Restorer Plants

Abstract

Hybrid cereals are described which are obtained by restoring the pollen fertility for the Pampa cytoplasmic male sterility (P-CMS) and which are characterized by a reduced linkage drag. Plants are provided, in particular rye, which, as the male pollen parent, are capable of restoring the pollen fertility for the P-CMS. Furthermore, the nucleic acid molecule which carries the necessary information for restoring the P-CMS, DNA and vectors which contain such a nucleic acid molecule, corresponding host cells as well as a protein which can be encoded by the nucleic acid molecule and antibodies directed against it are also described. Furthermore, methods for the production of corresponding hybrid plants and transgenic plants are provided.

Claims

1. A plant from the gramineous order (Poales suitable, as a male pollen parent, for restoring the pollen fertility for the Pampa cytoplasmic male sterility (CMS), wherein a) in the plant or in a hybrid plant from a cross with a female CMS parent, a linkage drag otherwise coupled with the restoration property is reduced or completely eliminated, and b) the plant comprises a chromosomal segment which has at least one nucleic acid molecule which is capable of mediating the restoration property, and the at least one nucleic acid molecule has a nucleotide sequence which is selected from the group consisting of: (i) a nucleotide sequence with one of SEQ ID NO: 1 or SEQ ID NO: 28 or a functional fragment thereof, (ii) a nucleotide sequence which codes for an amino acid sequence with one of SEQ ID NO: 2 or SEQ ID NO: 29 or a functional fragment thereof, (iii) a nucleotide sequence which is complementary to a nucleotide sequence in accordance with (i) or (ii), (iv) a nucleotide sequence which hybridizes with a sequence in accordance with (iii) under stringent conditions, (v) a nucleotide sequence which has an identity of at least 70% with the nucleotide sequence in accordance with (i) or (ii), (vi) a nucleotide sequence which codes for an amino acid sequence which has an identity of at least 65% with the SEQ ID NO: 2 or SEQ ID NO: 29, (vii) a nucleotide sequence which codes for an amino acid sequence which, compared with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 29, exhibits discrepancies in the amino acid sequence in the form of amino acid deletions, substitutions, additions and/or insertions in the amino acid sequence.

2. The plant as claimed in claim 1, wherein the chromosomal segment is an interval between the marker loci tc256739, ctg32 or ctg24met2a5 and tc300731 or 7_01_H_1441 on chromosome 4R from a donor selected from the group consisting of IRAN IX, Pico Gentario and Altevogt 14160.

3. The plant as claimed in claim 1, wherein the chromosomal segment has one or more of the following marker loci of the donor: ctg2 (amplification product of the primer with SEQ ID NOs: 4 and 5), P20 (amplification product of the primer with SEQ ID NOs: 6 and 7), 72F13_c2_mTERF (amplification product of the primer with SEQ ID NOs: 8 and 9) or ctg16b (amplification product of the primer with SEQ ID NOs: 10 and 11).

4. The plant as claimed in claim 1, wherein the chromosomal segment is characterized by the absence of one or more of the following marker loci of the donor: 7_01_H_1441 (amplification product of the primer with SEQ ID NOs: 12 and 13), ctg24met2a5 (amplification product of the primer with SEQ ID NOs: 14 and 15) or ctg32 (amplification product of the primer with SEQ ID NOs: 16 and 17).

5. The plant as claimed in claim 1, wherein the chromosomal segment is no larger than 190 kb.

6. The plant as claimed in claim 1, wherein the plant is an inbred plant, a plant, a double haploid plant or a hybrid plant.

7. The plant as claimed in claim 1, which has an enhanced resistance against a pathogen, preferably against a fungus.

8. The plant as claimed in claim 1, wherein the plant is of the genus Secale, Hordeum or Triticale.

9. A seed or descendant of the plant as claimed in claim 1, wherein the seed or the descendant comprises the chromosomal segment.

10. An organ, plant portion, tissue or cell of the plant as claimed in claim 1.

11. An oligonucleotide which has one of the following nucleotide sequences: (i) SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18 or a complement thereof, or (ii) SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19 or a complement thereof.

12. A nucleic acid molecule which comprises a nucleotide sequence selected from the group consisting of: (i) a nucleotide sequence with one of SEQ ID NO: 1 or SEQ ID NO: 28 or a functional fragment thereof, (ii) a nucleotide sequence which codes for an amino acid sequence with one of SEQ ID NO: 2 or SEQ ID NO: 29 or a functional fragment thereof, (iii) a nucleotide sequence which is complementary to a nucleotide sequence in accordance with (i) or (ii), (iv) a nucleotide sequence which hybridizes with a sequence in accordance with (iii) under stringent conditions, (v) a nucleotide sequence which has an identity of at least 70% with the nucleotide sequence in accordance with (i) or (ii), (vi) a nucleotide sequence which codes for an amino acid sequence which has an identity of at least 65% with the SEQ ID NO: 2 or SEQ ID NO: 29, (vii) a nucleotide sequence which codes for an amino acid sequence which, compared with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 29, exhibits discrepancies in the amino acid sequence in the form of amino acid deletions, substitutions, additions and/or insertions in the amino acid sequence.

13. An expression cassette, recombinant DNA or vector comprising a nucleic acid molecule as claimed in claim 12.

14. A host cell or plant cell comprising the expression cassette, the recombinant DNA as a transgene or a vector as claimed in claim 13.

15. A transgenic plant or seeds thereof, comprising a plant cell as claimed in claim 14.

16. A protein which is coded by a nucleic acid molecule as claimed in claim 13, an amino acid sequence with one of SEQ ID NO: 2 or SEQ ID NO: 29 or an amino acid sequence which has an identity of at least 65% with the SEQ ID NO: 2 or SEQ ID NO: 29.

17. A method for the production of the plant as claimed in claim 1, comprising either the removal of one or more chromosomal intervals which contains one or more of the marker loci of the donor selected from 7_01_H_1441, ctg24met2a5 or ctg32 from the genome of a plant, or the introduction of the chromosomal segment, comprising the following steps: (I) providing a portion of a plant as the target structure containing a target nucleic acid region; (II) providing one or more recombinant constructs which together comprise or code for the components of the genome editing tool; (III) providing at least one vector for introducing the recombinant construct/constructs; (IV) providing at least one further recombinant construct comprising the nucleic acid molecule, the recombinant DNA comprising the nucleic acid molecule, the expression cassette comprising the nucleic acid molecule or the chromosomal segment, for targeted homology-directed repair of the target nucleic acid region in the target plant structure or insertion into the target nucleic acid region in the target plant structure; (V) introducing the recombinant constructs from (II) and (IV) into the target plant structure; (VI) cultivating the target plant structure under conditions which activate the components of the genome editing tool and thereby allow a targeted modification of the target nucleic acid region in the target plant structure, in order to obtain a target plant structure comprising at least one cell which comprises the targeted modification of the target nucleic acid region; and (VII) regenerating a plant from the at least one cell, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence with one of SEQ ID NO: 1 or SEQ ID NO: 28 or a functional fragment thereof, (b) a nucleotide sequence which codes for an amino acid sequence with one of SEQ ID NO: 2 or SEQ ID NO: 29 or a functional fragment thereof, (c) a nucleotide sequence which is complementary to a nucleotide sequence in accordance with (i) or (ii), (d) a nucleotide sequence which hybridizes with a sequence in accordance with (iii) under stringent conditions, (e) a nucleotide sequence which has an identity of at least 70% with the nucleotide sequence in accordance with (i) or (ii), (f) a nucleotide sequence which codes for an amino acid sequence which has an identity of at least 65% with the SEQ ID NO: 2 or SEQ ID NO: 29, and (g) a nucleotide sequence which codes for an amino acid sequence which, compared with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 29, exhibits discrepancies in the amino acid sequence in the form of amino acid deletions, substitutions, additions and/or insertions in the amino acid sequence.

18. A method for the production of a transgenic plant which has a newly-mediated restoration property for the pollen fertility for the Pampa cytoplasmic male sterility (CMS) or an improved restoration property for the pollen fertility for the Pampa cytoplasmic male sterility (CMS) compared with a non-mutated wild type plant which is otherwise isogenic, and/or which has a newly-mediated resistance against a pathogen, preferably against a fungus, in particular against the fungus Claviceps purpurea (Fr.), or an enhanced resistance against a pathogen, preferably against a fungus, in particular against the fungus Claviceps purpurea (Fr.) compared with a non-mutated wild type plant which is otherwise isogenic the method comprising the following steps: A) providing the nucleic acid molecule as claimed in claim 12, the expression cassette or the recombinant DNA comprising the nucleic acid molecule, or providing the vector comprising the nucleic acid molecule, B) transforming at least one plant cell by introducing the nucleic acid molecule, the expression cassette, the recombinant DNA or the vector from A), and C) regenerating transgenic plants from the at least one transformed plant cell from B).

19. Use of the plant as claimed in claim 1, the descendant, wherein the seed or the descendant comprises the chromosomal segment or a transgenic plant, for the production of a hybrid plant, preferably of the genus Secale or Triticale, preferably a plant of the species Secale cereale, which has a pollen fertility for the Pampa CMS which has been restored and/or which has an enhanced resistance against a fungal pathogen, in particular against the fungus Claviceps purpurea (Fr.), wherein the transgenic plant comprises the expression cassette, the recombinant DNA as a transgene or a vector comprising a nucleotide sequence selected from the group consisting of: (i) a nucleotide sequence with one of SEQ ID NO: 1 or SEQ ID NO: 28 or a functional fragment thereof, (ii) a nucleotide sequence which codes for an amino acid sequence with one of SEQ ID NO: 2 or SEQ ID NO: 29 or a functional fragment thereof, (iii) a nucleotide sequence which is complementary to a nucleotide sequence in accordance with (i) or (ii), (iv) a nucleotide sequence which hybridizes with a sequence in accordance with (iii) under stringent conditions, (v) a nucleotide sequence which has an identity of at least 70% with the nucleotide sequence in accordance with (i) or (ii), (vi) a nucleotide sequence which codes for an amino acid sequence which has an identity of at least 65% with the SEQ ID NO: 2 or SEQ ID NO: 29, or (vii) a nucleotide sequence which codes for an amino acid sequence which, compared with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 29, exhibits discrepancies in the amino acid sequence in the form of amino acid deletions, substitutions, additions and/or insertions in the amino acid sequence.

Description

[0094] Embodiments and implementations of the present invention will now be described by way of example with reference to the accompanying figures and sequences:

[0095] FIG. 1: Genetic and physical map of the Rfp1 locus. A) High resolution genetic map of the Rfp1 locus on the long arm of the rye chromosome 4R. The numbers under the uppermost horizontal line describe the number of recombination events observed between the associated markers among 4563 individual test plants. Information regarding the marker codings are listed in Table 2. B) Rfp1-spanning contig of BAC clones from the Sce-B-R05104 library. C) Predicted genes on the Rfp1 locus. The bold boxes represent exons of functional genes or gene fragments, pseudogenes or mutated genes. The orientation of the genes is indicated by horizontal arrows. The vertical line in the mTERF gene 175O19_c7 describes an early stop codon in the gene sequence. The abbreviations F and C indicate that for the marker concerned, a dominant restorer genotype specific for fertility (F) or a co-dominant inheritance (C) has been observed.

[0096] FIG. 2: Mapping of functional restorer genes on Rfp1 locus. With the aid of molecular selection markers, in two exemplary test series, recombinant individual plants with different lengths of donor chromosomal segments (D) were identified in the genetic background of a pollen parent line (E). The expression of the functional restorer genes Rfp1a and Rfp1b was determined in test crossing descendencies for each recombinant plant with the highly diagnostic male-sterile tester genotype Lo6-P(SR). Table 2 lists the marker haplotype of the NIB partner D which carries the donor introgression segment. .sub.E-D: difference between the means of test crossings from NIB partners which each homozygously carry the elite allele (E) or the donor allele (D). This difference in the mean over 7 locations determines the linkage drag effect for grain yield absolutely in dt/ha and as a percentage of NIB partner E. LSD5%: limiting difference, 5% level of significance.

[0097] FIG. 3: Mapping of restorer gene Rfp1 b. From 13 recombinant plants, the allele of the donor genotype IR9 could be unambiguously determined between the markers P20 and 7_01_H_1441 at the marker locus 72F13_c2_mTERF in 4 plants. The Rfp1b phenotype was detected in test crossing descendencies of the recombinant genotypes with the CMS tester Lo6-P(SR) and matched perfectly with the marker genotypes of the mitochondrial transcription tERmination factor (mTERF) mapped by means of 72F13_c2_mTERF [A=homozygous carrier of elite allele; H=heterozygous carrier of elite or donor allele; Rfp1b*=Rfp1 or elite phenotypes were detected using the pollen shedding of respectively 15 individual test crossing descendants of the recombinant genotype and the highly diagnostic tester from Lo6-P(SR).]

[0098] FIG. 4: Shows the linkage drag effect for grain yield (.sub.E-D) for the introgression segment 455 and 765 (y-axis), plotted against the mean linkage drag effect for each of the seven locations (x-axis). The recombinants with the short introgression segment 455 exhibited a low linkage drag effect, and the recombinants 765 with the long introgression segment exhibited a large linkage drag effect. It was also clear from the experimental data that the linkage drag effects for the seven environments were very different. Clearly, adverse weather conditions during the shooting phase were responsible for the stress conditions.

[0099] FIG. 5: Production of test crossing seed with Near-Isogenic Bulk partners as the pollen parent and CMS single cross tester T911 as the female parent. The use of NIB partners (NIB pairs) is shown in isolation parcels which acted for seed production from test crossing seed. In this regard, NIB partners dusted a CMS single cross tester which represented the opposing heterotic pool. Seed which was harvested on the CMS testers was then sown in field experiments with multiple environments in order to determine the linkage drag effect phenotypically.

[0100] FIG. 6: Expression cassette in the vector pYFrfp1 containing the restoration gene rfp1b (SEQ ID NO: 1) under the control of the ubiquitin promoter from maize with the first intron and nos-terminators.

[0101] FIG. 7: Comparison of the nucleotide sequence of the wild type rfp1a gene (Wildtyp-rfp1a) (SEQ ID NO: 32) with the nucleotide sequence of the rfp1a gene from IRAN9 (Iran9_rfp1a) (SEQ ID NO: 28).

[0102] FIG. 8: Comparison of the nucleotide sequence of the wild type rfp1b gene (Wildtyp-rfp1b) (SEQ ID NO: 30) with the nucleotide sequence of the rfp1b gene from IRAN9 (Iran9_rfp1b) (SEQ ID NO: 1).

[0103] FIG. 9: Comparison of the amino acid sequence of the wild type rfp1a protein (Wildtyp-rfp1a) (SEQ ID NO: 33) with the amino acid sequence of the rfp1a protein from IRAN9 (Iran9_rfp1a) (SEQ ID NO: 29).

[0104] FIG. 10: Comparison of the amino acid sequence of the wild type rfp1b protein (Wildtyp-rfp1b) (SEQ ID NO: 31) with the amino acid sequence of the rfp1b protein from IRAN9 (Iran9_rfp1b) (SEQ ID NO: 2).

[0105] The following examples illustrate the invention without in any way limiting the subject matter of the invention. Unless stated otherwise, standard methods were employed.

EXAMPLES

Example 1: Exemplary Near Isogenic BulkDevelopment of Rye Line 455 in Lo310 Background

[0106] As can be seen in FIG. 5, for all recombinant genotypes, NIB D and E partners were produced in which bulks each of more than 100 BC.sub.6S.sub.1 plants, which were homozygous carriers or non-carriers of the Rfp1, were outcrossed with the single cross CMS tester T911. Boundary isolation walls ensured that no foreign pollination occurred. The test crossing seed produced in this manner was then used in field trials in multiple environments. Test crossing plants were verified for the correct pedigree by (i) subsequent marker analysis and (ii) evaluation of the pollen shedding in the field trials. All of the evaluated test crossing plants which were generated from the NIB D partners exhibited full pollen shedding, while those which originated from the E partners exhibited a very significantly reduced and only partially restored male fertility.

Example 2: Field Trials

[0107] The yield evaluation trials were carried out at locations with different environmental conditions. Thus, for example, in 2012, there were seven locations in Germany (D) and Poland (PL). As can be seen in Table 1, the locations were selected so that they represented agricultural conditions in Central Europe with, additionally, different stress conditions (drought stress and nitrogen deficiency). In the low nitrogen regime, nitrogen was applied in quantities which were substantially below the usual doses. In an unwatered trial, natural precipitation constituted the only source of water, while in the watered trials, an additional quantity of water of approximately 25 mm per week was applied. In this manner, it was possible to measure effects of the Rfp1 introgression segments in very different environments. The results were then used (1) to determine the introgression segment-specific linkage drag effect, (2) to identify introgression segments with high environmental stability, and (3) to identify diagnostic environments which make the linkage drag discernible to a greater extent.

TABLE-US-00001 TABLE 1 Description of the trial locations and the applied treatments in 2012 (BEK = Bekedorf (Lower Saxony); KON = Kondratowice (Lower Silesia); BBG = Bernburg (Saxony-Anhalt); KO2 and KO3 = Bergen (Lower Saxony); PET_I and PET_N = Petkus with watering (I) and nitrogen variants (N) (Brandenburg); Ground points: index measuring the quality of an area of farmland. The scale of possible values extends from 1 (very poor) to 100 (very good).) Ground Precipitation Agronomic Location State points mean [mm] regime BEK D 51 769 KON PL 55 581 local agricultural practice BBG D 93 469 KO2 D 43 769 low nitrogen KO3 D 43 769 not watered PET_I D 28 636 watered PET_N D 28 636 not watered

[0108] A split plot trial design was used for all environments. The main plots used the test crossings of the recombinant BC.sub.6S.sub.1 lines. The subplots were the respective near-isogenic D and E bulk NIB pairs. The NIB D partner was the homozygous carrier of the donor introgression segment, while the NIB E partner was the homozygous carrier of the corresponding elite line segment. The corresponding D and E partners were sown directly adjacent to each other in order to minimize environmental differences and thus to be able to measure the differences due to the introgression segment with more accuracy. Trial units of the yield experiments were the test crossings from 7 BC.sub.6S.sub.1 lines, which themselves represented four different haplotypes. As an example, the results for the recombinant with the shortest introgression segment (455) are shown compared with that with the longest introgression segment (765) in detail. The latter is already significantly shorter than the segments which are currently available for hybrid varieties which have already been approved.

[0109] The preparation and implementation of the field trials were in accordance with the general rules and are well known to the person skilled in the art. The statistical analysis of the data was carried out in two steps: firstly, at each individual location, a variance analysis was calculated for all repeats with the aim of determining the accuracy of the trial and to determine respective location-specific yield averages for the recombinant lines and their introgression segments. In a second step, said averages were then used for the analysis regarding the environments.

[0110] Drastic and statistically significant differences (t-test) for the linkage drag effect were detected, for example, between the recombinant genotypes 455 and 765. As can be seen in FIG. 2, the linkage drag effect averaged over the locations (.sub.E-D) was 3.7 dt/ha for haplotype 455, while it was nearly twice that (7.0 dt/ha) for haplotype 765. The differences between the two recombinants manifested themselves particularly clearly at location PET_N under high stress due to spring drought. Here, the linkage drag effect (.sub.E-D) for recombinant 765 rose to 18 dt/ha, while it remained at only 3 dt/ha for haplotype 455. At another location (BBG) under moderate stress conditions, the linkage drag effect (.sub.E-D) dropped to 11 dt/ha for the haplotype 765, which, however, was a multiple of that shown by haplotype 455 with only about 3 dt/ha. Fundamentally similar relationships were found in the experiments carried out in 2014. Here again, shortening of the introgression segment corresponded to a reduction in linkage drag for yield. In order to be able to compare the experiments in 2012 and 2014 with each other, it was recommended that the linkage drag effect be standardized as a percentage of the performance of the NIB partner. FIG. 2 illustrates that the linkage drag for the recombinants with the shortest introgression segments (1120 and 455) were only between 3.9% and 4.7%, while the recombinants with the longest introgression segments (1110 and 765), with 6.2% and 7.1%, suffered substantial performance losses. However, the yield reductions cited latterly are still relatively small when set in context, i.e. currently known introgression segments which contain the two markers tc256739 and tc300731 cause linkage drag effects of more than 10%.

[0111] The locations differ in their diagnostic value for detection of linkage drag (see FIG. 4). Means for .sub.E-D over all tested introgression segments in 2012 (Series 018/2012) were from 3.2 (PET_I), 3.3 (KON), 4.1 (KO2), 4.6 (BBG), 5.7 (Ko3), 6.7 (BEK) to 10.0 (PET_N) dt/ha. The smallest mean linkage drag effect was observed in the watered trials in Petkus (PET_I), in which the availability of water was not limited. In contrast, the unwatered trials in the same macro-environment (PET_N) were very strongly influenced by drought in the pre-flower phase. It can be seen (FIG. 4) that the segment from 765 reacted significantly to environmental stress (regression coefficient on the mean linkage drag effect: 1.6 dt/ha). In contrast to this, the segment from 455 exhibited a very high environmental stability which could be confirmed in the PET-N stress environment.

Example 3: Identification of Recombinant Genotypes

[0112] In order to identify recombinant genotypes and in order to describe the respective remaining introgression segments, the following markers were used: ctg24, ctg32, ctg16b, P20, c40745, wherein the marker P20 played the most significant role in all of the subsequent studies. From a publicly available rye BAC library developed from cv. Blanco, which is not a carrier of the Rfp1 gene (Shi B J, et al. (2009): Physical analysis of the complex rye (Secale cereale L.) Alt4 aluminium (aluminum) tolerance locus using a whole-genome BAC library of rye cv. Blanco. Theor Appl Genet. 119(4):695-704), and with the aid of marker P20, BACs could be identified as a source for further marker sequences. It was possible to isolate and sequence a highly promising BAC. This opened up the possibility of providing a BAC library of restorer gene-carrying genotypes (denoted here as IR9 or ROS104), which can be viewed with specific DNA probes using PCR. Although no Rfp1 locus-spanning BAC contig could be produced, the locus flanking BAC clones could be identified with the aid of this library. Multiple marker combinations could be designed using the sequences: see Table 2. These were used for the selection of new recombinants and partially converted into a new marker system (SNP-based).

[0113] Furthermore, with the aid of the investigations with mTERF, a novel Rf gene could be identified which until now has not been described as being of relevance to fertility restoration for any plant species. For the first time it has been shown that at the 4R introgression segment, two standalone and also equal-valued Rf genes are effective having regard to restoration.

[0114] With the aid of close-flanking markers and a phenotyping test, for both Rf genes involved, it could be shown that the respective donor introgression segments could be made even smaller and the restoration capability could be maintained in full.

Example 4: Development of Close-Coupled Markers

[0115] In order to develop close-coupled markers for the Rfp1 locus in rye, as well as in order to isolate the functional restorer gene, a Rfp1 allele from the exotic breed IRAN IX was used as the most efficient source of fertility restoration. Bound up with this very efficient restoration performance, however, is a linkage drag which can cause a significant reduction in yield, depending on the respective location.

[0116] In addition to the close-coupled marker P20, for fine mapping of the Rfp1 region, further proximal close-coupled markers were provided. Essentially, this was carried out using two strategies which enabled one recombinatorily shortened genomic interval per molecular marker to be selected and thus, finally, to enable the unwanted linkage drag to be identified and reduced.

[0117] 1) The first strategy is based on the exploitation of conserved synteny between rye and Brachypodium as well as rye and barley. In this manner, novel close-coupled markers were derived using gene information from the two cited model grass/cereals varieties.

[0118] 2) The second strategy starts from the assumption that the close coupling of the marker P20 also indicates a close physical coupling, and is based on the chromosome walking method. This means that, by means of close-coupled markers, a freely available rye BAC library was searched (population variety Blanco (Shi et al., Theor Appl Genet 119 (2009), 695-704), in order to produce an initial BAC contig as the starting point for a contig analysis of the Rfp1 locus. For this, a newly established BAC library of the restorer gene-carrying genotype (described here as IR9 or ROS104) could be viewed with specific DNA probes using PCR.

[0119] With the aid of these libraries, BAC clones could be identified from which new markers could be derived which finally authorized selection of a smaller interval about Rfp1.

Example 5: Mapping of New Markers in the Population ROS13024-BC1 and Identification of Two Independent but Equivalently-Acting Loci for the Restoration Property (Rfp1a and Rfp1b)

[0120] As a supplement to the marker P20, in the context of the present invention, individual new markers suitable for selection were developed on the basis of the isolated BAC clones from the ROS104 BAC library. The markers obtained using the isolated bac clones were used for high resolution mapping in advanced breeding material, whereupon finally, the target interval could be further resolved. The mapping of these markers in the target interval as well as relative to the target gene was carried out in multiple experiments on internally developed, splitting populations. The markers and associated primer sequences, with the aid of which the loci for the restoration property could be identified in plants, are summarized in Table 2 below.

[0121] With the aid of the newly established selection markers, surprisingly, for the first time it was possible to show, in the mapping studies that were carried out, that the restoration property can be associated with two independent but closely coupled and almost equivalently acting restorer genes (Rfp1a and Rfp1 b) at the Rfp1 locus (FIG. 1). In addition, it was shown that one of the Rf genes involved, namely the Rfp1b gene, is a gene which codes for an mTERF protein. In addition, Rfp1a has a very high sequence agreement with and can most probably be denoted as an mTERF gene. Because until now it was not known that such a gene was relevant in cereals for fertility restoration and/or pollen shedding, this result was completely unexpected.

[0122] As a consequence, with the aid of the present invention and the associated experiments, it has been shown for the first time that two independent and also almost equally-acting Rf genes having regard to restoration are located in the 4R introgression segment. Moreover, these two genes can now, for example with the aid of the markers described in this invention, also be separately evaluated for breeding purposes and can be used separately or in combination with each other. Thus, one aspect of the present invention concerns the use of the Rf gene Rfp1a alone or in combination with Rfp1 b. In a further embodiment, the Rf gene Rfp1b may be used independently of Rfp1a. Preferably, both of the equivalently acting loci cited above lead to a restoration of fertility.

TABLE-US-00002 TABLE 2 Marker overview Forward Reverse primer primer (5-3) (5-3) Product Derived from [SEQ ID [SEQ ID Tm size Marker ID BAC NO] NO] [ C.] [bp] Performance Category tc256739* Barley EST 21 22 60 200/300 codominant COS #1: 541014 16 17 60 371 fertile pool gene based ctg32 contig32 specific STS #2: 541O14 14 15 60 1148 codominant gene based ctg24met2a5 contig24 STS #3: 541014 4 5 60 221 codominant ISBP ctg2 contig2 #4: 541014 10 11 60 516 codominant gene based ctg16b contig16 STS #5: SceAssembly02 18 19 60 675 codominant gene based c40745_1 STS #6: 72F13 6 7 65 424 fertile pool gene based P20 contig2 specific STS #7: 72F13 8 9 68 475 fertile pool gene based 72F13_c2_mTERF contig2 specific STS #8: 72F13 12 13 60 480 fertile pool STS 7_01_H_1441 contigl specific tc300731* Wheat EST 23 24 55 340/300 codominant COS (Tm = melting temperature; *described in Hackauf et al, 2012)

[0123] In one of the experiments which were carried out (Ro14037), almost 5000 individual plants of a BCxS1 population were genotyped. In this regard, a genetic polymorphism between the Rfp1 donor chromosomal segment and the pollen parent line Lo727 could be detected. The genetic fingerprint produced on the basis of this marker enabled a reliable identification to be carried out of only approximately 20 plants which could be characterized by recombination in the region of the valuable Rfp1 gene variant. In this manner, the genetic interval around Rfp1 in the genetic background of the line Lo727 was defined by the flanking markers ctg2 and 7_01_H_1441, for which a genetic separation of approximately 0.2 cM or approximately 120 kb could be calculated (FIG. 1). The genetic map produced documented that the target interval around Rfp1 could be resolved in the desired manner with the aid of the newly developed marker. Firstly, the first gene-based markers as well as the marker c40745_1 were used for selection on the genetic background of an elite pollen parent genotype. The marker P20 was employed to detect the segment with the restorer gene Rfp1. In a test series (018/2012), it was then possible to observe the expression of Rfp1 and, connected with it, the complete restoration of male fertility for different lengths of Rfp1 introgression segments (bottom of FIG. 2) using test crossings with the male stamp CMS tester Lo6-P(SR).

[0124] This discovery proves (1) coupling between Rfp1 and P20, as well as (2) the value of the developed selection marker for recombinatorial reduction of the donor chromosomal segment.

[0125] Building on this result, in further experiments (for example Ro12011), further cleaving BCx families were initially genotyped with the marker P20. In an experiment denoted test series 12-1-23, approximately 3200 individual plants were identified which inherited pure for the allele for the elite line Lo310. With the gene-based markers defined above, 4 recombinant plants with different lengths of Rfp1 introgression segments were identified in this material group (top of FIG. 2). In test crossings with these 4 lines as well as the control genotype #1058 without Rfp1 donor segment with the male-sterile CMS tester Lo6-P(SR), the expression of Rfp1 could be observed in 3 entirely male-fertile descendants of the lines 1110, 1039 and 1120. The genetic constitution of the recombinants led to the conclusion that a further, independent and equivalently acting restorer gene was located in the region of the target interval. This restorer gene coupled with the ctg2 marker was denoted Rfp1a, while the restorer gene coupled with P20 was given the notation Rfp1b (see also FIG. 1).

[0126] For the exact localization of the restorer gene Rfp1b, additional mapping experiments were carried out (for example Ro13030). In analogous manner to the experiments above, BCx interval plants in which the donor chromosomal segment had already been recombinatorially shortened with the aid of the gene-based marker from BAC clone 541014 were initially genotyped with the marker P20. In this manner, almost 4300 genotypes were identified which inherited pure for the elite allele of the pollen parent line Lo310 at this marker gene site. With the aid of the marker 7_01_H_1441, for example, a total of 13 recombinants to marker P20 could be detected in this material group (FIG. 3). In 4 of these 13 recombinants, the donor allele from the genetic source could be observed at the marker locus 72F13_c2_mTERF. For 3 of these 4 recombinants, test crossing descendants were established in which the male fertility had been completely restored. In contrast, the test crossing descendants of the 9 carriers of the non-restorer marker allele of mTERF exhibited a completely male-sterile phenotype.

[0127] By matching the observed phenotypes with the marker genotypes of a mitochondrial transcription tERmination factor (mTERF), it was possible to calculate a genetic separation between P20 and Rfp1b of r=0.094 cM. This recombination estimate was in very good agreement with the recombination estimate of r=0.011 cM calculated for the earlier experiments between P20 and the mTERF gene.

Example 6: Rfp1 Contig Production with the Aid of the BAC Library ROS104

[0128] BAC clones selected from the ROS104 BAC library acted as the basis for the development of probes and primers to continue the chromosome walking. An approximately 350 kbp contig was derived in this manner. By means of the markers and the mapping thereof in the advanced breeding material, it was shown that this contig carried markers which flanked the two restorer loci (FIG. 1 and Table 2). Experiments showed that there was no PPR protein-coding gene in this interval, but in it there were 3 so-called mTERF (mitochondrial transcription termination factor) genes or gene fragments which were therefore clearly to be seen as candidate genes for Rfp1.

[0129] On the basis of the earlier work, a BAC contig of the Rfp1 locus in the background of a restorer genotype (elite inbred line Lo310 from the pollen parent pool) was constructed and the presence of two Rf genes was demonstrated by analyses of recombinant descendants.

Example 7: Validation of Results

[0130] In addition to the detection of the identified Rfp1b gene by genetic recombination in Example 5, the functionality of the gene was also tested in a transgenic approach. To this end, the protocol for Agrobacterium tumefaciens-mediated rye transformation by Herzfeld (2002. Development of a genetic transformation protocol for rye (Secale cereale L.) and characterisation of transgene expression after biolistic or Agrobacterium-mediated gene transfer. Dissertation, IPK, Germany) was used. To this end, donor plants from the inbred line L22 were cultivated in a greenhouse at approximately 20 C. with 16 h of light up to the flowering point, and then immature caryopses were surface-sterilized and immature embryos were prepared. These were placed with the scutellum side uppermost onto callus-inducing medium (containing MS salts (Murashige and Skoog, 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia plantarum 15.3: 473-497.), 100 mg/l caseine hydrolysate, 500 mg/l glutamine, 30 g/l saccharose, 2.5 mg/l 2.4-D, pH 5.8, 3.0 g/l phytagel) and pre-cultivated in darkness at 25 C. over a period of 5 days before transformation. For the purposes of the transformation, following earlier precultivation, the immature embryos were placed on 6 microplates (Greiner Cellstar) and suspended in 10 ml of liquid callus-inducing medium. For the osmotic treatment, the liquid medium was exchanged against 10 ml of osmotic medium (containing MS salts (Murashige and Skoog, 1962), 100 mg/l caseine hydrolysate, 500 mg/l glutamine, 30 g/l saccharose, 6.0 mg/l 2.4-D, 72.9 g/l mannitol, pH 5.8) and the explants were plasmolysed over a period of 4-6 h. Next, the osmotic medium was removed again and the calluses were inoculated with approximately 300 l of agrobacterium suspension. Next, a vacuum treatment at 500 mbar was carried out over one minute followed by an incubation for 10 min. The explants were washed twice in 10 ml of infection medium (containing MS salts (Murashige and Skoog, 1962), 100 mg/l caseine hydrolysate, 500 mg/l glutamine, 15 g/l saccharose, 15 g/l glucose, 6.0 mg/l 2.4D, pH 5.2, 200 M acetosyringone) and co-cultivated overnight at 22 C. After 14-16 h, the explants were again washed several times in infection medium and finally transferred to solid co-cultivation medium (infection medium supplemented with 3.0 g/l phytagel), keeping the scutellum side directed upwards. The explants were cultivated for two more days and then transferred to solid callus-inducing medium which had been enriched with 150 mg/l of timentin to inhibit the growth of agrobacteria.

[0131] After 14 days, the calluses were transferred onto selective regeneration medium (containing MS salts (Murashige and Skoog, 1962), 100 mg/l caseine hydrolysate, 500 mg/l glutamine, 30 g/l saccharose, pH 5.8, 5.0 g/l agarose type I, 150 mg/l timentin, 30 mg/l paromomycin). After a further three weeks, the calli were transferred into suitable cultivation receptacles which contained selective regeneration medium with 50 mg/l of paromomycin sulphate for shoot lengthening.

[0132] The vector pYFrfp1 (FIG. 6) containing the restoration gene rfp1b (SEQ ID NO: 1) under the control of the ubiquitin promoter from maize with the first intron and the 35-S terminator inserted into the vector pPZP111 were introduced by electroporation (Mersereau et al., 1990. Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene 90.1: 149-151) into the agrobacterium strain AGLO (Lazo et al., 1991. A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Nature Biotechnology 9.10 (1991): 963-967). An AGLO (pYFrfp1) culture was cultivated overnight in 50 mg/l LB medium to saturation (OD660 2-2.5). 2 ml was centrifuged at 5000 rpm for 5 min and the pellet was dissolved in 1 ml of LB medium as well as 1 ml of infection medium. Prior to infection of the implants, the bacteria were incubated for approximately two hours (OD660 1.5-2.0).

[0133] In order to analyse the tDNA, the binding region of the tDNA border and the rye genome was amplified using inverse PCR (Ochman et al., 1990. Amplification of flanking sequences by inverse PCR. PCR protocols: A guide to methods and applications: 219-227). To this end, the DNA of the transgenic rye plants was digested with BamHI or BglII, circularized with T4 DNA-Ligase and then used as the template for the PCR. The amplification was carried out in the context of a nested PCR with the GeneAmp-PCR System 9700 (Perkin Elmer). The reaction conditions corresponded to those recommended by the manufacturer, wherein 200 ng of template DNA was used in the first reaction and 0.5 l from the first reaction was used as the template for the second reaction, so that the final volume was 25 l.

[0134] For the right border (RB) for the first reaction (28 cycles at 94 C. for 30 s, 48 C. for 60 s and 72 C. for 2 min), the following primers were used: RB1R 5-CTG AAT GGC GAA TGC TAG AGC AG-3 (LacZ region) and UBIF 5-CTG CAG TGC AGC GTG ACC CG-3 (3 region of maize ubiquitin promoter). For the second reaction (32 cycles at 94 C. for 30 s, 52 C. for 60 s and 72 C. for 2 min) the following primers were used: RB2R 5-CGT TTC CCG CCT TCA GTT TAA AC-3 and UBIF primer. PCR amplification products with blunt ends were obtained in which pwo DNA polymerase was added to the second reaction mixture. These amplification products were cloned into the PCR vector (Invitrogen, San Diego, Calif.) and then a sequence analysis was carried out on it.

[0135] Successfully transformed rye plants were propagated and crossed with Pampa male sterile inbred lines. Descendants which carried and expressed the restoration gene rfp1b as a transgene exhibited a restoration of male sterility.

[0136] As an alternative to the transgenic approach described above, the gene function can also be produced by knockout of the restoration gene in a restorer line. To this end, the person skilled in the art could, for example, also employ TILLING or genome editing (for example TALENs or CRISPR/Cas) in order, for example, to introduce an early stop codon into the coding sequence or to displace the reading frame by insertion/deletion. The result would be a non-functional mTERF protein and a loss of restoration capability.

Example 8: Characterization of Plant Material with Regard to Pollen Shedding

[0137] The above results now enable a plant breeder to use the desired restoration for Pampa CMS together with an excellent pollen shedding in the development of new cereal plants, in particular rye and barley. During the course of this, negative agronomic traits on the yield have been significantly reduced and the risk of ergot infestation has simultaneously been minimized. The degree of pollen shedding which is obtained with a male pollen parent in accordance with the invention can be determined on a scale of 1 to 9 (Geiger H H, Morgenstern K (1975) Angewandt-genetische Studien zur cytoplasmatischen Pollensterilitat bei Winterroggen [Applied genetic studies on cytoplasmic pollen sterility in winter rye]. Theor Appl Genet 46:269-276). In this regard, values of 1 to 3 mean non-dehiscent, empty anthers with a small amount of degeneration; values of 4 to 6 indicate a partially removed male sterility with <10% to >50% fertile anthers; values from 7 to 8 denote pollen-shedding anthers with increased anther size; and a value of 9 corresponds to a completely male-fertile plant like that expected in normal cytoplasm. Test crossings produced plants in accordance with the invention which had a value of 7 or higher, preferably even a value of 8 or higher or, almost regularly, a value of 9.

[0138] In Germany, ergot susceptibility of new rye varieties has been tested in field trials with artificial inoculation over several years and in different locations. The evaluation of the ergot susceptibility in this regard is based on a score system of 1 (very slightly susceptible) to 9 (very strongly susceptible). As can be seen in Table 3, hybrid varieties which carry a restoration gene from the donors IRAN IX, Pico Gentario or Altevogt 14160 (#1-#4), because of the excellent pollen shedding, exhibit a significantly reduced infestation with ergot pathogens (Claviceps purpurea).

TABLE-US-00003 TABLE 3 Stages of expression for ergot susceptibility for four hybrid varieties which carry restoration genes for the donors IRAN IX, Pico Gentario or Altevogt 14160 (left hand half; #1 to #4) and for four hybrid varieties with other restoration systems (right hand half). Hybrid varieties which carry restoration genes from donors Hybrid varieties with other IRAN IX, Pico Gentario or restoration genes or Altevogt 14160 Value restoration systems Value Visello 3 SU Drive 6 Minello 4 SU Forsetti 5 Palazzo 4 SU Performer 6 KWS Bono 4 SU Mephisto 6

[0139] In the context of the particular harvest results, the MRI (Max Rubner-Institut, Bundesforschungsinstitut fr Ernhrung and Lebensmittel [Federal Research Institute for Nutrition and Foodstuffs]) regularly collates ergot infestation data from the rye harvest in German agriculture. An evaluation of this data shows that the occurrence of ergot can be more than halved if, instead of hybrid varieties with a stage of expression of 5 to 6, varieties are used which, with a stage of expression of 3-4, are significantly less susceptible as regards ergot.

Example 9: Structural Comparison of rfp1a and rfp1b on a DNA and Amino Acid Level

[0140] Structural comparisons of rfp1a and rfp1b on a DNA (Table 4) and amino acid level (Table 5) show a comparatively high agreement between non-restoring wild type and restoring IRAN9. Surprisingly, however, rfp1a and rfp1b from IRAN9 exhibit a very low agreement with only 76% on a DNA level and only 66% or 68% on a protein level, although both have a restoration-mediating action. This shows that the tendency of mTERF proteins to restore male fertility is possible over a wide structural variability.

TABLE-US-00004 TABLE 4 Comparison of identities of cDNAs of rfp1a and rfp1b rfp1a Wild rfp1b type Iran9 Wild type Iran9 rfp1b Wild type 97% 76% 76% Iran9 76% 76% rfp1b Wild type 95% Iran9

TABLE-US-00005 TABLE 5 Comparison of identities of cDNAs of rfp1a and rfp1b rfp1a Wild rfp1b type Iran9 Wild type Iran9 rfp1b Wild type 96% 67% 68% Iran9 66% 67% rfp1b Wild type 90% Iran9

Example 10: Detection of Restoration Capability of rfp1a and rfp1b Genes Alone and in Combination as Well as from Different Sources

[0141] Table 6 clearly shows that test crossing plants which are equipped with only one copy, rfp1a or rfp1b, have a slightly smaller but on the whole entirely sufficient pollen shedding and anther size when compared with plants which have both copies.

TABLE-US-00006 TABLE 6 Anther score, according to Geiger & Morgenstern (1975), of test cross plants (Tx . . .) with different rfp1 copy configurations: Mean of restored test cross plants Anther rfp1 copy Anther length Test crosses configuration score (mm) TxBC7(Lo310) 1120 rfp1a 8 7 TxBC7S1(Lo310) 3308 rfp1a 8 7 TxBC6S1(Lo310) 455 rfp1b 8 7 TxBC6S1(Lo310) 217 rfp1a and rfp1b 9 8 TxBC6S1(Lo310) 765 rfp1a and rfp1b 9 8 TxBC4(Lo316 IRAN IX) rfp1a and rfp1b 9 8 TxBC2(Lo316 Altevogt) rfp1a and rfp1b 9 8 TxLo310 (original line) 3