A detection method and application of yak Wnt7A gene CNV markers

Abstract

The present invention belongs to the technical field of molecular biology detection, and specifically relates to a method for detecting a CNV marker of the yak Wnt7A gene and its application. The present invention provides a CNV marker related to the growth traits of yaks: the copy number variation of the candidate region NW_005394031.1:534971-621416 of the yak Wnt7A gene; and provides a method for detecting the CNV marker: using the genomic DNA of yaks as a template, and using primer pairs P1 and P2 to amplify the CNV region of the Wnt7A gene and the internal reference gene by RT-qPCR respectively, to quantify the copy number variation of the yak Wnt7A gene. Among them, the chest circumference of 6-month-old individuals with the Wnt7A gene deletion type is significantly higher than that of the insertion type and normal type individuals, and it can be used as an important candidate molecular marker for marker-assisted selection of yak growth traits; the method is accurate, reliable, and easy to operate, and is used for the early breeding of yaks.

Description

Technical Field

[0001] The present invention relates to the field of livestock molecular biology detection, and in particular to a method for detecting CNV markers of the yak Wnt7A gene based on RT-qPCR technology and an application thereof. Background Art

[0002] Since the 1980s, with the rapid development of molecular biotechnology and bioinformatics, research in animal molecular breeding has achieved a series of breakthroughs, and animal breeding technology is evolving from conventional phenotypic selection to molecular breeding. Molecular marker-assisted breeding (MAS) utilizes molecular markers associated with quantitative traits to accurately and rapidly analyze the genetic composition of individuals at the molecular level, thereby enabling genotypic selection. Compared with conventional breeding methods, this method provides richer information and higher selection accuracy.

[0003] Copy number variations (CNVs) are a new form of genomic variation, typically referring to duplications and deletions of large genomic segments ranging from 1 kilobase (Kb) to several megabases. For well-defined CNVs, methods based on PCR and hybridization techniques are typically used, such as RT-qPCR, MLPA, FISH, and digital PCR. RT-qPCR is currently the most widely used detection method, owing to its simple operation, rapid detection, and minimal contamination, making it suitable for typing large sample populations.

[0004] Wnt7A is a secretory signaling protein that belongs to the Wnt gene family. Wnt7A can play a regulatory role in many biological processes. Many studies have shown that Wnt7A plays an important role in the occurrence and development of animal skeletal muscle. It can stimulate the symmetrical division of muscle satellite cells and has an important impact on the stability of the number of skeletal muscle satellite cells. The present invention unexpectedly found that the Wnt7A gene is located in the CNV region, thus providing a CNV marker related to the growth traits of yaks, and providing a convenient, accurate, and large-sample detection method for detecting yak Wnt7A-CNV, which provides a basis for early selection and breeding of yaks for growth traits. Summary of the Invention

[0005] To address the problems of the existing technology, the present invention provides a method and application for CNV markers of the yak Wnt7A gene, and in particular, a method and application for detecting CNV markers of the yak Wnt7A gene based on RT-qPCR technology. Specifically, the method includes the following:

[0006] In a first aspect, the present invention provides a CNV marker associated with yak growth traits, wherein the CNV marker is a copy number variation of the candidate region NW_005394031.1:534971-621416 of the yak Wnt7A gene.

[0007] In a second aspect, the present invention provides a reagent for detecting CNV markers related to yak growth traits described in the first aspect, and uses the reagent in detecting yak growth traits or in early yak breeding.

[0008] Preferably, the detection of yak growth traits or early yak breeding is specifically: according to 2 -ΔΔCt The copy number of CNV marker fragment of Wnt7A gene was quantified by the method; according to Log22 -ΔΔCt The quantitative results were divided into three types of copy number variation: -ΔΔCt >0.5 is the insertion type; when Log22 -ΔΔCt When <-0.5, it is missing type; -0.5≤Log22 -ΔΔCt When the copy number variation is ≤0.5, it is normal; among them, the chest circumference of 6-month-old yaks with deletion type is better than that of insertion type and normal type.

[0009] Preferably, the reagents include a target gene primer pair P1 and a reference gene BTF3 primer pair P2;

[0010] The target gene primer pair P1 is:

[0011] Upstream primer F1: 5′-TCAGGACCAGTGACCCACAG-3′;

[0012] Downstream primer R1: 5′-GCAGACGGGAAGTTGTGACC-3′;

[0013] The reference gene primer pair P2 is:

[0014] Upstream primer F2: 5′-GGCAGCAAACACTTTCACCATT-3;

[0015] Downstream primer R2: 5′-AGCAAAACCGCTACTAGCAAACTC-3′.

[0016] In a third aspect, the present invention provides a primer pair for detecting CNV markers related to yak growth traits, the primer pair comprising a target gene primer pair P1 and a reference gene BTF3 primer pair P2;

[0017] The target gene primer pair P1 is:

[0018] Upstream primer F1: 5′-TCAGGACCAGTGACCCACAG-3′;

[0019] Downstream primer R1: 5′-GCAGACGGGAAGTTGTGACC-3′;

[0020] The reference gene BTF3 primer pair P2 is:

[0021] Upstream primer F2: 5′-GGCAGCAAACACTTTCACCATT-3;

[0022] Downstream primer R2: 5′-AGCAAAACCGCTACTAGCAAACTC-3′.

[0023] In a fourth aspect, the present invention provides a kit for detecting the CNV marker described in the first aspect using RT-qPCR technology, characterized in that the kit contains the primer pair described in the third aspect.

[0024] Preferably, the kit further comprises qPCR Master Mix, deionized water, or control sample.

[0025] In a fifth aspect, the present invention provides a method for detecting CNV markers related to yak growth traits, the method comprising the following steps:

[0026] Using yak genomic DNA as a template, the CNV region of the yak Wnt7A gene and the reference gene BTF3 were amplified by RT-qPCR using the primer pair described in the third aspect above;

[0027] According to 2 -ΔΔCt The copy number of CNV marker fragment of Wnt7A gene was quantified by the method; according to Log22 -ΔΔCt The quantitative results were divided into three types of copy number variation; among them, when Log22 -ΔΔCt >0.5 is the insertion type; when Log22 -ΔΔCt When <-0.5, it is missing type; -0.5≤Log22 -ΔΔCt When the copy number variation is ≤0.5, it is normal; the chest circumference of 6-month-old yaks with deletion type is better than that of insertion type and normal type.

[0028] Preferably, the RT-qPCR amplification system is calculated as follows in 20 μL: 1 μL of 50 ng / μL template DNA, 1 μL of 10 μmol / L upstream and downstream primers, qPCR Master Mix 10 μL, deionized water 7 μL.

[0029] Preferably, the reaction procedure used in the RT-qPCR is:

[0030] (1) Pre-denaturation: 95°C for 30 seconds;

[0031] (2) Amplification reaction: denaturation at 95°C for 30 s, annealing at 62°C for 10 s, and extension at 72°C for 20 s, for 45 cycles;

[0032] (3) Draw the melting curve: 95℃ for 10s, from 65℃ to 97℃, +0.5℃ for 5s.

[0033] The beneficial effects of the present invention are as follows: the present invention detects the copy number variation of the Wnt7A gene at this site in the yak population through RT-qPCR technology, and performs association analysis with important economic traits such as weight, body height, body oblique length and chest circumference at 6 months, 12 months, 18 months and 30 months of age; if the copy number variation type of the Wnt7A gene is detected to be a deletion type, the chest circumference of the tested cattle at 6 months of age is better; according to the results of the association analysis between the corresponding Wnt7A gene copy number variation type and growth traits, a theoretical basis can be provided for the molecular breeding of yaks in my country, which is convenient for marker-assisted selection of growth traits of Chinese yaks and the rapid establishment of a yak population with excellent genetic resources.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] The present invention provides a CNV marker related to yak growth traits, wherein the CNV marker is a copy number variation of the candidate region NW_005394031.1:534971-621416 of the yak Wnt7A gene;

[0036] The present invention provides a method for detecting CNV markers, using yak blood whole genome DNA as a template, respectively amplifying the CNV region of the yak Wnt7A gene by RT-qPCR method and using the BTF3 gene as a reference, and then analyzing the CNV region of the yak Wnt7A gene according to Log22. -ΔΔCt The quantitative results were divided into three types of copy number variation; among them, when Log22 -ΔΔCt >0.5 is the insertion type; when Log22 -ΔΔCt When <-0.5, it is missing type; -0.5≤Log22 -ΔΔCt When the copy number variation is ≤0.5, it is normal. The chest circumference of 6-month-old yaks with deletion type is better than that of insertion type and normal type. It can be used as an important candidate molecular marker for marker-assisted selection of yak growth traits.

[0037] The present invention uses the CNV of the yak Wnt7A gene as a candidate site, detects the copy number variation of this site in the yak population through RT-qPCR technology, and performs association analysis with important economic traits such as weight, height, body length and chest circumference at 6 months, 12 months, 18 months and 30 months of age; if the copy number variation type of the Wnt7A gene is detected to be deletion type, the chest circumference of the tested cattle at 6 months of age will be better and have better growth performance; studying the CNV of this gene and analyzing its association with important growth traits of yaks is of great importance, which can provide a theoretical basis for the molecular breeding of yaks in my country, facilitate marker-assisted selection of yak growth traits, and quickly establish a yak population with excellent genetic resources.

[0038] The method for detecting copy number variation of yak genes provided by the present invention can be used for early breeding of yaks. The method for detecting copy number variation of the Wnt7A gene is accurate, reliable, and easy to operate. The detection of copy number variation sites of the Wnt7A gene provides a scientific basis for molecular marker-assisted selection of yaks. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0040] Figure 1 The Wnt7A gene amplification curve drawn by RT-qPCR in the present invention;

[0041] Figure 2 The BTF33 gene amplification curve drawn by RT-qPCR in the present invention;

[0042] Figure 3 The Wnt7A gene dissolution curve drawn by RT-qPCR in the present invention;

[0043] Figure 4 The present invention performs RT-qPCR to draw the BTF33 gene dissolution curve. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0045] The present invention uses RT-qPCR to detect the copy number variation of the yak Wnt7A gene. The present invention is described in detail below with reference to the accompanying drawings and examples, which are intended to explain rather than limit the present invention.

[0046] The present invention relates to a method for detecting CNV markers of the yak Wnt7A gene based on RT-qPCR technology and using it for molecular breeding, comprising the following steps:

[0047] (1) Using the yak Wnt7A gene sequence from the NCBI database, primers were designed using the Primer-BLAST website and tested by PCR;

[0048] (2) RT-qPCR technology was used to detect the copy number variation of candidate loci in the population;

[0049] (3) SPSS 23.0 software was used to perform association analysis between copy number variation types and growth traits of yaks at different age stages, and CNV markers related to yak growth traits were screened;

[0050] (4) Select yak populations with excellent growth traits based on copy number variation types for breeding.

[0051] Example Detection of CNV markers in the Wnt7A gene of Ashdan yak

[0052] 1. Yak sample collection

[0053] The present invention uses Ashdan yaks as detection objects, and collects blood samples from a total of 213 yaks with growth trait records.

[0054] 2. Extraction of blood genomic DNA

[0055] (1) Add 5 ml of cell lysis buffer CL to 5 ml of blood containing anticoagulant, mix by inversion 5 times, centrifuge at 3,600 rpm (2,000 × g) for 2 min, and discard the supernatant;

[0056] (2) Add 7.5 ml of cell lysis buffer CL, mix thoroughly by inversion five times, centrifuge at 3,600 rpm (2,000 × g) for 2 min, discard the supernatant, and invert the centrifuge tube on clean absorbent paper for 2 min to ensure that the precipitate remains in the tube;

[0057] (3) Prepare a mixture of buffer FG and Proteinase K;

[0058] (4) Add 2.5 ml of a mixture of buffer FG and Proteinase K and immediately vortex mix until the solution is free of lumps;

[0059] (5) 65°C water bath for 30 min, inverting several times to mix;

[0060] (6) Add 2.5 ml of isopropanol and mix thoroughly by inversion until filamentous or clustered genomic DNA appears;

[0061] (7) Centrifuge at 3,600 rpm (~2,000 × g) for 8 min and discard the supernatant. Invert the centrifuge tube onto clean absorbent paper to ensure that the pellet remains in the tube.

[0062] (8) Add 2.5 ml of 70% ethanol, vortex for 5 seconds, centrifuge at 3,600 rpm (~2,000 × g) for 3 minutes, and discard the supernatant;

[0063] (9) Repeat step H;

[0064] (10) Place the centrifuge tube upside down on clean absorbent paper for at least 5 minutes to ensure that the precipitate remains in the tube;

[0065] (11) Air-dry the DNA pellet until all the liquid evaporates;

[0066] (12) Add 500 μl of buffer TB, vortex at low speed for 5 seconds, and heat at 65°C for 1 hour to dissolve the DNA, flicking the tube several times to aid dissolution.

[0067] 3. Genomic DNA concentration and purity detection

[0068] OD was measured using a Nanodrop 2000 ultra-micro spectrophotometer. 260 ,OD 280 and OD 260 / OD 280 Ratio, determine the concentration and purity of DNA, high-quality DNAOD 260 / OD 280 The readings are between 1.7-1.9.

[0069] 4. Target gene and reference gene amplification

[0070] Using the yak Wnt7A gene (GenBank Accession No. NW_005394031.1) published in the NCBI database (http: / / www.ncbi.nlm.nih.gov / ) as a reference sequence, RT-qPCR primers (primer pair P1) were designed using Primer 5.0 to detect Wnt7A gene copy number variation. Simultaneously, using the yak BFT3 gene sequence (GenBank Accession No. NW_005393074.1) published in the NCBI database as a reference sequence, RT-qPCR primers (primer pair P2) were designed using the same method to amplify a 163-bp sequence within the reference gene. The primer pair sequence information is shown in Table 1.

[0071] Table 1 Primer information for real-time fluorescence quantitative PCR

[0072]

[0073] Note: F represents the upstream primer, and R represents the downstream primer.

[0074] The suitability of primers for RT-qPCR analysis is determined by plotting amplification curves and melting peaks. A smooth amplification curve indicates good quality of RT-qPCR reagents and appropriate amplification system and conditions. Figure 1 and Figure 2 ); The melting curves of the samples were drawn in a consistent manner, with smooth curves, high and sharp peaks, and no miscellaneous peaks caused by primer dimers or non-specific amplification, indicating that the primers were of good quality ( Figure 3 and Figure 4 ).

[0075] The amplification system used for RT-qPCR is as follows: 1 μL of 50 ng / μL template DNA, 1 μL of 10 μmol / L upstream and downstream primers, qPCR Master Mix 10 μL, deionized water 7 μL.

[0076] The reaction procedure for PCR amplification is:

[0077] (1) Pre-denaturation: 95°C for 30 seconds;

[0078] (2) Amplification reaction: denaturation at 95°C for 30 s, annealing at 62°C for 10 s, and extension at 72°C for 20 s, for 45 cycles;

[0079] (3) Draw the melting curve: 95℃ for 10s, from 65℃ to 97℃, +0.5℃ for 5s.

[0080] 4. Inference of copy number variation

[0081] Each sample was amplified using primers for the target sequence (primer pair P1) and the reference sequence (primer pair P2), and three replicates were performed for each primer pair. -ΔΔCt The method calculates the copy number of the target sequence. CT stands for cycle threshold, which is the number of amplification cycles required for the fluorescence signal of the amplified product to reach the set threshold during PCR amplification. ΔΔCt = (CT target gene - CT reference gene) experimental group - (CT target gene - CT reference gene) control group. The experimental group is the sample to be tested for CNVs, and the control group is the sample known to have no copy number variation. -ΔΔCt It represents the multiple of the target sequence copy number in the experimental group relative to the control group. Then the copy number change fold is logarithmically transformed (base 2 -ΔΔCt The logarithm of the data was used to make it conform to the normal distribution, and after the variance homogeneity test, the differences between the groups were statistically tested.

[0082] According to Log22 -ΔΔCt Calculate the normalized value and divide the quantitative results into three categories: insertion type, Log22 -ΔΔCt >0.5, missing type, Log22 -ΔΔCt <-0.5, normal type, -0.5≤Log22 -ΔΔCtt ≤0.5.

[0083] 5. Association analysis between CNV sites of the yak Wnt7A gene and growth traits

[0084] Production data: weight, height, length and chest circumference at 6 months, 12 months, 18 months and 30 months of age.

[0085] Association analysis model: First, descriptive analysis was performed on the data to determine whether there were outliers, and then the data were corrected using least squares analysis. Based on the data characteristics, SPSS23.0 software was used to analyze the production trait effects between genotypes. A linear model was used to analyze the genotype effect:

[0086] Yij=μ+Ai+Gij+Eij

[0087] Where: Yij represents the observed value of the growth trait, μ represents the population mean, Ai represents the age effect, Gj represents the fixed effect of genotype, and Eij represents the random residual. Differences between data groups were tested using LSD multiple comparisons, and the results are presented as mean ± SE. The results of the association analysis between Wnt7A gene CNV and growth traits in yak breeds are shown in Table 2. The calculated values ​​in the table are mean ± standard error. Values ​​in the same row marked with different lowercase letters in the upper right corner indicate significant differences between data in the same row at a P < 0.05 level.

[0088] The results of association analysis showed that the CNV site of the Wnt7A gene in yaks can significantly affect the chest circumference at 6 months of age, and the dominant copy number variation type is deletion type, indicating that the CNV site of the Wnt7A gene can be used as a candidate molecular genetic marker for growth traits in yaks.

[0089] Table 2 Association analysis between CNV of Wnt7A gene and growth traits of yak breeds

[0090]

[0091] 6. Application of the above CNV markers in yak breeding

[0092] The obtained CNVs can be used as candidate molecular genetic markers to find quantitative trait loci that are related to or closely linked to them and affect cattle growth traits, so as to carry out molecular marker-assisted selection of yaks, thereby accelerating the breeding process of yak breed improvement.

Claims

1. Use of a reagent for detecting a CNV marker associated with growth traits of Ashdan yaks for detecting growth traits of Ashdan yaks or in early breeding of Ashdan yaks; the CNV marker is a copy number variation in the candidate region NW_005394031.1:534971-621416 of the yak Wnt7A gene; According to 2 -ΔΔCt The copy number of CNV marker fragment of Wnt7A gene was quantified by the method; according to Log22 -ΔΔCt The quantitative results were divided into three types of copy number variation; When Log22 -ΔΔCt >0.5 is the insertion type; when Log22 -ΔΔCt When <-0.5, it is missing type; -0.5≤Log22 -ΔΔCt When the copy number variation is ≤0.5, it is normal. The chest circumference of 6-month-old Ashdan yaks with deletion type is better than that of insertion type and normal type. The reagents include a target gene primer pair P1 and a reference gene BTF3 primer pair P2; The target gene primer pair P1 is: Upstream primer F1: 5′-TCAGGACCAGTGACCCACAG-3′; Downstream primer R1: 5′-GCAGACGGGAAGTTGTGACC-3′; The reference gene primer pair P2 is: Upstream primer F2: 5′-GGCAGCAAACACTTTCACCATT-3; Downstream primer R2: 5′-AGCAAAACCGCTACTAGCAAACTC-3′.

2. A method for detecting CNV markers related to growth traits of Ashdan yaks, characterized in that: The method comprises the following steps: Using the genomic DNA of the Ashdan yak as a template, the CNV region of the Wnt7A gene of the Ashdan yak and the reference gene BTF3 were amplified by RT-qPCR using the primer pair described in claim 1; According to 2 -ΔΔCt The copy number of CNV marker fragment of Wnt7A gene was quantified by the method; according to Log22 -ΔΔCt The quantitative results were divided into three types of copy number variation; among them, when Log22 -ΔΔCt >0.5 is the insertion type; when Log22 -ΔΔCt When <-0.5, it is missing type; -0.5≤Log22 -ΔΔCt When the copy number variation is ≤0.5, it is normal; the chest circumference of 6-month-old Ashdan yaks with deletion type is better than that of insertion type and normal type.

3. The detection method according to claim 2, wherein The RT-qPCR amplification system in 20 μL is as follows: 1 μL of 50 ng / μL template DNA, 1 μL each of 10 μmol / L upstream and downstream primers, 10 μL of GoTaq® qPCR Master Mix, and 7 μL of deionized water.

Images