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

Young-Sun Yim,Zheiwei Fang,Hector Sanchez-Villeda,Georgia L Davis,John E Mullet,Pamela Close,Patricia E Klein,Mary L Schaeffer,Jack M Gardiner,Edward H Coe,Theresa A Musket,Michael D Mcmullen,Patricia Moak

doi:10.1186/1471-2164-8-47

Abstract

BackgroundMolecular 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.ResultsA total of 110,592 maize BAC clones (~ 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 [1,2].ConclusionThis 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.

Highlights

Molecular markers serve three important functions in physical map assembly
A total of 110,592 BAC clones were pooled in six distinct directions to generate 288 unique pools (48 per dimension) containing 2,304 clones
Contig assembly and anchoring via high-throughput PCR screening of pools DNA pools were screened with 254 PCR-based STS markers to identify overlapping BAC clones

Summary

Introduction

Molecular markers serve three important functions in physical map assembly They provide anchor points to genetic maps facilitating functional genomic studies. They reduce the overlap required for BAC contig assembly from 80 to 50 percent. Overgo hybridization probes resulting from annealing of two overlapping oligonucleotides (followed by a fill in reaction) have been employed to generate BAC contig maps [9,10]. This technique has an advantage over the conventional oligonucleotide probe hybridization method, because the probes are slightly larger, providing improved hybridization kinetics and specificity. The presence of repeat elements in the labeled probe often confounds hybridization results, [11] and the procedures are more cumbersome since they involve radioactive material

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Discussion

Conclusion