A BAC pooling strategy combined with PCR-based screenings in a large, highly repetitive genome enables integration of the maize genetic and physical maps.

BACKGROUND: Molecular markers serve three important functions in physical map assembly. First, they provide anchor points to genetic maps facilitating functional genomic studies. Second, they reduce the overlap required for BAC contig assembly from 80 to 50 percent. Finally, they validate assemblies based solely on BAC fingerprints. We employed a six-dimensional BAC pooling strategy in combination with a high-throughput PCR-based screening method to anchor the maize genetic and physical maps. RESULTS: A total of 110,592 maize BAC clones (approximately 6x haploid genome equivalents) were pooled into six different matrices, each containing 48 pools of BAC DNA. The quality of the BAC DNA pools and their utility for identifying BACs containing target genomic sequences was tested using 254 PCR-based STS markers. Five types of PCR-based STS markers were screened to assess potential uses for the BAC pools. An average of 4.68 BAC clones were identified per marker analyzed. These results were integrated with BAC fingerprint data generated by the Arizona Genomics Institute (AGI) and the Arizona Genomics Computational Laboratory (AGCoL) to assemble the BAC contigs using the FingerPrinted Contigs (FPC) software and contribute to the construction and anchoring of the physical map. A total of 234 markers (92.5%) anchored BAC contigs to their genetic map positions. The results can be viewed on the integrated map of maize 12. CONCLUSION: This BAC pooling strategy is a rapid, cost effective method for genome assembly and anchoring. The requirement for six replicate positive amplifications makes this a robust method for use in large genomes with high amounts of repetitive DNA such as maize. This strategy can be used to physically map duplicate loci, provide order information for loci in a small genetic interval or with no genetic recombination, and loci with conflicting hybridization-based information.

Quantitative trait loci and comparative genomics of cereal cell wall composition.

Quantitative trait loci (QTLs) affecting sugar composition of the cell walls of maize (Zea mays) pericarp were mapped as an approach to the identification of genes involved in cereal wall biosynthesis. Mapping was performed using the IBM (B73 x Mo17) recombinant inbred line population. There were statistically significant differences between B73 and Mo17 in content of xylose (Xyl), arabinose (Ara), galactose (Gal), and glucose. Thirteen QTLs were found, affecting the content of Xyl (two QTLs), Ara (two QTLs), Gal (five QTLs), Glc (two QTLs), Ara + Gal (one QTL), and Xyl + Glc (one QTL). The chromosomal regions corresponding to two of these, affecting Ara + Gal and Ara on maize chromosome 3, could be aligned with a syntenic region on rice (Oryza sativa) chromosome 1, which has been completely sequenced and annotated. The contiguous P1-derived artificial chromosome rice clones covering the QTLs were predicted to encode 117 and 125 proteins, respectively. Two of these genes encode putative glycosyltransferases, displaying similarity to carbohydrate-active enzyme database family GT4 (galactosyltransferases) or to family GT64 (C-terminal domain of animal heparan synthases). The results illustrate the potential of using natural variation, emerging genomic resources, and homeology within the Poaceae to identify candidate genes involved in the essential process of cell wall biosynthesis.

Characterization of three maize bacterial artificial chromosome libraries toward anchoring of the physical map to the genetic map using high-density bacterial artificial chromosome filter hybridization.

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the 185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage lambda. The results indicate that the libraries are of high quality with low contamination by organellar and lambda-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6x coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 x Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 +/- 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction.