Saline-alkali tolerant soybean liquid phase chip as well as design method and application thereof

Abstract

The invention relates to the technical field of molecular detection, and particularly discloses a saline-alkaline tolerant soybean liquid phase chip and a design method and application thereof, and genetic typing sites of the saline-alkaline tolerant soybean liquid phase chip comprise 232 SNP sites and 2 Indel sites; the physical positions of the 232 SNP loci and the two Indel loci are determined based on the comparison of the whole genome sequence of the soybean reference genome, and the version number of the whole genome sequence of the soybean reference genome is Glycine max Wm82. A2. V1; wherein the 232 SNP loci and the 2 Indel loci are as shown in a table 1. The screened 232 SNP loci and 2 Indel loci can realize genetic typing of the saline-alkaline tolerant soybeans so as to realize saline-alkaline tolerant character identification of the soybeans, and can be used for soybean molecule assisted breeding to breed the saline-alkaline tolerant soybeans.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection, and particularly relates to a saline-alkali tolerant soybean liquid-phase chip, a design method thereof, and an application thereof. Background Art

[0002] As an important food crop and oil source in China, the problem of insufficient self-sufficiency rate of soybeans has long troubled agricultural production. In view of this situation, breeding of saline-alkali tolerant soybeans has become one of the key ways to solve the soybean supply problem.

[0003] With the sharp decline in the cost of high-throughput sequencing, SNP markers, as the third-generation molecular markers, are increasingly used in the identification and evaluation of phenotypes in assisted breeding. In the process of soybean breeding, by identifying the saline-alkali tolerance performance of soybeans, it is possible to quickly select saline-alkali tolerant soybeans.

[0004] However, in the field of developing saline-alkali tolerant soybean breeding chips, China is still blank. Summary of the Invention

[0005] The purpose of the present invention is to provide a saline-alkali tolerant soybean liquid-phase chip, a design method thereof, and an application thereof. The liquid-phase chip can be used for identifying the saline-alkali tolerance traits of soybeans to achieve molecular assisted breeding of soybeans.

[0006] The present invention is achieved by the following technical solutions: A saline-alkali tolerant soybean liquid-phase chip, wherein the genotyping sites of the saline-alkali tolerant soybean liquid-phase chip include 232 SNP sites and 2 Indel sites; the physical positions of the 232 SNP sites and 2 Indel sites are determined based on the whole-genome sequence alignment of the soybean reference genome, and the version number of the whole-genome sequence of the soybean reference genome is Glycine max Wm82.a2.v1; among them, the 232 SNP sites and 2 Indel sites are shown in Table 1; among them, the numbers s112 and s118 are Indel sites, and the rest are SNP sites.

[0007] The 232 SNP sites and 2 Indel sites screened by the present invention can achieve genotyping of saline-alkali tolerant soybeans, and further achieve identification of the saline-alkali tolerance traits of soybeans, and can be used for molecular assisted breeding of soybeans to select saline-alkali tolerant soybeans.

[0008] Further, the saline-alkali tolerant soybean liquid-phase chip includes a probe combination for identifying the genotypes of 232 SNP sites and 2 Indel sites. [[ID=X]]

[0009] A design method of a saline-alkali tolerant soybean liquid-phase chip, comprising the following steps: S1. Obtain the original sites of soybeans through whole-genome resequencing; S2. Align the original loci with the target reference genome, remove duplicates, and obtain filtered loci. S3. Screen out salt - and - alkali - tolerant associated loci from the filtered loci. The associated loci include 232 SNP loci and 2 Indel loci. S4. Design nucleotide probes based on the 232 SNP loci and 2 Indel loci screened out, and prepare a liquid chip.

[0010] Furthermore, in step S1, the source of the original loci is the GWAS association interval related to salt - tolerance traits.

[0011] Furthermore, step S1 includes the following steps: S11. Sample collection: Collect DNA samples of soybeans. S12. Resequence the DNA samples obtained in step S11 to obtain the original sequencing data. S13. Perform quality control on the original sequencing data to obtain the filtered original loci.

[0012] Furthermore, in step S4, the screening conditions include locus filtering. The conditions for locus filtering are: MAF≥0.35, evenly distributed on the chromosome, missing rate≤0.1, and heterozygosity rate≤0.3.

[0013] Application of the liquid chip for salt - and - alkali - tolerant soybeans in soybean genotyping.

[0014] Application of the liquid chip for salt - and - alkali - tolerant soybeans in the association analysis of soybean salt - and - alkali - tolerance traits.

[0015] Application of the liquid chip for salt - and - alkali - tolerant soybeans in molecular marker - assisted breeding or genome - wide selection breeding of salt - and - alkali - tolerant soybeans.

[0016] Application of a soybean molecular marker combination. The soybean molecular marker combination consists of 232 SNP loci and 2 Indel loci in Table 1. The application of the soybean molecular marker combination includes soybean genotyping, association analysis of soybean salt - and - alkali - tolerance traits, and soybean breeding.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: Through genome-wide association analysis of salinity-alkalinity tolerance-related traits in 399 natural soybean populations, thousands of salinity-alkalinity tolerance-associated loci were obtained. Then, the salinity-alkalinity tolerance-associated loci in soybeans were screened, and 232 SNP loci and 2 Indel loci were obtained as the associated loci of soybean salt tolerance genes. Based on the 232 SNP loci and 2 Indel loci, a probe combination was synthesized according to the base complementary principle to prepare a liquid chip. Using this liquid chip, genotype typing of salinity-alkalinity tolerance in soybeans, molecular marker-assisted breeding of salinity-alkalinity tolerance soybeans, and genome-wide selection breeding can be achieved. Brief Description of the Drawings

[0018] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, form a part of this application, and do not limit the embodiments of the present invention. In the drawings: Figure 1 It is a statistical chart of the distribution information of the 232 SNP loci and 2 Indel loci screened in Example 2. Detailed Embodiments

[0019] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with the embodiments. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and do not limit the present invention. The following described embodiments are part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0020] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present invention. However, it is obvious to those of ordinary skill in the art that: these specific details do not have to be adopted to implement the present invention. In other embodiments, well-known structures, materials, or methods are not specifically described to avoid obscuring the present invention. The materials, instruments, and reagents used in the following embodiments, unless otherwise specified, can be obtained from commercial sources. The technical means used in the embodiments, unless otherwise specified, are conventional means well-known to those skilled in the art.

[0021] Example 1: Screening of Molecular Marker Combinations for Genotyping Salt-Tolerant Soybeans Based on the original loci (original sequencing data) obtained from genome-wide association analysis of salinity-alkalinity tolerance-related traits in 399 natural soybean populations, the associated loci of salinity-alkalinity tolerance were selected from the original data, and finally 232 SNP loci and 2 Indel loci shown in Table 1 were obtained. The specific screening process includes the following steps: S1. Obtain the original loci of soybeans through whole-genome resequencing; S11. Sample collection: Collect DNA samples from 399 natural soybean populations (source of the 399 natural soybean populations: collected by the laboratory, mainly cultivated soybeans in China); S12. Re-sequence the DNA samples obtained in step S11 to obtain the original sequencing data; The basic process of re-sequencing is as follows: First, use the CWseq Universal DirectFast DNA Library Prep Kit to construct a re-sequencing library for the DNA with qualified quality inspection. After the library construction is completed. After the library quality inspection is qualified, use the BGI MGI-T7 sequencing platform for sequencing, and the sequencing mode is PE150 mode.

[0022] S13. Perform quality control on the original sequencing data to obtain the filtered original loci (filtered fragments); Filter the original data, mainly to remove adapter contamination and low-quality sequencing fragments (reads), and obtain high-quality filtered data (clean reads) for subsequent alignment analysis.

[0023] The filtering is performed using the SOAPnuke (v1.5.6) (https: / / github.com / BGI-flexlab / SOAPnuke) software independently developed by BGI. The specific filtering conditions are as follows: (1) Remove the sequencing fragments (reads) containing adapters; (2) Remove low-quality sequencing fragments (reads), where low quality means that the proportion of bases with a base quality value less than or equal to 15 is 10%; (3) Remove reads with a proportion of N bases greater than 10%.

[0024] Using the above filtering conditions, obtain the filtered SNP loci and Indel loci for subsequent alignment analysis.

[0025] S2. Align and deduplicate the original loci with the target reference genome to obtain the filtered loci, ensuring that the original loci are consistent with the target reference genome (soybean reference genome). The version number of the whole genome sequence of the soybean reference genome is Glycine max Wm82.a2.v1. The specific steps are as follows: S21. Alignment: Use the "mem" algorithm of the short sequence alignment software BWA (Version: 0.7.12-r1039) [1] to align the quality-controlled and filtered fragments (clean reads) to the reference genome (Glycine max Wm82.a2.v1) (the alignment parameters are: -t8 -k19 -M –R). Use samtools (v1.9) [2] to convert the alignment results in sam format to bam format. Then use the SortSam.jar tool of Picard (v1.117) (https: / / broadinstitute.github.io / picard / ) software to remove duplicates and sort the alignment files to obtain a bam file for subsequent analysis.

[0026] S22. Bam Correction: Select the reads with a mapQ value greater than 20 and properly paired for subsequent variant detection analysis (the filtering parameters are: samtools view -f2 -q 20). To improve the accuracy of SNP sites around indels, use the RealignerTargetCreator and IndelRealigner tools of GATK (v3.3.0) [3] software to realign and correct the sequences near indels. The processed bam file can be used for variant detection.

[0027] S33. Use the HaplotypeCaller module of GATK (3.30) to perform variant detection on multiple samples of the processed alignment files to obtain the detected SNP sites and Indel sites; use the VariantFiltration module of GATK (3.30) to perform hard filtering on SNPs and Indels. The specific parameters are: QD < 2.0, FS > 60.0, MQ < 40.0, MQRankSum < -12.5, ReadPosRankSum < -8.0; perform strict filtering on SNPs: SNP cluster filtering (no two SNPs within 5bp), SNP filtering near indels (SNPs within 5bp of indels are filtered out), and adjacent indel filtering (the distance between two indels cannot be less than 10bp); also include filtering out sites with a GQ (Genotype Quality) less than 20.0 and marking the genotyping quality in the sample as lowGQ.

[0028] Using the above filtering conditions, a total of 1.3×10 7 SNP sites and 1.2×10 6 Indel sites are obtained as filtered sites for subsequent associated site mining.

[0029] S3. Screen out the salinity-alkalinity tolerant associated loci from the filtered loci, and the screening conditions are: MAF ≥ 0.35, evenly distributed on the chromosome, deletion rate ≤ 0.1, and heterozygosity rate ≤ 0.3. Finally, 232 SNP loci and 2 Indel loci are obtained as the associated loci, as shown in Table 1; Table 1

[0030]

[0031]

[0032]

[0033] Among them, the numbers s112 and s118 are Indel loci, and the rest are SNP loci.

[0034] Note: The physical position represents a point on the reference genome, that is, a base. The first base in the corresponding reference column of the Indel locus.

[0035] Note: The number before the "_" in the physical position represents the chromosome, and the number after the "_" represents the physical position of the locus on the corresponding chromosome.

[0036] Example 2: A salinity-alkalinity tolerant soybean liquid chip, which includes a probe combination for identifying the genotypes of 232 SNP loci and 2 Indel loci.

[0037] The purpose of this example is to apply the 232 SNP loci and 2 Indel loci in Table 1 to probe design. The probe design principle is as follows: Probes are designed within 110 bp on both sides of all target loci. The probe length is generally 110 bp, and the GC content is between 30% and 70%.

[0038] Based on the above probe design principle, Shijiazhuang Borui Di synthesized liquid-phase capture probes, with a total of 918 capture probes synthesized (probes designed for long-fragment Indel sites will be superimposed. According to the length of the Indel, the number of probes for Indel sites is 2 - 3. Probes designed for SNP sites generally do not overlap, and adjacent sites will share one probe). A precise positioning sequencing genotyping technology (cGPS) based on liquid-phase capture of the target region genomic sequence was used to form a liquid-phase chip system for salt-tolerant soybeans. Among them, cGPS designs probes for genomic sequences in different target regions based on an optimized thermodynamic stability algorithm model, uses the synthesized specific probes to perform liquid-phase hybridization capture and enrichment on multiple different target sequences located at different genomic positions, and then constructs a sequencing library and performs high-throughput sequencing on the captured and enriched target genomic sequences to obtain the genotypes of all SNP / Indel sites within the target region. According to the probe design results, probes that cannot be uniquely aligned on the genome and those with repetitive sequences in the flanking sequences were removed, and the designed probes were applied to the preparation of the liquid-phase new chip, and finally a liquid-phase chip for salt-tolerant soybeans was obtained. The statistical information of the locus distribution in this chip is as Figure 1 shown; the probes corresponding to 232 SNP sites and 2 Indel sites are shown in SEQ ID NO.1 - SEQ ID NO.234 respectively.

[0039] Example 3: Application of the liquid-phase chip for salt-tolerant soybeans in soybean genotyping: (1) Extraction and detection of soybean genomic DNA samples: Select 237 soybeans as the verification samples for the liquid-phase chip for salt-tolerant soybeans prepared in Example 2. Collect the tissues of 237 soybeans, and use a high-throughput DNA extraction kit (GenPrep Conventional Plant DNA Rapid Extraction Kit (Magnetic Bead Method) Mix_V4.0, specification: 960 rxn) to extract the sample DNA. The extracted DNA samples are subjected to two types of detections: (1) Use 1% agarose gel electrophoresis to analyze the purity and integrity of the DNA; (2) Use Qubit to accurately quantify the DNA concentration.

[0040] The 237 soybeans are from the F3 generation of 5 hybrid combinations. The method is conventional hybridization. For the F1 generation, all single plants plant all the pods at the same leaf position. The F2 generation is all planted, and each single plant takes one pod and is all planted to form the F3 generation population. These 237 soybeans are randomly selected from the F3 generation. The hybrid combinations are H12*PI538401, H12 *Fen Dou 92, Shan Da 5 Hao × JACK2-3, Shan Da 3 Hao × W82, and Shan Da 1 Hao × W82.

[0041] (2) GenoBaits experimental procedure: Quantitatively take the qualified DNA through quality inspection and randomly physically fragment it using an ultrasonic crusher, controlling the peak of the fragmented segments at 200 - 300 bp. After end repair of the fragmented DNA, add an A tail. Use ligase to ligate the A-tailed DNA fragments with the sequencing adapters, and then use carboxyl-modified magnetic beads to purify and select fragments of the library, retaining the ligation products with inserted fragments of 200 - 300 bp. Add the sequencing primers with Barcode and a high-fidelity PCR reaction system to the ligation products for PCR amplification. Different Barcodes are used to distinguish different samples. After purification of the amplification products with carboxyl magnetic beads, they can be used for probe hybridization experiments.

[0042] Take 500 ng of the completed sequencing library, freeze-dry it, and then add the probe (or the salt-tolerant soybean liquid chip containing the probe) and hybridization reagents (blocking reagent, RNase inhibitor (Rnase Block)). After denaturation, incubate at 65 °C for 2 hours to complete the hybridization reaction. After washing the hybridization products with the washing solution, perform another round of PCR to complete the construction of the hybridization capture library.

[0043] (3)Library construction and on-machine sequencing: For the samples with qualified DNA quality through the above methods, use the corresponding products to construct a targeted sequencing library, and finally complete the library preparation for all samples of this project. After the library construction is completed, first perform a preliminary quantification using Qubit 2.0, and use the qPCR method to accurately quantify the effective concentration of the library to ensure the library quality. After the library passes the inspection, use the BGI sequencing platform for PE150 sequencing.

[0044] (4)Information analysis process: The raw data after high-throughput sequencing is processed through quality control filtering, etc. Use the fastp software to remove the Reads contaminated with adapters and low-quality Reads, and use the BWA software to align with the target genome, and then use the GATK software to analyze the variant sites of the sequencing results to obtain the genotype typing results of the target sites.

[0045] Example 4: Effect evaluation of the salt-tolerant soybean liquid chip in soybean genotyping Use the salt-tolerant soybean liquid chip prepared in Example 2 to perform genotype detection on 13 soybean samples and 13 duplicate soybean samples with reference to the method in Example 3. After sequencing and data analysis (performing genome sequencing and alignment using the above chip sites), the detection rate results of the 13 soybean samples are shown in Table 2, and the genotype results of the 13 duplicate soybean samples are compared, and the comparison results are shown in Table 3.

[0046] Table 2

[0047] As can be seen from the data in Table 2, the uniformity of the salt-tolerant soybean liquid-phase chip prepared in Example 2 is above 99%, indicating that it has the advantage of high detection stability. Table 3

[0048] As can be seen from Table 3: The genotype consistency rate of the salt-tolerant soybean liquid-phase chip prepared in Example 2 is above 99%, indicating that it has the advantage of high accuracy.

[0049] Example 5: Association analysis of the molecular marker combination (232 SNP loci and 2 Indel loci) with soybean salt tolerance traits The main salt tolerance traits of soybeans include 4 traits: fresh seedling weight under salt stress (NFW), emergence rate in saline-alkali land (SE), germination rate under salt stress (GR), and root length under salt stress (NRL).

[0050] The 232 SNP loci and 2 Indel loci screened in the present invention are respectively related to fresh seedling weight under stress (NFW), emergence rate in saline-alkali land (SE), germination rate under salt stress (GR), and root length under salt stress (NRL). The association between the loci and the corresponding traits is reflected by the p-value (P.value) and the effect value (Effect). The p-values (P.value) and effect values (Effect) of the 232 SNP loci and 2 Indel loci with the corresponding traits are shown in Table 4.

[0051] Table 4

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] The p-values and effect values are from performing GWAS using GAPIT (http: / / zzlab.net / GAPIT / ). The MLM (PCA+Kinship) model in GAPIT was used to perform association analysis on the salt tolerance phenotypic indicators.

[0058] Determination of significance threshold: Using the GEC software, the number of independent and effective SNPs (Ne) was estimated based on linkage disequilibrium. According to the adjusted Bonferroni method, the threshold for significant association was calculated: p = 1 / Ne (Li et al , 2012).

[0059] After evaluation, the number of effective independent SNPs among 589,608 SNP data across the genome was 123,364. Determine -log 10 P = 5.090 as the threshold for significant association of P - value. The provided threshold is the threshold for GWAS to obtain associated markers. As can be seen from Table 4, the P - values of the 234 loci screened by the present invention are all greater than this threshold.

[0060] It can be proved by this example that the 232 SNP loci and 2 Indel loci screened by the present invention are closely associated with the salt - alkali tolerance trait of soybeans, that is, the salt - alkali - tolerant soybean liquid - phase chip prepared in Example 2 of the present invention can be used to detect whether the target gene (salt - alkali - tolerant gene) exists. During the soybean backcross breeding process and in the hybrid offspring, by detecting the genotypes of the offspring, it can be judged whether the offspring contains the target gene. The salt - alkali - tolerant soybean liquid - phase chip prepared in Example 2 can be used for molecular marker - assisted breeding and genomic selection breeding.

[0061] Example 6: Application of the salt - alkali - tolerant soybean liquid - phase chip in breeding In the F3 generation of the hybrid population, the salt - alkali - tolerant soybean liquid - phase chip prepared in Example 2 was used to detect the genotypes of the F3 generation plants. Based on the detection results, 8 individuals with the top - ranked predicted values of single - plant yield in saline land were selected (that is, 8 individuals with the largest number of dominant allelic variations related to 4 salt - tolerance traits were selected; the 4 salt - tolerance traits are fresh seedling weight under stress (NFW), emergence rate in saline - alkali land (SE), germination rate under salt stress (GR), and root length under salt stress (NRL)). 8 soybean lines with better breeding values of single - plant yield in saline land (salinity about 0.3%, pH about 8) were obtained. The single - plant yields of these 8 soybean lines after planting were statistically analyzed, and the statistical results are shown in Table 5. Finally, 8 soybean lines with high breeding values of single - plant yield in saline land shown in the following table were selected. Taking the single - plant weight as an example, the average breeding value of single - plant yield in saline land is 14.86, which is 7.56 higher than the average value of the breeding value of single - plant yield in saline land of the population, which is 7.30. This confirms that the 234 loci screened by the present invention are related to the salt - alkali tolerance performance of soybeans.

[0062] Table 5

[0063] In the above specific embodiments, the object, technical solution and beneficial effects of the present invention have been further described in detail. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A saline-alkali tolerant soybean liquid chip, characterized in that The genotyping sites of the salt-tolerant soybean liquid chip include 232 SNP sites and 2 Indel sites; The physical positions of the 232 SNP sites and 2 Indel sites are determined based on the whole-genome sequence alignment of the soybean reference genome, and the version number of the whole-genome sequence of the soybean reference genome is Glycine max Wm82.a2.v1; among them, the 232 SNP sites and 2 Indel sites are shown in Table 1: Table 1 ； ； ； ； Among them, the numbers s112 and s118 are Indel sites, and the rest are SNP sites.

2. The liquid-phase chip for salt-alkali tolerant soybean according to claim 1, wherein The salt-tolerant soybean liquid chip includes a probe combination for identifying the genotypes of 232 SNP sites and 2 Indel sites.

3. The design method of the salt-tolerant and alkali-tolerant soybean liquid-phase chip according to claim 1 or 2, characterized in that, It includes the following steps: S1. Obtain the original sites of soybeans through whole-genome resequencing; S2. Align and deduplicate the original sites with the target reference genome to obtain filtered sites; S3. Screen out the salt-tolerant associated sites from the filtered sites, and the associated sites include 232 SNP sites and 2 Indel sites; S4. Design nucleotide probes based on the 232 SNP sites and 2 Indel sites screened out, and prepare a liquid chip.

4. The design method according to claim 3, characterized in that In step S1, the source of the original sites is the GWAS association interval related to the salt-tolerance trait.

5. The design method according to claim 3, characterized in that Step S1 includes the following steps: S11. Sample collection: Collect the DNA samples of soybeans; S12. Resequence the DNA samples obtained in step S11 to obtain the original sequencing data; S13. Perform quality control on the original sequencing data to obtain the filtered original sites.

6. The design method according to claim 3, wherein In step S4, the screening conditions include site filtering, and the conditions for site filtering include: MAF≥0.35, evenly distributed on the chromosome, missing rate≤0.1, and heterozygosity rate≤0.

3.

7. The application of the salt-tolerant soybean liquid chip according to claim 1 or 2 in soybean genotyping.

8. The application of the salt-tolerant soybean liquid chip according to claim 1 or 2 in the association analysis of soybean salt-tolerance traits.

9. The application of the salt-tolerant soybean liquid chip according to claim 1 or 2 in molecular marker-assisted breeding of salt-tolerant soybeans or in genome-wide selection breeding.

10. Application of a soybean molecular marker combination, characterized in that, The soybean molecular marker combination is composed of 232 SNP sites and 2 Indel sites in Table 1 according to claim 1, and the application of the soybean molecular marker combination includes soybean genotyping, association analysis of soybean salt-tolerance traits, and soybean breeding.

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