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markers closer to the gene were initiated, result-
ing in the identification of another SSR marker,
NS158. With this result, two SSR markers:
SSRY28 and NS158 were selected as the clos-
est to the
CMD2
gene, at genetic distances
of 9 and 6.7 cM respectively (Akano et al.
2002; CIAT 2002). To improve on these mark-
ers, fine mapping of the
CMD2
region of the
genome was initiated by searching for recombi-
nants between
CMD2
and the two SSR markers
closely linked to the gene (SSRY28 and NS158),
using a large full-sib population followed by an
analysis of the recombinants with thousands of
readily assayed polymorphic markers to identify
additional markers more closely linked to the
gene. The fine-mapping population consisted of
1,690 individuals from a cross between TME3,
the source of
CMD2,
and the improved cultivar
TMS30572. The population had initially been
intended to improve CMD resistance by com-
bining both sources of resistance but was found
to be suitable for fine mapping studies, given that
cassava is highly heterozygous and as expected
segregated for CMD expression as revealed by
the phenotypic data for the disease. Beyond the
need to improve resolution of genetic mapping of
markers in the genomic region of interest and to
identify new markers tightly linked to the
CMD2
gene, which, being a dominant gene, was easily
evaluated for MAS efficiency in the population.
The cross was evaluated for CMD resistance at
IITA, Nigeria, under heavy natural pressure of
the disease. DNA was isolated from the indi-
viduals of the cross, using the method formu-
lated by Dellaporta and colleagues (1983). The
population was then evaluated with SSRY28 and
NS158, described by Mba and colleagues (2001),
and 112 recombinants between the markers and
CMD2
were identified.
DNA from ten resistant recombinants and
ten susceptible recombinants was combined
to form two bulks, which were then eval-
uated with several marker systems, includ-
ing AFLPs, ISTRs (inverse sequence-tagged
repeats), RAPDs, SCARs (sequence character-
ized amplified regions), and SSRs, using a mod-
ified bulk segregant analysis (BSA) method
(Michelmore et al. 1991). Evaluation with AFLP
markers (Vos et al. 1995) was done using a
commercial AFLP (Invitrogen Life Technolo-
gies, Gaithersburg, MD), following the manu-
facturer's instructions. All 64 possible combi-
nations were used in the evaluation. For ISTRs
(inverse sequence-tagged repeats), the method
described by Rohde and colleagues (1996) was
used, with all possible 64 combinations of the
8 F and 8 B universal retro-elements (retro-
transposons) sequence primers. Evaluation with
RAPD markers was done using 768 commercial
primers (Operon Technologies Inc, CA) and a
modified protocol of that developed by Williams
and colleagues (1990). Markers that were poly-
morphic in the recombinant bulks were then
analyzed in individuals of the bulks. Analysis
with RAPD markers produced three polymor-
phic candidate markers, AC-15, RME-1, and
RME-2, that remained consistent in the individ-
uals of the bulks (Figure 15.4). Evaluation of the
three markers in the entire fine-mapping progeny
revealed that the
CMD2
gene is flanked by
NS158 and RME-1, with RME-1 being the clos-
est marker, at 4.5 cM. The polymorphic fragment
in RME-1, a band of 800 bp was cloned into the
pGEMT-easy and sequenced. A SCAR primer
pair was designed from the cloned sequence in
order to permit the application of the new marker
in marker-assisted selection (MAS) programs.
The above result provided a molecular marker
with a closer link to the
CMD2
to be used for
screening of a cassava BAC library and construc-
tion of BAC contigs, toward positional cloning
of the gene.
(c) CMD3
TMS97/2205 is a top elite line developed at IITA
and has high resistance to CMD, with sever-
ity symptoms being low or near immunity. Dis-
ease incidence in this cultivar is also very low
(
<
1%) in high CMD pressure zones in Nige-
ria. The resistance profile provided a basis to
evaluate this cultivar with molecular markers for
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