(Triticum aestivum) - Nucleic Acids Research

© 2000 Oxford University Press

Nucleic Acids Research, 2000, Vol. 28, No. 24 e106

Non-gridded library: a new approach for BAC (bacterial artificial chromosome) exploitation in hexaploid wheat (Triticum aestivum) Zhiying Ma1,2, Song Weining3, Peter J. Sharp4 and Chunji Liu1,* 1CSIRO

Plant Industry, 306 Carmody Road, St Lucia, Queensland 4067, Australia, 2Faculty of Agriculture, Hebei Agricultural University, Baoding, Hebei, People’s Republic of China, 3Leslie Research Centre, 13 Holberton Steet, Toowoomba, Queensland 4350, Australia and 4Plant Breeding Institute, Cobbitty, NSW 2570, Australia

Received August 29, 2000; Revised and Accepted October 31, 2000

ABSTRACT The feasibility of exploiting non-gridded bacterial artificial chromosome (BAC) libraries and some major factors affecting the efficiency of handling such libraries were studied in hexaploid wheat. Even for a bacterial culture containing only 55% recombinants, some 2000 BAC clones with inserts ranging from 45 to 245 kb could be pooled. The pooled BAC clones could be amplified by culturing for up to 6 h without losing any target clones. These results imply that even for hexaploid wheat, which has an extremely large genome, some 250 pools are sufficient for a BAC library that should satisfy many research objectives. This non-gridded strategy would dramatically reduce the cost and make robotic equipment non-essential in exploiting BAC technology. To construct a representative library and to minimise clone competition, thawing and re-freezing ligation mixtures and bacterial cultures should be avoided in BAC library construction and application. INTRODUCTION Libraries of large DNA inserts are essential for physical mapping, map-based gene cloning and gene structure and function analyses in complex genomes. Due to its overwhelming advantages over cosmid and yeast artificial chromosome (YAC) libraries (1), bacterial artificial chromosome (BAC) libraries have rapidly become the technique of choice. Since the first report in plants (2,3), BAC libraries for many plant species have been constructed during the last few years. However, a BAC library for bread wheat (Triticum aestivum, 2n = 6× = 42) is still not available, despite its agronomic importance. The genome size of bread wheat is extremely large (16 700 Mb/1C; 4). Therefore, some 500 000 clones with an average insert size of 150 kb would be required to achieve a genome coverage of five genome equivalence, which is needed for a >99% probability of recovering any specific sequence of interest (5).

The large number of clones necessary for a bread wheat BAC library makes it very difficult to exploit BAC techniques in this species if the existing technology is used. This is because: (i) gridding of the BAC clones is time consuming and expensive; (ii) storage and maintenance of these clones require large freezer space; (iii) robotics are essential. These factors are preventing widespread application of this technology in the research community. Even for the elite few it would be difficult to construct BAC libraries for a number of different genotypes, which is essential as all genes of interest cannot be found in any single genotype. One of the possible alternatives is to construct BAC libraries for the diploid progenitors of bread wheat. In fact, BAC libraries for the A (6) and D (7) genome donors have been constructed recently. These libraries will undoubtedly contribute considerably to hexaploid wheat research. However, the genome sizes of these diploid progenitors themselves, although smaller than that of the hexaploids, are also huge and hundreds of thousands of BAC clones are needed for each of these libraries. In addition, the diploid libraries have other inherited limitations that do not allow them to replace hexaploid wheat libraries. Firstly, a diploid B genome donor has not been identified. Secondly, an important application of a BAC library is to develop SSR markers for targeted chromosome regions (8), a large proportion of which were found to be genome-specific in wheat (9). Thus a hexaploid wheat library will be essential for developing SSR markers for all three wheat genomes. Thirdly, a detailed comparative analysis of the three component genomes of hexaploid wheat would be highly desirable not only for understanding the evolution of this important crop but also for designing strategies for gene isolation and marker development. Again, these could only be done using hexaploid wheat genotypes. Some research projects, such as physical mapping of a whole plant genome, would require a large number of BAC clones from one or more libraries. Many other projects, however, require only a small number of clones. Examples of the latter include map-based gene cloning (10,11) and development of genetic markers for targeted genome regions (8). For these purposes a few relevant clones could be isolated without gridding the whole library. The BAC library could be collected and stored as BAC pools. The pools would be screened to identify those containing the target sequences and the desired

*To whom correspondence should be addressed. Tel: +61 7 3214 2223; Fax: +61 7 3214 2848; Email: [email protected]

e106 Nucleic Acids Research, 2000, Vol. 28, No. 24

clone(s) isolated from the positive pools by filter lifts or a similar technique. Such non-gridded methods have been successfully used for small insert size as well as large insert size libraries. Isolation of target sequences from pooled large insert size libraries has been successfully achieved in plant (12) as well as in animal (13) species. However, no data are available on the number of BAC clones that could be bulked nor on conditions under which to amplify pooled BAC clones. These factors, together with the effects of storing ligation mixtures and bacterial cultures, on the efficiency of BAC clone recovery have been investigated and the results are reported in this paper. MATERIALS AND METHODS BAC vector preparation pBeloBAC II (developed by Drs H. Shizuya and M. Simon, California Institute of Technology; unpublished results) was used as the cloning vector for the generation of BAC clones used in this study. The vector DNA was isolated from 4 l of LB culture using Qiagen tips (Qiagen) following the manufacturer’s instructions. The vector DNA was digested with 2 U restriction enzyme (either BamHI or HindIII) per µg DNA at 37°C for 2 h. The digested vector was purified by phenol/chloroform extraction and this was followed by dephosphorylation with shrimp alkaline phosphatase according to the manufacturer’s instructions (Boehringer Mannheim). Isolation and partial digestion of high molecular weight (HMW) genomic DNA embedded in plugs The hexaploid wheat cultivar Hartog was used for HMW DNA extraction. Fifty grams of leaf tissue was harvested from 4-weekold plants grown in pots. Agarose plugs containing HMW DNA were prepared based on the methods of Zhang et al. (14) and stored in 0.05 M EDTA, pH 8.0, at 4°C. Agarose plugs were dialysed against 0.5× TBE buffer on ice for at least 3 h. The whole plugs were then loaded into a 1% agarose gel and purified by pulsed field gel electrophoresis (PFGE) with 0.5× TBE buffer at 4 V/cm, 5 s pulses and 11°C for 5 h. After electrophoresis the plugs were recovered from the gel and dialysed against 1× TE for at least 3 h before being used for partial digestion. To determine the optimal conditions for partial digestion of HMW DNA, one plug was divided into 12 mini-plugs. These mini-plugs were washed in a buffer containing 1× restriction buffer (Gibco BRL) and 4 mM spermidine for 3 × 30 min on ice. Two of the washed mini-plugs were then allocated to separate 1.5 ml Eppendorf tubes. To each of the tubes was added 500 µl of restriction buffer plus 0.0, 0.2, 0.5, 1.0, 3.0 or 6.0 U restriction enzyme (BamHI or HindIII). These reaction mixtures were incubated on ice for 1 h and restriction digestion was then carried out at 37°C for 10 min. The reactions were stopped by adding 1/10 vol 0.5 M EDTA, pH 8.0, and the mini-plugs containing partially digested HMW DNA were analysed by PFGE in 0.5× TBE buffer at 6 V/cm, 11°C, with 90 s pulses for 18 h. Conditions that generated a majority of restricted DNA fragments ranging from 150 to 400 kb in size were used for large-scale partial digestion.

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Separation of partially digested DNA by PFGE and size fractionation A double size fractionation method was employed for separation of HMW DNA after the large-scale partial digestion. The first size fractionation was carried out utilising 10 plugs. The plugs containing partially digested DNA were applied to the centre of a 1% agrose gel, and the DNA size marker (λ concatemer DNA; BioLabs) was applied to flanking wells. The gel was run at 6 V/cm, 11°C, with 90 s pulses for 18 h. After electrophoresis the gel piece containing DNA ranging from 150 to 400 kb in size was excised and divided into five slices. Partially digested HMW DNA in each of these slices was recovered by electroelution based on the method of Osoegawa et al. (15) using dialysis tubes (¾ inch diameter; Life Technologies). The eluted DNA was subjected to a second size selection by PFGE in 0.5× TBE buffer at 3 V/cm, 11°C, with 5 s pulses for 8 h. Under these conditions the majority of the HMW DNA formed a sharp band. The band obtained was excised and the DNA was eluted from the gel in 0.5× TBE buffer at 6 V/cm, with 30 s pulses for 3 h in a ¼ inch diameter dialysis tube (Life Technologies). After electroelution the DNA solution was dialysed against ice-cold 1× TE for 3 h with the gel slices still contained within the dialysis tubes. DNA was recovered using a wide bore pipette tip. Ten microlitres of this solution was used for estimating the DNA concentration using electrophoresis with a 0.7% agrose gel and λ DNA as standard. Ligation and transformation The electroeluted DNA was ligated to BamHI- or HindIII-digested and dephosphorylated pBeloBAC II vector (1:10 molar ratio) at 15°C overnight. The ligation reaction was dialysed against water and then 30% PEG 8000 and transformed into ElectroMAX DH10B competent cells (Life Technologies) based on the methods described by Osoegawa et al. (15). Collection and storage of BAC clones in pools BAC clones used in this study were derived from eight ligation reactions. The BAC clones were collected, together with nonrecombinants (blue colonies), directly from overnight cultured LB plates. Depending on the number of colonies on a plate, the ratio of recombinants (white colonies) to non-recombinants (blue colonies) and the size of each pool, clones forming each of the pools were collected by scraping colonies from between one and eight plates. The collected clones for each of the pools were suspended in 2 ml of LB medium containing 12.5 µg/ml chloramphenical. A single colony for each of eight target clones (see below) was added to each of these pools. Glycerol was added to a final concentration of 7–25% (see Results and Discussion) and the glycerol stock from each of these pools was then aliquoted into five tubes and stored at –70°C. For each of the BAC pools one tube (400 µl) of glycerol stock was thawed on ice. Twenty-five microlitres of the stock was used to inoculate each of a series of 15 ml of LB medium containing chloramphenical. They were then cultured in 50 ml tubes at 37°C for different lengths of time (see below). Plasmid DNA was extracted from these cultures based on the method of Zhang et al. (14). Two to five micrograms of DNA from each of these cultures were digested with BamHI and separated on 0.8% agrose gels in 0.5× TBE buffer. The separated DNA was


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results indicated that the empty clones in these transformations varied between 0 and 12%. Table 1. Effects of frozen ligation mixtures on BAC clone recovery Ligation

Treatment

Recombinant (% loss)

Non-recombinant (% loss)

1

Fresh

425 (0.0%)

225 (0.0%)

Frozen

380 (10.6%)

205 (8.9%)

2

3

Re-frozen

106 (75.1%)

106 (52.9%)

Fresh

836 (0.0%)

520 (0.0%)

Frozen

755 (9.7%)

470 (28.8%)

Re-frozen

140 (83.3%)

135 (74.0%)

Fresh

375 (0.0%)

250 (0.0%)

Frozen

252 (32.8%)

190 (24.0%)

Re-frozen

104 (72.3%)

83 (66.8%)

Number of clones that could be pooled and time of culture Figure 1. The eight target BAC clones used and their estimated sizes.

transferred to Hybond N+ nylon membranes according to methods described by Liu and Musial (16). Isolation of DNA probes from target BAC clones Insert size was the only criterion used for selecting target BAC clones. To cover the spectrum of insert sizes reported in the majority of BAC libraries, we selected eight target clones with inserts ranging from 45 to 245 kb (Fig. 1). Insert DNA of the eight targets was released by NotI restriction, separated by PFGE and then purified with a Qiaex II Gel Extraction Kit (Qiagen). Purified insert DNA was digested with Sau3AI and ligated into BamHI-restricted pBluescript II SK+ vector. Fifteen subclones, with insert sizes ranging from 350 to 900 bp, from each of the eight target clones were dot-blotted onto Hybond N+ nylon membranes. The subclones were probed with Sau3AI-restricted total genomic DNA from the hexaploid wheat genotype Hartog. Those clones giving strong signals were believed to contain highly repeated sequences and were thus discarded and five of the remaining putative low copy clones for each of the target clones were PCR amplified as probes using the M13/M17 primers. Two of these low copy probes were used (separately) to detect each of the eight target clones. Methods for probe labelling and hybridisation were as described by Liu and Musial (16). RESULTS AND DISCUSSION BAC clones used in this study came from eight separate ligation reactions. These ligation mixtures produced clones with average inserts ranging from 125 to 187 kb (data not shown), and a white/blue ratio of 55–67% when fresh ligation mixtures were used for transformation (Tables 1 and 2 and below). For each transformation 10–40 white clones were analysed to estimate the percentage of empty clones (white clones without inserts). The

For long-term storage and utilisation of a library, clone amplification is essential. In contrast to a fully gridded library where clones are kept as individuals, competition among clones would occur when pooled clones were amplified. The effects of two of the major factors affecting clone competition, the number of clones in a pool and time of culture, were studied with pools of four different sizes (containing 500, 1000, 1500 and 2000 recombinants, respectively) and three culture times (4, 6 and 8 h). To reduce the possibility of false positives in dot-blot analysis we adopted the methods of digesting and separating BAC DNA using electrophoresis as described by Salimath and Bhattacharyya (12). Southern analysis showed that, as expected, the detectable signal became weaker as the number of clones in a pool increased. This was obviously caused by a dilution effect. The effect of the culture time on signal strength varies between targets, indicating the possibility of clone competition. In the largest pools (6 h culture) the presence of a target clone could still be unambiguously detected (Fig. 2). The same result was given by each of the eight target clones tested in this study. It is important to note that the BAC pools used in this study contain some 33–45% non-recombinant cells (blue colonies). It is not unrealistic to predict that these non-insert-carrying cells might successfully compete with those carrying large inserts. Thus, it would seem likely that many more BAC clones could be used to form each pool when ligation mixtures producing higher proportions of recombinant clones were used. As shown in Figure 2, a common fragment was detected from all the targets and pooled clones. This was most likely caused by the T7 sequence, which is shared between the probes obtained by PCR (see Materials and Methods) and the PBeloBAC II vector. Effects of frozen ligation mixtures on efficiency of BAC clone recovery Due to the large number of clones required for a given library and the limited number of clones that could be generated from

e106 Nucleic Acids Research, 2000, Vol. 28, No. 24

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Figure 2. Results from Southern-based screening of the BAC pools with two of the eight target clones. Four different sized pools with two replicates each were used and they were cultured for three different times. The specific fragments detected by probes isolated from two (T2 and T5) of the target BAC clones were marked.

Table 2. Effects of different glycerol stocks on BAC clone recovery Culture

Glycerol stocka

Final glycerol (%)

A

A fresh

N/A

755 (100%)

370 (100%)

A with 10% G

10

612 (81.1%)

310 (83.8%)

A with 15% G

15

705 (93.4%)

330 (89.2%)

A with 20% G

20

560 (74.2%)

285 (77.0%)

A with 25% G

25

490 (64.9%)

225 (60.8%)

B fresh

N/A

1980 (100%)

1450 (100%)

B with 20% GW

10

1930 (97.5%)

1290 (89.0%)

B with 30% GW

15

1920 (97.0%)

1350 (93.1%)

B with 40% GW

20

1810 (91.4%)

1200 (82.2%)

B with 50% GW

25

1690 (85.4%)

1220 (84.1%)

C fresh

N/A

572 (100%)

456 (100%)

C with 22% GLB

7

440 (76.9%)

150 (32.9%)

C with 33% GLB

10

500 (87.4%)

380 (83.3%)

C with 50% GLB

15

530 (92.6%)

405 (88.8%)

C with 60% GLB

18

535 (93.5%)

430 (94.3%)

B

C

aG,

Recombinant (%)

Non-recombinant (%)

100% glycerol; GW, 50% glycerol in water; GLB, 30% glycerol in LB medium.

a single transformation, storage of ligation mixtures is often necessary in generating BAC libraries. It was observed in our work, however, that only a proportion of clones from the stored ligation mixtures could be recovered when compared to the use of fresh ligation mixtures. To quantify this we tested three ligations. The results (Table 1) showed clearly that storage of ligation mixtures at –20°C, even for a few days, could cause a significant reduction in BAC clone recovery. When re-frozen ligation mixture was used the majority of the clones were lost.

It was not clear whether clones with some specific sequences and/or larger inserts would be more susceptible to the frozen/ re-frozen cycles. However, it was clear that, whenever possible, fresh ligation mixtures should be used. Otherwise, ligation mixtures should be stored in small aliquots. After being thawed these aliquots should be used up and re-freezing should be avoided. The feasibility of keeping ligation mixtures in ice-cold water during the time taken to check their quality (insert sizes and the

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number and ratio of recombinants versus non-recombinants) was also tested with two ligations. When fresh ligation mixture was used the first ligation produced ∼800 recombinant clones/µl mixture with a white/blue ratio of 65% and the second ligation produced ∼550 white clones/µl mixture with a white/blue ratio of 57%. After 5 days storage in ice-cold water no white clones were detected from either of these ligation mixtures. Clearly, this is not an option for keeping ligation mixtures.

ACKNOWLEDGEMENTS

Effects of glycerol storage on bacterial culture

1. Zhang,H.B. and Wing,R.A. (1997) Physical mapping of the rice geome with BACs. Plant Mol. Biol., 35, 115–127. 2. 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. 3. Tao,Q., Zhao,H., Qiu,L. and Hong,G. (1994) Construction of a full bacterial artificial chromosome (BAC) library of Oryza sativa genome. Cell Res., 4, 127–133. 4. Bennett,M.D. and Leitch,I.J. (1995) Nuclear DNA amounts in Angiosperms. Ann. Bot., 76, 113–176. 5. Clarke,L. and Carbon,J. (1976) A colony bank containing synthetic ColEi hybrid plasmids representative of the entire E. coli genome. Cell, 9, 91–100. 6. Lijavetzky,D., Muzzi,G., Wicker,T., Keller,B., Wing,R. and Dubcovsky,J. (1999) Construction and characterization of a bacterial chromosome (BAC) library for the A genome of wheat. Genome, 42, 1176–1182. 7. Moullet,O., Zhang,H.B. and Lagudah,E.S. (1999) Construction and characterization of a large DNA insert library from the D genome of wheat. Theor. Appl. Genet., 99, 305–313. 8. Cregan,P.B., Mudge,J., Fickus,E.W., Marek,L.F., Danesh,D., Denny,R., Shoemaker,R.C., Matthews,B.F., Jarvik,T. and Young,N.D. (1999) Targeted isolation of simple sequence repeat markers through the use of bacterial artificial chromosomes. Theor. Appl. Genet., 98, 919–928. 9. Devos,K.M., Bryan,G.J., Collins,A.J., Stephenson,P. and Gale,M.D. (1995) Application of two microsatellite sequences in wheat storage proteins as molecular markers. Theor. Appl. Genet., 90, 247–252. 10. Danesh,D., Peñuela,S., Mudge,J., Denny,R.L., Nordstrom,H., Martinez,J.P. and Young,N.D. (1998) A bacterial artificial chromosome library for soybean and identification of clones near a major cyst nematode resistance gene. Theor. Appl. Genet., 96, 196–202. 11. Nam,Y.-W., Penmetsa,R.V., Endre,G., Uribe,P., Kim,D. and Cook,D.R. (1999) Construction of a bacterial artificial chromosome library of Medicago truncatula and identification of clones containing ethylene-response genes. Theor. Appl. Genet., 98, 638–646. 12. Salimath,S.S. and Bhattacharyya,M.K. (1999) Generation of a soybean BAC library and identification of DNA sequences tightly linked to the Rps1-k disease resistance gene. Theor. Appl. Genet., 98, 712–720. 13. Pierce,J.C., Sternberg,N. and Sauer,B. (1992) A mouse genomic library in the bacteriophage P1 cloning system: organization and characterization. Mamm. Genome, 3, 550–558. 14. Zhang,H.-B., Zhao,X., Ding,X., Paterson,A.H. and Wing,R.A. (1996) Preparation of megabase-sized DNA from plant nuclei. Plant J., 7, 175–184. 15. Osoegawa,K., Woon,P.Y., Zhao,B., Frengen,E., Tateno,M., Catanese,J.J. and de Jong,P.J. (1998) An improved approach for construction of bacterial artificial chromosome libraries. Genomics, 52, 1–8. 16. Liu,C.J. and Musial,J.M. (1995) Restriction fragment length polymorphism detected by cDNA and genomic DNA clones in Stylosanthes. Theor. Appl. Genet., 91, 1210–1213.

Glycerol stocks are commonly used for storing libraries of all types and are also convenient in BAC library construction when used for storing bacterial cultures (15). To reduce the possibility of uneven loss of different clones in non-gridded BAC libraries, it is important to minimise cell loss during storage. To this end we compared the effects of different percentages of glycerol stock on the recovery of recombinant clones. The results showed that, as expected, all glycerol stocks caused a proportion of cell death. The optimal glycerol concentration seemed to vary depending on how the glycerol stocks were prepared (Table 2). When re-frozen bacterial culture was used only some 60% of recombinants could be recovered (data not shown). Thus, similar to the situation for ligation mixtures, it is important to avoid thawing/re-freezing of stored BAC pools or bacterial cultures. CONCLUSION By employing eight target clones with insert sizes ranging from 45 to 245 kb we demonstrated that a non-gridded BAC library could be constructed with pools each containing at least 2000 BAC clones. Such BAC pools could be amplified by culturing for up to 6 h without significantly changing the structure of the pooled clones. Thus some 250 pools are all that is needed for a hexaploid wheat library of 500 000 clones. Such non-gridded BAC libraries would satisfy the requirements of many research projects, dramatically reduce the cost of constructing and applying BAC libraries, offer the possibility of allowing BAC technology to be exploited for different varieties and make BAC technology accessible to research groups without robotic equipment. To minimise the effects of selective clone loss, fresh ligation mixtures should be used in constructing non-gridded BAC libraries. Otherwise, aliquots should be made and re-freezing ligation mixtures should be avoided. Similarly, re-freezing bacterial cultures and thawing glycerol stocks of BAC pools should be avoided.

We thank Drs Chris Grof and Malcolm Livingstone for their critical reading of the manuscript. Z.Y.M. is grateful to the China Scholarship Council for their financial support. The experiments comply with the current laws of Australia. REFERENCES