Deep-coverage Bacterial Artificial Chromosome Research Articles

A BAC based physical map and genome survey of the rice false smut fungus Villosiclava virens

BackgroundRice false smut caused by Villosiclava virens is a devastating fungal disease that spreads in major rice-growing regions throughout the world. However, the genomic information for this fungal pathogen is limited and the pathogenic mechanism of this disease is still not clear. To facilitate genetic, molecular and genomic studies of this fungal pathogen, we constructed the first BAC-based physical map and performed the first genome survey for this species.ResultsHigh molecular weight genomic DNA was isolated from young mycelia of the Villosiclava virens strain UV-8b and a high-quality, large-insert and deep-coverage Bacterial Artificial Chromosome (BAC) library was constructed with the restriction enzyme HindIII. The BAC library consisted of 5,760 clones, which covers 22.7-fold of the UV-8b genome, with an average insert size of 140 kb and an empty clone rate of lower than 1%. BAC fingerprinting generated successful fingerprints for 2,290 BAC clones. Using the fingerprints, a whole genome-wide BAC physical map was constructed that contained 194 contigs (2,035 clones) spanning 51.2 Mb in physical length. Bidirectional-end sequencing of 4,512 BAC clones generated 6,560 high quality BAC end sequences (BESs), with a total length of 3,030,658 bp, representing 8.54% of the genome sequence. Analysis of the BESs revealed general genome information, including 51.52% GC content, 22.51% repetitive sequences, 376.12/Mb simple sequence repeat (SSR) density and approximately 36.01% coding regions. Sequence comparisons to other available fungal genome sequences through BESs showed high similarities to Metarhizium anisopliae, Trichoderma reesei, Nectria haematococca and Cordyceps militaris, which were generally in agreement with the 18S rRNA gene analysis results.ConclusionThis study provides the first BAC-based physical map and genome information for the important rice fungal pathogen Villosiclava virens. The BAC clones, physical map and genome information will serve as fundamental resources to accelerate the genetic, molecular and genomic studies of this pathogen, including positional cloning, comparative genomic analysis and whole genome sequencing. The BAC library and physical map have been opened to researchers as public genomic resources (http://gresource.hzau.edu.cn/resource/resource.html).

Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries

BackgroundEucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing.ResultsWe describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1) digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes.ConclusionsThe two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae, including genome sequencing, gene isolation, functional and comparative genomics. Because they have been constructed using the same tree (E. grandis BRASUZ1) whose full genome is being sequenced, they should prove instrumental for assembly and gap filling of the upcoming Eucalyptus reference genome sequence.

A deep-coverage BAC library for Ponkan mandarin and its further use in screening ethylene regulated genes

A deep-coverage bacterial artificial chromosome (BAC) library of Yanxiwanlu Ponkan, a late maturing cultivar, was constructed and evaluated. Results showed that the library contained a total of 61,000 clones. Restriction analysis indicated that the DNA insert sizes ranged from 30 to 170 kb with an average of ∼82 kb. No empty clone was found. Therefore, the library should cover ∼13.6-fold of the citrus genome. Continuous sub-cultivation of 5 randomly chosen clones for up to 100 generations showed no detectable change in their restriction profiles. To demonstrate the application of the library in discovery of genes, 7 ethylene-regulated genes were screened by PCR amplification from hierarchically pooled library clones with gene-specific primers. Five positive clones, each representing a different gene, were thus identified from 3840 clones. Sequencing of the clones showed that all of them matched the expected genes, indicating that the library was highly representative.

Construction and characterization of twoCitrusBAC libraries and identification of clones containing the phytoene synthase gene

Two deep-coverage Bacterial Artificial Chromosome (BAC) libraries of Citrus sinensis (L.) Osbeck 'Cara Cara' navel orange and Citrus reticulata (L.) Blanco 'Egan No. 1' Ponkan mandarin, which belong to the two most important species of the Citrus genus, have been constructed and characterized to facilitate gene cloning and to analyze variety-specific genome composition. The C. sinensis BAC library consists of 36 000 clones with negligible false-positive clones and an estimated average insert size of 126 kb covering ~4.5 x 109 bp and thus providing an 11.8-fold coverage of haploid genome equivalents, whereas the C. reticulata library consists of 21 000 clones also with negligible false-positive clones and an estimated average of 120 kb covering ~2.5 x 109 bp representing a 6.6-fold coverage of haploid genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBAC536 vector, and organellar DNA sequences. Screening has been performed by Southern hybridization of BAC filters, which results in <0.5% chloroplast DNA contamination and no mitochondrial DNA contamination in both libraries. Eight and five positive clones harboring the gene encoding Phytoene synthase (Psy (EC 2.5.1.32)) were identified from the C. sinensis and C. reticulata libraries, respectively, using the filter hybridization procedure. These results suggest that the two BAC libraries are useful tools for the isolation of functional genes and advanced genomics research in the two important species C. sinensis and C. reticulata. Resources, high-density filters, individual clones, and whole libraries are available for public distribution and are accessible at the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.

A novel manual pooling system for preparing three‐dimensional pools of a deep coverage soybean bacterial artificial chromosome library

Compared with hybridization-based techniques, polymerase chain reaction-based screening of large insert libraries has been used widely as it is fast, easy and sensitive. However, various pooling strategies are needed to ensure efficient screening. It is time-consuming and labourious to prepare three-dimensional pools for a deep coverage bacterial artificial chromosome (BAC) library of soybean (1.12×10(9) bp) in the absence of robotic facility. In the present study, we describe a novel manual pooling system for preparing three-dimensional pools of a soybean BAC library. This simple technique enables a single researcher to construct three-dimensional pools for a deep-coverage (12 haploid genome equivalents) BAC library of soybean in less than 2 months without any robotic manipulation. When the prepared three-dimensional pools were screened with 29 polymerase chain reaction-based markers, an average of 9.2 clones per marker were identified. These identified clones will be useful either in quantitative trait loci gene isolation or in synteny study between soybean and other legumes including Lotus japonicus. This efficient pooling system could be applied to any other BAC libraries without the need for robotic manipulation.

Two highly representative rice BAC libraries of japonica cv Tainung 67 suitable for rice structural and functional genomic research

Two deep-coverage bacterial artificial chromosome (BAC) libraries of Oryza sativa japonica cv. Tainung 67 (TNG 67), a popular genetic stock in breeding programs and scientific research in Taiwan, have been constructed to facilitate positional cloning of rice genes and to analyze variety-specific genome composition, toward rice structural and functional genomic studies. Good high-molecular-weight DNA was produced well by nuclei preparation from two- to three-week-old seedlings, partially digested by either HindIII or EcoRI, and two cycles of size selection by pulsed-field gel electrophoresis (PFGE). The HindIII library consists of 45,312 clones, near lack of false positive clones, with an estimated average insert size of 138.4 kb and coverage of 15.1× haploid genome equivalents. The EcoRI library consists of 9984 clones, 2% of false positive clones, with an estimated average insert size of 137.8 kb and coverage of 3.2× haploid genome equivalents. Nine single-copy sequence tagged site (STS) markers, located on different chromosomes, were used to screen the two libraries, which a single BAC clone double-spotted on three filters, and hybridized 16–29, with an average of 21.2 BACs. As a result, these two libraries can complement each other and cover nearly 100% of the rice genome, consequently providing efficient tools to isolate any genes of interest. Resources, high-density filters, individual clones, and whole libraries, are available for public distribution and may be accessed at the Institute of Plant and Microbial Biology, Academia Sinica, or Arizona Genomic Institute (AGI).

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

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

Construction and Characterization of a Deep-Coverage Bacterial Artificial Chromosome Library for Maize

Construction and Characterization of a Deep-Coverage Bacterial Artificial Chromosome Library for Maize

High throughput direct end sequencing of BAC clones.

Libraries constructed in bacterial artificial chromosome (BAC) vectors have become the choice for clone sets in high throughput genomic sequencing projects primarily because of their high stability. BAC libraries have been proposed as a source for minimally over-lapping clones for sequencing large genomic regions, and the use of BAC end sequences (i.e. sequences adjoining the insert sites) has been proposed as a primary means for selecting minimally overlapping clones for sequencing large genomic regions. For this strategy to be effective, high throughput methods for BAC end sequencing of all the clones in deep coverage BAC libraries needed to be developed. Here we describe a low cost, efficient, 96 well procedure for BAC end sequencing. These methods allow us to generate BAC end sequences from human and Arabidoposis libraries with an average read length of >450 bases and with a single pass sequencing average accuracy of >98%. Application of BAC end sequences in genomic sequen-cing is discussed.