CN112795668B - Application of goat CFAP43 gene insertion/deletion marker in early selection of characters - Google Patents
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Abstract
The invention discloses an application of a goat CFAP43 gene insertion/deletion marker in early selection of characters. The full-genome DNA of the goat is taken as a template, the partial fragment of the CFAP43 gene of the goat is amplified through PCR, and the polymorphism of the 6-bp insertion mutation site of the gene in the goat population is identified after agarose gel electrophoresis. According to the correlation analysis result, the genotype of the insertion/deletion polymorphic site of the CFAP43 gene is obviously related to the chest circumference and the tube circumference of the goat, and can be used for quickly establishing a goat population with excellent genetic resources, so that the breeding process can be accelerated by the early marker-assisted selection of the goat traits.
Description
Technical Field
The invention belongs to the field of biotechnology and livestock breeding, and relates to typing detection of goat CFAP43 gene insertion/deletion (InDel) polymorphism and application of the typing detection in molecular marker-assisted selective breeding.
Background
The cashmere and meat dual-purpose goat variety, such as the white cashmere goat in northern Shaanxi, has an important role in driving the animal husbandry and the economic development, but the problems of uneven individual body type distribution, unstable genetic performance and the like of the goat variety are one of the key problems limiting the industrial upgrading of the goat variety. At present, genetic breeding of goat growth traits by using modern molecular biotechnology, such as a marker-assisted selection (MAS) method, has become a hotspot of research. Among MAS, the candidate gene method is one of the major research methods at present, and therefore, the identification of important candidate genes related to growth traits is a prerequisite for the improvement of goat growth traits and the development of goat industry.
Multiple morphological abnormalities of the sperm flagellate (MMAF) can significantly affect the reproductive performance of males and can lead to sterility. Cilia and flagella associated protein (cfap43) genes, which are major genes related to MMAF, are expressed in various tissues such as ovary and placenta, thereby affecting the reproduction of animals.
In the aspect of growth traits of livestock, researches find that the key QTL positioned in the IGF2 gene has large influence on the muscle content of pigs, and prove that the IGF2 gene is a key candidate gene for controlling the muscle content of the pigs. By utilizing an InDel detection method, researches show that the IGF2BP1 gene has effective molecular markers which can be used for breeding the growth traits of goats, and the combined genotype analysis of two sites of the IGF2BP1 gene shows that the IGF2BP1 gene can significantly influence the weight traits of goats. Similarly, as a candidate gene of the growth trait, the deletion mutation of 12-bp existing in the PRDM6 gene can obviously influence the growth and development of the goat. The research shows that the deletion mutation of the Myostatin (Myostatin) gene, also known as GDF8 gene, can cause the muscular hypertrophy phenotype of cattle, so the gene has important value for the growth trait genetic breeding of cattle, goats and other livestock.
Compared with the SNP (single nucleotide polymorphism) detection method, the InDel detection method has the advantages of strong operability, convenience and quickness in detection and the like in MAS breeding. However, reports on the relation between CFAP43 gene polymorphism and goat growth traits are not found at present.
Disclosure of Invention
The invention aims to provide application of a goat CFAP43 gene insertion/deletion marker in early selection of characters so as to quickly establish a goat genetic resource population with excellent growth characters.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting insertion/deletion polymorphism of a goat CFAP43 gene comprises the following steps:
the method comprises the steps of taking the whole genome DNA of a goat to be detected (such as an individual Shanxi Bairong goat) as a template, amplifying partial fragments of the goat CFAP43 gene by PCR, carrying out agarose gel electrophoresis on the amplified product, and identifying the genotype of the insertion/deletion polymorphic site of the goat CFAP43 gene according to the agarose gel electrophoresis result, wherein the insertion/deletion polymorphic site is selected from 6-bp insertion mutation sites of the goat CFAP43 gene NC _030833 g.27012996-27012997.
Preferably, the amplification primer pair of the PCR is:
an upstream primer: 5'-GGACAGAGAGACAGAGTTTCAGGT-3'
A downstream primer: 5'-CAAGACTCCCCTATCTTCAGATTA-3'.
Preferably, the reaction procedure adopted by the PCR is as follows: pre-denaturation at 95.0 deg.C for 5min; denaturation at 94.0 ℃ for 30s, renaturation at 68.0 ℃ for 30s, and extension at 72.0 ℃ for 20s, wherein the renaturation temperature is reduced by 1 ℃ in each cycle, and the cycle time is 18; 30 cycles of denaturation at 94.0 ℃ for 30s, renaturation at 50.0 ℃ for 30s, and extension at 72.0 ℃ for 20 s; extension for 10min at 72.0 ℃.
Preferably, the insertion/deletion polymorphic site has three genotypes according to an agarose gel electrophoresis result, wherein the insertion/insertion genotype is represented by 168bp one stripe, the insertion/deletion genotype is represented by 162bp and 168bp two stripes, and the deletion/deletion genotype is represented by 162bp one stripe.
A kit for detecting the insertion/deletion polymorphism of a goat CFAP43 gene comprises a primer pair (such as the upstream primer and the downstream primer) for PCR amplification of the insertion mutation site 6-bp of the goat CFAP43 gene NC _030833 from g.27012996-27012997.
The detection method of the goat CFAP43 gene insertion/deletion polymorphism is applied to the goat molecular marker-assisted selective breeding.
Preferably, the deletion/deletion genotype of the goat CFAP43 gene NC _030833 at the 6-bp insertion mutation site of the goat from 27012996-27012997 is a DNA marker (specifically an InDel marker) for improving the chest circumference and/or the tube circumference of the goat.
The invention has the beneficial effects that:
the invention takes goat genome DNA as a template, and can detect the genotype of the goat CFAP43 gene intron region insertion/deletion polymorphic site (NC _030833 g.27012996-27012997) in a goat (for example, shanxi white cashmere goat) population efficiently, accurately and at low cost through sequence amplification and electrophoretic identification. According to the correlation analysis of the insertion/deletion polymorphic site of the goat CFAP43 gene and the important body size characters of the goat, the result shows that the detected insertion/deletion polymorphic site of the goat CFAP43 is used as a molecular marker (InDel marker) for improving the chest circumference and the tube circumference characters of the goat, can be used for early selection of the chest circumference, the tube circumference and other growth characters of the goat, and can be used for quickly establishing a goat population with excellent growth characters, thereby accelerating the speed of fine breed breeding.
Drawings
FIG. 1 shows the result of agarose gel electrophoresis of PCR amplification product (target fragment: 6-bp insertion mutation site) of goat CFAP43 gene; wherein: m represents Marker I.
FIG. 2 is a sequence diagram of PCR amplification products of goat CFAP43 gene, wherein: the part marked by black boxes represents the 6-bp insertion sequence (NC _030833 g.27012996-27012997 insAGTTGG).
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
The invention utilizes a PCR method to detect the insertion/deletion polymorphism of the 6-bp insertion mutation site of the goat CFAP43 gene (reference sequence: NC-030833) in Shanxi white cashmere goat population, and performs correlation analysis on different genotypes and goat growth traits to verify whether the insertion/deletion polymorphism can be used as a molecular marker for auxiliary selection in goat molecular breeding, and the specific description is as follows.
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); (3) marker I (available from tiangen biochemistry technologies (beijing) ltd).
1.2 general reagents: the common reagent is purchased from Huamei bioengineering company, and is imported and subpackaged with products, including citric acid, sodium citrate, glucose, tris, EDTA, naCl, naOH, KCl and Na 2 HPO 4 、KH 2 PO 4 Tris-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 solution and buffer: all solutions and buffers were prepared using deionized ultrapure water. The reagents were prepared according to the molecular cloning protocol described in Sambrook et al. The autoclaving condition is 15bf/in (1.034X 10) 5 Pa)、25min。
1) Solution for extracting tissue-like DNA:
in addition to the common solutions for genomic DNA extraction, the following reagents were prepared:
(1) 2mol/L NaCl:11.688g was dissolved in water to 100mL and autoclaved.
(2) Tissue DNA extract (100 mL): l mol/L Tris-HCl (pH 8.0) L mL, 0.5mol/L EDTA (pH 8.0) 20mL, and 2mol/L NaCl 5mL, to a volume of 100mL.
2) Solutions for agarose gel electrophoresis analysis
(1) 0.5 × TBE buffer: take 10 XTBE 50mL to 1000mL.
(2) 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. Synthetic goat CFAP43 gene 6-bp insertion mutation site amplification primer
Based on the reference sequence, a primer pair capable of amplifying a DNA fragment containing a 6-bp insertion mutation (NC _030833 g.27012996-27012997 insAGTTGG) site of the goat CFAP43 gene is synthesized, and the sequence of the primer pair is as follows:
an upstream primer: 5'-GGACAGAGAGACAGAGTTTCAGGT-3' (24 bp),
a downstream primer: 5'-CAAGACTCCCCTATCTTCAGATTA-3' (24 bp).
The primer is used for amplifying a goat genome, an amplification product comprises a 6-bp insertion mutation area of a 12 th intron region of a goat CFAP43 gene (NC-030833), the insertion/insertion genotype can be amplified to obtain 168bp one stripe, the insertion/deletion genotype can be amplified to obtain 162bp and 168bp two stripes, and the deletion/deletion genotype can be amplified to obtain 162bp one stripe.
3.PCR amplification of goat CFAP43 Gene target fragment
3.1 goat tissue sample Collection and Property data Collection
Adult Shanxi white cashmere goat individuals with consistent feeding and management conditions are selected, ear tissue samples are collected and stored in 70% ethanol, and the ice boxes are brought back to a laboratory at low temperature and then placed at-80 ℃ for cryopreservation. The sampling information is as follows: 885 parts of Shaanbei white cashmere goat ear tissue samples are collected in Shaanxi province Yulin city Yangshan county cashmere goat farm by adopting a colony random sampling mode (6 months to 9 months in 2017);
the growth data of the Shanxi white cashmere goat comprise: height, length, height of cross, chest circumference, tube circumference, chest depth and chest width, and total 7 individual ruler characters.
3.2 extraction and isolation of genomic DNA from tissue samples
Reference is made to molecular cloning, a laboratory Manual (2002) written by Sambrook et al and to the following references: lan Xianyong genetic variation of important functional gene of goat and its relation with economic traits [ D. ] in doctor's academic thesis of university of agriculture and forestry, northwest, 2007, shanxi Yang Ling.
3.3 agarose gel electrophoresis detection of DNA
Reference is made to molecular cloning, A laboratory Manual (2002) 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. Calculation of DNA content and OD 260 /OD 280 The ratio of (a) to (b). Such as OD 260 /OD 280 The ratio is less than 1.6, which indicates that the sample 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 XOD 260 Value x dilution factor.
After the DNA detection, a certain amount of the DNA was taken out and diluted to 30 ng/. Mu.L as a template, stored at-20 ℃ for later use, and the rest 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 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.
And (3) PCR reaction system: 2 XTaq PCR Supermix (including Taq DNA polymerase, dNTPs and reaction buffer, concentration of 2 ×) 6.5. Mu.L, upstream primer 0.5. Mu.L, downstream primer 0.5. Mu.L (upstream and downstream primer concentrations of 10 pmol/. Mu.L); 0.6 mu L of template DNA (the concentration is 30 ng/. Mu.L of goat genome DNA) and 4.9 mu L of deionized water; a total of 13. Mu.L.
3.7 procedure for PCR reaction
1) Pre-denaturation at 95.0 ℃ for 5min, and then entering step 2);
2) Denaturation at 94.0 ℃ for 30s, renaturation at 68.0 ℃ for 30s (1 ℃ per cycle), extension at 72.0 ℃ for 20s for 18 cycles, and then step 3);
3) 30 cycles of denaturation at 94.0 ℃ for 30s, renaturation at 50.0 ℃ for 30s and extension at 72.0 ℃ for 20 s;
4) After step 3), extension was carried out at 72.0 ℃ for 10min.
4. Agarose gel electrophoresis detection
Agarose gel electrophoresis detection is divided into 3 steps: 1) Preparing 3.5% agarose gel, dyeing with nucleic acid dye, spotting 5.0 μ L, and performing 120V electrophoresis for 80-100min; 2) When the DNA fragments with different molecular weights are clearly separated, imaging in a BIO-RAD Gel Doc 2000 Gel imaging system; 3) And analyzing the InDel polymorphism according to the agarose gel electrophoresis result.
The electrophoresis detection result of the amplified fragment is shown in FIG. 1, and the lane 1 is Marker I (which is sequentially 600bp, 500bp, 400bp, 300bp, and 200bp from large to small). Lanes 2-8 show the detection results of the amplification products: II is an insertion/insertion genotype which is expressed as a striae of 168 bp; ID is insertion/deletion genotype and shows two bands of stripes of 162bp and 168 bp; DD is deletion/deletion genotype, and shows a 162bp stripe. After the amplified fragment was sequenced and identified, the 6-bp insertion sequence was found to exist, and the sequencing peak is shown in FIG. 2.
5. Frequency statistical analysis of goat CFAP43 gene 6-bp insertion mutation site
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:
P YY ＝N YY /N
in the formula, P YY Represents the YY genotype frequency of a certain locus; n is a radical of YY Representing the number of individuals in the population having a YY genotype; and N is the total number of the 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:
P Y ＝(2N YY +N Ya1 +N Ya2 +N Ya3 +N Ya4 +……+N Yan )/2N
in the formula, P Y Indicating allele Y frequency, N YY Representing the number of individuals in the population having the YY genotype, N Yai Representing the number of individuals in the population having the Yai genotype, a1-an are n mutually different multiple alleles of allele Y. The statistical results are shown in table 1.
TABLE 1 population genetic parameters of goat CFAP43 gene 6-bp insertion mutation site
According to the genotype and gene frequency analysis (Table 1) of the CFAP43 gene of the Shanxi white cashmere goat, a 6-bp insertion mutation site (NC _030833, g.27012996-27012997) of the CFAP43 gene of the Shanxi white cashmere goat is an insertion/deletion (InDel) polymorphic site.
6. Association analysis of gene effect of InDel locus of goat CFAP43 gene
Genotype data: carrying out agarose gel electrophoresis on the genotype identified after PCR amplification;
goat growth trait data: height, length, height of cross, bust, circumference of tube, depth of chest, and width of chest.
And (3) association analysis model: the SPSS (24.0) software is used for analyzing the correlation between different factors of the variety and the growth 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 model or t-analysis based on the characteristics of the data. In the data processing process, the fixed model is used for carrying out correlation analysis in consideration of the effect of individuals, the interaction between genes and the effect of genotypes, and the specific analysis result is shown in table 2.
Wherein, the complete model is as follows according to the practical conditions:
Y＝μ+G+E
wherein Y represents an individual phenotype record; u represents the overall mean; g represents a marker genotype effect; e denotes a random error.
TABLE 2 correlation analysis of the growth characteristics of the CFAP43 gene InDel locus and the southern Shaanxi white cashmere goat (mean. + -. Standard error)
Note: the average values of the same row of data are different in letters on shoulders, which indicates that the data have statistical differences; the data units are all centimeters
As can be seen from Table 2, the growth trait association analysis result of the Shanxi white cashmere goats shows that the 6-bp insertion mutation site of the CFAP43 gene (NC _030833 g.27012996-27012997) is significantly related to the chest circumference (P < 0.05) and the tube circumference (P < 0.05), and the DD genotype individual trait is superior to that of the ID and II genotype individuals. Therefore, the deletion/deletion (DD) genotype of the insertion mutation site 6-bp at position 5363 of the goat CFAP43 gene NC _030833 g.27012996-27012997 can be used as a DNA molecular marker for improving the properties of the chest circumference and the tube circumference of the goat, and can be used for early marker-assisted selection of the goat properties.
In a word, the invention utilizes a PCR amplification method to detect the insertion/deletion polymorphic sites of the NC _030833 of the CFAP43 gene of the goat, i.e., the sites from 27012996 to 27012997, and carries out correlation analysis on the insertion/deletion polymorphic sites and the growth traits of the white cashmere goat in northern Shaanxi, finds that the insertion/deletion polymorphic sites exist in the molecular breeding of the goat for auxiliary selection, can be used for quickly establishing a goat population with excellent growth traits, and further accelerates the breeding speed of improved varieties.
<110> northwest agriculture and forestry science and technology university
<120> application of goat CFAP43 gene insertion/deletion marker in early selection of traits
<160> 3
<210> 1
<211> 24
<212> DNA
<213> upstream primer
<400> 1
ggacagagag acagagtttc aggt 24
<210> 2
<211> 24
<212> DNA
<213> downstream primer
<400> 2
caagactccc ctatcttcag atta 24
<210> 3
<211> 20
<212> DNA
<213> NC _030833 g.27012996-27012997 insertion sequence
<400> 3
agttgg 6
Claims (5)
1. The application of the goat CFAP43 gene NC _030833 g.27012996-27012997 bit 6-bp insertion mutation site in goat molecular marker-assisted selection breeding is characterized in that: the deletion/deletion genotype of the insertion mutation site 6-bp at the position 5363 of the goat CFAP43 gene NC _030833 g.27012996-27012997 is a DNA marker for improving the chest circumference and/or the tube circumference of a goat selected from the group consisting of Shanxi white cashmere goats.
2. An application of a detection method of goat CFAP43 gene insertion/deletion polymorphism in goat molecular marker-assisted selective breeding is characterized in that: the detection method of the goat CFAP43 gene insertion/deletion polymorphism comprises the following steps:
using goat genome DNA as a template, amplifying partial fragments of the goat CFAP43 gene by PCR, then carrying out agarose gel electrophoresis on the amplified product, and identifying the genotype of the goat CFAP43 gene insertion/deletion polymorphic site according to the electrophoresis result, wherein the insertion/deletion polymorphic site is selected from 6-bp insertion mutation sites of the goat CFAP43 gene NC _030833 g.27012996-27012997;
the deletion/deletion genotype of the goat CFAP43 gene NC _030833 g.27012996-27012997 6-bp insertion mutation site is a DNA marker for improving the chest circumference and/or the tube circumference of the goat; the goat is selected from Shanxi white cashmere goat.
3. Use according to claim 2, characterized in that: the PCR amplification primer pair comprises:
an upstream primer: 5'-GGACAGAGAGACAGAGTTTCAGGT-3'
A downstream primer: 5'-CAAGACTCCCCTATCTTCAGATTA-3'.
4. Use according to claim 2, characterized in that: the reaction procedure adopted by the PCR is as follows: pre-denaturation at 95.0 deg.C for 5min; denaturation at 94.0 ℃ for 30s, renaturation at 68.0 ℃ for 30s, and extension at 72.0 ℃ for 20s, wherein the renaturation temperature is reduced by 1 ℃ in each cycle, and the cycle time is 18; 30 cycles of denaturation at 94.0 ℃ for 30s, renaturation at 50.0 ℃ for 30s, and extension at 72.0 ℃ for 20 s; extension at 72.0 ℃ for 10min.
5. Use according to claim 2, characterized in that: according to the electrophoresis result, the insertion/deletion polymorphic site has three genotypes, wherein the insertion/insertion genotype is represented by one stripe of 168bp, the insertion/deletion genotype is represented by two stripes of 162bp and 168bp, and the deletion/deletion genotype is represented by one stripe of 162 bp.
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