CN115927668A - Method for auxiliary detection of wool characters of alpine merino by CCND1 gene CNV marker and application thereof - Google Patents

Abstract

The invention belongs to the technical field of molecular biology detection, and particularly relates to a method for assisting in detecting wool traits of alpine merino sheep by using a CCND1 gene CNV marker and application thereof, wherein the CNV marker related to the wool traits of the alpine merino sheep is positioned in a chr21 exon region of the CCND1 gene of the alpine merino sheep: 46061901-46064400, and provides a method for detecting CNV markers: the method is characterized in that genomic DNA of the alpine merino is used as a template, a CNV region and an internal reference gene of a CCND1 gene are amplified through qPCR by using a primer pair P1 and P2, and copy number variation of the alpine merino CCND1 gene is quantified. The method can detect the CNV marker closely related to the wool character of the alpine merino sheep, is used for early selection of the alpine merino sheep, improves the accuracy of seed selection, shortens the cultivation period and accelerates the cultivation process.

Description

Method for detecting wool characters of alpine merino sheep in auxiliary mode through CCND1 gene CNV marker and application of method

Technical Field

The invention belongs to the technical field of molecular biology detection, and particularly relates to a method for auxiliary detection of wool traits of alpine merino sheep by using a CCND1 gene CNV marker and application thereof.

Background

Wool is a layer of textile-value fiber covering the surface of sheep body, and is a derivative of skin. Wool is the main raw material in the wool spinning industry, accounts for about 97% of the raw material of wool spinning, and is mainly used for processing products such as garment materials, wool, blankets and the like. In wool production and breeding practice, wool characteristics are important economic characteristics, mainly comprise multiple indexes such as wool length, wool yield, average fiber diameter, flexibility, breaking strength, elongation and net wool rate, and are closely related to weaving products and economic benefits. As the population grows, the demand for wool has exceeded the supply. With the change of market demands, the production of high-quality wool has become a main target of goat breeding.

The alpine merino sheep is a new merino sheep variety which is bred by taking Gansu alpine fine wool sheep as a female parent and Australian merino sheep as a male parent through 20 years by comprehensively utilizing modern advanced biotechnology and breeding technology, adapts to alpine frigid-drought ecological areas with the altitude of 2400-4070 m in the first example of the world, and has the main body of the fiber diameter of 19.0-21.5 mu m. Wool shape is also an important index determining economic value, and objective inspection of wool shape is increasingly emphasized in wool sales, which is increasingly combined with textile performance, breeding and production.

Compared with the traditional breeding method, the molecular marker assisted selection method has many advantages, such as obviously shortening generation intervals, improving selection accuracy, advancing selection time, and simultaneously has good selection effect on low heritability, early unexplained characters, characters which are difficult to measure or have high measurement difficulty and high cost. Copy Number Variations (CNVs), a newly discovered type of genomic sub-microscopic level structural variation, refers to the phenomenon of deletion or duplication of larger fragments in genomic DNA, involving fragment sizes ranging from 50bp to Mb, including increased Copy Number (Copy gain) and decreased Copy Number (Copy Number loss).

The common detection methods for CNV are mainly divided into two categories, that is, detection of unknown CNV in the genome-wide range and detection for site-specific detection or verification of known CNV. Common detection methods for the genome unknown CNV include a chip method and a sequencing method, but the two methods are limited by a detection platform and are expensive; for the detection of established CNVs, several methods based on PCR and hybridization techniques are generally employed. Among them, real-time fluorescence Quantitative PCR (QPCR) is most commonly used. QPCR was performed by relative quantification of the gene of interest (with copy number variation) and the reference gene (without copy number variation), according to 2 ^-ΔΔCt The method is used for counting the copy number of the candidate genes of the detection sample. The method has the advantages of simple operation, high universality, high speed and high acceptance degree.

Cyclin D1 (CCND 1) belongs to a highly conserved cell cycle family, the abundance of which varies periodically in the whole cell cycle, is a key cell cycle regulatory protein, has been proved to play an important role in the occurrence and development of malignant tumors, and is closely related to the occurrence, metastasis and prognosis of tumors. However, at present, no literature report on the correlation study of CCND1 gene CNV and sheep wool traits is found.

Disclosure of Invention

The invention provides a method for detecting wool characters of alpine merino by using CCND1 gene CNV marker in an auxiliary manner and application thereof. The method specifically comprises the following steps:

in a first aspect, the present invention provides a CNV marker associated with the lateral hair growth of a mountain merino goat, wherein the CNV marker is located in chr21:46061901-46064400.

In a second aspect, the present invention provides a use of a reagent for detecting a CNV marker associated with the shape of lateral hair of a alpine merino goat in detecting lateral hair of the alpine merino goat; the reagent amplifies the CNV marker and reference gene ANKRD1 of claim 1.

Preferably, the amplification result is according to 2X 2 ^-ΔΔCt The method defines the copy number variation type: 2X 2 ^-ΔΔCt =2 as normal, 2 × 2 ^-ΔΔCt >2 is insertion type, 2X 2 ^-ΔΔCt <2 is a deletion type; the body-side hair length of the normal type is significantly higher than that of the insertion type and the deletion type.

Preferably, the reagents comprise the CNV-labeled amplification primer pair P1:

an upstream primer F1:5 'CCCCGCCGCCGAGTTTGAATTCCTG-3';

a downstream primer R1:5 'ATTTCCCCCAGACGTCATC-3';

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

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

a downstream primer R2:5 'TTCACTCGTTATTGGGAT-3'.

In a third aspect, the present invention provides an application of a reagent for detecting the CNV marker related to the lateral hair growth status of the alpine merino sheep in the early breeding of the alpine merino sheep; the reagent amplifies the CNV marker and reference gene ANKRD1 of claim 1.

Preferably, the amplification result is according to 2 × 2 ^-ΔΔCt The method defines the copy number variation type: 2X 2 ^-ΔΔCt =2 normal type, 2 × 2 ^-ΔΔCt >2 is insertion type, 2X 2 ^-ΔΔCt <2 is a deletion type; the body-side hair length of the normal type is significantly higher than that of the insertion type and the deletion type.

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

an upstream primer F1:5 'CCCCGCGCCGCCGAGTTTGAATTCCTG-3';

a downstream primer R1:5 'ATTTCCCCCAGACGTCATC-3';

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

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

a downstream primer R2:5 'TTCACTCGTTATTGGGAT-3'.

In a fourth aspect, the present invention provides a primer pair for detecting CNV markers associated with lateral hair growth of alpine merino sheep, the primer pair including the CNV-labeled amplification primer pair P1:

an upstream primer F1:5 'CCCCGCCGCCGAGTTTGAATTCCTG-3';

a downstream primer R1:5 'ATTTCCCCCAGACGTCATC-3';

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

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

a downstream primer R2:5 'TTCACTCGTTATTGGGAT-3'.

In a fifth aspect, the present invention provides a kit for QPCR detection of CNV markers as described in the first aspect above, the kit comprising a primer pair as described in the fourth aspect above.

Preferably, the kit further comprises

One or more of Top Green qPCR Supermix, deionized water and a control sample.

In a sixth aspect, the present invention provides a method for detecting a CNV marker associated with lateral hair growth of a alpine merino sheep, the method comprising the steps of:

using genome DNA of a high mountain merino sheep as a template, and respectively amplifying the CNV marker and the reference gene ANKRD1 in the first aspect by QPCR;

according to 2X 2 ^-ΔΔCt The quantitative results classify the copy number variation into three categories: 2X 2 ^-ΔΔCt =2 as normal, 2 × 2 ^-ΔΔCt >2 is insertion type, 2X 2 ^-ΔΔCt <2 is deletion type; the body-side hair length of the normal type is significantly higher than that of the insertion type and the deletion type.

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

an upstream primer F1:5 'CCCCGCCGCCGAGTTTGAATTCCTG-3';

a downstream primer R1:5 'ATTTCCCCCAGACGTCATC-3';

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

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

a downstream primer R2:5 'TTCACTCGTTATTGGGAT-3'.

Preferably, the QPCR amplification system comprises:

top Green qPCR Supermix 10. Mu.L, upstream and downstream primers 0.4. Mu.L each, genomic DNA 1. Mu.L, nucleic-free Water 8.2. Mu.L.

Preferably, the reaction procedure used for QPCR is:

(1) Pre-denaturation: 30s at 94 ℃;

(2) And (3) amplification reaction: 94 ℃,5s; then, the temperature is 58 ℃ and 15s;72 ℃ for 10s;45 cycles;

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

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

the invention provides a CNV marker related to the lateral hair length character of a alpine merino, wherein the CNV marker is positioned in a chr21 exon region of a CCND1 gene of the alpine merino: 46061901-46064400;

the invention provides a method for detecting CNV (CNV) marker, which comprises the steps of respectively amplifying CCND1 gene CNV regions of alpine merino by using whole genome DNA of blood of the alpine merino as a template through a real-time fluorescence quantitative PCR (polymerase chain reaction) method, and taking ANKRD1 gene as reference and 2 multiplied by 2 ^-ΔΔCt The quantitative results classify the copy number variation into three categories: 2X 2 ^-ΔΔCt =2 normal type, 2 × 2 ^-ΔΔCt >2 is insertion type, 2X 2 ^-ΔΔCt <2 is a deletion type; the body side hair length of the normal type is obviously higher than that of the insertion type and the deletion type; the invention detects the CNV marker closely related to the lateral hair growth of the alpine merino sheep on the DNA level and can be used as the markerAn important candidate molecular marker for marker-assisted selection of the mountain merino sheep body-side hair length character;

the CNV of the CCND1 gene of the alpine merino is used as a candidate site, the copy number variation condition of the site in the alpine merino group is detected by a real-time fluorescent quantitative PCR technology, and correlation analysis is carried out on important economic characters such as side hair length and the like; if the copy number variation type of the candidate locus of the CCND1 gene is detected to be a normal type, the body side hair length is longer; the gene CNV is researched and is important to be associated and analyzed with important economic traits of the alpine merino, so that a theoretical basis can be provided for molecular breeding of the alpine merino in China, marker-assisted selection of the alpine merino side wool length trait is facilitated, and a alpine merino population with excellent genetic resources is quickly established;

the method for detecting the copy number variation of the alpine merino gene can be used for early breeding of the alpine merino; the method for detecting the copy number variation of the CCND1 gene is accurate and reliable and is simple and convenient to operate; the detection of the CCND1 gene copy number variation site provides scientific basis for the molecular marker-assisted selection of the alpine merino.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used 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 it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a curve of amplification of the CCND1 gene of a mountain merino sheep;

FIG. 2 is a dissolution curve of the CCND1 gene of the alpine merino sheep;

FIG. 3 the melting curve of reference gene ANKRD1.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1 detection of CNV marker of CCND1 Gene of merino sheep in alpine

1. Sample collection

The sample comes from a sheep breeding technology promotion station in Gansu province, 152 alpine merino sheep blood samples with production performance records are collected, 5mL of blood is collected from veins of each sheep in a blood collection tube added with EDTA-K2 anticoagulant, the blood samples are quickly and uniformly mixed after being collected, the blood samples are put into a sampling box containing an ice bag for temporary storage, and the blood samples are transported back to a laboratory and then are frozen and stored in a refrigerator at the temperature of-20 ℃ for extraction of DNA. The record of the wool characteristics (body side wool length, wool fiber diameter, shearing quantity and net wool rate) of each sheep is provided by a sheep breeding technology promotion station in Gansu province.

2. Main reagent and instrument

The EDTA-K2 vacuum blood collection tube is purchased from Jiangsu Yuli medical instruments GmbH; the blood genome extraction kit is purchased from Tiangen Biochemical technology (Beijing) Co., ltd; nanoDrop2000 Spectrophotometer Thermo Fisher Scientific, USA;

top Green qPCR SuperMix was purchased from Beijing Quanyujin Biotechnology Ltd; real-time quantitative fluorescent PCR instruments were purchased from Roche.

3. Extraction of blood genomic DNA

Extracting genome DNA from blood samples by adopting a blood genome extraction kit of Tiangen Biochemical technology (Beijing) Limited company, detecting the concentration and the purity of the extracted DNA under an ultraviolet spectrophotometer, wherein the concentration is more than 20 ng/mu L, and the OD260/OD280 is between 1.7 and 1.9, thus meeting the experimental requirements, and storing the DNA at the temperature of minus 20 ℃ for later use.

4. Primer design

Referring to the international sheep genome Oar _ v4.0 version 21 chromosome (GenBank accession number: NC-019478.2) CCND1 gene sequence as a reference sequence, a pair of specific primers are designed in a CNV region (chr 21: 46061901-46064400) by using primer premier5.0 software, a partial fragment of an amplified reference gene ANKRD1 is designed by the same method, the sequence information of the primers is shown in Table 1, and the primers are synthesized by Beijing Okagaku Biotechnology Ltd.

TABLE 1qPCR primer information Table

5. Real-time quantitative PCR amplification

qPCR amplification system 20 μ L:

top Green qPCR Supermix 10. Mu.L, upstream and downstream primers 0.4. Mu.L each, genomic DNA 1. Mu.L, nucleic-free Water 8.2. Mu.L.

PCR amplification procedure: pre-denaturation at 94 ℃ for 30s; amplification reactions are carried out for 94 ℃ for 5s,58 ℃ for 15s,72 ℃ for 10s and 45 cycles; melting curves were plotted at 95 ℃ for 5s, from 65 ℃ to 95 ℃ and +0.5 ℃ for 5s.

And determining that the primer is suitable for fluorescent quantitative PCR analysis by drawing an amplification curve and a dissolution curve. Wherein the amplification curve is smooth, which indicates that the fluorescent quantitative PCR amplification system and conditions are appropriate (the result is shown in figure 1); the melting curves of the target gene and the reference gene have obvious single peaks at the same position on the abscissa, which indicates that the amplification 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 delta Ct = (CT target gene-CT reference gene) _{Experimental group} - (CT target gene-CT reference gene) _{Control group} (ii) a The experimental group is an individual sample to be detected whether copy number variation exists or not; the control group is a sample of individuals known to have no copy number variation, and reference individuals selected in a re-sequencing test are used. 2 ^-ΔΔCt Expressed is the fold of the copy number of the target gene in the experimental group relative to the control group, according to 2X 2 ^-ΔΔCt The results are divided into three categories: 2X 2 ^-ΔΔCt =2 normal type, 2 × 2 ^-ΔΔCt >2 is insertion type, 2X 2 ^-ΔΔCt <2 is a deletion type.

Correlation analysis of CCND1 gene CNV and wool traits

The correlation between the CCND1 gene copy number variation site of the alpine merino and wool characters is analyzed by adopting a general linear model in IBM SPSS statics 22 software, the difference among various groups of data is tested by LSD multiple comparison, and the result is expressed as the average value +/-standard error. The data processing results are shown in table 2, the mutation site of the alpine merino CCND1 gene copy number has a significant correlation (p < 0.05) with the body-side hair length, wherein the body-side hair length of the normal type is significantly higher than that of the deletion type and the insertion type.

TABLE 2 Association analysis of the CCND1 gene copy number variation and growth traits of alpine merino sheep

Note: the same row of data is marked with different lower case letters to indicate significant difference (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 CCND1 gene influences the wool property of the alpine merino, the CNV detection is used for carrying out early selection of the alpine merino wool length, the cultivation period is shortened, the cultivation process is accelerated, the breeding cost is reduced, and the application value is high.

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. A CNV marker related to the lateral hair growth of a mountain merino sheep, wherein the CNV marker is located in the chr21 region of the exon of the CCND1 gene of the mountain merino sheep: 46061901-46064400.

2. Use of a CNV-labeled reagent for detecting a lateral wool length trait-related protein in a alpine merino goat as claimed in claim 1 for detecting lateral wool length in a alpine merino goat or early breeding of a alpine merino goat; the reagent amplifies the CNV marker and reference gene ANKRD1 of claim 1.

3. Use according to claim 2, wherein the amplification result is based on 2 x 2 ^-ΔΔCt The method defines the copy number variation type: 2X 2 ^-ΔΔCt =2 normal type, 2 × 2 ^-ΔΔCt >2 is insertion type, 2X 2 ^-ΔΔCt <2 is deletion type; the body-side hair length of the normal type is significantly higher than that of the insertion type and the deletion type.

4. The use of claim 2, wherein the reagents comprise the CNV-labeled amplification primer pair P1:

an upstream primer F1:5 'CCCCGCCGCCGAGTTTGAATTCCTG-3';

a downstream primer R1:5 'ATTTCCCCCAGACGTCATC-3';

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

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

a downstream primer R2:5 'TTCACTCGTTATTGGGAT-3'.

5. A primer pair for detecting CNV markers associated with lateral trichome character in alpine merino sheep, comprising the CNV-labeled amplification primer pair P1:

an upstream primer F1:5 'CCCCGCCGCCGAGTTTGAATTCCTG-3';

a downstream primer R1:5 'ATTTCCCCCAGACGTCATC-3';

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

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

a downstream primer R2:5 'TTCACTCGTTATTGGGAT-3'.

6. Kit for QPCR detection of CNV markers according to claim 1, characterized in that it contains a primer pair according to claim 5.

7. A method for detecting a CNV marker associated with the lateral trichome status of merino sheep in alpine, said method comprising the steps of:

amplifying the CNV marker and the reference gene ANKRD1 of claim 1 by QPCR using genomic DNA of merino sheep in mountain as a template;

according to 2X 2 ^-ΔΔCt The quantitative results classify the copy number variation into three categories: 2X 2 ^-ΔΔCt =2 normal type, 2 × 2 ^-ΔΔCt >2 is insertion type, 2X 2 ^-ΔΔCt <2 is a deletion type; the body-side hair length of the normal type is significantly higher than that of the insertion type and the deletion type.

8. The method of claim 7, wherein the CNV-labeled amplification primer pair P1 is:

an upstream primer F1:5 'CCCCGCGCCGCCGAGTTTGAATTCCTG-3';

a downstream primer R1:5 'ATTTCCCCCAGACGTCATC-3';

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

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

a downstream primer R2:5 'TTCACTCGTTATTGGGAT-3'.

9. The detection method according to claim 8, wherein the QPCR amplification system comprises:

top Green qPCR Supermix 10. Mu.L, upstream and downstream primers 0.4. Mu.L each, genomic DNA 1. Mu.L, nucleic-fre Water 8.2. Mu.L. />

10. The detection method according to claim 8, wherein the QPCR is performed by a reaction procedure comprising:

(1) Pre-denaturation: 30s at 94 ℃;

(2) And (3) amplification reaction: 94 ℃,5s; then, the temperature is 58 ℃ and 15s;72 ℃ for 10s;45 cycles;

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

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