阅读说明：本技术 一种绵羊chchd7基因插入/缺失多态性的检测方法及其应用 (Detection method of sheep CHCHCHD 7 gene insertion/deletion polymorphism and application thereof ) 是由 蔡勇 徐红伟 李海霞 蓝贤勇 阿依木古丽 杨具田 臧荣鑫 于 2020-04-23 设计创作，主要内容包括：本发明涉及生物技术与家畜育种技术领域,具体是公开了一种绵羊CHCHD7基因插入/缺失多态性的检测方法及其应用,该方法包括以下步骤,以包含CHCHD7基因的待测绵羊全基因组DNA为模板,利用PCR扩增包含绵羊CHCHD7基因3’侧翼区插入/缺失多态性位点的片段,对PCR扩增产物进行电泳,根据电泳结果鉴定所述插入/缺失多态性位点的基因型。本发明检测的插入/缺失多态性位点能够作为滩羊公羊体长性状、胸深性状的分子标记,有利于快速建立体尺性状优良的滩羊种群,加快良种选育速度。(The invention relates to the technical field of biotechnology and livestock breeding, and particularly discloses a detection method of sheep CHCHCHD 7 gene insertion/deletion polymorphism and application thereof. The insertion/deletion polymorphic site detected by the invention can be used as a molecular marker for body length traits and chest depth traits of Tan sheep rams, is beneficial to quickly establishing Tan sheep populations with excellent three-dimensional size traits and quickens the breeding speed of improved varieties.)

1. A method for detecting sheep CHCHHD 7 gene insertion/deletion polymorphism is characterized in that: the method comprises the following steps:

taking the whole genome DNA of a sheep to be detected containing a CHCHD7 gene as a template, amplifying a fragment containing the insertion/deletion polymorphic site in the 3' flanking region of the CHCHCHD 7 gene of the sheep by using PCR, carrying out electrophoresis on a PCR amplification product, and identifying the genotype of the insertion/deletion polymorphic site according to an electrophoresis result;

the primer pair (P1) adopted by the PCR is as follows:

an upstream primer: 5'-TGGCCTTGACAGACACATCC-3', respectively;

a downstream primer: 5'-CCTGCAGAGCTTCCCTTTCT-3' are provided.

2. The method for detecting an insertion/deletion polymorphism of a sheep CHCHHD 7 gene according to claim 1, wherein the polymorphism is as follows: the insertion/deletion polymorphic site is selected from 8-bp insertion/deletion polymorphic sites of sheep CHCHCHD 7 gene NC-040260.1: g 36219994-3622000.

3. The method for detecting the sheep CHCHHD 7 gene insertion/deletion polymorphism and the application thereof according to claim 2, are characterized in that: according to the electrophoresis result, the insertion/insertion genotype of the insertion/deletion polymorphic site is represented by 156bp one stripe, the insertion/deletion genotype is represented by 156bp and 148bp and three stripes of a heterologous double-chain band, and the deletion/deletion genotype is not existed.

4. The method for detecting an insertion/deletion polymorphism of a sheep CHCHHD 7 gene according to claim 1, wherein the polymorphism is as follows: the reaction procedure adopted by the PCR is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 68 ℃ for 30s, extension at 72 ℃ for 20s, and 18 cycles, wherein the annealing temperature is reduced by 1 ℃ after each cycle; annealing at 50 ℃ for 30s, extending at 72 ℃ for 12s, and performing 30 cycles; extension at 72 ℃ for 10 min.

5. The method for detecting an insertion/deletion polymorphism of a sheep CHCHHD 7 gene according to claim 1, wherein the polymorphism is as follows: the electrophoresis is performed by using agarose gel with the mass concentration of 3.5%.

6. The use of the method for detecting an insertion/deletion polymorphism of the sheep CHCHCHD 7 gene according to any one of claims 1-5 in sheep molecular marker-assisted selective breeding.

7. Use according to claim 6, characterized in that: the insertion/insertion genotype of the insertion/deletion polymorphic site can be used as a DNA marker for improving the body length and chest depth traits of ram rams.

8. Use according to claim 6, characterized in that: the sheep is selected from Tan sheep.

Technical Field

The invention relates to the technical field of biotechnology and livestock breeding, and particularly belongs to rapid and accurate typing detection of an 8-bp insertion/deletion polymorphism (indel) site of a sheep CHCHCHD 7 gene NC-040260.1: g 36219994-3622000 site and application thereof in molecular marker-assisted selective breeding.

Background

Since the proposal of MAS in 1990, more than twenty genetic markers including RF L P, RAPD, SSCP, RAMP, DDRT and the like have been used in MAS research, which is a method commonly used in molecular breeding to realize direct selection of genotypes, to shorten breeding cycles, accelerate animal breeding speeds, and has superiority in overcoming difficulties in phenotypic identification, early selection, performing harmless trait evaluation and selection, improving breeding efficiency and the like.

Insertion/deletion polymorphism (indel) is a molecular marker, which is an alteration at the DNA or protein sequence level that occurs only second in frequency to residue substitutions, and is expressed as a small fragment of DNA of different size inserted or deleted in the genome. Compared with SNP, indels are all derived from single mutation events, have lower mutation frequency and are relatively stable, structurally belong to the allelic gene polymorphism, and can be amplified by a very small amplicon (<50 bp).

indels are roughly classified into the following 5 major categories: (1) insertions/deletions of a single base pair; (2) single base insertions/deletions; (3) the repetitive unit is a multi-base pair insertion/deletion of 2-15 bases; (4) transposon insertion/deletion; (5) insertion/deletion polymorphism of an arbitrary DNA sequence. With the intensive research of comparative genomics, indels provide a great deal of biological information for theoretical research and genetic breeding application research, and the indels serve as a new generation of genetic identification markers and have the advantages of SNP. indels are mostly focused on genome research of human beings and various crops (such as rice, corn and the like), and are focused on research on growth traits of chickens in livestock and poultry, and research and application on ruminants are few. Therefore, indel marker research on functional genes of ruminant livestock is urgently needed to be developed and advanced.

With the improvement of living standard of people, the demand of society for sheep products is continuously strengthened, but in recent years, sheep products such as mutton, goat milk, cashmere and the like are in serious shortage. On the high-yield, high-quality and high-efficiency sheep breeding targets, the method has the advantages that the genetic polymorphism of DNA markers which are screened on the DNA level and are closely related to sheep growth and development traits is detected, and the association analysis of the genetic polymorphism and the body size traits is carried out, so that the establishment of excellent body size trait sheep populations accelerated by using MAS is always a focus.

The gene CHCHCHD 7 (coded-Coil-Helix-coded-Coil-Helix Domain containment 7), also known as COX23(Cytochrome C Oxidase Assembly) or MGC2217, is located on sheep chromosome 9, has 5 exons and 4 introns, is 7139bp in length, and the cDNA thereof can code for 91 amino acids (Westerman et al, 2004; Banci et al 20152012). CHCHHD 7 protein contains conserved twins Cc9C structural motif, is a conserved factor essential for Cytochrome C Oxidase Assembly in the mitochondrial membrane space (Barros, 2004; Cavallaro, 2010; Banci et al, 2012), may be related to copper delivery during Cytochrome C Oxidase biogenesis, copper homeostasis and to the hemachrome heme during heme transfer (Delala, 20111; bovine hemoglobin, 2018, and bovine cattle growth hormone receptor tyrosine kinase coding for the gene has more and more significant polymorphisms in the growth traits of cattle tissues such as the growth of cattle, cattle.

Disclosure of Invention

The invention aims to provide a detection method of sheep CHCHHD 7 gene insertion/deletion polymorphism and application thereof, which overcome the defects of the prior art.

In order to solve the problems, the technical scheme adopted by the invention is as follows:

a method for detecting sheep CHCHHD 7 gene insertion/deletion polymorphism is characterized in that: the method comprises the following steps:

taking the whole genome DNA of a sheep to be detected containing a CHCHD7 gene as a template, amplifying a fragment containing the insertion/deletion polymorphic site in the 3' flanking region of the CHCHCHD 7 gene of the sheep by using PCR, carrying out electrophoresis on a PCR amplification product, and identifying the genotype of the insertion/deletion polymorphic site according to an electrophoresis result;

the primer pair (P1) adopted by the PCR is as follows:

an upstream primer: 5'-TGGCCTTGACAGACACATCC-3' (20 nt);

a downstream primer: 5'-CCTGCAGAGCTTCCCTTTCT-3' (20 nt).

Further, the insertion/deletion polymorphic site is selected from 8-bp insertion/deletion polymorphic sites at positions NC-040260.1: g 36219994-3622000 of the sheep CHCHCHD 7 gene.

Further, according to the electrophoresis result, the insertion/insertion genotype of the insertion/deletion polymorphic site is represented by 156bp one stripe, the insertion/deletion genotype is represented by 156bp and 148bp and three stripes including a heterologous double-chain band, and the deletion/deletion genotype is not existed.

Further, the reaction procedure adopted by the PCR is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 68 ℃ for 30s, extension at 72 ℃ for 20s, and 18 cycles, wherein the annealing temperature is reduced by 1 ℃ after each cycle; annealing at 50 ℃ for 30s, extending at 72 ℃ for 12s, and performing 30 cycles; extension at 72 ℃ for 10 min.

Further, the electrophoresis was performed using agarose gel with a mass concentration of 3.5%.

The detection method of the sheep CHCHHD 7 gene insertion/deletion polymorphism is applied to sheep molecular marker-assisted selective breeding.

Furthermore, the insertion/insertion genotype of the insertion/deletion polymorphic site can be used as a DNA marker for improving the body length and chest depth traits of ram rams.

Further, the sheep is selected from Tan sheep.

Compared with the prior art, the invention has the following implementation effects:

the invention relates to a detection method of sheep CHCHHD 7 gene insertion/deletion polymorphism and application thereof, wherein a primer is designed according to the insertion/deletion polymorphism site (reference sequence NC-040260.1: g 36219994-3622000 delGTTACAAG) of the 3' flanking region of a sheep CHHD 7 gene, and the genotype of the insertion/deletion polymorphism site can be simply, quickly, at low cost and accurately detected by sequence amplification and electrophoretic identification by taking sheep genome DNA as a template.

The invention analyzes the genotype and the gene frequency of the sheep (such as Tan sheep) CHCHCHHD 7 gene insertion/deletion polymorphic site (reference sequence NC-040260.1: g 36219994-3622000 delGTTACAAG), and performs the correlation analysis of the insertion/deletion polymorphic site and the sheep ram multi-individual size character, and the result shows that the insertion/deletion polymorphic site detected by the invention can be used as the molecular markers of the Tan sheep body length character (P <0.05) and the chest depth character (P <0.05), thereby being beneficial to quickly building a Tan sheep population with excellent three-dimensional size character and accelerating the fine breed breeding speed.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis of the product of primer pair P1 amplified Tan sheep CHCHCHHD 7 gene; m represents Marker; a represents a heterologous double-stranded band.

FIG. 2 is a sequencing diagram of PCR amplification products of the sheep CHCHHD 7 gene, wherein: the part marked by the black box represents the 8-bp insert: NC-040260.1: g 36219994-3622000 delGTTACAAG.

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples, and any modification is within the scope of the present invention without departing from the spirit of the present invention.

The invention utilizes a PCR method to detect the insertion/deletion polymorphism possibly generated by the 9906-9913 site (reference sequence: NC-040260.1) mutation of the sheep CHCHCHD 7 gene, and performs correlation analysis on the polymorphism and the sheep related body size character to verify whether the polymorphism can be used as a molecular marker for auxiliary selection in sheep molecular breeding.

1. Experimental drugs and reagents

1.1 Biochemical and biological reagents ① Taq DNA polymerase (available from Fermantas, MBI), ② proteinase K (available from Huamei bioengineering Co., Ltd.) ③ Marker I (available from Tiangen Biotechnology, Beijing, Ltd.).

1.2 general reagents: the general reagent is purchased from Huamei bioengineering company and is an imported split charging product: citric acid, sodium citrate, glucose, Tris, EDTA, NaCl, NaOH, KCl, Na2HPO4, KH2PO4, Tris saturated phenol, chloroform, isoamyl alcohol, absolute ethanol, sodium acetate, Sodium Dodecyl Sulfate (SDS), Ethidium Bromide (EB), bromophenol blue, dimethyl benzonitrile FF, acetic acid, sucrose, boric acid, agarose and the like.

1.3 solutions and buffers, all prepared with deionized ultrapure water, autoclaving at 15bf/in (1.034 × 105Pa) and 25min, and the preparation of reagents is described in the molecular cloning protocols of Sambrook et al.

1) Solution for extracting tissue-like DNA:

in addition to the common solution for genome DNA extraction, L02 mol/L NaCl 11.688g dissolved in water to 100m L volume, and autoclaved L1 tissue DNA extract (100m L), l mol/L Tris-HCl (pH 8.0) lm L, 0.5 mol/L EDTA (pH 8.0)20m L, 2 mol/L NaCl 5m L to 100m L volume are prepared.

2) Solutions for agarose gel electrophoresis analysis

① 0.5 × TBE buffer solution, 10 × TBE 50m L is taken to be constant volume to 1000m L, ② sample loading buffer solution contains 0.25% bromophenol blue and 0.25% xylene blue FF, and the solvent is 40.0% (w/v) sucrose aqueous solution.

2. Design of sheep CHCHCHD 7 gene indel primer

The sequence (NC-040260.1) of the sheep HIAT1 gene is searched at NCBI, and primers capable of amplifying DNA fragments of a plurality of candidate indel sites of the CHCHCHD 7 gene are designed by using Primer5.0, wherein a PCR primer pair capable of amplifying the 9906 rd and 9913 th indel sites of the 3' flanking region of the sheep CHCHCHD 7 gene is P1 (the primer design time is: 2019, 6 and 30 days). Primer sequences are shown in table 1:

TABLE 1 sheep CHCHCHD 7 Gene candidate indel site amplification primer table

Referring to FIG. 2, the above primer pair P1 amplified the sheep genome, enabling amplification of a fragment containing the sheep CHCHHD 7 gene (NC-040260.1: g 36219994-3622000 delGTTACAAG). Theoretically, when the GTTACAAG between positions 9906 and 9913 is deleted, the PCR product is followed by a band of 148bp size by agarose gel electrophoresis; when GTTACAAG between positions 9906 and 9913 is present (insertion), the PCR product is followed by a band of 156bp size by agarose gel electrophoresis. When an insertion occurs in the GTTACAAG between positions 9906 and 9913 on one allele and a deletion occurs on the other allele, the PCR product is detected by agarose gel electrophoresis to be 156bp +148bp double-stripe and a heterogenous double-chain stripe. Therefore, according to the theoretical analysis result, the insertion/insertion genotype (II) corresponding to the insertion/deletion polymorphic site amplified by the primer pair P1 shows 156bp one-stripe, the insertion/deletion genotype (ID) shows 156bp and 148bp and three-stripe of heterologous double-chain band, and the deletion/deletion genotype (DD) shows 148bp one-stripe.

3. PCR amplification of sheep CHCHCHD 7 gene fragment to be detected by primer pair P1

3.1 Collection of sheep ear tissue samples

The animals used in the experiment were 458 samples in total, and the specific information is shown in table 2. The body size character data is measured by the personnel in the seed storage field or the farm, the individual ear tissue samples are adopted in the sampling mode, the samples are stored by 70 percent ethanol, and the ice box is placed at minus 80 ℃ for freezing storage after being brought back to the laboratory at low temperature.

TABLE 2 sampling information Table

3.2 extraction and isolation of genomic DNA from tissue samples

Reference is made to the molecular cloning guidelines (2002) compiled by Sambrook et al and to the following: lanxian warrior, genetic variation of important functional genes of goats and the relationship between the genetic variation and economic traits [ D. ] in doctor academic thesis of university of agriculture and forestry in northwest, 2007, Shaanxi Yangling.

3.3 agarose gel electrophoresis detection of DNA

Reference is made to the molecular cloning guidelines (2002) compiled by Sambrook et al.

3.4 purification of DNA

Reference is made to the molecular cloning guidelines (2002) compiled by Sambrook et al.

3.5 spectrophotometric detection of DNA

The OD values of the DNA samples at 260nm and 280nm were measured by an ultraviolet photometer. The DNA content and the ratio OD260/OD280 were calculated. If the ratio of OD260/OD280 is less than 1.6, indicating that the sample contains more protein or phenol, then purification is carried out; if the ratio is greater than 1.8, then RNA purification removal should be considered.

The DNA concentration (ng/. mu. L) was 50 × OD260 value × dilution.

After the DNA detection was complete, a certain amount was taken out and diluted to 50 ng/. mu. L, and stored at-20 ℃ for further use, while the remainder were stored at-80 ℃.

3.6PCR amplification

The PCR reaction system adopts a mixed sample adding method, namely the total amount of various reaction components is calculated according to the quantity of various components required by each reaction system and the quantity of PCR reactions required by 1 reaction, the reaction components are added into 1 centrifugal tube with the diameter of 1.5m L, the centrifugal tubes are mixed fully and evenly and are subjected to instantaneous centrifugation, the centrifugal tubes are subpackaged into each PCR tube with the diameter of 0.2m L Eppendorf, then template DNA is added, the PCR amplification is carried out after the instantaneous centrifugation, the PCR reaction system comprises 2 × Taq PCR Supermix (comprising Taq DNA polymerase, dNTPs and optimized reaction buffer solution with the concentration of 2 ×)6.5 mu L0, an upstream primer of 0.5 mu L1, a downstream primer of 0.5 mu L (the concentration of the upstream primer and the downstream primer of 10 pmol/mu L), genomic DNA (the concentration of 50 ng/mu L sheep genomic DNA)0.6 mu L, deionized water of 4.9 mu L and a PCR amplification system with the volume of 13 mu L.

3.7 procedure for PCR reaction

The PCR amplification reaction program of the primer pair P1 is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 68 ℃ for 30s, extension at 72 ℃ for 12s, and 18 cycles, wherein the annealing temperature is reduced by 1 ℃ after each cycle; annealing at 50 ℃ for 30s, extending at 72 ℃ for 12s, and performing 25 cycles; extension at 72 ℃ for 10 min.

4. Agarose gel electrophoresis detection analysis after PCR product amplification

The agarose gel electrophoresis detection comprises 3 steps of 1) preparing 3.5 percent agarose gel, dyeing by using nucleic acid dye, spotting 4.5 mu L, and carrying out electrophoresis at 120V for 1.0-1.2h after spotting, 2) imaging in a BIO-RADGel Doc 2000 gel imaging system when DNA fragments with different molecular weights are clearly separated, and 3) analyzing indel polymorphism according to the agarose gel electrophoresis result;

polymorphisms of indels were judged by photographic analysis using the BIO-RAD Gel Doc 2000 Gel imaging system (see FIG. 1):

for the 8bp insertion/deletion polymorphism (8bp-indel) existing at the NC-040260.1: g 36219994-3622000 site of the sheep CHCHCHD 7 gene, the II genotype shows a 156bp one-strip line, the ID genotype shows a 156bp +148bp and a heterologous double-strand strip three-strip line, and no DD genotype exists. The analysis results were verified by sequencing.

5. Frequency statistical analysis of sheep CHCHHD 7 gene indel locus

1) Gene and genotype frequency

Genotype frequency refers to the ratio of the number of individuals with a certain genotype for a trait to the total number of individuals in a population. PYY NYY/N, where PYY represents the YY genotype frequency at a site; NYY represents the number of individuals in the population having a YY genotype; and N is the total number of detection groups.

Gene frequency refers to the relative ratio of a certain number of genes in a population to the total number of its alleles. The formula for the calculation can be written as: PY ═ 2N (2NYY + NYa1+ NYa2+ NYa3+ NYa4+ … … + NYan)/2N

In the formula, PY represents allele Y frequency, NYY represents the number of individuals having YY genotype in the population, NYAI represents the number of individuals having Yai genotype in the population, and a 1-an are n different multiple alleles of allele Y.

The genotype frequencies and allele frequencies of the CHCHD7 gene at the 8-bp insertion/deletion polymorphic sites are shown in Table 3.

TABLE 3 frequency distribution table of 9906-9913-th indel gene of sheep CHCHCHHD 7 gene

6. Association analysis of sheep CHCHCHD 7 gene indel site gene effect

Genotype data: carrying out agarose gel electrophoresis on the genotype identified after PCR amplification;

production data: the body size (weight (kg), body length (cm), body height (cm), chest width (cm), chest depth (cm), chest circumference (cm), tube circumference (cm) and cross height (cm)) of Tan sheep are 8 characters.

And (3) correlation analysis model: the variety was analyzed using SPSS (24.0) software, with different factors being correlated with body size traits. The resulting data is first analyzed descriptively by statistics to determine if outliers exist. The effect of the genotype is then further analyzed using analysis of variance, multivariate linear models, or t-analysis, based on the characteristics of the data. During the data processing, a fixed model is used for correlation analysis in consideration of the individual effects, the interaction between genes and the genotype effects. In addition, the complete model is as follows according to the practical conditions: y ═ μ + G + E, where Y: (ii) an individual phenotype record; u: an overall mean; g: a marker genotype effect; e: random error.

As can be seen from Table 4, in the body size character study of 458 Tan sheep, the polymorphism of CHCHD7 gene 8bp indel has significant influence on body length and chest depth (P <0.05), and the II genotype individual character is superior to that of the ID genotype individual. Therefore, the sheep CHCHCHD 7 gene NC-040260.1 g 36219994-3622000 site 8-bp insertion/deletion polymorphic site can be used as a DNA molecular marker for body length and chest depth traits of Tan sheep.

TABLE 4 sheep CHCHCHD 7 Gene P1-8bp indel associated with Tan sheep body size trait analysis (Mean + -SE)

Note: mean shoulder letters a and b represent significant differences (P <0.05)

The results show that: different genotypes of the 8bp-indel locus of the sheep CHCHHD 7 gene have obvious association on body length and chest depth in Tan sheep.

In a word, the invention detects the 8-bp insertion/deletion polymorphic site of the sheep CHCHCHHD 7 gene NC-040260.1: g 36219994-3622000 by using a PCR amplification method, and performs correlation analysis on the polymorphism site and the growth traits (8 traits including weight, body height, body length, chest circumference, chest depth, chest width, tube circumference and cross height) of the Tan sheep to find that the polymorphism site can be used as a molecular marker for auxiliary selection in Tan sheep molecular breeding, thereby accelerating the speed of fine breed breeding. The detection method of the sheep CHCHCHD 7 gene NC-040260.1: g 36219994-3622000 site 8-bp insertion/deletion polymorphism site, which is established by the invention, provides theoretical and practical basis for realizing the marker-assisted selection (MAS) application of sheep body length and chest depth characters by using indels.

The foregoing is merely exemplary and illustrative of the present inventive concept and various modifications, additions and substitutions of similar embodiments may be made to the specific embodiments described by those skilled in the art without departing from the inventive concept or exceeding the scope of the claims as defined in the accompanying claims.