Construction and characterization of a bacterial artificial chromosome library for the hexaploid wheat line 92R137

Abstract

For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome (BAC) library, including two sublibraries, was constructed using the genomic DNA of 92R137 digested with restriction enzymes HindIII and BamHI. The BAC library was composed of total 765,696 clones, of which 390,144 were from the HindIII digestion and 375,552 from the BamHI digestion. Through pulsed-field gel electrophoresis (PFGE) analysis of 453 clones randomly selected from the HindIII sublibrary and 573 clones from the BamHI sublibrary, the average insert sizes were estimated as 129 and 113 kb, respectively. Thus, the HindIII sublibrary was estimated to have a 3.01-fold coverage and the BamHI sublibrary a 2.53-fold coverage based on the estimated hexaploid wheat genome size of 16,700 Mb. The 765,696 clones were arrayed in 1,994 384-well plates. All clones were also arranged into plate pools and further arranged into 5-dimensional (5D) pools. The probability of identifying a clone corresponding to any wheat DNA sequence (such as gene Yr26 for stripe rust resistance) from the library was estimated to be more than 99.6%. Through polymerase chain reaction screening the 5D pools with Xwe173, a marker tightly linked to Yr26, six BAC clones were successfully obtained. These results demonstrate that the BAC library is a valuable genomic resource for positional cloning of Yr26 and other genes of interest.

Figure 1

Partial digestion of DNA in half plugs. Lanes 1–8 contain DNA samples digested with restriction enzyme at the increasingly higher concentrations. (a) Partial digestions of half DNA plugs with serial dilutions of HindIII at 37°C for 30 min. (b) Partial digestions of half DNA plugs with serial dilutions of BamHI at 37°C for 30 min. Plug pieces were separated on 1% agarose gel in 0.5x TBE and run in the CHEF DR-III System (Bio-Rad) at 6 V/cm, 1–50 s switch time, linear ramp, 120° angle at 14°C for 18 h. M: the λ PFG marker.

Figure 2

Size selections of the partially digested products of genomic DNA of wheat line 92R137. (a) The first size selection of the partially digested products of genomic DNA. Plug pieces were separated on 1% agarose gel in 0.5x TBE buffer and run in a CHEF DR-III System (Bio-Rad) at 6 V/cm, 1–50 s switch time, linear ramp, 120° angle at 14°C for 18 h. (b) The second size selection of the partially digested products of genomic DNA. The first size selected fractions were separated on 1% agarose gel in 0.5x TBE buffer and run at 6 V/cm 3–5 s switch time, linear ramp, 120° angle at 14°C for 18 h. M: the λ PFG marker.

Figure 3

NotI digests of plasmids from randomly selected white colonies. The enzyme-digested products of selected plasmids with NotI were loaded onto 1% agarose in 0.5x TBE CHEF gel, 6 V/cm, 5–15 s pulse time for 16 h at 14°C. M: the λ PFG marker.

Figure 4

Insert size distribution of randomly selected BAC clones including 453 clones constructed with HindIII (open bars) and 573 clones constructed with BamHI (solid bars).

Figure 5

Screening of the BAC library with the Yr26-linked marker Xwe173. (a) Screening of BAC plate pools. A 451-bp band was amplified in positive control 92R137 with Xwe173. Two positive BAC plates, H186 (constructed with HindIII) and B634 (constructed with BamHI), were identified. (b) Six BAC clones (H186H13, H186E15, B634C10, B634C11, B634D10, and B634E10), which were identified from plates H186 and B634, were confirmed to contain Xwe173.