A Whole Genome Chip for Germplasm Traceability of Yellow-Feathered Broilers and Its Application

Abstract

The present invention belongs to the technical field of molecular biology, and particularly relates to a whole-genome chip for germplasm traceability of yellow-feathered broilers and its application. It is prepared from probes composed of 9,737 germplasm traceability sites of yellow-feathered broilers and 261 disease resistance-related sites, with a total of 9,998 SNP molecular markers. The physical position information on the chicken reference genome is shown in Table 1. The chip sites cover all chromosomes, are evenly distributed on each chromosome, with an average distance of 95 Kb, having the advantage of low marker density. The cost of the liquid-phase chip directly depends on the marker density, enabling genomic breeding related to chicken disease resistance at a relatively low cost. Moreover, the chip product has good stability, with a genotype consistency rate of >99% for repeated samples, and can reliably achieve germplasm traceability for local chicken breeds, commercial chicken breeds, and wild red jungle fowl populations at home and abroad, as well as commercial broilers of yellow-feathered broilers with highly similar population genetic backgrounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a whole-genome chip for germplasm traceability of yellow-feathered broilers and its application. Background Art

[0002] In the yellow-feathered broiler industry, due to the lack of reliable low-cost germplasm traceability technology methods, there is still a large amount of counterfeiting production and sales of parent stock and commercial stock seedlings. While seriously damaging the interests of relevant breeding enterprises, it also greatly restricts the vitality of the original innovation in the yellow-feathered broiler seed industry.

[0003] The core of intellectual property protection in the yellow-feathered broiler seed industry lies in establishing a set of reliable low-cost germplasm traceability technology methods to clarify the genetic relationship (whether directly derived from the target population) between suspected infringing individuals or groups and suspected infringed target groups, and to determine whether the germplasm property rights have been violated. At the technical level, the germplasm traceability technology methods for livestock and poultry are already very mature, and the key affecting their wide application lies in the cost of molecular markers used for traceability. In the broiler field, due to the low economic value of individual commercial units and the germplasm traceability scenario of identifying the genetic relationship between suspected infringing individuals or groups and suspected infringed target reference groups, it is necessary to genotype the target reference groups. Therefore, the cost factor of molecular markers is particularly prominent. In chickens, molecular markers commonly used for germplasm traceability include microsatellites and SNPs. In terms of applicability and cost performance, the low-density whole-genome SNP genotyping chip is the best. How to use as few SNP locus markers as possible to reduce the cost of gene chip genotyping while achieving reliable traceability of broiler germplasm is of great significance for effectively protecting the intellectual property rights of the yellow-feathered broiler seed industry.

[0004] Patent CN114480673A discloses a chicken low-density SNP liquid chip based on targeted capture sequencing and its application (5K density), and patent CN116377086A discloses a chicken whole-genome low-density chip and its manufacturing method and application (11K density). However, the former technology mainly focuses on the identification of genetic relationships of local chicken breeds in chicken germplasm traceability, rather than commercial yellow-feathered broilers with a highly similar genetic background in a large number of populations; while the loci of the latter come from breed-specific SNPs of local chicken breeds in Shandong Province and introduced breeds (7 local chicken breeds in Shandong Province and 1 introduced breed), and the genetic diversity information of chicken populations included is relatively limited, and this technology cannot effectively distinguish / trace commercial yellow-feathered broiler chickens with a highly similar genetic background in actual measurement. Summary of the Invention

[0005] Aiming at the above problems, the purpose of the present invention is to provide a whole-genome chip for germplasm traceability of yellow-feathered broilers and its application.

[0006] The technical content of the present invention is as follows:

[0007] The present invention provides a whole-genome chip for germplasm traceability of yellow-feathered broilers. The chip is a liquid-phase chip, and the chip is prepared from probes composed of 9,737 germplasm traceability sites of yellow-feathered broilers and 261 disease resistance-related sites, with a total of 9,998 SNP molecular markers. The physical position information of the SNP molecular markers on the chicken reference genome bGalGal1.mat.broiler.GRCg7b is shown in Table 1;

[0008] The whole-genome chip of yellow-feathered broilers is a low-density chip with a size less than 10 Kb;

[0009] The site information in the whole-genome chip is all in the bGalGal1.mat.broiler.GRCg7b version.

[0010] The present invention also provides a probe for the whole genome of yellow-feathered broilers for germplasm traceability. The probe includes 9,737 germplasm traceability sites of yellow-feathered broilers and 261 disease resistance-related sites, with a total of 9,998 SNP molecular markers. The physical position information of the SNP molecular markers on the chicken reference genome bGalGal1.mat.broiler.GRCg7b is shown in Table 1.

[0011] The present invention also provides an application of the whole-genome chip of yellow-feathered broilers, including one of the following:

[0012] (1) Application in the screening and identification of commercial yellow-feathered broilers;

[0013] (2) Application in the traceability of commercial yellow-feathered broilers;

[0014] (3) Application in the germplasm traceability of Chinese indigenous chicken breeds;

[0015] (4) Application of genomic breeding in disease resistance breeding of yellow-feathered broilers;

[0016] (5) Application in the construction of the population genetic pedigree of yellow-feathered broilers;

[0017] (6) Application in the genome-wide association analysis of yellow-feathered broilers;

[0018] (7) Application in the QTL mapping analysis of target traits of yellow-feathered broilers.

[0019] The present invention also provides a preparation method of the whole-genome chip of yellow-feathered broilers, including the following steps:

[0020] 1) Obtain indigenous chicken breeds at home and abroad for whole-genome resequencing, and use GATK to construct a high-quality digital gene bank to obtain high-quality autosomal SNPs;

[0021] 2) Filter the obtained high-quality autosomal SNPs through LD-pruning and physical distance dilution to obtain candidate loci for the germplasm traceability of yellow-feathered broilers;

[0022] Meanwhile, referring to the chicken disease susceptibility-related QTLs in the Animal QTL database, extract candidate loci for disease resistance traits from the locus library after LD-pruning of high-quality autosomal SNPs;

[0023] 3) Combine the candidate loci for the germplasm traceability of yellow-feathered broilers and the candidate loci for disease resistance traits obtained above, design probes, and a total of 9,998 high-quality custom chip loci are selected for chip probe synthesis to generate the final germplasm traceability chip for yellow-feathered broilers. The chip contains 9,737 germplasm traceability loci and 261 disease resistance trait loci respectively.

[0024] The present invention also provides an evaluation method for the germplasm traceability of yellow-feathered broilers, including the following steps:

[0025] 1) Extract DNA and construct a liquid-phase capture library

[0026] ① Fragment the extracted genomic DNA, repair the ends and add A;

[0027] ② Screen the range of fragmented DNA through magnetic beads, remove too large and too small fragments, and make the DNA fragments concentrated as much as possible at 200-300 bp;

[0028] ③ Connect sequencing adapters and perform PCR amplification enrichment to construct a whole-genome library;

[0029] ④ Pool the whole-genome libraries of each sample, concentrate and hybridize the pooled libraries with probes, capture the fragments in the target region from the whole-genome library, and finally perform PCR amplification to obtain a hybrid capture library;

[0030] 2) Library quality inspection and sequencing

[0031] Perform quantitative and sequencing detection on the hybrid capture library;

[0032] 3) Liquid-phase capture analysis

[0033] Including data filtering and statistics, alignment, and target site analysis;

[0034] 4) Evaluation of the germplasm traceability efficacy

[0035] Using the liquid-phase chip prepared above, genotype the genomes of yellow-feathered broilers with a highly similar population genetic background. Use VCF2Dis to construct a population genetic distance matrix, and then use ATGC:FastME to construct a phylogenetic tree. Observe the results to see if yellow-feathered broilers with a highly similar population genetic background can be effectively distinguished.

[0036] The beneficial effects of the present invention are as follows:

[0037] The whole-genome chip for tracing the germplasm of yellow-feathered broilers of the present invention is prepared from probes composed of 9,737 yellow-feathered broiler germplasm tracing loci and 261 disease resistance-related loci, with a total of 9,998 SNP molecular markers. The physical position information of the SNP molecular markers on the chicken reference genome bGalGal1.mat.broiler.GRCg7b is shown in Table 1. The chip loci cover all chromosomes, are evenly distributed on each chromosome, and the average distance is 95 Kb, having the advantage of low marker density (9,998 marker loci, less than 10 K). The cost of the liquid-phase chip directly depends on the marker density. It can perform genomic breeding related to chicken disease resistance at a lower cost, and the chip product has good stability, with a genotype consistency rate of >99% for repeated samples. It can reliably trace the germplasm of local chicken breeds, commercial chicken breeds, and wild red jungle fowl populations at home and abroad, as well as yellow-feathered broiler commercial broilers with a highly similar population genetic background; it can efficiently and reliably identify the population genetic relationships of local chicken breeds and yellow-feathered broiler commercial broilers with a highly similar population genetic background, and realize the tracing of the germplasm of local chicken breeds and yellow-feathered broilers. Description of the Drawings

[0038] Figure 1 For the germplasm tracing loci (9,737 SNPs) of the present invention ( Figure 1 C), whole-genome loci (17,110,868 SNPs) ( Figure 1 A), and loci after LD-pruning of the whole-genome loci (6,005,803 SNPs) ( Figure 1 B), the simulation comparison results of the germplasm tracing efficacy in 28 local chicken breeds, commercial chicken breeds, and red jungle fowl populations;

[0039] Figure 2 For the efficacy evaluation results when the present invention and whole-genome resequencing trace the germplasm of slow-growing broilers of 3 different Qingyuan partridge chicken breeding companies;

[0040] Figure 3 For the distribution map of the 9,998 SNPs loci of the whole-genome chip of yellow-feathered broilers of the present invention on chromosomes.

[0041] Figure 4This is the result graph of the genotyping consistency rate of the whole-genome chip and whole-genome resequencing (10×) of the same samples for the yellow-feathered broiler of the present invention. Specific embodiments

[0042] The present invention will be further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention. After reading the present invention, various equivalent modifications made by those skilled in the art to the present invention fall within the scope defined by the appended claims of this application.

[0043] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents on the conventional market.

[0044] Example 1

[0045] A whole-genome chip for tracing the germplasm of yellow-feathered broilers

[0046] 1) A total of 281 chickens, including 28 local chicken breeds, commercial chicken breeds, and red jungle fowl populations from China and abroad (White Ear Yellow Chicken, White Leghorn, Dong Tao Chicken, Hetian Chicken, Red Jungle Fowl, Huaixiang Chicken, Huiyang Bearded Chicken, Jining Hundred-Day Chicken, Laotian Fighting Chicken, Liyang Chicken, Lindian Chicken, Ross 308, Rhode Island Red Chicken, Luoba Chicken, Qilin Chicken, 3 Qingyuan Partridge Chicken populations, Queshan Chicken, Turpan Fighting Chicken, Wenchang Chicken, Wuhua Sanhuang Chicken, Xianju Chicken, Xiangdong Chicken, Yangshan Chicken, Yao Chicken, Recessive White Rock Chicken, Yunyang Big Chicken), etc., were selected to conduct high-depth (average sequencing depth > 30×) whole-genome resequencing to construct a high-quality digital gene bank of chickens at home and abroad, obtaining 17,110,168 high-quality autosomal SNPs (after obtaining individual g.vcf by single calling using the GATK HaplotypeCaller engine and merging them into a population vcf file; using default parameters, performing hard filtering using GATK to obtain a preliminarily quality-controlled population vcf file; further filtering the preliminarily quality-controlled population vcf file using VCFtools, retaining autosomal biallelic loci, and removing loci with a minor allele frequency less than 0.02, a locus deletion rate greater than 0.02, and a locus quality lower than 15 to obtain a high-quality autosomal population SNP set). Further, through LD-pruning (Plink; parameters: --indep-pairwise 50 50.5) and physical distance (85Kb) dilution, the obtained 17,110,168 high-quality autosomal SNPs were filtered to obtain 11,028 candidate loci for germplasm tracing of yellow-feathered broilers; meanwhile, referring to the chicken disease susceptibility-related QTLs in the Animal QTL database (https: / / www.animalgenome.org / cgi-bin / QTLdb / GG / ontrait?class_ID=1), relevant SNP loci were extracted from the locus library after LD-pruning of the 17,110,168 high-quality autosomal SNPs, and the extracted loci were regionally associated with the 11,028 germplasm tracing candidate loci, removing the loci located within the 10Kb region upstream and downstream of the 11,028 loci to obtain 382 candidate loci for disease resistance traits;

[0047] For the obtained 11,028 germplasm tracing candidate loci and 382 disease resistance trait candidate locus sets, probe design was carried out according to the evaluation results such as the complexity of the upstream and downstream sequences of the target loci and GC content. The designed probes were 120bp in length. According to the design results, a total of 9,998 high-quality chip custom loci were selected for chip probe synthesis to generate the final yellow-feathered broiler germplasm tracing chip, which contains 9737 and 261 germplasm tracing loci and disease resistance trait loci respectively, as shown in Table 1:

[0048] Table 1 Physical location information of the loci included in the whole-genome chip for the germplasm traceability of yellow-feathered broilers

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] Note: All the physical location information marked is in the bGalGal1.mat.broiler.GRCg7b version.

[0082] 2) Genomic DNA extraction

[0083] The genomic DNA extraction kit based on magnetic beads (CW2361S from CWBIO) was used to extract the sample DNA.

[0084] 3) Construction of liquid-phase capture library (enzymatic fragmentation)

[0085] 3.1) Fragmentation, end repair and A-tailing of genomic DNA

[0086] Add 4 μL of Smearase Buffer and 2 μL of Smearase Enzymes to 300 ng of DNA sample, with a total volume of 24 μL. Then place the reaction plate in a PCR instrument and execute the program: 4°C for 1 min → 30°C for 10 min → 72°C for 20 min → hold at 4°C.

[0087] 3.2) Fragment screening

[0088] The fragmented DNA was screened by magnetic beads to remove overly large and small fragments, making the DNA fragments concentrated at 200 - 300 bp as much as possible. The final volume of the fragment screening was 25 μL and stored in a 96-well PCR plate.

[0089] 3.3) Adapter ligation and enrichment

[0090] Add 5 μL CAGT Universal Adapters and 20 μL Ligation Master Mix to the 96-well PCR plate in the above step, vortex and mix, centrifuge briefly to collect the reaction solution to the bottom of the tube, react at 20°C for 15 minutes in a PCR instrument to complete the sequencing adapter connection. After the connection product is purified, PCR amplification and enrichment are performed, 15 μL of the connection purified product is taken, 10 μL CAGT UDI Primer and 25 μL Equinox Library Amp Mix (2x) are added, mixed, and 35 μL of the reaction solution is aspirated in a PCR instrument for library amplification. After the whole genome library is constructed, the library is quantified using the dsDNA HS Assay Kit for Qubit kit; at the same time, the main peak of the library fragment is detected by electrophoresis to see if it is in the range of 300-500 bp.

[0091] 3.4) Liquid-phase chip hybridization capture

[0092] The whole genome library of each sample was mixed, and the total amount of the final hybrid capture library was 4μg. The mixed pool library was concentrated and probe hybridized to capture the fragments of the target region from the whole genome library. The excess probes, hybridization reagents and other reagent components were removed through the elution step. Finally, the target region was enriched through PCR amplification after hybridization to obtain the machine library.

[0093] 4) Library quality inspection and sequencing

[0094] After the hybrid capture library was constructed, the dsDNA HS Assay Kit for Qubit was used to quantify the library; at the same time, electrophoresis was used to detect whether the main peak size of the library was in the range of 300-500bp. The constructed library was sequenced on a DNBSEQ-T7 sequencer.

[0095] 5) Analysis process

[0096] The liquid phase capture analysis process mainly includes data filtering statistics, alignment and target site analysis, as follows:

[0097] 5.1) Data filtering and statistics

[0098] After the raw data is downloaded, it will contain reads with adapters or low quality. Before using fastp for subsequent analysis, we need to filter the raw data. The filtering conditions are as follows:

[0099] A. Remove reads with adapters;

[0100] B. When the N content in the sequencing read exceeds 10% of the base number of the read, the paired reads are removed;

[0101] C. When the number of low-quality (Q<=5) bases in a sequencing read exceeds 50% of the number of bases in the read, the paired reads are removed.

[0102] The raw data were filtered through the above steps and the data volume before and after filtering was counted to obtain the parameters used by the Clean ReadsFastp software -u50-n 1-q 5-l 30.

[0103] 5.2) Comparison with reference genome

[0104] After data filtering, an index was built according to the provided reference genome, and then the CleanReads were aligned to the reference genome using the BWA0.7.17 software. Samtools 1.7 was used to sort and build the index, and the Bam file was deduplicated using the module provided by the GATK 4.1.8.0 software. Then, the sequencing depth, genome coverage and other information of each sample were statistically analyzed based on the Bam file to prepare for subsequent variant detection.

[0105] 5.3) Target site analysis

[0106] According to the comparison results of Clean Reads in the reference genome, the GenotypeGVCFs module in the software GATK 4.1.8.0 was used to generate the variant site file, and the written script was used to extract the target site typing according to the provided site file. Then, minDP=5X filtering was used, 100% missing, and the sites that did not meet the requirements were marked as . / ., and then the site detection rate and site depth were statistically analyzed.

[0107] 6) Evaluation of the effectiveness of liquid microarray in tracing the germplasm of slow-growing broilers from three different Qingyuan Silkie chicken breeding companies with highly similar population genetic backgrounds

[0108] As one of the representative varieties of yellow-feathered broilers in China, Qingyuan Ma chicken has an annual output of more than 200 million, most of which are slow-breeding Qingyuan Ma chickens. In order to ensure the high quality of broilers, each breeding company usually selects 2 or even 3 specialized strains of Qingyuan Ma chicken original species for matching production of slow-breeding yellow-feathered broilers, resulting in the commercial generation populations of different Qingyuan Ma chicken breeding companies having highly similar genetic backgrounds, making it difficult to distinguish them from the appearance, and it is difficult to effectively distinguish them using the characteristic markers of the Qingyuan Ma chicken breed.

[0109] Using the liquid-phase chip obtained according to the above steps 1) to 5), genotype the genomes of slow-growing broilers (8 individuals / company) from 3 different Qingyuan partridge chicken breeding companies with highly similar population genetic backgrounds. Use VCF2Dis to construct a population genetic distance matrix, and further use ATGC:Fast ME (http: / / www.atgc-montpellier.fr / fastme / ) to construct a phylogenetic tree. The results are shown by Figure 1 It can be seen that the population clustering results of the 9,737 SNPs loci involved in the present invention are highly similar to those of the whole-genome loci and the loci after whole-genome LD-pruning. The correlations between the population genetic distance matrices constructed by them and the whole-genome loci and the loci after whole-genome LD-pruning reach 0.9640 and 0.9843 respectively, and can reliably trace the origin of local chicken breeds, commercial chicken breeds and red jungle fowl populations at home and abroad.

[0110] At the same time, it can effectively distinguish the commercial broilers of 3 different slow-growing Qingyuan partridge chicken breeding companies with highly similar population genetic backgrounds and achieve reliable traceability ( Figure 2 ). Compared with the whole-genome loci (the whole-genome sequencing depth is 10×; 11,458,125 SNPs) ( Figure 2 A), the 9,737 germplasm traceability loci of yellow-feathered broilers in the present invention, in the simulation results (the intersection of 9,737 SNPs and 11,458,125 SNPs loci of the whole genomes of slow-growing broiler populations of 3 different Qingyuan partridge chicken breeding companies, that is, 6,686 SNPs loci are used for population clustering analysis) ( Figure 2 B) and the measured results (the chip probes are used to sequence the slow-growing broiler populations of 3 different Qingyuan partridge chicken breeding companies, and 6,604 high-quality SNPs are obtained after quality control for population clustering analysis) ( Figure 2 C), their population clustering shows high consistency.

[0111] Figure 3 The results show that the chip loci cover all autosomes and the Z chromosome of chickens, and the loci on each autosome are relatively evenly distributed, with an average distance of 95 Kb.

[0112] Figure 4 In, for sample 01, sample 02 and sample 03, genotype their whole genomes respectively using the yellow-feathered broiler whole-genome chip and whole-genome resequencing (10×) of the present invention. The results show that the common genotyped loci of the two genotyping methods in individuals of sample 01, 02 and 03 are 5,608. The genotype consistencies of the two whole-genome genotypings in 3 individuals are 99.96%, 100% and 99.98% respectively. The chip product has good stability, and the genotype consistency rate of repeated samples > 99%.

[0113] In summary, the whole-genome chip for germplasm traceability of yellow-feathered broilers of the present invention can efficiently and reliably identify the population genetic relationships of local chicken breeds and commercial yellow-feathered broilers with a highly similar genetic background in a large number of populations, and realize the germplasm traceability of local chicken breeds and yellow-feathered broilers.

Claims

1. A whole genome chip for tracing the germplasm of yellow-feathered broiler chickens, characterized in that: The chip is a liquid phase chip, which is made of probes combining 9737 yellow-feathered broiler germplasm traceability sites and 261 disease resistance-related sites, with a total of 9998 SNP molecular markers. The physical location information of the SNP molecular markers on the chicken reference genome bGalGal1.mat.broiler.GRCg7b is shown in Table 1.

2. The whole genome chip for tracing the germplasm of yellow-feathered broiler chickens according to claim 1, characterized in that: The yellow-feathered broiler germplasm traceability whole genome chip is a low-density chip of less than 10 Kb.

3. An application of the yellow-feathered broiler germplasm tracing whole genome chip according to claim 1, characterized in that: Include one of the following: (1) Application in the screening and identification of yellow-feathered commercial broiler chickens; (2) Application in the traceability of commercial yellow-feathered broiler chickens; (3) Application in the construction of genetic pedigree of yellow-feathered broiler chicken population; (4) Application in whole-genome association analysis of yellow-feathered broiler chickens; (5) Application in QTL location analysis of target traits in yellow-feathered broiler chickens.

4. A method for evaluating the germplasm traceability of yellow-feathered broiler chickens, characterized in that: The steps include: 1) Extract DNA and construct liquid capture library 2) Library quality inspection and sequencing Quantification and sequencing of hybrid capture libraries; 3) Liquid phase capture analysis Includes data filtering statistics, alignment, and target site analysis; 4) Evaluation of germplasm traceability effectiveness The liquid phase chip as described in claim 1 prepared above was used to perform genome typing on yellow-feathered broiler chickens from populations with highly similar genetic backgrounds, and a population genetic distance matrix was constructed using VCF2Dis. Then, a phylogenetic tree was constructed using ATGC:FastME to observe the results.

5. The method for evaluating the origin of yellow-feathered broiler germplasm according to claim 4, characterized in that: The operations of step 1) include the following: ① Fragment, end-repair and add A to the extracted genomic DNA; ② Use magnetic beads to screen the fragment range and remove fragments that are too large or too small; ③ Connect the sequencing adapters and perform PCR amplification enrichment to construct a whole genome library; ④ Take the whole genome library of each sample and mix it into a pool, concentrate and hybridize the pooled library with probes, capture the fragments of the target region from the whole genome library, and finally perform PCR amplification to obtain the hybrid capture library.

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