RU2716116C1 - METHOD FOR CATTLE GENOTYPING BY 1401G/T ALLELES lhcgr (ss52050737) GENE BY PCR IN REAL TIME - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, molecular-genetic diagnostics, genetics and breeding of farm animals. Method of genotyping cattle by 1401G/T alleles of lhcgr gene (ss52050737) is carried out by PCR method in real time using forward primer 5'-TGAACTCTCTGTCTACACCCTCACA-3', reverse primer 5'-GCATGACTGGAATGGCATGTT-3', lhcgr-T-allele-specific fluorescently-labeled probe 5'-(FAM)-CACTAGAAAGATGTCACACC-(BHQ1)-3', lhcgr-G-allele-specific fluorescently-labeled probe, 5'-(VIC)-CTAGAAAGATGGCACACC-(BHQ1)-3', wherein for homozygous samples on allele T (genotype 1401T/T) fluorescence increase is detected on FAM channel, for homozygous samples on allele G (genotype 1401G/G) signal is detected via VIC channel, for heterozygous sample (genotype 1401T/G) there is an increase fluorescence on both detection channels, and the presence of two dyes, FAM and VIC, enables unambiguous detection of the presence of each of the analyzed alleles of the lhcgr gene in the analyzed DNA sample and, accordingly, the animal's genotype.

EFFECT: invention simplifies the method of cattle genotyping by allelic versions 1401G/T (ss52050737) of the lhcgr gene, and reduces the analysis time.

1 cl, 2 dwg

Description

The invention relates to the field of biotechnology, molecular genetic diagnosis, genetics and breeding of farm animals, in particular to the assessment of the 1401G / T allelic polymorphism (ss52050737) of the lhcgr gene encoding the luteinizing hormone / choriogonadotropin receptor (luteinizing hormone / choriogonadotropinGRorhor), molecular genetic research method.

The study of the process of embryo transplantation began about a hundred years ago and already in the 1970s passed into the commercial sphere of activity at the international level. So, in 2016, 632,638 embryos were received from 93,815 donor cows, of which 196 thousand were transplanted immediately, and the rest was cryopreserved. For many years, North America has remained the leader in the field of embryo transplantation - 52.5% of the total number of embryos received in vivo was obtained in the USA and Canada, while in Europe only 20.4% [G.A. Waugh, R.J. Mapletoft, "Embryo Transfer Technology in Cattle", Animal Biotechnology 1. - Springer, Cham, pp. 107-133. 2018; G. Perry. "Statistics of embryo collection and transfer in domestic farm animals", Embryo Transfer Newsletter, vol. 32, pp. 14-26. 2014]. Due to the widespread use of technology for embryo transplantation, it became necessary to select donor cows according to genetic indicators of sensitivity to the procedure for stimulating ovulation with gonadotropic hormones for further assessment by the number of mature oocytes received [G.A. Waugh, R.J. Mapletoft, "Historical perspectives and recent research on superovulation in cattle", Theriogenology, vol. 81, No. 1, pp 38-48. 2014]. The superovulation process is based on the administration of gonadotropic drugs to animals, one of which is luteinizing hormone [W.K. Kroeze, D.J. Sheffler, B.L. Roth, "G-protein-coupled receptors at a glance", Journal of cell science, vol. 116, No. 24, pp. 4867-4869, 2003].

The luteinizing hormone / choriogonadotropin receptor (LHCGR) belongs to the class of membrane receptors associated with G-proteins [M.L. Dufau, S.N. Tsai-Morris, Z.Z. Hu, E. Buczko, "Structure and regulation of the luteinizing hormone receptor gene", The Journal of steroid biochemistry and molecular biology, vol. 53, No. 1-6, pp. 283-291, 1995]. In cattle (RED), the LHCGR gene is located on chromosome 11 and consists of 11 exons [O.J. Ginther, D.R. Bergfelt, M.A. Beg, K. Kot, "Follicle selection in cattle: role of luteinizing hormone", Biology of reproduction, vol. 64, No. 1, pp. 197-205, 2001]. The polymorphism of this gene may be closely related to the process of superovulation [W.C. Yang, K.Q. Tang, S.J. Li, L.M. Chao, L.G. Yang, "Polymorphisms of the bovine luteinizing hormone / choriogonadotropin receptor (LHCGR) gene and its association with superovulation traits", Molecular biology reports, vol. 39, No. 3, pp. 2481-2487, 2012; Y. Yu, Y. Pang, H. Zhao, X. Xu, Z. Wu, L. An, J. Tian, "Association of a missense mutation in the luteinizing hormone / choriogonadotropin receptor gene (LHCGR) with superovulation traits in Chinese Holstein heifers ", Journal of animal science and biotechnology, 3:35. 2012]. The lhcgr gene is considered as a candidate gene for predicting a response to superovulation induction. And the polymorphic variants identified in the gene are considered in connection with cattle fertility [N. Hastings, S. Donn, K. Derecka, A.P. Flint, J.A. Woolliams, "Polymorphisms within the coding region of the bovine luteinizing hormone receptor gene and their association with fertility traits", Animal genetics, vol. 37, No. 6, pp. 583-585, 2006; S.D. Cochran, J.B. Cole, D.J. Null, P.J. Hansen, "Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle", BMC genetics, 14: 49,2013; W.K. Santos-Biase, F.H. Biase, J.Jr. Buratini, J. Balieiro, Y.F. Watanabe, M.F. Accorsi, C.R. Ferreira, P. Stranieri, A.R. Caetano, F.V. Meirelles, "Single nucleotide polymorphisms in the bovine genome are associated with the number of oocytes collected during ovum pick up", Animal reproduction science, vol. 134, No. 3-4, pp. 141-149, 2012; H. Berg, "Restriction Fragment Length Polymorphism Analysis of PCR-Amplified Fragments (PCR-RFLP) and Gel Electrophoresis - Valuable Tool for Genotyping and Genetic Fingerprinting", in: Gel Electrophoresis - Principles and Basics: IntechOpen, 315-334, 2012] . The most significant single nucleotide substitutions (SNPs) are nonsynonymous mutations located in the exons of the gene, since they lead to the replacement of amino acid residues, which in turn changes the primary structure of the protein and can affect its function. Of all the SNPs found to date, the most interesting is the replacement 1401G> T (ss52050737), since it was found that animals with the GG or GT genotypes are characterized by higher rates in the total number of eggs, and in the number of surviving embryo transplants, the best indicators are in animals with the genotype GG. Also, animals with the GG genotype have the smallest number of unfertilized eggs compared to carriers of two other genotypes [N. Hastings, S. Donn, K. Derecka, A.P. Flint, J.A. Woolliams, "Polymorphisms within the coding region of the bovine luteinizing hormone receptor gene and their association with fertility traits", Animal genetics, vol. 37, No. 6, pp. 583-585, 2006]. Thus, the lhcgr gene encoding LHCGR is one of the promising genetic markers of the reproductive status of cattle.

The prior art method for the genotyping of cattle by the 1401G / T alleles (ss52050737) of the lhcgr gene, providing their identification based on a combination of three methods - polymerase chain reaction (PCR), analysis of conformational polymorphism of single-stranded DNA based on electrophoretic separation of the obtained polymorphic fragments of the lhcgrr gene (single-stranded conformation polymorphism - SSCP) and direct DNA sequencing (prototype - Y. Yu, Y. Pang, N. Zhao, X. Xu, Z. Wu, L. An, J. Tian, "Association of a missense mutation in the luteinizing hormone / choriogonadotropin receptor gene (LHCGR) with superovulation traits in Chinese Holstein heifers ", Journ al of animal science and biotechnology, 3:35. 2012]. The method consists in the fact that in order to detect 1401G / T alleles of the lhcgr gene (ss52050737), at the first stage of the analysis, the lhcgr gene region containing them is amplified by PCR using sense: 5 'primers -GCTCTACCTGCTGCTCAT-3 'and anti-sense: 5'-TAATTGCTGACACCCACA-3'. The resulting amplicons were then analyzed by SSCP, namely, mixed with 8 μl of denaturing solution (95% formamide, 25 mmol / L EDTA, 0.025% xylene-cyanol, 0.025% bromophenol blue), heated for 10 min at 98 ° C and cooled on ice. Denatured DNA was subjected to electrophoresis in 13% polyacrylamide gel (39: 1 acrylamide / bisacrylamide) in 1 × TBE buffer at constant voltage (120 V) for 8-10 hours. The gel was stained with 0.1% silver nitrate solution. At the last stage, PCR products with different electrophoretic mobility are sequenced in both directions.

The main disadvantages of the prototype are the multi-stage method for cattle-assisted allelic variants 1401G / T (ss52050737) of the lhcg gene based on a combination of three methods (amplification of the polymorphic region of the gene by PCR, electrophoretic separation of the obtained polymorphic fragments of the lhcgr gene, DNA sequencing, its duration) labor input.

The technical result provided by the invention is to simplify the method of genotyping cattle according to the allelic variants of 1401G / T (ss52050737) of the lhcgr gene, in reducing the time of analysis.

The technical result is achieved by the fact that the method of genotyping cattle by the 1401G / T alleles of the lhcgr gene (ss52050737) by real-time PCR consists in the following:

direct primer 5'-TGAACTCTCTGTCTACACCCTCTCACA-3 '

reverse primer 5'-GCATGACTGGAATGGCATGTT-3 '

lhcgr-T-allele-specific fluorescently-labeled probe

5 '- (FAM) -CACTAGAAAGATGTCACACC- (BHQ1) -3'

lhcgr-G - allele-specific fluorescently-labeled probe

5 '- (VIC) -CTAGAAAGATGGCACACC- (BHQ1) -3'

The list of graphic materials.

In FIG. 1 shows the nucleotide sequences of a 113 bp lhcgr gene fragment encoding 1401G / T alleles (ss52050737). Primer sequences are marked in bold; in bold and italics are sequences of probes.

In FIG. 2 presents: A - fluorescence curves for homozygous samples along the T allele (TT genotype). B - fluorescence curves for homozygous samples along the G allele (genotype GG). C - fluorescence curves for heterozygotes (TG genotype). D is a graph of the distribution of genotypes (ss52050737) of the lhcgr gene.

The patented method consists in real-time PCR for genotyping cattle according to the allelic variants of 1401G / T (ss52050737) of the lhcgr gene. The claimed method differs in that two common primers for allelic variants are used: the forward primer lhcgr-F: 5'-TGAACTCTCTGTCTACACCCTCTCACA-3 'and the reverse primer lhcgr-R: 5'-GCATGACTGGAATGGCATGTT-3' flanking the 113hr gene. n. and two allele-specific fluorescently-labeled probes lhcgr-T: 5 '- (FAM) -CACTAGAAAGATGTCACACC- (BHQ1) -3' and lhcgr-G: 5 '- (VIC) -CTAGAAAGATGGCACACC- (BHQ1) '(Fig. 1).

The mutation points are the same as in the prototype, however, the detection approach is different: in the claimed invention polymorphism is identified by real-time PCR using fluorescently-labeled allele-specific probes.

In the patented method for identifying 1401G / T allelic variants (ss52050737) of the lhcgr gene based on real-time PCR, two primers are used that are common to both alleles of the gene and two allele-specific fluorescently-labeled probes of the TaqMan type (Fig. 1). Primers lhcgr-F and lhcgr-R initiate amplification of a 113 bp length section of the cattle lhcgr gene The amplification reaction was carried out in 10 μl of a PCR mixture containing 5 μl of LightCycler® 480 Probes Master reagent (Roche, Switzerland), 0.4 μM lhcgr-F: 5′-TGAACTCTCTGTCTACACCCTCACA-3 ′ forward primer and lhcgr-R: 5 reverse primer '-GCATGACTGGAATGGCATGTT-3', 0.2 μM allele-specific probes lhcgr-T: 5 '- (FAM) -CACTAGAAAGATGTCACACC- (BHQ1) -3' (0.2 μM) and lhcgr-G: 5 '- (VIC) -CGCAGA - (BHQ1) -3 ', 10 ng of DNA. PCR was performed using a LightCycler 96 instrument (Roche, Switzerland) under optimized conditions (95 ° C for 10 min; 95 ° C for 15 sec., 58 ° C for 30 sec., 72 ° C for 20 sec., 40 cycles ) Fluorescence detection was carried out at the stage of elongation through the FAM and VIC channels. To analyze the results of endpoint genotyping, the LightCycler® 96 version SW1.1 software was used. The identification of the 1401G / T alleles (ss52050737 polymorphism) is based on a comparison of the fluorescence intensities of the VIC and FAM dyes, respectively. To determine the genotype of the animal, the final fluorescence values of the FAM and VIC dyes are used. LightCycler® 96 Amplifier Software Version SW1.1. presents the results of genotyping in the form of a distribution of genotypes (Fig. 2D). For homozygous samples, the T allele (genotype 1401T / T) shows an increase in fluorescence through the FAM channel (Fig. 2A). For homozygous samples, a signal is detected on the G allele (genotype 1401G / G) via the VIC channel (Fig. 2B). For a heterozygous sample (genotype 1401T / G), an increase in fluorescence is observed along both detection channels (Fig. 2C). The presence of two dyes, FAM and VIC, make it possible to unambiguously determine the presence of each of the studied alleles of the lhcgr gene in the analyzed DNA sample and, accordingly, the genotype of the animal.

The developed method based on real-time PCR was validated on 195 DNA samples of black-mottled Holsteinized cattle. Genotyping results showed that 43.2% of the animals were carriers of both alleles (GT genotype), 15.2% of the cows were homozygotes for the T allele (TT genotype) and 41.6% of animals were carriers of the G allele homozygotes (GG genotype). Thus, in the studied cattle population, the frequency of the G allele, associated with higher rates of the total number of oocytes and the number of surviving embryos after transplantation, as well as with the lowest number of unfertilized oocytes, was 63.2%.

Validation of the method was performed using the prototype assay (Y. Yu, Y. Pang, N. Zhao, X. Xu, Z. Wu, L. An, J. Tian, "Association of a missense mutation in the luteinizing hormone / choriogonadotropin receptor gene (LHCGR) with superovulation traits in Chinese Holstein heifers ", Journal of animal science and biotechnology, 3:35. 2012). The results of both genotyping methods are completely consistent. However, the patented method allows to simplify and significantly (up to 1 hour) reduce the analysis time by simultaneously amplifying the polymorphic region of the lhcgr gene and detecting its 1401G / T allelic variants (ss52050737) by real-time PCR, which distinguishes it from analysis used in the prototype.

Thus, an effective and reliable method has been developed for the rapid identification of 1401G / T allelic variants (ss52050737) of the bovine lhcgr gene by real-time PCR using allele-specific fluorescently-labeled probes. The developed method allows genotyping of up to 480 animals according to the 1401G / T allelic variants (ss52050737) of the lhcgr gene (depending on the model of the amplifier) for 1 hour and can be used to select promising donor cows for embryos.

Claims (7)

Method for genotyping cattle by the 1401G / T alleles of the lhcgr gene (ss52050737) by real-time PCR, characterized in that they are used: direct primer 5'-TSAASTSTSTSTSTASASSSTSASA-3 ', reverse primer 5'-GCATGACTGGAATGGCATGTT-3 ', lhcgr-T-allele-specific fluorescently-labeled probe 5 '- (FAM) -CACTAGAAAGATGTCACACC- (BHQ1) -3', lhcgr-G-allele-specific fluorescently-labeled probe 5 '- (VIC) -CTAGAAAGATGGCACACC- (BHQ1) -3'.

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