GrainGenes

Progress towards the construction of a Bacterial Artificial Chromosome
Library in Barley
Wayne Kennard and Nora Lapitan
Dept. of Soil and Crop Sciences, Colorado State University, Fort Collins, CO 80523

Introduction
BACs (bacterial artificial chromosomes) are F-based plasmid vectors that harbor large inserts (>200 Kbp) (Shizuya et al., 1992). A BAC library, like a YAC (yeast artificial chromosome) library, can provide the means for physical mapping and map-based cloning. BACs hold advantages over YACs by a relatively higher cloning efficiency and easier clone screening and manipulation. Recently, BAC libraries have been applied for the physical isolation of genes, chromosome walking, and flourescence in-situ hybridization in sorghum (Woo et al., 1994). We have adapted and developed methods for cloning high molecular weight (HMW) barley DNA into BAC vectors. In this report we describe progress and outline procedures for the construction of a BAC library in barley.

Materials and Methods
HMW DNA was purified from modification of a protoplast isolation procedure (Ganal et al., 1989). Ten day-old Morex or CM67 barley leaves were cut longitudinally and incubated with 2% cellulase and 0.05% pectolyase in 0.5% M sorbitol for four hours. Protoplasts were then embedded in low-melting-point (LMP) agarose microbeads (Wing et al., 1993) at a concentration of 2.0 - 4.0 X 10^7^ embedded cells/ml = 275ug/ml - 550 ug/n-A (assuming 11.0 pg/diploid cell). DNA embedded in microbeads was predominantly > 1 mega-base-pair. Digestion with ESP (0.5 M EDTA, 1 % sarkosyl, and 0.2mg/ml proteinase K), incubation with PMSF (phenlymethylsulfonylflouride, 0.1mM, in 10mM Tris pH 8.0, 10 mM EDTA), and dialysis (10 mM TrispH 8.0, 1mM EDTA) rendered the plugs susceptible to restriction enzyme digestion.

HindIII partial digestions were performed by adding serially diluted enzyme into 50 ul aliquots of microbeads. Prior to digestion, microbeads were equilibrated with digestion buffer (50mM NaCl, 6mM MgCl, 6mM Tris pH 7.6, 2mM spermidine, 1mM DTT). Serially diluted enzyme was added to microbeads on ice, allowed to equilibrate for 30 minutes and subsequently digested at 37C for 30 minutes. Generally, 2.0-5.0 units of HindIII yielded the desired fragment range between 100-500 Kbp. Partially digested DNA was selected by CHEF (1% LMP agarose, IXTAE buffer; run 6v/cm, 90 sec switch time, 20-24 hours. Electrophoresed DNA fragments were physically isolated from LMP agarose gels in 150 Kbp increments between 150-750 Kbp.

The BAC vector, pBeloBACII, was isolated by column chromatagraphy followed by two CsCl bandings to remove bacterial chromosomal DNA. The vector was digested with HindIII, phosphatased, phenol:chlorofrom extracted, precipitated, and washed with 70% EtoH. Prior to ligation DNA equilibrated > 6 hours with 0.75mM spermidine, 0.35mM spermidine, equilibrated one hour with GELase buffer for I hour, and melted at 70C 15 min. The liquified agarose was digested with GELase for two hours 42C and DNA concentration was estimated by an ethidium bromide spot staining technique. The partially digested barley DNA was ligated to Hind III-digested vector in a 10: 1 vector insert ratio (i.e.. 50ng vector: 100 ng insert) with 100 units of T4 DNA ligase in a 100 ul volume. Two ul of a ligation were added to 35 ul of DH10B competent cells for transformation. Transformations were performed with standard E. coli electroporation protocols.

Transformations were plated on LB/chloramphenicol (12.5ug/ml) supplemented with X-gal and IPTG. Recombinant (cream colored) colonies were picked to gridded plates and then two copies of storage microtitre plates supplemented with storage media (LB, 12.5ug/ml chloramphenicol, 36 mM K/2/HPO/4/,13.2 mM KH/2/PO/4/, 1.7 mMNaCitrate, 0.4mM MgSO/4/, 6.8 MM (NH4)/2/SO/4/ and 4.4% glycerol).

BAC plasmids were isolated with a modification of an alkaline lysis mini-prep (Sambrook et al, 1989). Plasmids were digested with NotI restriction enzyme and electrophoresed on CHEF (1 % agarose, 0.5XTBE, 125 volts, 5 - 15 ramped switch time for 16 hours). An insert band was measured if the vector band (7.4 Kbp) was apparent. Insert sizes were estimated in comparison to lambda concatamers. If more than one NotI fragment was released, the fragments were added up to a total insert size. Digested plasmid DNA was transferred to Hybond N+ membrane after depurination (0.25 M HCl, 20 min) with 1.5 M NaCl, 0.4 M NaOH by capillary Southern transfer for 15 - 17 hours. Colony transfers were performed by 15 hour growth of colonies on Hybond N+, denaturing 7 min (1.5 M NaCl, 0.4 M NaOH), neutralizing 2 X 3 min (1.5 M NaCl, 0.5 M Tris 7.5, 1 mM EDTA). Total genomic and chloroplast DNA was labeled with ^32^P by random priming (Feinberg and Vogelstein 1984), hybridized to Southern or colony transfers 65C in solution (5XSSC, 0.6%SDS, 50mM NaPO4, 10% Dextran Sulfate, 5X Denhardts, 2.5mM EDTA) and washed successively in 2xSSC, 0.1 %SDS; 1XSSC, 0.05% SDS; and 0.2XSSC, 0.01 % SDS at 65C.

Results and Discussion
HMW barley DNA was successfully cloned into the BAC vector, pBeloBAC11. Average insert sizes varied among ligations (45-103 Kbp) with individual clones ranging in size from 2 to > 200 Kbp. Optimizing the cloning system requires good partial digests (the majority of barley DNA to 100-500 Kbp), adding sufficiently concentrated DNA (>1ug/ml) to achieve 10: 1 insert vector ratios, and titering enzyme amounts in vector preparations and ligations.

Recombination frequency varied among the different size selection steps. For a typical 150-300 Kbp size selection, transformation with 2 ul of a ligation would yield 50-200 recombinant colonies on a plate (5 X 10^4^ - 2 X 10^5^ recombinants/ug vector).Recombinants to nonrecombinants were found in a frequency of 5 - 42 %. Size selections of 300-450 Kbp and 450-600 Kbp gave recombination frequencies of 0 - 33% and 0 - 4% respectively. The reduced recombination frequency may be due to the inefficiency of HMW insert cloning.

Sizes of cloned BAC inserts varied within and among size selections. Average insert sizes cloned from 150 - 300 Kbp selections varied between 45 and 103 Kbp. Insert analysis of a typical ligation with 68 ± 34 Kbp inserts is illustrated in Figure 1A. The variation within and among size selections may be due to differences in DNA concentration, physical size selection, or CHEF run differences. The CHEF size fractionation may not be efficient as lower molecular weight DNA may be trapped among expected sizes in a selection. Variation of insert size from different size selections may be due to different degrees of trapping.

Confirmation of the inserts containing barley DNA was performed via Southern hybridization. The majority of inserts indicated strong hybridization to total barley DNA upon overnight exposure (Fig 1B). Weaker hybridization of some clones may indicate absence of repetitive sequences. Moderate to strong hybridization of total genomic DNA to

Figure 1. Analysis of randomly chosen BAC clones from a typical cloning event by pulsed-field gel electrophoresis. (A) Ethidium bromide stained DNA mini preparations digested with NotI. The gel was loaded with lambda concatamer and lambda/HindIII size standards (first and second lanes left to right respectively), 20 random BAC clone isolates (third through twenty-second lanes), and lambda/hindIII (twenty-third lane). CHEF gel parameters were 1 % agarose, 1XTBE running buffer, 6 volts/cm, 5 -15 sec ramped switch time. (B) Southern-blot of gel in A showing hybridization to total genomic barley DNA .

colony transfers was evident among 89% of clones. The frequency of chloroplast clones was 5.1 % as determined through colony hybridization.

Currently we have 10,752 recombinant BAC clones with an average size of 78.4 Kbp. This represents 15 % of a haploid genome equivalent of barley. Size of clones ranged from 2 Kbp to greater than 20OKbp among cloning experiments. These clones represent the beginning of a genome equivalent of a BAC library. We hope to improve size selection and cloning techniques to increase insert size, frequency, and recombination efficiency. The library will be an asset to those interested in physical mapping of barley.

References

Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. In: Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laoratory Press, Cold Spring Harbor, NY.

Shizuya, H., Birren B., Kim UJ, Mancino V, Slepak, T., Tachiiri, Y., and Simon, M. 1992. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. 89:8794-8797.

Wing, R.A., Rastogi, V.K., Zhang H.B., Paterson, A. H., Tanksley S.D. 1993. An improved method of plant megabase DNA isolafion in agarose microbeads suitable for physical mapping and YAC cloning. The Plant J. 4:893-898.

Woo S.-S., Jiang, J., Gill, B.S., Paterson, A.H., and Wing, R.A. 1994. Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor. Nucleic Acids Res. 22:4922-4931.

Ganal, M., Young, N.D., Tanksley, S.D. 1989. Pulsed field gel electrophoresis and phyiscal mapping of large DNA fragments in the Tm-2a region of chromsosome 9 in tomato. 215:395-400.