An Integrated Map of Arabidopsis thaliana for Functional Analysis of Its Genome Sequence

Corresponding author: Hong-Bin Zhang, Department of Soil and Crop Sciences and Crop Biotechnology Center, 2123 TAMUS, Texas A&M University, College Station, TX 77843-2123., hbz7049{at}pop.tamu.edu (E-mail)

Communicating editor: C. S. GASSER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

The genome of the model plant species Arabidopsis thaliana has recently been sequenced. To accelerate its current genome research, we developed a whole-genome, BAC/BIBAC-based, integrated physical, genetic, and sequence map of the A. thaliana ecotype Columbia. This new map was constructed from the clones of a new plant-transformation-competent BIBAC library and is integrated with the existing sequence map. The clones were restriction fingerprinted by DNA sequencing gel-based electrophoresis, assembled into contigs, and anchored to an existing genetic map. The map consists of 194 BAC/BIBAC contigs, spanning 126 Mb of the 130-Mb Arabidopsis genome. A total of 120 contigs, spanning 114 Mb, were anchored to the chromosomes of Arabidopsis. Accuracy of the integrated map was verified using the existing physical and sequence maps and numerous DNA markers. Integration of the new map with the sequence map has enabled gap closure of the sequence map and will facilitate functional analysis of the genome sequence. The method used here has been demonstrated to be sufficient for whole-genome physical mapping from large-insert random bacterial clones and thus is applicable to rapid development of whole-genome physical maps for other species.

ARABIDOPSIS thaliana is a model system for genomic studies of plant species (MEINKE et al. 1998 ). To facilitate genome research of the species, chromosome- and genome-wide physical maps were developed from large-insert yeast artificial chromosome or bacterial artificial chromosome (BAC) libraries (HWANG et al. 1991 ; SCHMIDT et al. 1995 ; ZACHGO et al. 1996 ; CANILLERI et al. 1998 ; MARRA et al. 1999 ; MOZO et al. 1999 ). Using these maps as frameworks, the genome of A. thaliana was sequenced (ARABIDOPSIS GENOME INITIATIVE 2000). However, a number of gaps still exist in the sequence map, especially in the heterochromatic regions surrounding the centromeres of its chromosomes. These gaps are intractable to closure by the conventional chromosome walking approach using the ends of BACs adjacent to the gaps as probes because of repetitive sequences. This piecemeal approach is also time-consuming. Therefore, to rapidly close most, if not all, of the gaps in the sequence map, it is necessary to construct a whole-genome physical map from a new DNA library that complements the Texas A&M University (TAMU) and the Institut für Genbiologische Forschung (IGF) BAC libraries of the sequence map and to integrate the new map with the existing sequence map.

Sequence analysis has indicated that the genome of A. thaliana contains 25,498 genes. However, the functions of >90% of the predicted genes remain to be characterized experimentally (ARABIDOPSIS GENOME INITIATIVE 2000). Experimental determination of the functions of these genes and related sequences has been targeted as a goal for the coming decade (SOMERVILLE and DANGL 2000 ). To this end, several methods have been developed, including T-DNA-based (AZPIROZ-LEEHAN and FELDMANN 1997 ; SUSSMAN et al. 2000 ) or transposon-based (MARTIENSSEN 1998 ) gene tagging, DNA microarray or gene chip analysis (SCHENA et al. 1995 ; DESPREZ et al. 1998 ; RUAN et al. 1998 ), and genetic transformation (FELDMANN and MARKS 1987 ; CHANG et al. 1994 ; LIU et al. 1999 ). Because transformation of A. thaliana via Agrobacterium is efficient and can be accomplished without tissue culture procedures (FELDMANN and MARKS 1987 ; KONCZ et al. 1989 ; BECHTOLD et al. 1993 ), this method facilitates functional analysis of the genome sequence. Therefore, a whole-genome, binary, clone-based map that is integrated with the existing sequence map will be significant for accelerated experimental determination of the functions of every segment of the genome sequence. However, none of the Arabidopsis physical and sequence maps developed to date contains clones that can be directly transformed in plants. The TAMU and IGF BAC clones of the existing physical and sequence maps were cloned in general DNA cloning BAC vectors (CHOI et al. 1995 ; MOZO et al. 1998 ), which are incompetent for direct transformation in plants via A. tumefaciens. For functional analysis of the genome sequence by genetic transformation, these clones must be subcloned into a plant-transformation-competent binary vector. However, the process of subcloning is often tedious. Furthermore, the goals of Arabidopsis genome research are to identify every gene and determine the function(s) of every gene of this model species. Although individual clones for functional analysis by genetic transformation could be isolated from a plant-transformation-competent binary bacterial artificial chromosome (BIBAC) library using the corresponding sequences as probes, this process would be inefficient for isolation of a large number of BIBAC clones for functional analysis of different segments of the genome sequence. Therefore, it is desirable to develop a physical map from a large-insert BIBAC library that is competent for plant transformation and to integrate it with the existing sequence map.

In this study, we developed a whole-genome integrated physical and genetic map of the A. thaliana ecotype Columbia from a new plant-transformation-competent BIBAC library and integrated it with the existing sequence map of the species. The integration of the new map with the sequence map will significantly accelerate genome research of the model species in many aspects. The complementarity of the new BIBAC library to the source libraries of the sequence map and the competency of the BIBACs for plant transformation will facilitate gap closure of the sequence map and large-scale functional analysis of the genome sequence. Furthermore, because the new map was constructed using a DNA sequencing gel-based fingerprinting method (TAO et al. 2001 ) that differs from those used for construction of the existing physical maps of Arabidopsis (MARRA et al. 1999 ; MOZO et al. 1999 ), it may provide a tool to further verify the existing physical and sequence maps in which errors have been recently reported (STUPAR et al. 2001 ). This study has also demonstrated that the DNA sequencing gel-based fingerprinting method is powerful for rapid development of whole-genome physical maps from large-insert random bacterial clones.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

BAC and BIBAC libraries:
A new plant-transformation-competent binary library (the clones are hereafter referred as to BIBACs; Y.-L. CHANG, K. MEKSEM, H.-W. CHUANG, C. SCHEURING and H.-B. ZHANG, unpublished data) and the TAMU (CHOI et al. 1995 ) and IGF (MOZO et al. 1998 ) BAC libraries of the A. thaliana ecotype Columbia were used to develop the integrated physical map. The average insert sizes of the BIBAC library and the TAMU and IGF BAC libraries are 110, 100, and 100 kb, respectively. These BAC and BIBAC libraries are publicly available at the GENEfinder Genomic Resources (formerly the Texas A&M BAC Center; http://hbz.tamu.edu).

The TAMU and IGF BAC libraries are the source libraries of the existing physical and sequence maps of A. thaliana (MARRA et al. 1999 ; MOZO et al. 1999 ; ARABIDOPSIS GENOME INITIATIVE 2000). The BIBAC library was cloned in the bacterial P1-based binary vector pCLD04541, which was designed for Agrobacterium-mediated transformation in plants (JONES et al. 1992 ; TAO and ZHANG 1998 ). The ability of BIBACs to transform plants via Agrobacterium (JONES et al. 1992 ; BECHTOLD et al. 1993 ; BENT et al. 1994 ; WU et al. 2000 ; Y.-L. CHANG, K. MEKSEM, H.-W. CHUANG, C. SCHEURING and H.-B. ZHANG, unpublished data) will facilitate functional analysis of the genome sequence by genetic transformation. Furthermore, the BIBAC library was constructed with a restriction enzyme (BamHI) differing from those used for the TAMU (HindIII; CHOI et al. 1995 ) and IGF (EcoRI; MOZO et al. 1998 ) BAC libraries. The BamHI sites are G/C-rich, whereas the HindIII and EcoRI sites are A/T rich. These differences were expected to allow cloning of the regions that are not represented in the existing BAC libraries and thus to close the gaps in the existing Arabidopsis sequence map.

Fingerprinting and contig assembly:
BAC and BIBAC DNA were isolated and fingerprinted according to TAO et al. 2001 with modifications in which [³³P]dATP was used to label the digested BAC DNA fragments and 3.5% (w/v) denaturing DNA sequencing gels were used to fractionate the DNA fragments. The fingerprints were scanned into image files using a UMAX Mirage D-16L scanner and edited using Image 4.0 of the FingerPrinted Contig (FPC) package (SODERLUND et al. 1997 ). The resolvable bands of each fingerprint ranged from 58 to 2225 nucleotides in size. Considering the lower resolution (probably >1 nucleotide) of the bands in the higher-molecular-weight portion at the top of each autoradiograph, only the fragments ranging from 58 to 773 bases were used for contig assembly, on average, 36 bands per fingerprint. The bands derived from the BAC vectors were deleted from the data files, while the bands of the BIBAC vector pCLD04541 were not present in the fingerprint range (e.g., see Fig 1). The clones that had no inserts or produced four or fewer bands were excluded during fingerprint image editing because the number of bands per fingerprint was insufficient to be included for contig assembly. Consequently, 9389 of the clones, being equivalent to 7.2 x Arabidopsis haploid genomes, were used to assemble the physical map contigs. To construct the physical map, we first assembled automatic contigs using the FPC3.8 program (SODERLUND et al. 1997 ) and then merged the automatic contigs or singletons with the automatic contigs using a lower comparison stringency. The automatic contigs were assembled at tolerance 2, cutoff 10^-12, DiffBury 0.1, and MinBands 8. The mergence of contig-contig and singleton-contig was conducted at tolerance 2, cutoff 10^-7, DiffBury 0.1, and MinBands 8.

View larger version (55K): In this window In a new window Download PPT slide   Figure 1. Autoradiograph of the Arabidopsis BIBAC fingerprints used for contig assembly of the new BAC/BIBAC-based map. DNA was isolated, double digested with HindIII/HaeIII, end labeled with [33P]dATP, electrophoresed on a 3.5% (w/v) denaturing DNA sequencing gel, and exposed to an X-ray film. DNA/Sau 3AI markers labeled with [33P]dATP were used in the first lane and every seventh lane thereafter. Note that the bands derived from the cloning vector of the new BIBAC library, pCLD04541 (JONES et al. 1992 ; TAO and ZHANG 1998 ), were not present in the fingerprint range, which facilitated the map contig assembly. However, one or two bands derived from the cloning vectors of TAMU and IGF BACs were present in the range and were deleted manually from the fingerprint image files. The fragments ranging from 58 to 773 bases were used for contig assembly.

Library screening:
The source clones of the physical map were double spotted on Hybond N+ membrane in a format of 3 x 3 by using the Biomek 2000 Robotic Workstation (Beckman, Fullerton, CA), and the high-density colony filters were prepared according to ZHANG et al. 1996 . To determine the genome origin of the BAC/BIBAC contigs, three chloroplast DNA probes, adhA, psbA, and rbcL, which are 50 kb apart on the chloroplast genome, were used to screen the source BAC and BIBAC libraries, and the IGF BACs derived from mitochondrial DNA were used to search the database of the new physical map. To verify the accuracy of the contigs and anchor them to Arabidopsis genetic maps, 77 restriction fragment length polymorphism (RFLP) markers were selected from the Arabidopsis genetic map (LIU et al. 1996 ; see Table 2), obtained from the Arabidopsis Biological Resource Center (Ohio State University), and used as probes to hybridize the high-density BAC and BIBAC filters. The probes were prepared using the PCR Dig-Probe synthesis kit as described by its manufacturer (Roche Molecular Biochemicals, Indianapolis). In addition, nine Arabidopsis cDNAs were selected from the Arabidopsis expressed sequence tagged (EST) set obtained from the Arabidopsis Biological Resource Center (Ohio State University) and positioned to the integrated map by colony hybridization.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Fingerprinting the BAC and BIBAC clones:
To construct the whole-genome physical map that is integrated with the existing sequence map (ARABIDOPSIS GENOME INITIATIVE 2000), we selected 1536 IGF BACs (4 384-well plates), 3072 TAMU BACs (8 384-well plates), and 6144 BIBACs (16 384-well plates) from their libraries. The 10,752 BAC and BIBAC clones were fingerprinted on 224 autoradiographs using the DNA sequencing gel-based restriction fingerprinting method (TAO et al. 2001 ). Fig 1 shows an autoradiograph of the BAC and BIBAC fingerprints.

Assembling the BAC/BIBAC map:
We scanned the clone fingerprints into image files and edited with the Image program of the FPC package (SULSTON et al. 1988 ; SODERLUND et al. 1997 ). During editing (see MATERIALS AND METHODS), 1363 clones were deleted from the data files because they had no inserts or produced four or fewer bands in their fingerprints, which were insufficient to be included in contig assembly. As a result, the data from 9389 clones with an average of 36 bands per clone were used to assemble contigs with the FPC program (SODERLUND et al. 1997 ). Of the 9389 clones (7.2 x genome equivalents), the clones equivalent to 2.1 x haploid genomes were from the TAMU BAC library, the clones equivalent to 1.0 x haploid genomes were from the IGF BAC library, and the clones equivalent to 4.1 x haploid genomes were from the new BIBAC library. A total of 196 contigs were assembled, each consisting of at least 2 clones (Table 1 and Fig 4), whereas 279 clones remained as singletons due to the insufficient numbers of bands (<10 bands) in their fingerprints.

View larger version (50K): In this window In a new window Download PPT slide   Figure 2. Comparison of a BAC/BIBAC contig (ctg1026) of the integrated map developed in this study (A) vs. the corresponding BAC contig of the AGI sequence map (B; ARABIDOPSIS GENOME INITIATIVE 2000) anchored to chromosome 1. The clones highlighted in green are the positive clones of the DNA marker mi425. The IGF and TAMU BAC clones shared by these two contigs are highlighted in blue. Note that the content and order of the clones in the two contigs are identical, indicating that the contigs assembled from the fingerprints generated by the DNA sequencing gel-based restriction fingerprinting method are accurate.

View larger version (64K): In this window In a new window Download PPT slide   Figure 3. BAC/BIBAC contig of the Arabidopsis BAC/BIBAC-based, integrated genetic, physical, and sequence map (ctg4011 in Table 1). (A) Integration of the new map with the existing physical and sequence maps of A. thaliana (http://www.arabidopsis.org) and distribution of TAMU BACs, IGF BACs, and BIBACs in the map. The contig (ctg4011) contains 230 clones and has a length of 1084 unique bands, being equivalent to 3255 kb (see Table 1). The clones prefixed with letter T were from the TAMU BAC library, the clones prefixed with F from the IGF BAC library, and the clones prefixed with B from the new BIBAC library (see http://hbz.tamu.edu). Each clone is specified with a library letter (T, F, or B), plate number (two-digit number), row letter, and column number (two-digit number). For instance, the clone B07M01 was from the new BIBAC library, plate 7, row M, and column 1. (B) Accuracy verification and anchoring of the BAC/BIBAC contigs to the Arabidopsis chromosomes. The contig 4011 was anchored to chromosome 4 by seven linked-DNA markers (mi465, mi128, mi279, mi260, mi112, mi330, and mi32) mapped to linkage group 4 of the Arabidopsis genetic map (LIU et al. 1996 ). The clones highlighted in blue indicate the positive clones of each marker below and above them. Note that all positive clones of each marker were located to a single location of the contig and the order of the seven markers in the contig is completely the same as that in the genetic map (see Fig 4). Asterisk (*) indicates a parent clone that covers one or more clones.

View larger version (48K): In this window In a new window Download PPT slide   Figure 4. The BAC/BIBAC-based, integrated genetic, physical, and sequence map of A. thaliana ecotype Columbia. Since the clones of the map equivalent to 3.1 x Arabidopsis haploid genomes were from the TAMU and IGF BAC libraries used in the sequence map (http://www.arabidopsis.org), the new map is integrated with the sequence map (see Fig 3). The contigs containing DNA markers were anchored to the chromosomes of their origin using the screening results of the contig BACs and BIBACs with the DNA markers. The contigs not containing DNA markers were anchored to the chromosomes of their origin by using the database of the TAMU and IGF BAC-based maps of the IGF (MOZO et al. 1999 ) and the AGI (ARABIDOPSIS GENOME INITIATIVE 2000). The collective length of the contigs anchored to each chromosome is given below its physical map (PM) in mega base pairs (Mb) in parentheses. The sizes of gaps between neighboring contigs were not determined in this study; most of the neighboring contigs may overlap even though the extent of their overlaps could not be detected by the FPC program under the conditions used in this study. Although the orders of the four DNA markers mi19, mi467, mi357, and mi358, highlighted in red on the BAC/BIBAC-based physical map, are different from those on the genetic map (GM; LIU et al. 1996 ), they are consistent with their orders on the physical map of the IGF and the AGI (http://www.arabidopsis.org).

Origin of the 196 contigs was investigated by colony hybridization using chloroplast DNA as probes and by analyzing the mitochondrial DNA-derived clones of the IGF library (MOZO et al. 1998 ) against the BAC/BIBAC contigs. Contig 1 (ctg1 in Table 1) was shown to derive from chloroplast DNA. It consists of 161 clones and was estimated to be 162 kb in length. The size of the contig is close to the 153-kb chloroplast genome size (PALMER et al. 1994 ). Contig 2 (ctg2 in Table 1) was shown to derive from the mitochondrial DNA. It consists of 108 clones and was estimated to be 564 kb in length. The length of the contig is much greater than the size of either the cytoplasmic mitochondrial genome (372 kb; KLEIN et al. 1994 ) or the mtDNA inserted into the nuclear genome revealed by sequencing (270 kb; LIN et al. 1999 ), but close to the size of the mtDNA inserted into the nuclear genome revealed by fiber-fluorescence in situ hybridization (618 ± 42 kb; STUPAR et al. 2001 ). STUPAR et al. 2001 demonstrated that the size of the mtDNA inserted into the nuclear genome was underestimated in the sequence map (LIN et al. 1999 ) due to errors of the physical and sequence map assembly. This result indicated that it is necessary to further verify the existing physical and sequence maps using an approach differing from those used in the development of the existing maps. The 194 remaining contigs were derived from nuclear DNA (Table 1). The FPC program showed that these 194 contigs consisted of 42,119 unique bands, each band representing a 3.0-kb fragment. Therefore, the nuclear DNA clone contigs were estimated to span >126 Mb in length.

Verifying the map:
To test the accuracy of the new map, we compared the BAC/BIBAC contigs constructed in this study with the BAC contigs of the existing physical (MOZO et al. 1999 ) and sequence (ARABIDOPSIS GENOME INITIATIVE 2000; http://www.arabidopsis.org) maps using the TAMU and IGF BACs shared between the contigs. The comparison showed that 95% of the new BAC/BIBAC contigs were consistent with the contigs of the existing physical and sequence maps in both clone content and order (Fig 2), but 5% of the contigs were different. To further verify the accuracy of the new BAC/BIBAC contigs, we screened them with 77 DNA markers selected from the genetic map (LIU et al. 1996 ) and nine cDNA clones selected from the Arabidopsis EST set. The results showed that all positive clones of every marker or cDNA probe used were located at a restricted fragment of a single contig (see Table 2 and Fig 3). This indicates that the contigs assembled in this study are accurate. The cause of the 5% difference between the contigs of the new map and the existing physical and sequence maps needs to be further investigated as errors were recently reported in the existing physical and sequence maps (STUPAR et al. 2001 ). We have also investigated the orders of markers in the contigs that contained three or more of the DNA markers. The result showed that they were consistent with those in the genetic map (see Fig 4), except for four DNA markers (mi19, mi467, mi357, and mi358). To explain this inconsistency, we further investigated the orders of the four DNA markers in the existing physical and sequence maps (MOZO et al. 1999 ; http://www.arabidopsis.org). The investigation showed that the orders of the four DNA markers in the new physical map were consistent with their orders in the existing physical and sequence maps, indicating that these DNA markers might be mismapped in the genetic map. All above results strongly indicate that the BAC/BIBAC contigs constructed in this study are accurate and that the fingerprinting method used is reliable for whole-genome physical mapping from random BACs and/or BIBACs.

Anchoring the physical map contigs to the genetic map:
To anchor the BAC/BIBAC contigs to a public genetic map of A. thaliana, we used the above screening results of the contig BACs and BIBACs with the 77 mapped DNA markers and the database of the TAMU and IGF BAC-based maps of the IGF (MOZO et al. 1999 ) and the Arabidopsis Genome Initiative (AGI; ARABIDOPSIS GENOME INITIATIVE 2000). Of the 194 nuclear DNA clone contigs, 120 were anchored to the five chromosomes of Arabidopsis (Fig 4 and Table 2). These 120 contigs collectively span 114 Mb in length. Of them, 27 were anchored to chromosome 1, spanning 31.5 Mb in length; 20 to chromosome 2, spanning 18.2 Mb; 22 to chromosome 3, spanning 22.5 Mb; 17 to chromosome 4, spanning 17.0 Mb; and 34 to chromosome 5, spanning 25.2 Mb (see Table 1 and Fig 4).

Potential applications of the new integrated map for accelerated genome research of Arabidopsis:
This new integrated map has provided a platform for accelerated genome research of Arabidopsis in many aspects. To test the utility of the integrated map for gap closure in the sequence map, we attempted to close four gaps in the sequence map using the new map. One of the gaps was between clones T2P3 and F2G19 in the sequence map of chromosome 1, and three gaps were between ctg714-ctg719-ctg731-ctg11 in the sequence map of Arabidopsis chromosome 3 (http://genome.wustl.edu/gsc/arab/arabidopsis.html; communicated with Dr. Christopher Town, The Institute for Genome Research). The TAMU and IGF BACs in the contigs were used to search the new BAC/BIBAC contigs. One contig (ctg1024) was identified from the new map to span the gap between T2P3 and F2G19 by two BIBACs (B05G22 and B09C04). The two BIBACs have been used to close the gap of the existing sequence map (C. TOWN, personal communication). Similarly, we searched the contigs of the new map that span the three gaps between ctg714-ctg719-ctg731-ctg11 in chromosome 3. As a result, from the new map we identified three contigs, ctg3010 (810 kb), ctg3011 (345 kb), and ctg3012 (1308 kb). To further determine whether ctg3011 and ctg3012 overlap, we further analyzed the fingerprints at the contig ends under less comparison stringency and screened the BACs and BIBACs of the new map using one IGF BAC clone (F03O21) at a ctg3012 end as a probe. As a result, eight positive BIBAC clones at one of the ctg3011 ends were identified. The hybridization and fingerprint analysis results suggested that the ctg3011 and ctg3012 overlapped and thus were merged. The merged contig (1618 kb) seems to span the gaps between ctg719, ctg731, and ctg11. Therefore, of the four gaps in the AGI sequence map, three were likely to be closed using the new map. Whether the gap between ctg714 and ctg719 in the sequence map can be closed by ctg3010 and ctg3011 remains to be determined. Similarly, other gaps in the existing sequence map could also be closed using the new map.

Experimental determination of the function of the genes and related sequences predicted by the genome sequence analysis of A. thaliana will be a significant challenge. Since A. thaliana can be readily transformed via Agrobacterium (FELDMANN and MARKS 1987 ; KONCZ et al. 1989 ; BECHTOLD et al. 1993 ), genetic transformation and subsequently transgenic plant analysis will provide an alternative tool for experimental determination of the functions of the predicted genes and related sequences in the genome sequence map. The binary vector pCLD04541 used for the new BIBAC library was designed for plant transformation via Agrobacterium (JONES et al. 1992 ). It has been widely used in Arabidopsis genetic complementation studies (e.g., BENT et al. 1994 ; http://www.jic.bbsrc.ac.uk/staff/ian-bancroft/vectorspage/htm). Although further investigation will be needed to transform large DNA fragments using this vector in Arabidopsis and other plant species, it has been shown recently that a 135-kb clone of Brassica DNA in pCLD04541 was stable in Agrobacterium (WU et al. 2000 ) and transformed into Brassica (Y.-Z. WU and Y.-P. ZHANG, personal communication). We transformed a 120-kb clone of soybean DNA in the vector into A. thaliana by the vacuum-infiltration method (BECHTOLD et al. 1993 ; Y.-L. CHANG, K. MEKSEM, H.-W. CHUANG, C. SCHEURING and H.-B. ZHANG, unpublished data). Furthermore, using the same vacuum-infiltration method and a similar vector, LIU et al. 1999 successfully transformed a DNA fragment of 80 kb into A. thaliana and showed that the transformation efficiency was not affected substantially by the sizes of introduced T-DNA. By transforming two clones of 75 and 80 kb carrying the SGR1 locus into Arabidopsis, LIU et al. 1999 were able to complement the sgr1 mutant and thus identify the functional sequence of the SGR1 gene. Transformation of DNA fragments of 150 kb via Agrobacterium was also documented in tobacco (HAMILTON et al. 1996 ) and tomato (HAMILTON et al. 1999 ). These studies have indicated that high-molecular-weight DNA could be transformed into plants via Agrobacterium. Since the BAC/BIBAC-based map developed here is integrated with the existing sequence map, the BIBAC sequences could be deduced from their overlapping BACs and used for large-scale functional analysis of the genome sequence by genetic transformation.

In addition to their utility in gap closure and functional analysis of the Arabidopsis genome sequence, the new integrated map and fingerprint database have provided a platform for numerous other studies of not only A. thaliana, but also of many other plant species. We have already received a number of inquiries, including those from the laboratories of the AGI. These studies include gap closure in the sequence map (see above), isolation of genes by positional cloning, identification of the functional sequence of centromeres, studies of gene regulation, engineering of a cluster of genes at a locus, and comparative genomics research between A. thaliana and crop plants. Since errors have recently been identified in the existing sequence map (STUPAR et al. 2001 ), this new integrated map may provide a tool to identify and correct the errors in the sequence map because it was developed with a method different from those used for development of the existing maps. Furthermore, this study, along with the recent development of the whole-genome physical map of rice (TAO et al. 2001 ), has demonstrated that DNA sequence electrophoresis-based restriction fingerprint analysis is a reliable and high-throughput method for rapid genome-wide physical mapping of large, complex genomes from large-insert random bacterial clones.

Accessing the integrated map:
The integrated map and the new BIBAC library have been posted at http://hbz.tamu.edu (Physical Mapping-Arab Map) and made available to the public. Users can access the map using any of the following approaches: clone-FPC hitting; clone-graphic contig map; clone-fingerprint map; contig no.-graphic contig map; or marker/EST-positive clones-contig/PFC hit/ fingerprint matches. The contigs, clones, and libraries can be requested at http://hbz.tamu.edu-BAC Library-Library List.

	FOOTNOTES

¹ These authors contributed equally to this study.

	ACKNOWLEDGMENTS

We thank Dr. Christopher Town for kindly providing the gap information in the Arabidopsis sequence map of AGI. We thank Drs. Huey-Wen Chuang and David A. Lightfoot for critically reading the manuscript. This study was supported in part by Texas Agricultural Experiment Station (8536-203104) and the Texas Higher Education Coordinating Board (999902-042).

Manuscript received July 11, 2001; Accepted for publication August 13, 2001.

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