Haploidization in sorghum

Abstract

Sorghum plants are provided which are capable of inducing haploidy by modifications in the genome related to a pollen-specific expressed patatin phospholipid producing haploid offspring and can be produced for hybrid breeding in short time by chromosome doubling inbred lines, that is, homozygous father and mother lines. In addition, methods are provided for producing transgenic and non-transgenic plant haploid inducers and improving the induction performance of plants.

1 - 15 . (canceled) 
     
     
         16 . A sorghum plant which is capable of inducing haploidy, wherein the plant has one or more modifications relating to an endogenous gene encoding a patatin phospholipase,
 wherein the patatin phospholipase is encoded by the nucleotide sequence according to SEQ ID NO: 1 or 2 or by a nucleotide sequence which is at least 95% identical to SEQ ID NO: 1 or 2, or which comprises the amino acid sequence shown in SEQ ID NO: 3 or a homologous amino acid sequence,   wherein the one or more modifications are one or more mutations in the endogenous gene encoding the patatin phospholipase, and   wherein the one or more mutations result in an amino acid exchange at one or more of amino acid positions 53-85, 150-192 and/or 285-311 according to SEQ ID NO: 3.   
     
     
         17 . The plant according to  claim 16 , wherein the one or more mutations results in an amino acid exchange at the amino acid position 59, 162 and/or 291 according to SEQ ID NO: 3. 
     
     
         18 . The plant according to  claim 16 , wherein the modified patatin phospholipase
 (i) comprises an amino acid sequence according to SEQ ID NO: 3 or a homologous amino acid sequence in which at least one amino acid exchange is present, wherein arginine (R) at position 59, valine (V) at position 162, and/or serine (S) at position 291 according to SEQ ID NO: 3 is replaced by another amino acid;   (ii) is encoded by a nucleotide sequence comprising the coding sequence of the DNA sequence according to SEQ ID NO: 1 or a DNA sequence which is at least 95% identical to SEQ ID NO: 1, in which at least one nucleotide exchange is present, resulting in an amino acid exchange, wherein one or more nucleotides are exchanged at positions 421-423, 815-817, and/or 1420-1422 according to SEQ ID NO: 1;   (iii) comprises an amino acid sequence according to SEQ ID NO: 6, 9 or 12; or   (iv) is encoded by a nucleotide sequence comprising the coding sequence of the DNA sequence according to SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10.   
     
     
         19 . A nucleic acid molecule which encodes the mutated patatin phospholipase characterized in  claim 18 , wherein the one or more mutations results in an amino acid exchange at the amino acid position 59, 162 and/or 291 according to SEQ ID NO: 3. 
     
     
         20 . The nucleic acid molecule according to  claim 19 , wherein the presence of the nucleic acid molecule in a plant in the absence of a wild-type patatin phospholipase results in the plant being able to induce haploidy. 
     
     
         21 . A nucleic acid molecule of at least 15, 16, 17, 18, 19 or 20 nucleotides in length that specifically hybridizes to a nucleotide sequence as defined in  claim 18 , and comprises one of the mutations or a pair of nucleic acid molecules that is suitable for a range containing at least one of the mutations, to amplify in a polymerase chain reaction (PCR). 
     
     
         22 . A plant cell containing the nucleic acid molecule according to  claim 19 , a vector comprising the nucleic acid molecule or the nucleic acid molecule as a transgene, optionally under the control of a heterologous promoter. 
     
     
         23 . A method for obtaining a plant capable of inducing haploidy or having an increased induction rate with respect to the wild type, comprising the following steps:
 (a) (i) mutagenizing plant cells and then regenerating plants from the mutagenized plant cells or mutagenizing plants;
 (ii) identifying a plant from (i) having one or more mutations in an endogenous DNA sequence which correspond to one or more of the mutations identified in  claim 32  and/or which result in aspartic acid (D) at position 75, glycine (G) at position 79, and/or proline (P) at position 203 of the amino acid sequence according to SEQ ID NO: 3 is replaced by another amino acid, preferably by asparagine (N) at position 75, arginine (R) at position 79 and/or leucine (L) at position 203, or which corresponds to these, and capable of inducing the obtaining of haploid offspring at an increased rate compared to a non-mutagenized plant; or 
   (b) (i) introducing the nucleic acid molecule having one or more mutations which correspond to the one or more identified mutations characterized in  claim 32  and/or which result in aspartic acid (D) at position 75, glycine (G) at position 79, and/or proline (P) at position 203 of the amino acid sequence according to SEQ ID NO: 3 being replaced by another amino acid,
 preferably by asparagine (N) at position 75, arginine (R) at position 79 and/or leucine (L) at position 203 or which correspond to them, in a plant cell, and 
   (ii) regenerating a plant from the plant cell (i),   preferably wherein the endogenous DNA sequence or the nucleic acid molecule is a coding nucleotide sequence which comprises amino acid sequences shown in SEQ ID NO: 3 for sorghum ( Sorghum bicolor ), SEQ ID NO: 18 for sunflower ( Helianthus annuus ), SEQ ID NO: 21 for barley ( Hordeum vulgare ), or in SEQ ID NO: 24 or 27 for sugar beet ( Beta vulgaris ).   
     
     
         24 . A plant containing the plant cell according to  claim 22 . 
     
     
         25 . An organ, plant part, tissue or cell of the plant according to  claim 16  or seeds or offspring of the plant, wherein
 (i) the seed or the offspring has one or more mutations in the endogenous gene encoding the patatin phospholipase; and/or 
 (ii) the seed or the offspring comprises a nucleic acid molecule according to or a vector comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes a mutated patatin phospholipase comprising one or more mutations resulting in an amino acid exchange at the amino acid position 59, 162 and/or 291 according to SEQ ID NO: 3. 
 
     
     
         26 . A method for obtaining a haploid plant, comprising the following steps:
 (a) crossing a plant according to  claim 16  with a plant of the same genus, preferably of the same species,   (b) selecting a fertilized haploid seed or embryo, and   (c) producing a haploid plant from the seed or embryo from (b).   
     
     
         27 . A haploid plant, haploid fertilized seed or embryo obtainable by the method according to  claim 26 , the haploid plant, haploid fertilized seed or embryo comprising a patatin phospholipase encoded by the nucleotide sequence according to SEQ ID NO: 1 or 2 or by a nucleotide sequence which is at least 95% identical to SEQ ID NO: 1 or 2, or which comprises the amino acid sequence shown in SEQ ID NO: 3 or a homologous amino acid sequence,
 wherein the one or more modifications are one or more mutations in the endogenous gene encoding the patatin phospholipase, and   wherein the one or more mutations result in an amino acid exchange at one or more of amino acid positions 53-85, 150-192 and/or 285-311 according to SEQ ID NO: 3.   
     
     
         28 . The organ, plant part, tissue, cell, seed or offspring of the plant according to  claim 27 . 
     
     
         29 . The plant according to  claim 16 , wherein the one or more mutations in the endogenous gene encoding the patatin phospholipase is a substitution which results in one or more amino acid exchanges. 
     
     
         30 . The plant according to  claim 16 , wherein the one or more mutations results in an amino acid exchange
 a) in the range of amino acid positions 55 to 75 according to SEQ ID NO: 3, preferably wherein this region corresponds to the functional domain of the patatin phospholipase; and/or   b) in the range of amino acid positions 157 to 167 according to SEQ ID NO: 3, preferably wherein this region corresponds to the functional domain of the patatin phospholipase; and/or   c) in the range of amino acid positions 285 to 298 according to SEQ ID NO: 3.   
     
     
         31 . The plant according to  claim 24 , wherein the plant is sorghum, sunflower, rye, wheat, potato, barley, or sugar beet. 
     
     
         32 . The plant according to  claim 18 , wherein the arginine (R) at position 59 is replaced by glutamine (Q), the valine (V) at position 162 is replaced by isoleucine (I), and/or the serine (S) at position 291 is replaced by leucine (L).