Two broad-spectrum blast resistance genes,<Emphasis Type="Italic"> Pi9</Emphasis>(<Emphasis Type="Italic">t</Emphasis>) and<Emphasis Type="Italic"> Pi2</Emphasis>(<Emphasis Type="Italic">t</Emphasis>), are physically linked on rice chromosome 6

To understand the molecular basis of broad-spectrum resistance to rice blast, fine-scale mapping of the two blast resistance (R) genes, Pi9( t) and Pi2( t), was conducted. These two genes were introgressed from different resistance donors, previously reported to confer resistance to many blast isolates in the Philippines, and were mapped to an approximately 10-cM interval on chromosome 6. To further test their resistance spectrum, 43 blast isolates collected from 13 countries were used to inoculate the Pi2( t) and Pi9( t) plants. Pi9( t)-bearing lines were highly resistant to all isolates tested, and lines carrying Pi2( t) were resistant to 36 isolates, confirming the broad-spectrum resistance of these two genes to diverse blast isolates. Three RAPD markers tightly linked to Pi9( t) were identified using the bulk segregant analysis technique. Twelve positive bacterial artificial chromosome (BAC) clones were identified and a BAC contig covering about 100 kb was constructed when the Pi9( t) BAC library was screened with one of the markers. A high-resolution map of Pi9( t) was constructed using BAC ends. The Pi2( t) gene was tightly linked to all of the Pi9( t) markers in 450 F(2) plants. These data suggest that Pi9( t) and Pi2( t) are either allelic or tightly linked in an approximately 100-kb region. The mapping results for Pi9( t) and Pi2( t) provide essential information for the positional cloning of these two important blast resistance genes in rice.     

High-resolution mapping of the leaf rust disease resistance gene <Emphasis Type="Italic">Lr1</Emphasis> in wheat and characterization of BAC clones from the <Emphasis Type="Italic">Lr1</Emphasis> locus

Leaf rust is the most common disease in wheat production. There are more than 45 specific resistance genes described and used in wheat breeding to control epidemics of leaf rust, but none of them has been cloned. The leaf rust disease resistance gene 1 ( Lr1) is a good model gene for isolation by map-based cloning because it is a single, dominant gene which is located in the distal region of chromosome 5DL of wheat. As the first step towards the isolation of this gene we constructed a high-resolution genetic map in the region of the Lr1 locus by saturation mapping of two large segregating F(2) populations (Thatcher Lr1 x Thatcher, Thatcher Lr1 x Frisal). The resistance gene Lr1 was delimited in a 0.16-cM region between the RFLP markers ABC718 and PSR567 (0.12 cM from ABC718 and 0.04 cM from PSR567). A genomic BAC library of Aegilops tauschii (D genome) was screened using the RFLP markers ABC718 and PSR567. Five positive BAC clones were identified by ABC718 and four clones by PSR567. Two NBS-LRR type of resistance gene analogs, which encode proteins highly homologous to the bacterial blight disease resistance protein Xa1 of rice, were identified on BAC clones isolated with PSR567. Polymorphic BAC end probes were isolated from both ends of a 105-kb large BAC clone identified by ABC718. The end probes were mapped at the same locus as ABC718, and no recombination event was found within 105 kb around ABC718 in our analysis of more than 4,000 gametes.     

Estimates of conserved microsynteny among the genomes of <Emphasis Type="Italic">Glycine max</Emphasis>, <Emphasis Type="Italic">Medicago truncatula</Emphasis> and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A growing body of research indicates that microsynteny is common among dicot genomes. However, most studies focus on just one or a few genomic regions, so the extent of microsynteny across entire genomes remains poorly characterized. To estimate the level of microsynteny between Medicago truncatula (Mt) and Glycine max (soybean), and also among homoeologous segments of soybean, we used a hybridization strategy involving bacterial artificial chromosome (BAC) contigs. A Mt BAC library consisting of 30,720 clones was screened with a total of 187 soybean BAC subclones and restriction fragment length polymorphism (RFLP) probes. These probes came from 50 soybean contig groups, defined as one or more related BAC contigs anchored by the same low-copy probe. In addition, 92 whole soybean BAC clones were hybridized to filters of HindIII-digested Mt BAC DNA to identify additional cases of cross-hybridization after removal of those soybean BACs found to be repetitive in Mt. Microsynteny was inferred when at least two low-copy probes from a single soybean contig hybridized to the same Mt BAC or when a soybean BAC clone hybridized to three or more low-copy fragments from a single Mt BAC. Of the 50 soybean contig groups examined, 54% showed microsynteny to Mt. The degree of conservation among 37 groups of soybean contigs was also investigated. The results indicated substantial conservation among soybean contigs in the same group, with 86.5% of the groups showing at least some level of microsynteny. One contig group was examined in detail by a combination of physical mapping and comparative sequencing of homoeologous segments. A TBLASTX similarity search was performed between 1,085 soybean sequences on the 50 BAC contig groups and the entire Arabidopsis genome. Based on a criterion of sequence homologues <100 kb apart, each with an expected value of < or =1e-07, seven of the 50 soybean contig groups (14%) exhibited microsynteny with Arabidopsis.     

A bacterial artificial chromosome (BAC) library of sugar beet and a physical map of the region encompassing the bolting gene <Emphasis Type="Italic"> B</Emphasis>

In sugar beet (Beta vulgaris L.), early bolting is caused by a single dominant gene, designated B. Twenty AFLP markers selected from a 7.8-cM segment of the B region on chromosome 2 were used to screen a YAC library, and a first-generation physical map including the B gene, made up of 11 YACs, was established. Because the genome coverage of the YAC library was low, a BAC library was constructed in the vector pBeloBAC11. This library consists of 57,600 clones with an average insert size of 116 kb, corresponding to 8.8 genome equivalents. Screening of the BAC library with chloroplast and mitochondrial DNA probes indicated that less than 0.1% of the clones contained organelle-derived DNA. To fill the gaps in the physical map around the B gene, the BAC library was screened with four AFLP markers and 10 YAC-derived probes. In total, 54 different BACs were identified. Overlaps between BACs were detected by using BAC termini amplified by PCR as probes, and by RFLP fingerprinting. In this way, a minimal tiling path of the central 4.6-cM region was constructed, which consists of 14 BACs. The B locus was localized to a 360-kb contig, a size which makes positional cloning of the gene feasible.     

Development of SNP markers linked to the <Emphasis Type="Italic">L</Emphasis> locus in <Emphasis Type="Italic">Capsicum</Emphasis> spp. by a comparative genetic analysis

In pepper, the TMV resistance locus L is syntenic to the tomato I2 and the potato R3 loci on chromosome 11. In this report, we identified pepper bacterial artificial chromosome (BAC) clones corresponding to the I2 and R3 loci and developed L-linked markers using the BAC sequence information. A BAC library was screened using the tomato I2C-1 gene as a probe. The resulting clones were sorted further by PCR screening, sequencing, and genetic mapping. A linkage analysis revealed that BAC clone 082F03 could be anchored to the target region near TG36 on chromosome 11. Using the 082F03 sequence, more BAC clones were identified and a BAC contig spanning 224 kb was constructed. Gene prediction analysis showed that there were at least three I2/R3 R gene analogs (RGAs) in the BAC contig. Three DNA markers closely linked (about 1.2 cM) to the L ⁴ gene were developed by using the BAC contig sequence. The single nucleotide polymorphism marker 087H3T7 developed in this study was subjected to linkage analysis in L ⁴ - and L ³ -segregating populations together with previously developed markers. The 189D23M marker, which is known to co-segregate with L ³ , was located on the opposite side of 087H3T7, about 0.7 cM away from L ⁴ . This supports the idea that L ³ and L ⁴ may be different genes closely linked within the region instead of different alleles at the same locus. Finally, use of flanking markers in molecular breeding program for introgression of L ⁴ to elite germplasm against most aggressive tobamoviruses pathotype P_1,2,3 is discussed.     

Identification of candidate genes at the <Emphasis Type="Italic">Dp-fl</Emphasis> locus conferring resistance against the rosy apple aphid <Emphasis Type="Italic">Dysaphis plantaginea</Emphasis>

The cultivated apple is susceptible to several pests including the rosy apple aphid (RAA; Dysaphis plantaginea Passerini), control of which is mainly based on chemical treatments. A few cases of resistance to aphids have been described in apple germplasm resources, laying the basis for the development of new resistant cultivars by breeding. The cultivar ‘Florina’ is resistant to RAA, and recently, the Dp-fl locus responsible for its resistance was mapped on linkage group 8 of the apple genome. In this paper, a chromosome walking approach was performed by using a ‘Florina’ bacterial artificial chromosome (BAC) library. The walking started from the available tightly linked molecular markers flanking the resistance region. Various walking steps were performed in order to identify the minimum tiling path of BAC clones covering the Dp-fl region from both the “resistant” and “susceptible” chromosomes of ‘Florina’. A genomic region of about 279 Kb encompassing the Dp-fl resistance locus was fully sequenced by the PacBio technology. Through the development of new polymorphic markers, the mapping interval around the resistance locus was narrowed down to a physical region of 95 Kb. The annotation of this sequence resulted in the identification of four candidate genes putatively involved in the RAA resistance response.     

Construction of a<Emphasis Type="Italic"> Hin</Emphasis>dIII Bacterial Artificial Chromosome library and its use in identification of clones associated with disease resistance in chickpea

A chickpea (Cicer arietinum L.) Bacterial Artificial Chromosome (BAC) library from germplasm line, FLIP 84-92C, was constructed to facilitate positional cloning of disease resistance genes and physical mapping of the genome. The BAC library has 23,780 colonies and was calculated to comprise approximately 3.8 haploid-genome equivalents. Studies on 120 randomly chosen clones revealed an average insert size of 100 kb and no empty clones. Colony hybridization using the RUBP carboxylase large subunit as a probe resulted in a very low percentage of chloroplast DNA contamination. Two clones with a combined insert size of 200 kb were isolated after the library was screened with a Sequence Tagged Microsatellite Site (STMS) marker, Ta96, which is tightly linked to a gene (Foc3) for resistance to fusarium wilt caused by Fusarium oxysporum Schlechtend.: Fr. f. sp. ciceris (Padwick) race 3 at a genetic distance of 1 cM. Also, these two clones were analyzed with several resistance gene analog (RGA) markers. End sequencing of these clones did not identify repetitive sequences. The development of the BAC library will facilitate isolation of Foc3 and allow us to perform physical mapping of this genomic region where additional R genes against other races of the wilt causing pathogen are positioned.Communicated by C. Möllers     

Construction of a BAC library and a physical map of a major QTL for CBB resistance of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

A major quantitative trait loci (QTL) conditioning common bacterial blight (CBB) resistance in common bean (Phaseolus vulgaris L.) lines HR45 and HR67 was derived from XAN159, a resistant line obtained from an interspecific cross between common bean lines and the tepary bean (P. acutifolius L.) line PI319443. This source of CBB resistance is widely used in bean breeding. Several other CBB resistance QTL have been identified but none of them have been physically mapped. Four molecular markers tightly linked to this QTL have been identified suitable for marker assisted selection and physical mapping of the resistance gene. A bacterial artificial chromosome (BAC) library was constructed from high molecular weight DNA of HR45 and is composed of 33,024 clones. The size of individual BAC clone inserts ranges from 30 kb to 280 kb with an average size of 107 kb. The library is estimated to represent approximately sixfold genome coverage. The BAC library was screened as BAC pools using four PCR-based molecular markers. Two to seven BAC clones were identified by each marker. Two clones were found to have both markers PV-tttc001 and STS183. One preliminary contig was assembled based on DNA finger printing of those positive BAC clones. The minimum tiling path of the contig contains 6 BAC clones spanning an estimated size of 750 kb covering the QTL region.     

Construction and characterization of a half million clone BAC library of durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis>)

Durum wheat (Triticum turgidum ssp. durum, 2n = 4x = 28, genomes AB) is an economically important cereal used as the raw material to make pasta and semolina. In this paper we present the construction and characterization of a bacterial artificial chromosome (BAC) library of tetraploid durum wheat cv. Langdon. This variety was selected because of the availability of substitution lines that facilitate the assignment of BACs to the A and B genome. The selected Langdon line has a 30-cM segment of chromosome 6BS from T. turgidum ssp. dicoccoides carrying a gene for high grain protein content, the target of a positional cloning effort in our laboratory. A total of 516,096 clones were organized in 1,344 384-well plates and blotted on 28 high-density filters. Ninety-eight percent of these clones had wheat DNA inserts (0.3% chloroplast DNA, 1.4% empty clones and 0.3% empty wells). The average insert size of 500 randomly selected BAC clones was 131 kb, resulting in a coverage of 5.1-fold genome equivalents for each of the two genomes, and a 99.4% probability of recovering any gene from each of the two genomes of durum wheat. Six known copy-number probes were used to validate this theoretical coverage and gave an estimated coverage of 5.8-fold genome equivalents. Screening of the library with 11 probes related to grain storage proteins and starch biosynthesis showed that the library contains several clones for each of these genes, confirming the value of the library in characterizing the organization of these important gene families. In addition, characterization of fingerprints from colinear BACs from the A and B genomes showed a large differentiation between the A and B genomes. This library will be a useful tool for evolutionary studies in one of the best characterized polyploid systems and a source of valuable genes for wheat. Clones and high-density filters can be requested at Communicated by P. LangridgeThe first two authors contributed equally to the investigation     

Sugar beet BAC library construction and assembly of a contig spanning <Emphasis Type="Italic">Rf1</Emphasis>, a restorer-of-fertility gene for Owen cytoplasmic male sterility

Rf1 is a nuclear gene that controls fertility restoration in cases of cytoplasmic male sterility caused by the Owen cytoplasm in sugar beet. In order to isolate the gene by positional cloning, a BAC library was constructed from a restorer line, NK198, with the genotype Rf1Rf1. The library contained 32,180 clones with an average insert size of 97.8 kb, providing 3.4 genome equivalents. Five AFLP markers closely linked to Rf1 were used to screen the library. As a result, we identified eight different BAC clones that were clustered into two contigs. The gap between the two contigs was filled by chromosome walking. To map the Rf1 region in more detail, we developed five cleaved amplified polymorphic sequence (CAPS) markers from the BAC DNAs identified, and carried out genotyping of 509 plants in the mapping population with the Rf1-flanking AFLP and CAPS markers. Thirteen plants in which recombination events had occurred in the vicinity of the Rf1 locus were identified and used to map the molecular markers relative to each other and to Rf1. In this way, we were able to restrict the possible location of the Rf1 gene to a minimum of six BAC clones spanning an interval of approximately 250 kb. The first two authors contributed equally to this work.     

Creation of BAC genomic resources for cocoa (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) for physical mapping of RGA containing BAC clones

We have constructed and validated the first cocoa (Theobroma cacao L.) BAC library, with the aim of developing molecular resources to study the structure and evolution of the genome of this perennial crop. This library contains 36,864 clones with an average insert size of 120 kb, representing approximately ten haploid genome equivalents. It was constructed from the genotype Scavina-6 (Sca-6), a Forastero clone highly resistant to cocoa pathogens and a parent of existing mapping populations. Validation of the BAC library was carried out with a set of 13 genetically-anchored single copy and one duplicated markers. An average of nine BAC clones per probe was identified, giving an initial experimental estimation of the genome coverage represented in the library. Screening of the library with a set of resistance gene analogues (RGAs), previously mapped in cocoa and co-localizing with QTL for resistance to Phytophthora traits, confirmed at the physical level the tight clustering of RGAs in the cocoa genome and provided the first insights into the relationships between genetic and physical distances in the cocoa genome. This library represents an available BAC resource for structural genomic studies or map-based cloning of genes corresponding to important QTLs for agronomic traits such as resistance genes to major cocoa pathogens like Phytophthora spp (palmivora and megakarya), Crinipellis perniciosa and Moniliophthora roreri.Communicated by J.W. Snape     

Chromosome landing at the bacterial blight resistance gene<Emphasis Type="Italic"> Xa4</Emphasis> locus using a deep coverage rice BAC library

Xa4 is a dominantly inherited rice gene that confers resistance to Philippine race 1 of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae in rice. In order to isolate the gene by positional cloning, a bacterial artificial chromosome (BAC) library was constructed from genomic DNA isolated from an Xa4-harboring accession, IRBB56. The library contains 55,296 clones with an average insert size of 132 kb, providing 14 rice genome equivalents. Three DNA markers closely linked to Xa4 were used to screen the library. The marker RS13, a resistance gene analogue that co-segregates with Xa4, identified 18 clones, of which four and six, respectively, were simultaneously detected by the other two markers, G181 and L1044. Fingerprinting and Southern analysis indicated that these clones overlapped and define an interval spanning 420 kb. In an F2 population derived from an indica variety, IR24, and its Xa4-containing near isogenic line (NIL), IRBB4, the susceptible plants were screened in order to map the Xa4 gene genetically and physically. Out of 24 insert ends isolated from the BACs in the contig, three revealed polymorphisms between IR24 and IRBB4. Two insert ends, 56M22F and 26D24R, flanked Xa4 on each side. Based on the overlap of the BACs, six overlapping clones were considered to include the Xa4 allele, one of which, 106P13, was chosen for further investigation.     

Construction of two BAC libraries from the wild Mexican diploid potato,<Emphasis Type="Italic"> Solanum pinnatisectum,</Emphasis> and the identification of clones near the late blight and Colorado potato beetle resistance loci

To facilitate isolation and characterization of disease and insect resistance genes important to potato, two bacterial artificial chromosome (BAC) libraries were constructed from genomic DNA of the Mexican wild diploid species, Solanum pinnatisectum, which carries high levels of resistance to the most important potato pathogen and pest, the late blight and the Colorado potato beetle (CPB). One of the libraries was constructed from the DNA, partially digested with BamHI, and it consists of 40,328 clones with an average insert size of 125 kb. The other library was constructed from the DNA partially digested with EcoRI, and it consists of 17,280 clones with an average insert size of 135 kb. The two libraries, together, represent approximately six equivalents of the wild potato haploid genome. Both libraries were evaluated for contamination with organellar DNA sequences and were shown to have a very low percentage (0.65–0.91%) of clones derived from the chloroplast genome. High-density filters, prepared from the two libraries, were screened with ten restriction fragment length polymorphism (RFLP) markers linked to the resistance genes for late blight, CPB, Verticillium wilt and potato cyst nematodes, and the gene Sr1 for the self-incompatibility S-locus. Thirty nine positive clones were identified and at least two positive BAC clones were detected for each RFLP marker. Four markers that are linked to the late blight resistance gene Rpi1 hybridized to 14 BAC clones. Fifteen BAC clones were shown to harbor the PPO (polyphenol oxidase) locus for the CPB resistance by three RFLP probes. Two RFLP markers detected five BAC clones that were linked to the Sr1 gene for self-incompatibility. These results agree with the librarys predicted extent of coverage of the potato genome, and indicated that the libraries are useful resources for the molecular isolation of disease and insect resistance genes, as well as other economically important genes in the wild potato species. The development of the two potato BAC libraries provides a starting point, and landmarks for BAC contig construction and chromosome walking towards the map-based cloning of agronomically important target genes in the species.Communicated by H.F. Linskens     

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.     

Organization of phenylalanine ammonia lyase (<Emphasis Type="Italic">PAL</Emphasis>), acidic <Emphasis Type="Italic">PR-5</Emphasis> and osmotin-like (<Emphasis Type="Italic">OSM</Emphasis>) defence-response gene families in the potato genome

Defence-response (DR) genes are candidates for the genetic functions underlying quantitative resistance to plant pathogens. The organization of three DR gene families encoding phenylalanine ammonia lyase (PAL), acidic PR-(pathogenesis-related) protein 5, and basic PR-5, or osmotin-like (OSM), proteins was studied in the potato genome. A bacterial artificial chromosome (BAC) library containing ~50,000 clones was constructed from high-molecular weight genomic DNA of the diploid potato clone PD59, a hybrid between Solanum tuberosum and S. phureja. BAC clones carrying one or more copies of the DR genes were identified and characterized by Southern hybridization, sequence analysis and genetic mapping. PAL, acidic PR-5 and OSM (basic PR-5) genes were all organized into gene families of varying complexity. The PAL gene family consisted of at least 16 members, several of which were physically linked. Four acidic PR-5 homologous were localized to a 45-kb segment on potato chromosome XII. One of these, PR-5/319, codes for the acidic thaumatin-like protein C found in intercellular fluids of potato. Nine OSM genes were organized at two loci: eight form a 90-kb cluster on chromosome VIII, and a single gene was found on chromosome XI. The topology of a phylogenetic tree based on PR-5 and OSM protein sequences from Solanaceae suggests a mode of evolution for these gene families. The results will form the basis for further studies on the potential role of these defence-related loci in quantitative resistance to pathogens.     

Construction and characterisation of a BAC library for genome analysis of the allotetraploid coffee species (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

In order to promote genome research on coffee trees, one of the most important tropical crops, a bacterial artificial chromosome (BAC) library of the coffee allotetraploid species, Coffea arabica, was constructed. The variety IAPAR 59, which is widely distributed in Latin America and exhibits a fair level of resistance to several pathogens, was chosen. High-efficiency BAC cloning of the high molecular weight genomic DNA partially digested by HindIII was achieved. In total, the library contains 88,813 clones with an average insert size of 130 kb, and represents approximately eight C. arabica dihaploid genome equivalents. One original feature of this library is that it can be divided into four sublibraries with mean insert sizes of 96, 130, 183 and 210 kb. Characterisation of the library showed that less than 4.5% of the clones contained organelle DNA. Furthermore, this library is representative and shows good genome coverage, as established by hybridisation screening of high-density filters using a number of nuclear probes distributed across the allotetraploid genome. This Arabica BAC library, the first large-insert DNA library so far constructed for the genus Coffea, is well-suited for many applications in genome research, including physical mapping, map-based cloning, functional and comparative genomics as well as polyploid genome analyses.Communicated by J.W. Snape     

Concomitant reiterative BAC walking and fine genetic mapping enable physical map development for the broad-spectrum late blight resistance region,<Emphasis Type="Italic"> RB</Emphasis>

The wild potato species Solanum bulbocastanum is a source of genes for potent late blight resistance. We previously mapped resistance to a single region of the S. bulbocastanum chromosome 8 and named the region RB (for "Resistance from S. Bulbocastanum "). We now report physical mapping and contig construction for the RB region via a novel reiterative method of BAC walking and concomitant fine genetic mapping. BAC walking was initiated using RFLP markers previously shown to be associated with late blight resistance. Subcontig extension was accomplished using new probes developed from BAC ends. Significantly, BAC end and partial BAC sequences were also used to develop PCR-based markers to enhance map resolution in the RB region. As they were developed from BAC clones of known position relative to RB, our PCR-based markers are known a priori to be physically closer to the resistance region. These markers allowed the efficient screening of large numbers of segregating progeny at the cotyledon stage, and permitted us to assign the resistance phenotype to a region of approximately 55 kb. Our markers also directed BAC walking efforts away from regions distantly related to RB in favor of the 55-kb region. Because the S. bulbocastanum genotype used in BAC library construction is heterozygous for RB (RB/rb), codominant PCR-based markers, originally developed for fine-scale mapping, were also used to determine homolog origins for individual BAC clones. Ultimately, BAC contigs were constructed for the RB region from both resistant (RB) and susceptible (rb) homologs.Communicated by R. Hagemann     

<Emphasis Type="Italic">STAYGREEN</Emphasis> (<Emphasis Type="Italic">CsSGR</Emphasis>) is a candidate for the anthracnose (<Emphasis Type="Italic">Colletotrichum orbiculare</Emphasis>) resistance locus <Emphasis Type="Italic">cla</Emphasis> in Gy14 cucumber

Key message

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants.

Abstract

Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14?×?9930 recombinant inbred lines and 1043 F₂ individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

     

Construction and characterization of a bacterial artificial chromosome library of banana (<Emphasis Type="Italic">Musa acuminata</Emphasis> Colla)

A bacterial artificial chromosome (BAC) library for banana was constructed from leaves of the wild diploid 'Calcutta 4' clone (Musa acuminata subsp. Burmannicoides 2n = 2 x = 22). 'Calcutta 4' is widely used in breeding programs for its resistance to the current major disease of banana and is being used to build a genetic reference map of banana. As banana leaves are particularly rich in polyphenols and polysaccharides a protocol was adapted to isolate intact nuclei and high-molecular-weight (HMW) DNA. A total of 55,152 clones with an average insert size of 100 kb were picked. The frequency of BAC clones carrying inserts derived from chloroplast and mitochondrial DNA was estimated to be 1.5%. The coverage of the library is equivalent to 9.0-times the haploid genome. The BAC library was screened with 13 RFLP probes belonging to the 8 linkage groups of the consensus molecular map of banana. A total of 135 clones were identified giving an average of 10.38 clones for each locus. This BAC library will be a valuable starting tool for many of the goals of the recently emerged International Musa Genomic Consortium. One of our initial objectives will be to develop a banana physical map by BAC-FISH (fluorescent in situ hybridization) viewing the characterization of translocation break points.     

Physical analysis of the complex rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.) <Emphasis Type="Italic">Alt4</Emphasis> aluminium (aluminum) tolerance locus using a whole-genome BAC library of rye cv. Blanco

Rye is a diploid crop species with many outstanding qualities, and is important as a source of new traits for wheat and triticale improvement. Rye is highly tolerant of aluminum (Al) toxicity, and possesses a complex structure at the Alt4 Al tolerance locus not found at the corresponding locus in wheat. Here we describe a BAC library of rye cv. Blanco, representing a valuable resource for rye molecular genetic studies, and assess the library’s suitability for investigating Al tolerance genes. The library provides 6 × genome coverage of the 8.1 Gb rye genome, has an average insert size of 131 kb, and contains only ~2% of empty or organelle-derived clones. Genetic analysis attributed the Al tolerance of Blanco to the Alt4 locus on the short arm of chromosome 7R, and revealed the presence of multiple allelic variants (haplotypes) of the Alt4 locus in the BAC library. BAC clones containing ALMT1 gene clusters from several Alt4 haplotypes were identified, and will provide useful starting points for exploring the basis for the structural variability and functional specialization of ALMT1 genes at this locus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.