Plant Genome II Conference
Town & Country Conference Center, San Diego, CA, January, 1994.
PG-II: USE OF DIGOXIGENIN-LABELED PROBES FOR RFLP MAPPING OF POLYPLOID
SACCHARUM SPECIES
USE OF DIGOXIGENIN-LABELED PROBES FOR RFLP MAPPING OF POLYPLOID
SACCHARUM SPECIES.
Maureen M.M. Fitch 1, Elizabeth A. Ferrero 2, Paul H. Moore 1,
and K.K. Wu 3, 1 USDA,ARS, Aiea, Hi 96701; 2 University of
Hawaii, Honolulu, HI 96822; 3 Hawaiian Sugar Planters'
Association, Aiea, HI 96701.
Genome analysis by RFLPs depends on visualizing DNA
fragments hybridizing to labeled probes. The usual method of
labeling probes with radioactive 32P can be difficult in
localities where tropical crops such as sugarcane are grown and
analyzed. Digoxigenin-labeled DNA probes are widely used for
detecting single genes in simple genomes, but their applications
to mapping of complex genomes have been limited. We report
procedures that allow us to use chemiluminescence to RFLP map
Saccharum species having a haploid genome of about 3500 Mbp/1C.
Anion exchange chromatography was essential for quickly isolating
plasmids and purifying probe DNAs. The nonradioactive probes
were resin-purified as well in order to minimize background
interference. Use of the chemiluminescent substrate Lumi-phos
530 (1,2-dioxetanephosphate + a fluorescence enhancer) allowed
for quick exposure and re-probing of membranes at least three
times. Probes of 300 to 3000 bp in size were used at
concentrations of 10-25 ng/ml or 60 ng/80 cm^2 blot. Signals
were identical to those obtained with radiolabeled probes, while
detection time was significantly shorter. Probes prepared from
PstI-digests of genomic sugarcane clones and from cDNA clones
from bud, root, and callus libraries appear useful for mapping.
A progeny population (S. officinarum x S. robustum) screened with
21 probes produced 148 bands. About 100 were polymorphic and 58
were single dose. Approximately half of the single dose bands
were from S. officinarum. Two linkage groups have been detected
in S. officinarum and three in S. robustum. The probes were also
used to develop fingerprint identities of the 80 progeny and two
parents. Four probes were sufficient to fingerprint the entire
population. Initial difficulties in adapting chemiluminescence
labeling for RFLP mapping in sugarcane were resolved by using
anion exchange for purifying the plasmid and probe DNAS. With
this modification, the nonradioactive probes are being used in
the RFLP mapping of Saccharum species and to fingerprint
sugarcane.
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