A SNP molecular marker related to cadmium absorption characteristics of rice, primer set, kit and application thereof

Abstract

The application discloses a SNP molecular marker related to cadmium absorption characteristics of rice, a primer group, a kit and application thereof, and belongs to the field of agricultural biotechnology. OsNramp5 The application claims a SNP molecular marker located at the 8875111 locus of chromosome 7 of a rice gene, wherein when the locus lacks base A and the genotype is T / T, the rice exhibits low cadmium accumulation. The application further provides a primer group for specifically amplifying the marker and a detection kit containing the primer group. The application can be used for quickly and accurately identifying the genotype of the rice through KASP fluorescence PCR, realizing seedling stage screening, and greatly improving the breeding efficiency of the rice with low cadmium accumulation. The marker is specific, simple to detect, stable in result, and can be widely used for screening, identifying and molecular marker assisted breeding of the rice with low cadmium accumulation, and has important application value for solving cadmium pollution of rice.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology and relates to molecular breeding, specifically to an SNP molecular marker, primer set, reagent kit, and its application related to cadmium absorption characteristics in rice. Background Technology

[0002] Rice is one of the most important food crops in my country and even globally. More than 60% of my country's population relies on rice as their staple food, making the quality and safety of rice crucial. Rice itself has a strong capacity to accumulate heavy metals, and it is among the cereal crops with the highest capacity to accumulate cadmium. Excessive accumulation of cadmium in rice plants not only inhibits rice growth and development, reducing yield and quality, but also leads to unsold rice and serious economic losses. Cadmium is a non-essential element for the human body, but it can enter the human body through the food chain and accumulate continuously. It has significant toxicity, and the harm to the body intensifies with increasing accumulation, potentially inducing various serious diseases such as cancer. Breeding and promoting low-cadmium-accumulation rice varieties is the most economical, efficient, and feasible technical approach to solving the "cadmium rice" problem.

[0003] OsNramp5 It is a key transport protein that mediates the entry of cadmium into rice. The loss of function of this gene can directly reduce the absorption efficiency of cadmium by rice roots and significantly reduce the cadmium content of rice. It is the core genetic locus for breeding low-cadmium rice varieties at home and abroad.

[0004] Currently, there are few molecular markers available for breeding new varieties with low cadmium accumulation, which greatly limits the breeding process. Summary of the Invention

[0005] The first objective of this invention is to provide a SNP molecular marker related to cadmium absorption characteristics in rice; the second objective is to provide a primer set for amplifying the aforementioned SNP molecular marker; the third objective is to provide a kit containing the aforementioned primer set; and the fourth objective is to provide the application of the aforementioned SNP molecular marker, primer set, or kit in screening / breeding / detecting cadmium-low accumulating rice.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: A SNP molecular marker associated with cadmium absorption characteristics in rice, the SNP molecular marker being located in rice OsNramp5 The gene at chromosome 7, position 8875111 (located at position 1032 of the nucleotide sequence shown in SEQ ID NO. 1), exhibits a polymorphism with a deletion of base A. When the genotype is T / T, rice exhibits the trait of low cadmium accumulation.

[0007] A primer set for amplifying the above-mentioned SNP molecular markers.

[0008] In one specific embodiment, the primer set includes two specific primers, 820C7-8875111-wF2 and 820C7-8875111-mH2, and one universal primer, 820C7-8875111-C2; wherein the nucleotide sequence of primer 820C7-8875111-wF2 is shown in SEQ ID NO.2, the nucleotide sequence of primer 820C7-8875111-mH2 is shown in SEQ ID NO.3, and the nucleotide sequence of primer 820C7-8875111-C2 is shown in SEQ ID NO.4.

[0009] A kit comprising the above-described primer set.

[0010] Furthermore, the kit also contains one or more of the following: 2×Flu-ASPCR Mix for KASP, dNTP, MgCl2, PCR buffer, and deionized water.

[0011] The following are applications of any one of (1) to (3) in screening / breeding / detecting low cadmium accumulation rice: (1) The above-mentioned SNP molecular markers; (2) The primer set mentioned above; (3) The above-mentioned kit.

[0012] Furthermore, the genomic DNA of rice is detected using any one of the methods described in (1) to (3), based on... OsNramp5 Genotyping / breeding / detection of cadmium-low accumulating rice at locus 8875111 on chromosome 7; rice with the T / T genotype at this locus is cadmium-low accumulating rice.

[0013] Furthermore, the above application includes the following steps: Step 1: Obtain the genomic DNA of the rice to be tested; Step 2: Perform PCR amplification on the genomic DNA using the primer set or the kit described above to obtain the amplification product; Step 3: Obtain from the amplification products OsNramp5 The genotype at locus 8875111 on chromosome 7; Step 4, according to OsNramp5 The genotype at locus 8875111 on chromosome 7 is used to determine whether the rice being tested is a low-cadmium-accumulating rice variety.

[0014] In one specific embodiment, the PCR amplification program is as follows: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 20 s, annealing and extension at 65℃~57℃ for 40 s for 10 cycles, with the annealing and extension temperature decreasing by 0.8℃ in each cycle, 94℃ denaturation for 20 s, 57℃ annealing and extension for 40 s for 28 cycles.

[0015] Furthermore, based on the amplification products, OsNramp5 Methods for genotyping the gene at locus 8875111 on chromosome 7 include KASP fluorescence scanning genotyping.

[0016] Beneficial effects: 1. High molecular marker specificity and accurate typing: The SNP molecular marker of this invention is located at the 8875111 site on chromosome 7 of the rice OsNramp5 gene. It is closely linked to the trait of low cadmium accumulation. It has no high frequency distribution in natural rice populations, has high specificity and low background interference. Using KASP technology, homozygous mutant, heterozygous and wild type can be clearly distinguished. The detection results are stable, reliable and reproducible.

[0017] 2. The detection method is rapid, simple, high-throughput, and efficient: The primer set of this invention is specially designed for KASP fluorescence typing. It does not require enzyme digestion, electrophoresis, or complex sequencing. Genotype determination can be completed directly through real-time fluorescence PCR. The detection time for a single plant is short, the throughput is high, and the operation is simple, which can meet the rapid screening needs of large-scale breeding populations.

[0018] 3. Significantly improves the breeding efficiency of low cadmium accumulation rice: This invention can complete genotyping in the early seedling stage, accurately screen T / T homozygous low cadmium accumulation single plants, greatly reduce the scale of field planting, shorten the breeding cycle, reduce breeding costs, and effectively solve the problems of traditional breeding relying on phenotypic identification, long cycle, low accuracy, and great influence from the environment.

[0019] 4. Significant effect on low cadmium accumulation and high application value: The T / T genotype at the target locus of this invention can still control the cadmium content of grains at an extremely low level in heavily cadmium-contaminated soil, with a very significant cadmium reduction effect. It can be directly used as a core donor for the improvement of backbone parents, providing a key molecular tool for the breeding of cadmium-low accumulation rice varieties.

[0020] 5. High practicality and wide applicability: The primer set and kit of this invention are highly versatile and can be applied to genotype detection, germplasm resource identification and molecular marker-assisted breeding of rice materials with different genetic backgrounds. They are easy to standardize and promote industrialization. Attached Figure Description

[0021] Figure 1 The major cadmium-absorbing gene of cyclone 820S-N5-24-1 OsNramp5 Mutation type; Figure 2 The strains are Radiation Cyclone 820S-N5-24-1, Ronten 819S, and their hybrid F1 generation. OsNramp5 Genotyping diagram; Figure 3 For hurricane 820S-N5-24-1 and natural populations OsNramp5Genotyping diagram. Detailed Implementation

[0022] The substantive content of the present invention will be described in detail below with reference to specific embodiments. However, those skilled in the art should know that the scope of protection of the present invention should not be limited to these specific embodiments.

[0023] Example 1: Obtaining a new cadmium-low accumulating rice germplasm, Radiation Mutagenesis 820S-N5-24-1, using radiation mutagenesis + targeted sequencing screening technology. 1. Construction of the Hurricane 820S radiation-induced mutation population 500g of seeds from the temperature-sensitive nuclear male-sterile line Ju 820S were selected, soaked in tap water for 24 hours, drained, and then treated with 400Gy. 60 Co-γ ray radiation mutagenesis treatment. After soaking and germination, the treated seeds were sown (M1). After 25 days of seedling age, individual seedlings were transplanted and managed with standard fertilizer and water. Twenty days after transplanting, each seedling was numbered, and one leaf was taken from each tiller of each seedling, for a total of 1216 seedlings and 7503 leaves. Equal amounts of leaves were mixed, with approximately 500 leaves mixed into one pool, which was then divided into 15 pools for DNA extraction. The DNA samples were numbered 820-1 to 820-15.

[0024] 2. OsNramp5 Targeted sequencing of genes Using the DNA from each pool as a sequencing sample, targeting OsNramp5 The gene samples were sent to Beijing Qingke Biotechnology Co., Ltd. for targeted high-depth sequencing, with a sequencing depth >100,000 nucleotides. The high-depth targeted sequencing data were compared with Nipponbare sequences to obtain data from 15 mixed pool samples. OsNramp5 Indel data on gene mutations. Analysis of the Indel data revealed that in pool 820-3, [the following was observed / discussed / discussed]... OsNramp5 A deletion of base A exists at position 8875111 of the gene, resulting in a [A / T] base difference at this site. A KASP molecular marker was designed based on this base difference. 820C7-8875111-wF2: 5'-GAAGGTGACCAAGTTCATGCTAGCTCCACTATTACCGGCACA-3' (SEQ ID NO.2) 820C7-8875111-mH2: 5'-GAAGGTCGGAGTCAACGGATTAGCTCCACTATTACCGGCACT-3' (SEQ ID NO.3) 820C7-8875110-C2: 5'-TAATTACCTGCATGATGTACTGTCC-3' (SEQ ID NO.4) Using this KASP molecular marker, 500 leaves from the 820-3 mixed-pond sample were analyzed. It was found that 2 out of the 7 tillers of the first rice plant in the 24th row of the corresponding field planting material were... OsNramp5 A variation was found at position 8875111 of the gene. Further sequencing analysis of this site using first-generation sequencing revealed that both tillers of the first plant in row 24 were... OsNramp5 A bimodal pattern appeared at position 8875111 of the gene, indicating a heterozygous mutation at this site. This mutant was named Radiation 820S-N5-24-1. Seeds from tillers of the Radiation 820S-N5-24-1 mutant (M2 generation) were sown, and the M2 generation plants were genotyped using the aforementioned KASP primers, combined with first-generation sequencing, to screen for... OsNramp5 The homozygous mutant with a deletion of base A at position 8875111 has the mutation located at... OsNramp5 Exon 10 ( Figure 1 ).

[0025] 3. Detection of cadmium content in grains of the homozygous mutant of cyclone 820S-N5-24-1 We selected 20 homozygous mutants of Fuju 820S-N5-24-1 and 20 grains from the parent Fuju 820S to detect cadmium content in the grains. Under severe cadmium pollution conditions (soil cadmium concentration of 2.16 mg / kg, pH of 6.15), the average cadmium content in the grains of the homozygous mutant of Fuju 820S-N5-24-1 was 0.053 ± 0.007 mg / kg, significantly lower than the cadmium content of the parent Fuju 820S grains (2.226 ± 0.205 mg / kg). Given the importance of low-cadmium-accumulating rice in ensuring national food security, we used Fuju 820S-N5-24-1 as a cadmium-low-accumulation donor to improve the core parents of hybrid rice. We also used the developed KASP molecular marker for marker-assisted selection breeding to improve the breeding efficiency of low-cadmium-accumulating rice varieties.

[0026] Example 2: Polymorphism identification of the F1 generation of the cyclone 820S-N5-24-1 hybrid marked by the present invention 1. Biomaterials This embodiment describes the F1 strain constructed by hybridizing Radiation Cyclone 820S-N5-24-1, Ronten 819S, and Radiation Cyclone 820S-N5-24-1 with Ronten 819S.

[0027] 2. Genotyping Genomic DNA from the rice sample was extracted and used as a template. KASP reaction detection was performed using the primer combination of this invention. The PCR amplification reaction system (10 μl) consisted of: 5 μl of 2×Flu-ASPCR Mix for KASP, 0.15 μl each of 10 μM primers 820C7-8875111-wF2 and 820C7-8875111-mH2, 0.3 μl of 10 μM primer 820C7-8875111-C2, 2 μl of the rice sample DNA template, and the remainder was ultrapure water, totaling 10 μl.

[0028] The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 20 s, annealing and extension at 65℃~57℃ for 40 s for 10 cycles, with the annealing and extension temperature decreasing by 0.8℃ in each cycle, and 94℃ denaturation for 20 s, 57℃ annealing and extension for 40 s for 28 cycles.

[0029] The obtained PCR products were scanned and genotyped using a Bio-Rad CFX96 Touch real-time quantitative PCR instrument. The specific procedure was: 37℃, 1 min, Plate Read, End.

[0030] 3. Results Analysis Using the above method, 30 samples of each of the following F1 hybrids (Radiation Cyclone 820S-N5-24-1, Ronten 819S, and Radiation Cyclone 820S-N5-24-1 and Ronten 819S) were tested. The results are as follows: Figure 2 As shown, the 820S-N5-24-1 gene was detected as a T-type base (deletion type), and the 30 F1 lines constructed by crossing the 820S-N5-24-1 gene with the Longteng 819S gene were A / T heterozygous. The parent gene of the Longteng 819S gene was an A-type base (non-deletion type), which proves that the molecular marker detection results provided by this invention are accurate and can effectively distinguish different allelic types.

[0031] 4. Analysis of Cadmium Content in Grains To further verify the KASP molecular marker pair OsNramp5 To assess the effectiveness of genotyping, the cadmium content of grains from 30 F1 lines constructed by crossing Fuju 820S-N5-24-1, Longteng 819S, and Fuju 820S-N5-24-1 with Longteng 819S was determined. As shown in Table 1, under soil cadmium concentration of 2.16 mg / kg and pH of 6.15, the cadmium content of Fuju 820S-N5-24-1 grains was significantly lower than that of Longteng 819S and the F1 lines, demonstrating that the molecular marker detection provided by this invention can effectively distinguish between different genotypes. OsNramp5Different alleles can be identified, and individual plants with low cadmium content in grains can be accurately screened based on genotype. This can be used for molecular-assisted breeding of rice with low cadmium accumulation, thereby improving breeding efficiency.

[0032] Table 1. Cadmium content in planted grains under soil cadmium concentration of 2.16 mg / kg and pH of 6.15 ; Example 3: Application of the present invention's markers in natural population detection 1. Biomaterials In this embodiment, using Radiation Cyclone 820S-N5-24-1 as a control, the marker of this invention was validated in a natural population using 95 rice accessions from different countries and regions in the 3K rice core germplasm.

[0033] 2. Genotyping Same as the genotype detection steps in Example 2.

[0034] 3. Results Analysis This marker appears in natural population typing results such as Figure 3 As shown, the test results clearly distinguish the types. The control sample 820S-N5-24-1 showed a T-type base (deletion type), while all 95 samples showed an A-type base (non-deletion type). These results indicate that 820S-N5-24-1 contains... OsNramp5 The mutation type of gene with a deletion of base A at position 8875111 on chromosome 7 is lacking in natural populations. This allele has great application potential in the breeding of low cadmium accumulation varieties. It also shows that the marker of the present invention has good specificity among different rice materials and can be used in molecular marker-assisted selection breeding of low cadmium accumulation varieties using Fuju 820S-N5-24-1 as the donor.

[0035] In summary: 1. The SNP molecular marker of this invention is located at the 8875111 site on chromosome 7 of the rice OsNramp5 gene. It is closely linked to the trait of low cadmium accumulation. It has no high frequency distribution in natural rice populations, has high specificity and low background interference. Using KASP technology, homozygous mutant, heterozygous and wild type can be clearly distinguished. The detection results are stable, reliable and reproducible.

[0036] 2. The primer set of this invention is designed specifically for KASP fluorescence typing. It requires no enzyme digestion, no electrophoresis, and no complex sequencing. Genotype determination can be completed directly through real-time fluorescence PCR. Single-plant detection is time-saving, high-throughput, and simple to operate, which can meet the rapid screening needs of large-scale breeding populations.

[0037] 3. This invention enables genotyping to be completed in the early seedling stage, accurately screening T / T homozygous cadmium-low-accumulation individual plants, significantly reducing the scale of field planting, shortening the breeding cycle, and lowering breeding costs, effectively solving the problems of traditional breeding relying on phenotypic identification, long cycle, low accuracy, and great susceptibility to environmental influences.

[0038] 4. The T / T genotype at the target locus of this invention can still control the cadmium content of grains at an extremely low level in heavily cadmium-contaminated soil, with a very significant cadmium reduction effect. It can be directly used as a core donor for the improvement of backbone parents, providing a key molecular tool for the breeding of cadmium-low accumulation rice varieties.

[0039] 5. The primer set and kit of this invention are highly versatile and can be applied to genotype detection, germplasm resource identification and molecular marker-assisted breeding of rice materials with different genetic backgrounds. They are easy to standardize and promote industrialization.

[0040] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. A SNP molecular marker related to cadmium absorption characteristics in rice, characterized in that: The SNP molecular marker is located in rice. OsNramp5 The gene is located at chromosome 7, at locus 8875111. This locus exhibits a polymorphism with a deletion of base A. When the genotype is T / T, rice exhibits a low cadmium accumulation trait.

2. A primer set for amplifying the SNP molecular marker of claim 1.

3. The primer set according to claim 2, characterized in that: It includes two specific primers, 820C7-8875111-wF2 and 820C7-8875111-mH2, and one universal primer, 820C7-8875111-C2; wherein, the nucleotide sequence of primer 820C7-8875111-wF2 is shown in SEQ ID NO.2, the nucleotide sequence of primer 820C7-8875111-mH2 is shown in SEQ ID NO.3, and the nucleotide sequence of primer 820C7-8875111-C2 is shown in SEQ ID NO.

4.

4. A reagent kit, characterized in that: It includes the primer set as described in claim 2 or 3.

5. The reagent kit according to claim 4, characterized in that: It also contains one or more of the following: 2×Flu-ASPCR Mix for KASP, dNTP, MgCl2, PCR buffer, and deionized water.

6. Any one of the following (1) to (3) can be used in screening / breeding / detecting rice with low cadmium accumulation: (1) The SNP molecular marker as described in claim 1; (2) The primer set as described in claim 2 or 3; (3) The kit according to claim 4 or 5.

7. The application according to claim 6, characterized in that: Using any one of (1) to (3) to detect the genomic DNA of rice, according to OsNramp5 Genotyping / breeding / detection of cadmium-low accumulating rice at locus 8875111 on chromosome 7; rice with the T / T genotype at this locus is cadmium-low accumulating rice.

8. The application according to claim 7, characterized in that, Includes the following steps: Step 1: Obtain the genomic DNA of the rice to be tested; Step 2: Perform PCR amplification on the genomic DNA using the primer set described in claim 2 or 3 or the kit described in claims 4-5 to obtain the amplification product; Step 3: Obtain from the amplification products OsNramp5 The genotype at locus 8875111 on chromosome 7; Step 4, according to OsNramp5 The genotype at locus 8875111 on chromosome 7 is used to determine whether the rice being tested is a low-cadmium-accumulating rice variety.

9. The application according to claim 8, characterized in that, The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 20 s, annealing and extension at 65℃~57℃ for 40 s for 10 cycles, with the annealing and extension temperature decreasing by 0.8℃ in each cycle, 94℃ denaturation for 20 s, 57℃ annealing and extension for 40 s for 28 cycles.

10. The application according to claim 8, characterized in that: Based on the amplification products OsNramp5 Methods for genotyping the gene at locus 8875111 on chromosome 7 include KASP fluorescence scanning genotyping.

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