This study compared the field performance of red clover germplasm UK2014, selected for 2,4-D tolerance, to Kenland, a standard variety grown in the transition zone of the United States. UK2014 and Kenland were seeded in the spring of 2017 and 2018. Single applications of 0, 1.12, or 2.24 kg ae ha⁻¹ 2,4-D-amine were made in June, August, or October. One week after the treatments, yields were determined. Visible herbicide injury ratings were made prior to harvest and regrowth was visibly assessed 1 wk after harvest. Red clover stands were visibly assessed the following spring. Kenland, across all application timings, was injured by 2,4-D more than UK2014, with mean injury ratings of 39% and 63% compared with 26% and 37% at 1.12 and 2.24 kg 2,4-D ae ha⁻¹, respectively. At equivalent rates, Kenland regrowth was less than UK2014 at all application timings. UK2014 regrowth after 2,4-D treatment ranged from 65% to 91%, whereas Kenland regrowth ranged from 12% to 72%. Applications of 2,4-D in October were the most damaging to stands of both UK2014 and Kenland the following spring, but Kenland stands were reduced much more than those of UK2014. Kenland and UK2014 had similar season total yields when not treated with 2,4-D (means of 7,550 and 7,880 dry matter kg ha⁻¹, respectively in 2017 and 5,280 dry matter kg ha⁻¹ for both in 2018). Kenland season total yield in 2017 was reduced by both 2,4-D rates applied in June or August and at all timings in 2018. UK2014 season total yield in 2017 was reduced only when 2.24 kg 2,4-D ae ha⁻¹ was applied in August. In 2018, 2.24 kg ae ha⁻¹ 2,4-D resulted in reduced UK2014 season total yield across application timings. UK2014 has greater 2,4-D tolerance than Kenland, but additional selection might be beneficial.

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.