Next generation sequencing as a tool in modern pest risk analysis: a casestudy of groundnuts (Arachis hypogaea) as a potential host of new virusesin western Kenya

Abstract

Groundnut (Arachis hypogaea, L.) is grown in diverse environments throughout the semi-arid and sub-tropical regions of the world. Poor yields of 500-800kg/ha are attributed to poor agronomic practices, pests and diseases. The major disease reported in Kenya is Groundnut rosette disease (GRD). But recent observations in the field showed that the crop has varied and severe symptoms in addition to those caused by GRD. This required deeper analysis to establish the causal agents. Groundnut samples with virus-like symptoms were collected from western Kenya in 2016. Total RNA was extracted using All Prep RNA Mini Kit. Five mRNA libraries were prepared using the Illumina TrueSeq stranded mRNA library Prep Kit and pooled for multiplexed sequencing using an Illumina HiSeq 2500 to generate paired end reads (FastQ Sanger). The reads were analysed in the Galaxy project platform (customized). Quality reads were first mapped onto plant genome Refseq and unmapped reads isolated and mapped onto virus Refseq using Bowtie 2 (v2.2.3). Groundnut rosette virus satellite RNA, Groundnut rosette virus, Groundnut rosette assistor virus, Ethiopian tobacco bushy top virus, Cowpea polerovirus 2, Chickpea chlorotic stunt virus, Melon aphid-borne yellow virus, Phasey bean mild yellow virus, Beet mild yellowing virus, White clover mottle virus and Cotton leafroll dwarf virus were identified in four libraries. Other viruses (with less than 100 reads) including Bean common mosaic virus, Bean common mosaic necrosis virus, Cowpea chlorotic mottle virus RNA 3, Broad bean mottle virus RNA 3, Passion fruit woodiness virus among others were also mapped. Some of the viruses common in western Kenya were confirmed by PCR. The presence of at least three viruses in groundnuts in Western Kenya highlights the importance of starting a germplasm clean-up program of the plant material used as seed in this crop.