Molecular marker, detection reagent and method for breeding of seven hundred wonder rooster and application thereof

Abstract

The application discloses a molecular marker, a detection reagent, a method and application thereof for Qibanlong cock breeding. A SNP site and a haplotype molecular marker which are significantly related to the growth trait of Qibanlong cock are screened from an exon of a HTR1B gene of the Qibanlong cock, and the molecular marker can be applied to early assisted selection of Qibanlong cock breeding. The application further provides a detection reagent, a kit and a detection method for detecting the SNP site and the haplotype molecular marker, and the growth trait of the Qibanlong cock can be quickly, effectively and accurately predicted, so that the production cost is saved and the breeding process is accelerated, and the application has great economic application value and scientific research value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular breeding, in particular, relates to a molecular marker for Qianbannong rooster breeding, a detection reagent, a method and application thereof. BACKGROUND

[0002] Qianbannong chicken is a characteristic geographical indication breed in Dahua Yao Autonomous County of Hechi City, Guangxi Zhuang Autonomous Region. The rooster has a unique and bright feather color with golden yellow snowflake as the main color, and the hen has a unique feather color of flaxen snowflake. Because it also has the advantages of strong adaptability, tolerance to roughage, strong disease resistance, small body size, less subcutaneous fat, firm and elastic muscle, delicious and sweet meat, and rich nutrition, it is deeply favored by local breeders and consumers.

[0003] The temperament of livestock and poultry refers to the reaction of livestock and poultry to the operation of the manager, mainly the fear reaction, i.e., the stress reaction. In the daily production of animal husbandry, the temperament of livestock and poultry not only affects the management, but also affects the yield and quality of livestock and poultry products. 5-hydroxytryptamine (5-HT) is an important neurotransmitter in the regulation of animal behavior, which can regulate various behaviors including sleep, fear, aggression, emotion and feeding. 5-hydroxytryptamine 1B receptor (5-HT1B) is a G protein-coupled receptor that binds to serotonin, activates second messengers, inhibits the activity of adenylyl cyclase, and controls the release of serotonin, dopamine and acetylcholine in the brain, and is believed to be related to various emotional and mental problems. Mice with a deletion of 5-hydroxytryptamine 1B receptor gene (HTR1B) have increased aggression and reactivity. Continuous intraperitoneal injection of HTR1B antagonist GR-127935 into 24-week-old hens for 5 days can increase the frequency of feather pecking and aggressive behavior. Beef cattle with a calm temperament have higher average daily gain and slaughter rate than those with a temperamental temperament. In addition, when livestock and poultry experience stress, the body consumes a large amount of energy to cope with the stimulus, resulting in a decrease in body weight and feed conversion rate of livestock and poultry. Therefore, HRT1B may play an important role in the regulation of animal growth, slaughter performance and meat quality.

[0004] Single nucleotide polymorphism (SNP) belongs to the third generation of molecular markers, which has the characteristics of wide distribution, large density, strong representation and genetic stability, and is widely used in plant and animal breeding, disease resistance gene marker, superior variety screening and disease related gene identification. The chicken HTR1B gene (GenBank: NC_052534.1) is located on chromosome 3, and the full length of the gene is 3049bp, and there are two exons, and the full length of the exon is 2437bp. At present, the research on the polymorphism of HTR1B gene mainly focuses on human, mouse and cow, and the research on chicken has not been reported so far. SUMMARY

[0005] In order to overcome the above-mentioned defects and deficiencies existing in the prior art, the present application provides a molecular marker for Qianbai Nong cock breeding, a detection reagent, a method and application thereof.

[0006] The first object of the present application is to provide a molecular marker for Qianbai Nong cock breeding.

[0007] The second object of the present application is to provide a detection reagent for detecting the above-mentioned molecular marker.

[0008] The third object of the present application is to provide the application of the above-mentioned molecular marker and / or detection reagent in the preparation of Qianbai Nong cock breeding products.

[0009] The fourth object of the present application is to provide a kit for Qianbai Nong cock breeding.

[0010] The fifth object of the present application is to provide a method for Qianbai Nong cock breeding.

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

[0012] A molecular marker for Qianbai Nong cock breeding, which is composed of five single nucleotide polymorphism sites SNP1, SNP2, SNP3, SNP4 and / or SNP5:

[0013] SNP1 is located at position 79776832 on NC_052534.1 gene, which is C or T polymorphism, and there are three genotypes of CC, CT and TT;

[0014] SNP2 is located at position 79776916 on NC_052534.1 gene, which is G or A polymorphism, and there are three genotypes of GG, GA and AA;

[0015] SNP3 is located at position 79777422 on NC_052534.1 gene, which is T or C polymorphism, and there are three genotypes of TT, TC and CC;

[0016] SNP4 is located at position 79778162 of NC_052534.1 gene, which is A or G polymorphism, and there are three genotypes of AA, AG and GG;

[0017] SNP5 is located at position 79778500 of NC_052534.1 gene, which is A or C polymorphism, and there are three genotypes of AA, AC and CC.

[0018] Preferably, the single nucleotide polymorphism site SNP1 and the single nucleotide polymorphism site SNP2 construct three haplotypes:

[0019] The H1 haplotype is CG, the H2 haplotype is CA, and the H3 haplotype is TG.

[0020] The haplotypes construct haplotype combinations, and the haplotype combinations are respectively:

[0021] The H1H1 haplotype combination is CG / CG, the H1H2 haplotype combination is CG / CA, the H1H3 haplotype combination is CG / TG, and the H2H2 haplotype combination is CA / CA.

[0022] Preferably, the single nucleotide polymorphism site SNP3, the single nucleotide polymorphism site SNP4 and the single nucleotide polymorphism site SNP5 construct four haplotypes:

[0023] The H4 haplotype is CAA, the H5 haplotype is TAA, the H6 haplotype is TGA, and the H7 haplotype is TAC.

[0024] The haplotypes construct haplotype combinations, and the haplotype combinations are respectively:

[0025] The H4H4 haplotype combination is CAA / CAA, the H5H4 haplotype combination is TAA / CAA, the H5H5 haplotype combination is TAA / TAA, the H5H6 haplotype combination is TAA / TGA, and the H5H7 haplotype combination is TAA / TAC.

[0026] The detection reagent for detecting the above-mentioned molecular markers is a primer with a nucleotide sequence as shown in SEQ ID NO: 3-8;

[0027] The primer with a nucleotide sequence as shown in SEQ ID NO: 3-4 is used for detecting the SNP1 and / or SNP2 single nucleotide polymorphism site, the primer with a nucleotide sequence as shown in SEQ ID NO: 5-6 is used for detecting the SNP3 single nucleotide polymorphism site, and the primer with a nucleotide sequence as shown in SEQ ID NO: 7-8 is used for detecting the SNP4 and / or SNP5 single nucleotide polymorphism site.

[0028] The application of the molecular marker and / or detection reagent in the preparation of Qianbannong cock breeding products.

[0029] A kit for Qianbannong cock breeding, containing the detection reagent described above.

[0030] A method for Qianbannong cock breeding, detecting one or more of the single nucleotide polymorphism sites described above.

[0031] As an implementable way, detecting the SNP1, SNP2, SNP3 and / or SNP4 single nucleotide polymorphism sites described above;

[0032] The body weight of an individual with the genotype CT or TT at the SNP1 site is significantly higher than that of an individual with the genotype CC, the shank circumference of an individual with the genotype TT is extremely significantly higher than that of an individual with the genotype CC, the shank circumference of an individual with the genotype TT is significantly higher than that of an individual with the genotype CT, and the chest depth of an individual with the genotype TT is significantly higher than that of an individual with the genotype CC;

[0033] The chest width of an individual with the genotype GG at the SNP2 site is significantly higher than that of an individual with the genotype GA;

[0034] The body weight or body length of an individual with the genotype TT or TC at the SNP3 site is significantly higher than that of an individual with the genotype CC, the chest width of an individual with the genotype TT or TC is extremely significantly higher than that of an individual with the genotype CC, the cannon bone length of an individual with the genotype TC is significantly higher than that of an individual with the genotype CC, and the pelvic width of an individual with the genotype TT is significantly higher than that of an individual with the genotype CC;

[0035] The body weight of an individual with the genotype AG at the SNP4 site is extremely significantly higher than that of an individual with the genotype AA, the shank circumference of an individual with the genotype GG is extremely significantly higher than that of an individual with the genotype AA, and the shank length, chest width or chest depth of an individual with the genotype AG is significantly higher than that of an individual with the genotype AA.

[0036] As an implementable way, detecting the haplotype combination described above;

[0037] The body weight of an individual with the H1H3 haplotype combination is significantly higher than that of an individual with the H1H1 or H1H2 haplotype combination;

[0038] The body length of an individual with the H1H3 haplotype combination is significantly higher than that of an individual with the H1H1 haplotype combination;

[0039] The cannon bone length of an individual with the H1H3 haplotype combination is significantly higher than that of an individual with the H1H1 haplotype combination or H2H2 haplotype combination.

[0040] As an implementable way, detecting the haplotype combination described above;

[0041] the individual with the H5H6 haplotype combination has a body weight that is significantly higher than the individual with the H4H4 haplotype combination;

[0042] the individual with the H5H6 haplotype combination has a body weight that is significantly higher than the individual with the H5H4 haplotype combination;

[0043] the individual with the H5H4, H5H5 or H5H6 haplotype combination has a body length that is significantly higher than the individual with the H4H4 haplotype combination;

[0044] the individual with the H5H6 haplotype combination has a shank length that is significantly higher than the individual with the H5H5 haplotype combination;

[0045] the individual with the H5H6 or H5H7 haplotype combination has a shank girth that is significantly higher than the individual with the H4H5 or H5H5 haplotype combination;

[0046] the individual with the H5H4, H5H5, H5H6 or H5H7 haplotype combination has a chest width that is extremely significantly higher than the individual with the H4H4 haplotype combination;

[0047] the individual with the H5H6 haplotype combination has a chest width that is significantly higher than the individual with the H5H5 haplotype combination;

[0048] the individual with the H5H6 haplotype combination has a chest depth that is significantly higher than the individual with the H4H4, H5H4 or H5H7 haplotype combination;

[0049] the individual with the H5H6 or H5H7 haplotype combination has a pelvic width that is significantly higher than the individual with the H4H4 haplotype combination.

[0050] Preferably, the detection is performed using the detection reagent described above.

[0051] As an implementable mode, the method comprises the following steps:

[0052] S1. extracting genomic DNA of a Qianbailong rooster to be tested;

[0053] S2. performing PCR amplification on the genomic DNA obtained in step S1 using the detection reagent described above, and then performing sequencing on the PCR amplification product to obtain sequencing data;

[0054] S3. analyzing the sequencing data obtained in step S2 to determine the traits of the Qianbailong rooster to be tested.

[0055] Preferably, the traits in step S3 include body weight, shank length, shank girth, chest depth, chest width, body length, keel length and / or pelvic width.

[0056] As an implementable mode, the sequencing is Sanger sequencing.

[0057] Compared with the prior art, the application has the following beneficial effects:

[0058] The application discloses a molecular marker, a detection reagent, a method and application thereof for Qibanlong cock breeding. The application screens a SNP site and a haplotype molecular marker which are significantly related to growth traits of Qibanlong cocks in exons of HTR1B genes of the Qibanlong cocks, and the molecular marker can be applied to early assisted selection of Qibanlong cock breeding. The application further provides a detection reagent, a kit and a detection method for detecting the SNP site and the haplotype molecular marker, and the growth traits of the Qibanlong cocks can be quickly, effectively and accurately predicted, so that production cost is saved and breeding progress is accelerated, and the application has great economic application value and scientific research value. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 Fig. 1 is an agarose gel electrophoresis result of PCR amplification products, wherein lane 1 is a Marker, and lanes 2-6 are PCR product amplification results of HTR1B-Exon1-1, HTR1B-Exon1-2, HTR1B-Exon1-3, HTR1B-Exon2-1 and HTR1B-Exon2-2 respectively.

[0060] Figure 2 Fig. 4 is a sequencing peak chart of six SNP sites of the HTR1B gene.

[0061] Figure 3 Fig. 5 is a linkage disequilibrium analysis result of the six SNP sites of the HTR1B gene; A is a LD linkage chart, and the color of the block from light to dark represents the linkage degree from low to high, and the numerical value represents the strength of linkage disequilibrium between sites (numerical value = D' value x 100), B is haplotype analysis, and the thicker the line is, the more likely the adjacent haplotypes are combined. DETAILED DESCRIPTION

[0062] The application will be further described below in combination with specific embodiments, but the embodiments do not limit the application in any form. Unless otherwise specified, the reagents, methods and devices used in the application are conventional reagents, methods and devices in the technical field.

[0063] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0064] In the example, a healthy Qibanlong chicken population (♂=193, ♀=156) of the same age was selected as the research object, and all the chickens came from the poultry test base of the Guangxi Vocational and Technical University of Animal Husbandry. During the test, the chickens were raised in a three-dimensional cage mode, and the chickens were free to eat and drink water, the temperature was suitable, the indoor ventilation was good, the chickens were routinely immunized, and the chickens were fed until 120 days old.

[0065] Example 1 Amplification of HTR1B gene exons of Qianbannong chicken

[0066] I. Experimental methods

[0067] 1. Extraction and purity detection of blood DNA

[0068] 2 mL of blood was collected from the wing vein of Qianbannong chicken using a vacuum blood collection tube with EDTA anticoagulant. DNA was extracted using a blood genomic DNA extraction kit, and the integrity of the DNA was detected by 1% agarose gel electrophoresis (w / v). The OD value was measured by spectrophotometer, and the OD 260 : OD 280 value was used to determine the purity and concentration of the DNA, and the DNA of Qianbannong chicken was obtained.

[0069] 2. Primer design

[0070] Using the chicken HTR1B gene sequence (GenBank: NC_052534.1) in the NCBI database as the reference sequence, the exons were designed for specific amplification primers using Viewer 2.3.4 software, and the primer sequences are shown in Table 1. The primers were synthesized by Shanghai Bioengineering (Shengong) Technology Service Co., Ltd.

[0071] Table 1 Primer sequences of HTR1B gene

[0072]

[0073] 3. PCR amplification

[0074] After diluting the DNA samples of different Qianbannong chickens to the same concentration, 1 μL of each was taken into a 1.5 mL centrifuge tube to make a DNA pool.

[0075] The PCR amplification reaction was carried out using this as the template, and the PCR reaction system was as follows: PCR SuperMix 10 μL, 1 μL of each upstream and downstream primer (10 μmol·L -1 ), 1 μL of pool genomic DNA template, and Nuclease-free Water to 20 μL.

[0076] PCR reaction conditions: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 1 min, 35 cycles, 72°C extension for 10 min, and the PCR product was stored at 4°C and used for subsequent sequencing.

[0077] The PCR amplification product was detected by agarose gel electrophoresis, if the electrophoresis band was consistent with the size of the target band, the obtained PCR product was sent to the company for one-way sequencing. The sequencing results were compared with the reference fragment on NCBI using DNA MAN Version 9.0 software, and the peak chart in the sequencing results was observed using Chromas Version 2.3 software, and then the mutation site was screened out.

[0078] II. Experimental results

[0079] The extracted DNA was detected by spectrophotometer, and the OD 260 : OD 280 was between 1.8 and 2.0, indicating that the quality of the extracted DNA reached the standard and could be used for subsequent experiments.

[0080] The PCR amplification product was detected by 1.5% agarose gel electrophoresis (w / v), and the electrophoresis band was bright, specific and had no mixed band Figure 1 , which met the requirements of direct determination of nucleotide sequence by PCR product.

[0081] Example 2 Detection of SNP site of Qibannong chicken HTR1B gene

[0082] I. Experimental method

[0083] The sequencing results were compared with the reference group sequence on NCBI using software, the peak chart in the pool sequencing results was observed, and the single base containing overlapping peaks was screened out as the mutation site. Subsequently, individual amplification was carried out to verify the mutation site and genotyping, single peak was wild homozygous or mutant homozygous, and double peak was mutant heterozygous.

[0084] II. Experimental results

[0085] After comparing the sequencing results with the reference fragment on NCBI, the SNP site of Qibannong chicken HTR1B gene was screened out, and the Sanger sequencing peak of the SNP site of Qibannong chicken HTR1B gene was seen Figure 2The information of the 6 SNP sites detected is shown in Table 2. The results show that a total of 6 SNP sites are detected on the 2 exons, which are g.79776832C>T (HTR1B-Exon1-2), g.79776916G>A (HTR1B-Exon1-2), g.79777422T>C sites (HTR1B-Exon1-3) on the first exon, g.79778162A>G (HTR1B-Exon2-1), g.79778500A>C (HTR1B-Exon2-1), g.79778967A>G sites (HTR1B-Exon2-2) on the second exon. Among them, the amino acid mutation types of g.79776832C>T and g.79776916G>A sites are synonymous mutations, g.79777422T>C and g.79778162A>G sites are missense mutations, and g.79778500A>C and g.79778967A>G are located in 3'UTR.

[0086] Table 2 Information of HTR1B gene mutation sites of Qibaolong chicken

[0087]

[0088] Note: 1 indicates that the first base of Exon1 of the chicken HTR1B gene sequence (GenBank: NC_052534.1) in the NCBI database is the starting site. - indicates no amino acid coding.

[0089] Example 3 Genetic characteristics analysis of HTR1B gene SNP sites

[0090] I. Experimental method

[0091] The different genotypes obtained by sequencing were calculated by using Excel 2020 software to calculate the allele frequency, genotype frequency, genetic heterozygosity, effective allele number, polymorphic information content, χ 2 value and P value.

[0092] II. Experimental results

[0093] The genetic characteristics of the SNP sites of HTR1B gene in Qibannong chicken were analyzed, and the results are shown in Table 3. The results show that the dominant genotypes of the six SNP sites of HTR1B gene are CC (0.774), GA (0.458), TT (0.507), AA (0.756), AA (0.825) and AA (0.851) respectively, and the corresponding dominant alleles are C (0.877), G (0.645), T (0.708), A (0.871), A (0.908) and A (0.921) respectively. The PIC calculation results show that the g.79776916 G>A and g.79777422 T>C sites are moderately polymorphic, and the other sites are low polymorphic. After chi-square test, the χ 2 values of the six SNP sites of HTR1B gene are all less than 5.99, indicating that these SNP sites are in Hardy-Weinberg equilibrium state in Qibannong chicken population (P>0.05).

[0094] Table 3 Genetic characteristics analysis results of SNP sites of HTR1B gene

[0095]

[0096]

[0097] Note: 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 Hardy-Weinberg equilibrium, df=2, χ 2 0.05 =5.99, χ 2 0.01 =9.21, P<0.05 indicates significant difference; deviates from Hardy-Weinberg equilibrium, P>0.05 indicates no significant difference, and does not deviate from Hardy-Weinberg equilibrium.

[0098] Example 4 Analysis of the correlation between HTR1B gene polymorphism and growth performance

[0099] I. Experimental method

[0100] The IBM SPSS Statistics 26.0 software was used to perform correlation analysis between different genotypes of SNP sites of HTR1B gene and growth performance of Qibannong chicken, and the model used is as follows:

[0101] Y ijl = u + G i + e ijl

[0102] In the formula, Y ijlYi is the i th genotype effect of the SNP site or the effect of haplotype; e i Yi is the i th genotype effect of the SNP site or the effect of haplotype; e ijl The results were expressed as "mean ± standard error", and the significance of difference was tested by Duncan method, P < 0.05 represented significant difference, P < 0.01 represented extremely significant difference. For the traits with heterogeneous variance, the "Tukey HSD" method was used for multiple comparison, and for the traits with only two genotypes, the independent sample t test was used.

[0103] II. Results

[0104] The association analysis of different genotypes of 6 SNP sites of HTR1B gene and growth performance of 700-nong chickens was carried out, and the results were shown in Table 4 and Table 5.

[0105] As shown in Table 4, among the 6 SNP sites of HTR1B gene of 193 700-nong male chickens, the CT genotype and TT genotype of g.79776832 C > T were significantly higher than the CC genotype in body weight, the TT genotype was extremely significantly higher than the CC genotype and the TT genotype was significantly higher than the CT genotype in shank circumference, and the TT genotype was significantly higher than the CC genotype in breast depth; the GG genotype of g.79776916 G > A was significantly higher than the GA genotype in breast width; the TT genotype and TC genotype of g.79777422 T > C were extremely significantly higher than the CC genotype in body weight, body slant length and breast width, the TC genotype was significantly higher than the CC genotype in keel length, and the TT genotype was significantly higher than the CC genotype in pelvic width; the AG genotype of g.79778162 A > G was extremely significantly higher than the AA genotype in body weight, the GG genotype was extremely significantly higher than the AA genotype in shank circumference, and the AG genotype was significantly higher than the AA genotype in shank length, breast width and breast depth; the growth performance of each genotype of g.79778500 A > C and g.79778967 A > G sites was not significantly different.

[0106] Table 4 Growth performance of each genotype of SNP site of HTR1B gene of 700-nong male chickens

[0107]

[0108] Note: The data in the table was compared between the same column of the same site, and the different lower case letters in the superscript represented significant difference (P < 0.05), the different capital letters in the superscript represented extremely significant difference (P < 0.01), and the absence of letters in the superscript represented no significant difference (P > 0.05), and the genotypes with less than 5 individuals were not counted.

[0109] As shown in Table 5, among the 6 SNP sites of HTR1B gene of 156 700-nong female chickens, the growth performance of each genotype of all sites was not significantly different.

[0110] Table 5 Growth performance of each genotype of SNP site of HTR1B gene of 700-day-old Qianhong hens

[0111]

[0112] Note: The data in the table are compared between the same columns of the same sites, and the different lower case letters in the superscript represent significant difference (P <0.05), the different upper case letters in the superscript represent extremely significant difference (P <0.01), and the superscript without letters represents no significant difference (P >0.05). The genotypes with less than 5 individuals are not counted.

[0113] Example 5 Linkage disequilibrium and haplotype analysis of SNP site of HTR1B gene

[0114] I. Experimental method

[0115] The SNP site of HTR1B gene is subjected to linkage disequilibrium and haplotype analysis using Haploview software, Block block is drawn, and the haplotype type and frequency are counted.

[0116] The haplotype type is combined according to the Block block, and the haplotype combination type and frequency of HTR1B gene are counted. The haplotype combination and growth performance association analysis method are the same as those in Example 4.

[0117] II. Experimental results

[0118] The linkage disequilibrium analysis results are shown in the values in the A box in Figure 3 , Figure 3 The values in the A box in Figure 3 and Table 6 are obtained by multiplying the D' value by 100. It can be seen from B in Figure 3 and Table 6 that Block 1 is produced by strong linkage of g.79776832C>T and g.79776916G>A, and 3 haplotypes are produced, and the frequency of the dominant haplotype H1(CG) is 0.521; Block 2 is produced by strong linkage of g.79777422T>C, g.79778162A>G and g.79778500A>C, and 4 haplotypes are produced, and the frequency of the dominant haplotype H5(TAA) is 0.492.

[0119] Table 6 Haplotype statistical results of SNP site of HTR1B gene

[0120]

[0121]

[0122] From Table 7, haplotype combination was carried out based on haplotype analysis results, Block 1 combined H1HI (CG / CG), H1H2 (CG / CA), H1H3 (CG / TG) and H2H2 (CA / CA), and the frequencies were 0.272, 0.375, 0.123 and 0.126 respectively; Block 2 combined H4H4 (CAA / CAA), H5H4 (TAA / CAA), H5H5 (TAA / TAA), H5H6 (TAA / TGA) and H5H7 (TAA / TAC), and the frequencies were 0.089, 0.275, 0.223, 0.155 and 0.103 respectively.

[0123] Table 7 Statistical results of haplotype combination of SNP sites of HTR1B gene

[0124]

[0125] The association analysis of 6 haplotype combinations of HTR1B gene of Qibannong chicken and growth performance at 120 days of age was carried out, and the results are shown in Tables 8 and 9.

[0126] From Table 8, among the haplotype combinations of HTR1B gene of Qibannong cock, H1H3 was significantly higher than H1H1 and H1H2 in body weight, H1H3 was significantly higher than H1H1 in body slant length, H1H3 was significantly higher than H1H2 and H2H2 in keel length, and H1H3 was extremely significantly higher than H1H2 in chest width.

[0127] H5H6 was extremely significantly and significantly higher than H4H4 and H5H4 in body weight, H5H4, H5H5 and H5H6 were significantly higher than H4H4 in body slant length, H5H6 was significantly higher than H5H5 in shank length, H5H6 and H5H7 were significantly higher than H4H4 and H5H5 in shank circumference, H5H4, H5H5, H5H6 and H5H7 were extremely significantly higher than H4H4 in chest width, and H5H6 was significantly higher than H5H5, H5H6 was significantly higher than H4H4, H5H4 and H5H7 in chest depth, and H5H6 and H5H7 were significantly higher than H4H4 in pelvic width.

[0128] Table 8 Association analysis of haplotype combination of HTR1B gene of Qibannong cock and growth performance

[0129]

[0130] Note: In the table, the data in the same column of the same site are compared, and the lower case letters with different superscripts represent significant difference (P<0.05), the upper case letters with different superscripts represent extremely significant difference (P<0.01), and the absence of letters with different superscripts represents no significant difference (P>0.05). The genotypes with less than 5 individuals are not counted.

[0131] From Table 9, the growth performance of all haplotype combinations of HTR1B gene of Qibannong hens was not significantly different.

[0132] Table 9 Correlation analysis of haplotype combinations of HTR1B gene of Qibannong hen with growth performance

[0133]

[0134] Note: The data in the table is compared between the same columns in the same site. Different lower case letters in the superscript indicate significant difference (P < 0.05), different capital letters in the superscript indicate extremely significant difference (P < 0.01), and no letters in the superscript indicate no significant difference (P > 0.05). The genotypes with less than 5 individuals are not counted.

[0135] Example 6 A method for breeding Qibannong rooster

[0136] Single nucleotide polymorphism sites:

[0137] SNP1 is located at position 79776832 of NC_052534.1 gene, which is C or T polymorphism, and there are three genotypes of CC, CT and TT;

[0138] SNP2 is located at position 79776916 of NC_052534.1 gene, which is G or A polymorphism, and there are three genotypes of GG, GA and AA;

[0139] SNP3 is located at position 79777422 of NC_052534.1 gene, which is T or C polymorphism, and there are three genotypes of TT, TC and CC;

[0140] SNP4 is located at position 79778162 of NC_052534.1 gene, which is A or G polymorphism, and there are three genotypes of AA, AG and GG;

[0141] SNP5 is located at position 79778500 of NC_052534.1 gene, which is A or C polymorphism, and there are three genotypes of AA, AC and CC.

[0142] 1. Extract the DNA of the sample to be tested by using the blood genomic DNA extraction kit.

[0143] 2. Use the DNA of the sample to be tested extracted in step 1 as a template to perform PCR amplification of SNP1-SNP5 single nucleotide polymorphism sites;

[0144] The PCR reaction system is as follows: PCR SuperMix 10 μL, 1 μL (10 μmol·L of primer with nucleotide sequence as shown in SEQ ID NO: 3-8 each -1), DNA template 1 μL, and adding Nuclease-free Water to 20 μL;

[0145] The primer with the nucleotide sequence as shown in SEQ ID NO: 3-4 is used to detect SNP1 and SNP2 single nucleotide polymorphism sites, the primer with the nucleotide sequence as shown in SEQ ID NO: 5-6 is used to detect SNP3 single nucleotide polymorphism site, and the primer with the nucleotide sequence as shown in SEQ ID NO: 7-8 is used to detect SNP4 and SNP5 single nucleotide polymorphism sites.

[0146] PCR reaction conditions: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles, and 72℃ extension for 10 min.

[0147] 3. The PCR amplification product obtained in step 2 is detected by agarose gel electrophoresis, and if the electrophoresis band is consistent with the size of the target band and clear and bright, the PCR amplification product obtained in step 2 is sequenced.

[0148] 4. Result judgment:

[0149] SNP1, SNP2, SNP3 and / or SNP4 single nucleotide polymorphism sites can be detected, wherein:

[0150] The body weight of an individual with the genotype of CT or TT at the SNP1 site is significantly higher than that of an individual with the genotype of CC, the tibial circumference of an individual with the genotype of TT is extremely significantly higher than that of an individual with the genotype of CC, the tibial circumference of an individual with the genotype of TT is significantly higher than that of an individual with the genotype of CT, and the chest depth of an individual with the genotype of TT is significantly higher than that of an individual with the genotype of CC.

[0151] The chest width of an individual with the genotype of GG at the SNP2 site is significantly higher than that of an individual with the genotype of GA.

[0152] The body weight or body oblique length of an individual with the genotype of TT or TC at the SNP3 site is significantly higher than that of an individual with the genotype of CC, the chest width of an individual with the genotype of TT or TC is extremely significantly higher than that of an individual with the genotype of CC, the keel length of an individual with the genotype of TC is significantly higher than that of an individual with the genotype of CC, and the pelvic width of an individual with the genotype of TT is significantly higher than that of an individual with the genotype of CC.

[0153] The body weight of an individual with the genotype of AG at the SNP4 site is extremely significantly higher than that of an individual with the genotype of AA, the tibial circumference of an individual with the genotype of GG is extremely significantly higher than that of an individual with the genotype of AA, and the tibial length, chest width or chest depth of an individual with the genotype of AG is significantly higher than that of an individual with the genotype of AA.

[0154] Or a haplotype combination composed of SNP1, SNP2, SNP3, SNP4 and / or SNP5 single nucleotide polymorphism sites can be detected;

[0155] SNP1 site and SNP2 site construct 3 haplotypes:

[0156] H1 haplotype is CG, H2 haplotype is CA, and H3 haplotype is TG;

[0157] SNP3 site, SNP4 site and SNP5 site construct 4 haplotypes:

[0158] H4 haplotype is CAA, H5 haplotype is TAA, H6 haplotype is TGA, and H7 haplotype is TAC;

[0159] The haplotypes are constructed into haplotype combinations, and the haplotype combinations are respectively:

[0160] H1H1 haplotype combination is CG / CG, H1H2 haplotype combination is CG / CA, H1H3 haplotype combination is CG / TG, and H2H2 haplotype combination is CA / CA;

[0161] H4H4 haplotype combination is CAA / CAA, H5H4 haplotype combination is TAA / CAA, H5H5 haplotype combination is TAA / TAA, H5H6 haplotype combination is TAA / TGA, and H5H7 haplotype combination is TAA / TAC;

[0162] The body weight of the individual with H1H3 haplotype combination is significantly higher than that of the individual with H1H1 haplotype combination or H1H2 haplotype combination;

[0163] The body length of the individual with H1H3 haplotype combination is significantly higher than that of the individual with H1H1 haplotype combination;

[0164] The carapace length of the individual with H1H3 haplotype combination is significantly higher than that of the individual with H1H1 or H2H2 haplotype combination;

[0165] The chest width of the individual with H1H3 haplotype combination is extremely significantly higher than that of the individual with H1H2 haplotype combination;

[0166] The body weight of the individual with H5H6 haplotype combination is extremely significantly higher than that of the individual with H4H4 haplotype combination;

[0167] The body weight of the individual with H5H6 haplotype combination is significantly higher than that of the individual with H5H4 haplotype combination;

[0168] The body length of the individual with H5H4, H5H5 or H5H6 haplotype combination is significantly higher than that of the individual with H4H4 haplotype combination;

[0169] tibia length of the individual with the H5H6 haplotype combination is significantly higher than that of the individual with the H5H5 haplotype combination;

[0170] tibia girth of the individual with the H5H6 or H5H7 haplotype combination is significantly higher than that of the individual with the H4H5 or H5H5 haplotype combination;

[0171] chest width of the individual with the H5H4, H5H5, H5H6 or H5H7 haplotype combination is extremely significantly higher than that of the individual with the H4H4 haplotype combination;

[0172] chest width of the individual with the H5H6 haplotype combination is significantly higher than that of the individual with the H5H5 haplotype combination;

[0173] chest depth of the individual with the H5H6 haplotype combination is significantly higher than that of the individual with the H4H4, H5H4 or H5H7 haplotype combination;

[0174] pelvis width of the individual with the H5H6 or H5H7 haplotype combination is significantly higher than that of the individual with the H4H4 haplotype combination.

[0175] Embodiment 7 A kit for breeding of seven hundred wonder rooster

[0176] I. Composition

[0177] a primer with nucleotide sequence as shown in SEQ ID NO: 3-8, PCR SuperMix and Nuclease-free Water.

[0178] II. Method of use

[0179] The detection and result interpretation are performed according to Embodiment 6.

[0180] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement manners, and all shall be included in the protection scope of the present application.

Claims

1. The use of a detection reagent for a SNP molecular marker for Qianbailong rooster breeding in the preparation of a Qianbailong rooster breeding product, characterized in that, The SNP molecular marker is composed of 5 single nucleotide polymorphism sites SNP1, SNP2, SNP3, SNP4 and / or SNP5 SNP1 is located at position 79776832 on NC_052534.1, which is a C or T polymorphism, and exists in three genotypes of CC, CT and TT; SNP2 is located at position 79776916 on NC_052534.1, which is a G or A polymorphism, and exists in three genotypes of GG, GA and AA; SNP3 is located at position 79777422 on NC_052534.1, which is a T or C polymorphism, and exists in three genotypes of TT, TC and CC; SNP4 is located at position 79778162 on NC_052534.1, which is an A or G polymorphism, and exists in three genotypes of AA, AG and GG; SNP5 is located at position 79778500 on NC_052534.1, which is an A or C polymorphism, and exists in three genotypes of AA, AC and CC; The single nucleotide polymorphism site SNP1 and the single nucleotide polymorphism site SNP2 construct 3 haplotypes: The H1 haplotype is CG, the H2 haplotype is CA, and the H3 haplotype is TG; The haplotypes construct haplotype combinations, and the haplotype combinations are respectively: The H1H1 haplotype combination is CG / CG, the H1H2 haplotype combination is CG / CA, the H1H3 haplotype combination is CG / TG, and the H2H2 haplotype combination is CA / CA; The body weight of the individual with the H1H3 haplotype combination is significantly higher than that of the individual with the H1H1 or H1H2 haplotype combination; The body length of the individual with the H1H3 haplotype combination is significantly higher than that of the individual with the H1H1 haplotype combination; The body length of the individual with the H1H3 haplotype combination is significantly higher than that of the individual with the H1H1 or H2H2 haplotype combination; The chest width of the individual with the H1H3 haplotype combination is extremely significantly higher than that of the individual with the H1H2 haplotype combination; The single nucleotide polymorphism site SNP3, the single nucleotide polymorphism site SNP4 and the single nucleotide polymorphism site SNP5 construct 4 haplotypes: The H4 haplotype is CAA, the H5 haplotype is TAA, the H6 haplotype is TGA, and the H7 haplotype is TAC; The haplotypes construct haplotype combinations, and the haplotype combinations are respectively: The H4H4 haplotype combination is CAA / CAA, the H5H4 haplotype combination is TAA / CAA, the H5H5 haplotype combination is TAA / TAA, the H5H6 haplotype combination is TAA / TGA, and the H5H7 haplotype combination is TAA / TAC; The body weight of the individual with the H5H6 haplotype combination is extremely significantly higher than that of the individual with the H4H4 haplotype combination; The body weight of the individual with the H5H6 haplotype combination is significantly higher than that of the individual with the H5H4 haplotype combination; The body length of the individual with the H5H4, H5H5 or H5H6 haplotype combination is significantly higher than that of the individual with the H4H4 haplotype combination; The shank length of the individual with the H5H6 haplotype combination is significantly higher than that of the individual with the H5H5 haplotype combination; ​ The tibial circumference of the individual with the H5H6 or H5H7 haplotype combination is significantly higher than that of the individual with the H4H4 or H5H5 haplotype combination; The chest width of the individual with the H5H4, H5H5, H5H6 or H5H7 haplotype combination is extremely significantly higher than that of the individual with the H4H4 haplotype combination; The chest width of the individual with the H5H6 haplotype combination is significantly higher than that of the individual with the H5H5 haplotype combination; The chest depth of the individual with the H5H6 haplotype combination is significantly higher than that of the individual with the H4H4, H5H4 or H5H7 haplotype combination; The pelvic width of the individual with the H5H6 or H5H7 haplotype combination is significantly higher than that of the individual with the H4H4 haplotype combination.

2. Use according to claim 1, characterized in that, The detection reagent is a primer with a nucleotide sequence as shown in SEQ ID NO: 3-8.

3. A method for breeding a seven-hundred wonder rooster, characterized in that, SNP1, SNP2, SNP3 and / or SNP4 single nucleotide polymorphism sites are detected; SNP1 is located at position 79776832 on NC_052534.1, and is a C or T polymorphism, which exists in three genotypes of CC, CT and TT; SNP2 is located at position 79776916 on NC_052534.1, and is a G or A polymorphism, which exists in three genotypes of GG, GA and AA; SNP3 is located at position 79777422 on NC_052534.1, and is a T or C polymorphism, which exists in three genotypes of TT, TC and CC; SNP4 is located at position 79778162 on NC_052534.1, and is an A or G polymorphism, which exists in three genotypes of AA, AG and GG; The body weight of the individual with the genotype of CT or TT at the SNP1 site is significantly higher than that of the individual with the genotype of CC, the tibial circumference of the individual with the genotype of TT is extremely significantly higher than that of the individual with the genotype of CC, the tibial circumference of the individual with the genotype of TT is significantly higher than that of the individual with the genotype of CT, and the chest depth of the individual with the genotype of TT is significantly higher than that of the individual with the genotype of CC; The chest width of the individual with the genotype of GG at the SNP2 site is significantly higher than that of the individual with the genotype of GA; The body weight or body length of the individual with the genotype of TT or TC at the SNP3 site is significantly higher than that of the individual with the genotype of CC, the chest width of the individual with the genotype of TT or TC is extremely significantly higher than that of the individual with the genotype of CC, the carapace length of the individual with the genotype of TC is significantly higher than that of the individual with the genotype of CC, and the pelvic width of the individual with the genotype of TT is significantly higher than that of the individual with the genotype of CC; The body weight of the individual with the genotype of AG at the SNP4 site is extremely significantly higher than that of the individual with the genotype of AA, the tibial circumference of the individual with the genotype of GG is extremely significantly higher than that of the individual with the genotype of AA, and the tibial length, chest width or chest depth of the individual with the genotype of AG is significantly higher than that of the individual with the genotype of AA.

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