A liquid phase chip for the protection of local chicken genetic resources and variety identification and its application

Abstract

The present invention discloses a liquid phase chip for the protection and variety identification of local chicken genetic resources and its application, relating to the field of poultry genetic resource protection, evaluation, and identification. The present invention selects 26 representative local chicken resources and integrates the sequence information of red chicken from a public database. Based on the key application directions of resource protection and variety identification, the present invention prioritizes the evenly distributed site 23K on the chromosome and develops the "Youxin No. 1" liquid phase chip for the protection and variety identification of local chicken genetic resources. This chip has the characteristics of good data coverage and uniformity in the target area, high genotyping detection rate, and flexible chip data upgrade. This greatly reduces the cost of genome typing and is conducive to the large-scale application of the chip. In particular, the standardization of the chip data analysis method significantly improves the accuracy and scientificity of the evaluation and identification of local chicken resources.

Description

Technical Field

[0001] The present invention relates to the field of poultry genetic resource protection, evaluation and identification, and in particular to a liquid phase chip for local chicken genetic resource protection and variety identification and its application. Background Art

[0002] my country is one of the countries with the richest poultry genetic resources in the world. Due to its diverse geographical and ecological environment, after long-term domestication and selection, it has formed a rich and colorful local chicken breed resource. There are currently 115 local chicken breeds (National List of Livestock and Poultry Genetic Resources Varieties (2021 Edition)). These excellent breeds and the huge genetic resources they contain are an important material basis for seed industry innovation and sustainable development of the industry.

[0003] Governments at all levels have always prioritized resource conservation. Due to the large number and widespread distribution of local chicken breeds, my country implements a tiered approach to conservation, primarily employing two in-situ conservation approaches: "place of origin (breeding farm) conservation" and "ex-situ conservation (gene bank) conservation." Currently, 28 chicken breeds are listed as key nationally protected breeds, with 24 national-level chicken breeding farms and three national-level chicken gene banks established. Each province (autonomous region, and municipality) has also established a number of breeding farms. However, for a long time, methods for evaluating the conservation effectiveness of local chicken breeding farms and gene banks at all levels in my country have varied. These methods include comparative analysis of conventional phenotypic data (growth performance, reproductive performance, meat and egg quality, etc.), and calculation of genetic statistics using pedigree records or molecular markers such as microsatellite DNA and mitochondrial mtDNA. However, conventional phenotypic data are susceptible to environmental, batch, and nutritional status variations, and the available indicators are limited. The FAO recommends approximately 30 pairs of microsatellite DNA markers for chickens, and the mtDNA sequence is only approximately 1200 base pairs long, representing extremely limited genomic information. This makes it difficult to scientifically assess population status and changes using a unified standard. Similarly, in terms of breed identification, due to the widespread disorderly hybridization among local chicken breeds and the impact of foreign breeds, some breeds have become seriously hybridized. Therefore, how to accurately identify local chicken breed resources is also a difficult problem that urgently needs to be solved.

[0004] With the rapid development of high-throughput sequencing technology, high-throughput detection of single nucleotide polymorphisms (SNPs) at the whole-genome level has been achieved, and a variety of sequencing strategies have been developed based on different research objectives, including whole-genome resequencing, simplified genome sequencing, and exon sequencing. Using genome sequencing technology to screen out the target SNP site set and design probes to develop chips is an efficient means to achieve high-throughput typing of genomic SNPs. Currently, two chicken breeding-specific chips, "Jingxin No. 1" and "Fengxin No. 1", have been released in China. Their site design mainly considers the functional gene effect sites related to important economic traits in breeding, such as reproduction, meat and egg quality, feed conversion rate, disease resistance and stress resistance; foreign commercial chicken chips are based on foreign breed resources and inbred line genomic information, and are mainly used for breed selection; dedicated chips for the protection of local chicken genetic resources and breed identification have always been blank. Summary of the Invention

[0005] The purpose of the present invention is to provide a liquid phase chip for the protection of local chicken genetic resources and variety identification and its application to solve the problems existing in the above-mentioned prior art. The liquid phase chip can significantly improve the accuracy and scientificity of local chicken resource evaluation and identification, and provide strong core technical support and guarantee for the protection and accurate identification of local chicken genetic resources.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a SNP site combination for local chicken genetic resource protection and variety identification, including the SNP sites shown in Table 2.

[0008] The present invention also provides the use of the above-mentioned SNP site combination in preparing a liquid chip for protecting local chicken genetic resources and identifying varieties.

[0009] The present invention also provides a liquid phase chip for protecting local chicken genetic resources and identifying varieties. The liquid phase chip includes a probe combination, and the probe combination is used to identify the genotype of each SNP site in the above-mentioned SNP site combination.

[0010] The present invention also provides the application of the above-mentioned SNP site combination or liquid phase chip in the protection of local chicken genetic resources or variety identification.

[0011] The present invention also provides a method for evaluating the protection of local chicken genetic resources, comprising the following steps:

[0012] (1) Obtaining genomic DNA from the chicken to be tested;

[0013] (2) constructing a sequencing library based on the genomic DNA;

[0014] (3) performing probe hybridization reaction between the sequencing library and the liquid phase chip;

[0015] (4) extracting genotyping information from the sequence captured by the liquid chip after sequencing to form a genotyping file;

[0016] (5) Calculate heterozygosity, nucleotide polymorphism, molecular inbreeding coefficient, population effective content and differentiation coefficient based on the genotyping file to obtain evaluation results.

[0017] The present invention also provides a method for identifying local chicken breeds, comprising the following steps:

[0018] (1) Obtaining genomic DNA from individual chickens in the flock to be tested;

[0019] (2) constructing a sequencing library based on the genomic DNA;

[0020] (3) performing probe hybridization reaction between the sequencing library and the liquid phase chip;

[0021] (4) extracting genotyping information from the sequence captured by the liquid chip after sequencing to form a genotyping file;

[0022] (5) According to the genotyping file, the genome score of the chicken group to be tested is calculated by comparing with other chicken species in different branches. When the genome score of the chicken group to be tested is greater than 0.90, it indicates that the homozygosity of the chicken group to be tested is high.

[0023] The present invention discloses the following technical effects:

[0024] The present invention selects 26 representative local chicken resources (including new genetic resources) and integrates the original chicken sequence information in the public database; based on the key application directions of resource protection and variety identification, the site 23K evenly distributed on the chromosome is preferred to develop the local chicken genetic resource protection and variety identification liquid phase chip "Youxin No. 1", which has the characteristics of good target area data coverage and uniformity, high genotyping detection rate, and flexible chip data upgrade, which greatly reduces the genome typing cost and is conducive to the large-scale application of the chip; especially the standardization of chip data analysis methods, which significantly improves the accuracy and scientificity of local chicken resource evaluation and identification, and provides strong core technical support and guarantee for the protection and precise identification of local chicken genetic resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 It is a standard framework for the genetic structure of 26 chicken breed resources (with the Red Junglefowl RJF as the root);

[0027] Figure 2 The distribution of the 23K core SNP marker set on different chromosomes;

[0028] Figure 3 is the LD linkage disequilibrium decay diagram of two populations; where A is LS and B is RD;

[0029] Figure 4 is the historical fluctuation of the effective content of the two groups; A is LS and B is RD;

[0030] Figure 5 Signal analysis was selected for both population genomes;

[0031] Figure 6 Cluster diagram constructed for Example 3. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] In the following examples, the molecular statistics calculation method used for population evaluation and identification is as follows:

[0038] Genetic diversity and genetic differentiation indicators: heterozygosity Ho / HE, nucleotide polymorphism π, differentiation coefficient Fst, selection signal detection (Fst and θπ test), etc. were calculated using PopGen and PLINK software with a 100 kb window and 10 kb step.

[0039] Pedigree inbreeding coefficient: Pedigree inbreeding coefficient FPED is calculated according to conventional quantitative genetics path theory.

[0040] Molecular Inbreeding Coefficient: Based on the principles of long-range homozygous ROH and site homozygosity, two inbreeding coefficients, FROH and FHOM, were calculated using PLINK software. FROH calculation parameters were set as follows: length 100–1000 kb, number of SNPs / ROH >30, minimum SNP density >500 kb / SNP, and maximum interval length <1000 kb. FHOM was calculated based on the actual proportion of homozygous sites.

[0041] Analysis of the correlation between the molecular inbreeding coefficient (FROH>100), FHOM, and the pedigree inbreeding coefficient (FPED) showed a highly significant correlation (P<0.01). FROH, FHOM, and FPED were also highly significant (P<0.01). However, FROH>100 and FHOM are calculated based on different principles, making their values ​​incomparable. Therefore, data standardization was performed using a fitting method, allowing for direct comparison and evaluation.

[0042] Molecular kinship coefficient: VCFtool software was used to calculate two molecular kinship coefficients, kin1 and kin2. There was a very significant correlation between kin1 and kin2 (P<0.01). By comparing their detection effects on pedigree inherent kinship combinations, it was found that kin1 had the highest detection rate (>90.0%) for full-sib and half-sib combinations in the Langshan high inbreeding experimental group, indicating that kin1 is more accurate in estimating kinship.

[0043] LD linkage disequilibrium analysis and population effective content Ne: LD analysis was performed using the PopLDdecay software (https: / / github.com / BGI-shenzhen / PopLDdecay). The parameter setting was: -Out Pair LD 5. The Pairwise Sequential Markov Chain Model (PSMC) method was used to infer the effective population size and historical fluctuations based on genomic fragments with different heterozygous site densities. The generation interval and nucleic acid mutation rate parameters were set as: g = 1, u = 0.2 × 10 -8 .

[0044] Genetic cluster analysis: The maximum likelihood method ML of FASTtree software or the Bayesian inference algorithm of the unsupervised mixed model of STRUCTURE software were used to construct a genetic evolutionary tree that was consistent with the geographical distribution and formation history of the variety.

[0045] Genome fraction (GF): The unsupervised mixed model mode of Admixture or STRUCTURE software was used, and the Bayesian inference algorithm was adopted. The parameters were set as follows: After the K value was set, the run was repeated 500 times to obtain the individual genome fraction matrix. The genome fraction (eigenvalue) of all individuals in their respective varieties was greater than 0.90, with a high individual variety identification rate.

[0046] Example 1

[0047] 1. Screening of SNP sites on liquid microarray

[0048] (1) Collection of blood samples from representative chicken breed resources. Taking into account the ecological and geographical distribution of the breeds, economic types and special traits, 26 representative local chicken breed resources, including Wenchang chicken, Tibetan chicken, Camellia chicken, Bian chicken, Jingyuan chicken, Beijing oil chicken, Xianju chicken, Langshan chicken, Jinhu Wufeng chicken, Wenshang Luhua chicken, Dongxiang green-shelled laying hen, and Henan fighting chicken, were selected as the basic materials for chip development, which greatly represent the genetic characteristics of my country's local chicken breed resources. Among the 26 chicken breeds, 30 standard individuals were selected for each breed of 24 chicken breeds, 8 individuals for Yuanbao chicken, and 10 individuals for Dulong chicken. The Dulong chicken sequence was derived from the NCBI database (see Table 1). Except for Dulong chicken, the remaining local chicken breed resources were all derived from the purebred conservation population (experimental determination population) of the National Local Chicken Breed Gene Bank (Jiangsu) to ensure that there would be no interference from hybrid populations.

[0049] 1 mL of blood was collected from the wing vein of the above experimental individuals using sterile methods, and sodium citrate anticoagulant was added and mixed, and stored at -80°C for later use.

[0050] Table 1 Chicken breed resource information included in liquid chip design

[0051]

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[0053] (2) DNA sample extraction. The genomic DNA of all species was extracted using the conventional phenol-chloroform method. The obtained DNA was subjected to quality control, including detection of DNA sample concentration by fluorescence spectrometer and detection of DNA integrity by electrophoresis, including whether the DNA was degraded and whether there was contamination by other impurities such as protein and RNA. The DNA samples were accurately quantified using Qubit 2.0, and samples with a mass of ≥1 μg were selected for the next step of library construction and sequencing. Qualified samples were stored at -80°C for use in library construction and sequencing.

[0054] (3) Genomic RAD-seq library construction and sequencing. DNA samples that passed the quality inspection were constructed using the ddRAD library construction method to construct a paired-end library with a length range of 300 to 500 bp. The simplified genomic RAD-seq sequencing was performed using EcoRI (G^AATTC) and NlaIII (Hin1IICATG^) double enzyme digestion. The protocol is as follows: (1) Take 500 ng of genomic DNA, add 0.6U EcoRI (NEB), T4 DNA ligase (NEB), ATP (NEB) and EcoRI adapter (containing the index sequence to distinguish samples) and react at 37°C for 3 hours, and anneal at 65°C for 1 hour. Then add the restriction endonuclease NlaIII (NEB) and NlaIII adapter and react at 37°C for 3 hours. After the reaction is completed, place it in a 65°C PCR instrument for 30 minutes to inactivate the endonuclease. (2) Use agarose gel electrophoresis to select the fragments of the ligation products, and select 400 to 600 bp to recover the enzyme digestion products. (3) DNA quantification of the recovered products was performed using Qubit 3.0 (Life Technology), and equal amounts of each sample were mixed. (4) DNA library construction was performed on the mixed products using the Illumina TruSeq kit.

[0055] (4) Sequencing data quality control. The raw sequences (double-end sequences) obtained by sequencing were evaluated to obtain the raw reads of each sample. The reads were aligned to the chicken reference genome (GRCg6a, Gallus_gallus.GRCg6a.dna.toplevel.fa) using BWA MEM0.7.15 software. The raw read sequencing data were quality controlled using the GATK Samtools program, and the base number ratio Q20 ≥ 95% with an accuracy rate of 99% was filtered; SNP detection was performed using GATK software, and the coverage depth of double enzyme digestion genome sequencing was ≥ 60%, the SNP single nucleotide polymorphism call rate (Call rate) in all samples was ≥ 70%, and the minimum allele frequency MAF was ≥ 0.05; on this basis, the SNP detection rate in a single chicken breed was further ≥ 90%.

[0056] Based on the above quality control, about 4.7M SNP markers of 26 chicken breed resource genomes were screened.

[0057] 2. Screening of core SNP loci for variety protection and identification

[0058] (1) Fine genetic structure analysis of 26 breeds. Based on all 4.7M genomic SNP marker sites of the 26 selected chicken breed resources, PCA principal component analysis was performed on all individuals using GCTA software. At the same time, MEGA, STRUCTURE, PHYLIP and other software were used to construct genetic evolutionary trees based on genetic distance (DA, Dr) method, maximum likelihood method ML, model clustering method Model-based, Bayesian inference and neighbor-joining method NJ. Through comprehensive analysis, the genetic structure map constructed by maximum likelihood method ML and Bayesian inference is consistent with the geographical distribution, gene flow and formation history of the breed. Based on this, a "standard framework for breed genetic structure" was constructed, which finely positioned the genetic structure and branch composition among the breeds ( Figure 1 ). 7 major branches, including Branch 1: Dulong Chicken, Daweishan Miniature Chicken, Piao Chicken, Tibetan Chicken, Camellia Chicken, Branch 2: Wenchang Chicken, Huiyang Bearded Chicken, Branch 3: Jinhu Black Phoenix Chicken, Branch 4: Xiaoshan Chicken, Luyuan Chicken, Branch 5: Tianjin Monkey Chicken, Wenshang Reed Chicken, Yuanbao Chicken, Langya Chicken, Langshan Chicken, Shouguang Chicken, Branch 6: Jingyuan Chicken, Beijing Oily Chicken, Bian Chicken, Big Bone Chicken, Branch 7: Dongxiang Green-shelled Layer Chicken, Anyi Gray Chicken, White-eared Yellow Chicken, Xianju Chicken, Gushi Chicken, Henan Fighting Chicken.

[0059] (2) Screening of a “core SNP marker set” for 23K varieties. In chip design, if all 4.7M loci are used for resource conservation and identification, the detection cost will be too high. Therefore, it is necessary to screen out a core SNP marker set to facilitate the large-scale development and efficient use of chip products.

[0060] 10.1K shared loci were selected based on an even distribution across chromosomes: Miss rate <0.1, Het <0.3, Minimum allele frequency (Maf) >0.1, and Genome fraction >0.8 for each individual in their respective clade. Genetic structure analysis based on these 10.1K loci revealed no changes in the clade's genetic structure or clades.

[0061] We then calculated the top 100 (900) ΔMAF loci for each of the seven genetically close varieties and verified them using sequencing data. Using this 0.9K locus for genetic structure analysis allowed us to finely differentiate the seven genetically close varieties. The 0.9K locus was selected as a supplement for variety identification purposes.

[0062] Specific sites were further screened for the seven branches, and 12K homozygous inconsistent sites were found in pairwise comparisons between the branches, which enabled fine differentiation of the branches.

[0063] Further targeting the screened 23K core SNP marker set, some individuals (30% to 60%) from 26 varieties were randomly selected for genetic structure and cluster analysis. There was still no change in the genetic structure and branch composition among varieties, and no individuals were separated from the variety branch to which they belonged.

[0064] Therefore, the 23K core SNP marker set screened was confirmed to be the characteristic alleles of 26 varieties (including branch composition). The distribution of the 23K core SNP marker set on different chromosomes is shown in Figure 2 , the specific location information is shown in Table 2.

[0065] Table 2 Position information of the 23K core SNP marker set

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[0153] 3. Preparation and use of liquid phase chips

[0154] (1) Liquid phase probe design and synthesis: For the selected 23K core SNP marker set, two probe sequences were designed based on the upstream and downstream sequences of each SNP site. Starting from 100bp upstream of the core site, every 10bp was used as a step, and 110-120bp was used as a probe to construct a probe pool for this site. The probe sequences in the probe pool were homologously aligned on the chicken reference genome (GRCg6a) using the hidden Markov rule, and the GC content of each probe was calculated. The two probes with a GC content between 30% and 70% and the lowest homology on the chicken reference genome were selected as candidate probes for this site to capture the core SNP with 2× coverage. All probes for the 23K SNPs were synthesized using a nucleic acid synthesizer to form the "Unitary Core No. 1" liquid phase chip.

[0155] (2) Extraction of sample genomic DNA: Use a conventional phenol-chloroform (or high-throughput) DNA extraction kit to extract sample DNA.

[0156] (3) Genomic DNA fragmentation and end repair: Add A: 20 ng DNA, 4 μL GenoBaits End Repair Buffer, 2.6 μL GenoBaits End Repair Enzyme, and add Nuclease-free water to a total of 20 μL. Vortex to mix and briefly centrifuge. Incubate at 37°C for 20 min, then at 72°C for 20 min, and store at 4°C.

[0157] (4) Construction of sequencing library: Remove the tube from the PCR instrument and add 2μL of GenoBaitsUltraDNAligase, 8μL of GenoBaitsUltraDNALigaseBuffer and 4μL GenoBaitsAdapter. Add water to 40μL and place it on the PCR instrument for reaction at 22℃ for 60min to complete the connection of sequencing adapters. Add 48μL of GenoPrepDNACleanBeads to the connection product to purify the connection product. After purification, use 0.65+0.2 times magnetic beads for fragment screening, and retain the connection product with insert fragments between 300 and 350bp. Add 10μL of GenoBaits PCR MasterMix and 10μL of Barcode to the reaction system, and then amplify the library. Place the amplified product on the magnetic stand and let it stand for 3min. Add 100μL of 80% ethanol by volume to the reaction system, let it stand for 30 seconds, then remove the supernatant and complete the library purification after the ethanol is completely evaporated.

[0158] (5) Liquid chip hybridization capture: Take 500 ng of the constructed sequencing library, add 5 μL GenoBaits Block I and 2 μL GenoBaits Block II, and place it on an Eppendorf Concentrator plus vacuum concentrator at ≤70°C to dry powder. Add 8.5 μL of GenoBaits 2×HybBuffer, 2.7 μL GenoBaits HybBuffer Enhancer, and 2.8 μL Nuclease-Free Water to the dry powder tube, use a pipette to mix, and place it on a PCR instrument and incubate at 95°C for 10 minutes. Take out the PCR tube and add 3 μL of the synthesized local chicken genetic resource protection and identification chip "Youxin No. 1" liquid phase probe, vortex to mix, and place it on an ABI 9700 PCR instrument and incubate at 65°C for 2 hours to complete the probe hybridization reaction.

[0159] (6) Library quality control and sequencing: DNA concentration was measured using the Hanchen Guangyi Arrayer500, and agarose gel electrophoresis was used to check whether the fragment size of the library DNA was between 300 and 400 bp. The constructed library was sequenced using the BGI-T7 sequencer.

[0160] (7) Genotyping data analysis: After the sequencing data were quality controlled by FastQC (www.bioinformatics.babraham.ac.uk / project), they were mapped to the chicken reference genome (GRCg6a) using the inherent parameters of BWA (bio-bwa.sourceforge.net). SNPs were identified using GATK software (software.broadinstitute.org / gatk), and the genotyping information of the 23K core SNP set was extracted using a Perl script to generate the final genotyping file results.

[0161] (8) Chip detection rate test: 19 breeds were randomly selected (of which 14 were breeds included in the chip design and 5 were breeds not used in the chip design (experimental group)), with 10 to 30 individuals of each breed, totaling 378 samples (see Table 3), all from the National Local Chicken Gene Bank (Jiangsu). After blood was collected from the wing vein, genomic DNA was extracted, and liquid probe hybridization and library sequencing were performed according to the above method. The results are shown in Table 3. The results showed that the average detection rate of SNP sites was 99.95%. This fully confirmed the high detection rate and high reliability of the "Youxin No. 1" liquid chip sites.

[0162] Table 3 “Youxin No. 1” liquid phase chip site detection rate test

[0163] Variety name Liquid phase chip covers varieties Number of samples (pieces) Detection rate (%) BE White-eared Yellow Chicken yes 20 99.89 BJ Chicken yes 10 99.97 CH Camellia Chicken yes 20 99.96 DG Big Bone Chicken yes 20 99.97 GS Gushi Chicken yes 10 99.98 HX Huiyang Bearded Chicken yes 10 99.97 WC Wenchang Chicken yes 20 99.96 WH Anyi Gray Chicken yes 20 99.97 WS Wenshang Reed Chicken yes 20 99.97 WX Daweishan Miniature Chicken yes 20 99.91 XS Xiaoshan Chicken yes 10 99.90 JH Jinhu Black Phoenix Chicken yes 10 99.95 PJ Scoop Chicken yes 10 99.97 SG Shouguang Chicken yes 30 99.96 LSI Langshan Inbreeding Experiment Group no 30 99.96 QY Qingyuan Ma Chicken no 29 99.96 MC Macheng green shell laying hens no 30 99.92 CR Chongren Ma Chicken no 29 99.96 SY Silky Feather Chicken no 30 99.94 / / Total 378 Average 99.95%

[0164] (9) Standardization of molecular statistics calculation methods: The LSI of the Langshan chicken highly inbred experimental population established by the National Local Chicken Gene Bank (Jiangsu) was selected for standardization of molecular statistics calculation. The Langshan chicken highly inbred experimental population was propagated by full-sib or half-sib mating in each generation, and each individual had a complete pedigree record for nearly 10 consecutive generations. 30 individuals were selected, wing vein blood was collected, and genomic DNA was extracted. Liquid probe hybridization and library sequencing were performed according to the above method. The detection rate of the locus was 99.96% (see Table 3: LSI).

[0165] Example 2 Evaluation of the protective effect of the Langshan chicken conservation population in the origin conservation farm and gene bank

[0166] (1) Sample collection: Ten roosters and 10–20 females were selected from two Langshan chicken conservation populations, one at the origin and the other at the gene bank, according to their family lineage. 1.0 mL of blood was collected from the wing vein, anticoagulated with sodium citrate (ACD), and stored at -20°C until use.

[0167] (2) DNA extraction: A high-throughput DNA extraction kit was used to extract sample DNA.

[0168] (3) Genomic DNA fragmentation and end repair: Add A: 20 ng DNA, 4 μL GenoBaits End Repair Buffer, 2.6 μL GenoBaits End Repair Enzyme, and add Nuclease-free water to a total of 20 μL. Vortex to mix and briefly centrifuge. Incubate at 37°C for 20 min, then at 72°C for 20 min, and store at 4°C.

[0169] (4) Construction of sequencing library: Remove the tube from the PCR instrument and add 2μL of GenoBaitsUltraDNAligase, 8μL of GenoBaitsUltraDNALigaseBuffer and 4μL GenoBaitsAdapter. Add water to 40μL and place it on the PCR instrument for reaction at 22℃ for 60min to complete the connection of sequencing adapters. Add 48μL of GenoPrepDNACleanBeads to the connection product to purify the connection product. After purification, use 0.65+0.2 times magnetic beads for fragment screening, and retain the connection product with insert fragments between 300 and 350bp. Add 10μL of GenoBaits PCR MasterMix and 10μL of Barcode to the reaction system, and then amplify the library. Place the amplified product on the magnetic stand and let it stand for 3min. Add 100μL of 80% ethanol by volume to the reaction system, let it stand for 30 seconds, then remove the supernatant and complete the library purification after the ethanol is completely evaporated.

[0170] (5) Liquid phase chip hybridization capture: Take 500 ng of the constructed sequencing library, add 5 μL GenoBaits Block I and 2 μL GenoBaits Block II, and place it on an Eppendorf Concentrator plus vacuum concentrator at ≤70°C to dry powder. Add 8.5 μL GenoBaits 2×HybBuffer, 2.7 μL GenoBaits HybBuffer Enhancer, and 2.8 μL Nuclease-Free Water to the dry powder tube, use a pipette to mix, and place it on a PCR instrument and incubate at 95°C for 10 minutes. Take out the PCR tube and add 3 μL of the synthesized local chicken genetic resource protection and identification chip "Youxin No. 1" liquid phase probe, vortex to mix, and place it on an ABI9700 PCR instrument and incubate at 65°C for 2 hours to complete the probe hybridization reaction.

[0171] (6) Library quality control and sequencing: DNA concentration was measured using the Hanchen Guangyi Arrayer500, and agarose gel electrophoresis was used to check whether the fragment size of the library DNA was between 300 and 400 bp. The constructed library was sequenced using the BGI-T7 sequencer.

[0172] (7) Genotyping data analysis: After the sequencing data were quality controlled by FastQC (www.bioinformatics.babraham.ac.uk / project), they were mapped to the chicken reference genome (GRCg6a) using the inherent parameters of BWA (bio-bwa.sourceforge.net). SNPs were identified using GATK software (software.broadinstitute.org / gatk), and the genotyping information of the 23K core SNP set was extracted using a Perl script to generate the final genotyping file results.

[0173] Statistical analysis and conservation population evaluation: According to the established “molecular statistical standardization method”, the heterozygosity Ho, nucleotide polymorphism π, molecular inbreeding coefficient FROH and differentiation coefficient Fst were calculated. The results are shown in Table 4.

[0174] The results showed that the detection rate of SNP sites of "Youxin No. 1" liquid phase chip was high (>99.90%) in both Langshan chicken populations of the gene bank and the breeding farm. The observed heterozygosity Ho was 0.2310 and 0.2209, respectively. The nucleotide polymorphism π was 0.2479 and 0.2473, respectively. The genetic diversity was moderately polymorphic, and the difference was not significant (P>0.05). The inbreeding coefficient FROH was 0.0769 and 0.1087, respectively, which was at a low level, which was consistent with the results of LD linkage disequilibrium analysis ( Figure 3 ) are consistent, indicating that the seed conservation effect of the two populations is good and the seed conservation technology adopted effectively controls the inbreeding level. The effective content Ne of the two populations fluctuated significantly during the continuation process ( Figure 4 ), in the near future, the effective content of the group remained at a relatively stable state. The average differentiation coefficient Fst of the two Langshan chicken groups was 0.0040, which was at a low level, and no significant differentiation occurred overall; further selection signal analysis was performed using Fst and θπ tests, and the results were shown in Figure 5 The threshold of the selected genomic region was Fst>0.048, log2θπratio<-1.37, and a significantly selected region was detected on chromosome 18, involving three selected candidate functional genes, namely MCHR1, CASKIN2 and TMEM94, which provided a scientific basis for carrying out blood source supplementation between the two conservation populations.

[0175] Table 4 Calculation of genetic statistics of two conservation populations

[0176]

[0177] Example 3 Identification of local chicken resources

[0178] To identify the purity of a free-range black-feather chicken flock in the main Langshan chicken producing area.

[0179] (1) Sample collection: Ten males and ten females were randomly selected from a free-range black-feathered chicken population. 1.0 mL of blood was collected from the wing vein, anticoagulated with sodium citrate (ACD), and stored at -20°C until use.

[0180] (2) DNA extraction: A high-throughput DNA extraction kit was used to extract sample DNA.

[0181] (3) Genomic DNA fragmentation and end repair: Add A: 20 ng DNA, 4 μL GenoBaits End Repair Buffer, 2.6 μL GenoBaits End Repair Enzyme, and add Nuclease-free water to a total of 20 μL. Vortex to mix and briefly centrifuge. Incubate at 37°C for 20 min, then at 72°C for 20 min, and store at 4°C.

[0182] (4) Construction of sequencing library: Remove the tube from the PCR instrument and add 2μL of GenoBaitsUltraDNAligase, 8μL of GenoBaitsUltraDNALigaseBuffer and 4μL GenoBaitsAdapter. Add water to 40μL and place it on the PCR instrument for reaction at 22℃ for 60min to complete the connection of sequencing adapters. Add 48μL of GenoPrepDNACleanBeads to the connection product to purify the connection product. After purification, use 0.65+0.2 times magnetic beads for fragment screening, and retain the connection product with insert fragments between 300 and 350bp. Add 10μL of GenoBaits PCR MasterMix and 10μL of Barcode to the reaction system, and then amplify the library. Place the amplified product on the magnetic stand and let it stand for 3min. Add 100μL of 80% ethanol by volume to the reaction system, let it stand for 30 seconds, then remove the supernatant and complete the library purification after the ethanol is completely evaporated.

[0183] (5) Liquid phase chip hybridization capture: Take 500 ng of the constructed sequencing library, add 5 μL GenoBaits Block I and 2 μL GenoBaits Block II, and place it on an Eppendorf Concentrator plus vacuum concentrator at ≤70°C to dry powder. Add 8.5 μL GenoBaits 2×HybBuffer, 2.7 μL GenoBaits HybBuffer Enhancer, and 2.8 μL Nuclease-Free Water to the dry powder tube, use a pipette to mix, and place it on a PCR instrument and incubate at 95°C for 10 minutes. Take out the PCR tube and add 3 μL of the synthesized local chicken genetic resource protection and identification chip "Youxin No. 1" liquid phase probe, vortex to mix, and place it on an ABI9700 PCR instrument and incubate at 65°C for 2 hours to complete the probe hybridization reaction.

[0184] (6) Library quality control and sequencing: DNA concentration was measured using the Hanchen Guangyi Arrayer500, and agarose gel electrophoresis was used to check whether the fragment size of the library DNA was between 300 and 400 bp. The constructed library was sequenced using the BGI-T7 sequencer.

[0185] (7) Genotyping data analysis: After the sequencing data were quality controlled by FastQC (www.bioinformatics.babraham.ac.uk / project), they were mapped to the chicken reference genome (GRCg6a) using the inherent parameters of BWA (bio-bwa.sourceforge.net). SNPs were identified using GATK software (software.broadinstitute.org / gatk), and the genotyping information of the 23K core SNP set was extracted using a Perl script to generate the final genotyping file results.

[0186] Variety identification analysis: The Langshan chicken included in the "Youxin No. 1" liquid phase chip was compared with other chicken breeds from different branches (20 individuals were randomly selected) to calculate the individual genome scores (see Table 5). The average genome score of all 20 individuals in the free-range black-feathered chicken group in the "Youxin No. 1" Langshan chicken branch was 0.908. The cluster diagram constructed ( Figure 6 ) is also the case. All individuals in the black-feathered chicken group are clustered into one category in the "Youxin No. 1" Langshan chicken branch, and no individual is separated, indicating that it is a purebred Langshan chicken and no hybridization has occurred.

[0187] Table 5 Distribution of genome scores of black-feathered chicken populations in standard breeds (branches)

[0188]

[0189] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A liquid phase chip for the protection of local chicken genetic resources and variety identification, characterized in that: The liquid phase chip includes a probe combination, and the probe combination is used to identify the genotype of each SNP site in the SNP site combination; The SNP site combination includes the SNP sites located in the chicken reference genome GRCg6a as shown in the following table:

2. An application of the liquid phase chip as claimed in claim 1 in the protection of local chicken genetic resources or variety identification.

3. A method for evaluating the protection of local chicken genetic resources, characterized in that: The following steps are involved: (1) Obtaining genomic DNA from the chicken to be tested; (2) constructing a sequencing library based on the genomic DNA; (3) performing a probe hybridization reaction between the sequencing library and the liquid phase chip according to claim 1; (4) extracting genotyping information from the sequence captured by the liquid chip after sequencing to form a genotyping file; (5) Calculate heterozygosity, nucleotide polymorphism, molecular inbreeding coefficient, population effective content and differentiation coefficient based on the genotyping file to obtain evaluation results.

4. A method for identifying local chicken breeds, characterized in that: The following steps are involved: (1) Obtaining genomic DNA from individual chickens in the flock to be tested; (2) constructing a sequencing library based on the genomic DNA; (3) performing a probe hybridization reaction between the sequencing library and the liquid phase chip according to claim 1; (4) extracting genotyping information from the sequence captured by the liquid chip after sequencing to form a genotyping file; (5) According to the genotyping file, the genome score of the chicken group to be tested is calculated by comparing with other chicken species in different branches. When the genome score of the chicken group to be tested is greater than 0.90, it indicates that the homozygosity of the chicken group to be tested is high.

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