WO2016156407A1

WO2016156407A1 - Hybrid barley plant

Info

Publication number: WO2016156407A1
Application number: PCT/EP2016/056930
Authority: WO
Inventors: Anne-Marie Bochard; Thomas Jolliffe; Mark Glew
Original assignee: Limagrain Europe
Priority date: 2015-03-31
Filing date: 2016-03-30
Publication date: 2016-10-06

Abstract

The invention is in the field of plant genetics. The invention more specifically relates to a method to produce in the field new hybrid two-row barley plants with good yield performance. The invention also relates to the obtained hybrid two-row barley plant per se.

Description

HYBRID BARLEY PLANT

The invention is in the field of plant genetics. The invention more specifically relates to a method to produce new hybrid two-row barley plants with good yield performance. The invention also relates to the obtained hybrid two-row barley plant per se.

BACKGROUND OF THE INVENTION

Barley (e.g. Hordeum vulgare L.) is one of the major cereals in the world from an economic perspective and more specifically in Europe. The main use of barley varieties are for the production of malt, brewing, distilling and in the food and feed industries. For these applications, it is a perennial challenge to concurrently improve agronomic, morphological and industrial traits, affecting crop performance, yield, and grain and industrial quality.

Barley breeding programs have to take into account not only the quality traits required for malting, brewing and distilling, but also agronomic and morphological traits, particularly those affecting plant phenotype and yield. Yield is a very complex trait which can be affected by many factors such as earliness of flowering, spikelet formation and fertility, period of grain filling, grain maturity and harvest date, average kernel weight, size and uniformity, plant height, brackling, lodging and harvestability, tillering, number of ears per unit area, pest and disease resistance, tolerance and avoidance, pre-harvest grain loss and spoilage etc.

Barley forms three single-flowered spikelet per rachis node: Barley varieties in which all three spikelets, one central and two lateral, produce seeds are called six-rowed (six-row) barley, whereas varieties in which the lateral spikelets are infertile or vestigial and only the central spikelets produce seeds are generally called two-rowed (two-row) barley (Ramsay et al, 201 1 ).

Cultivated two-row barleys usually have Vrsl.b or vrsl.t alleles, vrsl.t being a post- domestication mutation resulting in deficiens phenotype with vestigial lateral spikelets. Cultivated six-row barleys usually have one of the three six-row vrsl.a mutations: vrsl.al, vrs1.a2 or vrs1.a3. The VRS1 gene encodes a homeodomain-leucine zipper l-class homeobox gene located on chromosome 2HL (EP1970447). This transcriptional repressor inhibits the development of fertile lateral spikelets and results in a two-row type barley. Loss of function of VRS1 is sufficient to convert the rudimentary (infertile or vestigial) spikelets of two-rowed barley into the fertile spikelets of six-rowed barley. This loss of function has occurred independently several times during barley domestication. Komatsuda et al (PNAS, 2007, 104, pp1424-1429) have further identified several mutations in VRS1 gene capable of altering a two-row type in a six-row type.

INTERMEDIUM-SPIKE-C {INT-C) gene can also affect spike morphology as it is involved in the lateral spikelet development. The effects of these two genes (VRS1 and INT-C) are complimentary to one other in the sense that the vrsl alleles are involved in the formation of lateral spikelets, whereas the int-c alleles are involved in development of lateral spikelets and affect floret fertility and seed set. Most two-row type barley plants carry the int-c.b allele while most of the six-row type barley plants carry Int-c.a allele. Two-row type Vrs1.b/int-c.b have small and non-fertile lateral spikelets while six-row type vrsl. al Int-c.a has large and fertile lateral spikelets (Lundqvist, 1997).

Two-row plants (Vrsl.b or vrsl.t) carrying the six-row Int-c.a allele, have enlarged and partially sterile lateral spikelets. This intermediate state between the standard two- and six-row types is a characteristic of the Intermedium phenotype (Ramsay et al, 201 1 ) and leads to reduced yield.

In hybrid combinations of two-row (Vrsl.b int-c.b) and six-row barley (vrsl.a Int-c.a), the Vrsl.b dominance is compensated by INT-C gene to give intermediate phenotype with reduced level of fertility of lateral spikelets. This lack of fertility in such cross can be seen as a significant and substantial restriction in the industrial exploitation of the large genetic variability currently existing between two-row and six-row barley types barley.

One important challenge for barley crop development is to find a hybrid system that can be used in industrial hybrid seed production based on the combination of genetic backgrounds provided by different barley types and on a good level of heterosis between parent lines that can lead to hybrid varieties with high yield. Heterosis has been reported in the literature when the hybrid shows trait value above the midparent value or the highest parent trait value or above the best commercial inbred cultivars trait value. Many attempts have been made to explore heterosis effect in barley hybrids (Ramage, 1983). Publications describe experiments notably based on crosses between six-row and two-row type barley plants. Most of these examples are based on yield trials made with hand-cross seeds, with low seeding rates, in greenhouse conditions and the yield complex trait is measured indirectly through yield components (like number of ears per plant, grain number per ear and thousand kernel weight). However, it is known that compensation effects can exist between yield components, which might lead to negative correlations between them (Adams, 1967).

Carleton and Foote (1968) showed that there were no significant increases in the yield components when comparing hybrid to parent lines. The authors conclude on a general lack of heterosis when considering complex traits like yield.

Tseng and Poehlman (1974) showed that the best expression of heterosis in a six-row by two-row or a two-row hybrid was reported for kernel weight out of the different yield components analyzed. For grain yield per plant or per plant and grain weight, the authors state that most of the hybrid crosses were not significantly higher than the midparent.

More recently, Rugen et al (2004) assessed heterosis performance in barley. The authors indicate that yield components such as thousand kernel weight (TKW) and kernel yield per plant (KY) were measured and conclude that stronger heterosis was shown by crossing six-row by six-row or two-row by two-row, in contrast to those exhibited by six- row by two-row.

Gymer (1976 and 1977) concluded that there was significant heterosis in six-row by two- row type crosses for grain yield/plot. However, the author noted that experiments are made in 25 X25 cm plots in a greenhouse under favourable conditions, which are therefore not comparable with field hybrid seed production. Moreover, the seeds used in this experiment were obtained by hand-crossing, which can lead to seeds having different behaviour due to the stress caused by hand-crossing, compared to seeds produced in natural conditions.

Besides, only yield components were measured and only the grain yield per plot of hybrids showed an increase compare to the parents. For all of the other components, the parental performances were better than those of the hybrids. Komatsuda et al (2004) described different recombinant populations in order to map the six-row spike locus vrsl. Consequently, this document does not deal at all with the production of hybrid plants (F1 ).The purpose of the analysis was only to map the location of vrsl locus using six different segregating F2 populations (see table 1 ). The row type phenotype was assessed in the F2 plants and the genotype was assessed in F3 plants (p69 "Genotyping and linkage analysis'). Besides, this document does not describe the use of a sterility/restoration system to allow industrial hybrid variety production.

Accordingly, there is still a need to provide hybrid barley plants with improved yield, in particular two-row hybrid barley plants, and to provide industrial process in order to be able to combine different genetic pools or backgrounds to increase the genetic variability used in commercial barley hybrids.

The present invention provides a method to produce in the field new two-row barley hybrids exhibiting higher heterosis and increased yield compared to the parent lines and compared to other hybrids. SUMMARY

Thus, a first aspect relates to a method to obtain a two-row barley hybrid plant comprising crossing a first parent six-row barley plant with a second parent two-row deficiens type barley plant, and obtaining a hybrid two-row barley plant from said crossing, wherein the first or second parent plant is male sterile. In specific embodiments, the two-row deficiens type barley parent plant is characterized by the presence of the vrsl.t and int-c.b alleles.

In other specific embodiments that may be combined with previous embodiments, the six- row type barley parent plant is characterized by the presence of vrsl.a and Int-c.a alleles.

In one preferred embodiment that may be combined with previous embodiments, the first or second parent plant comprises a cytoplasmic control of male sterility and the other parent plant comprises a dominant fertility restoration locus, and wherein said crossing is obtained in open fields. For example, one parent plant is a male sterile barley plant comprising an msm-1 cytoplasm and the second plant is a fertile barley plant comprising the Rfm-1 fertility restoration locus. Another aspect relates to a method for improving the yield of a two-row barley plant, said method comprising crossing a first six-row barley parent plant with a second two-row deficiens type barley plant and obtaining a hybrid two-row barley plant from said crossing, wherein the yield of said hybrid two-row barley plant is improved as compared to the yield of either of the parent plants.

The invention also relates to a two-row barley hybrid plant obtainable or obtained by the methods described herein. In specific embodiments, a two-row barley hybrid plant according to the invention is heterozygous with vrsl.t I vrsl.a alleles, and said hybrid plant further comprises a dominant fertility restoration gene. Advantageously, the two-row barley hybrid plant of the invention has an improved yield in open field as compared to the yield of the parent plants under similar environmental conditions.

In specific embodiments that may be combined with the previous embodiments, the two- row barley hybrid plant of the invention is further heterozygous for int-c alleles, for example int-c. b / Int-c. a.

In a preferred embodiment, the two-row barley hybrid plant of the invention is characterized by the presence of a male sterile cytoplasm and a fertility restoration locus. For example, the two-row hybrid barley plant is characterized by the presence of msml and/or msm2 male sterile cytoplasm and the presence of Rfmla fertility restoration locus. In specific embodiments that may be combined with the previous embodiments, the barley hybrid plant of the invention further comprises dwarf or semi-dwarf alleles.

In particular, the hybrid barley of the invention is a winter barley.

Another aspect of the invention relates to a method of identifying the two-row barley hybrid of invention including the use of markers linked to VRS1, INT-C and/ Rfmla alleles, thereby identifying said two-row barley hybrid plant.

DETAILED DESCRIPTION

Method of obtaining a two-row barley hybrid plant with improved yield

One of the objectives of the present invention was to provide a method for producing a two-row barley hybrid plant exhibiting higher heterosis and increased yield compared to the parent lines and/or compared to other hybrids known in the art. Another objective of the present invention was to obtain such two-row hybrid plants in open fields, without human intervention, i.e. by using hybrid large-scale production systems available in the art, such as male cytoplasmic sterility systems.

Accordingly, in one aspect, the invention relates to a method to obtain a two-row barley hybrid plant comprising crossing a first parent six-row barley plant with a second parent two-row deficiens type barley plant, and obtaining a hybrid two-row barley plant from said crossing, wherein at least one of the parent plants is male sterile, thereby reducing self- fertilization of said parent plants.

As used herein, the term "barley plant" comprises plants of the genus Hordeum, preferably of the species Hordeum vulgare. The plant may belong to cultivated barley, i.e. Hordeum vulgare ssp. Vulgare or to wild barley, i.e. Hordeum vulgare ssp. spontaneum. Hybrid or crossbred varieties between cultivar and wild barley are also acceptable.

"Barley plant" may comprise at least three different classes of barley, generally delineated according to their response to day length and need for vernalization and affecting growth habit: winter, facultative and spring barley. For example, in Europe and North America winter barley is often planted in late autumn and is harvested in the following summer whereas spring barley is often planted in spring and facultative barley is often planted in either late autumn or early spring. However, in some regions of the world these classes of barley is planted in different seasons depending on the local climate. Winter and facultative barleys tend to be more cold tolerant than spring barley.

In a particular embodiment, the barley plant of the invention is a winter barley. However the present invention could be applied to any other class of barley. In specific embodiments, the barley parent plants are derived from elite plants, for example msm1 elite plants, or Rfmla elite plants. An "elite plant" is a plant within the meaning of the present invention which is sufficiently homogenous to be used for commercial grain production, but which might also be used for further breeding steps. Examples of elite plants of the species Hordeum vulgare are the European cultivars "Ketos", "Captain", "California" and "Caribic". As used herein, the "two-row deficiens type barley plant" refers to a two-row plant with vestigial lateral spikelets (Pourkheirandish and Komatsuda, 2007). Specific examples of barley plant with two-row deficiens type are the varieties "California", "Campanile" and "Calypso". As used herein, the "six-row phenotype" (hereinafter referred to as "six-row type" or "6R type") refers to a panicle phenotype in which the degree of lateral spikelet development is comparable to that of the central spikelet. Specific examples of barley plant with six-row type are the varieties "Ketos" and "Rafaela".

As used herein, the term "hybrid" refers to the progeny of two genetically non-identical, parents which are produced by cross-pollination of genetically different parental lines. The hybrid barley plant of the invention usually shows the so-called "heterosis effect", which means that they display superior plant growth, feed yield and/or a pronounced stress tolerance in comparison to both parental lines.

As used herein the term "hybrid large-scale production system" refers to any system known in the art, for favouring, increasing, or ameliorating cross-pollinating plants instead of self-pollinating plants, in field conditions, for example by reducing, decreasing or avoiding self-fertilization of at least one of the parent plant.

Hybrid large-scale production systems include, mechanical, chemical or male sterility.

Typically, hybrid large-scale production system for producing barley hybrid plants may be based on cytoplasmic or nuclear male sterility systems. Male sterility systems usually comprises a cytoplasmic male sterility gene and a dominant nuclear fertility restoration gene (cytoplasmic male sterility system) or a recessive nuclear male sterile gene (genie male sterility system).

Accordingly, in one specific embodiment of the method, either the first or the second parent plant comprise cytoplasmic male sterility resulting in a male sterile cytoplasm, and the other parent plant comprises a dominant nuclear fertility restoration gene, said crossing being obtained in open fields.

Cytoplasmic male sterility systems have been described in the literature and used for the production of barley hybrid crop. For example, Schooler and Foster (1968) and Foster and Schooler (1970) described a cytoplasmic male sterility and fertility restoration genes derived from crosses with Hordeum jubatum. Ahokas (1979, 1982a and 1982b) described a cytoplasmic male sterility and fertility restoration genes derived from crosses with Hordeum spontaneum.

This system uses the two CMS cytoplasms known msml and msm2. The fertility restoration gene for both sterility cytoplasms is known as Rfm1 and the locus has been characterized molecularly (Matsui K., 2001 ). Accordingly, in one specific embodiment, one parent plant used in the method of the invention is a male sterile barley plant comprising an msml and/or msm2 cytoplasm and the second plant is a male fertile barley plant comprising the Rfm1 fertility restoration locus. Many male sterility genes have been identified in barley and are described in Ahokas H. (1998).

As used herein, the term "open field" is used as opposed to culture in a greenhouse. In particular, in open fields, the temperature, day length and humidity cannot be controlled.

Genetic DNA markers are available for most if not all these mutations, but phenotypic (i.e. visible) markers can also be used to follow, for example, alleles that compensate for the presence of a genie male sterile gene. Other markers, for example, associated with the blue pericarp or red colour, can be used to facilitate the recovering of sterile or fertile seeds. Chromosome Addition Lines can be used to compensate for genie male sterility. This way was the first developed to create hybrids in barley (Ramage R. T.,1965). Male sterile parent lines used for the method of the invention may advantageously be dwarf, semi-dwarf or double-dwarf plants or otherwise and under whatever genetic control of reduced or enhanced stature (see e.g. WO2015135940 for a description of the method using such plants for hybrid production).

The height difference between the two sets of parent plants allows an optimization of the pollen diffusion from taller plants to the shorter set of parent plants, female can be shorter; however standard female and taller male plants may be used as well, as may be shorter males and even shorter females or any other combination that creates a high difference and a desirable hybrid issue.

In order to prevent or reduce the production of self-fertilized seeds by fertile plants between anthesis, preferably near the end of anthesis and harvest, a tool, such as a weed wiper, applying a chemical, preferably a herbicide, preferably a herbicide that is systemic can be used. This treatment allows the harvest in a field comprising shorter female (male sterile) plants and taller fertile plants for producing hybrid barley seeds, wherein the chemical, e.g. a herbicide is applied, at least once, to the taller fertile plants extending above the height of the shorter female plants between anthesis and harvest. Alternatively, a tool can be used, for example, to cut taller plants between anthesis and harvest. In barley, many mutations affected in plant height are known, see Franckowiak et al., (1987). The most frequently used dwarfism genes are:

- Ari-e GP (Braumann et ai, 2014)

Sdw1, allelic to denso

- Sdw3 (Gottwald et al., 2004).

- Uzu

Dwarf barley plants can also be obtained by simple selection by choosing short plants in plant breeding populations or by pyramiding height QTL (Quantitative Trait Loci), or GMO (Genetically Modified Organism) strategies. The above male sterility systems are examples of the hybridization system that can be used to implement the present invention and shall not be limited to the systems provided above.

In a specific embodiment of the invention, the two-row deficiens type barley parent plant is characterized by the presence of vrsl.t and int-c.b alleles. In other specific embodiment, the six-row barley parent plant is characterized by the presence of vrsl.a and Int-c.a alleles.

For example, the method of the invention comprise crossing a two-row deficiens type barley parent plant characterized the presence of vrsl.t and int-c.b alleles with a six-row barley plant characterized by the presence of vrsl.a and Int-c.a alleles. The hybrid barley plant

The present invention also relates to the hybrid barley plant obtained or obtainable by the method described above. Said hybrid barley plants are characterized in that

(i) they are two-row barley plants,

(ii) they include at least a vrsl.t allele (obtained from the parent two-row deficiens barley plant), for example, they are heterozygous with vrs1.t/vrs1.a alleles,

(iii) they comprise a fertility restoration locus (obtained from one of the parent barley plant) and a male sterile cytoplasmic locus (obtained from the other parent barley plant), for example, they are heterozygous with Rfm1a/rfm1a alleles, (iv) optionally, they are further heterozygous for int-c alleles, for example int-c.b and Int-c.a alleles.

In a specific embodiment, the hybrid barley plant obtainable by the method includes msml and/or msm2 male sterile cytoplasm and Rfm1 fertility restoration allele. The hybrid barley plant has advantageously an improved yield in open field compared to the yield of parent lines.

As used herein, the yield is expressed as ql/ha (or equivalently 100kg/ha) and corresponds to the adjusted mean of yield determined at harvest from at least four different locations as described in the Example 1 below at the "phenotypic data" Section. In a specific embodiment, the yield is at least 2%, preferably at least 3%, and more preferably 4% higher in a hybrid barley plant according to the present invention than corresponding yield determined for either of the parent plant.

The invention also relates to plant material of the plant according to the invention. Such plant material may include seeds, grains, fruits, buds, (viable or not) kernels, embryos, leaves, stems, roots, flowers and fractions thereof. It also includes a fraction of a homogenate or milled barley plant or kernel. It may also relate to cells of barley plant, preferably viable cells, which may be propagated in tissue cultures in vitro.

The hybrid barley plant of the present invention, their progeny or parts thereof may be used in various ways, e.g. for the manufacture of malt or beverages, as ornamental plant, for animal feed, and for the manufacture of food such as soups and stews.

In particular, the beverage manufactured using the barley plant of the present invention may be beer, whisky, barley water, mogicha and coffee substitutes, or in fermentation processes utilizing other grain of other crops for alcohol production.

The invention thus further relates to all material for malt alcoholic beverage, as obtained from the barley plant of the invention. Said material for malt alcoholic beverage include without limitation, a seed, a malt, a malt extract, a barley decomposition product. Methods for making malt alcoholic beverages from malt extract of barley plant are well- known in the art. Markers for identifying the barley hybrid plant of the invention

The invention also relates to the use of specific markers of vrsl, int-c and rfm1 alleles for identifying a two-row barley plant of the invention as defined above. In one specific embodiment, said markers specific of int-c gene allele a or b are detected by primers or probes.

Allelic differences between Int-c-a and int-c-b at position 124, 189, 235 and 284 are described in Ramsay et al, 201 1 . Table 1 below shows a set of primers used to identify the Int-c-a or int-c-b alleles at the different allelic positions.

ID Primer AlleleX Primer AlleleY Primer Common

Table 1 : sequence of primers used to identify int-c alleles

The invention also relates to the use of specific markers of vrsl alleles for identifying a barley plant of the invention as defined above.

Methods and markers to identify vrsl alleles, including Vrsl alleles involved in two-row types barley as described in EP1970447 or vrsl alleles involved in six-row type barley are as described in Komatsuda et al, 2007. Accordingly the invention further relates to a method for identifying a hybrid barley plant of the invention comprising:

a. extracting DNA and/or proteins from said hybrid barley plant,

b. determining whether vrsl alleles are heterozygous vrsl.t/vrsl.a, using markers specific of vrsl alleles,

c. optionally, determining whether int-c alleles are heterozygous Int-c.a/int-c.b or, d. optionally, determining whether rfmla alleles are heterozygous Rfm1a/rfm1a, and,

e. identifying said barley plant comprising heterozygous vrsl .t/vrs1.a and optionally, heterozygous Int-c.a/int-c.b and, further optionally heterozygous Rfm1a/rfm1a.

The invention further relates to a method for selecting a hybrid barley plant of the invention comprising:

a. carrying out the method for obtaining a hybrid two-row barley plant as described above,

b. optionally, extracting DNA and/or proteins from said hybrid two-row barley plant obtained from a method according to the invention,

c. determining whether said vrsl alleles are heterozygous Vrs1 .t/vrs1.a, using markers specific of vrsl allele,

d. optionally, determining whether said int-c alleles are heterozygous Int-c.a/int- c.b optionally, determining whether rfmla alleles are heterozygous Rfm1a/rfm1a,

e. identifying said barley plant comprising heterozygous vrsl .t/vrs1.a and, optionally, heterozygous Int-c.a/int-c.b, and, further optionally, heterozygous Rfm1a/rfm1a.

The invention also pertains to the kit for carrying out the above methods, comprising at least:

specific probes or primers for detecting markers of vrsl.t and vrsl.a alleles, and/or Int-c alleles, and/or Rfmla alleles,

instructions for use, optionally, appropriate reagents and buffering agents.

The present invention is further illustrated by the following examples which are not intended to limit the scope of the invention. EXAMPLES:

Example 1 : heterosis in two-row deficiens Iyrsl.t, int-c.b) x six-row (yrsl.a, Int-c.a ) barley hybrid plant (first year assay on one hybrid).

Material:

Female lines were obtained using two backcrosses on a msm7-line with elite lines and three generations of selfing. msm-1 lines carry rudimentary anthers and are male sterile (Ahokas, 1978 and Ahokas, 1979), this sterility being maternally inherited.

Male lines were obtained using two backcrosses on a Rfm1-\^' e with elite lines and three generations of selfing. Rfm-1 lines can restore fertility due to msm1 (Ahokas, 1979 and Ahokas, 1982). In 201 1 , two-row deficiens male lines (vrsl.t, int-c.b ) and six-row female lines (vrsl.a, Int- c.a) were drilled in two meters-wide strips. Each female strip was surrounded by two male strips. Pollen coming from males pollinated male-sterile females and allowed hybrid seed production.

Phenotypic data: In 2012, these hybrids (vrsl .t/vrs1.a, Int-c.a/int-c.b) were drilled in four environments in Germany and Belgium and their yield was evaluated at harvest 2013. Experimental design was an alpha with two replicates per location. Each replicate was divided into 7 blocks of 7 lines. Parents were also included. Plots were 6 meters long and 1 .5 meter wide. Grain yield were measured on a plot basis in the 4 locations (2 replicates).

Adjusted means have been first computed per location, with a linear mixed model:

Vij = μ + a + η + n:b_k + e,, where y is the adjusted entry mean of the ith barley line at the jth replicate, μ the intercept term, g, the genetic effect of the ith barley line, the effect of the jth replicate, Tjibk the effect of the kth block in the jth replicate and ey the error term.

Then adjusted means have been computed for each genotype, using a fixed linear model: y = [i + g + \ + e ,

i j where yy is the adjusted entry mean of the ith barley line at the jth location, μ the intercept term, g, the genetic effect of the ith barley line, l_j the effect of the jth location, and ey the error term. Replicates, locations and genotypes were modeled as fixed effects and blocks as a random effect. Variance components were determined by the restricted maximum likelihood (REML) method.

Heritability on an entry-mean basis was calculated as the ratio of genotypic to phenotypic variance according to Piepho & Mohring, 2007. Results:

Yield results of one hybrid and its two parents are shown in Table 2. Yield is expressed in ql/ha and has been obtained on 4 locations. Adjusted mean of the four locations is given for one year and is computed with the fixed linear model described above.

The six-row x two-row deficiens hybrid had a two-row phenotype and it had an increased yield (1 15.4 ql/ha) compared to its parents (1 1 1 .3 and 1 1 1 .7 ql/ha) (Table 2).

Adjusted

Location 1 Location 2 Location 3 Location 4

mean 2013

Hybrid six-row x

two-row deficiens 1 15.2 1 18.4 108.4 1 19.2 1 15.4

Male two-row

deficiens 1 13.7 1 18.3 109.5 105.0 1 1 1.7

Female six-row 123.0 108.4 99.4 1 14.0 1 1 1.3 Table 2 : yield results (ql/ha) for a six-row x two-row deficiens hybrid, its male (two-row deficiens) and its female (six-row) parents, obtained in 4 locations and the adjusted mean computed from these 4 locations data.

The hybrid six-row x two-row deficiens plants showed heterosis compared to its parents. Example 2: comparison of two-row deficiens Iyrsl.t, int-c.b) x six-row (yrsl.a, Int-c.a ) and two-row IVrsl.b, int-c.b) x six-row (yrsl.a, Int-c.a ) barley hybrids,

Material:

Male lines were obtained using two backcrosses on a Rfmla line with elite lines and three generations of selfing. Rfm-1a lines can restore fertility due to msm1 (Ahokas, 1982).

In 201 1 , 1 1 hybrids have been produced using two-row deficiens male lines (vrsl.t, int- c.b) and one unique six-row female line (vrsl.a, Int-c.a) with the strip method. Parents were drilled in two meter -wide strips. Each female strip was surrounded by two male strips. Pollen coming from males pollinated male-sterile female and allowed hybrid seed production.

Using the same method, 4 hybrids have been produced using two-row male lines (Vrsl.b, int-c.b) and the same six-row female line (vrsl.a, Int-c.a) as for the 1 1 previous hybrids. Phenotypic data:

In 2012, these hybrids (vrsl .t/vrs1.a, Int-c.a/int-c.b) and (Vrs1.b/vrs1.a, Int-c.a/int-c.b) were drilled in four environments in Germany and Belgium and their yield was evaluated at harvest 2013. Experimental design was an alpha with two replicates per location. Each replicate was divided into 7 blocks of 7 lines. Parents were also included. Plots were 6 meters long and 1.5 meter wide.

Grain yield were measured on a plot basis in the 4 locations (2 replicates).

Adjusted means have been first computed per location, with a linear mixed model: y₄ ^■ = μ + g, + η + η κ + β₉, where y is the adjusted entry mean of the ith barley line at the jth replicate, μ the intercept term, g, the genetic effect of the ith barley line, the effect of the jth replicate, Tjibk the effect of the kth block in the jth replicate and ey the error term.

i j

where y is the adjusted entry mean of the ith barley line at the jth location, μ the intercept term, g, the genetic effect of the ith barley line, l_j the effect of the jth location, and ey the error term. Replicates, locations and genotypes were modeled as fixed effects and blocks as a random effect. Variance components were determined by the restricted maximum likelihood (REML) method.

Yield results of 15 hybrids, 1 1 being two-row deficiens crossed with six-row and 4 being two-row crossed with six-row are shown in Table 3. Yield is expressed in ql/ha and adjusted mean from 4 locations is computed with a fixed linear model. The two-row deficiens crossed to six-row hydrids show a significant higher yield compared to the two- row crossed to six-row.

Difference observed between two-row deficiens (vrsl.t,, int-c.b) x six-row (vrsl.a, Int-c.a ) hybrids and two-row (Vrs1.b„ int-c.b) x six-row (vrsl.a, Int-c.a ) hybrids has been tested for significance with an unpaired Student t-test. The difference is highly significant (PO.001 ).

Adjusted yield

Hybrids Parent 1 Parertt2 mean (ql/ha)

HI 6 (vrsl.a / Int-c.a) 2R deficiens (vrsl.t / int-c.b) 115.36

H2 6R (vrsl.a / Int-c.a) 2R deficiens (vrsl.t / int-c.b) 111.97

H3 6R (vrsl.a / Int-c.a) 2R deficiens (vrsl.t / int-c.b) 107.69 H4 6 (vrsl.a / Int-c.a) 2R deficiens (vrsl.t / int-c.b) 107.18

H5 6R (vrsl.a / Int-c.a 2R deficiens (vrsl.t / int-c.b) 106.46

H6 6R (vrsl.a / Int-c.a) 2R deficiens (vrsl.t / int-c.b) 106.38

H7 6R (vrsl.a / Int-c.a) 2R deficiens (vrsl.t / int-c.b) 106.38

H8 6R (vrsl.a / Int-c.a) 2R deficiens (vrsl.t / int-c.b) 105.27

H9 6R (vrsl.a / Int-c.a) 2R deficiens (vrsl.t / int-c.b) 104.13

H10 6R (vrsl.a / Int-c.a) 2R deficiens (vrsl.t / int-c.b) 101.24

Hll 6R (vrsl.a / Int-c.a) 2R deficiens (vrsl.t / int-c.b) 99.38

H12 6R (vrsl.a / Int-c.a) 2R (Vrsl.b / int-c.b) 96.28

H13 6R (vrsl.a / Int-c.a) 2R (Vrsl.b / int-c.b) 89.61

H14 6R (vrsl.a / Int-c.a) 2R (Vrsl.b / int-c.b) 89.17

H15 6R (vrsl.a / Int-c.a) 2R (Vrsl.b / int-c.b) 81.19

Table 3 : adjusted mean yield results (ql/ha) for 1 1 six-row x two-row deficiens hybrids and 4 six- row x two-row deficiens hybrids, The hybrid six-row x two-row deficiens plants showed a significantly higher heterosis compared to the six-row x two-row hybrids.

Example 3: heterosis in two-row deficiens (yrsl.t, int-c.b) x six-row (yrsl.a, Int-c.a ) barley hybrid plants (second year assays on 4 different hybrids).

Material:

Female lines were obtained using four backcrosses on a msm7-line with elite lines and three generations of selfing. msm-1 lines carry rudimentary anthers and are male sterile (Ahokas, 1978 and Ahokas, 1979), this sterility being maternally inherited. Male lines were obtained using four backcrosses on a Rfm1-\^' e with elite lines and three generations of selfing. Rfm-1 lines can restore fertility due to msm1 (Ahokas, 1979 and Ahokas, 1982).

In 2013, two-row deficiens female lines (vrsl.t, int-c.b ), six-row female lines (vrsl.a, Int- c.a), two-row deficiens male lines (vrsl.t, int-c.b ) and six-row male lines (vrsl.a, Int-c.a) were drilled in crossing blocks. Each crossing block was containing a two-row deficiens female line and six-row male line or a two-row deficiens male line and six-row female line. In each crossing block, one female plot (1 .5 meter-wide) was surrounded by male plots, and the crossing block was isolated from other crossing blocks by wheat strips (30 meter- wide) to avoid pollen flow.

Pollen coming from males pollinated male-sterile females and allowed hybrid seed production. Phenotypic data:

In 2014, these hybrids (vrsl .t/vrs1.a, Int-c.a/int-c.b) were drilled in three environments in Germany and France and their yield was evaluated at harvest 2015. Experimental design was an alpha with two replicates per location. Parents were also included. Plots were 6 meters long and 1.5 meter wide. Grain yield adjusted means and heritability are assessed as described in example 1 . Results:

Yield results of four hybrid and their two parents are shown in Table 4. Yield is expressed in ql/ha and has been obtained on 3 locations. Adjusted mean of the three locations is given for one year and is computed with the fixed linear model described above. The six-row x two-row deficiens hybrids had a two-row phenotype, and the best hybrid had a heterosis of +13% compared to its best parent and +17% compared to the mid- parent value (Table 4).

Male yield Mid-parent

Female yield

Female type Male type Hybrid Yield (ql/ha) value

(ql/ha)

Two-row

Hybrid 1 Six-row deficiens 74.28 65.303 75.69 70.5

Two-row

Hybrid 2 Six-row deficiens 72.82 69.76 70.19 69.97

Two-row

Hybrid 3 deficiens Six-row 86.73 76.28 71.93 74.1 1

Two-row

Hybrid 4 deficiens Six-row 73.1 1 79.17 68.77 73.97 Table 4: adjusted mean yield results (ql/ha) for 4 six-row x two-row deficiens hybrids and their male and female parents, obtained in 3 locations. Three hybrids six-row x two-row deficiens plants showed heterosis compared to their mid-parent values and two hybrids showed heterosis compared to their best parent. REFERENCES

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Carleton and Foote (1968), heterosis for grain yield and leaf area and their components in two-X six-rowed barely crosses. Crop Sc. vol 8,554-557. Franckowiak et al., (1987), Coordinator's report on the semi-dwarf barley collection, Barley Genet News 17: 1 14-1 15.

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Komatsuda, T. et al. (2007), Six-rowed barley originated from a mutation in a homeodomain-leucine zipper l-class homeobox gene. Proc. Natl. Acad. Sci. USA 104, 1424-1429.

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Claims

1 . Method to obtain a two-row barley hybrid plant comprising crossing a first parent six-row barley plant with a second parent two-row deficiens type barley plant, and obtaining a hybrid two-row barley plant from said crossing, wherein the first or second parent plant is male sterile.

2. The method of claim 1 , wherein the two-row deficiens type barley parent plant is characterized by the presence of the vrsl.t and int-c.b alleles.

3. The method of Claim 1 or 2, wherein the six-row type barley parent plant is characterized by the presence of vrsl.a allele and Int-c.a.

4. The method of claim 1 , 2 or 3, wherein the first or second parent plant comprises a cytoplasmic control of male sterility and the other parent plant comprises a dominant fertility restoration gene, and wherein said crossing is obtained in open fields.

5. The method of any one of the preceding claims, wherein one parent plant is a male sterile barley plant comprising a msml cytoplasm and the second plant is a fertile barley plant comprising the Rfm1 fertility restoration gene.

6. A method for improving the yield (ql/ha) of a two-row barley plant, said method comprising crossing a first six-row barley parent plant with a second two-row deficiens type barley plant and obtaining a hybrid two-row barley plant from said crossing, wherein the yield of said hybrid two-row barley plant is improved as compared to the yield of either of the parent plants.

7. A two-row barley hybrid plant obtainable or obtained by the method of any one of Claims 1 to 5.

8. A two-row barley hybrid plant which is heterozygous with vrsl .t/vrs1.a alleles, wherein said hybrid plant further comprises a dominant fertility restoration gene.

9. The two-row barley hybrid plant of Claim 7 or 8, wherein said dominant fertility restoration gene is Rfm1.

10. The two-row barley hybrid plant of any one of Claims 7 to 9, which has an improved yield in open field as compared to the yield of any of the parent plants under similar environmental conditions.

1 1 . The two-row barley hybrid plant of any one of Claims 7 to 10, which is further heterozygous with int-c.b/lnt-c.a alleles.

12. The two-row barley hybrid plant of any one of Claims 7 to 1 1 , characterized by the presence of a male sterile cytoplasm and a fertility restoration locus.

13. The two-row barley plant of any one of Claims 7 to 12, characterized by the presence of msml and/or msm2 male sterile cytoplasm and the presence of Rfm1 fertility restoration locus.

14. The two-row barley hybrid plant of any one of claims 7 to 13, further comprising dwarf or semi-dwarf alleles.

15. A method of identification of the two-row barley hybrid of any one of claims 7 to 14 including the use of markers linked to vrsl, int-c and/ Rfm1 alleles, thereby identifying said two-row barley hybrid plant.