Kit and method for detecting genes related to wool quality traits of fine-wool sheep

Abstract

The invention provides a fine-wool sheep wool quality character related gene detection kit and method, and belongs to the technical field of animal molecular breeding. The kit is based on 15 genetic variation sites of eight key functional genes such as KRTAPs, FGF5 and BMP2, target fragments are amplified through multiple PCR, genetic typing is achieved by combining high-resolution melting curve analysis, Sanger sequencing or CRISPR / Cas12a lateral flow detection, and wool quality genetic indexes are constructed for comprehensive evaluation. The kit adopts preloaded freeze-dried microspheres and partitioned sealing, so that the transportation stability and the operation convenience are ensured. The method can complete detection in the early stage of lambs, significantly shortens the breeding cycle, and improves the accuracy of seed selection. The method has the advantages of multi-gene joint detection, flow standardization, result visualization, wide application range and the like, and is suitable for genetic improvement and efficient breeding popularization of fine-wool sheep.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular, to a fine wool sheep wool quality trait-related gene detection kit and method. BACKGROUND

[0002] Fine wool sheep is an important economic livestock resource in China, and its wool has excellent characteristics such as fine fiber, good elasticity, and strong luster, and is widely used in high-end textile industry. Fine wool sheep breeds represented by Chinese Merino are large-scale bred in Xinjiang, Inner Mongolia, Gansu and other places in China, and are an important industry to promote the economic development of pastoral areas and the income increase of farmers and herdsmen. Traditional fine wool sheep breeding mainly relies on phenotypic selection, that is, selecting and mating according to wool fineness, wool length, net wool rate and other traits of adult individuals. However, this method has long cycle, high cost, and is easily disturbed by environmental factors, and it is difficult to accurately reflect the true genetic potential of individuals.

[0003] With the development of molecular biology and genomics, more and more studies have shown that the wool quality traits of fine wool sheep are jointly regulated by multiple functional genes and their genetic variations (such as single nucleotide polymorphism SNP, insertion and deletion InDel). For example: multiple members of the KRTAPs (keratin-associated protein gene) family (such as KRTAP1-1, KRTAP6-1) are closely related to wool fiber diameter; mutation of FGF5 (fibroblast growth factor 5) gene can significantly prolong the anagen phase and increase wool length; BMP2, EDAR and STAT3 genes have also been confirmed to affect hair follicle density and wool yield.

[0004] Based on the above findings, some studies have begun to try to use molecular marker-assisted selection (MAS) to guide breeding by detecting individual gene sites. At present, some laboratories have used PCR-RFLP, AS-PCR or sequencing methods to genotype specific SNPs, and have verified their correlation with phenotypes in a small range.

[0005] However, the existing technology still has the following significant deficiencies: most of the existing methods only detect 1-2 gene sites, and cannot cover the key gene network affecting wool quality, which cannot comprehensively evaluate the comprehensive genetic potential of individuals, resulting in low selection accuracy and difficulty in meeting the needs of modern efficient breeding.

[0006] Traditional PCR detection requires multiple steps of pipetting, centrifugation, electrophoresis, etc., and the reagents need to be prepared and used immediately, with strict storage conditions, and the result interpretation depends on large instruments such as gel imaging system or sequencer, which is not suitable for popularization and application in grass-roots breeding farms or remote pastoral areas.

[0007] At present, there is no commercial and integrated fine wool sheep special gene detection kit, most of which are self-built systems by research institutions, and the reagent ratio is not uniform, the batch difference is large, which leads to unstable detection results, and it is difficult to realize cross-regional data comparison and breeding resource sharing.

[0008] Due to the complex process and dependence on external sequencing, it usually takes 7-14 days from sampling to obtaining results, which cannot complete rapid screening at the lamb stage, misses the best selection opportunity, and still needs to be raised to adulthood to eliminate low-efficiency individuals according to phenotypes, causing waste of feed, manpower and time resources.

[0009] The existing technology generally requires constant-temperature PCR instrument, electrophoresis device and power supply support, and cannot be operated under conditions without electricity and laboratory, which seriously restricts the popularization and application of molecular breeding technology in remote pastoral areas.

[0010] Most of the detection only provides genotype results, and does not establish a genetic score model linked to production performance, so that farmers are difficult to convert detection data into actual selection scheme, which limits the transformation of molecular detection results to productivity. Therefore, a fine wool sheep wool quality trait related gene detection kit and method are proposed. SUMMARY

[0011] The purpose of the present application is to solve the problems existing in the background art. In order to achieve the above-mentioned purpose of the application, the present application provides the following technical scheme: a fine wool sheep wool quality trait related gene detection kit, the kit comprises an integrated plastic shell container, a plurality of functional area separation cavities are arranged in the container, and the following components are arranged in the cavities respectively: A genomic DNA extraction component, comprising a lysis solution, a binding solution, a washing solution, an elution solution and a silica gel membrane centrifugal column or a magnetic bead; A multiplex PCR amplification reagent group, comprising a freeze-dried primer mixture, dNTPs, a buffer and a high-fidelity Taq enzyme microsphere pre-packaged in a 96-well PCR plate; A genotyping detection module selected from Sanger sequencing reaction solution, HRM premix or CRISPR / Cas12a lateral flow detection card; A positive control DNA sample (containing known KRTAP1-1 GG, FGF5 TT and other high-quality genotypes) and a negative control (no nucleic acid water); The primer mixture comprises specific primer pairs for 15 SNP / InDel sites in KRTAP1-1, KRTAP6-1, KRTAP7-1, FGF5, BMP2, EDAR, STAT3 and LHX2 genes; The shell is provided with a barcode label and a counterfeit-proof sealing film, and the inside is provided with a desiccant bin and a temperature indicating label for transportation and storage monitoring.

[0012] As a preferred technical scheme of the present application, the primer pair in the primer mixture can specifically amplify the following: KRTAP1-1 g.123A>G, KRTAP6-1 g.456T>C, KRTAP7-1 15bp InDel, FGF5 g.890C>T, BMP2 g.1023G>A, EDAR g.1501T>C, STAT3 g.2100G>A, LHX2 g.3055C>T and all primers are confirmed by BLAST alignment to have unique specificity in the sheep genome, the annealing temperature is uniformly 60±1℃, and the GC content is between 45% and 60%.

[0013] As a preferred technical scheme of the present application, the genotyping detection module is a CRISPR / Cas12a lateral flow detection card, which is embedded in a separate sealed chamber of the kit and includes: a reaction pad, pre-fixed with a Cas12a-crRNA complex; a sample addition hole connected to a filter membrane channel; a detection line (T line) coated with anti-FAM antibody, and a control line (C line) coated with IgG antibody; a reporter probe which is a single-stranded RNA / DNA hybrid molecule labeled with 5'-FAM-rU-Biotin-3', and after the crRNA recognizes the target sequence and activates the Cas12a, transcleavage occurs, releasing the FAM-labeled fragment to form a T line color development; color development time ≤30 minutes, visible to the naked eye.

[0014] As a preferred technical scheme of the present application, the multiplex PCR amplification reagent is pre-disposed in each well of a 96-well plate in the form of freeze-dried microspheres, and each well contains: 1 pmol of primer mixture; 200 μM of dNTPs final concentration; 1.5 mM of Mg²⁺ final concentration; 0.5 U of Q5 High-Fidelity DNA Polymerase; trehalose and BSA as stabilizing protective agents; When used, only 2 μL of template DNA and 8 μL of ddH2O need to be added to start the reaction.

[0015] A fine wool sheep wool quality trait-related gene detection method, comprising the following steps: step 1, collecting ear tissue samples, using magnetic bead method to extract genomic DNA, concentration ≥ 30 ng / μL, A260 / A280=1.8-2.0; Step 2, add the DNA to the PCR plate of the kit of claim 1, and perform the following program amplification: 95℃ pre-denaturation 5 min; 35 cycles: 95℃ 30 s→ 60℃ 30 s→ 72℃ 45 s; 72℃ final extension 5 min; Step 3, after PCR product purification, Sanger sequencing or HRM analysis is performed; Step 4, the genotyping results are input into the WQGI scoring model, and the individual genetic potential grade is output.

[0016] As a preferred technical solution of the present application, the WQGI wool quality genetic index is calculated by the following weighted linear model: Wherein: The genotype score of the i-th locus: homozygous dominant allele = 2, heterozygous = 1, homozygous inferior = 0; The weight coefficient is standardized according to the GWAS effect size, for example: Finally The full score is 15.0, ≥12.0 is determined as a high genetic potential individual, which is suitable for core sheep breeding.

[0017] As a preferred technical solution of the present application, the HRM analysis uses EvaGreen dye, runs the melting program on a real-time fluorescent PCR instrument, the heating rate is 0.1℃ / s, from 65℃ to 95℃, and the difference in the second derivative curve peak value is determined by difference; Wherein the typical melting temperature (Tm) of FGF5 g.890C>T site is: CC type: Tm=78.3±0.2℃ CT type: double peak, Tm1=76.1℃, Tm2=78.4℃ TT type: Tm=76.0±0.3℃.

[0018] As a preferred technical solution of the present application, the detection performance of the kit meets the following technical indicators: Sensitivity: can detect as low as 10 pg / μL of genomic DNA; Specificity: no cross amplification with other common livestock (cattle, pigs, goats) DNA; Accuracy: genotype consistency ≥ 96.5% (n=200) compared with the gold standard Sanger sequencing; Batch difference CV <5%, good repeatability; Shelf life: 18 months at -20℃, 7 days at 4℃ for short-term stability.

[0019] As a preferred technical solution of the present application, for predicting individual adult wool traits, including: Average fiber diameter (AFD) prediction error ≤1.2 μm; Hair length prediction correlation coefficient R²≥0.75; Wool density improvement rate: preferably the average of individual offspring is increased by 12.3% (P<0.01); Applied to early screening within 30 days after lamb birth, shortening the generation interval by more than 30%.

[0020] A fine wool sheep wool quality prediction system, comprising the genetic detection kit of claim 1; Mobile terminal or PC terminal data analysis software, built-in WQGI algorithm engine and database, supporting code scanning to read sample information; The software automatically outputs: Each trait prediction value: , wherein (A) is the regression coefficient ; Breeding recommendation report, recommending the optimal mating combination; data uploaded to the cloud breeding management platform.

[0021] Compared with the prior art, the present application has the following beneficial effects: the present application can complete genetic detection and genetic potential evaluation within 30 days after lamb birth, without waiting for the animal to be adult and obtain phenotype data by shearing, shortening the traditional 4-5 year selection period to within 1 year, significantly improving breeding efficiency, and accelerating the diffusion speed of excellent genes in the population.

[0022] The kit covers 8 key functional genes such as KRTAPs, FGF5, BMP2, and EDAR closely related to wool fineness, length, and density, synchronously detects 15 verified SNP and InDel sites, breaks through the limitation of single site detection, realizes systematic prediction of wool quality through a multi-gene comprehensive scoring model, and is significantly superior to single marker assisted selection.

[0023] The kit adopts modular design, all key reagents are pre-packaged and freeze-dried, and clear operation instructions are provided. Users only need to add DNA templates to start the reaction, which greatly reduces the technical threshold and is suitable for operation by technical personnel of primary livestock stations and farms, promoting the popularization of molecular breeding technology to the front line. ​

[0024] Three typing methods of Sanger sequencing, high-resolution melting curve (HRM) analysis and CRISPR / Cas12a lateral flow visual detection are provided: The CRISPR test strip can be used on the pasture to realize integrated rapid detection of sampling-extraction-interpretation, and the result can be obtained within 30 minutes without complex instruments, so that the field laboratory is truly realized.

[0025] The kit adopts a multi-cavity sealed plastic shell, and is provided with a desiccant bin, a temperature indicating label and a tamper-proof sealing film, so that moisture, high temperature deterioration and cross contamination in the transportation and storage process are effectively prevented; the PCR plate is preloaded with freeze-dried microspheres, which have strong heat resistance, can be stably stored at 4°C for 7 days, and have a shelf life of 18 months at-20°C, and are convenient for distribution and use in remote areas.

[0026] Based on the wool quality genetic index (WQGI) of the detection result, complex genetic information is converted into intuitive scores, and individual genetic potential reports and breeding suggestions are output in combination with the supporting software, so as to help breeding units to scientifically select core breeding sheep and optimize breeding schemes, avoid inbreeding degeneration, and improve the overall wool production performance of the population.

[0027] The population subjected to molecular breeding by the application has offspring with an average fiber diameter of 1.5-2.5 μm, an increase of 1.0-2.0 cm in hair length, and an increase of more than 10% in wool density, which significantly improves the proportion of high-quality wool and market price. According to the statistics of the pilot farm, each high genetic potential breeding ram can drive a hundred ewe population to increase annual income of more than ten thousand yuan, and the economic benefit is outstanding.

[0028] The detection process does not involve radioactive isotopes or toxic chemical reagents, and the CRISPR detection product is a conventional biodegradable material, so that the waste disposal is simple and meets the requirements of green agricultural development. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The kit provided by the application is schematically shown in the whole composition; Figure 2 The core component data block diagram of the detection card provided by the application is shown; Figure 3 The multiple PCR freeze-dried microsphere formula and use data block diagram provided by the application is shown. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the embodiments of the application clearer, the technical scheme in the embodiments of the application will be described clearly and completely below with reference to the drawings. Obviously, the described embodiments are part of the embodiments of the application, rather than all the embodiments.

[0031] Therefore, the following detailed description of the embodiments of the application is not intended to limit the scope of the application as claimed, but merely represents some embodiments of this application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application. It should be noted that the embodiments in the present application and the features and technical solutions in the embodiments can be combined with each other without conflict, and similar reference numerals and letters represent similar items in the following drawings, therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0032] Embodiment 1: A fine wool sheep wool quality trait-related gene detection kit, the kit comprising an integrated plastic shell container, the container being provided with a plurality of functional area partition cavities, respectively accommodating the following components: genomic DNA extraction components, including lysis solution, binding solution, washing solution, elution solution and silica gel membrane centrifugal column or magnetic beads; multiplex PCR amplification reagent group, including pre-packaged in a 96-well PCR plate freeze-dried primer mixture, dNTPs, buffer and high-fidelity Taq enzyme microspheres; Genotyping detection module, selected from Sanger sequencing reaction solution, HRM premix or CRISPR / Cas12a lateral flow detection card; Positive control DNA sample (containing known KRTAP1-1 GG, FGF5 TT, etc. High-quality genotypes) and negative control (no nucleic acid water); The primer mixture contains specific primer pairs for 15 SNP / InDel sites in KRTAP1-1, KRTAP6-1, KRTAP7-1, FGF5, BMP2, EDAR, STAT3 and LHX2 genes; The shell is provided with a barcode label and a tamper-proof sealing film, and the inside is provided with a desiccant compartment and a temperature indicating label for transportation and storage monitoring.

[0033] The primer pairs in the primer mixture can specifically amplify: KRTAP1-1 g.123A>G, KRTAP6-1 g.456T>C, KRTAP7-1 15bp InDel, FGF5 g.890C>T, BMP2 g.1023G>A, EDAR g.1501T>C, STAT3 g.2100G>A, LHX2 g.3055C>T And all primers are confirmed to have unique specificity in the sheep genome by BLAST alignment, the annealing temperature is uniformly 60±1℃, and the GC content is between 45%-60%.

[0034] The genotyping detection module is a CRISPR / Cas12a lateral flow detection card embedded in a separate sealed chamber of the kit, including: a reaction pad for pre-fixing a Cas12a-crRNA complex; a sample addition hole connected to a filter membrane channel; a detection line (T line) coated with anti-FAM antibody, and a control line (C line) coated with IgG antibody; a reporter probe is a single-stranded RNA / DNA hybrid molecule labeled with 5'-FAM-rU-Biotin-3', which is trans-cleaved after the crRNA recognizes the target sequence and activates the Cas12a, releasing the FAM-labeled fragment to form a T line color; color development time ≤ 30 minutes, visible to the naked eye.

[0035] The multiplex PCR amplification reagent is pre-disposed in each well of a 96-well plate in the form of freeze-dried microspheres, and each well contains: 1 pmol of primer mixture; dNTPs at a final concentration of 200 μM; Mg²⁺ at a final concentration of 1.5 mM; Q5 High-Fidelity DNA Polymerase 0.5 U; trehalose and BSA as stabilizing and protective agents; When used, only 2 μL of template DNA and 8 μL of ddH2O need to be added to start the reaction.

[0036] A fine wool sheep wool quality trait-related gene detection method, comprising the following steps: step 1, collecting ear tissue samples, using magnetic bead method to extract genomic DNA, concentration ≥ 30 ng / μL, A260 / A280=1.8–2.0; Step 2, add the DNA to the PCR plate of the kit of claim 1, and perform the following program amplification: 95℃ pre-denaturation for 5 min; 35 cycles: 95℃ for 30 s, 60℃ for 30 s, and 72℃ for 45 s; 72℃ final extension for 5 min; Step 3, after purification of the PCR product, Sanger sequencing or HRM analysis is performed; Step 4, input the genotyping results into the WQGI scoring model, and output the individual genetic potential grade.

[0037] The WQGI wool quality genetic index is calculated by the following weighted linear model: Wherein: Genotype score for the ith locus: homozygous dominant allele = 2, heterozygous = 1, homozygous recessive = 0; is a weight coefficient, which is normalized according to the GWAS effect size, for example: Final The full score is 15.0, and ≥12.0 is determined as a high genetic potential individual, which is suitable for core sheep breeding.

[0038] HRM analysis uses EvaGreen dye, and the melting program is run on a real-time fluorescence PCR instrument with a heating rate of 0.1℃ / s from 65℃ to 95℃, and the difference in peak value of the second derivative curve is used for judgment; The typical melting temperature (Tm) of the FGF5 g.890C>T site is: CC type: Tm=78.3±0.2℃ CT type: double peak, Tm1=76.1℃, Tm2=78.4℃ TT type: Tm=76.0±0.3℃.

[0039] The detection performance of the kit meets the following technical indicators: Sensitivity: can detect as low as 10 pg / μL of genomic DNA; Specificity: no cross amplification with other common livestock (cattle, pigs, goats) DNA; Accuracy: compared with the gold standard Sanger sequencing, the genotype consistency is ≥96.5% (n=200); Batch difference CV<5%, good reproducibility; Shelf life: stored at -20℃ for 18 months, and at 4℃ for short-term stability for 7 days.

[0040] For predicting individual adult sheep wool traits, including: The average fiber diameter (AFD) prediction error is ≤1.2μm; The correlation coefficient R² of wool length prediction is ≥0.75; Wool density improvement range: the average of the offspring of the preferred individual is increased by 12.3% (P<0.01); Applied to early screening within 30 days after lamb birth, shortening the generation interval by more than 30%.

[0041] A fine wool sheep wool quality prediction system, comprising the genetic detection kit of claim 1; Mobile terminal or PC terminal data analysis software, built-in WQGI algorithm engine and database, supporting code scanning to read sample information; The software automatically outputs according to the input 15-locus genotypes: The predicted value of each trait: , wherein (a) is a regression coefficient ; Breeding recommendation report, recommending the optimal mating combination; Data is uploaded to the cloud breeding management platform.

[0042] Test Example 1: Test purpose To evaluate the detection accuracy and batch / intra-batch repeatability of the kit for key gene loci.

[0043] Test materials and methods 50 healthy adult fine-wool sheep were selected, and ear tissue samples were collected; The kit was used for DNA extraction, multiplex PCR amplification and HRM typing detection; At the same time, the PCR products were sent to a third-party sequencing agency for Sanger sequencing as a gold standard comparison; Three independent tests were completed by different operators at different times (7 days apart) to evaluate repeatability.

[0044] Test results All 15 target loci were successfully amplified, without non-specific bands or amplification failure; Compared with the Sanger sequencing results, the genotype consistency was 96.8% (718 / 742 loci consistent); Among them, the consistency of 12 loci was 100%, and 3 loci (KRTAP7-1 InDel, EDAR g.1501T>C, STAT3 g.2100G>A) had individual heterozygote interpretation deviation, which was improved to more than 98.5% after optimizing the HRM program; The intra-batch CV value was 3.2%, and the inter-batch CV value was 4.7%, indicating good detection stability.

[0045] Conclusion The kit has high accuracy and good repeatability, meeting the needs of large-scale breeding detection.

[0046] Test Example 2: Test purpose To verify the correlation between the detected gene loci and the actual wool quality traits.

[0047] Test materials and methods ​Select 120 2-year-old ewes, use the kit to complete all 15 loci genotyping; The wool phenotype indicators were measured after shearing in the spring of the following year: average fiber diameter (AFD, unit μm), wool length (cm), wool density (root / cm²), and net wool rate (%). The differences in each trait were compared according to genotype grouping, and the "wool quality genetic index" (WQGI) was calculated to analyze its correlation with the phenotype.

[0048] Test results The average wool length of FGF5 g.890 TT type individuals was 9.8 cm, which was significantly higher than that of CC type (8.0 cm, P<0.01); The average fineness of KRTAP1-1 g.123 GG type wool was 17.3 μm, which was 2.3 μm thinner than that of AA type (19.6 μm) (P<0.001); The wool density of BMP2 g.1023 GA / AA type individuals increased by an average of 13.5%; The comprehensive wool quality of the top 30% individuals (score ≥11.0) in WQGI score was significantly better than that of the bottom 30% individuals (score ≤8.0), with finer AFD (1.8 μm lower), 1.5 cm longer wool length, and 4.2 percentage points higher net wool rate; WQGI was negatively correlated with AFD (r=-0.72) and positively correlated with wool length (r=0.68), and had strong predictive ability.

[0049] Conclusion The key gene loci are highly associated with wool phenotype, and WQGI can effectively reflect the genetic potential of individuals.

[0050] The above examples are only used to illustrate the present application and not to limit the technical solutions described in the present application. Although the present application has been described in detail with reference to the above embodiments, the present application is not limited to the above specific embodiments, and therefore any modification or equivalent replacement of the present application; all technical solutions and improvements that do not deviate from the spirit and scope of the application are encompassed in the scope of the claims of the present application.

Claims

1. A fine-wool sheep wool quality trait-associated gene detection kit, characterized in that: The kit comprises an integrated plastic shell container, which is provided with multiple functional area separation cavities for containing the following components respectively: Genomic DNA extraction components, including lysis solution, binding solution, washing solution, elution solution, and silica gel membrane centrifugal column or magnetic beads; Multiplex PCR amplification reagent group, including pre-packaged lyophilized primer mix, dNTPs, buffer, and high-fidelity Taq enzyme microspheres in a 96-well PCR plate; Genotyping detection module, selected from Sanger sequencing reaction solution, HRM premix, or CRISPR / Cas12a lateral flow detection card; Positive control DNA sample (containing known KRTAP1-1 GG, FGF5 TT, etc. superior genotypes) and negative control (no nucleic acid water); The primer mix contains specific primer pairs for 15 SNP / InDel sites in KRTAP1-1, KRTAP6-1, KRTAP7-1, FGF5, BMP2, EDAR, STAT3, and LHX2 genes; The shell is provided with a barcode label and a tamper-proof sealing film, and is internally provided with a desiccant compartment and a temperature indicating label for transportation and storage monitoring.

2. The fine wool sheep wool quality trait related gene detection kit according to claim 1, characterized in that: The primer pairs in the primer mix can specifically amplify KRTAP1-1 g.123A>G, KRTAP6-1 g.456T>C, KRTAP7-1 15bp InDel, FGF5 g.890C>T, BMP2 g.1023G>A, EDAR g.1501T>C, STAT3 g.2100G>A, and LHX2 g.3055C>T All primers are confirmed by BLAST alignment to have unique specificity in the sheep genome, with annealing temperature of 60±1℃ and GC content between 45% and 60%.

3. The fine wool sheep wool quality trait related gene detection kit according to claim 2, characterized in that: The genotyping detection module is a CRISPR / Cas12a lateral flow detection card, which is embedded in a separate sealed chamber of the kit, including: Reaction pad, pre-fixed Cas12a-crRNA complex; Sample addition hole, connected to the filter membrane channel; Detection line (T line) coated with anti-FAM antibody, control line (C line) coated with IgG antibody; Report probe is a 5'-FAM-rU-Biotin-3' labeled single-stranded RNA / DNA hybrid molecule, which undergoes transcleavage after crRNA recognizes the target sequence and activates Cas12a, releasing the FAM labeled fragment to form the T line color; Color development time ≤ 30 minutes, visible to the naked eye.

4. The fine wool sheep wool quality trait related gene detection kit according to claim 3, characterized in that: The multiplex PCR amplification reagent is pre-disposed in the form of lyophilized microspheres in each well of a 96-well plate, each well containing: Total amount of primer mix 1 pmol; Final concentration of dNTPs 200 μM; Final concentration of Mg²⁺ 1.5 mM; Q5 High-Fidelity DNA Polymerase 0.5 U; Trehalose and BSA as stabilizing protective agents; When used, only 2 μL of template DNA and 8 μL of ddH2O are needed to start the reaction.

5. A method for detecting a fine-wool sheep wool quality trait-related gene, characterized in that, The fine wool sheep wool quality trait-related gene detection kit according to any one of claims 1-4 comprises the following steps: Step 1, collecting ear tissue samples, using magnetic bead method to extract genomic DNA, concentration ≥ 30 ng / μL, A260 / A280 = 1.8-2.0; Step 2, adding DNA to the PCR plate of the kit of claim 1, and performing the following program amplification: 95℃ pre-denaturation for 5 min; 35 cycles: 95℃ for 30 s→60℃ for 30 s→72℃ for 45 s; 72℃ final extension for 5 min; Step 3, after PCR product purification, Sanger sequencing or HRM analysis is performed; Step 4, input the genotyping results into the WQGI scoring model, and output the individual genetic potential grade.

6. The fine wool sheep wool quality trait-related gene detection method according to claim 5, characterized in that: The WQGI wool quality genetic index is calculated by the following weighted linear model: Wherein: Genotype score for the ith locus: homozygous dominant allele = 2, heterozygous = 1, homozygous recessive = 0; are weight coefficients, normalized according to GWAS effect size, for example: Finally The full score is 15.0, ≥12.0 is determined as high genetic potential individual, suitable for core sheep breeding.

7. The method for detecting fine wool sheep wool quality trait related genes according to claim 5, characterized in that: The HRM analysis uses EvaGreen dye, and the melting program is run on a real-time fluorescent PCR instrument with a heating rate of 0.1℃ / s from 65℃ to 95℃, and the difference in peak value of the second derivative curve is used for typing; Wherein the typical melting temperature (Tm) of FGF5 g.890C>T site is: CC type: Tm=78.3±0.2℃ CT type: double peak, Tm1=76.1℃, Tm2=78.4℃ TT type: Tm=76.0±0.3℃.

8. The method for detecting fine wool sheep wool quality trait related genes according to claim 7, characterized in that: The detection performance of the kit meets the following technical indicators: Sensitivity: can detect as low as 10 pg / μL of genomic DNA; Specificity: no cross amplification with DNA of other common livestock (cattle, pigs, goats); Accuracy: compared with the gold standard Sanger sequencing, the genotype consistency is ≥96.5% (n=200); Batch difference CV<5%, good reproducibility; Shelf life: stored at -20℃ for 18 months, stable at 4℃ for 7 days.

9. Use of the kit according to claim 1 in the genetic improvement of fine-wool sheep, characterized in that: For predicting individual adult wool traits, including: Average fiber diameter (AFD) prediction error ≤1.2μm; Correlation coefficient R²≥0.75 for wool length prediction; Wool density improvement range: preferred individual offspring average increase of 12.3% (P<0.01); Applied to early screening within 30 days after lamb birth, shortening the generation interval by more than 30%.

10. A fine wool sheep wool quality prediction system characterised by, Including: The gene detection kit of claim 1; Mobile terminal or PC terminal data analysis software, with built-in WQGI algorithm engine and database, supporting code scanning to read sample information; The software automatically outputs: Predicted values for each trait: where (a is the regression coefficient ; Breeding recommendation report, recommending the optimal mating combination; Data uploaded to the cloud breeding management platform.

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