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WO2018104724A1 - Procédés destinés à augmenter la fréquence d'enjambement méiotique dans des végétaux - Google Patents

Procédés destinés à augmenter la fréquence d'enjambement méiotique dans des végétaux Download PDF

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WO2018104724A1
WO2018104724A1 PCT/GB2017/053667 GB2017053667W WO2018104724A1 WO 2018104724 A1 WO2018104724 A1 WO 2018104724A1 GB 2017053667 W GB2017053667 W GB 2017053667W WO 2018104724 A1 WO2018104724 A1 WO 2018104724A1
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hei10
recombination
plant
protein
seq
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Ian Henderson
Piotr ZIÓLKOWSKI
Charles UNDERWOOD
Robert Martienssen
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Cambridge Enterprise Limited
Cold Spring Harbor Laboratory
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    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • 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
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to methods and agents for increasing recombination such as meiotic recombination in plants or animals.
  • polymorphism in the PRDM9 histone methyltransferase gene acts in trans to determine crossover hotspot locations in mammalian genomes.
  • Total crossover levels in mammals are controlled in trans by polymorphisms associated with RNF212, which belongs to a conserved family of SUMO/ubiquitin E3 ligases that regulate meiotic recombination.
  • Polymorphism per se can also locally inhibit crossovers in cis, likely via an effect of heteroduplex mismatches following strand invasion.
  • Heterozygous structural polymorphisms, such as inversions and translocations suppress crossovers in cis at larger physical scales. Evidence for both cis and trans crossover modification exists in plants.
  • WO03/104451 describes altering the expression of MutS and its homologues in order to increase recombination in a eukaryotic cell such as a plant cell.
  • WO03/104451 also refers to altering expression of other recombination factors in combination with MutS homologues, for example factors such as SP01 1 , MRE1 1 , RAD50, XRS2/NBS1 , RecA homologues, RAD52, RAD54, TID1 , MSH4, MSH5, BLM, WRN, SGS1 , MCM2-7, histone acetylases, synaptonemal complex proteins and any combination thereof.
  • WO2007/030014 describes altering the expression level of MLH1 to modify homologous recombination in plants, while US2014/0289902 suggests deactivation of the FANCM protein to increase meiotic recombination in plants.
  • a method for obtaining a plant cell with increased recombination or increased recombination potential comprising a step of increasing the activity and/or levels of an HEI10 protein in the plant cell, wherein the HEI10 protein has at least 50% sequence identity to the HEM 0 protein of any of SEQ ID NOs 1 -4 (see below).
  • the recombination may be meiotic recombination.
  • a plant cell obtained or obtainable according to the above method wherein the plant cell has increased recombination or increased recombination potential.
  • a plant comprising the plant cell of the invention, wherein the plant has increased recombination or increased recombination potential.
  • the plant may for example be a transgenic plant.
  • the invention also encompasses a non-transgenic plant derived from the transgenic plant, wherein the non- transgenic plant is obtained by screening for loss of increased activity and/or levels of the HEM 0 protein.
  • Another aspect of the invention provides an expression cassette comprising a promoter active during plant meiosis and operably linked to a polynucleotide encoding an HEI10 protein as defined herein, wherein the polynucleotide when expressed during plant meiosis is capable of increasing recombination.
  • a plant transformed with the above expression cassette is also encompassed by the invention.
  • the invention further comprises a method for obtaining a plant with increased frequency of homologous or homeologous recombination, wherein the method comprises the steps of:
  • the CSL genotype of each cross is indicated for the five Arabidopsis chromosomes, with 'C indicating Col and 'L' indicating Ler genotypes. Data from replicate F1 individuals are shown by black dots and mean values by grey dots, (d) As for (c), but measuring crossover frequency (cM) within the I2f FTL interval.
  • Fig. 2 Genetic mapping of Col/Ler recombination QTLs.
  • LOD Logarithm of the odds
  • cM Marker positions along the chromosome genetic maps
  • the vertical lines labelled "ATG” and “TAG” indicate the positions of HEI10 AJG and TAG codons, respectively.
  • the unlabelled vertical lines indicate the position of the HEI10 genomic clone amplified for transformation experiments. Beneath are ticks showing the position of polymorphisms in Ler-0, Bur-0 and Ct-1 accessions, identified by Sanger sequencing.
  • X-axis shows chromosome 1 coordinate (bp), (b) 420 recombination rates (cM) from individual plants in a Col-420xBur-O F 2 population. Individuals are grouped according to Col versus Bur-0 genotype at marker 19,917,692 bp.
  • X-axis represents genotype as indicated.
  • Fig. 5 Immunocytological analysis of meiocytes with varying rQTL genotypes.
  • Fig. 7. Increased HEI10 dosage increases euchromatic crossover frequency
  • Y-axis shows individuals,
  • X-axis shows proportion of chromosome arm.
  • Lower panels show top and middle panels superimposed. Mean values are shown by the dotted horizontal lines and the centromeres indicated by vertical dotted lines.
  • x-axis shows coordinates (Mbp) and y-axis shows crossovers
  • e Quantification of MLH1 foci on pachytene stage meiotic chromosomes in wild type and HEI10 Co1 C2 T 3 .
  • Y-axis shows MLH1 foci per cell
  • a magnified view of a wild type 'rod' bivalent is shown (right, upper panel) with homologues outlined and chiasma marked with 'X'.
  • a magnified view of a HEI10 Co1 ring bivalent (right, lower panel) is also shown.
  • Fig. 8 Crossovers identified by genotyping by sequencing in wild type and HEHOCol F2 populations, (a) Histograms showing the number of crossover events mapped by genotyping by sequencing in the Col/Ler F 2 population (see Table 1 ). Mean values are indicated by the vertical dotted lines. "WT” denotes wild type, (b) As for (a), but showing data from the HEUOCol F 2 population. In all panels, x-axis shows crossovers, and y-axis represents individuals. "G” represents genome. Fig. 9. Overlap of wild type and HEUOCol crossovers with genomic features, (a)
  • Y- axis shows overlap (base pairs)
  • the invention provides in one aspect a method for obtaining a plant cell with increased recombination or increased recombination potential, comprising a step of increasing the activity and/or levels of an HEM 0 protein in the plant cell, wherein the HEM 0 protein has at least 50% sequence identity to the HEI10 protein of any of SEQ ID NOs 1 -4.
  • the recombination may be meiotic recombination.
  • meiotic recombination causes reciprocal crossovers between chromosomes. Variation within and between species modifies crossover frequency, acting in cis and trans.
  • rQTL trans recombination quantitative trait loci
  • Semidominant HEI10 polymorphisms underlie rQTL 1, which encodes a conserved crossover-regulatory E3 SUMO/ubiquitin ligase. Haploinsufficiency of hei10 null alleles was observed, indicating dosage-sensitivity.
  • Example 1 we show in Example 1 that transformation of additional HEI10 copies is sufficient to more than double crossovers throughout the Vietnameseromatic chromosome arms.
  • HEI10 and its involvement as one of many proteins in meiosis has been identified previously, for example in mice (Qiao, H. et al., 2014, Nat. Genet. 46: 194-199) and in Arabidopsis (Chelysheva, L. et al. (2012, PLoS Genet. 8, e1002799), where heterozygous or null HEI10 mutants were shown to have reduced crossover levels.
  • the present invention is based on the unexpected finding that HEI10 is a limiting factor for plant crossover levels such that increases in HEI10 levels or activity can be used in methods to increase recombination or recombination potential.
  • Increasing HEM 0 dosage or activity to boost meiotic recombination is broadly applicable in breeding programs of different plant species. Applying this strategy in crop breeding programs, for example, could have a plethora of benefits, and could be employed in different ways depending on the breeding scheme of choice. For instance in pedigree breeding, one of the greatest costs is the requirement to grow sufficiently large F 2 populations in the field in order to observe and select for useful recombinant plants that combine desirable characteristics from both parents. If recombination is boosted, there will be more novel genetic combinations and greater variation between F 2 plants, and therefore fewer plants would need to be grown and analysed, leading to reduced costs.
  • the increased meiotic recombination or increased potential for recombination may be achieved by an increase in meiotic crossover frequency or an increase in potential for meiotic crossover frequency.
  • the recombination may be between homologous chromosomes.
  • the activity and/or levels of the HEI10 protein may be increased by enhancing gene expression of the HEI10 protein. Additionally or alternatively, activity and/or levels of the HEI10 protein may be increased by increasing copy number of a gene expressing the HEM 0 protein.
  • the plant cell may be transformed with a polynucleotide encoding an HEI10 protein as defined herein, thereby increasing the activity and/or levels of the HEI10 protein.
  • the HEI10 protein may be defined as an HEI10 protein with at least 50% sequence identity, for example at least 55%, 60%, 65%, 67% 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the HEM 0 protein of SEQ ID NO: 1 .
  • the HEI10 protein may for example have at least 95% or 99% sequence identity to the HEI10 protein of SEQ ID NO: 1 .
  • the Arabidopsis thaliana HEI10 protein of SEQ ID NO: 1 has been described in Chelysheva, L. et al. (2012, supra).
  • HEI10 proteins examples include the homologous wheat HEM 0 proteins Traes_6AL_B13BFCFF8.2 (SEQ ID NO: 2), Traes_6DS_18307F490.2 (SEQ ID NO: 3) and Traes_6BS_27266CEFE.1 (SEQ ID NO: 4), as well as proteins with at least 50% sequence identity, for example at least 55%, 60%, 65%, 67% 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to these homologues.
  • the HEI10 protein defined by SEQ ID NO: 1 has 67% sequence identity to the HEM 0 protein defined by SEQ ID NO: 2.
  • the HEI10 protein have at least 50% sequence identity, for example at least 55%, 60%, 65%, 67% 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the HEI10 protein of SEQ ID NO: 2.
  • the HEI10 protein may for example have at least 95% or 99% sequence identity to the HEM 0 protein of SEQ ID NO: 2.
  • the HEI10 protein may have at least 50% sequence identity, for example at least 55%, 60%, 65%, 67% 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the HEM 0 protein of SEQ ID NO: 3.
  • the HEI10 protein may for example have at least 95% or 99% sequence identity to the HEI10 protein of SEQ ID NO: 3.
  • the HEI10 protein may have at least 50% sequence identity, for example at least 55%, 60%, 65%, 67% 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the HEM 0 protein of SEQ ID NO: 4.
  • the HEI10 protein may for example have at least 95% or 99% sequence identity to the HEI10 protein of SEQ ID NO: 4.
  • the term ⁇ 10 protein" encompasses an HEI10 polypeptide, which as used herein refers to a polymer of amino acids.
  • polypeptide may include polypeptides with post-expression modifications, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition of "polypeptide” are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), polypeptides with substituted linkages, as well as other modifications known in the art both naturally occurring and non-naturally occurring.
  • Sequence identity between amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps.
  • sequence comparisons may be undertaken using the "needle" method of the EMBOSS Pairwise Alignment Algorithms, which determines an optimum alignment (including gaps) of two sequences when considered over their entire length and provides a percentage identity score.
  • Default parameters for amino acid sequence comparisons (“Protein Molecule” option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: Blosum 62.
  • Default parameters for nucleotide sequence comparisons (“DNA Molecule” option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: DNAfull.
  • the HEI10 protein may comprise an amino acid analogue.
  • amino acid analogue may be defined as any of the amino acid-like compounds that are similar in structure and/or overall shape to one or more of the twenty L-amino acids commonly found in naturally occurring proteins.
  • L-amino acids are defined and listed in WIPO Standard ST.25 (1998), Appendix 2, Table 3 as alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), phenylalanine (Phe or F), glutamate (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (lie or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y).
  • An amino acid analogue may thus include natural amino acids with modified side chains or backbones.
  • the analogue may share backbone structures, and/or even the most side chain structures of one or more natural amino acids, with the only difference(s) being containing one or more modified groups in the molecule.
  • modification may include substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl, or hydroxyl group, etc.) or an atom (such as CI or Br, etc.), deletion of a group (supra), substitution of a covalent bond (single bond for double bond, etc.), or combinations thereof.
  • Amino acid analogues may include a-hydroxy acids, and b-amino acids, and can also be referred to as "modified amino acids”. Amino acid analogues may either be naturally occurring or unnaturally occurring (e.g. synthesised). As will be appreciated by those skilled in the art, any structure for which a set of rotamers is known or can be generated can be used as an amino acid analogue.
  • the side chains may be in either the (R) or the (S) configuration (or D- or L-configuration).
  • recombination or recombination potential may be increased by at least 10%, or by at least 200%, 150%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20%, compared with a plant cell not exhibiting an increase in the activity and/or levels of the HEI10 protein.
  • a plant cell obtained or obtainable according to the method described above, wherein the plant cell has increased recombination or increased recombination potential.
  • the plant of the invention may be a transgenic plant.
  • non-transgenic plant which may for example be derived from the transgenic plant.
  • the non-transgenic plant may be obtained by screening for loss of increased activity and/or levels of the HEI10 protein as defined herein.
  • the plant cell or plant of the invention may be selected from the group consisting of: acacia, alfalfa, aneth, apple, apricot, Arabidopsis, artichoke, arugula, asparagus, aspen, avocado, bamboo, banana, barley, beans, beech, beet, Bermuda grass, bent grass, blackberry, blueberry, Blue grass, broccoli, Brussels sprouts, cabbage, cannabis, canola, cantaloupe, carinata, carnation, carrot, cassava, cauliflower, celery, cherry, chicory, cilantro, citrus, Clementine, cocoa, coffee, corn, cotton, cucumber, duckweed, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, fescue, figs, fir, flax, forest trees, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, lupin,
  • the plant cell or plant may be wheat, maize, soybean, oilseed rape, rice, barley, tomato, potato or Arabidopsis.
  • an expression cassette comprising a promoter active during plant meiosis and operably linked to a polynucleotide encoding an HEI10 protein as defined herein, wherein the polynucleotide when expressed during plant meiosis is capable of increasing recombination.
  • a plant transformed with the expression cassette as defined above is also encompassed by the invention. Further provided is a method for obtaining a plant with increased frequency of homologous or homeologous recombination, wherein the method comprises the steps of:
  • Each aspect of the invention as described herein may also be applicable to other eukaryotic organisms such as a yeast or animal (such as a non-human animal).
  • Crossover frequency was analysed using seed-based fluorescent reporters as described in Ziolkowski, P. A. et al. (2015, supra) and in Melamed-Bessudo et al. (2005, Plant J. 43, 458-66). Pollen-based fluorescent crossover reporters were used as described in Ziolkowski et al. (2015, supra) and Yelina et al. (2013, Nat. Protoc. 8, 21 19-2134).
  • SSLP Simple Sequence Length Polymorphism
  • TTGGATCCTAAGCCTTCAATGAACATCAC-3' SEQ ID NO: 16.
  • Amplification products were cloned into the pGREEN-0029 binary vector using Xba ⁇ and BamYW restriction enzymes. This vector was transformed into Agrobacterium strain GV3101 and used for floral dipping experiments. Plants segregating for the Col-420 FTL interval were used for transformation. Ti seeds were selected using kanamycin and then transfered to soil. Immature flower buds from Ti transformants were collected for HEI10 mRNA expression analysis. After harvesting, the seeds were analysed using the 420 fluorescent reporter to measure crossover frequency. Empty vector pGREEN-0029 transformants were generated and scored alongside.
  • the meiosis-specific gene DMC1 was amplified as a control for ACt calculations, using the following primers 5'- TGAAGAAACGAGCCAGATGC-3' (SEQ ID NO: 19) and 5'- GCGTTTATACCTTGTGCGATCA-3' (SEQ ID NO: 20).
  • the 2- AAC ⁇ method was used to quantify relative transcript levels in comparison with untransformed Col-420 plants. Immunostaining and cytology
  • microsphere bead adjacent to the cell was captured as Z-stacks of 10 optical sections of 0.4 ⁇ each.
  • the measurement of the fluorescence bead intensity was performed using the same procedure as for HEM 0 signal intensity measurement. HEM 0 signal intensity was then divided by the bead signal intensity to obtain the relative intensity of HEI10.
  • the same normalization procedure was applied for each meiotic cell analysed. Microscopy was conducted using a DeltaVision Personal DV microscope (Applied precision/GE Healthcare) equipped with a CDD Coolsnap HQ2 camera (Photometries). Image capture was performed using SoftWoRx software version 5.5 (Applied precision/GE Healthcare).
  • DNA was extracted using CTAB and used to generate sequencing libraries as described in Rowan, B. A. et al. (2015, G3 (Bethesda) 5, 385-98) and Yelina et al. (2015, Genes Dev. 29, 2183-202) with the following modifications.
  • DNA was extracted from 3 rosette leaves of 5 week old plants and 150 ng of DNA used as input for each library. DNA shearing was carried out for 20 minutes at 37°C with 0.4U of DNA
  • chromosomes after smoothing using the R function filter.
  • the coordinates of crossover intervals called by TIGER were used for subsequent analysis. Crossovers were counted and compared between the populations using 2x2 contingency tables and chi- square tests. To test for overlap with genome annotations the base pair coordinates within TIGER crossover intervals were overlapped with TAIR10 representative genes, transposons or other sequences. These analyses were performed separately for chromosome arms, pericentromeres and centromeres, and wild type and HEI10 Co1 crossovers compared. For each population a random set of intervals that matched the crossover widths were analysed in the same way, for each chromosome and region. To analyse relationship with DNA methylation the crossover data were compared to published bisulfite sequencing data (Stroud et al., 2013, supra).
  • Chromosome substitution lines identify a semidominant trans recombination modifier
  • Col/Ler chromosome substitution lines generated via reverse breeding; with the exception of LCCCC which was obtained from an esd7-1 backcross line, where LCCCC denotes Ler (L) and Col (C) genotypes for each of the 5 Arabidopsis chromosomes.
  • CSLs were crossed to Col-420 (chromosome 3) and Co ⁇ - I2f (chromosome 2) FTLs and replicate Fi measurements collected (Fig. 1 b-1 d).
  • Fig. 1 b-1 d Co ⁇ - I2f
  • rQTL trans recombination QTL
  • rQTL1 is caused by polymorphism in the HEI10 E3 ligase gene
  • FIG. 4b We Sanger sequenced HEI10 Bur and observed 30 polymorphisms, 29 of which were shared with HEUO 1 - 8 ", including R264G (Fig. 4a). In contrast, we previously observed an absence of trans rQTLs in ColxCt populations, and sequencing showed 1 3 /-/E/70 Ci polymorphisms, of which 4 were shared with ⁇ ⁇ ' (Fig. 4a). This analysis leads us to consider 25 polymorphisms, including R264G, that were present in HEI10 Bur , but absent in HEI10 ct , as candidates for the rQTL 1 causal genetic change (Fig. 4a).
  • We also performed qRT-PCR analysis of HEI10 transcripts and did not observe significant differences between Col, Ler and Col/Ler Fi genotypes (ANOVA P 0.127). Therefore, the HEI10 causal rQTL 1 polymorphism is either the R264G substitution, or may occur via a change in expression timing during meiosis.
  • the highest recombining HEI10 Co ' ⁇ showed a 420 genetic distance of 47.49 cM, compared to the mean Col-420 rate of 19.08 cM (Fig. 6d).
  • Variation in HEI10 Ti recombination rates likely results from the varying expression levels typically observed between independent T-DNA transformant lines.
  • HEI10/RNF212 E3 ligases are critical, dosage-sensitive regulators of genetic map length in diverse eukaryotes.
  • HEI10 and RNF212 show dynamic association with meiotic chromosomes and typically form numerous early foci at leptotene-stage, associated with DSB repair. During meiotic progression, HEI10 persists at a smaller number of foci that co-localise with MLH1 , and represent interfering crossover sites.
  • HEI10/RNF212 are proposed to promote stable accumulation of MSH4/MSH5 (MutSy) heterodimers on meiotic chromosomes, which are mismatch repair homologues with a specialized function in ZMM pathway crossover repair.
  • MSH4/MSH5 MSH4/MSH5
  • Arabidopsis hei10 and msh4/msh5 differ in dosage-sensitivity, indicating that HEI10 specifically is limiting for crossovers.
  • TTGTGGTCCCTGGCTAATCA SEQ ID NO:-10655-F 230 167 1 10655852 At1g30270 27
  • CAGTGACGAATTCCAAAACGA SEQ ID-10655-R 230 167 1 10655852 At1g30270 NO: 28
  • GCATCGTTAGACGGTTTGCT SEQ ID NO:-17135-R 482 398 1 17135019 At1g45211 34
  • AATTGTTGCCTTGTTGGATGT (SEQ ID-20606-F 157 203 1 20606439 At1g55240 NO: 43)
  • CAATGAGCCCTCTACGCTCT SEQ ID NO:-21236-F 476 340 1 21236506 At1g56650 45
  • AAGCCCATCATATCCCAACA (SEQ ID NO:-21236-R 476 340 1 21236506 At1g56650 46)
  • TGAAAACCGTTACCCCCATA (SEQ ID NO:-19621 -R 335 241 1 19620819 At1g52690 54)
  • ATCTTCGTAATGAAATGAACTGAGC SEQ-20072-F 120 120 1 20072074 At1g53770 ID NO: 63
  • AATTGTTGCCTTGTTGGATGT (SEQ ID-20606-F 157 203 1 20606439 At1g55240 NO: 67)
  • GTTCCCCGATTCATGTGAGA SEQ ID NO:-19540-F 221 286 1 19539729 At1g52440 69
  • CAAAAAGGGAAAAGCCCACT SEQ ID NO:-19540-R 221 286 1 19539729 At1g52440 70
  • GCGTTTTGTATCATCAAAGGTTCC SEQ-6789-F 112 82 2 6789815 At2g 15560 ID NO: 75
  • GGTTCCGTCAACTTCGAAAA SEQ ID NO:-11443-F 200 141 2 11443153 At2g26830 79
  • CAGTCATTAGAAATCGATCCCACA SEQ-11443-R 200 141 2 11443153 At2g26830 ID NO: 80
  • CAGAGGGACTTGACGAAAGAG SEQ ID-14714-R 125 89 2 14714870 AT2G34880 NO: 82
  • CAGCGCTGATGCAAAGGTAA SEQ ID NO:-19311 -R 140 101 2 19311521 At2g47000 84
  • GGATTCAATCACATTTCTTTTCAA SEQ ID-2450-R 242 184 4 2450565 At4g04840 NO: 88
  • TTCCCTTCTTTTGTGGCTTC SEQ ID NO:-10847-F 254 196 4 10846502 AT4G20030 95
  • CTCCAAGCTCCTTGTTTTGG SEQ ID NO:-12848-F 138 100 4 12848948 AT4G24980 99
  • AAATCAAAACCCCATGAAAGG SEQ ID-14558-F 168 136 4 14558575 AT4G29730 NO: 103
  • GACGAACAAGGCAACCCATT SEQ ID-18526-F 478 319 4 18526361 At4g39950 NO: 105)
  • ATGGTGGACCTGGGGGTAAC (SEQ ID-3750-F 137 97 5 3750331 At5g11660 NO: 109)
  • CAAACCAAAACCTACTTTTTCCAA (SEQ ID-19994-R 169 109 5 19994907 At5g49320 NO: 118)
  • ACCATCTACTTCCATTCAAATAACG (SEQ-26907-R 270 200 5 26907352 At5g67420 ID NO: 122)
  • dCAPS and CAPS primers used for rQTLI mapping start with letter 'd', and CAPS primers starts with letter 'c'.
  • TGTGAGT (SEQ ID NO: 125)
  • AAAGAT SEQ ID NO: 131
  • SEQ ID NO: 1 Arabidopsis thaliana HEI10 protein (from UniProtKB - F4HRI2 - See Chelysheva, L. et al. (2012, supra)).
  • SEQ ID NO: 2 Triticum aestivum HEI10 omologue l (Traes_6AL_B13BFCFF8.2) MKCNACWRELEGQAITTTCGHLLCTEDAKKILSNDGACP ICDQVLSKSHMKPTDINPSDE WTDMSMTGVSPQILMKSAYRSVMFYIGQKDLEMQYKMNRIVGQCRQKCEVMQAKFTEKLE EVHAAYQKMAKRCQLMEQEIENLTRDKQELQEKFAEKSRQKRKLDEMYDQLRNEYESVKR SAIQPANNYFPRAQPDLFSGMPNILDSGDPLRQGS IDPPETPGRRDEGWAPQPRQRRENS GPFELSGGSPGHTAAPPMDMRPRQPPRSVFGANMNNSSTALRNMI I SPVKRPQPRNRPQM FTL .

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Abstract

L'invention concerne des procédés et des agents destinés à augmenter la fréquence d'événements d'enjambement au cours de la méiose dans des cellules végétales. Ainsi, le procédé selon l'invention comprend une étape d'augmentation de l'activité et/ou des niveaux d'une protéine HEI10 dans la cellule végétale.
PCT/GB2017/053667 2016-12-05 2017-12-05 Procédés destinés à augmenter la fréquence d'enjambement méiotique dans des végétaux WO2018104724A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111172179A (zh) * 2020-01-19 2020-05-19 武汉艾迪晶生物科技有限公司 泛素连接酶基因OsNLA2、蛋白及其在水稻选育中的应用
WO2023164606A1 (fr) * 2022-02-25 2023-08-31 The University Of North Carolina At Chapel Hill Composés pour modifier la recombinaison méiotique et procédés associés

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007030014A2 (fr) * 2005-09-09 2007-03-15 Keygene N.V. Recombinaison homologue dans les plantes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007030014A2 (fr) * 2005-09-09 2007-03-15 Keygene N.V. Recombinaison homologue dans les plantes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHLYSHEVA L. ET AL.: "The Arabidopsis HEI10 us a new ZMM protein related to Zip3", PLOS GENETICS, vol. 8, no. 7, E1002799, July 2012 (2012-07-01), XP002777205 *
MURPHY D.J.: "People, Plants & Genes", 2007, OXFORD UNIVERSITY PRESS, Oxford, UK, ISBN: 978-0-19-920713-8, article "Chapter 6: The domestication of cereal crops; Chapter 7: The domestication on non-cereal crops", pages: 78 - 105, XP002777204 *
QIAO H. ETAL.: "Antagonistic roles of ubiquitin ligase HEI10 and SUMO ligase RNF212 regulate meiotic recombination", NATURE GENETICS, vol. 46, no. 2, February 2014 (2014-02-01), pages 194 - 200, XP002777206 *
ZIOLKOWSKI P.A. ET AL.: "Natural variation and dosage of the HEI10 meiotic E3 ligase control Arabidopsis crossover recombination", GENES & DEVELOPMENT, vol. 31, 2017, pages 306 - 317, XP002777207 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111172179A (zh) * 2020-01-19 2020-05-19 武汉艾迪晶生物科技有限公司 泛素连接酶基因OsNLA2、蛋白及其在水稻选育中的应用
CN111172179B (zh) * 2020-01-19 2020-09-08 武汉艾迪晶生物科技有限公司 泛素连接酶基因OsNLA2、蛋白及其在水稻选育中的应用
WO2023164606A1 (fr) * 2022-02-25 2023-08-31 The University Of North Carolina At Chapel Hill Composés pour modifier la recombinaison méiotique et procédés associés

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