**5. Confirming recovery of DH tall fescue lines**

There are two methods that can be performed to indicate or 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 grow outs. If the offspring from the selfing do not segregate for size, inflorescence morphology, maturity or other obvious phenotypic characteristics, they are likely DHs (Figure 5).

A second approach is to utilize molecular markers known to exhibit a banding pattern that is consistent with disomic inheritance and co-dominant expression. That is, each marker exhibits a maximum of two bands for each individual when two alleles at a particular locus are present in a heterozygous state. When one band is present, the allele at that locus would be in a homozygous condition. These markers will be most useful since segregation will be predictable and such markers are easy to score (Stift et al., 2008). Tall fescue, having a polyploid genome constitution (Alderson and Sharp, 1994), has 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 site in the tall fescue genome. When such markers are utilized, only a single amplification product will be formed at that locus since that locus would be homozygous. If the site were heterozygous, the amplification of multiple alleles would be visualized. Following the approach of Saha et al. (2004), selected DH lines were evaluated utilizing a set of twelve EST-SSR markers with the analyses being performed by DNA LandMarks (Quebec, Canada). The results of the analysis indicated that DH47 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 Laboratory.


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.
