A kit and method for assessing the growth traits of dwarf chickens

Abstract

This invention discloses a kit and method for evaluating the growth traits of Dwarf Chickens. Using Dwarf Chickens as the research subject, this invention identifies six SNP loci on the IGF2BP3 gene of Dwarf Chickens that are significantly associated with growth traits such as body weight, keel length, chest width, chest depth, shank length, or body oblique length. These SNP loci can serve as molecular breeding markers for Dwarf Chicken growth traits. By detecting these SNP loci, the body weight and body shape of Dwarf Chickens can be predicted rapidly, effectively, and accurately at an early stage, shortening breeding time and accelerating the breeding process. This invention provides fundamental data for future molecular breeding and breed improvement of Dwarf Chickens, and can significantly improve the efficiency of Dwarf Chicken germplasm resource improvement.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and more specifically, to a kit and method for evaluating the growth traits of dwarf chickens. Background Technology

[0002] The Dwarf Chicken is a dual-purpose breed, raised for both eggs and meat, with a long history of breeding. According to the 1941 "Pengxian Local Chronicles," the Dwarf Chicken has been raised in my country for over 200 years, mainly distributed in Xingyi City, Guizhou Province, with the central production areas being Pengxian, Qishe, Canggeng, and Nihan in Xingyi City. The Dwarf Chicken has a symmetrical body, short shanks, and a creeping posture. It is characterized by its docile temperament, strong adaptability, resistance to dampness, high egg production, and delicious meat. It also exhibits strong adaptability to harsh climates and extensive breeding management.

[0003] In the 1980s, with the introduction of foreign breeds, the lack of adequate protective measures for dwarf chickens led to quality degradation, resulting in problems such as long rearing cycles, changes in meat quality, low slaughter rates, and low economic benefits. Therefore, it is essential to accelerate the improvement of the dwarf chicken breed to enhance its production performance and develop local specialty breed resources. Dwarf chickens differ significantly from other breeds, and selection and breeding primarily rely on analysis of appearance and body phenotype. Therefore, molecular marker analysis is an effective method in the genetic selection and breeding of dwarf chickens.

[0004] IGF2BP3 (Insulin-like growth factor 2 mRNA binding protein 3) belongs to the insulin-like growth factor II mRNA binding protein family. It is a secreted protein that can bind to insulin-like growth factors (IGFs), specifically binding to the mRNA encoding IGF2. It participates in the regulation of IGF2 mRNA localization, stability, reverse transcription, and translation, playing a crucial role in RNA transport, stability, cell proliferation, and migration. Currently, regarding... IGF2BP3 Research on genetic polymorphism has primarily focused on human diseases, with relatively little research on poultry. In particular, no literature has been found on research specifically targeting the dwarf chicken breed. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a kit and method for evaluating the growth traits of dwarf chickens.

[0006] The first objective of this invention is to provide a kit for evaluating the growth traits of dwarf chickens.

[0007] The second objective of this invention is to provide a kit for detecting growth traits in dwarf chickens and its application in dwarf chicken growth trait-assisted selection and / or molecular breeding of dwarf chickens.

[0008] The third objective of this invention is to provide a method for detecting the growth traits of dwarf chickens.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0010] A kit for assessing growth traits in dwarf chickens, the kit comprising reagents for detecting the genotype of SNP loci in dwarf chickens, the SNP loci being located in the dwarf chicken... IGF2BP3 Genetically, the reagent detects one or more of the following SNP sites:

[0011] SNP1 is located at position 64077 on the NC_052533.1 gene and is a C or T polymorphism;

[0012] SNP2 is located at position 64405 on the NC_052533.1 gene and is a G or A polymorphism;

[0013] SNP3 is located at position 80306 on the NC_052533.1 gene and is an A or G polymorphism;

[0014] SNP4 is located at position 80431 on the NC_052533.1 gene and is an A or G polymorphism;

[0015] SNP5 is located at position 80488 on the NC_052533.1 gene and is an A or G polymorphism;

[0016] SNP6 is located at position 80635 on the NC_052533.1 gene and is either a G or A polymorphism.

[0017] Preferably, the growth trait is chest width: individuals with the SNP1 genotype CC have a significantly higher chest width than individuals with the genotype TT.

[0018] Preferably, the growth trait is keel length: the keel length of individuals with the SNP2 genotype GA is significantly higher than that of individuals with the genotype AA; the keel length of individuals with the SNP3 genotype GG is significantly higher than that of individuals with the genotype AG.

[0019] Preferably, the growth trait is body oblique length: the body oblique length of individuals with the SNP3 genotype GG is significantly higher than that of individuals with the genotype AG.

[0020] Preferably, the growth trait is tibia length: the tibia length of individuals with the SNP3 genotype GG is significantly higher than that of individuals with the SNP4 genotype AG; the tibia length of individuals with the SNP4 genotype AA is significantly higher than that of individuals with the SNP4 genotype AG.

[0021] Preferably, the growth trait is chest depth: individuals with the SNP3 genotype GG have a significantly higher chest depth than individuals with the SNP5 genotype GG; individuals with the SNP6 genotype GG or AA have a significantly higher chest depth than individuals with the SNP6 genotype GA.

[0022] Preferably, the growth trait is body weight: individuals with the SNP3 genotype GG have a significantly higher body weight than individuals with the genotype AG; individuals with the SNP5 genotype GG have a significantly higher body weight than individuals with the genotype AG; and individuals with the SNP6 genotype GG or AA have a significantly higher body weight than individuals with the genotype GA.

[0023] Preferably, the reagent comprises 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.

[0024] Specifically, primers with nucleotide sequences as shown in SEQ ID NO: 1-2 are used to amplify the SNP1 and / or SNP2;

[0025] The primers with nucleotide sequences as shown in SEQ ID NO: 3-4 are used to amplify the SNP3, SNP4, SNP5 and / or SNP6.

[0026] The application of the kit in the assisted selection of growth traits and / or molecular breeding of Dwarf Chickens should also be within the scope of protection of this invention.

[0027] Preferably, the kit further includes PCR amplification reagents.

[0028] More preferably, the kit further includes one or more of dNTPs, PCR reaction buffer, and / or DNA polymerase.

[0029] A method for detecting growth traits in dwarf chickens, using the aforementioned kit to detect the SNP sites.

[0030] Preferably, the method includes the following steps:

[0031] S1. Extract genomic DNA from the chicken to be tested;

[0032] S2. Use the kit described above to detect the SNP sites in the genomic DNA extracted in step S1, and obtain sequencing data after sequencing.

[0033] S3. Analyze the sequencing data obtained in step S2, and determine the body shape traits of the chickens to be tested based on the SNP sites.

[0034] More preferably, in step S2, the sequencing is Sanger sequencing.

[0035] More preferably, the body shape characteristics mentioned in step S3 include body weight, keel length, chest width, chest depth, tibia length and / or body oblique length.

[0036] The application of the method in assisted selection of growth traits and / or molecular breeding of dwarf chickens should also be within the scope of protection of this invention.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] Through in-depth analysis of the dwarf chicken IGF2BP3 This invention identifies six single nucleotide polymorphism (SNP) sites that are significantly associated with the growth traits of dwarf chickens. These SNP sites can serve as molecular markers for precise breeding of dwarf chicken growth traits, providing a scientific basis for early-stage assisted selection of dwarf chicken growth traits. This invention provides a complete set of primers, kits, and operating methods for detecting these molecular markers. Using the kits and / or methods described in this invention, the genotypes of these six SNP sites can be rapidly and accurately detected, predicting key growth traits such as body weight, shank length, keel length, and chest width in dwarf chickens in the early stages of breeding. This provides strong technical support for the targeted breeding of dwarf chickens with different body shape characteristics. The detection method provided by this invention has high operability, ensuring rapid and accurate prediction results. This not only improves the efficiency of dwarf chicken germplasm resource improvement but also provides a solid data foundation for future molecular breeding and breed improvement of local varieties. Attached Figure Description

[0039] Figure 1 Electrophoresis results of single-sample PCR products from primer sets IGF2BP3-Exon5-6, IGF2BP3-Exon7, and IGF2BP3-Exon11.

[0040] Figure 2 Electrophoresis results of mixed-pool genomic DNA PCR products from primer sets IGF2BP3-Exon5-6, IGF2BP3-Exon7, and IGF2BP3-Exon11.

[0041] Figure 3 This is a schematic diagram of the primer regions for the IGF2BP3-Exon5-6 primer set and the IGF2BP3-Exon11 primer set.

[0042] 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.

[0043] 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.

[0044] Figure 6 The sequence peak diagram shows the genotype mutation locations of SNP sites: g.64420T>C, g.64447C>T, and g.80086A>C.

[0045] Figure 7 The sequence peak diagram shows the genotype mutation locations of SNP sites: g.80306A>G, ​​g.80431A>G, g.80447T>C, and g.80469G>T.

[0046] Figure 8 The sequence peak diagram shows the genotype mutation locations of SNP sites: g.80488A>G, g.80604C>T, and g.80635G>A.

[0047] 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.

[0048] Figure 10 For dwarf chickens 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.

[0049] Figure 11 For dwarf chickens IGF2BP3 Linkage disequilibrium analysis of 17 SNP sites in the gene 2 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

[0050] 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.

[0051] Example 1: Extraction and PCR amplification of dwarf chicken genomic DNA

[0052] I. Feeding and Management of Experimental Animals and Sample Collection

[0053] Using dwarf chickens as the research subject, they were fed according to broiler feeding standards, with free access to feed and water during the experiment, and were fed normally until 60 days of age. Ninety-three 60-day-old dwarf chickens were randomly selected, and their weight and growth traits were measured and recorded. The growth traits measured included: weight, keel length, shank length, shank circumference, body length, chest width, and chest depth. Three ml of blood was collected from the wing vein of each of the 93 selected 60-day-old dwarf chickens using a vacuum blood collection tube, anticoagulated with EDTA, and stored at -20℃ for later use.

[0054] II. Extraction and Purity Testing of Dwarf Chicken Blood DNA

[0055] (1) Experimental methods

[0056] Using a blood genomic DNA extraction kit, DNA was extracted from the blood samples collected in Section I, "Feeding and Management of Experimental Animals and Sample Collection," to obtain DNA samples. The integrity of the DNA samples was detected by gel electrophoresis, and the concentration and purity of the DNA samples were determined by a spectrophotometer (model ND-LITE, purchased from Genesys Pharma Ltd.).

[0057] (2) Experimental results

[0058] Gel electrophoresis results showed that the DNA sample bands were neat and free of tails and stray bands. Spectrophotometry results showed that the extracted DNA OD... 260 :OD 280 All values ​​are between 1.8 and 2.0, indicating that the DNA sample quality and purity meet the standards and can be used for subsequent experiments.

[0059] III. Design of Specific Primers

[0060] (1) Experimental methods

[0061] 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. The primer information is shown in Table 1. The primer sequences were synthesized by Shanghai Bioengineering (Sangon) Technology Service Co., Ltd. The primers shown in Table 1 were used to perform PCR on the DNA samples extracted in "II. Extraction and Purity Detection of Blood DNA from Dwarf Chickens" to test the primer specificity.

[0062] Table 1 IGF2BP3 Gene primer information

[0063]

[0064] (2) 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] like Figure 1 As shown, Figure 1 Lanes 1-6 in the middle of the gel electrophoresis diagram show the PCR products of DNA samples with primer set IGF2BP3-Exon5-6, lanes 7-12 show the PCR products of DNA samples with primer set IGF2BP3-Exon7, and lanes 13-18 show the electrophoresis results of DNA samples with PCR products with primer set IGF2BP3-Exon11. The PCR products were detected by 1.0% agarose gel electrophoresis, showing bright bands, good specificity, and no extraneous bands, meeting the requirements for directly determining the nucleotide sequence using PCR products. Primer sets IGF2BP3-Exon5-6, IGF2BP3-Exon7, and IGF2BP3-Exon11 are specific.

[0067] Example 2: Construction, PCR amplification, and sequencing of a mixed pool of dwarf chicken DNA.

[0068] I. Construction of DNA Mixture Pool and PCR Amplification

[0069] (1) Experimental methods

[0070] The DNA samples extracted in Example 1, "II. Extraction and Purity Detection of Blood DNA from Dwarf Chickens," were sorted from highest to lowest concentration. 5 μl of each extracted DNA sample was drawn into a 1.5 ml centrifuge tube to prepare a mixed pool. Ten DNA samples were used as one mixed pool, resulting in a total of nine mixed pools (each mixed pool contained 13 DNA samples). These mixed pools were then used as templates for PCR amplification. The amplification system is shown in Table 2.

[0071] 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.

[0072] 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 amplification products were then subjected to Sanger sequencing.

[0073] Table 2 PCR amplification system

[0074]

[0075] (2) Experimental results

[0076] like Figure 2 As shown, Figure 2 Lanes 1-5 in the middle section are electrophoretic bands of PCR amplification products of primer set IGF2BP3-Exon5-6, lanes 6-10 are electrophoretic bands of PCR amplification products of primer set IGF2BP3-Exon7, and lanes 11-15 are electrophoretic bands of PCR amplification products of primer set IGF2BP3-Exon11. The electrophoretic bands are bright, free of impurities, and have good specificity, meeting the requirements for Sanger sequencing using PCR amplification products.

[0077] II. Sanger sequencing and analysis of sequencing results

[0078] (1) Experimental methods

[0079] The PCR amplification products obtained in "I. Construction of DNA Mixture and PCR Amplification" were subjected to Sanger sequencing, using 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.

[0080] (2) Experimental results

[0081] The primer regions of the IGF2BP3-Exon5-6 primer set and the IGF2BP3-Exon11 primer set are as follows: Figure 3 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... IGF2BP3 Part of the gene consists of intron 10, part of intron 11, and exon 11.

[0082] 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 the 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.

[0083] Analysis results show that in dwarf 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 .

[0084] Table 3 Information of IGF2BP3 gene mutation sites

[0085]

[0086] Example 3 Analysis of the genetic structure of the Bantam chicken population

[0087] I. Experimental method

[0088] Use Excel in WPS Office software to calculate allele frequency, genotype frequency, genetic heterozygosity, effective number of alleles, polymorphism information content, and χ 2 value to detect whether the SNP locus is in Hardy-Weinberg (H-W) equilibrium. The calculation method refers to the literature (Liu Mei, Zhu Yixuan, Wang Xiaoyi, et al. SNP detection in exon 3 of the PID1 gene of Xuanhe pigs and its association analysis with meat quality traits [J]. Southwest China Journal of Agricultural Sciences, 2022(007):035.).

[0089] II. Experimental method

[0090] Perform genetic characteristic analysis on the SNP loci of the Bantam chicken IGF2BP3 gene. 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.

[0091] As can be seen from Table 4: IGF2BP3The dominant genotypes at the 17 SNP loci were TT (0.581), CC (0.828), GG (0.699), GG (0.925), TT (0.871), CC (0.935), AA (0.398), AA (0.720), AA (0.634), TT (0.624), GG (0.989), AA (0.720), CC (0.946), GG (0.710), AA (0.409), and AA (0.387). ) and TT (0.398); the corresponding dominant alleles are T (0.731), C (0.898), G (0.812), G (0.962), T (0.919), C (0.968), A (0.516), A (0.785), A (0.758), T (0.742), G (0.989), A (0.796), C (0.962), G (0.790), A (0.527), A (0.511) and T (0.505).

[0092] The PIC calculation values ​​indicate that among the 17 SNP sites, g.64077C>T, g.64412G>T, g.64420T>C, g.64447C>T, g.80469G>T, and g.80604C>T are low-degree polymorphisms, while the remaining sites are moderately polymorphic.

[0093] IGF2BP3 The Hardy-Weinberg equilibrium test results of gene SNP sites showed that the g.80086A>C and g.80644A>G sites did not deviate from Hardy-Weinberg equilibrium (P>0.05), while the remaining sites deviated significantly from Hardy-Weinberg equilibrium (P<0.05).

[0094] Table 4. Dwarf Chicken IGF2BP3 Analysis results of genetic characteristics of 17 SNP loci in the gene

[0095]

[0096]

[0097] Example 4: Dwarf Chicken IGF2BP3 Linkage disequilibrium and haplotype analysis of gene SNP sites

[0098] I. Experimental Methods

[0099] For dwarf chickens IGF2BP3 The 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.

[0100] II. Experimental Results

[0101] Dwarf Chicken IGF2BP3 The linkage disequilibrium 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.

[0102] As shown in Table 5: IGF2BP3 Of the 17 SNP sites in the gene,

[0103] g.64017T>G and g.64405G>A's r 2 The value is 0.630, r 2 The value is greater than 0.330, so there is a strong linkage disequilibrium between the sites g.64017T>G and g.64405G>A;

[0104] r of g.64077C>T with g.64405G>A and g.64420T>C 2 The values ​​are 0.491 and 0.555 respectively, r 2 The values ​​are all greater than 0.330, so there is a strong linkage disequilibrium between the sites g.64077C>T and g.64405G>A and g.64420T>C;

[0105] g.64412G>T and g.64447C>T's r 2 The value is 0.852, r 2 The value is greater than 0.330, so there is a strong linkage disequilibrium between the g.64412G>T and g.64447C>T sites;

[0106] 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 0.340, 0.371, 0.958, 0.979, and 0.958, respectively, r 2The 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;

[0107] g.80306A>G and g.80488A>G and g.80635G>A r 2 The values ​​are 0.937 and 0.786, 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;

[0108] r of g.80431A>G and g.80447T>C and g.80656A>T 2 The values ​​are 0.918 and 0.333, r 2 The values ​​are all greater than 0.330, so there is a strong linkage disequilibrium between the sites g.80431A>G and g.80447T>C and g.80656A>T;

[0109] g.80447T>C and g.80656A>T and g.80696T>G r 2 The values ​​are 0.363 and 0.355, r 2 The values ​​are all greater than 0.330, so there is a strong linkage disequilibrium between the g.80447T>C site and the g.80656A>T and g.80696T>G sites;

[0110] g.80488A>G and g.80635G>A's r 2 The value is 0.842, r 2 The value is greater than 0.330, so there is a strong linkage disequilibrium between the sites g.80488A>G and g.80635G>A;

[0111] r of g.80644A>G and g.80656A>T and g.80696T>G 2 The values ​​are 0.937 and 0.917, 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;

[0112] g.80656A>T and g.80696T>G's r 2 The value is 0.936, 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;

[0113] There were no strong linkage disequilibrium among the remaining sites.

[0114] Table 5. Dwarf Chicken IGF2BP3 Linkage disequilibrium analysis results among 17 SNP sites of the gene

[0115]

[0116] As shown in Table 6, the dwarf chickens shown in Table 5... IGF2BP3 At 17 specific SNP loci of the gene, 33 different haplotypes can be combined. These 33 haplotypes are named H1 to H33 in order of the number of haplotypes from most to least. Among them, there are 5 effective haplotypes (H1, H2, H3, H4 and H5) with a frequency greater than 0.03 in the population, with frequencies of 0.397, 0.135, 0.120, 0.042 and 0.030, respectively.

[0117] Table 6. Dwarf Chicken IGF2BP3 Haplotype analysis results of 17 SNP sites in the gene

[0118]

[0119] Example 5: Dwarf Chicken IGF2BP3 Association analysis between different gene genotypes and growth traits of dwarf chickens

[0120] I. Experimental Methods

[0121] IBM SPSS Statistics 26.0 software was used to analyze the dwarf chickens. IGF2BP3 Association analysis was performed on different genotypes of 17 SNP loci of the gene with growth traits of dwarf chickens.

[0122] II. Experimental Results

[0123] For dwarf chickens IGF2BP3 Association analysis was performed on different genotypes at 17 SNP loci of the gene and the growth traits of dwarf chickens. The results are shown in Table 7. In Table 7, data are compared between the same columns at the same locus. Different lowercase letters on the shoulder indicate significant differences (P<0.05), different uppercase letters on the shoulder indicate extremely significant differences (P<0.01), and no letters on the shoulder indicate no significant differences (P>0.05). Genotypes with fewer than 5 individuals were not counted.

[0124] As shown in Table 7, the 93 dwarf chickens selected in Example 1... IGF2BP3 Of the 17 SNP sites in the gene,

[0125] The three genotypes of g.64077C>T 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.64405G>A showed differences in keel length, with the GA genotype being significantly longer than the AA genotype (P<0.05).

[0127] The three genotypes of g.80306A>G showed differences in body weight, keel length, tibia length, body length, and chest depth. The GG genotype was significantly higher than the AG genotype in body weight, keel length, tibia length, body length, and chest depth (P<0.05).

[0128] The three genotypes of g.80431A>G showed differences in tibia length, with the AA genotype being significantly longer than the AG genotype (P<0.05).

[0129] The three genotypes of g.80488A>G showed differences in body weight and chest depth, with the GG genotype being significantly higher than the AG genotype in both (P<0.05).

[0130] The three genotypes of g.80635G>A showed differences in body weight and chest depth. The GG and AA genotypes were significantly higher than the GA genotype in both body weight and chest depth (P<0.05), while there was no significant difference between the GG and AA genotypes.

[0131] There were no significant differences in growth performance among the genotypes at other loci (P>0.05).

[0132] Table 7 Dwarf Chicken IGF2BP3 Growth traits of each genotype at 17 SNP loci of the gene

[0133]

[0134]

[0135] According to Table 7, the dwarf chicken... IGF2BP3 Six SNP loci associated with the growth traits of dwarf chickens were screened from 17 SNP loci in the gene (GenBank accession number: NC_052533.1).

[0136] SNP1 is located in IGF2BP3 At position 64077 (g.64077C>T) on the gene, which is a C or T polymorphism, the body weight of dwarf chickens with the SNP1 genotype CC is significantly higher than that of individuals with the SNP1 genotype TT.

[0137] SNP2 is located in IGF2BP3 At position 64405 of the gene (g.64405G>A), which is a G or A polymorphism, the keel length of dwarf chickens with the GA genotype at the SNP2 locus is significantly higher than that of individuals with the AA genotype at the SNP2 locus.

[0138] SNP3 is located in IGF2BP3 At position 80306 (g.80306A>G), which is an A or G polymorphism, the weight, keel length, shank length, body length and chest depth of the dwarf chickens with the SNP3 genotype GG are significantly higher than those with the SNP3 genotype AG.

[0139] SNP4 is located in IGF2BP3 At position 80431 of the gene (g.80431A>G), which is an A or G polymorphism, the shank length of dwarf chickens with the genotype AA at SNP4 is significantly longer than that of individuals with the genotype AG at SNP4.

[0140] SNP5 is located in IGF2BP3 At position 80488 (g.80488A>G), which is an A or G polymorphism, the body weight and chest depth of the dwarf chickens with the SNP5 genotype GG are significantly higher than those with the SNP5 genotype AG.

[0141] SNP6 is located in IGF2BP3 At position 80635 (g.80635G>A), which is a G or A polymorphism, the body weight and chest depth of dwarf chickens with the genotype GG or AA at SNP6 locus are significantly higher than those with the genotype GA at SNP6 locus.

[0142] Example 6: A kit for detecting growth traits of dwarf chickens

[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. Using the DNA extracted in step S1 as a template, perform a PCR reaction to obtain the PCR amplification product:

[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 64077 (g.64077C>T) on the gene. IGF2BP3 SNP3 is located at position 64405 on the gene (g.64405G>A). IGF2BP3 SNP4 is located at position 80306 on the gene (g.80306A>G). IGF2BP3 SNP5 is located at position 80431 on the gene (g.80431A>G). IGF2BP3 SNP6 is located at position 80488 on the gene (g.80488A>G). IGF2BP3 Position 80635 on the gene (g.80635G>A).

[0153] Dwarf chickens with the SNP1 genotype CC had significantly higher body weights than those with the SNP1 genotype TT.

[0154] The keel length of dwarf chickens with the GA genotype at the SNP2 locus was significantly longer than that of individuals with the AA genotype at the SNP2 locus.

[0155] The body weight, keel length, shank length, body length, and chest depth of the dwarf chickens with the SNP3 locus genotype GG were significantly higher than those with the SNP3 locus genotype AG.

[0156] The shank length of dwarf chickens with the genotype AA at the SNP4 locus was significantly longer than that of individuals with the genotype AG at the SNP4 locus.

[0157] The body weight and chest depth of dwarf chickens with the SNP5 genotype GG were significantly higher than those with the SNP5 genotype AG.

[0158] Dwarf chickens with the SNP6 genotype GG or AA had significantly higher body weight and chest depth than individuals with the SNP6 genotype GA.

[0159] Example 7: A method for detecting growth traits of dwarf chickens

[0160] A method for detecting growth traits in dwarf chickens, the specific steps of which are as follows:

[0161] S1. Extract DNA from the sample to be tested;

[0162] S2. Using the DNA extracted in step S1 as a template, perform a PCR reaction to obtain the PCR amplification product:

[0163] 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;

[0164] 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.

[0165] 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.

[0166] S4. Result Judgment: SNP1 is located in IGF2BP3 SNP2 is located at position 64077 (g.64077C>T) on the gene. IGF2BP3 SNP3 is located at position 64405 on the gene (g.64405G>A). IGF2BP3 SNP4 is located at position 80306 on the gene (g.80306A>G). IGF2BP3 SNP5 is located at position 80431 on the gene (g.80431A>G). IGF2BP3 SNP6 is located at position 80488 on the gene (g.80488A>G). IGF2BP3 Position 80635 on the gene (g.80635G>A).

[0167] Dwarf chickens with the SNP1 genotype CC had significantly higher body weights than those with the SNP1 genotype TT.

[0168] The keel length of dwarf chickens with the GA genotype at the SNP2 locus was significantly longer than that of individuals with the AA genotype at the SNP2 locus.

[0169] The body weight, keel length, shank length, body length, and chest depth of the dwarf chickens with the SNP3 locus genotype GG were significantly higher than those with the SNP3 locus genotype AG.

[0170] The shank length of dwarf chickens with the genotype AA at the SNP4 locus was significantly longer than that of individuals with the genotype AG at the SNP4 locus.

[0171] The body weight and chest depth of dwarf chickens with the SNP5 genotype GG were significantly higher than those with the SNP5 genotype AG.

[0172] Dwarf chickens with the SNP6 genotype GG or AA had significantly higher body weight and chest depth than individuals with the SNP6 genotype GA.

[0173] 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 use of reagents for detecting SNP loci associated with growth traits of Silkie chickens in the assisted selection of growth traits of Silkie chickens and / or the molecular breeding of Silkie chickens, characterized in that, The SNP site is located in the IGF2BP3 gene of the Silkie chicken, and the reagent detects one or more of the following SNP sites: SNP1 is located at position 31100862 of NC_052533.1, and is a C or T polymorphism; SNP2 is located at position 31100534 of NC_052533.1, and is a G or A polymorphism; SNP3 is located at position 31084633 of NC_052533.1, and is an A or G polymorphism; SNP4 is located at position 31084508 of NC_052533.1, and is an A or G polymorphism; SNP5 is located at position 31084451 of NC_052533.1, and is an A or G polymorphism; SNP6 is located at position 31084304 of NC_052533.1, and is a G or A polymorphism; The growth trait is chest width: the chest width of an individual with SNP1 genotype CC is significantly higher than that of an individual with genotype TT; The growth trait is keel length: the keel length of an individual with SNP2 genotype GA is significantly higher than that of an individual with genotype AA, and the keel length of an individual with SNP3 genotype GG is significantly higher than that of an individual with genotype AG; The growth trait is body slant length: the body slant length of an individual with SNP3 genotype GG is significantly higher than that of an individual with genotype AG; The growth trait is tibia length: the tibia length of an individual with SNP3 genotype GG is significantly higher than that of an individual with genotype AG, and the tibia length of an individual with SNP4 genotype AA is significantly higher than that of an individual with genotype AG; The growth trait is chest depth: the chest depth of an individual with SNP3 genotype GG is significantly higher than that of an individual with genotype AG, the chest depth of an individual with SNP5 genotype GG is significantly higher than that of an individual with genotype AG, and the chest depth of an individual with SNP6 genotype GG or AA is significantly higher than that of an individual with genotype GA; The growth trait is body weight: the body weight of an individual with SNP3 genotype GG is significantly higher than that of an individual with genotype AG, the body weight of an individual with SNP5 genotype GG is significantly higher than that of an individual with genotype AG, and the body weight of an individual with SNP6 genotype GG or AA is significantly higher than that of an individual with genotype GA.

2. Use according to claim 1, characterized in that, The reagent comprises 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.

3. A method of detecting a growth trait in a bantam chicken, the method comprising, The reagent of claim 1 is used to detect the SNP site described in claim 1; The growth trait is chest width: the chest width of an individual with SNP1 genotype CC is significantly higher than that of an individual with genotype TT; The growth trait is keel length: the keel length of an individual with SNP2 genotype GA is significantly higher than that of an individual with genotype AA, and the keel length of an individual with SNP3 genotype GG is significantly higher than that of an individual with genotype AG; The growth trait is body slant length: the body slant length of an individual with SNP3 genotype GG is significantly higher than that of an individual with genotype AG; The growth trait is shank length: the shank length of individuals with the SNP3 genotype of GG is significantly higher than that of individuals with the genotype of AG, and the shank length of individuals with the SNP4 genotype of AA is significantly higher than that of individuals with the genotype of AG; The growth trait is chest depth: the chest depth of individuals with the SNP3 genotype of GG is significantly higher than that of individuals with the genotype of AG, the chest depth of individuals with the SNP5 genotype of GG is significantly higher than that of individuals with the genotype of AG, and the chest depth of individuals with the SNP6 genotype of GG or AA is significantly higher than that of individuals with the genotype of GA; The growth trait is body weight: the body weight of individuals with the SNP3 genotype of GG is significantly higher than that of individuals with the genotype of AG, the body weight of individuals with the SNP5 genotype of GG is significantly higher than that of individuals with the genotype of AG, and the body weight of individuals with the SNP6 genotype of GG or AA is significantly higher than that of individuals with the genotype of GA.

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