Method for detecting growth traits of f2 generation of hybrid of yue-xi roll feather chicken and dwarf chicken and application thereof

Abstract

The application discloses a method for detecting growth traits of F2 generation chickens crossed by Guangxi coiled-tail chicken and dwarf chicken and application, the F2 generation chickens are F2 generation hens crossed by Guangxi coiled-tail chicken and dwarf chicken, the method is a combination of molecular markers SNP sites related to growth traits of the F2 generation hens crossed by Guangxi coiled-tail chicken and dwarf chicken, the molecular markers SNP sites are located on an IGF2BP3 gene and are significantly related to body weight, keel length and / or tibia length, and the method provided by the application provides a theoretical basis for early selection of high-quality F2 offspring and lays a foundation for constructing F2 generation hens crossed by Guangxi coiled-tail chicken and dwarf chicken with different body shapes. The application also establishes an evaluation kit for growth traits of the F2 generation hens crossed by Guangxi coiled-tail chicken and dwarf chicken by using the molecular markers SNP sites, and the kit is applied to the assistance of molecular breeding, so as to more conveniently provide basic data for future molecular selection of local breed chickens.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular breeding, and more particularly to a method for detecting the growth traits of F2 generation chickens of a cross between Yuexi juan'yan chickens and aiju chickens and application thereof. BACKGROUND

[0002] Some local chicken breeds have the traits of small body size and curly feathers, such as Yuexi juan'yan chickens and aiju chickens, which have unique genetic traits and genetic resources: Yuexi juan'yan chickens are adapted to high-temperature environments and have strong disease resistance and high roughage tolerance, and aiju chickens have high feed utilization rate and high egg production rate and delicious meat. By crossing Yuexi juan'yan chickens and aiju chickens, the excellent traits of Yuexi juan'yan chickens and aiju chickens can be combined to produce offspring with better growth performance and adaptability, create new breed resources, provide more choices for breeding work, and also provide better breeds for the market. However, the growth traits of the offspring are difficult to predict, and therefore it is not possible to guarantee stable production of high-quality F2 generation populations.

[0003] In the process of genetic breeding, molecular genetic markers are an effective method for improving breeding efficiency, and detecting single nucleotide polymorphisms (SNPs) is a key molecular genetic marker method. SNPs refer to the diversity of DNA sequences caused by the variation of a single nucleotide base at the genomic level. With the rapid development of animal molecular breeding technology and high-throughput sequencing technology, more and more genes have been discovered and applied to the regulation of reproductive traits and growth traits of livestock and poultry. The application of these technologies has greatly promoted the progress of livestock and poultry breeding work and provided strong support for improving breeds and production performance.

[0004] The insulin-like growth factor 2 mRNA binding protein family is a highly conserved post-transcriptional regulatory factor, and its family members include IGF2BP1, IGF2BP2 and IGF2BP3, and mainly participate in the processing, localization, translation and stability of RNA. IGF2 is considered to be a key gene related to growth and reproductive traits in many agricultural animals, and research has found that IGF2BP3 plays an important role in bone muscle development by regulating IGF2. However, there is currently little research on the polymorphism of IGF2BP3 genes in poultry, and there is no report on the F2 generation population of a cross between Yuexi juan'yan chickens and aiju chickens. SUMMARY

[0005] The purpose of the present application is to overcome the deficiencies of the prior art and provide a method for detecting the growth traits of F2 generation chickens of a cross between Yuexi juan'yan chickens and aiju chickens and application thereof.

[0006] The first object of the present application is to provide a method for detecting the growth traits of F2 generation hens of a cross between a Youxi coiled-tail chicken and a Silkie chicken.

[0007] The second object of the present application is to provide the use of a reagent for detecting the IGF2BP3 gene of F2 generation hens of a cross between a Youxi coiled-tail chicken and a Silkie chicken in evaluating, detecting and / or predicting the growth traits of the F2 generation hens.

[0008] The third object of the present application is to provide the use of a kit for detecting the growth traits of F2 generation hens of a cross between a Youxi coiled-tail chicken and a Silkie chicken in evaluating, detecting and / or predicting the growth traits of the F2 generation hens.

[0009] In order to achieve the above objects, the present application is implemented by the following technical solutions:

[0010] A method for detecting the growth traits of F2 generation hens of a cross between a Youxi coiled-tail chicken and a Silkie chicken, detecting the genotype of a single nucleotide polymorphism site of the F2 generation hens, the single nucleotide polymorphism site being located on the IGF2BP3 gene of the F2 generation hens, comprising single nucleotide polymorphism sites SNP1, SNP2 and / or SNP3, wherein,

[0011] the SNP1 is located at position 64405 on the NC_052533.1 gene, and is a G or A polymorphism;

[0012] the SNP2 is located at position 80656 on the NC_052533.1 gene, and is an A or T polymorphism;

[0013] the SNP3 is located at position 80696 on the NC_052533.1 gene, and is a T or G polymorphism.

[0014] Preferably, the genotype of the single nucleotide polymorphism site SNP1 of the F2 generation hens is detected, and the individual with the SNP1 genotype of GG has a body weight significantly higher than the individual with the genotype of AA.

[0015] Preferably, the genotype of the single nucleotide polymorphism site SNP2 of the F2 generation hens is detected, and the individual with the SNP2 genotype of AA has a body weight, keel length and shank length significantly higher than the individual with the genotype of TT; the individual with the SNP2 genotype of AT has a keel length and shank length significantly higher than the individual with the genotype of TT.

[0016] Preferably, the genotype of the single nucleotide polymorphism site SNP3 of the F2 generation hens is detected, and the individual with the SNP3 genotype of TT has a body weight, keel length and shank length significantly higher than the individual with the genotype of GG; the individual with the SNP3 genotype of TG has a shank length significantly higher than the individual with the genotype of GG.

[0017] Preferably, the method comprises the following steps:

[0018] S1. extracting genomic DNA of the F2 generation hens to be detected;

[0019] S2. sequencing the IGF2BP3 gene of the genomic DNA extracted in step S1 to obtain sequencing data;

[0020] S3. analyzing the sequencing data obtained in step S2 to determine the growth traits of the F2 generation hens to be detected, the growth traits including body weight, keel length and / or shank length.

[0021] More preferably, in step S2, the sequencing is Sanger sequencing.

[0022] More preferably, in step S3, the body shape traits include body weight, keel length and / or shank length.

[0023] The application of the method in the assisted selection or molecular breeding of the growth traits of the F2 generation hens of the cross between the Western Guangdong fowl and the Silkie chicken should also be within the protection scope of the present application.

[0024] The application of a reagent for detecting the IGF2BP3 gene of the F2 generation hens of the cross between the Western Guangdong fowl and the Silkie chicken in evaluating, detecting and / or predicting the growth traits of the F2 generation hens, the reagent detecting the single nucleotide polymorphism sites SNP1, SNP2 and / or SNP3.

[0025] Preferably, the reagent is a primer.

[0026] More preferably, the reagent detects the single nucleotide polymorphism site SNP1, and the reagent is a primer with a nucleotide sequence as shown in SEQ ID NO: 1-2.

[0027] More preferably, the reagent detects the single nucleotide polymorphism site SNP2 and / or SNP3, and the reagent is a primer with a nucleotide sequence as shown in SEQ ID NO: 3-4.

[0028] The application of a kit for detecting the growth traits of the F2 generation hens of the cross between the Western Guangdong fowl and the Silkie chicken in evaluating, detecting and / or predicting the growth traits of the F2 generation hens, the kit containing the reagent.

[0029] Preferably, the kit comprises a primer with a nucleotide sequence as shown in SEQ ID NO: 1-2 and / or a primer with a nucleotide sequence as shown in SEQ ID NO: 3-4.

[0030] More preferably, the kit further comprises a PCR amplification reagent.

[0031] Most preferably, the kit further comprises one or several of dNTPs, PCR reaction buffer and / or DNA polymerase.

[0032] The application of the kit in the assisted selection or molecular breeding of the growth traits of the F2 generation hens of the cross between the Western Guangdong fowl and the Silkie chicken should also be within the protection scope of the present application.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] The present application discloses a molecular marker SNP site combination closely related to the growth traits of the F2 generation hens of the cross between the Western Guangdong fowl and the Silkie chicken, which is located on the IGF2BP3 gene and has a significant correlation with the body weight, keel length and tibia length of the F2 generation hens.

[0035] Based on these findings, the present application establishes a method for detecting the growth traits of the F2 generation hens of the cross between the Western Guangdong fowl and the Silkie chicken. The method not only provides a basis for evaluating the growth traits, but also provides a solid theoretical support for the early selection of high-quality F2 offspring. It lays a solid foundation for constructing the F2 generation hens of the cross between the Western Guangdong fowl and the Silkie chicken with different ideal body shapes.

[0036] Using the molecular marker SNP site, the present application also develops a kit for the growth traits of the F2 generation hens of the cross between the Western Guangdong fowl and the Silkie chicken. The kit can be applied to evaluate, detect and / or predict the growth traits of the F2 generation hens, providing an auxiliary tool for the molecular breeding of the F2 generation hens of the cross between the Western Guangdong fowl and the Silkie chicken, making the molecular breeding process more convenient, providing more abundant basic data for the molecular selection of local chicken breeds, and helping to promote the development of related fields. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The electrophoresis result diagram of the PCR product of the individual blood DNA sample of the primer group IGF2BP3-Exon5-6, the primer group IGF2BP3-Exon7 and the primer group IGF2BP3-Exon11.

[0038] Figure 2 The electrophoresis result diagram of the PCR product of the pooled genomic DNA of the primer group IGF2BP3-Exon5-6, the primer group IGF2BP3-Exon7 and the primer group IGF2BP3-Exon11.

[0039] Figure 3 The A diagram in FIG. 1 is the pooled sequencing peak diagram of the primer group IGF2BP3-Exon7; Figure 3 The B diagram in FIG. 1 is the single sample sequencing peak diagram of the primer group IGF2BP3-Exon7.

[0040] Figure 4 Schematic diagram of the primer ranges of the IGF2BP3-Exon5-6 primer set and the IGF2BP3-Exon11 primer set.

[0041] Figure 5 The sequence peak diagram of the genotype mutation position of SNP sites: g.64017T>G, g.64077C>T and g.64405G>A.

[0042] Figure 6 The sequence peak diagram of the genotype mutation position of SNP sites: g.64420T>C, g.80086A>C and g.80306A>G.

[0043] Figure 7 The sequence peak diagram of the genotype mutation position of SNP sites: g.80431A>G, g.80447T>C and g.80635G>A.

[0044] Figure 8 The sequence peak diagram of the genotype mutation position of SNP sites: g.80644A>G, g.80656A>T and g.80696T>G.

[0045] Figure 9 These are the D′ values ​​of linkage disequilibrium analysis of 12 SNPs in the IGF2BP3 gene of F2 hens derived from a cross between a western Guangdong Dalmatian and a bantam. The colors of the blocks range from light to dark, indicating a low to high degree of linkage, with dark indicating complete linkage. The numbers in the blocks represent the logarithm of the probability of linkage disequilibrium between a given pair of markers.

[0046] Figure 10 Linkage disequilibrium analysis of 12 SNPs in the IGF2BP3 gene of F2 hens derived from a cross between Yuexi Dalmatian and Bantam chickens 2 The color of each square indicates the degree of linkage from low to high, with dark representing complete linkage. The numbers in the squares represent the logarithm of the probability of linkage disequilibrium between a given pair of markers. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0048] Example 1 Genetic polymorphism of F2 generation hens derived from hybridization of Yuexi Dalmatian and Bantam chickens

[0049] 1. Experimental Methods

[0050] The blood of 60-day-old F2 generation hens of Guangxi coiled chicken and dwarf chicken cross was collected, 3 ml of blood of each F2 generation hen was taken to extract DNA template; the IGF2BP3 gene was amplified by primer PCR, and the polymorphism of IGF2BP3 gene of F2 generation hens of Guangxi coiled chicken and dwarf chicken cross was found by Sanger sequencing technology.

[0051] (1) Sample

[0052] 82 60-day-old F2 generation hens of Guangxi coiled chicken and dwarf chicken cross were randomly selected to determine and record their body weight and growth traits, and the detected growth traits included body weight, keel length, shank length, shank circumference, body slant length, chest width and chest depth. The blood of the 82 60-day-old F2 generation hens was taken, 3 ml of blood was taken from each hen by intravenous vacuum blood collection tube, and EDTA was anticoagulated to obtain blood samples, which were stored at -20℃.

[0053] (2) Primer design

[0054] The chicken IGF2BP3 gene sequence (GenBank accession number: NC_052533.1) was obtained in the NCBI database, the primer for PCR was designed, and Shanghai Bioengineering (Shengong) Technology Service Co., Ltd. was responsible for synthesis, and the primer sequence is shown in Table 1.

[0055] Table 1 IGF2BP3 gene primer information

[0056]

[0057]

[0058] (3) Extraction of genomic DNA of blood samples of F2 generation hens of Guangxi coiled chicken and dwarf chicken cross

[0059] The DNA of the blood sample obtained in the present embodiment "(1) sample" was extracted by using a DNA kit, and after the individual blood DNA sample was extracted, the concentration and purity of the blood DNA sample were detected by using a spectrophotometer, and the blood DNA sample with required purity was selected for subsequent experiment.

[0060] According to the order of concentration from large to small, 5 μl to 1.5 ml of each of the individual blood DNA samples was taken into a centrifugal tube to make a pool, 10 blood DNA samples were a pool, and 8 pools (a certain pool contained 12 blood DNA samples) were prepared, which were stored at -20℃.

[0061] (4) PCR amplification and PCR product purification and sequencing

[0062] The individual blood DNA sample obtained in the present example "(3) Genomic DNA extraction from blood samples of F2 generation hens of hybridization of Yuexi coiled-tail chicken and Scruff chicken" was used as a template for PCR amplification to verify the specificity of the primers obtained in the present example "(2) Primer design". The PCR amplification system is shown in Table 2.

[0063] PCR reaction conditions: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 59°C annealing for 30 s, 72°C extension for 1 min, 32 cycles, 72°C extension for 10 min.

[0064] The PCR amplification product was detected by agarose gel electrophoresis. If the electrophoresis band is consistent with the size of the target band and clear and bright, it indicates that the primer has good specificity.

[0065] The primers with good specificity were selected, and the pool obtained in the present example "(3) Genomic DNA extraction from blood samples of F2 generation hens of hybridization of Yuexi coiled-tail chicken and Scruff chicken" was used as a template for PCR amplification. The PCR amplification system is shown in Table 2.

[0066] PCR reaction conditions: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 59°C annealing for 30 s, 72°C extension for 1 min, 32 cycles, 72°C extension for 10 min.

[0067] The PCR amplification product was detected by agarose gel electrophoresis. If the electrophoresis band is consistent with the size of the target band and clear and bright, the obtained PCR amplification product is subjected to Sanger sequencing.

[0068] Table 2 PCR amplification system

[0069]

[0070] II. Experimental results

[0071] The three primer groups IGF2BP3-P1, IGF2BP3-P2 and IGF2BP3-P3 designed for the 2000 bp promoter region of IGF2BP3 gene did not run the target band, and were discarded.

[0072] The primer specificity is shown in Figure 1 Figure 1 ​Lane 1-4: PCR products of individual blood DNA samples of primer set IGF2BP3-Exon5-6, Lane 5-8: PCR products of individual blood DNA samples of primer set IGF2BP3-Exon7, Lane 9-12: PCR products of individual blood DNA samples of primer set IGF2BP3-Exon11, The electrophoresis results of the PCR products were detected by 1.0% agarose gel electrophoresis, which showed that the electrophoresis bands were bright, specific and without impurity bands, and the primer set IGF2BP3-Exon5-6, primer set IGF2BP3-Exon7 and primer set IGF2BP3-Exon11 were specific.

[0073] The electrophoresis bands of the PCR amplification products of the primer set IGF2BP3-Exon5-6, primer set IGF2BP3-Exon7 and primer set IGF2BP3-Exon11 using the pool as a template were as shown in Figure 2 Figure 2 Lane 1 and Lane 2: PCR amplification product electrophoresis bands of primer set IGF2BP3-Exon5-6, Lane 3 and Lane 4: PCR amplification product electrophoresis bands of primer set IGF2BP3-Exon7, Lane 5 and Lane 6: PCR amplification product electrophoresis bands of primer set IGF2BP3-Exon11, as shown in Figure 2 , the electrophoresis bands were bright, without impurity bands and specific, which met the requirements of Sanger sequencing using the PCR amplification products.

[0074] As shown in Figure 3 , the sequencing peak chart of the primer set IGF2BP3-Exon7 (A in Figure 3 ) and the sequencing peak chart of the single blood DNA sample (B in Figure 3 ) were chaotic, so the results were discarded, and only the amplification results of the primer set IGF2BP3-Exon5-6 and primer set IGF2BP3-Exon11 were analyzed.

[0075] The primer intervals of the primer set IGF2BP3-Exon5-6 and primer set IGF2BP3-Exon11 were as shown in Figure 4 , the amplification fragment of the primer set IGF2BP3-Exon5-6 included part of the 4th intron, 5th intron, 6th intron, 5th exon and 6th exon of IGF2BP3 gene, and the amplification fragment of the primer set IGF2BP3-Exon11 included part of the 10th intron, 11th intron and 11th exon of IGF2BP3 gene.

[0076] ​Sanger sequencing analysis results showed that 12 SNP mutation sites were detected on the 4th, 5th, 6th, 10th, 11th introns and 11th exon of chicken IGF2BP3 gene, and the mutation site information is shown in Table 3; the sequence peak map of different genotype mutation positions is shown in Figures 5 to 8 .

[0077] Table 3 IGF2BP3 gene mutation site information

[0078]

[0079] Example 2 Genetic property analysis of IGF2BP3 gene related SNP sites of F2 generation hens of Guangxi coiled feather chicken and dwarf chicken cross

[0080] I. Experimental method

[0081] The allele frequency, genotype frequency, genetic heterozygosity, effective allele number, polymorphic information content and χ 2 value were calculated by using Excel in WPS Office software to detect whether the SNP site was in Hardy-Weinberg (H-W) equilibrium state, and the calculation method was referred to the literature (Liu M, Zhu YX, Wang XY, et al. SNP detection of exon 3 of Xuanhe pig PID1 gene and its correlation analysis with meat quality traits [J]. Southwestern Agricultural Sciences, 2022(007):035.).

[0082] II. Experimental results

[0083] The genetic property analysis of IGF2BP3 gene related SNP sites of F2 generation hens of Guangxi coiled feather chicken and dwarf chicken cross was carried out, and the results are shown in Table 4. PIC>0.5 is high polymorphism, 0.25<PIC<0.5 is moderate polymorphism, and PIC<0.25 is low polymorphism; χ 2 value test of 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.

[0084] From Table 4, the dominant genotypes of the 12 SNP sites of IGF2BP3 gene were TG (0.512), CT (0.524), GA (0.378), TC (0.561), CC (0.488), AA (0.963), AG (0.500), TC (0.488), GG (0.963), AG (0.476), TT (0.476) and GG (0.476), respectively; and the corresponding dominant alleles were G (0.634), C (0.726), A (0.530), T (0.683), C (0.683), A (0.982), G (0.591), C (0.598), G (0.982), G (0.652), T (0.665) and G (0.665), respectively.

[0085] PIC calculation results showed that g.80306A>G and g.80635G>A sites were low polymorphism, and the rest were moderate polymorphism.

[0086] Hardy-Weinberg equilibrium test results of the SNP sites of IGF2BP3 gene showed that g.64017T>G, g.64405G>A, g.80447T>C and g.80696T>G sites did not deviate from Hardy-Weinberg equilibrium (P>0.05), and the rest sites significantly deviated from Hardy-Weinberg equilibrium (P<0.05).

[0087] Table 4 Genetic characteristics analysis results of the SNP sites of IGF2BP3 gene of F2 generation hens of Yuxi coiled-tail chicken and dwarf chicken cross

[0088]

[0089]

[0090] Example 3 Linkage disequilibrium and haplotype analysis of the SNP sites of IGF2BP3 gene of F2 generation hens of Yuxi coiled-tail chicken and dwarf chicken cross

[0091] I. Experimental method

[0092] The linkage disequilibrium and haplotype of the 12 SNP sites of IGF2BP3 gene of F2 generation hens of Yuxi coiled-tail chicken and dwarf chicken cross were analyzed by using online software SHEsis, and the haplotype type and its frequency of IGF2BP3 gene were obtained.

[0093] II. Experimental results

[0094] The linkage disequilibrium results of the 12 SNP sites of IGF2BP3 gene of F2 generation hens of Yuxi coiled-tail chicken and dwarf chicken cross were as follows:Figure 9 , Figure 10 and Table 5, Figure 9 and Figure 10 The color of each block in the table from light to dark indicates the degree of linkage from low to high, and the dark color indicates complete linkage. The number in the block indicates the logarithm of the probability of linkage disequilibrium between the given markers, Figure 9 D' value, Figure 10 r 2 value, indicating the strength of linkage disequilibrium between two loci; in Table 5, the upper triangle is the D' value, and the lower triangle is the r 2 value; r 2 <0.330 indicates the absence of strong linkage disequilibrium.

[0095] From Table 5, among the 12 SNP loci of the IGF2BP3 gene,

[0096] The r 2 values of g.64017T>G and g.64405G>A, g.80086A>C, g.80431A>G, g.80447T>C, g.80644A>G, g.80656A>T and g.80696T>G are 0.607, 0.515, 0.644, 0.618, 0.625, 0.492 and 0.492, respectively, and the r 2 values are all greater than 0.330, so there is strong linkage disequilibrium between g.64017T>G and g.64405G>A, g.80086A>C, g.80431A>G, g.80447T>C, g.80644A>G, g.80656A>T and g.80696T>G loci;

[0097] The r 2 value of g.64077C>T and g.64420T>C is 0.649, and the r 2 value is greater than 0.330, so there is strong linkage disequilibrium between g.64077C>T and g.64420T>C loci;

[0098] The r 2 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 are 0.361, 0.400, 0.421, 0.393, 0.407 and 0.406, respectively, and the r 2 values are all greater than 0.330, so there is 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 loci;

[0099] g.80086A>C and g.80431A>G, g.80447T>C, g.80644A>G, g.80656A>T and g.80696T>G 2 The values ​​are 0.672, 0.689, 0.767, 0.866 and 0.866 respectively, r 2 The values ​​were all greater than 0.330, so there was strong linkage disequilibrium between g.80086A>C and g.80431A>G, g.80447T>C, g.80644A>G, g.80656A>T, and g.80696T>G loci;

[0100] g.80306A>G and g.80635G>A 2 The value is 1.000, r 2 The value is greater than 0.330, so there is strong linkage disequilibrium between the g.80306A>G and g.80635G>A loci;

[0101] g.80431A>G and g.80447T>C, g.80644A>G, g.80656A>T and g.80696T>G 2 The values ​​are 0.925, 0.771, 0.683 and 0.683 respectively, r 2 The values ​​were all greater than 0.330, so there was strong linkage disequilibrium between the g.80431A>G and g.80447T>C, g.80644A>G, g.80656A>T, and g.80696T>G loci;

[0102] g.80447T>C and g.80644A>G, g.80656A>T and g.80696T>G 2 The values ​​are 0.742, 0.749 and 0.749 respectively, r 2 The values ​​were all greater than 0.330, so there was strong linkage disequilibrium between the g.80447T>C and g.80644A>G, g.80656A>T, and g.80696T>G loci;

[0103] g.80644A>G and g.80656A>T and g.80696T>G 2 The values ​​are all 0.791, r 2 The values ​​are all greater than 0.330, so there is strong linkage disequilibrium between the g.80644A>G and g.80656A>T and g.80696T>G loci;

[0104] g.80656A>T and g.80696T>G 2 The value is 0.946, r2 The value is greater than 0.330, so there is strong linkage disequilibrium between the g.80656A>T and g.80696T>G sites;

[0105] There is no strong linkage disequilibrium between the remaining sites.

[0106] Table 5 Analysis results of linkage disequilibrium between 12 SNP sites of IGF2BP3 gene of F2 generation hens of Guangxi roll-feather chicken and dwarf chicken cross

[0107]

[0108]

[0109] As shown in Table 6, 27 different haplotypes can be combined from the 12 specific SNP sites of IGF2BP3 gene of F2 generation hens of Guangxi roll-feather chicken and dwarf chicken cross shown in Table 5, which are sequentially named H1-H27 from the most to the least in terms of quantity, and there are 5 effective haplotypes (with a frequency greater than 0.03 in the population) H1, H2, H3, H4 and H5, and their frequencies are 0.261, 0.231, 0.205, 0.070 and 0.036, respectively.

[0110] Table 6 Analysis results of haplotype of 12 SNP sites of IGF2BP3 gene of F2 generation hens of Guangxi roll-feather chicken and dwarf chicken cross

[0111]

[0112] Example 4 Correlation analysis of different genotypes of IGF2BP3 gene and growth traits of F2 generation hens of Guangxi roll-feather chicken and dwarf chicken cross

[0113] I. Experimental method

[0114] IBM SPSS Statistics 26.0 software was used to perform correlation analysis of different genotypes of 12 SNP sites of IGF2BP3 gene and growth traits of F2 generation hens of Guangxi roll-feather chicken and dwarf chicken cross.

[0115] II. Experimental results

[0116] The correlation analysis results of different genotypes of 12 SNP sites of IGF2BP3 gene and growth traits of F2 generation hens of Guangxi roll-feather chicken and dwarf chicken cross are shown in Table 7, and the data in the same column of the same site in Table 7 are compared, and the different lower letters in the superscript represent significant differences (P<0.05), the different upper letters in the superscript represent extremely significant differences (P<0.01), and the absence of letters in the superscript represents no significant difference (P>0.05), and the genotypes with less than 5 individuals are not counted.

[0117] From Table 7, among the 82 F2 generation hens selected from Example 1, 12 SNP sites of the IGF2BP3 gene,

[0118] g. The three genotypes of g.64405G>A had significant differences in body weight, and the GG genotype was significantly higher than the AA genotype (P<0.05);

[0119] g. The three genotypes of g.80656A>T had significant differences in body weight, keel length and shank length, and the AA genotype was significantly higher than the TT genotype in body weight (P<0.05), and the AA and AT genotypes were significantly higher than the TT genotype in keel length and shank length (P<0.05), and there was no significant difference between the AA and AT genotypes;

[0120] g. The three genotypes of g.80696T>G had significant differences in body weight, keel length and shank length, and the TT genotype was significantly higher than the GG genotype in body weight and keel length (P<0.05), and the TT and TG genotypes were significantly higher than the GG genotype in shank length (P<0.05), and there was no significant difference between the TT and TG genotypes;

[0121] There was no significant difference in growth traits between genotypes of the remaining sites (P>0.05).

[0122] Table 7 Growth traits of each genotype of 12 SNP sites of IGF2BP3 gene in F2 generation hens of cross between Youxi coiled-tail chicken and dwarf chicken

[0123]

[0124]

[0125] According to Table 7, among the 12 SNP sites of IGF2BP3 gene (GenBank accession number: NC_052533.1) of F2 generation hens of cross between Youxi coiled-tail chicken and dwarf chicken, 3 SNP sites related to growth traits of F2 generation hens were screened out, wherein,

[0126] SNP1 is located at position 64405 (g.64405G>A) of IGF2BP3 gene, which is a G or A polymorphism, and the body weight of individuals with SNP1 genotype of GG is significantly higher than that of individuals with SNP1 genotype of AA;

[0127] SNP2 is located at position 80656 (g.80656A>T) of IGF2BP3 gene, which is an A or T polymorphism, and the body weight, keel length and shank length of individuals with SNP2 genotype of AA are significantly higher than those of individuals with genotype of TT; the keel length and shank length of individuals with SNP2 genotype of AT are significantly higher than those of individuals with SNP2 genotype of TT;

[0128] SNP3 is located at g.80696T>G of IGF2BP3 gene, which is T or G polymorphism, the body weight, sitting height and tibia length of individuals with TT genotype at SNP3 site are significantly higher than those of individuals with GG genotype; the tibia length of individuals with TG genotype at SNP3 site is significantly higher than that of individuals with GG genotype at SNP3 site.

[0129] Example 5 A kit for evaluating, detecting and / or predicting the growth traits of F2 generation hens of Guangxi roll feather chicken and dwarf chicken cross

[0130] I. Composition

[0131] The primer with the nucleotide sequence as shown in SEQ ID NO: 1-2, the primer with the nucleotide sequence as shown in SEQ ID NO: 3-4, Taq PCR Mix and Nuclease-free Water.

[0132] II. Method of use

[0133] S1. Extract DNA from the sample to be tested;

[0134] S2. Perform PCR reaction with the DNA extracted in step S1 as a template to obtain PCR amplification product:

[0135] PCR amplification system (total volume is 20 μl): Taq PCR Mix 10 μl, primer with nucleotide sequence as shown in SEQ ID NO: 1-2 or primer with nucleotide sequence as shown in SEQ ID NO: 3-4 each 0.5 μl, DNA extracted in step S1 1 μl, Nuclease-free Water 8 μl;

[0136] 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;

[0137] S3. Perform agarose gel electrophoresis detection on the PCR amplification product obtained in step S2, if the electrophoresis band is consistent with the size of the target band and clear and bright, then perform sequencing on the PCR amplification product obtained in step S2.

[0138] III. Result judgment

[0139] SNP1 is located at g.64405G>A of IGF2BP3 gene, SNP2 is located at g.80656A>T of IGF2BP3 gene, and SNP3 is located at g.80696T>G of IGF2BP3 gene.

[0140] Individuals with SNP1 genotype of GG have significantly higher body weight than individuals with SNP1 genotype of AA.

[0141] Individuals with SNP2 genotype of AA have significantly higher body weight, keel length and shank length than individuals with SNP2 genotype of TT.

[0142] Individuals with SNP3 genotype of TT have significantly higher body weight, keel length and shank length than individuals with SNP3 genotype of GG.

[0143] Individuals with SNP2 genotype of TT have significantly lower keel length and shank length than individuals with SNP2 genotype of AT.

[0144] Individuals with SNP3 genotype of GG have significantly lower shank length than individuals with SNP3 genotype of TG.

[0145] A method for evaluating, detecting and / or predicting the growth traits of F2 generation hens of Guangxi roll chicken and dwarf chicken cross

[0146] A method for evaluating, detecting and / or predicting the growth traits of F2 generation hens of Guangxi roll chicken and dwarf chicken cross, the specific steps are as follows:

[0147] S1. Extract the DNA of the sample to be tested;

[0148] S2. Perform PCR reaction with the DNA extracted in step S1 as template to obtain PCR amplification product:

[0149] PCR amplification system (total volume is 20 μl): Taq PCR Mix 10 μl, primers with nucleotide sequence as shown in SEQ ID NO: 1-2 or primers with nucleotide sequence as shown in SEQ ID NO: 3-4 each 0.5 μl, 1 μl of DNA extracted in step S1, Nuclease-free Water 8 μl;

[0150] 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;

[0151] S3. Perform agarose gel electrophoresis detection on the PCR amplification product obtained in step S2, if the electrophoresis band is consistent with the size of the target band and clear and bright, then perform sequencing on the PCR amplification product obtained in step S2;

[0152] S4. Result judgment: SNP1 is located at the 64405th position (g.64405G>A) of IGF2BP3 gene, SNP2 is located at the 80656th position (g.80656A>T) of IGF2BP3 gene, and SNP3 is located at the 80696th position (g.80696T>G) of IGF2BP3 gene.

[0153] The body weight of the individual with SNP1 genotype of GG is significantly higher than that of the individual with SNP1 genotype of AA.

[0154] The body weight, keel length and tibia length of the individual with SNP2 site genotype of AA are significantly higher than those of the individual with SNP2 site genotype of TT.

[0155] The body weight, keel length and tibia length of the individual with SNP3 site genotype of TT are significantly higher than those of the individual with SNP3 site genotype of GG.

[0156] The keel length and tibia length of the individual with SNP2 site genotype of TT are significantly lower than those of the individual with SNP2 site genotype of AT.

[0157] The tibia length of the individual with SNP3 site genotype of GG is significantly lower than that of the individual with SNP3 site genotype of TG.

[0158] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A method for detecting growth traits of F2 generation hens hybridized from Yuexi Dalmatian chickens and bantam chickens, characterized in that: Detect the genotype of the single nucleotide polymorphism site of the F2 generation hen, wherein the single nucleotide polymorphism site is located at the F2 generation hen IGF2BP3 Genetically, it is single nucleotide polymorphism site SNP1, SNP2 and / or SNP3, wherein, The SNP1 is located at position 31100534 on NC_052533.1 and is a G or A polymorphism; The SNP2 is located at position 31084283 on NC_052533.1 and is an A or T polymorphism; The SNP3 is located at position 31084243 on NC_052533.1 and is a T or G polymorphism; The weight of individuals with SNP1 genotype GG is significantly higher than that of individuals with genotype AA; The body weight, keel length and tibia length of individuals with the SNP2 genotype of AA were significantly higher than those with the genotype of TT; The keel length and tibia length of individuals with the SNP2 genotype of AT were significantly longer than those with the genotype of TT; The body weight, keel length and tibia length of individuals with the SNP3 genotype TT were significantly higher than those with the genotype GG; The tibia length of individuals with the SNP3 genotype of TG was significantly longer than that of individuals with the SNP3 genotype of GG.

2. The method according to claim 1, characterized in that The following steps are involved: S1. Extracting genomic DNA from the F2 generation hen to be tested; S2. The genomic DNA extracted in step S1 IGF2BP3 The gene is sequenced to obtain sequencing data; S3. Analyze the sequencing data obtained in step S2 to determine the growth traits of the F2 generation hens to be tested, where the growth traits are body weight, keel length and / or tibia length.

3. Use of a reagent for detecting growth traits of F2 generation hens hybridized from Yuexi Dalmatian chickens and bantam chickens in evaluating, detecting and / or predicting the growth traits of the F2 generation hens, characterized in that: The reagent detects single nucleotide polymorphism sites SNP1, SNP2 and / or SNP3; The SNP1 is located at position 31100534 on NC_052533.1 and is a G or A polymorphism; The SNP2 is located at position 31084283 on NC_052533.1 and is an A or T polymorphism; The SNP3 is located at position 31084243 on NC_052533.1 and is a T or G polymorphism; The weight of individuals with SNP1 genotype GG is significantly higher than that of individuals with genotype AA; The body weight, keel length and tibia length of individuals with the SNP2 genotype of AA were significantly higher than those with the genotype of TT; The keel length and tibia length of individuals with the SNP2 genotype of AT were significantly longer than those with the genotype of TT; The body weight, keel length and tibia length of individuals with the SNP3 genotype of TT were significantly higher than those with the genotype of GG; The tibia length of individuals with the SNP3 genotype of TG was significantly longer than that of individuals with the SNP3 genotype of GG.

4. The use according to claim 3, characterized in that The reagent is a primer.

5. The use according to claim 4, characterized in that The reagent detects the single nucleotide polymorphism site SNP1, and the reagent is a primer having a nucleotide sequence as shown in SEQ ID NO: 1-2.

6. The use according to claim 4, characterized in that The reagent detects single nucleotide polymorphism sites SNP2 and / or SNP3, and the reagent is a primer having a nucleotide sequence as shown in SEQ ID NOs: 3-4.

7. A kit for detecting growth traits of F2 generation hens hybridized from Yuexi Dalmatian chickens and bantam chickens, for use in evaluating, detecting and / or predicting growth traits of the F2 generation hens, characterized in that: The kit detects single nucleotide polymorphism sites SNP1, SNP2 and / or SNP3; The SNP1 is located at position 31100534 on NC_052533.1 and is a G or A polymorphism; The SNP2 is located at position 31084283 on NC_052533.1 and is an A or T polymorphism; The SNP3 is located at position 31084243 on NC_052533.1 and is a T or G polymorphism; The weight of individuals with SNP1 genotype GG is significantly higher than that of individuals with genotype AA; The body weight, keel length and tibia length of individuals with the SNP2 genotype of AA were significantly higher than those with the genotype of TT; The keel length and tibia length of individuals with the SNP2 genotype of AT were significantly longer than those with the genotype of TT; The body weight, keel length and tibia length of individuals with the SNP3 genotype of TT were significantly higher than those with the genotype of GG; The tibia length of individuals with the SNP3 genotype of TG was significantly longer than that of individuals with the SNP3 genotype of GG.

Images