(19)
(11)EP 2 975 927 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
02.10.2019 Bulletin 2019/40

(21)Application number: 14710304.8

(22)Date of filing:  17.03.2014
(51)International Patent Classification (IPC): 
A01H 1/02(2006.01)
A01H 5/10(2018.01)
A01H 1/04(2006.01)
C12N 15/82(2006.01)
(86)International application number:
PCT/EP2014/055249
(87)International publication number:
WO 2014/147005 (25.09.2014 Gazette  2014/39)

(54)

METHODS OF SEPARATING HYBRID SEED FROM A MIXTURE OF SEEDS

VERFAHREN ZUM TRENNEN DER HYBRIDSAAT AUS EINER MISCHUNG AUS SAATGUT

PROCÉDÉS POUR SÉPARER UNE SEMENCE HYBRIDE D'UN MÉLANGE DE SEMENCES


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 18.03.2013 EP 13159765

(43)Date of publication of application:
27.01.2016 Bulletin 2016/04

(73)Proprietor: BASF SE
67056 Ludwigshafen (DE)

(72)Inventors:
  • WEISSMANN, Sigrid
    78224 Singen-Bohlingen (DE)
  • WEISSMANN, Elmar Alfons
    78224 Singen-Bohlingen (DE)
  • JACOBS, John
    NL-9820 Merelbeke (NL)

(74)Representative: BASF IP Association 
BASF SE G-FLP-C006
67056 Ludwigshafen
67056 Ludwigshafen (DE)


(56)References cited: : 
WO-A1-2012/038350
CN-A- 102 475 055
US-A- 3 842 538
US-A1- 2011 232 247
CN-A- 1 442 038
CN-A- 102 948 361
US-A- 6 005 167
  
  • SIMONS KRISTIN J ET AL: "Molecular characterization of the major wheat domestication gene Q", GENETICS, vol. 172, no. 1, January 2006 (2006-01), pages 547-555, ISSN: 0016-6731
  • SOOD S ET AL: "The major threshability genes soft glume (sog) and tenacious glume (Tg), of diploid and polyploid wheat, trace their origin to independent mutations at non-orthologous loci", THEORETICAL AND APPLIED GENETICS JULY 2009 SPRINGER VERLAG DEU, vol. 119, no. 2, July 2009 (2009-07), pages 341-351, DOI: 10.1007/S00122-009-1043-0
  • JANTASURIYARAT C ET AL: "Identification and mapping of genetic loci affecting the free-threshing habit and spike compactness in wheat (Triticum aestivum L.)", THEORETICAL AND APPLIED GENETICS JANUARY 2004 SPRINGER VERLAG DE, vol. 108, no. 2, January 2004 (2004-01), pages 261-273, DOI: 10.1007/S00122-003-1432-8
  • VAMSI J NALAM ET AL: "Map-based analysis of genetic loci on chromosome 2D that affect glume tenacity and threshability, components of the free-threshing habit in common wheat (Triticum aestivum L.)", THEORETICAL AND APPLIED GENETICS ; INTERNATIONAL JOURNAL OF PLANT BREEDING RESEARCH, SPRINGER, BERLIN, DE, vol. 116, no. 1, 2 October 2007 (2007-10-02), pages 135-145, XP019558624, ISSN: 1432-2242, DOI: 10.1007/S00122-007-0653-7
  • SEBASTIAN FÃRSTER ET AL: "Discrimination of alleles and copy numbers at thelocus in hexaploid wheat using quantitative pyrosequencing", EUPHYTICA, KLUWER ACADEMIC PUBLISHERS, DO, vol. 186, no. 1, 30 October 2011 (2011-10-30), pages 207-218, XP035061121, ISSN: 1573-5060, DOI: 10.1007/S10681-011-0561-4
  • SIMONETTI M C ET AL: "Quantitative trait loci influencing free-threshing habit in tetraploid wheats", GENETIC RESOURCES AND CROP EVOLUTION, vol. 46, no. 3, June 1999 (1999-06), pages 267-271, ISSN: 0925-9864
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description


[0001] The present invention relates to the field of agriculture, more particular hybrid wheat seed production and separation of hybrid wheat seed from a wheat seed mixture using separation methods based on differences in the phenotype of the seeds comprised in said seed mixture. Separation of hybrid wheat seeds takes place based on e. g. differences in size or specific weight of the hybrid seeds as compared to other seeds comprised in the mixture, e. g. inbred seeds.

[0002] Hybrid plants are bred to improve the characteristics of the resulting plants, such as better yield or yield stability, greater uniformity, improved color, disease resistance, etc.. Today, hybrid seed production is predominant in agriculture and home gardening, and is one of the main contributing factors to the dramatic rise in agricultural output during the last half of the 20th century. In the US, the commercial hybrid seed market was launched in the 1920s, with the first hybrid maize. All of the hybrid seeds planted by the farmer will be the same hybrid while the seeds from the hybrids planted will not consistently have the desired characteristics.

[0003] Heterosis, or hybrid vigor (as in the familiar F1 hybrids of maize), occurs in common (hexaploid) wheat, but it is difficult to produce seed of hybrid cultivars on a commercial scale (as is done with maize) because wheat flowers are bi-sexual and normally self-pollinate. Commercial hybrid wheat seed has been produced using chemical hybridizing agents; these chemicals selectively interfere with pollen development, or naturally occurring cytoplasmic male sterility systems.

[0004] Hybrid plants are created by crossing a pollen parent to a seed parent of a different line or cultivar. In most cases, the male fertile pollen parent is also female fertile resulting in the production of inbred seed in addition to the hybrid seed produced on the seed parent. In most production methods this is solved by planting and harvesting the pollen parent and seed parent in physically separated strips or rows. However, hybrid seed yield in such a strip planting method is markedly lower compared to hybrid seed yield in a mixed planting of pollen parent and seed parent. With a field design where seed and pollen parent are planted in a mixed interplanting fashion a mixture of hybrid and inbred seed is obtained. With techniques known today, a separation of the two different kinds of seed in order to obtain hybrid seed with a purity of greater than 90% is cumbersome. The use of biotechnologically introduced phenotypic markers (e.g. fluorescent seed color) enabling for seed separation without resulting in a phenotype visible to the grower or consumer is not generally accepted. Therefore, there is the need of new methods of separating hybrid seeds from inbred seeds based on natural traits. This problem is addressed by the present invention.

Figure legends



[0005] 

Figure 1: Kernel phenotypes in cereals. Upper panel (wheat and oats): Free threshing seed with a softened, easily separable hull (left) and non-free threshing seed with a tenacious, tightly enclosing hull (right). The free-threshing phenotype is maternally determined. Lower panel (barley): free-threshing (naked) (left) and nonfree threshing (covered) seed (right), where the absence or presence of a sticky substance exuded from the seed (as indicated by the arrows) is suspected to determine the free-threshing phenotype.

Figure 2: Inheritance of threshability in cereals (wheat and oats) when seed parent is homozygous for one or more dominant free threshing (FT) allele(s) and the pollen parent is homozygous for one or more recessive hulled (h) allele(s), and the free-threshing phenotype is maternally expressed. The harvested seed from an F1 seed production field is a combination of free threshing hybrid seed from the seed parent and hulled inbred seed from the pollen parent, which can be separated by sorting, as indicated by the two-way arrow. The F2 grain resulting from planting of the hybrid seed is all free threshing.

Figure 3: Inheritance of threshability in cereals (wheat and oats) when seed parent is homozygous for one or more recessive hulled (h) allele(s) and the pollen parent is homozygous for one or more dominant free threshing (FT) allele(s), and the free-threshing phenotype is maternally expressed. The harvested seed from an F1 seed production field is a combination of hulled hybrid seed from the seed parent and free threshing inbred seed from the pollen parent, which can be separated by sorting, as indicated by the two-way arrow. The F2 grain resulting from planting of the hybrid seed is all free threshing.

Figure 4: Inheritance of threshability in cereals (wheat and oats) when the seed parent is homozygous for one or more dominant free threshing (FT) allele(s) and the pollen parent is homozygous for one or more recessive hulled (h) allele(s), and the free-threshing phenotype is expressed in the embryo or endosperm (principle). The harvested seed from an F1 seed production field is a combination of free threshing hybrid seed from the seed parent and hulled inbred seed from the pollen parent, which can be separated by sorting, as indicated by the two-way arrow. The F2 grain resulting from planting of the hybrid seed is a combination of free threshing and hulled seeds.

Figure 5: Inheritance of threshability in cereals (barley) when the seed parent is homozygous for one or more dominant covered (H) allele(s) and the pollen parent is homozygous for one or more recessive naked (ft) allele(s), and the free-threshing phenotype is expressed in the embryo or endosperm. Harvested seed from an F1 seed production field is a combination of covered hybrid seed from the seed parent and naked inbred seed from the pollen parent, which can be separated by sorting, as indicated by the two-way arrow. The F2 grain resulting from planting of the hybrid seed is a combination of covered and naked seed.

Figure 6: Inheritance of threshability in cereals (barley) when the seed parent is homozygous for one or more dominant covered (H) allele(s) and the pollen parent is homozygous for one or more recessive naked (ft) allele(s), and the free-threshing phenotype is maternally expressed (principle). The harvested seed from an F1 seed production field is a combination of covered hybrid seed from the seed parent and naked inbred seed from the pollen parent, which can be separated by sorting, as indicated by the two-way arrow. The F2 grain resulting from planting of the hybrid seed is all covered.

Figure 7: Inheritance of threshability in cereals (barley) when the seed parent is homozygous for one or more recessive naked (ft) allele(s) and the pollen parent is homozygous for one or more dominant covered (H) allele(s), and the free-threshing phenotype is maternally expressed (principle). The harvested seed from an F1 seed production field is a combination of covered hybrid seed from the seed parent and naked inbred seed from the pollen parent, which can be separated by sorting, as indicated by the two-way arrow. The F2 grain resulting from planting of the hybrid seed is all covered.


Detailed description



[0006] The present invention relates to a method of separating hybrid wheat seeds from a mixture comprising said hybrid wheat seeds and inbred seeds, comprising (a) obtaining or providing a mixture of seeds obtained or produced by interplanting a combination of plants comprising a male sterile or conditionally male sterile seed parent and a male fertile pollen parent, wherein said seed parent and pollen parent produce at least two types of seeds, at least one of which being said hybrid seeds, said at least two types of seed having different phenotypes allowing separation of said hybrid seed with non-invasive methods based on the differences in said phenotypes, said different phenotypes being determined by an allele or a combination of alleles at one or more genetic loci, wherein said seed parent is homozygous for a first allele or combination of alleles at said one or more genetic loci resulting in a first phenotype and said pollen parent is homozygous for a second allele or combination of alleles at said one or more genetic loci resulting in a second phenotype; (b) separating said hybrid seeds using a separation method based on differences in said phenotypes.

[0007] Hybrid seeds are generally produced by cross-pollinated plants. In a more particular sense, the term refers to the seed resulting from specific and controlled crossing of two inbred lines. The embryo of said hybrid seed comprises one haploid genome copy of each parent. On the other hand, inbred seed is produced when the egg inside the ovary of a plant is fertilized by pollen originating from anthers borne in the same flower (in cereals called spikelets) as the ovary, anthers from other flowers of the same plant or anthers from the flowers of another plant of the same parental origin.

[0008] The term "cereal" relates to members of the monocotyledonous family Poaceae which are cultivated for the edible components of their grain. These grains are composed of endosperm, germ and bran. Maize, wheat and rice together account for more than 80% of the worldwide grain production. Other members of the cereal family comprise rye, oats, barley, triticale, sorghum, wild rice, spelt, einkorn, emmer, durum wheat and kamut.

[0009] Upon producing hybrid seed, a mixture of hybrid and inbred seed is obtained if the pollen parent is female fertile. Accordingly, a mixture of seeds comprising hybrid seeds can be obtained by interplanting a seed parent and a pollen parent. Possible scenarios of interplanting comprise strip or row planting where pollen and seed parent are planted in alternate rows. A more desirable type of interplanting comprises mixed interplanting, where a true mixture of pollen and seed parent is sown so that the pollen parent which is sown as a lower proportion than the seed parent is distributed evenly among the male sterile seed parent so that optimal cross-pollination can occur. This design is only feasible if the male sterile trait is inherent to the seed parent or can be induced due to a specific property of the seed parent.

[0010] Male sterile in connection with the present invention refers to the failure of plants to produce functional anthers, pollen or male gametes. This can be due to natural or artificially introduced genetic predispositions or to human intervention on the plant in the field.

[0011] Male fertile on the other hand relates to plants being capable of producing normal functional anthers, pollen and/or male gametes.

[0012] A pollen parent, is a parent plant that provides the male gametes (pollen) for fertilization, while a seed parent is the plant that provides the female gametes for fertilization, resulting in that plant bearing the seeds.

[0013] Both seed and pollen parent should be planted so that they flower at the same time, i.e. pollen of the pollen parent and the ovules of the seed parent are ripe at the same time. If there are differences in flowering times, planting times are to be adapted accordingly.

[0014] A seed parent can be male sterile and hence incapable of self-fertilization. A male-sterile seed parent can thus only produce seed when fertilized by pollen from a male fertile plant, i.e. the pollen parent. Vice versa, a pollen parent can be female sterile, thereby being incapable of self-fertilization. A female sterile pollen parent is thus only capable of fertilizing another female fertile plant, i.e. the seed parent. Reliably male sterile seed parents can be created in many crops. However, pollen parents in crops with "perfect" (bisexual) flowers, i.e. flowers comprising male and female organs are usually also female fertile and can therefore self-fertilize. In the present invention, the desired hybrid seed grows on the seed parent, whereas undesired contaminating inbred seed grows on the pollen parent.

[0015] At least two types of seeds, as used herein, relates to different parental origin and hence genetic constitution (which may translate into differences in phenotype) of the seeds resulting from (cross-)fertilization of types of genetically equal or different parent plants. For example, when both parents are fertile, the following types of seeds can be produced; seeds resulting from self-fertilization of the first parent, seeds resulting from self-fertilization of the second parent and seeds resulting from cross-fertilization (i.e. hybrid seed) borne by either the first or the second parent. In case of one parent being male sterile (i.e. the seed parent) and the other parent being completely fertile (the pollen parent), two types of seeds can arise, i.e. seeds resulting from cross-fertilization (i.e. hybrid seed) borne by the male sterile seed parent and seeds resulting from self-fertilization of the pollen parent. In case of a male sterile seed parent and female sterile pollen parent only one type of seed can arise, the hybrid seed.

[0016] Hybrid seed, as used herein, refers to seed resulting from a cross-fertilization of two parent plants that are not genetically identical, thus leading to seeds originating from the cross-fertilization having a different genetic constitution than either of the two parents.

[0017] Different phenotypes allowing separation of hybrid seed with non-invasive methods based on the differences in said phenotypes relates to methods that do not require removing parts of the seed or damaging the seeds in any way to establish said phenotypic differences, thereby ensuring that the seeds, upon planting, germinate and develop normally. Such methods include separation based on relative size of the seeds by sieving, separation based on specific weight by gravity sorting on a table sorter, separation based on specific weight by wind sieving, separation based on differences in color using non-invasive imaging techniques. Such methods are for example described in Dessai et al. (1997, Seeds handbook: biology, production, processing, and storage. Marcel Dekker, Inc., New York.), Harmond et al. (1968, Mechanical seed cleaning and handling. USDA Agricultural Handbook No. 354.Washington, D.C.), Jorgensen et al. (2004, Seed collection, cleaning, and storage, Chap. 24. In: Monsen et al. Restoring Western Ranges and Wildlands. USDA Forest Service Gen. Tech. Rep. RMRS-GTR-136. Ft. Collins, Colorado), Vaughan et al (1968, Seed processing and handling. Seed Technology Laboratory, Mississippi State Univ., State College, Mississippi, see also mcia.msstate.edu/pdf/seed-processing-and-handling.pdf) Emberson et al. (Optical sorting (2011): Grain, Feed and Milling technology, Nov-Dec 2011, p. 22-24), Delwiche et al. (2005, Applied Engineering in Agriculture, Vol. 21(4): 681-688), Dowell et al. (2002, Use of optical sorting to detect karnal bunt-infected wheat kernels. ASAE Paper No. 023007. St. Joseph, Mich.: ASAE. The methods of the invention may comprise more than one separation step, for example two sieving steps with different cut-offs (see also Example 3).

[0018] As used herein, the term "allele(s)" means any of one or more alternative forms of a gene at a particular locus. In a diploid cell of an organism, alleles of a given gene are located at a specific location or locus (plural: loci) on a chromosome. One allele is present on each chromosome of the pair of homologous chromosomes or possibly on homeologous chromosomes.

[0019] As used herein, the term "locus" (plural: loci) means a specific place or places or a site on a chromosome where for example a gene or genetic marker is found.

[0020] As used herein, the term "homologous chromosomes" means chromosomes that contain information for the same biological features and contain the same genes at the same loci but possibly different alleles of those genes. Homologous chromosomes are chromosomes that pair during meiosis. "Non-homologous chromosomes", representing all the biological features of an organism, form a set, and the number of sets in a cell is called ploidy. Diploid organisms contain two sets of non-homologous chromosomes, wherein each homologous chromosome is inherited from a different parent. In tetraploid species, two sets of diploid genomes exist, whereby the chromosomes of the two genomes are referred to as "homeologous chromosomes" (and similarly, the loci or genes of the two genomes are referred to as homeologous loci or genes). Likewise, hexaploid species have three sets of diploid genomes, etc. A diploid, tetraploid or hexaploid plant species may comprise a large number of different alleles at a particular locus. The ploidy levels of domesticated wheat species range from diploid (Triticum monococcum, 2n = 14, AA), tetraploid (T. turgidum, 2n = 28, AABB) to hexaploid (T. aestivum,2n = 42, AABBDD).

[0021] As used herein, the term "heterozygous" means a genetic condition existing when two different alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell. In other words, "heterozygous" means a genetic condition existing when two different alleles reside at a specific locus of the two members of a pair of homologous chromosomes in the cell. Conversely, as used herein, the term "homozygous" means a genetic condition existing when two identical alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell. In other words, the term "homozygous" means a genetic condition existing when two identical alleles reside at a specific locus on both members of a pair of homologous chromosomes in the cell.

[0022] In the present invention, alternate alleles of one or more than one gene may determine the phenotype of the seed of inbred parent plants, whereby one allele or set of alleles determines one manifestation of the seed phenotype and another allele or set of alleles determines another manifestation of the seed phenotype and whereby one allele or set of alleles is dominant or partially dominant and the other allele or set of alleles is recessive or partially recessive. Accordingly, possible allele combinations specifying a particular seed phenotype include one dominant allele and one recessive allele as well as two dominant alleles, two recessive alleles, three dominant alleles, three recessive alleles, two dominant and one recessive allele, one dominant and two recessive alleles and all possible iterations for four, five, six or more genes involved in a specific phenotype according to the present invention. The same holds true for partially dominant and partially recessive alleles.

[0023] Disclosed herein is a method of producing hybrid seeds of a cereal comprising (a) interplanting a combination of plants comprising a male sterile or conditionally male sterile seed parent and a male fertile pollen parent, wherein said seed parent and pollen parent produce at least two types of seeds, at least one of which being said hybrid seeds, said at least two types of seed having different phenotypes allowing separation of said hybrid seed with non-invasive methods based on the differences in said phenotypes, said different phenotypes being determined by an allele or a combination of alleles at one or more genetic loci, wherein said seed parent is homozygous for a first allele or combination of alleles at said one or more genetic loci resulting in a first phenotype and said pollen parent is homozygous for a second allele or combination of alleles at said one or more genetic loci resulting in a second phenotype.

[0024] Said method of producing hybrid seeds of a cereal as disclosed herein further comprises separating the hybrid seeds using a non-invasive separation method, said planting comprises row or strip planting or mixed interplanting, said planting comprises sowing the seed of or planting young plants of said seed parent and said pollen parent into the soil, said method of producing hybrid seeds of a cereal further comprises harvesting the seeds produced prior to separating.

[0025] Harvesting may be effected, inter alia, by harvesting machines common in the art. In case of row or strip planting, harvesting machines are preferably designed so that the rows or strips of pollen plant can be harvested separately thus enabling for a pre-sorting of the resulting seeds.

[0026] In a preferred embodiment, said seed parent is homozygous for one or a combination of alleles at one or more genetic loci, wherein at least one of said alleles in said seed parent is dominant or partially dominant, and said pollen parent is homozygous for one or a combination of alleles at one or more genetic loci, wherein at least one of said alleles in said pollen parent is recessive or partially recessive.

[0027] The term "partial dominance" refers to the situation where the heterozygote displays a phenotype that is an intermediate between the phenotypes of both homozygote parent plants (one of which is homozygous (partially) dominant and the other is homozygous recessive). This intermediate phenotype is a demonstration of partial or incomplete dominance. When partial dominance occurs, a range of phenotypes is usually observed among the offspring. The same applies to partially recessive alleles. As used here, these terms imply that all alleles responsible for expression of hulled or free-threshing traits as explained further below, even if partially dominant or partially recessive, result in the expression of the desired phenotype, i.e. hulled or free-threshing seed.

[0028] The above combination of the genetic constitutions of the parents results in two types of seed being generated, wherein said two types of seed can be separated based on phenotype, as described above. When the seed parent is homozygous for one or more dominant alleles, the resulting hybrid seed also carries the maternal (dominant) phenotype; either because the phenotype of the seed is directly determined by genotype of the plant bearing the seeds (in this case the seed parent), as will be explained further below, or because of the genotype of the seed itself determining the seed phenotype. The inbred seed resulting from self-fertilization of the homozygous recessive pollen parent carries the (recessive) paternal phenotype, either because the phenotype of the seed is directly determined by genotype of the plant bearing the seeds (in this case the pollen parent), or because of the genotype of the seed itself determining the seed phenotype. This difference in the genotype of the plant bearing the seeds determining the seed phenotype vs. the genotype of the seed itself determining the seed phenotype is referred to as maternal expression vs. endosperm/embryonic expression.

[0029] In a more preferred embodiment, said combination of plants is characterized in that said one allele or combination of alleles at one or more genetic loci in said seed parent manifests in seeds with a free-threshing phenotype and wherein said one allele or combination of alleles at one or more genetic loci in said pollen parent manifests in seeds with a hulled phenotype.

[0030] Angiosperm seeds consist of three genetically distinct constituents: (1) the embryo formed from the zygote, (2) the endosperm, which is normally triploid, (3) the seed coat from tissue derived from the maternal tissue of the ovule. The seed coat is also referred to as husk or hull. The four wild species of wheat, along with the domesticated varieties einkorn, emmer and spelt, have hulls. This in evolutionary terms more primitive morphology which is also characteristic for barley consists of toughened or tenacious glumes that tightly enclose the grains, and, in domesticated wheats, a rachis that breaks easily on threshing. The result is that when threshed, the wheat ear breaks up into spikelets (also known as "speltoid phenotype"). To obtain the grain of hulled seed, further processing, such as milling or pounding, is needed to remove the hulls or husks. Hulled wheat seeds are often stored as spikelets because the toughened glumes give good protection against pests of stored grain. In contrast, in free-threshing (or naked) forms such as durum wheat and common wheat, the glumes are fragile and the rachis tough. On threshing, the chaff breaks up, releasing the grains.

[0031] Accordingly, in one embodiment, the seed parent is homozygous for a dominant or partially dominant allele or a combination of such alleles conferring the free-threshing phenotype, while the pollen parent is homozygous for a recessive allele or a combination of such alleles conferring the hulled phenotype. As a result, the hybrid seed will also be free-threshing whereas the selfed (inbred) seed is non-free-threshing (hulled).

[0032] In hexaploid wheat, the glume tenacity gene Tg along with the major domestication locus Q control threshability. The Q gene is a gene located on the long arm of wheat chromosome 5A (the Q locus) that is largely responsible for the domestication of wheat, as it confers the free threshing character by suppressing the speltoid phenotype. In addition, several positive pleiotropic effects are linked to Q, for example softening of the glumes, reduction of ear length, more spikelets per ear and toughness of the rachis, all of those features being favorable to harvesting (Salamini et al., 2002, Nature Reviews Genetics 3:429-441). The Q gene was cloned and molecularly characterized as an APETALA2 (AP2) like transcription factor. Of the two alleles, the Q allele appeared to be more abundantly transcribed than q, and can be differentiated by an A/G SNP at position 2123 of the Q gene, leading to a single amino acid difference between the Q and q protein. Accordingly, the Q protein is characterized by an isoleucine at position 329, while q has a valine at this position, the latter amino acid appearing to limit homodimer formation. The effect of Q mainly depends on its dosage. Based on dosage experiments of Q and q alleles in various backgrounds it was established that q is hypomorphic to Q and 2.5 doses of q equaled 1 dose of Q. In plants transformed with the Q allele, Q transcript levels were found to inversely correlate with the speltoid phenotype (Simons et al. 2006, Genetics 172: 547-555). Recently, a fast and reliable pyrosequencing application was developed to differentiate 5Aq and 5AQ alleles and determine 5AQ gene copy numbers in a single assay (Foerster et al., 2012, Euphytica 186:207-218).

[0033] Further determinant of the free threshing phenotype are the tenacious glume (Tg) gene and the soft glume (Sog) gene, that have been mapped to chromosomes 2DS and 2AS respectively, although the genes involved have not been identified yet. The Tg gene is a main threshability gene in hexaploid wheat, of which the Tg allele is partially dominant, while the free-threshing character of tg is recessive in nature. Sog is the main threshability gene in diploid wheat, the mutant allele (sog) being a recessive allele that confers a free threshing phenotype (Sood et al., 2009, Theor Appl Genet 199:341-351). Although in sog mutant plants the glumes that encase the grain are soft, the ears are very compact, which is a negative pleiotropic effect (Salamini et al., 2002, Nature Reviews Genetics 3:429-441)

[0034] Simonetti et al (1999, Genet Res Crop Evol 46:267-271) found in tetraploid wheat four QTLs influencing the threshability trait, associated with chromosomes 2BS, 5AL, 5AS, and 6AS. The 2BS and 5AL QTL corresponded to the homoeologous genes Tg and Q of hexaploid wheats, respectively, where Tg2 the putative ortholog ofTg, was located on chromosome 2BS (hereinafter referred to as Tg2B, Q5Aand Tg2B). Thus, the fact that QTLs affecting the threshability of wheat have been identified on chromosome 6A and 6D (Simonetti et al., supra; Jantasuriyarat et al. 2004, Theor Appl Genet 108:261-273), suggests that loci other than those on group-2 chromosomes and chromosome 5A (Q gene) also may be involved in controlling the threshability trait in wheat (Sood et al., 2009, supra).

[0035] For example, free-threshing durum wheat thus can have the genotype tgtg2BQQ5A, while free-threshing bread wheat can have the genotype tgtg2B tgtg2D QQ5A.

[0036] Accordingly, hulled durum wheat can have the genotype tgtg2Bqq5A, while hulled bread wheat can have the genotype tgtg2Btgt2Dqq5A.

[0037] In wheat, the seed phenotype in terms of hulls is maternally determined as the seed coat is in fact maternal tissue. This means that the genotype of the parent plant bearing the seeds determines whether the seed borne by that plant (i.e. the F1 seed) is either hulled of free-threshing. Consequently, the genotype of the F1 seed parent determines the phenotype of the seed born by that plant, i.e. of the F2 seed, etc. In connection to the present invention, the threshability phenotype of hybrid seed borne by the seed parent is determined by the genotype of the seed parent, while seed borne by the pollen parent as a result of self-fertilization of thus has the phenotype as determined by the genotype of the pollen parent. This principle of the genotype of plant bearing the seed directly determining the seed phenotype is referred to as maternal expression of the seed phenotype.

[0038] In a more preferred embodiment, said combination of plants is characterized in that said one allele or combination of alleles at one or more genetic loci in said seed parent manifests in seed with a hulled phenotype and wherein said one allele or combination of alleles at one or more genetic loci in said pollen parent manifests in seeds with a free-threshing phenotype.

[0039] In a preferred embodiment, said seed parent is homozygous for one or a combination of alleles at one or more genetic loci, wherein at least one of said alleles is recessive or partially recessive, and said pollen parent is homozygous for one or a combination of alleles at one or more genetic loci, wherein at least one of said alleles is dominant or partially dominant. In this embodiment, the seed phenotype should be maternally expressed.

[0040] In another preferred embodiment, said combination of plants is characterized in that said one allele or combination of alleles at one or more genetic loci in said seed parent manifests in seeds with a hulled phenotype and said one allele or combination of alleles at one or more genetic loci in said pollen parent manifests in seeds with a free-threshing phenotype.

[0041] An overview of the genetic determinants and inheritance of hulled vs. free threshing phenotype in various cereals is given below in table 1 and is schematically depicted in figures 1-7.
Table 1: Constitution of the parental and F1 grain and inheritance (simplified)
Cereal speciesPhenotype of seed parentPhenotype of pollen parentTarget fraction Phenotype of harvested F1 (hybrid) seedPhenotype of F2 seedInheritance and expression of the free threshing type
Barley Hulled Free threshing Hulled Hulled Recessive; endosperm/embryo
Oats Free threshing Hulled Free threshing Free threshing Dominant (partial); maternal
Wheat Free threshing Hulled+non-brittle Free threshing Free threshing Dominant for Q-factor/recessive for all others (tg, sog); maternal


[0042] Said non-invasive separation method can be based on differences in size, surface condition or specific weight of said at least two types of seed as described above.
For embodiments where the difference in phenotypes is expressed via a hulled and free-threshing phenotype, separation can be effected based on different sizes of the grains with and without hulls. Whereas the grains themselves usually have the same or a comparable size, the presence of the hulls tightly attached to the grains of one phenotype makes the volume of this type of seed larger than that of the free-threshing grains alone. An alternative separation method may be based on specific weight of the seed. Hulled seed, due to its bigger volume which is partly filled only by air, has a lower specific weight than free-threshing seed.

[0043] The present invention also relates to a method of separating hybrid wheat seed from a mixture of hybrid seed and inbred seed comprising obtaining or providing said mixture, wherein embryos of said hybrid wheat seeds have a genotype comprising one haploid genome copy of the seed parent and one haploid genome copy of the pollen parent, wherein embryos of said inbred seeds have a genotype comprising two haploid genome copies of the pollen parent, wherein said seed parent is male sterile or conditionally male sterile and wherein said pollen parent is male fertile, characterized in that (a) said seed parent is homozygous for seed with a free-threshing phenotype and said pollen parent is homozygous for seed with a hulled phenotype, or (b) said seed parent is homozygous for seed with a hulled phenotype and said pollen parent is homozygous for seed with a free-threshing phenotype; comprising applying said mixture of seeds to a means for separation according to size, or specific weight of the grains.

[0044] In a preferred embodiment, the ratio of pollen parent to seed parent is 1:100 to 25:100. Depending on the type of interplanting, different ratios of pollen parent to seed parent may be necessary. For the case of row or strip planting, the ratio of pollen parent to seed parent may be ranging from 10:100 to 25:100, such as 15:100 or 20:100. For mixed interplanting, less pollen parent is needed to ensure fertilization of the seed parent. Commonly used ratios range between 1:100 and 15:100, such as 2:100, 3:100, 5:100, 8:100, 10:100 or 12:100.

[0045] Said cereal may be selected from wheat, durum, and triticale.

[0046] In a more preferred embodiment, said cereal is wheat and said seed parent is selected from Triticum aestivum L. ssp. Vulgare Host, Triticum aestivum L. ssp. Compactum, Triticum aestivum L. ssp. Sphaerococcum, Triticum aestivum L. ssp. Petropavlovskyi and Triticum aestivum L. ssp. Tibetianum.

[0047] In another more preferred embodiment, said cereal is wheat and said pollen parent is selected from Triticum aestivum L. ssp. Spelta, Triticum aestivum L. ssp. Macha, Triticum aestivum L. ssp. Vavilovii, Triticum aestivum L. ssp. Yunnanense and a semi-wild form of Triticum aestivum L. ssp. Tibetianum.

[0048] In a more preferred embodiment, the cereal is wheat and seed parent and pollen parent are combined from the above listed cultivars.

[0049] In a preferred embodiment, said cereal is durum wheat and said seed parent is selected from T. turgidum L. ssp. Durum Desf. And T. turgidum L. ssp. Parvicoccum. In another preferred embodiment, said cereal is durum wheat and said pollen parent is selected from T. turgidum L. ssp. Dicoccum (emmer) and T. turgidum L. ssp. Dicoccoides (wild emmer). In a more preferred embodiment, the cereal is durum wheat and seed parent and pollen parent are combined from the above listed cultivars.

[0050] In another preferred embodiment, the genes determining said hulled phenotype are recessively inherited.

[0051] In a further preferred embodiment, said male sterility is based on cytoplasmic male sterility, nuclear male sterility or a trait introduced into said plant by genetic engineering or mutation, or induced using a gametocide.

[0052] During wheat hybrid seed production, different methods can be used to prevent self-pollination of the female line: application of male-specific gametocides on the seed parent, use of genetic cytoplasmic or nuclear-encoded male sterility or use of a trait introduced into the plant by genetic engineering or mutation (for a review see also Perez-Prat and van Lookeren Campagne, 2002, Trends Plant Science 7(5), 199-203).

[0053] Cytoplasmic male-sterile (CMS) lines have one or more mutations in their mitochondrial genome (termed "sterile cytoplasm") resulting in male sterility, and the male sterility is inherited as a dominant, maternally transmitted trait. For cytoplasmic male sterility to be used in hybrid seed production, the seed parent must contain a sterile cytoplasm and the pollen parent must contain (nuclear) restorer genes to restore the fertility of the hybrid plants grown from the hybrid seed harvested from the seed parent; and the CMS mutation should not be associated with a yield penalty.

[0054] Biotechnology has added new possibilities to obtain male-sterile plants, i.e. by introducing genes resulting in traits that confer (conditional) male sterility. For example, a gene, or set of genes may be introduced via genetic engineering, said genes being able to alter the levels of metabolites needed for the production of viable pollen, such as amino acids (WO94/29465), sugars (Goetz et al., 2001, PNAS 98, 6522-6527), flavonols (Derksen et al., 1999, Planta 207, 575-581), jasmonic acid (WO97/10703, McConn and Browse, Plant Cell 8, 403-416; Sanders et al., Plant Cell 12, 1041-1061), biotin (WO99/04023) or auxins (Spena et al., 1992, Theor. Appl. Genet. 84, 520-527). Inducible fertility can be achieved for some of these systems by application of the missing metabolite (see e. g. WO97/10703, McConn and Browse, Plant Cell 8, 403-416; Sanders et al., Plant Cell 12, 1041-1061, WO99/04023, WO93/18142, EP0513884).

[0055] Another approach to general male sterility is effected by the combined action of two (trans)genes brought together into the same plant by crossing two different parental lines each expressing one of the genes (see. E. g. US 5,426,041 or WO98/32325). An example of this approach is the reconstitution of an active toxin from two inactive toxin fragments each encoded by a separate transgene. The combined expression of both transgenes in the male reproductive tissues can result in male sterility. This strategy requires a pollination control system to cross the two fertile grandparent lines to produce the male-sterile female parent line for hybrid seed production.

[0056] Systems that rely on the selective removal of tissues needed for the production of functional pollen by the tissue-specific expression of a gene encoding a protein able to disrupt cell function can be based on both natural traits and genetically engineered traits (see. e. g. Mariani et al., 1990, Nature 347, 737-741; Koltunow et al, 1990, Plant Cell 2, 1201-1224; US 6,072,102 and US5,880,333). Some of these offer the possibility for chemical control of sterility or of fertility. For example, inducible sterility can be obtained by expressing a gene encoding a protein that catalyzes the conversion of a pro-herbicide into a herbicide only in male reproductive tissues (see e. g. O'Keefe et al., 1994, Plant Physiol. 105, 473-482; Kriete et al., 1996, Plant J. 9, 809-818; Dotson et al., 1996, Plant J. 10, 383-392 and US 6,177,616), or by engineering the male sterility gene in such a way that its expression can be induced by the application of a chemical. Similarly, chemical regulation of the expression of a gene that can complement the male sterility (fertility restorer gene) or of a repressor of the male sterility gene can be used to induce fertility (see e. g. US 6,072,102; Ward et al., 1993, Plant Mol Biol 22, 361-366, WO99/42598; US 6,172,279 and WO93/18171).

[0057] Finally, the possibility of making use of natural or induced mutations that cause male sterility has also been described. In this case, the wild-type allele is used as a fertility restorer gene, and its expression is controlled by the application of chemicals.

[0058] One of the most straightforward ways to propagate male-sterile plants is to link a gene conferring dominant male sterility to an herbicide tolerance gene. The heterozygous male-sterile plants are crossed with a wild-type line in the same (isogenic) genetic background. The male-sterile progeny of this cross is selected by herbicide application (see e. g. WO89/10396). This strategy is used successfully for the commercial production of canola hybrids (Brassica napus) in Canada.

[0059] The examples illustrate the present invention.

Example 1: Separation of artificial seed mixtures



[0060] Commercial hulled and free threshing varieties from three cereal species (table 2) were grown at two locations (D-78224 Bohlingen (BO), D-06466 Gatersleben (GA)) and harvested. Two locations where chosen in order to consider influence of environment on plant development and grain sizes. Harvested seed was cleaned from dirt and other unwanted material by wind sieving. The thousand kernel weight (TKW) of the different seed samples was determined (table 3). Seed mixtures were produced in order to have 85% of seed parent seeds and 15% of pollen parent seeds (table 3). Subsamples of 100 g of the mixtures were exposed to simple sieving, using a "Pfeuffer Sortimat" laboratory sieving machine equipped with different sieves of 2.8, 2.7, 2.6, 2.5, and 2.2 mm broadness respectively and 20mm length. Sieving fractions were weighed and sorted by hand in order to determine the number of hulled and free threshing seeds respectively. Results are reported on the sieve which resulted in optimal purity of the target fraction after one sieving step.
Table 2: Sample varieties
EntrySample noSpeciesSeed parentPollen parent
Example varietyExample variety
1 1 Barley Henrike (hulled) Pastello (free threshing)
2 2 Barley Westminster (hulled) Pastello (free threshing)
3 10 Oats Samuel (free threshing) Scorpion (hulled)
4 21 Wheat Tabasco (free threshing) Badenstern (hulled)
5 22 Wheat Genius (free threshing) Badenkrone (hulled)
Table 3: mixing procedure (mixture in % by number of seeds)
 TKW (g)Mixture % by number ofMixture weight (g)
EntrySpeciesMixture partnersLocationhulledfree threshinghulledfree threshinghulledfree threshing
1 Barley Henrike-Pastello BO 55,5 40,5 85,0 15,0 472,0 61,0
1 Barley Henrike-Pastello GA 51,0 49,0 85,0 15,0 130,1 22,1
2 Barley Westminster-Pastello BO 53,5 40,0 85,0 15,0 455,0 60,0
2 Barley Westminster-Pastello GA 49,3 48,0 85,0 15,0 125,7 21,6
3 Oats Scorpion-Samuel BO 36,0 28,6 15,0 85,0 27,0 121,6
3 Oats Scorpion-Samuel GA 39,7 26,6 15,0 85,0 29,8 113,1
4 Wheat Badenstern-Tabasco GA 168,0 47,0 15,0 85,0 126,0 199,8
5 Wheat Badenkrone-Genius GA 160,6 47,6 15,0 85,0 120,5 202,3


[0061] Table 4 shows the target fractions after sieving a sample of 100 g. The number of hulled and free threshing seeds was counted in the target fraction. It was possible to enrich the target fraction in all species and environments higher than the required threshold of 90%. In barley the purity of the target fraction varied from 90% to 95%. The variation of the purity in oats varied between 94% and 96%. The best results for separating hulled and free threshing seeds could be found for wheat. The purity of the target fraction in wheat was in both entries Badenstern-Tabasco and Badenkrone-Genius more than 99%.
Table 4: Purity of the target fraction by separation of hulled and free threshing mixtures after one single sieving step. Sieving of 100 g of seed mixture. Oberfraktion= target fraction in barley: seeds not gone through the sieve have been collected. Unterfraktion= target fraction in wheat and oats: seeds gone through the sieve have been collected.
EntrySpeciesMixture PartnersLocationSieve and fractionWeight (9)Number of seeds hulledNumber of seeds free threshing% of purity of the target fraction after
1 Barley Henrike BO 2.7mm Oberfraktion 89,2 1465 90 94,2%
2 Barley Westminster-Pastello BO 2.7mm Oberfraktion 90,5 1568 133 92,2%
3 Oats Scorpion-Samuel BO 2.6mm Unterfraktion 79,2 162 2663 94,3%
1 Barley Henrike - GA 2,6mm Oberfraktion 83,9 1482 111 93,0%
2 Barley Westminster-Pastello GA 2.7mm Oberfraktion 69,3 1213 61 95,2%
2 Barley Westminster-Pastello GA 2.6mm Oberfraktion 81,9 1408 155 90,1%
3 Oats Scorpion GA 2.6mm Unterfraktion 79,7 116 2913 96,2%
4 Wheat Badenstern-Tabasco GA 3.0mm Unterfraktion 61,4 2 1305 99,8%
5 Wheat Badenkrone-Genius GA 3.0mm Unterfraktion 62,7 5 1313 99,6%


[0062] Table 5 shows the purity of the target fraction before and after sieving the mixed samples.
Table 5: Efficiency of sieving a mixture of hulled and free threshing seed sample in different species
 % of purity of thetargetfraction (number of seeds)
EntrySpeciesMixture PartnersLocationSieve and fractionBefore sievingAfter sieving
1 Barley Henrike BO 2.7mmOberfraktion 85% 94,2%
2 Barley " Westminster-Pastello BO 2.7mmOberfraktion 85% 92,2%
3 Oats Scorpion BO 2.6 mm U nterfraktion 85% 94,3%
1 Barley Henrike-Pastello GA 2,6 mm Oberfraktion 85% 93,0%
2 Barley Westminster-Pastello GA 2.7 mm Oberfraktion 85% 95,2%
2 Barley Westminster-Pastello GA 2.6 mm Oberfraktion 85% 90,1%
3 Oats Scorpion GA 2.6mmUnterfraktion 85% 96,2%
4 Wheat badenstern-Tabasco GA 3.0mmUnterfraktion 85% 99,8%
5 Wheat badenkrone - Genius GA 3.0mmUnterfraktion 85% 99,6%


[0063] The hulled and free threshing seed show different colors. The difference in seed color would allow the seed processor to remove further pollen parent seeds by color sorting machines.

[0064] In conclusion, by combining simple sieving techniques with standard color sorting techniques it is possible to enrich the fraction of F1 seeds harvested from a mixed planting to more than 99%.

Example 2: Separation of harvested seed mixtures from strip planting



[0065] Hulled and free threshing commercial wheat varieties (Table 6) were grown at the location D-78224 Bohlingen. The field design is shown in table 7. For an effective gametocide application, row planting is necessary. The seed parent row was flanked by rows of the pollen parent. As this was a small scale demonstration, rows of 1,0 m length were planted with a plant breeder's sowing machine. One plot therefore consisted of three rows.
Table 6: parents
Seed parent wheat (Triticum aestivum) free threshingPollen parent spelt wheat (Triticum spelta) hulled
Egoist Badenstern
Meister
Tobak
Table 7: field design
Row number123
Spacing (m) 0.2 0.4 0.2
Row length (m) 1 1 1
Treatment with Gametocide no yes no
Species Triticum spelta Triticum aestivum Triticum spelta
Parent Pollen parent Seed parent Pollen parent
Combination 1 Badenstern Egoist Badenstern
Combination 2 Badenstern Meister Badenstern
Combination 3 Badenstern Tobak Badenstern


[0066] In due time, male sterility of the seed parent (middle row) was introduced via treatment with a gametocide (e.g. Genesis) according to the gametocide's instruction manual. Successful introduction of male sterility of the seed parent was checked by bagging some single ears within the seed parent row. Bagged single ears showed no seed set.

[0067] All three rows were harvested together. Harvested seed was cleaned from dirt and other unwanted material by wind sieving.

[0068] Subsamples of 80 g of the plot mixtures were exposed to simple sieving, using a "Pfeuffer Sortimat" laboratory sieving machine equipped with different sieves of 3.0, 2.8, 2.7, and 2.5 mm broadness respectively and 20mm length. Sieving fractions were weighed and sorted by hand in order to determine the number of hulled and free threshing seeds respectively. Due to the architecture of the hulled versus the free threshing wheat seed, separation was already successful with a 3.0 mm sieve after one sieving step, and a more that 99% purity of seed harvested from the seed parent could be achieved.
Table 8: Sieving of harvested F1 seed mixtures
CombinationSeed parentPollen parentYield mixture of seed parent and pollen parent (g)Weight of pollen parent after sieving (3.0 mm sieve) (g)Weight of seed parent after sieving (3.0 mm sieve) (g)Purity of seed parent after sieving (% number of seeds)
1 Egoist Badenstern 164 123 41 99,1
2 Meister Badenstern 141 114 27 99,8
3 Tobak Badenstern 180 136 44 99,4


[0069] Thus, by simple sieving techniques it is possible to enrich the fraction of F 1 wheat seeds harvested in a mixture from row planting to more than 99%.

Example 3: Production of wheat hybrid seed by using hulled pollen parents, where the seed parent exhibits cytoplasmatic male sterility (CMS) and is a free threshing hexaploid winter wheat.



[0070] The pollen sterile A-line "CMS-WW005" (Triticum aestivum) was used as a seed parent. This line exhibits cytoplasmatic male sterility (CMS). This line is a free threshing hexaploid winter wheat. Hybrid production was performed in mixed planting in the greenhouse at Gatersleben. Seed parent was pollinated by open pollination with the pollen parents of the variety Badenstern or Badenkrone (Triticum spelta). Badenstern or Badenkrone are fully fertile pollen parents with non-free threshing (hulled) grain set. Another hybrid seed production was performed in mixed planting in the counter season nursery in Pochefstroom /Rep. South Africa. Mixing rate was 50% seed parent and 50% pollen parent in order to have enough pollen. Plot size at all locations was 1 m 1.2 m, with four rows in Gatersleben and three rows in Pochefstroom.
Table 1: parents
Seed parent wheat (Triticum aestivum) free threshingPollen parent spelt wheat (Triticum spelta) hulledGrown in Gatersleben (green house)Grown in Pochefstroom
CMS-WW005 Badenstern Yes sample 1 Yes sample 2
CMS-WW005 Badenkrone no Yes, but no F1 seeds produced sample 4
- Badenstern Yes sample 3 no


[0071] In due time, male sterility of the seed parent was checked by bagging some single ears of seed parent plants. Bagged single ears showed no seed set.

[0072] At both locations, plots were harvested and threshed. Harvested seed was cleaned from dirt and other unwanted material by wind sieving. Seeds from the counter season nursery was sent to Gatersleben for further examination. Sample 4, i.e. the harvest from the production of CMS-WW005 Badenkrone at Pochefstroom, did not contain F1 seeds but only inbred seeds from the pollen parent, as the nicking was insufficient (flowering time of seed and pollen parent differed too much) and seed parent plants were not pollinated. Sample 3 only contained inbred seed from the pollen parent Badenstern and was harvested in Gatersleben for comparison purposes.

[0073] Subsamples of 80 g of the samples were exposed to simple sieving, using a "Pfeuffer Sortimat" laboratory sieving machine equipped with different sieves of 3.0, 2.8, 2.7, and 2.5 mm width respectively and 20mm length. Sieving fractions were weighed and sorted by hand in order to determine the number of hulled and free threshing seeds respectively.

[0074] Due to the architecture of the hulled versus the free threshing seed, separation was very successful with a 3.0 mm sieve after one sieving step, and a fraction of more than 90% of free threshing seeds was obtained (Table 2). That fraction still contained broken husks of hulled seed, which were easily removed by a seed cleaner (aspirator) in a second step (Table 2). This second step resulted in 100% seeds of free threshing phenotype. A closer visual inspection of the free threshing seeds revealed that some of the seeds had a slimmer shape, thus resembling dehulled T. spelta grains (25 and 6 seeds in sample 1 and sample 2 respectively, data not shown). This led to the conclusion that a too harsh threshing process partly disrupted some of the pollen parent husks and caused the release of some spelt grains. This finding was confirmed by evaluating the sieving of seeds harvest from pure pollen parent Badenstern or Badenkrone as shown in sample 3 and sample 4 (Table 3). Among the 1877 hulled seeds from T.spelta (pollen parent) 30 free threshing grains where found (1.6%). Therefore, sorting a mixture by sieves, aspirator and color sorter would result in a purity rate of 97-98%. This is far above the recommended hybridity of at least 90% (OECD SEED SCHEMES 2014: OECD Schemes for the Varietal Certification or the Control of Seed Moving in International Trade, ANNEX VIII, pages 99-101, OECD 2014, http://www.oecd.org/tad/code/1_compIete%20document.pdf).
Table 2: Sorting of harvested F1 seed mixtures.
Results of first sieving step, where fraction 2 contained free threshing and hulled types (*), results of second sorting step fraction FT.
sampleYield mixture of seed parent and pollen parent (g)weight offraction 1 Elements larger than 3.0mm diameter (hulled seeds) (g)weight offraction 2 elements smaller than 3.0 mm diameter (mainly free threshing seeds) (g)purity of fraction 2* after first step %weight offraction FT after second sorting step: elements smaller than 3.0 mm diameter (only free threshing seeds) (g) 46purity of fraction FT after second step %
Sample 1 245.6 178.6 67 90% 10.2 97.8%
Sample2 124.1 108.7 15.4 90%   98.5%
N.b.: at Pochefstroom, TKW was only 26 g, whereas at in Gatersleben TKW was 40.3 g.
Table 3: Sorting the pollen parent seed by sieve, wind and color sorter
sampleyield of pollen parentnumber of hulled seedsnumber of free threshing elementspercentage false positives free threshing
Sample 3 201 1877 30 1.6%
Sample 4 49.5 515 25 4.9%


[0075] Thus, by simple sorting techniques it is possible to enrich the fraction of F1 wheat seeds harvested in a mixture to more than 97%.


Claims

1. Method of separating hybrid wheat seeds from a mixture comprising said hybrid wheat seeds and inbred wheat seeds, comprising

(a) providing a mixture of wheat seeds, wherein said mixture of seeds has been obtained by interplanting a combination of plants comprising a male sterile or conditionally male sterile seed parent and a male fertile pollen parent, wherein said seed parent and pollen parent produce at least two types of seeds, at least one of which being said hybrid seeds, said at least two types of seed having different phenotypes allowing separation of said hybrid seed with non-invasive methods based on the differences in said phenotypes, said different phenotypes being determined by an allele or a combination of alleles at one or more genetic loci, wherein said allele or combination of alleles at one or more genetic loci are selected from alleles of the Q, Tg or Sog gene, wherein said seed parent is homozygous for a first allele or combination of alleles at said one or more genetic loci resulting in a first phenotype and said pollen parent is homozygous for a second allele or combination of alleles at said one or more genetic loci resulting in a second phenotype; wherein said seed parent is homozygous for one or a combination of alleles at one or more genetic loci, wherein at least one of said alleles in said seed parent is dominant or partially dominant, and said pollen parent is homozygous for one or a combination of alleles at said one or more genetic loci, wherein at least one of said alleles in said pollen parent is recessive or partially recessive; and wherein said combination of plants is characterized in that

(i) said one allele or combination of alleles at one or more genetic loci in said seed parent manifests in seeds with a free-threshing phenotype and wherein said one allele or combination of alleles at one or more genetic loci in said pollen parent manifests in seeds with a hulled phenotype; or

(ii) said one allele or combination of alleles at one or more genetic loci in said seed parent manifests in seed with a hulled phenotype and wherein said one allele or combination of alleles at one or more genetic loci in said pollen parent manifests in seeds with a free-threshing phenotype;

(b) separating said hybrid seeds using a separation method based on differences in size or specific weight of grains.


 
2. The method of claim 1, wherein said planting comprises row or strip planting or mixed interplanting.
 
3. Method of separating hybrid wheat seed from a mixture of said hybrid wheat seed and inbred wheat seed comprising providing said mixture, wherein embryos of said hybrid seeds have a genotype comprising one haploid genome copy of the seed parent and one haploid genome copy of the pollen parent, wherein embryos of said inbred seeds have a genotype comprising two haploid genome copies of the pollen parent, wherein said seed parent is male sterile or conditionally male sterile and wherein said pollen parent is male fertile, characterized in that

(a) said seed parent is homozygous for seed with a free-threshing phenotype and said pollen parent is homozygous for seed with a hulled phenotype, or

(b) said seed parent is homozygous for seed with a hulled phenotype and said pollen parent is homozygous for seeds with a free-threshing phenotype;
comprising applying said mixture of seeds to a means for separation according to size or specific weight of the grains, wherein said free-threshing or hulled phenotype is characterized by the presence of one or a combination of alleles at one or more genetic loci which are selected from alleles of the Q, Tg or Sog gene.


 
4. The method of any one of claims 1 to 3, wherein

(a) said cereal is wheat and wherein

(i) said seed parent is selected from Triticum aestivum L. ssp. vulgare Host, Triticum aestivum L. ssp. compactum, Triticum aestivum L. ssp. sphaerococcum, Triticum aestivum L. ssp. petropavlovskyi and Triticum aestivum L. ssp. tibetianum and/or

(ii) said pollen parent is selected from Triticum aestivum L. ssp. spelta, Triticum aestivum L. ssp. macha, Triticum aestivum L. ssp. vavilovii, Triticum aestivum L. ssp. yunnanense and a semi-wild form of Triticum aestivum L. ssp. Tibetianum.


 
5. The method of any one of claims 1 to 4, wherein the genes determining said hulled phenotype are recessively inherited.
 


Ansprüche

1. Verfahren zum Trennen von Hybridweizensaat aus einer Mischung, die die Hybridweizensaat und Inzuchtweizensaat aufweist, aufweisend:

(a) Bereitstellen einer Mischung aus Weizensaat, wobei die Mischung aus Saat durch Interplantieren einer Kombination aus Pflanzen erzielt wurde, die ein männliches steriles oder konditionell männliches steriles Saatelternteil und ein männliches fertiles Pollenelternteil aufweist, wobei das Saatelternteil und das Pollenelternteil mindestens zwei Arten von Saat produzieren, wobei mindestens eine davon die Hybridsaat ist, wobei die mindestens zwei Arten von Saat verschiedene Phänotypen aufweisen, die die Trennung der Hybridsaat mit nichtinvasiven Verfahren basierend auf den Unterschieden der Phänotypen ermöglichen, wobei die verschiedenen Phänotypen durch ein Allel oder eine Kombination von Allelen an einem oder mehr genetischen Orten bestimmt werden, wobei das Allel oder die Kombination von Allelen an einem oder mehr genetischen Orten aus Allelen des Q-, Tg- oder Sog-Gens ausgewählt werden, wobei das Saatelternteil homozygot für ein erstes Allel oder Kombination von Allelen an dem einen oder mehr genetischen Orten ist, was zu einem ersten Phänotyp führt, und das Pollenelternteil homozygot für ein zweites Allel oder Kombination von Allelen an dem einen oder mehr genetischen Orten ist, was zu einem zweiten Phänotyp führt; wobei das Saatelternteil homozygot für ein oder eine Kombination von Allelen an einem oder mehr genetischen Orten ist, wobei mindestens eines der Allele im Saatelternteil dominant oder teilweise dominant ist, und das Pollenelternteil homozygot für ein oder eine Kombination von Allelen an dem einen oder mehr genetischen Orten ist, wobei mindestens eines der Allele im Pollenelternteil rezessiv oder teilweise rezessiv ist; und wobei die Kombination von Pflanzen dadurch gekennzeichnet ist, dass

(i) sich das eine Allel oder die Kombination von Allelen an einem oder mehr genetischen Orten im Saatelternteil in Saat mit einem freidreschenden Phänotyp manifestiert, und wobei sich das eine Allel oder die Kombination von Allelen an einem oder mehr genetischen Orten im Pollenelternteil in Saat mit einem enthülsten Phänotyp manifestiert; oder

(ii) sich das eine Allel oder die Kombination von Allelen an einem oder mehr genetischen Orten im Saatelternteil in Saat mit einem enthülsten Phänotyp manifestiert, und wobei sich das eine Allel oder die Kombination von Allelen an einem oder mehr genetischen Orten im Pollenelternteil in Saat mit einem freidreschenden Phänotyp manifestiert;

(b) Trennen der Hybridsaat unter Verwendung eines Trennungsverfahrens basierend auf Unterschiedenen in der Größe oder im spezifischen Gewicht von Körnern.


 
2. Verfahren nach Anspruch 1, wobei das Pflanzen Reihen- oder Streifenpflanzen oder gemischtes Interplantieren aufweist.
 
3. Verfahren zum Trennen von Hybridweizensaat aus einer Mischung der Hybridweizensaat und Inzuchtweizensaat, aufweisend das Vorsehen der Mischung, wobei Embryonen der Hybridsaat einen Genotyp aufweisen, der eine haploide Genomkopie des Saatelternteils und eine haploide Genomkopie des Pollenelternteils aufweist, wobei Embryonen der Inzuchtsaat einen Genotyp aufweisen, der zwei haploide Genomkopien des Pollenelternteils aufweist, wobei das Saatelternteil männlich steril oder konditionell männlich steril ist, und wobei das Pollenelternteil männlich fertil ist, dadurch gekennzeichnet, dass

(a) das Saatelternteil homozygot für Saat mit einem freidreschenden Phänotyp ist und das Pollenelternteil homozygot für Saat mit einem enthülsten Phänotyp ist, oder

(b) das Saatelternteil homozygot für Saat mit einem enthülsten Phänotyp ist und das Pollenelternteil homozygot für Saat mit einem freidreschenden Phänotyp ist;

aufweisend das Anwenden der Mischung aus Saat auf ein Mittel zur Trennung gemäß Größe oder spezifischem Gewicht der Körner, wobei der freidreschende oder enthülste Phänotyp durch die Anwesenheit von einem oder einer Kombination von Allelen an einem oder mehr genetischen Orten gekennzeichnet ist, die aus Allelen des Q-, Tg- oder Sog-Gens ausgewählt werden.
 
4. Verfahren nach einem der Ansprüche 1 bis 3, wobei

(a) das Getreide Weizen ist, und wobei

(i) das Saatelternteil aus Triticum aestivum L. ssp. vulgare Host, Triticum aestivum L. ssp. compactum, Triticum aestivum L. ssp. sphaerococcum, Triticum aestivum L. ssp. petropavlovskyi und Triticum aestivum L. ssp. tibetianum ausgewählt wird, und/oder

(ii) das Pollenelternteil aus Triticum aestivum L. ssp. spelta, Triticum aestivum L. ssp. macha, Triticum aestivum L. ssp. vavilovii, Triticum aestivum L. ssp. yunnanense und einer halbwilden Form von Triticum aestivum L. ssp. Tibetianum ausgewählt wird.


 
5. Verfahren nach einem der Ansprüche 1 bis 4, wobei die Gene, die den enthülsten Phänotyp bestimmen, rezessiv ererbt sind.
 


Revendications

1. Procédé de séparation de semences de blé hybrides à partir d'un mélange comprenant lesdites semences de blé hybrides et des semences de blé consanguines, comprenant les étapes consistant à :

(a) fournir un mélange de semences de blé, ledit mélange de semences ayant été obtenu par inter-plantation d'une combinaison de plantes comprenant un parent de semence mâle stérile ou mâle stérile conditionnellement et un parent de pollen mâle fertile, dans lequel lesdits parent de semence et parent de pollen produisent au moins deux types de semences, au moins l'un d'entre eux étant lesdites semences hybrides, lesdits au moins deux types de semences ayant différents phénotypes qui permettent d'obtenir une séparation de ladite semence hybride avec des procédés non invasifs basés sur les différences dans lesdits phénotypes, lesdits phénotypes différents étant déterminés par un allèle ou une combinaison d'allèles au niveau d'un ou de plusieurs locus/loci génétique(s), dans lequel ledit allèle ou ladite combinaison d'allèles au niveau d'un ou de plusieurs locus/loci génétique(s) est choisi(e) parmi les allèles des gènes Q, Tg ou Sog, dans lequel ledit parent de semence est homozygote pour un premier allèle ou une première combinaison d'allèles au niveau dudit un ou desdits plusieurs locus/loci génétique(s) conduisant à un premier phénotype et ledit parent de pollen est homozygote pour un deuxième allèle ou une deuxième combinaison d'allèles au niveau dudit un ou desdits plusieurs locus/loci génétique(s) conduisant à un deuxième phénotype ; dans lequel ledit parent de semence est homozygote pour un ou une combinaison d'allèle(s) au niveau d'un ou de plusieurs locus/loci génétique(s), dans lequel au moins un desdits allèles dans ledit parent de semence est dominant ou partiellement dominant, et dans lequel ledit parent de pollen est homozygote pour un ou une combinaison d'allèle(s) au niveau dudit un ou desdits plusieurs locus/loci génétique(s), dans lequel au moins un desdits allèles dans ledit parent de pollen est récessif ou partiellement récessif ; et dans lequel ladite combinaison de plantes est caractérisée en ce que

(i) ledit un allèle ou ladite combinaison d'allèles au niveau d'un ou de plusieurs locus/loci génétique(s) dans ledit parent de semence se manifeste dans les semences par un phénotype sans battage et dans lequel ledit un allèle ou ladite combinaison d'allèles au niveau d'un ou de plusieurs locus/loci génétique(s) dans ledit parent de pollen se manifeste dans les semences par un phénotype décortiqué ; ou

(ii) ledit un allèle ou ladite combinaison d'allèles au niveau d'un ou de plusieurs locus/loci génétique(s) dans ledit parent de semence se manifeste dans les semences par un phénotype décortiqué et dans lequel ledit un allèle ou ladite combinaison d'allèles au niveau d'un ou de plusieurs locus/loci génétique(s) dans ledit parent de pollen se manifeste dans les semences par un phénotype sans battage ;

(b) séparer lesdites semences hybrides en utilisant un procédé de séparation basé sur des différences de taille ou de poids spécifiques des grains.


 
2. Procédé de la revendication 1, dans lequel ladite plantation comprend une plantation en rangées ou en bandes ou bien une inter-plantation mélangée.
 
3. Procédé de séparation d'une semence de blé hybride à partir d'un mélange de ladite semence de blé hybride et d'une semence de blé consanguine comprenant la fourniture dudit mélange, dans lequel des embryons desdites semences hybrides ont un génotype comprenant une copie du génome haploïde du parent de semence et une copie du génome haploïde du parent de pollen, dans lequel des embryons desdites semences consanguines ont un génotype comprenant deux copies du génome haploïde du parent de pollen, dans lequel ledit parent de semence est mâle stérile ou mâle stérile conditionnellement et dans lequel ledit parent de pollen est mâle fertile, caractérisé en ce que

(a) ledit parent de semence est homozygote pour une semence avec un phénotype sans battage et ledit parent de pollen est homozygote pour une semence avec un phénotype décortiqué ou

(b) ledit parent de semence est homozygote pour une semence avec un phénotype décortiqué et ledit parent de pollen est homozygote pour une semence avec un phénotype sans battage ;
comprenant une application dudit mélange de semences à un moyen destiné à une séparation selon la taille ou le poids spécifique des grains, dans lequel ledit phénotype sans battage ou décortiqué est caractérisé par la présence d'un ou d'une combinaison d'allèle(s) au niveau d'un ou de plusieurs locus/loci génétique(s) qui est choisi(e) parmi les allèles des gènes Q, Tg ou Sog.


 
4. Procédé de l'une quelconque des revendications 1 à 3, dans lequel

(a) ladite céréale est le blé et dans lequel

(i) ledit parent de semence est choisi parmi Triticum aestivum L. ssp. vulgare Host, Triticum aestivum L. ssp. compactum, Triticum aestivum L. ssp. sphaerococcum, Triticum aestivum L. ssp. petropavlovskyi et Triticum aestivum L. ssp. tibetianum et/ou

(ii) ledit parent de pollen est choisi parmi Triticum aestivum L. ssp. spelta, Triticum aestivum L. ssp. macha, Triticum aestivum L. ssp. vavilovii, Triticum aestivum L. ssp. yunnanense et une forme semi-sauvage de Triticum aestivum L. ssp. Tibetianum.


 
5. Procédé de l'une quelconque des revendications 1 à 4, dans lequel les gènes déterminant ledit phénotype décortiqué sont hérités de manière récessive.
 




Drawing


























Cited references

REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description




Non-patent literature cited in the description