The wheat genome is large (1.6×10 10 bp) and complex (hexaploid with the A,B and D genomes). Map based cloning in such genomes requires at least one, but frequently several walking steps on a chromosome to reach ...The wheat genome is large (1.6×10 10 bp) and complex (hexaploid with the A,B and D genomes). Map based cloning in such genomes requires at least one, but frequently several walking steps on a chromosome to reach the gene of interest, even if very closely linked markers are available for a “chromosome landing” approach. Chromosome walking in wheat has often been considered to be very difficult or impossible due to size and complexity of the wheat genome and the high content of repetitive sequences. We are interested to clone two genes on chromosome 1AS by map information only: the Lr10 leaf rust resistance gene and the Pm3 powdery mildew resistance gene. As no large insert library of wheat was available at that time, a collaborative effort of several research groups was started to create a BAC library of T.monococcum ,a cultivated diploid with a close relative of the A genome in hexaploid wheat. The BAC library contains more than six genome equivalents and is double spotted on filters which are available from our lab. A mapping population of 3150 F2 plants segregating for the Lr10 gene has been established and a marker closely linked to the gene (0.1 cM) was found. This marker was the starting point for the assembly of a physical contig in T.monococcum .The use of subcloned BAC ends for mapping was only successful in a few cases but in general was problematic. To derive probes from BAC clones for genetic mapping we developed a rapid “low pass” sequencing protocol. Shotgun DNA libraries from BAC clones were generated and sequenced at 1.5×genome equivalents. The obtained sequence data were sufficient to identify coding regions (usually good probes for mapping) as well as non coding, non repetitive sequences which sometimes can also be mapped and used as probes for further walking steps. Probes derived from sequencing have also to be physically mapped on the BAC clones to identify sequences close to the ends of the BACs. Four walking steps have been completed until now using these approaches. This resulted 展开更多
文摘The wheat genome is large (1.6×10 10 bp) and complex (hexaploid with the A,B and D genomes). Map based cloning in such genomes requires at least one, but frequently several walking steps on a chromosome to reach the gene of interest, even if very closely linked markers are available for a “chromosome landing” approach. Chromosome walking in wheat has often been considered to be very difficult or impossible due to size and complexity of the wheat genome and the high content of repetitive sequences. We are interested to clone two genes on chromosome 1AS by map information only: the Lr10 leaf rust resistance gene and the Pm3 powdery mildew resistance gene. As no large insert library of wheat was available at that time, a collaborative effort of several research groups was started to create a BAC library of T.monococcum ,a cultivated diploid with a close relative of the A genome in hexaploid wheat. The BAC library contains more than six genome equivalents and is double spotted on filters which are available from our lab. A mapping population of 3150 F2 plants segregating for the Lr10 gene has been established and a marker closely linked to the gene (0.1 cM) was found. This marker was the starting point for the assembly of a physical contig in T.monococcum .The use of subcloned BAC ends for mapping was only successful in a few cases but in general was problematic. To derive probes from BAC clones for genetic mapping we developed a rapid “low pass” sequencing protocol. Shotgun DNA libraries from BAC clones were generated and sequenced at 1.5×genome equivalents. The obtained sequence data were sufficient to identify coding regions (usually good probes for mapping) as well as non coding, non repetitive sequences which sometimes can also be mapped and used as probes for further walking steps. Probes derived from sequencing have also to be physically mapped on the BAC clones to identify sequences close to the ends of the BACs. Four walking steps have been completed until now using these approaches. This resulted
