CN113005201B - Method for detecting goat FecB gene CNV marker and application thereof - Google Patents
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Abstract
The invention discloses a detection mountainA method for labeling sheep FecB gene CNV and application thereof. Based on real-time fluorescent quantitative PCR technology, using goat genome DNA as a template, using one pair of specific primers to amplify partial fragments of copy number variation regions of goat FecB gene, using another pair of specific primers to amplify partial fragments of goat MC1R gene as a reference, and then using 2 x 2‑ΔCtThe method calculates the copy number variation type of the individual, and according to the correlation analysis result of the copy number variation and the reproduction traits and the growth traits of the goats, the method provided by the invention lays a foundation for establishing a dominant trait population by using the CNV (CNV) marker of the FecB gene of the goats, and is favorable for accelerating the breeding process of the goats.
Description
Technical Field
The invention belongs to the field of molecular genetic breeding, and particularly relates to a method for detecting copy number variation of a goat FecB gene, which utilizes a real-time quantitative PCR technology, takes goat genome DNA as a template, takes a single-copy MC1R gene as a reference, and takes 2 x 2 as the reference-ΔCtThe values determine the type of FecB gene copy number variation for the individual.
Background
Copy Number Variation (CNV) is a complex multiallelic variation that exists in different individuals or populations and constitutes a polymorphism of genomic genetic variation. CNVs are variations in microscopic and submicroscopic structure resulting from large-scale fragment rearrangements of the genome, including repeats, insertions and deletions in the range of 1Kb to several Mb. Unlike Single Nucleotide Polymorphisms (SNPs) and Structural Variations (SVs), CNVs are mainly genomic sequence changes of large fragments, which affect gene expression levels, phenotypic differences and phenotypic adaptation by changing gene Structure and dosage, changing gene regulation and exposing recessive alleles, etc. In recent years, copy number variation has been found in the genomes of a variety of organisms, including humans, pigs, chickens, cattle and sheep. Most CNVs usually occur in gene-rich regions, mainly associated with some complex traits and evolution.
At present, there are five specific methods for detecting known Copy Number Variation (CNV). (1) Southern hybridization and Fluorescence In Situ Hybridization (FISH) are two classical experimental methods based on hybridization technology, the operation of Southern hybridization is relatively complicated and time-consuming, FISH has the advantages of safety, rapidness, high sensitivity and the like, but the method has high cost, high technical requirement, small flux and long time, and can not accurately quantify Copy Number Variation (CNV) with adjacent position repetition and has higher requirement on samples. (2) The Real-time quantitative PCR (qPCR) technology is a detection method based on the PCR technology, and the method is simple, easy to operate, high in sensitivity, good in repeatability, high in speed and less in pollution, but is not suitable for high-throughput detection of large samples. (3) The short-fragment multiplex quantitative technique (QMPSFQ) can simultaneously perform multiplex PCR, and thus the detection throughput is improved, but the absolute value of the copy number cannot be determined when the copy number is increased more. (4) Multiplex Amplification Probe Hybridization (MAPH) can detect copy number variation of multiple sites rapidly and reliably. (5) Direct detection by PCR.
The FecB gene expresses an important transmembrane receptor protein in sheep reproductive organs such as ovary, granulosa cell, oocyte, follicle and corpus luteum. The gene is mainly involved in transforming growth factor beta (TGF-beta) pathway, plays an important role in regulating osteogenic differentiation, ovarian follicle development and other processes, and directly influences the reproductive traits of animals.
The FecB gene is mutated from the coding region of the BMPR1B gene. A method for rapidly identifying FecB genes by utilizing a sheep structural variation region is disclosed in Chinese patent CN 111560441A, and the method can rapidly identify FecB genes carried by sheep individuals based on the discovered structural variation region of a sheep reference genome chr6:29413436-29413526bp, thereby assisting in breeding sheep individuals with high reproductive performance, but does not relate to breeding of growth traits. At present, no report is found about the analysis and application of the FecB gene CNV.
Disclosure of Invention
The invention aims to provide a method for detecting a goat FecB gene CNV marker and application thereof, which can quickly establish a goat population with excellent genetic resources.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting copy number variation of goat FecB genes comprises the following steps:
using goat (for example, Shanxi white cashmere goat individual) genome DNA as a template, using a primer pair P1 and a primer pair P2 as primers, amplifying partial fragments of the FecB gene copy number variation region and partial fragments of the MC1R gene as internal references by real-time fluorescent quantitative PCR, and then identifying the copy number variation type of the goat individual FecB gene according to the quantitative result;
the primer pair P1 is:
the upstream primer F1: 5'-CAGATTTCAGCCTTTGCGGG-3'
The downstream primer R1: 5'-TTGGGGCAGTCAGGAAAGAG-3'
The primer pair P2 is:
the upstream primer F2: 5'-GGCCTGAGAGGGGAATCACA-3'
The downstream primer R2: 5'-AGTGGGTCTCTGGATGGAGG-3' are provided.
Preferably, the copy number variation region of the FecB gene is positioned in a goat reference genome sequence NC-030813.130116801-30118800 bp.
Preferably, said copy number variation pattern is according to 2 x 2-ΔCtDividing the quantitative results intoThree types of (c): insertion type, 2 x 2-ΔCt>2.0; deletion form, 2 x 2-ΔCt<2.0; normal type, 2 x 2-ΔCt＝2.0。
Preferably, the amplification system used in the real-time fluorescent quantitative PCR comprises 1. mu.L of 50 ng/. mu.L template DNA and 0.5. mu.L of each of the upstream and downstream primers corresponding to 10pmol/L primer pair P1 or P2.
Preferably, the reaction procedure for the real-time fluorescent quantitative PCR comprises the following steps: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 30s for a total of 39 cycles.
Preferably, the size of the amplified fragment is 111bp based on the primer pair P1 and 126bp based on the primer pair P2.
The method for detecting the copy number variation of the goat FecB gene is applied to the goat molecular marker-assisted selective breeding.
Preferably, in the goats, individuals with normal copy number variation types are superior to individuals with deletion type or insertion type copy number variation types in the characters such as lambing number, chest depth, tube circumference and the like.
A kit for detecting goat FecB gene copy number variation comprises the primer pair P1 and a primer pair P2.
The invention has the following beneficial effects:
the method takes the DNA of the goat genome to be detected as a template, utilizes the real-time fluorescent quantitative PCR detection result to identify the copy number variation type of the FecB gene in the genome, and shows that the method can be used for detecting the CNV marker of the FecB gene according to the correlation analysis result of the copy number variation of the FecB gene in the goat population, the number of lambs and the individual important economic characters, thereby quickly establishing the goat population with the dominant reproductive characters and the growth characters (such as the number of lambs, the chest depth and the tube circumference), being convenient for popularization and application and being beneficial to accelerating the breeding process of the goat.
Compared with the prior art, the invention has the following advantages:
(1) the method can be used for quickly, accurately and reliably detecting the copy number variation of the goat FecB gene, and is simple and convenient to operate;
(2) the detection of the goat FecB gene CNV marker is not limited by age, can be used for the early breeding of goat individuals, and can be selected even just after birth;
(3) the detection of the goat FecB gene CNV marker provides scientific basis for the molecular marker-assisted selection of the breeding and growth traits of goats.
Drawings
FIG. 1 is a graph of amplification curves plotted against qPCR (FecB gene) in an example of the present invention.
FIG. 2 is a melting profile plotted against qPCR (FecB gene) in an example of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
The invention utilizes real-time fluorescent quantitative PCR to detect the copy number variation of goat FecB gene and is used for molecular breeding, and the method comprises the following steps:
(1) designing a primer according to a goat FecB gene sequence of an NCBI database, and detecting the primer by using a common PCR;
(2) detecting the copy number variation condition of the candidate sites in the population by adopting a real-time fluorescent quantitative PCR (qPCR) technology;
(3) performing correlation analysis on the copy number variation type and goat lambing and growth traits by using SPSS 23.0 software, and screening a CNV (CNV) mark related to the goat lambing number and growth traits;
(4) selecting goat individuals according to copy number variation.
The detection of copy number variation of the goat FecB gene and the like will be specifically described below.
1. Goat sample Collection
The method specifically takes 313 Shanxi white cashmere goats as detection objects, and adopts Shanxi white cashmere goat ear tissue samples from cultivation bases in Yangshan county of Ullin City of Shaanxi province, wherein the sampling time is 6 months to 9 months in 2017.
2. Extraction of genomic DNA from an ear tissue sample
(1) Approximately 10mg of the tissue sample of the ear tissue sample was placed in a 1.5mL centrifuge tube and cut as much as possible with small scissors.
(2) Add 600. mu.L of tissue extract, 10% SDS to a final concentration of 1%, proteinase K to a final concentration of 100ng/mL and digest overnight at 55 ℃ to ensure that the tissue sample is more evenly distributed in the tissue extract.
(3) Cooling the solution to room temperature, adding equal volume of Tris saturated phenol, tightly covering the tube cap, slowly reversing the centrifuge tube back and forth, at least continuously for more than 10min, and centrifuging at 12000r/min for 15 min.
(4) Taking the supernatant, adding equal volume of phenol: chloroform (1: 1), covering the tube cap tightly, slowly reversing the centrifuge tube back and forth, at least lasting for more than 10min, and centrifuging at 12000r/min for 15 min.
(5) Taking supernatant, adding equal volume of chloroform isoamyl alcohol (24:1), covering the tube cover tightly, slowly reversing the centrifuge tube back and forth for at least more than 10min, and centrifuging at 12000r/min for 15 min.
(6) The supernatant was taken, 2 volumes of ice-cold absolute ethanol and 1/10 volumes of 3mol/L sodium acetate were added, the tube cap was closed, and the centrifuge tube was slowly reversed back and forth until the liquid was clear and white flocculent DNA appeared.
(7) Picking out DNA, putting the DNA into a 1.5mL centrifuge tube, adding 500 mu L70% ethanol, covering the tube cap tightly, slowly reversing the centrifuge tube back and forth, then centrifuging at 12000r/min for 3-5 min, carefully pouring off the ethanol, and pouring the tube on absorbent paper.
(8) And adding 500 mu L of 70% ethanol into the centrifuge tube again, tightly covering the tube cover, slowly reversing the centrifuge tube back and forth, then centrifuging at 12000r/min for 3-5 min, carefully pouring off the ethanol, and pouring the tube on absorbent paper.
(9) After drying, 60. mu.L of sterilized ultrapure water was added thereto, and the DNA was stored at 4 ℃ overnight and examined.
3. Amplification of target and reference sequences
A goat FecB gene sequence (GenBank accession NC-030813.1) published by NCBI database (http:// www.ncbi.nlm.nih.gov /) is used as a reference sequence, a real-time fluorescent quantitative PCR Primer for detecting FecB gene copy number variation is designed by using Primer-BLAST of an NCBI website according to the position (located at NC-030813.130116801 bp-30118800bp) of a candidate CNV region in a reference genome, and a fragment (target sequence) with the size of 111bp can be amplified in a FecB gene candidate region by using the Primer. The internal reference sequence is a sequence without copy number variation, namely a 126bp sequence in the MC1R gene, and the sequence information of the primer is shown in Table 1.
TABLE 1 primer information Table
The amplification system used for qPCR was calculated at 10.0 μ L as: 50 ng/. mu.L of template DNA (genomic DNA extracted from an ear tissue-like sample), 1. mu.L of 10.0pmol/L of upstream primer, 0.5. mu.L of 10.0pmol/L of downstream primer, 2 XSSYBR Green qPCR Mix 5.0. mu.L, and ddH2O 3.0μL。
The reaction procedure for qPCR amplification was: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30s, Plate reading (Plate Read), for a total of 39 cycles; melting curves were made starting at 65 ℃ and increasing by 0.5 ℃ per cycle, up to 95 ℃ for 5s, and plates were read.
Referring to fig. 1 and 2, primers were determined to be suitable for qPCR analysis by plotting amplification and melting curves. According to the drawn melting curve, the curves of all samples are matched together, the curves are smooth in trend, high and sharp in peak height, and no primer dimer or hybrid peak caused by non-specific amplification exists.
4. Determination of Copy Number Variation (CNV) type
Each sample was amplified with primers for the target sequence and the internal reference sequence, respectively, and 3 replicates were performed for each pair of primers. According to 2 x 2-ΔCtThe method performs analysis of copy number variation. Where Δ Ct ═ CtTarget sequence-CtInternal reference sequence。2*2-ΔCtThe number of copies is indicated. Ct, Cycle threshold, is the number of amplification cycles that pass when the fluorescence signal of the amplified product reaches a set threshold during PCR amplification.
According to 2 x 2-ΔCtThe quantitative results were classified into three categories: the insertion type is that the insertion type,2*2-ΔCt>2.0; deletion type, 2 x 2-ΔCt<2.0; normal type, 2 x 2-ΔCt＝2.0。
Correlation analysis of FecB Gene CNV with number of lambs and growth traits
Idiotype (CNV type): obtained by calculation of quantitative results of qPCR
Individual traits: lambing number, height, length, height of cross, bust, chest depth, chest width, and tube circumference
Firstly, performing description analysis on data to determine whether an outlier exists, and then correcting the data by using least square analysis; according to the data characteristics, the SPSS 23.0 software was used to analyze the effect of production traits among genotypes. The correlation analysis model used in the analysis of genotype effects was a fixed model:
Yijk＝μ+Ai+CNVj+eijk
wherein Y isijkFor trait observations, μ is the overall mean, AiIs age of the ith individual, CNVjAs a fixed effect of the jth copy number variation type, eijkIs a random error. The variability between each set of data was examined using multiple comparisons of LSDs and the results were expressed as Mean ± SE. The correlation analysis results are shown in tables 2 and 3.
TABLE 2 correlation analysis between FecB gene CNV and number of lambs born by Shaanxi Bairong goats
Note: the same row data mean value is different from the shoulder letter, which indicates that there is a statistical difference
TABLE 3 correlation analysis between FecB gene CNV and Shaanxi white cashmere goat growth traits
Note: the same row data mean value is different from the shoulder letter, which indicates that there is a statistical difference
The correlation analysis results show that individuals with CNV type in the FecB gene NC-030813.130116801 bp-30118800bp of the Shanxi white cashmere goat are obviously superior to individuals with insertion type and deletion type in lambing number and growth traits (chest depth and tube circumference). The results show that the normal type of the CNV locus of the FecB gene can be used as a candidate molecular genetic marker (CNV marker) for improving the goat lambing number and growth traits.
6. Application of CNV marker in early-stage molecular breeding of goats
The obtained CNV candidate molecular genetic marker can be used for searching quantitative trait loci which are related to the CNV candidate molecular genetic marker or are closely linked with the CNV candidate molecular genetic marker and influence the reproductive traits and the growth traits of goats. The method can also be used for molecular marker-assisted selection of goats (such as Shanxi white cashmere goats), namely, normal goat individuals are selected for reservation and propagation by detecting the copy number variation type of the CNV locus of the goat FecB gene, so that the breeding process of goat variety improvement can be accelerated.
<110> northwest agriculture and forestry science and technology university
<120> method for detecting goat FecB gene CNV marker and application thereof
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Claims (6)
1. Detection goatFecBA method of gene copy number variation, characterized by: the method comprises the following steps:
taking goat genome DNA as a template and a primer pair P1 and a primer pair P2 as primers, and respectively carrying out real-time fluorescent quantitative PCR amplificationFecBCopy number variation region of gene and its use as referenceMC1RPartial fragment of gene, and identifying goat according to quantitative resultFecBCopy number variation type of gene;
the primer pair P1 is:
the upstream primer F1: 5'-CAGATTTCAGCCTTTGCGGG-3'
The downstream primer R1: 5'-TTGGGGCAGTCAGGAAAGAG-3'
The primer pair P2 is:
the upstream primer F2: 5'-GGCCTGAGAGGGGAATCACA-3'
The downstream primer R2: 5'-AGTGGGTCTCTGGATGGAGG-3', respectively;
saidFecBThe copy number variation region of the gene is positioned in a goat reference genome sequence NC-030813.130116801-30118800 bp;
said copy number variation pattern is according to 2 x 2-ΔCtThe quantitative results were divided into three categories: insertion type, 2 x 2-ΔCt>2.0; deletion form, 2 x 2-ΔCt<2.0 of the total weight of the mixture; normal type, 2 x 2-ΔCt =2.0；
The goat is selected from Shanxi white cashmere goat.
2. The detection goat of claim 1FecBA method of gene copy number variation, characterized by: the real-time fluorescent quantitative PCR amplification system comprises 1 mu L of template DNA with the concentration of 50 ng/mu L and 0.5 mu L of upstream primer and downstream primer corresponding to 10pmol/L of primer pair P1 or primer pair P2 respectively.
3. The detection goat of claim 1FecBA method of gene copy number variation, comprising: the reaction program of the real-time fluorescence quantitative PCR comprises the following steps: 95oC, performing pre-denaturation for 1 min; 95oDenaturation with C15 s, 60oC anneal 15s, 72oC extends for 30s for 39 cycles.
4. The detection goat of claim 1FecBA method of gene copy number variation, characterized by: the size of the amplified fragment was 111bp based on primer pair P1 and 126bp based on primer pair P2.
5. The use of the method of any one of claims 1-4 in goat molecular marker assisted selection breeding, wherein: individuals with normal copy number variation types are superior to individuals with deletion type or insertion type copy number variation types in reproduction traits and growth traits;
the reproductive traits are lambing number and the growth traits are chest depth and/or tube circumference.
6. Detection goatFecBA kit for gene copy number variation, comprising: the kit comprises a PCR amplification kit for respectively carrying out real-time fluorescence quantification PCR amplificationFecBCopy number variation region of gene and its use as referenceMC1RPrimer pair P1 and primer pair P2 of a partial fragment of a gene;
the primer pair P1 is:
the upstream primer F1: 5'-CAGATTTCAGCCTTTGCGGG-3'
The downstream primer R1: 5'-TTGGGGCAGTCAGGAAAGAG-3'
The primer pair P2 is:
the upstream primer F2: 5'-GGCCTGAGAGGGGAATCACA-3'
The downstream primer R2: 5'-AGTGGGTCTCTGGATGGAGG-3', respectively;
saidFecBCopy number variation region of geneThe domain is located in a goat reference genome sequence NC _030813.130116801-30118800 bp;
the copy number variation type is according to 2 x 2-ΔCtThe quantitative results were divided into three categories: insertion type, 2 x 2-ΔCt>2.0; deletion form, 2 x 2-ΔCt<2.0; normal type, 2 x 2-ΔCt =2.0；
The goat is selected from Shanxi white cashmere goat.
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