A saline-alkali-resistant soybean liquid phase chip, a design method and application thereof

Abstract

The application relates to the technical field of molecular detection, and particularly discloses a salt and alkali tolerant soybean liquid phase chip as well as a design method and application thereof. The genotyping sites of the salt and alkali tolerant soybean liquid phase chip include 232 SNP sites and two Indel sites. The physical positions of the 232 SNP sites and the two Indel sites are determined based on whole genome sequence alignment of a soybean reference genome. The version number of the whole genome sequence of the soybean reference genome is Glycine max Wm82.a2.v1. The 232 SNP sites and the two Indel sites are shown in Table 1. The 232 SNP sites and the two Indel sites screened by the application can realize genotyping of salt and alkali tolerant soybeans, and further realize soybean salt and alkali tolerance trait identification. The application can be used for soybean molecular assisted breeding, and salt and alkali tolerant soybeans can be bred.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular detection technology, in particular to a salt-tolerant soybean liquid chip and a design method and application thereof. BACKGROUND

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

[0003] With the sharp decline in the cost of high-throughput sequencing, SNP markers as the third generation of molecular markers are increasingly applied to the identification and evaluation of phenotypes in assisted breeding. In the process of soybean breeding, by identifying the salt-tolerant performance of soybean, salt-tolerant soybean can be quickly bred.

[0004] However, in the field of salt-tolerant soybean breeding chip development, China is still a blank. SUMMARY

[0005] The purpose of the present application is to provide a salt-tolerant soybean liquid chip and a design method and application thereof, which can be used for soybean salt-tolerant trait identification to realize molecular assisted breeding of soybean.

[0006] The present application is realized by the following technical solutions:

[0007] A salt-tolerant soybean liquid chip, 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 the 2 Indel sites are determined based on whole genome sequence alignment of a soybean reference genome, the version number of the whole genome sequence of the soybean reference genome is Glycine max Wm82.a2.v1; wherein, the 232 SNP sites and the 2 Indel sites are shown in Table 1; wherein, the numbers s112 and s118 are Indel sites, and the others are SNP sites.

[0008] The 232 SNP sites and the 2 Indel sites screened out by the present application can realize genotyping of salt-tolerant soybean, and then realize soybean salt-tolerant trait identification, which can be used for molecular assisted breeding of soybean to breed salt-tolerant soybean.

[0009] Further, the salt-tolerant soybean liquid chip includes a probe combination for identifying the genotypes of the 232 SNP sites and the 2 Indel sites.

[0010] A design method of a salt-tolerant soybean liquid chip, comprising the following steps:

[0011] S1, obtaining original sites of soybean through whole genome resequencing;

[0012] S2, aligning the original sites with a target reference genome, and removing duplicates to obtain filtered sites;

[0013] S3, screening salt-tolerant associated sites from the filtered sites, the associated sites including 232 SNP sites and 2 Indel sites;

[0014] S4, designing nucleotide probes based on the 232 SNP sites and 2 Indel sites screened out, and preparing a liquid chip.

[0015] Further, in step S1, the original sites are derived from GWAS associated intervals related to salt tolerance traits.

[0016] Further, step S1 includes the following steps:

[0017] S11, sample collection:

[0018] Collecting DNA samples of soybean;

[0019] S12, resequencing the DNA samples obtained in step S11 to obtain original sequencing data;

[0020] S13, quality control of the original sequencing data to obtain filtered original sites.

[0021] Further, in step S4, the screening conditions include site filtering, and the conditions of site filtering include: MAF is greater than or equal to 0.35, is uniformly distributed on a chromosome, deletion rate is less than or equal to 0.1, and heterozygosity is less than or equal to 0.3.

[0022] Application of the salt-tolerant soybean liquid chip in soybean genotyping.

[0023] Application of the salt-tolerant soybean liquid chip in salt-tolerant soybean trait association analysis.

[0024] Application of the salt-tolerant soybean liquid chip in salt-tolerant soybean molecular marker assisted breeding or in whole genome selection breeding.

[0025] Application of a soybean molecular marker combination, the soybean molecular marker combination is composed of 232 SNP sites and 2 Indel sites in Table 1, and the application of the soybean molecular marker combination includes soybean genotyping, soybean salt-tolerant trait association analysis, and soybean breeding.

[0026] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0027] The application obtains thousands of salt and alkali tolerance associated sites through 399 soybean natural population salt and alkali tolerance related trait whole genome association analysis, reselects soybean salt and alkali tolerance associated sites, obtains 232 SNP sites and 2 Indel sites as soybean salt tolerance gene associated sites, and synthesizes probe combination based on 232 SNP sites and 2 Indel sites according to base complement principle to prepare a liquid phase chip, and the liquid phase chip can realize soybean salt and alkali tolerance genotyping, salt and alkali tolerance soybean molecular marker assisted breeding and whole genome selection breeding. BRIEF DESCRIPTION OF DRAWINGS

[0028] The drawings described herein are used to provide further understanding of the embodiments of the application, form a part of the application, and do not constitute a limitation on the embodiments of the application. In the drawings:

[0029] Figure 1 The distribution information statistical diagram of the 232 SNP sites and 2 Indel sites screened out in Example 2. DETAILED DESCRIPTION

[0030] In order to make the objects, technical schemes and advantages of the present application clearer, below, combined with examples, the present application is further described in detail, the illustrative embodiments of the present application and the description thereof are only used to explain the present application, and do not constitute a limitation on the present application, the examples described below are a part of the examples of the present application, rather than all the examples. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0031] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present application. However, it is apparent to those skilled in the art that the present application can be practiced without these specific details. In other embodiments, in order to avoid obscuring the present application, well-known structures, materials or methods are not specifically described. The materials, instruments and reagents used in the following examples, etc. can be obtained from commercial channels if not specifically stated. The technical means used in the examples, if not specifically stated, are conventional means known to those skilled in the art.

[0032] Example 1:

[0033] Screening of molecular marker combination for salt and alkali tolerance soybean genotyping

[0034] This example is based on the original sites (original sequencing data) obtained through 399 soybean natural population salt and alkali tolerance related trait whole genome association analysis, and the salt and alkali tolerance associated sites are selected based on the original data, and finally 232 SNP sites and 2 Indel sites as shown in Table 1 are obtained. The specific screening process includes the following steps:

[0035] S1, obtaining original sites of soybean by whole genome resequencing;

[0036] S11, sample collection:

[0037] Collecting DNA samples of 399 soybean natural populations (the sources of 399 soybean natural populations: collected in the laboratory, mainly Chinese cultivated soybeans);

[0038] S12, resequencing the DNA samples obtained in step S11 to obtain original sequencing data;

[0039] The basic process of resequencing is as follows:

[0040] First, the CWseq Universal DirectFast DNA Library Prep Kit kit is used to construct the resequencing library of the qualified DNA. After the library construction is completed, the library quality is detected. After the library quality is detected, the MGI-T7 sequencing platform of Huada is used for sequencing, and the sequencing mode is PE150 mode.

[0041] S13, quality control of original sequencing data, obtaining filtered original sites (filtered fragments);

[0042] The original data is filtered and processed, mainly to remove adapter contamination and low-quality sequencing fragments (reads), and to obtain high-quality filtered data (clean reads) for subsequent alignment analysis.

[0043] The filtering is performed by using the SOAPnuke (v1.5.6) software independently developed by Huada (https: / / github.com / BGI-flexlab / SOAPnuke), and the specific filtering conditions are as follows:

[0044] (1) removing sequencing fragments (reads) containing adapters;

[0045] (2) removing low-quality sequencing fragments (reads), low quality refers to that the proportion of bases with base quality value less than or equal to 15 is 10%;

[0046] (3) removing reads containing N bases with a proportion greater than 10%.

[0047] Using the above filtering conditions, the filtered SNP sites and Indel sites are obtained for subsequent alignment analysis.

[0048] S2, aligning the original sites with the target reference genome, deduplicating, obtaining filtered sites, ensuring that the original sites 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. Specifically, the following steps are included:

[0049] S21, alignment: using the "mem" algorithm of the short sequence alignment software BWA (Version: 0.7.12-r1039) [1] to align the quality-filtered fragments (clean reads) to the reference genome (Glycine max Wm82.a2.v1) (alignment parameters: -t8-k19-M-R), using 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 deduplicate and sort the alignment file, obtain the bam file, and use it for subsequent analysis.

[0050] S22, Bam correction: selecting mapQ value greater than 20 and properly paired reads for subsequent variant detection analysis (filtering parameters: samtools view-f2-q 20). In order to improve the accuracy of the SNP sites around indel, using the RealignerTargetCreator and IndelRealigner tools of GATK (v3.3.0) [3] software to realign and correct the sequence around indel, the processed bam file can be used for variant detection.

[0051] S33, using the HaplotypeCaller module of GATK (3.30) to perform multiple sample variant detection on the processed alignment file to obtain detected SNP sites and Indel sites; using the GATK (3.30) VariantFiltration module to perform hard filtering on SNPs and Indels, with specific parameters: QD < 2.0, FS > 60.0, MQ < 40.0, MQRankSum < -12.5, ReadPosRankSum < -8.0; strict filtering on SNPs: SNP cluster filtering (no more than 2 SNPs within 5bp), Indel nearby SNP filtering (SNPs within 5bp of Indels are filtered out), and adjacent Indel filtering (the distance between two Indels cannot be less than 10bp); and filtering out sites with GQ (Genotype Quality) less than 20.0, with the sample typing quality marked as lowGQ.

[0052] Using the above filtering conditions, a total of 1.3 x 10 7 SNP sites and 1.2 x 10 6 Indel sites were obtained as filtered sites for subsequent associated site mining.

[0053] S3, screening salt-tolerant associated sites from the filtered sites, with screening conditions being: MAF ≥ 0.35, uniformly distributed on chromosomes, deletion rate ≤ 0.1, and heterozygosity rate ≤ 0.3, and finally obtaining associated sites including 232 SNP sites and 2 Indel sites, as shown in Table 1.

[0054] Table 1

[0055]

[0056]

[0057]

[0058]

[0059] Among them, s112 and s118 are Indel sites, and the rest are SNP sites.

[0060] Note: The physical position represents a point on the reference genome, i.e. a base, and the Indel site corresponds to the first base in the reference column.

[0061] Note: The number before the underscore in the physical position represents the chromosome, and the number after the underscore represents the physical position of the site on the corresponding chromosome.

[0062] Example 2

[0063] The salt-tolerant soybean liquid chip comprises a probe combination for identifying the genotypes of 232 SNP sites and 2 Indel sites.

[0064] The purpose of this embodiment is to apply the 232 SNP sites and 2 Indel sites in Table 1 to probe design, and the principle of probe design is as follows: probes are designed within 110 bp on the left and right of all target sites, and the length of the probes is generally 110 bp, and the GC content is between 30 ~ 70%.

[0065] Based on the above probe design principle, 918 capture probes were synthesized by Shijiazhuang Borui Di (the probes designed for long fragment Indel sites will be overlapped, according to the length of the Indel, the number of probes for Indel sites is 2 ~ 3, and the probes designed for SNP sites are generally not overlapped, and adjacent sites will share a probe), and the precision positioning sequencing typing technology (cGPS) based on the target region genome sequence liquid capture was used to form the system of the salt-tolerant soybean liquid chip, wherein the cGPS is based on an optimized thermodynamic stability algorithm model, and probes are designed for different target region genome sequences, and specific probes are synthesized to capture and enrich multiple different target sequences located in different genome positions by liquid hybridization, and then sequencing library construction and high-throughput sequencing are performed on the captured and enriched target genome sequences, so as to obtain the genotypes of all SNP / Indel sites in the target region. According to the probe design results, the probes that cannot be uniquely aligned on the genome and contain repeat sequences in the flanking sequence are removed, the designed probes are applied to the preparation of the new liquid chip, and finally the salt-tolerant soybean liquid chip is obtained, and the distribution information statistics of each site in the chip are shown in Table 1. Figure 1 The probes corresponding to the 232 SNP sites and 2 Indel sites are shown as SEQ ID NO. 1 ~ SEQ ID NO. 234.

[0066] Example 3

[0067] Application of the salt-tolerant soybean liquid chip in soybean genotyping:

[0068] (1) Extraction and detection of soybean genomic DNA sample:

[0069] Select 237 soybeans as the verification sample of salt-tolerant soybean liquid chip prepared in Example 2. The tissues of 237 soybeans were collected, and high-throughput DNA extraction kit (GenPrep conventional plant DNA rapid extraction kit (magnetic bead method) Mix_V4.0, specification: 960 rxn) was used to extract sample DNA. The extracted DNA samples were detected in two ways: (1) 1% agarose gel electrophoresis method was used to analyze the purity and integrity of DNA; (2) Qubit was used to accurately quantify the concentration of DNA.

[0070] The 237 soybeans were from F3 generation of 5 hybrid combinations. The method was conventional hybridization. All single plants of F1 generation were sowed with a pod at the same leaf position, F2 was sowed, and each single plant was sowed with a pod to form F3 generation population. The 237 soybeans were randomly selected from F3 generation. The hybrid combinations were H12*PI538401, H12*Fendou 92, Shandaxian No. 5*JACK2-3, Shandaxian No. 3*W82 and Shandaxian No. 1*W82.

[0071] (2) GenoBaits experimental process:

[0072] The qualified DNA for quantitative quality inspection was randomly physically broken by ultrasonic wave crusher, and the peak value of the broken fragments was controlled at 200-300 bp. The broken DNA was end-repaired and connected with A tail. The DNA fragments with A were connected with sequencing adapters by using ligase, and then the library was purified and fragment selected by using carboxyl-modified magnetic beads, and the connection product with inserted fragments at 200-300 bp was reserved. The connection product was added with sequencing primer with Barcode and high-fidelity PCR reaction system for PCR amplification. Different Barcodes were used to distinguish different samples. After purification by carboxyl magnetic beads, the amplification product can be used for probe hybridization experiment.

[0073] 500 ng of the constructed sequencing library was taken, freeze-dried, and then added with probes (or salt-tolerant soybean liquid chip containing probes) and hybridization reagents (blocking reagent, Rnase Block). After denaturation, it was incubated at 65°C for 2 hours to complete the hybridization reaction. The hybridization product was washed with washing solution, and then a round of PCR was performed to complete the construction of hybridization capture library.

[0074] (3) Library construction and sequencing:

[0075] The samples qualified by the above DNA quality inspection were used to construct the targeted sequencing library using the corresponding products. Finally, the library preparation of all samples in this project was completed. After the library construction, Qubit 2.0 was used for preliminary quantification, and qPCR was used for accurate quantification of the effective concentration of the library to ensure the quality of the library. After the library was qualified, the PE150 sequencing was performed on the Huada sequencing platform.

[0076] (4) Information analysis process:

[0077] After the raw data of high-throughput sequencing was processed by quality control and filtration, the fastp software was used to remove the Reads containing adapter contamination and low-quality Reads, and the BWA software was used for alignment with the target genome. Then, the GATK software was used for variant site analysis of the sequencing results to obtain the genotyping results of the target site.

[0078] Example 4:

[0079] Evaluation of the effect of the salt-tolerant soybean liquid chip on soybean genotyping

[0080] The salt-tolerant soybean liquid chip prepared in Example 2 was used to detect 13 soybean samples and 13 repeated soybean samples according to the method of Reference Example 3. After sequencing and data analysis (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 repeated soybean samples were compared. The comparison results are shown in Table 3.

[0081] Table 2

[0082]

[0083] From the data in Table 2, it can be seen that the uniformity of the salt-tolerant soybean liquid chip prepared in Example 2 is more than 99%, which indicates that it has the advantage of high detection stability

[0084] Table 3

[0085]

[0086] From Table 3, it can be seen that:

[0087] The genotype consistency rate of the salt-tolerant soybean liquid chip prepared in Example 2 is more than 99%, which indicates that it has the advantage of high accuracy.

[0088] Example 5:

[0089] Association analysis of molecular marker combination (232 SNP sites and 2 Indel sites) and soybean salt-tolerant traits

[0090] The salt-tolerant traits of soybean mainly include four shapes: fresh weight of seedling under salt stress (NFW), emergence rate in saline-alkali land (SE), germination rate under salt (GR), and root length under salt (NRL).

[0091] The 232 SNP sites and 2 Indel sites screened in the application are respectively related to fresh weight of seedling under stress (NFW), emergence rate in saline-alkali land (SE), germination rate under salt (GR), and root length under salt (NRL), and the association of the sites with the corresponding traits is reflected by p value (P.value) and effect value (Effect). The p value (P.value) and effect value (Effect) of the 232 SNP sites and 2 Indel sites corresponding to the shapes are shown in Table 4.

[0092] Table 4

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] The p value and effect value are obtained by GWAS using GAPIT (http: / / zzlab.net / GAPIT / ). The MLM (PCA+Kinship) model in GAPIT is used for association analysis of salt-tolerant phenotype indexes.

[0100] Significance threshold determination: the number of independent effective SNPs (Ne) is estimated based on linkage disequilibrium by using GEC software, and the threshold of significant association is calculated according to the adjusted Bonferroni method: p = 1 / Ne (Li et al , 2012).

[0101] The effective independent SNP number of 589,608 SNP data in the whole genome is 123,364 after evaluation. The -log 10 P = 5.090 is the threshold of significant association of P value, and the threshold provided is the threshold of GWAS for obtaining associated markers. As shown in Table 4, the P values of the 234 sites screened in the application are all greater than the threshold.

[0102] It can be proved by the embodiment that the 232 SNP sites and 2 Indel sites screened by the application are closely related to the salt-tolerant trait of soybean, that is, the salt-tolerant soybean liquid chip prepared in the embodiment 2 can be used to detect whether the target gene (salt-tolerant gene) exists, and the genotype of the offspring can be detected in the process of backcross breeding of soybean and in the hybrid offspring, so as to determine whether the offspring contains the target gene, and the salt-tolerant soybean liquid chip prepared in the embodiment 2 can be used for molecular marker assisted breeding and genomic selection breeding.

[0103] Embodiment 6

[0104] Application of salt-tolerant soybean liquid chip in breeding

[0105] In the F3 generation of the hybrid population, the salt-tolerant soybean liquid chip prepared in the embodiment 2 is used for genotype detection of the F3 generation plants, based on the detection results, 8 individuals with high predicted values of single plant yield in saline-alkali land (that is, 8 individuals with the largest number of superior allelic variations related to 4 salt-tolerant traits are selected; the 4 salt-tolerant traits are fresh weight of seedling under stress (NFW), emergence rate in saline-alkali land (SE), germination rate under salt (GR), and root length under salt (NRL), 8 soybean lines with high breeding values of single plant yield in saline-alkali land (salinity of about 0.3% and pH of about 8) are obtained, the single plant yield of the 8 soybean lines after planting is counted, and the statistical results are shown in Table 5, and finally 8 soybean lines with high breeding values of single plant yield in saline-alkali land are selected as shown in the table below, taking single plant weight as an example, the average breeding value of single plant yield in saline-alkali land is 14.86, which is 7.56 higher than the average value of 7.30 of the breeding value of single plant yield in saline-alkali land of the population, which confirms that the 234 sites screened by the application are related to the salt-tolerant performance of soybean.

[0106] Table 5

[0107]

[0108] The above detailed description further illustrates the purpose, technical solutions and beneficial effects of the application, and it should be understood that the above is only a specific embodiment of the application and is not used to limit the protection scope of the application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the application shall be included in the protection scope of the application.

Claims

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

2. The salt- and alkali-resistant soybean liquid phase chip according to claim 1, characterized in that, The salt-tolerant soybean liquid phase chip includes a probe combination for identifying genotypes at 232 SNP sites and 2 Indel sites.

3. The design method of the salt- and alkali-resistant soybean liquid phase chip as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Obtain the original loci of soybeans through whole-genome resequencing; S2. Align the original loci with the target reference genome and remove duplicates to obtain the filtered loci; S3. Select salt-alkali tolerant associated sites from the filter sites. The associated sites include 232 SNP sites and 2 Indel sites. S4. Based on the 232 SNP sites and 2 Indel sites selected, nucleotide probes were designed and liquid-phase chips were prepared.

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

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

6. The design method according to claim 3, characterized in that, In step S4, the screening criteria include site filtering, which includes the following criteria: MAF ≥ 0.35, uniform distribution on chromosomes, deletion rate ≤ 0.1, and heterozygosity rate ≤ 0.

3.

7. The application of the salt-tolerant soybean liquid phase chip as described in claim 1 or 2 in soybean salt-tolerant genotyping.

8. The application of the salt-alkali tolerant soybean liquid phase chip as described in claim 1 or 2 in the correlation analysis of salt-alkali tolerance traits in soybeans.

9. The application of the salt-tolerant soybean liquid phase chip as described in claim 1 or 2 in marker-assisted breeding of salt-tolerant soybeans or in whole-genome selection breeding of salt-tolerant soybeans.

10. An application of a soybean molecular marker combination, characterized in that, The soybean molecular marker combination consists of 232 SNP sites and 2 Indel sites as described in Table 1 as claimed in claim 1. The applications of the soybean molecular marker combination include soybean salt-alkali tolerance genotyping, soybean salt-alkali tolerance trait association analysis, and salt-alkali tolerant soybean breeding.

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