A bacterial artificial chromosome library for 'Jefferson' hazelnut and identification of clones associated with eastern filbert blight resistance and pollen-stigma incompatibility

European hazelnut (Corylus avellana L.) is the only economically important nut crop in the family Betulaceae. Because of its small genome size (~385 Mb / 1C), relatively short life cycle, availability of a dense linkage map, and amenability to transformation by Agrobacterium, the European hazelnut could serve as a model plant for the Betulaceae. Here we report the construction of a bacterial artificial chromosome (BAC) library for 'Jefferson' hazelnut using the cloning enzyme MboI and the vector pECBAC1 (BamHI site). The library consists of 39,936 clones arrayed in 104,384-well microtitre plates with a mean insert size of 117 kb. The genomic coverage of the library is estimated to be about 12 genome equivalents. This library provides a valuable resource for the map-based cloning of two important genes, the resistance gene from 'Gasaway' that confers resistance to eastern filbert blight caused by the fungus Anisogramma anomala (Peck) E. Müller and the S locus that controls pollen-stigma incompatibility. Fine-resolution mapping near the two loci was carried out using random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers. Fine mapping at the disease resistance locus showed that markers W07-375 and X01-825 flanked the resistance locus at distances of 0.06 and 0.05 cM, respectively. The S locus is flanked by markers 204-950 and KG819-200 at distances of 0.14 and 0.24 cM, respectively. Assuming that 1 cM corresponds to a physical distance of 430 kb, it will take approximately two to three chromosome walks to assemble BAC contigs that span both loci.     

BAC contig development by fingerprint analysis in soybean.

We constructed a soybean bacterial artificial chromosome (BAC) library suitable for map-based cloning and physical mapping in soybean. This library consists of approximately 40 000 clones (4-5 genome equivalents) stored individually in 384-well microtiter dishes. A random sampling of 224 clones yielded an average insert size of 150 kb, giving a 98% probability of recovering any specific sequence. We screened the library for seven single or very low copy genie or genomic sequences using the polymerase chain reaction (PCR) and found between one and seven BACs for each of the seven sequences. When testing the library with a portion of the soybean psbA chloroplast gene, we found less than 1% chloroplast DNA representation. We also screened the library for eight different classes of disease resistance gene analogs (RGAs) and identified BACs containing all RGAs except class 8. We arranged nine of the class 1 RGA BACs and six of the class 3 RGA BACs into individual contigs based on fingerprint patterns observed after Southern probing of restriction digests of the member BACs with a class-specific sequence. This resulted in the partial localization of the different multigene family sequences without precise definition of their exact positions. Using PCR-based end rescue techniques and RFLP mapping of BAC ends, we mapped individual BACs of each contig onto linkage group J of the soybean public map. The class 1 contig mapped to the region on linkage group J that contains several disease resistance genes. The class 1 contig extended approximately 400 kb. The arrangement of the BACs within this contig has been confirmed using PCR. One end of the class 1 contig core BAC mapped to two positions on linkage group J and cosegregated with two class 1 RGA loci, suggesting that this segment is within an area of regional duplication.     

Physical mapping of resistant and susceptible soybean genomes near the soybean cyst nematode resistance gene Rhg4.

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is the foremost pest of soybean (Glycine max L. Merr.). The rhg1 allele on linkage group (LG) G and the Rhg4 allele on LG A2 are important in conditioning resistance. Markers closely linked to the Rhg4 locus were used previously to screen a library of bacterial artificial chromosome (BAC) clones from susceptible 'Williams 82' and identified a single 150-kb BAC, Gm_ISb001_056_G02 (56G2). End-sequenced subclones positioned onto a restriction map provided landmarks for identifying the corresponding region from a BAC library from accession PI 437654 with broad resistance to SCN. Seventy-three PI 437654 BACs were assigned to contigs based upon HindIII restriction fragment profiles. Four contigs represented the PI 437654 counterpart of the 'Williams 82' BAC, with PCR assays connecting these contigs. Some of the markers on the PI 437654 contigs are separated by a greater physical distance than in the 'Williams 82' BAC and some primers amplify bands from BACs in the mid-portion of the connected PI 437654 BAC contigs that are not amplified from the 'Williams 82' BAC. These observations suggest that there is an insertion in the PI 437654 genome relative to the 'Williams 82' genome in the Rhg4 region.     

Fine genetic mapping and BAC contig development for the citrus tristeza virus resistance gene locus in Poncirus trifoliata (Raf.)

A map-based cloning strategy has been employed to isolate Ctv, a single dominant gene from Poncirus trifoliata that confers resistance to citrus tristeza virus (CTV), the most important viral pathogen of citrus. Cloning of this gene will allow development of commercially acceptable, virus-resistant cultivars. A high-resolution genetic linkage map of the Ctv locus region was developed using a backcross population of 678 individuals. Three DNA markers that were closely linked or co-segregated with Ctv were identified and used to screen BAC libraries derived from an intergeneric hybrid of Poncirus and Citrus. Through chromosome walking and landing, two BAC contigs were developed: one encompassing the Ctv region, and the other spanning the allelic susceptibility gene region. The resistance gene contig consists of 20 BAC clones and is approximately 550 kb in length; the susceptibility gene contig consists of 16 BAC clones and extends about 450 kb. The Ctv locus was localized within a genomic region of approximately 180 kb by genetic mapping of BAC insert ends. The BAC contigs were integrated with the genetic map; variation in the ratio of genetic to physical distance was observed in the vicinity of Ctv. Southern hybridization data indicated that a few copies of NBS-LRR class sequences are distributed at or around the Ctv locus. Efforts are being made to assign the Ctv locus to a smaller genomic fragment whose function can be confirmed through genetic complementation of a CTV susceptible phenotype. These results indicate that map-based gene cloning is feasible in a woody perennial.     

Construction of a bacterial artificial chromosome (BAC) library and identification of overlapping BAC clones with chromosome 4-specific RFLP markers in rice

 To facilitate construction of physical map of the rice genome, a bacterial artificial chromosome (BAC) library of IR64 genomic DNA was constructed. It consists of 18 432 clones and contains 3.28 rice genomic equivalents. The insert size ranged from 37 to 364 kb with an average of 107 kb. We used 31 RFLP markers on chromosome 4 to screen the library by colony hybridization. Sixty eight positive clones were identified with 2.2 positive clones per RFLP marker. The positive clones were analyzed to generate 29 contigs whose sizes ranged from 50 to 384 kb with an average of 145.6 kb. Chromosome walking was initiated for ten contigs linked to resistance genes. Thirty eight BAC clones were obtained and two contigs were integrated. Altogether, they covered 5.65 Mb (15.1%) of chromosome 4. These contigs may be used as landmarks for physical mapping of chromosome 4, and as starting points for chromosome walking towards the map-based cloning of disease resistance genes which were located nearby. Received: 15 November 1996 / Accepted: 24 January 1997     

Identification of the sex-determining locus of Atlantic salmon (Salmo salar) on chromosome 2

We have integrated data from linkage mapping, physical mapping and karyotyping to gain a better understanding of the sex-determining locus, SEX, in Atlantic salmon (Salmo salar). SEX has been mapped to Atlantic salmon linkage group 1 (ASL1) and is associated with several microsatellite markers. We have used probes designed from the flanking regions of these sex-linked microsatellite markers to screen a bacterial artificial chromosome (BAC) library, representing an 11.7x coverage of the Atlantic salmon genome, which has been HindIII fingerprinted and assembled into contigs. BACs containing sex-linked microsatellites and their related contigs have been identified and representative BACs have been placed on the Atlantic salmon chromosomes by fluorescent in situ hybridization (FISH). This identified chromosome 2, a large metacentric, as the sex chromosome. By positioning several BACs on this chromosome by FISH, it was possible to orient ASL1 with respect to chromosome 2. The region containing SEX appears to lie on the long arm between marker Ssa202DU and a region of heterochromatin identified by DAPI staining. BAC end-sequencing of clones within sex-linked contigs revealed five hitherto unmapped genes along the sex chromosome. We are using an in silico approach coupled with physical probing of the BAC library to extend the BAC contigs to provide a physical map of ASL1, with a view to sequencing chromosome 2 and, in the process, identifying the sex-determining gene.     

Integrated physical,genetic and genome map of chickpea (Cicer arietinum L.)

Physical map of chickpea was developed for the reference chickpea genotype (ICC 4958) using bacterial artificial chromosome (BAC) libraries targeting 71,094 clones (~12× coverage). High information content fingerprinting (HICF) of these clones gave high-quality fingerprinting data for 67,483 clones, and 1,174 contigs comprising 46,112 clones and 3,256 singletons were defined. In brief, 574 Mb genome size was assembled in 1,174 contigs with an average of 0.49 Mb per contig and 3,256 singletons represent 407 Mb genome. The physical map was linked with two genetic maps with the help of 245 BAC-end sequence (BES)-derived simple sequence repeat (SSR) markers. This allowed locating some of the BACs in the vicinity of some important quantitative trait loci (QTLs) for drought tolerance and reistance to Fusarium wilt and Ascochyta blight. In addition, fingerprinted contig (FPC) assembly was also integrated with the draft genome sequence of chickpea. As a result, ~965 BACs including 163 minimum tilling path (MTP) clones could be mapped on eight pseudo-molecules of chickpea forming 491 hypothetical contigs representing 54,013,992 bp (~54 Mb) of the draft genome. Comprehensive analysis of markers in abiotic and biotic stress tolerance QTL regions led to identification of 654, 306 and 23 genes in drought tolerance “QTL-hotspot” region, Ascochyta blight resistance QTL region and Fusarium wilt resistance QTL region, respectively. Integrated physical, genetic and genome map should provide a foundation for cloning and isolation of QTLs/genes for molecular dissection of traits as well as markers for molecular breeding for chickpea improvement.     

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     

Eastern filbert blight disease resistance from <Emphasis Type="Italic">Corylus americana</Emphasis> ‘Rush’ and selection ‘Yoder #5’ maps to linkage group 7

Eastern filbert blight (EFB), caused by the pyrenomycete Anisogramma anomala, is a serious threat to the hazelnut industry in the Pacific Northwest. EFB is endemic in eastern North America where it occasionally produces small cankers on the wild American hazelnut (Corylus americana). In contrast, most cultivars of European hazelnut (Corylus avellana) are susceptible. Genetic resistance is the most promising disease control method and is an objective of the Oregon State University hazelnut breeding program. ‘Gasaway’ resistance, which is governed by a dominant allele at a single locus, has been extensively used in the program. However, ‘Gasaway’ and some of its offspring have been infected by EFB isolates from New Jersey, Minnesota, and Michigan. Efforts to create new cultivars with durable EFB resistance include identifying and studying new resistance sources. In this study, resistant accessions C. americana ‘Rush’ and interspecific hybrid selection ‘Yoder #5’ were crossed with susceptible C. avellana selections and the resulting segregating seedling populations were inoculated by either exposure of potted trees under a structure topped with diseased branches or field exposure supplemented by tying diseased branches to each tree. Disease response was scored when cankers were visible 20 months after inoculation. Resistance from both sources segregated in a 1:1 ratio, indicating control by a single locus and a dominant allele for resistance. DNA extracted from the seedlings was amplified with previously mapped microsatellite markers. Resistance from both C. americana ‘Rush’ and ‘Yoder #5’ was placed on linkage group 7 in the same position as resistance from C. avellana ‘Ratoli.’ Linked microsatellite markers B753, GB372, and B509 will be useful for marker-assisted selection and the pyramiding of genes for durable EFB resistance. Assessing response to EFB is challenging, whether the plants are inoculated under a structure topped with diseased wood or in a humidity chamber in the greenhouse, or by exposure in the field. The pathogen has a 2-year life cycle, and there is a 15-month wait between inoculation and symptom expression. A small number of escapes is commonly encountered, and resistant plants occasionally develop small cankers. Our approach of studying segregation ratios and then mapping with microsatellite markers should be a useful approach for disease resistance studies in many tree crops.     

Integration of chicken genomic resources to enable whole-genome sequencing

Different genomic resources in chicken were integrated through the Wageningen chicken BAC library. First, a BAC anchor map was created by screening this library with two sets of markers: microsatellite markers from the consensus linkage map and markers created from BAC end sequencing in chromosome walking experiments. Second, HINdIII digestion fingerprints were created for all BACs of the Wageningen chicken BAC library. Third, cytogenetic positions of BACs were assigned by FISH. These integrated resources will facilitate further chromosome-walking experiments and whole-genome sequencing.     

Fine genetic mapping and physical delimitation of the lesion mimic gene Spl11 to a 160-kb DNA segment of the rice genome

The rice lesion mimic mutant spl11 was previously found to confer broad-spectrum disease resistance to both Magnaporthe grisea and Xanthomonas oryzae pv. oryzae. To better understand the molecular basis underlying cell death and disease resistance in rice, a map-based cloning strategy has been employed to isolate Spl11. Five Spl11-linked RAPD markers were developed and four of them were mapped to rice chromosome 12. A high-resolution genetic map was developed using a segregating population consisting of 1138 lesion mimic individuals. Recombination suppression was observed in the vicinity of Spl11. Three molecular markers tightly linked to Spl11 were identified and used to screen a BAC library. A contig spanning the Spl11 locus was constructed and physical mapping delimited Spl11 to a 160-kb DNA segment within a single BAC clone. These results provide the essential information for the final isolation of this important gene in the rice defense pathway.     

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     

Integration of physical and genetic maps in apple confirms whole-genome and segmental duplications in the apple genome

A total of 355 simple sequence repeat (SSR) markers were developed, based on expressed sequence tag (EST) and bacterial artificial chromosome (BAC)-end sequence databases, and successfully used to construct an SSR-based genetic linkage map of the apple. The consensus linkage map spanned 1143 cM, with an average density of 2.5 cM per marker. Newly developed SSR markers along with 279 SSR markers previously published by the HiDRAS project were further used to integrate physical and genetic maps of the apple using a PCR-based BAC library screening approach. A total of 470 contigs were unambiguously anchored onto all 17 linkage groups of the apple genome, and 158 contigs contained two or more molecular markers. The genetically mapped contigs spanned ～421 Mb in cumulative physical length, representing 60.0% of the genome. The sizes of anchored contigs ranged from 97 kb to 4.0 Mb, with an average of 995 kb. The average physical length of anchored contigs on each linkage group was ～24.8 Mb, ranging from 17.0 Mb to 37.73 Mb. Using BAC DNA as templates, PCR screening of the BAC library amplified fragments of highly homologous sequences from homoeologous chromosomes. Upon integrating physical and genetic maps of the apple, the presence of not only homoeologous chromosome pairs, but also of multiple locus markers mapped to adjacent sites on the same chromosome was detected. These findings demonstrated the presence of both genome-wide and segmental duplications in the apple genome and provided further insights into the complex polyploid ancestral origin of the apple.     

Exploitation of a marker dense linkage map of potato for positional cloning of a wart disease resistance gene

A marker-saturated linkage map of potato was used to genetically map a locus involved in the resistance against wart disease Synchytrium endobioticum race 1. The locus mapped on the long arm of chromosome 4 and is named Sen1-4 in contrast to a Sen1 locus on chromosome 11. The AFLP markers from the Sen1-4 interval enabled the isolation of BAC clones from an 11 genome equivalent BAC library. This was achieved via fingerprinting of BAC pools with the AFLP primer pairs that resemble the genetic marker loci. With non-selective AFLP primers, fingerprints of individual BAC clones were generated to analyse the overlap between BAC clones using FPC. This resulted in a complete contig and a minimal tiling path of 14 BAC clones enclosing the Sen1-4 locus. The BAC contig has a genetic length of ~6 cM and a physical length of ~1 Mb. Our results demonstrate that map-based cloning of Sen1-4 can be pursued on the basis of a strategy of marker saturation alone. Genetic resolution achieved by screening large numbers of offspring for recombination events may not be required. Together with the construction of the BAC contig, a physical map with the position of the markers is accomplished in one step. This provides proof of concept for the utility of the marker saturation that is offered by the ultra dense AFLP map of potato for gene cloning.     

Use of the IGF BAC library for physical mapping of the Arabidopsis thaliana genome

In order to generate a physical map of the Arabidopsis thaliana genome based on bacterial artificial chromosome clones (BACs), an iterative high throughput hybridisation strategy was applied and its efficiency was evaluated. Thus, probes generated from both ends of 500 BAC clones selected from the Arabidopsis –IGF–BAC library were hybridised to the entire library gridded on high density filters. The 1000 hybridisation reactions identified 4496 clones (41.8% of the complete library, or 50.3% if organellar, centromeric, and ribosomal DNA carrying clones are excluded) which were assembled into a minimum of 220 contigs. These results demonstrate the viability of the applied ‘double-end clone-limited/sampling without replacement’ hybridisation strategy for the generation of a high resolution physical map, and provide a highly useful resource for map-based gene cloning approaches and further genome analysis.     

Construction and characterization of a bacterial artificial chromosome library of apple

 A bacterial artificial chromosome (BAC) library has been constructed from apple (Malus×domestica Borkh.) using the variety “Florina”, which is resistant to scab (Venturia inaequalis) by virtue of the Vf gene. Since apple leaves are rich in polyphenols, high-molecular-weight DNA was extracted from leaf nuclei with a protocol adapted to apple. The nuclei were then embedded in agarose microbeads and partially digested by varying ratios of EcoRI to EcoRI methylase. The resulting DNA fragments were size-selected by pulsed-field gel electrophoresis, ligated to the BAC cloning vector pECBAC1 and transformed into Escherichia coli cells by electroporation. A total of 36 864 recombinant clones (BACs) were obtained. The library has an average insert size of 120 kb and represents approximately 5×apple haploid-genome equivalents. It was screened with six cDNA probes using the chemiluminescent DIG system. An average of 4.4 clones was detected for each locus. The apple BAC library will be used to isolate the Vf scab resistance gene through map-based cloning. In this connection the library was screened with a marker closely linked to the Vf gene and six positive clones have been isolated. This library should thus be well suited for map-based gene cloning, in particular for the isolation of the Vf gene and for the construction of a physical map of the apple genome. Received: 19 February 1998 / Accepted: 30 April 1998     

A sunflower BAC library suitable for PCR screening and physical mapping of targeted genomic regions

A sunflower BAC library consisting of 147,456 clones with an average size of 118 kb has been constructed and characterized. It represents approximately 5× sunflower haploid genome equivalents. The BAC library has been arranged in pools and superpools of DNA allowing screening with various PCR-based markers. Each of the 32 superpools contains 4,608 clones and corresponds to a 36 matrix pools. Thus, the screening of the entire library could be accomplished in less than 80 PCR reactions including positive and negative controls. As a demonstration of the feasibility of the concept, a set of 24 SSR markers covering about 36 cM in the sunflower SSR map (Tang et al. in Theor Appl Genet 105:1124–1136, 2002) have been used to screen the BAC library. About 125 BAC clones have been identified and then organized in 23 contigs by HindIII digestion. The contigs are anchored on the SSR map and thus constitutes a first-generation physical map of this region. The utility of this BAC library as a genomic resource for physical mapping and map-based cloning in sunflower is discussed.     

DNA markers linked to eastern filbert blight resistance in ��Ratoli�� hazelnut (Corylus avellana L.)

Eastern filbert blight (EFB), caused by the pyrenomycete Anisogramma anomala (Peck) E. Müller, is a major disease problem and production constraint in orchards of European hazelnut (Corylus avellana L.) in Oregon’s Willamette Valley. Host genetic resistance is viewed as the most economical means of controlling this disease. A dominant resistance gene from “Gasaway” has been used extensively in the hazelnut breeding program at Oregon State University, but concern about the durability of a single resistance gene stimulated a search for new sources of resistance. “Ratoli,” a minor cultivar from Spain, showed no signs or symptoms of the fungus following a series of inoculations. The objective of this study was to study segregation for disease response in two progenies from crosses of Ratoli with susceptible selections and identify linked DNA markers. About half of the seedlings were resistant, suggesting control by a dominant allele at a single locus. A total of 900 random amplified polymorphic DNA (RAPD) primers and 64 amplified fragment length polymorphism (AFLP) primer combinations were screened. Four RAPD markers and two ALFP markers were identified and a linkage map constructed. On this map, disease resistance was flanked by AFLP marker C4-255 and RAPD marker G17-800 at distances of 0.4 cM and 2.8 cM, respectively. Based on co-segregation with SSR markers, Ratoli resistance was assigned to linkage group 7 while Gasaway resistance is on linkage group 6. Ratoli provides a novel source of EFB resistance, and robust RAPD marker G17-800 is useful for marker-assisted selection.