Detection reagents, kits, detection methods and applications of SNP molecular markers related to body shape traits in western Guangdong Dalmatian chickens

Abstract

This invention discloses detection reagents, kits, detection methods, and applications for SNP molecular markers related to the body shape traits of the Guangdong Western Dalmatian chicken. By analyzing SNP sites on the IGF2BP3 gene of the Guangdong Western Dalmatian chicken, this invention identified eight SNP sites significantly associated with the body shape traits. Detection of the IGF2BP3 gene in the Guangdong Western Dalmatian chicken allows for rapid, effective, and accurate early prediction of the chicken's weight and body shape. This enables the selection of Guangdong Western Dalmatian chickens with different body shape traits according to breeding needs, thereby shortening breeding time and accelerating the breeding process. This invention provides primers, kits, and methods for detecting SNP sites on the IGF2BP3 gene of the Guangdong Western Dalmatian chicken, showing broad application prospects in improving the genetic breeding efficiency of the Guangdong Western Dalmatian chicken.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and more specifically, to detection reagents, kits, detection methods, and applications of SNP molecular markers related to body shape traits of the Guangdong West Dalmatian chicken. Background Technology

[0002] With the continuous development of China's economy and the continuous improvement of people's living standards, China's poultry farming industry has developed rapidly, and its comprehensive production capacity has been significantly strengthened. The increasing demand for meat and dairy products has driven the rapid development of the poultry farming industry. my country has abundant poultry breed resources, a large-scale poultry farming industry, and rapid industrial development. Chicken has become the second largest source of meat consumption for residents, and consumer demand for chicken has shifted from quantity to quality. However, local breeds are underutilized due to their long breeding cycle, poor meat quality, low slaughter rate, and low economic benefits. Therefore, the main goal of breeding at this stage is to improve chicken breeds and enhance their production performance.

[0003] The Western Guangdong Curly-feathered Chicken is a local specialty chicken, mainly produced in Maoming and Zhanjiang areas of Guangdong Province. It gets its name from its upward-curling feathers. Its feathers, feet, and skin are yellow, exhibiting typical characteristics of the "Three Yellow Chicken" (a type of yellow-feathered chicken). It is known for its tolerance to roughage, strong disease resistance, and relatively large size, while also boasting relatively high survival and slaughter rates.

[0004] Members of the insulin-like growth factor 2 mRNA-binding protein family IGF2BP3 It is a typical multi-domain RNA-binding protein, exhibiting specificity and diversity in target recognition, and providing a good example of multivalent interactions among multi-domain RNA-binding proteins. Currently, regarding... IGF2BP3 Research on genetic polymorphism has mainly focused on human diseases, with relatively little research on poultry, and no literature reports on the local breed, the Western Guangdong Dwarf Chicken. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide detection reagents, kits, detection methods and their applications for SNP molecular markers related to the body shape traits of Dalmatian chickens in western Guangdong.

[0006] The first objective of this invention is to provide a detection reagent for detecting molecular markers in the Dalmatian chicken of western Guangdong, and its application in the detection of body shape traits in this chicken.

[0007] The second objective of this invention is to provide a kit for detecting the body shape characteristics of the Dalmatian chicken from western Guangdong.

[0008] The third objective of this invention is to provide a method for detecting the body shape characteristics of the Dalmatian chicken from western Guangdong.

[0009] A fourth objective of this invention is to provide the application of the aforementioned detection reagent, the aforementioned kit, and / or the method described therein in molecular-assisted breeding of the Guangdong West Dalmatian chicken.

[0010] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0011] Application of a detection reagent for molecular markers in the detection of body shape traits in the Guangdong Western Dalmatian chicken, wherein the molecular markers are located in the Guangdong Western Dalmatian chicken. IGF2BP3 Genetically, this includes single nucleotide polymorphism sites SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and / or SNP8, among which...

[0012] The SNP1 is located at position 64412 on the NC_052533.1 gene and is a G or T polymorphism;

[0013] The SNP2 is located at position 64017 on the NC_052533.1 gene and is a T or G polymorphism;

[0014] The SNP3 is located at position 64405 on the NC_052533.1 gene and is a G or A polymorphism;

[0015] The SNP4 is located at position 80644 on the NC_052533.1 gene and is an A or G polymorphism;

[0016] The SNP5 is located at position 80656 on the NC_052533.1 gene and is an A or T polymorphism;

[0017] The SNP6 is located at position 80696 on the NC_052533.1 gene and is a T or G polymorphism;

[0018] The SNP7 is located at position 80431 on the NC_052533.1 gene and is an A or G polymorphism;

[0019] The SNP8 is located at position 80447 on the NC_052533.1 gene and is a T or C polymorphism.

[0020] Specifically, the single nucleotide polymorphism sites are detected.

[0021] Chickens with the GT genotype at the SNP1 locus had significantly higher body weights than those with the GG genotype at the SNP1 locus.

[0022] Chickens with the SNP2 genotype TG or GG had significantly longer keel bones than those with the SNP2 genotype TT.

[0023] Chickens with the genotype AA at the SNP3 locus had significantly longer keel bones and significantly wider chests than those with the genotype GG at the SNP3 locus.

[0024] Chickens with the SNP4 genotype GG had significantly longer keel and tibia than those with the SNP4 genotype AA.

[0025] Chickens with the TT genotype at the SNP5 locus had significantly longer keel bones and significantly deeper chests than those with the AA genotype at the SNP5 locus.

[0026] Chickens with the SNP6 genotype GG had significantly longer keel bones and significantly deeper chests than those with the SNP6 genotype TT.

[0027] Chickens with the SNP7 genotype GG had significantly wider breasts than those with the SNP7 genotype AA.

[0028] Chickens with the SNP8 genotype CC had significantly wider breasts than those with the SNP8 genotype TT.

[0029] Preferably, the detection reagent is a primer.

[0030] A kit for detecting body shape traits of the Dalmatian chicken from western Guangdong, comprising the aforementioned detection reagent.

[0031] Preferably, the kit includes primers with nucleotide sequences as shown in SEQ ID NO: 1-2 and / or primers with nucleotide sequences as shown in SEQ ID NO: 3-4.

[0032] Specifically, the kit includes primers for amplifying the nucleotide sequences of the single nucleotide polymorphism sites SNP1 to SNP3 as shown in SEQ ID NO: 1 to 2;

[0033] And / or primers used to amplify the nucleotide sequences of the single nucleotide polymorphism sites SNP4 to SNP8 as shown in SEQ ID NO: 3 to 4.

[0034] Preferably, the kit further includes PCR amplification reagents.

[0035] More preferably, the kit further includes one or more of dNTPs, PCR reaction buffer, and / or DNA polymerase.

[0036] A method for detecting body shape traits in Dalmatian chickens from western Guangdong, comprising detecting one or more single nucleotide polymorphism sites.

[0037] Specifically, the method includes the following steps:

[0038] S1. Extract genomic DNA from the chicken to be tested;

[0039] S2. The genomic DNA extracted in step S1 IGF2BP3 Genes are sequenced to obtain sequencing data;

[0040] S3. Analyze the sequencing data obtained in step S2, and determine the body shape traits of the chicken to be tested based on molecular markers.

[0041] Preferably, the sequencing in step S2 is Sanger sequencing.

[0042] Preferably, the body shape traits described in step S3 include weight, keel length, chest width, chest depth, and / or tibia length.

[0043] The application of the aforementioned detection reagents, the aforementioned kits, and / or the method described in molecular-assisted breeding of Dalmatian chickens in western Guangdong should also be within the scope of protection of this invention.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] This invention analyzes the analysis of the western Guangdong dwarf chicken IGF2BP3 Eight SNP loci on the gene were identified that are significantly associated with the body shape traits of the Yuexi Dalmatian chicken. These SNP loci can serve as molecular markers for breeding Yuexi Dalmatian chickens and can be applied to early auxiliary selection of body shape traits. This invention provides primers, kits, and detection methods for detecting these molecular markers. Using these primers, kits, and / or detection methods, the genotypes of these SNP loci can be rapidly detected, aiming to quickly and effectively predict the body weight and body shape traits of Yuexi Dalmatian chickens in the early stages of breeding. This allows for targeted selection of Yuexi Dalmatian chicken germplasm resources with different body shape characteristics, providing basic data for future molecular breeding and breed improvement of local breeds. The detection method described in this invention is highly operable and can rapidly and effectively predict the body shape traits of Yuexi Dalmatian chickens in the early stages of breeding, showing broad application prospects in the improvement of Yuexi Dalmatian chicken germplasm resources. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the primer regions for the IGF2BP3-Exon5-6 primer set and the IGF2BP3-Exon11 primer set.

[0047] Figure 2 Figure A in the figure shows the electrophoresis results of single-sample PCR products of primer set IGF2BP3-Exon5-6 and primer set IGF2BP3-Exon11; Figure 2Figure B in the diagram shows the electrophoresis results of a single sample PCR product of primer group IGF2BP3-Exon7.

[0048] Figure 3 Figure A in the figure shows the electrophoresis results of the mixed-pool genomic DNA PCR products of primer set IGF2BP3-Exon5-6 and primer set IGF2BP3-Exon11; Figure 3 Figure B in the diagram shows the electrophoresis results of the mixed-pool genomic DNA PCR products of primer set IGF2BP3-Exon7.

[0049] Figure 4 Figure A in the diagram shows the mixed-pool sequencing peaks of primer set IGF2BP3-Exon7; Figure 4 Figure B in the diagram shows the sequencing peaks of a single sample from primer group IGF2BP3-Exon7.

[0050] Figure 5 The sequence peak diagram shows the genotype mutation locations of SNP sites: g.64017T>G, g.64077C>T, g.64405G>A, and g.64412G>T.

[0051] Figure 6 The sequence peak diagram shows the genotype mutation locations of SNP sites: g.64420T>C, g.64447C>T, g.64449T>C, and g.80086A>C.

[0052] Figure 7 This is a sequence peak diagram showing the genotype mutation locations of SNP sites: g.80306A>G, ​​g.80431A>G, and g.80447T>C.

[0053] Figure 8 The sequence peak diagram shows the genotype mutation locations of SNP sites: g.80488A>G, g.80537T>C, and g.80635G>A.

[0054] Figure 9 This is a sequence peak diagram showing the genotype mutation locations of SNP sites: g.80644A>G, g.80656A>T, and g.80696T>G.

[0055] Figure 10 For Western Guangdong Dwarf Chicken IGF2BP3 The results of D′ values ​​for linkage disequilibrium analysis of 17 SNP sites of the gene are shown. The color of each square from light to dark indicates the degree of linkage from low to high, and dark color indicates complete linkage. The number in the square represents the logarithm of the linkage disequilibrium probability between a given marker pair.

[0056] Figure 11 For Western Guangdong Dwarf Chicken IGF2BP3 Linkage disequilibrium analysis of 17 SNP sites in the gene2 The result is given by the color of each square, from light to dark, indicating the degree of chainage from low to high, with dark colors indicating complete chainage. The number in the square represents the logarithm of the chain imbalance probability between a given pair of tags. Detailed Implementation

[0057] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0058] Example 1 Primer Design

[0059] I. Experimental Methods

[0060] chicken IGF2BP3 The gene is located on chromosome 2, with a total length of 107,425 bp and 18 exons, each exon being 4,552 bp in length. (Based on the chicken data in the NCBI database...) IGF2BP3 Using the gene sequence (GenBank accession number: NC_052533.1) as the target gene, six pairs of specific primers were designed, and the primer information is shown in Table 1.

[0061] Table 1 IGF2BP3 Gene primer information

[0062]

[0063] The primer sequences were synthesized by Shanghai Bioengineering (BioGen) Technology Service Co., Ltd. The primers shown in Table 1 were used to perform PCR on the blood DNA of the Guangdong Dalmatian chickens previously preserved by the inventors to test the primer specificity.

[0064] II. Experimental Results

[0065] IGF2BP3 The three primer sets IGF2BP3-P1, IGF2BP3-P2 and IGF2BP3-P3 designed for the 2000 bp promoter region of the gene did not produce the target band and were discarded.

[0066] The primer regions of the IGF2BP3-Exon5-6 primer set and the IGF2BP3-Exon11 primer set are as follows: Figure 1 As shown, the amplified fragment of the IGF2BP3-Exon5-6 primer set includes IGF2BP3 The gene contains partial introns 4 and 5, partial intron 6, exon 5, and exon 6; the amplified fragment of the IGF2BP3-Exon11 primer set includes... IGF2BP3Part of the gene contains intron 10, part of intron 11, and exon 11.

[0067] Figure 2 In Figure A, lanes 1-5 are electrophoresis results of single-sample PCR products of primer set IGF2BP3-Exon5-6, and lanes 6-10 are electrophoresis results of single-sample PCR products of primer set IGF2BP3-Exon11. Figure 2 In Figure B, lanes 1-5 show the electrophoresis results of single-sample PCR products from primer set IGF2BP3-Exon7. Figure 2 As shown, the PCR products of a single sample were detected by 1.0% agarose gel electrophoresis. The electrophoretic bands were bright, with good specificity and no impurities, which meets the requirements for directly determining the nucleotide sequence using PCR products. Primer sets IGF2BP3-Exon5-6, IGF2BP3-Exon7, and IGF2BP3-Exon11 were specific.

[0068] Example 2 DNA pooling sequencing and SNP site analysis

[0069] I. Experimental Methods

[0070] Using the Guangdong Western Dalmatian chicken as the research subject, it was fed according to the feeding standards for broiler chickens, with free access to feed and water during the experiment, and was fed normally until 60 days of age. Ninety-five 60-day-old Guangdong Western Dalmatian chickens were selected. After production data were tested, 3 ml of blood was collected from the wing vein using vacuum blood collection tubes. EDTA was added to the blood collection tubes as an anticoagulant, and the samples were stored at -20℃ to obtain the blood samples to be tested.

[0071] DNA was extracted from the blood sample using a DNA kit. After extraction, the concentration and purity of the blood DNA were measured using a spectrophotometer. Blood DNA samples that met the purity requirements were selected for subsequent experiments.

[0072] Blood DNA samples that meet the purity requirements are sorted from highest to lowest concentration. 5 μl of each blood DNA sample is extracted into a 1.5 ml centrifuge tube to prepare a mixed pool. Ten blood DNA samples are used as one mixed pool, and a total of nine mixed pools are prepared (each mixed pool contains the 15 blood DNA samples). These mixed pools are then used as templates for PCR amplification. The amplification system is shown in Table 2.

[0073] Table 2 PCR amplification system

[0074]

[0075] PCR reaction conditions: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 1 min, 32 cycles, 72℃ extension for 10 min, PCR products were stored at 4℃ and used for subsequent sequencing.

[0076] The PCR amplification products were detected by agarose gel electrophoresis. If the electrophoretic bands were the same size as the target band and were clear and bright, the obtained PCR products were sent to the company for Sanger sequencing. (The last sentence appears to be incomplete and possibly refers to a different product, "NCBI chicken..."). IGF2BP3 The gene sequence (GenBank accession number: NC_052533.1) was used as a reference sequence. The sequencing results were compared with the reference fragment on NCBI using analysis software to find peaks in the sequencing results and then screen out SNP sites.

[0077] II. Experimental Results

[0078] Spectrophotometer results showed that the OD260 / OD280 of the blood DNA were both between 1.8 and 2.0, indicating that the quality and purity met the standards and could be used for subsequent experiments.

[0079] Electrophoretic bands of amplification products obtained using primer sets IGF2BP3-Exon5-6, IGF2BP3-Exon7, and IGF2BP3-Exon11 as templates are shown below. Figure 3 As shown, Figure 3 In Figure A, lanes 1-3 are the electrophoretic bands of the amplification products of primer set IGF2BP3-Exon5-6, and lanes 4-6 are the electrophoretic bands of the amplification products of primer set IGF2BP3-Exon11. Figure 3 In Figure B, lanes 1-3 are the electrophoretic bands of the amplification products of primer set IGF2BP3-Exon7. Figure 3 As shown, the electrophoretic bands of the amplified products are bright, free of impurities, and have good specificity, meeting the requirements for Sanger sequencing using PCR products.

[0080] like Figure 4 As shown, the mixed-pool sequencing peak diagram of primer set IGF2BP3-Exon7 is shown. Figure 4 Figure A in the diagram) and sequencing peak diagram of a single blood DNA sample ( Figure 4 The result in Figure B was disordered, so it was discarded. The amplification results of primer sets IGF2BP3-Exon5-6 and IGF2BP3-Exon11 were analyzed.

[0081] Sanger sequencing analysis results showed that in chickens IGF2BP3A total of 17 SNP mutation sites were detected on introns 4, 5, 6, 10, 11 and exon 11 of the gene. The information of the mutation sites is shown in Table 3; the sequence peak maps of the mutation positions of different genotypes are shown in Figures 5-9 .

[0082] Table 3 Information of IGF2BP3 gene mutation sites

[0083]

[0084] Example 3 Analysis of genetic characteristics of SNP sites

[0085] I. Experimental method

[0086] Use Excel in WPS Office software to calculate allele frequency, genotype frequency, genetic heterozygosity, effective number of alleles, polymorphic information content, and χ 2 value to detect whether the SNP site is in Hardy-Weinberg (H-W) equilibrium. The calculation method refers to the literature (Liu Mei, Zhu Yixuan, Wang Xiaoyi, et al. SNP detection of exon 3 of PID1 gene in Xuanhe pigs and its association analysis with meat quality traits [J]. Southwest China Journal of Agricultural Sciences, 2022(007):035.).

[0087] II. Experimental results

[0088] Perform genetic characteristic analysis on the SNP sites of IGF2BP3 gene in Yuexi Frizzle Chicken. The results are shown in Table 4. PIC>0.5 indicates high polymorphism, 0.25<PIC<0.5 indicates moderate polymorphism, and PIC<0.25 indicates low polymorphism; use χ 2 value to test Hardy-Weinberg equilibrium, df = 1, χ 2 0.05 = 5.99, χ 2 0.01 = 9.21, P<0.05 indicates significant difference; deviation from Hardy-Weinberg equilibrium, P>0.05 indicates no significant difference and no deviation from Hardy-Weinberg equilibrium.

[0089] As can be seen from Table 4: IGF2BP3The dominant genotypes among the 17 SNP loci were TG (0.547), CT (0.474), GA (0.526), ​​GG (0.958), TC (0.547), CC (0.968), TT (0.905), CC (0.421), AA (0.832), AG (0.389), TC (0.389), AA (0.916), TT (0.989), GG (0.768), GG (0.411), AG (0.411), TT (0.421), and GG (0.421). These genotypes may... These alleles are associated with certain specific phenotypic traits; the corresponding dominant alleles are G (0.611), C (0.584), A (0.547), G (0.979), T (0.705), C (0.984), T (0.953), C (0.621), A (0.884), A (0.511), T (0.511), A (0.947), T (0.995), G (0.853), G (0.616), T (0.616), and G (0.616). These alleles occur frequently and may be associated with certain specific phenotypic traits.

[0090] The PIC calculation values ​​indicate that among the 17 SNP sites, g.64412G>T, g.64447C>T, g.64449T>C, g.80306A>G, ​​g.80488A>G, g.80537T>C, and g.80635G>A are low-degree polymorphisms, while the remaining sites are moderately polymorphic.

[0091] IGF2BP3 The Hardy-Weinberg equilibrium test results for gene SNP loci showed that the loci g.64017T>G, g.64077C>T, g.64405G>A, and g.80696T>G did not deviate from Hardy-Weinberg equilibrium (P>0.05), and the gene frequencies of these loci remained stable in the population, unaffected by genetic factors such as mutation, selection, or migration; the remaining loci all significantly deviated from Hardy-Weinberg equilibrium (P<0.05).

[0092] Table 4. Results of genetic characteristics analysis of 17 SNP loci of the IGF2BP3 gene in the Western Guangdong Dwarf Chicken.

[0093]

[0094] Example 4: SNP linkage disequilibrium and haplotype analysis

[0095] I. Experimental Methods

[0096] For the western Guangdong dwarf chicken IGF2BP3The 17 SNP loci of the gene were analyzed for linkage disequilibrium and haplotype using the online software SHEsis, resulting in... IGF2BP3 Haplotype types and their frequencies of genes.

[0097] II. Experimental Results

[0098] Western Guangdong Dwarf Chicken IGF2BP3 The linkage disequilibrium analysis results of 17 SNP sites in the gene are as follows: Figure 10 , Figure 11 As shown in Table 5, Figure 10 and Figure 11 The colors of the squares, from light to dark, represent the degree of chaining from low to high, with darker colors indicating complete chaining. The numbers in the squares represent the logarithm of the probability of chain imbalance between a given pair of markers. Figure 10 The value of D′ Figure 11 For r 2 The value represents the chain imbalance strength between the two points; in Table 5, the upper triangle represents the D′ value, and the lower triangle represents the r value. 2 Value; r 2 A value <0.330 indicates that there is no strong linkage disequilibrium.

[0099] As shown in Table 5: IGF2BP3 Of the 17 SNP sites in the gene:

[0100] The r values ​​of g.64017T>G and g.64077C>T, g.64405G>A, g.80086A>C, g.80431A>G, g.80447T>C, g.80644A>G, g.80656A>T and g.80696T>G 2 The values ​​were 0.383, 0.650, 0.663, 0.392, 0.392, 0.683, 0.683, and 0.683, respectively. 2 The values ​​are all greater than 0.330, so there is a strong linkage disequilibrium between g.64017T>G and g.64077C>T, g.64405G>A, g.80086A>C, g.80431A>G, g.80447T>C, g.80644A>G, g.80656A>T and g.80696T>G sites;

[0101] g.64077C>T and g.64405G>A's r 2 The value is 0.339, r 2 The value is greater than 0.330, so there is a strong linkage disequilibrium between the sites g.64077C>T and g.64405G>A;

[0102] The r values ​​of g.64405G>A and g.80086A>C, g.80431A>G, g.80447T>C, g.80644A>G, g.80656A>T and g.80696T>G 2 The values ​​are 0.620, 0.460, 0.460, 0.636, 0.636, and 0.636, respectively, r 2 The values ​​are all greater than 0.330, so there is a strong linkage disequilibrium between g.64405G>A and g.80086A>C, g.80431A>G, g.80447T>C, g.80644A>G, g.80656A>T and g.80696T>G sites;

[0103] g.64420T>C and g.80431A>G and g.80447T>C r 2 The values ​​are all 0.362, r 2 The values ​​are all greater than 0.330, so there is a strong linkage disequilibrium between the sites g.64420T>C and g.80431A>G and g.80447T>C;

[0104] The r values ​​of g.80086A>C and g.80431A>G, g.80447T>C, g.80644A>G, g.80656A>T and g.80696T>G 2 The values ​​were 0.585, 0.585, 0.934, 0.934, and 0.934, respectively, r 2 The values ​​are all greater than 0.330, so there is a strong linkage disequilibrium between g.80086A>C and g.80431A>G, g.80447T>C, g.80644A>G, g.80656A>T and g.80696T>G sites;

[0105] g.80306A>G and g.80488A>G and g.80635G>A r 2 The values ​​are 0.424 and 0.758 respectively, r 2 The values ​​are all greater than 0.330, so there is a strong linkage disequilibrium between the sites g.80306A>G and g.80488A>G and g.80635G>A;

[0106] The r values ​​of g.80431A>G and g.80447T>C, g.80644A>G, g.80656A>T, and g.80696T>G 2 The values ​​are 1.000, 0.598, 0.598, and 0.598, respectively, r 2The values ​​are all greater than 0.330, so there is a strong linkage disequilibrium between g.80431A>G and g.80447T>C, g.80644A>G, g.80656A>T and g.80696T>G sites;

[0107] The r values ​​of g.80447T>C, g.80644A>G, g.80656A>T, and g.80696T>G 2 The values ​​are all 0.598, r 2 The values ​​are all greater than 0.330, so there is a strong linkage disequilibrium between g.80447T>C and g.80644A>G, g.80656A>T and g.80696T>G sites;

[0108] r of g.80644A>G and g.80656A>T and g.80696T>G 2 The values ​​are all 0.956, r 2 The values ​​are all greater than 0.330, so there is a strong linkage disequilibrium between the sites g.80644A>G and g.80656A>T and g.80696T>G;

[0109] g.80656A>T and g.80696T>G's r 2 The value is 1.000, r 2 The value is greater than 0.330, so there is a strong linkage disequilibrium between the g.80656A>T and g.80696T>G sites, that is, there is a certain linkage relationship between these sites and they are related in terms of genetic variation. There is no strong linkage disequilibrium between the other sites.

[0110] Table 5. Western Guangdong Dwarf Chicken IGF2BP3 Linkage disequilibrium analysis results of 17 SNP sites in the gene

[0111]

[0112] As shown in Table 6, in Table 5... IGF2BP3 At 17 specific SNP loci of a gene, 32 different haplotypes can be combined. These 32 haplotypes are named H1 to H32 in order of frequency. Among them, there are 7 effective haplotypes (with a frequency greater than 0.03 in the population) (H1, H2, H3, H4, H5, H6, and H7), with frequencies of 0.313, 0.227, 0.085, 0.052, 0.047, 0.045, and 0.037, respectively. The presence of these 7 haplotypes in the population is significant and can serve as effective genetic markers for genetic research and association analysis.

[0113] Table 6. Haplotype analysis results of 17 SNP sites in the IGF2BP3 gene of the Dalmatian chicken from western Guangdong.

[0114]

[0115] Example 5: Association Analysis between Gene Polymorphism and Body Shape Traits

[0116] I. Experimental Methods

[0117] IBM SPSS Statistics 26.0 software was used to analyze the data of the western Guangdong dwarf chicken. IGF2BP3 Association analysis was performed between different genotypes at 17 SNP loci of the gene and body shape traits.

[0118] II. Experimental Results

[0119] For the western Guangdong dwarf chicken IGF2BP3 Association analysis was performed on different genotypes and body shape traits at 17 SNP loci of the gene. The results are shown in Table 7. Data in Table 7 are compared between the same columns at the same locus. Different lowercase letters on the scapula indicate significant differences (P<0.05), different uppercase letters on the scapula indicate extremely significant differences (P<0.01), and no letters on the scapula indicate no significant differences (P>0.05). Genotypes with fewer than 5 individuals were not included in the statistics.

[0120] Table 7 shows that among 95 Guangdong West Dwarf Chickens IGF2BP3 Of the 17 SNP sites in the gene:

[0121] The three genotypes of g.64017T>G showed differences in keel length, with the GG genotype being significantly longer than the TT genotype and the TG genotype being significantly longer than the TT genotype (P<0.05).

[0122] The three genotypes of g.64405G>A showed differences in keel length and chest width. The AA genotype was significantly longer than the GG genotype in keel length, and the AA genotype was significantly longer than the GA genotype in chest width (P<0.05).

[0123] The two genotypes of g.64412G>T showed differences in body weight, with the GT genotype being significantly higher than the GG genotype (P<0.05).

[0124] The three genotypes of g.80431A>G showed differences in chest width, with the GG genotype being significantly higher than the AA genotype (P<0.05).

[0125] The three genotypes of g.80447T>C showed differences in chest width, with the CC genotype being significantly higher than the TT genotype (P<0.05).

[0126] The three genotypes of g.80644A>G showed differences in keel length and tibia length, with the GG genotype being significantly higher than the AA genotype in both keel length and tibia length (P<0.05).

[0127] The three genotypes of g.80656A>T showed differences in keel length and chest depth. In terms of keel length, the TT genotype was significantly higher than the AA genotype (P<0.05); in terms of chest depth, the TT genotype was significantly higher than the AA and AT genotypes, and there was no significant difference between the AA and AT genotypes.

[0128] The three genotypes of g.80696T>G showed differences in keel length and chest depth. In terms of keel length, the GG genotype was significantly longer than the TT genotype (P<0.05). In terms of chest depth, the GG genotype was significantly longer than both the TT and TG genotypes, and there was no significant difference between the TT and TG genotypes.

[0129] There were no significant differences in growth performance among the genotypes at the remaining loci (P>0.05).

[0130] Table 7. Western Guangdong Dwarf Chicken IGF2BP3 Body shape traits of each genotype at 17 SNP loci of the gene

[0131]

[0132]

[0133] According to Table 7, the western Guangdong dwarf chicken... IGF2BP3 Eight SNP loci associated with body shape traits in the Guangdong West Dalmatian chicken were screened from 17 SNP loci in the gene (GenBank accession number: NC_052533.1).

[0134] SNP1 is located in IGF2BP3 At position 64412 (g.64412G>T), which is a G or T polymorphism, the body weight of the Yuexi Dalmatian chicken with the genotype GT at the SNP1 locus is significantly higher than that of the individual with the genotype GG at the SNP1 locus.

[0135] SNP2 is located at IGF2BP3 At gene position 64017 (g.64017T>G), which is a T or G polymorphism, the keel length of the Yuexi Dalmatian chicken with the SNP2 locus genotype TG or GG is significantly higher than that of individuals with the SNP2 locus genotype TT.

[0136] SNP3 is located in IGF2BP3 At gene position 64405 (g.64405G>A), which is a G or A polymorphism, the keel length of the Yuexi Dalmatian chicken with the SNP3 locus genotype AA is significantly greater than that of the individual with the SNP3 locus genotype GG, and the chest width is significantly greater than that of the individual with the SNP3 locus genotype GA.

[0137] SNP4 is located in IGF2BP3At gene position 80644 (g.80644A>G), which is an A or G polymorphism, the keel length and tibia length of the Yuexi Dalmatian chicken with the SNP4 genotype GG are significantly higher than those of individuals with the SNP4 genotype AA.

[0138] SNP5 is located in IGF2BP3 At gene position 80656 (g.80656A>T), which is an A or T polymorphism, the keel length of the Yuexi Dalmatian chicken with the SNP5 genotype TT is significantly higher than that of individuals with the SNP5 genotype AA, and the chest depth is significantly higher than that of individuals with the SNP5 genotype AA or AT.

[0139] SNP6 is located in IGF2BP3 At gene position 80696 (g.80696T>G), which is a T or G polymorphism, the keel length of the Yuexi Dalmatian chicken with the genotype GG at SNP6 is significantly higher than that of the individual with the genotype TT at SNP6, and the chest depth is significantly higher than that of the individual with the genotype TT or TG at SNP6.

[0140] SNP7 is located in IGF2BP3 At position 80431 of the gene (g.80431A>G), which is an A or G polymorphism, the breast width of the Yuexi Dalmatian chicken with the genotype GG at SNP7 is significantly higher than that of the individual with the genotype AA at SNP7.

[0141] SNP8 is located in IGF2BP3 At position 80447 of the gene (g.80447T>C), which is a T or C polymorphism, the breast width of the Yuexi Dalmatian chicken with the SNP8 genotype CC is significantly higher than that of individuals with the SNP8 genotype TT.

[0142] Example 6: A kit for detecting body shape traits of Dalmatian chickens in western Guangdong

[0143] I. Composition

[0144] Primers with nucleotide sequences as shown in SEQ ID NO: 1-2, primers with nucleotide sequences as shown in SEQ ID NO: 3-4, Taq PCR Mix, and Nuclease-free Water.

[0145] II. Instructions for Use

[0146] S1. Extract DNA from the sample to be tested;

[0147] S2. Use the DNA extracted in step S1 as a template for PCR reaction:

[0148] PCR amplification system (total volume 20 μl): 10 μl Taq PCR Mix, 0.5 μl each of primers with nucleotide sequences as shown in SEQ ID NO: 1-2 or primers with nucleotide sequences as shown in SEQ ID NO: 3-4, 1 μl DNA extracted in step S1, and 8 μl Nuclease-free Water;

[0149] PCR amplification program: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 1 min, 32 cycles, 72℃ extension for 10 min.

[0150] S3. Perform agarose gel electrophoresis on the PCR amplification products obtained in step S2. 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 S2.

[0151] III. Result Judgment

[0152] SNP1 is located in IGF2BP3 SNP2 is located at position 64412 (g.64412G>T) on the gene. IGF2BP3 SNP3 is located at position 64017 on the gene (g.64017T>G). IGF2BP3 SNP4 is located at position 64405 on the gene (g.64405G>A). IGF2BP3 SNP5 is located at position 80644 on the gene (g.80644A>G). IGF2BP3 SNP6 is located at position 80656 (g.80656A>T) on the gene. IGF2BP3 SNP7 is located at position 80696 on the gene (g.80696T>G). IGF2BP3 SNP8 is located at position 80431 on the gene (g.80431A>G). IGF2BP3 At position 80447 on the gene (g.80447T>C).

[0153] The body weight of the Guangdong West Dalmatian chickens with the SNP1 genotype GT was significantly higher than that of individuals with the SNP1 genotype GG.

[0154] The keel length of the Dalmatian pheasant from western Guangdong with the SNP2 locus genotype TG or GG was significantly longer than that of individuals with the SNP2 locus genotype TT.

[0155] The keel length of the Dalmatian pheasant with the SNP3 genotype AA was significantly longer than that of the individual with the SNP3 genotype GG, and the chest width was significantly longer than that of the individual with the SNP3 genotype GA.

[0156] The keel length and tibia length of the Dalmatian pheasant with the SNP4 locus genotype GG were significantly higher than those of the individual with the SNP4 locus genotype AA.

[0157] The keel length of the Dalmatian pheasant with the SNP5 genotype TT was significantly longer than that of individuals with the SNP5 genotype AA, and the chest depth was significantly greater than that of individuals with the SNP5 genotype AA or AT.

[0158] The keel length of the Dalmatian pheasant with the SNP6 genotype GG was significantly longer than that of individuals with the SNP6 genotype TT, and the chest depth was significantly greater than that of individuals with the SNP6 genotype TT or TG.

[0159] The breast width of the Dalmatian chickens from western Guangdong with the genotype GG at the SNP7 locus was significantly greater than that of individuals with the genotype AA at the SNP7 locus.

[0160] The breast width of the Dalmatian chickens from western Guangdong with the SNP8 genotype CC was significantly higher than that of individuals with the SNP8 genotype TT.

[0161] Example 7: A method for detecting body shape traits in Dalmatian chickens from western Guangdong

[0162] A method for detecting body shape traits in Dalmatian chickens from western Guangdong, comprising the following steps:

[0163] S1. Extract DNA from the sample to be tested;

[0164] S2. Using the DNA extracted from the sample in step S1 as a template, perform a PCR reaction:

[0165] PCR amplification system (total volume 20 μl): 10 μl Taq PCR Mix, 0.5 μl each of primers with nucleotide sequences as shown in SEQ ID NO: 1-2 or primers with nucleotide sequences as shown in SEQ ID NO: 3-4, 1 μl DNA of the sample to be tested obtained in step S1, and 8 μl Nuclease-free Water;

[0166] PCR amplification program: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 1 min, 32 cycles, 72℃ extension for 10 min.

[0167] S3. Perform agarose gel electrophoresis on the PCR amplification products obtained in step S2. 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 S2.

[0168] S4. Result Judgment:

[0169] SNP1 is located in IGF2BP3SNP2 is located at position 64412 (g.64412G>T) on the gene. IGF2BP3 SNP3 is located at position 64017 on the gene (g.64017T>G). IGF2BP3 SNP4 is located at position 64405 on the gene (g.64405G>A). IGF2BP3 SNP5 is located at position 80644 on the gene (g.80644A>G). IGF2BP3 SNP6 is located at position 80656 (g.80656A>T) on the gene. IGF2BP3 SNP7 is located at position 80696 on the gene (g.80696T>G). IGF2BP3 SNP8 is located at position 80431 on the gene (g.80431A>G). IGF2BP3 At position 80447 on the gene (g.80447T>C);

[0170] The body weight of the Guangdong West Dalmatian chickens with the SNP1 genotype GT was significantly higher than that of individuals with the SNP1 genotype GG.

[0171] The keel length of the Dalmatian pheasant from western Guangdong with the SNP2 locus genotype TG or GG was significantly longer than that of individuals with the SNP2 locus genotype TT.

[0172] The keel length of the Dalmatian pheasant with the SNP3 genotype AA was significantly longer than that of the individual with the SNP3 genotype GG, and the chest width was significantly longer than that of the individual with the SNP3 genotype GA.

[0173] The keel length and tibia length of the Dalmatian pheasant with the SNP4 locus genotype GG were significantly higher than those of the individual with the SNP4 locus genotype AA.

[0174] The keel length of the Dalmatian pheasant with the SNP5 genotype TT was significantly longer than that of individuals with the SNP5 genotype AA, and the chest depth was significantly greater than that of individuals with the SNP5 genotype AA or AT.

[0175] The keel length of the Dalmatian pheasant with the SNP6 genotype GG was significantly longer than that of individuals with the SNP6 genotype TT, and the chest depth was significantly greater than that of individuals with the SNP6 genotype TT or TG.

[0176] The breast width of the Dalmatian chickens from western Guangdong with the genotype GG at the SNP7 locus was significantly greater than that of individuals with the genotype AA at the SNP7 locus.

[0177] The breast width of the Dalmatian chickens from western Guangdong with the SNP8 genotype CC was significantly higher than that of individuals with the SNP8 genotype TT.

[0178] 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 for SNP molecular markers in the Yuexi Dalmatian chicken in the detection of body shape traits of the Yuexi Dalmatian chicken, characterized in that, The molecular markers are located on the IGF2BP3 gene of the Guangdong Western Dalmatian chicken and are single nucleotide polymorphism sites SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and / or SNP8, among which, The SNP1 is located at bit 31100527 of NC_052533.1 and is a G or T polymorphism; The SNP2 is located at bit 31100922 of NC_052533.1 and is a T or G polymorphism; The SNP3 is located at bit 31100534 of NC_052533.1 and is a G or A polymorphism; The SNP4 is located at bit 31084294 of NC_052533.1 and is an A or G polymorphism; The SNP5 is located at bit 31084283 of NC_052533.1 and is an A or T polymorphism; The SNP6 is located at bit 31084243 in NC_052533.1 and is a T or G polymorphism; The SNP7 is located at bit 31084508 of NC_052533.1 and is an A or G polymorphism; The SNP8 is located at bit 31084492 of NC_052533.1 and is a T or C polymorphism; Chickens with the GT genotype at the SNP1 locus had significantly higher body weights than those with the GG genotype at the SNP1 locus. Chickens with the SNP2 genotype TG or GG had significantly longer keel bones than those with the SNP2 genotype TT. The keel length of the tested chickens with the SNP3 genotype AA was significantly longer than that of the individuals with the SNP3 genotype GG, and the breast width of the tested chickens with the SNP3 genotype AA was significantly longer than that of the individuals with the SNP3 genotype GA. Chickens with the SNP4 genotype GG had significantly longer keel and tibia than those with the SNP4 genotype AA. Chickens with the TT genotype at SNP5 had significantly longer keel lengths than those with the AA genotype at SNP5, and chickens with the TT genotype at SNP5 had significantly longer breast depths than those with the AA or AT genotypes at SNP5. Chickens with the SNP6 genotype GG had significantly longer keel lengths than those with the SNP6 genotype TT, and chickens with the SNP6 genotype GG had significantly longer breast depths than those with the SNP6 genotype TT or TG. Chickens with the SNP7 genotype GG had significantly wider breasts than those with the SNP7 genotype AA. Chickens with the SNP8 genotype CC had significantly wider breasts than those with the SNP8 genotype TT.

2. The application according to claim 1, characterized in that, The detection reagent is a primer.

3. The application according to claim 2, characterized in that, The detection reagent includes primers with nucleotide sequences as shown in SEQ ID NO: 1-2 and primers with nucleotide sequences as shown in SEQ ID NO: 3-4.

4. A method for detecting body shape traits of the Guangdong West Dalmatian chicken, characterized in that, Detect one or more single nucleotide polymorphism sites as described in claim 1; Chickens with the GT genotype at the SNP1 locus had significantly higher body weights than those with the GG genotype at the SNP1 locus. Chickens with the SNP2 genotype TG or GG had significantly longer keel bones than those with the SNP2 genotype TT. The keel length of the tested chickens with the SNP3 genotype AA was significantly longer than that of the individuals with the SNP3 genotype GG, and the breast width of the tested chickens with the SNP3 genotype AA was significantly longer than that of the individuals with the SNP3 genotype GA. Chickens with the SNP4 genotype GG had significantly longer keel and tibia than those with the SNP4 genotype AA. Chickens with the TT genotype at SNP5 had significantly longer keel lengths than those with the AA genotype at SNP5, and chickens with the TT genotype at SNP5 had significantly longer breast depths than those with the AA or AT genotypes at SNP5. Chickens with the SNP6 genotype GG had significantly longer keel lengths than those with the SNP6 genotype TT, and chickens with the SNP6 genotype GG had significantly longer breast depths than those with the SNP6 genotype TT or TG. Chickens with the SNP7 genotype GG had significantly wider breasts than those with the SNP7 genotype AA. Chickens with the SNP8 genotype CC had significantly wider breasts than those with the SNP8 genotype TT.

5. The method according to claim 4, characterized in that, Includes the following steps: S1. Extract genomic DNA from the chicken to be tested; S2. Sequencing the IGF2BP3 gene of the genomic DNA extracted in step S1 to obtain sequencing data; S3. Analyze the sequencing data obtained in step S2. According to the molecular marker described in claim 1, the body shape characteristics of the chicken to be tested are determined.

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

7. The method according to claim 5, characterized in that, The body shape traits mentioned in step S3 are weight, keel length, chest width, chest depth, and / or tibia length.

8. The application of the detection reagent described in claim 1 and / or the method described in claim 4 in molecular-assisted breeding of the Guangdong Western Dalmatian chicken, characterized in that, Chickens with the GT genotype at the SNP1 locus had significantly higher body weights than those with the GG genotype at the SNP1 locus. Chickens with the SNP2 genotype TG or GG had significantly longer keel bones than those with the SNP2 genotype TT. The keel length of the tested chickens with the SNP3 genotype AA was significantly longer than that of the individuals with the SNP3 genotype GG, and the breast width of the tested chickens with the SNP3 genotype AA was significantly longer than that of the individuals with the SNP3 genotype GA. Chickens with the SNP4 genotype GG had significantly longer keel and tibia than those with the SNP4 genotype AA. Chickens with the TT genotype at SNP5 had significantly longer keel lengths than those with the AA genotype at SNP5, and chickens with the TT genotype at SNP5 had significantly longer breast depths than those with the AA or AT genotypes at SNP5. Chickens with the SNP6 genotype GG had significantly longer keel lengths than those with the SNP6 genotype TT, and chickens with the SNP6 genotype GG had significantly longer breast depths than those with the SNP6 genotype TT or TG. Chickens with the SNP7 genotype GG had significantly wider breasts than those with the SNP7 genotype AA. Chickens with the SNP8 genotype CC had significantly wider breasts than those with the SNP8 genotype TT.

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