**6. Conclusion**

280 Genetic Diversity in Plants

exhibited a high level of allelic variability when compared to the other DH recoveries (Table 2). From this analysis, DH47 is indicated to be non-homologous for all marker loci and is likely a rogue. DH lines 41, 42, 44 and 57 are indicated to be true DH recoveries, homozygous at the evaluated marker loci. Similar approaches may be equally effective in polyploids thought to possess a high degree of genome duplication (Esselink et al., 2004).

Fig. 5. A plot in a DH phenotypic uniformity trial at the USDA-ARS, Grazinglands Research

**EST-SSR DH41 DH42 DH44 DH47 DH57 NFA004** 285 285 285 298 285 **NFA030** 199 199 199 199 199 **NFA039** 293 293 293 291 293 **NFA047** 244 244 244 241/244/250 244 **NFA067** 130 151 175 147 136 **NFA068** 254 254 254 251 254 **NFA073** 236 236 246 236 246 **NFA074** 244 244 244 241/244/250 244 **NFA095** 187 187 187 182/187 187 **NFA104** 218 218 218 238 218 **NFA115** 236 236 236 220/230 236 **NFA133** 150 150 150 145/155 150 Table 2. Base-pair marker size segregation of twelve EST-SSR markers across five potential dihaploid lines. The absence of allelic variation across markers indicates a dihaploid recovery, homozygous for the locus (e.g. DH41, DH42, DH44, DH57). An excessive level of

allelic marker variability suggests a non-homozygous condition and the lack of a

homozygous DH recovery (DH47). The DH47 line would be discarded.

Laboratory.

The generation of haploid or dihaploid lines through a gamete selection approach can be of critical importance in breeding and genetic analysis research (Duwell, 2010). The materials and procedures described above apply directly to the breeding and selection of superior tall fescue germplasm and could be expanded to other fescue species. The expansion of research programs utilizing this approach should find the availability of DH lines useful when objectives are focused on plant genomics, fine mapping of DH derived populations and/or marker-assisted selection of genes that confer agronomically favorable traits. One advantage to the described method is that microspore methods are not utilized and the researcher is not limited to genotypes amenable to microspore culture techniques. The sampling of hundreds of thousands of 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 diverse environments. The simultaneous selection of multiple traits or complementary traits can be applied quite effectively and the prescribed approach also does not require any prior genetic information regarding the inheritance or expression of the quantitative trait. Though molecular markers can be applied, the approach does not require the utilization of molecular markers for marker-assisted selection. Generated DH lines possessing superior genotypes are maintained through selfing, retaining the fixation of the qualitative or quantitative trait of interest.

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 hybrids are abundant. When numerous F1 inflorescences are utilized from the F1, the recovery of DH lines is quite efficient. Depending on the quality and number of inflorescences, 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 line 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. It is anticipated that when applied correctly, this approach would be effective for development of tall fescue lines exhibiting quality traits such as days to flowering, drought tolerance, grazing persistence, CP, NDF, ADF, RFQ, lignin, forage yield, etc. The gamete selection approach should also be advantageous to tall fescue breeders requiring efficient and rapid methods of developing tall fescue germplasm adapted to the unknown parameters of global climate change (Humphreys et al, 2006).
