As part of an initiative to develop Brachypodium distachyon as a genomic "bridge" species between rice and the temperate cereals and grasses, a BAC library has been constructed for the two diploid (2n = 2x = 10) genotypes, ABR1 and ABR5. The library consists of 9100 clones, with an approximate average insert size of 88 kb, representing 2.22 genome equivalents. To validate the usefulness of this species for comparative genomics and gene discovery in its larger genome relatives, the library was screened by PCR using primers designed on previously mapped rice and Poaceae sequences. Screening indicated a degree of synteny between these species and B. distachyon, which was confirmed by fluorescent in situ hybridization of the marker-selected BACs (BAC landing) to the 10 chromosome arms of the karyotype, with most of the BACs hybridizing as single loci on known chromosomes. Contiguous BACs colocalized on individual chromosomes, thereby confirming the conservation of genome synteny and proving that B. distachyon has utility as a temperate grass model species alternative to rice.

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TEMPERATE grasses diverged from rice almost 50 million years ago (GAUT 2002) and, although the rice genome sequence has proved useful in the analysis of the larger genomes of the temperate cereals, it has limited value for the exploration of many agronomic traits of interest in temperate grasses. Brachypodium is a genus of temperate grasses that is more closely related to the temperate cereals and forage grasses than is rice (CATALAN et al. 1995, 1997; DRAPER et al. 2001; KELLOGG 2001). It contains species that have small genomes, with sizes and proportions of repetitive DNA comparable to the model plants Arabidopsis thaliana and rice (MOORE et al. 1993a; CATALAN et al. 1995). The perennial, outbreeding species Brachypodium sylvaticum was adopted initially (MOORE et al. 1993a,b; ARAGON-ALCAIDE et al. 1996) as a species of choice for development as a genomic "bridge" to agronomically important cereals, such as wheat, barley, and the forage grasses, the rationale being that its close phylogenetic proximity to this group would be reflected in similar gene repertoires and synteny and would enable comparative genomic approaches for gene isolation and discovery. As part of this study, a bacterial artificial chromosome (BAC) library of B. sylvaticum was constructed, the analysis of which demonstrated synteny among this species, rice, and wheat (FOOTE et al. 2004). BACs are the large insert library tool of choice because, compared to the main alternative, yeast artificial chromosomes, they are easy to handle, and the clones are stable and less likely to be chimeric (SHIZUYA et al. 1992; WOO et al. 1994; YU et al. 2000; PETERSON et al. 2002). In addition to providing a mechanism for map-based cloning, a BAC library is also an important resource for physical mapping, genome structural analysis, comparative genomics, and genome sequencing.

The only annual species in the genus B. distachyon was proposed more recently as an alternative "bridge" species on the basis of its highly desirable biological features that make it suitable for functional genomics studies (DRAPER et al. 2001). B. distachyon has one of the smallest genomes (355 Mb) described in grasses to date (BENNETT and LEITCH 2005) and only five pairs of readily identifiable chromosomes in diploid ecotypes. Knowledge of the genomic infrastructure of this species has continued to develop, with advances in the cytogenetics of this species and its relatives (HASTEROK et al. 2004), its interaction with pathogens (DRAPER et al. 2001; ROUTLEDGE et al. 2004; JENKINS et al. 2005), its tissue culture and genetic transformation (BABLAK et al. 1995; CHRISTIANSEN et al. 2005), and mutagenesis (ENGVILD 2005). As part of this ongoing initiative, this article reports on the construction and analysis of two BAC libraries of two diploid (2n = 2x = 10) ecotypes of B. distachyon, ABR1 and ABR5. To validate the usefulness of B. distachyon as a "bridge" species, synteny to rice and some other members of the Poaceae was assayed by marker screening of the BAC libraries, coupled with the "landing" of selected BACs onto chromosomes of B. distachyon, other near relatives in the Brachypodium genus, as well as rice and Triticale.

The fluorescent tagging of BACs and their hybridization in situ to chromosome substrates has been used effectively in a number of ways to improve our understanding of the organization of a variety of plant genomes. In the Poaceae, BAC landing has been used in studies of structural genomics, integrated karyotyping, and chromosomal mapping in species as diverse as barley (LAPITAN et al. 1997), sorghum (ISLAM-FARIDI et al. 2002; KIM et al. 2002), rice (JIANG et al. 1995), and wheat (ZHANG et al. 2004a,b). The method has also been used for the positional cloning and mapping of particular genes, such as the bacterial blight resistance gene in rice (JIANG et al. 1995), and for the integration of genetic, cytogenetic, and physical maps, such as in rice (CHENG et al. 2001). BAC landing is an integral part of comparative genomics and assays of colinearity, for example, between A. thaliana and Brassicae species (JACKSON et al. 2000; ZIOLKOWSKI and SADOWSKI 2002), and has been used to "paint" chromosome arms in Arabidopsis (LYSAK et al. 2001, 2003). In addition to the primary aim of determining to what extent the genome of B. distachyon is colinear to its relatives, the mapping of single-locus BACs in this study also has utility in determining the pattern of divergence of the genomes of related cereals and grasses, the reconstruction of the archetypal grass genome, and the assembly of chromosome "paints" in this species for molecular cytogenetic investigations of chromosome-specific structure and function. In this study we demonstrate the potential of BAC landing to develop rapidly tiles of clones syntenic to important regions of much larger Gramineae genomes.

Plant material:

All Brachypodium ecotypes were sourced from the collection held by Brachyomics of the University of Wales, Aberystwyth. Diploid ecotypes ABR1 and ABR5 of B. distachyon (2n = 2x = 10) were collected from Kaman, Kiresihir (Turkey), and Huesca, Jaca (Spain), respectively. Accession ABR114 was collected from Formenterra (Spain) and originally classified as a cytotype of B. distachyon with 2n = 4x = 20 (ROBERTSON 1981). However, a recent study has shown that it is a distinctly different and unknown diploid with 20 chromosomes (HASTEROK et al. 2004). ABR113 was collected from Lisbon (Portugal) and originally classified as an autohexaploid cytotype of B. distachyon (ROBERTSON 1981). It has subsequently been shown to be an allotetraploid (2n = 4x = 30) with two diploid genomes similar to ABR1 and ABR114 (HASTEROK et al. 2004). Triticale cv. Lasko (2n = 6x = 42; AABBRR), an intergeneric hybrid between tetraploid wheat and rye, and the standard rice genotype Oryza sativa ssp. indica IR64 were used as substrates for chromosomal mapping of Brachypodium BAC clones.

Preparation of high-molecular-weight genomic DNA:

High-molecular-weight (HMW) DNA was isolated, in parallel, from two ecotypes (ABR1 and ABR5) of B. distachyon (2n = 2x = 10). Approximately 20 g of young leaves were harvested from glasshouse-grown plants and ground to a powder in liquid nitrogen. Nuclei were isolated using the method of ZHANG et al. (1995) and embedded in agarose plugs. The nuclei containing plugs were subjected to pre-electrophoresis in a Bio-Rad (Hercules, CA) CHEF-DR II pulsed-field gel electrophoresis (PFGE) apparatus as described by O'SULLIVAN et al. (2001).

Preparation of insert DNA and partial digestion:

The B. distachyon HMW DNA was partially digested using HindIII (1.25 units of enzyme for one plug in 0.5 ml of reaction buffer at 37° for 1 hr). The partially digested DNA was subjected to a single-step separation by PFGE (170 V for 16 hr, linear pulse ramp from 0.5 to 40 sec). After migration, the sides of the gel-containing HMW markers (New England Biolabs, Beverly, MA) were stained with ethidium bromide. This was used to determine the size of the partially digested DNA in the unstained part of the gel. One to two gel slices containing DNA of sizes ranging from 70 to 100 kb and from 100 to 130 kb were excised. The DNA was electroeluted into dialysis tubing and drop dialyzed on ice as described by O'SULLIVAN et al. (2001).

Ligation of size-selected DNA to a vector and transformation:

The purified DNA was ligated into the HindIII-digested pBeloBAC11 vector. The vector/insert ratio used was either 5:1 or 10:1. Ligation was carried out as described by O'SULLIVAN et al. (2001). BAC clones were grown overnight at 37° on LB–agar containing 12.5 µg/ml of chloramphenicol in 220- x 220-mm plates (Genetix). BAC clones were picked into 96-well microtiter plates filled with 100 µl of LB with chloramphenicol and incubated at 37° overnight; glycerol was then added (to 25% of final volume) and the plates were stored at –80°. For quality control purposes, a representative subset of 48 clones from the entire library was selected. The clones were cultured overnight, and BAC DNA was isolated and then restricted with NotI. After PFGE separation, the approximate sizes of the genomic DNA inserts were estimated.

Identification of BAC clones containing repetitive DNA:

High-density colony filters were prepared using a robotic workstation [QIAGEN (Chatsworth, CA) Bio-Robot 3000] custom programmed to array using a 96-pin replicating tool (V&P Scientific) as described by DONNISON et al. (2005). BAC clones were gridded in duplicate in a 5 x 5 array on Hybond-N⁺ membrane (Amersham, Buckinghamshire, UK), which allowed 1200 clones to be represented on one 120- x 80-mm filter. The library screening was performed by Southern analysis of eight filters representing 9100 clones.

Genomic DNA of B. distachyon (ABR1) was isolated from fresh tissue using the DNeasy maxi kit (QIAGEN), labeled with [³²P]dCTP by random priming using the Hi-Prime method (Stratagene, La Jolla, CA) and added to the hybridization buffer. Hybridization was performed overnight at 65° with gentle shaking. Filters were washed stringently (2x SSC + 0.1% SDS; 1x SSC + 0.1% SDS; 0.1x SSC + 0.1% SDS, each washed twice for 15 min). Hybridization was detected both by using a Typhoon PhosphorImager and by exposing X-ray film. A range of exposure times were used and BAC clones were categorized into three classes: highly repetitive (H), moderately repetitive (M), and low or nonrepetitive (L).

The pooling strategy for PCR-based screening:

DNA pools of BAC clones were generated to enable a PCR-based screen of the library. The entire libraries of ABR1 and ABR5 genotypes were replicated in 90, 96-well microtiter plates with each well containing 200 µl of LB and chloramphenicol at a concentration of 12.5 µg/ml. The BAC clones were grown overnight at 37° with gentle agitation at 200 rpm. The cultures from each microtiter plate were pooled into a 50-ml tube and centrifuged at 5000 rpm in an Eppendorf (Madison, WI) 5403 centrifuge. The supernatants were discarded and the pellets were frozen at –80°. Plasmid DNA was isolated using the alkaline lysis method as described by SAMBROOK and RUSSEL (2001). After screening the DNA pools with PCR primers, positive plates were identified and new plasmid DNA pools were created for the rows and columns of the microtiter plate. Finally, plasmid DNA was isolated for the individual clone identified and this DNA was rescreened by PCR to confirm the process.

Screening the BAC library for chloroplast DNA contamination:

To evaluate the contamination of BAC libraries with chloroplast DNA, both BAC libraries were screened by PCR using primers for three chloroplast genes (ndh, rbcL, psb). PCR amplification products were electrophoresed and bands of the predicted size for each gene were excised. The DNA was purified with the QIAquick PCR gel purification method (QIAGEN) and sequenced using an ABI 3100 DNA analyzer. Sequences were characterized by BLAST analysis (http://www.ncbi.nlm.nih.gov).

PCR screening of the B. distachyon BAC libraries:

Both BAC libraries were screened with 13 primer pairs designed to identify single gene sequences from the region of rice chromosome 6, which had previously been identified as containing the rice Hd3 QTL and Hd3a gene (MONNA et al. 2002). Each primer pair (Table 1) was tested for amplification on B. distachyon ABR1 and ABR5 genomic DNA prior to BAC library screening. In addition, both BAC libraries were screened with another nine primer pairs designed to amplify marker sequences previously genetically mapped in Lolium perenne, Triticeae species, and rice (Tables 1 and 2; http://www.gramene.org). Thermal cycling was performed with 1 min at 94° followed by 10 cycles of 1 min at 94°, 1 min at 60° (with the temperature reduced by 1°/cycle), and 3 min at 72°, followed by 30 cycles of 1 min at 94°, 1 min at 50°, and 3 min at 72°. Amplification products were electrophoresed on agarose gels and positive BAC clones were identified as described previously. The identity of the amplification products produced by markers LpF1-LpF4, LpHd3a, and B139776 and by the markers described in Table 2 was confirmed by direct DNA sequencing of identified BACs.

View this table: In this window In a new window   TABLE 1 Primer sequences used in BAC library screening

 

View this table: In this window In a new window   TABLE 2 Genetic map positions in L. perenne and rice of markers targeted by selected primers in the B. distachyon BAC library PCR screen

 

View this table: In this window In a new window   TABLE 5 Selected clones from B. distachyon ABR1/5 library used for physical mapping in chromosomes of related Brachypodium species, Triticale, and O. sativa

 

Preparation of root meristems:

The somatic chromosome preparations were made as described by HASTEROK et al. (2004). Briefly, whole seedlings with roots 1.0–2.0 cm long were immersed in ice-cold water for 24 hr, fixed in 3:1 (v/v) methanol:glacial acetic acid, and stored at –20°. After several washes in 0.01 M citric acid–sodium citrate buffer ("citric buffer," pH 4.8), excised roots were digested in an enzyme mixture comprising 20% (v/v) pectinase (Sigma, St. Louis), 1% (w/v) cellulase (Calbiochem, La Jolla, CA), and 1% (w/v) cellulase Onozuka R-10 (Serva) for 2 hr at 37°. Prior to squashing in a drop of 45% acetic acid, meristems were dissected out from root tips. After freezing, coverslips were removed and the preparations were postfixed in 3:1 ethanol:glacial acetic acid, dehydrated in absolute ethanol, and air dried.

Preparation of anther cells:

ABR1 and ABR5 were sown at high density in compost and vernalized for 6 weeks at 5° to ensure synchronous induction of flowering. They were then transferred to a glasshouse. For meiotic chromosome preparations, immature inflorescences (spikes) of different sizes were fixed in 3:1 ethanol:glacial acetic acid and stored at –20° until required. Because of the minute size (150–250 µm) of the anthers compared to the rest of the floret, the anthers were individually dissected and collected into a container with a citric acid–sodium citrate buffer (0.01 M, pH 4.8). Enzymatic digestion was carried out for 2 hr 15 min at 37° in a mixture of 10% (v/v) pectinase (Sigma), 0.65% (w/v) cellulase Onozuka R-10 (Serva), 0.5% (w/v) cellulase (Calbiochem), 0.15% (w/v) cytohelicase (Sigma), and 0.15% (w/v) pectolyase (Sigma) in 10 mM citric buffer (pH 4.8). After washing in citric buffer, the anthers were individually transferred onto a slide and gently homogenized in a drop of 45% acetic acid. For one preparation, 10–20 anthers of different sizes were used. The remainder of the procedure is the same as for somatic chromosome preparations. For high-resolution FISH mapping, only meiotic chromosome preparations at late zygotene and pachytene were used.

DNA probes and fluorescence in situ hybridization:

The following DNA probes were used in this study:

1. DNAs isolated from BACs were labeled with digoxigenin-11-dUTP or tetramethyl-rhodamine-5-dUTP (Roche, Indianapolis) by nick translation as described by HASTEROK et al. (2002).
   
2. A 2.3-kb ClaI subclone of the 25S rDNA coding region of A. thaliana (UNFRIED and GRUENDLER 1990) was labeled combinatorially with digoxigenin-11-dUTP and tetramethyl-rhodamine-5-dUTP by nick translation and used to detect 18S-5.8S-25S rDNA loci (45S rDNA).
   
3. Wheat clone pTa794 (GERLACH and DYER 1980) was amplified and labeled by PCR with digoxigenin-11-dUTP and used to detect 5S rDNA loci. The sequences of oligonucleotide primers and conditions for this reaction are described in detail by HASTEROK et al. (2004).
   

The FISH procedure was adopted from HASTEROK et al. (2002) with some modifications described below. The general conditions of the FISH procedure were as follows. Two kinetically different hybridization mixtures were used: (i) a low-stringency (65%) mixture consisting of inter alia 30% deionized formamide, 2x SSC, salmon sperm blocking DNA in 75–100x excess of labeled probe and 2.5–3.0 ng/µl of each DNA probe and (ii) a high-stringency (77%) mixture in which the concentration of formamide was increased to 50%. Chromosome preparations and denatured (80° for 10 min) hybridization mixtures were denatured together for 4.5 min at 70° and allowed to hybridize overnight in a humid chamber at 37°. Posthybridization washes were carried out for 10 min either in 20% deionized formamide in 2x SSC at 37° (which is equivalent to 59% stringency) or in 10% deionized formamide in 0.1x SSC at 42° (which is equivalent to 79% stringency). Lower stringency was used to localize heterologous BAC clones (i.e., those from B. distachyon library) onto targets from related species of Brachypodium as well as onto Triticale and rice. Immunodetection of digoxigenated probes was performed according to standard protocols using FITC-conjugated antidigoxigenin antibodies (Roche). The chromosomes were mounted and counterstained in Vectashield (Vector Laboratories, Burlingame, CA) containing 2.5 µg/ml of 4',6-diamidino-2-phenylindole (DAPI).

All fluorescent images were digitally captured, either using a Hamamatsu ORCA monochromatic CCD camera attached to a Zeiss Axioplan epifluorescence microscope and tinted using Wasabi software or using a Hamamatsu (Bridgewater, NJ) C5810 color CCD camera attached to an Olympus Provis AX microscope. All images were processed uniformly and superimposed using Micrografx (Corel) Picture Publisher software.

BAC library construction and quality control:

The library was constructed in two separate size-selection experiments. A total of 9100 BAC clones were "picked" into 96-well plates, and with an estimated average insert size of 88 kb/clone, the coverage is of >800 Mb. In general, slightly larger inserts were observed for BACs derived from the higher-molecular-weight gel slices (size selection 2; Table 3; Figure 1) compared to those derived from the lower-molecular-weight gel slices (size selection 1; Table 3). However, in the experiments where higher-molecular-weight gel slices of 130 kb and above were selected, the number of colonies derived was very low and a high proportion of the clones contained very short or no inserts. The library was therefore generated from the region 70–130 kb.

View this table: In this window In a new window   TABLE 3 General characteristics of B. distachyon BAC library

 

View larger version (68K): In this window In a new window Download PPT slide   FIGURE 1.— DNA from B. distachyon BAC clones was NotI restricted and the fragments were separated by PFGE. BACs were obtained by partial restriction of B. distachyon ABR5 genomic DNA with HindIII, followed by PFGE and cloning of the size-separated genomic DNA from a gel slice in the region of 100–130 kb (size selection 2 in Table 3). The average insert size of the BAC inserts in this size selection experiment is estimated to be 92.3 kb. M, -ladders (New England Biolabs and Roche); V, the position of the 7.0-kb pBeloBAC11 NotI vector fragment.

 

BAC library characterization:

To estimate the number of clones containing repetitive DNA, the library was hybridized with radioactively labeled genomic DNA of B. distachyon. BAC clones were categorized as highly (H) or moderately repetitive (M) after hybridization to the total genomic DNA probe. This provided an estimate of 2.9% of clones, which were measurably repetitive in the library. Chloroplast contamination as measured by PCR screening using primers for a number of chloroplast sequences was estimated as 3%.

Physical mapping of B. distachyon BACs targeted to rice chromosome 6:

All of the markers used in the PCR screen described in Table 4 were targeted to a contiguous region of 884 kb on rice chromosome 6 from 2,284,490 bp (LpF1) to 3,168,414 bp (LpF4) (rice 6 pseudomolecule; http://www.tigr.org; Table 4). All of these markers produced amplification products from genomic DNA derived from ABR1 and ABR5 except for B139345, which amplified only from ABR1. In addition, two other primer pairs, B29794 and B139282, failed to amplify a product from either ABR1 or ABR5 genomic DNA. The presence of a degree of conserved physical synteny between this region in rice and the identified region(s) in B. distachyon is indicated in the results of this study. Of the 13 markers that could be amplified from the BAC libraries, only 2 could not be physically associated with at least one other marker. Four markers (LpC764, B29786, B29797, and LpF3) could be associated in a single contiguous region spanning the equivalent distance of 140 kb in the rice genome and tiled by a minimum of two B. distachyon BACs (Table 4).

View this table: In this window In a new window   TABLE 4 Physical position of target sequences on rice 6 pseudomolecule

 

Chromosomal mapping of BAC clones:

Table 5 lists the physical features of selected BAC clones used in this study, together with their chromosomal map positions determined by FISH. The majority of the clones either are syntenic to the region of rice chromosome 6 described in Table 4 or are selected on the basis of genetic map position on chromosomes of L. perenne and Triticeae species (Table 2).

Of 39 BAC clones, 32 hybridize in situ to single loci in the genome of the diploid ABR1. Since all five pairs of chromosomes are identifiable on the basis of morphology and relative size (JENKINS et al. 2005), each of these single-locus clones was assigned to individual chromosomes. Furthermore, by cross-referencing the map positions of these clones to those already anchored to particular chromosome arms, it was possible to build up linkage groups of clones for every chromosome arm of the complement. In this way, each arm was designated "p" and "q" in the conventional way, although the symmetry about the centromere of chromosomes 1–3 precludes identification of actual long and short arms. Figure 2, A–F, shows the precision of the BAC "landing" on 9 of 10 chromosome arms of ABR1. It is worth emphasizing not only that coverage of all chromosome arms of the complement was achieved with 30 clones and one additional rDNA-based landmark, but also that the high resolution of BAC mapping was achieved without customary blocking with genomic or C₀t-1 DNA and is unaffected by lower stringency conditions.

View larger version (33K): In this window In a new window Download PPT slide   FIGURE 2.— Identification of B. distachyon ABR1 (2n = 2x = 10) chromosomes by dual-color FISH with BAC clones. (A) 1p, ABR1-63-E11 (red); 1q, ABR1-26-H1 (green). (B) 2p, ABR1-41-E10 (green); 2q, ABR5-1-H3 (red). (C) 3p, ABR5-33-F12 (red); 3q, ABR1-56-H6 (green). (D) 4p, ABR5-33-F2 (green); 4q, ABR5-32-C1 (red). (E) Multicolor FISH with three different BAC clones and 25S rDNA (yellow): 1q (subterminal), ABR1-41-A8 (green); 1q (interstitial), ABR1-59-F9 (red); 4q, ABR1-47-F4 (green); 5p (NOR), 25S rDNA. (F) Multicolor FISH with one BAC clone (red), 5S rDNA (green), and 25S rDNA (yellow). 4q (interstitial), 5S rDNA; 5p (NOR), 25S rDNA; 5q (subterminal), ABR1-63-E2. Color symbols in A–E describe the localization of landmarks in particular chromosome arms of ABR1 (p, short arm; q, long arm). (G) Ideogram of B. distachyon ABR1 (n = 5) chromosomes showing physical localization of all mapped BAC clones and the two auxiliary landmarks (5S and 25S rDNA). Asterisks indicate the clones from B. distachyon ABR5 library. Blue fluorescence, DAPI. Bar, 5 µm.

 
Several of the BAC clones do not map to single loci, but rather occupy pericentromeric regions or "paint" the entire chromosome complement (Table 5; Figure 4G). A representation of the physical map of all clones, together with the 25S and 5S rDNA loci, is shown in Figure 2G. Because of the small size of the somatic chromosomes of this species, it is not surprising that a number of clones map to the same chromosomal positions. Figure 3A shows two such clones colocalizing to the distal tip of chromosome 1q. However, if the resolution of mapping is enhanced through the use of longer chromosomes at zygotene of meiosis, the two loci are clearly separable (Figure 3, B and C). A similar example of improved mapping is shown in Figure 3, D–F. The closer proximity of the signals in Figure 3E compared to Figure 3B probably reflects the greater degree of chromosome compaction at the pachytene stage of meiosis.

View larger version (37K): In this window In a new window Download PPT slide   FIGURE 4.— Comparative analysis of (A, D, and G) B. distachyon ABR1 (2n = 2x = 10), (B, E, and H) ABR114 (2n = 2x = 20), (C, F, and I) ABR113 (2n = 4x = 30), and (L) Triticale (2n = 6x = 42) genomes by FISH of the B. distachyon ABR1/5 BAC clones. (A–C) ABR1-32-C1 (green), 5S rDNA (red). (D–F) ABR1-32-C1 (green); ABR5-33-F2 (red). Color symbols on A and D describe the localization of landmarks on particular chromosome arms. (G–I) ABR1-63-E6 (green). (L) ABR1-41-H4 (green). (J and K) Selected chromosomes from putative ancestral diploid species (B. distachyon ABR1 and ABR114) and an interspecific hybrid (ABR113) hybridizing with the BAC clones or a 5S ribosomal DNA probe. The chromosomes were extracted from (A and D) B. distachyon ABR1, (B and E) ABR114, and (C and F) ABR113. Bars: (A–I) 5 µm; (L) 10 µm.

 

View larger version (25K): In this window In a new window Download PPT slide   FIGURE 3.— High-resolution FISH mapping of closely linked on mitotic metaphase preparations (A and D) pairs of B. distachyon BAC clones on B. distachyon (2n = 2x = 10) zygotene (B) and pachytene (E) chromosomes. (A and B) ABR1-26-H1 (green) and ABR1-41-A8 (red) apparently colocalizing (yellow) in the distal part of the long arm of chromosome pair 1 at mitotic metaphase (A), while, on the zygotene chromosomes, ABR1-41-A8 is clearly more distal than ABR1-26-H1. (D) ABR1-56-H6 (red) and ABR1-54-D7 (green) at mitotic metaphase colocalizing (yellow) in the distal part of the long arm of chromosome pair 3 at mitotic metaphase. (E) BAC–FISH of the same clones to the pachytene chromosome reveals that ABR1-54-D7 (green) is located more distally than ABR1-56-H6. (C and F) Diagrams showing different resolution of BAC–FISH mapping on A and B and D and E. Blue fluorescence, DAPI. Bar, 5 µm.

 
Table 5 shows 30 clones that were hybridized in situ to chromosomes of related genotypes of Brachypodium, to Triticale, and to rice. ABR114 was originally classified as a cytotype of B. distachyon with 20 chromosomes (ROBERTSON 1981). The notion that ABR114 was an autotetraploid was refuted by HASTEROK et al. (2004) on the basis of its unique chromosome size and morphology and the fact that its chromosomes were not labeled by genomic DNA of ABR1. Interestingly, only 15 of the clones mapping to single loci of ABR1 map to single sites in ABR114, albeit with relatively lower signal intensity. Figure 4 shows one such clone (ABR1-32-C1) that maps together with 5S rDNA onto one arm of chromosome 4 of ABR1 (Figure 4A), which together map on a single arm of a pair of unidentifiable chromosomes of ABR114 (Figure 4B). By contrast, two clones (ABR1-32-C1 and ABR5-33-F2) map to opposite arms of chromosome 4 of ABR1 (Figure 4D), but land on two different pairs of chromosomes of ABR114 (Figure 4E). The contrasting map positions of these clones in ABR1 and ABR114 are reproduced for clarity in Figure 4, J and K, which have been assembled by extracting the relevant chromosomes from Figure 4, A, B, D, and E. The two single-locus clones (ABR1-41-E10 and ABR5-1-H3) that map to opposite arms of chromosome 2 of ABR1 (Figure 2B) also land on two different pairs of chromosomes in ABR114 (data not shown). The clone ABR1-63-E6 with a pericentromeric/dispersed distribution in ABR1 has a similar but significantly less distinct distribution in ABR114 (Figure 4, G and H).

ABR113 (Table 5) was considered to be an autohexaploid form of B. distachyon until HASTEROK et al. (2004) showed that it comprises two genomes of 10 and 20 chromosomes, each bearing close similarity to ABR1 and ABR114, respectively. The mapping data of this study largely reinforce these conclusions. All the single-locus clones of ABR1 map to the same positions and with the same intensity to the ABR1-like chromosomes of this allotetraploid. Most of the single-locus clones hybridizing to ABR114 have counterparts in ABR113 (Figure 4, C, F, J, and K). Furthermore, dispersed repeats in ABR1, such as ABR1-63-E6, depict 10 chromosomes in this hybrid and effectively "paint" one of the genomes (Figure 4I).

Despite numerous attempts, no single-locus clone was successfully hybridized to the chromosomes of either Triticale or rice, even under low-stringency conditions. Two dispersed, repetitive clones highlighted primarily the centromere in Triticale only (Table 5; Figure 4L).

This article describes the construction and exploitation of the first reported BAC library of B. distachyon and we believe this to be the smallest monocot genome for which a BAC library is available. There have been conflicting reports of genome size in B. distachyon (e.g., DRAPER et al. 2001; BENNETT and LEITCH 2005). This situation may reflect the use of nuclei from different species to produce nuclear DNA content calibration curves and, particularly, the frequent occurrence of polyploid nuclei in Arabidopsis is likely to have led to an underestimate in size in our previous study (DRAPER et al. 2001). Indeed, recent comparisons of mitotic metaphase karyotypes (data not shown) suggested that diploid (2n = 2x = 10) B. distachyon chromosomes were similar in size to those of B. sylvaticum, thus negating any suggestion (FOOTE et al. 2004) that B. distachyon may have deleted many of the genes that would have been found in other grasses. The B. distachyon BAC library will therefore provide a useful tool for grass comparative genomics to complement the existing BAC libraries of cereal and forage grass crops, including rice (ZHANG et al. 1996), bread wheat (ALLOUIS et al. 2003), durum wheat (CENCI et al. 2003), barley (YU et al. 2000), sorghum (WOO et al. 1994), maize (O'SULLIVAN et al. 2001; TOMKINS et al. 2002), and Festuca pratensis (DONNISON et al. 2005). Moreover, our investigation illustrates the relative ease with which BAC clones can be hybridized to single loci of the 10 chromosomes of B. distachyon. It is not considered likely that this efficiency is due entirely to the selection of clones on the basis of their synteny to gene-rich regions of the genomes of rice and other members of the Poaceae, since 19 of the 24 clones selected randomly from the library also "landed" at discrete loci. Rather, it is a consequence of the compactness and economy of the genome of B. distachyon, in which genes are not spaced out by large tracts of repetitive DNA as in other temperate grasses and cereals.

Clones ABR1-13-A2, ABR1-58-H2, and ABR1-59-F9 were identified by three markers as having sequences syntenic to a 69-kb section of rice chromosome 6 (Table 4). The latter two of these hybridize in situ to chromosome 1q of ABR1 (Table 5; Figure 2G). This indicates not only sequence conservation between rice and Brachypodium in this area, but also some degree of conservation of relative map positions. In addition, BAC clones ABR1-41-E4 and ABR1-43-E8 also colocalize with ABR1-58-H2 and ABR1-59-F9 on 1q and were identified by markers targeted to this same region on rice chromosome 6 (Table 4). While it was not established that ABR1-41-E4 and ABR1-43-E8 were directly contiguous with ABR1-58-H2/59-F9 or with each other in B. distachyon, this does indicate that they are located in approximately the same physical position on chromosome 1. Clone ABR1-13-A2 did not hybridize in situ to chromosome 1q but instead highlighted pericentromeric regions of all five chromosomes of the complement. Clearly, this clone contains a repetitive element that confounds its map position relative to its contigs.

Another two clones, ABR1-26-H1 and ABR1-41-E10, were identified by marker LpHd3a but found to be false positives on sequencing. End sequencing of ABR1-26-H1 showed that it had a weak correspondence with a different region of the rice 6 pseudomolecule at 12 Mb. Physical mapping of this clone in B. distachyon showed it to be associated with chromosome 1q, along with ABR1-58-H2, ABR1-59-F9, ABR1-41-E4, and ABR1-43-E8, but located subterminally rather than interstitially (Table 5). Clone ABR1-41-E10 mapped to chromosome 2p of B. distachyon ABR1 and could not be further associated with a region of the rice genome.

A previous comparative genome analysis between B. sylvaticum and rice using cross-hybridization of BAC clones on filters concentrated on clones syntenic to chromosome 9 of rice (FOOTE et al. 2004). Using BAC landing as an alternative approach in B. distachyon, we have extended this analysis and demonstrated that, excepting marker CHO15851, an STS marker mapped only in L. perenne, the primer pairs used successfully in the PCR screen described in Table 2 identified sequences associated with RFLP markers with known map positions on different chromosomes within L. perenne, Triticeae species, and rice. Interestingly, with the exception of LpCDO580, all of the markers that mapped to different chromosomes in L. perenne identified BACs that likewise hybridized to different chromosomes in B. distachyon (the BAC identified by LpCDO580 showed nonspecific hybridization in the B. distachyon genome). Conversely, all of the markers that mapped to the same chromosome in L. perenne identified BACs that hybridized to the same chromosome in B. distachyon. Additionally, if the physical positions on the same chromosome in B. distachyon of the BACs identified by the markers that map to L. perenne C5 reflect the complex conserved syntenic relationship seen between L. perenne C5 and rice C9/11/12, the implication is that there may also be conservation between B. distachyon and rice in this region (ARMSTEAD et al. 2002, 2006; JONES et al. 2002).

No single-locus clone of ABR1 hybridized in situ to the chromosomes of Triticale or rice, even under conditions of very low stringency. This is unlikely to be due in all cases to the absence of homologous sequences, since markers targeted to the rice sequence identify syntenic BACs from the Brachypodium library. It is more likely that syntenic regions in rice and Triticale are interspersed with genome-specific repeats that interfere with hybridization in situ. Subcloning of fragments of the large inserts of the BACs may be a means of circumventing this problem. Alternatively, it might be instructive to attempt a BAC "landing" on species that are phylogenetically more closely related to Brachypodium than rice, wheat, or rye, such as species of Lolium, Festuca, or Bromus. Using this approach, it may be possible to define a phylogenetic distance beyond which a heterologous BAC "landing" becomes unprofitable.

BAC "landing" also enabled further investigation into the phylogeny of the cytotypes of B. distachyon. The hybridization of single-locus clones and rDNA probes has confirmed that ABR114 is an unknown diploid species with 20 chromosomes in its own right and that ABR113 is an allotetraploid comprising two genomes that are similar to ABR1 and ABR114. Furthermore, Figure 4, A, B, and J, shows that the 5S rDNA probe and the clone ABR1-32-C1 mapping to the same chromosome arm of chromosome 4 of ABR1 land on a single pair of chromosomes in ABR114, albeit in reverse orientation to the centromere. However, by contrast, two clones (ABR1-32-C1 and ABR5-33-F2) mapping to opposite arms of chromosome 4 in ABR1 land on two different pairs of chromosomes in ABR114 (Figure 4, D, E, and K). Similarly, clones ABR1-41-E10 and ABR5-1-H3 map to opposite arms of chromosome 2 of ABR1 (Figure 2B) and land on two different pairs of chromosomes in ABR114 (data not shown). Although few clones have been compared in this way, the inference is that ABR1 and ABR114 may be related by multiple centric fission/fusion events. This would be entirely consistent with the observation that ABR1 has 10 large, mostly metacentric chromosomes, compared with 20 small, subtelocentric chromosomes of ABR114. This is not the complete story, however. In situ hybridization of ABR114 with genomic DNA of ABR1 does not label the complement to any great extent (HASTEROK et al. 2004). It is necessary, therefore, to invoke at least some sequence divergence of repetitive DNA to explain the observations.

Although the intention of this study was not to establish a detailed alignment among the genomes of B. distachyon, L. perenne, and rice, these data do demonstrate the potential of B. distachyon as a monocot comparative genomics resource. Linking and calibrating sequence data and chromosome map positions at this level of resolution is fraught with difficulties, but may be ameliorated by the careful selection of a small set of tiles from the BAC library and their "landing" on meiotic chromosomes or extended DNA fibers. A previous study (FOOTE et al. 2004) has suggested that B. sylvaticum, a related perennial species with a similar genome size, is also a useful "bridge" species in the grasses. However, the annual, self-fertile, fast-cycling B. distachyon has many biological features to recommend it as a model for future functional genomics studies (DRAPER et al. 2001).

The authors acknowledge financial support from the Royal Society (Joint Project in 2001–2002 to R.H. and G.J.) and the Biotechnology and Biological Sciences Research Council (International Scientific Interchange Scheme award to R.H. and to J.D. in 2003–2004).

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