Anchoring 9,371 maize expressed sequence tagged unigenes to the bacterial artificial chromosome contig map by two-dimensional overgo hybridization

Abstract

Our goal is to construct a robust physical map for maize (Zea mays) comprehensively integrated with the genetic map. We have used a two-dimensional 24 x 24 overgo pooling strategy to anchor maize expressed sequence tagged (EST) unigenes to 165,888 bacterial artificial chromosomes (BACs) on high-density filters. A set of 70,716 public maize ESTs seeded derivation of 10,723 EST unigene assemblies. From these assemblies, 10,642 overgo sequences of 40 bp were applied as hybridization probes. BAC addresses were obtained for 9,371 overgo probes, representing an 88% success rate. More than 96% of the successful overgo probes identified two or more BACs, while 5% identified more than 50 BACs. The majority of BACs identified (79%) were hybridized with one or two overgos. A small number of BACs hybridized with eight or more overgos, suggesting that these BACs must be gene rich. Approximately 5,670 overgos identified BACs assembled within one contig, indicating that these probes are highly locus specific. A total of 1,795 megabases (Mb; 87%) of the total 2,050 Mb in BAC contigs were associated with one or more overgos, which are serving as sequence-tagged sites for single nucleotide polymorphism development. Overgo density ranged from less than one overgo per megabase to greater than 20 overgos per megabase. The majority of contigs (52%) hit by overgos contained three to nine overgos per megabase. Analysis of approximately 1,022 Mb of genetically anchored BAC contigs indicates that 9,003 of the total 13,900 overgo-contig sites are genetically anchored. Our results indicate overgos are a powerful approach for generating gene-specific hybridization probes that are facilitating the assembly of an integrated genetic and physical map for maize.

Figure 1.

Flowchart for determination of overgo-BAC addresses. Approximately 70,716 maize ESTs were clustered to derive 10,723 EST unigene contigs, which were masked for repeat sequences prior to the identification of 10,642 40-bp regions that were suitable for overgo development. A total of 165,888 BACs evenly divided between HindIII and EcoRI libraries were spotted in duplicate to four high-density nylon filters. A two-dimensional, 24 × 24 overgo pooling strategy was used to allow identification of 576 overgo-BAC addresses upon deconvolution of the pooled hybridization data. For further details, see “Materials and Methods.”

Figure 2.

Distribution of BAC hits per overgo. Both the EcoRI (162 kb; 5.4×) and HindIII (137 kb; 4.6×) libraries are shown to illustrate the distribution of BAC hits per overgo for 9,371 overgo hybridizations across the two libraries. Pooled EcoRI and HindIII BAC libraries (10×) are also shown and reveal 345 overgos that identify only one BAC in the combined libraries. Overall, repeat masking of EST assemblies was effective, as only approximately 5% of the overgo probes hit more than 25 BACs in either the EcoRI or HindIII libraries. In the pooled libraries, only 13% and 4% of the overgos hit more than 25 or 50 BACs, respectively.

Figure 3.

Distribution of overgo-BAC hits across multiple contigs. A total of 292,039 HindIII BAC fingerprints (approximately 15×) and overgo markers identifying 25 or fewer BACs were assembled into 4,518 contigs using the automated assembly feature of FPC. Cutoff values used were 10⁻¹², 10⁻¹¹, 10⁻¹⁰, and 10⁻⁹ for zero, one, two, and three shared overgo markers, respectively. Contigs with greater than 5 Questionable clones were tested at higher stringencies of 10⁻¹³ and 10⁻¹⁴. Manual editing of the 4,518 contigs using the FPC contig end joining tool in conjunction with shared markers and selectively reduced cutoffs down to 10⁻⁸ reduced the contigs number to 3,488. To establish a robust data set, overgos were required to hit two or more BACs in a given contig, thereby removing single BAC hits in contigs. Using these filtering criteria, 13,900 overgo-contig associations were derived for 8,353 overgos. Approximately 88% of the overgo markers hybridized to BACs contained within one or two contigs, while only 7% hybridized to four or more contigs.

Figure 4.

Distribution of overgo density in BAC contigs The 2,005 contigs containing one or more overgos were evaluated for overgo density, placed in density categories (x axis), and cumulative sums calculated (y axis) to generate a distribution of overgo density in BAC contigs. Overgo density is defined as the number of overgos per megabase of contig. Overgo density varied up to 10-fold across contigs, with some contigs containing less than three overgos per megabase, while others contained 30 to 40 overgos per megabase. A small group of 16 contigs totaling approximately 9 Mb (data not shown) was very overgo dense with more than 40 overgos per megabase.

Figure 5.

Overgo distribution across maize chromosomes. A total of 13,900 overgo locations are distributed across 2,005 contigs encompassing 1,792 Mb. All overgo locations are confirmed by at least two BAC hits in a contig. There are 9,003 overgo locations (65%) distributed across 735 genetically anchored BAC contigs encompassing 1,053 Mb. Genetically anchored overgo locations are distributed across all 10 chromosomes. There are 4,897 of the 13,900 overgo locations on BAC contigs that remain unanchored to a chromosome. These overgo locations represent sites for targeted genetic marker development that will allow a rational approach toward genetic anchoring of unanchored BAC contigs.