SNP molecular marker combination for identifying AA broiler chicken, detection kit, and use thereof

Abstract

An SNP molecular marker combination for identifying AA broiler chicken, a detection kit, and use thereof. The SNP molecular marker combination for identifying AA broiler chicken consists of 25 SNP molecular markers, the SNP site of molecular markers 1-25 being located at position 51 of the nucleotide sequences shown in SEQ ID NO. 1-25. The SNP molecular marker combination has significant AA broiler chicken species specificity, is the determination site for variations and important traits unique to AA broiler chicken, can quickly determine the authenticity of AA broiler chicken using less genotype information, and provides a new technical reference for the future identification, breed conservation, and genetic breeding of the AA broiler chicken breed.

Description

A SNP molecular marker combination for identifying AA broiler chickens, a detection kit and its application Technical Field

[0001] The present invention relates to a SNP molecular marker combination for identifying AA broiler chickens, a detection kit and application thereof, and belongs to the field of biotechnology. Background Art

[0002] Single nucleotide polymorphisms (SNPs) refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. Due to their numerous and stable inheritance advantages, SNPs have gradually become a new generation of molecular markers and have been widely used in fields such as biology, agriculture, medicine, and biological evolution. Molecular marker technology can quickly, accurately, and efficiently identify breeds, which is of great significance for ensuring the correct breed in poultry production. With the continuous development of poultry breeding and molecular genetics, researchers have found that classifying target populations at the genetic level can provide important information for breed preservation and commercial utilization. In addition, the optimal combination of SNP markers can produce stable results in validation studies analyzing unknown samples.

[0003] AA broilers, also known as Aibo Yijia broilers, are a four-line, white-feathered hybrid chicken. They are a fast-growing, large-scale breed characterized by rapid growth, strong adaptability, high feed conversion efficiency, well-developed broilers, well-developed breast and leg muscles, and excellent carcass quality. Currently, conventional poultry breed identification relies solely on traditional appearance, such as body shape, plumage color, shank color, and comb shape. This is inevitably influenced by environmental and other subjective factors. Furthermore, morphological characteristics cannot be used to identify chicks with unclear breed characteristics or marketed meat products. Accurately identifying poultry strains at the genetic level is an effective solution to this problem. Therefore, identifying the characteristic markers of different strains is a pressing issue.

[0004] Summary of the Invention

[0005] The first object of the present invention is to provide a SNP molecular marker combination for identifying AA broiler chickens, so as to solve the problem that there is no SNP molecular marker for identifying AA broiler chickens in the prior art.

[0006] The second object of the present invention is to provide a detection kit to solve the problem in the prior art that it is impossible to detect SNP molecular markers characteristic of AA broiler breeds.

[0007] The third object of the present invention is to provide a SNP molecular marker combination or detection kit for identifying AA broiler chickens for use in the identification of AA broiler chicken germplasm resources, which solves the problem in the prior art that it is impossible to accurately identify whether the chicken sample to be tested is an AA broiler chicken based on morphology.

[0008] In order to achieve the above object, the present invention provides a technical solution for identifying a SNP molecular marker combination of AA broiler chickens:

[0009] A SNP molecular marker combination for identifying AA broiler chickens, the SNP molecular marker combination consisting of 25 SNP molecular markers; the SNP sites of the molecular markers 1 to 25 are located at position 51 of the nucleotide sequences shown in SEQ ID NOs. 1 to 25.

[0010] The beneficial effect of the above technical solution is that the present invention obtains whole-genome sequencing data of 30 AA broiler chickens through whole-genome resequencing. By comparing and analyzing the published whole-genome sequencing data of 336 individuals from 27 other chicken breeds, the present invention optimizes and combines 25 SNP loci that distinguish AA broilers from non-AA broilers, forming a SNP molecular marker combination for identifying AA broilers. The SNP molecular marker combination for identifying AA broilers of the present invention has obvious species specificity for AA broilers and can quickly authenticate AA broilers using less genotypic information, providing a new technical reference for future chicken breed identification, seed conservation, and genetic breeding.

[0011] In order to achieve the above-mentioned purpose, the technical solution of a detection kit in the present invention is:

[0012] A detection kit comprises the PCR primer set for identifying the SNP molecular marker combination genotype of AA broiler chickens.

[0013] The beneficial effect of the above technical solution is that: the PCR primer set of the above SNP molecular marker combination genotype is designed and synthesized, and after PCR amplification, combined with gene sequencing and other means, the genotype information of the SNP molecular marker can be quickly and accurately obtained, laying the foundation for subsequent analysis and verification.

[0014] As a further improvement, the PCR primer set is a competitive allele-specific PCR primer set.

[0015] The beneficial effect of the above technical solution lies in the fact that competitive allele-specific PCR (KSAP) is an effective means of SNP typing and indel detection by using specific matching of primer terminal bases. This method does not require the synthesis of specific fluorescent primers for each SNP locus. Based on its unique ARM PCR principle, all sites tested are ultimately amplified using universal fluorescent primers, achieving genotyping detection with the advantages of rapidity, accuracy, and high throughput.

[0016] As a further improvement, the competitive allele-specific PCR primer set includes the first primer set to the twenty-fifth primer set corresponding to the detection of the SNP molecular markers 1 to 25; the nucleotide sequences of the first primer set to the twenty-fifth primer set are shown in sequences SEQ ID NO. 26 to 100.

[0017] As a further improvement, the detection kit further comprises KASP reaction buffer, DNA polymerase and dNTPs.

[0018] In order to achieve the above-mentioned object, the technical solution of the present invention for the application of the SNP molecular marker combination or detection kit for identifying AA broiler chickens in the identification of AA broiler chicken germplasm resources is as follows:

[0019] Application of SNP molecular marker combinations or detection kits for identifying AA broiler chickens in the identification of AA broiler chicken germplasm resources.

[0020] The beneficial effect of the above technical solution lies in that it detects the genotype of the SNP molecular marker combination used to identify AA broiler chickens, enabling rapid and accurate identification of AA broiler chickens in test samples. The blind test accuracy rate reaches 99.47%, filling the gap in the current lack of genetic identification methods for AA broiler chickens. It can be used for traceability identification and protection of AA broiler chicken germplasm, which is of great significance for promoting the healthy development of AA broiler chicken germplasm resources.

[0021] As a further improvement, the genotypes of SNP molecular markers 1 to 25 of the sample to be tested are detected. If the genotypes of the 25 SNP molecular markers of the sample to be tested are consistent with the genotypes shown in Table 1, the sample to be tested is an AA broiler chicken.

[0022] Table 1

[0023] As a further improvement, the following method is used to detect the genotype of the molecular marker: using the extracted DNA of the sample to be tested as a template, the detection kit performs a PCR amplification reaction, and obtains the fluorescent signal for typing. DETAILED DESCRIPTION

[0024] The purpose, technical solutions and beneficial effects of the present invention are further described below in conjunction with the examples. The described embodiments are intended to help those skilled in the art better understand the invention and are not intended to limit the present invention. Unless otherwise specified, all reagents and instruments used in the examples are commercially available products.

[0025] Example 1 of a SNP molecular marker combination for identifying AA broiler chickens

[0026] The SNP molecular marker combination for identifying AA broiler chickens in this embodiment consists of 25 SNP molecular markers; the SNP sites of the molecular markers 1 to 25 are located at position 51 of the nucleotide sequences shown in SEQ ID NOs. 1 to 25.

[0027] Example 1 of a detection kit

[0028] The detection kit of this embodiment includes a competitive allele-specific PCR primer set for detecting the genotype of the SNP molecular marker combination used to identify AA broiler chickens; the competitive allele-specific PCR primer set includes a first primer set to a twenty-fifth primer set corresponding to detecting the SNP molecular markers 1 to 25; the nucleotide sequences of the first primer set to the twenty-fifth primer set are shown in sequences SEQ ID NO. 26-100, and the specific correspondence between the SNP molecular markers and the primer sets is shown in Table 2; the kit also includes a KASP reaction buffer, a DNA polymerase, and dNTPs.

[0029] Table 2 Specific correspondence between SNP molecular markers and primer pairs

[0030] Example 1 of the application of a SNP molecular marker combination or detection kit for identifying AA broiler chickens in the identification of AA broiler chicken germplasm resources

[0031] The use of the SNP molecular marker combination or detection kit for identifying AA broiler chickens in this embodiment in the identification of AA broiler chicken germplasm resources includes the following steps:

[0032] (1) Extraction of genomic DNA from chicken samples;

[0033] (2) PCR amplification: using the genomic DNA extracted in step (1) as a template and competitive allele-specific PCR primers with nucleotide sequences as shown in SEQ ID NO. 26-100, PCR amplification was performed;

[0034] (3) Fluorescence signal acquisition and genotyping: SNP typing is performed based on the fluorescence signal ratio. If the genotypes of the 25 SNP molecular markers of the sample to be tested are consistent with the genotypes shown in Table 1, the sample to be tested is an AA broiler chicken.

[0035] Experimental Example 1 Screening of SNP molecular marker combinations for identifying AA broiler chickens

[0036] In this experiment, whole-genome resequencing was used to obtain whole-genome sequencing data from 30 AA broiler chickens. Comparison and analysis with publicly available whole-genome sequencing data from 336 individuals from 27 other chicken breeds revealed 50 SNPs that distinguish AA broilers from non-AA broilers. Furthermore, through optimization analysis of these 50 distinguishing SNPs, a combination of 25 SNPs with clear species-specificity for AA broilers was obtained. The specific steps are as follows:

[0037] Sequencing: Genomic DNA was extracted from the blood of 30 AA broiler chickens, and the genomic DNA was used to perform whole-genome resequencing of the 30 AA broiler chickens using the Illumina Nova Seq platform. The average sequencing depth reached 10X, and a total of 262.21GB of raw sequencing data was obtained with a coverage rate of up to 97.61% (at least 1 base coverage).

[0038] The whole genome sequencing data of 336 individuals of the other 27 chicken breeds were downloaded from the NCBI website. The specific breeds, number of individuals and SRA accession numbers are shown in Table 3.

[0039] Table 3 Whole genome sequencing data of 336 individuals of 27 other chicken breeds obtained by NCBI

[0040] Data quality control and filtering: Fastp software (https: / / github.com / Open Gene / fastp) was used to merge and quality control (QC) the raw unprocessed data obtained from the whole-genome sequencing of 30 AA broiler chickens and the whole-genome sequencing data of 336 individuals of 27 other chicken breeds obtained by NCBI. High-quality data were ensured by trimming adapters and removing low-quality nucleotides, unknown nucleotides (NS), and reads containing more than 10% NS.

[0041] Analysis and Alignment: Filtered reads from all individuals were aligned to the chicken whole-genome reference standard sequence (version GRCg7b) using Burrow-Wheeler aligner software (BWA, version 0.7.17). Sambamba software (https: / / github.com / biod / sambamba) was used to discard duplicates and remove unmapped or low-mapping quality reads from the alignment results. The remaining reads were defined as good reads and used for further analysis. All parameters used the default settings. SNP calling was performed using GATK (version 4.0.3.0), and the Variant Filtration module was used for filtering. The filtering parameters were set to "QD < 2.0', 'QUAL < 30.0', 'FS > 60.0', 'MQ < 40.0' --cluster-window-size 5 - cluster-size 2", meaning that sites with a variation quality (Quality) / depth of coverage (Depth) ratio less than 2.0, a quality value less than 30, a value converted from the Fisher test P-value greater than 60, a read alignment quality value root mean square less than 40, and more than 2 variants within a 5 bp window were filtered out. Fifty SNP sites that differed between AA broilers and non-AA broilers were obtained. The location information of these 50 SNP sites on the chicken genome is as follows:

[0042] NC_052572.1.70865596、NC_052572.1.70866183、NC_052572.1.70881246 、NC_052572.1.70893429、NC_052572.1.70894238、NC_052572.1.7088657 1、NC_052572.1.85904837、NC_052572.1.10525401、NC_052546.1.772828 、NC_052546.1.601275、NC_052546.1.4650059、NC_052553.1.594633、NC_ 052553.1.3944064、NC_052541.1.4669367、NC_052541.1.4864127、NC_05 2541.1.5135554、NC_052541.1.5254806、NC_052541.1.4633170、NC_0525 41.1.4669367、NC_052572.1.10910679、NC_052572.1.12398194、NC_0525 72.1.53321700、NC_052572.1.83262553、NC_052550.1.5129655、NC_05253 2.1.82042978、NC_052557.1.2763553、NC_052555.1.594633、NC_052546. 1.4864127、NC_052546.1.10525401、NC_052546.1.11161858、NC_052536. 1.11174102、NC_052551.1.13425197、NC_052551.1.13438787、NC_052551 .1.13448044、NC_052551.1.13578084、NC_052551.1.13902189、NC_05255 1.1.14131279、NC_052536.1.10910679、NC_052536.1.11109988、NC_0525 36.1.11161858、NC_052544.1.7566196、NC_052532.1.120589548、NC_052 532.1.2223786、NC_052532.1.4650059、NC_052535.1.873526、NC_052535 .1.963782、NC_052535.1.3628194、NC_052536.1.12561592、NC_052536.1.57411559, NC_052549.1.8580169; the version number of the chicken whole genome standard sequence is GCA_016699485.1bGalGal1.mat.broiler.GRCg7b (https: / / www.ncbi.nlm.nih.gov / genome / 111?genome_assembly_id=1543395).

[0043] Further screening: The features of the 50 SNPs obtained above were used as classification features (independent variables), ensuring that the training set obtained by Bootstrap resampling contained data for each SNP. The random forest algorithm was used to construct a classification model using the R language package random Forest. The parameters were set as follows: the number of trees (ntree) was 1000, the number of variables selected for each branch (mtry) was 4, and the proximity matrix was calculated. Other parameters were default. The average OOB error rate was used to evaluate the generalization ability of the model. The MDSplot function was used to output the three-dimensional coordinate data generated by the standardized proximity matrix, and the rgl package was used to draw the distribution map of the samples in three-dimensional space to graphically display the classification effect. The predict function was used to identify the variety, setting the parameter type = "prob" to output the accuracy estimate of each identification result. The final optimization resulted in a SNP molecular marker combination consisting of 25 SNP molecular markers (or sites), which has obvious species specificity for AA broiler chickens. The genotype information of the SNP molecular markers used to identify AA broiler chickens is shown in Table 1 in the instructions. The positions and polymorphism information of SNP molecular markers 1 to 25 in the chicken genome in the SNP marker combination are shown in Table 4:

[0044] Table 4 Specific locations of 25 SNP sites and their deoxynucleotide information

[0045] Experimental Example 2: Blind test of the accuracy of 25 SNP loci combination in identifying AA broiler chickens

[0046] This experimental example used the detection kit in the example to perform 25 SNP molecular marker genotyping on 378 unknown chicken samples to be tested, and identified whether the unknown chicken samples to be tested were AA broiler chickens based on the genotype of the SNP molecular markers. The specific operation is as follows:

[0047] Samples to be tested:

[0048] There are 378 chickens of 9 breeds, including AA broilers, Hubbard broilers, Cobb broilers, Gushi chickens, Xichuan black-bone chickens, Lushi green-shell laying hens, Fufeng hemp chickens, Guifei chickens and Hailan laying hens.

[0049] Experimental methods:

[0050] (1) Genomic DNA extraction and PCR amplification

[0051] Blood was extracted from 378 chickens of the above 9 breeds, and genomic DNA was extracted. PCR amplification of 25 SNP sites was performed using the detection kit in Example 2. The PCR system for KASP amplification is shown in Table 5.

[0052] Table 5 PCR system for KASP amplification (10 μL)

[0053] The reaction conditions were as follows: 94°C for 15 min; 94°C for 20 s, 61°C for 60 s, ramping at 0.6°C / cycle for 10 cycles; 94°C for 20 s, 55°C for 60 s, for 26 cycles. If no fluorescence signal was detected at the end of the initial reaction, additional steps could be added: 94°C for 20 s, 57°C for 60 s, for 3 cycles.

[0054] Note: The reaction parameters in the reaction program can be adjusted appropriately according to the different PCR amplification instrument models, enzymes, primers, etc.

[0055] (2) Fluorescence signal and genotyping

[0056] The PCR amplification products were detected using a fluorescence microplate reader with a platform capable of detecting FAM and VIC fluorescence wavelengths. The detection data were then read using the SNP viewer 2.0 software developed by LGC. SNP typing was performed based on the fluorescence signal ratio to obtain the genotype information of the 25 SNP sites for each individual.

[0057] (3) Comparison

[0058] The genotype information of each individual's 25 SNP loci was compared with the genotypes of the 25 SNP loci in Table 1. Chickens that met the conditions were identified as AA broilers. The identification results were compared with the actual chicken breed to verify the accuracy of the identification.

[0059] Experimental results:

[0060] A total of 50 AA broiler chickens and 328 chickens of the other seven breeds were identified in the blind test. The identification results were compared with the actual breeds, and the identification accuracy was calculated to be 99.47%. The identification results are shown in Table 6.

[0061] Table 6 Accuracy statistics using 25 SNP blind test samples

[0062] In summary, the present invention obtained whole-genome sequencing data for 30 AA broiler chickens through whole-genome resequencing. By comparing and analyzing the published whole-genome sequencing data for 336 individuals from 27 other chicken breeds, 50 SNP loci that distinguish AA broilers from non-AA broilers were identified. Combinatorial optimization and verification of these 50 SNP loci using a random forest algorithm yielded 25 SNP loci, forming a SNP molecular marker combination for identifying AA broilers. Furthermore, the use of a random forest algorithm can effectively account for the interconnections between individual SNP loci, creating correlations between the characteristics of each locus, thereby improving the accuracy of AA broiler breed identification. The present invention achieved an accuracy of 99.47% in testing.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A SNP molecular marker combination for identifying AA broiler chickens, characterized in that: The SNP molecular marker combination consists of 25 SNP molecular markers; the SNP sites of the molecular markers 1 to 25 are located at the 51st position of the nucleotide sequence shown in SEQ ID NO.1 to 25.

2. A detection kit, characterized in that: The detection kit comprises a PCR primer set for detecting the SNP molecular marker combination genotype for identifying AA broiler chickens as described in claim 1.

3. The detection kit according to claim 2, characterized in that: The PCR primer set is a competitive allele-specific PCR primer set.

4. The detection kit according to claim 3, characterized in that: The competitive allele-specific PCR primer set includes the first primer set to the twenty-fifth primer set corresponding to the detection of the SNP molecular markers 1 to 25; the nucleotide sequences of the first primer set to the twenty-fifth primer set are shown in sequences SEQ ID NOs. 26 to 100.

5. The detection kit according to any one of claims 2 to 4, characterized in that: The detection kit also includes KASP reaction buffer, DNA polymerase and dNTPs.

6. Use of the SNP molecular marker combination for identifying AA broiler chickens as claimed in claim 1 or the detection kit as claimed in any one of claims 2 to 4 in identifying AA broiler chicken germplasm resources.

7. Use of the SNP molecular marker combination or detection kit for identifying AA broiler chickens according to claim 6 in identifying AA broiler chicken germplasm resources, characterized in that: The genotypes of SNP molecular markers 1 to 25 of the sample to be tested are detected. If the genotypes of the 25 SNP molecular markers of the sample to be tested conform to the genotypes shown in Table 1, the sample to be tested is an AA broiler chicken. Table 1 8. Use of the SNP molecular marker combination or detection kit for identifying AA broiler chickens according to claim 7 in identifying AA broiler chicken germplasm resources, characterized in that: The following method is used to detect the genotype of the molecular marker: using the extracted DNA of the sample to be tested as a template, using the detection kit described in claim 5 to perform PCR amplification reaction, and obtaining the fluorescent signal for typing.

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