Plant Genome II Conference
Town & Country Conference Center, San Diego, CA, January, 1994.
PG-II: ISOLATION OF MARKERS TIGHTLY TO A VIRUS RESISTANCE GENE (I) OF
THE COMMON BEAN
ISOLATION OF MARKERS TIGHTLY TO A VIRUS RESISTANCE GENE (I) OF
THE COMMON BEAN
I.V. Degremont and C. E. Vallejos, Dept. of Hort. Sci, and
Plant Molec. and Cell. Biology Program, University of Florida,
Gainesville, FL 32611
Dominant resistance to bean common mosaic virus (BCMV) is
controlled by the I gene in common beans. This gene is also
responsible for a hypersensitive response. The I gene has been
mapped to a distal location on linkage group D of the P. vulgaris
map [Vallejos et al., Genetics 131, 733 (1992)]. The objective
of this project is to construct a molecular marker-based high
density map around the I gene as a prerequisite to map-based
cloning. We are using a 'reverse genetics' approach to "bulked
segregant analysis' (Michelmore et al., PNAS 88, 9828 (1991)] for
the isolation of molecular markers tightly linked to the I gene.
Two methods have been used to isolate these markers: a)
subtractive hybridizations of size-selected restriction fragments
[Lisitsyn et al., Science 259, 946 (1993)]; and b) selective
amplification of short polymorphic restriction fragments. We
are using a segregating progeny between the Mesoamerican line
Jamapa (II) and the Andean line Calima (ii). F2-derived F3
families were used for genotyping at the I locus with the
necrotic strain NL3. Nuclear DNA was extracted from 12 to 16
plants per F3 family, and DNA pools of each homozygous class
were obtained (12-15 F3 families/pool). HindIII digests were
size fractionated by agarose gel electrophoresis and fragments
from a predetermined size range were recovered from the gel and
purified. These fragments were ligated to HindIII adapters and
amplified via PCR prior to subtraction. Subtractive
hybridizations of parental DNAs have been carried out to assess
the effectiveness of the method. Subtractions of the bulked DNAs
are now in progress. DNA fragments ligated to adapters were
amplified in the presence of 35S-dATP using a set of variable
primers; polymorphisms are resolved in denaturing gels and
visualized by autoradiography.
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