SNP (Single Nucleotide Polymorphism) molecular marker combination for identifying AA broiler chicken, detection kit and application of SNP molecular marker combination

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) molecular marker combination for identifying AA broiler chicken, a detection kit and application of the SNP molecular marker combination. The SNP molecular marker combination for identifying the AA broiler chicken is composed of 25 SNP molecular markers; and the SNP sites of the molecular markers 1-25 are located at the 51st site of the nucleotide sequences as shown in SEQ ID NO.1-25. The SNP molecular marker combination disclosed by the invention has obvious species specificity of the AA broiler chicken, is a specific variation and important character determination site of the AA broiler chicken, can quickly identify the authenticity of the AA broiler chicken by utilizing less genotype information, and provides a new technical reference for the identification, breed conservation and genetic breeding of the AA broiler chicken variety in the future.

Description

Technical Field

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

[0002] Single nucleotide polymorphism (SNP) refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. Due to its advantages of large number and stable inheritance, it has gradually become a new generation of molecular markers and has been widely used in biology, agriculture, medicine and biological evolution. Molecular marker technology can quickly, accurately and efficiently identify varieties, which is of great significance to ensure the correctness of varieties 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 the preservation and commercial use of varieties. In addition, the optimal combination of SNP markers can produce stable results in validation studies of unknown samples.

[0003] AA broiler, also known as Ai Bo Yi Jia broiler, is a four-line matching white-feathered hybrid chicken. It is a fast-growing large breed with the advantages of fast growth, strong adaptability, high feed conversion rate, neat development, plump chest and leg muscles, and good carcass quality. At present, conventional identification of poultry breeds is only based on traditional appearance identification such as body shape, feather color, shank color, and crown shape, which is inevitably affected by the environment and other subjective factors. In addition, chicks with unclear breed characteristics or meat products that have been on the market cannot be identified by morphological characteristics. Accurate identification of poultry strains at the genetic level is an effective way to solve this problem. Therefore, mining the characteristic markers of different strains is an urgent problem to be solved. Summary of the invention

[0004] 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.

[0005] 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 the characteristic SNP molecular markers of the AA broiler breed.

[0006] The third object of the present invention is to provide a SNP molecular marker combination or a 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 from the morphology.

[0007] In order to achieve the above object, a technical solution for identifying a combination of SNP molecular markers of AA broilers in the present invention is:

[0008] A SNP molecular marker combination for identifying AA broilers, the SNP molecular marker combination consisting of 25 SNP molecular markers; the SNP sites of the molecular markers 1-25 are located at the 51st position of the nucleotide sequence shown in SEQ ID NO.1-25.

[0009] The beneficial effect of the above technical solution is that: the present invention obtains the whole genome sequencing data of 30 AA broiler chickens through whole genome resequencing, and compares and analyzes the whole genome sequencing data of 336 individuals of 27 other chicken breeds, and optimizes the combination to obtain 25 SNP sites that are different from AA broilers and 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 of AA broilers, and can quickly identify the authenticity of AA broilers with less genotype information, providing a new technical reference for the identification, seed preservation and genetic breeding of chicken breeds in the future.

[0010] In order to achieve the above object, a technical solution of a detection kit in the present invention is:

[0011] A detection kit comprises the PCR primer set for identifying the SNP molecular marker combination genotype of AA broiler chickens.

[0012] 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 the genotype information of the SNP molecular marker can be quickly and accurately obtained by means of gene sequencing after PCR amplification, laying the foundation for subsequent analysis and verification.

[0013] As a further improvement, the PCR primer set is a competitive allele-specific PCR primer set.

[0014] The beneficial effect of the above technical solution is that competitive allele-specific PCR (KSAP) is an effective means of SNP typing and detecting Indels by using the specific matching of primer terminal bases. This method does not require the synthesis of specific fluorescent primers for each SNP site. It is based on its own unique ARM PCR principle, so that all site detections are ultimately amplified using universal fluorescent primers to achieve genotyping detection, which has the advantages of being fast, accurate, and high-throughput.

[0015] 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.26-100.

[0016] As a further improvement, the detection kit also includes KASP reaction buffer, DNA polymerase and dNTPs.

[0017] In order to achieve the above-mentioned object, the technical scheme of 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:

[0018] Application of SNP molecular marker combination or detection kit for identifying AA broiler chickens in the identification of AA broiler chicken germplasm resources.

[0019] The beneficial effect of the above technical solution is that the present invention detects the genotype of the SNP molecular marker combination for identifying AA broilers, and realizes rapid and accurate identification of whether the sample to be tested is an AA broiler, with a blind detection accuracy rate of up to 99.47%, filling the gap of the current method for identifying AA broilers at the genetic level. It can be used for the traceability identification and protection of AA broiler germplasm, which is of great significance for promoting the healthy development of AA broiler germplasm resources.

[0020] 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 conform to the genotypes shown in Table 1, the sample to be tested is an AA broiler chicken.

[0021] Table 1

[0022]

[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 scheme and beneficial effects of the present invention are further described below in conjunction with the examples. The described embodiments are helpful for those skilled in the art to better understand the invention and do not constitute a limitation of the present invention. Unless otherwise specified, all kinds of reagents, instruments, etc. 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 the 51st position of the nucleotide sequence 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 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 the sequence 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]

[0031]

[0032] Example 1 of application of a SNP molecular marker combination or detection kit for identifying AA broiler chickens in identifying AA broiler chicken germplasm resources

[0033] The application 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:

[0034] (1) Extraction of genomic DNA from chicken samples to be tested;

[0035] (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 is performed;

[0036] (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.

[0037] Experimental Example 1 Screening of SNP molecular marker combinations for identifying AA broiler chickens

[0038] In this experiment, whole genome sequencing data of 30 AA broiler chickens were obtained through whole genome resequencing. By comparing and analyzing the whole genome sequencing data of 336 individuals of 27 other chicken breeds, 50 SNP sites that are different from AA broilers and non-AA broilers were obtained. Then, through optimization analysis of the 50 different SNP sites, 25 SNP site combinations with obvious species specificity of AA broilers were obtained. The specific operations are as follows:

[0039] 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 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 with a coverage rate of up to 97.61% (at least 1 base coverage) was obtained.

[0040] The whole genome sequencing data of 336 individuals of 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.

[0041] Table 3 Whole genome sequencing data of 336 individuals of 27 other chicken breeds obtained by NCBI

[0042]

[0043]

[0044] 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, removing low-quality nucleotides, unknown nucleotides (NS), and reads containing more than 10% NS.

[0045] Analysis and alignment: The filtered reads of all individuals were aligned with the chicken reference whole genome standard sequence (version number GRCg7b) by 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 score reads from the alignment results. The remaining reads were defined as good reads and used for further analysis. All parameters used default settings. GATK (version 4.0.3.0) was used for SNP calling, and the VariantFiltration module was used for filtering. The screening parameters were set to "QD<2.0','QUAL<30.0','FS>60.0','MQ<40.0'--cluster-window-size5-cluster-size 2", which means that the variation quality (Quality) / coverage depth (Depth) ratio is less than 2.0, the quality value is less than 30, the value converted from the Fisher test P-value is greater than 60, the root mean square of the read alignment quality value is less than 40, and the number of variants in the 5bp window exceeds 2 sites will be filtered out. 50 SNP sites that are different between AA broilers and non-AA broilers were obtained, and the location information of the 50 SNP sites on the whole chicken genome is as follows:

[0046] 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). .

[0047] Further screening: The features of the 50 SNPs obtained above were used as classification features (independent variables) to ensure that the training set obtained by Bootstrap resampling contained data for each SNP. The random forest algorithm was used to construct the 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 misclassification 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, and the parameter type = "prob" was set to output the accuracy estimate of each identification result. Finally, a SNP molecular marker combination consisting of 25 SNP molecular markers (or loci) was optimized, which had obvious species specificity for AA broilers. The genotype information of the SNP molecular markers used to identify AA broilers is shown in Table 1 in the manual. The positions and polymorphism information of SNP molecular markers 1 to 25 in the SNP marker combination in the whole chicken genome are shown in Table 4:

[0048] Table 4 Specific locations of 25 SNP sites and their deoxynucleotide information

[0049] SNP molecular markers chromosome Specific location Deoxyribonucleotides SNP molecular markers chromosome Specific location Deoxyribonucleotides 1 Z 70865596 bits A or T 14 15 4864127 A or C 2 Z 70866183 T or C 15 5 11174102 A or G 3 Z 70893429 C or G 16 20 13438787 A or G 4 Z 70894238 G or A 17 20 13448044 bits C or T 5 Z 85904837 C or T 18 20 13578084 G or A 6 15 4650059 G or A 19 20 14131279 A or G 7 22 3944064 bits G or A 20 5 11109988 C or T 8 10 4633170 A or T 21 5 11161858 A or G 9 10 4669367 G or A 22 13 7566196 T or C 10 19 5129655 G or A 23 1 120589548 G or A 11 1 82042978 bits C or G 24 5 57411559 G or A 12 36 2763553 C or T 25 18 8580169 C or A 13 24 594633 A or G

[0050] Experimental Example 2: Blind test of the accuracy of 25 SNP loci combined to identify AA broiler chickens

[0051] In this experimental example, the detection kit in the embodiment was used to perform 25 SNP molecular marker genotype detection on 378 unknown chicken samples to be tested, and the genotype of the SNP molecular marker was used to identify whether the unknown chicken samples to be tested were AA broilers. The specific operation is as follows:

[0052] Samples to be tested:

[0053] 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 Ma chickens, Guifei chickens and Hailan laying hens.

[0054] Experimental methods:

[0055] (1) Genomic DNA extraction and PCR amplification

[0056] Blood was extracted from 378 chickens of the above 9 breeds, and genomic DNA was extracted. PCR amplification was performed on 25 SNP sites using the detection kit in Example 2. The PCR system for KASP amplification is shown in Table 5.

[0057] Table 5 PCR system for KASP amplification (10 μL)

[0058] Components Addition amount 2×KASP Master Mix 5μL primer mix 2.5μL Template DNA (10~20ng·μL-1) 2.5μL

[0059] The reaction conditions were as follows: 94°C for 15 min; 94°C for 20 s, 61°C for 60 s, ramping at a rate of 0.6°C / cycle, 10 cycles; 94°C for 20 s, 55°C for 60 s, 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, 3 cycles.

[0060] Note: The reaction parameters in the reaction program can be adjusted appropriately according to the different PCR amplification instrument models, enzymes, primers, etc.

[0061] (2) Fluorescence signal and genotyping

[0062] The PCR amplification products were detected using a platform capable of detecting FAM and VIC fluorescence wavelengths and a fluorescence microplate reader. 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.

[0063] (3) Comparison

[0064] The genotype information of the 25 SNP loci of each individual was compared and analyzed with the genotypes of the 25 SNP loci in Table 1. Those that met the conditions were AA broilers. The identification results were compared with the real chicken breeds to test the accuracy of the identification.

[0065] Experimental results:

[0066] A total of 50 AA broilers 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.

[0067] Table 6 Accuracy statistics of 25 SNP blind test samples

[0068] variety Number of blind test individuals Identification of accurate number Accuracy % AA broiler 50 50 100 Hubbard Broiler Chicken 50 50 100 Cobb broiler chicken 38 36 94.73 Gushi Chicken 50 50 100 Xichuan Black-bone Chicken 50 50 100 Lushi Green Shell Egg Chicken 50 50 100 Fufeng Chicken 30 30 100 Royal Chicken 30 30 100 Hy-Line Egg Chicken 30 30 100 total 378 376 99.47

[0069] In summary, the present invention obtains the whole genome sequencing data of 30 AA broiler chickens through whole genome resequencing, and obtains 50 SNP sites that are different from AA broilers and non-AA broilers by comparing and analyzing the whole genome sequencing data of 336 individuals of 27 other chicken breeds that have been disclosed. The 50 SNP sites obtained are combined and optimized by the random forest algorithm to obtain 25 SNP sites, which constitute the SNP molecular marker combination for identifying AA broilers. Moreover, the use of the random forest algorithm can well take into account the mutual connection between each SNP site, so that the characteristics of each site are correlated, and the accuracy rate in the identification process of AA broiler chickens is improved. The present invention achieves an accuracy rate of 99.47% in the test.

[0070] Finally, it is 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned 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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