De novo transcriptome analysis of finger millet and identification of genes involved in Striga infestation
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Erick Mikwa, Mark Wamalwa, Francesca Stomeo, Richard Oduor, Mercy Macharia, Damaris Odeny. (1/3/2018). De novo transcriptome analysis of finger millet and identification of genes involved in Striga infestation.
Background: Finger millet is a highly nutritious annual cereal grass mainly cultivated in the semi-arid tropics of the world. Despite its high yielding potential, finger millet production is mainly affected by Striga hermonthica in Sub-Saharan Africa. The survival of Striga on finger millet depends on a complex host-parasite interaction delineated by three critical infestation time points, 3 days’ post-inoculation (dpi), 5 dpi and 7 dpi. Using RNA-seq it is possible to identify functional transcripts in finger millet and to profile immunity related genes at the specific time points. Methods: In this study, the transcriptome of two resistant and two susceptible finger millet genotypes were sequenced upon infecting with Striga hermonthica seeds. The main aim of the study was to elucidate molecular responses to Striga in finger millet and to identify some of the genes acting at each time point. Roots of finger millet seedlings were infected with Striga seeds and whole seedling samples were collected after 3, 5 and 7 dpi for RNA extraction and sequencing. The resulting reads from each genotype were assembled using Shannon assembler and analyze separately. Results: The sequencer generated slightly over 67.5 million clean reads from the four genotypes, which were assembled per genotype to produce a total of 139,162 non-redundant transcripts. The transcripts were annotated by querying against NCBI RefSeq plant, Uniprot plant, Pfam and COG databases. A total of 76,415, 78,825, 2049 and 17930 transcripts mapped to RefSeq plant, Uniprot plant, Pfam and COG databases, respectively. 33, 527 Gene Ontology (GO) terms and 459 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were annotated using BLAST homologies from NCBI databases at an e-value of 1e-5. Further classification of Uniprot protein homologies produced about 2713 Panther protein classes. A total of 407 transcripts were differentially expressed (DE) at least 4-fold at a false discovery rate (FDR) of 0.1% in a pairwise comparison of all samples. Of the DE of 320 transcripts were down-regulated and 97 up-regulated between Striga infested Eleusine coracana and their respective controls. In all the genotypes a majority of genes were up-regulated at 5 dpi as compared to 3 dpi and 7 dpi. Conclusion: The results showed that a large number of photosynthesis related genes and housekeeping genes were expressed after 3 dpi. Each genotype had a partial stage-specific tolerance to Striga, as opposed to the overall compatibility or incompatibility revealed by the previous screening field methods.