Patent Application
20230089653
The contents of the electronic sequence listing (2022-11-10-Sequence Listing ST26-UGRF-026US2.xml; Size: 115,032 bytes; and Date of Creation: Nov. 10, 2022) is herein incorporated by reference in its entirety.
The invention disclosed herein generally relates to genes involved in plant reproduction and methods of using the same.
Apomixis is a naturally occurring mode of asexual reproduction in flowering plants; this process results in seed formation without the involvement of meiosis or fertilization of the egg. Apomictic processes bypass meiosis and fertilization, leading directly to clonal embryo formation. Apomictic hybrids are true-breeding hybrids because seed-derived progeny of an apomictic plant are genetically identical to the maternal parent. In other words, apomictic hybrids are clonal in origin.
Apomixis is characterized by: 1) apomeiosis, which refers to the formation of unreduced embryo sacs derived from nucellar cells of the ovary, and 2) parthenogenesis, which refers to the development of the unreduced egg into an embryo. Many types of plant species feature apomictic reproduction and can be propagated asexually.
Embodiments of the invention include a method of propagating one or more gametophytic or sporophytic cells in an ovule of a plant in the absence of egg cell fertilization. The method can include: transforming a plant with an BBML gene construct comprising a nucleic acid encoding a polypeptide having at least 40% sequence similarity to the polypeptide of SEQ ID NO: 4, wherein the nucleic acid is operably-linked to a promoter effective within an egg cell; and/or growing and selecting a progeny plant from the one or more gametophytic or sporophytic cells, wherein the progeny plant contains one or more sets of chromosomes from the transformed plant, wherein propagation of the plant occurs in the absence of egg cell fertilization.
In some embodiments, the BBML gene construct further can include one or more untranslated region (UTR).
In some embodiments, the BBML gene construct can further include one or more UTR has at least 70% sequence identity to SEQ ID NO: 1 or a fully complementary strand thereof.
In some embodiments, the promoter can include a nucleotide having at least 70% sequence identity to SEQ ID NO: 5 or a fully complementary strand thereof.
In some embodiments, the ASGR-BBML gene construct has at least 70% sequence identity to SEQ ID NO: 3 or a fully complementary strand thereof.
In some embodiments, the gene construct can encode a polypeptide having that has at least 40% sequence identity to Seita.8G107100.1.p (SiBBM1), Sieta.5G415800.1.p (SiBBM2) or Seita.1G232200.1.p (SiBBM3).
In some embodiments, an embryo can be formed from an unreduced egg.
In some embodiments, an embryo can be formed from a reduced egg.
In some embodiments, an embryo can be formed from a somatic cell.
In some embodiments, a polyploid plant can be transformed to produce a diploid or dihaploid progeny plant.
In some embodiments, a diploid plant can be transformed to produce a haploid progeny plant.
In some embodiments, the haploid progeny plant can be treated to achieve chromosome doubling and production of a homozygous plant.
In some embodiments, the progeny plant can be obtained via culturing.
In some embodiments, the plant can be a monocot.
In some embodiments, the plant can be a dicot.
In some embodiments, the plant can include a grass or a leguminous plant.
In some embodiments, the grass can be a species of millet, rice, maize, wheat, sorghum, switchgrass, and/or the like.
In some embodiments, the plant can be heterozygous and can be transformed to produce a clonal offspring.
In some embodiments, the plant can be heterozygous and can be transformed to produce a haploid offspring.
In some embodiments, the method can be used to propagate one or more heritable traits in the plant.
Some embodiments of the invention relate to a plant or plant part produced by any of the methods disclosed herein.
Some embodiments of the invention relate to a method of producing a plant capable of being reproduced in the absence of egg cell fertilization. The method can include: transforming a plant with an BBML gene construct encoding a polypeptide having at least 40% sequence similarity to the polypeptide of SEQ ID NO: 4, thereby producing a plant capable of being reproduced in the absence of egg cell fertilization.
Embodiments of the invention encompass methods of achieving propagation from one or more gametophytic or sporophytic cells in an ovule of a flowering plant in the absence of egg cell fertilization, the methods including: transforming a flowering plant with an ASGR-BBML gene construct capable of encoding a polypeptide having at least 75% sequence identity to the polypeptide of SEQ ID NO: 4; obtaining one or more gametophytic or sporophytic cells from an ovule from the transformed plant in the absence of egg cell fertilization; and deriving a progeny plant from the one or more gametophytic or sporophytic cells, wherein the progeny plant contains one or more sets of chromosomes from the transformed plant, thereby achieving propagation of the flowering plant in the absence of egg cell fertilization.
In some embodiments, the ASGR-BBML gene construct further includes one or more untranslated region (UTR). In some embodiments, the ASGR-BBML gene construct further including one or more UTR can have at least 70% sequence identity to SEQ ID NO: 1 or a fully complementary strand thereof. In some embodiments, the ASGR-BBML gene construct further includes a promoter. In some embodiments, the promoter is capable of regulating the expression of a polypeptide having at least 75% sequence identity to the polypeptide of SEQ ID NO: 4. In some embodiments, the promoter includes a nucleotide having at least 70% sequence identity to SEQ ID NO: 5 or a fully complementary strand thereof. In some embodiments, the ASGR-BBML gene construct further including one or more UTR and a promoter can have at least 70% sequence identity to SEQ ID NO: 3 or a fully complementary strand thereof.
In some embodiments, the embryo can be formed from an unreduced egg. In some embodiments, the embryo can be formed from a reduced egg. In some embodiments, the embryo can be formed from a somatic cell.
In some embodiments, a polyploid plant can be transformed to produce a diploid or dihaploid progeny plant. In some embodiments, a diploid plant can be transformed to produce a haploid progeny plant. In some embodiments, the haploid progeny plant can be treated to achieve chromosome doubling and production of a homozygous plant. In some embodiments, the progeny plant can be obtained via culturing.
In some embodiments, the flowering plant can be a monocot. In some embodiments, the flowering plant can be a dicot.
In some embodiments, the flowering plant can be a grass or a leguminous plant. In some embodiments, the grass can be a species of millet, rice, maize, wheat, sorghum, or switchgrass.
In some embodiments, the flowering plant can be heterozygous and can be transformed to produce a clonal offspring. In some embodiments, the flowering plant can be heterozygous and can be transformed to produce a haploid offspring.
In some embodiments, the methods of the present invention can be used to propagate one or more heritable traits in the flowering plant.
The invention also encompasses plants or plant parts produced by methods of achieving propagation from one or more gametophytic or sporophytic cells in an ovule of a flowering plant in the absence of egg cell fertilization, the methods including: transforming a flowering plant with an ASGR-BBML gene construct capable of encoding a polypeptide having at least 75% sequence identity to the polypeptide of SEQ ID NO: 4; obtaining one or more gametophytic or sporophytic cells from an ovule from the transformed plant in the absence of egg cell fertilization; and deriving a progeny plant from the one or more gametophytic or sporophytic cells, wherein the progeny plant contains one or more sets of chromosomes from the transformed plant, thereby achieving propagation of the flowering plant in the absence of egg cell fertilization.
The invention also encompasses methods of obtaining a flowering plant capable of being reproduced in the absence of egg cell fertilization, the method including: transforming a flowering plant with an ASGR-BBML gene construct capable of encoding a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 4, thereby obtaining a flowering plant capable of being reproduced in the absence of egg cell fertilization.
The invention also encompasses plants or plant parts produced by methods of obtaining a flowering plant capable of being reproduced in the absence of egg cell fertilization, the method including: transforming a flowering plant with an ASGR-BBML gene construct capable of encoding a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 4, thereby obtaining a flowering plant capable of being reproduced in the absence of egg cell fertilization.
The invention also encompasses methods of producing seed of a flowering plant in the absence of egg cell fertilization, the method including: transforming a flowering plant with an ASGR-BBML gene construct; obtaining one or more embryos from the transformed plant in the absence of egg cell fertilization; and producing seed from the one or more embryos which contains one or more sets of chromosomes which are derived solely from the transformed mother plant, thereby obtaining seed propagation from the flowering plant in the absence of egg cell fertilization.
The invention also encompasses seeds produced by methods of producing seed of a flowering plant in the absence of egg cell fertilization, the method including: transforming a flowering plant with an ASGR-BBML gene construct; obtaining one or more gametophytic or sporophytic cells from an ovule from the transformed plant in the absence of egg cell fertilization; and deriving seed from the one or more gametophytic or sporophytic cells, wherein the seed contains one or more sets of chromosomes from the transformed plant, thereby producing seed of the flowering plant in the absence of egg cell fertilization.
The invention also encompasses ASGR-BBML gene constructs capable of encoding a polypeptide having at least 75% sequence identity to the polypeptide of SEQ ID NO: 4, wherein the polypeptide encoded by the ASGR-BBML gene construct, when expressed in one or more gametophytic or sporophytic cells in an ovule of a flowering plant, can allow propagation to be achieved in the absence of egg cell fertilization. In some embodiments, the ASGR-BBML gene constructs can further include one or more UTR and a promoter, wherein the ASGR-BBML gene construct has at least 70% sequence identity to SEQ ID NO: 3 or a fully complementary strand thereof.
Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
As used herein, the terms “plant” and “plant part” can include inclusively, as context indicates, plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, flowers, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like.
The term polynucleotide can include the terms “nucleic acid”, “nucleic acid sequence”, and “oligonucleotide”, as those terms are generally understood in the art. What is included in a specific instance will be appreciated by a person of skill in the art as indicated by the context, but where no particular context limits the scope, then the term will be understood to be broadly inclusive. Therefore, the term polynucleotide can also include DNAs or RNAs that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides”, as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.
As used herein, the term “transgenic” describes a non-naturally occurring plant that contains a genome modified by man, wherein the plant includes in its genome an exogenous nucleic acid molecule, which can be derived from the same or a different species, including non-plant species. The exogenous nucleic acid molecule can be a gene regulatory element such as a promoter, enhancer, or other regulatory element, or can contain a coding sequence, which can be linked to a native or heterologous gene regulatory element. Transgenic plants that arise from sexual cross or by selfing are descendants of such a plant.
As used herein, “polymorphism” means the presence of one or more variations of a nucleic acid sequence at one or more loci in a population of one or more individuals. The variation can comprise, but is not limited to, one or more base changes, the insertion of one or more nucleotides, or the deletion of one or more nucleotides. A polymorphism includes a single nucleotide polymorphism (SNP), a simple sequence repeat (SSR), indels (insertions and deletions), a restriction fragment length polymorphism, a haplotype, and a tag SNP. In addition, a polymorphism can include a genetic marker, a gene, a DNA-derived sequence, a RNA-derived sequence, a promoter, a 5′ untranslated region of a gene, a 3′ untranslated region of a gene, microRNA, siRNA, a quantitative trait locus (QTL), a satellite marker, a transgene, mRNA, ds mRNA, a transcriptional profile, or a methylation pattern. A polymorphism can arise from random processes in nucleic acid replication, through mutagenesis, as a result of mobile genomic elements, from copy number variation and during the process of meiosis, such as unequal crossing over, genome duplication and chromosome breaks and fusions. The variation can be commonly found or can exist at low frequency within a population, the former having greater utility in general plant breeding and the latter can be associated with rare but important phenotypic variation.
As used herein, a “marker” can refer to a polymorphic nucleic acid sequence or nucleic acid feature. In a broader aspect, a “marker” can be a detectable characteristic that can be used to discriminate between heritable differences between organisms. Examples of such characteristics can include genetic markers, protein composition, protein levels, oil composition, oil levels, carbohydrate composition, carbohydrate levels, fatty acid composition, fatty acid levels, amino acid composition, amino acid levels, biopolymers, pharmaceuticals, starch composition, starch levels, fermentable starch, fermentation yield, fermentation efficiency, energy yield, secondary compounds, metabolites, morphological characteristics, and agronomic characteristics.
As used herein, a “genotype” can refer to the genetic component of the phenotype, and this can be indirectly characterized using markers or directly characterized by nucleic acid sequencing. Suitable markers include a phenotypic character, a metabolic profile, a genetic marker, or some other type of marker. A genotype can constitute an allele for at least one genetic marker locus or a haplotype for at least one haplotype window. In some embodiments, a genotype can represent a single locus, and in others it can represent a genome-wide set of loci. In some embodiments, the genotype can reflect the sequence of a portion of a chromosome, an entire chromosome, a portion of the genome, and the entire genome.
As used herein, a “construct” or “gene construct” can refer to a polynucleotide which codes for the particular gene of the gene construct. Such polynucleotides can be operably linked to one or more untranslated regions (UTRs), and/or one or more transcriptional initiation regulatory sequence/promoter regulatory region which is capable of directing the transcription of the polynucleotide in the intended host cell, such as tissues of a transformed plant, thereby regulating expression of a given gene. Expression of a given gene can be determined in terms of the amount of gene product or protein expressed, and a variety of methods can be used for detecting protein expression levels, including, for example, enzyme linked immunosorbent assays (ELISA), Western blots, immunoprecipitations, and immunofluorescence, and the like.
The construction of such gene constructs which can be employed in conjunction with the present invention is well known to those of skill in the art in light of the present disclosure. See, e.g., Sambrook et al.; Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, N.Y. (1989); Gelvin et al.; Plant Molecular Biology Manual (1990). Plant Biotechnology: Commercial Prospects and Problems, eds. Prakash et al.; Oxford & IBH Publishing Co.; New Delhi, India; (1993); and Heslot et al.; Molecular Biology and Genetic Engineering of Yeasts; CRC Press, Inc., USA; (1992). For example, plant expression vectors may include (1) a cloned plant gene under the transcriptional control of 5′ and 3′ regulatory sequences and (2) a dominant selectable marker. Such plant gene constructs may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal. Constitutive, tissue-preferred, or inducible promoters can be employed. For example, certain promoters are known to those in the art to be capable of initiating transcription preferentially in certain tissues, such as leaves, roots, fruit, seeds, or flowers.
As used herein, a “vector” can refer to any nucleic acid construct which is able to enter a plant cell, including circular or linear nucleic acids, and/or bacterial, viral fungal, plant and synthesized nucleic acids, as well as homologous or heterologous nucleic acid constructs.
As used herein, the terms “transform”, “transformed”, and “transforming” can refer to the introduction of a foreign gene into a plant. Numerous methods for introducing foreign genes into plants are known and can be used to insert nucleic acid sequences into a plant host, including biological and physical plant transformation protocols. See, for example, Miki et al. (1993) “Procedure for Introducing Foreign DNA into Plants,” in Methods in Plant Molecular Biology and Biotechnology, ed. Glick and Thompson (CRC Press, Inc., Boca Raton), pages 67-88. The methods chosen can vary with the host plant, and many such methods are known to those in the art; these include chemical transfection methods such as calcium phosphate, microorganism-mediated gene transfer such as Agrobacterium (Horsch et al. (1985) Science 227:1229-1231), electroporation, micro-injection, and biolistic bombardment. Expression cassettes and vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are known and available. See, for example, Gruber et al. (1993) “Vectors for Plant Transformation,” in Methods in Plant Molecular Biology and Biotechnology, ed. Glick and Thompson (CRC Press, Inc., Boca Raton), pages 89-119.
Once a single transformed plant has been obtained, e.g., a plant transformed with a desired gene, conventional plant breeding methods can be used to transfer the structural gene and associated regulatory sequences via crossing and backcrossing. In general, such plant breeding techniques are used to transfer a desired gene into a specific plant, e.g., a crop plant or another type of plant used for commercial purposes. Accordingly, the methods of the claimed invention can be used in, for example, plant breeding, plant improvement, propagation of unstable and/or recessive genotypes, seed production, and trait propagation, as well as other purposes involving the reproduction of plants.
As used herein, the terms “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are often said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
Those in the art recognize that the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
The percentage of sequence identity/similarity is an integer selected from the group consisting of from 50 to 99. Exemplary sequence identity/similarity values include 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%. Sequence identity can be determined using, for example, the GAP, CLUSTALW, or BLAST algorithms, preferably BLAST. Substantial identity of nucleotide or amino acid sequences for these purposes normally means sequence identity of at least 60%, 70%, 80%, 90%, and 95% or higher.
Moreover, one of skill in the art will recognize that the sequence identity values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Polypeptides that are “substantially similar” share sequences as noted above except that residue positions, which are not identical, can differ by conservative amino acid changes. Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5° C. to about 20° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent wash conditions are those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 50, 55, or 60° C. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
For a description of various libraries, vectors, nucleic acids, etc., see, for example, Stratagene Cloning Systems, Catalogs 1999 (La Jolla, Calif.); and, Amersham Life Sciences, Inc, Catalog '99 (Arlington Heights, Ill.).
The specification and claims use the singular forms “a”, “an”, and “the”. These terms are intended to not exclude a plural interpretation, and may preferably include a plural interpretation, depending on the context. Thus, for example, reference to “a compound” may include a variety of such compounds, or several of those same compounds, unless the interpretation is contrary to the context in which it is used.
Apomixis is a naturally occurring developmental process of asexual reproduction wherein the offspring produced are genetically identical to the mother plant, thereby resulting in clonal propagation of the maternal plant through seed. Multiple forms of apomixis exist in nature. For example, gametophytic apomixis is characterized by apomeiosis, which is the formation of unreduced embryo sacs derived from nucellar cells of the ovary, as well as by parthenogenesis, which is the development of the unreduced egg into an embryo. The ability to accomplish apomixis via genetic engineering or introgression can have a major economic impact for agricultural crops, as breeding programs would have the ability to transmit apomixis through the paternal gametes, then subsequently fix hybrid genotypes/vigor through clonal propagation of the maternal seed.
Apospory is one form of apomixis, which is prevalent in the grass family. This gametophytic form of apomixis is characterized by the failure of meiosis (apomeiosis) or by the degeneration of meiotic products and the development of embryo sacs from chromosomally unreduced nucellar cells. In these embryo sacs, the unreduced egg develops parthenogenetically. Therefore, apomeiosis and parthenogenesis are two fundamental components of apomixis. The results described herein demonstrate the molecular basis for apomixis in a plant species where the reproductive process has naturally evolved. This discovery can be utilized to facilitate the propagation of superior gene combinations in crop plants. A gene that induces the apomixis trait has been highly sought after for decades. Such a gene can be of great value in plant breeding.
The present work describes apomictic species in the genera Pennisetum and Cenchrus; both genera are in the grass family, which is the single most important family of cultivated plants. One of the members of this family is pearl millet (Pennisetum glaucum syn. Cenchrus americanus), which is a sexual diploid grain crop with significant production in the semi-arid tropics. The genus Pennisetum also has many apomictic species, most of which have been crossed with pearl millet in an attempt to introduce gene(s) for apomixis; however, male sterility during backcrossing has heretofore limited their use for conventional transfer of the apomixis trait (Dujardin M., Hanna W., J Hered. 74:277-279 (1983a); Dujardin M., Hanna W., Crop Sci. 23:156-160 (1983b); Dujardin M., Hanna W., Theor. Appl. Genet. 69:97-100 (1984); Dujardin M., Hanna W., Crop Sci. 25:59-62 (1985)). Some progress with conventional transfer has been achieved via the use of apomictic P. squamulatum (2n=8x=56) (Akiyama Y. et al., J Hered. 97:521-524 (2006)) as the male parent in crosses with artificially induced tetraploid pearl millet (Dujardin M., Hanna W., J Genet. Breed. 43:145-151 (1989)).
Apospory is the type of apomictic reproduction in Pennisetum/Cenchrus (Ozias-Akins P. et al., Funct. Integr. Genomics 3:94-104 (2003)), i.e., one or more somatic cells of the nucellus begins to enlarge, and the nucleus of each aposporous initial undergoes two successive mitotic divisions to produce a four-nucleate embryo sac. The four nuclei comprise the egg, two synergids, and one polar nucleus, or, alternatively, the egg, one synergid, and two polar nuclei. The uni- or bi-nucleate central cell must be fertilized in pseudogamous species in order for endosperm to develop (Kojima A., Nagato Y., Sex. Plant Reprod. 5:79-85 (1992); Naumova T. et al., Acta Botanica Neerlandica 42:299-312 (1993); Nogler G., Gametophytic apomixis, in: B. M. Johri (Ed.), Embryology of Angisoperms, Springer, Berlin. pp. 475-518. (1984)). Thus, there is selection pressure for normal microsporogenesis resulting in viable pollen even though female meiosis can be irregular. The unreduced egg cell begins parthenogenetic development (Vielle J. et al., Link. Plant J 8:309-316 (1995)) either slightly before or soon after single fertilization of the central cell in apomictic Cenchrus ciliaris.
The reproductive phenotype of species and hybrids in a genus usually can be determined unambiguously based on screening of a relatively small number of cleared pistils (which have been made optically transparent through chemical treatment). Phenotype in the genus Pennisetum can also be established via a dominant, red marker gene (Hanna W., Burton G., J Heredity 83:386-388 (1992)) that, when present in the pollen parent of a testcross, will allow the classification of red progeny as having arisen through sexual reproduction.
The Pennisetum squamulatum origin of apomixes has been previously described (U.S. Pat. No. 5,811,636). In addition, a nucleic acid marker has been used to identify a clone containing the gene of interest (U.S. Pat. No. 6,028,185), and some apospory genes have been previously characterized (Huo H. “Genetic analysis of the apospory-specific genomic region (ASGR) in Pennisetum squamulatum: from mapping to candidate gene” (Doctoral dissertation) (2008). Retrieved from http <colon slash slash> dbs <dot> galib <dot> uga <dot> edu <slash> cgi-bin <slash> getd <dot> cgi?userid=galileo&servemo=9&instcode=ugal; Zeng Y. “Identification and characterization of apospory candidate genes in Pennisetum and Cenchrus” (Doctoral dissertation) (2008). Retrieved from http <colon slash slash> dbs <dot> galib <dot> uga <dot> edu <slash> cgi-bin <slash> getd <dot> cgi?userid=galileo&servemo=9&instcode=ugal)). Apomixis is transmitted by a physically large, non-recombining chromosomal region in P. squamulatum; this region is known as the apospory-specific genomic region (ASGR) (P. Ozias-Akins, D. Roche, W. W. Hanna, Proc Natl Acad Sci USA 95:5127-5132 (1998)), and the PsASGR-BabyBoom-Like (PsASGR-BBML) genes reside within this region (G. Gualtieri et al., Plant Physiology 140:963-971 (2006)).
Apomixis in Pennisetum has been mapped molecularly via RAPD, RFLP, AFLP, and SCAR markers conducted in backcross populations as well as through a pseudo-testcross between the heterozygous apomict P. squamulatum (as male parent) and sexual pearl millet (Dujardin M., Hanna W., J Genet. Breed. 43:145-151 (1989); Goel S. et al., Genetics 163:1069-1082 (2003); Ozias-Akins P. et al., Proc. Natl. Acad. Sci. USA 95:5127-5132 (1998); Ozias-Akins P. et al., Theor. Appl. Genet. 85:632-638 (1993)). It was determined that a single linkage group from the apomictic parent was necessary and sufficient for the transmission of apomixis, and a single, intact chromosome has been transmitted to the BC8 generation where it has been found to reside in a tetraploid pearl millet background (Singh et al., Crop Sci. 50:892-902 (2011)). Mapping in a second species, namely C. ciliaris, has yielded similar results (Jessup R. et al., Crop Sci. 42:1688-1694 (2002); Roche D. et al., Plant J 19:203-208 (1999)). In both species, high-resolution mapping is prevented by a non-recombining chromosomal block, although the size of the ASGR has been reduced by recombination to ˜¼ of a chromosome or ˜50 Mb. A recombinant in buffelgrass was recently recovered that demonstrates separation of apomeiosis and parthenogenesis (Conner et al., Planta 238:51-63 (2013)). This recombinant retained the portion of the ASGR required for aposporous embryo sac formation; however, the portion of the ASGR necessary for parthenogenesis was lost. Comparison of the ASGR between buffelgrass and P. squamulatum shows conservation both on the macrosyntenic and microsyntenic level (Goel S. et al., Genetics 173:389-400 (2006); Gualtieri G. et al., Plant Physiol 140 963-971 (2006))
DNA sequence analysis of bacterial artificial chromosome clones (Roche D. et al., Theor. Appl. Genet. 104:804-812 (2002)) mapped to the ASGR has been conducted to search for synteny between rice and the two apomictic species under study. While small regions of microsynteny were identified whereas macrosynteny was not established, 25 C. ciliaris and 23 P. squamulatum ASGR genes were identified (Conner J. et al., Plant Physiol. 147:1396-1411 (2008)), with the sequencing data showed that overall the ASGR is a gene-poor and transposable element rich region. The identification of a highly abundant long terminal repeat (LTR) sequence within the ASGR sequencing data has allowed for the development of sequence-specific amplified polymorphism (SSAP) markers, which have been used to efficiently target the ASGR (Huo H. et al., Theor. Appl. Genet. 119:199-212 (2009)).
As described herein, in Pennisetum squamulatum (L.) R.Br., apomixes has been found to segregate as a single dominant locus, namely the ASGR, which contains multiple copies of the PsASGR-BBML gene. The present study investigated the function of PsASGR-BBML in sexual tetraploid pearl millet and apomictic F1 plants. PsASGRBBML was found to be expressed in egg-cells prior to fertilization, and expression of PsASGR-BBML in sexual pearl millet was found to induce parthenogenesis and the production of haploid offspring. Reduced PsASGR-BBML expression in apomictic F1 transgenic plants inheriting a PsASGR-BBML silencing construct was found to correspond with fewer parthenogenetic embryos and cell number in parthenogenetic embryos. These data demonstrate the key role of the PsASGR-BBML gene in parthenogenesis in the apomictic pathway.
The results described herein describe a gene found in tightly-linked ASGR bacterial artificial chromosome (BAC) vectors of both apomictic species, but lost in the recombinant C. ciliaris plant lacking parthenogenesis; this gene has high protein similarity to the BABY BOOM (BBM) gene of Brassica napus. BBM originally was coined as the gene name for a cDNA transcript that was induced in microspore cultures of Brassica napus (BnBBM) undergoing somatic embryogenesis (Boutilier K. et al., Plant Cell 14:1737-1749 (2002)). BnBBMJ and BnBBM2 predicted proteins are 98% similar to one another and are also 85% similar to their Arabidopsis ortholog.
These three proteins have the key feature of an AP2-domain region. AP2 domains, named for an amino acid repeat identified in the APETALA2 gene of Arabidopsis (Jofuku K. et al., Plant Cell 6:1211-1225 (1994)), a flower development gene that also was shown to be involved in regulation of other floral homeotic genes, are 60-70 amino acid DNA-binding domains (Pfam-PF00847). These AP2 domains are characteristic of a large family of transcription factor proteins that cluster due to their similarity in this conserved region, which is highly conserved among not only BBM but other related developmental genes in this class.
The APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) DNA-binding domain family has been identified in a wide group of plants, such as mosses, algae, gymnosperms, and angiosperms. The AP2 gene family is divided into two groupings, namely the ERP-like (ethylene response element binding factor) and AP2-like groups (Weigel D. Plant Cell 7:388-389 (1995); Ohme-Takagi M. and Shinshi H. Plant Cell 7:173-182 (1995); Okamuro J. et al., Proceedings of the National Academy of Sciences 94:7076-7081 (1997)). The ERP-like group is characterized as having only a single AP2 domain, in this group, functions tend to be related to stress responses (biotic and abiotic) (Riechmann J and Meyerowitz E., Biol. Chem. 379:633-646 (1998)). The AP2-like genes contain two AP2 domains (repeat 1 and 2) that have similarity to one another and a short interdomain linker region; this group can be further divided into two groups, namely eu-AP2 (which includes APETELA2) and ANT (for AINTEGUMENTA) lineages. The ANT lineage can itself be divided between basalANT and euANT lineages, which contain specific motifs euANT1 thru 4 (S. Kim, P. S. Soltis, K. Wall, D. E. Soltis, Mal. Biol. Evol. 23:107-120 (2006)), which contain the PLETHORA-like, AINTEGUMENTA-like, AINTEGUMENTA-like1, AINTEGUMENTA-like5 and BBM-like subgroups. Proteins within these subgroups have critical roles in meristem maintenance, cell proliferation, organ initiation and growth, embryogenesis, and root formation (A. Horstman, V. Willemsen, K Boutilier, R. Heidstra, Trends in Plant Science 19:146-157 (2014)). The ANT lineage is required for integument initiation and female gametophyte development in addition to early growth of other primordia, with the exception of roots (Elliott R. et al., Plant Cell 8:155-168 (1996); Klucher K. et al., The Plant Cell 8:137-153 (1996)). ANT, AtBBM, and BnBBM genes fall within the ANT Glade (Kim S. et al., Mal. Biol. Evol. 23:107-120 (2006)).
A BBM-like gene from Zea mays called ODP2 has previously been described (International Patent No. WO2005075655; U.S. Pat. No. 8,420,893). However, the apomixis-specific BBM differs substantially from the maize BBM genes outside of the AP2-domain. The BBM, BBM-like, and ASGR-BBM-like proteins share a conserved bbm-1 domain that has not been identified in other members of the euANT lineage. Deletion of the bbm-1 domain has eliminated the ability of transgenic plants to induce somatic embryogenesis on cotyledons (S. El Ouakfaoui et al., Plant Mal Biol 74:313-326 (2010)). A published phylogenetic study of the BBM-like proteins (S. El Ouakfaoui et al., Plant Mal Biol 74:313-326 (2010)) was extended to include BBM-like proteins from newly sequenced monocots. A distinct clade of proteins from Oryza sativa, including the ASGRBBMs, BBM1 (0511g19060), and proteins from Setaria italica and Panicum virgatum were found to be formed in the majority of phylogenic trees constructed (
While BBM genes have been expressed from sexual species in ovules prior to fertilization, neither embryo development nor an apomixis phenotype has been observed to date. The expression of BnBBM has been observed in microspores 3-4 days post-induction (at the time they were destined to become embryogenic), persisting throughout the time frame tested (28 days post-induction) (Boutilier K. et al., Plant Cell 14:1737-1749 (2002); Malik M. et al., Plant Physiology 144:134-154 (2007)). BnBBM expression, as determined by RT-PCR, has also been observed in 3-day-old seeds/globular embryos, and expression was found to persist throughout embryo development (Malik M. et al., Plant Physiology 144:134-154 (2007)). BnBBM and BnLECJ (LEAFY COTYLEDON 1), whose expression primarily occurs during microspore and zygotic embryogenesis, are considered to be markers for embryogenesis. BBM was also found to be detectable in Arabidopsis ovules in free-nuclear endosperm, as established by in situ hybridization (Boutilier K. et al., Plant Cell 14:1737-1749 (2002)). While expression was found to decline in endosperm once cellularization was initiated, to date there has been no published evidence for expression of BBM in egg or zygote cells within ovules. BnBBM was originally considered to provide a route to induction of adventitious embryony in seeds, hence maternally derived embryos as a form of apomixis (U.S. Pat. No. 7,151,170). In adventitious embryony, no alteration in embryo sac development occurs; rather, somatic cells of the ovule, usually nucellus, directly divide to form embryos. Adventitious embryony is therefore sporophytic apomixis.
While amino acid similarity between the AP2 regions of ASGR-BBML and BnBBM is high (96%), the similarity declines significantly outside of this region (35% similarity upstream and 27% similarity downstream). Three highly conserved genomic duplications of PsASGR-BBML have been identified from ASGR-linked BACs p203, p207 (J. A. Conner et al., Plant Physiol. 147:1396-411 (2008)), and p208 to date. The p208 PsASGR-BBML sequence is identical to the p207 PsASGR-BBML2 sequence (EU559277.1), with the exception of the number of AT repeats (11 vs. 17) found in intron 1. The conservation of gene sequence means that it is not clear which of the PsASGR-BBML genomic regions are transcribed. The PsASGR-BBML transcript encodes a 545 amino acid protein derived from the splicing of 8 exons, a 73 bp 5′ UTR, and multiple 3′ UTRs, with lengths ranging from 30 to 258 bp. The PsASGR-BBML gene contains two AP2 DNA-binding domains and is therefore predicted to function as a transcription factor (
Due to the protein similarity of the ASGR-BBM-LIKE (ASGR-BBML) gene to BBM, it was evaluated as to its use as an “apomixis” gene. As described herein, the ASGR-BBML gene was identified, and its role in parthenogenesis was characterized. ASGR-BBML was found to have an important role as a key gene in the induction of parthenogenesis in the apomictic pathway.
These results demonstrate that ASGR-BBML can be used to achieve in parthenogenesis in plants that do not normally show this phenotype. The seed set of the transformed lines was low, and plant offsprings can be screened for ploidy level. Parthenogenetic transgenic lines can thereby produce offspring that are reduced to the diploid level (or haploid within the context of the cell cycle). As haploid induction followed by chromosome doubling is a breeding practice followed in many cereal crops to more rapidly obtain homozygous lines, these findings can be used for haploid induction in sexual plants or clonal reproduction through an apomictic pathway dependent on chromosomal non-reduction in egg cells.
The gene construct used in the invention can include a sequence from the phylogenetic clades provided in
In some embodiments, the gene construct can include a gene selected from Seita.8G107100 (SiBBM1), Sieta.5G415800 (SiBBM2) and Seita.1G232200 (SiBBM3). In some embodiments, the gene construct can include a sequence that has 50, 55, 60, 65, 70, 75, 80 or more percent similarity to Seita.8G107100.1.p (SiBBM1), Sieta.5G415800.1.2 (SiBBM2) and Seita.1G232200.1.p (SiBBM3).
Having described the invention in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the invention defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.
The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
DNA Extraction
DNA extraction was accomplished usmg a cetyltrimethyl ammomum bromide (CTAB) protocol (J. Conner, G. Gunawan, P. Ozias-Akins, Planta 238:51-63 (2013)).
PCR Amplification
Different PCR conditions were required based on primer combinations and amplicon length. Primer and PCR information for all unpublished amplicons appears in Table 1. PCR reactions were performed according to manufacturer recommendations unless noted otherwise. Reactions were performed in a GeneAmp 9700 Thermocycler (Applied Biosystems, Foster City, Calif., USA).
Non-Quantitative RT-PCR Tissue Expression of ASGR-BBML
Expression of ASGR-BBML was confirmed by RT-PCR using RNA isolated from various tissues, including the following: ovaries isolated one day prior to anthesis, through seed development, and up to five days after anthesis; anthers at 1 day prior to anthesis; root tips collected from greenhouse-grown potted plants; newly emerging leaf tissue; and embryogenic callus derived from apomictic Pennisetum BC8 lines (M. Singh et al., Crop Science 50:892-902 (2010)). Total RNA was extracted from the various tissues via the RNeasy plant mini kit (QIAGEN, Valencia, Calif., USA) and subjected to DNAse treatment (Invitrogen, Carlsbad, Calif., USA). Three to five micrograms of total RNA was used for first-strand cDNA synthesis via oligo-dT and SuperScriptIII (Invitrogen). Two μL of the first-strand cDNA synthesis reaction was used for PCR amplification using ASGR-BBML specific primers p779/80 (Y. Akiyama et al., BMC Evolutionary Biology, 11:289 (2011)) or actin depolymerizing factor 3 (ADF3) primers p1127/1128 which were used as a control. PCR products were run on a 1.25% agarose gel, stained with ethidium bromide, and imaged with the Molecular Imager Gel Doc XR System (Bio-Rad Laboratories, Hercules, Calif., USA).
Rapid Amplification of cDNA Ends (RACE) of PsASGR-BBML
RACE products for the 5′ and 3′ UTRs for PsASGR-BBML were generated via the GeneRacer RLM-RACE kit (Invitrogen) and total RNA extracted from spikelet tissues from apomictic BC7 Line 58 (M. Singh et al., Crop Science 50:892-902 (2010)). The PCR primers used are listed in Table 1. The amplified PCR products were gel purified with QIAquick gel extraction kit (QIAGEN) and cloned into either a PCR4-TOPO (Invitrogen) or a pGEM-T easy (Promega, Madison, Wis., USA) vector. Nucleotide sequencing was carried out with a CEQ 8000 Genetic Analysis System (Beckman Coulter, Fullerton, Calif., USA), and sequences were processed with Vector NTI (Invitrogen).
Sequence Analysis of PsASGR-BBML Trans Gene
Total RNA was extracted from ovaries at the day of anthesis for g3f offspring, namely 105, 123, 144, and 159. Three μg of RNA was subjected to DNAse treatment (Invitrogen) and used for first-strand cDNA synthesis using oligo-dT and SuperScriptIII (Invitrogen). Two μL of the first-strand cDNA synthesis reaction was used for PCR amplification via transgene specific primers p1792/p1801 and p2347/p423 Amplified PCR products were gel purified with QIAquick gel extraction kit (QIAGEN) and cloned into PCR4-TOPO (Invitrogen). Nucleotide sequencing was conducted at the Laboratory for Genomics and Bioinformatics (University of Georgia, Athens, Ga.). Vector and bad quality sequences were removed, and trimmed sequences were then assembled using Geneious Pro 5.6.6 (Biomatters Limited, Auckland, New Zealand).
Transforming Constructs
PsASGR-BBMpromoter-GUS construct. pCambia3301 was digested with BamHI/NcoI to remove the CaMV 35S promoter, which was replaced with a 2,074 bp PCR-generated BamHI/NcoI PsASGR-BBMLpromoter fragment, which was amplified from BAC p208 (D. Roche et al. Theor Appl Genet., 104:804-812 (2002)) using primers p690 and p692. The BamHI site is endogenous to the promoter and is located just downstream of p690. A Neal site was incorporated into p692. The PsASGR-BBMLpromoter-GUS-Nos polyA cassette in pCambia3301 was removed by digestion with BamHI/BseYI, blunted, and then placed into pACH20 (A. H. Christensen, P. H. Quail, Transgenic Research, 5:213-218 (1996)) at a blunted and dephosphorylated HindIII site to create plasmid 2BE.
gPsASGR-BBML construct. The pBluescript vector was engineered with 2,074 bp of the PsASGRBBMLpromoter, 3540 bp of the 8 exon, 7 intron PsASGR-BBM coding region from BAC p208 (D. Roche et al. Theor Appl Genet., 104:804-812 (2002)), and 609 bp 3′ of the stop codon, including the predicted poly(A) signal.
RNAi-BBM-3p construct. The binary expression vector pMCG161 (http<colon slash slash> www <dot> chromdb <dot> org) was used for RNAi vector construction. A 425 bp amplicon covering 52% of the last exon of PsASGRBBML and incorporating restriction enzymes for cloning into pMCG161 was generated via primers p966 and p967 and inserted into pMCG161 following instructions available at http <colon slash slash> www<dot> chromdb <dot> org.
Ubi-Hygro construct. pCB13 (H. Yang et al., Plant Cell Reports, 17:693-699 (1998)) was digested with HindIII/BamHI to remove the CaMV 35S promoter and replaced with the HindIII/BamHI maize ubiquitin (Ubi-1) promoter fragment from pAHC20 (A. H. Christensen, P. H. Quail, Transgenic Research, 5:213-218 (1996)).
Plant Transformation
Embryogenic callus generated from 7-10 day old immature embryos of tetraploid sexual IA4X plants were bombarded, selected (25 mg/l Hygromycin B or 15 mg/l PPT) and regenerated according to a previous protocol (J. J. Goldman, W. W. Hanna, G. Fleming, P. Ozias-Akins, Plant Cell Rep., 21:999-1009 (2003)). The PsASGR-BBMpromoter-GUS lines were bombarded with a mix of plasmids 2BE and p524EGFP.1 (G. H. Fleming, O. Olivares-Fuster, S. Del-Bosco, J. W. Grosser, In Vitro Cell Dev Biol Plant 36:450-455 (2000)), the gPsASGR-BBML lines were bombarded with a mix of plasmids gPsASGR-BBML, p524EGFP.1 and Ubi-Hygro, and the RNAi lines were bombarded with plasmid RNAi-BBM-3p.
Embryo Rescue
Media for embryo rescue were based on previous work for zygotic Pennisetum embryos at 5 DAPS (C. Nitsch et al., “Production of haploid plants of Zea mays and Pennisetum through androgenesis.” Variability in plants regenerated from tissue culture. Praeger, N.Y. (1982): 69-91) and used IX Nitsch & Nitsch Basal Medium w/vitamins (PhytoTechnology Laboratories, Shawnee Mission, Kans.), 1% sucrose, gibberellic acid (1 mg/L), indoleacetic acid (0.03 mg/L), 0.75% agar, pH 5.8 with 0.2% Plant Preservative Mixture (PPM) (Plant Cell Technology, Inc., Washington, D.C.). Developing (10 to 15 days after pollination) and mature seed were sterilized according to a previous protocol (J. J. Goldman, W. W. Hanna, G. Fleming, P. Ozias-Akins, Plant Cell Rep., 21:999-1009 (2003)). Embryos were surgically removed, placed scutellum side up on the media, and monitored for root and shoot elongation. Embryos were left on the GA/IAA media for up to 10 days and were then discarded if no root/shoot growth occurred. Offspring with root and shoot growth were moved to a IX Murashige & Skoog (MS) medium w/vitamins (PhytoTechnology Laboratories) supplemented with 3% sucrose, 0.75% agar pH 5.8 with PPM until growth permitted hardening and movement into the greenhouse.
X-Gluc Staining for B-Glucuronidase Activity for PsASGR BBMpromoter-GUS Lines
Eight independent PsASGR_BBMpromoter-GUS lines were produced which contained a full-length transgene based on overlapping PCR amplifications using transgene-specific primers, namely p2354/p2355; p2349/p2350; and p2885/p2886 (Table 1). The PsASGR_BBMpromoter-GUS To lines were either allowed to self-pollinate or were cross-pollinated with apomictic BC8-Line 63 pollen. Seeds from 5 lines were germinated, and then genotyped for inheritance of the ASGR if crossed and the PsASGR_BBMpromoter-GUS transgene. Initially, PsASGR_BBMpromoter-GUS gene expression patterns were examined from ovaries dissected one day prior to anthesis or on the day of anthesis from heads bagged prior to stigma exsertion; stigmas were mechanically removed prior to anthesis.
New PsASGR_BBMpromoter-GUS plants were germinated and isolated to a single greenhouse in order to further control for unintended pollination. All heads were continually cut back prior to pollen shed in order to keep the greenhouse free of millet pollen. Florets were collected one day prior to anthesis, with the anthers being manually removed. The emasculated florets were then placed on IX MS media, 0.75% agar pH 5.8 with 0.2% PPM and incubated in a growth chamber under long-day light condition at 27° C. for 24 hours. Ovaries were the dissected and incubated in an X-Gluc reaction solution (100 mM sodium phosphate/pH 7.0, 10 mM EDTA, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 0.1% Triton X-100, and 1 mM 5-bromo-4-chloro-3-D-glucuronide) at 37° C. for 48 hours. X-Gluc-stained ovaries were dehydrated in 15, 30, and 50% ethanol at room temperature for 1 hour each and were then fixed in FAA (50% ethanol, 3.7% formaldehyde, and 5% acetic acid) overnight. FAA-fixed ovaries were further dehydrated in 70, 85, 90, 100, and 100% ethanol at room temperature for 1 hour each., then the dehydrated ovaries were cleared by a series of ethanol:methyl salicylate solutions (3:1, 1:1, and 1:3 for 1 hour each). Cleared ovaries were stored in 100% methyl salicylate for 1 hour. Phase-contrast images of the ovaries acquired using an Axioskop 2 Plus microscope, AxioCam camera and Axio Vision software 4.8 (Zeiss, Thornwood, N.Y.).
Genotyping of gPsASGR BBMLI Ubi Hvgo Transgenic Lines
Nine independent lines were recovered which contained both the gPsASGR_BBML and Ubi_Hygo constructs, based on results from PCR amplification using p1800/01 and p2971298, respectively.
Flow Cytometry
Samples were processed to run on a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, Calif. USA) or a Partee Ploidy analyzer (Partee, Munster, Germany) The process involved chopping young leaf tissue of sorghum (control) and samples (unknown) together in 1000 μL of Tris-MgCl2 nuclei extraction buffer (0.2 M Tris-HCl, pH 7.5, 4 mM MgCl2×6H20, 0.5% Triton X-100) and passing the mixture through a 30 μm CellTries disposable filter (Partee). 500 μL of RNAse/propidium iodide solution (BD Biosciences) was added to the filtered samples, which were then incubated on ice for 15 minutes. For the BD Accuri C6 flow cytometric analysis, gating was set by the selection of objects that exhibited a strong correlation between the FL2 and FL3 signals using a flow rate of 14 μL sample per minute. At least 3000 events were collected for each sample within the gated region. Samples were run on the Partee Ploidy analyzer until peaks could be confidently called. Ploidy level was determined based on the G1 and G2 sample peaks relative to the sorghum control G1 and G2 peaks.
Root Tip Chromosomal Counts
If available, six healthy root tips were harvested from g3f offspring, cleaned of soil, placed in Eppendorf tubes with 300 μl dH20, treated with nitrous oxide for 2 to 2½ hours under 150 psi, and finally fixed in fresh ethanol:acetic acid (3:1) for a minimum of 2 days. The meristematic region of the root was incubated in 0.3% cellulase RS (Karlan Research, Torrance, Calif.), 0.3% pectolyase Y23 (Karlan Research), and 0.3% cytohelicase (Sigma-Aldrich, St. Louis) in 30 mM citrate buffer, at a pH of 4.5 at 37 C for 60-90 minutes. After enzymatic digestion, a minimum of 3 root tips from each plant was spread individually on glass slides. Chromosomal spreads were located, digitally photographed, and chromosome number was then counted using Adobe Photoshop CS6. Ploidy level of each plant was determined using a minimum of 4 spreads originating from at least 2 slides.
RNAi Screening
Sixteen independent sexual tetraploid IA4X lines were recovered which contained the RNAi_BBM_3p construct based on PCR amplification for the BAR gene and the RNAi sense and antisense inserts using the following primers: p992/p993, p1222/p1223 and p1224/p1125, respectively. Eight lines were crossed with P. squamulatum pollen, thereby generating F1 offspring. The F1 offspring plants from each line were screened with P. squamulatum-specific primers p1032/1035, RNAi_BBM_3p primers p992/p993, p1222/p1223 and p1224/p1125, and ASGR-specific primers Ugt197 (P. Ozias-Akins, D. Roche, W. W. Hanna, Proc Natl Acad Sci USA 95:5127-5132 (1998)). ASGRpositive/RNAi-positive and ASGR-negative/RNAi-positive plants were subsequently tested for expression of the RNAi transgene using RT-PCR analysis of total RNA from leaf tissue and primers p1226/p1227 derived from the octopine synthase terminator region of the transgene. Twenty-five plants from 5 lines were chosen for further analysis; these included 14 ASGR-positive/RNAi-positive, 5 ASGRpositive/RNAi-negative and 6 ASGR-negative/RNAi-positive plants.
Semi-Quantitative Analysis of PsASGR-BBML Expression
Semi-quantitative RT-PCR quantification of PsASGR-BBML was based on a previous protocol (E. Albertini et al., Plant Molecular Biology 56:879-894 (2004)). ADF3 primers p1127/1128 were used as an internal control, along with PsASGR-BBML specific primers, namely p779/p780 (Y. Akiyama et al., BMC Evolutionary Biology, 11:289 (2011)). Each assay used 3.3 μg of total RNA extracted from ovaries at the day of anthesis via the RNeasy plant mini kit (QIAGEN), and the samples were subjected to DNAse treatment (Invitrogen, Carlsbad, Calif., USA) for first-strand cDNA synthesis using oligodT and SuperScriptIII (Invitrogen). Two μl of the cDNA synthesis reaction were used in all PCR reactions; all reactions were run in triplicate. The PCR cycle number determined empirically for ADF3 and PsASGR-BBML was found to be 26 and 40 cycles, respectively. Hybridization imaging was conducted with a Storm phosphorimager (Amersham Biosciences, Pittsburgh, Pa., USA), and bands were quantified using ImageQuant v5.0 following the manufacturers instructions (Amersham Biosciences).
Histological Analysis of Embryo Development in RNAi Lines
Heads were bagged prior to stigma exertion, and stigmas were removed prior to anthesis to prevent pollination. Spikelets from heads were collected 2 days after anthesis, fixed in FAA, and then subjected to a 30-min vacuum treatment at 15 mm Hg prior to a 24-hour fixation at room temperature. Dehydration was initiated with TBA1 (40% ethanol, 10% tertiary butyl alcohol (TBA), 50% distilled water) for 2 hours, then transferred through TBA2 (50% ethanol, 20% TBA, 30% distilled water) for 8 hours, TBA3 (50% ethanol, 35% TBA, 15% distilled water) for 1 hr, TBA 4 (45% ethanol, 55% TBA) for 1 hour, TBA 5 (25% ethanol, 75% TBA) for 1 hour, and TBA 6 (100% TBA) for 1 hour. Ovaries were then transferred to fresh 100% TBA for another 8 hours prior to being transferred to TBA: paraffin oil (Fisher, Pittsburgh, Pa., USA) (1:1) at 58° C. overnight. Ovaries were taken through three changes of pure Paraplast X-tra (Fisher, Pittsburgh, Pa.) for 48 hours each before embedding. Sectioning was then carried out for all samples at 9 μm. Samples were stained with safranin 0/fast green using a modification to a previously described protocol (Jensen 1962) wherein embedded and sectioned samples were de-waxed in ethanol, then coated with 0.05% nitrocellulose (diluted from collodion, Fisher, Pittsburgh, Pa.) in ether-alcohol (50% diethyl ether, 50% ethanol) for 30 seconds. Rehydration of samples was accomplished by transfer through 70% ethanol, 30% ethanol, and distilled water for 5 minutes each. Staining was performed first in Safranin 0 solution (4 g Safranin 0, 100 ml distilled water, 100 ml 95% ethanol, 4 g sodium acetate) for 5 minutes. Samples then were subjected to dehydration in the following series of solutions for 5 minutes each: two changes of distilled water, 50% ethanol, 95% ethanol, 100% ethanol. Samples subsequently were stained in fast green solution (1 g fast green, 100 ml 100% ethanol, 100 ml cellosolve and 100 ml clove oil) for 4 seconds, then immediately placed into pure clove oil for 10 seconds, prior to being quickly transferred into clearing mix (50% clove oil, 25% ethanol, 25% histoclear). Slides were finally cleared in histoclear for 5 minutes and were then mounted with permount.
Phylogenetic Analysis
The proteins identified in the BBM-like clade (S. El Ouakfaoui et al., Plant Mal Biol 74:313-326 (2010)) were downloaded from NCBI. Each member of the Grass clade in Phytozome was then individually searched using BLASTP and the entire PsASGR-BBML protein. All proteins with similarity to PsASGR-BBML extending past the AP2 domains were downloaded, while truncated proteins and those without a bbm-1 domain (S. El Ouakfaoui et al., Plant Mal Biol 74:313-326 (2010)) were removed. The phytozome target databases employed were the Sorghum bicolor v1.4 proteome; Zea mays proteome, Setaria italica proteome, Panicum virgatum v0.0 proteome, Oryza sativa proteome, and Brachypodium distachyon proteome. Proteins that were found to be identical between the BBM-like clade (S. El Ouakfaoui et al., Plant Mal Biol 74:313-326 (2010)) and Phytozome were removed. Twenty-six amino acid sequences were aligned using multiple web-based alignment programs; the results were that both T-Coffee (http <colon slash slash> tcoffee<dot> erg <dot> cat <slash> apps <slash> tcoffee <slash> do<dot> regular) and Mafft (http <colon slash slash> mafft <dot> cbrc <dot> jp<slash> alignment <slash> server) alignments identified the most conserved domains the BBM-like clade, especially within the C terminus of the proteins without editing. The T-Coffee alignment produced a 1,067 amino acid consensus sequence length with 283 conserved positions for all proteins (
A full-length ASGR-BBML cDNA from P. squamulatum was identified via 5′ and 3′ RACE. The cDNA was found to be overexpressed in Arabidopsis using the CaMV35S promoter, with pleiotropic effects of overexpression such as the formation of ectopic shoots, projections with trichomes, and incomplete flowers (
Transformation of sexual tetraploid pearl millet with the genomic copy of the ASGR-BBML gene construct, including a promoter and 3′UTR, was found to induce the formation of embryos in meiotically-derived embryo sacs in the absence of corresponding endosperm fertilization. Fertilization was prevented by bagging of heads prior to stigma exsertion and subsequent removal of stigma/style prior to anther exsertion, with evidence for lack of fertilization in ovules being the persistence of two polar nuclei in the central cell and absence of endosperm formation. Expression of ASGR-BBML was previously observed by RT-PCR in two apomictic species (P. squamulatum and C. ciliaris) and in apomictic Pennisetum backcross 7 and 8 lines (M. Singh et al., Crop Science 50:892-902 (2010)) in ovaries starting I-day prior to anthesis and continuing through early seed development, in anthers I-day prior to anthesis and roots.
PsASGR-BBML was found to be expressed in embryogenic callus derived from apomictic Pennisetum BC8 lines. In the apomictic species and the apomictic backcrosses, embryo development in aposporous embryo sacs can initiate prior to pollen shed, continuing for several days after pollen shed when pollination is prevented, and no endosperm is formed (
Unpollinated pistils were examined both for transcription of ASGR-BBML as well as for embryo development prior to the day of pollen shed and up to two days after pollen shed; pollination was prevented by removing the stigmas. Expression of the ASGR-BBML gene was demonstrated to correlate with parthenogenetic development of the unreduced egg in apomictic P. squamulatum and C. ciliaris; that is, expression was detected in the absence of pollination as ensured by removal of stigmas prior to anther dehiscence and pollen shed (DAP0 in
Tetraploid pearl millet was transformed with a portion of the ASGR-BBML coding region in an inverted repeat (IR) construct via microprojectile bombardment and selection on phosphinothricin. Transcription of the IR proceeded via the CaMV 35S promoter. Homology-dependent gene silencing depends on the formation of hairpin RNA and triggering of the RNA interference where endogenous gene sense transcripts are targeted for degradation (Ossowski S. et al., Plant Journal 53:674-690 (2008); Eamens A. et al., Plant Physiology 147:456-468 (2008)). According to Southern blot hybridization, the ASGR-BBML IR did not show significant homology with pearl millet.
RNAi lines were generated by transformation of tetraploid IA4X, but no RNAi phenotype was expected until the IR was combined with the ASGR by crossing. Crosses of IA4X RNAi lines with P. squamulatum produced progeny that combined both genes and were then screened for transcription of the transgene, changes in transcription of ASGR-BBML, and changes in reproductive development. Transcription of ASGR-BBML as determined by semi-quantitative RT-PCR varied in the progeny of several events, as follows: a reduction in ASGR-BBML signal (
Since no complete knockdown was obtained from either of these experiments, these data did not correlate ASGR-BBML expression with the extent of embryo development vs causality (ASGR-BBML expression was responsible for embryo induction); therefore, additional experiments were undertaken to test the role of ASGR-BBML in parthenogenesis. These consisted of the following steps: 1) transformation of sexual pearl millet with beta-glucuronidase (GUS) as a reporter gene, expressed under the control of the native BBM promoter, and 2) transformation of sexual pearl millet with ASGR-BBML, either as cDNA or as genomic DNA under control of its native promoter.
Although the organ-specific expression pattern of ASGR-BBML has been extensively characterized by RT-PCR in two apomictic species (P. squamulatum and C. ciliaris) and apomictic Pennisetum backcrosses, this does not enable the cellular location of expression to be determined. Expression in apomicts was observed in gynoecia (pistils) up to 2 days prior to pollination; expression was also observed after pollination as embryos began to develop and continued to grow. These analyses were conducted by RT-PCR because ASGR-BBML transcripts are of low abundance in gynoecia at anthesis; therefore, transcripts were not detected by in situ hybridization.
The pattern of PsASGR-BBML expression was investigated at the cellular level in ovaries prior to fertilization via a PsASGR-BBML promoter-GUS construct. In apomictic lines recovered from crosses with transgenic pearl millet, dividing unreduced egg cells expressing GUS were identified in aposporous embryo sacs (
This pattern of expression indicates that BBM is cell autonomous and expressed within a developmental time frame that is consistent a role in parthenogenesis. To obtain more definitive evidence, sexual pearl millet was transformed with ASGR-BBML either as cDNA or genomic DNA under control of its native promoter. The two versions of the construct are as described below according to the sequences provided in Table 2 below, which provides the cDNA, genomic DNA, amino acid sequence, and promoter sequence of ASGR-BBML. Genomic construct: 2074 bp ASGR-BBM promoter (p208) (containing a 6-residue GGATCC BamHI restriction site sequence) upstream of the 3,540 bp coding region (exons:introns) as well as 610 bp of the 3′UTR. cDNA construct: 2074 bp ASGR-BBM promoter (containing a 6-residue GGATCC BamHI restriction site sequence) fused to the 1,638 bp cDNA coding region as well as 610 bp of the 3′ UTR region. The alignment of the cDNA to the genomic DNA is depicted in
ATGGGTTCCACCAACAACTGGCTGCGCTTCGCCTCGTTCTCCGGCGGCGGCGGCGCCAAGGATGCCGC
Transgenic lines were regenerated from these experiments, and two plates of pearl millet embryogenic tissues were bombarded for each construct. For these constructs, the marker gene selected was for hygromycin resistance rather than phosphinothricin resistance. Of 5 cDNA construct lines, consisting of 13 plants total, that were regenerated and screened by ovule clearing, 3 plants showed a low frequency of small groups of densely cytoplasmic dividing cells in the nucellus; however, none of these was clearly interpreted as representing an organized embryo structure beyond the globular stage.
Of the nine independent transgenic lines (18 plants) containing the transgene, gPsASGR-BBML, generated from sexual tetraploid pearl millet, four lines, consisting of six plants, were not analyzed as a result of lack of flowering or demise of the plant. Four plants within 3 lines showed evidence of embryo formation from egg cells in the absence of fertilization, and 3 plants died prior to ovary analysis. Evidence for lack of fertilization was the persistence of polar nuclei in the central cell of the embryo sac. Parthenogenesis was assayed two days after anthesis for the remaining transgenic plants using a cleared-pistil technique (B. A. Young, R. T. Sherwood, E. C. Bashaw, Can. J Bot. 57:1668-1672 (1979)), and observation was conducted via differential interference contrast (DIC) or phase contrast optics. Fertilization was prevented by bagging heads prior to stigma exsertion and removing stigmas/styles prior to anthesis (since the plants are protygynous, meaning that stigmas exsert prior to anthers).
As shown in Table 3, the presence of embryos in the same embryo sac where polar nuclei persist (embryo+PN) provides evidence that the embryo sac was not fertilized and that the egg is developing parthenogenetically. Examples of embryo formation in embryo sacs with polar nuclei are shown in
All lines contained structurally mature embryo sacs (
As a result of low germination rates and low seed set for lines glia and g52, embryo rescue was employed on developing seed 10 to 15 days after pollination and on non-germinating mature seed in order to recover offspring from the 3 lines. Seed analyzed via flow cytometry demonstrated the production of reduced offspring (
Pollination with Red IA4X plants, which are sexual tetraploid lines containing a dominant Rpl allele which confers a dark red pigmentation in the midrib and sheath of leaves (W. W. Hanna, G. W. Burton, J Hered. 83:386-388 (1992)), over multiple heads and days was used to compensate in part for potential pollen sterility of the transgenic lines. Plant g11a set a total of nine seed, two offspring of which survived to greenhouse planting. Plant g52 set 97 seed 31 offspring of which survived to greenhouse planting. Plant g3f set hundreds of seed of which 194 were randomly selected, with 107 surviving to greenhouse planting.
All offspring were analyzed for the inheritance of a 3,694 bp amplicon covering the gPsASGR-BBML open reading frame starting five base pairs downstream from the start codon and amplifying into the 3′ UTR (ORF amplicon). The two g11a offspring showed no inheritance of the transgene. All offspring from g52 showed red pigmentation of the midrib; these were derived from the fertilization of g52 sexual embryo sacs with Red IA4X pollen. Nine of the offspring plants carried at least one copy of the ORF amplicon. The g3f offspring plants had a mix of both green and red pigmentation of the midrib. Twenty-six g3f offspring plants carried at least one copy of the ORF amplicon.
All offspring plants from line g52 were assayed for parthenogenesis (Table 4,
A subset of offspring plants generated from the g3f line was assayed for parthenogenesis (Table 5,
Because g3f offspring were a mix of both red and green pigmentation of the midrib, determination of ploidy level of the green phenotypes was required (Table 6). Six offspring were diploid/dihaploid in genome size as analyzed via flow cytometry using sorghum as the genome size reference (
Analysis of sexual transgenic lines which carry the gPsASGR-BBML transgene and produce fertilization-independent embryo formation and diploid/dihaploid offspring, albeit at somewhat low penetrance, demonstrates that the PsASGR-BBML gene alone can promote parthenogenesis in sexual tetraploid pearl millet, which are plants which do not normally show this trait. None of the TO lines or offspring from the TO lines showed complete penetrance of the trait; this incomplete penetrance within the original TO lines and offspring of lines g3f and g52 is likely due to transgene segregation, transgene expression levels, and/or unknown genetic factors interacting with PsASGRBBML. Although transcription of the transgene was identified in offspring that did not demonstrate parthenogenesis, quantifying levels of transgene expression is difficult due to variation in percentage of structurally mature sexual embryo sacs and the inability to verify that RNA is being extracted from ovaries that are at the same developmental stage. Generation of inbred lines containing a single copy of the gPsASGR-BBML transgene or the expression of the gPsASGRBBML transgene using egg-specific promoters with different expression levels would be advantageous to confirm these issues.
Ploidy level was not found to be critical for PsASGR-BBML transgene-induced parthenogenesis, as the four chromosomally reduced diploid/dihaploid offspring generated from g3f showed a similar range of parthenogenesis levels as the unreduced tetraploid offspring. While most natural apomicts are polyploid, natural populations and experimentally recovered apomictic diploids/dihaploids from several species have been identified. For example, polyploid apomictic plants can produce diploids/dihaploids offspring either through the genetic separation of the apomeiosis and parthenogenesis loci (R. D. Noyes, J. D. Wagner, American Journal of Botany 101:1-10 (2014)) or through the parthenogenetic development of a reduced egg carrying an apomixis locus (M. Dujardin, W. W. Hanna, Theor Appl Genet 72:33-36 (1986)).
The lack of an F1 aposporous transgenic line showing complete knock-down of PsASGRBBML precludes a determination of whether PsASGR-BBML is the sole gene required to induce parthenogenesis in plants programmed for the apomixis pathway through the inheritance of the ASGR. However, given that the number of ovaries showing parthenogenetic embryo development and that the number of cells in those embryos at 2 days after anthesis were significantly reduced in the F1 PsASGR-BBML-reduced-expression lines, PsASGRBBML clearly has an important role in parthenogenesis in the apomictic reproductive pathway.
While members of the BBM-like clade of AP2 transcription factors have noted roles in somatic embryogenesis and cell proliferation, these results have uncovered for the first time the role for PsASGRBBML in parthenogenesis. It is worth noting that neither maize nor sorghum have a PsASGR-BBML protein more related to PsASGR-BBML than does the more distantly related species rice. Therefore, this newly discovered role can have a major impact on the ability to genetically engineer apomixis into crop species This technique therefore can also be used as an alternative method for haploid induction in order to rapidly obtain homozygous lines for breeding.
0plants with the gASGR-BBM transgene not showing expected parthenogenesis.
The genomic PsASGR-BBM construct used in pearl millet transformation, which included A 2074 bp ASGR-BBM promoter (p208) (containing a 6-residue GGATCC BamHI restriction site sequence) upstream of the 3,540 bp coding region (exon:introns) plus 610 bp of the 3′UTR and residing in a blue script plasmid, was transferred using enzymes at the multiple cloning site to pCambia1300 for transformation of rice (Oryza sativa Japonica cv. Nipponbare). Sixteen different rice lines were found to contain the complete coding region of the PsASGR-BBM construct based on PCR analysis. RNA was isolated from four different lines of developing rice seed, and expression of the PsASGR-BBM transgene was assayed by non-quantitative RT-PCR. All four lines showed PsASGR-BBM transgene expression. Flow cytometry using ˜5 dissected developing rice embryos was used to determine whether haploid (In/Ie) offspring were generated from rice transgenic lines carrying the PsASGR-BBM transgene (
The present invention can be used as an alternative method for haploid induction in maize and other cereals. Specific lines of maize have been identified that, when used as pollinators, result in a low (2-8%) frequency recovery of haploid offspring from seed of the maternal parent (Chang M. and Coe E. H., “Doubled haploids,” pp 127-142, in Molecular Genetic Approaches to Maize Improvement, Kritz A. L. and Larkins B, Eds, Springer). These lines are extensively used in commercial maize breeding programs in North America and Europe.
The advantage of haploid induction in breeding is that different gene combinations can be fixed in each line once the chromosome number is doubled from haploid to diploid. This rapid recovery of homozygous inbred lines allows selection of inbred parents that can generate high-yielding, high-quality hybrids. Multiple inducer lines of maize have been identified, and the mechanism of haploid induction reported for at least one of them is chromosome elimination (Zhang Z. et al., Plant Cell Reports 27: 1851-1860 (2008)). Chromosome elimination post-fertilization is more likely to result in male contribution to haploid offspring than the invention described herein, where fertilization of the egg is avoided.
Haploid induction using the present invention can be of use in crops other than maize where inducer lines are not known. Any heterozygous individual will generate unique gene combinations in each egg if eggs are being formed from meiotically-derived products and are haploid. Each unique haploid individual will become homozygous and fertile once chromosomes are artificially doubled by chemical treatment. Homozygous lines can be more quickly generated by the method described herein for testing in the field.
A further use of this invention is as a component of apomixis. Both apomeiosis and parthenogenesis are required for functional gametophytic apomixis. Apomeiosis can be achieved by a combination of mutations affecting meiosis (Crismani W. et al., J. Exp. Bot. 64:55-65 (2013)), with the outcome of chromosomal non-reduction in megaspores, i.e., mitosis rather than meiosis. Somatic cells that assume a gametophytic fate through epigenetic alterations (Grimanelli D., Curr. Opin. Plant Biol. 15:57-62 (2012)) also result in unreduced spore-like cells that potentially can give rise to unreduced gametes (eggs). By whatever means unreduced eggs are formed, proper temporal and spatial expression of ASGR-BBML can induce the egg cells to behave as zygotes and divide in the absence of fertilization. The division of an unreduced egg to form the embryo component of a seed satisfies the conditions for apomixis provided that the endosperm of the seed can also complete development to yield a viable propagule.
Apomixis results in genetic identity of the offspring of a mother plant. The mother plant can be highly heterozygous, but since apomixis bypasses meiosis, there is no segregation of traits among seed-derived progeny. Apomixis can be used for more efficient hybrid seed production in hybrid crops, such as maize and the like, eliminating the need to use separate male and female parents grown in isolation to generate hybrid seed. Apomixis can also be used for seed propagation of heterozygous crops, such as potato and the like, that typically are vegetatively propagated through tubers, organs that can harbor and transmit diseases across generations. Apomixis can also promote the development of hybrids in crops where hybrids currently are not available due to the lack of parental lines that can be easily crossed on a commercial scale. Apomixis can be used as a breeding tool to increase and test large numbers of novel hybrids generated by sexual reproduction but increased through apomictic reproduction. The present invention, egg development into an embryo without fertilization, is an essential component of apomixis or clonal reproduction through seeds.
Materials and Methods
Transgene cassettes shown in
Transgene specific primers were used to screen transgene carrying plantlets. Pre-anthesis ovaries were collected and RNA extracted using Qiagen RNeasy plant mini kit and cDNA synthesis was done using Superscript III first-strand synthesis system. RT-PCR was performed to check the expression.
Induction of parthenogenesis was checked by doing flow cytometry on embryos dissected from the mature seeds, through germination of T1 seed and flow cytometry on germinated seedlings to determine ploidy and through phase contrast microscopy on fixed and cleared pre-anthesis ovaries.
Results
All transgenes showed expression in rice ovaries before anthesis.
pDD45:: PsASGR-BBML-ZsGreen1 lines show parthenogenetic embryo development as indicated by the haploid signal in flow cytometry plots (
pDD45::SiBBBM3 lines showed developmental abnormalities pertaining to vegetative as well as reproductive development (
pDD45::SiBBM1 and pDD45:: PsASGR-BBML-ZsGreen1 lines showed that both transgenes interfere with the normal embryo and/or seed development causing the lower seed set in transgenic lines (0-86%) compared to untransformed lines (85.6%).
In pDD45:: PsASGR-BBML-ZsGreen1 and pDD45::SiBBM3, transgene expression was observed in young leaf tissues and in tissue containing shoot meristems.
All transgenes (Seita.8G107100 (SiBBM1), Sieta.5G415800 (SiBBM2), Seita.1G232200 (SiBBM3) and PsASGR-BBML-Zsgreen1 driven by pDD45) induced parthenogenesis.
The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described are achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by including one, another, or several other features.
Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.
Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.
In some embodiments, any numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the disclosure are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and any included claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are usually reported as precisely as practicable.
In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of certain claims) are construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.
Variations on preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.
All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting effect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.
In closing, it is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.
The present application is a continuation-in-part of U.S. Non-provisional patent application Ser. No. 16/821,755 filed on Mar. 17, 2020, which is a continuation of U.S. Non-Provisional patent application Ser. No. 15/031,019 filed on Apr. 21, 2016 (now U.S. Pat. No. 10,633,672), which is a U.S. National Stage entry under 35 U.S.C. § 371 of International Application No. PCT/US2014/061630, filed on Oct. 21, 2014, designating the United States of America and published in English on Apr. 30, 2015, which in turn claims the benefit of priority 35 U.S.C. § 119(e) to U.S. Provisional Application No. 61/893,741, filed on Oct. 21, 2013, and U.S. Provisional Application No. 62/059,842, filed on Oct. 3, 2014; each of which is hereby incorporated by reference in its entirety. The present application also claims priority to U.S. Provisional Application No. 63/221,312, filed on Jul. 13, 2021; which is hereby incorporated by reference in its entirety.
This invention was made with government support under Grant No. 2010-65116-20449 awarded by the United States Department of Agriculture, National Institute of Food and Agriculture, Agriculture and Food Research Initiative (USDA-NIFA-AFRI), and Grant No. DBI-0115911 awarded by the National Science Foundation (NSF). In addition, this invention was made with government support under grant number 2017-06368 awarded by USDA NIFA. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63221312 | Jul 2021 | US | |
| 61893741 | Oct 2013 | US | |
| 62059842 | Oct 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15031019 | Apr 2016 | US |
| Child | 16821755 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16821755 | Mar 2020 | US |
| Child | 17812022 | US |
