The selection and use of sorghum (Sorghum propinquum) bacterial artificial chromosomes as cytogenetic FISH probes for maize (Zea mays L.)

Abstract

The integration of genetic and physical maps of maize is progressing rapidly, but the cytogenetic maps lag behind, with the exception of the pachytene fluorescence in situ hybridization (FISH) maps of maize chromosome 9. We sought to produce integrated FISH maps of other maize chromosomes using Core Bin Marker loci. Because these 1 Kb restriction fragment length polymorphism (RFLP) probes are below the FISH detection limit, we used BACs from sorghum, a small-genome relative of maize, as surrogate clones for FISH mapping. We sequenced 151 maize RFLP probes and compared in silico BAC selection methods to that of library filter hybridization and found the latter to be the best. BAC library screening, clone verification, and single-clone selection criteria are presented along with an example of transgenomic BAC FISH mapping. This strategy has been used to facilitate the integration of RFLP and FISH maps in other large-genome species.

Figure 1

EtBr-stained agarose gel of CBM inserts. PCR amplification products for individual prepared CBM RFLP probes (indicated above the lanes), loaded from left to right in order of their predicted size. Mass standards (HindIII-digested λ DNA) are labeled, and the total amounts loaded appear above the lanes. Size standards (100-bp marker) are included, and selected band sizes are indicated at right.

Figure 2

BAC filter hybridization, scoring of hits, and location in fingerprint contig (FPC) map for maize CBM1.11 (umc161). A representative filter-screen hybridization (Table 2, step 2) and the autoradiograph from one of the two filters are shown (top). A total of 5 positive signals are seen (circled or boxed), and the scoring scheme for one of them (a0074A24) is illustrated at the right. The autoradiograph is labeled to show the location of the 384-well plate fields 1–6 (F1–6), and the geometry duplicate spotting of the eight individual plates per field is indicated at the right for quadrant A24. A close-up of one hit (position 2, field 3) is shown with the resulting decoded BAC ID of plate 74 position A-24 (a0074A24). This BAC, along with four other contiguous (arrows) BACs, is indicated within contig number 113 of the Sorghum propinquum FPC map (screen capture bottom left). Four additional BACs (asterisks) were detected, but not found to belong to a contig.

Figure 3

Southern blot confirmation of sorghum BACs after filter hybridization. Step 5 (Table 2) of the BAC selection procedure is illustrated with CBM1.11 (umc161a). (a) Sorghum propinquum FPC contig map showing contig number 147 and the five contiguous BACs (boxed) detected by filter hybridization with the CBM1.11 probe. Lane numbers are indicated beside the BACs. (b) Ethidium bromide-stained agarose gel of HindIII-digested BAC minipreps. This gel includes the BACs under investigation (lanes 9–13) along with another set (lanes 1–8) serving as negative controls. For each confirmation blot, a positive control lane [CBM1.11 (umc161a)] is included. It contains a trace amount of the same insert preparation that was used in this experiment and in the preceding filter hybridization. (c) Autoradiograph after hybridization with the CBM1.11 probe. The asterisk indicates the BAC that was eventually selected as part of step 6 (Table 2).

Figure 4

FISH mapping of maize CBM1.10 with sorghum BAC a0053G04. (a) A DAPI-stained image of spread pachytene chromosomes from OMAd1.36. (b) FITC image showing maize chromosome 1 direct-labeled with Alexa-488-dUTP-KWF total maize DNA. (c) Rhodamine image from direct-labeled sorghum BAC FISH signals (green arrows). (d) Cy-5 image of centromere FISH signal (blue arrowhead) with direct-labeled CentC. (e–g) Three-color overlay of the FITC (red), rhodamine (green), and Cy-5 (blue) images. (f) Straightened projection of the maize chromosome from panel (e). The locations of the centromere (blue arrowhead) and CBM1.10 BAC FISH signals (green bracket) are indicated along with the resulting cytogenetic locus name (boxed). (g) Straightened projections of six additional chromosomes aligned at their centromeres for comparison. All scale bars represent 5 μm.