CN113897451B - Rice cadmium low-accumulation molecular marker, mutant gene, detection kit, rapid identification method and application thereof, and typing primer - Google Patents

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

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CN113897451B
CN113897451B CN202111210318.9A CN202111210318A CN113897451B CN 113897451 B CN113897451 B CN 113897451B CN 202111210318 A CN202111210318 A CN 202111210318A CN 113897451 B CN113897451 B CN 113897451B
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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
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Abstract

The invention discloses a rice cadmium low accumulation molecular marker, the nucleotide sequence of which takes a genome of Japanese sunny rice as a reference genome, which comprises a deletion of three bases of CCA at a 8874042-8874040 position of chromosome 7, and finally a rice lcd100-OsNramp5 mutant gene is obtained, and a 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 can reduce the workload of cultivating the rice with low cadmium accumulation and shorten the cultivation period, and has the advantages of simplicity, rapidness, high flux and the like.

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, and a parting primer for identification.
Background
In the southern areas of China, rice is mainly used as main 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 danger to national food safety. At present, the problem of exceeding cadmium in rice can be relieved to a certain extent by a heavy metal soil restoration and cadmium pollution paddy field cultivation matching technology, but the rice is difficult to popularize in a large range due to high cost and multiple technical links. Therefore, the method for creating the rice with low cadmium accumulation and the new variety with low cadmium accumulation is the most economical and effective method for solving the problem of the cadmium rice in China.
The existing rice natural resources have very deficient low cadmium accumulation germplasm, and the gene editing technology can be utilized to efficiently and directionally create the low cadmium accumulation rice germplasm, but industrialization of gene editing products faces the problems of 'transgenic safety management' policy obstacle, foreign intellectual property barriers and the like, so that the method is difficult to popularize and apply in a short period. The non-transgenic means is utilized to create a novel rice germplasm with low cadmium accumulation and complete independent intellectual property rights, and is the most feasible way to thoroughly solve the problem of cadmium rice in China at the present stage.
Physical and chemical mutagenesis of crops is a traditional germplasm innovation technology, and mutagenesis can generate a large amount of genetic variation and plays an important role in crop improvement worldwide. According to incomplete statistics of the FAO/IAEA database, from 1930 to date, more than 60 countries worldwide release 3200 mutant varieties of 214 crop varieties, which make an important contribution to the world food safety. Therefore, the crop physical and chemical mutagenesis technology is a safe germplasm innovation mode, and the product has high acceptance in the masses and can be directly applied on a large scale. The novel rice germplasm with low cadmium accumulation and independent intellectual property rights in China is created by utilizing a physicochemical mutagenesis mode, is hopeful 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 by overcoming the defects and shortcomings in the background art, providing a rice cadmium low accumulation molecular marker and a rice lcd100-OsNramp5 mutant gene for assisting in selecting the rice with low cadmium accumulation, providing a detection kit and a corresponding rapid identification method for carrying out molecular breeding of the rice with low cadmium accumulation, and correspondingly providing an application of the rice lcd100-OsNramp5 mutant gene in crop molecular assisted breeding or improving the rice variety with low cadmium accumulation.
In order to solve the technical problems, the technical scheme provided by the invention is that the rice cadmium low accumulation molecular marker (LC 100) is a nucleotide sequence on a rice genome, the rice cadmium low accumulation molecular marker is an Indel marker, the Indel marker is obviously related to rice grain cadmium low accumulation characters, the nucleotide sequence takes a genome of Nipponbare rice as a reference genome, the nucleotide sequence comprises a section of base sequence (particularly preferably a sequence of an 11 th exon region of an OsNramp5 gene) on an antisense strand of a 8871643-8878905bp interval of a chromosome 7, and the base sequence has a deletion of three bases of CCA at a 8874042-8874040 position of the chromosome 7.
In the above rice cadmium low accumulation molecular marker, preferably, the deficiency of three bases of CCA of the rice cadmium low accumulation molecular marker exists in the 11 th exon region of the OsNramp5 gene. Our study shows that the deletion of CCA does not cause frame shift mutation of OsNramp5 gene, but only causes two amino acids 372Ala and 373Ile to be 372Val one amino acid.
Preferably, the rice cadmium low accumulation molecular marker is obtained by a cadmium low accumulation mutant (cadmium low accumulation mutant lcd 100) created by radiation mutagenesis.
Preferably, the rice cadmium low accumulation molecular marker comprises a nucleotide sequence shown as SEQ ID No. 7.
Preferably, the rice cadmium low accumulation molecular marker is obtained by amplifying the following primer pairs LC100-F and LC100-R:
LC100-F:5'-CGAAGCGATGATGATGAGGCG-3'; (see SEQ ID No. 6)
LC100-R:5'-GGTTTCTTGGACATCAGGATGAGG-3'. (see SEQ ID No. 8)
As a general technical concept, the invention also provides a rice lcd100-OsNramp5 mutant gene, wherein the sequence of the rice lcd100-OsNramp5 mutant gene takes the OsNramp5 gene sequence of a chromosome 7 of Nipponbare rice as a basic sequence, and the basic sequence comprises the following base deletion segments, namely:
there is a CCA three base deletion at position 8874042-8874040 of exon 11 of the above OsNramp5 gene sequence (physical position is IRGSP-1.0 in Japanese sunny reference genome, sequence version number is https:// rapdb. Dna. Affrc. Go. Jp/download/IRGSP1.Html, on antisense strand of 8871643-8878905bp interval of chromosome 7);
the CDS region of the rice lcd100-OsNramp5 mutant gene comprises a nucleotide sequence shown as SEQ ID No. 1.
As a general technical concept, the invention also provides the rice lcd100-OsNramp5 mutant gene, and the amino acid sequence coded by the rice lcd100-OsNramp5 mutant gene is shown as SEQ ID No. 2.
The rice lcd100-OsNramp5 mutant gene is positioned in the 11 th exon region of the OsNramp5 gene (refer to figure 1), and the deletion of CCA leads to two amino acids 372Ala and 373Ile of the gene to be 372Val one amino acid at the 11 th exon after the transcription of the OsNramp 5.
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 lcd100-OsNramp5 mutant, wherein the detection kit comprises the following primer pairs of LC100-F and LC100-R:
LC100-F：5’-CGAAGCGATGATGATGAGGCG-3’；
LC100-R：5’-GGTTTCTTGGACATCAGGATGAGG-3’。
the above detection kit preferably further comprises 2X PCR Master Mix (Thermo Scientific) and ddH 2 O; the volume ratio of the 2X PCR Master Mix to the solution containing LC100-F/LC100-R is controlled to be 20-30:1, and the ddH is controlled to be the same as the solution containing LC100-F/LC100-R 2 The volume ratio of O to the solution containing LC100-F/LC100-R is controlled at 18-28:1.
The detection kit described above, a particularly preferred detection configuration system is: 5. Mu.L 2 XPCR Master Mix, 0.2. Mu.L LC100-F (10. Mu.M), 0.2. Mu.L LC100-R (10. Mu.M) and 4.6. Mu.L ddH 2 O。
As a general technical concept, the invention also provides a rapid identification method for genotyping the rice lcd100-OsNramp5 mutant by using the detection kit, which comprises the following steps:
(1) Taking a cadmium low accumulation mutant lcd100 of a mutant gene of oryza sativa lcd100-OsNramp5 as a donor, hybridizing with non-cadmium low accumulation oryza sativa to obtain a hybrid F1, performing F1 generation selfing to obtain an F2 separation population, or performing double crossing of F1 and other oryza sativa materials to obtain a double crossing offspring separation population;
(2) Collecting single plant leaves in the selfing F2 or double-crossing offspring separation group, separating single plants to extract DNA, and performing amplification reaction by using the detection kit to obtain an amplification product;
(3) Identifying the length of the amplification product by gel electrophoresis, and identifying the amplification product as a cadmium high accumulation plant if the amplification product contains only one 132bp band or two bands, and the size of the amplification product is 132bp and 129 bp; if only one 129bp band was contained, it was identified as a cadmium low accumulating plant.
Preferably, the conditions of the amplification reaction in step (2) are as follows: 3min at 95 ℃;95 ℃ for 30s,60 ℃ for 30s,72 ℃ for 10s,30 cycles; and at 72℃for 5min.
As a general technical concept, the invention also provides an application of the detection kit in rapid rice molecular breeding with low cadmium accumulation, which is characterized in that the following operation steps are further carried out after the rapid identification method of the invention:
(a) Carrying out comprehensive agronomic character evaluation on heterozygous strip plants in the obtained high-cadmium accumulation plants and homozygous strip plants in the low-cadmium accumulation plants, and selecting excellent single plants to carry out selfing continuously;
(b) Continuously selecting single plants with good comprehensive agronomic characters in the selfing F3 generation or the double-crossing offspring, taking leaf extraction DNA, carrying out genotyping by using the detection kit, and selecting seeds of which the heterozygous stripe plants of 132bp and 129bp are selfed with the homozygous stripe plants of 129 bp;
(c) Repeating the step (b) until the population genetic stability (generally preferably at least to F5 generation), the comprehensive agronomic characters are orderly and consistent, and the typing results of the detection kit are 129bp homozygous strip plants, so as to obtain the low-isolation improved line.
As a general technical conception, the invention also provides application of the rice lcd100-OsNramp5 mutant gene in crop molecular assisted breeding or improvement of cadmium low accumulation properties of rice varieties.
The application of the above-mentioned rice variety to the improvement of the cadmium low accumulation trait preferably comprises the following steps:
(1) The method comprises the steps of hybridizing a cadmium low accumulation mutant lcd100 containing a rice lcd100-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) Performing prospect selection on the BC1F1 population by using KASP typing primers, and selecting Chu 7:8874042-8874040CCA deleted genotype individuals; then, selecting the background, and selecting the single plant with the highest similarity with the genetic background of the conventional rice variety to continuously backcross with recurrent parent;
(4) And (3) continuing to select multiple generations through foreground selection and background selection according to the step (3) to obtain a low-cadmium improved line with genetic background consistent with the conventional rice variety and Chr7:8874042-8874040CCA deletion genotype.
In the above application, preferably, the conventional rice variety is a non-cadmium low-accumulation rice variety, and in the step (4), the foreground selection and the background selection are carried out for multiple generations, specifically 3-4 generations.
As a general technical idea, the present invention also provides a KASP typing primer for identifying, screening or applying the above rice lcd100-OsNramp5 mutant gene, the primer sequence of the KASP typing primer being (see SEQ ID No. 3-5):
FAM5’-GAAGGTGACCAAGTTCATGCTCCTGATGACAAGAACCATCGCCA-3’；
HEX5’-GAAGGTCGGAGTCAACGGATTCCTGATGACAAGAACCATCGTCG-3’；
COMMON5’-ATGCTGACCGAAGCGATGATGA-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 utilizes a 3bp base sequence deleted by mutant lcd100 to design a specific length polymorphism molecular marker for rapidly identifying the low-cadmium-accumulation rice, reduces the workload of cultivating the low-cadmium-accumulation rice and shortens the cultivation period, and provides technical support for simply, rapidly and highly flux application of a molecular marker assisted breeding technology to improve the low-cadmium-accumulation property;
2) The invention provides a detection kit for rice genotyping, which can directionally improve the cadmium low accumulation character of rice with a lcd100 mutant as a donor and provides a stable, efficient and low-cost molecular auxiliary breeding tool;
3) Based on the identification and screening of the invention, we creatively obtain a rice lcd100-OsNramp5 mutant gene, the mutant gene does not shift the frame of the translated amino acid, only has two amino acid differences with the wild type, and two amino acids 372Ala and 373Ile are found for the first time to be important sites of proteins involved in cadmium accumulation of the OsNramp5 protein, and the original structure of the wild type protein is maintained while the mutant type of the low-cadmium accumulation protein is obtained. Meanwhile, the application of the lcd100-OsNramp5 mutant gene of the rice in crop molecular assisted breeding and in breeding, preparing and improving rice varieties with low cadmium absorption phenotype is provided, the obvious technical effect is achieved, and finally the rice variety improvement line with low cadmium absorption phenotype is obtained;
4) The KASP typing primer for detecting, screening or applying the rice lcd100-OsNramp5 mutant gene can be used for carrying out accurate typing on indels found 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 cost of identification time, but also facilitates the improvement of crops and the germplasm innovation of low-cadmium accumulation varieties, and has remarkable 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 that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram showing comparison of mutation sites, mutation types and transcribed base sequences of a mutant gene of lcd100-OsNramp5 of rice.
FIG. 2 is a map of the localization of the mutant lcd100 cadmium low accumulation gene in the examples of the present invention. Wherein a is a BSA localization map; b is a schematic diagram of fine positioning by using marks.
FIG. 3 is a distribution plot of the distribution points of genotyping of the isolated populations using KASP typing primers in an embodiment of the present invention; wherein: the upper left scattered point is a non-mutation pure line, the middle scattered point is a CCA deletion mutation and non-mutation heterozygous line, the lower left scattered point is NTC, and the lower right scattered point is a CCA deletion mutation pure line.
FIG. 4 is a graph showing the comparative measurement results of the cadmium content of the brown rice of the China-occupied low-cadmium modified system compared with the wild type China-occupied brown rice in the embodiment of the invention.
FIG. 5 is a photograph showing an amplified product of the present invention after polyacrylamide gel electrophoresis and silver staining.
Detailed Description
The present invention will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown, for the purpose of illustrating the invention, but the scope of the invention is not limited to the specific embodiments shown.
Unless defined otherwise, all technical and scientific terms 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 be limiting of the scope of the present invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
Example 1:
the rice cadmium low accumulation molecular marker is a segment of intron nucleotide sequence on a rice genome, is Indel marker, is obviously related to rice grain cadmium low accumulation characters, takes a genome of Nipponbare rice as a reference genome, comprises a segment of base sequence on an 11 th exon on an antisense strand of a 8871643-8878905bp interval of a No.7 chromosome, and has a deletion of three bases of CCA at a 8874042-8874040 position of the No.7 chromosome in the base sequence.
The deletion of three bases of CCA of the rice cadmium low accumulation molecular marker exists in an exon region of an OsNramp5 gene, the deletion is obviously related to the cadmium content of rice seeds, and in a separation population hybridized with a rice cadmium low accumulation mutant lcd100, the cadmium content of rice seeds with Indel (Chr 7: 8874042-8874040) loci CCA (+/+, +/-) is obviously higher than that of a rice single plant with homozygous deletion CCA (-/-) 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 a cadmium low accumulation mutant created by radiation mutagenesis. The mutant contains the rice lcd100-OsNramp5 mutant gene, the sequence of the rice lcd100-OsNramp5 mutant gene takes the OsNramp5 gene sequence of a Japanese sunny rice chromosome 7 as a basic sequence, and the basic sequence contains the following base deletion segments, namely: on the antisense strand of the 8871643-8878905bp interval of chromosome 7 (physical position uses Japanese sunny as reference genome, sequence version number is IRGSP-1.0, database website is https:// rapdb. Dna. Affrc. Go. Jp/download/IRGSP1. Html), there are CCA three base deletions at 8874042-8874040 of exon 11 of the above-mentioned OsNramp5 gene sequence.
The CDS region of the rice lcd100-OsNramp5 mutant gene of this example contains the nucleotide sequence shown in SEQ ID No. 1.
As 3 bases are deleted in the 11 th exon region of the OsNramp5 mutant gene of the rice lcd100-OsNramp5 gene (see figure 1), the deletion of three bases of CCA leads to two amino acids 372Ala and 373Ile to be changed into 372Val, and the amino acid sequence coded by the rice lcd100-OsNramp5 mutant gene of the embodiment is shown as SEQ ID No. 2.
The rice lcd100-OsNramp5 mutant gene of the embodiment is specifically obtained by screening and identifying by the following method:
1. obtaining a rice cadmium low accumulation mutant lcd100.
Using high-energy heavy ion beam 12 C6+) irradiation treatment of high-quality rice strain 44-5 with a treatment dose of 120Gy. In mutagenesis M 2 In the generation group, the rice nutrient solution disclosed by International Rice is used for culturing for 2 weeks and then is placed in the final concentration of 0.5uMCdCl 2 After medium stress treatment for 2 weeks, cleaning with pure water, taking roots, drying, measuring the cadmium content by ICP-MS, identifying a cadmium low accumulation mutant, and naming the cadmium low accumulation mutant as a cadmium low accumulation mutant lcd100.
2. Localization of the cadmium low accumulation mutant lcd100 mutant gene.
Hybridization of cadmium Low accumulation mutant lcd100 with wild type 44-5, random selection of 300 seedlings in F2 population, laboratory hydroponic 0.5uMCdCl 2 After medium stress treatment for 2 weeks, detecting the cadmium content in roots by utilizing ICP-MS, wherein the ratio of a high cadmium accumulation plant to a low cadmium accumulation plant in 300F 2 groups is close to 3:1, and preliminarily considering that the low cadmium accumulation character of the low cadmium accumulation mutant lcd100 is from recessive single gene mutation.
And selecting 20 plants of the cadmium low accumulation plants and 20 plants of the cadmium high accumulation plants from the F2 population, and respectively constructing a cadmium high/low accumulation DNA sequencing mixed pool to generate 34G second-generation sequencing data. And comparing reads to a Japanese reference genome by BWA software, carrying out mutation detection by samtools, filtering out indel sites with a QUAL value smaller than 30, an MQ quality minimum smaller than 30 and a DP value smaller than 2 by using bcftools software, then reserving homozygous, amphiphilically homozygous and different indel sites in a cadmium low accumulation DNA sequencing mixed pool, and calculating the indel-index values of the cadmium high accumulation DNA sequencing mixed pool and the cadmium low accumulation DNA sequencing mixed pool to obtain the difference delta indel-index of the cadmium high accumulation DNA sequencing mixed pool and the cadmium low accumulation DNA sequencing mixed pool. And carrying out sliding window analysis by taking the physical distance 1M as a window and taking 10Kb as a step length to obtain a delta index-index mean value in each window. Through computer simulation experiments, a preset value corresponding to each sequencing depth is obtained, after the preset value is matched with each index site one by one, the same sliding window analysis is also carried out, the delta index-index mean value and the preset value are respectively plotted (figure 2A), the delta index-index value distribution is observed, and the fact that a peak interval which is obviously higher than a preset value line is 7Mb to10Mb is found on chromosome 7.
Based on the variation information of this interval, we developed 8 pairs of molecular marker primers, statistics of the marker band pattern and phenotype corresponding to 8 pairs of molecular marker primers, statistically mapping the recombinant individuals, and found that the last interval was located at 260kb between 8755509 and 9022510 (FIG. 2B).
3. Identification and confirmation of cadmium low accumulation mutant lcd100 mutant gene
Analysis of the variation in the interval located in the above step revealed that there was only one Indel variation that falls within the gene and may affect the function of the gene: 8874042-8874040 position CCA was deleted. The deletion is located within the OsNramp5 gene, within exon 11, and does not cause a frame shift mutation in the protein.
Designing KASP typing primer according to the [ CCA/- ] deletion, wherein the primer sequence is as follows:
FAM5’-GAAGGTGACCAAGTTCATGCTCCTGATGACAAGAACCATCGCCA-3’；
HEX5’-GAAGGTCGGAGTCAACGGATTCCTGATGACAAGAACCATCGTCG-3’；
COMMON5’-GATAAATCATCATCATGTCGCGTTG-3’。
genotyping of the isolate was performed using the KASP typing primer, 10. Mu.L KASP PCR amplification system: KASP M later mix (LGC Co., KBS-1016-016) 5. Mu.L, FAM-Primer (10. Mu.M) 0.15. Mu.L, HEX-Primer (10. Mu.M) 0.15. Mu.L, COMMON-Primer (10. Mu.M) 0.3. Mu.L, template DNA (50 ng/. Mu.L to 100 ng/. Mu.L) 1. Mu.L, ddH 2 O2.4. Mu.L. The prepared KASP reaction system was put into Roche LightCycler and subjected to PCR as follows: pre-denaturation at 94℃for 15min; denaturation at 94 ℃,20s, annealing at 65 ℃,1min, 10 cycles of-0.8 ℃ per cycle; denaturation at 94 ℃,20s, annealing at 57 ℃,1min, 30 cycles total; signal acquisition at 37℃for 1min. The typing results are shown in FIG. 3, which, in combination with the phenotypic results, found that the CCA-deleted homozygous genotype was fully linked to the low cadmium accumulation phenotype in the segregating population.
According to the typing result, 100 plants are randomly extracted from each of the three genotypes of CCA/CCA, CCA/- - - - - - - - - - - - - - - - - - - - - - -, and planted in a heavily cadmium-polluted field (the concentration of cadmium in the soil is 1.5mg/kg, and the pH is 5.7), and in order to enable the rice to accumulate as much cadmium as possible, cultivation and management are carried out in a dry-wet alternating mode. After the rice is fully mature, the rice is singly separated for seed collection, threshing and grinding of brown rice. Referring to the national standard measurement method (GB/T5009.15-2003), the cadmium content in brown rice was measured by ICP-MS, the results are shown in the following Table 1, KASP-Indel8874042-8874040 typing statistics seed cadmium content is shown in the following Table 2, and experiments find that the CCA deletion homozygous genotype is co-separated from the low cadmium accumulation phenotype in the segregating population.
Table 1: determination of cadmium content in brown rice of different genotypes
Table 2: KASP-Indel8874042-8874040 typing statistics of cadmium content in seeds
And Trizol (Invitrogen) is utilized to extract RNA of the cadmium low accumulation mutant lcd100 and wild type Huahui 8612. Reverse transcription of RNA into cDNA, reaction system: 30ng of RNA was used to prepare the DNA,All-in-One First-Strand cDNA Syn thesis SuperMix for qPCR (One-Step gDNA Removal) 4. Mu.L, gDNA Remove 1. Mu.L, RNas e-free Water to total volume 20. Mu.L; the reaction procedure: 15min at 42℃and 10s at 85 ℃. PCR amplification was performed using cDNA as a template and OsNramp5 full-length primers, which had the following sequences:
F5’-ATGGAGATTGAGAGAGAGAGCAGTG-3’；
R5’-CTACCTTGGGAGCGGGATGT-3’。
after the PCR product was sent to Sanger sequencing, obtained as a result of sequencing, the OsNramp5 transcript of the cadmium low accumulation mutant lcd100 and the wild type 44-5 was compared, and it was found that lcd100 lacked 3bp in the 11 th exon region (see FIG. 1), two amino acids 372Ala and 373Ile were changed to 372Val, and the amino acid sequence of the mutant final OsNramp5 was SEQ2.
Example 2:
a detection kit for amplifying the rice cadmium low accumulation molecular marker of example 1 or for genotyping the rice lcd100-OsNramp5 mutant of example 1, the detection kit comprising the following primer pairs LC100-F and LC100-R:
LC100-F：5’-CGAAGCGATGATGATGAGGCG-3’；
LC100-R：5’-GGTTTCTTGGACATCAGGATGAGG-3’。
the detection kit also comprises 2X PCR Master Mix (Thermo Scientific) and ddH 2 O; the volume ratio of 2X PCR Master Mix to the LC100-F/LC100-R containing solution is controlled at 25:1, ddH 2 The volume ratio of O to the LC100-F/LC100-R containing solution was controlled at 23:1. The configuration system is as follows: 5. Mu.L 2 XPCR Master Mix, 0.2. Mu.L LC100-F (10. Mu.M), 0.2. Mu.L LC100-R (10. Mu.M) and 4.6. Mu.L ddH 2 O。
The application of the detection kit in rapid breeding and identification of rice molecules with low cadmium accumulation, wherein the acceptor object is Huazhan, and the donor is a mutant lcd100 with low cadmium accumulation of a mutant gene of oryza sativa lcd100-OsNramp5, and the detection kit specifically comprises the following steps:
(1) Taking Huazhan as a female parent, hybridizing with a donor material cadmium low accumulation mutant lcd100 after artificial emasculation to obtain a hybrid F1 generation, and carrying out selfing on the F1 generation to obtain an F2 separation population or carrying out double cross on the F1 and another good variety strain (such as R900) to obtain a triple cross F1.
(2) 600 single plant leaves are taken from the selfing F2 generation and the three-way F1 separation group, and DNA is extracted from the single plant. The genome DNA of the detected rice is used as a template, the detection kit is used for PCR amplification, and a PCR reaction system is as follows: mu.L 2 XPCR Master Mix, 0.2. Mu.L LC100-F (10. Mu.M), 0.2. Mu.L LC100-R (10. Mu.M) and 4.6. Mu.L ddH2O, the total system was 10. Mu.L. The PCR reaction procedure was: 3min at 95 ℃;95 ℃ for 30s,60 ℃ for 30s,72 ℃ for 10s,30 cycles; and at 72℃for 5min.
(3) Carrying out polyacrylamide gel electrophoresis and silver staining color development on the obtained amplification product, carrying out genotype analysis on the corresponding plant under a white light lamp, and if the amplification product contains only one 132bp band and is a homozygous wild type plant, simultaneously containing two bands and heterozygous mutant genotype plants with the sizes of 132bp and 129bp respectively; if only one 129bp band was contained, homozygous lcd100-OsNramp5 mutant genotype plants were identified (see FIG. 5).
(4) And (3) carrying out comprehensive agronomic trait evaluation on plants with the 132bp heterozygous bands, the 129bp heterozygous bands and the 129bp homozygous bands, and selecting excellent single plants to carry out selfing continuously.
(5) And continuously selecting single plants with good comprehensive agronomic characters in the selfing F3 generation or the three-way cross generation, taking leaf extraction DNA, carrying out genotyping by using the detection kit, and selecting seeds of which the heterozygous bands of 132bp and 129bp and the homozygous band plants of 129bp are selfed.
(6) And (3) repeating the step (5) until the F5 generation, wherein the whole offspring population is genetically stable, the comprehensive agronomic characters are orderly and consistent, and the parting results of the detection kit are all 129bp homozygous bands, so as to obtain the Huazhan low-isolation improved line.
(7) The obtained modified Huazhan low-isolation line and wild Huazhan are planted in a test field (about 1.0mg/Kg of total cadmium in paddy field and pH=5.3) in a certain place in Hunan, and paddy is taken after the paddy is ripe; husking rice, pulverizing brown rice, sieving with 100 mesh sieve, and collecting HNO 3 -HClO 4 Digestion is carried out, the cadmium content in the brown rice is measured by ICP-MS, and compared with the wild type brown rice, the cadmium content in the brown rice of the Huazhan low-cadmium modified system is obviously reduced.
Example 3:
breeding application of cadmium low accumulation mutant lcd100
The cadmium accumulation property of the rice variety Huazhan is improved by using the cadmium low accumulation mutant lcd100 of the embodiment 1 as a donor parent, which comprises the following steps:
(1) Hybridization of cadmium low accumulation mutant lcd100 of example 1 with Huazhan to obtain F1;
(2) Backcrossing the Huazhan as recurrent parent with the F1 single plant to obtain BC1F1;
(3) Performing foreground selection on the BC1F1 population by using the KASP typing primer developed in the embodiment, selecting the Chr7 8874042-8874040CCA deletion genotype single plant, performing background selection, and selecting the single plant with the highest similarity with the Huazhan genetic background to continuously backcross with the recurrent parent Huazhan;
(4) Selecting 3-4 generations through foreground selection and background selection continuously according to the step (3), so as to obtain a Huazhan low-cadmium improved system with genetic background consistent with Huazhan and Chr7:8874042-8874040CCA deletion genotype;
(5) The modified Huazhan low-isolation line and the wild Huazhan are planted in a test field (about 1.0mg/Kg of total cadmium in rice field and pH=5.3) in a certain place in Hunan, and the rice is taken after the rice is ripe; husking rice, pulverizing brown rice, sieving with 100 mesh sieve, and collecting HNO 3 -HClO 4 Digestion is carried out, and the cadmium content in brown rice is measured by ICP-MS, so that the cadmium content in brown rice of the Huazhan low-cadmium modified system is obviously reduced compared with that of wild Huazhan brown rice, and other agronomic traits are not obviously different as shown in a figure 4.
Sequence listing
<110> Hunan hybrid Rice research center
<120> a rice cadmium low accumulation molecular marker, mutant gene, detection kit, rapid identification method and application thereof, and parting primer
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1614
<212> DNA
<213> Rice (Oryza sativa L.)
<400> 1
atggagattg agagagagag cagtgagaga gggagcatca gctggagagc tagtgcggca 60
catgatcaag atgccaagaa gctcgacgca gatgatcagc tgctaatgaa ggagcctgca 120
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 gtatggcgtg gcactgttgg catctgggca gagctccact 1020
attaccggca catacgctgg acagtacatc atgcagggtt tcttggacat caggatgagg 1080
aagtggcttc ggaacctgat gacaagaacc atcgtcgcgc cgagcctcat cgtctccatc 1140
atcggcggct ccaggggcgc cggccgcctc atcatcatcg cttcgatgat actgtccttc 1200
gagctgccgt ttgctctcat ccctcttctc aagttcagca gcagtaagag caagatgggg 1260
ccccacaaga actctatcta tataatagtg ttctcgtggt tcctggggct gctcatcatc 1320
ggcatcaaca tgtacttcct gagcacgagc ttcgtcggct ggctcatcca caacgacctc 1380
cccaagtacg ccaacgtgct cgtcggcgcc gccgtcttcc cgttcatgct cgtctacatc 1440
gtcgccgtcg tctacctcac catcaggaag gactccgtcg tcaccttcgt cgccgactcc 1500
tccctcgccg ccgtcgtcga cgccgagaag gccgacgccg gcgacctcgc cgtcgacgac 1560
gacgagccct tgccgtaccg cgacgacctg gccgacatcc cgctcccaag gtag 1614
<210> 2
<211> 537
<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
Gly Phe Leu Asp Ile Arg Met Arg Lys Trp Leu Arg Asn Leu Met Thr
355 360 365
Arg Thr Ile Val Ala Pro Ser Leu Ile Val Ser Ile Ile Gly Gly Ser
370 375 380
Arg Gly Ala Gly Arg Leu Ile Ile Ile Ala Ser Met Ile Leu Ser Phe
385 390 395 400
Glu Leu Pro Phe Ala Leu Ile Pro Leu Leu Lys Phe Ser Ser Ser Lys
405 410 415
Ser Lys Met Gly Pro His Lys Asn Ser Ile Tyr Ile Ile Val Phe Ser
420 425 430
Trp Phe Leu Gly Leu Leu Ile Ile Gly Ile Asn Met Tyr Phe Leu Ser
435 440 445
Thr Ser Phe Val Gly Trp Leu Ile His Asn Asp Leu Pro Lys Tyr Ala
450 455 460
Asn Val Leu Val Gly Ala Ala Val Phe Pro Phe Met Leu Val Tyr Ile
465 470 475 480
Val Ala Val Val Tyr Leu Thr Ile Arg Lys Asp Ser Val Val Thr Phe
485 490 495
Val Ala Asp Ser Ser Leu Ala Ala Val Val Asp Ala Glu Lys Ala Asp
500 505 510
Ala Gly Asp Leu Ala Val Asp Asp Asp Glu Pro Leu Pro Tyr Arg Asp
515 520 525
Asp Leu Ala Asp Ile Pro Leu Pro Arg
530 535
<210> 3
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
gaaggtgacc aagttcatgc tcctgatgac aagaaccatc gcca 44
<210> 4
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
gaaggtcgga gtcaacggat tcctgatgac aagaaccatc gtcg 44
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
cgaagcgatg atgatgaggc g 21
<210> 7
<211> 132
<212> DNA
<213> Rice (Oryza sativa L.)
<400> 7
cgaagcgatg atgatgaggc ggccggcgcc cctggagccg ccgatgatgg agacgatgag 60
gctcggcgcg atggcgatgg ttcttgtcat caggttccga agccacttcc tcatcctgat 120
gtccaagaaa cc 132
<210> 8
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
ggtttcttgg acatcaggat gagg 24

Claims (9)

1. The rice lcd100-OsNramp5 mutant gene is characterized in that the amino acid sequence encoded by the rice lcd100-OsNramp5 mutant gene is shown as SEQ ID No. 2.
2. A rapid identification method for typing of a rice lcd100-OsNramp5 mutant gene by using a detection kit is characterized by comprising the following steps of:
(1) Taking a cadmium low accumulation mutant lcd100 containing a rice lcd100-OsNramp5 mutant gene as a donor, hybridizing with non-cadmium low accumulation rice to obtain a hybrid F1 generation, and carrying out continuous selfing on the F1 generation to obtain an F2 segregation population or carrying out double crossing on the F1 generation and other rice materials to obtain a double crossing offspring segregation population;
(2) Collecting single plant leaves in the F2 or the double crossing offspring separation group, separating single plants to extract DNA, and performing amplification reaction by using the detection kit to obtain an amplification product;
(3) Identifying the length of the amplification product by gel electrophoresis, and identifying the amplification product as a cadmium high accumulation plant if the amplification product contains only one homozygous band of 132bp or contains two heterozygous bands of 132bp and 129bp in size; if only one homozygous band of 129bp is contained, the strain is identified as a cadmium low accumulation plant;
the detection kit comprises the following primer pairs LC100-F and LC100-R:
LC100-F：5’-CGAAGCGATGATGATGAGGCG-3’；
LC100-R：5’-GGTTTCTTGGACATCAGGATGAGG-3’。
3. the rapid identification method according to claim 2, which comprisesCharacterized in that the detection kit also comprises 2X PCRMaster Mix (Thermo Scientific) and ddH 2 O; the volume ratio of the 2X PCR Master Mix to the solution containing LC100-F/LC100-R is controlled to be 20-30:1, and the ddH is controlled to be the same as the solution containing LC100-F/LC100-R 2 The volume ratio of O to the solution containing LC100-F/LC100-R is controlled at 18-28:1.
4. The rapid identification method according to claim 2, wherein the conditions of the amplification reaction in step (2) are: 3min at 95 ℃;95 ℃ for 30s,60 ℃ for 30s,72 ℃ for 10s,30 cycles; and at 72℃for 5min.
5. The application of the lcd100-OsNramp5 mutant gene of the rice in the improvement of the cadmium low accumulation property of rice varieties is characterized in that:
the amino acid sequence of the rice lcd100-OsNramp5 mutant gene is shown as SEQ ID No. 2.
6. The use according to claim 5, characterized by the steps of:
(1) The method comprises the steps of hybridizing a cadmium low accumulation mutant lcd100 containing a rice lcd100-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) Performing prospect selection on the BC1F1 population by using KASP typing primers, and selecting Chu 7:8874042-8874040CCA deleted genotype individuals; then, selecting the background, and selecting the single plant with the highest similarity with the genetic background of the conventional rice variety to continuously backcross with recurrent parent;
the primer sequence of the KASP typing primer is as follows:
FAM 5’-GAAGGTGACCAAGTTCATGCTCCTGATGACAAGAACCATCGCCA-3’；
HEX 5’-GAAGGTCGGAGTCAACGGATTCCTGATGACAAGAACCATCGTCG-3’；
COMMON 5’-ATGCTGACCGAAGCGATGATGA-3’；
(4) And (3) continuing to select 3-4 generations through foreground selection and background selection according to the step (3), so as to obtain a low-cadmium improved line with genetic background consistent with the conventional rice variety and Chr7:8874042-8874040CCA deletion genotype.
7. A KASP typing primer for identifying or screening a rice lcd100-OsNramp5 mutant gene according to claim 1, wherein the primer sequence of the KASP typing primer is:
FAM 5’-GAAGGTGACCAAGTTCATGCTCCTGATGACAAGAACCATCGCCA-3’；
HEX 5’-GAAGGTCGGAGTCAACGGATTCCTGATGACAAGAACCATCGTCG-3’；
COMMON 5’-ATGCTGACCGAAGCGATGATGA-3’。
8. use of a detection kit for rapid breeding of low cadmium accumulation rice molecules, characterized in that the following steps are continued after the rapid identification method according to claim 2:
(a) Carrying out comprehensive agronomic character evaluation on heterozygous strip plants in the obtained high-cadmium accumulation plants and homozygous strip plants in the low-cadmium accumulation plants, and selecting excellent single plants to carry out selfing continuously;
(b) Continuously selecting single plants with good comprehensive agronomic characters in the selfing F3 generation or the double-crossing offspring, taking leaf extraction DNA, carrying out genotyping by using the detection kit, and selecting seeds of which the heterozygous stripe plants of 132bp and 129bp are selfed with the homozygous stripe plants of 129 bp;
(c) Repeating the step (b) until the population genetic stability and the comprehensive agronomic characters are orderly and consistent, and the typing results of the detection kit are 129bp homozygous strip plants, so as to obtain a low-cadmium improved line;
the detection kit comprises the following primer pairs LC100-F and LC100-R:
LC100-F：5’-CGAAGCGATGATGATGAGGCG-3’；
LC100-R：5’-GGTTTCTTGGACATCAGGATGAGG-3’。
9. the use according to claim 8, wherein the detection kit further comprises a 2X PCR Master Mix (Thermo Scienteric) and ddH 2 O; the volume ratio of the 2X PCR Master Mix to the solution containing LC100-F/LC100-R is controlled to be 20-30:1, and the ddH is controlled to be the same as the solution containing LC100-F/LC100-R 2 The volume ratio of O to the solution containing LC100-F/LC100-R is controlled at 18-28:1.
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