A bacterial artificial chromosome (BAC) library of sugar beet and a physical map of the region encompassing the bolting gene B

Abstract

In sugar beet (Beta vulgaris L.), early bolting is caused by a single dominant gene, designated B. Twenty AFLP markers selected from a 7.8-cM segment of the B region on chromosome 2 were used to screen a YAC library, and a first-generation physical map including the B gene, made up of 11 YACs, was established. Because the genome coverage of the YAC library was low, a BAC library was constructed in the vector pBeloBAC11. This library consists of 57,600 clones with an average insert size of 116 kb, corresponding to 8.8 genome equivalents. Screening of the BAC library with chloroplast and mitochondrial DNA probes indicated that less than 0.1% of the clones contained organelle-derived DNA. To fill the gaps in the physical map around the B gene, the BAC library was screened with four AFLP markers and 10 YAC-derived probes. In total, 54 different BACs were identified. Overlaps between BACs were detected by using BAC termini amplified by PCR as probes, and by RFLP fingerprinting. In this way, a minimal tiling path of the central 4.6-cM region was constructed, which consists of 14 BACs. The B locus was localized to a 360-kb contig, a size which makes positional cloning of the gene feasible.

Conservation and divergence of autonomous pathway genes in the flowering regulatory network of Beta vulgaris

“…Probe fragments were generated by PCR amplification using primer combinations A039–A064 for BvFLK (717 bp), A066–A042 for BvFVE1 (1089 bp), A043–A067 for BvLD (589 bp), and A033–A065 for BvLDL1 (678 bp), and genomic DNA of A906001 as template, and purified using the Montage PCR 96 Cleanup Kit (Millipore Corporation, Bedford, CA, USA). The probes were labelled and hybridized to high-density BAC filters essentially as described by Hohmann et al (2003). Positive clones were verified by PCR analysis using the same primer combinations as for PCR amplification of probe fragments.…”

Haplotype divergence in Beta vulgaris and microsynteny with sequenced plant genomes

“…The average number was 29.8 matched Arabidopsis genes per interval, suggesting that 30 matches in the interval of our study can be considered as representative. For each of the 30 sugar beet ESTs, we determined the exon structure, designed 35mer oligonucleotides from exons (one for each EST) and used them as hybridization probes against two sugar beet BAC libraries, called ZR (Hohmann et al, 2003) and SBI (B. Schulz, unpublished data), representing two different (Jansen et al, 2006;The Angiosperm Phylogeny Group, 2003). haplotypes (for macroarray preparation and image analysis see Appendix S1). A total of 27 648 clones were screened per library, corresponding to 4.4-fold genome coverage for the ZR library (average insert size 110 kbp) and 5.5-fold genome coverage for the SBI library (average insert size 130 kbp).…”

“…Two BAC libraries, ZR (Hohmann et al, 2003) and SBI (B. Schulz, unpublished data) were used. Library ZR had been constructed from B. vulgaris ssp.…”

“…A comprehensive study on systematic cDNA sequencing resulted in more than 22 000 public sugar beet ESTs (Herwig et al, 2002). Several genomic bacterial artificial chromosome (BAC) libraries have been constructed (Gindullis et al, 2001;Hohmann et al, 2003;McGrath et al, 2004;B. Schulz, unpublished data), providing useful resources for genomewide studies in sugar beet.…”

An EMS mutagenesis protocol for sugar beet and isolation of non-bolting mutants

“…Consequently, there is great interest in this gene which is presently cloned from its position in the genome. High density physical and genetic maps around this gene have been established (El-Mezawy et al 2002, Hohmann et al 2003. For the identification of the bolting gene among candidate sequences from a physical map, mutants with deficient bolting alleles are needed.…”