Report: Advancement of molecular markers and crop improvement in plant breeding

1. Progress in Agricultural Science and Technology
A Report
on
Advancement of molecular markers and crop
improvement in plant breeding
Submitted by:
Ripon Kumar Sikder, PhD Fellow
Institute of Cotton Research, CAAS
ID No. 2017Y90100144
Graduate School of Chinese Academy of Agricultural Sciences
12, Zhongguancun Nadajie, Haidian District, Beijing 100081, P. R. China

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Introduction
Plant Breeding is a dynamic area of applied science. It relies on genetic variation and uses
selection to gradually improve plants for traits and characteristics. The way of improvement is
the introduction of new genetic material (e.g. genes for disease resistance) from other sources,
such as gene bank accessions and related plant species. Although current breeding practices
have been very successful in producing a continuous range of improved varieties, recent
developments in the field of biotechnology and molecular biology can be employed to enhance
plant breeding efforts and to speed up the creation of cultivars.
Molecular breeding implies molecular marker-assisted breeding (MAB) and is defined as the
application of molecular biotechnologies, specifically molecular markers, in combination with
linkage maps and genomics, to alter and improve plant or animal traits on the basis of genotypic
assays. Genetic markers represent genetic differences between individual organisms or species.
Generally, they do not represent the target genes themselves but act as ‘signs’ or ‘flags’.
Genetic markers that are located in close proximity to genes (i.e. tightly linked) may be referred
to as gene ‘tags’. Such markers themselves do not affect the phenotype of the trait of interest
because they are located only near or ‘linked’ to genes controlling the trait. All genetic markers
occupy specific genomic positions within chromosomes (like genes) called ‘loci’ (singular
‘locus’).
Different Genetic Markers in Plant Breeding
Genetic markers are the biological features that are determined by allelic forms of genes or
genetic loci and can be transmitted from one generation to another, and thus they can be used
as experimental probes or tags to keep track of an individual, a tissue, a cell, a nucleus, a
chromosome or a gene. There are three major types of genetic markers:
(1) Morphological (also ‘classical’ or ‘visible’) markers which themselves are phenotypic traits
or characters. Morphological markers are usually visually characterized phenotypic characters
such as flower color, seed shape, growth habits or pigmentation.
(2) Biochemical markers, which include allelic variants of enzymes called isozymes. Isozyme
markers are differences in enzymes that are detected by electrophoresis and specific staining.
(3) DNA (or molecular) markers, which reveal sites of variation in DNA (Jones et al., 1997;
Winter & Kahl, 1995). DNA markers have developed into many systems based on different
polymorphism-detecting techniques or methods (southern blotting – nuclear acid hybridization,

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PCR – polymerase chain reaction, and DNA sequencing) (Collard et al., 2005), such as RFLP,
AFLP, RAPD, SSR, SNP, etc.
DNA markers are the most widely used type of marker predominantly due to their abundance.
They arise from different classes of DNA mutations such as substitution mutations (point
mutations), rearrangements (insertions or deletions) or errors in replication of tandemly
repeated DNA (Paterson, 1996a). These markers are selectively neutral because they are
usually located in non-coding regions of DNA. Unlike morphological and biochemical
markers, DNA markers are practically unlimited in number and are not affected by
environmental factors and/or the developmental stage of the plant (Winter & Kahl, 1995).
Apart from the use of DNA markers in the construction of linkage maps, they have numerous
applications in plant breeding such as assessing the level of genetic diversity within germplasm
and cultivar identity (Baird et al., 997; Henry, 1997; Jahufer et al., 2003; Weising et al., 1995;
Winter & Kahl, 1995).
DNA markers may be broadly divided into three classes based on the method of their detection:
(1) hybridization-based; (2) polymerase chain reaction (PCR)-based and (3) DNA sequence-
based (Gupta et al., 1999; Jones et al., 1997; Joshi et al., 1999; Winter & Kahl, 1995).
Essentially, DNA markers may reveal genetic differences that can be visualized by using a
technique called gel electrophoresis and staining with chemicals (ethidium bromide or silver)
or detection with radioactive or colorimetric probes. DNA markers are particularly useful if
they reveal differences between individuals of the same or different species. These markers are
called polymorphic markers, whereas markers that do not discriminate between genotypes are
called monomorphic markers. Polymorphic markers may also be described as co-dominant or
dominant.
Role of Molecular Markers in Crop Improvement
Conventional crop improvement has developed a large number of varieties. Genotypic and
environmental effects produce phenotypic values over genotypes in plants and other living
organisms. Strategies using best phenotypes to infer the best genotypes are commonly less
efficient than the schemes performing progeny tests. These strategies spend more time on
selection, and consequently it is necessary to invest major economic resources.

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On the other hand, it is convenient to recognize the effects of quantitative or qualitative genes
on phenotypes. In general, it can be assumed that if the substitution effect of an allele on the
phenotypic expression is small, the trait is classified as quantitative, in turn, if the effect is large
the characteristic is considered as qualitative. The selection using molecular markers can be
enhanced, and the efficiency of the molecular marker will be higher if the markers are closely
linked to the genes controlling quantitative and qualitative characters (Appels et al., 1998).
Progress in crop improvement has been reached by manipulating the genetic variation within
populations. Currently, novel molecular tools as the molecular markers have started to
demonstrate their usefulness into practical plant breeding facilitating the identification,
characterization, and manipulation of the genetic variation on important agronomic traits
(Sorrells and Wilson, 1997). Molecular markers are being used for crop improvement to
achieve different objectives such as measuring genetic diversity among corn lines. Results
indicated that when inbreed lines were unrelated, a measurement of relative relationship based
on proportion of homomorphic marker loci, was significantly correlated with a measure of
relationship based on yield. Conversely, when lines were related the correlations were low
(Dudley et al., 1991). Smith and Smith (1989) proposed that problems related to the property
and registration of lines and control of genetic purity in seed production can be solved by use
of molecular markers. SSR markers are being applied to identify and validate pedigree of
genotypes. For this purpose different lines of maize were compared by using SSR and RFLP
technologies and found that SSR markers have potential advantages of reliability,
reproducibility, discrimination, standardization, and cost of effectiveness over RFLPs (Smith
et al., 1997). All these uses have contributed to the modernization of plant improvement;
however, one of the most important uses has been the application of molecular markers to
increase effectiveness of selection. This application called Marker-Assisted Selection (MAS)
and playing important role in crop improvement.
Plant improvement, either by natural selection or through the efforts of breeders, has always
relied upon creating, evaluating and selecting the right combination of alleles. The
manipulation of a large number of genes is often required for improvement of even the simplest
of characteristics (Flavell, 1995). With the use of molecular markers it is now a routine to trace
valuable alleles in a segregating population and mapping them. These markers once mapped
enable dissection of the complex traits into component genetic units more precisely (Hayes,
1993), thus providing breeders with new tools to manage these complex units more efficiently
in a breeding program.

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The very first genome map in plants was reported in maize (Helentjaris et al., 1986 and
Gardiner et al. 1993), followed by rice (McCouch et al. 1988), Arabidopsis (Chang et al., 1988
and Nam et al., 1989) etc. using RFLP markers. Maps have since then been constructed for
several other crops like potato, barley, banana, members of Brassicaceae, etc (Winter et al.,
1995). Once the framework maps are generated, a large number of markers derived from
various techniques are used to saturate the maps as much as is possible. Microsatellite markers,
especially STMS markers, have been found to be extremely useful in this regard. Owing to
their quality of following clear Mendelian inheritance, they can be easily used in the
construction of index maps, which can provide an anchor or reference point for specific regions
of the genome. About 30 microsatellites have already been assigned to five linkage groups in
Arabidopsis, while their integration into the genetic linkage maps is still in progress in rice,
soybean, maize, etc. The very first attempt to map microsatellites in plants was made by Zhao
and Kochert (Zhao et al., 1992) in rice using (GGC)n, followed by mapping of (GA)n and
(GT)n by Tanksley et al and (GA/AG)n, (ATC) 10 and (ATT) 14, by Panaud et al., 1995 in
rice. The most recent microsatellite map has been generated by Milbourne et al., 1998 for
potato. Similar to microsatellites, looking at the pattern of variation, generated by
retrotransposons, it is now proposed that apart from genetic variability, these markers are ideal
for integrating genetic maps (Ellis et al., 1998).
Once mapped, these markers are efficiently employed in tagging several individual traits that
are extremely important for a breeding program like yield, disease resistance, stress tolerance,
seed quality, etc. A large number of monogenic and polygenic loci for various traits have been
identified in a number of plants, which are currently being exploited by breeders and molecular
biologists together, so as to make the dream of marker-assisted selection come true. Tagging
of useful genes like the ones responsible for conferring resistance to plant pathogen, synthesis
of plant hormones, drought tolerance and a variety of other important developmental pathway
genes, is a major target. Such tagged genes can also be used for detecting the presence of useful
genes in the new genotypes generated in a hybrid program or by other methods like
transformation, etc. RFLP markers have proved their importance as markers for gene tagging
and are very useful in locating and manipulating quantitative trait loci (QTL) in a number of
crops. The very first reports on gene tagging were from tomato (Paterson et al., 1988, Weller
et al., 1988 and Williamson, 1994), availing the means for identification of markers linked to
genes involved in several traits like water use efficiency (Martin et al., 1989), resistance
to Fusarium oxysporum (the 12 gene) (Sarfatti et al., 1989), leaf rust resistance genes LR 9 and

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24 (Schachermayr et al., 1994 and Schachermayr et al., 1995), and root knot nematodes
(Meliodogyne sp.) (mi gene) (Klein-Lankhorst et al., 1991 and Messeguer et al., 1991).
Recently, Xiao et al., 1998 have shown the utility of RFLP markers in identifying the trait
improving QTL alleles from wild rice relative O. rufipogon.
Allele-specific associated primers have also exhibited their utility in genotyping of allelic
variants of loci that result from both size differences and point mutations. Some of the genuine
examples of this are the waxy gene locus in maize (Shattuck-Eidens et al., 1991), the Glu D1
complex locus associated with bread making quality in wheat (D’Ovidio et al., 1994),
the Lr1 leaf rust resistance locus in wheat (Feuillet et al., 1995), the Gro1 and H1 alleles
conferring resistance to the root cyst nematode Globodera rostochiensis in potato (Niewohner
et al., 1995), and allele-specific amplification of polymorphic sites for detection of powdery
mildew resistance loci in cereals (Mohler et al., 1996). A number of other traits have been
tagged using ASAPs in tomato, lettuce, etc. (Olson et al., 1989, Klein-Lankhorst et al., 1991
and Paran et al., 1991). Besides ASAPs, AFLP and SSR markers have been identified to be
associated with quantitative resistance to Globodera pallida (stone) in tetraploid potato, which
can be very well employed in marker-assisted selection (Bradshaw et al., 1998).
STMS markers have displayed a potential use as diagnostic markers for important traits in plant
breeding programs, e.g. (AT) 15 repeat has been located within a soybean heat shock protein
gene which is about 0.5 cM from (Rsv) a gene conferring resistance to soybean mosaic virus
(Yu et al., 1994). Several resistance genes including peanut mottle virus (Rpv), phytopthera
(Rps3) and Javanese rootknot nematode are clustered in this region of the soybean genome.
Similar to specific markers like RFLPs, STMS and ASAPs, arbitrary markers like RAPDs have
also played important role in saturation of the genetic linkage maps and gene tagging. Their
use in mapping has been especially important in systems, where RFLPs have failed to reveal
much polymorphism that is so very important for mapping. One of the first uses of RAPD
markers in saturation of genetic maps was reported by Williams et al (Williams et al., 1991).
They have proven utility in construction of linkage maps among species where there has been
inherent difficulty in producing F2 segregating populations and have large genome size, e.g.
conifers (Carlson et al., 1991 & Chaparoo et al., 1992). RAPD markers in near isogenic lines
can be converted into SCARs and used as diagnostic markers. SCAR/STS marker linked to the
translocated segment on 4 AL of bread wheat carrying the Lr28 gene has been tagged by Naik et
al., 1998. Recently, ISSRs, which too belong to the arbitrary marker category, but are found to

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be devoid of many of the drawbacks shared by RAPD class of markers, have been employed
as a reliable tool for gene tagging. An ISSR marker (AG) 8YC has been found to be linked
closely (3.7 ± 1.1 cM) to the rice nuclear restorer gene, RF1 for fertility. RF1 is essential for
hybrid rice production and this marker would be useful not only for breeding both restorer and
maintainer lines, but also for the purity management of hybrid rice seeds (Akagi et al., 1996).
Similarly ISSR marker (AC)8 YT has been found to be linked to the gene for resistance to
fusarium wilt race 4 in repulsion at a distance of 5.2 cM in chickpea (Ratnaparkhe et al., 1998).
Apart from mapping and tagging of genes, an important utility of RFLP markers has been
observed in detecting gene introgression in a backcross breeding program (Jena et al., 1990),
and synteny mapping among closely related species (Gale et al., 1998). Similar utility of STMS
markers has been observed for reliable pre-selection in a marker assisted selection backcross
scheme (Ribaut et al 1997). Apart from specific markers, DAMD-based DNA fingerprinting
in wheat has also been useful for monitoring backcross-mediated genome introgression in
hexaploid wheat (Somers et al., 1996).
In plant breeding, MAS is a relatively new concept, nevertheless the original selection concept
has not changed, that is, the purpose of the selection is to search and preserve the best
genotypes, but using molecular markers. MAS can be used for manipulating both qualitative
and quantitative traits. A highly saturated marker linkage map is necessary for effective marker
based selection. Basically, MAS consists of identifying association between molecular markers
and genes controlling agronomic traits, and using these to improve lines or populations
(Dudley, 1993). Otherwise, based on the heritability of molecular markers, which is essentially
100 %, when the selection is performed for a low heritable trait, it will be more effective and
potentially less expensive than phenotypic selection (Winter and Kahl, 1995). Concerning
MAS applied to qualitative traits, Huang et al. (1997) using DNA marker-assisted selection
developed rice lines containing two, three, and four resistance genes to the bacterial blight
pathogen (Xanthomonas oryzae pv. orizae). Moreover, these researchers consider that a way
to maintain resistance in rice for long time is use of pyramiding, nevertheless the accumulation
of resistant genes and the epistasis and/or the masking effect of genes can be difficult or
impossible to manipulate in conventional plant improvement. A molecular marker was detected
in wheat associated with GBSS 4a locus, which affects the texture for Udon noodles.
Application of this result can reduce breeding cost for production of wheat varieties with this
characteristic (Briney et al., 1998).

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With respect to the application of MAS for selecting quantitative traits, some researchers have
used molecular markers to locate loci associated with quantitative characters. Osmotic
adjustments were identified to assist the selection for drought tolerance in rice by use of
molecular markers linked to root traits (Nguyen et al., 1997). Likewise in maize quantitative
trait loci (QTLs) for yield components under two water conditions and for drought tolerance
were detected by using molecular markers (Frova et al., 1999). In wheat utilizing RFLP
markers Shah et al. (1999) determined important association with QTLs influencing
characteristics such as plant height, 1000 kernel weight, and kernel number. spike-1
. On the
other hand, in maize crop QTLs have been detected for plant height and European corn borer
tunneling length (Lee et al., 1991), other morphological traits with polygenic control have also
been studied in corn for example; Godshalk et al. (1990) identified loci linked to grain yield,
moisture, ear height, and root lodging.
Marker-Based Breeding and Conventional Breeding: Challenges and Perspectives
Marker-assisted breeding became a new member in the family of plant breeding as various
types of molecular markers in crop plants were developed during the 1980s and 1990s. The
extensive use of molecular markers in various fields of plant science, e.g. germplasm
evaluation, genetic mapping, map-based gene discovery, characterization of traits and crop
improvement, has proven that molecular technology is a powerful and reliable tool in genetic
manipulation of agronomically important traits in crop plants. Compared with conventional
breeding methods, MAB has significant advantages:
 MAB can allow selection for all kinds of traits to be carried out at seedling stage and
thus reduce the time required before the phenotype of an individual plant is known. For
the traits that are expressed at later developmental stages, undesirable genotypes can be
quickly eliminated by MAS. This feature is particularly important and useful for some
breeding schemes such as backcrossing and recurrent selection, in which crossing with
or between selected individuals is required.
 MAB can be not affected by environment, thus allowing the selection to be performed
under any environmental conditions (e.g. greenhouse and off-season nurseries). For
low-heritability traits that are easily affected by environments, MAS based on reliable
markers tightly linked to the QTLs for traits of interest can be more effective and
produce greater progress than phenotypic selection.

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 MAB using co-dominance markers (e.g. SSR and SNP) can allow effective selection of
recessive alleles of desired traits in the heterozygous status. No selfing or test crossing
is needed to detect the traits controlled by recessive alleles, thus saving time and
accelerating breeding progress.
 For the traits controlled by multiple genes/QTLs, individual genes/QTLs can be
identified and selected in MAB at the same time and in the same individuals, and thus
MAB is particularly suitable for gene pyramiding. In traditional phenotypic selection,
however, to distinguish individual genes/loci is problematic as one gene may mask the
effect of additional genes.
 Genotypic assays based on molecular markers may be faster, cheaper and more accurate
than conventional phenotypic assays, depending on the traits and conditions, and thus
MAB may result in higher effectiveness and higher efficiency in terms of time,
resources and efforts saved.
However, MAS and MABC were and are primarily constrained to simply-inherited traits, such
as monogenic or oligogenic resistance to diseases/ pests, although quantitative traits were also
involved (Collard and Mackill, 2008; Segmagn et al., 2006; Wang and Chee, 2010). The
application of molecular markers in plant breeding has not achieved the results as expected
previously in terms of extent and success (e.g. release of commercial cultivars). Improvement
of most agronomic traits that are of complicated inheritance and economic importance like
yield and quality is still a great challenge for MAB including the newly developed GS. The
application of molecular technologies to plant breeding is still facing the following drawbacks
and/or challenges:
 Not all markers are breeder-friendly and not all markers can be applicable across
populations due to lack of marker polymorphism or reliable marker-trait
association.
 False selection may occur due to recombination between the markers and the genes/
QTLs of interest.
 Imprecise estimates of QTL locations and effects result in slower progress than
expected.
 A large number of breeding programs have not been equipped with adequate
facilities and conditions for a large-scale adoption of MAB in practice.

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 The methods and schemes of MAB must be easily understandable, acceptable and
implementable for plant breeders, unless they are not designed for a large scale use
in practical breeding programs.
 Higher startup expenses and labor costs.
With a long history of development, especially since the fundamental principles of inheritance
were established in the late 19th
and early 20th
centuries, plant breeding has become an
important component of agricultural science, which has features of both science and arts.
Conventional breeding methodologies have extensively proven successful in development of
cultivars and germplasm. However, subjective evaluation and empirical selection still play a
considerable role in conventional breeding. Scientific breeding needs less experience and more
science. MAB has brought great challenges, opportunities and prospects for conventional
breeding. As a new member of the whole family of plant breeding, however, MAB, as
transgenic breeding or genetic manipulation does, cannot replace conventional breeding but is
and only is a supplementary addition to conventional breeding. High costs and technical or
equipment demands of MAB will continue to be a major obstacle for its large-scale use in the
near future, especially in the developing countries (Collard and Mackill, 2008; Ribaut et al.,
2010). Therefore, integration of MAB into conventional breeding programs will be an
optimistic strategy for crop improvement in the future. It can be expected that the drawbacks
of MAB will be gradually overcome, as its theory, technology and application are further
developed and improved.
Conclusion
Molecular markers as new tools in crop improvement have demonstrated usefulness, especially
with genes controlling qualitative traits, whereas successful results are inconsistent with
application to quantitative characteristics. However, the value, ease, and cost of measurement,
and nature of genetic control of agronomic traits will determine the way in which molecular
markers may be effectively used in a breeding program. In addition, these new tools will have
a better opportunity for demonstrating their true values for crop improvement, when the
techniques used by molecular genetics reach a higher degree of automation; then it will be
possible to use molecular markers leading to a new “green revolution” in the world of
agriculture.

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