ГЕНЕТИКА, 2012, том 48, № 4, с. 503-507
ГЕНЕТИКА ЖИВОТНЫХ
УДК 575.174.015.3:599.75
EFFECT OF POLYMORPHIC VARIANTS OF GH,, Pit-1, AND ß-LG GENES ON MILK PRODUCTION OF HOLSTEIN COWS © 2012 M. Heidari1, M. A. Azari1, S. Hasani1, A. Khanahmadi2, S. Zerehdaran1
1 Department of Animal Sciences, Faculty of Agriculture, Gorgan University of Agricultural Sciences and Natural Resources,
P.O. Box 49189-43464, Gorgan, I.R. Iran e-mail: Mojtaba_9@yahoo.com 2 Department of Animal Sciences, Faculty of Agriculture, P.O. Box 163, Gonbad, I.R. Iran Received February 08, 2011; in final form, August 08, 2011
Effect of polymorphic variants of growth hormone (GH), ß-lactoglobulin (ß-LG), and Pit-1 genes on milk yield was analyzed in a Holstein herd. Genotypes of the cows for these genes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Allele frequencies were 0.884 and 0.116 for L and Vvariants of GH, 0.170 and 0.830 for A and Bvariants of Pit-1, and 0.529 and 0.471 for A and Bvariants of ß-LG, respectively. GLM procedure of SAS software was used to test the effects of these genes on milk yield. Results indicated significant effects of these genes on milk yield (P < 0.05). Cows with LL genotype of GH produced more milk than cows with LVgenotype (P < 0.05). Also, for Pit-1 gene, animals with AB genotype produced more milk than BB genotype (P < 0.05). In the case of ß-LG gene, milk yield of animals with AA genotype was more than BB genotype (P < 0.01). Therefore, it might be concluded that homozygote genotypes of GH (LL) and ß-LG (AA) were superior compared to heterozygote genotypes, whereas, the heterozygote genotype of Pit-1 gene (AB) was desirable.
Growth hormone gene is a member of multi-gene family approximately 1800bp in length with 4 intervening sequences [1] and assigned to chromosome region 19q26 in bovine genome [2]. This hormone plays an important role in biological processes such as mammary development, lactation, growth, and metabolism regulation [3].
Association of GH gene polymorphism with production traits in dairy cattle have been studied by many researchers [4—9].
Pit-1 is a pituitary-specific transcription factor that is responsible for pituitary development and hormone secreting gene expression in mammals [10] and it has been mapped by linkage analysis of bovine chromosome 1 [11]. There are a few reports on the relationships of Pit-1 genotypes with productive traits in dairy cattle [12-15].
p-LG is the major whey protein of ruminant species, and also presents in the milk of many, but not all, other species [16], and it accounts for approximately 10 to 15% of total milk proteins [17]. p-LG gene is located on the chromosome 11 in cattle, and has a 4.7kb transcriptional unit, including seven exons and six in-trons [18].
The effects of bovine p-LG variants on milk components, milk production, and cheese manufacturing have been extensively studied [19-26].
So these genes were considered promising candidate markers for economically important quantitative traits, and the purpose of this study was to investigate
their effect on milk yield in an industrial Holstein herd.
MATERIALS AND METHODS
The current study was carried out from July 2008 to May 2009. About 100 Holstein cows were genotyped for GH, Pit-1, and p-LG loci. The blood samples were collected randomly from dairy cows with at least one lactation milk production record. The animals belonged to Behin Talise farm in Golestan Province. DNA was extracted using modified salting out extraction protocol [27]. Polymorphism of the genes was determined by PCR-RFLP method. The primers sequences, region, and size of the amplified fragments for these genes are shown in Table 1.
PCR for all of the genes was carried out using Personal Cycler™ amplificator (Biometra, Germany) and PCR Master Kit (CinnaGen Inc., Iran). The Kit contained master mix including 0.04 u/|l Taq DNA polymerase, PCR buffer, 3mM MgCl2, and 0.04 mM of each dNTP. In each reaction, 12.5 |l master mix, 1 |l of DNA (50 to 100 ng/|l), 2-4 |l primers (5 pmol/|l), and some deionized water till final volume of 25 |l were used. Amplification programs for amplificating the investigated genes were as follow:
GH. The amplification program consisted of an initial denaturation at 94°C for 2 min, then 30 cycles of 94°C for 45 sec, 62°C for 1 min and 72°C for 1 min, and a final extension of 72°C was maintained for 3 min.
Table 1. Sequence of the primers, size and region of the amplified fragments in PCR
Gene Primer sequence Size (bp) Amplified region Reference
GH 5'-GTGGGCTTGGGGAGACAGAT-3' 5'-GTCGTCACTGCGCATGTTTG-3' 282 intron 4, exon 5 [30]
Pit-1 5'-CAA TGA GAA AGT TGG TGC-3' 5'-TCT GCA TTC GAG ATG CTC-3' 1355 intron 5, exon 5 and exon 6 [31]
P-LG 5'-TGTGCTGGACACCGACTACAAAAAG-3' 5'-GCTCCCGGTATATGACCACCCTCT-3' 247 exon 4 , intron 4 [32]
Table 2. Genotypes and size of the fragments of digestion reaction for the studied genes [8, 15, 18]
Gene Endonuclease Genotypes and size of the fragments (bp)
GH AluI LL VV L V
1 50, 82, 50 1 50, 132 1 50, 132, 8 2, 5 0
Pit-1 Hinfl AA BB AB
660, 425 , 270 660, 38 5 , 270, 40 660, 425 , 385, 270, 40
P-LG HaeIII AA BB AB
1 48, 99 9 9, 74 1 48, 99, 74
Pit-1. For this gene conditions were 95°C for 2 min and an initial annealing of 55°C for 1 min, then an initial extension of 72°C for 2 min, followed by 29 cycles of 94°C for 45 sec, 55°C for 1 min, and 72°C for 1 min. The final step was at 72°C for 2 min.
p-LG. PCR condition consisted of an initial dena-turation at 94°C for 5 min, then 35 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 1 min, and a final extension of 72°C was maintained for 5 min.
The PCR products were digested by restriction enzymes following their protocol: four units of AluI for GH gene, five units of Hinfl for Pit-1 gene, and ten units of Haelll for p-LG gene. After digestion, the fragments were separated on 3% (for GH and Pit-1 genes) and 2.5% (for p-LG gene) agarose gel electrophoresis. Determination of gene and genotype frequencies was carried out using POP Gene 1.31 software [28].
A fixed model was used to test the effect of GH, Pit-1, and p-LG genotypes on mature equivalent milk yield records. Statistical analysis was performed using GLM procedure of SAS software, 2002 [29], and comparisons of least squares means were carried out using Tukey-Kramer test at 5% probability level. The following linear model was used for analysis:
yijkLm = ^ + Ai + Bj + Ck + DL + where
yijkLm —milk record of the cow;
^ — population mean;
Aj — fixed effect of calving year (i = 1,..., 3);
Bj — fixed effect of calving season (j = 1,., 4);
Ck — fixed effect of parity (k = 1,., 8);
Dl — fixed effect of genotype (L = 1,., 3, GH, Pit-1, P-LG);
2
eykLm — random residual error with 0 mean and ae variance.
RESULTS
PCR of the investigated genes was without contamination and resulted in clear bands. Digestion of the PCR products with AluI, Hinfl, and Haelll showed three genotypes for each locus. Genotypes of the investigated loci accompanied by size of the fragments are shown in Table 2.
The frequencies of L and V variants of GH gene were 0.884 and 0.116, respectively. Also, allele frequencies were 0.170 and 0.830 for A and B variants of Pit-1 gene, and 0.529 and 0.471 for A and B variants of p-LG, respectively.
Table 3 presents results of comparison of milk yield squares means for the GH, Pit-1, and p-LG genotypes.
As Table 3 shows cows with the LL genotype of GH gene produced more milk than cows with the LV genotype (P < 0.05). Although VVgenotype presents more milk than other genotypes of this locus, but it's differences with them was not significant. Cows with the AB genotype of Pit-1 gene had more milk than the cows with BB genotype (P < 0.05). There were not any significant differences between other genotypes of these genes. In the case of p-LG gene, the effect of AA and BB genotypes on milk yield was significant (P < 0.01) but it was not significant with ABB genotype (P > 0.05).
EFFECT OF POLYMORPHIC VARIANTS
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DISCUSSION
In present study, the frequency of GH L allele (0.884) was more than V allele (0.116) that was in agreement with other studies. For example, the frequency of L allele was reported 0.93 in Danish Holstein; 0.85 in Danish Red; 0.51 in Danish Jersey [30], 0.909 in Hungarian Holstein Friesian [6], 0.936 in Iranian Holstein bulls [8], 0.85 in Holstein breed [33], 0.52 in Jersey breed [7]. Studies on Gyr breed showed that the breed was monomorphic for this allele [15]. Based on the reports, the Holstein breed have had the maximum frequency for L allele [8] and the minimum frequency for this allele have been observed in Jersey breed [7].
Because of the important effects of GH gene in growth and lactation, association of GH polymorphism with productive traits has been considered by many researches. In present investigation, the cows with LL genotype had significantly more milk yield than LV genotype (Table 3) and it confirmed the results of other researchers [7, 8]. Many authors found significant association between LL genotype and higher fat yield [4, 5, 8, 9].
There are some contradictory reports on association between LV genotype and milk composition. Some authors reported significant association between LV genotype and milk yield [4—6], While Dario et al. [7] reported higher fat yield with this genotype. Some researchers observed the superiority of this genotype in protein yield [4, 7]. Differences of least squares means of VVgenotype with LL and LVwere not significant in our study (Table 3).
Generally, for Pit-1 gene, in previous studies on Holstein breed and other breeds, the frequency of B allele was more than A allele. A allele frequency in different breeds was as follows: in Holstein breed, 0.15 [34], in Polish Black and White cows, 0.25 [13, 31], in dairy Gyr breed, 0.05 [15], and in Italian Holstein cows, 0.188 [12]. These results were in agreement w
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