InDEL molecular marker associated with holstein cow hh2 genetic defect and application thereof
By identifying a 1bp deletion mutation site in the IFT80 gene through whole-genome sequencing, a PCR typing method was designed, which solved the problem of high cost and time consumption in detecting HH2 genetic defects in Holstein cattle. This method is a rapid and low-cost detection method suitable for detection and breeding applications in ordinary molecular laboratories.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-02-07
- Publication Date
- 2026-03-27
AI Technical Summary
Existing technologies are difficult to use quickly and cost-effectively to detect HH2 genetic defects in Holstein cattle, resulting in high testing costs, long processing times, and limited applicability in ordinary molecular laboratories.
A candidate mutation site with a 1bp deletion in exon 12 of the IFT80 gene was identified through whole-genome sequencing. A PCR typing method was designed, and rapid detection was performed using specific primers and Sanger sequencing.
It enables rapid, low-cost, and convenient detection of HH2 genetic defects, can be applied in ordinary molecular laboratories, effectively screens and eliminates recessive harmful HH2 genes, and has important value in breeding and genetic disease screening.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of molecular markers, and particularly relates to an InDEL molecular marker related to a Holstein HH2 genetic defect and application thereof. BACKGROUND
[0002] A genetic defect refers to a disease caused by changes in genetic material, which affects the growth and development of animals, mainly manifested as structural defects or dysfunction, thereby affecting production performance, and even leading to death. In recent years, the application of modern molecular breeding techniques such as genome selection has greatly improved the production performance of dairy cows, and excellent family sire bulls are widely used, and milk production has increased significantly. At the same time, the increase in the use frequency of some bulls leads to the reduction of the dairy cow gene pool, the increase of the population inbreeding coefficient, and the increase of the probability of homozygosity of recessive harmful genes, thereby increasing the risk of genetic defects. Many bulls with excellent production performance but carrying genetic defect genes are widely used in the population, and the genetic defect genes are rapidly spread in the population. Common genetic defects in dairy cows are autosomal recessive inheritance, and heterozygotes (i.e. carriers) have no phenotypic difference from healthy individuals, and only recessive homozygotes show obvious symptoms.
[0003] HH2 (Lethal Holstein Haplotype 2) is a genetic defect of Holstein breed, which is in a recessive inheritance mode. In clinical manifestations, HH2 carriers have no phenotypic difference from healthy individuals, and recessive homozygotes die at about 56 days of embryonic period, showing cow abortion (VanRaden et al. 2011; McClure et al. 2014). Previous studies have located the HH2 defect gene on bovine chromosome 1. Since the pathogenic site and its molecular mechanism have not been found, the HH2 carrier needs to be indirectly inferred by a haplotype composed of 74 SNP marker sites (VanRaden et al. 2011; McClure et al. 2014). This process usually relies on SNP chip detection technology, which needs a special chip detection device, and not only has high detection cost but also takes a long time, and is difficult to apply in ordinary molecular laboratories. SUMMARY
[0004] In order to solve the above technical problems, the present application provides an InDEL molecular marker related to a Holstein HH2 genetic defect and application thereof.
[0005] In order to achieve the object of the present application, the present application takes Chinese Holstein as a resource group, and uses whole genome sequencing technology to find a 1bp deletion in the 12th exon of IFT80 gene as a candidate mutation site of HH2 genetic defect through genome sequencing original Reads comparison, genome variation screening, functional mutation mining and other processes, and designs a PCR typing method.
[0006] The present application first finds a candidate mutation site of Holstein HH2 genetic defect, which is a frame shift mutation in IFT80 gene, and the frame shift mutation is located in the 12th exon region of the gene, and the nucleotide sequence of the fragment is shown as SEQ ID NO. 1, and the base T at the 401st position of the sequence is deleted.
[0007] Specifically, it is the deletion of the 107172616th base on the 1st chromosome of the bovine reference genome (GCF_002263795.1). The experiment finds that the frame shift mutation in IFT80 gene is a candidate mutation site of HH2 genetic defect, the heterozygote of the mutation is a HH2 carrier, and the homozygote dies in the early embryonic development.
[0008] According to the above finding, the present application provides an InDEL molecular marker related to Holstein HH2 genetic defect, which is the deletion of the 107172616th base on the 1st chromosome of the bovine reference genome GCF_002263795.1.
[0009] Further, the InDEL molecular marker is the deletion of the 401st base of the nucleotide sequence shown as SEQ ID NO. 1.
[0010] Further, the InDEL molecular marker is amplified by the primers shown as SEQ ID NO. 2 and SEQ ID NO. 3.
[0011] The present application also provides a primer combination for amplifying the InDEL molecular marker.
[0012] As a preferred, when the primer combination includes the primers shown as SEQ ID NO. 2 and SEQ ID NO. 3, it is more conducive to screening Holstein IFT80 gene defect.
[0013] The present application also provides a reagent or kit containing the primer combination.
[0014] Further, as an embodiment, the working steps of the kit of the present application are as follows:
[0015] (1) Extract the genomic DNA of the sample to be tested, and use it as a template to perform PCR amplification with the primers shown in SEQ ID NO. 2-3 as amplification primers. The 25 μL reaction system of PCR: 21 μL of gold medal Mix rapid PCR premix solution (concentration 1.1X, containing DNA polymerase, dNTP, buffer and other components; Beijing Qikexinye Biotechnology Co., Ltd.), 1 μL of each of the two primers (concentration 10 μM), 2 μL of genomic DNA template (about 50 ng).
[0016] (2) Perform gel electrophoresis detection on the PCR amplification product, and after determining that a 387 bp band is amplified, perform bidirectional Sanger sequencing. If the sequencing of the amplified fragment is successful and there is no double peak, then the sample is from a Holstein IFT80 gene wild type homozygote; if the sequencing of the amplified fragment is successful and there is a double peak, then the sample is from a Holstein IFT80 gene defect carrier.
[0017] PCR reaction conditions: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s; 55℃ annealing for 15 s; 72℃ extension for 10 s; repeat for 35 cycles; finally 72℃ extension for 3 min.
[0018] The application also provides the application of the InDEL molecular marker, the primer combination or the reagent or kit in the detection of the Holstein HH2 genetic defect.
[0019] The application also provides the application of the InDEL molecular marker, the primer combination or the reagent or kit in the breeding of cattle.
[0020] Specifically, the application is to select Holstein cattle that do not carry IFT80 gene defects, or to eliminate Holstein cattle that carry IFT80 gene defects, or to avoid mating cattle that carry IFT80 gene defects.
[0021] The application also provides the application of the InDEL molecular marker, the primer combination or the reagent or kit in the inspection and quarantine of cattle genetic material at the port. The cattle genetic material includes live cattle, frozen semen and embryos.
[0022] The application further provides a method for detecting the Holstein HH2 genetic defect, which uses the genomic DNA of the cattle to be tested as a template and performs PCR amplification with the primers. If the sequencing of the amplified fragment is successful and there is no double peak, then the cattle to be tested is a Holstein IFT80 gene wild type homozygote; if the sequencing of the amplified fragment is successful and there is a double peak, then the cattle to be tested is a Holstein IFT80 gene defect carrier.
[0023] As a preferred, the method comprises the following steps:
[0024] (1) with the genomic DNA of the to-be-tested cow as a template, using the primers shown in SEQ ID NO. 2-3 as amplification primers, performing PCR amplification;
[0025] (2) performing gel electrophoresis detection on the PCR amplification product, determining that a 387bp band is amplified, and then performing bidirectional Sanger sequencing, if the sequencing of the amplified fragment is successful and there is no double peak, then the to-be-tested cow is a wild type homozygote of the Holstein cow IFT80 gene; if the sequencing of the amplified fragment is successful and there is a double peak, then the to-be-tested cow is a Holstein cow IFT80 gene defect carrier.
[0026] The beneficial effects of the present application are at least embodied in the following aspects:
[0027] (1) The present application takes the HH2 wild type homozygote Holstein cow and the HH2 carrier Holstein cow as reference samples, finds a frame shift mutation site caused by 1bp deletion of the IFT80 gene exon in the candidate region, and determines that the InDEL molecular marker is associated with HH2 genetic defects.
[0028] (2) The present application designs specific primers for the InDEL molecular marker, and realizes rapid and accurate typing of the wild type and mutant of the Holstein HH2 genetic defect.
[0029] (3) Compared with the traditional method, the method of the present application has the characteristics of low cost, rapid and efficient, and simple operation. Before this, the identification of HH2 carriers needs to be detected by SNP chip, which has high technical difficulty, long time consumption and high cost. The method of the present application can be typed by ordinary PCR and Sanger sequencing, and the detection method is simple and efficient, and can be completed in an ordinary molecular genetic laboratory, which provides a technical means for gradually eliminating the HH2 recessive harmful gene in the Chinese Holstein cow population in the future, and has important application value in the screening and breeding process of genetic diseases of cattle. BRIEF DESCRIPTION OF DRAWINGS
[0030] Figure 1 It is a schematic diagram of the frame shift mutation caused by 1bp deletion of the Holstein IFT80 gene.
[0031] Figure 2 It is an Integrative Genomics Viewer (IGV) image of the whole genome sequencing Reads of the HH2 carrier at the IFT80 gene frame shift mutation site position. The gray horizontal line represents the Reads band aligned to the vicinity of the site, and the middle black horizontal line represents the missing base.
[0032] Figure 3 It is a sequence characteristic and primer design schematic diagram of the HH2 wild type and mutant, the underlined part is the missing base, and the square box is the forward and reverse primers.
[0033] Figure 4 Sanger sequencing peak chart of wild type homozygote and IFT80 gene defect carrier heterozygote of Holstein cow at the frameshift mutation site, Figure (1) is the mutant carrier sample, the double-peak base is marked as N; Figure (2) is the wild type sample, the arrow in the figure points to the deletion site. DETAILED DESCRIPTION
[0034] The following examples are used to illustrate the present application, but not to limit the scope of the present application.
[0035] Unless otherwise specified in the examples, the techniques or conditions are carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels.
[0036] Example 1 Mining of molecular markers associated with HH2 genetic defects of Holstein cow
[0037] 1. Sample collection and extraction of genomic DNA
[0038] In the present application, 8 cows were selected for whole genome sequencing. These samples were first detected and haplotype analyzed by bovine genome SNP chip (NEOGEN GGP Bovine 100K) to determine 4 as HH2 genetic defect carriers and 4 as wild type Holstein cow individuals. Venous blood was collected, and genomic DNA was extracted by phenol-chloroform method.
[0039] 2. Genome sequencing and variation detection
[0040] Whole genome sequencing was performed on 4 HH2 carriers and 4 wild type Holstein cows by high-throughput second-generation sequencing platform. After quality control, the sequencing data was about 443 Gb, including 2951802390 Reads. The BWA software MEM method was used to align the sequencing Reads to the bovine reference genome (ARS-UCD1.2; GCF_002263795.1), and the GATK software was used to complete the variation detection and quality control. Previous SNP chip-based studies preliminarily located the HH2 genetic defect gene to the 94860836bp-96553339 region of chromosome 1 (bovine UMD 3.1 reference genome) (McClure et al.2014). Given that whole genome sequencing technology can completely screen functional mutations affecting protein-coding genes, the present application expands the candidate genomic region based on the reported genomic region, detects variation sites in the 82693722bp-107343500bp region of chromosome 1 (newly published bovine reference genome GCF_002263795.1), and obtains 102671 SNPs and 18003 Indels for later analysis.
[0041] 3. Mining of functional mutations
[0042] According to the recessive genetic characteristics of the Holstein HH2 defective gene, the causal mutation site is a mutant heterozygote in carriers and a wild-type homozygote in normal cows. According to the above characteristics, 392 SNP sites and 52 INDEL sites were screened in the 24.64 Mb (chromosome 1 82693722bp-107343500bp) candidate region. Using snpEFF software to annotate the screened variation sites, it was found that the above 392 SNP sites and 51 INDEL sites all exist in the non-coding region of the gene, and only the deletion of base T at position 107172616 (g.107172616delT, as shown in Figure 1 and Figure 2 located in the 12th exon of the coding region of the IFT80 gene, which belongs to the frameshift mutation type that makes the gene lose function. The cell cilia inner transport protein encoded by the IFT80 gene plays an important role in the Hedgehog signaling process in mice. The loss of its function will inhibit the transmission of Hedgehog signal in target cells, thereby preventing the differentiation of osteoblasts, leading to embryonic death during the development period (Wang et al, 2013). It is thus speculated that the frameshift mutation on the exon of the IFT80 gene is a candidate causal mutation site of the Holstein HH2 genetic defect haplotype.
[0043] Example 2 Detection and typing of the frameshift mutation site of the Holstein IFT80 gene
[0044] Design specific primers for PCR amplification and Sanger sequencing, and detect and type the mutation site by sequence difference.
[0045] 1. Design of specific primers
[0046] Referring to the reference genome sequence (GCF_002263795.1) of the bovine IFT80 gene and the position of the gene frameshift mutation site on the gene, a pair of specific PCR primers (as shown in Figure 3 ) were designed near the variation site using Primer Premier 5 software:
[0047] Forward primer 5'-TTTCTTTTCCTACCTCCA-3',
[0048] Reverse primer 5'-TTCATCAGCTTTGTCTTT-3'.
[0049] 2. PCR amplification and agarose gel electrophoresis
[0050] PCR reaction system: total volume 25 μL, 21 μL of PCR premix (Gold Mix, Beijing Chengke New Industry Biotechnology Co., Ltd.), 1 μL of each of the forward and reverse primers (both at a concentration of 10 μM), and 2 μL of template DNA (about 50 ng). PCR reaction conditions: 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s; 55°C annealing for 15 s; 72°C extension for 10 s; repeat for 35 cycles; and finally 72°C extension for 3 min.
[0051] The amplified PCR product was subjected to 2% agarose gel electrophoresis, and the gel was detected by a gel imaging system. If the amplified main band image is clear, the brightness is moderate, and there is basically no mixed band, it indicates that the concentration and specificity of the amplified gene sequence are good.
[0052] The PCR instrument used was Bio-Rad T100, the electrophoresis instrument was Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd. JY600+, and the gel imaging instrument was from Alpha Innotech.
[0053] 3. Sanger sequencing
[0054] After the PCR product was verified by electrophoresis to have good specificity, bidirectional Sanger sequencing was performed using PCR primers, and the sequencing results were observed and analyzed on the software Chromas.
[0055] Since the genotype of the mutation site in the carrier is heterozygous, the Sanger sequencing peak graph after the mutation site is double-peak shaped, while the wild type does not have the mutation site, and the peak graph is a normal single-peak shape. The comparison of Sanger sequencing results of IFT80 gene wild type and mutant near the site is shown in Figure 4 .
[0056] 8 whole genome sequencing samples (derived from 8 cows in Example 1) were verified by PCR product Sanger sequencing, and the results were completely consistent, i.e., the HH2 carrier had a 1 bp deletion at position 107172616 on chromosome 1, causing a frameshift mutation of the IFT80 gene.
[0057] Through statistical testing of the 8 sequencing samples (Fisher's exact test, R software fisher.test function), the results are shown in Table 1 below. The association of the IFT80 gene frameshift mutation site with HH2 reached a significant level (P = 0.02857).
[0058] Table 1
[0059]
[0060] As can be seen, the above PCR primers combined with Sanger sequencing can be used to screen for HH2 genetic defect carriers.
[0061] 4. Mutation detection method application
[0062] 26 heads of Chinese Holstein bull frozen semen were newly collected, and the genomic DNA in the frozen semen was extracted by high salt method. Detection by bovine SNP chip and haplotype analysis showed that 10 heads of HH2 carriers and 16 heads of wild type were included in the 26 heads of Holstein bulls. The IFT80 gene 1bp deletion mutation site (g.107172616delT) of the above-mentioned 26 heads of bulls was detected by the PCR primer combined with Sanger sequencing method disclosed in the application, and the sequencing typing result was completely consistent with the SNP chip analysis result (see Table 2), and Fisher exact test (R software fisher.test function) showed that the site was associated with HH2 at a very significant level (P = 1.883 x 10 -07 ).
[0063] Table 2
[0064]
[0065] Although the present application has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed. SEQUENCE LISTING <110> China Agricultural University <120> InDEL molecular marker related to Holstein cattle HH2 genetic defect and application thereof <130> KHP211111021.0 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1001 <212> DNA <213> Artificial Sequence <400> 1 taatactgct tcataaaaca aatagagtct gtcattaaaa gattcactga attgtaaatt 60 acatgattat ttattgcttt tgtatagata tgcttcttta ggaaaggaaa tgaattataa 120 tattaaagga atcaaaactt tatagggtgg tgattatgaa cattttcatt tttcttttcc 180 tacctccatc aaacaacaaa ggcctaggaa acaaatcaga acaaccaagt gttgactttt 240 caggttgttt ttatatttcg ttctttatat atctatgtct tattattgtt ttaattgtaa 300 gatttagata tttccaaagc ataaaatatt ttctaatatg aaaatgaaat tatagtaaat 360 cctacttgag aatgcatctt tattctgtat tttttagaca ttttcttctt gtagatggtg 420 gtggtatcta tttatattct tatgaagggc gcttcctttc ctctccaaaa tttcctggaa 480 tgagaacaga tattctaaat gcacagactg tctctctgag taatgatacc atagcaataa 540 aagacaaagc tgatgaaaaa agtaagtgca tttttataat tttcaatgaa atttattaaa 600 attttcatca attgactttc attttctcat ctattaatat attgacttta tatggaagaa 660 tgatgagcta atttgtccta atttgtccta atcatgaatt ctaattctaa tttgtcctaa 720 tcatgaatta aagaactaaa gttgtagtat catttccagt tgatggaaca aataaatatt 780 gtcacaacat caaagcttag ttgtaagaag taaatctata tattcagtag ctgttggtat 840 agttttaatt caagactgtg agtcctaaaa atgtatgtta atttttaagt ttattgaaag 900 tatttccttt ttctttattt agtaacatag taaatgttgt gcttcattgt actatttatt 960 ttgcttatgt atattcatgt attttttata cttacatatt t 1001 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 tttcttttcc tacctcca 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <400> 3 ttcatcagct ttgtcttt 18
Claims
1. The application of the InDEL molecular marker or primer combinations for detecting the InDEL molecular marker in the preparation of reagents for detecting the HH2 genetic defect in Holstein cattle, characterized in that, The InDEL molecular marker is a deletion of the base at position 107172616 on chromosome 1 of the bovine reference genome GCF_002263795.1, specifically a deletion of the base at position 401 in the nucleotide sequence shown in SEQ ID NO.1; The primer combination includes the primers shown in SEQ ID NO.2 and SEQ ID NO.3; The application method is as follows: using the genomic DNA of the bovine to be tested as a template, PCR amplification is performed using the primer combination; If the amplified fragment is successfully sequenced and there are no double peaks, then the cattle being tested are wild-type homozygous for the Holstein IFT80 gene. If the amplified fragment is successfully sequenced and shows a double peak, the tested cattle are Holstein cattle IFT80 gene defect carriers.