Molecular marker associated with yak immune index and use thereof in breeding

Abstract

The present invention belongs to the technical field of molecular biological detection, and particularly relates to a molecular marker associated with a yak immune index and the use thereof in breeding. In the present invention, an SNP molecular marker associated with a yak immune trait is obtained by means of screening. The marker is located at base position 26593056 on chromosome 3 of the yak reference genome assembly LU_Bosgru_v3.0, with the mutant base being G or A. In the present invention, the contents of immunoglobulin A, immunoglobulin G and immunoglobulin M in a yak individual can be obtained by using the SNP molecular marker, which provides a new SNP molecular marker resource for marker-assisted selection of yak immune traits for non-diagnostic purposes, thereby providing a basis for the breeding of yaks with high immunity.

Description

A molecular marker associated with yak immune indicators and its application in breeding. Technical Field

[0001] This invention relates to the field of molecular biology detection technology, and in particular to a molecular marker related to yak immune indicators and its application in breeding. Background Technology

[0002] Yaks, having long inhabited high-altitude regions, face harsh environmental conditions and require high immunity and disease resistance to adapt to the stresses of high altitude, low air pressure, strong ultraviolet radiation, hypoxia, and malnutrition. In yak farming, disease poses a serious threat to their health and leads to significant economic losses. Although improved feeding management and the use of medications and vaccines can prevent disease to some extent, these methods have not effectively controlled the spread of infectious diseases. Furthermore, the widespread use of drugs may lead to the development of drug resistance, affecting the safety of livestock products. Therefore, in the long run, conducting disease-resistant breeding through gene screening to enhance the disease resistance of yaks is the fundamental way to solve this problem.

[0003] Immune indicators are crucial indicators of a yak's disease resistance, depending on the health and function of its immune system. The primary function of the immune system is to identify and eliminate invading pathogens. When pathogens invade, the immune system reacts rapidly, initiating a series of immune processes to resist and clear them. Immune indicators, such as antibody levels and immunoglobulin counts, are important references for assessing an animal's health and disease resistance. By monitoring changes in immune indicators, immune problems in yaks can be identified promptly, allowing for measures to improve their disease resistance and prevent and treat diseases.

[0004] Immunoglobulins are a group of proteins with antibody activity, mainly found in blood plasma, but also in other body fluids, tissues, and some secretions. Immunoglobulins are classified into five classes: immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin E (IgE). Immunoglobulins are closely related to an animal's disease resistance. For example, a decrease in IgG and IgA levels in the body can easily lead to weakened immune function. Conversely, an increase in IgG and IgA levels enhances the body's disease resistance. Increased disease resistance can reduce the use of drugs, lower production costs, and increase breeding efficiency. Therefore, immunoglobulins can serve as immune indicators; changes in immunoglobulin levels can help detect immune problems in yaks in a timely manner, allowing for appropriate measures to improve their disease resistance and prevent and treat diseases.

[0005] With the rapid development of molecular marker technology, early selection in yak breeding using molecular markers to screen for individuals with high disease resistance has become an important means to improve breeding efficiency and accuracy. By screening for gene-level variant sites and conducting association analysis with disease resistance traits, the relationship between related genes and immune traits can be discovered, promoting the development of disease-resistant breeding. This provides new pathways and resources for the breeding of highly disease-resistant yaks and has significant application value. Summary of the Invention

[0006] The purpose of this invention is to provide SNP molecular markers related to yak immune indicators and their applications.

[0007] To achieve the above objectives, the present invention proposes the following technical solution:

[0008] This invention provides a SNP molecular marker associated with immune traits in yaks. The SNP molecular marker is located at the 26,593,056th base on chromosome 3 of the yak reference genome LU_Bosgru_v3.0, with the mutated base being G or A.

[0009] As preferred, the genotype of yak with the mutated base G is GG or GA; the genotype of yak with the mutated base A is AA; the immunoglobulin A content in yak individuals with genotype GG is higher than that in yak individuals with genotype GA or AA; the content of immunoglobulin G and immunoglobulin M in yak individuals with genotypes GG and GA is higher than that in yak individuals with genotype AA.

[0010] This invention also provides the application of the aforementioned SNP molecular marker in the preparation of products for detecting yak immunity or yak assisted breeding.

[0011] The present invention also provides primer pairs for amplifying the SNP molecular markers, the sequences of which are shown in SEQ ID NO:1-2.

[0012] The present invention also provides the application of the primer pair described herein in the preparation of products for detecting yak immunity or products for yak assisted breeding.

[0013] The present invention also provides a kit for detecting yak immunity, comprising a reagent for detecting the SNP molecular marker or the primer pair.

[0014] The present invention also provides a kit for yak assisted breeding, comprising reagents for detecting the SNP molecular markers or the primer pairs described herein.

[0015] This invention also provides a method for non-diagnostic selection of yak immune trait markers, comprising the following steps:

[0016] (1) Extracting yak genomic DNA;

[0017] (2) Using the yak genomic DNA obtained in step (1) as a template, amplification is performed using the primer pair to obtain the amplification product;

[0018] (3) Perform genotyping analysis on the amplification products to obtain yaks with different genotypes; associate the genotypes of yaks with immune indicators; the immunoglobulins are one or more of immunoglobulin A, immunoglobulin G and immunoglobulin M.

[0019] Preferably, the amplification system described in step (2) consists of 25 μL, including: 12.5 μL of 2×L-Exp Taq Master Mix, 8.5 μL of RNase-free water, 1 μL of upstream primer, 1 μL of downstream primer, and 2 μL of template;

[0020] As a preferred option, the amplification program in step (2) is: 94℃ for 1 min, 98℃ for 10 s, 58℃ for 30 s, 72℃ for 1 min, for a total of 35 cycles; extension at 72℃ for 2 min.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention provides a SNP molecular marker related to yak immune indicators and its role in breeding. Through research, this invention discovered that the SNP locus related to yak immunity is located at the 26,593,056th base on chromosome 3 of the yak reference genome LU_Bosgru_v3.0, with a variant type of G / A. There are three genotypes: when the 26,593,056th base on chromosome 3 is G, the genotype is GG or GA; when the 26,593,056th base on chromosome 3 is A, the genotype is AA.

[0023] Association analysis between different genotypes and the levels of immunoglobulin A, immunoglobulin G, and immunoglobulin M revealed that the immunoglobulin A level in yak individuals with genotype GG was higher than that in yak individuals with genotypes GA or AA (p<0.05); and the levels of immunoglobulin G and immunoglobulin M in yak individuals with genotypes GG and GA were higher than those in yak individuals with genotype AA (p<0.05).

[0024] This invention, by detecting the bases at nucleotide site 26,593,056 on chromosome 3 of yaks, can determine the levels of immunoglobulin A, immunoglobulin G, and immunoglobulin M in an individual yak. ​​This invention provides a new SNP molecular marker resource for non-diagnostic marker-assisted selection of yak immune traits, and provides a basis for breeding yaks with high immunity. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0026] Figure 1 shows the PCR amplification products: where M represents the marker; 1, 2, and 3 represent three replicates.

[0027] Figure 2 shows the peak diagram and sequence obtained after sequencing the PCR products. Detailed Implementation

[0028] This invention provides a SNP molecular marker associated with immune traits in yaks. The SNP molecular marker is located at the 26,593,056th base on chromosome 3 of the yak reference genome LU_Bosgru_v3.0, with the mutated base being G or A.

[0029] In this invention, the genotype of yak with a mutated base G is GG or GA; the genotype of yak with a mutated base A is AA; the immunoglobulin A content in yak individuals with genotype GG is higher than that in yak individuals with genotype GA or AA; the contents of immunoglobulin G and immunoglobulin M in yak individuals with genotypes GG and GA are higher than those in yak individuals with genotype AA.

[0030] This invention also provides the application of the aforementioned SNP molecular marker in the preparation of products for detecting yak immunity or screening yak breeds with high immunity.

[0031] The present invention also provides primer pairs for amplifying the SNP molecular marker gene fragments described above, the sequences of which are shown in SEQ ID NO:1-2.

[0032] SEQ ID NO:1 is the upstream primer, and its sequence is:

[0033] 5'-CACACAGGAAGCAAAGGCAC-3';

[0034] SEQ ID NO:2 is the downstream primer, and its sequence is:

[0035] 5'-TAGGACAGCAAAGCCAGCAA-3'.

[0036] The present invention also provides the application of the primer pair described herein in the preparation of products for detecting yak immunity or products for yak assisted breeding.

[0037] The present invention also provides a kit for detecting yak immunity, comprising a reagent for detecting the SNP molecular marker or the primer pair.

[0038] The present invention also provides a kit for yak assisted breeding, comprising reagents for detecting the SNP molecular markers or the primer pairs described herein.

[0039] This invention also provides a method for non-diagnostic selection of yak immune trait markers, comprising the following steps:

[0040] (1) Extracting yak genomic DNA;

[0041] (2) Using the yak genomic DNA obtained in step (1) as a template, amplification is performed using the primer pair to obtain the amplification product;

[0042] (3) Perform genotyping analysis on the amplification products to obtain yaks with different genotypes; associate the genotypes of yaks with immune indicators; the immunoglobulins are one or more of immunoglobulin A, immunoglobulin G and immunoglobulin M.

[0043] In this invention, the amplification system described in step (2) is 25 μL in total, including: 12.5 μL of 2×L-Exp Taq Master Mix, 8.5 μL of RNase-free water, 1 μL of upstream primer, 1 μL of downstream primer, and 2 μL of template;

[0044] In this invention, the amplification program in step (2) is as follows: 94℃ for 1 min, 98℃ for 10 s, 58℃ for 30 s, 72℃ for 1 min, for a total of 35 cycles; extension at 72℃ for 2 min.

[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1

[0047] 1 Sample Collection

[0048] The invention uses the Nya yak breed as the testing subject. 5 mL of blood samples were collected from 192 fasting yaks in a pasture in Jiali County, Nagqu City, Tibet Autonomous Region, in clean, anticoagulant vacuum blood collection tubes. The samples were allowed to stand for 30 minutes, then centrifuged at 3500 rpm for 10 minutes. The supernatant was collected into PE tubes, sealed, and stored at -20°C. Another 5 mL blood sample was collected in blood collection tubes containing EDTA-K2 anticoagulant. After collection, the samples were quickly mixed, placed in a sampling box with ice packs for temporary storage, and then transported back to the laboratory and frozen at -20°C for genomic DNA extraction.

[0049] 2. Main Reagents and Instruments

[0050] EDTA-K2 vacuum blood collection tubes were purchased from Jiangsu Yuli Medical Instrument Co., Ltd.; the blood genomic DNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; the Nano Drop 2000 spectrophotometer was purchased from Thermo Fisher Scientific, USA; DL2000 Marker, agarose, and nucleic acid dyes were purchased from Beijing Solarbio Science & Technology Co., Ltd.; 2×L-Exp Taq MasterMix (dye plus) was purchased from Hunan Aikerui Biotechnology Co., Ltd.; the electrophoresis apparatus was purchased from Beijing Liuyi Instrument Factory; the PCR instrument was purchased from BioRad. IgA (MB-4907A), IgG (MB-4616A), and IgM (MB-4908A) detection kits were purchased from Jiangsu Enzyme-Label Biotechnology Co., Ltd.

[0051] 3 methods

[0052] 3.1 Detection of immunoglobulins IgA, IgG, and IgM

[0053] The IgA, IgG, and IgM detection kits from Jiangsu Enzyme Biotechnology Co., Ltd. were used for assays using a one-step sandwich method with double antibodies. First, the required strips were removed from the aluminum foil bag after equilibration at room temperature for 20 minutes. The remaining strips were sealed in a resealable bag and returned to 4°C. Standard and sample wells were prepared. 50 μL of different concentrations of standard were added to each standard well. 10 μL of the test sample was added to each sample well, followed by 40 μL of sample diluent. No diluent was added to the blank wells. Except for the blank wells, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each of the standard and sample wells. The reaction wells were sealed with sealing film and incubated at 37°C in a water bath or incubator for 60 minutes. The liquid was discarded, and the plates were patted dry on absorbent paper. Washing buffer was added to each well, and the plates were allowed to stand for 1 minute. The washing buffer was then discarded, and the plates were patted dry on absorbent paper. This washing process was repeated 5 times (or a plate washer could be used). 50 μL each of substrates A and B were added to each well, and the plates were incubated at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well, and measure the OD value of each well at 450 nm within 15 min. Finally, plot the standard curve: In an Excel worksheet, plot the standard concentration on the x-axis and the corresponding OD value on the y-axis to create a linear regression curve of the standard. Calculate the IgA, IgG, and IgM concentrations of each sample according to the curve equation.

[0054] 3.2 Extraction of genomic DNA from blood

[0055] Genomic DNA was extracted from blood samples using the blood genomic extraction kit from Tiangen Biotech (Beijing) Co., Ltd. The extracted DNA was then analyzed for concentration and purity using a UV spectrophotometer. Concentrations >20 ng / μL and OD values ​​>20 were acceptable. 260 / OD 280 A pH between 1.7 and 1.9 is sufficient for experimental needs; store at -20°C for future use.

[0056] 3.3 Primer Design

[0057] Based on the gene sequence of chromosome 3 of the yak genome LU_Bosgru_v3.0 (GenBank accession number: GCA_005887515.1), specific primers containing the g26593056G>A SNP site were designed using the Pick Primers online tool provided by NCBI.

[0058] Primer sequence

[0059] F: 5'-CACACAGGAAGCAAAGGCAC-3' (SEQ ID NO: 1);

[0060] R: 5'-TAGGACAGCAAAGCCAGCAA-3' (SEQ ID NO: 2).

[0061] The amplified fragment was 389 bp in length, and the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0062] 3.4 PCR Amplification and Sequencing

[0063] PCR amplification system 25μL: 2×L-Exp Taq Master Mix (dye plus) 12.5μL, RNase free water 8.5μL, upstream primer 1μL, downstream primer 1μL, template 2μL.

[0064] PCR amplification program: 94℃ for 1 min, 98℃ for 10 s, 58℃ for 30 s, 72℃ for 1 min, for a total of 35 cycles; extension at 72℃ for 2 min.

[0065] PCR products were analyzed by 1% agarose gel electrophoresis. After passing the agarose gel electrophoresis, the PCR products were sequenced using direct sequencing, which was performed by Beijing Qingke Biotechnology Co., Ltd. The agarose gel electrophoresis results are shown in Figure 1. Figure 1 shows that the PCR-amplified sequence was 389 bp in length. Sequencing revealed a G / A mutation at position 189 (nucleotide 26593056 on chromosome 3 of the LU_Bosgru_v3.0 genome). The amplified product band was clear and free of extraneous bands, showing good specificity. This site was preliminarily identified as a yak SNP marker site and named g26593056G>A SNP. The PCR-amplified sequence is shown in SEQ ID NO:3, and position 189 of this sequence shows a G mutation. The size of the PCR-amplified product fragment met the expected size and could be used for further experiments.

[0066] SEQ ID NO:3

[0067] The sequencing results of PCR products were compared using the bioanalysis software MEGA 11.0, and the sequencing peak diagrams were analyzed to complete the typing.

[0068] 4. Statistical Analysis

[0069] According to the genotyping results, the number of individuals with different genotypes at each locus was counted. The Popgen32 software was used to calculate the gene frequency, genotype frequency, effective number of alleles (Ne), locus heterozygosity (He), and Hardy-Weinberg equilibrium test of the g26593056G>A gene, and the polymorphism information content (PIC) was calculated using the PIC (polymorphism information content) calculation software. The general linear model in IBM SPSS Statistics 26 software was used to analyze the association between different genotypes of yaks and immunoglobulins IgA, IgG, and IgM, and the results were expressed as "mean ± standard error".

[0070] 5 Results

[0071] 5.1 PCR Amplification and Sequencing Results

[0072] The amplification products of the g26593056G>A SNP locus on chromosome 3 of yaks were detected using 1% agarose gel (see Figure 1). The bands were clear without heterologous bands, and the specificity was good. The fragment size of the PCR product was 389bp, which was consistent with the expected size, and the next experiment could be carried out.

[0073] The peak map and sequence obtained after purifying and sequencing the PCR product are shown in Figure 2. As can be seen from Figure 2, a G-A mutation occurred at the g26593056G>A SNP locus, and there were three genotypes: GG, GA, and AA.

[0074] 5.2 Statistical Analysis Results

[0075] The genotypes and allele frequencies of the g26593056G>A SNP locus on chromosome 3 of yaks were analyzed from the perspective of population genetics. As shown in Table 1, at the g26593056G>A SNP locus, the GA genotype frequency was the highest, which was the dominant genotype, and the G allele frequency was 54.2%, showing as the dominant allele. By χ 2 The adaptability test showed that the SNP locus conformed to the Hardy-Weinberg equilibrium state (P>0.05) (Table 1). The expected heterozygosity of this locus was 0.497, and the PIC was 0.373. 0.25<PIC<0.50, belonging to moderate polymorphism.

[0076] Table 1 Polymorphism of the g26593056G>A SNP Locus on Chromosome 3 of Yaks

[0077] 5.3 Association Analysis between Different Genotypes and Immunoglobulins IgA, IgG, and IgM

[0078] The association between different yak genotypes and the levels of immunoglobulins IgA, IgG, and IgM was analyzed using a general linear model in IBM SPSS Statistics 26 software. The results showed that the immunoglobulin A level was higher in yaks with genotype GG than in those with genotypes GA or AA (p<0.05); the levels of immunoglobulin G and immunoglobulin M were higher in yaks with genotypes GG and GA than in those with genotype AA (p<0.05). This indicates that the base at the g26593056G>A locus on yak chromosome 3 is significantly correlated with yak IgA, IgG, and IgM (p<0.05), and is a yak-related SNP marker. The results are shown in Table 2.

[0079] Table 2. Correlation analysis between different genotypes and immunoglobulins IgA, IgG, and IgM.

[0080] Note: Different lowercase letters in the intercalation of data in the same row indicate significant differences (P<0.05).

[0081] The SNP molecular marker described in this invention is located at the 26,593,056th base on chromosome 3 of the reference yak genome LU_Bosgru_v3.0. The variant type is G / A, named g26593056G>A, and there are three genotypes. When the 26,593,056th base on chromosome 3 is G, the genotype is GG or GA; when the 26,593,056th base on chromosome 3 is A, the genotype is AA. Through association analysis between different genotypes and the levels of IgA, IgG, and IgM, it was found that the immunoglobulin A content in yak individuals with the GG genotype was higher than that in yak individuals with the GA or AA genotypes (p<0.05); the immunoglobulin G and immunoglobulin M content in yak individuals with the GG and GA genotypes was higher than that in yak individuals with the AA genotype (p<0.05). This invention can determine the levels of immunoglobulin A, immunoglobulin G, and immunoglobulin M in an individual yak by detecting the bases at the 26,593,056th nucleotide site on chromosome 3 of the yak. ​​This invention provides a new SNP molecular marker resource for the selection of yak immune trait markers for non-diagnostic purposes.

[0082] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a reagent for detecting SNP molecular markers in the preparation of in vitro detection reagents for yak immune traits or in yak immune trait-assisted breeding detection reagents, characterized in that, The SNP molecular marker is located at the 26,593,056th base on chromosome 3 of the yak reference genome LU_Bosgru_v3.0, with the mutated base being G or A; Among them, the yak genotype with the mutated base A is AA; the immunoglobulin A content in yak individuals with genotype GG is higher than that in yak individuals with genotype GA or AA; the immunoglobulin G and immunoglobulin M content in yak individuals with genotypes GG and GA is higher than that in yak individuals with genotype AA.

2. The application according to claim 1, characterized in that, The primer pairs for detecting the SNP molecular markers are shown in SEQ ID NO.1-2.

3. A method for non-diagnostic selection of yak immune trait markers, characterized in that, Includes the following steps: (1) Extracting yak genomic DNA; (2) Using the yak genomic DNA obtained in step (1) as a template, amplification was performed using primer pairs as shown in SEQ ID NO.1-2 to obtain amplification products; (3) Perform genotyping analysis on the amplification products to obtain yaks with different genotypes; associate the genotypes of yaks with immune indicators; the immunoglobulins are one or more of immunoglobulin A, immunoglobulin G and immunoglobulin M; The primer pair is used to amplify the SNP molecular marker, which is located at the 189th base as shown in SEQ ID No. 3, with the mutated base being G or A; wherein, the yak genotype with the mutated base A is AA; the immunoglobulin A content in yak individuals with genotype GG is higher than that in yak individuals with genotype GA or AA; the immunoglobulin G and immunoglobulin M content in yak individuals with genotypes GG and GA is higher than that in yak individuals with genotype AA.

4. The method according to claim 3, characterized in that, The amplification system described in step (2) consists of 25 μL, including: 12.5 μL of 2×L-Exp Taq Master Mix, 8.5 μL of RNase-free water, 1 μL of upstream primer, 1 μL of downstream primer, and 2 μL of template.

5. The method according to claim 3, characterized in that, The amplification program in step (2) is as follows: 94℃ for 1 min, 98℃ for 10 s, 58℃ for 30 s, 72℃ for 1 min, for a total of 35 cycles; extension at 72℃ for 2 min.

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