KIF26B gene molecular marker primer related to chicken continuous production character and application of KIF26B gene molecular marker primer

Abstract

The invention relates to a KIF26B gene molecular marker primer related to chicken continuous laying traits and application of the KIF26B gene molecular marker primer, and belongs to the technical field of biology. The molecular marker of the KIF26B gene is located at the 34213811th base of chromosome 3 of a chicken reference genome GRCg7b version, the base is mutated into A or G, and the genotypes are A / A, A / G and G / G. The total continuous production length of the G / G genotype chicken is higher than that of the A / G and A / A genotype individuals, and the total continuous production length of the A / G genotype chicken is higher than that of the A / A genotype individuals. According to the present invention, the forward and reverse primer pair is used to amplify the fragment containing the molecular marker site, and the amplification product is subjected to Sanger sequencing so as to rapidly and accurately identify the genotype of the individual; the molecular marker can be used as a genetic marker for chicken breeding, can efficiently and accurately detect the continuous laying character of chicken, improves the total egg laying number of chicken flocks, and has important value for chicken breeding and production.

Description

TECHNICAL FIELD

[0001] The present application relates to a KIF26B gene molecular marker primer related to chicken continuous laying traits and its application, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Continuous laying traits are that hens lay eggs for 2 days or more, and the number of days of continuous laying is called "continuous laying length" (i.e. the number of continuous laying eggs), and the number of days of stop laying is called "laying interval" (i.e. the number of days of stop laying). These two indicators provide additional information for the laying pattern: multiple continuous laying lengths will appear in the entire laying period, and if the continuous laying length is continuously lengthened and the continuous laying interval is continuously shortened, the laying performance tends to be stable, and the nest inclination is also reduced. The total continuous laying length is the sum of the number of times of continuous laying of hens, and is one of the important indicators of poultry reproductive performance, which is affected by multiple factors such as genetics, endocrine, and neural regulation. The total continuous laying length represents the ability of hens to continuously lay eggs before and after the peak laying period, and can directly reflect the early laying persistence and nest inhibition level. It indicates the laying stability and total laying potential in the entire subsequent laying period. At present, there is a lack of gene molecular markers that can accurately identify the chicken total continuous laying length trait, and they are used in breeding selection to quickly identify the corresponding traits for early screening. SUMMARY

[0003] The purpose of the present application is to overcome the defects in the prior art, and to provide a KIF26B gene molecular marker primer related to chicken continuous laying traits and its application, which can quickly detect and screen the continuous laying traits of chickens.

[0004] The present application records the sum of the number of times of continuous laying of the recessive white Lohmann chicken from the onset of laying to 36 weeks of age, calculates the total continuous laying length, uses whole genome resequencing technology for SNP genotyping, and selects KIF26B gene molecular markers significantly related to chicken continuous laying traits through whole genome association analysis, thereby providing new genes and molecular marker resources for the selection of chicken continuous laying traits.

[0005] The protein encoded by the KIF26B gene is an intracellular motor protein, and its overexpression is closely related to the shorter overall survival of ovarian cancer patients, and may exacerbate disease progression by promoting cell migration and immune escape. As a member of the kinesin family, KIF26B is a downstream target of nuclear zinc finger proteins, and regulates the adhesion of embryonic kidney mesenchymal cells by interacting with non-muscle myosin, which is essential for maintaining glial cell-derived neurotrophic factor expression and ureteric bud attraction, and its absence will lead to renal agenesis or absence. Studies have shown that high expression of KIF26B is associated with ovarian cancer, and combined with its regulation of cell adhesion / migration, it can be inferred that elevated KIF26B levels may affect the total continuous laying length by interfering with ovarian or uterine homeostasis.

[0006] The application provides a primer of a KIF26B gene molecular marker related to a chicken continuous laying trait, the molecular marker is located at a base at position 34213811 of a chicken chromosome 3, the base is mutated to A or G, a base at position 213 in a sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, and nucleotide sequences of a chicken DNA specific primer pair required for molecular marker detection are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0007] The application further provides application of the specific primer, and the application includes detection of a SNP genotype related to a recessive white leghorn chicken continuous laying trait. A first step is to provide a chicken DNA sample to be detected, and the chicken DNA sample to be detected contains a SNP molecular marker at position 34213811 of a chicken chromosome 3, and the chicken DNA sample to be detected is subjected to PCR amplification by using a DNA specific primer pair to obtain an amplification product. A second step is to perform Sanger sequencing on the PCR product. A third step is to determine a SNP molecular marker genotype at position 34213811 of a chicken chromosome 3 according to a sequencing result of the second step.

[0008] The deoxyribonucleotide sequence of the chicken DNA specific primer pair in the first step is as follows: An upstream primer: 5'-TGTCACCGAATTCCTCCACA-3' (SEQ ID NO: 1) A downstream primer: 5'-TCGTGACTGGTAGCTAGCTG-3' (SEQ ID NO: 2) The amplification product in the first step has a length of 478 bp and contains a base at position 34213811 of a chicken chromosome 3.

[0009] A final reaction system volume (25 μl) is as follows: Chicken DNA to be detected: 50 ng 2 x Accurate Taq Master Mix: 12.5 μl An upstream primer: 1 μl A downstream primer: 1 μl Sterilized water: supplemented to 25 μl, Reaction conditions of the PCR amplification are as follows: 94 ℃ pre-denaturation for 5 min, 94 ℃ denaturation for 30 sec, 60 ℃ annealing for 30 sec, 72 ℃ extension for 60 sec, a total of 27 cycles, 72 ℃ extension for 5 min, and 4 ℃ preservation.

[0010] The nucleotide sequence of the amplification product is shown as SEQ ID NO: 3 or SEQ ID NO: 4.

[0011] In the third step, the judging standard is that the total laying length of the chicken with G / G genotype is higher than that of the chicken with A / G and A / A genotype, and the total laying length of the chicken with A / G genotype is higher than that of the chicken with A / A genotype.

[0012] The present application detects the genotype of the chicken laying traits by the KIF26B gene molecular marker, finds that the total laying length of the chicken with G / G genotype is higher than that of the chicken with A / G and A / A genotype, and the total laying length of the chicken with A / G genotype is higher than that of the chicken with A / A genotype. By taking the genomic DNA of the chicken to be tested as a template, the specific primer is used for PCR amplification, and then the PCR amplification product is subjected to Sanger sequencing and SNP molecular marker genotyping, and the genotype of the SNP molecular marker can realize the selection of the chicken laying traits. In breeding, according to the breeding target, the A / A and A / G genotype individuals are eliminated, and the G / G genotype individuals are reserved, and the beneficial effects are that the molecular marker can be used as a genetic marker for chicken breeding, can efficiently and quickly identify the total laying length traits of the chicken, and can improve the total number of eggs laid by the chicken population, provides a scientific basis for early selection, and has important value for chicken breeding. In addition, the detection method disclosed by the present application is simple and easy to operate, and can be carried out in the laboratory. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is a Manhattan plot of total laying length whole genome association analysis.

[0014] Figure 2 is the Sanger sequencing result of the PCR amplification product of three genotypes. DETAILED DESCRIPTION

[0015] The following examples are suitable for the breeding of chickens.

[0016] Example 1 In this example, the total laying length of 36-week-old recessive white Lohmann hens is counted, the second-generation sequencing technology is used for whole genome SNP genotyping, the KIF26B gene molecular marker significantly related to the laying traits is screened by whole genome association analysis, and the result is shown as Figure 1 .

[0017] In this example, the KIF26B gene molecular marker related to the laying traits of the chicken is identified and applied through the following experiments.

[0018] 1. Phenotype determination and genotype detection (1) Experimental materials and phenotype determination Select 2476 recessive white Loke hens as test animals, feed under the same conditions, free diet throughout the process, from the first egg laying record each chicken egg laying time, calculate the total length of continuous production as the phenotype data.

[0019] (2) Extraction of genomic DNA The test individual was collected by subcostal vein, anticoagulated, lysed, digested with proteinase K, extracted by saturated sodium chloride method, dissolved in TE and stored at -20℃.

[0020] (3) PCR amplification The above extracted genomic DNA was used as template to amplify the fragment containing the 34213811th base of chromosome 3.

[0021] Upstream primer: 5'-TGTCACCGAATTCCTCCACA-3' (SEQ ID NO: 1) Downstream primer: 5'-TCGTGACTGGTAGCTAGCTG-3' (SEQ ID NO: 2) The final volume of the reaction system (25 μl) is: Test DNA 50 ng 2x Accurate Taq Master Mix 12.5 μl Upstream primer 1 μl Downstream primer 1 μl Sterile water, supplemented to 25 μl, The reaction conditions of PCR amplification are: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 sec, 60℃ annealing for 30 sec, 72℃ extension for 60 sec, a total of 27 cycles; 72℃ extension for 5 min; 4℃ storage; take 10 μl for agarose detection, the length of the single band obtained by amplification is 478 bp, and the SNP molecular marker containing the 34213811th base of chicken chromosome 3, the product sequence is as follows: SEQ ID NO: 3 TGTCACCGAATTCCTCCACATGAAAAAACTGCACCCACTGACATTCATCAATACTTGCTGAATGTCAATGCAGACCAAACAGTGGCTGTGAGCACAGTGAGGCAGTGGGTAGTGCATTTCAGCAGTGGTGACAGTGATGTGAAAGACAAGCATTATGAAATGAAGAGTGTCTCAATCAGTTCATCCACACAGATGAATCATCTGTGGGTTACAACCAGGTAACTGTGTACAGAGCTGAATATTGGCTCCATTGTGGTGGAAACAATGGTGGCAACATCAGAATATCACAAAGTTTGCACGAGGTGGGTCCCACAGGTGTTTACGCAGGGACAGAAAGAACACAGCTTGCAAGTACATCAGGACCTATTGAACCAACATGAGGATAGGTACACTCCTTGCTGGGTGAAGAACTGCAAGGAAGGCCAGGCCCAGACAGTGATGGTGAATGGAGCTGAGTCCAGCTAGCTACCAGTCACGA SEQ ID NO: 4 TGTCACCGAATTCCTCCACATGAAAAAACTGCACCCACTGACATTCATCAATACTTGCTGAATGTCAATGCAGACCAAACAGTGGCTGTGAGCACAGTGAGGCAGTGGGTAGTGCATTTCAGCAGTGGTGACAGTGATGTGAAAGACAAGCATTATGAAATGAAGAGTGTCTCAATCAGTTCATCCACACAGATGAATCATCTGTGGGTTACGACCAGGTAACTGTGTACAGAGCTGAATATTGGCTCCATTGTGGTGGAAACAATGGTGGCAACATCAGAATATCACAAAGTTTGCACGAGGTGGGTCCCACAGGTGTTTACGCAGGGACAGAAAGAACACAGCTTGCAAGTACATCAGGACCTATTGAACCAACATGAGGATAGGTACACTCCTTGCTGGGTGAAGAACTGCAAGGAAGGCCAGGCCCAGACAGTGATGGTGAATGGAGCTGAGTCCAGCTAGCTACCAGTCACGA (4) Sanger sequencing and genotyping The PCR products of each sample were subjected to Sanger sequencing, respectively, to obtain different genotyping sequencing peak charts as shown in Figure 2 .

[0022] 2. Correlation analysis Select 2476 individuals of recessive white Lohmann hens with clear phenotype records for correlation analysis. The ANOVA test function of R4.2 statistical plotting software was used for statistical test, and the average value comparison mode between each other was selected for statistical test of the genotypes and total laying length of the test chickens, P < 0.05 indicating significant difference. The results are shown in Table 1, and the three genotypes are significantly different (P < 0.05). The average total laying length of G / G genotype individuals is 62.03 days, which is higher than that of A / G genotype individuals (58.10 days, P < 0.05) and A / A genotype individuals (53.70 days, P < 0.05). The total laying length of A / G genotype individuals is higher than that of A / A genotype individuals (P < 0.05). The results show that the chicken KIF26B gene molecular marker is significantly related to the total laying length phenotype, and G / G genotype individuals can be selected for breeding total laying length larger chickens to improve the overall total laying length and uniformity, and improve the breeding efficiency according to the actual breeding goal.

[0023] Table 1 Correlation analysis of KIF26B gene molecular marker and laying traits

[0024] Note: The same column data with the same letter indicates no significant difference, and the different letters indicate significant difference (P < 0.05).

[0025] Example 2 Genotype frequency of different breeds 1. Blood sample collection The blood samples of tea flower chicken, big bone chicken, Fujian Hetian chicken, Gushi chicken, Guangxi chicken, Daweishan miniature chicken, black-bone chicken and recessive white Lohmann were collected by subclavian vein blood collection method, and stored at -20℃ for standby.

[0026] 2. Extraction of genomic DNA The tissue samples obtained in the first step were used to extract genomic DNA using Omega's tissue genomic DNA extraction kit, and the specific method was referred to the standard operation procedure provided by Omega.

[0027] 3. Genotype detection Using the genomic DNA obtained in the second step as a template, PCR amplification and Sanger sequencing were performed using primer pairs consisting of the F nucleotide sequence (SEQ ID NO: 1) and the R nucleotide sequence (SEQ ID NO: 2) to obtain the individual's G / G genotype, A / G genotype, and A / A genotype.

[0028] 4. Results Analysis Camellia chicken, Dagu chicken, Fujian Hetian chicken, Guangxi Ma chicken, Daweishan miniature chicken, and Silkie chicken are local Chinese chicken breeds that have not undergone long-term selective breeding and are often used as breeding stock in yellow-feathered broiler breeding lines. Gushi chicken is a dual-purpose breed (meat and egg), characterized by rapid growth and good reproductive performance. Recessive White Rock chicken is often used as a specialized strain in yellow-feathered broiler breeding lines, with the main breeding objective being to improve growth rate and feed efficiency. Identification revealed that the KIF26B gene molecular marker exhibits genetic polymorphism across multiple breeds, with a higher frequency of the G allele in Chinese breeds and a lower frequency in recessive White Rock chickens. This SNP locus can be used as a molecular marker for total consecutive laying length in chickens to help improve the egg production performance of the flock. See Table 2.

[0029] Table 2. Allelic frequency distribution of KIF26B gene molecular marker in different varieties

[0030] In addition to the above-described embodiments, the present invention may have other implementations. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention.

Claims

1. A KIF26B gene molecular marker primer associated with chicken fecundity traits, characterized in that: The nucleotide sequence of the molecular marker primer is shown as SEQ ID NO: 1 and SEQ ID NO: 2, and the molecular marker site is located at the base at position 34213811 of chromosome 3 of chicken reference genome GRCg7b version 3, and the base is A or G, and the genotype is A / A, A / G and G / G.

2. The use of a KIF26B gene molecular marker primer associated with chicken fertility traits, characterized in that: The molecular marker primer is used for detecting the recessive white rock chicken continuous production trait, and the detection method comprises the following steps: In the first step, the DNA sample of the chicken to be tested is subjected to PCR amplification using the molecular marker primer shown as SEQ ID NO: 1-2 to obtain an amplification product, the length of the amplification product is 478 bp, and the amplification product contains the base at position 34213811 of chromosome 3 of chicken reference genome GRCg7b version 3; In the second step, the PCR product is subjected to Sanger sequencing; In the third step, the chicken reference genome GRCg7b version 3 chromosome 3 base at position 34213811 SNP molecular marker genotype is determined according to the sequencing result of the second step.

3. The application of KIF26B gene molecular marker primer related to chicken fertility traits according to claim 2, characterized in that: The PCR reaction system is 25 μl, and the system is as follows: 50 ng of DNA of the chicken to be tested 2x Accurate Taq Master Mix 12.5 μl 1 μl of upstream primer 1 μl of downstream primer Sterile water is supplemented to 25 μl; The reaction conditions of the PCR amplification are as follows: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 sec, 60 ℃ annealing for 30 sec, 72 ℃ extension for 60 sec, a total of 27 cycles; 72 ℃ extension for 5 min; 4 ℃ storage; and the nucleotide sequence of the amplification product is shown as SEQ ID NO: 3 or SEQ ID NO:

4.

4. The application of the KIF26B gene molecular marker primer related to the chicken fertility trait according to claim 2, characterized in that: The judgment standard of the third step is that the total continuous production length of the G / G genotype chicken is higher than that of the A / G and A / A genotype individuals, and the total continuous production length of the A / G genotype chicken is higher than that of the A / A genotype individual.

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