CN111763755B

CN111763755B - SNP molecular marker of rice cadmium absorption related gene OsNRAMP5 and application thereof

Info

Publication number: CN111763755B
Application number: CN201911296360.XA
Authority: CN
Inventors: 唐丽; 赵炳然; 何含杰; 李曜魁; 董家瑜; 吕启明; 毛毕刚; 韶也; 胡远艺
Original assignee: Hunan Hybrid Rice Research Center
Current assignee: Hunan Hybrid Rice Research Center
Priority date: 2019-12-16
Filing date: 2019-12-16
Publication date: 2021-05-18
Anticipated expiration: 2039-12-16
Also published as: CN111763755A

Abstract

The invention discloses an SNP molecular marker of a rice cadmium absorption related gene OsNRAMP5, wherein SNP sites of the SNP molecular marker are positioned at the +938 site nucleotides downstream of the ATG (initiation codon) of a coding region of a rice OsNRAMP5 gene, and C/T polymorphism exists. The SNP molecular marker is related to the cadmium content of rice grains, the cadmium content of the rice grains can be conveniently and quickly predicted, low-cadmium rice germplasm resources are screened, the rice variety with the locus genotype TT has the characteristic of low cadmium accumulation, and the manganese content reduction amplitude is relatively small. The invention also discloses application of the SNP molecular marker in identifying cadmium content in rice grains and cultivating low cadmium accumulation rice varieties, and a specific primer for detecting polymorphism or genotype of the SNP molecular marker contained in a rice genome, so that the low cadmium accumulation rice varieties can be identified or bred quickly and accurately, and a technical basis is provided for realizing safe production of rice in fields with light and medium cadmium pollution.

Description

SNP molecular marker of rice cadmium absorption related gene OsNRAMP5 and application thereof

Technical Field

The invention belongs to the field of crop genetic breeding, and particularly relates to a SNP molecular marker of a rice cadmium uptake related gene OsNRAMP5 and application thereof.

Background

Single Nucleotide Polymorphism (SNP) refers to a variation of a single nucleotide in a genome, including a transition or transversion of a single base, and also includes an insertion or deletion. The genetic markers formed by the SNPs are large in number, rich in polymorphism and high in density on the whole genome. The SNP marker of the target gene can be used for carrying out the precise breeding of the related characters in the breeding process.

The rice is staple food for more than half of the population in the world and is also a crop with high cadmium accumulation. For cadmium-polluted areas with rice as the staple food, rice is the main cadmium intake source of human bodies. The rice area in south China is not only the main production area of rice, but also the serious disaster area of cadmium pollution, and the problem of excessive cadmium in rice in parts of south China becomes the food safety problem which causes wide social attention.

In recent years, the transport proteins responsible for absorption, transport and cadmium distribution of rice are identified in sequence, which provides clues for low-cadmium rice molecular breeding. The OsHMA3 can isolate cadmium in root cells in vacuoles, so that the transport of cadmium in root systems to the overground part is reduced, and the cadmium accumulation of grains is further reduced. Most japonica rice OsHMA3 can normally play a role, but most indica rice varieties OsHMA3 are lack of functions and can not effectively seal cadmium in root cell vacuoles, so that the cadmium accumulation of grains of most indica rice varieties is higher than that of most japonica rice varieties. OsNRAMP5, OsNRAMP1, OsIRT1 and OsIRT2 all participate in absorption of cadmium by rice, wherein OsNRAMP5 is a major transport protein for absorbing manganese and cadmium by rice roots. Among the reported cadmium transporter coding genes, the cadmium reduction effect of plant grains caused by the OsNRAMP5 mutation is the largest. Compared with wild type contrast grain cadmium content of the OsNRAMP5 function-deficient mutant obtained by mutagenesis or genome editing at present can be reduced by more than 90%, rice can be safely produced in a field heavily polluted by cadmium to a certain degree, but simultaneously, as the content of manganese in the overground part of the mutant is greatly reduced, the mutant has the hidden troubles of growth and development obstruction and stress resistance reduction when the concentration of manganese in the environment is very low. At present, the excellent allelic form of OsNRAMP5 and the corresponding natural variant rice material which can reduce the cadmium content and the manganese content in rice grains and have relatively small reduction amplitude are not found.

Because the cadmium content of rice grains can not be directly observed in the process of variety breeding and is not easy to be accurately determined under the influence of environment, the identified functional genetic sites and developed molecular markers for assisting the breeding of the low-cadmium-accumulation rice are few, so that the cultivation process of the low-cadmium-accumulation rice variety is greatly limited. Therefore, the development of molecular markers related to the cadmium content of rice grains can conveniently predict the cadmium content of the grains and quickly screen rice varieties with low cadmium accumulation; on the other hand, the method can be used for molecular marker-assisted selective breeding, can quickly improve the cadmium accumulation character of rice and can cultivate rice varieties with low cadmium accumulation, thereby having very important application value.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings in the background technology and provides an SNP molecular marker of a rice cadmium absorption related gene OsNRAMP5 and application thereof in identification or auxiliary identification of cadmium content in rice grains and cultivation of rice varieties with low cadmium accumulation.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

an SNP molecular marker of a rice cadmium absorption related gene OsNRAMP5 is characterized in that an SNP locus of the SNP molecular marker is located at the +938 bit nucleotide downstream of the ATG (initiation codon) of a coding region of a rice OsNRAMP5 gene, SNP, particularly C/T polymorphism exists, the SNP molecular marker is obviously related to the cadmium content of rice grains, and the cadmium content of indica rice grains with TT genotypes of the SNP locus is obviously lower than the cadmium content of indica rice grains with CC genotypes.

In the SNP molecular marker, the nucleotide sequence of the coding region of OsNRAMP5 is shown as SEQ ID NO.1, the sequence shown as SEQ ID NO.1 consists of 1617 nucleotides and is an antisense strand positioned in the interval of 8878905bp of No. 7 chromosome of Nipponbare rice, wherein the antisense strand is positioned in Nipponbare. The SNP site of the SNP molecular marker is specifically positioned at the 938 th nucleotide from the 5' end of the sequence shown in SEQ ID NO.1, namely TagSNP-8875486, and the base of the TagSNP-8875486 is T or C.

The physical position of the TagSNP-8875486 is determined based on the genome sequence of the rice variety Nipponbare, and the TagSNP-8875486 corresponds to the 8875486 th antisense strand nucleotide on the 7 th chromosome of the rice variety Nipponbare, and the base of the TagSNP-8875486 is C or T.

The amino acid sequence coded by the OsNRAMP5 is shown as SEQ ID No.2, and the 313 th amino acid of the amino acid sequence shown as the SEQ ID No.2 is serine or phenylalanine.

The SNP locus (TagSNP-8875486) of the SNP molecular marker is obviously related to the cadmium content of rice roots, stems and leaves and grains, in most rice varieties, the nucleotide base of the SNP locus is C, and in very few rice varieties, the nucleotide base of the SNP locus is T, so that the 313 rd serine of the coded OsNRAMP5 amino acid sequence (shown as SEQ ID NO. 2) is changed into phenylalanine.

Based on a general inventive concept, the invention also provides an application of the SNP molecular marker in identification or auxiliary identification of cadmium content in rice grains, the genotype of TagSNP-8875486 in genome DNA of rice to be detected is detected, and the cadmium content of indica rice grains with TT genotypes is obviously lower than that of indica rice grains with CC genotypes.

Based on a general inventive concept, the invention also provides an application of the SNP molecular marker in the cultivation of the low cadmium accumulation rice variety, the genotype of TagSNP-8875486 in the genome DNA of the rice to be detected is detected, the rice variety of which the genotype of TagSNP-8875486 is TT is selected as a donor parent for breeding, and the acceptor parent is enabled to have the genotype of TT through hybridization and continuous backcross, so that the low cadmium accumulation rice variety can be obtained.

In the application, the low cadmium content in the low cadmium accumulation rice variety is relative to the hybrid parent rice, if the cadmium content in rice grains of the two hybrid parents is different, the cadmium content in the rice grains of the low cadmium accumulation rice variety can be equal to or lower than that in the hybrid parent rice with lower cadmium content in the rice grains of the two hybrid parents; if the cadmium content in the two hybrid parent rice seeds is the same, the cadmium content in the seeds of the low-cadmium rice variety can be equal to or lower than the cadmium content in the hybrid parent rice seeds. More preferably, the low cadmium accumulation rice variety specifically comprises: when the cadmium content of the rice seeds is planted in a rice field with mild or moderate cadmium pollution, the cadmium content of the soil of the rice seeds is equal to or lower than 0.9mg/kg, and the cadmium content of the rice seeds is less than the national limit standard of 0.2 mg/kg.

Further, the method for detecting the genotype of TagSNP-8875486 in the genomic DNA of the rice to be detected specifically comprises the following steps:

(1) extracting the genome DNA of the rice to be detected;

(2) detecting the genotype of TagSNP-8875486 in the genomic DNA of the rice to be detected, wherein if the base type detection result of TagSNP-8875486 is TT genotype, the genotype of TagSNP-8875486 of the rice to be detected is homozygote of T; if the detection result is CC genotype, the genotype of the rice TagSNP-8875486 to be detected is homozygote of C; if the detection result is TC genotype, the genotype of the rice TagSNP-8875486 to be detected is a heterozygous type of T and C.

Further, the specific operation of the step (2) specifically includes the following steps: the DNA sequence containing the TagSNP-8875486 is amplified by PCR through specific primers shown in SEQ ID NO.3 and SEQ ID NO.4, the PCR product obtained through amplification is sequenced and is compared with the sequence shown in SEQ ID NO.5, the TagSNP-8875486 is located at the 515 th site of the sequence shown in SEQ ID NO.5, and the genotype of the TagSNP-8875486 in the genome DNA can be determined through MAN DNAsoftware analysis.

In the above applications, the polymorphism or genotype of TagSNP-8875486 in the genome can also be determined by any one of the following methods: restriction enzyme fragment length polymorphism, high resolution solubility curve, single strand conformation polymorphism, denaturing high performance liquid chromatography, and SNP chip. The SNP chip comprises a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on one-step reaction, a chip based on primer connection reaction, a chip based on restriction enzyme reaction, a chip based on protein DNA binding reaction and a chip based on fluorescent molecule DNA binding reaction.

Based on a general inventive concept, the invention also provides a specific primer for detecting whether the TagSNP-8875486 in the rice genome has polymorphism or genotype, wherein the specific primer consists of an upstream primer with a nucleotide sequence shown in SEQ ID NO.3 and a downstream primer with a nucleotide sequence shown in SEQ ID NO. 4.

Experiments prove that under the field conditions that the soil cadmium concentration is 0.9mg/kg and the pH value is 5.7, the cadmium content of the indica rice variety with 100 percent of TT genotype (TagSNP-8875486 is homozygote of T) grains is lower than that of the indica rice variety with 100 percent of CC genotype (TagSNP-8875486 is homozygote of C).

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

1. the SNP molecular marker is related to the cadmium content of rice grains, the polymorphism of the SNP locus is directly expressed in a DNA form, the root, stem leaf and grain of the rice variety with the locus genotype TT have the characteristic of low accumulation of cadmium, and the manganese content of the stem leaf is higher than that of osnramp5 function deletion mutant.

2. The method for identifying or assisting in identifying the cadmium content in the rice grains can detect DNA in each tissue and each development stage of the rice, can conveniently and quickly predict the cadmium content of the rice grains, and can quickly screen the germplasm resources of the low-cadmium rice.

3. The method for cultivating the low cadmium accumulation rice variety has the advantages that the gene type of TagSNP-8875486 in the genome DNA of the rice to be detected is detected, the rice variety with the TagSNP-8875486 gene type TT is selected from the detected gene type, the rice variety is used as a breeding parent, stable and hereditary low cadmium accumulation rice variety can be quickly and accurately bred by combining SNP molecular markers, the safe production of the rice in the field with light and medium cadmium pollution is expected to be realized, the time cost, the land cost and the labor cost required by the traditional breeding method are saved, and the method has obvious progress significance.

4. The specific primer for detecting whether the TagSNP-8875486 in the rice genome has polymorphism or genotype can quickly and accurately identify the SNP, and can improve the accuracy and efficiency of screening and identifying the TagSNP-8875486.

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.

FIGS. 1-4 show the comparison of cadmium and manganese content of the overground part and root system of 22 rice varieties with TagSNP-8875486 genotype of CC, 5 rice varieties with TagSNP-8875486 genotype of TT and 10 different genetic background osnramp5 function-deletion mutants, wherein:

FIG. 1 shows the distribution of the cadmium content in the aerial parts of three genotype rice varieties;

FIG. 2 shows the distribution of the manganese content in the aerial parts of three genotype rice varieties;

FIG. 3 shows the distribution of cadmium content in the root system of three types of rice;

FIG. 4 shows the distribution of manganese content in the root system of three genotype rice varieties;

FIG. 5 shows cadmium content of brown rice of Huazhan low cadmium improved line and original variety Huazhan;

FIG. 6 shows the manganese content of brown rice of Huazhan low-cadmium improved line and original variety Huazhan;

FIG. 7 is a plant morphology chart of Huazhan low cadmium improved line and original variety Huazhan;

different lower case letters indicate significant differences at the 0.05 level.

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 (b):

1. discovery of SNP marker (TagSNP-8875486) of rice cadmium uptake related gene OsNRAMP5

The inventor inquires a rice resource resequencing database, analyzes sequence polymorphism of 7000 rice varieties OsNRAMP5 at home and abroad, does not find that the coding region of OsNRAMP5 has variation such as deletion mutation, frameshift mutation and the like, and only finds that the coding region has 20 SNP. The coding region of OsNRAMP5 of Nipponbare of rice variety is taken as a reference sequence, different haplotypes of the coding region of OsNRAMP5 are classified and translated into amino acid sequences, and the result shows that the haplotypes of the 11 coding regions of OsNRAMP5 are nonsynonymous mutated, and only 1 amino acid of all the haplotypes is changed.

The rice varieties corresponding to the haplotypes are provided by the institute of crop science of Chinese academy of agricultural sciences and Huazhi Rice biotechnology, Inc. Further, PCR amplification and sequencing were carried out for OsNRAMP5 gene of each variety. Soaking and germinating haplotype rice variety and osnramp5 function-deficient mutant with different genetic backgrounds, culturing in artificial climate chamber for 14 days with rice nutrient solution formula of International Rice institute, and culturing with 0.5 μ MCdCl₂Treating for 14 days, washing with deionized water, separating aerial parts and root system, cutting, weighing, and adding HNO₃:HClO₄Mixed acid (volume ratio 6:1) digestion, 1% HNO₃And (3) performing constant volume measurement, measuring the cadmium and manganese contents of roots, overground parts and stems of each material by ICP-MS, and screening SNP (single nucleotide polymorphism) related to the cadmium contents of rice roots and stems, wherein the SNP site is positioned at the downstream +938 site of an ATG (start codon ATG) of a CDS (nucleotide sequence shown as SEQ ID NO. 1) region of an OsNRAMP5 gene, is positioned at the 8875486 site on the 7 th chromosome of Nipponbare of a rice variety, and is named as TagSNP-8875486. The antisense strand nucleotide corresponding to TagSNP-8875486 has C/T polymorphism. The homozygous TagSNP-8875486 genotype of the sequence shown in SEQ ID NO.1 with the position 938 being T is named TT, the homozygous TagSNP-8875486 genotype of the sequence shown in SEQ ID NO.1 with the position 938 being C is named CC, and the heterozygous TagSNP-8875486 genotype of the sequence shown in SEQ ID NO.1 with the position 938 being T and C is named TC.

In most rice varieties, the nucleotide base of TagSNP-8875486 is C, and in a few rice varieties, the nucleotide base at the site is mutated into T, so that the 313 rd serine of the encoded OsNRAMP5 amino acid sequence (shown as SEQ ID NO. 2) is changed into phenylalanine.

The results of comparing the cadmium and manganese contents of the aerial parts and the root systems of 22 rice varieties with the TagSNP-8875486 genotype of CC, 5 rice varieties with the TagSNP-8875486 genotype of TT and 10 osnramp5 function-deficient mutants with different genetic backgrounds are shown in figures 1-4.

As can be seen from FIGS. 1 to 4, the cadmium and manganese contents of the overground part and the root system of the rice variety with the TagSNP-8875486 genotype TT are lower than those of the rice variety with the TagSNP-8875486 genotype CC, which shows that the TagSNP-8875486 is obviously related to the overall cadmium and manganese accumulation amount of the rice. The cadmium content of the overground part of the rice variety with TT gene type of TagSNP-8875486 is not obviously different from that of osnramp5 function deletion mutant, but the manganese content of the overground part is obviously higher than that of osnramp5 function deletion mutant, which suggests that the SNP of TT gene type has great utilization value in cadmium accumulation rice breeding.

2. Identifying the TagSNP-8875486 genotypes of 60 rice varieties and the cadmium content of rice

The identification of the TagSNP-8875486 genotypes of 60 rice varieties comprises the following steps:

1) 60 Rice varieties at home and abroad are collected, and germplasm resource information can be obtained from national Rice data centers (http:// www.ricedata.cn /) and Rice SNP-Seek Database (https:// SNP-Seek.

2) The DNA of the 60 rice varieties is extracted by taking rice leaves as materials, and a plurality of reported methods for extracting the DNA are provided, and the invention adopts a CTAB method for extraction;

3) using DNA of 60 rice materials as a template, using specific primers shown in SEQ ID NO.3 and SEQ ID NO.4 as an upstream primer and a downstream primer respectively, and amplifying a DNA fragment containing TagSNP-8875486 by using high-fidelity enzyme PCR;

the reaction system is as follows: mu.L of DNA template, 1. mu.L of each forward and reverse primer, 4. mu.L of dNTP, 15. mu.L of 2 XBuffer, 0.5. mu.L of KOD enzyme, plus ddH₂O to the total volume of 30 mu L; book (I)The invention uses KOD high fidelity enzyme kit of TOYOBO company;

the reaction procedure is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 98 deg.C for 10s, annealing at 60 deg.C for 30s, extension at 68 deg.C for 1min, and 30 cycles;

4) sequencing the PCR product, comparing the sequencing result with the sequence shown in SEQ ID NO.5, determining the 515 th site of the SEQ ID NO.5, namely the base type of TagSNP-8875486, and determining the genotype of the rice according to the base type of TagSNP-8875486 in each rice variety; the invention adopts DNAMAN software comparison analysis sequencing results, and the genotype results of all varieties are shown in table 1.

The method for identifying the cadmium content of the TagSNP-8875486 rice of 60 rice varieties comprises the following steps:

1) planting the 60 rice varieties in a field with heavy cadmium pollution (the concentration of cadmium in soil is 0.9mg/kg, the pH value is 5.7), wherein the field irrigation mode is dry-wet alternation, so that the rice grains can accumulate cadmium element to the maximum extent, each variety is planted with 24 plants in a row, 8 plants are repeated, and 3 plants are repeated;

2) after the rice is ripe, repeatedly collecting seeds of 3 rice plants in the middle, threshing, shelling to obtain brown rice, crushing to obtain rice flour, and weighing;

3) measuring the cadmium content of brown rice by reference to national standard measurement method (GB/T5009.15-2003), specifically weighing 2 g of rice flour for each material, and using HNO₃:HClO₄Mixed acid (volume ratio 6:1) digestion, 1% HNO₃The volume is fixed, and the content of cadmium is measured by ICP-MS, and the result is shown in Table 1.

As can be seen from table 1, of the 60 rice varieties tested, 8 indica rice varieties having genotypes TT (homozygous for TagSNP-8875486T), 42 indica rice varieties having genotypes CC (homozygous for TagSNP-8875486C), and 10 japonica rice varieties having genotypes CC were tested.

The cadmium content of the grains of 60 rice varieties counted according to the TagSNP-8875486 genotypes is shown in Table 2. As can be seen from Table 2, the cadmium content of the brown rice of 8 rice varieties with the genotype TT is lower than that of the rice varieties with the genotype CC of 52.

The results in table 1 also show that the cadmium content of brown rice of 8 indica type rice varieties with the gene type TT is lower than 0.2mg/kg, the cadmium content of brown rice of 42 indica type rice varieties with the gene type CC is higher than 0.5mg/kg, the cadmium content of brown rice of 5 japonica type rice varieties with the gene type CC is lower than 0.2mg/kg, and the cadmium content of brown rice of 5 japonica type rice varieties with the gene type CC is higher than 0.2 mg/kg. This suggests that 100% of the indica rice varieties with the TT genotype have brown rice cadmium content lower than 100% of the indica rice varieties with the genotype CC, 100% of the indica rice varieties with the TT genotype have brown rice cadmium content lower than 50% of the japonica rice varieties with the genotype CC, and the brown rice cadmium content is equivalent to that of the other 50% of the japonica rice varieties with the CC genotype. The cadmium content of the indica type rice brown rice with the TT genotype marker can be reduced to the level of japonica rice, and the TagSNP-8875486 is an SNP molecular marker related to the cadmium content of rice grains and can be used for early prediction and screening of rice grain cadmium low-accumulation varieties.

Table 1: cadmium content of grains and TagSNP-8875486 genotype of 60 rice varieties

Table 2: the cadmium content of the grains of 60 rice varieties is counted according to the TagSNP-8875486 genotype

3. Application of TagSNP-8875486 in breeding of rice variety with low cadmium accumulation

The method for improving the cadmium accumulation character of the indica rice variety Huazhan by utilizing TagSNP-8875486 comprises the following specific steps:

1) selecting a rice variety LAYANDABU with the genotype of TagSNP-8875486 TT as a donor parent, and taking Huazhan with the genotype of CC as a receptor parent for hybridization;

2) backcrossing by taking Huazhan as a recurrent parent, performing foreground selection by using specific primers of SEQ ID NO.3 and SEQ ID NO.4, selecting a single plant with the genotype of TC, performing background selection, and selecting the single plant with the highest similarity to Huazhan for backcrossing;

3) backcrossing for 3-4 generations according to the step 2), selfing, performing foreground selection by using specific primers of SEQ ID NO.3 and SEQ ID NO.4, selecting a single plant with the genotype of TT, performing background selection, and selecting the single plant with the highest similarity to recurrent parents to obtain the Huazhan low-cadmium improved line.

Planting the Huazhan improved line with low cadmium and the original variety Huazhan in a heavy cadmium polluted field with the soil cadmium concentration of 0.9mg/kg and the pH value of 5.7, and planting and detecting the content of the brown rice element according to the method for identifying the cadmium content of the TagSNP-8875486 rice in 60 rice varieties.

The result in fig. 5 shows that the cadmium content of the brown rice of the low cadmium improved line is reduced by 85% compared with the control, and the manganese content and the plant growth of the brown rice of the low cadmium improved line are not obviously different from the original variety Huazhan as shown in fig. 6 and 7.

The method for cultivating the low cadmium accumulation rice variety can quickly and accurately breed the stable genetic low cadmium accumulation rice variety, provides a technical basis for realizing the safe production of the rice in the field with light low manganese and moderate cadmium pollution, saves the time cost, the land cost and the labor cost required by the traditional breeding method, and has remarkable progress significance.

Sequence listing

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Phe Glu Leu Pro Phe Ala Leu Ile Pro Leu Leu Lys Phe Ser Ser Ser

405 410 415

Lys Ser Lys Met Gly Pro His Lys Asn Ser Ile Tyr Ile Ile Val Phe

420 425 430

Ser Trp Phe Leu Gly Leu Leu Ile Ile Gly Ile Asn Met Tyr Phe Leu

435 440 445

Ser Thr Ser Phe Val Gly Trp Leu Ile His Asn Asp Leu Pro Lys Tyr

450 455 460

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

465 470 475 480

Ile Val Ala Val Val Tyr Leu Thr Ile Arg Lys Asp Ser Val Val Thr

485 490 495

Phe Val Ala Asp Ser Ser Leu Ala Ala Val Val Asp Ala Glu Lys Ala

500 505 510

Asp Ala Gly Asp Leu Ala Val Asp Asp Asp Glu Pro Leu Pro Tyr Arg

515 520 525

Asp Asp Leu Ala Asp Ile Pro Leu Pro Arg

530 535

<210> 3

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tctgttcttg cattctgcct tg 22

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gcatgatgta ctgtccagcg t 21

<210> 5

<211> 912

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 5

tctgttcttg cattctgcct tggtgctatc gaggaagaca ccggcatcag tcagaggaat 60

caaggtagct atattcagcc tgggcagatc gagataggta tagggtagct aggacatgaa 120

ctcgatctag tcctgtctct taaatcttgt ttaaatttta ttaaattaac atgtaaattt 180

catagaaaac caatacttta ttagctaagt tttattttag cactaatctt ctataatttc 240

gggctccagt accactggat tcagcttaga attttttcat gcatggaaag aaaccgaaca 300

aagatccagg ctaataatta atatgtacta tatggtgtgt gaattgccac gtaggacggg 360

tgcaggttct tcctgtacga gagcgggttc gcgctgttcg tggcgctgct gataaacatc 420

gccgtcgtct ccgtctccgg caccgcctgc tcctccgcca acctctccca agaggacgcc 480

gacaagtgcg ccaacctcag cctcgacacc tcctccttcc ttctcaaggt cattcattca 540

ttattacttc ctactcctta atattaaaaa tttttaaatg gattagacgt atcctagtac 600

tacagataga aatctattca gattcgtagt actagcttgt atgtgttcaa tttaatcaat 660

atttaattaa ccacaataat accgaattag tctacacgat ttctccgtcc taaaatataa 720

taactttatt agatatgttt atatttttga ataggaggag taactaattt atggtcaaat 780

tgtgacgtca atttttttat gtggttcaga acgtgctggg caagtcgagt gcgatcgtgt 840

acggcgtggc actgttggca tctgggcaga gctccactat taccggcaca tacgctggac 900

agtacatcat gc 912

Claims

1. Rice cadmium absorption related geneOsNRAMP5The application of the SNP molecular marker in identifying or assisting in identifying the cadmium content in rice grains is characterized in thatOsNRAMP5The nucleotide sequence of the coding region of the SNP molecular marker is shown as SEQ ID number 1, the SNP locus of the SNP molecular marker is positioned at the 938 th nucleotide from the 5' end of the nucleotide sequence shown as SEQ ID NO.1, and the base of the SNP molecular marker is T or C; the application comprises the following steps: and (3) detecting the genotype of the SNP locus in the genome DNA of the rice to be detected, wherein the cadmium content of the indica rice grains with the TT genotype is obviously lower than that of the indica rice grains with the CC genotype.

2. Rice cadmium absorption related geneOsNRAMP5The SNP molecular marker is used for cultivating cadmiumUse in low accumulation rice varieties characterized in thatOsNRAMP5The nucleotide sequence of the coding region of the SNP molecular marker is shown as SEQ ID number 1, the SNP locus of the SNP molecular marker is positioned at the 938 th nucleotide from the 5' end of the nucleotide sequence shown as SEQ ID NO.1, and the base of the SNP molecular marker is T or C; the application comprises the following steps: detecting the genotype of the SNP locus in the genome DNA of the rice to be detected, and selecting the rice with TT genotype as a donor parent for breeding; the cadmium low-accumulation rice variety specifically comprises the following components: when the cadmium content of the rice seeds is planted in a rice field with mild or moderate cadmium pollution, the cadmium content of the soil of which is equal to or lower than 0.9mg/kg, the cadmium content of the rice seeds is less than the national limit standard of 0.2 mg/kg; the rice is indica rice.

3. The use according to claim 1 or 2, wherein the detecting the genotype of the SNP site in the genomic DNA of the rice to be detected specifically comprises the following steps:

(1) extracting the genome DNA of the rice to be detected;

(2) detecting the genotype of the SNP locus in the genomic DNA, and if the detection result is TT genotype, indicating that the genotype of the SNP locus in the genomic DNA of the rice to be detected is homozygous for T; if the detection result is CC genotype, the genotype of the SNP locus in the genome DNA of the rice to be detected is homozygote of C; and if the detection result is the TC genotype, the genotype of the SNP site in the genome DNA of the rice to be detected is the heterozygous type of T and C.

4. The application of claim 3, wherein the specific operation of step (2) comprises the following steps: amplifying a DNA sequence containing the SNP locus by using specific primers shown in SEQ ID NO.3 and SEQ ID NO.4 through PCR, sequencing the PCR product obtained through amplification, comparing the sequence with the sequence shown in SEQ ID NO.5, wherein the SNP locus is located at the 515 th position of the sequence shown in SEQ ID NO.5, and determining the genotype of the SNP locus in the genome DNA through sequence comparison.

5. A specific primer for detecting the genotype of the SNP molecular marker in the rice genome, wherein the specific primer is composed of an upstream primer of a nucleotide sequence shown by SEQ ID number 3 and a downstream primer of a nucleotide sequence shown by SEQ ID number 4.