Sesame and sesame products with improved taste and enhanced nutritional value from crush-resistant sesame plants.

Abstract

The present invention provides a sesame plant having a capsule that is resistant to crushing, a high protein content and / or an improved taste, as well as its seeds and food products made therefrom. [Solution] Phenotypic and genotypic analyses were performed on numerous sesame varieties, followed by analyses to derive markers of phenotypic traits contributing to high protein content and / or improved taste. Breeding simulations were used to identify the most common and stable markers. After verifying the stability of the traits over several generations, the markers and marker cassettes were defined as being uniquely present in the developed sesame line. Various foods made from the seeds of the disclosed varieties have higher protein content and improved taste compared to products made from commercially available sesame varieties.

Description

Technical Field

[0001] Background of the Invention 1. Technical Field The present invention relates to the field of sesame breeding, and more specifically, to sesame protein composition and quantitative trait loci (QTLs) associated with sesame flavor.

Background Art

[0002] 2. Description of Related Art Sesame (Sesamum indicum) is an oilseed crop typically cultivated in a wide range of soil and climate conditions in subtropical climates and used for seeds, oil, and paste products.

[0003] U.S. Patent No. 10,577,623, which is incorporated herein by reference in its entirety, discloses the improvement of genes for shattering-resistant pods, more specifically, novel QTLs conferring shattering resistance, and methods for introgressing and combining novel QTLs into breeding germplasm in a breeding program for shattering-resistant pods.

[0004] Boydak et al. 2008 ("Effect of varieties and years on seed composition of sesame (Sesamum indicum L.) grown in semi-arid area", Asian Journal of Chemistry 20(5): 3907-3912), which is incorporated herein by whole reference in its entirety, discloses the effects of variety and growing years on the oil, protein, and fatty acid composition of five sesame cultivars. Of the five sesame cultivars, the Muganli-57 variety studied in Boydak et al. 2008 does not contain the genetic markers (and their respective results) disclosed below, but has been verified to possess the performance described in Boydak et al. 2008 and is used herein as a reference. The inventors note that the Muganli-57 variety already has an improved taste compared to a common commercial variety (the Ethiopian strain "Humera"). Humera received a rating nearly two grades lower than the Muganli-57 variety in the taste test. [Overview of the Initiative]

[0005] Summary of the Invention The following is a simplified summary to provide an initial understanding of the invention. This summary does not necessarily identify essential elements or limit the scope of the invention, but serves only as an introduction to the following description.

[0006] One aspect of the present invention provides a sesame plant having a crush-resistant capsule, its offspring, seeds and / or parts thereof, wherein the sesame plant comprises multiple quantitative trait loci (QTLs) having multiple corresponding nucleic acid gene markers associated with multiple phenotypic traits relating to the taste and nutritional content of the seeds, wherein the QTLs are combined in the sesame plant from multiple sesame varieties by computationally assisted breeding, wherein the QTLs comprise QTL1 having the corresponding marker described in Sequence ID No. 1 or 2 and associated with improved taste, wherein the sesame plant or part thereof relates to Sequence ID No. 1 Cassette 5 comprises at least one of cassettes 1, 2, 3, or 4, which is homozygous or heterozygous and includes at least one additional QTL, where the marker is at least QTL1 and QTL2, which has the corresponding marker described in SEQ ID NO: 3 or 4 and is associated with reduced ash content and increased protein levels, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 3; Cassette 5 comprises at least QTL1 and QTL8 and 9, which are associated with high protein content, where QTL8 has the corresponding marker described in SEQ ID NO: 15 or 16, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 15, where QTL9 has the corresponding marker described in SEQ ID NO: 17 or 18, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 17; Cassette 6 comprises at least QTL1 and QTL10, which has the corresponding marker described in SEQ ID NO: 19 or 20 and is associated with improved taste, wherein the sesame plant or part thereof is distributed With respect to sequence number 19, it is homozygous or heterozygous; and / or, cassette 7 comprises at least QTL1, and QTL11 and QTL12 associated with high protein content, wherein QTL11 has the corresponding marker described in SEQ ID NO: 21 or 22, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 21, wherein QTL12 has the corresponding marker described in SEQ ID NO: 23 or 24, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 23.

[0007] One aspect of the present invention provides a sesame plant having a crush-resistant capsule, its offspring, seeds and / or parts thereof, the sesame plant comprising: a plurality of quantitative trait loci (QTLs) having a plurality of corresponding nucleic acid gene markers associated with a plurality of phenotypic traits relating to the taste and nutritional content of the seeds, wherein the QTLs are combined in the sesame plant from a plurality of sesame varieties by computationally assisted breeding, wherein the QTLs include QTL2 having the corresponding marker described in SEQ ID NO: 3 or 4 and associated with reduced ash content, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 3 and comprises at least one additional QTL, and wherein the markers are arranged in a cassette comprising at least one of cassettes 1, 2, 3 or 4, which includes at least QTL2 and QTL1 having the corresponding marker described in SEQ ID NO: 1 or 2 and associated with improved taste, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 1.

[0008] One aspect of the present invention provides a food prepared using the disclosed sesame plant seeds and comprising at least one of the markers, the food comprising infertile sesame seeds, hulled seeds, sesame seeds or hulled seeds and / or baked or fried good, seed-containing foods and protein bars, seed-containing granola mixes and food bars, tahini and spreads, dips and sauces made from or containing seeds, seed-derived or seed-containing dairy alternatives, and at least one of seed-derived or seed-containing halba, candies and confectionery.

[0009] These additional and / or other aspects and / or advantages of the present invention are described in the following detailed description, can be inferred from the detailed description, and / or can be learned by carrying out the present invention.

[0010] Brief explanation of the drawing To better understand embodiments of the invention and to show how they can be implemented, refer to the accompanying drawings simply as examples. In the drawings, the same numbers indicate corresponding elements or sections throughout. In the accompanying drawings: [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a high-level schematic diagram of the sesame chromosome showing the loci of relevant markers according to several embodiments of the invention.

[0012] [Figure 2A] Figure 2A is a high-level schematic diagram of a computationally assisted breeding method according to several embodiments of the invention.

[0013] [Figure 2B-1] Figure 2B provides a non-limiting example of a breeding process and lineage of one of the disclosed varieties developed using the disclosed methodology, according to several embodiments of the invention. [Figure 2B-2] Figure 2B provides a non-limiting example of a breeding process and lineage of one of the disclosed varieties developed using the disclosed methodology, according to several embodiments of the invention.

[0014] [Figure 3A] Figure 3A shows the effect of the marker status of QTL2 on protein levels in the disclosed varieties according to several embodiments of the invention.

[0015] [Figure 3B] Figure 3B shows the effect of the marker status of QTL3 on methionine levels in the disclosed varieties according to several embodiments of the invention.

[0016] [Figure 4A-B] Figures 4A-4D illustrate the relationship between multiple parameters and bitterness according to several embodiments of the invention. [Figure 4C-D]Figures 4A - 4D show the relationships between a plurality of parameters and bitterness according to some embodiments of the invention.

[0017] [Figure 4E-F] Figures 4E and 4F show the corresponding effects of QTL1 on the taste of seeds and tahini prepared from the seeds of the disclosed variety according to some embodiments of the invention.

[0018] [Figure 5A-C] Figures 5A - 5H show the improved taste of seeds from the disclosed variety having each marker cassette according to some embodiments of the invention. [Figure 5D-F] Figures 5A - 5H show the improved taste of seeds from the disclosed variety having each marker cassette according to some embodiments of the invention. [Figure 5G-H] Figures 5A - 5H show the improved taste of seeds from the disclosed variety having each marker cassette according to some embodiments of the invention.

[0019] [Figure 6] Figure 6 shows the relative protein content of the disclosed sesame lines compared to the Muganli - 57 sesame variety according to some embodiments of the invention.

[0020] For the sake of brevity and clarity of the illustration, it will be understood that the elements shown in the figures are not necessarily drawn to scale. For example, some dimensions of the elements may be exaggerated relative to other elements for clarity. Further, reference numbers may be repeated between the figures where appropriate to indicate corresponding or similar elements.

Mode for Carrying Out the Invention

[0021] Detailed Description of the Invention The following description illustrates various aspects of the present invention. For explanatory purposes, specific configurations and details are described in order to provide a complete understanding of the invention. However, it will also be apparent to those skilled in the art that the invention can be implemented without the specific details presented herein. Furthermore, well-known features may be omitted or simplified in order to avoid obscuring the invention. In particular, referring to the drawings, it is emphasized that the details shown are illustrative and for illustrative purposes only, and are presented to provide what is considered to be the most useful and easily understandable explanation of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than necessary for a basic understanding of the invention, and by describing it together with the drawings, it will be apparent to those skilled in the art how some forms of the invention can actually be embodied.

[0022] Before describing in detail at least one embodiment of the invention, it should be understood that the invention is not limited to the details of the structure and arrangement of the components described or shown in the drawings. The invention is applicable to other embodiments that can be carried out or performed in various ways, as well as combinations of the disclosed embodiments. It should also be understood that the expressions and terms used herein are for illustrative purposes only and should not be considered limiting.

[0023] Unless otherwise specified, as will be apparent from the following description, throughout this specification, any use of terms such as “processing,” “calculating,” “calculating,” “determining,” “enhancing,” and “deriving” is understood to refer to the operation and / or process of a computer or computing system or similar electronic computing device that manipulates data represented as physical quantities, such as electron quantities, in the registers and / or memory of a computing system, and / or converts it into other data represented as similar physical quantities in the memory, registers, or other such information storage, transmission, or display devices of a computing system.

[0024] Sesame plants with high protein content and / or improved taste, as well as their seeds and foods made therefrom, are provided. Phenotypic and genotypic analyses were performed on numerous sesame varieties, followed by analyses to derive markers of phenotypic traits contributing to high protein content and / or improved taste. Breeding simulations were used to identify the most common and stable markers. After verification of trait stability over several generations, the markers and marker cassettes were defined as being uniquely present in the developed sesame lines. Various foods made from the seeds of the disclosed varieties have higher protein content and improved taste compared to products made from commercially available sesame varieties.

[0025] Embodiments of the present invention include a variety of foods prepared from sesame of the disclosed sesame varieties. The inventors have found that the flavor advantages achieved through breeding of the varieties are also present in foods prepared from sesame seeds. The inventors have found that in most foods, the disclosed markers are still present and detectable in the foods. Non-limiting examples of foods include sesame and hulled (impotent and non-germinating) sesame seeds, foods and protein bars, granola mixes and granola bars, various types of candy, various types of spreads (e.g., tahini, hummus, etc.), confectionery foods (e.g., halba, etc.), ground or crushed sesame, sesame flour and / or sesame oil, and various foods prepared from sesame hulls (foul) and sesame oil itself, packaged and sold as seeds, used as toppings and / or added to various baked or fried foods (e.g., bread, bagels, snacks, etc.). Following the initial investigation, all processed sesame seeds, with the exception of presumably heat-treated products (e.g., sesame oil), retained the detectable genetic markers disclosed herein. This is because heat is expected to degrade the genetic material.

[0026] Advantageously, sesame flour and sesame protein concentrates or isolates contain large amounts of sulfur-containing amino acids (e.g., methionine and cysteine), while most legumes and cereal crops such as peas, soybeans, wheat, barley, rice, and corn are low in sulfur-containing amino acids. Therefore, sesame can be used as a supplement to cereal and legume protein. For example, the disclosed sesame variety product can be mixed with products from legumes and / or cereal crops to produce a product with improved nutritional value and improved quantitative relationships between nutrients. Furthermore, mixing sesame products with cereal / legume products can improve taste and other culinary traits.

[0027] U.S. Patent No. 10,577,623 and U.S. Patent Application Publication No. 2020 / 0093087 (both incorporated herein by reference) teach QTLs that confer crush-resistant capsules and selected sesame varieties having crush-resistant capsules. Crush-resistant lines have been used at least partially to further derive the high-protein sesame lines disclosed herein.

[0028] Crush-resistant capsules are characterized by fully developed capsules with a maximum seed moisture content of 10%, and are characterized by at least one of the following features: (i) at least 80% of seeds are retained after the plant is shaken; (ii) at least 80% of seeds are retained after the capsule is turned upside down; (iii) the ratio of the total length of the capsule to the length of the open area at the tip of the capsule is at least 5:1; and / or 20-30% of the capsule retains 90-95% of the seeds in a fully developed green capsule before drying.

[0029] Various embodiments include sesame plants having crush-resistant capsules or portions thereof, comprising multiple loci associated with multiple quantitative trait loci (QTLs) that have multiple nucleic acid gene markers associated with multiple phenotypic traits of the sesame plant. The QTLs are combined in sesame plants from multiple sesame varieties using computationally assisted breeding tools. Phenotypic and genotypic analyses were performed on many sesame varieties to derive markers of phenotypic traits that contribute to improved taste, high protein, and / or specific amino acid composition, and the most common and stable markers were identified using breeding simulations. Examples of such phenotypic traits include protein content, amino acid composition (e.g., the proportion of specific amino acids such as methionine, cysteine, and phenylalanine), and taste traits that are semi-quantitatively evaluated in taste tests. After verification of trait stability over several generations, the markers and marker cassettes were defined as existing independently in the developed sesame lines. The resulting high-protein sesame lines can be used to improve the quality of sesame products and can be combined with other plant products to create a variety of textured vegetable protein (TVP) products with required compositions that may not be available from other plants. For example, high-protein sesame can be used to enhance the protein levels of other plant products, such as pea concentrate, to create products with specific nutritional value (e.g., meat substitutes). Details regarding QTLs and markers are provided in Table 1 below, and the methods used for developing and selecting varieties are disclosed in Figure 2A below.

[0030] Furthermore, various sesame products were tested and characterized in terms of the trait improvements provided by the disclosed varieties. Examples of sesame products include seeds and hulled seeds for various uses (e.g., roasted and fried), tahini spreads and other spreads, tahini-based dips and sauces, halba condiments, protein bars and sports bars, granola mixes, dairy alternatives, confectionery, etc.

[0031] It should be noted that the disclosed sesame plants with crush-resistant capsules are hybrids in that none of the disclosed varieties occur in nature or in any globally known sesame varieties. The sesame plants with crush-resistant capsules are characterized by disclosed QTL markers that were carefully detected and selected in other varieties and gradually introduced in disclosed combinations to produce the disclosed sesame plants with crush-resistant capsules. Once a specific disclosed sesame plant with crush-resistant capsules was achieved, further breeding was used to stabilize the variety, ensure a consistent phenotype for sesame production, and make the variety a pure line. In this specification, the terms “hybridized” or “introduced” are used to define disclosed varieties having QTL markers and traits collected during the breeding process from different varieties of sesame determined and hybridized in the highly complex computationally assisted breeding method described below, where the genotypes of multiple sesame varieties were carefully combined and analyzed to discover the described QTL markers and corresponding phenotypic traits, which were accumulated in the disclosed sesame plants having crush-resistant capsules. The described sesame plants are not genetically modified by sequences derived from other species, but rather cannot be achieved through mere natural processes, as evidenced by the detailed and deliberate breeding program applied to specifically measure the required characteristics, detect the corresponding markers using bioinformatics, and combine the QTLs detected in the selected varieties by classical breeding approaches (e.g., artificial pollination hybridization and single-plant selection). Further generations derived from the disclosed sesame plants having crush-resistant capsules may also be understood as sesame plants having crush-resistant capsules.

[0032] As described herein, six unique combinations of QTLs, known as QTL cassettes, were detected to distinguish the disclosed breeding material (having crush-resistant capsules) from sesame lines worldwide. The discovery of the cassettes was based on a set of approximately 600 selected crush-resistant lines (e.g., disclosed in U.S. Patent No. 10,577,623) and approximately 200 world accessions. Following the discovery of the cassettes, the disclosed germplasm was tested against world accessions and demonstrated their uniqueness as clearly distinct from known sesame varieties (see Table 5). The six cassettes share one common marker (QTL1) associated with the taste evaluation of sesame seeds. Furthermore, other markers in the cassettes (QTL2-10) were found to be associated with various protein content, amino acid composition, and taste components.

[0033] Figure 1 is a high-level schematic diagram of sesame chromosomes showing the loci of relevant markers according to several embodiments of the invention. Figure 1 schematically shows seven of the thirteen sesame chromosomes and the locations of the markers indicated along them. Table 3 below lists all loci, their markers, and their respective traits. Further details of the cassette composition are provided below. Chromosome locations refer to Genome V2.0 version (Wang et al. 2016, Updated sesame genome assembly and fine mapping of plant height and seed coat color QTLs using a new high-density genetic map, BMC Genomics 17:31).

[0034] Table 1 provides derived genetic markers, QTLs, corresponding traits, and resulting marker cassettes according to several embodiments of the invention. Protein, ash, carbohydrate, and amino acid levels were measured using a Perten Instruments DA 7200 NIR analysis system calibrated for sesame seeds. Protein, ash, and carbohydrate content were measured as grams per 100 grams of sesame seeds (raw percentage), while amino acid content was measured as amino acid content relative to protein level (grams / 100 grams) (representing the proportion of amino acids from protein content and revealing the correlation between amino acid levels and total protein content). Taste components (general taste grade and bitterness) were measured by physical tasting by at least five tasters per test. For each taste component, the tasters scored the specific seed variety on a relative scale of taste and bitterness. Taste was semi-quantitatively evaluated on a scale ranging from 1 to 9, where 1 indicates the worst-tasting seed relative to the reference variety, and 9 indicates the best-tasting seed relative to the reference variety. Bitterness was semi-quantitatively evaluated on a scale ranging from 1 to 9, where 1 indicates the least bitter or least bitter seed compared to the reference variety, and 9 indicates the most bitter or very bitter seed compared to the reference variety. The reference variety, the Ethiopian strain "Humera," was scored 4 for each component. Table 1: Genetic markers, QTLs, corresponding traits, and marker cassettes with corresponding high protein levels, enhanced amino acid content, and / or improved taste components. [Table 1-1] Table 1 (continued): [Table 1-2]

[0035] The disclosed sesame plants with crush-resistant capsules were derived by computationally assisted breeding, producing distinct plants different from prior art sesame varieties. Specifically, the disclosed sesame plants all share a common QTL1 and are grouped according to the combinations of QTLs shown herein as Cassettes 1 through 7 in Table 1.

[0036] Tables 2 and 3 show all analyzed gene markers (including QTLs 1-10) having their respective discovered phenotypic traits according to several embodiments of the invention. The traits are numbered 1-16 and associated with loci numbered 1-56 in the sesame genome. Table 2 is roughly arranged in order of QTL order. Table 3 is arranged in order of locus number with respect to the chromosomal location of the loci, as schematically shown in Figure 1. Phenotypic traits were measured as described above with respect to Table 1. Table 2: Phenotypic traits associated with high protein content, amino acid ratio in protein composition, ash, carbohydrates, and taste components, and the loci of their respective markers. [Table 2]

[0037] Some of the gene markers disclosed herein have been identified in previous applications (for example, locus 9 disclosed herein is similar to QTL5 in U.S. Patent No. 11,044,884; locus 35 disclosed herein is similar to locus 33 in U.S. Application No. 17 / 474,944 and QTL3 in U.S. Patent No. 10,577,623; and locus 1, 3, 4 (QTL7), 24 (QTL2), 28, 29, 31, 33, 34, 36, 38, 50 (QTL4) and 55 are similar to locus 8, 9, 10, 21, 25, 26, 31, 32, 34, 35, 60 and 63 in U.S. Application No. 17 / 474,944, respectively). However, it should be noted that none of the previous applications taught any association between each locus and any of the currently disclosed traits of protein content, amino acid content, and / or taste characteristics. Table 3: Analyzed gene markers (including QTL1-9), their loci, and their respective traits. [Table 3-1] [Table 3-2]

[0038] Figure 3A shows the effect of QTL2 marker status on protein levels in the disclosed varieties according to several embodiments of the invention. The QTL2 status (sequence number 3 of homozygotes marked as AA, sequence number 4 of homozygotes of Muganli-57 sesame varieties marked as GG, and heterozygotes marked as AG) correlated with protein content in two different environments (marked as Saad and EnZurim), showing enhanced protein content correlated with QTL2 status in both environments (e.g., 26.2% of homozygote sequence number 3 compared to 25.3% of homozygote sequence number 4 in Saad [p: 0.0001], and 25.2% of homozygote sequence number 3 compared to 24.9% of homozygote sequence number 4 in EnZurim [not significant]).

[0039] Figure 3B shows the effect of the marker status of QTL3 on methionine levels in the disclosed varieties according to several embodiments of the invention. The QTL3 status (sequence number 5 of homozygotes marked as AA, sequence number 6 of homozygotes of Muganli-57 sesame varieties marked as GG, and heterozygotes marked as AG) correlate with methionine content in two environments (Saad and EnZurim in Figure 3B), showing enhanced methionine content that correlates with the QTL3 status in both environments (e.g., 0.0252 for homozygote sequence number 5 in EnZurim compared to 0.0246 for homozygote sequence number 6 [p value: 0.002], and 0.0243 for homozygote sequence number 5 in Saad compared to 0.240 for homozygote sequence number 6 [p value: 0.062]).

[0040] Figures 4A–4D show the relationships between several seed parameters found to correlate with bitterness, according to several embodiments of the invention. Note that the correlations shown in Figures 4A–4D were detected from three different independent datasets, each reflecting three breeding cycles performed in several different environments.

[0041] Figure 4A shows the relationship between methionine levels and bitterness, illustrating a negative correlation between methionine levels and bitterness in two independent datasets (sesame grown in 2017 and 2018, respectively). This is demonstrated by providing a bivariate fit of the percentage of methionine to bitterness grade (the ellipse shows a p-value of 0.95, and the correlation coefficient is -0.5 for p<0.05). Thus, the inventors note that the taste of the resulting sesame can be improved by increasing methionine levels through the selection of varieties having respective QTLs for a high methionine percentage (e.g., QTLs 3, 4, 5, and 7), in addition to directly selecting varieties with QTLs for improved taste (QTLs 1 and 10, etc.).

[0042] Figure 4B shows the relationship between general sensory grade and bitterness grade by providing a bivariate approximation of general sensory grade to bitterness grade (the ellipse shows a p-value of 0.95, and the correlation coefficient is -0.95 for p<0.0001). The inventors note that the strong correlation indicates that general sensory grade is directly improved by reducing the bitterness level (due to several factors). Therefore, additional factors are being evaluated with respect to bitterness grade (which strongly correlates with general sensory grade). Note that data from seeds containing any of the marker cassettes (shown as cassette-Y in the figure) are scored significantly higher on general sensory grade and lower on the bitterness scale (by approximately 1-4 grades on each scale) compared to prior art seeds (including Muganli-57 and Humera varieties).

[0043] Figures 4C and 4D show the correlation between two such factors, namely carbohydrate and ash levels, and bitterness grade. Carbohydrate and ash content were measured by NOR. The obtained correlations were clear and significant (ANOVA bivariate approximation, the ellipse indicates a p-value of 0.95, with correlation coefficients of 0.49 for carbohydrate content at p=0.0075 (Figure 4C) and 0.59 for ash content at p=0.0009 (Figure 4D)), indicating that markers associated with low ash and carbohydrate content in seeds (e.g., QTL2 and 6, respectively) also contribute to improved taste. Note that these data are accumulated from multiple cultivation cycles and several years during the breeding program (regardless of specific genetic composition).

[0044] Figures 4E and 4F show the corresponding effects of QTL1 on the taste of the disclosed varieties (taste of seeds in Figure 4E, and taste of tahini prepared from seeds in Figure 4F) according to several embodiments of the invention. Figure 4E provides a correlation between taste grade and QTL1 status (homozygous for SEQ ID NO: 1 in the disclosed varieties compared to the wild type which is homozygous for SEQ ID NO: 2), showing a significant difference between the average taste grade of the disclosed varieties (6.5) and the average taste grade of the Muganli-57 sesame variety (6.0) (Student's t-test applied at significance level 0.05). Note that both the Muganli-57 sesame variety with the G allele in QTL1 and the disclosed varieties with the A allele in QTL1 have improved taste relative to the Ethiopian line "Humera," a common commercial variety rated 4 on the taste scale.

[0045] As shown in Figure 4F, the disclosed varieties having QTL1, a homozygous strain for Sequence ID No. 1, also exhibited a higher success rate in taste tests conducted using tahini prepared from each seed compared to Muganli-57 sesame varieties (75% success rate for the disclosed varieties compared to 40-45% success rate for Muganli-57 sesame varieties). Subjectively, the taste tests showed a statistically significant improvement in the taste of tahini made from the disclosed sesame varieties (p=0.0413).

[0046] Figures 5A–5H show the improved taste of seeds of disclosed varieties having each marker cassette according to several embodiments of the invention. The first data provide a comparison of seeds of several disclosed lines having each marker cassette with a prior art line, Muganli-57 sesame variety (grown under the same conditions), as a control. Table 4 (below) provides one-way analysis of variance (ANOVA) results showing improved taste compared to the prior art, with significant results for cassettes 3, 4, and 5 (p-values ​​of 0.039, 0.0094, and 0.0003, respectively; p-values ​​for cassettes 1, 2, and 6 were 0.0018, 0.0139, and <0.0001, respectively). For sesame lines having cassette 7, a taste and bitterness comparison was performed with the prior art Humera variety, and the comparison included a total of 10 additional samples: 5 from lines containing cassette 7 and 5 from prior art lines not containing cassette 7. Samples were randomized and the results are shown using a scale between +3 (best tasting) and -3 (worst tasting) (Figure 5G) and a scale between +3 (very bitter) and -3 (least bitter) (Figure 5H) against the results of the Humera strain (assigned a zero value). The results showed a significant effect on taste and bitterness of varieties containing the Cassette 7 marker, with the strain labeled ES309 (containing the Cassette 7 marker) being rated as the best variety in terms of overall taste and bitterness. The effect of Cassette 7 was measured as adding 1.24 on the taste scale (p=0.013, significant) and reducing bitterness by 1.1 on the bitterness scale (p=0.005, significant). Different hatching / shading indicates different examiners. Note that all comparisons refer to the disclosed varieties compared to prior art varieties grown under the same conditions.

[0047] Figure 6 shows the relative protein content of the disclosed sesame varieties compared to the Muganli-57 sesame variety, according to several embodiments of the invention. Variability is associated with different field and growing conditions, resulting in different outcomes for seed protein content. Nevertheless, when averaged across a range of varieties due to growing conditions, Figure 6 shows that the disclosed sesame varieties, including the disclosed markers, produce an average relative increase of 10% in protein content compared to the Muganli-57 sesame variety.

[0048] Table 4 provides the average effect of different cassette combinations on the protein content and taste grade of sesame varieties, compared to lines that do not express any cassettes (specifically, Muganli-57 sesame varieties grown under the same conditions). Note that since the nutrient content in the seeds depends on the growing environment, using Muganli-57 sesame varieties for comparison under similar conditions provides a reliable reference for the improvements achieved by the disclosed sesame varieties. Therefore, Table 4 provides non-limiting examples of some of the improved performance of the disclosed varieties. The values ​​are averaged across multiple lines grown under different environmental conditions, and a particular line with the disclosed cassettes may be selected to improve protein levels by, for example, 5–10% compared to Muganli-57 sesame varieties (see, e.g., Figure 6), and taste by, for example, at least one step on the rating scale compared to Muganli-57 sesame varieties (see, e.g., Figures 5A–5F). It should be noted that the taste was improved by modifying several parameters, such as the levels of ash, carbohydrates, and methionine, as shown herein (see, for example, Figures 4A–4D). Furthermore, it should be noted that with respect to sesame varieties lacking the disclosed cassettes, strains having specific combinations of cassettes may be selected to provide a particular increase in protein content and / or taste parameters. Table 4: Enhanced protein content and flavor in sesame varieties with different cassette combinations compared to lines that do not express any cassette. [Table 4]

[0049] It should be noted that each cassette offers a specific combination of QTLs associated with a particular trait. For example, cassettes 1, 2, and 3 contain QTLs 3, 4, 5, and 7 (see Table 1 for exact combinations) associated with high methionine levels and reduced bitterness (improved taste); cassettes 1, 2, 3, 4, and 5 contain QTLs 2, 6, 8, and 9 associated with high protein levels; and cassettes 1-6 contain QTLs 1-7 and 10 directly or indirectly associated (via increased methionine, reduced ash, or reduced carbohydrates) with better taste and reduced bitterness.

[0050] Table 5 shows the uniqueness of the disclosed varieties, characterized by cassettes 1-6 and combinations thereof, according to several embodiments of the invention, with respect to the global variety. The number of disclosed varieties is indicated with respect to the combination of marker cassettes that characterize each variety. Table 5: Comparison of disclosed varieties included in the disclosed cassette with varieties from around the world (prior art). [Table 5]

[0051] Therefore, a total of 281 varieties are shown to be included in one of the disclosed cassette combinations, while the global varieties surveyed are not included in any of the cassette combinations. All disclosed varieties are shatter-resistant and characterized by improved taste, increased protein content and / or improved flavor (e.g., increased methionine content and / or reduced ash or carbohydrate content).

[0052] Advantageously, the disclosed embodiments provide high-protein sesame plants that can improve the nutritional value of sesame seeds, for example, with respect to protein content and / or nutritional value with respect to specific amino acids (see Table 2). These varieties and products derived therefrom (e.g., isolates and / or concentrates) can be used to supplement products made from most legumes and cereal crops such as peas, soybeans, wheat, barley, rice, corn and others (low in sulfur-containing amino acids such as methionine and cysteine). Thus, the disclosed sesame plants and their products can be used as supplemental supplements to the proteins of cereals and legumes.

[0053] As used herein, sesame plants with high protein content offer a relative increase in protein content of at least 5% (e.g., typically between 5% and 10%) compared to Muganli-57 sesame varieties that do not express any of the marker cassettes described herein (and are grown under the same conditions). Alternatively or supplementally, compared to 22% for Muganli-57 sesame varieties (grown under the same conditions) that showed a relative increase in protein of 13%, the disclosed sesame varieties have at least 10% more protein, which corresponds to (e.g.) an NIR value of 25%. In various embodiments, the protein levels of the disclosed sesame varieties measured by NIR yield values ​​of 24–26%.

[0054] In various embodiments, sesame plants having a high methionine content and / or low ash or carbohydrate levels provide improved taste and reduced bitterness compared to Muganli-57 sesame varieties (grown under the same conditions) that do not express any of the marker cassettes described herein. As shown in Table 4, the improvement in taste is typically due to a tasting scale grade of 1–2. In various embodiments, sesame plants having improved taste are defined as those that provide an improvement of at least half of the evaluation stages in the tasting test compared to Muganli-57 sesame varieties (grown under the same conditions) that do not express any of the marker cassettes described herein (see Figures 5A–5F and Table 4). Alternatively or supplementally, sesame plants having improved taste are defined as those having at least a test grade of 6.5.

[0055] Muganli-57 is a commonly used sesame variety in Turkey and is available from the West Mediterranean Agricultural Research Institute (Demircikara Mahallesi Pasha Kavaklari Cad. No:11 MURATPASHA / ANTALYA, located in Turkey), a public agricultural research institute. Muganli-57 is registered with the institute, as described in Yol et al., “A high-density SNP genetic map construction using ddRAD-Seq and mapping of capsule shattering trait in sesame,” Frontiers in Plant Science, 12:679659 (2021).

[0056] As used herein, QTL1 refers to a polymorphic locus linked to a genetic marker at position 4959888 on chromosome 3 of sesame. The two alleles of the QTL1 genetic marker have the SNP base "A" or "G," as described in the nucleic acid sequences of SEQ ID NOs. 1 and 2, respectively. In all cassettes (1-6), QTL1 is either homozygous for the allele "A" (SEQ ID NOs. 1) or heterozygous for the allele "A" (including both SEQ ID NOs. 1 and 2). Sequence ID 1 (SNP base in bold): [ka] Sequence ID 2 (SNP base in bold): [ka]

[0057] As used herein, QTL2 refers to a polymorphic locus linked to a genetic marker at position 2595046 on chromosome 6 of sesame. The two alleles of the QTL2 genetic marker have the SNP base "A" or "G," as described in the nucleic acid sequences of SEQ ID NOs. 3 and 4, respectively. In cassettes 1, 2, 3, and 4, QTL2 is either homozygous for the allele "A" (SEQ ID NOs. 3) or heterozygous for the allele "A" (including both SEQ ID NOs. 3 and 4). Sequence ID 3 (SNP base in bold): [ka] Sequence ID 4 (SNP base in bold): [ka]

[0058] As used herein, QTL3 refers to a polymorphic locus linked to a genetic marker at position 2777176 on chromosome 4 of sesame. The two alleles of the QTL3 genetic marker have the SNP base "A" or "G," as described in the nucleic acid sequences of SEQ ID NOs. 5 and 6, respectively. In cassettes 1 and 2, QTL3 is homozygous for allele "A" (SEQ ID NOs. 5) or heterozygous for allele "G" (including both SEQ ID NOs. 5 and 6), while in cassettes 3 and 4, QTL3 is homozygous for allele "G" (SEQ ID NOs. 6) or heterozygous for allele "G" (including both SEQ ID NOs. 5 and 6). Sequence ID 5 (SNP base in bold): [ka] Sequence ID 6 (SNP base in bold): [ka]

[0059] As used herein, QTL4 refers to a polymorphic locus linked to a genetic marker at position 5809997 on chromosome 13 of sesame. The two alleles of the QTL4 genetic marker have the SNP base "A" or "G," as described in the nucleic acid sequences of SEQ ID NOs. 7 and 8, respectively. In cassette 1, QTL4 is homozygous for allele "A" (SEQ ID NOs. 7) or heterozygous for allele "G" (including both SEQ ID NOs. 7 and 8), while in cassette 2, QTL4 is homozygous for allele "G" (SEQ ID NOs. 8) or heterozygous for allele "G" (including both SEQ ID NOs. 7 and 8). Sequence ID 7 (SNP base in bold): [ka] Sequence ID 8 (SNP base in bold): [ka]

[0060] As used herein, QTL5 refers to a polymorphic locus linked to a genetic marker at position 8636698 on chromosome 4 of sesame. The two alleles of the QTL5 genetic marker have the SNP base "C" or "T", as described in the nucleic acid sequences of SEQ ID NOs. 9 and 10, respectively. In cassette 3, QTL5 is homozygous for the allele "C" (SEQ ID NOs. 9) or heterozygous for the allele "T" (including both SEQ ID NOs. 9 and 10), while in cassette 4, QTL5 is homozygous for the allele "T" (SEQ ID NOs. 10) or heterozygous for the allele "T" (including both SEQ ID NOs. 9 and 10). Sequence ID 9 (SNP base in bold): [ka] Sequence ID 10 (SNP base in bold): [ka]

[0061] As used herein, QTL6 refers to a polymorphic locus linked to a genetic marker at position 10189573 on chromosome 4 of sesame. The two alleles of the QTL6 genetic marker have the SNP base "A" or "G," as described in the nucleic acid sequences of SEQ ID NOs. 11 and 12, respectively. In cassette 4, QTL6 is either homozygous for the allele "A" (SEQ ID NOs. 11) or heterozygous for the allele "A" (including both SEQ ID NOs. 11 and 12). Sequence ID 11 (SNP base in bold): [ka] Sequence ID 12 (SNP base in bold): [ka]

[0062] As used herein, QTL7 refers to a polymorphic locus linked to a genetic marker at position 20018096 on chromosome 1 of sesame. The two alleles of the QTL7 genetic marker have the SNP base "T" or "C" as described in the nucleic acid sequences of SEQ ID NOs. 13 and 14, respectively. In cassette 2, QTL7 is either homozygous for the allele "T" (SEQ ID NOs. 13) or heterozygous for the allele "T" (including both SEQ ID NOs. 13 and 14). Sequence ID 13 (SNP base in bold): [ka] Sequence ID 14 (SNP base in bold): [ka]

[0063] As used herein, QTL8 refers to a polymorphic locus linked to a genetic marker at position 8384304 on chromosome 4 of sesame. The two alleles of the QTL8 genetic marker have the SNP base "T" or "C," as described in the nucleic acid sequences of SEQ ID NOs. 15 and 16, respectively. In cassette 5, QTL8 is either homozygous for the allele "T" (SEQ ID NOs. 15) or heterozygous for the allele "T" (including both SEQ ID NOs. 15 and 16). Sequence ID 15 (SNP base in bold): [ka] Sequence ID 16 (SNP base in bold): [ka]

[0064] As used herein, QTL9 refers to a polymorphic locus linked to a genetic marker at position 16918017 on chromosome 8 of sesame. The two alleles of the QTL9 genetic marker have the SNP base "G" or "A," as described in the nucleic acid sequences of SEQ ID NOs. 17 and 18, respectively. In cassette 5, QTL9 is either homozygous for the allele "G" (SEQ ID NOs. 17) or heterozygous for the allele "G" (including both SEQ ID NOs. 17 and 18). Sequence ID 17 (SNP base in bold): [ka] Sequence ID 18 (SNP base in bold): [ka]

[0065] As used herein, QTL10 refers to a polymorphic locus linked to a genetic marker at position 304411 on chromosome 11 of sesame. The two alleles of the QTL10 genetic marker have an SNP base "T" or "C", as described in the nucleic acid sequences of SEQ ID NOs. 19 and 20, respectively. In cassette 6, QTL10 is either homozygous for the allele "T" (SEQ ID NOs. 19) or heterozygous for the allele "T" (including both SEQ ID NOs. 19 and 20). Sequence ID 19 (SNP base in bold): [ka] Sequence ID 20 (SNP base in bold): [ka]

[0066] As used herein, QTL11 refers to a polymorphic locus linked to a genetic marker at position 11699161 on chromosome 6 of sesame. The two alleles of the QTL11 genetic marker have an SNP base "T" or "C", as described in the nucleic acid sequences of SEQ ID NOs. 21 and 22, respectively. In cassette 7, QTL11 is either homozygous for the allele "T" (SEQ ID NOs. 21) or heterozygous for the allele "T" (including both SEQ ID NOs. 21 and 22). Sequence ID 21 (SNP base in bold): [ka] Sequence ID 22 (SNP base in bold): [ka]

[0067] As used herein, QTL12 refers to a polymorphic locus linked to a genetic marker at position 13967471 on chromosome 3 of sesame. The two alleles of the QTL11 genetic marker have the SNP base "C" or "T", as described in the nucleic acid sequences of SEQ ID NOs. 23 and 24, respectively. In cassette 7, QTL12 is either homozygous for the allele "C" (SEQ ID NOs. 23) or heterozygous for the allele "C" (including both SEQ ID NOs. 23 and 24). Sequence ID 23 (SNP base in bold): [ka] Sequence ID 24 (SNP base in bold): [ka]

[0068] A sesame plant having a crush-resistant capsule, its offspring and / or parts thereof, is provided, which includes multiple quantitative trait loci (QTLs) having multiple nucleic acid gene markers associated with multiple phenotypic traits of the sesame plant, including at least one of taste traits, protein content traits, and amino acid composition traits, wherein the QTLs are combined in sesame plants from multiple sesame varieties by computationally assisted breeding.

[0069] The disclosed QTLs of the sesame plant, its offspring and / or parts thereof may include QTL1 having the corresponding marker described in SEQ ID NO: 1 or 2 and associated with improved taste, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 1.

[0070] A disclosed sesame plant (and / or its offspring, seeds and / or parts thereof) having a crush-resistant capsule may contain at least one additional QTL in addition to QTL1, the markers may be arranged in a cassette comprising at least one of the following: (i) Cassettes 1, 2, 3, or 4 comprising at least QTL1 and QTL2 having the corresponding marker described in SEQ ID NO: 3 or 4, and associated with reduced ash content and increased protein levels, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 3; (ii) Cassettes 5 comprising at least QTL1 and QTL8 and 9 associated with high protein content, wherein QTL8 has the corresponding marker described in SEQ ID NO: 15 or 16, and wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 15, and where QTL9 has the corresponding marker described in SEQ ID NO: 17 or Cassette 6 comprising QTL10 having the corresponding marker described in 18, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 17; and / or Cassette 7 comprising QTL11 and QTL11 and QTL12 having the corresponding marker described in SEQ ID NO: 19 or 20 and associated with improved taste, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 19; and / or Cassette 7 comprising QTL11 and QTL11 and QTL12 associated with high protein content, wherein QTL11 has the corresponding marker described in SEQ ID NO: 21 or 22, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 21, and QTL12 has the corresponding marker described in SEQ ID NO: 23 or 24, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 23.

[0071] The disclosed QTLs of the sesame plant, its offspring and / or parts thereof may have the corresponding marker described in SEQ ID NO: 3 or 4 and may include QTL2 associated with high protein content, a dry matter weight percentage greater than 29.8%, and low ash content, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 3.

[0072] The disclosed QTLs of the sesame plant, its offspring and / or parts thereof may include QTL3 associated with high methionine content, having the corresponding marker described in SEQ ID NO: 5 or 6, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 5.

[0073] The disclosed QTLs of the sesame plant, its offspring and / or parts thereof may have the corresponding marker described in SEQ ID NO: 7 or 8 and may include QTL4 associated with high methionine content, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 7.

[0074] In some embodiments, the disclosed sesame plant, its offspring and / or parts thereof may include the disclosed marker cassette 1, which includes QTL1, 2, 3 and 4 disclosed herein.

[0075] The disclosed QTLs of the sesame plant, its offspring and / or parts thereof may contain QTL7 having the corresponding marker described in SEQ ID NO: 13 or 14 and associated with high protein content, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 13.

[0076] In some embodiments, the disclosed sesame plant, its offspring and / or parts thereof may include the disclosed marker cassette 2 containing QTL1, 2, 3, 4 and 7 disclosed herein.

[0077] The disclosed QTLs of the sesame plant, its offspring and / or parts thereof may have the corresponding marker described in SEQ ID NO: 9 or 10 and may include QTL5 associated with high methionine content, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 9.

[0078] In some embodiments, the disclosed sesame plant, its offspring and / or parts thereof may include the disclosed marker cassette 3, which contains QTL1, 2, 3 and 5 disclosed herein.

[0079] The disclosed QTLs of the sesame plant, its offspring and / or parts thereof may include QTL6 having the corresponding marker described in SEQ ID NO: 11 or 12 and associated with high protein content, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 11.

[0080] In some embodiments, the disclosed sesame plant, its offspring and / or parts thereof may include the disclosed marker cassette 4, which includes QTL1, 2, 3, 5 and 6 disclosed herein.

[0081] The disclosed QTLs of the sesame plant, its offspring and / or parts thereof may contain QTL8 associated with high protein content, having the corresponding marker described in SEQ ID NO: 15 or 16, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 15.

[0082] The disclosed QTLs of the sesame plant, its offspring and / or parts thereof may contain QTL9 having the corresponding marker described in SEQ ID NO: 17 or 18 and associated with high protein content, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 17.

[0083] In some embodiments, the disclosed sesame plant, its offspring and / or parts thereof may include the disclosed marker cassette 5, which includes QTL1, 8 and 9 disclosed herein.

[0084] The disclosed QTLs of the sesame plant, its offspring and / or parts thereof may further comprise QTL10 having the corresponding marker described in SEQ ID NO: 19 or 20 and associated with improved taste, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 19.

[0085] In some embodiments, the disclosed sesame plant, its offspring and / or parts thereof may include the disclosed marker cassette 6 containing QTL1 and 10 as disclosed herein.

[0086] In some embodiments, the disclosed sesame plant, its offspring and / or parts thereof may include the disclosed marker cassette 7, which includes QTL1, 11 and 12 disclosed herein.

[0087] In various embodiments, the disclosed sesame plant, its offspring and / or parts thereof may include any of the QTLs disclosed herein or combinations thereof. In various embodiments, the disclosed sesame plant, its offspring and / or parts thereof may include any of the QTL cassettes disclosed herein or combinations thereof.

[0088] Figure 2A is a high-level schematic diagram of a computationally assisted breeding method 200 according to several embodiments of the invention. Method 200 can be carried out at least partially by at least one computer processor. The computationally assisted breeding method 200 is used to detect and combine QTLs from multiple sesame varieties to develop a disclosed high-protein sesame plant having a crush-resistant capsule that is distinct from any of the parent varieties thanks to the phenotypic features achieved.

[0089] The computationally assisted breeding method 200 includes, as described in detail below, a stage of trait discovery by cultivating a wide range of varieties and determining their phenotypes (stage 210), a stage of trait mixing by crossing selected lines to mix and combine traits and developing hybrid lines by self-pollinating the offspring in subsequent generations (stage 220), a stage of discovering the target product genome code (TPGC) by linking phenotypes and genotypes using a derivative linkage map (stage 230), an in silico selection stage to suggest candidate varieties (stage 240), a stage of breeding candidate varieties and selecting varieties based on the potential for the best TPGC (stage 250), and a stage of discovering the genome code (GC) to identify the most stable QTL in the hybrid offspring generation (stage 260). TPGC Discovery 230, In Silico Confirmation 250, and GC Discovery 260 are based on computational algorithms that cannot be performed manually and provide computational support for careful selection of varieties that are generated during the development process and further crossbred to produce disclosed high-protein sesame plants with crush-resistant capsules. Note that during the discovery stage, QTLs are derived and combined (by crossbreeding) to create unique combinations of QTLs that do not exist in known world lines.

[0090] In certain embodiments, sesame lines were bred to reach high protein levels by collecting various sesame lines from around the world, creating F2 linkage populations, applying intensive phenotypic and genotyping of thousands of sesame lines, predicting QTLs that affect protein levels and / or compositional traits, and establishing unique combinations of markers referred to herein as “marker cassettes,” thereby characterizing novel high-protein lines provided by the methods described herein that are not present in commercially available or naturally occurring lines.

[0091] The breeding method was based on an algorithm for deriving a Target Product Genomic Code (TPGC) to associate (i) a Target Product (TP) containing a set of desired attributes (traits) that are predefined based on market requirements and available through natural genetic mutations, and (ii) a Genomic Code (GC) containing a set of genomic regions that influence and are linked to the TP traits, including quantitative trait loci (QTLs). The algorithm could be configured to compute multiple genomic interactions and maximize the potential of a particular plant's genome for the development of new varieties. The breeding program was constructed to derive the TPGC and then, through crossbreeding and self-pollination, achieve a product containing a specific GC corresponding to the desired TP.

[0092] A specific embodiment of the breeding process for developing lines through successive generations of hybridization and self-pollination includes the following stages: (i) Trait discovery, where a wide range of varieties from different regional and global sources are cultivated to determine phenotypes and discover new traits that can potentially be combined to create new varieties; (ii) Trait mixing, where hybridization cycles are performed based on phenotypic assumptions, and different traits are mixed and combined. This is followed by additional cycles to create F2 (and possibly more generations) populations that provide the basis for algorithmic analysis to construct the TPGC; (iii) TPGC discovery, where plants are phenotyped, genotyped to generate linkage maps, relevant QTLs are discovered, and the TPGC is derived; (iv) Multiple line confirmation stages over several years, where sesame lines based on millions of in silico-calculated variations (and / or selections) are cultivated and used to define the first variety; (v) Trait TPGC mixing, where precise hybridization is performed to calculate the most efficient method to arrive at the best TPGC. The crosses are performed after in silico selection from millions of combinations and are at least partially based on phenotypic assumptions; and (vi) a sequential algorithm-based GC discovery stage applied to F2 (or higher generations) populations grown in additional cycles.

[0093] Defining the protein profile (TP) of high-protein, shatter-resistant sesame varieties involves developing high-throughput methods for identifying high protein levels.

[0094] In the following non-limiting example of the process, trait discovery (i) was based on the owner's germplasm, including hundreds of selected varieties and thousands of F2 individual plants, as well as approximately 200 different sesame lines obtained from the U.S. National Plant Germplasm System (NPGC) and provided from Professor Amram Ashri's sesame germplasm collection (see Ashri, A. 1998, Sesame Breeding. In: Janick J. (ed.), Plant Breeding Reviews Vol. 16. John Wiley and Sons, Somerset, NJ, pp. 179-228). These lines were used in the trait mixing stage (ii), where crosses were performed based on the potential to enhance genomic diversity as the first step in a TP-oriented breeding program for high-protein sesame lines, creating new complexes of high-protein level traits. The resulting F1 hybrids were then self-crossed to create F2 linkage populations exhibiting phenotypic segregation. Next, the F2 population was planted in three different environments to discover TPGC(iii), including high protein, amino acid composition, and taste traits. After screening 2500 individuals and determining their detailed phenotypes, a set of approximately 300 representative individuals was selected. Individuals selected from the F2 population underwent further extensive phenotyping for traits related to protein levels, amino acid composition, and taste, as detailed in the following description. The measurement results were summarized into representative high protein traits.

[0095] TPGC discovery (iii) involved determining the genotypes of approximately 2500 individual plants selected from six populations. The analysis was performed using a panel of 1000 markers based on single nucleotide polymorphisms (SNPs), directly designed based on polymorphisms found in the parental lines of the populations, which were analyzed in detail using high-throughput DNA sequencing techniques. The panel was designed to maximize the chance of having the largest number of common segregating SNPs to create highly similar linkage maps across all observed populations. Linkage map calculations were performed for each linkage F2 population based on the genotype determination results. Linkage maps were calculated using MultiPoint, an interactive package for ordering multilocus gene maps, and map validation was based on resampling techniques. QTL discovery for high protein levels was performed using the MultiQTL package with multiple interval mapping (MIM) based on linkage map and F2 population phenotypic data integrated by Multipoint. The importance and co-occurrence of high protein levels and protein content markers were evaluated for populations in different environments using an algorithm that associated the genotypic stage of each marker with its respective QTL and trait in linkage maps of six F2 populations (hereinafter also referred to as “linked F2 populations”) within each population. The importance of QTLs was calculated using permutations, bootstrap tools, and FDR (false detection rate) for the overall analysis. Information on high protein, amino acid composition, and taste traits across all linkage maps of the six F2 populations and all genotypic plants belonging to those populations was analyzed and used to predict QTLs using a “one marker, one trait” model. In this model, each trait was tested independently for each marker for all markers constituting the linkage map. Specifically, subsets of two populations (approximately 800 F2 individuals) were tasted for taste QTL discovery, and then confirmed within advanced lines of a smaller subset by applying high-resolution taste testing (using more tasters for fewer lines). In the provided examples, a total of 10 markers were found to be associated with traits related to protein-related components (see Table 1 above).One taste marker (QTL1) was common to all cassettes, and another high-protein marker (QTL2) was common to all four cassettes. The appearance of high-protein level markers (repeated markers) in two or more linkage maps of the F2 population reinforced their importance as representative of high-protein level QTLs.

[0096] Generally, the six linkage F2 populations exhibited different markers related to high protein levels. However, a subset of common markers was found to be shared by multiple populations, which we refer to as marker cassettes in this specification.

[0097] The breeding process is described using non-limiting examples from specific parts of the breeding program, and it is emphasized that it is not limited to the specific populations and varieties derived from this particular part of the breeding program. For example, it may be used to breed different F2 populations and derive additional varieties characterized by one or more of the disclosed QTLs.

[0098] Following the discovery of TPGCs (iii), an in silico breeding program (iv) was established to simulate and predict the genotypic status of self, hybrid-self, and hybrid plants in terms of their predicted effects on each stage of markers for QTLs and high protein level traits by processing TPGC mixtures (including combinations of QTLs from different plants). The in silico breeding program was constructed to generate millions of in silico self-pollination combinations, which were bred and evaluated in silico up to F8, measuring the likelihood that each genotyped plant would acquire high protein levels in the appropriate combination at the appropriate stage. The analysis resulted in the identification of approximately 300 F2 plants with the highest scores for high protein levels, and these plants were therefore selected for actual self-pollination and hybrid-self-pollination procedures. F3 seeds from these selected F2 plants were sown in plots during the subsequent growing season. In this procedure, QTLs from different populations were combined to produce F3 plants containing novel and unique cassettes of QTLs that resulted in high protein levels.

[0099] Next, the high-protein sesame lines were genotyped for F3 and several subsequent generations of offspring, and their retention of the identified marker cassette was verified to confirm that they maintained their traits in subsequent generations. Specifically, the parent lines of the linked F2 population were genotyped based on high-protein level markers in all populations, along with approximately 200 different sesame cultivars (traditional and older commercial varieties). The cassette, detailed in Table 1, was found to completely distinguish the developed high-protein lines from the rest of the screened sesame cultivars.

[0100] Figure 2B provides a non-limiting example of the breeding process and lineage of one of the disclosed varieties developed using the disclosed methodology (see the annotated steps) according to several embodiments of the invention. Specifically, Figure 2B shows the first descendant lines (denoted by accession serial numbers 107, 517, 48, and 11, corresponding to USDA identifiers PI 599447, PI 173955, PI 254699, and PI 157158) used in breeding one of the varieties characterized above (denoted as ES309), including marker cassette 7, and subsequent descendant lines (denoted by accession serial numbers 101, 334, and 266, corresponding to USDA identifiers PI 599438, PI 532845, and PI 263470). It should be noted that the breeding process was carried out according to Method 200 and included an extensive process of trait discovery and mixing by in silico selection from a very large number of possible combinations, as well as judicial crossing based on computer analysis and stabilization of the selected varieties, and actual long-term breeding over eight years.

[0101] The inventors note that none of the high-protein sesame plants with crush-resistant capsules bred according to the methods described herein occurred naturally. In fact, they were derived from the highly complex computationally assisted breeding method 200 described above, which involved carefully combining and analyzing the genotypes of multiple sesame varieties to discover and accumulate the aforementioned QTL markers and corresponding phenotypic traits. The aforementioned sesame plants were not genetically modified by sequences derived from other species, but they could not be achieved through mere natural processes, as is evident from the detailed and deliberate breeding program applied to specifically measure the required characteristics, detect the corresponding markers using bioinformatics methods, and combine the QTLs detected in the selected varieties using a classical breeding approach. The inventors note that, given the extreme complexity of the breeding program, which involves cultivating, selecting, and breeding hundreds of varieties over many generations in the field, and based on the genetic analysis of the relationship between varieties, markers, and phenotypic traits, this breeding process could not occur by mere natural means and is therefore not considered a natural phenomenon. Furthermore, it should be noted that due to their resistance to crushing, the disclosed varieties are significantly hindered from natural plant propagation. Finally, while the disclosed QTL markers are not heterologous to sesame as a species, it should be noted that the identified QTLs are not present in any combination described in any of the more than 200 prior art varieties used as the initial breeding stock, and that the QTL genome of sesame plants has been significantly and carefully modified by breeding programs. Therefore, at the taxonomic level of the varieties, high-protein sesame plants are considered hybridized in that QTL markers are mixed and introduced from other sesame varieties.

[0102] In the above description, embodiments are examples or practices of the invention. The various expressions “one embodiment,” “embodiment,” “specific embodiment,” or “several embodiments” do not necessarily all refer to the same embodiment. Various features of the invention may be described in the context of a single embodiment, but features may also be provided separately or in any preferred combination. Conversely, the invention may be described herein in the context of separate embodiments for clarity, but the invention may also be practiced in a single embodiment. A specific embodiment of the invention may include features from different embodiments disclosed above, and a specific embodiment may incorporate elements from other embodiments disclosed above. The disclosure of elements of the invention in the context of a specific embodiment should not be construed as limiting their use in that particular embodiment alone. Furthermore, it should be understood that the invention can be performed or practiced in a variety of ways, and that the invention can be practiced in specific embodiments other than those outlined in the above description.

[0103] The invention is not limited to these figures or corresponding descriptions. For example, the flow does not have to move through each box or state shown, or in the exact same order as shown and described. Unless otherwise defined, the meanings of the technical and scientific terms used herein will be generally understood by those skilled in the art to which the invention pertains. Although the invention is described in relation to a limited number of embodiments, these should not be construed as limiting the scope of the invention, but rather as examples of some embodiments. Other possible variations, modifications, and applications are also within the scope of the invention. Accordingly, the scope of the invention should not be limited by what has been described herein, but rather by the appended claims and their legal equivalents.

Claims

1. Sesame plants having crush-resistant capsules, their offspring, seeds and / or parts thereof, wherein the sesame plants include: Multiple quantitative trait loci (QTLs) having multiple corresponding nucleic acid gene markers associated with multiple phenotypic traits relating to the taste and nutritional content of the seeds, wherein the QTLs are combined in sesame plants from multiple sesame varieties by computationally assisted breeding. Here, the QTL includes QTL1 associated with improved taste, having the corresponding marker described in SEQ ID NO: 1 or 2, wherein the sesame plant or portion thereof is homozygous or heterozygous with respect to SEQ ID NO: 1, and includes at least one additional QTL, Here, the marker is placed in a cassette that includes at least one of the following: Cassettes 1, 2, 3, or 4, each comprising at least QTL1 and QTL2 having the corresponding marker described in Sequence ID No. 3 or 4, and associated with reduced ash content and increased protein levels, wherein the sesame plant or portion thereof is homozygous or heterozygous with respect to Sequence ID No. 3; Cassette 5 comprising at least QTL1 and QTL8 and 9 associated with high protein content, wherein QTL8 has the corresponding marker described in SEQ ID NO: 15 or 16, wherein the sesame plant or portion thereof is homozygous or heterozygous with respect to SEQ ID NO: 15, wherein QTL9 has the corresponding marker described in SEQ ID NO: 17 or 18, wherein the sesame plant or portion thereof is homozygous or heterozygous with respect to SEQ ID NO: 17; Cassette 6 comprising QTL1 and QTL10 having at least QTL1 and the corresponding marker described in SEQ ID NO: 19 or 20, and associated with improved taste, wherein the sesame plant or portion thereof is homozygous or heterozygous with respect to SEQ ID NO: 19; and Cassette 7 comprising at least QTL1, and QTL11 and QTL12 associated with high protein content, wherein QTL11 has the corresponding marker described in SEQ ID NO: 21 or 22, wherein the sesame plant or portion thereof is homozygous or heterozygous with respect to SEQ ID NO: 21, wherein QTL12 has the corresponding marker described in SEQ ID NO: 23 or 24, wherein the sesame plant or portion thereof is homozygous or heterozygous with respect to SEQ ID NO:

23. Sesame plants having crush-resistant capsules, their offspring, seeds and / or parts thereof.

2. The cassette comprises cassette 1, 2, 3, or 4, and is a sesame plant having a crush-resistant capsule as described in claim 1, its offspring, seeds, and / or parts thereof.

3. The QTL further comprises a QTL3 having a corresponding marker as described in SEQ ID NO: 5 or 6 and associated with a high methionine content, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 5, according to claim 2, a sesame plant having a crush-resistant capsule, its offspring, seeds and / or parts thereof.

4. A sesame plant having a crush-resistant capsule, its offspring, seeds and / or parts thereof, according to claim 3, comprising a cassette 1 having a QTL further comprising a QTL4 having the corresponding marker described in Sequence ID No. 7 or 8 and associated with a high methionine content, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to Sequence ID No.

7.

5. A sesame plant having a crush-resistant capsule according to claim 4, its offspring, seeds and / or parts thereof, which, when grown under the same conditions, has at least 5% higher protein content and at least one grade improved taste compared to the Muganli-57 sesame variety.

6. A sesame plant having a crush-resistant capsule, its offspring, seeds and / or parts thereof, according to claim 3, comprising a cassette 2 having QTLs further comprising QTLs 4 and 7 associated with high methionine content, wherein QTL 4 has the corresponding marker described in SEQ ID NO: 7 or 8, the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 8, and QTL 7 has the corresponding marker described in SEQ ID NO: 13 or 14, and wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO:

13.

7. A sesame plant having a crush-resistant capsule according to claim 6, which, when grown under the same conditions, has at least 5% higher protein content and at least one grade improved taste compared to the Muganli-57 sesame variety.

8. The QTL further comprises a QTL3 having a corresponding marker as described in SEQ ID NO: 5 or 6 and associated with a high methionine content, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 6, the sesame plant having a crush-resistant capsule, its offspring, seeds and / or parts thereof according to claim 2.

9. A sesame plant having a crush-resistant capsule, its offspring, seeds and / or parts thereof, according to claim 8, comprising a cassette 3 having a QTL further comprising a QTL 5 having a corresponding marker as described in Sequence ID No. 9 or 10, and wherein the sesame plant or part thereof is homozygous or heterozygous with respect to Sequence ID No.

9.

10. A sesame plant having a crush-resistant capsule according to claim 9, which, when grown under the same conditions, has at least 5% higher protein content and at least one grade improved taste compared to the Muganli-57 sesame variety.

11. A sesame plant having a crush-resistant capsule, its offspring, seeds and / or parts thereof, according to claim 8, comprising a cassette 4 having a QTL further comprising QTL 5 having a corresponding marker described in SEQ ID NO: 9 or 10 and associated with high methionine content, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 10, and QTL 6 having a corresponding marker described in SEQ ID NO: 11 or 12 and associated with low carbohydrate content, wherein the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO:

11.

12. A sesame plant having a crush-resistant capsule according to claim 11, which, when grown under the same conditions, has at least 5% higher protein content and at least one grade improved taste compared to the Muganli-57 sesame variety.

13. A sesame plant having a crush-resistant capsule as described in claim 1, including a cassette 5, its offspring, seeds and / or parts thereof.

14. A sesame plant having a crush-resistant capsule according to claim 13, which, when grown under the same conditions, has at least 5% higher protein content and at least one grade improved taste compared to the Muganli-57 sesame variety.

15. A sesame plant having a crush-resistant capsule as described in claim 1, including a cassette 6, its offspring, seeds and / or parts thereof.

16. A sesame plant having a crush-resistant capsule according to claim 15, which has a taste that is at least two grades better than the Muganli-57 sesame variety when grown under the same conditions, its offspring, seeds and / or parts thereof.

17. A sesame plant having a crush-resistant capsule as described in claim 1, including a cassette 7, its offspring, seeds and / or parts thereof.

18. The sesame plant having a crush-resistant capsule according to any one of claims 1 to 17, wherein the portion comprises any of seeds, endosperm, ovules, pollen, cells, cell cultures, tissue cultures, plant organs, protoplasts, meristematic tissue, embryos, or combinations thereof.

19. A food prepared using the seeds of the sesame plant as described in any one of claims 1 to 17.

20. The food according to claim 19, comprising: infertile sesame seeds; hulled seeds; baked or fried products containing and / or topped with the sesame seeds or hulled seeds; foods and protein bars containing the seeds; granola mixes and food bars containing the seeds; tahini and spreads; dips and sauces made from or containing the seeds; dairy alternatives made from or containing the seeds; and at least one halba, candy and confectionery made from or containing the seeds.

21. The food according to claim 19, comprising at least one of the markers.

22. Sesame plants having crush-resistant capsules, their offspring, seeds and / or parts thereof, wherein the sesame plants include: Multiple quantitative trait loci (QTLs) having multiple corresponding nucleic acid gene markers associated with multiple phenotypic traits relating to the taste and nutritional content of the seeds, wherein the QTLs are combined in sesame plants from multiple sesame varieties by computationally assisted breeding. Here, the QTL includes a QTL2 associated with the reduced ash content, having the corresponding marker described in SEQ ID NO: 3 or 4, where the sesame plant or part thereof is homozygous or heterozygous with respect to SEQ ID NO: 3, and at least one additional QTL, and Here, the marker is arranged in a cassette comprising at least one of cassettes 1, 2, 3, or 4, which includes QTL1 associated with improved taste and has at least QTL2 and the corresponding marker described in Sequence ID No. 1 or 2, wherein the sesame plant or portion thereof is homozygous or heterozygous with respect to Sequence ID No.

1. Sesame plants having crush-resistant capsules, their offspring, seeds and / or parts thereof.

23. A sesame plant having a crush-resistant capsule according to claim 22, which, when grown under the same conditions, has at least 5% higher protein content and at least one grade improved taste compared to the Muganli-57 sesame variety.

24. A food prepared using the seeds of the sesame plant according to claim 23, comprising: infertile sesame seeds; hulled seeds; baked or fried products containing and / or topped with the sesame seeds or hulled seeds; foods and protein bars containing the seeds; granola mixes and food bars containing the seeds; tahini and spreads; dips and sauces made from or containing the seeds; dairy alternatives made from or containing the seeds; and at least one halba, candy and confectionery made from or containing the seeds, comprising at least one of the markers.

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