CN111733261A

CN111733261A - Detection method and application of goat AKAP12 gene InDel marker

Info

Publication number: CN111733261A
Application number: CN202010718092.2A
Authority: CN
Inventors: 宋晓越; 袁荣荣; 白洋洋; 屈雷; 朱海鲸; 蓝贤勇
Original assignee: Yulin University
Current assignee: Yulin University
Priority date: 2020-07-23
Filing date: 2020-07-23
Publication date: 2020-10-02
Anticipated expiration: 2040-07-23
Also published as: CN111733261B

Abstract

The invention discloses a detection method of a goat AKAP12 gene InDel marker and application thereof. The genotype of the goat AKAP12 gene NC-030816.1 at g.110266-110278 InDel locus is identified by taking the whole genome DNA of the goat to be detected as a template through PCR amplification and agarose gel electrophoresis. According to the correlation analysis result of the InDel locus of the AKAP12 gene and the body length of the Shanxi white cashmere goat, the genotype of the InDel locus of the AKAP12 gene is determined to be a DNA marker for improving the body length of the goat. The invention can quickly establish the goat dominant genetic resource population by quickly and accurately detecting the InDel marker closely related to the goat body length character, thereby accelerating the marker-assisted selective breeding process of the goat growth character.

Description

Detection method and application of goat AKAP12 gene InDel marker

Technical Field

The invention belongs to the field of biotechnology and livestock breeding, relates to detection of gene insertion/deletion polymorphism (InDel), and particularly relates to rapid and accurate detection of an AKAP12 gene InDel marker related to goat body length traits.

Background

Animal breeding techniques mainly include conventional breeding techniques based on phenotype and phenotypic value and molecular breeding techniques based on DNA polymorphisms. As an important component of a molecular breeding technology system, a marker-assisted selection (MAS) breeding technology carries out character selection according to DNA markers which are obviously related to genetic characters, and realizes direct selection of genotypes by analyzing the genetic composition of individuals on the DNA level, thereby improving the directionality of a breeding target. The method has superiority in the aspects of early selection, nondestructive character evaluation and selection, backcross breeding efficiency improvement and the like. Searching important functional genes, screening important gene genetic variation sites, and analyzing the correlation between the important functional gene genetic variation sites and growth traits are the precondition and the key of the application of the marker-assisted selection technology.

Insertion/deletion polymorphism (InDel) refers to the alteration of a DNA sequence by the insertion or deletion of a nucleotide fragment in the DNA sequence, the length of the inserted or deleted fragment being between 1 and 50 bp. The phenotypic difference of individuals can be better explained by analyzing the genome of the individuals by using InDel, so that InDel detection of the difference of small fragment nucleotides on the DNA level has important significance in animal molecular breeding.

With the intensive research of comparative genomics, InDel provides a great deal of genetic information for theoretical research and genetic breeding application, and the InDel is used as a new generation of genetic identification marker and has the advantages of SNP. Both InDel and SNP are from single mutation events, the mutation frequency is low, and the relative stability is high: structurally belonging to the bi-allelic polymorphism, the alleles are fixed and known and can be amplified by a very small amplicon (<50 bp). As an important source of genetic markers, InDel is spread throughout the genome, second only to SNPs, of which about one third is located in known gene regions, and some are located in critical regions determining gene function, such as promoter and exon regions.

In 2005, Schnabel et al combined SSR markers and InDel markers studied the control of milk yield of cows and successfully carried out fine positioning. The research of InDel mostly focuses on the genome research of human beings and various crops (such as rice, corn and the like), and the excavation and application of the InDel marker of the functional gene of ruminant livestock are urgently needed.

With the increasing social demand for goat products, in order to breed local goat breeds earlier and faster on the high-yield, high-quality and high-efficiency goat breeding targets, DNA markers closely related to goat growth traits need to be screened and detected on the DNA level. AKAP12 is the A kinase anchor protein 12 coding gene. At present, reports of DNA markers related to marker-assisted selection of growth traits in the goat AKAP12 gene are not found.

Disclosure of Invention

The invention aims to provide a detection method of a goat AKAP12 gene InDel marker and application thereof, so that the improved variety breeding speed of goats can be accelerated through molecular marker-assisted selection.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for detecting insertion/deletion polymorphism of a goat AKAP12 gene comprises the following steps:

taking the whole genome DNA of a goat individual to be detected as a template and a primer pair P1 as primers, and amplifying a fragment containing 13-bp insertion/deletion polymorphic sites (reference sequence NC-030816.1: g.110266-110278) in the 3' UTR region of AKAP12 gene by PCR; carrying out agarose gel electrophoresis on the PCR amplified fragment; and identifying the genotype of the goat individual at the insertion/deletion polymorphic site according to the agarose gel electrophoresis result.

Preferably, the primer pair P1 is:

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

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

Preferably, the PCR amplification reaction procedure 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 20s, and performing 30 cycles; extension at 72 ℃ for 10 min.

Preferably, the agarose gel electrophoresis is performed by using agarose gel with mass concentration of 3.5%.

Preferably, the insertion/deletion genotype (II) of the insertion/deletion polymorphic site exhibits a single band of 192bp, the insertion/deletion genotype (ID) exhibits two bands of 192bp and 179bp, and the deletion/deletion genotype (DD) exhibits a single band of 179bp, according to the result of agarose gel electrophoresis.

A kit for detecting goat AKAP12 gene insertion/deletion polymorphism comprises the primer pair P1.

The detection method of the goat AKAP12 gene insertion/deletion polymorphism is applied to goat molecular marker-assisted selective breeding. The insertion/insertion genotype (II) of the insertion/deletion polymorphic site can be used as a DNA marker (particularly an InDel marker) for improving the body length of a goat.

The invention has the beneficial effects that:

according to the invention, through PCR amplification and agarose gel electrophoresis, the gene typing identification can be carried out on 110266-110278 (NC-030816.1) insertion/deletion polymorphic sites of the goat AKAP12 gene, and according to the result of the correlation analysis of the insertion/deletion polymorphic sites and the growth traits of the Shanxi white down goat, the AKAP12 gene is firstly found to be obviously related to the growth traits of the goat and has a DNA marker capable of improving the growth traits of the goat. The detection method is simple, rapid and low in cost, and can be used for accurately establishing the goat population with excellent growth traits (such as body length), so that the improved variety breeding speed of the goats is accelerated.

Drawings

FIG. 1 shows the result of 3.5% agarose gel electrophoresis of PCR amplification product of goat AKAP12 gene (primer pair P1); in FIG. 1, A represents a heteroduplex or homoduplex band.

FIG. 2 is a sequence diagram of PCR amplification products of goat AKAP12 gene; the upper half indicates genotype II, the lower half indicates genotype DD, and the part marked with black boxes indicates 13-bp deletion sequence (NC-030816.1: g.110266-110278 delTGGTCTTTGTG).

Detailed Description

The present invention will be described in further detail with reference to the following drawings and examples, which are illustrative of the present invention and are not intended to limit the scope of the present invention.

The invention utilizes PCR and agarose gel electrophoresis methods to detect the insertion/deletion polymorphism generated at 110266-110278 (NC-030816.1) of the goat AKAP12 gene, and performs correlation analysis on the polymorphism and the goat growth character to determine a candidate molecular marker which can be used as an auxiliary selection in goat molecular breeding.

1. Experimental drugs and reagents

1.1 Biochemical and biological reagents

(ii) Taq DNA polymerase (available from Fermantas, MBI); ② proteinase K (from Huamei bioengineering company) and Marker I (from Tiangen Biotechnology (Beijing) Co., 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 and Na₂HPO₄、KH₂PO₄Tris-saturated phenol, chloroform, isoamyl alcohol, absolute ethyl alcohol, 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 solutions and buffers were prepared with deionized ultrapure water and autoclaved at 15bf/in (1.034 × 10)⁵Pa), 25 min. The preparation method is described in molecular cloning, a laboratory Manual, which is published by Sambrook et al.

1) Solution for extracting tissue-like DNA

(ii) 2mol/L NaCl: 11.688g of the extract was dissolved in water, and the volume was adjusted to 100mL, and the solution was autoclaved.

Tissue DNA extract (100 mL): l mol/L Tris-Cl (pH8.0) L mL, 0.5mol/L EDTA (pH8.0)20mL, 2mol/L NaCl 5mL, constant volume to 100 mL.

③ the common solution for extracting the genome DNA.

2) Solutions for agarose gel electrophoresis analysis

(ii) 0.5 × TBE buffer: take 10 XTBE 50mL to 1000 mL.

Sample loading buffer solution: 0.25% bromophenol blue and 0.25% dimethyl benzonitrile FF, 40.0% (w/v) sucrose in water.

2. Goat AKAP12 gene InDel site amplification primer

The sequence of the goat AKAP12 gene (NC-030816.1 is used as a reference sequence) is searched at NCBI, and a PCR Primer pair P1 capable of amplifying the InDel site of the 110266-110278 region of the goat AKAP12 gene is designed by using Primer 6.0, wherein the Primer sequence is as follows:

an upstream primer: 5'-CACTCATCCTACTGGCAT-3' (18bp)

A downstream primer: 5'-TGTTAATAGCGTTCCTCC-3' (18bp)

Theoretically, when the 13-bp sequence (TGGTCTTTTTGTG) at 110266-110278 of the AKAP12 gene is deleted, the result of agarose gel electrophoresis detection of a PCR product is a 179-bp band; when the 13-bp sequence (TGGTCTTTTTGTG) at the 110266-110278 site exists, the result of agarose gel electrophoresis detection of the PCR product is a 192-bp band. When the 13-bp sequence (TGGTCTTTTTGTG) at the 110266-110278 site has the deletion in only one of the two chromosomes, the agarose gel electrophoresis detection result of the PCR product is two stripes of 192bp and 179 bp. Therefore, three genotypes are theoretically available with the AKAP12 gene at positions 110266 and 110278 as mutation sites: the genotype II shows a single stripe of 192bp, the genotype ID shows two stripes of 192bp and 179bp, and the genotype DD shows a single stripe of 179 bp.

3, PCR amplifying the to-be-detected goat AKAP12 gene fragment

3.1 goat ear tissue sample Collection

The experiment collects the ear tissues of 1405 Shanxi white cashmere goat samples, the Shanxi white cashmere goat samples are collected from Taiwan county red Tai farms in Yulin City of Shanxi province from 7 months and 9 days to 30 days in 2019, and bred sheep with the same nutrition level and age are randomly selected.

Adopting a random sampling mode to take individual ear tissue samples, preserving the samples with 70 percent ethanol in a low-temperature ice box, taking the samples back to a laboratory, and then placing the samples at minus 80 ℃ for freezing storage.

3.2 extraction of genomic DNA from ear tissue samples

1) Approximately 10mg of ear tissue was placed in a 1.5mL centrifuge tube and minced as much as possible with scissors.

2) Add 600. mu.L of tissue DNA extract to ensure that the tissue sample is evenly distributed in the tissue extract, add 10% SDS to a final concentration of 1%, add proteinase K to a final concentration of 100. mu.g/mL, digest overnight at 55.0 ℃.

3) And cooling the digested solution to room temperature, adding equal volume of Tris saturated phenol, tightly covering a tube cover, slowly reversing the centrifuge tube back and forth for 10min, and centrifuging at 12000r/min for 15 min.

4) Taking the supernatant, adding equal volume of phenol-chloroform (1:1), covering the tube cap tightly, slowly reversing the centrifuge tube back and forth for 10min, and centrifuging at 12000r/min for 15 min.

5) Taking supernatant, adding equal volume of chloroform isoamyl alcohol (24:1), covering the tube cap tightly, slowly reversing the centrifuge tube back and forth for 10min, and centrifuging at 12000r/min for 15 min.

6) The supernatant was taken, 2 volumes of ice-cold absolute ethanol and 1/10 volumes of 3mol/L sodium acetate were added, the tube cap was closed, and the centrifuge tube was slowly reversed back and forth until the liquid was clear and white flocculent DNA appeared.

7) Picking out DNA, putting the DNA into a 1.5mL centrifuge tube, adding 500 mu L70% ethanol, covering the tube cap tightly, slowly reversing the centrifuge tube back and forth, then centrifuging at 12000r/min for 3-5 min, and carefully pouring off the ethanol.

8) And adding 500 mu L of 70% ethanol into the centrifuge tube again, tightly covering the tube cover, slowly reversing the centrifuge tube back and forth, then centrifuging at 12000r/min for 3-5 min, carefully pouring off the ethanol, and pouring the tube on absorbent paper.

9) After the DNA was dried, 60. mu.L of sterilized ultrapure water was added thereto, and the mixture was stored at 4 ℃ overnight to completely dissolve the DNA and was examined.

3.3 agarose gel electrophoresis detection of DNA

1) The electrophoresis tank is cleaned, the two ends are sealed by adhesive tape paper, and a comb is inserted.

2) Weighing 1.4g of agarose, transferring the agarose into a triangular flask, adding 40mL of 0.5 xTBE to suspend the agarose, heating the agarose by using a medium fire in a microwave oven, taking out the agarose after boiling for 2 times, adding EB with the final concentration of 0.5 mu g/mL when the agarose is cooled to be not too hot, and slightly shaking to prevent bubbles.

3) After mixing (about 60 ℃ C.), the agarose solution was immediately poured into an electrophoresis tank. If air bubbles appeared, they were immediately removed by pipette.

4) After the mixture is completely cooled and solidified (about 25-40 min), the comb is pulled out, and the adhesive tapes at the two ends are removed.

5) And adding 1 XTBE buffer solution into the electrophoresis tank to ensure that the liquid level is 2-5 mm higher than the glue surface.

6) And (3) taking 2-4 mu L of DNA solution, adding 2 mu L of loading buffer solution, mixing uniformly, loading, and adding a DNA Marker to one side.

7) Electrophoresis was carried out for 2h at 80V.

8) And (4) observing on an ultraviolet analyzer, if RNA exists, purifying, and if obvious degradation exists, re-extracting DNA of a corresponding sample.

3.4 purification of DNA

1) To 500. mu.L of the DNA solution, 10% SDS was added so that the final concentration of SDS was 0.1%, and proteinase K was added so that the final concentration reached 100. mu.g/mL.

2) Keeping the temperature at 55 ℃ for about 10 h.

3) Equal volumes of phenol, chloroform, isoamyl alcohol (25:24:1) and chloroform were extracted once respectively.

4) Centrifuging at 12000r/min for 5min, and sucking the upper water phase into another centrifuge tube.

5) 1/10 volumes of 3mol/L sodium acetate and 2 volumes of ice-cold absolute ethanol were added to precipitate the DNA.

6) The liquid was decanted, washed with 70% ethanol, and the DNA was air dried, dissolved in 60. mu.L of sterile ultrapure water, stored at 4 ℃ and examined.

3.5 spectrophotometric detection of DNA

The OD values of the DNA samples at 260nm and 280nm were measured by an ultraviolet photometer. Calculation of DNA content and OD₂₆₀/OD₂₈₀The ratio of (a) to (b). Such as OD₂₆₀/OD₂₈₀The ratio is less than 1.6, which indicates that the sample DNA contains more protein or phenol, and purification is required; if the ratio is greater than 1.8, then RNA purification removal should be considered.

DNA concentration (ng/. mu.L) ═ 50 × OD₂₆₀Value × dilution factor

After the DNA is qualified, a certain amount of DNA solution is taken out, diluted to 50 ng/. mu.L and stored at-20 ℃ for later use, and the rest is stored at-80 ℃.

3.6 PCR 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 reaction required by 1 reaction, the reaction components are added into 1 1.5mL centrifuge tube, the centrifuge tubes are mixed fully and evenly and then are subjected to instantaneous centrifugation, the reaction components are subpackaged into 0.2mL Eppendorf PCR tubes, template DNA is added, and PCR amplification is carried out after the instantaneous centrifugation.

PCR reaction (13. mu.L): 2 XTaq PCR Supermix (including Taq DNA polymerase, dNTPs and reaction buffer, concentration is 2X) 6.5. mu.L, upstream primer 0.5. mu.L, downstream primer 0.5. mu.L (upstream primer concentration and downstream primer concentration are respectively 10 pmol/. mu.L), template DNA (concentration is 50 ng/. mu.L goat genome DNA) 1.0. mu.L, and deionized water 4.5. mu.L.

Procedure of PCR reaction: 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 20s, and performing 30 cycles; extension at 72 ℃ for 10 min.

Agarose gel electrophoresis analysis of PCR amplification products

4.1 agarose gel electrophoresis detection

1) Preparing 3.5% agarose gel, dyeing by using EB nucleic acid dye, spotting 4 microliter, and performing electrophoresis at 120V for 1-1.5 h;

2) when the DNA fragments with different molecular weights are clearly separated, imaging in a BIO-RAD Gel Doc 2000 Gel imaging system;

3) analyzing indel polymorphism according to the agarose gel electrophoresis result;

4.2 identification of genotype at Indel site

Referring to the figures 1 and 2, the BIO-RAD Gel Doc 2000 Gel imaging system is used for photographic analysis of the electrophoresis result, and the analysis result shows that the primer pair P1 is used for amplifying the goat genome, and the agarose Gel electrophoresis is used for analyzing the amplification product, so that the rapid and accurate typing identification (genotype: II, ID, DD) of the 13-bp insertion/deletion polymorphic site of 110266-110278 (NC-030816.1) of the goat AKAP12 gene can be realized.

5. Frequency statistical analysis of goat AKAP12 gene indel locus

Genotype frequency refers to the ratio of the number of individuals of a certain genotype to the total number of individuals in a population.

The statistical results of the genotype frequencies of the AKAP12 gene at the 13-bp insertion/deletion polymorphic sites in the sample of the white cashmere goat in northern Shaanxi are shown in Table 1.

TABLE 1 frequency distribution of goat AKAP12 gene at 110266-110278 InDel locus gene

6. Association analysis of gene effect of InDel locus of goat AKAP12 gene

Genotype data: genotype (II, ID, DD) recognized by agarose gel electrophoresis of PCR amplification product

Body size data: shanxi white cashmere goat with long body, high body and high cross part

And (3) correlation analysis model: analysis of the correlation of InDel sites with goat growth traits using SPSS (23.0) software

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. The complete model is as follows:

Y＝μ+G+e

wherein, Y: (ii) an individual phenotype record; u: an overall mean; g: a marker genotype effect; e: random error.

Table 2. correlation analysis of InDel locus of AKAP12 gene and growth traits of Shaanxi white cashmere goats

As can be seen from Table 2, different genotypes of the insertion/deletion polymorphic sites (NC-030816.1: g.110266-110278) of the AKAP12 gene have significant influence on the body length of the bred sheep (P <0.05), and have insignificant influence on the body height and the cross height of the bred sheep (P > 0.05). Wherein, the individual character with the genotype II is better than that with the genotypes ID and DD. Therefore, the genotype II of the above insertion/deletion polymorphic site can be used as a candidate genetic marker for increasing the body length of a goat.

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Claims

1. A method for detecting the insertion/deletion polymorphism of a goat AKAP12 gene is characterized by comprising the following steps: the method comprises the following steps:

using goat genome DNA as a template, amplifying partial fragments of AKAP12 gene by PCR, and carrying out agarose gel electrophoresis on the amplified fragments; and (3) identifying the genotype of the insertion/deletion polymorphic site of the AKAP12 gene according to the agarose gel electrophoresis result, wherein the insertion/deletion polymorphic site is located at NC-030816.1: g.110266-110278 of the AKAP12 gene.

2. The method for detecting the insertion/deletion polymorphism of the goat AKAP12 gene according to claim 1, wherein the method comprises the following steps: the PCR adopts amplification primer pairs as follows:

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

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

3. The method for detecting the insertion/deletion polymorphism of the goat AKAP12 gene according to claim 1, wherein the method comprises the following steps: 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 20s, and performing 30 cycles; extension at 72 ℃ for 10 min.

4. The method for detecting the insertion/deletion polymorphism of the goat AKAP12 gene according to claim 1, wherein the method comprises the following steps: the agarose gel electrophoresis adopts agarose gel with mass concentration of 3.5%.

5. The method for detecting the insertion/deletion polymorphism of the goat AKAP12 gene according to claim 1, wherein the method comprises the following steps: according to the result of agarose gel electrophoresis, the insertion/insertion genotype of the AKAP12 gene insertion/deletion polymorphic site is represented by a 192bp stripe, the insertion/deletion genotype is represented by two 192bp and 179bp stripes, and the deletion/deletion genotype is represented by a 179bp stripe.

6. A detection kit for goat AKAP12 gene insertion/deletion polymorphism is characterized in that: the kit comprises a primer pair for amplifying the insertion/deletion polymorphic sites of the AKAP12 gene by PCR, wherein the insertion/deletion polymorphic sites are positioned at NC-030816.1 (sites NC-030816.1) of the AKAP12 gene at g.110266-110278.

7. The kit for detecting the insertion/deletion polymorphism of the goat AKAP12 gene according to claim 6, wherein the kit comprises: the primer pair is as follows:

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

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

8, the AKAP12 gene NC-030816.1, g.110266-110278 insertion/deletion polymorphism sites in the application of the goat molecular marker auxiliary selection breeding.

9. The application of the method for detecting the insertion/deletion polymorphism of the goat AKAP12 gene in the goat molecular marker-assisted selective breeding as claimed in claim 1.

10. Use according to claim 8 or 9, 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 of the goat.