WO2019056205A1 - 包含短n导入片段的抗tmv的烟草植株及其选育方法 - Google Patents

包含短n导入片段的抗tmv的烟草植株及其选育方法 Download PDF

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WO2019056205A1
WO2019056205A1 PCT/CN2017/102444 CN2017102444W WO2019056205A1 WO 2019056205 A1 WO2019056205 A1 WO 2019056205A1 CN 2017102444 W CN2017102444 W CN 2017102444W WO 2019056205 A1 WO2019056205 A1 WO 2019056205A1
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tobacco
primer pair
plants
tmv
genotype
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PCT/CN2017/102444
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English (en)
French (fr)
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刘勇
黄昌军
李永平
于海芹
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云南省烟草农业科学研究院
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Priority to US16/648,284 priority Critical patent/US11259473B2/en
Priority to PCT/CN2017/102444 priority patent/WO2019056205A1/zh
Publication of WO2019056205A1 publication Critical patent/WO2019056205A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
    • A01H1/126—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance for virus resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/12—Leaves
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • A01H6/823—Nicotiana, e.g. tobacco
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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

Definitions

  • the invention relates to the field of tobacco breeding, in particular to an anti-TMV tobacco plant comprising a short N introduced fragment and a breeding method thereof.
  • TMV Tobacco mosaic virus
  • the current TMV resistance source for growing tobacco is mainly derived from Nicotiana glutionsa, whose resistance is controlled by a dominant single gene (N).
  • the N gene was cloned in 1994 and is the first NBS resistance gene cloned in plants.
  • the genomic sequence size of the N gene is 6656 bp, including 5 exons and 4 introns, belonging to the TIR-NBS-LRR type disease resistance gene.
  • the mechanism of disease resistance of the N gene is the appearance of allergic necrotic spots (spotted spots) at the site of virus infection, and the movement of TMV in the plant body is restricted by the induced cell allergic death.
  • N gene is transferred from the heart leaf tobacco to the oriental tobacco and then transferred to the tobacco variety.
  • the resistance of the N gene is transferred by cross breeding, and the chromosome fragment of the heart leaf containing the N gene (abbreviated as N introgression segment) is actually transferred.
  • N introgression segment the chromosome fragment of the heart leaf containing the N gene
  • the N-introduced fragment of Coker 176 is a non-target gene component (abbreviated as a cumbersome gene component) linked to the N gene on the N-introduced fragment of the same length. Reducing the length of the non-target gene component linked to the N gene on the N-introduced fragment of Coker176 is expected to reduce the linkage of the N-introduced fragment.
  • the N-introduced fragment has low homology with the common tobacco chromosome, and the N-introduced fragment is difficult to exchange chromosomes with ordinary tobacco (a probability of about 1/2000). Even if exchange occurs, conventional breeding techniques cannot select a chromosome-exchanged plant with a short N-introduced fragment (Ns segment) in the breeding population, because the cumulative phenotype is susceptible to environmental conditions and is difficult to select in the early stage of breeding. In addition, the lack of technical means for detecting non-target gene components linked to the N gene on the N-introduced fragment leads to a lack of breakthrough in anti-TMV tobacco breeding. Therefore, how to overcome the shortcomings of the prior art is an urgent problem to be solved in the field of tobacco anti-TMV breeding technology.
  • the object of the present invention is to solve the deficiencies of the prior art and to provide an anti-TMV tobacco plant comprising a short N introduced fragment and a method for breeding the same.
  • the anti-TMV tobacco plant containing the short N introduced fragment lacks the accumulation gene component linked to the N gene, has the effect of reducing the linkage of the N-introduced fragment, and can be used for breeding high anti-TMV without obvious yield and quality disadvantage. Tobacco variety.
  • N-introduced fragment refers to a fragment of a wild smoke chromosome comprising a TMV resistance gene (N gene).
  • short N-introduced fragment refers to a DNA fragment in which a non-target gene component (abbreviated as a cumbersome gene component) linked to an N gene on a N-introduced fragment is partially deleted or deleted in its entirety, and retains the function of the intact N gene.
  • a non-target gene component abbreviated as a cumbersome gene component
  • complex gene component refers to a genomic component comprising an accumulated gene on the N-introduced fragment but not comprising the N gene.
  • Another object of the present invention is to provide a method of obtaining an anti-TMV-resistant tobacco plant comprising a short N introduced fragment.
  • a further object of the invention is the use of an anti-TMV tobacco plant comprising a short N introduced fragment for breeding a tobacco resistant TMV variety.
  • the present invention provides an TMV-resistant tobacco plant comprising a short N-introduced fragment that is reduced by at least 0.51 Mb, preferably at least 0.93 Mb, more preferably at least 1.56 Mb, compared to the N-introduced fragment of Coker 176 type tobacco. Cumulative gene components.
  • the short N introduced fragment reduces at least a partial or total sequence of the sequence set forth in SEQ ID No. 13 compared to the N introduced fragment of Coker 176 type tobacco.
  • the short N-introduced fragment reduces at least a sequence from 806912 base to 1404293 base of the sequence of SEQ ID No. 13 as compared to the N-introduced fragment of Coker 176 type tobacco. .
  • the TMV-resistant tobacco plant is selected from the group consisting of:
  • N1N2 marker detection was positive, and TN4.99 primer pair, TN5.20 primer pair, TN5.34 primer pair and TN5.51 primer pair were negative for the detection of tobacco plants.
  • the short N introduced fragment is obtained by a method of chromosome exchange, genome editing, chemical mutagenesis or physical mutagenesis.
  • the TMV-resistant tobacco plant is derived from Nicotiana, preferably from flue-cured tobacco, burley tobacco, oriental tobacco, sun-cured tobacco or cigar.
  • the present invention provides tobacco hybrids, varieties or strains selected using tobacco plants resistant to TMV containing short N introduced fragments.
  • the present invention provides seeds, pollen and ovules of tobacco plants resistant to TMV comprising short N-introduced fragments.
  • the invention also provides the use of an anti-TMV tobacco plant comprising a short N introduced fragment for breeding a tobacco resistant TMV variety.
  • the present invention also provides a method of breeding an anti-TMV-resistant tobacco plant comprising a short N introduced fragment, comprising the steps of:
  • step b) inoculation of TMV in the seedling stage of the population material obtained in step a), and screening the Nn genotype plants exhibiting plaques in the population material;
  • step b) genotyping the Nn genotypes selected by step b) using the molecular marker TN5.51 primer pair at the right end of the N-introduced fragment and the N gene-specific molecular marker N1N2, and screening for N1N2 molecular marker detection as positive and TN5 .51 Primer pairs were tested negative for plants.
  • the present invention also provides a method of breeding an anti-TMV-resistant tobacco plant comprising a short N introduced fragment, comprising the steps of:
  • step b) inoculation of TMV in the seedling stage of the population material obtained in step a), and screening the Nn genotype plants exhibiting plaques in the population material;
  • step b) genotyping the Nn genotypes selected by step b) using TN5.34 primer pair, TN5.51 primer pair, and N gene-specific molecular marker N1N2, screening for N1N2 molecular marker detection as positive, and TN5
  • the .34 primer pair and the TN5.51 primer pair were negative for the detection of the plant.
  • the present invention also provides a method of breeding an anti-TMV-resistant tobacco plant comprising a short N introduced fragment, comprising the steps of:
  • Tobacco plants homozygous with N-introduced fragments are crossed with tobacco plants of genotype nn to obtain F1 tobacco plants with genotype Nn, and then F1 tobacco plants and tobacco with genotype nn The grass plants are crossed to obtain a population material for screening short N-introduced fragments;
  • step b) inoculation of TMV in the seedling stage of the population material obtained in step a), and screening the Nn genotype plants exhibiting plaques in the population material;
  • step b) genotyping the Nn genotypes selected by step b) using TN5.20 primer pair, TN5.34 primer pair, TN5.51 primer pair and N gene specific molecular marker N1N2, screening N1N2 molecular marker detection Positive, and TN5.20 primer pair, TN5.34 primer pair and TN5.51 primer pair were negative for the detection of plants.
  • the present invention also provides a method of breeding an anti-TMV-resistant tobacco plant comprising a short N introduced fragment, comprising the steps of:
  • step b) inoculation of TMV in the seedling stage of the population material obtained in step a), and screening the Nn genotype plants exhibiting plaques in the population material;
  • step b) genotyping the Nn genotypes selected by step b) using TN4.99 primer pair, TN5.20 primer pair, TN5.34 primer pair, TN5.51 primer pair and N gene specific molecular marker N1N2, The N1N2 molecular marker was detected as positive, and the TN4.99 primer pair, TN5.20 primer pair, TN5.34 primer pair and TN5.51 primer pair were negative.
  • the invention also provides for the use of a short N introduced fragment in a tobacco plant, the short N introduced fragment having a reduced genome of at least 0.51 Mb, preferably at least 0.93 Mb, more preferably at least 1.56 Mb compared to the N introduced fragment of Coker 176 type tobacco. Minute.
  • the short N introduced fragment reduces at least a partial or total sequence of the sequence set forth in SEQ ID No. 13 compared to the N introduced fragment of Coker 176 type tobacco.
  • the application results in a tobacco plant selected from the group consisting of:
  • N1N2 marker detection was positive, and TN4.99 primer pair, TN5.20 primer pair, TN5.34 primer pair and TN5.51 primer pair were negative for the detection of tobacco plants.
  • the short N introduced fragment is obtained by a method of chromosome exchange, genome editing, chemical mutagenesis or physical mutagenesis.
  • the tobacco plant is derived from the genus Nicotiana, preferably from flue-cured tobacco, burley tobacco, oriental tobacco, sun-cured tobacco or cigar.
  • the present invention also provides tobacco hybrids, varieties or strains selected from tobacco plants obtained according to the above application.
  • the present invention also provides seeds, pollen and ovules of tobacco plants obtained according to the above application.
  • a N-introduced homozygous tobacco refers to a tobacco comprising an N-introduced fragment, preferably a N-introduced length of a common tobacco of the Coker 176 type, including but not limited to flue-cured tobacco, burley tobacco, sun-cured tobacco , tobacco types such as oriental tobacco and cigars.
  • the feature of the N-introduced fragment length type of Coker 176 type was that the GL4.06 primer pair was negative and the TN5.51 primer pair was positive.
  • N-introduced lengths of common tobacco of the Coker 176 type include, but are not limited to, 6349, 7402, 7915, 8100, 8211, 8212, 78-3013, 8902-42, B09, Burley 21, B22, B49, B64, Banket-A-1, BG4, BYS, CV85, CV87, Coker 86, Coker 51, Coker 176, EMH14, Ergo, ETWM10, GH12, K10, K14, Ky10, Ky12, Ky 41A, Ky14, Ky15, Ky17, Ky171, Ky56, Ky8959, Ky8959 ( BC4), Ky9, Ky907 (BC4), Ky908, MS ky17, MBN2, MS Burley 21 ⁇ Ky14, MRS-3, MS B21, MS Ky10, MSKy14 ⁇ L8, NC3, NC567, NC7, NC86, PVH01, PVH02, PVH05, PVH06, PVH07, PVH08, RGH04, SC71,
  • Tobacco with genotype nn refers to tobacco that does not contain the N gene.
  • the preferred scheme refers to common tobacco without N gene, including but not limited to common tobacco types such as flue-cured tobacco, burley tobacco, sun-cured tobacco, oriental tobacco and cigar. .
  • the phenotypic traits of tobacco with genotype nn include disease resistance, high yield, high index, easy roasting, leaf quality, plant height, mature characteristics, early maturity to late maturity, and the number of plant leaves is moderate to high.
  • the tobacco having a genotype of nn preferably includes, but is not limited to, K326, Yunyan 87, Yunyan 97, Yunyan 85, NC89, Zhongyan 100, Honghua Daijinyuan, and Cuibi No. 1.
  • Tobacco hybrids varieties or strains consist of plants containing short N introduced fragments. Compared with the plant containing the N-introduced fragment, the plant containing the short N-introduced fragment was reduced in anti-TMV and the non-target gene component linked to the N gene was reduced, and the plant of the short N-introduced fragment was at least deleted as shown in SEQ ID No. Part to all sequences of the sequence.
  • the plants of the short N-introduced fragments were negative for both the GL4.06 primer pair and the TN5.51 primer pair.
  • the plant is tested negative using a TN4.99 primer pair or a TN5.30 primer pair or a TN5.34 primer pair.
  • a method of obtaining at least part of the sequence of the sequence of SEQ ID No. 13 in which the plant of the short N introduced fragment is obtained comprises obtaining a Nn population material by a hybrid breeding method, and screening the chromosome exchange plant therefrom. Also included are genome editing methods using chemical mutagenesis methods or physical mutagenesis methods, and biotechnology. A chemical mutagenesis method or a physical mutagenesis method is used to screen a partial to all sequence-deleted mutant of the sequence of SEQ ID No. 13. The method of chemical mutagenesis includes treatment with a mutagen such as sodium azide, ethidium bromide or ethyl methanesulfonate.
  • a mutagen such as sodium azide, ethidium bromide or ethyl methanesulfonate.
  • Physical mutagenesis methods include X-rays, gamma rays, fast neutron radiation, heavy ion radiation, and ultraviolet radiation.
  • Biotechnology genome editing methods include the use of CRISPR/Cas9 technology, zinc finger endonuclease (ZFN) technology
  • ZFN zinc finger endonuclease
  • the transcriptional activator-like effector nuclease (TALEN) technique knocks out part to all of the sequence of the sequence shown in SEQ ID No. 13.
  • the length of the partial sequence is an appropriate length, and is not particularly limited, and is, for example, 1 bp, 10 bp, 100 bp, 1 Kb, 10 Kb, 100 Kb, 1000 Kb, or 2000 Kb in length.
  • Screening for anti-TMV-infected tobacco plants using hybridization and molecular marker selection techniques includes the following steps: Tobacco plants that are homozygous for the N-introduced fragment and that are negative for the GL4.06 primer pair and positive for the TN5.51 primer pair Hybridization with tobacco plants of genotype nn yielded progeny tobacco plants with genotype Nn. The progeny plants of Nn were crossed with the tobacco plants of genotype nn to obtain a population material for screening short N-introduced fragments. TMV was inoculated at the seedling stage, and Nn genotype plants showing plaques were screened in the population materials.
  • the DNA was extracted, and the Nn genotype was genotyped by the molecular marker TN5.51 primer pair and the N gene-specific molecular marker N1N2 at the right end of the N-introduced fragment.
  • the TN5.51 primer pair was screened negative and the N1N2 molecular marker was A positive plant is a plant with a short N introduced fragment.
  • the progeny of Nn are crossed with tobacco of genotype nn, and a population material for screening short N-introduced fragments is obtained.
  • the population material includes a breeding population material comprising the Nn genotype.
  • Preferred are backcross population materials BC1F1, BC2F1, BC3F1, BC4F1, BC5F1 ... BCnF1 materials.
  • n represents the number of generations of backcrossing, which can be 6, 7, 8, 9, 10, 15, 20.
  • Other group materials are also included, such as self-crossing population material F2 containing the Nn genotype.
  • Methods for screening Nn genotypes from breeding population materials containing Nn genotypes include artificial inoculation of TMV, selection methods based on disease resistance phenotypes, and molecular marker-assisted selection methods.
  • a preferred method for selecting disease resistance phenotypes comprises: inoculating TMV at seedling stage, screening out the representative plaque plants in the population material, and screening the plants for the short N introduced fragments.
  • the molecular marker-assisted selection method includes detecting the molecular marker of the N gene in the breeding population material or the molecular marker closely linked to the N gene, and selecting the N-positive plant for screening the short N-introduced plant according to the molecular marker detection result.
  • a method for screening a plant having a short N introduced fragment includes using a N-introduced fragment-specific molecular marker, or a molecular marker closely linked to the N-introduced fragment, or closely linked to the N-introduced fragment The phenotypic tag of the lock.
  • a preferred method is to use a molecular marker introduced into the end of the fragment. The preferred method is to use the TN5.51 primer pair to amplify the molecular marker.
  • Methods for detecting molecular markers include PCR detection methods, molecular hybridization, and the like.
  • a preferred method is a PCR detection method.
  • the PCR amplification product detection method includes an electrophoresis detection method.
  • Electrophoretic detection methods include agarose gel electrophoresis and fragment analyzer detection.
  • the molecular hybridization method comprises hybridizing a nucleic acid sample of a population material by using a TN5.51 primer pair to amplify a molecularly labeled partial nucleic acid fragment as a probe.
  • the invention also discloses a method of breeding a short N-introduced anti-TMV tobacco plant.
  • the short N-introduced fragment-resistant TMV tobacco plants were selected from the hybrid progeny by hybridization with the above-mentioned short N-introduced fragment anti-TMV tobacco plants and another tobacco plant having a genotype of nn. Selection of breeding lines containing short N-introduced anti-TMV tobacco lines or varieties is selected by conventional breeding methods.
  • the phenotype of another plant including but not limited to disease resistance, high yield.
  • Hybridization methods include cross-pollination or pollen donors to hybridize with pollen receptors (male fertile or male sterile) to obtain hybrid or male sterile hybrids. One or both of the pollen receptor or the pollen donor comprises a short N introduced fragment.
  • the DNA fragment of the nucleotide sequence shown by SEQ ID NO. 13 disclosed in the present invention is a heart leaf smoke chromosome fragment closely linked to the N gene.
  • the use of cross breeding to transfer the resistance of the N gene is actually the transfer of a chromosome fragment containing the N gene.
  • the sequence shown in SEQ ID NO. 13 is a partial sequence closely linked to the N gene on the N-introduced fragment. Specifically, it is a non-target gene component linked to the N gene.
  • the gene component comprising the sequence shown in SEQ ID NO. 13 brings a chain of entanglement in common tobacco. Knocking out part or all of the sequence of the sequence shown in SEQ ID NO. 13 reduces linkage cumber.
  • the length of the upstream and downstream sequences of the 5' end and/or the 3' end of SEQ ID NO. 13 is an appropriate length, and is not particularly limited, for example, a length greater than 1 Kb, greater than 10 Kb, greater than 50 Kp, greater than 100 Kb, greater than 500 Kb, greater than 1000 Kb, greater than 2000Kb or more than 5000Kb.
  • the invention also discloses that the above-mentioned tobacco plants resistant to TMV containing short N introduced fragments are selected. Application of breeding tobacco to TMV varieties.
  • the present invention provides an anti-TMV tobacco plant comprising a short N introduced fragment.
  • the molecular marker of the present invention is used to reduce the non-target gene component linked to the N gene on the N-introduced fragment, rather than to evaluate N-mediated resistance, compared to the N-gene resistance-related molecular marker that has been reported in the literature. Whether there is.
  • Conventional breeding techniques fail to select chromosome exchange plants for short N introduced fragments in the breeding population.
  • the invention can select a chromosome-exchanged plant with a short N-introduced fragment from a large number of breeding population materials by formulating a breeding and isolation population, using N right-end molecular marker TN5.51, N gene-specific molecular marker and SEQ ID NO.
  • the anti-TMV plant containing the short N introduced fragment obtained by the invention is used for creating a novel anti-TMV germplasm resource and breeding the tobacco anti-TMV variety, which is beneficial to reduce the cumbersome linkage with the N gene.
  • 1 is an electrophoretic detection result of an amplification product of a short N-introduced fragment plant using N1N2, TN4.99, TN5.20, TN5.34, and TN5.51 primer pairs;
  • the first, sixth, eleventh, sixteenth lane, and the 21st lane are 100 bp DNA Ladder; the second to fifth lanes are primer pair N1N2 amplification Coker 176, 15-7F, 24-8H, 99-2D; lanes 7 to 10 TN4.99 amplified Coker176, 15-7F, 24-8H, 99-2D; lanes 12 to 15 were primer pairs TN5.20 amplification Coker176, 15-7F, 24-8H, 99-2D; lanes 17 to 20 are primer pair TN5.34 amplification Coker176, 15-7F, 24-8H, 99-2D; lanes 22 to 25 are primer pairs TN5.51 Coker 176, 15-7F, 24-8H, 99-2D were amplified.
  • the above tobacco plants are common tobacco germplasm resources, and the public can obtain them from the tobacco germplasm resources conservation unit or the Yunnan Tobacco Agricultural Science Research Institute.
  • the DNA extraction kit was purchased from QIAGEN, and DNA Marker and Taq DNA polymerase were purchased from Dalian Bao Biotech Co., Ltd. Other chemical reagents are commercially available products. Table 1 lists the primers used in the present invention:
  • Coker176 (genotype is NN, GL4.06 primer pair is negative and TN5.51 primer pair is positive) and Yunyan 87 (genotype is nn).
  • Coker 176 pollen is collected during flowering to the emasculated Yunyan 87 flower.
  • F1 seeds were harvested (genotype is Nn).
  • DNA was extracted using the DNeasy plant 96 Plant Kit (QIAGEN, Catalogue no. 69181) with reference to the reagent instructions.
  • Tobacco cultivar Yunyan 87 with TMV was used as a susceptibility control, and the tobacco variety Coker176, which contains the N-introduced fragment anti-TMV, was used as a disease-resistant control; the TN5.51 primer pair was used to detect the tobacco genomic DNA and the susceptible control.
  • Tobacco genomic DNA and disease-resistant tobacco genomic DNA were used as templates, PCR amplification, and amplification products were detected by electrophoresis;
  • the PCR reaction system is as follows:
  • the total volume is 20 ⁇ L
  • primer primer 10umol / ⁇ L each 1.5 ⁇ L specifically: the concentration of the upstream and downstream primers in the primer pair is 10umol / ⁇ L, the amount is 1.5 ⁇ L.
  • the primer pair is a TN5.51 primer pair.
  • the reagents used were purchased from Bao Bio.
  • the PCR reaction procedure was as follows: predenaturation at 94 ° C for 5 minutes; denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 30 seconds, running for 35 cycles; and finally extending at 72 ° C for 10 minutes.
  • the PCR amplification product can be stored at 4 °C.
  • PCR product electrophoresis detection electrophoresis was carried out with a mass percentage of 1.2% agarose gel 120v for 25 min, EB staining for 10 min, and the gel was recorded and recorded. The results are shown in Fig. 1.
  • N gene-specific molecular marker N1N2 according to the literature method (Lewis, RS, SRMilla, and JS Levin. Molecular and genetic characterization of Nicotiana glutinosa L. chromosome segments in tobacco mosaic virus-resistant tobacco accessions. Crop Sci. 2005, 45: 2355–2362.) PCR amplification performed.
  • Plants with negative TN5.51 primer pair and positive N1N2 marker detection were selected from tobacco plants, and then TN4.99 primer pair and TN5.20 primer pair and TN5.34 primer pair were used to estimate the plant deletion gene component. size.
  • the TN5.51 primer pair was used for amplification.
  • the anti-disease control had 845 bp amplification product, and the susceptible control had no 845 bp amplification product, indicating that the PCR amplification was normal.
  • the chromosome exchange plants containing the short N introduced fragment were then screened according to the following criteria: N1N2 positive and no 845 bp amplification product, initially The plant was judged to be a chromosome exchange plant containing a short N introduced fragment; N1N2 was positive and had an 845 bp amplification product, and it was judged that the chromosome exchange of the N introduced fragment did not occur in the plant.
  • Tobacco variety Yunyan 87 with TMV was used as a susceptibility control
  • tobacco cultivar Coker176 which is known to contain N gene anti-TMV, was used as a disease-resistant control
  • 3 plant DNAs with N1N2 positive and no 845 bp amplification products, and susceptible tobacco Genomic DNA and disease-resistant tobacco genomic DNA are used as templates, PCR amplification, and amplification products for electrophoresis detection;
  • the PCR reaction system is as follows:
  • the total volume is 20 ⁇ L
  • the primer pair is a TN4.99 primer pair or a TN5.20 primer pair or a TN5.34 primer pair.
  • the reagents used were purchased from Bao Bio.
  • the PCR reaction procedure was as follows: predenaturation at 94 ° C for 5 minutes; denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 30 seconds, running for 35 cycles; and finally extending at 72 ° C for 10 minutes.
  • PCR The amplified product can be stored at 4 °C.
  • PCR product electrophoresis detection electrophoresis was carried out with a mass percentage of 1.2% agarose gel 120v for 25 min, EB staining for 10 min, and the gel was recorded and recorded. The results are shown in Fig. 1.
  • TN4.99 primer pair, TN5.20 primer pair, TN5.34 primer pair and N1N2 primer pair were amplified respectively.
  • the anti-disease control had the amplification products of the test primer pair, and the susceptible control did not expand the test primer pair. Add product. This indicates that the PCR amplification is normal.
  • the gene component of the chromosome exchange plant containing the short N introduced fragment is then estimated according to the following criteria: if the TN5.34 primer pair is negative, the plant lacks the genome between the TN5.34 corresponding marker and the TN5.51 corresponding marker. Minute. If both the TN5.20 primer pair and the TN5.4 primer pair were negative, the plant lacked the gene component between the TN5.20 corresponding marker and the TN5.51 corresponding marker. If both the TN4.99 and TN5.20 primer pairs and the TN5.4 primer pair were negative, the plant lacked the genetic component between the corresponding marker of TN4.99 and the corresponding marker of TN5.51.
  • the physical distance of chromosome 11 is estimated to be 3 times the size of the tomato genome by the size of the tobacco wild species, and the N-introduction of the 24-8H plant is shortened by at least 1.56 Mb.
  • the physical distance of the TN5.20 primer pair corresponding to tomato chromosome 11 was 5.20 Mb
  • the physical distance of TN5.51 corresponding marker to tomato chromosome 11 was 5.51 Mb
  • the 15-7F plant was estimated to be at least 0.31 Mb shorter (relative tomato).
  • the physical distance of chromosome 11 is estimated by the fact that the size of the tobacco wild species genome is 3 times that of the tomato genome, and the 15-7F plant N introduced fragment is shortened by at least 0.93 Mb.
  • the physical distance of the amplified marker of TN5.34 primer pair to tomato chromosome 11 is 5.34Mb, and TN5.51 is stained with tomato 11th.
  • the physical distance of the color body is 5.51Mb, and it is estimated that the 99-2D plant is shortened by at least 0.17Mb (relative to the physical distance of tomato chromosome 11).
  • the tomato genome size is estimated to be 3 times, 99- The 2D plant N introduced fragment was shortened by at least 0.51 Mb.
  • BC6F1 backcross six generation
  • Anti-TMV plants (genotype Nn) were screened in the 5F x Coker 176BC6F1 population according to the method of Example 1, and DNA was extracted.
  • the short N introduced fragment plants were screened according to the method of Example 2. The results showed that 9,984 strains showing plaques were selected from BC6F1 plants of 20160 strain 5F ⁇ Coker176 by inoculation with TMV. The leaves of the plants showing the plaques were collected and DNA was extracted. Using TN5.51 labeling and N1N2 labeling detection, 1-1002 plants were screened from 9612 copies of DNA to be N1N2 positive and no 845 bp amplification product. The plant is a chromosome exchange plant comprising a short N introduced fragment.
  • the number of non-target gene components linked to the N gene of 1-1002 plants was measured.
  • the results showed that the 1-1002 plants were negative by TN4.99 primer pair and TN5.20 primer pair and TN5.34 primer pair, and positive by N1N2 primer pair.
  • the physical distance of the amplified pair of TN4.99 primers relative to tomato chromosome 11 was 4.99 Mb
  • the physical distance of TN5.51 to tomato chromosome 11 was 5.51 Mb
  • the number of 1-1002 plants was estimated to be at least 0.52 Mb (relative to tomato).
  • the physical distance of chromosome 11 according to the large genome of tobacco wild species The small is 3 times the size of the tomato genome, and the 1-1-1 plant N-introduced fragment is shortened by at least 1.56 Mb.
  • the plant was self-bred and 5F backcrossed, and 5F ⁇ 1-1002BC7F1 seeds were harvested for the linkage ⁇ phenotypic detection of short N-introduced fragments, ie, the yield of the short N-introduced plants and the chlorophyll content of the upper leaves in the mature stage.
  • 5F ⁇ 1-1002BC7F1 strain 1/2 is a plant containing Ns segment
  • 5FN BC4F6 strain N segment homozygous
  • 5F nn
  • the N-positive plants that is, the plants containing the Ns segment in the 5F ⁇ 1-1002BC7F1 strain, were selected from the 5F ⁇ 1-1002BC7F1 strain by the N1N2 marker-assisted selection before transplanting. Planted in the field, topping the same day on flowering. The leaves of 18-21 leaves can be collected uniformly, and the middle leaves of the third leaf are sampled from top to bottom. The measurement and sampling were started every 7 days from the 5th week after the topping, and a total of 4 samples were taken.
  • the yield of 5F ⁇ 1-1002BC7F1 strain was equivalent to 5F, and the yield of 5F ⁇ 1-1002BC7F1 strain was higher than that of 5FN.
  • the chlorophyll content of the 5F ⁇ 1-1002BC7F1 strain before the upper leaves were the same as the 5F content.
  • the chlorophyll content of the 5F ⁇ 1-1002BC7F1 before the upper leaves were lower than that of the 5FN.
  • the linkage traits of the 1-1002 strain containing the short N-introduced anti-TMV were clearly improved.

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Abstract

包含短N导入片段的抗TMV的烟草植株及其选育方法，利用N导入片段纯合的烟草与基因型为nn的烟草杂交，获得基因型为Nn的F1子代烟草。利用F1子代烟草与基因型为nn的烟草杂交，获得用于筛选短N导入片段的群体材料。苗期接种TMV，在群体材料中筛选出表现枯斑的Nn基因型植株。采用N导入片段右末端的分子标记TN5.51引物对和N基因特异分子标记N1N2对Nn基因型植株进行基因型分型，TN5.51引物对检测为阴性且N1N2分子标记为阳性的植株，就是短N导入片段的植株。利用TN5.34和TN5.20和TN4.99引物对估算短N导入片段的植株缺失的非目的基因组分数量。获得的短N导入片段的植株可应用于烟草TMV抗病资源创制和品种选育，有利于减少与N基因连锁的累赘。

Description

包含短N导入片段的抗TMV的烟草植株及其选育方法 技术领域
本发明涉及烟草育种领域，特别涉及包含短N导入片段的抗TMV的烟草植株及其选育方法。
背景技术
烟草花叶病毒病(Tobacco mosaic virus,TMV)是烟草(Nicotiana tabacum L.)上的重要病害，种植TMV抗病品种依然是防控TMV最根本同时也是最经济有效的手段。推广的抗病品种需要为抗性高、且无产量劣势和农艺性状劣势的品种。
目前栽培烟草的TMV抗源主要来源于烟草野生种心叶烟(Nicotana glutionsa)，其抗性由一个显性单基因(N)控制。N基因在1994年被克隆，是植物中克隆的第一个NBS类抗病基因。N基因抗TMV-U1株系。N基因的基因组序列大小为6656bp，包括5个外显子和4个内含子，属于TIR-NBS-LRR类型抗病基因。N基因的抗病机理为在病毒侵染位点出现过敏性坏死斑(枯斑)，通过诱导产生的细胞过敏性死亡限制TMV在植物体内的移动。在介导了过敏反应后，烟草植株能够获得系统性抗性，对TMV或其它类似病原的再次入侵产生广谱抗性。通过一系列的常规杂交和回交转育，N基因的抗性从心叶烟转育至香料烟中，然后转育至烟草品种中。采用杂交育种转育N基因的抗性，实际上是转育包含N基因的心叶烟染色体片段(简称为N导入片段，N introgression segment)。世界上抗TMV烟草育种利用的几乎都是N导入片段。代表性品种是较早商业化种植的包含N导入片段的抗TMV烟草品种Coker176和Speight H20。由于产量较低、上部叶落黄较慢等连锁累赘，包含与Coker176的N导入片段长度相同的N导入片段的烟草品种不能满足生产的迫切需要。已有研究证明N基因本身无产量和产值的连锁累赘，连锁累赘来源于与 Coker176的N导入片段长度相同的N导入片段上与N基因连锁的非目的基因组分(简称累赘基因组分)。减少长度同Coker176的N导入片段上与N基因连锁的非目的基因组分，可望减少N导入片段的连锁累赘。但是N导入片段与普通烟草染色体的同源性较低，N导入片段难以与普通烟草发生染色体交换(约1/2000的概率)。即使发生了交换，常规育种技术也无法选择出育种群体中短N导入片段(Ns segment)的染色体交换植株，因为累赘表型为数量性状易受环境条件影响，在育种早期难以选择。另外，检测N导入片段上与N基因连锁的非目的基因组分的技术手段缺乏，导致抗TMV烟草育种缺乏突破性进展。因此如何克服现有技术的不足是目前烟草抗TMV育种技术领域亟需解决的问题。
发明内容
本发明的目的是为了解决现有技术的不足，提供包含短N导入片段的抗TMV的烟草植株及其选育方法。该包含短N导入片段的抗TMV的烟草植株缺失了与N基因连锁的累赘基因组分，具有减少N导入片段的连锁累赘的效果，可用于选育出高抗TMV，且无明显产量和质量劣势的烟草品种。
在本发明的上下文中，术语“N导入片段”是指包含TMV抗病基因(N基因)的野生烟染色体片段。
术语“短N导入片段”是指N导入片段上与N基因连锁的非目的基因组分(简称累赘基因组分)部分缺失或全部缺失，并保留完整N基因功能的DNA片段。
术语“累赘基因组分”是指N导入片段上包含累赘基因、但不包含N基因的基因组组分。
本发明的另一目的是提供获得包含短N导入片段的抗TMV的烟草植株的方法。
本发明的再一目的是包含短N导入片段的抗TMV的烟草植株在选育烟草抗TMV品种中的应用。
本发明提供一种抗TMV的烟草植株，其包含短N导入片段，所述短N导入片段与Coker176类型烟草的N导入片段相比减少至少0.51Mb、优选至少0.93Mb、更优选至少1.56Mb的累赘基因组分。
在本发明的一个优选实施方案中，所述短N导入片段与Coker176类型烟草的N导入片段相比至少减少SEQ ID No.13所示序列的部分序列或全部序列。
在本发明的另一个优选实施方案中，所述短N导入片段与Coker176类型烟草的N导入片段相比至少减少SEQ ID No.13所示序列的第806912位碱基至1404293位碱基的序列。
在本发明的另一个优选实施方案中，所述的抗TMV的烟草植株，其选自下列植株：
a)N1N2标记检测为阳性，且TN5.34引物对和TN5.51引物对检测均为阴性的烟草植株；
b)N1N2标记检测为阳性，且TN5.20引物对、TN5.34引物对和TN5.51引物对检测均为阴性的烟草植株；
c)N1N2标记检测为阳性，且TN4.99引物对、TN5.20引物对、TN5.34引物对和TN5.51引物对检测均为阴性的烟草植株。
在本发明的另一个优选实施方案中，所述短N导入片段通过染色体交换、基因组编辑、化学诱变或物理诱变的方法获得。
在本发明的又一个优选实施方案中，所述的抗TMV的烟草植株，其来源于烟草属，优选来源于烤烟、白肋烟、香料烟、晒晾烟或雪茄烟。
本发明提供利用包含短N导入片段的抗TMV的烟草植株选育出的烟草杂交种、品种或株系。
本发明提供包含短N导入片段的抗TMV的烟草植株的种子、花粉和胚珠。
本发明还提供了包含短N导入片段的抗TMV的烟草植株在选育烟草抗TMV品种中的应用。
本发明还提供了选育包含短N导入片段的抗TMV的烟草植株的方法，其包含以下步骤：
a)利用N导入片段纯合的烟草植株与基因型为nn的烟草植株杂交，获得基因型为Nn的F1烟草植株，再将F1烟草植株与基因型为nn的烟草植株杂交，获得用于筛选短N导入片段的群体材料；
b)在步骤a)获得的群体材料的苗期接种TMV，在群体材料中筛选出表现枯斑的Nn基因型植株；
c)采用N导入片段右末端的分子标记TN5.51引物对和N基因特异分子标记N1N2对步骤b)筛选出的Nn基因型植株进行基因型分型，筛选出N1N2分子标记检测为阳性且TN5.51引物对检测为阴性的植株。
本发明还提供了选育包含短N导入片段的抗TMV的烟草植株的方法，其包含以下步骤：
a)利用N导入片段纯合的烟草植株与基因型为nn的烟草植株杂交，获得基因型为Nn的F1烟草植株，再将F1烟草植株与基因型为nn的烟草植株杂交，获得用于筛选短N导入片段的群体材料；
b)在步骤a)获得的群体材料的苗期接种TMV，在群体材料中筛选出表现枯斑的Nn基因型植株；
c)采用TN5.34引物对、TN5.51引物对、和N基因特异分子标记N1N2对步骤b)筛选出的Nn基因型植株进行基因型分型，筛选出N1N2分子标记检测为阳性，且TN5.34引物对和TN5.51引物对检测均为阴性的植株。
本发明还提供了选育包含短N导入片段的抗TMV的烟草植株的方法，其包含以下步骤：
a)利用N导入片段纯合的烟草植株与基因型为nn的烟草植株杂交，获得基因型为Nn的F1烟草植株，再将F1烟草植株与基因型为nn的烟 草植株杂交，获得用于筛选短N导入片段的群体材料；
b)在步骤a)获得的群体材料的苗期接种TMV，在群体材料中筛选出表现枯斑的Nn基因型植株；
c)采用TN5.20引物对、TN5.34引物对、TN5.51引物对和N基因特异分子标记N1N2对步骤b)筛选出的Nn基因型植株进行基因型分型，筛选出N1N2分子标记检测为阳性，且TN5.20引物对、TN5.34引物对和TN5.51引物对检测均为阴性的植株。
本发明还提供了选育包含短N导入片段的抗TMV的烟草植株的方法，其包含以下步骤：
a)利用N导入片段纯合的烟草植株与基因型为nn的烟草植株杂交，获得基因型为Nn的F1烟草植株，再将F1烟草植株与基因型为nn的烟草植株杂交，获得用于筛选短N导入片段的群体材料；
b)在步骤a)获得的群体材料的苗期接种TMV，在群体材料中筛选出表现枯斑的Nn基因型植株；
c)采用TN4.99引物对、TN5.20引物对、TN5.34引物对、TN5.51引物对和N基因特异分子标记N1N2对步骤b)筛选出的Nn基因型植株进行基因型分型，筛选出N1N2分子标记检测为阳性，且TN4.99引物对、TN5.20引物对、TN5.34引物对和TN5.51引物对检测均为阴性的植株。
本发明还提供了短N导入片段在烟草植株中的应用，所述短N导入片段与Coker176类型烟草的N导入片段相比减少至少0.51Mb、优选至少0.93Mb、更优选至少1.56Mb的累赘基因组分。
在本发明的一个优选实施方案中，所述短N导入片段与Coker176类型烟草的N导入片段相比至少减少SEQ ID No.13所示序列的部分序列或全部序列。
在本发明的一个优选实施方案中，所述应用得到选自以下的烟草植株：
a)N1N2标记检测为阳性，且TN5.34引物对和TN5.51引物对检测均为阴性的烟草植株；
b)N1N2标记检测为阳性，且TN5.20引物对、TN5.34引物对和TN5.51引物对检测均为阴性的烟草植株；
c)N1N2标记检测为阳性，且TN4.99引物对、TN5.20引物对、TN5.34引物对和TN5.51引物对检测均为阴性的烟草植株。
在本发明的一个优选实施方案中，所述短N导入片段通过染色体交换、基因组编辑、化学诱变或物理诱变的方法获得。
在本发明的一个优选实施方案中，所述烟草植株其来源于烟草属，优选来源于烤烟、白肋烟、香料烟、晒晾烟或雪茄烟。
本发明还提供了根据上述应用而得到的烟草植株选育出的烟草杂交种、品种或株系。
本发明还提供了根据上述应用而得到的烟草植株的种子、花粉和胚珠。
在本发明的上下文中，N导入片段纯合的烟草是指包含N导入片段的烟草，优选方案是N导入片段长度同Coker176类型的普通烟草，包括但不限于烤烟、白肋烟、晒晾烟，香料烟和雪茄烟等烟草类型。N导入片段长度类型为Coker176类型的特征为GL4.06引物对检测为阴性且TN5.51引物对检测为阳性。N导入片段长度同Coker176类型的普通烟草包括但不限于6349，7402，7915，8100，8211，8212，78-3013，8902-42，B09，Burley21，B22，B49，B64，Banket-A-1，BG4，BYS，CV85，CV87，Coker 86，Coker 51，Coker 176，EMH14，Ergo，ETWM10，GH12，K10，K14，Ky10，Ky12，Ky 41A，Ky14，Ky15，Ky17，Ky171，Ky56，Ky8959，Ky8959(BC4)，Ky9，Ky907(BC4)，Ky908，MS ky17，MBN2，MS Burley21×Ky14，MRS-3，MS B21，MS Ky10，MSKy14ΧL8，NC3，NC567，NC7，NC86，PVH01，PVH02，PVH05，PVH06，PVH07，PVH08，RGH04，SC71，SC72，TN97，Vamoor48，Virginia 1，Virginia3160，Virginia645，Virginia770，Virginia80，Va 1048，WE-12，WB68，RT32，巴引一号，大白筋2518，辽烟9号，辽烟14号，牡单80-7，牡单81-56，台烟5号，台烟6号，梧桐烟1012，中卫一号，引巴1，云烟202402， 521，911，8358，Blue star100，Burley mammoth ky16，Burley21，C6160，CCC-h，CCC-L，Coker51，Ex12，Gen164，Gen224，Gr38a，Havana 425，Havana 426，Havana 503b，Havanan 503，Holmes breeding line-1，KHD926，KY165，KY21，KY22，KY35，KY8654，lancaster seed，M-1，Massck-1，MD A30，MD B100，MD40，MD402，Metacomet，MRS-1，MRS-3，NC102，NC297，NC-BMR 90，NIC 112，NIC 112B(PL10)，NIC 112b(PL11)，NIC112C-G(PL271)，NIC 112C-G(PL37)，NIC 117D-1B，OS 802，Poquonock，Southern beauty，Vamorr48，WB68。
基因型为nn的烟草是指不含有N基因的烟草，优选方案是指不含有N基因的普通烟草，包括但不限于烤烟、白肋烟、晒晾烟，香料烟和雪茄烟等普通烟草类型。基因型为nn的烟草具有的表型性状包括病害抗性、高产、高级指、易烤性、叶片品质、株高、成熟特性早熟至晚熟、植株叶片数中等至多。基因型为nn的烟草优选的包括但不限于：K326，云烟87，云烟97，云烟85，NC89，中烟100，红花大金元，翠碧1号。
烟草杂交种、品种或株系由包含短N导入片段的植株组成。与包含N导入片段的植株相比，包含短N导入片段的植株抗TMV、且与N基因连锁的非目的基因组分减少，所述短N导入片段的植株至少缺失了SEQ ID No.13所示序列的部分至全部序列。所述短N导入片段的植株采用GL4.06引物对和TN5.51引物对检测都为阴性。优选为所述植株采用TN4.99引物对或TN5.30引物对或TN5.34引物对检测为阴性。
获得所述短N导入片段的植株至少缺失了SEQ ID No.13所示序列的部分至全部序列的方法，包括采用杂交育种方法获得Nn群体材料，从中筛选染色体交换植株。也包括采用化学诱变方法或者物理诱变方法、生物技术的基因组编辑方法。化学诱变方法或者物理诱变方法，筛选出SEQ ID No.13所示序列的部分至全部序列缺失的突变体。化学诱变的方法包括叠氮化钠、溴化乙锭、甲基磺酸乙酯等诱变剂处理。物理诱变方法包括X-射线、伽马射线、快中子辐射、重离子辐射和紫外线辐射。生物技术的基因组编辑方法包括采用CRISPR/Cas9技术、锌指核酸内切酶(ZFN)技 术、类转录激活因子效应物核酸酶(TALEN)技术敲除SEQ ID No.13所示序列的部分至全部序列。所述部分序列的长度为适当长度，并不特别限定，例如长度为1bp、10bp、100bp、1Kb、10Kb、100Kb、1000Kb或者2000Kb全长。
采用杂交和分子标记选择技术筛选包含短N导入片段的抗TMV的烟草植株包括如下步骤：利用N导入片段纯合且GL4.06引物对检测为阴性且TN5.51引物对检测为阳性的烟草植株与基因型为nn的烟草植株杂交，获得基因型为Nn的子代烟草植株。利用Nn的子代植株与基因型为nn的烟草植株杂交，获得用于筛选短N导入片段的群体材料。苗期接种TMV，在群体材料中筛选出表现枯斑的Nn基因型植株。提取DNA，采用N导入片段右末端的分子标记TN5.51引物对和N基因特异分子标记N1N2对Nn基因型植株进行基因型分型，筛选出TN5.51引物对检测为阴性且N1N2分子标记为阳性的植株，就是短N导入片段的植株。
Nn的子代与基因型为nn的烟草杂交，获得用于筛选短N导入片段的群体材料。群体材料包括包含Nn基因型的育种群体材料。优选的为回交群体材料BC1F1，BC2F1，BC3F1，BC4F1，BC5F1……BCnF1材料。n表示回交的世代数，可以是6，7，8，9，10，15，20。也包括其他群体材料，比如包含Nn基因型的自交群体材料F2。
从包含Nn基因型的育种群体材料中筛选出Nn基因型植株的方法，包括人工接种TMV，根据抗病表型挑选方法，也包括分子标记辅助选择方法。优选的抗病表型挑选方法包括：苗期接种TMV，在群体材料中筛选出表现枯斑植株，用于筛选短N导入片段的植株。分子标记辅助选择方法包括检测育种群体材料中N基因的分子标记或者与N基因紧密连锁的分子标记，根据分子标记检测结果，挑选出N基因阳性的植株用于筛选短N导入片段的植株。
筛选短N导入片段的植株的方法包括采用N导入片段特异的分子标记，或者与N导入片段紧密连锁的分子标记，或者与N导入片段紧密连 锁的表型标记。优选方法为采用N导入片段末端的分子标记。优中选优的方法为采用TN5.51引物对扩增的分子标记。
分子标记的检测方法包括PCR检测方法、分子杂交等方法。优选方法为PCR检测方法。PCR扩增产物检测方法包括电泳检测方法。电泳检测方法包括琼脂糖凝胶电泳、片段分析仪检测。分子杂交方法包括采用TN5.51引物对扩增的分子标记的部分核酸片段作为探针，杂交筛选群体材料的核酸样品。
本发明还公开选育包含短N导入片段抗TMV烟草植株的方法。利用上述包含短N导入片段抗TMV烟草植株与另一基因型为nn的烟草植株杂交，在杂交后代中选择含短N导入片段抗TMV烟草植株。通过常规育种方法，选育出选育包含短N导入片段抗TMV烟草品系或品种。另一植株的表型，包括但不限于抗病、高产。杂交方法包括自花授粉或花粉供体与花粉受体(雄性可育或雄性不育)杂交，获得杂交种或雄性不育杂交种。花粉受体或者花粉供体二者之一或者二者包含短N导入片段。
在本发明公开的SEQ ID NO.13所示核苷酸序列的DNA片段，是与N基因紧密连锁的心叶烟染色体片段。采用杂交育种转育N基因的抗性，实际上是转育包含N基因的心叶烟染色体片段。SEQ ID NO.13所示序列为所述N导入片段上与N基因紧密连锁的部分序列。具体为与N基因连锁的非目的基因组分。包含SEQ ID NO.13所示序列的基因组分在普通烟草中带来连锁累赘。敲除SEQ ID NO.13所示序列的部分序列或全部序列，可减少连锁累赘。本领域技术人员可以理解，在敲除SEQ ID NO.13所示序列的部分序列或全部序列时，同时敲除SEQ ID NO.13的5’端和/或3’端的上下游序列同样能达到本发明的效果。SEQ ID NO.13的5’端和/或3’端的上下游序列的长度为适当长度，并不特别限定，例如长度大于1Kb、大于10Kb、大于50Kp、大于100Kb、大于500Kb、大于1000Kb、大于2000Kb或者大于5000Kb。
本发明还公开了上述的包含短N导入片段的抗TMV的烟草植株在选 育烟草抗TMV品种中的应用。
本发明提供了包含短N导入片段的抗TMV的烟草植株。与文献已报道的N基因抗性相关分子标记相比，本发明所述分子标记是用于减少N导入片段上与N基因连锁的非目的基因组分，而不是用于评价N基因介导抗性的有无。常规育种技术无法选择出育种群体中短N导入片段的染色体交换植株。本发明可通过配制育种分离群体，采用N右末端分子标记TN5.51、N基因特异分子标记和SEQ ID NO.13分子标记筛选，从大量育种群体材料中筛选出短N导入片段的染色体交换植株，并初步鉴定出减少的非目的基因组分数量。本发明获得的包含短N导入片段抗TMV植株应用于创制新型抗TMV种质资源、选育烟草抗TMV品种，有利于减少与N基因连锁的累赘。
附图说明
图1为本发明的包含短N导入片段植株利用N1N2、TN4.99、TN5.20、TN5.34、TN5.51引物对的扩增产物电泳检测结果；
其中，从左至右第1、第6、第11、第16泳道、第21泳道为100bp DNA Ladder；第2至第5泳道分别为引物对N1N2扩增Coker176、15-7F、24-8H、99-2D；第7至第10泳道TN4.99扩增Coker176、15-7F、24-8H、99-2D；第12至第15泳道分别为引物对TN5.20扩增Coker176、15-7F、24-8H、99-2D；第17至第20泳道分别为引物对TN5.34扩增Coker176、15-7F、24-8H、99-2D；第22至第25泳道分别为引物对TN5.51扩增Coker176、15-7F、24-8H、99-2D。
具体实施方式
为使本发明的目的、技术方案、及优点更加清楚明白，以下参照附图并举实施例，对本发明进一步详细说明。实施例中未注明具体技术或条件 者，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。所用试剂或仪器未注明生产厂商者，均为可以通过购买获得的常规产品。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
包含N导入片段的抗TMV烟草植株Coker176、未包含N导入片段的感TMV材料云烟87。以上烟草植株均为常见的烟草种质资源，公众可从烟草种质资源保存单位或云南省烟草农业科学研究院获得。
DNA提取试剂盒购自QIAGEN公司，DNA Marker、Taq DNA聚合酶均购自大连宝生物公司。其他化学试剂均为市售产品。表1列出了本发明所使用的引物：
表1.引物序列
Figure PCTCN2017102444-appb-000001
实施例1植物材料的制备及DNA提取
(1)N导入片段的回交转育
种植Coker176(基因型为NN，GL4.06引物对检测为阴性且TN5.51引物对检测为阳性)与云烟87(基因型为nn)，开花时采集Coker176花粉授粉至去雄的云烟87花朵上，收获F1种子(基因型为Nn)。种植F1植株，开花时采集F1花粉授粉至去雄的云烟87花朵上，收获BC1F1种子(基因型为Nn:nn＝1:1)。播种BC1F1，苗期接种TMV，筛选出抗TMV的植株(基因型Nn)。开花时采集花粉与云烟87回交，获得回交种子。通过连续回交，获得回交三代(BC3F1)种子5克。在BC3F1中选择一株N导入片段长度同Coker176类型的植株，命名为Y87N。
(2)抗TMV植株筛选
采用32孔盘定植BC3F1回交群体4-5片烟苗，每孔1株，人工接种TMV，接种后5-7d调查接种叶是否产生过敏反应(枯斑)，挑选产生枯斑个体5000株(基因型Nn)，采集幼嫩叶片，用于提取DNA，用于包含短N导入片段的染色体植株筛选。
(3)DNA提取
采用DNeasy plant 96Plant Kit(QIAGEN,Catalogue no.69181)，参考试剂说明书提取DNA。
实施例2短N导入片段植株筛选
以感TMV的烟草品种云烟87为感病对照，以已知包含N导入片段抗TMV的烟草品种Coker176为抗病对照；以TN5.51引物对，以待检测的烟草基因组DNA、感病对照的烟草基因组DNA、抗病对照的烟草基因组DNA为模板，进行PCR扩增，扩增产物进行电泳检测；
PCR反应体系如下：
Figure PCTCN2017102444-appb-000002
Figure PCTCN2017102444-appb-000003
总体积为20μL；
本领域技术人员应该理解，“引物对中的引物10umol/μL各1.5μL”的具体为：引物对中的上、下游引物的浓度均为10umol/μL，用量均为1.5μL。
所述的引物对为TN5.51引物对。
所用试剂均购自宝生物公司。
PCR反应程序如下：94℃预变性5分钟；94℃变性30秒，55℃退火30秒，72℃延伸30秒，运行35个循环；最后72℃延伸10分钟。PCR扩增产物可以在4℃保存。
PCR产物电泳检测：用质量百分浓度为1.2％的琼脂糖凝胶120v电泳25min，EB染色10min，照胶并记录，结果如图1所示。
N基因特异分子标记N1N2，按照文献的方法(Lewis,R.S.,S.R.Milla,and J.S.Levin.Molecular and genetic characterization of Nicotiana glutinosa L.chromosome segments in tobacco mosaic virus-resistant tobacco accessions.Crop Sci.2005,45:2355–2362.)进行的PCR扩增。
先筛选出烟草植株中TN5.51引物对检测为阴性且N1N2标记检测为阳性的植株，然后采用TN4.99引物对和TN5.20引物对和TN5.34引物对标记估算该植株缺失基因组分的大小。
采用TN5.51引物对进行扩增，抗病对照有845bp扩增产物，感病对照无845bp扩增产物，表明PCR扩增正常。然后按照如下的标准筛选包含短N导入片段的染色体交换植株：N1N2阳性且无845bp扩增产物，初 步判断该植株为包含短N导入片段的染色体交换植株；N1N2阳性且有845bp扩增产物，判断该植株未发生N导入片段的染色体交换。结果表明：从11000株云烟87×Coker176的BC3F1植株中，通过接种TMV挑选出5000株表现枯斑的植株。采集表现枯斑植株叶片，提取DNA。采用TN5.51标记和N1N2标记检测，筛选到15-7F和99-2D和24-8H等3个植株为N1N2阳性且无845bp扩增产物。这3个植株为包含短N导入片段的染色体交换植株。3个植株自交留种并且与云烟87回交，获得15-7F BC4F1株系和99-2D BC4F1株系和24-8H BC4F1株系种子。
实施例3与N基因连锁的非目的基因组分减少数量检测
以感TMV的烟草品种云烟87为感病对照，以已知包含N基因抗TMV的烟草品种Coker176为抗病对照；以N1N2阳性且无845bp扩增产物的3个植株DNA、感病对照的烟草基因组DNA、抗病对照的烟草基因组DNA为模板，进行PCR扩增，扩增产物进行电泳检测；
PCR反应体系如下：
Figure PCTCN2017102444-appb-000004
总体积为20μL；
所述的引物对为TN4.99引物对或TN5.20引物对或TN5.34引物对。
所用试剂均购自宝生物公司。
PCR反应程序如下：94℃预变性5分钟；94℃变性30秒，55℃退火30秒，72℃延伸30秒，运行35个循环；最后72℃延伸10分钟。PCR 扩增产物可以在4℃保存。
PCR产物电泳检测：用质量百分浓度为1.2％的琼脂糖凝胶120v电泳25min，EB染色10min，照胶并记录，结果如图1所示。
分别采用TN4.99引物对、TN5.20引物对、TN5.34引物对和N1N2引物对进行扩增，抗病对照有供试引物对的扩增产物，感病对照无供试引物对的扩增产物。表明PCR扩增正常。然后按照如下的标准估算包含短N导入片段的染色体交换植株缺失的基因组分：若TN5.34引物对检测为阴性，则该植株缺失了TN5.34对应标记至TN5.51对应标记之间的基因组分。若TN5.20引物对和TN5.4引物对检测都为阴性，则该植株缺失了TN5.20对应标记至TN5.51对应标记之间的基因组分。若TN4.99和TN5.20引物对和TN5.4引物对检测都为阴性，则该植株缺失了TN4.99对应标记至TN5.51对应标记之间的基因组分。
结果表明24-8H植株用TN4.99引物对和TN5.20引物对和TN5.34引物对检测为阴性，用N1N2引物对检测为阳性。TN4.99引物对扩增的标记相对番茄11号染色体的物理距离为4.99Mb，TN5.51相对番茄11号染色体的物理距离为5.51Mb，推算出24-8H植株至少缩短了0.52Mb(相对番茄11号染色体的物理距离)，按照烟草野生种基因组大小为番茄基因组大小的3倍推算，24-8H植株N导入片段至少缩短了1.56Mb。
结果表明15-7F植株用TN5.20引物对和TN5.34引物对检测为阴性，用N1N2引物对和TN4.99引物对检测为阳性。TN5.20引物对对应标记相对番茄11号染色体的物理距离为5.20Mb，TN5.51对应标记相对番茄11号染色体的物理距离为5.51Mb，推算出15-7F植株至少缩短了0.31Mb(相对番茄11号染色体的物理距离)，按照烟草野生种基因组大小为番茄基因组大小的3倍推算，15-7F植株N导入片段至少缩短了0.93Mb。
结果表明99-2D植株用TN5.34引物对检测为阴性，用N1N2引物对和TN4.99引物对和TN5.20引物对检测为阳性。TN5.34引物对扩增的标记相对番茄11号染色体的物理距离为5.34Mb，TN5.51相对番茄11号染 色体的物理距离为5.51Mb，推算出也就是99-2D植株至少缩短了0.17Mb(相对番茄11号染色体的物理距离)，按照烟草野生种基因组大小为番茄基因组大小的3倍推算，99-2D植株N导入片段至少缩短了0.51Mb。
实施例4 5FN-分离群体中短N导入片段单株筛选与的非目的基因组分减少数量检测
(1)包含N导入片段群体的回交转育
种植Coker176(基因型为NN，GL4.06引物对检测为阴性且TN5.51引物对检测为阳性)与5F(基因型为nn)，开花时采集Coker176花粉授粉至去雄的5F花朵上，收获F1种子(基因型为Nn)。种植F1植株，开花时采集F1花粉授粉至去雄的5F花朵上，收获BC1F1种子(基因型为Nn:nn＝1:1)。播种BC1F1，苗期接种TMV，筛选出抗TMV的植株(基因型Nn)。开花时采集花粉与5F回交，获得回交种子。通过连续回交，获得回交六代(BC6F1)种子5克。回交四代后，选择一株N导入片段长度同Coker176类型的植株自交，获得BC4F6种子，命名为5FN。
按照实施例1的方法，在5F×Coker176BC6F1群体中，筛选出抗TMV植株(基因型Nn)，提取DNA。
按照实施例2的方法，筛选短N导入片段植株。结果表明：从20160株5F×Coker176的BC6F1植株中，通过接种TMV挑选出9984株表现枯斑的植株。采集表现枯斑植株叶片，提取DNA。采用TN5.51标记和N1N2标记检测，从9612份DNA中筛选到1-1002植株为N1N2阳性且无845bp扩增产物。该植株为包含短N导入片段的染色体交换植株。
按照实施例3的方法，检测1-1002植株与N基因连锁的非目的基因组分减少数量。结果表明1-1002植株用TN4.99引物对和TN5.20引物对和TN5.34引物对检测为阴性，用N1N2引物对检测为阳性。TN4.99引物对扩增的标记相对番茄11号染色体的物理距离为4.99Mb，TN5.51相对番茄11号染色体的物理距离为5.51Mb，推算出1-1002植株至少缩短了0.52Mb(相对番茄11号染色体的物理距离)，按照烟草野生种基因组大 小为番茄基因组大小的3倍推算，1-1002植株N导入片段至少缩短了1.56Mb。
该植株自交留种并且5F回交，收获5F×1-1002BC7F1种子，用于短N导入片段植株的连锁累赘表型检测，即短N导入片段植株产量与成熟期的上部叶叶绿素含量检测。
实施例5短N导入片段植株产量与成熟期的上部叶叶绿素含量检测
播种5F×1-1002BC7F1株系(1/2为包含Ns segment的植株),5FN BC4F6株系(N segment纯合)，5F(nn)。移栽前通过N1N2标记的辅助选择，在5F×1-1002BC7F1株系中挑选出N基因阳性植株，即5F×1-1002BC7F1株系中包含Ns segment的植株。在田间种植，在开花时同一天打顶。统一留18-21片叶可采烤叶，从上往下数定位第3片叶的叶中部取样。打顶后第5周开始、每隔7天测量和取样一次，共取样4次。用刀片划取叶中脉两侧约1厘米宽叶组织，用铝箔纸包裹液氮冻存，于-80度冰箱保存。用于HPLC测定叶绿素a、叶绿素b、叶黄素、β-胡萝卜素含量。测定方法为烟草行业标准(YC/T 382-2010烟草及烟草制品质体色素的测定高压液相色谱法)。产量采用常规方法测定。结果表明：与未截短的N导入片段的抗TMV烟草植株5FN相比，5F×1-1002BC7F1株系的产量提高3％、上部叶成熟期叶绿素含量降低3％。5F×1-1002BC7F1株系的产量与5F相当，5F×1-1002BC7F1株系的产量高于5FN产量。上部叶采烤前5F×1-1002BC7F1株系的叶绿素含量与5F含量相当，上部叶采烤前5F×1-1002BC7F1的叶绿素含量低于5FN的叶绿素含量。表明包含短N导入片段抗TMV的1-1002株系的连锁累赘性状有明确改善。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。

Claims (20)

  1. 一种抗TMV的烟草植株，其包含短N导入片段，所述短N导入片段与Coker176类型烟草的N导入片段相比减少至少0.51Mb、优选至少0.93Mb、更优选至少1.56Mb的累赘基因组分。
  2. 根据权利要求1所述的抗TMV的烟草植株，所述短N导入片段与Coker176类型烟草的N导入片段相比至少减少SEQ ID No.13所示序列的部分序列或全部序列。
  3. 根据权利要求2所述的抗TMV的烟草植株，所述短N导入片段与Coker176类型烟草的N导入片段相比至少减少SEQ ID No.13所示序列的第806912位碱基至1404293位碱基的部分序列或全部序列。
  4. 根据权利要求1所述的抗TMV的烟草植株，其选自下列植株：
    a)N1N2标记检测为阳性，且TN5.34引物对和TN5.51引物对检测均为阴性的烟草植株；
    b)N1N2标记检测为阳性，且TN5.20引物对、TN5.34引物对和TN5.51引物对检测均为阴性的烟草植株；
    c)N1N2标记检测为阳性，且TN4.99引物对、TN5.20引物对、TN5.34引物对和TN5.51引物对检测均为阴性的烟草植株。
  5. 根据权利要求1-4中任一项所述的抗TMV的烟草植株，所述短N导入片段通过染色体交换、基因组编辑、化学诱变或物理诱变的方法获得。
  6. 根据权利要求1-4中任一项所述的抗TMV的烟草植株，其来源于烟草属，优选来源于烤烟、白肋烟、香料烟、晒晾烟或雪茄烟。
  7. 利用根据权利要求1-4中任一项所述的抗TMV的烟草植株选育出的烟草杂交种、品种或株系。
  8. 权利要求1-4中任一项所述的抗TMV的烟草植株的种子、花粉和胚珠。
  9. 根据权利要求1-4中任一项所述的抗TMV的烟草植株在选育烟草抗TMV品种中的应用。
  10. 一种烟草植株的选育方法，用于选育权利要求1所述的抗TMV的烟草植株，其包含以下步骤：
    a)利用N导入片段纯合的烟草植株与基因型为nn的烟草植株杂交，获得基因型为Nn的F1烟草植株，再将F1烟草植株与基因型为nn的烟草植株杂交，获得用于筛选短N导入片段的群体材料；
    b)在步骤a)获得的群体材料的苗期接种TMV，在群体材料中筛选出表现枯斑的Nn基因型植株；
    c)采用N导入片段右末端的分子标记TN5.51引物对和N基因特异分子标记N1N2对步骤b)筛选出的Nn基因型植株进行基因型分型，筛选出N1N2分子标记检测为阳性且TN5.51引物对检测为阴性的植株。
  11. 一种烟草植株的选育方法，用于选育权利要求3中c)所述的抗TMV的烟草植株，其包含以下步骤：
    a)利用N导入片段纯合的烟草植株与基因型为nn的烟草植株杂交，获得基因型为Nn的F1烟草植株，再将F1烟草植株与基因型为nn的烟草植株杂交，获得用于筛选短N导入片段的群体材料；
    b)在步骤a)获得的群体材料的苗期接种TMV，在群体材料中筛选出表现枯斑的Nn基因型植株；
    c)采用TN5.34引物对、TN5.51引物对、和N基因特异分子标记N1N2对步骤b)筛选出的Nn基因型植株进行基因型分型，筛选出N1N2分子标记检测为阳性，且TN5.34引物对和TN5.51引物对检测均为阴性的植株。
  12. 一种烟草植株的选育方法，用于选育权利要求3中b)所述的抗TMV的烟草植株，其包含以下步骤：
    a)利用N导入片段纯合的烟草植株与基因型为nn的烟草植株杂交， 获得基因型为Nn的F1烟草植株，再将F1烟草植株与基因型为nn的烟草植株杂交，获得用于筛选短N导入片段的群体材料；
    b)在步骤a)获得的群体材料的苗期接种TMV，在群体材料中筛选出表现枯斑的Nn基因型植株；
    c)采用TN5.20引物对、TN5.34引物对、TN5.51引物对和N基因特异分子标记N1N2对步骤b)筛选出的Nn基因型植株进行基因型分型，筛选出N1N2分子标记检测为阳性，且TN5.20引物对、TN5.34引物对和TN5.51引物对检测均为阴性的植株。
  13. 一种烟草植株的选育方法，用于选育权利要求3中a)所述的抗TMV的烟草植株，其包含以下步骤：
    a)利用N导入片段纯合的烟草植株与基因型为nn的烟草植株杂交，获得基因型为Nn的F1烟草植株，再将F1烟草植株与基因型为nn的烟草植株杂交，获得用于筛选短N导入片段的群体材料；
    b)在步骤a)获得的群体材料的苗期接种TMV，在群体材料中筛选出表现枯斑的Nn基因型植株；
    c)采用TN4.99引物对、TN5.20引物对、TN5.34引物对、TN5.51引物对和N基因特异分子标记N1N2对步骤b)筛选出的Nn基因型植株进行基因型分型，筛选出N1N2分子标记检测为阳性，且TN4.99引物对、TN5.20引物对、TN5.34引物对和TN5.51引物对检测均为阴性的植株。
  14. 短N导入片段在烟草植株中的应用，所述短N导入片段与Coker176类型烟草的N导入片段相比减少至少0.51Mb、优选至少0.93Mb、更优选至少1.56Mb的累赘基因组分。
  15. 根据权利要求14所述的应用，所述短N导入片段与Coker176类型烟草的N导入片段相比至少减少SEQ ID No.13所示序列的部分序列或全部序列。
  16. 根据权利要求14所述的应用，其得到选自以下的烟草植株：
    a)N1N2标记检测为阳性，且TN4.99引物对、TN5.20引物对、TN5.34引物对和TN5.51引物对检测均为阴性的烟草植株；
    b)N1N2标记检测为阳性，且TN5.20引物对、TN5.34引物对和TN5.51引物对检测均为阴性的烟草植株；
    c)N1N2标记检测为阳性，且TN5.34引物对和TN5.51引物对检测均为阴性的烟草植株。
  17. 根据权利要求14-16中任一项所述的应用，所述短N导入片段通过染色体交换、基因组编辑、化学诱变或物理诱变的方法获得。
  18. 根据权利要求14所述的应用，所述烟草植株其来源于烟草属，优选来源于烤烟、白肋烟、香料烟、晒晾烟或雪茄烟。
  19. 利用根据权利要求14所述的应用而得到的烟草植株选育出的烟草杂交种、品种或株系。
  20. 根据权利要求14所述的应用而得到的烟草植株的种子、花粉和胚珠。
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