CN111909988A - Method for detecting polymorphism locus typing of chicken green shin Id gene based on KASP - Google Patents
Method for detecting polymorphism locus typing of chicken green shin Id gene based on KASP Download PDFInfo
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Abstract
The invention provides a method for detecting polymorphism locus typing of chicken green shin Id gene based on KASP, which comprises the steps of designing primers according to green shin control gene locus, wherein the primers comprise a first primer, a second primer and a third primer; the nucleotide sequence of the first primer is as follows: SEQ ID No.1, and the nucleotide sequence of the second primer is as follows: SEQ ID No.2, and the nucleotide sequence of the third primer is as follows: SEQ ID No. 3; then, a PCR system is established; then setting a blank control, and replacing DNA in the PCR system by equal amount of ddH2O to serve as the blank control; amplifying the PCR; finally, scanning data to obtain the parting result of the green shin control gene; the present invention is useful for research of related traits and exploration of gene functions.
Description
Technical Field
The invention relates to the field of molecular marker breeding, in particular to a method for detecting polymorphism locus typing of chicken green shin Id gene based on KASP.
Background
The shank color of the chicken has diversity, and the shank color of the poultry mainly comprises white, yellow, cyan, bean green and the like. Like skin traits, chicken shank color traits are also important quality traits, and abundant and diverse shank color phenotypes are determined by gene regulation of deposition of melanin and carotenoid in shanks. Early studies found that the key gene controlling the shank color is the dermal melanin inhibiting gene, which is inherited in a non-dominant, sex-linked manner. When the gene is not expressed, the poultry individual exhibits the green-shin trait. Smyth et al found that the W locus on chromosome 24 and the Id locus on chromosome Z are the major genetic factors affecting the tibia, and that the W and Id interactions contribute to the diversity of the tibia. Siwek et al report consistent with the previous conclusions, and showed that the pale green shin phenotype has a significant relationship with recessive allelic sites W and Id by association analysis of single nucleotide polymorphisms GGA24 and GGAZ on chromosome 24 and chromosome Z.
The tibial trait related loci are a W locus and an Id gene, the W locus has been identified as BCDO2 gene on chromosome 24, but the specific position of the Id gene is again located in the plastication. The work on the location of the Id gene has been long, Bitgood et al, first in 1988, found that there is a linkage between the B, Id, TB, y loci on the Z chromosome, with four loci exhibiting a linear relationship of B-Id-TB-y. Using the physical map mapping method in 2004, the investigator found the Id site to be at the end of the long arm about 20c M away from ACO1 on the Z chromosome. In 2009, foreign researchers Dorshorst isolated the population using the F2 generation gene and applied the whole genome association analysis method to find that the Id gene was located in the physical interval between 67.1-72.3Mb on chromosome Z. In 2010, Dorshorst et al further studied the position of Id accurately, found a SNP (rs14686603) at the position 72.31Mb of Z chromosome that was significantly associated with the green shin trait, while predicting that the candidate gene for Id might be B4GALT 1. Subsequently, studies reported that the Id gene was closely linked to the 71,945,632-73,291,823bp region of chromosome Z. In 2014, researchers established a source group of F2 generation of Gushi-Anka chicken, collected blood samples of individuals with extreme shank phenotype to construct a DNA pool, and located Id genes more finely by using a target sequence capture sequencing method, and located in a section of 0.4Mb between 71.58 Mb and 72.18Mb of Z chromosome. Subsequent investigators will use Id by methods of high throughput sequencing and GWAS
The gene localization interval was narrowed and a large number of candidate genes were found. However, the specific location of the Id gene is still an important problem to be solved by academia, which is about the successful progress of the research tasks of related traits and the exploration of gene functions.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects in the prior art and provide a method
The invention is realized by the following technical scheme:
a method for detecting polymorphism locus typing of chicken green shin Id gene based on KASP comprises the following specific steps:
s1: designing primers according to the green shin control gene locus, wherein the primers comprise a first primer, a second primer and a third primer; the nucleotide sequence of the first primer is as follows: SEQ ID No.1, and the nucleotide sequence of the second primer is as follows: SEQ ID No.2, and the nucleotide sequence of the third primer is as follows: SEQ ID No. 3;
s2: establishing a PCR system;
s3: setting a blank control, and replacing DNA in the PCR system by equal amount of ddH2O to serve as the blank control;
s4: amplifying the PCR;
s5: the data were scanned to obtain the typing results for the green shin control gene.
Further, in the step S4, pre-denaturation at 94 ℃ is carried out for 15min, denaturation at 94 ℃ is carried out for 20S, annealing and extension at 55-61 ℃ are carried out for 60S, and 10 cycles are carried out; denaturation at 94 ℃ for 20s, annealing and extension at 55 ℃ for 60s, and performing 26 cycles; denaturation at 94 ℃ for 20s, annealing and extension at 57 ℃ for 60s, and 3 cycles;
further, in the step S2, the DNA in the established PCR system is chicken blood DNA, and the chicken blood DNA obtaining method is as follows:
s21: adding protease K into a centrifuge tube, and then adding chicken blood;
s22, adding Buffer ML into the centrifuge tube, performing vortex oscillation for 5 seconds, placing the centrifuge tube in a 56 ℃ water bath pot for incubation for 15 minutes, and performing vortex oscillation and uniform mixing for 2 times;
s23: taking out the centrifuge tube from the water bath, centrifuging, standing for 5 minutes at room temperature, adding the uniformly mixed isopropanol and Magbeads mixture, performing vortex oscillation and uniform mixing for 5 seconds, placing the centrifuge tube on a constant-temperature mixing instrument with the temperature of 25 ℃ and the rpm of 1600, and performing oscillation and uniform mixing for 5 minutes or continuously reversing the centrifuge tube and uniformly mixing for 10 minutes;
s24: placing the centrifuge tube on a magnetic frame for standing for 1 minute, and removing the solution after the Magbeads are adsorbed on the side wall of the centrifuge tube;
s25: taking the centrifugal tube off the magnetic frame, adding absolute ethyl alcohol, adding Buffer GW1, vibrating for 1 minute by a vortex point or vibrating for 5 seconds by a vortex, then placing the centrifugal tube on a constant-temperature mixing instrument with the temperature of 25 ℃ and the rpm of 1600, vibrating and mixing for 2 minutes, placing the centrifugal tube on the magnetic frame, standing for 1 minute, and after the Magbeads are adsorbed on the side wall of the centrifugal tube, reversing the magnetic frame to wash impurities on the cover of the centrifugal tube, and then thoroughly discarding the solution;
s26: repeating said step S25;
s27: taking the centrifugal tube off the magnetic frame, adding absolute ethyl alcohol, adding Buffer GW2, vibrating for 1 minute after a vortex point or vibrating for 5 seconds after vortex, placing the centrifugal tube on a constant-temperature mixing instrument with the temperature of 25 ℃ and the rpm of 1600, vibrating and mixing for 2 minutes, placing the centrifugal tube on the magnetic frame, standing for 1 minute, inverting the magnetic frame after the Magbeads are adsorbed on the side wall of the centrifugal tube, cleaning impurities on the cover of the centrifugal tube, and discarding the solution;
s28: repeating said step S27;
s29: keeping the centrifugal tube fixed on the magnetic frame, removing the solution on the tube bottom and the tube cover of the centrifugal tube by using a liquid transfer machine, and standing at room temperature for 5-10 minutes to fully volatilize ethanol;
s210: taking the centrifugal tube off the magnetic frame, adding Buffer EB, performing vortex oscillation to enable the magnetic beads to be fully suspended in eluent, placing the eluent on a constant-temperature mixing instrument with the temperature of 56 ℃ and the rpm of 1600, and performing the oscillation elution for 10 minutes, or placing the centrifugal tube in a water bath kettle with the temperature of 56 ℃ for incubation for 10 minutes, wherein the vortex oscillation is performed for 10 seconds every 3 minutes;
s211: and placing the centrifuge tube on the magnetic frame for standing for 2 minutes, and transferring the eluent into a new centrifuge tube by using a liquid transfer machine after the Magbeads are fully adsorbed on the side wall of the centrifuge tube for preservation at-20 ℃ for later use.
Further, in step S29, if there is a bead on the sidewall of the centrifuge tube, adding absolute ethanol into the centrifuge tube, keeping the centrifuge tube fixed on the magnetic rack, turning the centrifuge tube upside down after covering the centrifuge tube, and discarding all absolute ethanol.
Further, the PCR system comprises the chicken blood DNA, KASP Master Mix and a mixture of the first primer, the second primer and the third primer.
The invention has the beneficial effects that: the technology of the invention is a molecular identification method for chicken green shin gene, which comprises the steps of designing primers after comparing nucleotide sequences of the green shin gene and yellow (white) shin gene, specifically matching FAM marked primer 1 with the green shin gene, specifically matching HEX marked primer 2 with the yellow (white) shin gene, using a universal primer as a reverse primer, and extending a forward primer specifically combined with allele after combining the reverse primer. Specifically matching FAM-labeled primer 1 with the green shin gene, amplifying, and detecting only FAM fluorescence, namely obtaining the green shin gene homozygote, which is marked as tt; the primer 2 marked by HEX is specifically matched with the yellow (white) shin gene and then amplified, and only HEX fluorescence is detected, namely the yellow (white) shin gene homozygote (the phenotype is cyan yellow (white) shin) and is marked as TT; if FAM and HEX signals are detected simultaneously, the protein is a cyan-shin heterozygote and is marked as Tt. .
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a diagram of the chicken green shin genotyping results from the genotyping assay according to the invention.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention will be further described in detail with reference to the accompanying drawings and preferred embodiments.
The following describes in detail a specific embodiment of the present invention with reference to the drawings. In the drawings, the same reference numerals indicate the same or corresponding features. The drawings are only schematic and are not necessarily drawn to scale, and SNP typing can be achieved by a PCR method by using a general fluorescent probe in the KASP method. Allele 1 and allele 2 containing SNP are used as templates, two forward primers and a universal reverse primer are designed aiming at the SNP locus of the allele, and the tail part of each forward primer has a specific sequence and can be combined with a fluorescent label. In the first round of PCR, a forward primer which can be complemented with a template can be extended, and a forward primer which cannot be complemented with the template can not be extended; in the second round of PCR, the reverse primer binds and the forward primer that binds specifically to the allele is extended. As the number of PCR cycles increases, the number of amplicons increases exponentially, at which time the forward primer to which the FAM or HEX label specifically binds is no longer quenched and fluorescence is detected. Fluorescence of different colors reflects different SNP types, and the final experiment purpose can be achieved by detecting the experiment result by using the microplate reader. The technology of the invention is a molecular identification method for chicken green shin gene, which comprises the steps of designing primers after comparing nucleotide sequences of the green shin gene and yellow (white) shin gene, specifically matching FAM marked primer 1 with the green shin gene, specifically matching HEX marked primer 2 with the yellow (white) shin gene, using a universal primer as a reverse primer, and extending a forward primer specifically combined with allele after combining the reverse primer. Specifically matching FAM-labeled primer 1 with the green shin gene, amplifying, and detecting only FAM fluorescence, namely obtaining the green shin gene homozygote, which is marked as tt; the primer 2 marked by HEX is specifically matched with the yellow (white) shin gene and then amplified, and only HEX fluorescence is detected, namely the yellow (white) shin gene homozygote (the phenotype is cyan yellow (white) shin) and is marked as TT; if FAM and HEX signals are detected simultaneously, the protein is a cyan-shin heterozygote and is marked as Tt.
Primer 1: 5' -GAAGGTGACCAAGTTCATGCTCGGACCCAATTGCTGTTGGG-3' (SEQ ID No.1, the underlined part is the specific fluorescent sequence FAM);
primer 2: 5' -GAAGGTCGGAGTCAACGGATTCGGACCCAATTGCTGTTGGC-3' (SEQ ID No.2, the specific fluorescent sequence HEX is underlined);
primer 3: 5'-CAGAGGGAGGCTGCGGAAC-3' (SEQ ID No. 3).
Example (b):
s1, designing a primer according to the green shin control gene locus:
primer 1: 5' -GAAGGTGACCAAGTTCATGCTCGGACCCAATTGCTGTTGGG-3' (SEQ ID No.1, the underlined part is the specific fluorescent sequence FAM);
primer 2: 5' -GAAGGTCGGAGTCAACGGATTCGGACCCAATTGCTGTTGGC-3' (SEQ ID No.2, the specific fluorescent sequence HEX is underlined);
primer 3: 5'-CAGAGGGAGGCTGCGGAAC-3' (SEQ ID No. 3). (ii) a
S2, establishing a PCR system, wherein the system comprises 2.5uL DNA, 2.5uLKASP Master Mix (LGC Genomics, Hoddeson, UK) and more than 3 mixed primers of 0.07 uL.
S3: a blank control was set, and the DNA in the PCR system was replaced with an equal amount of ddH2O to serve as a blank control.
S4: performing PCR amplification
Pre-denaturation at 94 deg.C for 15min, denaturation at 94 deg.C for 20s, annealing and extension at 55-61 deg.C for 60s, and performing 10 cycles
Denaturation at 94 ℃ for 20s, annealing and elongation at 55 ℃ for 60s, and 26 cycles
Denaturation at 94 ℃ for 20s, annealing and extension at 57 ℃ for 60s, 3 cycles.
S5: data scanning was performed with reference to ABI7900HT Fast Real Time PCR System laboratory protocol to obtain the typing results of the green shin control gene.
In step S2, the DNA in the PCR system was derived from chicken blood, and in this example, genomic DNA was extracted from chicken blood using the kanji blood genomic DNA extraction kit CW 2361.
The method for obtaining the DNA in the chicken blood comprises the following steps:
s21: to a 1.5mL centrifuge tube was added 20. mu.L of protease K followed by 200. mu.L of blood.
S22: 200 μ L of Buffer ML was added to the tube, vortexed for 5 seconds to mix it thoroughly, and then the tube was incubated in a 56 ℃ water bath for 15 minutes while vortexed for 2 times.
S23: the tube was removed from the water bath, centrifuged briefly and allowed to stand at room temperature for 5 minutes. Adding a thoroughly mixed mixture of 320 mu L of isopropanol and Magbeads, vortexing, shaking and mixing for 5 seconds, placing the centrifugal tube on a constant-temperature mixer with the temperature of 25 ℃ and the rpm of 1600, shaking and mixing for 5 minutes, or continuously reversing the centrifugal tube and mixing for 10 minutes. S24: the centrifuge tube was placed on a magnetic rack and allowed to stand for 1 minute, and the solution was completely discarded after Magbeads were completely adsorbed to the side wall of the centrifuge tube (keeping the centrifuge tube fixed on the magnetic rack).
S25: taking the centrifugal tube off the magnetic frame, adding 750uL Buffer GW1 (before using, whether absolute ethyl alcohol is added or not is checked), then carrying out vortex point vibration for 1 minute or carrying out vortex vibration for 5 seconds, and then placing the tube on a constant temperature mixer with the temperature of 25 ℃ and the speed of 1600rpm to carry out vortex mixing for 2 minutes (ensuring that Magbeads are in a mixing state in the process of the vortex). And then placing the centrifuge tube on a magnetic frame for standing for 1 minute, and after the Magbeads are completely adsorbed on the side wall of the centrifuge tube, slightly reversing the magnetic frame to wash out impurities on the centrifuge tube cover, and then completely discarding the solution (keeping the centrifuge tube fixed on the magnetic frame).
S26: step S25 is repeated.
S27: taking the centrifugal tube off the magnetic frame, adding 750uL Buffer GW2 (before using, whether absolute ethyl alcohol is added or not is checked), then carrying out vortex point vibration for 1 minute or carrying out vortex vibration for 5 seconds, and then placing the tube on a constant temperature mixer with the temperature of 25 ℃ and the speed of 1600rpm to carry out vortex mixing for 2 minutes (ensuring that Magbeads are in a mixing state in the process of the vortex). And then placing the centrifuge tube on a magnetic frame for standing for 1 minute, slightly reversing the magnetic frame after the Magbeads are completely adsorbed on the side wall of the centrifuge tube, and completely discarding the solution after washing the impurities on the centrifuge tube cover (keeping the centrifuge tube fixed on the magnetic frame).
S28: step S27 is repeated.
S29: keeping the centrifugal tube fixed on the magnetic frame, further removing the solution on the tube bottom and the tube cover of the centrifugal tube by using a liquid transfer device, and then placing the centrifugal tube at room temperature for 5-10 minutes to ensure that the ethanol is completely volatilized.
Note that: if beads are on the side wall of the tube, 750. mu.L of absolute ethanol can be added to the tube. The tube was inverted after capping (keeping the tube fixed to the magnetic frame) and then the absolute ethanol was discarded completely.
S210: the centrifuge tube was removed from the magnetic stand and 50-200. mu.L of Buffer EB was added. After the magnetic beads are completely suspended in the eluent by vortex oscillation, the mixture is put on a constant-temperature mixing machine with the temperature of 56 ℃ and the rpm of 1600 and eluted by oscillation for 10 minutes, or the centrifugal tube is put in a water bath with the temperature of 56 ℃ for incubation for 10 minutes, and the vortex oscillation is carried out for 10 seconds every 3 minutes.
S211: and (3) placing the centrifuge tube on a magnetic frame for standing for 2 minutes, and transferring the eluent into a new centrifuge tube by using a liquid transfer machine for storage at-20 ℃ for later use after the Magbeads are completely adsorbed on the side wall of the centrifuge tube.
FIG. 1 is a diagram of chicken green shin genotyping according to the genotyping of the present invention, and it can be seen from FIG. 1 that the present invention using KASP for chicken green shin genotyping has low DNA sample requirements, is suitable for large-scale sample detection, has detection accuracy up to 100%, and supports low, medium or high throughput studies and single replicate experiments.
And (3) experimental verification conditions:
163 chickens were tested, and the results were determined as follows:
1. only FAM fluorescence was detected in 65 out of 12 fast-onset chickens, 130 silky-feather silky fowl and the hexox a line orthogonal F1 generation, and the FAM fluorescence was found to be a green-shin gene homozygote.
2. Only HEX fluorescence was detected in 21 hessex a line chickens, which were homozygous for the yellow (white) shank gene.
3. 130 silky feather black-bone chickens and 65 of the hexox a line orthogonal F1 generations detected FAM and HEX fluorescence at the same time, and were heterozygous for the cyan-shank gene (the phenotype is cyan-yellow (white) shank).
The detection results are all consistent with phenotypes, and the consistency rate reaches 100%.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A method for detecting polymorphism locus typing of chicken green shin Id gene based on KASP is characterized by comprising the following specific steps:
s1: designing primers according to the green shin control gene locus, wherein the primers comprise a first primer, a second primer and a third primer; the nucleotide sequence of the first primer is as follows: SEQ ID No.1, and the nucleotide sequence of the second primer is as follows: SEQ ID No.2, and the nucleotide sequence of the third primer is as follows: SEQ ID No. 3;
s2: establishing a PCR system;
s3: setting a blank control, and replacing DNA in the PCR system by equal amount of ddH2O to serve as the blank control;
s4: amplifying the PCR;
s5: the data were scanned to obtain the typing results for the green shin control gene.
2. The KASP-based method for detecting polymorphism site typing of chicken shin Id gene according to claim 1, wherein in the step S4,
pre-denaturation at 94 ℃ for 15min, denaturation at 94 ℃ for 20s, annealing and extension at 55-61 ℃ for 60s, and performing 10 cycles;
denaturation at 94 ℃ for 20s, annealing and extension at 55 ℃ for 60s, and performing 26 cycles;
denaturation at 94 ℃ for 20s, annealing and extension at 57 ℃ for 60s, 3 cycles.
3. The KASP-based method for detecting polymorphism site typing of chicken shin Id gene according to claim 1, wherein in the step S2, the DNA in the PCR system is chicken blood DNA, and the method for obtaining chicken blood DNA is as follows:
s21: adding protease K into a centrifuge tube, and then adding chicken blood;
s22, adding Buffer ML into the centrifuge tube, performing vortex oscillation for 5 seconds, placing the centrifuge tube in a 56 ℃ water bath pot for incubation for 15 minutes, and performing vortex oscillation and uniform mixing for 2 times;
s23: taking out the centrifuge tube from the water bath, centrifuging, standing for 5 minutes at room temperature, adding the uniformly mixed isopropanol and Magbeads mixture, performing vortex oscillation and uniform mixing for 5 seconds, placing the centrifuge tube on a constant-temperature mixing instrument with the temperature of 25 ℃ and the rpm of 1600, and performing oscillation and uniform mixing for 5 minutes or continuously reversing the centrifuge tube and uniformly mixing for 10 minutes;
s24: placing the centrifuge tube on a magnetic frame for standing for 1 minute, and removing the solution after the Magbeads are adsorbed on the side wall of the centrifuge tube;
s25: taking the centrifugal tube off the magnetic frame, adding absolute ethyl alcohol, adding Buffer GW1, vibrating at the vortex point for 1 minute or vibrating at the vortex for 5 seconds, and placing at 25 ℃,
Shaking and mixing the mixture for 2 minutes on a constant-temperature mixer at 1600rpm, placing the centrifuge tube on the magnetic frame for standing for 1 minute, and completely removing the solution after the Magbeads are adsorbed on the side wall of the centrifuge tube and the magnetic frame is reversed to wash impurities on the centrifuge tube cover;
s26: repeating said step S25;
s27: taking the centrifugal tube off the magnetic frame, adding absolute ethyl alcohol, adding Buffer GW2, vibrating for 1 minute after a vortex point or vibrating for 5 seconds after vortex, placing the centrifugal tube on a constant-temperature mixing instrument with the temperature of 25 ℃ and the rpm of 1600, vibrating and mixing for 2 minutes, placing the centrifugal tube on the magnetic frame, standing for 1 minute, inverting the magnetic frame after the Magbeads are adsorbed on the side wall of the centrifugal tube, cleaning impurities on the cover of the centrifugal tube, and discarding the solution;
s28: repeating said step S27;
s29: keeping the centrifugal tube fixed on the magnetic frame, removing the solution on the tube bottom and the tube cover of the centrifugal tube by using a liquid transfer machine, and standing at room temperature for 5-10 minutes to fully volatilize ethanol;
s210: taking the centrifugal tube off the magnetic frame, adding Buffer EB, performing vortex oscillation to enable the magnetic beads to be fully suspended in eluent, placing the eluent on a constant-temperature mixing instrument with the temperature of 56 ℃ and the rpm of 1600, and performing the oscillation elution for 10 minutes, or placing the centrifugal tube in a water bath kettle with the temperature of 56 ℃ for incubation for 10 minutes, wherein the vortex oscillation is performed for 10 seconds every 3 minutes;
s211: and placing the centrifuge tube on the magnetic frame for standing for 2 minutes, and transferring the eluent into a new centrifuge tube by using a liquid transfer machine after the Magbeads are fully adsorbed on the side wall of the centrifuge tube for preservation at-20 ℃ for later use.
4. The KASP-based method for detecting polymorphism site typing of chicken shin Id gene according to claim 3, wherein in step S29, if there is liquid bead on the sidewall of the centrifuge tube, absolute ethanol is added into the centrifuge tube, the centrifuge tube is kept fixed on the magnetic frame, the centrifuge tube is turned upside down after the cover is closed, and all absolute ethanol is discarded.
5. The KASP-based method for detecting polymorphism loci typing of chicken shin Id gene according to any one of claims 3 or 4, wherein said PCR system comprises said chicken blood DNA, KASP Master Mix and a mixture of said first primer, said second primer and said third primer.
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Denomination of invention: A method based on KASP for detecting polymorphic loci in chicken tibia Id gene typing Effective date of registration: 20231226 Granted publication date: 20220913 Pledgee: Bank of Hangzhou Limited by Share Ltd. Beijing Zhongguancun branch Pledgor: Beijing Compson Agricultural Technology Co.,Ltd. Registration number: Y2023110000549 |
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