Figure 1.
Phylogenetic tree of cloned and sequenced PCR products (individual clones labeled a-e) spanning two introns of the EPSPS gene in three glyphosate-resistant (R) and three susceptible (S) A. palmeri individuals supports the existence of two EPSPS loci in A. palmeri, and the amplification of one EPSPS locus in R genomes.
Scale bar represents the number of nucleotide substitutions per site.
Table 1.
EPSPS genomic copy number measured using qPCR primers within an intron is similar to copy number measured using qPCR primers within an exon for R and S individuals.
Table 2.
Hits to EPSPS and other genes encoding herbicide target sites from 454 genomic sequencing in A. palmeri. BLAST searches performed against all raw reads and unigene only (contigs+singletons) databases.
Table 3.
Sequences in genomic regions flanking EPSPS in A. palmeri have similarity to known transposable elements in rice [28].
Figure 2.
Consensus sequence of A. palmeri EPSPS from 16 MS-R fosmids.
A) exons for EPSPS (gray) and a putative Ac transposase (red), putative promoters (green; numbers indicate promoter prediction score, 1 is maximum possible), MITE-homologous sequence (blue), a putative transposon (purple), and a repetitive sequence motif (orange). B) Alignment of the consensus A. palmeri EPSPS and flanking genomic region sequences for 16 fosmids from MS-R. EPSPS exons (gray arrows) from 1 to 12,000 bp, and exons of a putative Ac transposase (red) from 16,000 to 19,000 bp. Green color in identity bar indicates identical sequences in all fosmids and brown indicates polymorphisms.
Figure 3.
Assembly of 454 contigs to the fosmid consensus sequence.
Contigs were identified by running a BLAST of the 454 data against the fosmid consensus sequence. Top sequence highlighted in yellow is the fosmid consensus sequence, remaining sequences are from the 454 data, and sequence at the top is the consensus of the fosmid and 454 data. The EPSPS exons (gray), MITE-homologous sequences (blue), a putative transposon (purple), a putative Ac transposase (red), putative promoters (green triangles), and a repetitive sequence motif (orange) are indicated.
Figure 4.
Southern blot analysis of EPSPS gene amplification in GA-S (S, 20 µg gDNA) and GA-R (R1, 1 µg gDNA and R10, 10 µg gDNA) A. palmeri.
Hybridizations with 33P-dCTP labeled probes for A) EPSPS first exon, B) 5′ MITE, C) EPSPS last exon, and D) 3′ MITE; E) probe and exon locations and expected restriction sites.