CN113969286A

CN113969286A - Rice cadmium low-accumulation molecular marker, mutant gene, detection kit, rapid identification method and application thereof, and typing primer

Info

Publication number: CN113969286A
Application number: CN202111192651.1A
Authority: CN
Inventors: 赵炳然; 韶也; 毛毕刚; 余丽霞; 彭彦; 柏连阳; 李文建; 袁智成; 唐丽; 李曜魁; 彭选明; 胡远艺; 张丹; 罗武中
Original assignee: Hunan Hybrid Rice Research Center
Current assignee: Hunan Hybrid Rice Research Center
Priority date: 2021-10-13
Filing date: 2021-10-13
Publication date: 2022-01-25
Anticipated expiration: 2041-10-13

Abstract

The invention discloses a rice cadmium low accumulation molecular marker, wherein the nucleotide sequence of the marker takes the genome of Nipponbare rice as a reference genome, the marker comprises the deletion of three basic groups of CTG at the position of a No.7 chromosome 8874101-8874103, and finally a rice R001-OsNramp5 mutant gene is obtained, and the CDS region of the gene comprises a specific nucleotide sequence or an amino acid sequence; the invention also provides a molecular marker detection kit, which comprises a designed primer pair; the invention also provides application of the molecular marker or mutant gene in molecular assisted breeding or variety improvement of crops and a corresponding KASP typing primer, and also provides a rapid identification method based on the detection kit.

Description

Rice cadmium low-accumulation molecular marker, mutant gene, detection kit, rapid identification method and application thereof, and typing primer

Technical Field

The invention belongs to the technical field of rice mutation breeding, and particularly relates to a rice cadmium low-accumulation molecular marker, a rice mutant gene, identification and application thereof, and a typing primer for identification.

Background

In southern areas of China, rice is mainly used as staple food, and in areas with cadmium pollution in farmlands, rice is a main source of dietary cadmium. The cadmium rice problem brings risks to the health of people and also causes hidden dangers to national grain safety. At present, the problem of excessive cadmium in rice can be relieved to a certain extent by the matching technology of heavy metal soil remediation and cadmium-polluted rice field cultivation, but the method is difficult to popularize in a large range due to high cost and multiple technical links. Therefore, the creation of a new variety with low cadmium accumulation of rice and the cultivation of a new variety with low cadmium accumulation are the most economical and effective methods for solving the problem of cadmium rice in China.

The existing rice natural resources are deficient in low cadmium accumulation germplasm, and the gene editing technology can be used for efficiently and directionally creating the low cadmium accumulation rice germplasm, but the gene editing product industrialization faces policy obstacles of 'transgene safety management' and foreign intellectual property barriers and the like, so that the method is difficult to popularize and apply in a short period. The method creates a new germplasm of cadmium low-accumulation rice with complete independent intellectual property rights by using a non-transgenic means, and is the most feasible way for thoroughly solving the cadmium rice problem in China at the present stage.

The physical and chemical mutagenesis of crops is a traditional germplasm innovation technology, can generate a large amount of genetic variation and plays an important role in crop improvement all over the world. According to the incomplete statistics of FAO/IAEA database, more than 3200 mutant varieties of 214 crop varieties are released from more than 60 countries in the world from 1930 to the present, and the mutant varieties make important contribution to world food safety. Therefore, the crop physicochemical mutation technology is a safe germplasm innovation mode, the product has high acceptance in the public, and can be directly applied in a large scale. A physicochemical mutagenesis mode is utilized to create a new germplasm of cadmium low-accumulation rice with independent intellectual property rights in China, so that the method is expected to directly solve the problem of cadmium rice in China and has great significance.

Disclosure of Invention

The invention aims to solve the technical problems that the defects and defects mentioned in the background technology are overcome, a rice cadmium low-accumulation molecular marker for assisting in selecting cadmium low-accumulation rice and a rice R001-OsNramp5 mutant gene are provided, a detection kit for carrying out molecular breeding on the cadmium low-accumulation rice and a corresponding rapid identification method are also provided, and the application of the rice R001-OsNramp5 mutant gene in crop molecular assisted breeding or cadmium low-accumulation character improvement on rice varieties is correspondingly provided.

In order to solve the technical problems, the technical scheme provided by the invention is a rice cadmium low accumulation molecular marker (LC1), wherein the rice cadmium low accumulation molecular marker is a section of nucleotide sequence on a rice genome, the rice cadmium low accumulation molecular marker is an Indel marker, the Indel marker is obviously related to the rice grain cadmium low accumulation property, the nucleotide sequence takes the genome of Nipponbare rice as a reference genome and comprises a section of base sequence (particularly preferably a sequence of a No. 10 intron region of an OsNramp5 gene) positioned on an antisense chain of a No.7 chromosome 8871643-88905 bp interval, and the base sequence has deletion of three bases of CTG at a position of 8874101-8874103 positioned on the No.7 chromosome.

Preferably, the deletion of three bases of CTG of the rice cadmium low accumulation molecular marker exists in an intron region of the OsNramp5 gene. Although the Indel molecular marker is not located in any gene coding region, but is located in an intron region of LOC _ Os07g15370 gene, the Indel molecular marker is obviously related to the cadmium content of rice grains, and in an isolated population after hybridization with a rice cadmium low accumulation mutant R001, the cadmium content of rice grains with the CTG (+/+, +/-) type of Indel (Chr7:8874101-8874103) sites is obviously higher than that of a rice single plant with homozygous deletion CTG (-/-) genotype.

The molecular marker for low accumulation of cadmium in rice is preferably obtained by mutating a created mutant for low accumulation of cadmium (cadmium low accumulation mutant R001) by radiation.

Preferably, the molecular marker for low accumulation of cadmium in rice comprises a nucleotide sequence shown as SEQ ID No. 7.

The molecular marker with low accumulation of cadmium in rice is obtained by amplification of the following primer pairs LC1-F and LC 1-R:

LC1-F：5’-CATCAGGTTCCGAAGCCACTTC-3’；

LC1-R：5’-GATAAATCATCATCATGTCGCGTTG-3’。

as a general technical concept, the present invention also provides a rice R001-OsNramp5 mutant gene, wherein the sequence of the rice R001-OsNramp5 mutant gene is based on the OsNramp5 gene sequence of chromosome 7 of fine japonica rice, and the base sequence comprises the following base deletion segments:

three bases of CTG are deleted at the position of 8874101-8874103 at the end of intron 10 of the OsNramp5 gene sequence (the physical position takes Nipponbare as a reference genome, the sequence version number is IRGSP-1.0, the database website address is https:// rapdb.dna.affrc.go.jp/download/IRGSP1.html, on the antisense strand of 8871643-8878905bp interval of chromosome 7);

the CDS region of the rice R001-OsNramp5 mutant gene comprises a nucleotide sequence shown in SEQ ID No. 1.

As a general technical concept, the invention also provides the rice R001-OsNramp5 mutant gene, and the amino acid sequence coded by the rice R001-OsNramp5 mutant gene is shown as SEQ ID No. 2.

The rice R001-OsNramp5 mutant gene is positioned in the No. 10 intron region of the OsNramp5 gene and is the last 3 bases of the No. 10 intron (refer to figure 1), the deletion of CTG causes the splicing error of the OsNramp5 at the No. 10 intron and the No. 11 exon after transcription, and the first 17 bases of the No. 11 exon are spliced out.

As a general technical concept, the invention also provides a detection kit for amplifying the rice cadmium low accumulation molecular marker or for genotyping the rice R001-OsNramp5 mutant type gene, wherein the detection kit comprises the following primer pairs LC1-F and LC 1-R:

LC1-F：5’-CATCAGGTTCCGAAGCCACTTC-3’；

LC1-R：5’-GATAAATCATCATCATGTCGCGTTG-3’。

preferably, the detection kit further comprises 2 XPCR Master Mix (Thermo Scientific) and ddH₂O; the 2X PCR Master Mix and the mixture containing LC1-F/LC1-R solutionThe product ratio is controlled to be 20-30: 1, and the ddH₂The volume ratio of O to the solution containing LC1-F/LC1-R is controlled to be 18-28: 1.

The detection kit has a particularly preferred detection configuration system as follows: 5 μ L of 2 XPCR Master Mix, 0.2 μ L LLC1-F (10 μ M), 0.2 μ L LC1-R (10 μ M) and 4.6 μ L ddH₂O。

As a general technical concept, the invention also provides a rapid identification method for rice R001-OsNramp5 mutant genotyping by using the detection kit, which comprises the following steps:

(1) taking a cadmium low-accumulation mutant R001 containing a rice R001-OsNramp5 mutant gene as a donor, hybridizing with non-cadmium low-accumulation rice to obtain a hybrid F1, selfing an F1 generation to obtain an F2 segregation population, or carrying out repeated crossing on F1 and other rice materials to obtain a repeated crossing progeny segregation population;

(2) collecting single plant leaves from the inbred F2 or the multiple-cross progeny segregation population, extracting DNA from the single plants, and performing amplification reaction by using the detection kit to obtain an amplification product;

(3) identifying the length of the amplification product through gel electrophoresis, and identifying a cadmium high-accumulation plant if the plant only contains one 100bp strip or simultaneously contains two strips, and the sizes of the strips are 100bp and 97bp respectively; if only one 97bp strip is contained, the plant is identified as a low cadmium accumulation plant.

In the above rapid identification method, preferably, the amplification reaction conditions in step (2) are: 3min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 10 s; 5min at 72 ℃.

As a general technical concept, the invention also provides an application of the detection kit in rapid breeding of the rice molecules with low cadmium accumulation, which is to continue the following operation steps after the rapid identification method of the invention:

(a) carrying out comprehensive agronomic character evaluation on the heterozygous stripe plants in the obtained cadmium high accumulation plants and the homozygous stripe plants in the cadmium low accumulation plants, and selecting excellent single plants for continuous selfing;

(b) continuously selecting single plants with excellent comprehensive agronomic characters from selfing F3 generations or compound cross progeny, taking leaves to extract DNA, carrying out genotyping by using the detection kit, and selecting selfed seeds of 100bp and 97bp heterozygous strip plants and 97bp homozygous strip plants;

(c) and (c) repeating the step (b) until the population is genetically stable (at least to F5 generation is generally preferred), the comprehensive agronomic traits are uniform, and the typing results of the detection kit are all 97bp homozygous strip plants, so as to obtain a low-septate improved line.

As a general technical concept, the invention also provides application of the rice R001-OsNramp5 mutant gene in molecular assisted breeding of crops or improvement of low cadmium accumulation traits of rice varieties.

The above application of improving the low cadmium accumulation trait of a rice variety preferably comprises the following steps:

(1) hybridizing a cadmium low-accumulation mutant R001 containing a rice R001-OsNramp5 mutant gene with a conventional rice variety to obtain F1;

(2) backcrossing the conventional rice variety serving as a recurrent parent with an F1 single plant to obtain a BC1F1 population;

(3) carrying out foreground selection on the BC1F1 population by using KASP typing primers, and selecting a Chr 78874101-8874103 CTG deletion genotype single plant; then background selection is carried out, and the single plant with the highest similarity to the genetic background of the conventional rice variety is selected to continue backcrossing with the recurrent parent;

(4) and (4) continuing to select multiple generations through the foreground selection and the background selection according to the step (3) to obtain the low-cadmium improved line with the genetic background consistent with the conventional rice variety and the CTG deletion genotype of Chr7: 8874101-8874103.

In the above application, preferably, the conventional rice variety is a non-cadmium low-accumulation rice variety (particularly preferably Huazhan), and in the step (4), the multi-generation is specifically 3-4 generations through foreground selection and background selection.

As a general technical concept, the invention also provides a KASP typing primer for identifying, screening or applying the rice R001-OsNramp5 mutant gene, wherein the primer sequence of the KASP typing primer is as follows:

FAM 5’-GAAGGTGACCAAGTTCATGCTCATCCTGATGTCCAAGAAACCCT-3’；

HEX 5’-GAAGGTCGGAGTCAACGGATTCATCCTGATGTCCAAGAAACCCA-3’；

COMMON 5’-GATAAATCATCATCATGTCGCGTTG-3’。

compared with the prior art, the invention has the beneficial effects that:

1) the invention provides a molecular marker for assisting in selecting low cadmium accumulation rice, which designs a specific length polymorphism molecular marker by utilizing a 3bp base sequence deleted from a mutant R001, is used for quickly identifying the low cadmium accumulation rice, reducing the workload of cultivating the low cadmium accumulation rice and shortening the cultivation period, and provides technical support for improving the low cadmium accumulation character by simply, quickly and high-flux applying a molecular marker assisted breeding technology;

2) the invention provides a detection kit for rice genotyping, which can directionally improve the low-cadmium-accumulation character of rice with an R001 mutant as a donor, and provides a stable, efficient and low-cost molecular assisted breeding tool;

3) based on the identification and screening of the invention, the mutant gene of the rice R001-OsNramp5 is creatively obtained, and the application of the mutant gene of the rice R001-OsNramp5 in crop molecule-assisted breeding and the application in breeding, preparation and improvement of cadmium low-absorption phenotype rice varieties are provided, so that the remarkable technical effect is achieved, and finally, the rice variety improvement line with the cadmium low-absorption phenotype is obtained;

4) the KASP typing primer for detecting, screening or applying the rice R001-OsNramp5 mutant gene designed by the invention can be used for accurately typing different individuals of indels discovered by sequencing, and can further improve the accuracy and efficiency of screening and identifying the existing innovative germplasm;

5) the technical scheme of the invention not only saves the identification time cost, but also facilitates the crop improvement and germplasm innovation of cadmium low accumulation varieties, and has obvious progress significance.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a schematic diagram showing the comparison of the mutation site, mutation type and transcribed base sequence of the mutant gene R001-OsNramp5 of rice of the present invention.

FIG. 2 is the map of the mutant R001 cadmium low accumulation gene in the example of the present invention. Wherein A is a BSA mapping map; b is a schematic diagram of fine positioning by using the mark.

FIG. 3 is a scatter plot of genotyping segregating populations using KASP typing primers in accordance with embodiments of the present invention; wherein: the upper left scatter point is a non-mutation pure sum line, the middle scatter point is a CTG deletion mutation and non-mutation hybrid line, the lower left scatter point is NTC, and the lower right scatter point is a CTG deletion mutation pure sum line.

FIG. 4 is a comparison test result of the cadmium content of the modified brown rice with low cadmium content in China compared with wild type China in example 3 of the present invention.

FIG. 5 is a photograph of the amplification product of example 2 of the present invention after polyacrylamide gel electrophoresis and silver staining for color development.

Detailed Description

In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

Example 1:

the invention relates to a molecular marker for low accumulation of cadmium in rice, which is a nucleotide sequence of an intron on a rice genome, namely an Indel marker, wherein the Indel marker is obviously related to the low accumulation of cadmium in rice grains, the nucleotide sequence takes the genome of Nipponbare rice as a reference genome, the nucleotide sequence comprises a base sequence on a No. 10 intron on an antisense strand of an interval of 8871643-8878905bp of a No.7 chromosome, and the base sequence has deletion of three bases of CTG at the position of 8874101-8874103 of the No.7 chromosome.

The deletion of three basic groups of CTG of the rice cadmium low accumulation molecular marker exists in an intron region of an OsNramp5 gene. Although the Indel molecular marker is not located in any gene coding region, but is located in an intron region of LOC _ Os07g15370 gene, the Indel molecular marker is obviously related to the cadmium content of rice grains, and in an isolated population after hybridization with a rice cadmium low accumulation mutant R001, the cadmium content of rice grains with the CTG (+/+, +/-) type of Indel (Chr7:8874101-8874103) sites is obviously higher than that of a rice single plant with homozygous deletion CTG (-/-) genotype.

The rice cadmium low accumulation molecular marker of the embodiment comprises a nucleotide sequence shown as SEQ ID No. 7.

The rice cadmium low accumulation molecular marker of the embodiment is obtained by using a cadmium low accumulation mutant created by radiation mutagenesis. The mutant contains a rice R001-OsNramp5 mutant gene, the sequence of the rice R001-OsNramp5 mutant gene takes the OsNramp5 gene sequence of the No.7 chromosome of Nipponbare rice as a basic sequence, and the basic sequence comprises the following base deletion sections, namely: on the antisense strand of the 8874101-8874103 bp interval of chromosome 7 (the physical position takes Nipponbare as a reference genome, the sequence version number is IRGSP-1.0, the database website address is https:// rapdb.dna.affrc.go.jp/download/IRGSP1.html), the position of 8874101-8874103 at the end of intron 10 of the OsNramp5 gene sequence has three-base deletion of CTG.

The CDS region of the mutant gene of rice R001-OsNramp5 in the embodiment comprises a nucleotide sequence shown in SEQ ID No. 1.

Since the mutant gene R001-OsNramp5 of the rice is located in the 10 th intron region of the OsNramp5 gene and is the last 3 bases of the 10 th intron (see figure 1), deletion of CTG causes that shearing of OsNramp5 at the 10 th intron and the 11 th exon after transcription is wrong, and the first 17 bases of the 11 th exon are sheared, so that the amino acid sequence coded by the mutant gene R001-OsNramp5 of the final mutant rice is shifted. The amino acid sequence encoded by the mutant gene of rice R001-OsNramp5 is shown in SEQ ID No. 2.

The rice R001-OsNramp5 mutant gene of the embodiment is obtained by screening and identifying through the following method:

1. obtaining the rice cadmium low accumulation mutant R001.

Using a high-energy heavy ion beam (¹²C6+) irradiation treatment of hybrid rice restorer line variety Huahui 8612, with the treatment dose of 120 Gy. In the mutagenesis of M₂In the generation group, the rice is cultured for 2 weeks by using a rice nutrient solution disclosed in international rice and then placed in CdCl with the final concentration of 0.5uM₂And (3) carrying out medium stress treatment for 2 weeks, washing with pure water, drying roots, measuring cadmium content by ICP-MS, identifying a cadmium low accumulation mutant, and naming the mutant as the cadmium low accumulation mutant R001.

2. And (3) positioning of a cadmium low accumulation mutant R001 mutant gene.

Hybridizing the cadmium low-accumulation mutant R001 with a wild type Huahui 8612, randomly selecting 300 seedlings in an F2 population, and carrying out water culture on 0.5uM CdCl in a laboratory₂After 2 weeks of medium stress treatment, the content of cadmium in roots is detected by ICP-MS, the ratio of cadmium high-accumulation plants to cadmium low-accumulation plants in 300F 2 populations is close to 3: 1, and the cadmium low-accumulation character of the cadmium low-accumulation mutant R001 is preliminarily considered to be from recessive single gene mutation.

20 plants with low cadmium accumulation and 20 plants with high cadmium accumulation are selected from the F2 population, and a DNA sequencing mixed pool with high cadmium accumulation and low cadmium accumulation is respectively constructed to generate 33G second-generation sequencing data. Comparing reads to a Nippon reference genome through BWA software, carrying out mutation detection on samtools, filtering out index sites with QUAL value less than 30, MQ quality minimum value less than 30 and DP value less than 2 by using bcfttools software, then reserving index sites which are homozygous, amphipathic and different in the cadmium low accumulation DNA sequencing mixed pool, and calculating the index-index values of the cadmium high accumulation DNA sequencing mixed pool and the cadmium low accumulation DNA sequencing mixed pool to obtain the difference value delta index-index of the cadmium high accumulation DNA sequencing mixed pool and the cadmium low accumulation DNA sequencing mixed pool. And performing sliding window analysis by taking the physical distance 1M as a window and 10Kb as a step length to obtain a delta index-index mean value in each window. Through computer simulation experiments, a pre-value corresponding to each sequencing depth is obtained, after the pre-value is matched with each index locus one by one, the same sliding window analysis is also carried out, the average value and the pre-value of the delta index are respectively mapped (figure 2A), the distribution of the delta index-index values is observed, and the fact that the peak interval which is obviously higher than the pre-value line on the chromosome 7 is 7Mb to 10Mb is found.

Based on the variation information in this interval, we developed 8 pairs of molecular marker primers, statistically mapped the marker bands and phenotypes for the 8 pairs of molecular marker primers, and found that the final interval was 214kb between 8824201 and 9038461 (FIG. 2B).

3. Identification and confirmation of R001 mutant gene of cadmium low accumulation mutant

Analysis of the variation within the interval located in the above steps revealed only one Indel variation that falls within the gene and may affect gene function: 8874101-8874103 site. The deletion is located in the OsNramp5 gene and is the transcription cutting position of the No. 10 intron and the No. 11 exon.

Designing KASP typing primer according to the [ CTG/- ] deletion, wherein the primer sequence is as follows:

FAM 5’-GAAGGTGACCAAGTTCATGCTCATCCTGATGTCCAAGAAACCCT-3’；

HEX 5’-GAAGGTCGGAGTCAACGGATTCATCCTGATGTCCAAGAAACCCA-3’；

COMMON 5’-GATAAATCATCATCATGTCGCGTTG-3’。

the isolated population was genotyped with the KASP typing primer (see FIG. 3), 100 individuals were randomly selected from each of the three genotype individuals CT G/CTG, CTG/- - -, - - -/- - -, according to the typing results and planted in a heavily cadmium-contaminated field (soil cadmium concentration 1.5mg/kg, pH 5.7), and cultivation management was performed in an alternate dry and wet manner in order to accumulate cadmium as much as possible in rice. After the rice is completely ripe, the rice is harvested, threshed and milled into brown rice by single plant. By referring to a national standard determination method (GB/T5009.15-2003), the ICP-MS is used for determining the cadmium content in the brown rice, the result is shown in the following table 1, the KASP-Indel8874101-8874103 typing statistics on the cadmium content in grains are shown in the following table 2, and experiments show that the CTG deletion homozygous genotype is co-separated from the low cadmium accumulation phenotype in an isolated population.

Table 1: determination of cadmium content in brown rice with different genotypes

Table 2: KASP-Indel8874101-8874103 typing and counting the cadmium content of grains

The RNA of the cadmium low accumulation mutant R001 and the wild type Huahui 8612 is extracted by using Trizol (Invitrogen). Reverse transcription of RN A into cDNA, reaction system: 30ng of RNA is added into the culture medium,

All-in-One First-Strand cDNA Synthesis Supermix for qPCR (One-Step gDNA Remove): 4. mu.L, gDNA Remove: 1. mu.L, RNase-free Water: to total volume 20. mu.L; reaction procedure: 42 ℃ for 15min, 85 ℃ for 10 s. Using cDNA as a template, carrying out PCR amplification by using an OsNramp5 full-length primer, wherein the sequence of the primer is as follows:

F 5’-ATGGAGATTGAGAGAGAGAGCAGTG-3’；

R 5’-CTACCTTGGGAGCGGGATGT-3’。

sending the PCR product to Beijing Ongzhike Biotechnology limited company for Sanger sequencing, obtaining a sequencing result, comparing the cadmium low accumulation mutant R001 with the OsNramp5 transcription product of wild Huahui 8612, finding that R001 lacks 17bp in the number 11 exon region compared with the wild type (see figure 1), leading to subsequent amino acid frame shift, wherein the final amino acid sequence of OsNr amp5 of the mutant is shown as SEQ ID No. 2.

Example 2:

a detection kit for amplifying the rice cadmium low accumulation molecular marker in example 1 or for genotyping of the R001-OsNramp5 mutant gene of the rice in example 1 comprises the following primer pairs LC1-F and LC 1-R:

LC1-F：5’-CATCAGGTTCCGAAGCCACTTC-3’；

LC1-R：5’-GATAAATCATCATCATGTCGCGTTG-3’。

the detection kit also comprises 2 XPCR Master Mix (Thermo Scientific) and ddH₂O; the volume ratio of the 2X PCR Master Mix to the solution containing LC1-F/LC1-R is controlled to be 25: 1, ddH₂The volume ratio of O to the solution containing LC1-F/LC1-R is controlled at 23: 1.

The invention relates to an application of a detection kit in rapid molecular breeding and identification of low-cadmium-accumulation rice, wherein a receptor object is Huazhan, a donor is a low-cadmium-accumulation mutant R001 containing a rice R001-OsNramp5 mutant gene, and the detection kit specifically comprises the following steps:

(1) the Huazhan is used as a female parent, and is hybridized with a donor material cadmium low accumulation mutant R001 after artificial emasculation to obtain a hybrid F1 generation, the F1 generation is continuously selfed to obtain an F2 segregating population, or the F1 is then repeatedly crossed with another excellent variety line (such as R900) to obtain a triple-cross F1.

(2) 600 individual leaves are taken from the inbred F2 generation and the three-way F1 segregation population, and DNA is extracted by individual plants. The genome DNA of the detected rice is taken as a template, the detection kit is utilized to carry out PCR amplification, and the PCR reaction system is as follows: mu.L of 2 XPCR Master Mix, 0.2. mu.L of LC1-F (10. mu.M), 0.2. mu.L of LC1-R (10. mu.M) and 4.6. mu.L of ddH2O, totaling 10. mu.L. The PCR reaction program is: 3min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 10 s; 5min at 72 ℃.

(3) Performing polyacrylamide gel electrophoresis and silver staining and color development on the obtained amplification product, and performing genotype analysis on corresponding plants under a white light lamp, wherein if only one 100bp strip is contained, the obtained amplification product is a homozygous wild-type plant, and if the obtained amplification product contains two strips and heterozygous mutant genotype plants with the sizes of 100bp and 97bp respectively; plants containing only one 97bp band were identified as homozygous R001-OsNramp5 mutant genotypes (see figure 5).

(4) And carrying out comprehensive agronomic character evaluation on the plants with the 100bp, 97bp heterozygous strips and 97bp homozygous strips, and selecting excellent single plants for continuous selfing.

(5) And continuously selecting single plants with excellent comprehensive agronomic characters from selfing F3 generations or three-way progeny, taking leaves to extract DNA, carrying out genotyping by using the detection kit disclosed by the invention, and selecting selfed seeds of plants with 100bp and 97bp heterozygous strips and 97bp homozygous strips.

(6) And (5) repeating the step (5) until F5 generations, wherein the whole progeny population is stable in heredity, the comprehensive agronomic characters are uniform, and the typing results of the detection kit are all 97bp homozygous bands, so that the Huazhan low-bay improved line is obtained.

(7) The obtained Huazhan low-isolation improved line and wild Huazhan seeds are planted in a test field (the total cadmium in the rice field is about 1.0mg/Kg, and the pH value is 5.3) of a certain place in Hunan, and rice is taken after the rice is mature; husking rice, pulverizing brown rice, sieving with 100 mesh sieve, and adding HNO₃-HClO₄And digesting, and measuring the cadmium content in the brown rice by ICP-MS (inductively coupled plasma-mass spectrometry), wherein the cadmium content of the Huazhan low-cadmium modified brown rice is obviously reduced compared with that of the wild Huazhan.

Example 3:

breeding application of cadmium low accumulation mutant R001

The cadmium accumulation character improvement of the rice variety Huazhan by using the cadmium low accumulation mutant R001 of the embodiment 1 as a donor parent concretely comprises the following steps:

(1) hybridizing the cadmium low accumulation mutant R001 of example 1 with Huazhan to obtain F1;

(2) backcrossing the Huazhan as a recurrent parent with an F1 single plant to obtain BC1F 1;

(3) carrying out foreground selection on a BC1F1 population by using the KASP typing primer developed in the embodiment, selecting a Chr 78874101-8874103 CTG deletion genotype individual plant, then carrying out background selection, and selecting the individual plant with the highest similarity with the Huazhan genetic background to continue backcrossing with the recurrent parent Huazhan;

(4) continuing to select 3-4 generations through the foreground selection and the background selection according to the step (3) to obtain a Huazhan low-cadmium modified line which has the same genetic background as the Huazhan and has a Chr7:8874101-8874103CTG deletion genotype;

(5) the Huazhan improved line and wild Huazhan seed are planted in a test field (the total cadmium in the rice field is about 1.0mg/Kg, and the pH value is 5.3) of a certain place in Hunan, and rice is taken out after the rice is mature; husking rice, pulverizing brown rice, sieving with 100 mesh sieve, and adding HNO₃-HClO₄Digesting, and measuring the cadmium content in the brown rice by ICP-MS, the result is shown in figure 4, the cadmium content of the Huazhan low cadmium modified brown rice is obviously reduced compared with the wild Huazhan, and other agronomic characters have no obvious difference.

Sequence listing

<110> research center for hybrid rice in Hunan province

<120> rice cadmium low accumulation molecular marker, mutant gene, detection kit, rapid identification method and application thereof, and parting primer

<160> 7

<170> SIPOSequenceListing 1.0

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<211> 1600

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<213> Rice (Oryza sativa L.)

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tggaaaaggt tccttgccca tgttggtcct ggattcatgg tgtctttagc ctacttggat 180

cctggcaatt tggaaaccga tctgcaagcc ggagccaacc acagatatga gctgctctgg 240

gtgattctga ttggactcat cttcgcactt atcatacagt cgctagcagc taatcttgga 300

gtggttacag ggaggcatct ggctgagatc tgcaagagtg agtaccccaa gttcgtcaag 360

attttcctat ggctgctggc agagttggcc gtcatcgctg cagatatccc agaagttata 420

gggacggcct ttgctttcaa catattgttc catattccgg tgtgggtcgg cgtcctcatc 480

accggcacca gcactctact gcttcttggc ctccaaaaat acggggtgag gaagctggag 540

tttctgatat cgatgctggt gttcgtgatg gcggcgtgct tcttcgggga gctgagcatc 600

gtgaagccgc cggcgaagga ggtgatgaag gggctcttca tccccaggct caacggcgac 660

ggcgccaccg ccgacgccat tgccctcctc ggagctcttg tcatgcccca caatctgttc 720

ttgcattctg ccttggtgct atcgaggaag acaccggcat cagtcagagg aatcaaggac 780

gggtgcaggt tcttcctgta cgagagcggg ttcgcgctgt tcgtggcgct gctgataaac 840

atcgccgtcg tctccgtctc cggcaccgcc tgctcctccg ccaacctctc ccaagaggac 900

gccgacaagt gcgccaacct cagcctcgac acctcctcct tccttctcaa gaacgtgctg 960

ggcaagtcga gtgcgatcgt gtacggcgtg gcactgttgg catctgggca gagctccact 1020

attaccggca catacgctgg acagtacatc atgcaggatg aggaagtggc ttcggaacct 1080

gatgacaaga accatcgcca tcgcgccgag cctcatcgtc tccatcatcg gcggctccag 1140

gggcgccggc cgcctcatca tcatcgcttc gatgatactg tccttcgagc tgccgtttgc 1200

tctcatccct cttctcaagt tcagcagcag taagagcaag atggggcccc acaagaactc 1260

tatctatata atagtgttct cgtggttcct ggggctgctc atcatcggca tcaacatgta 1320

cttcctgagc acgagcttcg tcggctggct catccacaac gacctcccca agtacgccaa 1380

cgtgctcgtc ggcgccgccg tcttcccgtt catgctcgtc tacatcgtcg ccgtcgtcta 1440

cctcaccatc aggaaggact ccgtcgtcac cttcgtcgcc gactcctccc tcgccgccgt 1500

cgtcgacgcc gagaaggccg acgccggcga cctcgccgtc gacgacgacg agcccttgcc 1560

gtaccgcgac gacctggccg acatcccgct cccaaggtag 1600

<210> 2

<211> 410

<212> PRT

<213> Rice (Oryza sativa)

<400> 2

Met Glu Ile Glu Arg Glu Ser Ser Glu Arg Gly Ser Ile Ser Trp Arg

1 5 10 15

Ala Ser Ala Ala His Asp Gln Asp Ala Lys Lys Leu Asp Ala Asp Asp

20 25 30

Gln Leu Leu Met Lys Glu Pro Ala Trp Lys Arg Phe Leu Ala His Val

35 40 45

Gly Pro Gly Phe Met Val Ser Leu Ala Tyr Leu Asp Pro Gly Asn Leu

50 55 60

Glu Thr Asp Leu Gln Ala Gly Ala Asn His Arg Tyr Glu Leu Leu Trp

65 70 75 80

Val Ile Leu Ile Gly Leu Ile Phe Ala Leu Ile Ile Gln Ser Leu Ala

85 90 95

Ala Asn Leu Gly Val Val Thr Gly Arg His Leu Ala Glu Ile Cys Lys

100 105 110

Ser Glu Tyr Pro Lys Phe Val Lys Ile Phe Leu Trp Leu Leu Ala Glu

115 120 125

Leu Ala Val Ile Ala Ala Asp Ile Pro Glu Val Ile Gly Thr Ala Phe

130 135 140

Ala Phe Asn Ile Leu Phe His Ile Pro Val Trp Val Gly Val Leu Ile

145 150 155 160

Thr Gly Thr Ser Thr Leu Leu Leu Leu Gly Leu Gln Lys Tyr Gly Val

165 170 175

Arg Lys Leu Glu Phe Leu Ile Ser Met Leu Val Phe Val Met Ala Ala

180 185 190

Cys Phe Phe Gly Glu Leu Ser Ile Val Lys Pro Pro Ala Lys Glu Val

195 200 205

Met Lys Gly Leu Phe Ile Pro Arg Leu Asn Gly Asp Gly Ala Thr Ala

210 215 220

Asp Ala Ile Ala Leu Leu Gly Ala Leu Val Met Pro His Asn Leu Phe

225 230 235 240

Leu His Ser Ala Leu Val Leu Ser Arg Lys Thr Pro Ala Ser Val Arg

245 250 255

Gly Ile Lys Asp Gly Cys Arg Phe Phe Leu Tyr Glu Ser Gly Phe Ala

260 265 270

Leu Phe Val Ala Leu Leu Ile Asn Ile Ala Val Val Ser Val Ser Gly

275 280 285

Thr Ala Cys Ser Ser Ala Asn Leu Ser Gln Glu Asp Ala Asp Lys Cys

290 295 300

Ala Asn Leu Ser Leu Asp Thr Ser Ser Phe Leu Leu Lys Asn Val Leu

305 310 315 320

Gly Lys Ser Ser Ala Ile Val Tyr Gly Val Ala Leu Leu Ala Ser Gly

325 330 335

Gln Ser Ser Thr Ile Thr Gly Thr Tyr Ala Gly Gln Tyr Ile Met Gln

340 345 350

Asp Glu Glu Val Ala Ser Glu Pro Asp Asp Lys Asn His Arg His Arg

355 360 365

Ala Glu Pro His Arg Leu His His Arg Arg Leu Gln Gly Arg Arg Pro

370 375 380

Pro His His His Arg Phe Asp Asp Thr Val Leu Arg Ala Ala Val Cys

385 390 395 400

Ser His Pro Ser Ser Gln Val Gln Gln Gln

405 410

<210> 3

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gaaggtgacc aagttcatgc tcatcctgat gtccaagaaa ccct 44

<210> 4

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gaaggtcgga gtcaacggat tcatcctgat gtccaagaaa ccca 44

<210> 5

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gataaatcat catcatgtcg cgttg 25

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

catcaggttc cgaagccact tc 22

<210> 7

<211> 97

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 7

catcaggttc cgaagccact tcctcatcct gatgtccaag aaacccacat acatggatca 60

tatcatatat atcaacgcga catgatgatg atttatc 97

Claims

1. The molecular marker is characterized in that the molecular marker is an Indel marker, the Indel marker is obviously related to the low cadmium accumulation trait of rice grains, the nucleotide sequence takes the genome of Nipponbare rice as a reference genome, the nucleotide sequence comprises a section of base sequence positioned on an antisense chain of an interval of 8871643-8878905bp of a No.7 chromosome, and three bases of CTG are deleted at the position of 8874101-8874103 of the No.7 chromosome.

2. The rice cadmium low accumulation molecular marker as claimed in claim 1, wherein the deletion of three bases of CTG of the rice cadmium low accumulation molecular marker exists in an intron region of OsNramp5 gene.

3. The rice cadmium low accumulation molecular marker as claimed in claim 1, which is obtained by radiation mutagenesis of a created cadmium low accumulation mutant.

4. The rice cadmium low accumulation molecular marker as claimed in claim 1, 2 or 3, which comprises the nucleotide sequence shown as SEQ ID No. 7.

5. The rice cadmium low accumulation molecular marker as claimed in claim 4, which is obtained by amplification of the following primer pairs LC1-F and LC 1-R:

LC1-F：5’-CATCAGGTTCCGAAGCCACTTC-3’；

LC1-R：5’-GATAAATCATCATCATGTCGCGTTG-3’。

6. a rice R001-OsNramp5 mutant gene is characterized in that the sequence of the rice R001-OsNramp5 mutant gene takes the OsNramp5 gene sequence of the No.7 chromosome of Nipponbare rice as a basic sequence, and the basic sequence comprises the following base deletion segments, namely:

three CTG bases are deleted at the position of 8874101-8874103 at the end of the 10 th intron region of the OsNramp5 gene sequence;

7. A rice R001-OsNramp5 mutant gene is characterized in that the amino acid sequence coded by the rice R001-OsNramp5 mutant gene is shown as SEQ ID No. 2.

8. A detection kit for amplifying the rice cadmium low accumulation molecular marker as defined in any one of claims 1 to 5 or for genotyping of the rice R001-OsNramp5 mutant as defined in claim 6 or 7, wherein the detection kit comprises the following primer pairs LC1-F and LC 1-R:

LC1-F：5’-CATCAGGTTCCGAAGCCACTTC-3’；

LC1-R：5’-GATAAATCATCATCATGTCGCGTTG-3’。

9. the assay kit of claim 8, wherein the assay kit further comprises 2X PCR Master Mix (Thermo Scientific) and ddH₂O; the volume ratio of the 2X PCR Master Mix to the solution containing LC1-F/LC1-R is controlled to be 20-30: 1, and the ddH₂The volume ratio of O to the solution containing LC1-F/LC1-R is controlled to be 18-28: 1.

10. A method for rapidly identifying the rice R001-OsNramp5 mutant type gene typing by using the detection kit as claimed in claim 8 or 9, which is characterized by comprising the following steps:

(1) taking a cadmium low accumulation mutant R001 containing a rice R001-OsNramp5 mutant gene as a donor, hybridizing with non-cadmium low accumulation rice to obtain a hybrid F1 generation, continuously selfing the F1 generation to obtain an F2 segregation population or carrying out repeated crossing on the F1 generation and other rice materials to obtain a multiple-cross progeny segregation population;

(2) collecting single plant leaves from the F2 or the multiple-cross progeny segregation population, extracting DNA from the single plants, and performing amplification reaction by using the detection kit to obtain an amplification product;

(3) identifying the length of the amplification product through gel electrophoresis, and identifying a cadmium high-accumulation plant if the plant only contains a 100bp homozygous band or contains two bands simultaneously and the sizes of the heterozygous bands are 100bp and 97bp respectively; if only one 97bp homozygous band is contained, the plant is identified as a cadmium low accumulation plant.

11. The rapid identification method according to claim 10, wherein the amplification reaction in step (2) is carried out under the following conditions: 3min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 10 s; 5min at 72 ℃.

12. The application of the rice cadmium low accumulation molecular marker as claimed in any one of claims 1 to 5 or the rice R001-OsNramp5 mutant gene as claimed in any one of claims 6 to 7 in crop molecule assisted breeding or improvement of cadmium low accumulation traits of rice varieties.

13. Use according to claim 12, characterized in that it comprises the following steps:

14. The use of claim 13, wherein the conventional rice variety is a non-cadmium low accumulation rice variety, and in step (4), the selection of multiple generations by foreground selection and background selection is specifically 3-4 generations.

15. A KASP typing primer for identifying, screening or applying the rice cadmium low accumulation molecular marker as claimed in any one of claims 1 to 5 or the rice R001-OsNramp5 mutant gene as claimed in any one of claims 6 to 7, wherein the primer sequence of the KASP typing primer is as follows:

FAM 5’-GAAGGTGACCAAGTTCATGCTCATCCTGATGTCCAAGAAACCCT-3’；

HEX 5’-GAAGGTCGGAGTCAACGGATTCATCCTGATGTCCAAGAAACCCA-3’；

COMMON 5’-GATAAATCATCATCATGTCGCGTTG-3’。

16. use of the detection kit according to claim 8 or 9 for rapid breeding of low cadmium accumulating rice molecules, wherein the following steps are further performed after the rapid identification method according to claim 10:

(c) and (c) repeating the step (b) until the population inheritance is stable, the comprehensive agronomic characters are neat and consistent, and the typing results of the detection kit are all 97bp homozygous strip plants, so as to obtain a low-septate improved line.