CN110564862A - Method for rapidly screening Holstein cattle HH5 genetic defect gene carrier - Google Patents
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Abstract
The invention provides a method for rapidly screening a Holstein cattle HH5 genetic defect gene carrier, which belongs to the technical field of gene mutation detection, and discovers that 9bp insertion of mutant alleles occurs at a fusion site by taking an HH5 wild type homozygote Holstein bull of a known genotype and an HH5 carrier Holstein bull as reference samples, wherein the nucleotide sequence of an InDEL molecular marker is shown as SEQ ID NO. 1. The invention designs specific primers aiming at the InDEL molecular marker, and realizes the rapid and accurate typing of the genetic defect wild type and the mutant type of the Holstein cattle HH 5. The method for rapidly screening the HH5 genetic defect gene carrier of the Holstein cattle is simple to operate, high in sensitivity, strong in accuracy and low in detection cost, and has important application value in the screening and breeding processes of genetic diseases of the cattle.
Description
Technical Field
The invention relates to the technical field of gene mutation detection, in particular to an InDEL molecular marker related to HH5 genetic defect of Holstein cattle and a method for rapidly screening HH5 genetic defect gene carriers of the Holstein cattle based on the molecular marker.
Background
Genetic deficiency diseases refer to diseases that affect the growth and development of animals due to changes in genetic material, mainly manifested as defects in body structure or dysfunction, thereby affecting productivity and even leading to death. In recent years, the wide application of the breeding biotechnology of cow artificial insemination, embryo transplantation, sex control semen and the like increases the use frequency of excellent breeding bulls, sharply reduces the effective content of the population, increases the population inbreeding coefficient, increases the homozygous probability of recessive harmful genes, and thus increases the risk of genetic defect. Many bulls, which are excellent in productivity but carry a gene with a genetic defect, are extremely common in the case of being widely used in the population, and therefore the gene with a genetic defect spreads rapidly in the population. Common genetic defects of dairy cows are usually autosomal recessive inheritance, heterozygotes have no phenotypic difference from healthy individuals, and recessive homozygotes show obvious symptoms.
HH5 is a newly discovered genetic defect in Holstein cattle, with the pathogenesis of 138Kb deletion in the region 93.233Mb to 93.371Mb on chromosome 9, the deletion region containing the entire dimethyladenosine transferase 1(TFB1M) gene. The TFB1M gene is important for starting protein translation in mitochondria, and the main function of the TFB1M gene is to dimethylate adenine residues in a hairpin loop at the 12S rRNA 3' end of mitochondria, which plays a decisive role in the synthesis and biological functions of small subunits of ribosomes. TFB1M knockout mice died at 8.5 days of embryonic development.
China introduces a large amount of frozen sperms, embryos and cows from abroad to improve the Chinese Holstein cattle every year, but the detection of strict genetic diseases is not carried out in the introduction process, so that genetic defects flow into the Chinese cattle. The main risk of recessive deleterious genetic defects is when a carrier cow mates with a carrier bull, the offspring are homozygous negative with a 1/4 probability, resulting in early embryonic death or calf birth death. Therefore, the development of an HH5 molecular detection method has important significance for timely understanding the frequency of HH5 in Holstein cattle in China and controlling the negative effects of the HH5 by adopting breeding measures.
schultz et al (2016) reported an HH5 detection method based on fluorescence quantitative PCR technology, but the detection cost is high due to the need of special reagents and instruments, and the method is difficult to apply in ordinary molecular laboratories.
Disclosure of Invention
The invention aims to provide a method for rapidly screening Holstein cattle HH5 genetic defect gene carriers.
In order to achieve the purpose, the invention takes 46 heads of HH5 wild homozygote Holstein bull with known genotype and 7 heads of HH5 carrier Holstein bull as reference samples, and finds that the mutant allele has 9bp insertion at the fusion site besides the known 138bp deletion large fragment. Based on the discovery, the invention provides an InDEL molecular marker related to the genetic defect of Holstein cattle HH5, which is a 9bp fragment inserted into the 41 th to 49 th positions of the nucleotide sequence shown as SEQ ID NO.1, and the sequence of the fragment is TGATTACAA.
Preferably, the InDEL molecular marker is detectable by specific primers, and the InDEL molecular marker is detectable by PCR using specific primer combinations, wherein the specific primer combinations are any one of the following:
(1) The nucleotide sequences are respectively shown as specific primer combinations of SEQ ID NO.2, SEQ ID NO.7 and SEQ ID NO. 8; or
(2) The nucleotide sequences are respectively shown as specific primer combinations of SEQ ID NO.2-3 and SEQ ID NO. 4-5.
Further, the invention provides a specific primer combination for detecting the InDEL molecular marker, which is a combination of any one of the following groups:
(1) The nucleotide sequences are respectively shown as SEQ ID NO.2, SEQ ID NO.7 and SEQ ID NO. 8; or
(2) the primer combination comprises 2 pairs of specific primer pairs, and the nucleotide sequences of the 2 pairs of specific primer pairs are respectively shown as SEQ ID NO.2-3 and SEQ ID NO. 4-5.
The invention provides a primer combination for screening Holstein cattle HH5 genetic defect gene carriers, which comprises 2 pairs of specific primer pairs, and the nucleotide sequences of the specific primer pairs are respectively shown as SEQ ID NO.2-3 and SEQ ID NO. 4-5.
The invention also provides a kit for screening the carrier of the HH5 genetic defect gene of the Holstein cattle, which contains the primer combination.
Further, the kit of the invention comprises the following working steps:
(1) Extracting genome DNA of a cattle sample to be detected, and carrying out double PCR reaction by taking the genome DNA as a template and primers shown in SEQ ID NO.2-3 and SEQ ID NO.4-5 as amplification primers;
(2) Carrying out gel electrophoresis detection on the PCR product, and carrying out genotype judgment according to an electrophoresis result: if only a 209bp band is amplified, the sample is from a Holstein bovine HH5 genetic defect gene wild-type homozygote; if two bands are amplified, namely 209bp and 349bp respectively, the sample to be detected is from a Holstein cow HH5 genetic defect gene carrier.
the PCR reaction system in the step (1): the total volume is 25 μ L, wherein 22 μ L of gold medal Mix rapid PCR premix (containing buffer, dNTP, Taq enzyme) (Beijing Optimala New technology Co., Ltd.), 0.5 μ L of each of 4 primers (concentration is 10 μ M), and 1 μ L (about 50ng) of genome DNA template.
And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; then 35 cycles, 94 ℃ denaturation 30sec, 56 ℃ renaturation 30sec, 72 ℃ extension 30 sec; finally, extension was carried out at 72 ℃ for 7 min.
the invention also provides a method for detecting a Holstein cow HH5 genetic defect gene carrier, which comprises a method 1 or a method 2, wherein the method 1 comprises the following steps:
(1) Extracting genome DNA of a cattle sample to be detected, and carrying out double PCR reaction by taking the genome DNA as a template and primers shown in SEQ ID NO.2-3 and SEQ ID NO.4-5 as amplification primers;
(2) Carrying out gel electrophoresis detection on the PCR product, and carrying out genotype judgment according to an electrophoresis result: if only one 209bp band is amplified, the sample to be detected comes from the wild homozygote of the Holstein cattle HH5 genetic defect gene; if two bands are amplified, namely 209bp and 349bp respectively, the sample to be detected is from a Holstein cow HH5 genetic defect gene carrier;
The method 2 comprises the following steps: extracting genome DNA of a cattle sample to be detected, and carrying out PCR reaction by taking the genome DNA as a template and primers shown in SEQ ID NO.2 and SEQ ID NO.7 as amplification primers; and sequencing the amplification product by using the inner primer shown by SEQ ID NO.8, wherein if a segment of 9bp in total of TGATTACAA is inserted into the 41 th to 49 th sites of the nucleotide sequence shown by SEQ ID NO.1, the sample to be detected is from a Holstein cow HH5 genetic defect gene carrier.
The method for detecting the HH5 genetic defect gene carrier of the Holstein cattle is suitable for detecting the normal HH5 genetic Holstein cattle, namely if the amplification band of the sample to be detected only has a 209bp band but does not have a 349bp band, the sample to be detected is the normal HH5 genetic Holstein cattle, and in the breeding work, the normal HH5 genetic Holstein cattle can be left, and the Holstein cattle carrying the HH5 genetic defect gene are eliminated.
the invention provides application of the InDEL molecular marker or the primer combination in cattle breeding.
Specifically, the application is breeding Holstein cattle not carrying the HH5 genetic defect genotype, or eliminating Holstein cattle carrying the HH5 genetic defect gene, or avoiding mating cattle carrying the HH5 genetic defect gene.
The invention provides application of the InDEL molecular marker or the primer combination in port quarantine to inspection and quarantine of bovine genetic materials, wherein the bovine genetic materials comprise live cows, frozen semen and embryos.
The invention has the advantages that at least the following aspects are realized:
(1) The invention provides a novel InDEL molecular marker for detecting HH5 genetic defect of Holstein cattle. The invention uses HH5 wild homozygote Holstein bull with known genotype and HH5 carrier Holstein bull as reference samples to find that the mutant allele generates 9bp insertion at the fusion site, and the nucleotide sequence of the InDEL molecular marker is shown in SEQ ID NO. 1.
(2) the primer designed by the invention cannot be combined with the wild allele, while the primer designed by Schultz et al (2016) aiming at the mutant allele is arranged at two ends of 138kb deletion, and although the primer can be combined with the wild allele, the wild allele cannot be amplified due to too long target fragment, and the mutant fragment is short and can be amplified.
(3) Schultz et al (2016) reports a high-resolution solution curve method based on a fluorescent quantitative PCR instrument, needs a special instrument and a corresponding kit, and has higher cost.
(4) the inventor tries to apply the primers in Schutz et al (2016) literature to common PCR and agarose gel electrophoresis, and finds that PCR of a single primer pair (respectively aiming at wild type or mutant type) can be successful, but double PCR amplification fails, which indicates that the interference of two pairs of primers in the literature in the same PCR system is serious, and the two pairs of primers designed by the invention can be subjected to double PCR amplification, so that the method can realize simple, sensitive and accurate detection.
Drawings
FIG. 1 is a comparison of the Sanger sequencing results for the wild type allele and mutant allele of HH 5. (a) A partial sequence of the wild type allele corresponding to the front end sequence of the mutant deletion fragment, (b) a sequence specific to the mutant allele shown shaded as a 9bp sequence inserted at the fusion site of the deletion fragment, and (c) a partial sequence of the wild type allele corresponding to the rear end sequence of the mutant deletion fragment.
FIG. 2 is a schematic representation of the sequence characteristics and primer design for the wild type allele and mutant allele of HH 5; the primers are underlined and the ellipses represent DNA sequences not shown in the figure. The HH5-1F (SEQ ID NO.2) and HH5-1mutR (SEQ ID NO.3) primer combinations were used to specifically amplify mutant alleles, and the HH5-2F (SEQ ID NO.4) and HH5-2Rshort (SEQ ID NO.5) primer combinations were used to specifically amplify wild type alleles.
FIG. 3 shows the results of allele-specific PCR amplification assays of wild-type homozygotes of HH5 and carriers of the genetic defect allele.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1 development of InDEL molecular markers associated with the genetic Defect Gene of Holstein cattle HH5
And (3) designing specific primers to perform PCR amplification and Sanger sequencing by taking an individual with a known genotype as a reference sample, comparing the sequence difference of the wild type allele and the mutant allele, and excavating a specific sequence.
1. Determination of the sequence of the wild type allele and of the mutant allele of HH5
(1) Sample(s)
HH5 wild type homozygote holstein bull of known genotype 46 heads and HH5 carrier holstein bull 7 heads. Extracting genome DNA from the frozen bovine semen by a high-salt method.
(2) Primer design
Based on the position of the known HH5 genetic defect gene mutation (138kb deletion) on the bovine reference genome (Bos taurus genome assembly UMD3.1), reference genome sequences near two break sites in front of and behind the deleted fragment were extracted, and specific primers were designed as follows.
TABLE 1 primer sequences and amplified fragment lengths
(3) PCR amplification
And (3) PCR reaction system: a total volume of 25. mu.L, 10 XBuffer 2.5. mu.L, 2. mu.L of dNTP mix (2.5 mM each), 0.5. mu.L of Taq enzyme (5U/. mu.L), 0.5. mu.L of upstream and downstream primers (20. mu.M each), and 1. mu.g of genomic DNA template.
And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; then 35 cycles, 94 ℃ denaturation for 30sec, 60 ℃ (SEQ ID NO.2 and SEQ ID NO.6 primer combination) or 55 ℃ (SEQ ID NO.4 and SEQ ID NO.7 primer combination, SEQ ID NO.2 and SEQ ID NO.7 primer combination) renaturation for 30sec, 72 ℃ extension for 30 sec; finally, extension was carried out at 72 ℃ for 7 min.
The instrument is an Applied Biosystems model 9700 PCR instrument.
(4) Sanger sequencing of PCR products
The PCR product of the HH5-1F (SEQ ID NO.2) and HH5-1RNEW (SEQ ID NO.6) primer combination was sequenced using the downstream primer HH5-1RNEW (SEQ ID NO. 6); the PCR product of the HH5-2F (SEQ ID NO.4) and HH5-2R (SEQ ID NO.7) primer combination was sequenced using the upstream primer HH5-2F (SEQ ID NO. 4). HH5-1F (SEQ ID NO.2) and HH5-2R (SEQ ID NO.7) primer combinations were sequenced using the inner primer HH5mutF (5'-AAAAT ACTGG TTTGT TTTGC-3', SEQ ID NO. 8). A comparison of Sanger sequencing results for the HH5 wild type allele and mutant allele is shown in fig. 1.
2. Discovery of HH5 mutant allele-specific DNA sequences
The partial sequence of the wild-type allele obtained by sequencing was aligned with the bovine reference genome (Bos taurus genome assembly UMD3.1) and sequence identity was confirmed. Comparing the mutant allele sequence with the wild type allele sequence, it was found that the mutant allele could be divided into three parts, the first part (represented by GGTTC TAAAA TGTCT TGTAGGTCTT CATAG AACCA TTCAA, SEQ ID NO. 9) corresponding to the head of the wild type allele sequence, the last part (represented by TTACA ATGAT GCTTC TGGAT GCTGA ATTTC CTTTT CTCTT TCTTC TCTTC TCTTT CTTTCTGTTC TGTTC TTCTT TCTTT CTTCT CCTTT TTTCT TTCTT TCTTC TCTTT TCCTT TTCAC G, SEQID NO. 10) corresponding to the tail of the wild type allele sequence, and the middle part being 1 sequence of 9bp (TGATTACAA). The Sanger sequencing peak pattern of the wild type sequence and the mutant sequence at the fusion site of the 138kb deletion is shown in FIG. 1.
the HH5 mutant allele was reported in the previous literature to be a 138kb fragment deletion (Schultz et al, 2016), whereas the present inventors have for the first time discovered that the mutant allele, in addition to the deletion of this long fragment, simultaneously undergoes an insertion of 9bp (TGATTACAA) at the fusion site, and that the 9bp insertion and the 138kb deletion occur simultaneously (up to 100% overlap), both of which are directly related to the HH5 genetic defect.
Example 2 establishment of a method for detecting InDEL molecular markers associated with the genetic defect of Holstein bovine HH5
Based on the sequence characteristics of the HH5 mutant allele which is discovered by the invention and has 9bp insertion, the inventor designs an allele-specific PCR primer, and can realize rapid and accurate typing of the wild type and the mutant.
(1) Sample(s)
DNA from 2 HH5 carriers and 2 HH5 wild-type homozygous samples of known genotypes.
(2) Primer design
Based on the characteristic sequence information of the wild type and mutant alleles at the mutation site, specific primers were designed as follows (5 '-3'). The relative positions of the primers are shown in FIG. 2.
TABLE 2 primer sequences and amplified fragment lengths
(2) PCR amplification
The wild-type allele and the mutant allele are amplified simultaneously in one PCR system by a double PCR method.
And (3) PCR reaction system: the total volume is 25 μ L, wherein 22 μ L of gold medal Mix rapid PCR premix (containing buffer, dNTP, Taq enzyme) (Beijing Optimala New technology Co., Ltd.), 0.5 μ L of each of 4 primers (concentration is 10 μ M), and 1 μ L (about 50ng) of genome DNA template.
And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; then 35 cycles, 94 ℃ denaturation 30sec, 56 ℃ renaturation 30sec, 72 ℃ extension 30 sec; finally, extension was carried out at 72 ℃ for 7 min. The instrument is an Applied Biosystems model 9700 PCR instrument.
(3) Gel electrophoresis detection and genotype determination of PCR product
A2% agarose gel was prepared, and 4. mu.L of the PCR product from each sample was spotted, followed by electrophoresis in TAE buffer at 110V for 20min, and the results of the electrophoresis were observed in a gel imaging system. The PCR product gel is shown in FIG. 3. Lanes 1 and 2 are wild-type homozygotes, lanes 3 and 4 are carriers, lane 5 is a negative control, lane 6M is DNA marker, there are 6 DNA fragments, and reference fragment (bp): 100, 200, 300, 400, 500, 600.
it can be seen that HH5 wild-type homozygote and carrier can be accurately typed by electrophoresis gel chart, the wild-type homozygote is a single 209bp band, and the carrier has 349bp specific band besides 209bp band. The length difference of the two bands is 140bp, the method established by the invention can realize the detection of the InDEL molecular marker determined in the embodiment 1 through common agarose gel electrophoresis, and further realize the screening and analysis of the Holstein bovine HH5 genetic defect gene carrier.
EXAMPLE 3 practical application of screening of carriers of the HH5 genetic Defect Gene
(1) sample(s)
From a suburban dairy farm in Beijing, 100 Hestein cow blood samples were randomly collected, and the genomic DNA extraction was performed using a blood genomic DNA extraction kit DP318 of Tiangen Biochemical technology (Beijing) Co., Ltd.
(2) Typing
PCR and agarose gel electrophoresis were performed and typed as established in example 2. As a result, 3 HH5 carriers were detected in total in 100 cows.
To verify the correctness of the assay results, Sanger sequencing was performed on the 3 detected carriers. First, the HH5-1F (SEQ ID NO.2) and HH5-2R (SEQ ID NO.7) primer combinations were subjected to PCR amplification, and the PCR products were sequenced using the internal primer HH5mutF (5'-AAAAT ACTGG TTTGT TTTGC-3', SEQ ID NO. 8). Sequence analysis verified that the 3 cows carried HH5 mutant gene with 9bp (tgatacaa) insertion. In addition, 20 non-carriers were randomly selected, PCR amplification was performed using the primer combination of HH5-1F (SEQ ID NO.2) and HH5-1RNEW (SEQ ID NO.6) and the primer combination of HH5-2F (SEQ ID NO.4) and HH5-2R (SEQ ID NO.7), and then the two PCR products were sequenced using HH5-1RNEW (SEQ ID NO.6) and HH5-2F (SEQ ID NO.4), respectively, and no insertion of 9bp (TGATTACA) was found, indicating that the detection method was accurate and no false negative was found.
(3) Applications of
The detected 3 HH5 carrier cows need to be scientifically matched in future breeding to avoid mating with bulls carrying HH5, so that early embryo death caused by generation of HH5 mutant homozygotes is avoided, economic loss of a cattle farm is avoided, and the breeding efficiency of cattle herds is improved.
Although the present invention and the embodiments thereof have been described in detail, it should be understood that modifications to the corresponding conditions and the like can be made by those skilled in the art without departing from the technical principle of the present invention, and the modifications should be construed as the scope of the present invention.
Sequence listing
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Claims (10)
1. An InDEL molecular marker related to Holstein cattle HH5 genetic defect is characterized in that a 9bp fragment is inserted into the 41 th to 49 th positions of a nucleotide sequence shown as SEQ ID NO.1, and the sequence of the fragment is TGATTACAA.
2. The InDEL molecular marker of claim 1, wherein the InDEL molecular marker is detectable by PCR using a specific primer set, wherein the specific primer set is any one of the following:
(1) The nucleotide sequences are respectively shown as specific primer combinations of SEQ ID NO.2, SEQ ID NO.7 and SEQ ID NO. 8; or
(2) The nucleotide sequences are respectively shown as specific primer combinations of SEQ ID NO.2-3 and SEQ ID NO. 4-5.
3. A specific primer combination for detecting InDEL molecular markers of claim 1 or 2, which is a combination of any one of the following groups:
(1) The nucleotide sequences are respectively shown as SEQ ID NO.2, SEQ ID NO.7 and SEQ ID NO. 8; or
(2) The primer combination comprises 2 pairs of specific primer pairs, and the nucleotide sequences of the 2 pairs of specific primer pairs are respectively shown as SEQ ID NO.2-3 and SEQ ID NO. 4-5.
4. a primer combination for screening Holstein cattle HH5 genetic defect gene carriers is characterized by comprising 2 pairs of specific primer pairs, and the nucleotide sequences of the specific primer pairs are respectively shown as SEQ ID NO.2-3 and SEQ ID NO. 4-5.
5. A kit for screening a carrier of the genetic defect gene of Holstein bovine HH5, comprising the primer combination according to claim 4.
6. The kit according to claim 5, characterized in that it comprises the following working steps:
(1) Extracting genome DNA of a cattle sample to be detected, and carrying out double PCR reaction by taking the genome DNA as a template and primers shown in SEQ ID NO.2-3 and SEQ ID NO.4-5 as amplification primers;
(2) Carrying out gel electrophoresis detection on the PCR product, and carrying out genotype judgment according to an electrophoresis result: if only a 209bp band is amplified, the sample is from a Holstein bovine HH5 genetic defect gene wild-type homozygote; if two bands are amplified, namely 209bp and 349bp respectively, the sample to be detected is from a Holstein cow HH5 genetic defect gene carrier.
7. The kit of claim 5, wherein the PCR reaction conditions in step (1): pre-denaturation at 94 ℃ for 5 min; then 35 cycles, 94 ℃ denaturation 30sec, 56 ℃ renaturation 30sec, 72 ℃ extension 30 sec; finally, extension was carried out at 72 ℃ for 7 min.
8. A method for detecting a carrier of a HH5 genetic defect gene of a holstein cow, comprising method 1 or method 2, wherein the method 1 comprises the steps of:
(1) Extracting genome DNA of a cattle sample to be detected, and carrying out double PCR reaction by taking the genome DNA as a template and primers shown in SEQ ID NO.2-3 and SEQ ID NO.4-5 as amplification primers;
(2) Carrying out gel electrophoresis detection on the PCR product, and carrying out genotype judgment according to an electrophoresis result: if only one 209bp band is amplified, the sample to be detected comes from the wild homozygote of the Holstein cattle HH5 genetic defect gene; if two bands are amplified, namely 209bp and 349bp respectively, the sample to be detected is from a Holstein cow HH5 genetic defect gene carrier;
The method 2 comprises the following steps: extracting genome DNA of a cattle sample to be detected, and carrying out PCR reaction by taking the genome DNA as a template and primers shown in SEQ ID NO.2 and SEQ ID NO.7 as amplification primers; and sequencing the amplification product by using the inner primer shown by SEQ ID NO.8, wherein if a segment of 9bp in total of TGATTACAA is inserted into the 41 th to 49 th sites of the nucleotide sequence shown by SEQ ID NO.1, the sample to be detected is from a Holstein cow HH5 genetic defect gene carrier.
9. Use of the InDEL molecular marker of claim 1 or 2 or the primer combination of claim 4 for breeding cattle, to detect carriers, to phase out carriers in cattle breeding populations, or to avoid mating carriers during breeding.
10. Use of the InDEL molecular marker of claim 1 or 2 or the primer combination of claim 3 or 4 for port quarantine for inspection and quarantine of bovine genetic material including live bovine, frozen semen, embryo.
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| CN106319088A (en) * | 2016-11-15 | 2017-01-11 | 北京奶牛中心 | Specific kit for detecting cow HH5 haplotype and detecting method thereof |
| CN108456729A (en) * | 2018-06-15 | 2018-08-28 | 中国农业大学 | Primer combination, kit and its application for detecting 8 kinds of genetic defect gene locis of milk cow simultaneously |
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| CN106319088A (en) * | 2016-11-15 | 2017-01-11 | 北京奶牛中心 | Specific kit for detecting cow HH5 haplotype and detecting method thereof |
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