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Molecular marker-assisted breeding options for maize
improvement in Asia
B. M. Prasanna •Kevin Pixley •
Marilyn L. Warburton •Chuan-Xiao Xie
Received: 13 July 2009 / Accepted: 31 December 2009 / Published online: 14 January 2010
ÓSpringer Science+Business Media B.V. 2010
Abstract Maize is one of the most important food
and feed crops in Asia, and is a source of income for
several million farmers. Despite impressive progress
made in the last few decades through conventional
breeding in the ‘‘Asia-7’’ (China, India, Indonesia,
Nepal, Philippines, Thailand, and Vietnam), average
maize yields remain low and the demand is expected
to increasingly exceed the production in the coming
years. Molecular marker-assisted breeding is accel-
erating yield gains in USA and elsewhere, and offers
tremendous potential for enhancing the productivity
and value of Asian maize germplasm. We discuss the
importance of such efforts in meeting the growing
demand for maize in Asia, and provide examples of
the recent use of molecular markers with respect to (i)
DNA fingerprinting and genetic diversity analysis of
maize germplasm (inbreds and landraces/OPVs), (ii)
QTL analysis of important biotic and abiotic stresses,
and (iii) marker-assisted selection (MAS) for maize
improvement. We also highlight the constraints faced
by research institutions wishing to adopt the available
and emerging molecular technologies, and conclude
that innovative models for resource-pooling and
intellectual-property-respecting partnerships will be
required for enhancing the level and scope of
molecular marker-assisted breeding for maize
improvement in Asia. Scientists must ensure that
the tools of molecular marker-assisted breeding are
focused on developing commercially viable cultivars,
improved to ameliorate the most important con-
straints to maize production in Asia.
Keywords Maize Breeding Markers
Genetic diversity QTL Stress resistance
Quality
Introduction
Maize (Zea mays L.) holds a unique position in world
agriculture as a food, feed and source of diverse
industrially important products. Although developed
countries, particularly the USA, contribute predom-
inantly to maize production, demand for maize in
developing countries is expected to surpass the
B. M. Prasanna (&)
Division of Genetics, Indian Agricultural Research
Institute (IARI), New Delhi 110012, India
e-mail: bmprasanna@gmail.com
K. Pixley
International Maize and Wheat Improvement Center
(CIMMYT), Texcoco, Mexico
K. Pixley
University of Wisconsin, Madison, WI, USA
M. L. Warburton
USDA-ARS CHPRRU, Mississippi State,
MS 39762, USA
C.-X. Xie
Institute of Crop Science, Chinese Academy
of Agricultural Sciences (CAAS), 100081 Beijing, China
123
Mol Breeding (2010) 26:339–356
DOI 10.1007/s11032-009-9387-3
demands for both wheat and rice by the year 2020
(Pingali and Pandey 2001). The seven major maize
producing countries in Asia (‘‘Asia-7’’) are China,
India, Indonesia, Nepal, the Philippines, Thailand and
Vietnam, and have progressed from being net
importers of maize in the mid-1990s to net exporters
in the mid-2000s (FAO and USDA, as cited by
Gerpacio and Pingali 2007; Wada et al. 2008). This
achievement was primarily due to the influence of
China, which during the past four decades sustained
an impressive 3.4% annual average growth rate in
maize yields and by 2002 had become the world’s
second leading exporter of maize, after the USA
(Dixon et al. 2008; Gerpacio and Pingali 2007; Wada
et al. 2008). Overall, the recent increase in maize
production in the Asia-7 is indeed a success story,
with 3.1% average annual growth in yields, which
significantly exceeded the 2% rate in the USA during
the 1960s to mid 2000s (Edgerton 2009; Gerpacio
and Pingali 2007; Phillips 2009). However, average
maize yields in many of Asian countries remain low,
with India, Nepal and the Philippines achieving
&2 t/ha, Indonesia and Vietnam &3.5 t/ha, Thai-
land almost 4 t/ha, and China 5 t/ha, compared to the
world average of 4.7 t/ha in 2005 (Wada et al. 2008)
and current USA average of 9.4 t/ha (Edgerton 2009).
Demand for maize in Asia is expected to increase
rapidly over the next few decades, driven largely by
the growth rate of per capita GDP (gross domestic
product), and potentially influenced by the use of
maize for biofuel production in some regions of the
world (Falcon 2008; Dixon et al. 2008). The link
between growing income (and to a lesser extent
urbanization) and changing dietary habits (mainly
increased poultry and pork consumption) that require
more maize feed is well known. For example, while
India, Indonesia and China averaged 5–9% growth in
GDP from 1975 to 2000 (Falcon 2008), average
annual demand for maize in Asia-7 rose by 7% for
feed use and by 1.6% for food (Gerpacio and Pingali
2007). Global use of maize grain for ethanol
production is expected to double from 2007 to
2017, with most of this occurring in the USA and
Europe, but China will also use an important amount
of maize to produce biofuel (Dixon et al. 2008;
Edgerton 2009). Although predictions vary consider-
ably in magnitude, there seems to be little doubt that
the rate of increased demand will soon out-pace the
rate of increased maize production in Asia, and that
China, Japan, South Korea, Malaysia, the Philippines,
and Thailand will import substantial amounts, while
most others (e.g., India, Indonesia, and Vietnam) will
have balanced trade or small net exports of maize by
2025 (Falcon 2008; Gerpacio and Pingali 2007).
The current low average maize yields, particularly
in India, which has the second largest maize growing
area in Asia-7, offer tremendous challenges and
opportunities to increase regional maize production.
Wisely focused, intensive research efforts will be
crucial to minimize or eliminate the expected maize
production shortfall in Asia. Gerpacio and Pingali
(2007) and Gulati and Dixon (2008) described the
major maize production environments and systems in
Asia-7, and further identified and prioritized their
researchable production constraints. Surprisingly,
seed is only the third most expensive input to maize
production almost everywhere in Asia-7, after fertil-
izers, which cost one-half to one-tenth as much as the
most expensive input, i.e., power (animal or tractor).
There are various socio-economic and policy con-
straints, which if ameliorated with interventions such
as improved access to credit, input subsidies and
effective trade tariffs, could result in average yield
gains estimated between 24 and 48%. However, the
focus here shall be on opportunities and constraints
that can be addressed through maize breeding, which
together with other interventions could result in 80–
90% yield gains (Gerpacio and Pingali 2007).
Drought is the most important constraint across the
rainfed lowland and upland environments, covering
about 70% of the maize production area in Asia.
Considering that drought is also the eighth highest
priority constraint for the commercial subtropical/
mid-altitude areas (mostly in India), there is little
doubt that drought is the most important constraint to
maize production in South and Southeast Asia, and its
amelioration is estimated to offer the greatest benefit
or return for research investment (Gerpacio and
Pingali 2007; Gulati and Dixon 2008). Alleviating the
effects of drought alone could increase average maize
yields by 35% across Asia-7 (excluding China), and
by 28% in Southwest China (Gerpacio and Pingali
2007). Given the rapidly growing demand for maize
in Asia, which will require intensified cropping
systems in a context of increasing scarcity and value
of agricultural water, and likely unfavorable climate
change scenarios, the value of drought tolerance in
maize is certain to increase.
340 Mol Breeding (2010) 26:339–356
123
Other abiotic and biotic constraints that have
widespread yield-reducing effects and should receive
high priority for maize breeding research include (not
in order of priority) acid soils, water logging, downy
mildews, post-flowering stalk rot, turcicum leaf
blight, banded leaf and sheath blight, stem borers
and weevils (Gerpacio and Pingali 2007). Some of
these, plus several other constraints (e.g., weed
control, erosion control and soil micronutrient defi-
ciencies) may also be effectively addressed through
improved agronomic technologies, which is beyond
the scope of this paper.
Molecular tools to enhance breeding efficiency
and effectiveness have become integral to many
maize research programmes worldwide. The volume
of molecular data for diverse populations and breed-
ing lines, in both public and private breeding
institutions, has been accumulating at a rapid pace,
particularly in the developed world. This is now
enabling the use of sophisticated biometric and
modeling tools to predict genotypic value and
conduct genotypic selection before evaluating
phenotypes in large private breeding institutions.
Eathington et al. (2007) reported that MAS has more
than doubled the rate of gain for an index including
yield, moisture content and stalk strength compared
to conventional breeding for 248 maize populations.
Although not the subject of this paper, transgenic
events are now routinely contributing 5% yield
advantages to maize hybrids in the USA, and their
contribution to maize yields is anticipated to further
increase, perhaps to 25% by 2030 (Edgerton 2009).
This article reviews the status of diversity analysis,
QTL mapping and molecular marker-assisted selec-
tion in maize breeding in Asia, and highlights the
opportunities for molecular marker-assisted breeding
to contribute toward the efforts to meet the growing
demand for maize in Asia.
Molecular markers and mapping populations
in maize
Advances in genomics led to the identification of
numerous DNA markers in maize during the last few
decades, including thousands of mapped microsatel-
lite or simple sequence repeat (SSR) markers, and
more recently, single nucleotide polymorphisms
(SNPs) and insertion-deletion (InDel) markers. In
addition to the SSRs and SNPs, a large number of
genes controlling various aspects of plant develop-
ment, biotic and abiotic stress resistance, quality
characters, etc. have been cloned and characterized in
maize, which are excellent assets for molecular
marker-assisted breeding.
At present, SSRs are the most widely used markers
by maize researchers due to their availability in large
numbers in the public domain (MaizeGDB;
http://www.maizegdb.org), simplicity and effective-
ness. These PCR-based, genetically co-dominant
markers are robust, reproducible, hypervariable,
abundant, and uniformly dispersed in plant genomes
(Powell et al. 1996). While both SSRs and SNPs can
be reliably applied on a large scale, SNPs are highly
amenable for automation, and therefore offer signif-
icant advantages for genetic and breeding purposes.
Compared with the genomes of other cultivated plant
species, SNP frequency in maize is high, with one
SNP being found every 28–124 bp (Tenaillon et al.
2001; Ching et al. 2002; Vroh Bi et al. 2006). A
database and resource for SNP discovery and trait
dissection has been established for maize in which
genotype, phenotype and polymorphism data can be
accessed for diverse maize inbreds and populations
(Zhao et al. 2006a,b:http://www.panzea.org). Sev-
eral high throughput genotyping platforms have been
developed that allow rapid and simultaneous geno-
typing of up to a million SNP markers. In addition, a
custom GoldenGate assay containing 1,536 SNPs has
been developed based on public SNP information for
maize (Yan et al. 2009a).
In addition to powerful marker systems, diverse
mapping populations are available in maize as
international maize genomic resources, including
the intermated B73 9Mo17 (IBM) population (Lee
et al. 2002), and intermated recombinant inbred lines
(IRILs) developed from the IBM population (Coe
et al. 2002; Cone et al. 2002), etc. A genetic map of
maize, ISU–IBM Map4, that integrates 2,029 existing
markers with 1,329 new insertion-deletion polymor-
phism (IDP) markers has been developed using the
IBM population (Fu et al. 2006).
The maize ‘‘nested association mapping’’ (NAM)
population, comprising 5,000 RILs (200 RILs from
each of 25 populations), is another important genetic
resource developed in recent years. The NAM
population is a novel approach for mapping genes
underlying complex traits, in which the statistical
Mol Breeding (2010) 26:339–356 341
123
power of QTL (quantitative trait loci) mapping is
combined with the high (potentially gene-level)
chromosomal resolution of association mapping (Yu
et al. 2008; discussed later). The RILs are ‘‘nested’’ in
the sense that they all share a common parent, but
each population has a unique second parent. The
common parental line used in all 25 families, B73, is
the most important US corn breeding line. Descen-
dents of B73 are widely deployed in US production
corn agriculture, and the B73 genome has been
recently sequenced (Schnable et al. 2009). The
remaining NAM parental inbred lines were chosen
either on the basis of their agronomic importance in
the U.S. or to capture as much of the genetic diversity
present in maize as possible based on analysis of a
worldwide collection using 94 SSRs (Liu et al., 2003;
Flint-Garcia et al., 2005). The Suwan (Thailand)-
based inbred lines, Ki3 (drought-sensitive) and Ki11
(drought-tolerant), besides eight maize lines devel-
oped at CIMMYT mostly using tropical maize
germplasm (CML52, CML69, CML103, CML228,
CML247, CML277, CML322, CML333), were
among the 25 founder lines. Thus, global diversity
has been captured in the NAM RIL germplasm
resource, which will provide the maize research
community with the opportunity to map genes
involved for an array of traits of agronomic or
scientific interest (Yu et al. 2008). Although sufficient
diversity must be present in each association mapping
panel, too much phenotypic diversity (or poor
adaptation to any specific growing environment)
may make it difficult to phenotype a panel in an
association study. Thus, more region-specific associ-
ation mapping panels may need to be created that
contain germplasm more suited to specific growing
regions. One such panel has already been created,
characterized for population substructure and inter-
relatedness, and released for public use (Yang et al.
2009).
In Asia, and more specifically in the public sector
Institutions of China and India, several mapping
populations have been developed and utilized for
mapping QTL for various traits (discussed later).
However, published literature on the availability of
immortal mapping populations (RILs, doubled hap-
loids, and QTL-NILs) is very limited. At the Indian
Agricultural Research Institute (IARI), New Delhi,
two RIL populations have been developed, one using
CM139 (downy mildew susceptible) and NAI116
(downy mildew resistant), and another using
CA00106 (BLSB-tolerant; drought-susceptible;
waterlogging-tolerant) and CM140 (BLSB-suscepti-
ble; drought-tolerant; waterlogging susceptible), as
parental lines.
A RIL population (234 families) was derived from
the cross X178 (drought tolerant) 9B73 (drought
susceptible) at the Chinese Academy of Agricultural
Sciences (CAAS). This population can also be useful
for mapping other agronomically important traits,
because the parents have contrasting phenotypes for
low nitrogen and phosphorus stresses, acid soil
tolerance, and other traits. Maize scientists from
Sichuan Agricultural University, China, also devel-
oped 239 RILs from the cross Huangzao 4 (MDMV-
resistant) and Mo17 (MDMV-susceptible) for
mapping QTLs for resistance to Maize Dwarf Mosaic
Virus (MDMV) disease (Liu et al. 2006). Two
additional RIL populations were also developed in
China, one comprising 190 RILs derived from
Zong3 987-1, a widely used elite hybrid in China,
and another comprising 174 RILs derived from
B73 9BY804, the latter being a line with high oil
content (Song et al. 2004). Both populations have
been intensively analysed using SSR and SNP
markers (Ma et al. 2007; Chander et al. 2008; Yan
et al. 2009a).
Application of molecular markers in the Asian
maize breeding programmes
The Asian Maize Biotechnology Network (AMBI-
ONET), which was coordinated by CIMMYT and
financially supported by the Asian Development
Bank (ADB) during 1998–2005 in six Asian coun-
tries (China, India, Indonesia, Philippines, Thailand
and Vietnam), provided an impetus for application of
molecular markers for maize improvement by public
sector institutions in Asia. AMBIONET helped to
strengthen the capacity of partner institutions, and
aided in undertaking collaborative research on (i)
DNA fingerprinting and analysis of genetic diversity
in important inbred lines; (ii) assigning inbred lines to
heterotic groups; (iii) QTL analyses of some impor-
tant biotic and abiotic stresses; and (iv) initiating
molecular marker-assisted selection (MAS) projects.
Several AMBIONET partner institutions intensified
their molecular marker-assisted breeding efforts in
342 Mol Breeding (2010) 26:339–356
123
maize by attracting funding from their host organi-
zations as well as through external funding (Pray
2006). We shall highlight here salient applications of
molecular markers in the maize genetics and breeding
programmes in Asia.
DNA fingerprinting and genetic diversity analysis
of Asian inbred lines
DNA fingerprinting and genetic diversity analysis
using molecular markers is useful in effective man-
agement of germplasm collections and breeding
materials. Accurate assessment of the levels and
patterns of genetic diversity using molecular markers
is particularly helpful in maize breeding for (i)
maintenance and broadening of the genetic base of
the elite germplasm; (ii) assignment of lines to
heterotic groups; (iii) selection of appropriate paren-
tal lines for hybrid combinations; and (iv) generation
of segregating progenies with maximum genetic
variability for further selection (Mohammadi and
Prasanna 2003).
SSR markers have been successfully used for
DNA fingerprinting and analysis of genetic diversity
in China (e.g., Yuan et al. 2000; Li et al. 2002,2007,
2008; Yu et al. 2007; Xie et al. 2007,2008), India
(e.g., Pushpavalli et al. 2002; Prasanna and Hoising-
ton 2003; Bantte and Prasanna 2003; Mohammadi
et al. 2002,2008), Indonesia (e.g., George et al.
2004a; Pabendon et al. 2007), Philippines (e.g., Sales
et al. 2004), Thailand (e.g., Phumichai et al. 2008)
and Vietnam (e.g., Cuong et al. 2007a,b). In addition,
using diverse Asian maize inbred lines and six
‘reference lines’ from CIMMYT, standard alleles
for *80 SSR loci were developed to aid in cross
comparisons of these markers in diverse germplasm
and placed in public domain (George et al. 2004a;
http://www.cimmyt.org/ambionet).
Marker studies suggest that there is much more
diversity in tropical than temperate lines (e.g., Reif
et al. 2004; Tarter et al. 2004). Many useful alleles
for improving temperate maize may be hidden in the
tropical germplasm and should be uncovered for
continued future improvement. For example, a recent
study identified a gene, lycopene epsilon cyclase
(LCYE), related with provitamin A content in the
maize kernel. The diversity of alleles of this gene was
investigated using an allele mining strategy, demon-
strating that the favourable allele is more common in
the tropical lines (Harjes et al. 2008). However, the
favourable allele for CrtR-B1 gene, another important
gene influencing provitamin A content, is more
common in the temperate germplasm (Yan et al.
2009b). Thus, the study also indicates the value of
judiciously using tropical germplasm in temperate
maize breeding programmes and vice versa.
While significant diversity exists in the maize
genotypes of different Asian countries, molecular
characterization of the Asian maize inbred lines
revealed a relatively narrow genetic base for the
CIMMYT lines developed for the Asian region
(George et al. 2004a). In contrast to the case in
southern China where 95% of the lines clustered
separately from the CIMMYT lines, lines in the
Indonesian breeding programme showed a closer
relationship with the CIMMYT lines, reflecting a
long history of germplasm exchange. Analysis of the
SSR diversity in 102 Asian inbred lines also revealed
the effect of selection for downy mildew resistance in
Asian genotypes (George et al. 2004b), besides
indicating that maize breeding activity in Asia had
not caused a decline in the overall amount of
diversity in the region. SSR markers have also been
deployed for analysis of genetic diversity of maize
inbred lines in Asian countries including Japan
(Enoki et al. 2002) and Iran (Choukan et al. 2006).
Our survey of Asian institutions indicated that
except for a few multinational concerns operating in
Asia which are using SNP-based platforms for DNA
fingerprinting and diversity analysis, most of the
leading institutions in both public and private sectors
engaged in maize breeding are employing SSR
markers. It is interesting to note that the multinational
concerns based in Asia have been undertaking geno-
typing through the platforms located in USA or
Europe, while the relevant phenotypic data is collected
locally or regionally. However, this situation may soon
change with the establishment of cost-effective, high
throughput SNP genotyping platforms in the region.
Among the Asian countries, China and India are
actively involved in DNA fingerprinting of hundreds
of elite inbred lines, assessing their genetic diversity,
and assigning lines to heterotic groups. Core collec-
tions of inbred lines and landraces in the Gene Bank
have been formulated based on geographic origin,
phenotypic data and molecular analyses in China
(e.g., Yu et al. 2007; Wang et al. 2008) and in India
(Prasanna 2009).
Mol Breeding (2010) 26:339–356 343
123
Molecular markers can also play an important role
in plant varietal protection, specifically as tools to
distinguish an EDV (Essentially Derived Variety)
from an initial (protected) variety, as these markers
allow tracing of chromosomal segments from the
parent to their progeny and make possible empirical
evaluation of genetic relatedness. Research at the
University of Hohenheim, Germany, clearly demon-
strated the suitability of SSRs in differentiating EDVs
from initial varieties in maize, and provided the
technical and statistical framework for determining
EDVs (Heckenberger et al. 2002,2003,2005).
Through a project commissioned by India’s Protec-
tion of Plant Varieties & Farmers’ Rights Authority
(PPV & FRA), studies are being undertaken by the
Indian Agricultural Research Institute (IARI) on
putative EDVs and initial varieties based on the
Indian maize germplasm, for developing a suitable
protocol for ascertaining the EDV status using SSR
and SNP/InDel markers. In addition, DNA finger-
printing (and thus, distinguishing) open-pollinated
varieties (OPVs) is possible using SSR markers based
on a population bulk DNA fingerprinting technique
developed at CIMMYT (Warburton et al. 2009). The
SNPs do not bulk well, but the high automation may
enable analysis of 15–30 individuals per OPV at a
time for the same cost, once the platform is set up.
Assigning inbred lines to heterotic groups
The utility of SSRs for assigning lines to heterotic
groups and relating the SSR-based genetic distance
with hybrid yield or heterotic performance in maize
has been explored by a few research groups in Asia
(e.g., Yuan et al. 2001; Teng et al. 2004; Xu et al.
2005; Mohammadi et al. 2002,2008; Prasanna and
Hoisington 2003; Choukan et al. 2006; Xie et al.
2007,2008). Based on the planting areas of hybrids in
1992–2001 in China, Teng et al. (2004) selected 84
parent lines of 71 widely used hybrids and analysed
their heterotic groups and patterns using SSR data.
The study led to identification of seven heterotic
groups, and also indicated that to a certain extent, a
change of position for major heterotic groups of
maize took place during the past decade in China.
The major heterotic groups were Lancaster, Reid,
TangSPT, Zi330 and E28 in the early 1990s, while
they were Reid, Tem-tropic I, M30, TangSPT and
Lancaster in the early twenty-first century. Tem-
tropic I was a new heterotic group of elite germplasm
used widely in China, which contained tropical maize
germplasm. In another study, 187 commonly used
maize inbreds in China were defined into six
subpopulations, namely PA, BSSS (includes Reid),
PB, Lan (Lancaster Sure Crop), LRC (Luda Reb Cob,
a Chinese landrace, and its derivatives), and SPT (Si-
ping-tou, a Chinese landrace and its derivatives),
based on polymorphic data from 70 loci (Xie et al.
2007). Forty of the 187 lines, which formerly had
unclear and/or miscellaneous pedigree records, were
assigned to one of the six groups inferred via
STRUCTURE analysis (Xie et al. 2008).
The genetic diversity grouping data of the Asian
maize inbreds could provide valuable information for
improving the efficiency of hybrid breeding. How-
ever, the SSR loci, in general, may not be useful for
predicting heterosis or hybrid performance among the
lines, since genetic distance estimated using SSR data
has so far met with limited success for maize (e.g.,
Dhliwayo et al. 2009; Melchinger 1999; Xie et al.
2007,2008). Breeding lines developed from natural
populations or non-hybrid material or those lines
derived from pools or breeding materials with no
clear information about their constitution of germ-
plasm, may not separate into clear-cut heterotic
groups. This could be one of the major reasons why
many of the tropical maize lines could not be
delineated to distinct heterotic groups. For example,
a CIMMYT study of tropical inbred lines and the
populations from which these lines were developed
revealed a large amount of diversity that made it
difficult to find a clear-cut structure of the inbred
lines (Warburton et al. 2002). A similar situation was
also observed in a regional diversity study of
representative inbred lines from several Asian coun-
tries (George et al. 2004b). Studies using more
informative and functional markers based on poly-
morphic sites in a large number of agronomically
important/yield-related genes based on modern geno-
typing approaches and genomic information may help
to define heterotic groups where it has been difficult
to do so.
Molecular diversity in Asian landraces/
populations
Although maize hybrids represent the most econom-
ically important portion of the species, breeding
344 Mol Breeding (2010) 26:339–356
123
populations, open pollinated varieties (OPVs), land-
races, and wild relatives contain the majority of the
allelic diversity, much of which has never been
incorporated into improved maize cultivars. Popula-
tions introduced into Asia, originally from the center
of origin in Central America but following a
complicated pattern of introductions, have become
adapted to many new growing conditions, local biotic
and abiotic stresses, and farmers’ practices, and thus
represent unique sources of diversity. Many favorable
alleles for useful traits, including tolerance to biotic
and abiotic stresses, have developed in these areas
following natural and farmer’s selections over the
decades/centuries. In addition, alleles found abun-
dantly in germplasm from other regions can now be
efficiently moved into Asian maize germplasm via
MAS, which highlights the utility of characterizing
maize diversity at the global as well as national
levels.
The analysis of genetically heterogeneous popula-
tions has been until recently very expensive and time
consuming because variation tends to be partitioned
within, rather than between, maize populations, and
levels of variation can be very high. This means that
at least 15 individuals must be characterized in order
to adequately represent the allelic diversity present in
a population. A new method for SSR analysis of
pools of individuals from a population has proved to
be much more efficient than genotyping multiple
individuals per population, and much more accurate
than genotyping only one individual per population
(Dubreuil et al. 2006). A recent project among
researchers from CIMMYT, INRA (France), and
national programmes of China, India, Indonesia,
Thailand and Vietnam characterized 140 Asian maize
landraces using SSR markers and determined rela-
tionships among landraces/populations within each
country, between different countries, and compared
to the country of origin (Mexico). The populations
containing the most unique alleles at the SSR loci are
now being characterized for markers associated with
drought tolerance and other agronomically important
traits, as these are the populations most likely to
contain new and useful alleles for high maize
productivity under resource limited conditions. The
genetic characterization data will provide useful
information for utilizing these populations in geno-
mic studies and breeding efforts to create new maize
varieties.
The vast genetic resources available in the North
Eastern Himalayan (NEH) region as well as other
regions in India are interesting from both breeding
and evolutionary viewpoints (Prasanna and Sharma
2005); however, limited efforts have been made to
characterize and use these important genetic
resources in maize breeding programmes. Using the
‘population bulk DNA fingerprinting’ strategy, nearly
250 selected maize landraces in India have been
characterized using 42 SSR markers. The study
revealed significant intra-population and inter-popu-
lation diversity in the Indian maize landraces, espe-
cially those from the NEH region, and highlighted
the genetic distinctiveness of ‘Sikkim Primitives’
(a landrace with high prolificacy) from the rest of the
accessions (Sharma et al. 2009). In addition, several
promising NEH and non-NEH accessions have been
identified based on extensive multi-location pheno-
typic evaluation. Liu et al. (2007) studied the genetic
diversity among Chinese maize OPVs using SSR
markers and the population bulk DNA fingerprinting
strategy.
QTL analysis
Following the first report on QTLs for yield-related
traits in maize (Stuber et al. 1987), maize researchers
worldwide have generated numerous reports of
molecular markers tagging genes/QTLs for diverse
traits of agronomic and scientific interest. QTLs for
several important traits affecting maize in Asia have
also been mapped, particularly in China and India.
These traits include plant height (Wang et al. 2006;
Zhang et al. 2007), downy mildew resistance (Agra-
ma et al. 1999; George et al. 2003; Nair et al. 2005;
Sabry et al. 2006), SCMV (Sugarcane Mosaic Virus)
resistance (Zhang et al. 2003), MDMV (Maize Dwarf
Mosaic Virus) resistance (Liu et al. 2006), common
smut resistance (Ding et al. 2008), head smut
resistance (Li et al. 2008), Fusarium moniliforme
ear rot resistance (Zhang et al. 2006), Banded leaf
and sheath blight (BLSB) resistance (Zhao et al.
2006a,b; Garg et al. 2009), drought stress tolerance
(Xiao et al. 2005; Lu et al. 2006; Hao et al. 2008;
Prasanna et al. 2009a), waterlogging tolerance (Qiu
et al. 2007), nutrient components under low nitrogen
stress (Liu et al. 2008a,b), high-oil content (Song
et al. 2004), popping ability (Babu et al. 2006;Li
et al. 2006), and CMS-S (Tie et al. 2006). Such
Mol Breeding (2010) 26:339–356 345
123
studies have contributed to a greater understanding of
the genetic architecture of various traits, including
disease resistance (Wisser et al. 2006) and drought
tolerance (e.g., Tuberosa et al. 2007) in maize. We
review briefly the work done with respect to QTL
analyses of two important biotic and abiotic con-
straints of maize in the Asian context.
Downy mildew resistance
A major emphasis in the Asian maize breeding
programmes has been the improvement for resistance
to downy mildews, specifically Peronosclerospora
sorghi (sorghum downy mildew; SDM) and P. hetero-
pogoni (Rajasthan downy mildew; RDM) in India,
P. maydis (Java downy mildew) in Indonesia, P. zeae in
Thailand and P. philippinensis in the Philippines.
Using a set of RILs derived using Ki3 (downy
mildew-resistant) and CML139 (downy mildew-sus-
ceptible) as parental lines, QTLs conferring resis-
tance to five different downy mildews in tropical
Asia, including SDM and RDM in India, Philippine
downy mildew in the Philippines, Java downy
mildew in Indonesia, and P. zeae in Thailand were
mapped through a collaborative study within the
AMBIONET project (George et al. 2003). The study
identified QTLs with significant effects for resistance
to the five important downy mildews of maize in
Asian; of particular significance was a QTL on chr. 6
(bin 6.05) that influenced resistance to all five downy
mildews and accounted for nearly 20 and 31% of the
phenotypic variance for P. sorghi (SDM) and P.
heteropogoni (RDM) disease resistance, respectively.
Two major QTLs (one each on Chr. 6 and Chr. 3)
were further validated in India using a backcross
population derived from CM139 (SDM-susceptible)
and NAI116 (SDM-resistant) (Nair et al. 2005). An
integrated strategy of MAS in BC
2
F
1
and BC
2
F
2
generations, with foreground selection for the two
major QTLs (using flanking markers) and back-
ground selection for the recurrent parent genome
(using 54 SSR markers), followed by phenotypic
selection in BC
2
F
3
at an SDM nursery using artificial
inoculation, was used to generate SDM-resistant
CM139 and several QTL-NILs (Prasanna 2009).
The MAS-derived lines, including QTL-NILs, have
been used to develop SDM-resistant maize hybrids,
and for transcriptome profiling under downy mildew
stress (Singh et al. 2009).
BLSB resistance
The banded leaf and sheath blight (BLSB) disease,
caused by Rhizoctonia solani Ku
¨hn in maize, is one
of the most destructive and important diseases of
maize in South and Southeast Asia. Very few sources
of resistance to this disease have been found. In
China, a mapping population consisting of 229 F
2
individuals derived from the cross of inbreds R15
(resistant) and 478 (susceptible) were used to map
QTLs conferring resistance to BLSB. Of the eleven
significant QTLs for resistance, only four (located on
chromosomes 2, 6 and 10) were stable across
locations, accounting for 3.72–10.35% of the pheno-
typic variation (Zhao et al. 2006a,b).
In another study in India, a F
2:3
mapping popula-
tion was generated using CA00106 (BLSB-tolerant)
and CM140 (BLSB-susceptible). Phenotyping was
undertaken using artificial BLSB inoculation at three
locations (Delhi, Pantnagar and Udaipur) which are
‘hot spots’ for the disease. QTL mapping revealed
location-specific QTLs for BLSB resistance, with
most of the favorable QTL alleles contributed by the
resistant parent CA00106. The study also led to
identification of three QTLs (on chr. 6, 8 and 9) with
significant epistatic interactions (Garg et al. 2009).
The studies on BLSB in Asia have so far revealed
a high degree of genotype x environment interaction,
and complex nature of inheritance of resistance to the
disease. It is important to intensify efforts to identify
stable and additional sources of resistance to BLSB
and improve the disease resistance of present maize
hybrids.
Drought tolerance
As previously discussed, drought is the most impor-
tant constraint to maize production and breeding for
drought tolerance is the topmost research priority for
maize in South and Southeast Asia. Maize is partic-
ularly sensitive to water deficit stress during the
reproductive stages. Substantial progress has been
made, particularly by CIMMYT and its collaborators
in Africa and Asia, in improving, via selection under
managed stress, tolerance to drought at flowering.
Simultaneously, advances in genotyping and pheno-
typing, QTL mapping, and gene expression analyses,
have significantly improved the prospects for identi-
fying alleles with major effects on drought tolerance
346 Mol Breeding (2010) 26:339–356
123
and using them in breeding programmes. QTL
mapping experiments on drought stress have been
undertaken in China (e.g., Xiao et al. 2005; Hao et al.
2008), India (Prasanna et al. 2009a) and Thailand
(Pichet Grudloyma, personal communication); most
of these studies were conducted in collaboration with
CIMMYT.
QTL mapping for drought tolerance of maize in
India identified major QTLs on chr. 1, 2, 8 and 10,
based on evaluation of a set of 230 CIMMYT-
developed RILs at two locations (Hyderabad and
Karimnagar) (Prasanna et al. 2009a). Analyses of the
RIL datasets identified QTLs influencing specific
traits under drought stress that co-localised on chr. 1,
2, 8, and 10. Also, a significant digenic epistatic QTL
effect, other than the main effect QTLs, was detected
for kernel number per ear under drought stress.
Similarly, analysis of an F
2:3
population derived from
the cross X178 (a widely planted, drought-tolerant
line in China) 9B73 at different locations in central
and southern China (Xiao et al. 2005; Hao et al.
2008) resulted in detection of a major QTL for ASI
(anthesis-silking interval) and ear number per plant
under drought stress on chr. 1 (bin 1.03) and chr. 9
(bins 9.03–9.05), which correspond to some major
QTLs identified in different experiments on drought
stress worldwide (see Tuberosa et al. 2007). The
‘consensus QTLs’ for drought tolerance in maize
identified through different experiments worldwide,
including India and China, using different mapping
populations, could serve as good candidates for use in
marker-assisted breeding to improve maize produc-
tion under water-limited conditions.
Low nitrogen stress tolerance and nitrogen
use efficiency
Developing maize varieties with tolerance to low soil
nitrogen stress and with high nitrogen use efficiency
is gaining importance in Asia. Association analysis in
this regard has been recently undertaken in China
(Wu et al. 2008,2009; Xie et al. 2008). The natural
variations of genes encoding two cytosolic members
of the glutamine synthetase gene family, Gln1-3 and
Gln1-4, were analyzed in a structured population
panel of 187 Chinese maize inbreds with phenotyping
tests in two environments each at three locations in
2 years. A list of beneficial haplotypes among the
lines was identified through this analysis. The most
favourable allele accounted for a phenotypic differ-
ence of 42.3% of grain yield under low N versus
well-fertilized. In another study in China, Liu et al.
(2008a,b) identified several QTLs that specifically
expressed under different nitrogen conditions and
could therefore help understand the genetic basis of
nitrogen-use efficiency.
Powerful analytical techniques are now available
to scan the genome for significant marker-trait
associations, to estimate epistatic effects among
QTLs, and to study QTL 9environment interactions.
The importance of epistasis and QTL 9environment
effects on trait expression has been demonstrated for
plant height (Zhang et al. 2007), common smut
resistance (Ding et al. 2008), drought tolerance
(Prasanna et al. 2009a), BLSB resistance (Garg
et al. 2009), and other traits.
Meta-analyses to integrate results from QTL
experiments undertaken in various environments/
locations assumes importance in understanding the
genetic basis of complex traits and devising suitable
strategies to utilize the information in breeding
programmes. Wang et al. (2006) constructed an
integrated QTL map, based on 1,201 published maize
QTLs affecting 68 traits, and showed that maize
QTLs for various traits are clustered in all ten
chromosomes. Twenty-two plant height QTLs of
maize were co-linear with 64 plant height QTLs of
rice, and 43 grain yield QTLs of maize were co-linear
with seven grain yield QTLs of rice.
The information on QTLs for resistance to various
biotic and abiotic stresses as well as for other
agronomically important traits using Asian maize
germplasm has steadily increased in the last one
decade, mainly using biparental mapping popula-
tions. However, many of the QTL-marker associa-
tions remain unvalidated, and also as elsewhere, not
much translation of this information into products
using MAS has taken place. This could be attributed
to various reasons, which have been well elaborated
by Xu and Crouch (2008). Nevertheless, the infor-
mation still holds significance, and new possibilities
to validate QTLs include corroboration of the results
by association mapping studies which some institu-
tions in China and India have initiated, and creation
of Near Isogenic Lines (NILs) to test the effect of
individual QTLs, which should now be done. From
the trait perspective, there is still a need for genetic
dissection and QTL analysis of traits, such as nutrient
Mol Breeding (2010) 26:339–356 347
123
use efficiency, waterlogging tolerance and resistance
to post-flowering stalk rots, which are important in
several Asian countries.
MAS for developing improved maize germplasm
in Asia
Significant progress has been made worldwide in
optimizing MAS for improvement of both qualita-
tively and quantitatively inherited traits using maize
as a model system. One successful example of MAS
for maize improvement, and of particular use to the
developing world, is the utilization of opaque2-
specific SSR markers in conversion of maize lines
into quality protein maize (QPM) lines with enhanced
nutritional quality (Prasanna et al. 2001; Morris et al.
2003; Babu et al. 2005). A MAS-derived QPM hybrid,
‘Vivek QPM Hybrid 9’ has been recently released by
the Vivekananda Parvatiya Krishi Anusadhan Sans-
than (VPKAS) in Almora, India. This QPM hybrid
was developed through marker-assisted transfer of the
o2 gene and phenotypic selection for endosperm
modifiers in the parental lines (CM145 and CM212) of
Vivek Hybrid 9 (Babu et al. 2005; Gupta et al. 2009).
The same approach was used to develop QPM
versions of several elite, early maturing inbred lines
adapted to the hill regions of India (Gupta et al. 2009)
and QPM versions of six elite inbred lines (CM137,
CM138, CM139, CM150 & CM151), which are the
parents of three single-cross hybrids, PEHM2
(CM137 9CM138), Parkash (CM139 9CM140)
and PEEHM5 (CM150 9CM151) (Khanduri et al.,
2009).
Scientists at IARI have pyramided major genes/
QTLs for resistance to turcicum leaf blight (Exserohi-
lum turcicum) and Polysora rust (Puccinia polysora)
in five elite Indian lines, CM137, CM138, CM139,
CM140 and CM212 (Prasanna et al. 2009b). Similar
efforts on MAS for generation of QPM lines and
transfer of major QTLs for SCMV resistance are being
implemented at CAAS, and the MAS products are in
pipeline (Shihuang Zhang, personal communication).
Another potential application of MAS in maize
could be for improving the provitamin A content of
grain. Quantifying the provitamin A carotenoid
content of maize samples is difficult, time-consuming
and expensive, and breeding programmes will there-
fore benefit greatly from use of MAS to reduce
the need for phenotypic assays. Following the
publication of results of association mapping studies
(Harjes et al. 2008), sequence-tagged, PCR-based
markers were developed and demonstrated for use in
selecting favorable alleles of LCYE (lycopene epsilon
cyclase), a crucial gene in the carotenoid pathway.
More recently, collaborative research between CI-
MMYT, the University of Illinois, and Cornell
University, led to detection of important allelic
variation and development of useful markers for
favourable alleles of LCYE and another critical gene
in the pathway, CrtR-B1 (carotene beta-hydroxylase
1) (Yan et al. 2009b). CIMMYT maize breeders are
using MAS to develop better source lines by com-
bining favorable alleles for both LCYE and CrtR-B1
in lines with above-average concentrations of provi-
tamin A; however, they are first validating the effect
of these two alleles on provitamin A concentrations
in various maize populations before investing in
widespread use of MAS in breeding programme.
Allele mining and marker development are also
underway for other genes of the carotenoid biosyn-
thetic pathway, including PSY (phytoene synthase)
and CCD (carotenoid cleavage dioxygenases), giving
hope that MAS will soon be possible for several
genes which together explain much of the variation
for provitamin A in maize.
Despite the complexities of improving polygenic
traits through MAS, there have been a few successful
examples, including MAS for the improvement of
drought tolerance of both tropical inbred lines and
populations at CIMMYT (Ribaut and Ragot 2006).
Recent efforts are focusing on strategies that combine
high-density genotyping with index-based selection
for drought tolerance. Marker-assisted recurrent
selection (MARS) refers to the improvement of an
F
2
population by one cycle of marker-assisted
selection (i.e., based on phenotypic data and marker
scores) followed commonly by two or three cycles of
marker-based selection (i.e., based on marker scores
only). Bernardo and Charcosset (2006) examined the
usefulness of having prior knowledge of QTLs under
genetic models that included different numbers of
QTLs, different levels of heritability, unequal gene
effects, linkage, and epistasis, and concluded that
with known QTL, MARS is most beneficial for traits
controlled by a moderately large number of QTL
(e.g., 40). Bernardo and Yu (2007) further analyzed
the prospects for genome-wide selection (GWS) for
improving quantitative traits in maize, and concluded
348 Mol Breeding (2010) 26:339–356
123
that this approach, although more expensive, is
superior to MARS for improving complex traits, as
GWS effectively avoids issues pertaining to the
number of QTL controlling a trait, the distribution of
effects of QTL alleles, and epistatic effects due to
genetic background.
Maize seed companies have successfully exploited
marker-QTL associations in population improvement
and cultivar development (e.g., Johnson 2001,2004;
Eathington et al. 2007). Some of the important factors
that contributed to effective use of MAS schemes in
maize breeding, specifically by the private sector,
have been the use of year-round nurseries or contin-
uous nurseries, high throughput genotyping and
phenotyping, and efficient integration of phenotypic
and genotypic datasets using bioinformatic tools for
decision making (Ragot and Lee 2007; Eathington
et al. 2007). The recent rates of conventional plus
molecular plus transgenic breeding progress, and the
solid prospects for important achievements in breed-
ing for enhanced yield potential, stress tolerance
(including drought tolerance) and nutrient use effi-
ciency, have led Monsanto to boldly claim that ‘‘as the
world faces continued and growing demands for
agricultural goods, Monsanto has committed to dou-
ble crop yields in corn, soybeans and cotton by 2030’’
(http://monsanto.mediaroom.com/index.php?s=43&
item=642); Edgerton (2009) and Fraley (2009) pre-
sented some evidences indicating that this goal is
achievable.
Opportunities for enhancing the level and scope
of molecular marker-assisted breeding
Association mapping or linkage disequilibrium
analysis
Association mapping through LD analysis has led to
identification of many genes controlling simply
inherited traits in various plant species. This approach
is now being applied to dissect complex traits and
identify superior alleles contributing to improved
phenotypes. Association mapping seeks to identify a
statistically significant genetic association between a
change in the DNA sequence and a change in a trait
of interest using a large population of diverse
individuals, to remove circumstantial correlations
(e.g., Thornsberry et al. 2001; Harjes et al. 2008). The
approach provides excellent mapping resolution and
the ability to investigate many alleles at the same
time. Although conventional linkage/QTL mapping
and association mapping should be considered com-
plimentary techniques to find and corroborate results,
researchers will find that establishing an association
mapping panel of fixed lines can be carried out more
quickly than the generation of a fixed population of
recombinant lines for linkage mapping, and the same
association mapping panel may be used for the study
of many different traits.
Association mapping has been used in many crop
species including maize, rice, wheat, barley, sor-
ghum, sugarcane, soybean, potatoes, tomato, and
trees such as eucalyptus, aspen and pine (Zhu et al.
2008). There are many reports of successful associ-
ation between DNA polymorphisms and qualitative
traits in plants, but fewer reports for complex traits.
However, the genetic, genomic, and statistical tools
are now at hand to successfully apply association
mapping for the dissection of complex traits in plants,
which will harness the natural diversity in the crop-
related gene pool to identify and use allelic variants
for crop improvement (Yu et al. 2006; Zhu et al.
2008).
Outcrossing species such as maize, with high
levels of diversity and recombination and, thus, a fast
breakdown of linked genomic regions generally rely
on the use of candidate gene sequences, although
genome-wide association analysis in maize using
250,000 random SNP markers has been successfully
demonstrated recently (Belo
´et al. 2008). CIMMYT
scientists, with partners in China, Thailand, and
Kenya, are using association mapping to study
drought tolerance in tropical maize germplasm. The
project evaluated 384 lines under drought stressed
and well-watered conditions at five global locations
over 2 years. Standard drought tolerance traits and
metabolic component traits from different tissues
were measured and associated with data for 1,536
SNP markers within 582 candidate genes. A total of
331 of the 1,536 SNPs were significantly associated
with drought tolerance or changes in levels of
metabolic products known to be linked to drought
tolerance, including genes for ABA and carbohydrate
metabolism, transcription and signaling factors, and
chromatin remodeling. Twenty-two of the 331 SNPs
were highly associated in multiple environments,
years, and tissues.
Mol Breeding (2010) 26:339–356 349
123
Although several recent publications have described
association mapping studies in various crop plants (Zhu
et al. 2008), experimental design and statistical meth-
ods associated with association mapping are still
evolving. It should also be noted that linkage or QTL
mapping and association mapping are complementary
and are best used in conjunction to increase statistical
power and mapping resolution (Myles et al. 2009). This
is particularly important for genome-wide association
studies, which can suffer high rates of false-positive
results (Manenti et al. 2009).
High throughput genotyping and phenotyping
The use of molecular markers in crop improvement has
been well established, but their application in routine
crop breeding activities in most Asian institutions is
still very limited, primarily due to inadequate access to
high throughput genotyping and phenotyping facili-
ties. There are excellent opportunities for undertaking
high throughput genotyping in maize. Nearly one
million maize SNPs are available in public databases
(http://www.panzea.org), and several high throughput
genotyping platforms have been developed for com-
mercial use (Flint-Garcia et al. 2005). In addition, the
cost and speed of sequencing have improved dramat-
ically in recent years. These new technologies, and
associated data handling and analysis tools, provide
opportunities for the maize community to speed up
research progress for large scale diversity analysis,
high density linkage map construction, high resolution
QTL mapping, linkage disequilibrium (LD) analysis
and genome-wide association studies. Because the
genomic sequence of maize is publically available
(Schnable et al. 2009), re-sequencing of individual
maize inbred lines can now give the entire genotype of
that individual, that is to say, the allelic state of every
SNP in the genome. Expected advances in this tech-
nology should soon make it widely accessible, with
SNPs available in every region of the genome, and
advancing enormously the possibilities for gene dis-
covery and selection.
A recent study by Hamblin et al. (2007) compared
analyses based on 89 SSRs to analyses based on 847
SNPs in the same maize collection of 259 inbred lines.
Although the resolution in measuring genetic distance
using SNPs based on allele-sharing was lower than the
more polymorphic SSRs, the possibilities to automate
SNPs will allow a much higher number of them to be
used cheaply in characterization studies, overcoming
this drawback. Jones et al. (2007) also reported on the
comparative utilities of SSR and SNP markers for
characterizing maize germplasm in terms of their
informativeness, levels of missing data, repeatability
and ability to detect expected alleles in hybrids and
DNA pools. However, it is important to recognize that
even in the SNP era, SSRs would continue to be
important for specific uses (e.g., population genetic
analysis). Also, in situations where closely linked or
flanking or gene-based SSRs are available (e.g., MAS
for conversion of non-QPM into QPM versions), or
when the gene of interest is not sequenced and
sequence-tagged markers are not an option, a public
sector institution or a small breeding company might
effectively use SSRs in MAS programmes.
Array-based, high throughput DNA markers will
further revolutionize genotyping in crop plants (Gupta
et al. 2008). New SNP assays in maize (Illumina
GoldenGate array of 1,536 SNPs; Infinium array with
up to 1 million SNPs; Flint-Garcia et al. 2005; Fan
et al. 2006; Jones et al. 2009; Yan et al. 2009a) enable
quick estimation of genetic structure in populations,
and LD structure in genomes, which in turn, will
greatly speed up the identification and use of new and
useful alleles for maize improvement. Although the
advanced genotyping tools and platforms are not yet
widely used in maize in the Asian countries, the
scenario might soon change as costs of genotyping
continue to decline. Because many of the institutions
in Asia, especially in the public sector and small
breeding companies, do not have the resources to
establish advanced genotyping platforms nor in-house
technologies that are price-, throughput-, or turn-
around-time-competitive as compared to companies
who specialize in service provision, outsourcing of
genotyping is, and will increasingly, be the norm.
It is now increasingly realized that high throughput
genotyping will be of little value without high
throughput precision phenotyping, on which there
has been considerable emphasis in recent years (e.g.,
Montes et al. 2007). As suggested by Myles et al.
(2009), researchers must be concerned with deter-
mining and using appropriate experimental designs
that maximize the reliability of their phenotyping
efforts. Selection of germplasm with appropriate
levels of relatedness to enable association mapping
and generation of high-quality and high throughput
phenotype data will be the critical factors for
350 Mol Breeding (2010) 26:339–356
123
developing efficient molecular marker-assisted breed-
ing programmes.
Xu and Crouch (2008) discussed in detail the
possible means to optimize molecular marker systems
and to increase the accessibility of MAS to public
sector breeders, including building skills and capacity
in developing countries, and developing molecular
breeding decision support tools. A single seed–based
DNA genotyping system, developed at CIMMYT
(Gao et al. 2008), has the potential to reduce costs by
enabling selection of genotypes before planting,
thereby increasing the scale and efficiency in crop
species like maize with relatively large seeds.
Although the technology has some constraints, such
as quality of DNA for analysis of specific markers
(e.g., LCYE), and seedling establishment under field
conditions (Xu and Crouch 2008), it could be
effectively tapped for specific applications in molec-
ular marker-assisted breeding.
Doubled haploid technology and MAS
The use of doubled haploid (DH) techniques to rapidly
develop inbred lines is widespread among commercial
maize breeding programmes particularly in Europe
and USA, and to a limited extent in Asia. Our survey
indicated that some of the leading public and private
institutions in Asia are using or have initiated
programmes to develop DH lines in maize using
haploidy inducers (e.g., Chen and Song 2003; Zhang
et al. 2008), with the exception of Vietnam’s National
Maize Research Institute (NMRI), which has devel-
oped a large number of stable DH lines in different
genetic backgrounds using the anther culture tech-
nique (Cuong et al. 2007a). Factors making DHs
increasingly attractive include the development of
better inducer lines, more efficient chromosome
doubling methods, and protocols to efficiently intro-
gress transgenes, especially stacked transgenes.
Unfortunately, the available inducer lines are of
temperate adaptation, so the development of haploidy
inducer lines in tropical genetic background, currently
ongoing under a CIMMYT collaborative project with
the University of Hohenheim (Germany), promises to
be extremely valuable to breeding programmes in
tropical and subtropical regions of Asia and elsewhere.
Although much has been written about the use of
DHs in maize breeding (e.g., Forster and Thomas
2005), there is very little published evidence that DHs
and MAS are commonly used together. A likely
application of MAS and DNA fingerprinting together
with DHs should be to select parents with comple-
mentary genotypes to form crosses for use in deriving
DH lines. Another application of the combined use of
MAS and DHs could be in recurrent selection
projects. Bouchez and Gallais (2000) demonstrated
with simulations that use of DH lines will theoreti-
cally enhance the efficiency of recurrent selection
schemes for traits with low heritability, particularly
for breeding programmes without access to off-
season nurseries. Similarly, for some traits (e.g., see
our discussion of molecular marker-assisted breeding
for provitamin A), MAS could be cheaper, faster or
more effective than phenotyping DH lines to select
parents for subsequent cycles of recurrent selection
projects.
A third application in which DH and MAS
complement each other is to derive DH lines from
bi-parental crosses when the objective is to obtain
inbred lines genetically similar to either parent of the
cross (Smith et al. 2008) or to identify recombinants
at or flanking specific loci. The most frequent
application of this approach would likely be the use
of DH line conversion protocols instead of slower
conventional backcrosses (Forster and Thomas 2005),
and application of MAS to identify the DH lines with
closest genetic similarity to one of the parent lines.
Finally, the most widespread combined use of DH
and MAS is probably for genetic studies such as
bulked segregant analysis and developing genetic
maps (Chang and Coe 2009; Forster and Thomas
2005). Because DHs offer a fast way to obtain
homozygous lines, they can save time and increase the
efficiency of projects designed to identify or map
marker-trait associations, leading to potential use of
markers in MAS breeding projects. A DH population
of 83 lines was developed in China from a cross
between Nongxi531 and Nongxi110, and was used to
map QTLs influencing grain quality; because of the
contrasting phenotypes of the parents, this population
can also be useful to map QTLs for yield components,
especially kernel row number (Zhang et al. 2008).
Synergy through networks
The national agricultural research systems (NARS) in
some of the Asian countries, particularly China and
India, have demonstrated the capacity to effectively
Mol Breeding (2010) 26:339–356 351
123
apply modern biotechnology, particularly molecular
markers, for maize improvement. However, cost-
effective application of molecular marker technology
to agriculturally important problems cannot be done
in isolation. Networking can facilitate development
of an integrated system for efficient application of
molecular tools and techniques, including QTL
mapping and MAS, as demonstrated by the success
of AMBIONET (Pray 2006). Public sector funding
for maize molecular marker-assisted breeding in
India and China has increased several-fold in the last
5 years. Equally important, a number of network
projects related to maize molecular marker-assisted
breeding have been initiated and/or implemented in
India, leading to generation of information on
marker-trait associations as well as development of
MAS products as discussed above. The same strategy
could pay dividends in other major maize-growing
countries in Asia.
The level of public–private collaborations in maize
breeding and/or biotechnology in Asian countries is
almost negligible at present, unlike the situation in
USA and several European countries. Considering the
high cost of molecular research platforms, the need
for extensive and precise phenotyping, the increasing
complexity of bioinformatics tools to manage and
interpret data, and the ever-growing intellectual
property rights restrictions to germplasm exchange,
such collaborative networks offer a synergistic way to
share scientific and infrastructure capacities on a
cost- and time-effective manner. This may be the
only viable mechanism by which most public and
modestly-sized private sector programmes can effi-
ciently undertake molecular marker-assisted breeding
activities, thereby realizing their potential contribu-
tion to increasing maize production.
Conclusions
The technological opportunities for implementing
molecular marker-assisted breeding in maize have
increased tremendously in recent years. We have
provided only a glimpse of these advances and the
efforts made in the Asian institutions, specifically in
the public sector, to utilize molecular markers for
diverse purposes in maize genetics and breeding.
Significant strides have been made in China and
India, particularly with regard to understanding the
phenotypic and molecular diversity in the maize
germplasm, identification of QTLs influencing
diverse traits, especially tolerance to important biotic
and abiotic stresses, and MAS for improving disease
resistance and nutritional quality. Yet, the application
of molecular marker-assisted breeding tools to accel-
erate gains in maize productivity has barely begun in
much of Asia, and there is vast potential and need to
expand the scope and impact of such operations.
Molecular marker-assisted breeding has special rel-
evance for rapidly improving the low current average
yields of maize in Asia by aiding breeders to address
the biotic and abiotic stresses that most constrain
productivity. Breeders will also want to avail molec-
ular tools to more efficiently add value to new maize
cultivars, e.g., by enhancing their nutritional or
biochemical qualities for use as food, feed, and
industrial material.
The scientific and economic capacity in Asia is
improving, which in turn, will enhance the rate and
efficiency of breeding progress using modern tools as
well as utilization of advanced genotyping platforms.
Thus, these efforts will have not only regional but
international impact in the years to come. A major
investment in MAS infrastructure, including year-
round nurseries, high throughput and precision phe-
notyping facilities, and dedicated personnel are
required for the Asian institutions to effectively
deploy MAS, maximize selection gains, and mini-
mize time required for cultivar development. A
further challenge is that, as mentioned above, the
investment needed to become and remain competent
in molecular marker-assisted breeding techniques
may require innovative models for resource-pooling,
intellectual-property-respecting partnerships. Indeed,
our survey indicated that even the largest commercial
seed companies regularly engage in partnerships for
implementing their molecular maize breeding pro-
grammes (e.g., out-sourcing of critical activities, and
alliances with other private- and to a lesser extent
public-sector institutions). Finally and crucially,
scientists must ensure that the tools of molecular
marker-assisted breeding are wisely and intensely
focused on developing practical solutions—i.e., com-
mercially viable, improved cultivars—to the most
important constraints to maize production in Asia.
Acknowledgments Our sincere thanks to the scientists from
the public and private sector institutions in India, China,
352 Mol Breeding (2010) 26:339–356
123
Thailand and Vietnam, for responding to the survey on maize
molecular marker-assisted breeding and providing relevant
information; their names are withheld to protect the
confidentiality of their responses.
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