Molecular marker, detection reagent and method for breeding Jiajian ducks and application of molecular marker, detection reagent and method

Abstract

The invention discloses a molecular marker, a detection reagent and a method for breeding jaboticaba ducks and application of the molecular marker and the detection reagent. According to the invention, seven single nucleotide polymorphic sites obviously related to the growth traits of Jiajian ducks are respectively screened in a 5 'UTR region, an intron region and an exon region of the Jiajian duck APOC3 gene, and can be used as molecular markers for genetic improvement of Jiajian duck varieties. By adopting the molecular markers to breed Jiajian ducks, the growth traits of Jiajian duck groups can be improved. The invention also provides a detection primer, a kit and a breeding method for detecting the molecular markers, can be used for early auxiliary selection of Jiajian duck breeding, and has important significance for breeding Jiajian ducks which are easier to meet marketing standards.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular breeding, and in particular to molecular markers, detection reagents, methods and applications thereof for Jiaji duck breeding. Background Art

[0002] The apolipoprotein (APO) superfamily is a group of proteins that play a key role in lipid metabolism. They can bind to blood lipids such as cholesterol and triglycerides to form lipoproteins, thereby transporting lipids to various tissues and organs in the body. According to the protein structure and lipid binding characteristics of each member of the APO superfamily, it is divided into APOA, APOB, APOC, APOE, and APOH families. APOC3 belongs to the APOC family. It is mainly synthesized and secreted by the liver, and a small part is synthesized in the intestine. It is a small molecular weight extracellular protein and mainly exists in triglyceride-rich lipoproteins such as very low density lipoproteins, high density lipoproteins and chylomicrons in plasma. APOC3 can reduce the body's hydrolysis of TC in plasma by inhibiting the activity of enzymes such as lipoprotein lipase and hepatic lipase on the surface of vascular endothelial cells, and participate in regulating the body's lipid metabolism. After infecting C57BL / 6 mice with adenovirus expressing human APOC3, it was found that APOC3 can regulate the structure and function of high density lipoproteins, and at the same time can selectively promote the body's energy metabolism of activating brown adipose tissue. APOC3-deficient mice were induced with a high-fat diet and found to have a higher sensitivity to obesity than normal mice. This indicates that the APOC3 gene can be used as one of the potential targets for the treatment of obesity and metabolic-related diseases. In addition, studies on livestock have shown that the amount of intramuscular fat deposition in cola pigs is linearly positively correlated with the transcription level of APOC3. Goat APOC3 can promote the differentiation of IMF cells by upregulating SREBP1, CEBPβ, PPARγ and downregulating Pref-1. At present, research on the APOC3 gene is mostly focused on human cardiovascular diseases, while there are few reports on poultry research.

[0003] Jiaji duck is an excellent meat duck breed in Hainan Province, with strong resistance to heat and humidity, roughage and other stresses. Its meat has a unique flavor, large breast, thin skin, soft bones, tender meat and rich taste. Jiaji duck is deeply loved by consumers at home and abroad, and is also listed as one of the varieties protected by global genetic diversity. However, as one of Hainan's "four famous dishes", Jiaji duck still has a large gap in industrial scale compared with Wenchang chicken, and its annual output is only more than 2 million. According to market surveys, animals with larger weight and good body shape are often more popular and easier to meet the standards for market release. Therefore, breeding Jiaji ducks with large body and high reproduction rate is a direction for future variety breeding, and molecular breeding can accelerate the breeding process, so it is of great significance to screen out suitable molecular markers. Summary of the invention

[0004] In order to overcome the above-mentioned defects and shortcomings in the prior art, the present invention provides molecular markers, detection reagents, methods and applications thereof for Jiaji duck breeding.

[0005] The first object of the present invention is to provide a molecular marker for Jiaji duck breeding.

[0006] The second object of the present invention is to provide a detection reagent for the above-mentioned molecular markers.

[0007] The third object of the present invention is to provide the use of the above-mentioned molecular markers and / or detection reagents in evaluating the growth traits of Jiaji ducks.

[0008] The fourth object of the present invention is to provide the use of the above-mentioned molecular markers and / or detection reagents in the preparation of Jiaji duck breeding products.

[0009] A fifth object of the present invention is to provide a test kit for Jiaji duck breeding.

[0010] The sixth object of the present invention is to provide a method for evaluating the growth traits of Jiaji duck.

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

[0012] A molecular marker for Jiaji duck breeding, the molecular marker consisting of 7 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 and is a C or T polymorphism, with three genotypes: CC, CT, and TT;

[0016] SNP4 is located at position 6555640 on the NC_051796.1 gene and is a C or A polymorphism, with 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 and is a C or T polymorphism, with three genotypes: CC, CT, and TT;

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

[0020] The keel length of individuals with GA genotype at SNP1 was significantly higher than that of individuals with GG genotype;

[0021] The keel length and tibia length of individuals with GA genotype at SNP2 were significantly higher than those with AA genotype.

[0022] The body weight of individuals with TT genotype at SNP3 was significantly higher than that of individuals with CC or CT genotype;

[0023] The body weight of individuals with AA genotype at SNP4 was significantly higher than that of individuals with CA genotype.

[0024] The chest depth of individuals with AA genotype at SNP5 was significantly higher than that of individuals with GG or GA genotype.

[0025] The tibia circumference of individuals with CC genotype at SNP6 was significantly higher than that of individuals with CT genotype.

[0026] The chest depth of individuals with GG genotype at SNP7 was significantly higher than that of individuals with GA or AA genotype, and the body oblique length of individuals with AA genotype was significantly higher than that of individuals with GG or GA genotype.

[0027] The above-mentioned molecular marker detection reagents, the detection reagents for SNP1, SNP2, SNP3 and / or SNP4 are primers with nucleotide sequences as shown in SEQ ID NOs: 1 to 2, the detection reagents for SNP5 and / or SNP7 are primers with nucleotide sequences as shown in SEQID NOs: 3 to 4, and the detection reagents for SNP6 are primers with nucleotide sequences as shown in SEQ ID NOs: 5 to 6.

[0028] Application of the above-mentioned molecular markers and / or detection reagents in evaluating the growth traits of Jiaji duck.

[0029] A kit for Jiaji duck breeding contains the above-mentioned detection reagent.

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

[0031] A method for evaluating the growth traits of Jiaji duck, using the above-mentioned detection reagent to detect the above-mentioned molecular markers.

[0032] Preferably, the steps include:

[0033] S1. Extracting genomic DNA of Jiaji duck to be tested;

[0034] S2. Performing PCR amplification on the genomic DNA obtained in step S1 using the above-mentioned detection reagent, and then sequencing the PCR amplification product 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 above-mentioned molecular markers.

[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] The present invention discloses molecular markers, detection reagents, methods and applications for Jiaji duck breeding. The present invention screens 7 single nucleotide polymorphism sites significantly associated with the growth traits of Jiaji duck in the 5'UTR region, intron region and exon region of the APOC3 gene of Jiaji duck, which can be used as molecular markers for genetic improvement of Jiaji duck varieties. The use of these molecular markers to select and breed Jiaji ducks is conducive to improving the growth traits of the Jiaji duck population. The present invention also provides detection primers, kits and breeding methods for detecting these molecular markers, which can be used for early auxiliary selection of Jiaji duck breeding, and are of great significance for breeding Jiaji ducks that are more likely to meet the market standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Are the agarose gel electrophoresis results of PCR amplification products, where lane 1 is the Marker, lanes 2 to 6 are the PCR product amplification results of primers APOC3-P1-F and APOC3-P1-R, lanes 7 to 11 are the PCR product amplification results of primers APOC3-P2-F and APOC3-P2-R, and lanes 12 to 16 are the PCR product amplification results of primers APOC3-P3-F and APOC3-P3-R.

[0041] Figure 2 This is the sequencing peak diagram of 11 SNPs sites in the APOC3 gene of Jiaji duck.

[0042] Figure 3Figure 3 is the mRNA secondary structure of APOC3 gene in different genotypes; A is the mRNA secondary structure of wild-type APOC3 gene; B, D and F are the mutations at g.6555465G>A, g.6555513C>T and g.6555842G>A, respectively, which cause local changes in the mRNA secondary structure of APOC3 gene; C is the mutation at g.6555494G>A, which causes a complete change in the mRNA secondary structure; E is the mutation at g.6555842G>A, which does not cause obvious changes in the mRNA secondary structure. DETAILED DESCRIPTION

[0043] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

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

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

[0046] The measurement methods of body weight and body size refer to "NY / T823-2020 Poultry Production Performance Terminology and Measurement Calculation Methods".

[0047] Example 1 Amplification of Jiaji duck APOC3 gene

[0048] 1. Experimental Methods

[0049] 1. Blood DNA extraction and purity testing

[0050] Blood was collected from the subwing vein of Jiaji duck using a vacuum blood collection tube with EDTA-disodium salt anticoagulant, and whole genome DNA was extracted from the Jiaji duck blood sample according to the instructions of the genomic DNA extraction kit. The quality of the extracted DNA was evaluated using 1.0% agarose gel (w / v) electrophoresis and a spectrophotometer, and qualified DNA samples were stored at -20°C for future use.

[0051] 2. Primer Design

[0052] The duck APOC3 gene sequence in the NCBI database (GenBank accession number: NC_051796.1) was used as the reference sequence. Specific amplification primers were designed based on the full gene sequence using the PCR Viewer software. The primer sequences are shown in Table 1. The primers were synthesized by Shanghai Bioengineering (Shengong) Technology Service Co., Ltd.

[0053] Table 1 Primer sequences of APOC3 gene

[0054]

[0055] 3. PCR Amplification

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

[0057] This was used as a template for PCR amplification reaction. The PCR reaction system was: 2× PCR SuperMix 10 μL, upstream and downstream primers 1 μL each (10 μmol·L -1 ), mix 1 μL of genomic DNA template and add Nuclease-free Water to 25 μL.

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

[0059] The PCR amplification products were tested by agarose gel electrophoresis. If the electrophoresis band was consistent with the target band in size, the obtained PCR products were sent to the company for unidirectional sequencing.

[0060] 2. Experimental Results

[0061] The quality of the extracted DNA was tested by spectrophotometer, OD 260nm :OD 280nm All of them were between 1.8 and 2.0, indicating that the quality of the extracted DNA met the standard. The electrophoresis results of the PCR amplification products of the APOC3 gene with three pairs of primers showed that all bands were clear and free of contamination and foreign bands, approximately 639, 415 and 666 bp ( Figure 1 ), which were consistent with the expected fragment sizes, meeting the requirements for directly determining the nucleotide sequence using PCR products.

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

[0063] 1. Experimental Methods

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

[0065] 2. Experimental Results

[0066] A total of 11 SNPs were found in the APOC3 gene of Jiaji duck (Table 2), including 4 SNPs in 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. These 11 SNPs have three genotypes ( Figure 2 ).

[0067] Table 2 Mutation site information of APOC3 gene in Jiaji duck

[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 ” refers to the first base of chromosome 25 where duck APOC3 gene (GenBank: NC_051796.1) in NCBI database is located as the starting site; “-” means no amino acid coding.

[0070] Example 3 Genetic polymorphism analysis of 11 SNPs of APOC3 gene in Jiaji duck

[0071] 1. Experimental Methods

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

[0073] 2. Experimental Results

[0074] As shown in Table 3, 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 types, which are GG (0.768), CC (0.795), GG (0.609), and CC (0.417), GG(0.563), CC(0.682), GG(0.457); the dominant genotypes of g.6555494G>A, g.6555516G>A, g.6555632C>T, g.6555640C>A were homozygous mutant types, namely AA(0.583), AA(0.464), TT(0.457), AA(0.550).

[0075] According to the inbreeding coefficient FIS = 1-(Ho / He), the FIS of 11 SNPs were all greater than 0, indicating that the homozygous type was 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, showing low polymorphism (PIC<0.25); the other loci showed moderate polymorphism (0.25≤PIC<0.5). According to the P value test, g.6555762G>A, g.6555842G>A, and g.6555964C>T were in Hardy-Weinberg equilibrium in Jiaji duck population (P>0.05), and the other 8 SNPs deviated from Hardy-Weinberg equilibrium (P<0.05).

[0076] Table 3 Genetic diversity analysis of 11 SNPs in 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. P value was used to test Hardy-Weinberg equilibrium, and P < 0.05 indicated deviation from Hardy-Weinberg equilibrium.

[0080] Example 4 Association analysis between APOC3 gene polymorphism and growth traits of Jiaji duck

[0081] 1. Experimental Methods

[0082] One-way ANOVA in IBM SPSS Statistics 27.0 software was used to analyze the association between different genotypes of APOC3 gene SNPs and growth and slaughter traits of Jiaji duck. The results were expressed as "mean ± standard error". P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference. Tamhane's T2 method was used for multiple comparisons of traits with uneven variance.

[0083] 2. Experimental Results

[0084] As shown in Table 4, 7 out of the 11 SNPs in the APOC3 gene of Jiaji duck had significant effects on growth traits, of which 2 SNPs were located in 5'UTR (g.6555465G>A and g.6555516G>A), 4 were located in introns (g.6555632C>T, g.6555640C>A, g.6555762G>A and g.6555964C>T), and 1 was located in an exon (g.6555842G>A).

[0085] At the site located in 5'UTR, the keel length of individuals with GA genotype (g.6555465G>A) was significantly longer than that of individuals with GG genotype (P<0.05); the keel length and tibia length of individuals with GA genotype (g.6555516G>A) were significantly longer than those of individuals with AA genotype (P<0.05).

[0086] At the sites located in the intron, the weight of individuals with TT genotype (g.6555632C>T) was significantly higher than that of CC and CT types (P<0.05); the weight of individuals with AA genotype (g.6555640C>A) was significantly higher than that of CA type (P<0.05); the chest depth of individuals with AA genotype (g.6555762G>A) was significantly higher than that of GG and GA types (P<0.05); the tibia circumference of individuals with CC genotype (g.6555964C>T) was significantly higher than that of individuals with CT genotype (P<0.05).

[0087] At the g.6555842G>A site located in the exon, the chest depth of the GG genotype individuals was significantly higher than that of the GA and AA types (P<0.05), and the body length of the AA genotype individuals was significantly higher than that of the GG and GA types (P<0.05). There were no significant differences in growth traits among the genotypes at the other sites (P>0.05).

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

[0089]

[0090]

[0091] Note: Data in the same column and at the same site with different letters on their shoulders indicate significant differences (P<0.05), while data with the same letters on their shoulders or no letters on their shoulders indicate no significant differences (P>0.05).

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

[0093] 1. Experimental Methods

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

[0095] 2. Experimental Results

[0096] The mRNA secondary structure prediction was performed for the wild type of APOC3 gene and the mutant types of SNPs in the 5'UTR and exon regions. The results are as follows Figure 3 As shown. Compared with the wild-type mRNA secondary structure of the APOC3 gene, the mutation of the g.6555516G>A site did not cause obvious changes in the structure, while the mutations of the other four SNPs all caused structural changes. Among them, the mutation of the g.6555494G>A site had the greatest impact on the mRNA secondary structure, resulting in changes in the overall structure, while the mutations of the other three sites only caused local changes in the mRNA secondary structure.

[0097] As shown in Table 5, compared with the minimum free energy of the wild-type mRNA secondary structure of APOC3 gene (-276.02 kcal / mol), the mutations of g.6555465G>A and g.6555494G>A reduced the minimum free energy of the mRNA secondary structure by 0.52 kcal / mol and 0.61 kcal / mol, respectively; the mutations of 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. Among them, the mutation of g.6555516G>A site had little effect on the free energy of the mRNA secondary structure of the gene, which was consistent with the predicted change degree of the mRNA secondary structure.

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

[0099]

[0100]

[0101] Changes in the secondary structure of mRNA play an important role in the evolution of species. Changes in the secondary structure of mRNA have two main effects. First, changes in its structure will affect the recognition and binding of ribosomes to mRNA. If the secondary structure hinders the ribosome binding site, it will reduce the efficiency of translation initiation and affect the speed of ribosome movement on mRNA, thereby changing the speed and efficiency of translation. Secondly, the stability of the secondary structure will affect its existence time in the cell, thereby affecting the expression of the protein.

[0102] The present application found that the site g.6555465G>A mutation will change the mRNA secondary structure and reduce the minimum free energy (from -276.02kcal / mol to -276.54kcal / mol), significantly affecting the keel length trait of Jiaji duck; the site g.6555842G>A mutation will change the mRNA secondary structure and increase the minimum free energy (from -276.02kcal / mol to -273.92kcal / mol), significantly affecting the chest depth and body oblique length traits of Jiaji duck. It is speculated that the above two sites regulate the growth traits of Jiaji duck by affecting the existence time and translation efficiency of mRNA in cells. The site g.6555516G>A mutation has little effect on the mRNA secondary structure and free energy (from -276.02kcal / mol to -276.00kcal / mol), but has a significant effect on the keel length trait of Jiaji duck. Through analysis, it was found that the SNPs may affect the translation of APOC3 gene by changing the binding site of microRNA on mRNA and then regulate the growth traits of Jiaji duck.

[0103] Example 6 A method for breeding Jiaji ducks

[0104] Single nucleotide polymorphism site:

[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 and is a C or T polymorphism, with three genotypes: CC, CT, and TT;

[0108] SNP4 is located at position 6555640 on the NC_051796.1 gene and is a C or A polymorphism, with 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 and is a C or T polymorphism, with three genotypes: CC, CT, and TT;

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

[0112] 1. Use a genomic DNA extraction kit to extract the DNA of the sample to be tested.

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

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

[0115] The primers shown in nucleotide sequences SEQ ID NOs: 1 to 2 are used to detect single nucleotide polymorphism sites of SNP1, SNP2, SNP3 and SNP4, the primers shown in nucleotide sequences SEQ ID NOs: 3 to 4 are used to detect single nucleotide polymorphism sites of SNP5 and SNP7, and the primers shown in nucleotide sequences SEQ ID NOs: 5 to 6 are used to detect single nucleotide polymorphism sites of SNP6;

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

[0117] 3. Perform agarose gel electrophoresis on the PCR amplification product obtained in step 2. If the electrophoresis band is consistent with the target band in size and is clear and bright, sequence the PCR amplification product obtained in step 2.

[0118] 4. Result judgment:

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

[0120] The keel length and tibia length of individuals with GA genotype at SNP2 were significantly higher than those with AA genotype.

[0121] The body weight of individuals with TT genotype at SNP3 was significantly higher than that of individuals with CC or CT genotype;

[0122] The body weight of individuals with AA genotype at SNP4 was significantly higher than that of individuals with CA genotype.

[0123] The chest depth of individuals with AA genotype at SNP5 was significantly higher than that of individuals with GG or GA genotype.

[0124] The tibia circumference of individuals with CC genotype at SNP6 was significantly higher than that of individuals with CT genotype.

[0125] The chest depth of individuals with GG genotype at SNP7 was significantly higher than that of individuals with GA or AA genotype; the body oblique length of individuals with AA genotype was significantly higher than that of individuals with GG or GA genotype.

[0126] Example 7 A kit for breeding Jiaji ducks

[0127] 1. Composition

[0128] The primers whose nucleotide sequences are shown in SEQ ID NOs: 1 to 6, PCR SuperMix and Nuclease-free Water.

[0129] 2. Usage

[0130] The detection and result interpretation were performed according to Example 6.

[0131] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A molecular marker for Jiaji duck breeding, characterized in that: The molecular markers are composed of 7 single nucleotide polymorphism sites SNP1, SNP2, SNP3, SNP4, SNP5, SNP6 and / or SNP7. composition: 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; 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 and is a C or T polymorphism, with three genotypes: CC, CT, and TT; SNP4 is located at position 6555640 on the NC_051796.1 gene and is a C or A polymorphism, with 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; SNP6 is located at position 6555964 on the NC_051796.1 gene and is a C or T polymorphism, with three genotypes: CC, CT, and TT; SNP7 is located at position 6555842 on the NC_051796.1 gene and is a G or A polymorphism, with three genotypes: GG, GA, and AA; The keel length of individuals with GA genotype at SNP1 was significantly higher than that of individuals with GG genotype; The keel length and tibia length of individuals with GA genotype at SNP2 were significantly higher than those with AA genotype. The body weight of individuals with TT genotype at SNP3 was significantly higher than that of individuals with CC or CT genotype; The body weight of individuals with AA genotype at SNP4 was significantly higher than that of individuals with CA genotype. The chest depth of individuals with AA genotype at SNP5 was significantly higher than that of individuals with GG or GA genotype. The tibia circumference of individuals with CC genotype at SNP6 was significantly higher than that of individuals with CT genotype. The chest depth of individuals with GG genotype at SNP7 was significantly higher than that of individuals with GA or AA genotype, and the body oblique length of individuals with AA genotype was significantly higher than that of individuals with GG or GA genotype.

2. The molecular marker detection reagent according to claim 1, characterized in that The detection reagents for SNP1, SNP2, SNP3 and / or SNP4 are primers with nucleotide sequences as shown in SEQ ID NOs: 1 to 2, the detection reagents for SNP5 and / or SNP7 are primers with nucleotide sequences as shown in SEQ ID NOs: 3 to 4, and the detection reagents for SNP6 are primers with nucleotide sequences as shown in SEQ ID NOs: 5 to 6.

3. Use of the molecular marker described in claim 1 and / or the detection reagent described in claim 2 in evaluating the growth traits of Jiaji duck.

4. Use of the molecular marker described in claim 1 and / or the detection reagent described in claim 2 in the preparation of Jiaji duck breeding products.

5. A kit for Jiaji duck breeding, characterized in that: Contains the detection reagent according to claim 2.

6. The kit according to claim 5, characterized in that The kit also contains 2× PCR Super Mix and ddH2O.

7. A method for evaluating the growth traits of Jiaji duck, characterized in that: The molecular marker of claim 1 is detected using the detection reagent of claim 2.

8. The method according to claim 7, characterized in that The following steps are involved: S1. Extracting genomic DNA of Jiaji duck to be tested; S2. Performing PCR amplification on the genomic DNA obtained in step S1 using the detection reagent described in claim 2, and then sequencing the PCR amplification product to obtain sequencing data; S3. Analyze the sequencing data obtained in step S2, According to the molecular marker described in claim 1, the growth traits of the Jiaji duck to be tested are determined.

9. The method according to claim 8, characterized in that The sequencing described in step S2 is Sanger sequencing.

10. The method according to claim 8, characterized in that The growth traits described in step S3 include keel length, tibia length, body weight, chest depth, tibia circumference and / or body oblique length.

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