Validation of QTL related to Soluble Solid Content and Quantitative behaviour of Sugar in a Tomato F2 population MSc. Thesis Report – Plant Breeding Lorena Guardia Velarde Daily supervisor: Dr. Yury Tikunov Supervisors: Dr. Arnaud Bovy Dr. ir. Sjaak van Heusden April, 2015 Confidential P a g e | 1 Table of Contents List of Tables _______________________________________________________________ 3 List of Figures ______________________________________________________________ 3 List of acronyms ____________________________________________________________ 4 Abstract ___________________________________________________________________ 5 Introduction _______________________________________________________________ 6 Tomato flavour _________________________________________________________________ 6 Wild tomato accessions related to flavour traits _______________________________________ 6 Soluble solids content trait (Brix content) ____________________________________________ 7 QTLs related to brix content _______________________________________________________ 7 Previous results on Brix QTL analysis in high Brix interspecific introgression lines ____________ 8 QTLs associated to brix content found by Petit (2014) in Chromosome 6 ___________________ 9 Aim _____________________________________________________________________ 10 Materials & Methods _______________________________________________________ 11 DNA extraction _________________________________________________________________________ 11 DNA quality analysis _____________________________________________________________________ 11 Genotyping by Sequencing _______________________________________________________ 11 Primers design _________________________________________________________________________ 11 PCR __________________________________________________________________________________ 12 Sequencing ____________________________________________________________________________ 12 QTL analysis ____________________________________________________________________________ 13 CAPS Assay ____________________________________________________________________ 13 Enzyme digestion _______________________________________________________________________ 13 KASP assay ____________________________________________________________________ 13 Metabolic profiling _____________________________________________________________ 14 Results ___________________________________________________________________ 15 Selection of plants from F2 lines __________________________________________________ 15 Verification of the introgression length and marker position in region VIIa and VIIb _________ 18 QTL and linkage-map of Chromosome 6 and 9________________________________________ 22 Genotyping the complete population by CAPS and KASP _______________________________ 24 CAPS asay _____________________________________________________________________________ 24 KASP assay _____________________________________________________________________________ 25 Metabolomic analysis of the predominant sugars and their comparison with brix content ____ 30 Wageningen UR P a g e | 2 Comparison between sugars ______________________________________________________________ 33 Discussion ________________________________________________________________ 39 Comparison between genotyping methods __________________________________________ 39 Confirmation of the location, length of the introgression and gene involved in the QTL related to brix content in Chromosome 6 ____________________________________________________ 39 Higher hexose content was found in samples with higher brix content ____________________ 40 Ratification of number of QTLs present in Chromosome 9 related to brix content ___________ 40 Acknowledgments _________________________________________________________ 42 References ________________________________________________________________ 43 Appendix _________________________________________________________________ 45 Sequencing data _______________________________________________________________ 45 Previous markers from Petits Thesis________________________________________________ 59 Sugars values __________________________________________________________________ 61 Wageningen UR P a g e | 3 List of Tables - Table 1. Description of agarose gel and master mix - Table 2. Reaction Setup - Table 3. Thermo-cycling conditions for a routine PCR - Table 4. Marker seq-rs9017_b - Table 5. Lines for sequencing - Table 6. Detail of 14 primers - Table 7. Positions of marker seq-rs9017 (region VIIa) - Table 8. Markers in region VIIb - Table 9. Final position of markers at region VIIb - Table 10. Alleles of the 25 selected plants and 7 controls - Table 11. Markers related to brix content in Chromosome 6 - Table 12. Kruskal-Wallis results, markers significantly linked to brix content trait - Table 13. Kruskal-Wallis results, markers significantly linked to brix content trait - Table 14. Samples for metabolomic study - Table 15. Selected samples for metabolomics with alleles and brix values - Table 16. List of the identity of the sugars and other metabolites present in tomato fruit samples - Table 17. Groups for the metabolomic assay - Table 18. T-test results between groups and sugar - Table 19. T-test results between alleles and sugars, brix List of Figures - Figure 1. KASP primers. - Figure 2. List of 14 positions found between seq-rs6813 and seq-rs6622 - Figure 3. Amplification of primers in region VIIa to evaluate their specificity - Figure 4. Regions selected for sequencing - Figure 5. Alignments for 4 markers in samples: MM8, S40, F1 and S ch - Figure 6. Analysis of marker seq-rs9017_a - Figure 7. Markers in Chromosome 9 - Figure 8. Markers in Chromosome 6 - Figure 9. Gel picture of the digestion - Figure 10. KASP assay results for the whole population - Figure 11. Complete set of samples with the values for each marker - Figure 12. Markers at Chromosome 9 - Figure 13. Representation of Chromosome 9 - Figure 14. Markers in Chromosome 6 - Figure 15. Main sugars by groups - Figure 16. Brix versus Fructose - Figure 17. Brix versus Glucose - Figure 18. Brix versus Sucrose - Figure 19. Average of sugars and brix content between the two groups and the standard errors (bars) - Figure 20. Correlation between KASP markers with Brix content Wageningen UR P a g e | 4 List of acronyms - bp: Base pairs - °C: Centigrade - CASP: Cleaved amplified polymorphic sequence - Chr: Chromosome - cM: centiMorgan - F1 population: First filial population - F2 population: Second filial population - Forward: Fw - g: Gram - IL: Introgression Line - KASP: Kompetitive Allele Specific PCR - Lin: Acid invertase gene - Mbp: Mega base pair - ml: Mili litre - mM: Mili Molar - MiliQ water: mq - MoneyMaker: MM - “N”: None call - Nitrogen: N - Non template control: NTC - PCR: Polymerase Chain Reaction - pH: Potential hydrogen - QTL: Quantitative Trait Locus - Reverse: Rv - RIL: Recombinant Inbreed Lines - rpm: Revolutions per minute - SNP: Single Nucleotide Polymorphism - Solanum chmielewskii: S ch - SSC: Soluble Solid Content - Sweet 40: S40 - µl: Micro litre - V: Voltage Wageningen UR P a g e | 5 Abstract Tomato fruit conformation involves several chemical compounds, which collaborate in the composition of the flavour. The sweetness depends on the type and amount of sugars present, also in soluble solid content, pH, titratable acidity and fruit size. To estimate soluble solid content, the refractive index (°Brix) of the fruit juice is measured. Improving brix levels is an interesting trait to breed and for QTL research. Studies based on RILs or ILs between wild type species and MoneyMaker var. have been done in order to elucidate the location of QTL related to brix content in the genome. A previous study done by Petit presents that in a F2 population, coming from the Sweet Line 40 and MoneyMaker, 2 QTLs linked to brix content were found in Chromosome 6 and 9. During the current thesis it was proven that the QTL found in Chromosome 6 is located in Chromosome 9. In addition, a correlation between sugars and alleles was found. Keywords: °Brix, sugars, QTL, Sweet40, MoneyMaker, Chromosome 6 and 9. Wageningen UR P a g e | 6 Introduction Tomato flavour A general consumer opinion is that tomato flavour is poor. This might be due to the fact that flavour has not been the main focus in tomato breeding and the emphasis has been mainly on processing performance, yield, fruit size, firmness, lack of defects and disease resistance (Bucheli et al., 1999). This has led to an erosion of flavour quality in tomato (Fulton et al., 2002). Tomato fruit composition consist of more than 400 different volatile and non-volatile chemical compounds, which contribute to its odour and taste, respectively (Tiemen et al., 2006). The most prominent taste-related compounds are sugars (sucrose, glucose and fructose), organic acids (citric and malic acid) and amino acids. Humans can perceive them by taste receptors located in the tongue and olfactory receptors in the nose. All these components together represent about 60% of the tomato dry matter weight (Causse et al., 2004). Soluble sugar content is an important factor of fruit quality, for processing and fresh market tomatoes. Firstly, the tomato paste yield depends on solid content. Fruits with high soluble solids content have less water and need less processing. Secondly, the flavour depends on the sugars and acids concentration (Chetelat et al., 1993; Baxter et al., 2005). In order to achieve an excellent acceptation in the market the tomato