Patent Description:
Field Pennycress Thlaspi arvense L. (common names: fanweed, stinkweed, field pennycress), hereafter referred to as Pennycress or pennycress, is a winter cover crop that helps to protect soil from erosion, prevent the loss of farm-field nitrogen into water systems, and retain nutrients and residues to improve soil productivity. While it is well established that cover crops provide agronomic and ecological benefits to agriculture and environment, only <NUM>% of farmers today are using them. One reason is economics - it requires on average ~$<NUM>-<NUM>/acre to grow a cover crop on the land that is otherwise idle between two seasons of cash crops such as corn and soy. In the last <NUM> years, it has been recognized that pennycress could be used as a novel cover crop, because in addition to providing cover crop benefits, it is an oilseed with its oil being useful as a biofuel. Extensive testing indicates that it can be interseeded over standing corn in early fall and harvested in spring prior to soybean planting (in appropriate climates). As such, its growth and development requires minimal incremental inputs (e.g., no/minimum tillage, no/low nitrogen, insecticides or herbicides). Pennycress also does not directly compete with existing crops when intercropped for energy production, and the recovered oil and meal can provide an additional source of income for farmers.

Pennycress is a winter annual belonging to the Brassicaceae (mustard) family. It's related to cultivated crops, rapeseed and canola, which are also members of the Brassicaceae family. Pennycress seeds are smaller than canola, but they are also high in oil content. They typically contain <NUM>% oil, which is roughly twice the level found in soybean, and the oil has a very low saturated fat content (~<NUM>%). Pennycress represents a clear opportunity for sustainable optimization of agricultural systems. For example, in the US Midwest, ~<NUM> acres that remain idle could be planted with pennycress after a corn crop is harvested and before the next soybean crop is planted. Pennycress can serve as an important winter cover crop working within the no/low-till corn and soybean rotation to guard against soil erosion and improve overall field soil nitrogen and pest management.

Pennycress has an oil content that makes it highly desirable as a biofuel, and potentially as a food oil. Once the oil is obtained from pennycress, either from mechanical expeller pressing or hexane extraction, the resulting meal has a high protein level with a favorable amino acid profile that could provide nutritional benefits to animals. However, studies of pennycress processing have consistently demonstrated that the meal produced has a high level of non-digestible fiber, and as a result, not enough metabolizable energy to be competitive with high-value products like soybean and canola meals as an animal feed.

<NPL>, discloses a composition comprising non-defattened pennycress seed meal with an ADF (acid detergent fiber) of <NUM>% to <NUM>% and NDF (neutral detergent fiber) of <NUM>% to <NUM>%.

<NPL>, discloses a defattened pennycress meal preparations.

<NPL>, discloses that yellow oilseed rape seeds show lower content of fibre in terms of AOF and NOF.

<NPL>, discloses colocalisation yellow seed color with a major QTL for lowered AOF, likewise in oilseed rape.

In a first aspect, the invention relates to a composition comprising non-defatted pennycress seed meal, wherein the non-defatted pennycress seed meal has an acid detergent fiber (ADF) content of less than <NUM>% by dry weight, and wherein the non-defatted pennycress seed meal is obtained from a seed lot comprising a loss-of function (LOF) homozygous mutation in an endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO: <NUM>, <NUM>, <NUM>, allelic variants having at least <NUM>% sequence identity with SEQ ID NO:<NUM> , <NUM> or <NUM>, or any combination thereof.

In a second aspect, the invention relates to a composition comprising defatted pennycress seed meal, wherein the defatted pennycress seed meal comprises an acid detergent fiber (ADF) content of <NUM>% to <NUM>% by dry weight, and wherein the defatted pennycress seed meal is obtained from a seed lot comprising a loss-of function (LOF) homozygous mutation in an endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO: <NUM>, <NUM>, <NUM>, allelic variants having at least <NUM>% sequence identity with SEQ ID NO:<NUM> , <NUM> or <NUM>, or any combination thereof.

In a third aspect, the invention relates to a pennycress seed meal comprising an acid detergent fiber (ADF) content of <NUM>% to <NUM>% by dry weight, wherein the seed meal is non-defatted, and wherein the non-defatted seed meal is obtained from a seed lot comprising a loss-of function (LOF) homozygous mutation in an endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO: <NUM>, <NUM>, <NUM>, allelic variants having at least <NUM>% sequence identity with SEQ ID NO:<NUM>, <NUM> or <NUM>, or any combination thereof.

In a fourth aspect, the invention relates to a seed lot comprising a population of pennycress seeds that comprise an acid detergent fiber (ADF) content of <NUM>% to <NUM>% by dry weight, wherein said population of pennycress seeds comprise seeds having at least one loss-of-function homozygous mutation in at least one endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO: <NUM>, <NUM>, <NUM>, and allelic variants thereof having at least <NUM>% sequence identity with SEQ ID NO: <NUM> , <NUM> or <NUM>, or comprise seeds having at least one transgene that produces an artificial miRNA that suppresses expression of at least one endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO: <NUM>, <NUM>, <NUM>, and allelic variants having at least <NUM>% sequence identity with SEQ ID NO:<NUM> , <NUM> or <NUM>, with the proviso that the seed lot is not exclusively produced by an essentially biological method.

In a fifth aspect, the invention relates to a method of making non-defatted pennycress seed meal having an acid detergent fiber (ADF) content of less than <NUM>% by dry weight, comprising the step of grinding, macerating, extruding, expanding, and/or crushing the seed lot of the fourth aspect thereby obtaining the non-defatted seed meal.

In a sixth aspect, the invention relates to a method of making defatted pennycress seed meal comprising an acid detergent fiber (ADF) content of <NUM>% to <NUM>% by dry weight, comprising the step of solvent extracting the seed lot of fourth aspect, separating the extracted seed meal from the solvent, thereby obtaining the defatted seed meal.

In a seventh aspect, the invention relates to a method of making pennycress seed cake comprising an acid detergent fiber (ADF) content of <NUM>% to <NUM>% by dry weight, comprising the step of crushing or expelling the seed of the seed lot of fourth aspect, thereby obtaining a seed cake.

The accompanying drawings, which are incorporated in and form a part of the specification, illustrate the embodiments of the present disclosure and together with the description, serve to explain the principles of the disclosure. In the drawings:.

The term "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the terms "include," "includes," and "including" are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

Where a term is provided in the singular, other embodiments described by the plural of that term are also provided.

To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.

Pennycress has value in both its oil and the resulting meal following the removal of oil. The meal is used for animal feed and is typically valued for its energy, protein and sometimes fiber. Fiber is usually delivered by forage elements (not protein supplements) and only a modest amount is desired. Fiber is measured by multiple measures including Crude Fiber (CF), Acid detergent Fiber (ADF) and Neutral detergent fiber (NDF). ADF is a useful determinant in estimating the energy available to animals. In certain embodiments, ADF can be measured gravimetrically using <NPL>". In certain embodiments, modifications of this method can include use of Sea Sand for filter aid as needed. NDF can be determined as disclosed in <NPL>. In certain embodiments, fiber (ADF and/or NDF), protein, and/or oil content can be determined by Near-infrared (NIR) spectroscopy.

Compositions comprising defatted- pennycress seed meal having less fiber than defatted control pennycress seed meal obtained from wild type pennycress seed, wherein the defatted pennycress seed meal comprises an acid detergent fiber (ADF) content of <NUM>% to <NUM>% by dry weight, and wherein the defatted pennycress seed meal is obtained from a seed lot comprising a loss-of function (LOF) homozygous mutation in an endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO: <NUM>, <NUM>, <NUM>, allelic variants having at least <NUM>% sequence identity with SEQ ID NO:<NUM> , <NUM> or <NUM>, or any combination thereof is hereby disclosed herein. In certain embodiments, the ADF content of defatted pennycress seed meal and compositions comprising the same that are disclosed herein is reduced from about <NUM>-, <NUM>-, <NUM>-, or <NUM>-fold to about <NUM>-, <NUM>-, <NUM>-, or <NUM>-fold in comparison to control defatted pennycress seed meal and compositions comprising the same obtained from control wild-type pennycress seeds. Typically, the level of acid detergent fiber (ADF) in wild-type pennycress seed varies from about <NUM> to about <NUM>% by dry weight. Defatted-pennycress meal is a product obtained from high-pressure crushing of seed, via mechanical pressing and/or expanding/extrusion, followed by a solvent extraction process, which removes oil from the whole seed. Solvents used in such extractions include, but are not limited to, hexane or mixed hexanes. The meal is the material that remains after most of the oil has been removed. During a typical oilseed processing procedure, extraction of the oil leads to concentration of fiber as a result of oil mass removal. The typical range of ADF in meal made from wild-type pennycress seed is <NUM>-<NUM>%. To be useful as a high protein animal feed, and competitive with other protein feedstuffs, the level of ADF level in meal should be less than <NUM>% by dry weight, less than <NUM>% by dry weight, or less than <NUM>% by dry weight of the meal. In certain embodiments, defatted pennycress seed meal having an ADF content of about <NUM>%, <NUM>%, or <NUM>% to <NUM>%, <NUM>%, <NUM>%, or <NUM> % by dry weight is disclosed herein.

Non-defatted pennycress seed meal having less fiber than non-defatted control pennycress seed meal obtained from wild type pennycress seed, wherein the non-defatted pennycress seed meal has an acid detergent fiber (ADF) content of less than <NUM>% by dry weight, and wherein the non-defatted pennycress seed meal is obtained from a seed lot comprising a loss-of function (LOF) homozygous mutation in an endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO: <NUM>, <NUM>, <NUM>, allelic variants having at least <NUM>% sequence identity with SEQ ID NO:<NUM> , <NUM> or <NUM>, or any combination thereof disclosed herein. In certain embodiments, the ADF content of non-defatted pennycress seed meal and compositions comprising the same that are disclosed herein is reduced from about <NUM>-, <NUM>-, <NUM>-, or <NUM>-fold to about <NUM>-, <NUM>-, <NUM>-, or <NUM>-fold in comparison to control non-defatted pennycress seed meal and compositions comprising the same obtained from control wild-type pennycress seeds. In certain embodiments, the non-defatted pennycress seed meal is obtained from pennycress seeds that have been crushed, ground, macerated, expelled, extruded, expanded, or any combination thereof. Typically, the level of acid detergent fiber (ADF) in wild-type pennycress seed and non-defatted seed meal obtained therefrom varies from about <NUM>% to about <NUM>% by dry weight. To be useful as a high protein animal feed, and competitive with other protein feedstuffs, the level of ADF level in non-defatted meal should be less than <NUM> % by dry weight, less than <NUM>% by dry weight, or less than <NUM>% by dry weight of the meal. In certain embodiments, non-defatted pennycress seed meal having an ADF content of less than <NUM>% by dry weight, less than <NUM>% by dry weight, less than <NUM>% by dry weight, or less than <NUM>% by dry weight of the meal is disclosed herein. In certain embodiments, non-defatted pennycress seed meal having an ADF content of about <NUM>%, <NUM>%, or <NUM>% to <NUM>%, <NUM>%, or <NUM> % by dry weight is disclosed herein. Compositions comprising such non-defatted pennycress seed meal are also disclosed herein.

In certain embodiments, pennycress seed lots comprising a population of seed having reduced fiber content, in comparison to fiber content of the control seed lots of wild-type pennycress seed according to the invention are provided. The aforementioned reductions in activity, specific activity, and/or transcript levels are provided by at least one homozygous LOF mutation in an endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO:<NUM>, <NUM>, <NUM>, allelic variants thereof, or any combination thereof. In certain embodiments, an endogenous wild-type pennycress gene can encode a polypeptide allelic variant having at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>, <NUM> or <NUM>. In certain aspects (aspects not covered by the invention as claimed), an endogenous wild-type pennycress gene can encode a polypeptide allelic variant having one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. In certain aspects (aspects not covered by the invention as claimed), the seed lots will comprise one or more transgenes that suppress expression of one or more genes, coding sequences, and/or proteins, thus resulting in reduced fiber content, reduced fiber content and increased protein content, reduced fiber content and increased oil content, or reduced fiber content, increased protein content, and increased oil content, all in comparison to control or wild-type pennycress seed lots. Transgenes that can provide for such suppression include, but are not limited to, transgenes that produce artificial miRNAs targeting a given gene or gene transcript for suppression. In certain aspects (aspects not covered by the invention as claimed), the transgenes that suppress expression will result in: (a) a reduction in the enzymatic or other biochemical activity associated with the encoded polypeptide in the plant comprising the transgene in comparison to a wild-type control plant; or (b) both a reduction in the enzymatic or other biochemical activity and a reduction in the amount of a transcript (e.g., mRNA) in the plant comprising the transgene in comparison to a wild-type control plant. Such reductions in activity and transcript levels can in certain aspects (aspects not covered by the invention as claimed) comprise a reduction of at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% of activity and/or transcript levels in the transgenic plant in comparison to the activity or transcript levels in a wild-type control plant. In certain aspects (aspects not covered by the invention as claimed), certain genes, coding sequences, and/or proteins that can be targeted for introduction of LOF mutations or that are targeted for transgene-mediated suppression are disclosed in the following Table <NUM> and accompanying Sequence Listing. In certain aspects (aspects not covered by the invention as claimed), allelic variants of the wild-type genes, coding sequences, and/or proteins disclosed in Table <NUM> and the sequence listing are targeted for introduction of LOF mutations or are targeted for transgene-mediated suppression. Allelic variants found in distinct pennycress isolates or varieties that exhibit wild-type seed fiber, protein, and or oil content can be targeted for introduction of LOF mutations or are targeted for transgene-mediated suppression to obtain seed lots having reduced fiber content, reduced fiber content and increased protein content, reduced fiber content and increased oil content, or reduced fiber content, increased protein, and increased oil content, all in comparison to fiber, protein, and oil content of the control seed lots of wild-type pennycress. Such allelic variants can comprise polynucleotide sequences that have at least <NUM>% , <NUM>%, <NUM>%, <NUM>%, or <NUM>% sequence identity across the entire length of the polynucleotide sequences of the wild-type coding regions or wild-type genes of Table <NUM> and the sequence listing. Such allelic variants can comprise polypeptide sequences that have at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% sequence identity across the entire length of the polypeptide sequences of the wild-type proteins of Table <NUM> and the sequence listing. Pennycress seed lots having reduced seed coat fiber, lighter-colored seed coat due to reduced proanthocyanidins content, increased protein content, and/or higher seed oil content as described herein can comprise one or more LOF mutations in one or more genes that encode polypeptides involved in seed coat and embryo formation or can comprise transgenes that suppress expression of those genes. Polypeptides affecting these traits include, without limitation, TRANSPARENT TESTA1 (TT1) through TRANSPARENT TESTA19 (TT19) (e.g., TT1, TT2, TT3, TT4, TT5, TT6, TT7, TT8, TT9, TT10, TT12, TT13, TT15, TT16, TT18, and TT19), TRANSPARENT TESTA GLABRA1 and <NUM> (TTG1 and TTG2), GLABROUS <NUM> (GL2), GLABROUS <NUM> (GL3), ANR-BAN, and AUTO INHIBITED H+-ATPASE <NUM> (AHA10) disclosed in Table <NUM>. In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots disclosed herein can comprise LOF mutations in any of the aforementioned wild-type pennycress genes disclosed in Table <NUM> or any combination of mutations disclosed in Table <NUM>. Compositions comprising defatted or non-defatted seed meal obtained from any of the aforementioned seed lots, defatted or non-defatted seed meal obtained from any of the aforementioned seed lots, and seed cakes obtained from any of the aforementioned seed lots are also disclosed herein. Methods of making any of the aforementioned seed lots, compositions, seed meals, or seed cakes are also disclosed herein. As used herein, the phrase "seed cake" refers to the material obtained after the seeds are crushed, ground, heated, and expeller pressed or extruded/expanded prior to solvent extraction.

In certain embodiments, reductions or increases in various features of seed lots, seed meal compositions, seed meal, or seed cake are in comparison to a control or wild-type seed lots, seed meal compositions, seed meal, or seed cake. Such controls include, but are not limited to, seed lots, seed meal compositions, seed meal, or seed cake obtained from control plants that lack the LOF mutations or transgene-mediated gene suppression. In certain aspects (aspects not covered by the invention as claimed), control plants that lack the LOF mutations or transgene-mediated gene suppression will be otherwise isogenic to the plants that contain the LOF mutations or transgene-mediated gene suppression.

In certain aspects (aspects not covered by the invention as claimed), the controls will comprise seed lots, seed meal compositions, seed meal, or seed cake obtained from plants that lack the LOF mutations or transgene-mediated gene suppression and that were grown in parallel with the plants having the LOF mutations or transgene-mediated gene suppression. Such features that can be compared to wild-type or control plants include, but are not limited to, ADF content, NDF fiber content, protein content, oil content, protein activity and/or transcript levels, and the like.

In certain aspects (aspects not covered by the invention as claimed), pennycress plants having reduced seed coat fiber, lighter-colored seed coat, and/or higher seed oil content as described herein can be from the Y1067, Y1126, BC38, BJ8, P32, J22, Q36, BD24, AX17, E5-<NUM>, E5-<NUM>, E5-<NUM>, E5-<NUM>, E5-<NUM>, E5-<NUM>, E5-<NUM>, E5-<NUM>, E5-<NUM>, E5-<NUM>, E5-<NUM>, D3-N10 P5, D5-<NUM>, A7-<NUM>, A7-<NUM>, or A7-<NUM> variant lines provided herein, or can be progeny derived from those lines.

A representative wild-type (WT) pennycress TT2 coding sequence is as shown in sequence listing (SEQ ID NO:<NUM>). In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT2 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a TT2 coding sequence allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. A representative wild-type pennycress TT2 polypeptide is shown in sequence listing (SEQ ID NO:<NUM>). In certain embodiments, a WT pennycress TT2 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM>) and is referred to as an allelic variant sequence.

In certain embodiments, a WT pennycress TT2 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM>), referred to herein as an allelic variant sequence, disclosed the polypeptide maintains its wild-type function. For example, a TT2 polypeptide can have at least <NUM>, at least <NUM>, or at least <NUM>) percent sequence identity to SEQ ID NO:<NUM>. A TT2 polypeptide of an allelic variant can have one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM>.

In certain embodiments, pennycress seed lots having reduced seed coat fiber, lighter-colored seed coat due to reduced proanthocyanidins content, and/or higher seed oil content as described herein can include at least one loss-of-function modification in a TT2 gene (e.g., in a TT2 coding sequence, in a TT2 regulatory sequence including the promoter, <NUM>' UTR, intron, <NUM>' UTR, or in any combination thereof) or a transgene that suppresses expression of the TT2 gene. As used herein, a loss-of-function mutation in a TT2 gene can be any modification that is effective to reduce TT2 polypeptide expression or TT2 polypeptide function. In certain embodiments, reduced TT2 polypeptide expression and/or TT2 polypeptide function can be eliminated or reduced in comparison to a wild-type plant. Examples of genetic modifications that can provide for a loss-of-function mutation include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, or any combination thereof.

In certain embodiments, pennycress seed lots having reduced seed coat fiber, lighter-colored seed coat, and/or higher seed oil and/or protein content as described herein can include a substitution (e.g., a single base-pair substitution) relative to the WT pennycress TT2 coding sequence. In certain aspects (aspects not covered by the invention as claimed), a modified TT2 coding sequence can include a single base-pair substitution of the cytosine (G) at nucleotide residue <NUM> in a WT pennycress TT2 coding sequence (e.g., SEQ ID NO:<NUM> or an allelic variant thereof). The G at nucleotide residue <NUM> can be substituted with any appropriate nucleotide (e.g., thymine (T), adenine (A), or cytosine (C)). For example, a single base-pair substitution can be a G to A substitution at nucleotide residue <NUM> in a WT pennycress TT2 coding sequence thereby producing a premature stop codon. A representative modified pennycress TT2 coding sequence having a loss-of-function single base pair substitution is presented in SEQ ID NO:<NUM>.

A modified pennycress TT2 coding sequence having a loss-of-function single base pair substitution (e.g., SEQ ID NO:<NUM>) can encode a modified TT2 polypeptide (e.g., a modified TT2 polypeptide having reduced TT2 polypeptide expression and/or reduced TT2 polypeptide function). For example, a modified pennycress TT2 coding sequence having a single base-pair substitution (e.g., SEQ ID NO:<NUM>) can encode a modified TT2 polypeptide. In certain aspects (aspects not covered by the invention as claimed), a modified TT2 polypeptide can include a truncation resulting from the introduction of a stop codon at codon position <NUM> within the TT2 open reading frame (e.g., SEQ ID NO:<NUM>). A representative truncated pennycress TT2 polypeptide is presented in SEQ ID NO:<NUM>. Representative pennycress varieties having a mutation in the TT2 gene include the tt2-<NUM>, tt2-<NUM>, BC38, and E5-<NUM> varieties.

A representative WT pennycress TRANSPARENT TESTA8 (TT8) coding region is presented in SEQ ID NO:<NUM>. Two protospacer locations and adjacent protospacer-adjacent motif (PAM) sites that can be targeted by, for example, CRISPR-SpCAS9 correspond to nucleotides <NUM>-<NUM> and <NUM>-<NUM> (protospacers) or <NUM>-<NUM> and <NUM>-<NUM> (PAM sites). In another aspects (aspects not covered by the invention as claimed), two separate examples of alternative protospacer locations and adjacent protospacer-adjacent motifs (PAM) sites are provided in <FIG>. In each case, two protospacer locations can be targeted by, for example, CRISPR-FnCpf1, CRISPR-SmCsm1 or a similar enzyme, correspond to nucleotides <NUM>-<NUM> and <NUM>-<NUM> (protospacers) or <NUM>-<NUM> and <NUM>-<NUM> (PAM sites); and nucleotides <NUM>-<NUM> and <NUM>-<NUM> (protospacers) or <NUM>-<NUM> and <NUM>-<NUM> (PAM sites), all of SEQ ID NO:<NUM>.

In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT8 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a TT8 coding sequence allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. A representative WT pennycress TT8 polypeptide is presented in SEQ ID NO:<NUM>.

In certain embodiments, a WT pennycress TT8 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM>) and is referred to as an allelic variant sequence. For example, a TT8 polypeptide can have at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. A TT8 polypeptide can have one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM>.

In certain embodiments, pennycress seed lots having reduced fiber content as described herein can include a loss-of-function modification in a TT8 gene (e.g., in a TT8 coding sequence) or a transgene that suppresses expression of the TT8 gene. As used herein, a loss-of-function mutation in a TT8 gene can be any modification that is effective to reduce TT8 polypeptide expression or TT8 polypeptide function. In certain embodiments, reduced TT8 polypeptide expression and/or TT8 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof. Representative TT8 gene mutations include the mutations shown in SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> that result in the TT8 mutant polypeptides of SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>, respectively. Representative pennycress varieties with TT8 gene mutations include the tt4-<NUM> tt8-<NUM>, tt8-<NUM>, tt8-<NUM>, tt8-<NUM>, tt8-<NUM>, tt8-<NUM>, tt8-<NUM>, I0193, E5-<NUM>, E5-<NUM>, D5-<NUM>, D3-N25P1, E5-<NUM>, A7-<NUM>, and D3-N10 P5 varieties.

In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT1 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:<NUM> or <NUM>), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a TT1 coding sequence allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM> or <NUM>. In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT1 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM> or <NUM>), and is referred to as an allelic variant sequence. For example, a TT1 polypeptide allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM> or <NUM>. A TT1 polypeptide allelic variant can have one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM> or <NUM>.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a TT1 encoding gene or a transgene that suppresses expression of the TT1 gene. As used herein, a loss-of-function mutation in a TT1 gene can be any modification that is effective to reduce TT1 polypeptide expression or TT1 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), reduced TT1 polypeptide expression and/or TT1 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a TT1 encoding gene, a promoter thereof, or a terminator, thereof, or a transgene that suppresses expression of the TT1 gene. As used herein, a loss-of-function mutation in a TT1 gene can be any modification that is effective to reduce TT1 polypeptide expression or TT1 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), reduced TT1 polypeptide expression and/or TT1 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof.

In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT4 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a TT4 coding sequence allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT4 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. For example, a TT4 polypeptide allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. A TT4 polypeptide allelic variant can have one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM>.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a TT4 encoding gene or a transgene that suppresses expression of the TT4 gene. As used herein, a loss-of-function mutation in a TT4 gene can be any modification that is effective to reduce TT4 polypeptide expression or TT4 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), reduced TT4 polypeptide expression and/or TT4 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof. Representative TT4 gene mutations include the mutation shown in SEQ ID NO:<NUM> that results in the truncated TT4 mutant polypeptide of SEQ ID NO:<NUM>. Representative TT4 gene mutations also include the mutations shown in SEQ ID NO:<NUM> and <NUM> that result in the TT4 mutant polypeptides of SEQ ID NO: <NUM> and <NUM>, respectively. Representative pennycress varieties with TT4 gene mutations include the tt4-<NUM>, tt4-<NUM>, tt4-<NUM>, A7-<NUM>, E5-<NUM> and E5-<NUM> varieties.

In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT5, TT9, TT15, TT18, or TT19 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, or <NUM>, respectively), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a TT5, TT9, TT15, TT18, or TT19 coding sequence allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, or <NUM>, respectively. In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT5, TT9, TT15, TT18, or TT19 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, or <NUM>, respectively), and is referred to as an allelic variant sequence. For example, a TT5, TT9, TT15, TT18, or TT19 polypeptide allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, or <NUM>, respectively. A TT5, TT9, TT15, TT18, or TT19 polypeptide allelic variant can have one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, or <NUM>, respectively.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a TT5, TT9, TT15, TT18, or TT19 encoding gene or a transgene that suppresses expression of the TT5, TT9, TT15, TT18, or TT19 gene. As used herein, a loss-of-function mutation in a TT5 gene can be any modification that is effective to reduce TT5, TT9, TT15, TT18, or TT19 polypeptide expression or TT5, TT9, TT15, TT18, or TT19 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), TT5, TT9, TT15, TT18, or TT19 polypeptide expression and/or TT5, TT9, TT15, TT18, or TT19 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof.

In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT6 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a TT6 coding sequence allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT6 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. For example, a TT6 polypeptide allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. A TT6 polypeptide allelic variant can have one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM>.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a TT6 encoding gene or a transgene that suppresses expression of the TT6 gene. As used herein, a loss-of-function mutation in a TT6 gene can be any modification that is effective to reduce TT6 polypeptide expression or TT6 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), reduced TT6 polypeptide expression and/or TT6 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof. Representative TT6 gene mutations include the mutation shown in SEQ ID NO: <NUM> that results in the TT6 mutant polypeptide of SEQ ID NO: <NUM>. Representative pennycress varieties with TT6 gene mutations mutants include the tt6-<NUM> and AX17 varieties. Representative TT6 gene mutations also include the mutation shown in SEQ ID NO:<NUM> that results in the TT6 mutant polypeptide of SEQ ID NO: <NUM>. Representative pennycress varieties with TT6 gene mutations mutants also include the tt6-<NUM>, tt6-<NUM> and Q36 varieties.

In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT7 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a TT7 coding sequence allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT7 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. For example, a TT7 polypeptide allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. A TT7 polypeptide allelic variant can have one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM>.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a TT7 encoding gene or a transgene that suppresses expression of the TT7 gene. As used herein, a loss-of-function mutation in a TT7 gene can be any modification that is effective to reduce TT7 polypeptide expression or TT7 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), reduced TT7 polypeptide expression and/or TT7 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof. Representative TT7 gene mutations include the mutation shown in SEQ ID NO:<NUM> that results in the TT7 mutant polypeptide of SEQ ID NO: <NUM>. Representative pennycress varieties with TT7 gene mutations include the tt7-<NUM>, A7-<NUM>, E5-<NUM>, E5-<NUM> P15, and E5-<NUM> P5 varieties.

In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TTG1 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a TTG1 coding sequence allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TTG1 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. For example, a TTG1 polypeptide allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM> or <NUM>. A TTG1 polypeptide allelic variant can have one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM>.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function (LOF) modification in a TTG1 encoding gene or a transgene that suppresses expression of the TTG1 gene. As used herein, a loss-of-function mutation in a TTG1 gene can be any modification that is effective to reduce TTG1 polypeptide expression or TTG1 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), reduced TTG1 polypeptide expression and/or TTG1 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof. In certain aspects (aspects not covered by the invention as claimed), a LOF mutation in a TTG1 gene can comprise a 21bp deletion in the TTG1 coding sequence as shown in SEQ ID NO:<NUM>. In other aspects (aspects not covered by the invention as claimed), a LOF mutation in a TTG1 gene can comprise ttg1-<NUM> and ttgl-<NUM> mutant alleles having single nucleotide substitutions that result in the substitution of a conserved amino acid residue in the TTG protein (SEQ ID NOs: <NUM>-<NUM>). Representative TTG1 gene mutations thus include the mutations shown in SEQ ID NO:<NUM>, <NUM>, and <NUM> that result in the TTG1 mutant polypeptides of SEQ ID NO: <NUM>, <NUM>, and <NUM>, respectively. Representative pennycress varieties with TTG1 gene mutations include the Y1067, Y1126, ttg1-<NUM>, E5-<NUM>, ttg1-<NUM>, and A7-<NUM> varieties.

In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT10 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a TT10 coding sequence allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT10 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. For example, a TT10 polypeptide allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. A TT10 polypeptide allelic variant can have one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM>.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a TT10 encoding gene or a transgene that suppresses expression of the TT10 gene. As used herein, a loss-of-function mutation in a TT10 gene can be any modification that is effective to reduce TT10 polypeptide expression or TT10 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), reduced TT10 polypeptide expression and/or TT10 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a TT10 encoding gene or a transgene that suppresses expression of the TT10 gene. As used herein, a loss-of-function mutation in a TT10 gene can be any modification that is effective to reduce TT10 polypeptide expression or TT10 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), reduced TT10 polypeptide expression and/or TT10 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof. Representative TT10 gene mutations include the mutations shown in SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, or <NUM> that result in the TT10 mutant polypeptides of SEQ ID NO: <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>, respectively. Representative pennycress varieties with TT10 gene mutations include the tt10-<NUM>, tt10-<NUM>, tt10-<NUM>, tt10-<NUM>, tt10-<NUM>, E5-<NUM>, E5-<NUM>, and E5-<NUM> varieties.

In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT12 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a TT12 coding sequence allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT12 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. For example, a TT12 polypeptide allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. A TT12 polypeptide allelic variant can have one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM>.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a TT12 encoding gene or a transgene that suppresses expression of the TT12 gene. As used herein, a loss-of-function mutation in a TT12 gene can be any modification that is effective to reduce TT12 polypeptide expression or TT12 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), reduced TT12 polypeptide expression and/or TT12 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a TT12 encoding gene or a transgene that suppresses expression of the TT12 gene. As used herein, a loss-of-function mutation in a TT12 gene can be any modification that is effective to reduce TT12 polypeptide expression or TT12 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), reduced TT12 polypeptide expression and/or TT12 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof. Representative TT12 gene mutations include the mutations shown in SEQ ID NO:<NUM> or <NUM> that result in the TT12 mutant polypeptides of SEQ ID NO:<NUM> or <NUM>, respectively. Representative pennycress varieties with TT12 gene mutations include the tt12-<NUM>, tt12-<NUM>, A7-<NUM>, and J22 varieties.

In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT13 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a TT13 coding sequence allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT13 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. For example, a TT13 polypeptide allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. A TT13 polypeptide allelic variant can have one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM>.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a TT13 encoding gene or a transgene that suppresses expression of the TT13 gene. As used herein, a loss-of-function mutation in a TT13 gene can be any modification that is effective to reduce TT13 polypeptide expression or TT13 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), reduced TT13 polypeptide expression and/or TT13 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof. Representative TT13 gene mutations include the mutations shown in SEQ ID NO:<NUM>, <NUM>, or <NUM> that result in the TT13 mutant polypeptides of SEQ ID NO:<NUM>, <NUM>, or <NUM>, respectively. Representative pennycress varieties with TT13 gene mutations include the tt13-<NUM>, tt13-<NUM>, tt13-<NUM>, aha10-<NUM>, J22, and P32 E5-<NUM> varieties.

In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT16 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a TT16 coding sequence allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. In certain aspects (aspects not covered by the invention as claimed), a WT pennycress TT16 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a TT16 polypeptide allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. A TT16 polypeptide allelic variant can have one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM>.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a TT16 encoding gene or a transgene that suppresses expression of the TT16 gene. As used herein, a loss-of-function mutation in a TT16 gene can be any modification that is effective to reduce TT16 polypeptide expression or TT16 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), reduced TT16 polypeptide expression and/or TT16 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a TT16 encoding gene or a transgene that suppresses expression of the TT16 gene. As used herein, a loss-of-function mutation in a TT16 gene can be any modification that is effective to reduce TT16 polypeptide expression or TT16 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), reduced TT16 polypeptide expression and/or TT16 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof. Representative TT16 gene mutations include the mutations shown in SEQ ID NO:<NUM>, <NUM>, or <NUM> that result in the TT16 mutant polypeptides of SEQ ID NO:<NUM>, <NUM>, or <NUM>, respectively. Representative pennycress varieties with TT16 gene mutations include the tt16-<NUM>, tt16-<NUM>, and tt16-<NUM> varieties.

In certain aspects (aspects not covered by the invention as claimed), a genome editing system such as a CRISPR-Cas9 system can be used to introduce one or more loss-of-function mutations into genes such as the TRANSPARENT TESTA (TT) and related genes disclosed herewith in Table <NUM> and the sequence listing that are associated with agronomically-relevant seed traits including reduced seed coat fiber, lighter-colored seed coat due to reduced proanthocyanidins content, increased protein content, and/or higher seed oil content. For example, a CRISPR-Cas9 vector can include at least one guide sequence specific to a pennycress TT2 sequence (see, e.g., SEQ ID NO:<NUM>) and/or at least one guide sequence specific to a pennycress TT8 sequence (see, e.g., SEQ ID NO:<NUM>). A Cas9 enzyme will bind to and cleave within the gene when the target site is followed by a PAM sequence. For example, the canonical SpCAS9 PAM site is the sequence <NUM>'-NGG-<NUM>', where N is any nucleotide followed by two guanine (G) nucleotides. The Cas9 component of a CRISPR-Cas9 system designed to introduce one or more loss-of-function modifications described herein can be any appropriate Cas9. In certain aspects (aspects not covered by the invention as claimed), the Cas9 of a CRISPR-Cas9 system described herein can be a Streptococcus pyogenes Cas9 (SpCas9). One example of an SpCas9 is described in (Fauser et al.

In certain aspects (aspects not covered by the invention as claimed), a WT pennycress GL3 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a GL3 coding sequence allelic variants can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. In certain aspects (aspects not covered by the invention as claimed), a WT pennycress GL3 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. For example, a GL3 polypeptide allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. A GL3 polypeptide allelic variant can have one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM>.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a GL3 encoding gene or a transgene that suppresses expression of the GL3 gene. As used herein, a loss-of-function mutation in a GL3 gene can be any modification that is effective to reduce GL3 polypeptide expression or GL3 polypeptide function. In certain aspects (aspects not covered by the invention as claimed), GL3 polypeptide expression and/or GL3 polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof. In certain aspects (aspects not covered by the invention as claimed), the GL3 mutation can comprise the coding sequence mutations of SEQ ID NO:<NUM>, <NUM>, <NUM> and/or the protein sequence mutation of SEQ ID NO:<NUM>, <NUM>, <NUM>. Representative pennycress varieties with GL3 gene mutations include the gl3-<NUM>, gl3-<NUM>, gl3-<NUM>, E5-<NUM>, E5-<NUM>, A7-<NUM>, E5-<NUM>, A7-<NUM>, and E5-<NUM> varieties.

In certain aspects (aspects not covered by the invention as claimed), a WT pennycress BAN-ANR (or BAN) coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. In certain aspects (aspects not covered by the invention as claimed), a BAN coding sequence allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. In certain aspects (aspects not covered by the invention as claimed), a WT pennycress BAN polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:<NUM>), and is referred to as an allelic variant sequence. For example, a BAN polypeptide allelic variant can have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to SEQ ID NO:<NUM>. A BAN polypeptide allelic variant can have one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:<NUM>.

In certain aspects (aspects not covered by the invention as claimed), pennycress seed lots having reduced fiber as described herein can include a loss-of-function modification in a BAN encoding gene or a transgene that suppresses expression of the BAN gene. As used herein, a loss-of-function mutation in a BAN gene can be any modification that is effective to reduce BAN polypeptide expression and/or BAN polypeptide function. In certain aspects (aspects not covered by the invention as claimed), BAN polypeptide expression and/or BAN polypeptide function can be eliminated or reduced. Examples of genetic modifications include, without limitation, deletions, insertions, substitutions, translocations, inversions, duplications, and any combination thereof. In certain aspects (aspects not covered by the invention as claimed), the BAN mutation can comprise the coding sequence mutation of SEQ ID NO:<NUM> and/or the protein sequence mutation of SEQ ID NO:<NUM>. Representative pennycress varieties with BAN gene mutations include the ban-<NUM>, BJ8, and BJ8D varieties.

In certain aspects (aspects not covered by the invention as claimed), pennycress seeds or seed lots having reduced fiber, as well as pennycress seed meal obtained therefrom (including both defatted and non-defatted seed meal), as described herein can include a loss-of-function mutation in more than one of the genes or coding sequences set forth in Table <NUM>. In certain aspects (aspects not covered by the invention as claimed), pennycress seeds or seed lots having reduced fiber can have a LOF mutation in the gene(s) and/or coding sequences of any combination of SEQ ID NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,<NUM>, <NUM>, and/or any allelic variants thereof. In certain aspects (aspects not covered by the invention as claimed), pennycress seed meal, including de-fatted and non-defatted forms) and having reduced fiber can comprise a detectable amount of any combination of nucleic acids having a LOF mutation in the gene(s) and/or coding sequences of any combination of SEQ ID NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and/or any allelic variants thereof.

The LOF mutations in any of the genes or coding sequences of Table <NUM> can be introduced by a variety of methods. Methods for introduction of the LOF mutations include, but are not limited to, traditional mutagenesis (e.g., with EMS or other mutagens), TILLING, meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease (e.g., S. pyogenes Cas9 and its variants, S. aureus Cas9 and its variants, eSpCas9, Cpf1, Cms1 and their variants) targetrons, and the like. Various tools that can be used to introduce mutations into genes have been disclosed in <NPL>. Methods for modifying genomes by use of Cpf1 or Csm1 nucleases are disclosed in <CIT>, and can be adapted for introduction of the LOF mutations disclosed herein. Methods for modifying genomes by use of CRISPR/CAS systems are disclosed in <CIT>, and can be adapted for introduction of the LOF mutations disclosed herein. The genome editing reagents described herein can be introduced into a pennycress plant by any appropriate method. In certain aspects (aspects not covered by the invention as claimed), nucleic acids encoding the genome editing reagents can be introduced into a plant cell using Agrobacterium or Ensifer mediated transformation, particle bombardment, liposome delivery, nanoparticle delivery, electroporation, polyethylene glycol (PEG) transformation, or any other method suitable for introducing a nucleic acid into a plant cell. In certain aspects (aspects not covered by the invention as claimed), the Site-Specific Nuclease (SSN) or other expressed gene editing reagents can be delivered as RNAs or as proteins to a plant cell and the RT, if one is used, can be delivered as DNA.

The disclosure will be further described in the following examples, which do not limit the scope of the disclosure described in the claims.

Higher dietary fiber results in lower net energy for swine (Kil et al. , <NUM>) and poultry (Meloche et al. It was also reported that hemicellulose displayed the strongest correlation with apparent metabolizable energy (AMEn), followed by neutral detergent fiber (NDF), total dietary fiber (TDF), and crude fiber (CF) in broilers fed corn co-products (Rochelle et al. Thus, a reduction in fiber will result in increased available energy to pigs and poultry.

When comparing mechanically expeller-pressed meals made from two USDA-developed pennycress varieties (Beecher and Ruby II) to mechanically expeller-pressed canola meal, the various fiber fractions when analyzed as crude fiber (CF), acid detergent fiber (ADF), neutral detergent fiber (NDF) and total dietary fiber (TDF) were <NUM>-<NUM> times the levels in canola meal (Table <NUM>). Similar levels were observed when comparing different lots of pennycress meal with canola meal (Table <NUM>). Analysis conducted by Arvegenix at University of Georgia showed similar results (Table <NUM>).

Total Metabolizable Energy (TMEn) corrected for nitrogen was measured in mechanically expeller-pressed pennycress meal and canola meal. TMEn was found to be <NUM>% or <NUM>% less in the pennycress meal as compared to the canola meal when fed to chickens due to the higher fiber content (Table <NUM>) and Metabolizable Energy (ME) was <NUM>% less in pennycress meal as compared to the canola meal when fed to pigs due to the higher fiber content (Table <NUM>).

In summary, Beecher and Ruby II varieties of pennycress meal contain between <NUM>× to <NUM>× the fiber content as compared to similarly processed canola meal resulting in <NUM>-<NUM>% less energy when fed to chickens and pigs. Reduction in the fiber content of pennycress to levels of those in canola should result in a significant increase in value and energy to poultry and pigs.

About <NUM> wildtype pennycress seed samples exhibited a dark-brown seed coat were collected. These wildtype samples were then cultivated as independent lines for over two seasons in over <NUM>,<NUM> unique and managed plots. Upon careful analysis of the harvests from these dark type plantings, a few individual seeds which were yellow in color were identified in only two of the <NUM> cultivated lines (Table <NUM>) and selected for further propagation and breeding. Certain selected pennycress variant lines Y1067 and Y1126 were isolated from a cultivated field in Grantfork IL. Certain selected pennycress Y1126 lines were isolated from a cultivated field in Macomb IL in <NUM>. As no yellow pennycress seeds were reported to date, initially, the isolates were first assumed to be weed seeds from a species other than pennycress. However, upon careful evaluations of plants grown from these seeds in the greenhouse, they were positively identified as pennycress using visual (plant morphology) and molecular (PCR/sequencing) inspections. The selected Y1067 and Y1126 lines were then carefully grown as single seed isolates to produce progeny lines which consisted of <NUM>% yellow seeds. The yellow seed coat trait in the selected Y1067 and Y1126 lines has now been confirmed to be stable for several generations in both greenhouse and field environments.

Seeds from the yellow-seeded lines (Y1067 and Y1126) were carefully bulked up and sent to an analytical lab (Dairyland Laboratories) for analysis. Upon removal of the oil using standard defatting procedure, a small amount of yellow pennycress meal was produced and determined to have an ADF level (adjusted for oil content) of <NUM>% and <NUM>% vs. <NUM>% in wild type, demonstrating <NUM>-<NUM>% reduction in ADF fiber. Other measurements of fiber content such as NDF and CF were also significantly (<NUM>-<NUM>%) lower in the yellow-seeded lines relative to wild type, while the protein level was significantly (~<NUM>%) higher. The composition of yellow and dark brown seeds is listed in Table <NUM>. The yellow Y1067 and Y1126 lines have since been crossed with "regular" dark brown-seeded pennycress and demonstrated a non-reciprocal pattern of inheritance indicating that yellow seed coat is a maternally inherited trait.

In order to determine molecular nature of the mutations responsible for the low fiber, high oil/high protein phenotype in Y1067 and Y1126 lines, a combination of a genetic method called bulk segregant analysis (Michelmore et. , <NUM>) and a next generation sequencing (NGS) method was used. In brief, for each of the yellow-seeded lines, a genetically close black-seeded relative line was identified and <NUM> individuals from each population were grown. They were harvested in bulk and used for DNA isolation that was subsequently used for preparation of NGS libraries and sequencing using standard Illumina technology. It was determined that Y1067 and Y1126 lines carry the same <NUM> bp deletion in TTG1 gene (Seq ID No. <NUM>) by analyzing the sequencing data through comparative bioinformatics techniques. Comparative bioinformatics tools that were used in part to analyze the data are disclosed in Magwene et. This mutation results in a deletion of <NUM> amino acids in the conserved area of TTG1 protein, likely leading to a complete loss of function. The definitive nature of this <NUM> bp deletion was confirmed in heterologous (black ♀ × yellow ♂) crosses, where only the progeny of F2 segregants carrying the described deletion displayed the yellow-seeded phenotype.

In addition to mutants carrying domestication enabling traits selected from natural isolates, light colored pennycress mutants were isolated from a mutant population created using chemical mutagen (EMS) using the protocol described in the Materials and Methods section below.

To identify useful domestication genes in pennycress plants, pennycress seeds were mutagenized with several different mutagens, including ethyl methanesulfonate (EMS), fast neutrons (FN) and gamma rays (γ rays). Treatment of dry plant seeds with mutagens results in the generation of distinct sets of mutations in a variety of cells in the seed. The fate of many of these cells can be followed when a mutation in one of these cells results in a visible phenotype creating a marked plant sector.

Pennycress plants exhibiting domestication enabling traits such as reduced seed coat fiber, lighter-colored seed coat due to reduced proanthocyanidins content, and/or higher seed oil content were analyzed and loss of function mutations in domestication genes were identified.

For <NUM> of <NUM> sodium phosphate buffer at pH <NUM>:.

Wild-type pennycress (Thlaspi arvense) seeds (Spring <NUM> ecotype) were surface sterilized for <NUM> minutes in a <NUM>% bleach, <NUM>% SDS solution before being rinsed 3X with sterile water. Sterilized seeds were immediately subjected to EMS treatment.

Sterilized pennycress seeds (<NUM>) were agitated in distilled water overnight. Four <NUM> Erlenmeyer flasks with <NUM> seed each, and <NUM> in a separate small flask as a control, were agitated. The water was decanted.

<NUM> mLs of <NUM>% EMS in <NUM> sodium phosphate buffer (pH <NUM>) was added. The control received only phosphate buffer with no EMS. The flasks were shaken in fume hood for <NUM> hours. The EMS solution was decanted off into an EMS waste bottle.

To rinse the seeds, <NUM> of dH<NUM>O was added to each flask, and the flasks were shaken for <NUM> minutes. The rinse water was decanted into the EMS waste bottle.

To deactivate the EMS, seeds were washed for <NUM> minutes in <NUM> sodium thiosulfate (pH <NUM>), rinsed <NUM> with dH2O for <NUM> minutes, suspended in <NUM>% agarose, and germinated directly in autoclaved Reddiearth soil at a density of approximately <NUM> seeds per <NUM>-inch pot.

EMS-treated pennycress seeds were germinated and grown in an environmental growth chamber at <NUM>, <NUM>:<NUM><NUM> fluorescent light/dark, <NUM>% humidity. Approximately <NUM> days after planting, plants were thinned and transplanted to a density of <NUM> plants per <NUM>-inch pot. These M<NUM>-generation plants showed telltale chlorotic leaf sectors that are indicative of a successful mutagenesis.

After dry down, these M<NUM>-generation plants were catalogued and harvested. The M<NUM>- and M<NUM>-generation seeds were surface sterilized, planted and grown according to the protocols previously described.

Light-colored seed coat mutants in the M<NUM>-generation were identified as those having mature seed coats of a lighter color relative to that of wild type. Seeds (M<NUM>-generation) from putative M<NUM>-generation mutants were planted and grown in potting soil-containing <NUM>-inch pots in a growth chamber and the seed coat color phenotype re-assessed upon plant senescence.

Near infrared (NIR) spectroscopic analysis was used to determine the fiber content of selected seed lines to compare the obtained values to the range of fiber in control dark brown seeds. The results are presented in Table <NUM> of Example <NUM> (five light-colored lines mentioned above vs. almost one hundred control dark brown seed lines). These results indicate that ADF and NDF fiber levels in certain selected light-colored seed lines are significantly lower and are outside of the corresponding ranges found in control dark-colored seeds, while oil and protein levels are often higher and are also outside of their corresponding ranges found in dark-colored control seeds.

EMS mutagenesis typically introduces single-nucleotide transition mutations (e.g. G to A, or C to T) into plant genomes. To identify the causative mutations in selected light seed colored plants, DNA was extracted from mutant and wild-type leaf tissue and used for NGS and comparative bioinformatics analysis as described in Example <NUM>. Underlying gene and protein mutations were identified (Table <NUM>, SEQ ID NO: <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM> and <NUM>-<NUM>) and confirmed using standard Sanger sequencing and genetic segregation analyses.

The constructs and cloning procedures for generation of the Thlaspi arvense (pennycress) TT2-, TT8-, TT10-, and TT16-specific CRISPR-SpCas9, CRISPR-SaCas9, CRISPR-Cpf1 and CRISPR-Cms1 constructs are described in Fauser et. , <NUM>, Steinert et. , <NUM> and Begemann et.

The plant selectable markers (formerly NPT) in the original pDe-SpCas9 and pDe-SaCas9 binary vectors were swapped for hygromycin resistance (Hygromycin phosphotransferase (HPT) gene.

Complementary oligo pairs described in Table <NUM> (Seq ID NO: <NUM>-<NUM>) were synthesized, annealed to create the <NUM>-mer protospacers specific to the designated pennycress genes and used for construction of gene-editing binary vectors as described (Fauser et. , <NUM>, Steinert et. , <NUM> and Begemann et.

The pDe-SpCas9_Hyg and pDe-SaCas9_Hyg and related vectors containing the CRISPR nuclease and guide RNA cassettes with the corresponding sequence-specific protospacers were transformed into Agrobacterium tumefaciens strain GV3101 using the freeze/thaw method (Holsters et al, <NUM>).

The transformation product was plated on <NUM>% agar Luria Broth (LB) plates with gentamycin (<NUM>µg/ml) rifampicin (<NUM>µg/ml) and spectinomycin (<NUM>µg/ml). Single colonies were selected after two days of growth at <NUM>.

DAY ONE: <NUM> of LB + 5uL with appropriate antibiotics (Rifampin (<NUM>), Spectinomycin (<NUM>), and/or Gentamycin (<NUM>)) were inoculated with Agrobacterium. The cultures were allowed to grow, with shaking, overnight at <NUM>.

DAY TWO (early morning): <NUM> of Luria Broth + 25uL appropriate antibiotics (Rifampin (<NUM>), Spectinomycin (<NUM>), and/or Gentamycin (<NUM>)) were inoculated with the initial culture from day one. The cultures were allowed to grow, with shaking, overnight at <NUM>.

DAY TWO (late afternoon): <NUM> of Luria Broth + 250uL appropriate antibiotic (Rifampin (<NUM>), Spectinomycin (<NUM>), and/or Gentamycin (<NUM>)) were inoculated with <NUM> culture. The cultures were allowed to grow, with shaking, overnight at <NUM>.

DAY THREE: When the culture had grown to an OD<NUM> of ~<NUM>, the culture was decanted into large centrifuge tubes and spun at <NUM>,<NUM> RPM at room temperature for <NUM> minutes to pellet cells. The supernatant was decanted off. The pelleted cells were resuspended in a solution of <NUM>% sucrose and <NUM>% Silwet L-<NUM>. The suspension was poured into clean beakers and placed in a vacuum chamber.

Newly flowering inflorescences of pennycress were fully submerged into the beakers and subjected to a negative vacuum pressure of <NUM>-<NUM> PSI for <NUM> minutes.

After pennycress plants were dipped, they were covered loosely with Saran wrap to maintain humidity and kept in the dark overnight before being uncovered and placed back in the environmental growth chamber.

Pennycress seeds were surface sterilized by first rinsing in <NUM>% ethanol then incubating <NUM> minutes in a <NUM>% bleach, <NUM>% SDS solution before being rinsed two times with sterile water and plated on selective plates (<NUM>% agar/one half-strength Murashige and Skoog salts with hygromycin B selection (<NUM> U/ml) or glufosinate (<NUM>µg/ml). Plates were wrapped in parafilm and kept in an environmental growth chamber at <NUM>, <NUM>:<NUM> day/night for <NUM> days until antibiotic or herbicide selection was apparent.

Surviving hygromycin or glufosinate-resistant T<NUM>-generation seedlings were transplanted into autoclaved Reddiearth soil mix and grown in an environmental growth chamber set to <NUM>-hour days/<NUM>-hour nights at <NUM> and <NUM>% humidity. T<NUM>-generation seeds were planted, and ~<NUM> of leaf tissue from each T<NUM>-generation plant was harvested with a <NUM>-mm hole punch, then processed using the Thermo Scientific™ Phire™ Plant Direct PCR Kit as per manufacturer's instructions. Subsequently, PCR reactions for genotyping (<NUM>µl volume) were performed.

Gene editing using Cas9, Cpf1 and Cms1 nucleases typically introduces a double-stranded break into a targeted genome area in close proximity to the nuclease's PAM site. During non-homologous end-joining process (NHEJ), these double-stranded breaks are repaired, often resulting in introduction of indel-type mutations into targeted genomes. To identify plants with small indels in genes of interest, standard Sanger sequencing or T7 endonuclease assay (Guschin et. , <NUM>) were employed. Sequence analysis revealed that multiple guide RNAs/CRISPR nuclease combinations were effective in generating loss-of-function (LOF) mutations in targeted genes, as described in Table <NUM> (Seq ID Nos. <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>). Plants carrying LOF mutations were grown to homozygosity, and the phenotypes were confirmed using visual and analytical assessments.

Homozygous light seed coat-colored mutants obtained from screening EMS populations or from gene editing were bulked up in the greenhouse or in the fields and their fiber composition was assessed using standard methods below at Dairyland Laboratories (Arcadia, Wisconsin).

Fiber (Acid Detergent) and Lignin in Animal Feed: AOAC Official Method <NUM> (<NUM>) (Modification includes use of Sea Sand for filter aid as needed).

Fiber (Crude) in Animal Feed and Pet Food (Fritted Glass Crucible Method): AOAC Official Method <NUM> ch4 p28 (<NUM>) (Modification includes use of Sea Sand for filter aid as needed).

Fiber (Acid Detergent) and Lignin in Animal Feed: AOAC Official Method <NUM> (<NUM>) (Modification includes use of Sea Sand for filter aid as needed, use of Whatman GF/C filter paper to collect residue, and holding crucibles in beakers to cover fiber with <NUM>% sulfuric acid for full time required).

Amylase-Treated Neutral Detergent Fiber in Feeds AOAC Official Method <NUM><NUM> (Modification includes use of Sea Sand for filter aid and Whatman GF/C filter paper for residue collection).

The results presented in Table <NUM> indicate that majority of the light-colored mutants have <NUM>-<NUM>% less fiber and its components relative to WT plants (MN106 and Beecher).

Approximately <NUM> lbs each of cleaned Y1126 yellow-seeded mutant and regular black-seeded pennycress seed were processed into oil and hexane-extracted meal at the Texas A&M Engineering Experiment Station's Process Engineering Research & Development Center (College Station, TX). The material was conditioned using a single deck of the French cooker for approximately <NUM> minutes at <NUM>°F ± <NUM>°F. Conditioned seed was processed using a Ferrel Ross flaking rolls to yield flakes with a thickness of approximately <NUM> inches or thinner.

The flakes were loaded into a cooker with the objective of inactivating lipases, myrosinases, and other hydrolytic enzymes to facilitate pre-pressing. Maximum steam was used to get the flakes to <NUM>°F without lingering to avoid activation of such enzymes. This was achieved in <NUM>-<NUM> minutes. The press (Rosedowns Mini <NUM>) was fed from a Wenger metered feeder with flake at a rate of <NUM>-<NUM> pounds per minute. The press operated best at <NUM>-<NUM>, which corresponds to <NUM>-<NUM> RPM.

The presscake was extracted in stainless batch cans using commercial hexane at a temperature of <NUM>-<NUM>°F ± <NUM>°F. Solvent was added and drained sequentially in <NUM> rounds of incubation, each of which was approximately <NUM> minutes. To remove residual hexane and yield desolventized meal, a batch-type desolventizer/toaster (DT) was heated, which showed a product temperature of <NUM>-<NUM>°F under vacuum. Crude oil was made by desolventizing using a Precision Scientific Evaporator. The hexane extracted meal was air dried overnight.

Samples of the hexane extracted meal were sent to Dairyland and DairyOne Laboratories for analysis. A sample of commercial canola meal was acquired from a feed plant in Wisconsin, which was also sent to DairyOne for comparison.

Samples of the meal made from Y1126 yellow-seeded mutant, regular black-seeded pennycress and commercial canola meal were sent to the University of Illinois (Urbana-Champaign, IL) for Total Metabolizable Energy corrected for nitrogen (TMEn) and digestible amino acid analysis. The University of Illinois utilized the cecectomized rooster assay to measure TMEn and the digestibility of amino acids.

Claim 1:
A composition comprising non-defatted pennycress seed meal, wherein the non-defatted pennycress seed meal has an acid detergent fiber (ADF) content of less than <NUM>% by dry weight, and wherein the non-defatted pennycress seed meal is obtained from a seed lot comprising a loss-of function (LOF) homozygous mutation in an endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO: <NUM>, <NUM>, <NUM>, allelic variants having at least <NUM>% sequence identity with SEQ ID NO:<NUM> , <NUM> or <NUM>, or any combination thereof.