An additional B73 library (ISUM6) was prepared using the same mRNAs as ISUM5-RN, but 21 specific 6-bp "bar-code" tags were added to each mRNA prior to cloning (Qiu, et al, submitted). These tags allow the particular mRNA source from which a given EST was derived to be determined, even though this library was prepared using multiple sources of mRNAs. Over 3,600 3 ESTs from ISUM6 have been sequenced and submitted to GenBank.
Over 1,000 3 ESTs from a Mo17 cDNA library (ISUM7) were sequenced and aligned with the 3UTRs of corresponding B73 alleles. At least one indel is present in 48% (277/572) of these alignments.
Development of IDP Markers and Genetic Mapping of ESTs. IDPs (InDel Polymorphisms) are a class of PCR-based, allele-specific genetic marker that detects insertion and deletions (indels) among maize alleles. Primer pairs were designed based on the 3 UTRs of ESTs and used to PCR amplify B73 and Mo17 alleles from genomic DNA. A useful primer pair can distinguish B73 and Mo17 alleles because it can amplify one allele but not the other, or because the PCR products from the two inbreds display a size polymorphism. A high-throughput primer design tool was developed. 531 out of 7,702 (6.9%) primers designed with this tool yielded validated +/- results or size polymorphisms between B73 and Mo17. About one third of the IDPs are polymorphic between any pair of inbreds tested, demonstrating the widespread utility of these genetic markers. The 531 IDP markers were genetically mapped using a panel of 94 RIs from the Intermated B73 X Mo17 (IBM) population. A set of 22 inbreds has been surveyed with 3,884 primer pairs. About 31% of these primers detect polymorphisms between at least one inbred and B73 or one inbred and Mo17. 88 F1BC populations are being developed and will be used to map an additional 3,000 IDPs and the corresponding cDNAs.
Rescue: a new tool for gene discovery. Because genes are present
at equimolar concentrations in genomic DNA, genomic sequencing provides
a means to uncover those genes that will be missed via EST projects as
a consequence of being expressed only under unusual conditions or at very
low levels. Unfortunately, it is not currently feasible/cost-effective
to sequence the entire crop genomes. Hence, biochemical approaches need
to be developed to filter out the non-coding regions of the genome so that
limited sequences resources can be focused on genic DNA. A novel expression
vector system has been developed to directly rescue open-reading frames
(ORFs) from genomic DNA. In a preliminary experiment, 250 maize genomic
fragments cloned into this vector were selected based on a colorimetric
screen. Sequence analysis of these clones revealed that 93.6% (234 out
of 250) contain an uninterrupted ORF and 55% (129 out of 234) exhibit significant
degrees of sequence similarity to entries in protein and EST databases.
Many of the remaining clones are thought to contain ORFs that have not
yet been discovered via EST sequencing.
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