A single molecule scaffold for the maize genome

Abstract

About 85% of the maize genome consists of highly repetitive sequences that are interspersed by low-copy, gene-coding sequences. The maize community has dealt with this genomic complexity by the construction of an integrated genetic and physical map (iMap), but this resource alone was not sufficient for ensuring the quality of the current sequence build. For this purpose, we constructed a genome-wide, high-resolution optical map of the maize inbred line B73 genome containing >91,000 restriction sites (averaging 1 site/ approximately 23 kb) accrued from mapping genomic DNA molecules. Our optical map comprises 66 contigs, averaging 31.88 Mb in size and spanning 91.5% (2,103.93 Mb/ approximately 2,300 Mb) of the maize genome. A new algorithm was created that considered both optical map and unfinished BAC sequence data for placing 60/66 (2,032.42 Mb) optical map contigs onto the maize iMap. The alignment of optical maps against numerous data sources yielded comprehensive results that proved revealing and productive. For example, gaps were uncovered and characterized within the iMap, the FPC (fingerprinted contigs) map, and the chromosome-wide pseudomolecules. Such alignments also suggested amended placements of FPC contigs on the maize genetic map and proactively guided the assembly of chromosome-wide pseudomolecules, especially within complex genomic regions. Lastly, we think that the full integration of B73 optical maps with the maize iMap would greatly facilitate maize sequence finishing efforts that would make it a valuable reference for comparative studies among cereals, or other maize inbred lines and cultivars.

Figure 1. A screenshot of an optical contig (OMcontig_31) showing a possible telomeric-end.

The Genspect viewer depicts each optical map, constructed from a genomic DNA molecule, as a horizontal track consisting of colored boxes. The length of each box represents the size (in kb) of a restriction fragment within a genomic molecule. The map information of the entire contig is combined into a single track (top; blue) called the consensus map. Restriction fragments are colored keyed for indicating their agreement with the consensus map; gold boxes show agreement, while red (false cut), blue (missing cut), and purple (false cut) indicate aforementioned restriction map differences. This deep contig shows a distinct edge, or end, populated by ∼40 optical maps indicating a telomeric region (Chr7).

Figure 2. BACop, an algorithm that anchors optical contigs onto the iMap.

BACop (Materials and Methods) employs four distinct steps for anchoring optical contigs; we illustrate the first three steps here: (A) Restriction fragments of an optical contig map are matched against BAC sequences comprising multiple sub-contigs. (B) Matching BAC sequence contigs are located along the optical contig map. (C) Dynamic programming places BACs onto optical map contigs. Seq. = sequence, frag = restriction fragment, and BAC = bacterial artificial chromosome.

Figure 3. Optical contig placements on iMap using BACop.

iMap chromosomes are numbered tracks showing the locations of FPC contigs and their subsidiary BACs. BACs (small boxes) are colored keyed according to their sequencing status: magenta [HTGS_FULLTOP - 2×384 paired-end attempts (6× coverage), completed shotgun phase, initial assembly]; lime [HTGS_PREFIN - completed automated improvement phase (AutoFinish)]; cyan [HTGS_ACTIVEFIN - active work being done by a finisher], yellow [HTGS_IMPROVED - finished sequence in gene regions; improved regions will be indicated, once order and orientation of improved segments are confirmed; a comment will be added to indicate this], and black [BACs with no usable or complete SwaI fragments]. The inset shows a zoomed view of a region (ctg146–153) on Chr 3. The blue tracks show optical map contigs anchored to the iMap by BACop. Optical contig identifiers are lettered in blue; pink lettering indicates that an OMcontig was split into two or more pieces for optimizing alignments. Black lettering and a “+,” indicate that two or more optical map contigs were joined. Vertical grey lines show placements of BACs onto optical map contigs. OM = optical map.

Figure 4. Comparative alignment of optical and pseudomolecule maps.

In silico restriction maps of pseudomolecules (Zm1S_supercontig and Zm9L_supercontig) were found to align (Materials and Methods) to optical contigs (OMcontig_15 and 23). This allowed the identification of common and discordant regions. SwaI restriction sites are depicted by vertical lines. Regions of the optical contig and the pseudomolecule that align are teal colored, and the aligned regions are pointed to with black connecting lines.

Figure 5. Optical maps guide ongoing construction of pseudomolecules.

The quality of a large pseudomolecule (ctg182; ∼21.8 Mb) was successively improved by alignment of provisional builds against OMcontig_1 (optical contig). The increase in aligned regions of ctg182V7 to OMcontig_1 compared to the earlier version, ctg182V3, to the OMcontig demonstrates the improvement in the quality of the build. Red highlighting in OMcontig_1 shows optical contig regions aligning to both pseudomolecule versions. Maps of the ∼1.3 M region boxed in green are shown below, with concordances and discordances illustrated between the optical contig and ctg182V3 and V7 on a per restriction fragment basis. Gold colored fragments (boxes) signify concordance, while other colors signify discordance. Sizes are in kb. Ctg182V3 contains a run of pink fragments, indicating discordance with OMcontig_1, which is partially mediated in ctg182V7 as evidenced by greater alignment (less pink and more gold boxes).

Figure 6. Alignment of unfinished BAC sequence contigs to optical contig maps.

Matching restriction fragments in the BAC sub-contigs and the optical contig map are indicated by yellow boxes connected by lines; numbers show fragment size (kb). These alignments illustrate how BAC sequence contigs can be ordered and oriented using optical map alignments.