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

Abstract

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 the185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage λ. The results indicate that the libraries are of high quality with low contamination by organellar and λ-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 6× coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 × 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.

Physical and Genetic Structure of the Maize Genome Reflects its Complex Evolutionary History

“…Integrated Physical and Genetic Map of the Maize B73 Genome A total of three deep-coverage large-insert BAC libraries covering ;30 genome equivalents were constructed using HindIII, EcoRI, and MboI digests of high molecular weight DNA isolated from maize inbred line B73 [24,25]. We used two different methods to fingerprint the same set of BAC clones, referred to as agarose [29] and HICF [30,31], to crossconfirm the assembled physical contigs of homoeologous regions and highly conserved sequence families.…”

“…The three BAC libraries (303 total coverage) used in this study were HindIII (136-kb average insert size), EcoRI (163-kb), and MboI (167-kb) libraries [24,25], with 14.23, 7.63, and 7.83 coverage respectively. The use of three BAC libraries helped to ensure the maize genome was fully represented.…”

“…mays cv. B73, which is comprised of many complementary resources including: (1) deep-coverage large insert BAC libraries [24,25]; (2) agarose and high information content fingerprint (HICF) datasets assembled with fingerprinted contig software (FPC) [26,27]; (3) a genetic marker dataset to anchor the physical map to the maize genetic map; and (4) sequence-based marker datasets (overgos [28] and BESs [21]) to infer contig position and orientation with respect to the rice reference sequence [3]. Alignment of the two genomes allowed us to shed new light on the organization of the chromosomes of the progenitors contributing to the maize polyploidization, unravel rearrangements associated with the subsequent diploidization of the maize genome, and infer the sizes and locations of homoeologous regions together with spatial patterns of genome size evolution.…”

Gene expression of a gene family in maize based on noncollinear haplotypes

“…If we could determine contiguous genomic sequences comprising the loci of z1C zein in B73 that we had already determined from inbred line BSSS53 (4), we could make a high-resolution analysis of two haplotypes of a gene family and a gene-dense chromosomal region in maize. Using the same probes as described for the corn-belt inbred lines, we screened a maize HindIII BAC library constructed from inbred line B73 (10). We identified 15 positive BAC clones containing z1C gene sequences and͞or the php200725 marker (Table 3, which is published as supporting information on the PNAS web site) and subjected them to DNA fingerprinting.…”

“…A HindIII BAC library constructed from maize inbred line B73 was used for screening clones containing z1C gene sequences (10). High-density filter screening and BAC clone characterization were conducted as described (3).…”