Molecular marker related to soybean salt-tolerant gene GmGA2ox16 and application of molecular marker

Abstract

The invention discloses a soybean salt-tolerant gene GmGA2ox16 related molecular marker and application thereof, and relates to the technical field of plant molecular breeding and biology, and the molecular marker is characterized in that a single nucleotide in a soybean GmGA2ox16 gene coding region has a polymorphic SNP (Single Nucleotide Polymorphism) site. The molecular marker is used for screening salt-tolerant soybeans. By utilizing the SNP marker provided by the invention, individuals carrying excellent haplotype GmGA2ox16-Hap1 can be quickly screened out in the soybean seedling stage through a genetic typing technology, so that the breeding process of salt-tolerant soybean varieties is accelerated, the efficiency and accuracy of salt-tolerant breeding are remarkably improved, and valuable gene resources are provided for molecular breeding of the salt-tolerant soybean varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding and biotechnology, specifically relating to a molecular marker related to soybean salt tolerance, the protein it encodes, and their application in molecular marker-assisted selection breeding. Background Technology

[0002] Soil salinization, as one of the major abiotic stresses facing global agricultural production, has a wide and far-reaching impact. Soybeans, as a crop with both food and economic value, are particularly sensitive to salt stress. When subjected to salt damage, the growth and development of soybeans are significantly hindered, and their yield and quality decline sharply, severely limiting the suitable planting areas for soybeans. Therefore, deeply exploring the inherent salt-tolerant genetic potential of soybeans and cultivating new varieties with salt-tolerant characteristics through scientific methods is undoubtedly a key strategy for ensuring the stability of soybean production and guaranteeing food security.

[0003] Traditional breeding methods primarily rely on phenotypic selection in the field to improve crop salt tolerance. However, this method has many drawbacks: salt stress phenotypes are easily affected by variable environmental factors, leading to lengthy identification cycles, low efficiency, and difficulty in guaranteeing the accuracy of results. Therefore, developing molecular markers closely linked to salt tolerance, enabling precise genotypic selection at the seedling stage, is of crucial significance for accelerating the breeding process of salt-tolerant soybeans.

[0004] Plant hormones play an indispensable role in the complex regulatory network of plant responses to salt stress. Gibberellins, as important growth-promoting hormones, are closely related to the plant's stress response mechanisms through their metabolic homeostasis. Extensive research has shown that GA2-dioxygenases are key enzymes in the gibberellin metabolic pathway. Specifically, the GA2ox subfamily acts as a "brake" in regulating the balance between plant growth and stress tolerance by specifically degrading biologically active gibberellin molecules. In some plant species, specific GA2ox genes have been shown to be directly involved in the response to and regulation of salt stress.

[0005] Although functional studies of the GA2ox gene family have shown some potential in crop salt tolerance, GA2ox gene resources with significant salt tolerance effects that can be directly applied to breeding practices remain relatively scarce in soybean. In particular, current research has not provided a systematic and effective solution regarding the natural variations of this gene family in soybean (e.g., single nucleotide polymorphisms, SNPs) and the precise association between these variations and salt tolerance phenotypes. Furthermore, how to fully utilize these natural variations to develop efficient molecular markers and successfully apply them to molecular breeding practices is also one of the key challenges that urgently needs to be overcome. Summary of the Invention

[0006] In order to solve the above problems, this invention provides a molecular marker related to the soybean salt tolerance gene GmGA2ox16 and its application.

[0007] The present invention discloses a molecular marker related to the soybean salt tolerance gene GmGA2ox16, wherein the nucleotide sequence of the molecular marker, as shown in SEQ ID NO:1, has A>G base mutation and G>C base mutation at positions 91 and 544.

[0008] This invention relates to the application of a molecular marker related to the soybean salt tolerance gene GmGA2ox16, wherein the molecular marker related to the soybean salt tolerance gene GmGA2ox16 is used for soybean-assisted breeding, and the assisted breeding is for screening salt-tolerant soybeans.

[0009] Furthermore, the aforementioned screening of salt-tolerant soybeans,

[0010] If the genotype is AG, then it is GmGA2ox16. -Hap1 The haplotype is the genotype for soybean salt tolerance;

[0011] If the genotype is GC, then it is GmGA2ox16. -Hap2 The haplotype is the genotype of soybean that is intolerant to salt.

[0012] Soybeans containing salt-tolerant genes were selected for breeding.

[0013] Furthermore, the salt-tolerant soybean gene is GmGA2ox16. -Hap1 Haploid soybeans.

[0014] Furthermore, primer pairs for detecting GmGA2ox16-related molecular markers:

[0015] Primer GmGA2ox16-F1:

[0016] 5'-GAGCTCGGTACCCGGGGATCCATGGTGTTGTTGTCCAAAG-3';

[0017] Primer GmGA2ox16-R1:

[0018] 5'-CACCATGGTGTCGACTCTAGACGAAGCTGCAATTCTTTCA -3'.

[0019] Furthermore, the method for screening salt-tolerant soybeans is as follows:

[0020] (1) Extract genomic DNA from the soybean plants to be tested; (2) PCR amplification was performed using primers GmGA2ox16-F1 and GmGA2ox16-R1; (3) Screening for genotype GmGA2ox16 -Hap1 Soybean plants were used as parents for breeding.

[0021] Primer GmGA2ox16-F1:

[0022] 5'-GAGCTCGGTACCCGGGGATCCATGGTGTTGTTGTCCAAAG-3';

[0023] Primer GmGA2ox16-R1:

[0024] 5'-CACCATGGTGTCGACTCTAGACGAAGCTGCAATTCTTTCA -3'.

[0025] This invention discloses a primer pair for identifying molecular markers associated with the soybean salt tolerance gene GmGA2ox16, wherein the primer pair is as follows:

[0026] Primer GmGA2ox16-F1:

[0027] 5'-GAGCTCGGTACCCGGGGATCCATGGTGTTGTTGTCCAAAG-3';

[0028] Primer GmGA2ox16-R1:

[0029] 5'-CACCATGGTGTCGACTCTAGACGAAGCTGCAATTCTTTCA -3'.

[0030] A kit containing the primer pairs described in this invention.

[0031] This invention was validated by RT-qPCR, yeast expression system, and soybean hairy root overexpression system, demonstrating that the GmGA2ox16 gene can significantly improve salt tolerance. Haplotype analysis was performed on wild soybean, local varieties, and improved varieties using 2,898 resequencing germplasm accessions. A key SNP site was found in the coding region of the GmGA2ox16 gene. This polymorphism led to amino acid substitutions (N31D and A182P) within key functional domains (DIOX_N and 2OG-FeII_Oxy), with amino acid sequences shown in SEQ ID NO. 3 and 4. This resulted in the formation of GmGA2ox16. -Hap1 (Salt-tolerant) and GmGA2ox16 -Hap2 Two haplotypes, GmGA2ox16 -Hap1 Associated with enhanced salt tolerance in soybeans, it was significantly selected during domestication. Using the SNP markers provided in this invention, genotyping technology can be used to rapidly screen for individuals carrying the superior haplotype GmGA2ox16 at the soybean seedling stage. -Hap1 This allows for the identification of individual individuals, thereby accelerating the breeding process of salt-tolerant soybean varieties, significantly improving the efficiency and accuracy of salt-tolerant breeding, and providing valuable genetic resources for the molecular breeding of salt-tolerant soybean varieties.

[0032] The present invention has the following beneficial effects:

[0033] 1. This invention provides an applicable molecular tool that solves a key technical problem in breeding: For the first time, this invention discovered and verified a critical SNP site located in the coding region of the GmGA2ox16 gene. Polymorphism at this site directly leads to amino acid substitutions within the protein's functional domain, and defines two haplotypes (GmGA2ox16 and GmGA2ox16) that exhibit significant differences in salt tolerance. -Hap1 With GmGA2ox16 -Hap2 Based on this SNP, a molecular marker was developed, providing the first tool for soybean salt-tolerant breeding that is closely related to the major gene GmGA2ox16 and can be directly used for genotyping. This overcomes the bottlenecks of traditional field phenotypic identification, which is characterized by long cycles, high costs, and susceptibility to environmental interference, and enables rapid and accurate screening of a large number of germplasms during the seedling stage.

[0034] 2. Clear breeding direction and significant application results: Through genotype-phenotype association analysis of a large-scale population (2,898 accessions), this invention clearly confirms that GmGA2ox16... -Hap1Haplotypes are superior alleles directly associated with enhanced salt tolerance, and they undergo significant selection during domestication and improvement, making them extremely valuable for breeding. Using this invention, breeders can directly target this genetic locus with a clearly defined function, upgrading traditional experience-based breeding, which relies on "phenotype," to precision breeding, which relies on "genotype," significantly improving selection efficiency and accuracy and shortening the breeding cycle.

[0035] 3. The technical solution has been verified by multiple systems and is scientifically reliable: The biological importance of the SNP marker and the salt tolerance function of its haplotypes described in this invention have been fully verified through multi-level and mutually corroborating functional experiments, such as RT-qPCR expression analysis, yeast heterologous expression system and soybean hairy root genetic transformation system. This ensures the reliability and causality of the association between the marker and the salt tolerance trait, and provides a solid scientific basis for its practical application in molecular breeding. Attached Figure Description

[0036] Figure 1 Figure showing the expression pattern analysis of the GmGA2ox16 gene under salt stress;

[0037] Figure 2 Growth of recombinant pYES2-GmGA2ox16 yeast strain under different NaCl concentrations;

[0038] Figure 3 Salt tolerance analysis of soybean hairy roots overexpressing the GmGA2ox16 gene;

[0039] Figure 4 A diagram showing the genetic variation, haplotype distribution, and selection analysis of the GmGA2ox16 gene;

[0040] Figure 5 The two haplotypes of GmGA2ox16 showed differences in wilting index under salt stress. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0042] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0043] The effectiveness of the present invention was verified through the following experiments:

[0044] (I) Analysis of the expression pattern of GmGA2ox16 gene under salt stress

[0045] The expression pattern of the GmGA2ox16 gene under salt stress was analyzed using the soybean variety Williams 82. Plants were treated with 0.9% NaCl to induce the stress condition. Seedlings were grown in pots in a greenhouse with a photoperiod of 16 hours light / 8 hours dark, a day / night temperature of 28 / 20°C, and a relative humidity of 60%. Seedling roots were sampled at 0, 1, 2, and 4 hours during the treatment period to extract total RNA.

[0046] 1. For RNA extraction methods, refer to the Invitrogen TRIzol kit manual. Use the All-in-One First-Strand cDNA Synthesis SuperMix kit to reverse transcribe RNA into cDNA.

[0047] 2. Take 1 µl of cDNA diluted 30-fold in step 1 as a qPCR template. Use qGmGA2ox16-F1 (Seq ID No: 5) and qGmGA2ox16-R1 (Seq ID No: 6) as primers, and the primer sequence for the soybean internal reference gene TUA5 is qTUA5 (Seq ID No: 7 and Seq ID No: 8). Follow the TransStart Top Green qPCR Super Mix Kit instructions, using SYBR Green I as dye, and employ a three-step PCR reaction. qPCR reaction conditions: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ annealing for 15 s, 72℃ extension for 20 s, 40 cycles, using 2... –ΔΔCT The algorithm calculates the relative expression level of the GmGA2ox16 gene, and the results are as follows: Figure 1 As shown, the GmGA2ox16 gene responds to salt stress, and its expression level is continuously upregulated with the extension of treatment time.

[0048] (II) Salt tolerance analysis of the GmGA2ox16 gene in heterologous yeast systems

[0049] The coding sequence of GmGA2ox16 was amplified by PCR using primers GmGA2ox16-F1 (Seq ID No: 9) and GmGA2ox16-R1 (Seq ID No: 10).

[0050] The PCR amplification system is as follows: 10 μL system

[0051]

[0052] The PCR reaction conditions are as follows:

[0053]

[0054] The PCR product was then inserted into the pYES2 vector. The resulting construct was subsequently introduced into the yeast strain INVSCI. Stress tolerance in yeast was tested according to Gautam's method. Transformants were cultured on SD / -Ura plates at 28°C for 5 days, single colonies were picked and cultured in SD / -Ura liquid medium for 24 hours, followed by galactose induction for 36 hours. For salt stress testing, each diluted culture was added to SD / -Ura medium supplemented with 0, 0.4, 0.8, and 1.5 M NaCl. Transgenic yeast carrying the empty pYES2 vector was used as a control. Results are as follows: Figure 2 As shown, on plates without NaCl, the growth of strains transformed with the GmGA2ox16 gene was almost identical to that of the control strain. However, on plates containing 0.4 M or 0.8 M NaCl, expression of the GmGA2ox16 gene significantly enhanced the salt tolerance of yeast cells. Nevertheless, under 1.5 M NaCl conditions, the growth of transformants was completely inhibited, indicating that this concentration exceeded the protective capacity of the expressed gene. These results demonstrate that the GmGA2ox16 gene can confer salt tolerance in yeast systems.

[0055] (III) Salt tolerance analysis of soybean hairy root system overexpressing GmGA2ox16 gene

[0056] As described in section (II), the coding sequence of GmGA2ox16 was amplified. BamHI was introduced upstream of the GmGA2ox16 gene, and the restriction enzyme site of XbaI was introduced downstream. The amplified PCR product and the vector pCAMBIA1300-GFP were simultaneously digested with BamHI and XbaI, and then ligated overnight with T4 DNA ligase to construct the pCAMBIA1300-GmGA2ox16-GFP vector, which was then transformed into Agrobacterium rhizogenes strain K599. To produce transformed soybean hairy roots, Agrobacterium rhizogenes-mediated transformation was performed using the soybean variety Williams 82. Seeds germinated in a humidified chamber with a 16-hour light / 8-hour dark photoperiod and a temperature of 28°C. One week later, healthy plants were injected with Agrobacterium rhizogenes strain K599 containing the pCAMBIA1300-GmGA2ox16-GFP construct. Infected plants were then transferred to a humidity chamber and kept at high humidity until hairy roots grew from the infected site to a length of 2-5 cm. The expression of the bar gene at the translational level was verified using LibertyLink test strips. Before salt stress treatment, the original roots were removed, and plants carrying either GmGA2ox16-overexpressing hairy roots or hairy roots transformed with an empty vector were treated with 200 mM NaCl, and observed at 0, 6, and 24 hours. Results showed that before salt stress treatment (0 hours), no phenotypic differences were observed between control plants and plants with GmGA2ox16-overexpressing hairy roots. Figure 3 A). Six hours after treatment, control plants began to show initial symptoms of wilting, while plants with hairy roots overexpressing GmGA2ox16 remained upright and did not show obvious stress phenotypes. Figure 3 B). After 24 hours of salt stress, control plants showed severe wilting and extensive leaf dehydration; in contrast, plants with hairy roots overexpressing GmGA2ox16 showed only mild wilting. Figure 3 C). Survival rates (the proportion of surviving plants to the total number of tested plants) were statistically analyzed. The results showed that the survival rate of plants carrying GmGA2ox16 overexpressing hairy roots was significantly higher than that of the empty vector control group after salt stress treatment. Figure 3 D).

[0057] To investigate the physiological mechanisms behind the aforementioned phenotypic differences, peroxidase activity assay kits (Boxbio, AKAO005C), superoxide dismutase activity assay kits (Boxbio, AKAO001C-50S), and malondialdehyde (MDA) content assay kits (Boxbio, AKFA013C) were used, and the levels of endogenous substances were determined spectrophotometrically. The activities of antioxidant enzymes, including superoxide dismutase (SOD) and peroxidase (POD), and the content of the oxidative damage marker malondialdehyde (MDA) in hairy roots were quantitatively analyzed. Consistent with phenotypic observations, at 6 hours after treatment, the activities of SOD and POD in hairy roots overexpressing GmGA2ox16 were significantly increased compared to the empty vector control, while the MDA content was significantly decreased. Figure 3 (EG). As salt stress persisted for up to 24 hours, these differences became more pronounced: roots overexpressing GmGA2ox16 maintained significantly higher antioxidant enzyme activity and lower MDA accumulation, consistent with the observed mild wilting phenotype. These results demonstrate that GmGA2ox16 enhances salt tolerance by strengthening the root's antioxidant defense system and mitigating oxidative damage under salt stress.

[0058] (iv) Haplotype analysis of GmGA2ox16 in different soybean populations

[0059] To investigate whether GmGA2ox16 underwent selection during domestication, this invention performed haplotype analysis on 2,898 materials covering wild soybean, local varieties, and improved varieties based on published genomic data (National Center for Biotechnology Information, https: / / ngdc.cncb.ac.cn / soyomics / genome_variation). This invention identified two non-synonymous single nucleotide polymorphisms in the first and second exons of GmGA2ox16, inducing amino acid substitutions at N31D and A182P, respectively, and accordingly classified the haplotype as GmGA2ox16. -Hap1 With GmGA2ox16 -Hap2 ( Figure 4 A, B). Allele frequency analysis showed that GmGA2ox16 -Hap1 Dominant in domesticated populations, present in 77.17% of local varieties and 78.10% of improved cultivars ( Figure 4 C). Further comparison using three-dimensional structural modeling revealed that both amino acid substitutions are located within the conserved DIOX_N and 2OG-FeII_Oxy domains, suggesting potential functional differences between the two haplotypes. Figure 4 D).

[0060] To search for selection signals at the genomic level, this invention analyzed the nucleotide diversity of a 400 kb region surrounding the GmGA2ox16 locus. Interpopulation fixation index analysis showed high genetic differentiation (Fm) between wild soybean and local varieties, as well as between wild soybean and improved varieties. st (0.36 and 0.45 respectively) Figure 4 E). Meanwhile, wild soybeans exhibited significantly higher nucleotide diversity in this region compared to domesticated populations, a result consistent with the expectation of "selective clearance" occurring during domestication. Figure 4 F). The above evidence collectively indicates that the GmGA2ox16 site underwent intense artificial selection.

[0061] To further investigate the biological effects of the two haplotypes, this invention evaluated 102 soybean materials (81 of which carried GmGA2ox16) according to the soybean salt tolerance rating criteria in Table 1. -Hap1 21 samples carrying GmGA2ox16 -Hap2 Phenotypic differences under salt stress. After stress treatment, those carrying GmGA2ox16... -Hap1 The proportion of materials rated as salt resistance grade 1 was significantly higher than that of GmGA2ox16. -Hap2 Materials to carry ( Figure 5 A, B). Quantitative analysis of the wilting index also consistently showed that GmGA2ox16 -Hap1 Carriers consistently exhibited stronger salt tolerance. Figure 5 C). These results confirm that GmGA2ox16 -Hap1 It is a dominant haplotype associated with enhanced salt tolerance in soybeans.

[0062] Table 1. Standards for evaluating the salt tolerance level of soybeans

[0063]

Claims

1. A molecular marker related to the soybean salt tolerance gene GmGA2ox16, characterized in that, The molecular marker's nucleotide sequence, as shown in SEQ ID NO:1, has A>G base mutations and G>C base mutations at positions 91 and 544.

2. The application of a molecular marker related to the soybean salt tolerance gene GmGA2ox16 as described in claim 1, characterized in that, The aforementioned soybean salt tolerance gene GmGA2ox16-related molecular markers are used for soybean-assisted breeding, which involves screening for salt-tolerant soybeans.

3. The application according to claim 2, characterized in that, The aforementioned screening of salt-tolerant soybeans, If the genotype is AG, then it is GmGA2ox16. -Hap1 The haplotype is the genotype for soybean salt tolerance; If the genotype is GC, then it is GmGA2ox16. -Hap2 The haplotype is the genotype of soybean that is intolerant to salt. Soybeans containing salt-tolerant genes were selected for breeding.

4. The application according to claim 3, characterized in that, The salt-tolerant soybean gene mentioned is GmGA2ox16. -Hap1 Haploid soybeans.

5. The application according to claim 2, 3 or 4, characterized in that, Primer pairs for detecting GmGA2ox16-related molecular markers: Primer GmGA2ox16-F1: 5'-GAGCTCGGTACCCGGGGATCCATGGTGTTGTTGTCCAAAG-3'; Primer GmGA2ox16-R1: 5'-CACCATGGTGTCGACTCTAGACGAAGCTGCAATTCTTTCA -3'.

6. The application according to claim 2, 3 or 4, characterized in that, The method for screening salt-tolerant soybeans is as follows: (1) Extract genomic DNA from soybean plants to be tested; (2) Perform PCR amplification using primers GmGA2ox16-F1 and GmGA2ox16-R1; (3) Screen for genotypes of GmGA2ox16. -Hap1 Soybean plants were used as parents for breeding. Primer GmGA2ox16-F1: 5'-GAGCTCGGTACCCGGGGATCCATGGTGTTGTTGTCCAAAG-3'; Primer GmGA2ox16-R1: 5'-CACCATGGTGTCGACTCTAGACGAAGCTGCAATTCTTTCA -3'.

7. A primer pair for identifying molecular markers associated with the soybean salt tolerance gene GmGA2ox16, wherein the primer pair is: Primer GmGA2ox16-F1: 5'-GAGCTCGGTACCCGGGGATCCATGGTGTTGTTGTCCAAAG-3'; Primer GmGA2ox16-R1: 5'-CACCATGGTGTCGACTCTAGACGAAGCTGCAATTCTTTCA -3'.

8. A kit comprising the primer pair of claim 7.

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