KASP primer set based on SNP for discriminating soybean salt-tolerant cultivar and uses thereof

Abstract

The present invention relates to a primer set for SNP-based KASP (kompetitive allele specific PCR) for identifying salt-tolerant soybean varieties, comprising a primer set consisting of oligonucleotides of SEQ ID NOs. 1 to 3. It is expected that by using the primer set of the present invention to select salt-tolerant soybean varieties, it will be possible to prevent a decrease in soybean yield caused by soil salinity.

Description

Technology Field

[0001] The present invention relates to a set of primers for SNP-based KASP for identifying salt-tolerant soybean varieties and the use thereof. Background Technology

[0002] Salinity is a major abiotic stress that affects soil fertility and reduces crop yields. Salinity stress is generally associated with the use of irrigation facilities and crops that absorb water from irrigated soil and leave most of the salt in the soil. Even relatively high-quality irrigation water contains significant amounts of salt, and over time, this salt hinders crop growth; it is estimated that 60 million hectares, or about 20% of the world's irrigated farmland, are affected.

[0003] The accumulation of salt induces osmotic stress in plants and disrupts cellular ion homeostasis. In particular, high concentrations of Na + and Cl - Ions interfere with plants' absorption of essential elements such as nitrogen and potassium, thereby inhibiting photosynthesis and protein synthesis.

[0004] Soybeans are a crop with moderate salinity sensitivity, and soil salinity has been found to inhibit germination, plant growth, nodule formation, and seed production. High concentrations of Na in the leaves + and Cl - If it accumulates, chloroplasts and other organelles are damaged, and if it accumulates in the roots, K + , Ca 2+ , NO3 - The absorption of essential elements such as is inhibited, and Na + and Cl - Ion toxicity negatively affects ion homeostasis and protein synthesis. Salt stress reduces photosynthetic pigment levels and Cl in chloroplasts - and Na + It accumulates and damages the thylakoid membrane, reducing the plant's photosynthetic capacity.

[0005] Salt-sensitive soybean crops have an average Cl level 18 times higher than salt-tolerant soybean varieties - It has been reported that the yield was 37% lower and that as salt stress increased, soybean yield decreased by 40%. Accordingly, it has become important to develop salt-tolerant soybean varieties to secure more stable yields in saline soils.

[0006] Meanwhile, over the past few decades, several studies have been conducted to understand the genetic mechanisms related to salt tolerance in soybeans. Ncl The gene is a single dominant gene involved in salt stress in soybeans, and Cl causes leaf yellowing and necrosis. - It was revealed to possess an ion-repelling function. 106 F, produced by crossing the salt-tolerant S-100 with the salt-sensitive Tokyo line. 2:5 Using recombinant inbred lines, QTLs related to salt tolerance on chromosome 3 (Chr. 3; LG-N) were identified, and these QTLs were determined through pedigree tracing Ncl It was presumed to be a locus. In addition, in a study that mapped the salt-tolerant QTL of PI483463 to chromosome 3 Ncl It suggested that the gene is closely related to salt tolerance in various soybean seeds. The genomic region of this major QTL is a putative candidate gene Glyma03g32900 It was further narrowed to 209kb next to markers Qs08064 and Barcsoyssr_3_1303 containing, and Glyma03g32900 After DNA sequencing analysis GmCHX1 It was named and was confirmed to encode an ion transporter. In addition, after map-based cloning GmSALT3 It was named as and Na + and Cl - It was identified as a joint transport vehicle.

[0007] Gamma-induced mutagenesis is a widely used method for breeding purposes to generate new mutations in plant species. For example, Wonryul, a new variety resistant to soybean mummification, was developed by irradiating the native soybean KAS360-22 with gamma rays (Kim et al. 2013). In 2012, two mutant rice lines with excellent salt tolerance were tested with 171 mM NaCl after irradiating the rice variety Dongan with gamma rays ( Oryza sativa ST-87 and ST-301 have been produced.

[0008] Against this backdrop, the inventors generated a salt-tolerant soybean mutant using gamma irradiation and developed an SNP marker system capable of distinguishing it, thereby completing the present invention. Prior art literature

[0009] Ulukapi K, Nasircilar AG (2015) Developments of gamma ray application on mutation breeding studies in recent years. International Conference on Advances in Agricultural, Biological & Environmental Sciences. International Institute of Chemical, Biological, and Environmental..., pp 31-34Song J, Kim D, Lee M, Lee K, Kim J, Kim S, Ha B, Yun S, Kang S (2012) Physiological characterization of gamma-ray induced salt tolerant rice mutants. Australian Journal of Crop Science 6:421-429 The problem to be solved

[0010] The present invention aims to create a new soybean variety resistant to salt stress and to construct a primer set for KSAP to identify it. The present invention created a new salt-tolerant mutant KA-1285 by irradiating soybeans with gamma rays, discovered a QTL associated with the salt-tolerant phenotype located on chromosome 3 of KA-1285, discovered a specific genetic variation near the QTL, and constructed a primer set for KASP to identify the salt-tolerant mutant allele based on this. means of solving the problem

[0011] To solve the above problem, the present invention provides a primer set for KASP (kompetitive allele specific PCR) for identifying salt-tolerant soybean varieties, comprising a primer set consisting of oligonucleotides of SEQ ID NOs 1 to 3.

[0012] In addition, the present invention provides a kit for identifying salt-tolerant soybean varieties comprising the primer set and a reagent for performing an amplification reaction.

[0013] In addition, the present invention provides a method for identifying a salt-tolerant mutant soybean variety from a wild-type parent variety, comprising the steps of: isolating genomic DNA from a soybean sample; performing KASP (kompetitive allele specific PCR) using the isolated genomic DNA as a template and the primer set; and analyzing the amplification product of the KASP reaction.

[0014] In the present invention, the salt-tolerant mutant soybean variety may be KA-1285, and the wild-type parent variety may be Gwangan. Effects of the invention

[0015] By using the primer set for KASP of the present invention, when a new soybean variety resistant to salt stress, such as KA-1285, is developed by gamma irradiation, a method is provided to accurately identify the salt-tolerant soybean variety in a simple and economical manner, so it can be usefully utilized to prevent yield reduction caused by soil salinity. Brief explanation of the drawing

[0016] Figure 1 shows the results of treating five genotypes of soybeans (Gwangan, Daepung, S100, PI483463, KA-1285) with 150 mM salt for two weeks. Figure 2 shows the results of comparing the dry weights of height (A), leaves (B), stems (C), and roots (D) for five salt-treated soybean genotypes (Gwang-an, Daepung, S100, PI483463, KA-1285) and their respective controls (* indicates p < 0.05, ** indicates p < 0.01, and ns indicates not significant). Figure 3 shows the Cl accumulated in the leaves, stems, and roots of five genotypes of soybeans (Gwang-an, Daepung, S100, PI483463, KA-1285) upon salt treatment. - and Na + This is the result of comparing the quantities. Figure 4 shows the effect of salt stress on photosynthetic chlorophyll fluorescence between the control and salt-treated groups for five genotypes of soybeans (Gwangan, Daepung, S100, PI483463, KA-1285) (A is SPAD value, B is Fv / Fm, C is rETR, * is p < 0.05, ** is p < 0.01, and ns means not significant). Figure 5 shows the F1 of crossing Daepung and KA-1285 2:3 LSS (A), SPAD value (B), and Cl when treating the system with 150mM NaCl - (C) and Na + This is the concentration distribution of (D). Figure 6 shows a gene linkage map created by mapping 1,452 SNP markers of the F2 lineage to 20 soybean chromosomes. Figure 7 shows the results of QTL mapping regarding salt tolerance, where A is F 2:3 This is the QTL peak map of chromosome 3 of the lineage, and B shows candidate genes for salt tolerance. Figure 8 shows the results of genotyping for a mutant (KA-1285) and a wild type (Gwangan, PI483463) using KASP markers. Figure 9 shows the results of analyzing the expression patterns of candidate genes (Glyma03g171500(A), Glyma03g171600(B), Glyma03g171700(C)) using qRT-PCR in the roots of Gwangan and KA-1285. Specific details for implementing the invention

[0017] To achieve the objective of the present invention, the present invention provides a primer set for KASP (kompetitive allele specific PCR) for identifying salt-tolerant soybean varieties, comprising a primer set consisting of oligonucleotides of SEQ ID NOs 1 to 3.

[0018] The term "KASP (kompetitive allele specific PCR)" of the present invention is one of the PCR-based analysis methods, a homogeneous and fluorescence-based genotyping technique. KASP is a technique based on fluorescence resonance energy transfer for allele-specific oligo extension and signal generation.

[0019] The primer set for KASP of the present invention is related to the resistance of soybeans to salt stress. Glyma171600It is a primer set capable of detecting polymorphisms in the upstream region of a gene. Specifically, the primer set for KASP is Glyma171600 It is possible to identify the presence or absence of a deletion mutation of -30 to -20 bp, or specifically -25 bp, upstream of the gene.

[0020] The primer set for KASP of the present invention can identify salt-tolerant soybean varieties by combining two forward primers (Sequence No. 1 and Sequence No. 2), which have base sequences that differ partially from the fluorescent sample, with the same reverse primer (Sequence No. 3). For example, using the reverse primer of Sequence No. 3 and the forward primer of Sequence No. 1 allows for the identification of a mutant soybean variety with high resistance to salt stress compared to the parent variety, while using the forward primer of Sequence No. 2 with the reverse primer of Sequence No. 3 allows for the identification of the parent wild-type soybean variety.

[0021] The primer for KASP of the present invention may include an oligonucleotide composed of a fragment of 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, or 31 or more consecutive nucleotides within SEQ ID NOs 1 to 3, depending on the sequence length of each primer. For example, the primer of SEQ ID NO. 1 (30 oligonucleotides) may include an oligonucleotide composed of a fragment of 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, or 29 or more nucleotides within the sequence of SEQ ID NO. 1. Additionally, the primer may also include a sequence in which the base sequences of SEQ ID NOs 1 to 3 have been added, deleted, or substituted.

[0022] In the present invention, "primer" refers to a single-stranded oligonucleotide sequence complementary to the nucleic acid strand to be replicated, and can serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primer must allow the synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the complexity of the required DNA or RNA target, as well as primer usage conditions such as temperature and ionic strength.

[0023] In this specification, the oligonucleotide used as a primer may also include a nucleotide analogue, for example, a phosphorothioate, an alkylphosphorothioate, or a peptide nucleic acid, or an intercalating agent.

[0024] The aforementioned salt-tolerant soybean variety refers to a soybean capable of growing even in soil containing salt. While wild-type parent varieties fail to perform normal photosynthesis and protein synthesis and experience reduced yields when salt accumulates in the soil above a certain level due to osmotic stress and disruption of cellular ion homeostasis, salt-tolerant soybean varieties possess resistance to salt, enabling cultivation even in soils with high salt concentrations.

[0025] The present invention also provides a kit for identifying salt-tolerant soybean varieties comprising the primer set and a reagent for performing an amplification reaction.

[0026] In the kit of the present invention, the reagent for performing the amplification reaction may include DNA polymerase, a coenzyme of DNA polymerase, dNTPs, a buffer, etc. Additionally, the kit of the present invention may additionally include an internal control primer pair in addition to the primer set.

[0027] The kit of the present invention may preferably be a kit for KASP, but is not limited thereto.

[0028] The present invention also,

[0029] Step of isolating genomic DNA from a soybean sample;

[0030] A step of performing KASP using the isolated genomic DNA as a template and the primer set; and

[0031] A method for identifying a salt-tolerant mutant soybean variety from a wild-type parent variety is provided, comprising the step of analyzing the amplification product of the above KASP.

[0032] In the method for determining a salt-tolerant soybean variety of the present invention, the parent variety is Gwangan, and the salt-tolerant mutant soybean variety may be KA-1285.

[0033] The method of the present invention comprises the step of isolating genomic DNA from a soybean sample. The method of isolating genomic DNA from the soybean sample may utilize methods known in the art, or may utilize an ethanol precipitation method following phenol:chloroform extraction. Using the isolated genomic DNA as a template, a target sequence may be amplified by performing an amplification reaction by adding DNA polymerase using an oligonucleotide primer set according to one embodiment of the present invention as primers. Methods for amplifying the target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, strand displacement amplification, or amplification via Qβ replicase, or any other suitable method for amplifying nucleic acid molecules known in the art. In the above, PCR is a method of amplifying a target nucleic acid from a primer pair that specifically binds to the target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

[0034] In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling substance may be a fluorescent, phosphorescent, or radioactive substance, but is not limited thereto. Preferably, the labeling substance may be FAM, HEX, VIC, JOE, ROX, TAMRA, Cy3, or Cy5, etc. When PCR is performed by labeling the 5'-terminus of a primer with the labeling substance during the amplification of the target sequence, the target sequence may be labeled with a detectable fluorescent labeling substance. The set of oligonucleotide primers used to amplify the target sequence is as described above.

[0035] The structure and effects of the present invention will be explained in more detail below through examples. These examples are solely for the purpose of illustrating the present invention, and the scope of the present invention is not limited by these examples.

[0036] Example 1: Securing a salt-tolerant mutant after gamma irradiation of soybeans

[0037] At the Korea Atomic Energy Research Institute, approximately 1,300 M2 mutants were generated by irradiating Gwangan, a salt-sensitive variety, with 250 Gy of gamma rays. M2 seeds were planted in 50-cell plastic trays and treated with 100 mM NaCl at the V2 stage to select salt-tolerant individuals. One salt-tolerant mutant line selected in the preliminary evaluation was advanced to the M7 generation and named KA-1285.

[0038] Example 2 Phenotypic analysis regarding salt stress tolerance

[0039] Tolerance to salt stress in a greenhouse was evaluated using salt-sensitive varieties Gwang-an and Daepung, and salt-tolerant varieties S100 (Lee et al. 2004), PI483463 (Ha et al. 2013), and KA-1285.

[0040] First, seeds of each genotype were planted in 50-cell plastic trays (27 x 53 x 11.2 cm) filled with a growing medium (coco peat, peat moss, zeolite, perlite, caldolomite, wetting agent, and fertilizer), with one seedling grown per hole, and the experiment was performed in two replicates. Five plants of each genotype were used per replicate, and at the V2 stage, they were treated with 0 (control) and 150 mM NaCl for two weeks.

[0041] After two weeks, the salt-sensitive Gwang-an and Daepung showed severe yellowing of leaves due to salt treatment, while the salt-tolerant genotype and KA-1285 showed relatively healthy leaves with less damage (Fig. 1).

[0042] Example 2-1 Comparison of Soybean Height and Dry Weight

[0043] Height and dry weight were measured and compared for five genotypes of soybeans, including KA-1285. Plant height was measured from the rim of a plastic tray to the top of the main stem. The dry weight of leaves, stems, and roots was measured after drying them in a 70°C oven for 3 days prior to measurement.

[0044] As a result, the height of plants of all genotypes decreased significantly due to salt treatment compared to the control group. Salt-sensitive Gwang-an and Daepung decreased by 25% and 22%, respectively, while salt-tolerant genotypes PI483463 and S100 decreased by 13% and 21%, respectively. In the case of KA-1285, the height decreased by 20% from 31.7 cm to 25.3 cm after salt treatment (Fig. 2A).

[0045] In addition, the dry leaf weight of Gwang-an and Daepung decreased by 36% and 34%, respectively, while PI483463 decreased by 10% and KA-1285 decreased by 41%. However, no significant decrease in dry leaf weight was observed in any of the five genotypes of soybeans (Fig. 2B).

[0046] In contrast, regarding stem dry weight, the salt-sensitive Gwang-an and Daepung were significantly lower under salt treatment, decreasing by 52% and 54%, respectively. On the other hand, no significant decrease was observed in the salt-tolerant genotypes except for S100 (Fig. 2C).

[0047] In addition, for all genotypes except Daepung, the decrease in dry root weight due to salt treatment was not significant compared to the control group (Fig. 2D).

[0048] The genotypes that did not show a significant decrease in plant height and dry weight of leaves, stems, and roots are PI483463 and KA-1285.

[0049] Example 2-2 Analysis of Sodium and Chloride Ion Accumulation

[0050] Leaves, stems, and roots of plants of each genotype were sampled after two weeks of salt treatment. The samples were dried in a 62°C oven for 24 hours and then ground using a grinder (Geno Grinder 2010, Horiba). Subsequently, 0.15 g of the ground sample was mixed with 30 mL of distilled water and shaken at 200 rpm for one hour. The suspension was then passed through filter paper (Whatman® qualitative filter paper, Grade 4) to produce a clear solution capable of measuring NaCl concentration.

[0051] Chlorine concentration was measured using an ion-selective electrode (perfection comb CL; METTLER TOLEDO, Schwerzenbach, Switzerland). The enclosed chloride ISE standard solution was diluted and measured at three points (1 x 10⁻⁶). -1 , 1x10 -2 , 1x10 -3 Calibration was performed at mol / L. The sodium concentration was Na + Measurements were taken using a measuring instrument (LAQUQtwin-NA-11, Horiba, Japan), and the enclosed Na +Calibration was performed at two points (10 mg / L and 150 mg / L) using standard solutions. For chlorine and sodium, respectively, a tenfold change in concentration was indicated for every change of -59.16 mV and 59.16 mV (based on the theoretical Nernstian slope at 25°C), and these measurements were converted to mg / g.

[0052] As a result, Gwangan and Daepung, which are sensitive to salt, have Cl in their leaves and stems - and Na + While the concentration was found to be higher after salt treatment, in the case of the salt-tolerant genotype, Cl - and Na + No significant difference in concentration was observed (Fig. 3). Cl of the leaves - The concentration was lowest for KA-1285 at 9.2 mg / g, followed by S100 (10.3 mg / g) and PI486463 (10.5 mg / g), and Na + The concentration was lowest for S100 at 4.5 mg / g, followed by KA-1285 (5.4 mg / g) and PI483463 (9.2 mg / g) (Fig. 3A, 3D).

[0053] In the stems, PI483463 had the lowest concentrations of Cl- and Na+, followed by KA-1285 and S100 (Fig. 3B, 3E).

[0054] In the roots, Cl of PI483463 - The concentration was lowest, and the concentration of KA-1285 was highest (Fig. 3C). Root Na + There was no significant difference in concentration between genotypes (Fig. 3F).

[0055] KA-1285 showed the lowest NaCl levels in the leaves but high levels in the roots, which is consistent with the Cl reported by Do et al. (2016). - ions NclThis result is consistent with the fact that, due to its influence, it accumulates more in the underground parts than in the above-ground tissues, and the salt tolerance mechanism of KA-1285 is that Cl accumulates instead of large amounts of NaCl in the roots. - It can be inferred that this prevents movement into the leaves. Since the accumulation of NaCl in leaves leads to a decrease in photosynthetic pigments and damage to the thylakoid membrane, resulting in reduced photosynthetic capacity (Ashraf and Harris 2013; Omoto et al. 2010; Wu and Zou 2009), it can be inferred that KA-1285 will also cause less damage to photosynthetic capacity in the leaves.

[0056] Example 2-3 Chlorophyll Fluorescence Analysis

[0057] To investigate the effects of salinity stress on photosynthetic efficiency, soybeans of five genotypes were treated with 150 mM NaCl for two weeks, after which fluorescence parameters were analyzed using a fluorescence meter (Monitoring Pen, MP 100-E, Photon Systems Instruments, Czech Republic). After measuring the SPAD values ​​of the mesophageal leaflets among the fully developed trifoliate leaves, the plants exhibiting the highest values ​​were dark-acclimatized for 30 minutes around 9 PM and observed using the LC2 protocol of the Monitoring Pen. SPAD was measured using a SPAD-502 meter (SPAD-502, Minolta Co. Ltd., Japan), with 10 measurements taken per individual.

[0058] Fluorescence parameters were calculated according to the following formula.

[0059] Fv / Fm = (Fm - Fo) / Fm

[0060] rETR = QY x PAR x 0.84 x 0.5

[0061] Here, Fo is the minimum chlorophyll fluorescence intensity measured in the dark acclimatization state when all photosystem II (PSII) reaction centers are open, Fm is the maximum chlorophyll fluorescence intensity measured in the dark acclimatization state under a saturated light pulse, Fv is the variable chlorophyll fluorescence (Fm - Fo) measured in the dark acclimatization state, and Fv / Fm is the maximum quantum yield of photosystem II in the dark acclimatization state. Fv / Fm is an indicator that decreases in stressful environments, and in healthy plants, it generally appears as about 0.83. rETR is the relative electron transport rate, which is proportional to CO2 assimilation in the leaf and is related to the photosynthetic process. QY is the quantum yield, and PAR is the photosynthetically active radiation.

[0062] As a result, the SPAD values ​​of Gwang-an (28%) and Daepung (33%) decreased significantly compared to the control group due to salt treatment. On the other hand, the salt-tolerant genotypes PI483463 and KA-1285 did not show a significant decrease in SPAD values, indicating that chlorophyll content was not affected by salt stress. However, the salt-tolerant genotype S100 (23%) showed a significant decrease in SPAD values ​​(Fig. 4A).

[0063] And regarding maximum quantum yield (Fv / Fm), the salt-sensitive Gwangan and Daepung decreased by 4–5%, but salt stress had almost no effect on the Fv / Fm of the salt-tolerant genotype (Fig. 4B).

[0064] In addition, similar to Fv / Fm, the rETR values ​​for salt-sensitive Gwangan and Daepung decreased by 15% and 8%, respectively, while those for PI483463 and KA-1285 increased by 2% each (Fig. 4C).

[0065] The combined results of Example 2 show that KA-1285 has resistance to very high salt stress.

[0066] Example 3: Identification of Salt Tolerance Candidate Genes and Analysis of Genetic Variations via QTL Mapping

[0067] Example 3-1 F 2:3 Analysis of the line's salt tolerance phenotype

[0068] The salt-tolerant mutant KA-1285, selected by the method of Example 1, was crossed with Daepung, a salt-sensitive variety, at Chonnam National University. The F1 generation was propagated to the F2 generation in a winter greenhouse, and F2 seeds were sown in a field at Chonnam National University. F2 plants were harvested individually to obtain F 2:3 Obtained a lineage. F 2:3 169 individuals of the lineage were obtained, and they were planted in 50-cell plastic trays and grown in a greenhouse.

[0069] A total of 169 Fs 2:3 The strain was used to investigate salt tolerance phenotypes. The experiment was conducted by treating with 150 mM NaCl for 2 weeks, identical to Example 2, and LSS (leaf scorch socre), SPAD values, and Cl - , Na + The phenotype was analyzed by measuring the concentration of.

[0070] LSS (leaf scorch score) is a traditional method for evaluating salt tolerance through visually observable damage, and was measured according to the method reported by Lee et al. (2008). LSS was visually evaluated on a 5-point scale (no yellowing of leaves = 1, 25% yellowing of leaves = 2, 50% yellowing of leaves = 3, 75% yellowing of leaves = 4, wilting = 5), and the average was calculated by evaluating 5 times per replicate.

[0071] The SPAD value is Cl in the same way as in Examples 2-3. - and Na + The concentration was measured using the same method as in Example 2-2. As can be seen in Table 1 and Figure 5 below, Daepung and KA-1285 showed LSS, SPAD values, and Cl - and Na + A significant difference was observed in the concentration of. F2:3 In the population of the lineage, all four traits showed a continuous distribution, and transgressive segregation was also observed (Fig. 5). F 2:3 The system's LSS range is 1.2 to 4.3 (average 2.9), the SPAD value range is 17.9 to 36.2 (average 28.5), Cl - The concentration range is 13.1 to 87.9 mg / g (average 47.3 mg / g), and Na + The concentration ranges from 0.8 to 24.8 mg / g (average 9.9 mg / g).

[0072]

[0073] Example 3-2 Gene Linkage Mapping and QTL Mapping

[0074] DNA extraction was performed on the leaves of each F2 plant using the DNeasy Plant Kit (Qiagen, Hilden, Germany). DNA concentration was measured using a Nano-MD spectrophotometer (SCINCO Co., Seoul, Korea). SNP (Single Nucleotide Polymorphism) genotyping was performed using the SoySNP6K Illumina BeadChip (Illumina, Inc., San Diego, CA, USA) at TNT Research Co. (Anyang, Korea). SNP alleles were obtained using Illumina's GenomeStudio (Illumina, Inc., San Diego, CA, USA).

[0075] Genetic association maps for the F2 population were constructed using the Kosambi mapping function of JoinMap 4.1 software (van Ooijen 2006). F 2:3QTL mapping was performed using phenotypic data and prepared gene maps. For QTL mapping, interval mapping (IM) and multi-QTL method (MQM) were performed using MapQTL 6.0 software (van Ooijen 2004). The Load Value (LOD) for each trait was determined through a 1,000-time permutation test.

[0076] Of the 6K SNP markers, 2,022 were identified as having polymorphisms between the two maternal lines and were used to construct gene linkage maps for 169 F2 lines derived from Daepung X KA-1285. Excluding segregation distortions and identical loci, 1,452 SNP markers were successfully mapped to 20 soybean chromosomes, with a size of 2,523 cM. The fewest SNP markers were found on chromosomes 16 and 17 (36 each), while the most were found on chromosome 18 (109). The average distance between two adjacent SNP markers was 1.7 cM (Table 2, Fig. 6).

[0077]

[0078] Based on a 1,000-times permutation test, LSS, SPAD, and Cl were used with threshold LODs of 4.2, 4.1, 4.0, and 4.1, respectively. - , Na + The existence of QTLs related to was verified. As a result of Interval Mapping (IM) and MQM analysis, LSS, SPAD, Cl -Only one genomic region associated with was detected (Table 3, Fig. 7A). The Gm03_40600088 marker located on chromosome 3 at 39.75 Mb was associated with LSS and SPAD; the LOD of the QTL for LSS was 17.18, explaining 38.3% of the total phenotypic variation. The QTL for SPAD had an LOD of 17.74, explaining 39.8% of the total phenotypic variation. F1 individuals possessing the chlortolerance KA-1285 allele 2:3 The line had an average LSS of 2.3 and a SPAD value of 31.1, and was a F1 line possessing the salinity-sensitive Daepung allele. 2:3 The system had an average LSS of 3.5 and a SPAD value of 25.0. Cl - The concentration-related QTL was closely associated with the Gm03_39796778 marker located on chromosome 3, 38.95 Mb, with an LOD of 12.33 and R 2 It was 29.3%. The average Cl- concentration of F2:3 lines carrying the KA-1285 allele was 35.6 mg / g, and that of F2:3 lines carrying the Daepung allele was 59.1 mg / g (Table 3). Na values ​​showing statistically significant values + No SNP markers associated with accumulation were found. Candidate genes Glyma03g171500, Glyma03g171600, Glyma03g171700 It was identified in the genomic region discovered through QTL analysis.

[0079]

[0080] Example 3-3 Confirmation of genetic variations in candidate genes through resequencing analysis

[0081] To identify genetic variations in candidate genes responsible for tolerance to salt stress in KA-1285, whole-genome sequencing of KA-1285 was performed. NGS analysis was commissioned to Seeders (Daejeon, KOR) and conducted using the Illumine Novaseq6000 platform. The library was constructed using DNA fragmentation 151bp pair-end reads with the DNA Sample Prep Kit (Illumina, Inc. San Diego, CA, USA). SNP analysis was performed on Gwang-an and PI483463. Sequence information for Gwang-an (SRX8153387[accn]) and PI483463 (SRX1149918[accn]) was obtained from NCBI SRA (https: / / www.ncbi.nlm.nih.gov / sra).

[0082] After removing PCR duplicate reads from three soybean datasets, adapter trimming and quality control were performed using the Trimmomatix-0.39 program with the SLIDINGWINDOW, LEADING, and TRAILING options under the following conditions: 1) a WINDOW size of 4 bp with an average quality score of 15 or higher, 2) LEADING and TRAILING scores of 3 or higher, and 3) a minimum read length of 36 bp or higher.

[0083] The preprocessed NGS sequence fragments were aligned to the soybean reference genome using the BWA-MEM (0.7.17-r1188) program, and a SAM format file for SNP discovery was generated. After removing non-compliant SNPs, an SNP matrix was constructed using the SAMtools (v.0.1.16) program. SNP annotation was performed based on gene location information within the soybean reference genome.

[0084] SNPs were classified as homozygous if the mapped read results were 90% or more identical at the corresponding locus, heterozygous if 40% or more but less than 60% identical, and 'other' otherwise. If no mapping was performed at a specific SNP locus, it was indicated by a lowercase 'n'. If mapping was performed but the mapped read results were mixed with various nucleotide sequences so that the nucleotide sequence at the SNP locus could not be identified, it was indicated by an uppercase 'N'.

[0085] Polymorphic SNPs and InDel loci were identified by comparing the nucleotide sequences of the same SNPs and InDel positions across various sample combinations using an integrated SNP and InDel matrix.

[0086] The total number of reads for KA-1285 was 231,945,086, and the total size was approximately 35,024 Mb. The total number of mapped reads was 220,303,485, accounting for 95.7% of the standard sequence. The average read length was 151 bp, and the sequencing depth was 31.8X. The total number of variants was 2,009,714, consisting of 1,675,675 SNPs and 334,039 InDels (Table 4).

[0087]

[0088] SNP / InDel analysis results of KA-1285, GmSALT3 The gene named [Name] Glyma03g171600 17 SNPs / InDels were discovered, Glyma03g171600 A cation / H+ exchanger identical to Glyma03g171500 class Glyma03g171700 17 and 4 SNPs / InDels were found, respectively. Among these, Glyma03g171500 Seven SNPs and two InDels were found in the exon and 3'UTR regions, and a missense mutation was identified in Gm03:38604206(G→A). In addition, Glyma03g171600Three SNPs were found in the exon and 5'UTR regions, and Glyma03g171700 Two SNPs were found in the exons (Table 5).

[0089] To identify SNPs / InDels found in the promoter and coding regions of the candidate gene, the nucleotide sequence of KA-1285 was compared with the sequences of Gwangan and PI483463, and deletion mutations (deletion of T in GT) were in KA-1285 Glyma03g171600 It was found at -25bp upstream but not in the salt-tolerant PI483463. Glyma03g171500 and Glyma03g171700 SNPs and InDels found in the exons were also identified, but they were all identical to PI483463 (Table 5).

[0090] In summary, when comparing the KA-1285 to the salt-sensitive Gwangan Glyma03g171500, Glyma03g171600, Glyma03g171700 While 8, 3, and 2 SNPs / InDels were identified in the coding / UTR regions, respectively, when compared to the inflammation-tolerant PI483463 Glyma03g171600 Only one single nucleotide deletion mutation (GT→G) was found in the -25 bp upstream region, confirming high similarity between KA-1285 and PI483463. This suggests that KA-1285 and PI483463 may possess the same salt tolerance gene.

[0091]

[0092] Example 4 Development of KASP markers for tolerance to salt stress

[0093] The KASP marker is a gene associated with tolerance to salt stress. Glyma171600It was developed to identify upstream -25bp deletion mutations. KASP genotyping analysis was based on competitive allele-specific PCR and was used to score SNPs at specific loci as bi-alleles. KASP primers were designed to detect mutant SNPs in the Glyma03g171600 gene of KA-1285 using LGC Primer Picker software (LGC Genomics) (Mutant forward / HEX: 5'-GAGAAAAACATATAAGTGCACTACTAAAAC-3', SEQ NO. 1; Wild-type forward / FAM: 5'-TGGAGAAAAACATATAAGTGCACTACTAAAAA-3', SEQ NO. 2; Common reverse: 5'-GACTCGCGAATAGATACCAGCAAATATTA-3', SEQ NO. 3). PCR reactions were performed in a total volume of 10 μL containing DNA (50 ng / 5 μL), KASP Assay Mix (0.14 μL), and KASP Master Mix. Genotyping reactions were performed using ABI StepOnePlus (Applied Biosystems, CA, USA) under the following cycle conditions: pre-reading 1 min at 30°C → holding 15 min at 94°C → [20 sec at 94°C → touchdown 60 sec at 65°C, -1°C per cycle] (10 cycles) → [20 sec at 94°C → 60 sec at 55°C] (26 cycles) → post-reading step 1 min at 30°C. End-point fluorescence data were analyzed using StepOne software V.2.1 (Applied Biosystems, CA, USA).

[0094] Among the two developed allele-specific forward primers, HEX was attached to the end of the mutation-specific forward primer, and FAM was attached to the end of the wild-type specific forward primer. As a result of genotyping using the KASP analysis technique, it was confirmed that KA-1285 could be distinguished from Gwangan and PI483463 (Fig. 8).

[0095] Example 5 qRT-PCR Analysis

[0096] qRT-PCR analysis was performed to investigate gene expression patterns in the roots of *Gwangan* and *KA-1285*. Total RNA was isolated from the roots of *Gwangan* and *KA-1285* after treatment with 200 mM salinity for 0 and 24 hours, and Ribospin TM Total RNA was extracted from 50–100 mg of roots using Plant (GeneAll, Korea). The concentration and quality of the purified RNA were measured using a Nano-MD spectrophotometer (SCINCO Co., Seoul, Korea), and verified by 1.5% agarose gel electrophoresis after staining with Dyne LoadingSTAR (DYNEBIO INC). The RNA concentration of all samples was set to 50 ng(ul). The first-strand cDNA was SuperScript TM III. It was synthesized from DNase-treated total RNA according to the instructions of First-Strand Synthesis SuperMix (Invitrogen, USA).

[0097] qRT-PCR is iQ TMThe procedure was performed using SYBR Green Supermix (Bio-RAD) on an ABI StepOnePlus system (Applied Biosystem, CA, USA). The reaction mixture (total volume 20 μl) contained 2 μl of cDNA equivalent to 100 ng, 10 μl of 2X SYBR® Green Supermix, 3 μl of gene-specific qRT-PCR primers equivalent to 300 nM, and 5 μl of distilled water. qRT-PCR was performed under the following conditions: denaturation for 3 minutes in a 96-well optical reaction plate (Applied Biosystem, USA) at 95°C, followed by 40 cycles of amplification at 95°C for 15 seconds and 60°C for 1 minute per cycle. The relative expression level of the target gene was calculated using the DD Ct method (Livak, K et al. 2001) and expressed as a change in the number of light rays. GmUKN1 was selected as the housekeeping gene (Hu et al. 2009). Each measurement was performed three times with two biological clones.

[0098] As a result, KA-1285's Glyma03g171700 The expression of was high immediately after salt treatment, but decreased sharply after 24 hours. On the other hand, KA-1285's Glyma03g171600(GMSALT3) There was no difference in the expression of before and after salt treatment, and Glyma03g171500 The expression of increased in both Gwangan and KA-1285 24 hours after salt treatment (Fig. 9). These results indicate that the component responsible for salt tolerance GmSALT3 The functional part of a gene Glyma03g171700 It can be inferred that it is related to (Figs. 7, 9).

[0099] Statistical analysis

[0100] The statistical analysis used in the above examples was performed using IBM SPSS Statistics 25 (IBM Corporation, Armonk, NY, USA), and the significance of the difference between means was tested using Tukey multiple tests following the analysis of variance (ANOVA) (P<0.05). In addition, Student's t-test was used to compare the control group and the experimental group.

[0101] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

Claim 1 A primer set for KASP (kompetitive allele specific PCR) for determining salt-tolerant soybean varieties, comprising primer pairs composed of oligonucleotides of SEQ ID NOs. 1 to 3, wherein the primer set is intended to determine KA-1285 among soybean varieties KA-1285, PI483463, and Gwangan. Claim 2 delete Claim 3 A kit for KASP for determining salt-tolerant soybean varieties, comprising the primer set of claim 1 and a reagent for performing an amplification reaction. Claim 4 A kit for KASP for determining salt-tolerant soybean varieties, wherein, in paragraph 3, the reagent for performing the amplification reaction comprises DNA polymerase, dNTPs, and a buffer. Claim 5 A method for identifying a salt-tolerant mutant soybean variety from a soybean sample, comprising: (a) isolating genomic DNA from a soybean sample; (b) performing KASP using the genomic DNA of step (a) as a template and the primer set of claim 1; and (c) analyzing the amplification product of the KASP, wherein the soybean sample of step (a) is KA-1285, PI483463, and Gwang-an, and the salt-tolerant mutant soybean variety is KA-1285. Claim 6 delete Claim 7 delete

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