Identification of single nucleotide polymorphisms (SNPs) associated with heat stress and milk production traits in Chinese holstein cows

Abstract

Background: Heat stress (HS) poses a significant challenge to the dairy industry, affecting both the health and productivity of dairy cows. Identifying candidate genes and single nucleotide polymorphisms (SNPs) associated with HS is critical to improving heat tolerance of dairy cows. In our previous work, the eukaryotic translation initiation factor 4E (EIF4E), heat shock protein family A member 4 (HSPA4), and inositol 1,4,5-trisphosphate receptor type 2 (ITPR2) genes were found to play critical roles in the HS response of dairy cows.

Results: In this study, we further validated the gene expression patterns and genetic effects of the three candidate genes on HS response and milk production in Chinese Holstein cows. A total of 21 SNPs were identified by sequencing the exon and 2000 bp flanking region of the EIF4E (4 SNPs), HSPA4 (8 SNPs), and ITPR2 (9 SNPs) in pooled DNA samples from 70 Holstein bulls. Among these, two SNPs (g.44653172A > G and g.44660065C > T) were located in the coding exon and the 3' untranslated region of the HSPA4 gene, respectively. Association analyses were conducted between identified SNPs and three HS traits and six milk production traits in a population of 1,160 Chinese Holstein cows. The SNP-based association analysis identified significant associations between ten SNPs and HS traits (P < 0.05), as well as eight SNPs and milk production traits (P < 0.05). In the HSPA4 gene, five SNPs (g.44618036G > A, g.44624256 A > C, g.44624428T > C, g.44653172 A > G, and g.44660065 C > T) were significantly associated with rectal temperature (RT; P < 0.05). Notably, no SNPs were associated with milk production traits. Haplotype blocks containing g.44,653,172 A > G and g.44,660,065 C > T also showed significant associations with RT (P < 0.05). Further analysis suggests that g.44,660,065 C > T affects the stability of the mRNA secondary structure, microRNA and transcription factor binding, thereby potentially influencing the gene expression of HSPA4.

Conclusion: In conclusion, we demonstrated that these three genes had significant genetic effects on HS and milk production traits. And g.44,660,065 C > T in the HSPA4 gene could be used as functional molecular markers for the genetic selection of dairy cows for improving heat tolerance without compromising their high milk performance.

Keywords: HSPA4 gene; Association analysis; Dairy cows; Heat stress; Milk production traits.

Fig. 1

Transcript abundance and relative gene expression levels in the heat stress group and the non-heat stress group. A: Gene expression level determined by RNA-seq; TPM: Transcripts per million. B, C: Relative gene expression levels determined by qRT-PCR. HS: Heat stress group sampled in summer; Non-HS: Non-heat stress group sampled in spring; *: indicates a significant difference between the two groups (P < 0.05)

Fig. 2

Haplotype block for three genes. A: EIF4E; B: HSPA4; C: ITPR2

Fig. 3

Association analysis results between 21 SNPs and nine traits. *: a significant association (P < 0.05). DS: Drooling score; RS: Respiratory score; RT: Rectal temperature; MY: 305-day milk yield (MY); FY: Fat yield; FP: Fat percentage; PY: Protein yield; PP: Protein percentage; SCS: somatic cell score

Fig. 4

De-regressed proofs of rectal temperature (RT) for different genotypes or haplotypes of the HSPA4 gene. *Significant association (P < 0.05). ns: Not significant

Fig. 5

Association analysis between haplotype blocks and traits. *: a significant association (P < 0.05). DS: Drooling score; RS: Respiratory score; RT: Rectal temperature; MY: 305-day milk yield (MY); FY: Fat yield; FP: Fat percentage; PY: Protein yield; PP: Protein percentage; SCS: somatic cell score

Fig. 6

Bioinformatics analysis results of the HSPA4 gene. A: Expression profiles of HSPA4 across various tissues in Holstein cattle. B HSPA4 gene expression levels across different genotypes (C: C, C: T, T: T) at g.44660065C > T. Error bars are omitted for the T: T group (n = 2). C Transcript-level expression of HSPA4-201 across genotypes at g.44660065C > T. No error bars are shown for TT (n = 2). HSPA4-202 was excluded due to low abundance in transcriptomic data. D: Predicted mRNA secondary structure of HSPA4, with the minimum free energy configuration labeled. MFE: minimum free energy. E Allele-specific effects of SNP g.44660065C > T on bta-miR-445-3p binding to the HSPA4 mRNA 3’ untranslated region. F: Identified motif and transcription factors (TFs) in the 3’ untranslated region of HSPA4, with the most matched TF being MA0032.2 (FOXC1)