A bacterial artificial chromosome library for barley (Hordeum vulgare L.) and the identification of clones containing putative resistance genes

Modern cultivated barley is an important cereal crop with an estimated genome size of 5000 Mb. To develop the resources for positional cloning and structural genomic analyses in barley, we constructed a bacterial artificial chromosome (BAC) library for the cultivar Morex using the cloning enzyme HindIII. The library contains 313344 clones (816 384-well plates). A random sampling of 504 clones indicated an average insert size of 106 kbp (range=30–195 kbp) and 3.4% empty vectors. Screening the colony filters for chloroplast DNA content indicated an exceptionally low 1.5% contamination with chloroplast DNA. Thus, the library provides 6.3 haploid genome equivalents allowing a >99% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT in a 4×4 double-spotted array on 22.5-cm² filters. Each set of 17 filters allows the entire library to be screened with 18432 clones represented per filter. Screening the library with 40 single copy probes identified an average 6.4 clones per probe, with a range of 1–13 clones per probe. A set of resistance-gene analog (RGA) sequences identified 121 RGA-containing BAC clones representing 20 different regions of the genome with an average of 6.1 clones per locus. Additional screening of the library with a P-loop disease resistance primer probe identified 459 positive BAC clones. These data indicate that this library is a valuable resource for structural genomic applications in barley. Received: 20 September 1999 / Accepted: 25 March 2000     

Bacterial artificial chromosome library for genome‐wide analysis of Chinese hamster ovary cells

Chinese hamster ovary (CHO) cell lines are widely used for scientific research and biotechnology. A CHO genomic bacterial artificial chromosome (BAC) library was constructed from a mouse dihydrofolate reductase (DHFR) gene‐amplified CHO DR1000L‐4N cell line for genome‐wide analysis of CHO cell lines. The CHO BAC library consisted of 122,281 clones and was expected to cover the entire CHO genome five times. A CHO chromosomal map was constructed by fluorescence in situ hybridization (FISH) imaging using BAC clones as hybridization probes (BAC‐FISH). Thirteen BAC‐FISH marker clones were necessary to identify all the 20 individual chromosomes in a DHFR‐deficient CHO DG44 cell line because of the aneuploidy of the cell line. To determine the genomic structure of the exogenous Dhfr amplicon, a 165‐kb DNA region containing exogenous Dhfr was cloned from the BAC library using high‐density replica (HDR) filters and Southern blot analysis. The nucleotide sequence analysis revealed a novel genomic structure in which the vector sequence containing Dhfr was sandwiched by long inverted sequences of the CHO genome. Biotechnol. Bioeng. 2009; 104: 986–994. © 2009 Wiley Periodicals, Inc.     

Gene content and density in banana (<Emphasis Type="Italic">Musa acuminata</Emphasis>) as revealed by genomic sequencing of BAC clones

The complete sequence of Musa acuminata bacterial artificial chromosome (BAC) clones is presented and, consequently, the first analysis of the banana genome organization. One clone (MuH9) is 82,723 bp long with an overall G+C content of 38.2%. Twelve putative protein-coding sequences were identified, representing a gene density of one per 6.9 kb, which is slightly less than that previously reported for Arabidopsis but similar to rice. One coding sequence was identified as a partial M. acuminata malate synthase, while the remaining sequences showed a similarity to predicted or hypothetical proteins identified in genome sequence data. A second BAC clone (MuG9) is 73,268 bp long with an overall G+C content of 38.5%. Only seven putative coding regions were discovered, representing a gene density of only one gene per 10.5 kb, which is strikingly lower than that of the first BAC. One coding sequence showed significant homology to the soybean ribonucleotide reductase (large subunit). A transition point between coding regions and repeated sequences was found at approximately 45 kb, separating the coding upstream BAC end from its downstream end that mainly contained transposon-like sequences and regions similar to known repetitive sequences of M. acuminata. This gene organization resembles Gramineae genome sequences, where genes are clustered in gene-rich regions separated by gene-poor DNA containing abundant transposons.Communicated by J.S. Heslop-Harrison     

Genome-wide BAC-end sequencing of <Emphasis Type="Italic">Cucumis melo</Emphasis> using two BAC libraries

Background

Although melon (Cucumis melo L.) is an economically important fruit crop, no genome-wide sequence information is openly available at the current time. We therefore sequenced BAC-ends representing a total of 33,024 clones, half of them from a previously described melon BAC library generated with restriction endonucleases and the remainder from a new random-shear BAC library.

Results

We generated a total of 47,140 high-quality BAC-end sequences (BES), 91.7% of which were paired-BES. Both libraries were assembled independently and then cross-assembled to obtain a final set of 33,372 non-redundant, high-quality sequences. These were grouped into 6,411 contigs (4.5 Mb) and 26,961 non-assembled BES (14.4 Mb), representing ~4.2% of the melon genome. The sequences were used to screen genomic databases, identifying 7,198 simple sequence repeats (corresponding to one microsatellite every 2.6 kb) and 2,484 additional repeats of which 95.9% represented transposable elements. The sequences were also used to screen expressed sequence tag (EST) databases, revealing 11,372 BES that were homologous to ESTs. This suggests that ~30% of the melon genome consists of coding DNA. We observed regions of microsynteny between melon paired-BES and six other dicotyledonous plant genomes.

Conclusion

The analysis of nearly 50,000 BES from two complementary genomic libraries covered ~4.2% of the melon genome, providing insight into properties such as microsatellite and transposable element distribution, and the percentage of coding DNA. The observed synteny between melon paired-BES and six other plant genomes showed that useful comparative genomic data can be derived through large scale BAC-end sequencing by anchoring a small proportion of the melon genome to other sequenced genomes.

     

Construction of a bacterial artificial chromosome library containing large Eco RI and Hin dIII genomic fragments of lettuce

 Existing bacterial artificial chromosome (BAC) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5 and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLP^TM markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA. Received: 5 August 1996 / Accepted: 23 August 1996     

A major invasion of transposable elements accounts for the large size of the <Emphasis Type="Italic">Blumeria graminis</Emphasis> f.sp. <Emphasis Type="Italic">tritici</Emphasis> genome

Powdery mildew of wheat (Triticum aestivum L.) is caused by the ascomycete fungus Blumeria graminis f.sp. tritici. Genomic approaches open new ways to study the biology of this obligate biotrophic pathogen. We started the analysis of the Bg tritici genome with the low-pass sequencing of its genome using the 454 technology and the construction of the first genomic bacterial artificial chromosome (BAC) library for this fungus. High-coverage contigs were assembled with the 454 reads. They allowed the characterization of 56 transposable elements and the establishment of the Blumeria repeat database. The BAC library contains 12,288 clones with an average insert size of 115 kb, which represents a maximum of 7.5-fold genome coverage. Sequencing of the BAC ends generated 12.6 Mb of random sequence representative of the genome. Analysis of BAC-end sequences revealed a massive invasion of transposable elements accounting for at least 85% of the genome. This explains the unusually large size of this genome which we estimate to be at least 174 Mb, based on a large-scale physical map constructed through the fingerprinting of the BAC library. Our study represents a crucial step in the perspective of the determination and study of the whole Bg tritici genome sequence.     

Physical map and gene survey of the Ochrobactrum anthropi genome using bacterial artificial chromosome contigs

Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This report describes research aimed at determining the genome structure of Ochrobactrum anthropi, an opportunistic human pathogen that has potential applications in biodegradation of hazardous organic compounds. A BAC library for O. anthropi was constructed that provides a 70-fold genome coverage based on an estimated genome size of 4.8 Mb. The library contains 3072 clones with an average insert size of 112 kb. High-density colony filters of the library were made, and a physical map of the genome was constructed using a hybridization without replacement strategy. In addition, 1536 BAC clones were fingerprinted with HindIII and analyzed using IMAGE and Fingerprint Contig software (FPC, Sanger Centre, U.K.). The FPC results supported the hybridization data, resulting in the formation of two major contigs representing the two major replicons of the O. anthropi genome. After determining a reduced tiling path, 138 BAC ends from the reduced tile were sequenced for a preliminary gene survey. A search of the public databases with the BLASTX algorithm resulted in 77 strong hits (E-value < 0.001), of which 89% showed similarity to a wide variety of prokaryotic genes. These results provide a contig-based physical map to assist the cloning of important genomic regions and the potential sequencing of the O. anthropi genome.     

Efficient gene targeting and removal of foreign DNA by homologous recombination in the picoeukaryote Ostreococcus

With fewer than 8000 genes and a minimalist cellular organization, the green picoalga Ostreococcus tauri is one of the simplest photosynthetic eukaryotes. Ostreococcus tauri contains many plant‐specific genes but exhibits a very low gene redundancy. The haploid genome is extremely dense with few repeated sequences and rare transposons. Thanks to the implementation of genetic transformation and vectors for inducible overexpression/knockdown this picoeukaryotic alga has emerged in recent years as a model organism for functional genomics analyses and systems biology. Here we report the development of an efficient gene targeting technique which we use to knock out the nitrate reductase and ferritin genes and to knock in a luciferase reporter in frame to the ferritin native protein. Furthermore, we show that the frequency of insertion by homologous recombination is greatly enhanced when the transgene is designed to replace an existing genomic insertion. We propose that a natural mechanism based on homologous recombination may operate to remove inserted DNA sequences from the genome.     

A BAC library of <Emphasis Type="Italic">Beta vulgaris</Emphasis> L. for the targeted isolation of centromeric DNA and molecular cytogenetics of <Emphasis Type="Italic">Beta</Emphasis> species

We constructed a sugar beet (Beta vulgaris) bacterial artificial chromosome (BAC) library of the monosomic addition line PAT2. This chromosomal mutant carries a single additional chromosome fragment (minichromosome) derived from the wild beet Beta patellaris. Restriction analysis of the mutant line by pulsed-field gel electrophoresis was used to determine HindIII as a suitable enzyme for partial digestion of genomic DNA to generate large-insert fragments which were cloned into the vector pCC1. The library consists of 36,096 clones with an average insert size of 120 kb, and 2.2% of the clones contain mitochondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents 5.7 genome equivalents providing the probability of 99.67% that any sequence of the PAT2 genome can be found in the library. Hybridization to high-density filters was used to isolate 89 BACs containing arrays of the centromere-associated satellite repeats pTS5 and pTS4.1. Using the identified BAC clones in fluorescent in situ hybridization experiments with PAT2 and Beta patellaris chromosome spreads their wild beet origin and centromeric localization was demonstrated. Multi-colour FISH with differently labelled satellite repeats pTS5 and pTS4.1 was used to investigate the large-scale organization of the centromere of the PAT2 minichromosome in detail. FISH studies showed that the centromeric satellite pTS5 is flanked on both sides by pTS4.1 arrays and the arms of the minichromosome are terminated by the Arabidopsis-type telomeric sequences. FISH with a BAC, selected from high-density filters after hybridization with an RFLP marker of the genetic linkage group I, demonstrated that it is feasible to correlate genetic linkage groups with chromosomes. Therefore, the PAT2 BAC library provides a useful tool for the characterization of Beta centromeres and a valuable resource for sugar beet genome analysis.     

A bacterial artificial chromosome library for sugarcane

Modern cultivated sugarcane is a complex aneuploid polyploid with an estimated genome size of 3000 Mb. Although most traits in sugarcane show complex inheritance, a rust locus showing monogenic inheritance has been documented. In order to facilitate cloning of the rust locus, we have constructed a bacterial artificial chromosome (BAC) library for the cultivar R570. The library contains 103,296 clones providing 4.5 sugarcane genome equivalents. A random sampling of 240 clones indicated an average insert size of 130 kb allowing a 98% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT in a 4 × 4 double-spotted array on 22.5-cm² filters. Each set of five filters provides a genome coverage of 4x with 18,432 clones represented per filter. Screening of the library with three different barley chloroplast gene probes indicated an exceptionally low chloroplast DNA content of less than 1%. To demonstrate the library’s potential for map-based cloning, single-copy RFLP sugarcane mapping probes anchored to nine different linkage groups and three different gene probes were used to screen the library. The number of positive hybridization signals resulting from each probe ranged from 8 to 60. After determining addresses of the signals, clones were evaluated for insert size and HindIII-fingerprinted. The fingerprints were then used to determine clone relationships and assemble contigs. For comparison with other monocot genomes, sugarcane RFLP probes were also used to screen a Sorghum bicolor BAC library and two rice BAC libraries. The rice and sorghum BAC clones were characterized for insert size and fingerprinted, and the results compared to sugarcane. The library was screened with a rust resistance RFLP marker and candidate BAC clones were subjected to RFLP fragment matching to identify those corresponding to the same genomic region as the rust gene. Received: 12 September 1998 / Accepted: 12 March 1999     

Construction of a BAC Library for a Defoliating Insect-Resistant Soybean and Identification of Candidate Clones Using a Novel Approach

Positional cloning of an insect-resistance quantitative trait locus (QTL) requires the construction of a large-insert genomic DNA library from insect-resistant genotypes. To facilitate cloning of a major defoliating insect-resistance QTL on linkage group M of the soybean genetic map, a bacterial artificial chromosome (BAC) library for PI 229358 was constructed and characterized. The HindIII BAC library contains 55,296 clones with an average insert size 131 kb. This library represents a 6-fold soybean haploid genome equivalents, allowing a 99.8% probability of recovering any specific sequence of interest in soybean. BAC filters were screened with a genomic DNA probe Sat_258sc2 obtained through genome walking from flanking sequences of a simple sequence repeat (SSR) marker, Sat_258, which links to the insect-resistance QTL. Thirteen BAC clones were identified positive for Sat_258sc2, and two of them were confirmed to carry Sat_258. The results suggest that this library is useful in positional cloning of the major insect-resistance QTL, and the approach presented here can be used to screen a BAC library for a SSR marker without requiring the creation of BAC pools.     

Chromosome‐level genome assembly of a cyprinid fish Onychostoma macrolepis by integration of nanopore sequencing,Bionano and Hi‐C technology

Onychostoma macrolepis is an emerging commercial cyprinid fish species. It is a model system for studies of sexual dimorphism and genome evolution. Here, we report the chromosome‐level assembly of the O.macrolepis genome obtained from the integration of nanopore long‐read sequencing with physical maps produced using Bionano and Hi‐C technology. A total of 87.9 Gb of nanopore sequence provided approximately 100‐fold coverage of the genome. The preliminary genome assembly was 883.2 Mb in size with a contig N50 size of 11.2 Mb. The 969 corrected contigs obtained from Bionano optical mapping were assembled into 853 scaffolds and produced an assembly of 886.5 Mb with a scaffold N50 of 16.5 Mb. Finally, using the Hi‐C data, 881.3 Mb (99.4% of genome) in 526 scaffolds were anchored and oriented in 25 chromosomes ranging in size from 25.27 to 56.49 Mb. In total, 24,770 protein‐coding genes were predicted in the genome, and ~96.85% of the genes were functionally annotated. The annotated assembly contains 93.3% complete genes from the BUSCO reference set. In addition, we identified 409 Mb (46.23% of the genome) of repetitive sequence, and 11,213 non‐coding RNAs, in the genome. Evolutionary analysis revealed that O. macrolepis diverged from common carp approximately 24.25 million years ago. The chromosomes of O. macrolepis showed an unambiguous correspondence to the chromosomes of zebrafish. The high‐quality genome assembled in this work provides a valuable genomic resource for further biological and evolutionary studies of O. macrolepis.     

Construction and characterization of a Coffea canephora BAC library to study the organization of sucrose biosynthesis genes

The first bacterial artificial chromosome (BAC) library of Robusta coffee (Coffea canephora) was constructed, with the aim of developing molecular resources to study the genome structure and evolution of this perennial crop. Clone 126, which is highly productive and confers good technological and organoleptic qualities of beverage, was chosen for development of this library. The BAC library contains 55,296 clones, with an average insert size of 135 Kb per plasmid, therefore representing theoretically nine haploid genome equivalents of C. canephora. Its validation was achieved with a set of 13 genetically anchored single-copy and 4 duplicated RFLP probes and yielded on average 9 BAC clones per probe. Screening of this BAC library was also carried out with partial cDNA probes coding for enzymes of sugar metabolism like invertases and sucrose synthase, with the aim of characterizing the organization and promoter structure of this important class of genes. It was shown that genes for both cell wall and vacuolar forms of invertases were probably unique in the Robusta genome whereas sucrose synthase was encoded by at least two genes. One of them (CcSUS1) was cloned and sequenced, showing that our BAC library is a valuable tool to rapidly identify genes of agronomic interest or linked to cup quality in C. canephora.     

Targeted isolation,sequence analysis,and physical mapping of nonTIR NBS-LRR genes in soybean

Most cloned plant disease resistance genes (R-genes) code for proteins belonging to the nucleotide binding site (NBS) leucine-rich repeat (LRR) superfamily. NBS-LRRs can be divided into two classes based on the presence of a TIR domain (Toll and interleukin receptor-like sequence) or a coiled coil motif (nonTIR) in their N-terminus. We used conserved motifs specific to nonTIR-NBS-LRR sequences in a targeted PCR approach to generate nearly 50 genomic soybean sequences with strong homology to known resistance gene analogs (RGAs) of the nonTIR class. Phylogenetic analysis classified these sequences into four main subclasses. A representative clone from each subclass was used for genetic mapping, bacterial artificial chromosome (BAC) library screening, and construction of RGA-containing BAC contigs. Of the 14 RGAs that could be mapped genetically, 12 localized to a 25-cM region of soybean linkage group F already known to contain several classical disease resistance loci. A majority of the genomic region encompassing the RGAs was physically isolated in eight BAC contigs, together spanning more than 1 Mb of genomic sequence with at least 12 RGA copies. Phylogenetic and sequence analysis, together with genetic and physical mapping, provided insights into the genome organization and evolution of this large cluster of soybean RGAs. Received: 8 May 2001 / Accepted: 30 June 2001     

Development of a BAC library for yellow-poplar (Liriodendron tulipifera) and the identification of genes associated with flower development and lignin biosynthesis

Liriodendron tulipifera L., a member of the Magnoliaceae, occupies an important phylogenetic position as a basal angiosperm that has retained numerous putatively ancestral morphological characters, and thus has often been used in studies of the evolution of flowering plants and of specific gene families. However, genomic resources for these early branching angiosperm lineages are very limited. In this study, we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from L. tulipifera. Flow cytometry estimates that this nuclear genome is approximately 1,802 Mbp per haploid genome (±16 SD). The BAC library contains 73,728 clones, a 4.8-fold genome coverage, with an average insert size of 117 kb, a chloroplast DNA content of 0.2%, and little to no bacterial sequences nor empty vector content clones. As a test of the utility of this BAC library, we screened the library with six single/low-copy genic probes. We obtained at least two positive clones for each gene and confirmed the clones by DNA sequencing. A total of 182 paired end sequences were obtained from 96 of the BAC clones. Using BLAST searches, we found that 25% of the BAC end sequences were similar to DNA sequences in GenBank. Of these, 68% shared sequence with transposable elements and 25% with genes from other taxa. This result closely reflected the content of random sequences obtained from a small insert genomic library for L. tulipifera, indicating that the BAC library construction process was not biased. The first genomic DNA sequences for Liriodendron genes are also reported. All the Liriodendron genomic sequences described in this paper have been deposited in the GenBank data library. The end sequences from shotgun genomic clones and BAC clones are under accession DU169330–DU169684. Partial sequences of Gigantea, Frigida, LEAFY, cinnamyl alcohol dehydrogenase, 4-coumarate:CoA ligase, and phenylalanine ammonia-lyase genes are under accession DQ223429–DQ223434. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Construction of a bacterial artificial chromosome (BAC) library for citrus and identification of BAC contigs containing resistance gene candidates

A BAC library was constructed from the genomic DNA of an intergeneric Citrus and Poncirus hybrid. The library consists of 24,576 clones with an average insert size of 115 kb, representing approximately seven haploid genome equivalents and is able to give a greater than 99% probability of isolating single-copy citrus DNA sequences from this library. High-density colony hybridization-based library screening was performed using DNA markers linked to the citrus tristeza virus (CTV) resistance gene and citrus disease resistance gene candidate (RGC) sequences. Between four and eight clones were isolated with each of the CTV resistance gene-linked markers, which agrees with the library’s predicted genome coverage. Three hundred and twenty-two clones were identified using 13 previously cloned citrus RGC sequences as probes in library screening. One to four fragments in each BAC were shown to hybridize with RGC sequences. One hundred and nine of the RGC BAC clones were fingerprinted using a sequencing gel-based procedure. From the fingerprints, 25 contigs were assembled, each having a size of 120–250 kb and consisting of 2–11 clones. These results indicate that the library is a useful resource for BAC contig construction and molecular isolation of disease resistance genes. Received: 22 May 2000 / Accepted: 25 September 2000     

Cross-species bacterial artificial chromosome (BAC) library screening via overgo-based hybridization and BAC-contig mapping of a yield enhancement quantitative trait locus (QTL) yld1.1 in the Malaysian wild rice Oryza rufipogon

The construction of BAC-contig physical maps is an important step towards a partial or ultimate genome sequence analysis. Here, we describe our initial efforts to apply an overgo approach to screen a BAC library of the Malaysian wild rice species, Oryza rufipogon. Overgo design is based on repetitive element masking and sequence uniqueness, and uses short probes (approximately 40 bp), making this method highly efficient and specific. Pairs of 24-bp oligos that contain an 8-bp overlap were developed from the publicly available genomic sequences of the cultivated rice, O. sativa, to generate 20 overgo probes for a 1-Mb region that encompasses a yield enhancement QTL yld1.1 in O. rufipogon. The advantages of a high similarity in melting temperature, hybridization kinetics and specific activities of overgos further enabled a pooling strategy for library screening by filter hybridization. Two pools of ten overgos each were hybridized to high-density filters representing the O. rufipogon genomic BAC library. These screening tests succeeded in providing 69 PCR-verified positive hits from a total of 23,040 BAC clones of the entire O. rufipogon library. A minimal tilling path of clones was generated to contribute to a fully covered BAC-contig map of the targeted 1-Mb region. The developed protocol for overgo design based on O. sativa sequences as a comparative genomic framework, and the pooled overgo hybridization screening technique are suitable means for high-resolution physical mapping and the identification of BAC candidates for sequencing.     

A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance

We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73728 clones stored in 192384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, and less than 4% of the clones do not contain inserts. Ninety percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 1115 Mb, library coverage is 9 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.85%). Library screening with three genomic RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI 437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analyzed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analyzed in three categories: (1) contigs of BAC clones which hybridized to forward subtracted cDNAs, (2) contigs of BAC clones which hybridized to reverse subtracted cDNAs, and (3) contigs of BAC clones which hybridized to both forward and reverse subtracted cDNAs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a pathogenic attack.