Rice sterile line cadmium low accumulation mutant gene, protein, detection method and application thereof

Abstract

The invention belongs to the field of plant genetic breeding, and discloses a rice sterile line cadmium low-accumulation mutant gene which is based on a Nipponbare reference genome IRGSP-1. 0, and a basic group T is deleted at the downstream of a No.7 chromosome 8875112 of rice. The rice sterile line cadmium low-accumulation mutant gene is derived from rice sterile line grain 68S, and a gene resource library of low-cadmium rice breeding is enriched. The invention also provides an efficient detection tool for the low-cadmium-accumulation mutant gene, has the advantages of high throughput, rapidness, accuracy and relatively low cost, can be used for early and large-scale molecular marker-assisted selection (MAS), remarkably improves the breeding efficiency and shortens the breeding period. According to the invention, low-cadmium mutation is also created on the basis of important backbone sterile line grain 68S, a parent foundation is laid for cultivating a new variety of high-yield, high-quality and safe hybrid rice, and improvement of an excellent background is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic breeding and molecular biology, and specifically relates to a new rice ( Oryza sativa L.) Cadmium (Cd) low accumulation mutant genes, proteins, detection methods and their applications. Background Art

[0002] Cadmium (Cd) is a non-essential heavy metal that is highly toxic to plants, animals, and humans. With the acceleration of industrialization and the impact of agricultural activities (such as sewage irrigation, cadmium-containing fertilizers, and pesticides), soil cadmium contamination is becoming an increasingly prominent problem. Rice is the staple food for more than half of the world's population, but compared to other major food crops, rice is more susceptible to cadmium absorption from the soil and accumulation in its grains.

[0003] Breeding and promoting rice varieties with low cadmium accumulation is considered the most economical, effective, and fundamental approach to addressing cadmium contamination in rice. Recent studies have shown that the uptake and transport of cadmium by rice roots is regulated by a series of genes. Among them, the OsNramp5 gene (e.g., gene identifier Os07g0257200 or LOC_Os07g15370) has been shown to be the primary transporter gene for divalent cations such as cadmium and manganese (Mn) in rice roots. It is primarily expressed in tissues such as the root epidermis and cortex. Studies have shown that inactivating or creating functionally defective mutations in the OsNramp5 gene significantly reduces the efficiency of cadmium uptake by rice roots, thereby significantly reducing cadmium levels in the aboveground parts of the rice plant (including the grain). For example, studies have successfully created low-cadmium rice varieties by targeted knockout of the OsNramp5 gene using gene editing technologies (such as CRISPR / Cas9). However, gene-edited crops still face policy and regulatory restrictions on "GMO biosafety management" in many countries and regions, and there are obstacles to their commercial promotion and application.

[0004] To circumvent regulatory restrictions on genetically modified organisms (GMOs), traditional mutagenesis breeding techniques (such as physical mutagenesis using gamma rays or heavy ion beams, or chemical mutagenesis using EMS) are widely used to create natural mutants of the OsNramp5 gene. While mutagenesis techniques can increase the frequency of genomic variation, the resulting mutations are random and non-directional. Therefore, screening for target mutants requires large populations of induced progeny. Traditional phenotypic screening methods, which directly measure cadmium levels in large numbers of plants or rice grains, are labor-intensive, costly, and time-consuming, resulting in low efficiency. To improve screening efficiency, researchers have developed a variety of molecular-based detection technologies, such as TILLING, high-throughput sequencing technologies (such as targeted deep sequencing), and molecular markers based on known mutation sites (such as indel markers, single-nucleotide polymorphisms, and KASP markers) to assist in screening.

[0005] Using various techniques, researchers have discovered and reported multiple loss-of-function mutants of the OsNramp5 gene, including deletions of varying lengths, insertions, and single-base substitutions (SNPs). These mutants all exhibit varying degrees of low grain cadmium accumulation, providing valuable genetic resources for breeding low-cadmium rice. However, different OsNramp5 mutation types may have varying degrees of impact on other agronomic traits of rice, such as growth and development, yield, and manganese absorption and homeostasis. Furthermore, existing methods for screening specific mutants still have room for improvement, and the continuous discovery and creation of new OsNramp5 low-cadmium mutant genotypes is necessary.

[0006] In recent years, hybrid rice combinations, exemplified by "Liliangyou 8022," have repeatedly set record yields. Their core parental lines, the sterile male line "Li 68S" and the restorer line "R8022," are invaluable breeding resources. However, low grain cadmium accumulation is often not a priority during the selection process for these super-high-yielding combinations, potentially leading to high cadmium accumulation risks. Maintaining or enhancing the high-yield and high-quality characteristics of these superior parents (such as Li 68S and R8022) while efficiently creating and selecting new sterile and restorer lines with low cadmium accumulation traits (e.g., through the generation of new functional mutations in OsNramp5) through modern biotechnology (e.g., heavy ion mutagenesis and molecular marker-assisted selection), thereby achieving a synergistic effect between high yield and safety, remains a crucial challenge and urgent need in rice breeding. Therefore, there is still an urgent need in this field to provide new rice mutant genotypes derived from specific excellent genetic backgrounds (such as Grain 68S, R8022), with excellent low-cadmium traits and little (or beneficial) impact on key agronomic traits, so as to enrich the gene resource library for low-cadmium breeding and provide core parent support for the cultivation of high-quality, high-yield and safe new hybrid rice varieties suitable for different production needs. Summary of the Invention This invention aims to address the potential for high cadmium accumulation in existing elite male sterile rice lines (such as Li 68S), as well as the limited availability of low-cadmium gene resources and the need to improve screening and identification efficiency. Specifically, it aims to provide a novel mutant genotype derived from the elite male sterile rice line Li 68S, with a well-defined genetic background and low cadmium accumulation characteristics; a KASP marker and its detection method that enable rapid, accurate, and high-throughput identification of this mutant genotype; and the application of this mutant genotype and marker in the selection of low-cadmium male sterile rice lines and hybrid rice breeding.

[0007] In order to solve the above technical problems, the technical solution proposed by the present invention is: A rice sterile line cadmium low accumulation mutant gene, based on the Nipponbare reference genome IRGSP-1.0, has a deletion of one base T downstream of 8875112 on rice chromosome 7.

[0008] The above-mentioned rice sterile line cadmium low accumulation mutant gene further has a CDS sequence as shown in SEQ No.2.

[0009] A rice sterile line cadmium low accumulation mutant protein, the protein sequence of which is shown in SEQ No.1.

[0010] A KASP marker primer combination for detecting the cadmium low accumulation mutant gene in the rice sterile line, which targets the specific sequence change site, includes (as shown in SEQ No. 3-5): Allele-specific forward primer lcd8875112-FAM: GAAGGTGACCAAGTTCATGCTCAGAGCTCCACTATTACCGGCA; Allele-specific forward primer lcd8875112-HEX: GAAGGTCGGAGTCAACGGATTCAGAGCTCCACTATTACCGGCC; Universal reverse primer lcd8875112-COMMON: GCATGATGTACTGTCCAGCGTA.

[0011] A method for detecting whether a rice material contains the cadmium low accumulation mutant gene comprises the following steps: extracting genomic DNA from a rice sample to be tested; performing a KASP reaction amplification on the DNA to be tested using the KASP labeled primer combination; detecting the fluorescence signal after the reaction, and when the result shows only FAM fluorescence, it indicates that the rice sample to be tested is a homozygous wild type; when the result shows only HEX fluorescence, it indicates that the rice sample to be tested is a homozygous mutant type; when the result shows both FAM and HEX fluorescence, it indicates a heterozygous genotype; The KASP labeled primer combination includes: Specific forward primer lcd8875112-FAM: GAAGGTGACCAAGTTCATGCTCAGAGCTCCACTATTACCGGCA; Specific forward primer lcd8875112-HEX: GAAGGTCGGAGTCAACGGATTCAGAGCTCCACTATTACCGGCC; Universal reverse primer lcd8875112-COMMON: GCATGATGTACTGTCCAGCGTA.

[0012] Based on a general inventive concept, the present invention also provides a rice sterile line cadmium low accumulation mutant gene, protein or the KASP marker primer combination, or the detection method for use in breeding low cadmium hybrid rice varieties.

[0013] The above application, further, the steps of breeding low-cadmium hybrid rice varieties are as follows: using materials containing the rice sterile line cadmium low accumulation mutation gene as donor parents, and other rice to be improved as recipient parents, hybridizing to obtain F1, planting F1 to obtain F2 generation, detecting whether individual plants in the F2 generation population contain the rice sterile line cadmium low accumulation mutation gene, repeating the above process until genetically stable individual plants containing the homozygous rice sterile line cadmium low accumulation mutation gene are obtained in the offspring, that is, the low-cadmium hybrid rice variety is obtained.

[0014] Furthermore, the donor parent is the low-cadmium mutant Li68S-lcd8875112, and the recipient parent is Y58S.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a new cadmium low accumulation mutant genotype derived from the 68S background of an excellent sterile grain line with a clear genetic variation site. This specific mutation can significantly reduce cadmium accumulation while better maintaining the excellent agronomic traits of the original parent, enriching the gene resource library for low-cadmium rice breeding; laying a parental foundation for the cultivation of high-yield, high-quality and safe new hybrid rice varieties, and realizing the improvement of the excellent background.

[0016] 2. The development of the KASP molecular marker for this specific mutation provides an efficient detection tool with the advantages of high throughput, rapidity, accuracy, and relatively low cost. It can be used for early, large-scale molecular marker-assisted selection (MAS), significantly improving breeding efficiency and shortening the breeding cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is a schematic diagram of the gene mutation between the mutant lcd8875112 and the original parent sterile line "Li 68S" in Example 1.

[0019] Figure 2 These are the genotyping results of 500 M1 generation individual plants in the mixed pool in Example 1.

[0020] Figure 3 This is the typing result of the M2 generation in Example 1.

[0021] Figure 4 It is the cadmium content of Li68S-lcd8875112 and the original parent sterile line "Li 68S" under CdCl2 treatment conditions in Example 1; where A is the cadmium content in stems and leaves, and B is the cadmium content in roots.

[0022] Figure 5 The cadmium content of the grains of Li68S-lcd8875112 and the original parent sterile line "Li 68S" in Example 1.

[0023] Figure 6 is the cadmium content of the grains of Y58S-lcd8875112 and the original control material Y58S in Example 2. DETAILED DESCRIPTION

[0024] To facilitate understanding of the present invention, the present invention will be described in more comprehensive and detailed form below in conjunction with the accompanying drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0025] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0026] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0027] The method for obtaining the rice sterile line cadmium low accumulation mutant gene of the present invention comprises the following steps: (a) Heavy ion irradiation mutagenesis treatment of seeds or plants of the rice sterile line Li 68S; (b) Planting the mutagenized material to obtain M1 generation plants; (c) Extracting genomic DNA from M1 plants (either individual plants or mixed pools) and performing targeted capture sequencing of the target gene (OsNramp5); (d) identifying, through bioinformatics analysis, an M1 chimeric plant carrying the rice sterile line cadmium low accumulation mutant gene; (e) Design and validate KASP markers targeting the mutant gene; (f) Planting the selfed progeny of the M1 generation plants (M2 generation); (g) extracting DNA from the M2 generation population and performing high-throughput genotyping using the KASP marker developed in step (e) to screen out individual plants carrying the homozygous mutant genotype; (h) Verification of the low cadmium accumulation phenotype of the screened homozygous mutant plants.

[0028] Example 1: Creation and screening of 68S mutants in rice sterile lines A rice sterile line cadmium low accumulation mutant gene, which is derived from the rice sterile line grain 68S, contains a specific genomic DNA sequence change. The change is located at the downstream of rice chromosome 7 8875112, with a deletion of 1bp (T), namely Chr07: g.8875112delT (Nipponbare reference genome IRGSP-1.0). The present invention names the strain containing this homozygous mutant genotype Li68S-lcd8875112.

[0029] Mutagenesis treatment: 500 g of dry seeds of the sterile rice line "Li 68S" were taken and subjected to heavy ion (¹²C 6 ⁺) Irradiation treatment. The irradiation dose was set at 150 Gy. The treated seeds were immediately soaked and germinated.

[0030] M1 generation planting and targeted sequencing: Mutagenized seeds were sown in experimental fields to obtain M1 generation plants. At the tillering stage, a pooling strategy was used, with samples collected every 500 plants. Genomic DNA was extracted using a modified CTAB method. Targeted deep sequencing was performed on the exonic regions of the target gene, OsNramp5. Sequencing was performed on the Illumina NovaSeq 6000 platform, with an average sequencing depth of >50,000x across the target region.

[0031] Mutation identification: The clean data obtained by sequencing was aligned with the Nipponbare reference genome (IRGSP-1.0), and SNP and Indel mutation detection was performed using bioinformatics software such as GATK, Samtools, and Freebayes. In pool 27, a T-base deletion mutation was identified in the OsNramp5 gene, specifically a 1 bp (T) deletion downstream of position 8875112 on rice chromosome 7, namely Chr07: g.8875112delT (e.g. Figure 1 ), and the mutant was named lcd8875112. This mutation results in a premature stop codon.

[0032] The mutated protein sequence (such as SEQ No.1 shown): MEIERESSERGSISWRASAAHDQDAKKLDADDQLLMKEPAWKRFLAHVGPGFMVSLAYLDPGNLETDLQAGANHRYELLWVILIGLIFALIIQSLAANLGVVTGRHLAEICKSEYPKFVKIFLWLLAELAVIAADIPEVIGTAFAFNILFHIPVWVGVLITGSTLLLLGLQKYGV RKLEFLISMLVFVMAACFFGELSIVKPPAKEVMKGLFIPRLNGDGATADAIALLGALVMPHNLFLHSALVLSRKTPASVRGIKDGCRFFLYESGFALFVALLINIAVVSVSGTACSSANLSQEDADKCANLSLDTSSFLLKNVLGKSSAIVYGVALLASGQSSTITGHTLDSTSCRVSWTSG*.

[0033] The CDS sequence after mutation (as shown in SEQ No. 2):

[0034] KASP marker development and validation: KASP primers were designed for the mutation site Chr07: g.8875112delT identified above (as shown in SEQ No. 3-5): lcd8875112-FAM: GAAGGTGACCAAGTTCATGCTCAGAGCTCCACTATTACCGGCA; lcd8875112-HEX:GAAGGTCGGAGTCAACGGATTCAGAGCTCCACTATTACCGGCC; lcd8875112-COMMON: GCATGATGTACTGTCCAGCGTA.

[0035] Primers were synthesized by Beijing Qingke Biotechnology. The KASP marker was used to genotype 500 M1 generation strains in pool 27. KASP reaction system (10µL): 5µL 2x KASP Master Mix, 0.14µL KASP Assay Mix, 1-3µL template DNA (10-50 ng), and nuclease-free water was added to 10µL. The reaction and signal reading were performed on a fluorescence quantitative PCR instrument: 94°C for 3-15min; 10 cycles [94°C for 20s, 65°C→57.8°C (-0.8°C / cycle) for 60s]; ≥28 cycles [94°C for 20s, 57°C for 60s]; 37°C for 1min (fluorescence collection); storage at 4-12°C. Figure 2 It was shown that the 9th strain in line 192 of population 27 carried the mutation.

[0036] M2 generation screening and homozygote acquisition: Harvest self-pollinated seeds (M2 generation) from M1 (192-9) plants carrying the target mutation. Sow the M2 generation seeds and grow them into seedlings. Take leaves from a large number of individual M2 generation plants and extract DNA. Perform high-throughput genotyping using the KASP markers verified in step 4. Figure 3 Based on the typing results, individual strains with homozygous HEX fluorescence mutant genotypes were screened. These homozygous strains were propagated and purified to establish a stable mutant strain, named Li68S-lcd8875112.

[0037] Identification of the low-cadmium accumulation phenotype of the mutant Li68S-lcd8875112: (1) Seeds of the genetically stable homozygous mutant Li68S-lcd8875112 and the original parental sterile line "Li 68S" (WT, wild-type control) were selected, disinfected, and germinated on moist filter paper. When the seedlings grew to approximately two leaves and one heart, seedlings with consistent growth were carefully transplanted into Yoshida rice nutrient solution. Two treatment groups were set up: a control group (CK, no Cd was added to the nutrient solution) and a cadmium treatment group (0.5 μM CdCl2 was added). In each treatment group, five biological replicates were set for each genotype (Li68S-lcd8875112 and Li 68S), with each replicate containing three seedlings. The plants were cultured in a light-controlled culture chamber for 14 days. After the culture period, the plants were carefully removed and rinsed with deionized water, especially the roots, which needed to be carefully cleaned to remove surface-adsorbed Cd. The roots and aerial parts were separated, sterilized at 105°C for 30 minutes, and then dried at 70°C to constant weight. Weigh the dry weight respectively. After the dried sample is crushed, it is digested by nitric acid-perchloric acid digestion method. After constant volume, the cadmium content in the sample is determined by ICP-MS (inductively coupled plasma mass spectrometry). Figure 4 The results showed that under the treatment of 0.5 μM CdCl2, the average cadmium content in the aboveground part of the wild-type 68S was 10.03 mg / kg, and the average cadmium content in the root was 269.00 mg / kg; while the cadmium content in the aboveground part of the homozygous mutant Li68S-lcd8875112 was significantly reduced to an average of 2.07 mg / kg, and the cadmium content in the root was also significantly reduced to an average of 45.67 mg / kg.

[0038] (2) The homozygous mutant Li68S-lcd8875112 obtained in this example and verified by KASP at the seedling stage was planted simultaneously with the original parent sterile line "Li 68S" under potted conditions (total cadmium content in the soil was 1.5 mg / kg, pH value was 5.6). Five replicates were set for each genotype (for potted plants, each pot was used as a replicate, with 3 plants of each wild type and mutant). Field management was carried out according to conventional rice cultivation management methods until the rice was fully mature. After maturity, the aboveground part of each replicate was harvested separately, and rice was obtained after threshing. A rice sample was taken, and the brown rice sample was dried at 70°C to constant weight and then crushed. A certain amount of brown rice flour was weighed and sample pretreatment and digestion were performed in accordance with the national standard "GB 5009.15 National Food Safety Standard Determination of Cadmium in Food". The cadmium content in the brown rice sample was determined by ICP-MS. The results are as follows. Figure 5The results show that under conditions where the soil available cadmium concentration was 1.5 mg / kg, the brown rice cadmium content of the wild-type Li68S averaged 0.51 mg / kg, significantly exceeding the cadmium limit for rice in the National Food Safety Standard (GB2762-2017) of 0.2 mg / kg. In contrast, the brown rice cadmium content of the homozygous mutant Li68S-lcd8875112 averaged only 0.07 mg / kg, far below the national limit, and the difference compared to the wild type was highly significant (P < 0.01). Furthermore, an investigation of key agronomic traits revealed no significant adverse differences between the Li68S-lcd8875112 mutant and the wild-type Li68S.

[0039] Comprehensive results of hydroponic culture at the seedling stage and grain measurement at the mature stage prove that the mutant genotype Li68S-lcd8875112 created by the present invention has a stable and significant low cadmium accumulation effect, has no effect on agronomic traits, and has important breeding application value.

[0040] Example 2: Application of a rice sterile line cadmium low accumulation mutant gene and a KASP marker primer combination in breeding low cadmium hybrid rice varieties The low-cadmium mutant Li68S-lcd8875112 obtained in Example 1 was used as a donor and hybridized with the target rice line Y58S (recipient) to be improved. DNA was extracted from leaves of a large number of individual plants in the F2 generation or subsequent backcross generations (BCnFm), and genotypes were determined using the KASP marker primer combination developed in Example 1. A result showing only FAM fluorescence indicates that the rice sample is homozygous wild-type; a result showing only HEX fluorescence indicates a homozygous mutant; and a result showing both FAM and HEX fluorescence indicates a heterozygous genotype. Based on the KASP typing results, combined with investigations of other target agronomic traits, superior individual plants carrying the homozygous low-cadmium mutant genotype (with HEX fluorescence) can be quickly and accurately selected to obtain low-cadmium hybrid rice varieties, significantly accelerating the introduction of the low-cadmium trait and the breeding of superior varieties.

[0041] Field experiments (soil available cadmium content of 0.53 mg / kg, pH 5.7) showed that Figure 6 The average cadmium content in the grains of the original control material Y58S was 0.34 mg / kg, while the average cadmium content in the grains of the low-cadmium improved line Y58S-lcd8875112 was 0.05 mg / kg.

Claims

1. A rice sterile line cadmium low accumulation mutant gene, characterized in that: Based on the Nipponbare reference genome IRGSP-1.0, a base T was deleted downstream of 8875112 on rice chromosome 7.

2. The rice sterile line cadmium low accumulation mutant gene according to claim 1, characterized in that Its CDS sequence is shown in SEQ No.

2.

3. A rice sterile line cadmium low accumulation mutant protein, characterized in that: Its protein sequence is shown in SEQ No.

1.

4. A KASP marker primer combination for detecting the cadmium low accumulation mutant gene of the rice sterile line, characterized in that: include: Specific forward primer lcd8875112-FAM: GAAGGTGACCAAGTTCATGCTCAGAGCTCCACTATTACCGGCA; Specific forward primer lcd8875112-HEX: GAAGGTCGGAGTCAACGGATTCAGAGCTCCACTATTACCGGCC; Universal reverse primer lcd8875112-COMMON: GCATGATGTACTGTCCAGCGTA.

5. A method for identifying whether a rice material contains the cadmium low accumulation mutant gene according to claim 1 or 2, characterized in that: The method comprises the following steps: extracting genomic DNA from a rice sample to be tested; performing a KASP reaction amplification on the extracted genomic DNA using a KASP labeled primer combination; detecting the fluorescence signal after the reaction, and when the result shows only FAM fluorescence, it indicates that the rice sample to be tested is a homozygous wild type; when the result shows only HEX fluorescence, it indicates that the rice sample to be tested is a homozygous mutant type; when the result shows both FAM and HEX fluorescence, it indicates a heterozygous genotype; The KASP labeled primer combination includes: Specific forward primer lcd8875112-FAM: GAAGGTGACCAAGTTCATGCTCAGAGCTCCACTATTACCGGCA; Specific forward primer lcd8875112-HEX: GAAGGTCGGAGTCAACGGATTCAGAGCTCCACTATTACCGGCC; Universal reverse primer lcd8875112-COMMON: GCATGATGTACTGTCCAGCGTA.

6. Use of the rice sterile line cadmium low accumulation mutant gene according to claim 1 or 2, the protein according to claim 3, the KASP marker primer combination according to claim 4, or the detection method according to claim 5 in breeding low-cadmium hybrid rice varieties.

7. The use according to claim 6, characterized in that The steps for breeding low-cadmium hybrid rice varieties are as follows: using materials containing the rice sterile line cadmium low accumulation mutation gene as donor parents and other rice to be improved as recipient parents, hybridizing to obtain F1, planting the F1 to obtain the F2 generation, detecting whether individual plants in the F2 generation population contain the rice sterile line cadmium low accumulation mutation gene, and repeating the above process until genetically stable individual plants containing the homozygous rice sterile line cadmium low accumulation mutation gene are obtained in the offspring, thus obtaining the low-cadmium hybrid rice variety.

8. The use according to claim 7, characterized in that The donor parent is the low-cadmium mutant Li68S-lcd8875112, and the recipient parent is Y58S.

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