Identification Of Microsatellite Markers For Finger Millet (Eleusine Coracana) By Analysis Of Roche 454 Gs-Flx Titanium Sequence Data

ABSTRACT

Finger millet is an important cereal cultivated in Eastern Africa as well as Southern India. It is a staple crop that is characterized by ability to thrive on a variety of environmental conditions, excellent grain storage quality and ability to withstand significant levels of salinity. Scientific research aimed at improving this important cereal has been negligible and it is regarded as one of the orphaned crop species. The aim of this project was to isolate microsatellite markers from finger millet by analyzing data provided following 454 GS-FLX Titanium sequencing. This is a next generation sequencing platform that confers the potential of isolating a greater number of microsatellites at a much lower cost than the conventional Sanger sequencing platform. These markers are hyper variable, and exhibit wide genomic distribution, co-dominant inheritance, reproducibility, multi-allelic nature and chromosome specific location. The 2 genotypes of finger millet studied were obtained from ICRISAT, Nairobi. These were KNE 755 and KNE 796. In silico tools were used to mine for the microsatellites from sequence data obtained after enrichment and 454 GS-FLX sequencing of finger millet genomic DNA. These tools included NGSQC, for quality screening, MIRA for sequence assembly, SciRoKo for SSR mining and BatchPrimer3 for primer design. In vitro analysis was undertaken to evaluate the markers supplied by Ecogenics. This involved PCR analysis followed by allele calling using the Gene Mapper software package. The allelic data generated was subjected to statistical analysis using PowerMarker to reveal polymorphism. The in silico study resulted in the identification of 92 primers that were unique from the published as well as the Ecogenics supplied primers. From the in vitro study, 49 markers were polymorphic and the average polymorphic information content (PIC) was 0.4153. These markers are a significant addition to the existing 82 SSRs. They are also valuable tools that will be useful for conducting further genomic studies in finger millet, including MAS, fingerprinting studies as well as assaying genetic diversity. This study has demonstrated the use of NGS to rapidly and cost-effectively generate genomic sequences containing SSR motifs. It points to the possibility of using cutting edge technology to advance research in underutilized crops such as finger millet by researchers in Kenya. It also shows that it is possible to develop abundant genomic resources for hitherto understudied crops which have great significance to the development of third world countries in Africa and Asia