RU2847660C1 - Method for detecting the hh2 fertility haplotype in holstein cattle - Google Patents

Abstract

FIELD: molecular genetics.

SUBSTANCE: invention relates to molecular genetics and is intended for the diagnosis of the mutant allele of the IFT80 gene associated with fertility haplotype 2 (HH2) in Holstein cattle. The technical result is achieved by a method for detecting the HH2 fertility haplotype in Holstein cattle, which includes isolating DNA from biological material, performing a real-time polymerase chain reaction using a reaction mixture consisting of the following components: dnA from the test sample with an initial concentration of 10 ng/μl, in an amount of 1 μl; 10x buffer for TaqPol, in an amount of 2.5 μl; a mixture of primer solutions, including the forward primer HH2-F 5'-AAATCCTACTTGAGAATGCA-3' and the reverse primer HH2-R 5'-GGAAGCGCCCTTCATAAGA-3', where the concentration of each primer in its solution is 10 μM, in a total amount of 2.5 μl; HH2-T solution - allele-specific fluorescently labelled probe FAM-TAG+ACATTT+TCTTC+TTGTAG-BHQ1 with a concentration in solution of 10 μM, in an amount of 1 μl; HH2-del solution - allele-specific fluorescently labelled probe VIC-TAG+ACATTTCTTC+TTGTAG-BHQ1 with a concentration of 10 μM in solution, in an amount of 1 μl; a solution of a mixture of deoxynucleoside triphosphates (dNTP) with a concentration of 10 μM, in an amount of 1 μl; TaqPol polymerase with an activity of 5 units/μl, in an amount of 0.5 μl; water for PCR to a total reaction volume of 25 μl, then evaluate the reaction results based on the presence and combination of fluorescence signals in the Green (FAM) and Yellow (VIC) channels to determine the genotype: the absence of a mutation (genotype T/T, wild type) is established by the presence of a fluorescence signal in the Green (FAM) channel and the absence of a fluorescence signal in the Yellow (VIC) channel; the presence of a heterozygous mutation (genotype T/del) is established by the presence of fluorescence signals in both channels – Green (FAM) and Yellow (VIC); The presence of a homozygous mutation (del/del genotype) is determined by the absence of a fluorescence signal in the Green (FAM) channel and the presence of a fluorescence signal in the Yellow (VIC) channel.

EFFECT: identification of a specific reading frame shift mutation in the IFT80 gene of chromosome 1 of cattle and detection of the point mutation rs523422030 (T>del) associated with fertility haplotype 2 (HH2) in Holstein cattle using real-time PCR in blood samples from Jersey cattle.

1 cl, 3 dwg, 1 tbl, 1 ex

Description

The invention relates to molecular genetics and is intended for diagnosing a mutant allele of the IFT80 gene associated with the fertility haplotype 2 (HH2) in Holstein cattle.

A diagnostic method for Holstein haplotype 2 (fertility haplotype HH2) is known, which leads to embryonic mortality in Holstein cattle at up to 100 days of gestation in embryos homozygous for the mutant allele. It has been established that the causative mutation of HH2 is a deletion of thymine at position 107172616 on chromosome 1, which results in a frameshift and premature stop codon formation, resulting in a shortening of the amino acid sequence from 760 to 383 amino acids, which significantly alters the structure and function of the IFT80 protein ( Ortega MS, Bickhart DM, Lockhart KN, Null DJ, Hutchison JL, McClure JC, Cole JB. Truncation of IFT80 causes early embryonic loss in Holstein cattle associated with Holstein haplotype 2. J Dairy Sci. 2022 Nov;105(11):9001-9011. doi: 10.3168/jds.2022-21853. Epub 2022 Sep 7. PMID: 36085107; Yang Y, Si J, Lv X, Dai D, Liu L, Tang S, Wang Y, Zhang S, Xiao W, Zhang Y. Integrated analysis of whole genome and transcriptome sequencing reveals a frameshift mutation associated with recessive embryonic lethality in Holstein cattle. Anim Genet. Feb 2022; 53(1):137-141). The Holstein haplotype 2 (HH2) allele, which causes annual fertility losses of more than $2 million in the United States alone, is present in 1.66% of the domestic Holstein population [Harmful recessive effects on fertility detected by absence of homozygous haplotypes / P. M. VanRaden, K. M. Olson, D. J. Null, J. L. Hutchison // Journal of Dairy Science, Volume 94, Issue 12, 6153 - 6161, doi: 10.3168/jds.2011-4624] and has a significant negative impact on fertility in the form of early embryonic losses.

In global practice, the whole genome sequencing method is used to diagnose the HH2 haplotype in cattle. For this purpose, custom (user-designed) biochips are used that scan SNPs, however, DNA diagnostics using this method requires expensive equipment and the high cost of biochips (VanRaden, P.M. & Olson, K.M. & Null, D.J. & Hutchison, Jana. (2011). Harmful recessive effects on fertility detected by absence of homozygous haplotypes. Journal of dairy science. 94. 6153-61. 10.3168/jds.2011-4624; McClure MC, Bickhart D, Null D, Vanraden P, Xu L, Wiggans G, Liu G, Schroeder S, Glasscock J, Armstrong J, Cole JB, Van Tassell CP, Sonstegard TS. Bovine exome sequence analysis and targeted SNP genotyping of recessive fertility defects BH1, HH2, and HH3 reveal a putative causative mutation in SMC2 for HH3. PLoS One. 2014 Mar 25; 9(3):e92769. doi: 10.1371/journal.pone.0092769. PMID: 24667746; PMCID: PMC3965462.).

The disadvantage of these technical solutions is that these studies used reagents and reagent mixtures of imported origin, which creates difficulties in conducting mass monitoring of the presence or absence of the specified genetic mutation in Russian Holstein breeding cattle.

The objective of the proposed invention is to identify the HH2 fertility haplotype in Holstein cattle using real-time PCR, detecting even minimal amounts of genetic material, making it a more sensitive method, and making test results available within one business day, which is significantly faster than traditional methods (PCR-RFLP) and sequencing; using the developed composition (test system) of reagents available in the Russian Federation for monitoring the spread of the HH2 genetic anomaly in Holstein cattle in breeding and commercial herds.

The technical result of the claimed invention consists in identifying a specific frameshift mutation in the IFT80 gene of chromosome 1 of cattle and detecting the point mutation rs523422030 (T>del), associated with the fertility haplotype 2 (HH2) in Holstein cattle, using the real-time PCR method in blood samples of Jersey cattle.

The technical result is achieved using a method for identifying the HH2 fertility haplotype in Holstein cattle, which includes isolating DNA from biological material and performing a real-time polymerase chain reaction using a reaction mixture consisting of the following components:

DNA of the test sample, initial concentration of 10 ng/μl, in the amount of 1 μl;

10x TaqPol buffer, 2.5 µl;

a mixture of primer solutions, including a forward primer HH2-F 5'- AAATCCTACTTGAGAATGCA -3' and a reverse primer HH2-R 5'- GGAAGCGCCCTTCATAAGA -3', where the concentration of each primer in its solution is 10 μM, in a total amount of 2.5 μl;

a solution of HH2-T - allele-specific fluorescently labeled probe FAM-TAG+ACATTT+TCTTC+TTGTAG-BHQ1 with a concentration in solution of 10 μM, in an amount of 1 μl;

a solution of HH2-del - allele-specific fluorescently labeled probe VIC-TAG+ACATTTCTTC+TTGTAG-BHQ1 with a concentration in solution of 10 μM, in an amount of 1 μl;

a solution of a mixture of deoxynucleoside triphosphates (dNTPs), with a concentration of 10 μM, in an amount of 1 μl;

TaqPol polymerase, activity 5 units/μl, in an amount of 0.5 μl;

water for PCR to a total reaction volume of 25 µl

The reaction results are then assessed based on the presence and combination of fluorescence signals in the Green (FAM) and Yellow (VIC) channels to determine the genotype: the absence of a mutation (T/T genotype, wild type) is determined by the presence of a fluorescence signal in the Green (FAM) channel and the absence of a fluorescence signal in the Yellow (VIC) channel; the presence of a heterozygous mutation (T/del genotype) is determined by the presence of fluorescence signals in both channels – Green (FAM) and Yellow (VIC); the presence of a homozygous mutation (del/del genotype) is determined by the absence of a fluorescence signal in the Green (FAM) channel and the presence of a fluorescence signal in the Yellow (VIC) channel.

Figure 1 - Primers and probes for amplification of target loci of Holstein cattle.

Figure 2 - Program for real-time PCR.

Figure 3 – Fluorescence accumulation curves obtained during the study of cattle blood samples for the presence of a single-nucleotide deletion in the IFT80 gene, which leads to embryonic mortality in Holstein cattle.

Primers and probes for the selected DNA targets were designed using the Primer BLAST service (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). A preliminary assessment of the specificity of primers, amplicons, and probes was performed using the BLAST online program. Construction of a reagent kit for the detection of genetic anomalies in cattle using real-time PCR.

Molecular targets and flanking DNA sequences were identified by searching for nucleotide sequences of gene loci carrying genetic abnormalities. Analytical work was performed by monitoring the GenBank annotated nucleotide sequence bank of the National Center for Biotechnology Information (NCBI, USA). Following an analysis of literary sources, a point mutation for the Holstein (HH2) breed in the IFT80 gene was selected as a target for the study: 1: g.107172616delT, c.1140del, p.(L381Ffs*3). Assembly: ARS-UCD1.3 (GCF_002263795.2).

Primers and probes for amplifying selected DNA targets were developed using quantitative polymerase chain reaction. The resulting reagents were used to monitor the presence or absence of the HH2 genetic abnormality in Holstein cattle.

1. DNA samples. Samples were obtained from the blood of Holstein cattle.

2. Primers and probes for the selected DNA targets were designed using the Primer BLAST service (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). A preliminary assessment of the specificity of primers, amplicons, and probes was performed using the online BLAST program.

3. Design of a reagent kit for detection of genetic abnormalities in cattle using real-time PCR

3.1 Determination of molecular targets and flanking DNA sequences

A search for nucleotide sequences of gene loci carrying genetic abnormalities was performed in the GenBank annotated nucleotide sequence database of the National Center for Biotechnology Information (NCBI, USA). A literature review identified a point mutation in the IFT80 gene in Holstein (HH2) cattle as a target for the study: 1: g.107172616delT, c.1140del, p.(L381Ffs*3). Assembly: ARS-UCD1.3 (GCF_002263795.2).

3.2. Design of primers and probes with fluorescent labels.

When developing the nucleotide sequences of primers and probes, the following parameters were taken into account: amplicon size (length), annealing temperature, nucleotide composition, nucleotide distribution along the primer length, primer and probe length, and the possibility of hairpin and dimer formation. The selected primer and probe sequences with fluorescent labels were tested for annealing specificity in silico using the NCBI online service Primer BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), which performs local pairwise alignment of each primer with the nucleotide sequences in the database.

Sequences of primers and probes with fluorescent labels to four specific targets for the detection of genetic abnormalities in Jersey and Holstein cattle are shown in Fig. 1.

4. Selection of the reaction mixture composition for PCR

The possible composition of the reaction mixture and the ratio of primer/probe concentrations for the target were determined, ensuring a stable increase in the fluorescence level in the specific detection channels.

In order to develop a PCR test system for detecting a selected genetic anomaly in the cattle genome, the following procedure for preparing reaction mixtures and recording the results is proposed, requiring further experimental testing and optimization.

To perform the analysis, it is necessary to prepare a working solution of a mixture of primers (F+R) and probes with a concentration of 10 μM. In addition, the following reagents are required for the PCR reaction: a 10 mM mixture of four deoxynucleoside triphosphates (dNTPs), Thermus aquaticus DNA polymerase (TaqPol), and 10x DNA polymerase buffer (MgCl2 concentration of 1.5-2 mM, presence of NH4+ ions). The reaction is carried out in a volume of 25 μl. The reaction mixture consists of the following components:

DNA of the test sample, initial concentration of 10 ng/μl, in the amount of 1 μl;

10x TaqPol buffer, 2.5 µl;

a mixture of primer solutions, including a forward primer HH2-F 5'- AAATCCTACTTGAGAATGCA -3' and a reverse primer HH2-R 5'- GGAAGCGCCCTTCATAAGA -3', where the concentration of each primer in its solution is 10 μM, in a total amount of 2.5 μl;

a solution of HH2-T - allele-specific fluorescently labeled probe FAM-TAG+ACATTT+TCTTC+TTGTAG-BHQ1 with a concentration in solution of 10 μM, in an amount of 1 μl;

a solution of HH2-del - allele-specific fluorescently labeled probe VIC-TAG+ACATTTCTTC+TTGTAG-BHQ1 with a concentration in solution of 10 μM, in an amount of 1 μl;

a solution of a mixture of deoxynucleoside triphosphates (dNTPs), with a concentration of 10 μM, in an amount of 1 μl;

TaqPol polymerase, activity 5 units/μl, in an amount of 0.5 μl;

water for PCR to a total reaction volume of 25 µl.

The results are recorded and analyzed using the instrument's software based on the absence or presence of a fluorescence signal in the test samples and control samples across the detected channels. The reaction results are interpreted in accordance with the following algorithm:

1. Registration of an exponential increase in the fluorescence level only in the FAM channel indicates the presence of a T/T homozygote.

2. Registration of an exponential increase in the fluorescence level only in the HEX channel indicates the presence of a del/del homozygote.

3. Registration of an exponential increase in the fluorescence level simultaneously in two channels indicates the presence of a T/del heterozygote.

An example of the method for identifying the HH2 fertility haplotype in Holstein cattle.

In the experiment, the presence of a mutant allele of the IFT80 gene associated with the fertility haplotype 2 (HH2) in Holstein cattle was determined in whole blood samples from cattle.

DNA extraction was performed by alcohol precipitation using the RIBO-prep reagent kit (InterLabService LLC, Russia), according to the manufacturer's instructions. The blood sample volume for DNA extraction was 100 µl. Bovine blood samples were stored at -80°C until DNA extraction. DNA samples were stored at -20°C until analysis.

To carry out the amplification reaction, a reaction mixture was prepared, the composition of the reaction mixture (per 1 reaction):

10x TaqPol buffer, 2.5 µl;

a mixture of primer solutions, including a forward primer HH2-F 5'- AAATCCTACTTGAGAATGCA -3' and a reverse primer HH2-R 5'- GGAAGCGCCCTTCATAAGA -3', where the concentration of each primer in its solution is 10 μM, in a total amount of 2.5 μl;

a solution of HH2-T - allele-specific fluorescently labeled probe FAM-TAG+ACATTT+TCTTC+TTGTAG-BHQ1 with a concentration in solution of 10 μM, in an amount of 1 μl;

a solution of HH2-del - allele-specific fluorescently labeled probe VIC-TAG+ACATTTCTTC+TTGTAG-BHQ1 with a concentration in solution of 10 μM, in an amount of 1 μl;

a solution of a mixture of deoxynucleoside triphosphates (dNTPs), with a concentration of 10 μM, in an amount of 1 μl;

TaqPol polymerase, activity 5 units/μl, in an amount of 0.5 μl;

water for PCR to a total reaction volume of 25 µl.

The prepared mixtures were pipetted, 20 µl at a time, into the bottom of amplification tubes, and 5 µl of the test sample's DNA was added. The total volume of the reaction mixture was 25 µl.

To assess the correctness of the reaction mixture preparation, control amplification reactions were carried out:

a) negative amplification control (K-) – 5 µl of type 1 water was added to a test tube or well.

b) positive amplification control (K+T) – 5 µl of K+T (a preparation of recombinant plasmid DNA containing the wild-type allele of the IFT80 gene) were added to a test tube or well.

c) positive amplification control (K+del) – 5 µl of K+del (a preparation of recombinant plasmid DNA containing the allele of the IFT80 gene containing the desired mutation) were added to a test tube or well.

PCR was carried out in plate-type and rotary-type amplifiers with real-time detection of results in accordance with the amplification program:

The results were calculated based on an analysis of fluorescence accumulation in the reaction mixtures via the FAM and VIC channels. The results were interpreted based on the presence or absence of an intersection of the S-shaped (sigma-shaped) fluorescence curve with a threshold line set at the appropriate level.

The threshold line level for the FAM and VIC channels was set at 10–20% of the maximum fluorescence level of the positive control sample in the last amplification cycle.

The results of the analysis were taken into account if:

- there was no exponential increase in the fluorescence level in two channels in the negative control tube;

- an exponential increase in the fluorescence level was recorded in the FAM channel in a test tube with a positive control K+T.

- an exponential increase in the fluorescence level was recorded in the HEX channel in a test tube with a positive control K+ del.

The detection of an exponential increase in the fluorescence level only in the FAM channel indicated the presence of a T/T homozygote (wild type).

The detection of an exponential increase in the fluorescence level only in the HEX channel indicated the presence of a del/del homozygote (mutation).

The registration of an exponential increase in the fluorescence level simultaneously in two channels indicated the presence of a T/del heterozygote.

The results obtained from the study of samples are presented in Table 1.

Table 1 Ct values of the studied samples

Hole Try Ct (FAM) Ct (VIC) Genotype H01 1 30.93 - T/T (wild type) D07 2 30.27 - T/T (wild type) G06 3 30.01 - T/T (wild type) T/T (wild type) G07 4 29.96 - T/T (wild type) C07 5 29.88 - T/T (wild type) H12 K- - - OK H10 K+ 13.43 - OK H11 K+(del) - 14.80 OK

The fluorescence curves obtained during the study of the samples are shown in Fig. 3.

Compared to the prototype, the declared technical solution has the following advantages:

- the duration of the amplification program is 1.5 hours;

- the ability to examine biological material from cattle for the presence of a specific mutation without sequencing.

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Claims (10)

A method for identifying the HH2 fertility haplotype in Holstein cattle, including DNA extraction from biological material and real-time polymerase chain reaction using a reaction mixture consisting of the following components: DNA of the test sample with an initial concentration of 10 ng/μl, in the amount of 1 μl; 10x TaqPol buffer, 2.5 µl; a mixture of primer solutions comprising a forward primer HH2-F 5'-(SEQ ID NO:1)-3' and HH2-R 5'-(SEQ ID NO:2)-3', where the concentration of each primer in its solution is 10 μM, in a total amount of 2.5 μl; a solution of HH2-T - allele-specific fluorescently labeled probe FAM-(SEQ ID NO:3)-BHQ1 with a concentration in solution of 10 μM, in an amount of 1 μl; a solution of HH2-del - allele-specific fluorescently labeled probe VIC-(SEQ ID NO:4)-BHQ1 with a concentration in solution of 10 μM, in an amount of 1 μl; a solution of a mixture of deoxynucleoside triphosphates (dNTP) with a concentration of 10 μM, in an amount of 1 μl; TaqPol polymerase with an activity of 5 units/μl, in an amount of 0.5 μl; water for PCR to a total reaction volume of 25 µl, The reaction results are then assessed based on the presence and combination of fluorescence signals in the Green FAM and Yellow VIC channels to determine the genotype: the absence of a mutation (T/T genotype, wild type) is determined by the presence of a fluorescence signal in the Green FAM channel and the absence of a fluorescence signal in the Yellow VIC channel; the presence of a heterozygous mutation, the T/del genotype, is determined by the presence of fluorescence signals in both channels – Green FAM and Yellow VIC; the presence of a homozygous mutation, the del/del genotype, is determined by the absence of a fluorescence signal in the Green FAM channel and the presence of a fluorescence signal in the Yellow VIC channel.