Quality assessment of maize assembled genomic islands (MAGIs) and large-scale experimental verification of predicted genes

Abstract

Recent sequencing efforts have targeted the gene-rich regions of the maize (Zea mays L.) genome. We report the release of an improved assembly of maize assembled genomic islands (MAGIs). The 114,173 resulting contigs have been subjected to computational and physical quality assessments. Comparisons to the sequences of maize bacterial artificial chromosomes suggest that at least 97% (160 of 165) of MAGIs are correctly assembled. Because the rates at which junction-testing PCR primers for genomic survey sequences (90-92%) amplify genomic DNA are not significantly different from those of control primers ( approximately 91%), we conclude that a very high percentage of genic MAGIs accurately reflect the structure of the maize genome. EST alignments, ab initio gene prediction, and sequence similarity searches of the MAGIs are available at the Iowa State University MAGI web site. This assembly contains 46,688 ab initio predicted genes. The expression of almost half (628 of 1,369) of a sample of the predicted genes that lack expression evidence was validated by RT-PCR. Our analyses suggest that the maize genome contains between approximately 33,000 and approximately 54,000 expressed genes. Approximately 5% (32 of 628) of the maize transcripts discovered do not have detectable paralogs among maize ESTs or detectable homologs from other species in the GenBank NR nucleotide/protein database. Analyses therefore suggest that this assembly of the maize genome contains approximately 350 previously uncharacterized expressed genes. We hypothesize that these "orphans" evolved quickly during maize evolution and/or domestication.

Fig. 1.

Illustrations of computational and wet-laboratory strategies used for MAGI validation. (A) The consistency of MAGIs was assayed via alignment to maize B73 BACs. A set of potential MAGI/BAC alignments was identified by using blast (see Materials and Methods). The dashed lines mark portions of the MAGI that fail to match the BAC sequence. MAGIs were deemed to be inconsistent if they had a total overhang length (combined length of dashed lines) of >20 bp. The overhangs associated with four of the six consistent MAGI/BAC pairs that have sizes of between 6 and 20 bases can be recognized as incompletely trimmed vector sequences on a terminal GSS of a MAGI (Table 5). Four of the consistent MAGI/BAC pairs have overhangs of <6 bases, which may also be derived from incompletely trimmed vector. Terminal MAGI/BAC alignments of the type shown on the right do not provide evidence of inconsistency. Six such cases were identified. (B) Comparison of genomic PCR success rates: Within a MAGI, each primer pair annealed to the same GSS (set 1), two GSSs from the same clone (set 2), or two GSSs from different clones (set 3). Set 1 primer pairs served as a control to assess the success of primer design and PCR. Sets 2 and 3 primer pairs were used to validate the structure of MAGIs.

Fig. 2.

Collapse of NIPs in MAGI_89783. Mismatches among GSSs are highlighted.