An SNP locus related to yak milk density and milk fat content and its application

Abstract

The present invention belongs to the fields of molecular biotechnology and molecular marker technology, and specifically relates to an SNP locus related to yak milk density and milk fat content and its application. The SNP locus is located at the 13,729,718th position of chromosome 5 of the yak reference genome Bosgu_v3.0 version with the Ensemble accession number ENSBGRG00000003042, and the mutated base is C or T. The present invention also provides the application of a reagent for detecting the above SNP locus in the auxiliary breeding for detecting yak milk density and milk fat content. This molecular marker can be applied to the auxiliary selection of yak milk density and milk fat content traits, and the detection method is rapid and accurate; by screening the genotypes of this SNP molecular marker, the accuracy and effectiveness of the selection of yak milk density and milk fat content traits can be accelerated, and the economic benefits of yak breeding can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology detection, and particularly relates to an SNP locus related to yak milk density and milk fat content and its application. Background Art

[0002] Yaks (Bos grunnies) are distributed in the alpine regions above 3000 meters above sea level on the Qinghai-Tibet Plateau and its surrounding areas in China. They mainly rely on natural grassland grazing to obtain the nutrients they need for growth and rarely receive artificial supplementary feeding. Yak meat and milk are truly pollution-free green foods and have high development and utilization value. They are an important source of living and production materials for local people. Yak milk has a low yield but is rich in nutrients. Compared with cow's milk, yak milk has obvious advantages in terms of milk protein, milk fat and amino acid content. The commercialization and industrialization of yaks directly affect the economic sources and development levels of plateau animal husbandry and are an important support for the economy of the Qinghai-Tibet Plateau. Therefore, it is very necessary to breed yak breeds with excellent milk quality.

[0003] Single nucleotide polymorphism (SNP) is a DNA sequence polymorphism caused by single nucleotide variations at the genomic level. Such variations include single base transitions, inversions, insertions and deletions. SNPs that occur in the coding region can affect gene function and lead to changes in biological traits, so they can be used as biological markers related to certain traits. SNPs play an important role in many aspects such as livestock and poultry breed identification, genetic breeding, genetic resource protection, and disease diagnosis. Using SNPs for molecular marker-assisted breeding can not only achieve precise and directional improvement of excellent traits of livestock and poultry, improve breeding efficiency, but also ensure the genetic stability and persistence of excellent traits of the bred breeds, and promote the innovation and progress of animal husbandry.

[0004] The Phosphodiesterase 3A (PDE3A) gene belongs to the PDE3 family. The PDE3 family is generally recognized as a cAMP-hydrolyzing PDE inhibited by cGMP, which means that the main role of PDE3 in cells is to regulate the level of cAMP. The carboxyl terminus of the PDE3A gene contains a catalytic domain and an amino-terminal domain, and the amino-terminal domain is important for the enzyme to localize to the particulate part of the carboxyl terminus and the catalytic domain. The protein encoded by the PDE3A gene can initiate 3',5'-cyclic AMP phosphodiesterase activity and participate in the negative regulation of the apoptosis process by hydrolyzing cAMP; negatively regulate cAMP-mediated signals. The PDE3A gene plays an important role in maintaining cell homeostasis and regulating cell function. Summary of the Invention

[0005] Based on the above technical problems, the object of the present invention is to provide an SNP molecular marker related to yak milk density and milk fat content, which is fast, accurate, and has a low detection cost.

[0006] Specifically, it includes the following content:

[0007] In the first aspect, the present invention provides an SNP molecular marker related to yak milk density and milk fat content. The SNP molecular marker is located at the 13,729,718th position on chromosome 5 of the yak reference genome Bosgu_v3.0 version with the Ensemble accession number ENSBGRG00000003042, and the mutated base is C or T.

[0008] In the second aspect, the present invention provides the application of a reagent for detecting an SNP molecular marker related to yak milk density and milk fat content in detecting the quality traits of yak milk. The SNP molecular marker is located at the 13,729,718th position on chromosome 5 of the yak reference genome Bosgu_v3.0 version with the Ensemble accession number ENSBGRG00000003042, and the mutated base is C or T.

[0009] Preferably, according to the mutated base of the SNP molecular marker, the yak individual genotypes are divided into CC, CT, and TT; the milk fat content of the genotype TT yak individuals is significantly higher than that of the genotype CC; the milk density of the genotype CC yak individuals is significantly higher than that of the genotype TT.

[0010] Preferably, the reagent includes a primer pair for amplifying a nucleotide sequence containing the SNP molecular marker.

[0011] Preferably, the nucleotide sequence containing the SNP molecular marker is as shown in SEQ ID NO.1, and the SNP molecular marker is located at the 401st position.

[0012] In the third aspect, the present invention provides the application of a reagent for detecting an SNP molecular marker related to yak milk density and milk fat content in the early selection of yak milk quality traits. The SNP molecular marker is located at the 13,729,718th position on chromosome 5 of the yak reference genome Bosgu_v3.0 version with the Ensemble accession number ENSBGRG00000003042, and the mutated base is C or T.

[0013] Preferably, according to the mutated base of the SNP molecular marker, the yak individual genotypes are divided into CC, CT, and TT; the milk fat content of the genotype TT yak individuals is significantly higher than that of the genotype CC; the milk density of the genotype CC yak individuals is significantly higher than that of the genotype TT.

[0014] Preferably, the reagent includes a primer pair for amplifying a nucleotide sequence containing the SNP molecular marker.

[0015] Preferably, the nucleotide sequence containing the SNP molecular marker is as shown in SEQ ID NO.1, and the SNP molecular marker is located at position 401.

[0016] Fourthly, the present invention provides an application of a specific primer pair for amplifying a nucleotide sequence containing the SNP molecular marker described in the first aspect above in the early breeding for detecting yak milk density and milk fat content traits.

[0017] Preferably, the sequence of the specific primer pair is as shown in SEQ ID NO.2-3.

[0018] Preferably, the method for detecting yak milk density and milk fat content traits includes:

[0019] (1) Extracting yak genomic DNA as template DNA;

[0020] (2) Using the specific primer pair to perform PCR amplification on the genomic DNA of the tested yak obtained in step (1) to obtain a PCR amplification product;

[0021] (3) Purifying the PCR amplification product obtained in step (2), performing genotyping detection, and classifying the yak individual genotypes into CC, CT, and TT; the milk fat content of the yak individuals with genotype TT is significantly higher than that of genotype CC; the milk density of the yak individuals with genotype CC is significantly higher than that of genotype TT.

[0022] Preferably, the method for early breeding of yak milk density and milk fat content traits includes:

[0023] (1) Extracting yak genomic DNA as template DNA;

[0024] (2) Using the specific primer pair to perform PCR amplification on the genomic DNA of the tested yak obtained in step (1) to obtain a PCR amplification product;

[0025] (3) Purifying the PCR amplification product obtained in step (2), performing genotyping detection, and classifying the yak individual genotypes into CC, CT, and TT; the milk fat content of the yak individuals with genotype TT is significantly higher than that of genotype CC; the milk density of the yak individuals with genotype CC is significantly higher than that of genotype TT; selecting yak individuals with genotype TT for early breeding of milk fat content traits; selecting yak individuals with genotype CC for early breeding of milk density traits.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] By analyzing the correlation between locus genotypes and yak milk density and milk fat content, the present invention discovers SNP loci related to yak milk quality traits. The SNP molecular marker is located at position 13,729,718 on chromosome 5 of the yak reference genome Bosgu_v3.0 with the Ensemble accession number ENSBGRG00000003042, and the mutant base is C or T. According to genotyping detection, yak individual genotypes are divided into CC, CT, and TT. The milk fat of yak individuals with the genotype TT is significantly (p<0.05) higher than that of individuals with the genotype CC. The milk fat of yak individuals with the genotype TT and that of yak individuals with the genotype CT are not significant (p>0.05); the milk fat of yak individuals with the genotype CT and that of yak individuals with the genotype CC are not significant (p>0.05); the milk density of yak individuals with the genotype CC is significantly higher than that of individuals with the genotype TT (P<0.05); the milk density of yak individuals with the genotype CC and that of yak individuals with the genotype CT are not significant (P>0.05); the milk density of yak individuals with the genotype CT and that of yak individuals with the genotype TT are not significant (P>0.05). Corresponding traits can be quickly identified by PCR and gene sequencing methods, which can be used for yak molecular marker-assisted breeding and are not restricted by yak breeds and ages. Therefore, in production, individuals with the TT genotype at this locus can be preferentially selected as parents for large-scale breeding, greatly accelerating the accuracy and effectiveness of the selection of yak milk quality traits and improving the economic benefits of yak breeding. Brief Description of the Drawings

[0028] Figure 1 It is the sequencing peak map of three genotypes in the embodiment of the present invention. Detailed Embodiments

[0029] Single nucleotide polymorphism (SNP) is the most basic form of variation in genomic DNA sequences, caused by single base mutations in the DNA sequence. By determining nucleotide variations at specific positions in the genome and performing genotype typing, it provides an efficient and accurate means for the selection of breeding stock. By using SNPs for livestock and poultry genetic breeding research, we can gain a deeper understanding of the genetic background of livestock and poultry, reveal the genetic mechanisms of their complex traits, and thus provide strong support for optimizing livestock and poultry breeds, improving production performance, and accelerating the protection and utilization of livestock and poultry genetic resources. With the continuous progress of technology and the continuous expansion of application fields, SNPs will demonstrate greater potential and value in the field of livestock and poultry genetic breeding research.

[0030] The present invention designs multiple pairs of primers continuously on the yak PDE3A gene fragment (the sequence is SEQ ID NO.1) to perform PCR amplification on yak DNA and conduct gene sequencing. When analyzing the genotype of the target fragment amplified by one pair of primers (SEQ ID NO.2 and SEQ ID NO.3), a SNP site is found, and there are a total of three genotypes. Through the analysis of MEGA7.0 and BioEdit software, a SNP site is screened out at the 401st base of the fragment of sequence SEQ ID NO.1. Then, through the analysis of SPSS23.0 software on the correlation between the genotype of the mutation site and milk quality traits, it is found that the milk fat content of TT-type individuals is significantly higher than the phenotypic values of milk quality traits of CT and CC genotype individuals (p<0.05); the milk density of CC-type individuals is significantly higher than the phenotypic values of milk quality traits of CT and TT genotype individuals (p<0.05).

[0031] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the technical solutions of the present invention will be described in detail below with reference to the embodiments. It should be noted that the following embodiments are given only for the purpose of illustration and are not used to limit the scope of the present invention. The reagents not specifically described in detail in the present invention are all conventional reagents and can be obtained from commercial channels; the methods not specifically described in detail are all conventional experimental methods and can be known from the prior art. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0032] Example 1 Identification of SNP Mutation Sites

[0033] (1) Collection of Gannan Yak Samples

[0034] The present invention uses the Gannan yak breed as the detection object, and 162 yak milk samples and ear tissue samples are collected from the pasture in Xiahe County, Gannan Tibetan Autonomous Prefecture, Gansu Province. The parity of lactating yaks is all between 2 and 3 times. The collected yak milk is used for milk composition analysis. The analysis includes the determination of casein, milk density, protein, fat, total solids (TS), skim milk solids (SNF) and lactose. The determination is carried out using a MilkoScanTM FT120 type milk composition analyzer (Danish FUCHS Analytical Instruments Ltd., Hellerup, Denmark).

[0035] (2) Isolation, Extraction and Purification of Genomic DNA

[0036] Genomic DNA was extracted from the ear tissue samples of Gannan yaks using a magnetic bead method animal tissue genomic DNA extraction kit. The concentration of the DNA sample was detected by a Qubit fluorescence quantitative instrument. The integrity of the DNA sample was detected by 3% agarose gel electrophoresis.

[0037] (3) Primer design and screening

[0038] Based on the yak PDE3A gene (accession number: ENSBGRG00000003042) published by Ensemble, multiple pairs of primers were designed on its DNA sequence using the primer design software Primer 5.0. The yak DNA sample was subjected to PCR amplification, and the gene sequencing results were analyzed to screen out a pair of primers with SNP sites. The primer sequence information is as follows:

[0039] F: 5’-TATGGGAAAGGAAGAGTGGC-3’ (shown in SEQ ID NO.2);

[0040] R: 5’-TCGTTCGTATTCGCTTGGTG-3’ (shown in SEQ ID NO.3).

[0041] (4) PCR amplification of the target gene fragment

[0042] The PCR reaction system was 40 μL: 2×Accurate Taq Master Mix (dye plus) 20 μL, DNA template (100 ng / μL) 1 μL, upstream and downstream primers (10 μmol / L) 1 μL each, and enzyme-free and sterile water 17 μL. The PCR amplification program: pre-denaturation at 94 °C for 30 s; denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, 35 cycles; extension at 72 °C for 2 min, cooling at 4 °C. After the amplification, the amplification product was detected by electrophoresis on 3% agarose gel.

[0043] (5) Gene sequencing

[0044] The qualified PCR reaction solution was sent to Xi'an Qingke Jersey Biotechnology Co., Ltd. for bidirectional Sanger sequencing. The amplified target sequence is shown in SEQ ID NO.1, and the SNP site is located at position 401 of the sequence shown in SEQ ID NO.1. The sequencing peak map at the mutation site is as Figure 1 shown.

[0045] Example 2 Correlation between different genotypes of SNP molecular marker sites and milk quality traits

[0046] (1) Genotyping

[0047] All individuals repeated steps (4) and (5) in Example 1, and the specific genotypes of different individuals were determined according to the gene sequencing results. Three genotypes were detected in the test population, and the genotype frequencies and allele frequencies are shown in Table 1.

[0048] Genotyping of 162 yak ear tissue DNA samples by PCR and gene sequencing revealed that there were three genotypes at the SNP molecular marker locus of the yak PDE3A gene, namely the homozygous CC, the heterozygous CT, and the homozygous TT. The frequencies of the three genotypes were 0.290 (CC), 0.475 (CT), and 0.235 (TT).

[0049] Table 1 Genotype and allele frequencies of the SNP locus of the yak PDE3A gene

[0050]

[0051] (2) Association analysis between SNP genotypes and phenotypic values of milk quality traits

[0052] To determine whether the SNP markers prepared by the present invention are related to the differences in yak milk quality traits, SPSS 223.0 software was used to perform least-squares statistical analysis and association analysis on the three genotypes at the 401st SNP locus on the fragment of SEQ ID NO.1 with the phenotypic values of yak casein, milk density, protein, fat, total solids (TS), skim milk solids (SNF), and lactose, respectively, to calculate the association between the genotypes at this SNP locus and milk quality traits. The results are shown in Table 2.

[0053] The model used was as follows:

[0054] Yj = μ + Gj + ej; where Yj represents the measured milk quality trait value; μ represents the total mean of each trait; Gj represents the genetic effect of genotype j; ej represents the random error effect. The differences between groups of data were tested by LSD multiple comparison, and the experimental results were expressed as Mean ± SE.

[0055] Table 2 Association analysis between the polymorphism of the yak PDE3A gene and milk quality traits

[0056]

[0057] Note: Different lowercase letters with superscripts indicate significant differences (P < 0.05), and * indicates significant differences (P < 0.05)

[0058] As can be seen from Table 2, the milk fat content of individuals with the TT homozygous genotype was significantly higher than the phenotypic values of milk quality traits of individuals with the CT and CC genotypes (p < 0.05). The milk fat of yak individuals with the TT genotype was not significantly different from that of yak individuals with the CT genotype (p > 0.05); the milk fat of yak individuals with the CT genotype was not significantly different from that of yak individuals with the CC genotype (p > 0.05); the milk density of yak individuals with the CC genotype was significantly higher than that of the TT genotype (P < 0.05); the milk density of yak individuals with the CC genotype was not significantly different from that of yak individuals with the CT genotype (P > 0.05); the milk density of yak individuals with the CT genotype was not significantly different from that of yak individuals with the TT genotype (P > 0.05).

[0059] In this embodiment, an SNP marker significantly related to yak milk quality traits was identified. Therefore, the selection of individuals with dominant genotypes can be helpful to improve the milk quality traits of yaks.

[0060] Based on the above results, the mutation site of the present invention can be used as a potential genetic marker for improving the milk production performance of yaks for the assisted selection of yaks.

[0061] The above is only the specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention by those skilled in the art within the technical scope disclosed by the present invention shall be covered by the protection scope of the present invention.

Claims

1. Use of a reagent for detecting SNP molecular markers related to yak milk density and milk fat content in detecting yak milk density and milk fat content, characterized in that, The SNP molecular marker is located at position 13,729,718 on chromosome 5 of the yak reference genome Bosgu_v3.0 with the Ensemble accession number ENSBGRG00000003042, and the mutant base is C or T; according to the mutant base of the SNP molecular marker, the yak individual genotypes are divided into CC, CT, and TT; the milk fat content of the genotype TT yak individuals is significantly higher than that of the genotype CC; the milk density of the genotype CC yak individuals is significantly higher than that of the genotype TT.

2. Use of a reagent for detecting SNP molecular markers related to yak milk density and milk fat content in early breeding of yak milk density and milk fat content traits, characterized in that, The SNP molecular marker is located at position 13,729,718 on chromosome 5 of the yak reference genome Bosgu_v3.0 with the Ensemble accession number ENSBGRG00000003042, and the mutant base is C or T; according to the mutant base of the SNP molecular marker, the yak individual genotypes are divided into CC, CT, and TT; the milk fat content of the genotype TT yak individuals is significantly higher than that of the genotype CC; the milk density of the genotype CC yak individuals is significantly higher than that of the genotype TT.

3. The application according to claim 1 or 2, characterized in that, The reagent includes a primer pair for amplifying the nucleotide sequence containing the SNP molecular marker.

4. The application according to claim 3, characterized in that, The nucleotide sequence containing the SNP molecular marker is as shown in SEQ ID NO.1, and the SNP molecular marker is located at position 401.

5. Application of the specific primer pair for amplifying the nucleotide sequence containing the SNP molecular marker in detecting yak milk density, milk fat content or early breeding of yak milk density and milk fat content traits; the SNP molecular marker is located at position 13,729,718 on chromosome 5 of the yak reference genome Bosgu_v3.0 with the Ensemble accession number ENSBGRG00000003042, and the mutant base is C or T; according to the mutant base of the SNP molecular marker, the yak individual genotypes are divided into CC, CT, and TT; the milk fat content of the genotype TT yak individuals is significantly higher than that of the genotype CC; the milk density of the genotype CC yak individuals is significantly higher than that of the genotype TT.

6. The application according to claim 5, characterized in that The sequence of the specific primer pair is as shown in SEQ ID NO.2-3.

7. The application according to claim 6, wherein The method for realizing the detection of yak milk density and milk fat content traits includes: (1) Extracting yak genomic DNA as template DNA; (2) Using the specific primer pair to perform PCR amplification on the genomic DNA of the tested yak obtained in step (1) to obtain a PCR amplification product; (3) Purifying the PCR amplification product obtained in step (2), performing genotyping detection, and dividing the yak individual genotypes into CC, CT, and TT; the milk fat content of the genotype TT yak individuals is significantly higher than that of the genotype CC; the milk density of the genotype CC yak individuals is significantly higher than that of the genotype TT.

8. The application according to claim 6, characterized in that, The method for realizing the early breeding of yak milk density and milk fat content traits includes: (1) Extracting yak genomic DNA as template DNA; (2) Using the specific primer pair to perform PCR amplification on the genomic DNA of the tested yak obtained in step (1) to obtain a PCR amplification product; (3) Purify the PCR amplification product obtained in step (2), conduct genotyping detection, and classify the yak individual genotypes into CC, CT, and TT; the milk fat content of the yak individuals with genotype TT is significantly higher than that of genotype CC; the milk density of the yak individuals with genotype CC is significantly higher than that of genotype TT; select yak individuals with genotype TT for early breeding of milk fat content traits; select yak individuals with genotype CC for early breeding of milk density traits.

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