Method of generating plants having white foliage

Abstract

The disclosure relates to a method for the generation of plants, such as Euphorbia pulcherrima, having a dysfunctional glutathione S-transferase (GST), and the seeds, plant parts or plant cells derived therefrom. The disclosure further relates to a molecular marker capable of identifying a dysfunctional GST gene, to isolated DNA encoding such a dysfunctional GST gene and to the use of such DNA for the preparation of a molecular marker and for use in methods of targeted mutagenesis to inactivate the GST gene to generate plants with a white foliage phenotype.

What is claimed is: 
     
         1 . A method for the generation of a  Euphorbia pulcherrima  (Poinsettia) plant having a white foliage phenotype comprising the steps of:
 a. providing a target  E. pulcherrima  plant without a white foliage phenotype comprising in its genome at least one functional allele of a glutathione S-transferase gene (EpGST) comprising a simple sequence repeat (SSR) in the region encoding amino acids at positions 40-50 of the protein of SEQ ID NO: 3, comprising a stretch of 12 nucleotides consisting of a threefold CTTC repeat;   b. subjecting said  E. pulcherrima  plant to a mutagenesis treatment to produce a mutant  E. pulcherrima  plant;   c. selecting a mutant  E. pulcherrima  plant, wherein at least one allele of the EpGST gene comprises a CTTC deletion in said SSR motif;   d. repeating steps b. and c. until all alleles of the EpGST gene in the plant genome comprise said CTTC deletion in said SSR motif; and   e. selecting a  E. pulcherrima  plant having a white foliage phenotype, wherein said plant is homozygous for said CTTC deletion in said SSR motif.   
     
     
         2 . The method of  claim 1 , further comprising at least one of propagating said  E. pulcherrima  plant being homozygous for the EpGST gene comprising said CTTC deletion in said SSR motif and, crossing said  E. pulcherrima  plant being homozygous for the EpGST gene comprising said CTTC deletion in said SSR motif with another  Euphorbia  sp. plant. 
     
     
         3 . The method of  claim 1 , wherein said mutant  E. pulcherrima  plant without a white foliage phenotype is selected by a molecular marker suitable for the detection of said CTTC deletion in said SSR motif of the EpGST gene. 
     
     
         4 . The method of  claim 1 , wherein the mutagenesis treatment is a human-induced random mutagenesis treatment selected from the group consisting of agents which cause a DNA double-strand break, ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues. 
     
     
         5 . The method of  claim 1 , wherein the functional EpGST gene in said target  E. pulcherrima  is selected from the group consisting of:
 a. an EpGST gene encoding the protein of SEQ ID NO: 3 and functional homologs or variants thereof having at least 60%, amino acid identity to SEQ ID NO: 3, wherein said homologs or variants have a first domain at positions 11-13 of SEQ ID NO: 3 being AAC, AGC or AAN, where N can be any amino acid (SEQ ID NO: 62), a second domain at positions 53-56 of SEQ ID NO: 3 being LVPA, QVPA (SEQ ID NO: 63) or QPVP (SEQ ID NO: 64), and a third amino acid domain at positions 65-68 of SEQ ID NO: 3 being FESR;   b. a gene encoding an mRNA corresponding to the cDNA of SEQ ID NO: 2 and functional homologs or variants thereof having at least 90% nucleotide identity to SEQ ID NO: 2, wherein said homologs or variants comprises a stretch of 12 nucleotides consisting of a threefold CTTC repeat in the region of positions 118-150 of SEQ ID NO: 2;   c. the EpGST gene of SEQ ID NO: 1 and functional homologs or variants thereof having at least 90% nucleotide identity to SEQ ID NO: 1, wherein said homolog or variant comprises a stretch of 12 nucleotides consisting of a threefold CTTC repeat in the region of positions 128-139 of SEQ ID NO: 1; and   d. the EpGST gene of SEQ ID NO: 61 and functional homologs or variants thereof having at least 90% nucleotide identity to SEQ ID NO: 61, wherein said homolog or variant comprises a stretch of 12 nucleotides consisting of a threefold CTTC repeat in the region of positions 155-187 of SEQ ID No 61.   
     
     
         6 . The method of  claim 5 , wherein the functional homolog or variant of the protein of SEQ ID NO: 3 further has at least one of a V on position 2 of SEQ ID NO: 3, a F or an L on position 62 of SEQ ID NO: 3, a LE on positions 90-91 of SEQ ID NO: 3, and an S on position 153 of SEQ ID NO: 3. 
     
     
         7 . A plant or plant part having white foliage produced by the method of  claim 1 , wherein said plant has all of the essential morphological and physiological traits of the target  E. pulcherrima  plant. 
     
     
         8 . A white-foliaged  E. pulcherrima  plant derived from a non-white foliaged cultivated  E. pulcherrima  plant, wherein said non-white plant comprises in its genome a gene encoding a homolog or variant having at least 60% amino acid identity to SEQ ID NO: 3, said homolog or variant having a SSR comprising a stretch of 12 nucleotides consisting of a threefold CTTC repeat at positions 40-50 of the protein of SEQ ID NO: 3, wherein said derived white-foliaged  E. pulcherrima  plant comprises a CTTC deletion in said SSR, and wherein said white-foliaged  E. pulcherrima  plant is at least 99.9% genetically identical to said non-white foliaged  E. pulcherrima  plant. 
     
     
         9 . A white-foliaged  E. pulcherrima  plant of  claim 8 , wherein said derived from non-white foliaged cultivated  E. pulcherrima  plant comprises in its genome a first domain at positions 11-13 of SEQ ID NO: 3 being AAC, AGC or AAN, where N can be any amino acid (SEQ ID NO: 62), a second domain at positions 53-56 of SEQ ID NO: 3 being LVPA, QVPA (SEQ ID NO: 63) or QPVP (SEQ ID NO: 64) and a third amino acid domain at positions 65-68 of SEQ ID NO: 3 being FESR. 
     
     
         10 . A seed, plant part, plant cell, or a plant population of the white-foliage  E. pulcherrima  plant of  claim 8 . 
     
     
         11 . The method of  claim 1 , wherein said target  E. pulcherrima  plant without a white foliage phenotype comprises in its genome at least one dysfunctional allele of EpGST. 
     
     
         12 . The method of  claim 11 , further comprising at least one of propagating said  E. pulcherrima  plant having at least one allele of EpGST comprising said CTTC deletion and, crossing said  E. pulcherrima  plant having at least one allele of EpGST comprising said CTTC deletion with another  Euphorbia  sp. plant. 
     
     
         13 . The method of  claim 11 , wherein said mutant  E. pulcherrima  plant having a white foliage phenotype is selected by a molecular marker suitable for the detection of said CTTC deletion in said EpGST gene. 
     
     
         14 . The method of  claim 11 , wherein the mutagenesis treatment is a human-induced random mutagenesis treatment selected from the group consisting of agents which cause a DNA double-strand break, ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues. 
     
     
         15 . The method of  claim 11 , wherein the functional EpGST gene in said target  E. pulcherrima  is selected from the group consisting of:
 a. a EpGST gene encoding the protein of SEQ ID NO: 3 and functional homologs or variants thereof having at least 60%, amino acid identity to SEQ ID NO: 3, wherein said homologs or variants have a first domain at positions 11-13 of SEQ ID NO: 3 being AAC, AGC or AAN, where N can be any amino acid (SEQ ID NO: 62), a second domain at positions 53-56 of SEQ ID NO: 3 being LVPA, QVPA (SEQ ID NO: 63) or QPVP (SEQ ID NO: 64), and a third amino acid domain at positions 65-68 of SEQ ID NO: 3 being FESR;   b. a gene encoding an mRNA corresponding to the cDNA of SEQ ID NO: 2 and functional homologs or variants thereof having at least 90% nucleotide identity to SEQ ID NO: 2, wherein said homologs or variants comprises a stretch of 12 nucleotides consisting of a threefold CTTC repeat in the region of positions 118-150 of SEQ ID NO: 2;   c. the EpGST gene of SEQ ID NO: 1 and functional homologs or variants thereof having at least 90% nucleotide identity to SEQ ID NO: 1, wherein said homolog or variant comprises a stretch of 12 nucleotides consisting of a threefold CTTC repeat in the region of positions 128-139 of SEQ ID NO: 1; and   d. the EpGST gene of SEQ ID NO: 61 and functional homologs or variants thereof having at least 90% nucleotide identity to SEQ ID NO: 61, wherein said homolog or variant comprises a stretch of 12 nucleotides consisting of a threefold CTTC repeat in the region of positions 155-187 of SEQ ID No 61.   
     
     
         16 . The method of  claim 15 , wherein the functional homolog or variant of the protein of SEQ ID NO: 3 further has at least one of a V on position 2 of SEQ ID NO: 3, a F or an L on position 62 of SEQ ID NO: 3, a LE on positions 90-91 of SEQ ID NO: 3, and an S on position 153 of SEQ ID NO: 3. 
     
     
         17 . A plant or plant part having white foliage produced by the method of  claim 11 , wherein said plant has all of the essential morphological and physiological traits of the target  E. pulcherrima  plant. 
     
     
         18 . A  E. pulcherrima  plant with a white foliage phenotype, wherein said plant is derived from the white foliaged plant of  claim 11  as first donor plant by breeding technologies with one or more non-white foliaged second donor  E. pulcherrima  plants comprising one or more elite properties, wherein said derived plant comprises one or more elite properties from the one or more second donor plants. 
     
     
         19 . An isolated nucleic acid of the EpGST gene described by SEQ ID NO: 61 or a variant thereof having at least 80% identity to the sequence described by SEQ ID NO: 1 and encoding a functional homolog or variant of the protein of SEQ ID NO: 3, wherein said homolog or variant has a first domain at positions 11-13 of SEQ ID NO: 3 being AAC, AGC or AAN, where N can be any amino acid (SEQ ID NO: 62), a second domain at positions 53-56 of SEQ ID NO: 3 being LVPA, QVPA (SEQ ID NO: 63) or QPVP (SEQ ID NO: 64), and a third amino acid domain at positions 65-68 of SEQ ID NO: 3 being FESR, and further comprising, in the region encoding amino acids at positions 40-50 of the protein of SEQ ID NO: 3, a stretch of 12 nucleotides consisting of a threefold CTTC repeat. 
     
     
         20 . The isolated nucleic acid of  claim 19  selected from the group consisting of SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, or variants thereof with at least 95% identity. 
     
     
         21 . A method of use of the isolated nucleic acid of  claim 19  for the preparation of a molecular marker or for a method for targeted mutagenesis of said EpGST gene. 
     
     
         22 . The method of  claim 21 , wherein a continuous stretch of at least 17 nucleotides from any of the isolated DNA sequences is used to produce a guide RNA or an expression construct therefore for a CRISPR/Cas-based method of gene editing or to produce a silencing RNA or an expression construct therefore for a method of RNA-mediated gene silencing. 
     
     
         23 . The method of  claim 22 , wherein the targeted mutagenesis is introduced by a DNA modification enzyme selected from the group consisting of meganucleases (MNs), zinc-finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs), Cas9 nuclease, Cpf1 nuclease (Cas12a), dCas9-FokI, dCpf1-FokI, chimeric Cas9-cytidine deaminase, chimeric Cas9-adenine deaminase, chimeric FEN1-FokI, and Mega-TALs, a nickase Cas9 (nCas9), chimeric dCas9 non-FokI nuclease, dCpf1 non-FokI nuclease, chimeric Cpf1-cytidine deaminase, and Cpf1-adenine deaminase. 
     
     
         24 . The method of  claim 22 , wherein said DNA sequence used to design a guide RNA is an 18-21 nucleotide sequence and is at least 90% identical to a target sequence. 
     
     
         25 . The method of  claim 24 , wherein the target sequence is SEQ ID: 61. 
     
     
         26 . A method of use of the isolated nucleic acid of  claim 19  for the generation of a molecular marker, wherein said marker is capable of identifying a dysfunctional EpGST allele. 
     
     
         27 . The molecular marker of  claim 26 , wherein said marker identifies a CTTC deletion within positions 128-139 of the EpGST gene of SEQ ID NO: 1. 
     
     
         28 . A method for producing a  E. pulcherrima  plant having a white foliage phenotype comprising:
 a. screening a population of  E. pulcherrima  plants for dysfunctional GST using the marker of  claim 27 ;   b. selecting a first  E. pulcherrima  plant having at least one dysfunctional GST allele;   c. crossing said first selected  E. pulcherrima  plant having at least one dysfunctional GST allele with a second  E. pulcherrima  plant having at least one dysfunctional GST allele or itself to produce F 1  progeny; and   d. screening said F 1  progeny  E. pulcherrima  plants using said marker for homozygous dysfunctional GST alleles.   
     
     
         29 . A plant or plant part having white foliage produced by the method of  claim 28 . 
     
     
         30 . A method for producing a plant having reduced levels of anthocyanins comprising:
 a. providing a plant comprising in its genome at least one functional allele of a glutathione S-transferase gene;   b. subjecting said plant to targeted mutagenesis treatment to produce a mutant GST gene therein, wherein said mutation is selected from the group consisting of loss-of-function, partial loss-of-function, a restored frameshift, an in-frame deletion, or a promoter deletion, and wherein said targeted mutagenesis uses at least one of the sequences of SEQ ID NO: 44-49, or variants thereof having at least 95% identity, to produce a guide RNA; and;   c. selecting a plant having reduced levels of anthocyanins.