Overview of the BAP mapping method.

<p>(a) A genomic BAC library is pooled in a 3-dimensional fashion (1^st D for plate ID; pool all unique clones for each plate and create overlapping superpools of plate pools; 2^nd D for row ID: pool all clones in identical rows over several plates [3.5 plates for <i>Arabidopsis</i>] and create overlapping ‘superpools’ of row pools to allow row identification; 3^rd D pool for column ID: pool all clones in identical columns over several plates [3.5 plates for <i>Arabidopsis</i>] and create overlapping ‘superpools’ of column pools to allow column identification) to generate the BAC-range panel. (b) Of 96 wells of a plate, 88 contain random aliquots of a BAC library (8 wells are reserved for controls), each BAC aliquot contains DNA corresponding 0.6–0.7 fold coverage of the genome. The presence of single copy STS markers (M1-My) is indicated by various colours in the aliquots. (c) The presence of markers found after MT-PCR-HRM. (d) The BAC range panel mapping results allow creation of short linkage maps, and (e) at the same time, to establish a corresponding BAC tiling path. (f) To link the shorter contigs obtained from the BAC panel and to close the gaps between contigs, markers chosen from the ends of the contigs are mapped by the long-range (large size) DNA panel. (g) The merged linkage and physical map as the final result.</p

Comparison of BAP mapping and conventional HAPPY mapping.

<p>Comparison of BAP mapping and conventional HAPPY mapping.</p

The physical map of FCA locus in comparison to its sequence.

<p>The sequence position indicated by the first nucleotide of the 40 markers belonging to the FCA locus (a) is reflected by the physical map (b – enframed and c) after BAP mapping. With the BAC range panel, the BACs (blue rectangles) harbouring the 40 markers are sorted into 8 contigs (c) and assembled by means of the long-range panel into a single linkage map (b) spanning the entire region of 1.8 Mbp.</p

Marker typing in the BAC-range panel by MT-PCR-HRM.

<p>Each individual marker, which is uniquely amplified from genomic DNA, is mapped by genotyping in the mapping panel of 88 aliquots (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009089#pone-0009089-g001" target="_blank">Fig. 1b,f</a>). This MT-PCR-HRM typing identifies the aliquots within the panel that are positive (green) or negative (red) for a particular marker by melting curve analysis using a fluorescent dye. If the PCR product is present in aliquots, the ‘positive’ green trace is detected and scored as 1. The absence of PCR product in aliquot (red line) is scored as 0. With a series of 1 and 0 for all aliquots in the panel, the marker is scored.</p

Species distribution of annotated <i>Rafflesia cantleyi</i> flower transcripts.

<p>Species distribution of annotated <i>Rafflesia cantleyi</i> flower transcripts.</p

Statistical summary of <i>Rafflesia cantleyi</i> sequence data.

<p>Statistical summary of <i>Rafflesia cantleyi</i> sequence data.</p

Comparison of <i>de novo</i> assembly data using Oases, Trinity and CLC Genomics Workbench.

<p>Comparison of <i>de novo</i> assembly data using Oases, Trinity and CLC Genomics Workbench.</p

Perigone Lobe Transcriptome Analysis Provides Insights into <i>Rafflesia cantleyi</i> Flower Development

<div><p><i>Rafflesia</i> is a biologically enigmatic species that is very rare in occurrence and possesses an extraordinary morphology. This parasitic plant produces a gigantic flower up to one metre in diameter with no leaves, stem or roots. However, little is known about the floral biology of this species especially at the molecular level. In an effort to address this issue, we have generated and characterised the transcriptome of the <i>Rafflesia cantleyi</i> flower, and performed a comparison with the transcriptome of its floral bud to predict genes that are expressed and regulated during flower development. Approximately 40 million sequencing reads were generated and assembled <i>de novo</i> into 18,053 transcripts with an average length of 641 bp. Of these, more than 79% of the transcripts had significant matches to annotated sequences in the public protein database. A total of 11,756 and 7,891 transcripts were assigned to Gene Ontology categories and clusters of orthologous groups respectively. In addition, 6,019 transcripts could be mapped to 129 pathways in Kyoto Encyclopaedia of Genes and Genomes Pathway database. Digital abundance analysis identified 52 transcripts with very high expression in the flower transcriptome of <i>R</i>. <i>cantleyi</i>. Subsequently, analysis of differential expression between developing flower and the floral bud revealed a set of 105 transcripts with potential role in flower development. Our work presents a deep transcriptome resource analysis for the developing flower of <i>R</i>. <i>cantleyi</i>. Genes potentially involved in the growth and development of the <i>R</i>. <i>cantleyi</i> flower were identified and provide insights into biological processes that occur during flower development.</p></div

The pathways and products involved in plant hormone signal transduction.

<p>The pathways and products involved in plant hormone signal transduction.</p

Heat map of selected genes that are differentially expressed between the developing flower and the floral bud of <i>Rafflesia cantleyi</i>.

<p>Heat map of selected genes that are differentially expressed between the developing flower and the floral bud of <i>Rafflesia cantleyi</i>.</p