Melon bacterial artificial chromosome (BAC) library construction using improved methods and identification of clones linked to the locus conferring resistance to melon Fusarium wilt (Fom-2).

Utilizing improved methods, two bacterial artificial chromosome (BAC) libraries were constructed for the multidisease-resistant line of melon MR-1. The HindIII library consists of 177 microtiter plates in a 384-well format, while the EcoRI library consists of 222 microtiter plates. Approximately 95.6% of the HindIII library clones contain nuclear DNA inserts with an average size of 118 kb, providing a coverage of 15.4 genome equivalents. Similarly, 96% of the EcoRI library clones contain nuclear DNA inserts with an average size of 114 kb, providing a coverage of 18.7 genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. High-density filters were screened with two genetic markers FM and AM that co-segregate with Fom-2, a gene conferring resistance to races 0 and 1 of Fusarium wilt. Fourteen and 18 candidate BAC clones were identified for the FM and AM probes, respectively, from the HindIII library, while 34 were identified for the AM probe from filters A, B, and C of the EcoRI library.     

Bacterial artificial chromosome-based physical map of Gibberella zeae (Fusarium graminearum).

Fusarium graminearum is the primary causal pathogen of Fusarium head blight of wheat and barley. To accelerate genomic analysis of F. graminearum, we developed a bacterial artificial chromosome (BAC)-based physical map and integrated it with the genome sequence and genetic map. One BAC library, developed in the HindIII restriction enzyme site, consists of 4608 clones with an insert size of approximately 107 kb and covers about 13.5 genome equivalents. The other library, developed in the BamHI restriction enzyme site, consists of 3072 clones with an insert size of approximately 95 kb and covers about 8.0 genome equivalents. We fingerprinted 2688 clones from the HindIII library and 1536 clones from the BamHI library and developed a physical map of F. graminearum consisting of 26 contigs covering 39.2 Mb. Comparison of our map with the F. graminearum genome sequence showed that the size of our physical map is equivalent to the 36.1 Mb of the genome sequence. We used 31 sequence-based genetic markers, randomly spaced throughout the genome, to integrate the physical map with the genetic map. We also end-sequenced 17 BamHI BAC clones and identified 4 clones that spanned gaps in the genome sequence. Our new integrated map is highly reliable and useful for a variety of genomics studies.     

Structural characterization of Brachypodium genome and its syntenic relationship with rice and wheat

Brachypodium distachyon (Brachypodium) has been recently recognized as an emerging model system for both comparative and functional genomics in grass species. In this study, 55,221 repeat masked Brachypodium BAC end sequences (BES) were used for comparative analysis against the 12 rice pseudomolecules. The analysis revealed that ~26.4% of BES have significant matches with the rice genome and 82.4% of the matches were homologous to known genes. Further analysis of paired-end BES and ~1.0 Mb sequences from nine selected BACs proved to be useful in revealing conserved regions and regions that have undergone considerable genomic changes. Differential gene amplification, insertions/deletions and inversions appeared to be the common evolutionary events that caused variations of microcolinearity at different orthologous genomic regions. It was found that ~17% of genes in the two genomes are not colinear in the orthologous regions. Analysis of BAC sequences also revealed higher gene density (~9 kb/gene) and lower repeat DNA content (~13.1%) in Brachypodium when compared to the orthologous rice regions, consistent with the smaller size of the Brachypodium genome. The 119 annotated Brachypodium genes were BLASTN compared against the wheat EST database and deletion bin mapped wheat ESTs. About 77% of the genes retrieved significant matches in the EST database, while 9.2% matched to the bin mapped ESTs. In some cases, genes in single Brachypodium BACs matched to multiple ESTs that were mapped to the same deletion bins, suggesting that the Brachypodium genome will be useful for ordering wheat ESTs within the deletion bins and developing specific markers at targeted regions in the wheat genome.     

Human BAC ends quality assessment and sequence analyses

End sequences from bacterial artificial chromosomes (BACs) provide highly specific sequence markers in large-scale sequencing projects. To date, we have generated >300,000 end sequences from >186,000 human BAC clones with an average read length of >460 bp for a total of 141 Mb covering approximately 4.7% of the genome. Over 60% of the clones have BAC end sequences (BESs) from both ends representing more than fivefold coverage of the human genome by the paired-end clones. Our quality assessments and sequence analyses indicate that BESs from human BAC libraries developed at The California Institute of Technology (CalTech) and Roswell Park Cancer Institute have similar properties. The analyses have highlighted differences in insert size for different segments of the CalTech library. Problems with the fidelity of tracking of sequence data back to physical clones have been observed in some subsets of the overall BES dataset. The annotation results of BESs for the contents of available genomic sequences, sequence tagged sites, expressed sequence tags, protein encoding regions, and repeats indicate that this resource will be valuable in many areas of genome research.     

Advanced resources for plant genomics: a BAC library specific for the short arm of wheat chromosome 1B

Common wheat (Triticum aestivum L., 2n = 6x = 42) is a polyploid species possessing one of the largest genomes among the cultivated crops (1C is approximately 17 000 Mb). The presence of three homoeologous genomes (A, B and D), and the prevalence of repetitive DNA make sequencing the wheat genome a daunting task. We have developed a novel 'chromosome arm-based' strategy for wheat genome sequencing to simplify this task; this relies on sub-genomic libraries of large DNA inserts. In this paper, we used a di-telosomic line of wheat to isolate six million copies of the short arm of chromosome 1B (1BS) by flow sorting. Chromosomal DNA was partially digested with HindIII and used to construct an arm-specific BAC library. The library consists of 65 280 clones with an average insert size of 82 kb. Almost half of the library (45%) has inserts larger than 100 kb, while 18% of the inserts range in size between 75 and 100 kb, and 37% are shorter than 75 kb. We estimated the chromosome arm coverage to be 14.5-fold, giving a 99.9% probability of identifying a clone corresponding to any sequence on the short arm of 1B. Each chromosome arm in wheat can be flow sorted from an appropriate cytogenetic stock, and we envisage that the availability of chromosome arm-specific BAC resources in wheat will greatly facilitate the development of ready-to-sequence physical maps and map-based gene cloning.     

Construction, characterization, and preliminary BAC-end sequencing analysis of a bacterial artificial chromosome library of white clover (Trifolium repens L.).

White clover (Trifolium repens L.) is a forage legume widely used in combination with grass in pastures because of its ability to fix nitrogen. We have constructed a bacterial artificial chromosome (BAC) library of an advanced breeding line of white clover. The library contains 37 248 clones with an average insert size of approximately 85 kb, representing an approximate 3-fold coverage of the white clover genome based on an estimated genome size of 960 Mb. The BAC library was pooled and screened by polymerase chain reaction (PCR) amplification using both white clover microsatellites and PCR-based markers derived from Medicago truncatula, resulting in an average of 6 hits per marker; this supports the estimated 3-fold genome coverage in this allotetraploid species. PCR-based screening of 766 clones with a multiplex set of chloroplast primers showed that only 0.5% of BAC clones contained chloroplast-derived inserts. The library was further evaluated by sequencing both ends of 724 of the clover BACs. These were analysed with respect to their sequence content and their homology to the contents of a range of plant gene, expressed sequence tag, and repeat element databases. Forty-three microsatellites were discovered in the BAC-end sequences (BESs) and investigated as potential genetic markers in white clover. The BESs were also compared with the partially sequenced genome of the model legume M. truncatula with the specific intention of identifying putative comparative-tile BACs, which represent potential regions of microsynteny between the 2 species; 14 such BACs were discovered. The results suggest that a large-scale BAC-end sequencing strategy has the potential to anchor a significant proportion of the genome of white clover onto the gene-space sequence of M. truncatula.     

Construction of a hexaploid wheat (Triticum aestivum L.) bacterial artificial chromosome library for cloning genes for stripe rust resistance.

A hexaploid wheat (Triticum aestivum L.) bacterial artificial chromosome (BAC) library was constructed for cloning Yr5 and other genes conferring resistance to stripe rust (Puccinia striiformis f. sp. tritici). Intact nuclei from a Yr5 near-isogenic line were used to isolate high molecular weight DNA, which was partially cleaved with HindIII and cloned into pECBAC1 and pIndigoBAC-5 vectors. The wheat BAC library consisted of 422,400 clones arrayed in 1100 micro-titer plates (each plate with 384 wells). Random sampling of 300 BAC clones indicated an average insert size of 140 kb, with a size range from 25 to 365 kb. Ninety percent of the clones in the library had an insert size greater than 100 kb and fewer than 5% of the clones did not contain inserts. Based on an estimated genome size of 15,966 Mb for hexaploid wheat, the BAC library was estimated to have a total coverage of 3.58x wheat genome equivalents, giving approximately 96% probability of identifying a clone representing any given wheat DNA sequence. Twelve BAC clones containing an Yr5 locus-specific marker (Yr5STS7/8) were successfully selected by PCR screening of 3-dimensional BAC pools. The results demonstrated that the T. aestivum BAC library is a valuable genomic resource for positional cloning of Yr5. The library also should be useful in cloning other genes for stripe rust resistance and other traits of interest in hexaploid wheat.     

Characterizing the walnut genome through analyses of BAC end sequences

Persian walnut (Juglans regia L.) is an economically important tree for its nut crop and timber. To gain insight into the structure and evolution of the walnut genome, we constructed two bacterial artificial chromosome (BAC) libraries, containing a total of 129,024 clones, from in vitro-grown shoots of J. regia cv. Chandler using the HindIII and MboI cloning sites. A total of 48,218 high-quality BAC end sequences (BESs) were generated, with an accumulated sequence length of 31.2?Mb, representing approximately 5.1% of the walnut genome. Analysis of repeat DNA content in BESs revealed that approximately 15.42% of the genome consists of known repetitive DNA, while walnut-unique repetitive DNA identified in this study constitutes 13.5% of the genome. Among the walnut-unique repetitive DNA, Julia SINE and JrTRIM elements represent the first identified walnut short interspersed element (SINE) and terminal-repeat retrotransposon in miniature (TRIM) element, respectively; both types of elements are abundant in the genome. As in other species, these SINEs and TRIM elements could be exploited for developing repeat DNA-based molecular markers in walnut. Simple sequence repeats (SSR) from BESs were analyzed and found to be more abundant in BESs than in expressed sequence tags. The density of SSR in the walnut genome analyzed was also slightly higher than that in poplar and papaya. Sequence analysis of BESs indicated that approximately 11.5% of the walnut genome represents a coding sequence. This study is an initial characterization of the walnut genome and provides the largest genomic resource currently available; as such, it will be a valuable tool in studies aimed at genetically improving walnut.     

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.     

Chromosome bin map of expressed sequence tags in homoeologous group 1 of hexaploid wheat and homoeology with rice and Arabidopsis

A total of 944 expressed sequence tags (ESTs) generated 2212 EST loci mapped to homoeologous group 1 chromosomes in hexaploid wheat (Triticum aestivum L.). EST deletion maps and the consensus map of group 1 chromosomes were constructed to show EST distribution. EST loci were unevenly distributed among chromosomes 1A, 1B, and 1D with 660, 826, and 726, respectively. The number of EST loci was greater on the long arms than on the short arms for all three chromosomes. The distribution of ESTs along chromosome arms was nonrandom with EST clusters occurring in the distal regions of short arms and middle regions of long arms. Duplications of group 1 ESTs in other homoeologous groups occurred at a rate of 35.5%. Seventy-five percent of wheat chromosome 1 ESTs had significant matches with rice sequences (E < or = e(-10)), where large regions of conservation occurred between wheat consensus chromosome 1 and rice chromosome 5 and between the proximal portion of the long arm of wheat consensus chromosome 1 and rice chromosome 10. Only 9.5% of group 1 ESTs showed significant matches to Arabidopsis genome sequences. The results presented are useful for gene mapping and evolutionary and comparative genomics of grasses.     

A human-horse comparative map based on equine BAC end sequences

In an effort to increase the density of sequence-based markers for the horse genome we generated 9473 BAC end sequences (BESs) from the CHORI-241 BAC library with an average read length of 677 bp. BLASTN searches with the BESs revealed 4036 meaningful hits (E 相似文献   

The nuclear genome of Brachypodium distachyon: analysis of BAC end sequences

Due in part to its small genome (~350 Mb), Brachypodium distachyon is emerging as a model system for temperate grasses, including important crops like wheat and barley. We present the analysis of 10.9% of the Brachypodium genome based on 64,696 bacterial artificial chromosome (BAC) end sequences (BES). Analysis of repeat DNA content in BES revealed that approximately 11.0% of the genome consists of known repetitive DNA. The vast majority of the Brachypodium repetitive elements are LTR retrotransposons. While Bare-1 retrotransposons are common to wheat and barley, Brachypodium repetitive element sequence-1 (BRES-1), closely related to Bare-1, is also abundant in Brachypodium. Moreover, unique Brachypodium repetitive element sequences identified constitute approximately 7.4% of its genome. Simple sequence repeats from BES were analyzed, and flanking primer sequences for SSR detection potentially useful for genetic mapping are available at . Sequence analyses of BES indicated that approximately 21.2% of the Brachypodium genome represents coding sequence. Furthermore, Brachypodium BES have more significant matches to ESTs from wheat than rice or maize, although these species have similar sizes of EST collections. A phylogenetic analysis based on 335 sequences shared among seven grass species further revealed a closer relationship between Brachypodium and Triticeae than Brachypodium and rice or maize. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. N. Huo and G.R. Lazo contributed equally to this work.     

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Zhikong Scallop, Chlamys farreri Jones et Preston, and Identification of BAC Clones Containing the Genes Involved in Its Innate Immune System

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.     

Construction and characterization of the first bacterial artificial chromosome library for the cotton species Gossypium barbadense L.

As the second most widely cultivated cotton, Gossypium barbadense is well known for its superior fiber properties and its high levels of resistance to Fusarium and Verticillium wilts. To enhance our ability to exploit these properties in breeding programs, we constructed the first bacterial artificial chromosome (BAC) library for this species. The library contains 167 424 clones (49 920 BamHI and 117 504 HindIII clones), with an estimated average insert size of 130 kb. About 94.0% of the clones had inserts over 100 kb, and the empty clones accounted for less than 4.0%. Contamination of the library with chloroplast clones was very low (0.2%). Screening the library with locus-specific probes showed that BAC clones represent 6.5-fold genome equivalents. This high-quality library provides an additional asset with which to exploit genetic variation for cotton improvement.     

Construction of a subgenomic BAC library specific for chromosomes 1D, 4D and 6D of hexaploid wheat

The analysis of the hexaploid wheat genome (Triticum aestivum L., 2n=6x=42) is hampered by its large size (16,974 Mb/1C) and presence of three homoeologous genomes (A, B and D). One of the possible strategies is a targeted approach based on subgenomic libraries of large DNA inserts. In this work, we purified by flow cytometry a total of 10⁷ of three wheat D-genome chromosomes: 1D, 4D and 6D. Chromosomal DNA was partially digested with HindIII and used to prepare a specific bacterial artificial chromosome (BAC) library. The library (designated as TA-subD) consists of 87,168 clones, with an average insert size of 85 kb. Among these clones, 53% had inserts larger than 100 kb, only 29% of inserts being shorter than 75 kb. The coverage was estimated to be 3.4-fold, giving a 96.5% probability of identifying a clone corresponding to any sequence on the three chromosomes. Specificity for chromosomes 1D, 4D and 6D was confirmed after screening the library pools with single-locus microsatellite markers. The screening indicated that the library was not biased and gave an estimated coverage of sixfold. This is the second report on BAC library construction from flow-sorted plant chromosomes, which confirms that dissecting of the complex wheat genome and preparation of subgenomic BAC libraries is possible. Their availability should facilitate the analysis of wheat genome structure and evolution, development of cytogenetic maps, construction of local physical maps and map-based cloning of agronomically important genes.     

One large-insert plant-transformation-competent BIBAC library and three BAC libraries of Japonica rice for genome research in rice and other grasses

We report one large-insert BIBAC library and three BAC libraries for japonica rice cv Nipponbare. The BIBAC library was constructed in the HindIII site of a plant-transformation-competent binary vector (pCLD04541) and the three BAC libraries were constructed in the BamHI, HindIII and EcoRI sites of a BAC vector (pECBAC1), respectively. Each library contains 23,040 clones, has an average insert size of 130 kb, 170 kb, 150 kb and 156 kb, and covers 6.7x, 8.7x, 7.7x and 8.0 x rice haploid genomes, respectively. The combined libraries contain 92,160 clones in total, covering 31.1 x rice haploid genomes. To demonstrate their utility, we screened the libraries with 55 DNA markers mapped to chromosome 8 of the rice genetic maps and analyzed a number of clones by the restriction fingerprinting and contig assembly method. The results indicate that the libraries completely cover the rice genome and, thus, are well-suited for genome research in rice and other gramineous crops. The BIBAC library represents the first plant-transformation-competent large-insert DNA library for rice, which will streamline map-based cloning, functional analysis of the rice genome sequence and molecular breeding in rice and other grass species. These libraries are being used in the development of a whole-genome, BAC/BIBAC-based, integrated physical, genetic and sequence map of rice and in the research of genome-wide comparative genomics of grass species.     

Strategy to sequence the genome of Corynebacterium glutamicum ATCC 13032: use of a cosmid and a bacterial artificial chromosome library

The initial strategy of the Corynebacterium glutamicum genome project was to sequence overlapping inserts of an ordered cosmid library. High-density colony grids of approximately 28 genome equivalents were used for the identification of overlapping clones by Southern hybridization. Altogether 18 contiguous genomic segments comprising 95 overlapping cosmids were assembled. Systematic shotgun sequencing of the assembled cosmid set revealed that only 2.84 Mb (86.6%) of the C. glutamicum genome were represented by the cosmid library. To obtain a complete genome coverage, a bacterial artificial chromosome (BAC) library of the C. glutamicum chromosome was constructed in pBeloBAC11 and used for genome mapping. The BAC library consists of 3168 BACs and represents a theoretical 63-fold coverage of the C. glutamicum genome (3.28 Mb). Southern screening of 2304 BAC clones with PCR-amplified chromosomal markers and subsequent insert terminal sequencing allowed the identification of 119 BACs covering the entire chromosome of C. glutamicum. The minimal set representing a 100% genome coverage contains 44 unique BAC clones with an average overlap of 22 kb. A total of 21 BACs represented linking clones between previously sequenced cosmid contigs and provided a valuable tool for completing the genome sequence of C. glutamicum.     

Analysis of expressed sequence tag loci on wheat chromosome group 4

A total of 1918 loci, detected by the hybridization of 938 expressed sequence tag unigenes (ESTs) from 26 Triticeae cDNA libraries, were mapped to wheat (Triticum aestivum L.) homoeologous group 4 chromosomes using a set of deletion, ditelosomic, and nulli-tetrasomic lines. The 1918 EST loci were not distributed uniformly among the three group 4 chromosomes; 41, 28, and 31% mapped to chromosomes 4A, 4B, and 4D, respectively. This pattern is in contrast to the cumulative results of EST mapping in all homoeologous groups, as reported elsewhere, that found the highest proportion of loci mapped to the B genome. Sixty-five percent of these 1918 loci mapped to the long arms of homoeologous group 4 chromosomes, while 35% mapped to the short arms. The distal regions of chromosome arms showed higher numbers of loci than the proximal regions, with the exception of 4DL. This study confirmed the complex structure of chromosome 4A that contains two reciprocal translocations and two inversions, previously identified. An additional inversion in the centromeric region of 4A was revealed. A consensus map for homoeologous group 4 was developed from 119 ESTs unique to group 4. Forty-nine percent of these ESTs were found to be homoeologous to sequences on rice chromosome 3, 12% had matches with sequences on other rice chromosomes, and 39% had no matches with rice sequences at all. Limited homology (only 26 of the 119 consensus ESTs) was found between wheat ESTs on homoeologous group 4 and the Arabidopsis genome. Forty-two percent of the homoeologous group 4 ESTs could be classified into functional categories on the basis of blastX searches against all protein databases.     

Construction and characterization of a bacterial artificial chromosome (BAC) library for the A genome of wheat.

A genomic bacterial artificial chromosome (BAC) library of the A genome of wheat has been constructed. Triticum monococcum accession DV92 was selected for this purpose because it is a cultivated diploid wheat and one of the parental lines used in the construction of a saturated genetic map. Leaves from this accession were used to isolate high-molecular-weight DNA from nuclei. This DNA was partially digested with restriction enzyme Hind III, subjected to double size selection, electroeluted and cloned into the pINDIGO451 BAC vector. The library consists of 276,480 clones with an average insert size of 115 kb. Excluding the 1.33% of empty clones and 0.14% of clones with chloroplast DNA, the coverage of this library is 5.6 genome equivalents. With this genome coverage the probability of having any DNA sequence represented in this library is higher than 99.6%. Clones were sorted in 720,384-well plates and blotted onto 15 high-density filters. High-density filters were screened with several single or low-copy clones and five positive BAC clones were selected for further analysis. Since most of the T. monococcum BAC ends included repetitive sequences, a modification was introduced into the classical end-isolation procedure to select low copy sequences for chromosome walking.     

Construction and characterization of a bacterial artificial chromosome (BAC) library of hexaploid wheat (Triticum aestivum L.) and validation of genome coverage using locus-specific primers.

A BAC library of hexaploid wheat was constructed using the spring wheat cultivar Triticum aestivum L. 'Glenlea'. Fresh shoot tissue from 7- to 10-day-old seedlings was used to obtain HMW DNA. The library was constructed using the HindIII site of pIndigoBAC-5 and the BamHI site of pIndigoBAC-5 and pECBAC1. A total of 12 ligations were used to construct the entire library, which contains over 650 000 clones. Ninety-six percent of the clones had inserts. The insert size ranged from 5 to 189 kb with an average of 79 kb. The entire library was gridded onto 24 high-density filters using a 5 x 5 array. A subset of these membranes was hybridized with two intergenic chloroplast probes and the percentage of clones containing chloroplast DNA (cpDNA) was calculated to be 2.2%. The genome coverage was estimated to be 3.1 x haploid genome equivalents, giving a 95.3% probability of identifying a clone corresponding to any wheat DNA sequence. BAC pools were constructed and screened using markers targeting the Glu-B1 locus (1BL), the hardness loci (5AS, 5BS, 5DS), the leaf rust resistance locus Lr1 (5DL), and the major fusarium head blight QTL locus located on 3BS. These markers were either locus-specific amplicons or microsatellites. A total of 49 BAC clones were identified for 14 markers giving an average of 3.5 clones/marker, thereby corroborating the estimated 3.1x genome coverage. An example using the gene encoding the HMW glutenin Bx7 is illustrated.