Transcript Genetic Positioning of Centromeres Using Half

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)
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
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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)
BIOSYNTHESIS PATHWAY
GGPP =
geranylgeranylpyrophosphate
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
EIGHT ACCESSIONS
• W266Drprp (reduced pigment)
– Recessive allele for reduced pigment
– Shown to reduce carotenoid conc. by
up to 92%
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W266DRpRp (orange)
W276B (orange)
Danvers (orange)
HCM (orange)
Beta III (dark orange)
Okuzawa (red)
Yellow type (yellow)
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
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]
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]
α- and β-carotene
• High range within roots
– Artificially selected for human consumption
• Lower range within leaves
– Naturally selected for because prevent photooxidative damage in leaves
– Lack of artificial selection
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
TOCOPHEROL
• No patterns between orange and nonorange
• Much higher levels in leaves than in roots
– Perhaps it aids in photosynthesis
• Surprising ratios of [root] : [leaves]
BIOSYNTHESIS PATHWAY
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)
*minimal lycopene detected in all other accessions
**minimal phytoene detected in W276B and Danvers
PHYTOENE
PHYTOENE, LYCOPENE*
PHYTOENE
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
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 βcarotene
• 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.
rprp vs. RpRp
Carotenoids
Phytoene
[rprp] = 0.04*[RpRp]
[rprp] = 476.36mAu
[RpRp] = not detectable
BIOSYNTHESIS PATHWAY
•Recessive mutation
reported to cause 93%
loss of root pigmentation
•Simultaneous decrease
in levels of α- and βcarotene suggests allele
blocks carotenoid
pathway at step
immediately following
phytoene
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)
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
RESEARCH GOALS
• Identify putative carotenoid biosynthetic
gene sequences in carrot
• Place as STS markers on carrot linkage
map from Santos and Simon (2004)
METHODS
• Map population, and extract DNA
– B493 x QAL F2
• 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
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
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
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
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
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