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 Review TRENDS in Biotechnology
Genic microsatellite markers in plants:
features and applications
Rajeev K. Varshney1, Andreas Graner1 and Mark E. Sorrells2
1
Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany
2
Department of Plant Breeding, Cornell University, Ithaca, NY14853, USA
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 inex-
pensive, 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 transfer-
ability to related species, and they can often be used as
anchor markers for comparative mapping and evol-
utionary studies. They have been developed and
mapped in several crop species and could prove useful
for marker-assisted selection, especially when the
markers reside in the genes responsible for a phenotypic
trait. Applications and potential uses of EST-SSRs in
plant genetics and breeding are discussed.
The analysis of DNA sequence variation is of major
importance in genetic studies. In this context, molecular
markers are a useful tool for assaying genetic variation,
and have greatly enhanced the genetic analysis of crop
plants. A variety of molecular markers, including restric-
tion fragment length polymorphisms (RFLPs), random
amplification of polymorphic DNAs (RAPDs), amplified
fragment length polymorphisms (AFLPs) and microsatel-
lites or simple sequence repeats (SSRs), have been
developed in different crop plants [1,2]. Among different
classes of molecular markers, SSR markers are useful for
a variety of applications in plant genetics and breeding
because of their reproducibility, multiallelic nature,
codominant inheritance, relative abundance and good
genome coverage [3]. SSR markers have been useful for
integrating the genetic, physical and sequence-based physi-
cal maps in plant species, and simultaneously have provided
breeders and geneticists with an efficient tool to link
phenotypic and genotypic variation (for review, see [4]).
With the establishment of expressed sequence tag
(EST) sequencing projects for gene discovery programs
in several plant species, a wealth of DNA sequence
information has been generated and deposited in online
databases [5]. In addition, sequence data for many fully
Corresponding author: Rajeev K. Varshney (rajeev@ipk-gatersleben.de or
rajeevkvarshney@hotmail.com).
Available online 25 November 2004
www.sciencedirect.com 0167-7799/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.11.005
Vol.23 No.1 January 2005
characterized genes and full-length cDNA clones have
been generated for some plant species such as rice [6]. By
using some computer programs, the sequence data for
ESTs, genes and cDNA clones can be downloaded from
GenBank and scanned for identification of SSRs, which
are typically referred to as EST-SSRs or genic micro-
satellites (Figure 1). Subsequently, locus-specific primers
flanking EST- or genic SSRs can be designed to amplify the
microsatellite loci present in the genes. Thus, the
generation of (genic) SSR markers is relatively easy and
inexpensive because they are a byproduct of the sequence
data from genes or ESTs that are publicly available.
However, the generation of genic SSR markers is largely
limited to those species or close relatives for which there is
a sufficiently large number of ESTs available. Genic SSRs
have some intrinsic advantages over genomic SSRs
because they are quickly obtained by electronic sorting,
and are present in expressed regions of the genome. The
usefulness of these genic SSRs also lies in their expected
transferability because the primers are designed from the
more conserved coding regions of the genome. Because of
the advantages of genic SSR markers over genomic SSR
markers and the public availability of large quantities of
sequence data, genic SSRs have been identified, developed
and used in a variety of studies, for several plant species.
In this article, we review the current status of research on
genic microsatellites in plants and present a critical
appraisal of the relative use of genic SSRs and genomic
SSRs for specific purposes, showing a shifting paradigm in
microsatellite research for crop breeding with a particular
emphasis on cereals.
Identification, frequency and distribution of genic SSRs
Identification of SSRs in gene sequences of plant species was
carried out as early as 1993 by Morgante and Olivieri [7].
However, at that time the volume of sequence data available
for SSR analysis was limited (!5000 kb) and therefore only
a few genic SSRs were reported. Only one SSR per 64.6 kb in
monocotyledonous and one per 21.2 kb in dicotyledonous
species were identified [8]. Subsequently, the sudden
increase in the volume of sequence data generated from
EST projects in several plant species facilitated the
identification of genic SSRs in large numbers. For the
identification of SSRs in publicly available EST and gene
sequences, ‘regular expression matching’ or BLASTN tools
were initially used in the FASTA or BLAST2 formatted
sequences [9,10]. Subsequently, several Perl scripts, search
 Review TRENDS in Biotechnology Vol.23 No.1 January 2005 49

Singletons
Characterized Full-length Shotgun
and annotated cDNA sequencing Unigenes
genes clones (ESTs) Tentative
consensi

Public databases such as NCBIa, EMBLb

Available sequence data from genes or ESTs

Database mining:
Identification of SSR in sequence data of ESTs or genes

Primer designing for genic SSRs

Amplification of genic loci

Applications

Transferability
Functional Association Diversity Genome Gene tagging
and comparative
genomics mapping analysis mapping and QTL analysis
mapping

TRENDS in Biotechnology

a
Figure 1. A schematic representation of the development and application of genic simple sequence repeat (SSR) markers. NCBI, National Center for Biotechnology
Information, Bethesda, MD, USA (http://www.ncbi.nih.gov/); bEMBL, European Molecular Biology Laboratory, Heidelberg, Germany (http://www.embl-heidelberg.de/). These
databases can be used to download publicly available ESTs or sequence data for a plant species available in the public domain. Abbreviation: QTL, quantitative trait loci.

modules or programs have been developed for recognition of
SSR patterns in the sequence files (Table 1). Among different
programs available in the public domain, the MIcroSAtellite
(MISA) search module has some features that are useful for
EST quality control and for designing the primer pairs for
EST-SSRs in a batch file [11] (see also http://pgrc.ipk-
gatersleben.de/misa/). MISA has been used in several
studies, in different laboratories [11–15]. Another SSR
finder, called Sputnik, has the useful feature of enabling
the user to specify the percent imperfection allowed in the
SSR [16] (see also C. Abajian; http://abajian.net/sputnik/
index.html), and Perl scripts have been written to facilitate
routing the output to a relational database and batch primer
design for Primer3 (http://wheat.pw.usda.gov/ITMI/EST-
SSR/LaRota/).
Because limited genomic sequence data are available
for many plant species, EST databases have been screened
for the development of genic SSRs. For example, ESTs
have been scanned for the presence of SSRs in Arabidopsis
[16], cotton [17,18], Festuca species [19], grapes [9],
Medicago species [20], soybean [21], sugarcane [22],
spruce [23] and cereals including barley [11–13,17,24],
maize [13,16,21,24], rice [10,13,16,17,24], rye [13,15,25],
sorghum [13,24] and wheat [13,16,17,21,24,26,27].
The abundance of SSRs (perfect and imperfect) in
unigenes can range from 1 in every 100 to 1 in every 2
unigenes of rice, depending on the minimum length of the
SSR repeat motif (M. La Rota et al., unpublished).
Varshney et al. [13] estimated the density of SSRs in
expressed regions for 75.2 Mb of barley, 54.7 Mb of maize,

Table 1. Tools for database mining

Script or program Refs
MIcroSAtellite (MISA) http://pgrc.ipk-gatersleben.de/misa/; [11]
SSRFinder [21]
BuildSSR [23]
SSR Identification Tool (SSRIT) [24]
Tandem Repeat Finder (TRF) [59]
Tandem Repeat Occurrence Locator (TROLL) [60]
CUGIssr http://www.genome.clemson.edu/projects/ssr/
Sputnik C. Abajian; http://abajian.net/sputnik/index.html
Modified Sputnik [16]
Modified Sputnik II http://wheat.pw.usda.gov/ITMI/EST-SSR/LaRota/
SSRSEARCH ftp://ftp.gramene.org/pub/gramene/software/scripts/ssr.pl
www.sciencedirect.com
50 Review TRENDS in Biotechnology
43.9 Mb of rice, 3.7 Mb of rye, 41.6 Mb of sorghum and
37.5 Mb of wheat and found the overall average density of
(redundant) SSRs to be 1 per 6.0 kb. Higher frequencies,
however, were reported by Morgante et al. [16], with 2.1,
1.1 and 1.3 per kb for rice, maize and wheat, respectively.
In this context, it is important to note that the overall
frequency and the frequency of different lengths of SSRs
and repeat motifs depend on the criteria used to identify
SSRs in the database mining, and therefore varied widely
in different studies. In wheat, for example, the frequency
of SSRs in ESTs has been reported as 1 in 6.2 kb [13], 1 in
0.74 kb [16], 1 in 17.42 kb [21], 3.2% or w1 in 1 kb [24], 1 in
9.2 kb [26] and 7.5% of the contigs [27]. In some of these
studies, a redundant set of SSRs (see below) was taken into
account for estimating the SSR frequencies [13,21,24,26].
Furthermore, different SSR search tools (MISA [13];
Sputnik [16]; SSRFinder [21]; SSRIT [24]; macro [26];
SSRSEARCH, TRF and RepeatMasker [27]) with different
search SSR criteria and different datasets were used for
EST database mining. In general, when the minimum
repeat length is 20 bp, SSRs of various plant species are
present in w5% of the ESTs (for examples, see http://www.
genome.clemson.edu/projects/ssr).
Trinucleotide repeats (TNRs) are the most common,
followed by either dinucleotide repeats (DNRs) or tetra-
nucleotide repeats (TTNRs), depending on the report. For
example, Varshney et al. [13] reported that among cereal
species, TNRs were the most frequent (54–78%) followed
by DNRs (17.1–40.4%) and TTNRs (3–6%). Frequencies
and distribution of different repeat motifs varied substan-
tially in studies by Morgante et al. [16], Gao et al. [21] and
Kantety et al. [24]. In these reports, wheat TNRs ranged
from 49% to 83% but DNRs and TTNR frequencies were
similar. Proportions of the different rice SSRs were similar
in these reports but for maize there were large differences,
many of which could be attributed to different methods of
screening and analyzing SSRs and to differences in the
sources of DNA sequence. In a recent survey, the
proportions of DNRs, TNRs and TTNRs and motifs
observed varied with the length of the SSRs within and
among barley, wheat and rice [M. La Rota et al.,
unpublished]. Yu et al. [14] reported that 74% of the
TNRs were found in coding regions, 20% in 5 0 UTRs and
6% in 3 0 UTRs. By contrast, only 19% of the DNRs were in
coding regions and 42% and 39% were in 5 0 and 3 0 UTRs,
respectively. The abundance of trimeric SSRs in ESTs was
attributed to the absence of frameshift mutations in
coding regions when there is length variation in these
SSRs [28]. Also, among the TNRs, codon repeats corre-
sponding to small hydrophilic amino acids are perhaps
more easily tolerated, and selection pressure probably
eliminates codon repeats encoding hydrophobic and basic
amino acids [29].
Some inherent issues of genic SSRs
Redundancy
Large-scale EST sequencing projects have been performed
for several plant species [5,30]. However, random or
shotgun sequencing within cDNA libraries leads to a
high proportion of redundant ESTs [31]. For development
of unique genic SSR markers, a nonredundant EST
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Vol.23 No.1 January 2005
dataset (after clustering the redundant set of ESTs and
defining the ‘unigene’ set) should be used for identification
and development of EST-SSR markers. In some studies,
the redundant EST dataset has been scanned first for
the presence of ESTs containing SSRs (SSR-ESTs) and
then the smaller dataset of redundant SSR-ESTs has been
used to identify nonredundant SSR-ESTs or EST-SSRs
after clustering and defining the unigene SSR-ESTs
[11–13,24]. The frequency of SSRs in nonredundant ESTs
(or SSR-ESTs) more accurately reflects the density of
SSRs in the transcribed portion of the genome. Alterna-
tively, all available ESTs can be assembled and consensus
sequences from unigene datasets such as the gene indices
at The Institute for Genome Research (TIGR; http://www.
tigr.org/tdb/tgi/) or other sources can be used for proper
development of nonredundant marker sets [24,27].
Robustness and high-quality markers
The practical use of polymerase chain reaction-based
markers, especially in germplasm analysis, in which data
integration and comparison are crucial, requires that each
SSR marker be validated for quality and robustness of the
amplification product. However, a portion of genomic
SSRs, developed in the past, have produced faint
bands or stuttering, as observed in wheat [32] and
barley [33]. By contrast, SSR markers derived from
genes have produced a high proportion of high-quality
markers with strong bands and distinct allelic peaks in
most reports [11,12,14,19,27,34,35]. High quality and
robustness of amplification patterns, along with other
merits (see later) associated with EST-SSR markers,
enhance their value, especially for germplasm
characterization.
Amplification rate and null alleles
Primer design is not an exact science, and a success rate of
60–90% amplification for both genomic and EST-SSRs has
been reported in different studies [11,12,14,19,22,26].
Possible explanations include: (i) one or both primers of
the EST-SSR extend across a splice site; (ii) the presence of
large introns in genomic DNA sequence; (iii) the use of
questionable sequence information for primer develop-
ment; and (iv) primers were derived from chimeric cDNA
clones. Thus, the quality of the SSR-EST sequence for
designing the primer pairs is important. In a survey, up to
9% of cereal ESTs were of low quality [30] and should be
rejected for designing primer pairs for EST-SSRs [11].
Furthermore, compared with genomic SSRs, ampli-
con size more frequently deviated from expectation
[11,12,14,22,27]. This result is probably a result of the
presence of introns and insertions-deletions(in-dels) in the
corresponding genomic sequence, as was substantiated by
sequence analysis [19]. Large in-dels (20Cbp) in the
SSR-ESTs can alter amplicon size sufficiently to enable
visualization of polymorphism on agarose gels, which,
compared with the use of acrylamide gels, significantly
reduce costs and increase throughput [14].
Null alleles (alleles that do not give a polymerase chain
reaction product) were observed by using EST-SSR
markers in studies on kiwifruit [36], rice [34], spruce [23]
and wheat [26,35]. In wheat, occurrence of null alleles is
 Review TRENDS in Biotechnology
common and has been reported earlier using genomic
SSRs (for references, see [4]). Occurrence of null alleles
can be explained by: (i) the deletion of microsatellite at a
specified locus [37]; (ii) mutations (in-dels or substi-
tutions) in the primer binding site [38]. Occurrence of
null alleles complicates the interpretation of segregation
data because heterozygotes cannot be identified and
reaction failures cannot be detected. The latter can result
in deviation from the expected Mendelian segregation
ratios [36].
Level of polymorphism
EST-SSR primers have been reported to be less poly-
morphic compared with genomic SSRs in crop plants
because of greater DNA sequence conservation in tran-
scribed regions [9,23,34,35,39,40]. It is noteworthy that
for detection of polymorphism, EST-SSRs derived from
3 0 ESTs were found to be superior to those derived from
5 0 -ESTs [9,21,26,41]. Owing to the process of cDNA
generation (polyT priming), there is a preferential selec-
tion of untranslated regions (UTRs) within 3 0 -ESTs,
resulting in more variation than in 5 0 -ESTs. Scott et al.
[9] also reported that there were polymorphism differ-
ences among microsatellites derived from the 3 0 UTR
(most polymorphic at cultivar level), the 5 0 UTR (most
polymorphic between cultivar and species) and micro-
satellites within the coding sequence (most polymorphic
between species and genera).
Interestingly, in a recent study on identification and
genome mapping of EST-SSRs in kiwifruit (Actinidia
spp.), 93.5% of the markers were polymorphic and
segregating in a mapping population derived from the
intraspecific cross between two genotypes of diploid
Actinidia chinensis [36]. Saha et al. [19] reported that
66% of the tall fescue-derived EST-SSR primer pairs were
polymorphic between parents of tall fescue and ryegrass
populations, and 43% and 38% of these were polymorphic
in rice and wheat, respectively.
Applications
Genetic mapping
Microsatellite markers, developed from genomic libraries,
can belong to either the transcribed region or the
nontranscribed region of the genome, and rarely is there
information available regarding their functions. By con-
trast, genic microsatellite markers often have known or
‘putative’ functions and are gene targeted markers with
the potential of representing functional markers in those
cases where polymorphisms in the repeat motifs affect the
function of the gene in which they reside [42]. Putative
functions for a significant proportion of EST-SSR markers
have been reported [11,14,18,43]. EST-SSR markers are
one class of marker that can contribute to ‘direct allele
selection’, if they are shown to be completely associated or
even responsible for a targeted trait [44]. For example,
recently, a Dof homolog (DAG1 gene that showed a strong
effect on seed germination in Arabidopsis [45]) has been
mapped on chromosome 1B of wheat by using wheat
EST-SSR primers [43]. Similarly, Yu et al. [14] identified
two EST-SSR markers linked to the photoperiod response
gene (ppd) in wheat. Finally, mapping candidate genes can
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Vol.23 No.1 January 2005 51
facilitate genome alignment across distantly related
species [46,47].
In recent years, the EST-SSR loci have been integrated,
or genome-wide genetic maps have been prepared, in
several plant (mainly cereal) species (Table 2). A large
number of genic SSRs have been placed on the genetic
maps of wheat [14,27,41,43]. EST-SSRs have been mapped
as a part of the transcript map of barley (R.K. Varshney
et al., unpublished) [11]. Unlike genomic SSRs, genic
microsatellite markers were not clustered around the
centromere but, as expected, were concentrated in gene-
rich regions [11,14,43]. It is believed that the distribution
of genic SSRs in the genetic maps mirrors the distribution
of genes along the genetic map. In some earlier reports
dealing with genomic SSRs, microsatellite markers were
associated with repetitive DNA or retrotransposons [4,48];
however, recent reports indicated that they are predomin-
ately associated with nonrepetitive DNA (M. La Rota
et al., unpublished) [16,49].
Functional diversity
Characterization of genetic variation within natural
populations and among breeding lines is crucial for
effective conservation and exploitation of genetic
resources for crop improvement programs. Molecular
markers have proven useful for assessment of genetic
variation in germplasm collections [50]. Evaluation of
germplasm with SSRs derived from genes or ESTs might
enhance the role of genetic markers by assaying the
variation in transcribed and known-function genes,
although there is a higher probability of bias owing to
selection. Expansion and contraction of SSR repeats in
genes of known function can be tested for association with
phenotypic variation or, more desirably, biological func-
tion [51]. The presence of SSRs in the transcripts of genes
suggests that they might have a role in gene expression or
function; however, it remains to be seen whether any
unusual phenotypic variation might be associated with
the length of SSRs in coding regions, as was reported for
several diseases in humans [49,52]. It has been shown that
variation in repeat units of SSRs: (i) present in 5 0 UTR
affects the gene transcription and/or translation;
(ii) present in coding region inactivate or activate genes
or truncate protein; and (iii) present in 3 0 UTR might be
responsible for gene silencing or transcription slippage.
However, the function of genes that contain SSRs and the
role of the SSR motif in the function of the plant genes are
poorly understood. In a computational study, microsatel-
lite markers in the transcribed regions of rice and
Arabidopsis were more frequently found in the 5 0 UTRs
than in coding regions or 3 0 UTRs, suggesting that they
can potentially function as factors in regulating gene
expression [53]. In an experimental study in rice,
variation in the number of GA or CT repeats in the
5 0 UTR of the waxy gene was correlated with amylose
content [51,54]. Similarly, microsatellite markers (CCG)n
in 5 0 UTRs of some ribosomal protein genes of maize were
believed to be involved in the regulation of fertilization
[55]. Thus, the mechanisms found in human or animal
systems might also have a role in generating phenotypic
diversity in plant species. However, the variation associated
52 Review TRENDS in Biotechnology Vol.23 No.1 January 2005

Table 2. Genome mapping using genic simple sequence repeat (SSR) markersa
Plant species Number of genic SSR loci mapped Mapping population used Refs
Barley 185 3 DHsa (Igri!Franka, Steptoe!Morex, OWBDom!OWBRec) [11], R.K. Varshney
et al. unpublished
39 F2s (Lerche!BGR41936), DHs (Igri!Franka), wheat–barley [56]
addition lines
Cotton 111 BCb1 lines (TM1!Hai7124)!TM1) [18]
Kiwifruit 138 Intraspecific cross [36]
Raspberry 8 Full-sib family (Glen Moy!Latham) [61]
Rice 91 DHs (IR64!Azucena), RILsc (Milyang 23!Gihobyeo, Lemont! [10]
Teqing, BS125!WL02)
Rye 39 4 mapping populations derived from reciprocal crosses (P87! [15]
P105, N6!N2, N7!N2, N7!N6)
Ryegrass 91 Three-generation population (Floregon!Manhattan) [62]
Tall fescue 91 Pseudo-test cross-population (HD28–56!R43–64) [57]
(Festuca spp.)
Wheat 149 RILs (W7984!Opata85) [14]
126 RILs (W7984!Opata85) [27]
101 RILs (W7984!Opata85, Wenmai 6!Shanhongmai), DHs [43]
(Lumai14!Hanxuan 10)
White clover 449 Pseudotest cross-population (6525/5!364/7) [63]
a
Abbreviations: BC1, backcross population; DHs, doubled haploids; RILs, recombinant inbred lines.

with deleterious characters is less likely to be represented in
the germplasm collections of crop species than among
natural populations because undesirable mutations are
commonly culled from agricultural populations [34].
Several studies have found that genic SSRs are useful
for estimating genetic relationship (Table 3), and at the
same time provide opportunities to examine functional
diversity in relation to adaptive variation [35,40]. In
comparison to genomic SSRs, genic SSRs revealed less
polymorphism (low polymorphic information content
value) in germplasm characterization and genetic diver-
sity studies [9,11,34,35,39,40,56].
Transferability and comparative mapping
Perhaps the most important feature of the genic SSR
markers is that these markers are transferable among
distantly related species, whereas the genomic SSRs are
not suitable for this purpose. Transferability of such
markers to related species or genera has been demon-
strated in several studies (Table 4). Recently, the potential
use of EST-SSRs developed for barley and wheat has been
demonstrated for comparative mapping in wheat, rye and
rice [46,47]. These studies suggested that EST-SSR
markers could be used in related plant species for which
little information is available on SSRs or ESTs. In
addition, the genic SSRs are good candidates for the
development of conserved orthologous markers for genetic
analysis and breeding of different species. For example, a
set of 12 barley EST-SSR markers was identified that
showed significant homology with the ESTs of four
monocotyledonous species (wheat, maize, sorghum and
rice) and two dicotyledonous species (Arabidopsis and
Medicago) and could potentially be used across these
species [47].
Two issues of importance for cross-species utilization
are frequency of amplification for a given set of primers

Table 3. Utilization of genic simple sequence repeat (SSR) markers for estimation of genetic diversitya
Plant species Number of EST-SSR Details of genotypes used Average PIC Refs
markers used
Alpine lady-fern 10 186 individuals (6 populations) 0.49 [64]
Barley 38 54 cultivars 0.45 [11]
75 7 genotypes (parents of 3 DH mapping populations) – [12]
10 23 genotypes representing different geographic regions 0.60 [39]
8 8 spring barley cultivars, 8 Jordan and Syrian landraces and 8 0.38 [40]
wild barley lines
17 (barley and wheat) 11 varieties 0.36 [41]
22 28 Germany barley cultivars and 2 wild barley accessions 0.38 [56]
Coffea spp. 9 15 C. arabica and 8 C. robusta species 0.32 [65]
Fescue spp. 145 5 Fescue genotypes and 2 genotypes each of wheat and rice – [57]
Medicago spp. 39 24 species and subspecies of Medicago 0.66 [19]
Rice 129 14 genotypes (parents of six intersubspecific crosses and one 0.46 [34]
interspecific cross)
Rye 100 15 accessions (13 inbred lines and two open-pollinated – [25]
cultivars)
Sugarcane 21 5 genotypes 0.62 [22]
Wheat 20 52 elite exotic wheat genotypes 0.44 [26]
22 64 durum wheat accessions 0.62 [35]
10 (wheat and barley) 15 varieties 0.45 [41]
52 68 advanced wheat lines Average alleles 3.3 [66]
20 56 old and new UK wheat varieties 0.40 [67]
64 18 species of Triticum–Aegilops complex Average alleles 6.8 [68]
a
Abbreviations: EST, expressed sequence tag; DH, doubled haploid; PIC, polymorphic information content (unless otherwise specified).

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 Review TRENDS in Biotechnology Vol.23 No.1 January 2005 53

Table 4. Interspecific and generic transferability of genic simple sequence repeat (SSR) markers
Plant species, genic SSRs developed Species, transferability recorded Refs
Alpine lady-fern 9 species from Woodsiaceae [64]
Apricot 21 Prunus accessions, one pear and six apple cultivars [69]
Barley Wheat, rye, rice [11,47]
Barley and wheat Wheat and barley [41]
Coffee 12 Coffea species and 4 Psilanthus species [65]
Cotton 2 cotton species [17]
Grape 7 species from 2 Vitaceae genera [9]
46 species from Vitaceae family [69]
25 species from 5 Vitaceae genera [70]
8 species from 4 Vitaceae genera [71]
Loblolly pine Different subspecies and species of pine [58]
Medicago (M. truncatula) 6 Medicago species [20]
Rice Wild species of rice [34]
Spruce (Picea spp.) 23 spruce species [23]
Sugarcane (Sachharum spp.) Erianthus and Sorghum [22]
Tall fescue (Festuca arundinacea) Lolium spp., rice, wheat [19]
Wheat Barley, maize, rice [14,46]
Barley, maize, rice, rye and oats [26]
Rice, maize and soybean [43]
Aegilops and Triticum species [68]

and probability of amplifying the same (orthologous) gene
in multiple species. Studies have estimated that 44% to
60% of EST-SSR primer pairs designed for wheat or barley
will also yield amplicons in rice [11,14,21,41,46,47]. Of tall
fescue primers, 59% successfully amplified rice and 71%
amplified wheat DNA [57]. Similarly, 96% of the primers
designed for Medicago truncatula produced amplicons in
six other Medicago species [19]. In a study of the
transferability of Loblolly pine SSR markers to other
pine species, Liewlaksaneeyanawin et al. [58] compared
microsatellite markers developed from ESTs, unscreened
genomic DNA, low-copy genomic DNA and undermethy-
lated genomic DNA. Although all eight of the EST-SSR
markers produced amplicons on all four species, the three
groups of genomic SSR markers were only evaluated for
transferability to Pinus contorta ssp. latifolia and 29%,
23% and 30% produced amplicons, respectively. In a
comparison of methods for primer design, Yu et al. [46]
found that aligning consensus sequences from two or more
species to identify conserved regions for primer design was
less efficient than designing species–specific primers and
then testing them on other species.
Orthology can only be determined by comparing both
similarity of amplicon sequences and genome location
across species [46,47]. For example, Saha et al. [19]
sequenced the products of one EST-SSR primer pair for
three fescue species, ryegrass, rice and wheat, and all
sequences were O85% similar. Sequence-based compari-
son of mapped barley SSR-ESTs with genetically and/or
physically mapped markers in wheat, rye and rice
revealed several markers that showed an orthologous
relationship between examined cereal species [47]. Com-
parison of genome locations of polymorphic EST-SSR
markers mapped in both wheat and rice also confirmed
previously known genome relationships with most of the
markers examined [46]. However, the assessment of
colinearity was complicated by the detection of multiple
polymorphic loci in either wheat or rice by 85% of the
primer pairs. The tendency of EST-SSR primer pairs to
detect more loci than genomic SSRs was also reported for
tall fescue [57].
www.sciencedirect.com
Comparative account on genic and genomic
microsatellite markers
A comparative analysis of genomic SSRs and genic SSRs
reveals advantages to both; however, because of lower
polymorphism, EST-SSRs are not as efficient as genomic
SSRs for distinguishing the closely related genotypes
(for references, see [4]). Furthermore, the development of
genic SSRs is restricted to those species for which there
are sufficient sequence data (for ESTs or genes) available
because SSRs are present in only 2% to 5% of the unigenes
examined. Nevertheless, EST-SSR markers developed for
a given species can successfully be used in a related
species for a variety of purposes, including fingerprinting
or diversity studies, comparative mapping and marker-
assisted selection. Genic SSR- and genomic SSR markers
tend to be complementary for genome mapping, with genic
microsatellites being less polymorphic but concentrated in
the gene-rich regions. For assessment of functional
diversity, the genic SSRs are useful; however, because of
higher polymorphism, genomic SSRs are superior for
fingerprinting or varietal identification studies.
Future directions of microsatellite marker research
With more DNA sequence data being generated daily, the
trend is towards cross-referencing genes and genomes
using sequence- and map-based tools. Because polymorph-
ism is a major limitation for many species, microsatellite
markers are a valuable tool for plant genetics and
breeding.
Clearly, the most significant application of EST-SSRs is
for comparative mapping, with good examples in grami-
naceous and leguminous species. A database of EST-SSR
primer pairs that would amplify orthologous loci across
species and that are uniformly distributed over the rice,
Medicago and Arabidopsis genomes would be very useful
to breeders and geneticists, especially for minor or under-
funded crop species.
In the longer term, development of allele-specific
markers for the genes controlling agronomic traits will
be important for advancing the science of plant breeding.
In this context, genic microsatellites are but one class of
54 Review TRENDS in Biotechnology
marker that can be deployed, along with single nucleotide
polymorphisms and other types of markers that target
functional polymorphisms within genes. The choice of the
most appropriate marker system needs to be decided upon
on a case by case basis and will depend on many issues,
including the availability of technology platforms, costs for
marker development, species transferability, information
content and ease of documentation.
References
1 Philips, R.L. and Vasil, I.K. eds (2001) DNA-Based Markers in Plants,
Kluwer Academic Publishers
2 Varshney, R.K. et al. (2004) Molecular maps in cereals: methodology
and progress. In Cereal Genomics (Gupta, P.K. and Varshney, R.K.
eds), pp. 35–82, Kluwer Academic Publishers
3 Powell, W. et al. (1996) Polymorphism revealed by simple sequence
repeats. Trends Plant Sci. 1, 215–222
4 Gupta, P.K. and Varshney, R.K. (2000) The development and use of
microsatellite markers for genetic analysis and plant breeding with
emphasis on bread wheat. Euphytica 113, 163–185
5 Rudd, S. (2003) Expressed sequence tags: alternative or complement
to whole genome sequences? Trends Plant Sci. 8, 321–329
6 Kikuchi, S. et al. (2003) Collection, mapping, and annotation of over
28,000 cDNA clones from japonica rice. Science 301, 376–379
7 Morgante, M. and Olivieri, A.M. (1993) PCR-amplified microsatellites
as markers in plant genetics. Plant J. 3, 175–182
8 Wang, Z. et al. (1994) Survey of plant short tandem DNA repeats.
Theor. Appl. Genet. 88, 1–6
9 Scott, K.D. et al. (2000) Analysis of SSRs derived from grape ESTs.
Theor. Appl. Genet. 100, 723–726
10 Temnykh, S. et al. (2000) Mapping and genome organization of
microsatellite sequences in rice (Oryza sativa L.). Theor. Appl. Genet.
100, 697–712
11 Thiel, T. et al. (2003) Exploiting EST databases for the development of
cDNA derived microsatellite markers in barley (Hordeum vulgare L.).
Theor. Appl. Genet. 106, 411–422
12 Kota, R. et al. (2001) Generation and comparison of EST-derived SSRs
and SNPs in barley (Hordeum vulgare L.). Hereditas 135, 145–151
13 Varshney, R.K. et al. (2002) In silico analysis on frequency and
distribution of microsatellites in ESTs of some cereal species. Cell.
Mol. Biol. Lett. 7, 537–546
14 Yu, J.K. et al. (2004) Development and mapping of EST-derived simple
sequence repeat (SSR) markers for hexaploid wheat. Genome 47,
805–818
15 Khlestkina, E. et al. (2004) Mapping of 99 new microsatellite-derived
loci in rye (Secale cereale L.) including 39 expressed sequence tags.
Theor. Appl. Genet. 109, 725–732
16 Morgante, M. et al. (2002) Microsatellites are preferentially present
with non-repetitive DNA in plant genomes. Nat. Genet. 30, 194–200
17 Saha, S. et al. (2003) Simple sequence repeats as useful resources to
study transcribed genes of cotton. Euphytica 130, 355–364
18 Han, Z.-G. et al. (2004) Genetic mapping of EST-derived microsatel-
lites from the diploid Gossypium arboreum in allotetraploid cotton.
Mol. Gen. Genom. 272, 308–327
19 Saha, M.C. et al. (2004) Tall fescue EST-SSR markers with transfer-
ability across several grass species. Theor. Appl. Genet. 109, 783–791
20 Eujayl, I. et al. (2004) Medicago truncatula EST-SSRs reveal cross-
species genetic markers for Medicago spp. Theor. Appl. Genet. 108,
414–422
21 Gao, L.F. et al. (2003) Analysis of microsatellites in major crops
assessed by computational and experimental approaches. Mol. Breed.
12, 245–261
22 Cordeiro, G.M. et al. (2001) Microsatellite markers from sugarcane
(Saccharum spp.) ESTs cross transferable to erianthus and sorghum.
Plant Sci. 160, 1115–1123
23 Rungis, D. et al. (2004) Robust simple sequence repeat markers for
spruce (Picea spp.) from expressed sequence tags. Theor. Appl. Genet.
109, 1283–1294
24 Kantety, R.V. et al. (2002) Data mining for simple sequence repeats in
expressed sequence tags from barley, maize, rice, sorghum and wheat.
Plant Mol. Biol. 48, 501–510
www.sciencedirect.com
Vol.23 No.1 January 2005
25 Hackauf, B. and Wehling, P. (2002) Identification of microsatellite
polymorphisms in an expressed portion of the rye genome. Plant
Breed. 121, 17–25
26 Gupta, P.K. et al. (2003) Transferable EST-SSR markers for the study
of polymorphism and genetic diversity in bread wheat. Mol. Genet.
Genomics 270, 315–323
27 Nicot, N. et al. (2004) Study of simple sequence repeat (SSR) markers
from wheat expressed sequence tags (ESTs). Theor. Appl. Genet. 109,
800–805
28 Metzgar, D. et al. (2000) Selection against frameshift mutations limits
microsatellite expansion in coding DNA. Genome Res. 10, 72–80
29 Katti, M.V. et al. (2001) Differential distribution of simple sequence
repeats in eukaryotic genome sequences. Mol. Biol. Evol. 18,
1161–1167
30 Sreenivasulu, N. et al. (2002) Mining functional information from
cereal genomes – the utility of expressed sequence tags. Curr. Sci. 83,
965–973
31 Varshney, R.K. et al. (2004) A simple hybridization-based strategy for
the generation of non-redundant EST collections – a case study in
barley (Hordeum vulgare L.). Plant Sci. 167, 629–634
32 Stephenson, P. et al. (1998) Fifty new microsatellite loci for the wheat
genetic map. Theor. Appl. Genet. 97, 946–949
33 Ramsay, L. et al. (2000) A simple sequence repeat-based linkage map
of barley. Genetics 156, 1997–2005
34 Cho, Y.G. et al. (2000) Diversity of microsatellites derived from
genomic libraries and GenBank sequences in rice (Oryza sativa L.).
Theor. Appl. Genet. 100, 713–722
35 Eujayl, I. et al. (2001) Assessment of genotypic variation among
cultivated durum wheat based on EST-SSRs and genomic SSRs.
Euphytica 119, 39–43
36 Fraser, L.G. et al. (2004) EST-derived microsatellites from Actinidia
species and their potential for mapping. Theor. Appl. Genet. 108,
1010–1016
37 Callen, D. et al. (1993) Incidence and origin of null alleles in the (AC)n
microsatellite markers. Am. J. Hum. Genet. 52, 922–927
38 Lehman, T. et al. (1996) An evaluation of evolutionary constraints on
microsatellite loci using null alleles. Genetics 144, 1155–1163
39 Chabane, K. et al. (2005) EST versus genomic derived microsatellite
markers for genotyping wild and cultivated barley. Genet. Resour.
Crop Evol. (in press)
40 Russell, J. et al. (2004) A comparison of sequence-based polymorphism
and haplotype content in transcribed and anonymous regions of the
barley. Genome 47, 389–398
41 Holton, T.A. et al. (2002) Identification and mapping of polymorphic
SSR markers from expressed gene sequences of barley and wheat.
Mol. Breed. 9, 63–71
42 Anderson, J.R. and Lubberstedt, T. (2003) Functional markers in
plants. Trends Plant Sci. 8, 554–560
43 Gao, L.F. et al. (2004) One hundred and one new microsatellite loci
derived from ESTs (EST-SSRs) in bread wheat. Theor. Appl. Genet.
108, 1392–1400
44 Sorrells, M.E. and Wilson, W.A. (1997) Direct classification and
selection of superior alleles for crop improvement. Crop Sci. 37,
691–697
45 Papi, M. et al. (2000) Identification and disruption of an Arabidopsis
zinc finger gene controlling seed germination. Genes Dev. 14, 28–33
46 Yu, J.K. et al. (2004) EST-derived SSR markers for comparative
mapping in wheat and rice. Mol. Genet. Genomics 271, 742–751
47 Varshney, R.K. et al. (2005) Interspecific transferability and compara-
tive mapping of barley EST-SSR markers in wheat, rye and rice. Plant
Sci. 168, 195–202
48 Schulman, A. et al. (2004) Organization of retrotransposons and
microsatellites in cereal genomes. In Cereal Genomics (Gupta, P.K.
and Varshney, R.K. eds), pp. 83–118, Kluwer Academic Publishers
49 Li, Y.C. et al. (2004) Microsatellites within genes: structure, function,
and evolution. Mol. Biol. Evol. 21, 991–1007
50 Mohammadi, S.A. and Prasanna, B.M. (2003) Analysis of genetic
diversity in crop plants – salient statistical tools and considerations.
Crop Sci. 43, 1235–1248
51 Ayers, N.M. et al. (1997) Microsatellites and a single nucleotide
polymorphism differentiate apparent amylose classes in an extended
pedigree of US rice germplasm. Theor. Appl. Genet. 94, 773–781
 Review TRENDS in Biotechnology Vol.23 No.1 January 2005 55

52 Cummings, C.J. and Zoghbi, H.Y. (2000) Trinucleotide repeats:
mechanisms and pathophysiology. Annu. Rev. Genomics Hum.
Genet. 1, 281–328
53 Fujimori, S. et al. (2003) A novel feature of microsatellites in plants: a
distribution gradient along the direction of transcription. FEBS Lett.
554, 17–22
54 Bao, S. et al. (2002) Microsatellites in starch-synthesizing genes in
relation to starch physicochemical properties in waxy rice (Oryza
sativa L.). Theor. Appl. Genet. 105, 898–905
55 Dresselhaus, T. et al. (1999) Novel ribosomal genes from maize are
differentially expressed in the zygotic and somatic cell cycles. Mol.
Gen. Genet. 261, 416–427
56 Pillen, K. et al. (2000) Mapping new EMBL-derived barley micro-
satellites and their use in differentiating German barley cultivars.
Theor. Appl. Genet. 101, 652–660
57 Saha, M.C. et al. (2004) A high-density linkage map of tall fescue
based on SSR and AFLP markers. Theor. Appl. Genet. (in press)
58 Liewlaksaneeyanawin, C. et al. (2004) Single-copy, species-transfer-
able microsatellite markers developed from loblolly pine ESTs. Theor.
Appl. Genet. 109, 361–369
59 Benson, G. (1999) Tandem repeats finder: a program to analyze DNA
sequences. Nucleic Acids Res. 27, 573–580
60 Castelo, A.T. et al. (2002) TROLL – Tandem Repeat Occurrence
Locator. Bioinformatics 18, 634–636
61 Graham, J. et al. (2004) The construction of a genetic linkage map of
red raspberry (Rubus idaeus subsp. idaeus) based on AFLPs, genomic-
SSR and EST-SSR markers. Theor. Appl. Genet. 109, 740–749
62 Warnke, S.E. et al. (2004) Genetic linkage mapping of an annual x
perennial ryegrass population. Theor. Appl. Genet. 109, 294–304
63 Barrett, B. et al. (2004) A microsatellite map of white clover. Theor.
Appl. Genet. 109, 596–608
64 Woodhead, M. et al. (2003) Development of EST-SSRs from the alpine
lady-fern, Athyrium distentifolium. Mol. Ecol. Notes 3, 287–290
65 Bhat, P. et al. (2004) Identification and characterization of gene (EST)-
derived SSR markers from robusta coffee variety ‘CxR’ (an inter-
specific hybrid of Coffea canephora!C. congensis). Mol. Ecol. Notes
DOI:10.1111/j-1471-8286.2004.00839
66 Dreisigacker, S. et al. (2003) SSR and pedigree analyses of genetic
diversity among CIMMYT wheat lines targeted to different mega-
environments. Crop Sci. 44, 381–388
67 Leigh, F. et al. (2003) Assessment of EST- and genomic microsatellite
markers for variety discrimination and genetic diversity studies in
wheat. Euphytica 133, 359–366
68 Bandopadhyay, R. et al. (2004) DNA polymorphism among 18 species
of Triticum-Aegilops complex using wheat EST-SSRs. Plant Sci. 166,
349–356
69 Decroocq, V. et al. (2003) Development and transferability of apricot
and grape EST microsatellite markers across taxa. Theor. Appl. Genet.
106, 912–922
70 Arnold, C. et al. (2002) The application of SSRs characterized for grape
(Vitis vinifera) to conservation studies in Vitaceae. Am. J. Bot. 89,
22–28
71 Rossetto, M. et al. (2002) Evaluating the potential of SSR: flanking
regions examining taxonomic relationships in the Vitaceae. Theor.
Appl. Genet. 104, 61–66

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