Soybean snp molecular marker combination, chip and application thereof

Abstract

The application discloses a soybean SNP molecular marker combination, a chip and application thereof, relates to the field of soybean molecular genetics and breeding. The SNP molecular marker combination developed by the application is uniformly distributed on 20 chromosomes of the whole genome of soybean, and can improve the accuracy of related research and application of gene positioning. The SNP molecular marker combination provided by the application has rich information, and has important value in variety identification, correlation analysis and molecular marker assisted breeding. Based on this, a SNP typing chip, a kit and other detection products with high capture rate, high density, high polymorphism and uniform distribution in the genome can be developed.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of soybean molecular genetics and breeding, in particular, to a soybean SNP molecular marker combination, a chip and application thereof. BACKGROUND

[0002] Traditional morphological markers have limited distinguishing ability and are greatly affected by the environment, and have the disadvantages of large identification workload, long period, season limitation, etc. Molecular markers based on DNA polymorphism have become a powerful tool for analyzing biological genetic diversity. These molecular markers have the advantages of being unaffected by the environment, having a large number of markers to choose from, and being able to perform high-throughput testing and analysis, and have become an important means for analyzing the genetic relationship of crops, identifying seed purity, and detecting the authenticity of varieties.

[0003] Single-nucleotide polymorphism (SNP) refers to the polymorphism of DNA sequence caused by the variation of a single nucleotide at the genomic level. Compared with SSR markers, SNP markers have more advantages: first, SNP sites are more abundant and more evenly distributed in the genome; second, since SNP does not use DNA length difference as a detection method, it is not limited by gel electrophoresis, and is easy to realize high-throughput and automation of analysis; third, SNP sites usually have only two alleles, making it easy to integrate data between platforms.

[0004] Soybean originated in China and is a major source of human plant protein and fat, and is one of China's major crops. Many of the stored germplasm resources have only phenotypic trait records, and lack in-depth genetic identification. The large number of varieties increases the difficulty of soybean variety management, and the analysis of soybean genetic resources, variety identification and functional genomics research are heavily dependent on the development of high-density molecular markers. With the completion of soybean whole genome sequencing and the development of resequencing technology, the development of SNP sites has made important progress in soybean. Through resequencing of wild soybean and cultivated soybean, millions of SNP sites can be screened from the soybean genome. However, due to limitations in computing resources, chip throughput, etc., blindly increasing density will not only not improve accuracy, but will also lead to rising costs. Therefore, it is urgent to develop a SNP typing chip with high capture rate, high density, high polymorphism, and uniform distribution in the genome.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The present application aims to provide a soybean SNP molecular marker combination, a chip and application thereof to solve the above technical problems.

[0007] The present application is implemented as follows:

[0008] Noun explanation:

[0009] SNP (Single Nucleotide Polymorphism): Single nucleotide polymorphism refers to DNA sequence polymorphism caused by single nucleotide variation at the genomic level.

[0010] Bi-allelic SNP: Bi-allelic SNP site refers to a SNP site with only two alleles.

[0011] MAF (Minor Allele Frequency): Minor allele frequency (minor allele frequency) usually refers to the frequency of rare alleles of a site in a specific population.

[0012] SNP site detection rate: The ratio of samples in which a SNP site is successfully detected to all samples, generally ≥ 90%, the specific situation will be adjusted according to the target data and the number of target sites. When analyzing SNP genotype, the minimum depth is generally ≥ 10X, that is, the target site is covered by at least 10 reads.

[0013] In a first aspect, the present application provides a soybean SNP molecular marker combination, which comprises 35196 SNP molecular markers, and the information of the 35196 SNP molecular markers is as follows:

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[0106] Wherein, 1-20 on the left side of the site number represents the number of the chromosome where the site is located, the middle value represents the position of the site on the chromosome of the reference genome, the base type in the site number represents the SNP base type of the site in the reference genome, and the version number of the whole genome sequence of the reference genome is Glycine_max_v4.0.

[0107] The present application analyzes 2040 soybean resequencing samples, and a total of 43,377,445 SNP sites are obtained. Through filtering of double-allele SNP sites, sequencing depth ≥10, MAF ≥0.01, single-copy region of the genome and retaining one effective marker every 5kb, a total of 42,450 high-quality SNP marker combinations are finally obtained. And the 42,450 high-quality SNP marker combinations are evaluated for subsequent probe design, and a total of 35,196 sites are evaluated.

[0108] Through detection, the SNP molecular marker combination has a site detection rate of more than 96% for 20 soybean samples with a resequencing data amount of 2Gb. The SNP molecular marker combination developed by the present application is uniformly distributed on the 20 chromosomes of the soybean whole genome, which can improve the accuracy of related research applications such as gene positioning. The SNP molecular marker combination provided by the present application has rich information, and has important value in variety identification, association analysis and molecular marker assisted breeding. Based on this, a SNP typing chip, kit and other detection products with high capture rate, high density, high polymorphism and uniform distribution in the genome can be developed.

[0109] In a second aspect, the present application provides a soybean whole genome SNP detection product, comprising: nucleotide sequences for detecting the soybean SNP molecular marker combination described above.

[0110] In a preferred embodiment of the application, the detection product is selected from a chip, a reagent or a kit.

[0111] The chip can also be called a suspension array or a liquid array. It includes a carrier and nucleic acid molecules (such as primers and / or probes) bound on the surface of the carrier.

[0112] The aforementioned carrier can be of various materials and forms, for example, can be preferably selected from containers with flat bottoms. A more typical preferred example is a multi-well plate, a microplate, a microfluidic-based device (e.g., a microfluidic chip), a petri dish-like container, and the like, and is not limited thereto.

[0113] The microfluidic chip is selected from a PDMS chip of a T-shaped chip, a flow focusing type chip, or a coaxial flow type chip, or a PMMA microfluidic chip.

[0114] The soybean whole genome SNP detection product can further detect the soybean whole genome SNP by means of high-throughput sequencing or imaging detection, and is not limited thereto.

[0115] In a preferred embodiment of the application, the nucleotide sequence is a primer sequence and / or a probe sequence.

[0116] Further, the kit can further comprise an instruction manual, a detection reagent, and the like. A more typical detection reagent includes at least one of a detection antibody or a ligand labeled with a fluorescent dye, a detection antibody or a ligand labeled with biotin, an avidin-fluorescent protein (preferably, phycoerythrin) conjugate, an avidin-horseradish peroxidase complex, a chemiluminescent reagent (e.g., luminol, a luminol derivative, acridinium ester, luciferase, and an oxidizing agent such as hydrogen peroxide), and the like, and is not limited thereto.

[0117] In an alternative embodiment, the fluorescent dye includes, but is not limited to, a fluorescein-based dye and a derivative thereof (e.g., including, but not limited to, fluorescein isothiocyanate (FITC), FAM, tetra-chloro fluorescein (TET), and the like or an analog thereof), a rhodamine-based dye and a derivative thereof (e.g., including, but not limited to, red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like or an analog thereof), a Cy series dye and a derivative thereof (e.g., including, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, and the like or an analog thereof), an Alexa series dye and a derivative thereof (e.g., including, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like or an analog thereof), and a protein-based dye and a derivative thereof (e.g., including, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), and the like).

[0118] Further, the kit can further comprise at least one of a buffer, a detection reagent, a diluent, a washing solution, and the like, and is not limited thereto.

[0119] In a third aspect, the present application provides application of at least one soybean SNP molecular marker in the soybean SNP molecular marker combination or the above-mentioned soybean whole-genome SNP detection product in soybean genotyping.

[0120] In a preferred embodiment of the application, the soybean germplasm resources or genetic population is genotyped.

[0121] In a fourth aspect, the present application provides application of at least one soybean SNP molecular marker in the soybean SNP molecular marker combination or the above-mentioned soybean whole-genome SNP detection product in soybean whole-genome association analysis.

[0122] In a fifth aspect, the present application provides application of at least one soybean SNP molecular marker in the soybean SNP molecular marker combination or the above-mentioned soybean whole-genome SNP detection product in soybean cluster analysis and kinship identification.

[0123] In a sixth aspect, the present application provides application of at least one soybean SNP molecular marker in the soybean SNP molecular marker combination or the above-mentioned soybean whole-genome SNP detection product in establishing DNA fingerprint of soybean germplasm resources or varieties, soybean genetic diversity analysis or soybean molecular marker assisted breeding.

[0124] In a seventh aspect, the present application provides application of at least one soybean SNP molecular marker in the soybean SNP molecular marker combination in preparing a soybean SNP site detection product, and the detection product is selected from a chip, a reagent or a kit.

[0125] In an eighth aspect, the present application provides a method for genotyping soybean germplasm resources or genetic population, comprising the following steps: designing detection primers and / or probes based on at least one soybean SNP molecular marker in the above-mentioned soybean SNP molecular marker combination, respectively, performing PCR amplification on DNA of a test soybean sample based on the detection primers and / or probes or using the above-mentioned soybean whole-genome SNP detection product; and detecting the PCR amplification product.

[0126] In a preferred embodiment of the application, a DNA library of the test soybean sample is first constructed, and then the probe is incubated with the DNA library; after incubation, the DNA hybridization product library is captured; and then the captured DNA hybridization library is sequenced.

[0127] For example, the probe is incubated with the DNA library at 65-66℃ for 2-3 hours.

[0128] In a preferred embodiment of the application, the library capturing comprises: incubating the DNA hybridization product with magnetic beads, and then performing library amplification on the combination of the magnetic beads and the DNA hybridization product; and performing treatment on the library amplification product to obtain the DNA hybridization library.

[0129] The treatment on the library amplification product comprises: performing a 65℃ heat washing on the combination of the magnetic beads and the DNA hybridization product, and then performing normal-temperature washing on the magnetic beads with a magnetic bead washing solution respectively. The washed magnetic beads are resuspended with water.

[0130] Further, after sequencing the captured DNA hybridization library, the method further comprises performing FastQC (www.bioinformatics.babraham.ac.uk / project) quality control on the sequencing data.

[0131] In a ninth aspect, the application provides a method for constructing a DNA fingerprint of a soybean germplasm or variety, comprising the following steps:

[0132] Based on at least one of the above-mentioned combinations of soybean SNP molecular markers, detection primers and / or probes are designed, and the genotypes of the SNP molecular markers are detected based on the detection primers and / or probes or by using the above-mentioned soybean whole-genome SNP detection product. The genotypes of the SNP molecular markers of each soybean germplasm or variety are arranged in order of site number to form a specific sequence combination, and the specific sequence combination is the DNA fingerprint of the soybean germplasm or variety.

[0133] In an alternative embodiment, when constructing the DNA fingerprint of a soybean germplasm or variety, the above-mentioned 35,196 SNP molecular markers are optimized or screened or classified, and a part or all of the SNP molecular markers are selected for the fingerprint design of wild or cultivated soybeans. Thus, a more accurate and brief fingerprint or a more accurate and detailed fingerprint is constructed, which is adjusted by a person skilled in the art according to needs.

[0134] In an alternative embodiment, the method for constructing the DNA fingerprint of a soybean germplasm or variety comprises the following steps: S1. collecting a soybean variety, tissue or DNA sample; S2. performing whole-genome scanning analysis on the above-mentioned 35,196 soybean SNP molecular markers of the sample by using the chip product in the application; S3. analyzing the genetic differences between the soybean samples according to the genotyping results; S4. verifying the accuracy of part of the SNP markers by using the competitive allele-specific PCR (KASP) technology; and S5. constructing a high-density fingerprint of different varieties.

[0135] The application has the following beneficial effects:

[0136] The SNP molecular marker combination developed by the application is uniformly distributed on 20 chromosomes of the whole genome of soybean, has the advantages of high accuracy, rich variation, simple operation, etc., and the developed molecular marker is not affected by environmental factors and can directly reflect the difference of the gene level of soybean, so it can be widely applied to the whole genome correlation analysis of soybean, genetic map construction, soybean population typing and identification, and soybean resource protection. Through sample test verification, the inventors found that the SNP molecular marker combination has a site detection rate of more than 96% when the data amount of the soybean sample is about 2 Gb, indicating that the SNP molecular marker combination provided by the application has a large amount of useful information and has great utilization value. In addition, the SNP molecular marker combination developed by the application is uniformly distributed on 20 chromosomes of the whole genome of soybean, which can improve the accuracy of related research applications. Based on this, a SNP typing chip, kit and other detection products with high capture rate, high density, high polymorphism and uniform distribution in the genome can be developed. BRIEF DESCRIPTION OF DRAWINGS

[0137] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some of the embodiments of the application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0138] Figure 1 Process diagram for identification and screening of SNP markers;

[0139] Figure 2 Probe design and coverage statistical result diagram;

[0140] Figure 3 Site detection rate statistical table of 20 samples;

[0141] Figure 4 Distribution diagram of SNP marker number on different chromosomes;

[0142] Figure 5 Uniform distribution diagram of SNP markers on different chromosomes;

[0143] Figure 6 MAF distribution statistical result diagram;

[0144] Figure 7 Distribution diagram of SNP markers on gene structure. DETAILED DESCRIPTION

[0145] Reference will now be made in detail to embodiments of the application, one or more examples of which are described hereinbelow. Each example is provided as an explanation and not a limitation of the application. Indeed, it will be apparent to one of ordinary skill in the art that numerous modifications and variations of the present application are possible in light of the above teachings. For example, features described or illustrated as part of one embodiment can be used with another embodiment to yield still a further embodiment.

[0146] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. The specific conditions not noted in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not noted by the manufacturers are all conventional products that can be obtained by market purchase.

[0147] The features and performances of the present application are further described in detail below in combination with the embodiments.

[0148] Embodiment 1

[0149] The present embodiment provides a method for developing and screening a combination of soybean 35K SNP molecular markers.

[0150] (1) 2040 soybean resequencing samples were analyzed, and a total of 43,377,445 SNP sites were obtained. By filtering double-allele SNP sites, sequencing depth ≥10, MAF ≥0.01, and retaining one effective marker per 5 kb in the single-copy region of the genome, a total of 42,450 high-quality SNP marker combinations were finally obtained. The identification and screening process of the SNP markers is referred to Figure 1 .

[0151] (2) Chip site evaluation

[0152] Through the above selection process, a total of 42,450 background sites were finally obtained for subsequent probe design evaluation. Among them, a total of 35,196 sites were evaluated, and 63,352 probes were used for subsequent test analysis. Figure 2 ).

[0153] Experimental Example 1

[0154] 20 random soybean samples were detected, and the specific method was as follows: library construction, hybridization capture, library quality inspection and sequencing analysis.

[0155] 1. Library construction

[0156] The construction of the DNA library was carried out according to the method of the GenoBaits library construction kit.

[0157] 2. Library hybridization capture

[0158] The genotype of the target sample at the 35K SNP site is determined by liquid phase gene chip.

[0159] (1) DNA hybridization: 500 ng of the constructed sample genomic DNA sequencing library was taken, 5 μL of GenoBaits Block I and 2 μL of GenoBaits Block II were added, and the mixture was placed on an Eppendorf Concentrator plus vacuum concentrator and evaporated to dry powder at a temperature of ≤70°C. 8.5 μL of GenoBaits 2xHyb Buffer, 2.7 μL of GenoBaits Hyb Buffer Enhancer, and 2.8 μL of Nuclease-Free Water were added to the dry powder tube, mixed by pipetting, and then placed in an ABI 9700 PCR instrument for incubation at 95°C for 10 min. Then, 3 μL of the synthesized probe (the concentration of the probe was 60 ng / μL) was added to the PCR tube, vortexed to mix, and then placed in an ABI 9700 PCR instrument for incubation at 65°C for 2 hours to complete the probe hybridization reaction.

[0160] (2) DNA capture

[0161] 100 μL of GenoBaits DNA Probe Beads was added to the reaction system after the hybridization was completed in the previous step, and the mixture was pipetted up and down for 10 times, and then placed in an ABI 9700 PCR instrument for incubation at 65°C for 45 min to allow the magnetic beads to bind to the probe. The magnetic beads after binding to the probe were washed at 65°C with 100 μL of GenoBaits Wash Buffer I and 150 μL of GenoBaits Wash Buffer II, respectively, and then washed at room temperature with 100 μL of GenoBaits Wash Buffer I, 150 μL of GenoBaits Wash Buffer II, and 150 μL of GenoBaits Wash Buffer III. The washed beads were resuspended with 20 μL of Nuclease-Free Water.

[0162] 13 μL of the resuspended DNA (with magnetic beads) was taken and added to a new 0.2 mL PCR tube, then 15 μL of GenoBaits PCR Master Mix and 2 μL of GenoBaits Primer Mix were added to configure the post-PCR system, and the library was amplified using an ABI 9700 PCR instrument. The amplification program was as follows: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 60°C annealing for 30 s, and 72°C extension for 30 s; repeat 2-4 steps for a total of 15 cycles; and 72°C extension for 5 min.

[0163] Add 45 μL BeckmenAMPure XP Beads (Beackman) to the post-PCR product and mix well with pipette, then place the 0.2 mL PCR tube on the magnetic stand until the solution is clear, discard the supernatant and wash the magnetic beads twice with 75% ethanol, and elute the library DNA with Tris-HCl (pH 8.0). Complete the hybridization capture work of the probe.

[0164] (3) DNA hybridization capture library quality control

[0165] Determine the DNA concentration of the library by Qubit Fluorometric Quantitation (Thermo Fisher), and then detect the fragment size of the library DNA by agarose gel electrophoresis to determine whether the fragment size is between 300-400 bp.

[0166] (4) DNA hybridization capture library sequencing

[0167] The constructed DNA library is sequenced by Shijiazhuang Boruitai Biotechnology Co., Ltd.

[0168] (5) Genotype data analysis

[0169] After the sequencing data is subjected to

[0170] FastQC (www.bioinformatics.babraham.ac.uk / project) quality control, the data is aligned to the reference genome using BWA software (v0.7.17), and SNP identification is performed on the sequencing data using GATK software. The site test results are shown in Figure 3 .It is shown that the site detection rate of the SNP molecular marker provided by the present application exceeds 96% in 20 soybean populations. It is shown that the SNP molecular marker combination provided by the present application has a large amount of useful information and has a large utilization value. Figure 3

[0171] Figure 4 is a distribution diagram of 35,196 SNP markers on different chromosomes, and the number of target sites on different chromosomes is shown. The horizontal coordinate is the chromosome ID; the left example Count is the number of sites / number of segments; and the right example Length is the length of the chromosome (unit: bp). The results show that the SNP sites are evenly distributed on the 20 chromosomes.

[0172] Figure 5 is a diagram showing the uniform distribution of SNP markers on different chromosomes, showing 20 soybean chromosomes, and the drawing unit in the diagram is 1,000,000 bp.

[0173] Figure 6 Figure 5 is a statistical result chart of MAF distribution, Figure 6 is a statistical result chart of MAF distribution, Figure 6 Figure 5 shows that the SNP combination provided by the present application has a large amount of information, and the number of markers greater than 0.1 accounts for 67%. The statistical value of MAF of the core site in the section, the greater the MAF value of the target site, the better the polymorphism of the site. The abscissa MAF is different ranges of MAF values, and the ordinate Count is the number of core sites in the MAF range.

[0174] Figure 7 Figure 6 is a distribution chart of SNP markers in gene structure, which shows that the SNP in the intergenic sequence is the most, followed by intron, upstream and downstream of gene, CDS region and UTR region. The abscissa is the type of gene structure, and the ordinate is the number of target sites in the type.

[0175] The present application screens 35,196 sites and 63,352 corresponding probes which meet the evaluation requirements for subsequent test analysis. When the data amount of 20 samples used for testing is 2 Gb, the site detection rate is between 96.3% and 97.6%.

[0176] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A soybean whole-genome SNP chip, characterized in that, It includes: a probe for detecting soybean SNP molecular marker combinations, which consist of 35,196 SNP molecular markers. The information of the 35,196 SNP molecular markers is as follows: In this context, the numbers 1 to 20 on the left indicate the number of chromosomes on which the locus is located, the middle number indicates the position of the locus on the chromosome of the reference genome, the base type in the locus number indicates the SNP base type of the locus in the reference genome, and the version number of the whole genome sequence of the reference genome is: Glycine_max_v4.

0.

2. A soybean whole-genome SNP detection kit, characterized in that, It includes: Nucleotides used for detecting the soybean SNP molecular marker combination as described in claim 1, wherein the nucleotides are primer sequences and probe sequences.

3. The application of the soybean whole genome SNP chip of claim 1 or the soybean whole genome SNP detection kit of claim 2 in soybean genotyping; genotyping of soybean germplasm resources or genetic populations.

4. The application of the soybean whole genome SNP chip of claim 1 or the soybean whole genome SNP detection kit of claim 2 in soybean genome-wide association analysis.

5. The application of the soybean whole genome SNP chip as described in claim 1 or the soybean whole genome SNP detection kit as described in claim 2 in soybean cluster analysis and kinship identification.

6. The application of the soybean whole genome SNP chip as described in claim 1 or the soybean whole genome SNP detection kit as described in claim 2 in establishing DNA fingerprinting of soybean germplasm resources or varieties, analyzing soybean genetic diversity, or using soybean molecular marker-assisted breeding.

7. A method for genotyping soybean germplasm resources or genetic populations, characterized in that, The process includes the following steps: designing detection primers and probes based on the soybean SNP molecular markers in the soybean SNP molecular marker combination described in claim 1; performing PCR amplification on the DNA of the soybean sample based on the detection primers and probes or the soybean whole genome SNP detection kit described in claim 2; and detecting the PCR amplification products. The amplification products were subjected to fluorescence scanning, and the fluorescence signals were read and analyzed to obtain the genotyping results of the DNA of the tested soybean sample at the SNP sites.

8. The method for genotyping soybean germplasm resources or genetic populations according to claim 7, characterized in that, First, a DNA library of the soybean sample to be tested is constructed. Then, the probe is mixed with the DNA library and incubated. After incubation, the DNA hybridization product library is captured. Then, the captured DNA hybridization library is sequenced.

9. The method for genotyping soybean germplasm resources or genetic populations according to claim 8, characterized in that, The library capture includes: incubating the DNA hybridization product with magnetic beads, then amplifying the combination of the magnetic beads and the DNA hybridization product into a library; and processing the amplified library to obtain a DNA hybridization library.

10. A method for constructing DNA fingerprints of soybean germplasm resources or varieties, characterized in that, Includes the following steps: Based on the soybean SNP molecular markers in the soybean SNP molecular marker combination described in claim 1, detection primers and probes are designed respectively. Based on the detection primers and probes, or using the soybean whole genome SNP chip of claim 1 or the soybean whole genome SNP detection kit of claim 2, genotyping of soybean germplasm resources or varieties is performed. The genotype of the SNP molecular marker combination is detected. For each soybean germplasm resource or variety, the genotypes of the SNP molecular markers are combined and arranged in the order of the locus number to form a specific sequence combination. The specific sequence combination is the DNA fingerprint of the soybean germplasm resource or variety.

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