**2. Methodology**

Pollinations are generated by hand utilizing the appropriate IL genetic stock as the maternal parent. Hybrid seed is easily generated in quantity. Following germination of the hybrid seed, numerous ryegrass-tall fescue hybrids are now available with each F1 hybrid being derived from a single tall fescue pollen grain sperm nucleus fertilizing the IL egg. The chromosome number of the hybrids is typically 2n=2x=28 with each hybrid possessing one genomic contribution from each parent (ryegrass (n=7) and tall fescue (n=21). Hybrids are generally sterile, but a low incidence of fertility can occasionally occur (Buckner, 1960b; Buckner et al., 1961). The generated seed can be sown in low density space planting nurseries, grown in the greenhouse, or planted to a spaced planted nursery where various induced or natural selection pressures can be applied to the hybrid individuals (Figures 1a, 1b, 1c). Multiple locations are desirable to focus selection on the particular attributes such as stress or rust tolerance in a region where the germplasm is planned to be released (Figures 2a, 2b). If disease tolerance is a selection criterion, then the hybrids should be grown in a region where the specific disease under study is prevalent. Once exceptional hybrid individuals are identified, the F1's can be transferred to the greenhouse to exclude any chance of cross-pollination with any tall fescue pollen in the field. If an abundance of tall fescue pollen is not a problem in the nursery, then the hybrids can remain in the nursery for a future harvest of each selected plant inflorescence. Hybrid individuals that do not possess an appropriate genotype contribution from the paternal tall fescue parent are culled from the nursery. If multiple years of selection are to be performed, seed heads are not to be retained until the final year of selection.

Fig. 1. a (left) Low density nursery planting of IL X tall fescue seed; USDA-ARS, Grazinglands Research Laboratory, El Reno, OK, USA. Figure 1b (center) Space planted nursery of IL x tall fescue hybrids. Figure 1c (right) Stress tolerant and stress intolerant hybrids from the IL x tall fescue hybrid space planted nursery.

effects in the F1 that may occur between the *Lolium* and *Festuca* genomes. The primary focus of the approach is placed on the gametic contribution of the paternal tall fescue parent and its expression in the hybrid sporophyte. Through this process, the breeder or geneticist is essentially sampling the genetic diversity of the paternal individual on a single pollen grain

Pollinations are generated by hand utilizing the appropriate IL genetic stock as the maternal parent. Hybrid seed is easily generated in quantity. Following germination of the hybrid seed, numerous ryegrass-tall fescue hybrids are now available with each F1 hybrid being derived from a single tall fescue pollen grain sperm nucleus fertilizing the IL egg. The chromosome number of the hybrids is typically 2n=2x=28 with each hybrid possessing one genomic contribution from each parent (ryegrass (n=7) and tall fescue (n=21). Hybrids are generally sterile, but a low incidence of fertility can occasionally occur (Buckner, 1960b; Buckner et al., 1961). The generated seed can be sown in low density space planting nurseries, grown in the greenhouse, or planted to a spaced planted nursery where various induced or natural selection pressures can be applied to the hybrid individuals (Figures 1a, 1b, 1c). Multiple locations are desirable to focus selection on the particular attributes such as stress or rust tolerance in a region where the germplasm is planned to be released (Figures 2a, 2b). If disease tolerance is a selection criterion, then the hybrids should be grown in a region where the specific disease under study is prevalent. Once exceptional hybrid individuals are identified, the F1's can be transferred to the greenhouse to exclude any chance of cross-pollination with any tall fescue pollen in the field. If an abundance of tall fescue pollen is not a problem in the nursery, then the hybrids can remain in the nursery for a future harvest of each selected plant inflorescence. Hybrid individuals that do not possess an appropriate genotype contribution from the paternal tall fescue parent are culled from the nursery. If multiple years of selection are to be performed, seed heads are not to be

Fig. 1. a (left) Low density nursery planting of IL X tall fescue seed; USDA-ARS,

hybrids from the IL x tall fescue hybrid space planted nursery.

Grazinglands Research Laboratory, El Reno, OK, USA. Figure 1b (center) Space planted nursery of IL x tall fescue hybrids. Figure 1c (right) Stress tolerant and stress intolerant

basis and examining each contribution in the hybrids.

retained until the final year of selection.

**2. Methodology** 

Fig. 2. a (left) IL x tall fescue F1 drought selection nursery at he Mound Valley Unit, Kansas State University, Southeast Agricultural Research Center, KS in July, 2010. Circled individuals identify F1 plants exhibiting superior drought tolerance and plant vigor. Figure 2b (right) IL x tall fescue F1 rust tolerance selection nursery at Barenbrug Seeds, West Coast Research Center, Albany, OR in August, 2011.

The surviving hybrids are allowed to flower; then inflorescences are gathered at maturity, broken up by hand or machine and sown to trays. A light cleaning is applied to remove stems. The cleaned seed heads are then placed in germination trays for identification and selection of either recovered ryegrass or tall fescue seedlings. Typically, following two weeks of germination, a few seedlings will appear and are allowed to grow to appropriate size for transplanting (Figure 3). The germinating seedlings will generally be ryegrass recoveries possessing a chromosome number of 2n=2x=14, tall fescue recoveries possessing a chromosome number of 2n=6x=42, or various tall fescue DH recoveries with ryegrass introgression or ryegrass recoveries with tall fescue introgression. Since the seedlings obtained from the sterile F1 hybrids are generated through parthenogenic development following spontaneous chromosome doubling, each recovery will possess a full genome contribution of either the ryegrass or tall fescue parent. Genome loss, spontaneous doubling followed by parthenogenic development represent the important and essential contributions of the IL1 and IL2 lines. All recovered lines derived from this process will have all genes, alleles or quantitative loci conferring a trait fixed in the DH recovery (Kindiger and Singh, 2011; Kindiger 2011). Essentially, the process generates homozygous or DH lines from the IL x tall fescue F1 hybrids.

Each recovered individual will be free of the *Lolium* sp. fungal endophytes since the IL1 and IL2 lines do not possess endophyte. Recovered tall fescue DH lines can then be evaluated under additional selection schemes and eventually inter-crossed to perhaps generate a synthetic possessing various advantageous attributes, or be utilized as breeding lines for the development of cultivars.

#### **3. Example 1: Gamete selection for crude protein**

The development of forages with superior nutritional qualities is an ongoing and time consuming breeding effort often hindered by the complex genetics associated with each quality trait (Casler, 2001; Bouton, 2009). Since grazing animals have specific and differing

Fig. 3. Germination of dihaplod seedlings from a selected IL x tall fescue F1 hybrid. These individuals will represent DH tall fescue recoveries.

nutritional requirements, the qualities of the forage should be adjusted to fit that particular need (Brummer and Casler, 2009). Intergeneric hybridization between *Lolium* and fescue

has been a common method to understand and improve the genetics underlying the forage quality (Naganowska et al., 2001; Cogan et al., 2005). In addition, applying approaches such as introgression mapping facilitated with either genomic *in situ* hybridization techniques or molecular markers, provide an additional set of powerful tools for forage quality improvements (Cardinal et al., 2003). The close homology between the *Lolium* sp. and *Festuca* sp. genomes can also allow either recombination or a full substitution of a *Lolium* chromosome for a *Festuca* chromosome (Kopecký et al., 2009) and such events could affect the expression of a forage quality. It may also be useful to incorporate molecular markers during the breeding process or during backcross generations to identify any ryegrass segments that might have been introgressed into the fescue genome (Humphreys, 2004).

In a preliminary demonstration utilizing the gamete selection approach, 14 IL x tall fescue hybrids were evaluated for their crude protein (CP) quantity via available nitrogen in plant leaf tissue. The need for forages to possess adequate levels of CP must complement the grazing animal's nutritional requirements. Typically, CP estimates reflect the level of nitrogen and amino acids in forages. CP content is considered a quantitative trait in most forages (Fei et al., 2006.) and is typical of a trait exhibiting low heritability and is, as a consequence, difficult to transfer rapidly and effectively (Vogel et al., 1981). Leaf samples

Fig. 3. Germination of dihaplod seedlings from a selected IL x tall fescue F1 hybrid. These

nutritional requirements, the qualities of the forage should be adjusted to fit that particular need (Brummer and Casler, 2009). Intergeneric hybridization between *Lolium* and fescue

has been a common method to understand and improve the genetics underlying the forage quality (Naganowska et al., 2001; Cogan et al., 2005). In addition, applying approaches such as introgression mapping facilitated with either genomic *in situ* hybridization techniques or molecular markers, provide an additional set of powerful tools for forage quality improvements (Cardinal et al., 2003). The close homology between the *Lolium* sp. and *Festuca* sp. genomes can also allow either recombination or a full substitution of a *Lolium* chromosome for a *Festuca* chromosome (Kopecký et al., 2009) and such events could affect the expression of a forage quality. It may also be useful to incorporate molecular markers during the breeding process or during backcross generations to identify any ryegrass segments that might have been introgressed into the fescue genome (Humphreys, 2004).

In a preliminary demonstration utilizing the gamete selection approach, 14 IL x tall fescue hybrids were evaluated for their crude protein (CP) quantity via available nitrogen in plant leaf tissue. The need for forages to possess adequate levels of CP must complement the grazing animal's nutritional requirements. Typically, CP estimates reflect the level of nitrogen and amino acids in forages. CP content is considered a quantitative trait in most forages (Fei et al., 2006.) and is typical of a trait exhibiting low heritability and is, as a consequence, difficult to transfer rapidly and effectively (Vogel et al., 1981). Leaf samples

individuals will represent DH tall fescue recoveries.

from 14 IL x tall fescue F1 hybrids were obtained from the nursery in March 2011 and were run on an Elementar varioMacro flash combustion instrument (Elementar Americas, Inc., Mt. Laurel, NJ). In addition, DH lines derived from particular IL x tall fescue hybrids were assayed for N, along with the check tall fescue cultivars Nanyro, Retu, Drover and Barcarella. CP concentration of samples was estimated by multiplying total nitrogen (N) concentration by 6.25 (Hersom, 2007)(Table 1). Multiplying the N concentration by 6.25 to estimate CP level is performed because protein molecules contain an average of 16% N (1/16 = 6.25). This N and CP estimation represents the standard approach utilized for evaluating beef cattle protein requirements (Hersom, 2007). Results presented in Table 1 indicate that hybrids high in CP content provide DH recoveries high in CP content. DH23A, a DH exhibiting low CP content, was obtained from a hybrid exhibiting low CP content. A


Table 1. Crude protein estimates across IL x tall fescue hybrids (LF), their dihaploid recoveries (DH) and standard cultivar checks.

program focused on developing a high CP cultivar would simply require the identification of agronomically superior DH lines exhibiting high CP estimations. This approach is superior to traditional recurrent selection as there are no segregating alleles that could confer lower or segregating CP levels. All lines utilized in the breeding process are fixed for the high or low CP genes. This approach removes much of the random segregation of alleles governing CP or other traits of low heritability. Following identification of high CP materials, eight high CP DH lines were combined to generate an experimental synthetic (Syn1). In limited performance trials, Syn1 has demonstrated itself to be a DH synthetic exhibiting high CP and superior agronomic attributes. As a consequence, a selection program focused on identifying hybrid genotypes exhibiting high CP content, then deriving high CP content DH lines from those hybrids should be an efficient and effective method to concentrate quantitative trait loci defining elevated CP content via gamete selection.

### **4. Example 2: Gamete selection for stress tolerance**

A primary objective of the forage research program at the USDA-ARS, Grazinglands Research Laboratory is the development of tall fescue forage possessing tolerance to the environmental extremes of the Southern Plains Region. Typically, these environmental extremes involve heat, drought and low nitrogen inputs. To achieve this goal, a three year program of natural selection was conducted with numerous IL x tall fescue hybrids. Selection criteria were to evaluate the hybrids in a high stress environment on the Southern and Central Plains of the USA consisting of high summer temperatures, high wind, nonirrigated, low nitrogen input (40 lbs/ac) conditions (Figures 1a, 1b, 2a, 2b). Trials were conducted at the USDA-ARS, Grazinglands Research Laboratory, El Reno, OK and at the Mound Valley Research Unit, Kansas State University, Southeast Agricultural Research Center, Parsons, KS. Seed generated from the initial IL x tall fescue crosses were sown in a greenhouse and individuals were transferred to spaced plot field selection nurseries in the fall of 2006 (El Reno, OK) and 2008 (Mound Valley, KS). IL x tall fescue hybrids that did not possess a genotype adaptable to these conditions either died or, if the individual hybrids exhibited unsatisfactory agronomics, were physically removed from the nursery on a year to year basis (Figure 4). In the fall of 2009, seed heads were removed from the El Reno, OK nursery and in the fall of 2010, seed heads were removed from the surviving IL x tall fescue hybrids at the Mound Valley, KS location. Seed heads were threshed as described and sown to germination trays in the greenhouse.

Recovered DH tall fescue lines were removed from the germinating trays and transplanted to pots. In some instances, DH lines were selfed for seed increase. Though tall fescue is considered an obligate out-crossing species we have observed that this is not the case for many recovered DH lines and successful selfing of tall fescue has been observed and utilized in prior tall fescue research programs (Buckner and Fergus, 1960a). Seed generated through selfing recovered DH lines were sown in small, unreplicated plots in the nurseries at the USDA-ARS, Grazinglands Research Laboratory, El Reno, OK. Single DH plant selections were transplanted to the Kansas State University, Southeast Agricultural Research Center, Parsons Unit, Mound Valley, KS in 2010. The selection criteria remained unchanged at both locations for DH evaluation. During the summer of 2011 both locations experienced extended severe to extreme drought and heat conditions (National Oceanic and Atmospheric Administration, National Climatic Data Center, 2011). The DH tall fescue

program focused on developing a high CP cultivar would simply require the identification of agronomically superior DH lines exhibiting high CP estimations. This approach is superior to traditional recurrent selection as there are no segregating alleles that could confer lower or segregating CP levels. All lines utilized in the breeding process are fixed for the high or low CP genes. This approach removes much of the random segregation of alleles governing CP or other traits of low heritability. Following identification of high CP materials, eight high CP DH lines were combined to generate an experimental synthetic (Syn1). In limited performance trials, Syn1 has demonstrated itself to be a DH synthetic exhibiting high CP and superior agronomic attributes. As a consequence, a selection program focused on identifying hybrid genotypes exhibiting high CP content, then deriving high CP content DH lines from those hybrids should be an efficient and effective method to

concentrate quantitative trait loci defining elevated CP content via gamete selection.

A primary objective of the forage research program at the USDA-ARS, Grazinglands Research Laboratory is the development of tall fescue forage possessing tolerance to the environmental extremes of the Southern Plains Region. Typically, these environmental extremes involve heat, drought and low nitrogen inputs. To achieve this goal, a three year program of natural selection was conducted with numerous IL x tall fescue hybrids. Selection criteria were to evaluate the hybrids in a high stress environment on the Southern and Central Plains of the USA consisting of high summer temperatures, high wind, nonirrigated, low nitrogen input (40 lbs/ac) conditions (Figures 1a, 1b, 2a, 2b). Trials were conducted at the USDA-ARS, Grazinglands Research Laboratory, El Reno, OK and at the Mound Valley Research Unit, Kansas State University, Southeast Agricultural Research Center, Parsons, KS. Seed generated from the initial IL x tall fescue crosses were sown in a greenhouse and individuals were transferred to spaced plot field selection nurseries in the fall of 2006 (El Reno, OK) and 2008 (Mound Valley, KS). IL x tall fescue hybrids that did not possess a genotype adaptable to these conditions either died or, if the individual hybrids exhibited unsatisfactory agronomics, were physically removed from the nursery on a year to year basis (Figure 4). In the fall of 2009, seed heads were removed from the El Reno, OK nursery and in the fall of 2010, seed heads were removed from the surviving IL x tall fescue hybrids at the Mound Valley, KS location. Seed heads were threshed as described and sown

Recovered DH tall fescue lines were removed from the germinating trays and transplanted to pots. In some instances, DH lines were selfed for seed increase. Though tall fescue is considered an obligate out-crossing species we have observed that this is not the case for many recovered DH lines and successful selfing of tall fescue has been observed and utilized in prior tall fescue research programs (Buckner and Fergus, 1960a). Seed generated through selfing recovered DH lines were sown in small, unreplicated plots in the nurseries at the USDA-ARS, Grazinglands Research Laboratory, El Reno, OK. Single DH plant selections were transplanted to the Kansas State University, Southeast Agricultural Research Center, Parsons Unit, Mound Valley, KS in 2010. The selection criteria remained unchanged at both locations for DH evaluation. During the summer of 2011 both locations experienced extended severe to extreme drought and heat conditions (National Oceanic and Atmospheric Administration, National Climatic Data Center, 2011). The DH tall fescue

**4. Example 2: Gamete selection for stress tolerance** 

to germination trays in the greenhouse.

recoveries have exhibited good to excellent tolerance, superior adaptation to and persistence under these record-setting drought conditions at both locations. This study indicates that the gamete selection approach is effective and efficient in identifying genotypes that can tolerate high environmental stress conditions such as drought, heat and low nitrogen inputs. These and additional DH lines generated from this gamete selection approach will be utilized to form a foundation of tall fescue germplasm with particular adaptation to high stress sites across the Southern Plains and Midwest regions of the USA.

Fig. 4. Example of a stress intolerant IL x tall fescue F1 hybrid (left) and a stress tolerant hybrid (right).
