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

Abstract

This invention relates to a combination of SNP molecular markers for identifying AA broiler chickens, a detection kit, and their applications. The SNP molecular marker combination for identifying AA broiler chickens of this invention consists of 25 SNP molecular markers; the SNP sites of molecular markers 1-25 are located at position 51 of the nucleotide sequences shown in SEQ ID NO. 1-25. The SNP molecular marker combination of this invention exhibits significant species specificity for AA broiler chickens, representing unique variation and important trait-determining sites specific to AA broiler chickens. It enables rapid identification of genuine AA broiler chickens using relatively little genotypic information, providing a new technical reference for future identification, preservation, and genetic breeding of AA broiler chicken breeds.

Description

Technical Field

[0001] This invention relates to a combination of SNP molecular markers for identifying AA broiler chickens, a detection kit, and their applications, belonging to the field of biotechnology. Background Technology

[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 stably inherited characteristics, SNPs have gradually become a new generation of molecular markers and have been widely applied in biology, agriculture, medicine, and evolutionary biology. Molecular marker technology can rapidly, accurately, and efficiently identify breeds, which is of great significance for ensuring the correctness of breeds 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. Furthermore, the optimal combination of SNP markers can produce stable results in validation studies analyzing unknown samples.

[0003] AA broiler chickens, also known as Albert Ibrahim broiler chickens, are four-line crossbred white-feathered chickens. They are fast-growing breeds with advantages such as rapid growth, strong adaptability, high feed conversion ratio, uniform development, well-developed breast and leg muscles, and good carcass quality. Currently, conventional poultry breed identification relies solely on traditional appearance assessments such as body shape, feather color, shank color, and comb type. This method is inevitably influenced by environmental and other subjective factors, and chicks or already marketed meat products with indistinct breed characteristics cannot be identified through morphological features alone. Accurate identification of poultry strains at the genetic level is an effective way to solve this problem; therefore, identifying the characteristic markers of different strains is a pressing issue that needs to be addressed. Summary of the Invention

[0004] The first objective of this invention is to provide a combination of SNP molecular markers for identifying AA broiler chickens, thereby addressing the problem that there are no SNP molecular markers for identifying AA broiler chickens in the prior art.

[0005] The second objective of this invention is to provide a detection kit to address the problem in the prior art that it is impossible to detect SNP molecular markers characteristic of AA broiler breeds.

[0006] The third objective of this invention is to provide the application of SNP molecular marker combinations or detection kits for identifying AA broiler chickens in the identification of AA broiler chicken germplasm resources, thereby solving the problem in the prior art that it is impossible to accurately identify whether a chicken sample to be tested is an AA broiler chicken from a morphological perspective.

[0007] To achieve the above objectives, the present invention provides a technical solution for identifying SNP molecular marker combinations in AA broiler chickens:

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

[0009] The beneficial effects of the above technical solution are as follows: This invention obtains whole-genome sequencing data from 30 AA broiler individuals through whole-genome resequencing. By comparing and analyzing this data with the whole-genome sequencing data of 336 individuals from 27 other chicken breeds, 25 SNP loci that distinguish AA broilers from non-AA broilers are obtained through combination optimization, forming a combination of SNP molecular markers for identifying AA broilers. The SNP molecular marker combination for identifying AA broilers in this invention has obvious species specificity for AA broilers, enabling rapid identification of AA broilers with less genotypic information, and providing a new technical reference for future chicken breed identification, preservation, and genetic breeding.

[0010] To achieve the above objectives, the technical solution of a detection kit in this invention is as follows:

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

[0012] The beneficial effects of the above technical solution are as follows: designing and synthesizing PCR primer sets for the above SNP molecular marker combination genotypes, and then using gene sequencing and other methods after PCR amplification, can quickly and accurately obtain the genotype information of SNP molecular markers, 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 advantages of the above technical solution are as follows: Competitive allele-specific PCR (KSAP) is an effective means of SNP genotyping 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 site. Based on its unique ARM PCR principle, it allows all site detection to ultimately use universal fluorescent primers for amplification, achieving genotyping detection with the advantages of speed, accuracy, and high throughput.

[0015] As a further improvement, the competitive allele-specific PCR primer set includes a first primer set to a 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 SEQ ID NO. 26 to 100.

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

[0017] To achieve the above objectives, the technical solution of this invention for the application of SNP molecular marker combinations or detection kits for identifying AA broiler chickens in the identification of AA broiler chicken germplasm resources is as follows:

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

[0019] The beneficial effects of the above technical solution are as follows: This invention targets SNP molecular marker combinations used to identify AA broiler chickens, detects the genotype of these SNP molecular markers, and achieves rapid and accurate identification of whether a sample is an AA broiler chicken. The blind test accuracy rate is as high as 99.47%, filling the current gap in the lack of methods for identifying AA broiler chickens at the gene level. It can be used for the 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.

[0020] As a further improvement, the genotypes of SNP molecular markers 1 to 25 in the sample to be tested are detected. If the genotypes of the 25 SNP molecular markers in the sample to be tested match the genotypes shown in Table 1, then the sample to be tested is an AA broiler.

[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, a PCR amplification reaction is performed using a detection kit, and the fluorescence signal is obtained for typing. Detailed Implementation

[0024] The objectives, technical solutions, and beneficial effects of the present invention will be further explained below with reference to embodiments. The described embodiments help those skilled in the art to better understand the invention and do not constitute a limitation thereof. Unless otherwise specified, all reagents, instruments, etc., used in the embodiments are commercially available products.

[0025] Example 1: A combination of SNP molecular markers for identifying AA broiler chickens

[0026] The SNP molecular marker combination used to identify AA broiler chickens in this embodiment consists of 25 SNP molecular markers; the SNP sites of molecular markers 1 to 25 are located at position 51 of the nucleotide sequence shown in SEQ ID NO. 1 to 25.

[0027] Example 1 of a test kit

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

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

[0030]

[0031]

[0032] Example 1: Application of SNP molecular marker combinations or detection kits for identifying AA broiler chickens in the identification of AA broiler chicken germplasm resources

[0033] 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 in this embodiment includes the following steps:

[0034] (1) Extraction of genomic DNA from chickens 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 sequence SEQ ID NO. 26-100, PCR amplification was performed;

[0036] (3) Fluorescence signal acquisition and genotyping: SNP genotyping is performed based on the fluorescence signal ratio. If the genotypes of the 25 SNP molecular markers of the sample to be tested match the genotypes shown in Table 1, then the sample to be tested is an AA broiler.

[0037] Example 1: Screening for SNP molecular marker combinations in AA broiler chickens

[0038] This experiment obtained whole-genome sequencing data from 30 AA broiler chicken individuals through whole-genome resequencing. Comparative analysis with publicly available whole-genome sequencing data from 336 individuals of 27 other chicken breeds identified 50 distinguishing SNP loci between AA and non-AA broilers. Further optimization analysis of these 50 distinguishing SNP loci yielded a combination of 25 SNP loci exhibiting significant species specificity for AA broilers. The specific procedures are as follows:

[0039] Sequencing: Genomic DNA was extracted from the blood of 30 AA broiler chickens. The whole genome of the 30 AA broiler chickens was resequencing using the Illumina Nova Seq platform. The average sequencing depth reached 10X, and a total of 262.21 GB of raw sequencing data with a coverage of up to 97.61% (at least 1 base coverage) was obtained.

[0040] Whole genome sequencing data of 336 individuals from 27 other 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 shows the whole-genome sequencing data of 336 individuals from 27 other chicken breeds obtained from NCBI.

[0042]

[0043]

[0044] Data quality control and filtering: The raw, unprocessed data obtained from whole-genome sequencing of 30 AA broiler chickens and the whole-genome sequencing data of 336 individuals from 27 other chicken breeds obtained from NCBI were merged and quality controlled (QC) using the fastp software (https: / / github.com / Open Gene / fastp). 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: Filtered reads from all individuals were aligned with the chicken reference whole-genome 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-mapped quality reads from the alignment results. The remaining reads were defined as good reads and used for further analysis. All parameters were set to default. SNP calling was performed using GATK (version 4.0.3.0), and filtering was performed using its VariantFiltration module. The filtering parameters were set to "QD<2.0','QUAL<30.0','FS>60.0','MQ<40.0'--cluster-window-size5-cluster-size 2", meaning that sites with a quality / depth ratio less than 2.0, a quality value less than 30, a Fisher test p-value greater than 60, a root mean square of read alignment quality less than 40, and more than two variants within a 5bp window were filtered out. Fifty SNP sites distinguishing AA broilers from non-AA broilers were obtained. The location information of these 50 SNP sites in the 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: Using the features of the 50 SNPs obtained above as classification features (independent variables), it was ensured that the training set obtained through Bootstrap resampling contained data for each SNP. A random forest algorithm was employed, and the classification model was constructed using the R language package `randomForest`. Parameters were set as follows: the number of trees (`ntree`) was 1000, the number of variables selected in each branch (`mtry`) was 4, and the proximity matrix was calculated; other parameters remained at their default values. The average out-of-bounds (OOB) false positive rate was used to evaluate the model's generalization ability. 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 plot the distribution of samples in three-dimensional space, graphically demonstrating the classification effect. The `predict` function was used to identify the breed, with the parameter `type="prob"` set, outputting an accuracy estimate for each identification result. Finally, a combination of 25 SNP molecular markers (or loci) was optimized, exhibiting significant species specificity for AA broiler chickens. The genotypic information of the SNP molecular markers used to identify AA broiler chickens is shown in Table 1 of the instruction manual. The locations and polymorphism information of SNP molecular markers 1-25 in the chicken genome of the SNP marker combination are shown in Table 4:

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

[0049] SNP molecular markers chromosome Specific location Deoxynucleotides SNP molecular markers chromosome Specific location Deoxynucleotides 1 Z 70,865,596 A or T 14 15 4,864,127 people A or C 2 Z 70,866,183 T or C 15 5 11,174,102 A or G 3 Z 70,893,429 C or G 16 20 13,438,787 A or G 4 Z 70,894,238 G or A 17 20 13,448,044 C or T 5 Z 85,904,837 C or T 18 20 13,578,084 G or A 6 15 4,650,059 G or A 19 20 14,131,279 A or G 7 22 3,944,064 G or A 20 5 11,109,988 C or T 8 10 4,633,170 people A or T 21 5 11,161,858 A or G 9 10 4,669,367 people G or A 22 13 7,566,196 T or C 10 19 5,129,655 G or A 23 1 120,589,548 G or A 11 1 82,042,978 C or G 24 5 57,411,559 G or A 12 36 2,763,553 C or T 25 18 8,580,169th C or A 13 24 594,633 A or G

[0050] Experiment Example 2: Blind test to evaluate the accuracy of identifying AA broiler chickens using combinations of 25 SNP loci.

[0051] This experimental example utilizes the detection kit described in the previous example to perform genotyping of 25 SNP molecular markers on 378 unknown chicken samples. The genotype of the SNP molecular markers is used to determine whether the unknown chicken samples are AA broiler chickens. The specific operation is as follows:

[0052] Sample to be tested:

[0053] A total of 378 chicken individuals from 9 breeds, including AA broiler chickens, Hubbard broiler chickens, Cobb broiler chickens, Gushi chickens, Xichuan black-bone chickens, Lushi green-shelled egg chickens, Fufeng Ma chickens, Guifei chickens, and Hailan egg chickens.

[0054] Experimental methods:

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

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

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

[0058] Components Added amount 2×KASP Master Mix 5μL primer mix 2.5μL Template DNA (10–20 ng / μL) 2.5μL

[0059] The reaction conditions are as follows: 94℃ for 15 min; 94℃ for 20 s, 61℃ for 60 s, decreasing at a rate of 0.6℃ / cycle, for 10 cycles; 94℃ for 20 s, 55℃ for 60 s, for 26 cycles; if no fluorescence signal is detected after the initial reaction, the following step can be added: 94℃ for 20 s, 57℃ for 60 s, for 3 cycles.

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

[0061] (2) Fluorescence signal and genotyping

[0062] The PCR amplification products were detected using a fluorescence microplate detector on a platform capable of detecting FAM and VIC fluorescence wavelengths. The detection data were then read using SNP viewer 2.0 software developed by LGC, and SNP genotyping was performed based on the fluorescence signal ratio to obtain the genotype information of 25 SNP loci for each individual.

[0063] (3) Comparison

[0064] The genotype information of 25 SNP loci for each individual was compared with the genotypes of 25 SNP loci in Table 1. Those meeting the criteria were identified as AA broiler chickens. The identification results were compared with actual chicken breeds to verify the accuracy of the identification.

[0065] Experimental results:

[0066] A total of 50 AA broiler chickens and 328 chickens of 7 other breeds were identified in the blind test. The identification results were compared with the actual breeds, and the accuracy rate of identification was calculated to be 99.47%. The identification results are shown in Table 6.

[0067] Table 6 shows the accuracy statistics using 25 SNP blind test samples.

[0068] variety Number of individuals in blind test Accuracy of identification accuracy % AA broiler chickens 50 50 100 Hubbard broiler chickens 50 50 100 Kobo broiler chickens 38 36 94.73 Gushi Chicken 50 50 100 Xichuan Black-boned Chicken 50 50 100 Lushi Green-shelled Chicken 50 50 100 Fu Feng Ma Chicken 30 30 100 Imperial Concubine Chicken 30 30 100 Hy-Line Egg Chicken 30 30 100 total 378 376 99.47

[0069] In summary, this invention obtained whole-genome sequencing data from 30 AA broiler individuals through whole-genome resequencing. By comparing and analyzing this data with the whole-genome sequencing data of 336 individuals from 27 other chicken breeds, 50 SNP loci distinguishing AA broilers from non-AA broilers were identified. Furthermore, the 50 SNP loci were combined and optimized using a random forest algorithm to obtain 25 SNP loci, forming a combination of SNP molecular markers for identifying AA broilers. Moreover, the random forest algorithm effectively considers the interrelationships between SNP loci, ensuring correlation between the characteristics of each locus and improving the accuracy of AA broiler breed identification. This invention achieved an accuracy of 99.47% in testing.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions 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 combination of SNP molecular markers for identifying AA broiler chickens, characterized in that: The SNP molecular marker combination consists of 25 SNP molecular markers; the nucleotide sequence of molecular marker 1 is shown in SEQ ID NO.1, with the 51st base from the 5' end being A or T; the nucleotide sequence of molecular marker 2 is shown in SEQ ID NO.2, with the 51st base from the 5' end being T or C; the nucleotide sequence of molecular marker 3 is shown in SEQ ID NO.3, with the 51st base from the 5' end being C or G; the nucleotide sequence of molecular marker 4 is shown in SEQ ID NO.4, with the 51st base from the 5' end being G or A; the nucleotide sequence of molecular marker 5 is shown in SEQ ID NO.5, with the 51st base from the 5' end being C or T; the nucleotide sequence of molecular marker 6 is shown in SEQ ID NO.6, with the 51st base from the 5' end being G or A; the nucleotide sequence of molecular marker 7 is shown in SEQ ID NO.7, with the 51st base from the 5' end being G or A; the nucleotide sequence of molecular marker 8 is shown in SEQ ID NO. As shown in SEQ ID NO. 8, the 51st base from the 5' end is A or T; the nucleotide sequence of molecular marker 9 is shown in SEQ ID NO. 9, with the 51st base from the 5' end being G or A; the nucleotide sequence of molecular marker 10 is shown in SEQ ID NO. 10, with the 51st base from the 5' end being G or A; the nucleotide sequence of molecular marker 11 is shown in SEQ ID NO. 11, with the 51st base from the 5' end being C or G; the nucleotide sequence of molecular marker 12 is shown in SEQ ID NO. 12, with the 51st base from the 5' end being C or T; the nucleotide sequence of molecular marker 13 is shown in SEQ ID NO. 13, with the 51st base from the 5' end being A or G; the nucleotide sequence of molecular marker 14 is shown in SEQ ID NO. 14, with the 51st base from the 5' end being A or C; the nucleotide sequence of molecular marker 15 is shown in SEQ ID NO.

14. As shown in SEQ ID NO. 15, the 51st base from the 5' end is A or G; the nucleotide sequence of molecular marker 16 is shown in SEQ ID NO. 16, with the 51st base from the 5' end being A or G; the nucleotide sequence of molecular marker 17 is shown in SEQ ID NO. 17, with the 51st base from the 5' end being C or T; the nucleotide sequence of molecular marker 18 is shown in SEQ ID NO. 18, with the 51st base from the 5' end being G or A; the nucleotide sequence of molecular marker 19 is shown in SEQ ID NO. 19, with the 51st base from the 5' end being A or G; the nucleotide sequence of molecular marker 20 is shown in SEQ ID NO. 20, with the 51st base from the 5' end being C or T; the nucleotide sequence of molecular marker 21 is shown in SEQ ID NO. 21, with the 51st base from the 5' end being A or G; the nucleotide sequence of molecular marker 22 is shown in SEQ ID NO.As shown in SEQ ID NO. 22, the 51st base from the 5' end is T or C; the nucleotide sequence of molecular marker 23 is shown in SEQ ID NO. 23, the 51st base from the 5' end is G or A; the nucleotide sequence of molecular marker 24 is shown in SEQ ID NO. 24, the 51st base from the 5' end is G or A; the nucleotide sequence of molecular marker 25 is shown in SEQ ID NO. 25, the 51st base from the 5' end is C or A.

2. A test kit for identifying AA broiler chickens, characterized in that: The detection kit includes a PCR primer set for detecting the SNP molecular marker combination genotype of 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 SEQ ID NO. 26 to 100.

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

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

7. The application of the SNP molecular marker combination or detection kit for identifying AA broiler chickens according to claim 6 in the identification of AA broiler chicken germplasm resources, characterized in that: The genotypes of SNP molecular markers 1-25 in the sample to be tested are detected. If the genotypes of the 25 SNP molecular markers in the sample match the genotypes shown below, then the sample is an AA broiler chicken. 。 8. The application of the SNP molecular marker combination or detection kit for identifying AA broiler chickens according to claim 7 in the identification of AA broiler chicken germplasm resources, characterized in that: The genotype of the molecular marker was detected using the following method: using the extracted DNA from the sample to be tested as a template, a PCR amplification reaction was performed using the detection kit described in claim 5, and the fluorescence signal was obtained for genotyping.

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