A detection method and application of yak PCSK1 gene CNV markers

Abstract

The present invention belongs to the technical field of molecular biology detection, and specifically relates to a method for detecting CNV markers of yak PCSK1 gene and its application. The present invention provides a CNV marker related to yak growth traits: copy number variation in the candidate region NW_005394088.1: 602936-646787 of yak PCSK1 gene; and provides a method for detecting CNV markers: using the genomic DNA of yak as a template, and respectively amplifying the CNV region of PCSK1 gene and the reference gene by RT-qPCR with primer pairs P1 and P2 to quantify the copy number variation of yak PCSK1 gene. Among them, the chest circumferences of 12-month-old and 30-month-old of yak individuals with the inserted type and normal type of PCSK1 gene are significantly higher than those of the deletion type individuals, which can be used as an important candidate molecular marker for the assisted selection of yak growth traits; the method is accurate, reliable and easy to operate, and is used for the early breeding of yak.

Description

Technical Field

[0001] The present invention relates to the field of livestock molecular biology detection, and particularly relates to a method for detecting the CNV marker of the yak PCSK1 gene based on RT-qPCR technology and its application. Background Art

[0002] With the development of disciplines such as molecular biology and genomics, animal breeding technology is evolving from conventional phenotypic selection to molecular breeding. Molecular marker-assisted breeding (MAS) is one of the important methods for cultivating excellent animal and plant varieties by combining modern molecular biology with traditional breeding techniques. This method directly selects individuals with alleles or genotypes having trait advantages through DNA molecular markers closely related to traits, aiming to achieve early seed selection and improve the accuracy of breeding values.

[0003] Copy Number Variations (CNVs) is a newly discovered type of genomic submicroscopic structural variation, referring to microstructural variations derived from duplications, deletions, and mutual combinations of DNA fragments with a length greater than 50 bp occurring in the genome. For CNVs with a clear position, some methods based on PCR technology and hybridization technology are usually used, such as RT-qPCR, MLPA, FISH, and digital PCR. Among them, RT-qPCR is the most commonly used. The fluorescent dye added to the RT-qPCR reaction system can specifically penetrate into the DNA double strand and emit a fluorescent signal, and the quantity of genomic DNA is reflected by detecting the intensity of the fluorescent signal. The theoretical basis for detecting CNV using RT-qPCR technology is to perform relative quantification on the target gene (with copy number variation) and the reference gene (without copy number variation), and use the 2 -ΔΔCt method to statistically detect the copy number of the candidate gene in the test sample. The advantages of this method are low experimental cost, no need to design and synthesize probes, convenient use, and can detect the absolute copy number of the target fragment.

[0004] PCSK1 is a serine endoprotease, which is mainly involved in the processing and biological activation of various proprotein hormones such as insulin, cholecystokinin, and adrenocorticotropic hormone in animals, as well as the energy metabolism process. The present invention unexpectedly discovers that the PCSK1 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 method for detecting the CNV of the yak PCSK1 gene, laying a foundation for the early selection of the growth traits of yaks. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a method for detecting copy number variation (CNV) markers of yak PCSK1 gene and its application. Specifically, it includes the following contents:

[0006] In the first aspect, the present invention provides a CNV marker related to yak growth traits, and the CNV marker is a copy number variation in the candidate region NW_005394088.1: 602936-646787 of yak PCSK1 gene.

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

[0008] Preferably, the detection of yak growth traits or early yak breeding specifically includes: quantitatively detecting the copy number of the CNV marker fragment of PCSK1 gene according to...; classifying the quantitative results into three types of copy number variation types according to Log2... -ΔΔCt Quantitatively detecting the copy number of the CNV marker fragment of PCSK1 gene; according to Log2... -ΔΔCt Dividing the quantitative results into three types of copy number variation types: when Log2... -ΔΔCt > 0.5, it is the insertion type; when Log2... -ΔΔCt <-0.5, it is the deletion type; when -0.5 ≤ Log2... -ΔΔCt ≤ 0.5, it is the normal type; among them, the chest circumferences of 12-month-old and 30-month-old yaks with the copy number variation type of normal type or insertion type are better than those of the deletion type.

[0009] Preferably, the reagent includes the target gene primer pair P1 and the reference gene BTF3 primer pair P2;

[0010] The target gene primer pair P1 is:

[0011] Forward primer F1: 5’-CCCCCAAATGTTCTGAAATCGT-3’;

[0012] Reverse primer R1: 5’-CTGGTCCCAAGGGGATTTCG-3’;

[0013] The reference gene BTF3 primer pair P2 is:

[0014] Forward primer F2: 5’-GGCAGCAAACACTTTCACCATT-3;

[0015] Reverse primer R2: 5’-AGCAAAACCGCTACTAGCAAACTC-3’.

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

[0017] The target gene primer pair P1 is as follows:

[0018] The target gene primer pair P1 is as follows:

[0019] Forward primer F1: 5’-CCCCCAAATGTTCTGAAATCGT-3’;

[0020] Reverse primer R1: 5’-CTGGTCCCAAGGGGATTTCG-3’;

[0021] The reference gene BTF3 primer pair P2 is as follows:

[0022] Forward primer F2: 5’-GGCAGCAAACACTTTCACCATT-3;

[0023] Reverse primer R2: 5’-AGCAAAACCGCTACTAGCAAACTC-3’.

[0024] Fourthly, the present invention provides a kit for detecting the CNV marker described in the first aspect by RT-qPCR technology, characterized in that the kit contains the primer pair described in the third aspect.

[0025] Preferably, the kit further includes one or several of qPCR Master Mix, deionized water, and control samples.

[0026] Fifthly, the present invention provides a method for detecting a CNV marker related to yak growth traits, the method comprising the following steps:

[0027] Using the genomic DNA of yak as a template, the CNV region of the yak PCSK1 gene and the reference gene BTF3 are amplified by RT-qPCR technology respectively using the primer pair described in the third aspect;

[0028] According to 2 -ΔΔCt Quantify the copy number of the PCSK1 gene CNV marker fragment; According to Log22 -ΔΔCt Classify the quantification results into three copy number variation types: when Log22 -ΔΔCt > 0.5, it is an insertion type; when Log22 -ΔΔCt < - 0.5, it is a deletion type; - 0.5 ≤ Log22 -ΔΔCt ≤ 0.5, it is a normal type; among them, the chest circumferences of 12-month-old and 30-month-old yaks with a normal type or insertion type of copy number variation are better than those of the deletion type.

[0029] Preferably, the RT-qPCR amplification system is as follows in a 20 μL volume: 1 μL of 50 ng / μL template DNA, 1 μL each of 10 μmol / L upstream and downstream primers, 10 μL of qPCR Master Mix, and 7 μL of deionized water.

[0030] Preferably, the reaction program used in the RT-qPCR is as follows:

[0031] (1) Pre-denaturation: 95°C for 30 s;

[0032] (2) Amplification reaction: denaturation at 95°C for 30 s, annealing at 62°C for 10 s, extension at 72°C for 20 s, for 45 cycles;

[0033] (3) Melting curve plotting: 95°C for 10 s, from 65°C to 97°C, +0.5°C for 5 s.

[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, and the CNV marker is a copy number variation in the candidate region NW_005394088.1: 602936 - 646787 of the yak PCSK1 gene;

[0036] The present invention provides a method for detecting the CNV marker. Using the whole-genome DNA of yak blood as a template, the CNV region of the yak PCSK1 gene is amplified by real-time fluorescence quantitative PCR method and the BTF3 gene is used as a reference. According to Log22 -ΔΔCt The quantitative results are divided into three copy number variation types; among them, when Log22 -ΔΔCt > 0.5, it is the insertion type; when Log22 -ΔΔCt < - 0.5, it is the deletion type; when - 0.5 ≤ Log22 -ΔΔCt ≤ 0.5, it is the normal type; the chest circumferences of 12-month-old and 30-month-old yaks with the copy number variation types of normal type and insertion type are better than those of the 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 takes the copy number variation (CNV) of the yak PCSK1 gene as a candidate locus, detects the copy number variation of this locus in the yak population by RT-qPCR, and conducts an association analysis with important economic traits such as body weight, body height, body slant length, and chest girth at 6 months, 12 months, 18 months, and 30 months of age; if the normal type and insertion type of the copy number variation of the PCSK1 gene are detected, the chest girth of the tested yaks at 12 months and 30 months of age is more excellent, and they have better growth performance; studying the CNV of this gene and conducting an association analysis with important growth traits of yaks is crucial, which can provide a theoretical basis for molecular breeding of yaks in China, facilitate marker-assisted selection of yak growth traits, and quickly establish a yak population with excellent genetic resources.

[0038] The method for detecting the copy number variation of the yak gene provided by the present invention can be used for early selection of yaks; the method for detecting the copy number variation of the PCSK1 gene is accurate, reliable, and easy to operate; the detection of the copy number variation locus of the PCSK1 gene provides a scientific basis for 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 will briefly introduce the drawings required to be used in the embodiments of the present invention. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0040] Figure 1 The amplification curve of the PCSK1 gene drawn by RT-RT-qPCR in the present invention;

[0041] Figure 2 The amplification curve of the BTF3 gene drawn by RT-RT-qPCR in the present invention;

[0042] Figure 3 The melting curve of the PCSK1 gene drawn by RT-RT-qPCR in the present invention;

[0043] Figure 4 The melting curve of the BTF3 gene drawn by RT-RT-qPCR in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the following further describes the present invention in detail with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0045] The present invention uses RT-qPCR to detect the copy number variation of the yak PCSK1 gene. The following will explain the present invention in detail with reference to the accompanying drawings and embodiments, which are explanations of the present invention rather than limitations.

[0046] A method for detecting CNV markers of the yak PCSK1 gene based on RT-qPCR technology and used for molecular breeding according to the present invention includes the following steps:

[0047] (1) Using the yak PCSK1 gene sequence in the NCBI database, designing primers with the Primer-BLAST website, and testing the primers with PCR;

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

[0049] (3) Using SPSS 23.0 software to conduct correlation analysis between the copy number variation types and the growth traits of yaks at each age stage, and screening CNV markers related to the growth traits of yaks;

[0050] (4) Selecting yak populations with excellent growth traits according to the copy number variation types for breeding.

[0051] Example: Detecting CNV markers of the PCSK1 gene in Asidan yaks

[0052] 1. Collection of yak samples

[0053] In the present invention, Asidan yaks were used as the detection objects, and blood samples of 213 yaks with growth trait records were collected.

[0054] 2. Extraction of blood genomic DNA

[0055] (1) Adding 5 ml of cell lysate CL to 5 ml of anticoagulated blood, inverting and mixing 5 times, centrifuging at 3,600 rpm (2,000×g) for 2 min, and discarding the supernatant;

[0056] (2) Then adding 7.5 ml of cell lysate CL to it, inverting and mixing 5 times, centrifuging at 3,600 rpm (2,000×g) for 2 min, discarding the supernatant, inverting the centrifuge tube on a clean absorbent paper for 2 min to ensure that the precipitate is in the tube;

[0057] (3) Preparing a mixed solution of buffer FG and Proteinase K;

[0058] (4) Adding 2.5 ml of the mixed solution of buffer FG and Proteinase K, and immediately vortexing and mixing until there are no lumps in the solution;

[0059] (5) Incubate in a 65°C water bath for 30 min, and invert and mix several times during this period;

[0060] (6) Add 2.5 ml of isopropanol, invert and mix thoroughly 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 on a clean absorbent paper to ensure that the precipitate remains in the tube;

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

[0063] (9) Repeat operation step H;

[0064] (10) Invert the centrifuge tube on a clean absorbent paper for at least 5 min to ensure that the precipitate remains in the tube;

[0065] (11) Air-dry the DNA precipitate until all the liquid has evaporated completely;

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

[0067] 3. Detection of genomic DNA concentration and purity

[0068] Use a Nanodrop2000 ultra-micro spectrophotometer to detect OD 260 、OD 280 and OD 260 / OD 280 ratio to determine the concentration and purity of DNA. High-quality DNA OD 260 / OD 280 readings are between 1.7 - 1.9.

[0069] 4. Amplification of target gene and reference gene

[0070] Using the yak PCSK1 gene (GenBank Accession No. NW_005394088.1) published in the NCBI database (http: / / www.ncbi.nlm.nih.gov / ) as the reference sequence, RT-qPCR primers (primer pair P1) were designed using Primer 5.0 to detect copy number variations of the PCSK1 gene. At the same time, using the yak BFT3 gene sequence (GenBank Accession No. NW_005393074.1) published by NCBI as the reference sequence, RT-PCR primers (primer pair P2) were designed using the same method to amplify a 163-bp sequence in the reference gene. The primer pair sequence information is shown in Table 1.

[0071] Table 1 Primer Information for Real-Time Fluorescent Quantitative PCR

[0072]

[0073] Note: F represents the forward primer and R represents the reverse primer.

[0074] The suitability of the primers for RT-qPCR analysis was determined by plotting the amplification curve and melting peak. A smooth amplification curve indicates good quality of the RT-qPCR reagents and appropriate amplification system and conditions ( Figure 1 and Figure 2 ); for the plotted melting curve, the curves of each sample coincide, and the curve trend is smooth, the peak is high and sharp, and there are no miscellaneous peaks caused by primer dimers or non-specific amplification, indicating good primer quality ( Figure 3 and Figure 4 ).

[0075] Among them, the amplification system used for RT-qPCR was calculated as 20 μL: 1 μL of 50 ng / μL template DNA, 1 μL each of 10 μmol / L forward and reverse primers, 10 μL of qPCR Master Mix, and 7 μL of deionized water.

[0076] The reaction program for PCR amplification was:

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

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

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

[0080] 4. Inference of Copy Number Variation

[0081] Each sample was amplified with primers for the target sequence (primer pair P1) and the reference sequence (primer pair P2) separately, and each pair of primers had 3 replicates. The copy number of the target sequence was calculated according to 2 -ΔΔCt The method. CT, that is, Cycle threshold, is the number of amplification cycles experienced when the fluorescence signal of the amplification product reaches the set threshold during the PCR amplification process. Among them, ΔΔCt = (CT of the target gene - CT of the reference gene) experimental group - (CT of the target gene - CT of the reference gene) control group. The experimental group is the sample to be detected for CNVs, and the control group is the sample known to have no copy number variation. 2 -ΔΔCt It represents the multiple of the copy number of the target sequence in the experimental group relative to the control group. Then, the change multiple of the copy number was logarithmically transformed (logarithm to the base 2 2 -ΔΔCt to conform to the normal distribution. After performing the homogeneity of variance test, the differences between groups were statistically tested.

[0082] According to Log2 2 -ΔΔCt The normalization value was calculated and the quantitative results were divided into three categories: insertion type, Log2 2 -ΔΔCt > 0.5, deletion type, Log2 2 -ΔΔCt < - 0.5, normal type, - 0.5 ≤ Log2 2 -ΔΔCtt ≤ 0.5.

[0083] 5. Association analysis between CNV loci of yak PCSK1 gene and growth traits

[0084] Production data: body weight, body height, body length and chest girth 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 was corrected using least squares analysis; according to the data characteristics, SPSS 23.0 software was used to analyze the production trait effects among different genotypes. When analyzing the genotype effects, a linear model was used:

[0086] Yij = μ + Ai + Gij + Eij

[0087] Where: Yij is the observed value of the growth trait, μ is the population mean, Ai is the age effect, Gj is the fixed effect of the genotype, and Eij is the random residual. The differences between groups of data were tested using LSD multiple comparison, and the experimental results were expressed in the form of Mean ± SE. The results of the association analysis between PCSK1 gene CNV and yak breed growth traits are shown in Table 2. Among them, the calculated value Mean ± SE in the table is the mean ± standard error; the different lowercase letters superscripted at the upper right corner of the values in the same row represent significant differences (P < 0.05) between the data in the same row.

[0088] The results of association analysis showed that the CNV locus of the yak PCSK1 gene could significantly affect the chest girth at 12 and 30 months of age, and the dominant copy number variation types were normal type and deletion type, indicating that the CNV locus of the PCSK1 gene could be used as a candidate molecular genetic marker for yak growth traits.

[0089] Table 2 Association analysis of the CNV of the PCSK1 gene and the growth traits of yak breeds

[0090]

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

[0092] The obtained CNV can be used as a candidate molecular genetic marker to search for quantitative trait loci related to or tightly linked to it that affect cattle growth traits, so as to conduct molecular marker-assisted selection for yaks, thereby accelerating the breeding process of yak breed improvement.

Claims

1. Use of a reagent for detecting CNV markers related to the growth traits of Ashidan yaks in detecting the growth traits of Ashidan yaks or in the early breeding of Ashidan yaks, wherein the CNV marker is a copy number variation in the candidate region NW_005394088.1: 602936-646787 of the yak PCSK1 gene; characterized in that, According to 2 -ΔΔCt Quantify the copy number of the PCSK1 gene CNV marker fragment; According to Log22 -ΔΔCt Classify the quantitative results into three types of copy number variations: When Log22 -ΔΔCt > 0.5, it is the insertion type; When Log22 -ΔΔCt < -0.5, it is the deletion type; -0.5 ≤ Log22 -ΔΔCt ≤ 0.5, it is the normal type; Among them, the chest circumferences of 12-month-old and 30-month-old Qaidam yaks with the normal or insertion type of copy number variation are better than those of the deletion type; The reagent includes the target gene primer pair P1 and the reference gene BTF3 primer pair P2; The target gene primer pair P1 is: Forward primer F1: 5’-CCCCCAAATGTTCTGAAATCGT-3’; Reverse primer R1: 5’-CTGGTCCCAAGGGGATTTCG-3’; The reference gene primer pair P2 is: Forward primer F2: 5’-GGCAGCAAACACTTTCACCATT-3; Reverse primer R2: 5’-AGCAAAACCGCTACTAGCAAACTC-3’.

2. A detection method for CNV markers related to the growth traits of Asidan yaks, characterized in that, The method includes the following steps: Using the genomic DNA of Qaidam Cashmere Goat as a template, the CNV region of the PCSK1 gene and the reference gene BTF3 of the yak are amplified by RT-qPCR using the primer pairs described in claim 1 respectively; According to 2 -ΔΔCt Quantify the copy number of the PCSK1 gene CNV marker fragment; according to Log22 -ΔΔCt Classify the quantitative results into three types of copy number variations: when Log22 -ΔΔCt > 0.5, it is an insertion type; when Log22 -ΔΔCt < -0.5, it is a deletion type; -0.5 ≤ Log22 -ΔΔCt ≤ 0.5, it is a normal type; among them, the chest circumferences of 12-month-old and 30-month-old Qaidam yaks with a normal or insertion type of copy number variation are superior to those of the deletion type.

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

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