Biotechnology & Biotechnological Equipment

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Marker-assisted selection and gene pyramiding for

resistance to bacterial leaf blight disease of rice
(Oryza sativa L.)

Samuel Chibuike Chukwu, Mohd Y. Rafii, Shairul Izan Ramlee, Siti Izera
Ismail, Yussuf Oladosu, Emmanuel Okporie, Godwin Onyishi, Emeka Utobo,
Lynda Ekwu, Senesie Swaray & Momodu Jalloh

To cite this article: Samuel Chibuike Chukwu, Mohd Y. Rafii, Shairul Izan Ramlee, Siti Izera
Ismail, Yussuf Oladosu, Emmanuel Okporie, Godwin Onyishi, Emeka Utobo, Lynda Ekwu, Senesie
Swaray & Momodu Jalloh (2019): Marker-assisted selection and gene pyramiding for resistance to
bacterial leaf blight disease of rice (Oryza�sativa L.), Biotechnology & Biotechnological Equipment,
DOI: 10.1080/13102818.2019.1584054

To link to this article: https://doi.org/10.1080/13102818.2019.1584054

© 2019 The Author(s). Published by Taylor & Published online: 23 Feb 2019.
Francis Group on behalf of the Academy of
Forensic Science.

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BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT
https://doi.org/10.1080/13102818.2019.1584054

Marker-assisted selection and gene pyramiding for resistance to bacterial

leaf blight disease of rice (Oryza sativa L.)
Samuel Chibuike Chukwu a,d, Mohd Y. Rafii a,b, Shairul Izan Ramleeb, Siti Izera Ismailc,
Yussuf Oladosue, Emmanuel Okporied, Godwin Onyishie, Emeka Utobod, Lynda Ekwud,
Senesie Swaraya and Momodu Jalloha

a
Laboratory of Climate-Smart Food Crop Production, Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia
(UPM), Selangor, Malaysia; bDepartment of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia (UPM), Selangor, Malaysia;
c
Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia (UPM), Selangor, Malaysia; dDepartment of Crop
Production and Landscape Management, Faculty of Agriculture and Natural Resources Management, Ebonyi State University,
Abakaliki, Nigeria; eDepartment of Crop Science and Technology, School of Agriculture and Agricultural Technology, Federal
University of Technology, Owerri, Nigeria

ABSTRACT ARTICLE HISTORY

Marker-assisted selection and gene pyramiding are very important breeding strategies for confer- Received 17 December 2018
ring broad spectrum and durable resistance against diseases causing yield loss in rice. One such dis- Accepted 14 February 2019
ease causing major set backs in rice production is bacterial leaf blight (BLB) caused by the
KEYWORDS
pathogen Xanthomonas oryzae pv. oryzae. Molecular markers are very essential in both marker-
Marker-assisted selection;
assisted selection and pyramiding of genes, hence, many molecular marker techniques have already gene; pyramiding; BLB;
been developed. Presently, the most commonly used ones are DNA-based markers also known as bacterial leaf blight disease;
molecular markers. The molecular markers are classified into two major categories based on the rice; Oryza sativa L.;
techniques used for detecting them. These are hybridization and polymerase chain reaction-based molecular marker
markers. Other types of markers available include the morphological (traditionally based) and bio-
chemical (enzyme-based) markers. Host plant/varietal resistance is the most suitable means for con-
trolling BLB disease of rice. Marker-assisted gene pyramiding has the potential to accelerate the
breeding programmes and guarantee the durability of resistance conferred in the host plant.
Therefore, this paper uncovers the utilization, economic importance, limitations and future pros-
pects of marker-assisted selection and gene pyramiding for resistance to BLB disease of rice.

Introduction resistance genes. It is also important to consider pyra-

Rice serves as staple food crop and source of energy-
giving food for more than half of the world’s popula-
tion, which is over 3.5 billion people. The demand for
rice is still increasing in Asia as the consumption rate
is at least 90% and it is globally projected that the
demand for rice will rise up to 650 million tonnes by
2050 [1]. At present, the population of the world is
increasing geometrically and the demand for staple
food like rice is also increasing, so bacterial leaf blight
(BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo)
constitutes a major threat to rice production and
invariably, to food security [2]. In order to guard
against the threat posed by the disease, it is important
to make use of varieties of rice incorporated with
miding of several genes for durable resistance to BLB
of rice in order to guarantee long-term resistance [3].
Biotechnology makes use of two strategies in molecu-
lar breeding of rice. This involves marker assisted
selection also known as marker assisted breeding,
while the other one would require development of
genetically modified crops (GMOs) [4]. A genetic
marker is any assayable phenotype or visible trait, for
which alleles at individual loci segregate following the
laws of Mendel. Marker assisted selection is a tech-
nique that helps to make conventional breeding more
efficient but not a substitute for conventional breed-
ing [1]. It offers genetic tools for selection of target
genes from the existing germplasm that would be
used in breeding activities but does not include

CONTACT Mohd Y. Rafii mrafii@upm.edu.my Laboratory of Climate-Smart Food Crop Production, Institute of Tropical Agriculture and Food
Security, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
ß 2019 The Author(s). Published by Taylor & Francis Group on behalf of the Academy of Forensic Science.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
2 S. C. CHUKWU ET AL.
genetic engineering which involves the transfer of iso-
lated gene sequences [5,6].
Molecular or genetic markers are the stable and
inheritable variation at DNA sequence level, which
could be measured or detected by using appropriate
methods and subsequently used to identify a specific
genotype [7]. Molecular/DNA-based markers are
regarded as steady landmarks in the genetic make-up
of plants. They are identifiable sequences of DNA that
are found at specific locations of the genome and are
transmitted by the standard laws of inheritance from
one generation to the next. Molecular markers are
tools used to study diversity at DNA level (polymorph-
ism) and help breeders to identify specific chromo-
some segments that contain genes of interest.
Genetic basis of bacterial leaf blight resistance
in rice
The BLB pathogen Xoo belongs to the c-division of
Gram-negative proteobacteria. This pathogen grows in
the vascular system until the xylem vessels are
clogged with bacterial cells and extracellular polysac-
charides. There are several races of Xoo. All the races
secrete race-specific effectors in the xylem which acti-
vate individual response leading to infection. Xoo also
release avirulence factors that bind and activate the
transcription of genes known as resistance genes,
which activate a resistance response [8]. The host spe-
cificity is determined by avirulence factors through the
interaction of genes. It also reduces the virulence of
Xoo as the host recognizes them. Resistance genes
have evolved to give resistance to specific Xoo races
due to the fact that each race of Xoo produces differ-
ent virulence and avirulence factors. The hypersensi-
tive response and pathogenicity genes are used to
determine the interactions of Xoo with plants. These
are required for hypersensitive response (HR) in resist-
ant/non-host plants and for pathogenicity in suscep-
tible/host plants [9]. Substantial attempt has been
made to identify genes responsible for pathogenesis
of Xoo and roles of each gene in causing disease. The
genome sequence of Xoo strains like KACC10331,
MAFF311018 and PXO99A has made the knowledge of
pathogenesis genes clearer [10].
The inheritance of BLB has been critically studied.
Plants usually contain a single locus which confers
resistance against a complementary avirulence. This
has been shown from genetic analysis of many plant
pathogen interactions [10]. Japan, IRRI, Sri Lanka, India
and China were among the first to study the inherit-
ance of BLB resistance in rice. Researchers found it
difficult to characterize and differentiate the R-genes
due to diversity of Xoo strains in various countries. The
identical differential standard was set up to compare the
genes identified [10]. Currently, 42 BLB R-genes have
been discovered in rice [10–12]. These genes were iden-
tified from mutant populations, cultivated and wild rice
species and confer resistance against different Xoo
strains (Table 1). Out of the 42 genes, 14 are recessive
while nine have been cloned and characterized. The
genes encode different types of proteins which suggest
multiple mechanisms of R-gene mediated Xoo resistance
[13–15]. Majority of these genes give complete race-spe-
cific resistance to the pathogen. The genes have either
been introgressed singly or pyramided in breeding for
BLB resistance in rice [16]. Due to artificial and natural
selection of BLB resistant genes, the bacterial races keep
evolving. Therefore, it would be required to critically
explore the new resistant resources to guard against the
evolved races.
Development of molecular markers is one of the
most recent advances in DNA technologies and has
made it possible to reveal a large number of genetic
variations within a population [45]. Consequently, they
are used in evaluating the genetic basis of the pheno-
typic variability. Molecular markers are more or less
DNA loci with no function and no impact on plants’
performance. However, they are located either near or
within major or functional genes so that their pres-
ence indicates to a large extent the presence of the
functional gene [46,47].
Types of markers
Generally, there are three types of markers according
to Akhtar et al. [7].
Morphological markers
The morphological markers are known as the trad-
itional markers. The morphological mutant characters
in the given population are mapped. Linkage to a
desirable or non-desirable character is determined
while indirect selection is done using the physically
identifiable mutant for the trait [48,49]. Morphological
markers have many non-desirable factors associated
with them. Hence, the use of such markers in breed-
ing is labour intensive, space and time consuming.
Biochemical markers
These markers are superior to traditional/morpho-
logical markers. Generally, unlike morphological
 BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT 3

Table 1. BLB resistance genes in rice.

S. no. R-gene Chromosome Donor Source Remark Reference
1 Xa-1 4 Temperate japonica Japan Dominant, cloned and characterized [17]
2 Xa-2 4 Indica Vietnam Dominant [18]
3 Xa-3/Xa26 11 Japonica Japan Dominant, cloned and characterized [19]
4 Xa-4 11 Indica India Dominant [20]
5 xa-5 5 Aus Bangladesh Recessive, cloned and characterized [21]
6 Xa-6/xa-3 11 – USA Dominant [22]
7 Xa-7 6 Aus Bangladesh Dominant [23]
8 xa-8 7 USA Recessive [24]
9 Xa-9 11 – Laos Dominant [25]
10 Xa-10 11 Senegal Dominant, cloned and characterized [18]
11 Xa-11 3 Indica Philippines Dominant [18]
12 Xa-12 4 Japonica Japan Dominant [26]
13 xa-13 8 – India Recessive, cloned and characterized [18]
14 Xa-14 4 Japonica Taiwan Dominant [18]
15 xa-15 – – – Recessive [27]
16 Xa-16 – Indica Vietnam Dominant [18]
17 Xa-17 – Japonica South Korea Dominant [18]
18 Xa-18 – Indica, japonica Philippines, Japan Dominant [18]
19 xa-19 – – – Recessive [18]
20 xa-20 – – Recessive [18]
21 Xa-21 11 Oryza wild spp. Africa, Mali Dominant, cloned and characterized [28]
22 Xa-22(t) 11 – China Dominant [18]
23 Xa-23 11 Oryza wild spp. China/Cambodia Dominant, cloned and characterized [29]
24 xa-24 2 – Bangladesh Recessive [30]
25 xa-25(t) 12 Indica China Recessive, cloned and characterized [15]
26 xa-26(t) – Indica China Recessive [31]
27 Xa-27(t) 6 Oryza wild spp. Philippines Dominant, cloned and characterized [32]
28 xa-28(t) – Indica Bangladesh Recessive [31]
29 Xa-29(t) 1 Oryza wild spp. – Dominant [33]
30 Xa-30(t) 11 Oryza wild spp. India Dominant [12]
31 xa-31(t) 4 Japonica China Recessive [34]
32 xa-32(t) 11 Oryza wild spp. – Recessive [35]
33 xa-33 6 Oryza wild spp. – Recessive [36]
34 Xa-33(t) – – Thailand Dominant [37]
35 xa-34(t) – Indica Sri Lanka Recessive [38]
36 Xa-35(t) 11 Oryza wild spp. Philippines Dominant [39]
37 Xa-36(t) – – China Dominant [40]
38 Xa-38 – Oryza wild spp. – Dominant [41]
39 Xa39 11 FF329 Chinese, Philippines Dominant [42]
40 Xa40(t) 11 IR65482-7-216-1-2 Korea Dominant [43]
41 xa41(t) – Rice germplasm – Dominant [44]
42 xa42 3 XM14, a mutant of IR24 Japan Dominant [11]

markers, they are independent of growth conditions in
the environment. Biochemical markers involve the use
of isozymes (enzymes) that are expressed in the plants
cells [46]. After extraction, the enzymes are run on
denaturing electrophoresis gels while the denaturing
component eliminates the enzyme’s secondary and
tertiary structure and separates the enzymes based on
net charge and mass [50]. Polymorphism takes place
at the amino acid level enabling the detection of sin-
gular peptide polymorphism which is used as a
marker (polymorphic biochemical marker). However,
the major limitation of biochemical markers is that iso-
zymes do not produce sufficient polymorphism since
most commercial breeds of plants are genetically very
similar [7,51].
Molecular markers
Molecular markers are DNA-based markers. Molecular
markers are practically unlimited and also
phenotypically neutral. The use of such markers has
enabled scanning of the entire rice genome and
assigning landmarks in high density on every chromo-
some of the plant [7,52]. Examples of molecular
markers are restriction fragment length polymorphism
(RFLP), cleaved amplified polymorphic sequences
(CAPS), sequence characterized amplified regions
(SCARs) and other polymerase chain reaction (PCR)-
based markers. A suitable molecular marker must be
polymorphic, randomly and frequently distributed
throughout the genome, co-dominant in inheritance,
reproducible, easy and cheap to detect.
A rice marker should be closely linked to target
loci. Less than 5 cM genetic distance is preferable [53].
The reliability of a rice marker in predicting the
phenotype would increase when flanking or intragenic
markers are used. Although it may be difficult to
obtain, some marker techniques require large quantity
and high quality of DNA [54]. This increases the cost
of the scheme. The simplicity and the time
4 S. C. CHUKWU ET AL.
requirement of the marker technique are important
considerations. Highly desirable methods should be
very simple and quick. An ideal rice marker is also
expected to be highly polymorphic. This implies that
it should be able to discriminate or separate between
different genotypes [55]. The cost effectiveness of the
marker assay is also important for the marker assisted
selection to be feasible. Simple sequence repeats
(SSRs) or microsatellites are often used in rice and
other cereal crops [56]. The major problem of micro-
satellite markers is that they particularly need poly-
acrylamide gel electrophoresis and generally provide
information only for a single locus per assay. Breeders
have been able to overcome the challenge by select-
ing SSR markers that have very large size differences
for detection in agarose gels. Multiplexing several
markers in a single reaction is also helpful. The micro-
satellites also require enough time and money to
develop, and often, some orphan crop species lack
sufficient numbers for high density mapping. Other
DNA-based markers like the RFLPs that are linked to a
target gene or QTL such as SCAR, sequence tagged
site (STS) or single nucleotide polymorphism (SNP)
markers are also properly utilized for marker assisted
selection [57,58].
On the basis of the techniques used for their detec-
tion, molecular markers are classified into two major
categories: hybridization-based markers and PCR-based
markers [46].
Hybridization-based markers
The traditional, or first-generation, RFLPs require the
use of an appropriately labelled DNA probe to select
the gene of interest from a digested DNA sample by
hybridization. The generated DNA fragments are sepa-
rated by gel electrophoresis and polymorphism, or
genetic variation among genotypes, is visualized as
hybridization bands, wherein identical genotypes pro-
duce similar banding patterns while different geno-
types have different banding patterns. Thus, the
process involves DNA extraction, digestion of the iso-
lated DNA with restriction enzymes, hybridization of
the digested DNA fragments with a labelled DNA
probe, separation of the hybridization products on
agarose or polyacrylamide gel and analysis of the
hybridization bands for DNA variation [59].
PCR-based markers
PCR-based markers are molecular markers that do not
require the probe hybridization step. Their
development has led to the discovery of several useful
and easy-to-screen new generation markers such as
random amplified polymorphic DNA (RAPD), amplified
fragment length polymorphism (AFLP), microsatellites
or SSRs, SNP, expressed sequence tags (EST), etc.
[46,60]. Being PCR-based, this category of markers
requires the use of primer pairs to select specific
regions of the DNA for which genetic variation (poly-
morphism) is measured [61]. Primers are short sequen-
ces of nucleotides designed to be complementary to
specific regions of DNA. Consequently, primers are
used to select, by base pairing, a specific segment of
DNA for amplification by PCR and sequence analysis
[38]. They, therefore, initiate the amplification of a par-
ticular DNA segment. The amplification products of
DNA from different genotypes are separated on a gel
to observe size-dependent variation in banding pat-
tern and may be further sequenced to identify
sequence variations [62].
PCR-based molecular marker analysis involves DNA
extraction from the source material, determination of
quantity and quality of the isolated DNA, amplification
of the isolated DNA using PCR and gel electrophoresis
[63]. If sequencing is required, the amplification prod-
ucts are usually purified, subjected to sequencing PCR
and further purified before sequencing [56,64].
DNA-based markers for rice
There are several advantages of the use of DNA
markers for trait selection. Collard and Mackill [65]
reviewed the application of DNA-based markers in the
breeding of BLB disease resistant rice varieties. They
both emphasized the application of the markers and
their limitations in crop improvement. Akhtar et al. [7]
listed the following advantages of molecular markers:
Time saving: Any part of the rice crop can be
sampled for isolation of genomic DNA and information
about the trait of choice can be obtained using tightly
linked polymorphic markers before fertilization,
thereby facilitating a more informed genetic crossing
by breeders.
Consistency: Molecular markers are not affected by
variation in the environment. Environmental factors
usually affect the phenotypic screening of gen-
etic traits.
Biosafety: Diagnostic tests to confirm the absence
or presence of disease resistance traits can be carried
out using molecular markers that are tightly linked to
the gene of interest without adopting inoculation of
pathogen in the greenhouse or field. Also, DNA-based
markers would facilitate the introgression of genes

into elite cultivars before the epidemics of cer-
tain pathogens.
Efficiency: Early generation evaluation of breeding
lines with molecular markers can guide breeders in
rejecting undesired progenies while improving the
genetic quality of desirable breeding materials.
High accuracy in complex traits selection: The use of
DNA-based markers makes it easier to select multi-
genic traits. Molecular markers that are linked to QTLs
enable them to be treated like single Mendelian fac-
tors. McCouch et al. [66] initially developed RFLP-
framework map with 250 markers which was later sup-
plemented with additional markers. The additional
markers include RAPD, SSR or microsatellite markers,
STS and AFLP markers [67]. Tightly linked markers
have been used to map numerous agronomically
important genes of rice. About 18,828 class-I SSR
sequences were listed by the International Rice
Genome Sequencing Project [68]. Dai et al. [69]
reported that several BLB disease resistance genes
were isolated from the plant’s genome with the aid of
saturated rice molecular map and genome sequencing
information. The BLB resistance genes include Xa1,
xa5, xa13, Xa21, Xa26 and Xa27 [17,28,70,71].
Simple sequence repeats (SSRs) markers
Microsatellites, also known as SSRs, are tandemly
arranged repeats of short DNA motifs (1–6 bp in
length) that frequently exhibit variation in the number
of repeats at a locus. Litt and Lutty [72] first coined
the term ‘microsatellite’. SSR markers have high poly-
morphism and abundance in nature; hence these
markers have become a valuable source of genetic
information. Microsatellites are ubiquitous in eukary-
otic genomes and exhibit highly variable numbers of
repeats at a locus, and polymorphism in the repeat
motif is referred to as simple sequence length poly-
morphism (SSLP). Their hyper variability, abundance,
co-dominant and multi-allelic nature make them valu-
able as genetic markers. Using PCR, microsatellites can
be amplified by the use of primers that are comple-
mentary to the flanking region. When PCR products
from different plants vary in length due to variation in
the number of repeat units in the SSR, it is taken as
length polymorphism [73,74]. Microsatellites, or SSR
markers, are regarded as the ideal markers (genetic)
for rice out of the several available DNA-based
markers. Candit and Hubbell [75] observed heavy pres-
ence of SSRs in plants, and that was the first pub-
lished study on microsatellites in plants. Subsequently,
microsatellites have been developed for different plant
BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT 5
species including barley [76], rice [77,78], soybean
[79], etc.
Initial effort to map microsatellites in rice was made
by Zhao and Kochert [78] using (GGC)n, followed by
mapping of (GA/AG)n, (ATC)10 and (ATT)14 microsatel-
lites by Panaud et al. [80]. Presently, over 20,000 SSR
markers (microsatellites), which are well distributed
landmarks all over the genome of rice, have been
developed [68,81–83]. Significantly greater allelic diver-
sity of microsatellites over random fragment length
polymorphism (RFLPs) has been reported [84].
Research has also shown that genetically mapped
microsatellite markers cover the entire rice genome
with at least one microsatellite per 157 kb (<1 cM)
[67,85]. A study comparing 12 Japonica cultivars found
microsatellite’s mean polymorphism of at least 1.5
times higher than AFLP and RAPD markers [86]. Intra
and inter cultivar polymorphism between the culti-
vated rice and the landrace was detected by Provan
et al. [87] using SSR markers. Xu et al. [88] found that
genetic diversity is present to a greater extent in land-
races compared to improved cultivars in their molecu-
lar diversity studies of Philippine landraces and
cultivars. Enoki et al. [89] reported the use of SSRs in
estimating the diversity among parental lines, inbreds
and varieties of rice. SSR markers have also been used
for tagging and mapping of target genes and/or major
QTL associated with Xa30t for resistance to BLB [12],
Xa7 and xa13 gene from Basmati-48, Basmati-51A,
Basmati-334, and Basmati-370A [90], dominant Rsb1
for resistance to sheath blight [91], sub1A for tolerance
of submergence [92,93], restorer gene for HL type
cytoplasmic male sterile lines [94] and fragrance gene
in rice [95].
Marker-assisted selection
Marker-assisted selection is the selection for a trait on
the basis of genotype using associated markers rather
than the phenotype of the trait. Thus, marker-assisted
selection of breeding materials involves cultivar identi-
fication, assessing the genetic diversity and purity,
selection of parents, identifying the genomic regions
under selection and study of heterosis [96]. It makes
use of DNA-based markers that are tightly linked to
the gene of interest to either assist or replace pheno-
typic evaluation. Identification of plants that carry spe-
cific genes or QTLs is based on their genotype and
this is done by determining the allele of the molecular
(DNA-based) marker [54]. Marker-assisted selection is
very promising in the development of rice varieties
resistant to BLB disease [5,97]. The use of molecular
6 S. C. CHUKWU ET AL.
marker information to identify and select specific gen-
otypes also gives a clear understanding of the molecu-
lar marker. Before now, breeders relied on information
about how plants or animals perform to make infer-
ences about how good their genes were, a practice
that is time consuming, laborious and less effective
[98–100]. A direct handle on the genes controlling the
traits of interest could lead to much faster progress.
Some traits are controlled by single genes, but most
traits of economic importance are controlled by a
large number of genes [4,101]. Such traits are called
quantitative traits and the genes locations on the
chromosomes are quantitative trait loci (QTL).
However, some of the genes in the QTL have larger
effects than others. Such genes are called major
genes. In effect, QTL actually refers to the major genes
following the pattern of inheritance of the QTL or the
major genes that might be helpful in selection. In
practice, it is not easy to observe inheritance at QTL
itself, but it is possible to observe the inheritance of
marker sequences located close to or within the QTL.
Molecular markers are chosen for their proximity to
QTL. Well-designed experiments can be used to iden-
tify marker sequences located close to or within major
genes on the chromosomes or genomes [102,103].
The molecular markers should be able to predict
the phenotype reliably. A marker can either be located
within the gene of interest or be linked to a gene
determining a trait of interest, which is the most com-
mon case. Molecular markers can also be used in con-
firmation of true F1, testing the seeds for genetic
purity, protecting the cultivar, establishment of breed-
ing strategies, linkage map construction, gene map-
ping and also mapping of QTLs associated with
biological processes [5]. The following considerations
are necessary in applying DNA-based markers in
marker-assisted selection [96]:
i. Reliability: for a molecular marker to be reliable,
it should be tightly linked to target traits (<5 cM
genetic distance is preferable). The use of intra-
genic or flanking markers would increase the
reliability of the markers to predict pheno-
type rapidly.
ii. Quantity and quality of DNA: The techniques of
some markers require DNA in a large amount
with high quality. This increases the cost of the
procedure and is sometimes difficult to obtain
in practice.
iii. Technical procedure: it is required that molecular
markers should be transferable among research-
ers and also have high reproducibility across
laboratories. Time and level of simplicity are to
be considered. Therefore, a high throughput sim-
ple and quick methods are desirable factors to
be considered.
iv. Polymorphism: DNA-based markers should have
a high level of polymorphism, hence, be hyper-
variable. Such markers should also possess co-
dominant inheritance of homozygotes and het-
erozygotes in progenies that are still
segregating.
v. Affordability: the cost of DNA markers ought to
be affordable. This implies that the assay should
be cheap, user-friendly and easy to apply for effi-
cient screening of large samples/populations. The
cost effectiveness of the marker assay should be
considered in order to guarantee the feasibility of
the marker-assisted selection.
Importance of quantitative trait loci (QTLs)
mapping for marker-assisted selection
QTL is a gene or chromosomal region that affects a
quantitative trait. The QTLs are regarded as the under-
lying genes controlling quantitative traits. With the aid
of QTL mapping experiments, the breeder would be
able to identify the DNA-markers that are linked to
specific genes and/or QTLs [104]. Although, it was pre-
viously thought that markers could be used directly in
marker-assisted selection, but research has shown that
QTL mapping is actually the basis for developing
markers in marker-assisted selection [105]. However,
environmental effects, population size and type, num-
ber of replication of phenotypic data, genotyping
errors, etc. are some of the factors that affect the QTL
mapping accuracy [106–108]. These factors are very
relevant for more complex polygenes which controls
quantitative characters comprising several QTLs each
with relatively small effects. Therefore, it has been
widely accepted in recent years that QTL confirmation,
validation and/or additional marker testing steps are
necessary after QTL mapping and prior to marker-
assisted selection [105,109,110]. These steps may
include conversion of the marker which is required to
improve the reliability and make marker genotyping
simpler for marker assisted selection. Confirmation of
QTL is necessary to ascertain the accuracy of the
results obtained from the primary QTL mapping study
[111,112]. In order to verify the effectiveness of the
QTLs in different genetic backgrounds, validation of
QTL is done. The validation of a marker involves test-
ing the level of polymorphism of most tightly-linked

markers and the reliability of markers for predicting
phenotype [84,113].
Application of marker-assisted selection
schemes in plant breeding
Marker-assisted introgression
Introgression simply means the transfer a specific
gene of interest from one species to another through
hybridization and repeated backcrossing. Hospital
[114] defined introgression as the process where a tar-
get gene or QTL from a plant in population ‘A’ is
inserted to another plant in population ‘B’ by crossing
the two of them and then repeatedly backcrossing to
‘B’ which is known as the recipient and/or recurrent
parent. In this case, DNA markers are useful in control-
ling the presence of the target gene or QTL. This is
also useful in accelerating the recovery of the back-
ground genome to the recipient type. Introgression
using molecular markers is very effective in incorporat-
ing genes or QTLs from landraces, because the time
required to produce an improved variety and the issue
of linkage drag are reduced [60].
Marker-assisted backcross breeding (MABB)
Markers can be applied in backcross breeding at three
stages of foreground, recombinant and background
selections. At the first stage of foreground selection,
markers are used to select for the target trait. The use
of markers here are very effective because some traits
have laborious phenotypic selection procedures or
recessive alleles whose effects could have been
masked by the dominant genes. The recombinant
selection is the second stage which involves selecting
backcross progeny with the specific gene and tightly-
linked flanking markers so that linkage drag can be
reduced. The third stage is referred to as the back-
ground selection and it involves selecting backcross
progeny (i.e. progeny already selected for the target
trait) using background markers. However, the back-
ground markers must not be tightly linked markers
but should be ideal rice markers [53,56]. This implies
that markers can be used to select against the donor
genome, i.e. reduce the genetic contribution of the
donor parent while accelerating the recovery ratio of
the recurrent parent genome [101].
Marker-assisted pyramiding
The process of simultaneously incorporating multiple
genes or QTLs into a single genotype is known as
BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT 7
pyramiding [115]. Pyramiding of genes is very essential
for broad spectrum resistance to BLB of rice and to
guarantee the durability of the disease resistance.
Molecular markers facilitate selection because molecu-
lar or DNA-based marker assays are non-destructive
and markers encoding for several target genes can be
tested using a single DNA sample without phenotyp-
ing. Combining multiple disease resistance genes or
QTLs so as to provide durable disease resistance has
been the most widespread application of pyramiding
in plant breeding [60,116]. Although it is still possible
to use conventional breeding, it is very difficult or
nearly impossible at the early generations due to the
need to phenotypically screen each plant for all traits
being tested. This makes it very hard to evaluate
plants from some populations like the F2 or for traits
with destructive bioassays [4,117].
Early generation marker-assisted selection
Early generation stages are the most important stages
to use markers for selection of plants. The essence of
this is such that the plants that do not carry required
genes can be deselected. In breeding of plants for
resistance to BLB, the plants to be selected must carry
the disease resistance genes in addition to other use-
ful agronomic traits. The implication of this is that
there would be an efficient evaluation of fewer
breeder lines with a cost effective design at the later
stage of breeding [118].
Combined approaches
Sometimes, phenotypic screening can be combined
with marker-assisted selection in order to maximize
the genetic gain especially where QTL mapping could
not identify some QTLs. A combined approach is also
useful to find the level of recombination between a
marker and a QTL. It also reduces the size of the
population for traits [105]. Although marker genotyp-
ing seems to be easier and cheaper compared to phe-
notyping, marker-assisted phenotyping (tandem
selection) can be utilized where it is obvious that
marker genotyping alone is not completely accurate
or that phenotyping alone would cost more
money [1,119].
Advantages of marker-assisted selection
Plants can be selected at their seedling stages. There
is high reliability of single plant selection. On average,
the marker-assisted selection is a simpler process
8 S. C. CHUKWU ET AL.
compared to phenotypic evaluation [53]. Given these
advantages, marker-assisted selection is capable of
increasing the efficiency and effectiveness of a breed-
ing programme. For instance, marker genotyping or
DNA marker tests can be used to replace large popu-
lation field trials. This saves the breeder time and diffi-
cult labour thereby saving costs as well. Also, the
molecular marker based selection of plants is more
reliable due to the variability in the environmental fac-
tors which affect field trials [5,120]. The use of fore-
ground markers in marker-assisted selection enables
the breeder to discard plants that are not carrying the
target genes at the early stage of breeding. This is
another advantage of marker-assisted selection
because the number of plants to be tested is reduced,
thereby increasing the efficiency of target trait selec-
tion with effective breeding design. In addition,
marker-assisted selection enables the acceleration of
recurrent parent genome recovery ratio with the aid
of background markers during marker-assisted back-
cross breeding [7,51,64].
Limitations of marker-assisted selection
The prohibitive cost is one of the limitations of using
marker-assisted selection in plant breeding today. In
addition, two more factors limiting the use of marker-
assisted selection in plant breeding are the equipment
and consumables. These two are required to establish
and maintain a molecular marker laboratory and
therefore, their costs should be considered [7,121].
Development of markers also requires large initial
costs and should be put into consideration. In some
developing nations, electricity supply poses a major
challenge to preserve the markers at a steady tem-
perature. Although, few numbers of reports are avail-
able in literature on the economics of marker-assisted
selection compared to conventional breeding, the cost
effectiveness of the two approaches considerably
varies in different studies. In marker assisted backcross
breeding (MABB), the initial cost implication of adopt-
ing markers may appear to be higher in the short run.
In the long run, however, the release of newly devel-
oped varieties in a short time as a result of marker-
assisted selection could turn into higher profits com-
pared to the cost of production [122,123].
Markers have low reliability to determine the
phenotype. This is another limitation of using marker-
assisted selection for line development. The thorough-
ness of the primary QTL mapping study is always
responsible for this. Sampling bias mostly in small
populations can still affect the QTLs that are detected
with high LOD scores which explain a large proportion
of the phenotype, hence, may not be utilized for
marker-assisted selection. Also, the genetic make-up of
the plant may determine the effect of a QTL. This
explains why the effects of QTL and the reliability of
markers, otherwise referred to as QTL/marker valid-
ation, should be tested before embarking on marker-
assisted selection [109,124].
Recombination or crossing-over may occur during
DNA copying and this is a major problem of marker-
assisted selection technology, because with recombin-
ation, one cannot be sure of which marker variant or
allele is associated to each gene variant or allele. This
is why molecular markers may be described as direct
or indirect markers. A marker is a direct one when it is
located within the major gene, and an indirect or
linked when it is located near the major gene.
Recombination is a function of the distance between a
gene or QTL and the marker linked to it. The larger
the distance between a maker and a major gene is,
the greater the problem of recombination [104,105].
If a marker is located within a major gene, recombin-
ation will not occur at all. In this case, knowledge of
the marker allele tells us directly about the gene vari-
ant. Use of direct markers for selection is straightfor-
ward. The problem is how to decide based on linked or
indirect markers. To do this, the pattern of inheritance
of the major gene and its associated marker is first
established. Consequently, progenies that perform well
phenotypically may lack the preferred marker allele,
while progenies that perform poorly may have the
desired marker variant. Generally, linked markers with
high recombination frequency (more than 25%) should
be avoided, while those with 75% fidelity and above
are efficient [7,106]. This means that if 75% or more of
the genotypes that performed well phenotypically
show the preferred marker allele, the marker is useful
in marker-assisted breeding. If the genotypes are less
than 75%, the marker is inefficient [52].
Gene pyramiding
Gene pyramiding can be defined as a process of com-
bining two or more genes from multiple parents to
develop superior lines and varieties. Pyramiding
involves combining or stacking multiple genes leading
to the simultaneous expression of more than one
gene in a variety. Molecular markers aid in selecting
the best plants with which to proceed. The term gene
pyramiding is used in agricultural research to describe
a breeding approach to achieve disease control and
higher crop yield. The development of molecular

genetics and associated technology like marker-
assisted selection has led to the emergence of a new
field in plant breeding called gene pyramiding. The
gene pyramiding scheme can be distinguished into
two parts [116]. The first part is called a pedigree,
which aims at cumulating all the target genes in a sin-
gle genotype called the root genotype. The second
part is called the fixation step which aims at fixing the
target genes into a homozygous state, i.e. to derive
the ideal genotype from the one single genotype.
Molecular marker genotyping can facilitate the gene
pyramiding process by reducing the number of gener-
ations that breeders must evaluate to ensure they
have the desired gene combination. Gene pyramiding
is an important strategy for germplasm improvement
[116,117,125,126].
Main factors affecting gene pyramiding
Characteristics of the target trait/gene
Gene pyramiding will prove to be more successful
when the genes to be stacked are characterized well
functionally, using perfect markers. The presence or
absence of the gene of interest can be traced using
one or two markers per gene. It is preferable to use
the bulk segregant analysis (BSA) method to identify
tightly linked markers to a major gene [119,127].
Reproductive traits
The number of seeds produced by a plant determines
its propagation capability. Collection of seeds from
many F1 plants from a cross involving two homozy-
gous parents would produce a fairly large F2 popula-
tion. The F3 generation requires that seeds be
collected from only a single plant. However, the effi-
ciency of cross fertilization could largely constraint
some species of crop. It is also important to note that
some other reproduction related problems such as
incompatibility in crossing may arise when wild rela-
tives are used as donor parent contributing the desir-
able genes into the cultivated rice variety [128,129].
It is difficult to pyramid genes using only conven-
tional breeding due to the effect of linkage drag,
which is not easy to break after repeated backcross-
ing. With the introgression of two or more genes, phe-
notyping alone cannot clearly separate the effect of
each individual gene. Each gene confers resistance to
several races of the same pathogen. There is also the
issue of the masking effect where dominant and reces-
sive genes are involved. Marker assisted pyramiding
enables the use of DNA-based markers that are tightly
BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT 9
linked with each of the disease resistance genes for
selection of plants with multiple genes [4].
Success has been recorded in earlier attempts to gen-
erate three-gene pyramids in Pusa Basmati, PR106 and
Samba Mahsuri cultivars [129–131]. Although susceptible
to various pests and disease, hybrid rice is suitable for
increasing the productivity of rice. There is a longer
period for BLB susceptibility in a hybrid rice compared
to rice varieties. ‘Kresek’ at seedling stage, defoliation
and death of the entire plant under severe infection at
the stage of maximum tillering are some symptoms of
BLB. Some management practices, such as leaf cutting,
also make the plant more vulnerable. KMR3 is among
the best restorer lines for breeding hybrid rice and it is
used as the donor parent of KRH2 (a non-aromatic
hybrid). On the other hand, PRR78 is the donor parent
of Pusa RH10, an aromatic hybrid which is well known
for its aroma and superfine grain quality. Research has
shown that these two lines are highly susceptible to BLB
causing a yield reduction up to 40% [64]. Many hybrids
have been developed using IR58025A, a well known
cytoplasmic male sterile (CMS) line, as female line.
Examples are KRH2, Pusa 6A and Pusa RH10 [64].
Pyramiding of resistant genes alone in the restorer lines
would not be sufficient since the hybrid will carry the
resistance genes in a heterozygous state. This would
reduce the level of resistance incorporated. Therefore,
the recessive xa5 and xa13 genes should also be intro-
gressed in the recipient line. Currently, it is impossible to
transfer the BLB resistance genes directly into the ‘A’
lines because the genes are available in partial restorer
backgrounds. This requires transferring the genes to the
maintainer backgrounds first. They can be easily trans-
ferred to the male sterile background after stabilization.
When the genes have been successfully transferred to
the CMS and the maintainer line base, it would be easier
to transfer multiple resistance to every other CMS line
without encountering fertility restoration problems. It is
also important to insulate both parents in order to get
resistance in the hybrid. This requires that BLB resistance
genes be introgressed into the genetic background of
the maintainer and the restorer lines. Shanti et al. [64]
reported that a four-gene combination (Xa4, xa5, xa13
and Xa21) was the most effective gene combination that
confers broad spectrum resistance. The result showed
that it did not show any sign of loss of resistance to
varying strains of the BLB pathogen.
Marker-assisted pyramiding
The process of introgressing various genes into a sin-
gle cultivar is known as pyramiding. Pyramiding can
10 S. C. CHUKWU ET AL.
be done through conventional techniques, but it is
difficult to identify plants containing multiple genes
using that approach [132]. Evaluation of all traits
tested is required in conventional phenotypic selec-
tion. Therefore, it could be difficult to assess traits
with destructive bioassays on plants from populations
like the F2. Molecular markers can enhance selection
because molecular markers are not destructive and
markers for multiple specific genes could be tested
using a single DNA sample without phenotyping [133].
Introgressing various disease resistance genes into a
particular variety is the most wide utilization of pyra-
miding. The aim of pyramiding has been to develop
varieties with stable and durable resistance to dis-
eases, since pathogens usually break single gene
resistance after some time due to mutations or emer-
gence of new races of pathogen. Previous studies
have shown that incorporating multiple genes can
guarantee broad spectrum resistance [134].
Initially, it was not easy to pyramid multiple genes
of resistance because they displayed a phenotype,
which led to progeny tests to confirm the particular
plant with multiple genes. Now, by the use of linked
molecular markers, the breeder can identify the num-
ber of resistance genes in the plant. Pyramiding may
involve incorporating resistance genes sourced from
over two parents. For instance, Pradhan et al. [117]
pyramided three BLB (xa5þxa13þXa21) resistance
genes for broad-spectrum resistance in deepwater rice
variety, Jalmagna. Three major genes (Pi1, Piz-5 and
Pita) were pyramided by Hittalmani et al. [135] using
RFLP markers. Hittalmani et al. [135] and Castro et al.
[134] combined genes originating from three parents
for rice blast and stripe rust in barley, respectively.
Marker assisted pyramiding was also proposed as an
effective approach to produce three-way F1 cereal
hybrids possessing durable resistance. Strategies for
marker assisted pyramiding of linked genes of interest
have also been evaluated [61]. It is preferable to pyra-
mid over successive generations in terms of minimal
marker genotyping for many linked target loci. In the-
ory, marker-assisted selection could be used to pyra-
mid genes from different parents.
Marker-assisted selection in rice breeding for
bacterial leaf blight resistance
Marker-assisted selection enhances the identification
of rice cultivar with multiple resistance genes. For
example, the IRBB60 has four BLB resistance genes,
and namely Xa4, xa5, xa13 and Xa21 [1,62]. There are
about 40 known Xa-genes conferring resistance
against the BLB of rice and each of them can be singly
identified using marker assisted selection. Major genes
conferring resistance to various diseases in several
crops have also been mapped with linked DNA
markers, thereby facilitating marker-assisted selection
for disease resistance in such species. The marker-
assisted selection scheme has also been used success-
fully in selection for resistance when pathogens were
absent [136], multiple genes pyramiding for durable
resistance against rice BLB [137] and in developing
multiple disease-resistant cultivars [138,139]. Many rice
BLB resistance genes have tightly-linked primers while
some are already cloned for developing BLB resistant
rice varieties. Some examples are Xa1, xa5, xa13, Xa21,
Xa26 and Xa27. Excluding recessive xa-5 and xa-13
genes, the bacteria leaf blight disease resistant genes
mentioned here are dominant genes. The markers are
developed from the primer sequence information,
which is broadly utilized in marker-assisted selection
[70]. Due to the presence of molecular markers devel-
oped from the BLB resistance genes, the breeder can
now pyramid multiple genes of resistance into a sus-
ceptible rice variety with good agronomic value.
The pyramiding of dominant Xa4 and Xa21 genes led
to development of an improved ‘indica’ rice variety with
‘broad spectrum durable resistance’ to BLB. Pyramiding
of Xa4 þ xa5 þ Xa21 BLB resistance genes expressed
effective resistance to virulent BLB isolates of Korea in
comparison to single resistance genes which had their
resistance broken after a short period of time and had
become susceptible [140]. The xa5þxa13þXa21 resist-
ance genes were pyramided through marker-assisted
selection into an indica rice (PR106) cultivar, which
exhibited effective resistance to Indian races of BLB
[129]. Angke and Conde are two commercially cultivated
rice cultivars released for cultivation in Indonesia in 2002
with gene pyramids Xa4 þ xa5 and Xa4 þ Xa7. NSIC-
Rc142 and NSIC-Rc154 rice cultivars were also released
in the Philippines and possess the genes
Xa4 þ xa5 þ Xa21 pyramid. The BLB resistance genes
were introgressed into the genetic background of the
susceptible IR64 cultivar through marker-assisted selec-
tion [141]. Genes for basmati quality from PB-1 and BLB
resistance from IRBB55 xa13þXa21 have been pyra-
mided. Rice lines pyramided with BLB disease resistance
genes and their reactions to various races of Xoo are pre-
sented in Table 2.
Conclusions
The future prospects of marker-assisted selection in
rice breeding are very promising. The adoption of
 BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT 11

Table 2. Rice lines pyramided with BLB disease resistance genes and their reactions to various races of the pathogen
(Xanthomonas oryzae pv. oryzae).
Reaction to Xoo
S. no. Rice line Resistance genes Cross R1 R2 R3 R4
1. IRBB50 Xa4þxa5 IR248/O BARTHII HR MR MR R
2. IRBB51 Xa4þxa13 IRBB4XIR66699-9-1-1-5-2 MS MR MR MR
3. IRBB52 Xa4þXa21 IRBB4/66700-3-3-3-4-2 MR MR R MR
4. IRBB53 Xa5þxa13 IRB4/IR66699-9-1-1-5-2 MR S MR S
5. IRBB56 Xa4þxa5þxa13 AY4 þ 5/IR68311-13-3-42 MR MS MR HR
6. IRBB57 Xa4þxa5þXa21 AY4 þ 5/IR66700-4-2-9-5-2 MR MR MR HR
7. IRBB58 Xa4þxa13þXa21 NH11-35/NH9-53 R MR MR MS
8. IRBB59 xa5þxa13þXa21 NH11-35/NH9-53 R MR MR R
9. IRBB60 Xa4þxa5þxa13þXa21 NH11-35/NH9-53 R HR R R
10. IRBB61 Xa4þxa5þXa7 IR-BB7/IR-BB60 MS MR MS R
11. IRBB62 Xa4þXa7þXa21 IR-BB7/IR-BB60 MR MS MR MR
12. IRBB64 Xa4þxa5þXa7þXa21 IR-BB7/IR-BB60 MR HR R R
13. IRBB65 Xa4þXa7þxa13þXa21 IR-BB7/IR-BB60 MR R MS R
14. IRBB66 Xa4þxa5þXa7þxa13þXa21 IR-BB7/IR-BB60 R R HR R
Table modified from Arshad et al. [142].
HR, highly resistant; R, resistant; MR, moderately resistant; S, susceptible; MS, moderately susceptible; HS, highly susceptible; R1, Race1; R2, Race2; R3,
Race3; R4, Race4.

markers in rice breeding programmes is expected to ORCID

increase in the future. The successes and challenges
recorded in using marker-assisted selection would be
critical to determine the future endeavours in marker-
assisted selection. The major challenges for increased
adoption and impact of marker-assisted selection for
breeding rice for BLB resistance include the cost-
effectiveness. However, developing effective strategies
for using markers would facilitate a greater level of
the adoption. Establishment of facilities for marker
genotyping as well as training the members of staff in
rice breeding stations in different countries are import-
ant strategies for future prospects. Identification of
tightly-linked markers, establishment and curation of
public databases for QTL/marker data would also help
to increase the adoption of marker-assisted selection
in breeding for BLB resistance in rice. Hence, the avail-
able resources for developing new markers from DNA
sequence data as a result of rice genome sequencing
and research in functional genomics would be helpful.
Acknowledgements
We appreciate the useful comments, suggestions and discus-
sions from our colleagues in different universities that con-
tributed to the success of this review.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was supported by the Higher Institution Centre of
Excellence (HiCoE) Research Grant (Vot number 6369105),
Ministry of Education, Malaysia.
Samuel Chibuike Chukwu http://orcid.org/0000-0002-
8041-8479
Mohd Y. Rafii http://orcid.org/0000-0003-4763-6367
References
[1] Chukwu SC, Rafii MY, Ramlee SI, et al. Bacterial leaf
blight resistance in rice: a review of conventional
breeding to molecular approach. Mol Biol Rep. 2019.
doi:10.1007/s11033-019-04584-2
[2] Roy S, Banerjee A, Bhattacharya S. Chapter 1. Omics-
based approaches for rice improvement. In: Barh D,
editor. OMICS applications in crop science. New York
(NY): CRC Press; 2013.
[3] Zhang J, Li X, Jiang G, et al. Pyramiding of Xa7 and
Xa21 for the improvement of disease resistance to
bacterial blight in hybrid rice. Plant Breed. 2006;125:
600–605.
[4] Miah G, Rafii MY, Ismail MR, et al. A review of micro-
satellite markers and their applications in rice breed-
ing programs to improve blast disease resistance.
IJMS. 2013;14:22499–22528.
[5] Oladosu Y, Rafii MY, Magaji U, et al. Genotypic and
phenotypic relationship among yield components in
rice under tropical conditions. Biomed Res Int. 2018;
2018:1.
[6] Mannan S, Hameed S. A molecular tool for differen-
ciation of Xanthomonas oryzae pathovars isolated.
Gene. 2013;51:1–7.
[7] Akhtar S, Bhat MA, Wani SA, et al. Marker assisted
selection in rice. J. Phytol. 2010;2:66–81.
[8] Hummel AW, Doyle EL, Bogdanove AJ. Addition of
transcription activator-like effector binding sites to a
pathogen strain-specific rice bacterial blight resist-
ance gene makes it effective against additional
strains and against bacterial leaf streak. New Phytol.
2012;195:883–893.
12 S. C. CHUKWU ET AL.
[9] Gill US, Lee S, Mysore KS. Host versus nonhost resist-
ance: distinct wars with similar arsenals. Phytopathol.
2015;105:580–587.
[10] Vikal Y, Bhatia D. Genetics and genomics of bacterial
blight resistance in rice. In: Li JQ, editor. Advances in
international rice research. London (UK): InTech;
2017. p. 175–213.
[11] Busungu C, Taura S, Sakagami J-I, et al. Identification
and linkage analysis of a new rice bacterial blight
resistance gene from XM14, a mutant line from IR24.
Breed Sci. 2016;66:636–645.
[12] Cheema KK, Grewal NK, Vikal Y, et al. A novel bacter-
ial blight resistance gene from Oryza nivara mapped
to 38 Kb region on chromosome 4L and transferred
to Oryza sativa L. Genet Res. 2008;90:397–407.
[13] Tian D, Wang J, Zeng X, et al. The rice TAL effector-
dependent resistance protein Xa10 triggers cell
death and calcium depletion in the endoplasmic
reticulum. Plant Cell. 2014;26:497–515.
[14] Wang C, Zhang X, Fan Y, et al. Xa23 is an executor R
protein and confers broad spectrum disease resist-
ance in rice. Mol Plant. 2015;8:290–302.
[15] Liu Q, Yuan M, Zhou Y, et al. A paralog of the MtN3/
saliva family recessively confers race-specific resist-
ance to Xanthomonas oryzae in rice. Plant Cell
Environ. 2011;34:1958–1969.
[16] Pandey MK, Rani NS, Sundaram RM, et al.
Improvement of two traditional Basmati rice varieties
for bacterial blight resistance and plant stature
through morphological and marker-assisted selec-
tion. Mol Breed. 2013;31:239–246.
[17] Yoshimura S, Yamanouchi U, Katayose Y, et al.
Expression of Xa1, a bacterial blight-resistance gene
in rice, is induced by bacterial inoculation. Proc Natl
Acad Sci USA. 1998;95:1663–1668.
[18] Kurata N, Yamazaki Y. Oryzabase. An integrated bio-
logical and genome information database for rice.
Plant Physiol. 2006;140:12–17.
[19] Xiang Y, Cao Y, Xu C, et al. Xa3, conferring resistance
for rice bacterial blight and encoding a receptor kin-
ase-like protein, is the same as Xa26. Theor Appl
Genet. 2006;113:1347–1355.
[20] Wang W, Zhai W, Luo M, et al. Chromosome landing
at the bacterial blight resistance gene Xa4 locus
using a deep coverage rice BAC library. Mol Genet
Genomics. 2001;265:118–125.
[21] Petpisit V, Khush GS, Kauffman HE. Inheritance of
resistance to bacterial blight in rice. Crop Sci. 1977;
17:551–554.
[22] Sidhu GS, Khush GS, Mew TW. Genetic analysis of
bacterial blight resistance in seventy-four cultivars of
rice, Oryza sativa L. Theor Appl Genet. 1978;53:
105–111.
[23] Lee KS, Khush GS. Genetic analysis of resistance to
bacterial blight, Xanthomonas oryzae pv. oryzae rice.
Rice Genet Newsl. 2000;17:72–78.
[24] Singh K, Vikal Y, Singh S, et al. Mapping of bacterial
blight resistance gene xa8 using microsatellite
markers. Rice Genet Newsl. 2002;19:94–96.
[25] Ogawa T, Yamamoto T, Khush GS, et al. Near-iso-
genic lines as international differentials for resistance
to bacterial blight of rice. Rice Genet Newsl. 1988;5:
106–109.
[26] Ogawa T, Lin L, Tabien RE, et al. A new recessive
gene for resistance to bacterial blight of rice. Rice
Genet Newsl. 1987;4:98–100.
[27] Ogawa T. Monitoring race distribution and identifica-
tion of genes for resistance to bacterial leaf blight.
In: Khush GS, editor. Rice genetics III. Proceedings of
the 3rd International Rice Genetics Symposium; 1995
Oct 16–20. Manila, Philippines: International Rice
Research Institute; 1996. p. 456–459.
[28] Song WY, Wang GL, Chen LL, et al. A receptor kin-
ase-like protein encoded by the rice disease resist-
ance gene, Xa21. Science. 1995;279:1084–1086.
[29] Zhang Q, Wang CL, Zhao KJ, et al. The effectiveness
of advanced rice lines with new resistance gene
Xa23 to rice bacterial blight. Rice Genet Newsl. 2001;
18:71.
[30] Khush GS, Angeles ER. A new gene for resistance to
race 6 of bacterial blight in rice, Oryza sativa. Rice
Genet Newsl. 1999;16:92–93.
[31] Lee KS, Rasabandith S, Angeles ER, et al. Inheritance
of resistance to bacterial blight in 21 cultivars of
rice. Phytopathology. 2003;93:147–152.
[32] Gu K, Yang B, Tian D, et al. R gene expression
induced by a type-III effector triggers disease resist-
ance in rice. Nature. 2005;435:1122–1125.
[33] Tan GX, Ren X, Weng Q, et al. Mapping of a new
resistance gene to bacterial blight in rice line intro-
gressed from O. officinalis. J Genet Genomics. 2004;
31:724–729.
[34] Wang CT, Wen GS, Lin XH, et al. Identification and
fine mapping of the new bacterial blight resistance
gene, Xa31(t), in rice. Eur J Plant Pathol. 2009;23:
235–240.
[35] Zheng CK, Wang CL, Yu YJ, et al. Identification and
molecular mapping of Xa32(t), a novel resistance
gene for bacterial blight (Xanthomonas oryzae pv.
oryzae) in rice. Acta Agron Sin. 2009;35:1173–1180.
[36] Natarajkumar P, Sujatha K, Laha GS, et al.
Identification of a dominant bacterial blight resist-
ance gene from Oryza nivara and its molecular map-
ping. Rice Genet Newsl. 2010;25:54–56.
[37] Korinsak S, Sriprakhon S, Sirithanya P, et al.
Identification of microsatellite markers (SSR) linked
to a new bacterial blight resistance gene xa33(t) in
rice cultivar ‘Ba7’. Maejo Int J Sci Technol. 2009;3:
235–247.
[38] Chen W, Yao X, Cai K, et al. Silicon alleviates drought
stress of rice plants by improving plant water status,
photosynthesis and mineral nutrient absorption. Biol
Trace Elem Res. 2011;142:67–76.
[39] Guo SB, Zhang DP, Lin XH. Identification and map-
ping of a novel bacterial blight resistance gene
Xa35(t) originated from Oryza minuta. Sci Agric Sin.
2010;43:2611–2618.
[40] Miao LL, Wang CL, Zheng CK, et al. Molecular map-
ping of a new gene for resistance to rice bacterial
blight. Sci Agric Sin. 2010;43:3051–3058.
[41] Bhasin H, Bhatia D, Raghuvanshi S, et al. New PCR-
based sequence tagged site marker for bacterial

blight resistance gene Xa38 in rice. Mol Breed. 2012;
30:607–611.
[42] Zhang F, Zhuo D-L, Zhang F, et al. Xa39, a novel
dominant gene conferring broad-spectrum resist-
ance to Xanthomonas oryzae pv. oryzae in rice. Plant
Pathol. 2015;64:568–575.
[43] Kim SM, Suh JP, Qin Y, et al. Identification and fine-
mapping of a new resistance gene, Xa40, conferring
resistance to bacterial blight races in rice (Oryza sat-
iva L.). Theor Appl Genet. 2015;128:1933–1943.
[44] Hutin M, Sabot F, Ghesqui
ere A, et al. A knowledge-
based molecular screen uncovers a broad-spectrum
OsSWEET14 resistance allele to bacterial blight from
wild rice. Plant J. 2015;84:694–703.
[45] Arif IA, Bakir MA, Khan HA, et al. A brief review of
molecular techniques to assess plant diversity. Int J
Mol Sci. 2010;11:2079–2096.
[46] Yang HB, Kang WH, Nahm SH, et al. Methods for
developing molecular markers. In: Current technolo-
gies in plant molecular breeding, Chapter 2,
Dordrecht: Springer; 2015, p. 15–50.
[47] Andersen JR, Lu €bberstedt T. Functional markers in
plants. Trends Plant Sci. 2003;8:554–560.
[48] Govindaraj M, Vetriventhan M, Srinivasan M.
Importance of genetic diversity assessment in crop
plants and its recent advances: an overview of its
analytical perspectives. Genet Res Int. 2015;2015:
1–14.
[49] Jonah PM, Bello LL, Lucky O, et al. The importance
of molecular markers in plant breeding programmes.
Glob J Sci Front Res. 2011;11:5–12.
[50] Ewing E. In vacuo glycation of enzymes: a novel
approach for increasing enzyme stability [disserta-
tion]. Canada: University of Ottawa; 2006.
[51] Sattari A, Fakheri B, Noroozi M. Blast resistance in
ricew: a review of breeding and biotechnology. Int J
Agric Crop Sci. 2014;7:329–333.
[52] Sorrells ME, La-Rota M, Bermudez-Kandianis CE, et al.
Comparative DNA sequence analysis of wheat and
rice genomes. Genome Res. 2003;13:1818–1827.
[53] Jena KK, Mackill DJ. Molecular markers and their use
in marker-assisted selection in rice. Crop Sci. 2008;
48:1266–1276.
[54] Ramalingam J, Savitha P, Alagarasan G, et al.
Functional marker assisted improvement of stable
cytoplasmic male sterile lines of rice for bacterial
blight resistance. Front Plant Sci. 2017;8:1–9.
[55] Swapna L, Khurana R, Vijaya-Kumar S, et al. Pollen-
specific expression of Oryza sativa indica pollen aller-
gen gene (OSIPA) promoter in rice and arabidopsis
transgenic systems. Mol Biotechnol. 2011;48:49–59.
[56] Ashkani S, Rafi MY, Sariah M, et al. Analysis of simple
sequence repeat markers linked with blast disease
resistance genes in a segregating population of rice
(Oryza sativa). Genet Mol Res. 2011;10:1345–1355.
[57] Ates D, Aldemir S, Alsaleh A, et al. A consensus link-
age map of lentil based on DArT markers from three
RIL mapping populations. PLoS One. 2018;13:
e0191375.
[58] Mulualem T, Bekeko Z. Advances in quantitative trait
loci, mapping and importance of markers assisted
BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT 13
selection in plant breeding research. Int J Plant
Breed Genet. 2016;10:58–68.
[59] Basavaraj SH, Singh VK, Singh A, et al. Marker-
assisted improvement of bacterial blight resistance
in parental lines of Pusa RH10, a superfine grain aro-
matic rice hybrid. Mol Breed. 2010;26:293–305.
[60] Dwivedi S, Tripathi RD, Srivastava S, et al. Growth
performance and biochemical responses of three
rice (Oryza sativa L.) cultivars grown in fly-ash
amended soil. Chemosphere. 2007;67:140–151.
[61] Servin B, Martin OC, Mezard M, et al. Toward a the-
ory of marker-assisted gene pyramiding. Genetics.
2004;168:513–523.
[62] Perumalsamy S, Bharani M, Sudha M, et al.
Functional marker-assisted selection for bacterial leaf
blight resistance genes in rice (Oryza sativa L.). Plant
Breed. 2010;129:400–406.
[63] He B, Huang X, Li D, et al. The cDNA cloning of a
novel bacterial blight-resistance gene ME137. Acta
Biochim Biophys Sin. 2013;45:422–424.
[64] Shanti ML, Shenoy VV, Devi GL, et al. Marker-assisted
breeding for resistance to bacterial leaf blight in
popular cultivar and parental lines of hybrid rice. J
Plant Pathol. 2010;92:495–501.
[65] Collard BCY, Mackill DJ. Conserved DNA-derived
polymorphism (CDDP): a simple and novel method
for generating DNA markers in plants. Plant Mol Biol
Rep. 2009;27:558–562.
[66] McCouch SR, Kochert G, Yu ZH, et al. Molecular
mapping of rice chromosomes. Theor Appl Genet.
1988;76:815–829.
[67] McCouch SR, Teytelman L, Xu Y, et al. Development
and mapping of 2240 new SSR markers for rice
(Oryza sativa L.). DNA Res. 2002;9:199–207.
[68] International Rice Genome Sequencing Project
(IRGSP): BiOS; 2019. Available from https://rgp.dna.
affrc.go.jp/IRGSP/
[69] Dai L, Liu X, Xiao Y, et al. Recent advances in cloning
and characterization of disease resistance genes in
rice. J Integr Plant Biol. 2007;49:112–119.
[70] Chu Z, Yuan M, Yao J, et al. Promoter mutations of
an essential gene for pollen development result in
disease resistance in rice. Genes Dev. 2006;20:
1250–1255.
[71] Sun X, Cao Y, Yang Z, et al. Xa26, a gene conferring
resistance to Xanthomonas oryzae pv. oryzae in rice,
encodes an LRR receptor kinase-like protein. Plant J.
2004;37:517–527.
[72] Litt M, Luty JA. A hypervariable microsatellite
revealed by in vitro amplification of a dinucleotide
repeat within the cardiac muscle actin gene. Am J
Hum Genet. 1989;44:397
[73] Narina SS, Orgeix CA, Sayre BL, et al. Optimization of
PCR conditions to amplify microsatellite loci in the
bunchgrass lizard (Sceloporus slevini) genomic DNA.
BMC Res Notes. 2011;4:26.
[74] Park YJ, Lee JK, Kim NS. Simple sequence repeat
polymorphisms (SSRPs) for evaluation of molecular
diversity and germplasm classification of minor
crops. Molecules. 2009;14:4546–4569.
14 S. C. CHUKWU ET AL.
[75] Candit R, Hubbel SP. Abundance and DNA sequence
of two base repeat regions in tropical genomes.
Genome. 1991;34:66–71.
[76] Maroof MS, Biyashev RM, Yang GP, et al.
Extraordinarily polymorphic microsatellite DNA in
barley: species diversity, chromosomal locations, and
population dynamics. Proc Natl Acad Sci USA. 1994;
91:5466–5470.
[77] Wu KS, Tanksley SD. Abundance, polymorphism and
genetic mapping of microsatellites in rice. Mol Gen
Genet. 1993;241:225–235.
[78] Ashkani S, Rafii MY, Shabanimofrad M, et al.
Molecular progress on the mapping and cloning of
functional genes for blast disease in rice (Oryza sat-
iva L.): current status and future considerations. Crit
Rev Biotechnol. 2016;36:353–367.
[79] Akkaya MS, Bhagwat AA, Cregan PB. Length poly-
morphisms of simple sequence repeat DNA in soy-
bean. Genetics. 1992;132:1131–1139.
[80] Panaud O, Chen X, McCouch SR. Development of
microsatellite markers and characterization of simple
sequence length polymorphism (SSLP) in rice (Oryza
sativa L.). Mol Gen Genet. 1996;252:597–607.
[81] Temnykh S, Park WD, Ayres N, et al. Mapping and
genome organization of microsatellite sequences in
rice (Oryza sativa L.). Theor Appl Genet. 2000;100:
697–712.
[82] Temnykh S, DeClerck G, Lukashova A, et al.
Computational and experimental analysis of microsa-
tellites in rice (Oryza sativa L.): frequency, length
variation, transposon associations, and genetic
marker potential. Genome Res. 2001;11:1441–1452.
[83] Gramene [Internet]: a comparative resource for
plants; 2019. Available from www.gramene.org
[84] McCouch SR, Chen X, Panaud O, et al. Microsatellite
marker development, mapping and applications in
rice genetics and breeding. Plant Mol Biol. 1997;35:
89–99.
[85] Chen X, Temnykh S, Xu Y, et al. Development of a
microsatellite framework map providing genome-
wide coverage in rice (Oryza sativa L.). Theor Appl
Genet. 1997;95:553–567.
[86] Mackill DJ, Zhang Z, Redon ~a ED, et al. Level of poly-
morphism and genetic mapping of AFLP markers in
rice. Genome. 1996;39:969–977.
[87] Provan J, Corbett G, Powell W, et al. Chloroplast
DNA variability in wild and cultivated rice (Oryza
spp.) revealed by polymorphic chloroplast simple
sequence repeats. Genome. 1997;40:104–110.
[88] Xu W, Virmani SS, Hernandez JE, et al. Genetic diver-
sity in the parental lines and heterosis of the tropical
rice hybrids. Euphytica. 2002;127:139–148.
[89] Enoki H, Sato H, Koinuma K. SSR analysis of genetic
diversity among maize inbred lines adapted to cold
regions of Japan. Theor Appl Genet. 2002;104:
1270–1277.
[90] Ullah I, Jamil S, Iqbal MZ, et al. Detection of bacterial
blight resistance genes in basmati rice landraces.
Genet Mol Res. 2012;11:1960–1966.
[91] Chen H, Wang S, Xing Y, et al. Comparative analyses
of genomic locations and race specificities of loci for
quantitative resistance to Pyricularia grisea in rice and
barley. Proc Natl Acad Sci USA. 2003;100:2544–2549.
[92] Neeraja CN, Maghirang-Rodriguez R, Pamplona A,
et al. A marker-assisted backcross approach for
developing submergence-tolerant rice cultivars.
Theor Appl Genet. 2007;115:767–776.
[93] Xu K, Deb R, Mackill DJ. A microsatellite marker and
a codominant PCR-based marker for marker-assisted
selection of submergence tolerance in rice. Crop Sci.
2004;44:248–253.
[94] Wang Z, Zou Y, Li X, et al. Cytoplasmic male sterility
of rice with boro II cytoplasm is caused by a cyto-
toxic peptide and is restored by two related PPR
motif genes via distinct modes of mRNA silencing.
Plant Cell. 2006;18:676–687.
[95] Cordeiro GM, Christopher MJ, Henry RJ, et al.
Identification of microsatellite markers for fragrance
in rice by analysis of the rice genome sequence. Mol
Breed. 2002;9:245–250.
[96] Collard BC, Mackill DJ. Marker-assisted selection: an
approach for precision plant breeding in the twenty-
first century. Philos Trans R Soc B: Biol Sci. 2008;363:
557–572.
[97] Das G, Rao GJN. Molecular marker assisted gene
stacking for biotic and abiotic stress resistance genes
in an elite rice cultivar. Front Plant Sci. 2015;6:1–18.
[98] Poczai P, Varga I, Laos M, et al. Advances in plant
gene-targeted and functional markers: a review.
Plant Methods. 2013;9:6.
[99] Okporie EO, Chukwu SC, Onyishi GC. Phenotypic
recurrent selection for increase yield and chemical
constituents of maize (Zea mays L.). World Appl Sci
J. 2013;21:994–999.
[100] Ross-Ibarra J, Morrell PL, Gaut BS. Plant domestica-
tion, a unique opportunity to identify the genetic
basis of adaptation. Proc Natl Acad Sci USA. 2007;
104:8641–8648.
[101] Kumar S, Rao M. Conventional and molecular breed-
ing for bacterial leaf blight and blast resistance in
rice. Res Rev J Ecol. 2014;3:1–3.
[102] Dossa GS, Sparks A, Cruz CV, et al. Decision tools for
bacterial blight resistance gene deployment in rice-
based agricultural ecosystems. Front Plant Sci. 2015;
6:1–5.
[103] Singh BD. Plant breeding: principles and methods.
New Delhi: Kalyani Publishers; 2015.
[104] Ashkani S, Rafii MY, Rahim HA, et al. Genetic dissec-
tion of rice blast resistance by QTL mapping
approach using an F 3 population. Mol Biol Rep.
2013;40:2503–2515.
[105] Collard BCY, Jahufer MZZ, Brouwer JB, et al. An
introduction to markers, quantitative trait loci (QTL)
mapping and marker-assisted selection for crop
improvement: the basic concepts. Euphytica. 2005;
142:169–196.
[106] Wang GL, Paterson AH. Assessment of DNA pooling
strategies for mapping of QTLs. Theor Appl Genet.
1994;88:355–361.
[107] DeVicente MC, Tanksley SD. QTL analysis of trans-
gressive segregation in an interspecific tomato cross.
Genetics. 1993;134:585–596.

[108] Tanksley SD. Mapping polygenes. Annu Rev Genet.
1993;27:205–233.
[109] Gelli M, Konda AR, Liu K, et al. Validation of QTL
mapping and transcriptome profiling for identifica-
tion of candidate genes associated with nitrogen
stress tolerance in sorghum. BMC Plant Biology.
2017;17:1–18.
[110] Du Q, Gong C, Wang Q, et al. Genetic architecture of
growth traits in Populus revealed by integrated
quantitative trait locus (QTL) analysis and association
studies. New Phytol. 2016;209:1067–1082.
[111] Manly KF, Cudmore RH, Jr, Meer JM. Map Manager
QTX, cross-platform software for genetic mapping.
Mamm Genome. 2001;12:930–932.
[112] Melchinger AE, Utz HF, Scho €n CC. Quantitative trait
locus (QTL) mapping using different testers and
independent population samples in maize reveals
low power of QTL detection and large bias in esti-
mates of QTL effects. Genetics. 1998;149:383–403.
[113] Doerge RW. Mapping and analysis of quantitative
trait loci in experimental populations. Nat Rev
Genet. 2002;3:43–52.
[114] Hospital F. Challenges for effective marker-assisted
selection in plants. Genetica. 2009;136:303–310.
[115] Ejeta G. Breeding for Striga resistance in sorghum:
exploitation of an intricate host–parasite biology.
Crop Sci. 2007;47:S-216.
[116] Ji Z, Shi J, Zeng Y, et al. Application of a simplified
marker-assisted backcross technique for hybrid
breeding in rice. Biologia (Poland). 2014;69:463–468.
[117] Pradhan SK, Nayak DK, Mohanty S, et al. Pyramiding
of three bacterial blight resistance genes for broad-
spectrum resistance in deepwater rice variety,
Jalmagna. Rice. 2015;8:19.
[118] Xu Y, Crouch JH. Marker-assisted selection in plant
breeding: from publications to practice. Crop Sci.
2008;48:391–407.
[119] Chukwu SC, Okporie EO, Onyishi GC, et al.
Application of diallel analyses in crop improvement.
Agric Biol J North Am. 2016;7:95–106.
[120] Bishwas N, Sharma M, Hasan A, et al. Improvement
of rice crop by Marker-assisted Backcross method.
Magnesium. 2016;8:2.
[121] Das G, Patra JK, Baek KH. Insight into MAS: a
molecular tool for development of stress resistant
and quality of rice through gene stacking. Front
Plant Sci. 2017;8:985.
[122] Amagai Y, Watanabe N, Kuboyama T. Genetic map-
ping and development of near-isogenic lines with
genes conferring mutant phenotypes in Aegilops
tauschii and synthetic hexaploid wheat. Euphytica.
2015;205:859–868.
[123] Hasan MM, Rafii MY, Ismail MR, et al. Marker-assisted
backcrossing: a useful method for rice improvement.
Biotechnol Biotechnol Equip. 2015;29:237–254.
[124] Tuberosa R, Forster BP, Ellis RP, et al. The develop-
ment and application of molecular markers for abi-
otic stress tolerance in barley. J Exp Bot. 2000;51:
19–27.
[125] Pinta W, Toojinda T, Thummabenjapone P, et al.
Pyramiding of blast and bacterial leaf blight resistance
BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT 15
genes into rice cultivar RD6 using marker assisted
selection. Afr J Biotechnol. 2013;12:4432–4438.
[126] Rajpurohit D, Kumar R, Kumar M, et al. Pyramiding
of two bacterial blight resistance and a semidwarf-
ing gene in Type 3 Basmati using marker-assisted
selection. Euphytica. 2011;178:111–126.
[127] Michelmore RW, Paran I, Kesseli RV. Identification of
markers linked to disease-resistance genes by bulked
segregant analysis: a rapid method to detect
markers in specific genomic regions by using segre-
gating populations. Proc Natl Acad Sci USA. 1991;88:
9828–9832.
[128] Sharma P, Dubey RS. Drought induces oxidative
stress and enhances the activities of antioxidant
enzymes in growing rice seedlings. Plant Growth
Regul. 2005;46:209–221.
[129] Singh S, Sidhu JS, Huang N, et al. Pyramiding three
bacterial blight resistance genes (xa5, xa13 and
Xa21) using marker-assisted selection into indica rice
cultivar PR106. Theor Appl Genet. 2001;102:
1011–1015.
[130] Joseph M, Gopalakrishnan S, Sharma RK, et al.
Combining bacterial blight resistance and Basmati
quality characteristics by phenotypic and molecular
marker-assisted selection in rice. Mol Breed. 2004;13:
377–387.
[131] Sundaram RM, Vishnupriya MR, Biradar SK, et al.
Marker assisted introgression of bacterial blight
resistance in Samba Mahsuri, an elite indica rice var-
iety. Euphytica. 2008;160:411–422.
[132] Pradhan SK, Nayak DK, Pandit E, et al. Incorporation
of bacterial blight resistance genes into lowland rice
cultivar through marker-assisted backcross breeding.
Phytopathology. 2016;106:710–718.
[133] Arunakumari K, Durgarani CV, Satturu V, et al.
Marker-assisted pyramiding of genes conferring
resistance against bacterial blight and blast diseases
into Indian rice variety MTU1010. Rice Sci. 2016;23:
306–316.
[134] Castro AJ, Capettini F, Corey AE, et al. Mapping and
pyramiding of qualitative and quantitative resistance
to stripe rust in barley. Theor Appl Genet. 2003;107:
922–930.
[135] Hittalmani S, Parco A, Mew TV, et al. Fine mapping
and DNA marker-assisted pyramiding of the three
major genes for blast resistance in rice. Theor Appl
Genet. 2000;100:1121–1128.
[136] Melchinger AE. Use of molecular markers in breed-
ing for oligogenic disease resistance. Plant Breed.
1990;104:1–19.
[137] Huang N, Angeles ER, Domingo J, et al. Pyramiding
of bacterial resistance genes in rice: marker aided
selection using RFLP and PCR. Theor Appl Genet.
1997;95:313–320.
[138] Swamy BPM, Kumar A. Genomics-based precision
breeding approaches to improve drought tolerance
in rice. Biotechnol Adv. 2013;31:1308–1318.
[139] Kelly JD, Miklas PN. The role of RAPD markers in
breeding for disease resistance in common bean.
Mol Breed. 1998;4:1–11.
[140] Jeung JU, Heu SG, Shin MS, et al. Dynamics of
Xanthomonas oryzae pv. oryzae populations in Korea
16 S. C. CHUKWU ET AL.
and their relationship to known bacterial blight
resistance genes. Phytopathology. 2006;96:867–875.
[141] Toenniessen GH, O’Toole JC, DeVries J. Advances in
plant biotechnology and its adoption in developing
countries. Curr Opin Plant Biol. 2003;6:191–198.
[142] Arshad HMI, Sahi ST, Atiq M. Appraisal of resistant
genes and gene pyramid lines of rice against indi-
genous pathotypes of Xanthomonas oryzae pv. ory-
zae in Punjab, Pakistan. Pak J Agric Sci. 2016;53:
365–370.