WO2023165128A1 - Chicken low-density snp liquid-phase chip based on target capture sequencing and use thereof - Google Patents

Abstract

Provided are a chicken low-density SNP liquid-phase chip based on target capture sequencing and the use thereof. The chip contains 5912 SNP loci, 2582 of which are remarkably associated with economic traits of slaughter-type chicken breeds and are derived from whole genome resequencing data and related research of 33 representative local chicken breeds in China. The chip is suitable for resource evaluation and paternity test of local chicken breeds, and use in genetic improvement of economic traits of slaughter-type broiler breeds. In addition, the chip can adjust a target SNP locus by directly adding or reducing a probe, so that the chip has better flexibility compared with a solid-phase chip.

Description

A chicken low-density SNP liquid chip based on targeted capture sequencing and its application technical field

The application belongs to the field of gene chip technology, and relates to a chicken low-density SNP liquid chip based on targeted capture sequencing and its application.

Background technique

Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP), as the latest generation of molecular markers, has been widely used in medical diagnosis, agricultural breeding and other fields due to its advantages of large number, wide distribution, rich polymorphism and easy detection. . With the rapid development of molecular biology and related disciplines, genome sequencing of more and more species has been completed one after another, and high-throughput SNP detection technology has emerged to meet the growing demand for genome-level SNP detection.

At present, high-throughput SNP detection technology mainly adopts two strategies: solid-phase chip and sequencing. Solid-phase chips are based on the complementary hybridization of probes and DNA sequences, and the typing is performed through the fluorescent signal of the marker. It has the advantages of high typing accuracy and short cycle time, but there are also disadvantages such as high cost of single-point typing and not easy to customize. .

Existing commercialized chicken SNP chips mainly include Illumina 60K, Axiom 600K, Pheno ixChip-I (Illumina 50K) and IASCHICK (Illumina 50K), which have been widely adopted by breeding companies and research institutes, and are mainly used in chicken genome selection, germplasm Resource genetic diversity analysis, genetic relationship identification, genome association analysis, etc. The SNP density of these solid-phase chips is generally high, so the cost of typing is high, and there is a lack of low- and medium-density SNP chips on the market. At the same time, because these chips are mainly designed based on foreign high-yield breeds, there are some limitations in the application of local poultry breeds in my country.

The yellow-feather broiler industry is a traditional industry with Chinese characteristics. Traditional broiler consumption is mainly based on live chickens on the market. As governments at all levels close down the live poultry market and gradually intensify, yellow-feather broiler slaughtered and listed will become the main consumption in the future. trend. Yellow-feathered broiler breeds suitable for slaughter and market generally have better development of breast and leg muscles and better meat quality, have an earlier market age, and must have obvious carcass appearance characteristics. In 2021, the Jiangsu Institute of Poultry Science and Jiangsu Lihua Animal Husbandry Co., Ltd. successfully bred the first yellow-feathered broiler chicken in China - "Huashan Chicken" (http://xh.xhby.net/pc/ con/202112/10/content_1006062.html). At present, the economic traits related to slaughter breeds such as chicken carcass appearance are still dominated by conventional selection, and the research and application of related molecular markers need to be improved, and there is no gene chip suitable for slaughter breed selection.

Contents of the invention

In order to overcome the above technical problems, the present invention provides a chicken low-density SNP liquid chip based on targeted capture sequencing, including 5912 SNP sites. The chicken low-density liquid-phase chip of the invention can be used for genome selective breeding, genetic diversity analysis, variety identification, genetic relationship identification and genome-wide association analysis of local chicken slaughter traits in my country.

The first aspect of the present invention provides a chicken whole genome SNP molecular marker combination, the chicken whole genome SNP molecular marker combination is composed of 5912 SNP molecular markers, the position of the SNP molecular marker on the chicken reference genome GRCg6a is as shown in the table No.001 to NO.5912 in 1.

Table 1 5912 SNP molecular marker positions

The SNP molecular markers are associated with slaughter-type chicken breed economic traits, and the economic traits are feed remuneration and body weight at the age of 90 days, slaughter product yield (breast muscle, leg muscles, wings), comb development, skin pore density, five toe , skin color, shin color, gutter length, muscle fiber area, muscle fiber type distribution.

The SNP molecular markers described in the present invention comprise two classes: one is based on genome-wide association analysis, comparative genomics research and literature data, and screens and excavates 2582 SNP loci (hereinafter referred to as functional loci) that are significantly associated with the economic traits of slaughter-type chicken breeds. The second category is to use the whole genome resequencing data of 33 representative local chicken breeds in China, and use the screening principles of high polymorphism and uniform genome distribution to screen 3330 SNP sites (hereinafter referred to as background sites) .

The SNP molecular markers of the present invention, of which 1254 are newly discovered SNP sites, which have not been included in the European Variation Archive database (https://www.ebi.ac.uk/eva), and 4121 sites have not been included by other commercial Included in Huaji SNP chip.

The second aspect of the present invention provides a chicken whole genome low-density SNP chip, the chicken whole genome low-density SNP liquid chip is composed of chicken whole genome SNP molecular markers shown in Table 1 from NO.001 to NO.5912 be made of.

In some embodiments, the chip is a liquid phase chip.

In the third aspect of the present invention, the chicken whole genome low-density SNP chip described in the second aspect of the invention is provided for the evaluation of chicken genetic diversity, the evaluation of local chicken breed conservation effect, the identification of chicken kinship, the identification of chicken breeds and the association of chicken whole genome applications in analysis.

The fourth aspect of the present invention provides the application of the chicken whole genome low-density SNP chip described in the second aspect of the present invention in genetic improvement of economic traits of slaughter broiler breeds.

In certain embodiments, the economic traits include, but are not limited to, feed remuneration and body weight at 90 days of age, slaughter product yield, comb development, skin pore density, five toes, skin color, shank color, pitch length, Muscle fiber area, muscle fiber type distribution.

In some embodiments, the slaughter product yield includes breast muscle, leg muscle, and wing slaughter yield.

Compared with the prior art, the present invention has the following advantages: firstly, the SNP sites contained in the liquid-phase chip are derived from the whole-genome resequencing data of 33 representative local chicken breeds in China, so there are relatively abundant polymorphisms in local chicken populations, Therefore, it is more suitable for genetic improvement and resource evaluation of local chicken breeds. Second, the SNP sites contained in this chip are excavated for the key traits of slaughtered breeds such as carcass appearance, so it is very suitable for the improvement of slaughtered breeds and the slaughtered selection of local chicken breeds. The third is that the liquid-phase chip is based on targeted capture sequencing technology, which can not only type the target site, but also type the SNPs within a certain range near the target site, so more typing information can be obtained. Fourth, the liquid-phase chip can adjust the target SNP site by directly adding or subtracting probes, which is more flexible than the solid-phase chip. Fifth, the liquid-phase chip relies on the next-generation sequencing platform, which has a lower typing cost and a higher cost performance.

Description of drawings

In order to more clearly illustrate specific embodiments of the present invention or technical solutions in the prior art, the following briefly introduces the accompanying drawings that are used in the description of specific embodiments.

Figure 1 shows the distribution of 5912 SNPs on each chromosome in Example 1 of the present invention.

Fig. 2 is a diagram showing the results of PCA clustering of 8 local chicken breeds by using a liquid phase chip in Example 3 of the present invention.

Fig. 3 is a clustering diagram of the kinship relationship of 648 chickens in the same group by using the liquid chip in Example 4 of the present invention.

Detailed ways

Embodiments of the technical solution of the present invention will be described in detail below. The following examples are only used to illustrate the technical solution of the present invention more clearly, so they are only examples, and should not be used to limit the protection scope of the present invention. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application shall have the usual meanings understood by those skilled in the art to which the present invention belongs.

Embodiment 1 Design and preparation of chicken low-density SNP liquid chip

1. Establishment of local chicken SNP background database

The present invention utilizes the whole genome resequencing data of 475 individuals of 33 local chicken breeds in China, and obtains highly credible SNP sites through data analysis for subsequent screening.

Blood samples of local chicken breeds were obtained from the Poultry Genetic Material Bank of Jiangsu Poultry Research Institute. Firstly, the whole genome of 475 local chickens (Table 2) was resequenced using the Hiseq X-ten sequencer (Illumina), and the average genome coverage of each sample was above 10×. After preliminary quality control of the off-machine data, the sequencing data was compared to the chicken reference genome (GRCg6a) by BWA software (Li and Durbin, 2010), and the SNP mutation identification was carried out using the Haplotype caller module of the GATK4 software. A total of 17,464,141 high-quality mutation sites were obtained.

Table 2 Chicken breeds and quantities used for whole genome resequencing

serial number	Variety	feature		quantity
1	Deer Park Chicken	large size	10

2	Liyang Chicken	large size	10
3	big bone chicken	large size	15
4	Turpan cockfight	large size	10
5	wolf pheasant	blue shins	10
6	Yao chicken	blue shins	20
7	Longsheng Phoenix Chicken	blue shins	20
8	Chongren No-Ma Chicken	blue shins	20
9	Dried silky chicken		upi	10
10	Dehua black chicken		upi	10
11	Jiangshan silky chicken		upi	10
12	Wuliangshan silky chicken		upi	10
13	Golden Lake Black Phoenix Chicken		upi	10
14	Wumeng Silky Chicken		upi	10
15	Xuefeng Silky Chicken		upi	10
16	silky chicken	Upi, five toes	10
17	Peking Fried Chicken	five toes	20
18	Camellia Chicken		Small size	10
19	Daweishan Micro Chicken		Small size	15
20	Qiandongnan Small Fragrant Chicken		Small size	20
twenty one	Taihu Chicken	the	10
twenty two	Rugao Yellow Chicken	the	10
twenty three	Guangxi No-Ma Chicken	the	20
twenty four	ladle chicken	the	12
25	Tengchong Snow Chicken	the	18
26	Wuding Chicken	the	20
27	Xishuangbanna cockfighting	the	20
28	Yunlong Bantam	the	15
29	Lanping fluffy chicken	the	20
30	Xiayan Chicken	the	20
31	Guangxi Three Yellow Chicken	the	20
32	Wenchang Chicken	the	20
33	Tibetan chicken	the	10
the	total	the	475

2. Screening of functional sites

Step 1, using the genome-wide association analysis strategy to screen and obtain the SNP loci related to the economic traits of slaughter-type varieties.

The experimental chickens were from the Chongren Ma chicken resource group in the Chongren Ma chicken original breeding farm in Chongren County, Jiangxi Province, with a total of 718 individuals. The phenotypic measurements are all performed using well-known methods, and the indicators include body weight at 90 days, feed conversion ratio at 90 days, chest muscle rate, leg muscle rate, wing rate, comb height, skin pore density on the back, chicken gutter length, leg muscle fiber area, The distribution ratio of oxidized and glycolytic muscle fibers in leg muscles.

Wing vein blood collection for genomic DNA extraction. The IASCHICK 50K chip was used for genomic SNP detection (Liu et al., 2019). After quality control, the remaining 31,628 SNP sites were used for subsequent analysis. Using the GLM model of Plink V1.9b software for genome-wide association analysis (Purcell et al., 2007), after correcting the p-value by the Bonferroni method, 1,893 loci with the significance of the Top% association with 12 phenotypic indicators were obtained as Candidate SNP sites.

Step 2, using comparative genomics method to screen and obtain the SNP loci related to the economic traits of slaughter-type varieties.

Using the whole genome resequencing data of 33 local chicken breeds, according to the performance characteristics of each breed (Table 2), the functional loci were mined by comparative genomics methods. VCFtools software (Danecek et al., 2011) was used to count the differences between large and small chickens, black and non-black chickens, five-toed and non- The ratio of group fixation index (FST) and nucleic acid diversity index (θπ) between five-toed chickens, between blue-shank chickens and non-blue-shank chickens, the intersection of the top 1% window with the highest ratio of FST and θπ in the genome range, They were respectively used as selected areas for chicken body weight, skin color, five toes and shank color traits.

After merging each selected region, the sites with minimum allele frequency (MAF)>0.1 and deletion rate<0.1 were retained. Then Haploview software (Barrett et al., 2005) was used for linkage analysis, and a SNP located in the gene region in the longest linkage block was selected for each region as a candidate site for the region. A total of 1,253 candidate sites were obtained.

Step 3, based on the applicant's previous research and the animalQTL database (www.animalgenome.org), a total of 2,218 SNP loci related to economic traits of slaughter broilers were obtained (Table 3). After integrating with the loci obtained in steps 1 and 2, compare the candidate loci with the whole-genome sequencing data of 33 local chicken breeds, and use MAF>0.2, deletion rate<0.1 and heterozygosity rate<0.5 as screening criteria to eliminate Sites that do not meet the requirements; then select the sites within 500bp, and retain the sites with good polymorphism and located on the gene; finally retain 2,582 SNP sites as the final functional SNP sites.

Table 2 Sources of SNP sites based on previous research and databases

3. Screening of background loci

According to the principle of uniform distribution of the selected SNP sites in the genome, based on the resequencing data of 33 local chicken breeds, the background SNP sites were supplemented on the chip. Divide the genome into several intervals based on 200kb. If there is a functional site in the interval, no site will be added. If there is no functional site in the interval, a background site will be added. The screening principles of background loci were MAF>0.35, deletion rate<0.1 and heterozygosity rate<0.5.

Through the above steps, a total of 5,981 functional sites and background sites were selected and submitted to Shijiazhuang Bo Ruidi Biotechnology Co., Ltd. for evaluation, and sites that could not be uniquely compared on the genome and sites containing repetitive sequences in the flanking sequences were removed and adjacent sites with a distance of less than 500 bp. Finally, a total of 5,912 SNP sites evenly distributed in the chicken genome were obtained, and the specific genome coordinates are shown in Table 1. The distribution of all sites on each chromosome of the chicken genome is shown in Figure 1.

According to the position and flanking sequence information of 5,912 SNP sites, Shijiazhuang Boruidi Biotechnology Co., Ltd. used targeted capture sequencing technology to design primers and synthesize probes to obtain chicken low-density SNP liquid-phase chips.

Example 2 Application method of chicken low-density SNP liquid chip in chicken DNA sample detection

1. Extraction of chicken genomic DNA samples: blood was collected from chicken wing veins, and DNA was extracted using the phenol-chloroform method.

2. DNA sample quality detection: Agilent 2100 bioanalyzer (Agilent) was used to measure the DNA concentration, and 1% agarose gel electrophoresis was used to detect the integrity of the DNA. Qualified samples were stored in a 4°C refrigerator for later use.

3. Liquid phase chip detection: operate according to the standard procedure of liquid phase chip detection (http://www.molbreeding.com/index.php/Technology/GenoBaits.html).

4. Data analysis: The obtained raw data was quality controlled using fastp software (Chen et al., 2018), and then the sequencing data was compared to the chicken reference genome GRCg6a using BWA software (Li and Durbin, 2010), using GATK4 software (Auwera et al., 2013) standard procedure to detect SNPs and perform genotyping.

Example 3 Application of chicken low-density SNP liquid-phase chip in the detection of genetic diversity of local chicken breeds

The present invention uses the chicken liquid chip prepared in Example 1 to perform genotyping of 8 local chicken breeds (see Example 2 for the specific operation method). Specific local chicken breeds include Jinhu Wufeng chicken (JH), Langshan chicken (LS), Langya chicken (LY), Wenshang reed chicken (WS), silky silkie (SK), bantam (AJ) ), Dongxiang Green-shell Chicken (DX), Gushi Chicken (GS). The samples were all from the National Gene Bank of Local Chicken Breeds of the Jiangsu Institute of Poultry Science, with 10 chickens for each breed. Among the tested varieties, the detection rate of the target SNP loci was above 98%, the loci with MAF>0.05 exceeded 91% of all the target loci, and the average MAF within the variety was between 0.34 and 0.41. In addition to the target SNP, 14,895 sites around the target SNP were detected. After quality control, sites with MAF<0.05 and deletion rate>0.1 were removed, leaving 12,918 valid sites.

The data was analyzed with reference to the method of Example 2, and the PCA results of local chicken breeds are shown in Figure 2. The results show that the chip can be used to distinguish various varieties very well. The genetic distance between each breed is close to the geographical distance of the main producing areas of each breed. For example, Langya chicken and Wenshang reed chicken are distributed in North China, so they have a relatively close genetic distance. In addition, there is a relatively long genetic distance between some local chickens with obvious characteristics and other breeds. For example, the silky chicken with silk feathers has the characteristics of "crested head, silky feathers, five toes, mulberry crown, and black bones", which is in line with previous research. Results (Liu et al., 2021).

The above results confirmed that the chicken low-density SNP liquid-phase array has high polymorphism in local chicken breeds, which is very suitable for the evaluation of local chicken germplasm resources, genetic improvement and related research. Even without genotyping, more genotyping results than target SNPs can be obtained using this liquid chip. Compared with the existing chicken solid-phase chip, chicken breeding and research work can be carried out at a lower cost.

Example 4 Application of chicken low-density SNP liquid-phase chip in identification of chicken kinship

The accuracy of livestock and poultry pedigree information is of great significance to the progress of breeding or the effect of conservation. However, due to the introduction of individuals of unknown blood relationship, human error in recording and other reasons, errors in pedigree records are common in the process of production, breeding and conservation. Therefore, it is necessary to carry out genetic relationship identification work on chicken flocks.

In this example, 648 chickens in a group of Guangxi Fufeng Farming Co., Ltd., including 98 roosters and 550 hens, were used to analyze the genetic relationship and construct a molecular pedigree using the chicken liquid chip prepared in Example 1. Through genomic kinship analysis and cluster analysis based on G matrix, it was found that the existing rooster samples could be divided into 37 families (Figure 3), and hens were divided into different families according to the kinship between hens and roosters. Compared with the original genealogy, the accuracy rate reaches 94%. The inbreeding coefficient statistics based on ROH were also carried out for all individuals in the group, and the average inbreeding coefficient of the group was 0.1754. Individuals with a higher degree of inbreeding should be avoided in subsequent breeding.

The above results suggest that the application of the liquid-phase chip can obtain a more refined kinship relationship at a lower cost, and at the same time correct pedigree errors, eliminate inbred individuals, and provide support for improving breeding progress or conservation effects.

Example 5 Application of Chicken Low Density SNP Liquid Chip in Genetic Improvement of Slaughter Chicken Breeds

Genome-wide selection technology is a breeding method that has just emerged in the field of animal husbandry in recent years. Compared with conventional methods, genomic selection adds genomic data based on the use of pedigree and phenotypic data, greatly increasing the accuracy of selection. Skin pore density affects the aesthetics of broiler carcasses, which is one of the important breeding traits for slaughter broiler breeds.

In this example, the male line of a slaughter-type breed of a broiler breeding enterprise was used as the object, and the chicken low-density SNP liquid chip was used to perform genomic selection on the pore density. At the same time, conventional selection based on pedigree was carried out on this strain, and an unselected control group was set up. In the first generation, 824 breeder chickens were selected as a reference group, all of which were genotyped by liquid chip, and slaughtered at 90 days old, and the pore density (2cm×2cm) on the back of the individual was measured. In the second generation, 500 individuals were selected for genotyping by liquid-phase chips, and the SS-GBLUP method was used to estimate the genome breeding value (GEBV) of the candidate population for the obtained genotyping results, so as to select excellent individuals for pore density traits. Keep. In the third generation, the phenotypes of each group of individuals were determined, and the genetic progress was counted. It was found that the genetic progression of pore density in the population applying genomic selection was 6.31% higher than that of the conventional selection group and 8.28% higher than that of the control group. The above results suggest that the liquid-phase chip is very suitable for the genomic selection of pore density traits, and the application of the liquid-phase chip can significantly accelerate the progress of breeding of slaughter-type chicken breeds.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. All of them should be covered by the scope of the claims and description of the present invention.

Claims (7)

1. A chicken whole genome SNP molecular marker combination is characterized in that the chicken whole genome SNP molecular marker combination is composed of 5912 SNP molecular markers, and the position of the SNP molecular marker on the chicken reference genome GRCg6a is as NO in Table 1 of the description .001 to NO.5912 shown.
2. A chicken whole genome low-density SNP chip, characterized in that the chicken whole genome low-density SNP liquid chip is made of the chicken whole genome SNP molecular marker combination according to claim 1.
3. The chicken whole genome low-density SNP chip according to claim 2, wherein the chip is a liquid phase chip.
4. The application of the whole genome low-density SNP chip of chicken according to any one of claims 2-3 in the genetic improvement of economic traits of slaughter-type broiler chicken breeds.
5. The application according to claim 4, wherein the economic traits include, but are not limited to, feed remuneration and body weight at the age of 90 days, slaughter product yield, comb development, skin pore density, five toes, skin color, shins Color, gutter length, muscle fiber area, muscle fiber type distribution.
6. The application according to claim 5, characterized in that said slaughter product yield includes breast muscle, leg muscle, and wing slaughter yield.
7. The application of the whole-genome low-density SNP chip of chicken according to any one of claims 2-3 in evaluation of chicken genetic diversity, evaluation of conservation effect of local chickens, identification of chicken kinship, identification of chicken breeds and association analysis of chicken whole genome.

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