CN114908174A - InDEL molecular marker related to Holstein cattle HH2 genetic defect and application thereof - Google Patents
InDEL molecular marker related to Holstein cattle HH2 genetic defect and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of molecular markers, in particular to an InDEL molecular marker related to Holstein cattle HH2 genetic defect and application thereof. The invention provides an InDEL molecular marker related to HH2 genetic defect of Holstein cattle, which is the deletion of 107172616 th base on No.1 chromosome of cattle reference genome GCF _ 002263795.1. 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 2. Compared with the traditional method, the method has the characteristics of low cost, rapidness, high efficiency and simple and convenient operation, provides a technical means for gradually eliminating the HH2 recessive harmful gene in the Chinese Holstein cattle herd in future, and has important application value in the process of screening genetic diseases and breeding of cattle.
Description
Technical Field
The invention relates to the technical field of molecular markers, in particular to an InDEL molecular marker related to Holstein cattle HH2 genetic defect and application thereof.
Background
Genetic defects refer to a class of diseases that affect the growth and development of animals due to changes in genetic material, and are mainly manifested as structural defects or dysfunction of the body, which in turn affects productivity and even causes death. In recent years, the application of modern molecular breeding technologies such as genome selection greatly improves the production performance of dairy cows, so that excellent family breeding bulls are widely used, the milk yield is greatly increased, and meanwhile, the increase of the use frequency of partial bulls causes the reduction of a dairy cow gene bank, the increase of population inbred coefficients, the increase of the probability of recessive harmful gene homozygosis, and the incidence risk of genetic defects is increased. 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 (i.e. carriers) have no phenotypic difference from healthy individuals, and recessive homozygotes show obvious symptoms.
HH2(Lethal Holstein Haplotpype 2) is a genetic defect of Holstein cattle breed, presents a recessive genetic pattern, and has no phenotype difference between HH2 carrier and healthy individuals on clinical presentation, and recessive homozygote dies about 56 days in embryonic period, and presents as cow abortion (VanRaden et al 2011; McClure et al 2014). The HH2 defect gene is positioned on a cattle No.1 chromosome in earlier research, because a pathogenic site and a molecular mechanism thereof are not found yet, an HH2 carrier (VanRaden et al 2011; McClure et al 2014) is indirectly presumed through a haplotype consisting of 74 SNP marker sites, the process usually depends on an SNP chip detection technology, special chip detection equipment is needed, the detection cost is high, the time consumption is long, and the application in a common molecular laboratory is difficult.
Disclosure of Invention
In order to solve the technical problems, the invention provides an InDEL molecular marker related to Holstein cattle HH2 genetic defect and application thereof.
In order to realize the purpose of the invention, the invention takes Chinese Holstein cattle as a resource population, utilizes the whole genome sequencing technical means, finds that 1bp deletion exists on the 12 th exon of IFT80 gene as a candidate mutation site of HH2 genetic defect through the processes of genome sequencing original Reads comparison, genome variation screening, functional mutation excavation and the like, and designs a PCR typing method on the basis.
The invention firstly discovers a candidate mutation site of HH2 genetic defect of Holstein cattle, wherein the candidate mutation site is a frame shift mutation on IFT80 gene, the frame shift mutation is positioned in the 12 th exon region of the gene relative to the DNA sequence of IFT80 gene of healthy Holstein cattle, the nucleotide sequence of the fragment is shown as SEQ ID NO.1, and the 401 th base T of the sequence is deleted.
Specifically, the nucleotide sequence is 107172616 th base deletion on chromosome 1 of a bovine reference genome (GCF _ 002263795.1). The frame shift mutation on IFT80 gene is found to be the candidate mutation site of HH2 genetic defect, the heterozygote of the mutation is the HH2 carrier, and the homozygote dies in early embryonic development.
Based on the above findings, the present invention provides an InDEL molecular marker associated with a genetic defect of holstein cow HH2, which is a deletion of the 107172616 th base on chromosome 1 of the bovine reference genome GCF _ 002263795.1.
Further, the InDEL molecular marker is a 401 th base deletion of the nucleotide sequence shown as SEQ ID NO. 1.
Further, the InDEL molecular marker is obtained by amplifying primers shown in SEQ ID NO.2 and SEQ ID NO. 3.
The invention also provides a primer combination for amplifying the InDEL molecular marker.
Preferably, when the primer combination comprises primers shown in SEQ ID NO.2 and SEQ ID NO.3, the method is more favorable for screening Holstein cattle IFT80 gene defects.
The invention also provides a reagent or a kit containing the primer combination.
Further, as an embodiment, the working steps of the kit of the present invention are as follows:
(1) extracting the genome DNA of a cattle sample to be detected, and carrying out PCR amplification by using the genome DNA as a template and using a primer shown in SEQ ID NO.2-3 as an amplification primer. 25 μ L reaction for PCR: gold medal Mix rapid PCR premix (concentration 1.1X, containing components such as DNA polymerase, dNTP, Buffer, etc.; Beijing Optimaku New Biotechnology Co., Ltd.) 21. mu.L, 1. mu.L each of 2 primers (concentration 10. mu.M), 2. mu.L of genomic DNA template (about 50 ng).
(2) Carrying out gel electrophoresis detection on the PCR amplification product, carrying out bidirectional Sanger sequencing after determining that a 387bp band is amplified, and if the sequencing of the amplified fragment is successful and no double peaks exist, obtaining a sample from a Holstein cattle IFT80 gene wild homozygote; if the amplified fragment was sequenced successfully and there were double peaks, the sample was from a carrier of the Holstein bovine IFT80 gene defect.
And (3) PCR reaction conditions: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 s; renaturation at 55 ℃ for 15 sec; extension at 72 ℃ for 10 sec; repeat for 35 cycles; finally, extension is carried out for 3min at 72 ℃.
The invention also provides application of the InDEL molecular marker or the primer combination or the reagent or the kit in detection of genetic defects of Holstein cattle HH 2.
The invention also provides application of the InDEL molecular marker or the primer combination or the reagent or the kit in cattle breeding.
Specifically, the application is to breed Holstein cattle which do not carry IFT80 gene defects, or to eliminate Holstein cattle which carry IFT80 gene defects, or to avoid cattle which carry IFT80 gene defects from mating.
The invention also provides application of the InDEL molecular marker or the primer combination or the reagent or the kit in port quarantine to bovine genetic material inspection and quarantine. The bovine genetic material comprises live cattle, frozen semen and embryo.
The invention further provides a method for detecting HH2 genetic defect of Holstein cattle, which comprises the steps of taking the genome DNA of cattle to be detected as a template and utilizing the primer to carry out PCR amplification; if the sequencing of the amplified fragment is successful and no double peaks exist, determining that the cattle to be detected is a Holstein cattle IFT80 gene wild homozygote; if the sequencing of the amplified fragment is successful and the amplified fragment has double peaks, the cattle to be detected is a carrier of the Holstein cattle IFT80 gene defect.
Preferably, the method comprises the steps of:
(1) taking the genome DNA of a cattle to be detected as a template, and taking a primer shown in SEQ ID NO.2-3 as an amplification primer to carry out PCR amplification;
(2) performing gel electrophoresis detection on the PCR amplification product, performing bidirectional Sanger sequencing after determining that a 387bp band is amplified, and if the sequencing of the amplified fragment is successful and no double peaks exist, determining that the cattle to be detected is a Holstein cattle IFT80 gene wild type homozygote; if the sequencing of the amplified fragment is successful and double peaks exist, the cattle to be detected are carriers of the Holstein cattle IFT80 gene defect.
The invention has the advantages that at least the following aspects are reflected:
(1) the method takes HH2 wild homozygous Holstein cattle and HH2 carrier Holstein cattle as reference samples, discovers a frame shift mutation site caused by 1bp deletion on an exon of an IFT80 gene in a candidate region, and determines that the InDEL molecular marker is associated with HH2 genetic defect.
(2) 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 2.
(3) Compared with the traditional method, the method has the characteristics of low cost, rapidness, high efficiency and simple and convenient operation. Heretofore, the identification of the HH2 carrier required SNP chip detection, which is difficult, time-consuming and expensive. The method can be typed by using common PCR and Sanger sequencing, has simple, convenient and efficient detection method, can finish detection in a common molecular genetic laboratory, provides a technical means for gradually eliminating the recessive harmful gene HH2 in the Hestein cattle herd in China in the future, and has important application value in the screening and breeding process of the genetic diseases of cattle.
Drawings
FIG. 1 is a schematic diagram of a frame shift mutation caused by deletion of 1bp of Holstein bovine IFT80 gene.
FIG. 2 is an image of Integrated Genomics Viewer (IGV) of whole genome sequencing Reads of HH2 carrier at IFT80 gene frameshift mutation site. The grey horizontal line represents the Reads bands aligned near the site, and the middle black horizontal line represents the missing base.
FIG. 3 is a schematic diagram showing the sequence characteristics and primer design of the wild type and mutant HH2, with the deletion of the base underlined and the positive and negative primers boxed.
FIG. 4 is a Sanger sequencing peak diagram of a Holstein cattle wild type homozygote and an IFT80 gene defect carrier heterozygote at a frame shift mutation site, and FIG. 1 is a mutant carrier sample, wherein a bimodal base is marked as N; FIG. 2 shows a wild-type sample, in which the arrow points to the deletion site.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.
Example 1 mining of Histeinman HH2 genetic Defect associated molecular markers
1. Sample collection and genomic DNA extraction
The invention selects 8 cattle to carry out whole genome sequencing. These samples were first tested by a Bovine genomic SNP chip (NEOGEN GGP Bovine 100K) and haplotype analysis to determine 4 carriers of the HH2 genetic defect and 4 individuals of the wild type Holstein cattle. Venous blood was collected and genomic DNA was extracted by phenol-chloroform method.
2. Genome sequencing and variation detection
Whole genome sequencing was performed on 4 HH2 carriers and 4 wild-type holstein cattle by a high throughput next generation sequencing platform. The sequencing data after quality control was about 443Gb, including 2951802390 Reads. The Reads obtained by sequencing was aligned to the bovine reference genome (ARS-UCD 1.2; GCF-002263795.1) using the BWA software MEM, and mutation detection and quality control were performed using the GATK software. The HH2 genetic defect gene was previously mapped primarily to chromosome 1, 94860836bp-96553339 region (bovine UMD 3.1 reference genome), based on SNP chip studies (McClure et al 2014). In view of the fact that the whole genome sequencing technology can completely screen functional mutations affecting protein coding genes, the invention expands candidate genome regions on the basis of reported genome regions, detects variation sites in 82693722bp-107343500bp regions (newly published bovine reference genome GCF _002263795.1) of chromosome 1, and obtains 102671 SNPs and 18003 indels for later analysis.
3. Excavation of functional mutations
According to the recessive inheritance characteristic of the HH2 defect gene of Holstein cattle, the causal mutation sites are all mutant heterozygotes in carriers, and are wild homozygotes in normal cattle. Based on the characteristics, mutation sites are screened in a candidate region of 24.64Mb (No. 1 chromosome 82693722bp-107343500bp), and 392 SNP sites and 52 INDEL sites are screened out. The screened mutation sites were annotated by snpEFF software, and it was found that the 392 SNP sites and the 51 INDEL sites both exist in the non-coding region of the gene, and that only the 107172616 th base T deletion on chromosome 1 (g.107172616delT, shown in FIGS. 1 and 2) is located in the 12 th exon of the coding region of IFT80 gene, and they belong to the type of frame-shift mutation that makes the gene non-functional. The intracellular transport protein of cellular cilia encoded by IFT80 gene is involved in the important loop of mouse Hedgehog signaling, and the loss of its function can inhibit the conduction of Hedgehog signal in target cells, thus preventing the differentiation of osteoblasts and leading the embryo to die during development (Wang et al, 2013). It is therefore assumed that frame shift mutations in the exons of IFT80 gene are candidate causal mutation sites for the H.holstein cattle HH2 genetically deficient haplotype.
Example 2 detection and typing of frameshift mutation site of Holstein cattle IFT80 Gene
Designing specific primers to carry out PCR amplification and Sanger sequencing, and carrying out detection and typing on mutation sites through sequence difference.
1. Specific primer design
Referring to the reference genomic sequence (GCF _002263795.1) of bovine IFT80 gene and the position of the frameshift mutation site on the gene, 1 pair of specific PCR primers (shown in fig. 3) were designed near the mutation site using Primer Premier 5 software:
the forward primer 5'-TTTCTTTTCCTACCTCCA-3' is the primer that is used for the forward primer,
the reverse primer 5'-TTCATCAGCTTTGTCTTT-3'.
2. PCR amplification and agarose gel electrophoresis
And (3) PCR reaction system: a total volume of 25. mu.L was obtained, wherein the PCR master Mix was 21. mu.L (gold medal Mix, Beijing Optimalaceae, New Biotechnology Co., Ltd.), 1. mu.L each of the forward and reverse primers (concentration: 10. mu.M), and 2. mu.L (about 50ng) of the template DNA. And (3) PCR reaction conditions: performing pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 s; renaturation at 55 ℃ for 15 sec; extension at 72 ℃ for 10 sec; repeat 35 cycles; finally, extension is carried out for 3min at 72 ℃.
And (3) carrying out 2% agarose gel electrophoresis on the PCR product after amplification, and detecting by using a gel imaging system for gel illumination, wherein if the amplified main strip has clear images, moderate brightness and basically no miscellaneous bands, the concentration and the specificity of the amplified gene sequence are good.
The PCR instrument used was of the Bio-Rad T100 type, the electrophoresis instrument was of the JY600+ type, a Photogel instrument, from Alpha Inotech, Beijing Junyi Oriental electrophoresis apparatus Co.
3. Sanger sequencing
After the PCR product is verified to have good specificity through electrophoresis, bidirectional Sanger sequencing is carried out by utilizing the PCR primer, and the sequencing result is observed and analyzed on software Chromas.
Since the mutation site genotype in the carrier is heterozygote, the Sanger sequencing peak pattern thereof is in a bimodal shape after the mutation site, while the wild type does not have the mutation site, and the peak pattern is in a normal monomodal shape. A comparison of Sanger sequencing results of wild type and mutant versions of IFT80 gene near the site is shown in fig. 4.
The 8 whole genome sequencing samples (from 8 cattle in example 1) were verified by Sanger sequencing, which is a PCR product, and the results are completely consistent, namely that the HH2 carrier has 1bp deletion at 107172616 th base on chromosome 1, which causes IFT80 gene frame shift mutation.
The IFT80 gene frameshift mutation site correlated with HH2 to a significant level (P0.02857) as a result of statistical tests on 8 sequenced samples (Fisher exact test, R software Fisher. test function) as shown in table 1 below.
TABLE 1
It can be seen that the PCR primers described above in combination with Sanger sequencing can be used to screen carriers of the genetic defect of HH 2.
4. Mutation detection method application
The frozen semen of 26 Chinese Holstein bulls is newly collected, and the genome DNA in the frozen semen is extracted by a high-salt method. Detection by a bovine SNP chip and haplotype analysis showed that 10 HH2 carriers and 16 wild types were included in the 26 holstein bulls. The PCR primer disclosed by the invention is combined with a Sanger sequencing method to detect the 1bp deletion mutation site (g.107172616delT) of the IFT80 gene of the 26-head bulls, the sequencing and typing result is completely consistent with the SNP chip analysis result (see table 2), and Fisher precise test (R software Fisher. test function) shows that the relevance of the site and HH2 reaches a very significant level (P-1.883 × 10) -07 )。
TABLE 2
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (10)
1. An InDEL molecular marker associated with a genetic defect of Holstein cattle HH2, characterized in that it is a deletion of base 107172616 on chromosome 1 of the bovine reference genome GCF _ 002263795.1.
2. The InDEL molecular marker according to claim 1, which is a base deletion at position 401 of the nucleotide sequence shown in SEQ ID No. 1.
3. The InDEL molecular marker according to claim 1, which is amplified by primers shown in SEQ ID No.2 and SEQ ID No. 3.
4. A primer combination for amplifying an InDEL molecular marker of any one of claims 1-3.
5. The primer combination of claim 4, wherein the primer combination comprises the primers shown in SEQ ID No.2 and SEQ ID No. 3.
6. A reagent or kit comprising the primer combination according to claim 4 or 5.
7. Use of the InDEL molecular marker of any one of claims 1 to 3 or the primer combination of claim 4 or 5 or the reagent or kit of claim 6 for the detection of a genetic defect of holstein cow HH 2.
8. Use of the InDEL molecular marker of any one of claims 1-3 or the primer combination of claim 4 or 5 or the reagent or kit of claim 6 for cattle breeding.
9. Use of the InDEL molecular marker of any one of claims 1-3 or the primer combination of claim 4 or 5 or the reagent or kit of claim 6 for port quarantine against bovine genetic material inspection and quarantine.
10. A method for detecting HH2 genetic defect of Holstein cattle, which is characterized in that genome DNA of cattle to be detected is used as a template, and the primer of claim 4 or 5 is used for PCR amplification; if the sequencing of the amplified fragment is successful and no double peaks exist, the cattle to be detected is a Holstein cattle IFT80 gene wild type homozygote; if the sequencing of the amplified fragment is successful and the amplified fragment has double peaks, the cattle to be detected is a carrier of the Holstein cattle IFT80 gene defect.
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