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.     

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 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.     

Construction and Identification of Bacterial Artificial Chromosome Library for 0-613-2R in Upland Cotton

A bacterial artificial chromosome （BAC） library containing a large genomlc DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To Isolate genes via a map-based cloning strategy and to perform physical mapping of the cotton genome, a high-quality BAC library containing large cotton DNA Inserts Is needed. We have developed a BAC library of the restoring line 0-613-2R for Isolating the fertility restorer （Rf1） gene and genomic research in cotton （Gossypium hirsutum L.）. The BAC library contains 97 825 clones stored In 255 pieces of a 384-well mlcrotiter plate. Random samples of BACs digested with the Notl enzyme Indicated that the average Insert size Is approximately 130 kb, with a range of 80-275 kb, and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 × haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with Notl and Hlndiii enzymes. Thus, the atabiiity of a single BAC clone can be sustained at iesat for 100 generations. Eight simple sequence repeat （SSR） markers flanking the Rf; gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker.     

A Sorghum propinquum BAC library,suitable for cloning genes associated with loss-of-function mutations during crop domestication

A large insert Sorghum propinquum BAC library has been constructed to analyze the physical organization of the sorghum genome and to facilitate positional cloning of sorghum genes and QTLs associated with the early stages of grain crop domestication. This library was established from 12 different ligations using high-molecular-weight DNA generated from either one cycle or two cycles of size selection. This library consists of 38 016 BAC clones with an estimated average insert size of 126 kb and coverage of 6.6 genome equivalents. The 6.6 genome-equivalent BAC library of S. propinquum provides a 99.7% probability of finding one or more BACs that contain genes of interest. Twenty mapped DNA probes, ten homologous and ten heterologous, were used to screen the library, and 121 positive clones were identified, 6.05 per locus or 6.37 per probe.     

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     

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.     

Generation of a soybean BAC library, and identification of DNA sequences tightly linked to the Rps1-k disease resistance gene

 A soybean bacterial artificial chromosome (BAC) library, comprising approximately 45 000 clones, was constructed from high-molecular-weight nuclear DNA of cultivar Williams 82, which carries the Rps1-k gene for resistance against Phytophthora sojae. The library is stored in 130 pools with about 350 clones per pool. Completeness of the library was evaluated for 21 random sequences including four markers linked to the Rps1 locus and 16 cDNAs. We identified pools containing BACs for all sequences except for one cDNA. Additionally, when screened for possible contaminating BAC clones carrying chloroplast genes, no sequences homologous to two barley chloroplast genes were found. The estimated average insert size of the BAC clones was about 105 kb. The library comprises about four genome equivalents of soybean DNA. Therefore, this gives a probability of 0.98 of finding a specific sequence from this library. This library should be a useful resource for the positional cloning of Rps1-k, and other soybean genes. We have also evaluated the feasibility of an RFLP-based screening procedure for the isolation of BAC clones specific for markers that are members of repetitive sequence families, and are linked to the Rps1-k gene. We show that BAC clones isolated for two genetically linked marker loci, Tgmr and TC1-2, are physically linked. Application of this method in expediting the map-based cloning of a gene, especially from an organism, such as soybean, maize and wheat, with a complex genome is discussed. Received: 12 May 1998/Accepted: 24 August 1998     

高覆盖率水稻ＢＡＣ库的构建及抗病基因相关克隆的筛选

利用含Ｘa4、xa5和xa13 3个水稻白叶枯病抗性基因的累加系ＩＲＢＢ５６构建了一个水稻细菌人工染色体文库,该文库包含５５２９６个克隆,平均插入升段为１３２kb。按水稻基因组为４５０Ｍb计,该文库覆盖１４倍基因组,筛选出任一水稻基因或序列的概率为９９．９９％。用均匀分布的３个叶绿体基因和４个线粒体基因克隆作探针筛选文库,结果显示该文库中含细菌器基因组ＤＮＡ同源序列的克隆数小于１％、用分布于水稻３条不同染色体、分别与Ｘa4、xa5和xa13连锁的ＤＮＡ标记筛选文库,分别检测出１１－１０６个阳性克隆,为克隆这些基因打下了基础。该文库对水稻基因组的高度覆盖率和较大的插入片段,非常适合于物理作图和基因的分离和克隆。     

A ‘Chinese Spring’ wheat (Triticum aestivum L.) bacterial artificial chromosome library and its use in the isolation of SSR markers for targeted genome regions

A bacterial artificial chromosome (BAC) library was constructed from the bread wheat (Triticum aestivum L.) genotype ‘Chinese Spring’ (‘CS’). The library consists of 395,136 clones with an estimated average insert size of 157 kb. This library provides an estimated 3.4-fold genome coverage for this hexaploid species. The genome coverage was confirmed by RFLP analysis of single-copy RFLP clones. The CS BAC library was used to develop simple sequence repeat (SSR) markers for targeted genome regions using five sequence-tagged-site (STS) markers designed from the chromosome arm of 3BS. The SSR markers for the targeted genome region were successfully obtained. However, similar numbers of new SSR markers were also generated for the other two homoeologous group 3 chromosomes. This data suggests that BAC clones belonging to all three chromosomes of homoeologous group 3 were isolated using the five STS primers. The potential impacts of these results on marker isolation in wheat and on library screening in general are discussed.     

Construction of a deep coverage BAC library from Capsicum annuum, 'CM334'

A bacterial artificial chromosome (BAC) library consisting of 235,000 clones with an average insert size of 130 kb was constructed from Capsicum annuum, 'CM334'. Based on a pepper haploid genome size of 2,702 Mbp/C, the BAC library is estimated to contain approximately 12 genome equivalents and represents at least 99% of the pepper genome. Screening of the library with mitochondrial DNA probes (coxII, coxIII, atp6 and atp9) and chloroplast DNA probes (atpB, rbcL) indicated that contamination with cytoplasmic DNA was less than 0.5%. To estimate the possibility of isolating a specific clone, the library was screened with single or low-copy gene-specific probes and RFLP probes. Screening of high density BAC filters with RFLP markers linked to L (TMV resistance), y (fruit color), C2 (fruit color) and C (pungency) loci under high stringency conditions revealed that at least three positive BAC clones were found per each probe. This fact indicates that the library is highly reliable and represents a resource for map-based cloning, physical mapping, and characterization of upstream and downstream regulations of the chili pepper genes.     

Bacterial artificial chromosome (BAC) library resource for positional cloning of pest and disease resistance genes in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Pest and disease problems are important constraints of cassava production and host plant resistance is the most efficient method of combating them. Breeding for host plant resistance is considerably slowed down by the crop’s biological constraints of a long growth cycle, high levels of heterozygosity and a large genetic load. More efficient methods such as gene cloning and transgenesis are required to deploy resistance genes. To facilitate the cloning of resistance genes, bacterial artificial chromosome (BAC) library resources have been developed for cassava. Two libraries were constructed from the cassava clones, TMS 30001, resistant to the cassava mosaic disease (CMD) and the cassava bacterial blight (CBB), and MECU72, resistant to cassava white fly. The TMS30001 library has 55 296 clones with an insert size range of 40–150 kb with an average of 80 kb, while the MECU72 library consists of 92 160 clones and an insert size range of 25–250 kb average of 93 kb. Based on a genome size of 772 Mb, the TMS30001 and MECU72 libraries have a 5 and 11.3 haploid genome equivalents and a 95 and 99 chance of finding any sequence, respectively. To demonstrate the potential of the libraries, the TMS30001 library was screened by southern hybridization using a cassava analog (CBB1) of the Xa21 gene from rice that maps to a region containing a QTL for resistance to CBB as probe. Five BAC clones that hybridized to CBB1 were isolated and a Hind III fingerprint revealed 2–3 copies of the gene in individual BAC clones. A larger scale analysis of resistance gene analogs (RGAs) in cassava has also been conducted in order to understand the number and organization of RGAs. To scan for gene and repeat DNA content in the libraries, end-sequencing was performed on 2301 clones from the MECU72 library. A total of 1705 unique sequences were obtained with an average size of 715 bp. Database homology searches using BLAST revealed that 458 sequences had significant homology with known proteins and 321 with transposable elements. The use of the library in positional cloning of pest and disease resistance genes is discussed.     

Direct targeting and rapid isolation of BAC clones spanning a defined chromosome region

To isolate genes of interest in plants, it is essential to construct bacterial artificial chromosome (BAC) libraries from specific genotypes. Construction and organisation of BAC libraries is laborious and costly, especially from organisms with large and complex genomes. In the present study, we developed the pooled BAC library strategy that allows rapid and low cost generation and screening of genomic libraries from any genotype of interest. The BAC library is constructed, directly organised into a few pools and screened for BAC clones of interest using PCR and hybridisation steps, without requiring organization into individual clones. As a proof of concept, a pooled BAC library of approximately 177,000 recombinant clones has been constructed from the barley cultivar Cebada Capa that carries the Rph7 leaf rust resistance gene. The library has an average insert size of 140 kb, a coverage of six barley genome equivalents and is organised in 138 pools of about 1,300 clones each. We rapidly established a single contig of six BAC clones spanning 230 kb at the Rph7 locus on chromosome 3HS. The described low-cost cloning strategy is fast and will greatly facilitate direct targeting of genes and large-scale intra- and inter-species comparative genome analysis.Edwige Isidore and Beatrice Scherrer contributed equally to the work.     

A bacterial artificial chromosome (BAC) library for sunflower, and identification of clones containing genes for putative transmembrane receptors

Sunflower (Helianthus annuus L.) is an economically important oil seed crop with an estimated genome size of 3000 Mb. We have constructed a bacterial artificial chromosome (BAC) library for sunflower, which represents an estimated 4- to 5-fold coverage of the genome. Nuclei isolated from young leaves were used as a source of high-molecular-weight DNA and a partial restriction endonuclease digestion protocol was used to cleave the DNA. A random sample of 60 clones indicated an average insert size of 80 kb, implying a 95% probability of recovering any specific sequence of interest. The library was screened with chloroplast DNA probes. Only 0.1% of the clones were identified to be of chloroplast origin, indicating that contamination with organellar DNAs is very low. The utility of the library was evaluated by screening for the presence of genes for putative transmembrane receptors sharing epidermal growth factor (EGF) and integrin-like domains. First, a homologous sunflower EST (HaELP1) was obtained by degenerate RT-PCR cloning, using Arabidopsis thaliana genes (AtELP) as a source of consensus sequences. Three different BACs yielded positive hybridization signals when HaELP1 was used as a probe. BAC subcloning and sequencing demonstrated the presence of two different loci putatively homologous to genes for transmembrane proteins with EGF- and integrin-like domains from sunflower. This work demonstrates the suitability of the library for homology map-based cloning of sunflower genes and physical mapping of the sunflower genome.     

Construction of a bacterial artificial chromosome library, determination of genome size, and characterization of an Hsp70 gene family in Phytophthora nicotianae

The oomycete plant pathogen Phytophthora nicotianae causes diseases on a wide range of plant species. To facilitate isolation and functional characterization of pathogenicity genes, we have constructed a large-insert bacterial artificial chromosome (BAC) library using nuclear DNA from P. nicotianae H1111. The library contains 10,752 clones with an average insert size of 90 kb and is free of mitochondrial DNA. The quality of the library was verified by hybridization with 37 genes, all of which resulted in the identification of multiple positive clones. The library is estimated to be 10.6 haploid genome equivalents based on hybridization of 23 single-copy genes and the genome size of P. nicotianae was estimated to be 95.5 Mb. Hybridization with a nuclear repetitive DNA probe revealed that 4.4% of clones in the library contained 28S rDNA. Hybridization of total genomic DNA to the library indicated that at least 39% of the BAC library contains repetitive DNA sequences. A BAC pooling strategy was developed for efficient library screening. The library was used to identify and characterize BAC clones containing an Hsp70 gene family whose four members were identified to be clustered within approximately 18 kb in the P. nicotianae genome based on the physical mapping of eight BACs spanning a genomic region of approximately 186 kb. The BAC library created provides an invaluable resource for the isolation of P. nicotianae genes and for comparative genomics studies.     

Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population

Rice is a leading grain crop and the staple food for over half of the world population. Rice is also an ideal species for genetic and biological studies of cereal crops and other monocotyledonous plants because of its small genome and well developed genetic system. To facilitate rice genome analysis leading to physical mapping, the identification of molecular markers closely linked to economic traits, and map-based cloning, we have constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs). Lemont (japonica) and Teqing (indica) represent the two major genomes of cultivated rice, both are leading commercial varieties and widely used germplasm in rice breeding programs. The Lemont library contains 7296 clones with an average insert size of 150 kb, which represents 2.6 rice haploid genome equivalents. The Teqing library contains 14208 clones with an average insert size of 130 kb, which represents 4.4. rice haploid genome equivalents. Three single-copy DNA probes were used to screen the libraries and at least two overlapping BAC clones were isolated with each probe from each library, ranging from 45 to 260 kb in insert size. Hybridization of BAC clones with chloroplast DNA probes and fluorescent in situ hybridization using BAC DNA as probes demonstrated that both libraries contain very few clones of chloroplast DNA origin and are likely free of chimeric clones. These data indicate that both BAC libraries should be suitable for map-based cloning of rice genes and physical mapping of the rice genome.     

Construction and characterization of a bacterial artificial chromosome library of maize inbred line 77Ht2

Maize inbred line 77Ht2 contains agriculturally important genes and has been widely used in corn breeding in China. A bacterial artificial chromosome (BAC) library of 77Ht2 has been constructed in order to identify useful genes and to facilitate the study of the maize genome. The library contains 175104 clones with an average insert size of 57 kb and represents about 4 maize haploid genome equivalents. Characterization of the library showed less than 0.5% of clones to not contain large inserts. Significant contamination of chloroplast and mitochondria DNA was not detected. BAC clones (152 arrays) were stored in 96 microtiter plates, with each well containing 12 clones. This is the first maize BAC library constructed in China. It is well suited for map-based cloning of maize genes and genome physical mapping.     

A maize bacterial artificial chromosome (BAC) library from the European flint inbred line F2

We report here the construction and characterisation of a BAC library from the maize flint inbred line F2, widely used in European maize breeding programs. The library contains 86,858 clones with an average insert size of approximately 90 kb, giving approximately 3.2-times genome coverage. High-efficiency BAC cloning was achieved through the use of a single size selection for the high-molecular-weight genomic DNA, and co-transformation of the ligation with yeast tRNA to optimise transformation efficiency. Characterisation of the library showed that less than 0.5% of the clones contained no inserts, while 5.52% of clones consisted of chloroplast DNA. The library was gridded onto 29 nylon filters in a double-spotted 8 × 8 array, and screened by hybridisation with a number of single-copy and gene-family probes. A 3-dimensional DNA pooling scheme was used to allow rapid PCR screening of the library based on primer pairs from simple sequence repeat (SSR) and expressed sequence tag (EST) markers. Positive clones were obtained in all hybridisation and PCR screens carried out so far. Six BAC clones, which hybridised to a portion of the cloned Rp1-D rust resistance gene, were further characterised and found to form contigs covering most of this complex resistance locus. Received: 30 August 2000 / Accepted: 6 December 2000     

A BAC Library Constructed to the Rice Cultivar

"Minghui 63" is the restorer line for a number of the most important commercial rice hybrids varieties in China. To facilitate long-term commitment in genetic analysis and molecular cloning of the superior genes in the genome of "Minghui 63", the authors have constructed a largeinsert genomic DNA library using the bacterial artificial chromosome (BAC) cloning vector (pBe- loBAC 11). Size fractionated Hind m digest of genomic DNA was ligated to the BAC vector, and the ligation mixture was used to transform the bacterial strain DH10B. A total of over 26 000 clones were obtained with the average insert size of about 150 kb, ranging from 90 to 240 kb. These clones thus represent 9 x rice haploid genome equivalents. The library is now being used for physical mapping of several genomic regions for map-based gene cloning.     

A bacterial artificial chromosome (BAC) library of Malus floribunda 821 and contig construction for positional cloning of the apple scab resistance gene Vf.

The apple scab resistance gene Vf, originating from the wild species Malus floribunda 821, has been incorporated into a wide variety of apple cultivars through a classical breeding program. With the aim of isolating the Vf gene, a bacterial artificial chromosome (BAC) library consisting of 31 584 clones has been constructed from M. floribunda 821. From the analysis of 88 randomly selected BAC clones, the average insert size is estimated at 125 kb. If it is assumed that the genome size of M. floribunda 821 is 769 Mb/haploid, the library represents about 5x haploid genome equivalents. This provides a 99% probability of finding any specific sequence from this library. PCR-based screening of the library has been carried out using eight random genomic sequence-characterized amplified regions (SCARs), chloroplast- and mitochondria-specific SCARs, and 13 high-density Vf-linked SCAR markers. An average of five positive BAC clones per random SCAR has been obtained, whereas less than 1% of BAC clones are derived from the chloroplast or mitochondrial genomes. Most BAC clones identified with Vf-linked SCAR markers are physically linked. Three BAC contigs along the Vf region have been obtained by assembling physically linked BAC clones based on their fingerprints. The overlapping relatedness of BAC clones has been further confirmed by cytogenetic mapping using fiber fluorescence in situ hybridization (fiber-FISH). The M. floribunda 821 BAC library provides a valuable genetic resource not only for map-based cloning of the Vf gene, but also for finding many other important genes for improving the cultivated apple.