| isolation and growth condition | Seeds of the B. rapa subsp. trilocularis var. R-o-18 were previously subjected to 0.3% ethyl methane sulfonate (EMS) mutagenesis. Seeds of 3,464 M2 genotypes were acquired and sown directly into fine-grade compost-based growing media in 1 L pots and established in a single-skinned polytunnel with no additional lighting or heating. Pots were arranged in a randomised block design with 20 plants per tray, 23 trays per block, two blocks per bed, and four beds per run. There were three runs in total, with each mutant genotype represented once per run (n = 10,392) along with 100 R-o-18 wild-type control plants per run (n = 300). Plants were covered by 380 x 900 mm micro-perforated pollination bags once inflorescences began to show to prevent cross pollination. Three leaves (typically one each of old, mid-age and young) were sampled from each M2 plant at the rosette stage (typically 6-8 true-leaves showing) and ionomic analysis was performed to determine leaf Mg concentration. Genotypes with leaf Mg concentrations over three standard deviations above the population-wide mean Mg concentration were considered as candidates. This amounted to 83 genotypes, of which seven set seed. Between two and ten M3 plants of each of these lines (based on seed yield of M2 plants) were subsequently grown and leaf element concentrations were quantified as described above. Two and one genotype, respectively, maintained the high Mg trait in generations M3 and M4. Plants of the genotype that retained the high Mg trait to M4, were backcrossed to wild-type R-o-18 plants via emasculation of wild-type plants and hand-pollination with mutant pollen, to reduce background mutation load typically associated with EMS mutagenised plants. Resulting plants were maintained by single-seed descent from generations BC1F1-BC1F5. In each generation, leaf element concentrations of all plants were quantified by ICP-MS in order to track the high Mg trait. The BC1F2 population was observed to be segregating for the high Mg trait, so individual high and low Mg lines were derived by self-fertilising plants of contrasting phenotypes. Additional ionome profiling took place on BC1F3 plants. Six plants of each of the low Mg segregants and wild-type R-o-18 genotypes of this generation were sampled at the rosette stage and roots were hand-washed in deionised H2O and dried in an incubator set to 60�C for 24 hours. These were grouped into three pools of two roots each, to attain an adequate sample weight for ionomic analysis. An additional six plants of each genotype were grown to maturity and the 1st, 8th and cauline leaves, immature pods/flowers, mature pods, stem, and mature seeds were sampled at appropriate points in the plants� life cycle. Each sample was microwave digested and analysed by ICP-MS. Plants of the BC1F4 population were once more backcrossed to wild-type R-o-18 via emasculation and hand-pollination. Plants of the resulting BC2F1 population were grown in glasshouse conditions and made to self-fertilise by covering inflorescences with 380 x 900 mm micro-perforated pollination bags. BC2F2 seed from a single sibling plant was harvested and designated for use in bulked-segregant analysis experiments. A total of 200 BC2F2, 10 wild-type R-o-18, and 10 BC1F4 high Mg reference seeds were sown directly into homogenised, high nutrient compost in two litre pots distributed in a random blocked design such that there were 10 blocks each comprising 20 BC2F2 plants, one wild-type R-o-18 plant and one BC1F4 high Mg reference plant. At 21 days after germination, third and fourth leaves were taken from each plant and placed into individual paper bags. Leaves were dried for five days in an incubator set to 50 �C prior to element concentration analysis by ICP-MS, to identify segregants with the high Mg trait. First and second leaf tips (first 2 cm) were separately sampled from all plants, snap frozen in liquid nitrogen (N) in individual Eppendorf tubes and stored at -80�C prior to high molecular weight (HMW) DNA extraction.Low Mg BC2F2 segregants were identified. |
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