Sweet potato snp molecular marker combination, snp chip and application thereof

Abstract

This invention discloses a combination of SNP molecular markers for sweet potato, an SNP chip, and their applications, relating to the fields of plant biotechnology and plant molecular breeding. The chip contains 16,730 SNP loci located on chromosome 15 of the sweet potato reference genome "Y22". This sweet potato 16K liquid-phase SNP chip, SweetpotatoGBTS16K, is applied to genotyping, variety identification, gene mapping, and genome-wide association analysis of sweet potato varieties. The SNP loci on the liquid-phase chip of this invention were screened from large-scale sweet potato genome resequencing data, totaling 16,730 SNP loci, encompassing associated loci for major agronomic traits such as yield, quality, and resistance in sweet potato, and has broad application prospects in multiple fields of sweet potato breeding.

Description

Technical Field

[0001] This invention relates to the fields of plant biotechnology and plant molecular breeding, specifically to a sweet potato SNP molecular marker combination, SNP chip, and their applications. Background Technology

[0002] Sweet potatoes originated in tropical America and are an important global crop for food, feed, energy, and industrial raw materials. Characterized by stable and high yields, strong resistance to adverse conditions, and wide adaptability, sweet potatoes were once a crucial crop for disaster relief and famine relief, and remain an important food security reserve crop today. More than 110 countries worldwide cultivate sweet potatoes, covering a total area of ​​approximately 9 million hectares, with an annual total output of about 130 million tons, ranking seventh in global food production. They are mainly grown in developing countries in Asia and Africa. China is the world's largest producer and consumer of sweet potatoes.

[0003] With the continuous development of molecular detection technology, modern biotechnology and molecular genetic improvement techniques have become powerful tools for future molecular design breeding. The design and development of high-density, low-cost molecular markers are a prerequisite for molecular design breeding. Traditional molecular markers, such as restriction fragment length polymorphisms (RFLPs) and simple sequence repeats (SSRs), have already played an important role in marker-assisted breeding of crops. However, these molecular markers are limited in number across the genome, have low throughput, are technically complex to operate, and are costly for large-scale genotyping, making large-scale commercial application difficult. With the development of de novo genome sequencing and large-scale resequencing, single nucleotide polymorphism (SNP) markers have become the most important markers in modern molecular breeding. Single nucleotide polymorphisms mainly refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. SNPs are caused by the conversion or transversion of a single base, or by the insertion or deletion of a base, and can occur either within the gene sequence or in non-coding sequences outside the gene. SNPs are widely distributed in the genomes of plants and animals, and are characterized by high density, genetic stability and ease of automated typing. They have become common molecular markers in the study of genetic variation in plants and animals.

[0004] Currently, gene chip technologies used for SNP locus genotyping mainly include solid-phase chips and liquid-phase chips. Solid-phase chips are generally based on complementary hybridization between probes and DNA sequences, and genotyping is performed through the fluorescent colorimetric signal of the marker. Mainstream chip detection methods include Illumina SNP chips, Affymetrix SNP chips, and Agilent chips. Solid-phase chips have high development costs, and their core technologies rely on foreign countries, which limits their application to some extent. Liquid-phase chips are a technology system based on genotypig by taget sequencing (GBTS). This is a newly developed genotyping technology. Compared with solid-phase chips, it has high flexibility and scalability, allowing the size and number of target regions to be adjusted according to specific research needs. It has already been applied in major food crops and animal husbandry. Several liquid phase chips have been developed for crops such as cucumber (Yang et al., 2019), pepper (Du et al., 2019), maize (Gao et al., 2019), wheat (Hou et al., 2020), rice (Li et al., 2020), and broccoli (Shen et al., 2021), and have been applied to genetic mapping, genome-wide association studies, and genome-wide selection breeding. However, there are currently no reports on the development of liquid phase chips for sweet potatoes, leaving the field unexplored.

[0005] Currently, there are no SNP chips related to sweet potatoes. Therefore, selecting suitable SNPs based on sweet potato genome information to create low-density liquid phase chips is one of the urgent problems that my country's sweet potato industry needs to solve. Summary of the Invention

[0006] This invention aims to at least partially address one of the problems in related technologies. Therefore, the object of this invention is to provide a sweet potato SNP molecular marker combinatorial array, an SNP chip, and its applications.

[0007] This invention aims to develop a sweet potato SNP chip based on GenoBaits technology (based on targeted sequencing genotyping), a technology system of GBTS (Genotyping by target sequencing). This chip is the first high-density liquid phase chip, and this application labels it as SweetpotatoGBTS16K. This chip can be applied to sweet potato genotyping, variety identification, gene mapping, genome-wide association analysis, and genome-wide selection breeding, thereby solving the problems in the aforementioned existing technologies. GenoBaits technology is based on the complementary binding of target probes and target sequences. Through site-specific capture under liquid phase conditions, the captured target sequences are eluted, amplified, library constructed, and sequenced to finally obtain the genotype of the target SNP. This method can detect a large number of target sites and markers under economical and efficient conditions, and is suitable for genotyping analysis of various plants.

[0008] Therefore, this invention proposes a combination of SNP molecular markers for sweet potatoes.

[0009] This invention provides a set of probes targeting the aforementioned combinations of SNP molecular markers in sweet potato. These probes are used to detect SNP site combinations associated with sweet potato agronomic, quality, and resistance traits. In some embodiments, the probes are nucleotide probes.

[0010] The present invention also proposes an SNP chip for detecting the above-mentioned sweet potato SNP molecular marker combinations.

[0011] This invention also proposes a kit for detecting the above-mentioned combinations of sweet potato SNP molecular markers. The kit includes an SNP chip.

[0012] This invention also proposes an application of the above-mentioned sweet potato SNP molecular marker combination, SNP chip, or reagent kit.

[0013] This invention also proposes a method for breeding sweet potatoes.

[0014] According to one aspect of the present invention, a sweet potato SNP molecular marker combinatorial system is proposed, comprising 16,730 SNP molecular markers. The site information of these 16,730 SNP molecular markers was obtained based on sequence screening of the sweet potato reference genome "Y22," and the site information is shown in Table 2. The SNP molecular markers are distributed across 15 chromosomes of the sweet potato reference genome "Y22." Information on the sweet potato reference genome "Y22" can be found in the NCBI database, specifically at https: / / ngdc.cncb.ac.cn; accession number PRJCA015454.

[0015] In a second aspect of the invention, a sweet potato SNP chip is provided, the sweet potato SNP chip comprising a primer set and / or probes for detecting the above-mentioned combinations of sweet potato SNP molecular markers.

[0016] In some embodiments of the present invention, the sweet potato SNP chip is a solid-phase chip or a liquid-phase chip.

[0017] In some embodiments of the present invention, the sweet potato SNP chip is a liquid phase chip.

[0018] In some embodiments of the present invention, the sweet potato SNP chip is a 16K liquid phase SNP chip.

[0019] In a third aspect of the invention, a kit is provided comprising the above-described sweet potato SNP chip.

[0020] In a fourth aspect of the present invention, the application of at least one of the above-mentioned sweet potato SNP molecular marker combinations, sweet potato SNP chips, and kits is proposed.

[0021] In some embodiments of the present invention, the application can be implemented through the following methods: S1. Use at least one of the above-mentioned targeted sweet potato SNP molecular marker combinations and kits to perform SNP site typing detection on the sample to be tested, and obtain SNP site typing results. S2. Analyze the SNP locus typing results obtained in step S1.

[0022] In some embodiments of the present invention, the SNP site genotyping detection is performed based on liquid-phase probe capture sequencing genotyping technology.

[0023] In some embodiments of the present invention, the application is in the analysis of sweet potato agronomic traits.

[0024] In some embodiments of the present invention, the sweet potato agronomic traits include sweet potato yield, sweet potato quality, and sweet potato resistance.

[0025] In some embodiments of the present invention, the application is in the genotyping of sweet potatoes.

[0026] In some embodiments of the present invention, the application is in the identification of sweet potato varieties.

[0027] In some embodiments of the present invention, the application is an application in the genetic mapping of sweet potatoes.

[0028] In some embodiments of the present invention, the application is in the detection of SNP sites in sweet potatoes.

[0029] In some embodiments of the present invention, the application is in genome-wide association analysis of sweet potato varieties.

[0030] In some embodiments of the present invention, the application is an application in the analysis of genetic diversity in sweet potatoes.

[0031] In some embodiments of the present invention, the application is in sweet potato breeding or assisted breeding.

[0032] In some embodiments of the present invention, the application is in the whole-genome selection breeding of sweet potatoes.

[0033] In some embodiments of the present invention, the application is in the genetic background selection and breeding analysis of sweet potatoes.

[0034] In a fifth aspect of the present invention, a method for breeding sweet potatoes is proposed, comprising the following steps: using at least one of the above-mentioned probes targeting sweet potato SNP molecular marker combinations, sweet potato SNP chips, and kits to detect the DNA of the sweet potato to be tested, and selecting suitable sweet potatoes for subsequent breeding.

[0035] Compared with the prior art, this application has the following advantages and beneficial effects: (1) The sweet potato SNP molecular marker combinations provided in this embodiment of the invention are evenly distributed on chromosomes, with good locus representativeness, strong specificity, and high polymorphism. By identifying the genotypes of the corresponding sweet potato SNP molecular marker combinations in sweet potatoes, molecular marker fingerprinting analysis of sweet potato varietal resources, genotype identification 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 can be performed. It can be widely used for the detection and application of different types of sweet potatoes, such as wild varieties, local varieties, and cultivated varieties, and provides an indispensable and important tool for molecular breeding research such as high-throughput genotyping, gene mapping, fingerprinting, and genome-wide selection of sweet potatoes.

[0036] (2) The sweet potato SNP molecular marker combination provided in this embodiment of the invention was obtained by screening from large-scale sweet potato genome resequencing data. There are a total of 16,730 SNP sites, which include the associated sites of major agronomic traits such as yield, quality and resistance of sweet potato. It has broad application prospects in many fields of sweet potato breeding.

[0037] (3) The sweet potato SNP liquid phase chip provided in this embodiment of the invention is a high-density liquid phase chip; based on the targeted capture sequencing technology, it can genotype the target site, and at the same time, it can also accurately genotype the SNPs of the flanking sequences of the target site, and can obtain more SNP genotyping information than the labeled site; the chip site can be flexibly added or deleted, and the site selection is highly flexible, breaking through the technical limitations of solid phase chips; the liquid phase chip can utilize the second-generation sequencing platform, and the genotyping cost is low and the throughput is high. Attached Figure Description

[0038] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0039] 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, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Attached image:

[0041] Figure 1 This is a distribution map of sweet potato liquid phase chip sites on the genome in an embodiment of the present invention; Figure 2 This is a distribution map of the detection rate of loci in 181 sweet potato F1 (Mianzishu 9 × Daja) in an embodiment of the present invention; where Mianzishu 9 × Daja is the variety name; Figure 3 This is a genetic mapping map of sweet potato QTL (quantitative trait locus) in an embodiment of the present invention. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. It should be noted that any processes or parameters not specifically described in detail below are those that can be understood or implemented by those skilled in the art with reference to the prior art.

[0043] Example 1 The SNP chip provided in this embodiment of the invention is the SweetpotatoGBTS16K liquid-phase SNP chip, as described in the above invention description. The following are specific embodiments of the invention.

[0044] Terminology Explanation: In this embodiment of the invention, "SweetpotatoGBTS16K" is the chip's name.

[0045] Example 1: Design and fabrication of a "SweetpotatoGBTS16K" liquid phase chip Establishment of a sweet potato SNP database This invention utilizes whole-genome resequencing data from 294 sweet potato samples to obtain highly reliable SNP sites for subsequent screening through sequencing analysis.

[0046] The test samples consisted of 294 DNA samples from sweet potato materials. First, whole-genome resequencing was performed on all 294 samples using a DNBSEQ-T7 sequencer (BGI Genomics). The results were then analyzed using the wild sweet potato relative Y22 (… Ipomoea trifida Using the Y22 genome as a reference genome, sequencing data were aligned to the reference genome using BWA software. SNP mutations were identified using the Haplotypecaller module of GATK software, yielding 6,828,068 mutation sites with a minimum allele frequency ≥0.05 and a deletion rate ≤0.5%. This invention utilized 6.81 million SNPs to conduct genome-wide association analysis (GWAS) on 82 major agronomic, quality, and resistance traits of sweet potato (as shown in Table 1 below), obtaining 6,467 significant association QTLs (Quantitative Trait Loci).

[0047] In this embodiment of the invention, a total of 16,730 target SNPs were screened, including 4,490 functional SNPs and 12,240 background SNPs. These target SNPs were used for liquid-phase chip design and fabrication, ensuring that the loci were basically uniformly distributed across the genome with a distance of <1 Mb between adjacent loci. Furthermore, they needed to include important SNPs significant in genome-wide association analysis (as shown in Table 1 below) and important trait-related functional SNPs. Finally, after removing duplicate sites from all candidate loci, a total of 16,730 SNPs were selected as target SNPs for liquid-phase chip design (information on the 16,730 SNP loci is shown in Table 2 in the appendix). Based on the locations of these 16,730 SNP loci and their flanking 200 bp sequences, primers were designed and probes synthesized using targeted capture sequencing technology by Shijiazhuang Borui Biotechnology Co., Ltd., resulting in a sweet potato SNP liquid-phase chip named "SweetpotatoGBTS16K". Figure 1 This is a map showing the distribution of SNP sites on the genome in a sweet potato SNP liquid-phase chip according to an embodiment of the present invention; by Figure 1 It can be seen that SNP sites are relatively evenly distributed on chromosomes.

[0048] Table 1. Number of significant SNP loci for important agronomic traits

[0049] Table 2. Information on 16,730 SNP sites

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[0101] Example 2: Genotyping method of sweet potato liquid phase chip "SweetpotatoGBTS16K" The method for genotyping using the "SweetpotatoGBTS16K" liquid phase chip mainly includes three steps: sample preparation, DNA extraction (refer to the instructions for the GenoPrep® Magnetic Bead Genomic DNA Extraction Kit from Shijiazhuang Borui Biotechnology Co., Ltd.), and DNA library construction and hybridization capture (refer to the GenoBaits® DNA Library Construction & Hybridization Capture Operation Manual from Shijiazhuang Borui Biotechnology Co., Ltd.).

[0102] Specifically, the procedures include: sample DNA extraction, sample DNA fragmentation, sample end repair, sample sequencing adapter ligation, sample DNA purification, sample library amplification, DNA hybridization, DNA capture, quality control of the DNA hybridization capture library, and sequencing of the DNA hybridization capture library. For detailed steps and standard operating procedures, please refer to the Experimental Technology Manual of Shijiazhuang Borui Biotechnology Co., Ltd. (https: / / www.molbreeding.com / ). The instruments and equipment used are shown in Table 3 below.

[0103] Table 3 List of Instruments and Equipment

[0104] The method for genotyping using the "SweetpotatoGBTS16K" liquid phase microarray includes the following steps: (1) Sample preparation The test samples are sweet potato seedlings, leaves, and other tissues or organs, and the test samples must comply with the provisions of GB / T 3543.2.

[0105] (2) DNA extraction Thirty leaves (approximately 200 mg) were randomly selected from the sample and placed in 2.0 mL centrifuge tubes. Polyvinylpyrrolidone (PVPP) was added, and the mixture was thoroughly ground with liquid nitrogen. 700 μL of preheated CTAB extraction solution (65 °C) was added to each tube, and the mixture was thoroughly mixed and incubated in a 65 °C water bath for 30 min, gently inverting the tube during the incubation. An equal volume of premixed chloroform:isoamyl alcohol (24:1) was added to each tube, and the mixture was thoroughly mixed and allowed to stand for 10 min. The mixture was then centrifuged at 10,000 r / min for 10 min at 4 °C. The supernatant was transferred to a new centrifuge tube, and an equal volume of pre-cooled isopropanol was added. The mixture was gently inverted and incubated at -20 °C for 30 min, then centrifuged at 10,000 r / min for 10 min at 4 °C. The supernatant was discarded, and the tubes were washed twice with 70% ethanol solution, air-dried at room temperature, and then dissolved thoroughly in 200 μL of ddH₂O.

[0106] The extracted DNA was detected by electrophoresis on a 1% agarose gel, and the images were analyzed using a gel imaging system. The DNA samples were required to have complete electrophoretic bands without tailing, and the Qubit concentration was greater than 10 ng / uL. The absorbance ratio at 260 nm to 280 nm was between 1.8 and 2.0, and the absorbance ratio at 260 nm to 230 nm was between 1.8 and 2.2.

[0107] The quality requirements for extracted DNA are as follows: DNA must be free from degradation, and the concentration measured by Qubit must be greater than 10 ng / uL.

[0108] DNA extraction was performed using the GenoPrep® Genomic DNA Extraction Kit (provided by Shijiazhuang Borui Biotechnology Co., Ltd.). Specific operating procedures can be found in the kit's instruction manual. The kit components are shown in Table 4 below.

[0109] Table 4. Kit Components

[0110] DNA extraction includes the following steps: 1. Take 20mg of dried plant leaves and crush them thoroughly into powder. Transfer the powder to a centrifuge tube.

[0111] 2. Add 500 μL of PL2 Buffer and 30 μL of Proteinase KSolution 20 mg / mL to the centrifuge tubes respectively, and vortex to mix.

[0112] 3. Incubate in a water bath at 65℃ for 30 minutes, shaking and mixing every 10 minutes. After the water bath, centrifuge at 10,000 rpm for 5 seconds.

[0113] 4. Add 5 μL of RNase Solution (5 mg / mL) to the centrifuge tube, vortex to mix, let stand at room temperature for 10 min, and centrifuge at 10000 rpm for 5 s.

[0114] 5. Add 500 μL of phenol, chloroform and isoamyl alcohol to the centrifuge tube, shake to mix, let stand at room temperature for 5 min, and centrifuge at 12000 rpm for 10 min.

[0115] 6. Transfer 300 μL of the supernatant from step 5 to a new centrifuge tube, add 300 μL of PW Buffer and 360 μL of pre-cooled isopropanol, vortex to mix, let stand at room temperature for 5 min, and add 20 μL of Magnetic Beads Solution.

[0116] 7. Shake to mix for 30 seconds, let stand at room temperature for 1 minute, briefly centrifuge, place the centrifuge tube on a magnetic rack and let stand for 3 minutes until the magnetic beads are completely adsorbed, then discard the supernatant.

[0117] 8. Remove the centrifuge tube from the magnetic rack, add 600 μL of WBI Buffer, vortex to mix for 30 sec, let stand at room temperature for 1 min, centrifuge for 3 sec, place the centrifuge tube on the magnetic rack and let stand for 3 min until the magnetic beads are completely adsorbed, then discard the supernatant.

[0118] 9. Repeat step 8 once.

[0119] 10. Remove the centrifuge tube from the magnetic rack, add 600 μL of WBII Buffer, vortex to mix for 30 sec, let stand at room temperature for 1 min, centrifuge briefly, place the centrifuge tube on the magnetic rack and let stand for 3 min until the magnetic beads are completely adsorbed, then discard the supernatant.

[0120] 11. Repeat step 10 once.

[0121] 12. Keep the centrifuge tubes on the magnetic rack to further remove any remaining liquid inside the tubes. Let them air dry at room temperature for 5-10 minutes until no ethanol remains.

[0122] 13. Remove the centrifuge tube from the magnetic rack, add 100 μL of 1×TE Buffer, vortex to mix for 30 sec, let stand at room temperature for 5-10 min, centrifuge at 10000 rpm for 5 s, transfer the centrifuge tube to the magnetic rack and let stand for 3 min until the magnetic beads are completely adsorbed, then transfer the DNA elution buffer to a new centrifuge tube and store it properly.

[0123] (3) DNA library construction and hybridization capture The components of the kit used for DNA library preparation are shown in Table 5 below. The kit was purchased from Shijiazhuang Borui Biotechnology Co., Ltd.

[0124] Table 5 Components of the DNA Library Kit

[0125] DNA library preparation procedure Step 1: DNA fragmentation and end repair with A (30~50 min) 1. Prepare the end-of-life repair reaction system according to the reagents and dosages shown in Table 6.

[0126] Table 6 End-of-Life Remediation System

[0127] 2. Vortex the above reaction system to mix it thoroughly, and then briefly centrifuge.

[0128] 3. Place the PCR tubes / plates containing the reaction system into the PCR instrument, and set the PCR amplification program according to Table 7, with the hot cap temperature set to 82 ℃.

[0129] Table 7 End-of-Life Repair System Procedures

[0130] 4. Once the above procedure reaches 4 ℃, immediately proceed to step two: connector connection.

[0131] Step 2: Connector Connection (70 min) 1. Add the connector connection system directly to the system in step 3 of step 1 according to the reagents and dosages shown in Table 8.

[0132] Table 8 Connector Connection System

[0133] 2. Vortex the above reaction system to mix it thoroughly, and centrifuge at 10,000 rpm for 10 seconds.

[0134] 3. Place the PCR tubes / plates containing the reaction system into the PCR instrument, set the PCR amplification program according to Table 9, and remove the heat cap.

[0135] Table 9 Connector Connection System Procedure

[0136] 4. Once the above procedure reaches 4 ℃, immediately proceed to step three: purification of the connector connection system.

[0137] Step 3: Purification of the adapter ligation system (30-65 min) 1. Add 48 μL of GenoPrep® DNA Clean Beads to the ligation system in step 3 of step 2, vortex to mix, being careful not to generate air bubbles during vortexing, let stand at room temperature for 5 min, and then centrifuge at 10000 rpm for 10 s.

[0138] 2. Place the PCR tube / plate on a magnetic rack and let it stand at room temperature for at least 3 minutes until the solution is clear. Carefully remove the supernatant (keeping the magnetic beads), making sure not to pick up the magnetic beads.

[0139] 3. Keep the PCR tube / plate on the magnetic rack, add 100 μL of 80% ethanol, let stand at room temperature for 30 seconds, carefully remove all supernatant (keeping the magnetic beads), and let stand at room temperature for at least 5 minutes until the ethanol has completely evaporated.

[0140] 4. Remove the PCR tube / plate from the magnetic rack, add 32 μL of 10 mM Tris-HCl, vortex to mix, let stand at room temperature for 5 min, and then centrifuge at 10000 rpm for 10 s.

[0141] 5. Place the PCR tube / plate on a magnetic rack and let it stand at room temperature for at least 3 minutes until the solution becomes clear.

[0142] 6. Transfer 30 μL of supernatant to a new 0.2 mL low-adsorption PCR tube / plate (retain the supernatant), making sure not to aspirate the magnetic beads.

[0143] 7. Add 16.5 μL (0.55×) of GenoPrep® DNA Clean Beads to the supernatant, vortex to mix the amplification product with the GenoPrep® DNA Clean Beads thoroughly, let stand at room temperature for 5 min, and centrifuge at 10000 rpm for 10 s.

[0144] 8. Place the PCR tube / plate on a magnetic rack and let it stand at room temperature for at least 3 minutes until the solution becomes clear.

[0145] 9. Carefully aspirate the supernatant into a new 0.2 mL low-adsorption PCR tube / plate (retain the supernatant), making sure not to aspirate the magnetic beads.

[0146] 10. Add 4.5 μL (0.15×) of GenoPrep® DNA Clean Beads to the fresh supernatant, vortex to thoroughly mix the amplification product with the GenoPrep® DNA Clean Beads. Incubate at room temperature for 5 min, then centrifuge at 10,000 rpm for 10 s.

[0147] 11. Place the PCR tube / plate on a magnetic rack and incubate at room temperature for at least 2 minutes, or until the solution is clear. Carefully remove the supernatant (keeping the magnetic beads in place), making sure not to pick up the magnetic beads.

[0148] 12. Keep the PCR tube / plate on the magnetic rack, add 100 μL of 80% ethanol, and let stand at room temperature for 30 seconds. Carefully remove the supernatant (keeping the magnetic beads), and let stand at room temperature for 5 minutes until the ethanol has completely evaporated.

[0149] 13. Remove the PCR tubes / plates from the magnetic rack and immediately resuspend the magnetic beads according to the PCR system in step 1 of step four.

[0150] Step 4: Library amplification (30 min) 1. Prepare the PCR amplification system in the PCR tubes / plates according to Table 10 in step 3, step 13.

[0151] Table 10 PCR Amplification System

[0152] 2. Vortex the above reaction system to mix it thoroughly, and centrifuge at 10,000 rpm for 10 seconds.

[0153] 3. Place the PCR tubes / plates containing the reaction system into the PCR instrument and set the PCR amplification program according to Table 11. The hot cap should be set to 105 °C.

[0154] Table 11 PCR amplification system cycling program

[0155] 4. Select the appropriate number of cycles based on Table 12. Table 12 DNA Input Amount and Recommended Amplification Cycles

[0156] 5. Once the above procedure reaches 4℃, the PCR amplification system can be stored at -20℃ overnight (12 hours).

[0157] Step 5: Purification (40 min) 1. Add 40 μL of GenoPrep® DNA Clean Beads to the system in step 3 of step 4, vortex to mix, being careful not to generate air bubbles during vortexing. After standing at room temperature for 5 min, centrifuge at 10000 rpm for 10 s.

[0158] 2. Place the PCR tube / plate on a magnetic rack and incubate at room temperature for at least 3 minutes, until the solution becomes clear. Carefully remove the supernatant (keeping the magnetic beads in place), making sure not to pick up the magnetic beads.

[0159] 3. Keep the PCR tube / plate on the magnetic rack, add 100 μL of 80% ethanol, and let stand at room temperature for 30 seconds. Carefully remove the supernatant (keeping the magnetic beads), and let stand at room temperature for 5 minutes until the ethanol has completely evaporated.

[0160] 4. Remove the PCR tube / plate from the magnetic rack, add 45 μL of 10 mM Tris-HCl, vortex to mix, let stand at room temperature for 5 min, and then centrifuge at 10000 rpm for 10 s.

[0161] 5. Place the PCR tube / plate on a magnetic rack and let it stand at room temperature for at least 3 minutes until the solution becomes clear.

[0162] 6. Transfer the supernatant to a new 0.2 mL low-adsorption PCR tube / plate (retain the supernatant), making sure not to aspirate the magnetic beads.

[0163] 7. The purified DNA library can be stored at -20°C until hybridization or sequencing is required.

[0164] Step Six: Document Quality Inspection 1. Use the Qubit detection reagent or dsDNA fluorescence quantitative reagent (GenoPrep® dsDNA Quantitation Kit, Cat.no.DN000109) to accurately quantify the DNA library.

[0165] 2. The size distribution of the DNA library was examined using agarose gel electrophoresis. Evaluation was performed using an Agilent Technology 2100 Bioanalyzer. The main size range of the DNA fragments used for hybridization capture was 250–500 bp.

[0166] DNA library hybridization capture Step 1: Mixing and drying of reagents 1. Calculate the mixing volume of the libraries based on the measured library concentrations. For example, if the input volume of a single library is 200 ng, and the library concentration is 20 ng / μL, then 10 μL of that library is required. Ensure that the input volume for each library is consistent.

[0167] 2. Mix the library into a new 1.5 mL EP tube according to the calculated volume.

[0168] 3. Add the reagents shown in Table 13 to the tubes above.

[0169] Table 13 Reagents that need to be mixed and dried

[0170] 4. Seal the tube opening with sealing film, make 3-5 small holes, and concentrate it to dryness using a vacuum concentrator at a temperature ≤30℃; 5. After concentration, centrifuge at 12,000 rpm for 1 minute before proceeding with subsequent operations.

[0171] Step 2: DNA library hybridization capture 1. After dissolving the hybridization reagents in Table 14, shake to mix and centrifuge. Add the prepared reaction system to the test tube in step 5 of step one.

[0172] Table 14 Hybridization Capture Reaction Solution

[0173] 2. Mix thoroughly by suction or vortexing, centrifuge at 12000 rpm for 1 min, and let stand at room temperature for 5 min.

[0174] 3. Mix again by suction or vortexing, and centrifuge at 12000 rpm for 5 seconds.

[0175] 4. Transfer the entire 16 μL hybridization capture mix into a 0.2 mL PCR tube.

[0176] 5. Set the programs for the two PCR instruments according to Table 15.

[0177] 6. Thermal cycling incubation conditions: 95 ℃ for 10 min (heat cap temperature 105 ℃).

[0178] 7. Once the PCR amplification instrument has cooled down to 65°C, transfer it to another PCR instrument that is at 65°C (heat cover temperature 75°C), and start timing. The hybridization time is 2 hours or more.

[0179] Table 15 Procedures for hybridization incubation

[0180] Step 3: Prepare the wash buffers 1. Single capture system, dilute GenoBaits® Wash Buffers to 1× working system according to Table 16.

[0181] Table 16 Dilution method for 1X Wash Buffer (amount used per hybridization reaction)

[0182] 2. 100 μL of GenoBaits® 1× Wash Buffer I and 300 μL of GenoBaits® 1× Stringent Wash Buffer need to be preheated at 65 °C.

[0183] 3. Store the remaining GenoBaits® 1× Wash Buffer at room temperature.

[0184] Step 4: Prepare GenoBaits® DNA Probe Beads 1. Vortex the GenoBaits® DNA Probe Beads, which have been equilibrated at room temperature, for 15 seconds to mix thoroughly.

[0185] 2. For each reaction, transfer 50 μL of GenoBaits® DNA Probe Beads into a new 0.2 mL low-adsorption PCR tube.

[0186] 3. Place the PCR tube on a magnetic rack until the solution becomes clear.

[0187] 4. Remove the supernatant with a pipette, keeping the magnetic beads. Remove the PCR tube from the magnetic rack.

[0188] 5. Add 150 μL of GenoBaits® 1X Beads Wash Buffer and vortex for 10 seconds. Place the PCR tube on a magnetic rack until the solution is clear. Carefully remove the supernatant, keeping the magnetic beads.

[0189] 6. Repeat step 5 of step four twice, for a total of three washes.

[0190] Step 5: The hybridization fragment binds to GenoBaits® DNA Probe Beads 1. After the hybridization procedure is complete, open the PCR instrument cap, open the PCR tube cap, and transfer 16 μL of hybridization capture solution into the prepared GenoBaits® DNA Probe Beads.

[0191] 2. Vortex for 10 seconds to mix thoroughly, then centrifuge momentarily.

[0192] 3. Place the PCR tube into the PCR instrument, incubate at 65°C for 45 minutes, with the hot cap at 75°C.

[0193] 4. Vortex for 5 seconds every 12 minutes, then centrifuge briefly.

[0194] Step Six: Wash away unbound DNA 1. Add 100 μL of preheated GenoBaits® 1× WashBuffer Ⅰ to the PCR tube in step 4 of step 5.

[0195] 2. Vortex oscillation for 5 seconds, followed by instantaneous centrifugation.

[0196] 3. Place the PCR tube on a magnetic rack until the solution is clear, then carefully remove the supernatant (keeping the magnetic beads in place), making sure not to pick up the magnetic beads.

[0197] 4. Add 150 μL of GenoBaits® 1X Stringent Wash Buffer preheated to 65 °C, and slowly pump up and down 10 times to thoroughly mix the magnetic beads.

[0198] 5. After the last batch of samples is mixed, place the PCR tube in a PCR instrument at 65 ℃ for 2 min.

[0199] 6. Place the PCR tube on the magnetic rack until the magnetic beads are completely separated from the solution. Quickly remove the supernatant with a pipette, keeping the magnetic beads.

[0200] 7. Repeat steps 4 through 6 of step six.

[0201] 8. Transfer the PCR tube from the magnetic rack, add 150 μL of room temperature GenoBaits® 1× WashBuffer Ⅰ, vortex for 2 min, and then centrifuge briefly.

[0202] 9. Place the PCR tube on a magnetic rack until the solution is clear. Carefully remove the supernatant (keeping the magnetic beads in place), making sure not to pick up the magnetic beads.

[0203] 10. Transfer the PCR tube from the magnetic rack, add 150 μL of room temperature GenoBaits® 1× WashBuffer II, vortex for 1 min, and then centrifuge briefly.

[0204] 11. Place the PCR tube on a magnetic rack until the solution is clear, then carefully remove the supernatant (keeping the magnetic beads in place), making sure not to pick up the magnetic beads.

[0205] 12. Add 150 μL of GenoBaits® 1× Wash Buffer Ⅲ at room temperature, vortex for 30 sec, and then centrifuge briefly.

[0206] 13. Place the PCR tube on a magnetic rack until the solution is clear, then carefully remove the supernatant (keeping the magnetic beads in place), making sure not to pick up the magnetic beads.

[0207] 14. Remove the test tube containing the captured DNA from the magnetic rack and add 20 μL of Nuclease-Free Water.

[0208] 15. Use a pipette to slowly aspirate 10 times to ensure all magnetic beads are resuspended.

[0209] Step 7: PCR enrichment 1. Prepare the reaction system in a 0.2 mL low-adsorption PCR tube according to Table 17.

[0210] Table 17 PCR enrichment system (one-hybrid system)

[0211] 2. Shake to mix and centrifuge briefly to ensure the magnetic beads remain in the solution.

[0212] 3. Place the PCR tubes into the PCR instrument and set the PCR program according to Table 18, with the hot cap temperature set to 105 ℃.

[0213] Table 18 PCR Enrichment Procedure

[0214] Step 8: Purification of PCR Products 1. Place the PCR tube on a magnetic rack until the solution becomes clear, expecting to stand at room temperature for at least 3 minutes.

[0215] 2. Transfer the supernatant to a new PCR tube.

[0216] 3. Add 45 μL of GenoPrep® DNA Clean Beads and vortex to mix.

[0217] 4. After standing at room temperature for 5 minutes, briefly centrifuge.

[0218] 5. Place the PCR tube on a magnetic rack until the solution becomes clear, expecting at least 3 minutes at room temperature. Carefully remove the supernatant (keeping the magnetic beads in place), making sure not to pick up the magnetic beads.

[0219] 6. Keep the PCR tube on the magnetic rack, add 100 μL of 80% ethanol, and let it stand at room temperature for 30 seconds. Carefully remove the supernatant (keeping the magnetic beads), and let it stand at room temperature for 5 minutes until the ethanol has completely evaporated.

[0220] 7. Remove the PCR tube from the magnetic rack to room temperature, add 35 μL of 10 mM Tris-HCl, and vortex to mix. After standing at room temperature for 5 min, briefly centrifuge.

[0221] 8. Place the PCR tube on a magnetic rack until the solution becomes clear, expecting to stand at room temperature for at least 3 minutes.

[0222] 9. Transfer the supernatant to a new 0.2 mL low-adsorption tube, ensuring no magnetic beads are aspirated. The purified DNA hybridization library can be stored at -20 °C until sequencing is required.

[0223] Step Nine: Quality control and sequencing of DNA hybridization capture library 1. It is recommended to use the Qubit detection reagent or the dsDNA fluorescence quantitative reagent (GenoPrep® dsDNAQuantitation Kit, Cat.no.DN000109) to accurately quantify the hybridization library.

[0224] 2. It is recommended to use agarose gel electrophoresis to examine the size distribution of the hybridization library. The main size range of the hybridization library fragments is approximately 250~500 bp.

[0225] 3. Perform sequencing according to the instructions for your Illumina® or MGI® sequencer.

[0226] Data analysis is performed using software: After the obtained genotype sequencing data is quality controlled by FastQ software, the sequencing data is aligned to the "Y22" reference genome using the default parameters of BWA software. SNP identification is performed on the sequencing data using GATK software. Finally, the genotyping information of the probe-captured sequencing is extracted to form the final genotyping file.

[0227] Example 3: Application of the sweet potato liquid phase chip "SweetpotatoGBTS16K" in genotyping. This embodiment is based on the sweet potato liquid phase chip "SweetpotatoGBTS16K" provided in Embodiment 1, and follows the method provided in Embodiment 2 to process 181 sweet potato materials from the F1 (Mianzishu 9 × Daja) population (the 181 sweet potato materials are numbered N1 to N). 182) Perform genotyping testing.

[0228] Using the "SweetpotatoGBTS16K" liquid chromatography chip, and following the genotyping procedure in Example 2, genotyping was performed on 181 sweet potato samples (all of which were Mianzishu 9 × Daja). Data analysis (which can be performed using software as described in Example 2) yielded genotypes that formed a DNA fingerprint database. The results showed that the "SweetpotatoGBTS16K" liquid chromatography chip achieved a detection rate of 97.55%-98.97% for the samples, indicating that this liquid chromatography chip can efficiently perform genotyping of sweet potato samples (e.g., ...). Figure 2 (As shown). Figure 2 The information of the horizontal coordinate from left to right is shown in Table 19 below. Figure 2 The information on the horizontal axis from left to right corresponds to the information from top to bottom in Table 19.

[0229] Table 19 M145 M178 M167 M158 M223 M199 M244 M263 M161 M119 M130 M100 M225 M160 M186 M227 M182 M79 M210 M124 M190 M206 M157 M291 M293 M150 M175 M152 M273 M7 M99 M271 M137 M270 M102 M237 M44 M138 M15 M288 M82 M214 M220 M248 M139 M192 M16 M19 M43 M45 M215 M257 M128 M17 M177 M204 M258 M274 M33 M21 M4 M5 MZ9 M209 M171 M189 M298 M252 M133 M170 M191 M195 M232 M93 M1 M113 M142 M269 M172 M241 M218 M101 M166 M118 M136 M282 M285 M11 M9 M156 M187 M75 M165 M168 M110 daja M205 M23 M250 M297 M299 M20 M229 M106 M211 M22 M266 M90 M13 M155 M181 M255 M92 M230 M251 M176 M228 M267 M116 M202 M91 M208 M140 M292 M261 M164 M233 M286 M284 M256 M123 M132 M254 M277 M197 M280 M27 M163 M141 M289 M87 M122 M28 M238 M114 M131 M14 M103 M245 M104 M154 M240 M151 M272 M188 M290 M143 M243 M185 M260 M98 M134 M25 M125 M279 M236 M275 M249 M126 M296 M264 M262 M278 M235 M265 M6 M112 M109 M120 M203 M115 Example 4: Application of the sweet potato liquid phase chip "SweetpotatoGBTS16K" in gene mapping Genotyping was performed on the F1 (Mianzi Sweet Potato No. 9 × Daja) population in Example 3 to obtain the genotype of each individual. Polymorphic markers in the population were rigorously screened: (1) bases with read coverage less than 40 were filtered out and considered deletions (NA); (2) markers whose genotypes covered at least 80% of all offspring individuals were screened; (3) polymorphic markers with severe segregation were filtered out, ultimately yielding 15,940 high-quality markers. Combining genotype and leaf shape traits of the F1 population, GWASpoly analysis was used to locate a total of 30 significant SNP loci on 15 chromosomes, with 1, 1, 27, and 1 (…) on chromosomes 3, 5, 7, and 9, respectively. Figure 3 (Table 20). The above results show that the "SweetpotatoGBTS16K" liquid phase chip can be used in the genetic mapping of sweet potato genes. It has low genotyping cost, high accuracy, and simple and easy-to-operate data analysis process, and has broad prospects for development and application.

[0230] Table 20 Important SNP sites regulating sweet potato leaf shape

[0231] In summary, through the above embodiments of the present invention, the "SweetpotatoGBTS16K" liquid phase chip of the present invention was obtained by screening from large-scale sweet potato genome resequencing data, with a total of 16,730 SNP loci, including associated loci of major agronomic traits such as sweet potato yield, quality, and resistance, and has broad application prospects in multiple fields of sweet potato breeding; the "SweetpotatoGBTS16K" liquid phase chip of the present invention is a high-density liquid phase chip for sweet potato; based on targeted capture sequencing technology, it can genotype target loci, and at the same time, it can also accurately genotype SNPs in the flanking sequences of target loci, and can obtain more SNP genotyping information than labeled loci; chip loci can be flexibly added or deleted, and the site selection flexibility is high, breaking through the technical limitations of solid phase chips; the liquid phase chip can utilize a second-generation sequencing platform, with low genotyping cost and high throughput.

[0232] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A combination of sweet potato SNP molecular markers, characterized in that, It includes 16,730 SNP molecular markers. The site information of these 16,730 SNP molecular markers was obtained by sequence screening based on the sweet potato reference genome "Y22". The site information is shown in Table 2.

2. A sweet potato SNP chip, characterized in that, The sweet potato SNP chip includes a primer set and / or probes for detecting the sweet potato SNP molecular marker combination of claim 1.

3. A reagent kit, characterized in that, The kit contains the sweet potato SNP chip as described in claim 2.

4. The application of at least one of the sweet potato SNP molecular marker combination of claim 1, the sweet potato SNP chip of claim 2, and the kit of claim 3 in the analysis of sweet potato agronomic traits.

5. The application of at least one of the sweet potato SNP molecular marker combination of claim 1, the sweet potato SNP chip of claim 2, and the kit of claim 3 in the genotyping of sweet potatoes.

6. The application of at least one of the sweet potato SNP molecular marker combination of claim 1, the sweet potato SNP chip of claim 2, and the kit of claim 3 in the identification of sweet potato varieties.

7. The application of at least one of the sweet potato SNP molecular marker combination of claim 1, the sweet potato SNP chip of claim 2, and the kit of claim 3 in the genetic mapping of sweet potato genes.

8. The application of at least one of the sweet potato SNP molecular marker combination of claim 1, the sweet potato SNP chip of claim 2, and the kit of claim 3 in genome-wide association analysis of sweet potato.

9. The application of at least one of the sweet potato SNP molecular marker combination of claim 1, the sweet potato SNP chip of claim 2, and the kit of claim 3 in the whole genome selection breeding of sweet potato.

10. A method for breeding sweet potatoes, characterized in that, The process includes the following steps: using at least one of the sweet potato liquid phase chip of claim 2 and the kit of claim 3 to detect the DNA of the sweet potato to be tested, and selecting suitable sweet potatoes for subsequent breeding.

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