Construction of BAC and BIBAC libraries from sunflower and identification of linkage group-specific clones by overgo hybridization

Complementary BAC and BIBAC libraries were constructed from nuclear DNA of sunflower cultivar HA 89. The BAC library, constructed with BamHI in the pECBAC1 vector, contains 107,136 clones and has an average insert size of 140 kb. The BIBAC library was constructed with HindIII in the plant-transformation-competent binary vector pCLD04541 and contains 84,864 clones, with an average insert size of 137 kb. The two libraries combined contain 192,000 clones and are equivalent to approximately 8.9 haploid genomes of sunflower (3,000 Mb/1C), and provide a greater than 99% probability of obtaining a clone of interest. The frequencies of BAC and BIBAC clones carrying chloroplast or mitochondrial DNA sequences were estimated to be 2.35 and 0.04%, respectively, and insert-empty clones were less than 0.5%. To facilitate chromosome engineering and anchor the sunflower genetic map to its chromosomes, one to three single- or low-copy RFLP markers from each linkage group of sunflower were used to design pairs of overlapping oligonucleotides (overgos). Thirty-six overgos were designed and pooled as probes to screen a subset (5.1×) of the BAC and BIBAC libraries. Of the 36 overgos, 33 (92%) gave at least one positive clone and 3 (8%) failed to hit any clone. As a result, 195 BAC and BIBAC clones representing 19 linkage groups were identified, including 76 BAC clones and 119 BIBAC clones, further verifying the genome coverage and utility of the libraries. These BAC and BIBAC libraries and linkage group-specific clones provide resources essential for comprehensive research of the sunflower genome.     

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

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

A plant-transformation-competent BIBAC library from the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Landsberg ecotype for functional and comparative genomics

The genome of the model plant Arabidopsis thaliana has been sequenced to near completion. To facilitate experimental determination of the function of every gene in the species, we constructed a large-insert library from the Landsberg ecotype using a plant-transformation-competent binary BAC vector, BIBAC2. The library contains 11,520 clones with an estimated average insert size of 162 kb. Of a sample of 102 clones, 17.6% had no inserts; further, in the library as a whole, 287 clones contained chloroplast DNA, and 25 contained mitochondrial DNA. Thus it is estimated that 9,295 clones originated from the nuclear genome, representing a 11.5 x coverage. The library was further characterized by screening with probes corresponding to 180-bp repeats, 5S rDNA, 18S-25S rDNA and 23 single-copy RFLP markers. The results showed that 92 clones contained 180-bp centromeric repeats, 78 contained 5S rDNA and 95 contained 18S-25S rDNA, approximately 1%, 0.8% and 1%, respectively, of the nuclear clones in the library. Screening the library with the 23 RFLP markers showed that each one hybridized to an average of seven clones. This library is the first large-insert DNA library for the widely studied Landsberg erecta strain. It will greatly facilitate gene identification by complementation screening, and will enhance analysis of the structure, organization and evolution of the A. thaliana genome.     

Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor.

The construction of representative large insert DNA libraries is critical for the analysis of complex genomes. The predominant vector system for such work is the yeast artificial chromosome (YAC) system. Despite the success of YACs, many problems have been described including: chimerism, tedious steps in library construction and low yields of YAC insert DNA. Recently a new E.coli based system has been developed, the bacterial artificial chromosome (BAC) system, which offers many potential advantages over YACs. We tested the BAC system in plants by constructing an ordered 13,440 clone sorghum BAC library. The library has a combined average insert size, from single and double size selections, of 157 kb. Sorghum inserts of up to 315 kb were isolated and shown to be stable when grown for over 100 generations in liquid media. No chimeric clones were detected as determined by fluorescence in situ hybridization of ten BAC clones to metaphase and interphase S.bicolor nuclei. The library was screened with six sorghum probes and three maize probes and all but one sorghum probe hybridized to at least one BAC clone in the library. To facilitate chromosome walking with the BAC system, methods were developed to isolate the proximal ends of restriction fragments inserted into the BAC vector and used to isolate both the left and right ends of six randomly selected BAC clones. These results demonstrate that the S. bicolor BAC library will be useful for several physical mapping and map-based cloning applications not only in sorghum but other related cereal genomes, such as maize. Furthermore, we conclude that the BAC system is suitable for most large genome applications, is more 'user friendly' than the YAC system, and will likely lead to rapid progress in cloning biologically significant genes from plants.     

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.     

Construction and characterization of a peanut HindIII BAC library

Bacterial artificial chromosome (BAC) libraries have been an essential tool for physical analyses of genomes of many crops. We constructed and characterized the first large-insert DNA library for Arachis hypogaea L. The HindIII BAC library contains 182,784 clones; only 5,484 (3%) had no inserts; and the average insert size is 104.05 kb. Chloroplast DNA contamination was very low, only nine clones, and r-DNA content was 1,208, 0.66% of clones. The depth of coverage is estimated to be 6.5 genome-equivalents, allowing the isolation of virtually any single-copy locus. This rate of coverage was confirmed with the application of 20 overgos, which identified 305 positive clones from the library. The identification of multiple loci by most probes in polyploids complicates anchoring of physical and genetic maps. We explored the practicality of a hybridization-based approach for determination of map locations of BAC clones in peanut by analyzing 94 clones detected by seven different overgos. The banding patterns on Southern blots were good predictors of contig composition; that is, the clones that shared the same size bands and ascribed to the same overgos usually also located in the same contigs. This BAC library has great potential to advance future research about the peanut genome.Requests for the BAC library (or subsets) should be directed to Dr. A. Paterson (paterson@uga.edu).     

Construction of tomato genomic DNA libraries in a binary-BAC (BIBAC) vector

This is the first report of large insert genomic DNA libraries constructed in a binary-BAC (BIBAC) vector. Genomic DNA libraries containing approximately 4.6 haploid nuclear genomic equivalents were constructed for Lycopersicon esculentum (cv. Mogeor) and Lycopersicon pennellii (LA716) in the BIBAC2 vector. The L. esculentum library has an average insert size of 125 kb and is comprised of 42 272 individual colonies stored as frozen cultures in a 384-well format (108 plates). The L. pennellii library has an average insert size of 90 kb and is comprised of 53 760 individual clones (140 384-well plates). In each of the libraries, it is estimated that 90% of the colonies contain genomic DNA inserts. The composition of the L. esculentum and L. pennellii libraries was determined by analyzing a series of randomly selected clones. The L. esculentum library was surveyed for clones containing chloroplast DNA (1.4%), mitochondrial DNA (0.012%) and repetitive DNA motifs. BIBAC clones that may contain a gene of interest can be identified from these libraries by colony hybridization with homologous or heterologous probes or by PCR pooling techniques. Once identified, BIBAC genomic DNA library clones are immediately suitable for Agrobacterium tumefaciens-mediated plant transformation.     

Construction of BAC and BIBAC libraries and their applications for generation of SSR markers for genome analysis of chickpea, Cicer arietinum L.

Large-insert bacterial artificial chromosome (BAC) libraries, plant-transformation-competent binary BAC (BIBAC) libraries, and simple sequence repeat (SSR) markers are essential for many aspects of genomics research. We constructed a BAC library and a BIBAC library from the nuclear DNA of chickpea, Cicer arietinum L., cv. Hadas, partially digested with HindIII and BamHI, respectively. The BAC library has 14,976 clones, with an average insert size of 121 kb, and the BIBAC library consists of 23,040 clones, with an average insert size of 145 kb. The combined libraries collectively cover ca. 7.0× genomes of chickpea. We screened the BAC library with eight synthetic SSR oligos, (GA)₁₀, (GAA)₇, (AT)₁₀, (TAA)₇, (TGA)₇, (CA)₁₀, (CAA)₇, and (CCA)₇. Positive BACs were selected, subcloned, and sequenced for SSR marker development. Two hundred and thirty-three new chickpea SSR markers were developed and characterized by PCR, using chickpea DNA as template. These results have demonstrated that BACs are an excellent source for SSR marker development in chickpea. We also estimated the distribution of the SSR loci in the chickpea genome. The SSR motifs (TAA)_n and (GA)_n were much more abundant than the others, and the distribution of the SSR loci appeared non-random. The BAC and BIBAC libraries and new SSR markers will provide valuable resources for chickpea genomics research and breeding (the libraries and their filters are available to the public at ).J. Lichtenzveig and C. Scheuring contributed equally to this study.     

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.     

Construction and characterization of a bacterial artificial chromosome library from chili pepper

A library of the bacterial artificial chromosome (BAC) that consisted of a total of 78,336 clones with an average insert size of 80 kb was constructed from Capsicum annuum, 'CM334', which is resistant to Phytophthora capsici and PVY. Based on a haploid genome size of pepper of 2,702 Mbp/C, the BAC library was estimated to contain approximately three genome equivalents and represented at least 90% of the pepper genome. In order to determine the percentage of BAC clones that contained mitochondrial DNAs, the entire library was screened with probes of chili pepper mitochondrial DNAs. The result showed that only twenty-five clones, which is 0.03% of the total BAC clones, were hybridized to mitochondrial gene probes. This indicates that the library is comprised predominantly of the nuclear sequences. The library was also tested for isolating specific clones by screening with a few known genes from the chili pepper, phytoene synthase gene, and two MADS genes--HpMADS1 and HpMADS3. The result showed that the three clones for phytoene synthase and the two clones for each MADS gene were positively hybridized to the specific probes. This indicates that the library is highly reliable and represents a resource for initiating map-based cloning and contig mapping in chili pepper.     

Construction and Characterization of aMagnaporthe griseaBacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of aMagnaporthe griseabacterial artificial chromosome library.Fungal Genet. Biol.20,280–288. A bacterial artificial chromosome (BAC) library ofMagnaporthe griseacontaining 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents ofM. griseaand has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using alys1-3auxotroph, we have shown that BAC clones at least 113 kb can be transformed intoM. griseato screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.     

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.     

Two large-insert soybean genomic libraries constructed in a binary vector: applications in chromosome walking and genome wide physical mapping

Large DNA insert libraries in binary T-DNA vectors can assist in the isolation of the gene(s) underlying a quantitative trait locus (QTL). Binary vectors facilitate the transfer of large-insert DNA fragments containing a QTL from E. coli to Agrobacterium sp. and then to plants. We constructed two soybean large-insert libraries from cv. Forrest in the pCLD04541 (V41) binary vector after partial digestion of genomic high-molecular-weight DNA with BamHI or HindIII. The libraries contain 76,800 clones with an average insert size of 125 kb, and therefore represent 9.5-fold haploid genome equivalents. Colony hybridization using a chloroplast-specific probe infers that the libraries contain less than 0.5% clones of chloroplast DNA origin. These two libraries have provided clones for physical mapping of the soybean genome and for isolation of a number of disease resistance genes. One microsatellite marker was identified from the clone that hybridized to the Bng122 RFLP probe. The sequence-tagged site was used for genetic mapping and marker-assisted selection for genes underlying resistance to the soybean cyst nematode and sudden death syndrome. Received: 7 May 1999 / Accepted: 18 February 2000     

A Large-Insert (130 kbp) Bacterial Artificial Chromosome Library of the Rice Blast FungusMagnaporthe grisea:Genome Analysis, Contig Assembly, and Gene Cloning

Magnaporthe grisea(Hebert) Barr causes rice blast, one of the most devastating diseases of rice (Oryza sativa) worldwide. This fungus is an ideal organism for studying a number of aspects of plant–pathogen interactions, including infection-related morphogenesis, avirulence, and pathogen evolution. To facilitateM. griseagenome analysis, physical mapping, and positional cloning, we have constructed a bacterial artificial chromosome (BAC) library from the rice infecting strain 70-15. A new method was developed for separation of partially digested large-molecular-weight DNA fragments that facilitated library construction with large inserts. The library contains 9216 clones, with an average insert size of 130 kbp (>25 genome equivalents) stored in 384-well microtiter plates that can be double spotted robotically on to a single nylon membrane. Several unlinked single-copy DNA probes were used to screen 4608 clones in the library and an average of 13 (minimum of 6) overlapping BAC clones was found in each case. Hybridization of total genomic DNA to the library and analysis of individual clones indicated that ≈26% of the clones contain single-copy DNA. Approximately 35% of BAC clones contained the retrotransposon MAGGY. The library was used to identify BAC clones containing a adenylate cyclase gene (mac1). In addition, a 550-kbp contig composed of 6 BAC clones was constructed that encompassed two adjacent RFLP markers on chromosome 2. These data show that the BAC library is suitable for genome analysis ofM. grisea.Copies of colony hybridization membranes are available upon request.     

Artificial chromosome libraries of Streptomyces coelicolor A3(2) and Planobispora rosea

Using an Escherichia coli-Streptomyces shuttle vector derived from a bacterial artificial chromosome (BAC), we developed methodologies for the construction of BAC libraries of filamentous actinomycetes. Libraries of Streptomyces coelicolor, the model actinomycete, and Planobispora rosea, a genetically intractable strain, were constructed. Both libraries have an average insert size of 60 kb, with maximal insert larger than 150 kb. The S. coelicolor library was evaluated by selected hybridisations to DraI fragments and by end sequencing of a few clones. Hybridisation of the P. rosea library to selected probes indicates a good representation of the P. rosea genome and that the library can be used to facilitate the genomic analysis of this actinomycete.     

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

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

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     

Construction and Characterization of a Magnaporthe grisea Bacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of a Magnaporthe grisea bacterial artificial chromosome library. Fungal Genet. Biol. 20, 280-288. A bacterial artificial chromosome (BAC) library of Magnaporthe grisea containing 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents of M. grisea and has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using a lys1-3 auxotroph, we have shown that BAC clones at least 113 kb can be transformed into M. grisea to screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.     

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Grass Carp

Bacterial artificial chromosome (BAC) library is an important tool in genomic research. We constructed two libraries from the genomic DNA of grass carp (Ctenopharyngodon idellus) as a crucial part of the grass carp genome project. The libraries were constructed in the EcoRI and HindIII sites of the vector CopyControl pCC1BAC. The EcoRI library comprised 53,000 positive clones, and approximately 99.94% of the clones contained grass carp nuclear DNA inserts (average size, 139.7 kb) covering 7.4× haploid genome equivalents and 2% empty clones. Similarly, the HindIII library comprised 52,216 clones with approximately 99.82% probability of finding any genomic fragments containing single-copy genes; the average insert size was 121.5 kb with 2.8% insert-empty clones, thus providing genome coverage of 6.3× haploid genome equivalents of grass carp. We selected gene-specific probes for screening the target gene clones in the HindIII library. In all, we obtained 31 positive clones, which were identified for every gene, with an average of 6.2 BAC clones per gene probe. Thus, we succeeded in constructing the desired BAC libraries, which should provide an important foundation for future physical mapping and whole-genome sequencing in grass carp.     

SPC-P1: a pathogenicity-associated prophage of Salmonella paratyphi C

Background

The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy.

Results

The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome BAC libraries.

Conclusions

The negligibly low level of incorporation of clones from homoeologous chromosome arms into a contig during contig assembly suggested that it is feasible to construct contigs and physical maps using global BAC libraries of wheat and almost certainly also of other plant polyploid species with genome sizes comparable to that of wheat. Because of the high purity of the resulting assembled contigs, they can be directly used for genome sequencing. It is currently unknown but possible that equally good BAC contigs can be also constructed for polyploid species containing smaller, more gene-rich genomes.