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Association of single nucleotide polymorphisms in candidate genes previously related to genetic variation in fertility with phenotypic measurements of reproductive function in Holstein cows

Open ArchivePublished:March 03, 2017DOI:https://doi.org/10.3168/jds.2016-12260

      ABSTRACT

      Many genetic markers related to health or production traits are not evaluated in populations independent of the discovery population or related to phenotype. Here we evaluated 68 single nucleotide polymorphisms (SNP) in candidate genes previously associated with genetic merit for fertility and production traits for association with phenotypic measurements of fertility in a population of Holstein cows that was selected based on predicted transmitting ability (PTA) for daughter pregnancy rate (DPR; high, ≥1, n = 989; low, ≤ −1.0, n = 1,285). Cows with a high PTA for DPR had higher pregnancy rate at first service, fewer services per conception, and fewer days open than cows with a low PTA for DPR. Of the 68 SNP, 11 were associated with pregnancy rate at first service, 16 with services per conception, and 19 with days open. Single nucleotide polymorphisms in 12 genes (BDH2, BSP3, CAST, CD2, CD14, FUT1, FYB, GCNT3, HSD17B7, IBSP, OCLN, and PCCB) had significant associations with 2 fertility traits, and SNP in 4 genes (CSPP1, FCER1G, PMM2, and TBC1D24) had significant associations with each of the 3 traits. Results from this experiment were compared with results from 2 earlier studies in which the SNP were associated with genetic estimates of fertility. One study involved the same animals as used here, and the other study was of an independent population of bulls. A total of 13 SNP associated with 1 or more phenotypic estimates of fertility were directionally associated with genetic estimates of fertility in the same cow population. Moreover, 14 SNP associated with reproductive phenotype were directionally associated with genetic estimates of fertility in the bull population. Nine SNP (located in BCAS, BSP3, CAST, FUT1, HSD17B7, OCLN, PCCB, PMM2, and TBC1D24) had a directional association with fertility in all 3 studies. Examination of the function of the genes with SNP associated with reproduction in more than one study indicates the importance of steroid hormones and immune function as determinants of reproductive function. All but 1 of the 68 evaluated SNP were variable in 11 breeds besides Holstein, indicating the potential effects of these SNP on reproductive function across breeds of cattle.

      Key words

      INTRODUCTION

      The use of genomics has improved response to selection for functional traits with low heritability such as daughter pregnancy rate (DPR) and productive life (
      • García-Ruiz A.
      • Cole J.B.
      • VanRaden P.M.
      • Wiggans G.R.
      • Ruiz-López F.J.
      • Van Tassell C.P.
      Changes in genetic selection differentials and generation intervals in US Holstein dairy cattle as a result of genomic selection.
      ). Much of the work on fertility traits has been performed through use of genome-wide association studies (GWAS) to identify genetic loci associated with reproductive traits (
      • Cole J.B.
      • Wiggans G.R.
      • Ma L.
      • Sonstegard T.S.
      • Lawlor T.J.
      • Crooker B.A.
      • Van Tassell C.P.
      • Yang J.
      • Wang S.
      • Matukumalli L.K.
      • Da Y.
      Genome-wide association analysis of thirty one production, health, reproduction and body conformation traits in contemporary U.S. Holstein cows.
      ;
      • Minozzi G.
      • Nicolazzi E.L.
      • Stella A.
      • Biffani S.
      • Negrini R.
      • Lazzari B.
      • Ajmone-Marsan P.
      • Williams J.L.
      Genome wide analysis of fertility and production traits in Italian Holstein cattle.
      ;
      • Nayeri S.
      • Sargolzaei M.
      • Abo-Ismail M.K.
      • May N.
      • Miller S.P.
      • Schenkel F.
      • Moore S.S.
      • Stothard P.
      Genome-wide association for milk production and female fertility traits in Canadian dairy Holstein cattle.
      ). One outcome has been the identification of haplotypes affecting fertility in dairy breeds (
      • VanRaden P.M.
      • Olson K.M.
      • Null D.J.
      • Hutchison J.L.
      Harmful recessive effects on fertility detected by absence of homozygous haplotypes.
      ;
      • Larkin D.M.
      • Daetwyler H.D.
      • Hernandez A.G.
      • Wright C.L.
      • Hetrick L.A.
      • Boucek L.
      • Bachman S.L.
      • Band M.R.
      • Akraiko T.V.
      • Cohen-Zinder M.
      • Thimmapuram J.
      • Macleod I.M.
      • Harkins T.T.
      • McCague J.E.
      • Goddard M.E.
      • Hayes B.J.
      • Lewin H.A.
      Whole-genome resequencing of two elite sires for the detection of haplotypes under selection in dairy cattle.
      ;
      • Sahana G.
      • Nielsen U.S.
      • Aamand G.P.
      • Lund M.S.
      • Guldbrandtsen B.
      Novel harmful recessive haplotypes identified for fertility traits in Nordic Holstein cattle.
      ;
      • Cooper T.A.
      • Wiggans G.R.
      • Null D.J.
      • Hutchison J.L.
      • Cole J.B.
      Genomic evaluation, breed identification, and discovery of a haplotype affecting fertility for Ayrshire dairy cattle.
      ;
      • Cuyabano B.C.
      • Su G.
      • Lund M.S.
      Genomic prediction of genetic merit using LD-based haplotypes in the Nordic Holstein population.
      ) and identification of loss-of-function mutations that are embryo lethal (
      • Fritz S.
      • Capitan A.
      • Djari A.
      • Rodriguez S.C.
      • Barbat A.
      • Baur A.
      • Grohs C.
      • Weiss B.
      • Boussaha M.
      • Esquerré D.
      • Klopp C.
      • Rocha D.
      • Boichard D.
      Detection of haplotypes associated with prenatal death in dairy cattle and identification of deleterious mutations in GART, SHBG and SLC37A2..
      ;
      • Sonstegard T.S.
      • Cole J.B.
      • VanRaden P.M.
      • Tassell C.P.V.
      • Null D.J.
      • Schroeder S.G.
      • Bickhart D.
      • McClure M.C.
      Identification of a nonsense mutation in CWC15 associated with decreased reproductive efficiency in Jersey cattle.
      ).
      The basis for GWAS is the assumption that the SNP on the panel are in linkage disequilibrium with causative mutations. In many cases, identification of the causative mutation is difficult because an associated genetic marker can often be located in an intergenic region and can be in linkage disequilibrium with variants in several nearby genes. Another approach is to identify the causative SNP in the regulatory or coding region of a gene that is responsible for genetic variation in biological function. The causative allelic variant is expected to be more strongly associated with a trait than other SNP in linkage disequilibrium. Moreover, the allelic association between a functional mutation and a genetically controlled trait would be more likely to extend across breeds than a genetic marker based on linkage disequilibrium (
      • Zhu M.
      • Zhao S.
      Candidate gene identification approach: Progress and challenges.
      ;
      • Weller J.I.
      • Ron M.
      Invited review: Quantitative trait nucleotide determination in the era of genomic selection.
      ). Understanding the biological basis of genetic variation could also lead to insights into the underlying physiology controlling a trait. One approach to identify causative mutations is the candidate gene approach. Among the genes with SNP associated with reproductive traits in cattle are DGAT1, CAST, GHR, and LEPR for services per conception and DPR (
      • Garcia M.D.
      • Michal J.J.
      • Gaskins C.T.
      • Reeves J.J.
      • Ott T.L.
      • Liu Y.
      • Jiang Z.
      Significant association of the calpastatin gene with fertility and longevity in dairy cattle.
      ;
      • Schneider A.
      • Corrêa M.N.
      • Butler W.R.
      Association between growth hormone receptor AluI polymorphism and fertility of Holstein cows.
      ;
      • Hill R.
      • Canal A.
      • Bondioli K.
      • Morell R.
      • Garcia M.D.
      Molecular markers located on the DGAT1, CAST, and LEPR genes and their associations with milk production and fertility traits in Holstein cattle.
      ); IGF1 for resumption of ovarian cyclicity (
      • Nicolini P.
      • Carriquiry M.
      • Meikle A.
      A polymorphism in the insulin-like growth factor 1 gene is associated with postpartum resumption of ovarian cyclicity in Holstein-Friesian cows under grazing conditions.
      ); and HSPA1L, STAT1, STAT3, PARM1, and WBP1 for fertilization and embryonic development during the preimplantation period (
      • Khatib H.
      • Maltecca C.
      • Monson R.L.
      • Schutzkus V.
      • Rutledge J.J.
      Monoallelic maternal expression of STAT5A affects embryonic survival in cattle.
      ;
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Single nucleotide polymorphisms in candidate genes associated with fertilizing ability of sperm and subsequent embryonic development in cattle.
      ).
      For many genetic markers, SNP have not been independently evaluated in separate populations. When they are, replication of the effects is often poor (
      • Ioannidis J.P.A.
      • Ntzani E.E.
      • Trikalinos T.A.
      • Contopoulos-Ioannidis D.G.
      Replication validity of genetic association studies.
      ;
      • Siontis K.C.M.
      • Patsopoulos N.A.
      • Ioannidis J.P.A.
      Replication of past candidate loci for common diseases and phenotypes in 100 genome-wide association studies.
      ;
      • Littlejohn M.
      • Grala T.
      • Sanders K.
      • Walker C.
      • Waghorn G.
      • Macdonald K.
      • Spelman R.
      • Davis S.
      • Snell R.
      Non-replication of genome-wide-based associations of efficient food conversion in dairy cows.
      ). Confidence in the relationship between a genetic mutation and phenotype is increased by replication of the allelic relationship in separate populations and by demonstrating that phenotype is also associated with the mutation. Here we evaluated the effect of 68 SNP in candidate genes previously associated with genetic merit for fertility and production traits in Holstein cattle (
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      ;
      • Ortega M.S.
      • Denicol A.C.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows.
      ) on phenotypic measurements of fertility and production in a population of Holstein cows. A fraction of the SNP was similarly associated with fertility traits in both studies (
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      ;
      • Ortega M.S.
      • Denicol A.C.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows.
      ). The majority of the 68 SNP (64 of 68) are located in coding regions of genes and result in a change in the amino acid sequence of the encoded protein. We also evaluated whether the SNP were variable only in Holsteins or were common among multiple cattle breeds.

      MATERIALS AND METHODS

      Phenotypic Measurements for Fertility and Milk Production

      Collection of Phenotypic Data from Genotyped Animals

      Details of the animals included in the study and methods for genotyping were detailed in
      • Ortega M.S.
      • Denicol A.C.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows.
      . Briefly, Holstein cows with a high (≥1.5) or low (≤−1.0) PTA for DPR and located on 6 dairies in Florida and 5 in California were used. The high DPR group had 989 cows, and the low DPR group had 1285. Phenotypic data were collected for up to 5 lactations from each farm and combined with records from the national genetic evaluation system. Data for pregnancy rate at first service, services per conception, and days open (i.e., interval from calving to conception) were evaluated. Cows were genotyped for each of 68 SNP using a Sequenom MassARRAY system (iPLEX GOLD; Sequenom, San Diego, CA). The SNP were also previously described by
      • Ortega M.S.
      • Denicol A.C.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows.
      . Of the 68 SNP, 48 were associated with 1 or more fertility traits [DPR, cow conception rate (CCR) or heifer conception rate (HCR)] by
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      , and the remaining SNP were associated with milk production traits by
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      .

      Data Analysis

      The association of each genetic variant with phenotypic traits was performed by ANOVA using the Statistical Analysis System v 9.4 (SAS Institute Inc., Cary, NC). Days open and pregnancy rate were analyzed with the MIXED procedure. Days open were log-transformed before analysis to establish normality. The number of services per conception was analyzed with the GLIMMIX procedure using a negative binomial distribution for the responses and a logarithmic link function (
      • Dobson A.J.
      ).
      In all analyses, genotype was considered a categorical variable. The full model was as follows:
      Yijkl=μ+αi+gj+lk+fl+eijkl,


      where Yijkl is the value of the trait of interest for the ith cow (i = 1, 2, …, n), ai is the random polygenic effect (including all available pedigree information) for the ith cow, gj is the fixed effect of SNP genotype (j = 1,…, 3 such that g1 is the genotypic value of AA homozygotes, g2 is the genotypic value of AB heterozygotes, and g3 is the genotypic value of BB homozygotes), lk is the fixed effect of lactation number (k = 1,…, 5), fl is the fixed effect of farm (l = 1,…, 6), and eijkl is the random residual effect. We assume that random polygenic effects aN(0, Aσa2) and residuals e ∼N(0, σe2), where A is the numerator relationship matrix, σa2 is the additive genetic variance of the trait of interest, and σe2 is the residual error variance. All of the available pedigree information for each cow was used to generate A, which models the covariance among the polygenic effects. Following
      • Falconer D.S.
      • MacKay T.F.C.
      , we estimated the a and d parameters for each locus as (g3g1)/2, and g2 − (g1 + g2)/2, respectively. Effects of P < 0.05 were considered significant.

      Concordance of SNP Effects Across Studies

      Results on significant SNP effects associated with phenotype from the current study were compared with results from 2 other studies examining the effects of these 68 markers on estimates of genetic merit for reproductive and production traits. One study involved an independent population of 550 Holstein bulls (
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      ), and the other evaluated genetic merit from the same cow population as in the present experiment (
      • Ortega M.S.
      • Denicol A.C.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows.
      ).

      Marker Frequencies in Other Breeds

      The variability of the 68 SNP evaluated in the present study was determined in other cattle breeds. The frequency of the genetic variants was determined in a total of 203 sequenced animals from 11 Bos taurus breeds: 109 Angus, 10 Beefmaster, 12 Charolais, 8 Gelbvieh, 18 Hereford, 3 Jersey, 9 Limousin, 5 Maine-Anjou, 14 Red Angus, 4 Romagnola, and 11 Simmental.

      RESULTS AND DISCUSSION

      Effect of PTA for DPR on Phenotypic Measurements of Reproductive Function

      Pregnancy rate at first service, services per conception, and days open were all affected by DPR (P < 0.0001, Table 1). In particular, cows with a high PTA for DPR had a higher pregnancy rate at first service, fewer services per conception, and fewer days open than cows with a low PTA for DPR. Note that the effect of DPR class was observed on pregnancy rate at first service, services per conception, and days open for each of the 5 examined lactations. These results confirm the usefulness of selection for DPR for improving reproductive function despite the low heritability associated with reproductive traits (
      • VanRaden P.M.
      • Sanders A.H.
      • Tooker M.E.
      • Miller R.H.
      • Norman H.D.
      • Kuhn M.T.
      • Wiggans G.R.
      Development of a national genetic evaluation for cow fertility.
      ;
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      ). Similar results have been obtained for a pasture-based production system (
      • Cummins S.B.
      • Lonergan P.
      • Evans A.C.O.
      • Berry D.P.
      • Evans R.D.
      • Butler S.T.
      Genetic merit for fertility traits in Holstein cows: I. Production characteristics and reproductive efficiency in a pasture-based system.
      ).
      Table 1Phenotypes for fertility and production for animals classified based on predicted transmitting ability for daughter pregnancy rate (DPR)
      TraitLactationRecords, no.Least squares means (SEM)P-value
      TotalHigh DPRLow DPRHigh DPRLow DPR
      Pregnancy rate at first service, %12,2459601,28553.1 (1.69)28.6 (2.32)<0.0001
      22,1189281,19043.9 (1.77)23.0 (2.38)<0.0001
      31,74382192241.0 (1.88)25.0 (2.53)<0.0001
      41,06758947838.8 (2.11)25.0 (2.40)<0.0001
      548430517938.3 (2.94)15.1 (4.00)<0.0001
      Services per conception, no.12,2749891,2851.93 (0.06)3.26 (0.07)<0.0001
      22,1189281,1902.09 (0.07)3.30 (0.07)<0.0001
      31,7438219222.20 (0.08)3.20 (0.10)<0.0001
      41,0675894782.34 (0.12)3.22 (0.28)0.0125
      54843051792.56 (0.12)3.55 (0.20)0.0960
      Days open, d12,2749891,28598 (2.59)163 (2.94)<0.0001
      22,1189281,190112 (2.80)167 (3.13)<0.0001
      31,743821922110 (3.24)158 (3.81)<0.0001
      41,067589478123 (3.43)170 (3.90)<0.0001
      5484305179133 (5.12)174 (6.90)<0.0001
      The proportion of animals at later lactations was higher for the high DPR group than for the low DPR group. For example, the proportion of records represented from cows in fifth lactation was 305/3,603 (8.5%) for the high DPR group versus 179/4,054 (4.4%) for the low DPR group. This result probably reflects culling for reproductive reasons, which has been estimated to represent 13 to 20% of all culling decisions (
      • Hadley G.L.
      • Wolf C.A.
      • Harsh S.B.
      Dairy cattle culling patterns, explanations, and implications.
      ). Indeed, the genetic correlation between productive life and DPR is positive (
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      ;
      • VanRaden P.M.
      • Tooker M.E.
      • Wright J.R.
      • Sun C.
      • Hutchison J.L.
      Comparison of single-trait to multi-trait national evaluations for yield, health, and fertility.
      ).

      SNP Associated with Fertility Traits

      Twenty-six SNP were associated with one or more phenotypic measures of fertility, with 11 SNP being associated with pregnancy rate at first service (Table 2), 16 being associated with services per conception (Table 3), and 19 being associated with days open (Table 4). Most SNP effects indicated a difference between homozygotes (a > 0) or else both a and d effects were significant. Genes for which heterozygote deviations were found were FSHR, IBSP, and SERPINE2 for pregnancy rate at first service; FYB for services per conception; and FYB and IBSP for days open.
      Table 2Single nucleotide polymorphisms associated with pregnancy rate at first service
      SNP IDChromosomeLocationGeneCopies of minor allele
      Values are least squares means (SEM).
      EffectP-value
      A = one-half difference between homozygotes; D = deviation of heterozygotes from homozygote midpoint.
      012AD
      rs41857027182783606CFDP20.34 (0.01)0.35 (0.02)0.43 (0.04)0.040.03060.1718
      rs1094435821433342060CSPP10.35 (0.01)0.35 (0.02)0.68 (0.10)−0.170.00110.0019
      rs10913798238308678FCER1G0.34 (0.01)0.36 (0.02)0.55 (0.06)0.110.00070.0178
      rs437452341131176783FSHR0.33 (0.01)0.36 (0.01)0.34 (0.02)−0.010.60690.0346
      rs1098308801050709147GCNT30.36 (0.01)0.33 (0.02)0.25 (0.05)−0.050.04500.4352
      rs11078909863809790IBSP0.37 (0.01)0.33 (0.01)0.35 (0.02)−0.010.37350.0168
      rs412568481130824442LHCGR0.36 (0.01)0.36 (0.01)0.30 (0.02)−0.030.00570.0224
      rs109629628257716425PMM20.33 (0.01)0.34 (0.01)0.38 (0.02)0.020.04150.1719
      rs433211882112900094SERPINE20.34 (0.01)0.36 (0.01)0.32 (0.03)−0.010.32680.0429
      rs419122901935248180SREBF10.33 (0.01)0.36 (0.01)0.37 (0.02)0.020.02300.8057
      rs110660625252007163TBC1D240.32 (0.01)0.36 (0.01)0.40 (0.02)0.040.00210.7620
      1 Values are least squares means (SEM).
      2 A = one-half difference between homozygotes; D = deviation of heterozygotes from homozygote midpoint.
      Table 3Single nucleotide polymorphisms associated with services per conception
      SNP IDChromosomeLocationGeneCopies of minor allele
      Values are least squares means (SEM).
      EffectP-value
      A = one-half difference between homozygotes; D = deviation of heterozygotes from homozygote midpoint.
      012AD
      rs133674837623051485BDH22.71 (0.05)2.75 (0.05)2.54 (0.06)0.030.02200.0268
      rs1102178521851919757BSP32.66 (0.05)2.73 (0.05)2.87 (0.10)0.040.03250.5794
      rs137601357798485273CAST2.63 (0.06)2.78 (0.05)2.79 (0.07)0.030.03310.2267
      rs109621328753448291CD142.72 (0.04)2.59 (0.07)2.18 (0.22)−0.110.03100.3017
      rs133747802326593448CD22.75 (0.05)2.93 (0.21)2.60 (0.06)−0.030.02130.2112
      rs1094435821433342060CSPP12.70 (0.04)2.74 (0.08)1.84 (0.33)−0.190.03200.0268
      rs10913798238308678FCER1G2.75 (0.05)2.69 (0.07)2.04 (0.20)−0.150.00180.0176
      rs418937561855831611FUT12.68 (0.05)2.78 (0.06)2.99 (0.16)0.050.03930.5256
      rs1092623552035249040FYB2.75 (0.05)2.64 (0.05)2.86 (0.09)0.020.23220.0081
      rs1098308801050709147GCNT32.70 (0.05)2.79 (0.07)3.10 (0.21)0.070.04410.3481
      rs11082805336630548HSD17B72.74 (0.05)2.67 (0.06)2.35 (0.12)−0.080.00290.0938
      rs1110159122841679976LDB32.71 (0.05)2.73 (0.06)3.07 (0.16)0.060.01500.0704
      rs1342645632010167825OCLN2.78 (0.05)2.70 (0.05)2.56 (0.08)−0.040.01910.6011
      rs1098138961134130474PCCB2.78 (0.06)2.69 (0.05)2.56 (0.08)−0.040.00730.6954
      rs109629628257716425PMM22.79 (0.06)2.71 (0.05)2.61 (0.07)−0.030.02310.8280
      rs110660625252007163TBC1D242.74 (0.05)2.72 (0.05)2.56 (0.08)−0.030.03760.2119
      1 Values are least squares means (SEM).
      2 A = one-half difference between homozygotes; D = deviation of heterozygotes from homozygote midpoint.
      Table 4Single nucleotide polymorphisms associated with days open
      SNP IDChromosomeLocationGeneCopies of minor allele
      Values are least squares means (SEM).
      EffectP-value
      A = one-half difference between homozygotes; D = deviation of heterozygotes from homozygote midpoint.
      012AD
      rs1096695731382164839BCAS1141.5 (2.3)142.7 (2.2)147.7 (3.2)3.080.04780.4033
      rs133674837623051485BDH2143.7 (2.5)146.0 (2.3)137.1 (3.3)−3.330.03870.0416
      rs1102178521851919757BSP3141.4 (2.1)144.5 (2.3)150.2 (4.0)4.380.04810.7073
      rs137601357798485273CAST141.0 (2.6)145.7 (2.4)150.2 (3.2)4.620.00660.8920
      rs109621328753448291CD14144.7 (2.1)158.3 (9.5)139.1 (3.0)−2.800.00890.0769
      rs133747802326593448CD2144.8 (2.0)156.8 (8.4)139.0 (2.8)−2.900.03820.0811
      rs1094435821433342060CSPP1141.4 (1.7)146.5 (3.3)111.5 (17.1)−4.220.01930.0069
      rs10913798238308678FCER1G144.7 (2.3)143.5 (2.4)140.7 (4.1)−14.940.00180.0199
      rs418937561855831611FUT1142.4 (2.1)146.3 (2.7)157.2 (7.6)−2.000.04740.2983
      rs1092623552035249040FYB143.1 (2.1)139.9 (2.1)152.2 (3.6)7.420.20290.0255
      rs11082805336630548HSD17B7144.2 (2.1)144.3 (2.7)130.8 (6.4)4.540.03880.1411
      rs11078909863809790IBSP141.2 (2.5)147.8 (2.3)140.5 (3.6)−6.730.86260.0180
      rs1093837581862241722NLRP9137.9 (2.9)144.5 (2.2)145.5 (3.1)−0.340.04280.3361
      rs1342645632010167825OCLN147.4 (2.3)143.7 (2.2)135.8 (3.7)3.810.01080.4494
      rs1098138961134130474PCCB146.0 (2.5)142.7 (2.3)134.8 (3.7)−5.790.02000.9398
      rs109629628257716425PMM2145.1 (2.2)141.3 (2.0)135.4 (2.8)−5.60<0.0010.0667
      rs1337291052526182660RABEP2141.1 (2.4)144.8 (2.2)149.6 (3.2)−4.850.02520.9928
      rs110660625252007163TBC1D24145.1 (2.4)143.8 (2.4)132.3 (3.8)4.240.00080.1144
      rs132789482328420362TSHB143.7 (2.2)147.6 (3.1)116.7 (10.4)−6.390.02670.0114
      1 Values are least squares means (SEM).
      2 A = one-half difference between homozygotes; D = deviation of heterozygotes from homozygote midpoint.
      For 12 SNP, the same allele was positively and significantly associated with 2 fertility traits (BDH2, BSP3, CAST, CD14, CD2, FUT1, FYB, GCNT3, HSD17B7, IBSP, OCLN, and PCCB), and 4 SNP had significant allelic associations with each of the 3 traits (CSPP1, FCER1G, PMM2, and TBC1D24).

      SNP with Effects Concordant in More than One Study

      Results from the current experiment were compared with results from earlier studies by
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      and
      • Ortega M.S.
      • Denicol A.C.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows.
      to identify genes containing SNP in which (1) a significant association between the SNP and 1 or more reproductive traits was observed in at least 2 studies and (2) the allele associated with superior reproduction was the same in each case. Results are shown in Table 5, and 26 SNP met these criteria. A total of 13 SNP associated with phenotypic measurements of fertility were also found to be directionally associated in genetic estimates of fertility using the same study population (
      • Ortega M.S.
      • Denicol A.C.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows.
      ). Moreover, 14 SNP associated with genetic estimates of fertility in the cow population (
      • Ortega M.S.
      • Denicol A.C.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows.
      ) were directionally associated with genetic estimates of fertility in an independent population of bulls (
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      ). Nine SNP (located in BCAS, BSP3, CAST, FUT1, HSD17B7, OCLN, PCCB, PMM2, and TBC1D24) had a directional association with fertility in all 3 studies.
      Table 5Single nucleotide polymorphisms associated with fertility traits in more than one study
      Shown are genes containing SNP in which a significant association between the SNP and one or more reproductive traits was observed in at least 2 studies. The letter represents the allele associated with superior reproduction. SNP significant in more than one study but where different alleles were associated with superior reproduction are not included in the table. CCR = cow conception rate; DO = days open; DPR = daughter pregnancy rate; HCR = heifer conception rate; PR = pregnancy rate; SPC = services per conception.
      SNP IDGeneCow phenotype
      Based on a population of 2,273 Holstein cows [Ortega et al. (2016) and present study].
      Cow genotype
      Based on a population of 2,273 Holstein cows [Ortega et al. (2016) and present study].
      Bull genotype
      Based on a population of 550 Holstein bulls (Cochran et al., 2013a).
      PRSPCDODPRHCRCCRDPRHCRCCR
      rs109967779ACAT2CCCC
      rs41766835APBB1GGGG
      rs133700190AP3B1TTTTTT
      rs109669573BCAS1CCC
      rs110217852BSP3AAAA
      rs109332658C7H19orf60CCC
      rs135744058CACNA1DGGG
      rs137601357CASTTTTTTT
      rs109621328CD14CCCC
      rs41711496CD40GGG
      rs133449166CSNK1ECCCCC
      rs109137982FCER1GAAAA
      rs43745234FSHRCC
      rs41893756FUT1AAAAAA
      rs109262355FYBAAA
      rs109830880GCNT3TT
      rs109711583HSD17B12GGGG
      rs110828053HSD17B7CCCCCCCC
      rs110789098IBSPTTT
      rs111015912LDB3TTTT
      rs41256848LHCGRGG
      rs134264563OCLNGGGGGG
      rs109813896PCCBCCCCC
      rs109629628PMM2GGGGGGG
      rs133729105RABEP2GGG
      rs110660625TBC1D24AAAAAA
      1 Shown are genes containing SNP in which a significant association between the SNP and one or more reproductive traits was observed in at least 2 studies. The letter represents the allele associated with superior reproduction. SNP significant in more than one study but where different alleles were associated with superior reproduction are not included in the table. CCR = cow conception rate; DO = days open; DPR = daughter pregnancy rate; HCR = heifer conception rate; PR = pregnancy rate; SPC = services per conception.
      2 Based on a population of 2,273 Holstein cows [
      • Ortega M.S.
      • Denicol A.C.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows.
      and present study].
      3 Based on a population of 550 Holstein bulls (
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      ).
      The agreement of SNP effects between 2 independent populations compares favorably with other SNP associated with reproduction in which the degree of replication of SNP effects from one population to another ranged from 18% (
      • Höglund J.K.
      • Sahana G.
      • Guldbrandtsen B.
      • Lund M.S.
      Validation of associations for female fertility traits in Nordic Holstein, Nordic Red and Jersey dairy cattle.
      ) to 0% (
      • Pryce J.E.
      • Bolormaa S.
      • Chamberlain A.J.
      • Bowman P.J.
      • Savin K.
      • Goddard M.E.
      • Hayes B.J.
      A validated genome-wide association study in 2 dairy cattle breeds for milk production and fertility traits using variable length haplotypes.
      ). In a recent study, 93 of 245 QTL regions identified by GWAS as being related to cow fertility were repeatable in 2 independent GWAS (
      • Moore S.G.
      • Pryce J.E.
      • Hayes B.J.
      • Chamberlain A.J.
      • Kemper K.E.
      • Berry D.P.
      • McCabe M.
      • Cormican P.
      • Lonergan P.
      • Fair T.
      • Butler S.T.
      Differentially expressed genes in endometrium and corpus luteum of Holstein cows selected for high and low fertility are enriched for sequence variants associated with fertility.
      ). The consistency of the effect of SNP described in Table 5 across studies provides confidence that associations between many of the SNP and reproductive function reported by
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      and
      • Ortega M.S.
      • Denicol A.C.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows.
      are real and not false positives. Moreover, a few of the genes found to be associated with reproductive traits across studies evaluated here have also been reported to be associated with reproductive traits in other reports. The SNP in CAST has been associated with genetic merit for DPR, days open, and productive life (
      • Garcia M.D.
      • Michal J.J.
      • Gaskins C.T.
      • Reeves J.J.
      • Ott T.L.
      • Liu Y.
      • Jiang Z.
      Significant association of the calpastatin gene with fertility and longevity in dairy cattle.
      ;
      • Hill R.
      • Canal A.
      • Bondioli K.
      • Morell R.
      • Garcia M.D.
      Molecular markers located on the DGAT1, CAST, and LEPR genes and their associations with milk production and fertility traits in Holstein cattle.
      ). The SNP in PCCB, PMM2, and TBC1D24 have previously been associated with percentage of cleaved embryos that develop to the blastocyst stage (
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Single nucleotide polymorphisms in candidate genes associated with fertilizing ability of sperm and subsequent embryonic development in cattle.
      ).

      Functional Ontology of SNP

      Examination of the function of genes that were repeatedly associated with reproductive traits (Table 5) provides an indication of physiological processes important for variation among cows in reproductive function. In earlier studies of these SNP (
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      ;
      • Ortega M.S.
      • Denicol A.C.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows.
      ), pathway analysis of genes associated with fertility traits using Ingenuity Pathway Analysis indicated that 14 of these genes were regulated by estradiol and 6 were regulated by progesterone. Most of these steroid-regulated genes were among the list of 26 genes found to be associated with reproductive function in more than one study. The list included 9 estradiol-regulated genes (APBB1, BCAS1, CAST, HSD17B12, HSD17B7, LHCGR, OCLN, PMM2, and RABEP2) and 4 progesterone-regulated genes (CD40, LHCGR, PMM2, and RABEP2). Besides steroid hormones being essential for reproduction in mammals, data indicate the importance of variation in their circulating concentrations for cow fertility. In beef cattle, preovulatory concentrations of estradiol are related to subsequent fertility (
      • Perry G.A.
      • Smith M.F.
      • Lucy M.C.
      • Green J.A.
      • Parks T.E.
      • MacNeil M.D.
      • Roberts A.J.
      • Geary T.W.
      Relationship between follicle size at insemination and pregnancy success.
      ;
      • Jinks E.M.
      • Smith M.F.
      • Atkins J.A.
      • Pohler K.G.
      • Perry G.A.
      • MacNeil M.D.
      • Roberts A.J.
      • Waterman R.C.
      • Alexander L.J.
      • Geary T.W.
      Preovulatory estradiol and the establishment and maintenance of pregnancy in suckled beef cows.
      ). For Holstein cows on pasture, those with high genetic merit for fertility had a larger corpus luteum and higher circulating concentrations of progesterone than cows with lower genetic merit (
      • Cummins S.B.
      • Lonergan P.
      • Evans A.C.O.
      • Butler S.T.
      Genetic merit for fertility traits in Holstein cows: II. Ovarian follicular and corpus luteum dynamics, reproductive hormones, and estrus behavior.
      ;
      • Moore S.G.
      • Scully S.
      • Browne J.A.
      • Fair T.
      • Butler S.T.
      Genetic merit for fertility traits in Holstein cows: V. Factors affecting circulating progesterone concentrations.
      ). Progesterone concentrations on d 4 to 7 after AI were positively associated with pregnancy rate in Holstein heifers (
      • Parr M.H.
      • Mullen M.P.
      • Crowe M.A.
      • Roche J.F.
      • Lonergan P.
      • Evans A.C.O.
      • Diskin M.G.
      Relationship between pregnancy per artificial insemination and early luteal concentrations of progesterone and establishment of repeatability estimates for these traits in Holstein-Friesian heifers.
      ). Steroid hormones may be an especially important determinant of reproduction in lactating cows because of increased catabolism of steroids associated with lactation (
      • Wiltbank M.
      • Lopez H.
      • Sartori R.
      • Sangsritavong S.
      • Gümen A.
      Changes in reproductive physiology of lactating dairy cows due to elevated steroid metabolism.
      ,
      • Wiltbank M.C.
      • Souza A.H.
      • Carvalho P.D.
      • Cunha A.P.
      • Giordano J.O.
      • Fricke P.M.
      • Baez G.M.
      • Diskin M.G.
      Physiological and practical effects of progesterone on reproduction in dairy cattle.
      ).
      The other function that was well represented in genes containing SNP identified earlier (
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      ;
      • Ortega M.S.
      • Denicol A.C.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows.
      ) and in the present study was immune function. Of the 10 genes containing SNP related to reproductive traits that were involved in immune function as identified by
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      or
      • Ortega M.S.
      • Denicol A.C.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows.
      , 6 (CD14, CD40, FCER1G, FUT1, GCNT3, and RABEP2) were found to be related to fertility traits in more than one study (Table 5). Immune function is an important determinant of reproductive function, and dairy cows that experience one or more diseases postpartum have reduced reproductive function (
      • Santos J.E.P.
      • Bisinotto R.S.
      • Ribeiro E.S.
      • Lima F.S.
      • Greco L.F.
      • Staples C.R.
      • Thatcher W.W.
      Applying nutrition and physiology to improve reproduction in dairy cattle.
      ). Genes involved in inflammation are among those whose expression in endometrium, liver, and muscle differed between Holstein cows that were genetically divergent in fertility (
      • Moran B.
      • Butler S.T.
      • Moore S.G.
      • MacHugh D.E.
      • Creevey C.J.
      Differential gene expression in the endometrium reveals cytoskeletal and immunological genes in lactating dairy cows genetically divergent for fertility traits.
      ,
      • Moran B.
      • Cummins S.B.
      • Creevey C.J.
      • Butler S.T.
      Transcriptomics of liver and muscle in Holstein cows genetically divergent for fertility highlight differences in nutrient partitioning and inflammation processes.
      ).

      Variability of the SNP in Other Breeds

      The variability of the 68 SNP studied here was evaluated in 11 B. taurus breeds (Table 6). All but one SNP was variable in each of the examined breeds. The exception was for SEC14L1, which was variable only in Holstein. The variability of these SNP in other breeds indicates that they arose before the Holstein separated from other B. taurus breeds and that the SNP could be associated with reproductive function in breeds other than Holstein.
      Table 6Frequency of the SNP in selected breeds of cattle
      SNP IDGeneStudyBreed (no.)
      Minor allele
      Based on a population of 2,798 Holsteins [combined data of Cochran et al. (2013a) and Ortega et al. (2016)].
      FrequencyAngus (109)Beef- master (10)Charolais (12)Gelbvieh (8)Hereford (18)Jersey (3)Limousin (9)Maine- Anjou (5)Red Angus (14)Romagnola (4)Simmental (11)
      rs109967779ACAT2G0.370.480.450.250.440.250.830.330.300.350.750.41
      rs133700190AP3B1T0.230.350.600.080.250.250.330.500.000.310.380.27
      rs41766835APBB1G0.210.000.050.090.000.190.000.000.100.040.250.05
      rs110541595ARL6IP1A0.390.080.250.000.250.500.330.330.500.120.250.14
      rs110127056ASLG0.440.100.050.040.190.220.000.170.300.000.000.14
      rs43114141AVPA0.450.090.220.380.190.250.000.750.300.000.000.20
      rs109669573BCAS1T0.400.320.550.501.000.921.000.500.000.310.330.41
      rs133674837BDH2A0.410.580.350.330.310.280.330.390.700.120.000.59
      rs109032590BOLA-DMBT0.290.720.950.500.441.001.000.750.600.850.370.23
      rs110217852BSP3G0.290.060.100.130.250.060.830.720.000.040.250.27
      rs133455683C17H22orf25A0.430.600.700.130.500.390.000.500.900.540.500.05
      rs109332658C7H19orf60T0.280.500.250.250.690.500.000.400.600.270.500.50
      rs135744058CACNA1DG0.300.520.700.420.560.500.000.190.100.420.000.55
      rs137601357CASTC0.430.200.250.500.560.420.000.190.200.210.000.50
      rs109447102CCDC86C0.200.240.550.330.630.360.500.750.400.380.750.64
      rs137673698CCT8T0.320.991.001.001.001.001.000.941.000.881.001.00
      rs109621328CD14C0.080.090.000.040.440.000.000.060.100.120.000.05
      rs41711496CD40A0.470.220.800.330.370.531.000.420.400.230.170.55
      rs41857027CFDP2T0.160.000.100.250.310.170.170.110.000.000.000.14
      rs133449166CSNK1EC0.370.610.450.550.250.361.000.300.800.810.750.36
      rs109443582CSPP1A0.060.400.150.290.000.110.000.310.200.230.250.14
      rs110270752DEPDC7A0.240.420.250.540.190.220.170.440.400.380.250.32
      rs110629231DNAH11T0.330.700.400.330.370.690.500.440.300.190.870.45
      rs109503725DSC2C0.450.240.300.920.500.640.500.890.600.350.750.41
      rs109561866DYRK3T0.120.160.600.580.380.250.330.750.500.270.380.41
      rs133175991DZIP3C0.200.770.900.710.810.920.830.941.000.851.000.64
      rs43676052EPAS1G0.260.060.150.080.120.110.000.190.100.040.250.09
      rs135071345FAM5CC0.111.000.951.000.940.781.001.001.000.920.750.91
      rs109137982FCER1GA0.100.330.100.420.190.470.000.170.300.040.250.14
      rs110937773FGF2A0.310.490.750.460.870.750.500.780.500.540.750.64
      rs43745234FSHRG0.320.340.700.630.810.470.500.440.700.420.880.82
      rs109247499FSTC0.450.000.050.040.060.000.000.060.100.000.000.05
      rs41893756FUT1G0.200.120.150.500.380.580.500.430.200.121.000.23
      rs109262355FYBG0.340.800.950.751.001.000.501.000.800.581.001.00
      rs109830880GCNT3C0.140.070.000.380.250.060.170.000.100.190.380.05
      rs42339105GOLGA4G0.080.200.500.420.060.250.330.110.500.190.370.32
      rs109516714GPLD1C0.380.590.650.630.560.421.000.830.600.690.750.36
      rs109711583HSD17B12G0.430.310.300.540.250.750.170.390.100.460.250.36
      rs43079452HSD17B3C0.130.950.950.961.000.721.001.001.001.001.001.00
      rs109769865HSD17B6T0.130.920.950.790.630.250.670.861.000.810.880.55
      rs110828053HSD17B7T0.180.700.450.580.500.330.500.170.500.690.370.05
      HSPA1LDeletion0.290.010.150.210.190.190.170.000.000.000.000.14
      rs110789098IBSPC0.400.250.250.040.000.080.500.170.200.080.000.05
      rs111015912LDB3T0.190.290.000.500.940.331.000.250.100.350.330.36
      rs41256848LHCGRT0.440.781.000.921.000.971.000.891.000.961.000.95
      rs41859871MON1BT0.130.950.951.000.940.891.001.001.001.001.001.00
      rs109248655MRGPRFT0.040.170.200.210.060.220.000.200.000.150.000.27
      rs43703916MRPL48G0.400.630.450.290.310.140.000.670.500.730.750.36
      rs109761676MS4A8BG0.250.170.250.330.250.060.000.280.100.040.380.14
      rs133497176NFKBIL1T0.150.030.150.170.000.080.000.250.200.120.330.18
      rs109383758NLRP9C0.470.560.850.710.500.360.670.360.300.731.000.68
      rs134264563OCLNG0.320.080.350.040.000.170.170.060.000.040.250.09
      rs111027720PARM1G0.480.760.500.290.310.861.000.641.000.810.000.32
      rs109813896PCCBC0.360.380.200.290.690.421.000.000.000.460.500.23
      rs109629628PMM2G0.390.510.700.460.120.560.830.560.300.880.620.05
      rs133729105RABEP2A0.380.600.650.500.880.220.000.700.600.420.000.91
      rs43572154ROR2A0.150.860.901.000.750.501.000.601.000.650.500.77
      rs136746215SEC14L1G0.050.000.000.000.000.000.000.000.000.000.000.00
      rs43321188SERPINE2T0.260.110.450.000.060.280.000.130.400.040.500.14
      rs110365063SLC18A2A0.170.140.200.250.130.030.170.290.200.120.000.18
      rs41912290SREBF1C0.390.570.950.950.750.751.000.420.800.651.000.82
      rs42158454SYTL2A0.020.150.050.080.130.390.000.060.200.210.130.05
      rs110660625TBC1D24A0.350.950.750.670.880.811.001.000.600.811.000.68
      rs110805802TDRKHT0.120.010.150.170.000.000.330.000.000.040.000.00
      rs132789482TSHBT0.160.080.200.330.310.170.000.440.300.040.750.41
      rs134031231TXN2G0.400.500.850.120.250.860.000.580.500.500.670.27
      rs137248155VCANT0.270.180.150.000.130.030.250.220.200.420.130.00
      rs110883602ZP2C0.400.130.000.290.560.310.170.110.100.350.250.27
      1 Based on a population of 2,798 Holsteins [combined data of
      • Cochran S.D.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.
      and
      • Ortega M.S.
      • Denicol A.C.
      • Cole J.B.
      • Null D.J.
      • Hansen P.J.
      Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows.
      ].

      CONCLUSIONS

      This study demonstrated that genetic differences in fertility are associated with differences in phenotype. This finding was true in comparisons of cows that diverged in predicted transmitting ability for DPR or in examination of the effects of specific SNP on reproductive function. A total of 26 SNP were identified for which the same allele was associated with increases in at least one reproductive trait in 2 separate studies. Examination of the function of genes represented by the 26 SNP with concordant effects indicates the importance of steroid hormones and immune function as determinants of reproductive function. All but one of the studied SNP were variable in 11 breeds besides Holstein, indicating the potential association of these SNP with reproductive function in cattle in general.

      ACKNOWLEDGMENTS

      Research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2013-68004-20365 from the USDA National Institute of Food and Agriculture (Washington, DC) , a grant from the Southeast Milk Inc. Milk Check-off Program , and funds from the L.E. “Red” Larson Endowment . Cole and Null were supported by appropriated project 1265-31000-096-00 , “Improving Genetic Predictions in Dairy Animals Using Phenotypic and Genomic Information,” of the Agricultural Research Service of the USDA . The authors thank the dairies that formed part of the study: Davie 1 and 2 (Okeechobee, FL), University of Florida Dairy (Hague, FL), Genasci Brothers Dairy (Modesto, CA), Larson Dairy Barn 5 (Okeechobee, FL), Maddox and Ruann Dairies (Riverview, CA), North Florida Holsteins (Bell, FL), PH Dairies 1 and 2 Dairies (Winton, CA), and Shenandoah Dairy (Live Oak, FL). The authors also thank GeneSeek for outstanding cooperation in completion of the study. Thanks are also extended to the following for assistance with obtaining samples and data: Dr. Alvaro Magalhaes, Lander Veterinary Clinic (Turlock, CA), and Kelly Buchanan, Serdal Dikmen, Kyle Dobbs, Javier Juarez, James Moss, Antonio Ruiz de King, and Paula Tribulo from the University of Florida. The Council on Dairy Cattle Breeding (Reynoldsburg, OH) and the Cooperative Dairy DNA Repository (Columbia, MO) are acknowledged for providing data used in this study. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.

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