Comparative Gene Expression following 2,4-D Treatment in Two Red Clover (Trifolium pratense L.) Populations with Differential Tolerance to the Herbicide

Incorporation of red clover (Trifolium pratense L.) into grass pastures can reduce the need for nitrogen fertilizer applications and increase the nutritional value of the forage. However, red clover cultivars available for Kentucky producers are highly susceptible to herbicides, such as 2,4-D (2,4-dichlorophenoxy acetic acid), used for pasture broadleaf weed control. To overcome this problem, ‘UK2014’ red clover was selected for increased tolerance to 2,4-D. We employed a transcriptome analysis approach to compare the gene expression response following 2,4-D treatment of ‘UK2014’ to that of ‘Kenland’, a 2,4-D sensitive red clover and one of the parents of ‘UK2014’. The objectives were to first determine if the increased 2,4-D tolerance in ‘UK2014’ is reflected in a change of transcription response and/or a quicker recovery of a transcriptional response following 2,4-D treatment, and second, to identify genes, whether constitutively expressed or induced by 2,4-D, which could be the basis for the increased 2,4-D tolerance. Leaf tissue from the two red clovers grown in the field was collected at 4, 24, and 72 h after 2,4-D (1.12 kg 2,4-amine a.e. ha⁻¹) treatment from both untreated and treated plants. Global gene expression was determined with reads from Illumina Hiseq 2500 mapped against the red clover draft genome, Tpv2.1 (GenBank Accession GCA_900079335.1). Genes that displayed differential expression (DEGs) following 2,4-D treatment were selected for further analysis. The number of DEGs was higher for ‘Kenland’ than for ‘UK2014’, suggesting that a lower transcriptional response corresponds with the higher 2,4-D tolerance in the ‘UK2014’ line. Similarly, gene ontology enrichment analysis revealed that expression of photosynthesis-related genes was less affected by 2,4-D in the ‘UK2014’ line than ‘Kenland’. Although we were not able to identify any specific genes that are the basis for the increased 2,4-D tolerance of ‘UK2014’, we concluded that the increased 2,4-D tolerance of ‘UK2014’ correlates with a decreased transcription response to 2,4-D. Additionally, expression of several cytochrome P450 genes that had different isoforms between ‘UK2014’ and ‘Kenland’ increased significantly in both following 2,4-D treatment, one or more of these P450s could be mediators of 2,4-D metabolism and tolerance in red clover.