Fine mapping of the bolting gene of sugar beet (Beta vulgaris L.) using BAC-derived sequences
In sugar beet, bolting is considered to be an important trait. The presence of the dominant allele B of the bolting gene in annual beets causes bolting without vernalization. It is known that the bolting locus B is located on chromosome 2. In cultivated sugar beet, bolting is an undesirable phenomenon because it creates troubles during harvesting and sugar extraction as well as it reduces the yield. Owing to the incomplete penetrance of the B allele and its environmental dependence, closely linked molecular markers are needed to screen its presence in breeding lines. The main goals of this study were to fine map the B gene using BAC-derived markers from the B gene region and to screen the breeding and wild accessions, in order to conduct a preliminary association mapping study of the B allele. From an uncomplete physical map (four different contigs), eighteen BACs were selected for marker development around the B gene. In the GABI BOLT project, three BACs were shotgun sequenced and 61 open reading frames (ORF) were identified. From 104 BAC ends, 37 BAC ends and 12 ORF single or low copy sequences were chosen for marker development. Thirty-one percent of the BAC-end sequences were found to be single copy and 24% were low. From all selected BAC sequences, 17 markers from 10 different BACs were developed. Ten polymorphisms were determined by simple agarose gel electrophoresis of either restricted or non-restricted PCR products. Another 7 markers were mapped by SNAP (single nucleotide amplified polymorphism) or tetra-primer ARMS (amplification refractory mutation system)-PCR. A prerequisite for map-based cloning of the B gene is a high-resolution genetic map with two flanking markers tightly linked to the gene and a large insert genomic library. In order to select candidate BACs for cloning the B gene, genetic linkage was performed using two different populations: 1) A genetic linkage map (using MAPMAKER) surrounding the B locus was constructed with 8 BAC-derived, one RFLP-derived, and one YAC-derived marker locus. 4 previously mapped AFLP markers were also included. Based on 299 plants (with verified phenotype in F3) from a segregating population, the 14 marker loci mapped to a region spanning 28.3 cM around the B gene. Two markers (GJ01co36F4 and GJ10SP6) mapped at the same distance of 0.2 cM around the B gene. 2) Another genetic linkage analysis based only on the non-bolting plants from a F2 population (1617 plants) segregating for early bolting was performed. 8 markers (7 BAC-derived and one RFLP-derived) were used and their distances to the bolting gene were calculated. The recombination values ranged between 0.0033 and 0.0201. Both analyses revealed that the B locus is located between two markers (GJ01co36F4 and GJ10SP6). Unfortunately, these two markers originated from two non-overlapping BAC contigs (Contig 3 and Contig 2). Comparison between the physical and genetic distances showed high recombination in the B locus region. Physical to genetic ratios of 240 and 554 kb/cM were estimated when comparing physical and genetic distances of markers in contigs Contig 2 and Contig 1 and 4, respectively. In addition, a set of 41 wild and cultivated Beta accessions, differing in their character for early bolting, was genotyped with seven markers. A common haplotype encompassing two marker loci and the b allele found in all sugar beet varieties indicating linkage disequilibrium between these loci. This suggests that the bolting gene might be located in close vicinity to these markers and the corresponding BACs can be used for cloning the gene. However, there was a disagreement between results from association mapping and linkage mapping that might be due to population structure or environmental factors. Using gene sequences from Arabidopsis, three PCR-based sugar beet markers for VRN1, VRN2, and CO were developed. After mapping of these markers in an F2 population segregating for early bolting, VRN1 and VRN2 derived markers were found to be in a distance of 44.9 and 88.8 cM from the B locus, respectively. These markers could b e useful in the isolation of sugar beet vernalization and flowering time orthologous genes.