**1. Introduction**

270 Genetic Diversity in Plants

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Origin of resistance to plum pox virus in apricot: what new AFLP and targeted SSR

Tall fescue (*Lolium arundinaceum* (Schreb.) S.J. Darbyshire) (2n=6x=42) represents the predominant perennial cool-season grass forage in the USA. Its wide adaptation, excellent spring, summer and fall production, deep root system, tolerance to heat and persistence over summer conditions makes this a highly desirable species for hay, pasture and turf. Tall fescue tolerates short-term flooding, moderate drought and heavy livestock grazing and machinery traffic. It responds well to fertilizer but can maintain itself under limited fertility conditions and is adapted to moderately acid and wet soils (Jennings et al., 2008). Approximately two-thirds of the total annual growth of tall fescue occurs during the spring and about one-third occurs during summer and fall making it nearly a full season forage. A wide array of phenotypic and genotypic variation exists in tall fescue and cultivar development has traditionally used recurrent selection techniques. For this review, we focus primarily on tall fescue as a forage or hay producing species; however, methods discussed can be equally applicable to turf programs. Although tall fescue is a complex hexaploid species, it can be hybridized with annual (2n=2x=14) or perennial ryegrass (2n=4x=28) (*Lolium perenne* L. subsp. *multiflorum* (Lam.) Husnot (syn. *Lolium multiflorum* Lam.) for the development of festuloliums (Yamada and Takamizo, 2004). In some instances, induced androgenesis has been utilized to generate useful germplasm (Vagera et al., 1998).

Gamete selection as originally defined by Stadler (1944) is based on the principal that selection exerted at the gametophytic level can increase desirable allelic frequencies detectable at the sporophytic level. If superior gametes can be recognized with certainty through a selection cycle, then such a system would be theoretically more efficient than one based on zygotic selection (Richey, 1947). In practice, gamete selection ordinarily involves two steps: 1) selection on the basis of outcross performance testing of individual plants of a variety or population, and 2) a similar controlled selection for outstanding individuals exhibiting desirable agronomic attributes. Following the identification of superior genotypes, such individuals would undergo continued selfing, followed by phenotypic selection, to generate a homozygous line, fixed for the desired agronomic characteristics. In instances where haploids can be generated through microspore culture, followed by genome doubling, homozygous or dihaploid lines can be obtained.

Breeding methods that utilize haploids can be varied and often provide materials exhibiting particular usage in genetic analysis and breeding systems when properly exploited (Dunwell, 2010). In addition, gametophytic selection approaches can lead to a correlated sporophytic response that can involve the segregation of important agronomic traits (Mulcahy, 1971). When applicable, gamete selection has emerged as a superior and efficient form of selection and the feasibility of this approach appears to be a promising method for facilitating the incorporation of desirable alleles within a short period of time. Stadler (1944) is among the earliest who reviewed its potential application in corn breeding. More recently, the importance of a gamete form of selection, utilizing early generation selection, has demonstrated successful simultaneous selection of multiple traits, including quantitative trait loci for characteristics such as seed yield, maturity, and tolerance to disease (Singh, 1994; Ravikumar et al., 2004). In these instances, gamete selection was found to be superior in its efficiency to conventional pedigree, single seed descent, and mass selection methods (Singh, 1994). A gamete selection approach may also be equally effective on complex traits such as biotic and abiotic stress, herbicide tolerance, general and specific combining ability, and the selection of complex quantitative traits such as yield, drought tolerance, disease resistance, etc. Traditionally applied to commercial grain crops such as corn (Gordilli and Geiger, 2008; Chang and Coe, 2009), wheat (Barclay, 1975), barley (Kasha and Kao, 1970; Shugar, 1989) and beans (Singh et al., 1998) gamete selection often utilizes the induction of haploids and the subsequent generation of double haploid breeding approaches. With the use of these methods, various degrees of success in gamete selection have been attained. The generation of haploid or monoploid individuals is typically achieved through microspore culture methods (Mohan et al., 1997; Maheshwari et al., 1980; Tuvesson et al., 2008), wide hybridization (Inagaki, 1997), or through a pollen/sperm nucleus utilizing a haploid inducing system (Chang and Coe, 2009). This approach has even been applied to tall fescue (Kasperbauer et al., 1980; Bai & Qu, 2000). Each of these methods relies on the haploid individual normalizing its chromosome number either through the utilization of mitotic inhibitors such as colchicine, or through spontaneous doubling. This chromosome doubling event results in the generation of individuals that are homozygous for the genotype conferred by the single contributing gamete. By using a paternal monoploid or dihaploid generation process, gamete selection is a proven and efficient method of selection that has yet to be applied to forage grass breeding (Stadler 1944; Lu, et al., 1996; Rotarenco and Chalyk, 2000). Dihaploid (DH) breeding methods have been suggested and employed as a means to develop new germplasm in the *Festuca*-*Lolium* complex of polyploid grasses (Humphreys et al., 2003; Guo and Yamada, 2004; Guo et al., 2005). Previous research utilizing homozygous lines to study the inheritance of palatability has suggested that selection within homozygous lines can represent a useful methodology for the improvement of tall fescue (Henson and Buckner, 1957; Buckner and Fergus, 1960a). As a consequence, when the gamete selection approach is utilized in a breeding program, the generation of DH tall fescue lines should result in the development of efficient breeding advances and superior germplasm (Bouchez and Gallais, 2000). Additional research also indicates that genetic mapping studies focused on the elucidation of a variety of complex genetic traits can be more readily accomplished through the use of homozygous lines (Riera-Lizarazu et al., 2010). The application of gamete selection to tall fescue could be accomplished if an efficient system of haploid induction followed by parthenogenesis were available.

Breeding methods that utilize haploids can be varied and often provide materials exhibiting particular usage in genetic analysis and breeding systems when properly exploited (Dunwell, 2010). In addition, gametophytic selection approaches can lead to a correlated sporophytic response that can involve the segregation of important agronomic traits (Mulcahy, 1971). When applicable, gamete selection has emerged as a superior and efficient form of selection and the feasibility of this approach appears to be a promising method for facilitating the incorporation of desirable alleles within a short period of time. Stadler (1944) is among the earliest who reviewed its potential application in corn breeding. More recently, the importance of a gamete form of selection, utilizing early generation selection, has demonstrated successful simultaneous selection of multiple traits, including quantitative trait loci for characteristics such as seed yield, maturity, and tolerance to disease (Singh, 1994; Ravikumar et al., 2004). In these instances, gamete selection was found to be superior in its efficiency to conventional pedigree, single seed descent, and mass selection methods (Singh, 1994). A gamete selection approach may also be equally effective on complex traits such as biotic and abiotic stress, herbicide tolerance, general and specific combining ability, and the selection of complex quantitative traits such as yield, drought tolerance, disease resistance, etc. Traditionally applied to commercial grain crops such as corn (Gordilli and Geiger, 2008; Chang and Coe, 2009), wheat (Barclay, 1975), barley (Kasha and Kao, 1970; Shugar, 1989) and beans (Singh et al., 1998) gamete selection often utilizes the induction of haploids and the subsequent generation of double haploid breeding approaches. With the use of these methods, various degrees of success in gamete selection have been attained. The generation of haploid or monoploid individuals is typically achieved through microspore culture methods (Mohan et al., 1997; Maheshwari et al., 1980; Tuvesson et al., 2008), wide hybridization (Inagaki, 1997), or through a pollen/sperm nucleus utilizing a haploid inducing system (Chang and Coe, 2009). This approach has even been applied to tall fescue (Kasperbauer et al., 1980; Bai & Qu, 2000). Each of these methods relies on the haploid individual normalizing its chromosome number either through the utilization of mitotic inhibitors such as colchicine, or through spontaneous doubling. This chromosome doubling event results in the generation of individuals that are homozygous for the genotype conferred by the single contributing gamete. By using a paternal monoploid or dihaploid generation process, gamete selection is a proven and efficient method of selection that has yet to be applied to forage grass breeding (Stadler 1944; Lu, et al., 1996; Rotarenco and Chalyk, 2000). Dihaploid (DH) breeding methods have been suggested and employed as a means to develop new germplasm in the *Festuca*-*Lolium* complex of polyploid grasses (Humphreys et al., 2003; Guo and Yamada, 2004; Guo et al., 2005). Previous research utilizing homozygous lines to study the inheritance of palatability has suggested that selection within homozygous lines can represent a useful methodology for the improvement of tall fescue (Henson and Buckner, 1957; Buckner and Fergus, 1960a). As a consequence, when the gamete selection approach is utilized in a breeding program, the generation of DH tall fescue lines should result in the development of efficient breeding advances and superior germplasm (Bouchez and Gallais, 2000). Additional research also indicates that genetic mapping studies focused on the elucidation of a variety of complex genetic traits can be more readily accomplished through the use of homozygous lines (Riera-Lizarazu et al., 2010). The application of gamete selection to tall fescue could be accomplished if an efficient

system of haploid induction followed by parthenogenesis were available.

Sporophytically expressed traits are transmitted as genetic information through the gametes (sperm or egg nuclei) and contain half the information that is contained in the sporophytic tissue. In some instances, gametophytic genes can regulate both gamete and sporophytic traits and this is called genetic overlap. In the presented gamete selection approach, a slight modification of the methodology as presented by Stadler and his contemporaries is utilized. Employing the method of gamete selection considered in this text, traits transmitted by the gamete are under the control of genes that are expressed specifically in the sporophyte. A single gamete provided by the tall fescue parent fertilizes the egg of an inducer line (IL) that results in genome loss and a parthenogenic response. Individuals derived from this hybridization event, followed by genome loss and the parthenogenic response, are recovered ryegrass, tall fescue or varied intermediate genotypes of ryegrass-fescue (e.g. festulolium). Selection applied at the F1 sporophyte results in a form of gamete selection.

The USDA-ARS has recently developed an approach utilizing *Lolium multiflorum* lines (IL1 and IL2) that are uniquely characterized by their ability to loose either a ryegrass or tall fescue genome following hybridization and the expression of a low frequency of parthenogenic behavior (Kindiger, 2009). Parthenogenesis involves development of the egg cell within the embryo sac without involvement of the sperm nucleus and such behavior has been characterized across numerous species, especially following wide hybridizations (Kendall, 1934). In this review, a low frequency parthenogenic response represents the first reported incidence of such behavior in ryegrass x tall fescue hybrids. The frequency of this behavior is difficult to determine, however, it is estimated to occur less than 1% which is typical for other species (Kendall, 1934). This frequency can and does vary across tall fescue genotypes hybridized with the IL lines.

Two ryegrass (*Lolium perenne* L. subsp. *multiflorum* (Lam.) Husnot (syn. *Lolium multiflorum*  Lam.) (2n=2x=14) genetic stocks, identified as IL1 and IL2 (Kindiger and Singh, 2011; Kindiger, 2011), are characterized by a genome loss phenomenon following hybridization with tall fescue (*Lolium arundinaceum* (Schreb.)) S.J. Darbyshire (syn. *Festuca arundinacea* Schreb.) (2*n* = 6*x* = 42)\, followed by a low level of parthenogenic development. The IL1 and IL2 genetic stocks exhibit few advantageous agronomic characteristics and are essentially notable only for their ability to induce chromosome or genome loss following hybridization. Both lines are free of the fungal endophytes *Epichloë* sp. or *Neotyphodium* sp. (Carroll, 1988; Moon et al., 1994; Pederson & Sleper, 1988). Recurrent selection remains a widely utilized approach for breeding most cereal and forage species (Gallais, 1993) and has also been applied utilizing DH lines (Bouchez and Gallais, 2000). Incorporation of a gamete selection methodology should provide an equally successful approach for tall fescue improvement.

In the approach, hybrids are generated utilizing genetic stocks IL1 or IL2 as the maternal parent and a single tall fescue individual or population as the paternal parent. Varietal or individual plant sampling within improved tall fescue cultivars or populations will yield a myriad of segregating gametes possessing a wide array of advantageous and disadvantageous alleles. Each pollen sperm nuclei will contribute a unique genotype to each hybrid and as a result, each hybrid represents a random genotypic combination of the IL maternal line and tall fescue pollen parent. Since the IL1 and IL2 lines are narrow based, agronomically inferior, unimproved genetic stocks, any potential advantageous contribution they may provide is considered minimal, notwithstanding unknown potential heterotic 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 basis and examining each contribution in the hybrids.
