CONTROL OF MEIOTIC CROSSOVER IN MAIZE

Abstract

The present disclosure provides methods for increasing meiotic recombination in crop plants, as well as plants and seeds produced by such methods.

Claims

1. An F.sub.1 hybrid maize seed, comprising an expression cassette comprising a maize promoter that is active during meiosis and operably linked to a nucleic acid encoding an artificial microRNA (amiRNA) that targets ZMET2, ZMET5 or KRYPTONITE.

2. An F.sub.1 hybrid maize seed produced by crossing two different maize inbred lines, one of which comprises an expression cassette that includes a maize promoter that is active during meiosis and is operably linked to a nucleic acid encoding an artificial microRNA (amiRNA) that targets ZMET2, ZMET5 or KRYPTONITE.

3. The F.sub.1 hybrid maize seed of claim 1 or 2, wherein the maize promoter is specifically active during meiosis.

4. The F.sub.1 hybrid maize seed of claim 3, wherein the maize promoter is a AMEIOTIC1 promoter, a AFD1/REC8 promoter, a DMC1 promoter, or a PHS1 promoter.

5. The F.sub.1 hybrid maize seed of any one of claims 1-4, wherein the amiRNA has a length of 15-30 nucleotides.

6. The F.sub.1 hybrid maize seed of any one of claims 1-5, wherein the amiRNA targets ZMET2.

7. The F.sub.1 hybrid maize seed of claim 6, wherein the amiRNA is encoded by a nucleic acid comprising at least one nucleic acid sequence selected from SEQ ID NO: 1-SEQ ID NO: 10.

8. The F.sub.1 hybrid maize seed of any one of claims 1-5, wherein the amiRNA targets ZMET5.

9. The F.sub.1 hybrid maize seed of claim 8, wherein the amiRNA is encoded by a nucleic acid comprising at least one nucleic acid sequence selected from SEQ ID NO: 11-SEQ ID NO: 20.

10. The F.sub.1 hybrid maize seed of any one of claims 1-5, wherein the amiRNA targets KRYPTONITE.

11. The F.sub.1 hybrid maize seed of claim 10, wherein the amiRNA is encoded by a nucleic acid comprising at least one nucleic acid sequence selected from SEQ ID NO: 21-SEQ ID NO: 30.

12. An F.sub.1 hybrid maize plant produced by growing the F.sub.1 hybrid maize seed of any one of claims 1-11.

13. A BC.sub.1 maize seed produced by crossing the F.sub.1 hybrid maize plant of claim 12 to a parental line or other line.

14. An F.sub.2 maize seed produced by self-pollinating the F.sub.1 hybrid maize plant of claim 12.

15. An F.sub.2 maize plant produced by growing the F.sub.2 maize seed of claim 13 or 14.

16. An F.sub.1 hybrid maize plant, comprising an expression cassette comprising a maize promoter that is active during meiosis and operably linked to a nucleic acid encoding an artificial microRNA (amiRNA) that targets ZMET2, ZMET5 or KRYPTONITE.

17. The F.sub.1 hybrid maize plant of claim 16, wherein the maize promoter is specifically active during meiosis.

18. The F.sub.1 hybrid maize plant of claim 17, wherein the maize promoter is a AMEIOTIC1 promoter, a AFD1/REC8 promoter, a DMC1 promoter, or a PHS1 promoter.

19. The F.sub.1 hybrid maize plant of any one of claims 16-18, wherein the amiRNA is encoded by a nucleic acid comprising at least one nucleic acid sequence selected from SEQ ID NO: 1-30.

20. A method, comprising: (a) introducing into an embryo of an elite maize inbred plant or a cell of an elite maize inbred plant an expression cassette that includes a maize promoter that is active during meiosis and is operably linked to a nucleic acid encoding an artificial microRNA (amiRNA) that targets ZMET2, ZMET5 or KRYPTONITE to produce an embryo comprising the expression cassette or a cell comprising the expression cassette; (b) regenerating the embryo comprising the expression cassette or the cell comprising the expression cassette to produce a T.sub.0 maize plant; and (c) crossing the T.sub.0 maize plant to an exotic maize inbred plant to produce F.sub.1 maize seed.

21. The method of claim 20 further comprising growing the F.sub.1 maize seed to produce an F.sub.1 maize plant.

22. The method of claim 21 further comprising crossing the F.sub.1 maize plant to a parental line to produce BC.sub.1 maize seed, or self-pollinating the F.sub.1 maize plant to produce F.sub.2 maize seed.

23. The method of claim 22 further comprising growing the BC.sub.1 maize seed to produce a BC.sub.1 maize plant, or growing the F.sub.2 maize seed to produce an F.sub.2 maize plant.

24. The method of claim 23 further comprising screening the BC.sub.1 maize plant or the F.sub.2 maize plant for at least one desirable trait.

25. A progeny maize seed produced by a subsequent generation of the F.sub.1 hybrid maize plant of any one of claims 16-19.

26. The progeny maize seed of claim 25, wherein subsequent generation is produced by self-pollinating an F.sub.1 hybrid maize plant or by backcrossing an F.sub.1 hybrid maize plant to a parental line or other line.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGS. 1A-1G show genome-wide increases in pericentromeric crossover frequency in cmt3 mutants. FIG. 1A, Crossing scheme: Col chromosomes are black and Ler chromosomes are blue. FIG. 1B, Heterochromatic CEN3 crossover frequency (cM) in wildtype and cmt3, in Col/Col inbreds, or Col/Ler F1 hybrids. Replicate measurements are shown in black and mean values in red. FIG. 1C, Euchromatic 420 crossover frequency in wildtype and cmt3, with Col/Col homozygosity, or Col/Ler heterozygosity, shown as for FIG. 1B. FIG. 1D, Crossover frequency measured in wildtype (blue) and cmt3 (red) F2 populations. Mean values are indicated by the horizontal dotted lines and centromeres by the vertical dotted lines. The heterochromatic knob inversion on the short arm of chromosome 4 is indicated by the black box and INV. FIG. 1E, Crossovers from wildtype and cmt3 (black) plotted as in FIG. 1D, overlaid with published BS-seq data (Stroud H, et al. Cell 2013; 152: 352-64) shown in CG (red), CHG (blue) or CHH (green) sequence contexts. FIG. 1F, Distribution of crossovers per F2 individual for wildtype and cmt3 populations. Red dotted lines indicate mean values. FIG. 1G, Normalized crossover distributions analyzed along chromosome telomere (TEL) to centromere (CEN) axes in wildtype (blue) or cmt3 (red) populations.

[0015] FIG. 2 shows a flow diagram of a timeline of a maize transformation protocol (Ishida Y et al. Nature Protocols 2007: 2: 1614-21).

[0016] FIGS. 3A-3C show schematics of the protocols described in Example 3.

[0017] FIGS. 4A-4B show schematics of the protocols described in Example 7.

DESCRIPTION

[0018] The centromeres of eukaryotic chromosomes are surrounded by repetitive heterochromatic regions, which are suppressed for both meiotic crossover and gene transcription. In fission yeast, histone 3 lysine 9 dimethylation (H3K9me2) is guided to heterochromatin by RNA interference (RNAi), and genetic ablation of the pathway induces mitotic homologous recombination and meiotic crossover in pericentromeric heterochromatin. In addition, plants and mammals silence heterochromatin by DNA cytosine methylation, which can occur in CG and non-CG (CHG and CHH) sequence contexts (where H=A, C or T). Data provided herein shows that the downregulation of the interlinked H3K9me2 and non-CG DNA methylation pathway in Arabidopsis increases meiotic crossover within pericentromeric heterochromatin. Reduction of these epigenetic marks causes increases in pericentromeric crossover frequency. The present disclosure provides results showing that activation of recombination occurs in both inbred and hybrid backgrounds. Suppression of pericentromeric recombination severely limits plant breeding. As described herein, mutation of the H3K9me2 and non-CG DNA methylation pathway presents an attractive solution to unlock genetic diversity in hybrid crops, such as hybrid maize (corn).

[0019] Provided herein, in some embodiments, are F.sub.1 hybrid maize plants and seeds that include an expression cassette that functions as a dominant negative suppressor of a gene in the H3K9me2 and non-CG DNA methylation pathway. In some embodiments, F.sub.1 hybrid maize plants and seeds include an expression cassette that includes a maize promoter that is active during meiosis and is operably linked to a nucleic acid encoding an amiRNA that targets a gene in the H3K9me2 and non-CG DNA methylation pathway. In some embodiments, F.sub.1 hybrid maize plants and seeds include an expression cassette that includes a maize promoter that is active during meiosis and is operably linked to a nucleic acid encoding an antisense RNA that targets a gene in the H3K9me2 and non-CG DNA methylation pathway. In some embodiments, F.sub.1 hybrid maize plants and seeds include an expression cassette that includes a maize promoter that is active during meiosis and is operably linked to a nucleic acid encoding virus induced gene silencing vector. In some embodiments, F.sub.1 hybrid maize plants and seeds include an expression cassette that includes a maize promoter that is active during meiosis and is operably linked to a nucleic acid encoding long double stranded RNA (which are processed into small RNA molecules).

[0020] Also provided herein are F.sub.1 hybrid maize seeds produced by crossing two different maize lines (e.g., maize inbred lines), one of which comprises expression cassette that functions as a dominant negative suppressor of a gene in the H3K9me2 and non-CG DNA methylation pathway (e.g., an expression cassette that includes a maize promoter that is active during meiosis and is operably linked to a nucleic acid encoding an amiRNA that targets a gene in the H3K9me2 and non-CG DNA methylation pathway).

[0021] The H3K9me2 and non-CG DNA methylation pathway, as discussed above, is an epigenetic pathway in flowering plants that silences gene expression and meiotic recombination. Genes of the maize H3K9me2 and non-CG DNA methylation pathway that may be targeted, as provided herein, include, but are not limited to, homologs of the Arabidopsis chromomethylases (e.g., CMT1, CMT2 and CMT3), such as ZMET2, ZMET5 and homologs of the Arabidopsis histone H3K9 methyltransferases, such as KRYPTONITE. Other genes of the maize H3K9me2 and non-CG DNA methylation pathway may also be targeted, alone or in combination with ZMET2, ZMET5 and KRYPTONITE.

[0022] An inbred plant is a plant having a genome that is genetically homozygous (there are few genetic polymorphismsfor example three or less, including nogenetic polymorphisms between homologous chromosomes). Inbred plants are usually produced by multiple generations of self-pollination. If an individual plant is heterozygous at a locus, approximately seven generations of self-pollination are needed until nearly all the progeny generated from that single plant are homozygous. After seven consecutive generations of self-pollination, more than 99% of the progeny are expected to be homozygous at any one locus. This process of repeated self-pollinations is known as inbreeding.

[0023] An F.sub.1 hybrid plant is a first-generation plant produced by crossing two genotypically different plants (e.g., plants from different inbred lines). The genome of an F.sub.1 hybrid plant is genetically heterozygous (there are many, for example more than three, genetic polymorphisms between the homologous chromosomes).

[0024] An F.sub.2 plant is a second-generation plant produced by inbreeding an F.sub.1 hybrid plant. By comparison, a BC.sub.1 hybrid plant is a second-generation plant produced by crossing an F.sub.1 hybrid plant to a parental plant or a plant from a parental line (a line used to produce the F.sub.1 hybrid plant).

[0025] Inbred parental plants, in some embodiments, carry an expression cassette that comprises a maize promoter that is active during meiosis. An expression cassette refers to a nucleic acid (e.g., DNA) that comprises a promoter operably linked to a sequence of nucleotides encoding a molecule of interest (e.g., RNA or protein). A promoter is a control region of a nucleic acid at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter is considered to be operably linked when it is in a correct functional location and orientation relative to a sequence of nucleic acid that it regulates (e.g., to control (drive) transcriptional initiation and/or expression of that sequence). Expression cassettes of the present disclosure, in some embodiments, use maize promoters, which are promoters active in maize plants. Maize promoters (maize promoter sequences) for use as provided herein may be derived in whole or in part from a maize plant, a plant other than maize, or a combination thereof (a recombinant maize promoter), provided the promoter is active in maize plants. A maize promoter that is active during meiosis refers to a maize promoter that controls transcriptional initiation and/or expression of a downstream sequence during any one or more stages of maize meiosis. A maize promoter that is active during meiosis may also be active during other stages of development. By comparison, a maize promoter that is specifically active during meiosis refers to a maize promoter that is active only during meiosis, or is active primarily during meiosis. Examples of such meiosis-specific maize promoters include, but are not limited to, AMEIOTIC1 (GRMZM5G883855; Pawlowski W P, et al. PNAS 2008; 106(9): 3603-08), ABSENCE OF FIRST DIVISION (AFD1/REC8) (GRMZM2G059037; Golubovskaya I N, et al. Journal of Cell Science 2006; 119: 3306-3315), and DMC1(GRMZM2G109618; Etedali F, International journal of Agronomy and Plant Production 2012; 3(3): 100-108), and PHS1(GRMZM2G100103; Ronceret A, et al. PNAS 2009; 106(47): 20121-26). Other meiosis-specific maize promoters are encompassed by the present disclosure.

[0026] Gene silencing nucleic acids, generally, are nucleic acids that are capable of reducing expression of genes during meiosis, during gene transcription or during post-transcriptional processes. Well-known examples of approaches by which gene silencing can be carried out include RNA interference (RNAi) and RNA silencing. RNAi, for example, leads to post transcriptional gene silencing triggered by double-stranded RNA molecules. Other examples of gene silencing molecules for use in plants include antisense RNA, nucleic acids encoding virus induced gene silencing (VIGS) vectors, and long double stranded RNA, which are processed into small RNA molecules.

[0027] Artificial microRNAs (amiRNAs) are small RNAs that can be genetically engineered and function to specifically silence single or multiple genes of interest (Tiwari M, et al. Plant Mol. Biol. 2014; 86(1-2): 1-18). An amiRNA is complementary to and binds to a target RNA (e.g., mRNA), the binding of which elicits a reduction (e.g., 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 20%-95%, 30%-95%, 40%-95%, 50%-95%, 60%-95%, 70%-95%, 20%-90%, 30%-90%, 40%-90%, 50%-90%, 60%-90% or 70%-90% reduction) in expression of the target RNA. The length of an amiRNA may vary. In some embodiments, an amiRNA has a length of 15-50 nucleotides, or 15-30 nucleotides. For example, an amiRNA may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In some embodiments, an amiRNA has a length of 21 nucleotides. Examples of amiRNA targets are provided as SEQ ID NO: 1-10 (targeting maize ZMET2), SEQ ID NO: 11-20 (targeting maize ZMET5), and SEQ ID NO: 21-30 (targeting maize KRYPTONITE).

[0028] An amiRNA may be expressed, for example, as part of a backbone having a sequence based on the maize microRNA gene zma-miR396h (Meng X, et al. The Plant Cell 2011; 23(3): 942-60). The amiRNA sequence may replace the endogenous miR396 sequence. As another example, the amiRNA may be expressed as part of a backbone based on a rice microRNA gene Oryza sativa MIR528 (osa-miR528) that has been modified for amiRNA production (Yan F, et al. J of Biotech. 2012; 160(3-4): 146-50).

[0029] Virus-induced gene silencing (VIGS) refers to sequence-specific reduction in target gene expression by infection of a plant with a virus vector containing fragments of the target gene (see, e.g., Ruiz M T et al. Plant Cell 1998; 10(6): 937-46; and Baulcombe D C, Current Opinion in Plant Biology 1999; 2(2): 109-13, each of which is incorporated by reference).

[0030] A V.sub.0 plant is a plant infected with virus.

[0031] A V.sub.1 plant is a progeny plant of a virus-infected V.sub.0 plant.

[0032] Also provided herein are methods of producing maize plants that carry (e.g., chromosomally or episomally) an expression cassette that has a maize promoter that is active during meiosis and operably linked to a nucleic acid encoding an amiRNA (or other dominant negative suppressor, such as an antisense RNA or virus induced gene silencing vector) that targets a gene in the H3K9me2 and non-CG DNA methylation pathway. The methods may comprise, for example, (a) introducing into (transforming into) an embryo of a maize inbred plant or a cell of a maize inbred plant an expression cassette that includes a maize promoter that is active during meiosis and is operably linked to a nucleic acid encoding an amiRNA that targets ZMET2, ZMET5 or KRYPTONITE to produce an embryo comprising the expression cassette or a cell comprising the expression cassette, (b) regenerating the embryo comprising the expression cassette or the cell comprising the expression cassette to produce a T.sub.0 maize plant, and (c) crossing the T.sub.0 maize plant to another inbred plant to produce maize seed (e.g., F.sub.1 hybrid seed).

[0033] Methods of transforming plants and plant parts with nucleic acids are known, any of which may be used to introduce an expression cassette into a plant or plant part, as provided herein. Examples include, but are not limited to agrobacterium-mediated transformation of maize embryos (Gelvin S B Microbiol Mol Biol Rev. 2003; 67(1): 16-37) followed by plant regeneration (Ishida Y et al. Nature Protocols 2007: 2: 1614-21), by particle bombardment (Kikkert J R et al. Methods Mol Biol. 2005; 286:61-78) or similar approaches, such as PEG-mediated protoplast transformation, silicon carbide whisker, or electroporation (see, e.g., Que Q, et al. Front Plant Sci 2014; 5: 379).

[0034] A To plant is a plant regenerated following transformation of plant embryos/cells with a nucleic acid construct. A plant may be regenerated, for example, from immature embryos (Green C E, Philips R L. Crop Sci. 1975; 15:417-21; Duncan D R, et al. Planta. 1985; 165:322-32; Bohorova N E, et al. Maydica. 1995; 40:275-81; Ishida Y, et al. Nat Biotechnol. 1996; 14:745-50; and Aguado-Santacruz G A, et al. In Vitro Cell Dev Biol Plant. 2007; 43:215-24), mature embryos (Huang X Q, Wei Z M. Plant Cell Rep. 2004; 22:793-800; and Al-Abed D, et al. Planta. 2006; 223:1355-60), nodal regions (Sidorov V, et al. Plant Cell Rep. 2006; 25:320-8), leaf tissues (Conger B V, et al. Plant Cell Rep. 1987; 6:345-7; and Ahmadabadi M, et al. Transgenic Res. 2007; 16:437-48), anthers (Ting Y C, et al. Plant Sci Lett. 1981; 23:139-45; and Barloy D, Beckert M. Plant Cell Tissue Org. Cult. 1993; 33:45-50), tassel and ear meristems (Pareddy D R, Petolino J F. Plant Sci. 1990; 46:225-32), protoplast (Morocz C, et al. Theor Appl Genet. 1990; 80:721-6), and shoot meristems (Sairam R V, I. 2003; 46:323-9).

[0035] For example, maize callus from embryo scutellar tissues may be initiated and maintained on MS medium inorganic components, Straus medium vitamins and amino acids, 20 g sucrose and 8 g agar per liter, and 2 mg 2,4-dichlorophenoxyacetic acid (2,4-D)/liter. Callus may be maintained in subculture every 21 to 28 days and remained capable of differentiation for 9 months. Regeneration of complete plants may be accomplished by subculture of callus to 0.25 mg 2,4-D/liter for 30 days followed by transfer to 2,4-D-free culture medium. At 0.25 mg 2,4-D/liter numerous curled and wrinkled leaves typically develop. Complete plants differentiate after transfer to 2,4-D-free medium. After transplantation to soil, plants survive and grow normally. The optimum embryo age for scutellar callus initiation is, in some embodiments, 18 days post-pollination. Hormone combinations such as 1 mg 2,4-D, 4 mg a-naphthaleneacetic acid (NAA), and 0.05 mg 6-(,-dimethyl allylamino)-purine (2iP)/liter may increase the efficiency of scutellar callus initiation (Green C E, Philips R L. Crop Sci. 1975; 15:417-21).

[0036] Maize seeds (e.g., F1 hybrid seeds) may be grown to produce F.sub.1 maize plants. In some embodiments, an F.sub.1 maize plant (comprising the amiRNA expression cassette) is backcrossed to a plant from a parental line or other line to produce BC.sub.1 maize seed. The backcross breeding method is typically used to incorporate specific traits into elite lines. This method works by crossing a transgenic inbred line (e.g., carrying the amiRNA expression cassette) with an elite inbred line. The offspring of this cross, referred to as the F.sub.1 hybrid generation, should have 50% of the transgenic line's alleles and 50% of the elite line's alleles. An elite maize inbred line generally refers to a maize line that has many alleles that confer many strong agronomic traits resulting in high yields in a particular environment. An exotic maize inbred line refers to a non-elite line that contains a particular favorable trait, e.g., drought tolerance, but otherwise has poor characteristics compared to elite lines. Exotic lines can be a useful source of novel genetic variants that can be introgressed into elite lines to confer the useful trait. The F.sub.1 plants are crossed back to the elite inbred again. These offspring are referred to as the BC.sub.1 generation (backcross 1). The BC.sub.1 offspring may be genotyped by extracting DNA and performing an amplification assay (e.g., PCR using cassette-specific primers) on the extracted DNA to determine which of the BC.sub.1 offspring are transgenic (e.g., which of the offspring carry the amiRNA expression cassette).

[0037] In some embodiments, an F.sub.1 maize plant is self-pollinated to produce F.sub.2 maize seed. Under self-pollination, the silks of an ear are pollinated by pollen from the same plant.

[0038] The BC.sub.1 maize seed, or F.sub.2 maize seed, may then be grown to produce a BC.sub.1 maize plant, or an F.sub.2 maize plant, respectively. The mature plants may then be screened for desirable traits. Examples of such desirable traits include, but are not limited to, herbicide tolerance, insecticide tolerance, increased starch, insect resistance, disease resistance, and drought tolerance, salt tolerance, waterlogging tolerance and soil pH tolerance.

[0039] Epigenetic drift in plants could affect crossover distribution over evolutionary time, reminiscent of genetic drift in mammalian systems. Suppression of recombination in repetitive regions is a major barrier to the introduction of genetic diversity, causing significant linkage drag in elite maize and wheat varieties, where most of the chromosome is composed of pericentromeric heterochromatin. H3K9me2 and non-CG DNA methylation mutants should unlock pericentromeric crossover in crop breeding programmes.

EXAMPLES

Example 1. Epigenetic Control of Meiotic Crossover in Arabidopsis Pericentromeric Heterochromatin

[0040] To measure recombination frequency H3K9me2 and DNA methylation mutants were combined with fluorescent crossover reporter lines (Fluorescent Tagged Lines, FTLs) (Berchowitz L E, Copenhaver G P. Nat. Protoc. 2008; 3: 41-50). In order to test whether pericentromeric crossovers were increased in hybrid backgrounds, the CEN3 FTL pollen line with cmt3 alleles in Col (cmt3-11) and Ler (cmt3-7) accessions were used (Lindroth A M, et al. Science 2001; 292: 2077-80) (FIG. 1A). CEN3 crossover frequency significantly increased in both cmt3 inbreds (X.sup.2 P=3.0010.sup.143) and, unexpectedly, in F.sub.1 hybrids (X2 P=1.2710.sup.86) (FIG. 1B). In contrast, the euchromatic 420 FTL interval on chromosome 3 did not significantly change in cmt3 inbreds (X.sup.2 P=0.651), and slightly decreased in hybrids (X.sup.2 P=1.9610.sup.3) (FIG. 1C). Crossovers genome-wide were then mapped by generating large wildtype and cmt3 F.sub.2 populations (FIG. 1A). In order to map crossover distributions genome-wide at high resolution, genotyping-by-sequencing was performed on 192 F.sub.2 individuals from wildtype and cmt3 populations (Yelina N E, et al. Genes Dev. 2015; 29: 2183-202) (FIGS. 1D, 1E, 1F and 1G). The mean number of crossovers per wildtype F.sub.2 individual (mean=6.41) was comparable to that observed in similar populations (Giraut L, et al. PLoS Genet. 2011; 7: e1002354), and a significant increase in total crossovers in the cmt3 population was observed (mean=7.34) (Mann-Whitney-Wilcoxon test P=8.8610.sup.5) (FIGS. 1D, 1E).

[0041] Crossover distributions were analysed according to their location in the euchromatic arms, heterochromatic pericentromeres and centromeres (FIGS. 1D, 1E). Centromeres were defined as the regions that surround centromeric genome sequence assembly gaps (version TAIR10) that fail to undergo recombination in wildtype (Copenhaver G P, et al. Science 1999; 286: 2468-74; and Giraut L, et al. PLoS Genet. 2011; 7: e1002354). Pericentromeric heterochromatin was defined as regions flanking the centromeres with higher than average DNA methylation, and the euchromatic arms were defined as the remainder of the chromosomes. Crossovers were significantly increased in cmt3 pericentromeres (X.sup.2 P=6.5010.sup.5), which are strongly depleted of CHG DNA methylation (FIGS. 1D-1F). We also observed centromeric crossovers in cmt3 but not wildtype controls (18 versus 0) (X.sup.2 P=1.8510), but crossovers were still suppressed in centromeres, either because of residual non-CG DNA methylation and H3K9me2, other chromatin modifications, or structural polymorphisms (Ito H, et al. Genet. Genomics 2007; 277: 23-30). For example, the 1.17 Mb heterochromatic knob inversion on chromosome 4 prevents crossover in both wildtype and cmt3 populations (Fransz P F, et al. Cell 2000; 100: 367-76) (FIG. 1D). The chromosome arms showed a significant decrease in crossovers in cmt3 (X.sup.2 P=2.6110.sup.6), which is consistent with a compensatory homeostatic adjustment (FIGS. 1D, 1E, 1G). The location of crossover events was analyzed in relation to gene, levels of CHG DNA methylation and H3K9me2 (Stroud H, et al. Cell 2013; 152: 352-64; and Inagaki S, et al. EMBO J. 2010; 29: 3496-506). Pericentromeric crossovers in both wildtype and cmt3 occurred in regions with lower DNA methylation and H3K9me2 than expected at random.

[0042] The following Examples described how to modify H3K9me2 and non-CG DNA methylation during meiosis using artificial microRNA transgenes in maize F.sub.1 hybrids, or maize lines with mixed germplasm, in order to elicit recombination in ordinarily recombination-suppressed regions.

Example 2. Artificial microRNA (amiRNA) Targeting Components of the H3K9Me2 and Non-CG DNA Methylation Pathway

[0043] Maize components of the H3K9me2 and non-CG DNA methylation pathway include, but are not limited to, the chromomethylase genes ZMET2 (GRMZM2G025592), ZMET5 (GRMZM2G005310) and the maize homologue of KRYPTONITE (GRMZM2G336909). Artificial microRNAs were designed against these genes from the sequences in the reference maize genome (B73 background) using Web MicroRNA designer 3. The Zea mays genome version used was Zea Mays ZmB73 v5b (MGC). See SEQ ID NO: 1-30, below.

Example 3. Constructs that Specifically Express amiRNA in Meiotic Tissues

[0044] amiRNAs that specifically target components of the H3K9me2 and non-CG DNA methylation pathway are expressed under a promoter such that the amiRNAs are active during meiosis in order to reduce expression of the target gene during meiosis. This can be accomplished using a constitutively active promoter or a promoter specifically active (primarily only active) during meiosis. Down regulation of these factors specifically in meiosis, in some embodiments, is preferred, because down regulation during other stages of plant development may have undesirable phenotypic effects (Li Q, et al. The Plant Cell, 2015; 26(12): 4602-16). The amiRNA may be expressed under maize promoters that are specifically active in meiosis, including, but not limited to, AMEIOTIC1 (GRMZM5G883855; Pawlowski W P, et al. PNAS 2008; 106(9): 3603-08), ABSENCE OF FIRST DIVISION (AFD1/REC8) (GRMZM2G059037; Golubovskaya I N, et al. Journal of Cell Science 2006; 119: 3306-3315), and DMC1 (GRMZM2G109618; Etedali F, International journal of Agronomy and Plant Production 2012; 3(3): 100-108), and PHS1 (GRMZM2G100103; Ronceret A, et al. PNAS 2009; 106(47): 20121-26).

[0045] The amiRNA may be expressed, for example, as part of a backbone based on the maize microRNA gene zma-miR396h (Meng X, et al. The Plant Cell 2011; 23(3): 942-60). The amiRNA sequence may be inserted into the zma-miR396h gene in place of the endogenous miR396 sequence. As another example, the amiRNA may be expressed as part of a backbone based on a rice microRNA gene Oryza sativa MIR528 (osa-miR528) that has been modified for amiRNA production (Yan F, et al. J of Biotech. 2012; 160(3-4): 146-50).

Example 4. Transform amiRNA Expression Cassette into Maize to Downregulate H3K9Me2 and Non-CG DNA Methylation in Maize Meiosis

[0046] Constructs that express an amiRNA targeting a component of the H3K9me2 and non-CG DNA methylation pathway under a meiotic promoter are transformed into maize using standard approaches (e.g., by agrobacterium mediated transformation of maize embryos followed by plant regeneration (Ishida Y et al. Nature Protocols 2007: 2: 1614-21), by particle bombardment or similar approaches). FIG. 2 shows a flow diagram of a timeline of a maize transformation protocol (Ishida Y et al. Nature Protocols 2007: 2: 1614-21).

[0047] The transformation may be carried out on maize inbreds, F.sub.1 hybrids or maize lines with mixed germplasm (e.g., F.sub.2 generation or a later generation). In some embodiments, inbred lines (e.g., B73) are preferred.

[0048] A F.sub.1 hybrid plant, or plant with mixed germplasm, where an amiRNA targets a component of the H3K9me2 and non-CG DNA methylation pathway is the key intermediate where novel recombination events (in ordinarily recombination suppressed regions) may occur during meiosis. The seeds that are produced from this plant are the final product of sexual reproduction and they can be used to screen for novel recombination events, which are unlikely to occur in wild type control crosses.

Protocol for using an inbred for transformation (FIG. 3A): [0049] a) An amiRNA targeting H3K9me2 and non-CG DNA methylation pathway component is transformed into inbred maize variety (e.g., B73) and a T.sub.0 plant is regenerated. [0050] b) A T.sub.0 plant, or derivative, is crossed to a divergent maize variety (e.g., Missouri 17). [0051] c) The product of the cross, the F.sub.1 seed, is harvested and then planted and grown to maturation. [0052] d) A F.sub.1 hybrid plant is self-pollinated to produce F.sub.2 seed (or is backcrossed to a parental line or outcrossed to a non-parental inbred line). [0053] e) F.sub.2 seeds (or equivalent seeds derived from a backcross or outcross) are used to develop a large mapping population (e.g., of 100 individuals or more) by germinating seed, growing plants and extracting DNA. [0054] f) Novel recombination events, in ordinarily recombination suppressed regions, are screened. Genotyping of a mapping population may be carried out using, for example, a maize SNP array (Ganal M W, et al. PLOS One 2011; 6(12): e28334) or by ILLUMINA-based genotyping by sequencing.
Protocol for using a hybrid for transformation (FIG. 3B): [0055] a) An amiRNA targeting H3K9me2 and non-CG DNA methylation pathway component is transformed directly into F.sub.1 hybrid maize (e.g., B73Missouri 17 F.sub.1) and a T.sub.0 plant is regenerated. [0056] b) The F.sub.1T.sub.0 plant is grown to maturation. [0057] c) A F.sub.1T.sub.0 plant is self-pollinated to produce F.sub.2 seed (or is backcrossed to a parental line or outcrossed to a non-parental inbred line). [0058] d) F.sub.2 seeds (or equivalent seeds derived from outcross) are used to develop a large mapping population (e.g., of 100 individuals or more) by germinating seed, growing plants and extracting DNA. [0059] e) Novel recombination events, in ordinarily recombination suppressed regions, are screened. Genotyping of a mapping population may be carried out using, for example, a maize SNP array (Ganal M W, et al. PLOS One 2011; 6(12): e28334) or by ILLUMINA-based genotyping by sequencing.
Protocol for using a later generation (e.g., F.sub.2 or later) for transformation (FIG. 3C): [0060] a) An amiRNA targeting a H3K9me2 and non-CG DNA methylation pathway component is transformed into a plant with mixed germplasm (e.g., a B73Missouri 17 F.sub.2 linea second generation post crossing) and a T.sub.0 plant regenerated. [0061] b) A T.sub.0 plant is grown to maturation. [0062] c) A T.sub.0 plant is be self-pollinated to produce seed (or is backcrossed to a parental line or outcrossed to a non-parental inbred line). [0063] d) Seeds from self-pollination (or equivalent seeds derived from outcross) are used to develop a large mapping population (e.g., of 100 individuals or more) by germinating seed, growing plants and extracting DNA. [0064] e) Novel recombination events, in ordinarily recombination suppressed regions, are screened. Genotyping of a mapping population may be carried out using, for example, a maize SNP array (Ganal M W, et al. PLOS One 2011; 6(12): e28334) or by ILLUMINA-based genotyping by sequencing.

[0065] The following Examples described how to modify H3K9me2 and non-CG DNA methylation during meiosis using virus-induced gene silencing (VIGS) in maize F.sub.1 hybrids, or maize lines with mixed germplasm, in order to elicit recombination in ordinarily recombination-suppressed regions.

Example 5. Virus-Induced Gene Silencing (VIGS) in Maize to Downregulate H3K9Me2 and Non-CG DNA Methylation in Maize Meiosis

[0066] Maize components of the H3K9me2 and non-CG DNA methylation pathway include the chromomethylase genes ZMET2 (GRMZM2G025592), ZMET5 (GRMZM2G005310) and the maize homologue of KRYPTONITE (GRMZM2G336909). Sequences for introduction into a VIGS vector were designed against these genes using the cDNA sequences in Gramene's B73 RefGen_v3 sequence: GRMZM2G025592_T01; GRMZM2G005310_T01; and GRMZM2G336909_T01. These cDNA sequences were inputted into a VIGS prediction software (vigs.solgenomics.net) that minimizes off target effects, by comparing to the reference Maize transcriptome Zea mays B73 v5a. Parameters included: fragment size, 300; n-mer, 21; mismatches, 0; and database, Zea_mays_B73_v5a. See SEQ ID NO: 31-33, below. Also described below is a sequence (SEQ ID NO: 34) derived from ZMET2 (GRMZM2G025592_T01), which when inserted into a VIGS vector also targets ZMET5 (GRMZM2G005310_T01). A VIGS vector using this sequence may lead to concurrent knockdown of both ZMET2 and ZMET5.

Example 6. VIGS Vectors for Infection of Maize to Initiate Knockdown of the H3K9Me2 and Non-CG DNA Methylation Pathway

[0067] VIGS vectors are based on the maize-infecting cucumber mosaic virus (CMV) strain, ZMBJ-CMV (Wang R, et al. Plant J. 2016; 86(1):102-15, incorporated by reference). ZMBJ-CMV has been modified to facilitate efficient VIGS in maize (Wang R, et al. Plant J. 2016; 86(1):102-15). This method modifies the CMV RNA2 genome by introducing 100-500 nucleotide fragments of the target gene downstream of the 3 of the open reading frame (ORF) expressing the 2b protein. Other viruses may be modified in a similar manner to include sequences corresponding to ZMET2 (GRMZM2G025592), ZMET5 (GRMZM2G005310) and KRYPTONITE (GRMZM2G336909).

Example 7. Infect Maize with Virus Designed to Elicit Knockdown of H3K9Me2 and Non-CG DNA Methylation

[0068] DNA constructs that contain the modified viral genome are transformed into Agrobacterium tumefaciens (Wang R, et al. Plant J. 2016; 86(1):102-15). This Agrobacterium is used to inoculate maize directly at kernel, seedling, plantlet or other stage. The Agrobacterium inoculation leads to virus production, which elicits VIGS of the target gene.

[0069] The Agrobacterium is also used to inoculate N. benthamiana, a plant species that is capable of high viral titers. Crude sap from the inoculated N. benthamiana is then prepared (4 days after agro infiltration), which is used to infect maize at kernel, seedling, plantlet or other stage. This elicits VIGS of the target gene.

[0070] Viral infection and VIGS is carried out on maize F.sub.1 hybrids or maize lines with mixed germplasm (e.g., F.sub.2 generation or later).

[0071] The F.sub.1 hybrid plant, or plant with mixed germplasm, where a VIGS vector targets a component of the H3K9me2 and non-CG DNA methylation pathway, is the key intermediate where novel recombination events (in ordinarily recombination suppressed regions) occur during meiosis. The seeds that are produced from this plant are the final product of sexual reproduction and they are used to screen for novel recombination events, which are unlikely to occur in wild type control crosses.

Protocol for using a hybrid for infection (see FIG. 4A): [0072] a) A VIGS virus targeting H3K9me2 and non-CG DNA methylation pathway component is used to infect F.sub.1 hybrid maize (e.g., B73Missouri 17 F.sub.1). [0073] b) The F.sub.1 (V.sub.0) plant is grown to maturation. [0074] c) The F.sub.1 (V.sub.0) plant is self-fertilized to produce F.sub.2 seed (or is outcrossed to a parental or non-parental inbred line). [0075] d) The F.sub.2 seeds (or equivalent seeds derived from outcross) are used to develop a large mapping population (e.g., of 100 individuals or more) by germinating seed, growing plants up and extracting DNA. [0076] f) Novel recombination events, in ordinarily recombination suppressed regions, are screened. Genotyping of a mapping population may be carried out using, for example, a maize SNP array (Ganal M W, et al. PLOS One 2011; 6(12): e28334) or by ILLUMINA-based genotyping by sequencing.
Protocol for using a plant with mixed germplasm (e.g., F.sub.2 generation or later) for infection) (see FIG. 4B): [0077] a) A VIGS virus targeting H3K9me2 and non-CG DNA methylation pathway component is used to infect plant with mixed germplasm (e.g., a B73Missouri 17 F.sub.2 linei.e. second generation post crossing). [0078] b) The V.sub.0 plant is grown to maturation. [0079] c) The V.sub.0 plant is self-fertilized to produce seed (or is to a parental or non-parental inbred line). [0080] d) Seeds from self-fertilization (or equivalent seeds derived from outcross) are used to develop a large mapping population (e.g., of 100 individuals or more) by germinating seed, growing plants up and extracting DNA. [0081] e) Novel recombination events, in ordinarily recombination suppressed regions, are screened. Genotyping of a mapping population may be carried out using, for example, a maize SNP array (Ganal M W, et al. PLOS One 2011; 6(12): e28334) or by ILLUMINA-based genotyping by sequencing.

Example 8. Detecting Meiotic Crossovers by Examining Chiasmata in Maize Meiotic Cells

[0082] Chiasmata are the physical manifestations of meiotic crossovers and can be detected by mounting and spreading meiotic cells in the presence of a DNA stain (e.g., DAPI) followed by visualization of the chromosomes by fluorescence microscopy. In maize, most chiasmata are located at the tips (subtelomeric regions) of the chromosomes, where gene density is highest (Anderson L K et al. Genetics. 2003; 165: 849-65; Sidhu G K, et al. PNAS 2015; 112: 15982-87; and Rodgers-Melnick E, et al. PNAS USA 2015: 112: 3823-28). In contrast, the central parts of the chromosomes tend not to recombine, due to their highly repetitive nature. Therefore, chiasmata are generally not found in central parts of maize chromosomes in what is known as pericenteromeric heterochromatin (Sidhu G K, et al. PNAS 2015; 112: 15982-87; and Rodgers-Melnick E, et al. PNAS USA 2015: 112: 3823-28).

[0083] A detailed protocol that makes it possible to examine chiasmata in maize has recently been described (Sidhu G K, et al. PNAS 2015; 112: 15982-87).

[0084] In order to assay whether knockdown of H3K9me2 or non-CG DNA methylation factors has an effect on crossover distribution directly in an F.sub.1 hybrid plant, the spatial distribution of chiasmata along the lengths of chromosomes is examined in wild type and knock down meiotic cells (Sidhu G K, et al. PNAS 2015; 112: 15982-87). It is possible to determine chiasmata location along the length of a chromosome by visual inspection of meiotic bivalents.

[0085] In wild type plants, almost all chiasmata are present at the tips of chromosomes (subtelomeric regions), and not in central chromosomal regions (pericentromeric heterochromatin) (Sidhu G K, et al. PNAS 2015; 112: 15982-87).

[0086] In the H3K9me2/non-CG DNA methylation knockdown lines, chiasmata may be detected in central chromosomal regions (pericentromeric heterochromatin).

[0087] For further validation of location of chiasmata relative to maize centromeres, chiasmata detection may be combined with Fluorescence In Situ Hybridisation (FISH) to locate the position of centromeres on the chromosomes. By using fluorescently labeled oligonucleotide probes against the maize centromeric repeats, CentC and CRM (Jin W, et al. PLANT CELL ONLINE 2004; 16: 571-81), maize centromeres can be specifically labeled. Proximity of chiasmata location in wild type and H3K9me2/non-CG DNA methylation knockdown lines to centromeres shows whether the desired increase in pericenteromeric recombination has occurred in the H3K9me2/non-CG DNA methylation knockdown lines.

Example 9. Quantification of Knockdown Efficiency of H3K9Me2/Non-CG DNA Methylation Factor Gene Expression in Maize Anthers

[0088] To have an effect on meiotic recombination, H3K9me2/non-CG DNA methylation factor gene expression should be reduced in meiotic cells. Maize anthers, the male sexual organs, can contain a high proportion of meiotic cells, when the correct stage of anther development is analyzed. Reduction in gene expression of a H3K9me2/non-CG DNA methylation factor, in maize anthers that harbor a gene-silencing cassette, may be assessed using the following approach:

[0089] 1) Extract RNA from developmentally staged maize anthers;

[0090] 2) Produce complementary DNA (cDNA) based on the RNA, using a reverse transcriptase reaction; and [0091] 3) Quantify gene expression using quantitative reverse transcript polymerase chain reaction (qRT-PCR)
RNA Extraction from Maize Anthers

[0092] Maize anthers between 1000-1200 m are dissected and collected, as these anthers are most enriched for pollen mother cells (meiotic cells) (Kelliher T and Walbot V, Dev. Biol. 2011; 350: 32-49). Collected tissue is frozen in liquid nitrogen and ground to a fine powder using a pre-frozen pestle and mortar. RNA extraction is carried out using Plant RNA reagent (Invitrogen), according to manufacturer's protocol, including a DNase treatment step. RNA concentration is quantified (e.g., by using a Qubit fluorometer (ThermoFisher), in accordance with the manufacturers protocol).

Complementary DNA (cDNA) Preparation

[0093] Equal amounts of RNA are used to prepare cDNA by incubating RNA in the presence of a reverse transcriptase enzyme (e.g., SuperScript (ThermoFisher), in accordance with the manufacturers protocol). The reaction is primed with either random hexamer primers or oligo(dT) primers. Control reactions are carried out without reverse transcriptase present.

Gene Expression Quantification by Quantitative Reverse Transcript PCR (qRT-PCR)

[0094] qRT-PCR is used to quantify gene expression of the target gene. Primers that specifically amplify the target gene and a housekeeping gene are. cDNA is PCR amplified in separate reactions containing either the target gene specific primers or housekeeping gene specific primers. Amplification is carried out in a quantitative PCR (qPCR) machine, in the presence of a fluorescent DNA stain (e.g., SYBR Green reagent (ThermoFisher), according to manufacturer's protocol). Ct values are calculated and used to determine changes of H3K9me2/non-CG DNA methylation factor gene expression in knockdown and control samples.

TABLE-US-00001 SEQUENCES MET2(GRMZM2G025592_T01) TTTCTGTACCGATTAAACCCG(SEQIDNO:1) TTTCTGTACCGATAAAACCCG(SEQIDNO:2) TTAAATGTTACCTGGTGACTG(SEQIDNO:3) TTTTAGATTAGATACCGTCAC(SEQIDNO:4) TTTTTTAACGGACTCGGTCAA(SEQIDNO:5) TCATAGTATAGGTTGCGACTC(SEQIDNO:6) TTAGTATAGGTCGTACCTCTC(SEQIDNO:7) TTAACATATCCCGAGAAGCTT(SEQIDNO:8) TTAAATGTTACCTGATGACTC(SEQIDNO:9) TACGAAGCATCAATACGGCTG(SEQIDNO:10) ZMET5(GRMZM2G005310_T01) TATGTTAGTAACACGTGGCCG(SEQIDNO:11) TTCAAGAGCTGTAATTGACTT(SEQIDNO:12) TATGTTAGTAACACATGGCCG(SEQIDNO:13) TTCAAGAGCTGTAATTGGCTT(SEQIDNO:14) TCTATGGGTCTAAAGTACCTA(SEQIDNO:15) TCTATGGGTCTAAAGCGCCTA(SEQIDNO:16) TTTAACAGCTGACAATTGCTA(SEQIDNO:17) TTTAACAGCTGACAACCGCTA(SEQIDNO:18) TTTAACAGCTGACAACTCCGA(SEQIDNO:19) TTGTTAGTAACACAGCGCCGT(SEQIDNO:20) KRYPTONITE(GRMZM2G336909_T01) TTAATCGAAGATTGTCCGCAA(SEQIDNO:21) TTAATCGAAGATTGTTGGCAA(SEQIDNO:22) TATTGTCGGCAAAATATGCTG(SEQIDNO:23) TAGTTAATCGAAGACTGTCGG(SEQIDNO:24) TAGTTAATCGAAGATTCTCGG(SEQIDNO:25) TATTGTCGGCAAAATACGCTA(SEQIDNO:26) TTACAATATAAGCTGTCTCTT(SEQIDNO:27) TTACAATATAAGCTCTTTCCT(SEQIDNO:28) TTAGAGTGAGCGTCTATCCAA(SEQIDNO:29) TATTGTCGGCAAAATATGCTA(SEQIDNO:30) GRMZM2G025592_T01(1297-1596) CGATGAGTTTCTTGCCCTCCTTAAGGAATGGGCAGTTCTATGCAAAAAAT ATGTCCAAGATGTGGATTCAAATTTAGCAAGCTCAGAGGATCAAGCGGAT GAAGACAGCCCTCTTGACAAGGACGAATTTGTTGTAGAGAAGCTTGTCGG GATATGTTATGGTGGCAGTGACAGGGAAAATGGCATCTATTTTAAGGTCC AGTGGGAAGGATACGGCCCTGAGGAGGATACATGGGAACCGATTGATAAC TTGAGTGACTGCCCGCAGAAAATTAGAGATTTTGTACAAGAAGGGCACAA (SEQIDNO:31) GRMZM2G005310_T01(1234-1533) TGATGAGTTTCTTGCTCTCCTTAAGGAATGGGCAGTCTTATGTGAAAAAT ATGTTCATCAAGACGTGGATTCGAATTTAGCAGGTTCAGAGGATCAAGAG GATGCTGACACTCTTGACAAGGATGAATTTGTTGTACAGAAGCTTATTGG AATACGCTATGATGGCACTGGAAGGAAAAAAGGCGTATATTTTAAGGTCC AATGGGAAGGATATGGTCCTGAAGAGGATACATGGGAACCTATTGATAAC CTGAGTGACTGTCCGCTGAAAATTAGAGAATTTGTACAAGAAGGGCGCAA (SEQIDNO:32) GRMZM2G336909_T01(1947-2246) AAAAATTGTGAAGCTGGCCTGCCACTGTGGTGCTCCTGATTGCCGGAAGC GCCTCTACTAACCTGCAGTGTGTTTATTTCTATTTGGATTTTTGAGGAAA CAGCTTATATTGTAGGAAAAACAGTCATGGTACCTATGTAGTTGACATGC TCTTGATCATATATAGCAAAAGAGGAGCAATTTATGGGGTGTTCGGTTTA AGAATGAAATTAGAGATTTAGAGGACATGGTGGTCATGGTAGCATATTTT GCCGACAATCTTCGATTAACTGATATATCTGGACATCTGGTCCTGCAACA (SEQIDNO:33) GRMZM2G025592_T01(960-1296) GATAGAGAGTTGCGACCTATACTATGACATGTCTTACTCTGTTGCATATT CTACATTTGCTAATATCTCGTCTGAAAATGGGCAGTCAGACAGTGATACC GCTTCGGGTATTTCTTCTGATGATGTGGATCTGGAGACGTCATCTAGTAT GCCAACGAGGACAGCAACCCTTCTTGATCTGTATTCTGGCTGTGGGGGCA TGTCTACTGGTCTTTGCTTGGGTGCAGCTCTTTCTGGCTTGAAACTTGAA ACTCGATGGGCTGTTGATTTCAACAGTTTTGCGTGCCAAAGTTTAAAATA TAATCATCCACAGACTGAGGTGCGAAATGAGAAAGC(SEQID NO:34)

[0095] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0096] The indefinite articles a and an, as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean at least one.

[0097] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0098] In the claims, as well as in the specification above, all transitional phrases such as comprising, including, carrying, having, containing, involving, holding, composed of, and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases consisting of and consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.