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

A method for detection of wool traits assisted by AMH gene CNV markers in Alpine Merino sheep and applications

technical field

The invention belongs to the technical field of molecular biology detection, and in particular relates to a method and an application of an assisted detection method and application of the CNV marker of the AMH gene of alpine merino sheep.

Background technique

Wool refers to a layer of fibers with textile value covering the surface of sheep, which is a derivative of skin. Wool is the main raw material of the wool spinning industry, accounting for about 97% of wool spinning raw materials, and is mainly used to process clothing fabrics, wool, blankets and other products. In wool production and breeding practice, wool traits are important economic traits, mainly including multiple indicators such as wool length, wool yield, average fiber diameter, curvature, breaking strength, elongation, net wool rate, etc., which are related to weaving products and Economic benefits are closely related. Demand for wool has outstripped supply due to population growth. As market demands change, producing high-quality wool has become the main goal of goat breeding.

Alpine Merino sheep is the female parent of Gansu alpine fine-wool sheep and Australian Merino sheep is the male parent. It is the first in the world to adapt to the cold and drought at an altitude of 2400-4070m after 20 years of breeding by comprehensively utilizing modern advanced biotechnology and breeding technology. In the ecological zone, a new breed of Merino sheep with wool fiber diameters ranging from 19.0 to 21.5 μm. Wool shape is also an important indicator to determine its economic value, and the objective inspection of wool shape is getting more and more attention in wool sales, and it is more and more closely combined with textile performance, breeding and production.

Compared with traditional breeding methods, the molecular marker-assisted selection method has many advantages, such as significantly shortening the generation interval, improving the selection accuracy, and advancing the selection time. Traits that are difficult to measure and costly to measure have a good selection effect. Copy number variation (CopyNumber Variations, CNVs), as a newly discovered type of genome submicroscopic 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 gain and copy number loss.

Commonly used CNV detection methods are mainly divided into two types: detecting unknown CNVs in the whole genome and using for fixed-point detection or verification of known CNVs. Commonly used detection methods for genome-unknown CNVs include chip method and sequencing method, but these two methods are limited by the detection platform and are expensive; for the detection of confirmed CNVs, some methods based on PCR technology and hybridization technology are usually used . Among them, real-time fluorescent quantitative PCR (QPCR) is the most commonly used. QPCR performs relative quantification of the target gene (with copy number variation) and reference gene (without copy number variation), and statistically detects the copy number of the candidate gene in the sample according to the 2 ^-ΔΔCt method. The method is simple to operate, highly universal, fast, and highly recognized.

Anti-Mullerian hormone (Anti-Mullerian hormone, AMH) is a glycoprotein expressed only in gonads secreted by Sertoli cells of male testes and granulosa cells of female ovaries, and is a member of the TGFβ superfamily (Transforming growthfactor beta superfamily, TGFβ superfamily). family), it has the functions of regulating cell differentiation and development, and promoting the degeneration of Müllerian ducts. At the same time, AMH can also be used as a predictor of superovulation in ruminants, and it plays a good role in ruminant breeding. However, there is no literature report on the relationship between AMH gene CNV and sheep wool traits.

Contents of the invention

The invention provides a method and application for detecting the CNV marker of the AMH gene of alpine merino sheep, and in particular relates to a method for detecting the CNV marker of the AMH gene of alpine merino sheep based on QPCR technology and its application. Specifically include the following:

In a first aspect, the present invention provides a CNV marker related to the traits of wool net wool of Alpine Merino sheep, and the CNV marker is located at chr5: 18901101-18905300 in the exon region of the AMH gene of Alpine Merino sheep.

In the second aspect, the present invention provides the application of the reagents for detecting the CNV markers related to the wool yield traits of Alpine Merino sheep described in the first aspect above in detecting the wool yield of Alpine Merino sheep; The CNV marker and internal reference gene ANKRD1 described in claim 1 are added.

Preferably, the amplification result defines the copy number variation type according to the 2×2 ^-ΔΔCt method: 2×2 ^-ΔΔCt =2 is normal type, 2×2 ^-ΔΔCt >2 is insertion type, 2×2 ^-ΔΔCt <2 It is a deletion type; the net wool rate of the normal type and the deletion type is significantly higher than that of the insertion type, and the wool net rate of the normal type is significantly higher than that of the deletion type.

Preferably, the reagents include the CNV-labeled amplification primer pair P1 according to claim 1:

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

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

And the amplification primer pair P2 of the internal reference gene ANKRD1:

Upstream primer F2: 5'-TCTTGTACCGATTCAGCC-3';

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

In the third aspect, the present invention provides the application of the reagent for detecting the CNV markers related to the wool yield traits of Alpine Merino sheep described in the first aspect above in the early breeding of Alpine Merino sheep; the reagent expands the claims 1 The CNV marker and internal reference gene ANKRD1.

Preferably, the amplification result defines the copy number variation type according to the 2×2 ^-ΔΔCt method: 2×2 ^-ΔΔCt =2 is normal type, 2×2 ^-ΔΔCt >2 is insertion type, 2×2 ^-ΔΔCt <2 It is a deletion type; the net wool rate of the normal type and the deletion type is significantly higher than that of the insertion type, and the wool net rate of the normal type is significantly higher than that of the deletion type.

Preferably, the reagents include the CNV-labeled amplification primer pair P1 according to claim 1:

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

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

And the amplification primer pair P2 of the internal reference gene ANKRD1:

Upstream primer F2: 5'-TCTTGTACCGATTCAGCC-3';

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

In the fourth aspect, the present invention provides a pair of primers for detecting CNV markers related to the wool yield traits of Alpine Merino sheep, the primer pair includes the amplification primer pair of CNV markers described in the first aspect above P1:

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

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

And the amplification primer pair P2 of the internal reference gene ANKRD1:

Upstream primer F2: 5'-TCTTGTACCGATTCAGCC-3';

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

In the fifth aspect, the present invention provides a kit for detecting the CNV marker of claim 1 by QPCR, characterized in that the kit contains the primer pair described in the fourth aspect above.

Preferably, the kit also includes One or more of Top Green qPCR SuperMix, deionized water, and control samples.

In the sixth aspect, the present invention provides a method for detecting CNV markers related to the wool yield traits of Alpine Merino sheep, the method comprising the following steps:

Using the genomic DNA of Alpine Merino sheep as a template, amplify the CNV marker and the reference gene ANKRD1 described in the first aspect above by QPCR;

According to the quantitative results of the 2×2 ^-ΔΔCt method, the copy number variation is divided into three types: 2×2 ^-ΔΔCt = 2 is normal type, 2×2 ^-ΔΔCt >2 is insertion type, and 2×2 ^-ΔΔCt <2 is Deletion type; the net wool rate of the normal type and the deletion type is significantly higher than that of the insertion type, and the wool net rate of the normal type is significantly higher than that of the deletion 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:

Upstream primer F2: 5'-TCTTGTACCGATTCAGCC-3';

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

Preferably, the QPCR amplification system includes: Top Green qPCR SuperMix 10 μL, upstream and downstream primers 0.4 μL each, genomic DNA 1 μL, Nuclease-free Water 8.2 μL.

Preferably, the reaction procedure used in the QPCR is:

(1) Pre-denaturation: 94°C, 30s;

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

(3) Draw the melting curve: 95°C, 5s, then from 65°C to 95°C, +0.5°C, 5s.

In combination with all the above-mentioned technical solutions, the advantages and positive effects of the present invention are:

The present invention provides a CNV marker related to the traits of wool net wool of Alpine Merino sheep, and the CNV marker is located in chr5: 18901101-18905300 of the exon region of AMH gene of Alpine Merino sheep;

The invention provides a method for detecting CNV markers, using the whole genome DNA of alpine merino sheep as a template, amplifying the CNV region of the AMH gene of alpine merino sheep through real-time fluorescent quantitative PCR method, and using the ANKRD1 gene as a reference, The quantitative results of the 2×2 ^-ΔΔCt method divide the copy number variation into three types: 2×2 ^-ΔΔCt = 2 is normal type, 2×2 ^-ΔΔCt >2 is insertion type, and 2×2 ^-ΔΔCt <2 is deletion type; the wool net rate of the normal type and the missing type is higher, the wool net rate of the normal type and the missing type is significantly higher than that of the insertion type, and the wool net rate of the normal type is significantly higher than that of the missing type ; The present invention detects CNV markers closely related to the wool yield traits of Alpine Merino sheep at the DNA level, and can be used as an important candidate molecular marker for marker-assisted selection of the wool yield traits of Alpine Merino sheep;

The present invention takes the CNV of Alpine Merino sheep AMH gene as a candidate site, and detects the copy number variation of the site in the Alpine Merino sheep population by real-time fluorescent quantitative PCR technology, and compares it with important economic factors such as wool net wool rate. traits; if the copy number variation type of the AMH gene candidate locus is detected as normal type and deletion type, it will have a higher wool clean rate, especially the normal type wool has the highest clean rate; study the CNV of the gene and The analysis of its association with important economic traits of alpine merino sheep is very important, which can provide a theoretical basis for the molecular breeding of alpine merino sheep in China, facilitate the marker-assisted selection of alpine merino wool wool yield traits, and quickly establish genetic resources An excellent alpine merino sheep herd.

The copy number variation detection method of Alpine Merino sheep gene provided by the present invention can be used for early breeding of Alpine Merino sheep; the method for detecting AMH gene copy number variation is accurate, reliable, and easy to operate; AMH gene copy number variation site provide a scientific basis for the molecular marker-assisted selection of alpine merino sheep.

Description of drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings that need to be used in the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For Those of ordinary skill in the art can also obtain other drawings based on these drawings without making creative efforts.

Fig. 1 Amplification curve of Alpine Merino sheep AMH gene;

Fig. 2 Melting curve of Alpine Merino sheep AMH gene;

Fig. 3 Melting curve of internal reference gene ANKRD1.

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

Example 1 Detection of Alpine Merino sheep AMH gene CNV markers

1. Sample collection

The samples of Alpine Merino sheep came from the Sheep Breeding Technology Promotion Station of Gansu Province. The blood samples of 152 Alpine Merino sheep with production performance records were collected. 5 mL of venous blood was collected from each sheep in a blood collection tube added with EDTA-K2 anticoagulant. The blood samples After the collection is completed, mix them quickly, put them into a sampling box containing ice packs for temporary storage, and transport them back to the laboratory and store them in a -20°C refrigerator for DNA extraction. The records of wool traits of each sheep (length of side hair, diameter of wool fiber, amount of sheared wool, net wool rate) were provided by Gansu Sheep Breeding Technology Extension Station.

2. Main reagents and instruments

EDTA-K2 vacuum blood collection tubes were purchased from Jiangsu Yuli Medical Instrument Co., Ltd.; blood genome extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; NanoDrop2000 spectrophotometer was purchased from Thermo Fisher Scientific, USA; Top Green qPCR SuperMix was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; real-time quantitative fluorescent PCR instrument was purchased from Roche Company.

3. Extraction of Genomic DNA from Blood

Using the blood genome extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract genomic DNA from the blood sample, put the extracted DNA under the ultraviolet spectrophotometer to detect the concentration and purity, the concentration is >20ng/μL, OD260/OD280 Between 1.7-1.9, it meets the needs of the experiment, and it is stored at -20°C for future use.

4. Primer Design

Referring to the AMH gene sequence of chromosome 5 (GenBank accession number: NC_019462.2) of the International Sheep Genome Oar_v4.0 version as the reference sequence, a pair of specific primers were designed in the CNV region (chr5: 18901101-18905300) using primer premier5.0 software At the same time, the same method was used to design and amplify a partial fragment of the internal reference gene ANKRD1. The sequence information of the primers is shown in Table 1. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

Table 1 qPCR primer information table

5. Real-time quantitative PCR amplification

qPCR amplification system 20μL: Top Green qPCR SuperMix 10 μL, upstream and downstream primers 0.4 μL each, genomic DNA 1 μL, Nuclease-free Water 8.2 μL.

PCR amplification program: pre-denaturation at 94°C for 30s; amplification reaction at 94°C for 5s, 58°C for 15s, 72°C for 10s, 45 cycles; draw a melting curve at 95°C for 5s, from 65°C to 95°C, +0.5°C for 5s.

The primers were determined to be suitable for fluorescent quantitative PCR analysis by drawing amplification curves and melting curves. Among them, the amplification curve is smooth, indicating that the fluorescent quantitative PCR amplification system and conditions are suitable (results are shown in Figure 1); the melting curves of each sample of the target gene and the internal reference gene have obvious single peaks at the same position on the abscissa, indicating that the amplification product is single ( The results are shown in Figure 2 and Figure 3, respectively).

6. Calculation of copy number variation

Each individual was amplified with the primers of the target gene and the internal reference gene, and each individual was set up with 3 technical replicates. Analysis of copy number was performed according to the 2x2 ^-ΔΔCt method. Among them, ΔΔCt=(CT target gene-CT internal reference gene) _{experimental group-} (CT target gene-CT internal reference gene) _{control group} ; the experimental group is the individual sample to be detected with or without copy number variation; the control group is known to have no copy number The individual sample with variation can be the reference individual selected in the resequencing test. 2 ^-ΔΔCt indicates the multiple of the copy number of the target gene in the experimental group relative to the control group. According to 2×2 ^-ΔΔCt , the results are divided into three categories: 2×2 ^-ΔΔCt = 2 is normal type, 2×2 ^-ΔΔCt >2 It is an insertion type, and 2×2 ^-ΔΔCt <2 is a deletion type.

7. Association analysis of AMH gene CNV and wool traits

The general linear model in IBM SPSS Statistics 22 software was used to analyze the correlation between the AMH gene copy number variation sites of Alpine Merino sheep and the wool traits. The differences between the data of each group were tested by LSD multiple comparisons. ±standard error” means. The data processing results are shown in Table 2. Alpine Merino sheep AMH gene copy number variation sites have a significant correlation with the wool clean rate (p<0.05), and the normal type wool clean rate is significantly higher than that of the insertion type.

Table 2 Correlation analysis between AMH gene copy number variation and growth traits in Alpine Merino sheep

Note: Different lowercase letters in the same row of data indicate significant differences (P<0.05), and n indicates the number of individuals with the same copy number.

In conclusion, the above results show that the copy number variation of AMH gene affects the wool traits of Alpine Merino sheep. CNV detection can be used to carry out early selection of Alpine Merino sheep wool net wool rate, shorten the breeding cycle, speed up the breeding process, and reduce the breeding risk. cost, and has high application value.

The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Anyone familiar with the technical field within the technical scope disclosed in the present invention, whoever is within the spirit and principles of the present invention Any modifications, equivalent replacements and improvements made within shall fall within the protection 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℃.