Utilizing a dihaploid-gamete selection strategy for tall fescue development

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1 Utilizing a dihaploid-gamete selection strategy for tall fescue development Bryan Kindiger USDA-ARS, Grazinglands Research Laboratory, 7207 West Cheyenne St., El Reno, OK USA Introduction Gamete selection as originally defined by Stadler (1944) is based on the principle 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, or some other method to induce homozygosity, homozygous or dihaploid lines will result. Dihaploid (DH) selection methods can result in more rapid and efficient gains than other forms of selection (Niroula and Bimb, 2009). Briefly, when utilizing a DH approach, in a diploid organism, only two types of genotypes occur for a pair of alleles, A and a, with the frequency of ½ AA and ½ aa. When utilizing a selfing or backcross approach, three genotypes occur with the frequency of ¼ AA, ½ Aa, ¼ aa. Therefore, if AA represents a desirable genotype, the probability of obtaining the desirable AA genotype is higher when 1

2 utilizing a DH approach. Also, if n advantageous loci are segregating in a population, the probability of obtaining that desirable genotype is (1/2)n when employing a DH approach, and (1/4)n when utilizing selfing or backcrossing methods. As a consequence, the efficiency of a DH selection approach will be higher when the number of genes governing a trait are quantitative in their inheritance and expression (Kotch et al., 1992). When applied to a polyploid species such as tall fescue, the gain in efficiency is exponential. Stadler (1944) is among the earliest who reviewed the potential application of gamete selection 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 effective selection for quantitative trait loci governing characteristics such as seed yield, maturity, and tolerance to disease (Singh, 1994; Ravikumar et al., 2004). In these instances, gamete selection has emerged as a superior and efficient form of selection and the feasibility of this approach appeared to be a promising method for facilitating the incorporation of desirable alleles within a shortened breeding period (Singh, 1994). Through the utilization of a paternal monoploid or dihaploid generation process, gamete selection is a proven and efficient method of selection for several species (Stadler 1944; Fehr, 1984; Snape el al., 1986; Schon et al., 1990; Lu, et al., 1996; Rotarenco and Chalyk, 2000). Though DH breeding methods have been suggested and occasionally employed as a means to develop new festulolium lines within the Festuca-Lolium complex of polyploid grasses (Guo and Yamada, 2004; Guo et al., 2005), there have been few commercially successful cultivar releases. In addition, the time consuming and genotypic specific method of microspore culture were employed to generate the new festulolium s (Humphreys et al., 2003). Previous research utilizing homozygous tall fescue derivatives, generated through a traditional program of selfing, were used to study the inheritance of palatability. These 2

3 studies 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 a gamete selection approach can be applied 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). 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, genetic overlap, a situation where gametophytic genes can regulate both gamete and sporophytic traits can occur. 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 from the tall fescue parent fertilizes the egg of an inducer line (IL) which generates an F1 hybrid. Within the F1, genome loss and a parthenogenic response give rise to DH recoveries. Essentially this protocol constitutes a gamete based dihaploid technology that greatly enhances the production of complete homozygous dihaploid lines in a single generation that increases the precision and efficacy of selection in tall fescue breeding and genetic studies. This approach for generating DH is more efficient for producing recovered dihaploid lines than typical microspore culture approaches. It is also simple, less genotype dependent, less labor intensive and less time consuming than other breeding approaches. The discussed approach represents a dihaploid inducement technology 3

4 that is similar to the wheat x maize DH inducer technology (Niroula and Bimb; 2009). In this approach, embryo culture or rescue is not necessary and selection is based on the F1 hybrid, prior to DH generation. In the described method, selection applied upon the F1 sporophyte and the tall fescue genome that it possesses constitutes a form of gamete selection. DH individuals derived from these events are generally recovered ryegrass, tall fescue or varied intermediate genotypes of ryegrass-fescue (e.g. festuloliums). A few amphidiploid selections are also generated. 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 make 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). Gamete selection, as applied here, will be more efficient and effective in developing superior tall fescue cultivars than traditional recurrent selection techniques. Methods The USDA-ARS has recently released two ryegrass (Lolium perenne L. subsp. multiflorum (Lam.) Husnot (syn. Lolium multiflorum Lam.) (2n=2x=14) genetic stocks, identified as IL1 and IL2. Each is characterized by a genome loss phenomenon following hybridization with tall fescue (Lolium arundinaceum (Schreb.)) S.J. Darbyshire (syn. Festuca arundinacea Schreb.) (2n = 6x = 42), then followed by a low level of parthenogenic development. The application of the gamete selection breeding approach using these lines is 4

5 relatively straightforward and efficient. 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). Pollinations are generated by hand or in field or greenhouse isolations utilizing the IL genetic stock as the maternal parent. Following germination of the hybrid seed, numerous ryegrass-tall fescue F1 hybrids are generated, with each F1 hybrid being derived from a single tall fescue pollen grain sperm nucleus fertilizing the IL egg. Hybrids are generally sterile; however, a low incidence of fertility can occasionally occur (Buckner, 1960b; Buckner et al., 1961). Due to chromosome or genome loss, F1 hybrids will occasionally generate ryegrass or tall fescue sectors and exhibit chimeras. Possible outcomes of an IL x tall fescue hybridization are illustrated in figure 1. Each type can be identified through phenotypic evaluations, chromosome counts or molecular marker analysis. The DH ryegrass and DH tall fescue recoveries represent the most abundant types (>90%). IL (2n=2x=14) x Tall Fescue (2n=6x=42) F1 (7R + 21TF) Types of Individuals Recovered from the F1: Recovered dihaploid ryegrass with 14 chromosomes Recovered dihaploid tall fescue with 42 chromosomes Recovered amphidiploids (possessing 14 ryegrass + 42 tall fescue chromosomes) Recovered 28 chromosomes (the original F1 plants, 7R + 21 TF chromosomes) Recovered 35 chromosomes individuals (possess balanced but uncharacterized doses of the ryegrass and tall fescue genomes) Aneuploids (individuals with infinite combinations of unbalanced chromosome numbers. Generally die as seedlings) Possible festulolium with varying degrees of ryegrass/tall fescue introgression Figure 1. Critique of various outcomes from IL x tall fescue F1 hybrids. 5

6 Principles of Plant Genetics and Breeding The generated F1 (IL x tall fescue) hybrid seed can be sown in low density space planting nurseries or grown in the greenhouse where various induced or natural selection pressures can be applied to the hybrid individuals to identify superior genotypes (Figure 2). Multiple locations are desirable to apply an appropriate level of selection on a plant weakness such as stress or rust tolerance in the region where the germplasm is to be eventually released. 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 crosspollination 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. Figure 2. F1 evaluations for rust susceptibility at the Barenbrug USA Research Center, Albany, OR, USA. Hybrid individuals that do not possess a viable or tolerant genotypic contribution from the paternal tall fescue parent are culled from the nursery. If multiple years of selection are to 6

7 be performed, seed heads are not to be retained until the final year of selection. The surviving hybrids are allowed to flower; then inflorescence 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. 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. Occasional amphidiploids (2n=8x=56) possessing full ryegrass (2n=2x=14) and full tall fescue (2n=6x=42) complements are generated. 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 followed by spontaneous doubling and subsequent 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). Each recovered individual will be free of the Lolium sp. fungal endophyte since the IL1 and IL2 lines do not possess an 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. Seed increase of superior DH lines can be accomplished by selfing. Since tall fescue is generally an outcrossing species, varying levels of self-incompatibility are observed; however, appropriate 7

8 Principles of Plant Genetics and Breeding selection for recovered DH lines having low or no levels of self-incompatibility can be identified and seed generated (Figure 3). Figure 3. Uniformity growout trials of offspring generated by selfing DH lines at the Barenbrug USA Research Center, Albany, OR, USA. Though IL1 and IL2 can be utilized to generate DH lines, the frequency of generation is low, less than 1%. However, the ability to generate IL x tall fescue hybrids is rapid and inflorescences on the F1 hybrids are abundant. When numerous F1 inflorescence are harvested from the F1, the recovery of DH lines is quite efficient. Depending on the quality and number of inflorescence, it is not unusual to obtain one to eight seedlings from each IL x tall fescue hybrid. When multiple hybrids are screened and placed in a commercial DH production situation, hundreds of DH recoveries may be obtained each season. What the approach may lack in DH generation frequency, is compensated by its efficiency in recovering DH lines. The actual mechanism of genome loss and subsequent parthenogenic behavior is unknown. Assumptions based on the occasional generation of chimeral sectors in F1 individuals (Kindiger, 2012) indicate some form of mitotic nuclear fusion events (SeguiSimarro and Nuez, 2008). 8

9 There are two methods utilized to verify that the recovered tall fescue materials are DH homozygous recoveries. The first step is to self-pollinate the particular DH individual for seed increase and perform nursery or greenhouse grow outs (Figure 3, 4a). If the offspring from the selfing do not segregate for size, inflorescence morphology, maturity or other obvious phenotypic characteristics, they are likely DHs. A second approach is to utilize molecular markers known to exhibit a banding pattern that is consistent with disomic inheritance and co-dominant expression. Since tall fescue possesses a polyploid genome constitution (Alderson and Sharp, 1994) the species will possess a considerable level of genome duplication. As a consequence, it is important to utilize markers that are known to amplify only a single or specific locus within the tall fescue genome. When such markers are utilized on offspring generated by selfing a presumed DH line, only a single amplification product will be formed at that locus since that locus would be homozygous (Figure 4b). Such markers will be most useful since segregation will be predictable and these markers will be easy to score (Stift, et al., 2008). Once DH lines are confirmed, they can be utilized in the development of new wide- or narrow-based cultivars or even hybrids. Selection of superior 9

10 Figure 4a. Offspring generated by selfing DH recovery R4P14. Seedlings phenotypes such as size, leaf angle, color, etc. are an indicator of uniformity. Figure 4b. Replicated EST-SSR marker NF24 evaluation of 6 offspring generated by selfing DH recovery R4P15. Size markers are in lanes 1, 7, 14 and 21. DH lines exhibiting superior trait qualities can be followed by the performance of a genetic distance analysis (GDA) through the use of molecular markers. DNA markers provide a powerful tool for genetic evaluation and marker-assisted breeding of crops and especially for cultivar identification. The greater the GDA, the greater the potential for advantageous genome interactions and an advantageous heterotic response. Recent studies have suggested that advances can be made in yield performance of forages, including tall fescue, through the use of molecular markers (Brummer and Casler, 2009; Amini et al., 2011). A preliminary analysis of forty DH lines is provided in a GDA dendogram tree and subjected to a principal component analys based on 40 EST-SSR markers (Figures 5a, 5b). The observed differences across the DH lines can be utilized to combine agronomically superior DH lines to maximize the potential for heterosis. The squared-euclidean distance (horizontal axis) between objects was used as the genetic distance measure and the dendogram tree clustering was performed following the method of Ward (1963). 10

11 Figure 5a. A dendogram illustrating the genetic variability or genetic distance of 40 DH tall fescue lines. DH lines with greater distances between branches or within branches represent a visual indicator of genetic distance. Principal Coordinates. 2 rd o C Coord Series1 Conclusions Figure 5b. Principal coordinate analysis of forty DH lines evaluated across 40 EST-SSR markers. Dihaploid breeding methods provide an alternative approach to the traditional backcross, selfing or recurrent selection breeding programs since they can greatly simplify 11

12 gene or trait inheritance ratios and intralocus effects (Maluszynski et al., 2003). The generation of haploid or dihaploid lines through a gamete selection approach represents an under-utilized but important breeding approach for plant breeding and genetic analysis research (Dunwell, 2010). The materials and procedures described apply directly to the breeding and selection of superior tall fescue germplasm. However, they could be expanded to ryegrass or additional fescue species. The sampling of hundreds of thousands of gametes via the pollen grains, each segregating for a myriad of genotypes from a single tall fescue individual or population, requires less labor input and represents a low cost, rapid selection strategy that can be implemented across a diversity of environments. More importantly, the breeder applying this technique to generate tall fescue DH recoveries will only rely on the potential genotypic contributions of the paternal parent. The simultaneous selection of multiple traits or complementary traits can be applied quite effectively and the prescribed approach does not require any prior genetic information regarding the inheritance or expression of the quantitative trait. When applying this method to improved or previously selected populations or cultivars, the breeder can essentially skim the cream from these germplasm resources. The retention and fixation of the qualitative or quantitative traits of interest are easily maintained by selfing the DH line. It is anticipated that when applied correctly, this approach will be effective for selection of both qualitative and quantitative traits such as maturity, drought tolerance, disease resistance, grazing persistence, forage yield and superior forage quality. References Alderson, J. & Sharp, W. C Grass varieties in the United States. Agric. Handbook 170. Soil Conserv. Serv., USDA. Washington, D.C. 12

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