A gene-enriched BAC library for cloning large allele-specific fragments from maize: isolation of a 240-kb contig of the bronze region

Abstract

A generic bacterial artificial chromosome (BAC) library from a complex plant genome like maize may not be suitable for some types of genomic analysis, for example, for establishing correlations between the genetic and the physical organization of a given chromosome region. Previously, we carried out extensive genetic analysis of the bronze (Bz) region in Zea mays using a W22 inbred line carrying the Bz-McC allele; however, BAC libraries of that line are neither available nor under construction. Here, we report the isolation of large, adjacent BAC clones of this region from a partial BAC library of W22. We developed a BAC vector suitable for cloning NotI fragments and used it to clone size-fractionated genomic DNA that had been cut to completion with the methylation-sensitive, rare-cutting enzyme NotI. This strategy resulted in a very significant enrichment of large genic DNA. From a library of about 20,000 BACs, containing just two-thirds of a maize genome, we isolated 16 BAC clones of the 110-kb distal Bz fragment and 10 BAC clones of the 130-kb proximal Bz fragment. This recovery means that our strategy resulted in a 15- to 24-fold enrichment of specific sequences. The order of the BAC clones in the 240-kb contig, predetermined from an internal NotI site in the Bz-McC allele was confirmed by hybridization with sequences from sites previously mapped proximal and distal to Bz and by sequencing. To show the general utility of our approach and the value of our partial BAC library, we also isolated BAC clones of other sequences, such as tub4 and the complex R-r allele, contained in the same size fraction of DNA. This is the first report of the use of a BAC vector to clone allele-specific large DNA fragments from a plant with a large genome, circumventing the need to construct a complete BAC library.

Figure 1

Schematic representation of the pNOBAC 1 vector. pNOBAC 1 is a NotI cloning BAC vector derived from pBeloBAC 11 (Kim et al. 1996b). It lacks the two NotI sites located on either side of the lacZ gene in pBeloBAC 11, has a new NotI cloning site between BamHI and HindIII in the polylinker, and retains the blue-white selection feature of its progenitor.

Figure 2

CHEF gel electrophoresis of maize genomic DNA. (A) Ethidium bromide-stained gel of maize genomic DNA that was not digested (lane 2) or was digested with either NotI (lane 3) or Sfi I (lane 4). (Lane 1) DNA size markers. (B) Autoradiogram of a different gel containing NotI-digested maize DNA. The membrane was hybridized with the probe Bz-528 (see Fig. 3).

Figure 3

Partial restriction map of the Bz-McC and Ac2094 λ genomic clones (Ralston et al. 1988, 1989). (Large arrow) Location and orientation of Bz transcript; (double-headed arrows) locations and extents of the three probes used in this work, Bz-528, STC-323, and tac2094. The NotI site in the Bz-528 fragment divides the Bz-McC gene into a proximal and a distal fragment, as shown (Dooner et al. 1985).

Figure 4

Analysis of BAC clones that hybridized to the probe Bz-528. BAC DNA from positive clones was extracted, digested with NotI, and separated by CHEF gel electrophoresis. (A) Ethidium bromide-stained gel containing DNA from five positive clones (lanes 2–6), one negative clone (lane 7), and high-molecular-weight markers (lane 1). The gel was transferred to a nylon membrane and hybridized successively with three different probes, as shown in B–D. (B) Hybridization with probe Bz-528; (C) hybridization with probe STC-323, distal to Bz; (D) hybridization with a tac2094 probe, proximal to Bz.

Figure 5

Southern blot analysis of high-molecular-weight maize genomic DNA. High-molecular-weight DNA was digested with NotI, separated by CHEF gel electrophoresis, and hybridized to different cDNA probes. (Lane 1) wx; (lane 2) enod93; (lane 3) tub4; (lane 4) R; (lane 5) sh2.

Figure 6

Analysis of R and tub4 BAC clones. BAC DNA was extracted, digested with NotI, and separated by CHEF gel electrophoresis. (A) Ethidium bromide-stained gel. (Lane 1) R clone; (lane 2) tub4 clone. (B) Autoradiogram of the gel blot in A hybridized to R cDNA (lane 1) and tub4 cDNA (lane 2).