An integrated BAC and genome sequence physical map of Phytophthora sojae

Phytophthora spp. are serious pathogens that threaten numerous cultivated crops, trees, and natural vegetation worldwide. The soybean pathogen P. sojae has been developed as a model oomycete. Here, we report a bacterial artificial chromosome (BAC)-based, integrated physical map of the P. sojae genome. We constructed two BAC libraries, digested 8,681 BACs with seven restriction enzymes, end labeled the digested fragments with four dyes, and analyzed them with capillary electrophoresis. Fifteen data sets were constructed from the fingerprints, using individual dyes and all possible combinations, and were evaluated for contig assembly. In all, 257 contigs were assembled from the XhoI data set, collectively spanning approximately 132 Mb in physical length. The BAC contigs were integrated with the draft genome sequence of P. sojae by end sequencing a total of 1,440 BACs that formed a minimal tiling path. This enabled the 257 contigs of the BAC map to be merged with 207 sequence scaffolds to form an integrated map consisting of 79 superscaffolds. The map represents the first genome-wide physical map of a Phytophthora sp. and provides a valuable resource for genomics and molecular biology research in P. sojae and other Phytophthora spp. In one illustration of this value, we have placed the 350 members of a superfamily of putative pathogenicity effector genes onto the map, revealing extensive clustering of these genes.     

Piggy-BACing the human genome I: constructing a porcine BAC physical map through comparative genomics

Availability of the human genome sequence and high similarity between humans and pigs at the molecular level provides an opportunity to use a comparative mapping approach to piggy-BAC the human genome. In order to advance the pig genome sequencing initiative, sequence similarity between large-scale porcine BAC-end sequences (BESs) and human genome sequence was used to construct a comparatively-anchored porcine physical map that is a first step towards sequencing the pig genome. A total of 50,300 porcine BAC clones were end-sequenced, yielding 76,906 BESs after trimming with an average read length of 538 bp. To anchor the porcine BACs on the human genome, these BESs were subjected to BLAST analysis using the human draft sequence, revealing 31.5% significant hits (E < e(-5)). Both genic and non-genic regions of homology contributed to the alignments between the human and porcine genomes. Porcine BESs with unique homology matches within the human genome provided a source of markers spaced approximately 70 to 300 kb along each human chromosome. In order to evaluate the utility of piggy-BACing human genome sequences, and confirm predictions of orthology, 193 evenly spaced BESs with similarity to HSA3 and HSA21 were selected and then utilized for developing a high-resolution (1.22 Mb) comparative radiation hybrid map of SSC13 that represents a fusion of HSA3 and HSA21. Resulting RH mapping of SSC13 covers 99% and 97% of HSA3 and HSA21, respectively. Seven evolutionary conserved blocks were identified including six on HSA3 and a single syntenic block corresponding to HSA21. The strategy of piggy-BACing the human genome described in this study demonstrates that through a directed, targeted comparative genomics approach construction of a high-resolution anchored physical map of the pig genome can be achieved. This map supports the selection of BACs to construct a minimal tiling path for genome sequencing and targeted gap filling. Moreover, this approach is highly relevant to other genome sequencing projects.     

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.     

BAC library construction and BAC end sequencing of five Drosophila species: the comparative map with the D. melanogaster genome

We constructed and characterized arrayed bacterial artificial chromosome (BAC) libraries of five Drosophila species (D. melanogaster, D. simulans, D. sechellia, D. auraria, and D. ananassae), which are genetically well characterized in the studies of meiosis, evolution, population genetics, and developmental biology. The BAC libraries comprise 8,000 to 12,500 clones for each species, estimated to cover the most of the genomes. We sequenced both ends of most of these BAC clones with a success rate of 91%. Of these, 53,701 clones consisting of non-repetitive BAC end sequences (BESs) were mapped with reference of the public D. melanogaster genome sequences. The BES mapping estimated that the BAC libraries of D. auraria and D. ananassae covered 47% and 57% of the D. melanogaster genome, respectively, and those of D. melanogaster, D. sechellia, and D. simulans covered 94-97%. The low coverage by BESs of D. auraria and D. ananassae may be due to the high sequence divergence with D. melanogaster. From the comparative BES mapping, 111 possible breakpoints of chromosomal rearrangements were identified in these four species. The breakpoints of the major chromosome rearrangement between D. simulans and D. melanogaster on the third chromosome were determined within 20 kb in 84E and 30 kb in 93E/F. Corresponding breakpoints were also identified in D. sechellia. The BAC clones described here will be an important addition to the Drosophila genomic resources.     

Anchoring of rice BAC clones to the rice genetic map in silico

A wealth of molecular resources have been developed for rice genomics, including dense genetic maps, expressed sequence tags (ESTs), yeast artificial chromosome maps, bacterial artificial chromosome (BAC) libraries and BAC end sequence databases. Integration of genetic and physical maps involves labor-intensive empirical experiments. To accelerate the integration of the bacterial clone resources with the genetic map for the International Rice Genome Sequencing Project, we cleaned and filtered the available EST and BAC end sequences for repetitive sequences and then searched all available rice genetic markers with our filtered databases. We identified 418 genetic markers that aligned with at least one BAC end sequence with >95% sequence identity, providing a set of large insert clones with an average separation of 1 Mb that can serve as nucleation points for the sequencing phase of the International Rice Genome Sequencing Project.     

A set of BAC clones spanning the human genome

Using the human bacterial artificial chromosome (BAC) fingerprint-based physical map, genome sequence assembly and BAC end sequences, we have generated a fingerprint-validated set of 32855 BAC clones spanning the human genome. The clone set provides coverage for at least 98% of the human fingerprint map, 99% of the current assembled sequence and has an effective resolving power of 79 kb. We have made the clone set publicly available, anticipating that it will generally facilitate FISH or array-CGH-based identification and characterization of chromosomal alterations relevant to disease.     

白氏文昌鱼单个体基因组BAC文库的构建

隶属于头索动物亚门的文昌鱼是现存生物中最近似于脊椎动物亚门直接祖先的一个类群, 具有重要的进化地位, 是研究脊椎动物原始祖先的重要材料和模式动物。随着文昌鱼实验室连续繁殖的成功, 全基因组测序成为中国文昌鱼模式化急需完成的工作之一。文章从单条雄性白氏文昌鱼精巢组织中提取高质量的基因组DNA, 经EcoRⅠ限制性内切酶和EcoRⅠ甲基化酶酶切, 脉冲场电泳选择合适酶切DNA片段, 连接线性磷酸化的载体pCC1BAC, 转化大肠杆菌EPI300 E. coli, 构建了含有44 706个克隆的全基因组BAC( Bacterial artificial chromosome)文库, 该文库平均插入片段80 kb,具有9倍的基因组覆盖率, 基本能够满足功能基因等研究需要, 为中国文昌鱼全基因组测序打下基础。     

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety

Background

Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.

Principal Findings

We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).

Conclusions

Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.     

Construction and characterization of a sheep BAC library of three genome equivalents

A sheep BAC library of over three genome equivalents was constructed and arrayed in superpools and row, column, and plate pools. The library contains 90,000 clones distributed in 39 superpools. The average insert size was estimated at 123 kb. The library was screened by PCR with 77 primer pairs corresponding to ovine microsatellites distributed throughout the genome. The probability of finding a random sequence in the library could be estimated at 0.96. Received: 2 November 1998 / Accepted: 29 January 1999     

BAC end sequences and a physical map reveal transposable element content and clustering patterns in the genome of Magnaporthe grisea

Transposable elements (TEs) are viewed as major contributors to the evolution of fungal genomes. Genomic resources such as BAC libraries are an underutilized resource for studying genome-wide TE distribution. Using the BAC end sequences and physical map that are available for the rice blast fungus, Magnaporthe grisea, we describe a likelihood ratio test designed to identify clustering of TEs in the genome. A significant variation in the distribution of three TEs, MAGGY, MGL, and Pot2 was observed among the fingerprint contigs of the physical map. We utilized a draft sequence of M. grisea chromosome 7 to validate our results and found a similar pattern of clustering. By examining individual BAC end sequences, we found evidence for 11 unique integrations of MAGGY or MGL into Pot2 but no evidence for the reciprocal integration of Pot2 into another TE. This suggests that: (a) the presence of Pot2 in the genome predates that of the other TEs, (b) Pot2 was less transpositionally active than other TEs, or (c) that MAGGY and MGL have integration site preference for Pot2. High transition/transversion mutation ratios as well as bias in transition site context was observed in MAGGY and MGL elements, but not in Pot2 elements. These features are consistent with the effects of a Repeat-Induced Point (RIP) mutation-like process occurring in MAGGY and MGL elements. This study illustrates the general utility of a physical map and BAC end sequences for the study of genome-wide repetitive DNA content and organization.     

A physical map of the bovine genome

Background

Cattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The BAC libraries and clone maps are essential for the hybrid clone-by-clone/whole-genome shotgun sequencing approach taken by the bovine genome sequencing project.

Results

A bovine BAC map was constructed with HindIII restriction digest fragments of 290,797 BAC clones from animals of three different breeds. Comparative mapping of 422,522 BAC end sequences assisted with BAC map ordering and assembly. Genotypes and pedigree from two genetic maps and marker scores from three whole-genome RH panels were consolidated on a 17,254-marker composite map. Sequence similarity allowed integrating the BAC and composite maps with the bovine draft assembly (Btau3.1), establishing a comprehensive resource describing the bovine genome. Agreement between the marker and BAC maps and the draft assembly is high, although discrepancies exist. The composite and BAC maps are more similar than either is to the draft assembly.

Conclusion

Further refinement of the maps and greater integration into the genome assembly process may contribute to a high quality assembly. The maps provide resources to associate phenotypic variation with underlying genomic variation, and are crucial resources for understanding the biology underpinning this important ruminant species so closely associated with humans.     

Construction of a BAC library and generation of BAC end sequence-tagged connectors for genome sequencing of the African malaria mosquito Anopheles gambiae

A Bacterial Artificial Chromosome (BAC) genomic DNA library of Anopheles gambiae, the major human malaria vector in sub-Saharan Africa, was constructed and characterized. This library (ND-TAM) is composed of 30,720 BAC clones in eighty 384-well plates. The estimated average insert size of the library is 133 kb, with an overall genome coverage of approximately 14-fold. The ends of approximately two-thirds of the clones in the library were sequenced, yielding 32,340 pair-mate ends. A statistical analysis (G-test) of the results of PCR screening of the library indicated a random distribution of BACs in the genome, although one gap encompassing the white locus on the X-chromosome was identified. Furthermore, combined with another previously constructed BAC library (ND-1), ~2,000 BACs have been physically mapped by polytene chromosomal in situ hybridization. These BAC end pair mates and physically mapped BACs have been useful for both the assembly of a fully sequenced A. gambiae genome and for linking the assembled sequence to the three polytene chromosomes. This ND-TAM library is now publicly available at both http://www.malaria.mr4.org/mr4pages/index.html/ and http://hbz.tamu.edu/, providing a valuable resource to the mosquito research community.     

Construction of a California condor BAC library and first-generation chicken-condor comparative physical map as an endangered species conservation genomics resource

To support genomic analysis of the endangered California condor (Gymnogyps californianus), a BAC library (CHORI-262) was generated using DNA from the blood of a female. The library consists of 89,665 recombinant BAC clones providing approximately 14-fold coverage of the presumed approximately 1.48-Gb genome. Taking advantage of recent progress in chicken genomics, we developed a first-generation comparative chicken-condor physical map using an overgo hybridization approach. The overgos were derived from chicken (164 probes) and New World vulture (8 probes) sequences. Screening a 2.8x subset of the total library resulted in 236 BAC-gene assignments with 2.5 positive BAC clones per successful probe. A preliminary comparative chicken-condor BAC-based map included 93 genes. Comparison of selected condor BAC sequences with orthologous chicken sequences suggested a high degree of conserved synteny between the two avian genomes. This work will aid in identification and characterization of candidate loci for the chondrodystrophy mutation to advance genetic management of this disease.     

Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex PCR

Despite their numerous advantages, the use of microsatellites as genetic markers could be limited because of the low number of loci that can be simultaneously analysed per experiment. To increase the information per simple sequence repeat (SSR) assay in the grapevine, we developed a large set of new markers suitable for multiplexing and multi-loading. We produced microsatellite motif-enriched genomic libraries containing preferentially large size inserts which allowed us to design primers generating a wide range of allele sizes in a very standard and unique PCR condition. Three hundred and fifty clones were sequenced and 190 of them (54%) contained microsatellite motifs with suitable flanking regions for primer design. We developed 169 new SSR markers giving suitable signal with fluorescent-based DNA detection. The total number of alleles detected varied from 1 to 8 per locus with an average of 3.5 and the mean expected heterozygosity was 0.544 (range: 0 0.86). Sixty-eight loci (40%) were perfect types, 73 (43%) were imperfect and 28 (17%) were compound or imperfect-compound. The number of alleles generated by perfect and imperfect type loci was positively correlated to the length of the microsatellite motif. Forty-six multiplex sets based on 125 selected loci were developed. Considering their allele size range, up to four PCR multiplex were pooled together for multi-loading. The 169 SSR loci developed in this study represent a new and informative set of markers easy to combine for multiplexing and multi-loading according to the needs of any user and suitable for large scale genetic analyses in grapevine.     

Cytogenetical anchoring of sheep linkage map and syntenic groups using a sheep BAC library

In order to simultaneously integrate linkage and syntenic groups to the ovine chromosomal map, a sheep bacterial artificial chromosome (BAC) library was screened with previously assigned microsatellites using a sheep-hamster hybrid panel and genetic linkage. Thirty-three BACs were obtained, fluorescently labelled and hybridised on sheep-goat hybrid metaphases (2n = 57). This study allowed us, (i), to anchor all linkage groups on sheep chromosomes, (ii), to give information on the probable position of the centromere on the linkage map for the centromeric chromosomes, (iii), to contradict the previous orientation of the ovine × linkage group by the mapping of BMS1008 on OARXq38. Concerning our somatic cell hybrid panel, this study resulted in the assignment of all the previously unassigned groups to ovine chromosomes and a complete characterisation of the hybrid panel. In addition, since hybridisations were performed on a sheep-goat hybrid, new marker/anchoring points were added to the caprine cytogenetic map.     

Construction and characterization of a papaya BAC library as a foundation for molecular dissection of a tree-fruit genome

A bacterial artificial chromosome (BAC) library was constructed from high-molecular-weight DNA isolated from young leaves of papaya (Carica papaya L.). This BAC library consists of 39168 clones from two separate ligation reactions. The average insert size of the library is 132 kb; 96.5% of the 18700 clones from the first ligation contained inserts that averaged 86 kb in size, 95.7% of the 20468 clones from the second ligation contained inserts that averaged 174 kb in size. Two sorghum chloroplast probes hybridized separately to the library and revealed a total of 504 chloroplast clones or 1.4% of the library. The entire BAC library was estimated to provide 13.7× papaya-genome equivalents, excluding the false-positive and chloroplast clones. High-density filters were made containing 94% or 36864 clones of the library with 12.7× papaya-genome equivalents. Eleven papaya-cDNA and ten Arabidopsis-cDNA probes detected an average of 22.8 BACs per probe in the library. Because of its relatively small genome (372 Mbp/1 C) and its ability to produce ripe fruit 9 to 15 months after planting, papaya shows promise as a model plant for studying genes that affect fruiting characters. A rapid approach to locating fruit-controlling genes will be to assemble a physical map based on BAC contigs to which ESTs have hybridized. A physical map of the papaya genome will significantly enhance our capacity to clone and manipulate genes of economic importance. Received: 11 April 2000 / Accepted: 28 July 2000     

A physical map of the Mycoplasma genitalium genome

We report the construction of a physical map of the genome of the human pathogen Mycoplasma genitalium through the use of pulse-field gel electrophoresis. The small size and relative simplicity of this genome permit the arrangement of restriction fragments without having to construct linking clones. The size of the genome has been calculated to be approximately 600 kb and several important genetic determinants have been assigned specific loci on the map.     

A BAC-based physical map of the apple genome

Genome-wide physical mapping is an essential step toward investigating the genetic basis of complex traits as well as pursuing genomics research of virtually all plant and animal species. We have constructed a physical map of the apple genome from a total of 74,281 BAC clones representing approximately 10.5x haploid genome equivalents. The physical map consists of 2702 contigs, and it is estimated to span approximately 927 Mb in physical length. The reliability of contig assembly was evaluated by several methods, including assembling contigs using variable stringencies, assembling contigs using fingerprints from individual libraries, checking consensus maps of contigs, and using DNA markers. Altogether, the results demonstrated that the contigs were properly assembled. The apple genome-wide BAC-based physical map represents the first draft genome sequence not only for any member of the large Rosaceae family, but also for all tree species. This map will play a critical role in advanced genomics research for apple and other tree species, including marker development in targeted chromosome regions, fine-mapping and isolation of genes/QTL, conducting comparative genomics analyses of plant chromosomes, and large-scale genomics sequencing.