Method for detecting 17 bp InDel mutation of intron region of SPATS2L gene of Anhui white goat and application
By designing specific primers and using agarose gel electrophoresis, the detection challenge of the 17 bp InDel mutation in the intron region of the SPATS2L gene in Anhui white goats was solved, realizing a rapid and low-cost detection method. This provides molecular markers related to growth traits and promotes the goat breeding process.
Patent Information
- Application Number
- CN202511217419.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-28
- Publication Date
- 2025-11-18
AI Technical Summary
Existing technologies are insufficient for efficient and low-cost detection of the 17 bp InDel mutation in the intron region of the SPATS2L gene in Anhui white goats, and its relationship with growth traits is unclear, affecting the breeding process.
Specific PCR amplification primers were designed and combined with agarose gel electrophoresis detection. The 17 bp InDel mutation in the intron region of the SPATS2L gene was identified by the number and size of the electrophoretic bands. Genotype determination was performed using the electrophoresis results, and association analysis was conducted to screen candidate markers related to growth traits.
It enables rapid, low-cost, and accurate detection of InDel mutations, provides molecular markers that are significantly associated with goat growth traits, offers valuable data for genetic breeding, and advances the process of selecting superior goat breeds.
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Figure CN120967006A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of molecular genetics and breeding, and particularly relates to a method for detecting a 17 bp InDel mutation in an intron region of a SPATS2L gene of Anhui white goat and application thereof. BACKGROUND
[0002] With the rapid development of society, people's living standards are constantly improving, and the demand for livestock products is rapidly increasing. The quality requirements for livestock products are constantly changing, thus promoting the continuous development and improvement of breeding goals. Ancient humans began breeding activities when they began to domesticate livestock. They selected individuals that met people's needs for breeding by observing phenotypes, thus improving the production performance of livestock generation after generation. In the 20th century, people realized the importance of genes on biological phenotypes, so they gradually began to consider how to improve the production performance of livestock from a genetic perspective. Gradually, breeding through hybridization and other means was developed to combine the excellent performance of different breeds.
[0003] In recent years, the breeding process has become more and more systematic, not only limited to livestock phenotypes, but also involving economic benefits and environmental protection, so as to maximize the production performance of livestock under the condition of maximizing economic benefits and not affecting the environment. At the same time, in the process of breeding, not only single production performance is considered, but also growth, reproduction, meat quality and other indicators are considered, so as to develop a reasonable breeding plan. With the continuous development of molecular and cellular biology technology, more and more modern biological technologies are applied to livestock breeding, such as marker-assisted selection (MAS) breeding, which is based on known key variations, that is, in practical application, it is necessary to first find a quantitative trait locus (QTL) or key genetic variation that controls a certain key trait as a genetic marker, such as copy number variation (CNV), short fragment insertion / deletion, single nucleotide polymorphism (SNP), etc., and then select individuals with excellent performance in certain traits by selecting genetic markers.
[0004] InDel genetic variation refers to the collective term of insertion and deletion mutations in the genome, and such genetic variation is caused by the insertion (Insertion) or deletion (Deletion) of a certain length of nucleotide at a certain position of DNA sequence. Compared with single nucleotide polymorphism (SNP), the distribution density of InDel variation in the genome is lower, but the individual identification rate is higher. Researchers have found that there is an InDel variation in the human genome every 3000 bases on average, which makes it unique in genetic analysis and identification, therefore, Indel can also provide a basis for improving marker-assisted selection and molecular breeding of important economic traits.
[0005] With the rapid development of whole genome sequencing technology, a large number of Indels have been mined. However, so far, the relationship between most InDel mutations and traits is still unknown. Therefore, it is necessary to detect the InDel of individuals and explore the relationship between them and traits. At present, the methods for identifying InDel mainly include DNA sequence determination method, PCR-SSCP (single-stranded conformation polymorphism) method, and direct agarose gel electrophoresis method after PCR; among them, the DNA sequence determination method is accurate and intuitive, but the cost is relatively high when applied in a population, while the direct agarose gel electrophoresis method after PCR can separate the amplification products of different lengths by electrophoresis, which is simple to operate and low in cost, and is suitable for identifying a certain population.
[0006] At present, many studies have used whole genome association analysis (GWAS) to mine key variation sites or regions related to livestock economic traits. Some studies have found that the genetic variation of rs4673912 about 1.6kb upstream of the gene is significantly related to the muscle mass of individuals Cordero A I H et al. Combined with the previous sequencing of the research group, it is found that the high expression in the embryonic period of Anhui White Goat is closely related to the economic traits of individuals. Therefore, it is of great theoretical and practical significance to study the genetic variation and molecular genetic characteristics of Chinese local goat genes. SUMMARY
[0007] The purpose of the present application is to provide a method for detecting 17 bp InDel mutation in the intron region of SPATS2L gene of Anhui White Goat and its application, which is simple, fast, low in cost, high in detection accuracy, and lays a foundation for the relationship between SPATS2L gene InDel mutation and goat growth traits.
[0008] In one aspect of the present application, a method for detecting a 17 bp InDel mutation in the intron region of a SPATS2L gene of Anhui white goat is provided. According to an embodiment of the present application, a full-genome DNA of the Anhui white goat to be tested is used as a template, a third intron fragment of the SPATS2L gene of the Anhui white goat is amplified by PCR, and the number and size of bands are detected by electrophoresis to identify one 17 bp InDel mutation site in the intron region of the SPATS2L gene of the Anhui white goat (XM_018061185.1 transcript), which is located at NC_030809.1:g.46990698_46990714.
[0009] In addition, the method for detecting a 17 bp InDel mutation in the intron region of a SPATS2L gene of Anhui white goat according to the above-mentioned embodiments of the present application can further have the following additional technical features.
[0010] In some embodiments of the present application, the amplification primers used in the PCR amplification are as follows:
[0011] the upstream primer: 5'-CCTTCCTCATACAAGCAGCA-3';
[0012] the downstream primer: 5'-GATACTCATGGTACTTTGTGGAG-3'.
[0013] In some embodiments of the present application, the amplification reaction program of the PCR is as follows: pre-denaturation at 93-95 ℃ for 3-5 min; denaturation at 95-98 ℃ for 10-30 s, recombination at 55-60 ℃ for 30-50 s, extension at 65-72 ℃ for 1-2 min, and cycling for 25-30 times; extension at 65-72 ℃ for 3-5 min, and preservation at 4 ℃.
[0014] In some embodiments of the present application, the reaction system of the PCR amplification comprises 2x PCR MasterMix, 25-50 ng of goat genomic DNA, and 2.5-5 pmol of each amplification primer.
[0015] In some embodiments of the present application, the electrophoresis uses an agarose gel with a mass fraction of 3.5%-4.0%. According to the electrophoretic results, an individual with a normal genotype (i.e., a wild-type homozygous genotype DD) shows a 201 bp band, an individual with an insertion genotype (i.e., a mutant homozygous genotype II) shows a 218 bp band, and an individual with a heterozygous genotype (i.e., a mutant heterozygous genotype ID) shows 201 bp and 218 bp bands.
[0016] In another aspect of the present application, the present application provides an application of the method for detecting the 17 bp InDel mutation in the intron region of the SPATS2L gene of the Anhui white goat in goat marker-assisted selection breeding.
[0017] In addition, the application according to the above-mentioned embodiments of the present application can also have the following additional technical features:
[0018] In some embodiments of the present application, the InDel mutation site is a molecular marker related to the growth traits of the Anhui white goat individual. The genotype DD of the individual goat at the corresponding 17 bp InDel mutation (NC_030809.1:g.46990698_46990714dup) in the intron region of the SPATS2L gene can be used as a DNA marker (specifically, an InDel marker) for improving the growth traits of the goat.
[0019] In some embodiments of the present application, the growth traits include body weight, body height, body length, chest circumference, pipe circumference, and rump width.
[0020] Compared with the prior art, the present application has the following beneficial effects:
[0021] The present application designs specific primers for different Indel alleles of the SPATS2L gene insertion / deletion site, and through PCR, agarose gel electrophoresis detection, and gel imaging system analysis, the genotype of the individual can be determined (the genotype of the individual can be distinguished according to the size difference of the bands in the electrophoresis result). At the same time, the insertion / deletion genetic variation detected by the method can be associated with the phenotype, and a candidate marker with significant correlation with the growth traits of the goat is screened, which provides valuable data for marker-assisted selection (MAS) in the field of genetic breeding, and can be used to quickly establish a goat population with excellent genetic resources, thereby facilitating the process of breeding of fine goat breeds. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 For the 3.5% (W / V) agarose gel electrophoresis map of the PCR amplification product of part of the goat SPATS2L gene in the embodiments of the present application, the genotype of the individual can be distinguished according to the presence or absence of the 201 bp and 218 bp fragments;
[0023] Figure 2 For the sequencing map of the different genotypes of the 17 bp Indel site of part of the goat SPATS2L gene in the embodiments of the present application. DETAILED DESCRIPTION
[0024] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work are within the protection scope of the present application.
[0025] A method for detecting a 17 bp InDel mutation in the intron region of SPATS2L gene of Anhui white goat:
[0026] 1. Discovery of 17 bp InDel mutation in goat gene intron region (located at position 46990698-46990714 of chromosome 2):
[0027] An InDel site of SPATS2L gene was searched from an Ensembl database (http: / / lasia.ensembl.org / index.html), and primers were designed for identifying the genetic variation site. The primers were designed by using NCBI primer Blast (https: / / www.ncbi.nlm) with goat SPATS2L gene (Gene ID: 102186063) as a reference sequence. Anhui white goat whole genome DNA was used as a template for PCR amplification, and the amplified product was subjected to high-concentration 3.5% w / v agarose gel electrophoresis. According to the number and size of the electrophoretic bands, it was identified that there was a possible 17 bp InDel variation site in the third intron (XM_018061185.1 transcript) region of SPATS2L gene. Then, part of the PCR amplification products with different band numbers and sizes were subjected to DNA sequencing, and according to the sequencing peak graph, it was determined that there was indeed a 17 bp InDel mutation site (i.e. NC_030809.1: g.46990698_46990714 insertion sequence is GCCTCCACTAGGTACCA) in the third intron (XM_018061185.1 transcript) region of SPATS2L gene of Anhui white goat.
[0028] After obtaining the above finding, in order to determine the association of the 17 bp InDel mutation in the third intron (XM_018061185.1 transcript) region of SPATS2L gene with the growth traits of Anhui white goat population, 185 Anhui white goat DNAs preserved in the laboratory were used as templates, PCR technology and high-concentration 3.5% w / v agarose gel electrophoresis were used for population detection, and the population detection was associated with the growth traits of Anhui white goat to determine that the corresponding InDel site can be used as a molecular genetic marker for assisted selection in goat molecular breeding.
[0029] 2. The experimental reagents and materials are as follows:
[0030] (1) Biochemical reagents and biological reagents
[0031] ① PCR MasterMix (purchased from Biotech Inc., China); ② Proteinase K (purchased from Huamei Biotechnology Co., Ltd.); ③ DNA Marker (purchased from Tiangen Biotech (Beijing) Co., Ltd.).
[0032] (2) Common reagents
[0033] Common reagents were purchased from Huamei Bioengineering Co., Ltd., and were imported and repackaged products: Tris, EDTA, NaCl, NaOH, KCl, Na2HPO4, KH2PO4, Tris saturated phenol, chloroform, isoamyl alcohol, anhydrous ethanol, sodium acetate, sodium dodecyl sulfonate (SDS), ethidium bromide (EB), bromophenol blue, xylene cyanide FF, acetic acid, sucrose, boric acid, agarose, etc.
[0034] (3) Solutions and buffer solutions
[0035] All solutions and buffer solutions were prepared using deionized ultrapure water and autoclaved at 15 bf / in (1.034 × 10⁻⁶). 5 Pa), 25 min. Reagent preparation methods were all based on *Molecular Cloning: A Laboratory Manual* edited by Sambrook et al. Specific solutions and buffers are as follows:
[0036] A. Solution used for separating genomic DNA from blood samples
[0037] ①PBS buffer: Add 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 to ultrapure water to 1000 mL, adjust pH to 7.4, autoclave, and store at 4 ℃.
[0038] ②10% SDS: Dissolve 10 g SDS in 90 mL of ultrapure water, dissolve in a 68 ℃ water bath, adjust the pH to 7.2 with HCl, and bring the volume to 100 mL.
[0039] ③ 0.5 mol / L EDTANa2: 186.1 g of EDTA Na2 was dissolved in 800 mL of ultrapure water, the pH was adjusted to 8.0 with NaOH, the volume was brought to 1000 mL, and the mixture was autoclaved and stored at 4 °C.
[0040] ④1 mol / L Tris·HCl: 121.14 g Tris dissolved in 800 mL of ultrapure water, pH adjusted to 8.0 with HCl, volume brought to 1000 mL, autoclaved, and stored at 4 ℃.
[0041] ⑤ 5 mol / L NaCl: NaCl 292.2 g dissolved in 1000 mL ultrapure water.
[0042] ⑥ DNA extraction buffer: 0.5 mol / L EDTA Na2 4 mL, 1 mol / L Tris-HCl 10 mL, 5 mol / L NaCl 4 mL, and 10% SDS 20 mL were mixed, and the volume was made up to 100 mL, and then 20 μg / mL RNase was added.
[0043]
[0044] ⑦ NaAc buffer: NaAc-3H2O 20.4 g was mixed with 40 mL ultrapure water, dilute HAc was used to adjust the pH to 7.4, and the volume was made up to 50 mL.
[0045] ⑧ TE buffer: 1 M Tris-HCl (pH 8.0) 1 mL and 0.5 M EDTA buffer (pH 8.0) 0.2 mL were added to 100 mL ultrapure water, high-pressure sterilized, and stored at 4 ℃.
[0046] ⑨ Proteinase K: 20 mg / mL was prepared with ultrapure water and stored at -20 ℃.
[0047] B. Solutions used for agarose gel electrophoresis analysis
[0048] 1×TBE buffer: 10×TBE 50 mL was made up to 1000 mL.
[0049] 3. Designing genotype-specific primers for different alleles
[0050] The sequence of the third intron region of the goat gene was searched on NCBI, and the amplification primers were designed using NCBI Primer Blast. The primer sequences are as follows.
[0051] Upstream primer: 5'-CCTTCCTCATACAAGCAGCA-3';
[0052] Downstream primer: 5'-GATACTCATGGTACTTTGTGGAG-3'.
[0053] The goat genome was amplified by PCR using the above primers, and the electrophoretic detection of the amplification product is as follows Figure 1 Marker lane is Marker I (from large to small, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp). Lanes 1-10 are the amplified fragments (201 bp or 218 bp) of different samples.
[0054] 4. Detection of individual genotype by PCR amplification with primers
[0055] 4.1 Collection of goat samples
[0056] The goat used in the experiment is Anhui White Goat, and a total of 500 samples are collected. The blood samples of male and female Anhui White Goats are collected from the neck vein. After the blood samples are collected, ACD anticoagulation is used, and the ice box is low-temperature taken back to the laboratory and stored at -80℃. The goat samples are from Hefei Boda Animal Husbandry Science and Technology Development Co., Ltd.
[0057] 4.2 Isolation and extraction of blood genomic DNA
[0058] 1) Freeze the blood sample (mainly blood cells) at room temperature, transfer to a 1.5 mL Eppendorf centrifuge tube, add an equal volume of PBS buffer, mix well, centrifuge at 12000 r / min for 10 min (4℃), discard the supernatant, and repeat the above steps until the supernatant is transparent and the precipitate is light yellow;
[0059] 2) Add DNA extraction buffer 500 μL in the centrifuge tube, shake to separate the blood cell precipitate from the centrifuge tube wall, and incubate at 37℃ water bath for 1 h;
[0060] 3) Add 3 μL of proteinase K (20 mg / mL) to the mixture obtained in step 2 and mix well, incubate at 55℃ overnight until clear; if not clear, add 1 μL of proteinase K and mix well, then continue to digest until clear;
[0061] 4) Cool the reaction solution to room temperature, add Tris-saturated phenol 500 μL, gently shake the centrifuge tube for 20 min to mix well; centrifuge at 4℃, 12000 r / min for 10 min, transfer the supernatant to another 1.5 mL centrifuge tube, and repeat once;
[0062] 5) Add chloroform 500 μL, mix well for 20 min, centrifuge at 4℃, 12000 r / min for 10 min, transfer the supernatant to another 1.5 mL centrifuge tube;
[0063] 6) Add chloroform:isopropyl alcohol mixture (24:1) 500 μL, mix well for 20 min, centrifuge at 4℃, 12000 r / min for 10 min, transfer the supernatant to another 1.5 mL centrifuge tube;
[0064] 7) Add 0.1 volume of NaAc buffer and 2 volumes of ice-cold anhydrous ethanol, mix the centrifuge tube until white flocculent precipitate is precipitated, store at -20℃ for 30-60 min;
[0065] 8) Centrifuge at 4℃, 12000 r / min for 10 min, discard the supernatant, rinse the DNA precipitate with 70% ice-cold ethanol twice, centrifuge at 4℃, 12000 r / min for 10 min, discard the supernatant, and evaporate the ethanol at room temperature until dry;
[0066] 10) Dissolve the dried DNA in 80-100 μL ultrapure water, store at 4℃ until the DNA is completely dissolved, detect the quality by 0.8% agarose gel electrophoresis, and store at -80℃.
[0067] 4.3 Agarose gel electrophoresis detection of DNA
[0068] 1) Wash the gel box and gel plate for making agarose gel, dry, place the washed and dried gel-making bottom plate into the gel box, and insert the comb.
[0069] 2) Weigh 0.9 g of agarose, transfer into a triangular flask, add 1×TBE buffer 30 mL to suspend, heat in a microwave oven, take out after boiling twice, and add nucleic acid dye with a final concentration of 0.5 μg / mL when it is cooled to not hot. Gently shake to prevent bubbles.
[0070] 3) After mixing (about 60℃), immediately pour the agarose solution into the slot; if bubbles appear, immediately remove them with a pipette.
[0071] 4) After complete cooling and solidification (about 25-40 min), remove the comb, and move the gel into the electrophoresis tank.
[0072] 5) Add 1×TBE buffer to the electrophoresis tank to make the liquid level higher than the gel surface by 2-5 mm.
[0073] 6) Take 2-4 μL of DNA sample, mix after adding 2 μL of loading buffer, uniformly load (note that the order of the gun head should correspond to the front and back), and add DNA Marker to one side.
[0074] 7) Electrophorese at 110 V for 40 min.
[0075] 8) Observe on an ultraviolet analyzer, if there is RNA, it needs to be purified, and if there is obvious degradation, the corresponding sample needs to be re-extracted.
[0076] 4.4 Spectrophotometric detection of DNA
[0077] The OD values of DNA samples at 260 nm and 280 nm were measured by a UV spectrophotometer. The DNA content and OD 260 / OD 280 ratio were calculated. If the OD 260 / OD 280 ratio is less than 1.6, it indicates that the sample contains more protein or phenol, and then purification should be performed; if the ratio is greater than 1.8, it should be considered to remove RNA purification. Among them, the DNA concentration (ng / μL) = 50 x OD 260 value x dilution factor. After DNA detection, a certain amount was diluted to 50 ng / μL and stored at -20℃ for standby, and the rest was stored at -80℃.
[0078] 4.5 PCR amplification
[0079] The PCR reaction system adopts a mixed sample method. According to the amount of various components required for each reaction system and the number of PCR reactions required for one reaction, the total amount of various reaction components is calculated, added to a 1.5 mL centrifuge tube, mixed thoroughly, then centrifuged, then divided into each 0.2 mL Eppendorf PCR tube, then added template DNA (genomic DNA extracted from goat blood sample), then centrifuged, and then PCR amplification was performed;
[0080] The PCR reaction system is: PCR MasterMix (including Taq DNA polymerase, dNTPs and reaction buffer, concentration is 2x) 13.0 μL; upstream primer 1.0 μL; downstream primer 1.0 μL (the concentration of upstream and downstream primers is 10 pmol / μL); genomic DNA (the concentration of genomic DNA is 50 ng / μL) 1.0 μL; deionized water 9 μL; total 25.0 μL.
[0081] The PCR reaction program is: 94℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 57℃ annealing for 40 s (the annealing temperature is determined according to the annealing temperature of the primer), 68℃ extension for 1 min (the extension time is determined according to the maximum fragment), and the cycle is repeated for 25 times; 68℃ extension for 5 min, 4℃ storage.
[0082] 4.6 Agarose gel electrophoresis detection, DNA sequencing and individual genotype analysis
[0083] 1) Make a 3.5% agarose gel (add nucleic acid dye), 120 V voltage electrophoresis for 50 min.
[0084] 2) Image in Bio-Rad gel imaging system, and analyze the fragment size according to the image, such as Figure 1As shown, there is a 201 bp electrophoresis band consistent with the length of the designed primer amplification product in lanes 1, 5, 6, 9, a 218 bp electrophoresis band inconsistent with the length of the designed primer amplification product in lane 10, and two 201 bp and 218 bp electrophoresis bands in lanes 2, 3, 4, 7, 8.
[0085] 3) The part individual samples with different band sizes and quantities were sent to Shenguo Bioengineering (Shanghai) Co., Ltd. for DNA sequencing, and the sequencing peak maps of different genotypes are shown in Figure 2 As shown, the sequencing peak map identifies that the 218 bp electrophoresis band is the insertion genotype (i.e. mutant homozygous genotype II), and the 201 bp electrophoresis band is the normal genotype (i.e. wild homozygous genotype DD).
[0086] 4) In the detected Anhui white goat population, when the amplification product only shows a 201 bp band, it represents an individual with normal genotype DD; when the amplification product only shows a 218 bp band, it represents an individual with insertion genotype II; and when the amplification product shows 201 bp and 218 bp bands, it represents an individual with heterozygous genotype ID.
[0087] 5, Frequency statistical analysis of genetic variation of 17 bp Indel site in the third intron of Anhui white goat gene
[0088] 5.1 Gene and genotype frequency
[0089] Genotype frequency refers to the ratio of the number of individuals with a certain genotype in a population to the total number of individuals:
[0090] P YY = N YY / N
[0091] In the formula, P YY represents the frequency of YY genotype at a certain site; N YY represents the number of individuals with YY genotype in the population; and N represents the total number of the detected population.
[0092] Gene frequency refers to the relative ratio of the number of a certain gene to the total number of alleles in a population, and the formula for calculation is as follows:
[0093] P Y =(2N Y +N Ya1 +N Ya2 +N Ya3 +N Ya4 +……+N Yan ) / 2N
[0094] In the formula, P Y represents the frequency of allele Y, and N YYi represents the number of individuals with genotype Y in the population, N Yai Yi represents the number of individuals with genotype Y in the population, N i Yi represents the number of individuals with genotype Y in the population, N n Yi represents the number of individuals with genotype Y in the population, N
[0095] 5.2 Results
[0096] The allele frequency and genotype frequency of the 17 bp Indel site in the third intron of the gene in Anhui White Goats are shown in Table 1. This site has three genotypes in Anhui White Goats, namely II, ID and DD, and the frequencies of the three genotypes in the Anhui White Goat population are 0.06, 0.39 and 0.54, respectively. Analysis of Hardy-Weinberg disequilibrium found that this site is in Hardy-Weinberg equilibrium.
[0097] Table 1. Distribution of allele frequencies of the 17 bp Indel site in the gene in Anhui White Goats
[0098]
[0099] 6. Association analysis of the genetic effect of the 17 bp Indel site in the third intron of the gene in Anhui White Goats
[0100] Genotype data: II, ID and DD three genotypes;
[0101] The growth trait data of Anhui White Goats include body weight, body height, body length, chest circumference, pipe circumference and rump width of individuals at different ages.
[0102] Association analysis model: SPSS 25.0 software was used to analyze the correlation between breed, environment, gene site and growth traits.
[0103] First, descriptive statistical analysis of the data obtained was performed to determine whether there were outliers. Then, according to the characteristics of the data, variance analysis (when there are three genotypes, and the number of individuals of different genotypes is greater than 3), multivariate linear model or t analysis (when there are only two genotypes, and the number of individuals of different genotypes is greater than 3) were used to analyze the effect of genotypes. In the process of data processing, according to the different factors affecting the indicators of growth traits, the effects of age and genotype were considered, and the following fixed effect statistical model was established:
[0104] Y ijk = μ + Age j + Marker k + e ijk
[0105] Y ijk: individual phenotype record; μ: population mean; Age j : age effect; Marker k : marker genotype effect; e ijk : random error.
[0106] The results show (Table 2) that the 17 bp Indel mutation in the third intron region of the gene, i.e. NC_030809.1:g.46990698_46990714ins GCCTCCACTAGGTACCA, has a certain influence on the growth traits of the Anhui white goat.
[0107] Table 2 Association analysis of the 17 bp Indel polymorphism of the gene of the Anhui white goat with the growth traits of the goat
[0108]
[0109] Note: The same row data shoulder mark the same letter or no letter mark indicates that the difference is not significant (P>0.05).
[0110] As can be seen from Table 2, the different genotypes of the 17 bp Indel site in the intron of the gene are significantly related to the body weight, body length, chest circumference and pipe circumference of the Anhui white goat (P<0.05), wherein the body weight, body length and chest circumference of the individual with the normal genotype (DD) are significantly better than those of the individual with the insertion genotype (II) and the individual with the heterozygous genotype (ID), but the pipe circumference trait of the individual with the normal genotype (DD) is the best. The above results show that the 17 bp Indel in the third intron region of the gene is a harmful mutation, and the genotype DD is a candidate marker for early selection of the growth traits of the goat, which can be applied in marker-assisted selection and molecular breeding of the goat.
[0111] The above content is merely an example and a description of the present application, and those skilled in the art can make various modifications or supplements to the described specific embodiments or use similar ways to replace, as long as the modifications or supplements do not deviate from the structure of the present application or exceed the scope defined by the present claims, and should belong to the protection scope of the present application.
Claims
1. A method for detecting a 17 bp InDel mutation in an intron region of a SPATS2L gene of Anhui white goat, characterized in that: A 17 bp InDel mutation site in the intron region of SPATS2L gene of Anhui White Goat is identified by PCR amplification of a 17 bp InDel mutation site in the intron region of SPATS2L gene of Anhui White Goat using the whole genome DNA of the goat as a template, and electrophoretic detection of the number and size of bands. 2.The method for detecting the 17 bp InDel mutation in the intron region of SPATS2L gene of Anhui white goat according to claim 1, characterized in that, The amplification primers used in the PCR amplification are as follows: upstream primer: 5'-CCTTCCTCATACAAGCAGCA-3'; downstream primer: 5'-GATACTCATGGTACTTTGTGGAG-3'.
3. The method for detecting the 17 bp InDel mutation in the intron region of SPATS2L gene of Anhui white goat according to claim 1, characterized in that, The amplification reaction program of the PCR is as follows: pre-denaturation at 93-95 ℃ for 3-5 min; denaturation at 95-98 ℃ for 10-30 s, recombination at 55-60 ℃ for 30-50 s, extension at 65-72 ℃ for 1-2 min, and cycling for 25-30 times; extension at 65-72 ℃ for 3-5 min, and preservation at 4 ℃.
4. The method for detecting the 17 bp InDel mutation in the intron region of SPATS2L gene of Anhui white goat according to claim 1, characterized in that, The reaction system of the PCR amplification comprises 2x PCR MasterMix, 25-50 ng of goat genomic DNA, and 2.5-5 pmol of each amplification primer.
5. The method for detecting the 17 bp InDel mutation in the intron region of SPATS2L gene of Anhui white goat according to claim 1, characterized in that: The electrophoresis is performed using an agarose gel with a mass fraction of 3.5%-4.0%.
6. The method for detecting the 17 bp InDel mutation in the intron region of SPATS2L gene of Anhui white goat according to claim 1, characterized in that: According to the electrophoretic results, a normal genotype individual shows a 201 bp band, an insertion genotype individual shows a 184 bp band, and a heterozygous genotype individual shows 201 bp and 218 bp bands.
7. Use of a method for detecting a 17 bp InDel mutation in the intron region of SPATS2L gene of Anhui White Goat according to claim 1 in goat marker-assisted selection breeding.
8. Use according to claim 7, characterized in that: The InDel mutation site is a molecular marker related to growth traits of Anhui White Goat individuals.
9. Use according to claim 8, characterized in that: The growth traits include body weight, body height, body length, chest circumference, pipe circumference, and rump width.