Determination of genetic effects of ATF3 and CDKN1A genes on milk yield and compositions in Chinese Holstein population.

BACKGROUND: Our previous RNA-sequencing study revealed that the ATF3 and CDKN1A genes were remarkably differentially expressed between the mammary glands of lactating Holstein cows with extremely high and low milk protein and fat percentage so that both of them were considered as candidates for milk composition. Herein, we further verified whether these genes have genetic effects on milk production traits in a Chinese Holstein cow population.
RESULTS: By re-sequencing the entire coding and regulatory regions, we identified four SNPs in 5'promoter region, two in exons, seven in 3' un-translated region (UTR), and six in 3'flanking region of ATF3 gene, and one SNP in exon 5, two in 3'UTR, and two in 3'flanking region of CDKN1A gene. Of these, only the SNP, c.271C > T (rs442346530), in exon 5 of CDKN1A gene was predicted to result in an amino acid replacement from arginine to tryptophan. Subsequent genotype-phenotype association analysis revealed that 19 SNPs in ATF3 and 5 SNPs in CDKN1A were evidently associated with 305-days milk yield, fat yield, protein yield, or protein percentage (P = < 0.0001 ~ 0.0494). Whilst, no significant SNPs in ATF3 gene were associated with fat percentage in both first and second lactations (P > 0.05), and only two SNPs of CDKN1A gene, c.271C > T (P = 0.0377) and c.*654C > T (P = 0.0144), were markedly associated with fat percentage in the first lactation. Further, linkage disequilibrium (LD) analyses were conducted among the identified SNPs in ATF3 and/or CDKN1A genes to further confirm the association results. We also observed that the four SNPs, g.72834301C > A, g.72834229C > A, g.72833969A > G, and g.72833562G > T altered the specific transcription factor (TF) binding sites in ATF3 promoter, and one SNP, c.271C > T, changed the CDKN1A protein secondary structure, suggesting they might be the promising potential functional mutations.
CONCLUSION: Our findings first profiled the genetic effects of ATF3 and CDKN1A genes for milk production traits in dairy cattle and will be available for marker-assisted breeding in dairy cattle.

Background

Milk production traits are the most important economic traits in dairy development, including milk yield, fat yield, protein yield, fat percentage and protein percentage [1]. As we know, most economic traits of livestock are quantitative traits, caused by minor genes. In the past several decades, quantitative trait locus (QTL) mapping, candidate gene analysis, and genome-wide association study (GWAS) have been used to identify the causal genes or mutations for milk production in dairy cattle [2-4]. However, only the causative mutations in DGAT1, GHR, and ABCG2 gene, have been confirmed to date [5-7].

RNA sequencing (RNA-Seq) has become a comprehensive and accurate tool for analyzing the gene expression pattern [8]. Our previous RNA-Seq study has identified 31 differentially expressed genes between the mammary glands of lactating Chinese Holstein cows with the high and low milk protein percentage and fat percentage, of which, 17 genes are located within the known QTL regions for milk protein and fat traits and 21 contain or are near to the significant SNPs identified by previous GWAS, including ATF3 and CDKN1A [9]. The ATF3 gene (chr16: 72,820,025–72,832,974) were 1.9 Mb or 3.3 Mb close to the two SNPs, ARS-BFGL-NGS-70836 (chr16: 74,741,746) associated with milk protein percentage (P < 3.58E-07) and ARS-BFGL-NGS-85980 (chr16: 76,091,078) associated with fat percentage (P < 6.46E-07) and protein percentage (P < 8.33E-09) detected by a previous GWAS in dairy cattle [10]. ATF3 (activating transcription factor 3) is a transcription factor belonging to the mammalian activation transcription factor/cAMP responsive element-binding (CREB) protein family [11]. Our GO and Ingenuity Pathway Analysis (IPA) also found that ATF3 was involved in the lipid metabolism pathway, accumulation of glycoproteins, apoptosis, and regeneration of epithelial cells so that it is thought to be related to milk fat formation and development of mammary epithelial cells [9]. In addition, the CDKN1A gene (chr23: 10,560,498–10,568,780) is located within the known QTL regions (BTA23: 12.4 cM) that were confirmed to have large genetic effects on milk protein percentage [12]. CDKN1A (cyclin dependent kinase inhibitor 1A) encodes a 21-kD protein (p21) that is a cyclin-dependent kinase inhibitor, which is a rate-limiting regulator in the transition from G1 to S phase [13], and is also a key mediator of growth arrest that induced by the tumor suppressor protein p53 [14]. So far, there is no study revealing the associations of ATF3 and CDKN1A genes with milk production in dairy cattle.

Based on our previous RNA-Seq study [9], the purpose of this study was to identify whether ATF3 and CDKN1A genes have a genetic effect on milk production traits in dairy cows. Herein, we detected the SNPs in ATF3 and CDKN1A genes, and analyzed the association between these polymorphisms with five milk production traits in a Chinese Holstein population. In addition, bioinformatics analysis was performed to profile the promoter activity and protein structure variation of ATF3 and CDKN1A genes.

Methods

Animals and phenotypic data

A total of 1093 Chinese Holstein cows from 40 sire families were used in this study, and each sire had between 6 and 70 daughters with approximately 27 daughters on average per sire. These cows were from 22 dairy farms belonging to the Sanyuanlvhe Dairy Farming Center, where standard performance testing for dairy herd improvement (DHI) has been regularly conducted since 1999. Estimate breeding values (EBVs) for 305-days milk yield, fat yield, fat percentage, protein yield, and protein percentage during the first and second lactations were provided by the Beijing Dairy Cattle Center (http://www.bdcc.com.cn/). The descriptive statistics of phenotypic values for dairy production traits in two lactations were shown in Additional file 1. All protocols for sample collections of experimental individuals and phenotypic observations were approved by the Institutional Animal Care and Use Committee (IACUC) at China Agricultural University, and the permit number is DK996.

DNA extraction

The whole blood samples of 1093 Chinese Holstein cows were extracted by TIANamp Blood DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The genomic DNA from semen samples of the sires were extracted using the standard salt-out procedures. The quantity and quality of the extracted DNA were respectively measured using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, DE, USA) and by the gel electrophoresis.

SNP identification and genotyping

We designed a total of 35 primers (Additional file 2) using Primer 3.0 (http://primer3.wi.mit.edu/) and Oligo 6.0 (Molecular Biology Insights, Inc., CO, USA) to amplify the entire coding region and 2000 bp of flanking sequences based on the genomic sequence of the bovine ATF3 (GenBank accession no.: AC_000173.1) and CDKN1A (GenBank accession no.: AC_000180.1) genes, and the primers were synthesized by the Beijing Genomics Institute (BGI, Beijing, China). We randomly divided the DNA samples of the 40 sires into two equal groups, and diluted all the DNA samples into the concentration of 50 ng/μL. Subsequently, we constructed two DNA pools (50 ng/mL) as the templates for the PCR amplification. The amplifications were performed using the PCR system or the touch-down PCR (Additional file 2). The purified PCR products were directly sequenced by ABI3730XL DNA analyzer (Applied Biosystems, Foster City, CA, USA), and the sequences were compared by DNAMAN 6.0 (Lynnon Biosoft, USA) and NCBI-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to search potential SNPs. The identified SNPs were further individually genotyped for all the 1093 Chinese Holstein cows using the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS, Sequenom MassARRAY, Bioyong Technologies Inc. HK).

Association analyses

Association analyses between SNP genotypes and/or haplotypes and the five milk traits were conducted by SAS 9.13 (SAS Institute Inc., Cary, NC, USA), based on the following animal model:

where, Y was the phenotypic values of each trait for the cows; μ was the overall mean; hys was the fixed effect of farm, year, and season; b was the regression coefficient of covariant M; M was the fixed effect of calving month; G was the SNP genotype or haplotype effect; a was the individual random additive genetic effect, distributed as , with the additive genetic variance ; and e was the random residual, distributed as , with identity matrix I and residual error variance . Bonferroni correction was performed for multiple testing, and the significant level of the multiple tests was equal to the raw P value divided by number of tests. Currently, we considered the statistically significant association from being null effect if a raw P value is less than 0.05/N, where N represents the number of SNP loci.

We calculated the additive effect (a), dominant effect (d), and substitution effect (α) using SAS 9.13, and the computational formula was as follows: . Where, AA, BB, and AB were the least squares mean of the milk production traits in the corresponding genotype.

Haplotype analysis

The linkage disequilibrium (LD) extent between all SNPs and haplotype blocks were estimated using Haploview 4.2 (Broad Institute of MIT and Harvard, Cambridge, MA, USA).

Bioinformatics analysis

To analysis the biological functions of the SNPs, we used the JASPAR database (http://jaspar.binf.ku.dk/cgi-bin/jaspar_db.pl?rm=browse&db=core&tax_group=vertebrates) to profile the genetic variants of the transcription factor binding sites (TFBSs) of the SNPs associated with milk production traits in the 5′ promoter region (relative score > 0.85). In addition, we utilized NPSA SOPMA SERVER (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html) to predict the variations of protein secondary structure caused by missense mutation in the coding regions, and the parameters were window width (17), similarity threshold (8), and number of states (4).

Results

SNPs identification

After sequencing the entire coding and up/downstream regions, we totally identified 19 and five SNPs for ATF3 and CDKN1A genes, respectively, and all the identified SNPs have been reported in the NCBI database (Table 1). There were four SNPs in the 5’promoter region, two in exons, seven in the 3’UTR, and six in the 3’flanking region of ATF3 gene. For CDKN1A gene, one SNP, c.271C > T, was located in the exon 5, c.*9C > G and c.*654C > T in the 3’UTR, and g.10569766 T > C and g.10569779 T > C in the 3’flanking region. Of these, only the SNP in exon 5 of CDKN1A gene (c.271C > T) was predicted to result in an amino acid replacement from arginine (CGG) to tryptophan (UGG), and the two SNPs of ATF3 gene in the coding region (c.291G > A and c.489G > A) were synonymous mutations.

Table 1

Information about identified SNPs of ATF3 and CDKN1A genes

Gene	Gene region	SNPs	Position (UMD 3.1)	Allele	Amino acid missence	GenBank no.	Origin
ATF3	5′ promoter region	g.72834301C > A	chr16:72,834,301	C/A		rs41634778	NCBI
		g.72834229C > A	chr16:72,834,229	C/A		rs41634777	NCBI
		g.72833969A > G	chr16:72,833,969	A/G		rs41823579	NCBI
		g.72833562G > T	chr16:72,833,562	G/T		rs41823578	NCBI
	exon 4	c.291G > A	chr16:72,822,913	G/A		rs209694892	NCBI
	exon 5	c.489G > A	chr16:72,821,334	G/A		rs207598277	NCBI
	3’UTR	c.*190A > G	chr16:72,821,087	A/G		rs136869959	NCBI
		c.*321G > C	chr16:72,820,956	G/C		rs135704995	NCBI
		c.*326A > G	chr16:72,820,951	A/G		rs137084971	NCBI
		c.*640G > A	chr16:72,820,637	G/A		rs210941907	NCBI
		c.*685G > C	chr16:72,820,592	G/C		rs209048412	NCBI
		c.*735 T > C	chr16:72,820,542	T/C		rs210174407	NCBI
		c.*1064G > A	chr16:72,820,213	G/A		rs211390316	NCBI
	3′ flanking region	g.72819977 T > C	chr16:72,819,977	T/C		rs208866610	NCBI
		g.72819850A > G	chr16:72,819,850	A/G		rs211185269	NCBI
		g.72818819A > G	chr16:72,818,819	A/G		rs41823568	NCBI
		g.72818818C > T	chr16:72,818,818	C/T		rs41823569	NCBI
		g.72818292 T > C	chr16:72,818,292	T/C		rs209896483	NCBI
		g.72818161 T > C	chr16:72,818,161	T/C		rs210138925	NCBI
CDKN1A	exon 5	c.271C > T	chr23:10,565,335	C/T	p.Arg91Trp	rs442346530	NCBI
	3’UTR	c.*9C > G	chr23:10,567,031	C/G		rs210197882	NCBI
		c.*654C > T	chr23:10,567,676	C/T		rs110284249	NCBI
	3’flanking region	g.10569766 T > C	chr23:10,569,766	T/C		rs133659402	NCBI
		g.10569779 T > C	chr23:10,569,779	T/C		rs110283961	NCBI

In addition, in this study, we performed the pooled sequencing method to identify the allelic variants across the entire coding and regulatory regions of ATF3 and CDKN1A genes, and detected a total of 24 SNPs. However, this method has disadvantage that it may miss rare allelic variants due to sequencing cannot catch the very low fluorescent signal of the alleles with very low frequencies. Further, the allelic frequencies cannot be obtained merely by pooled sequencing, hence, we further genotyped 1093 cows and performed association analysis.

Associates between SNPs and five milk traits

Associations between the total 24 SNPs of ATF3 and CDKN1A genes and five milk production traits were presented in Tables 2 and 3. Using single-SNP association analysis, we found that 14, 16, 16 and 3 SNPs of ATF3 gene were significantly associated with milk yield (P = < 0.0001 ~ 0.031), fat yield (P = 0.0002 ~ 0.0197), protein yield (P = < 0.0001 ~ 0.0331), and protein percentage (P = 0.0108 ~ 0.0494) in the first lactation, respectively. For example, the cows with allele C in g.72834301C > A showed higher milk yield, milk protein yield and fat yield than that with allele A (Table 2 ). Similarly, 13, 14, 11 and 8 SNPs were associated with milk yield (P = < 0.0001 ~ 0.0078), fat yield (P = < 0.0001 ~ 0.0395), protein yield (P = < 0.0001 ~ 0.0461), and protein percentage (P = 0.0003 ~ 0.0371) in the second lactation, respectively (Table 2). Of these, 13 SNPs of ATF3 gene (g.72834301C > A, g.72834229C > A, g.72833969A > G, g.72833562G > T, c.291G > A, c.489G > A, c.*685G > C, c.*1064G > A, g.72819850A > G, g.72818819A > G, g.72818818C > T, g.72818292 T > C, and g.72818161 T > C) were identified significantly associated with at least one milk trait in both the first and second lactations. Four SNP, c.*190A > G, c.*326A > G, c.*640G > A, and g.72819977 T > C, were only found evidently associated with the milk traits in the first lactation, and the other two SNPs, c.*321G > C and c.*735 T > C, were markedly associated with at least one milk trait in the second lactation (Table 2), however, the allelic effects of these six SNPs showed almostly same directions between the first and second lactations although some associations did not reach statistical significance of 0.05 (Table 2). Interestingly, no significant SNPs were associated with fat percentage in both first and second lactations.

Table 2

Associations of 19 SNPs of ATF3 gene with milk production traits in Chinese Holstein cattle during two lactations (LSM ± SE)

SNPs	Lactation	Genotype (No.)	Milk yield (kg)	Fat yield (kg)	Fat percentage (%)	Protein yield (kg)	Protein percentage (%)
g.72834301C > A	1	CC(222)	10,305 ± 73.52a	346.63 ± 3.19Aa	3.37 ± 0.03	306.89 ± 2.32Aa	2.98 ± 0.01
		CA(553)	10,339 ± 62Aa	343.59 ± 2.77Aa	3.34 ± 0.026	305.6 ± 2.01Aa	2.96 ± 0.008
		AA(299)	10,146 ± 68.79Bb	336.42 ± 3.01B	3.33 ± 0.028	300.12 ± 2.19B	2.96 ± 0.009
		P	0.001**	0.0002**	0.2065	0.0003**	0.0737
	2	CC(170)	10,707 ± 78.61ab	385.28 ± 3.39ab	3.61 ± 0.032	315.96 ± 2.47Aa	2.96 ± 0.011Aa
		CA(371)	10,785 ± 64.99Aa	390.15 ± 2.89Aa	3.63 ± 0.027	322.03 ± 2.1B	2.99 ± 0.009B
		AA(212)	10,567 ± 73.79Bb	379.59 ± 3.21Bb	3.59 ± 0.03	312.96 ± 2.34Aa	2.96 ± 0.01Aa
		P	0.0051**	0.0004**	0.2901	<.0001**	0.0005**
g.72834229C > A	1	CC(221)	10,318 ± 73	346.25 ± 3.17Aa	3.36 ± 0.03	306.34 ± 2.31Aa	2.97 ± 0.01a
		CA(553)	10,315 ± 61.81	341.19 ± 2.75b	3.32 ± 0.026	303.89 ± 2.01ab	2.95 ± 0.008b
		AA(299)	10,194 ± 68.95	337.13 ± 3.02Bb	3.31 ± 0.028	301.01 ± 2.2Bb	2.96 ± 0.009ab
		P	0.0523	0.003**	0.1234	0.0212*	0.0494*
	2	CC(169)	10,691 ± 78.17a	383.01 ± 3.37a	3.6 ± 0.032	314.66 ± 2.45Aa	2.95 ± 0.011Aa
		CA(369)	10,802 ± 64.49Aa	388.4 ± 2.86Aa	3.61 ± 0.027	321.87 ± 2.08B	2.99 ± 0.009B
		AA(211)	10,493 ± 73.62Bb	374.92 ± 3.19Bb	3.58 ± 0.03	309.72 ± 2.32Ab	2.96 ± 0.01Aa
		P	<.0001**	<.0001**	0.5118	<.0001**	0.001**
g.72833969A > G	1	GG(300)	10,147 ± 68.91Aa	333.48 ± 3.02Aa	3.3 ± 0.028	299.66 ± 2.2A	2.96 ± 0.009
		GA(551)	10,318 ± 61.59Bb	338.88 ± 2.74b	3.31 ± 0.025	304.12 ± 2Ba	2.95 ± 0.008
		AA(222)	10,273 ± 72.9ab	342.05 ± 3.16Bb	3.35 ± 0.03	304.89 ± 2.3Ba	2.97 ± 0.010
		P	0.0049**	0.0033**	0.1466	0.0057**	0.053
	2	GG(211)	10,466 ± 75.09A	372.1 ± 3.27A	3.56 ± 0.031	309.3 ± 2.38Aa	2.96 ± 0.010Aa
		GA(369)	10,793 ± 65.5Ba	388.2 ± 2.91Ba	3.61 ± 0.027	321.77 ± 2.12B	2.99 ± 0.009B
		AA(169)	10,700 ± 78.46Ba	382.28 ± 3.38Bb	3.59 ± 0.032	315.32 ± 2.46Ab	2.95 ± 0.011Aa
		P	<.0001**	<.0001**	0.1837	<.0001**	0.0015**
g.72833562G > T	1	TT(221)	10,340 ± 73.11	347.61 ± 3.17Aa	3.37 ± 0.03	306.84 ± 2.31a	2.97 ± 0.01
		TG(552)	10,360 ± 62.09	343.16 ± 2.77a	3.33 ± 0.026	305.18 ± 2.02a	2.95 ± 0.008
		GG(299)	10,234 ± 68.85	338.54 ± 3.02Bb	3.32 ± 0.028	301.92 ± 2.2b	2.95 ± 0.009
		P	0.0512	0.0029**	0.097	0.0275*	0.0779
	2	TT(169)	10,769 ± 78.92Aa	386.76 ± 3.41Aa	3.6 ± 0.032	316.78 ± 2.48Aa	2.95 ± 0.011Aa
		TG(370)	10,824 ± 64.37Aa	391.65 ± 2.85Aa	3.63 ± 0.027	322.38 ± 2.07B	2.98 ± 0.009B
		GG(210)	10,528 ± 74.13B	377.34 ± 3.22B	3.59 ± 0.03	310.91 ± 2.34Ab	2.96 ± 0.01Aa
		P	<.0001**	<.0001**	0.2651	<.0001**	0.0014**
c.291G > A	1	AA(31)	9757.9 ± 144.69A	326.63 ± 5.95Aa	3.37 ± 0.058	288.83 ± 4.34A	2.98 ± 0.021
		AG(345)	10,202 ± 66.08Ba	339.19 ± 2.91b	3.34 ± 0.027	301.61 ± 2.12Ba	2.96 ± 0.009
		GG(697)	10,268 ± 60.55Ba	343.04 ± 2.71Bc	3.35 ± 0.025	303.66 ± 1.98Ba	2.96 ± 0.008
		P	0.0007**	0.0034**	0.6488	0.0008**	0.752
	2	AA(25)	10,866 ± 165.07	400.94 ± 6.75Aa	3.68 ± 0.066	319.04 ± 4.93	2.94 ± 0.024
		AG(226)	10,557 ± 73.54	382.33 ± 3.2Bb	3.62 ± 0.03	313.83 ± 2.33	2.98 ± 0.01
		GG(503)	10,683 ± 61.69	385.82 ± 2.77b	3.62 ± 0.026	317.26 ± 2.01	2.98 ± 0.008
		P	0.0504	0.0172*	0.6436	0.147	0.3771
c.489G > A	1	AA(31)	9774.7 ± 144.35A	326.99 ± 5.93Aa	3.36 ± 0.058	289.58 ± 4.32A	2.98 ± 0.021
		AG(344)	10,239 ± 65.93Ba	338.76 ± 2.9c	3.32 ± 0.027	302.48 ± 2.11Ba	2.96 ± 0.009
		GG(709)	10,294 ± 60.82Ba	342.86 ± 2.72Bb	3.34 ± 0.025	304.28 ± 1.98Ba	2.96 ± 0.008
		P	0.0006**	0.0033**	0.4033	0.0009**	0.5871
	2	AA(25)	10,971 ± 164.92ab	399.91 ± 6.75a	3.63 ± 0.066	322.88 ± 4.92ab	2.94 ± 0.024
		AG(225)	10,657 ± 73.09Aa	386.05 ± 3.17b	3.61 ± 0.03	318.62 ± 2.31a	2.98 ± 0.01
		GG(510)	10,847 ± 60.46Bb	390.87 ± 2.71ab	3.6 ± 0.025	323.16 ± 1.97b	2.97 ± 0.008
		P	0.005**	0.0395*	0.8064	0.0461*	0.1971
c.*190A > G	1	AA(584)	10,178 ± 62.42a	338.65 ± 2.78Aa	3.35 ± 0.026	300.85 ± 2.02	2.96 ± 0.008
		AG(444)	10,217 ± 63.84Aa	338.37 ± 2.83Aa	3.33 ± 0.026	301.91 ± 2.06	2.96 ± 0.009
		GG(56)	9939.48 ± 113.17Bb	323.63 ± 4.7B	3.28 ± 0.046	294.8 ± 3.43	2.98 ± 0.016
		P	0.031*	0.0014**	0.2273	0.0705	0.6105
	2	AA(423)	10,792 ± 63.54	389.14 ± 2.82	3.61 ± 0.026	321.2 ± 2.05	2.98 ± 0.008
		AG(295)	10,671 ± 67.97	387.7 ± 2.98	3.63 ± 0.028	318.92 ± 2.17	2.99 ± 0.009
		GG(42)	10,736 ± 131.2	385.86 ± 5.42	3.58 ± 0.053	316.43 ± 3.95	2.94 ± 0.019
		P	0.1326	0.7237	0.5687	0.249	0.0664
c.*321G > C	1	CC(53)	10,076 ± 114.66	329.19 ± 4.76	3.29 ± 0.046	298.66 ± 3.47	2.97 ± 0.016
		CG(435)	10,277 ± 63.65	339.07 ± 2.82	3.32 ± 0.026	303.06 ± 2.05	2.96 ± 0.009
		GG(587)	10,217 ± 62.36	338.28 ± 2.78	3.33 ± 0.026	301.31 ± 2.02	2.96 ± 0.008
		P	0.1191	0.0698	0.5569	0.2362	0.583
	2	CC(41)	10,584 ± 131.99	380.38 ± 5.45	3.6 ± 0.053	311.06 ± 3.97	2.94 ± 0.019a
		CG(290)	10,690 ± 69.23	388.18 ± 3.04	3.64 ± 0.028	319.27 ± 2.21	2.99 ± 0.009b
		GG(424)	10,706 ± 64.21	385.69 ± 2.86	3.62 ± 0.026	317.31 ± 2.08	2.97 ± 0.009ab
		P	0.6383	0.2827	0.6702	0.0947	0.0371*
c.*326A > G	1	AA(587)	10,223 ± 62.03Aa	337.47 ± 2.77Aa	3.32 ± 0.026	301.41 ± 2.01Aa	2.96 ± 0.008
		AG(431)	10,295 ± 63.85Aa	338.53 ± 2.83Aa	3.31 ± 0.026	303.33 ± 2.06Aa	2.95 ± 0.009
		GG(57)	9904.58 ± 111.55B	322.04 ± 4.64B	3.28 ± 0.045	292.88 ± 3.38B	2.97 ± 0.016
		P	0.0008**	0.0004**	0.5042	0.0026**	0.4722
	2	AA(425)	10,789 ± 64.31	389.29 ± 2.86	3.62 ± 0.027	319.31 ± 2.08	2.97 ± 0.009
		AG(285)	10,706 ± 68.79	387.76 ± 3.02	3.63 ± 0.028	318.88 ± 2.2	2.98 ± 0.009
		GG(43)	10,572 ± 129.83	382.68 ± 5.36	3.62 ± 0.052	310.75 ± 3.91	2.95 ± 0.019
		P	0.1309	0.3978	0.9601	0.0689	0.0988
c.*640G > A	1	AA(31)	9805.53 ± 145.34A	328.47 ± 5.97Aa	3.36 ± 0.058	290.54 ± 4.36A	2.97 ± 0.021
		AG(340)	10,228 ± 66.3Ba	341.02 ± 2.92c	3.34 ± 0.027	302.39 ± 2.13Ba	2.95 ± 0.009
		GG(709)	10,299 ± 60.44Ba	345.29 ± 2.7Bb	3.36 ± 0.025	304.73 ± 1.97Ba	2.96 ± 0.008
		P	0.0009**	0.0018**	0.5681	0.0009**	0.655
	2	AA(25)	10,910 ± 164.71	396.49 ± 6.74	3.63 ± 0.066	2.93 ± 0.024	2.93 ± 0.024
		AG(221)	10,653 ± 73.17	381.19 ± 3.18	3.58 ± 0.03	2.97 ± 0.01	2.97 ± 0.01
		GG(510)	10,763 ± 61.24	384.41 ± 2.75	3.58 ± 0.025	2.97 ± 0.008	2.97 ± 0.008
		P	0.107	0.0533	0.7617	0.2326	0.2326
c.*685G > C	1	CC(30)	9978.84 ± 146.26	328.2 ± 6	3.32 ± 0.059	294.37 ± 4.38a	2.96 ± 0.021
		CG(337)	10,272 ± 66.72	336.86 ± 2.94	3.3 ± 0.027	303.87 ± 2.14b	2.96 ± 0.009
		GG(713)	10,302 ± 60.16	339.63 ± 2.69	3.31 ± 0.025	304.85 ± 1.96b	2.96 ± 0.008
		P	0.0617	0.0588	0.6272	0.0331*	0.9781
	2	CC(25)	11,131 ± 164.65Aa	398.09 ± 6.74a	3.57 ± 0.066	323.53 ± 4.92ab	2.92 ± 0.024
		CG(221)	10,600 ± 73.44B	383.03 ± 3.19b	3.61 ± 0.03	314.94 ± 2.33a	2.97 ± 0.01
		GG(511)	10,774 ± 61.52Ab	388.76 ± 2.77a	3.61 ± 0.025	319.05 ± 2.01b	2.97 ± 0.008
		P	0.0008**	0.0168*	0.8299	0.0384*	0.05
c.*735 T > C	1	CC(30)	10,031 ± 146.66	330.74 ± 6.02	3.32 ± 0.059	296.27 ± 4.39	2.96 ± 0.021
		CT(338)	10,256 ± 66.98	337.13 ± 2.95	3.3 ± 0.028	303.04 ± 2.15	2.96 ± 0.009
		TT(712)	10,306 ± 60.38	339.99 ± 2.7	3.32 ± 0.025	304.31 ± 1.97	2.96 ± 0.008
		P	0.1031	0.1125	0.7774	0.1133	0.9598
	2	CC(25)	11,178 ± 166.12Aa	397.7 ± 6.82a	3.56 ± 0.067	326.05 ± 4.97a	2.92 ± 0.024
		CT(222)	10,608 ± 73.59B	383.22 ± 3.2b	3.61 ± 0.03	315.95 ± 2.33b	2.98 ± 0.01
		TT(511)	10,791 ± 61.3Ab	388.01 ± 2.75ab	3.6 ± 0.025	320.43 ± 2a	2.97 ± 0.008
		P	0.0003**	0.0358*	0.7454	0.0166*	0.0571
c.*1064G > A	1	AA(30)	9789.35 ± 145.71A	324.33 ± 5.97Aa	3.34 ± 0.058	290.87 ± 4.35A	2.98 ± 0.021
		AG(346)	10,225 ± 66.48Ba	336.45 ± 2.93b	3.31 ± 0.027	302.73 ± 2.13Ba	2.96 ± 0.009
		GG(699)	10,290 ± 60.6Ba	340.29 ± 2.71Bb	3.33 ± 0.025	304.73 ± 1.97Ba	2.96 ± 0.008
		P	0.0011**	0.0046**	0.6409	0.0019**	0.5754
	2	AA(24)	11,065 ± 169.52a	411.69 ± 6.93A	3.7 ± 0.068	324.69 ± 5.05	2.94 ± 0.024
		AG(227)	10,639 ± 72.64b	386.11 ± 3.16Ba	3.62 ± 0.03	317.56 ± 2.3	2.98 ± 0.01
		GG(501)	10,797 ± 61.59a	391.84 ± 2.76Bb	3.63 ± 0.025	321.28 ± 2.01	2.98 ± 0.008
		P	0.0062**	0.0004**	0.5116	0.0823	0.1711
g.72819977 T > C	1	TT(709)	10,252 ± 60.58Aa	341.13 ± 2.71Aa	3.34 ± 0.025	303.11 ± 1.97Aa	2.96 ± 0.008
		TC(345)	10,190 ± 66.68Aa	336.44 ± 2.94b	3.32 ± 0.027	301.02 ± 2.14a	2.96 ± 0.009
		CC(31)	9810.39 ± 144.46B	326.61 ± 5.93Bb	3.35 ± 0.058	290.6 ± 4.33Bb	2.97 ± 0.021
		P	0.0037**	0.0034**	0.391	0.0044**	0.6802
	2	TT(510)	10,795 ± 60.71	388.17 ± 2.72	3.6 ± 0.025	321.11 ± 1.98	2.97 ± 0.008
		TC(225)	10,673 ± 72.24	385.16 ± 3.13	3.6 ± 0.03	318.52 ± 2.28	2.98 ± 0.01
		CC(25)	10,953 ± 165.5	399.73 ± 6.77	3.64 ± 0.066	321.39 ± 4.94	2.94 ± 0.024
		P	0.0686	0.0736	0.777	0.3541	0.2628
g.72819850A > G	1	GG(31)	9737.41 ± 143.71A	322.7 ± 5.9Aa	3.34 ± 0.058	287.79 ± 4.3A	2.97 ± 0.021
		GA(342)	10,221 ± 66.54Ba	336.58 ± 2.93b	3.3 ± 0.027	301.93 ± 2.13Ba	2.95 ± 0.009
		AA(706)	10,282 ± 60.43Ba	340.2 ± 2.7Bb	3.32 ± 0.025	303.63 ± 1.97Ba	2.95 ± 0.008
		P	0.0003**	0.0021**	0.6324	0.0003**	0.7239
	2	GG(25)	10,948 ± 164.92b	404.59 ± 6.75Aa	3.69 ± 0.066	321.58 ± 4.92ab	2.94 ± 0.024
		GA(224)	10,546 ± 73.43Aa	381.72 ± 3.19B	3.62 ± 0.03	313.96 ± 2.32Aa	2.98 ± 0.01
		AA(508)	10,773 ± 61.24Bb	388.75 ± 2.75Ab	3.61 ± 0.025	320.15 ± 2Bb	2.98 ± 0.008
		P	0.0005**	0.0004**	0.5079	0.003**	0.2881
g.72818819A > G	1	GG(305)	10,154 ± 68.15Aa	336.43 ± 2.98A	3.33 ± 0.028	299.24 ± 2.17A	2.95 ± 0.009a
		GA(557)	10,314 ± 61.4Bb	341.97 ± 2.74Ba	3.33 ± 0.025	303.62 ± 1.99Ba	2.95 ± 0.008Aa
		AA(218)	10,301 ± 73.25b	346.69 ± 3.17Bb	3.38 ± 0.03	305.87 ± 2.31Ba	2.97 ± 0.01Bb
		P	0.0068**	0.0004**	0.1048	0.0012**	0.0108*
	2	GG(216)	10,481 ± 73.65A	374.35 ± 3.2A	3.57 ± 0.03	309.56 ± 2.33A	2.96 ± 0.01Aa
		GA(373)	10,845 ± 64.47Ba	391.01 ± 2.86Ba	3.61 ± 0.027	323.76 ± 2.08B	2.99 ± 0.009B
		AA(167)	10,778 ± 79.59Ba	384.6 ± 3.44Bb	3.57 ± 0.033	317.22 ± 2.51C	2.95 ± 0.011Aa
		P	<.0001**	<.0001**	0.1906	<.0001**	0.0003**
g.72818818C > T	1	TT(305)	10,224 ± 68.77Aa	337.97 ± 3.01A	3.31 ± 0.028	301.6 ± 2.19A	2.95 ± 0.009ab
		TC(555)	10,382 ± 61.58Bb	344.22 ± 2.75Ba	3.33 ± 0.025	305.98 ± 2Ba	2.95 ± 0.008a
		CC(221)	10,330 ± 72.84ab	346.52 ± 3.16Ba	3.36 ± 0.03	306.65 ± 2.3Ba	2.97 ± 0.01b
		P	0.0106*	0.0019**	0.1484	0.0075**	0.0355*
	2	TT(216)	10,466 ± 74A	374.26 ± 3.22A	3.58 ± 0.03	308.75 ± 2.34A	2.96 ± 0.01Aa
		TC(372)	10,767 ± 64.62Ba	389.23 ± 2.87Ba	3.63 ± 0.027	320.3 ± 2.09Ba	2.99 ± 0.009B
		CC(169)	10,737 ± 78.69Ba	385.23 ± 3.4Ba	3.6 ± 0.032	315.35 ± 2.48Bb	2.95 ± 0.011Aa
		P	<.0001**	<.0001**	0.1591	<.0001**	0.0006**
g.72818292 T > C	1	TT(709)	10,300 ± 60.49Aa	341.5 ± 2.71a	3.33 ± 0.025	304.06 ± 1.97a	2.96 ± 0.008
		TC(336)	10,249 ± 66.63a	337.35 ± 2.93b	3.31 ± 0.027	302.39 ± 2.13a	2.95 ± 0.009
		CC(33)	9929.1 ± 140.09Bb	330.35 ± 5.74b	3.34 ± 0.056	294.03 ± 4.19b	2.97 ± 0.02
		P	0.0156*	0.0197*	0.453	0.0253*	0.632
	2	TT(509)	10,683 ± 61.62c	382.44 ± 2.77Aa	3.6 ± 0.026	316.43 ± 2.02a	2.97 ± 0.008
		TC(219)	10,522 ± 72.71Aa	377.12 ± 3.15Ab	3.6 ± 0.03	312.89 ± 2.3Aa	2.98 ± 0.01
		CC(27)	11,074 ± 159.67Bb	405.34 ± 6.54B	3.64 ± 0.064	326.97 ± 4.77Bb	2.95 ± 0.023
		P	0.0006**	<.0001**	0.7811	0.0057**	0.4872
g.72818161 T > C	1	TT(711)	10,326 ± 60.82Aa	343.62 ± 2.72A	3.34 ± 0.025	305.45 ± 1.98Aa	2.96 ± 0.008
		TC(322)	10,206 ± 67.05Ab	338.41 ± 2.95Ba	3.32 ± 0.028	302.22 ± 2.15Ab	2.96 ± 0.009
		CC(30)	9725.31 ± 145.87B	325.45 ± 5.98Bb	3.36 ± 0.059	287.93 ± 4.36B	2.97 ± 0.021
		P	<.0001**	0.0004**	0.7091	<.0001**	0.6543
	2	TT(511)	10,714 ± 61.04Aa	383.57 ± 2.74a	3.59 ± 0.025	317.97 ± 1.99	2.97 ± 0.008ab
		TC(208)	10,546 ± 73.84Bb	378.91 ± 3.19Aa	3.6 ± 0.03	315.19 ± 2.33	2.99 ± 0.01a
		CC(24)	10,933 ± 168.72a	397.23 ± 6.9Bb	3.63 ± 0.068	319.26 ± 5.03	2.93 ± 0.024b
		P	0.0078**	0.014*	0.7997	0.2957	0.0239*

* indicates P < 0.05; ** indicates P < 0.01; a, b, c within the same column with different superscripts means P < 0.05; A, B, C within the same column with different superscripts means P < 0.01.

Table 3

Associations of 5 SNPs of CDKN1A gene with milk production traits in Chinese Holstein cattle during two lactations (LSM ± SE)

SNPs	Lactation	Genotype (No.)	Milk yield (kg)	Fat yield (kg)	Fat percentage (%)	Protein yield (kg)	Protein percentage (%)
c.271C > T	1	CC(582)	10,249 ± 62.43a	339.12 ± 2.79	3.32 ± 0.026ab	302.37 ± 2.03	2.95 ± 0.008
		CT(412)	10,212 ± 65.4Aa	341.67 ± 2.89	3.36 ± 0.027a	301.44 ± 2.11	2.96 ± 0.009
		TT(76)	10,478 ± 99.65Bb	340.05 ± 4.18	3.27 ± 0.04b	308.04 ± 3.05	2.95 ± 0.014
		P	0.0162*	0.414	0.0377*	0.0522	0.8062
	2	CC(394)	10,564 ± 64.48Aa	378.99 ± 2.86Aa	3.6 ± 0.027	313.03 ± 2.08A	2.97 ± 0.009Aa
		CT(302)	10,772 ± 67.72Bb	387.31 ± 2.98Bb	3.6 ± 0.028	319.25 ± 2.17Ba	2.97 ± 0.009Aa
		TT(54)	10,819 ± 119.09b	388.01 ± 4.94ab	3.61 ± 0.048	327.12 ± 3.6Bb	3.03 ± 0.017B
		P	0.0012**	0.0018**	0.9967	<.0001**	0.0012**
c.*9C > G	1	CC(593)	10,229 ± 61.78a	338.35 ± 2.76	3.33 ± 0.026	301.99 ± 2.01	2.96 ± 0.008
		CG(415)	10,173 ± 64.52Aa	339.45 ± 2.85	3.35 ± 0.027	300.37 ± 2.08	2.96 ± 0.009
		GG(76)	10,428 ± 99.99Bb	338.21 ± 4.19	3.28 ± 0.04	306.56 ± 3.06	2.95 ± 0.014
		P	0.0193*	0.8322	0.0956	0.0613	0.8896
	2	CC(401)	10,603 ± 64.7A	380.64 ± 2.87Aa	3.6 ± 0.027	314.09 ± 2.09A	2.97 ± 0.009Aa
		CG(305)	10,811 ± 67.06Ba	389.97 ± 2.96Bb	3.61 ± 0.028	320.55 ± 2.15B	2.97 ± 0.009Aa
		GG(54)	10,922 ± 117.94Ba	391.5 ± 4.89b	3.59 ± 0.048	329.78 ± 3.56C	3.02 ± 0.017B
		P	0.0004**	0.0003**	0.874	<.0001**	0.0055**
c.*654C > T	1	CC(497)	10,276 ± 62.96	337.95 ± 2.8	3.3 ± 0.026Aa	303.16 ± 2.04	2.95 ± 0.008
		CT(479)	10,213 ± 63.44	340.69 ± 2.82	3.35 ± 0.026Bb	301.57 ± 2.05	2.96 ± 0.008
		TT(107)	10,298 ± 90.26	338.05 ± 3.83	3.29 ± 0.037ab	303.44 ± 2.79	2.95 ± 0.013
		P	0.3235	0.3355	0.0144*	0.4693	0.7739
	2	CC(350)	10,862 ± 65.32	388.92 ± 2.89	3.58 ± 0.027	323.75 ± 2.11Aa	2.98 ± 0.009
		CT(330)	10,755 ± 67.61	389.17 ± 2.98	3.61 ± 0.028	319.07 ± 2.17Bb	2.96 ± 0.009
		TT(79)	10,756 ± 102.96	384.73 ± 4.33	3.58 ± 0.042	316.84 ± 3.16b	2.95 ± 0.014
		P	0.1847	0.529	0.5	0.0092**	0.0922
g.10569766 T > C	1	CC(79)	10,394 ± 98.78	334.84 ± 4.14	3.26 ± 0.04	304.06 ± 3.02	2.94 ± 0.014
		CT(427)	10,192 ± 65.04	336.77 ± 2.88	3.32 ± 0.027	299.51 ± 2.09	2.95 ± 0.009
		TT(564)	10,191 ± 62.48	335.13 ± 2.79	3.31 ± 0.026	299.53 ± 2.03	2.95 ± 0.008
		P	0.0672	0.6615	0.1899	0.2059	0.878
	2	CC(60)	10,803 ± 113.4a	386.17 ± 4.72ab	3.59 ± 0.046	326.9 ± 3.44Aa	3.03 ± 0.016A
		CT(309)	10,790 ± 67.29Aa	388.76 ± 2.96Aa	3.61 ± 0.028	320.1 ± 2.16Ab	2.97 ± 0.009Ba
		TT(380)	10,560 ± 65.37Bb	379.99 ± 2.9Bb	3.61 ± 0.027	313.48 ± 2.11B	2.98 ± 0.009Ba
		P	0.0004**	0.0015**	0.8386	<.0001**	0.0009**
g.10569779 T > C	1	CC(559)	10,286 ± 61.82	339.28 ± 2.76	3.32 ± 0.026	303.96 ± 2.01	2.96 ± 0.008
		CT(434)	10,251 ± 65.2	339.93 ± 2.89	3.33 ± 0.027	302.21 ± 2.1	2.95 ± 0.009
		TT(87)	10,353 ± 95.78	337.22 ± 4.03	3.28 ± 0.039	305.05 ± 2.94	2.96 ± 0.013
		P	0.4605	0.7357	0.4159	0.3306	0.7423
	2	CC(374)	10,598 ± 64.94Aa	382.05 ± 2.88Aa	3.62 ± 0.027	314.19 ± 2.09A	2.97 ± 0.009Aa
		CT(316)	10,799 ± 66.94Bb	388.48 ± 2.95Bb	3.61 ± 0.028	319.81 ± 2.15Ba	2.97 ± 0.009Aa
		TT(65)	10,726 ± 108.83ab	381.24 ± 4.54ab	3.57 ± 0.044	323.07 ± 3.31Ba	3.02 ± 0.015B
		P	0.0041**	0.0197*	0.6029	0.001**	0.0035**

* indicates P < 0.05; ** indicates P < 0.01; a, b, c within the same column with different superscripts means P < 0.05; A, B, C within the same column with different superscripts means P < 0.01.

Regarding CDKN1A gene, it showed that c.271C > T (P = 0.0162) and c.*9C > G (P = 0.0193) were significantly associated with 305-days milk yield, and c.271C > T (P = 0.0377) and c.*654C > T (P = 0.0144) were markedly associated with fat percentage in the first lactation. Four identified SNPs of CDKN1A gene, c.271C > T, c.*9C > G, g.10569766 T > C, and g.10569779 T > C, were significantly associated with 305-days milk yield, fat yield, protein yield, and protein percentage (P = < 0.0001 ~ 0.0197), and c.*654C > T was only evidently associated with protein yield (P = 0.0092) in the second lactation (Table 3). Additionally, three SNPs of CDKN1A gene, c.271C > T, c.*9C > G, and c.*654C > T, were both significantly associated with milk traits in both first (P = 0.0144 ~ 0.0377) and second (P = < 0.0001 ~ 0.0197) lactations. The g.10569766 T > C and g.10569779 T > C were merely associated with milk traits in the second lactation (P = < 0.0001 ~ 0.0197), but the allelic effects of them were almost in the same direction between two lactations. Taken together, the identified SNPs of CDKN1A gene were mainly associated with the milk yield, fat yield, protein yield, and protein percentage in the second lactation of Chinese Holstein cattle in this study (Table 3).

The allele additive, dominant, and substitution effects of 19 SNPs identified in ATF3 gene were mainly significantly associated with milk yield, fat yield, and protein yield (P < 0.05), and the results in the first lactation were slightly different from that in the second lactation; nevertheless, it is interesting that only two SNPs, g.72833562G > T and g.72818819A > G, were evidently associated with fat percentage in the first lactation (P < 0.05) (Additional file 3). Nevertheless, the allele additive, dominant, and substitution effects of 5 SNPs of CDKN1A gene were mainly associated with milk yield, protein yield, and protein percentage (P < 0.05) (Additional file 4).

Associations between haplotypes and five milk traits

The pair-wise D’ measures indicated that all 19 SNPs in ATF3 gene were highly linked (D’ > 0.95), and one haplotype block comprising the 19 SNPs was inferred (Fig. 1a), in which four haplotypes were formed. The frequency of the four haplotypes, H1 (AAGTTAGTGGAGAGGATGG), H2 (AAACTAGTGGAGAGGCCTT), H3 (GGACCGACCAGCGAACCTT), and H4 (AAACTAGTGGGCGGGCCTT), were 46.3%, 28.0%, 17.7%, and 7.2%, respectively. Haplotype-based association analysis showed that the six haplotype combinations were all significantly associated with 305-days milk yield, fat yield, protein yield, and protein percentage in the first lactation (P = 0.0019 ~ 0.0398), and evidently associated with 305-days milk yield, fat yield, and protein yield in the second lactation (P < 0.0001; Table 4).

Fig. 1

Linkage disequilibrium estimated between SNPs in ATF3 (a) and CDKN1A (b) genes. The values in boxes are pair-wise SNP correlations (D’)

Table 4

Haplotypes analysis of ATF3 gene (LSM ± SE)

Lactation	Haplotype combination (No.)	Milk yield (kg)	Fat yield (kg)	Fat percentage (%)	Protein yield (kg)	Protein percentage (%)
1	H1H1 (220)	10,293 ± 73.81ab	344.45 ± 3.21Aa	3.36 ± 0.03	305.64 ± 2.34ABa	2.97 ± 0.01Aa
	H1H2 (286)	10,256 ± 70.59abc	337.6 ± 3.1bc	3.31 ± 0.029	301.1 ± 2.25bc	2.94 ± 0.01Bb
	H1H3 (184)	10,355 ± 77.21Aab	343.03 ± 3.34Aab	3.33 ± 0.032	305.97 ± 2.43Aa	2.96 ± 0.011ab
	H1H4 (84)	10,406 ± 100.1Aa	340.62 ± 4.21abc	3.29 ± 0.041	305.61 ± 3.07ab	2.95 ± 0.014b
	H2H2 (79)	10,165 ± 100.53bc	336.09 ± 4.22bc	3.32 ± 0.041	298.33 ± 3.08BCc	2.94 ± 0.014b
	H2H3 (125)	10,119 ± 86.34Bc	332.39 ± 3.69Bc	3.3 ± 0.035	297.98 ± 2.69Cc	2.95 ± 0.012ab
	P	0.0398*	0.0038**	0.3088	0.0019**	0.0334*
2	H1H1 (161)	10,740 ± 79.51Aab	389.62 ± 3.42Aa	3.64 ± 0.03	317.62 ± 2.49Ab	2.96 ± 0.011
	H1H2 (195)	10,813 ± 76.98Aa	389.73 ± 3.34Aa	3.62 ± 0.03	322.02 ± 2.43Aab	2.98 ± 0.011
	H1H3 (113)	10,625 ± 90.3b	390.54 ± 3.86Aa	3.68 ± 0.04	318.51 ± 2.81Aab	2.99 ± 0.013
	H1H4 (56)	10,887 ± 116.86Aa	392.75 ± 4.86Aa	3.61 ± 0.05	325.59 ± 3.54Aa	2.99 ± 0.017
	H2H2 (52)	10,293 ± 122.84Bc	369.21 ± 5.1Bb	3.6 ± 0.05	304.19 ± 3.72Bc	2.95 ± 0.017
	H2H3 (84)	10,405 ± 99.79Bc	374.45 ± 4.2Bb	3.6 ± 0.04	308.09 ± 3.06Bc	2.96 ± 0.014
	P	<.0001**	<.0001**	0.4721	<.0001**	0.0743

H means haplotype; H1: AAGTTAGTGGAGAGGATGG, H2: AAACTAGTGGAGAGGCCTT, H3: GGACCGACCAGCGAACCTT, and H4: AAACTAGTGGGCGGGCCTT; * indicates P < 0.05; ** indicates P < 0.01; a, b, c within the same column with different superscripts means P < 0.05; A, B, C within the same column with different superscripts means P < 0.01.

Regarding the CDKN1A haplotypes, one block was inferred (D’ > 0.98) as shown in Fig. 1b, consisting of H1 (CCCTC), H2 (CCTTC), H3 (TGCCT), and H4 (CCCCT) with frequency of 39.9%, 31.7%, 25.7%, and 1.5%, respectively. The associations (Table 5) were indicated that the haplotype combinations were markedly associated with fat percentage (P = 0.044) and protein percentage (P = 0.0351) in the first lactation, and remarkably associated with 305-days milk yield, fat yield, protein yield, and protein percentage in the second lactation (P = < 0.0001 ~ 0.0163).

Table 5

Haplotypes analysis of CDKN1A gene (LSM ± SE)

Lactation	Haplotype combination (No.)	Milk yield (kg)	Fat yield (kg)	Fat percentage (%)	Protein yield (kg)	Protein percentage (%)
1	H1H1 (175)	10,246 ± 78.7	337.02 ± 3.4	3.3 ± 0.032ab	301.01 ± 2.47	2.94 ± 0.011Bc
	H1H2 (279)	10,190 ± 69.46	339.81 ± 3.04	3.35 ± 0.029a	302.54 ± 2.22	2.97 ± 0.009Aa
	H1H3 (222)	10,179 ± 74.12	338.05 ± 3.22	3.33 ± 0.03ab	301.48 ± 2.35	2.97 ± 0.01ab
	H2H2 (107)	10,250 ± 89.88	337.17 ± 3.81	3.3 ± 0.036ab	302.85 ± 2.78	2.96 ± 0.013abc
	H2H3 (179)	10,140 ± 77.68	339.79 ± 3.35	3.36 ± 0.032Aa	298.23 ± 2.44	2.95 ± 0.011bc
	H3H3 (75)	10,418 ± 100.99	336.31 ± 4.24	3.25 ± 0.041Bb	306.5 ± 3.09	2.95 ± 0.014abc
	P	0.1065	0.8508	0.044*	0.0939	0.0351*
2	H1H1 (114)	10,555 ± 90.95Bc	378.57 ± 3.87Cc	3.59 ± 0.037	314.92 ± 2.82Bb	2.98 ± 0.013ABb
	H1H2 (174)	10,623 ± 79.4Bc	387.32 ± 3.43BCb	3.65 ± 0.032	315.26 ± 2.5Bb	2.97 ± 0.011Bb
	H1H3 (150)	11,061 ± 81.27Aa	399.1 ± 3.49Aa	3.62 ± 0.033	328.44 ± 2.54Aa	2.97 ± 0.011Bb
	H2H2 (78)	10,722 ± 104.5Bbc	383.32 ± 4.39BCbc	3.58 ± 0.042	316.19 ± 3.2Bb	2.95 ± 0.015Bb
	H2H3 (131)	10,717 ± 87.66Bbc	389.21 ± 3.74ABb	3.62 ± 0.036	317.87 ± 2.73Bb	2.96 ± 0.012Bb
	H3H3 (53)	10,884 ± 120.25ab	388.23 ± 4.99b	3.58 ± 0.048	329.01 ± 3.64Aa	3.02 ± 0.017Aa
	P	<.0001**	<.0001**	0.5127	<.0001**	0.0163*

H means haplotype; H1: CCCTC, H2: CCTTC, H3: TGCCT, and H4: CCCCT; * indicates P < 0.05; ** indicates P < 0.01; a, b, c within the same column with different superscripts means P < 0.05; A, B, C within the same column with different superscripts means P < 0.01.

Prediction of TFBSs variations in promoter region of ATF3 gene

With regard to the four SNPs on the 5’promoter region of ATF3 gene, we predicted the variation of TFBSs after mutation using JASPAR software. As the results shown in Table 6, the A allele in g.72834301C > A created the putative binding sites for the transcription factor E2F3 (E2F transcription factor 3; relative score = 0.88) and Zfp423 (zinc finger protein 423; relative score = 0.89). The binding sites for the transcription factor Bcl6 (B-cell CLL/lymphoma 6) was invented because of the A allele in g.72834229C > A (relative score = 0.87), and g.72833969A > G resulted in the appearing of the binding sites for transcription factor STAT3 (signal transducer and activator of transcription 3) after the substitution by G allele (relative score = 0.95). It was also indicated that the binding site for transcription factor Zfp423 was arisen due to the T allele in g.72833562G > T (relative score = 0.88).

Table 6

Transcription factor binding sites (TFBSs) prediction for ATF3 gene

SNPs	Allele	TFBSs
g.72834301C > A	C	-
	A	E2F3, Zfp423
g.72834229C > A	C	-
	A	Bcl6
g.72833969A > G	A	-
	G	STAT3
g.72833562G > T	G	-
	T	Zfp423

- means no predicted TFBSs.

Variant structure of CDKN1A protein caused by missense mutation

There was a missense mutation (p.Arg91Trp) on CDKN1A protein caused by c.271C > T. The results from the SOPMA SERVER revealed that α-helix was changed from 32.92% to 32.3%, β-turn from 4.35% to 5.59%, and random coil from 49.69% to 49.07% (Table 7), indicating that the CDKN1A protein secondary structure was altered from arginine to tryptophan.

Table 7

Alteration of CDKN1A protein caused by the mutation

SNPs	Allele	α-helix	Extended strand	β-turn	Random coil
c.271C > T	C	32.92	13.04	4.35	49.69
	T	32.3	13.04	5.59	49.07

Discussion

This study was a follow-up investigation of our previous RNA-Seq work, in which the ATF3 and CDKN1A genes were potentially associated with milk protein and fat percentage [9]. Here, we first determined that SNPs within the ATF3 and CDKN1A genes were significantly associated with milk production traits in dairy cattle. Of these, four SNPs, g.72834301C > A, g.72834229C > A, g.72833969A > G, and g.72833562G > T, potentially changing the ATF3 promoter activity, and one SNP, c.271C > T, potentially altering the CDKN1A protein secondary structure, might be potentially causal mutations.

With regard to the associations of ATF3 and CDKN1A with five milk traits, we found that eight SNPs showed different associations between the first and second lactations (Tables 2 and 3). The possible reason may be that different number of cows were used for association analysis (1093 cows in the first lactation versus 769 cows in the second lactation) because 324 cows merely completed their milking of first lactation, which could impact the statistical significance. In addition, the physiologic status of cows between the first and second lactations are different as well, generally, cows show higher milk production in the second lactation. Further, the directions of allelic effects of the eight SNPs on the milk traits were almost consistent between the two lactations. Studies revealed that haplotype blocks in human genome is independent of their surrounding areas with regard to LD [15-17], and the haplotype analyses were widely applied to the genetic variation studies [18, 19]. Our haplotypes analyses showed that the SNPs were highly linked, and the haplotypes were also significantly associated with milk yield, fat yield, fat percentage, protein yield, and protein percentage, which were consistent with the associations of SNPs with milk traits.

ATF3 is involved in the TLR4 (toll-like receptor 4) signaling pathway, and its expression was markedly increased causing by chemotaxis and diapedesis in vitro, indicating that ATF3 acts as the early adaptive-response gene having an important role in maintaining the cellular homeostasis [20]. It was reported that ATF3 activates the cyclin D1 expression, thereby stimulating the mouse hepatocellular proliferation [21]. As we know, liver plays a key role in lipid metabolism, including fatty acid uptake, synthesis and oxidation, glycerolipid synthesis, and triacylglycerol export [22]. Subsequently, Invernizzi et al. found ATF3 may modulate milk fat synthesis during lactation by participating in the endoplasmic reticulum stress pathway [23, 24]. Our results also showed a significant relationship between SNP polymorphism of ATF3 gene and fat yield, thus it can be seen that ATF3 may be associated with lipid metabolism. The CDKN1A (p21) protein regulates the cell cycle at G1 and S phase by inhibiting the activity of cyclin-CDK2, −CDK1, and –CDK4/6 complexes [25]. Li and Capuco conducted a systematic search for estrogen-responsive genes in bovine mammary gland, and identified 23 regulatory networks, of these, CDKN1A gene occupied a focal position in the network that functions as cell cycle, cellular movement and cancers, indicating that CDKN1A may play an important role in mammary gland development [26]. Together, ATF3 and CDKN1A were considered as important promising candidate genes for milk production traits.

As we know, SNPs in transcription factor binding sites could lead to allele-specific binding of transcription factors and enhancing or repressing the gene expression [27]. Studies have revealed that transcription factor E2F3 [28-31], Zfp423 [32, 33], and STAT3 [34, 35] could activate or repress the gene expression, and Bcl6 might play as a repressor for gene expression [36, 37]. Our results showed that the milk yield, fat yield, fat percentage, protein yield, and protein percentage were evidently decreased or had the downtrends in genotype AA, AA, and GG severally causing by g.72834301C > A, g.72834229C > A, and g.72833969A > G, indicating that the transcription factors E2F3, Zfp423, Bcl6, and STAT3, might prejudice the milk production by repressing the expression of ATF3 gene. On the contrary, the transcription factor Zfp423 might be also beneficial to the milk production through activating the expression of ATF3 gene, because that the genotype TT causing by g.72833562G > T increased the milk yield, fat yield, fat percentage, protein yield, and protein percentage in the two lactations. Overall, our findings suggested that the four SNPs, g.72834301C > A, g.72834229C > A, g.72833969A > G, and g.72833562G > T in ATF3 gene, might be the potential mutations in milk production traits by changing promoter activity in Holstein cattle.

In the present, we found the SNP c.271C > T of CDKN1A gene was a missense mutation that caused an amino acid substitution from arginine to tryptophan, and the association analyses showed that this SNP was remarkably associated with milk yield and fat percentage in the first lactation, and milk yield, fat yield, protein yield, and protein percentage in the second lactation. Importantly, the changes of the protein structure causing by this amino acid substitution showed that the α-helix changed from 32.92% to 32.3%. Generally, the α-helix was preferably located at the core of the protein and had important functions in proteins for flexibility and conformational changes [38], and it was presumed that the CDKN1A protein might be more stable in conformation when the base was C. Hence, the SNP c.271C > T in CDKN1A gene might be another potential functional mutation for milk production through changing the protein structure in dairy cattle. Further in-depth investigation should be performed to validate the biological functions of these SNPs.

Conclusions

In this study, we totally identified 19 and five SNPs in the ATF3 and CDKN1A genes, respectively, and observed their associations with milk yield and milk composition. Of these, four SNPs in ATF3 gene altered the specific TF binding sites thereby potentially changed promoter activities, and one SNP in CDKN1A gene changed the protein secondary structure, might be the potential causal mutations. In a word, our study first determined the significant genetic effects of ATF3 and CDKN1A genes on milk yield and composition traits in dairy cattle and will be available for marker-assisted breeding based on further validation.

Descriptive statistics of phenotypic values for dairy production traits in two lactations. (XLSX 9 kb)

Primers and procedures for PCR or touch-down PCR used in SNPs identification of ATF3 and CDKN1A genes. (XLSX 15 kb)

Additive, dominant and allele substitution effects of 19 SNPs on milk production traits of ATF3 gene in Chinese Holstein cattle during two lactations. (XLSX 17 kb)

Additive, dominant and allele substitution effects of 5 SNPs on milk production traits of CDKN1A gene in Chinese Holstein cattle during two lactations. (XLSX 10 kb)