ГЕНЕТИКА, 2009, том 45, № 7, с. 926-931
ГЕНЕТИКА ЖИВОТНЫХ
УДК 575.1
STUDY OF THE GENETIC STRUCTURE OF DAIRY CATTLE BASED ON POLYMORPHISM WITHIN THE AROMATASE GENE
© 2009 I. Kowalewska-Luczak
Department of Genetics and Animal Breeding, Westpomeranian University of Technology, Szczecin 71-466, Poland
e-mail: inga.kowalewska-luczak@zut.edu.pl Received September 12, 2007
The study induced 1083 Polish Holstein-Friesian strain Black-and-White cows. The genetic structure of the herd was determined on the basis of polymorphism within the aromatase gene (CYP19/Cfr 13I and CYP19/PvuII). Genotypes were identified by the PCR-RFLP method. The CYP19/Cfr 13I allele frequencies were as follows: A - 0.86 and B - 0.14. The CYP19/PvuII allele frequencies were as follows: A - 0.91 and B - 0.09. The highest average heterozygosity rate was found in herd A (0.2108). The largest genetic distance was between cows kept in farms A and C (0.00103).
Reproduction and lactation are closely connected and therefore all the physiological processes and hormones affecting the development of the reproductive system also affect lactation. Such broad-spectrum hormones include estrogens synthesized in the ovaries. These hormones prepare the structural determinants of the female reproductive system to reproduction by stimulating the maturation of primary reproductive cells, securing hormone levels appropriate for the ovulation process, creating environment necessary for pregnancy maintenance and inducing parturition and lactation [1]. Estrogens promote lobular proliferation in the mammary gland. Estrogen is formed by androgen aromatization [2].
The key enzyme responsible for the biosynthesis of estrogens from androgen precursors is an enzymatic complex known as aromatase [3]. Aromatase is encoded by the CYP19 gene, which is located in cattle on chromosome 10 [4]. The CYP19 gene expression is regulated by tissue-specific alternative promoter regions. Different promoter regions correspond to different untranslatable sequences (5'-UTR) but the encoding region is identical for all tissues where CYP19 expression takes place [5].
Within the CYP19 gene in cattle, several single nucleotide polymorphisms (SNP) were found, which were located mainly in the promoter regions. Namely, the P1.1 region contains three SNPs detectable by restriction enzymes PvuII, Cfr13I and BseNI whilst the P1.2 region contains two SNPs detectable by enzymes BseNI and TaiI [6, 7].
Based on an analysis of the current state of knowledge regarding aromatase, this study aimed to define the genetic structure of a herd of dairy cows on the basis of a polymorphism within the aromatase encoding gene.
MATERIALS AND METHODS
The study included 1083 Polish Holstein-Friesian strain Black-and-White cows kept in the West Pomera-nia region, Poland. The cows were kept in five herds: A (185 individuals), B (170 individuals), C (373 individuals), D (123 individuals) and E (232 individuals). The genotypes of particular individuals were determined by the PCR-RFLP method. The RFLP analysis included two promoter region fragments of the cytochrome P450 aromatase gene. The two polymorphic sites are located in the P I.1 region at positions 1044 (CYP19/PvuII) and 1902 (CYP19/Cfr13I). In both cases the polymorphism consists in a G —- A substitution. The positions of the polymorphic sites are based on the P I.1 sequence no. Z69241 (the European Molecular Biology Laboratory database).
The first aromatase gene fragment (CYP19/PvuII) of 288 base pairs (bp) was amplified using a pair of primers with the following nucleotide sequences: 5'-GCA TGG GCA CTT GCT CTC GAT-3' and 5'-TGA TTT CCA GGT TGT TAA GTG AAT GA-3' [6]. The sequences of the primers used to amplify the other 283-bp-long CYP19 gene fragment (CYP19/Cfr13I) were as follows: 5'-AAA GGC CGG TAT TGC TGC ATT T-3' and 5'-CGC AAG TTC CTC CAA GGC AAA T-3' [6].
The PCR reaction mix of 20 ^l contained Taq poly-merase buffer, 200 mM nucleotide mix, 2 mM MgCl2, 10 pM each primer (appropriate for a particular amplification reaction), 70-100 ng DNA, 0.5 U thermostable Taq polymerase and deionized water to volume.
The amplification reactions for both CYP19 gene fragments were performed in thermal cyclers by Whatman Biometra (TGradient, T3) under the following temperature profile: initial denaturation at 94°C for 2 min followed by 30 cycles of proper denaturation for 15 s at 94°C, primer annealing for 30 s at 55°C and DNA chain extension for 2 min at 70°C and final extension for 5 min also at 70°C. The PCR products were an-
alyzed electrophoretically on a 2% agarose gel containing ethidium bromide (0.5 Mg/ml) in lxTBE buffer at 90 V using DNA marker pUC19/MspI. In the next stage of the analysis, the obtained amplification products were digested with appropriate restriction enzymes at 37°C for at least 3 hours. The 288-bp fragment was digested with restrictase PvuII whilst the 283-bp fragment was digested with restrictase Cfr13I. The resulting restriction fragments were separated on a 2.5% agarose gel containing ethidium bromide (0.5 Mg/ml) in lxTBE buffer at 90V using DNA marker pUC19/MspI. All the obtained PCR products and restriction fragments were visualized and documented using an elec-trophoresis gel documentation and analysis system by Vilber Lourmat.
The obtained genotyping results were analyzed statistically. To define the genetic structure of the population under study, the following were calculated:
- the frequencies of the CYP19 genotype for both sites in the gene and their expected distributions,
- the frequencies of particular alleles,
- the frequencies of homo- and heterozygous genotypes and their expected frequencies, and
- homozygous mean coefficient and heterozygous mean coefficient for the herds.
The frequencies of particular genotypes and alleles were compared across the herds using the x2 test. On the basis of the allele frequencies, the genetic distance among the studied herds was estimated according to the following formula [8]:
2 2 1/2
Djk = -log e[(Z qijqik) / (Sq^) ],
i i i
where Djk - genetic distance between populations j and k; qij and qik - frequencies of allele i (allele A or B both polymorphism) in populations j and k.
All the calculations regarding the genetic structure of the population under study were made using the STAT-GeN calculation software package - version 1.2 [9].
Fig. 1. The bovine CYP19/Cfr13I product and genotypes observed. M - DNA marker pUC19/MspI, 1 - PCR product, 2 -homozygote AA, 3 - heterozygote AB, 4 - homozygote BB.
RESULTS
As a result of a restriction analysis of the 283-bp fragment with restrictase Cfr13I, the following three genotypes were identified: AA (235-bp and 48-bp fragments), AB (283-bp, 235-bp and 48-bp fragments) and BB (283-bp fragment). The occurrence of these three genotypes was controlled by two alleles: allele A (two restriction fragments of 235 bp and 48 bp) and allele B (no site recognized by enzyme Cfri3I due to a mutation) - Figure 1.
Table 1 shows the frequencies of individual CYP19/Cfr13I genotypes and alleles. The most frequent genotype in the studied herds of Polish Holstein-Friesian strain Black-and-White cows was genotype AA (0.74). The other two genotypes were much less frequent: AB - 0.23 and BB - 0.03.
Statistically significant differences (P < 0.05, P < 0.01) were found in the CYP19/Cfr13I genotype frequencies among individuals from particular herds. Genotype AA was found to be most frequent in herd D (0.86) and least frequent in herd B (0.63). The highest frequency of the heterozygous genotype was found in herd B (0.31) whilst the lowest in herd D (0.14). The
Table 1. Frequencies of alleles and genotypes and observed (ob) and expected (ex) numbers genotypes of CYP19/Cfr13I in studied herds of cows
Alleles Genotypes
Herd n A B AA AB BB
freq. ob ex S freq. ob ex S freq. ob ex S
A 185 0.82AB 0.18AB 0.67ab 124 123.2 n.s. 0.29AB 54 55.5 n.s. 0.04 7 6.2 n.s.
B 170 0.79a 0.21a 0.63CDE 108 105.6 n.s. 0.31CD 52 56.8 n.s. 0.06a 10 7.6 n.s.
C 373 0.85abC 0.15abC 0.73aCF 271 268.6 n.s. 0.24a 91 95.9 n.s. 0.03 11 8.6 n.s.
D 123 0.93AC 0.07AC 0.86ADF 106 106.6 n.s. 0.14AaC 17 15.8 n.s. 0.00 - 0.6 n.s.
E 232 0.90Bb 0.10Bb 0.81aBE 187 186.5 n.s. 0.18BD 42 43.0 n.s. 0.01a 3 2.5 n.s.
Total 1083 0.86 0.14 0.74 796 790.5 0.23 256 267 0.03 31 25.6
Notes (for Tables 1-4). Frequencies in columns with the same letter differ significantly; small letters P < 0.05, capitals letters < 0.01, n - number of individuals, S - significance of difference between observed (ob) and expected (ex) numbers genotypes, n. s. - non significant.
Table 2. Frequencies and observed (ob) and expected (ex) numbers of homo- and heterozygous genotypes of the CYP19/Cfr13I in analyzed herds of cows
Herd n Homozygotes Heterozygotes
frequency ob ex S frequency ob ex S
A 185 0.71AB 131 129.5 n.s 0.29AB 54 55.5 n.s
B 170 0.69CD 118 113.2 n.s 0.31CD 52 56.8 n.s
C 373 0.76a 282 277.1 n.s 0.24a 91 95.9 n.s
D 123 0.86AaC 106 107.2 n.s 0.14AaC 17 15.8 n.s
E 232 0.82BD 190 189.0 n.s 0.18BD 42 43.0 n.s
Total 1083 0.77 827 816.0 0.23 256 267.0
homozygous BB genotype was most frequent in herd B (0.06) and least frequent in herd E (0.01). No homozy-gous BB genotype was found in herd D.
In each of the studied herds of dairy cows, allele A was more frequent (0.86) than allele B (0.14). The highest frequency of allele A was characteristic of herd D (0.93) whilst the lowest frequency of this allele was found in herd B (0.79).
It was established that the herd of cows under study was in genetic equilibrium as the number of individuals observed in each CYP19/Cfr13I genotype group was not statistically significantly different compared with their expected number calculated according to the Har-dy-Weinberg law (Table 1).
An analysis of the studied herds in terms of homo-and heterozygous genotype frequencies showed that the homozygous genotypes were most frequent i
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