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A BAC pooling strategy combined with PCR-based screenings in a large, highly repetitive genome enables integration of the maize genetic and physical maps.

ABSTRACT: BACKGROUND: Molecular markers serve three important functions in physical map assembly. First, they provide anchor points to genetic maps facilitating functional genomic studies. Second, they reduce the overlap required for BAC contig assembly from 80 to 50 percent. Finally, they validate assemblies based solely on BAC fingerprints. We employed a six-dimensional BAC pooling strategy in combination with a high-throughput PCR-based screening method to anchor the maize genetic and physical maps. RESULTS: A total of 110,592 maize BAC clones (approximately 6x haploid genome equivalents) were pooled into six different matrices, each containing 48 pools of BAC DNA. The quality of the BAC DNA pools and their utility for identifying BACs containing target genomic sequences was tested using 254 PCR-based STS markers. Five types of PCR-based STS markers were screened to assess potential uses for the BAC pools. An average of 4.68 BAC clones were identified per marker analyzed. These results were integrated with BAC fingerprint data generated by the Arizona Genomics Institute (AGI) and the Arizona Genomics Computational Laboratory (AGCoL) to assemble the BAC contigs using the FingerPrinted Contigs (FPC) software and contribute to the construction and anchoring of the physical map. A total of 234 markers (92.5%) anchored BAC contigs to their genetic map positions. The results can be viewed on the integrated map of maize 12. CONCLUSION: This BAC pooling strategy is a rapid, cost effective method for genome assembly and anchoring. The requirement for six replicate positive amplifications makes this a robust method for use in large genomes with high amounts of repetitive DNA such as maize. This strategy can be used to physically map duplicate loci, provide order information for loci in a small genetic interval or with no genetic recombination, and loci with conflicting hybridization-based information.

SUBMITTER: Yim YS

PROVIDER: S-EPMC1821331 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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A BAC pooling strategy combined with PCR-based screenings in a large, highly repetitive genome enables integration of the maize genetic and physical maps.

Yim Young-Sun YS Moak Patricia P Sanchez-Villeda Hector H Musket Theresa A TA Close Pamela P Klein Patricia E PE Mullet John E JE McMullen Michael D MD Fang Zheiwei Z Schaeffer Mary L ML Gardiner Jack M JM Coe Edward H EH Davis Georgia L GL

BMC genomics 20070209

<h4>Background</h4>Molecular markers serve three important functions in physical map assembly. First, they provide anchor points to genetic maps facilitating functional genomic studies. Second, they reduce the overlap required for BAC contig assembly from 80 to 50 percent. Finally, they validate assemblies based solely on BAC fingerprints. We employed a six-dimensional BAC pooling strategy in combination with a high-throughput PCR-based screening method to anchor the maize genetic and physical m ...[more]

PMID: 17291341

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Project description:BACKGROUND: Cucumis melo (melon) belongs to the Cucurbitaceae family, whose economic importance among horticulture crops is second only to Solanaceae. Melon has a high intra-specific genetic variation, morphologic diversity and a small genome size (454 Mb), which make it suitable for a great variety of molecular and genetic studies. A number of genetic and genomic resources have already been developed, such as several genetic maps, BAC genomic libraries, a BAC-based physical map and EST collections. Sequence information would be invaluable to complete the picture of the melon genomic landscape, furthering our understanding of this species' evolution from its relatives and providing an important genetic tool. However, to this day there is little sequence data available, only a few melon genes and genomic regions are deposited in public databases. The development of massively parallel sequencing methods allows envisaging new strategies to obtain long fragments of genomic sequence at higher speed and lower cost than previous Sanger-based methods. RESULTS: In order to gain insight into the structure of a significant portion of the melon genome we set out to perform massive sequencing of pools of BAC clones. For this, a set of 57 BAC clones from a double haploid line was sequenced in two pools with the 454 system using both shotgun and paired-end approaches. The final assembly consists of an estimated 95% of the actual size of the melon BAC clones, with most likely complete sequences for 50 of the BACs, and a total sequence coverage of 39x. The accuracy of the assembly was assessed by comparing the previously available Sanger sequence of one of the BACs against its 454 sequence, and the polymorphisms found involved only 1.7 differences every 10,000 bp that were localized in 15 homopolymeric regions and two dinucleotide tandem repeats. Overall, the study provides approximately 6.7 Mb or 1.5% of the melon genome. The analysis of this new data has allowed us to gain further insight into characteristics of the melon genome such as gene density, average protein length, or microsatellite and transposon content. The annotation of the BAC sequences revealed a high degree of collinearity and protein sequence identity between melon and its close relative Cucumis sativus (cucumber). Transposon content analysis of the syntenic regions suggests that transposition activity after the split of both cucurbit species has been low in cucumber but very high in melon. CONCLUSIONS: The results presented here show that the strategy followed, which combines shotgun and BAC-end sequencing together with anchored marker information, is an excellent method for sequencing specific genomic regions, especially from relatively compact genomes such as that of melon. However, in agreement with other results, this map-based, BAC approach is confirmed to be an expensive way of sequencing a whole plant genome. Our results also provide a partial description of the melon genome's structure. Namely, our analysis shows that the melon genome is highly collinear with the smaller one of cucumber, the size difference being mainly due to the expansion of intergenic regions and proliferation of transposable elements.

Project description:Integrated cytogenetic pachytene fluorescence in situ hybridization (FISH) maps were developed for chromosomes 1, 3, 4, 5, 6, and 8 of maize using restriction fragment length polymorphism marker-selected Sorghum propinquum bacterial artificial chromosomes (BACs) for 19 core bin markers and 4 additional genetic framework loci. Using transgenomic BAC FISH mapping on maize chromosome addition lines of oats, we found that the relative locus position along the pachytene chromosome did not change as a function of total arm length, indicative of uniform axial contraction along the fibers during mid-prophase for tested loci on chromosomes 4 and 5. Additionally, we cytogenetically FISH mapped six loci from chromosome 9 onto their duplicated syntenic regions on chromosomes 1 and 6, which have varying amounts of sequence divergence, using sorghum BACs homologous to the chromosome 9 loci. We found that successful FISH mapping was possible even when the chromosome 9 selective marker had no counterpart in the syntenic block. In total, these 29 FISH-mapped loci were used to create the most extensive pachytene FISH maps to date for these six maize chromosomes. The FISH-mapped loci were then merged into one composite karyotype for direct comparative analysis with the recombination nodule-predicted cytogenetic, genetic linkage, and genomic physical maps using the relative marker positions of the loci on all the maps. Marker colinearity was observed between all pair-wise map comparisons, although marker distribution patterns varied widely in some cases. As expected, we found that the recombination nodule-based predictions most closely resembled the cytogenetic map positions overall. Cytogenetic and linkage map comparisons agreed with previous studies showing a decrease in marker spacing in the peri-centromeric heterochromatin region on the genetic linkage maps. In fact, there was a general trend with most loci mapping closer towards the telomere on the linkage maps than on the cytogenetic maps, regardless of chromosome number or maize inbred line source, with just some of the telomeric loci exempted. Finally and somewhat surprisingly, we observed considerable variation between the relative arm positions of loci when comparing our cytogenetic FISH map to the B73 genomic physical maps, even where comparisons were to a B73-derived cytogenetic map. This variation is more evident between different chromosome arms, but less so within a given arm, ruling out any type of inbred-line dependent global features of linear deoxyribonucleic acid compared with the meiotic fiber organization. This study provides a means for analyzing the maize genome structure by producing new connections for integrating the cytogenetic, linkage, and physical maps of maize.

Project description:Lactobacillus casei is a potential cell factory for the production of enzymes and bioactive molecules using episomal plasmids, which suffer from genetic instability. While chromosomal integration strategies can provide genetic stability of recombinant proteins, low expression yields limit their application. To address this problem, we developed a two-step integration strategy in Lb. casei by combination of the LCABL_13040-50-60 recombineering system (comprised of LCABL_1340, LCABL_13050, and LCABL_13060) with the Cre/loxP site-specific recombination system, with an efficiency of ∼3.7 × 103 CFU/µg DNA. A gfp gene was successfully integrated into six selected chromosomal sites, and the relative fluorescence intensities (RFUs) of the resulting integrants varied up to ∼3.7-fold depending on the integrated site, among which the LCABL_07270 site gfp integration showed the highest RFU. However, integrants with gfp gene(s) integrated into the LCABL_07270 site showed various RFUs, ranging from 993 ± 89 to 7,289 ± 564 and corresponding to 1 to 13.68 ± 1.08 copies of gfp gene integration. Moreover, the integrant with 13.68 ± 1.08 copies of the gfp gene had a more stable RFU after 63 generations compared to that of a plasmid-engineered strain. To investigate the feasibility of this system for bioactive molecules with high expression levels, the fimbrial adhesin gene, faeG, from Escherichia coli was tested and successfully integrated into the LCABL_07270 site with 5.51 ± 0.25 copies, and the integrated faeG achieved stable expression. All results demonstrate that this two-step integration system could achieve a high yield of heterologous gene expression by repetitive integration at a targeted chromosomal location in Lb. caseiIMPORTANCE Lactic acid bacteria (LAB), including Lactobacillus casei, have the potential for overexpression of heterologous proteins, such as bioactive molecules and enzymes. However, traditional genetic tools for expression of these proteins show genetic instability or low yields of the desired product. In this study, we provide a procedure for repetitive integration of genes at various chromosomal locations, achieving high-level and stable expression of proteins in Lb. casei without selective pressure. The protocol developed in this study provides an essential reference for chromosomal overexpression of proteins or bioactive molecules in LAB.

Dataset Information

A BAC pooling strategy combined with PCR-based screenings in a large, highly repetitive genome enables integration of the maize genetic and physical maps.

Publications

A BAC pooling strategy combined with PCR-based screenings in a large, highly repetitive genome enables integration of the maize genetic and physical maps.

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