RU2708559C1 - Method for real-time pcr for cattle genotyping by alleles 337c/g of fshr (rs43745234) gene - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention represents a real-time PCR method for cattle genotyping by alleles 337C/G of fshr (rs43745234) gene, consists in the following: forward primer 5'-GAATTGAAAAGGCCAACAACC-3', reverse primer 5'-CACCAGAATACAGAAGTTCTTACAGA-3', FSHR337C – allele-specific fluorescent-labeled probe, -5'-FAM-CATCGACCCTGATGCC BHQ1-3', FSHR337G – allele-specific fluorescent-labeled probe, 5'-VIC-CATCGACGCTGATGC-BHQ1-3'.EFFECT: invention increases reliability of detecting alleles of 337C/G (rs43745234) of the fshr gene, simplifies analysis and reduces the length of time to 1,5 hours.1 cl, 2 dwg

Description

The invention relates to the field of biotechnology, molecular genetic diagnostics, genetics and breeding of farm animals, in particular to the assessment of the 337C / G (rs43745234) allelic polymorphism of the fshr gene encoding the bovine follicle-stimulating hormone (FSHR) receptor by the molecular genetic research method.

One of the key steps in cattle accelerated reproduction technology is the selection of donor cows that are most sensitive to the hormonal stimulation of ovulation and are able to produce, as a result, the maximum number of mature oocytes [Bó G.A., Mapletoft R.J. Historical perspectives and recent research on superovulation in cattle. Theriogenology. 2014. vol. 81. no. 1. P. 38-48. doi: 10.1016 / j.theriogenology. 2013.09.020.]. Despite the fact that in recent decades a number of new high-tech methods have been introduced into the technology of embryo transplantation, the efficiency of the superovulation stimulation procedure in donor cows has not significantly increased. One of the main limiting factors in the development of technology is the high variability of the response from the body of donor cows to hormonal processing. The physiological effect of treating donor cows with gonadotropins is still individual and unpredictable [Palubinskas G. et al. Superovulatory response in relation to the size and side of ovary location in high yielding dairy cows on the first day of treatment protocol. Veterinarski arhiv. 2016. vol. 86. no. 1. P. 65-76]. However, studies have shown that the effectiveness of ovulation stimulation is closely associated with allelic variants of the FSHR gene encoding the follicle-stimulating hormone receptor [Fauser B., Diedrich K., Devroey P. Predictors of ovarian response: Progress towards individualized treatment in ovulation induction and ovarian stimulation. Human Reproduction Update. 2008. vol. 14. no. 1. P. 1-14. doi: 10.1093 / humupd / dmm034].

FSHR belongs to the class of membrane receptors associated with G proteins, whose activation triggers the signaling pathway of cyclic adenosine monophosphate (cAMP) [Katritch V., Cherezov V., Stevens R. Structure-function of the G protein-coupled receptor superfamily. Annual Review of Pharmacology and Toxicology. 2013. vol. 53. vo. 1. P. 531-556. doi: 10.1146 / annurev-pharmtox-032112-135923. In the Bos taurus genome, the FSHR gene is located on chromosome 11 and consists of 10 exons. The first 9 exons encode the extracellular domain of the receptor, exon 10 encodes the transmembrane domain [Minj A. et al. Molecular characterization of follicle stimulating hormone receptor (FSHR) gene in the Indian river buffalo (Bubalus bubalis). General and Comparative Endocrinology. 2008. vol. 158, no. 2, P. 147-153. doi: 10.1016 / J.YGCEN.2008.07.003]. The literature has accumulated data on the relationship of point mutations in the FSHR gene (SNP, single nucleotide polymorphism) with a superovulatory response to gonadotropin stimulation. In the FSHR gene, B. taurus SNPs were detected in the 5-UTR and coding parts by the SSCP method (Single-Strand Conformation Polymorphism) and examined for possible associations with reproductive potential in Holstein cattle [Sharifiyazdi N., Mirzaei A., Ghanaatian Z. Characterization of polymorphism in the FSH receptor gene and its impact on some reproductive indices in dairy cows. Animal Reproduction Science. 2018. vol. 188. P. 45-50. doi: 10.1016 / j.anireprosci.2017.11.006; Yang W.C., Li S.J., Chen L., Yang L.G. FSHR genotype affects estrogen levels but not pregnancy rates in Luxi cattle resulting to embryo transfer. Genetics and Molecular Research. 2014. vol. 13. No. 1. P. 1563-1569. doi: 10.4238 / 2014.March.12.8; Milazzotto M.P. et al. New molecular variants of hypothalamus-pituitary-gonad axis genes and their association with early puberty phenotype in Bos taurus indicus (Nellore). Livestock Science. 2008. vol. 114. vo. 2-3. P. 274-279. doi: 10.1016 / j.livsci.2007.05.00.00]. Of particular interest is the non-synonymous substitutions in exons, since such changes in the primary structure of the gene affect the sequence and conformation of the corresponding protein. In this context, the single nucleotide polymorphism 337C / G (rs43745234) in exon 4 of the FSHR gene corresponding to the amino acid substitution P113A is of particular interest. It was found that individuals homozygous for the G allele (genotype G337G) are characterized by a higher percentage of viable embryos, animals with the genotypes G337G and heterozygotes (genotype G337C) have fewer unfertilized oocytes compared to cows homozygous for the allele C (genotype Cory7C) A.T., Price S.A., Lefebvre R., Palin MF Identification of single nucleotide polymorphisms in the bovine follicle-stimulating hormone receptor and effects of genotypes on superovulatory response traits. Animal Genetics. 2013. vol. 44. no. 2. P. 197-201. doi: 10.1111 / j.l365-2052.2012.02380.x]. Thus, the fshr gene encoding FSHR is one of the promising genetic markers of the reproductive status of cattle.

To differentiate allelic variants of genes, the most widely used method is PCR-RFLP (polymerase chain reaction followed by analysis of restriction fragment length polymorphism).

и R:

с помощью эндонуклеазы рестрикции HgaI и последующим анализом полученных фрагментов ДНК методом электрофореза в 2%-ном агарозном геле.The prior art method for cattle genotyping by alleles 337C / G (rs43745234) of the fshr gene, which ensures their identification based on the PCR-RFLP method (prototype - [Cory A.T., Price S.A., Lefebvre R., Palin MF Identification of single nucleotide polymorphisms in the bovine follicle-stimulating hormone receptor and effects of genotypes on superovulatory response traits. Animal Genetics. 2013. vol. 44. no. 2. P. 197-201. Doi: 10.1111 / j.l365- 2052.2012.02380.x]. The method consists in the fact that the 337C / G alleles (rs43745234) are detected by processing the 286 base pairs of the fshr gene containing them obtained by PCR using F primers:

and R:

using HgaI restriction endonuclease and subsequent analysis of the obtained DNA fragments by electrophoresis in a 2% agarose gel.

The main disadvantages of this method are the multi-stage analysis (amplification of the polymorphic portion of the gene, restriction of the resulting amplicon by a specific endonuclease, electrophoretic separation of the resulting fragments), its duration (more than 6-8 hours), the probability of unreliable results in the case of non-optimal ratio of DNA amount, restriction enzyme and restriction time.

The technical result provided by the invention is to increase the reliability of detection of alleles 337C / G (rs43745234) of the fshr gene, to simplify the analysis and reduce its time to 1.5 hours.

The technical result is achieved by the fact that the method of real-time PCR for genotyping cattle according to the 337C / G alleles of the fshr gene (rs43745234) is different in that they are used:

direct primer;

reverse primer

FSHR337C - allele-specific fluorescently-labeled probe

FSHR337G - allele-specific fluorescently-labeled probe

The list of graphic materials.

In FIG. 1 shows the nucleotide sequences of a 96 bp fshr gene fragment encoding the 337C / G alleles (rs43745234). Primer sequences are marked in bold, probe sequences in italics.

In FIG. 2 presents: A - fluorescence curves for homozygous samples along the C allele (genotype C337C). B - fluorescence curves for homozygous samples along the G allele (genotype G337G). B - fluorescence curves for G337C heterozygotes. G is a graph of the distribution of the 337C / G alleles of the fshr gene.

и обратный праймер FSHR-R:

фланкирующие участок гена fshr длиной 96 п.н., и два аллель-специфичных флуоресцентно-меченых зонда типа TaqMan: FSHR337C:

и FSHR337G:

(фиг. 1).The patented method consists in real-time PCR for genotyping cattle according to allelic variants of 337C / G (rs43745234) of the fshr gene. The claimed method is characterized in that two common primers for allelic variants are used: direct primer FSHR-F:

and reverse primer FSHR-R:

96 bp flanking region of the fshr gene, and two allele-specific fluorescently-labeled probes of the TaqMan type: FSHR337C:

and FSHR337G:

(Fig. 1).

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

и обратного праймера FSHR-R:

с концентрацией 10 мкМ, 1 мкл зонда FSHR337C: '

(10 мкМ), 1 мкл зонда FSHR337G:

(10 мкМ), 1 мкл смесь дНТФ (5 мМ), 0,3 мкл HS TaqDNA полимеразы (Евроген, Россия), 10-30 нг ДНК. Программа для проведения ПЦР: 1-ый цикл: 95°С - 3 мин; далее 40 циклов при следующих условиях: 95°С - 20 сек, 55°С - 30 сек, 72°С - 20 сек. Детекция флуоресценции проводилась на стадии отжига праймеров и зондов по каналам FAM и VIC. ПЦР и анализ результатов генотипирования проводили с использованием амплификатора LightCycler® с программным обеспечением версии SW1.1. Идентификация аллелей 337C/G (rs43745234) проводится на основании сравнения интенсивности флуоресценции красителей FAM и VIC, соответственно. Для определения генотипа животного используются конечные значения флуоресценции красителей FAM и VIC. Программное обеспечение для амплификатора LightCycler® 96 версии SW1.1. представляет результаты генотипирования в виде распределения аллелей (Фиг. 2Г). Для гомозиготных образцов по аллелю С (генотип С337С) детектируется нарастание флуоресценции по каналу FAM (Фиг. 2А). Для гомозиготных образцов по аллелю G (генотип G337G) детектируется сигнал по каналу VIC (Фиг. 2Б). Для гетерозиготного образца (генотип G337C) наблюдается нарастание флуоресценции по обоим каналам детекции (Фиг. 2В). Наличие двух красителей, FAM и VIC, позволяют однозначно определить присутствие каждого из исследуемых аллелей гена DGAT1 в анализируемом образце ДНК и, соответственно, генотип животного.In the patented method for identifying allelic variants of 337C / G (rs43745234) fshr gene based on real-time PCR, two primers are used that are common to both alleles of the gene, and two allele-specific fluorescence-labeled probes of the TaqMan type (Fig. 1). Primers FSHR-F and FSHR-R initiate amplification of a 96 bp long bovine fshr gene region. The reaction was carried out in 25 μl of the reaction mixture containing 2.5 μl of 10x buffer for HS TaqDNA polymerase (Eurogen, Russia), 2.5 μl of a mixture of direct primer FSHR-F:

and reverse primer FSHR-R:

with a concentration of 10 μm, 1 μl of the probe FSHR337C: '

(10 μM), 1 μl of probe FSHR337G:

(10 μM), 1 μl dNTP mixture (5 mM), 0.3 μl HS TaqDNA polymerase (Eurogen, Russia), 10-30 ng DNA. Program for PCR: 1st cycle: 95 ° C - 3 min; further 40 cycles under the following conditions: 95 ° C - 20 sec, 55 ° C - 30 sec, 72 ° C - 20 sec. Fluorescence was detected at the stage of annealing of primers and probes through the FAM and VIC channels. PCR and analysis of the genotyping results were performed using a LightCycler® amplifier with software version SW1.1. The identification of 337C / G alleles (rs43745234) is based on a comparison of the fluorescence intensities of the FAM and VIC 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 distribution of alleles (Fig. 2G). For homozygous samples, the C allele (genotype C337C) detects an increase in fluorescence through the FAM channel (Fig. 2A). For homozygous samples, a signal is detected on the G allele (genotype G337G) via VIC channel (Fig. 2B). For a heterozygous sample (genotype G337C), an increase in fluorescence is observed along both detection channels (Fig. 2B). The presence of two dyes, FAM and VIC, make it possible to unambiguously determine the presence of each of the studied alleles of the DGAT1 gene in the analyzed DNA sample and, accordingly, the animal genotype.

The developed method was tested on 95 DNA samples of black-mottled Holsteinized cattle. According to the results of genotyping, 47.37% of the animals were heterozygotes (genotype G337C), 47.37% were homozygotes for the C allele (C337C genotype) and 5.26% of animals were homozygotes for the G allele (genotype G337G). Validation of the method was carried out using PCR-RFLP analysis according to the prototype (Cory A.T., Price S.A., Lefebvre R., Palin MF Identification of single nucleotide polymorphisms in the bovine follicle-stimulating hormone receptor and effects of genotypes on superovulatory response traits. Animal Genetics. 2013. vol. 44. no. 2. P. 197-201. doi: 10.1111 / j.l365-2052.2012.02380.x). The results of both methods of genotyping completely coincided. However, the patented method for genotyping cattle according to the 337C / G alleles (rs43745234) of the fshr gene significantly (up to 1.5 hours) reduces the analysis time, which compares favorably with PCR-RFLP analysis.

Thus, an effective and reliable method has been developed for the rapid identification of allelic variants of 337C / G (rs43745234) cattle fshr gene in real-time PCR using allele-specific fluorescently-labeled probes. The developed method allows genotyping of up to 480 animals according to allelic variants of 337C / G (rs43745234) of the fshr gene (depending on the model of the amplifier) for 1.5 hours and can be used to select promising donor cows for embryos.

Claims (7)

The method of real-time PCR for genotyping of cattle by alleles 337C / G of the fshr gene (rs43745234), characterized in that they are used: direct primer 5'-GAATTGAAAAGGCCAACAACC-3 ', reverse primer 5'-CACCAGAATACAGAAGTTCTTACAGA-3 ', FSHR337C - allele-specific fluorescently-labeled probe -5'-FAM-CATCGACCCTGATGCC BHQ1-3 ', FSHR337G - allele-specific fluorescently-labeled probe 5'-VIC-CATCGACGCTGATGC-BHQ1-3 '.

Images