Patent Publication Number: US-11647765-B2

Title: Low fiber pennycress meal, seeds, and methods of making

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This is a Division of U.S. Non-Provisional patent application Ser. No. 16/893,636, filed Jun. 5, 2020 and now U.S. Pat. No. 11,224,237, which is Division of U.S. Non-Provisional patent application Ser. No. 16/131,633, filed Sep. 14, 2018 and now U.S. Pat. No. 10,709,151, which claims the benefit of U.S. Provisional Patent Application Ser. No. 62/559,122, filed Sep. 15, 2017, all of which are incorporated herein by reference in its entirety. 
    
    
     STATEMENT REGARDING FEDERAL FUNDING 
     This invention was made with government support under Grant Number 2014-67009-22305 and 2018-67009-27374 awarded by the National Institute of Food and Agriculture, USDA. The government has certain rights in the invention. 
    
    
     INCORPORATION OF SEQUENCE LISTING 
     The sequence listing contained in the file named “2021_12_10_ULMASOV_P13415US04_ST25.txt” contains 183 sequences, is contemporaneously filed with this specification by electronic submission (using United States Patent Office EFS-Web filing system), and is incorporated herein by reference in its entirety. 
     BACKGROUND 
     Different plants have seed contents that make them desirable for feed compositions. Examples are soybean, canola, rapeseed and sunflower. After crushing the seeds and recovering the oil, the resulting meal has a protein content making the meal useful as a feed ingredient for ruminants, monogastrics, poultry, and aquaculture. Nevertheless, there remains a desire for improved plant seeds that can provide additional sources of nutrition to animals. 
     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 5% of farmers today are using them. One reason is economics—it requires on average ˜$30-40/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 5 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&#39;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 36% oil, which is roughly twice the level found in soybean, and the oil has a very low saturated fat content (˜4%). Pennycress represents a clear opportunity for sustainable optimization of agricultural systems. For example, in the US Midwest, ˜35M 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. 
     SUMMARY 
     Compositions comprising non-defatted pennycress seed meal comprising an acid detergent fiber (ADF) content of 5% to 20% by dry weight are provided herein. 
     Compositions comprising defatted pennycress seed meal comprising an acid detergent fiber (ADF) content of 7% to 25% by dry weight are provided herein. 
     Pennycress seed meals comprising an acid detergent fiber (ADF) content of 5% to 20% by dry weight, wherein the seed meal is non-defatted, are provided herein. 
     Pennycress seed meals comprising an acid detergent fiber (ADF) content of 7% to 25% by dry weight, wherein the seed meal is defatted, are provided herein. 
     Pennycress seed cakes comprising an acid detergent fiber (ADF) content of 7% to 25% by dry weight are provided herein. 
     In one embodiment, this disclosure provides a low fiber pennycress meal composition. 
     Seed lots comprising a population of pennycress seeds that comprise an acid detergent fiber (ADF) content of 5% to 20% by dry weight are provided herein. 
     Methods of making non-defatted pennycress seed meal comprising an acid detergent fiber (ADF) content of 5% to 20% by dry weight, comprising the step of grinding, macerating, extruding, and/or crushing the aforementioned seed lots, thereby obtaining the non-defatted seed meal, are provided herein. 
     Methods of making defatted pennycress seed meal comprising an acid detergent fiber (ADF) content of 7% to 25% by dry weight, comprising the step of solvent extracting the, separating the extracted seed meal from the solvent, thereby obtaining the defatted seed meal, are provided herein. 
     Methods of making pennycress seed cake comprising an acid detergent fiber (ADF) content of 7% to 25% by dry weight, comprising the step of crushing or expelling the seed of any of the aforementioned seed lots, thereby obtaining a seed cake, are provided herein. 
     Methods of making a pennycress seed lot comprising the steps of: (a) introducing at least one loss-of-function mutation in at least one endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO:2, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 172, and allelic variants thereof; (b) selecting germplasm that is homozygous for said loss-of-function mutation; and, (c) harvesting seed from the homozygous germplasm, thereby obtaining a seed lot, wherein said seed lot comprises an acid detergent fiber (ADF) content of 5% to 20% by dry weight, are provided herein. 
     Method of making a pennycress seed lot comprising the steps of: (a) introducing at least one transgene that suppresses expression of at least one endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO:2, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 172, and allelic variants thereof into a pennycress plant genome; (b) selecting a transgenic plant line that comprises said transgene and (c) harvesting seed from the transgenic plant line, thereby obtaining a seed lot, wherein said seed lot comprises an acid detergent fiber (ADF) content of 5% to 20% by dry weight, are provided herein. 
     In one embodiment, this disclosure provides a method for producing low fiber pennycress seeds and meal. The method comprises genetically modifying pennycress seed (e.g., using gene editing or transgenic approach) to modify expression of one or more genes involved in seed coat development. Genetically altered seed lots with improved composition, such as lower fiber content, increased oil content, and increased protein content, all in comparison to control seed lots that lack the genetic alteration can be obtained by these methods. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       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: 
         FIG.  1    A, B, C illustrate mutant pennycress seeds with varying seed color. Dark seeds in the center are representative of a wild-type genetic background. The seeds of two pennycress seed isolates (Y1126 and Y1067), along with 7 pennycress M3-generation EMS mutants in the Spring 32 background are shown. All mutant seeds exhibit light-colored seed coats compared to the dark color of typical wild-type pennycress seeds (wild-type Spring 32 seeds shown as an example). Examples of dark and light-colored seed and meal (non-defatted) are also shown. Panel A: Spectrum of seed coat color ranging from dark to light in wild type and mutant pennycress seeds. Panel B: Pennycress meal produced from wild type (Beecher). Panel C: Pennycress meal produced from one of the light-colored seed lines (Y1126). 
         FIG.  2 A , B illustrates pARV8 (SS51_Tt10),  Agrobacterium  CRISPR-Cas9 vector and its gene editing sgRNA cassette, for targeting pennycress homolog of  Transparent testa  10 (Tt10) gene. Panel A: Plasmid map of pARV8 (SS51_Tt10). Panel B: sgRNA cluster in pARV8, targeting nucleotides 341-360 and 382-401 of SEQ ID NO: 33. 
         FIG.  3    illustrates pARV187,  Agrobacterium  CRISPR-FnCpf1 base vector for editing plant genome. gRNA cassette stuffers are inserted at the dual AarI site, replacing a small fragment of the vector with synthetic gRNA cassette. 
         FIG.  4    illustrates pARV191,  Agrobacterium  CRISPR-SmCsm1 base vector for editing plant genome. gRNA cassette stuffers are inserted at the dual AarI site, replacing a small fragment of the vector with synthetic gRNA cassette. 
         FIGS.  5    A, B, C, D, E, F, G, gRNA cassettes targeting pennycress  Transparent testa  (Tt) genes.  FIG.  5 A  illustrates a gRNA cassette stuffer, designed for insertion into the AarI-digested plant genome editing vector (such as pARV187 or pARV191) for targeting pennycress Tt1 gene, nucleotides 59-81 and 307-329 of SEQ ID NO: 27;  FIG.  5 B : gRNA cassette stuffer for targeting pennycress Tt2 gene, nucleotides 177-199 and 240-262 of SEQ ID NO: 1;  FIG.  5 C : gRNA cassette stuffer for targeting pennycress Tt8 gene, nucleotides 261-283 and 153-175 of SEQ ID NO: 69;  FIG.  5 D : gRNA cassette stuffer for targeting pennycress Tt8 gene, nucleotides 145-167 and 274-296 of SEQ ID NO: 69;  FIG.  5 E : gRNA cassette stuffer for targeting pennycress Tt10 gene, nucleotides 304-326 and 415-437 of SEQ ID NO: 33;  FIG.  5 F : gRNA cassette stuffer for targeting pennycress Tt12 gene, nucleotides 399-421 and 450-472 of SEQ ID NO: 36;  FIG.  5 G : gRNA cassette stuffer for targeting pennycress Tt15 gene, nucleotides 255-277 and 281-303 of SEQ ID NO: 42. 
         FIG.  6    illustrates total oil content in seeds of selected yellow-seeded pennycress mutants measured using GC-chromatography analysis. 
     
    
    
     DETAILED DESCRIPTION 
     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 Association of Official Analytical Chemists (AOAC) Official Method 973.18 (1996): “Fiber (Acid Detergent) and Lignin in Animal Feed”. 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 JAOAC 56, 1352-1356, 1973. In certain embodiments, fiber (ADF and/or NDF), protein, and/or oil content can be determined by Near-infrared (NIR) spectroscopy. 
     Defatted-pennycress seed meal having less fiber than defatted control pennycress seed meal obtained from wild type pennycress seed is provided herein. In certain embodiments, the ADF content of defatted pennycress seed meal and compositions comprising the same that are provided herein is reduced from about 1.25-, 1.5-, 2-, or 3-fold to about 4-, 5-, 6-, or 7-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 25 to about 31% 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 35-45%. 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 20% by dry weight, less than 15% by dry weight, or less than 10% by dry weight of the meal. In certain embodiments, defatted pennycress seed meal having an ADF content of less than 25% by dry weight, less than 20% by dry weight, less than 15% by dry weight, less than 10% by dry weight, or less than 7% by dry weight of meal is provided herein. In certain embodiments, defatted pennycress seed meal having an ADF content of about 5%, 8%, or 10% to 15%, 18%, 20%, or 25% by dry weight is provided herein. Compositions comprising such defatted pennycress seed meal are also provided herein. 
     Non-defatted pennycress seed meal having less fiber than non-defatted control pennycress seed meal obtained from wild type pennycress seed is provided herein. In certain embodiments, the ADF content of non-defatted pennycress seed meal and compositions comprising the same that are provided herein is reduced from about 1.25-, 1.5-, 2-, or 3-fold to about 4-, 5-, 6-, or 7-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 20% to about 38% 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 20% by dry weight, less than 15% by dry weight, or less than 10% by dry weight of the meal. In certain embodiments, non-defatted pennycress seed meal having an ADF content of less than 20% by dry weight, less than 15% by dry weight, less than 10% by dry weight, or less than 7% by dry weight of the meal is provided herein. In certain embodiments, non-defatted pennycress seed meal having an ADF content of about 5%, 8%, or 10% to 15%, 18%, or 20% by dry weight is provided herein. Compositions comprising such non-defatted pennycress seed meal are also provided herein. 
     In certain embodiments, pennycress seed lots comprising a population of seed having reduced fiber content, reduced fiber content and increased protein content, reduced fiber content and increased oil content, or reduced fiber content and increased protein and oil content, all in comparison to fiber, protein, and oil content of the control seed lots of wild-type pennycress seed, are provided. In certain embodiments, the seed lots will comprise loss-of-function (LOF) mutations in one or more genes, coding sequences, and/or proteins that result in 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. Such LOF mutations include, but are not limited to, INDELS (insertions, deletions, and/or substitutions or any combination thereof), translocations, inversions, duplications, or any combination thereof in a promoter, a 5′ untranslated region, coding region, an intron of a gene, and/or a 3′ UTR of a gene. Such Indels can introduce one or more mutations including, but not limited to, frameshift mutations, missense mutations, pre-mature translation termination codons, splice donor and/or acceptor mutations, regulatory mutations, and the like that result in an LOF mutation. In certain embodiments, the LOF mutation will result in: (a) a reduction in the enzymatic or other biochemical activity associated with the encoded polypeptide in the plant comprising the LOF mutation 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 LOF mutation in comparison to a wild-type control plant. Such reductions in activity or activity and transcript levels can, in certain embodiments, comprise a reduction of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of activity or activity and transcript levels in the LOF mutant in comparison to the activity or transcript levels in a wild-type control plant. In certain embodiments, reductions in activity, specific activity, and/or transcript levels are provided by at least one LOF mutation in an endogenous wild-type pennycress gene, promoter, terminator, or protein set forth in Table 1. In certain embodiments, such aforementioned reductions in activity, specific activity and/or transcript levels are provided by at least one LOF mutation in an endogenous wild-type pennycress gene comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 171, 173, allelic variants thereof, or any combination thereof. In certain embodiments, such aforementioned reductions in activity, specific activity, and/or transcript levels are provided by at least one LOF mutation in an endogenous wild-type pennycress gene, promoter, or terminator comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 69, 71, 75, 77, 87, 88, allelic variants thereof, or any combination thereof. In certain embodiments, any of the aforementioned allelic variants of endogenous wild-type pennycress genes can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO: 1, 3, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 171, or 173. In certain embodiments, such aforementioned reductions in activity, specific activity, and/or transcript levels are provided by at least one LOF mutation in an endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO:2, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 172, allelic variants thereof, or any combination thereof. In certain embodiments, such aforementioned reductions in activity or activity and transcript levels are provided by at least one LOF mutation in an endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO: 70, 76, 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 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:2, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, or 172. In certain embodiments, an endogenous wild-type pennycress gene can encode a polypeptide allelic variant having one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:2, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, or 172. In certain embodiments, 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 embodiments, 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 embodiments comprise a reduction of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% 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 embodiments, 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 provided in the following Table 1 and accompanying Sequence Listing. In certain embodiments, allelic variants of the wild-type genes, coding sequences, and/or proteins provided in Table 1 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 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity across the entire length of the polynucleotide sequences of the wild-type coding regions or wild-type genes of Table 1 and the sequence listing. Such allelic variants can comprise polypeptide sequences that have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity across the entire length of the polypeptide sequences of the wild-type proteins of Table 1 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 2 (TTG1 and TTG2), GLABROUS 2 (GL2), GLABROUS 3 (GL3), ANR-BAN, and AUTOINHIBITED H+-ATPASE 10 (AHA10) disclosed in Table 1. In certain embodiments, pennycress seed lots provided herein can comprise LOF mutations in any of the aforementioned wild-type pennycress genes disclosed in Table 1 or any combination of mutations disclosed in Table 1. 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 provided herein. Methods of making any of the aforementioned seed lots, compositions, seed meals, or seed cakes are also provided 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 embodiments, 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 embodiments, 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. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Wild-type (WT) coding regions, encoded proteins, and genes that can be targeted  
               
               
                 for introduction of LOF mutations or transgene-mediated suppression, their mutant variants  
               
               
                 and representative genetic elements for achieving suppression of gene expression. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Other Names Used  
               
               
                   
                   
                   
                   
                 and Representative 
               
               
                   
                   
                   
                   
                 Pennycress LOF 
               
               
                 SEQ 
                 Sequence 
                   
                   
                 Mutants 
               
               
                 ID NO: 
                 Name 
                 Type 
                 Function/Nature of the mutation 
                 Disclosed Herein 
               
               
                   
               
               
                   1 
                 TT2 CDS 
                 WT Coding 
                 R2R3 MYB domain transcription 
                 MYB123, 
               
               
                   
                   
                 region 
                 factor, a key determinant in 
                 TRANSPARENT 
               
               
                   2 
                 TT2 ORF 
                 WT Protein 
                 proanthocyanidin accumulation 
                 TESTA 2 (TT2) 
               
               
                   3 
                 TT2 Ta 
                 WT Gene 
                   
                   
               
               
                   
                 locus 
                   
                   
                   
               
               
                   
               
               
                   4 
                 TT2 CDS- 
                 Mutant Coding 
                 Modified TT2 gene isolated from  
                 tt2-1, tt2-2, BC38, 
               
               
                   
                 Mut 
                 region 
                 an EMS-mutagenized population, 
                 E5-547 
               
               
                   
                   
                   
                 GAACCATTG   G   AACTCAAAC  
                   
               
               
                   
                   
                   
                 (nt 321-339 of SEQ ID NO: 1) →  
                   
               
               
                   
                   
                   
                 GAACCATTG   A   AACTCAAAC  
                   
               
               
                   
                   
                   
                 (nt 321-339 of SEQ ID NO: 4) 
                   
               
               
                   
               
               
                   5 
                 TT2 Mut  
                 Mutant Protein 
                 Truncated protein, due to Trp  
                   
               
               
                   
                 P1 
                   
                 (W) codon -&gt; Stop mutation 
                   
               
               
                   
               
               
                   6 
                 ATS-KAN4 
                 WT Coding 
                 Member of the KANADI family of 
                 ABERRANT TESTA  
               
               
                   
                 CDS 
                 region 
                 transcription factors, involved  
                 SHAPE, ATS, KAN4, 
               
               
                   7 
                 ATS-KAN4 
                 WT Protein  
                 in integument formation during  
                 KANADI 4 
               
               
                   
                 ORF 
                   
                 ovule development and expressed  
                   
               
               
                   8 
                 ATS-KAN4 
                 WT Gene 
                 at the boundary between the  
                   
               
               
                   
                 Ta locus 
                   
                 inner and outer integuments.  
                   
               
               
                   
                   
                   
                 Essential for directing laminar  
                   
               
               
                   
                   
                   
                 growth of the inner integument 
                   
               
               
                   
               
               
                   9 
                 BAN-ANR 
                 WT Coding 
                 Negative regulator of flavonoid 
                 BAN, BANYULS, 
               
               
                   
                 CDS 
                 region 
                 biosynthesis, putative  
                 NAD(P)-binding 
               
               
                  10 
                 BAN-ANR 
                 WT Protein  
                 oxidoreductase. Mutants  
                 Rossmann-fold 
               
               
                   
                 ORF 
                   
                 accumulate flavonoid pigments  
                 superfamily protein 
               
               
                  11 
                 BAN-ANR 
                 WT Gene 
                 in seed coat. Putative ternary  
                   
               
               
                   
                 Ta locus 
                   
                 complex composed of TT2, TT8  
                   
               
               
                   
                   
                   
                 and TTG1 is believed to be  
                   
               
               
                   
                   
                   
                 required for correct expression  
                   
               
               
                   
                   
                   
                 of BAN in seed endothelium 
                   
               
               
                   
               
               
                  12 
                 DTX35 CDS 
                 WT Coding 
                 Encodes a multidrug and toxin  
                 Detoxifying Efflux 
               
               
                   
                   
                 region 
                 efflux family transporter.  
                 Carrier 35, FFT, 
               
               
                  13 
                 DTX35 ORF  
                 WT Protein  
                 Involved in flavonoid  
                 FLOWER FLAVONOID 
               
               
                  14 
                 DTX35 Ta 
                 WT Gene 
                 metabolism, affecting root  
                 TRANSPORTER 
               
               
                   
                 locus 
                   
                 growth, seed development and  
                   
               
               
                   
                   
                   
                 germination, pollen  
                   
               
               
                   
                   
                   
                 development, release and  
                   
               
               
                   
                   
                   
                 viability 
                   
               
               
                   
               
               
                  15 
                 GL2 CDS 
                 WT Coding 
                 Glabra 2, a homeodomain protein 
                 Glabra 2, HD-ZIP IV 
               
               
                   
                   
                 region 
                 affects epidermal cell identity 
                 homeobox-leucine  
               
               
                  16 
                 GL2 ORF 
                 WT Protein  
                 including trichomes, root hairs,  
                 zipper protein with  
               
               
                  17 
                 GL2 Ta 
                 WT Gene 
                 and seed coat. Abundantly  
                 lipid-binding START 
               
               
                   
                 locus 
                   
                 expressed during early seed  
                 domain 
               
               
                   
                   
                   
                 development and in  
                   
               
               
                   
                   
                   
                 atrichoblasts. Directly  
                   
               
               
                   
                   
                   
                 regulated by WER 
                   
               
               
                   
               
               
                  18 
                 MUM4_like 
                 WT Coding 
                 Encodes a putative NDP-L- 
                 MUCILAGE-MODIFIED 4, 
               
               
                   
                 1 CDS 
                 region 
                 rhamnose synthase, an enzyme  
                 RHAMNOSE 
               
               
                  19 
                 MUM4_like 
                 WT Protein  
                 required for the synthesis of  
                 BIOSYNTHESIS 2,  
               
               
                   
                 1 ORF  
                   
                 the pectin rhamnogalacturonan I,  
                 RHM2, ATRHM2 
               
               
                  20 
                 MUM4_like 
                 WT Gene 
                 major component of plant   
                   
               
               
                   
                 1 Ta locus 
                   
                 mucilage. Involved in seed coat  
                   
               
               
                  21 
                 MUM4_like 
                 WT Coding 
                 mucilage cell development.  
                   
               
               
                   
                 2 CDS 
                 region 
                 Required for complete mucilage  
                   
               
               
                  22 
                 MUM4_like 
                 WT Protein 
                 synthesis, cytoplasmic  
                   
               
               
                   
                 2 ORF 
                   
                 rearrangement and seed coat  
                   
               
               
                  23 
                 MUM4_like 
                 WT Gene 
                 development 
                   
               
               
                   
                 2 Ta locus 
                   
                   
                   
               
               
                   
               
               
                  24 
                 MYB61 
                 WT Coding 
                 Putative transcription factor.  
                 MYB DOMAIN PROTEIN  
               
               
                   
                 CDS 
                 region 
                 Mutants are deficient in  
                 61, ATMYB61 
               
               
                  25 
                 MYB61 
                 WT Protein  
                 mucilage extrusion from the  
                   
               
               
                   
                 ORF 
                   
                 seeds during imbibition,  
                   
               
               
                  26 
                 MYB61 Ta 
                 WT Gene 
                 resulting in reduced deposition  
                   
               
               
                   
                 locus 
                   
                 of mucilage during development  
                   
               
               
                   
                   
                   
                 of the seed coat epidermis in  
                   
               
               
                   
                   
                   
                 myb61 mutants 
                   
               
               
                   
               
               
                  27 
                 TT1_like1 
                 WT Coding 
                 Encodes a zinc finger protein; 
                 WIP DOMAIN PROTEIN  
               
               
                   
                 CDS 
                 region 
                 involved in photomorphogenesis, 
                 1, WIP1 
               
               
                  28 
                 TT1_like1 
                 WT Protein 
                 flavonoid biosynthesis, flower  
                   
               
               
                   
                 ORF 
                   
                 and seed development 
                   
               
               
                  29 
                 TT1_like1 
                 WT Gene 
                   
                   
               
               
                   
                 Ta locus 
                   
                   
                   
               
               
                  30 
                 TT1_like2 
                 WT Coding 
                   
                   
               
               
                   
                 CDS 
                 region 
                   
                   
               
               
                  31 
                 TT1_like2 
                 WT Protein 
                   
                   
               
               
                   
                 ORF 
                   
                   
                   
               
               
                  32 
                 TT1_like2 
                 WT Gene 
                   
                   
               
               
                   
                 Ta locus 
                   
                   
                   
               
               
                   
               
               
                  33 
                 TT10 CDS 
                 WT Coding 
                 Protein similar to laccase-like 
                 ATLAC15, ATTT10,  
               
               
                   
                   
                 region 
                 polyphenol oxidases, with  
                 LAC15 (LACCASE-LIKE 
               
               
                  34 
                 TT10 ORF 
                 WT Protein  
                 conserved copper binding  
                 15), TRANSPARENT 
               
               
                  35 
                 TT10 Ta 
                 WT Gene 
                 domains. Involved in lignin and  
                 TESTA 10 (TT10) 
               
               
                   
                 locus 
                   
                 flavonoids biosynthesis.  
                   
               
               
                   
                   
                   
                 Expressed in developing testa,  
                   
               
               
                   
                   
                   
                 colocalizing with flavonoid end  
                   
               
               
                   
                   
                   
                 products proanthocyanidins and  
                   
               
               
                   
                   
                   
                 flavonols. Mutants exhibit delay  
                   
               
               
                   
                   
                   
                 in developmentally determined  
                   
               
               
                   
                   
                   
                 browning of the testa,  
                   
               
               
                   
                   
                   
                 characterized by the pale brown  
                   
               
               
                   
                   
                   
                 color of seed coat 
                   
               
               
                   
               
               
                  36 
                 TT12 CDS 
                 WT Coding 
                 Proton antiporter, involved in  
                 TRANSPARENT TESTA 12 
               
               
                   
                   
                 region 
                 the transportation of  
                 (TT12), ATTT12, 
               
               
                  37 
                 TT12 ORF  
                 WT Protein  
                 proanthocyanidin precursors  
                 MATE efflux family  
               
               
                  38 
                 TT12 Ta 
                 WT Gene 
                 into the vacuole. Loss-of- 
                 protein 
               
               
                   
                 locus 
                   
                 function mutation has strong  
                   
               
               
                   
                   
                   
                 reduction of proanthocyanidin  
                   
               
               
                   
                   
                   
                 deposition in vacuoles and  
                   
               
               
                   
                   
                   
                 reduced dormancy. Expressed in  
                   
               
               
                   
                   
                   
                 the endothelium of ovules and  
                   
               
               
                   
                   
                   
                 in developing seeds 
                   
               
               
                   
               
               
                  39 
                 TT13 CDS  
                 WT Coding 
                 Proton pump from the H + -ATPase 
                 AHA10 
               
               
                   
                   
                 region 
                 family, involved in  
                 (AUTOINHIBITED H(+)- 
               
               
                  40 
                 TT13 ORF 
                 WT Protein  
                 proanthocyanidin biosynthesis.  
                 ATPASE ISOFORM 10), 
               
               
                  41 
                 TT13 Ta 
                 WT Gene 
                 Mutations disturb vacuolar  
                 TRANSPARENT TESTA  
               
               
                   
                 locus 
                   
                 biogenesis and acidification  
                 13 (TT13) 
               
               
                   
                   
                   
                 process. The acidification of  
                   
               
               
                   
                   
                   
                 the vacuole provides energy for  
                   
               
               
                   
                   
                   
                 import of proanthocyanidins into  
                   
               
               
                   
                   
                   
                 the vacuole 
                   
               
               
                   
               
               
                  42 
                 TT15 CDS 
                 WT Coding 
                 Encodes a UDP-glucose:sterol- 
                 TRANSPARENT TESTA 15 
               
               
                   
                   
                 region 
                 glucosyltransferase. Mutants  
                 (TT15), TRANSPARENT 
               
               
                  43 
                 TT15 ORF 
                 WT Protein  
                 produce pale greenish-brown  
                 TESTA GLABROUS 15 
               
               
                  44 
                 TT15 Ta 
                 WT Gene 
                 seeds with slightly reduced  
                 (TTG15), UGT80B1,  
               
               
                   
                 locus 
                   
                 dormancy 
                 UDP- 
               
               
                   
                   
                   
                   
                 Glycosyltransferase 
               
               
                   
                   
                   
                   
                 superfamily protein 
               
               
                   
               
               
                  45 
                 TT16 CDS 
                 WT Coding 
                 MADS-box protein regulating 
                 ABS, AGAMOUS-LIKE 32  
               
               
                   
                   
                 region 
                 proanthocyanidin biosynthesis  
                 (AGL32), ARABIDOPSIS 
               
               
                  46 
                 TT16 ORF 
                 WT Protein  
                 and cell shape in the inner- 
                 BSISTER, TRANSPARENT 
               
               
                  47 
                 TT16 Ta 
                 WT Gene 
                 most cell layer of the seed  
                 TESTA16 (TT16) 
               
               
                   
                 locus 
                   
                 coat. Required for determining  
                   
               
               
                   
                   
                   
                 the identity of the endothelial  
                   
               
               
                   
                   
                   
                 layer within the ovule.  
                   
               
               
                   
                   
                   
                 Paralogous to GOA. Plays a  
                   
               
               
                   
                   
                   
                 maternal role in fertilization  
                   
               
               
                   
                   
                   
                 and seed development 
                   
               
               
                   
               
               
                  48 
                 TT18 CDS 
                 WT Coding 
                 Encodes leucoanthocyanidin 
                 ANS, ANTHOCYANIDIN  
               
               
                   
                   
                 region 
                 dioxygenase, which is involved  
                 SYNTHASE, LDOX, 
               
               
                  49 
                 TT18 ORF 
                 WT Protein  
                 in proanthocyanin biosynthesis.  
                 LEUCOANTHOCYANIDIN 
               
               
                  50 
                 TT18 Ta 
                 WT Gene 
                 Mutant analysis suggests that  
                 DIOXYGENASE, TANNIN 
               
               
                   
                 locus 
                   
                 this gene is also involved in  
                 DEFICIENT SEED 4  
               
               
                   
                   
                   
                 vacuole formation 
                 (TDS4), TT18 
               
               
                   
               
               
                  51 
                 TT19 CDS 
                 WT Coding 
                 Encodes glutathione transferase 
                 GLUTATHIONE S- 
               
               
                   
                   
                 region 
                 belonging to the phi class of  
                 TRANSFERASE PHI 12, 
               
               
                  52 
                 TT19 ORF 
                 WT Protein  
                 GSTs. Mutants display no  
                 ATGSTF12,GLUTATHIONE 
               
               
                  53 
                 TT19 Ta 
                 WT Gene 
                 pigments in the leaves or  
                 S-TRANSFERASE 26  
               
               
                   
                 locus 
                   
                 stems. Likely to function as a  
                 (GST26), GLUTATHIONE 
               
               
                   
                   
                   
                 carrier to transport anthocyanin  
                 S-TRANSFERASE PHI  
               
               
                   
                   
                   
                 from the cytosol to tonoplasts 
                 12, GSTF12, 
               
               
                   
                   
                   
                   
                 TRANSPARENT 
               
               
                   
                   
                   
                   
                 TESTA 19 (TT19) 
               
               
                   
               
               
                  54 
                 TT3 CDS 
                 WT Coding 
                 Dihydroflavonol reductase.  
                 DFR, DIHYDROFLAVONOL  
               
               
                   
                   
                 region 
                 Catalyzes conversion of  
                 4-REDUCTASE, M318, 
               
               
                  55 
                 TT3 ORF  
                 WT Protein  
                 dihydroquercetin to  
                 TRANSPARENT TESTA  
               
               
                  56 
                 TT3 Ta 
                 WT Gene 
                 leucocyanidin in the  
                 3, (TT3) 
               
               
                   
                 locus 
                   
                 biosynthesis of anthocyanins 
                   
               
               
                   
               
               
                  57 
                 TT4 CDS  
                 WT Coding 
                 Encodes chalcone synthase (CHS),  
                 ATCHS, CHALCONE 
               
               
                   
                   
                 region 
                 a key enzyme in biosynthesis of 
                 SYNTHASE, CHS, 
               
               
                  58 
                 TT4 ORF  
                 WT Protein 
                 flavonoids. Required for 
                 TRANSPARENT TESTA  
               
               
                  59 
                 TT4 Ta 
                 WT Gene 
                 accumulation of purple  
                 4 (TT4) 
               
               
                   
                 locus 
                   
                 anthocyanins in leaves, stems  
                   
               
               
                   
                   
                   
                 and seed coat. Also involved in  
                   
               
               
                   
                   
                   
                 regulation of auxin transport  
                   
               
               
                   
                   
                   
                 and root gravitropism 
                   
               
               
                   
               
               
                  60 
                 TT5 CDS 
                 WT Coding 
                 Another key enzyme in  
                 A11, ATCHI, CFI, 
               
               
                   
                   
                 region 
                 biosynthesis of flavonoids.  
                 CHALCONE FLAVANONE 
               
               
                  61 
                 TT5 ORF 
                 WT Protein  
                 Catalyzes the conversion of  
                 ISOMERASE, CHALCONE 
               
               
                  62 
                 TT5 Ta 
                 WT Gene 
                 chalcones into flavanones.  
                 ISOMERASE, CHI, 
               
               
                   
                 locus 
                   
                 Required for the accumulation  
                 TRANSPARENT 
               
               
                   
                   
                   
                 of purple anthocyanins leaves,  
                 TESTA 5 (TT5) 
               
               
                   
                   
                   
                 stems and seed coat. Co- 
                   
               
               
                   
                   
                   
                 expressed with CHS 
                   
               
               
                   
               
               
                  63 
                 TT6 CDS 
                 WT Coding 
                 Encodes flavanone 3-hydroxylase, 
                 F3′H, F3H, FLAVANONE  
               
               
                   
                   
                 region 
                 regulating flavonoid  
                 3-HYDROXYLASE, 
               
               
                  64 
                 TT6 ORF 
                 WT Protein  
                 biosynthesis. Coordinately  
                 TRANSPARENT 
               
               
                  65 
                 TT6 Ta 
                 WT Gene 
                 expressed with chalcone synthase  
                 TESTA 6 (TT6) 
               
               
                   
                 locus 
                   
                 and chalcone isomerases 
                   
               
               
                   
               
               
                  66 
                 TT7 CDS 
                 WT Coding 
                 Required for flavonoid 3′- 
                 F3′H CYP75B1, 
               
               
                   
                   
                 region 
                 hydroxylase activity. Enzyme 
                 CYTOCHROME P450  
               
               
                  67 
                 TT7 ORF 
                 WT Protein  
                 abundance relative to CHS 
                 75B1, D501, 
               
               
                  68 
                 TT7 Ta 
                 WT Gene 
                 determines Quercetin/Kaempferol 
                 TRANSPARENT 
               
               
                   
                 locus 
                   
                 metabolite ratio 
                 TESTA 7 (TT7) 
               
               
                   
               
               
                  69 
                 TT8 CDS 
                 WT Coding 
                 TT8 is a transcription factor  
                 ATTT8, BHLH42, 
               
               
                   
                   
                 region 
                 acting in concert with TT1, PAP1  
                 TRANSPARENT 
               
               
                  70 
                 TT8 ORF 
                 WT Protein  
                 and TTG1 on regulation of  
                 TESTA 8, (TT8) 
               
               
                  71 
                 TT8 Ta 
                 WT Gene 
                 flavonoid pathways, namely  
                   
               
               
                   
                 locus 
                   
                 proanthocyanidin and anthocyanin  
                   
               
               
                   
                   
                   
                 biosynthesis. Affects  
                   
               
               
                   
                   
                   
                 dihydroflavonol 4-reductase gene  
                   
               
               
                   
                   
                   
                 expression. It is believed that  
                   
               
               
                   
                   
                   
                 a ternary complex composed of  
                   
               
               
                   
                   
                   
                 TT2, TT8 and TTG1 is required  
                   
               
               
                   
                   
                   
                 for correct expression of BAN in  
                   
               
               
                   
                   
                   
                 seed endothelium. Interacts with  
                   
               
               
                   
                   
                   
                 JAZ proteins to regulate  
                   
               
               
                   
                   
                   
                 anthocyanin accumulation 
                   
               
               
                   
               
               
                  72 
                 TT9 CDS 
                 WT Coding 
                 Encodes a peripheral membrane 
                 GFS9, GREEN 
               
               
                   
                   
                 region 
                 protein localized at the Golgi 
                 FLUORESCENT 
               
               
                  73 
                 TT9 ORF 
                 WT Protein 
                 apparatus. Involved in membrane 
                 SEED 9, TRANSPARENT 
               
               
                  74 
                 TT9 Ta 
                 WT Gene 
                 trafficking, vacuole development  
                 TESTA 9, TT9 
               
               
                   
                 locus 
                   
                 and in flavonoid accumulation in  
                 CLEC16A-like protein 
               
               
                   
                   
                   
                 the seed coat. Mutant seed color  
                   
               
               
                   
                   
                   
                 is pale brown 
                   
               
               
                   
               
               
                  75 
                 TTG1 CDS  
                 WT Coding 
                 Part of a ternary complex  
                 TTG1, TTG, URM23, 
               
               
                   
                   
                 region 
                 composed of TT2, TT8 and TTG1  
                 ATTTG1, Transducin/ 
               
               
                  76 
                 TTG1 ORF  
                 WT Protein 
                 necessary for correct expression  
                 WD40-repeat- 
               
               
                  77 
                 TTG1 Ta 
                 WT Gene 
                 of BAN in seed endothelium.  
                 containing protein 
               
               
                   
                 locus 
                   
                 Required for the accumulation  
                   
               
               
                   
                   
                   
                 of purple anthocyanins in  
                   
               
               
                   
                   
                   
                 leaves, stems and seed coat.  
                   
               
               
                   
                   
                   
                 Controls epidermal cell fate  
                   
               
               
                   
                   
                   
                 specification. Affects  
                   
               
               
                   
                   
                   
                 dihydroflavonol 4-reductase gene  
                   
               
               
                   
                   
                   
                 expression. TTG1 was shown to  
                   
               
               
                   
                   
                   
                 act non-cell autonomously and  
                   
               
               
                   
                   
                   
                 to move via plasmodesmata  
                   
               
               
                   
                   
                   
                 between cells 
                   
               
               
                   
               
               
                  78 
                 TTG2 CDS  
                 WT Coding 
                 Belongs to a family of WRKY 
                 TRANSPARENT TESTA  
               
               
                   
                   
                 region 
                 transcription factors expressed  
                 GLABRA 2 (TTG2), 
               
               
                  79 
                 TTG2 ORF  
                 WT Protein 
                 in seed integument and  
                 AtWRKY44, DSL1  
               
               
                  80 
                 TTG2 Ta 
                 WT Gene 
                 endosperm. Mutants are defective  
                 (DR. STRANGELOVE 1) 
               
               
                   
                 locus 
                   
                 in proanthocyanidin synthesis   
                   
               
               
                   
                   
                   
                 and seed mucilage deposition.  
                   
               
               
                   
                   
                   
                 Seeds are yellow colored. Seed  
                   
               
               
                   
                   
                   
                 size is also affected; seeds are  
                   
               
               
                   
                   
                   
                 reduced in size but only when  
                   
               
               
                   
                   
                   
                 the mutant allele is transmitted  
                   
               
               
                   
                   
                   
                 through the female parent 
                   
               
               
                   
               
               
                  81 
                 TT1 
                 Artificial 
                 Artificial micro-RNA designed to 
                   
               
               
                   
                 aMIR319a 
                 miRNA 
                 reduce expression of TT1 in 
                   
               
               
                   
                 gene 
                   
                 corresponding cell layer of 
                   
               
               
                   
                   
                   
                 developing seed coat 
                   
               
               
                   
               
               
                  82 
                 TT10 
                 Artificial 
                 Artificial micro-RNA designed to 
                   
               
               
                   
                 aMIR319a 
                 miRNA 
                 reduce expression of TT10 in 
                   
               
               
                   
                 gene 
                   
                 corresponding cell layer of 
                   
               
               
                   
                   
                   
                 developing seed coat 
                   
               
               
                   
               
               
                  83 
                 TT2 
                 Artificial 
                 Artificial micro-RNA designed to 
                   
               
               
                   
                 aMIR319a 
                 miRNA 
                 reduce expression of TT2 in 
                   
               
               
                   
                 gene 
                   
                 corresponding cell layer of 
                   
               
               
                   
                   
                   
                 developing seed coat 
                   
               
               
                   
               
               
                  84 
                 TT8 
                 Artificial 
                 Artificial micro-RNA designed to 
                   
               
               
                   
                 aMIR319a 
                 miRNA 
                 reduce expression of TT8 in 
                   
               
               
                   
                 gene 
                   
                 corresponding cell layer of 
                   
               
               
                   
                   
                   
                 developing seed coat 
                   
               
               
                   
               
               
                  85 
                 TT1 
                 Promoter 
                 Genomic region of TT1 locus 
                   
               
               
                   
                 Promoter 
                   
                 upstream of TT1 start codon 
                   
               
               
                   
                   
                   
                 containing TT1 promoter  
                   
               
               
                   
                   
                   
                 regulatory elements 
                   
               
               
                   
               
               
                  86 
                 TT1 
                 Transcriptional 
                 Genomic region of TT1 locus 
                   
               
               
                   
                 Terminator 
                 terminator 
                 downstream of TT1 stop codon 
                   
               
               
                   
                   
                   
                 containing regulatory elements 
                   
               
               
                   
               
               
                  87 
                 TT8 
                 Promoter 
                 Genomic region of TT8 locus 
                   
               
               
                   
                 Promoter 
                   
                 upstream of TT8 start codon 
                   
               
               
                   
                   
                   
                 containing TT8 promoter  
                   
               
               
                   
                   
                   
                 regulatory elements 
                   
               
               
                   
               
               
                  88 
                 TT8 
                 Transcriptional 
                 Genomic region of TT8 locus 
                   
               
               
                   
                 Terminator 
                 terminator 
                 downstream of TT8 stop codon 
                   
               
               
                   
                   
                   
                 containing regulatory elements 
                   
               
               
                   
               
               
                  89 
                 TT2_CRISPR- 
                 Oligo- 
                 TT2 CDS targeted for cleavage by 
                   
               
               
                   
                 SpCAS9_F1 
                 nucleotide 
                 SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                  90 
                 TT2_CRISPR- 
                 Oligo- 
                 TT2 CDS targeted for cleavage by 
                   
               
               
                   
                 SpCAS9_R1 
                 nucleotide 
                 SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                  91 
                 TT2_CRISPR- 
                 Oligo- 
                 TT2 CDS targeted for cleavage by 
                   
               
               
                   
                 SaCAS9_F2 
                 nucleotide 
                 SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                  92 
                 TT2_CRISPR- 
                 Oligo- 
                 TT2 CDS targeted for cleavage by 
                   
               
               
                   
                 SaCAS9_R2 
                 nucleotide 
                 SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                  93 
                 TT2_CRISPR- 
                 Oligo- 
                 TT2 CDS targeted for cleavage by 
                   
               
               
                   
                 SaCAS9_F3 
                 nucleotide 
                 SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                  94 
                 TT2_CRISPR- 
                 Oligo- 
                 TT2 CDS targeted for cleavage by 
                   
               
               
                   
                 SaCAS9_R3 
                 nucleotide 
                 SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                  95 
                 TT8_CRISPR- 
                 Oligo- 
                 TT8 CDS targeted for cleavage by 
                   
               
               
                   
                 SpCAS9_F1 
                 nucleotide 
                 SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                  96 
                 TT8_CRISPR- 
                 Oligo- 
                 TT8 CDS targeted for cleavage by 
                   
               
               
                   
                 SpCAS9_R1 
                 nucleotide 
                 SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                  97 
                 TT8_CRISPR- 
                 Oligo- 
                 TT8 CDS targeted for cleavage by 
                   
               
               
                   
                 SpCAS9_F2 
                 nucleotide 
                 SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                  98 
                 TT8_CRISPR- 
                 Oligo- 
                 TT8 CDS targeted for cleavage by 
                   
               
               
                   
                 SpCAS9_R2 
                 nucleotide 
                 SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                  99 
                 TT8_CRISPR- 
                 Oligo- 
                 TT8 CDS targeted for cleavage by 
                   
               
               
                   
                 SpCAS9_F3 
                 nucleotide 
                 SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 100 
                 TT8_CRISPR- 
                 Oligo- 
                 TT8 CDS targeted for cleavage by 
                   
               
               
                   
                 SpCAS9_R3 
                 nucleotide 
                 SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 101 
                 TT10_CRISPR- 
                 Oligo- 
                 TT10 CDS targeted for cleavage  
                   
               
               
                   
                 SaCAS9_F1 
                 nucleotide 
                 by SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 102 
                 TT10_CRISPR- 
                 Oligo- 
                 TT10 CDS targeted for cleavage  
                   
               
               
                   
                 SaCAS9_R1 
                 nucleotide 
                 by SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 103 
                 TT10_CRISPR- 
                 Oligo- 
                 TT10 CDS targeted for cleavage  
                   
               
               
                   
                 SaCAS9_F2 
                 nucleotide 
                 by SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 104 
                 TT10_CRISPR- 
                 Oligo- 
                 TT10 CDS targeted for cleavage  
                   
               
               
                   
                 SaCAS9_R2 
                 nucleotide 
                 by SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 105 
                 TT16_CRISPR- 
                 Oligo- 
                 TT16 CDS targeted for cleavage  
                   
               
               
                   
                 SpCAS9_F1 
                 nucleotide 
                 by SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 106 
                 TT16_CRISPR- 
                 Oligo- 
                 TT16 CDS targeted for cleavage  
                   
               
               
                   
                 SpCAS9_R1 
                 nucleotide 
                 by SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 107 
                 TT16_CRISPR- 
                 Oligo- 
                 TT16 CDS targeted for cleavage  
                   
               
               
                   
                 SpCAS9_F2 
                 nucleotide 
                 by SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 108 
                 TT16_CRISPR- 
                 Oligo- 
                 TT16 CDS targeted for cleavage  
                   
               
               
                   
                 SpCAS9_R2 
                 nucleotide 
                 by SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 109 
                 TT8_CRISPR- 
                 Oligo- 
                 TT8 CDS targeted for cleavage by 
                   
               
               
                   
                 SpCAS9_F4 
                 nucleotide 
                 SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 110 
                 TT8_CRISPR- 
                 Oligo- 
                 TT8 CDS targeted for cleavage by 
                   
               
               
                   
                 SpCAS9_F5 
                 nucleotide 
                 SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 111 
                 TT8_CRISPR- 
                 Oligo- 
                 TT8 CDS targeted for cleavage by 
                   
               
               
                   
                 SaCAS9_F1 
                 nucleotide 
                 SaCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 112 
                 TT8_CRISPR- 
                 Oligo- 
                 TT8 CDS targeted for cleavage by 
                   
               
               
                   
                 SaCAS9_F2 
                 nucleotide 
                 SaCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 113 
                 TTG1_CRISPR- 
                 Oligo- 
                 TTG1 CDS targeted for cleavage  
                   
               
               
                   
                 SpCAS9_F1 
                 nucleotide 
                 by SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 114 
                 TTG1_CRISPR- 
                 Oligo- 
                 TTG1 CDS targeted for cleavage  
                   
               
               
                   
                 SpCAS9_F2 
                 nucleotide 
                 by SpCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 115 
                 TTG1_CRISPR- 
                 Oligo- 
                 TTG1 CDS targeted for cleavage  
                   
               
               
                   
                 SaCAS9_F1 
                 nucleotide 
                 by SaCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 116 
                 TTG1_CRISPR- 
                 Oligo- 
                 TTG1 CDS targeted for cleavage  
                   
               
               
                   
                 SaCAS9_F2 
                 nucleotide 
                 by SaCAS9 enzyme; part of gRNA 
                   
               
               
                   
                   
                   
                 cassette 
                   
               
               
                   
               
               
                 117 
                 TT4-1 CDS- 
                 Mutant Coding 
                 GTCTGCTCC   G   AGATCACAG  
                 tt4-1, A7-95 
               
               
                   
                 Mut 
                 region 
                 (nt 580-598 of SEQ ID NO: 57) → 
                   
               
               
                   
                   
                   
                 GTCTGCTCC   A   AGATCACAG  
                   
               
               
                   
                   
                   
                 (nt 580-598 of SEQ ID NO: 117) 
                   
               
               
                 118 
                 TT4 Mut P1 
                 Mutant Protein 
                 Presumed LOF due to E-&gt;K aa 
                   
               
               
                   
                   
                   
                 change 
                   
               
               
                   
               
               
                 119 
                 TT4-2 CDS- 
                 Mutant Coding 
                 AAGTGACTG   G   AACTCTCTC  
                 tt4-2, E5-549 
               
               
                   
                 Mut 
                 region 
                 (nt 894-912 of SEQ ID NO: 57) → 
                   
               
               
                   
                   
                   
                 AAGTGACTG   A   AACTCTCTC  
                   
               
               
                   
                   
                   
                 (nt 894-912 of SEQ ID NO: 119) 
                   
               
               
                 120 
                 TT4 Mut P2 
                 Mutant Protein 
                 Truncated protein, W-&gt;Stop  
                   
               
               
                   
                   
                   
                 change 
                   
               
               
                   
               
               
                 121 
                 TT6-1 CDS- 
                 Mutant Coding 
                 GAGACTGTG   C   AAGATTGGA  
                 tt6-1, AX17 
               
               
                   
                 Mut 
                 region 
                 (nt 364-382 of SEQ ID NO: 63) → 
                   
               
               
                   
                   
                   
                 GAGACTGTG   T   AAGATTGGA  
                   
               
               
                   
                   
                   
                 (nt 364-382 of SEQ ID NO: 121) 
                   
               
               
                 122 
                 TT6 Mut P1 
                 Mutant Protein 
                 Truncated protein, Q-&gt;Stop  
                   
               
               
                   
                   
                   
                 change 
                   
               
               
                   
               
               
                 123 
                 TT6-2 CDS- 
                 Mutant Coding 
                 TTCAGAATC   C   GGCGCAGGA  
                 tt6-2, Q36 
               
               
                   
                 Mut 
                 region 
                 (nt 872-890 of SEQ ID: 63) → 
                   
               
               
                   
                   
                   
                 TTCAGAATC   T   GGCGCAGGA  
                   
               
               
                   
                   
                   
                 (nt 872-890 of SEQ ID: 123) 
                   
               
               
                 124 
                 TT6 Mut P2 
                 Mutant Protein 
                 Presumed LOF due to P-&gt;L aa 
                   
               
               
                   
                   
                   
                 change 
                   
               
               
                   
               
               
                 125 
                 TT7-1 CDS- 
                 Mutant Coding 
                 CCAAATTCA   G   GAGCCAAAC  
                 tt7-1, A7-3, E5-586,  
               
               
                   
                 Mut 
                 region 
                 (nt 304-322 of SEQ ID: 66) → 
                 E5-484 P15, E5-484  
               
               
                   
                   
                   
                 CCAAATTCA   A   GAGCCAAAC  
                 P5 
               
               
                   
                   
                   
                 (nt 304-322 of SEQ ID: 125) 
                   
               
               
                 126 
                 TT7-1 Mut 
                 Mutant Protein 
                 Presumed LOF due to G-&gt;R aa 
                   
               
               
                   
                 P1 
                   
                 change 
                   
               
               
                   
               
               
                 127 
                 TT8-1 CDS- 
                 Mutant Coding 
                 TTTACGGCA   G   AGAAAGTGA  
                 tt8-1, D3-N10 P5 
               
               
                   
                 Mut 
                 region 
                 (nt 19-37 of SEQ ID: 69) → 
                   
               
               
                   
                   
                   
                 TTTACGGCA   A   AGAAAGTGA  
                   
               
               
                   
                   
                   
                 (nt 19-37 of SEQ ID: 127) 
                   
               
               
                 128 
                 TT8 Mut P1 
                 Mutant Protein 
                 Presumed LOF due to E-&gt;K aa 
                   
               
               
                   
                   
                   
                 change 
                   
               
               
                   
               
               
                 129 
                 TT8-2 CDS- 
                 Mutant Coding 
                 TCTTACATC   C   AATCATCAT  
                 tt8-2, D5-191, D3- 
               
               
                   
                 Mut 
                 region 
                 (nt 940-958 of SEQ ID: 69) → 
                 N25P1, E5-590, 
               
               
                   
                   
                   
                 TCTTACATC   T   AATCATCAT  
                 A7-191 
               
               
                   
                   
                   
                 (nt 940-958 of SEQ ID: 129) 
                   
               
               
                 130 
                 TT8 Mut P2 
                 Mutant Protein 
                 Truncated protein, Q-&gt;Stop  
                   
               
               
                   
                   
                   
                 change 
                   
               
               
                   
               
               
                 131 
                 TT8-3 CDS- 
                 Mutant Coding 
                 TGCCACATG   G   AAGGCTGAT  
                 tt8-3, I0193, E5- 
               
               
                   
                 Mut 
                 region 
                 (nt 960-978 of SEQ ID: 69) → 
                 542, E5-548 
               
               
                   
                   
                   
                 TGCCACATG   A   AAGGCTGAT  
                   
               
               
                   
                   
                   
                 (nt 960-978 of SEQ ID: 131) 
                   
               
               
                 132 
                 TT8 Mut P3 
                 Mutant Protein 
                 Truncated protein, W-&gt;Stop  
                   
               
               
                   
                   
                   
                 change 
                   
               
               
                   
               
               
                 133 
                 TT8-11 
                 Mutant Coding 
                 GCAATAAAGACGAGGAAGA  
                 tt8-11 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 172-190 of SEQ ID: 69) → 
                   
               
               
                   
                   
                   
                 GCAATAAAGA   A   CGAGGAAGA 
                   
               
               
                   
                   
                   
                 (nt 172-191 of SEQ ID: 133) 
                   
               
               
                 134 
                 TT8 Mut P4 
                 Mutant Protein 
                 Frameshift caused by 1bp  
                   
               
               
                   
                   
                   
                 insertion 
                   
               
               
                   
               
               
                 135 
                 TT8-12 
                 Mutant Coding 
                 GCAATAAAGACGAGGAAGA  
                 tt8-12 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 172-190 of SEQ ID: 69) → 
                   
               
               
                   
                   
                   
                 GCAATAAA--CGAGGAAGA  
                   
               
               
                   
                   
                   
                 (nt 172-188 of SEQ ID: 135) 
                   
               
               
                 136 
                 TT8 Mut P5 
                 Mutant Protein 
                 Frameshift caused by 2bp  
                   
               
               
                   
                   
                   
                 deletion 
                   
               
               
                   
               
               
                 137 
                 TT8-13 
                 Mutant Coding 
                 GCAATAAAGACGAGGAAGA  
                 tt8-13 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 172-190 of SEQ ID: 69) → 
                   
               
               
                   
                   
                   
                 GCAATAAAG   G   ACGAGGAAGA 
                   
               
               
                   
                   
                   
                 (nt 172-191 of SEQ ID: 137) 
                   
               
               
                 138 
                 TT8 Mut P6 
                 Mutant Protein 
                 Frameshift caused by 1bp  
                   
               
               
                   
                   
                   
                 insertion 
                   
               
               
                   
               
               
                 139 
                 TT10-1 
                 Mutant Coding 
                 GACTGTTTG   G   TGGCATGCG  
                 tt10-1, E5-539, 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 354-372 of SEQ ID: 33) → 
                 E5-543 
               
               
                   
                   
                   
                 GACTGTTTG   A   TGGCATGCG  
                   
               
               
                   
                   
                   
                 (nt 354-372 of SEQ ID: 139) 
                   
               
               
                 140 
                 TT10 Mut 
                 Mutant Protein 
                 Truncated protein, W-&gt;Stop  
                   
               
               
                   
                 P1 
                   
                 change 
                   
               
               
                   
               
               
                 141 
                 TT10-2 
                 Mutant Coding 
                 TACCGCATT   C   GGATGGTAA  
                 tt10-2, E5-545 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 646-664 of SEQ ID: 33) → 
                   
               
               
                   
                   
                   
                 TACCGCATT   T   GGATGGTAA  
                   
               
               
                   
                   
                   
                 (nt 646-664 of SEQ ID: 141) 
                   
               
               
                 142 
                 TT10 Mut 
                 Mutant Protein 
                 Presumed LOF due to R-&gt;W aa 
                   
               
               
                   
                 P2 
                   
                 change 
                   
               
               
                   
               
               
                 143 
                 TT10-11 
                 Mutant Coding 
                 GGACCAGTGTTAAGGGCT  
                 tt10-11 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 154-171 of SEQ ID: 33) → 
                   
               
               
                   
                   
                   
                 GGACCAGTG   T   TTAAGGGCT  
                   
               
               
                   
                   
                   
                 (nt 154-172 of SEQ ID: 143) 
                   
               
               
                 144 
                 TT10 Mut 
                 Mutant Protein 
                 Frameshift caused by 1bp  
                   
               
               
                   
                 P3 
                   
                 insertion 
                   
               
               
                   
               
               
                 145 
                 TT10-12 
                 Mutant Coding 
                 GGACCAGTGTTAAGGGCT  
                 tt10-12 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 154-171 of SEQ ID: 33) → 
                   
               
               
                   
                   
                   
                 GGACCAGTG   A   TTAAGGGCT  
                   
               
               
                   
                   
                   
                 (nt 154-172 of SEQ ID: 145) 
                   
               
               
                 146 
                 TT10 Mut 
                 Mutant Protein 
                 Frameshift caused by 1bp  
                   
               
               
                   
                 P4 
                   
                 insertion 
                   
               
               
                   
               
               
                 147 
                 TT10-13 
                 Mutant Coding 
                 TCCTGGACCAGTGTTAAGG  
                 tt10-13 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 150-168 of SEQ ID: 33) → 
                   
               
               
                   
                   
                   
                 TCCTGG--------TTAAGG  
                   
               
               
                   
                   
                   
                 (nt 150-161 of SEQ ID: 147) 
                   
               
               
                 148 
                 TT10 Mut 
                 Mutant Protein 
                 Frameshift caused by 7bp  
                   
               
               
                   
                 P5 
                   
                 deletion 
                   
               
               
                   
               
               
                 149 
                 TT12-1 
                 Mutant Coding 
                 AACCCTTTGGCTTACATGTC  
                 tt12-1, A7-261 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 604-623 of SEQ ID: 36) → 
                   
               
               
                   
                   
                   
                 AACCCTTT----TACATGTC  
                   
               
               
                   
                   
                   
                 (nt 604-619 of SEQ ID: 149) 
                   
               
               
                 150 
                 TT12 Mut 
                 Mutant Protein 
                 Frameshift caused by 4bp  
                   
               
               
                   
                 P1 
                   
                 deletion 
                   
               
               
                   
               
               
                 151 
                 TT12-2 
                 Mutant Coding 
                 ATTCTCTCT   G   GTGTTGCCA  
                 tt12-2, J22 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 1237-1255 of SEQ ID: 36) → 
                   
               
               
                   
                   
                   
                 ATTCTCTCT   A   GTGTTGCCA  
                   
               
               
                   
                   
                   
                 (nt 1237-1255 of SEQ ID: 151) 
                   
               
               
                 152 
                 TT12 Mut 
                 Mutant Protein 
                 Presumed LOF due to G→S aa 
                   
               
               
                   
                 P2 
                   
                 change 
                   
               
               
                   
               
               
                 153 
                 TT13-1 
                 Mutant Coding 
                 GCTCTTAAC   C   TTGGAGTTT  
                 tt13-1, aha10-1, J22 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 895-913 of SEQ ID: 39) → 
                   
               
               
                   
                   
                   
                 GCTCTTAAC   T   TTGGAGTTT  
                   
               
               
                   
                   
                   
                 (nt 895-913 of SEQ ID: 153) 
                   
               
               
                 154 
                 TT13 Mut 
                 Mutant Protein 
                 Truncated protein, L-&gt;F change 
                   
               
               
                   
                 P1 
                   
                   
                   
               
               
                   
               
               
                 155 
                 TT13-2 
                 Mutant Coding 
                 ACAGGAAGG   C   GACTTGGGA  
                 tt13-2, P32 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 958-976 of SEQ ID: 39) → 
                   
               
               
                   
                   
                   
                 ACAGGAAGG   T   GACTTGGGA  
                   
               
               
                   
                   
                   
                 (nt 958-976 of SEQ ID: 155) 
                   
               
               
                 156 
                 TT13 Mut 
                 Mutant Protein 
                 Truncated protein, R-&gt;Stop  
                   
               
               
                   
                 P2 
                   
                 change 
                   
               
               
                   
               
               
                 157 
                 TT13-3 
                 Mutant Coding 
                 GGAATGACC   G   GAGATGGTG  
                 tt13-3, E5-540 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 1144-1162 of SEQ ID: 39) → 
                   
               
               
                   
                   
                   
                 GGAATGACC   A   GAGATGGTG  
                   
               
               
                   
                   
                   
                 (nt 1144-1162 of SEQ ID: 157) 
                   
               
               
                 158 
                 TT13 Mut 
                 Mutant Protein 
                 Truncated protein, G-&gt;R change 
                   
               
               
                   
                 P3 
                   
                   
                   
               
               
                   
               
               
                 159 
                 TT16-1 
                 Mutant Coding 
                 TACTTGAAGACCAGTGGAAT  
                 tt16-1 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 211-230 of SEQ ID: 45) → 
                   
               
               
                   
                   
                   
                 TACTTGAAGAC   C   CAGTGGAAT 
                   
               
               
                   
                   
                   
                 (nt 211-231 of SEQ ID: 159) 
                   
               
               
                 160 
                 TT16 Mut 
                 Mutant Protein 
                 Frameshift caused by 1bp  
                   
               
               
                   
                 P1 
                   
                 insertion 
                   
               
               
                   
               
               
                 161 
                 TT16-2 
                 Mutant Coding 
                 TACTTGAAGACCAGTGGAAT  
                 tt16-2 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 211-230 of SEQ ID: 45) → 
                   
               
               
                   
                   
                   
                 TACTTGAAGAC   G   CAGTGGAAT 
                   
               
               
                   
                   
                   
                 (nt 211-231 of SEQ ID: 161) 
                   
               
               
                 162 
                 TT16 Mut 
                 Mutant Protein 
                 Frameshift caused by 1bp  
                   
               
               
                   
                 P2 
                   
                 insertion 
                   
               
               
                   
               
               
                 163 
                 TT16-3 
                 Mutant Coding 
                 TACTTGAAGACCAGTGGAAT  
                 tt16-3 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 211-230 of SEQ ID: 45) → 
                   
               
               
                   
                   
                   
                 TACTTGAAGAC   T   CAGTGGAAT 
                   
               
               
                   
                   
                   
                 (nt 211-231 of SEQ ID: 163) 
                   
               
               
                 164 
                 TT16 Mut 
                 Mutant Protein 
                 Frameshift caused by 1bp  
                   
               
               
                   
                 P3 
                   
                 insertion 
                   
               
               
                   
               
               
                 165 
                 TTG1 CDS- 
                 Mutant Coding 
                 GATC TCCTCGCTTCCTCCGGCGATT TCCT  
                 Y1067, Y1126 
               
               
                   
                 Mut 
                 region 
                 (nt 286-314 of SEQ ID: 75) →  
                   
               
               
                   
                   
                   
                 GATC---------------------TCCT  
                   
               
               
                   
                   
                   
                 (nt 286-293 of SEQ ID: 165) 
                   
               
               
                 166 
                 TTG1 Mut 
                 Mutant Protein 
                 LOF caused by 21bp/7aa deletion 
                   
               
               
                   
                 P1 
                   
                   
                   
               
               
                   
               
               
                 167 
                 TTG1-1 
                 Mutant Coding 
                 TCGCTTCCT   C   CGGCGATTT  
                 ttg1-1, E5-544 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 293-311 of SEQ ID: 75) → 
                   
               
               
                   
                   
                   
                 TCGCTTCCT   T     C GGCGATTT  
                   
               
               
                   
                   
                   
                 (nt 293-311 of SEQ ID: 167) 
                   
               
               
                 168 
                 TTG1 Mut 
                 Mutant Protein 
                 Presumed LOF due to S-&gt;F aa 
                   
               
               
                   
                 P2 
                   
                 change 
                   
               
               
                   
               
               
                 169 
                 TTG1-2 
                 Mutant Coding 
                 TCGCTTGGG   G   AGAAGCTAG  
                 ttg1-2, A7-187 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 542-560 of SEQ ID: 75) → 
                   
               
               
                   
                   
                   
                 TCGCTTGGG   A   AGAAGCTAG  
                   
               
               
                   
                   
                   
                 (nt 542-560 of SEQ ID: 169) 
                   
               
               
                 170 
                 TTG1 Mut 
                 Mutant Protein 
                 Presumed LOF due to G-&gt;E aa 
                   
               
               
                   
                 P3 
                   
                 change 
                   
               
               
                   
               
               
                 171 
                 GL3 CDS 
                 WT Coding 
                 Transcription activator of bHLH 
                 GL3, MYC6.2 basic  
               
               
                   
                   
                 region 
                 superfamily involved in cell  
                 helix-loop-helix  
               
               
                 172 
                 GL3 ORF 
                 WT Protein  
                 fate specification. In  
                 protein 
               
               
                 173 
                 GL3 Ta 
                 WT Gene 
                 association with TTG1, promotes  
                   
               
               
                   
                 locus 
                   
                 trichome formation. Together  
                   
               
               
                   
                   
                   
                 with MYB75/PAP1, plays a role  
                   
               
               
                   
                   
                   
                 in the activation of anthocyanin  
                   
               
               
                   
                   
                   
                 biosynthesis. Activates the  
                   
               
               
                   
                   
                   
                 transcription of GL2. 
                   
               
               
                   
               
               
                 174 
                 GL3-1 CDS- 
                 Mutant Coding 
                 CAACTTAGG   G   AGCTTTACG  
                 gl3-1, E5-541, E5- 
               
               
                   
                 Mut 
                 region 
                 (nt 241-259 of SEQ ID: 171) → 
                 559 
               
               
                   
                   
                   
                 CAACTTAGG   A   AGCTTTACG  
                   
               
               
                   
                   
                   
                 (nt 241-259 of SEQ ID: 174) 
                   
               
               
                 175 
                 GL3 Mut P1 
                 Mutant Protein 
                 Presumed LOF due to E-&gt;K aa 
                   
               
               
                   
                   
                   
                 change 
                   
               
               
                   
               
               
                 176 
                 GL3-2 CDS- 
                 Mutant Coding 
                 GCCGACACA   G   AGTGGTACT  
                 gl3-2, A7-92, E5- 
               
               
                   
                 Mut 
                 region 
                 (nt 358-376 of SEQ ID: 171) → 
                 444 
               
               
                   
                   
                   
                 GCCGACACA   A   AGTGGTACT  
                   
               
               
                   
                   
                   
                 (nt 358-376 of SEQ ID: 176) 
                   
               
               
                 177 
                 GL3 Mut P2 
                 Mutant Protein 
                 Presumed LOF due to E-&gt;K aa 
                   
               
               
                   
                   
                   
                 change 
                   
               
               
                   
               
               
                 178 
                 GL3-3 CDS- 
                 Mutant Coding 
                 GGTTTAACT   G   ATAATTTAA  
                 gl3-3, A7-229, E5- 
               
               
                   
                 Mut 
                 region 
                 (nt 1663-1681 of SEQ ID: 171) → 
                 582 
               
               
                   
                   
                   
                 GGTTTAACT   A   ATAATTTAA  
                   
               
               
                   
                   
                   
                 (nt 1663-1681 of SEQ ID: 178) 
                   
               
               
                 179 
                 GL3 Mut P3 
                 Mutant Protein 
                 Presumed LOF due to D-&gt;N aa 
                   
               
               
                   
                   
                   
                 change 
                   
               
               
                   
               
               
                 180 
                 BAN-1 
                 Mutant Coding 
                 ATCAAGCCA   G   GGATACAAG  
                 ban-1, BJ8, BJ8D 
               
               
                   
                 CDS-Mut 
                 region 
                 (nt 319-337 of SEQ ID: 9) → 
                   
               
               
                   
                   
                   
                 ATCAAGCCA   A   GGATACAAG  
                   
               
               
                   
                   
                   
                 (nt 319-337 of SEQ ID: 9 and  
                   
               
               
                   
                   
                   
                 SEQ ID: 180) 
                   
               
               
                 181 
                 BAN Mut  
                 Mutant Protein 
                 Presumed LOF due to G-&gt;R aa 
                   
               
               
                   
                 P1 
                   
                 change 
                   
               
               
                   
               
               
                 182 
                 TT4-3 CDS- 
                 Mutant Coding 
                 CTCACCCTG G AGGTCCTGC  
                 tt4-3, A7-229, E5- 
               
               
                   
                 Mut 
                 region 
                 (nt 923-941 of SEQ ID: 57) → 
                 582 
               
               
                   
                   
                   
                 CTCACCCTG A AGGTCCTGC  
                   
               
               
                   
                   
                   
                 (nt 923-941 of SEQ ID: 182) 
                   
               
               
                 183 
                 TT4-3 Mut 
                 Mutant Protein 
                 Presumed LOF due to G-&gt;R aa 
                   
               
               
                   
                 P1 
                   
                 change 
               
               
                   
               
            
           
         
       
     
     In certain embodiments, 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, E5-444, E5-540, E5-541, E5-542, E5-543, E5-544, E5-545, E5-547, E5-549, E5-582, E5-586, D3-N10 P5, D5-191, A7-95, A7-187, or A7-261 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:1). In certain embodiments, a WT pennycress TT2 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:1), and is referred to as an allelic variant sequence. In certain embodiments, a TT2 coding sequence allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:1. A representative wild-type pennycress TT2 polypeptide is shown in sequence listing (SEQ ID NO:2). In certain embodiments, a WT pennycress TT2 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:2) 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:2), referred to herein as an allelic variant sequence, provided the polypeptide maintains its wild-type function. For example, a TT2 polypeptide can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99) percent sequence identity to SEQ ID NO:2. A TT2 polypeptide of an allelic variant can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:2. 
     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, 5′ UTR, intron, 3′ 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 embodiments, a modified TT2 coding sequence can include a single base-pair substitution of the cytosine (G) at nucleotide residue 330 in a WT pennycress TT2 coding sequence (e.g., SEQ ID NO:1 or an allelic variant thereof). The G at nucleotide residue 330 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 330 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:4. 
     A modified pennycress TT2 coding sequence having a loss-of-function single base pair substitution (e.g., SEQ ID NO:4) 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:4) can encode a modified TT2 polypeptide. In certain embodiments, a modified TT2 polypeptide can include a truncation resulting from the introduction of a stop codon at codon position 110 within the TT2 open reading frame (e.g., SEQ ID NO:4). A representative truncated pennycress TT2 polypeptide is presented in SEQ ID NO:5. Representative pennycress varieties having a mutation in the TT2 gene include the tt2-1, tt2-2, BC38, and E5-547 varieties. 
     A representative WT pennycress TRANSPARENT TESTA8 (TT8) coding region is presented in SEQ ID NO:69. Two protospacer locations and adjacent protospacer-adjacent motif (PAM) sites that can be targeted by, for example, CRISPR-SpCAS9 correspond to nucleotides 164-183 and 287-306 (protospacers) or 184-186 and 284-286 (PAM sites). In another embodiment, two separate examples of alternative protospacer locations and adjacent protospacer-adjacent motifs (PAM) sites are provided in  FIGS.  3 - 5   . In each case, two protospacer locations can be targeted by, for example, CRISPR-FnCpf1, CRISPR-SmCsm1 or a similar enzyme, correspond to nucleotides 175-153 and 261-283 (protospacers) or 179-176 and 257-260 (PAM sites); and nucleotides 145-167 and 274-296 (protospacers) or 141-144 and 270-273 (PAM sites), all of SEQ ID NO:69. 
     In certain embodiments, a WT pennycress TT8 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:69), and is referred to as an allelic variant sequence. In certain embodiments, a TT8 coding sequence allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:69. A representative WT pennycress TT8 polypeptide is presented in SEQ ID NO:70. 
     In certain embodiments, a WT pennycress TT8 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:70) and is referred to as an allelic variant sequence. For example, a TT8 polypeptide can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:70. A TT8 polypeptide can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:70. 
     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:127, 129, 131, 133, 135, and 137 that result in the TT8 mutant polypeptides of SEQ ID NO:128, 130, 132, 134, 136, and 138, respectively. Representative pennycress varieties with TT8 gene mutations include the tt4-2 tt8-1, tt8-2, tt8-3, tt8-11, tt8-12, tt8-12, tt8-13, I0193, E5-542, E5-548, D5-191, D3-N25P1, E5-590, A7-191, and D3-N10 P5 varieties. 
     In certain embodiments, a WT pennycress TT1 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:27 or 30), and is referred to as an allelic variant sequence. In certain embodiments, a TT1 coding sequence allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:27 or 30. In certain embodiments, a WT pennycress TT1 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:28 or 31), and is referred to as an allelic variant sequence. For example, a TT1 polypeptide allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:28 or 31. A TT1 polypeptide allelic variant can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:28 or 31. 
     In certain embodiments, 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 embodiments, 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 embodiments, 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 embodiments, 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 embodiments, a WT pennycress TT4 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:57), and is referred to as an allelic variant sequence. In certain embodiments, a TT4 coding sequence allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:57. In certain embodiments, a WT pennycress TT4 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:58), and is referred to as an allelic variant sequence. For example, a TT4 polypeptide allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:58. A TT4 polypeptide allelic variant can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:58. 
     In certain embodiments, 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 embodiments, 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:119 that results in the truncated TT4 mutant polypeptide of SEQ ID NO:120. Representative TT4 gene mutations also include the mutations shown in SEQ ID NO:117 and 182 that result in the TT4 mutant polypeptides of SEQ ID NO: 118 and 183, respectively. Representative pennycress varieties with TT4 gene mutations include the tt4-1, tt4-2, tt4-3, A7-229, E5-582 and E5-549 varieties. 
     In certain embodiments, 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:60, 72, 42, 48, or 51, respectively), and is referred to as an allelic variant sequence. In certain embodiments, a TT5, TT9, TT15, TT18, or TT19 coding sequence allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:60, 72, 42, 48, or 51, respectively. In certain embodiments, 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:61, 73, 43, 49, or 52, 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 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:61, 73, 43, 49, or 52, respectively. A TT5, TT9, TT15, TT18, or TT19 polypeptide allelic variant can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:61, 73, 43, 49, or 52, respectively. 
     In certain embodiments, 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 embodiments, 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 embodiments, a WT pennycress TT6 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:63), and is referred to as an allelic variant sequence. In certain embodiments, a TT6 coding sequence allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:63. In certain embodiments, a WT pennycress TT6 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:64), and is referred to as an allelic variant sequence. For example, a TT6 polypeptide allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:64. A TT6 polypeptide allelic variant can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:64. 
     In certain embodiments, 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 embodiments, 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:121 that results in the TT6 mutant polypeptide of SEQ ID NO:122. Representative pennycress varieties with TT6 gene mutations mutants include the tt6-1 and AX17 varieties. Representative TT6 gene mutations also include the mutation shown in SEQ ID NO:123 that results in the TT6 mutant polypeptide of SEQ ID NO:124. Representative pennycress varieties with TT6 gene mutations mutants also include the tt6-1, tt6-2 and Q36 varieties. 
     In certain embodiments, a WT pennycress TT7 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:66), and is referred to as an allelic variant sequence. In certain embodiments, a TT7 coding sequence allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:66. In certain embodiments, a WT pennycress TT7 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:67), and is referred to as an allelic variant sequence. For example, a TT7 polypeptide allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:67. A TT7 polypeptide allelic variant can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:67. 
     In certain embodiments, 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 embodiments, 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:125 that results in the TT7 mutant polypeptide of SEQ ID NO:126. Representative pennycress varieties with TT7 gene mutations include the tt7-1, A7-3, E5-586, E5-484 P15, and E5-484 P5 varieties. 
     In certain embodiments, a WT pennycress TTG1 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:75), and is referred to as an allelic variant sequence. In certain embodiments, a TTG1 coding sequence allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:75. In certain embodiments, a WT pennycress TTG1 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:76), and is referred to as an allelic variant sequence. For example, a TTG1 polypeptide allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:28 or 31. A TTG1 polypeptide allelic variant can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:76. 
     In certain embodiments, 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 embodiments, 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 embodiments, a LOF mutation in a TTG1 gene can comprise a 21 bp deletion in the TTG1 coding sequence as shown in SEQ ID NO:165. In other embodiments, a LOF mutation in a TTG1 gene can comprise ttg1-1 and ttg1-2 mutant alleles having single nucleotide substitutions that result in the substitution of a conserved amino acid residue in the TTG protein (SEQ ID NOs:167-170). Representative TTG1 gene mutations thus include the mutations shown in SEQ ID NO:165, 167, and 169 that result in the TTG1 mutant polypeptides of SEQ ID NO:166, 1268, and 170, respectively. Representative pennycress varieties with TTG1 gene mutations include the Y1067, Y1126, ttg1-1, E5-544, ttg1-2, and A7-187 varieties. 
     In certain embodiments, a WT pennycress TT10 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:33), and is referred to as an allelic variant sequence. In certain embodiments, a TT10 coding sequence allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:33. In certain embodiments, a WT pennycress TT10 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:34), and is referred to as an allelic variant sequence. For example, a TT10 polypeptide allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:34. A TT10 polypeptide allelic variant can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:34. 
     In certain embodiments, 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 embodiments, 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 embodiments, 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 embodiments, 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:139, 141, 143, 145, or 147 that result in the TT10 mutant polypeptides of SEQ ID NO: 140, 142, 144, 146, or 148, respectively. Representative pennycress varieties with TT10 gene mutations include the tt10-1, tt10-2, tt10-1, tt10-12, tt10-13, E5-539, E5-543, and E5-545 varieties. 
     In certain embodiments, a WT pennycress TT12 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:36), and is referred to as an allelic variant sequence. In certain embodiments, a TT12 coding sequence allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:36. In certain embodiments, a WT pennycress TT12 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:37), and is referred to as an allelic variant sequence. For example, a TT12 polypeptide allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:37. A TT12 polypeptide allelic variant can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:37. 
     In certain embodiments, 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 embodiments, 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 embodiments, 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 embodiments, 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:149 or 151 that result in the TT12 mutant polypeptides of SEQ ID NO:150 or 152, respectively. Representative pennycress varieties with TT12 gene mutations include the tt12-1, tt12-2, A7-261, and J22 varieties. 
     In certain embodiments, a WT pennycress TT13 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:39), and is referred to as an allelic variant sequence. In certain embodiments, a TT13 coding sequence allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:39. In certain embodiments, a WT pennycress TT13 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:40), and is referred to as an allelic variant sequence. For example, a TT13 polypeptide allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:40. A TT13 polypeptide allelic variant can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:40. 
     In certain embodiments, 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 embodiments, 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:153, 155, or 157 that result in the TT13 mutant polypeptides of SEQ ID NO:154, 156, or 158, respectively. Representative pennycress varieties with TT13 gene mutations include the tt13-1, tt13-2, tt13-3, aha10-1, J22, and P32 E5-540 varieties. 
     In certain embodiments, a WT pennycress TT16 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:45), and is referred to as an allelic variant sequence. In certain embodiments, a TT16 coding sequence allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:45. In certain embodiments, a WT pennycress TT16 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:46), and is referred to as an allelic variant sequence. In certain embodiments, a TT16 polypeptide allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:46. A TT16 polypeptide allelic variant can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:46. 
     In certain embodiments, 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 embodiments, 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 embodiments, 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 embodiments, 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:159, 161, or 163 that result in the TT16 mutant polypeptides of SEQ ID NO:160, 162, or 164, respectively. Representative pennycress varieties with TT16 gene mutations include the tt16-1, tt16-2, and tt16-3 varieties. 
     In certain embodiments, 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 provided herewith in Table 1 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:1) and/or at least one guide sequence specific to a pennycress TT8 sequence (see, e.g., SEQ ID NO:5). 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 5′-NGG-3′, 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 embodiments, 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., 2014). 
     In certain embodiments, a WT pennycress GL3 coding sequence can have a sequence that deviates from the coding sequence set forth above (e.g., SEQ ID NO:171), and is referred to as an allelic variant sequence. In certain embodiments, a GL3 coding sequence allelic variants can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:171. In certain embodiments, a WT pennycress GL3 polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:172), and is referred to as an allelic variant sequence. For example, a GL3 polypeptide allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:160. A GL3 polypeptide allelic variant can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:172. 
     In certain embodiments, 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 embodiments, 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 embodiments, the GL3 mutation can comprise the coding sequence mutations of SEQ ID NO:174, 176, 178 and/or the protein sequence mutation of SEQ ID NO:175, 177, 180. Representative pennycress varieties with GL3 gene mutations include the g13-1, g13-2, g13-3, E5-541, E5-559, A7-92, E5-444, A7-229, and E5-582 varieties. 
     In certain embodiments, 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:9), and is referred to as an allelic variant sequence. In certain embodiments, a BAN coding sequence allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:9. In certain embodiments, a WT pennycress BAN polypeptide can have a sequence that deviates from the polypeptide sequence set forth above (SEQ ID NO:10), and is referred to as an allelic variant sequence. For example, a BAN polypeptide allelic variant can have at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99 percent sequence identity to SEQ ID NO:10. A BAN polypeptide allelic variant can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to SEQ ID NO:10. 
     In certain embodiments, 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 embodiments, 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 embodiments, the BAN mutation can comprise the coding sequence mutation of SEQ ID NO:180 and/or the protein sequence mutation of SEQ ID NO:181. Representative pennycress varieties with BAN gene mutations include the ban-1, BJ8, and BJ8D varieties. 
     In certain embodiments, 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 1. In certain embodiments, 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: 1, 3, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 171, 173, and/or any allelic variants thereof. In certain embodiments, 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: 1, 3, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 171, 173, and/or any allelic variants thereof. 
     The LOF mutations in any of the genes or coding sequences of Table 1 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 Guha et al. Comput Struct Biotechnol J. 2017; 15: 146-160. Methods for modifying genomes by use of Cpf1 or Csm1 nucleases are disclosed in US Patent Application Publication 20180148735, which is incorporated herein by reference in its entirety, 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 US Patent Application Publication 20180179547, which is incorporated herein by reference in its entirety, 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 embodiments, 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 embodiments, 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. 
     EXAMPLES 
     Example 1: Meal Made from Wild Type Pennycress Plants is High in Fiber, but Low in Metabolizable Energy 
     Higher dietary fiber results in lower net energy for swine (Kil et al., 2013) and poultry (Meloche et al., 2013). 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., 2011). 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 1.5-2 times the levels in canola meal (Table 2). Similar levels were observed when comparing different lots of pennycress meal with canola meal (Table 3). Analysis conducted by Arvegenix at University of Georgia showed similar results (Table 4). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Nutrient composition of mechanically expeller-pressed canola 
               
               
                 and pennycress meals produced at Dairyland by Arvegenix in 
               
               
                 August 2015. All numbers are in percent dry weight (% DW). 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Expeller- 
                 Pennycress 
                 Pennycress 
               
               
                   
                 Meal 
                 Pressed 
                 Meal 
                 Meal 
               
               
                   
                 Constituent 
                 Canola Meal 
                 (Beecher) 
                 (Ruby II) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Crude Protein 
                 38.7 
                 31.3 
                 31.1 
               
               
                   
                 Either extract 
                 11.2 
                 10.1 
                 10.6 
               
               
                   
                 Crude fiber 
                 10.9 
                 27.1 
                 27.9 
               
               
                   
                 ADF 
                 18.1 
                 35.6 
                 33.8 
               
               
                   
                 NDF 
                 22.7 
                 40.5 
                 36.8 
               
               
                   
                 Total 
                 29.5 
                 43.3 
                 37.8 
               
               
                   
                 Dietary Fiber 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Lot variation in proximate values in mechanically expeller-pressed pennycress 
               
               
                 meal, composite mechanically expeller-pressed pennycress meal blend (all produced 
               
               
                 by Arvegenix), and commercially available mechanically expeller-pressed canola 
               
               
                 (ME Canola). All numbers represent the average of duplicate analytical runs 
               
               
                 for mean and standard error measured in percent dry weight (% DW). 
               
            
           
           
               
               
            
               
                   
                 Meal Constituent Processing Date(s) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Blend* 
                 ME 
               
               
                   
                 Lot 1 
                 Lot 2 
                 Lot 3 
                 Lot 4 
                 22-27 
                 Canola 
               
               
                   
                 22 Jul. 2015 
                 23 Jul. 2015 
                 23 Jul. 2015 
                 23 Jul. 2015 
                 Jul. 2015 
                 N/A 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Moisture (% FW) 
                  2.12 ± 0.08 
                 6.10 ± 0.1 
                  5.20 ± 0.01 
                 4.06 ± 0.08 
                  3.36 ± 0.05 
                  4.41 ± 0.13 
               
               
                 Ash Content 
                  7.32 ± 0.06 
                 7.24 ± 0.1 
                  7.13 ± 0.01 
                 7.17 ± 0.02 
                  5.62 ± 2.38 
                  6.88 ± 0.02 
               
               
                 Carbohydrates 
                  51.4 ± 0.07 
                 50.9 ± 0.7 
                  50.9 ± 0.14 
                 49.7 ± 0.07 
                  49.8 ± 2.26 
                 40.7 ± 1.3 
               
               
                 Crude Fat 
                  8.99 ± 0.03 
                  10.3 ± 0.01 
                  10.6 ± 0.14 
                 11.1 ± 0.01 
                  11.6 ± 0.01 
                 13.5 ± 1.5 
               
               
                 Crude Protein 
                 32.2 ± 0.1 
                 31.6 ± 0.7 
                 31.4 ± 0.1 
                 32.0 ± 0.01 
                 33.1 ± 0.1 
                 38.9 ± 0.2 
               
               
                 Crude Fiber 
                 28.7 ± 1.2 
                 29.5 ± 2.1 
                 30.3 ± 0.2 
                 28.0 ± 0.1  
                 26.4 ± 0.6 
                 10.9 ± 0.5 
               
               
                 Acid Detergent 
                 37.9 ± 0.5 
                 38.7 ± 0.1 
                 36.7 ± 2.8 
                 36.8 ± 0.5  
                 32.1 ± 0.8 
                 18.25 ± 0.1  
               
               
                 Fiber 
               
               
                 Neutral Detergent 
                 39.8 ± 0.6 
                 39.9 ± 0.1 
                 39.5 ± 0.8 
                 38.5 ± 0.6  
                 34.8 ± 2.0 
                 23.3 ± 0.2 
               
               
                 Fiber 
               
               
                 Total Dietary 
                 41.6 ± 1.2 
                 41.2 ± 1.2 
                 41.0 ± 1.0 
                 39.0 ± 0.1  
                 42.2 ± 7.4 
                 29.7 ± 1.3 
               
               
                 Fiber 
               
               
                   
               
               
                 *The Blend sample, consisting of Lots 1-4 (~66% by weight) and Lot 5 (~33% by weight), was blended and analyzed for nutrition studies. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Proximate compositions (% as is) for canola 
               
               
                 meal (CM) and pennycress meal samples. 
               
            
           
           
               
               
               
            
               
                   
                 CM  1   
                 PM  2   
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Crude Protein 
                 36.7 
                 32.0 
               
               
                   
                 Fat 
                 11.4 
                 8.61 
               
               
                   
                 Crude Fiber 
                 9.27 
                 19.9 
               
               
                   
                 ADF  3   
                 18.3 
                 39.6 
               
               
                   
                 NDF  4   
                 22.7 
                 43.0 
               
               
                   
                 Ash 
                 6.51 
                 7.57 
               
               
                   
                 Dry Matter 
                 94.1 
                 94.4 
               
               
                   
                   
               
            
           
         
       
     
     Total Metabolizable Energy (TMEn) corrected for nitrogen was measured in mechanically expeller-pressed pennycress meal and canola meal. TMEn was found to be 18.2% or 18.9% less in the pennycress meal as compared to the canola meal when fed to chickens due to the higher fiber content (Table 5) and Metabolizable Energy (ME) was 16% less in pennycress meal as compared to the canola meal when fed to pigs due to the higher fiber content (Table 6). 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Total metabolizable energy corrected for nitrogen 
               
               
                 (TMEn) for mechanically expeller-pressed canola 
               
               
                 and pennycress meal when fed to chickens. 
               
            
           
           
               
               
               
               
            
               
                   
                 Mech Pennycress 
                 Mech 
                 Difference, 
               
               
                   
                 Meal (Beecher) 
                 Canola Meal 
                 % 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                 Energy 
                 Parsons 2015 
                 Parsons 2006 
                   
               
               
                 TMEn (kcal/g DM) 
                 2.455 
                 3 
                 −18.17 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Concentration of digestible energy (DE) and metabolizable 
               
               
                 energy (ME) in pennycress expeller and canola expellers when fed 
               
               
                 to pigs (data 1  produced at University of Illinois). 
               
            
           
           
               
               
               
            
               
                   
                 Ingredients 
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Pennycress 
                 Canola 
                   
                   
               
               
                 Item 
                 expellers 
                 expellers 
                 SEM 
                 P - value 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 DE, kcal/kg 
                 3,191 
                 3,582 
                 92.18 
                 0.009 
               
               
                 DE, kcal/kg of DM 
                 3,536 
                 3,833 
                 99.43 
                 0.053 
               
               
                 ME, kcal/kg 
                 2,652 
                 3,269 
                 143.98 
                 0.009 
               
               
                 ME, kcal/kg of DM 
                 2,938 
                 3,499 
                 158.17 
                 0.025 
               
               
                   
               
               
                   1 Data are means of 8 observations per treatment. SEM abbreviation stands for standard error of the mean. DM abbreviation is for Dry Matter. 
               
            
           
         
       
     
     In summary, Beecher and Ruby II varieties of pennycress meal contain between 1.5× to 2× the fiber content as compared to similarly processed canola meal resulting in 18-19% 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. 
     Example 2: Selection of Mutant Pennycress Plants Low in Fiber, High in Oil and Protein from Cultivated Isolates 
     About 850 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 10,000 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 850 cultivated lines (Table 2) and selected for further propagation and breeding. Certain selected pennycress variant lines Y1067 and Y1126 were isolated from a cultivated field in Grantfork Ill. Certain selected pennycress Y1126 lines were isolated from a cultivated field in Macomb Ill. in 2015. 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 100% 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 15.5% and 11.5% vs. 27.5% in wild type, demonstrating 43-58% reduction in ADF fiber. Other measurements of fiber content such as NDF and CF were also significantly (29-55%) lower in the yellow-seeded lines relative to wild type, while the protein level was significantly (˜50%) higher. The composition of yellow and dark brown seeds is listed in Table 7. 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. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 The composition of meal (adjusted for oil content) 
               
               
                 made from yellow and dark brown seeds (Dairyland 
               
               
                 Laboratories, Arcadia, Wisconsin). 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Pennycress 
                 Seed coat 
                 % mois- 
                 ADF 
                 NDF 
                 Crude 
                 Pro- 
               
               
                 line 
                 color 
                 ture 
                 fiber 
                 fiber 
                 fiber 
                 tein 
               
               
                   
               
               
                 Y1067 
                 yellow 
                 6.63 
                 15.5 
                 22.3 
                 15.5 
                 32.4 
               
               
                 Y1126 
                 yellow 
                 6.38 
                 11.5 
                 15.2 
                  9.9 
                 31.9 
               
               
                 1063 
                 dark brown 
                 7.39 
                 27.2 
                 30.6 
                 22.6 
                 21.3 
               
               
                 1067 
                 dark brown 
                 7.29 
                 26.6 
                 29.8 
                 19.9 
                 19.8 
               
               
                 1126 
                 dark brown 
                 6.43 
                 28.4 
                 33.7 
                 24.7 
                 24.6 
               
               
                 1139 
                 dark brown 
                 6.50 
                 26.4 
                 29.8 
                 19.9 
                 22.4 
               
               
                 1204 
                 dark brown 
                 6.58 
                 26.3 
                 28.9 
                 18.7 
                 20.9 
               
               
                 1228 
                 dark brown 
                 6.30 
                 28.8 
                 33.8 
                 25.4 
                 22.1 
               
               
                 1326 
                 dark brown 
                 6.47 
                 29.2 
                 32.6 
                 23.4 
                 21.7 
               
               
                 2032 
                 dark brown 
                 6.16 
                 24.7 
                 28.8 
                 17.6 
                 22.1 
               
               
                 2084 
                 dark brown 
                 6.89 
                 26.0 
                 29.0 
                 19.4 
                 22.2 
               
               
                 2116 
                 dark brown 
                 7.16 
                 30.4 
                 36.2 
                 24.4 
                 20.1 
               
               
                 2133 
                 dark brown 
                 6.64 
                 29.6 
                 34.4 
                 25.0 
                 21.5 
               
               
                 2206 
                 dark brown 
                 6.69 
                 25.5 
                 29.4 
                 18.1 
                 20.7 
               
               
                 2229 
                 dark brown 
                 6.61 
                 27.1 
                 32.5 
                 23.0 
                 21.9 
               
               
                 2253 
                 dark brown 
                 6.42 
                 24.0 
                 28.3 
                 17.8 
                 22.5 
               
               
                 2288 
                 dark brown 
                 6.28 
                 26.6 
                 33.0 
                 25.5 
                 N/A 
               
               
                 2329 
                 dark brown 
                 6.57 
                 26.6 
                 31.9 
                 18.8 
                 20.8 
               
               
                 2369 
                 dark brown 
                 6.05 
                 23.1 
                 26.7 
                 17.9 
                 23.2 
               
               
                 2458 
                 dark brown 
                 6.39 
                 25.4 
                 29.8 
                 18.8 
                 22.2 
               
               
                 2460 
                 dark brown 
                 6.49 
                 30.6 
                 36.3 
                 26.7 
                 21.2 
               
               
                 2369 
                 light brown 
                 6.50 
                 36.9 
                 45.8 
                 32.1 
                 19.1 
               
               
                 Average 
                 yellow 
                 6.51 
                 13.5 
                 18.7 
                 12.7 
                 32.2 
               
               
                 Average 
                 dark brown 
                 6.59 
                 27.5 
                 32.1 
                 22.0 
                 21.6 
               
               
                 % change 
                 yellow 
                 Y1067 
                 −43%  
                 −30%  
                 −29%  
                 50% 
               
               
                 % change 
                 yellow 
                 Y1126 
                 −58%  
                 −53%  
                 −55%  
                 48% 
               
               
                   
               
            
           
         
       
     
     Example 3: Identification of Mutated Gene in Pennycress Plants Low in Fiber, High in Oil and Protein from Cultivated Isolates 
     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. al., 1991) 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 200 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 21 bp deletion in TTG1 gene (Seq ID No. 165) 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. al., 2011. This mutation results in a deletion of 7 amino acids in the conserved area of TTG1 protein, likely leading to a complete loss of function. The definitive nature of this 21 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. 
     Example 4: Generation and Characterization of EMS-Mutagenized Light-Colored Seed Coat Mutant Lines BC38, BJ8, P32, J22, Q36, BD24, AX17, E5-444, E5-540, E5-541, E5-542, E5-543, E5-545, E5-547, E5-549, E5-582, E5-586, D3-N10 P5, D5-191, A7-95, A7-187 and A7-261 
     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. 
     Materials and Methods 
     Solutions: 
                                                    A)   0.2M sodium phosphate monobasic   6.9 g/250 mL               (NaH 2 PO 4 *H 2 O)           B)   0.2M sodium phosphate dibasic   7.1 g/250 mL               (NaH 2 PO 4  anhydrous)                        
For 50 mL of 0.1 M sodium phosphate buffer at pH 7:
 
                                    9.75 mL   A       15.25 mL    B       25.0 mL   dH 2 O                    
0.2% EMS in buffer:
         20 mL 0.1M Sodium Phosphate Buffer, pH 7   40 μL EMS liquid (Sigma #M0880-5G)
 
0.1 M sodium thiosulfate at pH 7.3:
   12.4 g sodium thiosulfate in 500 mL
 
Primary Seed Surface Sterilization
       

     Wild-type pennycress ( Thlaspi arvense ) seeds (Spring 32 ecotype) were surface sterilized for 10 minutes in a 30% bleach, 0.05% SDS solution before being rinsed 3× with sterile water. Sterilized seeds were immediately subjected to EMS treatment. 
     Ethyl Methane Sulfonate (EMS) Treatment of Pennycress Seeds 
     Sterilized pennycress seeds (41 g) were agitated in distilled water overnight. Four 250 mL Erlenmeyer flasks with 10 g seed each, and 1 g in a separate small flask as a control, were agitated. The water was decanted. 
     25 mLs of 0.2% EMS in 0.1M sodium phosphate buffer (pH 7) was added. The control received only phosphate buffer with no EMS. The flasks were shaken in fume hood for 18 hours. The EMS solution was decanted off into an EMS waste bottle. 
     To rinse the seeds, 25 ml of dH 2 O was added to each flask, and the flasks were shaken for 20 minutes. The rinse water was decanted into the EMS waste bottle. 
     To deactivate the EMS, seeds were washed for 20 minutes in 0.1M sodium thiosulfate (pH 7.3), rinsed 4 with dH2O for 15 minutes, suspended in 0.1% agarose, and germinated directly in autoclaved Reddiearth soil at a density of approximately 10 seeds per 4-inch pot. 
     Plant Growth Conditions 
     EMS-treated pennycress seeds were germinated and grown in an environmental growth chamber at 21° C., 16:8 6400K fluorescent light/dark, 50% humidity. Approximately 14 days after planting, plants were thinned and transplanted to a density of 4 plants per 4-inch pot. These M 1 -generation plants showed telltale chlorotic leaf sectors that are indicative of a successful mutagenesis. 
     After dry down, these M 1 -generation plants were catalogued and harvested. The M 2 - and M 3 -generation seeds were surface sterilized, planted and grown according to the protocols previously described. 
     Identification and Characterization of Light-Colored Seed Coat Mutant Lines 
     Light-colored seed coat mutants in the M 3 -generation were identified as those having mature seed coats of a lighter color relative to that of wild type. Seeds (M 3 -generation) from putative M 2 -generation mutants were planted and grown in potting soil-containing 4-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 8 of Example 5 (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 3. Underlying gene and protein mutations were identified (Table 1, SEQ ID NO: 117-132, 139-142, 149-158, 167-170 and 174-181) and confirmed using standard Sanger sequencing and genetic segregation analyses. 
     Example 5: Generation of Transgenic Pennycress Lines Harboring the CRISPR-Cas9 or CRISPR-Cpf1 or CRISPR-Cms1 Constructs 
     Materials and Methods 
     Construction of the  Thlaspi arvense  (Pennycress) TT1, TT2, TT8, TT10, and TT16 Gene-Specific CRISPR Genome-Editing Vectors. 
     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. al., 2014, Steinert et. al., 2015 and Begemann et. al., 2017. 
     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 1 (Seq ID NO: 89-116) were synthesized, annealed to create the 20-mer protospacers specific to the designated pennycress genes and used for construction of gene-editing binary vectors as described (Fauser et. al., 2014, Steinert et. al., 2015 and Begemann et. al., 2017). 
     Vector Transformation into  Agrobacterium    
     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, 1978). 
     The transformation product was plated on 1% agar Luria Broth (LB) plates with gentamycin (50 μg/ml) rifampicin (50 μg/ml) and spectinomycin (75 μg/ml). Single colonies were selected after two days of growth at 28° C. 
     Plant Transformation—Pennycress Floral Dip 
     DAY ONE: 5 mL of LB+5 uL with appropriate antibiotics (Rifampin (50), Spectinomycin (75), and/or Gentamycin (50)) were inoculated with  Agrobacterium . The cultures were allowed to grow, with shaking, overnight at 28° C. 
     DAY TWO (early morning): 25 mL of Luria Broth+25 uL appropriate antibiotics (Rifampin (50), Spectinomycin (75), and/or Gentamycin (50)) were inoculated with the initial culture from day one. The cultures were allowed to grow, with shaking, overnight at 28° C. 
     DAY TWO (late afternoon): 250 mL of Luria Broth+250 uL appropriate antibiotic (Rifampin (50), Spectinomycin (75), and/or Gentamycin (50)) were inoculated with 25 mL culture. The cultures were allowed to grow, with shaking, overnight at 28° C. 
     DAY THREE: When the culture had grown to an OD 600  of ˜1.0, the culture was decanted into large centrifuge tubes and spun at 3,500 RPM at room temperature for 10 minutes to pellet cells. The supernatant was decanted off. The pelleted cells were resuspended in a solution of 5% sucrose and 0.02% Silwet L-77. 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 25-30 PSI for 10 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. 
     Screening Transgenic Plants and Growth Condition 
     Pennycress seeds were surface sterilized by first rinsing in 70% ethanol then incubating 10 minutes in a 30% bleach, 0.05% SDS solution before being rinsed two times with sterile water and plated on selective plates (0.8% agar/one half-strength Murashige and Skoog salts with hygromycin B selection (40 U/ml) or glufosinate (18 μg/ml). Plates were wrapped in parafilm and kept in an environmental growth chamber at 21° C., 16:8 day/night for 8 days until antibiotic or herbicide selection was apparent. 
     Surviving hygromycin or glufosinate-resistant T 1 -generation seedlings were transplanted into autoclaved Reddiearth soil mix and grown in an environmental growth chamber set to 16-hour days/8-hour nights at 21° C. and 50% humidity. T 2 -generation seeds were planted, and ˜1.5 mg of leaf tissue from each T 2 -generation plant was harvested with a 3-mm hole punch, then processed using the Thermo Scientific™ Phire™ Plant Direct PCR Kit as per manufacturer&#39;s instructions. Subsequently, PCR reactions for genotyping (20 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&#39;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. al., 2010) 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 1 (Seq ID Nos. 133-138, 143-148, 159-164). Plants carrying LOF mutations were grown to homozygosity, and the phenotypes were confirmed using visual and analytical assessments. 
     Example 6. Selected Yellow-Seeded Pennycress Mutants Demonstrate Significant Reductions in Fiber and Fiber Components 
     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, Wis.). 
     ADF (Acid Detergent Fiber) 
     Fiber (Acid Detergent) and Lignin in Animal Feed: AOAC Official Method 973.18 (1996) (Modification includes use of Sea Sand for filter aid as needed). 
     Crude Fiber 
     Fiber (Crude) in Animal Feed and Pet Food (Fritted Glass Crucible Method): AOAC Official Method 978.10 ch4 p 28 (1979) (Modification includes use of Sea Sand for filter aid as needed). 
     Lignin 
     Fiber (Acid Detergent) and Lignin in Animal Feed: AOAC Official Method 973.18 (1996) (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 72% sulfuric acid for full time required). 
     NDF (Neutral Detergent Fiber) 
     Amylase-Treated Neutral Detergent Fiber in Feeds AOAC Official Method 2002.04 2005 (Modification includes use of Sea Sand for filter aid and Whatman GF/C filter paper for residue collection). 
     The results presented in Table 8 indicate that majority of the light-colored mutants have 35-60% less fiber and its components relative to WT plants (MN106 and Beecher). 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Composition of sixteen selected light-colored pennycress mutants vs. two wild type 
               
               
                 pennycress accessions measured using wet chemistry methods at Dairyland Laboratories 
               
               
                 (Arcadia, Wisconsin). The numbers represent percent of dry matter (% DM). 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 Mutated 
                 Seed 
                   
                 Crude 
                   
                   
                 Crude 
               
               
                 No. 
                 Name/ID 
                 Gene/Allele 
                 Coat 
                 Moisture 
                 Protein 
                 ADF 
                 aNDF 
                 fiber 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 Y1126 
                 ttg1 
                 light 
                 7.6 
                 28.1 
                 13.9 
                 16.6 
                 9.6 
               
               
                 2 
                 E5-543 
                 tt10-1 
                 light 
                 7.4 
                 26.5 
                 15.3 
                 19.7 
                 14.4 
               
               
                 3 
                 E5-542 
                 tt8 
                 light 
                 7.5 
                 30.6 
                  9.1 
                 17.5 
                 13.8 
               
               
                 4 
                 E5-547 
                 tt2-1 
                 light 
                 6.7 
                 28.1 
                 12.8 
                 17.2 
                 12.1 
               
               
                 5 
                 A7-63 
                 N/A 
                 light 
                 6.9 
                 28.7 
                 14.6 
                 20.5 
                 11.8 
               
               
                 6 
                 A7-187 
                 ttg1-2 
                 light 
                 7.5 
                 29.2 
                 12.9 
                 17.8 
                 13.1 
               
               
                 7 
                 E5-559 
                 gl3-1 
                 light 
                 7.0 
                 26.3 
                 21.8 
                 32.5 
                 22.5 
               
               
                 8 
                 E5-539 
                 tt10-1 
                 light 
                 7.5 
                 27.3 
                 13.9 
                 17.6 
                 12.0 
               
               
                 9 
                 A7-261 
                 tt12-1 
                 light 
                 6.6 
                 27.2 
                 14.9 
                 19.5 
                 13.6 
               
               
                 10 
                 E5-549 
                 tt4-2 
                 light 
                 7.4 
                 26.5 
                 16.2 
                 22.3 
                 12.7 
               
               
                 11 
                 E5-444 
                 gl3-2 
                 light 
                 7.8 
                 27.7 
                 14.6 
                 17.5 
                 10.8 
               
               
                 12 
                 D5-191 
                 tt8-2 
                 light 
                 6.5 
                 26.6 
                 13.3 
                 17.9 
                 13.0 
               
               
                 13 
                 E5-586 
                 tt7-1 
                 light 
                 7.4 
                 27.9 
                 12.6 
                 17.2 
                 11.3 
               
               
                 14 
                 E5-542 
                 tt8-3 
                 light 
                 6.9 
                 26.0 
                 13.5 
                 19.9 
                 16.2 
               
               
                 15 
                 E5-541 
                 gl3-1 
                 light 
                 6.8 
                 27.2 
                 15.1 
                 19.2 
                 13.2 
               
               
                 16 
                 E5-545 
                 tt10-2 
                 light 
                 6.7 
                 24.5 
                 14.8 
                 18.5 
                 12.9 
               
               
                 17 
                 MN106 
                 WT 
                 dark 
                 6.7 
                 25.2 
                 22.7 
                 25.8 
                 16.1 
               
               
                 18 
                 Beecher 
                 WT 
                 dark 
                 6.5 
                 25.6 
                 21.1 
                 23.9 
                 15.4 
               
               
                 19 
                 MIN of 
                 light-colored 
                 % of DM 
                 6.5 
                 24.5 
                  9.1 
                 16.6 
                 9.6 
               
               
                 20 
                 MAX of 
                 light-colored 
                 % of DM 
                 7.8 
                 30.6 
                 21.8 
                 32.5 
                 22.5 
               
               
                 21 
                 MIN of 
                 light-colored 
                 % of WT 
                 97%     
                 97% 
                 40% 
                 64% 
                 60% 
               
               
                   
               
            
           
         
       
     
     Example 7. Selected Yellow-Seeded Pennycress Mutants Demonstrate Significant Increases in Protein and Oil Composition 
     
       
         
           
               
             
               
                 TABLE 9 
               
               
                   
               
               
                 Composition of five selected light-colored pennycress mutants vs. 95 wild type pennycress accessions 
               
               
                 harvested at various locations across USA and measured using NIR spectroscopy analysis. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 % 
                 % Erucic 
                 % Total 
                 Sinigrin 
                 % ADF 
                 % NDF 
                 % 
               
               
                 No. 
                 Accession 
                 Color 
                 Moisture 
                 Acid 
                 Oil 
                 μmol/g 
                 Fiber 
                 Fiber 
                 Protein 
               
               
                   
               
               
                 1 
                 Y1067 
                 Yellow 
                 7.2 
                 25.1 
                 37.6 
                 149.1 
                 15.5 
                 16.2 
                 32.5 
               
               
                 2 
                 Y1126 
                 Yellow 
                 8.3 
                 31.1 
                 43.3 
                 49.9 
                 11.5 
                 14.9 
                 31.8 
               
               
                 3 
                 P32 
                 Light brown 
                 6.0 
                 39.5 
                 36.4 
                 180.2 
                 13.5 
                 18.0 
                 29.1 
               
               
                 4 
                 Q36.C 
                 Brown 
                 6.1 
                 22.8 
                 33.0 
                 196.2 
                 19.7 
                 24.1 
                 25.0 
               
               
                 5 
                 BJ.8 
                 Tan 
                 7.0 
                 39.0 
                 49.0 
                 107.4 
                 10.0 
                 13.1 
                 33.6 
               
               
                 6 
                 1126 
                 Dark brown 
                 10.2 
                 33.7 
                 30.8 
                 59.2 
                 27.6 
                 31.2 
                 22.2 
               
               
                 7 
                 Spring32 (WT) 
                 Dark brown 
                 8.6 
                 34.8 
                 30.6 
                 116.0 
                 27.6 
                 32.2 
                 22.0 
               
               
                 8 
                 1069 
                 Dark brown 
                 8.8 
                 32.9 
                 29.4 
                 103.4 
                 37.8 
                 35.1 
                 22.6 
               
               
                 9 
                 1096 
                 Dark brown 
                 8.4 
                 31.3 
                 26.0 
                 128.7 
                 32.9 
                 34.2 
                 20.1 
               
               
                 10 
                 2139 
                 Dark brown 
                 8.7 
                 29.6 
                 23.1 
                 147.0 
                 29.0 
                 33.9 
                 20.4 
               
               
                 11 
                 2057 
                 Dark brown 
                 8.2 
                 31.0 
                 23.7 
                 157.6 
                 31.5 
                 33.8 
                 18.7 
               
               
                 12 
                 1126 
                 Dark brown 
                 7.8 
                 29.2 
                 30.6 
                 117.4 
                 34.7 
                 31.1 
                 20.8 
               
               
                 13 
                 2066 
                 Dark brown 
                 8.7 
                 36.8 
                 35.2 
                 83.0 
                 26.2 
                 29.1 
                 22.4 
               
               
                 14 
                 2142 
                 Dark brown 
                 8.9 
                 32.6 
                 32.5 
                 85.5 
                 29.8 
                 32.7 
                 20.4 
               
               
                 15 
                 2170 
                 Dark brown 
                 8.8 
                 31.8 
                 29.4 
                 118.4 
                 30.6 
                 31.3 
                 22.3 
               
               
                 16 
                 2055 
                 Dark brown 
                 8.7 
                 30.8 
                 27.6 
                 87.1 
                 36.1 
                 34.0 
                 21.1 
               
               
                 17 
                 2065 
                 Dark brown 
                 9.0 
                 27.8 
                 29.7 
                 127.6 
                 30.0 
                 33.9 
                 19.7 
               
               
                 18 
                 2110 
                 Dark brown 
                 9.0 
                 27.3 
                 31.4 
                 85.3 
                 35.4 
                 33.1 
                 20.5 
               
               
                 19 
                 2154 
                 Dark brown 
                 8.7 
                 32.0 
                 34.6 
                 58.1 
                 33.2 
                 32.2 
                 20.1 
               
               
                 20 
                 2195 
                 Dark brown 
                 8.6 
                 32.3 
                 34.3 
                 61.6 
                 29.2 
                 32.5 
                 19.1 
               
               
                 21 
                 1311 
                 Dark brown 
                 8.3 
                 34.8 
                 30.1 
                 126.6 
                 26.7 
                 28.4 
                 25.0 
               
               
                 22 
                 2003 
                 Dark brown 
                 8.3 
                 33.4 
                 25.4 
                 79.5 
                 29.6 
                 29.6 
                 20.7 
               
               
                 23 
                 1065 
                 Dark brown 
                 8.7 
                 34.2 
                 29.6 
                 112.5 
                 29.2 
                 31.7 
                 23.5 
               
               
                 24 
                 2045 
                 Dark brown 
                 8.8 
                 33.9 
                 25.3 
                 122.0 
                 33.0 
                 31.9 
                 22.4 
               
               
                 25 
                 2128 
                 Dark brown 
                 8.5 
                 34.6 
                 29.5 
                 129.3 
                 23.4 
                 27.2 
                 25.2 
               
               
                 26 
                 2182 
                 Dark brown 
                 8.4 
                 32.7 
                 33.7 
                 81.6 
                 28.2 
                 29.6 
                 22.2 
               
               
                 27 
                 2030 
                 Dark brown 
                 7.7 
                 31.3 
                 33.2 
                 105.8 
                 24.0 
                 27.7 
                 20.3 
               
               
                 28 
                 2034 
                 Dark brown 
                 8.1 
                 32.4 
                 29.6 
                 116.9 
                 26.6 
                 30.0 
                 22.9 
               
               
                 29 
                 2072 
                 Dark brown 
                 8.2 
                 30.2 
                 27.8 
                 97.3 
                 30.8 
                 31.0 
                 21.3 
               
               
                 30 
                 2145 
                 Dark brown 
                 8.2 
                 33.1 
                 29.7 
                 119.0 
                 23.3 
                 28.6 
                 24.1 
               
               
                 31 
                 1027 
                 Dark brown 
                 8.0 
                 29.4 
                 30.6 
                 110.6 
                 30.5 
                 29.1 
                 23.4 
               
               
                 32 
                 1323 
                 Dark brown 
                 8.5 
                 31.2 
                 28.2 
                 115.3 
                 33.0 
                 32.2 
                 23.3 
               
               
                 33 
                 1340 
                 Dark brown 
                 8.0 
                 32.3 
                 29.2 
                 129.8 
                 28.5 
                 29.4 
                 22.9 
               
               
                 34 
                 2129 
                 Dark brown 
                 8.0 
                 33.1 
                 29.6 
                 109.4 
                 21.5 
                 27.4 
                 24.1 
               
               
                 35 
                 2167 
                 Dark brown 
                 8.5 
                 28.6 
                 34.8 
                 71.8 
                 34.4 
                 31.7 
                 21.5 
               
               
                 36 
                 2171 
                 Dark brown 
                 8.0 
                 33.4 
                 28.6 
                 108.1 
                 24.5 
                 28.5 
                 20.7 
               
               
                 37 
                 1054 
                 Dark brown 
                 8.3 
                 34.0 
                 29.0 
                 128.4 
                 29.4 
                 31.3 
                 22.2 
               
               
                 38 
                 1092 
                 Dark brown 
                 8.3 
                 36.6 
                 29.8 
                 131.6 
                 27.2 
                 30.1 
                 22.6 
               
               
                 39 
                 2196 
                 Dark brown 
                 9.2 
                 32.4 
                 32.5 
                 113.1 
                 22.7 
                 30.7 
                 21.2 
               
               
                 40 
                 2183 
                 Dark brown 
                 8.1 
                 33.4 
                 28.0 
                 111.7 
                 27.0 
                 30.0 
                 21.2 
               
               
                 41 
                 2020 
                 Dark brown 
                 8.5 
                 32.5 
                 31.9 
                 128.1 
                 22.5 
                 29.0 
                 21.4 
               
               
                 42 
                 2123 
                 Dark brown 
                 8.5 
                 34.9 
                 30.9 
                 122.3 
                 22.7 
                 27.1 
                 25.3 
               
               
                 43 
                 1296 
                 Dark brown 
                 8.0 
                 36.2 
                 30.6 
                 113.3 
                 25.9 
                 28.3 
                 23.7 
               
               
                 44 
                 2062 
                 Dark brown 
                 8.8 
                 31.6 
                 26.7 
                 117.5 
                 29.5 
                 31.7 
                 22.2 
               
               
                 45 
                 1167 
                 Dark brown 
                 8.0 
                 34.0 
                 28.3 
                 121.0 
                 31.7 
                 30.4 
                 22.3 
               
               
                 46 
                 1359 
                 Dark brown 
                 7.7 
                 33.4 
                 29.4 
                 125.9 
                 25.2 
                 27.2 
                 22.9 
               
               
                 47 
                 1265 
                 Dark brown 
                 8.4 
                 34.6 
                 32.2 
                 78.0 
                 29.6 
                 30.7 
                 22.8 
               
               
                 48 
                 1331 
                 Dark brown 
                 8.0 
                 37.6 
                 29.0 
                 112.3 
                 27.0 
                 28.3 
                 23.1 
               
               
                 49 
                 2002 
                 Dark brown 
                 7.9 
                 33.1 
                 27.4 
                 59.8 
                 28.6 
                 30.0 
                 20.6 
               
               
                 50 
                 2009 
                 Dark brown 
                 7.4 
                 35.9 
                 32.3 
                 67.1 
                 26.7 
                 26.9 
                 22.7 
               
               
                 51 
                 2079 
                 Dark brown 
                 8.0 
                 37.5 
                 29.3 
                 126.2 
                 21.0 
                 28.3 
                 22.5 
               
               
                 52 
                 2092 
                 Dark brown 
                 9.1 
                 32.3 
                 33.4 
                 89.7 
                 27.6 
                 33.4 
                 21.0 
               
               
                 53 
                 2107 
                 Dark brown 
                 8.8 
                 35.8 
                 29.7 
                 103.4 
                 21.3 
                 28.8 
                 21.5 
               
               
                 54 
                 2113 
                 Dark brown 
                 8.8 
                 31.9 
                 33.7 
                 83.4 
                 28.5 
                 30.3 
                 23.0 
               
               
                 55 
                 2117 
                 Dark brown 
                 8.2 
                 30.8 
                 26.6 
                 99.0 
                 23.7 
                 29.5 
                 20.9 
               
               
                 56 
                 2132 
                 Dark brown 
                 8.0 
                 36.1 
                 29.2 
                 121.4 
                 25.1 
                 27.9 
                 23.4 
               
               
                 57 
                 2137 
                 Dark brown 
                 7.9 
                 32.9 
                 28.8 
                 115.6 
                 27.7 
                 28.8 
                 22.2 
               
               
                 58 
                 2140 
                 Dark brown 
                 8.7 
                 32.0 
                 27.5 
                 103.9 
                 24.7 
                 31.2 
                 20.7 
               
               
                 59 
                 2008 
                 Dark brown 
                 7.7 
                 35.0 
                 29.7 
                 75.5 
                 23.8 
                 26.3 
                 22.1 
               
               
                 60 
                 2102 
                 Dark brown 
                 7.9 
                 18.3 
                 24.0 
                 193.8 
                 35.2 
                 32.3 
                 16.4 
               
               
                 61 
                 2021 
                 Dark brown 
                 9.0 
                 30.5 
                 28.1 
                 127.7 
                 26.4 
                 33.3 
                 19.7 
               
               
                 62 
                 2114 
                 Dark brown 
                 9.4 
                 30.6 
                 30.1 
                 114.7 
                 27.1 
                 32.2 
                 20.3 
               
               
                 63 
                 1022 
                 Dark brown 
                 8.7 
                 33.8 
                 28.4 
                 137.0 
                 26.6 
                 30.8 
                 22.3 
               
               
                 64 
                 2051 
                 Dark brown 
                 9.4 
                 34.8 
                 31.7 
                 73.9 
                 30.1 
                 32.7 
                 21.3 
               
               
                 65 
                 2073 
                 Dark brown 
                 9.8 
                 33.5 
                 27.6 
                 132.3 
                 27.3 
                 34.0 
                 20.2 
               
               
                 66 
                 2078 
                 Dark brown 
                 7.6 
                 37.1 
                 29.2 
                 74.5 
                 22.3 
                 27.4 
                 22.0 
               
               
                 67 
                 2209 
                 Dark brown 
                 8.1 
                 31.0 
                 28.4 
                 104.2 
                 27.3 
                 29.2 
                 22.1 
               
               
                 68 
                 2210 
                 Dark brown 
                 8.6 
                 32.5 
                 33.4 
                 86.3 
                 24.9 
                 29.4 
                 20.5 
               
               
                 69 
                 1332 
                 Dark brown 
                 7.9 
                 36.5 
                 30.1 
                 113.4 
                 24.1 
                 26.9 
                 23.8 
               
               
                 70 
                 2095 
                 Dark brown 
                 8.6 
                 31.0 
                 27.4 
                 114.6 
                 30.7 
                 31.2 
                 22.8 
               
               
                 71 
                 2143 
                 Dark brown 
                 9.0 
                 29.1 
                 33.1 
                 97.8 
                 23.7 
                 32.3 
                 21.5 
               
               
                 72 
                 2156 
                 Dark brown 
                 8.1 
                 35.5 
                 28.5 
                 144.4 
                 22.1 
                 28.7 
                 23.7 
               
               
                 73 
                 1235 
                 Dark brown 
                 8.1 
                 32.7 
                 27.8 
                 148.3 
                 27.4 
                 28.4 
                 23.0 
               
               
                 74 
                 2058 
                 Dark brown 
                 8.2 
                 31.1 
                 26.1 
                 142.6 
                 26.3 
                 28.8 
                 23.4 
               
               
                 75 
                 2151 
                 Dark brown 
                 8.7 
                 29.5 
                 33.2 
                 68.4 
                 37.3 
                 34.1 
                 20.4 
               
               
                 76 
                 1002 
                 Dark brown 
                 8.1 
                 29.2 
                 26.8 
                 141.7 
                 28.7 
                 31.1 
                 22.1 
               
               
                 77 
                 1218 
                 Dark brown 
                 8.0 
                 23.9 
                 26.6 
                 120.2 
                 37.9 
                 34.9 
                 18.3 
               
               
                 78 
                 1345 
                 Dark brown 
                 8.0 
                 36.1 
                 32.5 
                 99.1 
                 27.4 
                 27.9 
                 24.5 
               
               
                 79 
                 1366 
                 Dark brown 
                 8.0 
                 36.5 
                 31.3 
                 115.1 
                 26.9 
                 28.2 
                 22.4 
               
               
                 80 
                 2185 
                 Dark brown 
                 9.1 
                 32.9 
                 31.7 
                 97.0 
                 28.1 
                 32.4 
                 21.5 
               
               
                 81 
                 2221 
                 Dark brown 
                 7.7 
                 35.8 
                 29.9 
                 123.2 
                 23.3 
                 26.9 
                 23.2 
               
               
                 82 
                 2332 
                 Dark brown 
                 8.2 
                 30.6 
                 28.7 
                 70.4 
                 34.0 
                 31.9 
                 20.9 
               
               
                 83 
                 1149 
                 Dark brown 
                 8.2 
                 31.7 
                 29.8 
                 114.2 
                 30.5 
                 31.0 
                 23.1 
               
               
                 84 
                 1001 
                 Dark brown 
                 7.7 
                 30.4 
                 30.7 
                 124.6 
                 29.6 
                 28.2 
                 23.7 
               
               
                 85 
                 1082 
                 Dark brown 
                 8.1 
                 30.8 
                 30.7 
                 85.6 
                 33.3 
                 30.2 
                 22.4 
               
               
                 86 
                 2286 
                 Dark brown 
                 8.5 
                 34.2 
                 34.3 
                 74.7 
                 27.2 
                 30.7 
                 22.8 
               
               
                 87 
                 2298 
                 Dark brown 
                 8.0 
                 33.6 
                 27.5 
                 106.8 
                 25.2 
                 30.6 
                 20.8 
               
               
                 88 
                 2304 
                 Dark brown 
                 7.6 
                 33.5 
                 29.7 
                 108.0 
                 23.8 
                 26.9 
                 23.0 
               
               
                 89 
                 2308 
                 Dark brown 
                 8.7 
                 36.0 
                 29.0 
                 113.9 
                 27.0 
                 30.0 
                 22.8 
               
               
                 90 
                 2318 
                 Dark brown 
                 9.2 
                 31.4 
                 32.5 
                 90.6 
                 28.8 
                 32.3 
                 21.5 
               
               
                 91 
                 2319 
                 Dark brown 
                 9.0 
                 27.4 
                 32.2 
                 71.6 
                 31.1 
                 35.1 
                 20.2 
               
               
                 92 
                 2332 
                 Dark brown 
                 8.8 
                 25.0 
                 22.9 
                 169.3 
                 26.7 
                 31.5 
                 17.0 
               
               
                 93 
                 2338 
                 Dark brown 
                 8.0 
                 24.5 
                 24.1 
                 145.7 
                 20.8 
                 30.9 
                 15.3 
               
               
                 94 
                 2346 
                 Dark brown 
                 8.3 
                 31.7 
                 27.6 
                 140.9 
                 27.6 
                 30.4 
                 22.8 
               
               
                 95 
                 2347 
                 Dark brown 
                 8.8 
                 31.0 
                 34.4 
                 78.9 
                 27.8 
                 30.5 
                 22.9 
               
               
                 96 
                 2349 
                 Dark brown 
                 9.6 
                 31.2 
                 32.3 
                 88.0 
                 26.6 
                 32.2 
                 21.7 
               
               
                 97 
                 2354 
                 Dark brown 
                 8.3 
                 28.9 
                 27.2 
                 84.5 
                 30.4 
                 30.1 
                 21.7 
               
               
                 98 
                 2359 
                 Dark brown 
                 7.6 
                 29.3 
                 27.7 
                 101.4 
                 28.2 
                 30.2 
                 20.3 
               
               
                 99 
                 2362 
                 Dark brown 
                 8.7 
                 30.5 
                 28.6 
                 86.7 
                 30.1 
                 31.3 
                 22.7 
               
               
                 100 
                 2364 
                 Dark brown 
                 9.2 
                 31.4 
                 32.2 
                 89.6 
                 28.9 
                 34.4 
                 21.6 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 % 
                 % Erucic 
                 % Total 
                 Sinigrin 
                 % ADF 
                 % NDF 
                 % 
               
               
                   
                 Color 
                 Moisture 
                 Acid 
                 Oil 
                 μmol/g 
                 Fiber 
                 Fiber 
                 Protein 
               
               
                   
               
               
                 Minimum 
                 Light 
                 6.0 
                 22.8 
                 33.0 
                 49.9 
                 10.0 
                 13.1 
                 25.0 
               
               
                 Minimum 
                 Dark 
                 7.4 
                 18.3 
                 22.9 
                 58.1 
                 20.8 
                 26.3 
                 15.3 
               
               
                 Maximum 
                 Light 
                 8.3 
                 39.5 
                 49 
                 196.2 
                 19.7 
                 24.1 
                 33.6 
               
               
                 Maximum 
                 Dark 
                 10.2 
                 37.6 
                 35.2 
                 193.8 
                 37.9 
                 35.1 
                 25.3 
               
               
                   
               
            
           
         
       
     
     Example 8. Composition and Performance of Pennycress Meal Produced from Y1126 Yellow-Seeded Mutant is Superior Relative to Meal Made from Black-Seeded Pennycress and is Similar to Canola Meal 
     Approximately 13 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&amp;M Engineering Experiment Station&#39;s Process Engineering Research &amp; Development Center (College Station, Tex.). The material was conditioned using a single deck of the French cooker for approximately 5 minutes at 100° F.±10° F. Conditioned seed was processed using a Ferrel Ross flaking rolls to yield flakes with a thickness of approximately 0.012 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 190° F. without lingering to avoid activation of such enzymes. This was achieved in 10-15 minutes. The press (Rosedowns Mini 200) was fed from a Wenger metered feeder with flake at a rate of 3.5-4 pounds per minute. The press operated best at 50-55 Hz, which corresponds to 38-40 RPM. 
     The presscake was extracted in stainless batch cans using commercial hexane at a temperature of 110-140° F.±10° F. Solvent was added and drained sequentially in 6 rounds of incubation, each of which was approximately 12 minutes. To remove residual hexane and yield desolventized meal, a batch-type desolventizer/toaster (DT) was heated, which showed a product temperature of 150-175° 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. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 The meal produced from Y1126 yellow-seeded pennycress mutant is significantly more valuable 
               
               
                 (lower in fiber, higher in protein and available energy and nutrients) than regular pennycress 
               
               
                 meal and is closer in composition and predicted performance to canola meal. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Yellow 
                   
               
               
                   
                   
                   
                 Desired 
                   
                 seed 
               
               
                 Meal Component 
                 Type 
                 Unit 
                 Change 
                 Pennycress 
                 (Y1126) 
                 Canola 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 CP 
                 Crude Protein 
                 Protein 
                 % Dry Matter 
                 Increased 
                 31.9 
                 40.5 
                 41.4 
               
               
                 RUP 
                 Rumen Undegraded 
                 Protein 
                 % CP 
                 No change 
                 41.45 
                 42 
                 55 
               
               
                   
                 Protein 
               
               
                 Fat 
                 Oil 
                 Oil 
                 % Dry Matter 
                 No change 
                 1.17 
                 1.69 
                 3.6 
               
               
                 ADF 
                 Acid Detergent Fiber 
                 Fiber 
                 % Dry Matter 
                 Reduce 
                 41.7 
                 20.6 
                 22.9 
               
               
                 NDF 
                 Neutral Detergent Fiber 
                 Fiber 
                 % Dry Matter 
                 Reduce 
                 45.5 
                 27.2 
                 34.3 
               
               
                 Lignin 
                 indigestible cell wall 
                 Fiber 
                 % Dry Matter 
                 Reduce 
                 24.3 
                 7.7 
                 10 
               
               
                   
                 material 
               
               
                 Starch 
                 Starch 
                 Starch 
                 % Dry Matter 
                 No change 
                 0.5 
                 0.5 
                 0.3 
               
               
                 Sugar 
                 Sugar 
                 Sugar 
                 % Dry Matter 
                 No change 
                 6.5 
                 9.5 
                 8 
               
               
                 IVTD 24 
                 24 hour In Vitro Total 
                 Energy 
                 % Dry Matter 
                 Increase 
                 65 
                 89 
                 82 
               
               
                   
                 Digestibility 
               
               
                 TDN 
                 Total Digestible Nutrients 
                 Energy 
                 % Dry Matter 
                 Increase 
                 53 
                 68.5 
                 67 
               
               
                 ME, 1X 
                 Calculated Metabolizable 
                 Energy 
                 Mcal/lb 
                 Increase 
                 0.93 
                 1.33 
                 1.33 
               
               
                   
                 Energy, 1 X maintenance 
               
               
                 NEL, 1X 
                 Calculated Net Energy 
                 Energy 
                 Mcal/lb 
                 Increase 
                 1.08 
                 1.52 
                 1.55 
               
               
                   
                 Lactation, 1X 
               
               
                   
                 maintenance 
               
               
                 NEG, 1X 
                 Calculated Net Energy 
                 Energy 
                 Mcal/lb 
                 Increase 
                 0.32 
                 0.91 
                 0.93 
               
               
                   
                 Gain, 1X maintenance 
               
               
                 NEM, 1X 
                 Calculated Net Energy 
                 Energy 
                 Mcal/lb 
                 Increase 
                 0.86 
                 1.5 
                 1.52 
               
               
                   
                 Maintenance, 1X 
               
               
                   
                 maintenance 
               
               
                   
               
            
           
         
       
     
     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, Ill.) 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. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Y1126 yellow-seed mutant had increased TMEn as compared to 
               
               
                 the black-seeded pennycress and was comparable to canola. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Dry Matter (DM) 
                 TMEn 
               
               
                   
                 Feed 
                 % 
                 Kcal/g DM 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Pennycress 
                 97.0 
                 1.68 
               
               
                   
                 Yellow Seed (Y1126) 
                 97.6 
                 2.02 
               
               
                   
                 Canola 
                 89.1 
                 2.14 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Y1126 yellow-seeded mutant has increased true amino acid 
               
               
                 digestibility as compared to the black-seeded pennycress 
               
               
                 and was as digestible or more so than canola. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Amino 
                   
                   
                 Yellow Seed 
                   
               
               
                 No. 
                 Acid 
                 Unit 
                 Canola 
                 Y1126 
                 Pennycress 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 1 
                 ASP 
                 % 
                 77.6 
                 84.8 
                 79.6 
               
               
                 2 
                 THR 
                 % 
                 77.0 
                 79.2 
                 73.6 
               
               
                 3 
                 SER 
                 % 
                 76.7 
                 81.8 
                 81.8 
               
               
                 4 
                 GLU 
                 % 
                 87.5 
                 90.0 
                 82.6 
               
               
                 5 
                 PRO 
                 % 
                 76.0 
                 82.2 
                 66.0 
               
               
                 6 
                 ALA 
                 % 
                 76.9 
                 82.4 
                 76.1 
               
               
                 7 
                 CYS 
                 % 
                 76.6 
                 71.0 
                 63.7 
               
               
                 8 
                 VAL 
                 % 
                 75.5 
                 81.3 
                 72.9 
               
               
                 9 
                 MET 
                 % 
                 85.9 
                 84.9 
                 75.8 
               
               
                 10 
                 ILE 
                 % 
                 77.2 
                 82.2 
                 75.7 
               
               
                 11 
                 LEU 
                 % 
                 81.5 
                 86.1 
                 79.1 
               
               
                 12 
                 TYR 
                 % 
                 77.1 
                 83.8 
                 78.2 
               
               
                 13 
                 PHE 
                 % 
                 81.6 
                 87.1 
                 80.4 
               
               
                 14 
                 LYS 
                 % 
                 73.5 
                 76.7 
                 68.9 
               
               
                 15 
                 HIS 
                 % 
                 83.4 
                 86.6 
                 70.1 
               
               
                 16 
                 ARG 
                 % 
                 87.0 
                 93.0 
                 83.6 
               
               
                 17 
                 TRP 
                 % 
                 95.4 
                 93.2 
                 89.2 
               
               
                   
               
            
           
         
       
     
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     Other Embodiments 
     It is to be understood that while certain embodiments have been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages, and modifications are within the scope of the following embodiments and claims. 
     Embodiment 1. A composition comprising non-defatted pennycress seed meal comprising an acid detergent fiber (ADF) content of 5%, 8%, or 10% to 15%, 18%, or 20% by dry weight. 
     Embodiment 2. The composition of embodiment 1, wherein said composition has a protein content of 28%, 30%, 32%, or 34% to 38% or 40% by dry weight. 
     Embodiment 3. The composition of embodiment 1, wherein said composition has an oil content of 30%, 32%, or 34% to 40%, 42%, 46%, 48%, or 50% by dry weight. 
     Embodiment 4. The composition of embodiment 1, wherein said composition has a neutral detergent fiber (NDF) content of 10%, 12%, 14%, or 16% to 20%, 22%, 24%, or 25% by dry weight. 
     Embodiment 5. The composition of embodiment 1, wherein said composition has a protein content of 28%, 30%, 32%, or 34% to 38% or 40% by dry weight and an oil content of 30% to 50% by dry weight. 
     Embodiment 6. A composition comprising defatted pennycress seed meal comprising an acid detergent fiber (ADF) content of 7%, 8%, 10%, or 12% to 20%, 22%, 24%, or 25% by dry weight. 
     Embodiment 7. The composition of embodiment 6, wherein said composition has a protein content of 30%, 35%, 40%, or 45% to 55%, 60%, 65%, or 70% by dry weight. 
     Embodiment 8. The composition of embodiment 6, wherein said composition has an oil content of 0%, 2%, or 4% to 8%, 10%, or 12% by dry weight. 
     Embodiment 9. The composition of embodiment 6, wherein said composition has a neutral detergent fiber (NDF) content of 10%, 12%, or 15% to 20%, 25%, 28%, or 30% by dry weight. 
     Embodiment 10. The composition of embodiment 6, wherein said composition has a protein content of 30%, 35%, 40%, or 45% to 55%, 60%, 65%, or 70% by dry weight and an oil content of 0%, 2%, or 4% to 8%, 10%, or 12% by dry weight. 
     Embodiment 11. The composition of embodiment 6, wherein said composition has a protein content of 30%, 35%, 40%, or 45% to 55%, 60%, 65%, or 70% by dry weight and a neutral detergent fiber (NDF) content of 10%, 12%, or 15% to 20%, 25%, 28%, or 30% by dry weight. 
     Embodiment 12. The composition of any one of embodiments 1-11, wherein said composition further comprises a preservative, a dust preventing agent, a bulking agent, a flowing agent, or any combination thereof. 
     Embodiment 13. The composition of any one of embodiments 1-12, wherein said pennycress seed meal is obtained from pennycress seeds that have been crushed, ground, macerated, expelled, extruded, expanded, or any combination thereof. 
     Embodiment 14. The composition of any one of embodiments 1-13, wherein said pennycress seed meal is obtained from a population of pennycress seeds comprising seeds having at least one loss-of-function mutation in at least one endogenous wild-type pennycress gene comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 171, 173, and allelic variants thereof. 
     Embodiment 15. The composition of any one of embodiments 1-14, wherein said pennycress seed meal is obtained from a population of pennycress seeds comprising seeds having at least one loss-of-function mutation in at least one endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO:2, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 172, and allelic variants thereof. 
     Embodiment 16. The composition of any one of embodiments 1-15, wherein said composition comprises a detectable amount of a polynucleotide comprising at least one loss-of-function mutation in at least one endogenous wild-type pennycress gene comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 171, 173, and allelic variants thereof. 
     Embodiment 17. The composition of any one of embodiments 1-16, wherein said pennycress seed meal comprises: (i) pennycress variety Y1067, Y1126, BC38, BJ8, P32, J22, Q36, BD24, AX17, E5-444, E5-540, E5-541, E5-542, E5-543, E5-544, E5-545, E5-547, E5-549, E5-582, E5-586, D3-N10 P5, D5-191, A7-95, A7-187 or A7-261 seed meal; (ii) seed meal of hybrids of the varieties; (iii) seed meal from progeny of the varieties; (iv) seed meal from seed comprising germplasm from the varieties that provides seed comprising an acid detergent fiber (ADF) content of 5% to 20% by dry weight; or (v) seed meal of any combination of said varieties, hybrid varieties, progeny of said varieties, or seed comprising the germplasm. 
     Embodiment 18. The composition of any one of embodiments 1-17, wherein said pennycress seed meal comprises seed meal obtained from the seed lot of anyone of embodiments 43 to 62, or any combination thereof. 
     Embodiment 19. The composition of any one of embodiments 1 to 18, wherein the composition exhibits a lighter-color in comparison to a control composition comprising wild-type pennycress seed meal. 
     Embodiment 20. Pennycress seed meal comprising an acid detergent fiber (ADF) content of 5%, 8%, or 10% to 15%, 18%, or 20% by dry weight, wherein the seed meal is non-defatted. 
     Embodiment 21. The seed meal of embodiment 20, wherein said seed meal has a protein content of 28%, 30%, 32%, or 34% to 38% or 40% by dry weight. 
     Embodiment 22. The seed meal of embodiment 21, wherein said seed meal has an oil content of 30%, 32%, or 34% to 40%, 42%, 46%, 48%, or 50% by dry weight. 
     Embodiment 23. The seed meal of embodiment 21, wherein said seed meal has a neutral detergent fiber (NDF) content of 10%, 12%, 14%, or 16% to 20%, 22%, 24%, or 25% by dry weight. 
     Embodiment 24. The seed meal of embodiment 21, wherein said seed meal has a protein content of 28%, 30%, 32%, or 34% to 38% or 40% by dry weight and an oil content of 30%, 32%, or 34% to 40%, 42%, 46%, 48%, or 50% by dry weight. 
     Embodiment 25. Pennycress seed meal comprising an acid detergent fiber (ADF) content of 7%, 8%, 10%, or 12% to 20%, 22%, 24%, or 25% by dry weight, wherein the seed meal is defatted. 
     Embodiment 26. The seed meal of embodiment 25, wherein said seed meal has a protein content of 30%, 35%, 40%, or 45% to 55%, 60%, 65%, or 70% by dry weight. Embodiment 27. The seed meal of embodiment 25, wherein said seed meal has an oil content of 0%, 2%, or 4% to 8%, 10%, or 12% by dry weight. 
     Embodiment 27. The seed meal of embodiment 25, wherein said seed meal has a neutral detergent fiber (NDF) content of 10%, 12%, or 15% to 20%, 25%, 28%, or 30% by dry weight. 
     Embodiment 28. The seed meal of embodiment 25, wherein said seed meal has a protein content of 30%, 35%, 40%, or 45% to 55%, 60%, 65%, or 70% by dry weight and an oil content of 0%, 2%, or 4% to 8%, 10%, or 12% by dry weight. 
     Embodiment 29. The pennycress seed meal of any one of embodiments 20-28, wherein the meal comprises ground and/or macerated seed of the seed lot of any one of embodiments 43 to 62. 
     Embodiment 30. The pennycress seed meal of any one of embodiments 20-29, wherein said meal comprises a detectable amount of a polynucleotide comprising at least one loss-of-function mutation in at least one endogenous wild-type pennycress gene comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:1, 3, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 171, 173, and allelic variants thereof. 
     Embodiment 31. The pennycress seed meal of any one of embodiments 20-30, wherein said meal comprises ground and/or macerated seed of a population of pennycress seeds comprising seeds having at least one loss-of-function mutation in at least one endogenous wild-type pennycress gene comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 171, 173, and allelic variants thereof. 
     Embodiment 32. The pennycress seed meal of any one of embodiments 20-31, wherein said meal comprises ground and/or macerated seed of a population of pennycress seeds comprising seeds having at least one loss-of-function mutation in at least one endogenous pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO:2, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 172 and allelic variants thereof. 
     Embodiment 33. The pennycress seed meal of any one of embodiments 20-32, wherein said meal comprises ground and/or macerated seed of a population of pennycress seeds comprising seeds having at least one transgene that suppresses expression of at least one endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO:2, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 172, and allelic variants thereof. 
     Embodiment 34. The pennycress seed meal of any one of embodiments 20-33, wherein the meal exhibits a lighter-color in comparison to a control pennycress seed meal prepared from wild-type pennycress seed. 
     Embodiment 35. Pennycress seed cake comprising an acid detergent fiber (ADF) content of 7%, 8%, 10%, or 12% to 20%, 22%, 24%, or 25% by dry weight. 
     Embodiment 36. The seed cake of embodiment 35, wherein said seed meal has a protein content of 30%, 35%, 40%, or 45% to 55%, 60%, 65%, or 70% by dry weight. 
     Embodiment 37. The seed cake of embodiment 35, wherein said seed meal has an oil content of 0%, 2%, or 4% to 8%, 10%, or 12% by dry weight. 
     Embodiment 38. The seed cake of embodiment 35, wherein said seed meal has a neutral detergent fiber (NDF) content of 10%, 12%, or 15% to 20%, 25%, 28%, or 30% by dry weight. 
     Embodiment 39. The seed cake of embodiment 35, wherein said seed meal has a protein content of 30%, 35%, 40%, or 45% to 55%, 60%, 65%, or 70% by dry weight and an oil content of 0%, 2%, or 4% to 8%, 10%, or 12% by dry weight. 
     Embodiment 40. The pennycress seed cake of any one of embodiments 35 to 39, wherein the cake comprises crushed or expelled seed of the seed lot of any one of embodiments 43 to 62. 
     Embodiment 41. The pennycress seed cake of any one of embodiments 35 to 40, wherein the cake comprises a detectable amount of a polynucleotide comprising at least one loss-of-function mutation in at least one endogenous wild-type pennycress gene comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 171, 173, and allelic variants thereof. 
     Embodiment 42. The pennycress seed meal or pennycress seed meal cake of any one of embodiments 36 to 41, wherein the cake exhibits a lighter-color in comparison to a control pennycress seed meal cake prepared from wild-type pennycress seed. 
     Embodiment 43. A seed lot comprising a population of pennycress seeds that comprise an acid detergent fiber (ADF) content of 5%, 8%, or 10% to 15%, 18%, or 20% by dry weight. 
     Embodiment 44. The seed lot of embodiment 43, wherein said seed has a protein content of 28%, 30%, 32%, or 34% to 38% or 40% by dry weight. 
     Embodiment 45. The seed lot of embodiment 43, wherein said seed has an oil content of 30%, 32%, or 34% to 40%, 42%, 46%, 48%, or 50% by dry weight. 
     Embodiment 46. The seed lot embodiment 43, wherein said seed has a neutral detergent fiber (NDF) content of 10%, 12%, 14%, or 16% to 20%, 22%, 24%, or 25% by dry weight. 
     Embodiment 47. The seed lot of embodiment 43, wherein said seed has a protein content of 28%, 30%, 32%, or 34% to 38% or 40% by dry weight and an oil content of 30%, 32%, or 34% to 40%, 42%, 46%, 48%, or 50% by dry weight. 
     Embodiment 48. The seed lot of any one of embodiments 43 to 47, wherein the population comprises at least 10, 20, 50, 100, 500, or 1,000 seeds comprising said ADF content. 
     Embodiment 49. The seed lot of any one of embodiments 43 to 48, wherein at least 95% of the pennycress seeds in the seed lot are seeds comprising said ADF content and said protein content. 
     Embodiment 50. The seed lot of any one of embodiments 43 to 49, wherein less than 5% of the seeds in said seed lot have an ADF content of greater than 20% by dry weight. 
     Embodiment 51. The seed lot of any one of embodiments 43 to 50, wherein said seeds further comprise an agriculturally acceptable excipient or adjuvant. 
     Embodiment 52. The seed lot of any one of embodiments 43 to 51, wherein said seeds further comprise a fungicide, a safener, or any combination thereof. 
     Embodiment 53. The seed lot of any one of embodiments 43 to 52, wherein said population of pennycress seeds comprise seeds having at least one loss-of-function mutation in at least one endogenous pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO:2, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 172, and allelic variants thereof or comprise seeds having at least one transgene that suppresses expression of at least one endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO:2, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 172, and allelic variants thereof. 
     Embodiment 54. The seed lot of any one of embodiments 43 to 53, wherein said population of pennycress seeds comprise seeds having at least one loss-of-function mutation in an endogenous wild-type pennycress gene that encodes SEQ ID NO:2, 70, 76, or an allelic variant thereof. 
     Embodiment 55. The seed lot of embodiment 54, wherein the loss-of-function mutation in the gene encoding SEQ ID NO:2, 70, 76, or the allelic variant thereof comprises an insertion, deletion, or substitution of one or more nucleotides. 
     Embodiment 56. The seed lot of embodiment 54, wherein the loss-of-function mutation in the gene encoding SEQ ID NO:2 or the allelic variant thereof comprises a mutation that introduces a pre-mature stop codon or frameshift mutation at codon positions 1-108 of SEQ ID NO:1 or an allelic variant thereof, wherein the loss-of-function mutation in the gene encoding SEQ ID NO:70 or the allelic variant thereof comprises a mutation set forth in SEQ ID NO:127, 129, 131, 133, 135, or 137, or wherein the loss-of-function mutation in the gene encoding SEQ ID NO:76 or the allelic variant thereof comprises a mutation set forth in SEQ ID NO:165, 167, or 170. 
     Embodiment 57. The seed lot of any one of embodiments 54-56, wherein the loss-of-function mutation in the gene encoding SEQ ID NO:2 or the allelic variant thereof comprises a substitution of a guanine residue at nucleotide 491 of SEQ ID NO:1 with an adenine residue or a substitution of a guanine residue a nucleotide equivalent to nucleotide 491 of SEQ ID NO:1 in the allelic variant thereof with an adenine residue. 
     Embodiment 58. The seed lot of any one of embodiments 43 to 57, wherein said population of pennycress seeds comprise seeds having at least one loss-of-function mutation in at least one endogenous wild-type pennycress gene comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:1, 3, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 171, 173, and allelic variants thereof. 
     Embodiment 59. The seed lot of any one of embodiments 43 to 58, wherein said population of pennycress seeds comprising seeds having at least one transgene that suppresses expression of at least one endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO:2, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 172, and allelic variants thereof. 
     Embodiment 60. The seed lot of any one of embodiments 43 to 59, wherein said population of pennycress seeds comprise: (i) pennycress variety Y1067, Y1126, BC38, BJ8, P32, J22, Q36, BD24, AX17, AX17, E5-444, E5-540, E5-541, E5-542, E5-543, E5-544, E5-545, E5-547, E5-549, E5-582, E5-586, D3-N10 P5, D5-191, A7-95, A7-187 or A7-261 seed; (ii) hybrid seed of said varieties; (iii) seed from progeny of said varieties; (iv) seed comprising germplasm from said varieties that provides seed having an acid detergent fiber (ADF) content of 10% to 20% by dry weight; or (v) any combination of said seed, hybrid seed, seed from progeny of said varieties, or seed comprising said germplasm. 
     Embodiment 61. The seed lot of any one of embodiments 43 to 60, wherein the seeds in the population exhibit a lighter-colored seed coat in comparison to a wild-type pennycress seed. 
     Embodiment 62. A method of making non-defatted pennycress seed meal comprising an acid detergent fiber (ADF) content of 5%, 8%, or 10% to 15%, 18%, or 20% by dry weight, comprising the step of grinding, macerating, extruding, and/or crushing the seed lot of any one of embodiments 43 to 62, thereby obtaining the non-defatted seed meal. 
     Embodiment 63. The method of embodiment 62, wherein the seed meal has a protein content of 28%, 30%, 32%, or 34% to 38% or 40% by dry weight, or the combination thereof. 
     Embodiment 64. The method of embodiment 62, wherein said seed meal has an oil content of 30%, 32%, or 34% to 40%, 42%, 46%, 48%, or 50% by dry weight. 
     Embodiment 65. The method of embodiment 62, wherein said seed meal has a neutral detergent fiber (NDF) content of 10%, 12%, 14%, or 16% to 20%, 22%, 24%, or 25% by dry weight. 
     Embodiment 66. The method of embodiment 62, wherein said seed meal has a protein content of 28%, 30%, 32%, or 34% to 38% or 40% by dry weight and an oil content of 30%, 32%, or 34% to 40%, 42%, 46%, 48%, or 50% by dry weight. 
     Embodiment 67. A method of making defatted pennycress seed meal comprising an acid detergent fiber (ADF) content of 7%, 8%, 10%, or 12% to 20%, 22%, 24%, or 25% by dry weight, comprising the step of solvent extracting the seed lot of any one of embodiments 43 to 62, separating the extracted seed meal from the solvent, thereby obtaining the defatted seed meal. 
     Embodiment 68. The method of embodiment 67, wherein the seed meal has a protein content of 30%, 35%, 40%, or 45% to 55%, 60%, 65%, or 70% by dry weight. 
     Embodiment 69. The method of embodiment 67, wherein said seed meal has an oil content of 0%, 2%, or 4% to 8%, 10%, or 12% by dry weight. 
     Embodiment 70. The method of embodiment 67, wherein said seed meal has a neutral detergent fiber (NDF) content of 10% to 30% by dry weight. 
     Embodiment 71. The method of embodiment 67 wherein said seed meal has a protein content of 30%, 35%, 40%, or 45% to 55%, 60%, 65%, or 70% by dry weight and an oil content of 0%, 2%, or 4% to 8%, 10%, or 12% by dry weight. 
     Embodiment 72. The method of any one of embodiments 67 to 71, wherein the solvent is hexane or mixed hexanes. 
     Embodiment 73. A method of making pennycress seed cake comprising an acid detergent fiber (ADF) content of 7%, 8%, 10%, or 12% to 20%, 22%, 24%, or 25% by dry weight, comprising the step of crushing or expelling the seed of the seed lot any one of embodiments 43 to 62, thereby obtaining a seed cake. 
     Embodiment 74. The method of embodiment 73, wherein the seed cake has a protein content of 30%, 35%, 40%, or 45% to 55%, 60%, 65%, or 70% by dry weight. 
     Embodiment 75. The method of embodiment 74, wherein the seed cake has an oil content of 0%, 2%, or 4% to 8%, 10%, or 12% by dry weight. 
     Embodiment 76. A method of making a pennycress seed lot comprising the steps of: 
     (a) introducing at least one loss-of-function mutation in at least one endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO:2, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 172, and allelic variants thereof;
 
(b) selecting germplasm that is homozygous for said loss-of-function mutation; and,
 
(c) harvesting seed from the homozygous germplasm, thereby obtaining a seed lot, wherein said seed lot comprises an acid detergent fiber (ADF) content of 5%, 8%, or 10% to 15%, 18%, or 20% by dry weight.
 
     Embodiment 77. The method of embodiment 76, wherein said seed lot comprise the seed lot of any one of embodiments 43 to 61. 
     Embodiment 78. A method of making a pennycress seed lot comprising the steps of: 
     (a) introducing at least one transgene that suppresses expression of at least one endogenous wild-type pennycress gene encoding a polypeptide selected from the group consisting of SEQ ID NO:2, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 172, and allelic variants thereof into a pennycress plant genome;
 
(b) selecting a transgenic plant line that comprises said transgene; and,
 
(c) harvesting seed from the transgenic plant line, thereby obtaining a seed lot, wherein said seed lot comprises an acid detergent fiber (ADF) content of 5%, 8%, or 10% to 15%, 18%, or 20% by dry weight.
 
     Embodiment 79. The method of embodiment 78, wherein said harvested seed comprise a seed lot of any one of embodiments 43 to 61.