A set of SNP site probes for rubber tree genotyping and their applications

Abstract

The present invention discloses a set of SNP site probes for rubber tree genotyping and their applications, belonging to the field of agricultural biotechnology. The present invention screens out a set of 66K SNP site information covering the whole genome of rubber trees from the resequencing data of 400 wild rubber tree germplasms and 300 cultivated rubber tree germplasms and some publicly published gene data related to important traits of rubber trees. Based on this, a set of 66K SNP site probes for rubber tree genotyping is constructed. Through this probe, rubber tree genotyping can be realized. Compared with technologies such as whole genome resequencing, reduced-representation genome sequencing, and solid-phase chips for genotyping, the present invention has the advantages of high efficiency and accuracy, low cost, large throughput, flexible customization of probes, and wide application scenarios, providing technical support for rubber tree genetic breeding research.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and particularly to a set of SNP site probes for genotyping rubber trees and their applications. Background Art

[0002] In the genetic breeding research of rubber trees, genotyping of individual materials can analyze their genetic compositions, obtain accurate genetic information, facilitate subsequent related analyses, and improve breeding efficiency. Since the 20th century when protein (mainly isozyme) was used as a molecular marker for genotyping, a series of revolutionary changes have occurred in the molecular marker technologies used for genotyping in terms of quantity, type, throughput, analysis cost, etc. It has developed from RAPD, SSR, AFLP, etc. to SNP markers. SNP markers are mainly detected by methods such as whole-genome resequencing, reduced-representation genome sequencing, and solid-phase chip sequencing. Whole-genome sequencing can perform complete detection of an individual within the entire genome range, but its sequencing cost is relatively high. Especially for plant materials with a large genome, whole-genome sequencing requires a large amount of cost. Reduced-representation genome sequencing is to enzymatically digest the plant genome and then perform sequencing. Although the sequencing cost is reduced compared to whole-genome sequencing, the cost is still relatively high when a large number of detections are carried out. The sequencing data volumes of these two methods are relatively large, requiring high software and hardware for data storage, analysis, and calculation, and also requiring a large amount of effort in sequencing information processing and analysis. Solid-phase chips are based on the complementary hybridization of probes and DNA sequences, and are genotyped through the fluorescence color signal of markers. They can only genotype the SNP sites included on the chip, cannot be added or deleted, have poor flexibility, and have a relatively high genotyping cost, making it difficult to meet the requirements of different application scenarios.

[0003] The present invention uses a targeted sequencing technology based on liquid-phase chips to specifically capture each target site, which has the advantages of simple development technology, high detection accuracy, flexibility, large throughput, and low cost. Moreover, the same SNP markers can be stably obtained in different laboratories, providing a simple and reliable technical platform for the accumulation, sharing, comparison, and integration of detection data. The density of liquid-phase chips can be flexibly adjusted according to application needs, can be reduced to a low density of 1K, or can be extended to a high density of 200K or even higher, and there is no limit on the test sample size. This technology has a wide range of applications and has currently been widely used in plant genetic breeding research, such as genome-wide association analysis, DNA fingerprint identification, molecular genetic map construction, QTL mapping, marker-assisted selection, genome-wide selection, etc. Summary of the Invention

[0004] The object of the present invention is to provide a set of SNP site probes for rubber tree genotyping and their applications to solve the problems existing in the above-mentioned prior art. The probes are 66K SNP site probes constructed based on a liquid-phase chip, which can genotype rubber trees, and have the advantages of high detection efficiency and accuracy, low cost, large throughput, flexible customization of probes, and wide application scenarios, providing technical support for rubber tree genetic breeding research.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a set of SNP site probes for rubber tree genotyping, and the SNP site probes include 66,000 probes.

[0007] Preferably, the nucleotide sequence of each SNP site probe is 120 bp, and the SNP site probe is obtained by screening the nucleotide sequence with a GC content of 45% in 120 bp on both sides of the SNP site centered on the SNP site.

[0008] Preferably, the 5' end of the SNP site probe is modified with a biotin group, and the SNP site probe is coupled to the fluorescent microspheres through a C12 molecular arm and amino modification, and each fluorescent microsphere is coupled with one SNP site probe.

[0009] The present invention also provides the application of the SNP site probe in the preparation of a liquid-phase chip for rubber tree genotyping.

[0010] The present invention also provides a liquid-phase chip for rubber tree genotyping, and the liquid-phase chip includes the SNP site probe.

[0011] The present invention also provides the application of the SNP site probe or the liquid-phase chip in rubber tree genotyping.

[0012] The present invention also provides the application of the SNP site probe or the liquid-phase chip in rubber tree genetic diversity analysis, high-density genetic map construction, genome-wide association analysis, variety authenticity identification, genome-wide association analysis or genome-wide selection breeding.

[0013] The present invention also provides a method for rubber tree genotyping, including the steps of hybridizing the genomic DNA of the rubber tree to be tested with the SNP site probe and a hybridization reagent, and then sequencing.

[0014] Preferably, the conditions for the hybridization reaction are: incubation at 65 °C for 6 hours.

[0015] The present invention discloses the following technical effects:

[0016] (1) The present invention adopts a liquid-phase probe capture technology to design and synthesize a set of probes containing 66K SNP locus information, which can be used to detect the background loci of rubber trees. This set of probes can quickly and effectively trace the genetic information of rubber trees, facilitating the acceleration of the rubber tree breeding process and improving the efficiency of new variety selection.

[0017] (2) The SNP loci involved in the 66K SNP locus probes of the present invention are screened from the resequencing data of 400 wild rubber tree germplasms and 300 cultivated rubber tree germplasms, as well as some publicly published gene data related to important traits of rubber trees. The material backgrounds of these data cover a wider range of rubber tree germplasms, with high representativeness and rich diversity information.

[0018] (3) The SNP loci involved in the 66K SNP locus probes of the present invention are evenly distributed on the chromosomes.

[0019] (4) The 66K SNP locus probes of the present invention, compared with solid-phase chips, have high detection accuracy, a large amount of effective information, high flexibility and diversity, and effectively reduce the detection cost and improve the efficiency of genetic breeding research.

[0020] (5) In rubber tree genetic breeding research, compared with the existing resequencing technology, the 66K SNP locus probes of the present invention overcome the defects of large resequencing data volume, a large amount of useless information, and difficult analysis. On the premise of achieving the same results, it realizes the advantages conducive to industrial development such as rapid detection, cost reduction, and simple data analysis; compared with reduced-representation genome sequencing (GBS), this set of probes avoids the defects of excessive data volume during GBS analysis and the difficulty in comparing and accumulating GBS marker data obtained between different materials, laboratories, and platforms, resulting in the difficulty of long-term preservation and comprehensive utilization of GBS data; this set of probes has many advantages such as a high data output consistency rate, the data of the same material can be used for a long time, and relatively simple data analysis.

[0021] (6) The 66K SNP locus probes developed by the present invention can be applied to genetic diversity analysis, kinship identification, DNA fingerprint map construction, and genome-wide association analysis in rubber tree genetic breeding research, promoting the development of rubber tree genetic breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0023] Figure 1It is the uniform distribution map of 66K SNP loci of rubber tree on chromosomes (calculating the probe coverage density with a 200kb length as the observation window on chromosomes); that is, the statistical result of the number of target loci on different chromosomes; among them, the vertical coordinate is the chromosome ID; the horizontal coordinate is the chromosome length (unit: bp); the right example is the schematic diagram of locus coverage.

[0024] Figure 2 It is the distribution quantity of 66K SNP loci of rubber tree on different chromosomes; among them, the horizontal coordinate is the chromosome ID; the vertical coordinate is the number of SNP loci.

[0025] Figure 3 It is the distribution region of 66K SNP loci of rubber tree in the genome; among them, intergenic is the intergenic region, cds is the coding region of the gene, up_down_stream is the upstream or downstream region of the gene, intron is the intron region of the gene, and utr is the non-coding region of the gene. Detailed implementation manners

[0026] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and embodiments of the present invention are only exemplary.

[0030] For the terms "comprising", "including", "having", "containing", etc. used in this article, they are all open-ended terms, meaning including but not limited to.

[0031] Example 1 A set of SNP locus probes for rubber tree genotyping

[0032] In this example, a set of SNP locus probes for rubber tree genotyping was constructed, including a set of 66K SNP locus probes for rubber tree. The 66K SNP locus probes for rubber tree are single-stranded DNAs synthesized based on 66K SNP molecular markers of rubber tree.

[0033] The 5'-end of the probe is labeled with a biotin group. The probe is coupled to the fluorescent microspheres through a C12 molecular arm and amino modification, and each fluorescent microsphere is coupled with one kind of probe.

[0034] The preparation process of the above-mentioned 66K SNP locus probes for rubber tree is as follows:

[0035] (1) Screening 66K SNP loci of rubber tree, which are specifically screened and obtained by the following method:

[0036] Step 1: According to the resequencing data of 700 rubber tree germplasms (400 wild rubber tree germplasms and 300 cultivated rubber tree germplasms), SNP loci on the genome are detected by aligning with the rubber tree reference genome (GCF_030052815.1) using BWA for further selection; the above loci are selected according to the following criteria: MAF≥0.05, NA<5%, heterozygosity rate<5%, combined with the principle of uniform distribution on chromosomes, and each locus is evaluated by simulation.

[0037] Step 2: According to the gene data related to important traits of rubber tree published in the literature (Wang Aosheng et al., 2023; Feng Weiqiang et al., 2016; Lu Yijiang et al., 2022; Yang Jianghua et al., 2007), screening is carried out according to the selection method in Step 1.

[0038] (2) Designing and preparing 66K SNP locus probes for rubber tree, the specific design process is as follows:

[0039] Step 1: Determining the probe design principle

[0040] The probe length is 120bp, the GC content of the probe is between 30% and 65%, the average GC content is about 45%, the number of homologous regions ≤3, and the selected region maximally does not contain SSR and GAP regions; and the priority is: gene region>gene promoter region (2Kb)>gene downstream adjacent region (2Kb)>gene intergenic region, combined with the principle of uniform distribution on chromosomes.

[0041] Step 2: Then, centering around the SNP sites obtained from the above-mentioned "(1) Screening of 66K SNP sites in rubber trees", screen a 120bp nucleotide sequence with the GC content closest to 45% in the 120bp on its left and right as the probe.

[0042] Step 3: Synthesize a single-stranded DNA modified with a biotin group at the 5' end according to the 120bp nucleotide sequence screened in Step 2, which is called the 66K SNP site probe of rubber tree.

[0043] (3) Couple the probe prepared in the previous step to the fluorescence microspheres, and add the target capture reagent to obtain the 66K SNP liquid-phase probe of rubber tree.

[0044] The target capture reagents used specifically include the following reagents: Universal Block I, Universal Block II, TCGBS 2×Hyb Buffer, TCGBS Repeatseq Block, TCGBS 2×Beads Wash Buffer, TCGBS Wash Buffer I / Wash Buffer II / Wash Buffer III, TCGBS Stringent Wash Buffer.

[0045] The core site information in the obtained 66K SNP site probe of rubber tree is as follows:

[0046] Among them, chr1, chr2, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17, chr18 represent the rubber tree chromosome IDs where the SNP sites are located, and the numbers after the chromosome IDs represent the positions of the SNP sites on these chromosomes.

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[0244] Mapping based on the rubber tree reference genome in the NCBI database to analyze the distribution of SNP loci. The obtained 66K SNP locus probes of rubber tree, the distribution of SNP loci on different chromosomes is as Figure 1 shown, from Figure 1 it can be seen that the coverage rate of SNP loci in the 200Kb window of the genome is 93.5%, and the overall genome coverage is good, without large fragments not covered.

[0245] The number of SNP loci contained on different chromosomes is as Figure 2 shown, from Figure 2 it can be seen that the present invention designs different numbers of probes according to the length of chromosomes, so that the captured loci can cover the entire genome.

[0246] The distribution region of the obtained 66K SNP loci in the genome is as Figure 3 shown, from Figure 3 it can be seen that 72.20% of the SNP loci are located in the intergenic region, 11.69% of the SNP loci are located in the gene coding region, 9.18% of the SNP loci are located in the upstream or downstream region of the gene, 5.67% of the SNP loci are located in the gene intron region, and 1.27% of the SNP loci are located in the gene non-coding region.

[0247] Therefore, the 66K SNP molecular marker combination of the rubber tree of the present invention, as well as the probe for detecting the SNP molecular marker combination (rubber tree 66K SNP locus probe), can be applied to genetic diversity analysis, molecular genetic map construction, genome-wide association analysis, variety authenticity identification, molecular marker-assisted selection breeding, and genome-wide selection breeding in rubber tree genetic breeding research.

[0248] Example 2 Application method of the rubber tree 66K SNP locus probe, the steps are as follows:

[0249] (1) DNA extraction: Use a high-throughput DNA extraction kit or the CTAB method to extract DNA from rubber tree samples;

[0250] (2) DNA quality inspection: Use 1% agarose gel electrophoresis to analyze the purity and integrity of DNA; use Qubit to accurately quantify the DNA concentration; use Agilent 2100 to accurately detect the integrity. The standard for qualified quality inspection is DNA with a total amount of not less than 4 μg, a sample concentration of not less than 40 ng / μL, good sample integrity, high purity, and no impurity contamination;

[0251] (3) Library construction: Use an ultrasonic crusher to randomly fragment the qualified sample DNA, electrophoretically recover the DNA fragments of the required length, and add adapters to its ends to form a library;

[0252] (4) Sequencing library construction: Use LM-PCR to amplify the sample library and purify it, which is the sequencing library and can be used for probe hybridization experiments;

[0253] (5) Hybrid capture library construction: Take 300 ng of the completed sequencing library, lyophilize it, add the rubber tree 66K SNP locus probe and hybridization reagent, denature it, and incubate it at 65 °C for 6 hours to complete the hybridization reaction; after washing the hybridization product with the washing solution, perform another round of PCR to complete the construction of the hybrid capture library;

[0254] (6) Hybrid capture library quality inspection: Use Qubit2.0 for preliminary quantification, and use the qPCR method to accurately quantify the effective concentration of the library to ensure the library quality;

[0255] (7) Sequencing: Use an illumina sequencer and related reagents for sequencing;

[0256] (8) Analysis: After obtaining the off-machine data, use the FastQC software for quality control, use the BWA software to align the quality-controlled data to the reference genome, and use the GATK software to perform SNP identification to obtain the genotyping data of the sample to be tested.

[0257] Example 3

[0258] Using the obtained 66K SNP locus probes of rubber trees, 10 rubber tree germplasms were detected:

[0259] (1) A total of 15 rubber tree leaf samples were taken from 10 rubber tree germplasms. Among them, 10 samples contained 5 germplasms (2 replicates for each germplasm). Specifically, W-6, W-7, W-8, W-9, and W-10 were the replicates of W-6-2, W-7-2, W-8-2, W-9-2, and W-10-2 respectively; they were placed in a -20 °C refrigerator for storage, and DNA was extracted according to the method of Example 2.

[0260] (2) According to the method of Example 2, library construction was carried out, followed by sequencing on the machine, and the final SNP genotyping data was obtained;

[0261] (3) Calculate the detection rate. The locus detection rate is an important indicator to measure the quality of SNP locus probes. In plants, the detection rate is generally measured by the ratio of the number of detected loci in the sample to the number of developed loci; the average detection rate of the 15 samples in this example was greater than 98%. The locus deletion rate was 0.21% - 1.18%, indicating that the locus design quality and probe capture efficiency of the liquid-phase chip prepared by the present invention were very good. The specific detection rate results are shown in Table 1.

[0262] Table 1 Statistical table of detection results of 15 rubber tree samples

[0263] Sample Number of population loci Number of missing loci Missing rate Detection rate W-1 66000 237 0.36％ 99.64％ W-2 66000 152 0.23％ 99.77％ W-3 66000 323 0.49％ 99.51％ W-4 66000 508 0.77％ 99.23％ W-5 66000 139 0.21％ 99.79％ W-6 66000 330 0.50％ 99.50％ W-6-2 66000 205 0.31％ 99.69％ W-7 66000 568 0.86％ 99.14％ W-7-2 66000 779 1.18％ 98.82％ W-8 66000 601 0.91％ 99.09％ W-8-2 66000 376 0.57％ 99.43％ W-9 66000 198 0.30％ 99.70％ W-9-2 66000 165 0.25％ 99.75％ W-10 66000 356 0.54％ 99.46％ W-10-2 66000 568 0.86％ 99.14％

[0264] (4) Calculate the repeatability rate. Sample repeatability statistics were carried out. The overall average repeatability rate of the five groups of replicate samples was 99%, and the homozygous locus consistency rate was greater than 99%, indicating that the SNP locus probe detection had very good stability. The statistical results are shown in Table 2.

[0265] Table 2 Statistical detection of technical repeatability of test samples

[0266] Sample group Number of effective loci Consistent loci Duplication rate Heterozygous loci Homozygous consistency rate W-6 vs W-6-2 65570 65294 99.58％ 276 100％ W-7 vs W-7-2 64975 64241 98.87％ 712 99.97％ W-8 vs W-8-2 65248 65026 99.66％ 222 100％ W-9 vs W-9-2 65692 65508 99.72％ 184 100％ W-10 vs W-10-2 65329 65094 99.64％ 225 99.98％

[0267] Generally speaking, the 66K SNP locus probes of rubber trees developed based on the liquid-phase chip strategy of the present invention can be effectively applied to fields such as genetic diversity analysis of rubber trees, construction of high-density genetic maps, genome-wide association analysis, variety authenticity identification, genome-wide association analysis, and genome-wide selection breeding.

[0268] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A set of SNP locus probes for rubber tree genotyping, characterized in that: The site information of the SNP site probe is as follows: In the above site information, chr1, chr2, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17 and chr18 represent the chromosome IDs of the rubber tree where the SNP sites are located, and the number after the chromosome ID represents the position of the SNP site on the chromosome; The accession number of the reference genome where the SNP site is located on NCBI is GCF_030052815.

1.

2. The SNP site probe according to claim 1, wherein The nucleotide sequence of each SNP site probe is 120 bp. The SNP site probe is obtained by screening the nucleotide sequence with a GC content of 45% in the 120 bp around the SNP site with the SNP site as the center.

3. The SNP site probe according to claim 1, wherein The 5' end of the SNP site probe is modified with a biotin group, and the SNP site probe is coupled to a fluorescent microsphere through a C12 molecular arm and amino modification, and each fluorescent microsphere is coupled with a SNP site probe.

4. Use of the SNP locus probe as described in any one of claims 1 to 3 in preparing a liquid phase chip for rubber tree genotyping.

5. A liquid phase chip for rubber tree genotyping, characterized in that, The liquid phase chip comprises the SNP site probe according to any one of claims 1 to 3.

6. Use of the SNP locus probe according to any one of claims 1 to 3 or the liquid phase chip according to claim 5 in genotyping of rubber trees.

7. Application of the SNP site probe according to any one of claims 1 to 3 or the liquid phase chip according to claim 5 in genetic diversity analysis of rubber trees, construction of high-density genetic maps, whole genome association analysis, variety authenticity identification or whole genome selection breeding.

8. A method for genotyping of rubber trees, characterized in that, The method comprises the steps of carrying out hybridization reaction on the genomic DNA of the rubber tree to be tested, the SNP site probe according to any one of claims 1 to 3 and a hybridization reagent, and then sequencing.

9. The method according to claim 8, characterized in that The conditions of the hybridization reaction are: incubation at 65°C for 6 hours.

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