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Proc Natl Acad Sci U S A. Dec 27, 2005; 102(52): 19243–19248.
Published online Dec 15, 2005. doi:  10.1073/pnas.0509473102
PMCID: PMC1323192
Plant Biology

Analysis and mapping of randomly chosen bacterial artificial chromosome clones from hexaploid bread wheat

Abstract

The current view of wheat genome composition is that genes are compartmentalized into gene-rich and gene-poor regions. This model can be tested by analyzing randomly selected bacterial artificial chromosome (BAC) clones for gene content, followed by placement of these BACs onto physical and genetic maps. Map localization could be difficult for BACs that consist entirely of repeated elements. We therefore developed a technique where repeat junctions are used to generate unique markers. Four BAC clones from hexaploid wheat variety Chinese Spring were randomly selected and sequenced at 4- to 6-fold redundancy. About 50% of the BAC sequences corresponded to previously identified repeats, mainly LTR-retrotransposons, whereas most of the remaining DNA consisted of sequences with unknown origin or function. The average gene content was <1%, although each BAC contained one or two identified genes. Repeat boundaries were amplified and used to map each clone to a chromosome arm. Extrapolation from wheat-rice comparative knowledge suggests that three of the four BAC clones originate from “gene-rich” regions of the wheat genome. Nevertheless, because these BACs carry only a single gene (two BACs) or two genes (one BAC), the predicted gene density is ≈1 gene per 75 kb, which is considerably lower than previously estimated gene densities (one gene per 5-20 kb) for gene-rich regions in wheat. This analysis of randomly selected wheat BAC clones suggests that genes are more evenly distributed in wheat than previously believed and substantiates the need for large-scale random BAC sequencing to determine wheat genome organization.

Keywords: gene density, genome organization, repeat boundaries, repeat markers, Triticum aestivum

Now that sequencing of the rice genome has been nearly completed (1-3) and sequencing of the maize genome is in progress (4, 5), consideration of whether to comprehensively sequence other cereal genomes is warranted (6). Bread wheat ranks third in world production, after maize and rice. Its allohexaploid genome, however, is 40 times larger than that of rice and 6 times that of maize (7). In maize, sequencing efforts to date have concentrated on the gene-rich fraction of the genome (4, 5). Gene contigs can be ordered by using end sequences of methylation-spanning linker library clones, which consist of blocks of repetitive DNA flanked on either side by genic regions (8), by superimposing the contigs on a low-redundancy sequence of the entire genome or anchoring them to a genetic map. It is estimated that the gene discovery ability of these combined technologies is significantly >95% (9). Alternative strategies are based on the fact that genes are not evenly distributed over a genome (10-13). In Lotus japonicus, which has a 500-Mb genome, only bacterial artificial clones that contain ESTs are being sequenced (14, 15). The level of gene discovery provided by this approach depends on the EST coverage and the genome organization.

Sample sequence analysis of a few thousand randomly selected clones with inserts from Aegilops tauschii, the D genome donor of bread wheat, has shown that its nuclear DNA contains ≈91.6% repetitive sequences (16). How these repeats are associated with each other and with genes is not well understood. The little information we have to date on sequence interspersion in wheat has been gained primarily through sequencing of individual bacterial artificial chromosomes (BACs) containing genes of agronomic interest (17-23) and through large-scale physical mapping of ESTs to chromosome regions defined by deletions (“bins”; ref. 24). Most BACs preselected to contain at least one gene have been shown to be composed of one or two small gene islands that are separated by 5- to 150-kb blocks of repetitive DNA, primarily retroelements (17, 18, 20). The mapping of ESTs to sets of overlapping deletion lines further suggests that gene-containing BACs are clustered in the genome to form gene-rich regions that can be cytologically defined (24). A similar study, but using a larger number of deletion lines and combining the data across the three homoeologous genomes, has reported that 29% of the genome contains 94% of the genes, with 60% of the genes being concentrated in only 11% of the genome (25). Assuming that this is an accurate representation of the gene distribution in wheat, identification of 94% of the genes would require sequencing of some 5,000 Mb of DNA of the hexaploid wheat genome with a BAC by BAC approach. This is twice the size of the maize genome.

To make an informed decision on the best strategy to sequence the wheat genome, more hard data are needed on the distribution of genes. This can be best accomplished by sequencing a random selection of wheat BACs (26). One useful, but challenging, addition to such a project would be to link the BAC clones to previously established regions of high or low gene density. Based on the current perception of the organization of the wheat genome, more than half of the randomly selected BACs would contain only repeats, which are difficult to map, and no genes. This study develops the amplification of unique repeat boundaries as markers for BAC mapping, using sequences identified on four randomly chosen BAC clones from the hexaploid wheat variety Chinese Spring. Linking of these BACs to chromosome regions with known EST densities suggests that gene densities in “gene-rich” regions are considerably lower, and that gene distribution in wheat is more even, than calculated in previous publications.

Materials and Methods

BAC Clone Sequencing. Four BACs were chosen randomly from among 1,200,000 hexaploid wheat (Triticum aestivum L. cv. Chinese Spring) BAC clones (27). To reduce the chance that duplicate clones would be selected, the four clones were taken from different plates. Shotgun libraries for each of these four BACs were constructed as described (28). A total of 384 subclones were sequenced from both directions by using ABI PRISM BigDye Terminator Chemistry (Applied BioSystems, Foster, CA) and run on an ABI3730xl capillary sequencer. Base calling and quality assessment were done by using phred (29), and reads were assembled with phrap. The resulting data yielded 4- to 6-fold average sequence redundancy across the four BACs. The contigs for each BAC were ordered by using consed (30). phred, phrap, and consed were used under the default parameters, as described (28-31). Unordered contigs were concatenated at the end of each of the four “working draft” sequences. The sequences from BACs TA350B2, TA574A4, TA1353M1 and TA1426L2 have been deposited in GenBank under accession nos. AY772734, AY772735, AY772732, and AY772733, respectively.

Sequence Analysis. The gene prediction program fgenesh, with the monocot (maize, rice, wheat, and barley) training set (www.softberry.com), was used to predict genes. The entire sequence, including the gene candidates predicted by fgenesh, was subsequently investigated by cross_match searches against the Gramineae repeat databases (www.tigr.org/tdb/e2k1/plant.repeats; http://wheat.pw.usda.gov/ITMI/Repeats/index.shtml; http://data.genomics.purdue.edu/~pmiguel/projects/retros). Predicted genes that did not match retroelements or other transposons were used as queries in blastx searches against the GenBank protein database. They were considered “real” genes if they detected homology at an expect value of <E-10 in any species that was not a member of the Triticeae. Our previous studies have shown that this is a rigorous criterion for gene identification (31). The requirement of a low expect value excludes most transposable elements that are misannotated as genes, partly because these mobile DNAs evolve more rapidly in sequence than do the other coding sequences in plant nuclear genomes.

Primer Design. To establish the origin of BACs that consist mainly of repetitive DNA, primers were designed so that one of the primers or the amplification product spanned a repeat boundary (Fig. 1). Primers were designed across four, five, one, and four boundaries, respectively, for BACs TA350B2, TA574A4, TA1353M1, and TA1426L2. In addition, two sets of nested primers were designed for BAC TA1426L2. If genes were present, these were used as queries against the EST_others section of GenBank to establish the presence and position of introns. Where possible, primers against genes were made to span an intron. This process increased the likelihood that the A, B, and D genome products would be distinguishable in either length or base composition, and hence could be separated by single-strand conformation polymorphism (SSCP). The primers that were used to generate the BAC fragments that were mapped are given in Table 1.

Fig. 1.
Primer design across a repeat boundary on BAC TA1353M1 and an agarose gel showing the mapping of the corresponding amplification product on a set of 21 NT lines. The absence of the fragment in the line nullisomic for 3B (N3B) allocates BAC TA1353M1 to ...
Table 1.
Primer sequences that were used for mapping, the type of sequence in which they are located, and the chromosomal location of the amplification products

Mapping. DNA from nullisomic-tetrasomic (NT) and ditelosomic lines was kindly provided by P. Stephenson and J. Beales, John Innes Centre, Norwich, United Kingdom. PCRs were carried out in a total volume of 20 μl of 1× PCR buffer containing 100 ng of template DNA, 1.5 mM MgCl2, 200 μM dNTPs, 500 nM forward primer, 500 nM reverse primer, and 0.8 units of Taq DNA polymerase (Promega). PCR conditions consisted of an initial denaturation step of 3 min at 94°C, followed by 45 cycles of 94°C for 30 s, touchdown from 62°C to 55°C at a rate of 0.7°C per cycle, 72°C for 1 min, and a final extension of 3 min at 72°C. For nested PCR, conditions were as above but 1 μl of the previous amplification reaction mixture was used as DNA template. Amplification products were separated on 1.2% agarose gels and visualized after staining with ethidium bromide or separated by SSCP on 0.5× mutation detection enhancement (MDE) gels (Cambrex, East Rutherford, NJ) (32). The MDE gels were run overnight at a constant power of 8 W and silver-stained.

Results and Discussion

BAC Composition. Four BAC clones were randomly picked from four different plates of the 1.2 million clone library containing the Chinese Spring wheat hexaploid genome inserted into vector pIndigoBAC5 at 9× redundancy (27). For each BAC, 384 subclones with 3- to 5-kb inserts were selected for sequencing. This approach results in a 4-6× average coverage for BACs in the size range from 75,000 to 125,000 bp. After assembly, 117 kb of sequence with 13 gaps organized into three scaffolds was obtained for BAC TA350B2, 100 kb of sequence with 20 gaps in eight scaffolds for TA574A4, 75 kb of sequence with 9 gaps in two scaffolds for TA1353M1, and 124 kb of sequence with 20 gaps in seven scaffolds for TA1426L2 (see Fig. 3, which is published as supporting information on the PNAS web site). Previous reconstruction of sequence coverage in BACs that were completely sequenced from wheat, maize, and rice indicate that 3× coverage is sufficient to identify >95% of all genes and abundant repeats (W. Ramakrishna and J.L.B., unpublished observations). Hence, the 4-6× coverage in this study was sufficient to establish the repeat and genic content of each BAC (Table 2).

Table 2.
Sequence composition of four randomly selected BACs from hexaploid wheat cultivar Chinese Spring

The compositions of BACs TA350B2, TA574A4, and TA1426L2 were very similar. Each BAC carried one (TA350B2 and TA574A4) or two genes (TA1426L2), and ≈50% of the sequence consisted of previously characterized repeats, mainly members of LTR retrotransposon families (Table 2 and Table 5, which is published as supporting information on the PNAS web site). Given the many gaps derived from the low redundancy sequence across each of these BACs, efforts were not made to find new transposons or other repeat families by structural criteria (33, 34). The LTR retrotransposons found most frequently on these four BACs were Fatima, Sabrina, WIS, and Sukkula, with a respective four, four, five, and six copies, none of them solo LTRs. The most numerous DNA transposons identified were Jorge and Caspar (Table 5), CACTA elements that had been previously seen to be the most abundant large DNA elements in wheat (16, 34).

The remaining 50% of the BAC sequence had no hits against the databases used and remains uncharacterized. It is likely that much of this uncharacterized sequence is comprised of repeat families that have not yet been cataloged. BAC TA1353M1 also carried one gene but, in contrast to the other three BACs, only one LTR retrotransposon could be identified, so that ≈80% of the DNA remained uncharacterized (Tables (Tables22 and 5). The gene and DNA transposon content in TA1353M1 is similar to that of the other three BACs sequenced. We therefore hypothesize that TA1353M1 carries numerous, as yet uncharacterized, transposable elements. As previously noted in other studies of wheat and other members of the Triticeae, CACTA DNA transposons were found to be much more abundant on all four of these BACs than in those from other well characterized plant genomes, namely Arabidopsis, rice, and maize (34, 35).

A total of 71 genes were predicted by fgenesh to be present across the four BACs (Table 3). However, 54 matched transposable elements and only four fulfilled our stringent criterion for a “true” gene (31). This criterion is homology at the protein level to a predicted or known gene from a species outside the Triticeae tribe at an E value of <10-10 (Table 4). One predicted gene on BAC TA1426L2 showed homology to a rice hypothetical protein at an expect value of 3 E-5. Although this E value was higher than our cut-off value of 10-10, this gene was considered a true gene because (i) the gene structure predicted by fgenesh represented only the 3′ end of the gene, (ii) blastx analysis of the complete wheat gene derived from a wheat EST had homology to the rice hypothetical protein at an E value < 10-10, and (iii) this gene had homology to both Triticeae and non-Triticeae (sugarcane) ESTs. Furthermore, the rice genes orthologous to these two true genes on wheat BAC TA1426L2 were both located on the same rice BAC clone. The other 13 predicted gene models did not exhibit strong homology with any known gene or EST in rice, maize, or other monocotyledonous or dicotyledonous plant. Our previous experience with annotating rice and other cereal genomes (31, 36) has shown that all or most of these 13 candidates will eventually be characterized as portions of as-yet-uncharacterized transposons. Hence, we predict that these BACs encode five protein-encoding genes. The single “certified” genes on BACs TA574A4 and TA1353M1, and one of the genes on TA1426L2, are complete. The second gene on TA1426L2 and the gene on TA350B2, both of which are near the end of a contig, have been predicted to be complete genes by fgenesh. Comparative data, however, suggest that the TA1426L2 gene lacks the 5′ end and the TA350B2 gene lacks an intact 3′ end.

Table 3.
Predicted genes and their EST matches in cereals
Table 4.
Predicted genes in four sequenced BACs from Chinese Spring

Mapping. Because we wanted to develop a technology that could be used to locate on the genetic map any sequenced wheat BAC, even those that contained zero genes and perhaps no single copy DNA, we decided to see whether repeat boundaries could be used as unique PCR products. For virtually all plant transposable element insertions that have been investigated, the insertion site has been unique, although often into another repetitive DNA (e.g., another transposable element) (37). Hence, in theory, the use of primers that flank such an insertion point should yield a unique product, even when each primer is specific to a different DNA repeat. If successfully amplified, these markers should have a very good chance to detect polymorphism because the transposable element complement of any genome tends to be its most dynamic component.

blast alignments between the wheat BAC sequences and previously identified repeats were manually inspected to precisely locate the repeat boundaries/junctions. Primers were designed so that either one of the primers or the amplification product spanned a boundary. Primers were tested for amplification in Chinese Spring, the variety that was the DNA source for the sequenced BAC clones. Of the 14 primer sets made across repeat boundaries, two failed to amplify, nine produced a single amplification product, and two generated two or three bands as assessed by separation of the reaction products on a 1.2% agarose gel (data not shown). Primer pairs that produced the strongest signals were subsequently used for amplification in a set of 21 Chinese Spring NT lines. A NT line lacks one chromosome pair and its absence is compensated by the presence of an extra pair of homoeologous chromosomes. Such lines are available for each of the 21 wheat chromosomes (38). For BAC TA350B2, the primer sets TA350B2_18F1/R1 and F2/R2 that spanned different boundaries between a Fatima LTR retrotransposon and a Jorge CACTA transposon amplified a single product in all NT lines with the exception of the line nullisomic for 1B, indicating that the amplification product, and thus BAC TA350B2, originated from chromosome 1B (Table 1). BAC TA350B2 was further mapped to the long arm of 1B by using the ditelosomic lines 1BS and 1BL that carry the short and long arms of chromosome 1B, respectively. In a similar manner, BAC TA1353M1 was shown to originate from chromosome arm 3BL by using primer set TA1353M1_5F1/R1 that amplified the junction between two CACTA elements belonging to the Sherlock and TAT-1 families (Fig. 1). The chromosomal location of this BAC clone was confirmed by using a primer set designed against the single gene present in TA1353M1 (Table 1).

The three primer sets designed against BAC TA1426L2 that produced amplification products in Chinese Spring also generated fragments in all NT lines. To enhance the specificity of the reaction, nested PCR was carried out across two repeat boundaries, but failed to reveal the origin of BAC TA1426L2. In the course of the amplification experiments for BACs TA350B2 and TA1353M1, it had been noted that the absence of the “perfect” priming sites often resulted in the amplification of a secondary fragment (data not shown). To test whether a secondary fragment of similar molecular weight as the primary fragment had been generated in one of the NT lines, amplification products generated by three primer sets were separated by SSCP gel electrophoresis, a technology that detects tiny differences in size and nucleotide composition. All primer sets now unambiguously located BAC TA1426L2 to chromosome 5B (Fig. 2).

Fig. 2.
Separation of the amplification products generated by primer set TA1426L2_16F2/R2 in the group 5 NT lines by SSCP. A comparison of the banding patterns in the N5A, N5B, and N5D lines shows that specific amplification products are absent in the N5B line, ...

BAC TA574A4 was mapped to chromosome arm 3DL by using primers designed across the boundary of a Caspar CACTA element and a Wham LTR retrotransposon (Table 1). The 3D location was confirmed by using primer sets against the identified EST. Primers designed against exons often amplify fragments of similar molecular weight from two or three of the homoeologous wheat genomes. The homoeologous genes usually differ in a few base pairs, which allow their separation by SSCP. Primer set TA574A4_10F2/R2 indeed produced amplification products from both chromosomes 3B and 3D. TA574A4_10F1/R1, however, was specific for 3D and unambiguously located BAC TA574A4.

These experiments demonstrate that it is possible to map any BAC clone, regardless of the gene content. In fact, where primers designed against ESTs often amplify from multiple homoeoloci, allowing allocation of the marker only to a chromosome group in a polyploid rather than an individual chromosome, boundaries between repeats, even between high-copy-number LTR retrotransposons, are generally unique features in the genome that can be exploited to establish the chromosomal origin of a sequenced BAC clone. Although we only located BAC clones to chromosome arms, a more precise map position can be obtained by using either chromosome deletion lines (39) or segregation analysis. Physical mapping onto the deletion lines requires only intergenomic polymorphisms (between the A, B, and D genomes). The presence of intragenomic polymorphisms, however, is a prerequisite for genetic mapping. In maize, the organization of repeats in a genome is variety dependent (40, 41). If this is also the case in wheat, it should be possible to genetically map repeat boundaries in progeny derived from crosses in which one of the parents is the source of the BAC DNA.

Wheat Genome Organization. Previously reported BAC analyses, and consequently any conclusions regarding gene densities in wheat, have been based on BACs that had been preselected to contain a gene of agronomic importance (17-23, 42, 43). The sequence of randomly selected BACs, on the other hand, provides unbiased information on the organization of the wheat genome, at least to the degree in which the library itself provides an unbiased representation of the genome. To provide a comprehensive and statistically significant description of the entire hexaploid wheat genome, we have estimated that a minimum of ≈60 randomly chosen BACs would need to be sequenced from each of the A, B, and D genomes (unpublished observations). Hence, this preliminary study is not meant to describe the entire wheat genome, but only the properties of a few randomly chosen BACs that were subsequently mapped to known locations.

Although none of the four BACs in our study were preselected to contain a gene, they each carried a single (three BACs) or two (one BAC) well supported gene candidate(s). To correlate the observed gene density with previously established high and low gene-density regions of the wheat genome, the sequenced wheat BACs were located in silico to cytologically defined chromosome bins (39). We first identified the rice BAC/P1 artificial chromosome (PAC) clone that had the highest homology to the gene identified on each of the wheat BACs. A putative orthologous origin of the homologous wheat and rice genes was supported if they were located on syntenic chromosomes (44), which was the case for three of the wheat genes. The gene on BAC TA574A4, which originated from wheat chromosome 3D, identified a putative ortholog on rice chromosome 1 PAC P0503C12. Similarly, TA1353M1 (located on wheat chromosome 3B) had putative orthology to rice chromosome 1 PAC P0459B04 and TA1426L2 (5B) to rice chromosome 3 BAC OSJNBa0057G07. The highest homology of the gene on TA350B2 (1B) was with a region on rice chromosome 7 BAC P0045F02. As rice chromosome 7 is nonsyntenic to the wheat group 1 chromosomes (44), no in silico map position was obtained for TA350B2. The bin position of the other three rice BAC/PAC clones was derived from the available knowledge on the colinearity between deletion-mapped wheat ESTs and the rice genomic sequence (ref. 45 and www.tigr.org/tdb/e2k1/tae1/wheat_synteny.shtml). TA574A4 was located to deletion interval 3L-0.42-0.50, TA1353M1 to 3L-0.81-1.00, and TA1426L2 to 5L-0.76-0.79. All three bins represent regions of high gene density (46, 47). It thus appears that at least three of the randomly selected BACs are derived from gene-rich regions. Combined, these three BACs comprise 300 kb, yielding a calculated density of one gene per 75 kb. Bin 3L-0.42-0.50 comprises 8% of the long arm and corresponds to ≈40 Mb of DNA. Based on the amount of mapped ESTs in bin 3L-0.42-0.50 and a total gene number for wheat of 150,000, we expect, on average, a gene density of one gene per 62 kb in this gene-rich bin. The expected gene density in bin 3L-0.81-1.00 is one gene per 107 kb. One gene per BAC, as obtained in our study, is in line with these expectations. Interestingly, the gene density of these regions is <2-fold higher than the value of one gene per 113 kb expected for a random distribution of ≈150,000 genes in the 17,000-Mb hexaploid wheat genome.

The lower gene density on the randomly selected BACs in this study, compared with preselected gene-containing BACs, can be partly explained by the fact that most wheat BACs sequenced so far carried disease resistance and storage protein genes (18-20, 23, 43). These genes often occur in clusters, leading to an overestimation of overall gene densities in gene-rich regions of the wheat genome. Gene density around other genes such as the vernalization response gene Vrn-Am1 in Triticum monococcum, a close relative of T. aestivum, which is also located in a gene-rich portion of the genome, is in the expected range of one to two genes per BAC (21). These data suggest that the previous estimates of one gene per 5-20 kb in the area of disease resistance and storage protein genes do not accurately represent the organization of the gene-rich fraction of the genome. Sequencing and bin mapping of a larger number of randomly selected BAC clones is needed to establish in detail the overall organization of the wheat genome.

Supplementary Material

Supporting Information:

Acknowledgments

We thank G. Moore (John Innes Centre, Norwich, United Kingdom) for the wheat BAC clones; P. Stephenson and J. Beales for donation of DNA of the NT and ditelosomic lines; B. Keller and M. Sorrells for their helpful comments concerning this manuscript; and T. Thomas and M. Estep for expert technical assistance. This research was supported by the Doris and Norman Giles Professorship (J.L.B.).

Notes

Author contributions: K.M.D. and J.L.B. designed research; K.M.D., J.M., A.C.P., and L.H.P. performed research; L.H.P. contributed new reagents/analytic tools; K.M.D., J.M., A.C.P., and J.L.B. analyzed data; and K.M.D., J.M., A.C.P., L.H.P., and J.L.B. wrote the paper.

Conflict of interest statement: No conflicts declared.

Abbreviations: BAC, bacterial artificial chromosome; SSCP, single-strand conformation polymorphism; NT, nullisomic-tetrasomic.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AY772732-AY772735).

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