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

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. 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. 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.

Han B ，Liang W ，Liu L ，Li Y ，Sun D ... -  《BMC GENETICS》

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 association of the ACACB gene with milk yield and composition traits in dairy cattle.

Previously, we re-sequenced the whole genomes of eight Holstein bulls with high or low milk protein and fat percentage, and we detected two indels in the ACACB (acetyl-CoA carboxylase beta) gene that were polymorphic between the two groups. Thus, we considered ACACB as a promising candidate gene potentially affecting milk composition traits. Herein, we verified the genetic effects of ACACB on five milk traits in a Chinese Holstein population. We identified six SNPs in the 5'-promoter region, five in the 5'- untranslated region (UTR), 11 in exons, four in the 3'-UTR and three in the 3'-flanking region by re-sequencing the entire coding and regulatory regions of ACACB. One of these SNPs (ss1987461005) is reported here for the first time, and three of the SNPs (rs109482081, rs110819816 and rs109281947) were predicted to result in amino acid replacements. Genotype-phenotype association analyses showed that all the identified SNPs, except for ss1987461005, rs208919019 and rs134447911, were significantly associated with milk yield, fat yield, fat percentage, protein yield or protein percentage (P < 0.0001 to 0.0484). Linkage disequilibrium analyses were conducted among the identified SNPs to confirm the genetic associations. Two SNPs-rs135874354 (g.66218726T>C) and rs210928430 (g.66218117G>A)-were predicted to alter transcription factor binding sites in the 5'-promoter region of ACACB. A luciferase activity assay showed that the promoter activity of haplotype TG was significantly higher than that of CG (P = 0.0002) and that the promoter activity of haplotype TA was remarkably higher than that of CA (P = 7.4285E-09), showing that the T allele of rs135874354 increased promoter activity. Thus, rs135874354 was considered to be a potentially functional mutation. Our findings have, for the first time, profiled the genetic effect of ACACB on milk production traits in dairy cattle and revealed a potentially causal mutation that requires further the in-depth validation.

Han B ，Liang W ，Liu L ，Li Y ，Sun D ... -  《-》

Genetic association of DDIT3, RPL23A, SESN2 and NR4A1 genes with milk yield and composition in dairy cattle.

Previously, we identified by RNA sequencing that DDIT3, RPL23A, SESN2 and NR4A1 genes were significantly differentially expressed between the mammary glands of lactating Holstein cows with extremely high and low milk protein and fat percentages; thus, these four genes are considered as promising candidates potentially affecting milk yield and composition traits in dairy cattle. In the present study, we further verified whether these genes have genetic effects on milk traits in a Chinese Holstein population. By re-sequencing part of the non-coding and the entire coding regions of the DDIT3, RPL23A, SESN2 and NR4A1 genes, a total of 35 SNPs and three insertions/deletions were identified, of which three were found in DDIT3, 12 in RPL23A, 16 in SESN2 and seven in NR4A1. Moreover, two of the insertions/deletions-g.125714860_125714872del and g.125714806delinsCCCC in SESN2-were novel and have not been reported previously. Subsequent single SNP analyses revealed multiple significant association with all 35 SNPs and three indels regressed against the dairy production traits (P-value = <0.0001-0.0493). In addition, with a linkage disequilibrium analysis, we found one, one, three, and one haplotype blocks in the DDIT3, RPL23A, SESN2 and NR4A1 genes respectively. Haplotype-based association analyses revealed that some haplotypes were also significantly associated with milk production traits (P-value = <0.0001-0.0461). We also found that 12 SNPs and two indels (two in DDIT3, two in RPL23A, nine in SESN2 and one in NR4A1) altered the specific transcription factor binding sites in the promoter, thereby regulating promoter activity, suggesting that they might be promising potential functional variants for milk traits. In summary, our findings first determined the genetic associations of DDIT3, RPL23A, SESN2 and NR4A1 with milk yield and composition traits in dairy cattle and also suggested potentially causal variants, which require in-depth validation.

Li Y ，Han B ，Liu L ，Zhao F ，Liang W ，Jiang J ，Yang Y ，Ma Z ，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》