Manipulation of self-incompatibility in plants
11224181 · 2022-01-18
Assignee
Inventors
- Hiroshi Shinozuka (Kingsbury, AU)
- Noel Cogan (Macleod, AU)
- John White Forster (Diamond Creek, AU)
- German Carlos Spangenberg (Bundoora, AU)
- Nicola Patron (Norwich, GB)
- Yidong Ran (Bundoora, AU)
- Luke Pembleton (Diamond Creek, AU)
Cpc classification
C12N15/8218
CHEMISTRY; METALLURGY
International classification
A01H1/00
HUMAN NECESSITIES
A01H6/46
HUMAN NECESSITIES
Abstract
The present invention relates to methods for controlling hybridization in plants and producing hybrid plants. The present invention also relates to nucleic acids encoding amino acid sequences for self-incompatibility (SI) proteins in plants, and the use thereof for the manipulation of SI, including seed production, in plants, particularly of the Poaceae family. The present invention also relates to kits, compositions, constructs and vectors including such nucleic acids, and related polypeptides, regulatory elements and methods. The present invention also relates to expression of self-gamete recognition genes in plants and to related nucleic acids, constructs, molecular markers and methods.
Claims
1. A kit for hybridization or self-incompatibility (SI) control in plants, said kit including: a first nucleic acid or nucleic acid fragment included in a vector encoding a SI polypeptide, wherein said first nucleic acid or nucleic acid fragment is isolated from or corresponds to a gene from the Z locus of a plant of the genera Lolium or Festuca of the Poaceae family; and a second nucleic acid or nucleic acid fragment included in a vector encoding a SI polypeptide, wherein said second nucleic acid or nucleic acid fragment is isolated from or corresponds to a gene from the S locus of a plant of the genera Lolium or Festuca of the Poaceae family; wherein said first nucleic acids or nucleic acid fragment includes a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 39; (b) a nucleotide sequence encoding the polypeptide of SEQ ID NO: 109; (c) complements of the sequences recited in (a) and (b); (d) sequences antisense to the sequences recited in (a) and (b); (e) functionally active fragments of the sequences recited in (a), (b), (c) and (d); having a size of at least 100 nucleotides and (f) functionally active variants having at least 90% identity to any one of the sequences recited in (a), (b), (c), (d) and (e); wherein said second nucleic acids or nucleic acid fragments includes a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 59; (b) a nucleotide sequence encoding the polypeptide of SEQ ID NO: 129; (c) complements of the sequences recited in (a) and (b); (d) sequences antisense to the sequences recited in (a) and (b); (e) functionally active fragments of the sequences recited in (a), (b), (c) and (d) having a size of at least 100 nucleotides; and (f) functionally active variants having at least 90% identity to any one of the sequences recited in (a), (b), (c), (d) and (e).
2. A vector comprising an isolated nucleic acid or nucleic acid fragment encoding a plant SI protein, selected from the group consisting of: (a) a polynucleotide comprising SEQ ID NO: 39 and SEQ ID NO: 59; (b) a nucleotide sequence encoding the polypeptide of SEQ ID NO: 109 and SEQ ID NO: 129; (c) complements of the sequences recited in (a) and (b); (d) sequences antisense to the sequences recited in (a) and (b); (e) functionally active fragments of the sequences recited in (a), (b), (c) and (d); having a size of at least 100 nucleotides and (f) functionally active variants having at least 90% identity to any one of the sequences recited in (a), (b), (c), (d) and (e).
3. A plant cell, plant, plant seed or other plant part comprising the vector according to claim 2.
4. A method of manipulating self-incompatibility in a plant, said method including introducing into said plant an effective amount of the vector according to claim 2.
5. The method according to claim 4, wherein said method includes introducing into said plant said vector comprising; a first nucleic acid or nucleic acid fragment encoding a SI polypeptide, wherein said first nucleic acid or nucleic acid fragment is isolated from or corresponds to a gene from the Z locus of a plant of the Poaceae family; and a second nucleic acid or nucleic acid fragment encoding a SI polypeptide, wherein said second nucleic acid or nucleic acid fragment is isolated from or corresponds to a gene from the S locus of a plant of the Poaceae family, and wherein the plant of the Poaceae family is from the genera Lolium or Festuca.
6. The kit according to claim 1, wherein the plant of the Poaceae family is Lolium perenne L., Lolium multiflorum, or Festuca arundinaceum.
7. The vector comprising the isolated nucleic acid or nucleic acid fragment encoding a plant SI protein according to claim 2, wherein the plant is Lolium perenne L., Lolium multiflorum, or Festuca arundinaceum.
8. The method according to claim 5, wherein the method the plant of the Poaceae family is Lolium perenne L., Lolium multiflorum, or Festuca arundinaceum.
Description
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
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(23) Legend: Gateway attB1 site (bold underline); Zea mays Ubi promoter (italics)+intron (underlined italics); Lolium perenne coding region in antisense and sense orientations (underline); rga2 intron (bold); Nopaline synthase (nos) terminator (bold italics); Gateway attB2 site (bold underline)
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EXAMPLES
Example 1. Isolation of SI Genes
(34) Both the S and Z locus was delimited through comparative genomics and BAC clone and genomic sequencing. All genes within the sequence data were identified (Tables 1 and 2). Sequences were determined through the FGENESH prediction software. Expression profiles were determined for each gene as described in Example 6. Expression profiles were determined through BLAST analysis of sequence reads from multiple tissues of the Lolium perenne L. genotype Impact04, compared to the Brachypodium distachion CDS gene sequences used as orthologous templates (See
(35) TABLE-US-00001 TABLE 1 Genes identified within the S locus Nucleic acid Polypeptide Ryegrass gene SEQ ID SEQ ID identified Syn Predicted Gene Function NO: NO: LpOs05g0481800 Protein prenyltransferase domain 1 71 containing protein, pentatricopeptide repeat- containing protein LpOs05g0147700 Cyclin-like F-box domain 2 72 containing protein LpOs05g0148300 Ribosomal protein S27, 3 73 mitochondrial family protein LpOs01g0254300 Similar to Pectinesterase-1 4 74 precursor (EC 3.1.1.11) (Pectin methylesterase 1) (PE 1) LpOs08g0226800 TRAF-like domain containing 5 75 protein, BTB/POZ and MATH domain-containing protein LpOs05g0148400 Conserved hypothetical protein 6 76 LpOs05g0148500 Electron transport accessory 7 77 protein domain containing protein LpOs07g0118800 Conserved hypothetical protein 8 78 LpOs07g0118900 Cyclin-like F-box domain 9 79 containing protein LpOs05g0148600 Na+/H+ antiporter 10 80 LpOs05g0148700 Armadillo-like helical domain 11 81 containing protein, senescence associated protein LpOs01g0372700 hypothetical protein, putative 12 82 asparagine--tRNA ligase, cytoplasmic 1-like LpOs05g0148900 Glutathione-S-transferase 19E50 13 83 LpOs01g0369700 Similar to Glutathione S- 14 84 transferase GST 8 LpOs05g0149100 C2 calcium/lipid-binding region, 15 85 CaLB domain containing protein LpOs05g0149200 PWWP domain containing 16 86 protein LpOs05g0149300 1-aminocyclopropane-1- 17 87 carboxylate oxidase LpOs05g0149400 1-aminocyclopropane-1- 18 88 carboxylic acid oxidase LpOs05g0149500 Lipopolysaccharide-modifying 19 89 protein family protein, predicted: O-glucosyltransferase rumi homolog LpOs05g0149600 Cullin-1 20 90 LpOs05g0149800 EF-Hand type domain containing 21 91 protein, serine/threonine-protein phosphatase 2A regulatory subunit B″ subunit gamma-like LpOs05g0149900 Tetratricopeptide-like helical 22 92 domain containing protein LpOs05g0150000 putative proline synthetase 23 93 associated protein LpOs05g0150300 probable chromatin-remodelling 24 94 complex ATPase chain-like protein LpOs05g0150400 Double-stranded RNA binding 25 95 domain containing protein LpOs05g0150500 Conserved hypothetical protein, 26 96 putative transport inhibitor response TIR1 LpOs05g0150600 ATP-dependent DNA helicase 27 97 RecQ family protein LpOs10g0545800 Cytochrome biosynthesis 28 98 CcmE/CycJ protein family protein LpOs05g0150700 Heavy metal 29 99 transport/detoxification protein domain containing protein LpOs05g0150800 Similar to Plastid 5,10- 30 100 methylene-tetrahydrofolate dehydrogenase, LpOs05g0150900 Histidyl-tRNA synthetase 31 101 LpOs02g0508100 hypothetical protein containing 32 102 DUF3339 LpOs05g0151000 Lpbcd762 Similar to RNA polymerase II 33 103 largest subunit LpOs05g0151100 Conserved hypothetical protein, 34 104 ferritin domain LpOs05g0151300 Rubber elongation factor family 35 105 protein LpOs05g0151400 Chloroplast protein import 36 106 component Toc86/159 family protein LpSb07g026730 Putative uncharacterized protein 37 107 LpOs05g0152400 Glycosyl transferase, family 14 38 108 protein, xylosyltransferase-like LpOs06g0680500 Glutamate receptor 3.4 precursor 39 109 (Ligand-gated ion channel 3.4) LpOs05g0152900 Seven in absentia protein family 40 110 protein LpOs05g0153000 Gelsolin family protein, villin-1- 41 111 like LpOs05g0153200 Region of unknown function, 42 112 putative Zinc finger, XS and XH domain containing protein LpOs05g0153300 Lipase, class 3 family protein 43 113 LpOs05g0153400 predicted pentatricopeptide 44 114 repeat-containing protein LpOs05g0153600 FAR1 domain containing protein 45 115 LpOs05g0154500 Spc97/Spc98 family protein, 46 116 gamma-tubulin complex LpOs05g0154600 Similar to VIP2 protein, 47 117 Hypothetical RING domain containing protein LpOs01g0652800 Protein of unknown function 48 118 DUF231, leaf senescence like protein, yellow leaf specific - like protein LpOs07g0286100 Cyclin-like F-box domain containing protein;
(36) TABLE-US-00002 TABLE 2 Genes identified within the Z locus Nucleic Polypeptide Ryegrass gene acid SEQ SEQ ID identified Syn Predicted Gene Function ID NO: NO: LpOs04g0645100 LpTC101821 Tetratricopeptide-like 49 119 helical domain containing protein LpOs04g0645200 LpVQ VQ domain containing 50 120 protein LpOs07g0213300 pentatricopeptide repeat- 51 121 containing protein LpOs04g0645500 methyltransferase-like 52 122 protein 22-like LpOs04g0645600 Protein of unknown 53 123 function DUF6, transmembrane domain containing protein, vacuolar protein LpOs04g0647300 LpTC116908 Ubiquitin-specific protease 54 124 22 LpOs04g0647800 LpTC89057 Glycerol kinase 2 55 125 LpOs04g0647701 LpDUF247 Protein of unknown 56 126 function DUF247 LpOs03g0193400 Polyamine oxidase 57 127 precursor (EC1.5.3.11); LpOs06g0607800 Similar to 26S proteasome 58 128 regulatory complex subunit p42D LpOs06g0607900 C2 and GRAM domain 59 129 containing protein “No Pollen” LpOs11g0242400 Rieske [2Fe—2S] region 60 130 domain containing protein oxidoreductase LpOs04g0648400 Leucine rich repeat, N- 61 131 terminal domain containing protein LpOs04g0648500 Zinc Finger Protease 62 132 LpOs04g0648600 Conserved hypothetical 63 133 protein LpOs10g0419600 Chlorophyllase family 64 134 protein LpOs04g0648700 Conserved hypothetical 65 135 protein LpOs04g0274400 YL1 nuclear, C-terminal 66 136 domain containing protein LpOs04g0649200 Protein of unknown 67 137 function DUF869, filament-like plant protein 7-like LpOs04g0648800 RING-type domain 68 138 containing protein, zinc finger binding LpOs04g0648900 Dehydration responsive 69 139 element binding protein 2F, AP2 domain containing LpOs04g0649100 Pathogenesis-related 70 140 transcriptional factor and ERF domain containing protein, AP2 domain containing, Apetala 2 like LpOs04g0650000 Lpbcd266 Oryzain alpha chain precursor (EC 3.4.22.-)
Example 2. Resequencing Data Identified DNA Nucleotide Variance
(37) A cohort of 21 genes were selected as key candidates of the S and Z loci. The genes were selected based on expression profile as well as sequence annotation.
(38) The collection of 21 genes all had PCR primers designed to resequence the coding regions of the genes. The designed PCR amplicons were optimised to generate large genomic fragments. A total of 50 plant genotypes were used as the template DNA for resequencing. The 50 plants were chosen as a diverse spread of plants with a potential wide range of diversity to maximise allelic variation at the genic loci being resequenced.
(39) The amplicons were generated, then pooled from each genotype and physically sheared to smaller fragments. DNA bar codes and sequencing adaptors were ligated onto the sheared fragments to identify each sample and then all samples were combined and sequenced using a next-generation Illumina MiSeq platform with 300 bp×2 reads.
(40) The resulting sequence data was attributed back to the individual samples using the bar codes and was then checked for quality and low quality reads removed. The sequence reads were then reference aligned to the genes amplified and variant bases identified. The individual samples were then combined to give a dataset to identify all variant bases from the 50 samples, with potentially 100 different alleles.
(41) The variant bases were recorded for each gene to identify if the variation was synonymous or non-synonymous in nature. A minimalistic requirement for each of the genes under investigation would be to have 5 or more variant amino acids identified within the transcript. A total of 5 variant amino acids would enable a maximum of 32 potential haplotypes from the data set allowing complete random mating maximal recombination.
(42) As 100 haplotypes were resequenced, high levels of diversity are expected, however there could be a degree of overlap between the haplotypes from the plants chosen so the total number of unique haplotypes could be lower than the number sequenced.
(43) Perennial ryegrass has been characterised as having a high degree of sequence variation within its genome, with estimates ranging from 1 SNP every 20-30 bases within a gene bases on resequencing 2-4 haplotypes. With two exceptions all of the genes resequenced contained sufficient variation in the coding regions of the genes that would generate a sufficient diversity of polypeptides that could deliver the required allelic variability (See
(44) The genes LpOs05g0151300 and LpOs05g0152400 did not have sufficient diversity, with only 3 and 2 variant amino acids respectively.
Example 3—Isolation of SI Genes: Cloning of the Ryegrass LpOs06g0607800 26S Proteasome Gene
(45) In order to develop novel genetic markers for fine-scale genetic and physical mapping of the perennial ryegrass SI loci, linked heterologous cDNA-derived RFLP markers were selected for the S locus and Z on the basis of ortholocus co-segregation in cereal rye and/or blue canary grass. Molecular marker development, genetic mapping and region dissection is described in Shinozuka et al (2010). As a result of the assembled data sets fine-scale comparative sequence synteny with the model Poaceae species, specifically Oryza sativa and Brachypodium distachyon, was achieved for the delimited S and Z regions. Using the defined gene complement from the model Poaceae species, a BAC library was screened with primer pairs specific to the genes described and 39 specific clones were identified. The identity of the selected BAC clones was verified through direct sequencing of locus-specific amplicons. The specific BAC clones were then sequenced using Sanger and/or GSFLX technology and the resulting data was sequence assembled using the Newbler software package. Following sequencing and assembly gene-like nucleotide sequences were identified using BLAST and gene prediction software tools. Based on the derived information the reiteration of the procedure was performed for the selection of additional clones to further enhance the resolution and sequence data to assemble physical maps for the SI locus regions (
(46) Molecular markers were developed from resequencing of specific genic loci, identified from the BAC sequencing and genetically mapped in a segregating population of Lolium perenne L. to confirm the location of the generated sequence.
(47) The genome of a single Lolium perenne L. genotype (the plant—Impact04) has been sequenced to approximately 70× coverage, generating c. 2 billion sequencing reads of 100 bp paired-end sequence reads on the Illumina GA2X and HiSeq2000 platform. The sequence data was filtered for high quality reads before being assembled using the SOAPdenovo v. 1.05 software package. The sequence assembly has been empirically optimised through iterative assessment of performance based on a range of input kmer sizes, in terms of number of bases assembled, and the average length of assembled contigs and scaffolds. An optimal assembly has generated 1.9 million scaffolds covering c. 1.7 Gb, while all contigs and singletons cover c. 3.5 Gb.
(48) Comparison of contigs and scaffolds to the coding sequences of the model grass species Brachypodium distachyon L. permitted identification of putative perennial ryegrass orthologues to c. 86% of all predicted genes and alternate transcripts from the model grass species. A pipeline approach was implemented based on a highly parallel BLAST analysis method in order to group transcript and genomic sequences relevant to each individual Brachypodium gene sequence into individual local CAP3-based assemblies. This approach generated 23,285 genic files that were indexed to the corresponding Brachypodium gene. Development of the exome sequence library enabled identification of a large collection of genic contigs, along with the corresponding regulatory elements. The collection of contigs was then screened for the presence of the predicted genes within the S and Z loci that had not been identified through the BAC screening process.
(49) A novel Lolium perenne L. gene was identified from BAC clone-related sequence that displayed sequence similarity with the rice gene Os06g0607800, hence the ryegrass gene was designated LpOs06g0607800 (SEQ ID NO: 58 and
(50) In addition the ryegrass 26S proteasome subunit gene identified in the Z locus region through BAC sequencing, was compared through BLAST analysis to the genomic Impact04 sequence through BLAST analysis. The identification of the sequence from the BAC clones as well as the genomic sequence enabled the identification of variant sequence bases from the coding region of the gene (
(51) Intracellular proteolysis is mainly regulated and enabled through the ubiquitin-proteasome pathway or the autophagy-lysozome/vacuole pathway. Proteolytic events play significant roles in SI through self-pollen rejection. Ubiquitin-mediated proteolysis is involved in the SI mechanism of the Brassicaceae and the Solanaceae.
(52) The 26S proteasome consists of the 20S core proteasome (CP) element and the 19S regulatory particle (RP). Proteolysis occurs in the 20S compartment, while the 19S element confers ATP dependence and substrate specificity to the CP. The RP consists of two elements: a ring of six AAA-ATPase subunits (often abbreviated as RPT) that is expected to function in target unfolding and transport, and three non-ATPase subunits (often abbreviated as RPN). As the 26S Proteasome subunit gene LpOs06g0607800 contains the AAA-ATPase domain, it is of the class RPT.
(53) Arabidopsis thaliana L. mutant lines, in which the RTP2 subunit of the 26S proteasome gene is disrupted, have demonstrated that male and female gamete transmission require a normal copy of the RPT2 gene to avoid abortion and failure in gametogenesis. In tobacco (Nicotiana tabacum L.) the NtRpn3 gene was found to physically interact with a calcium-dependent protein kinase and become phosphorylated in a calcium dependent manner.
(54) While applicants do not wish to be restricted by theory, the LpOs06g0607800 26S Proteasome gene is hence proposed to be the female determinant of the Z locus.
Example 4—Generation of Transformation Vectors Containing an Inverted Hairpin Structure of the LpOs06g0607800 26S Proteasome Gene
(55) The LpOs06g0607800 expression cassette consists of the promoter, 5′ untranslated region and intron from the Ubiquitin (Ubi) gene from Zea mays (Toki et al 1992) followed by 500 bp of coding sequence of the LpOs06g0607800 gene from L. Perenne in an inverted repeat interrupted by intron 2 of the RGA2 gene from Triticum turgidum subsp. durum (Douchkov et al 2005). The hairpin cassette was terminated with the 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the nopaline synthase gene (nos) from A. tumefaciens pTi15955 (Fraley et al 1983).
(56) The selection cassette (delivered either in cis or trans) comprised of the promoter, 5′ untranslated region and intron from the Actin (Act1) gene from Oryza sativa (McElroy et al 1990) followed by a synthetic, version of hph gene from E. coli (Kaster et al 1983) codon-optimized for expression in monocots, which encodes a protein that confers resistance to the antibiotic hygromycin. This cassette was terminated with the 3′ UTR comprising the transcriptional terminator and polyadenylation sites from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault and Melcher 1993).
(57) The selection cassette was synthesized by a commercial gene synthesis vendor (GeneArt, Life Technologies) and cloned into a Gateway-enabled vector. The LpOs06g0607800 expression cassette was synthesized by a commercial gene synthesis vendor (GeneArt, Life Technologies) with flanking attB sites. For delivery in cis the LpOs06g0607800 expression cassette was sub-cloned into pDONR221 II (Invitrogen, Life Technologies) in a BP Clonase reaction. The resulting ENTRY clone was used in a LR Clonase II (Invitrogen, Life Technologies) reaction with the Gateway-enabled vector encoding the hph expression cassette. Colonies of all assembled plasmids were initially screened by restriction digestion of miniprep DNA. Restriction endonucleases were obtained from New England BioLabs (NEB; Ipswich, Mass.) and Promega (Promega Corporation, WI). Plasmid preparations were performed using the QlAprep Spin Miniprep Kit (Qiagen, Hilden) or the Pure Yield Plasmid Maxiprep System (Promega Corporation, WI) following the instructions of the suppliers. Plasmid DNA of selected clones was sequenced using ABI Sanger Sequencing and Big Dye Terminator v3.1 cycle sequencing protocol (Applied Biosystems, Life Technologies). Sequence data were assembled and analyzed using the SEQUENCHER™ software (Gene Codes Corporation, Ann Arbor, Mich.).
(58) An ideogram of the gene expression cassette is shown in
Example 5—Biolistic Transformation of Perennial Ryegrass (Lolium perenne) for Expression of dsRNA Products of the LpOs06g0607800 26S Proteasome Gene for RNAi-Mediated Down-Regulation of SI
(59) Biolistic co-transformation of perennial ryegrass with the vectors containing the LpOs06g0607800 26S Proteasome gene sequence, driving the expression of the RNAi cassette and the synthetic version of hph gene from E. coli for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass.
(60) The vector used along with the transformation protocol has previously been used successfully in plant transformation experiments (Bilang, et al., 1991; Spangenberg, et al., 1995a; Spangenberg, et al., 1995b; Ye, et al., 1997; Bai, et al., 2001). The perennial ryegrass biolistic transformation method is outlined in
(61) Phenotypic evaluation of the resulting transgenic plants was performed by growing the plants to maturity, then following a vernalisation period of at 5° C. with 12 hour lighting for ten weeks, the plants were subjected to 22° C. with 24 hour lighting. The change in temperature and perceived day length initiated flowering in the plants, which then occurred 2-3 weeks later. Before the ryegrass flowers opened the flowering spike was checked for morphological alterations or deformities and was then contained within a paper bag to isolate the flowers from other potential pollen donors. The flowering spike may be maintained in its isolated state until flowering is complete, at which time seed set may be assessed. Multiple spikes may be bagged per plant and each spikelet and flower assessed visually for seed production.
(62) Once a putative transgenic plant had regenerated on the selective medium, the plant was split into three single plant tillers. Each tiller was individually screened for the presence of the transgene through conventional PCR. Oligonucleotide primers had been designed to the promoter region that originated from the Ubiquitin (Ubi) gene from Zea mays as well as a second assay that targeted the RGA2 intron sequence from Triticum turgidum subsp, which interrupts the inverted ryegrass repeat of the target gene. These regions were chosen to minimise cross amplification from the endogenous ryegrass genome. The assays were developed and initially tested on untransformed ryegrass genomic DNA to confirm that cross amplification did not occur. Transgenic events were only selected when all three tillers returned a positive result for the presence of the transgene with both assays. If variable results were seen, the single tillers that were positive were returned to growth medium and grown further, until they could be resplit into three tillers and screened again.
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(64) An average of 12 different transgenic events per construct were generated, with a range of 3-19. The plants were transferred to soil and were maintained in an appropriate containment glasshouse.
(65) TABLE-US-00003 TABLE 3 Numbers of transgenic events generated for the seven candidate genes undergoing functional evaluation. Positive Transgenic Gene Number Gene Name Events Generated LpOs04g0648500 Zinc Finger Protease 3 LpOs05g0149600 Cullin 15 LpOs06g0607800 26S proteasome 8 LpOs06g0680500 Glutamate receptor 11 LpOs06g0607900 NoPollen Gram domain 11 LpOs05g0152900 SlAHa 18 LpOs04g0647300 TC116908 19
(66) Phenotypic evaluation was performed upon the plant reaching reproductive maturity. Initially pollen grains were assessed for viability to ensure a response would be seen. A common effect on plant cells passing through a transformation and tissue culture process is reduced fertility of the resultant whole plant. The viability was confirmed through staining with fluorescein diacetate (FDA). FDA is a lipophilic compound and is membrane-permeable and non-fluorescent. Viable pollen grains will have intracellular esterase activity and will be able to perform enzymatic hydrolysis of FDA upon its entry into the cell. Once FDA has been hydrolyzed within the viable pollen grain it will be a highly fluorescent compound that is unable to diffuse out of the cell and will be retained, producing an intense green fluorescence within the cytoplasm.
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(68) Once pollen viability was confirmed pollen-pistil interactions were assessed. Dissection of the floral tissues of flowering ryegrass plants were performed to microscopically assess pollen-pistil interactions.
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(70) Each transgenic event was represented by three plants as described in the PCR screening process. Multiple transgenic events are required as the insertion of the transgene SiRNA construct is likely to result in a range of expression levels. This difference in expression between the transgenic events is likely to lead to a range of phenotypes for the reaction. The pollen-pistil compatible/incompatible reaction can be visualised through pollen tube abortion upon contact with the stigmatic tissue, or pollen tube directed growth towards the ovary. Multiple flowers per plant are required to be assessed for confidence over the observed phenotype. Once pollinated stigmatic tissues were isolated, aniline blue staining was performed and the tissue visualised under an inverted fluorescent microscope. A range of reactions were observed. Incidences of self-incompatibility was seen for many plants, while instances of partial compatibility was also seen.
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Example 6—Expression Analysis of the LpOs06g0607800 26S Proteasome Gene
(75) A single genotype of perennial ryegrass was subjected to transcriptome analysis through deep-sequencing of cDNA samples derived from multiple distinct tissue types. A total of 19 different RNA samples were generated from vegetative tissues, including leaf, pseudostem and root samples for both terrestrial and subterranean aspects of gene expression (Table 4). In addition a collection of reproductive libraries were generated from anthers, pistils, stigmas and pollinated pistils. The libraries were prepared for Illumina-based sequencing using the RNASeq preparation method. Each library was internally bar-coded to permit discrimination following the sequencing process.
(76) A total of c. 0.6 billion sequencing reads were generated from the Illumina HiSeq2000 platform. Approximately 30 million sequence reads was generated from each tissue sample. The generated sequences were then filtered and quality trimmed to ensure <3 bases per sequence read were called as “N” and mean and local Phred quality was >30 in all instances.
(77) The quality filtered reads were then BLASTn analysed against the coding sequences of the Brachypodium distachion genome. The number of BLASTn matches per gene were counted per tissue type and tabulated. As the number of reads generated per sample varied, the BLASTn mapped read count was normailsed on the 75th percentile to generate normalised values for comparative analysis. The Brachypodium distachion genome was used as a whole genome reference in this analysis to mitigate issues of gene absence or incomplete assemblies of any de novo generated gene catalogue.
(78) TABLE-US-00004 TABLE 4 Gene expression analysis through RNA sequencing from different tissues of Lolium perenne L. Number of unique reads aligned Tissue name - to the Brachypodium library Description of tissue source CDS gene catalogue Tip 1 Tip of the youngest leaf from a 22,752,873 single tiller Tip 2 Tip of the second youngest leaf 18,298,951 from a single tiller Tip 3 Tip of the third youngest leaf from 14,929,714 a single tiller Mid 1 Mid section of the youngest leaf 17,729,494 from a single tiller Mid 2 Mid section of the second youngest 18,952,545 leaf from a single tiller Mid 3 Mid section of the third youngest 13,934,280 leaf from a single tiller Pseudo 1 Complete pseudostem from a single 9,887,218 tiller Pseudo 2 Lower portion of the pseudostem of 12,245,297 a single tiller Pseudo 3 Upper portion of the pseudostem of 12,594,959 a single tiller Root Mid Mid section of root mass 12,571,148 Root Tip Tip section of root mass 12,239,578 Flower Complete flower, (un)opened 9,920,235 Pollinated Pistil Self pollen added to pistil, then after 10,840,818 5 mins 5 minutes pistil excised and frozen Pollinated Pistil Self pollen added to pistil, then after 9,281,790 1 hour minus c. 1 hour pistil excised and frozen Pollinated Pistil Self pollen already added to pistil, 11,208,524 1 hour plus upon tissue harvest, for an undefined time greater than 1 hour prior to pistil being excised and frozen Stigma Harvested following pollination at 0 9,724,120 pollinated minutes 0 mins Stigma Harvested following pollination at 5 13,258,777 pollinated minutes 5 mins Pistil Complete pistil, without pollen 13,714,389 Anther Complete anther without(out) pollen 13,724,305 grains
(79) The nucleic acid sequence identified as the LpOs06g0607800 gene, when compared against the coding portion of the Brachypodium distachion genome sequence, identifies Bradi1g36400 as the closest matching gene sequence. The expression profile that has been generated from this analysis identifies a dramatic increase in gene expression over time in the pistil tissues upon self pollination (
Example 7—Isolation of SI Genes: Cloning of the Ryegrass LpOs05g0149600 Cullin Gene
(80) Using the methods outlined in Example 3, a novel Lolium perenne L. gene was identified from BAC clone-related sequence that displayed sequence similarity with the rice gene Os05g0149600, hence the ryegrass gene was designated LpOs05g0149600 (SEQ ID NO: 20 and
(81) In addition the ryegrass Cullin gene identified in the S locus region through BAC sequencing, was compared through BLAST analysis to the genomic Impact04 sequence through BLAST analysis. The identification of the sequence from the BAC clones as well as the genomic sequence enabled the identification of variant sequence bases from the coding region of the gene (
(82) Cullins are molecular scaffolds responsible for assembling RING-based E3 ubiquitin ligases. Within the Solanaceae, Rosaceae and Plantaginaceae families the SI mechanism involves the formation of a complex consisting of a Cullin gene, an F-box gene along with a suppressor of kinetochore protein. The complex possesses the ubiquitin E3 ligase activity that attaches polyubiquitin chains to target proteins, such that ubiquitinated proteins are degraded by the 26S proteasome. The Cullin gene within the complex plays a role in assembling the other sub-units, and links to a further compound that recruits ubiquitin proteins to attach to the target proteins.
Example 8—Generation of Transformation Vectors Containing an Inverted Hairpin Structure of the LpOs05g0149600 Gene
(83) The LpOs05g0149600 expression cassette consists of the promoter, 5′ untranslated region and intron from the Ubiquitin (Ubi) gene from Zea mays (Toki et al 1992) followed by 500 bp of coding sequence of the LpOs05g0149600 gene from L. Perenne in an inverted repeat interrupted by intron 2 of the RGA2 gene from Triticum turgidum subsp. durum (Douchkov et al 2005). The hairpin cassette was terminated with the 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the nopaline synthase gene (nos) from A. tumefaciens pTi15955 (Fraley et al 1983).
(84) The selection cassette (delivered either in cis or trans) comprised of the promoter, 5′ untranslated region and intron from the Actin (Act1) gene from Oryza sativa (McElroy et al 1990) followed by a synthetic, version of hph gene from E. coli (Kaster et al 1983) codon-optimized for expression in monocots, which encodes a protein that confers resistance to the antibiotic hygromycin. This cassette was terminated with the 3′ UTR comprising the transcriptional terminator and polyadenylation sites from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault and Melcher 1993).
(85) The selection cassette was synthesized, delivered and sequenced as described in Example 4.
(86) An ideogram of the gene expression cassette is shown in
Example 9—Biolistic Transformation of Perennial Ryegrass (Lolium perenne) for Expression of dsRNA Products of the LpOs05g0149600 Cullin Gene for RNAi-Mediated Down-Regulation of SI
(87) Biolistic co-transformation of perennial ryegrass with the vectors containing the LpOs05g0149600 gene sequence, driving the expression of the RNAi cassette and the synthetic version of hph gene from E. coli for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass, as described in Example 5.
Example 10—Expression Analysis of LpOs05g0149600 Cullin Gene
(88) Using the methods outlined in Example 6, the nucleic acid sequence identified as the LpOs05g0149600 gene, when compared against the coding portion of the Brachypodium distachion genome sequence, identifies Bradi2g35830 as the closest matching gene sequence. The expression profile that has been generated from this analysis identifies a constitutive level of gene expression in all tissues (
Example 11—Isolation of SI Genes: Cloning of the Ryegrass LpOs06g0680500 Glutamate Receptor Gene
(89) Using the methods outlined in Example 3, a novel Lolium perenne L. gene was identified from BAC-clone related sequence that displayed sequence similarity with the rice gene Os06g0680500, hence the ryegrass gene was designated LpOs06g0680500 (
(90) Glutamate receptor genes have been identified in Arabidopsis thaliana and tobacco as forming influx channels in tip cell types that undergo directed patterns of growth such as those of pollen tubes, as well as root hairs. Studies on Arabidopsis root cells show that glutamate induces a sharp depolarization of the membrane potential, and a concomitant rise in intracellular calcium. Growth of tobacco pollen tubes in the presence of a glutamate receptor antagonist has been shown to be repressed, as is also the case for specific directed uptake of calcium. Gene knock-out experiments of pollen expressing glutamate receptor genes in Arabidopsis have documented reduction in growth rates as well as abnormal morphology of the tip and tube.
(91) While applicants do not wish to be restricted by theory, LpOs06g0680500 LpGlu1 Glutamate receptor gene is hence proposed to be the male determinant of the S locus.
Example 12—Generation of Transformation Vectors Containing an Inverted Hairpin Structure of the LpOs06g0680500 LpGlu1 Gene
(92) The nucleic acid sequence identified as LpOs06g0680500 gene has a 484 bp fragment selected as a design element for expression cassette. The Zea mays ubiquitin gene promoter (Christensen et al. 1992) was used to drive expression and the nopaline synthase (nos) gene terminator (Bevan, 1984; Rogers et al., 1985) was selected to arrest transcription.
(93) The LpGlu1 expression cassette consists of the promoter, 5′ untranslated region and intron from the Ubiquitin (Ubi) gene from Zea mays (Toki et al 1992) followed by 484 bp of coding sequence of the LpGlu1 gene from L. Perenne in an inverted repeat interrupted by intron 2 of the RGA2 gene from Triticum turgidum subsp. durum (Douchkov et al 2005). The hairpin cassette was terminated with the 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the nopaline synthase gene (nos) from A. tumefaciens pTi15955 (Fraley et al 1983).
(94) The selection cassette (delivered either in cis or trans) comprised of the promoter, 5′ untranslated region and intron from the Actin (Act1) gene from Oryza sativa (McElroy et al 1990) followed by a synthetic, version of hph gene from E. coli (Kaster et al 1983) codon-optimized for expression in monocots, which encodes a protein that confers resistance to the antibiotic hygromycin. This cassette was terminated with the 3′ UTR comprising the transcriptional terminator and polyadenylation sites from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault and Melcher 1993).
(95) The selection cassette was synthesized, delivered and sequenced as described in Example 4.
(96) An ideogram of the gene expression cassette is shown in
Example 13—Biolistic Transformation of Perennial Ryegrass (Lolium perenne) for Expression of dsRNA Products of the LpOs06g0680500 Glutamate Receptor Gene for RNAi-Mediated Down-Regulation of SI
(97) Biolistic co-transformation of perennial ryegrass with the vectors containing the LpOs06g0680500 LpGlu1 gene sequence, driving the expression of the RNAi cassette and the synthetic version of hph gene from E. coli for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass, as described in Example 5.
Example 14—Expression Analysis of the LpOs06g0680500 Glutamate Receptor Gene
(98) Using the methods outlined in Example 6, the nucleic acid sequence identified as the LpOs06g0680500 gene, when compared against the coding portion of the Brachypodium distachion genome sequence, identifies Bradi1g32800 as the closest matching gene sequence. The expression profile that has been generated from this analysis identifies high levels of gene expression in vegetative tissues and some gene expression in anthers (
Example 15—Isolation of SI Genes: Cloning of the Ryegrass LpOs04g0648500 Gene
(99) Using the methods outlined in Example 3, a novel Lolium perenne L. gene was identified from BAC clone-related sequence that displayed sequence similarity with the rice gene Os04g0648500, hence the ryegrass gene was designated LpOs04g0648500 (See
(100) The LpOs04g0648500 gene was annotated as an Ubiquitin-specific protease 22 gene through a BLASTx analysis (SEQ ID NO: 132). The gene contained a Znf-UBP and BRAP2 domains. The Znf-UBP domain exhibits the ubiquitin-specific protease activity and functions as protein stabiliser through target-specific de-ubiquitinylation. The human BRAP2 domain was originally identified as interacting with the BRCA1 (breast cancer 1) gene products. A sequence homology search indicated that this domain is also conserved in the Arabidopsis At2g26000 [zinc finger (ubiquitin-hydrolase) domain-containing protein] and At2g42160 [zinc finger (C3HC4-type RING finger) family protein] gene products, suggesting that this gene is involved in the ubiquitin-proteasome system.
(101) In the S-RNase-based and Brassicaceae-type SI systems, involvement of the ubiquitin-proteasome system has been suggested. The 26S proteasome complex is bound with a UBP, and the 19S regulatory particle of the 26S proteasome complex is activated by the UBP. In the Z locus-linked BAC clones, 26S proteasome-related genes (LpTC116908 and LpOs06g0607800) were identified, of which products may interact with the LpOs04g0648500 gene product.
(102) While applicants do not wish to be restricted by theory, the LpOs04g0648500 Ubiquitin-specific protease 22 gene is hence proposed to be one of the SI determinants in the Z locus
Example 16—Generation of Transformation Vectors Containing an Inverted Hairpin Structure of the LpOs04g0648500 Gene
(103) The nucleic acid sequence identified as LpOs04g0648500 gene has a 578 bp fragment selected as a design element for expression cassette. The Zea mays ubiquitin gene promoter (Christensen et al. 1992) was used to drive expression and the nopaline synthase (nos) gene terminator with (Bevan, 1984; Rogers et al., 1985) was selected to arrest transcription.
(104) The LpOs04g0648500 expression cassette consists of the promoter, 5′ untranslated region and intron from the Ubiquitin (Ubi) gene from Zea mays (Toki et al 1992) followed by 578 bp of coding sequence of the LpOs04g0648500 gene from L. Perenne in an inverted repeat interrupted by intron 2 of the RGA2 gene from Triticum turgidum subsp. durum (Douchkov et al 2005). The hairpin cassette was terminated with the 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the nopaline synthase gene (nos) from A. tumefaciens pTi15955 (Fraley et al 1983).
(105) The selection cassette (delivered either in cis or trans) comprised of the promoter, 5′ untranslated region and intron from the Actin (Act1) gene from Oryza sativa (McElroy et al 1990) followed by a synthetic, version of hph gene from E. coli (Kaster et al 1983) codon-optimized for expression in monocots, which encodes a protein that confers resistance to the antibiotic hygromycin. This cassette was terminated with the 3′ UTR comprising the transcriptional terminator and polyadenylation sites from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault and Melcher 1993).
(106) The selection cassette was synthesized, delivered and sequenced as described in Example 4.
(107) An ideogram of the gene expression cassette is shown in
Example 17—Biolistic Transformation of Perennial Ryegrass (Lolium perenne) for Expression of dsRNA Products of the LpOs04g0648500 Gene for RNAi-Mediated Down-Regulation of SI
(108) Biolistic co-transformation of perennial ryegrass with the vectors containing the LpOs04g0648500 gene sequence, driving the expression of the RNAi cassette and the synthetic version of hph gene from E. coli for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass, as described in Example 5.
Example 18—Expression Analysis of the LpOs04g0648500 Gene
(109) Using the methods outlined in Example 6, the nucleic acid sequence identified as the LpOs04g0648500 gene, when compared against the coding portion of the Brachypodium distachion genome sequence, identifies Bradi5g23970 as the closest matching gene sequence. The expression profile that has been generated from this analysis identifies a low level of constitutive expression in all tissues, with an increase in all of the reproductive samples. The highest level of gene expression was detected in the pistil samples (See
Example 19—Isolation of SI Genes: Cloning of the Ryegrass LpOs06g0607900 Gene
(110) Using the methods outlined in Example 3, a novel Lolium perenne L. gene was identified from BAC clone-related sequence that displayed sequence similarity with the rice gene Os06g0607900, and the ryegrass gene was hence designated LpOs06g0607900 (See
(111) The rice homologue of the novel gene identified from the BAC sequence characterisation has been partially described in its function and has been designated the “no-pollen” gene (Osnop). The rice Osnop gene was identified and characterised through a Ds transposon insertion strategy. The deleted gene displayed abnormal anthers and no pollen production. Through promoter fusions with the GUS reporter gene, the endogenous gene was characterised as showing gene expression late in pollen formation and in the germination of pollen tubes (Jiang et al. 2005).
(112) While applicants do not wish to be restricted by theory, the LpOs06g0607900 No-Pollen (LpNOP) gene is hence proposed to be the male determinant of the Z locus.
Example 20—Generation of Transformation Vectors Containing an Inverted Hairpin Structure of the LpOs06g0607900 LpNOP1 Gene
(113) The LpOs06g0607900 expression cassette consists of the promoter, 5′ untranslated region and intron from the Ubiquitin (Ubi) gene from Zea mays (Toki et al 1992) followed by 400 bp of coding sequence of the LpOs06g0607900 gene from L. Perenne in an inverted repeat interrupted by intron 2 of the RGA2 gene from Triticum turgidum subsp. durum (Douchkov et al 2005). The hairpin cassette was terminated with the 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the nopaline synthase gene (nos) from A. tumefaciens pTi15955 (Fraley et al 1983).
(114) The selection cassette (delivered either in cis or trans) comprised of the promoter, 5′ untranslated region and intron from the Actin (Act1) gene from Oryza sativa (McElroy et al 1990) followed by a synthetic, version of hph gene from E. coli (Kaster et al 1983) codon-optimized for expression in monocots, which encodes a protein that confers resistance to the antibiotic hygromycin. This cassette was terminated with the 3′ UTR comprising the transcriptional terminator and polyadenylation sites from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault and Melcher 1993).
(115) The selection cassette was synthesized, delivered and sequenced as described in Example 4.
(116) An ideogram of the gene expression cassette is shown in
Example 21—Biolistic Transformation of Perennial Ryegrass (Lolium perenne) for Expression of dsRNA Products of the LpOs06g0607900 Gene for RNAi-Mediated Down-Regulation of SI
(117) Biolistic co-transformation of perennial ryegrass with the vectors containing the LpOs06g0607900 gene sequence, driving the expression of the RNAi cassette and the synthetic version of hph gene from E. coli for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass, as described in Example 5.
Example 22—Expression Analysis of the LpOs06g0607900 Gene
(118) Using the methods outlined in Example 6, the nucleic acid sequence identified as the LpOs06g0607900 gene, when compared against the coding portion of the Brachypodium distachion genome sequence, identifies Bradi1g36390 as the closest matching gene sequence. The expression profile that has been generated from this analysis identifies high levels of gene expression almost exclusively in anthers. Limited expression has been detected in flowers and stigma pollinated at 0 minutes, which could result from anthers in the flower or initial germination of the pollen grain (See
Example 23—Isolation of SI Genes: Cloning of the Ryegrass LpOs05g0152900 a Seven-in-Absentia Homologue Gene
(119) Using the methods outlined in Example 3, from the exome sequencing of the genotype Impact04, a Lolium perenne L. gene was identified that displayed sequence similarity with the rice gene Os05g0152900 (
(120) SIAH proteins consist of a RING finger domain at the N-terminus and a Sina domain at the C-terminus, and have an ubiquitin-E3 ligase activity when a homodimer is formed (Den Herder et al. 2008). Suppression of SIAH protein function in plant species results in increased root systems, enlarged leaves and increased shoot number, suggesting that SIAH protein is involved in a wide range of plant developmental processes.
(121) Substrates of SIAH proteins are degraded in an ubiquitin-related pathway following interaction. Glutamate receptor proteins are substrates of SIAH proteins. The RING finger domain of SIAH protein and the Siah-interacting domain of glutamate receptor proteins are essential for interaction. Interaction of the SIAH and glutamate receptor proteins exerts effects on calcium current modulation. A glutamate receptor-like gene, LpGlu1, was identified as being located physically close to LpSIAH.
Example 24—Generation of Transformation Vectors Containing an Inverted Hairpin Structure of the LpOs05g0152900 LpSIAH Gene
(122) The LpSIAH expression cassette consists of the promoter, 5′ untranslated region and intron from the Ubiquitin (Ubi) gene from Zea mays (Toki et al 1992) followed by 400 bp of coding sequence of the LpSIAH gene from L. Perenne in an inverted repeat interrupted by intron 2 of the RGA2 gene from Triticum turgidum subsp. durum (Douchkov et al 2005). The hairpin cassette was terminated with the 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the nopaline synthase gene (nos) from A. tumefaciens pTi15955 (Fraley et al 1983).
(123) The selection cassette (delivered either in cis or trans) comprised of the promoter, 5′ untranslated region and intron from the Actin (Act1) gene from Oryza sativa (McElroy et al 1990) followed by a synthetic, version of hph gene from E. coli (Kaster et al 1983) codon-optimized for expression in monocots, which encodes a protein that confers resistance to the antibiotic hygromycin. This cassette was terminated with the 3′ UTR comprising the transcriptional terminator and polyadenylation sites from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault and Melcher 1993).
(124) The selection cassette was synthesized, delivered and sequenced as described in Example 4.
(125) An ideogram of the gene expression cassette is shown in
Example 25—Biolistic Transformation of Perennial Ryegrass (Lolium perenne) for Expression of dsRNA Products of the LpOs05g0152900 LpSIAH Gene for RNAi-Mediated Down-Regulation of SI
(126) Biolistic co-transformation of perennial ryegrass with the vectors containing the LpOs05g0152900 LpSIAH gene sequence, driving the expression of the RNAi cassette and the synthetic version of hph gene from E. coli for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass, as described in Example 5.
Example 26—Expression Analysis of the LpOs05g0152900 LpSIAH Gene
(127) Using the methods outlined in Example 6, the nucleic acid sequence identified as the LpOs05g0152900 gene, when compared against the coding portion of the Brachypodium distachion genome sequence, identifies Bradi2g35550 as the closest matching gene sequence. The expression profile that has been generated from this analysis identifies gene expression predominantly in anthers and the pollinated stigma at 0 minutes. Low or negligible expression in all other tissues was observed (See
Example 27—Isolation of SI Genes: Cloning of the Ryegrass LpTC116908 Gene
(128) Using the methods outlined in Example 3, a novel Lolium perenne L. gene was identified from BAC clone-related sequence that displayed sequence similarity with the rice gene Os04g0647300 and the barley gene TC116908, hence the ryegrass gene was designated LpTC116908. In rye (Secale cereale L.), a TC116908 derived genetic marker co-segregated with the rye Z locus. Close to the TC116908 derived marker, 4 genetic markers were located (Hackauf and Wehling 2005). The corresponding genetic markers were assigned to the lower part of LG2 of perennial ryegrass (Shinozuka et al. 2010). BAC clones containing the genetic marker-related sequences were sequenced to identify Lolium perenne L. genes encoded in the Z locus (See
(129) The LpTC116908 gene was annotated as an Ubiquitin-specific protease 22 gene through a BLASTx analysis (SEQ ID NO: 124). The gene contained a Znf-UBP (Zinc finger ubiquitin-specific processing protease) and peptidase C19 domains. The Znf-UBP domain exhibits the ubiquitin-specific protease activity and functions as protein stabiliser through target-specific de-ubiquitinylation. The peptidase C19 domain shares sequence similarity to the Znf-UBP-like domain and possesses ubiquitin-specific peptidase activity. The LpTC116908 gene was expressed in perennial ryegrass reproduction organs (Shinozuka et al. 2010).
(130) In the S-RNase-based and Brassicaceae-type SI systems, involvement of the ubiquitin-proteasome system has been suggested. The 26S proteasome complex is bound with a UBP, and the 19S regulatory particle of the 26S proteasome complex is activated by the UBP. In the Z locus-linked BAC clones, 26S proteasome-related genes (LpOs06g0607800 and LpOs04g0648800) were identified, of which products may interact with the LpTC116908 gene product.
(131) While applicants do not wish to be restricted by theory, the LpTC116908 Ubiquitin-specific protease 22 gene is hence proposed to be one of SI determinants of the Z locus
Example 28—Generation of Transformation Vectors Containing an Inverted Hairpin Structure of the LpTC116908 LpOs04g0647300 Gene
(132) The LpTC116908 expression cassette consists of the promoter, 5′ untranslated region and intron from the Ubiquitin (Ubi) gene from Zea mays (Toki et al 1992) followed by 492 bp of coding sequence of the LpTC116908 gene from L. Perenne in an inverted repeat interrupted by intron 2 of the RGA2 gene from Triticum turgidum subsp. durum (Douchkov et al 2005). The hairpin cassette was terminated with the 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the nopaline synthase gene (nos) from A. tumefaciens pTi15955 (Fraley et al 1983).
(133) The selection cassette (delivered either in cis or trans) comprised of the promoter, 5′ untranslated region and intron from the Actin (Act1) gene from Oryza sativa (McElroy et al 1990) followed by a synthetic, version of hph gene from E. coli (Kaster et al 1983) codon-optimized for expression in monocots, which encodes a protein that confers resistance to the antibiotic hygromycin. This cassette was terminated with the 3′ UTR comprising the transcriptional terminator and polyadenylation sites from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault et al 1993).
(134) The selection cassette was synthesized, delivered and sequenced as described in Example 4.
(135) An ideogram of the gene expression cassette is shown in
Example 29—Biolistic Transformation of Perennial Ryegrass (Lolium perenne) for Expression of dsRNA Products of the LpTC116908 Gene for RNAi-Mediated Down-Regulation of SI
(136) Biolistic co-transformation of perennial ryegrass with the vectors containing the LpTC116908 gene sequence, driving the expression of the RNAi cassette and the synthetic version of hph gene from E. coli for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass, as described in Example 5.
Example 30—Expression Analysis of the LpTC116908 Gene
(137) Using the methods outlined in Example 6, the nucleic acid sequence identified as the LpTC116908 gene, when compared against the coding portion of the Brachypodium distachion genome sequence, identifies Bradi5g23920 as the closest matching gene sequence. The expression profile that has been generated from this analysis identifies a low level of constitutive expression in all tissues, with a significant increase in both of the pollinated stigma samples. Slight increases in gene expression are also identified in pistil samples as well as pseudostems (
Example 31. Generation of Transformation Vectors Containing an Inverted Hairpin Structure
(138) SiRNA constructs were prepared for all 21 candidate genes, using the methods outlined in Example 4. From the resequencing data the most conserved 300-500 bp region of the gene was chosen for the design.
(139) S Locus
(140) LpOs05g0148600 LpOs01g0369700 LpOs05g0149100 LpOs05g0149500 LpOs05g0149600 LpOs05g0150400 LpOs05g0150500 LpOs05g0151300 LpOs05g0152400 LpOs06g0680500 LpOs05g0152900 LpOs05g0153200
Z Locus LpOs04g0645500 LpOs04g0645600 LpOs04g0647300 LpOs03g0193400 LpOs06g0607800 LpOs06g0607900 LpOs04g0648500 LpOs04g0648600 LpOs04g0648900
(141)
Example 30. Application of Genomic Data from the S and Z Interval in F1 Hybrid Grass Breeding
(142) Heterosis or hybrid vigour is the phenomenon where the performance of an F.sub.1 hybrid is greater than that of the parents. Ryegrass cultivars today are commonly bred from a limited number of elite parents (4-12), polycrossed together and then further polycrossed to bulk up seed numbers suitable for commercial sale. There are no commercial activities or schemes to capture heterosis in ryegrass breeding currently.
(143) The identification of genetic markers in linkage disequilibrium with S and Z loci enables haplotypic prediction. The ability to genotype individuals for S and Z haplotypes opens a new avenue for efficient F.sub.1 hybrid ryegrass production by selectively bottlenecking and combining haplotypes. The production of F.sub.1 hybrid ryegrass by selectively bottlenecking SI alleles using linked genetic markers displays the greatest potential for cost effective application to commercial ryegrass breeding in the near future. The application of SI genetic markers to bottleneck SI allows breeders to work with any germplasm at their disposal with no prior requirements. The haplotype defining SI markers will enable breeders to selectively bottleneck SI haplotypes (without self ing) within defined pools to reduce the within pool compatibility and then bring two pools together for random crossing, where the SI haplotypes ensure between pool compatibility is greater than within, resulting in an increased production of F.sub.1 progeny.
(144) For example, in the initial stages a breeding nursery of phenotypically elite plants would be genotyped with the SI linked molecular markers and haplotypic prediction would be performed. Pairs of individuals (termed parent pools) would then be identified where one individual is heterozygous at both S and Z and the other individual is homozygous at one locus, either S or Z, and heterozygous at the other locus (for the same haplotypes present in the heterozygous individual), for example: Ind x s1s2-z1z2 Ind y s1s2-z1z1
(145) Two parent pools where the S and Z haplotypes between pools are completely different are identified and taken forward to the next step. The two selected pools are referred to as pool A and B in the follow stages.
(146) Following from the initial parental pool development the seeds are multiplied. During this seed bulk stage, pool A and B are maintained in isolation to ensure no foreign SI haplotypes are introduced through pollen flow or external seed.
(147) One round of random mating within each pool will bring the S and Z haplotype frequencies to equilibrium, with 50% of individuals heterozygous at both the S and Z loci, 25% homozygous at S or Z for one of the haplotypes, and the remaining 25% of individuals homozygous at the same locus but for the opposing haplotype, for example: 25% inds—s1s1-z1z2 50% inds—s152-z1z2 25% inds—s2s2-z1z2
(148) Continued unselected random mating within the two pools will maintain the homozygous and heterozygous frequencies whilst increasing seed numbers. In every round of mating, the heterozygous locus will alternate, for example:
(149) TABLE-US-00005 25% - s1s1-z1z2 25% - s1s2-z1z1 25% - s1s1-z1z2 50% - s1s2-z1z2 .fwdarw. 50% - s1s2-z1z2 .fwdarw. 50% - s1s2-z1z2 25% - s2s2-z1z2 25% - s1s2-z2z2 25% - s2s2-z1z2
(150) Pollen within pools will never be compatible with individuals heterozygous at both the S and Z loci. Consequently those individuals (which make up 50% of the plants) will only be pollen donors, not producing any seed, resulting in a 50% seed production rate within pools.
(151) Once sufficient seed has been generated within pools, equal numbers of seed would be combined and sown out for F.sub.1 seed production. As pollen from within pools is not compatible with the respective S and Z heterozygous individuals, only pollen from between the two pools will fertilize those individuals, resulting in those plants yielding 100% F.sub.1 hybrid seed. The remaining individuals, which are compatible with pollen both from within and between pools will yield both F.sub.1 hybrid and within-pool seed. However, as there is a greater number of compatible haplotypic combinations from between-pools, than from within-pools, a higher proportion of the seed will be F.sub.1 hybrids.
(152) From simulation of all the haplotypic combinations between and within-pools, and the proportion of compatible combinations, the theoretical percentage of F.sub.1 hybrid seed produced following the described scheme is >83%. With hybrid individuals likely to be more vigorous and competitive than within-pool seed, the proportion of hybrids, >83%, is likely to increase on farm when grown under a competitive sward situation. In the proposed breeding design, pool A and B will reach haplotype frequency equilibrium after one round of crossing, meaning that breeders can bulk seed up within pools over as many generations as deemed necessary, as long as the pools are maintained in isolation from foreign pollen. This also allows breeders to perform small test crosses between pools with each seed bulk up to ensure heterosis still remains in the progeny. At no point during the breeding design is there a requirement for controlled pollination.
(153) This breeding design could be applied to any outbreeding grass species belonging to the Poaceae that has the S and Z loci regulating self-incompatibility without limitation. More preferably the grass species would be of the Bambusoideae, Ehrhartoideae (formerly Oryzoideae) or Pooideae clade. More preferably the grass species would be of the tribe Poeae. More preferably the grass species would be of the genera Lolium, Festuca, Poa, Dactylis, Bromus, Secale, Pennisetum and Panicum. More preferably the grass species would be of the genera Lolium and Festuca. More preferably the species would be of the genus Lolium. More preferably the Lolium species would be Lolium perenne (perennial ryegrass), Lolium multiflorum (Italian ryegrass), Lolium boucheanum (hybrid ryegrass) Lolium arundinaceum (tall fescue) and Lolium pratense (meadow fescue).
(154)
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