RNA-seq characterization of non-target-site mechanisms in imidazolinone resistant sunflower (Helianthus annuus L.)

FELITTI, S.; NESTARES, G.; GIL, MERCEDES; PICARDI, L.; VEGA, T.; BALZERGUE, S.

Praga

Congreso; 7th International Weed Science Congress (IWSC); 2016

Imidazolinone resistance has been found in a sunflower wild population and successfully transferred to a cultivated sunflower inbred line, HA425. This resistance has been reported to be genetically controlled by two genes: Imr1 (an allelic variant of ahasl1 locus that codes for acetohydroxyacid synthase catalytic subunit) and the modifier Imr2. The mechanism of the resistance endowed by Imr2 remains unknown but it could be related to non-target-site resistance such as xenobiotic metabolism. The objective of this study was to characterize HA425 resistant sunflower gene expression in response to imazethapyr using RNA-Seq. Previously, two assays were carried out to determine: (i) optimal herbicide treatment length (ii) acetohydroxyacid synthase in vitro activity to assess enzyme inhibition levels. Plants were grown in plastic pots (4cm x 5.5cm) filled with commercial perlite and watered by capillarity with 25% Murashige and Skoog?s salts solution, in growth chamber under controlled conditions. After 7 days, plants were treated with 0 (control) and 1μM imazethapyr during 12 hours. Leaf RNA was purified and quantified. Stranded, pair-ended cDNA libraries were constructed with Illumina TruSeq Stranded RNA-Seq protocol and sequencing was performed in Illumina HiSeq2000. Local, with and without multihits mapping was carried out using Bowtie2/Sam2Count over a reference de novo transcriptome. After normalization and FastQC quality assessment, expression levels were analysed with EdgeR. Seventeen interesting contigs were verified by RT-qPCR. An important number of xenobiotic metabolism genes were found: cytochromes P450s, ABC transporters, glycosyltransferases, UDPglucuronosil/glucosyltransferases and glutathione S-transferases. Tissue expression patterns were analyzed using HeliaGene tools. None of these genes showed differential expression between 1μM imazethapyr and control plants (P>0.05). These results suggest that non-target-site resistance mechanisms may contribute to herbicide resistance in sunflower and they could be related to the modifier Imr2. RNAseq analysis allowed to detect constitutively expressed detoxification genes potentially related to imidazolinone resistance in sunflower.