RU2317704C1 - Method for detecting genetic potential by milk quality in cattle - Google Patents

Abstract

FIELD: agricultural biotechnology, selection of farm animals.

SUBSTANCE: genetic potential in cattle should be evaluated due to DNA-testing animals' genotypes by RsaI-marker of prolactin gene and AluI-marker of growth hormone gene and detecting conjugated genotypes by the loci of prolactin and growth hormone that detect either increased or decreased content of fat and protein in milk according to the elaborated list of genotypes. The innovation enables to carry out early selection of perspective heifers for dairy animal science and conclude upon the purposefulness of applying stud bulls before the appearance of lactating offspring in them and, also, match the pairs in selection work on improving dairy breeds.

EFFECT: higher accuracy of detection.

1 ex, 6 tbl

Description

The invention relates to agricultural biotechnology and breeding of farm animals, in particular to a method for determining the genetic potential of cattle (cattle) by the content (in%) of fat and protein in milk for the purpose of early selection of heifers for dairy farming and solving the question of the advisability of using bulls manufacturers before the appearance of lactating offspring, as well as for the selection of pairs in breeding work to improve dairy breeds.

There is a method of selecting heifers with the desired content of protein and fat in milk of future lactation by pedigree, general development and exterior, selection of bulls - by indicators of milk production of daughters. However, these traditional methods of selecting animals are ineffective, associated with high time and material costs.

The development of molecular genetic methods of analysis based on the polymerase chain reaction (PCR) has allowed the development of new marker systems that determine the genotypes of animals directly at the level of the genetic material of the cell (at the DNA level) regardless of gender and age, reduce analysis time and increase its accuracy.

There is also a method for evaluating the genetic potential of cows for milk fat based on the analysis of DNA polymorphism of the growth hormone gene (MspI polymorphism). However, the correlation of the MspI (-) - allele with milk fat is contradictory: according to some authors, the MspI (-) - allele correlates with a high fat content in milk [Falaki M., Gengler N., Prandi A. et al. Relationships of polymorphism for growth hormon and growth hormone reseptor genes with milk production for Italian Holstein-Friesian bulls // Journal of Dairy Science. 1993. V.76, p.149], according to other authors - with a low [Yao J., Aggerey SE, Zadworny D. et al. Sequence variations in the bovine gene characterized by single-strand conformation polymorphism (SSCP) analysis and their association with milk production traits in Holsteins // Genetics. 1996. V.144. p.1809-1816], which does not allow us to consider the proposed marker as a reliable marker of milk fat in cattle. The same contradictory picture is observed when using a genetically determined level of protein and fat in cow's milk as a method based on the Rsal-polymorphism of the prolactin gene and AluI-polymorphism of the growth hormone gene [Chrenek P., Huba J., Hetenyi L., Peskovieova D ., Bulla J. Genotypes of bGH and bPRL genes in relationships to milk production // Proceedings of the 50 ^th Annual Meeting of the EAAP. Zuerich. Book of Abstracts. 1999, p.40; Dybus A. Assaciations of growth hormone (GH) and prolactin (PRL) genes polymorphisms with milk production traits in Plish Black-and-White cattle // Animal Skiense Papers and Reports. 2002. V.20. N.4, p.203-212].

More promising methods for assessing the genetic potential of cows in terms of milk productivity, taking into account the combined influence of two or more genes involved in the formation of a sign of milk productivity.

As a prototype, a method for determining animals with improved milk production indicators was selected based on the analysis of gene polymorphism of the transcription factor Pit-1 and kappa-casein. Genotypes providing an increased level of milk yield, fat and protein content in milk were identified for individual loci: TT and AA genotypes for the Pit-1 gene and BB genotype for the kappa-casein gene, as well as conjugated genotypes for two loci: TTBB and AAVB [WO 2003076657, 09/18/2003 Renaville, Robert et al. Method for identifying animals for milk production qualities by analysing the polymorphism of the Pit-1 and kappa-casein genes]. However, the patent does not provide data on the productivity of groups of animals with heterozygous genotypes, absolute values of productivity parameters for groups of animals with homozygous genotypes are not given (data are presented only in relative terms, as the difference in productivity between groups of homozygous animals). The patent also does not assess the significance of differences between indicators of milk productivity in animals with economically valuable homozygous genotypes and other groups of animals. All of the above does not allow to evaluate the diagnostic value of the proposed marker system. In addition, marker genes (kappa-casein and transcription factor of growth hormone P / M) do not belong to the same metabolic chain, which does not exclude the random nature of the relationship of these genes revealed by the authors and casts doubt on the validity of considering their genotypes as conjugated.

The objective of the invention is the creation of an effective method for determining the genetic potential according to the parameters of milk productivity of cattle (fat and protein content) for early detection of promising animals in dairy farming and breeding, significantly reducing time and economic costs.

The essence of the invention is a method for determining the genetic potential of cattle by the level of fat and protein in milk using a test system based on the RsaI variation of exon III growth hormone gene and AluI variation on exon V of the prolactin gene, and the identification of conjugated genotypes associated with these parameters milk production. The use in the proposed method of genes of prolactin and growth hormone involved in the same metabolic system, namely in the formation of milk productivity, more adequately reflects the existing associations with the parameters of milk productivity of cattle in comparison with the prototype.

The RsaI restriction polymorphism in exon III of the prolactin gene (bPRL) is due to silent A-G transition occurring in the 103 codon of the amino acid and leading to the appearance of the polymorphic RsaI site. Restriction AluI polymorphism in exon V of the growth hormone gene (bGH) is due to the CG conversion, which leads to the amino acid substitution at position 127 (2141 nucleotide positions) of the amino acid leucine for the amino acid valine (Leu for Val, CTG codon for GTG codon) in the protein product and the disappearance of the AluI site (AG | CT) in the nucleotide sequence of the gene. The amino acid leucine corresponds to AluI (+) (L allele), and valine corresponds to AluI (-) (V allele).

The method is based on PCR followed by restriction analysis of amplification products (PCR-RFLP method) and includes DNA extraction, amplification of the exon III fragment of the prolactin gene and exon V fragment of the growth hormone gene using specific primers, restriction analysis of the obtained amplicons RsaI- and AluI- endonucleases, respectively, the establishment of genotypes for each gene, followed by the identification of conjugated genotypes for both genes. The resulting conjugated genotypes are compared with a list of established genotypes that determine the increased (or reduced) content of fat and protein in milk (Table 1).

Table 1. The list of genotypes that determine the increased or decreased content of fat and protein in cow's milk. Genotypes that determine the increased or decreased fat content in milk Genotypes for high or low protein in milk increased low increased low aaVV aaLL abVV abLL abLL abVV bbVL bbVV bbLL bbVV Notes: a and b are the alleles of the prolactin gene, RsaI (+) and RsaI (-), respectively; V and L are alleles of the growth hormone gene, AluI (-) and AluI (+), respectively;

The method is as follows.

1. DNA is isolated from blood samples (200 μl) of the animal using the DIAtom ^™ DNA Prep reagent kit (Biocom, Moscow) or any other standard method. It is possible to use other tissues or animal cells (hair follicles, sperm, etc.).

2. Amplification of a fragment (156 bp) of exon III of the bPRL gene is carried out using primers PRL-1 (5'-CGAGTCCTTATGAGCTTGATTCTT-3 ') and PRL-2 (5'-GCCTTCCAGAAGTCGTTTGTTTTC-3') [Mitra A., Schte P., Balakrishnan CR, Pirshner F. Polymofphisms at growth hormone and prolactin loci in Indian cattle and buffalo // Journal of Animal Breeding and genetics. 1995. V.112, p.71-74] in the following mode: preliminary denaturation at 94 ° C for 4 min; 35 cycles according to the scheme: denaturation at 94 ° C for 30 s, annealing at 58 ° C for 30 s, synthesis 72 ° C for 30 s; final synthesis at 72 ° С - 7 min.

The amplification of the fragment (223 nucleotides) of the bGH gene is carried out using primers P1: 5'-GCTGCTCCTGAGGGCCCTTCG-3 'and P2: 5'-GCGGCGGCACTTCATGACCCT-3' in the following mode: preliminary denaturation at 94 ° С - 4 min, 30 cycles: C - 45 s, 67 ° C - 45 s, 72 ° C - 45 s; final synthesis at 72 ° C for 7 min [Mattos K.K., Lama S.N.D., Martinez M.L.M., Freitas A.F. Association of bGH and Pit-1 gene variants with milk production traits in dairy Gyr bulls // Pesq. agropec. bras. 2004. V.39. No. 2, p. 147-150; Khatami S.R., Lazebny O.E., Maksimenko V.F., Sulimova G.E. DNA polymorphism of growth hormone and prolactin genes in Yaroslavl and black-motley cattle in connection with milk production // Genetics. 2005.V.41. N 2, p. 229-236].

The size of the obtained fragments is determined by electrophoresis in 2% agarose gel.

3. The amplified fragment of exon III of the bPRL gene is restricted by the restriction endonuclease RsaI, and the bGH gene fragment is restricted by AluI restriction enzyme under standard conditions.

4. The size of the obtained restriction fragments is determined by electrophoresis in 6% polyacrylamide gel.

5. Determine the genotypes for each gene (according to table 2) and then the conjugated genotypes for both genes, possible variants of which are given in table 3.

Table 2. Genotyping of individuals according to the results of electrophoresis separately for each gene Sizes of fragments (bp) PCR-RsaI genotypes of exon III prolactin gene aa ab bb 156 156
82
74 82
74 PCR-AluI genotypes of exon V of the bGH gene Vv Vl LL 223 223
171
52 171
52

Table 3. The list of possible conjugated genotypes for loci of prolactin and growth hormone * aa vv ab VV bb vv aa vl ab VL bb VL aa LL ab LL bb LL

6. Compare conjugated genotypes detected in animals with genotypes that determine the increased or decreased content of fat and protein in milk (according to Table 1).

7. Decide on the advisability of using an animal with this conjugate genotype in dairy production and breeding.

Example 1. This method was used to analyze cows of the Yaroslavl breed (113 individuals from the farms "Mikhailovsky" and "Gorshikha" of the Yaroslavl region). Animal genotypes were determined for individual loci (prolactin and growth hormone) and conjugated genotypes for both loci.

The effect of prolactin (RsaI-marker) and growth hormone (AluI-marker) genes, as well as their combined effect (RsaI * AluI) on the parameters of milk production of Yaroslavl cattle was evaluated by two-way analysis of variance analysis (Table 4).

The value of the confidence level indicates the presence or absence of the influence of the studied factors (in this case, genotypes at individual loci and conjugate genotypes) on the parameters of milk productivity. Reliable values (shown in table 4 in bold) were obtained when assessing the effect on the content of protein and fat in milk only for conjugated genotypes. According to the genotypes of the separately examined genes, no reliable dependence was revealed. The influence of genotypes in individual studied loci and their conjugated genotypes on milk production of Yaroslavl cattle was also not revealed. In this regard, in further calculations, only the effect of certain conjugated genotypes on the content (in%) of protein and fat in cow's milk was considered.

A comparative analysis of the milk production parameters of the studied animals with different conjugated genotypes showed significant differences in the fat content (%) in milk between animals with genotypes 1 and 2 (see Table 5) and a group of animals with genotypes 3 and 4, and in protein content ( %) - between animals with conjugated genotypes indicated in the table by the numbers 1 *, 2 *, Z * and the group of genotypes 4 *, 5 *, 6 *. Based on the results obtained, groups of genotypes with increased and decreased indices of the studied characters were established, significantly differing from each other (Table 5).

Table 5. Significance of differences between the genotype determining the increased value of the sign of productivity (fat and protein in%) and the group of genotypes determining their low content Genotypes that determine the increased or decreased fat content in milk Genotypes for high or low protein in milk increased low increased low aaVV (1 *) P _{1 * (4 *, 5 *, 6 *)} = 0.017 aaLL (4 *) abVV (1) P _{1 (3, 4)} = 0.008 abLL (3) abLL (2 *) P _{1 * (4 *, 5 *, 6 *)} = 0.008 abVV (5 *) bbVL (2) P _{2 (3, 4)} = 0.009 bbVV (4) bbLL (3 *) P _{1 * (4 *, 5 *, 6 *)} = 0.020 bbVV (6 *) Note: P - significance of differences between the genotype, which determines the increased value of the productivity parameter (fat, protein) with the group of genotypes, which determine their low content, calculated using the F-criterion by the method of planned comparisons (analysis of contrasts).

Table 6 shows the average values of fat content (%) and protein (%) in cattle milk and their standard errors for these groups of genotypes.

Table 6. Average values (%) of fat and protein in cattle milk with different genotypes for prolactin and growth hormone loci with standard error Genotypes that determine the fat content in milk Genotypes that determine the protein content in milk increased low increased low aaVV aaLL 3.42 ± 0.08 3.22 ± 0.06 abVV abLL abLL abVV 4.72 ± 0.13 4.29 ± 0.09 3.39 ± 0.05 3.22 ± 0.08 bbVL bbVV bbLL bbVV 4.72 ± 0.13 4.26 ± 0.18 3.47 ± 0.10 3.18 ± 0.10

According to the data of table 6 in animals with genotypes that determine high fat content (%) in milk, the average values of this indicator differ from those in animals with genotypes that determine low fat content in milk by about four tenths of a percent, which is significant when calculating milk volume for all lactations. In terms of protein content (%), the difference between genotypes that determine its increased and decreased content in milk is about two tenths of a percent, which is also economically significant [Zhou G.L, Jin H.G., Liu C., Guo S. L., Zhu Q. and Wu Y.H. Association of genetic polymorphism in GH gene with milk production traits in Beijing Holstein cows // J. Biosci. 2005. V.30, p. 595-598; Zwierzchowski L., Krzyzewski J., Strzalkowska N., Siadkowska E., Ryniewicz Z. Effect of polymorphism of growth hormon (GH), Pit-1, and leptin (LEP) genes, cow's age, lactation stage and somatic cell count on milk yield and composition of Polish Black-and-White cows // Animal Science Papers and Reports. 2002. V.20. N4, p. 213-227].

Thus, conjugated genotypes were identified by loci of prolactin and growth hormone genes that determine the increased and decreased content of fat and protein in cattle milk. Using the proposed method allows to evaluate their potential in the early stages of animal life by signs of milk productivity (fat and protein), which significantly accelerates the selection of animals during the formation of highly productive milk herds, and also increases the efficiency of breeding work to improve milk breeds.

Claims (1)

A method for determining the genetic potential of cattle by milk quality, including DNA extraction, amplification using specific primers, restriction analysis of the resulting amplicons with the establishment of genotypes separately for each gene, and then the conjugated genotypes for both genes, which are compared with established genotypes that determine milk quality characterized in that as primers use primers specific for a fragment of exon III of the prolactin gene and a fragment of exon V of the hormone gene growth, the analysis of the resulting amplicons is carried out by RsaI and AluI restriction enzymes, respectively, and the genotypes abVV, bbVL and aaVV, abLL, bbLL, respectively, are used as the identified genotypes, which are responsible for the increased content of fat and protein in milk, and genotypes that cause a reduced content of fat and protein in milk, the genotypes abLL, bbVV and aaLL, abVV, bbVV, respectively.