Identification of genetic associations and functional SNPs of bovine KLF6 gene on milk production traits in Chinese holstein.

Our previous research identified the Kruppel like factor 6 (KLF6) gene as a prospective candidate for milk production traits in dairy cattle. The expression of KLF6 in the livers of Holstein cows during the peak of lactation was significantly higher than that during the dry and early lactation periods. Notably, it plays an essential role in activating peroxisome proliferator-activated receptor α (PPARα) signaling pathways. The primary aim of this study was to further substantiate whether the KLF6 gene has significant genetic effects on milk traits in dairy cattle. Through direct sequencing of PCR products with pooled DNA, we totally identified 12 single nucleotide polymorphisms (SNPs) within the KLF6 gene. The set of SNPs encompasses 7 located in 5' flanking region, 2 located in exon 2 and 3 located in 3' untranslated region (UTR). Of these, the g.44601035G > A is a missense mutation that resulting in the replacement of arginine (CGG) with glutamine (CAG), consequently leading to alterations in the secondary structure of the KLF6 protein, as predicted by SOPMA. The remaining 7 regulatory SNPs significantly impacted the transcriptional activity of KLF6 following mutation (P < 0.005), manifesting as changes in transcription factor binding sites. Additionally, 4 SNPs located in both the UTR and exons were predicted to influence the secondary structure of KLF6 mRNA using the RNAfold web server. Furthermore, we performed the genotype-phenotype association analysis using SAS 9.2 which found all the 12 SNPs were significantly correlated to milk yield, fat yield, fat percentage, protein yield and protein percentage within both the first and second lactations (P < 0.0001 ~ 0.0441). Also, with Haploview 4.2 software, we found the 12 SNPs linked closely and formed a haplotype block, which was strongly associated with five milk traits (P < 0.0001 ~ 0.0203). In summary, our study represented the KLF6 gene has significant impacts on milk yield and composition traits in dairy cattle. Among the identified SNPs, 7 were implicated in modulating milk traits by impacting transcriptional activity, 4 by altering mRNA secondary structure, and 1 by affecting the protein secondary structure of KLF6. These findings provided valuable molecular insights for genomic selection program of dairy cattle.

Liu Y ，Han B ，Zheng W ，Peng P ，Yang C ，Jiang G ，Ma Y ，Li J ，Ni J ，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》

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》

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》

Associations between polymorphisms of SLC22A7, NGFR, ARNTL and PPP2R2B genes and Milk production traits in Chinese Holstein.

Our preliminary work confirmed that, SLC22A7 (solute carrier family 22 member 7), NGFR (nerve growth factor receptor), ARNTL (aryl hydrocarbon receptor nuclear translocator like) and PPP2R2B (protein phosphatase 2 regulatory subunit Bβ) genes were differentially expressed in dairy cows during different stages of lactation, and involved in the lipid metabolism through insulin, PI3K-Akt, MAPK, AMPK, mTOR, and PPAR signaling pathways, so we considered these four genes as the candidates affecting milk production traits. In this study, we detected polymorphisms of the four genes and verified their genetic effects on milk yield and composition traits in a Chinese Holstein cow population. By resequencing the whole coding region and part of the flanking region of SLC22A7, NGFR, ARNTL and PPP2R2B, we totally found 20 SNPs, of which five were located in SLC22A7, eight in NGFR, three in ARNTL, and four in PPP2R2B. Using Haploview4.2, we found three haplotype blocks including five SNPs in SLC22A7, eight in NGFR and three in ARNTL. Single-SNP association analysis showed that 19 out of 20 SNPs were significantly associated with at least one of milk yield, fat yield, fat percentage, protein yield or protein percentage in the first and second lactations (P < 0.05). Haplotype-based association analysis showed that the three haplotypes were significantly associated with at least one of milk yield, fat yield, fat percentage, protein yield or protein percentage (P < 0.05). Further, we used SOPMA software to predict a SNP, 19:g.37095131C > T in NGFR, changed the structure of NGFR protein. In addition, we used Jaspar software to found that four SNPs, 19:g.37113872C > G,19:g.37113157C > T, and 19:g.37112276C > T in NGFR and 15:g.39320936A > G in ARNTL, could change the transcription factor binding sites and might affect the expression of the corresponding genes. These five SNPs might be the potential functional mutations for milk production traits in dairy cattle. In summary, we proved that SLC22A7, NGFR, ARNTL and PPP2R2B have significant genetic effects on milk production traits. The valuable SNPs can be used as candidate genetic markers for genomic selection of dairy cattle, and the effects of these SNPs on other traits need to be further verified.

Jia R ，Fu Y ，Xu L ，Li H ，Li Y ，Liu L ，Ma Z ，Sun D ，Han B ... -  《-》