Identification of single nucleotide polymorphisms of PIK3R1 and DUSP1 genes and their genetic associations with milk production traits in dairy cows.

Previously, phosphoinositide-3-kinase regulatory subunit 1 () and dual specificity phosphatase 1 () were identified as promising candidate genes for milk production traits due to their being differentially expressed between the dry period and the peak of lactation in livers of dairy cows. Hence, in this study, the single nucleotide polymorphisms (SNPs) of and genes were identified and their genetic associations with milk yield, fat yield, fat percentage, protein yield, and protein percentage, were investigated using 1067 Chinese Holstein cows from 40 sire families. By re-sequencing the entire coding region and 2000 bp of the 5' and 3' flanking regions of the two genes, one SNP in the 5' untranslated region (UTR), three in the 3' UTR, and two in the 3' flanking region of were identified, and one in the 5' flanking region, one in the 3' UTR, and two in the 3' flanking region of were found. Subsequent single-locus association analyses showed that five SNPs in , rs42590258, rs210389799, rs208819656, rs41255622, rs133655926, and rs211408208, and four SNPs in , rs207593520, rs208460068, rs209154772, and rs210000760, were significantly associated with milk, fat and protein yields in the first or second lactation ( values ≤ 0.0001 and 0.0461). In addition, by the Haploview 4.2 software, the six and four SNPs in and respectively formed one haplotype block, and the haplotype-based association analyses showed significant associations between their haplotype combinations and the milk traits in both two lactations ( values ≤ 0.0001 and 0.0364). One SNP, rs207593520(T/G), was predicted to alter the transcription factor binding sites (TFBSs) in the 5' flanking region of . Further, the dual-luciferase assay showed that the transcription activity of allele T in rs207593520 was significantly higher than that of allele G, suggesting the activation of transcriptional activity of gene by allele T of rs207593520. Thus, the rs207593520 SNP was highlighted as a potential causal mutation that should be further verified. We demonstrated novel and significant genetic effects of the and genes on milk production traits in dairy cows, and our findings provide information for use in dairy cattle breeding.

Han B ，Yuan Y ，Shi L ，Li Y ，Liu L ，Sun D ... -  《-》

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

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 () 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 ( value < 0.05). Hence, in this study, we detected the single nucleotide polymorphisms (SNPs) of 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 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 ( values <= 1 × 10 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 ( 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 , and considered that g.35735477C>T might affect the expression of 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 and its polymorphisms on milk traits. Our findings first revealed the genetic effects of 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.

Han B ，Yuan Y ，Li Y ，Liu L ，Sun D ... -  《Genes》

Identification of Genetic Effects of ACADVL and IRF6 Genes with Milk Production Traits of Holstein Cattle in China.

With the development of high-throughput sequencing, RNA sequencing has been widely used in the identification of candidate genes for complex traits in livestock, and the functional genes and mutations with large genetic effects on milk production traits can provide molecular information for marker-assisted selection to increase the selection accuracy and accelerate genetic gain in dairy cattle. Our previous study on the liver transcriptome of Holstein cows found that acyl-CoA dehydrogenase () and interferon regulatory factor 6 () are differentially expressed between dry and peak lactation periods, as well as that they are involved in lipid metabolism and the proliferation and differentiation of mammary epithelial cells. Thus, the two genes were considered candidates for milk traits. Hence, this study further collected 1186 Holstein cows from 110 sire families to investigate their genetic associations with milk yield and composition traits. By resequencing the entire exons and 2000 bp of the 5' and 3' flanking regions of the two genes, we identified eight SNPs in and eight SNPs in . Subsequent single-locus association analyses showed that the eight SNPs in were all significantly associated with milk fat yield, fat percentage, and protein yield ( values ≤ 0.0001-0.0414), and the eight SNPs in were associated with milk, fat, and protein yields in the first or second lactation ( values ≤ 0.0001-0.0467). Using Haploview 4.2, one haplotype block with eight of the SNPs in (D' = 0.99-1.00) and two haplotype blocks in with three of the SNPs in each were observed (D' = 0.98-1.00). Similarly, the haplotype combinations of were significantly associated with milk yield, fat percentage, fat yield, and protein yield in the two lactations ( values ≤ 0.0001-0.0125), and those of were associated with five milk traits ( values ≤ 0.0001-0.0263). Furthermore, with the JASPAR software, it was predicted that the SNPs 19:g.26933503T>C in and 16:g.73501985G>A in changed the transcription factor binding sites of ZEB1, PLAGL2, and RHOXF1, implying their impacts on the expressions of the corresponding genes. Our findings demonstrated that the and genes have significant genetic effects on milk yield and composition traits, and the valuable SNPs might be used as genetic markers for genomic selection programs in dairy cattle.

Peng P ，Liu Y ，Zheng W ，Han B ，Wang K ，Sun D ... -  《Genes》

Genetic Effects of LPIN1 Polymorphisms on Milk Production Traits in Dairy Cattle.

Our initial RNA sequencing work identified that lipin 1 () was differentially expressed during dry period, early lactation, and peak of lactation in dairy cows, and it was enriched into the fat metabolic Gene Ontology (GO) terms and pathways, thus we considered as the candidate gene for milk production traits. In this study, we detected the polymorphisms of and verified their genetic effects on milk yield and composition in a Chinese Holstein cow population. We found seven SNPs by re-sequencing the entire coding region and partial flanking region of , including one in 5' flanking region, four in exons, and two in 3' flanking region. Of these, four SNPs, c.637T > C, c.708A > G, c.1521C > T, and c.1555A > C, in the exons were predicted to result in the amino acid replacements. With the Haploview 4.2, we found that seven SNPs in formed two haplotype blocks (D' = 0.98-1.00). Single-SNP association analyses showed that SNPs were significantly associated with milk yield, fat yield, fat percentage, or protein yield in the first or second lactation ( = < 0.0001-0.0457), and only g.86049389C > T was strongly associated with protein percentage in both lactations ( = 0.0144 and 0.0237). The haplotype-based association analyses showed that the two haplotype blocks were significantly associated with milk yield, fat yield, protein yield, or protein percentage ( = < 0.0001-0.0383). By quantitative real-time PCR (qRT-PCR), we found that had relatively high expression in mammary gland and liver tissues. Furthermore, we predicted three SNPs, c.637T > C, c.708A > G, and c.1521C > T, using SOPMA software, changing the LPIN1 protein structure that might be potential functional mutations. In summary, we demonstrated the significant genetic effects of on milk production traits, and the identified SNPs could serve as genetic markers for dairy breeding.

Han B ，Yuan Y ，Liang R ，Li Y ，Liu L ，Sun D ... -  《Genes》

Genetic polymorphisms of PKLR gene and their associations with milk production traits in Chinese Holstein cows.

Our previous work had confirmed that pyruvate kinase L/R () gene was expressed differently in different lactation periods of dairy cattle, and participated in lipid metabolism through insulin, PI3K-Akt, MAPK, AMPK, mTOR, and PPAR signaling pathways, suggesting that is a candidate gene to affect milk production traits in dairy cattle. Here, we verified whether this gene has significant genetic association with milk yield and composition traits in a Chinese Holstein cow population. In total, we identified 21 single nucleotide polymorphisms (SNPs) by resequencing the entire coding region and partial flanking region of gene, in which, two SNPs were located in 5' promoter region, two in 5' untranslated region (UTR), three in introns, five in exons, six in 3' UTR and three in 3' flanking region. The single marker association analysis displayed that all SNPs were significantly associated with milk yield, fat and protein yields or protein percentage ( ≤ 0.0497). The haplotype block containing all the SNPs, predicted by Haploview, had a significant association with fat yield and protein percentage ( ≤ 0.0145). Further, four SNPs in 5' regulatory region and eight SNPs in UTR and exon regions were predicted to change the transcription factor binding sites (TFBSs) and mRNA secondary structure, respectively, thus affecting the expression of , leading to changes in milk production phenotypes, suggesting that these SNPs might be the potential functional mutations for milk production traits in dairy cattle. In conclusion, we demonstrated that had significant genetic effects on milk production traits, and the SNPs with significant genetic effects could be used as candidate genetic markers for genomic selection (GS) in dairy cattle.

Du A ，Zhao F ，Liu Y ，Xu L ，Chen K ，Sun D ，Han B ... -  《Frontiers in Genetics》