Biology Reference
In-Depth Information
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RME-1
NS158
Fig. 15.8. Fingerprinting of positive clones. RME-1 and NS158 clones were
digested with Hind III and resolved on 1.2% agarose gel.
PCR was conducted under standard conditions.
To develop allele-specific primers, the ampli-
fied bands from parentals and bulks were eluted,
cloned into the commercial vector pGemT-easy,
and sequenced at the Iowa State University facil-
ity center. Sequences obtained with each primer
were aligned using ClustalW (Thompson et al.
1994) to find single nucleotide polymorphism
(SNP) regions, and then allele-specific primers
were designed using the SNAPER 3 program.
To develop SSCP-SNP markers, the PCR ampli-
fication products of the parentals and bulks were
denatured and separated by single-strand con-
formational polymorphism (SSCP) gels using
a mutation detection enhancement (MDE) gel
solution, following the conditions recommended
for the author (Bertin et al. 2005). The candi-
date markers were evaluated in each individual
of each bulk (opened bulk) and the recombinant
susceptible individuals.
Two contigs from RME-1 positive clones and
one contig from NS158 positive clones were
obtained with the FPC program. Six BAC clones
(# 9, 18, 23, 33, 35, and 36) were identified as the
contigs' ends. BAC clones at the extremes of the
two contigs were end-sequenced and converted
into SSCP markers. The Clustal W analysis of
all sequences obtained for each primer permit-
ted us to identify an allele-specific region that
showed differences between resistant and sus-
ceptible individuals. This region was identified
when the sequences from clone # 9 were ana-
lyzed, and eight pairs of primers were designed
on this region. During the SSCP-SNP primers
evaluation, a clear difference between resistant
and susceptible bulks with the BAC#33b SSCP-
SNP was obtained (Figure 15.9). The SSCP
markers were then screened in resistant bulk,
susceptible bulk, and the parents of the map-
ping population, and then used in another round
of BAC pool screening. In addition, a sub-
library of the BAC#33 was constructed (Figure
15.10) by digestion with Hind III and cloning
into the pBluescript vector using standard pro-
tocols. The sequencing was conducted at The
Institute for Genomic Research (TIGR). Low
copy sequences were analyzed in the genome
public database and the biotechnology cassava
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