Molecular markers, detection reagents, methods and their applications in Jiaji duck breeding

Abstract

This invention discloses molecular markers, detection reagents, methods, and their applications for breeding Jiaji ducks. Seven single nucleotide polymorphism (SNP) sites significantly associated with growth traits in Jiaji ducks were screened in the 5'UTR, intron, and exon regions of the APOC3 gene. These SNP sites can serve as molecular markers for genetic improvement of the Jiaji duck breed. Using these molecular markers for selective breeding of Jiaji ducks is beneficial for improving the growth traits of the population. This invention also provides primers, kits, and breeding methods for detecting these molecular markers, which can be used for early-stage auxiliary selection in Jiaji duck breeding and are of great significance for breeding Jiaji ducks that are more likely to meet market standards.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and more specifically, to molecular markers, detection reagents, methods, and their applications for breeding Jiaji ducks. Background Technology

[0002] The apolipoprotein (APO) superfamily is a group of proteins that play a crucial role in lipid metabolism. They bind to blood lipids such as cholesterol and triglycerides to form lipoproteins, thereby transporting lipids to various tissues and organs throughout the body. Based on the protein structure and lipid-binding characteristics of its members, the APO superfamily is further divided into the APOA, APOB, APOC, APOE, and APOH families. APOC3 belongs to the APOC family. It is mainly synthesized and secreted by the liver, with a small portion synthesized in the intestine. It is a low-molecular-weight extracellular protein, primarily found in very low-density lipoproteins, high-density lipoproteins, and triglyceride-rich lipoproteins in plasma. APOC3 can participate in regulating lipid metabolism by inhibiting the activity of lipoprotein lipases and hepatic lipases on the surface of vascular endothelial cells, thereby reducing the hydrolysis of total cholesterol (TC) in plasma. Infection of C57BL / 6 mice with an adenovirus expressing human APOC3 revealed that APOC3 can regulate the structure and function of high-density lipoprotein and selectively promote energy metabolism in activated brown adipose tissue. High-fat diet induction in APOC3-deficient mice revealed a higher susceptibility to obesity compared to normal mice. This suggests that the APOC3 gene may serve as a potential therapeutic target for obesity and metabolic-related diseases. Furthermore, studies in livestock have shown a linear positive correlation between intramuscular fat deposition in cola pigs and APOC3 transcription levels. In goats, APOC3 can promote IMF cell differentiation by upregulating SREBP1, CEBPβ, and PPARγ, and downregulating Pref-1. Currently, research on the APOC3 gene primarily focuses on human cardiovascular diseases, with few reports on its application in poultry.

[0003] Jiaji duck is a superior meat duck breed from Hainan Province, characterized by its strong resistance to heat and humidity, and its ability to thrive on roughage. Its meat has a unique flavor, with a large breast, thin skin, soft bones, and tender meat, offering a rich taste. Jiaji duck is highly popular with consumers both domestically and internationally and is listed as a globally protected breed for genetic diversity. However, despite being one of Hainan's "Four Famous Dishes," the Jiaji duck industry lags significantly behind Wenchang chicken in scale, with an annual output of only over 2 million ducks. Market research indicates that larger animals with better body shape are more popular and easier to market. Therefore, breeding large-sized, high-fertility Jiaji ducks is a future direction for breed selection, and molecular breeding can accelerate this process; thus, identifying suitable molecular markers is of great importance. Summary of the Invention

[0004] To overcome the aforementioned defects and shortcomings in the existing technology, the present invention provides molecular markers, detection reagents, methods, and applications for breeding Jiaji ducks.

[0005] The first objective of this invention is to provide a molecular marker for breeding Jiaji ducks.

[0006] A second objective of this invention is to provide a detection reagent for the aforementioned molecular markers.

[0007] A third objective of this invention is to provide the application of the aforementioned molecular markers and / or detection reagents in evaluating the growth traits of Jiaji ducks.

[0008] A fourth objective of this invention is to provide the application of the aforementioned molecular markers and / or detection reagents in the preparation of Jiaji duck breeding products.

[0009] The fifth objective of this invention is to provide a kit for breeding Jiaji ducks.

[0010] The sixth objective of this invention is to provide a method for evaluating the growth traits of Jiaji ducks.

[0011] Therefore, the present invention claims protection for the following:

[0012] A molecular marker for breeding Jiaji ducks, said molecular marker consisting of seven single nucleotide polymorphism sites: SNP1, SNP2, SNP3, SNP4, SNP5, SNP6 and / or SNP7.

[0013] SNP1 is located at position 6555465 on the NC_051796.1 gene and is a G or A polymorphism, with three genotypes: GG, GA, and AA.

[0014] SNP2 is located at position 6555516 on the NC_051796.1 gene and is a G or A polymorphism, with three genotypes: GG, GA, and AA.

[0015] SNP3 is located at position 6555632 on the NC_051796.1 gene. It is a C or T polymorphism and has three genotypes: CC, CT, and TT.

[0016] SNP4 is located at position 6555640 on the NC_051796.1 gene. It is a C or A polymorphism and has three genotypes: CC, CA and AA.

[0017] SNP5 is located at position 6555762 on the NC_051796.1 gene and is a G or A polymorphism, with three genotypes: GG, GA, and AA.

[0018] SNP6 is located at position 6555964 on the NC_051796.1 gene. It is a C or T polymorphism and has three genotypes: CC, CT, and TT.

[0019] SNP7 is located at position 6555842 on the NC_051796.1 gene. It is a G or A polymorphism and has three genotypes: GG, GA, and AA.

[0020] The keel length of individuals with the GA genotype at the SNP1 locus was significantly longer than that of individuals with the GG genotype.

[0021] Individuals with the GA genotype at the SNP2 locus had significantly higher keel and tibia lengths than individuals with the AA genotype.

[0022] Individuals with the TT genotype at the SNP3 locus had significantly higher body weight than individuals with the CC or CT genotypes.

[0023] Individuals with the AA genotype at the SNP4 locus had significantly higher body weight than individuals with the CA genotype.

[0024] Individuals with the AA genotype at the SNP5 locus had significantly greater chest depth than individuals with the GG or GA genotypes.

[0025] Individuals with the CC genotype at the SNP6 locus had significantly higher tibia circumferences than those with the CT genotype.

[0026] Individuals with the GG genotype at SNP7 had significantly greater chest depth than those with the GA or AA genotypes, while individuals with the AA genotype had significantly greater body oblique length than those with the GG or GA genotypes.

[0027] The detection reagents for the aforementioned molecular markers are as follows: the detection reagents for SNP1, SNP2, SNP3 and / or SNP4 are primers with nucleotide sequences as shown in SEQ ID NO: 1-2; the detection reagents for SNP5 and / or SNP7 are primers with nucleotide sequences as shown in SEQ ID NO: 3-4; and the detection reagents for SNP6 are primers with nucleotide sequences as shown in SEQ ID NO: 5-6.

[0028] The above-mentioned molecular markers and / or detection reagents are used in evaluating the growth traits of Jiaji ducks.

[0029] A kit for breeding Jiaji ducks contains the above-mentioned detection reagents.

[0030] Preferably, the kit further contains 2× PCR SuperMix and ddH2O.

[0031] A method for evaluating the growth traits of Jiaji ducks, using the aforementioned detection reagents to detect the aforementioned molecular markers.

[0032] Preferably, it includes the following steps:

[0033] S1. Extract genomic DNA from the Jiaji duck to be tested;

[0034] S2. Use the above detection reagents to perform PCR amplification on the genomic DNA obtained in step S1, and then sequence the PCR amplification products to obtain sequencing data;

[0035] S3. Analyze the sequencing data obtained in step S2, and determine the growth traits of the Jiaji duck to be tested based on the molecular markers mentioned above.

[0036] Preferably, the sequencing in step S2 is Sanger sequencing.

[0037] Preferably, the growth traits described in step S3 include keel length, tibia length, body weight, chest depth, tibia circumference, and / or body oblique length.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] This invention discloses molecular markers, detection reagents, methods, and their applications for breeding Jiaji ducks. Seven single nucleotide polymorphism (SNP) sites significantly associated with growth traits in Jiaji ducks were screened in the 5'UTR, intron, and exon regions of the APOC3 gene. These SNP sites can serve as molecular markers for genetic improvement of the Jiaji duck breed. Using these molecular markers for selective breeding of Jiaji ducks is beneficial for improving the growth traits of the population. This invention also provides primers, kits, and breeding methods for detecting these molecular markers, which can be used for early-stage auxiliary selection in Jiaji duck breeding and are of great significance for breeding Jiaji ducks that are more likely to meet market standards. Attached Figure Description

[0040] Figure 1 The results are shown in the agarose gel electrophoresis of the PCR amplification products. Lane 1 is the marker, lanes 2-6 are the PCR amplification results of primers APOC3-P1-F and APOC3-P1-R, lanes 7-11 are the PCR amplification results of primers APOC3-P2-F and APOC3-P2-R, and lanes 12-16 are the PCR amplification results of primers APOC3-P3-F and APOC3-P3-R.

[0041] Figure 2 Sequencing peaks of 11 SNP sites in the APOC3 gene of Jiaji duck.

[0042] Figure 3The images show the mRNA secondary structures of different genotypes of the APOC3 gene; A represents the mRNA secondary structure of the wild-type APOC3 gene; B, D, and F represent localized changes in the mRNA secondary structure of the APOC3 gene caused by mutations at the g.6555465G>A, g.6555513C>T, and g.6555842G>A sites, respectively; C represents a complete change in the mRNA secondary structure caused by a mutation at the g.6555494G>A site; and E represents a mutation at the g.6555842G>A site that does not cause significant changes in the mRNA secondary structure. Detailed Implementation

[0043] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0044] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0045] The example selected 151 healthy adult Jiaji ducks as the research subjects, all of which came from the breeding duck farm of Hainan Chuanwei Muscovy Duck Breeding Co., Ltd.

[0046] The methods for measuring body weight and body size are as specified in "NY / T823-2020 Terminology and Measurement Calculation Methods for Poultry Production Performance".

[0047] Example 1: Amplification of the APOC3 gene in Jiaji ducks

[0048] I. Experimental Methods

[0049] 1. Extraction and purity testing of blood DNA

[0050] Blood was collected from the subwing vein of Jiaji ducks using vacuum blood collection tubes containing EDTA-disodium salt anticoagulant. Whole genomic DNA was extracted from the blood samples according to the instructions of the genomic DNA extraction kit. The quality of the extracted DNA was assessed using 1.0% agarose gel electrophoresis (w / v) and spectrophotometry. Qualified DNA samples were stored at -20℃ for later use.

[0051] 2. Primer design

[0052] Using the duck APOC3 gene sequence (GenBank accession number: NC_051796.1) in the NCBI database as a reference sequence, and utilizing... Viewer software was used to design specific amplification primers for the full gene sequence. The primer sequences are shown in Table 1. The primers were synthesized by Shanghai Bioengineering (Sangon) Technology Service Co., Ltd.

[0053] Table 1 Primer sequences for the APOC3 gene

[0054]

[0055] 3. PCR amplification

[0056] After diluting the DNA samples to the same concentration, 5 μL of each sample was transferred to a 1.5 mL centrifuge tube to prepare a DNA pool.

[0057] Using this as a template, a PCR amplification reaction was performed. The PCR reaction system was: 2× PCR SuperMix 10 μL, forward and reverse primers 1 μL each (10 μmol·L⁻¹) -1 Add 1 μL of genomic DNA template to the pool and bring the total volume to 25 μL with Nuclease-free Water.

[0058] PCR reaction conditions: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 61℃ annealing for 30 s, 72℃ extension for 30 s, 32 cycles; 72℃ final extension for 2 min. PCR products were stored at 4℃ and used for subsequent sequencing.

[0059] The PCR amplification products were detected by agarose gel electrophoresis. If the electrophoretic bands were the same size as the target bands, the obtained PCR products were sent to the company for one-way sequencing.

[0060] II. Experimental Results

[0061] The extracted DNA was analyzed for quality using a spectrophotometer, and the OD value was measured. 260nm OD 280nm All values ​​were between 1.8 and 2.0, indicating that the extracted DNA quality met the standard. Electrophoresis results of the APOC3 gene PCR amplification products using three primer pairs showed that all bands were clear, uncontaminated, and free of impurities, with values ​​of approximately 639, 415, and 666 bp, respectively. Figure 1 All of them were consistent with the expected fragment size, which meets the requirements for directly determining the nucleotide sequence using PCR products.

[0062] Example 2: Detection of APOC3 gene SNP sites in Jiaji ducks

[0063] I. Experimental Methods

[0064] SeqMAN software was used to compare the sequencing results with the reference fragment on NCBI, and the peaks in the sequencing results were observed to screen out mutation sites.

[0065] II. Experimental Results

[0066] Eleven SNPs were found in the APOC3 gene of Jiaji ducks (Table 2). Among them, there were 4 SNPs in the 5'UTR, namely g.6555465G>A, g.6555494G>A, g.6555513C>T, and g.6555516G>A; 2 SNPs in intron 1, namely g.6555632C>T and g.6555640C>A; 2 SNPs in intron 2, namely g.6555762G>A and g.6555787C>T; 1 SNP in exon 2, namely g.6555842G>A, which is a synonymous mutation; and 2 SNPs in intron 3, namely g.6555964C>T and g.6555984G>A. All 11 SNPs have 3 genotypes. Figure 2 ).

[0067] Table 2 Information on APOC3 gene mutation sites in Jiaji ducks

[0068] SNP name Mutation region <![CDATA[Chromosomal locus 1 > Base mutation Encoding amino acids Mutation type g.6555465G>A 5'UTR g.6555465 G→A - - g.6555494G>A 5'UTR g.6555494 G→A - - g.6555513C>T 5'UTR g.6555513 C→T - - g.6555516G>A 5'UTR g.6555516 G→A - - g.6555632C>T Intron1 g.6555632 C→T - - g.6555640C>A Intron1 g.6555640 C→A - - g.6555762G>A Intron2 g.6555762 G→A - - g.6555787C>T Intron2 g.6555787 C→T - - g.6555842G>A Exon2 g.6555842 G→A Thr Synonymous mutation g.6555964C>T Intron3 g.6555964 C→T - - g.6555984G>A Intron3 g.6555984 G→A - -

[0069] Note:" 1 "" indicates that the start site is the first base of chromosome 25 where the duck APOC3 gene (GenBank: NC_051796.1) is located in the NCBI database; "-" indicates that there is no amino acid encoding.

[0070] Example 3: Genetic polymorphism analysis of 11 SNPs in the APOC3 gene of Jiaji ducks

[0071] I. Experimental Methods

[0072] The different genotypes obtained from the sequencing results were used to calculate allele frequency, genotype frequency, genetic heterozygosity, effective number of alleles, and polymorphism information content using Excel software in WPS Office.

[0073] II. Experimental Results

[0074] Table 3 shows that the dominant genotypes of g.6555465G>A, g.6555513C>T, g.6555762G>A, g.6555787C>T, g.6555842G>A, g.6555964C>T, and g.6555984G>A are homozygous wild-type, namely GG (0.768), CC (0.795), GG (0.609), and CC (0.795), respectively. (0.417), GG(0.563), CC(0.682), GG(0.457); the dominant genotypes of g.6555494G>A, g.6555516G>A, g.6555632C>T, and g.6555640C>A are homozygous mutants, namely AA(0.583), AA(0.464), TT(0.457), and AA(0.550).

[0075] Based on the inbreeding coefficient FIS = 1 - (Ho / He), the FIS values ​​of all 11 SNPs were greater than 0, indicating that homozygous types are more adaptable to environmental selection. The polymorphic information content (PIC) of g.6555465G>A and g.6555964C>T were 0.232 and 0.248, respectively, indicating low polymorphism (PIC < 0.25); the remaining loci showed moderate polymorphism (0.25 ≤ PIC < 0.5). P-value tests showed that g.6555762G>A, g.6555842G>A, and g.6555964C>T were in Hardy-Weinberg equilibrium in the Jiaji duck population (P > 0.05), while the other 8 SNPs deviated from Hardy-Weinberg equilibrium (P < 0.05).

[0076] Table 3. Genetic diversity analysis of 11 SNPs in the APOC3 gene of Jiaji duck.

[0077]

[0078]

[0079] Note: PIC ≥ 0.5 indicates high polymorphism, 0.25 ≤ PIC < 0.5 indicates moderate polymorphism, and PIC < 0.25 indicates low polymorphism; the p-value is used to test Hardy-Weinberg equilibrium, and p < 0.05 indicates a deviation from Hardy-Weinberg equilibrium.

[0080] Example 4: Association Analysis of APOC3 Gene Polymorphism with Growth Traits of Jiaji Ducks

[0081] I. Experimental Methods

[0082] One-way ANOVA using IBM SPSS Statistics 27.0 software was used to perform association analysis between different genotypes of APOC3 gene SNPs in Jiaji ducks and growth and slaughter traits. Results are expressed as mean ± standard error, with P < 0.05 indicating statistical significance and P < 0.01 indicating highly statistical significance. For traits with unequal variances, Tamhane's T2 method was used for multiple comparisons.

[0083] II. Experimental Results

[0084] Table 4 shows that 7 out of the 11 SNPs in the APOC3 gene of Jiaji duck have a significant impact on growth traits. Among them, 2 SNPs are located in the 5'UTR (g.6555465G>A and g.6555516G>A), 4 are located in introns (g.6555632C>T, g.6555640C>A, g.6555762G>A and g.6555964C>T), and 1 is located in an exon (g.6555842G>A).

[0085] At the 5'UTR locus, individuals with the GA genotype g.6555465G>A had a significantly longer keel than those with the GG genotype (P<0.05); individuals with the GA genotype g.6555516G>A had a significantly longer keel and tibia than those with the AA genotype (P<0.05).

[0086] At the intron loci, individuals with the TT genotype (g.6555632C>T) had significantly higher body weight than those with the CC and CT genotypes (P<0.05); individuals with the AA genotype (g.6555640C>A) had significantly higher body weight than those with the CA genotype (P<0.05); individuals with the AA genotype (g.6555762G>A) had significantly higher chest depth than those with the GG and GA genotypes (P<0.05); and individuals with the CC genotype (g.6555964C>T) had significantly higher tibia circumference than those with the CT genotype (P<0.05).

[0087] At the g.6555842G>A locus in exon G, individuals with the GG genotype had significantly greater chest depth than those with the GA and AA genotypes (P<0.05), while individuals with the AA genotype had significantly greater body length than those with the GG and GA genotypes (P<0.05). No significant differences in growth traits were observed among the genotypes at other loci (P>0.05).

[0088] Table 4. Association analysis between different APOC3 gene genotypes and growth traits of Jiaji ducks.

[0089]

[0090]

[0091] Note: Different letters in the superscript of data at the same locus in the same column indicate significant differences (P<0.05), while the same letter in the superscript or no letter indicates no significant differences (P>0.05).

[0092] Example 5: Prediction of mRNA secondary structure of different APOC3 gene genotypes

[0093] I. Experimental Methods

[0094] The 5'UTR and exon sequences of the duck (Anas platyrhynchos) APOC3 gene were retrieved from NCBI. Combined with the above SNP test results, the effects of 5'UTR and exon SNP mutations on the secondary structure of duck APOC3 gene mRNA were analyzed using the RNAfold WebServer online website.

[0095] II. Experimental Results

[0096] mRNA secondary structure prediction was performed on wild-type APOC3 gene and mutant SNPs in the 5'UTR and exon regions. The results are as follows: Figure 3 As shown. Comparing the secondary structure of the wild-type APOC3 mRNA, the mutation at the g.6555516G>A site did not cause significant structural changes, while mutations at the other four SNPs all led to structural alterations. Among these, the mutation at the g.6555494G>A site had the greatest impact on the mRNA secondary structure, resulting in an overall structural change, while mutations at the other three sites only caused localized changes in the mRNA secondary structure.

[0097] As shown in Table 5, compared with the wild-type APOC3 mRNA minimum free energy of -276.02 kcal / mol, the mutations g.6555465G>A and g.6555494G>A decreased the minimum free energy of mRNA secondary structure by 0.52 kcal / mol and 0.61 kcal / mol, respectively; while the mutations g.6555513C>T, g.6555516G>A, and g.6555842G>A increased the minimum free energy by 1.64 kcal / mol, 0.02 kcal / mol, and 2.1 kcal / mol, respectively. The mutation at the g.6555516G>A site had a negligible impact on the free energy of the mRNA secondary structure, consistent with the predicted changes in mRNA secondary structure.

[0098] Table 5 Free energy of mRNA secondary structure before and after mutation.

[0099]

[0100]

[0101] Changes in mRNA secondary structure play a crucial role in species evolution. Alterations in mRNA secondary structure primarily affect two aspects. First, changes in its structure influence ribosome recognition and binding to mRNA. If the secondary structure obstructs ribosome binding sites, it reduces translation initiation efficiency and affects the speed of ribosome movement on the mRNA, thus altering the rate and efficiency of translation. Second, the stability of the secondary structure affects its duration of presence within the cell, thereby influencing protein expression levels.

[0102] This application found that the G>A mutation at site g.6555465 alters the mRNA secondary structure and decreases the minimum free energy (from -276.02 kcal / mol to -276.54 kcal / mol), significantly affecting the keel length trait of Jiaji ducks; the G>A mutation at site g.6555842 alters the mRNA secondary structure and increases the minimum free energy (from -276.02 kcal / mol to -273.92 kcal / mol), significantly affecting the chest depth and body oblique length trait of Jiaji ducks. It is speculated that these two sites regulate the growth traits of Jiaji ducks by affecting the duration of mRNA presence in cells and translation efficiency. However, the G>A mutation at site g.6555516 has little effect on mRNA secondary structure and free energy (from -276.02 kcal / mol to -276.00 kcal / mol), but significantly affects the keel length trait of Jiaji ducks. Analysis suggests that these SNPs may regulate the growth traits of Jiaji ducks by altering the microRNA binding site on mRNA, thereby affecting the translation of the APOC3 gene.

[0103] Example 6: A method for breeding Jiaji ducks

[0104] Single nucleotide polymorphism sites:

[0105] SNP1 is located at position 6555465 on the NC_051796.1 gene and is a G or A polymorphism, with three genotypes: GG, GA, and AA.

[0106] SNP2 is located at position 6555516 on the NC_051796.1 gene and is a G or A polymorphism, with three genotypes: GG, GA, and AA.

[0107] SNP3 is located at position 6555632 on the NC_051796.1 gene. It is a C or T polymorphism and has three genotypes: CC, CT, and TT.

[0108] SNP4 is located at position 6555640 on the NC_051796.1 gene. It is a C or A polymorphism and has three genotypes: CC, CA and AA.

[0109] SNP5 is located at position 6555762 on the NC_051796.1 gene and is a G or A polymorphism, with three genotypes: GG, GA, and AA.

[0110] SNP6 is located at position 6555964 on the NC_051796.1 gene. It is a C or T polymorphism and has three genotypes: CC, CT, and TT.

[0111] SNP7 is located at position 6555842 on the NC_051796.1 gene. It is a G or A polymorphism and has three genotypes: GG, GA, and AA.

[0112] 1. Extract DNA from the sample to be tested using a genomic DNA extraction kit.

[0113] 2. Using the DNA extracted from the sample in step 1 as a template, perform PCR amplification of SNP1 to SNP7 single nucleotide polymorphism sites;

[0114] The PCR reaction system is: 2× PCR SuperMix 10 μL, primers with nucleotide sequences as shown in SEQ ID NO: 1-6, 1 μL each (10 μmol·L⁻¹) -1 ), 1 μL of DNA template, add Nuclease-free Water to 25 μL;

[0115] The primers shown in SEQ ID NO: 1-2 are used to detect single nucleotide polymorphism sites of SNP1, SNP2, SNP3 and SNP4, the primers shown in SEQ ID NO: 3-4 are used to detect single nucleotide polymorphism sites of SNP5 and SNP7, and the primers shown in SEQ ID NO: 5-6 are used to detect single nucleotide polymorphism site of SNP6.

[0116] PCR reaction conditions: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 61℃ annealing for 30 s, 72℃ extension for 30 s, 32 cycles; 72℃ final extension for 2 min.

[0117] 3. Perform agarose gel electrophoresis on the PCR amplification products obtained in step 2. If the electrophoretic bands are the same size as the target bands and are clear and bright, then sequence the PCR amplification products obtained in step 2.

[0118] 4. Result Interpretation:

[0119] The keel length of individuals with the GA genotype at the SNP1 locus was significantly higher than that of individuals with the GG genotype.

[0120] Individuals with the GA genotype at the SNP2 locus had significantly higher keel and tibia lengths than those with the AA genotype.

[0121] Individuals with the TT genotype at the SNP3 locus have significantly higher body weight than those with the CC or CT genotype;

[0122] Individuals with the AA genotype at the SNP4 locus had significantly higher body weight than those with the CA genotype.

[0123] Individuals with the AA genotype at the SNP5 locus had significantly greater chest depth than those with the GG or GA genotypes.

[0124] Individuals with the CC genotype at the SNP6 locus had significantly higher tibia circumferences than those with the CT genotype.

[0125] Individuals with the GG genotype at SNP7 locus had significantly greater chest depth than those with the GA or AA genotypes; individuals with the AA genotype had significantly greater body oblique length than those with the GG or GA genotypes.

[0126] Example 7: A kit for breeding Jiaji ducks

[0127] I. Composition

[0128] Nucleotide sequences such as primers shown in SEQ ID NO: 1-6, PCR SuperMix and Nuclease-free Water.

[0129] II. Instructions for Use

[0130] The detection and result interpretation were carried out in accordance with Example 6.

[0131] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a detection reagent based on a molecular marker composition in evaluating the growth traits of Jiaji ducks, characterized in that, The molecular marker composition comprises one or more single nucleotide polymorphism sites SNP2, SNP3, SNP4, and / or SNP5. composition: SNP2 is located at position 6555516 on the NC_051796.1 gene and is a G or A polymorphism, with three genotypes: GG, GA, and AA. SNP3 is located at position 6555632 on the NC_051796.1 gene. It is a C or T polymorphism and has three genotypes: CC, CT, and TT. SNP4 is located at position 6555640 on the NC_051796.1 gene. It is a C or A polymorphism and has three genotypes: CC, CA and AA. SNP5 is located at position 6555762 on the NC_051796.1 gene and is a G or A polymorphism, with three genotypes: GG, GA, and AA. Individuals with the GA genotype at the SNP2 locus had significantly higher keel and tibia lengths than individuals with the AA genotype. Individuals with the TT genotype at the SNP3 locus had significantly higher body weight than individuals with the CC or CT genotypes. Individuals with the AA genotype at the SNP4 locus had significantly higher body weight than individuals with the CA genotype. Individuals with the AA genotype at the SNP5 locus had significantly greater chest depth than individuals with the GG or GA genotypes. The detection reagents for SNP2, SNP3 and / or SNP4 are primers with nucleotide sequences as shown in SEQ ID NO: 1-2, and the detection reagent for SNP5 is primers with nucleotide sequences as shown in SEQ ID NO: 3-4.

2. The application of the detection reagent of the molecular marker composition in the preparation of Jiaji duck breeding products, characterized in that, The molecular marker composition comprises one or more single nucleotide polymorphism sites SNP2, SNP3, SNP4, and / or SNP5. composition: SNP2 is located at position 6555516 on the NC_051796.1 gene and is a G or A polymorphism, with three genotypes: GG, GA, and AA. SNP3 is located at position 6555632 on the NC_051796.1 gene. It is a C or T polymorphism and has three genotypes: CC, CT, and TT. SNP4 is located at position 6555640 on the NC_051796.1 gene. It is a C or A polymorphism and has three genotypes: CC, CA and AA. SNP5 is located at position 6555762 on the NC_051796.1 gene and is a G or A polymorphism, with three genotypes: GG, GA, and AA. Individuals with the GA genotype at the SNP2 locus had significantly higher keel and tibia lengths than individuals with the AA genotype. Individuals with the TT genotype at the SNP3 locus had significantly higher body weight than individuals with the CC or CT genotypes. Individuals with the AA genotype at the SNP4 locus had significantly higher body weight than individuals with the CA genotype. Individuals with the AA genotype at the SNP5 locus had significantly greater chest depth than individuals with the GG or GA genotypes. The detection reagents for SNP2, SNP3 and / or SNP4 are primers with nucleotide sequences as shown in SEQ ID NO: 1-2, and the detection reagent for SNP5 is primers with nucleotide sequences as shown in SEQ ID NO: 3-4.

3. A method for evaluating the growth traits of Jiaji ducks, characterized in that, Detection of molecular marker compositions using detection reagents; The molecular marker composition comprises one or more single nucleotide polymorphism sites SNP2, SNP3, SNP4, and / or SNP5. composition: SNP2 is located at position 6555516 on the NC_051796.1 gene and is a G or A polymorphism, with three genotypes: GG, GA, and AA. SNP3 is located at position 6555632 on the NC_051796.1 gene. It is a C or T polymorphism and has three genotypes: CC, CT, and TT. SNP4 is located at position 6555640 on the NC_051796.1 gene. It is a C or A polymorphism and has three genotypes: CC, CA and AA. SNP5 is located at position 6555762 on the NC_051796.1 gene and is a G or A polymorphism, with three genotypes: GG, GA, and AA. Individuals with the GA genotype at the SNP2 locus had significantly higher keel and tibia lengths than individuals with the AA genotype. Individuals with the TT genotype at the SNP3 locus had significantly higher body weight than individuals with the CC or CT genotypes. Individuals with the AA genotype at the SNP4 locus had significantly higher body weight than individuals with the CA genotype. Individuals with the AA genotype at the SNP5 locus had significantly greater chest depth than individuals with the GG or GA genotypes. The detection reagents for SNP2, SNP3 and / or SNP4 are primers with nucleotide sequences as shown in SEQ ID NO: 1-2, and the detection reagent for SNP5 is primers with nucleotide sequences as shown in SEQ ID NO: 3-4.

4. The method according to claim 3, characterized in that, Includes the following steps: S1. Extract genomic DNA from the Jiaji ducks to be tested; S2. Perform PCR amplification on the genomic DNA obtained in step S1 using detection reagents, and then sequence the PCR amplification products to obtain sequencing data; S3. Analyze the sequencing data obtained in step S2 and determine the growth traits of the Jiaji duck to be tested based on the molecular marker composition.

5. The method according to claim 4, characterized in that, The sequencing described in step S2 is Sanger sequencing.

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