Genetic Markers Associated with Milk Production Traits in Dairy Cattle

: Increasing milk production is one of the key concerns in animal production. Traditional breeding has gotten limited achievement in the improvement of milk production because of its moderate heritability. Milk production traits are controlled by many genes. Thus, identifying candidate genes associated with milk production traits may provide information that can be used to enhance the accuracy of animal selection for moderately heritable traits like milk production. The genomic selection can enhance the accuracy and intensity of selection and shortening the generation interval. The genetic progress of economically important traits can be doubled with the accuracy of selection and shortening of generation interval. Genome-wide association studies (GWAS) have made possible the screening of several single nucleotide polymorphisms (SNPs) in genes associated with milk production traits in dairy cattle. In addition, RNA-sequencing is another well-established tool used to identify genes associated with milk production in dairy cattle. Although it has been widely accepted that these three methods (GWAS, RNA-seq and DNA sequencing) are considered the ﬁrst step in the screening of genes, however, the outcomes from GWAS, DNA-sequencing and RNA-seq still need further veriﬁcation for the establishment of bonaﬁde causal variants via genetic replication as well as functional validation. In the current review, we have highlighted genetic markers identiﬁed (2010-to date) for their associations with milk production traits in dairy cattle. The information regarding candidate genes associated with milk production traits provided in the current review could be helpful to select the potential genetic markers for the genetic improvement of milk production traits in dairy cattle.


Introduction
Milk production traits have fundamental roles in dairy development and related economy [1,2].The bovine milk production traits such as milk yield, fat content, protein content and somatic cell score (SCS) are the essential economic traits used to measure the quality of milk [3,4].Traditional breeding methods have achieved considerable success in many economic traits; however, milk production having moderate heritability, the gains was not fruitful with common traditional breeding [5].Being a polygenetic trait, milk production is controlled by many genes [6,7].Thus exploring the genetic changes underlying preferred phenotypes is the target of today's animal producers.It has been well-established that the production of milk can be enhanced through genetic marker-assisted selection [8,9].Various approaches such as mapping of quantitative trait loci (QTL), genome-wide association study (GWAS), RNA-sequencing, whole-genome sequencing and candidate gene analysis have been used to screen out the causal genes or their mutations associated with milk production traits [10][11][12][13][14].So far, many candidate genes or polymorphisms within these genes have been identified that have a positive correlation with milk production traits in dairy cattle [1,15,16].
Through genomic selection, we can identify genetically superior animals at a very early age.The DNA-tested animals can get accurate genomically enhanced breeding values before they enter into sexual maturity.In addition, because of the heavier use of young, genetically superior males and females in genomic selection, the generation interval can be decreased.The intensity of selection can be enhanced because the breeders use genomic testing to identify a larger group of potentially superior animals.Altogether by enhancing the accuracy and intensity of selection and decreasing the generation interval, the rate of genetic progress for economically essential dairy traits can be almost doubled.Keeping in view the importance of genomic selection, the current review was designed to highlight the possible development on genetic markers associated with milk production traits in dairy cattle.

Materials and Methods
The data were collected through authentic sources, such as PubMed, ScienceDirect, Web of Science, SpringerLink, Scopus, and Google Scholar, using polymorphism, genetic markers, GWAS, RNA-seq, DNA-sequencing (Whole genome sequencing) and dairy cattle milk production traits as major keywords.All the published studies that have discussed the polymorphisms in genes and their association with milk production traits in dairy cattle were included in the current review.Moreover, we also included the published studies that reported the direct effect of genes on milk production traits in dairy cattle.Similarly, all the published articles in the English language and scientific citation index (SCI) peerreviewed journals were incorporated for discussion in the current review.Furthermore, we considered articles (approximately 96%) published from 2010 onward in a present review article.Those genes from RNA-seq data associated with milk production traits and differentially expressed (p < 0.05, Q < 0.05) or validated through qPCR, were selected in the current review article.The present review article included all the polymorphisms in genes reported through GWAS or functional validation that were significantly associated with milk production traits in dairy cattle.Conversely, we excluded the data that was available in the form of conference papers, books, book chapters, thesis data and unpublished findings.
By using the GWAS study, Ariyarathne et al. [29] reported key genetic markers that were associated with FP (MGST1, DGAT1, CEBPD, SLC52A2, GPAT4, and ACOX3), PP (CSN1S1, GOSR2, HERC6, and IGF1R) and milk urea (GMDS, E2F7, SIAH1, SLC24A4, LGMN, and ASS1) of Holstein Friesian, Jersey or crossbred cows in New Zealand [29].Similarly, Bouwman et al. documented candidate genes (ABCG2, DGAT1 SCD1, ACLY, SREBF1, STAT5A, GH, PPARGC1A, ACSS2, AGPAT6 and FASN) that were significantly correlated with milk fatty acid traits in lactating Dutch Holstein Friesian at Netherlands [17].Li et al. conducted a GWAS study for milk fatty acid traits in 784 Chinese Holstein cows and found that polymorphisms in some key genes showed a link with milk fatty acid traits [30].Although we highlighted several genes documented through the GWAS study, however, the functional validation of these genes is highly warranted before adding them as genetic markers for milk improvement in dairy cattle breeding.

Transcriptomic Analysis for Screening of Genetic Markers Associated with Milk Production
RNA-sequencing has been a newly merged tool for screening genetic markers associated with milk production [2,10].Besides genetic data, the gene expression profile also plays a vital role in exploring the underlying mechanism for complex traits such as milk production in dairy cattle.Constantly, Cui et al. performed the transcriptomic profiling of the bovine mammary gland of four lactating Chinese Holstein cows with high and low phenotypic milk protein and fat percentage values.They reported some promising candidate genes (TRIB3, SAA1, M-SAA3.2,SAA3, VEGFA, PTHLH, HSPD1, KRT24, and RPL23A) that were significantly correlated with milk protein and milk fat percentage [10].Bagnato et al. also reported the significant association of HSPD1 and KRT24 genes with milk yield and protein percentage in Brown Swiss dairy cattle [41].Furthermore, Khan et al. by using RNA-seq analysis reported the number of genes (DGAT2, ALOX5, AGPAT4, GPAT3, GGH, ALDOA, TKT, SLC11A1 and LAP3) in response to folic acid treatment that were associated with milk protein, milk yield and milk fat in dairy cattle [2].Consistently, Ouattara et al. reported that vitamin B9 and B12 combine supplementation regulated key genes that were associated with milk production traits.Furthermore, the candidate genes (MYOM1, HP, CDK5R1, MEP1B, DLK1, PPP1R3B, GSTA5, HERC6, LOXL4, SAA3, FUT5, PYCR1 and CACNA2D1) they documented were linked to milk protein and milk fat traits [42].The genes associated with milk production traits screened out through RNA-seq by different studies have been summarized in Table 2.

Whole-Genome Sequencing for Screening of Genetic Marker Associated with Milk Production in Cattle
Whole-genome sequencing is one of the next-generation sequencing methods utilized to identify a large number of SNPs more quickly and inexpensively [50].The DNA-seq method has been widely excised in livestock genomics to identify the genetic markers associated with milk production traits [51,52].To perform whole-genome sequencing, the data composed of about 254 k milk, fat, and protein test-day records were collected from 7522 Holstein cows calved from 2006-2016 on two dairy farms in the state of Florida [53].They reported several genes (CDKN1B, DUSP16, HSF1, EEF1D, VPS28, TONSL, PEX16, MAPK8IP1, CREB3L1 and CRY2) on BTA5, BTA14 and BTA15 that were significantly associated with milk production in traits in dairy cattle.Interestingly, the reported genes in this study are involved in the inositol phosphate mediated signaling pathway, insulin receptor signaling pathway, JNK cascade, stress-activated MAPK cascade, and glutamine metabolic process.These pathways play a major role in maintaining milk production even under stressful condition to regulate the antioxidant system [53].Similarly, Nanaei et al. performed DNA sequencing for screening genetic makers associated with milk production traits by using the Illumina whole genomes of 21 cattle individuals, including 3 indigenous African breeds (Ankole n = 4, Kenana n = 4 and N'Daman = 6), and two commercial breeds (Polish Holstein-Friesian n = 3 and Hereford n = 4).Importantly, they documented some key genes (IGFBP2, B4GALT1, RORA, LPIN1, ATP2B, CSN3, NME1, ACACA, PDE3A, XP-CLR, KCNIP4, GHR, NF2, ABCC9, CD44, MACF1, IL15) involved in the regulation of biological function processes such as phosphorus metabolic process, phosphate-containing compound, metabolic process, phosphorylation, protein phosphorylation" and metal ion transport that were significantly related with milk production traits [54].Recently a study selected 45 blood samples from two-year-old animals and DNA-sequencing was carried to identify genetic markers associated with milk production traits [55].Interestingly they found nine genes (ADCY5, CACNA1A, CREB1, INHBA, INHBB, PIK3R1, PLCB1, PRKCE, and SMAD2) distributed in the ionotropic glutamate receptor pathway, the endothelin signaling pathway, and the gonadotropin-releasing hormone receptor pathway, which are involved in the hormonal regulation of lactation [55].Whole-genome sequencing for data obtained from 4280 progeny tested Nordic Red Cattle bulls was performed to identify the genetic markers for milk production [56].In addition, the genes related to milk production traits including DGAT1, HSF1, TRIM26, CLEC16A, NEURL1 (Fat yield), MKL1, CPSF1, ADCK5, LAX1, GHR (milk yield), DGAT1, HSF1, UNKL, PAM16, GLIS2 (protein yield) were documented in Nordic Red Cattle [56].

DNA Polymorphisms and Their Association with Milk Production Traits in Dairy Cattle
The correlation of DNA polymorphisms with milk production in dairy cattle has been studied for several genes, including SCD, prolactin, DGAT1, leptin, GHR, CSN1S1, ABCG2, GH etc.In Table 3, we have summarized all the major DNA polymorphisms in genes and their association with milk production traits in dairy cattle.
Stearoyl-CoA desaturase 1 (SCD1) located on chromosome 26 has been widely studied for its association with milk production traits in dairy cattle [57][58][59][60][61][62].Taniguchi et al. studied the polymorphisms of SCD in Holstein-Friesian, Jersey, Brown Swiss, and Japanese black cattle breeds and found their association with milk fat composition [60].Similarly, Kgwatala et al. documented the SNP at 3-UTR of SCD and their link with milk fatty acids in Canadian Holstein and Jersey breeds [62].Consequently, Macciotta et al. reported that the Italian Holsteins with VV genotypes produced more milk and protein than those with AA genotypes.In contrast it has been reported that cows with AA genotypes were producing more milk fat [61].Furthermore, they highlighted that because of the involvement of SCD gene in energetic pathways, it might be the reason for their association with milk production traits such as milk, yield, and protein [62].Mele et al. [59] studied the genotypic effect of SCD on milk fatty acids in 297 Italian Holstein Friesian cows.The genotypes in SCD were confirmed through the single-strand conformation polymorphism method.Interestingly, they found that cows having AA genotypes producing more milk fat compared to VV genotypes cows [60].The above results were also verified by a recent study who found that heterozygous genotypes Chinese Holsteins were producing more milk than the cows of homozygous genotypes [63].Similarly, Kesek-Wozniak et al. reported that heterozygous genotypes Polish Holstein-Friesian cows produced more milk fatty acids in milk compared to VV and AA genotypes cows [64].
Alim et al. [63] reported several SNPs in the SCD gene and their association with milk production traits in Chinese Holsteins.They documented that polymorphism g.10329C/T at exon 5 changed the amino acid alanine to valine.In addition, it was noticed that the two SNPs (g.6926A/G and g.8646A/G) at intron 3 and three polymorphisms (g.10153A/G, g.10213T/C and g.10329C/T) at exon 5 in SCD were significantly associated with milk fat, milk yield, protein yield and protein (%) in Chinese Holsteins [63].Recently, it has been documented that A293V (c.878C/T) mutation in SCD changed the amino acid alanine to valine and is associated with milk fatty acid in Polish Holstein-Friesian cows [64].Constantly, Bouwman et al. reported that the A allele of SNP in SCD was associated with higher milk fatty acids [17], while other studies found the less effect of V allele on milk fats in White Fulani and Borgou cattle breeds [65,66].
The polymorphism (DGAT1 K232A) in DGAT1 has been widely studied for its association with milk production traits particularly milk fatty acids in dairy cattle (Table 2) [64,66,67].In addition, it has been documented that K allele is linked to high milk fat yield, fat content, and protein content and lower milk production protein and lactose yield [68,69].While other studies reported that cows with AA genotypes have higher milk yield and lactose yield and low milk fat and protein contents [70][71][72].Based on the above findings, it can be concluded that the DGAT1 K232A can be a target as a useful genetic marker for milk production improvement in dairy cattle.
Fatty acid desaturase 2 (FADS2) is another promising candidate gene that influences milk fatty acid traits and is located on bovine chromosome 29, with 16 exons encoding 359 amino acid chains [75].The polymorphism in the FADS2 gene has been widely studied for its association with milk fatty acids (MFAs) in dairy cattle [43,76,77].Based on published data, it can be recommended that the FADS2 can be a useful candidate marker for milk fat traits improvement in dairy cattle.The detail of FADS2 gene and their polymorphisms has been given in Table 3.
Ahmed et al. documented few milk protein genes (CSN1S1, CSN2, CSN1S2, CSN3, LALBA, and LGB) that were linked to increase milk protein traits in Sudanese Butana cattle [78].Consistently, CSN1S1, CSN2CSN2, CSN2, CSN1S2, LALBA genes have been studied for their association with milk production traits in other Bos indicus breeds such as Sarabi, Sistani, Golpayegani and Gir in Iran and Brazil, respectively [79,80].Moreover, Miluchová et al. proved experimentally that the CSN3 gene was significantly associated with milk production traits in the Slovakian Holstein population [81].
Haruna et al. had documented that the myostatin gene was significantly associated with increasing the amount of milk unsaturated fatty acid and decreasing the amount of saturated fatty acid in New Zealand Holstein-Friesian cross Jersey-Cross Cows.Moreover, they reported that cows with AD genotypes were linked to decreased saturated fatty acid while cows with AA genotypes correlated with increased milk unsaturated fatty acid [82].Consequently, it has been documented that fatty acid-binding proteins (FABPs) is significantly associated with milk fatty acids synthesis in Holstein-Friesian × Jersey (HF × J) dairy cows [83].
The glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein1 (GPI-HBP1) is a key gene that has been studied for its association with milk fat (%) and milk protein yield [32,84]. Consistently, another study had reported that GPIHBP1 was significantly correlated with milk fat traits in Chinese Holsteins [85].They demonstrated that when the expression of GPIHBP1 was decreased, which decreased the LPL binding ability to GPIHBP1 and alternatively, the process of lipolysis was inhibited in mammary epithelial cells, resulting in increased fat in milk [85].Moreover, Dong et al. [86] illustrated that the decrease of the expression of GPIHBP1, result in an increase in milk protein genes (CSN1S1, CSN1S2, CSN2, and CSN3, lactoferrin) which were associated with the regulation of milk protein biosynthesis [86].
Long-chain acyl-CoA synthetase 1 (ACSL1) is located on chromosome 27 of cattle (Bos Taurus), having 20 exons, 19 introns with 64,883 bp length [87].The SNPs detected in the ACSL1 gene were genotyped in 992 Chinese Holstein cows and documented the significant association of these SNPs with milk production traits [87].Consistently a study also documented the up-regulation of sic genes (ACACA, GPAM, ACSL1, FASN, LPIN1 and ACSL6) in dairy cattle during lactation [88].Twenty candidate genes associated with milk fatty acid traits in Chinese Holstein cows were identified in a previous study, and ACSL1 was one of them [30].Furthermore, a study had documented that mutation in the ACSL1 gene plays a key role in the milk fat enhancement of Yak [89].Fan et al. experimentally proved that the expression (increase and decrease) of ASCL4 was significantly associated with milk fat synthesis in bovine mammary epithelial cells [90].The interaction of ASCL4 was reported with ASCL1, FADS2, FASN, PPARD, CPT1A, FABP3 and ELOVL6 which are key genes associated with milk production traits.Based on the above findings, we can conclude that ASCL1 can be considered a key regulator of milk fat synthesis.
Acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3), located on Bos taurus autosome 1 (BTA1) having eight exons encoding 376 amino acid chains, is a crucial acyltransferase that is involved in triglyceride (TG) and phospholipid biosynthesis [91].AGPAT3 has been identified through GWAS studies as a positional candidate gene affecting milk fatty acids in dairy cattle [30,91,92].A study by using GWAS study documented the AGPAT3 and was found to be significantly linked with milk fatty acid traits in Chinese and Danish Holstein populations [39].Recently, a study detected a SNP1 (g.12264 C > T) at promoter region, SNP2 (g.18852 C > T) in exon 5 and other six SNPs (g.18658 G > A, g.20046 G > A, g.23034 C > A, g.28332 C > T, g.28484 C > T, and g.28731A > G) on intronic regions of AGPAT3 in dairy cattle [93].All the SNPs reported by Sun et al. showed significant association with at least one phenotypic trait of milk production.Similarly, Shi et al. reported 17 SNPs in AGPAT3 that were associated with milk fatty acid traits in Chinese Holstein cows [94].Littlejohn et al. also documented several SNPs of AGPAT3 in Holstein-Friesian × Jersey crossbreed that were associated with milk fat synthesis [95].The detail of SNPs in AGPAT3 has been given in Table 3.

Conclusions
In the current review, we documented several genes associated with milk production traits in dairy cattle.Moreover, many SNPs within candidate genes were highlighted in the current review, which could be a useful addition to the genetic markers linked to the improvement of milk production traits in dairy cattle.There are still many candidate genes reported through GWAS studies, RNA-seq and DNA-seq need further validation in dairy cattle before selecting them as genetic markers in cattle breeding.

Table 1 .
GWAS study for screening genetic markers associated with milk production trait.

Table 1 . Cont. SNP (Gene) Production Traits Breed and Phenotypic Traits and Method Used for Association Country Author
[20]phenotypic data was collected from 2515 MON, 2203 NOR, and 6321 HOL bulls and verified in 23,926 MON, 9400 NOR, and 51,977 HOL cows Illumina Bovine SNP50 BeadChip (50K; Illumina Inc., San Diego, CA, USA) was used for genotyping France[20]

Gene) Production Traits Breed and Phenotypic Traits and Method Used for Association Country Author
MY: milk yield; FY: fat yield; FP: fat percentage; PY: protein yield; PP: protein percentage; MFAs: Milk fatty acids.

Table 2 .
Genes identified through the RNA-seq method.

Table 3 .
The variations in genes and their association with milk production traits in dairy cattle.