Gene chip for breed selection and breeding of local chickens in Guangxi Zhuang autonomous region and application of gene chip

Abstract

The invention discloses a gene breeding chip (Guangxin No.1) for selecting and breeding local chickens in Guangxi Zhuang autonomous regions and application of the gene breeding chip. The gene chip is designed aiming at the breeding condition of high-quality broiler chickens in Guangxi Zhuang Autonomous District, has lower typing cost, is beneficial to popularization and application of enterprises, has higher flexibility, can be used for carrying out iterative updating on chip sites according to application purposes and practical effects, and has a wide application prospect. The method can be effectively utilized in genetic diversity evaluation of local chickens in the Guangxi Zhuang Autonomous Region, group breed conservation effect identification, pedigree correction, whole genome association analysis and genome selection, the application of molecular biological breeding of high-quality broiler chickens in the Guangxi Zhuang Autonomous Region is further enhanced, and the breeding level of the high-quality broiler chickens in the Guangxi Zhuang Autonomous Region is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a gene chip used for selecting and breeding local chickens in Guangxi Zhuang Autonomous Region and an application thereof. Background Art

[0002] Guangxi Zhuang Autonomous Region is the main production area of ​​high-quality broiler chickens. The scale of the high-quality broiler industry ranks among the top in the country, and the development and utilization of local chickens is high. However, the current breeding technology of high-quality broiler chickens in Guangxi Zhuang Autonomous Region is mainly based on individual selection combined with family selection. The relevant molecular technology has not been effectively applied due to cost, technical level and other reasons, which seriously restricts the high-quality development of high-quality broiler breeding in Guangxi Zhuang Autonomous Region. Breeding efficiency has an important impact on seed selection and matching work and economic production benefits. A key link in the development of the high-quality broiler industry in Guangxi Zhuang Autonomous Region is to improve breeding efficiency. In order to promote the innovative application of high-quality broiler breeding technology in Guangxi Zhuang Autonomous Region, it is imperative to further improve the molecular breeding level of high-quality broilers in Guangxi Zhuang Autonomous Region.

[0003] Single nucleotide polymorphism (SNP) is widely present in the genome. Rational use of SNP information in the whole genome can effectively carry out the application and research of modern molecular biotechnology such as genome-wide association study (GWAS) of important traits, population diversity detection, population protection, molecular marker-assisted selection, and genomic selection (GS). Common methods of genotyping include whole genome resequencing, simplified genome sequencing, and gene chips. Currently, there are 4 mainstream chips used in chicken research, among which the 60K SNP chip has been discontinued, the 600K SNP chip is too expensive and has certain limitations in the research of local chickens; the "Phoenix Core No. 1" chip is mainly designed for laying hen breeding, and the SNP site information of local chickens in Guangxi Zhuang Autonomous Region is not included in the design; the "Beijing Core No. 1" chip comprehensively considers the genome information of Chinese and foreign broiler breeds, but its price is still relatively high in production applications. In addition, relevant domestic units have also developed related medium and low-density chips for local chicken breeds, which are used for diversity analysis of local chicken genetic resources, protection of genetic resources and genome selection, such as the "Youxin No. 1" chip developed by Jiangsu Poultry Research Institute and the "Luxin No. 1" series chip developed by Shandong Poultry Research Institute. Therefore, it is urgent to develop a low-density, low-priced gene breeding chip that is suitable for the conservation and selection of local chickens in Guangxi Zhuang Autonomous Region, and promote the application of molecular biotechnology in the selection and breeding of local chickens in Guangxi Zhuang Autonomous Region.

[0004] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0005] In order to achieve the technical purpose of the present invention, the present invention provides the following technical solutions:

[0006] A SNP site combination for selection and breeding of local chickens in Guangxi Zhuang Autonomous Region, characterized in that: the SNP site combination consists of 20,000 SNP sites, the physical positions of the 20,000 SNP sites are determined based on the chicken GRCg6a genome sequence alignment, and the physical positions of the 20,000 SNP sites are shown in Table 1.

[0007] Table 1 Physical location information of SNP sites

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[0070] The above SNP site combination is used in the preparation of gene chips for the selection and breeding of local chickens in Guangxi Zhuang Autonomous Region.

[0071] A gene chip for selecting and breeding local chickens in Guangxi Zhuang Autonomous Region, the gene chip is a liquid phase chip, comprising a probe combination, the probe combination is used to identify the genotype of each SNP site in the SNP site combination described in claim 1.

[0072] Application of the above SNP locus combination and gene chip in the selection and breeding of local chickens in Guangxi Zhuang Autonomous Region.

[0073] Preferably, the application is any of the following uses:

[0074] (1) Application in the breeding of local chickens in Guangxi Zhuang Autonomous Region;

[0075] (2) Application in identification of local chicken breeds in Guangxi Zhuang Autonomous Region;

[0076] (3) Application in genome-wide association analysis of native chickens in Guangxi Zhuang Autonomous Region;

[0077] (4) Application in genomic selection of local chickens in Guangxi Zhuang Autonomous Region;

[0078] (5) Application in the assessment of genetic diversity of local chickens in Guangxi Zhuang Autonomous Region;

[0079] (6) Its application in correcting the pedigree of local chickens in Guangxi Zhuang Autonomous Region.

[0080] A method for preparing the above gene chip comprises the following steps:

[0081] (1) Six local chicken breeds in Guangxi Zhuang Autonomous Region were selected for whole genome resequencing. After quality control, the original data were compared with the chicken reference genome GRCg6a (GCA_000002315.5). After removing low-quality SNPs, the SNP dataset of local chicken in Guangxi Zhuang Autonomous Region was obtained, namely the Guangxi Zhuang Autonomous Region local chicken genome database.

[0082] (2) Obtain phenotypic information of sequenced individuals and screen SNPs related to economic traits such as body size, slaughter, meat quality, feed efficiency, egg production, and egg quality of local chickens in Guangxi Zhuang Autonomous Region through genome-wide association analysis as the first type of targets for the above-mentioned chip.

[0083] (3) Based on the transcriptome, candidate genes related to the egg production, fat deposition, feed efficiency, and chicken spur of the Guangxi Zhuang Autonomous Region local chicken were screened. By locating the position of the candidate genes on the genome, the Guangxi Zhuang Autonomous Region local chicken genome database was used to select Tag SNPs within the candidate interval as the second type of targets of the above chip.

[0084] (4) Referring to the QTL information of chicken economic traits provided by the QTL database, relevant functional loci were screened as the third type of targets for the above chip.

[0085] (5) Using the Guangxi Zhuang Autonomous Region local chicken genome database, the genomic regions that cannot be covered by the above three types of targets are filled in as background SNPs, i.e., the fourth type of target of the above chip.

[0086] (6) Through the probe design system, qualified probes that meet the above four types of targets were screened, and finally a gene chip "Guixin No. 1" was created as described above for the selection and breeding of local chickens in Guangxi Zhuang Autonomous Region.

[0087] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a gene breeding chip (Guixin No. 1) for the selection and breeding of local chickens in Guangxi Zhuang Autonomous Region and its application. The chip is designed for the breeding of high-quality broilers in Guangxi Zhuang Autonomous Region, has a low typing cost, is conducive to the promotion and application of enterprises, and has high flexibility. The chip sites can be iteratively updated according to the application purpose and practical effect. It can be effectively used in the evaluation of genetic diversity of local chickens in Guangxi Zhuang Autonomous Region, identification of population conservation effects, pedigree correction, whole genome association analysis and genome selection, further strengthening the application of molecular biological breeding of high-quality broilers in Guangxi Zhuang Autonomous Region and improving the breeding level of high-quality broilers in Guangxi Zhuang Autonomous Region. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 This is a diagram showing the proportion of functional sites in the gene chip molecular marker combination of the present invention;

[0089] Figure 2 A distribution statistical diagram of the gene chip sites of the present invention on each chromosome;

[0090] Figure 3 This is a diagram showing the application of the gene chip described in the present invention in variety identification. DETAILED DESCRIPTION

[0091] The specific implementation modes of the present invention are described in detail below in conjunction with the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific implementation modes.

[0092] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0093] Example 1 Preparation method of low-density 20K gene breeding chip for local chickens in Guangxi Zhuang Autonomous Region

[0094] The present invention obtains the whole genome SNP information of 3469 individuals of six Guangxi Zhuang Autonomous Region chicken breeds through whole genome resequencing, and uses whole genome association analysis and transcriptome difference analysis to dig out functional sites related to important economic traits of Guangxi Zhuang Autonomous Region local chickens, combined with relevant functional sites in the chicken QTL database, and further fills the background sites through the resequencing data of Guangxi Zhuang Autonomous Region local chickens, and finally designs a 20,000 SNP molecular marker combination specific for Guangxi Zhuang Autonomous Region local chicken breeding, namely the "Guixin No. 1" gene breeding chip. The process of obtaining the above molecular markers is as follows:

[0095] 1. Construction of the Guangxi Zhuang Autonomous Region local chicken genome database

[0096] Blood was collected from 3469 individuals of six local chicken breeds in Guangxi Zhuang Autonomous Region (see Table 2 for breed number and information). Genomic DNA was extracted using the phenol / mimetic method, and the quality of genomic DNA was tested by electrophoresis. After the samples met the library construction requirements, they were handed over to a biological company for sequencing. The whole genome resequencing was completed using the Illumina PE150 double-end sequencing strategy, and the average sequencing data volume for each individual was 7.55Gb. The offline data needed to remove reads containing adapters and low quality reads. The genome information was compared using BWA and other software, and the reference genome version was the sixth version of the chicken reference genome (GRCg6a, https: / / ftp.ensembl.org / pub / release-104 / fasta / gallus_gallus / dna / ); the sam files were converted to bam files using SAMtools software and sorted using the sort function, and then duplicates were marked; the best variant detection process of GATK4 software was used to detect SNPs, and low-quality SNPs were eliminated. The filtering criteria included QualByDepth (QD) <2.0, Quality>30, MappingQuality (MQ) <40.0, MQRankSum <-12.5, ReadPosRankSum <-8.0, SOR>3.0 and FisherStrand (FS) >60.0. This SNP dataset provides sufficient information for locating important molecular markers for economic traits of local chickens in Guangxi Zhuang Autonomous Region, screening high-frequency SNP sites, and calculating Tag SNPs.

[0097] Table 2 Population information of Guangxi Zhuang Autonomous Region local chicken genome database

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[0099] 2. Screening of relevant functional sites through genome-wide association analysis. Based on the genotype and phenotypic data of 12 groups of six local chicken breeds in Guangxi Zhuang Autonomous Region collected by the inventors in the early stage, a genome-wide association study (GWAS) was performed using GCTA software. The phenotypic data included economic traits such as body size, slaughter, meat quality, feed efficiency, egg production and egg quality.

[0100] There are three strategies for screening functional SNP sites: ① Conduct GWAS on a single population and retain SNP sites that reach the potential association threshold for each phenotype in each variety; ② Conduct GWAS based on all varieties, that is, conduct association analysis on all varieties as a whole, add population structure information (the first five principal components) as covariates for population stratification correction during analysis, and retain SNP sites that reach the potential association threshold for each phenotype; ③ Meta-analysis based on the GWAS results of each population, using individual GWAS data and the fixed effect model provided by METAL software. This model is mainly based on the inverse variance weighting method for calculation to obtain candidate sites. The SNPs screened by the above three strategies are integrated, and the candidate SNPs obtained are used as the main components of the "Guixin No. 1" gene breeding chip, which is the first type of target of the chip.

[0101] 3. Screening related sites based on differentially expressed genes obtained from transcriptome analysis. Based on the inventors' previous research, candidate genes related to the phenotypes of egg production, fat deposition, feed efficiency, and chicken spurs in Guangxi Zhuang Autonomous Region were obtained through transcriptome differential analysis. The position of the candidate gene on the genome was anchored using the annotation file, and then the Tag SNPs in the target interval were selected using the PLINK software. If there is no Tag SNP in the candidate gene interval, a SNP site will be randomly selected. This part of the SNP constitutes the second type of target of the chip.

[0102] 4. Screen relevant loci based on the chicken QTL database (https: / / www.animalgenome.org / cgi-bin / QTLdb / GG / index). In order to further enhance the proportion of functional loci of the "Guixin No. 1" gene breeding chip, refer to some functional loci information provided by the chicken QTL database and integrate them into the combination of chip loci. The process includes: ① Download the statistical file in gff format from the chicken QTL database, extract the rs number (SNP ID) corresponding to the QTL interval with stress resistance, reproduction, feed utilization efficiency, egg production, growth rate and other related traits as the target; ② Based on the variation information file provided by ENSEMBL (http: / / ftp.ensembl.org / pub / release-103 / variation / vcf / gallus_gallus / ), extract the position information of the variation corresponding to the rs number; ③ Delete the adjacent SNP sites and InDel sites. The candidate sites obtained through the above screening are used as the third type of target for the SNP molecular marker combination.

[0103] 5. Remove the possible duplicate sites in the above three types of targets, and then use the Guangxi Zhuang Autonomous Region local chicken genome database constructed by the inventors to supplement the blank genomic regions where the above three types of targets are distributed as chip background sites, i.e., the fourth type of targets. The fourth type of targets must meet MAF>0.05, and Tag SNPs are preferred.

[0104] 6. Design probes for the screened SNP sites based on the probe design system of Beijing Compson Biotechnology Co., Ltd. Evaluate the GC content, complexity and other characteristics of the upstream and downstream sequences of the target site, and eliminate probes and sites with abnormal GC content (less than 35% and greater than 65%), the number of homology regions >10, and highly repeated SSRs. Complete the probe design based on the evaluation results. The designed probe is a double-stranded DNA with a length of 120bp, which meets the capture region length of about 200bp, and gives priority to placing the target site in the middle of the probe. After the probe sequence is determined, synthesize a probe modified with biotin at the 5' end, and adjust the concentration to ensure uniform capture. According to the scoring, 20,000 SNP sites were finally retained as the marker combination of the "Guixin No. 1" gene breeding chip (as shown in Table 1), of which there were 12,674 first-class targets, 537 second-class targets, and 2,262 fourth-class targets. A total of 15,473 sites were derived from local chicken breeds in Guangxi Zhuang Autonomous Region, accounting for 77.37% of the total number of chip sites. The proportion of functional sites is as follows Figure 1As shown in the figure, 34.90% of SNP loci are related to growth traits, 16.29% of SNP loci are related to appearance traits, 16.09% of SNP loci are related to reproduction traits, 10.79% of SNP loci are related to meat quality traits, 5.55% of SNP loci are related to feed efficiency, and a total of 17,700 loci are related to economic traits. The overall distribution of chip loci is shown in the figure. Figure 2 shown.

[0105] 7. Verification of the detection rate of the “Guixin No. 1” gene breeding chip

[0106] Twenty Guangxi Zhuang Autonomous Region Ma chicken samples were collected again, and genomic DNA was extracted using a special DNA extraction kit. Targeted hybridization capture sequencing was performed based on the "Guixin No. 1" chip. The HaplotypeCaller module in the GATK software was used to generate SNP variant site files. After extracting the target sites, 10× depth conditions were used for filtering. The results are shown in Table 3. The average detection rate of the 20 test samples was 99.6%.

[0107] Table 3. Detection rate of typing of test samples

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[0110] Example 2 Variety identification based on the typing results of the "Guixin No. 1" gene breeding chip

[0111] Blood samples of three local chicken breeds in Guangxi Zhuang Autonomous Region (Guangxi Zhuang Autonomous Region Sanhuang Chicken, Guangxi Zhuang Autonomous Region Ma Chicken, Yao Chicken) and two non-local chicken breeds in Guangxi Zhuang Autonomous Region (bearded chicken, recessive white-feathered chicken) were collected, with 10 chickens of each breed. Genomic DNA was extracted using the Ezup column blood genomic DNA extraction kit, and sequenced using the "Guixin No. 1" gene chip. The sequencing results were typed with reference to Example 1. Individuals with a missing rate greater than 10% and sites with a typing missing rate greater than 10% were filtered using PLINK software, and then principal component analysis (PCA) was performed on the test population using the --pca parameter. The results are shown in Figure 1. Figure 3 As shown in the figure, the local chicken breeds in Guangxi Zhuang Autonomous Region can be clearly distinguished from those in non-local chicken breeds in Guangxi Zhuang Autonomous Region; among the local chicken breeds in Guangxi Zhuang Autonomous Region, the distance between the Guangxi Zhuang Autonomous Region Sanhuang Chicken and the Guangxi Zhuang Autonomous Region Ma Chicken is relatively close. The above results show that the "Guixin No. 1" gene chip can be well applied to the identification of local chicken breeds in Guangxi Zhuang Autonomous Region.

[0112] Example 3 Application of "Guixin No. 1" 20K gene breeding chip in genome-wide association analysis

[0113] The comb length of 464 Nandan Yao chickens was measured at 18, 22, and 26 weeks of age, and blood samples of the test group were collected at the same time. Genomic DNA was extracted using the Ezup column blood genomic DNA extraction kit, and sequenced by the "Guixin No. 1" gene chip, and the sequencing results were typed with reference to Example 1. PLINK was used to retain SNP sites with minor allele frequency>0.05, missing rate<0.1, and Hardy-Weinberg equilibrium>1E-6, and individuals with SNP typing missing rate>0.1 were filtered. The mixed linear model of GEMMA software was used for genome-wide association analysis, y=Wα+xβ+u+e, where y is the phenotype vector, W is the fixed effect matrix (including sex and the first three principal components), α is the corresponding covariance vector, x is the SNP marker genotype matrix, β is the marker effect vector, u is the random effect vector, and e is the random residual. The ANNOVAR software was used to annotate the selected associated sites. The results are shown in Table 4. A total of 6 sites were associated with the comb length of Nandan Yao chicken, of which 5 sites were located in the introns of genes, including AKT3, EMC2, LYN and other genes. The AKT3 gene has been reported to be associated with the reproductive traits of ducks and the growth traits of chickens, and the LYN gene has been reported to be associated with growth traits in different livestock and poultry. The above results show that the use of the "Guixin No. 1" gene chip can obtain better association analysis results.

[0114] Table 4 Results of correlation analysis on comb length of Nandan Yao chicken

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[0116] Example 4 Evaluation of the application effect of "Guixin No. 1" 20K gene breeding chip in genomic selection

[0117] The phenotypic data and blood of 1303 chickens of a certain lineage were collected at 70 days of age. The 70-day-old phenotypes included body weight, body oblique length, keel length, hip width, shin length and shin circumference. After typing the test population using the "Guixin No. 1" chip, quality control was performed using PLINK software, retaining SNPs with an allele frequency greater than 0.05 and located on autosomes, while eliminating individuals with a site deletion rate greater than 10%. ABLUP and ssGBLUP were used to estimate breeding values, respectively: the calculation model of ABLUP is y=Xb+Zu+e; Where y is the phenotype vector, b is the fixed effect vector, u is the breeding value vector, and X and Z are the corresponding matrices. A is the kinship matrix constructed based on pedigree information. The model of ssGBLUP is consistent with ABLUP, but the A matrix needs to be replaced by the H matrix that integrates family information (A matrix) and genome information (G matrix). The genome relationship matrix G is constructed based on the results of typing of the "Guixin No. 1" gene chip. The accuracy was estimated by 5-fold cross validation, and the accuracy was calculated as the correlation coefficient r between the estimated breeding value and the corrected phenotypic value. The difference in the accuracy of ABLUP and ssBLUP was compared as the evaluation result of the genomic selection effect of the "Guixin No. 1" gene chip (Table 5). The results showed that the accuracy of ssGBLUP was improved to a certain extent, indicating that the application effect of the "Guixin No. 1" gene chip in genomic selection was good.

[0118] Table 5 Comparison of genome selection accuracy

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[0120] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A SNP locus combination for the selection and breeding of local chickens in Guangxi Zhuang Autonomous Region, Features: The SNP site combination consists of 20,000 SNP sites, and the physical positions of the 20,000 SNP sites are determined based on the chicken GRCg6a genome sequence alignment. The physical positions of the 20,000 SNP sites are shown in Table 1.

2. Use of the SNP locus combination as claimed in claim 1 in preparing a gene chip for selecting and breeding local chickens in Guangxi Zhuang Autonomous Region.

3. A gene chip for the selection and breeding of local chickens in Guangxi Zhuang Autonomous Region, It is characterized in that The gene chip is a liquid phase chip, comprising a probe combination, wherein the probe combination is used to identify the genotype of each SNP site in the SNP site combination described in claim 1.

4. Use of the SNP locus combination described in claim 1 or the gene chip described in claim 3 in the selection and breeding of local chickens in Guangxi Zhuang Autonomous Region.

5. The use according to claim 4, It is characterized in that The application is any of the following uses: (1) Application in the breeding of local chickens in Guangxi Zhuang Autonomous Region; (2) Application in identification of local chicken breeds in Guangxi Zhuang Autonomous Region; (3) Application in genome-wide association analysis of native chickens in Guangxi Zhuang Autonomous Region; (4) Application in genomic selection of local chickens in Guangxi Zhuang Autonomous Region; (5) Application in the assessment of genetic diversity of local chickens in Guangxi Zhuang Autonomous Region; (6) Its application in correcting the pedigree of local chickens in Guangxi Zhuang Autonomous Region.

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