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BMC Genomics
2013 Dec 16;14:883. doi: 10.1186/1471-2164-14-883.
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A BAC based physical map and genome survey of the rice false smut fungus Villosiclava virens.
Wang X
,
Liu Q
,
Wang H
,
Luo CX
,
Wang G
,
Luo M
.
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BACKGROUND: Rice 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.
RESULTS: High 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.
CONCLUSION: This 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).
Figure 1. Verification of the overlaps between clones in contig155. Section A: Location of the clones in contig155; In section B, C and D, the templates used in PCR, from left to right, are: no template, host cells, empty vector, U13J12 (contig6), U03H01 (contig133), U03J10 (contig53), U03A03 (contig100), U12J05, U08K05 and U15M06. B: The pair primers were derived from BES of U12J05.f; C: The pair primers were derived from BES of U15M06.f; D: The pair primers were derived from BES of U15M06.r.
Figure 2. Distribution of BAC end sequence lengths.
Figure 3. The BLAST hit distribution of UV-8b repeat-masked BESs in four related fungal genomes. The repeat-masked BESs were BLASTed against the four related fungal genomes using BLASTN program. A total of 1234, 779, 689 and 679 BESs hit to M. anisopliae, T. reesei, N. haematococca and C. militaris genome sequences, respectively. The shared BES hits are illustrated here; for example, 326 repeat-masked BESs hit to the four genomes simultaneously.
Figure 4. Mapping UV-8b PhaseIA contigs to the four related genome sequences. The UV-8b PhaseIA contigs were anchored to the four fungal genomes based on the repeat-masked BESs. The left bars represent UV-8b fingerprint contigs and the right boxes represent each of the four related genome sequences. The purple lines represent the BES alignments. A: Alignments between UV-8b contigs and the partial draft sequence of M. anisopliae (gi 322711353). B: Alignments between UV-8b contigs and the partial draft sequence of T. reesei (gi 340520780). C: Alignments between UV-8b contigs and the partial draft sequence of N. haematococca (gi 302917142). D: Alignments between UV-8b contigs and the partial draft sequence of C. militaris (gi 346324065).
Figure 5. The SSR content and distribution in different fungal genomes. The GSS sequence data of B. graminis, F. virguliforme, M. oryzae and T. reesei as well as UV-8b BESs were scanned for SSRs. The Y-axis represents the percentage of each SSR type to the total SSRs.
Figure 6. Distribution of the matched species in annotation step. The masked and non-redundant BESs were BLASTed against the NR database of NCBI using BLASTX program. A: All-Hit species distribution. All BLAST hits of the BESs were counted. B: Top-Hit species distribution. Only the best BLAST hit was counted.
Figure 7. Distribution of GO annotations of gene products predicted from UV-8b BESs.
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