Genetic variants in the serum amyloid A2 (SAA2) gene as a potential marker for milk production traits in Chinese Holstein cows

Abstract Background This study was conducted to detect potential polymorphisms of the serum amyloid A2 (SAA2) gene and explore their relationships with milk production traits in Chinese Holstein cows. Objectives: This study used sequencing technology conducted in 532 Chinese Holstein cows. Methods Three single nucleotide polymorphisms (SNPs) were identified within intron 1, named g.14061A>G, g.14072G>C and g.14819C>T. Eight estimated haplotypes were identified, of which three major haplotypes had a frequency of Hap3 (‐ACC‐), Hap5 (‐GCC‐) and Hap2 (‐AGT‐), with 17.9%, 12.30% and 8.10%, respectively. Results The association analysis of single markers (g.14061A>G and g.14819C>T) and combined genotypes (Hap1/5) revealed prominent effects on milk production traits in Chinese Holstein cows (p < 0.05). Conclusions Our results suggest that the SAA2 gene is associated with economic traits in Chinese Holstein cows and may be used as candidate gene for marker‐assisted selection and management in breeding programs.


INTRODUCTION
In mammals, serum amyloid A (SAA) is comprised of five isoforms with high conservation and various biological functions, designated as SAA1, SAA2, SAA3, M-SAA3.2 and SAA4, respectively (Kovačević-Filipović et al., 2012). SAA is synthesized primarily by hepatocytes, macrophages, vascular endothelial cells and adipocytes (Ahmed et al., 2012;Artl et al., 2000). Clinical studies confirmed that SAA is considered a biomarker of inflammation and angiocardiopathy, with dramatic increases in SAA plasma levels in response to inflammatory and atherosclerotic cardiovascular diseases (Smole et al., 2020). The SAA2 gene encodes the amphipathic alpha-helical apolipoprotein (Chait et al., 2005) that plays a pivotal role in mobilization of cholesterol during tissue repair and regeneration and was described as a 'gatekeeper' for its function in normal tissues (Urieli-Shoval et al., 2000). When released, the SAA2 gene readily bound to high-density lipoprotein (HDL), and then rerouted HDL transport (Benditt & Eriksen, 1977). Via modulating the ability of HDL, the SAA2 gene contributed to reverse transport of cholesterol and removed excess cholesterol from the body (De Beer et al., 2014). Compared to cows with lower milk protein and fat percentage, expression of the SAA2 gene was remarkably reduced in the mammary gland with extremely high milk protein and fat percentage (Cui et al., 2014). Therefore, these findings lend credence to the hypothesis that the SAA2 gene is an excellent candidate marker for milk composition traits in dairy cattle.
The purpose of the present research was to analyze the influence of SAA2 gene polymorphisms on the comparison of milk traits in Chinese Holstein cows and to determine the changes in milk traits during lactation depending on polymorphisms of the analyzed genes.

Animals and phenotypic data
A total of 532 Chinese Holstein dairy cows belonging to 24 sire families were collected from Xi'an Caotan Animal Husbandry Co. LTD (Shaanxi Province, China). All of the cows were fed with the same regular total mixed ration composed of concentrated feed and coarse fodder and were routinely milked three times a day (07:00, 15:00, 23:00). The milk yields were recorded with a computerized herd management system.
The dairy herd improvement (DHI) in milk samples were provided from Xi'an Caotan Animal Husbandry Co. LTD.

DNA isolation
Genomic DNA from blood samples were collected by TIANamp Blood DNA kit (Tiangen, Beijing, China). The quality of DNA was measured using a NanoDrop ND-2000c Spectrophotometer (Thermo Scientific).

Statistical analysis
The associations of the SNPs and milk production traits were analyzed by analysis of variance (ANOVA) the SPSS 16.0 software (IBM Company, NY, USA). The equation was as follows:

Polymorphisms and genetic diversity
The bovine SAA2 gene is located on chromosome 29 of the bovine genome. The total length of SAA2 is 3447 bp, comprising the genomic coordinates starting from 26458422 to 26461868 (NC_037356.1, Reference genome bos taurus published in GenBank in the NCBI database). This gene comprises 04 exons, the ORF that started from the start codon to the stop codon is 393 bp, and the putative protein contains 131 amino acids (Figure 1).
In the current study, three SNPs, including g.14061A>G, g.14072G>C and g.14819C>T, were identified by DNA direct sequencing. All three loci mapped on intron 1 of the SAA2 gene.
The genotype TT of g.14819C>T was not observed in the sampled population, probable explanations may be that (1) this genotype at g.14819C>T locus may never exist in the population and (2) the experimental population size was insufficient to capture full genetic variation .

Association analysis
So far, litter polymorphism on the SAA2 gene is reported regarding herbivore breeding and reproduction. Previously, two mutations (c.17G>C and c.114G>A) within the 5′-regulatory region of the SAA2 gene were found to be statistically significance for milk yield, fat yield and protein yield in Chinese Holstein cows as a result of alteration in SAA2 gene transcriptional activity (Yang et al., 2015). In addition, both c.-84 TA B L E 2 Population genetic analysis of single nucleotide polymorphisms (SNPs) in serum amyloid A2 (SAA2) gene  -Luczak et al., 2021). In the current study, association analysis between SNPs of the SAA2 gene and milk production traits (Table 4), as well as, the significant additive effects, dominant effects and allele substitution effects were observed (Table 5). Furthermore, individuals carrying GG genotype at g.14061A>G locus had significantly higher milk fat percentage and milk protein percentage than individuals with AA genotype (p < 0.05). Compared to animals with genotype TT, the role of CT genotype at g.3208C>T locus caused the highest average for lactose percentage (p < 0.05). Different from previous studies, the location of mutations was found on the introns in the current study. The mutation differences may be explained by the diversity of the sample population.

Loci (gene) Genotypic frequency (%)
Haplotypes composed of SNPs provided greater power than singlemarker analysis for associations of inheritable characters because of the ancestral structure captured in the distribution of haplotypes TA B L E 5 Genetic effects of the three single nucleotide polymorphisms (SNPs) in serum amyloid A2 (SAA2) gene on the milk production traits ( Akey et al., 2001). Currently, the analysis of combined haplotypes with milk production traits in Chinese Holstein dairy cows is listed in Table 6.
Correlation analysis revealed that both g.14061A>G and g.14819C>T were identified in the intron of the SAA2 gene and contributed to the genetic breeding of Chinese Holstein cows.
Although the structure of the encoded protein was not changed, intronic SNPs may influence assembly of spliceosome components, thereby maintaining the genetic stability (Wu et al., 2019). In addition, intronic SNPs affected the initiation and extension of transcription via binding to several enhancers or cis-acting elements (Chai et al., 2015).
In two Chinese indigenous beef cattle, the g.2694 C>T and g.3801 T>C mapping on intronic SNPs of max dimerization protein 3 (MXD3) influenced growth traits (Hao et al., 2020).
The intronic g.18341 C>T of the lipoprotein lipase (LPL) gene was significantly correlated with withers height and chest depth in Nanyang cattle (Gui et al., 2016). Currently, it was speculated that 14061A>G and g.3208C>T may impact the biological function, resulting in an alteration in phenotype of mammal.
Milk performance is considered to be primary economic trait in dairy cattle, which is regulated by a series of factors, such as inheritance, age, diet, location and other random environment factors. Accumulating genome-wide association studies (GWAS) indicated that a numbers of candidate genes (i.e., Toll-like receptor 4, UDP-glucose dehydrogenase, complement component 4) are involved in complex mechanisms, thereby altering milk performance traits (Gui et al., 2016;Li et al., 2015;Palombo et al., 2018), Here, we detected significant SNPs within the SAA2 gene that potentially influence milk composition and yield in Chinese dairy cattle, which could be used as a genetic marker aiming towards long-term improvement in production.

CONCLUSION
In summary, three SNPs (g.14061A>G, g.14072G>C and g.14819C>T) and eight haplotypes were discovered within the SAA2 gene of Chinese Holstein cows. The association analysis of single markers (g.14061A>G and g.14819C>T) and combined genotypes (Hap1/5) revealed prominent effects on comparison of milk traits in Chinese Holstein cows. Further research should be conducted in a large population before they could be applied as genetic markers for selection purposes.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.