Identification and Use of Rare Natural Genetic Sequence Variation ...

ISB NEWS REPORT JUNE 2010
Identification and Use of Rare Natural Genetic Sequence Variation to Improve
Levels of β-Carotene in Maize
Marilyn L. Warburton
Sufficient vitamin A is not provided by a diet based mainly on corn
Association mapping, validated by linkage mapping and expression studies, has been used to boost the nutritional value
of corn. Corn, or maize, is a staple crop for many people in developing countries, but lacks sufficient micronutrients
to provide a balanced diet by itself, particularly for growing children. Corn contains carotenoids, some of which the
body can convert to vitamin A. Beta-carotene is the best provitamin A (vitamin A precursor), but only a very small
percentage of corn varieties have naturally high beta-carotene levels. Markers found linked to genes encoding high
levels of provitamin A in corn could speed the efficient development of corn cultivars that have the potential to reduce
the number of children in developing countries who become blind, develop weakened immune systems, and die because
of vitamin A deficiencies (VAD). In Africa alone, this number stands in the hundreds of thousands every year. Improving
the micronutrient balance of staple crops such as maize through biofortification is an economically and socially sound
way to address micronutrient malnutrition, including VAD, on a global scale. Daily dietary intake of maize with 15
μg g -1 provitamin A carotenoids could greatly alleviate VAD. 1 Maize exhibits considerable natural variation for kernel
carotenoids, with total carotenoid content ranging from virtually zero to nearly the 15 μg g -1 suggested levels; however
most maize grown and consumed throughout the world has only 0.5 – 1.5 μg g -1 of β-carotene.
The carotenoid pathway makes provitamin A
The carotenoid pathway has been elucidated in model crop species, and with few exceptions, all plants use the same
pathway, although the number of genes mediating each step can vary. A limited number of carotenoids from the pathway
can be converted to vitamin A in the human body, including α-carotene (αC), β-carotene (βC), and β-cryptoxanthin (βCX),
although βC is converted to twice the amount of vitamin A than are the other two molecules. The pathway is shown
in Fig. 1, including the position of βC and the genes mediating each step in Arabidopsis and maize. The Arabidopsis
pathway was used to identify candidate genes and find the orthologous copies in maize for use in association analysis
studies to confirm which of the genes mediate the most important steps in the accumulation of high levels of βC. As can
be seen in Fig. 1, the pathway splits into two mutually exclusive branches, only one of which leads to βC; this branch
of the pathway then converts βC into other molecules, eventually leading to non-provitamin A structures. Ideally, maize
grains would shunt much of the carotenoid precursors down the right branch of the pathway and stop at βC; logically,
the genes lycopene epsilon cyclase (LcyE or B) and β-carotene hydroxylases (CRTRB), along with phytoene synthase
(PSY1), the gene encoding the first committed step to the pathway, should be the most important. This hypothesis was
tested using association mapping.
Association mapping of beta-carotene
Association mapping seeks statistically significant associations between a change in a DNA sequence and a change in
the phenotype of a trait in a large panel of unrelated lines of a species. Association mapping takes advantage of recent
breakthroughs that accelerate the genetic profiling of crops, including large scale Single Nucleotide Polymorphism
(SNP) discovery and characterization platforms, as well as high-throughput sequencing and the publication of the maize
genomic sequence. Association mapping has been widely used to study the genetic basis of complex traits in human and
animal systems, and is increasingly used in a wide range of plant species, although many of the first association studies
in plants focused on qualitative or single gene controlled traits. It is currently used to study quantitative traits as well.
Unlike more traditional linkage mapping studies of populations created by crossing two parents, association mapping
can explore all the recombination events and mutations in large and diverse populations, and with a higher resolution. 2
Association studies of provitamin A carotenoids, measured as a group (αC plus βC plus βCX) or singly, tested each
candidate gene from the carotenoid pathway, particularly those thought to control key steps in the pathway for βC
accumulation. Three independent populations, or association mapping panels, of maize lines were used in these studies,
and as diverse a range of maize as possible was surveyed to ensure that as many alleles of the tested genes were in the
study as possible.
These studies found that the allelic variation at the lcyE gene (Fig. 2a) explained over half the phenotypic variation in
provitamin A levels, 3 and that alleles at crtRB1 (Fig. 2b) explained 40% of the phenotypic variation in β-carotene level. 4
Significant associations for all alleles of each gene were seen in every panel and in nearly every year (of four years) that

ISB NEWS REPORT JUNE 2010
the traits were measured. Previous sequencing studies have also confirmed the role of the psy1 gene in the carotenoid
pathway, 5 where it behaves as a single gene controlling the presence or absence of any carotenoids. Once the carotenoid
pathway has been activated, it is significant that haplotypes in just two more genes explain most of the phenotypic
variation in the levels of carotenoid and βC levels, traits showing continuous variation, and this is very promising for the
ability to generate high βC maize lines via Marker Assisted Selection (MAS).
The combined effects on βC concentrations of one main functional polymorphism (allele) of lcyE (5′TE, Fig. 2a) and
two of crtRB1 (5′TE and/or 3′TE, Fig. 2b) were analyzed in the three association panels. These polymorphisms were
chosen because they had the highest estimated phenotypic effects and were physically farthest away from each other on
the chromosomes, thus avoiding the confounding effect of linkage on the analysis. A greater proportion of the phenotypic
variation in βC and the ratio of βC to all other carotenoids (βC/ALL) (52% and 65%, respectively) was explained by
the combined crtRB1/lcyE polymorphisms than by those of either gene alone. Combined lcyE and crtRB1 effects at
each polymorphism were largely additive. No inbred haplotype in the association panels combined the most favorable
haplotypes for crtRB1 5′TE, crtRB1 3′TE, and lcyE 5′TE, which is a problem when searching for specific combinations
of several alleles simultaneously, unless a prohibitively large association mapping panel is used. However, between the
existing common haplotypes predicted to yield the most and least βC, there was a 12-fold difference in βC concentration
and nearly a 20-fold difference in βC/ALL in two of the three association mapping panels. The third panel was not
polymorphic for one of the alleles and could not be included in the analysis, which is one advantage of using multiple
panels. Increases in βC and provitamin A derived from the more favorable haplotype in the two panels averaged 5.91 and
5.65 μg g -1 , or nearly 40% of the goal of nutritional breeders for human health.
Figure 1: Simplified carotenoid biosynthetic
pathway in maize and Arabidopsis.
CRTRB, in blue, represents the
nonheme di-iron β-carotene hydroxylase
(BCH) family in maize, which has at least
five members; the orthologous family in
Arabidopsis has two members (BCH1 and
BCH2). Carotenoid intermediates highlighted
in red were measured by HPLC for
association studies. GGPP, geranylgeranyl
pyrophosphate; PSY, phytoene synthase;
PDS, phytoene desaturase; Z-ISO,
ζ-carotene isomerase; ZDS, ζ-carotene desaturase;
CRTISO, carotenoid isomerase;
LCYE, lycopene ε-cyclase; LCYB, lycopene
β-cyclase; CRTRB, β-carotene hydroxylase
family; CYP97A, β-carotene hydroxylase
(P450); CYP97C, ε-carotene hydroxylase
(P450); ZEP1, zeaxanthin epoxidase;
VDE1, violaxanthin de-epoxidase; ABA,
abscisic acid.
Figure 2a: Polymorphisms significantly associated with carotenoid phenotypes in
Zea 4 LcyE. Putative promoters are depicted as orange arrows, exons as black squares, and
the sampled regions as gray boxes. Polymorphisms that significantly associated with changes
in flux between the lutein (left) and zeaxanthin (right) branches of the pathway in Figure 1 are
labeled with asterisks. The 5’ transposable element insertion(s) are represented by the white
triangles. Positions relative to the sequence alignment are indicated numerically above the
polymorphisms.

ISB NEWS REPORT JUNE 2010
Figure 2b: Polymorphisms significantly associated with carotenoid phenotypes in Zea mays crtRB1. The sequenced region
is framed in gray, translated exons are depicted as black boxes and the putative start of transcription (TSS) and poly(A) sites are indicated.
Polymorphisms found in one association mapping panel are marked in the diagram, and those that are significantly associated with changes in
βC, βC/βCX, βC/Z and βC/ALL are labeled with asterisks; abbreviations are the same as in Figure 1.
QTL mapping to confirm the role of the two genes on beta-carotene levels
The phenotypic effects of both genes on different carotenoid levels (particularly on the desired trait, βC) were confirmed
by linkage mapping. The lcyE gene mapped to chromosome 8 bin 5 in a QTL mapping population from B73 × Mo17,
where it co-localized with a previously mapped QTL that had no known underlying candidate gene. This QTL showed
significant effects for modification of the ratio of the left and right branch carotenoids and explained 31.7% of the
variation for lutein. 6 This QTL was not significant for total carotenoids, which further supports the conclusion that
variation within the lcyE gene underlies this QTL for carotenoid composition, since it influences the type of carotenoid,
rather than total amount.
QTL mapping of crtRB1 was carried out in three recombinant inbred lines (RILs) populations. 3 In all populations,
crtRB1 mapped to a genetic interval containing a principal QTL for βC concentration and βC/ALL, explaining 16.3%
and 32.8% of the trait variation, respectively. Favorable homozygous genotypes in the three mapping populations led
to β-carotene increases of 2.78 μg g -1 , 1.61 μg g -1 , and 0.53 μg g -1 over βC concentrations of the respective unfavorable
homozygotes, which averaged 2.79, 0.76, and 4.62 μg g -1 . Collectively, these results indicate that favorable crtRB1 alleles
can lead to higher βC concentrations across a range of segregating genetic backgrounds.
Expression analysis confirms the role of the two genes on beta-carotene levels
Expression analysis indicated that lcyE is preferentially expressed in the endosperm relative to the embryo. 2 Expression
profiling of kernels at 15 and 20 days after pollination (DAP) indicated that expression levels correlated well with the ratio
of carotenoids from each pathway branch, explaining 70 to 76% of the variance. Lines with transposon insertions near the
start site (Fig. 2b) had much lower expression levels (in 15 and 20 DAP, lower by a factor of 3.7 and 13, respectively).
crtRB1 transcripts from kernels harvested 15 DAP in six inbred lines (A619, B77, CI7, Hi27, NC320, and NC356),
which varied by an order of magnitude in their seed βC concentration, were examined. 3 In lines containing relatively high
concentrations of βC (CI7 and B77; identical crtRB1 haplotypes), crtRB1 transcripts accumulated to only 1/70 of the
level of crtRB1 transcripts in lines with low βC concentrations (NC356, Hi27, and NC320). Relative to all tested lines,
transcript and βC concentrations were both intermediate in A619, which carries only the 3′TE insertion (Fig. 2b). This
implies that polymorphisms associated with 5′TE, 3′TE, and InDel4 result in substantial transcript reduction in kernels
and probably decrease β-hydroxylase activity through reduction of CRTRB1 protein levels. The differences in expression
levels were very high in endosperm, not very different in embryos, and not at all different in leaves, which suggest tissuespecific
regulation of crtRB1. This is important and encouraging, as the carotenoid pathway later leads into the ABA
pathway, and stopping the production of carotenoids at βC could conceivably cause problems for the germinating embryo
or the maize plant, which requires ABA to properly respond to developmental and environmental growth signals.
Markers from two genes speed breeding progress for high beta-carotene maize lines
Inexpensive PCR-based markers were developed to differentiate all alleles of the two genes for use in MAS. The favorable

ISB NEWS REPORT JUNE 2010
alleles for both genes are rare in most maize germplasm and are especially rare in tropically adapted maize cultivars, which
are urgently needed to express high levels of βC if they are to benefit the people who need them most. Through the use
of donor lines and user-friendly PCR-based marker systems, introgression of the most favorable crtRB1 and lcyE alleles
into tropical germplasm has already achieved provitamin A target concentrations of 15 μg g -1 in preliminary evaluations
of breeding lines (data not shown), and the use of these markers in applied breeding programs throughout the world will
quickly bring more highly nutritious, well-adapted maize cultivars to farmers.
Concluding remarks
Profiling techniques offer a multidisciplinary approach to the safety assessment of GE plants, and provide a rigorous
scientific basis for the identification of any possible unintended health effect that could arise from genetic engineering. In
this study non-targeted molecular profiling technologies were used to provide insight into the extent of variation in the
maize transcriptome, proteome, and metabolome by analyzing three maize genotypes, two of them transgenic, grown in
the same location over three years/growing seasons. The observed variation was caused mainly by growing season, with
the associated environmental factors, and not due to genotype. Even though the environment was the dominant source
of variation, no common drivers of variation were identified.
This study also highlights the possibilities as well as the challenges of profiling analysis for food safety evaluation. A
big challenge of the ‘omics’ technologies is the vast amount of data generated, making it extremely complex to evaluate
individual GE lines and difficult to form a meaningful interpretation. Other challenges include the many gaps related to
the number of genes for which a function has been identified and the limited coverage of the proteome and metabolome.
Furthermore these technologies should not be limited to the safety assessment of GE-derived crops, which are already
subjected to extensive and costly pre-market analysis, but should be extended to plant varieties improved by mutagenesis.
Mutagenised rice plants are reported to have more changes in their transcript profiles than genetic engineered rice plants,
even though changes in the transcriptome do not necessarily correlate with risk; proteomic studies need to be performed
to provide information on the nature of the proteins. 5 These technologies still need to be validated before they can be
used on a case-by-case basis to confirm or supplement the current targeted analytical approaches. They are not intended
to replace existing analyses, but they may trigger the need for a more detailed and targeted analysis of specific groups of
genes, proteins, and metabolites.
References
1.
2.
3.
4.
5.
Pfeiffer WH & McClafferty B. ( 2007) HarvestPlus: breeding crops for better nutrition. Crop Sci. 47, S88–S105
Yu J, & Buckler ES. (2006) Genetic association mapping and genome organization of maize. Curr. Opin. Biotech. 17, 155–160
Harjes CE, et al. (2008) Natural genetic variation in lycopene epsilon cyclase tapped for maize biofortification. Science 319, 330–333
Yan J, et al. (2010) Rare genetic variation at Zea mays crtRB1 increases β-carotene in maize grain. Nature Genetics doi:10.1038/ng.551
Palaisa K, et al. (2004) Long-range patterns of diversity and linkage disequilibrium surrounding the maize Y1 gene are indicative of an asymmetric selective
sweep. Proc Natl Acad Sci USA 101, 9885–9890
6. Wong JC, et al. (2004) QTL and candidate genes phytoene synthase and ζ -carotene desaturase associated with the accumulation of carotenoids in
maize. Theor. Appl. Genet. 108, 349
Marilyn L. Warburton
USDA ARS Corn Host Plant Research Resistance Unit
Mississippi State University, MS 39759
Marilyn.warburton@ars.usda.gov