Systems and methods for sorting seeds

Abstract

Systems and methods of sorting a mixture of wheat seed comprising inbred pollinator wheat seed and F1 hybrid wheat seed are provided herein. The systems and methods may be used to sort the wheat seed into an enriched population of inbred pollinator wheat seed and / or an enriched population of F1 hybrid wheat seed.

Description

TITLE

[0001] SYSTEMS AND METHODS FOR SORTING SEEDSFIELD

[0002] The disclosure relates to the field of plant breeding and seed processing.CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 745,793, filed January 16, 2025, the entire contents of which is herein incorporated by reference.BACKGROUND

[0004] Commercial seed production is a process that involves many steps. In the case of wheat, for example, inbred plants must first be crossed in a field under conditions that allow the pollen from the male parent to fertilize the flower of the female parent in order to produce the F1 hybrid wheat seeds that will be sold to the farmer for planting.SUMMARY

[0005] The present disclosure provides methods for sorting and enriching F1 hybrid wheat seed from a wheat seed mixture. In one embodiment, a method of sorting F1 hybrid wheat seed is provided, the method includes sorting a wheat seed mixture including inbred pollinator wheat seed and F1 hybrid wheat seed using seed density and / or morphological characteristics, including, but not limited to, linear dimensions, including, but not limited to, seed size, seed shape, seed length, and seed thickness; geometric properties, including, but not limited to, seed surface area, calculated seed volume, seed perimeter, and seed circularity; geometric ratios, including, but not limited to, aspect ratio (length-to-width ratio) and surface-area-to-volume ratio; surface topography, including but not limited to, seed coat texture, wrinkling, or the presence of a crease, or combinations thereof to produce an enriched population of inbredpollinator wheat seed and / or an enriched population of F1 hybrid wheat seed.

[0006] The present disclosure provides methods of obtaining an enriched population of F1 hybrid wheat seed. In one embodiment, the method includes separating a wheat seed mixture including inbred pollinator wheat seed and F1 hybrid wheat seed by optically sorting the wheat seed mixture based on at least one identified phenotype to separate the F1 hybrid wheat seed and the inbred pollinator wheat seed from one another to produce an enriched population of F1 hybrid wheat seed and / or an enriched population of inbred pollinator wheat seed.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1 is a schematic representation of various embodiments of seed sorting methods for removal of inbred male seeds (inbred pollinator wheat seed) from F1 hybrid wheat seed production mixtures. Any combination of one or more seed sorting methods, e.g., density sorting, optical sorting, size sorting, and / or shape sorting, may be used to sort a seed mixture of inbred and hybrid wheat seeds into a more pure hybrid wheat seed population.

[0008] FIG. 2. shows the difference in thousand kernel weight (FIG. 2A, FIG. 2C, FIG. 2D) and test weight (FIG. 2B) between F1 hybrid wheat seeds (Hybrid) and inbred male wheat seeds (Male) (inbred pollinator wheat seed). F1 hybrid wheat seeds produced with the Ms45-Blue aleurone (Ms45-BA) male sterility system in Soft Red Winter Wheat (FIG.2A- FIG. 2B), F1 hybrid wheat seeds produced with the Ms45-Blue aleurone (Ms45-BA) male sterility system in in Hard Red Winter Wheat (FIG. 2C), F1 hybrid wheat seeds produced with the Triticum timopheevi cytoplasm (tCMS) in Hard Red Winter Wheat (FIG. 2D) are shown.Statistical significance across all pairwise comparisons was determined using the Tukey-Kramer Honestly Significant Difference (HSD) test at a significance level of alpha = 0.05.

[0009] FIG. 3. Shows the results of optical color sorting of a mixed hybrid and male inbred seeds (inbred pollinator wheat seeds). (A) Separation of a proxy F1 (genotype RRR x R) and Male (genotype Rrr) seeds at different ratios and in either one or two passes. (B) Separation of a proxy F1 (RRR x rrr)) and male inbred (rrr) pollinator wheat seeds. The ratio of male inbred pollinator wheat seeds is indicated by the percentages listed below each bar set, the number of passes through the seed sorter are indicated, and the percent of each genotype within the sorted seed is shown on the y-axis.

[0010] FIG. 4 shows a schematic representation of one embodiment of a wheat seed sorting method described in Example 4. The wheat seed mixture is first density sorted using a gravity table, and then color sorting is used to increase F1 hybrid seed purity. Inbred seed is discarded at each step. As used herein, hybridity and purity are used interchangeably.

[0011] FIG. 5 shows a schematic representation of sorting a mixture of wheat seed (inbred pollinator wheat seed and F1 hybrid wheat seed) in multiple steps. Sort steps can utilize any sorting method or combination of methods described herein. Sorting steps 2 - N (any number of sort steps needed) may be performed on any seed population created through prior sorting. The final seed product may be created by combining the desired seed fractions from among all of the sorting steps performed.

[0012] FIG. 6 shows a schematic representation of sorting of a mixture of wheat seed (inbred pollinator wheat seed and F1 hybrid wheat seed) in multiple steps. Sort steps may utilize any sorting method or combination of methods described herein. In this embodiment, the initial wheat seed mixture is separated into two fractions that enrich for F1 hybrid wheat seed and inbred pollinator wheat seed, respectively. The enriched inbred pollinator wheat seed fraction is then sorted a second time, resulting in a fraction with recovered F1 hybrid wheat seed separated from further enriched inbred seed. The enriched F1 hybrid wheat seed from Sort 1 andthe recovered F1 hybrid wheat seed from Sort 2 are then combined to produce a further enriched F1 hybrid wheat seed population.DETAILED DESCRIPTION

[0013] It is to be understood that this invention is not limited to particular embodiments, which can, of course, vary. All publications referred to herein are each incorporated by reference for the purpose cited to the same extent as if each was specifically and individually indicated to be incorporated by reference herein.

[0014] Different approaches can be used to produce F1 hybrid wheat seed. For example, in hybrid wheat production, fields may be planted by blending wheat seeds with male sterility with fertile pollen donor wheat seeds and planting them in the same row. This results in a field with intermixed wheat plants, with male-sterile wheat plants that will act as the female parent to produce F1 hybrid seeds and the male fertile plants that will act as the pollen donor for the F1 hybrid seeds but will themselves produce inbred seeds. The male and female wheat inbred parent plants are grown and the male-fertile pollinator wheat parent line fertilizes the female wheat inbred parent plants’ flowers to produce F1 hybrid wheat seed. To prevent self-pollination, the female wheat inbred parent used in F1 hybrid wheat production may be male sterile. Male-sterile female parent seed for use in F1 seed production may be maintained and increased using a hybrid wheat sterility system, such as a nuclear-genome based male-sterility system, such as Ms45, or cytoplasmic male sterility (CMS). See, for example, published application W02020056259, incorporated herein by reference in its entirety, Knudson, M. K., and Ruttan, V. W., “Research and Development of a Biological Innovation: Commercial Hybrid Wheat,” Food Research Institute Studies, Vol. XXI, No. 1, pp. 46-68, 1988. When the resulting seed is harvested from the F1 hybrid wheat production fields, the seed mixture will contain a mixture of both F1 hybrid and inbred pollinator wheat seed. F1 hybrid wheat seed or Fi hybrid wheat seed, as used interchangeably herein, refers to the first filial generation (Fi) progeny resulting from the cross-pollination of a male-sterile female wheat parent plant by a male-fertile pollinator wheat parent plant. As used herein, inbred pollinator wheat seed (also referred to herein interchangeably as male seed, inbred seed, inbred male seed, or inbred male seed) refers to the self-pollinated or sib-pollinated progeny of the male-fertile pollinator parent plant harvested from the F1 hybrid wheat production field. To maximize yield for the farmers who will grow the hybrid seeds, the inbred seeds are separated from the F1 hybrid wheat seed after harvest.

[0015] As disclosed herein, it was discovered that F1 hybrid wheat seeds produced using nuclear-genome based male-sterile females generated from a nuclear-based male-sterility system using the Ms45-blue aleurone (ms45-BA) platform described in published application W02020056259, resulted in F1 hybrid wheat seeds that were able to be separated from the male inbred pollinator seeds because they exhibit a distinct density and morphology compared to inbred pollinator wheat seeds, enabling separation of the two populations using various sorting methods. See, for example, FIG. 2, which demonstrates that Ms45-derived F1 hybrid seeds had a higher thousand kernel weight (TKW) and test weight than the inbred pollinator wheat seeds. In contrast, as shown in FIG. 2, the F1 hybrid wheat seeds produced using Timopheevi cytoplasmic male sterility (tCMS) exhibited a lower density and smaller seed size relative to the inbred pollinator wheat seeds.

[0016] The sorting methods disclosed herein may be used to sort F1 hybrid wheat seeds from male inbred pollinator seeds. The F1 hybrid wheat seeds may be produced from a cytoplasmic male-sterile female parent via a cytoplasmic male-sterility system (CMS), where the malesterility mechanism resides in the mitochondrial genome (see, for example, Knudson, M. K., and Ruttan, V. W., “Research and Development of a Biological Innovation: Commercial Hybrid Wheat,” Food Research Institute Studies, Vol. XXI, No. 1, pp. 46-68, 1988, which is herein incorporated by reference in its entirety); or from a nuclear-genome based male-sterile female parent via a nuclear-genome based male-sterility system, where the male-sterility mechanism resides in the nuclear genome and not in the mitochondrial genome. See, for example, publishedpatent application W02020056259, or WO2019043082, each of which is herein incorporated by reference each in its entirety Examples of wheat male-fertility genes include but are not limited to, Ms1, Ms2, MS3, Ms5, Ms9, Ms22, Ms26, or Ms45 male fertility genes. As a non-limiting example, the nuclear-based male-sterile female may be sterile due to genome editing of an endogenous wheat nuclear male fertility gene, including but not limited to Ms1, Ms2, Ms3, Ms5, Ms9, Ms22, Ms26, or Ms45 male fertility genes, to render the male-sterile female wheat plant homozygous recessive for the male fertility gene, see, for example, published patent application US 20190177722 and US Patent No.12,054,732, each of which is herein incorporated by reference each in its entirety. In some embodiments, the nuclear-genome based male-sterile female parent is a Ms45 male-sterile female wheat parent line, e.g., homozygous recessive for the Ms45 male fertility gene. In some embodiments, the nuclear-genome based male-sterile female parent is a Ms45 male-sterile female wheat parent line, e.g., homozygous recessive for the Ms45 male fertility gene. In some embodiments, the nuclear-genome based male-sterile female parent is a Ms1 male-sterile female wheat parent line, e.g., homozygous for a mutation in the endogenous Ms1 male fertility gene on chromosome 4BS (the B genome). The nuclear-based male-sterile female wheat parent may be increased using a hybrid wheat production platform, including, but not limited to, a Ms45-blue aleurone (Ms45-BA) or Ms1-blue aleurone system (Ms1-BA), including, but not limited to those described published patent application W02020056259, or WO2019043082, each of which is herein incorporated by reference each in its entirety.

[0017] The present disclosure relates to methods for sorting wheat seed to obtain an enriched population of F1 hybrid wheat seed. As used herein, enriched or enrichment refers to the concentration of a particular type of seed, for example, increased percentage of a particular seed type within a population. To illustrate using an example, a population of F1 hybrid seed may be considered enriched by increasing the concentration of the F1 seed in the population even if the absolute number of the F1 hybrid wheat seeds are not increased, for example, if a wheat seed mixture comprises70 F1 hybrid wheat seeds and 30 male pollinator inbred seeds, by removing 30 of the male pollinator inbred seeds, the concentration of the F1 hybrid wheat seeds has increased from 70% to 100% in the resulting population even though the number of seeds has not changed. The enrichment may be accomplished in any number of ways, including but not limited to, sorting a mixture of wheat seed of inbred pollinator wheat seed and F1 hybrid wheat seed using density sorting, optical sorting, seed morphological characteristic-based sorting, such as, size sorting, and / or shape sorting, or any combinations thereof. The methods disclosed herein may use one or more physical, morphological, and / or optical properties of wheat seed, including density, size, shape, and color, as well as compositional and spectral characteristics, to distinguish and separate inbred pollinator wheat seed from F1 hybrid wheat seed. The methods disclosed herein may include one or more of the following steps: sorting based on density, sorting based on a wheat seed morphological characteristic, such as seed size and / or seed shape, and sorting based on optical properties, such as color or spectral composition, or combinations thereof. These sorting approaches may be implemented individually or in combination. In some embodiments, use of these methods may improve sorting efficiency, enable the achievement of desired levels of hybrid ity, increase F1 hybrid wheat seed recovery, or combinations thereof. As used herein, hybridity means a measure of the proportion of true F1 hybrid wheat seeds within a population, representing the percentage of seeds that successfully resulted from the intended cross-pollination between two distinct parent lines.

[0018] In some examples, use of the disclosed methods enable high-throughput, industrial-scale separation of F1 hybrid wheat seed lots. In some examples, the disclosed methods enable high percentages of hybridity while achieving high F1 hybrid wheat seed recovery rates.

[0019] The present disclosure provides methods for sorting wheat seed into an enriched population of F1 hybrid seed based on density. In some examples, the methods include sorting wheat seed comprising a mixture of inbred pollinator wheat seed and F1 hybrid wheat seed using density to sort the wheat seed mixture into a population of inbred pollinator wheatseed and a population of F1 hybrid wheat seed. The methods may include sorting or separating the inbred pollinator wheat seed from the F1 hybrid wheat seed based on the density of the inbred pollinator wheat seed, or conversely, separating the F1 hybrid wheat seed from the inbred pollinator wheat seed based on the density of the F1 hybrid wheat seed.Accordingly, in certain embodiments, wheat seed comprising a mixture of inbred pollinator wheat seed and F1 hybrid wheat seed may be sorted based on density differences between the two seed types. Sorting may be implemented using a threshold density value or a range of values to partition and separate the seeds. For example, if the F1 hybrid wheat seed is produced using a nuclear-genome based female wheat parent, such as a Ms45 male-sterile female, the threshold density value or a range of values to partition for sorting may be higher than for the male inbred pollinator wheat seed. In some embodiments, the F1 hybrid wheat seed produced using a nuclear-genome male sterility system may be sorted from inbred pollinator wheat seed on the basis that the F1 hybrid wheat seed has a higher density, larger size, a distinct shape, or a higher thousand kernel weight (TKW) relative to the inbred pollinator wheat seed. For example, if the F1 hybrid wheat seed is produced using a CMS-female wheat parent it may be a threshold density value, the threshold density value or a range of values to partition for sorting may be lower than for the male inbred pollinator wheat seed. In some embodiments, the F1 hybrid wheat seed produced using a male-sterile female having mitochondrial-genome based male sterility is sorted from inbred pollinator wheat seed on the basis that the F1 hybrid wheat seed has a smaller size, a distinct shape, a lower density, and / or a lower thousand kernel weight (TKW) relative to the inbred pollinator wheat seed.

[0020] The sorting threshold may be determined by evaluating the density distributions of representative samples from both populations (inbred pollinator and F1 hybrid wheat seed) to identify the divergence point where the trait distributions of the two populations are most distinct. To maximize efficiency, the threshold density can be adjusted dynamically based on calibration targets, such as a specific hybridity percentage or a desired volume of seed recovery. The threshold value may vary based oncalibration, for example, whether the inbred seed population meets or exceeds a desired percentage or amount when sorted. Accordingly, the methods may also include adjusting the threshold density value for the inbred pollinator wheat seed and / or F1 hybrid wheat seed to improve the sorting efficiency, for example, to increase hybridity in the resulting population of F1 hybrid wheat seed and / or to increase F1 hybrid wheat seed recovery relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting or re-sorting. The mixture of F1 hybrid and inbred pollinator wheat seed may be sorted on any suitable instrument, including, but not limited to, a gravity separator, a terminal velocity sorter, an aspirator, a column separator, gravity table, fluidized bed separator, a destoner, or hydrocyclone. Additionally, optical sorters may be configured to perform density-proxy sorting by identifying morphological characteristics, including but not limited to seed volume, thickness, or plumpness, that correlate with the higher thousand kernel weight (TKW) of the F1 hybrid wheat seed population.

[0021] The density sorted population of F1 hybrid wheat seed may be subjected to additional density sorting using the same or different apparatuses, same or different density sorting threshold values, or other sorting approaches to further enrich the F1 hybrid wheat seed population. In some aspects, the inbred pollinator wheat seed population separated from the F1 hybrid wheat seed population may be retained and subjected to one or more additional sorting steps using the same or different sorting technologies to recover additional F1 hybrid wheat seeds contained therein. These additional F1 hybrid wheat seeds may be combined with the F1 hybrid wheat seed population from a previous sort to increase total F1 hybrid wheat seed recovery. For example, in some embodiments, the separated inbred pollinator wheat seed population may be re-sorted to recover residual F1 seeds, which may then be recombined with the F1 hybrid wheat seed from a previous sort to increase total recovery by 1 % to 10%. As non-limiting examples, additional steps may include re-sorting retained inbred pollinator wheat seed to recover residual F1 hybrid wheat seed or performing an initial density sort to remove inbred pollinator wheat seeds from the wheat seed mixture followed by optical sorting to separateadditional inbred pollinator wheat seeds from the F1 hybrid wheat seeds. These additional F1 hybrid wheat seeds may be combined with the F1 hybrid wheat seed population from a previous sort to increase total F1 hybrid wheat seed recovery. Accordingly, disclosed herein are methods of sorting a density-sorted population of F1 hybrid wheat seed using optical sorting to detect and sort out any contaminating inbred pollinator wheat seed from the density-sorted population of F1 hybrid wheat seed.

[0022] Disclosed herein are methods of sorting a mixture of wheat seed comprising F1 hybrid wheat seed and inbred pollinator wheat seed using optical sorting. The optical sorting may detect inbred pollinator wheat seed and separate it from the F1 hybrid wheat seed or conversely detect F1 hybrid wheat seed and separate it from the inbred pollinator wheat seed. Optical sorting may use one or more characteristics, for example, morphological, compositional, and spectral characteristics, to distinguish F1 hybrid wheat seed and inbred pollinator wheat seed from one another and use it for detection and separating the seed into populations.

[0023] Optical sorting may be used as an initial sorting technology in the enrichment process or as a subsequent step following seed density, seed size, or shape separation or other wheat seed morphological characteristics, to further enrich the F1 hybrid wheat seed population. In some aspects, the methods include subjecting the mixture of wheat seed comprising F1 hybrid wheat seed and inbred pollinator wheat seed, a partially enriched subpopulation thereof, or a separated inbred population to multiple passes of optical-based sorting, including hyperspectral, NIR, IR, visible light, and / or RGB optical sorting. These sorting steps may be carried out until the desired level of hybridity of the resulting F1 hybrid wheat seed population is achieved and / or until the F1 hybrid wheat seed recovery is increased to a desired amount or weight.

[0024] As mentioned elsewhere herein, the male-sterile female inbred used as the female parent in the production of the F1 hybrid wheat seed has a nuclear-genome based male-sterility or a mitochondrial-genome based male sterility. In certain embodiments, the parent lines used to produce the F1 hybrid wheat seed are selected so that each parent has different genotypes at one or more R gene loci controlling seed color tofacilitate separation. The R gene loci are located on homoeologous chromosomes 3A, 3B, and 3D. For example, the male-fertile pollinator may be homozygous for recessive white alleles (rr), while the male-sterile female is homozygous for dominant red alleles (RR). See Himi, Eiko, et al. "Development of PCR Markers for TamyblO Related to R-1, Red Grain Color Gene in Wheat." Theoretical and Applied Genetics, vol. 122, no. 8, 2011, pp. 1561-76. Consequently, in this example, the resulting F1 hybrid seed expresses a detectable red phenotype, while the self-pollinated inbred seed remains white. Due to the hexapioid nature of wheat and the maternal inheritance of seed color, the R gene expresses an additive phenotype based on the genetic dosage of dominant alleles across three homoeologous loci, whereby the homozygous dominant genotype (RRR, or R1R1, R2R2, R3R3) produces the darkest red color, the double-dominant allele dosage (RRr) results in a medium red shade, the single-dominant allele dosage (Rrr) results in a light red shade, and the genotype homozygous for recessive white alleles (rrr) produces white seed. These distinct phenotypes create a detectable color gradient that enables optical separation of F1 hybrid wheat seed from inbred pollinator wheat seed. In some embodiments, the seed color phenotype used for optical sorting is maternally inherited, including but not limited to, the red pigmentation conferred by the R gene alleles. In some examples, the method includes using color-based sorting to detect the inbred pollinator wheat seed based on the different number of copies of the dominant red allele from the R gene. In other examples, the inbred and F1 hybrid wheat seeds comprise a different number of copies of the recessive white allele from the R gene. This genetic dosage creates a detectable color gradient that the optical sorting apparatus may use to distinguish and separate the two populations. In some embodiments, the optical sorting uses a spectral signature or reflectance profile corresponding to the concentration of catechin, proanthocyanidin, or combinations thereof regulated by the R gene alleles.

[0025] In some embodiments, the methods disclosed herein may utilize optical sorting approaches to detect variations in the inbred pollinator wheat seed or F1 hybrid wheat seed characteristics such as moisture, fat(lipid), or protein content, or combinations thereof. In some examples, this includes implementing transmission Near-Infrared spectroscopy (NIR-T), reflectance Near-Infrared (NIR) spectroscopy, diffuse Reflectance NearInfrared (NIR) spectroscopy, transflectance Near-Infrared (NIR) spectroscopy, or fiber optic Near-Infrared (NIR) spectroscopy.

[0026] The methods disclosed herein may use optical sorting utilizing hyperspectral, NIR, IR, visible light, RGB, or combinations thereof to detect and distinguish between inbred pollinator wheat seed and F1 hybrid wheat seed within the mixture of wheat seeds or an enriched F1 hybrid wheat seed population to facilitate their separation. In some examples, the optical sorting may be used to identify the inbred pollinator wheat seed as the target for removal from the mixture or enriched F1 hybrid wheat seed population based on its specific color or compositional profile.

[0027] Alternatively, the methods disclosed herein may include identifying and selecting the F1 hybrid wheat seed from the mixture or a retained inbred pollinator wheat seed fraction to separate the F1 hybrid wheat seed from the inbred pollinator wheat seed. This selection may be based on a unique phenotypic or chemical signature indicative of the F1 hybrid wheat seed, allowing for targeted collection.

[0028] In some aspects, the specific detection mechanisms employed are dependent on the sorting technology used, where RGB sorting evaluates visible color intensity, while hyperspectral, IR, and NIR sorting analyze spectral reflectance or unique spectral signatures to differentiate between seed populations. Accordingly, the optical sorting threshold may be determined by identifying a divergence point where the trait distributions of the inbred and F1 hybrid wheat seed populations are distinct, based on a measurement of spectral reflectance, spectral signature, or color intensity depending on the specific sorting technology employed. This threshold determination may involve a training step where known populations of inbred and F1 hybrid wheat seeds are provided to the sorting apparatus to calibrate its sensors to the specific color or compositional gradient resulting from the R gene allele dosages or chemical composition of the specific wheat varieties being sorted. Accordingly, the methods include adjusting optical sorting parameters, such as color sensitivity, spectraldetection ranges, or detection thresholds, to improve sorting efficiency and achieve a desired balance between final F1 hybrid wheat population hybridity and total F1 hybrid wheat seed recovery. In some examples, the sorting is carried out until the desired level of accuracy or hybridity for the population of F1 hybrid wheat seed is achieved.

[0029] By targeting either the unique profile of the inbred pollinator wheat seed for rejection or the unique profile of the F1 hybrid wheat seed for collection, these methods enable efficient separation using hyperspectral, NIR, IR, visible light, or RGB-based sorting across one or more sorting passes. In some examples, the optical sorting apparatus is configured to identify the inbred pollinator wheat seed as the primary target for removal to produce the enriched population of F1 hybrid wheat seed. In other embodiments, such as a recovery pass of a separated inbred pollinator wheat seed population, the apparatus may be configured to identify the F1 hybrid wheat seed as the primary target for collection or rejection. In either instance, the sorting may be carried out to achieve the desired level of hybridity and / or F1 hybrid wheat seed recovery.

[0030] The methods disclosed herein may involve multiple stages or passes of optical sorting to achieve a desired balance of hybridity and / or F1 hybrid wheat seed recovery. In some examples, the initially sorted population of F1 hybrid wheat seed may be subjected to additional optical sorting using the same or different apparatuses, or the same or different optical sorting threshold values, to further enrich the F1 hybrid wheat seed population. This iterative process may include subjecting the enriched F1 hybrid wheat seed population to one or more secondary passes to remove residual inbred pollinator seeds and improve final hybridity levels.

[0031] Furthermore, the methods disclosed herein may include recovering F1 hybrid wheat seeds that are initially partitioned with the inbred pollinator wheat seed fraction. In some aspects, the inbred pollinator wheat seed population separated during a primary sort may be retained and subjected to one or more additional sorting steps using the same or different sorting technologies. In some examples, this re-sorting of the separated inbred pollinator wheat seed population is used to identify and recover residual F1 hybrid wheat seeds that were mischaracterized ormisdirected during the initial pass. These recovered F1 hybrid wheat seeds may then be combined with the F1 hybrid wheat seed population from a previous sort to increase the total F1 hybrid wheat seed recovery. In some embodiments, re-sorting the separated inbred pollinator wheat seed population facilitates increasing the total F1 hybrid wheat seed recovery by about 1% to about 10% relative to the total amount of F1 hybrid wheat seed present in the mixture or population prior to sorting or re-sorting as the case may be.

[0032] The mixture of F1 hybrid wheat seed and separated inbred pollinator wheat seed may be sorted on any suitable optical instrument, sorter, or apparatus, including, but not limited to, optical sorters, hyperspectral imaging systems, near-infrared (NIR) based separators, or machine-learning-enabled sorting platforms utilizing RGB, NIR, or IR sensor technologies. Non-limiting examples of suitable sorting systems include the Satake Evolution (Satake USA Inc., Stafford, TX), the Satake Enpresor (Satake USA Inc., Stafford, TX), the Buhler Sortex H (Buhler AG, Uzwil, Switzerland), the Vmek Profile 3D (Vmek LLC, Covington, LA), the BoMill InSight (BoMill AB, Vellinge, Sweden), the Satake Alpha (Satake USA Inc., Stafford, TX), the TOMRA IX (TOMRA Food, Leuven, Belgium), the Ishida IX-Series (Ishida Co., Ltd., Kyoto, Japan), the Cimbria SEA (Cimbria s.r.l., Imola, Italy), and the Vmek sorter (Vmek LLC, Covington, LA). In certain embodiments, the wheat seeds may be sorted for shape and color using high-resolution RGB platforms such as the Satake Evolution (Evolution, Satake USA Inc., Stafford, TX), the Satake Enpresor (Enpresor, Satake USA Inc., Stafford, TX), or the Buhler Sortex H (Sortex H, Buhler AG, Uzwil, Switzerland). In certain embodiments, sorters may be configured to sort seeds concurrently based on one or more physical, morphological, and / or optical characteristics.

[0033] In certain embodiments, the F1 hybrid wheat seed and inbred pollinator wheat seed are separated from one another using a directed pneumatic force, including but not limited to a pulse or plurality of pulses of air. This pneumatic separation may occur while the seeds are in-flight or free-falling within the sorting apparatus, allowing for high-throughput, contactless separation based on the previously identified density,morphological, or optical characteristics that distinguish the inbred pollinator wheat seed and the F1 hybrid wheat seed from one another.

[0034] As disclosed herein, the methods may include sorting or separating the separated inbred pollinator wheat seed from the F1 hybrid wheat seed based on wheat seed morphological characteristics. Such morphological characteristics, include, but are not limited to: linear dimensions, including, but not limited to, seed size, seed shape, seed length, and seed thickness; geometric properties, including, but not limited to, seed surface area, calculated seed volume, seed perimeter, and seedcircularity; geometric ratios, including, but not limited to, aspect ratio (length-to-width ratio) and surface-area-to-volume ratio; and surface topography, including but not limited to, seed coat texture, wrinkling, the presence of a crease, or combinations thereof. These physical and morphological dimensions of the inbred pollinator wheat seed and F1 hybrid wheat seed may used as a basis for sorting and population enrichment.

[0035] As an example, for the separation of F1 hybrid wheat seed and inbred pollinator wheat seed from one another based on thickness, slotted sieves may be used and for separation of F1 hybrid wheat seed and inbred pollinator wheat seed from one another based on width round-hold sieves may be used. Accordingly, for the separation of F1 hybrid wheat seed and inbred pollinator wheat seed from one another, sieves with intermediate apertures may be used, such as round-hole or slotted sieves with dimensions from 7.0 / 64 inch to 11.0 / 64 inch. In some aspects, seedsize and / or seed-shape sorting may be based on a threshold size and / or shape using a round-hole sieve or slotted sieve with a diameter selected from the range of 6.5 / 64 inches and 11.5 / 64 inches, including all 0.5 / 64-inch increments therein, such as 6.5 / 64, 7.0 / 64, 7.5 / 64, 8.0 / 64, 8.5 / 64, 9.0 / 64, 9.5 / 64, 10.0 / 64, 10.5 / 64, 11.0 / 64, and 11.5 / 64 inches.

[0036] In some embodiments, for the separation of F1 hybrid wheat seed and inbred pollinator wheat seed from one another based on length, sorting may be performed using an indent cylinder, trieur, or disc separator to distinguish and select the longer F1 hybrid wheat seeds from the shorter inbred pollinator wheat seeds if a nuclear-genome based male-sterile female is used as the female parent. In some embodiments, for the separation of F1 hybrid wheat seed and inbred pollinator wheat seed from one another based on length, sorting may be performed using an indent cylinder, trieur, or disc separator to distinguish and select the shorter F1 hybrid wheat seeds from the longer inbred pollinator wheat seeds if a mitochondrial-genome based male-sterile female is used as the female parent (from CMS-system). Accordingly, for the separation of F1 hybrid wheat seed and inbred pollinator wheat seed from one another based on length, an indent cylinder, trieur, or disc separator may be used with pocket diameters selected from the range of 12.0 / 64 inches to 24.0 / 64 inches. In some aspects, length sorting may be based on a threshold length using a pocket diameter selected from the range of 12.0 / 64 inches to 24.0 / 64 inches, including all 0.5 / 64-inch increments therein, such as 12.0 / 64, 12.5 / 64, 13.0 / 64, 13.5 / 64, 14.0 / 64, 14.5 / 64, 15.0 / 64, 15.5 / 64, 16.0 / 64, 16.5 / 64, 17.0 / 64, 17.5 / 64, 18.0 / 64, 18.5 / 64, 19.0 / 64, 19.5 / 64, 20.0 / 64, 20.5 / 64, 21.0 / 64, 21.5 / 64, 22.0 / 64, 22.5 / 64, 23.0 / 64, 23.5 / 64, and 24.0 / 64 inches.

[0037] In some embodiments, scalping or bottom screening or both of the wheat seed mixture may be performed as a pre-cleaning step prior to subjecting the wheat seed mixture to seed-size and / or seed-shape sorting for F1 hybrid wheat seed and inbred pollinator wheat seed separation, density sorting, or optical sorting to remove bulk debris and / or fine contaminants that may interfere with subsequent sorting efficiency. For scalping purposes to remove large debris, sieves of various shapes and sizes ranging from 12 / 64 inch to 3 / 8 inch may be used, including 12.0 / 64, 12.5 / 64, 13.0 / 64, 13.5 / 64, 14.0 / 64, 14.5 / 64, 15.0 / 64, 15.5 / 64, 16.0 / 64, 16.5 / 64, 17.0 / 64, 17.5 / 64, 18.0 / 64, 18.5 / 64, 19.0 / 64, 19.5 / 64, 20.0 / 64, 20.5 / 64, 21.0 / 64, 21.5 / 64, 22.0 / 64, 22.5 / 64, 23.0 / 64, 23.5 / 64, and 24.0 / 64 inches. To function as a bottom screen for removing fine contaminants, round-hole sieves, slotted sieves, or wire mesh shapes with apertures ranging from 4.0 / 64 inch to 6.0 / 64 inches may be used, including 4.0 / 64, 4.5 / 64, 5.0 / 64, 5.5 / 64, and 6.0 / 64 inches.

[0038] In some embodiments, the methods may include utilizing optical sorting to evaluate advanced physical and / or morphological dimensionssuch as seed surface area, calculated seed volume, seed circularity, and aspect ratio. In some examples, the optical sorting leverages real-time phenotypic analysis to identify morphological phenotypes that distinguish the F1 hybrid wheat seed from the inbred pollinator wheat seed.

[0039] To determine and apply the appropriate threshold or range for separation, the method may include evaluating the physical dimensions of representative samples from both the inbred pollinator wheat seed and F1 hybrid wheat seed populations to identify a distinct value or range where the trait distributions of the two populations diverge. This identified divergence point is then applied by selecting a specific sorting parameter, including but not limited to, sieve aperture size, indent pocket diameter, or precision machine setting that aligns with this divergence point to maximize the separation of the target and non-target fractions.

[0040] In some embodiments, the sorting based on physical, morphological, and / or optical characteristics of the seed, including seedsize, seed shape, seed density, and / or seed color, is contactless with respect to a solid mechanical barrier. This contactless sorting may be performed without relying on a solid mechanical barrier, such as a sieve, a screen, or a gate, to physically trap or redirect seeds. For example, optical sortng devices with sensors and imaging technologies may be used to identify the physical, morphological, and / or optical characteristics of individual seeds in-flight, and separation is executed using directed forces, such as a pulse or pulses of air. In some embodiments, the sorting methods and systems provided herein are automated, high-throughput, or both enabling industrial-scale enrichment of F1 hybrid wheat seed populations.

[0041] In some aspects, the size and / or shape sorting may be based on a certain range of seed-size and / or seed-shape. Determining this range may involve statistical or visual analysis of the seed lot to establish the upper and lower bounds of the target F1 hybrid wheat seed population.

[0042] Accordingly, the method also includes adjusting the threshold size and / or shape for the inbred pollinator wheat seed and / or F1 hybrid wheat seed to improve the sorting efficiency. In some aspects, the size and / or shape sorting may be based on a certain range of seed-size and / or seed-shape. Accordingly, the method also includes adjusting the range of size and / or shape for the inbred pollinator wheat seed and / or F1 hybrid wheat seed to improve the sorting efficiency. The mixture of F1 hybrid and inbred pollinator wheat seed may be sorted based on size and / or shape using any suitable instrument, including, but not limited to, an optical sorter, a cylindrical screen, a vibrating screen, a drum sorter, an indent cylinder, a trieur, a disc separator, a precision sizer, a roller sorter, or a gap sorter. The seed-size- and / or seed-shape-sorted population of F1 hybrid wheat seed may be subjected to additional seed-size and / or seedshape sorting using the same or different apparatuses, same or different size and / or shape sorting threshold or range of size and / or shape, or other sorting approaches.

[0043] Additionally, the inbred pollinator wheat seed population separated during the size and / or shape sorting may be retained and re-sorted using the same or different sorting technologies, such as density or optical sorting, to identify and recover additional F1 hybrid wheat seeds. The additional F1 hybrid wheat seeds recovered from the inbred pollinator wheat seed population may then be combined with the previously sorted F1 hybrid wheat seed population to increase the total F1 hybrid wheat seed recovery, including but not limited to, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. Accordingly, disclosed herein are methods of sorting a seed-size- and / or seed-shape-sorted population of F1 hybrid wheat seed using optical sorting to detect and sort out any inbred pollinator wheat seed from the seed-size- and / or seed-shape-sorted population of F1 hybrid wheat seed. In some examples, the optical sorting uses phenotypic analysis of individual seeds to identify at least one morphological, compositional, or spectral phenotype that distinguishes the F1 hybrid wheat seed from the inbred pollinator wheat seed. The at least one morphological phenotype may include: linear dimensions, including, but not limited to, seed size, seed shape, seed length, and seed thickness; geometric properties, including, but not limited to, seed surface area, calculated seed volume, seed perimeter, and seedcircularity; geometric ratios, including, but not limited to, aspect ratio (length-to-width ratio) and surface-area-to-volume ratio; and surfacetopography, including but not limited to, seed coat texture, wrinkling, or the presence of a crease, or combinations thereof. In some aspects, the optical sorting may identify the F1 hybrid wheat seed based on a higher surface area, a higher calculated volume, or a distinct topographic or geometric profile relative to the inbred pollinator wheat seed.

[0044] The methods disclosed herein may include selecting a male-sterile female wheat parent line and a male-fertile pollinator wheat parent line, where these parent lines possess different genotypes at one or more R gene loci controlling seed color to facilitate downstream optical sorting. In some examples, the male-sterile female parent line has a nuclear-based male-sterility where the male-sterility mechanism resides in the nuclear genome rather than the mitochondria. Examples of male fertility genes include but are not limited to, Ms1, Ms2, Ms3, Ms5, Ms9, Ms22, Ms26, or Ms45 male fertility genes. As a non-limiting example, the nuclear-based male-sterile female wheat parent line may be generated using any number of approaches, including but not limited to chemical mutagenesis or genome-editing technologies. Accordingly, the female plant may be rendered male sterile due to genome editing of a nuclear male fertility gene on each of the A, B, and D genomes, e.g., Ms1 , Ms2, Ms3, Ms5, Ms9, Ms22, Ms26, or Ms45 male fertility genes, to render the male-sterile female wheat parent line homozygous recessive for the male fertility gene across all three genomes, see, for example, published patent application US 20190177722 and US Patent No. 12,054,732 each of which is herein incorporated by reference each in its entirety. The nuclear-based male-sterile female parent may be increased using a hybrid wheat production platform, including those described published patent application W02020056259, or WO2019043082, each of which is herein incorporated by reference each in its entirety. The production process includes interplanting the parent lines in a field using configurations such as seed blends, intermixed rows, or strips to facilitate cross-pollination and produce a mixed population of wheat plants.

[0045] In some examples, the interplanting comprises planting a seed blend comprising from about 1% to about 30% by seed count or seed weight of the male-fertile pollinator wheat parent line seed and from about70% to about 99% by seed count or seed weight of the male-sterile female wheat parent line seed. For instance, the seed blend may comprise from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% of the male-fertile pollinator wheat parent line seed, with the remainder being the male-sterile female parent line seed. For example, the seed blend may include from about 1% to about 30% by seed count or seed weight of the male-fertile pollinator wheat parent line seed and from about 70% to about 99% by seed count or seed weight of the male-sterile female wheat parent line seed, for example, a seed blend comprising from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% by seed count or seed weight of the male-fertile pollinator wheat parent line seed and from about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by seed count or seed weight of the male-sterile female wheat parent line seed. Non-limiting examples of such blends include a ratio of 10% to 30% pollinator to 70% to 90% female, or a ratio of 1 % to 5% pollinator to 95% to 99% female.

[0046] Following planting, the seeds are grown into a mixed population of wheat plants, and the plants are allowed to pollinate such that the male-fertile pollinator plants cross-pollinate the male-sterile female wheat plants to produce F1 hybrid wheat seed. Simultaneously, the male-fertile pollinator wheat plants are allowed to i ntra-pol linate, including selfpollinating or sib-pollinating, to produce inbred pollinator wheat seed. The methods then include harvesting the wheat seed from the mixed population of plants to obtain a harvested wheat seed mixture comprising F1 hybrid wheat seed and inbred pollinator wheat seed, which is subsequently subjected to the density, size, shape, and / or optical sorting processes disclosed herein.

[0047] Hybridity may be evaluated using any suitable process or technique, for example, using a molecular or biochemical analysis toconfirm the genetic identity of the F1 hybrid wheat seeds, for example, detecting single nucleotide polymorphisms. In some examples, use of the sorting methods described herein results in an enriched F1 population of F1 hybrid wheat seed with a hybridity of at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some examples, use of the sorting methods described herein results in reducing the presence of the inbred pollinator wheat seed to less than 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some examples, use of the sorting methods described herein results population of F1 hybrid wheat seed having a hybridity of at least 70%, 71%, 72%, 73%, 74% 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% and a F1 hybrid wheat seed recovery of at least 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting.

[0048] As used herein, the term “wheat” refers to any species of the genus Triticum, including progenitors thereof, as well as progeny thereof produced by crosses with other species. Wheat includes “hexapioid wheat” which has genome organization of AABBDD, comprised of 42 chromosomes, and “tetraploid wheat” which has genome organization of AABB, comprised of 28 chromosomes. Hexapioid wheat includes T. aestivum, T. spelta, T. mocha, T. compactum, T. sphaerococcum, T. vavilovii, and interspecies cross thereof. Tetraploid wheat includes T. durum (also referred to as durum wheat or Triticum turgidum ssp. durum), T. dicoccoides, T. dicoccum, T. polonicum, and interspecies cross thereof. In addition, the term “wheat” includes possible progenitors of hexapioid or tetraploid Triticum sp. such as T. urartu, T.monococcum or T. boeoticum for the A genome, Aegilops speltoides for the B genome, and T. tauschii (also known as Aegilopssquarrosa or Aegilops tauschii) for the D genome. A wheat cultivar for use in the present disclosure may belong to, but is not limited to, any of the above-listed species. Also encompassed are plants that are produced by conventional techniques using Triticum sp. as a parent in a sexual cross with a non-Triticum species, such as rye (Secale cereale), including but not limited to Triticale. In some embodiments, the wheat plant is suitable for commercial production of grain, such as commercial varieties of hexapioid wheat or durum wheat, having suitable agronomic characteristics which are known to those skilled in the art. In some embodiments, the wheat seed, e.g., F1, inbred pollinator wheat seed, or the male-sterile female wheat parent seed, is Soft Red Winter Wheat, Hard Red Winter Wheat, Soft White Winter Wheat, or Hard White Winter Wheat.

[0049]

[0050] EMBODIMENT

[0051] The present disclosure is further illustrated in the following embodiments. It should be understood that these embodiments are given by way of illustration only.

[0052]

[0053] Embodiment 1. A method of sorting wheat seed, the method comprising:

[0054] sorting wheat seed comprising a mixture of inbred pollinator wheat seed and F1 hybrid wheat seed using density to sort the wheat seed into an enriched population of inbred pollinator wheat seed and an enriched population of F1 hybrid wheat seed.

[0055]

[0056] Embodiment 2. The method of embodiment 1 , the method further comprising:

[0057] sorting the inbred pollinator wheat seed based on the density of the inbred pollinator wheat seed.

[0058]

[0059] Embodiment 3. The method of embodiment 1 , the method further comprising:

[0060] sorting the F1 hybrid wheat seed based on the density of the F1 hybrid wheat seed.

[0061]

[0062] Embodiment 4. The method of embodiment 1 , the method further comprising:

[0063] sorting the inbred pollinator wheat seed based on the density of the inbred pollinator wheat seed using a threshold density value.

[0064]

[0065] Embodiment 5. The method of embodiment 1 , the method further comprising:

[0066] sorting the F1 hybrid wheat seed based on the density of the F1 hybrid wheat seed using a threshold density value.

[0067]

[0068] Embodiment 6. The method of embodiment 1 , the method further comprising sorting the wheat seed mixture based on density using a gravity table, fluidized bed separator, or hydrocyclone.

[0069]

[0070] Embodiment 7. The method of embodiment 1 , the method further comprising adjusting the threshold density value of the inbred pollinator wheat seed to improve the sorting efficiency.

[0071]

[0072] Embodiment 8. The method of embodiment 1 , the method further comprising adjusting the threshold density value of the F1 hybrid wheat seed to improve the sorting efficiency.

[0073]

[0074] Embodiment 9. The method of embodiment 1 , the method further comprising:

[0075] sorting the enriched population of F1 hybrid wheat seed using optical sorting.

[0076]

[0077] Embodiment 10. The method of embodiment 9, the method further comprising:

[0078] using optical-based sorting to detect inbred pollinator wheat seed and separate the inbred pollinator wheat seed from the enriched population of F1 hybrid wheat seed.

[0079]

[0080] Embodiment 11. The method of embodiment 9, wherein the optical-based sorting is color-based sorting, the method further comprising using color-based sorting to detect the inbred pollinator wheat seed in the enriched population of F1 hybrid wheat seed based on the color of the inbred wheat seed and separate the inbred pollinator wheat seed from the enriched population of F1 hybrid wheat seed.

[0081]

[0082] Embodiment 12. The method of embodiment 9, the method further comprising: wherein the optical-based sorting is color-based sorting, the method further comprising using color-based sorting to detect F1 hybrid wheat seed in the inbred pollinator wheat seed population based on the color of the F1 hybrid wheat seed and separate the inbred pollinator wheat seed from the enriched population of F1 hybrid wheat seed.

[0083]

[0084] Embodiment 13. The method of embodiment 11 or 12, wherein the inbred pollinator wheat seed and F1 hybrid wheat seed comprise different number of copies of the recessive white allele from the R gene or different number of copies of the dominant red allele from the R gene.

[0085]

[0086] Embodiment 14. The method of embodiment 9, the method further comprising:

[0087] using hyperspectral, NIR, IR, visible light-based sorting (or combinations thereof) to detect the inbred pollinator wheat seed in the enriched population of F1 hybrid wheat seed based on the color or composition of the inbred pollinator wheat seed and separate the inbred pollinator wheat seed from the enriched population of F1 hybrid wheat seed.

[0088]

[0089] Embodiment 15. The method of embodiment 14, the method further comprising:

[0090] using hyperspectral, NIR, IR, visible light-based sorting (or combinations thereof) to detect the F1 hybrid wheat seed in the enriched population of inbred pollinator wheat seed based on the color or composition of the F1 hybrid wheat seed.

[0091]

[0092] Embodiment 16. The method of embodiment 9, the method further comprising:

[0093] sorting inbred pollinator wheat seed from the enriched population of F1 hybrid wheat seed using RGB.

[0094]

[0095] Embodiment 17. The method of any of the embodiments from 1 to 16, the method further comprising: collecting or retaining the enriched F1 hybrid wheat seed population or a fraction of F1 hybrid wheat seed population, for example, as recovered from an enriched inbred pollinator population.

[0096]

[0097] Embodiment 18. The method of any of the embodiments from 1 to 19, the method further comprising: discarding or removing the sorted or separated inbred pollinator wheat seed population or a fraction of F1 hybrid wheat seed population, for example, as recovered or separated from an enriched F1 hybrid wheat seed population.

[0098]

[0099] Embodiment 19. The method of embodiment 10, the method further comprising: subjecting the population or subpopulation of F1 hybrid wheat seed mixture to multiple passes of optical-based sorting (including but not limited to hyperspectral, NIR, IR, visible light, and / or RGB) until the desired level of hybridity for the enriched population of F1 hybrid wheat seed and / or F1 hybrid wheat seed recovery relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting is achieved.

[0100]

[0101] Embodiment 20. The method of any one of embodiments 9-19, the method further comprising: (a) sorting the mixture of inbred pollinator wheat seed and F1 hybrid wheat seed based on density to enrich the population of F1 hybrid wheat seed; (b) sorting the population of F1 hybridwheat seed using optical sorting; and (c) sorting the population of F1 hybrid wheat seed based on size and / or shape; wherein steps (b) and (c) are performed in any order following step (a) to produce an enriched population of F1 hybrid wheat seed.

[0102]

[0103] Embodiment 21. The method of any one of embodiments 20, wherein two or more of the sorting based on density, the sorting based on size and / or shape, and the sorting based on an optical property are performed concurrently or simultaneously to produce the final enriched population of F1 hybrid wheat seed.

[0104]

[0105] Embodiment 22. The method of any one of embodiments 1 -21 , comprising sorting the inbred pollinator wheat seed and the F1 hybrid wheat seed from one another based on seed width, seed thickness, or seed length.

[0106]

[0107] Embodiment 23. The method of embodiment 22, wherein sorting based on width or thickness comprises using a round-hole sieve or a slotted sieve with intermediate apertures selected from the range of 7.0 / 64 inch to 11.0 / 64 inch.

[0108]

[0109] Embodiment 24. The method of embodiment 20, wherein the round-hole or slotted sieve has a diameter or slot-width selected from the range of 6.5 / 64 inches to 11.5 / 64 inches, including all 0.5 / 64-inch increments therein, such as 6.5 / 64, 7.0 / 64, 7.5 / 64, 8.0 / 64, 8.5 / 64, 9.0 / 64, 9.5 / 64, 10.0 / 64, 10.5 / 64, 11.0 / 64, and 11.5 / 64 inches.

[0110]

[0111] Embodiment 25. The method of embodiment 22, wherein sorting based on length comprises using an indent cylinder, trieur, or disc separator with pocket diameters selected from the range of 12.0 / 64 inches to 24.0 / 64 inches, including all 0.5 / 64-inch increments therein in ascending order.

[0112]

[0113] Embodiment 26. The method of any one of embodiments 1 -25, further comprising performing scalping or bottom screening or both as a pre-cleaning step prior to the sorting based on density, size, shape, or optical properties.

[0114]

[0115] Embodiment 27. The method of embodiment 26, wherein scalping comprises using sieves with sizes ranging from 12.0 / 64 inch to 24.0 / 64 inch (3 / 8 inch), and bottom screening comprises using sieves with apertures ranging from 4.0 / 64 inch to 6.0 / 64 inches.

[0116]

[0117] Embodiment 28. The method of any one of embodiments 1 -27, comprising evaluating physical dimensions of representative samples from both the inbred pollinator wheat seed and F1 hybrid wheat seed populations to identify a divergence point where trait distributions diverge to determine a sorting threshold or range.

[0118]

[0119] Embodiment 29. The method of any one of embodiments 1 -28, wherein the sorting is performed using an indent cylinder, a trieur, a disc separator, a precision sizer, a roller sorter, a gap sorter, or an aspirator.

[0120]

[0121] Embodiment 30. The method of embodiment 9, the method comprising selecting a male-sterile female wheat parent line and a male-fertile pollinator wheat parent line, wherein said parent lines have different genotypes at one or more R gene loci controlling seed color.

[0122]

[0123] Embodiment 31. The method of embodiment 30, the method comprising selecting a male-sterile female wheat parent line and a male-fertile pollinator wheat parent line, wherein said parent lines have different genotypes at one or more R gene loci controlling seed color, wherein the different genotypes confer a detectable difference in seed color phenotype between the resulting F1 hybrid wheat seed produced on the female parent and self-pollinated male-fertile pollinator wheat parent line.

[0124]

[0125] Embodiment 32. The method of embodiment 1 , the method comprising interplanting a male-sterile female wheat parent line with a male-fertile pollinator wheat parent lines in a field to produce a mixed population of wheat plants.

[0126]

[0127] Embodiment 33. The method of embodiment 32, where the interplanting comprises planting a seed blend comprising from about 1% to about 30% by seed count or seed weight of the male-fertile pollinator wheat parent line seed and from about 70% to about 99% by seed count or seed weight of the male-sterile female wheat parent line seed, for example, where the interplanting comprises planting a seed blend comprising from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% by seed count or seed weight of the male-fertile pollinator wheat parent line seed and from about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by seed count or seed weight of the male-sterile female wheat parent line seed. In a non-limiting example, the seed blend comprises from about 10% to about 30% by seed count or seed weight of the male-fertile pollinator wheat parent line seed and from about 70% to about 90% by seed count or seed weight of the male-sterile female wheat parent line seed. In a non-limiting example, the seed blend comprises from about 10% to about 30% by seed count or seed weight of the male-fertile pollinator wheat parent line seed and from about 70% to about 90% by seed count or seed weight of the male-sterile female wheat parent line seed, or from about 1 % to about 5% by seed count or seed weight of the male-fertile pollinator wheat parent line seed and from about 95% to about 99% by seed count or seed weight of the male-sterile female wheat parent line seed.

[0128]

[0129] Embodiment 34. The method of embodiment 32 or 33, the method further comprising: growing the seed into a mixed population of wheatplants, wherein the mixed population of wheat plants comprises male-sterile female wheat plants and male-fertile pollinator wheat plants.

[0130]

[0131] Embodiment 35. The method of embodiment 34, the method further comprising: allowing the male-fertile pollinator wheat plants to cross-pollinate the male-sterile female wheat plants to produce F1 hybrid wheat seed and male-fertile pollinator wheat plants to self-or sib-pol linate to produce inbred pollinator wheat seed .

[0132]

[0133] Embodiment 36. The method of embodiment 35, the method comprising harvesting wheat seed from the mixed population of wheat plants to obtain a wheat seed mixture comprising F1 hybrid wheat seed and inbred pollinator wheat seed prior to sorting the seed.

[0134]

[0135] Embodiment 37. The method of embodiment 32, wherein the male-sterile female wheat parent line and the male-fertile pollinator wheat parent lines comprise different genotypes at one or more R gene loci controlling seed color.

[0136]

[0137] Embodiment 38. The method of embodiment 37, wherein the male-fertile pollinator wheat parent line is homozygous for recessive white alleles (r) at its R gene loci, and the male-sterile female parent line comprises at least one dominant red allele (R) at its R gene loci.

[0138]

[0139] Embodiment 39. The method of embodiment 37, wherein the male-sterile female parent line is homozygous for recessive white alleles (r) at its R gene loci, and the male-fertile pollinator wheat parent line comprises at least one dominant red allele (R) at its R gene loci.

[0140]

[0141] Embodiment 40. The method of embodiment 38, optically sorting wheat seed comprising red F1 hybrid seed produced by the crosspollination of the male-sterile female wheat parent line with the male-fertile pollinator wheat parent and white inbred pollinator wheat seed produced by self-pollination of the male-fertile pollinator wheat parent line.

[0142]

[0143] Embodiment 41. The method of embodiment 39, optically sorting wheat seed comprising white F1 hybrid seed produced by the crosspollination of the male-sterile female wheat parent line with the male-fertile pollinator wheat parent and red inbred pollinator wheat seed produced by self-pollination of the male-fertile pollinator wheat parent line.

[0144]

[0145] Embodiment 42. The method of embodiment 9, wherein the optical sorting produces an enriched population of F1 hybrid wheat seed comprising a hybridity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0146]

[0147] Embodiment 43. The method of embodiment 20, wherein the combination of the size, shape, and / or density sorting step and the optical color sorting step results in an enriched population of F1 hybrid wheat seed with less than 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% inbred pollinator seed.

[0148]

[0149] Embodiment 44. The method of embodiment 43, wherein the sorting comprises adjusting one or more sorting parameters to produce a resulting enriched population of F1 hybrid wheat seed comprising a hybridity of at least 70% and a F1 hybrid wheat seed recovery of at least 45% relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting.

[0150]

[0151] Embodiment 45. The method of embodiment 44, wherein the resulting population of F1 hybrid wheat seed comprising a hybridity of at least 70%, 71%, 72%, 73%, 74% 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% and a F1 hybrid wheat seed recovery of at least 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting. In a non-limiting example, the resulting population of F1 hybrid wheat seed comprises a hybridity from about 75% to about 85% and a F1 hybrid wheat seed recovery from about 45% to about 65% relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting, or a hybridity from about 91% to about 100% and a F1 hybrid wheat seed recovery from about 66% to about 100% relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting.

[0152]

[0153] Embodiment 46. The method of embodiment 44, wherein the one or more sorting parameters includes but is not limited to a density threshold, a size threshold, a sorter speed, or a color sensitivity setting.

[0154]

[0155] Embodiment 47. The method of embodiment 44, wherein the sorting results in a population of F1 hybrid wheat seed comprising a hybridity of at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hybridity relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting.

[0156]

[0157] Embodiment 48. The method of embodiment 1 , the method further comprising: further separating the inbred pollinator wheat seed from the mixture of inbred pollinator wheat seed and F1 hybrid wheat seed.

[0158]

[0159] Embodiment 49. The method of embodiment 1 , the method further comprising: retaining the population of inbred pollinator wheat seed and re-sorting said inbred pollinator wheat seed population using the same or different sorting technology to identify and recover additional F1 hybrid wheat seeds contained therein.

[0160]

[0161] Embodiment 50. The method of embodiment 49, the method further comprising:

[0162] combining the recovered F1 hybrid wheat seeds with the enriched population of F1 hybrid wheat seed or with a subpopulation of F1 hybrid wheat seed after said population has been subjected to one or more additional sorting steps using the same or different sorting technology to increase total F1 hybrid wheat seed recovery or hybridity.

[0163]

[0164] Embodiment 51. The method of embodiment 1 or 49, comprising recovering additional F1 hybrid seed from the enriched inbred pollinator wheat seeds population or a fraction thereof to increase total F1 hybrid wheat seed recovery by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or higher.

[0165]

[0166] Embodiment 52. The method of embodiment 19, the method further comprising: separating by each pass inbred pollinator wheat seed from the population of F1 hybrid wheat seed.

[0167]

[0168] Embodiment 53. The method of any one of embodiments 1-52 and 54-55, wherein the F1 hybrid wheat seed is produced by pollen from the male-fertile pollinator wheat parent line fertilizing the nuclear-genome based male-sterile female wheat parent line, where the male-sterility mechanism resides in the nuclear genome rather than the mitochondria. For example, the nuclear-genome based male-sterile female wheat parent line may be male-sterile for a nuclear male fertility gene, including but not limited to, Ms1, Ms2, Ms3, Ms5, Ms9, Ms22, Ms26, or Ms45. As a nonlimiting example, the nuclear-based male-sterile female may be rendered sterile using any suitable approach including chemical mutagenesis or genome editing of an endogenous nuclear male fertility gene, e.g., Ms1 , Ms2, Ms3, Ms5, Ms9, Ms22, Ms26, or Ms45 male fertility genes, on each of the A, B, and D genomes to render the male-sterile female plant homozygous recessive for the male fertility gene, see, for example, published patent application See, published patent application US 20190177722 and US Patent No. 12,054,732 each of which is hereinincorporated by reference each in its entirety. Since Ms1 in wheat behaves as a single gene recessive, in some embodiments, only the Ms1 male-fertility polynucleotide or allele located on chromosome 4BS may need to be mutated to confer male-sterility to a wheat plant. Accordingly, in some examples, with respect to Ms1, genome-editing or mutating the endogenous Ms1 male fertility gene on the B genome is sufficient to render the nuclear-based male-sterile female parent male sterile. See, for example, US Patent No. 12,054,732. The nuclear-based male-sterile female parent, such as Ms1 or Ms45 male-sterile female parent, may be increased using a hybrid wheat production platform, such as Ms1-BA or Ms45-BA, including those described published patent application W02020056259, or WO2019043082, each of which is herein incorporated by reference each in its entirety.

[0169]

[0170] Embodiment 54. The method of any of the embodiments herein (1-157), wherein the seed color of the F1 hybrid wheat seed and / or inbred pollinator wheat seed used in sorting is maternally inherited.

[0171]

[0172] Embodiment 55. The method of any of the embodiments herein herein (1-157), wherein optical sorting utilizes a spectralsignature or reflectance profile corresponding to the concentration of catechin, proanthocyanidin, or combinations thereof regulated by the R gene alleles when the F1 hybrid wheat seed and / or inbred pollinator wheat seed comprises one or more R alleles.

[0173]

[0174] Embodiment 56. A method of sorting wheat seed, the method comprising:

[0175] sorting wheat seed comprising a mixture of inbred pollinator wheat seed and F1 hybrid wheat seed using seed size and / or seed shape to sort the wheat seed into an enriched population of inbred pollinator wheat seed and an enriched population of F1 hybrid wheat seed.

[0176]

[0177] Embodiment 57. The method of embodiment 56, the method further comprising:

[0178] sorting the inbred pollinator wheat seed based on the size and / or shape of the inbred pollinator wheat seed .

[0179]

[0180] Embodiment 58. The method of embodiment 56, the method further comprising:

[0181] sorting the F1 hybrid wheat seed based on the size and / or shape of the F1 hybrid wheat seed.

[0182]

[0183] Embodiment 59. The method of any one of embodiments 56 to 58, comprising sorting the inbred pollinator wheat seed and the F1 hybrid wheat seed from one another based on seed width, seed thickness, or seed length.

[0184]

[0185] Embodiment 60. The method of embodiment 59, wherein sorting based on width or thickness comprises using a round-hole sieve or a slotted sieve with intermediate apertures selected from the range of 7.0 / 64 inch to 11.0 / 64 inch.

[0186]

[0187] Embodiment 61. The method of embodiment 60, wherein the round-hole or slotted sieve has a diameter or slot-width selected from the range of 6.5 / 64 inches to 11.5 / 64 inches, including all 0.5 / 64-inch increments therein, such as 6.5 / 64, 7.0 / 64, 7.5 / 64, 8.0 / 64, 8.5 / 64, 9.0 / 64, 9.5 / 64, 10.0 / 64, 10.5 / 64, 11.0 / 64, and 11.5 / 64 inches.

[0188]

[0189] Embodiment 62. The method of embodiment 59, wherein sorting based on length comprises using an indent cylinder, trieur, or disc separator with pocket diameters selected from the range of 12.0 / 64 inches to 24.0 / 64 inches, including all 0.5 / 64-inch increments therein in ascending order.

[0190]

[0191] Embodiment 63. The method of any one of embodiments 56, further comprising performing scalping or bottom screening or both as a pre-cleaning step prior to the sorting based on density, size, shape, or optical properties.

[0192]

[0193] Embodiment 64. The method of embodiment 63, wherein scalping comprises using sieves with sizes ranging from 12.0 / 64 inch to 24.0 / 64 inch (3 / 8 inch), and bottom screening comprises using sieves with apertures ranging from 4.0 / 64 inch to 6.0 / 64 inches.

[0194]

[0195] Embodiment 65. The method of embodiment 56, the method further comprising:

[0196] sorting the inbred pollinator wheat seed based on the size and / or shape of the inbred pollinator wheat seed using a threshold size and / or shape value.

[0197]

[0198] Embodiment 66. The method of embodiment 56, the method further comprising:

[0199] sorting the F1 hybrid wheat seed based on the size and / or shape of the F1 hybrid wheat seed using a threshold size and / or shape value.

[0200]

[0201] Embodiment 67. The method of embodiment 56, the method further comprising sorting the wheat seed based on size and / or shape using a gravity table, indent cylinder separator, screens, sieves, optical sorters, roller sorters, or combinations thereof.

[0202]

[0203] Embodiment 68. The method of embodiment 65 or 66, the method further comprising adjusting the threshold size and / or shape value of the inbred pollinator wheat seed or F1 hybrid wheat seed to improve the sorting efficiency.

[0204]

[0205] Embodiment 69. The method of embodiment 56, the method further comprising:

[0206] sorting the enriched population of F1 hybrid wheat seed using density-based sorting, for example, as described elsewhere herein and in Embodiments 1 to 8.

[0207]

[0208] Embodiment 70. The method of embodiment 56, the method further comprising:

[0209] sorting the enriched population of F1 hybrid wheat seed using optical sorting.

[0210]

[0211] Embodiment 71. The method of embodiment 70, the method further comprising:

[0212] using optical-based sorting to detect inbred pollinator wheat seed and separate the inbred pollinator wheat seed from the enriched population of F1 hybrid wheat seed.

[0213]

[0214] Embodiment 72. The method of embodiment 70 to 72, wherein the optical-based sorting is color-based sorting, the method further comprising using color-based sorting to detect the inbred pollinator wheat seed in the enriched population of F1 hybrid wheat seed based on the color of the inbred pollinator wheat seed .

[0215]

[0216] Embodiment 73. The method of embodiment 73, the method further comprising: using color-based sorting to detect the inbred pollinator wheat seed in the enriched population of F1 hybrid wheat seed based on the color of the inbred pollinator wheat seed, wherein the inbred pollinator wheat seed and F1 hybrid wheat seed comprise different number of copies of the dominant red allele from the R gene.

[0217]

[0218] Embodiment 74. The method of embodiment 73, the method further comprising: using color-based sorting to detect the inbred pollinator wheat seed in the enriched population of F1 hybrid wheat seed based on the color of the inbred pollinator wheat seed, wherein the inbred pollinator wheat seed and F1 hybrid wheat seed comprise different number of copies of the recessive white allele from the R gene.

[0219]

[0220] Embodiment 75. The method of embodiment 70 or 71 , the method further comprising:

[0221] using NIR or IR-based sorting to detect the inbred pollinator wheat seed in the enriched population of F1 hybrid wheat seed based on the color or composition of the inbred pollinator wheat seed.

[0222]

[0223] Embodiment 76. The method of embodiment 70 or 71 , the method further comprising:

[0224] sorting inbred pollinator wheat seed from the enriched population of F1 hybrid wheat seed using hyperspectral, NIR, IR, visible light, or combinations thereof.

[0225]

[0226] Embodiment 77. The method of embodiment 70 or 71 , the method further comprising:

[0227] sorting inbred pollinator wheat seed from the enriched population of F1 hybrid wheat seed using RGB.

[0228]

[0229] Embodiment 78. The method of embodiment 70 or 71 , the method further comprising:

[0230] sorting the enriched population of F1 hybrid wheat seed using NIR or IR-based sorting based on the color or composition of the F1 hybrid wheat seed to further enrich the F1 hybrid wheat seed population.

[0231]

[0232] Embodiment 79. The method of embodiment 70 or 71 , the method further comprising:

[0233] sorting the enriched population of F1 hybrid wheat seed using hyperspectral, NIR, IR, visible light, or combinations thereof to further enrich the F1 hybrid wheat seed population.

[0234]

[0235] Embodiment 80. The method of embodiment 70 or 71 , the method further comprising:

[0236] sorting the enriched population of F1 hybrid wheat seed using RGB to further enrich the F1 hybrid wheat seed population.

[0237]

[0238] Embodiment 81. The method of any of the embodiments from 56 to 80 , the method further comprising: collecting or retaining the F1 hybridwheat seed from the sorting or separation step to enrich the F1 hybrid wheat seed population.

[0239]

[0240] Embodiment 82. The method of any one of embodiments 70 to 81 , the method further comprising:

[0241] subjecting the enriched population of F1 hybrid wheat seed to multiple passes of optical-based sorting (including but not limited to hyperspectral, NIR, IR, visible light, and / or RGB or combinations thereof) until the desired level of hybridity for the enriched population of F1 hybrid wheat seed and / or F1 hybrid wheat seed recovery relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting is achieved.

[0242]

[0243] Embodiment 83. The method of any one of embodiments 56, 69, or 70, the method further comprising: (a) sorting the mixture of inbred pollinator wheat seed and F1 hybrid wheat seed based on seed size or seed shape to enrich the population of F1 hybrid wheat seed; (b) sorting the enriched population of F1 hybrid wheat seed using optical sorting; and (c) sorting the enriched population of F1 hybrid wheat seed based on density; wherein steps (b) and (c) are performed in any order following step (a) to produce a further enriched population of F1 hybrid wheat seed.

[0244]

[0245] Embodiment 84. The method of any one of embodiments 69 or 70 or 83, wherein two or more of the sorting based on density, the sorting based on size and / or shape, and the sorting based on an optical property are performed concurrently or simulataneously to produce the final enriched population of F1 hybrid wheat seed.

[0246]

[0247] Embodiment 85. The method of any one of embodiments from 56 to 84, the method further comprising: separating inbred pollinator wheat seed from the mixture of inbred pollinator wheat seed and F1 hybrid wheat seed or the enriched F1 hybrid seed population or enriched inbred pollinator population as the case may be by performing additional sorting passes or steps.

[0248]

[0249] Embodiment 86. The method of embodiment 56 or 85, the method further comprising: retaining the enriched population of inbred pollinator wheat seed and re-sorting the enriched inbred pollinator wheat seed population using the same or different sorting technology to identify and recover additional F1 hybrid wheat seeds contained therein.

[0250]

[0251] Embodiment 87. The method of embodiment 86, the method further comprising: combining the recovered F1 hybrid wheat seeds with the enriched population of F1 hybrid wheat seed or with a subsequent population of F1 hybrid wheat seed after said population has been subjected to one or more additional sorting steps using the same or different sorting technology to increase total F1 hybrid wheat seed recovery.

[0252]

[0253] Embodiment 88. The method of embodiment 86 or 87, comprising recovering additional F1 hybrid seed from the inbred population to increase total F1 hybrid wheat seed recovery by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or higher.

[0254]

[0255] Embodiment 89. The method of embodiment 82, the method further comprising: separating by each pass inbred pollinator wheat seed from the enriched population of F1 hybrid wheat seed.

[0256]

[0257] Embodiment 90. The method of embodiment 56, the method comprising interplanting seeds of a male-sterile female wheat parent plant and a male-fertile pollinator wheat parent in a field to produce a mixed population of wheat plants, wherein the mixture of wheat seeds is obtained therefrom.

[0258]

[0259] Embodiment 91. The method of embodiment 90, the method comprising selecting a mitochondrial-genome based or nuclear-genome based male-sterile female wheat parent line and a male-fertile pollinatorwheat parent line, wherein said parent lines have different genotypes at one or more R gene loci controlling seed color.

[0260]

[0261] Embodiment 92. The method of embodiment 91 , the method comprising selecting a mitochondrial-genome based or a nuclear-genome based male-sterile female wheat parent line and a male-fertile pollinator wheat parent line, wherein said parent lines have different genotypes at one or more R gene loci controlling seed color, wherein the different genotypes confer a detectable difference in seed color phenotype between the resulting F1 hybrid wheat seed produced on the female parent and self-pollinated male-fertile pollinator wheat parent line.

[0262]

[0263] Embodiment 93. The method of embodiment 90, where the interplanting comprises planting a seed blend comprising from about 1% to about 30% by seed count or seed weight of the male-fertile pollinator wheat parent line and from about 70% to about 99% by seed count or seed weight of the male-sterile female wheat parent line seed, for example, where the interplanting comprises planting a seed blend comprising from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% by seed count or seed weight of the male-fertile pollinator wheat parent line seed and from about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by seed count or seed weight of the male-sterile female wheat parent line seed. In a non-limiting examples, the seed blend comprises from about 10% to about 30% by seed count or seed weight of the male-fertile pollinator wheat parent line seed and from about 70% to about 90% by seed count or seed weight of the male-sterile female wheat parent line seed, or from about 1 % to about 5% by seed count or seed weight of the male-fertile pollinator wheat parent line seed and from about 95% to about 99% by seed count or seed weight of the male-sterile female wheat parent line seed.

[0264]

[0265] Embodiment 94. The method of embodiment 90 to 93, the method further comprising: growing the seed into a mixed population of wheat plants, wherein the mixed population of wheat plants comprises male-sterile female wheat plants and male-fertile pollinator wheat plants.

[0266]

[0267] Embodiment 95. The method of embodiment 94, the method further comprising: allowing the male-fertile pollinator wheat plants to cross-pollinate the male-sterile female wheat plants to produce F1 hybrid wheat seed and male-fertile pollinator wheat plants to self-or sib-pol linate to produce inbred pollinator wheat seed.

[0268]

[0269] Embodiment 96. The method of embodiment 94 or 95, the method comprising harvesting wheat seed from the mixed population of wheat plants to obtain a seed mixture comprising F1 hybrid wheat seed and inbred pollinator wheat seed prior to sorting the seed.

[0270]

[0271] Embodiment 97. The method of embodiment 90, wherein the male-sterile female wheat parent line and the male-fertile pollinator wheat parent lines comprise different genotypes at one or more R gene loci controlling seed color.

[0272]

[0273] Embodiment 98. The method of embodiment 90, wherein the male-fertile pollinator wheat parent line is homozygous for recessive white alleles (r) at its R gene loci, and the male-sterile female parent line comprises at least one dominant red allele (R) at its R gene loci.

[0274]

[0275] Embodiment 99. The method of embodiment 90, wherein the male-sterile female parent line is homozygous for recessive white alleles (r) at its R gene loci, and the male-fertile pollinator wheat parent line comprises at least one dominant red allele (R) at its R gene loci.

[0276]

[0277] Embodiment 100. The method of any one of embodiment 98, optically sorting wheat seed comprising red F1 hybrid seed produced by the cross-pollination of male-sterile female wheat parent line with themale-fertile pollinator wheat parent line and white inbred pollinator wheat seed produced by self-pollination of the male-fertile pollinator wheat parent line.

[0278]

[0279] Embodiment 101. The method of any one of embodiments 99, optically sorting wheat seed comprising white F1 hybrid wheat seed produced by the cross-pollination of male-sterile female wheat parent line with the male-fertile pollinator wheat parent line and red inbred pollinator wheat seed produced by self-pollination of the male-fertile pollinator wheat parent line.

[0280]

[0281] Embodiment 102. The method of embodiment 70, wherein the optical sorting produces an enriched population of F1 hybrid wheat seed comprising a hybridity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0282]

[0283] Embodiment 103. The method of embodiment 56 or 69 or 70, wherein the sorting comprises adjusting one or more sorting parameters to produce a resulting population of F1 hybrid wheat seed comprising a hybridity of at least 70% and a F1 hybrid wheat seed recovery of at least 45% relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting. For example, where the resulting population of F1 hybrid wheat seed comprising a hybridity of at least 70%, 71%, 72%, 73%, 74% 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% and a F1 hybrid wheat seed recovery of at least 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting. In a non-limiting example, the resulting population of F1 hybrid wheat seed comprises a hybridity from about 75%to about 85% and a F1 hybrid wheat seed recovery from about 45% to about 65% relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting, or a hybridity from about 91 % to about 100% and a F1 hybrid wheat seed recovery from about 66% to about 100% relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting.

[0284]

[0285] Embodiment 104. The method of embodiment 56 or 69 or 70, wherein the combination of the size, shape, and / or density sorting step and the optical color sorting step results in an increased enriched population of F1 hybrid wheat seed with less than 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% inbred pollinator seed.

[0286]

[0287] Embodiment 105. The method of embodiment 56 or 69 or 70 or 104, wherein the one or more sorting parameters includes but is not limited to a density threshold, a size threshold, a sorter speed, or a color sensitivity setting.

[0288]

[0289] Embodiment 106. The method of any of the embodiments of 56 to 89 or 105, wherein the sorting results in a enriched population of F1 hybrid wheat seed comprising a hybridity of at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0290]

[0291] Embodiment 107. The method of any one of embodiments 56-106, wherein the F1 hybrid wheat seed is produced by pollen from the male-fertile pollinator wheat parent line fertilizing the nuclear-genome based male-sterile female wheat parent line, where the male-sterility mechanism resides in the nuclear genome rather than the mitochondria. For example, the nuclear-genome based male-sterile female wheat parent line may be male-sterile for a nuclear male fertility gene, including but not limited to,Ms1, Ms2, Ms3, Ms5, Ms9, Ms22, Ms26, or Ms45. As a non-limiting example, the nuclear-based male-sterile female may be rendered sterile using any suitable approach including chemical mutagenesis or genome editing of an endogenous nuclear male fertility gene, e.g., Ms1 , Ms2, Ms3, Ms5, Ms9, Ms22, Ms26, or Ms45 male fertility genes, on each of the A, B, and D genomes to render the male-sterile female plant homozygous recessive for the male fertility gene, see, for example, published patent application See, published patent application US 20190177722 and US Patent No. 12,054,732 each of which is herein incorporated by reference each in its entirety. Since Ms1 in wheat behaves as a single gene recessive, in some embodiments, only the Ms1 male-fertility polynucleotide or allele located on chromosome 4BS may need to be mutated to confer male-sterility to a wheat plant. Accordingly, in some examples, with respect to Ms1, genome-editing or mutating the endogenous Ms1 male fertility gene on the B genome may be sufficient to render the nuclear-based male-sterile female parent male sterile. See, published patent application W02020056259. The nuclear-based male-sterile female parent may be increased using a hybrid wheat production platform, including those described published patent application W02020056259, or WO2019043082, each of which is herein incorporated by reference each in its entirety.

[0292]

[0293] Embodiment 108. A method of obtaining an enriched population of F1 hybrid wheat seed, the method comprising:

[0294] (a) providing a hybrid wheat seed mixture comprising F1 hybrid wheat seed and inbred pollinator wheat seed; and

[0295] (b) separating the F1 hybrid wheat seed and inbred pollinator wheat seed from one another by optically sorting the seed mixture based on at least one identified phenotype for either the F1 hybrid wheat seed or the inbred pollinator wheat seed to produce the enriched population of F1 hybrid wheat seed.

[0296]

[0297] Embodiment 109. The method of embodiment 108, the method further comprising: analyzing wheat seeds from the seed mixture using anoptical sorting apparatus to identify at least one morphological, compositional, or spectral phenotype for the F1 hybrid wheat seed, inbred pollinator wheat seed, or both.

[0298]

[0299] Embodiment 110. The method of embodiment 109, wherein the at least one identified phenotype is a morphological, compositional, or spectral phenotype that distinguishes the F1 hybrid wheat seed and the inbred pollinator wheat seed from one another.

[0300]

[0301] Embodiment 111. The method of embodiment 109 or 110, wherein the at least one morphological phenotype comprises a physical geometry comprising seed length, seed width, seed surface area, calculated seed volume, seed circularity, or aspect ratio.

[0302]

[0303] Embodiment 112. The method of embodiment 108 or 110, wherein the at least one morphological phenotype identifies the F1 hybrid wheat seed based on a higher surface area or volume relative to the inbred pollinator wheat seed.

[0304]

[0305] Embodiment 113. The method of embodiment 110, wherein the at least one compositional phenotype comprises an infrared (IR) or nearinfrared (NIR) spectral signature indicative of an internal chemical difference between the F1 hybrid wheat seed and the inbred pollinator wheat seed.

[0306]

[0307] Embodiment 114. The method of embodiment 113, wherein the internal chemical difference comprises a difference in protein content, starch composition, lipid content, or moisture content in the F1 hybrid wheat seed.

[0308]

[0309] Embodiment 115. The method of embodiment 110, wherein the at least one spectral phenotype comprises visible color intensity, spectral reflectance, or a unique spectral signature resulting from different copy numbers of a dominant red allele (R) or a recessive white allele (r) at oneor more R gene loci in the F1 hybrid wheat seed and the inbred pollinator wheat seed.

[0310]

[0311] Embodiment 116. The method of embodiment 110 or 115, wherein the at least one spectral phenotype identifies a color or compositional gradient indicative of the genetic dosage of R gene alleles unique to the specific parental lines of the inbred pollinator wheat seed and the resulting F1 hybrid wheat seed.

[0312]

[0313] Embodiment 117. The method of any one of embodiments 108 to 116, wherein the separation is based on a combination of at least two or more of the morphological, compositional, and spectral phenotypes.

[0314]

[0315] Embodiment 118. The method of embodiment 115 or 116, wherein the inbred pollinator wheat seed and F1 hybrid wheat seed comprise different number of copies of the dominant red allele from the R gene.

[0316]

[0317] Embodiment 119. The method of embodiment 115 to 118, wherein the inbred pollinator wheat seed and F1 hybrid wheat seed comprise different number of copies of the recessive white allele from the R gene.

[0318]

[0319] Embodiment 120. The method of embodiment 108, further comprising prior to providing the wheat seed mixture:(a) planting seeds of a male-sterile female wheat parent line and seeds of a male-fertile pollinator wheat parent line;(b) growing the seeds into plants; and(c) allowing the plants to pollinate such that the male-fertile pollinator wheat plants cross-pollinate the male-sterile female wheat plants to produce F1 hybrid wheat seed and intra-pollinate, including self-pollinate or sib-poll inate, to produce inbred pollinator wheat seed, wherein, when harvested, gives rises to the wheat seed mixture comprising F1 hybrid wheat seed and inbred pollinator wheat seed.

[0320]

[0321] Embodiment 121. The method of embodiment 120, the method further comprising:

[0322] planting seeds of a male-sterile female wheat parent line and seeds of a male-fertile pollinator wheat parent line in a field in an arrangement comprising a seed blend (interplanting), intermixed rows, or strips to produce a population of wheat plants comprising: wheat seed mixture comprising F1 hybrid wheat seed and inbred pollinator wheat seed.

[0323]

[0324] Embodiment 122. The method of embodiment 120 or 121, further comprising:

[0325] planting a seed blend comprising from about 1% to about 30% by seed count or seed weight of the male-fertile pollinator wheat parent line and from about 70% to about 99% by seed count or seed weight of the male-sterile female wheat parent line seed, for example, where the interplanting comprises planting a seed blend comprising from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% by seed count or seed weight of the male-fertile pollinator wheat parent line and from about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by seed count or seed weight of the male-sterile female wheat parent line seed. In a non-limiting example, the seed blend comprises from about 10% to about 30% by seed count or seed weight of the male-fertile pollinator wheat parent line seed and from about 70% to about 90% by seed count or seed weight of the male-sterile female wheat parent line seed. In a non-limiting example, the seed blend comprises from about 10% to about 30% by seed count or seed weight of the male-fertile pollinator wheat parent line seed and from about 70% to about 90% by seed count or seed weight of the male-sterile female wheat parent line seed, or from about 1 % to about 5% by seed count or seed weight of the male-fertile pollinator wheat parent line seed and from about 95% to about 99% by seed count or seed weight of the male-sterile female wheat parent line seed.

[0326]

[0327] Embodiment 123. The method of any of the embodiments of 120 to 122, the method further comprising comprising selecting a male-sterile female wheat parent line and a male-fertile pollinator wheat parent line, wherein said parent lines have different genotypes at one or more R gene loci controlling seed color, wherein the different genotypes confer a detectable difference in seed color phenotype between the resulting F1 hybrid wheat seed produced on the female parent and self-pollinated male-fertile pollinator wheat parent line.

[0328]

[0329] Embodiment 124. The method of any of the embodiments of 120 to 122, wherein the male-fertile pollinator wheat parent line is homozygous for recessive white alleles (r) at its R gene loci, and the male-sterile female parent line comprises at least one dominant red allele (R) at its R gene loci or wherein the male-sterile female parent line is homozygous for recessive white alleles (r) at its R gene loci, and the male-fertile pollinator wheat parent line comprises at least one dominant red allele (R) at its R gene loci or wherein the male-fertile pollinator wheat parent line is homozygous for recessive white alleles (r) at its R gene loci, and the male-sterile female parent line comprises at least one dominant red allele (R) at its R gene loci.

[0330]

[0331] Embodiment 125. The method of embodiment 124, the method comprising:

[0332] the method comprising harvesting wheat seed to obtain a wheat seed mixture comprising F1 hybrid wheat seed and inbred pollinator wheat seed, wherein: (a) the F1 hybrid wheat seed is red when the male-sterile female wheat parent line comprises at least one dominant red allele (R) and the inbred pollinator wheat seed is white when the male-fertile pollinator wheat parent line is homozygous for recessive white alleles (rr); or (b) the F1 hybrid wheat seed is white when the male-sterile female wheat parent line is homozygous for recessive white alleles (rr) and the inbred pollinator wheat seed is red when the male-fertile pollinator wheat parent line comprises at least one dominant red allele (R).

[0333]

[0334] Embodiment 126. The method of embodiment 108, further comprising using NIR, IR, hyperspectral imaging, or RGB-based sorting to detect the inbred pollinator wheat seed in the mixture.

[0335]

[0336] Embodiment 127. The method of embodiment 126, the method further comprising using NIR or IR-based sorting to detect the inbred pollinator wheat seed in the enriched population of F1 hybrid wheat seed based on the color or composition of the inbred pollinator wheat seed.

[0337]

[0338] Embodiment 128. The method of embodiment 108, the method further comprising sorting inbred pollinator wheat seed from the enriched population of F1 hybrid wheat seed using hyperspectral, NIR, IR, visible light, or combinations thereof.

[0339]

[0340] Embodiment 129. The method of embodiment 108, the method further comprising sorting inbred pollinator wheat seed from the enriched population of F1 hybrid wheat seed using RGB.

[0341]

[0342] Embodiment 130. The method of embodiment 108, wherein the specific detection mechanisms employed are dependent on the sorting technology used, wherein RGB sorting evaluates visible color intensity, and hyperspectral, IR, and NIR sorting analyze spectral reflectance or unique spectral signatures to differentiate the F1 hybrid wheat seeds from the inbred pollinator wheat seeds.

[0343]

[0344] Embodiment 131. The method of embodiment 109 or 110, the method further comprising: identifying the morphological, compositional, or spectral phenotype through a training step using machine learning to analyze known subpopulations of inbred seeds and F1 hybrid wheat seeds.

[0345]

[0346] Embodiment 132. The method of embodiment 131, further comprising determining an optical sorting threshold for the known subpopulations by identifying a divergence point where the traitdistributions of the inbred pollinator and F1 hybrid populations are distinct based on a measurement of spectral reflectance, spectral signature, or color intensity.

[0347]

[0348] Embodiment 133. The method of embodiment 132, wherein the optical sorting threshold determination includes a training step to calibrate a sorting apparatus to the specific color or compositional gradient resulting from the R gene allele dosages or chemical composition unique to the specific parental lines and resulting F1 hybrid wheat seed being sorted.

[0349]

[0350] Embodiment 134. The method of embodiment 82 or 83, the method further comprising: adjusting optical sorting parameters, such as color sensitivity or detection thresholds, to improve sorting efficiency, for example, until the desired enriched F1 hybrid seed population hybridity and / or F1 hybrid wheat seed recovery is obtained.

[0351]

[0352] Embodiment 135. The method of embodiment 108, the method further comprising sorting the wheat seed mixture by a physical property prior to optically sorting the wheat seed.

[0353]

[0354] Embodiment 136. The method of embodiment 135, wherein the physical property includes but is not limited to seed size, seed shape, and / or seed density prior to optically sorting the wheat seed.

[0355]

[0356] Embodiment 137. The method of embodiment 135 or 136, the method comprising sorting by shape using one or more sieves to enrich the concentration of F1 hybrid seed prior to optical sorting.

[0357]

[0358] Embodiment 138. The method of embodiment 135 or 136, the method comprising sorting by size using one or more sieves to enrich the concentration of F1 hybrid seed prior to optical sorting.

[0359]

[0360] Embodiment 139. The method of embodiment 135 or 136, the method comprising sorting by density using a gravity table, a fluidized bedseparator, or hydrocyclone to enrich the concentration of F1 hybrid seed prior to optical sorting.

[0361]

[0362] Embodiment 140. The method of embodiment 136, the method comprising sorting the wheat seed mixture based on size and / or shape using an indent cylinder separator or roller sorters.

[0363]

[0364] Embodiment 141. The method of embodiment 136, the method further comprising sorting the wheat seed mixture using a threshold density, size, or shape value.

[0365]

[0366] Embodiment 142. The method of embodiment 141, the method further comprising adjusting the threshold density, size, or shape value of the wheat seed mixture to improve the sorting efficiency.

[0367]

[0368] Embodiment 143. The method of any one of embodiment 136, comprising sorting the inbred pollinator wheat seed and the F1 hybrid wheat seed from one another based on seed width, seed thickness, or seed length.

[0369]

[0370] Embodiment 144. The method of embodiment 108, the method further comprising subjecting the population or subpopulation of F1 hybrid wheat seed mixture to multiple passes of optical-based sorting (including but not limited to hyperspectral, NIR, IR, visible light, and / or RGB) until the desired level of hybridity for the population of F1 hybrid wheat seed and / or F1 hybrid wheat seed recovery relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting is achieved.

[0371]

[0372] Embodiment 145. The method of embodiment 144, the method further comprising: separating by each pass inbred pollinator wheat seed from the enriched population of F1 hybrid wheat seed.

[0373]

[0374] Embodiment 146. The method of embodiment 108, the method further comprising retaining the enriched population of inbred pollinatorwheat seed and re-sorting the enriched inbred pollinator wheat seed population using the same or different sorting technology to identify and recover additional F1 hybrid wheat seeds contained therein.

[0375]

[0376] Embodiment 147. The method of embodiment 146, the method further comprising

[0377] combining the recovered F1 hybrid wheat seeds with the enriched population of F1 hybrid wheat seed or with a subpopulation of F1 hybrid wheat seed after said population has been subjected to one or more additional sorting steps using the same or different sorting technology to increase total F1 hybrid wheat seed recovery.

[0378]

[0379] Embodiment 148. The method of embodiment 146 or 147, comprising recovering additional F1 hybrid seed from the inbred population to increase total F1 hybrid wheat seed recovery by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or higher.

[0380]

[0381] Embodiment 149. The method of embodiment 108, 147, or 148, the method further comprising:

[0382] (a) sorting the mixture of inbred pollinator wheat seed and F1 hybrid wheat seed using optical sorting to enrich the population of F1 hybrid wheat seed;

[0383] (b) sorting the population of F1 hybrid wheat seed based on seed size or seed shape or combinations thereof; and

[0384] (c) sorting the population of F1 hybrid wheat seed based on density; wherein steps (b) and (c) are performed in any order following step (a) to produce a further enriched population of F1 hybrid wheat seed.

[0385]

[0386] Embodiment 150. The method of any one of embodiments of 136 to 145 or 158, the method comprising performing any two or more sorting steps comprising sorting based on seed density, sorting based on seed size and / or seed shape or combinations thereof, and / or sorting based on an optical property simultaneously in a single sorting step using a singlesorting apparatus to produce a final enriched population of F1 hybrid wheat seed.

[0387]

[0388] Embodiment 151. The method of any of any one of embodiments 136 to 145, wherein the optical sorting and the sorting based on size and / or shape are performed simultaneously in a single sorting step using a single sorting device.

[0389]

[0390] Embodiment 152. The method of embodiment 69, wherein the enriched population of F1 hybrid wheat seed has a hybridity of at least 70%, 71%, 72%, 73%, 74% 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0391]

[0392] Embodiment 153. The method of embodiment 136 to 145, wherein the combination of the size, shape, and / or density sorting step and the optical color sorting step results in an enriched population of F1 hybrid wheat seed with less than 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% inbred pollinator seed.

[0393]

[0394] Embodiment 154. The method of embodiment 108 or 136 to 145, wherein the sorting comprises adjusting one or more sorting parameters to produce a resulting population of F1 hybrid wheat seed comprising a hybridity of at least 70% and a F1 hybrid wheat seed recovery of at least 45% relative to the total amount of F1 hybrid wheat seed present in the mixture, population, or subpopulation prior to sorting.

[0395]

[0396] Embodiment 155. The method of embodiment 154, wherein the resulting population of F1 hybrid wheat seed comprises a hybridity of at least 70%, 71%, 72%, 73%, 74% 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% and a F1 hybrid wheat seed recovery of at least 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting. In a non-limiting example, the resulting population of F1 hybrid wheat seed comprises a hybridity from about 75% to about 85% and a F1 hybrid wheat seed recovery from about 45% to about 65% relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting, or a hybridity from about 91% to about 100% and a F1 hybrid wheat seed recovery from about 66% to about 100% relative to the total amount of F1 hybrid wheat seed present in the mixture prior to sorting.

[0397]

[0398] Embodiment 156. The method of any one of the preceding embodiments, wherein one or more additional sorting steps comprises using a different sorting technology to sort based on the same physical property as a previous sorting step, wherein the physical property is selected from the group consisting of: size, shape, or density.

[0399]

[0400] Embodiment 157. The method of any one of the embodiments 108 to 156, wherein the F1 hybrid wheat seed is produced by pollen from the male-fertile pollinator wheat parent line fertilizing the nuclear-genome based male-sterile female wheat parent line, where the male-sterility mechanism resides in the nuclear genome rather than the mitochondria. For example, the nuclear-genome based male-sterile female wheat parent line may be male-sterile for a nuclear male fertility gene, including but not limited to, Ms1, Ms2, Ms3, Ms5, Ms9, Ms22, Ms26, or Ms45. As a nonlimiting example, the nuclear-based male-sterile female may be rendered sterile using any suitable approach including chemical mutagenesis or genome editing of an endogenous nuclear male fertility gene, e.g., Ms1 , Ms2, Ms3, Ms5, Ms9, Ms22, Ms26, or Ms45 male fertility genes, on each of the A, B, and D genomes to render the male-sterile female plant homozygous recessive for the male fertility gene, see, for example, published patent application See, published patent application US20190177722 and US Patent No. 12,054,732 each of which is herein incorporated by reference each in its entirety. Since Ms1 in wheat behaves as a single gene recessive, in some embodiments, only the Ms1 male-fertility polynucleotide or allele located on chromosome 4BS may need to be mutated to confer male-sterility to a wheat plant. Accordingly, in some examples, with respect to Ms1, genome-editing or mutating the endogenous Ms1 male fertility gene on the B genome may be sufficient to render the nuclear-based male-sterile female parent male sterile. See, published patent application W02020056259. The nuclear-based male-sterile female parent may be increased using a hybrid wheat production platform, including those described published patent application W02020056259, or WO2019043082, each of which is herein incorporated by reference each in its entirety.

[0401]

[0402] Embodiment 158. The method of any of the embodiments 1 -157, wherein the F1 hybrid wheat seed produced using nuclear-genome male sterility system system is sorted from inbred pollinator wheat seed on the basis that the F1 hybrid wheat seed has a larger size, a distinct shape, a higher density, or a higher thousand kernel weight (TKW) relative to the inbred pollinator wheat seed.

[0403]

[0404] Embodiment 159. The method of any of the preceding embodiments of 1-54, 56-106, or 109-157, where the F1 hybrid wheat seed is produced using cytoplasmic male sterility system (CMS) system.

[0405]

[0406] Embodiment 160. The method of any of the preceding embodiments of 1-54, 56-106, 109-157, or 159, wherein the F1 hybrid wheat seed produced using cytoplasmic male sterility system (CMS) system is sorted from inbred pollinator wheat seed on the basis that the F1 hybrid wheat seed has a smaller size, a distinct shape, a lower density, or a lower thousand kernel weight (TKW) relative to the inbred pollinator wheat seed.

[0407]

[0408] Embodiment 161. The method of any of the preceding embodiments, wherein the F1 hybrid wheat seed and inbred pollinator wheat seed are separated from one another using a directed pneumatic force, including but not limited to a pulse or plurality of pulses of air.

[0409]

[0410] Embodiment 162. The method of any of the preceding embodiments, wherein the sorting is automated, high-throughput or both.

[0411] Embodiment 163. The method of any of the preceding embodiments, wherein the seed-size, seed shape, or seed density-based sorting is contactless with respect to a solid mechanical barrier, where the solid mechanical barrier includes but is not limited to a sieve, screen, or gate.

[0412]

[0413] Embodiment 164. The method of any of the preceding embodiments, wherein the F1 hybrid wheat seed and inbred pollinator wheat seed are separated from one another while free-falling or in-flight using a directed pneumatic pulse.

[0414]

[0415] Embodiment 165. The method of any of the preceding embodiments, wherein the seed-size, seed shape, or seed density-based sorting is not performed using mechanical sorting.

[0416]

[0417] Embodiment 166. The method of any of the preceding embodiments, wherein the seed-size, seed shape, or seed density-based sorting is not performed using a mechanical sieve, a screen, or gravity table.

[0418]

[0419] Embodiment 167. The method of any of the preceding embodiments, wherein the wheat seed mixture comprises Soft Red Winter Wheat or Hard Red Winter Wheat or Soft White Winter Wheat or Hard White Winter Wheat.

[0420]

[0421] Embodiment 168. A system for sorting wheat seed comprising a mixture of inbred pollinator wheat seed and F1 hybrid wheat seed, the system comprising:

[0422] at least one or more sorting apparatus configured to perform one or more of the sorting operations in embodiments 1-167.

[0423]

[0424] Embodiment 169. A system for sorting wheat seed comprising a mixture of inbred pollinator wheat seed and F1 hybrid wheat seed, the system comprising:

[0425] at least one or more sorting apparatus configured to distinguish F1 hybrid wheat seed from inbred pollinator wheat seed based on at least one morphological, compositional, or spectral characteristic.

[0426]

[0427] Embodiment 170. The system of Embodiment 169, wherein the at least one sorting apparatus comprises a density-sorting apparatus or an optical sorting apparatus.

[0428]

[0429] Embodiment 171. The system of any of Embodiments 168-169, wherein the optical sorting apparatus comprises one or more RGB, visible-light, infrared (IR), near-infrared (NIR), or hyperspectral sensors.

[0430]

[0431] Embodiment 172. The system of any of Embodiments 168-170, wherein the phenotypic, compositional, or spectral characteristic comprises seed color, color intensity, spectral reflectance, moisture content, protein content, lipid content, calculated seed volume, circularity, or aspect ratio, or a combination thereof.

[0432]

[0433] Embodiment 173. The system of any of Embodiments 168-171, wherein the density-sorting apparatus comprises a gravity table, terminal velocity sorter, aspirator, column separator, fluidized bed separator, or hydrocyclone.

[0434]

[0435] Embodiment 174. The system of any of Embodiments 168-172, further comprising a controller programmed to adjust at least one sortingparameter selected from a density threshold, a size threshold, a shape threshold, a spectral detection threshold, or a color sensitivity value.

[0436]

[0437] Embodiment 175. The system of any of Embodiments 168-173, wherein the system is configured to perform one or more sorting steps concurrently.

[0438] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a seed” is understood to represent one or more seeds. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0439] Throughout this specification and the claims, the words “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise.

[0440]

[0441] EXAMPLES

[0442] The present disclosure is further illustrated in the following Examples. It should be understood that these Examples, while indicating embodiments of the invention, are given by way of illustration only.

[0443] Example 1 : Utilization of density sorting in wheat for F1 hybrid seed production

[0444] One method for separating seeds is based on the difference in density between F1 hybrid and inbred wheat seeds (inbred pollinator wheat seed) using density sorting. Thousand Kernel Weight (TKW) is higher for F1 hybrid seeds of Ms45 Soft Red Winter Wheat (FIG 2A) and Hard Red Winter Wheat (FIG 2C). In contrast, for tCMS Hard Red Winter Wheat, inbred seed has a higher TKW (FIG 2D). Furthermore, test weight, a measure of density measured in pounds per bushel, is also higher for F1 hybrid wheat seeds than for inbred male wheat seeds (FIG 2B). Typically, inbred wheat seeds have a thousand seed weight of 32-35 g and F1 hybrid wheat seeds have a thousand seed weight of 38 - 45 g, although different inbred and hybrid wheat varieties may have weights outside of these ranges.

[0445] To separate seeds based on density, a gravity sorter is used. In this example, the Cimbria GA31 gravity sorter is used, although other machines or technologies capable of sorting seeds by density may also be used such as a terminal velocity sorter, aspirator, or column separator.

[0446] Density filtering can be performed multiple times with the same or different cutoffs and settings to achieve the desired purity. Once the desired purity is reached, F1 hybrid wheat seeds may be packaged for distribution. Alternatively, this method may be combined with other sorting methods described in Example 2 and Example 3 for improved performance (see Example 4).

[0447] Example 1A

[0448] The methods described in Example 1 were applied to a seed source from a field planted with 80% a Ms45 male-sterile female wheat parent line and 20% male seeds (male-fertile pollinator wheat parent line). This trial resulted in a seed harvest with 51% hybridity.

[0449] Hybridity was measured using 12 TaqMan markers. Marker purity was first measured for each inbred parent, and then 92 seeds were genotyped for each seed population to determine the percentage of heterozygous seeds, male seeds, inbred seeds, or unknown seeds.

[0450] Gravity sorting was performed on the Cimbria GA31 system.Gravity sorting resulted in three seed populations: 1 Gravity (heaviest), 2 Gravity (intermediate), and 3 Gravity (lightest). The heaviest gravity population showed an increase in the percentage of F1 hybrid seeds, the intermediate population showed a marginal increase in F1 hybrid wheat seeds, and the lightest population showed a decrease in F1 hybrid wheat seeds (Table 1). This demonstrates that density can be used to enrich a seed population for F1 hybrid wheat seeds.

[0451]

[0452] Table 1 : Hybridity of seeds sorted by density.

[0453] Example 2: Utilizing optical sorting in wheat for male removal in F1 hybrid seed production

[0454] Optical color sorting can be used to separate F1 hybrid seeds from inbred seeds. Optical color sorting relies on visual differences in color and / or shape between inbred and F1 hybrid wheat seeds that can be detected by RGB, IR, including NIR, and / or hyperspectral imaging. In wheat, the R gene is the main driver of seed color. Wheat is genetically hexapioid with one copy of the red (R) gene on chromosome 3 of each sub-genome A, B, and D. Each red gene has two alleles, a recessive white allele and a dominant red allele. Each red allele contributes additively to the red color so that genotypes that are homozygous dominant at all three loci have the darkest red color, those that are homozygous recessive at all three loci have a white seed color, and seeds with intermediate copy numbers of the dominant red allele have intermediate red colors, resulting in a gradient of color dependent on R-gene genotype. Optical sorting may be used to distinguish between different copy numbers of dominant red alleles resulting from crosses between male and female genotypes with different copy numbers of the dominant red alleles at R genes.

[0455] Optical color sorting may be performed using SATAKE Optical Sorting Technology, although other platforms capable of sorting seeds by color may also be used. To train the SATAKE machine to separate seeds by color, the operator first inputs two populations of seeds: one from inbred pollinator wheat seeds and one from F1 hybrid sources. The inbred pollinator wheat seeds may be sourced from sorting using automated or manual methods. The machine uses internal machine learning software to identify color differences, and the machine is calibrated to enable sorting. The operator then loads the machine with the seed mixture to sort. F1 hybrid wheat seeds are retained, and inbred pollinator wheat seeds are discarded.

[0456] Optical color sorting may be repeated as many times as desired to achieve target purity. Once the desired purity is reached, F1 hybrid seeds may be packaged for distribution. Alternatively, this method may becombined with other sorting methods described in Example 1 and Example 3 for improved performance (see Example 4).

[0457]

[0458] Example 2A

[0459] In another experiment, color sorting was also performed using the Satake Enpresor sorter on three different speed settings. Sorting was performed in two passes to demonstrate the improvement in hybridity compared to a single pass. Hybridity was determined using markers as described in Example 1A.

[0460] The starting seed lot was a mixture of F1 hybrid wheat seeds and inbred pollinator wheat seeds that had a hybridity of 55% for F1 hybrid wheat seeds, and with a first pass of sorting, the hybridity increased to 66% for slow and medium sorting and to 56% for fast sorting. After a second round of sorting, the highest performing speed, slow, increased to 76% hybridity, with more moderate increases observed for other speed settings (Table 2).

[0461] F1 hybrid wheat seed recovery, a measure of the percentage of F1 hybrid wheat seeds remaining in the sorted seed mixture, showed higher rates of conversion for faster sorting speeds than slower sorting speeds, with only 45% of the initial F1 hybrid wheat seeds retained in the 2-pass slow sort.

[0462]

[0463] Table 2: F1 Seed sorting results for two rounds of optical sorting

[0464] Example 2B

[0465] The efficacy of optical sorting in differentiating between various dosages of the dominant Red allele in wheat seed populations was evaluated, simulating the separation of F1 hybrid wheat seeds from pollinator inbred wheat seeds using specific varieties as proxies.

[0466] In this experiment, optical sorting was applied to seed populations with known mixtures ranging from 10 to 30% male seed. Two different genetic combinations were made to determine if color sorting could distinguish between varying levels of red pigment / various dosages of the dominant R allele. In a Red / Red mixture, Shelly, a varietal wheat serving as a proxy for the male pollinator, contained one copy of the dominant R allele and Faller, a varietal wheat serving as a proxy for F1 hybrid wheat seed, contained three copies of the dominant R allele. (FIG. 3A)

[0467] In the Red / White mixture, Inbred A, a varietal wheat serving as a proxy for the male pollinator, contained zero copies of the dominant R allele and was mixed with Inbred B, a varietal wheat serving as a proxy for the F1 hybrid wheat seed, which contained two copies of the dominant R allele. (FIG. 3B) The seed populations were sorted through a Satake Evolution optical sorter either one or two times. After each sorting pass, the percentage of Inbred B seeds remaining in the population was verified using genetic markers.

[0468] Optical sorting resulted in a maximum of 95% Faller seeds in the 10 and 20% Red / Red mix scenarios. A single pass of sorting on the Red / White mixture captured 95% of Inbred B, the varietal wheat serving as F1 hybrid wheat seed proxy. (Table 2B). These results demonstrate that color sorting can be used for multiple color allele combinations.

[0469] Table 2B: Optical seed sorting results for different genetic combinations of red alleles.Male Sorting Percent PercentAllelesseed step hybrid maleRed / Red 10% Pass 1 95 5Red / Red 20% Pass 1 94 6Red / Red 20% Pass 2 95 5Red / Red 30% Pass 1 71 29Red / Red 30% Pass 2 84 16Red / White 10% Pass 1 95 5Red / White 20% Pass 1 91 9Red / White 30% Pass 1 90 10

[0470] Example 3: Utilizing size sorting in wheat for F1 hybrid seed production

[0471] In some cases, inbred (pollinator inbred) and F1 hybrid wheat seeds may be sufficiently different in size to allow for size separation. In this example, size separation is performed by sieves, although other methods of size and / or shape separation may also be implemented.

[0472] In this example, three sieves are used, with round hole sizes of 3 / 8 inch, 12 / 64 inch, and 5 / 64 inch, although other hole sizes and shapes may also be used to optimize seed separation. The sieves are stacked so that larger seeds remain on higher levels and smaller seeds and other contaminants fall through one or more sieve levels. Seeds with the desired size are retained, and contaminant seeds are discarded.

[0473] Size sorting may be repeated as many times as desired to achieve target hybridity. Once the desired hybridity is reached, F1 hybrid seeds may be packaged for distribution. Alternatively, this method may be combined with other sorting methods described in Example 1 and Example 2 for optimal performance.

[0474]

[0475] Example 3A

[0476] The methods described in Example 3 were applied to F1 hybrid wheat seed populations harvested from fields ranging from 10% to 30% male (male fertile pollinator wheat parent line) planted, where the pollinated male-sterile female wheat parent line was a Ms45 male-sterile female. Size separation was performed on each seed population by passing seeds through an 8.5 / 64 round sieve and keeping the large seed fraction. Hybridity was determined using genetic markers as described inExample 1A. Size sorting increased the marker-measured hybridity of the seed population from 13-30% to 27-66%, with the highest hybridity achieved in the populations with 10% male (male fertile pollinator wheat parent line) planted (Table 3). After size sorting, the F1 hybrid wheat seed from all planting ratios had a higher thousand kernel weight (TKW) than the pollinator inbred wheat seed (Table 4), further demonstrating the effectiveness of this method.

[0477] Table 3: Hybridity of F1 wheat seed sorted by size

[0478] Table 4: Thousand Kernel Weight (TKW) of seeds sorted by size.

[0479] Example 4: Two-types of seed sorting for improved seed hybridity

[0480] To optimize sorting for F1 hybrid seed hybridity, seed sortingprocess can be implemented. This may include any combination of sorting methods described in Examples 1-3 (FIG 1). In this example, a two-part method composed of first density sorting as described in Example 1 andthen color sorting as described in Example 2 is implemented (FIG 4).

[0481] First, gravity sorting as described in Example 1 is conducted. The weight cutoff is optimized for the specific seed source in order to bestretain heavier F1 hybrid wheat seeds (e.g., for F1 hybrid wheat seeds produced when using a nuclear-genome male-sterile female as the female wheat parent line) and exclude lighter inbred wheat seed contamination(inbred pollinator wheat seed). An aliquot of discarded lightweight inbred seeds is retained to train the color sorter.

[0482] The second sorting portion is performed by an optical color sorteras described in Example 2. The color sorter is trained by inserting thealiquot of discarded lightweight seeds in addition to full-color F1 hybridseeds. By using discard seeds for the inbred line (inbred pollinator wheat seeds), the machine is better able to detect color differences in thespecific lot of seeds it is sorting. The internal machine learning algorithmthen determines machine settings to optimize separation based on color, retaining, in this example, the darker F1 hybrid wheat seeds anddiscarding the lighter inbred wheat seeds (inbred pollinator wheat seed).

[0483] After both sorting parts are complete and the F1 hybrid wheat seed has reached the target hybridity, F1 hybrid wheat seeds are packaged for distribution.

[0484]

[0485] Example 4A

[0486] The methods described in Example 4 were applied to a seed lot starting with 51% F1 hybrid wheat seed produced on a Ms45 male-sterile female and 16% male seed (inbred pollinator wheat seed). In this experiment, mixture of wheat seeds were first sorted by gravity as described in Example 1A, resulting in 63% F1 hybrid seed and 22% male seed (inbred pollinator wheat seed) in the target fraction. A second sort was performed using optical sorting as described in Example 2 using the Satake Evolution using RGB technology, resulting in a final seed fraction with 78% hybridity and 5% male seed (inbred pollinator wheat seed) (Table 5), demonstrating the effectiveness combined methods in enriching for F1 hybrid wheat seeds and depleting for male seed (inbred pollinator wheat seed).

[0487]

[0488] Table 5: Hybridity and male fractions of seed sorted by gravity then color.

[0489] Example 4B

[0490] As an alternative two-step method, wheat seeds were sorted by size followed by optical sorting. The target seeds that were sorted by size in Example 3A were further sorted VMEK with RGB optical sorting as described in Example 2 to create target and non-target seed populations (Table 6).

[0491] With combined size sorting and optical sorting, the percent of hybridity increased from 26-46% in the harvested F1 seed to 74-87% in the final seed product (Table 6). The highest hybridity was achieved in a 10% male (male-fertile pollinator wheat parent line) planting scheme,although a hybridity above 85% was also achieved with a 15% male (male-fertile pollinator wheat parent line) planted. This experiment demonstrates that two-step seed sorting is an effective method for the production of F1 hybrid wheat seeds.

[0492]

[0493] Table 6: Seed sorting results from two-step process of size sorting followed by color sorting.

[0494] In a second experiment sorting a different F1 seed lot produced with a Ms45 male sterile female, size sorting was followed by optical sorting on a Vmek sorter using RGB. The initial seed mixture had a hybridity of 58.2%. After size sorting, the hybridity increased to 64%, and after optical sorting, hybridity increased to 82.1% (Table 7). In addition to tracking hybridity, the F1 hybrid wheat seed recovery was also measured as a percentage of the initial F1 hybrid wheat seed that was retained within the target seed fraction after sorting. After size sorting, 82.72% of the F1 hybrid wheat seed was retained, and after size plus optical sorting, 63.26% of the F1 hybrid wheat seed was retained. This shows that while sorting increases hybridity, some F1 hybrid seeds are lost through sorting.

[0495] Table 7: Hybridity of seed sorted by size then color

[0496] Together, these results demonstrate that the two-step seed sorting scheme is an effective strategy for enriching F1 hybrid wheat seeds within a mixed seed population.

[0497]

[0498] Example 5: Three-step seed sorting in F1 hybrid wheat seed production

[0499] All three seed sorting methods, size, optical, and density, can be combined to sort a single seed lot. Seed sorting can proceed in any order as needed. In this example, seeds that were previously sorted by size and optical sorting (Example 4A) are subsequently sorted by density.

Claims

We claim:

1. A method of sorting F1 hybrid wheat seed, the method comprising: sorting a wheat seed mixture comprising inbred pollinator wheat seed and F1 hybrid wheat seed using seed density and / or at least one morphological characteristic to sort the wheat seed mixture into an enriched population of inbred pollinator wheat seed and / or an enriched population of F1 hybrid wheat seed.

2. The method of claim 1 , further comprising:sorting the wheat seed mixture based on the density of the inbred pollinator wheat seed or F1 hybrid wheat seed; orsorting the wheat seed mixture based on the at least one morphological characteristic of the inbred pollinator wheat seed or F1 hybrid wheat seed.

3. The method of claim 1 , further comprising re-sorting the separated population of inbred pollinator wheat seed using a second sorting step to recover F1 hybrid wheat seed.

4. The method of claim 3, wherein the resorting increases the recovery of F1 hybrid wheat seed by at least 1%, 5%, 10%, or 20% relative to the total amount of F1 hybrid wheat seed present in the population prior to the resorting.

5. The method of claim 1 , further comprising re-sorting the separated population of F1 hybrid wheat seed using a second sorting step to enrich the separated population of F1 hybrid wheat seed for F1 hybrid wheat seed.

6. The method of claim 5, wherein the re-sorting increases the percentage of hybridity in the sorted population to at least 70%.

7. The method of claim 1 or 3, comprising subjecting the population of separated inbred pollinator wheat seed to optical sorting.

8. The method of claim 7, wherein the optical sorting increases the recovery of F1 hybrid wheat seed by at least 1 %, 5%, 10%, or 20% relative to the total amount of F1 hybrid wheat seed present in the population prior to optical sorting.

9. The method of claim 1 or 5, comprising subjecting the population of separated F1 hybrid wheat seed to optical sorting.

10. The method of claim 9, wherein the optical sorting increases the percentage of hybridity in the sorted population to at least 70%.

11. The method of claim 7, the method comprising performing multiple passes of optical sorting on the separated inbred pollinator wheat seed population to recover F1 hybrid wheat seed.

12. The method of claim 7, the method comprising performing multiple passes of optical sorting on the separated population of inbred pollinator wheat seed until a desired level of recovery is achieved for the F1 hybrid wheat seed.

13. The method of claim 9, wherein the hybridity is increased by at least 1%, 5%, 10%, or 20% relative to the total amount of F1 hybrid wheat seed present in the population prior to the multiple passes of optical sorting.

14. The method of claim 9, the method comprising performing multiple passes of optical sorting on the separated population of F1 hybrid wheat seed to further separate inbred pollinator wheat seed from the separated F1 hybrid wheat seed.

15. The method of claim 9, the method comprising performing multiple passes of optical sorting on the separated population of F1 hybrid wheat seed until a hybridity of at least 70% is achieved.

16. The method of claim 7, the method comprising optical sorting the separated inbred pollinator wheat seed population by detecting inbred pollinator wheat seed or F1 hybrid wheat seed in the separated inbred pollinator wheat seed population and separating the F1 hybrid wheat seed from, or retaining the inbred pollinator wheat seed, in the separated inbred pollinator wheat seed population.

17. The method of claim 9, the method comprising optical sorting the separated F1 hybrid wheat seed population by detecting inbred pollinator wheat seed or F1 hybrid wheat seed in the separated F1 hybrid wheat seed population and separating the inbred pollinator wheat seed from, or retaining the F1 hybrid wheat seed in, the separated F1 hybrid wheat seed population.

18. The method of claim 3, wherein the recovered F1 hybrid wheat seed is combined with the separated F1 hybrid wheat seed population.

19. The method of claim 9, wherein the optical-based sorting is colorbased sorting to detect the inbred pollinator wheat seed or F1 hybrid wheat seed based on a visible color or spectral reflectance.

20. The method of claim 7 or 9, wherein the optical-based sorting comprises detecting the inbred pollinator wheat seed or the F1 hybrid wheat seed based on a visible color, spectral reflectance, or a combination thereof.

21. The method of claim 7 or 9, wherein the optical-based sorting uses RGB, NIR, IR, or hyperspectral imaging to separate the inbred pollinator wheat seed and F1 hybrid wheat seed from one another.

22. The method of claim 7 or 9, comprising optical sorting the separated inbred pollinator wheat seed population to sort inbred pollinator wheat seed and F1 hybrid wheat seed from one another based on a maternally- inherited seed color phenotype.

23. The method of claim 7 or 9, wherein the combination of size, shape, and / or density sorting and optical color sorting results in an enriched population of F1 hybrid wheat seed with less than 20% inbred pollinator wheat seed.

24. The method of claim 1 , wherein the F1 hybrid wheat seed is produced by pollinating a nuclear-genome based male-sterile female wheat parent line or a mitochondrial-genome based male-sterile female wheat parent line.

25. A method of obtaining an enriched population of F1 hybrid wheat seed and / or an enriched population of inbred pollinator wheat seed, the method comprising: separating a wheat seed mixture comprising inbred pollinator wheat seed and F1 hybrid wheat seed by optically sorting the wheat seed mixture based on at least one identified phenotype to separate the F1 hybrid wheat seed and the inbred pollinator wheat seed from one another to produce the enriched population of F1 hybrid wheat seed and / or the enriched population of inbred pollinator wheat seed.

26. The method of claim 25, further comprising: analyzing wheat seed from the wheat seed mixture using optical sorting to identify the at least one phenotype, wherein the at least one phenotype is a morphological,compositional, or spectral phenotype that distinguishes the F1 hybrid wheat seed from the inbred pollinator wheat seed.

27. The method of claim 26, wherein the morphological phenotype comprises seed length, seed width, seed surface area, calculated seed volume, seed circularity, or aspect ratio, or combinations thereof.

28. The method of claim 26, wherein the spectral phenotype comprises: visible color intensity, spectral reflectance, a unique spectral signature, or a combination thereof.

29. The method of claim 26, wherein the optical sorting uses color-based sorting to detect the inbred pollinator wheat seed or F1 hybrid wheat seed based on a visible color or spectral reflectance.

30. The method of claim 28, wherein the spectral phenotype is a maternally inherited seed color phenotype.

31. The method of claim 26, wherein the optical sorting uses RGB, NIR, IR, or hyperspectral imaging to detect the inbred pollinator wheat seed or F1 hybrid wheat seed based on a visible color, spectral reflectance, or a combination thereof and separate the inbred pollinator wheat seed and F1 hybrid wheat seed from one another.

32. The method of claim 25, further comprising: performing multiple passes of optical sorting on the enriched population of F1 hybrid wheat seed until a hybrid ity of at least 70% is achieved.

33. The method of claim 25, further comprising: performing multiple passes of optical sorting on the separated population of inbred pollinator wheat seed to recover residual F1 hybrid wheat seed.

34. The method of claim 33, wherein the additional optical sorting pass increases the total recovery of F1 hybrid wheat seed by at least 1%, 5%, 10%, or 20% relative to the total amount of F1 hybrid wheat seed present in the population prior to the additional optical sorting pass or passes.

35. The method of claim 33, further comprising: combining the recovered residual F1 hybrid wheat seed with the enriched population of F1 hybrid wheat seed.

36. The method of claim 25, further comprising: sorting the separated enriched inbred pollinator wheat seed population based on seed size, seed shape, seed density, or a combination thereof, to recover F1 hybrid wheat seed.

37. The method of claim 25, further comprising: sorting the separated enriched population of F1 hybrid wheat seed based on seed size, seed shape, seed density, or a combination thereof, to enrich the concentration of F1 hybrid wheat seed.

38. The method of claim 25, wherein the optical sorting separates the inbred pollinator wheat seed and the F1 hybrid wheat seed in the wheat seed mixture based on at least one morphological characteristic.

39. The method of claim 25, wherein the optical sorting separates the inbred pollinator wheat seed and the F1 hybrid wheat seed in the wheat seed mixture based on at least two morphological, compositional, or spectral phenotypes comprising: seed color, seed density, at least one morphological characteristic, or a combination thereof, simultaneously in a single sorting step.

40. The method of claim 39, wherein the combination of optical color, seed size, seed shape, and / or density sorting results in an enriched population of F1 hybrid wheat seed with less than 20% inbred pollinator wheat seed.

41. The method of claim 25, wherein the F1 hybrid wheat seed is produced by pollinating a nuclear-genome based male-sterile female wheat parent line or a mitochondrial-genome based male-sterile female wheat parent line.

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