Sorghum whole genome SNP site combination, liquid chip and application thereof

Abstract

The application discloses a sorghum whole genome SNP site combination, a liquid phase chip and application thereof. The SNP site is obtained after filtering and evaluation of whole genome resequencing data of 445 sorghum inbred lines by comparison to a sorghum reference genome BTx623 (version 3.1.1). The SNP site combination is uniformly distributed in the whole genome of sorghum. The provided 9K liquid phase gene chip of sorghum can simultaneously detect 9654 SNP sites, and is the first liquid phase chip in sorghum. The application provides an efficient genome typing tool for genetic diversity analysis, variety identification, gene positioning, background screening and molecular marker assisted breeding of sorghum, and helps shorten the breeding cycle of sorghum and promote the breeding process of new sorghum varieties.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular breeding technology, specifically involving a combination of SNP sites in the whole genome of sorghum, a liquid phase chip, and their applications. Background Technology

[0002] To meet national energy and food security needs, there is an urgent need to develop high-yield, multi-purpose sorghum varieties suitable for marginal soil cultivation, serving as grain, sugar, feed, and fiber. Compared to the demands of production practices for sorghum varieties, sorghum breeding has lagged behind, necessitating a shift from traditional breeding methods to more efficient whole-genome selection breeding techniques. Whole-genome SNP genotyping is crucial for the transition from theoretical research to practical application of whole-genome selection technology. Therefore, developing a genome-wide representative SNP combination and utilizing the latest probe hybridization-based liquid-phase probe capture sequencing technology for high-throughput, low-cost genotyping of sorghum populations is essential. Summary of the Invention

[0003] The purpose of this invention is to provide a sorghum genome-wide SNP locus combination, a liquid-phase chip, and their applications. This allows for the rapid detection of SNPs in sorghum populations, enabling efficient and rapid sorghum genotyping and solving the problem that a large amount of existing sorghum genetic information cannot be applied to practical breeding.

[0004] To achieve the above objectives, the technical solution of the present invention is summarized as follows:

[0005] This technical solution provides a combination of SNP sites in the whole sorghum genome. Based on this combination, sorghum 9K probes can be designed. Furthermore, a sorghum whole genome liquid-phase microarray is disclosed. This microarray is a sorghum 9K liquid-phase gene microarray based on targeted capture sequencing. The sorghum 9K liquid-phase microarray consists of individually packaged sorghum 9K probe mixtures and hybridization capture reagents. The sorghum 9K probes are DNA double-stranded probes, which are nucleotide sequences designed and synthesized based on the screened SNP sites. The SNP sites were obtained by aligning 445 sorghum resequencing data to the BTx623 (v3.1.1) genome using bwa software, followed by filtering to obtain a dataset containing 9654 SNPs that are distributed as evenly as possible throughout the sorghum genome. Specifically, the location information of the SNP sites is shown in Table 1.

[0006] Table 1 shows the specific locations of 19,654 SNP loci on the sorghum reference genome “BTx623, v3.1.1”.

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[0033] In the table, No. represents the number, Chrom. represents the chromosome, and Position represents the specific location on the sorghum reference genome “BTx623, v3.1.1”.

[0034] This invention also provides a method for efficiently detecting sorghum SNPs and then performing genotyping, the method comprising the following steps:

[0035] The liquid probe described above is hybridized with the DNA library of the sample to be tested to capture the DNA library that has hybridized with the probe and the target region. The DNA library is then amplified by PCR, high-throughput sequencing, and SNP detection for genotyping.

[0036] The DNA library of the test sample is obtained by fragmentation and end repair, adapter ligation, library amplification and purification of the total DNA of the test sample.

[0037] The present invention also provides a genotyping kit for sorghum populations, the kit comprising the above-mentioned liquid phase probe set and / or the above-mentioned 9654 SNP markers.

[0038] When designing and screening relevant probes, efforts are made to ensure that SNP sites are evenly distributed throughout the sorghum genome. The probes are designed according to the principles of liquid-phase probe design. These principles include: 1) single copy on the reference genome; 2) GC content between 0.35 and 0.65; and 3) no repetitive sequences within the probe. Furthermore, in practical applications, commonly used probe design principles in existing technologies can be employed.

[0039] This invention also provides the above-mentioned sorghum whole genome SNP locus combinations and sorghum whole genome liquid phase chip applications in the genetic diversity analysis of sorghum germplasm resources, sorghum population structure analysis, sorghum kinship identification, gene mapping and molecular-assisted breeding.

[0040] Advantages of this invention:

[0041] (1) This invention provides a combination of SNP sites in the whole genome of sorghum. The combination of SNP sites is relatively evenly distributed in the whole genome of sorghum, which provides a basis for the development of liquid phase probes and liquid phase gene chips for the whole genome of sorghum.

[0042] (2) The provided sorghum 9K liquid phase gene chip can be applied to basic research and molecular breeding of sorghum. It can simultaneously detect 9654 SNP sites, making it the first liquid phase chip for sorghum. The number, density, and efficiency of the SNPs contained in this chip are also relatively high.

[0043] (3) This invention provides an efficient genomic typing tool for sorghum genetic diversity analysis, variety identification, gene mapping, background screening, and molecular marker-assisted breeding. Furthermore, based on existing research, this invention allows for the addition of new probes to existing loci at any time, without requiring large sample volumes, thus meeting the diverse usage scenarios and requirements of users. It has high application value in sorghum molecular breeding.

[0044] (4) The present invention also provides a method for efficient genotyping of sorghum populations. Due to the use of high-throughput sequencing genotyping technology, the procedure is simple and standardized, reducing individual operation errors and making the unit price of batch identification analysis extremely low. It has the characteristics of being economical and efficient, and is particularly suitable for large sample analysis, with good application prospects. Attached Figure Description

[0045] Figure 1 It is the distribution of 9654 SNPs on chromosomes used for probe design and genotyping;

[0046] Figure 2 It consists of 445 resequencing samples based on principal component analysis of genome-wide variation;

[0047] Figure 3 Principal component analysis of 445 resequencing samples based on 9K liquid-phase chip sites;

[0048] Figure 4 Principal component analysis of resequencing materials and test materials based on 9K liquid-phase chip capture sites (A1 R1 A2R2 B2 are test materials);

[0049] Figure 5 This is the background response rate distribution of 233 materials. Detailed Implementation

[0050] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.

[0051] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0053] I. Screening of SNP loci combinations across the entire sorghum genome, design of liquid-phase probes, and development of microarray-based methods.

[0054] Based on the sorghum reference genome (v3.1.1) and SNP marker combinations detected by resequencing 445 sorghum whole genomes (SNP dataset), a non-redundant SNP dataset of 9654 SNPs was obtained after filtering. The distribution of the 9654 SNPs dataset on chromosomes for probe design and genotyping is shown in [link to relevant documentation]. Figure 1 .

[0055] The method for designing liquid phase probes based on the aforementioned SNPs dataset includes the following steps:

[0056] (1) Genomic DNA was extracted from leaf tissues of 445 sorghum materials using the CTAB method. After the DNA passed quality inspection, it was used to construct a whole genome resequencing library. Fragments in the range of 260-350bp were selected, enriched and amplified. After the library passed inspection, it was sequenced using the Illumina Hi-seq 2000 sequencing platform.

[0057] (2) Using the BTx623 genome (v3.1.1) as a reference genome, the clean data obtained from sequencing in step (1) was aligned to the genome using bwa, and GATK variant detection was performed to obtain 23,992,689 high-quality SNP loci. The SNP filtering parameters were: QD<2.0, QUAL<30.0, SOR>3.0, FS>60.0, MQ<40.0, MQRankSum<-12.5, ReadPosRankSum<-8.0. The obtained SNP loci were converted to their original format using vcftools and plink software, and then principal component analysis and plotting were performed using gcta software. Figure 2 ).

[0058] (3) Using Perl scripts, marker combinations were screened according to the selection criteria, resulting in 9654 SNP marker combinations (SNPs2 dataset). The molecular markers for genotyping / population structure analysis of sorghum populations were successfully constructed. Marker information is shown in Table 1.

[0059] The principle for filtering SNPs using Perl scripts is as follows:

[0060] Chromosomes were evenly distributed, with one marker retained within 2kb. SNP sites and their upstream and downstream 100bp sequences were extracted using Bedtools software, retaining SNP sites with GC content between 35-65% and no N bases in the sequence. The extracted sequences were aligned to the sorghum BTx623 genome (v3.1.1) using blastn software, retaining only single-copy SNP sites. Sites meeting the following criteria were added: maximum deletion rate of 0.5, small allele greater than or equal to 0.05, and LD less than or equal to 0.2. Finally, one marker was retained for every 20kb.

[0061] (4) Analyze and plot the 9654 SNPs selected in step (3) against the composition of 445 resequencing materials. Figure 3 As shown in the figure, the principal component analysis results for different types of sorghum are basically consistent with the results obtained from whole-genome SNPs. Conclusion: 9654 SNP markers can be used for sorghum population structure analysis and genotyping.

[0062] (5) The 9654 SNP markers (SNPs2 dataset) obtained in step (3) were used to design liquid phase probe sequences for subsequent capture sequencing.

[0063] Finally, the liquid-phase probe containing the above 9654 SNP marker combinations was developed according to the conventional design principles of liquid-phase chips to obtain the Sorghum 9K liquid-phase chip based on targeted capture sequencing.

[0064] Example 2: Sorghum population structure analysis using the liquid phase probe corresponding to the SNPs2 dataset described in Example 1.

[0065] 1. Genomic DNA extraction

[0066] Sorghum leaves were collected, preserved with ice packs, and promptly transported back to the laboratory. Total DNA from the sorghum was extracted using the CTAB method.

[0067] 2. Genomic DNA fragmentation and end repair

[0068] Prepare the following reaction system in sterile PCR tubes placed on ice (Table 2).

[0069] Table 2 List of whole-genome DNA fragmentation systems

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[0072] Where X represents any volume not exceeding 20 μL, which is added together with 4.5 μL of buffer and 5 μL of enzymes. Any volume less than 30 μL is made up with 1×TE buffer. Use a pipette to gently pipette up and down or vortex to ensure thorough mixing, then briefly centrifuge. Immediately place the PCR tube on a preheated PCR instrument at 32°C and run the reaction program shown in Table 3. Set the PCR instrument's heating cap to 75°C. Once the temperature drops to 4°C, immediately place the tube on ice for the next experiment; do not maintain the temperature at 4°C. Specifically, the fragmentation and end-repair reaction programs are shown in Table 3.

[0073] Table 3 Fragmentation and Terminal Repair Response Procedures

[0074] temperature time 32℃ 20min 65℃ 30min 4℃ Proceed to the next step immediately after the temperature drops to 4°C.

[0075] Connect the connector and prepare the reaction system as shown in Table 4 below.

[0076] Table 4 Reaction System of Connector

[0077] reagents volume EndPrepReactionMix (Reaction product of step (2)) 30μL LigationBuffer 15μL ddH2O 2.5μL LigationEnzymes 5μL TruncatedAdaptor 2.5μL Total volume 55μL

[0078] All operations were performed on ice. The PCR tubes were placed on the PCR instrument, and the reaction program was 22°C for 15 minutes. The PCR instrument was not covered with a heat cap. Afterward, the tubes were stored at 4°C (the next step was performed immediately after the temperature dropped to 4°C).

[0079] 3. DNA sample purification

[0080] 3.1 Add 44 μL (0.8×) DNA Clean Beads to each sample, mix well, and incubate at room temperature for 5 min;

[0081] 3.2 Place the PCR tube on a magnetic rack and let it stand for 3 minutes until the solution becomes clear, then remove the supernatant;

[0082] 3.3 Add 180 μL of 80% ethanol to rinse the magnetic beads, incubate for 30 seconds, remove the supernatant, and repeat the operation once.

[0083] 3.4 Keep the PCR tube on the magnetic rack, use a 10 μL pipette to remove any residual ethanol from the bottom of the tube, and dry until no ethanol remains;

[0084] 3.5 Resuspend the magnetic beads in 21 μL ddH2O and let stand at room temperature for 1 min to allow the DNA on the magnetic beads to be fully released;

[0085] 3.6 Place the PCR tube on a magnetic rack for 2 min, then transfer 20 μL of supernatant to a new PCR tube for library amplification.

[0086] 4. Library amplification and purification

[0087] Configure the PCR reaction system as shown in Table 5 below, mix thoroughly by pipetting and then centrifuge briefly.

[0088] Table 5 PCR reaction system

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[0091] The prepared reaction system was placed under the PCR amplification program shown in Table 6 below for amplification;

[0092] Table 6 PCR Amplification Procedure

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[0094] Add 35 μL (1×) DNAClean Beads to the amplification product, mix well, and incubate at room temperature for 5 min. Place the sample on a magnetic rack for 2 min until the solution becomes clear, then remove the supernatant. Add 200 μL of 80% ethanol to rinse the magnetic beads, incubate for 30 s, then remove the supernatant. Repeat this step once. Keep the PCR tube on the magnetic rack, remove any residual ethanol from the bottom of the tube with a 10 μL pipette, and open the tube cap to dry until no ethanol residue remains. Resuspend the magnetic beads in 31 μL of ddH2O and incubate at room temperature for 1 min to allow the DNA on the magnetic beads to be fully released. Place the sample on the magnetic rack for 2 min, transfer 30 μL of supernatant to a new PCR tube, and store the library at -20℃ for subsequent library quality testing and sequencing.

[0095] 4. Library and probe hybridization

[0096] Add 750 ng of the library constructed in step (4) to a PCR tube and label it. Add purification magnetic beads to the library and gently mix with a pipette. Incubate at room temperature for 5 min, then place the PCR tube on a magnetic rack for 3 min to allow the solution to become clear. Remove the supernatant, continue to place the PCR tube on the magnetic rack, add 180 μL of 80% ethanol, and let it stand for 30 s. Remove the supernatant, add another 180 μL of 80% ethanol to the PCR tube, let it stand for 30 s, and then completely remove the supernatant. Place at room temperature for 5 min to allow the residual ethanol to evaporate completely. Add the hybridization reaction system to the PCR tube according to Table 7 below.

[0097] Table 7 Hybridization reaction system

[0098] reagents volume HybBuffer 13μL HybHumanBlock 5μL AdapterBlocker 2μL RnaseBlock 5μL NucleaseFreeWater 3μL Total volume 28μL

[0099] Gently pipette and mix well, let stand at room temperature for 3 minutes, and then briefly centrifuge. Place the PCR tube on a magnetic rack and let stand for 3 minutes. Pipe 28 μL of supernatant into a new PCR tube, add 2 μL of TargetProbe, gently pipette and mix well, and then briefly centrifuge. Set the PCR instrument parameters as follows: hot cap temperature: 85℃; 80℃ for 5 minutes; hold at 50℃. Place the PCR tube on the PCR instrument and run the above program to incubate overnight.

[0100] 5. Capture the target region DNA library

[0101] 5.1 Pretreatment of magnetic beads

[0102] 5.1.1 Remove the capturing magnetic beads from 4℃, vortex them to resuspend them, and let them equilibrate at room temperature for 30 min;

[0103] 5.1.2 Add 50 μL of magnetic beads to a new PCR tube, place it on a magnetic rack for 1 min until the solution becomes clear, and remove the supernatant;

[0104] 5.1.3 Remove the PCR tube from the magnetic rack, add 180 μL of Binding Buffer, gently aspirate and mix several times, then resuspend the magnetic beads;

[0105] Place on the magnetic rack for 1 minute, then remove the supernatant; repeat this step once.

[0106] 5.1.4 Remove the PCR tube from the magnetic rack, add 180 μL of Binding Buffer, gently pipette to resuspend and mix the magnetic beads, and set aside; 5.2 Capture the target region DNA library

[0107] 5.2.1 Keep the hybridization product on the PCR instrument, add 180 μL of the captured magnetic beads resuspended in step 5.1.1 to the hybridization product, mix with a pipette, and place on a rotary mixer to bind at room temperature for 30 min.

[0108] 5.2.2 Place the PCR tube on a magnetic rack for 2 minutes to allow the solution to clarify, then remove the supernatant;

[0109] 5.2.3 Add 150 μL of preheated Wash Buffer at 50 °C, gently aspirate and mix, then briefly centrifuge, and incubate at 50 °C for 10 min on a constant temperature shaking mixer;

[0110] 5.2.4 Briefly centrifuge, place the PCR tube on a magnetic rack for 2 minutes to allow the solution to clarify, and remove the supernatant; repeat this step twice, washing the magnetic beads a total of 3 times;

[0111] 5.2.5 Keep the sample on the magnetic rack, add 150 μL of 80% ethanol to the PCR tube, let it stand for 30 seconds, then completely remove the ethanol solution and let it air dry at room temperature;

[0112] 5.2.6 Add 24 μL of Nuclease-free Water to the PCR tube, remove the PCR tube from the magnetic rack, and gently resuspend and mix the magnetic beads with a pipette.

[0113] 5.3 Post-capture PCR amplification

[0114] Remove the PostPCR MasterMix and PostPCR Primer from the -20℃ freezer, place them on an ice box to thaw, mix well after thawing, and place on ice or at 4℃ for later use.

[0115] After capture, the DNA library needs to be amplified by PCR. Prepare the reaction system according to Table 8 below:

[0116] Table 8 PCR Reaction System

[0117] reagents volume Step (6) Capture the DNA library of the target region 24μL PostPCRMasterMix 25μL PostPCR Primer (select according to library type) 1μL Total volume 50μL

[0118] Adjust the pipette to 40 μL, gently aspirate and mix 6 times, then immediately place it on the PCR instrument; run the PCR program: hot lid temperature: 105℃ program: 95℃ 1 min; 98℃ 20 s; 60℃ 30 s N cycles; 72℃ 30 s; 72℃ 5 min; hold at 4℃;

[0119] After PCR, add 55 μL of purification magnetic beads to the sample, vortex or pipette to mix, and let stand at room temperature for 5 min; briefly centrifuge, and place the PCR tube on a magnetic rack for 3 min to allow the solution to clarify.

[0120] Keep the PCR tube on the magnetic rack, remove the supernatant, add 180 μL of 80% ethanol solution to the PCR tube, and let stand for 30 seconds;

[0121] Keep the PCR tube on the magnetic rack, remove the supernatant, add 180 μL of 80% ethanol solution to the PCR tube again, let it stand for 30 seconds, and then completely remove the supernatant; let it stand at room temperature for 5 minutes to allow the residual ethanol to evaporate completely.

[0122] Add 25 μL of Nuclease-free water, remove the PCR tube from the magnetic rack, vortex or pipette to mix 10 times, and let stand at room temperature for 2 minutes.

[0123] After a brief centrifugation, place the PCR tube on a magnetic rack for 2 minutes to allow the solution to clarify.

[0124] Use a pipette to transfer 23 μL of supernatant to a 1.5 mL centrifuge tube and label the sample information;

[0125] Take 1 μL of the library and quantify it using the Qubit dsDNAHS Assay Kit. Record the library concentration, which is approximately 1-20 ng / μL.

[0126] Take 1 μL of sample and use the Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit) to determine fragment length.

[0127] 5.4 High-throughput sequencing

[0128] The captured and amplified library was subjected to high-throughput sequencing using the BGI Genomics DNBSEQ-T7 sequencing platform to obtain the sequencing results of the tree species' genomic DNA, and the obtained data underwent basic cleaning.

[0129] 6. Evaluation of liquid phase probe capture rate

[0130] The cleaned sequencing data were aligned to the BTx623 v3.1.1 reference genome using bwa software, and SNP genotyping data of sorghum materials were obtained using GATK software. Genotyping of sorghum materials was completed using the SNP genotyping data (principal component analysis). Figure 3 Principal component analysis of the test material and resequencing material based on liquid-phase chip sites, such as... Figure 4 As shown. From Figure 4 The distribution of the tested materials within the natural population can be observed, thus aiding in the intuitive determination of the genetic range of these materials (the figure shows that these materials have a relatively narrow genetic distribution) and assisting in the selection of breeding materials. Furthermore, the results are consistent with expectations.

[0131] Example 3: Detecting background recovery rate of near-isogenic lines using the SNPs dataset contained in this chip.

[0132] 1. Obtaining the testing population

[0133] Using sweet sorghum E-tian and grain sorghum Ji2731 as the recurrent parent and donor parent, respectively, near isogenic lines (NILs) for the Dry gene were constructed. The Dry gene reported in the article "Sweet sorghum originated through selection of Dry, a plant-specific NACtranscription factor gene" published by Zhang et al. (2018) in our laboratory was used as a foreground marker. After crossing the two parents, the F1 generation was obtained, and then backcrossed with the E-tian parent multiple times to obtain the BC3F3 generation.

[0134] 2. Sample testing and SNP mutation acquisition

[0135] 233 leaf samples from BC3F3 generation were collected. DNA was extracted and purified, libraries were constructed and hybridized, high-throughput sequencing and data processing were performed using the same method as in Implementation Case 2 above. SNP variations in the 233 near-isogenous line materials were detected.

[0136] 3. Results Analysis

[0137] From the obtained SNP data of near-isogenic lines, 983 parental differential loci were selected, and the homozygous background reversion rate of recurrent parents was detected (the percentage of loci with genotypes consistent with the recurrent parent, inconsistent with the donor parent, and homozygous). The distribution of homozygous background reversion rates of 233 materials is shown below. Figure 5As shown, one material exhibited the highest homozygous reversion rate at 90.25%, which can be used for subsequent Dry gene functional analysis. The homozygous reversion rates of other materials were mostly around 80% (one material achieved 59.8%, while others ranged from 60.06% to 89.10%). These materials can be used for subsequent functional studies of other differentially expressed genes in the two parents, as well as for the creation and screening of materials with different combinations of differentially expressed genes, especially materials combining the Dry gene with its upstream and downstream genes in its pathway.

[0138] The above-described embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. The application of a sorghum genome-wide SNP locus combination detection reagent in the analysis of genetic diversity of sorghum germplasm resources, analysis of sorghum population structure, identification of sorghum kinship, gene mapping, and molecular-assisted breeding, characterized in that... The SNP loci combination consists of 9654 SNP loci in the sorghum reference genome "BTx623, version 3.1.1", and the locations of the 9654 SNP loci are shown in the table below: In the table, No. represents the number, Chrom. represents the chromosome, and Position represents the specific location on the sorghum reference genome "BTx623, v3.1.1".

2. A sorghum whole genome liquid-phase chip, characterized in that: The genotyping target of the liquid-phase chip is the combination of SNP sites in the whole genome of sorghum as described in claim 1.

3. The sorghum whole genome liquid chip according to claim 2, characterized in that: The chip is a sorghum 9K liquid phase chip based on targeted capture sequencing.

4. The application of the sorghum whole genome liquid phase chip as described in claim 2 or 3 in the analysis of genetic diversity of sorghum germplasm resources, analysis of sorghum population structure, identification of sorghum kinship, gene mapping and molecular-assisted breeding.

5. The application according to claim 4, characterized in that, The sorghum whole genome liquid phase chip was used to perform rapid genotyping of sorghum samples.

6. The application according to claim 4, characterized in that, The sorghum whole genome liquid chip contains a combination of sites used to determine the background recovery rate of near-isogenic lines.

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