Reference "Construction 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"

Reference ID

3069

Title

Construction 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

Source

Molecular & General Genetics, 1997, vol. 254, pp. 611-620

Authors (6)

Nakamura-S	Asakawa-S
Ohmido-N	Fukui-K
Shimizu-N	Kawasaki-S

Abstract

We constructed a rice Bacterial Artificial Chromosome (BAC) library from green
leaf protoplasts of the cultivar Shimokita harboring the rice blast resistance
gene Pi-ta. The average insert size of 155 kb and the library size of seven
genome equivalents make it one of the most comprehensive BAC libraries
available, and larger than many plant YAC libraries. The library clones were
plated on seven high density membranes of microplate size, enabling efficient
colony identification in colony hybridization experiments. Seven percent of
clones carried chloroplast DNA. By probing with markers close to the blast
resistance genes Pi-ta2(closely linked to Pi-ta) and Pi-b, respectively located
in the centromeric region of chromosome 12 and near the telomeric end of
chromosome 2, on average 2.2 +/- 1.3 and 8.0 +/- 2.6 BAC clones/marker were
isolated. Differences in chromosomal structures may contribute to this wide
variation in yield. A contig of about 800 kb, consisting of 19 clones, was
constructed in the Pi-ta2 region. This region had a high frequency of repetitive
sequences. To circumvent this difficulty, we devised a "two-step walking"
method. The contig spanned a 300 kb region between markers located at 0 cM and
0.3 cM from Pi-ta. The ratio of physical to genetic distances (> 1,000 kb/cM)
was more than three times larger than the average of rice (300 kb/cM). The low
recombination rate and high frequency of repetitive sequences may also be
related to the near centromeric character of this region. Fluorescent in situ
hybridization (FISH) with a BAC clone from the Pi-b region yielded very clear
signals on the long arm of chromosome 2, while a clone from the Pi-ta2 region
showed various cross-hybridizing signals near the centromeric regions of all
chromosomes.