Transgene stability, transposon activation and potential for gene tagging in peanut (Arachis hypogaea L)
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Crop improvement in peanut (Arachis hypogaea L.) is aimed at improving yield, product quality, reduction in seed allergen content and resistance to biotic and abiotic stresses. Application of RNA interference (RNAi) was successful in developing a transgenic line B11.1.1/11 with complete reduction of Ara h 2 and reduced levels of Ara h 6, two major seed allergens. The transgene was stable across three transgenic generations tested as Ara h 2 signal was absent and Ara h 6 signal was reduced in quantitative western blot assay. Stability of transgene silencing to altered soil sulfur, specifically on partially silenced Ara h 6, was tested by growing the transgenic line under three soil sulfur levels. Transgenic lines showed no Ara h 2 expression and significantly reduced Ara h 6 expression under different sulfur levels, providing further evidence for stability of the transgene and the effectiveness of RNAi even under nutritional conditions optimal for synthesis of sulfur-rich proteins. High soil sulfur concentrations significantly increased the levels of Ara h 2 and Ara h 6 in non-transgenic lines compared to low sulfur. Ara h 3 levels were significantly reduced under low sulfur in both transgenic and non-transgenic lines and Ara h 1 levels were unaffected by sulfur. Activation of the peanut transposon, AhMITE1, was studied under two stress situations, namely ethylmethane sulfonate (EMS) mutagenesis and prolonged tissue culture. Transposon display provided evidence that the peanut Miniature Inverted-repeat Transposable Element (MITE) was activated under both stresses and variation was observed with regard to the different stress situations and genotypes studied (‘Tifrunner’ and ‘Florunner’). Peanut MITE sequence capture, Sanger sequencing, and primer design to MITE and flanking DNA sequences enabled the development of 17 markers polymorphic across 29 peanut genotypes. Sequences flanking MITEs showed similarity to protein coding regions via BLAST searches. High throughput Illumina sequencing with five genotypes was carried out to recover additional flanking sequence for marker development and gene identification. Stability of protein silencing across generations and under different nutritional conditions showed that the polyploid peanut genome is amenable to stable transgene expression. Genome instability associated with MITE transposons may occur under conditions of stress and may provide a source of positive and negative variation for gene functional analysis and breeding.