Leaf rust, caused by Puccinia recondita Rocb. ex Desm. f. sp. tritici Eriks. & Henn, is one of the most important diseases in wheat worldwide. There are more than 40 resistance genes against wheat leaf rust and us...Leaf rust, caused by Puccinia recondita Rocb. ex Desm. f. sp. tritici Eriks. & Henn, is one of the most important diseases in wheat worldwide. There are more than 40 resistance genes against wheat leaf rust and used in wheat breeding. The Lr1 resistance gene is one of them, originates from hexaploid wheat and is present in a number of cultivars. It is a dominant gene located at the distal end of chromosome 5DL of wheat. We are working on isolation of the Lr1 gene using a map based gene cloning approach. Generation of a saturated map around the target gene is the first step of map based gene cloning. Two segregating F 2 populations Thatcher Lr1 ×Thatcher (2814 individual plants) and Thatcher Lr1 ×Frisal (832 plants) are used for fine mapping of the Lr1 gene. Three micro satellite markers (GWM654, GWM269 and GWM272) and four RFLP markers (BCD1421, Psr567, pTAG621 and ABC718) are used to analyze the two mapping populations. The micro satellite marker GWM272 and the RFLP marker ABC718 are tightly linked to Lr1 gene. The two markers are located at 0.1 cM from the Lr1 gene. For physical mapping of the Lr1 gene, genomic BAC and YAC libraries of barley and T.tauschii (D genome) have been screened with the RFLP marker ABC718. Five BAC clones from a genomic library of T.tauschii ,six from a genomic library of barley and one YAC clone from a YAC library of barley were isolated. All ends of BAC and YAC clones have been isolated and analyzed. The ends of BAC and YAC clones from barley could not be used for mapping because they are repetitive or did not hybridize with wheat DNA. Most of BAC ends from T.tauschii BAC clones showed repetitive sequences. Two BAC ends isolated from BAC clone L 1 121K23 (100 kb) are polymorphic and were mapped at the same position as the RFLP marker ABC718. We did not find any recombinants in the 100 kb region around the RFLP marker ABC718.展开更多
Comparative genomic analysis at the genetic map level has shown extensive conservation of the gene order between the different grass genomes in many chromosomal regions. However, little is known about the gene organiz...Comparative genomic analysis at the genetic map level has shown extensive conservation of the gene order between the different grass genomes in many chromosomal regions. However, little is known about the gene organization in grass genomes at the microlevel. Comparison of gene coding regions between maize, rice and sorghum showed that the distance between the genes is correlated with the genome size. We have investigated the microcolinearity at Lrk gene loci in the genomes of four grass species: wheat, barley, maize and rice. The Lrk genes, which encode receptor like kinases, were found to be consistently associated with another type of receptor like kinase ( Tak ) on chromosome groups 1 and 3 in Triticeae and on chromosomes homoeologous to Triticeae group 3 in the genomes in rice and maize. On Triticeae chromosome group 1, Tak and Lrk together with genes putatively encoding NBS/LRR proteins form a cluster of genes possibly involved in signal transduction. Comparison of the gene composition at orthologous Lrk loci in wheat, barley and rice revealed a maximal gene density of one gene per 4～5 kb, very similar to the gene density in Arabidopsis thaliana . We conclude that small and large grass genomes contain regions which are highly enriched in genes with very little or no repetitive DNA. The comparison of the gene organization suggested various genome rearrangements during the evolution of the different grass species, including a duplication of the Lrk region specific for the Triticeae on group 1 chromosomes. We are now analyzing the gene organization in the Lrk regions using BAC clones of the A genome (from T. monococcum ) and the D genome (from Ae.tauschii ). In addition, we are investigating the A, B and D genome in hexaploid wheat using a cosmid library. The accumulation of sequence information around the Lrk loci in several species (orthologs) and in the same species (paralogous genes) has allowed comparisons of genome relationships in the investigated regions.展开更多
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 in a physical contig spanning around 440 kb on chromosome 1AS. Progress will also be reported on the mapping of the Pm3b gene.展开更多
文摘Leaf rust, caused by Puccinia recondita Rocb. ex Desm. f. sp. tritici Eriks. & Henn, is one of the most important diseases in wheat worldwide. There are more than 40 resistance genes against wheat leaf rust and used in wheat breeding. The Lr1 resistance gene is one of them, originates from hexaploid wheat and is present in a number of cultivars. It is a dominant gene located at the distal end of chromosome 5DL of wheat. We are working on isolation of the Lr1 gene using a map based gene cloning approach. Generation of a saturated map around the target gene is the first step of map based gene cloning. Two segregating F 2 populations Thatcher Lr1 ×Thatcher (2814 individual plants) and Thatcher Lr1 ×Frisal (832 plants) are used for fine mapping of the Lr1 gene. Three micro satellite markers (GWM654, GWM269 and GWM272) and four RFLP markers (BCD1421, Psr567, pTAG621 and ABC718) are used to analyze the two mapping populations. The micro satellite marker GWM272 and the RFLP marker ABC718 are tightly linked to Lr1 gene. The two markers are located at 0.1 cM from the Lr1 gene. For physical mapping of the Lr1 gene, genomic BAC and YAC libraries of barley and T.tauschii (D genome) have been screened with the RFLP marker ABC718. Five BAC clones from a genomic library of T.tauschii ,six from a genomic library of barley and one YAC clone from a YAC library of barley were isolated. All ends of BAC and YAC clones have been isolated and analyzed. The ends of BAC and YAC clones from barley could not be used for mapping because they are repetitive or did not hybridize with wheat DNA. Most of BAC ends from T.tauschii BAC clones showed repetitive sequences. Two BAC ends isolated from BAC clone L 1 121K23 (100 kb) are polymorphic and were mapped at the same position as the RFLP marker ABC718. We did not find any recombinants in the 100 kb region around the RFLP marker ABC718.
文摘Comparative genomic analysis at the genetic map level has shown extensive conservation of the gene order between the different grass genomes in many chromosomal regions. However, little is known about the gene organization in grass genomes at the microlevel. Comparison of gene coding regions between maize, rice and sorghum showed that the distance between the genes is correlated with the genome size. We have investigated the microcolinearity at Lrk gene loci in the genomes of four grass species: wheat, barley, maize and rice. The Lrk genes, which encode receptor like kinases, were found to be consistently associated with another type of receptor like kinase ( Tak ) on chromosome groups 1 and 3 in Triticeae and on chromosomes homoeologous to Triticeae group 3 in the genomes in rice and maize. On Triticeae chromosome group 1, Tak and Lrk together with genes putatively encoding NBS/LRR proteins form a cluster of genes possibly involved in signal transduction. Comparison of the gene composition at orthologous Lrk loci in wheat, barley and rice revealed a maximal gene density of one gene per 4～5 kb, very similar to the gene density in Arabidopsis thaliana . We conclude that small and large grass genomes contain regions which are highly enriched in genes with very little or no repetitive DNA. The comparison of the gene organization suggested various genome rearrangements during the evolution of the different grass species, including a duplication of the Lrk region specific for the Triticeae on group 1 chromosomes. We are now analyzing the gene organization in the Lrk regions using BAC clones of the A genome (from T. monococcum ) and the D genome (from Ae.tauschii ). In addition, we are investigating the A, B and D genome in hexaploid wheat using a cosmid library. The accumulation of sequence information around the Lrk loci in several species (orthologs) and in the same species (paralogous genes) has allowed comparisons of genome relationships in the investigated regions.
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 in a physical contig spanning around 440 kb on chromosome 1AS. Progress will also be reported on the mapping of the Pm3b gene.
