Ginseng 40K SNP (Single Nucleotide Polymorphism) liquid phase breeding chip and application thereof

Abstract

The invention belongs to the technical field of plant biotechnology and molecular breeding, and relates to a ginseng 40K SNP liquid phase breeding chip and application thereof, the chip comprises 41684 SNP sites which are positioned on 24 chromosomes of a ginseng T2T reference genome (v2024), and the ginseng 40K SNP liquid phase breeding chip has the advantages of uniform site distribution, good representativeness, strong specificity and high polymorphism. The application range of the chip covers genetic diversity analysis of ginseng germplasm resources, population structure analysis, heredity and evolution analysis, genetic relationship identification, whole genome association analysis, genome selection and other molecular breeding related researches, the chip is developed based on a high-throughput sequencing technology, the detection throughput is high, detection of nearly thousands of materials can be covered at the same time, and the application range is wide. The method provided by the invention is suitable for mainstream second-generation sequencing platforms such as Illu mina and MGI, is simple, convenient and rapid to operate, provides key technical support for genetic resource innovation, excellent gene mining and precise molecular breeding of the ginseng, assists in cultivation of high-quality, high-yield and stress-resistant new varieties of the ginseng, and improves the core competitiveness of the ginseng industry.

Description

Technical Field

[0001] This invention relates to the fields of plant biotechnology and plant molecular breeding technology, specifically to a ginseng 40K SNP liquid phase breeding chip and its application. Background Technology

[0002] Ginseng (Panax ginseng CAMey.), a perennial herb belonging to the genus Panax in the family Araliaceae, is a traditional and precious Chinese medicinal herb. It possesses significant effects such as replenishing vital energy, restoring pulse and consolidating the body, invigorating qi and promoting body fluid production, and calming the mind and improving intelligence. It has wide applications in medicine, health products, and cosmetics. With a long history of cultivation and a wide distribution, it has developed abundant varietal resources through long-term artificial selection and natural evolution. However, the current development of the ginseng industry faces numerous bottlenecks, severely restricting the cultivation of high-quality varieties and the sustainable development of the industry.

[0003] In traditional breeding models, ginseng breeding mainly relies on phenotypic trait selection, which has inherent drawbacks such as long cycles, low efficiency, and poor selection accuracy. Ginseng has a growth cycle of 4-6 years, and traditional breeding methods often take more than ten or even decades from hybridization to the development of stable, superior varieties, making it difficult to meet the market's urgent demand for high-quality, high-yield, and stress-resistant ginseng varieties. At the same time, ginseng germplasm resources have a narrow genetic background, and existing cultivated varieties are mostly domesticated from local farms, resulting in unstable quality, large differences in the content of effective components, weak stress resistance (such as insufficient resistance to diseases, pests, cold, and drought), and germplasm degeneration. This leads to large fluctuations in yield during production, excessive use of pesticides and fertilizers, which increases production costs and brings ecological and environmental risks and product quality and safety hazards. With the increasing demands for ginseng quality and the promotion of standardized cultivation of Chinese medicinal herbs, accelerating the cultivation of superior new ginseng varieties, improving breeding strategies, and enhancing breeding efficiency have become key tasks for the development of the ginseng industry.

[0004] The emergence of molecular marker-assisted breeding technology has provided a completely new approach to crop breeding. By directly selecting genes or molecular markers associated with target traits, the breeding cycle can be significantly shortened and the selection accuracy improved. Currently, the molecular marker technologies used in crop breeding mainly include SSR (simple sequence repeat) markers, ISSR markers, and AFLP markers. However, these traditional molecular marker technologies have many limitations in ginseng breeding applications: although SSR markers have high polymorphism, they have low throughput, high development costs, and cumbersome detection processes, making it difficult to meet the needs of large-scale germplasm resource identification and commercial breeding; ISSR markers are mostly dominant markers, unable to distinguish between dominant homozygous and heterozygous genotypes, resulting in insufficient accuracy in genotype identification; AFLP markers have stringent reaction conditions, poor experimental reproducibility, and uneven distribution on chromosomes, making it difficult to achieve comprehensive coverage of the entire genome. These technical shortcomings make it difficult for traditional molecular marker technologies to play an effective role in precision ginseng breeding.

[0005] Single nucleotide polymorphisms (SNPs), as a new generation of molecular markers, have become a research hotspot in the field of molecular breeding due to their advantages such as large number, wide distribution, high stability, and ease of high-throughput detection. SNP liquid-phase chip technology, based on the principle of targeted capture sequencing, combines the high accuracy of SNP markers with the high efficiency of high-throughput sequencing. It features convenient detection, low cost, high throughput, high sensitivity, and strong specificity, enabling rapid and accurate genotyping of large numbers of samples. It has been successfully applied in various crops such as rice, maize, cucumber, and tomato, providing a powerful tool for molecular breeding research such as crop genetic diversity analysis, gene mapping, genome-wide association studies, and genome selection, significantly promoting the innovation and upgrading of breeding technologies for these crops.

[0006] While SNP liquid-phase microarray technology has achieved significant results in crop breeding, its development lags behind in ginseng research. Currently, the ginseng whole-genome sequencing has been preliminarily completed, but the development of genome-wide SNP markers and the fabrication of liquid-phase microarrays have not yet been reported. Molecular-level research on ginseng mainly focuses on the cloning and expression analysis of a few functional genes, lacking high-density SNP marker resources at the genome-wide level. This hinders a comprehensive analysis of the genetic background of ginseng germplasm resources and precise selection of target traits. Existing research is insufficient to meet the needs of molecular breeding work such as genetic diversity analysis of ginseng germplasm resources, variety authenticity identification, genotype screening of hybrid offspring, genome-wide association analysis, and genome selection, severely impeding the transformation of ginseng breeding technology from traditional phenotypic selection to precise and efficient molecular breeding.

[0007] Furthermore, as an outcrossing crop, ginseng has a complex genetic background with relatively small genetic differences among different varieties. Coupled with its long growth cycle and low genetic transformation efficiency, this places higher demands on the accuracy and density of molecular markers. Developing a high-density SNP liquid-phase chip that covers the entire genome, has good locus representativeness, high polymorphism, and strong specificity can provide key technical support for research on ginseng germplasm resource innovation, superior gene discovery, and molecular marker-assisted breeding. This is of great significance for accelerating the breeding of high-quality, high-yield, and stress-resistant new ginseng varieties and enhancing the core competitiveness of my country's ginseng industry. Therefore, addressing the current bottlenecks in ginseng breeding technology and the status quo of molecular marker technology, this study develops a ginseng 40K liquid-phase SNP chip, filling the research gap in high-density SNP chips for ginseng, meeting the practical needs of ginseng molecular breeding, and possessing significant theoretical value and application prospects. Summary of the Invention

[0008] The technical problem this invention aims to solve is to overcome the shortcomings and defects mentioned in the background art above, and to provide a ginseng 40K SNP liquid-phase breeding chip and its application. This chip features uniformly distributed loci on chromosomes, good loci representativeness, high specificity, and high polymorphism, making it a precise and efficient molecular breeding chip. It can be widely used for the detection and application of different types of ginseng materials, including local varieties and cultivated varieties. It provides an indispensable tool for molecular breeding research such as high-throughput genotyping, gene mapping, fingerprinting, and whole-genome selection in ginseng.

[0009] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0010] A ginseng 40K SNP liquid phase breeding chip, the genotyping sites of which include 41,684 SNP sites on the whole ginseng genome, the location information of the SNP sites is shown in Table 1 of the specification, and the reference genome of the SNP sites is the ginseng T2T reference genome (v2024).

[0011] Based on a general inventive concept, the present invention also provides an application of the ginseng 40K SNP liquid phase breeding chip in the analysis of genetic diversity, population structure, genetic and evolutionary analysis, kinship identification, genome-wide association analysis, or genome selection of ginseng germplasm resources.

[0012] In the above-mentioned application, preferably, the method of the application includes the following steps: using the ginseng 40K SNP liquid phase breeding chip to perform genotyping on ginseng samples.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] (1) The ginseng 40K liquid phase breeding chip has the characteristics of good site representativeness, strong specificity and high polymorphism. It is a precise and efficient molecular breeding chip that can be widely used for the detection of different varieties (lines) of ginseng materials.

[0015] (2) Based on high-throughput sequencing technology, this detection method has high throughput and produces a large amount of data at one time, which can cover the detection of nearly a thousand materials at the same time; it is also applicable to mainstream second-generation sequencing platforms such as Illumina and MGI, and has platform versatility.

[0016] (3) This chip can perform molecular marker fingerprint analysis of ginseng germplasm resources, genotyping of hybrid population offspring, identification of variety authenticity, genetic background analysis and screening of breeding materials, genome-wide association analysis, genetic diversity analysis of germplasm resources, and identification of kinship. It has wide adaptability, strong specificity, high polymorphism, and is simple and quick to operate. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a flowchart of the cGPS sequencing library construction process in Example 1;

[0019] Figure 2 This is a flowchart of the development process of the ginseng 40K SNP liquid phase breeding chip in Example 1;

[0020] Figure 3 This is a chromosome distribution map of the ginseng 40K SNP locus in Example 1;

[0021] Figure 4 This is a PCA scatter plot of the population structure analysis of ginseng breeding materials in Example 2;

[0022] Figure 5 This is a phylogenetic tree diagram of the population structure analysis of ginseng breeding materials in Example 2. Detailed Implementation

[0023] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0024] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0025] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0026] Example 1:

[0027] This embodiment provides a ginseng 40K SNP liquid phase breeding chip.

[0028] This embodiment obtained 2,690,340 SNP loci based on resequencing data from different types of ginseng materials (wild germplasm, local varieties, and cultivated varieties). Combined with 1011 pre-screened potential functional loci, 41,684 SNP loci with strong representativeness, good polymorphism, and even chromosome distribution were selected as the 40K liquid-phase SNP chip for ginseng. This chip was obtained using the following method:

[0029] 1. Collection of ginseng germplasm resources,

[0030] To ensure the representativeness of the ginseng materials and the universality of liquid chromatography-chip detection, we collected 401 ginseng diversity materials from different regions and varieties (wild germplasm, local varieties and cultivated varieties), and summarized 4 resequencing data.

[0031] 2. Ginseng whole genome resequencing,

[0032] The 405 ginseng samples collected were subjected to whole-genome resequencing. The specific steps included: (1) DNA extraction using magnetic beads. (2) Using the MGI library standard method, the quality-tested DNA was used for DNA-seq sequencing library construction. (3) After the library passed the quality test, it was sequenced using the BGI sequencing platform. The sequencing strategy was PE150, the sequencing depth was 10×, and 30Gb was sequenced for each strain.

[0033] The BWA and GATK tools were used to perform sequence alignment and variant detection on 405 resequencing data. The analysis process was as follows: (1) The BWA tool was used to align the reads to the corresponding ginseng T2T reference genome (v2024), orient the positions, and mark the duplicate reads. (2) The GATK tool was used to detect variant sites for each sample and obtain the gVCF of each sample. (3) The GATK tool was used to perform joint-calling and joint analysis of the gVCF of all samples to obtain the variant results of each individual in the population. To ensure the accuracy of SNPs, the SNP sites obtained after joint analysis were initially hard filtered (SNP hard filtering criteria: QD<2.0, FS>60.0, MQ<40.0, SOR>3.0, MQRankSum<-12.5, ReadPosRankSum<-8.0).

[0034] 3. Development of ginseng liquid phase breeding chip,

[0035] a. Candidate site screening: The quality indicators of the resequencing sites were calculated, and SNP polymorphism sites with heterozygosity ≤0.3, site deletion frequency ≤0.1, minimum allele frequency ≥0.1, and sequencing depth ≥8× were screened as candidate sites, resulting in a total of 446,664 SNP sites.

[0036] b. Probe Design and Screening: Probes were designed for each target site within a 100 bp range upstream and downstream. Probe lengths were approximately 100 bp, with GC content ranging from 20% to 80%. Based on the probe design results, probes that could not be uniquely aligned to the genome or contained repetitive sequences in their flanking sequences were removed. Based on the principle of uniform distribution, sites with high MAF values ​​were preferentially retained, ultimately yielding 41,368 highly polymorphic genome-wide background sites and 316 potential functional sites.

[0037] c. Locus density screening: Based on the principle of uniform locus distribution, SNP loci that are uniformly distributed on the ginseng chromosome are screened. The average distance between the loci is 82.79Kb, containing 41684 SNP loci.

[0038] 4. Develop ginseng 40K SNP liquid phase chip,

[0039] The 41,684 selected candidate SNP loci were used to develop a ginseng 40K SNP liquid-phase chip using Huazhi's independently developed liquid-phase probe precise localization sequencing and genotyping technology (cGPS). cGPS is based on an optimized thermodynamic stability algorithm model, which designs specific probes for target region sequences. Then, the synthesized specific probes are used to capture and enrich multiple different target sequences located at different genomic positions using liquid-phase hybridization. Sequencing libraries are then constructed and high-throughput sequencing is performed on the captured and enriched target genomic sequences to obtain the genotypes of all SNP / InDel loci within the target region.

[0040] This technology involves the following steps:

[0041] (1) Sample DNA extraction and quality control: DNA was extracted from the samples using the magnetic bead method. The concentration of DNA samples was detected using a Qubit fluorescence quantitative PCR instrument; the integrity of DNA samples was detected by 1% agarose gel electrophoresis, and samples that passed the quality control were used for library preparation.

[0042] (2) cGPS Library Construction and Quality Control: a. Take 200 ng of qualified genomic DNA, digest the DNA sample with fragmentation enzyme, repair the enzyme ends, add an A base to the 3' end, and detect the fragment size by agarose gel electrophoresis. b. Use T4 ligase to ligate sequencing adapter fragments to both ends of the DNA, and purify the ligation product using fragment sorting magnetic beads. Detect the concentration of the purified product using a Qubit real-time fluorescence instrument, and detect the fragment size by agarose gel electrophoresis. c. Perform PCR amplification on the purified ligation product, and screen the amplified product using magnetic beads. Detect the concentration of the screened product using a Qubit real-time fluorescence instrument, and detect the fragment size by agarose gel electrophoresis. d. Place the qualified library, blocking reagent, RNase inhibitor, and 50K liquid phase breeding chip probe on a PCR instrument for hybridization reaction, and incubate at 54℃ for 16-24 hours. e. The hybridization product was captured using streptavidin, and the captured library was amplified and enriched. PE150 sequencing was performed using the BGI sequencing DNBSEQ-T7 platform. The principle and procedure of cGPS library construction are described in [link to documentation]. Figure 1 .

[0043] (3) Bioinformatics Analysis: The raw data after high-throughput sequencing underwent quality control filtering and other processing. FASTP software was used to remove adapter fragments and low-quality reads, resulting in high-quality Clean Reads. BWA software was used to align the obtained Clean Reads with a reference genome and perform positional sorting to obtain the sorted BAM file. GATK software was used to analyze the sequencing results for variant sites, obtaining the genotyping results for the target loci.

[0044] Ultimately, high-throughput genotyping results for each target SNP in a specific individual can be obtained, achieving high-throughput SNP genotyping; information on these 41,684 SNP loci is shown in Table 1. The marker development process is described in [link to documentation]. Figure 2 .

[0045] Table 1: Label Sequence Information

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[0100]

[0101]

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[0135]

[0136] Based on the collection of multiple stable ginseng varieties, high-quality 40K sites were screened to construct a ginseng 40K liquid phase breeding chip, covering most ginseng varieties and possessing a wide range of chip application scenarios.

[0137] The distribution of the screened 40K chips on the 24 chromosomes was statistically analyzed. It was found that the 40K SNP sites were evenly distributed across the 24 ginseng chromosomes, with an average interchromosomal distance of 82.79 kb. The site distribution map is shown below. Figure 3 .

[0138] Example 2:

[0139] This embodiment provides the application of ginseng SNP liquid phase chip in the analysis of polymorphism and population structure of ginseng breeding materials.

[0140] Using the ginseng 40K liquid-phase breeding chip designed in Example 1, 74 ginseng samples were analyzed. Four samples were used for technical replication experiments, resulting in a total of 78 sample data sets. Sequencing and data analysis showed that the locus detection rate of the 78 samples ranged from 99.89% to 99.99%, with an average detection rate of 99.96%; the heterozygosity ranged from 0.25% to 40.32%, with an average heterozygosity of 8.46%; and the genotypic consistency rate of the replicate samples ranged from 99.93% to 99.98%, with an average consistency rate of 99.96%.

[0141] Genotyping was performed, and the PCA composition of the materials was analyzed using Plink software to construct PCA scatter plots. Each locus in the scatter plot represents a sample; the greater the distance between two samples, the greater the difference in their genetic background. Individuals with similar genetic backgrounds will cluster together in the plot. Statistical analysis revealed that the ginseng breeding materials were divided into two distinct populations, see [see...]. Figure 4 .

[0142] Genetic distance matrices were calculated and cluster analysis was performed using Plink software to construct phylogenetic trees, which can determine the kinship, evolutionary relationships, and compositional structure of different materials. Population analysis of 74 ginseng materials was conducted using a ginseng 40K liquid-phase breeding chip, and the results were consistent with the actual population divisions. This indicates that the screened SNP sites are highly representative. Figure 5 .

Claims

1. A ginseng 40K SNP liquid phase breeding chip, characterized in that, The genotyping sites of the liquid-phase breeding chip include 41,684 SNP sites, which are located on 24 chromosomes of the ginseng T2T reference genome v2024; the marker sequence information of the 41,684 SNP sites is shown in Table 1. Table 1:

2. The application of the ginseng 40K SNP liquid phase breeding chip as described in claim 1 in the analysis of genetic diversity, population structure, genetic and evolutionary analysis, kinship identification, genome-wide association analysis, or genome selection of ginseng germplasm resources.

3. The application according to claim 2, characterized in that, The method of the application includes the following steps: using the ginseng 40K SNP liquid phase breeding chip to perform genotyping on ginseng samples.

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