sunTILL: a TILLING resource for gene function analysis in sunflower

Abstract

BackgroundCultivated sunflower (Helianthus annus L.) is a globally important oilseed crop, subjected to intensive genetic and genomic studies. Although classical mutagenesis has successfully been applied to Helianthus genus in the past, we have developed the first sunflower TILLING resource.ResultsTo 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.ConclusionA 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.

Turning heads: The biology of solar tracking in sunflower

“…These resources will foster candidate gene identification through transcriptomic comparisons of lateral stem sections in tracking and top lit non-tracking plants. Recent advances facilitating reverse genetics through targeted induced lesions and germline transformation will allow targeted alteration of candidate genes [19]. In addition, recently generated germplasm resources -including advanced generation recombinant inbred lines and association mapping panels [18,20] -will provide powerful tools for connecting natural allelic variation to phenotypic variation in solar tracking.…”

“…Consequently, many aspects of the physiology, development, and ecological function of solar tracking remain unexplained. Recent genetic and genomic advances [e.g., [18][19][20] poise sunflower to be a strong, tractable model system for revealing the basic mechanisms underlying growth-mediated heliotropism. Here, we review the state of our knowledge regarding solar tracking in sunflower with the purpose of highlighting open questions and raising hypotheses to be addressed by future efforts that take advantage of these new experimental resources.…”

Targeted mutation breeding of horticultural plants

“…Changes in seed oil composition have obtained from mutations induced in the gene encoding Δ12 fatty acid desaturase (FAD2). TILLING for fad2 variants that increase oleic acid has been successful in soybean (Dierking and Bilyeu, 2009) and novel fad2 alleles are being pursued in peanut (Knoll et al, 2011) and sunflower (Sabetta et al, 2011).…”

Allele Mining Strategies: Principles and Utilisation for Blast Resistance Genes in Rice (Oryza sativaL.)

“…TILLING can help us to focus on specific genes or genomic regions, avoiding difficulties with other functional genomic methods (such seeing that T-DNA knock-outs or perhaps RNAi-based gene silencing) that need the generation of transgenic plants (Sabetta et al, 2011). TILLING strategy, with developed software, will work for productive reverse genetics in the near future.…”

“…Chemical mutagens produce predictable heritable nucleotide changes at high densities. The chemical mutagenic treatment providing an easy and costeffective way to saturate a genome is coupled with PCRbased mutation detection (Sabetta et al, 2011). Chemical mutagenic agents, such as Ethylmethane sulphonate (EMS), methylmethane sulphonate (MMS), hydrogen fluoride (HF), sodium azide, N-methyl-N-nitrosourea or methylnitrosourea (MNU), and hydroxylamine have been used to generate mutant populations for many years for forward genetics in various plant species (Parry et al, 2009).…”