This study was performed to look at three regions of the Pt genome that were hypothesized to be under selection pressure because of the presence of putative secreted proteins or loci associated with avirulence. To begin with, gene order is conserved between the Pt BACs and Pgt. However, there is a wide range of protein conservation. A previous comparison of ESTs of Pt and Pgt found a similar level of variation in sequence, but only 40% of the Pt EST unigenes had orthologs in Pgt. Many genes were likely missing in the unigene set because of the difficulty of sampling other Pt life stages to sufficient depth, affecting the percentage. Nevertheless, within the BAC clones, many protein identities were supported by ESTs and similar sequence variation was present . Some proteins were highly conserved between the two wheat rust fungi and had homologs in Mlp and Um.
The three genes used for identifying the BACs were of most interest, in particular, the amount of variation within the sequence. PgtRAD18 had been associated with an avirulence locus in Pgt. PtRAD18 protein length is relatively similar but the sequence has diverged from the PgtRAD18 with only 56% identity. Structurally, PtRAD18 is still closely associated with a predicted secreted protein. Pt has two genes similar to HESP-379 from M. lini. Two indels in PtHSP02-4 suggest a recombination event or splicing difference evolved since the two species diverged, while the sequence differences in the C-terminus of PtHSP02-5 suggest that this region could be very variable. PtHSP04 contained a four-gene locus predicted to code for secreted proteins. Two of them are unique while two are recently duplicated paralogs. Secreted proteins are believed to be most variable amongst fungal proteins because they are under the highest selection pressure to avoid recognition by the host [16, 19, 37]. At least with these examples, It can be said that sequence variation, recombination, and duplication are driving the changes in these proteins.
Numerous fungal genomes have recently been generated, analyzed, and published. Now comparisons can be made to find core gene families associated with specific life styles and cycles. In an extensive comparison, Duplessis et al.  identified core conserved genes needed for biotrophic life in both rust species. It appears that PtHSP02-6 may be one of those genes. PtHSP02-6 aligns with a G-protein beta subunit (GPBS) and no peptide differences were found between Pt and Pgt. Furthermore, there is little difference between Pt and Mlp suggesting that this protein is under strong purifying selection in rusts. Yet, the genes flanking PtHSP02-6 are relatively conserved indicating strong selection and the importance of this gene. In Verticillium dahliae, mutations in GPBS had reduced virulence, increased microsclerotia and conidiation and decreased ethylene production . GPBS is also involved in similar functions in F. oxysporum. In M. grisea, GPBS mutants could not form appresorium, and hyphae could not penetrate and grow in rice leaves . The authors also showed that by over expressing GPBS in the fungus, appressorium could form on a hydrophillic surfaces suggesting that GPBS is necessary for control of surface recognition, growth and appressorium formation . Surface recognition and appressorium formation are the key to rust fungal establishment. This suggests that PtHSP02-6 is indispensable for the biotrophic lifecycle and could be a regulating link in pathogenicity.
A strong correlation between genome size and repetitive element content has been found for many fungal genomes. Genome expansion is significant between Pt and Pgt, even though they are both closely related and are both dikaryotic. The assembled genome for Pgt is 89 Mb  while Pt is currently estimated to be 135 Mb (Broad Institute). The sequence analysis of the three BAC clones gives some indication on why the Pt genome may be larger than the Pgt genome. Pt1F16 had the least mobile element complexity, but had Gypsy elements within Copia elements, as did PtHSP02. PtHSP02 also harbored numerous TEs and LTRs in the region between PtHSP02-1 and 3. Meanwhile, PtHSP04 contains more non-TE repeat ORFs, its homologous genes are scattered across Pgt scaffolds, and its sequence reveals recombination and/or transposition events disrupting syntenic genes. There is also evidence of gene movement by active elements. PtHSP02-2 was directly flanked by LTRs and was not found in PgtSC7, PtHSP04-5 was also flanked by LTRs and could be found in PgtSC48, and PtHSP04-10 only had a single LTR flanking it, but was flanked on the opposite side by a partial Harbinger element. It is possible that since these regions are in repetitive sequence there are assembly errors in Pgt, however, each Pgt homolog are in high confidence scaffolds.
Most surprising are the non-transposable element, repeated sequences found in the Pt BACs (Table 2). Each had homologs throughout the Pgt genome. Most had conserved domains that were maintained, while flanking sequences were greatly diverged. Many were high in Lys suggesting a helix protein structure. Some are expressed, based on the presence of an aligning EST, and have homologs in Mlp, suggesting an importance. The helical nature of these proteins would suggest their involvement as nucleotide binding elements. Pt has five different spore types in its lifecycle involving two different hosts requiring a significant level of cell modifications and cell types. Sequences like these have not been described before and could represent undiscovered elements in the disease cycle.
This work has shown significant genome synteny between two closely related wheat rust fungi. Gene sequences confirmed previous findings of the existence of EST sequence variation between Pt and Pgt. Various levels of homologies are present, but many of the genes are diverging in a manner that is species specific . Both genomes have a significant amount of mobile elements. Some TE copies are conserved between the two species suggesting ancestral insertion. The insertion of TE sequences helps explain genome expansion, and their insertion near secreted protein genes may alter their regulation or cause their duplication and spread or deletion. Most surprising was the presence of small predicted non-TE genes with numerous homologs in Pgt. As many of the small repeated sequences are highly helical in predicted structure, one could suggest they are involved in DNA binding and regulation. Further work is needed to determine when they are expressed and at what stage of the life cycle. When analysis of the Pt and Pst genomes has been concluded, it can be determined if the repeated nature of these predicted genes is maintained within the wheat rust fungi.