Single Nucleotide Polymorphisms of NUCB2 and their Genetic Associations with Milk Production Traits in Dairy Cows

Abstract

We previously used the RNA sequencing technique to detect the hepatic transcriptome of Chinese Holstein cows among the dry period, early lactation, and peak of lactation, and implied that the nucleobindin 2 (NUCB2) gene might be associated with milk production traits due to its expression being significantly increased in early lactation or peak of lactation as compared to dry period (q value < 0.05). Hence, in this study, we detected the single nucleotide polymorphisms (SNPs) of NUCB2 and analyzed their genetic associations with milk yield, fat yield, fat percentage, protein yield, and protein percentage. We re-sequenced the entire coding and 2000 bp of 5' and 3' flanking regions of NUCB2 by pooled sequencing, and identified ten SNPs, including one in 5' flanking region, two in 3' untranslated region (UTR), and seven in 3' flanking region. The single-SNP association analysis results showed that the ten SNPs were significantly associated with milk yield, fat yield, fat percentage, protein yield, or protein percentage in the first or second lactation (p values <= 1 × 10^-4 and 0.05). In addition, we estimated the linkage disequilibrium (LD) of the ten SNPs by Haploview 4.2, and found that the SNPs were highly linked in one haplotype block (D' = 0.98-1.00), and the block was also significantly associated with at least one milk traits in the two lactations (p values: 0.0002-0.047). Further, we predicted the changes of transcription factor binding sites (TFBSs) that are caused by the SNPs in the 5' flanking region of NUCB2, and considered that g.35735477C>T might affect the expression of NUCB2 by changing the TFBSs for ETS transcription factor 3 (ELF3), caudal type homeobox 2 (CDX2), mammalian C-type LTR TATA box (VTATA), nuclear factor of activated T-cells (NFAT), and v-ets erythroblastosis virus E26 oncogene homolog (ERG) (matrix similarity threshold, MST > 0.85). However, the further study should be performed to verify the regulatory mechanisms of NUCB2 and its polymorphisms on milk traits. Our findings first revealed the genetic effects of NUCB2 on the milk traits in dairy cows, and suggested that the significant SNPs could be used in genomic selection to improve the accuracy of selection for dairy cattle breeding.

Keywords: SNP; TFBS; dairy cattle; genetic effect; milk yield and composition.

Figure 1

Allelic and genotypic frequencies of the ten single nucleotide polymorphisms (SNPs) in nucleobindin 2 (NUCB2). The “*” shows the SNPs located in the 3’untranslated region (UTR).

Figure 2

Linkage disequilibrium estimated among the SNPs of NUCB2 (D′ = 0.98–1.00). The blocks indicate haplotype blocks and the text above the horizontal numbers is the SNP names. The values in boxes are pairwise SNP correlations (D′), while the boxes without numbers indicate complete linkage disequilibrium (LD) (D′ = 1).

Figure 3

Transcription factor binding sites (TFBSs) in the 5′ flanking region of NUCB2 gene. The nucleotides represent the transcription factor binding site sequences of ETS transcription factor 3 (ELF3), caudal type homeobox 2 (CDX2), mammalian C-type LTR TATA box (VTATA), nuclear factor of activated T-cells (NFAT), and v-ets erythroblastosis virus E26 oncogene homolog (ERG) with their matrix similarity over 0.85 (in blue), and the underlined nucleotide in red was the SNP.

Figure 4

Expression of NUCB2 gene in bovine tissues. Relative mRNA expression of NUCB2 in eight tissues of lactating Holstein cows normalized by two reference genes, RPS9 and UXT.