Variability in Fusarium species Causing Wilt Disease in Crops: A Transcriptomic Approach to Characterize Dialogue Between Host and Pathogen - Crop Improvement: New Approaches and Modern Techniques - page 284

done in unigenes within the transcript assembly. The SSRs with a minimum repeat
count ( n ) threshold of n ≥ 5 can be selected for further analysis and EST-SSR marker
development. Flanking forward and reverse primers will be designed for SSRs in
unigenes using Primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.
cgi) and products can be genotyped by sequencing and the allele lengths can be as-
certained by gene mapper. The SSR markers can be screened for amplification and
length polymorphisms among Fusarium strains on agarose. The estimation of EST-
SSR individual markers heterozygosities ( H ) and Genetic distances ( G ) can be done
using the proportion of shared alleles estimator in Microsat, where G = (1 − p ) and p
is the proportion of shared alleles http://hpgl.stanford.edu/projects/microsat/. These
data offer an opportunity to identify single sequence repeats (SSRs) in expression
sequence tags (ESTs) by data mining. Such kind of studies shall give an insight into
the frequency, distribution and type of Fusarium EST-SSRs and demonstrate suc-
cessful development of EST-SSR markers in crop pathogenesis. These EST-SSR
markers would be enriching the current resources of molecular markers for the sci-
entific community and would be useful for Fusarium identification at species level
and breeding programs to develop resistant varieties. Further, the novel EST-SSRs
would be useful for comparative genetic mapping which shall give us information
about the genetic diversity and polymorphism among the Fusarium sp. All these
shall provide the novel insights and knowledge about Fusarium pathogenesis.
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