Detection of a salt tolerance related kasp molecular marker of rice, primer combination and application

Abstract

This invention discloses a KASP molecular marker and method for detecting salt tolerance in rice. The molecular marker is designed based on a G→A mutation occurring at a locus of 28,165,515 bp on chromosome 8. This locus is located in the promoter region of the salt tolerance gene BAHD and encodes an acyltransferase. This locus can serve as a molecular marker for the development of rice salt tolerance genes. Three primers are used for the molecular marker: two forward-specific primers and one reverse universal primer. Genotyping using the KASP marker provided by this invention helps to quickly determine the genotype and haplotype of this allele in rice segregating populations. This invention provides an efficient method for marker-assisted breeding of the rice salt tolerance gene BAHD, reducing time and labor costs, and offering significant application value for marker-assisted breeding of salt-tolerant germplasm.

Description

Technical Field

[0001] This invention belongs to the field of detection technology for rice salt tolerance genes, specifically involving a SNP site, molecular marker, primer combination and application related to rice salt tolerance traits. Background Technology

[0002] Soil salinization restricts crop growth and development, leading to reduced grain yields. This affects approximately 99.13 million hectares of land in my country. 2 Saline-alkali land, mainly distributed in 17 provinces and regions including Northwest, Northeast, North China, and coastal areas, can be divided into slightly saline-alkali land, moderately saline-alkali land, and severely saline-alkali land (Cheng Shih-hua, 2021). Saline-alkali land is a reserve agricultural land resource with huge potential for comprehensive utilization in my country. How to utilize these saline-alkali lands to grow crops and achieve increased grain production and income is a huge challenge we face today.

[0003] my country has been working on the screening and breeding of salt-tolerant rice varieties for over 70 years. This primarily involves traditional breeding methods such as screening and identifying salt-tolerant germplasm, and artificial hybridization or backcrossing to introduce salt-tolerant genes into superior rice varieties, followed by screening and identification through multiple generations of salt stress. Since 2017, a nationwide surge in the research and development of salt-tolerant (alkaline) rice has emerged. Several domestic consortia (such as the East China Coastal Indica Rice Group, the South China Coastal Indica Rice Group, the Yellow River Japonica Rice Group, the Northern Japonica Rice Group, and the Jiangsu Province Salt-Tolerant Japonica Rice Consortium) have been established to organize relevant trials. Several new salt-tolerant rice varieties, including Salt Rice 18, Salt Rice 21, and Zhongke Salt 4, have been approved by the national or provincial governments. These varieties all exhibit good salt tolerance, strong disease resistance, high and stable yields, and excellent rice quality. They are being widely promoted and applied on saline-alkali land with a salt content of 0.3%–0.5% along my country's coast, achieving significant socio-economic benefits (Sun Mingfa, 2022).

[0004] Compared to traditional breeding methods, my country currently lacks sufficient emphasis on collaborative innovation in the breeding of salt-tolerant rice varieties. In recent years, molecular marker-assisted breeding technology has developed rapidly, playing an increasingly important role in crop genetic improvement processes such as crop breeding and new variety selection, crop genotyping and identification. Research and breeding units in my country's eastern coastal provinces, represented by the Jiangsu Academy of Agricultural Sciences and the Jiangsu Coastal Agricultural Research Institute, have utilized the unique geographical conditions of their coastal locations and adopted a combination of conventional and molecular breeding methods to successively breed salt-tolerant rice varieties such as Salt-tolerant Rice 10, Salt-tolerant Rice 12, Nanjing 9108, and Yanfeng 47, which have been widely applied and have generated significant socio-economic benefits (Sun Mingfa, 2022).

[0005] Currently, there is limited research on natural variations of superior salt-tolerant alleles in rice. More molecular markers need to be developed to efficiently and accurately screen and discover superior allele variations in order to quickly screen and breed new salt-tolerant rice varieties. Summary of the Invention

[0006] The purpose of this invention is to provide a KASP molecular marker, primer combination, and application for SNP sites related to salt tolerance in rice.

[0007] To achieve the above objectives, this invention provides a method for detecting the KASP molecular marker in rice Chr8_28165515 and applying it to marker-assisted breeding and genotyping to accelerate molecular breeding progress and improve breeding efficiency. The technical solution adopted by this invention is as follows:

[0008] A SNP locus related to salt tolerance in rice, Chr8_28165515, is located at 28165515 bp on rice chromosome 8, and the bases at the locus are A or G.

[0009] A mutant of the BAHD promoter region of the rice salt tolerance gene, wherein the mutant is a mutation of G to A at 28165515bp on chromosome 8.

[0010] Products that detect the aforementioned SNP site Chr8_28165515 or the aforementioned mutant, wherein the products are selected from any one of (1) to (5):

[0011] (1) Detecting molecular markers at the relevant SNP sites;

[0012] (2) Primer combinations for detecting the molecular markers described in (1);

[0013] (3) Reagents, instruments, and software containing the primer combinations described in (2);

[0014] (4) Molecular marker probes for detecting the relevant SNP sites;

[0015] (5) A chip containing the probe described in (4).

[0016] Furthermore, (1) the molecular marker is a KASP marker.

[0017] Furthermore, (2) the primer combination includes:

[0018] Shared downstream primer (SEQ ID NO.3): 5'-gaggtggcgcatttagtgatg-3';

[0019] Upstream typing primer 1 (SEQ ID NO.4): 5'-GAAGGTGACCAAGTTCATGCTgcaattgcaagcaacgcag-3';

[0020] Upstream typing primer 2 (SEQ ID NO.5): 5'-GAAGGTCGGAGTCAACGGATTgcaattgcaagcaacgcaa-3'.

[0021] in:

[0022] The upstream typing primer 1 (SEQ ID NO.4) can be replaced with the upstream primer shown in SEQ ID NO.1: 5'-gcaattgcaagcaacgcag-3';

[0023] The upstream typing primer 2 (SEQ ID NO.5) can be replaced with the downstream primer shown in SEQ ID NO.2: 5'-gcaattgcaagcaacgcaa-3';

[0024] Furthermore, (4) the molecular marker probe is the 5' end of the upstream typing primer 1 shown in SEQ ID NO.4 modified with the specific fluorescent label FAM, and the 5' end of the upstream typing primer 2 shown in SEQ ID NO.5 modified with the specific fluorescent label HEX.

[0025] The aforementioned SNP site Chr8_28165515, or the aforementioned mutant, or the application of the aforementioned product, wherein the application includes at least one of (1) to (3):

[0026] (1) To identify or assist in the identification of salt tolerance traits in rice;

[0027] (2) Screening of rice germplasm resources and molecular marker-assisted breeding;

[0028] (3) Identify the mutants described in claim 2 for rapid typing;

[0029] When the genotype of the SNP locus is A:A, the rice exhibits a salt-tolerant phenotype; when the genotype of the molecular marker is G:G, the rice exhibits a salt-sensitive phenotype.

[0030] Furthermore, the application includes the following steps:

[0031] (1) Extract genomic DNA from the rice population to be tested;

[0032] (2) PCR detection of the SNP genotype at the locus;

[0033] Optionally, it also includes (3) classifying plants according to their genotypes and selecting plants with salt-tolerant genotypes as parents for hybridization breeding.

[0034] Furthermore, the PCR detection system in step (2) is as follows: 1 μL of DNA to be tested, 1 μL of KASP Primer mix (SEQ ID NO.4 0.1 μL, SEQ ID NO.5 0.1 μL, SEQ ID NO.3 0.267 μL, dd H2O 0.533 μL, the concentration of each primer is 0.1 nmol / μL), and 1 μL of KASP Master mix (0.5 μL of KASP Master mix stock solution, 0.5 μL of ddH2O);

[0035] The PCR detection procedure was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s; 61-55℃ annealing and extension for 60 s, 10 cycles, with the temperature decreasing by 0.6℃ per cycle; 94℃ denaturation for 20 s; 55℃ annealing and extension for 60 s, 40 cycles.

[0036] Furthermore, the genotyping method in step (3) is as follows: After the PCR amplification program is completed, genotyping is performed using Pherastar, the sample plate is placed in Stacker for detection, and the data is imported into Kraken software for data analysis and genotyping visualization. According to the visualization results, the genotype of the samples aggregated near the X-axis and showing blue is the G:G allele with FAM fluorescent tag sequence, the genotype of the samples aggregated near the Y-axis and showing red is the A:A allele with HEX fluorescent tag sequence, and the genotype of the samples showing green in the middle is the heterozygous type of the two alleles.

[0037] The beneficial effects of this invention are as follows:

[0038] This invention develops a method for detecting the Chr8_28165515 locus in rice and performing genotyping. This method can not only efficiently and accurately identify the salt tolerance of rice, but also use this marker for high-throughput molecular marker-assisted selection, genotyping, and visualization of genotyping results, effectively improving the efficiency of rice breeding. It has important practical significance and application value in molecular marker-assisted selection breeding for salt-tolerant rice varieties. Attached Figure Description

[0039] Figure 1 This is a genotyping diagram of 208 alleles in the F1 hybrid population using the KASP molecular marker Chr8_28165515.

[0040] Figure 2 This is a schematic diagram showing the results of haplotype analysis of different haplotypes in the BAHD promoter region of the rice salt tolerance gene. Figure 2A represents the haplotype analysis results of the gene promoter region. The part circled in red is the SNP at the Chr8_28165515 site. Figure 2 B is a box plot showing the fresh weight analysis of salt-tolerant seedlings based on this haplotype. Figure 2 C is a box plot of salt-tolerant seedling dry weight analysis based on this gene haplotype.

[0041] Figure 3 for Figure 2 Salt tolerance identification of seedlings of the haplotype described in the paper.

[0042] Figure 4 for Figure 3 The traits measured at the seedling stage after the identification of salt tolerance of the two haplotypes described in the article, among which, Figure 4 A represents the survival rate. Figure 4 B represents the water content. Figure 4 C represents chlorophyll content. Figure 4 D represents the fresh weight of the leaf. Detailed Implementation

[0043] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0044] Example:

[0045] This embodiment provides an SNP site, molecular marker, primer combination, and application of Chr8_28165515 related to the detection of salt tolerance traits in rice.

[0046] This embodiment provides a method for detecting the SNP site of Chr8_28165515, which is associated with salt tolerance in rice. The method includes: using the genomic DNA of the plant sample to be tested as a template, performing real-time PCR amplification on the template using a combination of KASP molecular marker primers linked to the site, and performing genotyping based on the amplification results.

[0047] 1. Test materials

[0048] Using the salt-tolerant rice germplasm CSSL140 (9311 background) as the donor parent, hybridization combinations were set up with Ningjing 7, Ningxiangjing 9, Wujing 2038, etc., to obtain the F1 hybrid population, which constituted the genetic mapping population.

[0049] 2. DNA extraction from rice leaves

[0050] Rice leaves are collected during the tillering stage. 100 mg of leaves are added to liquid nitrogen and ground thoroughly. The DNA concentration after grinding should be 10-40 ng / μL.

[0051] 3. PCR amplification of the target sequence

[0052] (1) The extracted DNA samples were aliquoted into LGC-1536 plates using the KRAKEN and LGC high-throughput SNP sample aliquoting system, 1 μL per well. The samples were then centrifuged at 3,000 rpm for 2 min and dried in an oven at 55 °C.

[0053] (2) The samples were dispensed into primers and KASP Master mix using an LGC micro-liquid dispenser, including 1 μL of KASP Primermix (0.1 μL of SEQ ID NO.4, 0.1 μL of SEQ ID NO.5, 0.267 μL of SEQ ID NO.3, and 0.533 μL of dd H2O, with each primer concentration being 0.1 nmol / μL) and 1 μL of KASP Master mix (0.5 μL of KASP Master mix stock solution and 0.5 μL of dd H2O). The reaction system was then used for PCR.

[0054] (3) The PCR amplification program is as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s; 61-55℃ annealing extension for 60 s, 10 cycles (each cycle decreasing by 0.6℃); 94℃ denaturation for 20 s; 55℃ annealing extension for 60 s, 40 cycles.

[0055] 4. Genotyping of the KASP molecular marker Chr8_28165515

[0056] After the reaction was complete, Pherastar was used for genotyping. The sample plates were placed in Stacker for detection, and the data were imported into Kraken for analysis. The genotypes of the samples that are blue near the X-axis (1 genotype) are linked to FAM fluorescent tag sequences. The genotypes of the samples that are red near the Y-axis (106 genotypes in total) are linked to HEX fluorescent tag sequences. The genotypes of the samples that are green in the middle (101 genotypes in total) are heterozygous for two alleles.

[0057] The 106 dominant SNP sites A:A selected from this population can be used as parental materials for the breeding of salt-tolerant japonica rice varieties (Table 1 below) and can be used for the breeding improvement of target varieties.

[0058] Table 1. Genotyping results of the Chr8_28165515 SNP locus in 208 rice samples from the F1 generation of the salt-tolerant rice germplasm CSSL140 (9311 background) as the donor parent, hybridized with Ningjing 7, Ningxiangjing 9, and Wujing 2038.

[0059]

[0060]

[0061] Genotyping of alleles in 208 individuals from the F1 hybrid population using the KASP molecular marker Chr8_28165515. (See attached image.) Figure 1 As shown: Each dot in the figure represents a sample. The genotypes of the F1 generation samples (1 in total) that are blue near the X-axis are linked to the FAM fluorescent tag sequence. The genotypes of the F1 generation samples (106 in total) that are red near the Y-axis are linked to the HEX fluorescent tag sequence. The genotypes of the samples (101 in total) that are green in the middle are heterozygous for two alleles.

[0062] Schematic diagram of different haplotype analysis results of the BAHD promoter region of the rice salt tolerance gene. Figure 2 As shown, where, Figure 2 The area circled in red in section A represents the Chr8_28165515 SNP site, where a G / A mutation is present. When the genotype is A:G, the salt tolerance level falls between that of A:A and G:G. Figure 2 B and Figure 2 C shows the salt tolerance identification results of the three haplotypes. HapT has a higher fresh weight and dry weight than HapM, while HapS has the lowest fresh weight and dry weight values, indicating that HapT has the strongest salt tolerance, followed by HapM, and HapS has the weakest salt tolerance.

[0063] Salt tolerance was assessed for haplotype HapS and its complementary family HapT, with HapS corresponding to the genotype G:G and HapT corresponding to the genotype A:A.

[0064] (1) The sample was: the BAHD promoter region SNP of the rice variety 'Dwarf Nantes' was used as an appendix. Figure 2 The haplotype HapS mentioned above, 'Dwarf Nantes-BAHD-T', is obtained by complementary haplotype HapT to 'Dwarf Nantes'.

[0065] (2) The experimental procedure was as follows: 90-100 seeds of the rice variety 'Aijiao Nante' and its complementary variety with relatively intact seed coats and no discoloration were randomly selected. The sterilized rice seeds were then placed in petri dishes with sterilized filter paper, completely moistened with ddH2O, and covered. The dishes were soaked in the dark at room temperature for 2 days, then transferred to a 30℃ incubator for 1 day of germination in the dark. After most seeds had germinated and shown white sprouts, the best-germinating and uniformly growing rice grains were carefully sown with tweezers into 12*8 (96-well) small black boxes and placed in an artificial climate chamber (30℃, 14 hours, light / 28℃, 8 hours, darkness). After 7 days of incubation, the nutrient solution was treated with 150mM NaCl or 15mM Na2CO3, with conventional culture medium used as a control. Photographs were taken 7 days after salt or alkali stress treatment to determine the phenotype and measure relevant physiological and biochemical indicators.

[0066] (3) Experimental results showed that after 7 days of treatment with NaCl and Na2CO3, the salt tolerance of 'Nant' 'Dwarf Nantes-BAHD-T' with genotype A:A was significantly higher than that of 'Nant' 'Dwarf Nantes' with genotype G:G. Figure 3 Post-seedling stage measurements of survival rate, water content, chlorophyll content, and leaf fresh weight showed that the A:A genotype 'Dwarf Nantes-BAHD-T' was significantly superior to the G:G genotype 'Dwarf Nantes'. Figure 4 It can be seen that when the genotype of the SNP is A:A, a salt-tolerant phenotype is exhibited; when the genotype of the molecular marker is G:G, a salt-sensitive phenotype is exhibited.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a KASP molecular marker primer combination in identifying salt tolerance traits in rice, characterized in that, When the genotype of the amplification product is A:A, the rice exhibits a salt-tolerant phenotype; when the genotype of the amplification product is G:G, the rice exhibits a salt-sensitive phenotype. The primer combination is as follows: The common downstream primer shown in SEQ ID NO.3 is: 5'-gaggtggcgcatttagtgatg-3'; The upstream typing primer 1 shown in SEQ ID NO.4 is 5'- GAAGGTGACCAAGTTCATGCTgcaattgcaagcaacgcag-3'; Upstream typing primer 2 shown in SEQ ID NO.5: 5'- GAAGGTCGGAGTCAACGGATTgcaattgcaagcaacgcaa -3'; The 5' end of upstream typing primer 1 shown in SEQ ID NO.4 is modified with the specific fluorescent label FAM, and the 5' end of upstream typing primer 2 shown in SEQ ID NO.5 is modified with the specific fluorescent label HEX.

2. The application according to claim 1, characterized in that, The application includes the following steps: (1) Extract genomic DNA from the rice population to be tested; (2) PCR detection of the genotype of the amplified product.

3. The application according to claim 2, characterized in that, It also includes (3) classifying plants according to their genotypes and selecting salt-tolerant genotype plants as parents for hybridization breeding.

4. The application according to claim 2, characterized in that, The PCR detection system in step (2) is as follows: 1 μL of DNA sample to be tested; 1 μL of KASP Primer mix, wherein the KASP Primer mix consists of 0.1 μL of upstream genotyping primer 1 shown in SEQ ID NO.4, 0.1 μL of upstream genotyping primer 2 shown in SEQ ID NO.5, 0.267 μL of the common downstream primer shown in SEQ ID NO.3, and 0.533 μL of dd H2O, with each primer having a concentration of 0.1 nmol / μL; 1 μL of KASP Master mix, wherein the KASP Master mix consists of 0.5 μL of KASP Master mix stock solution and 0.5 μL of dd H2O; The PCR detection procedure was as follows: 94 ℃ pre-denaturation for 15 min; 94 ℃ denaturation for 20 s; 61-55 ℃ annealing extension for 60 s, 10 cycles, with a decrease of 0.6 ℃ per cycle; 94 ℃ denaturation for 20 s; 55 ℃ annealing extension for 60 s, 40 cycles.

5. The application according to claim 2, characterized in that, The genotyping method in step (3) is as follows: After the PCR amplification program is completed, genotyping is performed using Pherastar. The sample plate is placed in Stacker for detection. The data is imported into Kraken software for data analysis and genotyping visualization. According to the visualization results, the genotype of the samples aggregated near the X-axis and showing blue is the G:G allele with FAM fluorescent tag sequence. The genotype of the samples aggregated near the Y-axis and showing red is the A:A allele with HEX fluorescent tag sequence. The genotype of the samples showing green in the middle is the heterozygous type of the two alleles.

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