Aplicación del procedimiento en la cadena de suministro de agua mineral

1.6.1 Initial Salt Stress Screening leading to Line Selection for main

Thesis.

52 brassica oleracea DFFS lines, (only the S1s), selfed from the original wild founder lines as described in Section 1.3.5.2 and Figure 1.3 were selected based on availability of seed. They were comprised of different Brassica oleracea species as listed in Table SII in Appendix I. In 2014 between May – September, a salt stress experiment was designed in conjunction with Dr Peter Walley. From the design, six replicates of each line (three control and three treated plants) were paired and randomly distributed. Seeds from the 52 lines selected were provided by G. Teakle and the GenBank sourced from the DEFRA funded VeGIN project. The initial experiment setup was executed by Christine Hicks assisted by Gary Grant, in the glasshouse Phytobiology unit. The plants were grown for 4 weeks in smaller plastic pots before being transplanted into bigger pots. LD50 was determined using some spare plants that were not included before the main experiment. Different concentrations of salt (NaCl) were prepared, 350 mM, 300 mM, and 250 mM respectively. Some plants died between 350 mM – 300 mM and the idea was not to kill the plants but observed some immediate salt stress response and by following the Systems biology approach to interpreting the data, therefore 250 mM salt concentration was adapted and mode of salt administration to be as shock (single dose) by feeding the treated lines with 250 mM NaCl.

In addition, plants were treated at week six (6), at least each with four completely expanded leaves. They were allowed to grow for three additional weeks. My role in this experiment started with the data collection and measuring morphological traits; plants dry weight, leaf dry weight, where plants height and leaf area were measured by using photographs of

individually between myself and Christine significant morphological variation was observed. Statistical analysis ANOVA and t-test carried out by myself compared treated and control plants. Plants were compared only within the line and not between species because of their morphologically diversity. Different lines were grouped and consider as being resilience (tolerance) and or susceptible. After the data was sorted and ranked according to the level of significance (p-values). Based on the ranking, an improvement in leaf area against the control was chosen as a marker to guide selection. This work led to the selection of 13 lines of susceptible and tolerant plants (26 in total) were selected from the 52 (Table 1.2). These were the lines selected to be taken forward within my main thesis. However, some of the wild S1 lines had issue with seeds availability and some on poor germination (see Table 2.1 in Chapter 2 for the list of the retained ones). These were subsequently dropped.

Table 1.2: List of some selected B. oleracea genotype lines from 2014 salt stress experiment

*Note: The listed B. oleracea in brown colour Geno ID are those that show an ability to grow (Tolerant) to short-term salt stress based on; Plant Height, Plant dry weight, leaf dry weight and leaf area as compared to the control While those in green colour are considered susceptible to short-term salt stress.

1.6.2 Salt Stress Screening Experiments in 2015

During the initial experiment work by Peter Walley and G. Teakle, generated DH lines derived from the original wild founder DFS (section 1.3.5.1) lines and related S1 lines (Table 2.3 in chapter 2). This offered a valuable resource and lines related to the wild lines selected were identified and seeds obtained. Another valuable addition enabling me to develop my work arose from transcriptomic profiling using RNA-Seq analysis conducted on three (3) of the DH lines and one parent line (DHSL150). Two DH lines, C13013 & C10128 were selected for resilience while C13001 & DHSL150 were considered as susceptible. Christine Hicks reported the data in her final thesis this is the reason I have not included this work in my main thesis although she used plant materials (DH lines and DHSL150), from my initial salt stress experiments. Based on this fact, it was recommended that I should review that aspect of her work and come up with my own hypothesis. After this review, I concentrated on the iron membrane proteins that were shown to be expressed in some DH lines and significantly different from the tolerant and susceptible lines. The

Tolerant Suscetible

line no. Geno ID Name line no. Geno ID Name

2 C04099 B. oleracea (DHSL150 C01001) 5 C07011 B. cretica 3 C07007 B. bourgaei 8 C07015 B. cretica 4 C07010 B. cretica 11 C07019 B. hilarionis 25 C07059 B. oleracea 12 C07020 B. hilarionis 26 C07060 B. oleracea 14 C07024 B. incana 32 C07073 B. oleracea 20 C07045 B. macrocarpa 33 C07075 B. oleracea 22 C07047 B. macrocarpa 35 C07078 B. oleracea 23 C07049 B. macrocarpa 36 C07079 B. oleracea 29 C07069 B. oleracea

41 C07091 B. incana (listed as villosa in seed book) 37 C07079A B. oleracea

43 C07094 B. incana 46 C07104 B. villosa bivoniana

47 C07113 B. villosa tinei 49 Early Big B. oleracea

oleracea genotypes tolerant lines enhances resilience as compared to susceptible genotypes. The 2017 the data (Fastq files) were re-analysed and the Fastq files were aligned to B. oleracea (To1000) genome. I kindly appreciate Luca Illing for his support in guiding me through this. Following the alignment, heat-reads (BAM files) were further processed using SeqMonk software, by using two statistical algorisms; DSeq2 and Edge R analyses. This formed the basis of the list of iron membrane transporters that were subsequently investigated (Chapter 5).

1.7 AIM AND OBJECTIVES