Journal of Biotechnology

& Crop Science Review

5(6): 4-31, 2016

Development of gene based markers for crop improvement

Rajesh Singh, Shiv Kuamr Agrawal
Received: 13 February 2016 Revised Accepted: 10 April 2016

ABSTRACT
The advancement in biological research encompassing: generation of huge amount of molecular-genetic data, development of
impressive methodological skills in molecular biology experimentation, and system and systems analyses, has set the stage to
search for ways/means to utilize the available resources to strengthen interdisciplinary efforts to find solutions to the
challenging goals in crop improvement. With the advent of molecular markers, a new generation of markers has been
introduced over the last two decades, which has revolutionized the entire scenario of biological sciences. A positive fall out of
such a realization and efforts has been the identification/development of a new class of very useful DNA markers called genic
molecular markers (GMMs) utilizing the ever-increasing archives of gene sequence information being accumulated under the
EST sequencing projects on a large number of plant species in the recent years. These markers being part of the cDNA/EST-
sequences, are expected to represent the functional component of the genome i.e. gene(s) in contrast to all other random DNA-
based markers (RDMs) that are developed/generated from the anonymous genomic DNA sequences/demonstrate large effects
on adaptive plant behavior remains fundamental to the development of GMMs. DNA-based molecular markers have acted as
versatile tools and have found their own position in various fields. They are no longer looked upon as simple DNA
fingerprinting markers in variability studies or as mere forensic tools. Ever since their development, they are constantly being
modified to enhance their utility and to bring about automation in the process of genome analysis. The discovery of PCR
(polymerase chain reaction) was a landmark in this effort and proved to be a unique process that brought about a new class of
DNA profiling markers. This facilitated the development of marker-based gene tags, map-based cloning of ergonomically
important genes, variability studies, phylogenic analysis, synteny mapping, marker-assisted selection of desirable genotypes,
etc. In this article, genic/ functional developed during the last two decades of molecular biology research and utilized for
various applications in the area of plant genome analysis are reviewed.

Key Words: Gene based markers, Molecular markers, Plant breeding, Simple equnce repeat

Plant Breeding and Molecular Marker

Conventional plant breeding is primarily based on
phenotypic selection of superior individuals among
segregating progenies resulting from hybridization.
Although significant strides have been made in crop
improvement through phenotypic selections for
agronomically important traits, considerable
difficulties are often encountered during this process,
primarily due to genotype-environment interactions.
Rajesh Singh ( )
Genetics and Plant Breeding, Institute of Agricultural Sciences,
BHU, Varanasi-221005 (UP), India
Email: rsingh6361@gmail.com
Shiv Kumar Agrawal
Lentil Breeder and Food Legumes Coordinator,
ICARDA/Rabat Office, PO box 6299, Rabat-Institutes, Rabat
10112, Morocco
Besides, testing procedures may be many times
ifficult, unreliable or expensive due to the nature of his
target traits (e.g. abiotic stresses) or the target
environment. With the advent of DNA marker
technology, several types of DNA markers and
molecular breeding strategies are now available to
plant breeders and geneticists, helping them to over
come many of them the problems faced during
conventional breeding. Markers that reveal
polymorphisms at the DNA level are known as
molecular markers. The last two decades have
witnessed a remarkable activity in the development
and use of molecular markers both in animal and plant
systems. This activity started with low-throughput

4

restriction fragment length polymorphisms and
culminated in recent years with single nucleotide
polymorphisms (SNPs), which are abundant and
uniformly distributed. There have been several reports
of the potential applications of molecular markers to
plant improvement (Burr et al 1983, Helentjaris et al
1985, Beckman and Soller 1986). A molecular marker
is defined as a particular segment of DNA that is
representative of the differences at the genome level.
Molecular markers may or may not correlate with
phenotypic expression of a trait. Molecular markers
offer numerous advantages over conventional
phenotype based alternatives as they are stable and
detectable in all tissues regardless of growth,
differentiation, development, or defense status of the
cell are not confounded by the environment,
pleiotropic and epistatic effects. The
identification/development of a new class of very
useful DNA markers called genic molecular markers
(GMMs) utilizing the ever-increasing archives of gene
sequence information being accumulated under the
EST sequencing projects on a large number of plant
species in the recent years. These markers being part
of the cDNA/EST-sequences, are expected to represent
the functional component of the genome i.e., gene(s),
in contrast to all other random DNA based markers
(RDMs) that are developed/generated from the
anonymous genomic DNA sequences/domains
irrespective of their genic content/information.
Therefore, identifying DNA sequences that
demonstrate large effects on adaptive plant behavior
remains fundamental to the development of GMMs
(Varshney et al 2007).
Molecular markers are now widely used to track loci
and genome regions in several crop breeding
programmes, as molecular markers tightly linked with
a large number of agronomic and disease resistance
traits are available in major crop species (Phillips and
Vasil 2001, Jain et al 2002, Gupta and Varshney
2004). These molecular markers include: (i)
hybridization-based markers such as restriction
fragment length polymorphism (RFLP), (ii) PCR-
based markers: random amplification of polymorphic
J of Biotech & Crop Sci (2016) 5(6): 4-31
DNA (RAPD), amplified fragment length
polymorphism (AFLP) and microsatellite or simple
sequence repeat (SSR), and (iii) sequence-based
markers: single nucleotide polymorphism (SNP). The
majority of these molecular markers has been
developed either from genomic DNA libraries (e.g.
RFLPs and SSRs) or from random PCR amplification
of genomic DNA (e.g. RAPDs) or both (e.g. AFLPs).
These DNA markers can be generated in large
numbers and can prove to be very useful for a variety
of purposes relevant to crop improvement. For
instance, these markers have been utilized extensively
for the preparation of saturated molecular maps
(genetical and physical). Their association with
genes/QTLs controlling the traits of economic
importance has also been utilized in some cases for
indirect marker-assisted selection (MAS) (e.g.
Koebner 2004, Korzun 2002). Other uses of molecular
markers include gene introgression through
backcrossing, germplasm characterization, genetic
diagnostics, characterization of transformants, study of
genome organization and phylogenetic analysis (Jain
et al 2002). For plant breeding applications, SSR
markers, among different classes of the existing
markers, have been proven and recommended as
markers of choice (Gupta and Varshney 2000). RFLP
is not readily adapted to high sample throughput and
RAPD assays are not sufficiently reproducible or
transferable between laboratories. While both SSRs
and AFLPs are efficient in identifying polymorphisms,
SSRs are more readily automated (Shariflou et al
2001). Although AFLPs can in principle be converted
into simple PCR assays (e.g. STSs), this conversion
can become cumbersome and complicated as
individual bands are often composed of multiple
fragments (Shan et al 1999), particularly in large
genome templates. An increasing number of
monogenic, race-specific genes showing a gene-for-
gene interaction have been mapped, and agronomically
important genes have been correlated to molecular
markers, as demonstrated for potato in Table 1. For
wheat, such validated markers are available for
resistance genes against powdery mildew (Pm1c,
Pm17, Pm24, mlRD30), the yellow dwarf virus, the
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J of Biotech & Crop Sci (2016) 5(6): 4-31

cyst nematodes (Cre1 and Cre3), and the rusts (Lr9,
Lr21, Lr24, Lr38, Lr47, Sr38, Yr5, Yr17) and
Fusarium head blight (Mohler and Singrun 2005).
Presently, the most powerful application of such
identified genes and molecular markers is opened up
by MAS. It offers the opportunity of combining
different genes for a given pathosystem in a single
genotype (gene pyramiding). A prerequisite for gene
pyramiding is that characters are not allelic.
Furthermore, knowledge on the gene distances in
genetic or better physical maps is very helpful. Using
such information, it was possible to combine three
race-specific powdery mildew genes (Pm) in a single
line which is now under variety test, hoping that such a
pyramided resistance will be rather durable (Figure 1).
The publication of Botstein et al (1980) about the
construction of genetic maps using restriction
fragment length polymorphism (RFLP) was the first
reported molecular marker technique in the detection
of DNA polymorphism. In RFLP, DNA polymorphism
is detected by hybridizing a chemically labelled DNA
probe to a Southern blot of DNA digested by
restriction endonucleases, resulting in differential
DNA fragment profile. This differential profile is
generated due to nucleotide substitutions or DNA
rearrangements like insertion or deletion or single
nucleotide polymorphisms. The RFLP markers are
relatively highly polymorphic, codominantly inherited
and highly reproducible. Because of their presence
throughout the plant genome, high heritability and
locus specificity the RFLP markers are considered
superior. The method also provides opportunity to
simultaneously screen numerous samples. The
technique is not very widely used because it is time
consuming, involves expensive and radioactive/toxic
reagents and requires large quantity of high quality
genomic DNA. After the invention of polymerase
chain reaction (PCR) technology (Mullis and Faloona
1987), a large number of approaches for generation of
molecular markers based on PCR were undertaken.
The RAPD technique is based on PCR amplification
of genomic DNA. It deduces DNA polymorphisms
produced by rearrangements or deletions at or
between oligonucleotide primer binding sites in the
genome using short random oligonucleotide
sequences (mostly ten bases long) (Williams et al
1991).

Table 1 Some important and mapped DNA markers on the example of potato (Wenzel 2006).
Trait Gene Chromosome
Potato virus Y Ryadg XI
Nytbr IV
Potato virus X Rx1, Rx2 XII, V
Na XI
Nb V
Nx IX
Potato leaf roll virus PLRV QTL XI
Globodera rostochiensis Gro III, VII, X, XI
H1 V
Globodera pallida Gpa QTL IV,V,IX,XII
Gpa2 XII
Phytophthora infestans RB X
R1 V
R2 IV
R3, R6, R7 XI
Rblc VIII
QTL V
Synchytrium endobioticum Sen1 XI
Erwinia carotovora QTL
Tuber starch, tuber yield QTL IXII
Cold sweetening QTL V, IX
Skin colour QTL X
Tuber flesh color QTL III

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As the approach requires no prior knowledge of the
genome that is being analyzed, it can be employed
across species using universal primers. The major
drawback of the method is that the profiling is
dependent on the reaction conditions so may vary
within two different laboratories and as several
discrete loci in the genome are amplified by each
primer, profiles are not able to distinguish
heterozygous from homozygous individuals (Bardakci
2001). Due to the speed and efficiency of RAPD
analysis, high-density genetic mapping in many plant
species such as alfalfa (Kiss et al 1993) faba bean
(Torress et al 1993) and apple (Hemmat et al 1994)
was developed in a relatively short time. The RAPD
analysis of NILs (non-isogenic lines) has been
successful in identifying markers linked to disease
resistance genes in tomato (Lycopersicon sp.) (Martin
et al 1991) lettuce (Lactuca sp.) (Paran et al 1991) and
common bean (Phaseolus vulgaris) (Adam-Blondon et
J of Biotech & Crop Sci (2016) 5(6): 4-31
al 1994). To overcome the limitation of reproducibility
associated with RAPD, AFLP technology (Vos et al
1995) was developed. It combines the power of RFLP
with the flexibility of PCR-based technology by
ligating primerrecognition sequences (adaptors) to the
restricted DNA and selective PCR amplification of
restriction fragments using a limited set of primers The
AFLP technique generates fingerprints of any DNA
regardless of its source, and without any prior
knowledge of DNA sequence. Most AFLP fragments
correspond to unique positions on the genome and
hence can be exploited as landmarks in genetic and
physical mapping. The technique can be used to
distinguish closely related individuals at the sub-
species level (Althoff et al 2007) and can also map
genes. Applications for AFLP in plant mapping
include establishing linkage groups in crosses,
saturating regions with markers for gene landing
efforts (Yin et al 1999) and assessing the degree of
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J of Biotech & Crop Sci (2016) 5(6): 4-31
relatedness or variability among cultivars (Mian et al
2002). Single nucleotide variations in genome
sequence of individuals of a population are known as
SNPs. They constitute the most abundant molecular
markers in the genome and are widely distributed
throughout genomes although their occurrence and
distribution varies among species. Maize has 1 SNP
per 60-120 bp (Ching et al 2002), while humans have
an estimated 1 SNP per 1,000 bp (Sachidanandam et al
2001). The SNPs are usually more prevalent in the
non-coding regions of the genome. Within the coding
regions, an SNP is either non-synonymous and results
in an amino acid sequence change (Sunyaev et al
1999), or it is synonymous and does not alter the
amino acid sequence. Synonymous changes can
modify mRNA splicing, resulting in phenotypic
differences (Richard and Beckman 1995).
Improvements in sequencing technology and
availability of an increasing number of EST sequences
have made direct analysis of genetic variation at the
DNA sequence level possible (Buetow et al 1999,
Soleimani et al 2003). Majority of SNP genotyping
assays are based on one or two of the following
molecular mechanisms: allele specific hybridization,
primer extension, oligonucleotide ligation and invasive
cleavage (Sobrino et al 2005). High throughput
genotyping methods, including DNA chips, allele-
specific PCR and primer extension approaches make
single nucleotide polymorphisms (SNPs) especially
attractive as genetic markers. They are suitable for
automation and are used for a range of purposes,
including rapid identification of crop cultivars and
construction of ultra high-density genetic maps.
However, with the availability of microarrays, SNP
platforms have been developed, which allow
genotyping of thousands of markers in parallel.
Besides SNPs, some other novel marker systems,
including single feature polymorphisms, diversity
array technology and restriction site-associated DNA
markers, have also been developed, where array-based
assays have been utilized to provide for the desired
ultra-high throughput and low cost. These micro array-
based markers are the markers of choice for the future
and are already being used for construction of high-
density maps, quantitative trait loci (QTL) mapping
(including expression QTLs) and genetic diversity
analysis with a limited expense in terms of time and
money.
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GENIC MOLECULAR MARKERS
Functional genomics have led and established several
gene discovery projects in the form of genome
sequencing, transcriptome sequencing or gene
expression studies since last five years. A large
number of genes have been identified through wet
lab as well as in silico studies and a wealth of
sequence data have been accumulated in public
domain (http://www.ncbi.nlm.nih.gov,
http://www.ebi.ac.uk) in the form of BAC (bacterial
artificial chromosome) clones, ESTs (expressed
sequence tags), full length cDNA clones and genes.
The availability of vast amount of information from
complete or partial genes has led to development of
the molecular markers directly from the parts of genes.
These markers are referred as genic molecular
markers (GMM). The majority of the markers,
developed and used in the past as described earlier, is
directly derived from the genomic DNA, and therefore
could belong to either the transcribed or the non-
transcribed part of the genome without any
information available on their functions. In contrast,
GMMs developed from coding sequences like ESTs or
fully characterized genes frequently have been
assigned known functions. Based on the site of
polymorphism and its expression of phenotype,
GMMs have been classified into two groups
(Anderson and Luebberstedt, 2003):
(a) Gene-targeted markers (GTMs): derived from
polymorphisms within genes, however not necessarily
involved in phenotypic trait variation, e.g. untranslated
regions (UTRs) of EST sequences (Schmitt et al 2006,
Aggarwal et al 2007).
(b) Functional markers (FMs): derived from
polymorphic sequences or sites within genes and, thus,
more likely to be causally involved in phenotypic trait
variation (e.g. candidate gene-based molecular
markers).
The FMs, depending on the involvement in the
phenotypic trait variation, are further classified into
two subgroups: (a) indirect functional markers (IFMs),
J of Biotech & Crop Sci (2016) 5(6): 4-31
for which the role for phenotypic trait variation is
indirectly known, and (b) direct functional markers
(DFMs), for which the role for the phenotypic trait
variation is well proven.
Functional markers: With the advent of high-
throughput sequencing technology, abundant
information on DNA sequences for the genomes of
many plant species has been generated (Goff et al
2002, The Arabidopsis Genome Initiative 2000, Yu et
al 2002). ESTs of many crop species have been
generated and thousands of sequences have been
annotated as putative functional genes using powerful
bioinformatics tools. To gain benefits from plant
genomics, new knowledge must be translated into
crop varieties with improved characteristics (Thro et al
2004). Functional markers (FMs) are a good
translator of gains from emerging technologies into
improved crop cultivars. FMs are derived from
polymorphic sites within genes causally involved in
phenotypic trait variation. Once genetic effects have
been assigned to functional sequence motifs, FMs
derived from such motifs are used for fixation of gene
alleles in a number of genetic backgrounds without
additional calibration. FM development requires (1)
functionally characterized genes, (2) allele sequences
from such genes, (3) identification of polymorphic,
functional motifs affecting plant phenotype within
these genes and (4) validation of associations between
DNA polymorphisms and trait variation (Chun et al.
2006).
In order to correlate DNA sequence information with
particular phenotypes, sequence-specific molecular
marker techniques have been designed. Microsatellite
or short tandem repeats or simple sequences repeats
are monotonous repetitions of very short (one to five)
nucleotide motifs, which occur as interspersed
repetitive elements in all eukaryotic genomes (Tautz
and Renz 1984). Variation in the number of tandemly
repeated units is mainly due to strand slippage during
DNA replication where the repeats allow matching via
excision or addition of repeats (Schlotterer and Tautz
1992). As slippage in replication is more likely than
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point mutations, micro satellite loci tend to be hyper
variable. Microsatellite assays show extensive inter-
individual length polymorphisms during PCR analysis
of unique loci using discriminatory primers sets.
Expressed sequence tag (EST) projects have generated
a vast amount of publicly available sequence data from
plant species; these data can be mined for simple
sequence repeats (SSRs). These SSRs are useful as
molecular markers because their development is
inexpensive, they represent transcribed genes and a
putative function can often be deduced by a homology
search. Because they are derived from transcripts, they
are useful for assaying the functional diversity in
natural populations or germplasm collections. These
markers are valuable because of their higher level of
transferability to related species, and they can often be
used as anchor markers for comparative mapping and
evolutionary studies. They have been developed and
mapped in several crop species and could prove useful
for marker-assisted selection. Functional Markers are
further classed as Direct Functional Markers and
Indirect Functional Markers.
Direct functional markers: Functional markers are
developed from gene sequences with known
expression and hence it is of prime importance to
establish proof of gene function affecting a particular
phenotype. The most direct means of obtaining proof
of sequence motif function is by comparing isogenic
genotypes differing in single sequence motifs. At
current, the most appropriate approach for generating
isogenic lines in crops is by targeting induced local
lesions in genomes (TILLING). TILLING provides
point mutant alleles that are usually induced by
chemical mutagens like ethyl methane sulphonate and
these mutations are used in functional genomics and
gene characterization. The ability to detect point-
mutations in specific genes within a large population
of mutagenized plants was first demonstrated by Claire
McCallum from the Henikoff and Comai laboratories
at the Fred Hutchinson Cancer Center and the
University of Washington in Seattle (Nat Biotechnol.
2000 Apr, 18(4): 455-7 and Plant Physiol, June 2000,
Vol 123, pp 439-442) and coined the word TILLING.
Since then TILLING has been developed in several
species including Arabidopsis, rice, maize, sorghum,
wheat, barley, tomato, soybean, rapeseed etc.
TILLING helps in generating a series of missense
mutations in the alleles of a target gene. The
polymorphisms that are generated by these mutations
are compared with phenotypic variation to provide
direct functional markers. Thus TILLING represents a
viable method by which spontaneous and induced
mutants help in the direct identification of beneficial
nucleotide and amino acid changes in genes with
known functions that can further be used in the
development of diagnostic functional markers for
selection.
Indirect functional markers: Association studies have
the potential to identify sequence motifs affecting trait
expression. They provide indirect evidence for the
function of a sequence motif. In association studies the
polymorphisms that exist within the gene such as a
few nucleotides differences or insertions/deletions, are
correlated to the phenotype of interest. With the advent
sequencing methods it is possible to find the SNP
variations that occur through out the gene and the
linkage disequilibrium (LD) that exists between these
SNPs. Based on the LD of the SNPs they can be
grouped as haplotype SNPs and those haplotype SNPs
that are involved in the functional variation of the gene
can be identified. This strategy would considerably
reduce the number of SNPs that has to be genotyped.
The selected SNPs which are functional variants can
be genotyped on a set of accessions to find
associations with the phenotype of interest. In one of
the pioneering studies, nine sequence motifs in the
dwarf8 gene of maize were shown to be associated
with variation for flowering time. A set of 92 inbred
lines were genotyped and associations between the
polymorphisms in the dwarf8 gene and flowering time
was established. These associations of the
polymorphisms in the dwarf 8 gene aided in the
selection of lines that exhibited early flowering by 7-
11 days (Thornsberry et al 2001). Similarly
associations of three haplotypes of the StVe1 locus of
potato that confers resistance to V. alboatrum were
10

used to genotype 30 potato cultivars and one haplotype
showed significant associations with V. alboatrum
resistance (Simko et al 2004). A recent study in grapes
showed that allelic variation in the gene VvmybA1 that
is responsible for transcriptional regulation of
anthocyanin biosynthesis, was associated with
multiple classes of fruit color in the 200 accessions of
cultivated grapevine that were studied. One SNP and
three insertion/deletions (indels) were found to be
significantly associated with fruit skin color in the
structured association analysis of pigmented
accessions. All four polymorphisms were associated
with genetic differences separating black or gray-
skinned accessions from red and pink skinned
accessions (This et al 2007). The use of functional
motifs to correlate with phenotypes requires
comprehensive allele sequencing, a relatively low LD
between haplotypes and a phenotypically well
characterized population. Several studies have been
carried out on LD decay and haplotype diversity
(Dvornyk et al 2002, van der Voort et al 2004, Oleson
et al 2004, Neale and Savolainen 2004). In crops with
low LD and high resolution of intragenic
polymorphisms, association studies have the potential
to identify sequence motifs that are correlated with
trait variation.
The molecular markers derived from anonymous
regions of the genome are called random DNA
markers (RDMs), which may or may not be developed
from the polymorphic site in gene or may not be
developed from a gene at all. Although genic markers
were developed earlier also, these were in the form of
cDNARFLP (Graner et al 1991, Causse et al 1994)
for which functions could not be predicted at that time.
However, some efforts were made to sequence these
early cDNA clones to determine the genes and their
functions (Michalek et al 1999). Compared to these
earlier efforts, development of genic markers have
become a reality only in recent years, because of
accumulation of large ESTs or gene sequences
resources resulting from EST and genome sequencing
projects in several crop species and also due to the
developments in the field of bioinformatics (Gupta and
J of Biotech & Crop Sci (2016) 5(6): 4-31
Rustgi 2004). For example, several transcriptome
resources have become available
(http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summar
y.html), and software tools or pearl scripts have been
developed to search for SSRs and SNPs from EST or
gene sequences (Varshney et al 2004, 2005a).
Although, whole genome sequencing and annotation is
the way to identify the entire gene repository of a
species, this has been possible only for a limited
number of crop species involving large scale
sequencing of their genome or gene space. On the
other hand, ESTs represent a basic commodity within
the analysis of genomes and their genes for a species
(Rudd et al 2003). Whereas the complete sequencing
of a genome may utilize either a clone-by-clone
approach or a whole genome shotgun approach to
acquire adequate coverage to assemble a meaningful
scaffold, EST sequencing is directed at the quick,
cheap and simple sequencing of partial gene
transcripts (Sreenivasulu et al 2002). As a result, a
significant redundancy can be observed in gene
sequence data obtained from EST sequencing projects
(see Varshney et al 2004). Therefore before
developing molecular markers from ESTs, it is
essential to define the unigenes after cluster analysis
of random ESTs using appropriate computer
programmes such as stack Pack (Miller et al 1999).
Once the unigene sequence data from EST analysis or
non-redundant set of genes are available, molecular
markers can be developed using two main approaches:
(1) Direct mapping: Under this approach, either the
cDNA clones corresponding to the ESTs of interest
can be used as RFLP probe or the PCR primers can
two common ways to develop GMMs are shown in the
figure. In the first method, the sequence data are used
to define the unigenes and then the cDNA clones or
genic clones corresponding to the unigenes can be
assayed as RFLPs or the unigene sequence data can be
used to design the primer pairs and assayed using
STS/CAPS or SNP assays. In the second method,
the sequence data can be mined by using some
computer programmes or scripts to identify the SSRs,
SNPs or COSs from given sequence data and hen these
markers, after defining the unigenes, can be assayed
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J of Biotech & Crop Sci (2016) 5(6): 4-31
Figure 2 A schemes for development of genic molecular
markers (GMMs).
using appropriate genotyping platforms designed for
the EST/gene and used as STS or CAPS marker.
Direct mapping approach should be undertaken with
the unigene set of ESTs or genes only.
(2) In silico mining: In this approach, the SSR or SNP
identification software tools are used to screen the
sequence data for ESTs/genes. For identification of
SNPs, the redundant set of EST data, generated from
more than one genotype of a given species, are used.
However, after identification of SNPs, only
nonredundant set of ESTs should be considered for
SNP mapping. A scheme for development of GMMs
has been shown in Figure 2. Development of FMs,
however, requires: (i) functionally characterized genes,
(ii) allele sequences from such genes, (iii)
identification of polymorphic, functional motifs
affecting plant phenotype within these genes, and (iv)
validation of associations between DNA
polymorphisms and trait variation. Therefore
depending on the objective as well as available
information or feasibility, the FMs, the special class of
GMMs, can also be generated.
EST programs: Apart from whole genome sequencing
projects and gene space sequencing, strategies to study
the transcriptome of several important crop species
have lead to the establishment of expressed sequence
tag (EST) sequencing projects for gene discovery.
Generation of ESTs is a quick and simple strategy
involving partial sequencing of 5 or 3 end of the
cDNAs. A wealth of DNA sequence information has
been generated from these projects and deposited in
online databases like http://www.ncbi.nlm.nih.go:
National Centre for Biotechnology Information,
http://www.tigr.org: the Institute of Genome Research,
http://www.ebi.ac.uk: European Bioinformatics
Institute. The plant EST database at EMBL has
exceeded over five million EST sequences. ESts have
been developed from more than 50 plant species and in
each species more than 5000 ESTs have been made
available. These species represent important crops and
their sequences are a potential source from which
several valuable molecular markers that are of interest
to plant breeders can be generated. Several crop
specific EST databases are available for crops like
wheat http://genome.arizona.edu,
http://wheat.pw.usda.gov/genome,
http://wheat.pw.usda.gov/NSF/ barley
http://hordeum.ipk-gatersleben.de/est/est.html,
sugarcane http://sucest.lad.ic.unicamp.br/en/ etc. In
species that lack EST resources comparative mapping
strategies have facilitated the isolation of genes from
these species. GRAMENE (http://www.gramene.org)
is a curated opensource database that is available for
comparative genome analysis for grasses. HarVEST
(http://harvest.ucr.edu) is software developed to enable
searching for differentially expressed ESTs among
cDNA libraries and is oriented towards comparative
genomics. However since the EST sequences are
generated through partial sequencing of the 3 or 5
ends of cDNAs there is redundancy in the genes
sequences that are obtained from the databases. The
EST data has to be clustered to identify unigenes from
random EST sequences using bioinformatics tools.
The NCBI UniGene Resources is an excellent system
for automatically partitioning GenBank sequences into
a nonredundant set of gene oriented clusters. UniGene
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Sets are available for wheat, barley and many other
crop plants in the NCBI database. The TIGR Gene
Indices includes ESTs clustered into Tentative
Consensi (TC), with top 5 peptide hits, and alignment
to rice BACs and Arabidopsis chromosomes. These
unigenes are then utilized to develop molecular
markers.
Approaches for the development of microsatellites
markers: SSRs are actually considered the most
efficient markers, but their use is still limited because
of the long and laborious steps to develop them. There
are two general strategies to access these regions and
create SSR markers: (1) searching for sequences
containing microsatellites in the available data bases;
or (2) constructing and screening the genomic (or
other) library with probes complementary to
microsatellite sequences. Exceptionally, some
strategies without library construction have been
developed.
Data base searching is a cost effective tool for the
development of SSRs: This strategy of developing
SSR markers is based on searching for sequences
containing microsatellites deposited in the data bases
(EMBL, GenBank). This method is cost-effective,
simple and relatively quick; however, it does show
some limitations. It should be underlined that when
exploring data from expressed sequences, a
considerable amount of potential polymorphism can be
lost, as microsatellites are broadly present in the non-
coding regions of genomes. Additionally, this strategy
is limited to plants with high economical or scientific
interest which are well represented in the databases. In
rice (Cho et al 2000), showed that microsatellites
derived from genomic libraries detected a higher level
of polymorphism than those derived from ESTs
contained in the GenBank database (83.8% vs. 54.0%).
The other measures of genetic variability, like the
number of alleles per locus, polymorphism
information content, and allele size ranges, were
higher in the case of the genomic library- than in that
of the EST-derived microsatellites. Conversely, in rye,
Hackauf and Wehling (2002) identified much more
J of Biotech & Crop Sci (2016) 5(6): 4-31
effective SSR loci when exploring EST data bases
than Saal and Wricke (1999) that searched the
genomic library. The authors examined more than
8000 rye cDNA sequences from anthers, cold-stressed
leaves, and aluminium-stressed and unstressed roots.
A total of 157 sequences out of 528 SSRs comprising
di-triand tetra-nucleotide motifs turned out to be useful
for primer design. One hundred EST-derived loci
displayed a length polymorphism among 15 rye
accessions.
Cross species amplification leads to the development
of SSR markers: Cross species amplification is also a
powerful approach to develop microsatellites markers
in plants. Database searching is an economic approach
for obtaining new microsatellite loci (Brown et al
1996). However, database searching alone is unlikely
to provide sufficient markers in plant species for
mapping or breeding applications. The application of
cross-species transfer of microsatellites was difficult to
predict (Brown et al 1996). The taxonomic distance of
the species of interest and conservation of the flanking
sequence determines whether the correct region is
amplified and how much is the variability in the
microsatellites.
The reaction conditions are often need to be optimized
the products sequenced to verify the presence of the
microsatellite region. Microsatellites have been
transferred between closely related plant species, but
there is not much information is available between the
genera.
Library construction strategy for development of
SSRs: This strategy is usually used for newly analyzed
species. The following steps are involved in generating
SSR markers from a library:
Isolation of DNA
Digestion with the appropriate restriction enzymes
Hybridization with probes composed of several
repeats
Sequencing of positive clones
Designing of primers complementary to both
flanking regions.
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J of Biotech & Crop Sci (2016) 5(6): 4-31
Although such an approach has been applied in many
cases (Roder et al 1995, Saal and Wricke 1999,
Ashkenazi et al 2001, Brown et al 1996, Panaud et al
1996, Taramino et al 1996) a number of disadvantages
seems to be common for research starting from library
construction, especially in species with large genomes.
The most often-admitted problems are the low
effectiveness and specificity of hybridization as well
as the presence of one-side flanks in sequenced
fragments. In rye, Saal and Wricke (1996) sequenced
seventy-four (40.7%) out of 182 positive clones, and
the primer pairs were designed for 57 (31.3%) of them.
Only 27 primer pairs resulted in specific SSR markers,
of which, 20 were mapped. From this calculation
comes the final efficiency of about 10%.
The sequencing of 1739 positive clones in wheat (511
for GT and 1228 for GA motifs) resulted in obtaining
70 primer pairs, among them only 25 (less than 2%)
gave amplified fragments with the expected length
(Roder et al 1995). In order to increase the amount of
successful sequencing, positive clones can be pre-
screened for insert length, repeat position and
orientation by the use of an anchor PCR technique
described by Rafalski et al 1996. In this technique, a
set of PCR reactions with a combination of four
primers (two vector and two degenerated primers
complementary to the repeat) is carried out. Clones
containing microsatellites positioned either too close
or too far from the cloning site are not amplified.
Enriched libraries are the rich source of SSR
markers: Different enrichment methods have been
developed to increase the efficiency of microsatellite
loci isolation from genomic DNA libraries. Recently,
the attractiveness of enriched protocols has
increased notably, especially in plants Zane et al
(2003). A standard method for the isolation of plant
microsatellite loci involves screening colonies/plaque
with oligonucleotide probes complementary to
microsatellite repeats. Enrichment by primer extention,
enrichment by hybridization and enrichment by
screening random amplified polymorphic DNA
(RAPD) profiles are other approaches for enrichment.
Different microsatellite enrichment methods have been
given in the table 2.
The most popular method of enriched library
construction is selective hybridization of DNA
fragments using streptavidin-coated magnetic beads or
nylon membranes. The procedure of the construction
of enriched libraries using streptavidin-coated
magnetic beads or nylon membranes comprises the
following steps:
DNA digestion and ligation of the resulting
fragments to double-stranded adaptors
Their hybridization to biotinylated microsatellite
probes, followed by binding to streptavidin-coated
magnetic beads
The elution of the DNA fragments from the beads,
and PCR amplification with primers
complementary to the adaptor sequence
Cloning of the amplified products into the vector
Transformation of Escherichia coli
Sequencing of the positive clones
Such an enrichment method has been successfully
applied to plants by several authors (Fischer et al.
1998, Hamilton et al 1999, Milbourne et al 1998,
Prochazka et al 1996) with minor modifications, such
as additional screenings for the presence of SSRs or
the use of l phagemids instead of E. coli. In spite of the
sufficient level of progress in the efficiency of positive
clone isolation, the procedure employing magnetic
beads allows enrichment in a single or, in the best
case, several SSR motifs. This problem can be solved
by using Nylon membranes with many bound
microsatellite oligonucleotides, as proposed by
Edwards et al (1996).
Other strategies without library construction: The
construction of genomic library for the development of
SSR is time consuming process and it usual takes up to
one month. To avoid this problem, several procedures
without library construction have been developed. One
group of protocols is based on the fact that RAPD
fragments contain SSRs more frequently than random
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 J of Biotech & Crop Sci (2016) 5(6): 4-31

genomic clones. This procedure starts with a random SSR probes and selective cloning of positive bands, or
PCR amplification (either with RAPD starters or by cloning and screening all the products (Lench et al
microsatelliteanchored random primers) followed 1996, Cifarelli et al 1995, Lunt et al 1999).
either by Southern hybridization of PCR products with

Table 2 Summary of microsatellite enrichment method (Adapted from Maguire TL 2001).

Enrichment Method Level of enrichment Reference
Enrichment by Primer extension
Microsatellite oligonucleotide 50-fold compared with un-nriched Ostander et al
24 positive clones sequenced, all contained (1992)
microsatellites Paetku (1999)
Degenerate oligonucleotide 15 positive clones sequenced, 13 contained Fisher et al (1996)
microsatellites 19 positive clones sequenced, all
contained microsatellites Koblizkova et al
(1998)
Enrichment by hybridization
Streptavidin-coated magnetic beads 48 positive clones sequenced, 29 contained Fisher and
microsatellites Bachmann (1998)
9 positive clones sequenced, 5 contained
microsatellites Prochazka (1996)
207 positive clones sequenced, 180 contained
microsatellites
20% positive clones compared with un-enriched
with no detectable positive clones
12 positive clones sequenced, 8 contained
microsatellites 120 positive clones sequenced, all Kijas et al (1994)
contained microsatellites
Connel et al (1998)
Hamilton et al
(1999)
Nylon membranes 50-70% clones randomly sequenced contained Edwards et al
microsatellites (1996)
Enrichment by screening RAPD 30 positive clones sequenced, 21 contained Ueno et al (1999)
profiles microsatellites
14 positive clones sequenced, 12 contained
microsatellites Lunt et al (1999)

An interesting nonlibrary protocol based on the
same idea was proposed by Zane et al. (2003). In this
protocol, called FIASCO (Fast Isolation by AFLP of
Sequences Containing repeats), products derived in a
fast and efficient digestion-ligation reaction of AFLP
were hybridized with biotinylated probes, followed by
selective capturing of microsatellites with streptavidin-
coated beads. The usefulness of SSR markers for
numerous purposes has been well documented for
plants; among such purposes, the construction of
molecular maps has a dominant position (Roder et al
1995, Saal and Wricke (1996), Taramino et al 1996,
Becker et al 1995, De la Rosa 2003, Hackenberger et
al 2003, Roder et al 1998, Tang et al 2002). Expressed
sequence tag derived microsatellite loci were detected
and mapped in many species, such as barley (De la
Rosa et al 2003), alfalfa (Mahalakshmi et al 2002),
maize (Senior et al 1993), and rice (Temnykh et al
2000). The SSRs are abundant, ubiquitous and
hypervariable in nature; this attracted the attention of
breeders who could utilize them for MAS, a modern
tool in breeding. Masojc et al 2002 listed four major

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J of Biotech & Crop Sci (2016) 5(6): 4-31
strategies for finding a molecular marker tightly linked
to a target gene of agronomic importance. The first
approach uses NILs which are differentiated only by
the allelic sets in the gene of interest and in the
adjacent chromosomal region. The second one
involves BSA. The third one comprises the
identification of QTLs, and the last strategy involves
computer databases. In the literature, there are several
examples of applying SSRs for these purposes.
Recently, by means of the BSA strategy, SSR markers
closely linked to genes conferring resistance against
sugarcane mosaic virus in maize Scmv1 and Scmv2
(Duble et al 2003), and leaf rust in barley Rph5
(Mammadov et al 2003) were identified. Zhou et al
2003, showed that the MAS for the major scab
resistance QTL with the SSR markers combined with
phenotypic selection were much more effective than
selection based only on phenotypic evaluation in an
early generation. The authors identified markers linked
to the major QTL on chromosome 3BS in the original
mapping population, these were closely associated
with scab resistance. Another interesting application of
SSRs in rice breeding was described by Liu and Wu
1998. The authors showed that it is possible to predict
heterosis and hybrid performance by the detection of
the chromosomal regions influencing yield. However,
the use of SSR markers is still relatively expensive for
application on a large scale in breeding programs.
Because of the possibility to detect several alleles at a
high frequency, SSRs turned out to be an ideal tool for
identifying individuals and for establishing genetic
diversity between them. It was well demonstrated in
the study by Prasad et al 2000, who examined 55 elite
wheat genotypes with SSR markers, and found that a
set of only 12 primer pairs allowed a maximum of 48
genotypes to be distinguished. In the study published
by Ashkenazi et al 2001, two SSR markers were
sufficient to discriminate between 12 potato cultivars.
SSRs have also been applied in phylogenetic
investigations for the construction of evolutionary
trees, in, among other species, melon (Monforte et al
2003) and barley (Provan et al 1999). Yaish and Perez
de la Vega 2003 were the first to identify (GA)n
microsatellite containing loci linked to a putative
MADS-box gene (PVMADS) in the common bean.
Afterwards, the authors constructed an un-rooted
phylogenetic tree of the MADS-box genes of
Arabidopsis and the common bean, which made it
possible to show that the PVMADS gene is closely
related to the AGL2 group of Arabidopsis, involved in
floral morphogenesis. It was demonstrated that
microsatellites in plants could even be up to ten-fold
more variable than other markers; thus, they are highly
recommended for genetic diversity analysis. Russell et
al 1997 compared the level of polymorphism in barley
as detected by four types of markers: RFLPs, AFLPs,
SSRs and RAPDs. Although all four assays were able
to detect the polymorphism between 18 cultivated
barley accessions, the similarity index was the lowest
in the case of SSRs for both the spring and winter
types while the diversity index calculated based on
SSR data was similar to that obtained for AFLPs. The
high level of DNA polymorphism of SSRs makes
them especially useful for self-pollinated species like
wheat (Roder et al 199)5 or barley (Becker et al 1995).
However, they have also been used successfully in
open-pollinated plants as rye (Saal and Wricke 1999)
or maize (Taramino et al 1996).
ISSR MARKERS
Microsatellites are usually more or less proportionally
dispersed in the genome. However, regions with a
greater abundance of these sequences have been found
and are named "SSR hot spots" (Bornet et al 2002a,
Bornet et al 2002b, Zietkiewicz et al 1994). such
regions can serve as a source of ISSR markers. The
ISSR technology is based on the amplification of
regions (100-3000 bp) between inversely oriented
closely spaced microsatellites (Zietkiewicz et al 1994).
Single primers (16-18 bp) consisting of several simple
sequence repeats used for an amplification of these
regions can be based on any SRR motif and be 5 or 3
anchored by 2-4 (usually) arbitrary selective
nucleotides. However, nonanchored primers have also
been used (Bornet et al 2002b). The resulting PCR
products are anonymous SSR loci. ISSRs usually
amplify 25 to 50 products in one reaction. The number
16

of bands produced may be negatively correlated with
the number of nucleotides in the repeat unit of the
motif, as shown by (Nagaraju et al 2002), who
investigated the genetic relationship between Basmati
and non-Basmati rice varieties. The major advantage
of this method is the fact that it does not require a
time-consuming (and expensive) step of genomic (or
other) library construction. In spite of the fact that
ISSRs are mostly inherited as dominant or rarely as
codominant genetic markers (if the length of the
intervening space between the microsatellites has
changed) and are random-type markers, they are
thought to be highly useful for many different
purposes. This has been confirmed in numerous
studies. They seem to be especially suitable for
phylogenetic studies, the evaluation of genetic
diversity and cultivar identification (Zietkiewicz et al
1994, Nagaraju et al 2002, Blair et al 1999, Cavan et
al 2000, Fang et al 1997, Gupta et al 1994, Jain et al
1999, Korbin et al 2002, Raina et al 2001, Wolfe
1998). The simplicity of ISSR markers predetermines
them for gene tagging. An excellent example was
reported on by (Ammiraju et al 2001), who tested the
association of ISSRs with seed size in wheat. The
authors found three markers for low seed weight and
four markers for high seed weight, and identified
QTL-associated ISSRs on three chromosomes. Other
examples of gene tagging by means of ISSRs are the
identification of a tight linkage between a marker and
nuclear restorer gene in rice (Agaki et al 1996), a gene
controlling Fusarium wilt resistance in chickpea
(Ratnaparkhe et al 1998), dominant allele Ns
confering resistance to Potato virus S in potato
(Marczewski et al 2002), and the Fgr major locus
modulating the fructose to glucose ratio in mature
tomato fruit (Levin et al 2000). ISSR marker also
turned out to be highly useful for monitoring
somaclonal variation (Albani et al. 1998, Leroy and
Leon 2000, Rostiana et al 1999). Leroy and Leon 2000
described the application of the ISSR technique for the
detection of differences between the hypocotyl-derived
calli and leaves of cauliflower. They found
polymorphic bands in callus tissues when using
primers (GACA)4 and (GATA)4, one of the
J of Biotech & Crop Sci (2016) 5(6): 4-31
sequenced bands showed a high similarity to the gene
coding for protein kinase of Arabidopsis thaliana,
which is involved in the regulation of cell
proliferation. The authors suggested the ISSR
technique to be a highly useful tool for the
investigation of genetic instabilities at early stages of
in vitro culture. Another benefit of ISSR markers is the
possibility to study SSR abundance and distribution in
genomes. The bands produced by an ISSR primer with
a given microsatellite repeat should reflect the relative
frequency of that motif in a given genome. This
approach was reported by Van der Nest et al 2000 who
used an inter-simple sequence repeat technique for an
access of microsatellite-rich regions in Eucalyptus
grandis. The amplification of the microsatellite-rich
regions using typical ISSR arbitrary primers was
followed by the cloning and sequencing of the PCR
products. This made it possible to design a set of SRR
primers amplifying mono-, di-, tri-, hexa-and nona-
nucleotide repeats, which were also able to generate
the corresponding microsatellite loci from other
Eucalyptus species (E. grandis, E. nitens, E. globulus,
E. camaldulensis and E. urophylla). ISSRs are
considered to be highly informative. In rice, a higher
percentage of polymorphic bands were produced with
the ISSR technique than with AFLP (Blair et al 1999).
Therefore, the ISSRs were more suitable to
discriminate between varieties and showed a lower
similarity than AFLP 55.5% vs. 73.3%. A similar
conclusion was drawn by Nagaoka and Ogihara 1997;
Korbin et al 2002 and Galvan et al 2003, who
respectively observed that ISSRs were more
informative than RAPDs in wheat, fruit plants
(strawberry, apple and Ribes species) and the common
bean for the evaluation of genetic diversity.
SAMPL MARKERS
SAMPL, another microsatellite-based marker system,
is a modification of the AFLP technique (Morgante et
al 1994, Vos et al 1995). The same template is used as
in the case of conventional AFLP restriction fragments
resulting from the digestion of genomic DNA with two
endonucleases, ligated with adaptors and preamplified
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using primers designed on the basis of the synthetic
adaptor plus the restriction site and carrying one
selective base. The selective amplification is achieved
using one of the standard AFLP primers with a
SAMPL primer. The design of the SAMPL primer
used in the original procedure was based only on
compound SSR sequences consisting of two different
adjacent dinucleotide repeats, i.e. G(TG)4(AG)4A.
Later protocols (Paglia et al 1998, Vivek et al 1999)
introduced primers complementary to microsatellites
and anchored at the 5end with a non microsatellite
sequence. Such primers allow the amplification of any
type of repeat structure (not only compound
microsatellites) and can be extended to different types
of tri-, tetra- and pentanucleotide repeats. 3-achored
SAMPL primers also proved to be useful in producing
clear and reproducible banding profiles, as shown for
rye (Bolibok et al 2003).
Because SAMPL analysis allows the amplification of
microsatellite regions without any previous
information on microsatellite flankig sequences and
has a high multiplex ratio, it is considered one of the
most efficient of all the molecular marker systems
known so far (Roy et al 2002). One of the problems
occuring while utilizing multiplex fingerprinting
techniques is the high complexity of amplification
profiles, especially in the case of plants with a large
genome size and a high proportion of repetitive DNA.
Several ways of dealing with this problem are reported
on in published SAMPL protocols. One of them is a
removal of restriction fragments with identical
adapters at both ends. It can be achieved via affinity
capture using streptavidin-coated magnetic beads as
was done in lettuce (Witsenboer et al 1997) or by
ligation of a special type of adapters and amplification
using suppression PCR technology (Paglia et al.
1998). To date, the SAMPL marker system has been
established for only a few plant species, namely carrot
(Vivek et al 1999), rye (Bolibok et al 2003), wheat
(Roy et al 2002), lettuce (Witsenboer et al 1997),
Conifer (Paglia et al 1998), chicory (De Simone et al
1997), neem (Singh et al 2002), sweet potato (Tseng
et al 2002) and cowpea (Tosti and Negri 2002), where
it was successfully utilized for studies involving
genetic diversity, genotype identification, gene tagging
and linkage mapping. As an arbitary multilocus
fingerprintig technique, SAMPL also turned out to be
a valuable tool for constructing genetic linkage maps,
especially for species for which no or only limited
previous DNA sequence information was available,
and it was used for this purpose on chicory (De
Simone et al 1997), conifer (Paglia et al 1998) and
lettuce (Witsenboer et al 1997). A summary of the
different applications of microsatellite-based markers
in plants is given in Table 3. However, each type of
microsatellite-based markers shows a set of
advantages and disadvantages, such as the mode of
inheritance, level of informativity and reproducibility,
or procedural complicacy, along with economical
aspects like costs and the time required to produce the
final result. Tab. 4 presents the main features of the
above-characterized microsatellite- based molecular
markers.
There are two main approaches for the isolation of
microsatellite loci from genomic libraries. One method
is to screen a large insert genomic library with an end
labeled microsatellite oligonucleotide probe. The
hybridized clones are purified and divided into
subclones. Selected clones are then sequenced and
flanking region of microsatellite repeats are used to
design PCR primers. Many blot hybridizations
requirement and sequencing of large subclones is the
draw back of this approach. The alternative is to
produce small insert genomic libraries constructed in a
plasmid or phage vector. These libraries are suitable
for sequencing of entire insert. They can also be highly
enriched for the desired microsatellite repeats using
enrichment strategies (Edwars et al 1996, Maguire et
al 2000).
Bioinformatics: The advances made in bioinformatics
have made it possible to acquire and organize large
amounts of information and also allows the
visualization of information from heterogeneous
datasets. It facilitates both the analysis of genomic and
postgenomic data, and the integration of data from the
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related fields of transcriptomics, proteomics,
metabolomics and phenomics. Improved algorithms
and increased computing power has helped to analyse
DNA sequences, marker discovery and analyzing the
information generated. It provides tools to intergrate
phenotypic and genotypic data and to derive
meaningful conclusions for important agronomic
traits. Development of bioinformatic tools, databases
and integration of information from different fields

Table 3 The application of microsatellite-based markers for different approaches in chosen plant species
(Rakoczy-Trojanowska and Bolibok, 2004).
Type of microsatellite Plant species Application References
marker
SSR winter rye linkage mapping, variability analysis Saal and Wricke 1999
wheat linkage mapping variability analysis Roder et al 1998
Roder et al. 1995
potato phylogenetic and fingerprinting analyses Ashkenazi et al 2001
linkage mapping Milbourne et al 1998
rice linkage mapping Panaud et al 1995, Vivek
allelic diversity analysis et al 1999 Panaud et al
analysis of allele variation 1995
Cho et al 2000
barley linkage mapping, analysis of allele variation Becker et al 1995
evaluation of genetic diversity
Russell et al 1997
sunflower linkage mapping Tang et al 2002
gene tagging Hongtrakul et al 1998
maize linkage mapping, analysis of allele variation Taramino et al 1996
olive linkage mapping De la Rosa et al 2003
maize linkage mapping Senior et al 1993
ISSR wheat gene tagging Ammiraju et al 2001
evaluation of genetic diversity Nagaoka and Ogihara.
1997
rice gene tagging Agaki et al 1996
fingerprinting Blair et al 1999
evaluation of genetic diversity Nagaraju et al 2002
potato gene tagging Marczewski et al 2002
evaluation of genetic diversity Bornet et al 2002a
tomato gene tagging Levin et al 2000
chickpea gene tagging Ratnaparkhe et al 1998
cauliflower detection of somaclonal variation Leroy and Leon 2000
horseradish Detection of somaclonal variation Rostiana et al1999
strawberry, apple evaluation of genetic diversity Korbin et al 2002
and Ribes species
common bean evaluation of genetic diversity Galvan et al 2003
peanut evaluation of genetic diversity, phylogenetic Raina et al 2001
analysis, cultivar identification
citrus cultivar identification Fang et al. 1997
SAMPL lettuce linkage mapping, Witsenboer et al 1997
evaluation of genetic diversity
Norway spruce linkage mapping Paglia et al 1998
carrot linkage mapping Vivek et al 1999
Kentucky linkage mapping Porceddu et al 2001
bluegrass
chicory linkage mapping De Simone et al 1997
wheat evaluation of genetic diversity, gene tagging Roy et al 2002
cowpea evaluation of genetic diversity Tosti and Negri 2002
sweet potato evaluation of genetic diversity Tseng et al 2002
winter rye evaluation of genetic diversity Bolibok et al 2003

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J of Biotech & Crop Sci (2016) 5(6): 4-31

enable the identification of genes and gene products, used foe developing CAPs from SNP markers,
and can elucidate the functional relationships between TASSEL which is a tool for microarray data analysis,
genotype and observed phenotype. Bioinformatic tools datamining and data visualization etc. Databases like
that are commonly used for datamining, gene NCBI, EMBL, Swissprot, AceDB, Plantmarkers, Harv
identification, analysis and molecular marker EST, PEDANT, tools for datamining,
development include MISA helps to identify SSRs, Bioinformatic.net etc also provides multiple
AutoSNP used for SNP identification, SNP2CAPS- bioinformatics tools.

Table 4 A comparisons of the main features of microsatellite-based markers.

Marker type
Feature SSR ISSR SAMPL
Abundance Locus specifity High High Medium/high
Nature of polymorphism yes No no
Variation in repeat length/number of Base changes base changes (insertions,
motifs (insertions, deletions) variation in deletions) variation in SSR repeat
SSR repeat length/number of length/number of motifs
motifs
Level of polymorphism High/very high High/medium high
Inheritance Codominance dominance codominance/dominance
Mode
Reproducibility high high/medium high
Sequence information yes no no
required
Technical demands medium/low (except for library low/medium medium
construction and screening)
Cost medium low medium
Labor high (a labor-consuming step of low medium
library construction and screening)
Time usually a time-consuming step of low medium
library construction and screening is
needed
Main applications Linkage mapping, studies on genetic Identification of cultivars studies on genetic diversity,
diversity, gene tagging phylogenetic studies linkage mapping
Main advantages high level of polymorphisms (up to 26 Mulitlocus and highly Amplification of many
alleles), co-dominant mode of polymorphic pattern production informative bands per reaction,
inheritance, very high reproducibility per reaction, technical simplicity, high reproducibility
low expenses
Problems Frequently a small number of band profiles cannot be Relatively time-consuming and
potential microsatellite loci are interpreted in terms of loci and labor-intensive procedure, high
identified, polymerase slippage when alleles, dominance of alleles complexity of amplification
analyzing mono-and di-nucleotide (frequently), similar-sized profiles may occur
repeats, co migrating fragments not fragments may not be
always are homologous homologous

Table 5 Databases related to crop genomics.

Barley Genomics Databases on molecular markers, QTLs http://barleygenomics.wsu.edu
ESTs, maps, mutants barley BACs http://uscrop.net/perl/ace/search/BarleyDB

MaizeDB Comprehensive information source on the genetics and molecular http://www.zmdb.iastate.edu

biology, analysis tool for sequence, expression and phenotype data, http://www.agron.missouri.edu
online ordering for ESTs, seed and microarrays http://www.zmdb.iastate.edu
MilletGenes
AceDB database with molecular markers, ESTs, QTL, maps

SorghumDB AceDB database with molecular markers, ESTs, QTL, maps http://uscrop.net/perl/ace/search/MilletGenes

Gene Index
http://algodon.tamu.edu/sorghumdb.html
ZmDB
compile and distribute SSR-containing ESTs
(RyeGI), Triticum SNP Development http://www.tigr.org
aestivum
Wheat http://wheat.pw.usda.gov/ITMI/EST-SSR/
http://wheat.pw.usda.gov/ITMI/WheatSNP

20

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