CN115851985B - Method for auxiliary detection of wool characters by AMH gene CNV (complementary deoxyribonucleic acid) markers of merino sheep at high mountain and application - Google Patents

Abstract

The invention belongs to the technical field of molecular biology detection, and particularly relates to a method for detecting wool characters with the assistance of an AMH gene CNV (complementary deoxyribonucleic acid) marker of a merino sheep at high mountain and application thereof, wherein the CNV marker related to the merino sheep wool characters at high mountain is positioned in an AMH gene exon region chr5:18901101-18905300 and provides a method of detecting a CNV marker: amplifying a CNV region of an AMH gene and an internal reference gene by qPCR with primer pairs P1 and P2 by taking genomic DNA of the alpine merino sheep as a template, and quantifying copy number variation of the alpine merino sheep AMH gene; the method is simple to operate, accurate and reliable. The invention can detect CNV marks closely related to the wool traits of the mountain merino sheep, is used for the early selection of the mountain merino sheep, improves the seed selection accuracy, shortens the cultivation period and accelerates the cultivation process.

Description

Method for auxiliary detection of wool characters by AMH gene CNV (complementary deoxyribonucleic acid) markers of merino sheep at high mountain and application

Technical Field

The invention belongs to the technical field of molecular biology detection, and particularly relates to a method for detecting wool characters with the aid of AMH gene CNV markers of merino sheep at high mountain and application thereof.

Background

Wool is a layer of textile-value fiber which is covered on the surface of sheep body and is a derivative of skin. Wool is a main raw material of the wool spinning industry, and accounts for about 97% of the wool spinning raw material, and is mainly used for processing products such as clothing fabrics, knitting wool, blanket and the like. In wool production and breeding practice, wool characteristics are important economic characteristics, and mainly comprise a plurality of indexes such as wool length, wool yield, average fiber diameter, bending degree, breaking strength, elongation, net wool rate and the like, and are closely related to woven products and economic benefits. As the population grows, the demand for wool has exceeded supply. With the change of market demands, the production of high-quality wool has become a main goal of goat breeding.

The alpine merino sheep is a new merino sheep variety which takes Gansu alpine fine wool sheep as a female parent and Australian merino sheep as a male parent, comprehensively utilizes modern advanced biotechnology and breeding technology, adapts to alpine arid ecological regions with the altitude of 2400-4070 m in the first example of the world bred for 20 years, and has the wool fiber diameter of 19.0-21.5 mu m. Wool shape is also an important index for determining economic value, and objective inspection of wool shape is increasingly paid attention to wool sales, and is more and more tightly combined with spinning performance, breeding and production.

Compared with the traditional breeding method, the molecular marker assisted selection method has a plurality of advantages, such as obviously shortening the generation interval, improving the selection accuracy, advancing the selection time, and simultaneously having good selection effect on the characteristics of low genetic strength, the characteristics which are not represented in early stage, the characteristics which are difficult to measure in living bodies or have larger measurement difficulty and higher cost. Copy number variation (Copy Number Variations, CNVs), a newly discovered type of genomic sub-microscopic structural variation, refers to the deletion or duplication of larger fragments in genomic DNA, involving fragments ranging in size from 50bp to several Mb, including Copy number increase (Copy number gain) and Copy number decrease (Copy number loss).

The detection methods commonly used for CNV are largely divided into two categories, detection of unknown CNV over the whole genome and for site-directed detection or validation of known CNV. The common detection methods of the unknown CNV of the genome comprise a chip method and a sequencing method, but the two methods are limited by a detection platform and have high price; for the detection of established CNV, methods based on PCR techniques and hybridization techniques are generally employed. Among them, real-time fluorescent Quantitative PCR (QPCR) is most commonly used. QPCR is performed on a target gene (withWith copy number variation) and a reference gene (without copy number variation) according to 2 ^-ΔΔCt The method is used for counting and detecting the copy number of the sample candidate genes. The method has the advantages of simple operation, high universality, high speed and high acceptance degree.

Anti-Miaole's tube hormone (Anti-Mullerian hormone, AMH) is a glycoprotein secreted by male testis support cells and female ovary granulosa cells and expressed only in gonads, is a member of TGF beta superfamily (Transforming growth factor beta superfamily, TGF beta superfamily), and has effects of regulating cell differentiation and development, promoting Miaole's tube degeneration, etc. Meanwhile, AMH can also be used as a prediction index of ruminant superovulation, plays a good role in ruminant breeding, but at present, no literature report on the research on the correlation of AMH gene CNV and sheep wool traits is seen.

Disclosure of Invention

The invention provides a method for detecting AMH gene CNV markers of merino alpine sheep and application thereof, in particular to a method for detecting AMH gene CNV markers of merino alpine sheep based on QPCR technology and application thereof. The method specifically comprises the following steps:

in a first aspect, the present invention provides a CNV marker associated with the merino alpine wool net hair rate trait, said CNV marker being located in the merino alpine AMH gene exon region chr5:18901101-18905300.

In a second aspect, the present invention provides an application of the CNV-labeled reagent related to the merino alpine wool net rate trait in the first aspect in detecting merino alpine wool net rate; the agent amplifies the CNV marker of claim 1 and the internal reference gene ANKRD1.

Preferably, the amplification result is according to 2X 2 ^-ΔΔCt The method defines copy number variation types: 2X 2 ^-ΔΔCt =2 is normal, 2×2 ^-ΔΔCt >2 is insertion type, 2×2 ^-ΔΔCt <2 is a deletion type; the normal and absent types of wool are significantly higher in net wool than the insert type, while the normal type of wool is significantly higher in net wool than the absent type.

Preferably, the reagent comprises the CNV-labeled amplification primer pair P1 of claim 1:

upstream primer F1:5'-AAGCCAGCACCTCCCTACTCC-3';

downstream primer R1:5'-CCAGCCTCACTATGCAGAACCAC-3';

and an amplification primer pair P2 of the reference gene ANKRD 1:

the upstream primer F2:5'-TCTTGTACCGATTCAGCC-3';

downstream primer R2:5'-TTCACTCGTTTATTGGGAT-3'.

In a third aspect, the present invention provides an application of the CNV-labeled reagent related to the merino alpine sheep wool net rate trait in the first aspect in early breeding of merino alpine sheep; the agent amplifies the CNV marker of claim 1 and the internal reference gene ANKRD1.

Preferably, the amplification result is according to 2X 2 ^-ΔΔCt The method defines copy number variation types: 2X 2 ^-ΔΔCt =2 is normal, 2×2 ^-ΔΔCt >2 is insertion type, 2×2 ^-ΔΔCt <2 is a deletion type; the normal and absent types of wool are significantly higher in net wool than the insert type, while the normal type of wool is significantly higher in net wool than the absent type.

Preferably, the reagent comprises the CNV-labeled amplification primer pair P1 of claim 1:

upstream primer F1:5'-AAGCCAGCACCTCCCTACTCC-3';

downstream primer R1:5'-CCAGCCTCACTATGCAGAACCAC-3';

and an amplification primer pair P2 of the reference gene ANKRD 1:

the upstream primer F2:5'-TCTTGTACCGATTCAGCC-3';

downstream primer R2:5'-TTCACTCGTTTATTGGGAT-3'.

In a fourth aspect, the present invention provides a primer pair for detecting a CNV marker associated with the merino wool net hair rate trait in alpine, the primer pair comprising the CNV-labeled amplification primer pair P1 of the first aspect described above:

upstream primer F1:5'-AAGCCAGCACCTCCCTACTCC-3';

downstream primer R1:5'-CCAGCCTCACTATGCAGAACCAC-3';

and an amplification primer pair P2 of the reference gene ANKRD 1:

the upstream primer F2:5'-TCTTGTACCGATTCAGCC-3';

downstream primer R2:5'-TTCACTCGTTTATTGGGAT-3'.

In a fifth aspect, the present invention provides a kit for QPCR detection of CNV markers according to claim 1, characterized in that the kit comprises a primer pair according to the fourth aspect.

Preferably, the kit further comprisesTop Green qPCR SuperMix, deionized water, control samples.

In a sixth aspect, the present invention provides a method for detecting a CNV marker associated with the merino wool net rate trait in alpine, the method comprising the steps of:

respectively amplifying the CNV marker and the reference gene ANKRD1 of the first aspect by QPCR with genomic DNA of the merino alpine as a template;

according to 2X 2 ^-ΔΔCt Quantitative results of the method divide the copy number variation into three categories: 2X 2 ^-ΔΔCt =2 is normal, 2×2 ^-ΔΔCt >2 is insertion type, 2×2 ^-ΔΔCt <2 is a deletion type; the normal and absent types of wool are significantly higher in net wool than the insert type, while the normal type of wool is significantly higher in net wool than the absent type.

Preferably, the CNV-labeled amplification primer pair P1 is:

upstream primer F1:5'-AAGCCAGCACCTCCCTACTCC-3';

downstream primer R1:5'-CCAGCCTCACTATGCAGAACCAC-3';

the amplification primer pair P2 of the internal reference gene ANKRD1 is as follows:

the upstream primer F2:5'-TCTTGTACCGATTCAGCC-3';

downstream primer R2:5'-TTCACTCGTTTATTGGGAT-3'.

Preferably, the QPCR amplification systemComprising the following steps:top Green qPCR SuperMix 10. Mu.L of each of the upstream and downstream primers was 0.4. Mu.L, 1. Mu.L of genomic DNA and 8.2. Mu.L of nucleic-free Water.

Preferably, the reaction procedure used for QPCR is:

(1) Pre-denaturation: 94 ℃ for 30s;

(2) Amplification reaction: 94 ℃ for 5s; then 58 ℃ for 15s;72 ℃,10s;45 cycles;

(3) Drawing a melting curve: 95℃for 5s, followed by from 65℃to 95℃and +0.5℃.

By combining all the technical schemes, the invention has the advantages and positive effects that:

the invention provides a CNV marker related to wool purification rate characteristics of merino sheep in alpine, which is positioned in an exon region chr5 of an AMH gene of merino sheep in alpine: 18901101-18905300;

the invention provides a method for detecting CNV markers, which uses blood whole genome DNA of alpine merino sheep as a template, respectively expands CNV regions of AMH genes of alpine merino sheep by a real-time fluorescent quantitative PCR method, and uses ANKRD1 genes as a reference, 2 multiplied by 2 ^-ΔΔCt Quantitative results of the method divide the copy number variation into three categories: 2X 2 ^-ΔΔCt =2 is normal, 2×2 ^-ΔΔCt >2 is insertion type, 2×2 ^-ΔΔCt <2 is a deletion type; the normal and missing type wool net rates are higher, the normal and missing type wool net rates are significantly higher than the insertion type wool net rate, and the normal type wool net rate is significantly higher than the missing type wool net rate; the invention detects CNV mark closely related to the wool net rate character of the mountain merino sheep on the DNA level, and can be used as an important candidate molecular mark for the mark auxiliary selection of the wool net rate character of the mountain merino sheep;

according to the invention, CNV of AMH gene of the merino alpine sheep is taken as a candidate site, the copy number variation condition of the site in the merino alpine sheep population is detected by a real-time fluorescent quantitative PCR technology, and the correlation analysis is carried out with important economic characters such as wool purification rate and the like; if the copy number variation type of the AMH gene candidate locus is detected to be normal type and deletion type, the AMH gene candidate locus has higher wool clearance, and especially the normal wool clearance is the highest; the research of the gene CNV and the correlation analysis of the gene CNV and important economic characters of the alpine merino sheep are important, so that a theoretical basis can be provided for molecular breeding of the alpine merino sheep in China, the marker-assisted selection of the wool purification rate characters of the alpine merino sheep is facilitated, and the alpine merino sheep population with excellent genetic resources is quickly established.

The copy number variation detection method of the gene of the merino alpine can be used for early breeding of the merino alpine; the method for detecting AMH gene copy number variation is accurate and reliable and is simple and convenient to operate; the detection of AMH gene copy number variation site provides scientific basis for molecular marker assisted selection of mountain merino sheep.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows an amplification curve of AMH gene of merino sheep at high mountain;

FIG. 2 dissolution profile of AMH gene of merino sheep at mountain;

FIG. 3 dissolution profile of reference gene ANKRD1.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Example 1 detection of AMH Gene CNV markers from mountain merino sheep

1. Sample collection

The method comprises the steps of collecting 152 merino sheep blood samples with production performance records from a breeding technology popularization station of Gansu sheep, taking 5mL of each merino sheep vein blood sample into a blood collection tube added with EDTA-K2 anticoagulant, quickly mixing the blood samples uniformly after the blood sample collection is finished, placing the blood samples into a sampling box containing an ice bag for temporary storage, transporting the blood samples back to a laboratory, and freezing the blood samples at the temperature of minus 20 ℃ for preservation for DNA extraction. The record of each sheep wool character (body side wool length, wool fiber diameter, wool shearing amount and net wool rate) is provided by a Gansu province sheep breeding technology popularization station.

2. Main reagent and instrument

EDTA-K2 vacuum blood collection tubes were purchased from Jiangsu Yuli medical instruments Co., ltd; blood genome extraction kit was purchased from tiangen biochemical technology (beijing) limited; nanoDrop2000 spectrophotometer us Thermo Fisher Scientific company;top Green qPCR SuperMix from Beijing all gold Biotechnology Co., ltd; real-time quantitative fluorescent PCR instruments were purchased from Roche company.

3. Extraction of blood genomic DNA

Extracting genome DNA from blood sample by adopting a blood genome extraction kit of Tiangen biochemical technology (Beijing) limited company, and placing the extracted DNA under an ultraviolet spectrophotometer to detect the concentration and purity, wherein the concentration is more than 20 ng/mu L, OD260/OD280 and is between 1.7 and 1.9, thus meeting the experimental requirement, and storing at-20 ℃ for standby.

4. Primer design

Referring to the chromosome 5 (GenBank accession number: NC-019462.2) AMH gene sequence of International sheep genome Oar_v4.0 as a reference sequence, a pair of specific primers was designed in CNV region (chr5: 18901101-18905300) by using primer premier5.0 software, and a partial fragment of the internal reference gene ANKRD1 was amplified by the same method, the sequence information of the primers is shown in Table 1, and the primers were synthesized by Beijing qing biological science and technology Co.

TABLE 1qPCR primer information Table

5. Real-time quantitative PCR amplification

qPCR amplification System 20. Mu.L:top Green qPCR SuperMix 10. Mu.L of each of the upstream and downstream primers was 0.4. Mu.L, 1. Mu.L of genomic DNA and 8.2. Mu.L of nucleic-free Water.

PCR amplification procedure: pre-denaturation at 94℃for 30s; amplification reaction at 94℃for 5s,58℃for 15s,72℃for 10s,45 cycles; the melting curve was drawn at 95℃for 5s, from 65℃to 95℃for 5s, +0.5℃.

The primer is determined by drawing an amplification curve and a dissolution curve, and is suitable for fluorescent quantitative PCR analysis. Wherein the amplification curve is smooth, which indicates that the fluorescent quantitative PCR amplification system and conditions are proper (the result is shown in FIG. 1); the dissolution curves of the target gene and the reference gene have obvious single peaks at the same positions on the abscissa, which indicates that the amplified products are single (the results are respectively shown in FIG. 2 and FIG. 3).

6. Calculation of copy number variation

Each individual was amplified with primers for the gene of interest and the reference gene, respectively, and 3 technical replicates were set for each individual. According to 2X 2 ^-ΔΔCt The method performs copy number analysis. Wherein ΔΔct= (Ct gene of interest-Ct reference gene) _{Experimental group} - (CT objective gene-CT reference gene) _{Control group} The method comprises the steps of carrying out a first treatment on the surface of the The experimental group is an individual sample of whether copy number variation exists or not to be detected; the control group is a sample of individuals known to have no copy number variation, and may be selected from reference individuals in a resequencing assay. 2 ^-ΔΔCt The copy number of the objective gene of the experimental group is shown as a multiple of that of the control group according to 2X 2 ^-ΔΔCt The results are divided into three categories: 2X 2 ^-ΔΔCt =2 is normal, 2×2 ^-ΔΔCt >2 is insertion type, 2×2 ^-ΔΔCt <2 is a deletion type.

Correlation analysis of AMH Gene CNV and wool traits

The correlation of AMH gene copy number variation sites and wool traits of the mountain merino sheep was analyzed by using a general linear model in IBM SPSS Statistics software, and the differences between the data groups were tested by using LSD multiple comparisons, and the results were expressed as average value.+ -. Standard error. The data processing results are shown in Table 2, the AMH gene copy number variation sites of the high mountain merino sheep have obvious correlation (p < 0.05) with wool net hair rate, wherein the wool net hair rate of the normal type is obviously higher than that of the insertion type.

TABLE 2 correlation analysis of AMH Gene copy number variation and growth Properties of mountain merino sheep

Note that: the different lower case letters between the same row of data indicate significant differences (P < 0.05), and n indicates the number of individuals with the same copy number.

In a word, the results show that the copy number variation of the AMH gene affects wool properties of the alpine merino sheep, CNV detection is used for carrying out early selection of wool purification rate of the alpine merino sheep, so that the cultivation period is shortened, the cultivation process is quickened, the breeding cost is reduced, and the method has high application value.

The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.

Claims (4)

1. The application of the CNV marked reagent for detecting the wool net rate of the mountain merino sheep or the breeding of the wool net rate of the mountain merino sheep is disclosed; the CNV marker is positioned in an exon region chr5 of AMH genes of the merino sheep in the mountain: 18901101-18905300 GenBank accession number of the reference sequence is NC_019462.2; amplifying the CNV marker and the internal reference gene ANKRD1 by the reagent;

the reagents include a CNV-labeled amplification primer pair P1:

upstream primer F1:5'-AAGCCAGCACCTCCCTACTCC-3';

downstream primer R1:5'-CCAGCCTCACTATGCAGAACCAC-3';

and an amplification primer pair P2 of the reference gene ANKRD 1:

the upstream primer F2:5'-TCTTGTACCGATTCAGCC-3';

downstream primer R2:5'-TTCACTCGTTTATTGGGAT-3';

amplification results according to 2X 2 ^-ΔΔCt The method defines copy number variation types: 2X 2 ^-ΔΔCt =2 is normal, 2×2 ^-ΔΔCt >2 is insertion type, 2×2 ^-ΔΔCt <2 is a deletion type; the normal and absent types of wool are significantly higher in net wool than the insert type, while the normal type of wool is significantly higher in net wool than the absent type.

2. A method for detecting a CNV marker associated with the merino wool net rate trait in alpine, said method comprising the steps of:

respectively amplifying CNV markers and internal reference genes ANKRD1 related to wool purification rate characters of the alpine merino sheep by QPCR by taking genomic DNA of the alpine merino sheep as a template; the CNV marker is positioned in an exon region chr5 of AMH genes of the merino sheep in the mountain: 18901101-18905300 GenBank accession number of the reference sequence is NC_019462.2;

the amplification primer pair P1 of the CNV label is as follows:

upstream primer F1:5'-AAGCCAGCACCTCCCTACTCC-3';

downstream primer R1:5'-CCAGCCTCACTATGCAGAACCAC-3';

the amplification primer pair P2 of the internal reference gene ANKRD1 is as follows:

the upstream primer F2:5'-TCTTGTACCGATTCAGCC-3';

downstream primer R2:5'-TTCACTCGTTTATTGGGAT-3';

according to 2X 2 ^-ΔΔCt Quantitative results divide copy number variation into three categories: 2X 2 ^-ΔΔCt =2 is normal, 2×2 ^-ΔΔCt >2 is insertion type, 2×2 ^-ΔΔCt <2 is a deletion type; the wool purifying rate of the normal type and the deletion type is obviously higher than that of the insertion type, and the method simultaneously comprises the following steps ofThe normal type wool net rate is significantly higher than the absent type.

3. The method of detection of claim 2, wherein the QPCR amplification system comprises:top Green qPCR SuperMix 10. Mu.L of each of the upstream and downstream primers was 0.4. Mu.L, 1. Mu.L of genomic DNA and 8.2. Mu.L of nucleic-free Water.

4. The method of claim 2, wherein the QPCR is performed using the following reaction procedure:

(1) Pre-denaturation: 94 ℃ for 30s;

(2) Amplification reaction: 94 ℃ for 5s; then 58 ℃ for 15s;72 ℃,10s;45 cycles;

(3) Drawing a melting curve: 95℃for 5s, followed by from 65℃to 95℃and +0.5℃.