CN108285927B - Intragenic molecular marker Caps7 of rice cadmium accumulation related gene OsHMA3 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of molecular biology, and provides an intragenic molecular marker Caps7 of a rice cadmium accumulation related gene OsHMA3 and application thereof, wherein a positive primer and a negative primer are designed and synthesized, rice DNA is amplified in a PCR system, then an amplified product is subjected to enzyme digestion, and finally, the rice to be detected contains HMA3-1 or HMA3-2 allele through agarose electrophoresis detection, so that the rice cadmium accumulation character is determined, and the breeding of a indica rice low-cadmium accumulation variety is assisted. The method can effectively identify the cadmium accumulation character of the rice, provides a new technical means for breeding indica rice cadmium low accumulation varieties and improves the breeding efficiency.

Description

Intragenic molecular marker Caps7 of rice cadmium accumulation related gene OsHMA3 and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to an intragenic molecular marker Caps7 of a rice cadmium accumulation related gene OsHMA3 and application thereof.

Background

Cadmium in rice field exceeds standard due to Cadmium (Cadmium, Cd) pollution, and food safety and grain safety in China are seriously affected. Therefore, in order to avoid the damage of cadmium pollution to human bodies, the reduction of cadmium accumulated in rice grains is urgently needed. The research on the genetic characteristics of cadmium accumulation of rice varieties shows that indica rice varieties often accumulate more cadmium in grains than japonica rice varieties. In the main rice production area in south China, a single indica rice planting structure, a slightly acidic soil background and a high cadmium pollution degree aggravate the risk that the cadmium content of the indica rice variety exceeds the standard. Therefore, before the cadmium pollution of the rice field is completely eradicated, planting indica rice varieties with low cadmium accumulation is the most economic and effective method for avoiding the cadmium pollution risk at present. However, the cadmium accumulation character of rice cannot be directly observed by breeders in the actual variety breeding process, so that the breeding of low-cadmium rice varieties needs to identify and utilize excellent gene resources and corresponding molecular auxiliary Means (MAS). The molecular auxiliary means capable of effectively identifying the cadmium accumulation character of rice is still in the stage to be developed.

Disclosure of Invention

Aiming at the technical problems, the invention provides an intragenic molecular marker Caps7 of a rice cadmium accumulation related gene OsHMA3 and application thereof, which can effectively identify the cadmium accumulation character of rice varieties and accelerate the breeding process of indica rice low-cadmium accumulation varieties.

At present, for the rice grain cadmium accumulation trait, researchers have identified a series of key quantitative trait loci and related genes for controlling rice cadmium absorption, transport and distribution, wherein a major QTL (quantitative trait locus) which is positioned at the short arm of the seventh chromosome of rice and controls the rice cadmium transport rate is separated and cloned and is named as OsHMA3(Ueno et al, 2010). The gene mainly isolates cadmium absorbed by the root system by controlling and influencing rice root system vacuole, and reduces cadmium transported to overground parts, thereby influencing the rice cadmium transport rate. The inventor researches cadmium accumulation of rice by using linkage analysis and correlation analysis, and finds that the gene OsHMA3 can influence the accumulation of cadmium absorbed by rice from soil in rice; meanwhile, two important allelic gene types of HMA3-1 and HMA3-2 mainly exist in the gene in a natural population, the nucleotide sequence of HMA3-2 is shown in SEQ ID NO:1, the two allelic gene types account for more than 85% of the natural population, and the cadmium content of rice of a rice variety containing HMA3-1 is obviously lower than that of a rice variety containing HMA3-2 (P is less than 0.01); the inventor further analyzes and finds that HMA3-1 and HMA3-2 have obvious characteristic of indica-japonica differentiation, namely japonica rice varieties tend to contain HMA3-1, while indica rice varieties mostly contain HMA3-2 and a small part of HMA 3-1.

The genetic background of the present invention is focused on: 1) the cadmium content of rice of most indica rice varieties is often at a higher level (indica rice tends to accumulate more under the same similar soil environment), which is probably due to the fact that the allelic type of OsHMA3 is a high cadmium accumulation genotype HMA 3-2; 2) the low cadmium accumulation allelic type HMA3-1 can be introduced into a high accumulation variety (replacing the high cadmium accumulation allelic type HMA3-2 of OsHMA 3) by a molecular auxiliary means, and an important genetic means, related varieties and lines are provided for improving the cadmium accumulation characteristics of the indica rice variety.

The technical scheme of the invention is as follows: provides an intragenic molecular marker Caps7 of rice cadmium accumulation related gene OsHMA3, wherein the gene OsHMA3 comprises allele HMA3-1 and allele HMA3-2, and the molecular marker Caps7 can distinguish the sequence difference of the allele HMA3-1 and the allele HMA 3-2.

Further, the PCR amplification primer pair of the molecular marker Caps7 is:

forward primer F (SEQ ID NO: 4): CTCGTCAGCGGCCTCAAGG, respectively;

reverse primer R (SEQ ID NO: 5): GCACGACGAGGAGGCACG, respectively;

the molecular marker Caps7 appeared as: when the primer pair is used for PCR amplification, the amplification product of the allele HMA3-1 has a nucleotide sequence shown by SEQ ID NO. 2, and the amplification product of the allele HMA3-2 has a nucleotide sequence shown by SEQ ID NO. 3; the allele HMA3-1 shows low cadmium accumulation, and the nucleotide sequence of the allele HMA3-2 is shown as SEQ ID NO. 1 and shows high cadmium accumulation.

The invention also provides a method for distinguishing alleles HMA3-1 and HMA3-2 by using the molecular marker Caps7, wherein the primer pair is used for carrying out PCR amplification on the genomic DNA of rice to be detected; carrying out enzyme digestion on the PCR product by using AscI endonuclease, detecting the amplified product after enzyme digestion by agarose electrophoresis, and if the agarose electrophoresis detection shows that the amplified product is a strip, determining that the rice to be detected has allele HMA 3-1; and if the agarose electrophoresis detection shows that the two bands exist, the rice to be detected has the allele HMA3-2, and if the agarose electrophoresis detection shows that the three bands exist, the gene OsHMA3 of the rice to be detected is in a heterozygous state.

Further, if agarose electrophoresis detection shows that the rice to be detected is a strip with the sequence length of 337bp, the rice to be detected has allele HMA3-1, which is expressed as low cadmium accumulation; if agarose electrophoresis detection shows that the two bands have sequence lengths of 196bp and 141bp respectively, the rice to be detected has allele HMA3-2 which shows high cadmium accumulation, and if the agarose electrophoresis detection shows that the three bands have sequence lengths of 337bp, 196bp and 141bp respectively, and the band with the sequence length of 337bp is brightest, the gene OsHMA3 of the rice to be detected is in a heterozygous state.

Further, the PCR system is as follows: 10-50 ng of template DNA, 0.25um each of a forward primer and a reverse primer, 1U of Taq DNA polymerase, 0.1mM each of four deoxyribonucleotides dATP, dTTP, dCTP and dGTP, 10ul of 2 XGC buffer I, and the balance of 20ul of deionized water;

the PCR reaction program is as follows: performing pre-denaturation at 94 ℃ for 4min, performing PCR (polymerase chain reaction) cyclic amplification, wherein the cyclic amplification comprises denaturation at 94 ℃ for 30sec, annealing at 60 ℃ or 62 ℃ for 30sec, and extension at 72 ℃ for 1min, three steps are performed, the cyclic operation is performed for 30-34 times, and finally, after extension at 72 ℃ for 10min, heat preservation is performed at 25 ℃ for 4min to obtain an amplification product;

the enzyme cutting system is a10 ul system, and the following components are mixed: 100-200 ng of PCR amplification product, 1ul of AscI endonuclease Buffer (10X), 2U of AscI endonuclease and the balance of deionized water; enzyme digestion program: and placing the mixed enzyme digestion system in a 37 ℃ thermostat to react for more than 8-12 h for enzyme digestion.

The invention also provides the application of the molecular marker Caps7 and the PCR amplification primer pair thereof in identifying the cadmium accumulation traits of rice.

The invention also provides the application of the molecular marker Caps7 and the PCR amplification primer pair thereof in breeding low-cadmium accumulation indica rice varieties.

The invention also provides the application of the method for distinguishing the alleles HMA3-1 and HMA3-2 in breeding indica rice varieties with low cadmium accumulation.

The invention also provides a method for identifying the cadmium accumulation character of rice, which comprises the steps of carrying out PCR amplification of molecular marker Caps7 on the genomic DNA of rice to be detected by utilizing the primer pair, carrying out enzyme digestion by using AscI endonuclease, and detecting the length of a PCR product by agarose electrophoresis to determine the cadmium accumulation character of rice;

if agarose electrophoresis detection shows that the sequence length is 337bp, the rice to be detected has allele HMA3-1, which is expressed as low cadmium accumulation; if agarose electrophoresis detection shows that the two bands have sequence lengths of 196bp and 141bp respectively, the rice to be detected has allele HMA3-2 which shows high cadmium accumulation, and if the agarose electrophoresis detection shows that the three bands have sequence lengths of 337bp, 196bp and 141bp respectively, and the band with the sequence length of 337bp is brightest, the gene OsHMA3 of the rice to be detected is in a heterozygous state.

The invention also provides a low-cadmium accumulation indica rice auxiliary breeding method, which comprises the following steps: the molecular marker Caps7 is detected by the method to determine the accumulation character of the indica rice cadmium to be detected.

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

1. according to the invention, through natural variation research on a major QTL gene OsHMA3 controlling and influencing rice cadmium accumulation, an intragenic molecular marker Caps7 capable of effectively distinguishing a low cadmium accumulation allelic type HMA3-1 from a high cadmium accumulation allelic type HMA3-2 is developed.

2. The intragenic molecular marker Caps7 can effectively identify the cadmium accumulation character of rice, provide a new technical means for breeding indica rice varieties with low cadmium accumulation and accelerate the breeding process.

3. The near-isogenic line material NIL (93-11) obtained in the embodiment 3 of the invention provides a good donor material for breeding low-cadmium accumulation indica rice varieties.

Drawings

FIG. 1 is a graph of natural variation and haplotype analysis of OsHMA3, wherein a is the natural variation analysis and haplotype analysis of OsHMA3, wherein the dark grey indicates indica genotype, the light grey indicates japonica genotype, and the number in the right hand box is the number of varieties of that genotype that occur in the natural population; b is the comparison of the cadmium accumulation of the rice in 105 rice varieties of HMA3-1 and HMA3-2, and the level of P <0.01 shows that the difference is extremely obvious; c is a frequency distribution histogram showing the distribution of HMA3-1 and HMA3-2 in 300 indica rice varieties, black shows the HMA3-2 allelic type, and light gray shows the HMA3-1 allelic type; d is the difference between the cadmium content of the rice in 300 parts of materials of HMA3-1 and HMA3-2, and the level of P <0.01 shows that the difference is extremely obvious;

FIG. 2 is a diagram of analysis of a weak functional allele of an OsHMA3 gene in HMA3-2, wherein a is the expression difference of the OsHMA3 allele HMA3-1 and HMA3-2 in yeast, Glucose is Glucose as a carbon source for inhibiting expression, galctose is galactose as a carbon source for inducing expression, the yeast grows on an SC-Ura (uracil) auxotrophic medium, and the cadmium stress concentration is 10 uM; b is a yeast growth curve under cadmium stress, and the culture medium is an SC-Ura (uracil) liquid culture medium; c is the yeast cell expression localization of GFP-HMA3 fusion protein, HMA3^CKKIndicates the absence of OsHMA3A functional allele type;

FIG. 3 is a PCR identification electrophoresis chart of HMA3-1 and HMA3-2 using developed OsHMA3 gene internal marker Caps7, the concentration of agarose gel is 2%; 9311 and Nip were control varieties, wherein 9311 contained HMA3-2 and Nip contained HMA 3-1. HMA3-1/HMA3-2 is shown in heterozygous state;

FIG. 4 is a PCR identification gel diagram of Caps7 for NIL (HMA3-1) and NIL (HMA3-2) of two near isogenic lines, the concentration of agarose gel being 3%; wherein the NIL (HMA3-1) comprises HMA3-1 with a single gel pattern, and the NIL (HMA3-2) comprises HMA3-2 with a double gel pattern.

Detailed Description

The technical solution of the present invention will be further described with reference to the accompanying drawings and examples.

Example 1: discovery of allele function difference of HMA3 in indica rice subgroup and japonica rice subgroup of rice

(1) Natural variation of the Gene HMA3

Sequence analysis was performed on the OsHMA3 gene sequences in 105 germplasm resource materials (collected in this house) for the rice cadmium accumulation-related QTL gene OsHMA 3. Haplotype analysis is carried out through MEGA sequence analysis software and STRUCTURE software, and the gene is found to coexist in 9 genotypes in the nature and is sequentially marked as HMA 3-1-HMA 3-9; wherein HMA 3-2-HMA 3-6 belong to indica rice genotypes uniformly, while HMA3-1, HMA 3-7-HMA 3-9 belong to japonica rice genotypes, as shown in figure 1 a. HMA3-1 and HMA3-2 are two major alleles, which together account for 83.8% of the total material; further analysis finds that most of HMA3-1 appears in japonica rice varieties and a small part appears in indica rice varieties, while HMA3-2 mainly appears in indica rice varieties, and haplotype analysis of OsHMA3 finds that the gene has a relatively obvious indica-japonica differentiation phenomenon. In combination with the cadmium content phenotype of rice, the average accumulated cadmium in rice containing HMA3-1 genotype was found to be significantly lower than in rice variety containing HMA3-2 allele, as shown in FIG. 1 b. In indica rice varieties, the OsHMA3 gene also has distribution between HMA3-1 and HMA3-2, but in indica rice varieties, the number of varieties occupied by HMA3-1 is small, as shown in figure 1c, and in indica rice, rice containing HMA3-1 genotype is also found to accumulate significantly less cadmium on average than rice containing HMA3-2 allele, as shown in figure 1 d.

(2) HMA3-2 is a weakly functional allele of OsHMA3

The wild type yeast BY4741 was used for functional difference analysis. Yeast strains containing HMA3-1 and HMA3-2 compared to empty vector and nonfunctional allelic HMA3 in 10uMCdSO4 stress culture^CKKAll grew less while the yeast strain containing HMA3-2 died more than the yeast strain containing HMA3-1, see fig. 2b, while yeast growth experiments showed that the growth rate of the yeast containing HMA3-2 was significantly lower than the yeast strain containing HMA3-1, see fig. 2 b; by yeast subcellular localization of the GFP fusion protein, it was found that either HMA3-1 or HMA3-2 was localized predominantly to the vacuolar membrane (with minor distribution of other plasma membranes), see FIG. 2 c. The results show that the HMA3-2 has the function of transferring cadmium outside the yeast into the yeast body like the HMA 3-1; because HMA3 has the ability to trap cadmium in cells in the vacuole, while HMA3-1 has stronger transport ability into the vacuole than HMA3-2 as a strongly functional allele of OsHMA3, the growth of yeast strains containing HMA3-2 is weaker in a cadmium stress environment than that of yeast strains containing HMA3-1, and thus HMA3-2 is a weakly functional allele of OsHMA 3.

Example 2: development of molecular marker Caps7 and genotype identification method

(1) Development of molecular marker Caps7

The molecular marker Caps7 of the invention has the principle of marker design as follows: sequence comparison is carried out on sequences of HMA3-1 and HMA3-2 by using a BLAST tool in a bioinformatics website NCBI (https:// www.ncbi.nlm.nih.gov /) to find a difference site, the difference site forms two genotypes, wherein the gene derived from japonica rice genotype HMA3-1 cannot be cut by restriction endonuclease AscI, after AscI enzyme digestion after PCR amplification, the gene is detected to be a single band through agarose electrophoresis, and the size of a PCR product is 337 bp; indica rice genotype HMA3-2 was cleaved by restriction enzyme AscI and detected as two bands by agarose electrophoresis, and the PCR products were 196bp and 141bp in size, as shown in FIG. 3.

(2) Genotype identification method

The PCR procedure was described in Sambruke et al, 2002, molecular cloning guidelines, third edition, Jindong goose, et al (translation), methods introduced by the scientific Press, and modified according to the conditions and requirements of the subtropical agroecological institute of Chinese academy of sciences.

Forward primer (F): CTCGTCAGCGGCCTCAAGG, respectively;

reverse primer (R): GCACGACGAGGAGGCACG are provided.

The specific steps of genotype identification are as follows:

1) extracting the genome DNA of the rice plant;

2) performing PCR amplification, namely adding the forward primer (F) and the reverse primer (R) into a PCR reaction system by taking the rice plant genome DNA obtained in the step S1 as a template, and amplifying the rice plant genome DNA to obtain an amplification product;

the PCR system is a 20ul system: 10-50 ng of genome DNA of a rice plant, 0.25um each of a forward primer (F) and a reverse primer (R), 1U of Taq DNA polymerase, 0.1mM each of four deoxyribonucleotides dATP, dTTP, dCTP and dGTP, 10ul of 2 XGC buffer I, and the balance to 20ul of deionized water. The 2 × GC buffer I is manufactured by TAKARA BAO bioengineering, Inc.;

the reaction procedure of the PCR is as follows: performing pre-denaturation at 94 ℃ for 4min, performing PCR (polymerase chain reaction) cyclic amplification, wherein the cyclic amplification comprises denaturation at 94 ℃ for 30sec, annealing at 60 ℃ or 62 ℃ for 30sec, and extension at 72 ℃ for 1min, three steps are performed, the cyclic operation is performed for 30-34 times, and finally, after extension at 72 ℃ for 10min, heat preservation is performed at 25 ℃ for 4min to obtain an amplification product;

3) and (3) carrying out enzyme digestion on the PCR product, wherein the enzyme digestion system is 10ul, and mixing the following components: 100-200 ng of PCR amplification product, 1ul of AscI endonuclease Buffer (10X), 2U of AscI endonuclease and the balance of deionized water; placing the mixed enzyme digestion system in a thermostat at 37 ℃ for reaction for more than 8-12 h;

4) carrying out 2% agarose gel electrophoresis detection, carrying out sample application, carrying out electrophoresis for 15-30 min at a direct current voltage of 250V, dyeing the sample by ethidium bromide for 1h, placing the sample under an ultraviolet lamp, and reading the PCR band type of each sample; judging the allelic type of OsHMA3 according to the PCR banding pattern, wherein two bands are HMA3-2, and one band is HMA 3-1; when the rice cadmium accumulation related gene OsHMA3 is in a heterozygous state, three bands are generated in agarose detection after the identification is carried out by an intragenic marker Caps7, the sizes of PCR products are 337bp, 196bp and 141bp respectively, and the 337bp band type is brightest, which is shown in figure 3. 9311 and Nip were control varieties, wherein 9311 contained HMA3-2 and Nip contained HMA 3-1. HMA3-1/HMA3-2 is shown in a heterozygous state.

Example 3:

(1) allele type of rice cadmium accumulation related gene OsHMA3 of 300 parts of rice indica rice material and validity test thereof

And identifying the allelic type of OsHMA3 of 300 parts of indica rice material by using the molecular marker Caps7 of the invention with the overall average value as a standard for distinguishing the cadmium content of the rice.

The cadmium accumulation phenotype of the rice with 300 parts of materials is collected in a contaminated field test, and the field planting mode is carried out in a dry-wet alternating mode, so that cadmium elements are accumulated in rice grains to the maximum extent (the cadmium concentration in the field is severe pollution, the concentration is 1.9mg/kg, and the pH value is 5.4). Genomic DNA was extracted from 300 parts of indica rice material of Table 1, and allelic type identification was performed according to the method shown in example 2 using the intragenic marker Caps7 (shown as SEQ ID NO:4 and SEQ ID NO:5 as PCR amplification primers) of the cadmium accumulation-associated gene OsHMA3 of rice.

The results in Table 1 show that the identification result of 49 parts of the rice and indica rice materials is 337bp, the allelic type of OsHMA3 is HMA3-1, and 45 parts of the 49 parts of the materials have rice cadmium content lower than the overall average level and account for 91.8%; the results of the 251 materials were 196bp and 141bp, indicating that they have HMA3-2 allele, and 151 of the 251 materials had a rice cadmium content higher than the overall average value, accounting for 60.2%. The molecular marker Caps7 is a molecular marker for identifying the cadmium accumulation varieties of rice, and can be used for predicting and screening the low cadmium accumulation varieties of rice.

Table 1300 shows OsHMA3 genotype of indica rice material and Cd content phenotype of rice

(2) Breeding of low-cadmium accumulated indica rice variety

According to the screening result in the step (1), the inventor selects indica rice variety 93-11 as an improved variety (containing HMA3-2 allelic type), the cadmium content of the variety is high, and the cadmium content of the grain rice is 3.6-4.5 mg/kg. Meanwhile, japonica rice Nipponbare is selected as a donor variety (containing HMA3-1 allelic gene types), and the high cadmium accumulation characteristic of 93-11 rice is improved by a backcross breeding mode.

After hybridization and subsequent three generations of backcross, F1 progeny with OsHMA3 allelic gene type being HMA3-1/HMA3-2 heterozygous state under the genetic background of 93-11 is obtained, and then F1 is separated after selfing.

Through the identification of molecular marker Caps7, two near isogenic line families of HMA3-1 homozygous and HMA3-2 homozygous in 93-11 genetic background are obtained respectively and are marked as NIL (HMA3-1) and NIL (HMA3-2), the Caps7 marker identification gel maps of the two near isogenic lines are shown in figure 4, the NIL (HMA3-1) contains HMA3-1, the gel map is a single band, and the NIL (HMA3-2) contains HMA3-2, the gel map is two bands. The yield and the cadmium accumulation phenotype of the two materials are inspected in a table 2, and the cadmium content of the rice of the NIL (HMA3-1) is remarkably lower than that of the NIL (HMA3-2) and is about 30.7 percent (P is less than 0.01); no obvious difference on the accumulation of zinc and manganese in rice (P > 0.05); the agronomic characters such as plant height, single plant yield and the like have no obvious difference (P is more than 0.05).

The results show that the intragenic molecular marker Caps7 of the rice cadmium accumulation related gene OsHMA3 is an effective molecular marker and can be used for breeding indica rice varieties with low cadmium accumulation.

TABLE 2 comparison of the cadmium accumulation in rice and other elements (Mn, Zn) and agronomic traits for NIL (HMA3-1) and NIL (HMA3-2)

And represent P <0.05, P <0.01 respectively

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

SEQUENCE LISTING

<110> research institute for subtropical agriculture ecology of Chinese academy of sciences

<120> intra-gene molecular marker Caps7 of rice cadmium accumulation related gene OsHMA3 and application thereof

<130> 2018

<160> 5

<170> PatentIn version 3.5

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<213> Rice (Oryza sativa)

<400> 3

ctcgtcggcg gcctcaaggc gaggttcggc ccgacgatga tggttggcga cgggatgaac 60

gacgcggcgg cgctggcggc ggcggacgtg ggcgtgtcca tgggcatctc cgggtcggcg 120

gcggcgatgg agaccagcca cgcgacgctc atgtcgagcg acgtgctcag ggtccccgag 180

gccgtcaggc tcggccggcg cgcccgccgg accatcgccg tcaacgtggc cggctcggtg 240

gccgtgaagg ccgccgtgct cgcgctggcc gcggcgtggc gccccgtgct ctgggccgcc 300

gtgctcgccg acgtcgggac gtgcctcctc gtcgtgc 337

<210> 4

<211> 19

<212> DNA

<213> Rice (Oryza sativa)

<400> 4

ctcgtcagcg gcctcaagg 19

<210> 5

<211> 18

<212> DNA

<213> Rice (Oryza sativa)

<400> 5

gcacgacgag gaggcacg 18

Claims (7)

1. An intragenic molecular marker Caps7 of rice cadmium accumulation related gene OsHMA3, which is characterized in that: the sequence of the molecular marker Caps7 is ctcgtcagcg gcctcaaggc gaggttcggc ccgacgatga tggtcgggga cgggatgaac gacgcggcgg cgctggcggc ggcggacgtg ggcgtgtcca tgggcatctc cgggtcggcg gcggcgatgg agaccagcca cgcgacgctc atgtcgagcg acgtgctcag ggtccccgag gccgtcaggc tcggccggtg cgcccgccgg accatcgccg tcaacgtggc cggctcggtg gccgtgaagg ccgccgtgct cgcgctggcc gcggcgtggc gccccgtgct ctgggccgcc gtgctcgccg acgtcgggac gtgcctcctc gtcgtgc or ctcgtcggcg gcctcaaggc gaggttcggc ccgacgatga tggttggcga cgggatgaac gacgcggcgg cgctggcggc ggcggacgtg ggcgtgtcca tgggcatctc cgggtcggcg gcggcgatgg agaccagcca cgcgacgctc atgtcgagcg acgtgctcag ggtccccgag gccgtcaggc tcggccggcg cgcccgccgg accatcgccg tcaacgtggc cggctcggtg gccgtgaagg ccgccgtgct cgcgctggcc gcggcgtggc gccccgtgct ctgggccgcc gtgctcgccg acgtcgggac gtgcctcctc gtcgtgc

A T or C mutation at position 199 thereof; the gene OsHMA3 comprises an allele HMA3-1 and an allele HMA3-2, and the PCR amplification primer pair of the molecular marker Caps7 can distinguish the sequence difference of the allele HMA3-1 and the allele HMA 3-2;

the PCR amplification primer pair of the molecular marker Caps7 is as follows:

a forward primer F: CTCGTCAGCGGCCTCAAGG, respectively;

reverse primer R: GCACGACGAGGAGGCACG, respectively;

the molecular marker Caps7 appeared as: when the primer pair is used for PCR amplification, the amplification product of the allele HMA3-1 has a nucleotide sequence shown by SEQ ID NO. 2, and the amplification product of the allele HMA3-2 has a nucleotide sequence shown by SEQ ID NO. 3; the allele HMA3-1 shows low cadmium accumulation, and the nucleotide sequence of the allele HMA3-2 is shown as SEQ ID NO. 1 and shows high cadmium accumulation.

2. A method of distinguishing between alleles HMA3-1 and HMA3-2 using the PCR amplification primer pair of molecular marker Caps7 of claim 1, characterized in that: carrying out PCR amplification on the genome DNA of the rice to be detected by using the PCR amplification primer pair of molecular marker Caps7 as claimed in claim 1, carrying out enzyme digestion on the PCR product by using AscI endonuclease, and detecting the amplified product after enzyme digestion by agarose electrophoresis, wherein if the agarose electrophoresis detection shows that the amplified product is a strip and the sequence length is 337bp, the rice to be detected has allele HMA3-1, which shows low cadmium accumulation; if agarose electrophoresis detection shows that the two bands have sequence lengths of 196bp and 141bp respectively, the rice to be detected has allele HMA3-2 which shows high cadmium accumulation, and if the agarose electrophoresis detection shows that the three bands have sequence lengths of 337bp, 196bp and 141bp respectively, and the band with the sequence length of 337bp is brightest, the gene OsHMA3 of the rice to be detected is in a heterozygous state.

3. The use of the PCR amplification primer pair of the molecular marker Caps7 according to claim 1 in the identification of rice cadmium accumulation traits.

4. Use of the PCR amplification primer pair of the molecular marker Caps7 according to claim 1 in breeding of low-cadmium-accumulation indica rice varieties.

5. Use of the method of differentiating between alleles HMA3-1 and HMA3-2 as claimed in claim 2 in the breeding of low cadmium accumulating indica rice varieties.

6. A method for identifying rice cadmium accumulation traits is characterized by comprising the following steps: carrying out PCR amplification on the genomic DNA of the rice to be detected by using the PCR amplification primer pair of the molecular marker Caps7 as claimed in claim 1, carrying out enzyme digestion by using AscI endonuclease, and detecting the length of a PCR product by agarose electrophoresis to determine the cadmium accumulation character of the rice;

if agarose electrophoresis detection shows that the sequence length is 337bp, the rice to be detected has allele HMA3-1, which is expressed as low cadmium accumulation; if agarose electrophoresis detection shows that the two bands have sequence lengths of 196bp and 141bp respectively, the rice to be detected has allele HMA3-2 which shows high cadmium accumulation, and if the agarose electrophoresis detection shows that the three bands have sequence lengths of 337bp, 196bp and 141bp respectively, and the band with the sequence length of 337bp is brightest, the gene OsHMA3 of the rice to be detected is in a heterozygous state.

7. An auxiliary breeding method of low-cadmium accumulation indica rice, which is characterized by comprising the following steps: detecting the molecular marker Caps7 of claim 1 by the method of claim 2 or 6 to determine the indica rice cadmium accumulation trait to be detected.

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