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Small Ruminant Research 90 (2010) 139–141

Contents lists available at ScienceDirect

Small Ruminant Research

journa l homepage: www.e lsev ier .com/ locate /smal l rumres

hort communication

novel single nucleotide polymorphism in the coding region of goatrowth hormone receptor gene and its association with lactoseontent and somatic cell count in milk

ndrzej Maja, Emilia Bagnickaa,∗, Jarosław Kabab, Mariusz Nowickib, Ewa Kosciuczuka,rzysztof Słoniewskia, Karina Horbanczuka, Lech Zwierzchowskia

Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, 05-552 Wólka Kosowska, PolandDivision of Infectious Diseases and Epidemiology, Department of Clinical Sciences Faculty of Veterinary Medicine, Warsaw Agricultural University,owoursynowska 159C, Warsaw, Poland

r t i c l e i n f o

rticle history:eceived 24 August 2009eceived in revised form 8 December 2009ccepted 10 December 2009vailable online 12 January 2010

a b s t r a c t

A novel single nucleotide polymorphism—a C/T transition (RFPL-MspI) was found uponsequencing of a 439 bp DNA fragment, comprising whole exon 4 and parts of adjacentintrons of the goat growth hormone receptor gene. This mutation was located at 8thnucleotide of exon 4 (position 94 according to GenBank Acc. No. AY739707). The nucleotide

eywords:rowth hormone receptorene polymorphism

substitution has no effect on the amino acid sequence of the GHR protein. Within the cohortof 227 Polish dairy goats three genotypes were found: CC (frequency 0.96), CT (0.036), andTT (0.004). The frequency of C and T alleles was 0.978 and 0.022, respectively. It was shownthat CC genotype goats had significantly higher lactose content and lower somatic cell count

e CT gen

ssociationilk traitsoat

than those with thtraits studied.

. Introduction

Hormones, growth factors, and other regulatory pro-eins associated with the so-called “somatotropic axis”re candidate markers for quantitative traits in farm ani-als (Parmentier et al., 1999). The biological effects of

rowth hormone (GH) concern a variety of tissues andhe metabolism of all nutrient classes. In farm ruminants,he GH actions play a key role in increasing growth per-ormance and milk yield (Etherton and Bauman, 1998).

herefore, there is a great interest in using growth hormoneo improve the production in farm animals.

Growth hormone’s actions on target cells depend on GHeceptor (GHR) (Burton et al., 1994). The GHR is a member

∗ Corresponding author at: Institute of Genetics and Animal Breeding,olish Academy of Sciences, Department of Animal Science, Postepu 1,astrzebiec, 05-552 Wólka Kosowska, Poland. Tel.: +48 22 756 17 11;ax: +48 22 756 16 99.

E-mail address: e.bagnicka@ighz.pl (E. Bagnicka).

921-4488/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.smallrumres.2009.12.006

otype. No association was found with the other milk production

© 2009 Elsevier B.V. All rights reserved.

of the cytokine/hematopoietin superfamily of receptors. Inmost mammalian species the gene encoding GHR consistsof nine exons (from 2 to 10) in the translated part. In allspecies studied so far, GHR gene characterizes a complexstructure of exon 1, coding for the 5′-untraslated region(5′-UTR) (Jiang and Lucy, 2001).

It has been reported that some productive traits of cat-tle, e.g. milk yield and composition, are associated with theGHR gene polymorphism (Aggrey et al., 1999; Blott et al.,2003; Maj et al., 2004). The objective of this study was tosearch for nucleotide sequence polymorphism in exon 4 ofthe goat GHR gene and for its possible association with milkproduction traits.

2. Materials and methods

2.1. Animals

Studies were conducted on 227 dairy goats—156 Polish WhiteImproved (PWI) and 71 Polish Fawn Improved (PFI) goats maintained inthree dairy herds. The mean productive values were as follows: daily milkyield: 2.36 kg, fat: 3.37%, and protein: 3.28%. The goats were kept in loose

ant Research 90 (2010) 139–141

Fig. 1. Agarose gel electrophoresis showing RFLP-MspI genotyping ofC/T single nucleotide polymorphism in the goat GHR gene exon 4:M—100–1000 bp DNA marker (Sigma); ND—non-digested 439 bp PCRproduct; CC, CT, TT—GHR genotypes. Digestion of the 439 bp PCR prod-

140 A. Maj et al. / Small Rumin

barns with outside run (except winter). During the test period the ani-mals were fed according to the INRA system (Jarrige, 1988). Water wasavailable ad libitum.

2.2. Genotype determination

Blood samples were collected from the jugular vein to the tubescontaining K3EDTA. DNA for GHR gene sequencing and genotyping wasisolated from blood by the method of Kanai et al. (1994).

The 439 bp DNA fragment, comprising 113 bp of intron 3, whole130-bp of exon 4, and 196 bp of intron 4, was amplified usingthe following primers: forward—GCCCA-GAGAAACAGCATTTCTA andreverse—TCACTGCCATATTTCCAGCATC. Polymerase chain reactions andDNA sequencing were performed as previously described (Maj andZwierzchowski, 2006).

For restriction fragment length polymorphism (RFLP) analyses theamplified DNA was digested with MspI endonuclease. Restriction prod-ucts were separated by electrophoresis in 2% agarose (Gibco-BRL, England)in 1× TBE buffer (0.09 M Tris-boric acid, 0.002 M EDTA) with 0.5 �g/mlethidium bromide (Et-Br), visualized under UV light, and scanned in anFX Phosphorimager apparatus (Bio-Rad, Hercules, CA, USA).

2.3. Analysis of milk composition

The experiment lasted during 6 successive lactations. The goats weremilked twice a day. Milk samples were taken from each goat once a monthduring the whole lactation period (270 days in average). The daily milkyield (kg) and content of the major milk components (%): fat, proteinand lactose, and also somatic cell count (SCC) were evaluated in eachmilk sample. The fat, protein and lactose content were estimated in themilk preserved with Broad Spectrum Microtabs II (Bentley, Poland), usingMilko Scan 104A/B (FOSS A/S, Hillerød, Denmark). Somatic cells werecounted by means of a Fossomatic apparatus (FOSS A/S). Somatic cellcount and lactose concentration in milk were used as indicators of thehealth status of the udder.

2.4. Statistical calculations

Altogether 2905 records about milk yield, fat and protein content and1181 records about lactose content and SCC, from 180 does were used instatistical analyses. The SCC values (expressed in thousands) were trans-formed to the natural logarithm scale (ln of SCC). In order to determineassociations between the GHR gene polymorphism and the investigatedtraits the multi-trait repeatability test-day model was used. The DMU pro-gram was used for computation (Madsen and Jansen, 2000). The modelincluded the animal’s genotype, breed, year of birth, herd-year-seasonof kidding and parity as fixed effects and the additive genetic effect,permanent environmental influence and the date of the test as randomeffects. Legendre polynomials nested within parity were applied takinginto account of stage of lactation effects (Brotherstone et al., 2000). Thedifferences between solutions for genotypes were checked using Student’st-test with Bonferroni adjustment. The significance of the differencesbetween observed and expected genotype frequencies was estimatedusing �2 test.

3. Results and discussion

In this study a 439 bp DNA fragment was sequenced,comprising whole exon 4 and parts of adjacent intronsof the goat growth hormone (GHR) gene to search fornucleotide sequence polymorphisms. Sequencing DNAsamples from 36 goats revealed one single nucleotidepolymorphism—a C/T transition at 8th nucleotide of exon4 (position 94 according to GenBank Acc. No. AY739707).Since the whole sequence of the goat GHR gene is not

known the PCR primers were used, designed previouslyby Blott et al. (2003) for the amplification of the corre-sponding fragment of the bovine GHR gene. In the bovinegene they match sequences between nucleotides 1594 and1615 (forward primer) and 2011 and 2032 (reverse) (Gen-

uct with MspI restriction endonuclease resulted in two DNA bands (318and 121 bp) for homozygote CC, three bands (439, 318 and 121 bp) forthe CT heterozygote, and one band (439 bp) for homozygous TT genotypeanimals.

Bank AM161140.1). With the caprine DNA as a templatethey gave a good quality PCR product of the expectedlength of 439 bp (Fig. 1). Known is the complete mRNAsequence (cds) of the goat GHR-exons 2–10 (GenBank Acc.No. EF559245). This allowed us to localise the C/T mutationat mRNA position 437. The nucleotide substitution has noeffect on the amino acid sequence of the GHR protein; bothtriplets—TCC and TCT encode serine.

As the C → T mutation removes the MspI restriction site,the polymorphism could be identified using RFLP tech-niques. After digestion with the MspI endonuclease threegenotypes were identified: homozygotes either digested(CC) or non-digested (TT) by MspI, and CT heterozy-gotes (Fig. 1). The genotype frequencies were as follows:CC = 0.96, CT = 0.036, TT = 0.004 (one animal only among227 genotyped). The frequency of C and T alleles was 0.978and 0.022, respectively. The distribution of genotypes fol-lowed the Hardy–Weinberg rule.

Maj and Zwierzchowski (2006) searched for sequencevariation in the GHR gene in different ruminantspecies—Bovidae and Caprine. Only two differenceswere found in 130 bp long exon 4 sequence of GHR genebetween bovine (Bos indicus) vs. (Capra hircus): T → C atposition 8 and C → T at position 16.

Associations were studied of the GHR polymorphismwith the goat’s milk traits. The mean values and standarddeviations of the investigated traits and the effects of GHRgenotypes are shown in Table 1. The animal carrying the TTgenotype was not included in the statistical analysis.

No associations were found between the C/T polymor-phism in GHR gene exon 4 and the milk yield and fat andprotein content. But there were differences found (p ≤ 0.01)between goat GHR genotypes as concerns lactose content

and somatic cell count (SCC) in the milk. The CC goats hada higher lactose content and lower SCC than those with theCT genotype. Both lactose content and SCC in milk are theindicators of health status of the goat’s mammary gland(Lerondelle et al., 1992; Leitner et al., 2004).

A. Maj et al. / Small Ruminant Research 90 (2010) 139–141 141

Table 1Means and standard deviations of investigated traits and estimates for, genotypes effects with their standard errors (SE).

Trait Overall Genotype

Mean SD CC CT

Estimate (N = 2821a) SE Estimate (N = 83a) SE

Milk yield [kg] 2.36 1.03 2.41 0.21 2.42 0.27Fat [%] 3.37 1.13 3.07 0.23 3.12 0.26Protein [%] 3.28 0.85 3.44 0.15 3.34 0.18

Trait Overall Genotype

Mean SD CC CT

Estimate (N = 1129a) SE Estimate (N = 44a) SE

Lactose [%] 4.38 0.34 4.21A 0.08 4.08B 0.11

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coding sequences of the growth hormone receptor (GHR) gene in thefamily bovidae. Folia Biologica (Kraków) 54, 31–36.

SCC [ln] 6.81 1.25 7.06A

a Number of milk samples analysed; the different letters within rows in

There is very little information about polymorphismf the goat GHR gene and its association with productionraits. The possible effects of the TG-repeat variants in theoat GHR gene 5′-noncoding region were studied with milkroduction traits but no association was found (Maj et al.,007).

More studies were conducted on the polymorphism ofhe bovine GHR gene. Association was established between/T polymorphism in the GHR gene exon 8 (position 914,ccording to GenBank AY748827), which resulted in aminocid substitution Phe279Tyr in the transmembrane domainf the receptor, and milk, fat and protein yield and fat androtein content in milk of Holstein-Friesian cows (Blott etl., 2003). Effects of two SNPs (RFLP-NsiI and -AccI) andheir combination were shown on milk production traitsf Holstein-Friesian cows (Maj et al., 2004). Moreover, the257G polymorphism was found in the GHR gene exon0 which was connected with milk fat and protein yieldKaminski et al., 2006).

. Conclusion

This study presents a novel polymorphism of theaprine GHR gene—the C/T transition in exon 4. An asso-iation was found between the GHR genetic polymorphismnd lactose concentration and somatic cell count in goat’silk, the traits which are highly related to udder health.

herefore, we suggest the potential use of C/T polymor-hism in exon 4 of the GHR as a genetic marker of goat’sesistance/susceptibility to mastitis. However, to provehis, further study must be done with more numerousnimal cohorts, including more animals representing theomozygous genotype TT.

cknowledgement

This study was supported by IGAB grant S.V.3.

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