WO2022048070A1 - 水稻镉积累分子标记及其在改良水稻籽粒镉积累上的应用 - Google Patents

水稻镉积累分子标记及其在改良水稻籽粒镉积累上的应用 Download PDF

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WO2022048070A1
WO2022048070A1 PCT/CN2020/139294 CN2020139294W WO2022048070A1 WO 2022048070 A1 WO2022048070 A1 WO 2022048070A1 CN 2020139294 W CN2020139294 W CN 2020139294W WO 2022048070 A1 WO2022048070 A1 WO 2022048070A1
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rice
seq
cadmium
dna molecule
stranded dna
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French (fr)
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李莉
王天抗
宋书锋
李懿星
余应弘
柏连阳
傅岳峰
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湖南杂交水稻研究中心
湖南省农业科学院
岳阳市农业科学研究院
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Priority to US18/024,593 priority Critical patent/US20230323479A1/en
Publication of WO2022048070A1 publication Critical patent/WO2022048070A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to a molecular marker of cadmium accumulation in rice and its application in improving cadmium accumulation in rice grains in the field of biotechnology.
  • Rice is one of the most important food crops in the world, and more than half of the world's population currently eats rice as the staple food.
  • Cadmium is carcinogenic to humans and has been classified as a Class I carcinogen by the International Agency for Research on Cancer.
  • the main sources of cadmium in soil are industrial waste gas, the deposition of automobile exhaust gas, the application of pesticides, fertilizers and agricultural film, and sewage irrigation.
  • Two transport pathways in the body reach the vascular bundle, then transport to the stem and leaves, and then migrate to the grain, and finally accumulate into the human body through the food chain cycle, thus causing serious harm to human health.
  • the first is to use physical and chemical methods to remove cadmium in the soil or to change the form of cadmium in the soil.
  • the second is to control the soil through cultivation measures such as water and fertilizer management
  • the third is to select low-accumulation varieties of grain cadmium from existing rice varieties, and the fourth is to obtain new materials with low grain cadmium accumulation through gene editing technology.
  • these four approaches all have major limitations in application.
  • the cost of remediation of cadmium-contaminated soil is high, and it is easy to cause secondary pollution to the soil; different soil types or rice varieties require different cultivation measures, and the application of The effect is greatly affected by environmental conditions; screening varieties with low cadmium accumulation requires multiple years of verification, and many of the screened varieties have high accumulation of cadmium in grains in heavily cadmium-contaminated fields; New materials with low accumulation of cadmium in grains of polluted fields, but the country has not released the application of gene editing technology in production.
  • Cadmium accumulation in rice grains is a complex trait, which is easily affected by external environment and cultivation measures. Therefore, it is necessary to verify whether rice grains have low cadmium accumulation through years of multi-point verification. Cadmium accumulation in new rice varieties brings great difficulty. Therefore, finding an economical, rapid and efficient method for breeding new rice varieties with low grain cadmium accumulation has become an urgent problem to be solved in production.
  • the technical problem to be solved by the present invention is how to breed new rice varieties with low grain cadmium accumulation economically, rapidly and efficiently.
  • the present invention first provides any of the following applications:
  • the molecular marker for cadmium accumulation in rice is the 8899129-9307609 region of rice chromosome 7 (the reference genome is CANU of Shuhui 498). Version (updated on November 23, 2018, URL: http://www.mbkbase.org/R498/) DNA fragment, the rice cadmium accumulation molecular marker is lcrf1 or lcrf2 or lcrf3, and the lcrf1 is M1) or M2):
  • M1 The DNA fragment shown in rice chromosome 7 8899129-9307609 (the reference genome is the CANU version of Shuhui 498 (updated on November 23, 2018, website: http://www.mbkbase.org/R498/), M2) M1) is subjected to the substitution and/or deletion and/or addition of one or several nucleotides or DNA fragments and has a DNA fragment of 75% or more identity with M1);
  • the lcrf2 is N1) or N2):
  • N1 The DNA fragment shown in SEQ ID No. 1 in the sequence listing, N2) N1) is subjected to substitution and/or deletion and/or addition of one or several nucleotides or DNA fragments and has 75% or DNA fragments that are more than 75% identical;
  • the lcrf3 is O1) or O2):
  • O1 The DNA fragment shown in SEQ ID No. 2 in the sequence listing, O2) O1) is subjected to substitution and/or deletion and/or addition of one or several nucleotides or DNA fragments and has 75% or DNA fragments that are more than 75% identical;
  • nucleotide sequences of M1), N1) and O1) of the present invention can easily mutate the nucleotide sequences of M1), N1) and O1) of the present invention using known methods such as directed evolution and point mutation.
  • Those artificially modified nucleotides having 75% or more identity with the nucleotide sequences of M1), N1) and O1) of the present invention are derived from the nucleotide sequences of the present invention and are equivalent to Sequences of the present invention.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes 75% or more, 80% or more, or 85% or more, or 90% or more with the nucleotide sequences of M1), N1) and O1) of the invention, or Nucleotide sequences of 95% or higher, or 97% or higher, or 98% or higher, or 99% or higher identity. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
  • the substance for detecting the molecular marker of cadmium accumulation in rice is a substance that can detect the molecular marker of cadmium accumulation in rice by conventional experimental methods and can specifically recognize the molecular marker of cadmium accumulation in rice, and the substance can be a substance that can detect the molecular marker of cadmium accumulation in rice.
  • the PCR primers can be part or all of the following (a1), (a2), (a3) and (a4):
  • (a1) is (b1) or (b2) or (b3) as follows:
  • (b2) a single-stranded DNA molecule having SEQ ID No.3 substituted and/or deleted and/or added by one or several nucleotides and having 75% or more identity with (b1);
  • (a2) is (c1) or (c2) or (c3) as follows:
  • (c2) a single-stranded DNA molecule having SEQ ID No.4 substituted and/or deleted and/or added by one or several nucleotides and having 75% or more identity with (c1);
  • (d2) a single-stranded DNA molecule having SEQ ID No.5 substituted and/or deleted and/or added by one or several nucleotides and having 75% or more identity with (d1);
  • (a4) is (e1) or (e2) or (e3) as follows:
  • (e2) a single-stranded DNA molecule with SEQ ID No. 6 subjected to substitution and/or deletion and/or addition of one or several nucleotides and having 75% or more identity with (e1);
  • nucleotide sequences of (b1), (c1), (d1) and (e1) of the present invention can easily mutate the nucleotide sequences of (b1), (c1), (d1) and (e1) of the present invention using known methods such as directed evolution and point mutation.
  • Those artificially modified nucleotides having 75% or more identity to the nucleotide sequences of (b1), (c1), (d1) and (e1) of the present invention are derived from the present invention Nucleotide sequences and are equivalent to the sequences of the present invention.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes 75% or more, 80% or more, or 85% or more with the nucleotide sequences of (b1), (c1), (d1) and (e1) of the invention, or Nucleotide sequences of 90% or higher, or 95% or higher, or 97% or higher, or 98% or higher, or 99% or higher identity. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
  • the stringency conditions may be as follows: 50°C, hybridization in a mixture of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO 4 and 1 mM EDTA, rinsed at 50°C, 2 ⁇ SSC, 0.1% SDS ; Also: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1 mM EDTA, rinse at 50°C, 1 ⁇ SSC, 0.1% SDS; also: 50°C, 7% Hybridization in a mixture of SDS, 0.5M NaPO 4 and 1 mM EDTA, rinsed in 0.5 ⁇ SSC, 0.1% SDS at 50°C; also: 50°C in a mixture of 7% SDS, 0.5M NaPO 4 and 1 mM EDTA Hybridization in solution, rinsed at 50°C, 0.1 ⁇ SSC, 0.1% SDS; alternatively: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M NaPO 4 and
  • the two single-stranded DNAs shown in SEQ ID No.3 and 4 can be amplified to a DNA fragment with a length of 483 bp, and the two single-stranded DNAs shown in SEQ ID No.5 and 6 can be used for amplification.
  • the rice to be tested that can be amplified to a DNA fragment with a length of 494bp is the hybrid rice containing the lcrf1 and the lcrf2; using the rice genomic DNA as a template, using the two single chains shown in SEQ ID No.
  • the tested rice whose DNA can be amplified to a DNA fragment with a length of 483 bp and cannot be amplified to a DNA fragment with a length of 494 bp using the two single-stranded DNAs shown in SEQ ID No. 5 and 6 is the homozygous rice containing the lcrf1 Rice; using rice genomic DNA as a template, the two single-stranded DNAs shown in SEQ ID No.
  • the tested rice whose strand DNA can be amplified to a DNA fragment with a length of 494 bp is the homozygous rice containing the lcrf2.
  • the present invention also provides a method for identifying or assisting in identifying a trait of low cadmium accumulation in rice grains.
  • the DNA fragment determines the grain low cadmium accumulation trait of the rice to be tested.
  • the DNA fragment in the 8899129-9307609 region of rice chromosome 7 is the lcrf1 or the lcrf2, and the cadmium content in the grain of the homozygous rice containing the lcrf2 is lower or is candidate lower than that of the hybrid containing lcrf1 and the lcrf2.
  • the cadmium content in the grain of the homozygous rice containing the lcrf2 is lower or candidate is lower than that of the homozygous rice containing the lcrf1.
  • the method for identifying or assisting the identification of the trait of low cadmium accumulation in rice grains or the application of the lcrf2 in rice breeding also belong to the protection scope of the present invention.
  • the present invention also provides a rice breeding method, which comprises detecting a DNA fragment in the 8899129-9307609 region of chromosome 7 in the rice genome, and selecting the DNA fragment in the 8899129-9307609 region of chromosome 7 as homozygous or heterozygous for the lcrf2 Type rice was bred as a parent.
  • the rice breeding is cultivating rice with low grain cadmium accumulation.
  • the lcrf2 can be introduced into other background rice by breeding methods such as hybridization or backcrossing to select rice with low grain cadmium accumulation.
  • the parent may be a rice male sterile line.
  • the parent is Luohong 3A or Luohong 4A.
  • X1 the substance that detects the molecular marker of cadmium accumulation in rice
  • the substance for detecting the molecular marker of cadmium accumulation in rice can be a reagent, a kit or a system.
  • the system may include a combination of reagents or kits, instruments and analysis software, such as products consisting of PCR primers, reagents for PCR amplification.
  • Figure 1 shows partial results of mf-F and mf-R PCR assays.
  • M is the DNA molecular weight standard (the size of the bands from top to bottom is: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp), and 1 to 24 are different individual plants of the F2 population.
  • Figure 2 shows the partial results of lcrf-F and lcrf-R PCR detection.
  • M is the DNA molecular weight standard band size from top to bottom: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp), 1-16 are different individual plants of the F2 population.
  • Luohong 3A in the following examples (Zhu Renshan, Liu Wenjun, Li Shaoqing, Zhu Yingying, the breeding and utilization of the red lotus-type hybrid rice sterile line Luohong 3A and its combination Luoyou No. 8, Journal of Wuhan University (Science Edition) ), Vol. 59, No. 1, February 2013), the public can obtain the biological material from the applicant, and the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
  • Luohong 4A in the following examples (Zhu Renshan, Huang Wenchao, Hu Jun, Liu Wenjun, Zhu Yingying, the breeding of the new sterile line of red lotus hybrid rice Luohong 4A, Wuhan University Journal (Science Edition), Volume 59 Issue 1, February 2013), the public can obtain the biological material from the applicant, and the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
  • Minghui 63 in the following examples (Wu Fangxi, Cai Qiuhua, Zhu Yongsheng, et al. Utilization and innovation of indica hybrid rice restorer line Minghui 63 [J]. Fujian Agricultural Journal, 2011(06): 1101-1112. ), the public can obtain the biological material from the applicant, and the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
  • Huang Huazhan in the following examples (Pan Fuhe, Wu Duoxin. High-yield cultivation technology of high-quality conventional rice variety Huang Huazhan [J]. Modern Agricultural Science and Technology, 2009, 000(001): 193-193.), the public can apply for The biological material is obtained from a human, and the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
  • the detection method of cadmium content in grains using nitric acid-perchloric acid (4:1, V/V) wet digestion method, using ultrapure water to dissolve, using AA240FS graphite furnace atomic absorption spectrophotometer (VARIAN, USA) , determination of metal content in rice grains.
  • the quality control was carried out with the Chinese national standard material GB W080684 as the internal standard, and blank experiments were carried out throughout the process. All utensils were soaked in 5% nitric acid solution overnight and rinsed with deionized water.
  • the cadmium content in the grains of male sterile lines Luohong 3A and Luohong 4A was extremely low, 0.01 mg/kg and 0.03 mg/kg, respectively, while the grain cadmium content of other rice materials was higher than 0.20 mg. /kg.
  • Luohong 3A, Luohong 4A and other materials were sown in heavily cadmium-contaminated fields (the total cadmium content in the soil was 5.0 mg/kg).
  • Luohong 3A and Luohong 4A still used other fertile materials as male parents. Pollination makes it fruity. Grain cadmium content of each material was determined after harvest. It was found that the grain cadmium content of Luohong 3A and Luohong 4A was also extremely low, 0.03 mg/kg and 0.06 mg/kg, respectively, while the grain cadmium content of other rice varieties was higher than 0.90 mg/kg.
  • the inventors performed third-generation high-depth sequencing and genome de novo assembly and comparative genomics analysis of Luohong 4A. It was found in the 8899129-9307609 region of chromosome 7 of Luohong 4A (the reference genome is the CANU version of Shuhui 498, updated on November 23, 2018, website: http://www.mbkbase.org/R498/), namely A total of 408481bp fragment (named lcrf1) was replaced by a 2980bp fragment (named lcrf2, whose sequence is SEQ ID No. 1 in the sequence listing). Using Sanger sequencing to amplify the 8899129-9307609 region of chromosome 7 of Luohong 3A and its upstream and downstream, it was found that lcrf1 was also replaced by lcrf2.
  • Luohong 3A (as the female parent) was crossed with Minghui 63, and sown after harvesting the seeds to obtain 52 F1 plants. The cadmium content was 0.8 mg/kg) to obtain the F2 population. After transplanting, 1,000 individual plants were taken to extract genomic DNA, and the obtained genomic DNA was used as a template for PCR amplification to detect the genotype of each individual plant, that is, the DNA fragment in the 8899129-9307609 region of chromosome 7.
  • the detection method is: design forward and reverse primers in the 8899129-9307609 region of chromosome 7 (mf-F: 5'-ACTTGACAATCGATCCAACTAGC-3' (SEQ ID No. 3 in the sequence table); mf-R: 5'-CGAAGCTTTGCTGATCGGG-3 ' (SEQ ID No. 4 in the sequence listing)), the full length of the amplified sequence is 483bp.
  • the PCR reaction system is 10 ⁇ l: Gold Mix 8.5 ⁇ l, mf-F 0.5 ⁇ l, mf-R 0.5 ⁇ l, template DNA 0.5 ⁇ l.
  • the PCR amplification program was as follows: pre-denaturation at 98 °C for 2 min; denaturation at 98 °C for 10 s, annealing at 54 °C for 10 s, extension at 72 °C for 10 s, 30 cycles, and extension at 72 °C for 5 min. Then electrophoresis was performed, and the electrophoresis results showed that some samples could amplify a 483bp band, while some samples could not amplify a 483bp band. Among them, the samples that can amplify a 483bp band are homozygous or lcrf1/lcrf2-containing heterozygous individual plants (part of the detection results are shown in Figure 1).
  • the next step is to test and verify, that is, design forward and reverse primers (lcrf-F: 5) on the lcrf2 fragment and the flanks (name the fragment lcrf3, SEQ ID No. 2 in the sequence listing).
  • lcrf-F design forward and reverse primers
  • lcrf-F design forward and reverse primers
  • the PCR reaction system is 10 ⁇ l: Gold Mix 8.5 ⁇ l, lcrf-F 0.5 ⁇ l, lcrf-R 0.5 ⁇ l, template DNA 0.5 ⁇ l.
  • the PCR amplification program was as follows: pre-denaturation at 98 °C for 2 min; denaturation at 98 °C for 10 s, annealing at 55 °C for 10 s, extension at 72 °C for 10 s, 30 cycles, and extension at 72 °C for 5 min. Then, electrophoresis was performed, and the sample that could amplify the 494bp band was a homozygous single plant containing the lcrf2 fragment (see Figure 2 for some detection results).
  • lcrf-F and lcrf-R to amplify the samples that can amplify a 483bp band by PCR.
  • the samples that can amplify a 494bp band are heterozygous single plants containing lcrf1 and lcrf2 fragments, but cannot amplify a 494bp band. Banded samples are homozygous individuals containing the lcrf1 fragment.
  • heterozygous individuals containing lcrf1 and lcrf2 fragments, and homozygous individuals containing lcrf1 fragments, 10 were randomly selected for the 8899129-9307609 region of chromosome 7 (reference genome). It is the CANU version of Shuhui 498, the update date is November 23, 2018, website: http://www.mbkbase.org/R498/) for sequencing, the results show that the homozygous single plant containing the lcrf2 fragment has two chromosomes.
  • This region is lcrf2 fragment, the region of the two chromosomes of the heterozygous single plant containing lcrf1 and lcrf2 fragments is lcrf2 fragment and the other is lcrf1 fragment, the region of the two chromosomes of the homozygous single plant containing lcrf1 fragment is lcrf1 Fragment.
  • F2 population 2 Another part of the F2 seeds were sown into highly cadmium-contaminated fields (the total cadmium content in the soil was 3.5 mg/kg) to obtain F2 population 2. After transplanting, 1000 individual plants were still taken to extract genomic DNA, and the genotype of each individual plant was detected according to the above method.
  • the results show that the homozygous single plant containing lcrf2 fragment has two chromosomes This region is lcrf2 fragment, the region of the two chromosomes of the heterozygous single plant containing lcrf1 and lcrf2 fragments is one lcrf2 fragment and the other is the lcrf1 fragment, and the region of the two chromosomes of the homozygous single plant containing lcrf1 fragment is both lcrf
  • the grains of the 1000 individual plants were harvested by individual plants, and then the cadmium content of the grains of the individual plants was detected.
  • the final results showed that among the 1000 individual plants, there were 255 homozygous individual plants containing lcrf2, and the results of the grain cadmium content per plant ranged from 0.01 to 0.07 mg/kg, with an average of 0.05 mg/kg; heterozygous lcrf1 and lcrf2 contained 502 individual plants, the results of cadmium content in grains per plant ranged from 0.89 to 6.57 mg/kg, with an average of 3.23 mg/kg, which was significantly higher than that of homozygous single plants containing lcrf2; 243 homozygous single plants containing lcrf1
  • the results of the grain cadmium content of each plant and individual plant ranged from 0.81 to 6.62 mg/kg, with an average of 3.27 mg/kg, which was significantly higher than that of the homozygous individual plant containing l
  • Luohong 4A (as the female parent) was crossed with Huanghuazhan; after harvesting, sowing seeds to obtain an F1 population, and backcrossing the F1 population with Huanghuazhan; after harvesting, sowing the seeds to obtain a BC1F1 population, extracting the genomic DNA of a single plant of the BC1F1 population, and using The primers lcrf-F/lcrf-R of Example 1 were used for PCR amplification, and the individual plant corresponding to the sample that could amplify the 494bp band was backcrossed with Huanghuazhan; after sowing, the BC2F1 population was obtained, and the individual plant genome of the BC2F1 population was extracted.
  • the DNA was amplified by PCR using the primers lcrf-F/lcrf-R of Example 1, and the individual plant corresponding to the sample that could amplify the 494bp band was backcrossed with Huang Huazhan; after the seeds were harvested, they were sown again to obtain the BC3F1 population, and extracted
  • the genomic DNA of the individual plant of the BC3F1 population was amplified by PCR using the primers lcrf-F/lcrf-R of Example 1, and the individual plant corresponding to the sample that could amplify the 494bp band was backcrossed with Huang Huazhan;
  • the BC4F1 population was obtained, the genomic DNA of the individual plant of the BC4F1 population was extracted, and the primers lcrf-F/lcrf-R of Example 1 were used for PCR amplification, and the single plant corresponding to the sample that could amplify the 494bp band was cultured for anther to obtain seedlings
  • PCR amplification of lcrf-F/lcrf-R can amplify the single plant corresponding to the sample with a 494bp band, which is a new rice diploid low cadmium material with homozygous lcrf2 fragment, stable comprehensive traits and similar to Huang Huazhan DGHJ.
  • the low-cadmium material DGHJ was sown in heavily cadmium-contaminated fields (the total cadmium content of the soil was 5.0 mg/kg), and Huang Huazhan was sown as a control. After maturity, the grains were harvested to detect the cadmium content. And Huang Huazhan's grain cadmium content was 2.15mg/kg.
  • Luohong 3A (as the female parent) was crossed with Longtefu B; sowing after harvesting to obtain F1 population; sowing after harvesting to obtain F2 population, extracting F2 population individual plant genomic DNA, using the primer lcrf- of Example 1 PCR amplification of F/lcrf-R was performed, and the single plant corresponding to the sample that could amplify the 494bp band was harvested; then sowing, the F3 population was obtained, and the genomic DNA of the individual plant of the F3 population was extracted, and the primer lcrf- of Example 1 was used.
  • F/lcrf-R was amplified by PCR, and the single plant corresponding to the sample that could amplify the 494bp band was harvested; then seeded again to obtain the F4 population, and the genomic DNA of the individual plant of the F4 population was extracted, and the primer lcrf of Example 1 was used.
  • -F/lcrf-R is amplified by PCR, and the single plant corresponding to the sample that can amplify the 494bp band will be harvested; then seeded again to obtain the F4 population, and the genomic DNA of the individual plant of the F4 population will be extracted.
  • the primers mf-F/mf-R were used for PCR amplification, and the samples that could not amplify the 483bp band were then PCR amplified with the primers lcrf-F/lcrf-R of Example 1, and the samples that could amplify the 494bp band were amplified by PCR.
  • the individual plant corresponding to the sample is a homozygous individual plant containing the lcrf2 fragment.
  • the homozygous individual plant containing the lcrf2 fragment is selfed to obtain a stable and comprehensive low-cadmium new material DGZJ.
  • the low-cadmium material DGZJ was sown in the heavily cadmium-contaminated fields (the total cadmium content of the soil was 5.0 mg/kg), and Longtefu B was sown as a control. After maturity, the grains were harvested to detect the cadmium content. /kg, while the grain cadmium content of Longtefu B was 2.73 mg/kg.
  • the present invention finds that the DNA fragment in the 8899129-9307609 region on rice chromosome 7 is related to the accumulation of cadmium in rice grains, and the DNA fragment sequence here is the homozygous rice of SEQ ID No. 1 in the sequence table. Planted in high cadmium polluted fields It has the characteristics of low accumulation of cadmium in grains. Therefore, new rice varieties with low grain cadmium accumulation can be selected by introducing the DNA fragment into other backgrounds by breeding methods such as hybridization and backcrossing.

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Abstract

本发明公开了水稻镉积累分子标记及其在改良水稻籽粒镉积累上的应用。本发明公开的水稻镉积累分子标记为水稻7号染色体8899129-9307609区域的DNA片段，其为lcrf1或lcrf2，lcrf1为水稻7号染色体8899129-9307609所示的DNA片段，所述lcrf2为序列表中SEQ ID No.1所示的DNA片段。本发明发现该水稻镉积累分子标记与水稻籽粒的镉积累有关，水稻镉积累分子标记为序列表中SEQ ID No.1的纯合水稻在高镉污染田种植具有籽粒镉低积累的特性。因此，可以通过杂交、回交等育种方法将该DNA片段导入其他背景来选育籽粒镉低积累的水稻新品种。

Description

水稻镉积累分子标记及其在改良水稻籽粒镉积累上的应用 技术领域
本发明涉及生物技术领域中，水稻镉积累分子标记及其在改良水稻籽粒镉积累上的应用。
背景技术
稻米是世界上最重要的粮食作物之一，目前世界上有一半以上的人口以稻米为主食。镉对人体具有致癌作用，已被国际癌症研究机构列为I类致癌物。土壤中镉的主要来源为工业废气、汽车尾气的沉降，农药、化肥及农膜的施用，污水灌溉等；水稻主要通过根部吸收土壤中的镉，镉被根吸收后通过共质体和质外体两种运输途径抵达维管束，随后向茎叶转运，再迁移到籽粒，最终通过食物链循环富集到人体，从而对人类健康造成严重的危害。
目前主要有以下四种途径来降低水稻籽粒的镉含量，第一是利用物理、化学等方法去除土壤中的镉或改变土壤镉的存在形态，第二是通过水分、肥料管理等栽培措施抑制土壤交换态镉的形成，第三是从现有的水稻品种中筛选出籽粒镉低积累品种，第四是通过基因编辑技术获得籽粒镉低积累新材料。不过这四种途径在应用上都存在较大的局限性，比如镉污染土壤修复的成本较高，而且易对土壤造成二次污染；不同的土壤类型或水稻品种需要不同的栽培措施，而且施用效果受环境条件影响比较大；筛选镉低积累品种，需要经过多年多点的验证，而且许多筛选出来的品种在重度镉污染田会出现籽粒镉高积累；虽然通过基因编辑技术能够获得在重度镉污染田籽粒镉低积累新材料，但是目前国家一直未放开基因编辑技术在生产上的应用。
水稻籽粒镉积累是一个复杂的性状，很容易受外界环境和栽培措施的影响，因此鉴定水稻籽粒是否为镉低积累需要通过多年多点的验证，这给通过杂交、回交等方法选育籽粒镉积累水稻新品种带来了极大的难度。因此，探索出一种能够经济、快速、高效的选育籽粒镉低积累水稻新品种的方法成为了生产上一个亟需解决的问题。
发明公开
本发明所要解决的技术问题是如何经济、快速、高效的选育籽粒镉低积累水稻新品种。
为解决上述技术问题，本发明首先提供了下述任一应用：
1、检测水稻镉积累分子标记的物质在鉴定或辅助鉴定水稻籽粒镉低积累性状中的应用，所述水稻镉积累分子标记为水稻7号染色体8899129-9307609区域(参考基因组为蜀恢498的CANU版本(更新日期为2018年11月23，网址：http://www.mbkbase.org/R498/)的DNA片段，所述水稻镉积累分子标记为lcrf1或lcrf2或lcrf3，所述lcrf1为M1)或M2)：
M1)水稻7号染色体8899129-9307609所示的DNA片段(参考基因组为蜀恢 498的CANU版本(更新日期为2018年11月23，网址：http://www.mbkbase.org/R498/)，M2)将M1)经过一个或几个核苷酸或DNA片段的取代和/或缺失和/或添加且与M1)具有75％或75％以上同一性的DNA片段；
所述lcrf2为N1)或N2)：
N1)序列表中SEQ ID No.1所示的DNA片段，N2)将N1)经过一个或几个核苷酸或DNA片段的取代和/或缺失和/或添加且与N1)具有75％或75％以上同一性的DNA片段；
所述lcrf3为O1)或O2)：
O1)序列表中SEQ ID No.2所示的DNA片段，O2)将O1)经过一个或几个核苷酸或DNA片段的取代和/或缺失和/或添加且与O1)具有75％或75％以上同一性的DNA片段；
2、检测所述水稻镉积累分子标记的物质在制备鉴定或辅助鉴定水稻籽粒镉低积累性状产品中的应用；
3、检测所述水稻镉积累分子标记的物质在水稻育种或制备水稻育种产品中的应用。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的M1)、N1)和O1)的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明的M1)、N1)和O1)的核苷酸序列75％或者更高同一性的核苷酸，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的M1)、N1)和O1)的核苷酸序列具有75％或更高，80％或更高，或85％或更高，或90％或更高，或95％或更高，或97％或更高，或98％或更高，或99％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述应用中，所述检测水稻镉积累分子标记的物质为能用常规的实验方法检测出所述水稻镉积累分子标记的且能特异识别所述水稻镉积累分子标记的物质，该物质可为能区分水稻镉积累分子标记为所述lcrf1和所述lcrf2的PCR引物和/或探针，只要能特异识别本发明的水稻镉积累分子标记，均属于本发明的保护范围。
所述PCR引物具体可为如下(a1)、(a2)、(a3)和(a4)中的部分或全部：
(a1)为如下(b1)或(b2)或(b3)：
(b1)序列表的SEQ ID No.3所示的单链DNA分子；
(b2)将SEQ ID No.3经过一个或几个核苷酸的取代和/或缺失和/或添加且与(b1)具有75％或75％以上同一性的单链DNA分子；
(b3)在严格条件下与(b1)或(b2)限定的核苷酸序列杂交的DNA分子；
(a2)为如下(c1)或(c2)或(c3)：
(c1)序列表的SEQ ID No.4所示的单链DNA分子；
(c2)将SEQ ID No.4经过一个或几个核苷酸的取代和/或缺失和/或添加且与(c1)具有75％或75％以上同一性的单链DNA分子；
(c3)在严格条件下与(c1)或(c2)限定的核苷酸序列杂交的DNA分子；
(a3)为如下(d1)或(d2)或(d3)：
(d1)序列表的SEQ ID No.5所示的单链DNA分子；
(d2)将SEQ ID No.5经过一个或几个核苷酸的取代和/或缺失和/或添加且与(d1)具有75％或75％以上同一性的单链DNA分子；
(d3)在严格条件下与(d1)或(d2)限定的核苷酸序列杂交的DNA分子；
(a4)为如下(e1)或(e2)或(e3)：
(e1)序列表的SEQ ID No.6所示的单链DNA分子；
(e2)将SEQ ID No.6经过一个或几个核苷酸的取代和/或缺失和/或添加且与(e1)具有75％或75％以上同一性的单链DNA分子；
(e3)在严格条件下与(e1)或(e2)限定的核苷酸序列杂交的DNA分子。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的(b1)、(c1)、(d1)和(e1)的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明的(b1)、(c1)、(d1)和(e1)的核苷酸序列75％或者更高同一性的核苷酸，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的(b1)、(c1)、(d1)和(e1)的核苷酸序列具有75％或更高，80％或更高，或85％或更高，或90％或更高，或95％或更高，或97％或更高，或98％或更高，或99％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
所述严格条件可为如下：50℃，在7％十二烷基硫酸钠(SDS)、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，2×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，0.5×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，0.1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在65℃，0.1×SSC，0.1％SDS中漂洗；也可为：在6×SSC，0.5％SDS的溶液中，在65℃下杂交,然后用2×SSC,0.1％SDS和1×SSC,0.1％SDS各洗膜一次；也可为：2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次， 每次15min；也可为：0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
以水稻基因组DNA为模板，利用SEQ ID No.3和4所示的两条单链DNA能扩增到长度为483bp的DNA片段且利用SEQ ID No.5和6所示的两条单链DNA能扩增到长度为494bp的DNA片段的待测水稻为含有所述lcrf1和所述lcrf2的杂合水稻；以水稻基因组DNA为模板，利用SEQ ID No.3和4所示的两条单链DNA能扩增到长度为483bp的DNA片段且利用SEQ ID No.5和6所示的两条单链DNA不能扩增到长度为494bp的DNA片段的待测水稻为含有所述lcrf1的纯合水稻；以水稻基因组DNA为模板，利用SEQ ID No.3和4所示的两条单链DNA不能扩增到长度为483bp的DNA片段且利用SEQ ID No.5和6所示的两条单链DNA能扩增到长度为494bp的DNA片段的待测水稻为含有所述lcrf2的纯合水稻。
本发明还提供了鉴定或辅助鉴定水稻籽粒镉低积累性状的方法，所述方法包括：检测待测水稻7号染色体8899129-9307609区域的DNA片段，根据待测水稻7号染色体8899129-9307609区域的DNA片段确定所述待测水稻的籽粒镉低积累性状。
上述方法中，水稻7号染色体8899129-9307609区域的DNA片段为所述lcrf1或所述lcrf2，含有所述lcrf2的纯合水稻的籽粒镉含量低于或候选低于含有lcrf1和所述lcrf2的杂合水稻，含有所述lcrf2的纯合水稻的籽粒镉含量低于或候选低于含有lcrf1的纯合水稻。
所述鉴定或辅助鉴定水稻籽粒镉低积累性状的方法或所述lcrf2在水稻育种中的应用，也属于本发明的保护范围。
本发明还提供了水稻育种方法，所述方法包括检测水稻基因组中7号染色体8899129-9307609区域的DNA片段，选择7号染色体8899129-9307609区域的DNA片段为所述lcrf2的纯合型或杂合型水稻作为亲本进行育种。
上述方法中，所述水稻育种为培育籽粒镉低积累水稻。
在进行育种时，可通过杂交或回交等育种方法将所述lcrf2导入其他背景水稻中来选育籽粒镉低积累的水稻。
所述亲本可为水稻雄性不育系。在本发明的一个实施例中，所述亲本为珞红3A或珞红4A。
下述X1或X2，也属于本发明的保护范围：
X1、所述检测水稻镉积累分子标记的物质；
X2、所述lcrf2。
本发明中，所述检测水稻镉积累分子标记的物质可为试剂或试剂盒或系统。所述系统可包括试剂或试剂盒、仪器和分析软件的组合产品，如由PCR引物、PCR扩增所用试剂组成的产品。
附图说明
图1为mf-F和mf-R PCR检测的部分结果。M为DNA分子量标准(条带大小从上到下依次为：2000bp,1500bp,1000bp,750bp,500bp,250bp,100bp)，1～24分别为F2群体的不同单株。
图2为lcrf-F和lcrf-R PCR检测的部分结果。M为DNA分子量标准条带大小从上到下依次为：2000bp,1500bp,1000bp,750bp,500bp,250bp,100bp)，1～16分别为F2群体的不同单株。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。下述实施例中，如无特殊说明，序列表中各核苷酸序列的第1位均为相应DNA/RNA的5′末端核苷酸，末位均为相应DNA/RNA的3′末端核苷酸。
下述实施例中的珞红3A(朱仁山，刘文军，李绍清，朱英国，红莲型杂交稻不育系珞红3A及其组合珞优8号的选育与利用，武汉大学学报(理学版)，第59卷第1期，2013年2月)，公众可从申请人处获得该生物材料，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
下述实施例中的珞红4A(朱仁山，黄文超，胡骏，刘文军，朱英国，红莲型杂交稻新不育系珞红4A的选育，武汉大学学报(理学版)，第59卷第1期，2013年2月)，公众可从申请人处获得该生物材料，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
下述实施例中的明恢63(吴方喜,蔡秋华,朱永生,et al.籼型杂交稻恢复系明恢63的利用与创新[J].福建农业学报,2011(06):1101-1112.)，公众可从申请人处获得该生物材料，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
下述实施例中的龙特甫B(胡德辉,刘开雨,周萍,等.分子标记辅助选择改良天B和龙特甫B稻米品质[J].分子植物育种印刷版,2013,11(005):486-493.)，公众可从申请人处获得该生物材料，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
下述实施例中的黄华占(潘甫河,吴多新.优质常规稻新品种黄华占高产栽培技术[J].现代农业科技,2009,000(001):193-193.)，公众可从申请人处获得该生物材料，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
籽粒镉含量的检测方法：采用硝酸-高氯酸(4：1，V/V)湿法消煮，用超纯水定溶后，利用AA240FS型石墨炉原子吸收分光光度计(VARIAN，USA)，测定水稻籽粒的金属含量。以中国国家标准物质GB W080684为内标进行质量控制，同时全程做空白实验。所用器皿均用5％硝酸溶液浸泡过夜，并用去离子水冲洗干净。
金牌Mix：北京擎科生物科技有限公司；货号：TSE101。
实施例1、水稻镉积累分子标记的发现
1、在现有水稻资源中筛选籽粒镉低积累材料
目前已有多个应急性镉低积累水稻杂交组合被报道，但是这些应急性组合在中高镉污染土壤种植籽粒仍然为高积累(高于国家标准上限0.2mg/kg)，为了寻找在中高镉污染土壤种植籽粒仍为低积累的水稻材料，发明人将收集到的来自国内外的275份水稻资源(其中，恢复系162份，不育系35份，国外材料78份)播种到中度镉污染田(土壤全镉含量为0.8mg/kg)，来筛选籽粒镉低积累品种。对于不育系，用其他可育材料作为父本对其授粉使其结实。收获后测定每个材料的籽粒镉含量。发现相对于其他亲本，雄性不育系珞红3A和珞红4A所结籽粒镉含量极低，分别为0.01mg/kg和0.03mg/kg，而其他水稻材料的籽粒镉含量均高于0.20mg/kg。
再将珞红3A和珞红4A及其他材料播种到重度镉污染田(土壤全镉含量为5.0mg/kg)，其中，珞红3A和珞红4A仍用其他可育材料作为父本对其授粉使其结实。收获后测定每个材料的籽粒镉含量。发现珞红3A和珞红4A的籽粒镉含量也极低，分别为0.03mg/kg和0.06mg/kg，而其他水稻品种的籽粒镉含量均高于0.90mg/kg。
2、对珞红4A进行全基因组测序
为了从基因水平上获得珞红4A和珞红3A在重度镉污染田籽粒镉低积累的原因，发明人对珞红4A进行了第三代高深度测序和基因组de novo组装及比较基因组学分析。发现在珞红4A的7号染色体8899129-9307609区域(参考基因组为蜀恢498的CANU版本，更新日期为2018年11月23，网址：http://www.mbkbase.org/R498/)，即共有408481bp的片段(将该片段命名为lcrf1)，被一条2980bp的片段(将该片段命名为lcrf2，其序列为序列表中SEQ ID No.1)替换。利用桑格测序对珞红3A的7号染色体8899129-9307609区域及其上下游进行扩增，发现同样也存在lcrf1被lcrf2替换。
3、lcrf2片段与水稻籽粒镉积累特性关联性分析
将珞红3A(作为母本)与明恢63杂交，收获种子后播种，获得52株F1植株，F1自交，混收获得F2种子，将部分F2种子播种到中度镉污染田(土壤全镉含量为0.8mg/kg)，获得F2群体。插秧后取1000个单株提取基因组DNA，以所得基因组DNA为模板进行PCR扩增检测每个单株的基因型，即7号染色体8899129-9307609区域的DNA片段情况。
检测方法为：在7号染色体8899129-9307609区域内设计正反引物(mf-F： 5'-ACTTGACAATCGATCCAACTAGC-3'(序列表中SEQ ID No.3)；mf-R：5'-CGAAGCTTTGCTGATCGGG-3'(序列表中SEQ ID No.4))，扩增序列全长为483bp。PCR反应体系为10μl：金牌Mix 8.5μl，mf-F 0.5μl，mf-R 0.5μl，模板DNA 0.5μl。PCR扩增程序为：98℃预变性2min；98℃变性10s，54℃退火10s，72℃延伸10s，30次循环，72℃延伸5min。然后进行电泳，电泳结果显示部分样品能扩增出483bp条带，而部分样品不能扩增出483bp条带。其中，能够扩增出483bp条带的样品为含有lcrf1的纯合或lcrf1/lcrf2的杂合单株(部分检测结果见附图1)。
对于不能扩出483bp条带的样品再进行下一步检测验证，即在lcrf2片段及侧翼(将该片段命名为lcrf3，序列表中SEQ ID No.2)分别设计正反引物(lcrf-F：5'-CGCCGAATTGTAGGAGTTG-3'(序列表中SEQ ID No.5)；lcrf-R：5'-GGATGGTTTAGGTGGATGG-3'(序列表中SEQ ID No.6))。扩增序列全长为494bp。PCR反应体系为10μl：金牌Mix 8.5μl，lcrf-F 0.5μl，lcrf-R 0.5μl，模板DNA 0.5μl。PCR扩增程序为：98℃预变性2min；98℃变性10s，55℃退火10s，72℃延伸10s，30次循环，72℃延伸5min。然后进行电泳，能够扩增出494bp条带的样品为含有lcrf2片段的纯合单株(部分检测结果见附图2)。
对能够扩增出483bp条带的样品利用lcrf-F与lcrf-R进行PCR扩增，能扩增出494bp条带的样品为含有lcrf1和lcrf2片段的杂合单株，不能扩增出494bp条带的样品为含有lcrf1片段的纯合单株。
对于所检测出的含有lcrf2片段的纯合单株、含有lcrf1和lcrf2片段的杂合单株、含有lcrf1片段的纯合单株各随机选取10株对其7号染色体8899129-9307609区域(参考基因组为蜀恢498的CANU版本，更新日期为2018年11月23，网址：http://www.mbkbase.org/R498/)进行测序，结果显示，含有lcrf2片段的纯合单株两条染色体的该区域均为lcrf2片段，含有lcrf1和lcrf2片段的杂合单株两条染色体的该区域一条为lcrf2片段，一条为lcrf1片段，含有lcrf1片段的纯合单株两条染色体的该区域均为lcrf1片段。
F2群体成熟后，分单株收获1000个单株的籽粒，然后对单株籽粒进行镉含量检测。结合基因型检测数据，最终结果表明，1000株单株中有含有lcrf2的纯合单株253株，单株籽粒镉含量的结果介于0.01～0.04mg/kg，平均为0.02mg/kg；含有lcrf1与lcrf2的杂合单株498株，单株籽粒镉含量的结果介于0.21～0.57mg/kg，平均为0.39mg/kg，显著高于含有lcrf2的纯合单株籽粒镉含量；含有lcrf1的纯合单株249株，单株籽粒镉含量的结果介于0.23～0.63mg/kg，平均为0.41mg/kg，显著高于含有lcrf2的纯合单株籽粒镉含量。含有lcrf1与lcrf2的杂合单株与含有lcrf1的纯合单株籽粒镉含量无显著差异。
之后将另一部分F2种子播种到高度镉污染田(土壤全镉含量为3.5mg/kg)， 获得F2群体2。插秧后依然取1000个单株提取基因组DNA，按照上述方法检测每个单株的基因型。而后对于所检测出的含有lcrf2片段的纯合单株、含有lcrf1和lcrf2片段的杂合单株、含有lcrf1片段的纯合单株各随机选取10株对其7号染色体8899129-9307609区域(参考基因组为蜀恢498的CANU版本，更新日期为2018年11月23，网址：http://www.mbkbase.org/R498/)进行测序，结果显示，含有lcrf2片段的纯合单株两条染色体的该区域均为lcrf2片段，含有lcrf1和lcrf2片段的杂合单株两条染色体的该区域一条为lcrf2片段，一条为lcrf1片段，含有lcrf1片段的纯合单株两条染色体的该区域均为lcrf1片段。F2群体2成熟后，分单株收获该1000个单株的籽粒，然后对单株籽粒进行镉含量检测。最终结果表明，1000株单株中，含有lcrf2的纯合单株255株，单株籽粒镉含量的结果介于0.01～0.07mg/kg，平均为0.05mg/kg；含有lcrf1与lcrf2的杂合单株502株，单株籽粒镉含量的结果介于0.89～6.57mg/kg，平均为3.23mg/kg，显著高于含有lcrf2的纯合单株籽粒镉含量；含有lcrf1的纯合单株243株，单株籽粒镉含量的结果介于0.81～6.62mg/kg，平均为3.27mg/kg，显著高于含有lcrf2的纯合单株籽粒镉含量。含有lcrf1与lcrf2的杂合单株与含有lcrf1的纯合单株籽粒镉含量无显著差异。
以上研究表明，7号染色体8899129-9307609区域lcrf1被lcrf2替换为水稻在重度镉污染田籽粒镉低积累的原因，这两个片段可以作为水稻镉积累分子标记用于水稻育种。
实施例2、水稻镉积累分子标记在水稻育种中的应用
1、水稻低镉材料DGHJ的制备
将珞红4A(作为母本)与黄华占杂交；收种后播种，获得F1群体，将F1群体与黄华占进行回交；收种后播种，获得BC1F1群体，提取BC1F1群体单株基因组DNA，利用实施例1的引物lcrf-F/lcrf-R进行PCR扩增，将能够扩增出494bp条带的样品对应的单株与黄华占回交；收种后播种，获得BC2F1群体，提取BC2F1群体单株基因组DNA，利用实施例1的引物lcrf-F/lcrf-R进行PCR扩增，将能够扩增出494bp条带的样品对应的单株与黄华占回交；收种后再次播种，获得BC3F1群体，提取BC3F1群体单株基因组DNA，利用实施例1的引物lcrf-F/lcrf-R进行PCR扩增，将能够扩增出494bp条带的样品对应的单株与黄华占回交；收种后再次播种，获得BC4F1群体，提取BC4F1群体单株基因组DNA，利用实施例1的引物lcrf-F/lcrf-R进行PCR扩增，将能够扩增出494bp条带的样品对应的单株进行花药培养，获得幼苗后，提取基因组DNA，对所得基因组DNA先用实施例1的引物mf-F/mf-R进行PCR扩增，将不能扩增出483bp条带的样品对应的单株再用实施例1的引物lcrf-F/lcrf-R进行PCR扩增，能够扩增出494bp条带的样品所对应的单株，即为lcrf2片段纯合、综合性状稳定且与黄华占相似的水稻二倍体低镉新材料DGHJ。
对低镉新材料DGHJ的7号染色体8899129-9307609区域(参考基因组为蜀 恢498的CANU版本，更新日期为2018年11月23，网址：http://www.mbkbase.org/R498/)进行测序，结果显示，其两条染色体的该区域均为lcrf2片段。
而后将低镉材料DGHJ播种在重度镉污染田(土壤全镉含量为5.0mg/kg)，同时播种黄华占作为对照，成熟后收获籽粒检测镉含量，发现DGHJ的籽粒镉含量为0.04mg/kg，而黄华占的籽粒镉含量为2.15mg/kg。
2、水稻低镉新材料DGZJ的制备
将珞红3A(作为母本)与龙特甫B杂交；收种后播种，获得F1群体；收种后播种，获得F2群体，提取F2群体单株基因组DNA，利用实施例1的引物lcrf-F/lcrf-R进行PCR扩增，将能够扩增出494bp条带的样品对应的单株收种；然后播种，获得F3群体，提取F3群体单株基因组DNA，利用实施例1的引物lcrf-F/lcrf-R进行PCR扩增，将能够扩增出494bp条带的样品对应的单株收种；然后再次播种，获得F4群体，提取F4群体单株基因组DNA，利用实施例1的引物lcrf-F/lcrf-R进行PCR扩增，将能够扩增出494bp条带的样品对应的单株收种；然后再次播种，获得F4群体，提取F4群体单株基因组DNA，先用实施例1的引物mf-F/mf-R进行PCR扩增，将不能扩增出483bp条带的样品再用实施例1的引物lcrf-F/lcrf-R进行PCR扩增，能够扩增出494bp条带的样品所对应的单株，即为含有lcrf2片段的纯合单株，将该含有lcrf2片段的纯合单株再通过自交，获得稳定的综合性状优异的低镉新材料DGZJ。
对低镉新材料DGZJ的7号染色体8899129-9307609区域(参考基因组为蜀恢498的CANU版本，更新日期为2018年11月23，网址：http://www.mbkbase.org/R498/)进行测序，结果显示，其两条染色体的该区域均为lcrf2片段。
而后将低镉材料DGZJ播种在重度镉污染田(土壤全镉含量为5.0mg/kg)，同时播种龙特甫B作为对照，成熟后收获籽粒检测镉含量，发现DGZJ的籽粒镉含量为0.05mg/kg，而龙特甫B的籽粒镉含量为2.73mg/kg。
各序列如下：
Figure PCTCN2020139294-appb-000001
Figure PCTCN2020139294-appb-000002
Figure PCTCN2020139294-appb-000003
Figure PCTCN2020139294-appb-000004
Figure PCTCN2020139294-appb-000005
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
工业应用
本发明发现在水稻7号染色体上的8899129-9307609区域的DNA片段与水稻籽粒的镉积累有关，此处的DNA片段序列为序列表中SEQ ID No.1的纯合水稻在高镉污染田种植具有籽粒镉低积累的特性。因此，可以通过杂交、回交等育种方法将该DNA片段导入其他背景来选育籽粒镉低积累的水稻新品种。

Claims (15)

  1. 检测水稻镉积累分子标记的物质在鉴定或辅助鉴定水稻籽粒镉低积累性状中的应用，所述水稻镉积累分子标记为水稻7号染色体8899129-9307609区域的DNA片段，所述水稻镉积累分子标记为lcrf1或lcrf2，所述lcrf1为M1)或M2)：
    M1)水稻7号染色体8899129-9307609所示的DNA片段，M2)将M1)经过一个或几个核苷酸或DNA片段的取代和/或缺失和/或添加且与M1)具有75％或75％以上同一性的DNA片段；
    所述lcrf2为N1)或N2)：
    N1)序列表中SEQ ID No.1所示的DNA片段，N2)将N1)经过一个或几个核苷酸或DNA片段的取代和/或缺失和/或添加且与N1)具有75％或75％以上同一性的DNA片段。
  2. 根据权利要求1所述的应用，其特征在于：所述检测水稻镉积累分子标记的物质为能区分水稻镉积累分子标记为所述lcrf1和所述lcrf2的PCR引物。
  3. 根据权利要求2所述的应用，其特征在于：所述PCR引物为如下(a1)、(a2)、(a3)和(a4)中的部分或全部：
    (a1)为如下(b1)或(b2)或(b3)：
    (b1)序列表的SEQ ID No.3所示的单链DNA分子；
    (b2)将SEQ ID No.3经过一个或几个核苷酸的取代和/或缺失和/或添加且与(b1)具有75％或75％以上同一性的单链DNA分子；
    (b3)在严格条件下与(b1)或(b2)限定的核苷酸序列杂交的DNA分子；
    (a2)为如下(c1)或(c2)或(c3)：
    (c1)序列表的SEQ ID No.4所示的单链DNA分子；
    (c2)将SEQ ID No.4经过一个或几个核苷酸的取代和/或缺失和/或添加且与(c1)具有75％或75％以上同一性的单链DNA分子；
    (c3)在严格条件下与(c1)或(c2)限定的核苷酸序列杂交的DNA分子；
    (a3)为如下(d1)或(d2)或(d3)：
    (d1)序列表的SEQ ID No.5所示的单链DNA分子；
    (d2)将SEQ ID No.5经过一个或几个核苷酸的取代和/或缺失和/或添加且与(d1)具有75％或75％以上同一性的单链DNA分子；
    (d3)在严格条件下与(d1)或(d2)限定的核苷酸序列杂交的DNA分子；
    (a4)为如下(e1)或(e2)或(e3)：
    (e1)序列表的SEQ ID No.6所示的单链DNA分子；
    (e2)将SEQ ID No.6经过一个或几个核苷酸的取代和/或缺失和/或添加且与(e1)具有75％或75％以上同一性的单链DNA分子；
    (e3)在严格条件下与(e1)或(e2)限定的核苷酸序列杂交的DNA分子。
  4. 检测权利要求1中所述水稻镉积累分子标记的物质在制备鉴定或辅助鉴定水稻籽粒镉低积累性状产品中的应用。
  5. 根据权利要求4所述的应用，其特征在于：所述检测水稻镉积累分子标记的物质为能区分水稻镉积累分子标记为所述lcrf1和所述lcrf2的PCR引物。
  6. 根据权利要求5所述的应用，其特征在于：所述PCR引物为为如下(a1)、(a2)、(a3)和(a4)中的部分或全部：
    (a1)为如下(b1)或(b2)或(b3)：
    (b1)序列表的SEQ ID No.3所示的单链DNA分子；
    (b2)将SEQ ID No.3经过一个或几个核苷酸的取代和/或缺失和/或添加且与(b1)具有75％或75％以上同一性的单链DNA分子；
    (b3)在严格条件下与(b1)或(b2)限定的核苷酸序列杂交的DNA分子；
    (a2)为如下(c1)或(c2)或(c3)：
    (c1)序列表的SEQ ID No.4所示的单链DNA分子；
    (c2)将SEQ ID No.4经过一个或几个核苷酸的取代和/或缺失和/或添加且与(c1)具有75％或75％以上同一性的单链DNA分子；
    (c3)在严格条件下与(c1)或(c2)限定的核苷酸序列杂交的DNA分子；
    (a3)为如下(d1)或(d2)或(d3)：
    (d1)序列表的SEQ ID No.5所示的单链DNA分子；
    (d2)将SEQ ID No.5经过一个或几个核苷酸的取代和/或缺失和/或添加且与(d1)具有75％或75％以上同一性的单链DNA分子；
    (d3)在严格条件下与(d1)或(d2)限定的核苷酸序列杂交的DNA分子；
    (a4)为如下(e1)或(e2)或(e3)：
    (e1)序列表的SEQ ID No.6所示的单链DNA分子；
    (e2)将SEQ ID No.6经过一个或几个核苷酸的取代和/或缺失和/或添加且与(e1)具有75％或75％以上同一性的单链DNA分子；
    (e3)在严格条件下与(e1)或(e2)限定的核苷酸序列杂交的DNA分子。
  7. 检测权利要求1中所述水稻镉积累分子标记的物质在水稻育种或制备水稻育种产品中的应用。
  8. 根据权利要求7所述的应用，其特征在于：所述检测水稻镉积累分子标记的物质为能区分水稻镉积累分子标记为所述lcrf1和所述lcrf2的PCR引物。
  9. 根据权利要求8所述的应用，其特征在于：所述PCR引物为为如下(a1)、(a2)、(a3)和(a4)中的部分或全部：
    (a1)为如下(b1)或(b2)或(b3)：
    (b1)序列表的SEQ ID No.3所示的单链DNA分子；
    (b2)将SEQ ID No.3经过一个或几个核苷酸的取代和/或缺失和/或添加且与(b1)具有75％或75％以上同一性的单链DNA分子；
    (b3)在严格条件下与(b1)或(b2)限定的核苷酸序列杂交的DNA分子；
    (a2)为如下(c1)或(c2)或(c3)：
    (c1)序列表的SEQ ID No.4所示的单链DNA分子；
    (c2)将SEQ ID No.4经过一个或几个核苷酸的取代和/或缺失和/或添加且与(c1)具有75％或75％以上同一性的单链DNA分子；
    (c3)在严格条件下与(c1)或(c2)限定的核苷酸序列杂交的DNA分子；
    (a3)为如下(d1)或(d2)或(d3)：
    (d1)序列表的SEQ ID No.5所示的单链DNA分子；
    (d2)将SEQ ID No.5经过一个或几个核苷酸的取代和/或缺失和/或添加且与(d1)具有75％或75％以上同一性的单链DNA分子；
    (d3)在严格条件下与(d1)或(d2)限定的核苷酸序列杂交的DNA分子；
    (a4)为如下(e1)或(e2)或(e3)：
    (e1)序列表的SEQ ID No.6所示的单链DNA分子；
    (e2)将SEQ ID No.6经过一个或几个核苷酸的取代和/或缺失和/或添加且与(e1)具有75％或75％以上同一性的单链DNA分子；
    (e3)在严格条件下与(e1)或(e2)限定的核苷酸序列杂交的DNA分子。
  10. 鉴定或辅助鉴定水稻籽粒镉低积累性状的方法，包括：检测待测水稻7号染色体8899129-9307609区域的DNA片段，根据待测水稻7号染色体8899129-9307609区域的DNA片段确定所述待测水稻的籽粒镉低积累性状。
  11. 权利要求10所述的方法或权利要求1中所述lcrf2在水稻育种中的应用。
  12. 水稻育种方法，包括：检测水稻基因组中7号染色体8899129-9307609区域的DNA片段，选择7号染色体8899129-9307609区域的DNA片段为权利要求1中所述lcrf2的纯合型或杂合型水稻作为亲本进行育种。
  13. 根据权利要求12所述的方法，其特征在于：所述水稻育种为培育籽粒镉低积累水稻。
  14. 权利要求1-3中任一所述检测水稻镉积累分子标记的物质。
  15. 权利要求1中所述lcrf2。
PCT/CN2020/139294 2020-09-04 2020-12-25 水稻镉积累分子标记及其在改良水稻籽粒镉积累上的应用 WO2022048070A1 (zh)

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