Sorghum silage hybrids with enhanced protein digestibility

Abstract

A forage sorghum plant or plant part comprising in its genome at least one mutant allele of a kafirin storage protein gene, which encodes a polypeptide comprising SEQ ID NO: 3. The mutant forage sorghum plant of the present disclosure has improved protein digestibility thus enhancing the quality of silage produced from the plant.

Description

SORGHUM SILAGE HYBRIDS WITH ENHANCED PROTEIN DIGESTIBILITYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application No. 63 / 570,914 filed on March 28, 2024, the disclosure of which is expressly incorporated herein.REFERENCE TO AN ELECTRONICALLY SUBMITTED SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (file name: 3220421652.xml; size: 13 kilobytes, created on March 27, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0003] Silage is a high moisture fodder used by farmers to feed their domestic animals, especially during the dry season. Silage is made from green foliage crops which have been preserved by fermentation to the point of souring, and is typically fed to cattle, sheep and other ruminants. The fermentation and storage process is called ensilage, ensiling, or silaging. Com has been used for preparing silage, but forage sorghum offers several advantageous as a crop for producing silage.

[0004] Forage sorghum has higher drought tolerance and slightly less nitrogen fertilizer requirements than corn. Additionally, forage sorghum yield is equivalent or slightly higher than com. Sorghum forage production systems are utilized across the United States, especially in regions that are too hot or dry to produce other crops. Sorghum generally requires about half the water to grow compared to corn and exhibits excellent performance in diverse production systems. The USDA reported that more than 5 million tons of sorghum silage were produced in 2022 (United States Department of Agriculture, National Agriculture Statistics Service). Most of these silage cultivars incorporate very little grain into the silage because digestibility of this fraction is often lower than in other crops.

[0005] Recent advancements in plant breeding and biotechnology are making substantial contributions to engineering new silage traits in sorghum, especially traits for improved nutritional value. Recent genetic studies identified a sorghum mutant, SbEMS3324, with highly digestible protein (HDP) compared to conventional sorghum. Whole genome resequencing coupled with genetic linkage analyses identified the causal mutation for the HDP trait as a single nucleotide polymorphism (SNP) in the coding sequence of Sobic.005Gl 89000,a kafirin storage protein (Diatta, 2018). The single modified nucleotide at position 61 of SEQ ID NO: 2 replacing the native guanine with adenosine results in the substitution of threonine residue for the native alanine residue in the expressed protein. Otherwise, native protein and the mutant SbEMS3324 protein are identical.SUMMARY

[0006] As disclosed herein a mutant kafirin storage protein gene is provided that encodes a highly digestible protein (HDP) compared to conventional sorghum. This highly digestible protein (HDP) mutation was incorporated into sorghum silage backgrounds to improve silage feed quality. Field trials showed that the HDP silage hybrids exhibited similar biomass yields to conventional sorghum hybrids; however, the HDP hybrids exhibited significantly improved protein content and protein digestibility. Protein digestibility in the HDP hybrids was 65% higher than the conventional silage check. Accordingly, the novel silage hybrids disclosed herein provide an improvement of sorghum silage quality. Furthermore, genetic markers and NIRS phenotyping technologies have been developed for this trait to facilitate easy manipulation in silage breeding programs.

[0007] In accordance with one embodiment an isolated polynucleotide is provided that encodes a polypeptide of SEQ ID NO: 3, or a polypeptide that differs from SEQ ID NO: 3 by one or more amino acid substitutions selected the group consisting of L5I, A7V, F29L, T35I, P36S, S41P, V42L, P44L, F45V, G46R, and Y47F. In accordance with one embodiment a polynucleotide is provided that encodes a polypeptide that differs from SEQ ID NO: 3 by two or more amino acid substitutions selected the group consisting of L5I, A7V, F29L, T35I, P36S, S41P, V42L, P44L, F45V, G46R, and Y47F. In accordance with one embodiment a polynucleotide is provided that encodes a polypeptide of SEQ ID NO: 3, or a polypeptide that differs from SEQ ID NO: 3 by 3, 4, 5, 6, 7 , 8 , 9, 10 or 11 or more amino acid substitutions selected the group consisting of L5I, A7V, F29L, T35I, P36S, S41P, V42L, P44L, F45V, G46R, and Y47F. In accordance with one embodiment a polynucleotide is provided that encodes a kafirin storage protein that comprises an amino acid sequence of of SEQ ID NO: 3. In a further embodiment the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 is operably linked to regulatory elements, including a promoter, that are functional in plant cells, and more particularly, is functional in sorghum plant cells. In one embodiment, the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 is part of a eukaryotic expression vector, optionally wherein the expression vector is designed for integration of the gene sequences encoding SEQ ID NO: 3 into the genome of a plant cell. In one embodiment the expression vector further comprises a selectable marker.

[0008] In one embodiment an isolated polynucleotide is provided wherein the polynucleotide comprises the sequence of SEQ ID NO: 1 or a sequence having at least 80%, 85%, 95% or 99% sequence identity to SEQ ID NO: 1. In a further embodiment a polynucleotide having at least 95% sequence identity to SEQ ID NO: 1 is operably linked to regulatory elements, including a promoter, that are functional in plant cells, and more particularly, functional in sorghum plant cells.

[0009] In one embodiment a sorghum plant cell is provided wherein the plant cell comprises a polynucleotide that encodes a modified kafirin storage protein that encodes a polypeptide of SEQ ID NO: 3, or a polypeptide that differs from SEQ ID NO: 3 by one or more amino acid substitutions selected the group consisting of L5I, A7V, F29L, T35I, P36S, S41P, V42L, P44L, F45V, G46R, and Y47F. In one embodiment the sorghum plant cell is a forage sorghum plant cell that comprises a polynucleotide that encodes a polypeptide of SEQ ID NO: 3. In one embodiment the sorghum plant cell is a forage sorghum plant that comprises a polynucleotide coding sequence of SEQ ID NO: 1 or a sequence having at least 80%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1. In one embodiment, the plant cell is homozygous for the the nucleic acid sequence encoding the mutant kafirin storage polynucleotide coding sequence of SEQ ID NO: 3.

[0010] In one embodiment a sorghum plant is provided wherein the cells of the plant comprise a polynucleotide that encodes a modified kafirin storage protein comprising the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that differs from SEQ ID NO: 3 by one or more amino acid substitutions selected the group consisting of L5I, A7V, F29L, T35I, P36S, S41P, V42L, P44L, F45V, G46R, and Y47F. In one embodiment the sorghum plant is a forage sorghum plant that comprises a polynucleotide that encodes a polypeptide of SEQ ID NO: 3. In one embodiment the sorghum plant is a forage sorghum plant that comprises a polynucleotide coding sequence of SEQ ID NO: 1 or a sequence having at least 80%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1. In one embodiment, the plant is homozygous for the the nucleic acid sequence encoding the mutant kafirin storage polynucleotide of SEQ ID NO: 3.

[0011] In one embodiment a plant part, progeny, or asexual propagate of a forage sorghum plant is provided, wherein the plant part, progeny, or asexual propagate comprises a polynucleotide that encodes a modified kafirin storage protein that encodes a polypeptide of SEQ ID NO: 3, or a polypeptide that differs from SEQ ID NO: 3 by one or more amino acid substitutions selected the group consisting of L5I, A7V, F29L, T35I, P36S, S41P, V42L, P44L, F45V, G46R, and Y47F. In one embodiment the plant part is a seed. In one embodiment a seed is provided for producing forage sorghum plants that are used to produce silage havingimproved protein digestibility, said seed comprising a mutant kafirin storage protein gene that encodes a protein comprising the sequence of SEQ ID NO: 3.

[0012] The present disclosure also is directed to methods for producing forage sorghum plants that have enhanced nutritional value. The method comprises introducing a gene encoding a modified kafirin storage protein of the present disclosure into existing forage sorghum lines. In one embodiment the method comprises introducing a polynucleotide that encodes a modified kafirin storage protein of SEQ ID NO: 3, or a polypeptide that differs from SEQ ID NO: 3 by one or more amino acid substitutions selected the group consisting of L5I, A7V, F29L, T35I, P36S, S41P, V42L, P44L, F45V, G46R, and Y47F into the genome of an existing forage sorghum plant line. In one embodiment the polynucleotide is introduced into the cell via transfection of a plant cell and regeneration of a plant from the transfected cell.

[0013] The insertion of the modified kafirin storage protein gene of the present disclosure into the genome of a sorghum plant cell can be conducted using any of the standard tools for transfection. In one embodiment the the native kafirin storage protein gene is targeted for modification / replacement with the modified kafirin storage protein gene disclosed herein. In one embodiment the modified kafirin storage protein gene is inserted into the plant's genome through the use of a site specific recombinase including for example: CRISPR-Cas9 endonuclease, Cpf 1 endonuclease, Zn finger nuclease, meganuclease, or TALEN. When the targeted site-directed modification is introduced into a cell, tissue or organ, the method can further comprise regenerating a plant therefrom. The resulting plant can be sexually propagated using traditional plant breeding techniques. The plant can be sexually propagated as a male or a female.

[0014] In an alternative embodiment the polynucleotide is introduced into an inbred sorghum plant line by traditional breeding, including the steps of: 1) crossing a first sorghum plant, comprising a polynucleotide encoding a modified kafirin storage protein of the present disclosure, with a second sorghum plant lacking said polynucleotide; 2) identifying progeny plants comprising said polynucleotide; and 3) selecting progeny plants comprising the polynucleotide. In one embodiment, the plant comprising the modified kafirin storage protein gene can be sexually propagated as a male or a female with an inbred sorghum line as a male or a female to produce a plant comprising all of the characteristics of the inbred line but exhibiting improved digestibility. Optionally the method can include one or more rounds of backcrossing of the progeny of the first cross with the second plant, and selecting progeny plants that comprise the polynucleotide.

[0015] In one embodiment the modified kafirin storage protein gene of the present disclosure encodes the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that isat least 95% or 99% identical to SEQ ID NO: 3, with the proviso that the amino acid at position 21 (relative to the numbering of SEQ ID NO: 5) is threonine.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Fig. 1 provides an alignment of the first 150 nucleotides of native cDNA acid sequence for kafirin storage protein (BTx623; SEQ ID NO: 4) relative to two known mutants SbEMS3324 (SEQ ID NO: 5) and P721Q (hl) (SEQ ID NO: 6) and the present mutant 3005G189000 (SEQ ID NO: 7).

[0017] Fig. 2 provides an alignment of the first 50 amino acids of native kafirin storage protein (BTx623; SEQ ID NO: 8) relative to mutant P721Q (hl) (SEQ ID NO: 10) and the present mutant 3005G189000 (SEQ ID NO: 9).

[0018] Fig. 3 is a photograph of sorghum plants growing in a field wherein near isogenic sorghum hybrids with and without the HDP mutation from SbEMS3324 are shown.

[0019] Fig. 4 is a bar graph of data showing the variation in protein digestibility of silage hybrids in trials conducted on conventional sorghum versus two near isogenic hybrids: HDP silage 1 and HDP silage 2.DETAILED DESCRIPTIONDEFINITIONS

[0020] In describing and claiming the disclosed embodiments, the following terminology will be used in accordance with the definitions set forth below.

[0021] The term "about" as used herein means greater or lesser than the value or range of values stated by 10 percent but is not intended to limit any value or range of values to only this broader definition. Each value or range of values preceded by the term "about" is also intended to encompass the embodiment of the stated absolute value or range of values.

[0022] The term "substantially" can allow for a degree of variability in a value or range, for example, within 90%, within 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more of a stated value or of a stated limit of a range.

[0023] As used herein, the term "purified" and like terms relate to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment. As used herein, the term "purified" does not require absolute purity; rather, it is intended as a relative definition. The term "purified polypeptide" is used herein to describe a polypeptide which has been separatedfrom other compounds including, but not limited to nucleic acid molecules, lipids and carbohydrates.

[0024] The term "isolated" requires that the referenced material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.

[0025] The term "mutant kafirin storage protein gene" as used herein without any further modification includes any polynucleotide that encodes the mutant kafirin protein of SEQ ID NO: 3.

[0026] Conservative amino acid substitutions include those set forth in the following Table 1:Table 1: Similar Amino Acid SubstitutionsEMBODIMENTS

[0027] The present disclosure is directed to a new mutant forage sorghum line having improved protein digestibility. The increased digestibility of the mutant line enhances the quality of silage produced from the plant. In accordance with one embodiment a forage sorghum plant or plant part is provided wherein the plant or plant part comprises in its genome at least one mutant allele of a kafirin storage protein gene, which encodes a polypeptide comprising SEQ ID NO: 3.

[0028] Also provided is a plant cell, tissue, organ, seed comprising the modified kafirin storage protein gene of the present disclosure, or progeny of an above-described sorghum plant or a hybrid thereof. In one embodiment a sorghum plant, or sorghum plant part thereof, is provided wherein the cells of the sorghum plant, or sorghum plant part thereof, comprise a polynucleotide that encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that differs from SEQ ID NO: 3 by one or more amino acid substitutions selected the group consisting of L5I, A7V, F29L, T35I, P36S, S41P, V42L, P44L, F45V, G46R, and Y47F.

[0029] Further provided is an isolated or purified nucleic acid molecule comprising a nucleotide sequence encoding a CDS of a modified kafirin storage protein gene. In one embodiment the CDS of the isolated or purified nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 1, wherein upon introduction of said isolated or purified nucleic acid molecule into a sorghum plant cell can regenerate a plant having improved digestibility relative to a sorghum plant lacking the modified kafirin storage protein disclosed herein.

[0030] In addition to the modified kafirin storage protein discussed above, other modifications, such as amino acid substitutions, deletions, truncations, and insertions can be introduced into the kafirin storage protein gene. In one embodiment the modifications include one or more conservative amino acid substitutions. Such methods of manipulation are known in the art. See, e.g., Kunkel, PNAS USA 82: 488-492 (1985); Kunkel et al., Methods in Enzymol 154: 367-382 (1987); and Walker and Gaastra, eds., Techniques in Molecular Biology (MacMillan Pub Co, New York (1983)). Guidance regarding appropriate amino acid substitutions that do not affect biological activity can be found in Dayhoff et al., Atlas of Protein Sequence and Structure (Nat’l Biomed Res Found, Washington, DC (1978)).

[0031] In accordance with one embodiment an isolated polynucleotide is provided that encodes a polypeptide of SEQ ID NO: 3, or a polypeptide that differs from SEQ ID NO: 3 by one or more amino acid substitutions selected the group consisting of L5I, A7V, F29L, T35I, P36S, S41P, V42L, P44L, F45V, G46R, and Y47F, or one or more conservative amino acid substitutions. In accordance with one embodiment a polynucleotide is provided that encodes a polypeptide that differs from SEQ ID NO: 3 by two or more amino acid substitutions selected the group consisting of L5I, A7V, F29L, T35I, P36S, S41P, V42L, P44L, F45V, G46R, and Y47F.

[0032] Even still further provided is a vector, which can be introduced into a plant, a plant cell, a plant tissue, or a plant organ, and which comprises an above-described isolated or purified nucleic acid molecule operably linked to a promoter (and, if desired, other regulatory sequences) that promotes expression of the CDS in the plant, the plant cell, the plant tissue, or the plant organ. Two or more elements can be operably linked, i.e., functionally linked, yet contiguous or non-contiguous. The promoter can be one that is not naturally operably linked to the nucleic acid, being one isolated or derived from another gene. The native promoter, however, can be used. The promoter can be inducible, tissue-preferred, tissue-specific, developmentally regulated, or constitutive. Examples of promoters include, but are not limited to, the cauliflower mosaic virus 35S (CaMV 35S) promoter, the maize glutathione-S-transferase isoform II (GST-II-27) promoter, the cauliflower meri-5 promoter, the Arabidopsis thaliana LEAFY promoter, rice actin, and ubiquitin.

[0033] Suitable vectors can be chosen, such as selected from commercially available sources, or constructed and can contain regulatory sequences, such as promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as appropriate. See, e.g., Molecular Cloning: A Laboratory Manual, 2nded., Sambrook et al., Cold Spring Harbor Laboratory Press (1989). Plant preferred codons can be used to improve expression (see, e.g., Murray et al., Nucl Acids Res 17: 477-498 (1989)). Likewise, the G-C content of the nucleotide sequence can be adjusted to levels characteristic of a given host plant.

[0034] There are many known techniques and protocols for manipulating nucleic acids, such as in the preparation of nucleic acid constructs / vectors, mutagenesis, sequencing, introduction of nucleic acids (e.g., DNA) into cells, gene expression, and protein analysis. See, e.g., Protocols in Molecular Biology, 2nded., Ausubel et al., eds. John Wiley & Sons (1992); Bevan, Nucl Acids Res 12: 8711-8721 (1984); and Guerineau and Mullineaux, “Plant transformation and expression vectors,” in Plant Molecular Biology Labfax (Croy RRD, Ed.), Oxford, BIOS Scientific Publishers, pp. 121-148 (1993), all of which are specifically incorporated by reference for their teachings regarding same.

[0035] A plant, or a cell, tissue, or organ thereof, which comprises the above-described vector is also provided. There may be more than one heterologous nucleotide sequence per haploid genome. The nucleotide sequence may be incorporated into the genome. Indeed, if the vector used to introduce an above-described isolated or purified nucleic acid molecule will recombine with the genome, the vector need not necessarily comprise an operably linked promoter or other regulatory sequence(s). Alternatively, the vector can be extra-genomic.

[0036] Any appropriate method of plant transformation can be used to generate plant cells comprising a nucleic acid, construct, or vector. Following transformation, plants may be regenerated from transformed plant cells and tissues. DNA can be transformed into plant cells using Ti-plasmid carried by Agrobacterium, particle or microprojectile bombardment, microinjection (see, e.g., Green et al., Plant Tissue and Cell Culture, Academic Press (1987)), electroporation, other forms of direct DNA uptake, liposome-mediated DNA uptake (see, e.g., Freeman et al., Plant Cell Physiol 29: 1353 (1984)), or vortexing (see, e.g., Kindle, PNAS USA 87: 1228 (1990)). See, also, Oard, Biotech Advn 9: 1-11 (1991), in re physical methods for plant transformation and Shimamoto, Curr Opi in Biotech 5: 158-162 (1994) in re a review of the generation of fertile transgenic plants in rice, maize, wheat, oat, and barley. A combination of different techniques can be employed to enhance the efficiency of transformation, e.g., bombardment with Agrobacterium-coated microparticles or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium.

[0037] Following transformation, a plant can be regenerated, for example, from single cells, callus tissue, or leaf discs. See, e.g., Vasil et al., Cell Culture and Somatic Cell Genetics of Plants (Vols. I-III); Laboratory Procedures and Their Applications, Academic Press (1984); and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press (1989).

[0038] In view of the above, a method of producing a sorghum plant comprising a polynucleotide encoding a modified kafirin storage protein of SEQ ID NO: 3 is further provided. The method comprises introducing an above-described vector into a plant, or a cell, tissue, or organ thereof, whereupon the CDS is stably expressed therein, and, when the nucleic acid molecule or the vector is introduced into a cell, tissue or organ, the method further comprises regenerating a plant therefrom. The method can further comprise sexually or asexually propagating the plant. The plant can be sexually propagated using plant breeding techniques. The plant can be sexually propagated as a male.

[0039] Still further provided is a method of transferring into a plant at least one mutant allele of a kafirin storage protein gene, wherein the gene encodes a protein comprising the sequence of SEQ ID NO: 3, optionally wherein the CDS of the gene comprises a nucleotide sequence of SEQ ID NO: 1 or a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 1. The method comprises:

[0040] (a) providing or generating a first sorghum plant comprising a polynucleotide that encodes the mutant kafirin protein of SEQ ID NO: 3; and

[0041] (b) crossing the first plant with a second sorghum plant, which does not comprise a polynucleotide that encodes the mutant kafirin protein of SEQ ID NO: 3 (i.e., the mutant kafirin storage protein gene; and

[0042] (c) identifying Fl plants comprising the mutant kafirin protein gene.

[0043] Optionally the method further comprises the steps of:

[0044] (d) backcrossing Fl plants comprising the mutant kafirin storage protein gene for at least one generation and collecting seeds from the backcrossed generation; and

[0045] (e) identifying in every generation backcrossed, plants comprising the mutant kafirin protein gene. In one embodiment the first plant is crossed as a male or a female. In one embodiment the first plant a forage sorghum plant comprising the mutant kafirin storage protein gene, and the second plant can be an inbred forage sorghum plant that lacks the mutant kafirin storage protein gene.

[0046] Even still further provided is a method of enhancing the nutritional and digestibility of a forage sorghum plant. The method comprises introducing at least one nucleotide modification through a targeted site-directed modification at a genomic locus of a plant, or a cell, tissue, or organ thereof, wherein the genomic locus comprises kafirin storageprotein gene. In one embodiment the kafirin storage protein gene is modified to encode a polypeptide comprising the sequence of SEQ ID NO: 3. In one embodiment targeted site- directed modification can involve the use of CRISPR-Cas9 endonuclease, Cpf 1 endonuclease, Zn finger nuclease, meganuclease, or TALEN (see, e.g., USPAPN 2020 / 0181623 and LJSPAPN 2020 / 0199609, both of which are hereby incorporated by reference for their teachings regarding same). When the targeted site-directed modification is introduced into a cell, tissue or organ, the method can further comprise regenerating a plant therefrom. The plant can be sexually propagated using plant breeding techniques. The plant can be sexually propagated as a male or a female.

[0047] In accordance with one embodiment a method of detecting a polynucleotide that encodes a polypeptide of SEQ ID NO: 3 in a sample comprising nucleic acids is provided wherein said method comprises contacting said sample with a polynucleotide segment of SEQ ID NO: 1 or the complement thereof, wherein the polynucleotide segment is unique to a polynucleotide that encodes a polypeptide of SEQ ID NO: 3, wherein detecting specific binding of the polynucleotide segment to the sample identifies the presence of a modified kafirin storage protein gene in said sample. In one embodiment the polynucleotide segment is a unique nucleic acid sequence of at least 10 nucleotides. In an alternative embodiment the method of detecting a polynucleotide that encodes a modified kafirin stage protein gene in a sample comprising nucleic acids is provided, wherein said method comprises contacting said sample with a. a first primer that binds to a sequence 5 ’ to a polynucleotide segment is unique to SEQ ID NO: 1; and b. a second primer that binds to a sequence 3’ to polynucleotide segment is unique to SEQ ID NO: 1; subjecting the sample to polymerase chain reaction; and assaying for an amplicon generated between the first and second primers.EXAMPLES

[0048] The following examples serve to illustrate the present disclosure. The examples are not intended to limit the scope of the claimed invention in any way.

[0049] The HDP trait was introgressed into forage sorghum by genetic crosses between SbEMS3324, the HDP trait donor line, and silage parent lines 03MN952 and MR732. Near isogenic hybrids contrasting for the HDP trait were produced for testing in field trials (Table 2).

[0050] Table 2: Near-isogenic silage hybrids contrasting for the Highly Digestible Protein trait.Code Entry GenerationConventional Silage (03MN952A x MR732J-F1 Fl hybridHDP Silage-1 (20WL1596A x 21WL21O2)-F1 Fl hybridHDP Silage-2 (20WL1599A x 21WL21O2)-F1 Fl hybrid

[0051] The HDP Silage-1 and HDP Silage-2 hybrids were contrasted with a closely related Conventional Silage hybrid in field trials in 2022 and 2023 using a randomized complete block design with entries in 4-row plots planted at a population of 60,000 plants per acre (Fig. 3). Differences in phonologic measurements were taken by direct measurement of these attributes on a plot basis. Biomass information was determined using a biomass harvester on a fresh weight basis (tonnes ha-1). The moisture content and lodging of the plots were also determined and used to adjust biomass yield to a dry weight basis. The HDP and Conventional Silage hybrids were phenotypically similar in appearance. The Silage Hybrids exhibited similar phenological characteristics with small differences in flowering time and plant height but similar biomass yields each year (Table 3).

[0052] Table 3: Agronomic characteristics of near-isogenic silage hybrids contrasting for the Highly Digestible Protein trait.

[0053] Biomass fractionation studies were conducted to determine the fraction of grain and forage in each of the silage samples. These studies showed that the Conventional and HDP Silage hybrids produced similar silage products with a high fraction of grain ranging from 46- 48% on a dry weight basis in silage from each of the hybrids (Table 4).

[0054] Table 4. Dry matter partitioning to grain for the Conventional and HDP Silages.

[0055] Near Infrared Spectroscopy (NIRS) was used to predict differences in grain and forage quality in samples pulled from the field trials. These studies showed that HDP Silage- 1 and HDP Silage-2 exhibited lower Acid Detergent Fiber content and higher Total Digestibly Nutrients and Net Energy Contents than the Conventional Silage (Table 5). Significant differences in grain compositions were also noted with the HDP Silage hybrids exhibiting lower oil and starch contents but significantly higher protein content compared to the normal protein hybrid (Table 5).

[0056] Table 5. Grain and forage quality traits for near isogenic hybrids contrasting for the HDP mutation.

[0057] Difference in protein digestibility among the Conventional Silage and HDP Silage- 1 and HDP Silage-2 was evaluated using an in vitro digestibility assay (Diatta- Holgate et al., 2023). Significant differences in protein digestibility among silage types were shown (Fig. 4). The protein digestibility of HDP hybrids was approximately 65% higher than in the conventional silage.SUMMARY

[0058] HDP Silages compare favorably with Conventional Silage with competitive biomass production, increased protein content, and substantially increased protein digestibility. Protein digestibility in the HDP hybrids was 65% higher than the conventional silage check. These characteristics are unique and indicate a step-change technology for improvement of sorghum silage quality. HDP hybrids should compete favorably with com silage, especially in water-limited and dryland production environments that are common in the South-Central region of the United States.

Claims

WHAT IS CLAIMED IS:

1. A method of enhancing the quality of silage produced by a forage sorghum plant, said method comprising providing a first inbred forage sorghum plant; crossing the first plant with a second plant, wherein said second plant comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 3 or a polypeptide that differs from SEQ ID NO: by one or more amino acid substitutions selected from group consisting of L5I, A7V, F29L, T35I, P36S, S41P, V42L, P44L, F45V, G46R, and Y47F; selecting Fl progeny plants that comprise said polynucleotide, and growing said Fl progeny plants, or progeny thereof, as a source for silage production.

2. The method of claim 1 further comprising a step of backcrossing said Fl plants for at least one generation and collecting seeds from the backcrossed generation; and identifying in every generation backcrossed, plants comprising said polynucleotide, and growing said identified plants, or progeny thereof, as a source for silage production.

3. A method of producing a forage sorghum plant having improved digestibility relative to plants lacking a gene encoding the polypeptide of SEQ ID NO: 3, said method comprising the steps of introducing a polynucleotide that encodes a polypeptide comprising SEQ ID NO: 3 into the cells of a forage sorghum plant.

4. The method of claim 3 wherein said polynucleotide is introduced into a forage sorghum plant cell by transfecting said cell and regenerating a plant from the transfected cell.

5. The method of claim 3 wherein said polynucleotide is introduced into the cells of a forage sorghum plant by traditional breeding comprising the steps ofcrossing a first forage sorghum plant comprising said polynucleotide with a second forage sorghum plant lacking said polynucleotide; identifying progeny plants comprising said polynucleotide; and selecting progeny plants comprising said polynucleotide.

6. The method of claim 5 further comprising the step of backcrossing said progeny plants for at least one generation and collecting seeds from the backcrossed generation; and identifying in every generation backcrossed, plants comprising said polynucleotide.

7. The method of any one of claims 1-6 wherein said polynucleotide comprises the coding nucleic acid sequence of SEQ ID NO: 1 or a sequence having at least 95% sequence identity to SEQ ID NO: 1.

8. A part, progeny, or asexual propagate of the plant of any one of claims 3-6, wherein said part, progeny, or sexual propagate comprises said polynucleotide.

9. A seed for producing forage sorghum plants that are used to produce silage having improved protein digestibility, said seed comprising a mutant kafirin storage gene that encodes a protein comprising the sequence of SEQ ID NO: 3.

10. A polynucleotide that encodes a polypeptide of SEQ ID NO: 3.

11. A polynucleotide that encodes a polypeptide that differs from SEQ ID NO: 3 by one or more amino acid substitutions selected the group consisting of I5L, V7A, L29F, I35T, S36P, P41S, L42V, L44P, V45F, R46G, and F47Y.

12. The polynucleotide of claim 10 or 11 further comprising a promoter, that is functional in sorghum plant cells, operably linked to the nucleic acid sequences encoding said polynucleotide.

13. An expression vector comprising the polynucleotide of claim 12, wherein said polynucleotide is operably linked to a promoter that is functional in sorghum plants and the vector further comprises a selectable marker.

14. A forage sorghum plant cell of comprising the polynucleotide of claim 10 or 11.

15. A forage sorghum plant comprising a plurality of the plant cell of claim 14.

16. A forage sorghum seed comprising a plurality of the plant cell claim 14.

17. A part, progeny, or asexual propagate of the plant of claim 15, wherein said part, progeny, or sexual propagate comprises said polynucleotide.

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