sunTILL: a TILLING resource for gene function analysis in sunflower

To balance the maximum mutation density with an acceptable plant survival rate, a 'kill curve' analysis was first conducted with different ethylmethanesulfonate (EMS) dosages and different exposure times. According to the germination rate, a treatment with 0.7% EMS for 6 h was chosen. An M2 progeny of 3,651 fertile plants was obtained. Totally, 4.79% of the whole population showed clear aberrant phenotypes. A microsatellite analysis on a representative sample of the original seed stock and mutant lines confirmed the uniformity of the genetic background of plant material. The TILLING procedure was successfully applied to sunflower genome, initially by a CelI-nuclease mismatch cleavage assay coupled with a DNA-pooling level test. To investigate the efficiency of the mutagenic treatment, a pilot screening was carried out on 1,152 M2 lines focusing on four genes, three involved in the fatty acid biosynthetic pathway and one for downy mildew resistance. A total of 9 mutant lines were identified and confirmed by sequencing; thereby, the estimated overall mutation frequency for the pilot assay resulted to be 1/475 kb.A first TILLING population for a high throughput identification of EMS-induced point mutations in sunflower genome has been successfully obtained. This represents a powerful tool to a better understanding of gene function in sunflower.Cultivated sunflower (Helianthus annuus L.) is a globally important oilseed crop subjected to intensive molecular genetic and genomic studies during recent decades [1]. Sunflower belongs to the genus Helianthus, which is native to temperate areas of North America and includes 12 annual and 37 perennial species [2,3]. The world production of sunflower is estimated to be 23.4 million tons [4]; seed production in the world increased by 24% (or 5 million metric tons) between 1993 and 2003 [5]. After maize, it is the second largest hybrid crop, and the fifth largest among the oilseed crops, after soybean, rapeseed, cottonseed, and gro