A whole genome 20k liquid breeding chip for apostichopus japonicus and application thereof

Abstract

This invention relates to the fields of genomics, molecular biology, bioinformatics, and genome-wide selection breeding, specifically a 20k liquid-phase breeding chip for the whole genome of *S. esculenta* and its applications. The liquid-phase chip contains background SNPs and functional SNPs located on the *S. esculenta* reference genome; wherein the background SNPs are uniformly distributed within the genome; and the functional SNPs are associated with important economic traits of *S. esculenta*; these important economic traits include one or more of the following: saponin content, polysaccharide content, and heat tolerance. The chip can be applied to the assessment of genetic diversity in *S. esculenta*, identification of germplasm resources and phylogenetic relationships, genome-wide association analysis of important economic traits, and genome-wide selection breeding. This chip has advantages such as high throughput, high region coverage, high locus detection rate, and high flexibility, providing powerful tool support for molecular breeding of *S. esculenta*.

Description

Technical Field

[0001] This invention relates to the fields of genomics, molecular biology, bioinformatics, and whole-genome selection breeding technology, specifically a 20k liquid phase breeding chip for sea cucumber whole genome and its application. Background Technology

[0002] sea ​​cucumber (imitation sea cucumber) Apostichopus japonicus It belongs to the phylum Echinodermata, class Holothuroidea, order Aspidochirotida, family Stichopodidae, genus Stichopodida. Apostichopus The sea cucumber (also known as the spiny sea cucumber) is rich in functional components such as polysaccharides, saponins, and collagen. It has the effects of tonifying the kidneys and replenishing essence, lowering blood sugar and lipids, nourishing blood, antibacterial, and anti-tumor. It has extremely high nutritional and medicinal value and is the most economically valuable and promising species among sea cucumbers.

[0003] Among the numerous nutrients in sea cucumber, saponins are the main secondary metabolites, possessing pharmacological activities such as antifungal, antiviral, antitumor, and immunomodulatory effects, and are an important material basis for the medicinal value of sea cucumber. Polysaccharides, on the other hand, have effects such as anticoagulation, lowering blood lipids, delaying aging, and enhancing immunity, directly determining the nutritional quality and economic value of sea cucumber. However, current breeding of sea cucumber farmed varieties mainly focuses on phenotypic traits such as growth rate, body color, and number of spines, with insufficient attention paid to the genetic selection of functional nutritional quality traits such as saponins and polysaccharides. This has led to a scarcity of high-nutritional-value germplasm resources, hindering the high-quality development of the sea cucumber "medicinal and edible homology" industry.

[0004] Furthermore, heat tolerance has become a key limiting factor affecting the stability and sustainability of the sea cucumber farming industry. Sea cucumbers are cold-water organisms, with an optimal growth temperature of 10–20℃. In recent years, global warming has led to frequent periods of sustained high temperatures in summer, causing widespread problems such as skin peeling, evisceration, and even death among farmed sea cucumbers, resulting in significant economic losses. Developing new heat-resistant sea cucumber varieties is an urgent need to fundamentally solve the problem of high-temperature disasters and ensure the safety of the industry.

[0005] In recent years, with the development of genomics and molecular marker technology, breeding chips have become a core tool for genomic selection breeding, mainly including two technical routes: solid-phase chips and liquid-phase chips. Among them, liquid-phase chips have been widely used in non-model organism breeding due to their advantages such as flexible design, relatively low cost, and ease of customization. Currently, 24K, 30K, and 50K liquid-phase breeding chips have been successfully developed for sea cucumbers and have been initially applied to the genetic analysis and breeding practices of phenotypic traits such as growth and stress resistance. However, the site design of existing sea cucumber breeding chips mainly focuses on conventional economic traits, and the molecular marker coverage for functional nutritional quality traits such as saponins and polysaccharides, as well as complex stress resistance traits such as heat resistance, is insufficient. A systematic dedicated chip resource has not yet been formed, which limits the in-depth development of multi-trait synergistic breeding of sea cucumbers.

[0006] Currently, only nine new sea cucumber varieties have been approved by the state. Breeding objectives primarily focus on phenotypic traits such as growth and stress resistance, while molecular breeding chips targeting key functional nutritional qualities like saponins and polysaccharides, as well as heat resistance and stress tolerance, are still lacking. The reasons for this are mainly as follows: First, the genetic regulatory mechanisms of quality traits such as saponins and polysaccharides are not yet clear, and the discovery of related synthetic pathways and key functional genes is insufficient, resulting in a lack of reliable SNP loci for chip design. Second, phenotypic determination of these traits relies on complex chemical analysis methods such as liquid chromatography, which are costly and have low throughput, making it difficult to accumulate phenotypic data from large-scale breeding populations for genome-wide association studies. Third, heat resistance and stress tolerance are controlled by multiple gene-environment interactions, resulting in a complex genetic basis, weak single-marker effects, and differences in stress resistance mechanisms among different geographical populations, leading to poor locus universality. Fourth, the existing sea cucumber breeding industry is limited in scale, with insufficient market driving force and relatively low investment in the research and development of dedicated chips. The aforementioned factors have collectively led to a severe lack of molecular breeding tools in this field, significantly hindering the breeding process of high-quality, high-yield, highly resistant, and highly nutritious sea cucumber varieties. Therefore, how to fully utilize existing liquid-phase breeding chip platforms for sea cucumbers, systematically mine molecular markers related to functional qualities and heat resistance traits, and establish efficient genomic selection methods has become an urgent technical problem to be solved. Summary of the Invention

[0007] The purpose of this invention is to provide a 20k liquid phase breeding chip for the whole genome of sea cucumber and its application.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A 20k liquid-phase breeding chip for the whole genome of *Astrophytum asterias* is disclosed. The liquid-phase chip contains background SNP sites and functional SNP sites located on the reference genome of *Astrophytum asterias*. The background SNP sites are sites that are uniformly distributed within the genome. The functional SNP sites are associated with important economic traits of *Astrophytum asterias*. The important economic traits include one or more of the following: saponin content, polysaccharide content, and heat resistance.

[0009] The background SNP sites are any one or more combinations of the 19,718 SNP sites evenly distributed in the genome of *Astrophytum asterias*; the functional SNP sites are any one or more combinations of the 408 SNP sites.

[0010] The 408 SNP sites were significantly associated with at least one of the traits of saponin content, polysaccharide content, and heat resistance, and the significance of the associations satisfied -log10(p-value)>5.

[0011] The functional SNP sites include any one or more combinations of extremely important functional SNP sites from the 325 SNP sites.

[0012] An application of the aforementioned 20k liquid phase breeding chip for the whole genome of sea cucumber, wherein the 20k liquid phase breeding chip for the whole genome of sea cucumber is used in the breeding of sea cucumber.

[0013] The 20k liquid phase breeding chip for the whole genome of *Stichopus japonicus* is used for gene mapping, genetic diversity analysis, germplasm resource improvement, germplasm resource protection, genome-wide association analysis, genome-wide selection breeding, or kinship identification of important economic traits (saponins, polysaccharides, heat resistance) of *Stichopus japonicus*.

[0014] Advantages of the present invention This invention utilizes whole-genome resequencing and genome-wide association analysis to screen for 20,000 high-quality SNP loci, including 19,718 high-quality background SNP loci evenly distributed throughout the genome, and 408 functional SNP loci significantly associated with saponins, polysaccharides, and heat resistance traits (of which 325 are highly significantly associated important functional SNP loci). Based on these loci, a liquid-phase breeding chip probe for sea cucumber-like genomes was directly synthesized. The chip provided by this invention has advantages such as high throughput, high region coverage, high locus detection rate, and high flexibility. It is the first to systematically incorporate functional nutritional quality traits such as saponins and polysaccharides, as well as heat resistance and stress tolerance traits, into a sea cucumber breeding chip, filling the current gap in the industry for molecular breeding chips targeting nutritional quality and heat resistance traits, and providing powerful tool support for molecular breeding of sea cucumber-like organisms. Attached Figure Description

[0015] Figure 1 The distribution of SNP sites in the genome provided in the liquid-phase chip of this invention. Detailed Implementation

[0016] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0017] Example 1: Site screening and chip fabrication of sea cucumber-like liquid-phase microarray 1. Construction of experimental populations for functional nutritional quality traits Based on previous surveys of saponin content in sea cucumbers in the Yellow and Bohai Seas, the Penglai Nanhuangcheng population, which has significantly enriched saponin content, was selected as the breeding parent to construct a high-saponin G1 generation sea cucumber strain. The Laizhou population, which has the highest polysaccharide content, was used as the parent to construct a high-polysaccharide G1 generation sea cucumber strain.

[0018] Two g of each of the randomly selected high-saponin G1 generation sea cucumber tissues were freeze-dried and extracted by reflux with 40 ml of ethanol. After evaporation, the extract was further extracted with 20 ml of water and 20 ml of n-butanol. The crude extract was then obtained by evaporation. The crude extracts of each strain were dissolved in 2 ml of trifluoroacetic acid, incubated at 120°C for 2 h, and then extracted with chloroform. This process was repeated three times to obtain a solution. PMP and NaOH were added to the solution in a 1:1 ratio, and the mixture was incubated in a water bath at 70°C for 30 min. After cooling, HCl was added to obtain a mixed solution. The mixed solution was extracted with chloroform three times, and then filtered through a 0.45 μm filter membrane. The filtrate was used for high-performance liquid chromatography (HPLC) analysis. The saponin content was calculated based on the peak area (Table 1).

[0019] Weigh 2 g of each of the randomly selected high-polysaccharide G1 generation sea cucumber tissues, freeze-dried, and mix with 5 ml of distilled water to form a homogenate. Add the enzyme mixture, mix well, and then enzymatically hydrolyze at 60°C for 1 h. Cool to room temperature. Take an appropriate amount of the enzymatic hydrolysate in a test tube, add water to a volume of 0.1 mL, then add 2.5 mL of colorimetric solution and shake well. After 15 min, measure the absorbance at 500 nm using a 1 cm cuvette. Calculate the polysaccharide content based on the absorbance (see Table 1).

[0020] Table 1. Descriptive statistics of functional nutritional quality traits of sea cucumber

[0021] 2. Construction of experimental populations for high-temperature resistance A total of 145 sea cucumber mutants were collected from eight regions in the Yellow and Bohai Seas of my country, including Dalian Bangchui Island, Dalian Lushun, Yantai Nanhuangcheng, Yantai Danzi Island, Yantai Laizhou, Weihai Liugong Island, Rizhao, and Qingdao Lingshan Island, to serve as a heat-resistant control population. The heat-resistant variety "Dongke No. 1" (GS-01-015-2017) was bred through population selection to obtain 230 individuals of the 7th generation (G7 generation) as a heat-resistant population.

[0022] 3. Whole-genome resequencing and association analysis of sea cucumber (Amorphophallus spp.) Genomic DNA was extracted from sea cucumber body wall tissue using the CTAB method. DNA integrity was assessed by 1% agarose gel electrophoresis, and concentration was determined using a Qubit v2.0 quantitative PCR system (Thermo Fisher Scientific, USA). DNA libraries were constructed following standard procedures: DNA fragment end repair, addition of poly-A tails, ligation of Illumina sequencing adapters, and library enrichment by PCR amplification. Sequencing platform: Illumina Novaseq X Plus (Illumina, CA, USA). Sequencing strategy: Paired-end 150 bp (PE150) sequencing. Sequencing depths were 10 × for quality traits and 15 × for heat resistance traits. The sequencing was performed by Novogene (Tianjin). Raw sequencing data underwent rigorous quality control, including removal of reads containing adapter sequences, low-quality sequences (quality value Q < 20), and reads containing unknown bases (N > 5%).

[0023] The high-quality sequencing data after quality control were aligned to the sea cucumber reference genome (https: / / figshare.com / articles / dataset / The_genome_annotation_files_of_Apostichopus_japonicus / 22140020) using BWA v0.7.13. Subsequently, SNP calling was performed using the mpileup module of SAMTOOLS (v1.16.1) (parameters: -q 1 -C 50 -t SP -t DP -m 2 -F 0.002). Quality control was performed using Plink v1.90: SNPs with a coverage depth ≥ 3 were retained; SNPs with a minor allele frequency (MAF) < 5% were removed; individuals with a missing sample rate > 10% were excluded; and the significance level of the Hardy-Weinberg equilibrium test was removed. p <1×10 -5 SNPs; SNPs with a genotyping detection rate of <90%.

[0024] Using Gapit software, a mixed linear model (MLM) was used to perform association analysis between high-quality SNP loci after quality control and phenotypic data such as nutritional quality and heat tolerance. To correct for population stratification, principal component analysis results were used as covariates, and the kinship matrix was used as a random effect. A genome-wide significance threshold of -log was established. 10 ( p The -value was set to an empirical value of 5, and the Manhattan plot of the correlation data was plotted using the R function "CMplot". SNP sites that reached a significant correlation were extracted, and 408 SNP sites associated with the nutritional quality and heat resistance of sea cucumber were selected.

[0025] 4. Probe Design and Synthesis Probe design is based on the principle of thermodynamic stability, comprehensively considering factors such as target site location and GC content near the target site to ensure 100% capture in conventional regions. For complex regions with high difficulty, a multi-layer "shingled" probe placement scheme is used to improve the effective coverage of these regions by adjusting probe positions. Probe preparation includes template synthesis and probe preparation. The synthesized DNA template (Oligo Pool) undergoes quality control and in vitro amplification experiments to finally prepare the finished DNA probes. The probe length range is 80-120 bp, with an overall average probe length of around 100 bp. Biotin-labeled NTPs and nucleotide analogs are used as synthetic raw materials in DNA probe preparation. Nucleotide analogs can increase the Tm value of probe-liquid binding, making the binding between probe and library more stable. At the same time, the biotin label on the synthesized probes can bind to streptavidin-coated magnetic beads in subsequent experiments. Finally, 20k SNP sites are evenly distributed throughout the genome. Figure 1 (Tables 2 and 3).

[0026] Table 2 Functional sites of the sea cucumber-inspired liquid-phase chip

[0027] Table 3 Background sites of the sea cucumber-like microarray

[0028] Example 2: Performance Testing of Sea Cucumber Imitation Liquid Chip Eleven sea cucumbers were randomly selected, and DNA was extracted according to the method described in Example 1. After quality testing by 1% agarose gel electrophoresis, the liquid-phase microarray constructed in Example 1 was used for library construction, target region capture, and high-throughput sequencing. The main objective of this example was to detect the genotyping detection rate of the constructed liquid-phase microarray. Statistical analysis of the microarray site detection showed that the detection rate was generally above 99% (Table 4).

[0029] Table 4. Detection rate of microarray sites at different sequencing depths

[0030] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A liquid-phase breeding chip for mimicking the whole genome of sea cucumber (Amorphophallus spp.) at 20kbps, characterized in that: The liquid-phase chip contains background SNP sites and functional SNP sites located on the reference genome of *Astrophytum asterias*. The background SNP sites are sites that are uniformly distributed within the genome. The functional SNP sites are associated with important economic traits of *Astrophytum asterias*. The important economic traits include one or more of the following: saponin content, polysaccharide content, and heat resistance.

2. The 20k liquid-phase breeding chip for the whole genome of the sea cucumber as described in claim 1, characterized in that: The background SNP sites are any one or more combinations of the 19,718 SNP sites evenly distributed in the genome of *Astrophytum asterias*; the functional SNP sites are any one or more combinations of the 408 SNP sites.

3. The 20k liquid-phase breeding chip for the whole genome of sea cucumber as described in claim 1 or 2, characterized in that: The 408 SNP sites were significantly associated with at least one of the traits of saponin content, polysaccharide content, and heat resistance, and the significance of the associations satisfied -log10(p-value)>5.

4. The 20k liquid-phase breeding chip for the whole genome of sea cucumber as described in claim 3, characterized in that: The functional SNP sites include important functional SNP sites that are any one or more combinations of 325 SNP sites.

5. The application of the 20k liquid phase breeding chip for the whole genome of the sea cucumber as described in claim 1, characterized in that: The 20k liquid phase breeding chip for the whole genome of the sea cucumber is used in the breeding of sea cucumbers.

6. The application according to claim 5, characterized in that: The 20k liquid phase breeding chip for the whole genome of *Astrophytum asterias* is used for gene mapping, genetic diversity analysis, germplasm resource improvement, germplasm resource protection, genome-wide association analysis, genome-wide selection breeding, or kinship identification of important economic traits of *Astrophytum asterias*.

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