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20151
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Subject: Agricultural Biotechnology × Author: Ambu, Vijayan × Author: KAU × Start date: 2015 × Type: Thesis ×
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    Molecular marker development for cassava mosaic disease resistance using bioinformatics tools
    (Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Ambu, Vijayan; KAU; Sreekumar, J
    The study entitled “Molecular marker development for cassava mosaic disease resistance using bioinformatics tools” was conducted at ICAR-CTCRI, Sreekariyam, Thiruvananthapuram during October 2104 to October 2015. The objectives of the study included development and evaluation of various SNP and SSR prediction pipelines, computational prediction and characterization of SNP and SSR in cassava, verification of SNP and SSR markers for cassava mosaic disease (CMD) resistant and susceptible breeding lines. The preliminary data set for the identification of SSR and SNP markers was obtained from the EST section of NCBI and the cassava transcript sequences from the Phytozome. A total of 120461 sequences was classified into 20 cultivars. The dataset was reduced to 14336 sequences after several pre-processing and screening steps. The resulting sequences were assembled and aligned using CAP3 and 2088 contigs were obtained. SNPs and SSRs were predicted from these datasets using respective prediction tools. The SNP prediction tools such as QualitySNP and AutoSNP were compared for their performance. Analysis was performed to identify the tool with the ability to annotate and identify more viable nonsynonymous and synonymous SNPs. The SSR prediction tools such as MISA and SSRIT was compared for their performance. Analysis was performed to identify the tool having the ability to predict more viable SSRs and the ability to classify them as mono, di, tri, tetra, penta, hexa and poly SSRs. Using QualitySNP, thirty nonsynonymous SNPs and twenty-six synonymous SNPs were identified. Using MISA, n 217 mono SSRs, 132 di SSRs, 139 tri SSRs, 3 tetra SSRs, 1 penta SSRs, 3 hexa SSRs and 42 complex SSRs were identified. Five sequences from identified SNPs and SSRs which have high hit percentage were selected for validation and primer designing for CMD resistant genes. These primers were validated using 5 resistant and 5 susceptible cassava varieties. Among the 10 primers after validation in wet lab, one SNP (SNP896) and one SSR (SSR 2063) primer was able to clearly differentiate between the resistant and susceptible varieties which can be used as potential markers in the breeding program for screening CMD resistance in cassava.