Soybean salt-tolerant molecular marker DNKA281 and primer thereof

Abstract

This invention discloses a soybean salt tolerance molecular marker DNKA281 and its primers, belonging to the field of plant stress resistance identification technology. This invention aims to provide a molecular marker for identifying soybean salt tolerance during the flowering and podding stage. The marker's gene is Glyma.04G018300, and the nucleotide position at 2804 bp is C or T. This marker can be used for genotyping in soybean materials, accurately and clearly distinguishing materials containing salt-tolerant genotypes. The average plant height of the selected materials after salt treatment during the flowering and podding stage is significantly higher than that of non-salt-tolerant materials. In actual production, this marker can be used to screen soybean breeding materials with salt tolerance potential.

Description

Technical Field

[0001] This invention belongs to the field of plant stress resistance identification technology, specifically involving a soybean salt tolerance molecular marker DNKA281 and its primers. Background Technology

[0002] Soil salinization is one of the main factors restricting agricultural development in my country. Developing new salt-tolerant soybean varieties and effectively utilizing saline-alkali land are potential ways to increase soybean planting area and total yield. Current conventional salt-tolerant breeding methods include seedling growth trials and screening by physiological and biochemical index determination.

[0003] Conventional breeding methods are time-consuming and slow to yield results, while developing molecular markers for molecular-assisted breeding can effectively overcome these drawbacks. Soybean salt tolerance is often controlled by different genes at different stages. Currently, a small number of soybean salt tolerance molecular markers have been developed, but these mainly target genes or loci at the bud and seedling stages. There is an urgent need for a method that can detect salt tolerance at any tissue stage. Summary of the Invention

[0004] The purpose of this invention is to provide a molecular marker for identifying salt resistance in soybeans during the flowering and podding stage.

[0005] This invention provides a molecular marker related to salt tolerance in soybeans. The gene of the molecular marker is Glyma.04G018300, and the nucleotide site at position 2804 bp is C or T.

[0006] The present invention provides a primer combination for amplifying the above-mentioned molecular marker, wherein the primer combination consists of an upstream primer as shown in SEQ ID NO.3 or SEQ ID NO.4 and a downstream primer as shown in SEQ ID NO.5.

[0007] This invention provides a kit for detecting salt tolerance in soybeans, the kit comprising the primer combination described above.

[0008] Furthermore, the kit also includes FLu-Arms 2X PCR Mix reagent.

[0009] This invention provides the application of the above-mentioned molecular markers or primer combinations in the preparation of a kit for identifying soybean salt tolerance.

[0010] This invention provides an application of the above-mentioned molecular marker or primer combination in the breeding of salt-tolerant soybeans.

[0011] This invention provides a method for identifying the salt tolerance of soybeans. The specific steps of the method are as follows:

[0012] Step 1: Extract DNA from the soybeans to be tested;

[0013] Step 2: Use the primer combination described above to perform a PCR reaction to detect the genotype of the soybean variety to be tested.

[0014] Further specifying the PCR reaction conditions in step 2, the PCR reaction program is as follows: PCR reaction program: 95℃ pre-denaturation for 10 min; Amplification cycle 1: 95℃ denaturation for 15 s, 61℃-55℃, -0.6℃ / cycles, annealing for 60 s, for a total of 10 cycles; Amplification cycle 2: 95℃ denaturation for 15 s, 55℃ annealing for 60 s, for a total of 32 cycles.

[0015] To further specify, if the soybean variety identified in step 3 is of the CC genotype, it is a soybean variety with low salt tolerance; if the soybean variety identified is of the TT genotype, it is a soybean variety with high salt tolerance.

[0016] To further specify, the SNP site to be tested for the CC genotype is site 1435281 on chromosome 4, which is C, and the SNP site to be tested for the TT genotype is site 1435281 on chromosome 4, which is T.

[0017] The SNP site to be detected for the CC genotype is at position 1664 bp of the CDS sequence of Glyma.04G018300, and the SNP site to be detected is C. The SNP site to be detected for the TT genotype is at position 1664 bp of the CDS sequence of Glyma.04G018300, and the SNP site to be detected is T.

[0018] Beneficial Effects: This invention relates to a novel soybean salt tolerance marker, DNKA281 (marked on soybean chromosome 7, with a G or T nucleotide at a 19,078,765 bp nucleotide position) and its specific primers. This marker is a KASP marker associated with salt tolerance during the flowering and podding stage of soybean. By identifying salt tolerance-related phenotypes during the flowering and podding stage in natural soybean populations, and combining data obtained from resequencing, genome-wide association analysis (GWAS) was performed to identify significantly associated SNP sites, and candidate genes were screened from genes linked to these sites. Furthermore, extremely resistant soybeans were selected from natural populations, and based on their phenotypic values ​​of salt tolerance-related traits, association analysis was performed on candidate genes using genome resequencing data. This identified the soybean salt tolerance gene during flowering and podding (Glyma.04G018300) and its functional SNP site. The KASP marker DNKA281 was developed targeting this site. This marker can be used for genotyping in soybean materials, accurately and clearly distinguishing materials containing salt-tolerant genotypes. The average plant height of the selected materials after salt treatment during the flowering and podding stages was significantly higher than that of the non-salt-tolerant materials. In actual production, this marker can be used to screen soybean breeding materials with salt tolerance potential. Attached Figure Description

[0019] Figure 1 This is a graph showing the frequency distribution of salt tolerance traits.

[0020] Figure 2 The graph shows the GWAS analysis results of soybean salt tolerance.

[0021] Figure 3 This is a graph showing the comparison results of soybean varieties;

[0022] Figure 4 The following diagram shows the typing results of the soybean molecular marker DNKA281; a: typing results of the first group of materials; b: typing results of the second group of materials;

[0023] Figure 5 The figure shows the results of the analysis of variance for salt tolerance levels of different genotypes. Detailed Implementation

[0024] 1. R1 - Initial Flowering Stage: One flower opens at any node on the main stem. At this time, soybeans begin to transition from vegetative growth to reproductive growth, marking the initial stage of reproductive growth and playing a crucial role in subsequent yield formation. During this period, the plant requires ample nutrients and water to ensure the normal development and opening of the flowers.

[0025] 2. R2 - Peak Flowering Period: Flowers bloom at any of the two nodes at the top of the main stem with fully grown leaves. At this time, the soybean plant opens a large number of flowers, entering its peak flowering period, which is a crucial stage for soybean pollination. Favorable weather conditions (such as suitable temperature, humidity, and light wind) are conducive to pollen dispersal and pollination, and are essential for improving the seed setting rate of soybeans.

[0026] 3. R3 - Initial Pod Formation Stage: At any of the top four nodes of the main stem with fully grown leaves, a 5mm long young pod is formed. After successful pollination, the ovary begins to develop into a young pod, marking the start of the pod-setting stage for soybeans. During this period, it is crucial to ensure the plant has sufficient nutrient supply to support the growth and development of the young pods.

[0027] 4. R4 - Peak Pod Development Stage: At any of the top four nodes of the main stem with fully grown leaves, there is a pod about 2 cm long. At this stage, the length and width of the pods are rapidly increasing, making it a crucial period for pod development. The plant still has high requirements for nutrients and water, and needs sufficient sunlight and suitable temperature to promote good pod development.

[0028] 5. R5 - Initial Seed Development Stage: At any of the top four nodes of the main stem with fully grown leaves, the seed length inside the pod reaches 3mm. Seeds begin to form and gradually grow within the pod, marking the initial stage of soybean seed development. Management during this period has a significant impact on the final yield and quality, requiring continued maintenance of a favorable growing environment and nutrient supply.

[0029] Example 1. Screening for molecular markers

[0030] 1. The method for treating soybeans during the flowering and podding stages (R1-R5) with a 150 mmol / L NaCl solution is as follows:

[0031] Ten plump, disease-free soybean seeds were sown in flowerpots, and the soil was thoroughly watered until it swelled. When the seedlings reached the two-leaf stage, thinning was performed, leaving three uniformly growing, healthy plants. Subsequent watering was adjusted according to the soybean plant growth to maintain suitable soil moisture. Due to the varying salt tolerance of soybeans at different stages, salt stress treatment was initiated from the R1 stage. Each flowerpot was watered with 1L of a 150mmol / L NaCl solution every 7 days, continuing until the R5 stage. During the salt stress period, the salt concentration in the control and treatment groups was measured using a soil EC analyzer at 8:00 AM daily, with the probe inserted 6-9 cm into the soil. The average EC value for the control group was approximately 0.60 ms / cm, and the average EC value for the treatment group was approximately 0.87 ms / cm.

[0032] 2. Salt tolerance evaluation:

[0033] At R5 stage, the height from the cotyledon scar to the top of the main stem of each plant was measured using a measuring tape. Three plants were measured per pot, and the average value was taken as one replicate, with three replicates. The salt tolerance of different germplasms was evaluated by relative plant height, which was calculated as treatment / control.

[0034] Results: Population salt tolerance assessment:

[0035] A natural population of soybean germplasm (170 accessions) was subjected to salt stress during the flowering and podding stage. Plant height was investigated in the treatment and control groups at stage R5, and relative plant height was calculated. Salt tolerance of the soybean germplasm was evaluated by relative plant height; a higher relative plant height value indicated stronger salt tolerance. Figure 1 As shown, under salt stress, the relative plant height of the population mainly ranged from 0.1 to 0.6, with an average relative plant height of 0.33. Furthermore, the relative plant height distribution exhibited a continuous unimodal pattern, consistent with quantitative trait characteristics.

[0036] 3. Selection of salt-tolerant functional sites:

[0037] Resequencing was performed on a natural population of soybean germplasm resources (hereinafter referred to as the population). The resulting whole-genome SNP data were used for GWAS analysis. GWAS analysis was performed using a mixed linear model (MLM) in TASSEL 5.0. The obtained SNPs (-log) were analyzed based on gene annotations from SoyBase (http: / / www.soybase.org). 10 Candidate genes within a 150kb range upstream and downstream of the physical location (where the P value is at its peak) are predicted.

[0038] Ten samples each of extreme resistance and susceptibility were selected from the population. Based on the phenotypic values ​​of soybean salt tolerance-related phenotypic traits, and combined with the polymorphic SNPs on candidate genes obtained by resequencing, candidate gene association analysis was performed using the MLM model in TASSEL 5.0 software. Superior allelic variants on candidate genes that were significantly associated with salt tolerance traits (P value greater than 0.05, i.e., considered significantly associated) and suitable for developing KASP markers were screened.

[0039] 4. Excellent allelic variant sequencing:

[0040] Using the Wm82.a2.v1 reference genome from the Phytozome (https: / / phytozome-next.jgi.doe.gov) database, the target genome sequence was downloaded. Using salt-tolerant SNP sites as target sites, a primer containing a sequence of the target sites was designed using Premier 5.0 software (Table 1).

[0041] Table 1 Primer Information

[0042]

[0043] A portion of the material in the population is randomly selected as the test sample. DNA is extracted from the test sample, and the concentration and quality of the DNA sample are tested. The reference standard is that the value range of OD 260 / OD 280 is 1.6-2.2, which is considered a qualified sample.

[0044] PCR amplification was performed using designed primers and DNA from the sample to be tested. The PCR amplification products were then subjected to first-generation sequencing to determine the authenticity of superior allelic variants.

[0045] The PCR program was as follows: pre-denaturation at 94℃ for 6 min; 38 cycles (denaturation at 94℃ for 30 s, annealing at 47℃ for 30 s, extension at 72℃ for 30 s); extension at 72℃ for 5 min; and storage at 4℃. The reaction system is shown in Table 2.

[0046] Table 2 Reaction system for target fragment amplification

[0047]

[0048] Results: First, genome-wide association analysis was performed based on the resequencing results of the population used (containing 1,355,930 SNPs) combined with salt tolerance phenotypic traits (relative plant height) during the flowering and podding stages. Figure 2 ), and explore SNPs (-log) associated with salt-tolerant plant height during the flowering and podding stage of soybean. 10 (P>6), and the peak SNP located on chromosome 4, namely rs1420272, was finally selected.

[0049] Subsequently, a 150kb range upstream and downstream of the rs1420272 locus was used as the confidence interval for this locus. Within this target interval, 32 genes were identified. Based on gene annotations in SoyBase (http: / / www.soybase.org), candidate genes within the interval were predicted, and 13 genes were predicted as candidate genes (Table 3). These genes include enzyme protein genes, genes affecting chloroplast synthesis, and plant hormone-related genes.

[0050] Table 3 Candidate Gene Prediction

[0051] Finally, candidate gene association analysis was performed on the predicted candidate genes based on the resequencing data. Glyma.04G018300 Five significantly associated SNPs (P<0.05) were found (Table 4), among which Chr.04:1435281 was highly significantly associated with salt tolerance during the flowering and podding stage of soybean. P The value is <0.01 and located on an exon; therefore, this position was chosen for further molecular marker development. Simultaneously, upstream and downstream primers were designed for this site, and PCR amplification of this site was successfully performed using these primers. The PCR product length was 598 bp, and verification was performed using first-generation sequencing, confirming the existence of this site. Figure 3 ).

[0052] Table 4. SNPs significantly associated with salt tolerance among candidate genes.

[0053]

[0054] 5. KASP tag development:

[0055] Download via Phytozome database Glyma.04G018300 The sequence was designed using Primer Premier 5.0 software to design primers for the KASP marker (DNK281), with the significant SNP site Chr.04:1435281 as the target site. The primers included two specific primers and one universal primer. The two specific primers had two allelic variations of the site at their terminal bases, and different fluorescent adapters were added to the 5′ end of the primers (Table 5).

[0056] Table 5 KASP-labeled primer sequences

[0057]

[0058] The effectiveness of the KASP markers was determined by genotyping the population using KASP markers and analyzing the salt tolerance of different types of germplasm.

[0059] Result: Based on Glyma.04G018300 A KASP marker was developed at the Chr.04:1435281 site and named DNKA281. Figure 4 As shown, this marker was used to genotype soybean materials (100 accessions, divided into two groups) used in a partial genome-wide association study. The marker clearly identified the C / T allele variation at the Chr.04:1435281 locus, accurately and clearly clustering experimental materials with the same genotype. Blue dots near the Y-axis represent homozygous CC genotypes, red dots near the X-axis represent homozygous TT genotypes, and green dots near the lower left corner represent heterozygous C / T genotypes. Among all experimental materials, 83% carried the CC genotype, and 16% carried the TT genotype. One-way ANOVA revealed that materials carrying the TT genotype had significantly higher resistance levels than those carrying the CC genotype. Figure 5 ).

[0060] Example 2. A reagent kit for identifying salt tolerance in soybeans.

[0061] 1. A primer combination: the upstream primer is shown in SEQ ID NO.3 or SEQ ID NO.4, and the downstream primer is shown in SEQ ID NO.5.

[0062] 2. Screening method: Step 1: Extract DNA from the soybeans to be tested (DNA from tissues at any stage is acceptable);

[0063] 3. Step 2: Use the primer combination from Step 1 to perform a PCR reaction to detect the genotype of the soybean variety to be tested;

[0064] 4. The PCR reaction program was as follows: pre-denaturation at 95℃ for 10 min; amplification cycle 1: denaturation at 95℃ for 15 s, annealing at 61℃-55℃ (-0.6℃ / cycles) for 60 s, for a total of 10 cycles; amplification cycle 2: denaturation at 95℃ for 15 s, annealing at 55℃ for 60 s, for a total of 32 cycles.

[0065] 5. Results: If the soybean variety is identified as having the CC genotype, it is a soybean variety with low salt tolerance; if the soybean variety is identified as having the TT genotype, it is a soybean variety with high salt tolerance.

[0066] For soybean samples with unknown salt tolerance, those labeled with the CC genotype using primers are classified as low-salt-tolerant soybean varieties, while those with the TT genotype are classified as highly salt-tolerant. Salt tolerance is then evaluated on the identified varieties, and the results are consistent with the genotype results.

Claims

1. The application of a KASP primer combination for amplifying molecular markers related to soybean salt tolerance in the preparation of a kit for identifying soybean salt tolerance during the flowering and podding stages, characterized in that, The primer combination consists of an upstream primer and a downstream primer. The nucleotide sequences of the upstream primer are shown in SEQ ID NO.3 and SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

5. If the soybean variety is identified as having the CC genotype, it is a low-salt-tolerant soybean variety. If the soybean variety is identified as having the TT genotype, it is a high-salt-tolerant soybean variety.

2. The application according to claim 1, characterized in that, The kit also includes FLU-Arms 2× PCR Mix reagent.

3. The application of the primer combination according to claim 1 in screening salt-tolerant soybeans during the flowering and podding stage.

4. A method for identifying salt tolerance of soybeans during the flowering and podding stage, characterized in that, The specific steps of the method are as follows: Step 1: Extract DNA from the soybeans to be tested; Step 2: Perform a PCR reaction using the primer combination described in claim 1 to detect the genotype of the soybean variety to be tested; if the soybean variety is identified as having the CC genotype, it is a low salt-tolerant soybean variety; if the soybean variety is identified as having the TT genotype, it is a high salt-tolerant soybean variety.

5. The method according to claim 4, characterized in that, The PCR reaction program in step 2 is as follows: pre-denaturation at 95℃ for 10 min; amplification cycle 1: denaturation at 95℃ for 15 s, 61℃-55℃, -0.6℃ / cycles, annealing for 60 s, for a total of 10 cycles; amplification cycle 2: denaturation at 95℃ for 15 s, annealing at 55℃ for 60 s, for a total of 32 cycles.

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