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

Abstract

The invention discloses a KASP molecular marker and a method for detecting the salt tolerance character of rice, the molecular marker is designed according to G-to-A mutation occurring at the site 28bp, 165 bp and 515bp of the No.8 chromosome, the site is located in a salt-tolerant gene BAHD promoter region, an acyltransferase is encoded, and the molecular marker can be used for detecting the salt tolerance character of rice. The site can be used as a molecular marker for developing a rice salt tolerance gene. The molecular marker comprises three primers, including two forward specific primers and one reverse universal primer, genetic typing is carried out according to the KASP marker provided by the invention, rapid genotype and haplotype typing of the allele in a rice segregation population is helped, and the genotype and haplotype typing of the allele in the rice segregation population is improved. An efficient method is provided for molecular marker-assisted breeding of the rice salt-tolerant gene BAHD, time cost and labor cost are reduced, and good application value is provided for molecular marker-assisted breeding of salt-tolerant germplasm.

Description

Technical Field

[0001] The invention belongs to the technical field of detection of rice salt-tolerance genes, and specifically relates to a SNP site, a molecular marker, a primer combination and an application related to the salt-tolerance trait of rice. Background Art

[0002] Soil salinization will limit the growth and development of crops, leading to a reduction in grain production. The total area in my country is about 99.13 million hm2. 2 Saline-alkali land is mainly distributed in 17 provinces and regions including Northwest, Northeast, North China and coastal areas. It can be divided into light saline-alkali land, moderate saline-alkali land and heavy saline-alkali land (Cheng Shihua, 2021). Saline-alkali land is a reserve agricultural arable land resource with great potential for comprehensive utilization in my country. How to use these saline-alkali lands to grow crops and increase grain production and income in saline-alkali land is a huge challenge facing us today.

[0003] my country has been working on the screening and breeding of salt-tolerant rice varieties for more than 70 years. Salt-tolerant genes are introduced into excellent rice varieties mainly through the screening and identification of salt-tolerant germplasm and traditional breeding methods such as artificial hybridization or back (repeated) cross, and then screened and identified through salt stress for many years and generations. Since 2017, there has been a wave of research and development of salt-tolerant (alkali) rice across the country. A number of domestic consortia have been established (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 Salt-tolerant Japonica Rice Consortium) to organize relevant experiments. Several new salt-tolerant rice varieties such as Salt Rice No. 18, Salt Rice No. 21, and Zhongke Salt No. 4 have been approved by the state or provincial level. They all have 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% to 0.5% along the coast of my country, and have achieved good social and economic benefits (Sun Mingfa, 2022).

[0004] Compared with traditional breeding methods, my country currently pays insufficient attention to joint innovation in the research on the selection and breeding of salt-tolerant rice varieties. In recent years, molecular marker-assisted breeding technology has developed rapidly, playing an increasingly important role in crop breeding and new variety selection, crop genotyping and identification and other crop genetic improvement processes. Scientific research and breeding units in the eastern coastal provinces of my country, represented by Jiangsu Academy of Agricultural Sciences and Jiangsu Coastal Agricultural Science Research Institute, have taken advantage of the unique geographical location in the coastal areas and adopted a combination of conventional and molecular breeding methods to successively breed new salt-tolerant rice varieties such as Salt Rice No. 10, Salt Rice No. 12, Nanjing 9108, and Yanfeng 47, which have been promoted and applied and have produced good social and economic benefits (Sun Mingfa, 2022).

[0005] At present, there is little exploration of natural variations in excellent alleles for rice salt tolerance. There is still a need to develop more molecular markers for efficient and accurate screening and exploration of excellent allele variations, so as to quickly screen and cultivate new salt-tolerant rice varieties. Summary of the invention

[0006] The purpose of the present invention is to provide a KASP molecular marker of a SNP site related to the salt tolerance trait of rice, a primer combination and an application thereof.

[0007] To achieve the above object, the present invention provides a KASP molecular marker for detecting rice Chr8_28165515 and applying it to molecular marker-assisted breeding and genotyping to accelerate the progress of molecular breeding and improve breeding efficiency. The present invention adopts the following technical solutions:

[0008] A SNP site Chr8_28165515 related to the salt tolerance trait of rice, wherein the SNP site is located at 28165515bp of rice chromosome 8, and the base of the site is A or G.

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

[0010] A product for detecting the aforementioned SNP site Chr8_28165515 or the aforementioned mutant, wherein the product is selected from any one of (1) to (5):

[0011] (1) detecting molecular markers of the relevant SNP sites;

[0012] (2) a primer combination for detecting the molecular marker described in (1);

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

[0014] (4) a molecular marker probe for detecting the relevant SNP site;

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

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

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

[0018] Common 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 by 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 by the downstream primer shown in SEQ ID NO.2: 5'-gcaattgcaagcaacgcaa-3';

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

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

[0026] (1) Identify or assist in identifying the salt tolerance traits of rice;

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

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

[0029] When the genotype of the SNP site 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 comprises the following steps:

[0031] (1) extracting rice genomic DNA from the test population;

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

[0033] Optionally, the method further includes (3) typing according to genotypes and selecting plants with determined salt-tolerant genotypes as parents for hybrid breeding.

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

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

[0036] Furthermore, the method of genotyping in step (3) is as follows: after the PCR amplification program is completed, Pherastar is used for genotyping, the sample plate is placed in a stacker for detection, and the data is imported into the software kraken for data analysis and typing visualization, and classified according to the visualization results. The genotype of the samples displayed in blue and clustered close to the X-axis is the G:G allele type connected to the FAM fluorescent label sequence, the genotype of the samples displayed in red and clustered close to the Y-axis is the A:A allele type connected to the HEX fluorescent label sequence, and the genotype of the sample displayed in green in the middle is the heterozygous type of the two alleles.

[0037] The beneficial effects of the present invention are:

[0038] The present invention develops a method for detecting the Chr8_28165515 locus of rice and performing genotyping, which can not only efficiently and accurately identify the salt tolerance of rice, but also use the marker to perform high-throughput molecular marker-assisted selection, perform genotyping, and visualize the typing results, thereby effectively improving the efficiency of rice breeding. The method has important practical significance and application value in molecular marker-assisted selection breeding for the cultivation of salt-tolerant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0040] Figure 2 It is a schematic diagram of the results of different haplotype analysis of the promoter region of the rice salt tolerance gene BAHD, wherein: Figure 2A is the result of haplotype analysis of the promoter region of the gene. The red circled part is the Chr8_28165515 site SNP. Figure 2 B is a box plot of fresh weight analysis of salt tolerance seedlings based on the haplotype of the gene. Figure 2 C is a box plot of dry weight analysis of salt-tolerant seedlings based on the haplotype of this gene.

[0041] Figure 3 for Figure 2 Identification of salt tolerance at the seedling stage of the haplotype described in .

[0042] Figure 4 for Figure 3 The traits measured at the seedling stage after the salt tolerance of the two haplotypes described in the above are identified, among which, Figure 4 A is the survival rate, Figure 4 B is the water content, Figure 4 C is the chlorophyll content, Figure 4 D is the fresh weight of leaves. DETAILED DESCRIPTION

[0043] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0044] Example:

[0045] This embodiment provides a SNP site, molecular marker, primer combination and application of Chr8_28165515 related to detecting the salt tolerance trait of rice.

[0046] This embodiment provides a method for detecting the Chr8_28165515 SNP site related to the salt tolerance trait of rice, comprising: using the genomic DNA of the plant sample to be tested as a template, using the KASP molecular marker primer combination linked to the site to perform fluorescent quantitative PCR amplification on the template, and using the amplification result to perform genotyping.

[0047] 1. Test materials

[0048] The salt-tolerant rice germplasm CSSL140 (9311 background) was used as the donor parent and hybridized with Ningjing 7, Ningxiangjing 9, Wujing 2038, etc. to obtain the hybrid F1 population, which constituted the genetic mapping population.

[0049] 2. DNA Extraction from Rice Leaves

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

[0051] 3. PCR amplification of target sequence

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

[0053] (2) The sample was dispensed into primers and KASP Master mix using an LGC microdispenser, including KASP Primermix 1 μL (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, where the concentration of each primer was 0.1 nmol / μL), KASP Master mix 1 μL (KASP Master mix mother solution 0.5 μL, dd H2O 0.5 μL). The reaction system was prepared for PCR reaction.

[0054] (3) The specific PCR amplification procedure was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s; annealing and extension at 61-55°C for 60 s, 10 cycles (0.6°C reduction per cycle); denaturation at 94°C for 20 s; annealing and extension at 55°C for 60 s, 40 cycles.

[0055] 4. Genotyping of KASP molecular marker Chr8_28165515

[0056] After the reaction is completed, Pherastar is used for genotyping, the sample plate is placed in stacker for detection, and the data is imported into Kraken for analysis. The genotype of the sample displayed in blue close to the X-axis (1) is connected to the FAM fluorescent label sequence, the genotype of the sample displayed in red close to the Y-axis (a total of 106) is connected to the HEX fluorescent label sequence, and the genotype of the sample displayed in green in the middle (a total of 101) is the heterozygous type of the two alleles.

[0057] The 106 dominant SNP loci A:A plants screened out in 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 breeding improvement of target varieties.

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

[0059]

[0060]

[0061] The genotyping diagram of 208 individual alleles in the hybrid F1 population using the KASP molecular marker Chr8_28165515. Figure 1 As shown: Each dot in the figure represents a sample. The genotype of the F1 generation sample shown in blue close to the X-axis (1) is connected to the FAM fluorescent label sequence, the genotype of the F1 generation sample shown in red close to the Y-axis (106 in total) is connected to the HEX fluorescent label sequence, and the genotype of the sample shown in green in the middle (101 in total) is the heterozygous type of the two alleles.

[0062] Schematic diagram of the results of different haplotype analysis of the promoter region of the rice salt tolerance gene BAHD Figure 2 As shown, Figure 2 The red circled part in A is the Chr8_28165515 site SNP, and it can be seen that there is a G / A mutation at this site. When the genotype is A:G, the salt tolerance is between A:A and G:G. Figure 2 B and Figure 2 C shows the salt tolerance identification results of the three haplotypes. The fresh weight and dry weight of HapT are higher than those of HapM, and the fresh weight and dry weight values ​​of HapS are the lowest, indicating that HapT has the strongest salt tolerance, followed by HapM, and HapS has the weakest salt tolerance.

[0063] The salt tolerance of haplotype HapS and its complementary lineage HapT were identified respectively. The genotype corresponding to HapS is G:G, and the genotype corresponding to HapT is A:A:

[0064] (1) Samples: The BAHD promoter region SNP of the rice variety 'Aijiao Nante' was used as the Figure 2 The haplotype HapS described in the 'BAHD-T' is obtained by complementation of 'BAHD' with the haplotype HapT.

[0065] (2) The experimental steps are as follows: 90-100 seeds of the rice variety 'Dwarf Nante' and its complementary variety with relatively intact seed coat and no color spots were randomly selected, and then the sterilized rice seeds were placed in a culture dish with sterilized filter paper, completely soaked with ddH2O, and covered with a culture dish lid. The seeds were soaked in the dark at room temperature for 2 days, and transferred to a 30℃ incubator for germination in the dark for 1 day. After most of the germinated seeds turned white, the rice grains with good germination degree and uniform growth were carefully sown in a 12*8 (96-well) small black box with tweezers, and placed in an artificial climate chamber (30℃, 14 hours, light / 28℃, 8 hours, dark). After incubation for 7 days, the nutrient solution was treated with 150mM NaCl or 15mM Na2CO3, and the conventional culture solution was used as a control. Photos were taken 7 days after salt or alkali stress treatment to determine the phenotype and measure relevant physiological and biochemical indicators.

[0066] (3) The experimental results showed that after 7 days of NaCl and Na2CO3 treatment, the salt tolerance of the genotype A:A 'Dwarf Nante-BAHD-T' was significantly higher than that of the genotype G:G 'Dwarf Nante' ( Figure 3 The survival rate, water content, chlorophyll content, and leaf fresh weight measured at the seedling stage showed that the genotype of A:A'Dwarf Nante-BAHD-T' was significantly better than the genotype of G:G'Dwarf Nante' ( Figure 4 ). It can be seen that when the genotype of the SNP is A:A, it exhibits a salt-tolerant phenotype; when the genotype of the molecular marker is G:G, it exhibits a salt-sensitive phenotype.

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A SNP site Chr8_28165515 related to rice salt tolerance, characterized in that: The SNP site is located at 28165515bp of rice chromosome 8, and the base of the site is A or G.

2. A rice salt tolerance gene BAHD promoter region mutant, characterized in that: The mutant is a mutation from G to A at 28165515bp of chromosome 8.

3. A product for detecting the SNP site Chr8_28165515 of claim 1 or the mutant of claim 2, characterized in that: The product is selected from any one of (1) to (5): (1) detecting molecular markers of the relevant SNP sites; (2) a primer combination for detecting the molecular marker described in (1); (3) Reagents, instruments, and software comprising the primer combination described in (2); (4) a molecular marker probe for detecting the relevant SNP site; (5) A chip comprising the probe described in (4).

4. The product according to claim 3, characterized in that (1) The molecular marker is a KASP marker.

5. The product according to claim 3, characterized in that (2) The primer combination comprises: Common downstream primer (SEQ ID NO.3): 5'-gaggtggcgcatttagtgatg-3'; Upstream typing primer 1 (SEQ ID NO. 4): 5′-GAAGGTGACCAAGTTCATGCT gcaattgcaagcaacgcag-3′; Upstream typing primer 2 (SEQ ID NO. 5): 5'-GAAGGTCGGAGTCAACGGATT gcaattgcaagcaacgcaa-3'.

6. The product according to claim 3 or 5, characterized in that (4) The molecular marker probe is the upstream typing primer 1 shown in SEQ ID NO.4, whose 5' end is modified with a specific fluorescent marker FAM, and the upstream typing primer 2 shown in SEQ ID NO.5, whose 5' end is modified with a specific fluorescent marker HEX.

7. Use of the SNP site Chr8_28165515 of claim 1, or the mutant of claim 2, or the product of claim 3, characterized in that: The application includes at least one of (1) to (3): (1) Identify or assist in identifying the salt tolerance traits of rice; (2) Screening of rice germplasm resources and molecular marker-assisted breeding; (3) identifying the mutants described in claim 2 for rapid typing; When the genotype of the SNP site in claim 1 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.

8. The use according to claim 7, characterized in that: The application comprises the following steps: (1) extracting rice genomic DNA from the test population; (2) PCR detection of the SNP genotype of the site; Optionally, the method further includes (3) typing according to genotypes and selecting plants with determined salt-tolerant genotypes as parents for hybrid breeding.

9. The use according to claim 8, characterized in that: The PCR detection system of step (2) is: 1 μL of the sample DNA to be tested, 1 μL of KASP Primer mix (0.1 μL of SEQ ID NO.4, 0.1 μL of SEQ ID NO.5, 0.267 μL of SEQ ID NO.3, 0.533 μL of dd H2O, where the concentration of each primer is 0.1 nmol / μL), 1 μL of KASP Master mix (0.5 μL of KASP Master mix stock solution, 0.5 μL of dd H2O); The PCR detection program was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s; annealing and extension at 61-55°C for 60 s, 10 cycles, with the temperature decreasing by 0.6°C each cycle; denaturation at 94°C for 20 s; annealing and extension at 55°C for 60 s, 40 cycles.

10. The use according to claim 8, characterized in that: The method of genotyping in step (3) is as follows: after the PCR amplification program is completed, Pherastar is used for genotyping, the sample plate is placed in a stacker for detection, and the data is imported into the software kraken for data analysis and typing visualization. According to the visualization results, the genotype of the samples displayed in blue near the X-axis is the G:G allele type connected to the FAM fluorescent label sequence, the genotype of the samples displayed in red near the Y-axis is the A:A allele type connected to the HEX fluorescent label sequence, and the genotype of the sample displayed in green in the middle is the heterozygous type of the two alleles.

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