WO2023165128A1 - Puce snp de poulet à faible densité en phase liquide reposant sur le séquençage par capture de cible et son utilisation - Google Patents

Puce snp de poulet à faible densité en phase liquide reposant sur le séquençage par capture de cible et son utilisation Download PDF

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WO2023165128A1
WO2023165128A1 PCT/CN2022/122546 CN2022122546W WO2023165128A1 WO 2023165128 A1 WO2023165128 A1 WO 2023165128A1 CN 2022122546 W CN2022122546 W CN 2022122546W WO 2023165128 A1 WO2023165128 A1 WO 2023165128A1
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chicken
snp
chip
whole genome
density
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PCT/CN2022/122546
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Chinese (zh)
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刘一帆
束婧婷
章明
邹剑敏
屠云洁
单艳菊
姬改革
巨晓军
盛中伟
贾晓旭
周成浩
李云雷
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江苏省家禽科学研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • 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.
  • Single nucleotide polymorphism 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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.
  • the chip is a liquid phase chip.
  • 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.
  • 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.
  • the slaughter product yield includes breast muscle, leg muscle, and wing slaughter yield.
  • 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 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.
  • 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.
  • the liquid-phase chip relies on the next-generation sequencing platform, which has a lower typing cost and a higher cost performance.
  • 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.
  • Embodiment 1 Design and preparation of chicken low-density SNP liquid chip
  • 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.
  • 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.
  • 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.
  • GLM model of Plink V1.9b software for genome-wide association analysis (Purcell et al., 2007)
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Liquid phase chip detection operate according to the standard procedure of liquid phase chip detection (http://www.molbreeding.com/index.php/Technology/GenoBaits.html).
  • 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).
  • JH Jinhu Wufeng chicken
  • LS Langshan chicken
  • LY Langya chicken
  • SK silky silkie
  • SK silky silkie
  • AJ bantam
  • DX Dongxiang Green-shell Chicken
  • GS Gushi Chicken
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • conventional selection based on pedigree was carried out on this strain, and an unselected control group was set up.
  • 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.
  • 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.
  • GEBV genome breeding value
  • 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.

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Abstract

La présente invention concerne une puce SNP de poulet à faible densité en phase liquide, reposant sur le séquençage par capture de cible, et l'utilisation de cette puce. La puce contient 5912 loci SNP, dont 2582 sont remarquablement associés aux caractéristiques génétiques à des fins économiques des races de poulets de chair d'abattage et sont dérivés des données de reséquençage du génome entier et de la recherche connexe de 33 races locales de poulets représentatives en Chine. La puce convient à l'évaluation des ressources et au test de paternité des races locales de poulets, ainsi qu'à l'amélioration génétique à des fins économiques des races de poulets de chair d'abattage. En outre, la puce peut ajuster un locus SNP cible en ajoutant ou en réduisant directement une sonde, ce qui lui confère une plus grande flexibilité qu'une puce en phase solide.
PCT/CN2022/122546 2022-03-04 2022-09-29 Puce snp de poulet à faible densité en phase liquide reposant sur le séquençage par capture de cible et son utilisation WO2023165128A1 (fr)

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CN117646075A (zh) * 2023-12-04 2024-03-05 江苏省家禽科学研究所 一种鸡繁殖性能分子微模块芯片及其应用
CN117701722A (zh) * 2024-01-16 2024-03-15 佛山科学技术学院 一种牛高原适应育种10k液相芯片及应用
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CN118389706A (zh) * 2024-06-24 2024-07-26 中国水产科学研究院珠江水产研究所 一种用于鳜鱼基因分型的标记组合及应用其的全基因组液相芯片

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