (Fig. 3). The non-synonymous g.16024G>A, g.17924G>A in KR and TE domains respectively resulted in a nonconservative substitution of alanine by threonine. The frequencies of G allele and GG genotype of g.16024 G>A and g.17924 G>A were higher in Chinese Holstein cows (Table II).   (Table III), which indicated that the genotype frequencies of the two loci were not affected by selection, mutation or migration. Generally, polymorphism information content (PIC) was classified into the following three types: low polymorphism (PIC value < 0.25), intermediate polymorphism (0.25 < PIC value < 0.5) and high polymorphism (PIC value > 0.5) (Serrote et al., 2020). According to this classification of PIC, the g.17924 G>A in Chinese Holstein belonged to the intermediate polymorphism level, which indicated that there was a greater variation and selective potentiality in this locus. It might obtain a greater genetic progress for selection.

Association of FASN gene polymorphisms with milk production traits
The least squares means and standard errors for the five milk production traits of different genotypes in Chinese Holstein cows were given in Table IV. The mutation g.16024 G>A had no significant effect on milk production traits (milk yield at 305 d, protein percentage, fat percentage, lactose percentage and dry matter percentage) (P > 0.05). Regarding to the mutation g.17924 G>A, the fat percentage of AA genotype was higher than the other genotypes (P < 0.05). There was no significant difference on fat percentage between genotypes GG and GA (P > 0.05) in Chinese Holstein cows (Table IV).

O n l i n e F i r s t A r t i c l e
Polymorphisms of Fatty Acid Synthase Gene

Polymorphisms of bovine FASN gene
In this study, we identified two SNPs by PCR-SSCP previously reported, g.16024 A>G (Abe et al., 2009), and g.17924 G>A (Abe et al., 2009;Morris et al., 2007;Zhang et al., 2008). It had been found that the g.17924 A>G SNP was located in the thioesterase domain of the FASN protein, and changed from polar, neutral, and hydrophilic to nonpolar, aliphatic, and hydrophobic, which had an effect on increasing fat deposition in Korean cattle (Oh et al., 2018). Morris et al. (2007) identified five mutations in bovine FASN gene, g.17250-17251 AT indel, g.15603 G>A in introns, g.16907 T>C, g.15531 C>A and g.17924 A>G in exons, of which only g.17924 A>G caused an amino acid.
Substitution of threonine to alanine in FASN protein. In the meantime, researchers detected three single nucleotide mutations, g.17924 A>G reported by Morris (Morris et al., 2007), and two silent mutations g.18663 T>C and g.18727 C>T in Angus cattle (Zhang et al., 2008). Previous studies had detected four known mutations (g.16907 T>C, g.17924 A>G, g.18663 T>C and g.18727 C>T) (Abe et al., 2009). The bovine FASN gene showed abundant polymorphisms affecting the fat related traits in different breeds. The difference of allele frequency may be due to the effect of artificial selection in different cattle breeds.

Association between FASN gene and milk production traits
Bovine FASN is located on BTA19 where several quantitative trait loci (QTL) affecting milk-fat content and related traits (Boichard et al., 2003;Bouwman et al., 2014;Uemoto et al., 2011;Bhuiyan et al., 2018). It had been reported that the frequencies of g.763 G>C and g.16009 A>G were significantly different in Holsteins with high and low breeding values for milk-fat content (Roy et al., 2006). The individuals with haplotype C-G were associated with a high production of fat, while those with haplotype G-A were associated with a low production of milk fat. Some researchers concluded that mutation g.763 G>C in bovine FASN exon 1 increased milk fat content in dairy cattle due to the decrease of bovine FASN promoter activity in vitro (Ordovas et al., 2008). Morris found that the substitution g.15531 C>A or g.15603 G>A had effects on the C14:0 percentage of adipose fat and milk fat in the opposite direction (Morris et al., 2007). Researchers also found that cows with the FASN GG genotype, the protein percentage was higher, but the A allele was associated with higher milk, protein and fat yields than the G allele (Čítek et al., 2021). It had been proved that g.17924 A>G of FASN gene was significantly associated with fatty acid composition of longissimus dorsi muscle of Angus bulls (Zhang et al., 2008) and significantly associated with the decreasing of milk fat percentage (P < 0.05) in Dutch Holstein-Friesian cows (Schennink et al., 2009). In addition, Ye's study in Mediterranean buffalo found that the two mutations g.7164 G>A and g.8927 T>C were also significantly related to peak milk production and protein percentage, respectively (Ye et al., 2021). Besides, it had been reported that the haplotypes (TW and AR) of g.16024 A>G and g.16039 T>C segregated in F2 individuals from Japanese Black and Limousin cattle and had a significant effect on the fatty acid composition of back fat, intermuscular fat, and intramuscular fat. The two mutations similarly determined the contribution to the fatty acid composition of intramuscular fat in two commercial Japanese Black half-sibling populations (Abe et al., 2009).

CONCLUSION
Our study indicated that allele A of g.17924 G>A of FASN gene was significantly associated with increased fat percentage in Chinese Holstein cows. The results of this study also showed that g.17924 G>A mutation might provide useful information in terms of predicting the milk fat percentage, or that allele A of g.17924 G>A of FASN gene may be applied as a potential genetic marker for improving milk fat percentage in dairy cattle.

O n l i n e F i r s t A r t i c l e O n l i n e F i r s t A r t i c l e
Polymorphisms of Fatty Acid Synthase Gene