1. Relationship of carotenoids and tecopherols in a sample of carrot root-color accessions and carrot germplasm carrying Rp and rp alleles Koch, T. C. and I. L. Goldman Journal of Agricultural and Food Chemistry 53: 325-331 (2005)

2. CAROTENOIDS & TOCOPHEROLS Role in plants: –Carotenoids prevent formation of oxygen radicals –Tocopherols protect membranes from oxidative stress Role in human diet: –Powerful antioxidants that prevent degenerative effects –Some convert to vitamin A α- and β-carotene  orange-colored roots Lycopene  red Anthocyanins  purple Low total carotenoids  white Tocopherols don’t contribute to color Carotene: origin 1860-1865 “carrot” + “-ene” (dictionary.com) Richest source of carotenoids in crude palm oil (wikipedia.org)

3. BIOSYNTHESIS PATHWAY GGPP = geranylgeranyl- pyrophosphate

4. FIELD EXPERIMENT Assess levels of major carotenoids and tocopherols in carrot roots & leaves Measure accumulation of compounds along carotenoid & tocopherol biosynthesis pathway –Explain color differences among 8 accessions 4 replications/accession 10 samples/replication 2 locations; 2 growing seasons

5. EIGHT ACCESSIONS W266Drprp (reduced pigment) –Recessive allele for reduced pigment –Shown to reduce carotenoid conc. by up to 92% W266DRpRp (orange) W276B (orange) Danvers (orange) HCM (orange) Beta III (dark orange) Okuzawa (red) Yellow type (yellow)

6. RESULTS: FIXED EFFECTS Accession, year, and location all interacted significantly Year and location effects: –Mainly resulted in change of data magnitude –Rarely changed accession ranks For analysis, years and locations were pooled, since ranks were rarely affected

7. RESULTS: α-carotene W266Drprp (reduced pigment) W266DRpRp (orange) W276B (orange) Danvers (orange) HCM (orange) Beta III (dark orange) Okuzawa (red) Yellow type (yellow) xylem and phloem: [orange] > [non-orange] [xylem] = 0.69*[phloem] [leaf] = 0.36*[phloem]

8. RESULTS: β-carotene W266Drprp (reduced pigment) W266DRpRp (orange) W276B (orange) Danvers (orange) HCM (orange) Beta III (dark orange) Okuzawa (red) Yellow type (yellow) xylem and phloem: [orange] > [non-orange] [xylem] = 0.67*[phloem] [leaf] = 0.33*[phloem]

9. α- and β-carotene High range within roots –Artificially selected for human consumption Lower range within leaves –Naturally selected for because prevent photo- oxidative damage in leaves –Lack of artificial selection

10. RESULTS: α-TOCOPHEROL W266Drprp (reduced pigment) W266DRpRp (orange) W276B (orange) Danvers (orange) HCM (orange) Beta III (dark orange) Okuzawa (red) Yellow type (yellow) HIGHEST AVERAGE FOR XYLEM AND PHLOEM

11. TOCOPHEROL No patterns between orange and non- orange Much higher levels in leaves than in roots –Perhaps it aids in photosynthesis Surprising ratios of [root] : [leaves]

12. BIOSYNTHESIS PATHWAY

13. PHYTOENE AND LYCOPENE: PRECURSORS TO CAROTENOIDS W266Drprp (reduced pigment) W266DRpRp (orange) W276B (orange)** Danvers (orange)** HCM (orange) Beta III (dark orange) Okuzawa (red) Yellow type (yellow) PHYTOENE PHYTOENE, LYCOPENE* PHYTOENE *minimal lycopene detected in all other accessions **minimal phytoene detected in W276B and Danvers

14. PHYTOENE AND LYCOPENE: PRECURSORS TO CAROTENOIDS Non-orange roots showed increased levels of precursors –Suggests reduction in production/efficiency of enzyme converting to α- and β-carotene Leaves didn’t contain the precursors –All leaves contained ample α- and β-carotene

15. SUMMARY OF CORRELATIONS Positive correlation (r=0.92) between α- and β-carotene –May be able to simultaneously select for both α- and β-carotene negatively correlated with phytoene and lycopene* –Possibly because phytoene and lycopene are precursors to α- and β- carotene Tycopherol negatively correlated with phytoene and lycopene* Xylem: tycopherol positively correlated with α- and β-carotene (r=0.65 and r=0.52) –Possibility of selecting for high levels of all three compounds Leaves: tycopherol positively correlated with α- and β-carotene (r=0.28 and r=0.65) –Possibly due to common origin of biosynthetic pathways *Correlations may be skewed due to small number of accessions with presence of phytoene or lycopene. Require more tests with more non-orange accessions.

16. rprp vs. RpRp Carotenoids [rprp] = 0.04*[RpRp] Phytoene [rprp] = 476.36mAu [RpRp] = not detectable

17. BIOSYNTHESIS PATHWAY Recessive mutation reported to cause 93% loss of root pigmentation Simultaneous decrease in levels of α- and β- carotene suggests allele blocks carotenoid pathway at step immediately following phytoene

18. Carotenoid biosynthesis structural genes in carrot (Daucus carota): isolation, sequence-characterization, single nucleotide polymorphism (SNP) markers and genome mapping Just, B.J., C.A.F. Santos, M.E.N. Fonseca, L.S. Boiteux, B.B. Oloizia, and P.W. Simon Theoretical Applied Genetics 114: 693-704 (2007)

19. HISTORY OF CARROT MAPPING: AN OVERVIEW Genetic linkage maps –Several have been published –Santos and Simon (2004) merged maps for six linkage groups in two populations PCR-based codominant markers –Several published, but limited usefulness across unrelated populations STS (sequence tagged sites) markers –Have not been developed for carrot –Used in other crops to create linkage maps from different crosses that can be compared

20. RESEARCH GOALS Identify putative carotenoid biosynthetic gene sequences in carrot Place as STS markers on carrot linkage map from Santos and Simon (2004)

21. METHODS Map population, and extract DNA –B493 x QAL F 2 B493: dark orange inbred, QAL: white wild carrot F1 plant self-pollinated to produce F2 183 F2 plants grown Target genes, design primers, and amplify initial PCR of putative carotenoid structural gene-containing genomic sequences Clone and sequence Design copy-specific primers, and identify polymorphism Genotype the population at each putative carotenoid biosynthetic gene and Y2mark Construct linkage map –Added to map consisting mostly of AFLP markers, generated by Santos (2001) Extracted RNA Performed RACE PCR and amplified full-length cDNA clones

22. RESULTS: mapping Placed 24 putative carrot carotenoid biosynthetic structural genes on carrot linkage map –2 genes omitted because lacked polymorphism or displayed severe segregation distortion Sequenced full-length transcript for 22 of the genes –15 new putative genes identified 24 genes studied are distributed over eight of the nine carrot linkage groups

23. B493 X QAL LINKAGE MAP QAL and B493 coupling linkage groups shown side by side Maps positions of putative carotenoid biosynthetic structural genes Codominant markers are connected with dotted lines between the two maps Other markers are dominant AFLP fragments from Santos (2001) Just one codominant marker  ambiguous orientation

24. RESULTS: QTLs 3 of the markers mapped to region of QTL clustering identified by Santos and Simon (2002) for major carotenoid pigments –Candidate genes for some of the QTLs

25. RESULTS: mRNA mRNA for all genes present in orange roots –Genes before and after α- and β-carotene in pathway are expressed –Need future research to elucidate extent of pathway regulation at transcription level

26. FUTURE RESEARCH Mapped genes will aid in identifying homologous groups across studies Future researchers now have tool to study functionality of the genes by producing their protein products