ГЕНЕТИКА, 2013, том 49, № 8, с. 981-985
ГЕНЕТИКА ЖИВОТНЫХ
УДК 575.113.1:636.2.034
FABP3, FABP4 AND ANXA9 SNP GENOTYPES IN RELATION TO BREEDING VALUES FOR MILK PRODUCTION TRAITS IN POLISH HOLSTEIN-FRIESIAN COWS © 2013 H. Kulig1, I. Kowalewska-Luczak1, K. Z ukowski2, W. Kruszynski3
department of Genetics and Animal Breeding, West Pomeranian University of Technology in Szczecin, 71-466Szczecin, Poland e-mail: hanna.kulig@zut.edu.pl 2Department of Animal Genetics and Breeding, National Research Institute of Animal Production, 32-083 Balice, Poland 3Department of Genetics, Wroclaw University of Environmental and Life Sciences, 51-631 Wroclaw, Poland
Received November 13, 2012
The aim of this study is to estimate the associations between the ANXA9, FABP3, and FABP4 genotypes and the breeding value for milk production traits (yields of milk, protein, and fat, as well as protein and fat percentage) in 975 Polish Holstein-Friesian cows. The frequencies of genotypes and alleles were determined. Statistically significant relations between the ANXA9 SNP and the breeding value for fat content, as well as the FABP4 SNP and the breeding value for protein content were found. The results indicated that selection for the ANXA9 GG and FABP4 GG animals might contribute to an decreased fat content and increased protein content in milk, respectively, in Polish Holstein-Friesian cows. Although verification in the further studies is needed.
DOI: 10.7868/S0016675813080080
The constituents of milk are synthesized in the mammary epithelial cells from blood plasma substrates. Milk lipids are synthesized from fatty acids that bind to appropriate proteins named fatty acid-binding proteins (FABPs). FABPs are a small family of cytoplasmic proteins which bind long chain fatty acids and other hydrophobic ligands. Nine different members of this family, FABP1—FABP9, have been identified so far. Their main functions include the capture, transport and metabolism of fatty acids. They are involved in the transport of fatty acids across the cytoplasmic membrane to the triglycerides or phospholipids p-ox-idation and synthesis sites. FABPs can modulate the concentration of the fatty acids in cells and thus affect various cellular processes, in particular lipid metabolism. They are present in various tissues where there is a high demand for fatty acids, e.g. in the mammary gland during lactation [1]. During the early period of lactation, a significant increase in FABP4 expression in the bovine mammary gland has been observed, but the expression of FABP4 is still less abundant than that of FABP3: during lactation, FABP3 mRNA represent over 75% of FABPs expressed. It has been found that FABP3 plays the most important role of all the FABP proteins in fat production in the mammary gland of cattle [2]. The FABP3 gene has been mapped in bovine chromosome 2 [3], where QTLs affecting the fat yield and fat content of milk have also been identified [4]. The FABP4 gene has been mapped in bovine chromosome 14 [5] which is extremely rich in QTLs for milk
traits such as milk, fat and protein yield, and the percentage of fat and protein in milk [4].
This fatty acid transport is supported by a specific transporter protein — annexin A9 (ANXA9), which belongs to the family of phospholipid-binding proteins and is considered to be a protein that is involved in transport through the cytoplasmic membrane [6]. It is expressed in various tissues, including the mammary gland [6, 7]. ANXA9 is encoded by a gene which has been mapped in bovine chromosome 3, in a region which contains QTLs for milk fat content and for the other milk traits listed above [1, 4].
The aim of this study was to determine the frequencies of FABP3, FABP4 and ANXA9 alleles and genotypes and to establish possible associations between SNP genotypes in these genes and the estimated breeding values (EBV) for milk production traits in a herd of Polish Holstein-Friesian cows.
MATERIALS AND METHODS
The study was carried out on 975 Polish Holstein-Friesian (Black & White strain) cows kept on one farm situated in the western part of Poland. The animals were born between 1998 and 2005, and came from 305 sires. The cows were fed standardized diets. They were milked twice a day, and their milk yield was evaluated using the A4 method according to ICAR (International Committee for Animal Recording) recommendations. Genomic DNA was extracted from the
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Table 1. The genotype and allele frequencies of the SNP studied
SNP Genotype, allele Frequencies Standard error
ANXA9 AA GA GG A G 0.5138 0.3867 0.0995 0.7072 0.2928 0.0068 0.0068 0.0068 0.0106 0.0106
FABP3 AA AG GG A G 0.0605 0.3908 0.5487 0.2559 0.7441 0.0060 0.0060 0.0060 0.0098 0.0098
FABP4 CC GC GG C G 0.5446 0.4133 0.0421 0.7513 0.2487 0.0055 0.0055 0.0055 0.0093 0.0093
cows' blood using the MasterPure™ Genomic DNA Purification Kit (Epicentre® Biotechnologies, Madison, USA) according to the procedure prescribed by the manufacturer.
The three following SNPs were analyzed: the AG transition in position 951 of the ANXA9 gene described by Calvo et al. [1], the AG transition in the FABP3 gene described by Wu et al. [8] and the GC transversion in the FABP4 gene described by Michal et al. [5]. The ANXA9 SNP is situated in the exon and it results in the H84R change in the mature protein. The FABP3 and FABP4 polymorphisms are located within non-coding gene sequences.
The genotypes were determined by the PCR-RFLP method. The ANXA9 genotypes were determined according to a previously developed method [9] by using primers as follows: 5'-TCC CAG ACC TTG TCA TTT CC-3' and 5'-CTC CTG GGA ATC AGT GTG GT-3'; and also by using the Nlalll restriction endonuclease. After digestion, the 241-bp amplicon revealed uncut fragments (allele G) and cut fragments were observed at 202 bp and 39 bp (allele A). The FABP3 and FABP4 genotypes were determined according to Wu et al. [8] and Michal et al. [5] respectively. Amplification of the desired gene fragments was performed with the following primer pairs: 5'-GTG AGT TGA GGA AGG GGC TGTG-3' and 5'-TAG GTC TCC ACC TCT TGT CCT TCA G-3', a 438 base pair-long fragment (for FABP3 polymorphism); and 5'-ATA TAG TCC ATA GGG TGG CAA AGA-3' and 5'-AAC CTC TCT TTG AAT TCT CCA TTC T-3', a 452 base pair-long fragment (for FABP4 polymorphism). The amplicons were then digested using the Acil and MspAI1 enzymes respectively. The re-
striction fragments obtained were described using the Vilber Lourmat® software for the photo documentation of electrophoretic separation, and image storage.
Statistical analysis was carried out to establish possible associations between the genotypes and the breeding values for milk yield (MY, kg), fat yield (FY, kg), fat content (FC, %), protein yield (PY, kg) and protein content (PC, %). Additionally index (I) was also taken into account; It is the sum of the FY (kg) and the doubled PY (kg). Breeding values for cows are expressed as lactational breeding values, which are obtained by adding up the breeding values for days 5 to 305. Variances in the second and third lactations are standardized to the variance in the first lactation, and then the mean lactational breeding value is calculated. This data was obtained from the official electronic documentation of the herd, and the data evaluation was carried out by the National Research Institute of Animal Production in Balice (Poland).
Association analysis was carried out as a regression of the EBVs for MY, FY, FC, PY and PC and the I of the ANXA9, FABP3 and FABP4 genotypes using the MIXED procedure implemented in SAS [10]. These associations were tested by using the i-test for multiple testing, including the Bonferroni correction for differences between means at the threshold 0.05. The following linear model was applied:
ttj = ^ + bi + Eij,
where y¡j — denotes the predicted breeding value of cow,
^ — the overall mean,
bi - the fixed effect of the SNP i-th genotypes ANXA9 (i = GG, GA, AA), FABP3 (i = AA, AG, GG) and FABP4 (i = GG, GC, CC),
Sij — an error.
The differences between the means were compared using Duncan's multiple range tests. We also considered models with the interaction effect between ANXA9*FABP3, ANXA9*FABP4, *FABP3*FABP4 and ANXA9*FABP3*FABP4.
RESULTS
Every possible SNP genotype was identified in the herd. The frequency of each genotype and allele is set out in Table 1. The highest frequency was observed for the ANXA9*A, FABP3*G and FABP4*C alleles.
The means of the EBVs for milk production traits in relation to the genotypes analyzed are given in Table 2. Statistical analysis revealed that the ANXA9 genotypes were associated with the breeding value for milk FC (%). This was also confirmed after the Bonferroni correction. The GG cows showed a significantly lower EBV for milk fat percentage than the GA (P < 0.01) and AA (P < 0.05) animals; this difference was 0.067% and 0.043% respectively.
FABP3, FABP4 AND ANXA9 SNP GENOTYPES
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Table 2. The means with standard error of the estimated breeding values for milk production traits in relation to the ANXA9, FABP3 and FABP4 genotypes
Genotype Trait
MY (kg) FY (kg) FC (%) PY (kg) PC (%) I
ANXA9
GG (n = 97) 363.54 (38.50) 9.08 (1.28) -0.085 (0.02)Aa 11.49 (1.16) -0.010 (0.01) 32.06 (3.39)
GA (n = 377) 282.04 (19.50) 10.35 (0.65) -0.018 (0.01)B 9.90 (0.58) 0.007 (0.01) 30.15 (1.72)
AA (n = 501) 299.06 (16.90) 9.44 (0.56) -0.042 (0.01)b 10.23 (0.51) 0.003 (0.01) 29.89 (1.49)
p--value 0.1686 0.4874 0.0034 0.4739 0.2461 0.8417
P-value Bonf 0.50 1 0.01 1 0.73 1
FABP3
AA (n = 59) 315.69 (49.39) 8.79 (1.63) -0.063 (0.02) 10.82 (1.48) 0.002 (0.00) 30.42 (1.44)
AG (n = 381) 278.50 (19.43) 8.40 (0.64)a -0.043 (0.01) 9.35 (0.58) 0.001 (0.00) 27.10 (1.70)
GG (n = 535) 311.57 (16.40) 10.83 (0.54)b 0.030 (0.01) 10.79 (0.49) 0.005 (0.00) 32.39 (1.44)
p-value 0.4041 0.0135 0.2850 0.1598 0.7759 0.0614
P-value Bonf 1 0.04 0.85 0.48 1 0.26
FABP4
GG (n = 41
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