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ARS Home » Southeast Area » Stoneville, Mississippi » Genomics and Bioinformatics Research » Research » Research Project #434492

Research Project: Genetic Diversity of Allium cepa and Its Pathogens

Location: Genomics and Bioinformatics Research

Project Number: 6066-21310-005-31-I
Project Type: Interagency Reimbursable Agreement

Start Date: Apr 1, 2018
End Date: Sep 30, 2018

Objective:
Develop genetic markers for Allium cepa and its pathogens.

Approach:
The process of genetic marker development includes extraction of DNA from subject species, generation of genomic DNA sequencing libraries, high-throughput sequencing of genomic DNA libraries, assembly of genomic scaffolds, bioinformatics of DNA sequences to identify polymorphic markers, development of primer pairs to amplify genomic DNA regions containing polymorphic markers, and validation of the selected markers on a broader population. Extraction of DNA in the home country of the Fellow prior to beginning of the fellowship will facilitate orderly cataloguing and preservation of the germplasm of cultivars and strains used during the fellowship and eliminate the quarantine requirements for transporting tissues. Indexed DNA sequencing libraries will be prepared using the Nextera (Illumina) high-throughput library construction kit. Library purification and quantification will be performed using standard procedures. Multiple indexed libraries will be pooled for sequencing on Illumina miSeq or HiSeq2500 instruments. DNA sequence data will be de-multiplexed and sequence reads from each individual will be assembled using CLC Genome Work Bench (Qiagen) v10. Assembled contigs and scaffolds will be used to identify simple sequence repeats (SSR) and single nucleotide polymorphisms (SNP) using bioinformatics software. PCR primers will be developed for verified polymorphic markers and tested on a broader population of the same species to empirically validate the markers.