A 30K liquid-phase breeding chip based on the whole genome of sea cucumber and its application

Abstract

The present invention relates to the fields of genomics, molecular biology, bioinformatics and whole genome selective breeding technology, and specifically to a 30K liquid phase breeding chip of a sea cucumber whole genome based on the precise positioning sequencing typing technology (Genotyping by Pinpoint Sequencing of captured targets, cGPS) of the target region genome sequence liquid phase capture and its application. The SNP molecular marker combination is any combination of 1-30781 SNP sites. The chip can be applied to the genetic diversity assessment of sea cucumbers, the identification of germplasm resources and kinship, the whole genome association analysis of important economic traits and the whole genome selective breeding; it has the advantages of high throughput, high interval coverage, high site detection rate, high flexibility, etc., and provides a powerful tool support for the molecular breeding of sea cucumbers.

Description

Technical Field

[0001] The present invention relates to the fields of genomics, molecular biology, bioinformatics and whole genome selection breeding technology, and specifically to a sea cucumber whole genome 30K liquid phase breeding chip based on the precise positioning sequencing typing technology (Genotyping by Pinpoint Sequencing of captured targets, cGPS) of target region genome sequences captured by liquid phase and its application. Background Art

[0002] As an important aquatic economic species of the genus Apostichopus in the order Synallactida, class Holothuroideaia, phylum Echinodermata, Apostichopus japonicus has led the fifth wave of marine aquaculture in my country, with an unprecedentedly fast industrial development speed and high economic benefits. However, with the expansion of the scale of the aquaculture industry, problems such as germplasm degradation, slow growth, frequent diseases, and poor ability to resist environmental changes have become increasingly prominent. Currently, there are only 8 new varieties of apostichopus japonicus in China, and the lack of original and improved varieties has restricted the sustainable and healthy development of the apostichopus japonicus industry.

[0003] Genetic markers are special identifiable and heritable manifestations of the genotype of an organism and are an indispensable tool for studying biological inheritance and evolution. Traditional genetic markers include morphological markers, cytological markers, and biochemical markers. These three types of markers indirectly reflect the variation of genetic material based on the results of gene expression. They are generally dominant and cannot reveal the variation information of recessive traits. Molecular markers represented by single nucleotide polymorphisms (SNPs) are usually co-dominantly inherited. Their number is almost distributed throughout the entire genome and they are highly polymorphic. They are currently the most widely used and most effective marker type.

[0004] The molecular marker detection platform has been updated and iterated with the rapid development of genetic marker types and detection equipment, and has gone through three development stages: gel electrophoresis, fluorescent gel electrophoresis, and gene chips. The current mainstream high-throughput genotyping platforms include two types: second-generation sequencing technology and gene chips. Gene chips can be divided into two types: solid phase and liquid phase. Liquid phase chip technology was developed in the mid-1990s and is known as the chip technology of the post-genomic era. It is one of the most popular high-throughput genotyping platforms. Genotyping by Pinpoint Sequencing of captured targets (cGPS) based on liquid phase capture of target interval genomic sequences is a targeted sequencing genotyping technology independently developed by Huazhi Bio. The principle is to use the optimized thermodynamic stability algorithm model to design characteristic probes for the target interval sequence, use synthetic specific probes to perform liquid phase hybridization capture and enrichment of multiple different target sequences located at different genomic positions, and then perform second-generation sequencing on the captured and enriched target intervals to obtain the genotypes of all SNP / InDel sites in the target interval.

[0005] The working principle of solid-phase chips is based on solid-phase hybridization between probes and DNA sequences. It relies on expensive foreign instruments and equipment, has a limited number of detectable molecular markers, and cannot detect haplotypes in intervals. However, liquid-phase chips can detect all genetic variations in tens of thousands of target intervals at a time, and the genotype detection rate and accuracy can usually reach more than 99%. The number of detection sites can be increased as needed. At the same time, it has autonomy and does not rely on expensive foreign experimental equipment. It has advantages that solid-phase chips cannot match, such as high information content, high accuracy, high detection rate, and high site flexibility. It has broad application prospects in many fields of life science research. Summary of the invention

[0006] The purpose of the present invention is to provide a 30K liquid-phase breeding chip imitating the whole genome of sea cucumber and its application.

[0007] To achieve the above purpose, the present invention adopts the following technical solution:

[0008] A SNP molecular marker combination for whole genome selection of sea cucumbers, wherein the SNP molecular marker combination is any combination of 1-30781 SNP sites; the 1-30781 SNP sites are shown in the following table.

[0009]

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043] The molecular markers are associated with the economic traits of the sea cucumber, and the economic traits are weight (Weight) and specific growth rate (SGR) related to growth traits; the number of thorns related to the thorny trait; and the skinning rate related to the quality trait.

[0044] An application of the SNP molecular marker combination according to claim 1, wherein the SNP molecular marker combination is used in sea cucumber breeding.

[0045] A 30K liquid-phase breeding chip of the whole genome of sea cucumber, comprising 30,781 SNP sites.

[0046] The method for preparing a sea cucumber whole genome liquid phase breeding chip comprises the following steps:

[0047] 1) Screening high-quality SNP sites from the resequencing data of sea cucumber;

[0048] 2) Screening SNP loci associated with growth, spininess and quality traits from genome-wide association analysis data of sea cucumber;

[0049] 3) Remove the SNP sites that cannot design probes at both ends from the above sites;

[0050] 4) Search for SNP intervals longer than 2000 bp and add SNPs that may cover important genes, repeating multiple times until no sites can be filled;

[0051] 5) The above SNP loci are combined to finally obtain the sea cucumber whole genome liquid phase breeding chip.

[0052] The above growth, thorniness and quality traits are related to the important economic traits of sea cucumbers, namely, weight and specific growth rate (SGR) related to growth traits; the number of thorns related to thorniness traits; and the skinning rate related to quality traits.

[0053] Application of a sea cucumber whole genome liquid phase breeding chip in detecting sea cucumber DNA samples.

[0054] Specifically:

[0055] (1) Extraction of genomic DNA from sea cucumbers: Genomic DNA was extracted from the body wall tissue of sea cucumbers using the CTAB method according to standard procedures;

[0056] (2) DNA quality detection: Detection by 1% agarose gel electrophoresis; use NanoDrop to detect genomic DNA concentration to ensure that the DNA concentration is ≥10 ng / μL, 1.8≤OD260 / OD280≤2.0, OD260 / OD230≥2.0; use gel imaging to detect the integrity of the DNA sample to ensure that the main band of the sample is clear and the integrity is good;

[0057] (3) Liquid chip detection: The sample is tested using the liquid chip to obtain the genotype of the sample to be tested.

[0058] Compared with other genotyping platforms, the present invention has the following advantages and effects: (1) Screening characteristic sites of sea cucumber populations in multiple sea areas in China, the site information has universality and effectiveness; (2) Screening sites that are significantly associated with important economic traits such as growth and quality, and have functional relevance of economic traits; (3) Screening sites according to the principle of uniform distribution in the genome, with an average spacing of 21.3kb, ensuring the accuracy of breeding value evaluation; (4) Compared with solid-phase chips, the above chip has high information content, high site detection rate, and high site flexibility, and does not need to be restricted by expensive foreign instruments and equipment; (5) Compared with the genotyping platform based on second-generation sequencing, the above chip detects fixed variant sites, can realize standardized automated detection and analysis, and data from different batches are comparable. In general, liquid phase chip technology has a wider application prospect in many fields of life science research. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Schematic diagram of the distribution of 30k sites of the "Shenxin No. 1" whole genome liquid phase breeding chip on the genome provided by the embodiment of the present invention

[0060] Figure 2 The site detection rate of the "Shenxin No. 1" whole genome liquid phase breeding chip provided in the embodiment of the present invention

[0061] Figure 3 The site consistency rate of the "Shenxin No. 1" whole genome liquid phase breeding chip provided in the embodiment of the present invention DETAILED DESCRIPTION

[0062] The following examples are used to illustrate the content of the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, the modification or replacement of the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0063] The technical solution of the present invention is to promote the innovation of sea cucumber germplasm and the breeding of improved varieties, and is assisted in verification by Huazhi Biotechnology Co., Ltd. The company uses liquid phase capture of the target region genome sequence in the chip designed by the applicant to accurately locate and sequence the genes to meet the needs of whole genome improved variety breeding of sea cucumber.

[0064] Example 1 Preparation method of the 1-cell whole genome liquid phase breeding chip

[0065] (1) Full genome acquisition:

[0066] The muscle tissue of a sea cucumber was selected, DNA was extracted, and 56.64G data was obtained by second-generation illunima sequencing after library construction, and 26.55G Hifi data was obtained by sequencing on the PacBio platform. Hifiasm was used to quickly construct haplotypes of PacBio Hifi reads data-de novo assembly, and the second-generation data was used for correction. Finally, a genome with a total length of 670Mb was constructed, and contigN50 reached 9.13Mbp (Marine Science Big Data Center, DOI code 10.12157 / IOCAS.20230105.001).

[0067] (2) Obtaining the first type of loci: Based on the whole genome resequencing technology, 30,410 SNP loci were screened from the resequencing data of the sea cucumber population and were evenly distributed in the genome, including 22,784 SNP loci from four sea cucumber families in Weihai and 7,626 SNP loci from wild sea cucumber populations in eight different sea areas in China (see Table 2).

[0068] Specifically, in 2018, four pairs of sea cucumbers with multi-thorny and fast-growing traits were selected from Rushan, Weihai as parents to construct four full-sib families. After being cultured to 30 months of age, they were used as experimental materials. The Illumina Nova 6000 platform was used to perform whole genome resequencing on 298 sea cucumbers from the four families (see Table 1). The total sequencing volume was 2962.48G, and the average sequencing depth of each sample was about 10×. The specific process is to construct a sequencing library for each sample, and all DNA libraries are sequenced on the IlluminaNova 6000 platform. 1) Low-quality reads (the number of bases with a quality value of Q≤5 accounts for more than 50% of the entire read); 2) Reads containing N (N means that the base information cannot be determined) with a ratio greater than 5%; 3) Reads with adapter contamination were filtered out, and finally 2586.98G clean data was obtained. All clean data were aligned to the above-mentioned sea cucumber reference genome sequence using BWA software, variant site detection and typing were performed using GATK software, and the retained variant sites were annotated using snpEff software, and 57,724,642 SNP sites were finally screened. SNP sites that could not design high-quality DNA probes on both sides of the above SNP sites were removed, and SNP sites were further screened based on the principle of uniform distribution on the genome, and a total of 22,784 high-quality SNP sites were finally obtained.

[0069] Table 1 Resequencing and SNP numbers of four sea cucumber families

[0070]

[0071] The whole genome of 145 wild sea cucumbers (see Table 2) from eight different sea areas in China was resequenced using the Illumina PE 150 platform, with a total sequencing volume of 1647.2G and an average sequencing depth of 10× for each sample. The specific process is to construct a sequencing library for each sample, sequence all DNA libraries on the Illumina PE 150 platform, filter out low-quality and adapter-contaminated reads, and finally obtain 1611.761G clean data. 1) Low-quality reads (the number of bases with a quality value Q≤5 accounts for more than 50% of the entire read) were filtered out; 2) reads containing N (N means that the base information cannot be determined) with a ratio greater than 5% were removed; 3) reads with adapter contamination were used, and all clean data were aligned to the sea cucumber reference genome sequence using BWA software, and variant site detection and typing were performed using GATK software. The retained variant sites were annotated using snpEff software, and 38,749,645 SNP sites were finally screened. SNP sites that could not design high-quality DNA probes on both sides were removed from the above SNP sites, and SNP sites were further screened based on the principle of uniform distribution on the genome, and finally a total of 7626 high-quality SNP sites were obtained.

[0072] Table 2 Resequencing results of eight wild sea cucumber populations

[0073]

[0074] (2) Obtaining the second type of loci: Based on genome-wide association analysis and SNP screening within related candidate genes, 73 SNP loci associated with the target economic traits were obtained.

[0075] Specifically, the four sea cucumber families F in Rushan, Weihai were used. 1 The phenotypic determination method is a well-known method, using an electronic balance to weigh the body weight, manually counting the number of spines, and calculating the peeling rate and specific growth rate (SGR) of the sea cucumber according to the following formula. 2 / W 1 ×100%; SGR(%d -1 )=100×[ln(W 1 )-ln(W 0 )] / T(W 0 , initial body weight; W 1 , final body weight; W 2, body wall weight; T, experimental period; ln, natural logarithm). After quality control, 57,724,642 SNP sites remained in the whole genome resequencing. The population structure Q matrix and kinship K matrix were calculated based on the obtained SNP sites, and the results were corrected according to the "Q+K" model. The EMMAX model was used to perform association analysis between phenotypic data and SNPs. After the Bonferroni method corrected the p-value, 73 SNP sites significantly associated with the target traits were obtained.

[0076] The process of liquid phase chip site screening is as follows:

[0077] (1) The first and second category sites were sent to Huazhi Biotechnology Co., Ltd. to remove sites where probes could not be designed (the specific conditions for probe design are: 1) the probe length is generally between 80-150bp; 2) the probes are arranged in a tiling manner; 3) the GC content of the probe sequence is about 50%; 4) some areas are not suitable for probe design, and probes can be designed around them.);

[0078] (2) After the selection of the first and second categories of sites is completed, for the gap regions that still lack site coverage, the third category of site data is obtained by screening from the sea cucumber reference genome.

[0079] Finally, 30781 SNP sites were evenly distributed in the whole genome (see Table 3 and the 1-30781 SNP site table, Figure 1 ).

[0080] Table 3 Distribution of 30k sites on each chromosome in liquid phase microarray

[0081]

[0082] Example 2 Application of the Shenxin No. 1 Whole Genome Liquid Phase Breeding Chip in Detecting Sea Cucumber DNA Samples

[0083] The development and verification was carried out using the "Shenxin No. 1" sea cucumber liquid phase breeding chip:

[0084] (1) Extraction of genomic DNA from sea cucumbers: The body wall tissues of sea cucumbers were dissected and sampled according to standard procedures, and genomic DNA was extracted using the CTAB method.

[0085] (2) DNA sample quality detection: Detection by 1% agarose gel electrophoresis; Use UV spectrophotometer to detect genomic DNA concentration to ensure that DNA concentration is ≥100 ng / μL, 1.8≤OD260 / OD280≤2.0, OD260 / OD230≥2.0; Use gel imaging to detect DNA sample integrity to ensure that the main band of the sample is clear and the integrity is good;

[0086] (3) Liquid phase chip testing: Through the standardized process operation and quality control of Huazhi Company, the samples are subjected to library construction, target region capture and high-throughput sequencing. (4) Bioinformatics analysis: Freebayes software is used to analyze the variant sites of the sequencing results to obtain the genotype of the target site; IGV software is used to view the variation between the sequencing results and the reference genome.

[0087] Among the 96 samples tested, the average detection rate of all loci was 98.38%, and the average consistency rate with the resequencing genotype was 89.63%. The above data show that the cGPS test results meet the development standards (see Figure 2 and Figure 3 ).

[0088] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A 30K liquid-phase breeding chip based on the whole genome of sea cucumber, characterized in that: The chip includes probes for detecting the SNP molecular marker combination of sea cucumber, and the SNP molecular marker combination consists of 30781 SNP sites, and the 30781 SNP sites are shown in the following table:

2. An application of the sea cucumber whole genome 30K liquid phase breeding chip according to claim 1 in sea cucumber breeding.

3. A method for preparing the sea cucumber whole genome 30K liquid phase breeding chip according to claim 1, characterized in that: The following steps are involved: 1) Screening high-quality SNP sites from the resequencing data of sea cucumber; 2) Screening SNP loci associated with growth, spininess and quality traits from genome-wide association analysis data of sea cucumber; 3) Remove the SNP sites that cannot design probes at both ends from the above sites; 4) Search for SNP intervals longer than 2000 bp and add SNPs that may cover important genes, repeating multiple times until no sites can be filled; 5) The above SNP loci are combined to finally obtain the sea cucumber whole genome liquid phase breeding chip.

4. An application of the sea cucumber whole genome 30K liquid phase breeding chip according to claim 1 in detecting sea cucumber DNA samples.

Images