Construction of the BAC contig encompassing the dl locus

VI. Gene and Genome Structure 39. Construction of the BAC contig encompassing the dl locus T. YAMAGUCFH�, S. KAWASAKI2, Y. NAGATO1, H.Y. Hnw�io� 1) Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, 113-8657 Japan 2) National Institute of Agrobiological Resources, Kannon-dal 2-1-2, Tsukuba, 305-0856 Japan Flower development is one of the most important events in plant development. We have attempted to elucidate the genetic and molecular mechanisms that control rice flower development. In rice, the DROOPING LEAF (DL) gene plays an important role in flower development. DL has a dual function that specifies the pistil identity and forms the leaf midrib (Nagasawa eta!. 1996). To understand the role of DL in flower development and the mechanisms how a single locus regulates the development of two different organs, we have attempted to isolate the DL gene by map-based cloning. In this study, we identified the molecular markers closely linked to the dl locus and constructed the BAC contig encompassing the dl locus. The dl locus has been mapped on chromosome 3 of the rice linkage map. Preliminary studies using several RFLP markers on chromosome 3 indicated that the marker C316 was close to the dl locus. The RFLP analysis usingl52 dl homozygous F2 plants from the cross between fm28 (Kinmaze), one of the dl mutants, and Kasalath, revealed that the dl locus was located near the marker C316 (Fig. 1A). No recombinant between dl and the markers C316 and R2479 was detected but one recombinant between dl and the marker Ri 76 were observed. The recombination between dl and the other markers was relatively high. Since tightly linked markers were identified, we tried to construct the BAC contig around the dl locus. Five clones were isolated by screening the rice BAC library (Nakamura et a!. 1997) with the marker C316, and a primary BAC contig was constructed. The DNA segment (A5L) at the proximal end of this contig was obtained by TAIL-PCR and was used as a probe in chromosome walking. As a result, four clones were isolated and the expanded BAC contig was constructed. The contiguity of these BAC clones was confirmed by the pattern of the DNA fragments digested with NotI and hybridization experiments probed with several BAC end segments. The resulting BAC contig consists of nine BAC clones, covering the region of about 250kb (Fig. 1B). Since relatively wide range was covered by this contig, we tested whether the dl locus was included in this BAC contig. The DNA segments at the both ends of this BAC contig were used for a RFLP analysis after the identification of several restriction enzymes that produced polymorphisms between fm28 and Kasalath. Among the 152 dl homozygous F2 plants, one recombinant with the segment (AJ5R) at the distal end and four recombinants with the segment (LJL) at the proximal end were found. It is therefore concluded that this BAC contig encompasses the dl locus. We are now trying to isolate the DL gene using this BAC contig. References Nagasawa, N., M. Miyoshi, Y. Sano and Y. Nagato, 1996. DL regulates both leaf and pistil development in rice. Rice Genet. Newslett. 13: 102-105. Nakamura, S., S. Asakawa, N. Ohmido, K. Fukui, N. Shimizu and S. Kawasaki, 1997. Constniction of an 800- kb contig in the near-centromeric region of the rice blast resistance gene Pi-ta2 using a highly representative rice BAC library. Mol. Gen. Genet. 254: 611-620.

39. Construction of the BAC contig encompassing the dl locus
       T. YAMAGUCFH�, S. KAWASAKI2, Y. NAGATO1, H.Y. Hnw�io�
       1) Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, 113-8657 Japan
       2) National Institute of Agrobiological Resources, Kannon-dal 2-1-2, Tsukuba, 305-0856 Japan

Flower development is one of the most important events in plant development. We have attempted to elucidate the genetic and molecular mechanisms that control rice flower development. In rice, the DROOPING LEAF (DL) gene plays an important role in flower development. DL has a dual function that specifies the pistil identity and forms the leaf midrib (Nagasawa eta!. 1996). To understand the role of DL in flower development and the mechanisms how a single locus regulates the development of two different organs, we have attempted to isolate the DL gene by map-based cloning. In this study, we identified the molecular markers closely linked to the dl locus and constructed the BAC contig encompassing the dl locus.

The dl locus has been mapped on chromosome 3 of the rice linkage map. Preliminary studies using several RFLP markers on chromosome 3 indicated that the marker C316 was close to the dl locus. The RFLP analysis usingl52 dl homozygous F2 plants from the cross between fm28 (Kinmaze), one of the dl mutants, and Kasalath, revealed that the dl locus was located near the marker C316 (Fig. 1A). No recombinant between dl and the markers C316 and R2479 was detected but one recombinant between dl and the marker Ri 76 were observed. The recombination between dl and the other markers was relatively high.

Since tightly linked markers were identified, we tried to construct the BAC contig around the dl locus. Five clones were isolated by screening the rice BAC library (Nakamura et a!. 1997) with the marker C316, and a primary BAC contig was constructed. The DNA segment (A5L) at the proximal end of this contig was obtained by TAIL-PCR and was used as a probe in chromosome walking. As a result, four clones were isolated and the expanded BAC contig was constructed. The contiguity of these BAC clones was confirmed by the pattern of the DNA fragments digested with NotI and hybridization experiments probed with several BAC end segments. The resulting BAC contig consists of nine BAC clones, covering the region of about 250kb (Fig. 1B).

Since relatively wide range was covered by this contig, we tested whether the dl locus was included in this BAC contig. The DNA segments at the both ends of this BAC contig were used for a RFLP analysis after the identification of several restriction enzymes that produced polymorphisms between fm28 and Kasalath. Among the 152 dl homozygous F2 plants, one recombinant with the segment (AJ5R) at the distal end and four recombinants with the segment (LJL) at the proximal end were found. It is therefore concluded that this BAC contig encompasses the dl locus. We are now trying to isolate the DL gene using this BAC contig.

References

Nagasawa, N., M. Miyoshi, Y. Sano and Y. Nagato, 1996. DL regulates both leaf and pistil development in rice. Rice Genet. Newslett. 13: 102-105.

Nakamura, S., S. Asakawa, N. Ohmido, K. Fukui, N. Shimizu and S. Kawasaki, 1997. Constniction of an 800- kb contig in the near-centromeric region of the rice blast resistance gene Pi-ta2 using a highly representative rice BAC library. Mol. Gen. Genet. 254: 611-620.