Haploid inducers
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.
1. A method for identification of a Zea mays plant which has at least one mutation in an endogenous DNA sequence encoding for a pollen-specific phospholipase, which endogenous pollen-specific phospholipase-encoding DNA sequence
(i) has the genomic sequence of SEQ ID NO: 8 and/or leads to a cDNA comprising the sequence of SEQ ID NO: 10, or
(ii) is complementary to the full length of a sequence from (i), or
(iii) is at least 90% identical to the full length of a sequence from (i), or
(iv) encodes for a protein with the amino acid sequence selected from the group consisting of SEQ ID NOs: 21 and 22;
comprising the step of:
(a) detecting in a pollen or a tissue of a pollen of the identified plant a change in the expression rate of the endogenous DNA sequence, in comparison to a non-mutagenized wild-type plant, or
(b) verifying the at least one mutation via sequencing, molecular markers for SNPs or length polymorphism-based markers for insertion or deletion mutants,
wherein the at least one mutation
i) leads to the sequence of SEQ ID NO: 9 or a cDNA comprising the sequence of SEQ ID NO: 11; and/or;
ii) leads to a protein comprising the amino acid sequence of SEQ ID NO: 23; and/or
iii) is an alteration in the encoding sequence of SEQ ID NO: 8 which causes an amino acid exchange between the amino acid positions 74 and 78 of SEQ ID NO: 21 or 22, and/or
iv) causes an amino acid exchange in the encoded amino acid at position 74 of SEQ ID NO: 21 or 22, wherein the aspartate is replaced by asparagine (D74N) as set forth in SEQ ID NO: 51, and/or
v) causes an amino acid exchange in the encoded amino acid at position 78 of SEQ ID NO: 21 or 22, wherein the glycine is replaced by arginine (G78R) as set forth in SEQ ID NO: 54,
and causes the property of a haploid inductor to be mediated or the induction capability of a haploid inductor to be increased in the identified plant.
2. The method of claim 1 , wherein the at least one mutation produces an alteration in the activity or stability of the pollen-specific phospholipase, in comparison to the wild-type pollen-specific phospholipase.
3. The method of claim 1 , wherein the molecular marker is a DNA primer or a pair of DNA primers.
4. The method of claim 3 , wherein a sample from the plant is contacted with the DNA primer or the pair of DNA primers in a polymerase chain reaction (PCR).
5. The method of claim 4 , wherein the PCR is reverse transcription PCR (RT-PCR).
6. The method of claim 1 , wherein the molecular markers demonstrate the presence or absence of the at least one mutation.
7. The method of claim 6 , wherein the molecular markers are KASPar or TaqMan markers.
8. The method of claim 4 , wherein the sample is a pollen sample.
9. The method of claim 3 , 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 the pollen-specific phospholipase.
10. The method of claim 3 , wherein the DNA primer or the pair of DNA primers are specific for a target gene.
11. The method of claim 1 , wherein the at least one mutation in the endogenous DNA sequence is a point mutation.
12. The method of claim 1 , further comprising developing a haploid inducer with the identified plants.
13. The method of claim 1 , further comprising using the identified plant as a pollen parent in a cross with a seed parent of the same species.
14. The method of claim 1 , further comprising isolating pollen from the identified Zea mays plant, and pollinating another plant with the isolated pollen.
15. The method of claim 1 , wherein the endogenous pollen-specific phospholipase-encoding DNA sequence has the genomic sequence of SEQ ID NO: 8 or leads to a cDNA comprising the sequence of SEQ ID NO: 10, wherein the at least one mutation causes an amino acid exchange in the encoded amino acid at position 74 of SEQ ID NO: 21 or 22, and wherein the aspartate is replaced by asparagine (D74N) as set forth in SEQ ID NO: 51.
16. The method of claim 1 , wherein the endogenous pollen-specific phospholipase-encoding DNA sequence has the genomic sequence of SEQ ID NO: 8 or leads to a cDNA comprising the sequence of SEQ ID NO: 10, wherein the at least one mutation causes an amino acid exchange in the encoded amino acid at position 78 of SEQ ID NO: 21 or 22, and wherein the glycine is replaced by arginine (G78R) as set forth in SEQ ID NO: 54.