A 60K SNP liquid breeding chip for broilers

Abstract

The present invention discloses a 60K SNP liquid breeding chip for broiler chickens, which relates to the field of biological detection technology. The 60K SNP liquid breeding chip for broiler chickens includes a probe combination, which is used to identify the genotypes of 61,219 SNP loci. The functional loci and gene region loci of the 60K SNP liquid breeding chip for broiler chickens provided by the present invention are rich, the chip loci have high polymorphism and are evenly distributed on chromosomes, and it can be widely used in different application scenarios such as the construction of genetic maps of broiler chickens, gene mapping, genome-wide association analysis, molecular marker-assisted selection, directional improvement, mining and identification of genes for important traits and functional analysis, and genome-wide selection breeding for different broiler chicken samples, which is of great significance for improving the breeding efficiency of broiler chickens and promoting the high-quality development of the broiler chicken industry in China.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and particularly to a 60K SNP liquid breeding chip for broiler chickens. Background Art

[0002] In 2001, Theo Meuwissen et al. first proposed the innovative idea of genomic selection, which first promoted the comprehensive application of genomic selection in dairy cattle breeding. Aviagen, a multinational company in poultry breeding, used genomic selection technology to increase the accuracy of breeding value assessment for feed conversion efficiency traits from 0.11 by traditional methods to 0.27, an increase of 145%. There are a wide variety of broiler chicken resources in China, with obvious regional characteristics. The rich local chicken breed resources and unique dietary and cultural habits have directly given rise to the distinctive yellow - feather broiler industry. Although yellow - feather broilers have excellent meat quality, they have the disadvantages of low feed conversion efficiency, poor reproductive performance, slow production speed, facing transformation and upgrading such as fresh - market listing, and poor carcass trait uniformity. The traditional breeding strategy targeting feather color and appearance can no longer meet the requirements, and there is an urgent need to develop a breeding chip suitable for genomic selection of yellow - feather broilers and rich in functional loci. Summary of the Invention

[0003] The purpose of the present invention is to provide a 60K SNP liquid breeding chip for broiler chickens to solve the problems existing in the above - mentioned prior art. The functional loci and gene - region loci of the 60K SNP liquid breeding chip for broiler chickens provided by the present invention are rich, the chip loci have high polymorphism and are evenly distributed on chromosomes, and can be widely used in different application scenarios such as construction of genetic maps of different broiler chicken samples, gene mapping, genome - wide association analysis, marker - assisted selection, directional improvement, mining and identification of genes for important traits and functional analysis, and genome - wide selection breeding, which is of great significance for improving broiler breeding efficiency and promoting the high - quality development of the broiler industry in China.

[0004] To achieve the above - mentioned purpose, the present invention provides the following solutions:

[0005] The present invention provides a combination of SNP loci for yellow - feather broiler breeding, including the SNP loci located on the reference genome GRCg7b shown in Table 2.

[0006] The present invention also provides the application of the above - mentioned combination of SNP loci in construction of genetic maps of broiler chickens, gene mapping, genome - wide association analysis, marker - assisted selection, directional improvement, mining and identification of genes for important traits and functional analysis, or genome - wide selection breeding of broiler chickens.

[0007] Further, the broiler chicken is a yellow - feather broiler chicken.

[0008] The present invention also provides the application of the above SNP locus combination in preparing a 60K SNP liquid breeding chip for broilers.

[0009] Furthermore, the broilers are yellow - feather broilers.

[0010] The present invention also provides a 60K SNP liquid breeding chip for broilers, which includes a probe combination for identifying the genotypes of each SNP locus in the above - mentioned SNP locus combination.

[0011] The present invention also provides the application of the above 60K SNP liquid breeding chip for broilers in genetic map construction, gene mapping, genome - wide association analysis, marker - assisted selection, directional improvement, mining and identification of genes for important traits and functional analysis, or genome - wide selection breeding of broilers.

[0012] Furthermore, the broilers are yellow - feather broilers.

[0013] The present invention also provides a method for evaluating the breed traits of broilers, comprising the following steps:

[0014] (1) Obtain the genomic DNA of the chicken individual to be tested;

[0015] (2) Based on the genomic DNA, construct a sequencing library;

[0016] (3) Perform a probe hybridization reaction between the sequencing library and the above - mentioned 60K SNP liquid breeding chip for broilers;

[0017] (4) Extract the genotyping information after sequencing the sequences captured by the liquid chip to form a genotyping file;

[0018] (5) Obtain the evaluation result according to the genotyping file.

[0019] Furthermore, the broilers are yellow - feather broilers.

[0020] The present invention discloses the following technical effects:

[0021] (1) The 60K SNP liquid breeding chip for broilers is based on the whole - genome data of multiple specialized lines such as Wen's N301, integrating 5658 significant association loci related to seven major categories of more than 160 important economic traits such as residual feed intake, daily weight gain, and body weight at 90 days of age, 2546 breeding loci, and 2755 loci on 1506 key genes. Finally, 61219 SNP loci with high quality, high polymorphism, and uniform distribution on chromosomes are screened, which is a set of accurate and efficient genotyping identification chips.

[0022] (2) The precise positioning sequencing and genotyping technology based on liquid-phase capture of genomic sequences in the target interval can not only genotype the target sites, but also detect all genetic variant sites within the target interval and a certain range around it, with a large amount of output result information. Based on high-throughput sequencing technology, this detection method has a high detection throughput, a large amount of data output at one time, and can cover the detection of nearly a thousand samples at the same time; it is also applicable to mainstream second-generation sequencing platforms such as Illumina and MGI, with wide platform applicability.

[0023] (3) The functional sites and gene region sites of the 60K SNP liquid-phase breeding chip for broilers provided by the present invention are rich, the chip sites have high polymorphism and are evenly distributed on the chromosomes, and can be widely used in different application scenarios such as the construction of genetic maps for different broiler samples, gene mapping, genome-wide association analysis, marker-assisted selection, directional improvement, the mining and identification of genes for important traits and functional analysis, and genome-wide selection breeding. It is of great significance for improving the breeding efficiency of broilers and promoting the high-quality development of the broiler industry in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 It is a distribution map of the marker sites of the 60K SNP liquid-phase breeding chip for broilers on the chromosomes;

[0026] Figure 2 It is a schematic diagram of the principle of the liquid-phase probe capture sequencing and genotyping technology;

[0027] Figure 3 It is a statistical chart of the genotype detection rate of the sites of the 60K SNP liquid-phase breeding chip for broilers in the samples;

[0028] Figure 4 It is a statistical chart of the genotype consistency rate of the repeated samples of the 60K SNP liquid-phase breeding chip for broilers. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, 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 related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0033] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0034] Example 1

[0035] This example provides a 60K SNP liquid breeding chip for broilers, which is obtained by the following method:

[0036] Based on the whole-genome data of multiple specialized strains such as Wen's N301, sequencing data analysis is performed to obtain highly reliable SNP locus information for subsequent screening. According to indicators such as the chromosomal position, minor allele frequency, missing rate, and heterozygosity rate of the SNP loci, 50,263 SNP loci with good polymorphism, strong representativeness, and uniform chromosomal distribution are screened. Integrate 5,658 significant association loci, 2,546 breeding loci, and 2,755 loci on 1,506 key genes related to seven major categories and more than 160 important economic traits such as residual feed intake, daily weight gain, and body weight at 90 days of age. Finally, 61,219 SNP markers for chip development that evenly cover the whole genome, have high polymorphism, and good generality are screened. Probes are designed for the 61,219 markers to obtain a 60K SNP liquid breeding chip for broilers.

[0037] The physical positions of these 61,219 SNP molecular markers were determined by sequence alignment based on the chicken reference genome GRCg7b. The specific information of these 61,219 SNP molecular markers is shown in Table 2.

[0038] The specific method for obtaining the broiler 60K SNP liquid breeding chip is as follows:

[0039] Collect the whole-genome data of representative broiler materials from genetic resources such as yellow-feathered broilers, white-feathered broilers, and local broiler breeds at home and abroad. The breed information is shown in Table 1. Calculate the quality indicators of the re-sequenced sites, and screen polymorphic SNP sites with quality indicators in each population that all meet the minimum allele frequency MAF≥0.1, deletion rate <0.1, heterozygosity rate ≤0.4, and sequencing depth ≥8X as the preliminary screening candidate sites.

[0040] Table 1 List of broiler breed categories

[0041] Serial number variety Number Serial number variety Number Serial number variety Number 1 Southeast Guizhou Small Fragrant Chicken 31 25 Guangxi Black Chicken 19 49 Apricot Chicken 9 2 Bantam 30 26 Yao Chicken 19 50 Xianju Chicken 7 3 Camellia Chicken 30 27 Silky Chicken 18 51 Jining Hundred Days Chicken 6 4 Changshun Green Shell Egg Chicken 30 28 Xiayan Chicken 17 52 Lindian Chicken 6 5 Daweishan Miniature Chicken 30 29 Huiyang Bearded Chicken 16 53 Liyang Chicken 6 6 Chicken on high legs 30 30 Niya Black Chicken 15 54 Rock Island Red Chicken 6 7 Langshan Chicken 30 31 Shalan Chicken 15 55 Pengxian Yellow Chicken 6 8 Lingkun Chicken 30 32 Xiushui Yellow Chicken 15 56 Turpan cockfighting 6 9 Qingyuan Chicken 30 33 Xichuan Black-bone Chicken 15 57 Yunyang Chicken 6 10 Wumeng Black-bone Chicken 30 34 Wenchang Chicken 12 58 Red Jungle Fowl 5 11 Recessive white chicken 30 35 Cockfighting in Xishuangbanna 12 59 Luning Chicken 5 12 Weining Chicken 29 36 White Leghorn Chicken 11 60 Nisi Chicken 3 13 Bamboo Chicken 28 37 Tibetan Chicken 11 61 Chicken 3 14 Yellow Lang Chicken 25 38 River Chicken 11 62 White Rock Chicken 2 15 Wuliangshan Black-bone Chicken 24 39 Longsheng Feng Chicken 11 63 Big bone chicken 2 16 Yangshan Chicken 23 40 Chicken 10 64 Emei Black Chicken 2 17 Ningdu Yellow Chicken 22 41 Cenxi Classical Chicken 10 65 Shouguang Chicken 2 18 Wuhua Chicken 22 42 Dulong Chicken 10 66 Dongxiang Green Shell Egg Chicken 1 19 Henan cockfighting 21 43 Guangxi Three Yellow Chicken 10 67 Royal Chicken 1 20 White-eared yellow chicken 20 44 Gushi Chicken 10 68 Cornish Chicken 1 21 Beijing Fried Chicken 20 45 Huaibei Ma Chicken 10 69 Taoyuan Chicken 1 22 Southern Anhui Three Yellow Chicken 20 46 Nostalgic Chicken 10 70 Wenshang Luhua Chicken 1 23 Zhengyang Three Yellow Chicken 20 47 Jianghan Chicken 10 71 Yanjin Black-bone Chicken 1 24 Guangxi Ma Chicken 19 48 Lushi Chicken 10

[0042] Based on the whole-genome data of multiple specialized lines such as Wen's N301, 5,658 significant associated sites related to seven major categories and more than 160 important economic traits such as residual feed intake, daily weight gain, and body weight at 90 days old were mined through GWAS analysis; according to the existing research results of functional genomics and multi-omics analysis, 2,546 breeding sites and 2,755 high-quality sites on 1,506 key genes were preferably obtained.

[0043] Design and synthesize liquid capture probes for all the above-mentioned candidate sites and functional sites through Huazhi Biotechnology Co., Ltd. The probe design method is to design probes within 100bp upstream and downstream of each site. Remove the probes that cannot be uniquely aligned on the genome and contain repetitive sequences in the flanking sequences. According to the principle of uniform distribution of physical positions, screen SNP sites that are evenly distributed on chicken chromosomes.

[0044] Finally, 61,219 SNP sites with strong representativeness, high polymorphism, and good universality were screened through the above methods. The average distance between the sites on the chromosome is 23.3Kb. The sites are evenly distributed on the chromosome. The distribution of the sites on the chromosome is shown in Figure 1 . The 61,219 SNP sites finally screened (Table 2) were developed into a broiler 60K liquid breeding chip using the targeted sequencing genotyping technology (Genobaits technology). Its principle is to design probes for the target interval sequence based on an optimized thermodynamic stability algorithm model, use the synthesized specific probes to perform liquid hybridization capture and enrichment on multiple different target sequences located at different genomic positions, and then construct a sequencing library and perform high-throughput sequencing on the captured and enriched target genomic sequences, so as to obtain the genotypes of all SNP / InDel sites within the target region.

[0045] Table 2 SNP Locus Information of 60K SNP Liquid Breeding Chip for Broiler Chickens

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[0174] Example 2

[0175] This example provides the application of the whole-genome liquid breeding chip for broilers in Example 1 to genotype broiler samples, and the steps are as follows:

[0176] (1) Extraction and quality control of genomic DNA

[0177] The magnetic bead method is used to extract sample DNA, and the quality of the DNA sample is detected. The quality detection includes measuring the DNA concentration with a Qubit fluorometer and detecting the integrity of the DNA by 1% agarose gel electrophoresis. The samples that pass the quality inspection are used for library preparation.

[0178] (2) Construction and quality control of the cGPS library

[0179] a. Use a fragmenting enzyme to digest the DNA sample, repair the ends of the digestion, add an A base at the 3' end, and detect the fragment size by agarose gel electrophoresis.

[0180] b. Use T4 ligase to ligate the sequencing adapter to the DNA fragment, and purify the ligation product using magnetic beads. The purified product is detected for concentration with a Qubit fluorometer and the fragment size is detected by agarose gel electrophoresis.

[0181] c. Perform PCR amplification on the purified ligation product, and use magnetic beads to screen the amplified product for fragments. The product after fragment screening is detected for concentration with a Qubit fluorometer and the fragment size is detected by agarose gel electrophoresis.

[0182] d. Take 200 ng of the completed library, add the probe and hybridization reagent, and incubate at 50 °C for 16 hours to complete the hybridization reaction. Use magnetic beads to capture the target region, wash the captured product with the washing solution to remove non-specific binding fragments, and then perform another round of PCR amplification. Use a Qubit fluorometer to detect the library concentration and agarose gel electrophoresis to detect the fragment size. After the concentration and fragment size are determined to be qualified, the construction of the cGPS sequencing library is completed. The prepared library is sequenced by high-throughput sequencing using a BGI sequencer, and the sequencing strategy is PE150. The principle and process of cGPS library construction are shown in Figure 2 .

[0183] (3) Data analysis

[0184] Perform quality control filtering and other processing on the raw data after high-throughput sequencing. Use the FASTP software to remove adapter fragments and low-quality reads to obtain high-quality Clean Reads. Use the BWA software to align the obtained Clean Reads with the reference genome and perform position sorting to obtain the sorted bam file of the sample. Use the GATK software to analyze the variant sites of the sequencing results to obtain the original vcf variant result file. Judge according to the proportion of the supporting Reads number of different Alles at the target site. When the mutation reads support ratio ≥ 0.8 or ≤ 0.2, the site is judged as a homozygous genotype. When the mutation reads support ratio is between 0.2 - 0.8, it is judged as a heterozygous genotype. Use an internally written perl script to process the original vcf variant result file to obtain the converted vcf variant result file.

[0185] Finally, genotype results of each target SNP in a specific individual are obtained through high throughput, realizing high-throughput SNP genotyping.

[0186] Example 3

[0187] To verify the genotyping effect of the 60K SNP liquid breeding chip for broilers, using the 60K SNP liquid breeding chip for broilers designed in Example 1, genotype detection was performed on 64 broiler samples (19 Qingyuan partridge chickens, 25 Wenchang chickens, 20 Xianju chickens), and technical replicate experiments were set up for 4 samples (2 Qingyuan partridge chickens, 1 Wenchang chicken, 1 Xianju chicken). The specific genotyping method is as shown in Example 2.

[0188] After sequencing and data analysis, the site detection rate of 64 broiler samples was between 99.41% - 99.83%, and the average detection rate was 99.69%. The results are as Figure 3 shown. For the comparison of the genotype results of 4 replicate samples, the average consistency rate of the genotypes of the technical replicate samples was 99.32% (in the consistency comparison, if one of the two results of a certain site is missing, this site is not included in the consistency statistics). The results are as Figure 4 shown. The results show that when the 60K SNP liquid breeding chip for broilers is used for genotyping the test materials, the target site detection rate is high, the stability is good, the genotyping results are accurate and reliable, and it can be fully used for SNP genotyping detection of different broiler samples.

[0189] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A 60K SNP liquid breeding chip for broilers, characterized in that The 60K SNP liquid breeding chip for broiler chickens includes a probe combination for identifying the genotypes of each SNP locus in the SNP locus combination; The SNP locus combination consists of SNP loci located on the reference genome GRCg7b as shown in the following table:

2. Use of the 60K SNP liquid breeding chip for broiler chickens according to claim 1 in the construction of a genetic map, gene mapping or genome-wide association analysis of broiler chickens.

3. The application according to claim 2, wherein The broiler chickens are yellow - feathered broiler chickens.

4. A method for evaluating the varietal traits of broiler chickens, characterized in that, It includes the following steps: (1) Obtain the genomic DNA of the chicken individual to be tested; (2) Based on the genomic DNA, construct a sequencing library; (3) Perform a probe hybridization reaction between the sequencing library and the 60K SNP liquid breeding chip for broiler chickens according to claim 1; (4) Extract the genotyping information after sequencing the sequences captured by the 60K SNP liquid breeding chip for broiler chickens to form a genotyping file; (5) Obtain an evaluation result according to the genotyping file.

5. The variety trait evaluation method according to claim 4, wherein The broiler chickens are yellow - feathered broiler chickens.

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