WO2008135296A2 - New hybrid system for brassica napus - Google Patents

New hybrid system for brassica napus Download PDF

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Publication number
WO2008135296A2
WO2008135296A2 PCT/EP2008/004762 EP2008004762W WO2008135296A2 WO 2008135296 A2 WO2008135296 A2 WO 2008135296A2 EP 2008004762 W EP2008004762 W EP 2008004762W WO 2008135296 A2 WO2008135296 A2 WO 2008135296A2
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WO
WIPO (PCT)
Prior art keywords
allele
brassica napus
plant
seed
male
Prior art date
Application number
PCT/EP2008/004762
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English (en)
French (fr)
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WO2008135296A3 (en
Inventor
Gunther Stiewe
Stephan Pleines
Marie Coque
Johannes Jacobus Ludgerus Gielen
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Syngenta Participations Ag
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Application filed by Syngenta Participations Ag filed Critical Syngenta Participations Ag
Priority to AU2008248878A priority Critical patent/AU2008248878B9/en
Priority to CA2690561A priority patent/CA2690561A1/en
Priority to EA200901631A priority patent/EA200901631A1/ru
Priority to CA2720634A priority patent/CA2720634C/en
Priority to BRPI0813445-6A2A priority patent/BRPI0813445A2/pt
Priority to CN200880020023A priority patent/CN101861390A/zh
Priority to US12/600,481 priority patent/US20100222605A1/en
Publication of WO2008135296A2 publication Critical patent/WO2008135296A2/en
Publication of WO2008135296A3 publication Critical patent/WO2008135296A3/en

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Classifications

    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • 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
    • A01H5/10—Seeds
    • 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
    • A01H6/20—Brassicaceae, e.g. canola, broccoli or rucola
    • A01H6/202—Brassica napus [canola]

Definitions

  • a simple and efficient pollination control system is the key step for utilizing heterosis in commercial hybrid seed production. If one of the parents is a Sl, CMS or NMS plant that is not able to self-pollinate or is incapable of producing pollen, only cross pollination will occur. By eliminating the pollen of one parental variety in a cross, a plant breeder is assured of obtaining hybrid seed of uniform quality, provided that the parents are of uniform quality and the breeder conducts a single cross.
  • CMS systems used in the commercial production of F1 hybrid of rapeseed plants are the Polima (pol; Fu 1 1981 ), Kosena, and the Ogura system (Ogura, 1968; Makaroff, 1989; Pellan- Delourme et al., 1987; US 5,254,802; US20040237141 , US20020032916, EP 0 599 042; US 6,229,072) .
  • hybrid in the context of plant breeding refer to a plant that is the offspring of genetically dissimilar parents produced by crossing plants of different lines or breeds or species, including but not limited to the cross between two inbred lines (e.g., a genetically heterozygous or mostly heterozygous individual).
  • single cross F 1 hybrid refers to an F 1 hybrid produced from a cross between two inbred lines.
  • the presently disclosed subject matter can also employ genetic markers that are physi- cally within the boundaries of a genetic locus (e.g., inside a genomic sequence that corresponds to a gene such as, but not limited to a polymorphism within an intron or an exon of a gene).
  • the one or more genetic markers comprise between one and ten markers, and in some embodiments the one or more genetic markers comprise more than ten genetic markers.
  • the target polynucleotides may be detected by hybridization with a probe polynucleotide which forms a stable hybrid with that of the target sequence under stringent to moderately stringent hybridization and wash conditions. If it is expected that the probes will be essentially completely complementary (i.e., about 99% or greater) to the target sequence, stringent conditions will be used.
  • PCR primer is preferably understood within the scope of the present invention to refer to relatively short fragments of single-stranded DNA used in the PCR amplification of specific regions of DNA.
  • Recombination is the exchange of information between two homologous chromosomes during meiosis.
  • the method of the present invention employs an up- scaling step, in which the conditionally male sterile (prebasic female) line is crossed with a maintainer line to provide again male sterile seed.
  • This seed is heterogeneous for the male sterility gene, but has still the conditionally male sterile phenotype.
  • This propagation has only become possible by providing a maintainer line, which does have neither a functional ms allele nor a functional restorer allele.
  • This up-scaling procedure is required for a commercially viable system.
  • the Ms allele is preferably characterized by conferring a conditional nuclear male sterile phenotype, which a) is restored temporarily to fertility by an exposure to a temperature of higher than 35°C, b) is restored to fertility in at least part of the F 1 plants obtained from crossing a male sterile plant with the genotype MsMsrfrf or Msmsrfrf with any Brassica napus plant comprising at least one dominant Rf allele, and c) is maintained in the Fi plants obtained from crossing a plant with a conditional male sterile phenotype referred by said Ms allele with the male fertile plants derived from seed deposited under Deposit Number NCIMB 41481.
  • conditionally male sterile phenotype and/or the Ms allele is linked to and/or associated with one or more marker (Ms allele marker) selected from the group ("MS gene marker group") consisting of I.
  • III. comprise at least 25 consecutive nucleotides of the marker sequences defined above in I. to V.
  • one or more marker of group V. above linked to and/or associated with the conditionally male sterile phenotype and/or the Ms allele can be linked to 1 , 2, 3, or all of the sequences set forth as SEQ ID NOs: 3, 6, 11 and 18.
  • Markers as described above may be used in various other aspects of the present invention. However, the aspects of the present invention are not limited to the use of the markers as disclosed in the present application. It is further emphasized that these aspects may also make use of markers not explicitly disclosed herein or markers yet to be identified.
  • SNP detection include the use of molecular beacons that make use of a specifically engineered single-stranded oligonucleotide probe comprising a fluorophore and a fluorescence quencher (Abravaya et al., 2003). SNPs can also be detected by high density oligonucleotide SNP arrays comprising hundreds of thousands of probes arrayed on a small chip, allowing for a large number of SNPs to be interrogated simultaneously (Rapley & Harbron, 2004). Furthermore, a broad range of enzymes including DNA ligase, DNA polymerase and nucleases have been employed to generate high-fidelity SNP genotyping methods.
  • a probe is added to the reaction, i.e., a single-stranded oligonucleotide complementary to a segment of 20-60 nucleotides within the DNA template and located between the two primers.
  • a fluorescent reporter or fluorophore e.g., 6-carboxyfluorescein, acronym: FAM, or tetrachlorofluorescein, acronym: TET
  • a quencher e.g., tetramethylrhodamine, acronym: TAMRA, or dihydrocyclopyrroloindole tripeptide "minor groove binder", acronym: MGB
  • FAM 6-carboxyfluorescein
  • TAMRA tetrachlorofluorescein
  • MGB dihydrocyclopyrroloindole tripeptide
  • variant when used with regard to the Ms or ms allele, also refers to variants of the Ms or ms alleles that map in the same region as the Ms or ms allele described above, i.e. the variant is also being localized on the Brassica napus chromosome N7, preferably between the marker sequences NR1116 (e.g., SEQ ID NO: 21 ) and NR2525 (e.g., SEQ ID NO: 22), more preferably with a distance of 2.8 cM to NR1116 and 6.0 cM to NR2525, respectively, even more preferably between the SNP markers SR0002A and SR20003B, most preferably with a distance of approximately 2.8 cM to SR0002A and 3.3 cM to SR0003B, respec- tively.
  • NR1116 e.g., SEQ ID NO: 21
  • NR2525 e.g., SEQ ID NO: 22
  • 'maintainer allele means the absence of the functional restorer allele (Rf allele) and - more specifically - a rf allele as obtainable from the Brassica napus seed deposited under Deposit Number NCIMB 41480 and/or 41481 and variants thereof.
  • This rf allele is preferably linked to and characterized by the phe- notypic properties of a) not being capable of reverting to fertility the male sterile phenotype conferred by the Ms allele, and b) being capable of maintaining the male sterile phenotype conferred by the Ms allele.
  • the maintainer allele (rf allele) is linked to a male sterility maintaining phenotype, which in a homozygous form allows the male sterile phenotype caused by the Ms to be expressed.
  • Said male sterility maintaining phenotype can by reversed by at least one functional dominant Rf allele.
  • Said Rf allele is obtainable from any non-Takagi based germplasm.
  • Other than a germplasm derived from the Takagi germplasm there are no known lines which would not comprise at least one functional copy of the Rf allele. As a consequence virtually all rapeseed lines available at the date of the invention are found to be restorer lines.
  • the female (male sterile) plants are delayed in the flowering as compared to the male fertile plants employed in these production steps.
  • the methods selected from the group consisting of: i. cut-back of the male fertile plant (i
  • ms allele means the absence of the functional male sterility allele (Ms allele or Ms gene) and - more specifically - a ms allele as obtainable from the Brassica napus seed deposited under Deposit Number NCIMB 41481 and variants of said allele conferring essentially the same phenotype (i.e. a male fertile phenotype).
  • SNP-Probe 1 SNP-probe 1
  • SNP-Probe 4 SNP probe
  • the markers selected from the group of SSR markers consisting of a) a PCR fragment with an apparent molecular weight selected from the group of apparent weights consisting of 94 (+/- 0.9) bp, 110.4 (+/- 0.5) bp, 112.3 (+/- 0.4) bp, and 116.3 (+/- 0.4) bp resulting from a PCR reaction with the primers having the sequence set forth as SEQ ID NOs: 1 and 2, b) a PCR fragment with an apparent molecular weight of 183.8 (+/- 0.4) bp or no fertile allele associated PCR fragment resulting from a PCR reaction with the primers having the sequence set forth as SEQ ID NOs: 4 and 5, wherein the one or more marker (Ms allele marker) also includes an isolated nucleotide sequence selected from the group consisting of sequences which
  • the fertility allele (dysfunctional male sterility allele; ms allele) is linked in a homozygous form to a male fertile phenotype, wherein said ms allele is preferably selected from the group consisting of a) the ms allele as present in the Brassica napus seed deposited under Deposit Number NCIMB 41481 , and b) variants thereof, which have the same phenotypic property.
  • a predominantly male fertile phenotype when exposed before and/or during flowering to a temperature of preferably between 35°C and 43°C, more preferably to a temperature of between 37 0 C and 4O 0 C, most preferably to a temperature of about 39°C; preferably with an exposure for the preferred heat treatment time as specified herein and a subsequent growing at ambient temperature, as defined herein (see iv. under step a) above).
  • NCIMB 41481 as a male fertile plant.
  • markers can be used for the visualization of differences in nucleic acid sequences. This visualization is possible - for example - due to DNA-DNA hybridization techniques after digestion with a restriction enzyme (RFLP) and/or due to techniques using the polymerase chain reaction (e.g. STS, microsatellites, AFLP).
  • RFLP restriction enzyme
  • STS microsatellites
  • AFLP polymerase chain reaction
  • the markers identified herein may be used in various aspects of the present invention as will be illustrated below. Aspects of the present invention are not limited to the use of the markers identified herein. It is stressed that the aspects may also make use of markers not explicitly disclosed herein or even yet to be identified.
  • Molecular markers i.e.
  • the plants of the present invention conferring male sterility or maintaining same may for instance have the genotype MsMsrfrf, Msmsrfrf, or msmsrfrf, while the hybrid plants of the invention have the genotype of MsmsRfrf, or msmsRfrf, wherein Ms, ms, rf, Rf have the mean- ing as described above.
  • the present invention thus also provides methods for selecting a plant of the species Brassica napus exhibiting a male sterility conferring or maintaining phe- notype by detecting in said plant the presence of the Ms and/or rf allele as defined herein.
  • the analysis may be conducted in various ways and may comprise for example the following steps: a) identifying the at least one marker locus using a pair of PCR oligonucleotide primers consisting of a forward primer and a reverse primer exhibiting a nucleotide sequence set forth in any of SEQ ID NOs: 1 , 2, 4, 5, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, and b) identifying the marker allele by determining the molecular weight of the PCR amplification product obtained in step a), and c) identifying a plant or plants with the desired profile using the data of the marker analysis.
  • step b may comprise the use of a set of bi-directional primers that were used in the SSR method to produce the amplification product that later proved to be a suitable marker for the Ms, ms, Rf, or rf allele.
  • a set of primers is herein referred to as the primers that define the SSR marker or marker-specific primers.
  • “Bi-directional" means that the orientation of the primers is such that one functions as the forward and one as the reverse primer in an amplification reaction of nucleic acid.
  • the method of detecting a Brassica plant (with the Ms, Rf, ms, or rf allele) according to the present invention further comprises step c) of selecting said Brassica plant, or a part thereof, containing said DNA fragment.
  • the method of detecting a Brassica plant according to the present invention further comprises step d) of selfing said Brassica plant containing said DNA fragment.
  • the method of detecting a Brassica plant according to the present invention further comprises step e) of crossing said Brassica plant with another Brassica plant. 5. Agricultural and industrial use of the plants of the present invention
  • the present invention relates to a method of using one or more Brassica napus plants of the present invention selected from the group consisting of a conditionally male sterile Brassica napus plant with the genotype MsMsrfrf (i.e. the prebasic female) and a male fertile Brassica napus plant with the genotype msmsrfrf (i.e. the maintainer) in a method of producing a conditionally male sterile Brassica napus plant with the genotype Msmsrfrf (i.e. the basic mother) or seed thereof.
  • a conditionally male sterile Brassica napus plant with the genotype MsMsrfrf i.e. the prebasic female
  • a male fertile Brassica napus plant with the genotype msmsrfrf i.e. the maintainer
  • Inbred male sterile basic female lines or maintainer lines can be developed using the techniques of recurrent selection and backcrossing, selfing and/or dihaploids or any other technique used to make parental lines.
  • the male sterility referring or maintaining genotype can be introgressed into a target recipient plant (the "recurrent parent") by crossing the recurrent parent with a first donor plant, which differs from the recurrent parent and is referred to herein as the "non-recurrent parent".
  • the recurrent parent is a plant that is lacking the male sterility conferring or maintaining properties and preferably possesses commercially desirable characteristics, such as, but not limited to (additional) disease resistance, insect resistance, valuable oil or meal characteristics, etc.
  • the SNPs at position 214 (T/C) and 218 (T/G) were selected to develop a TaqMan® assay using the Primer Express 2.0 software distributed by Applied Biosystems Inc. and following the corresponding instructions.
  • the sequences of primers and probes corresponding to this assay referred to as SR0002A are listed in Table 4.
  • SSR microsatellite markers
  • NR2219 forward primer sequence 5 ' -ATTATCCTCTCGCCATTTC -3 ' (SEQ ID NO: 19)
  • NR2219 reverse primer sequence 5 ' -ATTATCCTCTCGCCATTTC -3 ' (SEQ ID NO: 19)
  • the consolidated consensus sequences were obtained by merging the 3 assem- blies using the cd-hit program (http://bioinformatics.ljcrf.edu/cd-hi/) with a threshold of 98% sequence identity.
  • Each consensus sequence was divided into probe selection regions (PSR) of 150 bases long. On average, 4 probes per PSR, and 16 probes per transcript (perfect match probes only) were designed along the entire transcript, avoiding intron/exon boundaries.
  • the constraints applied for the probe design ensured uniqueness of the probe sequence, comparable hybridization efficiencies and removal of cross-hybridizing probes as recommended by Affymetrix. In order to enable estimating background signals, 17.000 antigenomic probes were included on the chip.
  • the primary PCR reactions were run in a final volume of 15 ⁇ l comprising 5 ⁇ l of genomic DNA at a concentration of 5 ng/ ⁇ l, 1.5 ⁇ l of 1OX reaction buffer, 1.2 ⁇ l of 1OmM dNTPs, 0.5 ⁇ l of 5OmM MgCI 2 , 0.3 ⁇ l of each primer at a concentration of 10 ⁇ M, 0.12 ⁇ l of Invitrogen Taq platinium (5U/ ⁇ l). All PCR reactions were performed at an ABI GeneAmp PCR System 9700.
PCT/EP2008/004762 2007-06-13 2008-06-13 New hybrid system for brassica napus WO2008135296A2 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
AU2008248878A AU2008248878B9 (en) 2007-06-13 2008-06-13 New hybrid system for Brassica napus
CA2690561A CA2690561A1 (en) 2007-06-13 2008-06-13 New hybrid system for brassica napus
EA200901631A EA200901631A1 (ru) 2007-06-13 2008-06-13 Новая гибридная система для brassica napus
CA2720634A CA2720634C (en) 2007-06-13 2008-06-13 Nuclear conditional male sterility system in brassica napus and its use for producing male fertile hybrid seed of brassica napus
BRPI0813445-6A2A BRPI0813445A2 (pt) 2007-06-13 2008-06-13 Sistema híbrido para brassica napus.
CN200880020023A CN101861390A (zh) 2007-06-13 2008-06-13 用于欧洲油菜的新杂交系统
US12/600,481 US20100222605A1 (en) 2007-06-13 2008-06-13 New hybrid system for brassica napus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP07290741A EP2016821A1 (en) 2007-06-13 2007-06-13 New hybrid system for Brassica napus
EP07290741.3 2007-06-13

Publications (2)

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WO2008135296A2 true WO2008135296A2 (en) 2008-11-13
WO2008135296A3 WO2008135296A3 (en) 2009-01-08

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Country Status (16)

Country Link
US (1) US20100222605A1 (zh-CN)
EP (3) EP2016821A1 (zh-CN)
CN (1) CN101861390A (zh-CN)
AT (1) ATE488131T1 (zh-CN)
AU (1) AU2008248878B9 (zh-CN)
BR (1) BRPI0813445A2 (zh-CN)
CA (2) CA2720634C (zh-CN)
CY (1) CY1111514T1 (zh-CN)
DE (2) DE602008003480D1 (zh-CN)
DK (1) DK2002711T3 (zh-CN)
EA (1) EA200901631A1 (zh-CN)
ES (1) ES2356491T3 (zh-CN)
HR (1) HRP20110111T1 (zh-CN)
PL (1) PL2002711T3 (zh-CN)
SI (1) SI2002711T1 (zh-CN)
WO (1) WO2008135296A2 (zh-CN)

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FR2948533A1 (fr) * 2009-08-03 2011-02-04 Limagrain Verneuil Holding Plante brassica restauratrice de la sterilite male cytoplasmique ogura, procede de production et utilisation de cette plante
WO2011020698A1 (fr) * 2009-08-03 2011-02-24 Nickerson International Research Plante brassica restauratrice de fertilite dans un systeme de sterilite mâle cytoplasmique ogura, procede de production et utilisation de cette plante
WO2011022792A1 (pt) * 2009-08-31 2011-03-03 Empresa Brasileira De Pesquisa Agropecuária - Embrapa Método para obtenção de linhagens parentais fêmeas de asteraceas a partir de híbridos
US10709079B2 (en) 2009-12-18 2020-07-14 Cargill, Incorporated Brassica plants yielding oils with a low total saturated fatty acid content
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CN107699631B (zh) * 2017-11-17 2021-09-10 青海省农林科学院 甘蓝型油菜雌雄不育系Bnmfs利用分子标记筛选的引物及方法
CN110804675A (zh) * 2019-11-21 2020-02-18 南京农业大学 不结球白菜微卫星dna标记指纹图谱及其应用
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