Goose whole genome liquid phase chip and application thereof

Abstract

The invention belongs to the field of biotechnology application, and particularly relates to a goose whole genome liquid phase chip and application thereof. The SNP sites on the liquid phase chip are located in a goose reference genome GCA013030995.1, and the number of the SNP sites is 30000. The chip can specifically identify the genetic relationship between commercial geese and local geese, and can also be applied to whole genome association analysis, whole genome selective breeding, target trait QTL positioning analysis, population genetics analysis and the like. According to the invention, by capturing the reference gene and loci in the real sequence thereof, the coverage of sequencing data is improved, and the application value of the chip is enhanced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology application, and specifically relates to a goose whole genome SNP chip and application thereof. Background Art

[0002] DNA molecular markers are genetic markers based on nucleotide sequence variation between individuals and are an important basis for molecular breeding. Compared with other genetic markers such as biochemical markers and cytological markers, DNA molecular markers are diverse in type and simple and rapid in detection methods, and have been widely used in animal and plant breeding.

[0003] Single Nucleotide Polymorphism (SNP) is formed by the variation of a single nucleotide in the genome (such as substitution, transversion, insertion or deletion). SNP has the characteristics of large number and high detection accuracy, and is the most common molecular marker.

[0004] Currently, the main methods used for SNP site typing are whole genome resequencing and gene chips. Whole genome resequencing is to randomly interrupt DNA at the level of the entire genome, then add adapters to the fragmented samples, and sequence the inserted fragments after amplification. Although the cost of sequencing has been significantly reduced, whole genome sequencing for hundreds or thousands of samples still faces huge challenges such as high costs and massive data processing.

[0005] In order to reduce costs and data volume, gene chips based on typing of partial sites or regions have emerged and are widely used, achieving detection of high-density molecular markers covering the entire genome with a smaller sequencing volume. Gene chips mainly include solid-phase chips and liquid-phase chips. Among them, solid-phase chips identify gene information based on the fluorescent color signal after hybridization of the marker sequence on the chip with the genome. Liquid-phase chips use biotin-labeled probes designed for the area near the target site, and after hybridization with the genome, amplification, library construction, and second-generation sequencing are performed to achieve typing of key regions of the genome. Compared with solid-phase chips, liquid-phase chips have the following advantages: based on the principle of amplification, liquid-phase chips can not only type the target site, but also other sites (such as SNPs) around the target site that are within the coverage range of the probe; secondly, the detection site flexibility of liquid-phase chips is strong and the typing cost is low.

[0006] Most of the loci information of commercial SNP chips for geese comes from the goose reference genome. However, the goose reference genome assembly is not complete. For example, some coding genes are missing, which has a certain impact on the mining and utilization of genetic loci related to important traits, so that its application is mainly concentrated in breed identification and kinship identification, and it is impossible to determine whether there are molecular marker sites related to economic traits in these sequences. Therefore, there is an urgent need for a goose whole genome SNP chip with a wide coverage and more comprehensive application range. Summary of the invention

[0007] The purpose of the present invention is to provide a goose liquid phase chip and its application, which can improve the coverage of sequencing data and enhance the application value of the chip by capturing sites in the reference genome and its missing sequences.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a goose whole genome SNP chip, which consists of 30,000 SNP molecular markers, and the chip is a liquid phase chip; the chromosome positions and numbers of the SNP molecular markers on the goose reference genome GCA_013030995.1 are shown in Table 1.

[0010] Table 130000 SNP molecular marker positions

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[0148] Preferably, the SNP chip is a liquid phase chip.

[0149] Preferably, the number of detection samples of the chip is 1 or more.

[0150] Preferably, the molecular marker is associated with goose disease resistance traits or goose economic traits.

[0151] More preferably, the disease resistance traits of the goose include gosling plague, goose influenza, goose Escherichia coli and fowl cholera; and the economic traits of the goose include growth rate, body weight and body size indicators and egg production quality.

[0152] The present invention also provides the application of the above-mentioned goose whole genome SNP chip in the identification of goose breeds or genetic relationships.

[0153] The present invention also provides the application of the above-mentioned goose whole genome SNP chip in improving goose breed quality and provenance.

[0154] The present invention also provides application of the goose whole genome SNP chip in goose breeding.

[0155] The present invention also provides the application of the goose whole genome SNP chip in the genetic diversity analysis of goose populations.

[0156] The present invention also provides the application of the above-mentioned goose whole genome SNP chip in the QTL positioning analysis of goose target traits.

[0157] Beneficial effects of the present invention:

[0158] (1) Compared with solid-phase chips with the same number of probes, the goose liquid-phase chip of the present invention can detect more SNP sites; the cost of detection reagents is significantly reduced, and the cost of single marker detection is advantageous.

[0159] (2) The goose liquid phase chip of the present invention has no sample size limit for a single test, and customization and use do not require a minimum sample size.

[0160] (3) The goose liquid phase chip designed in the present invention is suitable for the detection of both known functional loci and unknown loci, which is beneficial to the development of breeding work, germplasm resource evaluation, DNA fingerprinting and related research on QTL positioning. BRIEF DESCRIPTION OF THE DRAWINGS

[0161] 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 labor.

[0162] Figure 1 Flowchart for construction of the library of the present invention;

[0163] Figure 2 This is the information analysis flow chart of the present invention;

[0164] Figure 3 This is a flowchart of paternity testing technology. DETAILED DESCRIPTION

[0165] The present invention provides a goose whole genome SNP chip, which is composed of 30,000 SNP molecular markers, and the chromosome positions and numbers of the SNP molecular markers on the goose reference genome GCA_013030995.1 are shown in Table 1 of the specification. The goose whole genome SNP chip is a liquid phase chip.

[0166] The goose breed described in the present invention is preferably the Wanxi white goose.

[0167] The present invention optimizes the design of the goose whole genome chip by adding the missing sites in the goose reference genome as probe targets, so that the chip can capture more regions, improve the detection efficiency and reduce the detection cost of the goose whole genome chip, and has high application value in many aspects such as goose germplasm resource evaluation and improvement. The present invention screens out 30,000 SNP sites that can be used for chip design from large-scale sequencing data. Compared with the SNP sites designed in commercial chips, the site sources cover more goose breeds worldwide and are more versatile. At the same time, it can also promote goose germplasm resource evaluation and improvement by detecting sites that are correlated with important economic traits of geese.

[0168] According to the position of the site in Table 1, the corresponding goose reference genome deletion sequence is extended forward and backward by 50 bp, so that a total of 101 bp of bases including the site are used as the probe sequence of the site. The designed probe can be synthesized to prepare a goose liquid phase chip (belonging to a goose whole genome chip).

[0169] Furthermore, the goose whole genome chip of the present invention is suitable for identifying seed goose breeds, by screening specific SNP sites related to seed goose breed identification, detecting the joint genotype of the SNP sites combined with the genotype frequency of each SNP site, and using the Bayesian theorem to identify the seed goose breed.

[0170] Furthermore, the goose whole genome chip of the present invention is suitable for targeted capture sequencing technology (i.e., constructed as a liquid phase chip). Through the corresponding probe design, not only the probe target point can be typed, but also the amplification area around the probe target point can be typed. Therefore, compared with a solid phase chip with the same number of probes, more variant sites can be detected.

[0171] Furthermore, the goose whole genome chip of the present invention can realize genotyping. As a goose whole genome liquid phase chip, it provides an important tool for goose whole genome association analysis, whole genome selection, variety identification and breeding, genetic diversity assessment, germplasm resource and kinship identification, genetic map construction and gene positioning, molecular marker-assisted breeding, etc.

[0172] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0173] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0174] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0175] Example 1 Project Process

[0176] 1.1 Sample sequencing

[0177] The DNA samples were blood tissues of Wanxi white geese. After testing, amplification, fragment purification, library construction and other steps, the DNA samples were sequenced on the machine. The experimental process was strictly carried out in accordance with the standard protocol provided by the sequencing company. During the experiment, DNA sample testing mainly included the following three methods.

[0178] 1) Agarose gel electrophoresis analysis: detect the degree of DNA degradation and determine whether there is RNA contamination; 2) Nanodrop: detect the purity of DNA samples (OD 260 / OD 280 ratio); 3) Qubit: Accurately quantify DNA concentration.

[0179] Basic process: The genomic DNA is randomly broken into fragments of about 200 bp in length by a fragmentation instrument. After end repair, the sequencing adapter is connected and the probe is hybridized to obtain the target region capture library for sequencing. The flowchart of library construction is as follows Figure 1 shown.

[0180] 1.2 Information Analysis

[0181] The raw data was cleaned after quality control steps such as removing the linker and filtering the low-quality data for subsequent bioinformatics analysis. The clean data was compared with the reference genome GCA_013030995.1 for mutation detection, and the typing results of the target site were obtained from it. The specific process is as follows Figure 2 As shown:

[0182] 2. Data Quality Control

[0183] After the raw data is downloaded, it will contain short sequence fragments (reads) with adapters or low quality. Before subsequent analysis, the raw data needs to be filtered. The filtering conditions are as follows:

[0184] 1) Remove reads with adapters;

[0185] 2) When the N content in the sequencing read exceeds 10% of the base number of the read, the paired reads are removed;

[0186] 3) When the number of low-quality (Q<=5) bases in a sequencing read exceeds 50% of the number of bases in the read, the paired reads are removed.

[0187] 3. Alignment with reference genome

[0188] The clean data is aligned to the reference genome (goose) using the bwa software, and information such as the alignment rate and data duplication rate of each sample is counted to prepare for subsequent variant detection.

[0189] The sample alignment rate refers to the proportion of reads mapped to the reference genome. When the selected reference genome is appropriate and the experimental process is non-contaminating, the alignment rate is generally greater than 70%. The alignment rate is affected by multiple factors, such as the closeness of the relationship with the reference genome, the quality of the reference genome assembly, and the quality of the sequencing data.

[0190] 4 Target site analysis

[0191] According to the alignment results of Clean Reads on the reference genome, we used the HaplotypeCaller module in the GATK software to generate SNP and INDEL variant site files, extracted target sites, and then used 5X filtering to mark sites that did not meet the requirements as . / . This sequencing included a total of 30,000 sites, and the specific results are shown in Table 1.

[0192] Example 2 Application of goose whole genome liquid phase chip in genomic selection breeding - genetic evaluation

[0193] The whole genome liquid phase chip of goose provided by the present invention was used for genomic genetic evaluation. The DNA of 213 laying hens (113 F1 generation and 100 F2 generation) of three generations of Wanxi white geese was subjected to SNP genotyping. The flow chart of the parentage identification technology is as follows: Figure 3 shown.

[0194] Before analysis, Plink (V1.90) software was first used to perform quality control on the genotype data, and only the sites with the best typing quality were retained for subsequent analysis. For individuals with genotypes, Mendel's law of inheritance was used, that is, the alleles of each genetic locus were passed from parents to offspring in a Mendelian manner, and paternity testing was performed. Between the parent-offspring pairs to be tested, consisting of suspected parents and suspected offspring individuals, Mendelian error determination can be performed on each biallelic genetic locus.

[0195] Mendelian error means that an individual's allele does not come from either of its biological parents, and Mendelian error rate means the proportion of markers with Mendelian error to the total number of markers. The smaller the Mendelian error rate, the greater the probability of a true parent-child relationship.

[0196] Mendelian error analysis is performed on the individual to be tested and its candidate parents one by one, and the individual with the smallest Mendelian error is selected as its true parent. Generally speaking, the Mendelian error rate between individuals with parent-child relationships is less than 1%. If the Mendelian error rate of the candidate parents is above 1%, it is considered that their true parent is not in the candidate list.

[0197] Repeat sample test results: Repeat sample calculation method: the number of inconsistent sites between two individuals / the total number of comparisons between two individuals < threshold (default 0.01)

[0198] Among them: The number of inconsistent sites between the two individuals: For the same site, if both individuals are not missing at this site and the genotypes are inconsistent (for example, one is AA and the other is AT), it is considered an inconsistent site; The total number of comparisons between the two individuals: The total number of sites that are not missing in both individuals.

[0199] The results of principal component analysis of the analyzed samples show that the detection results of the goose liquid phase chip of the present invention for geese genome genetic evaluation are consistent with the original parent relationship. This also illustrates the applicability of the chip of the present invention in the family construction and pedigree combing of the Wanxi white goose population, and its identification results are accurate and reliable.

[0200] Example 3 Application of liquid phase chip in goose breed identification

[0201] In order to identify whether the sample to be tested is Wanxi white goose, blood samples of Wanxi white goose to be tested and Guangdong local geese (Lionhead goose, Magang goose, Wuzong goose, Yangjiang goose and Yangchun white goose) are collected respectively, and the goose 30K liquid phase chip of the present invention is used for genotyping, and then the SNP sets obtained by genotyping are subjected to principal component analysis and phylogenetic tree construction respectively. If the sample to be tested is clustered with Wanxi white goose in the principal component analysis result, and is clustered into a branch with Wanxi white goose in the phylogenetic tree, then the sample to be tested is determined to be Wanxi white goose, otherwise it is not a Wanxi white goose breed.

[0202] Chip advantages: excellent detection performance, blind sample test results show that the chip site detection rate is 96.5%, and the accuracy of variety identification results is more than 99.9%. High flexibility, high cost performance, and variety-specific sites can be appropriately added / deleted according to demand in the later stage to further meet different product application scenarios. Based on the localized design, detection and analysis process, the typing cost is greatly reduced; the automated detection process is adopted to improve the detection throughput and stability.

[0203] Example 4 Application of liquid phase chip in identifying SNP sites of excellent genetic traits of Wanxi white geese

[0204] The liquid phase chip of the present invention is a 30K targeted capture chip, which uses targeted probes to accurately capture the target site area, realizes sequencing of the target site, and ensures the accuracy of genotyping.

[0205] The 30K liquid phase chip of the present invention was used to perform genome-wide association analysis to obtain genotype data and phenotypic data. The genotype data quality control was performed to eliminate low-quality SNP sites and samples that did not meet the analysis standards, and missing data were processed to perform phenotypic descriptive statistics, normality tests, and Hardy-Weinberg equilibrium tests. And through population structure analysis and genome-wide association analysis, SNP sites significantly associated with five types of traits such as goose growth rate, weight, body size, meat quality, and reproductive egg production were obtained.

[0206] The Hardy-Weinberg equilibrium test is a statistical method used to assess whether genotype frequencies in population genetics conform to an expected equilibrium state.

[0207] The molecular markers of the breeding Wanxi white geese are tested, and the individuals with excellent genotypes obtained by the test are selected as breeding geese, and the male and female breeding geese are bred; the molecular markers of the geese born from the breeding are tested, and the genotypes with excellent alleles are detected for breeding; the individuals with excellent genotypes are retained, and the individuals with other genotypes are eliminated for breeding, so as to cultivate a high-production performance Wanxi white goose strain with fast growth rate, heavy weight, excellent body size indicators, good meat quality, and high breeding and egg production.

[0208] Chip advantages: The research and development of this chip has broken through the technical bottleneck of the small number and limited coverage of traditional molecular markers. It has the characteristics of high accuracy, comprehensive coverage of 30K targeted capture genome, low cost and good effect. It can be used for precise genetic analysis of local goose breed characteristics (growth rate, weight, body size, meat quality, reproduction and egg production, etc.), which has far-reaching significance for the protection and utilization of local goose resources.

[0209] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A goose whole genome SNP chip, characterized in that: The chip consists of 30,000 SNP molecular markers, and the chromosome positions and numbers of the SNP molecular markers on the goose reference genome GCA_013030995.1 are shown in Table 1 of the specification.

2. The SNP chip according to claim 1, characterized in that: The SNP chip is a liquid phase chip.

3. The SNP chip according to claim 1, characterized in that: The number of detection samples of the chip is greater than 1.

4. The goose whole genome SNP chip according to claim 1, characterized in that: The molecular marker is associated with goose disease resistance traits or goose economic traits.

5. The property according to claim 4, characterized in that: The disease resistance traits of the goose include gosling plague, goose influenza, goose Escherichia coli and fowl cholera; the economic traits of the goose include growth rate, body weight and body size indicators and egg production quality.

6. Use of the goose whole genome SNP chip according to claim 1 in the identification of goose breeds or genetic relationships.

7. Use of the goose whole genome SNP chip according to claim 1 in improving goose breed quality and provenance.

8. Use of the goose whole genome SNP chip according to claim 1 in goose breeding.

9. Use of the goose whole genome SNP chip according to claim 1 in genetic diversity analysis of goose populations.

10. Use of the goose whole genome SNP chip according to claim 1 in QTL location analysis of goose target traits.

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