TECHNICAL ADVANCE
Dissecting large and complex genomes: flow sorting and BAC
cloning of individual chromosomes from bread wheat
Jan Sˇafa´rˇ1, Jan Bartosˇ1, Jaroslav Janda1, Arnaud Bellec2, Marie Kubala´kova´1,3, Miroslav Vala´rik1, Ste´phanie Pateyron2,
Jitka Weiserova´1, Radka Tusˇkova´1, Jarmila Cˇı´halı´kova´1,3, Jan Vra´na1, Hana Sˇimkova´1, Patricia Faivre-Rampant2,
Pierre Sourdille4, Michel Caboche2, Michel Bernard4, Jaroslav Dolezˇel1,3and Boulos Chalhoub2,* 1
Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovska 6, CZ-77200 Olomouc, Czech Republic,
2
Laboratory of Genome organization, Unite´ de Recherches en Ge´nomique Ve´ge´tale (INRA-URGV), 2 rue Gaston Cre´mieux, CP 5708, F-91057 E´vry Cedex, France,
3
Department of Cell Biology and Genetics, Palacky´ University, Sˇlechtitelu˚ 11, Olomouc, Czech Republic, and
4
Ge´ne´tique Mole´culaire des Ce´re´ales, UMR INRA-UBP, Domaine de Crouelle, 234 Avenue du Bre´zet, F-63039 Clermont-Ferrand Cedex 2, France
Received 1 February 2004; revised 5 May 2004; accepted 11 May 2004. *
For correspondence (fax 33 1 60874549; e-mail chalhoub@evry.inra.fr).
Summary
The analysis of the complex genome of common wheat (Triticum aestivum, 2n¼ 6x ¼ 42, genome formula AABBDD) is hampered by its large size (17 000 Mbp) and allohexaploid nature. In order to simplify its analysis, we developed a generic strategy for dissecting such large and complex genomes into individual chromosomes. Chromosome 3B was successfully sorted by flow cytometry and cloned into a bacterial artificial chromosome (BAC), using only 1.8 million chromosomes and an adapted protocol developed for this purpose. The BAC library (designated as TA-3B) consists of 67 968 clones with an average insert size of 103 kb. It represents 6.2 equivalents of chromosome 3B with 100% coverage and 90% specificity as confirmed by genetic markers. This method was validated using other chromosomes and its broad application and usefulness in facilitating wheat genome analysis were demonstrated by target characterization of the chromosome 3B structure through cytogenetic mapping. This report on the successful cloning of flow-sorted chromosomes into BACs marks the integration of flow cytogenetics and genomics and represents a great leap forward in genetics and genomic analysis.
Keywords: wheat, large genomes, isolated chromosomes, flow sorting, BAC cloning, DNA library.
Introduction
Hexaploid wheat (Triticum aestivum L., 2n¼ 6x ¼ 42) is one of the most important crops in the world. It is a recently formed allopolyploid, with a huge nuclear genome (16 974 Mb/1C, Bennett and Smith, 1991), assembled from three homoeologous genomes (A, B and D). Wheat genome organization is very complex and consists of unique or low-copy sequences surrounded by regions of highly repetitive DNA which represent more than 75% of the genome (Vedel and Delseny, 1987; Wicker et al., 2003). Although early studies indicated gene clustering in ‘gene-rich islands’
located in distal parts of chromosomes, it is now believed that the islands are dispersed throughout the whole length of the chromosomes (Akhunov et al., 2003). These features hamper construction of physical maps and gene cloning in wheat and delay the development of wheat genome projects. Dissecting the wheat genome into single chromosomes and chromosome arms may represent a promising approach that could greatly simplify its analysis. First attempts to sort wheat chromosomes by flow cytometry were compromised by the low quality of chromosome
suspensions that had a negative impact on the resolution of flow karyotypes (histograms of relative fluorescence inten-sity) and the ability to sort specific chromosomes (Wang et al., 1992, Lee et al., 1997). The situation has changed since 2000 with a novel protocol for preparation of suspensions of intact wheat chromosomes (Vra´na et al., 2000) and the possibility of sorting individual chromosomes and chromo-some-arms by flow cytometry (Kubala´kova´ et al., 2002). The development of a method for isolation of high molecular weight (HMW) DNA from flow-sorted chromosomes (Sˇimkova´ et al., 2003) combined with the recent optimization of methods for efficient construction of bacterial artificial chromosome (BAC) libraries (Chalhoub et al., 2004; Osoegawa et al., 1998; Peterson et al., 2000; Shizuya et al., 1992; Zhang and Wu, 2001) indicated a potential for dissect-ing and clondissect-ing individual chromosome types from wheat.
In this study, we report the successful cloning of flow-sorted individual chromosomes into BACs. Wheat chromo-some 3B, which carries important genes and quantitative trait locus (QTL) for disease resistance and grain quality (Bo¨rner et al., 2002), was sorted using flow cytometry and successfully cloned into BACs using an adapted protocol developed for this purpose. Dissecting and cloning individ-ual chromosome types would largely facilitate genome analysis and gene cloning in wheat and other organisms with large and complex genomes.
Results
Flow cytometric analysis of wheat chromosomes
The analysis of DAPI-stained chromosome suspension prepared from a cultivar with normal karyotype (cv. ‘Chi-nese Spring’) resulted in a histogram of relative fluores-cence intensity (flow karyotype) with three composite peaks (I, II and III) representing groups of chromosomes and a well-discriminated peak corresponding to chromo-some 3B (Figure 1). Thus, chromochromo-some 3B could be easily sorted by flow cytometry and was used as a model in the present study.
Setting-up of a method for BAC libraries construction from flow-sorted chromosomes
The amount of HMW DNA recovered from flow-sorted chromosomes is low compared with that needed for BAC library construction using conventional protocols (Chalhoub et al., 2004; Osoegawa et al., 1998; Peterson et al., 2000; Zhang and Wu, 2001). Thus, as a first step in BAC library production, we optimized conditions for partial digestion, size selection, and the minimum amount of flow-sorted HMW DNA needed for BAC library construction. About 4· 106chromosomes from peaks I to III and 3B (Figure 1),
representing all wheat chromosomes, were sorted in
aliqu-ots of 1· 105
(equivalent to 0.165 lg DNA) and used. Pulsed field gel electrophoresis (PFGE) indicated that the DNA of sorted chromosomes was intact as most of it remained in the plug, with a smaller quantity in the compression zone (2 Mb). No degraded ‘smeared’ DNA was detected on the gel and the DNA was shown to be easily digestible by restriction enzyme (HindIII) (Figure 2). Optimum HindIII concentration Figure 1. Flow karyotype of the common wheat cultivar ‘Chinese Spring’ obtained after flow cytometric analysis of DAPI-stained chromosome sus-pension.
The karyotype consists of three composite peaks (I, II and III) representing groups of chromosomes (Vra´na et al., 2000) and a clearly discriminated peak representing chromosome 3B.
Figure 2. Pulse field gel electrophoresis showing HMW DNA prepared from flow-sorted wheat chromosomes.
Isolated DNA was intact as it remained in the sample well (line 1). Digestibility of HMW DNA was tested with HindIII. DNA was cut with 0.5 (line 2) and 15 (line 3) units of enzyme for 20 min at 37C, respectively. PFGE was run in 1% agarose gel at 6 V cm)1, 14C, 0.5· TBE for 18 h with 1–50 sec switching interval and an angle of 120. M ¼ k ladder for PFGE (New England Biolabs, Beverly, MA, USA).
units/tube that generate maximum amounts of partially digested DNA, suitable for BAC library construction (50– 300 kb size), were determined to range between 0.1 and 10 units ml)1 of digestion buffer, containing 0.2–0.3· 106 chromosomes.
As expected, a very faint smear of partially digested DNA ranging from 5 kb to 2 Mb was observed on the ethidium bromide stained edges of the gel. Partially digested DNA ranging in size from 50 to 300 kb was fractionated at 50 kb intervals and recovered by electroelution. Normal electro-phoresis conditions (Chalhoub et al., 2004) resulted in less than 0.2 ng DNA ll)1(in a total volume of 200 ll), which was not adequate for ligation and BAC cloning. Concentrating electroeluted DNA through extended electrophoresis and recovery in only the bottom 39 ll, but directly on the membrane yielded from 5 to 20 ng of partially digested DNA from each of the sized fractions (see Experimental procedures). This step was critical for successful ligations and BAC library construction. Interestingly, at equivalent amounts, partially digested DNA obtained from flow-sorted chromosomes was shown to be more efficient in overall BAC cloning compared with HMW DNA obtained by
con-ventional methods (Chalhoub et al., 2004; Osoegawa et al., 1998; Peterson et al., 2000; Zhang and Wu, 2001) (data not shown). Thus, 0.5· 106 flow-sorted metaphase
chromo-somes (equivalent to 0.825 lg DNA) were sufficient to obtain BAC clones with insert sizes ranging from 40 to 150 kb.
Flow-sorting of chromosome 3B and BAC library construction
In order to construct a chromosome-specific BAC library, 2· 106
3B chromosomes (approximately 4 lg DNA) were sorted. On average, 10 000 chromosomes could be purified from one sample. With the average sorting speed of 17 000 chromosomes per hour, the sorting took 18 working days. In total, 200 samples of chromosome suspensions were pre-pared from 4000 seedlings. The purity of the sorted fractions was monitored periodically during sorting by microscopic observation after fluorescent labeling of repetitive DNA sequences (Figure 3a). It was found that sorted 3B chromo-somes were contaminated at an average frequency of 11.4% by various chromosomes and chromosome-arms, without an apparent prevalence of certain chromosome types. As the Figure 3. Physical mapping of selected repetit-ive DNA sequences and BAC clones on wheat chromosome 3B.
The probes were detected either with fluorescein (yellow-green color) or Cy3 (red color). Chromo-somes were counterstained with DAPI (Figure 3b shown in red pseudocolor). For each probe, two representative examples are given.
(a) Localization of three repetitive DNA sequences. Localization of GAA microsatellite was sufficient to identify chromosome 3B unam-biguously.
(b) Localization of four ‘low copy’ BAC clones. Two clones, 63C11 and 63B13, localized to short arm of 3B; BAC clones 63N2 and 81B7 localized to the long arm of 3B.
(c) Relative arrangement of BAC 63C11 and three repetitive DNA sequences on chromosome 3B. (d) Ideogram showing genomic distribution of three repeats and BAC 63C11 on chromo-some 3B. Key to repetitive DNA probes: 119¼ pSc119.2; GAA¼ GAA microsatellite; Afa¼ Afa family repeat.
3B chromosome is the largest in the set, the contamination was probably the result of doublets of various chromo-somes and chromosome arms whose relative DNA content was close to that of 3B. However, as most of the doublets disintegrated because of mechanical shearing forces during the sorting process, their nature and frequency could not be established reliably.
Approximately 1.8· 1063B chromosomes embedded in
18 agarose miniplugs were partially digested using seven partial digestion conditions (see Experimental procedures), and partially digested chromosome 3B miniplugs were poured together and size-selected by PFGE. Partially diges-ted DNA ranging in size from 50 to 300 kb was fractionadiges-ted at 50-kb intervals and recovered by electroelution as described above and subsequently ligated to the BAC vector. On average, 500–3000 white colonies were obtained by elec-troporating 2 ll of the ligation mix. Less than 2% of the obtained colonies were empty.
Chromosome 3B BAC library characterization
The insert size of 144 randomly selected BAC clones, com-pletely digested with NotI, ranged from 40 to 170 kb (Figure 4). The chromosome 3B-specific BAC library (desig-nated as TA3B) consists of 67 968 BAC clones ordered in 177, 384-well plates that can be sorted into three sub-libraries based on mean insert size of the different ligations. The first sub-library contains 22 656 clones with an average insert
size of 55 kb and a chromosome 3B coverage of 1.1·. The second sub-library consists of 14 976 clones with an average insert size of 100 kb and a chromosome 3B coverage of 1.3·. The most abundant sub-library contains 30 336 clones with an average insert size of 140 kb and a chromosome 3B coverage of 3.8·. The average insert size of the whole library is 103 kb. Considering the presence of 11.4% of contamin-ating clones (7748 clones), the library represents 6.2 genome equivalents of chromosome 3B and 0.0499 genome equiv-alents of other chromosomes. Theoretically, any gene pre-sent on chromosome 3B should be recovered with a probability of 99.8%.
To analyze its coverage and specificity, the chromosome 3B BAC library was divided into pools of 384 clones each and PCR-screened with 38 microsatellite markers known to map to chromosome 3B. On average, the 3B microsatellites were detected 9.6 times on the BAC library (Table 1) indicating higher genome coverage than the 6.2· expected from mean insert size calculation. The chromosome 3B BAC library was also screened with 40 microsatellite markers specific for the remaining 20 wheat chromosomes (two for each of them) to test contamination with other chromosomes and chromo-some arms. Out of these, only four markers that map to chromosomes 1B, 2B, 4B and 5B, were detected (each of them only once), demonstrating the high specificity of the library (Table 2). No chloroplast or mitochondrial marker was detected (Table 2).
The use of chromosome 3B BAC resource for cytogenetic mapping
Hybridization of 3072 randomly chosen BAC clones with genomic DNA identified 36 clones (1.17%) with a low pro-portion of repetitive DNA (low hybridization signal). Seven clones were chosen for cytogenetic mapping using fluores-cent in situ hybridization (FISH). Three of them showed dispersed signals on all wheat chromosomes while the other four resulted in discrete signals on chromosome 3B only (Table 3). For the BAC clone 63C11, having the shortest insert size (18 kb), no blocking DNA was needed to suppress dispersed background hybridization, and direct FISH resul-ted in a double signal on the short arm of chromosome 3B (3BS) and very weak signals on the long arm of chromosome 3B (3BL). Under high stringency, only the distal loci on 3BS were detected with the BAC clone 63C11 (Figure 3b). The other three clones could be specifically located on 3B only when the C0t-1 fraction was used as a blocking DNA at a ratio
of 1:300 (probe: C0t-1). BAC clone 63B13 localized to the
interstitial region of 3BS, while BAC clones 63N2 and 81B7 were localized to distal and interstitial parts of 3BL, respectively (Figure 3b). None of the four BAC clones that could be mapped by FISH localized to other chromosomes. Further FISH analysis of the chromosome 3B structure was carried out through the analysis of location and distribution Figure 4. Insert sizes of 19 randomly selected BAC clones from the specific
chromosome 3B library.
The BAC DNA was completely digested with NotI enzyme and insert size was determined by PFGE (1% agarose, 0.5· TBE, 6 V cm)1, 1–40 sec switch time ramp, angle 120, 14 h at 14.0C along with a k marker for PFGE (M) (New England Biolabs). 7.5 kb band represent the BAC vector (V).
of a BAC clone 63C11, two repeated sequences (pSc119.2, Afa) and GAA microsatellite (Figure 3c,d). The 63C11 locus was shown to be flanked by the GAA and Afa repeats, and the whole cluster was bordered by two pSc119.2 clusters (Figure 3c). Genomic distribution of the repeats and location of the 63C11 clone are shown in the ideogram presented in Figure 3(d).
Discussion
Here we report on a chromosome-specific BAC library produced directly from flow-sorted chromosomes. Previous
attempts to clone flow-sorted chromosomes in human involved either cosmid or YACs cloning systems, and relied on various strategies that avoided partial digestion and/or gel-size selection of chromosomal DNA (Kouprina et al., 1998; McCormick et al., 1993; Nizetic et al., 1994; Van Dilla and Deaven, 1990). The protocol developed in this study overcomes previously encountered difficulties and repre-sents a generally applicable strategy that may assist in
Table 1 Results of library screening on pools of 384-well plates with the 3B-specifc PCR markers
Marker Number of positive pools Code Reference cfa2170 a 9 cfa2191 a 6 cfa2226 a 6 cfd4 a 4 gpw118 a 16 gpw1025 a 11 gpw1107 a 13 gpw1108 a 8 gpw1120 a 19 gpw1145 a 13 gpw1146 a 4 gpw3085 a 1 gpw3233 a 5 gpw3248 a 15 gpw4034 a 12 gpw4044 a 14 gpw5007 a 6 gpw5016 a 1 gpw7031 a 6 gpw7080 a 15 gpw8056 a 8 gpw8064 a 9 gpw8100 a 1 gwm72 b 7 gwm77 b 8 gwm131 b 9 gwm181 b 12 gwm247 b 11 gwm264 b 5 gwm284 b 14 gwm285 b 9 gwm299 b 15 gwm340 b 14 gwm376 b 14 gwm389 b 7 gwm493 b 5 gwm533 b 13 gwm566 b 8
a: P. Sourdille and M. Bernard (unpublished data); b: Ro¨der et al. (1998).
Table 2 Results of library screening on pools of 384-well plates with PCR markers specific for chromosomes other than 3B
Chromosome specificity Marker Number of positive pools Code Reference 1A Xgwm99 a 0 1A Xgwm135 a 0 2A Xgwm312 a 0 2A Xgmw372 a 0 3A Xgwm5 a 0 3A Xgwm480 a 0 4A Xgwm610 a 0 4A Xgwm601 a Not functional 5A Xgwm415 a 0 5A Xgwm186 a 0 6A Xgwm169 a 0 6A Xgwm427 a 0 7A Xgwm233 a 0 7A Xgwm260 a 0 1B Xgmw11 a 0 1B Xgwm413 a 1 2B Xgwm120 a 0 2B Xgwm257 a 1 4B Xgwm149 a 1 4B Xgwm251 a 0 5B Xgwm234 a 1 5B Xgwm408 a 0 6B Xgwm132 a 0 6B Xgwm219 a 0 7B Xgwm46 a 0 7B Xgwm400 a 0 1D Xgwm337 a 0 1D Xgwm642 a 0 2D Xgwm261 a 0 2D Xgwm539 a 0 3D Xgwm161 a 0 3D Xgwm664 a 0 4D Xgwm194 a 0 4D Xgwm609 a 0 5D Xgwm190 a 0 5D Xgwm272 a 0 6D Xgwm325 a 0 6D Xgwm469 a 0 7D Xgwm44 a 0 7D Xgwm437 a 0 Mitochondria mit 3 b 0 Mitochondria mit 4 b 0 Chloroplast CCM 2 b 0 Chloroplast CCM 6 b 0 a: Ro¨der et al. (1998); b: B. Chalhoub (unpublished data).
dissecting large and complex genomes of wheat and several other species. We have now validated our method by cloning into BACs other wheat chromosomes and chromo-some-arms (Janda et al., Institute of Experimental Biology, Olomouc University, Czech Republic, in preparation), using specific ditelosomic and isochromosomic lines (Sears, 1954) that represent a valuable resource for sorting any single chromosome or chromosome-arm (Kubala´kova´ et al., 2002). In animals and humans, a strong possibility is offered by the fact that specific chromosomes can be cloned in hybrid cells (e.g. hamster/human hybrid cells) and easily sorted by flow cytometry (Kouprina et al., 1998; McCormick et al., 1993; Nizetic et al., 1994; Van Dilla and Deaven, 1990).
Sorting, by flow cytometry, the required number of copies of a specific chromosome and construction of a BAC library from a small quantity of DNA were two major challenges of this project. Traditionally, higher amounts of DNA are needed to prepare BAC libraries as DNA is only partially digested and only a small portion is finally recovered after gel sizing and recovery (Osoegawa et al., 1998; Peterson et al., 2000; Zhang and Wu, 2001). In addition to the improvement of the yield of flow-sorted chromosomes and the superior quality of HMW DNA, we adapted the recently described efficient protocol for BAC library construction (Chalhoub et al., 2004) for lower amounts of DNA, which was shown to be more efficient in overall BAC cloning.
The insert size and coverage of the 3B BAC library compare well with the genomic BAC libraries obtained from diploid (Lijavetzky et al., 1999; Moullet et al., 1999), tetraploid (Cenci et al., 2003) and hexaploid (Allouis et al., 2003) wheat. The reason for the discrepancy between the chromosome 3B genome coverage estimated by BAC clones insert sizes (6.2·) and the markers (9.6·) may be the result of an overestimation of the real molecular size of chromosome 3B or to an underestimation of the average size of the clones as several bands were obtained after a NotI digestion of BAC clones, rendering calcula-tions relatively difficult. The chromosome 3B BAC library is free of chloroplast and mitochondrial DNA. This is an important advantage, especially in plants where contam-ination with chloroplast DNA was observed to be high,
ranging from 3 to 5% (Moullet et al., 1999; Peterson et al., 2000). The major source of low contamination with DNA from other chromosomes is the result of the formation, during the preparation of chromosome suspension, of doublets of various chromosomes, chromatids and chro-mosome fragments with a total DNA content close to that of chromosome 3B and which the cytometer misclassified for 3B types.
It is surprising that all low copy BAC clones (four clones) that could be mapped by FISH were localized exclusively on chromosome 3B, as the hexaploid wheat genome also contains the homoeologous chromosomes 3A and 3D. Quite interestingly, a chance to physically map a BAC clone by FISH did not show any bias toward long inserts, indicating that large stretches of wheat DNA do not contain significant amounts of repetitive DNA. These results confirm the degree of ‘rapid divergence’ between homoeologous chromo-somes (Gaut, 2002; Wicker et al., 2003) and indicate that the library could become a rich source of molecular cytogenetic markers. It should be relatively straightforward to select other BAC clones from the library to evenly cover the chromosome and produce molecular banding. A similar strategy was used to ‘paint’ chromosome 4 of Arabidopsis (Lysa´k et al., 2001). Many interesting genes and QTL have been mapped on 3B (Bo¨rner et al., 2002). The short arm of chromosome 3B carries the Sr2 gene conferring durable resistance to stem rust (Spielmeyer et al., 2003) and a major QTL for scab resistance (Anderson et al., 2001; Gervais et al., 2003). Construction of the 3B-specific BAC library now provides a means for its more efficient targeting and for mapping and cloning of other genes that would be discov-ered on 3B.
In addition to these ‘conventional’ uses, one may foresee several interesting applications based on the use of DNA isolated from a pooled chromosome specific library. Chro-mosome 3B-derived DNA could be used for rapid isolation of gene-coding sequences from 3B via methyl filtration (Rabinowicz et al., 2003) or Cot analysis (Peterson et al., 2002). Other applications of chromosome-derived DNA may involve HAPPY mapping (Thangavelu et al., 2003) and high-throughput physical mapping of ESTs and cDNAs on high-density arrays and large sequencing projects.
Table 3 Cytogenetic mapping of puta-tively ‘low copy’ clones selected from the 3B-specific BAC library as weakly hybrid-izing with genomic DNA
Clone
Insert size (kb)
Specificity
for chromosome 3B Remarks 63C11 18 3BS (3BL) No blocking DNA
63B13 150 3BS C0t-1 fraction used as a blocking DNA
63N2 50 3BL C0t-1 fraction used as a blocking DNA
81B7 70 3BL C0t-1 fraction used as a blocking DNA
54O8 80 – Dispersed signals on all chromosomes 54O12 40 – Dispersed signals on all chromosomes 63E4 110 – Dispersed signals on all chromosomes
To conclude, this work marks the first step in the integration of wheat flow cytogenetics with genomics. The possibility of preparing a high quality BAC library from flow-sorted wheat chromosomes was demonstrated and a chromosome 3B-specific BAC library was constructed. The TA-3B BAC library is publicly available and should provide a resource for cloning agronomically important genes, known to map on this chromosome. The results of this study, combined with the possibility to sort any single chromo-some or chromochromo-some-arm from wheat (Kubala´kova´ et al., 2002), using ditelosomic or isochromosomic lines (Sears, 1954), show the possibility of dissecting the whole-wheat genome into small and defined parts. We have now cloned the short arm of chromosome 1B from hexaploid wheat (Janda et al., in preparation) and we are going to sort and clone chromosomes 3A and 3D to complete the homology group 3 resources panel, which will be very valuable for genome initiatives that aim to decipher the hexaploid wheat genome (Gill and Appels, 2004).
The availability of chromosome and chromosome-arm specific BAC libraries will greatly facilitate genome analysis and cloning of useful genes from plants with large and complex genomes.
Experimental procedures Plant material
Seeds of hexaploid wheat cultivar ‘Chinese Spring’ were germina-ted in the dark at 25 0.5C on moistened filter paper in glass Petri dish for 3 days to achieve optimal root length (2–3 cm). In total, 4000 seeds were used and germinated in batches of 20, and used for preparation of chromosome suspensions.
Preparation of chromosome suspensions and chromosome sorting
Mitotic chromosomes were isolated from synchronized root tips according to Vra´na et al. (2000). Chromosome suspensions were stained with 2 lg ml)1 4¢,6-diamidino-2-phenylindole (DAPI) and chromosomes were sorted at a rate of about five per second using FACS Vantage flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) as described by Vra´na et al. (2000). Sorting gates were set on a dot plot of fluorescence pulse area versus fluor-escence pulse width to select either all wheat chromosomes or the chromosome 3B. Chromosomes were sorted in aliquots of 1.0· 105 into 160 ll of 1.5
· IB buffer (Sˇimkova´ et al., 2003). The identity and purity of the sorted chromosome fractions were determined microscopically after fluorescent labeling of GAA microsatellites, Afa family repeat and a pSc119.2 repeat according to Kubala´kova´ et al. (2003).
Preparation of high molecular weight DNA
Isolation of HMW DNA was carried out as described by Sˇimkova´ et al. (2003). Briefly, flow-sorted chromosomes were pelleted at 200 g for 30 min at 4C and resuspended in 7.5 ll of 1· IB at 50C, and mixed
with 4.5 ll pre-warmed 2% InCert low melting point agarose (GTG) in 1· IB. The mixture was poured into an 80-ll plug mold to form an agarose miniplug. The quality of HMW DNA was checked by PFGE.
Partial digestion, size-selection and recovery of HMW DNA
Agarose miniplugs were washed twice for 1 h in TE 10:10 buf-fer (10 mMTris, 10 mMEDTA) with 2% w/v polyvinylpyrrolidone (PVP-40). Subsequently, they were equilibrated on ice for 1 h in 10 ml of 1X HindIII buffer (Invitrogen, Carlsbad, CA, USA) supple-mented with 4 mMspermidine, 1 mMDTT and 0.1 mg ml)1BSA.
Restriction enzyme (HindIII) concentrations for optimal partial digestion (0.1–10 units for three miniplug ml)1total volume diges-tion) were initially determined using whole genomic DNA miniplugs (containing 105chromosomes each). For 3B flow-sorted
chromo-somes, three miniplugs at a time (of 105chromosomes each) were
partially digested in 1 ml of reaction buffer, using six conditions of HindIII enzyme concentration (0.2, 0.3, 0.5, 1, 2, and 10 units/tube), for 20 min at 37C. The digestion was stopped on ice by the addition of 200 ll 0.5MEDTA, pH 8.0, for 30 min. Partially digested DNA was
size-selected by PFGE in 1% Gold SeaKem agarose (GTG) gel at 6 V cm)1, 12C in 0.25· TBE for 14 h, with a 1.0–40 sec switching interval and an angle of 120. After the electrophoresis, the edges of the gel containing size markers were excised and stained with ethidium bromide. Five regions of the gel (50–100, 100–150, 150– 200, 200–250 and 250–300 kb) were excised and equilibrated with 1x TAE buffer, twice for 1 h each. The size-selected DNA was isolated by electroelution in 1X TAE, using a Bio-Rad Electroelution system (Model 422, Biorad, Hercules, CA, USA). The optimum electroelu-tion (electrophoresis) time for concentrating the partially digested DNA in the bottom 39 ll fraction, directly on the membrane CAPS was determined by conducting several monitoring experiments at different electrophoresis time, using whole genomic DNA. DNA concentration was estimated in 1% normal agarose gels using 6 ll of electroeluted DNA and a dilution series of HindIII-digested lambda as a standard.
Ligation and transformation
The remaining complete DNA fraction (33 ll) was ligated at 16C overnight in a 50 ll reaction with 4 units of T4 DNA ligase (Invi-trogen) and 4 ng of pIndigoBAC (Caltech, Pasadena, CA, USA) vector prepared for high efficiency cloning with HindIII. For this we performed digestion and dephosphorylation reactions in the same tube without any precipitation steps to avoid vector dam-age. Our previous experiments have shown that this method of vector preparation results in two- to threefold higher ligation efficiency, compared with conventionally described methods (Chalhoub et al., 2004). The ligation mix was dialyzed for 90 min at 4C (Allouis et al., 2003; Chalhoub et al., 2004). Fifteen microl-iters of dialyzed ligation was mixed with 110 ll ElectroMax DH10B electrocompetent cells, and incubated for 5 min at room tem-perature. Fifteen microliters of the mixture was electroporated using Gibco BRL Cell-Porator System (Life Technologies, Gai-tersburg, MD, USA) with the following settings: 350 V, 330 lF capacitance, low ohms impedance, fast charge rate, and 4 kX resistance. The electroporations were pooled in a tube containing 3 ml of SOC media (Sambrook et al., 1989) and incubated for 45 min at 37C on an orbital shaker at 180 rpm. Aliquots of the SOC medium with recombinant cells were plated on LB plates containing 12.5 lg ml)1 chloramphenicol, 50 lg ml)1 X-Gal and 25 lg ml)1IPTG, and incubated at 37C overnight. White recom-binant colonies were picked using a Q-bot (Genetix, Dorset, UK)
and transferred to 384-well plates containing 90 lL of LB freezing buffer (Peterson et al., 2000). The plates were incubated overnight at 37C, triplicated and stored at)80C.
Isolation of BAC DNA and insert analysis
Individual BAC clones were cultured overnight in deep 96-well plates containing 1.5 ml LB supplemented with 12.5 lg ml)1 chloramphenicol. BAC DNAs were isolated and digested to com-pletion with NotI according to Allouis et al. (2003). DNA fragments were size separated by PFGE in 1% Gold SeaKem agarose (GTG) gel at 6 V cm)1, with a 1–40 sec switch time ramp, angle 120, for 14 h at 14.0C in 0.5x TBE buffer.
Coverage and specificity of the BAC library
The 3B chromosome coverage was calculated based on average insert size, total number of clones and 11.4% contamination by other chromosomes. The genome size of chromosome 3B was determined as 995 Mb based on its relative length (5.86%, Gill et al., 1991) and nuclear genome size of common wheat (16,974 Mb/1C, Bennett and Smith, 1991). The probability of occurrence of DNA sequences from chromosome 3B in the library was determined according to Clarke and Carbon (1976), taking into account 11.4% contamination by other chromosomes. The same approach was used to determine the probability of occur-rence of DNA sequences from contaminating chromosomes. The specificity of the BAC library and the extent of contamination with other chromosomes and cytoplasmic DNA were estimated by PCR on pools of bacteria from the 384-well plates with specific primers (Tables 1 and 2). DNA was released from bacteria by a modified boiling method that consists of incubation in 10:10 Tris-EDT (TE) buffer at 95C for 30 min. Cell debris was pelleted at 3000 g for 60 min, the supernatant was diluted 20 times with deionized water and used for PCR. The PCR mixture consisted of 2 ll of template DNA, 1x PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs,
1.25 lM primers, 0.5 U of AmpliTaq DNA polymerase (Roche
Applied Science, Brancburg, NJ, USA) in a total volume of 10 ll. PCR was performed in Gene Amp PCR System 9700 (Applied Biosystems, Warrington, UK) as follows: 1 cycle of 5 min at 94C; 10 cycles of 1C ‘touch down’ PCR reactions consisting of 12 sec at 94C, 12 sec at 65–55C and 20 sec at 72C; 35 cycles of 12 sec at 94C, 12 sec at 55C, and 20 sec at 72C; and final extension cycle of 72C for 7 min. PCR products were separated on 4% MetaPhor agarose gel (FMC Bioproducts, Rockland, ME, USA).
Identification of BAC clones for FISH
BAC clones, 3072 in number, were doubly spotted onto two 8· 12-cm Hybond Nþ filters (AP Biotech, Buckinghamshire, UK) with the GeneTACTMG3 workstation (Genomic Solutions, Ann Arbor, MI, USA). Putative BAC clones for specific hybridization with chromosome 3B were selected based on weak Southern hybrid-ization signals with wheat ‘Chinese Spring’ genomic DNA labeled with digoxigenin. Hybridization was carried out at 65C overnight in 5 ml of hybridization buffer (5· SSC, 2% blocking reagent 0.1% so-dium N-lauroylsarcosine, 0.02% SDS) containing 400 ng of labeled probe. Stringent washing was carried out by incubation twice in 200 ml of 0.1· SSC, 0.1% SDS buffer at 68C. Hybridization signals were detected using anti-digoxigenin-AP (Roche Applied Science) and visualized after incubation with CDP Star chemiluminescent substrate (Roche Applied Science).
Chromosomal localization of BAC clones
Seven putative 3B-specific BAC clones were digoxigenin-labeled and localized on flow-sorted wheat chromosomes by fluorescence FISH according to Kubala´kova´ et al. (2003), with modifications. Labeled probe was used at 1 and 2 lg ml)1in combination with C0t-1 fraction
of wheat genomic DNA in various ratios (1:0, 50, 100, 200, 300) to suppress repetitive DNA hybridization. Hybridization was carried out at 37 or 45C (higher stringency) overnight. The sites of digoxigenin-labeled probe hybridization were detected using anti-digoxigenin-FITC and the chromosomes were counterstained with DAPI.
Fluorescence microscopy
The slides were examined with an Olympus BX60 epifluorescence microscope using 100· oil immersion objective. The images of DAPI, Cy3 and fluorescein fluorescence were acquired separately with a cooled high-resolution black and white CCD camera, inter-faced to a PC running ISIS software (Metasystems, Altlussheim, Germany). Image processing consisted exclusively of signal inten-sity, contrast, and background adjustments that affected the whole image.
Acknowledgements
We sincerely thank Dr Heather I. McKhann (INRA-Versailles/CNG) and Dr Sylvie Bernard (INRA-Clermont-Ferrand) for valuable com-ments and English correction of the paper. This work was supported by research grants from the Grant Agency of the Czech Republic (522/03/0354 and 521/04/0607), Ministry of Agriculture of the Czech Republic (QC 1336), research grants from the French Ministry of Research and Agriculture, and by a collaborative project ‘Barrande’ (reg. no. 2003-035-1) between the Ministry of Education, Youth and Sports of the Czech Republic and the French Ministry of Foreign Affairs and Education. J. Janda acknowledges the receipt of a Marie Curie PhD student Fellowship at URGV-INRA.
References
Akhunov, E.D., Goodyear, A.W., Geng, S. et al. (2003) The organ-ization and rate of evolution of wheat genomes are correlated with recombination rates along the chromosome arms. Genome Res. 13, 753–763.
Allouis, S., Moore, G., Bellec, A. et al. (2003) Construction and characterization of a hexaploid wheat (Triticum aestivum L.) BAC library from the reference germplasm ‘Chinese Spring’. Cereal Res. Comm. 31, 331–338.
Anderson, J.A., Stack, R.W., Liu, S. et al. (2001) DNA markers for Fusarium head blight resistance QTLs in two wheat populations. Theor. Appl. Genet. 102, 1164–1168.
Bennett, M.D. and Smith, J.B. (1991) Nuclear DNA amounts in angiosperms. Proc. R. Soc. Lond. B, 334, 309–345.
Bo¨rner, A., Schumann, E., Fu¨rste, A., Co¨ster, H., Leithold, B., Ro¨der, M.S. and Weber, W.E. (2002) Mapping of quantitative trait loci determining agronomic important characters in hexaploid wheat (Triticum aestivum L.). Theor. Appl. Genet. 105, 921–936. Cenci, A., Chantret, N., Kong, X., Gu, Y., Anderson, O.D., Fahima, T.,
Distelfeld, A. and Dubcovsky, J. (2003) Construction and charac-terization of a half million clone BAC library of durum wheat (Tridicum turgidum ssp. durum). Theor. Appl. Genet. 107, 931–939. Chalhoub, B., Belcram, H. and Caboche, M. (2004) Efficient cloning of plant genomes into bacterial artificial chromosome (BAC)
libraries with larger and more uniform insert size. Plant Biotech-nol. J. 2, 181–188.
Clarke, L. and Carbon, J. (1976) A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell, 9, 91–99.
Gaut, B.S. (2002) Evolutionary dynamics of grass genomes. New Phytologist, 154, 15–28.
Gervais, L., Dedryver, F., Morlais, J.Y., Bodusseau, V., Negre, S., Bilous, M., Groos, C. and Trottet, M. (2003) Mapping of quanti-tative trait loci for field resistance to Fusarium head blight in an European winter wheat. Theor. Appl. Genet. 106, 961–970. Gill, E.T. (2004) Workshop on wheat genome sequencing. Genetics,
in press.
Gill, B.S., Friebe, B. and Endo, T.R. (1991) Standard karyotype and nomenclature system for description of chromosome bands and structural aberrations in wheat (Triticum aestivum). Genome, 34, 830–839.
Kouprina, N., Campbell, M., Graves, J. et al. (1998) Construction of human chromosome 16- and 5- specific circular YAC/BAC librar-ies by in vivo recombination in Yeast (TAR cloning). Genomics, 53, 21–28.
Kubala´kova´, M., Vra´na, J., Cˇı´halı´kova´, J., Sˇimkova´, H. and Dolezˇel, J. (2002) Flow karyotyping and chromosome sorting in bread wheat (Triticum aestivum L.). Theor. Appl. Genet. 104, 1362–1372. Kubala´kova´, M., Vala´rik, M., Bartosˇ, J., Vra´na, J., Cˇı´halı´kova´, J., Molna´r-La´ng, M. and Dolezˇel, J. 2003. Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry. Genome, 46, 893–905.
Lee, J.H., Arumuganathan, K., Yen, Y., Kaeppler, S., Kaeppler, H. and Baenziger, P.S. (1997) Root tip cell cycle synchronization and metaphase-chromosome isolation suitable for flow sorting in common wheat (Triticum aestivum L.). Genome, 40, 633–638. Lijavetzky, D., Muzzi, G., Wicker, T., Keller, B., Wing, R. and
Dub-covsky, J. (1999) Construction and characterization of a bacterial artificial chromosome (BAC) library for the A genome of wheat. Genome, 42, 1176–1182.
Lysa´k, M.A., Fransz, P.F., Ali, H.B.M. and Schubert, I. (2001) Chro-mosome painting in Arabidopsis thaliana. Plant J. 28, 689–697. McCormick, M.K., Campbell, E., Deaven, L. and Moysis, R. (1993)
Low-frequency chimeric yeast artificial chromosome libraries from flow-sorted human chromosomes 16 and 21. Proc. Natl Acad. Sci. USA, 90, 1063–1067.
Moullet, O., Zhang, H.B. and Lagudah, E.S. (1999) Construction and characterisation of a large DNA insert library from the D genome of wheat. Theor. Appl. Genet. 99, 305–313.
Nizetic, D., Monard, S., Young, B., Cotter, F., Zehetner, G. and Lehrac, H. (1994) Construction of cosmid libraries from flow-sor-ted human chromosomes 1, 6, 7, 11, 13, and 18 for reference library resources. Mamm. Genome, 5, 801–812.
Osoegawa, K., Woon, P.Y., Zhao, B., Frengen, E., Tateno, M., Cat-anese, J.J. and de Jong, P.J. (1998) An improved approach for construction of bacterial artificial chromosome libraries. Genomics, 52, 1–8.
Peterson, D.G., Tomkins, J.P., Frisch, D.A., Wing, R.A. and Paterson, A.H. (2000) Construction of plant bacterial artificial chromosome (BAC) libraries in plants: An illustrated guide. J. Agric. Genom. 5. http://www.ncgr.org./research/jag.
Peterson, D.G., Wessler, S.R. and Paterson, A.H. (2002) Efficient capture of unique sequences from eukaryotic genomes. Trends Genet. 18, 547–550.
Rabinowicz, P.D., McCombie, W.R. and Martienssen, R.A. (2003) Gene enrichment in plant genomic shotgun libraries. Curr. Opin. Plant Biol. 6, 150–156.
Ro¨der, M.S., Korsun, V., Wendehake, K., Plaschke, J., Tixier, M.H., Leroy, P. and Ganal, M.W. (1998) A microsatellite map of the wheat genome. Genetics, 149, 2007–2023.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Clo-ning, a Laboratory Manual, 2nd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Sears, E.R. (1954) The aneuploids of common wheat. Univ. Missouri Res. Bull. 572, 1–58.
Shizuya, H., Birren, B., Kim, U.-J., Mancino, V., Slepak, T., Tachiiti, Y. and Simon, M. (1992) Cloning and stable maintenance of 300-kilobasepair fragment of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl Acad. Sci. USA, 89, 8794–8797. Sˇimkova´, H., Cˇı´halı´kova´, J., Vra´na, J., Lysa´k, M.A. and Dolezˇel, J.
(2003) Preparation of HMW DNA from plant nuclei and chromo-somes isolated from root tips. Biol. Plant. 46, 369–373.
Spielmeyer, W., Sharp, P.J, Lagudah, E.S. (2003) Identification and validation of markers linked to broad-spectrum stem rust resistance gene Sr2 in wheat (Triticum aestivum L.). Crop Sci. 43, 333–336.
Thangavelu, M., James, A.B., Bankier, A., Bryan, G.J., Dear, P.H. and Waugh, R. (2003) HAPPY mapping in a plant genome: recon-struction and analysis of a high-resolution physical map of a 1.9 Mbp region of Arabidopsis thaliana chromosome 4. Plant Biotech. J. 1, 23–31.
Van Dilla, M.A. and Deaven, L. (1990) Construction of gene libraries for each human chromosome. Cytometry, 11, 208–218. Vedel, F. and Delseny, M. (1987) Repetitivity and variability of
higher-plant genomes. Plant Physiol. Biochem. 25, 191–210. Vra´na, J., Kubala´kova´, M., Sˇimkova´, H., Cˇı´halı´kova´, J., Lysa´k, M.A.
and Dolezˇel, J. (2000) Flow-sorting of mitotic chromosomes in common wheat (Triticum aestivum L.). Genetics, 156, 2033–2041. Wang, M.L., Leitch, A.R., Schwarzacher, T., Heslop-Harrison, J.S. and Moore, G. (1992) Construction of a chromosome-enriched Hpall library from flow-sorted wheat chromosomes. Nucleic Acids Res. 20, 1897–1901.
Wicker, T., Yahyaoui, N., Gyuot, R., Schlagenhauf, E., Liu, Z.-D., Dubkovsky, J. and Keller, B. (2003) Rapid genome divergence at orthologous low molecular weight glutenin loci of the A and Am
genomes of wheat. Plant Cell, 15, 1186–1197.
Zhang, H.-B. and Wu, Ch. (2001) BAC as tools for genome sequen-cing. Plant Physiol. Biochem. 39, 195–209.
