A Potato 50K SNP Liquid Phase Gene Chip and Its Application

Abstract

The present invention belongs to the field of crop molecular breeding, discloses a potato 50K SNP liquid phase gene chip and application, uses 778 potato varieties to simplify sequencing results, identifies 16,531,339 single nucleotide polymorphism (SNP) sites, filters according to integrity>0.8, minor genotype frequency>0.05, obtains a total of 353,753 highly consistent population SNPs, and finally screens 50167 SNPs to make chips, and designs corresponding probe primers. The chip is named "Potato Core No. 1", and the chip contains a total of 50167 polymorphic SNP markers uniformly distributed throughout the genome, verified and preferred, and trait linkage or functional marker probes. The potato 50K liquid phase gene chip SNP proposed by the present invention has a rich genetic diversity basis of 778 materials from different regions at home and abroad, and has application value in potato breeding after population structure analysis, association analysis, core germplasm and core SNP screening.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the technical field of crop molecular breeding, and in particular relates to a potato 50KSNP liquid-phase gene chip and its application. Background Art

[0002] Potato (Solanum tuberosum L.) is the world's fourth largest food crop. Due to the complex genetic background of potatoes, conventional breeding efficiency is low. Selecting ideal individual plants through phenotypic observation requires a long and labor-intensive cycle. With the development of molecular technology, the application of molecular marker-assisted breeding, which changes phenotypic selection to genotypic selection, is considered an effective way to improve breeding efficiency. Molecular breeding requires the use of efficient genotyping technology to perform genotyping tests on massive germplasm resources, thereby enabling more efficient and accurate breeding. With the development of potato genomics and advancements in sequencing methods, a large number of SNPs (single nucleotide polymorphism) molecular markers have been developed, which provide sufficient molecular marker information for breeding. Single nucleotide polymorphism genotyping arrays (SNP chips) are powerful tools for studying crop genetic variation. They can simultaneously detect tens of thousands of SNPs in a single assay, providing a high-throughput, low-cost method for analyzing genetic diversity. Potato SNP genotyping enables researchers to quickly screen and identify genetic variations associated with important traits. This can be applied to studying resource system evolution, variety fingerprint identification, construction of genetic linkage maps, association analysis studies, breeding programs, and the development of breeding markers, especially in genomic selection, which helps promote the prediction and evaluation of quantitative traits.

[0003] Internationally, Illumina potato SNP solid-phase arrays have been commercialized. These arrays were developed based on SNPs identified through transcriptome sequencing of six cultivated North American potato varieties. The initial design was the Infinium SolCAP 8303V1 SNP array (Hamilton et al., 2011; Felcher et al., 2012). Subsequently, the array was validated in two diploid linkage populations, mapping over 4400 markers. Linkage maps from the two diploid populations were compared to the assembled potato genome sequence, covering 87% and 88% of the length of the two parental genomes, respectively. The Infinium 8303 potato array has subsequently been used in numerous studies. However, due to its narrow SNP collection, this array may be biased when applied to broader collections, such as those from other regions of the world or those containing wild-type introgression. To address this issue, Peter G. Vos (2015) combined 15,138 previously identified SNPs and 4,454 SNPs from the SolCAP project to develop the 20K SolSTW SNP chip. The chip used fitTetra software to design and analyze SNPs, and genotyped 569 potato genotypes from different countries, market segments, historical varieties and important parents. It mainly verified the population materials that formed the genetic composition of modern European potatoes. However, due to the manufacturer's quality standards for shelf life, quantity of synthetic materials and inventory willingness, this SNP chip is no longer used. Subsequently, Illumina launched the Infinium 12K V2, 22K V3, and other versions of the potato chip, which have been applied to detection in South American materials, including wild species (Ellis et al., 2018; Pandey et al., 2021; Selga et al., 2022; Pandey et al., 2023; Anglin et al., 2024). However, solid-phase chips have high requirements for sample processing and require pretreatment of the target DNA. The experimental operation is relatively complex, the chip cost is relatively high, and solid-phase chips have high requirements for the detection platform and are slow to update. Especially with the rapid identification of a large number of new SNP sites, solid-phase chips are difficult to update in a timely manner.

[0004] In view of the above analysis, the technical problems that need to be solved urgently in the existing technology are:

[0005] Conventional potato breeding is inefficient, and there is an urgent need to utilize a large number of SNP molecular markers for marker-assisted selection (MAS). However, potato solid-phase chips are updated slowly. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a potato 50K SNP liquid-phase gene chip and its application.

[0007] The present invention is achieved as follows: a potato 50K SNP liquid-phase gene chip, characterized in that the potato 50K SNP liquid-phase gene chip uses simplified sequencing results of 778 potato varieties to identify 16,531,339 SNP sites. Filtering based on completeness > 0.8 and minor genotype frequency > 0.05, a total of 353,753 highly consistent population SNPs were obtained. Finally, 50,167 SNPs were screened to make a chip, named "Shuxin No. 1". The chip contains a total of 50,167 evenly distributed whole-genome polymorphic SNP markers, trait linkage or functional marker probes for verification.

[0008] Furthermore, the chip includes a total of 50167 SNPs shown as ID No. 1 to 50167.

[0009] Furthermore, the chip includes a total of 50,167 probe sequences represented by ID No. 1 to 50,167.

[0010] Furthermore, the potato 50K SNP liquid phase gene chip capture sequencing process is as follows:

[0011] Utilizing the developed No. 1 to 50167 SNP information, the target region is captured for high-throughput second-generation sequencing using probe hybridization-based targeted capture technology under liquid phase reaction conditions. First, a gDNA library is constructed for the material to be tested. At the same time, based on the principle of DNA complementarity, a probe covering the target SNP is designed at each site to be tested, and the target probe is modified with biotin labeling. Then, the biotin-modified probe is hybridized with the target region of the genome in a liquid to form a double strand. Subsequently, streptavidin-coated magnetic beads are used to molecularly adsorb the biotin-modified probe, thereby capturing the target hybridized with the probe. Finally, the captured target sequence is eluted, amplified, and sequenced to ultimately obtain the genotype of the target SNP.

[0012] Another object of the present invention is to provide an application of a potato liquid-phase gene chip, including using the SNP chip provided by the present invention to perform genetic diversity, systematic evolution and kinship analysis of potato germplasm resources, genetic background analysis of breeding materials, variety fingerprinting and authenticity identification, population genetic structure analysis, genome-wide association study (GWAS), QTL / gene mapping, functional gene mining, molecular marker-assisted breeding, molecular design breeding-related phenotype prediction, GS whole-genome selection breeding applications and other different application scenarios.

[0013] The present invention also provides a genotype detection method based on a potato 50K SNP liquid phase gene chip, comprising the following steps:

[0014] (1) Designing probe sequences covering the target SNPs based on the SNP locus information described in ID No. 1 to 50167;

[0015] (2) constructing a genomic DNA (gDNA) library for the test material;

[0016] (3) Under liquid phase reaction conditions, a biotin-modified probe is used to hybridize with the target SNP region to form a double strand;

[0017] (4) The hybridization probe is molecularly adsorbed by streptavidin-coated magnetic beads to capture the target area.

[0018] Furthermore, after the captured targets are eluted by magnetic beads, the target sequences are amplified and sequenced to finally obtain the genotype information of the target SNP.

[0019] Furthermore, the gene chip capture sequencing technology is a high-throughput second-generation sequencing method based on probe hybridization targeted capture, which achieves accurate capture and analysis of targeted SNP sites through liquid phase hybridization.

[0020] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0021] 1. Potato 50K SNP liquid phase gene chip SNP has a rich genetic diversity basis of 778 materials from different regions at home and abroad.

[0022] The SNP sites of the "Shuxin No. 1" 50K SNP liquid-phase gene chip of the present invention were identified and screened from sequencing data of more than 778 global potato variety resources. The SNP sites were derived from the sequencing results of 778 variety resources from all over the world by the applicant unit. In 2000, the applicant unit began potato variety breeding and actively introduced potato germplasm resources from potato breeding units at home and abroad. Over the past two decades, more than 700 resources have been introduced and evaluated for resistance. Over the past 20 years, we have used resources for hybridization, formulated a total of 2,202 hybrid combinations, and obtained 1.6762 million seedlings. The resources and breeding materials have achieved extensive and dynamic improvement goals. Linkage disequilibrium analysis shows that the resources are haplotype-rich.

[0023] 2. Potato 50KSNP liquid phase gene chip SNP has application value in potato breeding through analysis of my country's resource population structure, association analysis, screening of core germplasm and core SNPs.

[0024] In the early stages of the project, further SNP marker identification was performed on the GBS data using a combination of restriction endonucleases (RsaI+HaeIII) and SLAF library construction. A total of 6,162.86 million reads were generated, with an average sequencing Q30 of 93.30% and an average GC content of 39.51%. A total of 1,198,928 SLAF tags were developed, with an average sequencing depth of 17.05x. Among these, 1,032,778 polymorphic SLAF tags were identified, yielding 16,531,339 SNP markers. Filtering based on completeness > 0.8 and minor genotype frequency > 0.05 yielded 353,753 highly concordant population SNPs. Core population and core SNP analysis further identified core SNPs. The project employed various statistical models to systematically evaluate the phenotypes of tetraploid potato resources. Genome-wide marker-trait association analysis was performed on key agronomic, tuber, yield, and quality traits. Some of the identified SNPs were also associated with breeding genetic traits. By designing microarrays targeting loci associated with potato traits and analyzing the associations between variation and phenotype, rapid and cost-effective detection of trait-related genotypes was achieved. Linkage disequilibrium (LD)-based association analysis, using existing natural populations and leveraging the recombination information accumulated over long-term evolution, yields high accuracy and allows for direct application of the identified molecular markers in breeding.

[0025] 3. We analyzed multiple high-quality genomes from the potato genome database Spud DB (http: / / spuddb.uga.edu / ) and performed liquid-phase SNP array verification.

[0026] Several potato genome resequencing projects are currently underway internationally. This application builds upon this experience by designing and developing liquid-phase microarrays. Integrating this with reference genome information and existing resequencing data, this application applies stringent screening criteria, including, but not limited to, SNPs with >5 reads, bi-allelicity, and detection in >90% of individuals within the population, to select for subsequent analysis. The resulting SNP set is further screened based on genomic location, removing SNPs located in repetitive regions and those that do not meet Harwin equilibrium or are highly linked. The remaining SNPs are then used for subsequent analysis. Priority is given to SNPs observed in multiple lines, observed on more than one NGS platform, obtained at high sequencing depth, with high quality scores on NGS platforms, validated on other genotyping platforms, and with no other polymorphisms within 35 or 50 bp. A comprehensive assessment of the sequence of the target region and adjacent regions is performed to ensure genome-wide uniqueness of the probes. Probe placement is carefully considered in low-complexity regions. Regions with a GC content of approximately 50% are preferred to minimize heterogeneity in PCR amplification. In regions with high GC and AT concentrations, the number of probes should be appropriately increased to compensate for competitive resistance and binding capacity disadvantages. Hairpin formation within the probes and dimer formation between probes should be avoided as much as possible. Probes were designed for the target region genome and synthesized to generate effective, specific probes. Liquid-phase hybridization with genomic DNA was then performed, and rigorous probe capture testing was performed to validate the probes. After capturing and enriching the target region sequence, high-throughput sequencing was performed on a sequencing platform. The validity of the population variation sites in potato was tested, and candidate genes or loci were verified.

[0027] 4. The potato 50K SNP liquid-phase gene chip performs targeted sequencing detection based on SNPs obtained from the previous simplified genome sequencing analysis screening, reducing costs and achieving standardization.

[0028] The potato 50K SNP liquid phase gene chip is a genotyping detection technology based on "targeted sequencing typing" (genotyping by targetsequencing, GBTS). After capturing specific sites or regional DNA fragments, the genotype of the target region is further obtained by second-generation sequencing. The SNP of the present invention was originally derived from simplified genome sequencing (genotyping by sequencing, GBS), but GBS has randomness. Targeted sequence capture captures the target region site in a targeted manner, separates a group of genes or genomic regions for sequencing, thereby reducing the sequencing amount, greatly simplifying bioinformatics analysis and data processing. Targeted sequence capture has a high tolerance for the GC content of the target sequence, there is no randomness in the detected region, and the experiment is standardized and reproducible. Compared with whole genome resequencing, the sequencing cost and data processing difficulty are reduced. For the vast majority of studies, the experimenter does not need to use the sequencing data of the whole genome, but only the sequencing data of certain sites or certain DNA regions. Through targeted sequence capture, only the target region needs to be sequenced, which greatly reduces the sequencing amount and data output, thereby reducing the sequencing cost. Compared with solid-phase chips, it has low cost and lower requirements for the detection platform, which solves the problem of slow updates and replacements of solid-phase chips.

[0029] Second, as auxiliary evidence for the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0030] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0031] In molecular breeding, scientists often need to use efficient genotyping technology to conduct genotyping tests on massive germplasm resources, so as to carry out more efficient and accurate breeding, and the application prospects are broad.

[0032] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad:

[0033] Internationally, the Illumina Infinium SolCAP SNP chip is mainly targeted at European and American materials. The technical solution of the present invention is developed based on international and domestic potato resource varieties, filling the gap in a chip platform that is more targeted at the genetic diversity of Chinese potato varieties.

[0034] (3) The technical solution of the present invention solves the technical problems that people have been eager to solve but have never been able to solve successfully:

[0035] The potato 50K liquid-phase gene chip achieves high-throughput genotyping by detecting genetic variations in specific genomic regions. It can be used for potato genome polymorphism analysis, variety identification, population genetics analysis, etc., solving the urgent need for molecular marker platform tools in potato genetic research and breeding.

[0036] (4) The technical solution of the present invention overcomes technical prejudice:.

[0037] The threshold for solid-phase chip technology is high internationally. It uses oligonucleotide probes fixed on a solid substrate (such as a glass slide, etc.). After sample processing and hybridization, single-base extension and fluorescent staining are performed, and the sample is scanned on the machine to directly detect the SNP site. It has high throughput, fast speed, and accurate data. However, for domestic users, the cost is high, and there is also the problem of sending samples abroad. The "liquid-phase chip" is a high-throughput genotyping based on the second-generation sequencing technology (NGS). After capturing the target area, it relies on the second-generation sequencing platform to complete the subsequent genotype detection, and the accuracy of the target sequence capture and the number of captured markers depend on the sequencing depth. However, with the development of second-generation sequencing technology, the "liquid-phase chip" has achieved low cost, high capture rate, high accuracy, and has completely independent intellectual property rights. It can also realize the localization of full-process detection equipment and reagent consumables, and can replace expensive imported solid-phase chips. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a genome-wide distribution map of potato 50K SNP liquid-phase gene chip sites provided in an embodiment of the present invention;

[0039] Figure 2 This is a gene annotation distribution map of the potato 50K SNP liquid phase gene chip sites provided in an embodiment of the present invention;

[0040] Figure 3 This is a flowchart of the potato 50K SNP liquid-phase gene chip capture sequencing provided by an embodiment of the present invention;

[0041] Figure 4 is the length distribution of the inserted fragments of the Control reads provided in the embodiment of the present invention;

[0042] Figure 5 This is a graph showing the repetition rate of the potato 50K SNP liquid-phase gene chip test results for samples provided by an embodiment of the present invention.

[0043] Figure 6 This is a SNP genetic evolutionary tree diagram of the sample detection data of the potato 50K SNP liquid phase gene chip provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0045] The present invention provides a potato 50K SNP liquid-phase gene chip. Using simplified sequencing results from 778 potato varieties, 16,531,339 SNP sites were identified. Filtering was performed based on completeness > 0.8 and minor genotype frequency > 0.05, resulting in a total of 353,753 highly consistent population SNPs. Finally, 50,167 SNPs were screened and used to make a chip. The chip, named "Shuxin No. 1," contains a total of 50,167 evenly distributed SNPs across the entire genome, validating preferred polymorphic SNP markers and trait linkage or functional marker probes.

[0046] like Figure 1-Figure 2 As shown, the chip includes a total of 50167 SNPs shown as ID No.1 to 50167.

[0047] like Figure 3 As shown, liquid-phase probe capture technology is used to design specific probes for the target region gene sequence based on the principle of complementary base pairing to achieve targeted capture of the target gene or site. The captured fragments are then subjected to high-throughput second-generation sequencing using a sequencing platform, thereby achieving accurate genotype identification of the target region or site. The specific experimental process is as follows:

[0048] 1. Preparation of Potato 50K SNP Liquid Phase Gene Chip

[0049] The preparation process includes template synthesis and probe preparation. The synthesized DNA template (Oligo Pool) undergoes quality control and in vitro amplification experiments before being converted into the final probe. This completes the production of the potato 50K liquid-phase gene chip capture kit ("Shuxin No. 1" 50K SNP liquid-phase gene chip).

[0050] 2. DNA library construction and capture

[0051] The DNA sample undergoes ultrasonic or enzyme digestion, end repair and 3'-end "A" addition, adapter ligation and purification, and pre-PCR library amplification to obtain the library required for hybrid capture. After library-probe hybridization, probe-magnetic bead binding, non-specifically bound library washing, post-capture PCR amplification, library quantification, and quality control, sequencing is performed.

[0052] 3. Library quality control and quantification

[0053] Library concentration was measured using a Qubit 4.0 Fluorometer (Thermofisher). A pre-capture concentration >25 ng / L was considered acceptable. Using an ABI Qsep 100 assay, the main peak of the library should be approximately 200-500 bp, with no additional peaks before or after the main peak.

[0054] 4. Sequencing

[0055] The BGI DNBSEQ-T7 second-generation sequencing platform was used for 2*150bp paired-end sequencing.

[0056] The data analysis method comprises:

[0057] 1. Raw sequencing data

[0058] Raw image data generated by high-throughput sequencing (such as the MGI platform and Nova platform) is converted into Fastq format raw sequencing data (referred to as raw data) through a specific program. The provided file format is generally: Project Number / Flowcell Number / Sample Name_Lane Number_I+Index Number.R1 or R2.clean.fastq.gz (e.g., 1_L5_I108.R1.clean.fastq.gz). Open it in Linux using the less command or other editor. For a detailed description of the data format, refer to the following website: http: / / en.wikipedia.org / wiki / FASTQ_format

[0059] FASTQ format files contain sequence information and sequencing quality information for sequencing data. Each read consists of four lines: the first line begins with an "@" sign, followed by the BGI sequencing identifier (Sequence Identifiers) and index sequence; the second line is the measured base sequence; the third line begins with a "+" sign, followed by the BGI sequencing identifier (optional part); and the fourth line is the sequencing quality of the corresponding base.

[0060] 2. Data filtering and quality control

[0061] By setting certain filtering criteria, we can remove adapter information, low-quality bases, unmeasured bases (indicated by N), etc. that may interfere with subsequent information analysis. The final data obtained is clean data or clean reads. The data filtering criteria are:

[0062] 1) Filter out reads containing adapter sequences;

[0063] 2) When the N content in a single-end sequencing read exceeds 3% of the read length, the paired reads are removed;

[0064] 3) When the number of low-quality (less than 3) bases in a single-end sequencing read exceeds 50% of the read length, the paired reads are removed.

[0065] 3. Data Analysis Process

[0066] The samtools / bcftools (https: / / github.com / samtools / samtools) pipeline was used for variant analysis. The filtered sequencing sequences were aligned to the reference genome. The main process is as follows:

[0067] Raw sequencing reads were processed using "Fastap" (version 0.12.4) to remove low-quality reads and adapters. High-quality reads were aligned to the Solanum tuberosum DM V6.1 genome using BWA (version 0.7.16) mem with default parameters. samtools (version 1.17) was used for bam sorting, removal of PCR duplicates of reads, and sample alignment statistics. Genomic variation analysis of bam was then performed using bcftools. The high-quality aligned read parameters for SNP acquisition were set to MQ>=30. bcftools (version 1.17) was used for SNP quality filtering with parameters set to "QUAL>30" and "DP>5". SNPs were annotated using snpEff based on a custom database containing potato DM V6.1 HC / LC genes and self-annotated genes.

[0068] The application of a potato liquid-phase gene chip provided in an embodiment of the present invention includes using the SNP chip provided by the present invention to perform different application scenarios, such as genetic diversity of potato germplasm resources, population genetic structure analysis, systematic evolution and kinship analysis, genetic background analysis of breeding materials, variety fingerprinting and authenticity identification, QTL / gene mapping, genome-wide association study (GWAS), functional gene / QTL positioning mining, molecular marker-assisted breeding, molecular design breeding-related phenotype prediction, and GS whole-genome selection breeding applications.

[0069] 1. Specific application fields or related products of the present invention.

[0070] A potato 50K SNP gene chip kit was developed and used to perform chip testing on 265 potato materials, including the Yunshu series of potato varieties. A SNP fingerprint database for the Yunshu series and other variety resources was established, laying the foundation for authenticity authentication of the Yunshu series varieties. Genetic evolutionary analysis of high-quality chip data was completed. Using MEGA X software, based on the neighbor-joining method, the Kimura 2-parameter model was adopted, and 100 bootstrap replicates were used to construct a phylogenetic tree for each sample. The SNP data provided by the present invention can be used for genetic diversity, phylogeny, and kinship analysis of potato germplasm resources, genetic background analysis of breeding materials, variety fingerprinting and authenticity authentication, population genetic structure analysis, genome-wide association study (GWAS), QTL / gene mapping, functional gene mining, molecular marker-assisted breeding, molecular design breeding-related phenotypic prediction, GS whole-genome selection breeding applications, and other different application scenarios.

[0071] 2. Relevant evidence of the technical effects obtained by the embodiments of the present invention.

[0072] Example 1: Acquisition and identification of target sites for potato 50K SNP liquid-phase gene chip

[0073] The diploid potato genome was selected as a reference genome for enzyme digestion prediction. The actual potato genome size is approximately 511.89 MB, with a GC content of 34.22%. Based on the principles of enzyme digestion protocol selection, the restriction endonuclease combination RsaI+HaeIII was determined. Sequences with a length of 314-414 bp were defined as SLAF tags, and 240,000 SLAF tags were predicted to be generated. A control sequencing run of Nipponbare rice, used to assess the accuracy of the experimental library construction, generated 1.32 million reads. This control sequencing data was used to monitor the progress of the experiment and confirm the effectiveness of the enzyme digestion protocol.

[0074] The sequencing reads of the control were aligned with its reference genome using SOAP software, which showed that the paired-end alignment efficiency of this experiment was 94.40%, which was basically normal.

[0075]

[0076] The proportion of residual enzyme cleavage sites in the inserted fragments of sequencing reads was counted. The enzyme cleavage efficiency of the control data was 92.34%, indicating that the enzyme cleavage reaction was normal.

[0077]

[0078] like Figure 5 As shown in the figure, the actual length of the SLAF tag is calculated according to the position of the control sequencing pair-end mapped reads in the genome, and the length distribution of the control reads inserted fragments is drawn.

[0079] Summary of experimental library construction evaluation: The paired-end alignment efficiency of the control data was 94.40%, the enzyme digestion efficiency was 92.34%, and the SLAF library construction was normal.

[0080] Further SNP marker identification was performed on the GBS data. A total of 6,162.86M reads were obtained in this project, with an average sequencing Q30 of 93.30% and an average GC content of 39.51%. A total of 1,198,928 SLAF tags were developed, and the average sequencing depth of the sample SLAF tags was 17.05x. Among them, there were 1,032,778 polymorphic SLAF tags. Using the diploid potato genome as the reference genome, BWA was used to align the sequencing reads to the reference genome, and SNP markers were developed using GATK and samtools. The intersection of the SNP markers obtained by the two methods was used as the final reliable SNP marker dataset, resulting in a total of 16,531,339 SNP markers. Based on the completeness > 0.8 and the minor genotype frequency > 0.05, a total of 353,753 highly consistent population SNPs were obtained.

[0081] A high-quality reference genome is the foundation for resequencing potato samples and detecting variant sites. The potato genome database, Spud DB (http: / / spuddb.uga.edu / ), contains multiple high-quality genomes. Therefore, detecting population variant sites in potato provides a strong genomic foundation, serving as a foundation for liquid chromatography array design and development. In this project, based on existing resequencing data, SNPs meeting stringent criteria, including but not limited to support read counts >5, bi-allelicity, and detection in >90% of individuals within the population, were selected for subsequent analysis. The resulting SNP set was further screened based on its genomic location, removing SNPs located in repetitive sequence regions. SNPs that did not meet Harwin-Wen equilibrium or were highly linked were also removed. The remaining SNPs were used for subsequent analysis. The project also conducted core population and core SNP analyses to further identify core SNPs.

[0082] Site selection should follow the following principles:

[0083] SNPs observed in multiple strains, SNPs observed on more than one NGS platform, SNPs obtained under higher sequencing depth conditions, SNPs with higher quality scores on the NGS platform, SNPs verified on other genotyping platforms, and SNPs with no other polymorphisms within the adjacent 35 bp or 50 bp should be given priority.

[0084] Probe design principles

[0085] Specificity: This refers to the uniqueness of the probe across the genome. Higher specificity results in higher probe capture efficiency. Probe design should be avoided in low-complexity regions. However, in special cases, such as when hotspots happen to be located in low-complexity regions, careful probe placement is necessary, after comprehensively evaluating the sequence of that region and adjacent regions.

[0086] Binding Capacity: Probes have the strongest capture capacity in regions with a GC content of approximately 50%. Probes in high-GC regions have strong binding capacity, but the DNA fragments themselves have stronger binding capacity (longer lengths), resulting in greater competitive resistance from the probes. High-AT regions have weaker binding capacity for DNA fragments themselves, but the probes also have weaker binding capacity to them. Therefore, the number of probes in these high-GC and high-AT regions needs to be appropriately increased to compensate for the disadvantages in competitive resistance and binding capacity.

[0087] Probe preparation: After high-throughput probe synthesis, the PCR process is required to increase the probe quantity. Since the amplification efficiency of probes with different GC contents is different, uneven amplification should be avoided as much as possible.

[0088] Secondary structure: The probe itself should be prevented from forming a hairpin structure and the probes should be prevented from forming dimers, otherwise the binding of the probe to the target DNA will be affected.

[0089] Example 2: Evaluation of target site capture efficiency of potato 50K liquid-phase gene chip

[0090] Testing of five randomly selected natural potato samples showed that 50K SNPs were consistently captured in nearly all samples. Using a sequencing depth of 1x, over 99.6% of the 50K SNPs were captured. Using a filtering criteria of 10x, the capture efficiency for all sites exceeded 98%. The remaining sites require verification based on the phenotypes and results of the actual samples. The potato 50K SNP liquid-phase gene array was designed based on pre-assessed design, with an additional 10% of target sites. The actual capture rate exceeded 98%, achieving consistent capture of all target sites exceeding 50K SNPs. This overall capture efficiency exceeded 98%, achieving the desired capture efficiency. This testing is suitable for scalable deployment.

[0091] Target site capture efficiency of potato 50K SNP liquid-phase gene array

[0092] Sample name / sequencing depth coverage_1x coverage_5x coverage_10x coverage_30x YN66 0.996771 0.988459 0.98090378 0.939921 He01 0.997289 0.992784 0.98786055 0.960133 N16 0.998924 0.995375 0.99017282 0.961229 YN2 0.999362 0.998027 0.99621265 0.961907 MP175 0.998246 0.992246 0.983794128 0.944186

[0093] Using five potato leaf samples, two DNA samples were randomly extracted from two leaves of each plant, and the potato 50K SNP liquid phase gene chip was used for experiments and testing. The consistency of the two DNA test results of the same plant sample was over 97%. This shows that the potato 50K SNP liquid phase gene chip test results have good reliability and stability. Figure 6 shown.

[0094] Test sample group He01|He01-1 MP175|MP175-1 N16|N16-1 YN2|YN2-1 YN66|YN66-1 Site repetition rate 97.76％ 98.08％ 97.21％ 96.92％ 98.27％

[0095] The present invention has better variety representativeness than the international Infinium 8303 potato SNP chip. The SNP sites of the "Shuxin No. 1" 50K liquid phase chip of the present invention are mined from the sequencing sequence information of 778 potato variety resources from representative regions around the world. Foreign variety resources include resource materials from the International Potato Center (CIP), Europe, the United States and Southeast Asia. The resources of the International Potato Center are divided into CIP-C, CIP-D and CIP-E, with 123, 201 and 55 genotypes respectively. There are 98 genotypes in European and American resources, including materials from Canada, the United States, the Netherlands, Germany, Belarus, Japan and other countries. Domestic resources include: 75 local varieties in Yunnan, 35 varieties of Yunnan potato, 80 varieties of Yunnan potato lines, 52 varieties in northern China (one-season cropping area in the north, two-season cropping area in the central plains), 27 varieties in the southwest (one-season and two-season mixed cropping area in the southwest and winter cropping area in the south), 6 materials in Southeast Asia, and 26 other materials.

[0096] Compared with the internationally available Infinium 8303 potato SNP chip, this method has better breeding application prospects. In the early stages of the project, SLAF-seq (Specific-Locus Amplified Fragment Sequencing), a genotyping by sequencing (GBS) method, was used to identify 16,531,339 SNP markers. After screening and filtering, a total of 353,753 highly consistent population SNPs were obtained. Based on the SNP markers, the population structure of 778 potato germplasm genotypes was clarified. Using different statistical models, a systematic phenotypic evaluation of tetraploid potato resources was conducted. Genome-wide marker-trait association analysis was performed on key agronomic traits, tuber traits, yield, and quality traits. The results provide technical support for improving potato genetic traits through molecular marker-assisted selection. As can be seen, compared with the Infinium 8303 SNP chip, the SNPs in this invention are derived from a wide range of natural potato populations and subjected to population structure analysis. Through association analysis of genetic traits, SNPs are further optimized, resulting in a higher proportion of excellent markers, which can facilitate more targeted breeding work.

[0097] 50K site:

[0098] 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[0099] 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[0100] 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[0101] 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Claims

1. A potato 50K SNP liquid phase gene chip, characterized in that: Based on the simplified sequencing results of 778 potato varieties, 16,531,339 SNP sites were screened out. Through the filtering criteria of completeness>0.8 and minor genotype frequency>0.05, 353,753 highly consistent population SNPs were obtained, and finally 50,167 SNPs were screened out for making a probe chip with uniform distribution throughout the genome. The chip uses probe hybridization targeted capture technology in liquid phase reactions to achieve high-throughput second-generation sequencing of the target area, and uses biotin-labeled probes and streptavidin-coated magnetic beads to capture the target area, and finally obtain the genotype information of the target SNP.

2. The potato 50K SNP liquid phase gene chip according to claim 1, characterized in that: Potato 50K SNP liquid-phase gene chip, using simplified sequencing results of 778 potato varieties, identified 16,531,339 SNP sites, filtered according to completeness>0.8, minor genotype frequency>0.05, and obtained a total of 353,753 highly consistent population SNPs. Finally, 50,167 SNPs were screened to make chips. The chip contains a total of 50,167 evenly distributed whole genomes to verify the preferred polymorphic SNP markers, trait linkage or functional marker probes; The chip includes a total of 50167 SNPs indicated by ID No. 1 to 50167.

3. The potato 50K SNP liquid phase gene chip according to claim 1, characterized in that: The chip includes a total of 50167 probe sequences shown as ID No.1-50167.

4. The potato 50K SNP liquid phase gene chip according to claim 1, characterized in that: The potato 50K SNP liquid phase gene chip capture sequencing process is: Utilizing the developed No. 1-50167 SNP information, the target region is captured for high-throughput second-generation sequencing using probe hybridization-based targeted capture technology under liquid phase reaction conditions. First, a gDNA library is constructed for the material to be tested. At the same time, based on the principle of DNA complementarity, a probe covering the target SNP is designed at each site to be tested, and the target probe is modified by biotin labeling; then, the biotin-modified probe is hybridized with the target region of the genome in a liquid state to form a double strand; subsequently, the biotin-modified probe is molecularly adsorbed on streptavidin-coated magnetic beads to capture the target hybridized with the probe; finally, the captured target sequence is eluted, amplified and sequenced, and the genotype of the target SNP is finally obtained.

5. A genotype detection method based on potato 50K SNP liquid phase gene chip, characterized in that: The following steps are involved: (1) Designing a probe sequence covering the target SNP according to the SNP site information described in ID No. 1 to 50167; (2) constructing a genomic DNA (gDNA) library for the material to be tested; (3) Under liquid phase reaction conditions, the biotin-modified probe is hybridized with the target SNP region to form a double strand; (4) The hybridization probe is molecularly adsorbed by streptavidin-coated magnetic beads to capture the target area.

6. The genotype detection method according to claim 5, characterized in that: After the captured targets are eluted by magnetic beads, the target sequences are amplified and sequenced to finally obtain the genotype information of the target SNP.

7. The genotype detection method according to claim 5, characterized in that: The gene chip capture sequencing technology is a high-throughput second-generation sequencing method based on probe hybridization targeted capture, which achieves accurate capture and analysis of targeted SNP sites through liquid phase hybridization.

8. An application of the potato liquid phase gene chip according to claims 1-4, characterized in that: Applications include using the SNP chip provided by the present invention to conduct genetic diversity, systematic evolution and kinship analysis of potato germplasm resources, genetic background analysis of breeding materials, variety fingerprint and authenticity identification, population genetic structure analysis, genome-wide association analysis (GWAS), QTL / gene positioning, functional gene mining, molecular marker-assisted breeding, molecular design breeding-related phenotype prediction, GS whole genome selection breeding application and other different application scenarios.

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