HAPLOID INDUCERS

Abstract

The present invention relates to the provision of technical means such as nucleic acids which, after transcription or after expression in a plant, are suitable for mediating the property of a haploid inductor or for increasing the induction capability of a haploid inductor, as well as methods and uses for the production and identification of non-transgenic and transgenic plant haploid inductors, as well as the improvement of existing plant haploid inductors.

Claims

1-18. (canceled)

19. A method for production of a plant which is suitable for use as a haploid inductor, the method comprising: mutagenizing plant cells; regenerating plants from the mutagenized plant cells; and identifying the regenerated plant which has at least one mutation in an endogenous DNA sequence which is identical to an isolated nucleic acid comprising a nucleotide sequence, which is (i) selected from the group consisting of SEQ ID Nos: 8, 9, 10 and 11, (ii) complementary to the sequence selected from the group consisting of SEQ ID Nos: 8, 9, 10 and 11, (iii) is at least 80% identical to the sequence selected from the group consisting of SEQ ID Nos: 8, 9, 10 and 11, (iv) hybridizes with a sequence at least 80% identical to the sequence selected from the group consisting of SEQ ID Nos: 8, 9, 10 and 11 under stringent conditions, or has at least one mutation in a regulatory sequence of the endogenous DNA sequence, which mutation produces a change in expression rate of the endogenous DNA sequence in the identified plant, in comparison to a non-mutagenized wild-type plant, or a change in the activity or stability of a protein or polypeptide encoded by the endogenous DNA sequence in the identified plant, in comparison to a non-mutagenized wild-type plant, wherein the at least one mutation causes a property of a haploid inducer to be mediated or the induction capability of a haploid inducer to be increased in the identified plant.

20. The method of claim 19, wherein the change in the expression rate of the endogenous DNA sequence in the identified plant is detected in a pollen or a tissue of a pollen of the identified plant.

21. The method of claim 19, wherein the change in the activity or stability of the protein or the polypeptide encoded by the endogenous DNA sequence is detected in a pollen or a tissue of a pollen of the identified plant.

22. The method of claim 19, wherein the plant comprises the at least one mutation in the regulatory sequence of the endogenous DNA sequence.

23. The method of claim 22, wherein the at least one mutation is the deletion of a nucleotide or the addition of a nucleotide.

24. The method of claim 22, wherein the regulatory sequence of the endogenous DNA sequence is a promoter.

25. The method of claim 24, wherein the promoter comprises a modification in a cis-regulatory element.

26. The method of claim 25, wherein the activity of the promoter is altered as compared to the activity of a promoter without the modification in the cis-regulatory element.

27. The method of claim 26, wherein the activity of the promoter is increased as compared to the activity of a promoter without the modification in the cis-regulatory element.

28. The method of claim 26, wherein the activity of the promoter is reduced as compared to the activity of a promoter without the modification in the cis-regulatory element.

29. The method of claim 19, wherein the at least one mutation is an exchange, addition or deletion of at least one nucleobase in the encoding region of the endogenous DNA sequence, and wherein the exchange, addition or deletion of the at least one nucleobase leads to an amino acid exchange in the encoded protein and produces an alteration in the activity or stability of the protein, in comparison to the wild-type protein.

30. The method of claim 19, wherein the identifying step comprises contacting a sample from the plant with a molecular marker, wherein the molecular marker is a DNA primer or a pair of DNA primers.

31. The method of claim 30, wherein a sample from the plant is contacted with the DNA primer or the pair of DNA primers in a polymerase chain reaction (PCR).

32. The method of claim 31, wherein the PCR is reverse transcription PCR (RT-PCR).

33. The method of claim 29, wherein the molecular marker demonstrates the presence or absence of the at least one mutation.

34. The method of claim 33, wherein the molecular markers are KASPar or TaqMan markers.

35. The method of claim 29, wherein the sample is a pollen sample.

36. The method of claim 31, wherein the DNA primer or the pair of DNA primers comprises a sequence for detection of the at least one mutation in a gene encoding for phospholipase.

37. The method of claim 31, wherein the DNA primer or the pair of DNA primers are specific for a transposon or a target gene.

38. The method of claim 31, wherein the at least one mutation in the endogenous DNA sequence is a point mutation.

39. A plant or plant part produced according to the method of claim 19.

40. A descendant of the plant according to claim 39.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG. 1: Genomic arrangement of the identified genes in comparison to B73 (AGPv02):

SNAREv 1 (GRMZM2G179789): increased expression in RWS pollen;
SNAREv 2 (GRMZM2G412426): increased expression in RWS pollen;
ITP (Inositol-1,4,5-triphosphate-5-phosphatase) (GRMZM2G106834): reduced expression in RWS pollen;
PL (Patatin phospholipase) (GRMZM2G471240): polymorphisms in encoding sequence;
MITO1 (Mitochondrial import receptor): present only in RWS;
MITO2: Homolog to MITO1, but shortened. Present only in RWS;
PGM (Phosphoglycerate mutase) (GRMZM2G062320): deleted in RWS;
lncRNA: Homolog of PL: deleted in RWS;
AC213048: anchor gene for comparison of the sequences;
MT (RNA methyl transferase) (GRMZM2G347808): polymorphisms in the regulatory region.
The GRMZM names relate to the annotation in AGPv02.

[0071] FIG. 2: RT-PCR of ripe pollen in inductor RWS and three non-inductor controls (NI1, NI2, NI3) across the genes SNAREv 1, RNA methyl transferase, and patatin phospholipase.

[0072] FIG. 3: RNASeq data of RWS pollen, projected onto artificial reference from AGPv02 with regions for SNARE and phospholipase loci substituted by RWS BAC's. (T1: Transcript 1. Homolog to SNARE2, but with altered exon-intron structure; T2: Homolog to SNARE1. Encoding for a protein of 131AA; T3: Homolog to SNARE1/2. RT-PCR fragment from FIG. 2).

QTL ANALYSIS AND IDENTIFICATION OF CANDIDATE GENES

[0073] In the maize haploid inductor RWS, which is to be ascribed to the inductor Stock6 (Coe, 1959), a main-QTL on chromosome 1 (bin 1.04) was identified and finely mapped. Based upon these works, the QTL in RWS should be verified and molecularly analyzed, in order to identify and functionally validate the underlying genes. A QTL mapping population from RWSControl1 (maternal inductornon-inductor) was tested for induction capability. It could thereby be shown that the known QTL is probably also present in the inductor RWS. However, it was further also achieved that a strong allele shift to the benefit of the non-RWS (Control 1) allele was discovered.

[0074] In order to molecularly describe the locus, various sequencing approaches to DNA and at the RNA level were selected. Due to structural differences between inductors and reference genome B73, only a small proportion of classical, reference-based sequencing approaches lead to success. Expanded and complicated bioinformatic analyses had the result that structural differences would then need to be reviewed via other technologies (FIG. 1).

[0075] Within the scope of a sequence capture approach, approximately three megabases around the identified QTL in three Stock6-derived inductors, as well as RWS and five non-inductor controls, were sequenced, and were analyzed on inductor-specific polymorphisms such as presence-absence variations, SNP's, and InDel's. Initially, 16 candidate genes were thereby identified, of which three genes were confirmed via post-sequencing and analysis of expression data: one gene that encodes for an anther-specific patatin phospholipase A2 which has an RWS inductor-specific haplotype; a phosphoglycerate mutase gene which is not present in the inductor RWS; and an RNA methyl transferase gene which has a mutation in a regulatory sequence (FIG. 2).

[0076] BAC banks were also developed for RWS, EMK (an additional inductor derived from Stock6), and Control 1 and screened with probes distributed over the identified QTL. For a target range of approximately 150 kB, which was mentioned by Dong et al. 2013 in inductor UH400 as possibly being inductor-relevant, BAC's of RWS, Control 1, and EMK were extracted and sequenced. The BAC sequences were annotated and compared with comprehensive transcriptome data which were created for RWS, Control 1, EMK, and B73.

[0077] As a result, the deletion in the inductor could here be confirmed. Accordingly, the examined maternal inductors lack a region of 100 kB between 68.26 and 68.36 MB (AGP Version 2 of the B73 reference sequence) on chromosome 1. Furthermore, an inversion in a gene-similar region and a large, repetitive sequence segment that is not comparable to the reference genome of B73 and to Control 1 appears outside of the target region in the inductors.

[0078] In spite of the deletion, the already identified phospholipase is still present in the inductors, but shows the aforementioned haplotype strongly deviating from the controls, and marked genetic variations in the promoter region. As a result of the deletion, the phosphoglycerate mutase that was already identified above is no longer present.

[0079] Furthermore, it is also achieved that a non-coding RNA (lncRNA) is identified in the 100 kb deletion. Like the phospholipase, it is pollen-specifically expressed and, moreover, shows a homology of 82% with the identified phospholipase. The sequence is inherently complementary, i.e., the lncRNA forms a hairpin structure. The very high expression rate, the significant homology with the phospholipase, and the low SNP density that was determined via Sanger sequencing indicate a regulatory function of this lncRNA for the phospholipase. Theoretically, an 88 amino acid-long, truncated version of the phospholipase protein could also be translated from this transcript.

[0080] In order to also be able to measure differences in the expression level of the identified gene from the region, in addition to measuring polymorphisms at the DNA level, RT-PCR and RNASeq experiments were implemented. In addition to RWP (a subline of RWS) as an inductor, three, genetically very different, control lines were used. From these plants, pollen was harvested, anthers without pollen, and embryos from 6-7 days after pollination by selfings or crossings. The phospholipase here showed a slightly increased expression in pollen from RWP. The methyl transferase shows a weak expression in the pollen of RWP and no expression in the pollen of the control. lncRNA is expressed and absent pollen-specifically, as also expected in RWP.

[0081] RNASeq was additionally applied to pollen of the same material in order to further verify the preceding results.

[0082] The transcriptome data (RNA-Seq at Pollen RNA of RWS) was projected on an artificial reference, in which the region of the QTL in B73 was replaced with RWS-BAC's. This analysis shows an expression of the phospholipase in pollen. The exon-intron structure of the gene corresponds to that of B73, but a deletion exists at the 5 end, which leads to a stop codon and therefore to a shortened protein. Furthermore, three additional RWS-specific transcripts were detected above and below the phospholipase. A region having two transcripts is located approximately 60 kb above the phospholipase. The first transcript is non-coding; the second encodes for a 192 amino acid-long protein that shows homologies with the mitochondrial import receptor (MITO1). In B73, this is situated only 15 megabases upstream of the QTL (GRMZM2G174696). Approximately 90 kilobases (kb) below the phospholipase is an additional transcript that in turn shows high homologies with the 192 amino acid-long transcript.

[0083] In order to also receive inductor-specific expression outside of the QTL, the RNASeq data were evaluated genome-wide. Unexpectedly, new candidate genes were identified outside of, but near, the finely-mapped region cited above, which probably could not previously be found due to the technical limitations of the SeqCapture approach. Approximately 400 kb upstream of the identified phospholipase from the finely-mapped region is a gene complex which, in pollen of RWP, is expressed distinctly differently (by at least a factor of 2) in comparison to the controls. This gene complex contains three genes: two genes annotated as SNAREv genes which have a high homology to one another and are over-expressed in RWP, and one gene that is annotated as inositol-1,4,5-trisphosphate-5-phosphatase and whose expression in RWP is reduced. Cloned transcripts of these genes distinctly deviate in part from the public annotation, such that they may also encode for proteins with deviating functions, or also may function as lncRNA's. A BAC made up of RWS could be isolated from this locus, and sequenced. This sequence was integrated into the artificial reference for re-analysis of the RNASeq data in AGPv02 (FIG. 3). In addition to a transposase, two RNA's (T1 (SEQ ID Nos: 55, 56, 57, and 63) and T3 (SEQ ID Nos: 60, 61, 62, and 65)) and an RNA with an ORF of 131 amino acids are expressed in this locus (T2 (SEQ ID Nos: 58, 59, and 64)). Except for the transposase, all transcripts are situated within or between the two SNAREv genes. Although they presumably have no SNARE function themselves, they could be involved in the regulation of homologous genes. The sequence capture data of this region show that there are distinct structural deviations between inductors, controls, and reference genome. The BAC sequencing confirms the absence of both inositol-1,4,5-trisphosphate-5-phosphatase gene at the genomic level in the inductor and the absence of an lncRNA from B73 that shares the transcription start with the inositol-1,4,5-trisphosphate-5-phosphatase, but is read from the counter-strand. The isolation of a cDNA from one of the SNARE genes (GRMZM2G179789) also indicates complex structural changes in the inductors, since one part of the cDNA corresponds to the plus strand and one part corresponds to the minus strand of the reference.

Gene Functionalities

[0084] Overall, seven genes could thus be identified which could be important for the in vivo haploid induction or the in vivo haploid induction capability in maize.

[0085] Among these four genes, which are of particular importance to pollen tube growth:

the two SNAREv genes encoding for proteins which are known to be involved in vesicle transport (literature). In the model plant Arabidopsis thaliana, SNAREv proteins have already been demonstrated at the tip of the pollen tube, where they are involved in the transport of phospholipids and pectins (literature). The over-expression of the SNAREv proteins that was observed in the examined maize inductors would lead to increased pollen tube growth.

[0086] That the phospholipase A2 also distinctly influences the pollen tube growth could be shown in the model plant Nicotiana tabacum. The inhibition of phospholipase A2 accordingly leads to a suppression of the pollen tube growth (Kim et al., 2011). In the examined maize inductors, the absence of the identified lncRNA having significant homology with the phospholipase may lead to a reduction in the expression or translation rate of the phospholipase gene, which would accelerate the growth speed of the pollen tube.

[0087] In a knockout mutant of inositol-polyphosphate-5-phosphatase in Arabidopsis thaliana, it appeared that the pollen tube grows uninhibitedly. In the examined maize inductors, the reduced expression level of the inositol-1,4,5-trisphosphate-5-phosphatase thus may likewise lead to an accelerated pollen tube growth. The identified lncRNA associated with inositol-1,4,5-trisphosphate-5-phosphatase could here have a regulatory effect on the expression rate.

[0088] The examined maize inductors thus show a modified regulation/expression rate of the four genes, in comparison to non-inductors. This disruption should lead to a markedly faster pollen tube growth, which is also promoted by a possibly increased energy metabolism, due to the expression of a mitochondrial transporter or its regulation. This could have the result of a decoupling of the transport of the generative cells in the pollen tube with its growth. As a result, an incomplete or incorrect pollination with subsequent chromosome elimination may occur.

[0089] It is known that active centromeres play a key role in chromosome distribution and are characterized and modified via chromatin modifications at the DNA or histone levelmoreover, by transcription, RNA interactions, and RNA binding. A change in the regulation of the methyl transferase gene may influence the activity of the inductor centromere during the early embryogenesis, which ultimately leads to the elimination of the inductor genome in the early seed development stage.

[0090] In the examined inductors, it could be shown that the phosphoglycerate mutase gene is no longer present. The absence of the gene may negatively affect the energy metabolism of the pollen, and therefore have effects on the pollination. Moreover, the energy metabolism may be influenced by the mitochondrial membrane protein.

[0091] Any gene individually, or any combination of the genes, may be responsible for the effect of the haploid induction.

Creation of New In Vivo Haploid Inductors

[0092] In order to develop a new inductor in other crop types or maize non-inductor genotypes, or to increase the induction capability of an inductor genotype, the following is to be performed:

[0093] Identification of the corresponding genes in other crop types or maize non-inductor genotypes: In single-cotyledon plants such as maize, rice, wheat, rye, or barley, the pollen-specific patatin phospholipases are strongly conserved, and, therefore, homologs of these are easy to identify. In contrast to this, regulatory lncRNA's are absent in most single-cotyledon plants. However, in the event that they are present, they may likewise be discovered using significant homologies, just as they also occurred in the examined maize inductors. In double-cotyledon plants, other phospholipase types take on the corresponding tasks in the pollen tube growth. In order to identify these, RNA banks of pollen or pollen tubes are to be created and screened for the specific phospholipase of the present invention. A patatin phospholipase that is strongly expressed in pollen could already be identified via RNASeq of sunflower pollen (SEQ ID Nos: 46-48).

[0094] The SNAREv genes and the methyl transferase gene do not need to be pollen-specific. For example, one of the identified SNAREv genes (SNAREv 1) in maize is also not expressed in a pollen-specific manner. SNAREv 1 is not expressed at all in wild-type pollen. In annotated genomes, homologous genes may be identified via BLASTP and the functional region of a SNAREv protein. In unannotated genomes, RNASeq data would need to be annotated and selected for SNARE genes.

[0095] Homologous inositol-1,4,5-trisphosphate-5-phosphatases and phosphoglycerate mutases must be expressed in pollen, in order to be used as candidate genes. The identification may take place as above, via BLASTP and subsequent RT-PCR in pollen or via annotation of RNASeq data of pollen.

Manipulation of the Candidate Gene:

[0096] Possible inductors or an increased induction capability may be achieved via transgenic expression of the phospholipases and/or SNARE's and/or methyl transferase and/or phosphoglycerate mutases and/or lncRNA's and/or of the mitochondrial import receptor described above. For this, the corresponding genesincluding their promotersare to be cloned from the inductor line RWS. These genes may be cloned in a suitable transformation vector and be transformed in the desired plant.

[0097] The pollen-expressed inositol-1,4,5-trisphosphate-5-phosphatase may be additionally or exclusively reduced in their activity via RNAi, for example. For example, for this, hairpin constructs are to be produced, which then include a suitable promoter and terminator which allow a transcription of the hairpin construct before or at the point in time of the pollen formation. These hairpin constructs would be cloned in a suitable transformation vector and be transformed in the desired plant.

[0098] Alternatively or additionally, plants having mutations (for example, in the identified genes) that stabilize the phospholipase and/or SNARE's and/or methyl transferase, amplify the expression, or increase the activity may be generated via TILLING, transposon mutagenesis or other mutagenesis methods, or genome editing. Structural analyses of secondary and tertiary structure of the mutated proteins may be helpful for this, which mutated proteins indicate denser structures, for example, and therefore fewer attack points for proteases. Moreover, the regions of the proteins that play a role in ubiquitin interactions may be considered. Mutants in the active center of the gene may be directly tested for their activity. For verification of the functionality of the phospholipase, various Tilling mutants have already been checked for induction capability. The exchange D74N (exchange of aspartate at Position 74 for asparagine) or G78R (exchange of glycine at Position 78 for arginine) lead to a maternal induction rate of 0.2-0.4%. In order to alternatively or additionally manipulate the inositol-1,4,5-triphosphate-5-phosphatase or the phosphoglycerate mutase, one must search for knockout mutants or for additional mutants that reduce the activity of the gene.

[0099] A Stock6-derived inductor may also be improved. This is possible via the above-described transgenic approach and via the introduction of mutations in the identified candidate genes. Insofar as they are expressed in pollen, it would additionally be possible to manipulate additional copies of the genes in the genome via transgenic or non-transgenic approaches.

[0100] Test of the induction capability: There are, for example, the following possibilities for testing the induction capability of a potential inductor:

1. Pollination of a line having a visual recessive marker (for example, glossy (Bordes et al., 1997) or liguleless (Sylvester et al., 1990), for maize). Descendants that express this feature are tested for haploidy via flow cytometry.
2. Pollination of a line that differs genetically from the inductoroptimally, via multiple markers. Use of these markers in order to identify homozygotic plants. These plants are tested for haploidy via flow cytometry.

[0101] Both possibilities were applied to test the induction capability.

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