Application of a combination of SNP sites in the genome of wheat stripe rust fungus in genotyping of wheat stripe rust fungus
By using a combination of 9365 SNP sites and liquid-phase chip technology, the problems of low efficiency, high cost and insufficient accuracy in wheat stripe rust genotyping detection have been solved, achieving efficient, economical and accurate genotyping detection that is applicable to multiple sequencing platforms.
Patent Information
- Application Number
- CN202310187152.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-03-01
- Publication Date
- 2026-01-06
- Estimated Expiration
- 2043-03-01
AI Technical Summary
Existing technologies for genotyping detection of wheat stripe rust fungus suffer from low efficiency, high cost, and insufficient accuracy, and different molecular marker technologies may lead to inconsistent research results.
Using a combination of 9365 SNP loci, the physical locations of wheat stripe rust were determined by alignment with the Pst_104E_v13_all_ctg reference genome. Combined with liquid-phase chip technology, genotyping detection of wheat stripe rust was achieved.
It improves the accuracy and efficiency of detection, reduces costs, and enables efficient, convenient, and economical genotyping of wheat stripe rust. It is applicable to multiple sequencing platforms and captures rich genomic variations.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of wheat stripe rust technology, specifically relating to the application of a combination of SNP sites in the genome of wheat stripe rust in genotyping of wheat stripe rust. Background Technology
[0002] Wheat stripe rust is an airborne fungal disease caused by *Strombus leuciscus*, which is devastating to wheat production. In years with an epidemic, it can lead to yield losses of over 30%, seriously threatening wheat production. Its effective control is a long-standing international challenge, attracting widespread attention from countries and international organizations worldwide. The Chinese government has always attached great importance to the research and control of wheat stripe rust. Due to the frequent mutations of *Strombus leuciscus*, its physiological races exhibit diversity. However, research on the virulence response of *Strombus leuciscus* has significant limitations; therefore, molecular marker-based research at the DNA level offers a new approach.
[0003] Currently, although molecular marker technology has been widely applied in the population genetics of wheat stripe rust, previous studies have mostly used only a few dozen molecular markers, failing to capture the rich variation in the wheat stripe rust genome. Furthermore, different molecular marker technologies may yield different experimental results, leading to conflicting findings despite extensive research on the prevalence of wheat stripe rust populations.
[0004] Therefore, developing a combination of SNP sites in the wheat stripe rust genome that can be applied efficiently, conveniently, accurately, with high coverage, and economically and quickly to the genotyping detection of wheat stripe rust is of great significance for population genetics research of wheat stripe rust. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of wheat stripe rust genome SNP site combination in wheat stripe rust genotyping, which can perform wheat stripe rust genotyping detection efficiently, conveniently, accurately, with high coverage, and economically and quickly.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0007] An application of a combination of SNP sites from the wheat stripe rust genome in wheat stripe rust genotyping, wherein the combination of SNP sites from the wheat stripe rust genome includes 9365 SNP sites, the physical locations of which are determined based on alignment with the wheat stripe rust reference genome Pst_104E_v13_all_ctg; the version number of the wheat stripe rust reference genome Pst_104E_v13_all_ctg is V13;
[0008] The physical location information of the 9365 SNP loci is shown in Table 1. The physical location information of the SNP loci is represented in the format of "chromosome number:physical location". In the chromosome number, p is the abbreviation of pcontig and h is the abbreviation of hcontig.
[0009] Table 1
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[0051] As a further technical solution, the wheat stripe rust fungus was genotyped by detecting 9365 SNP sites.
[0052] The application of a set of wheat stripe rust genome probes in wheat stripe rust genotyping: the probe set was used to detect 9365 SNP sites in wheat stripe rust. The physical locations of the 9365 SNP sites were determined based on the alignment of the wheat stripe rust reference genome Pst_104E_v13_all_ctg. The physical location information of the 9365 SNP sites is shown in Table 1.
[0053] Application of a liquid phase chip for wheat stripe rust genome in wheat stripe rust genotyping, wherein the liquid phase chip includes the probe combination described above.
[0054] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0055] 1. Highly representative and rich in diverse information
[0056] The 9365 SNP loci of this invention are derived from large-scale resequencing data and all currently available resequencing data. The background of these data covers wheat stripe rust samples from various endemic areas around the world, which are highly representative and rich in diversity information. They can capture the rich variation of the wheat stripe rust genome and improve the accuracy of detection.
[0057] 2. The markings are evenly distributed.
[0058] The 9365 SNP loci identified by the genotyping detection of wheat stripe rust fungus in this invention are evenly distributed throughout the entire genome of wheat stripe rust fungus.
[0059] 3. Platform adaptability
[0060] The marker detection of this invention does not require specific expensive equipment and can be adapted to various sequencing platforms to complete sequencing.
[0061] 4. High detection efficiency
[0062] This invention enables the mixed determination of samples, eliminating the cumbersome detection process and thus improving detection efficiency.
[0063] 5. Same marking density, lower price
[0064] This invention greatly reduces the cost of detecting mutation sites in wheat stripe rust, and there are currently no similar inventions. Attached Figure Description
[0065] Figure 1 This invention illustrates the distribution of 9365 SNP loci on the genome.
[0066] Figure 2 This is a statistical graph illustrating the SNP loci annotations of the present invention;
[0067] Figure 3 This is a statistical graph showing the alignment results of sequencing data with the reference genome in Example 5;
[0068] Figure 4 This is a statistical chart of sequencing results from Example 5; Detailed Implementation
[0069] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0070] Example 1
[0071] A combination of SNP loci for genotyping includes 9365 SNP loci, the physical locations of which are determined based on alignment with the wheat stripe rust reference genome Pst_104E_v13_all_ctg; the version number of the wheat stripe rust reference genome Pst_104E_v13_all_ctg is V13.
[0072] The physical location information of the 9365 SNP sites is shown in Table 1 (Invention Content).
[0073] The method for obtaining it includes the following steps:
[0074] Step 1: Target gene selection
[0075] Based on the gene function of wheat stripe rust fungus, target genes were selected. In this example, 1727 target genes were obtained, with a total length of 1.74 Mb.
[0076] Step 2: Foreground site information verification
[0077] Based on the NCBI database, 67 SNP sites of wheat stripe rust were screened, and KASP primer sequences were provided. The flanking sequences of these primers were used to verify the sites. In this example, 274 candidate sites were obtained.
[0078] Step 3: Background site information filtering
[0079] Based on the whole-genome resequencing data of 42 wheat stripe rust fungi, the loci (2,350,234 SNP loci) in the data were initially screened, retaining loci with an NA ratio of <30%, a heterozygosity frequency of less than 30%, and a minor allele frequency of greater than 0.05, totaling 1,843,846 loci. Using a sliding window of 110bp, segments with 2-7 SNP loci were selected. According to the principle of uniform distribution, segments with high PIC values (PIC value greater than 0.5) or high MAF values of target SNPs within the segment (MAF value greater than 0.05) were prioritized. In this example, a total of 2,146 candidate segments were obtained.
[0080] Step 4: Probe Design and Synthesis
[0081] Probes were designed based on the target genes, prospective loci, and candidate regions obtained from steps 1, 2, and 3. In this embodiment, a total of 1727 genes, 134 loci, and 1944 target regions were successfully evaluated.
[0082] Step 5: Site testing and probe adjustment
[0083] The probe designed in step 4 was tested using test samples. The test results showed that there were hollow areas at the sites in step 4. Sites with high polymorphism were selected from the candidate sites in step 2, and the probe was adjusted accordingly.
[0084] Step 6: Determine the final probe and capture site details.
[0085] The probe optimized in step 5 was tested again using test samples to finally determine the regions that could be stably captured. In this embodiment, 1727 genes, 41 SNP sites, and 6540 target segments were obtained that could be stably captured; the 6540 SNP segments covered 9324 SNP markers; therefore, this embodiment identified a total of 1727 genes and 9365 SNP sites.
[0086] The distribution of SNP sites on the genome in this embodiment Figure 1 The number of SNPs within a 100kb window sliding statistical window on the chromosome, and the distribution of the target SNP sites on the genome; the SNP site annotation statistics are shown in [link to SNP annotation statistics]. Figure 2 .
[0087] Example 2
[0088] A probe combination, using the probe combination obtained after adjustment in step 5 of Example 1, is used to capture 9365 SNP sites obtained in Example 1 of the present invention.
[0089] Example 3
[0090] A liquid-phase chip was prepared based on GenoBaits and GenoPlexs technologies. The liquid-phase chip included a probe combination as described in Example 2, which could be used to capture 9365 SNP sites obtained in Example 1 of the present invention.
[0091] The GenoBaits and GenoPlexs technologies of this invention are unique yet overlapping, and both technologies can achieve precise capture of any location and length of the genome.
[0092] Example 4
[0093] A method for genotyping 9365 SNP loci obtained in Example 1 using the probe combination described in Example 2 or the liquid-phase chip prepared in Example 3 includes the following steps:
[0094] Step 1: Extract DNA from the target sample;
[0095] Step 2: Library Construction
[0096] Add 100 ng DNA, 4 μL GenoBaits End Repair Buffer, and 3.1 μL GenoBaits End Repair Enzyme to a 0.2 μL PCR tube, and make up the volume to 20 μL with water. Place the reaction tube in a PCR instrument and incubate at 37℃ for 20 min, then at 72℃ for 20 min to complete enzyme digestion, end repair, and A addition.
[0097] Add 2 μL of GenoBaits MLtra DNA ligase, 8 μL of GenoBaits MLtra DNA Ligase Buffer, 2 μL of GenoBaits Adapter, and 8 μL of Nuclease-free water. Place the mixture in a PCR instrument at 22°C for 60 min to complete the ligation of the sequencing adapter.
[0098] Add 1.2 times the amount of GenoPrep DNA Clean Beads, mix thoroughly, place on a magnetic rack, let stand for 3 min, and remove the supernatant. Add 100 μL of freshly prepared 80% ethanol. Incubate at room temperature for 30 s, and remove the supernatant. Air dry until all the ethanol has evaporated, then add 10 μL of GenoBaits PCR Master Mix, 1 μL of GenoBaits Uni-oligo, 5 μL of GenoBaits DNAindex, and 4 μL of Nuclease-free water to the PCR instrument. Resuspend the magnetic beads, place in the PCR instrument, and incubate at 98℃ for 2 min; repeat 6-8 cycles of 98℃ for 30 s, 65℃ for 30 s, and 72℃ for 40 s; then incubate at 72℃ for 4 min.
[0099] Add 20 μL of GenoPrep DNA Clean Beads, mix thoroughly and let stand for 5 min. Remove the supernatant and add 100 μL of freshly prepared 80% ethanol. Incubate at room temperature for 30 s, then remove the supernatant. Add 30 μL of Tris-HCl to resuspend the library, mix thoroughly, place on a magnetic rack, let stand for 3 min, and remove the supernatant.
[0100] Step 3: Hybridization capture of the library;
[0101] Take 500 ng of purified DNA library, add 5 μL of GenoBaits Block I and 2 μL of GenoBaits Block II, and evaporate to dryness at ≤70℃ using an Eppendorf Concentrator plus vacuum concentrator. Add 8.5 μL of GenoBaits 2×Hyb Buffer, 2.7 μL of GenoBaits Hyb Buffer Enhancer, and 2.8 μL of Nuclease-Free Water to the dry powder tube, mix well with a pipette, and incubate at 95℃ for 10 min on an ABI 9700 PCR instrument. Remove the PCR tube, add 3 μL of the prepared liquid phase probe, vortex to mix, and incubate at 65℃ for 2 h on an ABI 9700 PCR instrument to complete the probe hybridization reaction. Use a washbuffer to elute the hybridized library.
[0102] Add 13 μL of resuspended DNA (with magnetic beads) to a new 0.2 mL PCR tube, add 15 μL GenoBaits PCR Master Mix and 2 μL GenoBaits Primer Mix to prepare a post-PCR system, and perform library amplification using an ABI 9700 PCR instrument. The amplification program is as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s; repeat steps 2-4 for a total of 15 cycles; 72℃ extension for 5 min.
[0103] Add 45 μL of Beckmen AMPure XP Beads to the post-PCR product, pipette and mix well, place the 0.2 PCR tube on a magnetic rack 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) to complete the liquid-phase chip hybridization capture.
[0104] Step 3: Quality control and sequencing of the library
[0105] DNA concentration was determined using qubit fluidometric quantitation (Thermo Fisher), and the fragment size of the library DNA was detected by agarose gel electrophoresis to ensure it was between 350-400 bp. The constructed library was sequenced using a BGI sequencer to obtain genotyping data for the target SNP loci.
[0106] Example 5
[0107] The application of the 9365 SNP sites obtained in Example 1, the probe combination described in Example 2, or the liquid phase chip prepared in Example 3 in wheat stripe rust genotyping includes the following steps:
[0108] Seventy-six wheat leaves infected with wheat stripe rust were collected, and genotyping of 9365 SNP loci was performed using the method described in Example 4. The genotyping results were then subjected to quality control, removing reads with N content exceeding 10% of the read length and reads with low-quality (Q≤20) bases exceeding 40% of the read length. The quality-controlled clean reads were then aligned with the reference genome sequence to locate their positions within the reference genome. The alignment results are shown below. Figure 3 The sample alignment rate reflects the similarity between the sample sequencing data and the reference genome.
[0109] Genotyping statistics of core SNP loci:
[0110] The alignment and mutation results were used to extract genotyping information for SNP loci using Perl scripts. If the coverage depth of a sample at a certain SNP locus was <5X, it indicated insufficient sequencing depth at that locus; to ensure accurate genotyping results, this locus was treated as a deletion. If a sample had a mutation at a certain SNP locus, the mutation frequency was used for assessment. If the mutation frequency was ≥0.8 or ≤0.2, the locus was homozygous. If the mutation frequency was between 0.2 and 0.8, each allele in the heterozygous genotype had at least 4 reads supporting it; otherwise, the locus was treated as a deletion. Finally, the genotyping results for 76 samples at the 9365 SNP loci were compiled. The deletion and mutation status of each sample was then statistically analyzed and plotted. Figure 4 The horizontal axis represents the sample name, and the vertical axis represents the percentage. NA_rate ≤ 10% indicates that the sample's missing detection rate meets the requirements; a lower NA_rate indicates fewer undetected sites in the sample. Het_alt_rate, Hom_alt_rate, and Ref_rate are sample-specific.
[0111] This invention utilizes the ANNOVAR software tool to perform functional annotation on detected gene variations. Based on the location of the variant site on the reference genome and the gene location information on the reference genome, the region in which the variant site occurs in the genome (intronic region, intergenic region, coding region, 5-terminal UTR region, 3-terminal UTR region, etc.) and the impact of the variation (synonymous and non-synonymous mutations, etc.) can be determined.
[0112] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.
Claims
1. Use of a combination of SNP loci of the genome of Puccinia striiformis f.sp. tritici in genotyping of Puccinia striiformis f.sp. tritici, characterized in that, The wheat stripe rust genome SNP site combination comprises 9365 SNP sites, physical positions of the 9365 SNP sites are determined based on wheat stripe rust reference genome Pst_104E_v13_all_ctg alignment; the physical position information of the 9365 SNP sites is shown in Table 1; wherein the physical position information of the SNP site is represented in the form of "chromosome number: physical position"; in the chromosome number, p is a short form of pcontig, and h is a short form of hcontig; Table 1 2. Use of a combination of SNP loci of the wheat stripe rust pathogen genome according to claim 1 for genotyping the wheat stripe rust pathogen, characterized in that, The wheat stripe rust is genotyped by detecting the 9365 SNP sites in the wheat stripe rust.
3. Use of a set of Puccinia striiformis f. sp. tritici genomic probes in Puccinia striiformis f. sp. tritici genotyping, characterized in that, The probe combination is used for detecting 9365 SNP sites in the wheat stripe rust, physical positions of the 9365 SNP sites are determined based on wheat stripe rust reference genome Pst_104E_v13_all_ctg alignment; the physical position information of the 9365 SNP sites is shown in the table 1 as claimed in claim 1.
4. Use of a liquid chip of the genome of Puccinia striiformis in genotyping of Puccinia striiformis, characterized in that, The liquid chip comprises the probe combination as claimed in claim 3.