Indian Journal of Biotechnology

Vol 2, January 2003, pp.85-98

Molecular Breeding for Maize Improvement: An Overview

B.M. Prasanna1* and D. Hoisington2
1
Division of Genetics, Indian Agricultural Research Institute, New Delhi 110012, India
2
International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641, 06600 Mexico, D.F., Mexico

The maize genome is one of the most extensively analyzed among the plant genomes. Consequently, maize has
been at the forefront in development and evaluation of an array of molecular markers for varied purposes in genetics
and breeding. Besides the well-demonstrated utility of molecular markers in genotype differentiation and analysis of
genetic diversity in maize germplasm, application of DNA-based markers is also of considerable significance to
tropical/sub-tropical maize production systems, such as in India, for mapping and marker-assisted selection for
resistance to major biotic/abiotic stresses affecting production and productivity. Significant impetus in this direction
has been provided in recent years through the Asian Maize Biotechnology Network (AMBIONET). This article
provides an overview of the recent efforts under AMBIONET in relation to: (i) the molecular characterization of
inbred lines developed by various public sector institutions in India; (ii) the analysis of genetic diversity in the Indian
maize germplasm using microsatellite markers; and (iii) the mapping of quantitative trait loci conferring resistance
to different downy mildews affecting maize in tropical Asia. Judicious integration of conventional and molecular
approaches in maize breeding programmes is vital for efficient utilization of genetic resources, and improving the
production and post-harvest characteristics of the elite germplasm. This shall, in turn, require further strengthening
of synergistic linkages and partnerships among national and international research institutions to harness the
rapidly emerging information and technologies related to molecular breeding in maize.

Keywords: Zea mays, markers, fingerprinting, genome mapping, marker-assisted selection

Introduction challenges are diverse and complex, and there is no

Maize (Zea mays Linn.) holds a unique position in single technological solution. While significant
world agriculture as a food, feed and industrial crop progress has been made in relation to maize
par excellence. Although the developed countries, improvement in India using traditional breeding
particularly USA, contribute predominantly to the strategies (Dhillon & Prasanna, 2001), considerable
maize production, demand for maize in developing scope exists to further enhance maize productivity.
countries is expected to surpass the demand for both Modern molecular tools and techniques can
wheat and rice by the year 2020 (Pingali & Pandey, complement conventional approaches to allow
2001). However, average productivity of maize in breeders to effectively address priority research areas.
several developing countries is still considerably low. The term ‘molecular breeding’ is now popularly
About 45 million hectares of maize is grown in the used for the utilization of molecular (DNA-based)
lowland tropics, where a range of climatic, biotic and tools, including markers, to enhance the efficiency of
abiotic constraints severely affect productivity. The the breeding process. DNA markers have the potential
____________ to aid plant breeding programmes through diverse
*Author for correspondence: ways, such as fingerprinting of elite genetic stocks,
Tel: 25824285; Fax: 25766420 analysis of genetic diversity, and increasing the
E-mail: prasanna@ndf.vsnl.net.in
efficiency of selection for difficult traits. Among the
Abbreviations
AFLP: Amplified fragment length polymorphism; EST: array of DNA-based markers available to plant
Expressed sequence tag; GS: Genetic similarity; MAS: Marker- scientists, the ones most commonly used are RFLPs,
assisted selection; PAGE: Polyacrylamide gel electrophoresis; RAPDs, SSRs and AFLPs. Excellent reviews are
PCR: Polymerase chain reaction; PIC: Polymorphism information available discussing the genetic bases of various
content; QPM: Quality protein maize; QTL: Quantitative trait
loci; RAPD: Random amplified polymorphic DNA; RFLP: DNA-based markers, the means for detecting
Restriction fragment length polymorphism; RILs: Recombinant molecular polymorphism, and the strengths and
inbred lines; SNP: Single nucleotide polymorphism; SSR: Simple constraints associated with different markers for
sequence repeats
 PRASANNA & HOISINGTON: MOLECULAR BREEDING IN MAIZE

various applications (Karp et al, 1997; Liu, 2002). basic and applied research areas relevant to
The most appropriate marker(s) for a particular agricultural production systems (Mohan et al, 1997;
application will depend on the target crop, it’s Prioul et al, 1997). DNA fingerprinting and genetic
breeding behaviour, specific objectives of the diversity analysis using molecular markers is of
experiment, the resolution required, and the significant utility in effective management of
operational/financial constraints, if any. A comparison germplasm collections (Warburton & Hoisington,
of the various marker systems in relation to their 2001). Increasing emphasis is also being placed on
characteristics and applicability is provided in Table comprehensive analysis of genetic diversity in
1. For example, for genetic linkage map development, breeding materials of major crops (Mohammadi &
any type of molecular marker may be used. However, Prasanna, 2002). Accurate assessment of the levels
codominant markers (e.g., RFLPs, SSRs or SNPs), and patterns of genetic diversity using molecular
provide more genetic information in F2 and backcross markers is particularly helpful in maize breeding for
generations than markers detecting predominantly (i) maintenance and broadening of the genetic base of
presence/absence or dominant polymorphisms (e.g., the elite germplasm; (ii) assignment of lines to
RAPDs or AFLPs). For comparative mapping within heterotic groups; (iii) selection of appropriate parental
and across crop species, the use of RFLP as anchor lines for hybrid combinations; and (iv) generation of
loci are the best choice as they detect evolutionarily segregating progenies with maximum genetic
conserved loci in a more predictable manner than loci variability for further selection.
detected by hypervariable SSRs and AFLPs. DNA-based markers are also being used to discover
Among the different types of PCR-based DNA and exploit the evolutionary relationships between
markers available for diverse applications in maize various genera within a family (e.g., the grass family,
breeding, SSR markers are often preferred for reasons Poaceae), and various species within a genus. Genetic
of cost, simplicity and effectiveness. SSR markers are mapping of members of the agriculturally-important
robust, codominant, hypervariable, abundant, and grasses, including rice, wheat, maize, sorghum and
uniformly dispersed in plant genomes (Powell et al, sugarcane, with common DNA probes has revealed
1996a,b). In maize, more than 1000 mapped SSR remarkable conservation of gene content and gene
markers are available in the public domain (MaizeDB; order (Devos & Gale, 2000), reinforcing the paradigm
http://www.agron.missouri.edu). Mogg et al (1999) of the “grasses as a single genetic system” (Bennetzen
showed that by sequencing the flanking regions of & Freeling, 1993; Freeling, 2001). Comparative
maize microsatellites, a SNP could be found every 40 genomics has significant implications for the
bp. Given that the maize genome is estimated to be application of genetic information generated in one
2.5 x 109 bp in size, there is a potential for up to 62 member of the grass family (such as rice or maize or
million SNPs in maize. With the recent initiation of a sorghum) to the potential improvement of
large-scale EST sequencing programme in maize agronomically important traits in other members.
(http://www.zmdb.iastate.edu/zmdb/EST_project.html), This article provides a brief overview of (i) the
a new and potentially rich source of SNPs has been application of molecular markers to characterize
uncovered (Edwards & Mogg, 2001). While both maize germplasm and analyze genetic diversity; and
SSRs and SNPs can be reliably applied on a large (ii) the mapping and marker-assisted selection for
scale with only small quantities of DNA required for specific agronomically important traits in maize. In
PCR amplification, SNPs are highly amenable for discussing the above, the focus will be on the recent
automation, and therefore, offer significant studies undertaken under the AMBIONET
advantages for plant breeding purposes. SSRs, programme in India, and the work being carried out at
however, are the preferred choice when codominant, CIMMYT’s Applied Biotechnology Center in
multiallelic information is required, or when the Mexico.
infrastructure and resources are limited.
Molecular Profiling of Maize Germplasm
Applications of Molecular Markers Maize breeders in India, as in most developing
Molecular markers are increasingly being adopted countries, have differentiated inbred lines mainly on
by researchers involved in crop improvement as an the basis of major morphological characters such as
effective and appropriate tool for addressing several plant height, anthocyanin colouration of various plant
 PRASANNA & HOISINGTON: MOLECULAR BREEDING IN MAIZE

parts, tassel type, tassel branching, days to flowering, descriptors’ (qualitative or visually assessed
ear characters, cob colouration, grain colour and grain quantitative characters) in 47 Indian inbred lines to
type (Virk & Witcombe, 1997). Although ascertain their utility in effective differentiation of
morphological descriptions are important for genotypes. The ‘categorical’ descriptors, however,
ascertaining the agronomic utility of germplasm, such revealed very low polymorphism in the Indian lines
descriptions are not very reliable because of complex analyzed, with a total of only 55 variants, highlighting
‘genotype x environment’ interactions that require the severe limitations of utilizing only morphological
assessment in multiple locations/environments (Smith data for establishing the identity or distinctness of
& Smith, 1989). Detailed studies in various crop genotypes. In contrast, molecular profiling of 69
species, particularly in maize, have established that inbred lines, comprising 58 Indian lines, 6 CIMMYT
methods that solely depend on morphological data are lines developed at Mexico (used as ‘reference
neither consistent nor effective in unambiguous genotypes’), and 5 lines from the CIMMYT-Asian
differentiation of elite breeding materials (Smith & Regional Maize Programme (CIMMYT-ARMP),
Smith, 1988, 1989; Bar-Hen et al, 1995). Genetic Thailand, using 58 polymorphic SSR markers
heterogeneity, different combinations of alleles revealed high levels of polymorphism (435 alleles)
producing similar phenotypes, and environmental (Fig. 1A). Identification of 109 unique/rare SSR
influence on genotypes, result in morphological alleles (found in not more than 1 or 2 of the genotypes
similarities or differences that may not be analyzed) facilitated effective discrimination of the
proportional to the underlying genetic differences. genotypes analyzed. The high level of polymorphism
In the past, isozyme and zein chromatographic data displayed by the SSR loci was also reflected by the
(Stuber & Goodman, 1983; Smith, 1988) have been average PIC value (0.70). On the basis of high PIC
used to characterize elite inbred lines and commercial values (>0.75) and distinct allelic size ranges, SSR
hybrids of maize (Bar-Hen et al, 1995). Isozyme markers such as dupssr17, bnlg1647, and bnlg198,
analysis is relatively simple and less costly in could be effectively used in differentiating the Indian
comparison with molecular marker analysis; however, maize inbred lines. Distinct and non-overlapping size
inadequate genomic coverage, relatively low levels of ranges of the amplification products of SSR loci with
polymorphism, developmental regulation and high PIC would also facilitate multiplexing for
pleiotropic effects impose major constraints in improving the assay efficiency (Fig. 1B), as suggested
effectively using these markers in genotype by Mitchell et al (1997).
differentiation and analysis of genetic diversity The AMBIONET study also revealed high level of
(Smith & Smith, 1986; Dubreuil et al, 1996). In SSR heterozygosity in some of the Indian maize
recent years, PCR-based SSR markers have been inbred lines. There could be various reasons including
effectively used for differentiation of US and residual heterozygosity due to inadequate cycles of
European maize germplasm, as they are particularly inbreeding, improper pollination control during seed
suited for genotype discrimination (Smith et al, 1997; multiplication, seed stock contamination or
Warburton et al, 2002). accumulation of mutations at diverse SSR loci.
In India, no systematic efforts were made to Amplification of similar sequences in different
effectively apply molecular markers for genetic genomic regions due to duplications is another
fingerprinting or analysis of genetic diversity in the possible reason for occurrence of double-band
maize inbred lines developed by public sector phenotypes. High levels of heterozygosity for some of
institutions, including those that are commonly used the inbred lines such as CM115, CM117, CM123,
for hybrid maize breeding. Recently, however, studies CM124, CM205 and CM208 were revealed earlier
have been carried out to profile a selected set of through isozyme analysis (Mauria et al, 2000). SSR
Indian maize genotypes, including inbred lines and profiling of these inbreds confirmed the above
single-cross hybrids, using both morphological and observation. However, some inbreds such as CM111
microsatellite markers, and to analyze the genetic and CM116, which were considered as 'genetically
diversity in the maize inbred lines that are commonly pure' based on isozyme analysis (Mauria et al, 2000),
used in the public sector institutions (Pushpavalli et showed high levels of heterozygosity in SSR analysis.
al, 2001, 2002; Mohammadi et al, 2002a). Such incongruities in data derived from biochemical
Observations were recorded on 20 ‘categorical versus DNA-based markers could result due to the
 PRASANNA & HOISINGTON: MOLECULAR BREEDING IN MAIZE

low level of isozyme polymorphisms and limited Indian maize germplasm provided valuable
genomic coverage. information about genetic relationships in the
A sequential method combining marker information breeding materials (Pushpavalli et al, 2001, 2002;
and agro-morphological description, proposed by Mohammadi et al, 2002a,b). Analysis of SSR allele
Smith et al (1991) suggests: (i) comparison of two frequencies revealed a reasonably broad genetic base
lines at marker loci and declaring them distinct only if in the Indian maize germplasm. However, much of
their genetic similarity (GS) value is below a the SSR allelic variation in the inbred lines analyzed
predetermined threshold; (ii) comparison of the two was contributed by the inbred lines developed at
lines for agro-morphological traits only if their GS Punjab Agricultural University (PAU), Ludhiana.
value is beyond this threshold. In the second step, the Cluster analysis of the genetic dissimilarity matrix for
environmental variation for the morphological traits the genotypes under study using Ward’s method,
allows construction of statistical tests to determine the besides application of ‘pattern-finding methods’ such
'minimum distance' between the two inbreds, which as principal coordinate analysis, aided in determining
assumes considerable significance in the context of genetic relationships, which were broadly in
plant variety protection. It would be interesting to agreement with the available pedigree data.
ascertain the broader applicability and effectiveness The cluster pattern based on the AMBIONET study
of this proposal for routine profiling of elite breeding revealed essentially three main groups having two
materials. sub-clusters each. The majority of the Indian inbred
With the availability of high throughput lines that were derived earlier from the Colombian
technologies that can make use of fluorescent-labeled germplasm (CM111, CM114, CM120, CM300) were
SSR markers through multiplexing, fingerprinting has clustered in Group I, while some Colombian lines
been extended to classification of genetically diverse (CM104, CM105 and CM115) were placed in Group
materials such as landraces, populations, open- II. Almost all of the early-maturing inbred lines
pollinated varieties, and germplasm accessions. developed at IARI, New Delhi, such as CM135,
Earlier studies on characterization of populations have CM136, CM137 and CM138 clustered in Group II.
relied on only a few individuals per population, as the Some of the inbreds developed at PAU, Ludhiana,
cost and time required to characterize each line tends and analyzed in this study (CM122, CM123, CM124,
to be the limiting factor. The efficiency and accuracy CM125, CM140) clustered in Group II. The validity
of population fingerprinting can be enhanced by using of the clusters was reflected when the patterns were
a bulking strategy for individuals of a specific analyzed in relation to the well-known pedigree
population, followed by analysis of the bulks using information for some inbred lines, particularly those
multiplexed SSR primers and semi-automated DNA developed by PAU, Ludhiana (Dhillon et al, 1998;
sequencing technology. A set of 7 tropical maize Saxena et al, 2000). For instance, based on pedigree
populations and 57 inbred lines at CIMMYT were data, CM122 must be highly related to CM140, and
recently fingerprinted using 85 multiplexed SSR both these genotypes should also show close genetic
primers, leading to identification of 53 highly relationship with CM124 and CM125. These expected
discriminatory SSR markers (Warburton et al, 2002). genetic relationships were clearly validated by the
results of various clustering procedures that formed
Analysis of Genetic Diversity in Indian Maize the basis for the consensus cluster pattern. Group II
Germplasm using Molecular Markers also included CM202 and its close derivatives,
Pedigree information provides a broad estimate of CM211 and CM208. These inbred lines also show
the expected genetic relatedness among lines, but for close genetic association primarily with CM122 and
allogamous crops such as maize, such information is CM140. This could be due to the fact that CM202 was
often unobtainable or unreliable especially when extensively utilized in the derivation of Makki Safed
inbred lines were derived from a broad base Pool C4, which also comprises CM122 and CM140.
population (Melchinger et al, 1991; Messmer et al, The study indicated the genetic distinctness of some
1993). DNA-based markers, particularly SSRs and of the Ludhiana inbred lines (LM5, LM6 and
AFLPs, have provided powerful tools for analyzing CM139), as they were placed in a distinct cluster
genetic diversity (Pejic et al, 1998; Vuylsteke et al, (Group III). LM5 and LM6, parental lines of the
2000). AMBIONET studies on molecular profiling of single-cross hybrid ‘Paras’, were derived from
 PRASANNA & HOISINGTON: MOLECULAR BREEDING IN MAIZE

Tuxpeno Pool and Makki Safed Pool C2, respectively. markers. Polymorphic profiles for 36 SSR loci have
Suwan-1, developed by Kasetsart University, aided in effectively differentiating the QPM inbred
Thailand, had also contributed to the development of lines. The study resulted in identification of SSR
Tuxpeno Pool (Dhillon et al, 1998; Saxena et al, markers, such as bnlg439, phi037, bnlg125, dupssr34
2000). AMB109, a line developed from Thailand and bnlg105, with high polymorphism information
germplasm (AMATL line) has shown close content in the selected QPM genotypes. Analysis
relationship with LM5, while other AMB lines (which using SSR markers indicated high levels of
mainly includes lines from Thailand and Philippines) heterozygosity in majority of the Indian QPM lines
displayed closer relationship with LM6 and CM139. and in one CIMMYT QPM inbred, CML188. Cluster
The semi-exotic Pools A and B have close analysis using SSR data, followed by canonical
correspondence with Makki Safed and Tuxpeno discriminant analysis, clearly distinguished the Indian
Pools, respectively. This was also reflected in the QPM inbreds from those developed at CIMMYT
placement of CM139 between LM5 (from Tuxpeno (Kassahun & Prasanna, 2003). The cluster patterns
Pool) and LM6 (from Makki Safed Pool). The were largely in congruence with the available
accuracy of the cluster pattern derived from this study pedigree information of the QPM inbreds studied. The
was reflected by the grouping of some of the BIO study demonstrated the utility of SSR markers in
lines, which served as ‘controls’. For instance, BIO5 analysis of genetic relationships among QPM lines,
(an advanced line from LM5) grouped closely with and shall aid in planned utilization of CIMMYT QPM
LM5; similarly, BIO7 (an advanced line from LM6) lines in QPM breeding programmes being undertaken
with LM6, and BIO2 (an advanced line from CM211) in India.
with CM211. One of the potential applications of molecular
The AMBIONET study in India also indicated close marker data of inbred lines is to identify parents
genetic associations among some downy mildew- useful for developing or improving single-cross
resistant lines such as AMB112, AMB119, AMB115 hybrid performance. Although it is unlikely that the
and AMB109 (obtained from the CIMMYT-ARMP, markers such as SSRs affect the phenotypic
Thailand) and NAI116, an unreleased Indian inbred expression of the targeted quantitative trait(s) directly,
that is highly resistant to sorghum downy mildew they can serve to identify adjacent (linked) genomic
(SDM; Peronosclerospora sorghi) and Rajasthan segments. In such a case, marker divergence of inbred
downy mildew (P. heteropogoni) (Nair et al, 2001). lines can be useful to predict hybrid performance.
This can be attributed to the utilization of SDM- This is of particular value in crops like maize where
resistant germplasm from Thailand in the significant effort and resources are devoted to field-
development of NAI116 and some AMB lines (e.g., testing of newly created lines in various single-cross
AMB112). The close clustering of the CIMMYT- combinations to identify lines with superior
ARMP lines and their genetic distinctness from a combining ability. A number of studies have been
majority of the Indian maize lines highlights the carried out in maize to ascertain the association
possibility for further expansion of the genetic base of between molecular marker divergence and hybrid
Indian maize germplasm through efficient use of these performance, leading to different results (Stuber et al,
genotypes. In contrast to the CIMMYT-ARMP lines, 1999). Majority of the studies, however, indicate that
the six inbred lines from CIMMYT, Mexico, showed genotypic differences may be useful for preliminary
dispersion in various clusters. The AMBIONET study selection of loci/alleles for possible improvement of
serves as an effective foundation for further analysis hybrids (Mohammadi et al, 2002b), but probably will
of genetic relationships of the inbred lines being not accurately reflect performance of a hybrid. Field
developed by the National Agricultural Research evaluation of nearly 92 hybrid combinations derived
System (NARS). from 48 Indian maize inbred lines in three
In another recent study at IARI, a set of 23 QPM seasons/environments, recently carried out by the
lines, including 13 inbreds developed by the national AMBIONET-India team, in conjunction with the SSR
programme as well as 10 selected tropical/sub- allele data for these inbred lines, indicated that
tropical QPM lines developed by CIMMYT were molecular marker divergence is not significantly
analyzed for their grain quality, agronomic correlated with the hybrid performance, reinforcing
performance and molecular polymorphism using SSR the conclusion made above. A consensus opinion is
 PRASANNA & HOISINGTON: MOLECULAR BREEDING IN MAIZE

now emerging on this key issue that: (i) the genome Mapping QTL Influencing Resistance to Downy
should be well-saturated with uniformly spaced Mildews in Asia A Case Study
markers and/or high level of linkage equilibrium must Downy mildews occur predominantly in
exist for marker data to be reasonably successful in tropical/sub-tropical regions of China, India,
predicting hybrid performance; and (ii) marker data Indonesia, Japan, Nepal, Pakistan, Philippines and
can be more useful for predicting hybrid performance Thailand in Asia, where the disease is an important
of lines that are related and from a narrow genetic factor limiting maize production (Pingali & Pandey,
base than those derived from highly divergent genetic 2001). The major downy mildews that infect maize in
backgrounds. the region include the sorghum downy mildew
[Peronosclerospora sorghi (Weston & Uppal)],
QTL Mapping in Maize Philippine downy mildew (P. philippinensis [Weston]
The discovery of extensive, yet easily visualized, Shaw), Java downy mildew [P. maydis (Raciborski)],
variability at the DNA level, coupled with the sugarcane downy mildew [P. sacchari (Miyabe)
development of statistical packages that can help in Shirai & Hara], and brown stripe downy mildew
analyzing variation in a quantitative trait in [Scleropthora rayssiae var. zeae Payak & Renfro].
congruence with molecular marker data in a While P. sorghi causes downy mildew in both
segregating population, led to mapping of QTL sorghum and maize, the maize strain of P. sorghi in
influencing an array of agronomically important traits Thailand, which is now reclassified as P. zeae, rarely
in diverse crop plants including maize. A QTL may infects sorghum. Different types of downy mildew are
be defined as a region of the genome that is reported in India, including sorghum downy mildew,
associated with an effect on a quantitative trait. brown stripe downy mildew and sugarcane downy
Conceptually, a QTL can be a single gene, or a cluster mildew. In Rajasthan (India), the downy mildew
of tightly linked genes that affect the trait. Excellent pathogen which forms oospores in the wild grass,
reviews dealing with various aspects of QTL mapping Heteropogon contortus (speargrass) was renamed P.
in crop plants are available (Beavis, 1998; Liu, 2002; heteropogoni (Siradhana et al, 1980) and the disease,
Hackett, 2002). QTL mapping and identification of caused when maize is infected by the conidial stage of
molecular markers closely linked to QTL with major the fungus, is now referred to as Rajasthan downy
effects on a target trait can permit MAS in backcross, mildew (White, 1999). Despite the introduction of
pedigree, and population improvement programmes downy mildew resistant cultivars and the use of
(Young, 1999; Ribaut et al, 2002a,b). This is metalaxyl fungicide, severe incidence of the downy
especially useful for crop traits that are otherwise mildews still occurs in localized areas (Dalmacio,
difficult or impossible to select for by conventional 2000). Cost concerns related to seed treatment with
means. fungicide, and the emerging problem of chemical
Molecular markers have been used to identify and resistance build-up in the pathogen (Raymundo,
characterize QTL associated with diverse traits in 2000), point to the use of resistant varieties as a more
maize including grain yield, characters concerned cost-effective and environmentally-safe alternative in
with domestication, environmental adaptation, disease controlling this disease.
and insect pest resistance, and drought and heat stress Identification of molecular markers linked to downy
tolerance (Stuber, 1995; Stuber et al, 1999). mildew resistance genes should have a major impact
Comprehensive information about such experiments on maize breeding across the tropical Asian region.
can be obtained from the MaizeDB As a Network activity under the AMBIONET
(http://agron.missouri.edu). A case study with programme, four countries (India, Indonesia, Thailand
potential utility in effective management of downy and Philippines) undertook a QTL mapping project
mildew diseases in maize in tropical Asian countries aimed at identifying downy mildew resistance genes
is discussed below. (George et al, 2002). The mapping was based on
evaluation of a set of recombinant inbred lines (RILs)
derived from the cross of Ki3 (resistant) by CML139
(susceptible). The downy mildew resistant parent,
Ki3, is a tropical yellow flint line with late maturity
from Suwan-1, a cultivar developed in Thailand
 PRASANNA & HOISINGTON: MOLECULAR BREEDING IN MAIZE

against P. zeae. The susceptible parent, CML139, is a The proportion of the phenotypic variance
subtropical yellow-red semi-flint line with explained by each of the five QTL (R2 values) varied
intermediate maturity, developed from CIMMYT across environments. Collectively, the five QTL
materials for tropical corn borer resistance. Groh et al identified in this study explained phenotypic variation
(1998) constructed a molecular map using 135 RILs in disease susceptibility ranging from 24% (Thailand)
developed from the Ki3 x CML139 cross and 143 to 54% (Udaipur, India). Most significantly, the QTL
RFLP markers, for QTL mapping of southwestern on chromosome 6 had the largest contribution,
corn borer (SWCB) resistance. In the AMBIONET accounting for nearly 20% and 31% of the phenotypic
study, the same 135 RIL families were evaluated for variance for P. sorghi and P. heteropogoni disease
downy mildew reaction (during 2000-2001) at susceptibility at Mandya and Udaipur, respectively,
Mandya in southern India against sorghum downy and explaining more than half of the total phenotypic
mildew (P. sorghi); at Udaipur in western India variance due to the five QTL in each of the four
against Rajasthan downy mildew (P. heteropogoni); environments. Significant QTL x E interactions and
at Maros in Indonesia against Java downy mildew (P. large estimates of σ2ge observed across the locations
maydis); at Farm Suwan in Thailand against sorghum indicated major influence of the environment,
downy mildew (P. zeae); and at Southern Mindanao particularly the characteristic pathogen populations on
in Philippines against Philippine downy mildew (P. the expression of downy mildew resistance.
philippinensis). The phenotypic data, thus, comprised Significantly, the major QTL on chromosome 6 (bin
downy mildew disease incidence data from individual 6.05) is located in a region holding clusters of
environments as well as pooled data across resistance genes in maize. Groh et al (1998) identified
environments. Composite interval mapping (Zeng, a QTL in Ki3 at an adjacent region on chromosome 6
1994) was carried out for joint analysis of data across conferring resistance to leaf feeding damage caused
environments to map QTL and to estimate their by south western corn borer. Other genes that have
genetic effects. been located on chromosome 6 in bin 6.01 include
The AMBIONET study led to the identification of mdm1 which confers resistance to the maize dwarf
QTL with significant effects on resistance to the five mosaic virus (MDMV) (Simcox et al, 1995); wsm1
important downy mildew diseases affecting maize which confers resistance to a related potyvirus, wheat
production in the Asian region. The QTL that were streak mosaic virus (WSMV) (McMullen & Louie,
detected highlighted differences in the pathogen 1991); rhm1, which confers resistance to the fungal
populations that characterize the four locations. pathogen Cochliolobus heterostrophus (Zaitlin et al,
Three QTL, two on chromosome 2 and one on 1993); and a QTL conferring resistance to sugarcane
chromosome 7, significantly influenced resistance mosaic virus (SCMV) (S. Zhang & X. Li, personal
only to particular pathogen populations. The first communication).
QTL on chromosome 2 (position 158 cM), with Selection for QTL using genetic markers can be
resistance due to alleles from the susceptible parent effective if a significant association is found between
CML139, was specific to sorghum downy mildew at the quantitative trait and the genetic markers. The
Mandya. The second QTL on chromosome 2 (position AMBIONET study identified three SSR markers
234 cM) and the QTL on chromosome 7, with umc11, umc23a, and umc113 tightly linked to the
resistance due to alleles from the resistant parent Ki3, QTL on chromosome 6 (George et al, 2002),
were found to influence specifically P. heteropogoni indicating their possible use for MAS. Beyond their
at Udaipur in India. The most important genomic possible use in MAS, another potential application of
region, having the highest LR values in the analysis of these results would be the identification and analysis
data from individual locations as well as in the joint of candidate genes to deduce information about the
analysis, and having a consistent expression against nature and function of the detected gene(s) in
the different downy mildews, was found on determining resistance to downy mildews in Asia.
chromosome 6. This QTL was consistently expressed Chances of successful application of MAS for
across environments despite the significant effect of downy mildew resistance are better when QTL are
the environments having distinct pathogen identified in the germplasm used in the national
populations. breeding programme. For this purpose, the
AMBIONET-India team also screened nearly 80
 PRASANNA & HOISINGTON: MOLECULAR BREEDING IN MAIZE

inbred lines, including 50 Indian genotypes, against P. MAS is now being routinely applied in the breeding
sorghi and P. heteropogoni at Mandya and Udaipur, programmes of several crops, including maize, for (1)
respectively. The study led to the identification of tracing favourable alleles in the genomic background
NAI116, an excellent source of resistance against of genotypes of interest; and (2) identifying individual
both the downy mildew diseases in India (Nair et al, plants in large segregating populations that carry the
2001). A backcross mapping population was favourable alleles. For instance, two of the prominent
developed using CM139 (elite susceptible inbred) and examples of utilization of molecular markers in line
NAI116. Analysis of the genotypic and phenotypic conversions through a BC approach being practiced in
data from this mapping population (Fig. 2 A & B) maize at CIMMYT are: (1) introgression of the
confirmed the effect of the major QTL detected on opaque2 (o2) gene on chromosome 7 for the
chromosome 6 (bin location 6.05) by the earlier development of QPM lines, and (ii) transfer of a
AMBIONET study of the RILs. Introgression of this major QTL identified on the short arm of
major QTL governing resistance to downy mildews chromosome 1 that is associated with maize streak
into CM139 using marker-assisted backcrossing is virus (MSV) resistance. The utility of MAS in QPM
now ready to be undertaken. breeding is particularly worth discussing as QPM has
considerable relevance to various maize-growing
Marker-assisted Selection (MAS) in Maize Breeding countries, particularly in the developing world,
MAS consists of identifying associations between including India.
markers and alleles of the gene/QTL of interest, and The maize grain accounts for about 15% to 56% of
then using these associations to develop improved the total daily calories in diets of people in about 25
lines or populations (Ribaut & Hoisington, 1998; developing countries, particularly in Africa and Latin
Knapp, 1998). Through marker-assisted backcrossing, America (FAO Agrostat, 1992), where animal protein
individuals can be backcrossed until they contain the is scarce and expensive and consequently, unavailable
particular genomic segment in the genetic background to a vast sector of the population. Maize seed-protein
of the recipient or recurrent parent. For MAS to be quality can be improved by selecting for the
effective, recombination between the marker and the homozygous recessive o2 allele state (Mertz et al,
gene/QTL must be minimal. This is achieved using 1964). The presence of the homozygous o2 allele state
closely linked flanking markers. is correlated with changes in the amino acid balance
The basic purpose of marker-assisted backcrossing within the endosperm, and more specifically, a
is to speed up line conversion, and to reduce the favourable increase in the proportion of lysine and
linkage drag of the transferred gene(s). Through tryptophan. Cloning and sequencing of the o2 gene
classical backcross (BC) breeding, the transfer of a (Schmidt et al, 1990) allowed detection of three SSR
single dominant gene would require six BC markers (phi057, phi112 and umc1066) within the
generations to recover 99% of the recurrent parent sequence of the gene itself. CIMMYT has been
genome. This procedure is time-consuming and routinely screening thousands of genotypes, using
labour-intensive for breeding of crops such as maize, these three SSRs, in segregating populations to
where turnover times of new lines and hybrids are identify genotypes that have one copy of the o2
fast. In a BC1 generation the proportion of the mutant allele (BC strategy) and those that have two
recurrent parent genome would be distributed copies (self-pollination strategy). Selection is
normally around a mean of 75% (in later BC conducted before flowering to allow the pollination of
generations, the distribution would become only the selected plants. Integration of MAS for o2 is
increasingly skewed) but given a sufficient sample a relatively simple and effective strategy for
size, it would contain plants with more than 85% accelerating QPM development, and this strategy is
recurrent parent genome. These plants can be currently being employed in various countries
identified with molecular markers to accelerate the including India (Prasanna et al, 2001).
breeding process (Tanksley et al, 1989). Without MAS can also be of great relevance to improvement
molecular markers flanking the target gene it is nearly of polygenic traits. By combining the QTL approach
impossible to remove the linkage drag coming as (selection for favourable QTL effects) with
"baggage" with the introgressed segment (Murray et backcrossing, useful genes that control quantitative
al, 1988). traits can be identified and transferred to advanced
 PRASANNA & HOISINGTON: MOLECULAR BREEDING IN MAIZE

breeding lines (Lande & Thompson, 1990; Tanksley Integrating MAS in Maize Breeding
& Nelson, 1996; Hospital & Charcosset, 1997). In Despite a wealth of published literature on QTL
maize, Stuber et al (1992, 1999) mapped QTL mapping, particularly in recent years, a number of
associated with seven major traits (including grain constraints have imposed severe limitations on
yield), and were also able to generate improved effective utilization of QTL information in plant
versions of inbred lines using obsolete inbreds as breeding through MAS. Salient among these
donors. The efficiency of using molecular markers for constraints are: (i) identification of a limited number
improvement of polygenic traits in maize breeding of major QTLs controlling target traits; (ii)
programmes was also demonstrated in a few other inadequacies/experimental deficiencies in QTL
studies (Ribaut et al, 2002a). Despite these examples, analysis leading to overestimation/underestimation of
MAS for polygenic traits in maize, as in most other the number and effects of QTLs; (iii) lack of
crop plants, is still in its infancy. Manipulating QTL/marker associations applicable over different
quantitative traits is difficult due to the involvement sets of breeding material; (iv) strong QTL x
of a large number of genes involved in trait environment interaction; and (v) difficulty in
expression, often with varying effects, interactions precisely evaluating epistatic effects. Recently, novel
between the genes (epistasis), and QTL x strategies have been proposed (Ribaut & Betran,
environment interactions (Beavis & Keim, 1996). 1999; Ribaut et al, 2002b), particularly using maize as
This implies that several regions/QTL must be a model system, to overcome some of these major
manipulated simultaneously to have a significant constraints. The efficacy of such strategies in
impact, and that the effect of individual regions is not improving the efficiency of gene introgression using
easily identified. molecular markers, and reducing the cost of MAS
CIMMYT researchers have devoted considerable experiments, is being analyzed at CIMMYT.
efforts during the past three decades to improve pre- The cost-effectiveness of using molecular markers
and post-flowering drought tolerance in maize. (SSRs) in MAS experiments in maize was also
Although significant progress has been achieved for estimated (Dreher et al, 2000). The study revealed
improving drought tolerance in CIMMYT maize that when only a few SSR markers are used and when
germplasm through conventional breeding (Bänziger several hundred genotypes are screened, MAS is cost-
et al, 2000), the approach is slow and time- effective. Using SSR markers to select for the
consuming. Use of molecular markers and QTL opaque2 gene during QPM development exemplifies
information based on carefully managed replicated the utility of MAS as an efficient substitute for
tests has the potential to alleviate the problems phenotypic selection, considering the recessive nature
associated with inconsistent and unpredictable onset of the gene, absence of obvious visual selection due to
of moisture stress or the confounding effect of other the interaction of this gene with modifiers involved in
stresses such as heat. The approach was primarily kernel vitreousness or hardness (an essential character
based on breaking down the complex trait of drought in QPM), and greater cost per sample when the
tolerance into simpler components, and to manipulate endosperm protein quality is analyzed through
genomic regions related to components like anthesis- chemical analysis. The cost-effectiveness of MAS
silking interval (ASI) that are closely associated with over phenotypic selection, particularly for complex
drought tolerance. To this end, CIMMYT conducted polygenic traits, is also likely to improve in the future,
several experiments on QTL analysis and MAS for with the availability of more efficient and high-
transfer of drought tolerance to tropical maize, and throughput techniques for detection of molecular
obtained encouraging results. An integrated strategy polymorphism.
of QTL mapping, MAS and functional genomics is
now being explored to further provide useful Future Prospects
information and tools to effectively complement Understanding the complex web of interactions
conventional selection for drought tolerance in maize between genes and environmental factors, and
(Ribaut et al, 2002a). effective application of this information for
plant/animal improvement is a challenging endeavour
for biologists. To obtain relevant information, it is
imperative to exploit the tools of both classical and
 PRASANNA & HOISINGTON: MOLECULAR BREEDING IN MAIZE

molecular genetics. The developments in the recent mapping populations based on parents that are
years in relation to molecular marker technology and adapted to the Indian agricultural production
QTL analysis have allowed identification of genomic system(s). The data already developed under the
regions involved in an array of agronomically AMBIONET programme in relation to molecular
important traits in diverse crop species, particularly profiling of Indian maize inbred lines, besides agro-
maize. Such information is also providing clues to morphological data already available from the
better understand genome organization as well as breeders, provide valuable information in clearly
genetic phenomena such as epistasis, pleiotropy and understanding the genetic diversity in the inbred
heterosis. However, the impact of marker-based QTL genetic base, thereby permitting more efficient
analysis on varietal development has been less than utilization of genetic resources, both indigenous and
expected, primarily due to two reasons: (i) exotic, in breeding programmes. In addition,
experiments related to QTL discovery and varietal application of optimized molecular marker technology
development have largely been independent, as will reduce the costs of genetic resource conservation
pointed out by Tanksley & Nelson (1996); and (ii) for and further improve their utilization. Towards this
traits such as grain yield, QTL expression is usually goal, core collections of maize in India, that include
dependent upon the genetic background, unlike traits maximum genetic diversity and best represent
such as disease or insect resistance which are usually existing variation, must be developed.
less complex in comparison with grain yield (Stuber Cost-effective application of molecular marker
et al, 1999). Development of high-throughput, technology to agriculturally important problems in
reproducible molecular marker technologies, coupled India cannot be done in isolation. Researchers in India
with advances in genomics research, are now can immensely gain by building effective linkages
promising to offer powerful tools to maize researchers with partners elsewhere to harness the synergy of
for more effective integration and utilization of MAS collective efforts in molecular breeding, as
for diverse applications in breeding programmes. exemplified by AMBIONET. Networking can
Besides some highly encouraging developments in facilitate development of an integrated system for
molecular breeding, structural and functional efficient application of molecular tools and
genomics research in maize is progressing at a healthy techniques, including QTL mapping and MAS, in
pace (Stuber et al, 1999; Coe et al, 2002). From a maize breeding programmes of the NARS. This
time when the maize genome was considered to be would, in turn, significantly aid in development of
too complex to consider large-scale physical mapping improved germplasm, including cultivars, with greater
and sequencing, we have now reached a point where yield potential and ability to overcome major biotic
several research teams in the developed world are and abiotic constraints limiting maize productivity, in
striving to generate contig maps for maize and the minimum possible time and with minimal
determine the sequence and function of the several operational expenses. Collaborative research under
thousands of ESTs that are already identified (Coe et AMBIONET is presently focused on the application
al, 2002). The advances in maize functional of molecular marker technology to problems of
genomics, including gene expression profiling and national and regional importance, such as molecular
proteomics (Lee et al, 2002), should allow us in the characterization of locally important maize lines,
near future to identify the key genes as well as mapping of QTLs for resistance to major diseases –
pathways involved in expression of traits such as downy mildews, SCMV and Banded leaf and sheath
biotic/abiotic stress tolerance (Cushman & Bohnert, blight – and tolerance to abiotic stresses (drought and
2000; Seki et al, 2001). low nitrogen conditions), and integration of MAS in
The NARS in India have demonstrated the the breeding programmes.
commitment and capacity to effectively apply modern
biotechnology, particularly molecular markers, for Acknowledgements
crop improvement. In maize, we should focus Research work related to molecular profiling,
primarily on three areas: assessment of genetic genetic diversity analysis of Indian maize germplasm,
diversity, application of marker-assisted selection and genome mapping for downy mildew resistance
using previously identified QTL and their flanking was facilitated by CIMMYT, Mexico, under
markers, and development of multiple trait-targeted AMBIONET, with financial support from Asian
 PRASANNA & HOISINGTON: MOLECULAR BREEDING IN MAIZE

Development Bank. We express our gratitude to other Consortium Agric Biotechnol Res, August 25-28, Ravello,
AMBIONET teams (Indonesia, Thailand and Italy.
Dubreuil P, Dufour P, Krejci E, Causse M, De Vienne D, Gallais
Philippines) for sharing unpublished results from the A & Charcosset A, 1996. Organization of RFLP diversity
network activity on QTL mapping for downy mildew among inbred lines of maize representing the most
resistance. Special thanks to AMBIONET resource significant heterotic groups. Crop Sci, 36, 790-799.
personnel from CIMMYT and India, particularly Drs. Edwards K J & Mogg R, 2001. Plant genotyping by analysis of
single nucleotide polymorphisms. in Plant Genotyping: The
M.L.C. George (AMBIONET Coordinator), N.N. DNA Fingerprinting of Plants, edited by R J Henry. CABI
Singh, R.S. Rathore. T.A.S. Setty, and the past and Publishing, Wallingford, UK. Pp 1-13.
present members of the AMBIONET-India Lab at FAO Agrostat, 1992. Food Balance Sheets. FAO, Rome, Italy.
IARI, New Delhi, for sharing of data and for Freeling M, 2001.Grasses as a single genetic system.
dedicated support to AMBIONET-India research Reassessment 2001. Plant Physiol, 125, 1191-1197.
George M L, Prasanna B M, Rathore R S, Setty T A S, Singh N
activities. N, Kasim F, Azrai M, Vasal S, Balla O, Regalado E, Vargas
M, Khairallah M, Jeffers D & Hoisington D, 2002.
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 PRASANNA & HOISINGTON: MOLECULAR BREEDING IN MAIZE

Table 1Characteristics and utility of different molecular markers for applied molecular genetics in crop plants.

RFLPs RAPDs SSRs AFLPs SNPs

Fingerprinting ++ −/+ ++ ++ +++

Genetic diversity ++ − ++ + ?
Tagging qualitative genes ++ ++ + +++ ++
Mapping polygenic traits ++ −/+ + ++ ++
Marker-assisted selection ++ − ++ +/++ ++
Comparative genome ++ − − − −
mapping

Principle Endonuclease DNA amplification PCR of simple Endonuclease DNA

restriction; with random sequence repeats restriction; sequencing
Southern blotting primers amplification using
adapters and
specific primers
Type of polymorphism Single base changes; Single base changes; Changes in Single base changes; Single base
Insertions/Deletions Insertions/Deletions length Insertions/Deletions differences
of repeats
Genomic abundance High Very high Medium Very high Very high
Level of polymorphism Medium Medium High High Very high
Inheritance Codominant Dominant Codominant Dominant..? Codominant
Detection of allelic variants Yes No Yes No Yes
No. of loci detected 1-5 1-10 1 30-100 1
Need for sequence No No Yes No Yes
information
Technical difficulty Medium Low Low Medium/High Medium/High
Reliability High Intermediate High Medium/High High
Quantity of DNA required 2-15µg 10-50 ng 2-15 ng 2-15 ng 2-15 ng
Use of radioisotopes Yes/No No Yes/No Yes/No No
Probes/primers required gDNA/cDNA Random 9- or Specific 16- Specific adapters Specific
10-mer 30-mer and primers primers
oligonucleotides primers
Start-up costs Medium Low Medium High High
Development costs Medium Low High Medium/High Medium/High
 PRASANNA & HOISINGTON: MOLECULAR BREEDING IN MAIZE

Fig. 1(A) SSR polymorphism revealed by bnlg439 (A) in Indian maize inbred lines using PAGE and silver staining technology. Φx-
174/Hinf1 digest (size range of 66-726 bp) was used as molecular weight standard (M). (B) Polymorphism in selected Indian maize
inbreds revealed by multiplexed, fluorescent-labeled SSR primers, ph084 (a), nc130 (b), phi308707 (c), and phi089 (d); the methodology,
using semi-automated DNA sequencers, facilitates accurate sizing of SSR alleles, besides enhancing the assay efficiency.

Fig. 2Genotyping of a panel of BC1F1 mapping population (for QTL mapping of downy mildew resistance in maize), for two
polymorphic SSR loci, bnlg490 (A) and bnlg1655 (B); the mapping population was derived using CM139 (P1) and NAI116 (P2) used as
recurrent (susceptible) and donor (resistant) parents, respectively. A 100-bp ladder was used as the molecular size standard (M) for the
gels run on a super-fine resolution 3.5% agarose.