Soybean salt-tolerant molecular marker DNKA281 and primer thereof

Abstract

The invention discloses a soybean salt-tolerant molecular marker DNKA281 and primers thereof, and belongs to the technical field of plant stress resistance identification. The invention aims to provide a molecular marker for identifying the salt resistance of soybeans in the flower and pod period of the soybeans. The invention provides a molecular marker related to salt resistance of soybeans, the gene of the molecular marker is Glyma. 04G018300, and the nucleotide site on the 2804bp site is C or T. The invention also provides a preparation method of the molecular marker. The marker can be used for genotyping in soybean materials, materials containing salt-tolerant genotypes can be accurately and clearly distinguished, and the average value of the plant height of the screened materials after salt treatment in the flower and pod period is obviously higher than that of non-salt-tolerant materials. In actual production, the marker can be used for screening soybean breeding materials with salt-tolerant potential.

Description

Technical Field

[0001] The invention belongs to the technical field of plant stress resistance identification, and specifically relates to a soybean salt-tolerance molecular marker DNKA281 and a primer thereof. Background Art

[0002] Land salinization is one of the main factors restricting the development of agriculture in my country. Cultivating new salt-tolerant soybean varieties and effectively utilizing salinized land are potential ways to increase soybean planting area and total yield. The current conventional salt-tolerant breeding methods include seedling growth test screening and physiological and biochemical index determination screening.

[0003] Conventional breeding methods have a long cycle and slow results, but the development of molecular markers for molecular-assisted breeding can effectively overcome this shortcoming. The salt tolerance of soybeans is often controlled by different genes at different stages. Currently, a small number of soybean salt tolerance molecular markers have been developed, but they are mainly targeted at genes or loci at the bud and seedling stages. A method that can detect any period of tissue is urgently needed. Summary of the invention

[0004] The purpose of the present invention is to provide a molecular marker for identifying the salt tolerance of soybean during the flowering and pod stage of soybean.

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

[0006] The present invention provides a primer combination for amplifying the above-mentioned molecular marker, wherein the primer combination comprises 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] The invention provides a kit for detecting soybean salt resistance, and the kit comprises the above primer combination.

[0008] It is further defined that the kit also includes FLu-Arms 2X PCR Mix reagent.

[0009] The present invention provides an application of the above molecular marker or the above primer combination in preparing a kit for identifying soybean salt tolerance.

[0010] The present invention provides an application of the above molecular marker or the above primer combination in screening soybean salt-tolerant breeding.

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

[0012] Step 1: Extract the DNA of the soybean to be tested;

[0013] Step 2: Perform a PCR reaction using the primer combination described in claim 2 to detect the genotype of the soybean variety to be detected.

[0014] It is further defined that the PCR reaction conditions in step 2 are as follows: the PCR reaction program is 95°C pre-denaturation for 10 min; amplification cycle 1: 95°C denaturation for 15 s, 61°C-55°C, -0.6°C / cycles, annealing for 60 s, for a total of 10 cycles; amplification cycle 2: 95°C denaturation for 15 s, 55°C annealing for 60 s, for a total of 32 cycles.

[0015] It is further defined that if the soybean variety is identified as CC genotype in step 3, it is a soybean variety with low salt tolerance, and if the soybean variety is identified as TT genotype, it is a soybean variety with high salt tolerance.

[0016] It is further defined that the SNP site to be detected for the CC genotype is the 1435281 site on chromosome 4, which is C, and the SNP site to be detected for the TT genotype is the 1435281 site on chromosome 4, which is T.

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

[0018] Beneficial effects: The present invention relates to a new soybean salt-tolerance marker DNKA281 (the marker chromosome is chromosome 7 of soybean, and the nucleotide position at 19078765bp is G or T) and its dedicated primers. The marker is a KASP marker related to the salt tolerance of soybean during the flowering and pod stage. The phenotypes related to the salt tolerance during the flowering and pod stage of the natural soybean population are identified, and a genome-wide association analysis (GWAS) is performed in combination with the data obtained by resequencing to mine significantly associated SNP sites, and candidate genes are screened from the genes linked to the site. Furthermore, extremely resistant materials are selected from the natural population, and based on the phenotypic values ​​of their salt-tolerance-related phenotypic traits, an association analysis of candidate genes is performed in combination with genome resequencing data, and a soybean salt-tolerance gene (Glyma.04G018300) during the flowering and pod stage and a functional SNP site on the gene are mined, and a KASP marker developed for the site is DNKA281. This marker can be used for genotyping in soybean materials, accurately and clearly distinguishing materials with salt-tolerant genotypes. The average plant height of the selected materials after salt treatment at the flowering and pod 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-tolerant potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the frequency distribution result of salt tolerance traits;

[0020] Figure 2 This is the result of GWAS analysis of soybean salt tolerance;

[0021] Figure 3 This is the soybean variety comparison result diagram;

[0022] Figure 4 The typing results of soybean molecular marker DNKA281 are shown in Figure 1; a: Typing results of the first group of materials; b: Typing results of the second group of materials;

[0023] Figure 5 The results of variance analysis of the salt resistance levels of different genotypes are shown in Figure 2. DETAILED DESCRIPTION

[0024] 1. R1-initial flowering stage: a flower blooms on any node of the main stem. At this time, soybeans begin to change from vegetative growth to reproductive growth. It is the initial stage of soybean reproductive growth and is of great significance to the subsequent yield formation. During this period, the plant needs sufficient nutrients and water supply to ensure the normal development and opening of the flowers.

[0025] 2. R2-full flowering period: Flowers bloom at any of the two nodes on the uppermost part of the main stem with fully grown leaves. At this time, the flowers on the soybean plants bloom in large numbers, entering the vigorous flowering period, which is the key stage of soybean pollination. Good weather conditions (such as suitable temperature, humidity and breeze) are conducive to the spread and pollination of pollen, which is crucial to improving the fruit setting rate of soybeans.

[0026] 3. R3-Initial pod stage: any of the four nodes on the top of the main stem with fully grown leaves has a 5mm long young pod. After the flower is successfully pollinated, the ovary begins to develop into young pods, marking the beginning of the soybean pod stage. During this period, it is necessary to ensure that the plant has sufficient nutrient supply to support the growth and development of young pods.

[0027] 4. R4-full pod stage: any of the four nodes on the top of the main stem with fully grown leaves has a 2cm long pod. At this time, the length and width of the pods are growing rapidly, which is a critical period for pod development. The plant still has a high demand for nutrients and water, and also needs sufficient light and suitable temperature to promote good pod development.

[0028] 5. R5-Initial seeding stage: In the top four nodes of the main stem with fully grown leaves, the length of the seeds in the pods at any node reaches 3mm. The seeds in the pods begin to form and gradually grow, which is the initial stage of soybean seed development. Management during this period has an important impact on the final yield and quality, and it is necessary to continue to maintain a good growth environment and nutrient supply.

[0029] Example 1. Screening of molecular markers

[0030] 1. Determine to use a NaCl solution with a concentration of 150mmol / L to treat soybeans at the flowering and pod stage (R1-R5) as follows:

[0031] 10 soybean seeds with full seeds and no pests and diseases were selected and sown in pots. The soil was fully irrigated to allow the soil to absorb water. When the two leaves and one heart stage came, the seedlings were thinned out, leaving 3 plants with uniform growth and good health. In the future, appropriate irrigation was carried out according to the growth of the soybean plants to keep the pot soil at a suitable humidity. Due to the difference in salt tolerance of soybeans at different stages, salt stress treatment was carried out from the R1 stage of soybeans. Each pot was irrigated with 1L of NaCl solution with a concentration of 150mmol / L each time, and the treatment was carried out once every 7 days until the R5 stage. During the salt stress period, the salt concentration in the potted plants of the control group and the treatment group was detected using a soil EC detector. The measurement time was 8 o'clock every morning, and the probe was inserted into the soil 6-9cm. The average EC value of the control group was about 0.60ms / cm, and the average EC value of the treatment group was about 0.87ms / cm.

[0032] 2. Salt tolerance evaluation:

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

[0034] Results: Identification of population salt tolerance:

[0035] A natural population of soybean germplasm (a total of 170 germplasms) was subjected to salt stress at the flowering and pod stage. The plant heights of the treatment and control groups were investigated at the R5 stage, and the relative plant heights were calculated. The relative plant height was used to evaluate the salt tolerance of soybean germplasm. The larger the relative plant height value, the stronger the salt tolerance. Figure 1 As shown in the figure, under salt stress, the relative plant height values ​​of the population used were mainly distributed between 0.1 and 0.6, with an average relative plant height of 0.33. At the same time, the relative plant height distribution was a continuous single peak, which was consistent with the characteristics of quantitative traits.

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

[0037] The natural population composed of soybean germplasm resources (hereinafter referred to as the population) was resequenced, and the whole genome SNP data obtained by sequencing was used for GWAS analysis. The GWAS analysis was performed using the mixed linear model (MLM) in TASSEL 5.0. The obtained SNPs (-log 10 The candidate genes within 150 kb upstream and downstream of the physical location (P value is at the peak) were predicted.

[0038] Ten extremely resistant and susceptible materials were selected from the population. Based on the phenotypic values ​​of soybean salt tolerance-related phenotypic traits and combined with the polymorphic SNPs located on the candidate genes obtained by resequencing, the MLM model in TASSEL 5.0 software was used to perform candidate gene association analysis, and excellent allele variations located on the candidate genes that were significantly correlated with the salt tolerance trait (P value greater than 0.05, which was considered to be significantly correlated) and suitable for developing KASP markers were screened.

[0039] 4. Excellent allelic variant sequencing:

[0040] The Wm82.a2.v1 reference genome in the Phytozome (https: / / phytozome-next.jgi.doe.gov) database was used to download the target genome sequence, and the salt-tolerant SNP site was used as the target site. The Premier5.0 software was used to design a primer containing the sequence of the target site (Table 1).

[0041] Table 1 Primer information

[0042]

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

[0044] PCR amplification was performed using the designed primers and the DNA of the sample to be tested, and the PCR amplification products were sequenced to determine the authenticity of the superior allele variation.

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

[0046] Table 2 Reaction system for target fragment amplification

[0047]

[0048]

[0049] Results: First, a genome-wide association analysis was performed based on the population resequencing results (including 1,355,930 SNPs) combined with the salt tolerance phenotypic traits (relative plant height) during the flowering and pod stage. Figure 2 ), mining SNPs associated with salt tolerance of soybean plant height during flowering and pod stage (-log 10 P>6), and finally the peak SNP located on chromosome 4, rs1420272, was selected.

[0050] Subsequently, the 150 kb range upstream and downstream of the rs1420272 site was used as the confidence interval of the site. There were 32 genes in the target interval. According to the gene annotation in SoyBase (http: / / www.soybase.org), the candidate genes in the interval were predicted, and a total of 13 genes were predicted as candidate genes (Table 3). These genes include enzyme protein genes, genes affecting chloroplast synthesis, and plant hormone-related genes.

[0051]

[0052] Table 3 Candidate gene prediction

[0053] Finally, candidate gene association analysis was performed on the predicted candidate genes based on the resequencing data. Glyma.04G018300 had 5 significantly (P<0.05) related SNPs (Table 4), among which Chr.04:1435281 was extremely significantly correlated with soybean salt tolerance during flowering and pod stage (P<0.01) and was located on the exon, so this position was selected for the next step of molecular marker development. At the same time, upstream and downstream primers were designed for this site, and the primers were used to successfully amplify the site by PCR. The length of the PCR product was 598bp, which was verified by first-generation sequencing to confirm that the site really existed ( Figure 3 ).

[0054] Table 4 SNPs significantly associated with salt tolerance in candidate genes

[0055]

[0056]

[0057] 5.KASP Marker Development:

[0058] The Glyma.04G018300 sequence was downloaded from the Phytozome database, and the significant SNP site Chr.04:1435281 was used as the target site. The primers for the KASP marker (DNK281) were designed using Primer Premier 5.0 software, including 2 specific primers and 1 universal primer. The terminal bases of the 2 specific primers included the 2 allelic variations of the site, and different fluorescent linkers were added to the 5′ end of the primers (Table 5).

[0059] Table 5 KASP marker primer sequences

[0060]

[0061] The population was genotyped by KASP markers and different types of germplasm were analyzed for salt tolerance to determine the effectiveness of KASP markers.

[0062] Results: A KASP marker was developed based on the Chr.04:1435281 locus of Glyma.04G018300 and named DNKA281. Figure 4 As shown, the marker was used to genotype some soybean materials used in whole genome association analysis (a total of 100 samples, divided into 2 groups). The marker can clearly identify the C / T allele variation at the Chr.04:1435281 site, accurately and clearly cluster the test materials with the same genotype. The blue dots close to the Y-axis represent homozygous genotype materials with a genotype result of CC, the red dots close to the X-axis represent homozygous genotype materials with a genotype result of TT, and the green dots close to the lower left corner represent heterozygous C / T. Among all the test materials, materials carrying the CC genotype accounted for 83%, and materials carrying the TT genotype accounted for 16%. Through one-way analysis of variance, it was found that the resistance level of materials carrying the TT genotype was significantly higher than that of materials carrying the CC genotype ( Figure 5 ).

[0063] Example 2. A kit for identifying soybean salt tolerance

[0064] 1. A primer combination: the upstream primer is shown as SEQ ID NO.3 or SEQ ID NO.4, and the downstream primer is shown as SEQ ID NO.5.

[0065] 2. Screening method: Step 1: Extract the DNA of the soybean to be tested (DNA from any tissue period is acceptable);

[0066] 3. Step 2: Perform PCR reaction using the primer combination in step 1 to detect the genotype of the soybean variety to be tested;

[0067] 4. The PCR reaction program was pre-denaturation at 95°C for 10 min; amplification cycle 1: denaturation at 95°C for 15 s, annealing at 61°C-55°C (-0.6°C / cycles) for 60 s, for a total of 10 cycles; amplification cycle 2: denaturation at 95°C for 15 s, annealing at 55°C for 60 s, for a total of 32 cycles.

[0068] 5. Result: If the soybean variety is identified as CC genotype, it is a soybean variety with low salt tolerance. If the soybean variety is identified as TT genotype, it is a soybean variety with high salt tolerance.

[0069] To detect unknown soybean salt tolerance samples, the CC genotype marked by primers is a low salt tolerance soybean variety. If the identified soybean variety is a TT genotype, it is a high salt tolerance soybean variety. The identified variety is evaluated for salt tolerance, and the test results are consistent with the genotype results.

Claims

1. A molecular marker related to soybean salt tolerance, characterized in that: The gene of the molecular marker is Glyma.04G018300, and the nucleotide site at the 2804 bp position is C or T.

2. A primer combination for amplifying the molecular marker according to claim 1, characterized in that: The primer combination is 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.

3. A kit for detecting soybean salt tolerance, characterized in that: The kit comprises the primer combination according to claim 2.

4. The kit according to claim 3, characterized in that The kit also includes FLu-Arms 2X PCRMix reagent.

5. Use of the molecular marker according to claim 1 or the primer combination according to claim 2 in preparing a kit for identifying soybean salt tolerance.

6. Use of the molecular marker according to claim 1 or the primer combination according to claim 2 in screening soybean salt-tolerant breeding.

7. A method for identifying the salt tolerance of soybeans, characterized in that: The specific steps of the method described are as follows: Step 1: Extract the DNA of the soybean to be tested; Step 2: Perform a PCR reaction using the primer combination described in claim 2 to detect the genotype of the soybean variety to be detected.

8. The method according to claim 7, characterized in that The PCR reaction conditions in step 2 are as follows: the PCR reaction program is 95°C pre-denaturation for 10 min; amplification cycle 1: 95°C denaturation for 15 s, 61°C-55°C, -0.6°C / cycles, annealing for 60 s, for a total of 10 cycles; amplification cycle 2: 95°C denaturation for 15 s, 55°C annealing for 60 s, for a total of 32 cycles.

9. The method according to claim 7, characterized in that: In step 3, if the soybean variety is identified as CC genotype, it is a soybean variety with low salt tolerance. If the soybean variety is identified as TT genotype, it is a soybean variety with high salt tolerance.

10. The method according to claim 9, characterized in that The SNP site to be detected for CC genotype is 1435281 on chromosome 4, which is C. The SNP site to be detected for TT genotype is 1435281 on chromosome 4, which is T.

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