A segment of the apospory-specific genomic region is highly microsyntenic not only between the apomicts Pennisetum squamulatum and buffelgrass, but also with a rice chromosome 11 centromeric-proximal genomic region

Abstract

Bacterial artificial chromosome (BAC) clones from apomicts Pennisetum squamulatum and buffelgrass (Cenchrus ciliaris), isolated with the apospory-specific genomic region (ASGR) marker ugt197, were assembled into contigs that were extended by chromosome walking. Gene-like sequences from contigs were identified by shotgun sequencing and BLAST searches, and used to isolate orthologous rice contigs. Additional gene-like sequences in the apomicts' contigs were identified by bioinformatics using fully sequenced BACs from orthologous rice contigs as templates, as well as by interspecies, whole-contig cross-hybridizations. Hierarchical contig orthology was rapidly assessed by constructing detailed long-range contig molecular maps showing the distribution of gene-like sequences and markers, and searching for microsyntenic patterns of sequence identity and spatial distribution within and across species contigs. We found microsynteny between P. squamulatum and buffelgrass contigs. Importantly, this approach also enabled us to isolate from within the rice (Oryza sativa) genome contig Rice A, which shows the highest microsynteny and is most orthologous to the ugt197-containing C1C buffelgrass contig. Contig Rice A belongs to the rice genome database contig 77 (according to the current September 12, 2003, rice fingerprint contig build) that maps proximal to the chromosome 11 centromere, a feature that interestingly correlates with the mapping of ASGR-linked BACs proximal to the centromere or centromere-like sequences. Thus, relatedness between these two orthologous contigs is supported both by their molecular microstructure and by their centromeric-proximal location. Our discoveries promote the use of a microsynteny-based positional-cloning approach using the rice genome as a template to aid in constructing the ASGR toward the isolation of genes underlying apospory.

Figure 1.

BAC composition and structure of contigs. The overlapping pattern of BACs in each contig assembly is shown for each species: P2, P. squamulatum contig; C1A, C1B, and C1C, buffelgrass contigs; RA, RB, and RC, rice contigs. Each BAC is represented by a black or gray line. The identity of BACs is given at the right of each contig. In the apomicts, black lines represent BACs initially isolated by screening the BAC libraries with the ugt197 marker (Roche et al., 2002) and assembly into contigs. BACs depicted as gray lines in the buffelgrass C1C contig were isolated by chromosome walking and represent a contig extension. All rice BACs were isolated by screening the rice BAC library with the AP2 and XYL sequences. A 10-kb scale bar is provided.

Figure 2.

Microsynteny between the ASGRs of P. squamulatum and buffelgrass at the ugt197-containing contigs and their rice orthologous contigs. The chromosomal region spanned by contigs is represented by black thick vertical lines. Gray lines at the right side of contigs represent HindIII restriction fragments to which the analyzed gene-like sequences and markers represented by colored arrows hybridize. A kilobase scale, color codes, and notes are given in the picture reference frame. Microsynteny patterns I and II referred to in the text are quoted and depicted by double-blue and double-green fine vertical lines beside contig C1C and contig P2, respectively. When these patterns show disruption in one or more sequences, the region of disruption is indicated by dashed double lines. For the contigs C1C and RA that show the highest microsynteny between the apomicts and rice, oblique colored lines are drawn that connect each syntenic gene-like sequence and marker. When these lines are dashed, they indicate a synteny disruption. Rice contig names are followed by a number between parentheses corresponding to the rice genome database FPC contigs to which they belong, according to the September 12, 2003, build (www.genome.arizona.edu). The chromosome location of each rice contig is indicated below the drawings.

Figure 3.

Comparative chromosomal location relative to centromeric sequences of the ASGRs from the two apomicts and their rice chromosome 11 orthologous genomic regions. Thick black vertical lines represent the relevant chromosomes in each species. Gray horizontal lines indicate chromosome centromeres and a centromere-like sequence on the distal side of the short arm of P. squamulatum chromosome, while black horizontal lines indicate the syntenic ugt197-containing ASGR segment in the apomicts and their orthologous rice genomic regions within FPC contig 77. The approximately 2.77-Mb FPC contig 77 is zoomed in next to the chromosome 11 scheme to show the relative orientation of contigs RA and RC (depicted by thin black vertical lines), and also to indicate in megabase (Mb) units the approximate sequence spanning (shown by dashed lines) in between these two contigs, and spanning from RA and RC to the centromere and to the distal end of FPC contig 77, respectively. The chromosome schemes for P. squamulatum and buffelgrass are representing the recently described localization of the ASGRs relative to centromeric sequences (Goel et al., 2003).