Constructing Physical and Genomic Maps for Puccinia striiformis f. sp. tritici, the Wheat Stripe Rust Pathogen, by Comparing Its EST Sequences to the Genomic Sequence of P. graminis f. sp. tritici, the Wheat Stem Rust Pathogen

Abstract

The wheat stripe rust fungus, Puccinia striiformis f. sp. tritici (Pst), does not have a known alternate host for sexual reproduction, which makes it impossible to study gene linkages through classic genetic and molecular mapping approaches. In this study, we compared 4,219 Pst expression sequence tags (ESTs) to the genomic sequence of P. graminis f. sp. tritici (Pgt), the wheat stem rust fungus, using BLAST searches. The percentages of homologous genes varied greatly among different Pst libraries with 54.51%, 51.21%, and 13.61% for the urediniospore, germinated urediniospore, and haustorial libraries, respectively, with an average of 33.92%. The 1,432 Pst genes with significant homology with Pgt sequences were grouped into physical groups corresponding to 237 Pgt supercontigs. The physical relationship was demonstrated by 12 pairs (57%), out of 21 selected Pst gene pairs, through PCR screening of a Pst BAC library. The results indicate that the Pgt genome sequence is useful in constructing Pst physical maps.

Figure 1

An example of physical maps for Pst ESTs based on corresponding sequence positions of homologous genes of Pgt. All 242 physical groups are presented in Supplementary file 1. The distance in mega base (Mb) is shown on the left. The clones in a group indicated by a vertical line are in the same contig and the start and end positions of the sequence matching the positions in the contig are shown in the “( )” following the clone identification number. The number after the “( )” indicates the number of the gene with multiple positions in the Pgt genome. An asterisk indicates that the number of matching base pairs is smaller than 100. The clones underlined were used in PCR amplification of the Pst BAC library.

Figure 2

Characterization of Pst physical groups corresponding to Pgt supercontigs. (a) Frequencies of genomic sequence sizes that were covered by Pst gene physical groups. (b) Frequencies of number of Pst genes in a physical group corresponding to a Pgt supercontig in the three Pst cDNA libraries and the total. (c) Gene densities (Kb/gene) in the Pst physical groups corresponding to Pgt supercontigs.

Figure 3

Agarose gels showing positive amplification of the Pst BAC clones using multiplex PCR with primer pairs PSTCY32GT1071F/R (upper bands) and PST78SP3H2F/R (lower bands). (a) Amplification of 58 BAC plate pools to identify positive pools. (b) Amplification of row and column pools of a positive plate to identify individual positive clones. The arrow in (a) indicates the bands amplified with Pst genomic DNA and in (b) indicates molecular size marker.