Multi-gene allelic variation detection method based on targeted sequencing

Abstract

The invention discloses a multi-gene allelic variation detection method based on targeted sequencing, and belongs to the technical field of rice breeding. The detection method comprises the following steps: (1) carrying out breaking, terminal repairing, linker connection and linker product purification on a rice genome to obtain a sequencing library; (2) amplifying and purifying a sequencing library; (3) carrying out hybrid capture on the purified library and a probe composition with a sequence as shown in SEQ ID NO.1-60, and then removing uncombined DNA (Deoxyribonucleic Acid); (4) carrying out PCR (Polymerase Chain Reaction) amplification and purification on the hybridized DNA; and (5) sequencing the purified PCR amplification product. According to the invention, the high-speed detection of the sample is realized, the efficiency is improved by at least 10 times compared with the traditional KASP genotyping technology through experimental operation, and the automatic and large-scale detection is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of rice breeding, and in particular relates to a multi-gene allele variation detection method based on targeted sequencing. Background Art

[0002] Rice (Oryza sativa L.) is one of the world's most important food crops, providing the primary source of energy for over half of the world's population. Traditional rice breeding methods, including hybridization and selective breeding, are limited by long production cycles, low efficiency, and difficulty in precisely controlling target traits.

[0003] GBTS (Genotyping-by-Targeted Sequencing) liquid phase array technology, an emerging genotyping targeted sequencing tool, combines the advantages of high-throughput sequencing with target capture technology, enabling simultaneous detection of multiple target loci in a single reaction. Compared to traditional SNP arrays, GBTS liquid phase arrays offer greater flexibility and precision, enabling flexible design of capture probes based on breeding objectives to precisely capture key loci associated with important agronomic traits.

[0004] Important agronomic traits of rice (such as yield, disease resistance, stress tolerance, and quality) are regulated by multiple genes and exhibit complex quantitative trait inheritance. Against this complex backdrop, the application of GBTS liquid phase array technology is particularly important. GBTS liquid phase array technology offers the advantages of high detection accuracy, high throughput, flexible probe design, and cost-effectiveness. By performing high-throughput and precise detection of key genes, researchers can rapidly obtain extensive genotyping data, providing a solid molecular foundation for breeding decisions. Summary of the Invention

[0005] This study utilizes GBTS (Genotyping-by-Targeted Sequencing) liquid microarray technology to design a targeted sequencing-based method for detecting multi-gene allelic variation. This method targets multiple rice traits, including yield, metabolism, abiotic stress, rice quality, biotic stress, appearance, and fertility. Sixty GBTS liquid microarray probes were developed based on 30 high-quality variant loci. These markers and corresponding probe pairs can be used in the subsequent development of integrated liquid microarrays for detecting multi-gene allelic variation in rice materials. The specific protocol is as follows:

[0006] 1. Probe Selection

[0007] (1) Select target genes

[0008] Rice has numerous traits. Based on rice breeding and cultivation experience, target genes for seven traits of greatest concern in rice breeding, including yield, metabolism, abiotic stress, biotic stress, rice quality, appearance, and fertility, were selected for subsequent GBTS liquid phase chip probe design.

[0009] This study collected reported, well-defined rice functional loci using both online databases and breeding population materials. Online databases included the National Rice Data Center (https: / / www.ricedata.cn / ), the IRIC SNP-seek database based on 3,000 rice accessions (https: / / snp-seek.irri.org / index.zul), and the pan-genome database constructed based on the 3K rice population (https: / / cgm.sjtu.edu.cn / 3kricedb / about.php). Breeding population materials were collected using whole-genome data from natural populations maintained by the National Engineering Research Center for Plant Space Breeding (Xie et al., 2023).

[0010] Using the 'Nipponbare' genome (NCBI No. GCF_001433935.1) as the reference genome, the collected sites were mapped to the reference genome, and the sites that could be mapped to the reference genome were retained. Such sites have a wider application value in rice breeding.

[0011] (2) Synthesis of probes

[0012] A probe covering the target SNP is designed at each site, and the target probe is labeled with biotin.

[0013] In order to better capture the target site sequence, the probe design process is based on 2X design.

[0014] The probe design principles are as follows: a. Select all probes that cover the target region to be 110 bp in length; b. Calculate the GC content of the probes that cover the target region; c. Calculate the number of homology regions in the probes. The probe selection criteria are as follows: a. Select probes with a GC content between 30% and 70%; b. Select probes with fewer than 5 homology regions; c. Select probes that do not contain SSR or N regions; d. Screen the probe sequences that meet the requirements (see Tables 1-3) to confirm coverage of the target site.

[0015] 2. Genomic DNA Sequencing Library Construction Process

[0016] The reagents used in the following steps are from DNA Hybridization kit for ILM hybridization kit, which can be purchased from Shijiazhuang Boruidi Biotechnology Co., Ltd.

[0017] 1. Sample DNA Extraction

[0018] The CTAB method was used to extract DNA from rice samples.

[0019] 2. Sample DNA Quality Inspection

[0020] The DNA concentration of the test samples was determined using Qubit Fluorometric Quantitation (Thermo Fisher), and the integrity of the DNA was checked using 1% agarose gel electrophoresis. Qualified samples were stored in a 4°C refrigerator for future use.

[0021] 3. Sample DNA Fragmentation

[0022] Take 12 μL of DNA that has passed the quality inspection and place it in a 0.2 μL PCR tube. Place the tube in an ultrasonic crusher to randomly physically break the DNA into fragments of 200 to 400 bp.

[0023] 4. Sample end repair

[0024] Add 4 μL GenoBaits End Repair Buffer and 2.7 μL GenoBaits End Repair Enzyme to the tube, add water to 20 μL, and place it in an ABI9700 PCR instrument. Incubate at 37°C for 20 minutes, and then at 72°C for 20 minutes to complete the end repair and A addition process of the broken fragments. When the above program reaches 4°C, immediately perform adapter ligation.

[0025] 5. Sample Sequencing Adapter Connection

[0026] Remove the tube from the PCR instrument and add 2 μL GenoBaits Ultra DNA ligase, 8 μL GenoBaits Ultra DNA Ligase Buffer and 2 μL GenoBaits Adapter, add water to 20 μL, shake to mix, and then place it on the ABI9700 PCR instrument at 22°C for 60 minutes to complete the connection of the sequencing adapter.

[0027] 6. Sample DNA Purification

[0028] 48 μL of GenoPrep DNACleanBeads was added to the ligation product to purify the ligation product. After purification, magnetic beads were used for fragment screening, and ligation products with insert fragments between 300 and 350 bp were retained.

[0029] 7. Sample Library Amplification

[0030] To the PCR tube from the previous step, add 5 μL of sequencing adapters with barcode sequences, 1 μL of P5 adapters, and 10 μL of GenoBaits PCR Master Mix. Make up to 20 μL with purified water. Amplify using an ABI 9700 PCR instrument using the following program: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. Repeat steps 2-4 for a total of 8 cycles, followed by extension at 72°C for 5 minutes. Different barcodes are used to distinguish different samples.

[0031] 8. Sample Library Purification

[0032] Add 20 μL of GenoPrep DNA Clean Beads to the second-round PCR product and vortex to mix. Place a 0.2 μL PCR tube on a magnetic stand until the solution is clear. Discard the supernatant and wash the beads once with 80% ethanol. Elute the library DNA with Tris-HCl (pH 8.0).

[0033] 9. DNA hybridization

[0034] Take 500ng of the constructed genomic DNA sequencing library, add 5μL of GenoBaits Block I and 2μL of GenoBaits Block II, place on an Eppendorf Concentratorplus vacuum concentrator, and evaporate to dry powder at ≤70°C. Add 8.5μL of GenoBaits 2xHyb Buffer, 2.7μL of GenoBaits HybBuffer Enhancer, and 2.8μL of Nuclease-Free Water to the dry powder tube. Mix thoroughly with a pipette, and incubate on an ABI9700 PCR instrument at 95°C for 10 minutes. Then remove the PCR tube and add 3μL of the synthesized probe (each probe concentration is 20ng / μL). Vortex to mix thoroughly, and incubate on an ABI9700 PCR instrument at 65°C for 2 hours to complete the probe hybridization reaction.

[0035] 10. DNA capture

[0036] Add 100 μL of GenoBaits DNA Probe Beads to the hybridization reaction system from the previous step. Pipette up and down 10 times and incubate at 65°C on an ABI9700 PCR instrument for 45 minutes to allow the beads to bind to the probe. Wash the probe-bound beads at 65°C with 100 μL of GenoBaits Wash Buffer I and 150 μL of GenoBaits Wash Buffer II, respectively. Then, wash the beads 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. Resuspend the washed beads in 20 μL of Nuclease-Free Water. This process is the elution of the target sequence.

[0037] Take 13 μL of resuspended DNA (with magnetic beads) and add it to a new 0.2 mL PCR tube. Then add 15 μL GenoBaits PCR Master Mix and 2 μL GenoBaits Primer Mix to prepare the post-PCR system.

[0038] Library amplification was performed on a 9700 PCR instrument using the following protocol: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. Steps 2-4 were repeated for 15 cycles, followed by extension at 72°C for 5 minutes. This process constitutes target amplification.

[0039] Add 45 μL of GenoPrep DNAClean Beads to the post-PCR product and pipette up and down to evenly distribute the solution. Place the 0.2 mL PCR tube on a magnetic stand until the solution is clear. Discard the supernatant and wash the beads twice with 75% ethanol. Elute the library DNA with Tris-HCl (pH 8.0). This completes the probe hybridization capture process. This process is called library DNA elution.

[0040] 11. DNA hybridization capture library quality control

[0041] The DNA concentration of the library was determined using Qubit Fluorometric Quantitation (ThermoFisher), and then agarose gel electrophoresis was used to check whether the fragment size of the library DNA was between 300 and 400 bp. This completed the DNA library construction process.

[0042] 3. DNA Hybridization Capture Library Sequencing

[0043] The constructed DNA library was sequenced using the BGI MGISEQ2000 sequencer.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) The variation sites used in the probe design of the present invention are widely distributed on each chromosome of the rice genome, so that the detection covers a wider range of the genome.

[0046] (2) The mutation sites designed in the present invention cover more trait types that are of high concern in production (such as yield, abiotic stress resistance and biotic stress resistance), and can focus on important traits during material testing.

[0047] (3) The probes designed in the present invention can detect a variety of secondary traits and can more comprehensively cover various sub-traits of rice.

[0048] (4) The present invention has a wide range of channels for collecting functional sites, and combined with the genome resequencing data of population materials in breeding work, it can more diversely mine various trait-related variation sites.

[0049] (5) The present invention combines the characteristics of GBTS liquid phase chip technology and utilizes the company's independently developed primer design software (GenoBaits Designer), multiplex PCR amplification technology and fully automatic gene sequencer to achieve high-speed detection of samples. It improves the efficiency by at least 10 times compared with the traditional KASP (Kompetitive Allele Specific PCR) genotyping technology through experimental operation, and realizes automated and large-scale detection. DETAILED DESCRIPTION

[0050] Example 1 Design of 30 pairs of GBTS liquid phase chip probes for 7 rice trait types

[0051] Targeting seven rice trait types, including yield, metabolism, abiotic stress, rice quality, biotic stress, appearance, and fertility, and 23 secondary traits, 30 pairs of GBTS liquid phase chip probes, totaling 60, were developed for high-quality variant sites on 30 functional genes (Tables 1, 2, and 3).

[0052] Table 1 Number of probe pairs corresponding to 17 trait types and 23 secondary traits

[0053]

[0054]

[0055] Table 2

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] Table 3 Mutation sites and probes on the same chromosome

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] Statistics revealed that 27 of the 30 loci were SNPs and 3 were InDel loci. These loci were distributed across chromosomes 1-8 and 11, with the highest number of loci on chromosome 1, at 20, and the lowest number on chromosome 11, at 2. The broad distribution of loci across chromosomes in the rice genome enabled the detection to cover a wider genomic range.

[0076] Yield, abiotic stress resistance, and biotic stress resistance (primarily pests and diseases) are the three most important agronomic traits of concern in rice breeding. This example focuses on developing probes for these three agronomic traits, developing 6, 5, and 7 pairs of probes, respectively, for a total of 18 pairs of probes, representing more than half of the 30 pairs of probes. This allows for focused testing of important traits during material testing. Furthermore, these 18 pairs of probes detect a wide variety of secondary traits, more comprehensively covering various rice traits. For example, yield covers six secondary traits: grain weight, grain length, number of grains per panicle, grain width, and grain shattering. This allows for more comprehensive testing of yield-related secondary traits, given the limited number of detection sites, thereby inferring the yield performance of the material.

[0077] Example 2 Detection of 30 rice varieties using 30 pairs of probes

[0078] In March 2024, seeds of 30 rice varieties planted in the 2023 late season at the Qilin North Agricultural Base of South China Agricultural University were collected, the seeds were used to germinate seedlings, and the leaves of the seedlings were taken.

[0079] All 30 rice varieties have passed national or provincial variety approval and are available on the official website of the National Rice Data Center. The names of the materials are as follows: Pohuxiang, Huajing, Yueyinruanzhan, Efengsi Miao, Tianhuang 101, Xing'anxiangzhan, Ivory Xiangzhan, Nanxiuruanzhan, Nanguizhan, Huahang 38, Jianghangsi Miao, Juxiangsi Miao, Yujingsi Miao, Yujingyouzhan, Guangjingyouzhan, Bahesi Miao, Fuxiangzhan, Xiangxiuzhan, Jiuxiangnian, Lixiangzhan, Guxiangyouzhan 1, Huahang 57, Huahang 58, Yuejingsi Miao 2, Yuehongbao, Taixiang 812, Shuangzhensi Miao, Meixiangzhan 2, Xiangzhuxiangsi Miao, and Guang 8B.

[0080] From March to April 2024, leaf samples were sent to Shijiazhuang Boridi Biotechnology Co., Ltd., which used the 30 pairs of probes designed in Example 1 and performed detection according to the steps of “(3) multiplex PCR amplification, target fragment recovery, and high-throughput sequencing”.

[0081] Table 4 GBTS liquid phase chip probe detection results of 30 rice varieties

[0082]

[0083]

[0084]

[0085] The 30 pairs of GBTS liquid-phase chip probes demonstrated excellent detection and high stability. A total of 900 samples were tested on the 30 materials, with only one unsuccessful test (the test of the Pi35 variant in "Lixiangzhan"), for a detection success rate of 99.89%. The 30 probe pairs effectively distinguished the genotypes of all materials, with three genotypes: "Ref homozygous," "heterozygous," and "Alt homozygous," representing "homozygous genotype identical to the wild type," "heterozygous genotype," and "mutant homozygous genotype," respectively. For example, at the Pi2 variant, the Ref homozygous genotype is TT, the heterozygous genotype is TA, and the mutant homozygous genotype is AA, corresponding to susceptible, partially resistant, and resistant phenotypes, respectively.

[0086] Through testing, a total of 661 Ref homozygous genotypes, 27 heterozygous genotypes, and 211 mutant homozygous genotypes were obtained. Generally, the mining of high-quality variants gives priority to genotype homozygous materials. Based on the correspondence between excellent phenotypes and genotypes, high-quality variant materials can be mined. For example, for the phenotype of "cadmium accumulation", the detection of the variant site CAL1 obtained a total of 5 homozygous materials with the "cadmium accumulation prevention" phenotype (Nan Guizhan, Yujing Youzhan, Yuejing Si Miao No. 2, Shuangzhen Si Miao, Meixiangzhan No. 2), which is conducive to the mining of materials that prevent cadmium accumulation and their in-depth utilization in breeding.

[0087] The detection results of the probes in the present invention can be supplemented and verified using KASP technology. For example, for the variant site "Badh2_1" in Table 4, KASP primers can be designed based on KASP technology (Kompetitive Allele-Specific PCR) (reference: Yang G, Chen S, Chen L, et al. Development of a core SNP arrays based on the KASP method for molecular breeding of rice [J]. Rice, 2019, 12: 1-18. The variant site "Badh2_1" in this patent is the variant site "Badh2" mentioned in the text). The KASP primers were used to detect 30 materials in this patent, and the results were compared with the results of the 30 materials detected using the probe for the variant site "Badh2_1" in this patent. The results are shown in Table 5. KASP is a genotyping technology with a high accuracy rate for SNP detection. The results show that the detection accuracy of the probe is as high as 80%, which also shows a high detection accuracy rate.

[0088] Table 5 Comparison of probe detection and KASP detection results of the variant site "Badh2_1"

[0089]

[0090]

[0091] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for detecting multi-gene allelic variation based on targeted sequencing, characterized in that: The following steps are involved: The probe combination with the sequence shown in SEQ ID NO. 1-60 is used to capture and sequence the rice gene to be tested.

2. The method for detecting multi-gene allelic variation based on targeted sequencing according to claim 1, characterized in that: The following steps are involved: (1) The rice genome is fragmented, end-repaired, adapter-ligated, and the adapter products are purified to obtain a sequencing library; (2) Sequencing library amplification and purification; (3) hybridizing and capturing the purified library with a probe composition having a sequence as shown in SEQ ID NO. 1-60, and then removing unbound DNA; (4) PCR amplification and purification of the hybridized DNA; (5) Sequence the purified PCR amplification products.

3. The method for detecting multi-gene allelic variation based on targeted sequencing according to claim 2, characterized in that: The step (1) also includes a rice genome extraction process.

4. The method for detecting multi-gene allelic variation based on targeted sequencing according to claim 2 or 3, characterized in that: The amount of each probe used in step (2) is the same.

5. The method for detecting multi-gene allelic variation based on targeted sequencing according to claim 4, characterized in that: The PCR amplification system in step (4) is 30 μL, specifically comprising: 13 μL of hybridized DNA, 15 μL of GenoBaits PCR MasterMix, and 2 μL of GenoBaits Primer Mix.

6. The method for detecting multi-gene allelic variation based on targeted sequencing according to claim 5, characterized in that: The PCR amplification procedure in step (4) is as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, for a total of 15 cycles, and extension at 72°C for 5 min.

7. The method for detecting multi-gene allelic variation based on targeted sequencing according to claim 6, characterized in that: In the step (5), the second generation sequencing technology is used for sequencing.

8. Use of the probe composition of claim 1, wherein the sequences are as shown in SEQ ID NOs. 1-60, in detecting multi-gene allelic variation in rice.

Images