Survey of Carotenoid ProVitamin A Variability in Maize

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Survey of Carotenoid (Pro-Vitamin A) Variability
in Maize
Sultana Islam Chandra Paul Ed Buckler Torbert
Rocheford University of Illinois
Urbana-Department of Crop Sciences
2

• HarvestPlus Program
• Micronutrient Biofortification of Staple Crops
• zinc, iron, and Pro-vitamin A (carotenoids)
• Add Nutritional value to Crops
• Food as a mechanism to improve
• Human Health

3

• Survey Carotenoid (Pro-vitamin A)
• Variability in Maize
• Facilitate Breeding for Higher
• levels of Pro-vitamin A for Human
• Nutrition

4
Carotenoids

• Carotenoids are a diverse class of hydrocarbon
  compounds
• Carotenes Lycopene, alpha (a)-carotene and beta
  (ß)-carotene
• Xanthophylls beta (ß)-cryptoxanthin, zeaxanthin
  and lutein
• Responsible for Red, orange
• and Yellow colors in fruits
• and vegetables

5

• In plants carotenoids are essential in
• Photosynthesis
• Dietary sources of vitamin A (retinol)
• and Antioxidants in Humans and
• Animals

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Pro-vitamin A

• Prominent Function of Carotenoids in Diet is as
  Pro-vitamin A
• Pro-vitamin A Carotenoids
• ß-carotene
• a-carotene
• ß-cryptoxanthin
• Converted to Vitamin A (retinol) in Liver

7

• In the U.S., 30 of total vitamin A
• intake provided by Pro-vitamin A
• from Plant sources
• ß-carotene is the most Effective
• Precursor to Vitamin A

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Chemical Structure of Vitamin A and Pro-vitamin A
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Vitamin A Activity

• In Humans important for
• Vision
• Bone Growth
• Reproduction
• Immune Function
• Gene Expression
• Embryonic Development
• Cell Division and Cell Differentiation
• Healthy Respiratory and Gastrointestinal systems

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• Prevalence of Vitamin A Deficiency
• ranges from 100 to 250 million
• Children
• An estimated 250,000 to 500,000
• Children go Blind each year from
• Vitamin A Deficiency

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Antioxidant Activity

• All function as Antioxidants in vitro
• Scavenge free radicals
• Protect cells from oxidative damage

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• Antioxidative Potential of Carotenoids
• may include
• Reduced risk of certain types of Cancer
• Enhanced Immune Response
• Protection against lipid peroxidation in
• Humans

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Other Benefits

• Lutein and Zeaxanthin
• Found in the macula of the human eye
• Actively involved in the maintenance of
• the visual system
• Sharp and Detailed Vision
• Age-related Macular Degeneration
• Cataract Development

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Economic Benefits???

• Carotenoid supplements are
• used in Animal Feed
• Protects broiler leg meat from oxidation
• Imparts desirable color to egg yolks

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Carotenoids in Maize

• 2 carotenes a-carotene and ß-carotene
• 3 xanthophylls ß-cryptoxanthin, lutein and
  zeaxanthin
• Carotenoids are present in
• horny endosperm (74-86)
• floury endosperm (9-23)
• rest in the germ and bran of the kernel

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Characterization of Carotenoid Biosynthetic
Pathway in Maize - Aided by White Endosperm
Mutants

• Cloned genes in the pathway
• Phytoene synthase (Psy)
• Phytoene desaturase (Pds)
• ?-carotene desaturase (Zds)
• PSY activity is an important control point for
  regulation of the flux
• (Fraser, et al., 2002)

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Carotenoid Biosynthetic Pathway
GGPP
Lycopene
PSY
y1
Phytoene
b- LCY
b- LCY e- LCY
PDS
2x
Phytofluene
vp5
a- carotene
b- carotene
z- carotene
ZDS
2x
Neurosporene
b-crytptoxanthin
vp9
Zeaxanthin
Lutein
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• Substantial variation in levels of specific
  isoforms and in levels of total carotenoids has
  been reported
• Weber (1987) evaluated 15 inbred lines
• Kurilich and Juvik (1999) evaluated 44 sweet and
  dent corn lines at 20 DAP

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Why Study?

• No recent reports on carotenoids in a Diverse set
  of lines at mature dry stage
• Analytical methods have changed dramatically
  since the early studies
• Survey of a Broad range of germplasm with modern
  analytical tools was needed

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Objectives

• Survey the Variation in a Diverse Set of Inbred
  Lines
• Evaluate F1 crosses
• Genotype the lines with SSR marker associated
  with Psy gene (Y1 locus)

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Genetic Materials

• Set of 102 inbred lines
• 2001 60 lines
• 2002 85 lines
• Expanded set of 300 inbred lines
• 2003 176 lines evaluated so far
• 17 F1 crosses (F1 Seed analyzed)
• Five reciprocal pairs

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Vitamin Extraction
16 Samples Extracted in Two Hours
Extract Upper Layer
Heat 5min at 86C
2X
Hexane (Extraction)
Heat 10 min at 86 C
dH20
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Waters Alliance HPLC System
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HPLC Chromatogram 450 nm
Zeaxanthin
AU
Lutein
AU
ß-cryptoxanthin
a-carotene
ß-carotene
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Lycopene
a-carotene
b-carotene
b-crytptoxanthin
OH
OH
OH
Lutein
OH
Zeaxanthin
OH
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Results

• 2002 Survey (85 lines)
• F1 Crosses
• 2003 Survey (176 lines)
• Vitamin A Equivalents
• Y1ssr Marker Analysis

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Means and Ranges from 2002 Survey
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Repeatability Estimates
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Correlations between a-carotene, ß-carotene,
ß-cryptoxanthin, lutein and zeaxanthin in 2002
and are significant at p lt.01 and p lt.0001,
respectively. ns, not significant at p lt.01
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, significant at p lt .0001
Lycopene
a-carotene
b-carotene
0.56
0.51
b-crytptoxanthin
OH
OH
OH
0.81
Lutein
Zeaxanthin
OH
OH
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F1 Crosses
X
X
Well known Dosage Effects
May be Dosage Effects at Y1 and other Loci
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• Seeds from 17 F1 Crosses analyzed
• Total Carotenoids
• Higher than mid Parental mean 3
• Equal to mid Parental mean 6
• Lower than mid Parental mean 8
• 5 Reciprocal Pairs
• 2 showed Reciprocal Effect for Total

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Carotenoid Profiles of the SC55 Crosses
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Lycopene
a-carotene
b-carotene
X
b-crytptoxanthin
OH
OH
OH
X
Lutein
OH
Zeaxanthin
OH
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Carotenoid Profiles of NC354, TZI18 and their
Reciprocal Crosses
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ß-carotene Profiles of NC354, TZI18 and their
Reciprocal Crosses
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Means and Ranges from 2003 Survey
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Dietary Reference Intakes for Vitamin A

• Children (lt 4 yrs) 300 µg
• Children (4 yrs) 400 - 600 µg
• Women 700 µg
• Men 600 µg
• Lactation 1200 µg

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Candidate Pro-vit A Lines
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Vitamin A Equivalents from CI.7, DE3, Mo17 and B73
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• DKXL 212 x GEM Line
• DE 3
• (L317 x 33-16) 33-16(2)
• CI.7

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Candidate Genes

• Psy and Zds may be candidate genes that are the
  basis of two QTL mapped for Quantitative
  Variation in Carotenoids
• (Wong et al., 2004)
• Candidate gene associated with Quantitative
  Variation for a trait may be validated with
  Associative Analyses
• (Thornsberry et al., 2001)

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Carotenoid Biosynthetic Pathway
GGPP
Lycopene
PSY
y1
Phytoene
b- LCY
b- LCY e- LCY
PDS
2x
Phytofluene
vp5
z- carotene
a- carotene
b- carotene
ZDS
2x
Neurosporene
b-crytptoxanthin
vp9
Zeaxanthin
Lutein
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Gel image of Y1ssr marker
227bp-
-224bp
216bp-
210bp-
-204bp
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Sample of Inbreds and Fragment Sizes
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Y1ssr Marker Allele Groups and Group Means
groups include yellow and orange lines only
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Multiple Regression of Means of Y1ssr Marker
Groups
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Single Factor Analysis between 210 bp (D) and 216
bp (F) groups and their Marker Means
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Single Factor Analysis between 210 bp (D) and 227
bp (G) groups and their Marker Means
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Carotenoid Biosynthetic Pathway
GGPP
Lycopene
PSY
y1
Phytoene
b- LCY
b- LCY e- LCY
PDS
2x
Phytofluene
vp5
z- carotene
a- carotene
b- carotene
ZDS
2x
Neurosporene
vp9
b-crytptoxanthin
Lutein
Zeaxanthin
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Pre-screening with TLC

• RAPID
• INEXPENSIVE!

Identification of the Top Lines for more detailed
HPLC analysis
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Carotenes
ß-Crypto.
Lutein/Zea.
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Summary

• Survey of diverse maize inbreds revealed
  considerable Variation in Carotenoids and Vitamin
  A equivalents
• Certain lines appear to Combine better with other
  lines for ß-carotene and total carotenoids in F1
  seed

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• An initial candidate gene analysis approach with
  y1ssr may be revealing an Association between Psy
  and ß-carotene and total carotenoids, will
  genotype more
• TLC or other assays may enable a Rapid screen of
  germplasm enabling identification of the top 20
  to keep and analyze by HPLC

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Acknowledgements

• Advisor Dr. Rocheford
• Committee Drs. Bohn, Briskin and Lambert
• Funded by US AID
• Dr. Darryl Clark
• Dr. Qiao Qingyan
• Dr. Peter Beyers Lab Patrick Schaub
• Field Technician Jerry Chandler
• Everyone at Dr. Rochefords Lab Dr. Chandra Paul
• Everyone at Dr. Juviks Lab
• Undergraduates Ryan Wilson, Rachel Hopkins,
  Esther Lee, Wanda Averhart