A 60K SNP liquid phase chip for broiler chickens and its application in breed identification

Abstract

The present invention discloses a broiler chicken 60K SNP liquid phase chip and its application in variety identification, and relates to the field of biotechnology. The broiler chicken 60K SNP liquid phase breeding chip includes a probe combination, and the probe combination is used to identify the genotype of 62013 SNP sites. The broiler chicken 60K SNP liquid phase chip can be widely used in different application scenarios such as germplasm resource identification, genetic diversity assessment, variety identification, kinship identification, purification and rejuvenation of local varieties of different broiler chicken varieties (Xinghua chicken, Qingyuan Ma chicken, Zhongshan Shalan chicken, etc.), providing an effective scientific basis for broiler chicken variety identification, improving the efficiency of variety identification and evaluation, and having significant application value.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a broiler 60K SNP liquid phase chip and application thereof in variety identification. Background Art

[0002] my country is the country with the richest livestock and poultry genetic resources in the world. It has the characteristics of vast territory, diverse climate, and great cultural differences, forming local broiler genetic resources with their own characteristics. However, for a long time, due to the difficulty in protecting resources in some places and the lack of effective integrated utilization, the germplasm resources have been degraded and some characteristic traits have been lost. The South China region (including Guangxi), mainly in Guangdong, is the main place for independent breeding of high-quality yellow-feathered broilers. In recent years, although my country's research teams have successively developed a number of local chicken genetic resource protection and variety identification chips, due to the small number of varieties included and the limited geographical areas covered, the excavation and integrated utilization of the excellent traits of local broiler genetic resources are still lacking. Therefore, it is very necessary to develop a genotyping product with a wide range of varieties and geographical areas, efficient and accurate. Summary of the invention

[0003] The purpose of the present invention is to provide a broiler chicken 60K SNP liquid phase chip and its application in variety identification to solve the problems existing in the above-mentioned prior art. The broiler chicken 60K SNP liquid phase chip can be widely used in different application scenarios such as germplasm resource identification, genetic diversity assessment, variety identification, kinship identification, purification and rejuvenation of local varieties of different broiler chicken varieties (Xinghua chicken, Qingyuan Ma chicken, Zhongshan Shalan chicken, etc.), providing an effective scientific basis for broiler chicken variety identification, improving the efficiency of variety identification and evaluation, and having significant application value.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a SNP site combination for broiler breed identification, including SNP sites located in the reference genome GRCg7b as shown in Table 2.

[0006] The present invention also provides the use of the above-mentioned SNP site combination in the identification of broiler germplasm resources, genetic diversity assessment, variety identification, kinship identification or variety purification and rejuvenation.

[0007] The present invention also provides the use of the above-mentioned SNP site combination in the preparation of a 60K SNP liquid phase chip for broiler breed identification.

[0008] The present invention also provides a 60K SNP liquid phase chip for broiler breed identification. The 60K SNP liquid phase chip comprises a probe combination, and the probe combination is used to identify the genotype of each SNP site in the above-mentioned SNP site combination.

[0009] The present invention also provides the use of the above-mentioned 60K SNP liquid phase chip in the identification of broiler germplasm resources, genetic diversity assessment, variety identification, kinship identification or variety purification and rejuvenation.

[0010] The present invention also provides a method for identifying the breed of broiler chickens, characterized in that it comprises the following steps:

[0011] (1) Obtaining genomic DNA from the chicken individual to be tested;

[0012] (2) constructing a sequencing library based on the genomic DNA;

[0013] (3) performing probe hybridization reaction between the sequencing library and the above-mentioned 60K SNP liquid phase chip;

[0014] (4) extracting genotyping information after sequencing of the sequence captured by the liquid phase chip to form a genotyping file;

[0015] (5) Obtaining variety identification results based on the genotyping file.

[0016] The present invention discloses the following technical effects:

[0017] (1) The broiler chicken 60K SNP liquid phase chip of the present invention is constructed based on the whole genome resequencing data of 71 broiler chicken breeds at home and abroad (Xinghua chicken, Huaixiang chicken, Huiyang bearded chicken, Qingyuan chicken, Yangshan chicken, Zhongshan Shalan chicken, etc., and the "Six Famous Chickens of Guangdong" are integrated for the first time). 62,013 SNP sites with high quality and high breed identification are screened and obtained. It has a wide variety of sources and high site versatility. It is a set of accurate and efficient genotyping identification chips.

[0018] (2) Based on the precise positioning sequencing typing technology of liquid phase capture of the target interval genome sequence, it can not only type the target site, but also detect all genetic variation sites within the target interval and a certain range around it, and the output result has a large amount of information. Based on high-throughput sequencing technology, this detection method has a high detection throughput and a large amount of one-time output data, which can cover the detection of nearly a thousand materials at the same time; it is also suitable for mainstream second-generation sequencing platforms such as Illumina and MGI, and has wide platform adaptability.

[0019] (3) The broiler 60K SNP liquid phase chip of the present invention can be widely used in different application scenarios such as germplasm resource identification, genetic diversity assessment, variety identification, kinship identification, purification and rejuvenation of local varieties of different broiler breeds (Xinghua chicken, Qingyuan Ma chicken, Zhongshan Shalan chicken, etc.), providing an effective scientific basis for broiler breed identification, improving the efficiency of breed identification and evaluation, and has significant application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0021] Figure 1 This is the distribution map of 60K SNP liquid phase chip marker loci on the chromosome of broiler chicken;

[0022] Figure 2 It is a schematic diagram of the principle of liquid-phase probe capture sequencing typing technology;

[0023] Figure 3 This is a statistical chart of the genotype detection rate of 60K SNP liquid phase chip sites in broiler chicken samples;

[0024] Figure 4 This is the statistical chart of genotype consistency of repeated samples of broiler 60K SNP liquid phase chip;

[0025] Figure 5 This is the PCA analysis diagram of broiler 60K SNP liquid phase chip on 314 broiler materials;

[0026] Figure 6 This is the cluster analysis diagram of broiler 60K SNP liquid phase chip on 402 broiler materials. DETAILED DESCRIPTION

[0027] 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] This embodiment provides a broiler 60K SNP liquid phase chip, which is obtained by the following method:

[0034] 71 broiler breeds from home and abroad (Xinghua chicken, Huaixiang chicken, Huiyang bearded chicken, Qingyuan Ma chicken, Yangshan chicken, Zhongshan Shalan chicken, etc., integrating the "Six Famous Chickens of Guangdong" for the first time), totaling 1019 broiler materials. The breeds come from a wide range of sources, covering more than 20 provincial administrative regions in my country and seven natural geographical divisions. Based on the whole genome resequencing data of 71 broiler breeds, the sequencing data were analyzed to obtain highly reliable SNP site information, from which 62013 high-quality and high-breed discrimination SNP sites were selected, and probes were designed and synthesized for 62013 markers, thus obtaining a broiler 60K SNP liquid phase chip.

[0035] The physical positions of the 62013 SNP molecular markers were determined by sequence alignment based on the reference genome GRCg7b of broiler chickens. The specific information of the 62013 SNP molecular markers is shown in Table 2.

[0036] The specific method for obtaining the broiler 60K SNP liquid phase chip is as follows:

[0037] 71 broiler breeds from home and abroad (Xinghua chicken, Huaixiang chicken, Huiyang bearded chicken, Qingyuan Ma chicken, Yangshan chicken, Zhongshan Shalan chicken, etc., the first integration of the "Six Famous Chickens of Guangdong") were collected. The broiler breed categories are shown in Table 1. A total of 1019 whole genome resequencing data of broiler materials were collected. VCFtools-0.1.13 was used to calculate the Fst values ​​between the SNP sites of 71 broiler breeds and other broiler breeds, and high Fst sites were screened for each breed. At the same time, the SNP sites with population quality indicators that met the minimum allele frequency MAF ≥ 0.05, missing rate < 0.1, heterozygosity rate ≤ 0.5, and sequencing depth ≥ 8X were selected as preliminary screening candidate sites. All the above candidate sites were used for probe design. The probe design method is to design probes within 100bp upstream and downstream of each site. Remove probes that cannot be uniquely aligned on the genome and contain repetitive sequences in the flanking sequence.

[0038] Table 1 List of broiler breeds

[0039] 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 100-day 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

[0040] The above methods finally screened 62,013 SNP loci with high quality and high variety identification ability. The distribution of the loci on the chromosome is shown in Figure 1 The 62,013 SNP sites finally screened were designed and synthesized by Huazhi Biotechnology Co., Ltd., and developed into a broiler 60K liquid phase chip using targeted sequencing genotyping technology (Genobaits technology). The principle is to design probes for target interval sequences based on an optimized thermodynamic stability algorithm model, and use synthetic specific probes to perform liquid phase hybridization capture enrichment on multiple different target sequences located at different genomic locations. Then, the captured and enriched target genomic sequences are sequenced by library construction and high-throughput sequencing to obtain the genotypes of all SNP / InDel sites in the target area.

[0041] Table 2 SNP loci information of broiler 60KSNP liquid phase chip

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[0159] Example 2

[0160] This example provides a method for using the broiler whole genome liquid phase chip in Example 1 to perform genotyping on broiler samples, and the steps are as follows:

[0161] (1) Extraction and quality control of genomic DNA

[0162] The sample DNA was extracted by magnetic bead method and the quality of DNA samples was tested. The quality test included measuring DNA concentration with Qubit fluorescence quantification instrument and testing DNA integrity with 1% agarose gel electrophoresis. Samples that passed the quality test were used for library preparation.

[0163] (2) GPS library construction and quality control

[0164] a. Use fragmentase to digest the DNA sample, repair the digested ends, add A base to the 3' end, and detect the fragment size by agarose gel electrophoresis.

[0165] b. Use T4 ligase to connect the sequencing adapter and DNA fragment, and use magnetic beads to purify the ligation product. The concentration of the purified product is detected by Qubit fluorescence quantification instrument, and the fragment size is detected by agarose gel electrophoresis.

[0166] c. Perform PCR amplification on the purified ligation product, and use magnetic beads to screen the amplified product. The concentration of the product after fragment screening is detected by Qubit fluorescence quantitative instrument, and the fragment size is detected by agarose gel electrophoresis.

[0167] d. Take 200ng of the constructed library, add probes and hybridization reagents, and incubate at 50°C for 16-24 hours to complete the hybridization reaction. Use magnetic beads to capture the target segment, use cleaning solution to clean the captured product, remove non-specific binding fragments, and then perform another round of PCR amplification. Use Qubit fluorescence quantification instrument to detect library concentration, and agarose gel electrophoresis to detect fragment size. After the concentration and fragment size are qualified, the construction of cGPS sequencing library is completed. Use BGI sequencer to perform high-throughput sequencing on the prepared library, and the sequencing strategy is PE150. The principle and process of GPS library construction can be found in Figure 2 .

[0168] (3) Data analysis

[0169] The raw data after high-throughput sequencing was processed by quality control filtering, and the adapter fragments and low-quality reads were removed using FASTP software to obtain high-quality Clean Reads. The obtained Cleanreads were aligned with the reference genome using BWA software, and the positions were sorted to obtain the bam file after the sample sorting. The sequencing results were analyzed for variant sites using GATK software to obtain the original vcf variant result file. According to the ratio of the number of supported reads of different Alles of the target site, when the mutation reads support ratio is ≥0.8 or ≤0.2, the site is judged as a homozygous genotype, and when the mutation reads support ratio is between 0.2-0.8, it is judged as a heterozygous genotype. The original vcf variant result file was processed using an internally written perl script to obtain a 28-converted vcf variant result file.

[0170] Ultimately, the genotyping results of each target SNP in a specific individual are obtained with high throughput, achieving high-throughput SNP genotyping.

[0171] Example 3

[0172] In order to verify the genotyping effect of the broiler 60K SNP liquid phase chip, the broiler 60KSNP liquid phase chip designed in Example 1 was used to perform genotyping detection on 64 broiler samples (19 Qingyuan Ma chickens, 25 Wenchang chickens, and 20 Xianju chickens). A technical replicate experiment was set up for 4 samples (2 Qingyuan Ma chickens, 1 Wenchang chicken, and 1 Xianju chicken). The specific usage method is shown in Example 2.

[0173] After sequencing and data analysis, the average detection rate of the loci in 64 broiler samples was 99.2%. Figure 3 As shown; the average genotype consistency of technical repeat samples was 99.5%, and the results were as follows Figure 4When the broiler 60K SNP liquid phase chip is used to perform genotyping on the materials to be tested, the target site detection rate is high, the stability is good, and the typing results are accurate and reliable. It can be fully used for SNP typing detection of different broiler samples.

[0174] Example 4

[0175] Application of 60K SNP liquid phase chip in broiler breed identification:

[0176] The genotyping results of 314 broiler materials of 18 broiler breeds were extracted. The 18 broiler breed categories are: Lingkun chicken, Henan fighting chicken, Shalan chicken, Camellia chicken, recessive white-feathered chicken, Langshan chicken, Daweishan miniature chicken, Silky-bone chicken, White Leghorn chicken, Luodao red chicken, Liyang chicken, Lindian chicken, Cenxi classical chicken, Longshengfeng chicken, Bian chicken, Huaibei Ma chicken, Dulong chicken, Qingyuan Ma chicken. The PCA components of the test materials were analyzed using Plink software to construct a PCA scatter plot. Each site in the scatter plot represents a sample. The farther the distance between the two samples in the figure, the greater the difference in the genetic background of the two samples. Individuals with similar genetic backgrounds will be clustered into one category in the figure. The PCA analysis results were compared with the actual grouping situation, and it was found that the 314 broiler samples were divided into 18 obvious groups, see Figure 5 .

[0177] The genotyping results of 402 broiler materials from 27 broiler breeds were extracted. The 27 broiler breeds were Gushi chicken, Lushi chicken, Xichuan black-bone chicken, Xinghua chicken, Jining 100-day chicken, Yunyang large chicken, Xianju chicken, Niya black chicken, Jianghan chicken, Lingkun chicken, Henan fighting chicken, Shalan chicken, Camellia chicken, recessive white-feathered chicken, Langshan chicken, Daweishan miniature chicken, Silky-feathered chicken, White Leghorn chicken, Luodao red chicken, Liyang chicken, Lindian chicken, Cenxi classical chicken, Longshengfeng chicken, Bian chicken, Huaibei Ma chicken, Dulong chicken, Qingyuan Ma chicken. The genetic distance matrix of 402 broiler materials was calculated using Plink software and cluster analysis was performed. The phylogenetic tree diagram was constructed to determine the kinship, evolutionary relationship and composition structure of different materials. The results showed that the clustering effect was consistent with the actual grouping, as shown in Figure 2. Figure 6 This indicates that the 60K SNP liquid phase chip loci of broiler chickens are highly representative and can be used to determine the kinship, evolutionary relationship, composition structure and variety identification of different materials.

[0178] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A 60K SNP liquid phase chip for broiler breed identification, characterized in that: The 60K SNP liquid phase chip includes a specific probe combination, and the specific probe combination is used to identify the genotype of each SNP site in the SNP site combination; The SNP site combination consists of SNP sites located in the reference genome GRCg7b as shown in Table 2 of the specification.

2. An application of the 60K SNP liquid phase chip as claimed in claim 1 in genetic diversity assessment, breed identification, and kinship identification of broiler chickens.

3. A method for identifying broiler breeds, 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 specific probe hybridization reaction between the sequencing library and the 60K SNP liquid phase chip according to claim 1; (4) extracting genotyping information after sequencing of the sequence captured by the liquid phase chip to form a genotyping file; (5) Obtaining variety identification results based on the genotyping file.

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