Assessment of Functional EST-SSR Markers (Sugarcane) in Cross-Species Transferability, Genetic Diversity among Poaceae Plants, and Bulk Segregation Analysis

Abstract

Expressed sequence tags (ESTs) are important resource for gene discovery, gene expression and its regulation, molecular marker development, and comparative genomics. We procured 10000 ESTs and analyzed 267 EST-SSRs markers through computational approach. The average density was one SSR/10.45 kb or 6.4% frequency, wherein trinucleotide repeats (66.74%) were the most abundant followed by di- (26.10%), tetra- (4.67%), penta- (1.5%), and hexanucleotide (1.2%) repeats. Functional annotations were done and after-effect newly developed 63 EST-SSRs were used for cross transferability, genetic diversity, and bulk segregation analysis (BSA). Out of 63 EST-SSRs, 42 markers were identified owing to their expansion genetics across 20 different plants which amplified 519 alleles at 180 loci with an average of 2.88 alleles/locus and the polymorphic information content (PIC) ranged from 0.51 to 0.93 with an average of 0.83. The cross transferability ranged from 25% for wheat to 97.22% for
Schlerostachya
, with an average of 55.86%, and genetic relationships were established based on diversification among them. Moreover, 10 EST-SSRs were recognized as important markers between bulks of pooled DNA of sugarcane cultivars through BSA. This study highlights the employability of the markers in transferability, genetic diversity in grass species, and distinguished sugarcane bulks.

Full text

Research Article
Assessment of Functional EST-SSR Markers
(Sugarcane) in Cross-Species Transferability, Genetic Diversity
among Poaceae Plants, and Bulk Segregation Analysis
Shamshad Ul Haq,1,2,3 Pradeep Kumar,1,4 R. K. Singh,1Kumar Sambhav Verma,5
Ritika Bhatt,2,3 Meenakshi Sharma,3Sumita Kachhwaha,3and S. L. Kothari2,3,5
1Biotechnology Division, UP Council of Sugarcane Research, Shahjahanpur 242001, India
2Interdisciplinary Programme of Life Science for Advance Research and Education, University of Rajasthan, Jaipur 302004, India
3Department of Botany, University of Rajasthan, Jaipur 302015, India
4School of Biotechnology, Yeungnam University, Gyeongsan 712-749, Republic of Korea
5Amity Institute of Biotechnology, Amity University Rajasthan, Jaipur 302006, India
Correspondence should be addressed to Shamshad Ul Haq; shamshadbiotech@gmail.com
Received  November ; Revised  April ; Accepted  April 
Academic Editor: Norman A. Doggett
Copyright ©  Shamshad Ul Haq et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Expressed sequence tags (ESTs) are important resource for gene discovery, gene expression and its regulation, molecular marker
development, and comparative genomics. We procured  ESTs and analyzed  EST-SSRs markers through computational
approach. e average density was one SSR/.kb or .% frequency, wherein trinucleotide repeats (.%) were the most
abundant followed by di- (.%), tetra- (.%), penta- (.%), and hexanucleotide (.%) repeats. Functional annotations were
done and aer-eect newly developed  EST-SSRs were used for cross transferability, genetic diversity, and bulk segregation
analysis (BSA). Out of  EST-SSRs,  markers were identied owing to their expansion genetics across  dierent plants which
amplied  alleles at  loci with an average of . alleles/locus and the polymorphic information content (PIC) ranged from
. to . with an average of .. e cross transferability ranged from % for wheat to .% for Schlerostachya, with an average
of .%, and genetic relationships were established based on diversication among them. Moreover,  EST-SSRs were recognized
as important markers between bulks of pooled DNA of sugarcane cultivars through BSA. is study highlights the employability
of the markers in transferability, genetic diversity in grass species, and distinguished sugarcane bulks.
1. Introduction
Sugarcane is a bioenergy crop belonging to the genus Sac-
charum L. of the tribe Andropogoneae (family: Poaceae).
is tribe comprises grass species which have high eco-
nomic value. e noble sugarcane varieties are developed
from interspecic hybridization of Saccharum ocinarum L.
(2𝑛 = ) which has high sugar content with less disease
tolerance and Saccharum spontaneum (2𝑛 =to)which
provides stress, disease tolerance, and high ber content for
biomass. e taxonomy and genetic constitution of sugarcane
are complicated due to complex interspecic aneupolyploid
genome which makes chromosome numbers range from 
to  []. Moreover, six Saccharum spp. (S. spontaneum, S.
ocinarum,S. robustum,S. edule,S. barberi, and S. sinense)
and four Saccharum related genera (Erianthus, Miscanthus,
Sclerostachya, and Narenga) have purportedly undergone
interbreeding, forming the “Saccharum complex” [, ]. e
interbreeding has made their genome more complex and
added to multigenic and/or multiallelic nature for most agro-
nomic traits that made sugarcane breeding a more dicult
task [].
A vast array of genomic tools has been developed which
has opened new ways to dene the genetic architecture of
sugarcane and helped to explore its functional system [,
]. Among the molecular markers, microsatellites are most
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Genetics Research International
Volume 2016, Article ID 7052323, 16 pages
http://dx.doi.org/10.1155/2016/7052323

Genetics Research International
favored for a variety of genetic applications due to their
multiallelic nature, high reproducibility, cross transferability,
codominant inheritance, abundance, and extensive genome
coverage [–]. Microsatellites or simple sequences repeats
(SSRs) are monotonous repetitions of very short (one to
six) nucleotide motifs, which occur as interspersed repet-
itive elements in all eukaryotic and prokaryotic genomes.
However, transcribed regions of the genome also contain
enormous range of microsatellites that correspond to genic
microsatellites or EST-SSRs. erefore, expressed sequence
tags (ESTs) are the short transcribed portions and involved
in the variety of metabolic functions. e presence of the
microsatellites in genes as well as ESTs unveils the biological
signicance of SSR distribution, expansion, and contraction
on the function of the genes themselves [].
Presently, huge amounts of expressed sequence tags
have been deposited in public database (NCBI). In silico
approaches to retrieve EST sequences from NCBI and func-
tional annotations provide more constructive EST-SSRs or
gene-based SSR (genic SSRs) marker development besides
own EST libraries development. is method of the EST-SSR
markers development provides the easiest way to reduce cost,
time, and labours along with more meaningful marker iden-
tications []. e presence of microsatellites in the genic
region is found to be more conserved due to which they pos-
sess high reproducibility and high interspecic/intraspecic
transferability. Hence, EST-SSR could be used for polymor-
phism, genetic diversity, cross transferability, and compara-
tive mapping in dierent plant species. Accordingly, several
genetic studies were done on sugarcane using microsatellite
markers to decipher polymorphism, cross transferability,
genetic diversity, informative marker detection through bulk
segregation analysis (BSA), and comparative genomics [,
–]. e objective of the present study was to retrieve
EST sequences for more informative EST-SSR development
andtheirgeneticassessmentwithinandacrossthetaxa
through cross transferability, genetic relationships, and bulk
segregation analysis.
2. Materials and Methods
2.1. EST Sequences Retrieving, ESTs Assembling, and
Microsatellites Identication. Total  EST sequences of
the Saccharum spp. were downloaded in Fasta format from
National Centre for Biotechnology Information (NCBI) for
microsatellites deciphering. Further, ESTs assembling was
carried out using CAP programme (http://mobyle.pasteur
.fr/cgi-bin/portal.pyforms::cap) for minimization of se-
quences redundancy. Microsatellite identication was carried
out using MISA soware (http://pgrc.ipk-gatersleben.de/
misa/) and the criteria for SSR detection were , , , ,
and  repeat units for di-, tri-, tetra-, penta-, and hexanu-
cleotides, respectively. SSR primer pairs (forward and
reverse) were designed for the selected EST sequences having
microsatellites using online web tool, batch primer  pipeline
[].
2.2. EST-SSR Sequences Annotation. Assessment of EST
sequences having SSR was done through blastn/blastx
analysis for homology search and against nonredundant (nr)
protein at the NCBI. Furthermore, functional annotation
pipeline was also run at online tool for gene ontology (GO)
which was intended for dierent GO functional classes
like biological process, cellular component, and molecular
function [].
2.3. PCR Amplication and Electrophoresis. PCR reactions
were carried out in a total of  𝜇L volume containing  ng
template DNA, . 𝜇L(pmol/𝜇L) of each forward and
reverse primer,  mM of dNTPs, . U of Ta q DNA poly-
merase, and . 𝜇L of x PCR buer with .mM of MgCl2.
Amplication was performed in a thermal cycler (Bio-Rad)
in the following conditions: initial denaturation at ∘Cfor
 min followed by  amplication cycles of denaturation for
min at 
∘C followed by annealing temperature (𝑇𝑎)for
 min and then extension for  min at ∘C; nal extension at
∘C for  min was allowed. e PCR conditions particularly
the annealing temperatures (varying from ∘Cto
∘C) for
each primer were standardized and amplied products were
stored at ∘C.ePCRproductswereanalyzedona%
native PAGE in vertical gel electrophoresis unit (Bangalore
Genei) using TBE buer. e sizes of amplied fragments
were estimated using  bp DNA ladder (Fermentas). Gels
were documented using ethidium bromide (EtBr) stained
dye.
2.4. Evaluation of Saccharum EST-SSR across the Taxa through
Cross Transferability. e cross transferability of Saccharum
derived EST-SSR markers was evaluated among the 
accessions comprising seven cereals (wheat, maize, barley,
rice,pearlmillet,oat,andSorghum), four Saccharum related
genera (Erianthus,Miscanthus,Narenga,andSclerostachya),
three Saccharum species (NG (S. robustum), N (S.
spontaneum), and two clones of S. ocinarum (Bandjermasin
Hitam and Gunjera)), and ve Saccharum commercial culti-
vars (CoS , CoS , UP , CoS , and CoS
). All genotypes were collected from the Sugarcane
Research Institute Farm, UPCSR, Shahjahanpur, India. Fur-
thermore, genomic DNA from young juvenile, disease-free,
immature leaves was isolated for each genotype using CTAB
(cetyl trimethylammonium bromide) method []. Isolated
DNA samples were treated with RNAase for  h at ∘Cand
puried by phenol extraction ( phenol :  chloroform : 
isoamyl alcohol, v/v/v) followed by ethanol precipitation []
and stored at −∘C. DNA was quantied on .% agarose gel
and the working concentration of  ng/𝜇Lwasobtainedby
making nal adjustment in  mM TE buer.
2.5. Genetic Diversity Analysis. e assessment of EST-SSRs
in genetic diversity analysis was done among  plants
belonging to distinct groups comprising cereals, Saccharum
related genera, Saccharum species, and Saccharum cultivars.
e allelic data of  EST-SSR primers were used to ascertain
the genetic relationships between  genotypes by clustering
analysis. Amplied bands were scored as binary data in the
form of present () or absent (). Dendrogram was con-
structed by neighbour-joining and Jaccard’s algorithm using

Genetics Research International 
FreeTree and TreeView soware [, ]. e polymorphic
information content (PIC) values were calculated for each
primer by using the online resource of PIC Calculator (http://
www.liv.ac.uk/∼kempsj/pic.html).
2.6. Informative Assessment of Functional EST-SSR Markers
between Bulks. Plant materials were used as F mapping pop-
ulation comprising  genotypes of the sugarcane cultivars
which were developed from cross between CoS  (Parent;
CoS  ×Co ) with CoS  (Parent; MS / ×
Co ) from September to March (-). Grouping
of genotypes was done according to their stem diameter
(contrasting high and low stem diameter genotypes) into
two sets. DNA extractions were carried out from both sets
and equal quantities of genomic DNA from  extreme high
stem diameter and  extreme low stem diameter genotypes
were pooled into two bulks. PCR amplication was done
in both bulks with newly developed EST-SSR primers for
informative markers identications through bulk segregation
analysis (BSA) [].
3. Results and Discussion
3.1. Mining of Microsatellites in EST Sequences and SSRs
Characterization. Total , EST sequences related to
Saccharum spp. were examined from NCBI for the simple
sequence repeat (SSR) identication and characterization
using computational approach. Prior to the marker decipher-
ing, sequence assembly was performed and  ( kb)
nonredundant sequences were detected comprising 
contigs and  singlets, wherein  SSRs were identied
with  perfect SSRs and  sequences containing more
than  SSR and  SSRs in compound formation. ere-
fore, computational and experimental approach to ascertain
microsatellites in EST libraries from public database (NCBI)
turned to be very cost eective and reduces time and labour
besides expense of own libraries development. EST-SSRs are
a more preferable DNA marker in the variety of genetic
analysis and found to be more conserved as present in the
transcribed region of the genome. ese were found to be
more transferable across the taxonomic boundaries and could
be evaluated as most informative markers for variety of
genomics applications [, ]. ese are more adapted in
plants comparative genetic analysis for gene identication,
gene mapping, marker-assisted-selection, transferability, and
genetic diversity [, –]. Also, a variety of studies have
been reported on sugarcane using EST-SSR markers for
desired genetic analysis [, , , ].
e frequency of SSR in EST sequences was .% includ-
ing all the repeats except mononucleotide repeats. is result
is comparatively higher compared to previous studies on
sugarcane [, –]. Contrary to this, Singh et al. []
reported higher frequency (.%) in sugarcane. Kumpatla
and Mukhopadhyay [] also observed high range (.% to
.%) of SSR frequency in dierent plant species. In general,
about%ofESTscontainedSSRwhichhasbeenreported
in many plant species []. ese variations in microsatellite
frequency could be attributed to the “search criteria” used,
type of SSR motif, size of sequence data, and the mining tools
used [, ]. In other words, the density of the microsatellites
was one SSR per . kb which is closely comparable to
earlier studies in sugarcane with densities  SSR/. kb []
and / kb SSR [].
Analysis revealed that trinucleotide repeats (.%)
were found to be more frequent followed by di- (.%),
tetra- (.%), penta- (.%), and hexanucleotide (.%)
repeats. Our observation of high frequency of trinu-
cleotide repeats is in agreement with previous reports on
sugarcane [, , –, ]. Several other studies have
also represented high frequency of trinucleotide repeats
in dierent plant species [, , –]. A total of 
dierent types of motifs were identied of which four
belonged to dinucleotide, eight belonged to trinucleotides,
twelve belonged to tetranucleotide, ve belonged to pen-
tanucleotide, and two belonged to hexanucleotide repeats
(Figure ). We observed that motifs AG/CT and AT/AT
were more frequent in dinucleotide repeat followed by
motifs CCG/CGG, AGC/CTG, AGG/CCT, and ACG/CGT in
trinucleotide repeat, motif AAAG/CTTT in tetranucleotide
repeats, motif ACAGG/CCTGT in pentanucleotide repeats,
and AACACC/GGTGTT in hexanucleotide repeats. e
presence of motif CCG/CGG was also observed in sugarcane
by dierent authors [, ]. Kantety et al. [] also reported
CCG/CGG motif as most abundant in wheat and Sorghum.
Similarly, both Lawson and Zhang [] and Da Maia et
al. [] also observed abundance of motif CCG/CGG in
dierent member of the grass family. Victoria et al. [] also
decoded motif CCG/CGG in the lower plants (C. reinhardtii
and P. p a t e n s ). us, this predominance of CCG/CGG motif
frequency has been related to a high GC-content []. Some
motifs which are responsible for making unusual DNA
folding structure (hairpin formed, bipartite triplex formed,
and simple loop folding) also have eect on gene expressions
and regulations mechanism, namely, CCT/AGG, CCG/GGC,
GGA/TTC, and GAA/TTC motifs [, ]. Moreover, the
presence of trinucleotide repeats in the coding region formed
a distinct group and encoded amino acid tracts within the
peptide []. We also observed predictable twenty dierent
types of amino acids including stop codon. Alanine, arginine,
glycine, proline, and serine were most frequent (Figure ).
isisinagreementwithpreviousstudiesthatreportedon
dierent plant species [, , ].
3.2. Expressed Sequence Tags Annotation and Primers
Development. All EST sequences having SSRs were examined
by functional annotation (blastn, blastx, and gene ontology).
Aer-eect, sixty-three ESTs having SSRs were successfully
identied on the basis of their involvement in the various
metabolic processes (Figure ). Aer-eect, sixty-three
EST-SSRs primer pairs were designed for polymorphic
nature, cross transferability, bulk segregation analysis, and
genetic diversity in the test plants (Table ). ese selected
EST-SSRs comprised all types of repeat motifs (excluding
mononucleotide repeat), and among trinucleotide repeats
they were highly frequent with GCT/CGA, TCC/AGG,
and GGT/CCA repeat motifs. Similarly, Sharma et al. []
also used functional annotation pipelines for the more
prominent molecular markers development related to gene

Genetics Research International
T : Details of selected  EST-SSR primer pairs used for cross transferability, genetic diversity, and bulks segregation analysis.
Serial
number Ty p e Primer sequence Annealing
temperature SSR motif PIC value 𝐸-value Putative identities
(blastn/blastx)
SYMS F GCGTCAGAGTGTTAAAACAAG  (GCT)4. .E− Protein transport protein
Sec beta
SYMS R GTGTAGAACTGGAGCATTGAG
SYMS F GGGCAAGCAAGAAACCAC  (TCC)4. .E− Protein translation factor
SUI
SYMS R GAAGAGGTCAACCAAGAACTC
SYMS F GCGTCAGAGTGTTAAAACAAG  (GCT)4. .E− Preprotein translocase Sec
SYMS R GTGTAGAACTGGAGCATTGAG
SYMS F GAAGCTCCCAAGCTGCTA  (AGCT)3. .E− Predicted:uncharacterized
protein
SYMS R CCTACAGGAAAGATTTTAGGG
SYMS F GTCTCTTCTCCAGTTCTCCTT  (TGCG)4. .E−
Predicted:
actin-depolymerizing
factor
SYMS R GCTCAACAAATGTCTCCCTA
SYMS F TGCACTAACATGGTTGATGT  (GAAG)3. .E− Hypothetical protein
SORBIDRAFT g
SYMS R GGTGATTGTAAGGGTCATCTT
SYMS F GTTAATGGTGGTTCCGTTC  (GGC)6. E− Predicted:uncharacterized
protein LOC
SYMS R ATTATCAGCGCAGAGACATC
SYMS F GCGTCAGAGTGTTAAAACAAG  (GCT)4. .E− Preprotein translocase
SYMS R GTGTAGAACTGGAGCATTGAG
SYMS F GGACTGTACAAGGACGACAG  (GCT)4. .E− Protein transport protein
Sec beta subunit
SYMS R TCTGCTTTCTTGGATATGGTA
SYMS F AAGAAGGATGCAAAGAAGAAG  (GAT)4. .E− Hypothetical protein
SORBIDRAFT g
SYMS R AGGCTTAGTAACAGCAGGTTT
SYMS F AAGAAGGATGCAAAGAAGAAG  (AGA)4. .E− Hypothetical protein
SYMS R AGGCTTAGTAACAGCAGGTT T
SYMS F GGACTGTACAAGGACGACAG —(GCT)
4—.E− Protein transport protein
SYMS R TCTGCTTTCTTGGATATGGTA
SYMS F GGACTGTACAAGGACGACAG —(GCT)
4—.E− Preprotein translocase
SYMS R TCTGCTTTCTTGGATATGGTA
SYMS F GGACTGTACAAGGACGACAG —(GCT)
4—.E− Protein transport protein
Sec beta subunit
SYMS R TCTGCTTTCTTGGATATGGTA
SYMS F CCAAAGAGATCTTGCAGACTA —(ATG)
4—.E− Jasmonate-induced protein
SYMS R CCCAACACAACAACCAAT
SYMS F CCACACAAGCAAGAAATAAAC — (GGT)4— .E− Di rige nt-like protein
SYMS R TCGAACACTATGGTAAAGGTG
SYMS F GGACTGTACAAGGACGACAG —(GCT)
4—.E− Homeodomain-like
transcription factor
SYMS R TCTGCTTTCTTGGATATGGTA
SYMS F GCGTCAGAGTGTTAAAACAAG  (GCT)4. .E− Protein transport protein

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SYMS R GACTCTGCTTTCTTGGATATG
SYMS F AGCTATCTTTAGTGGGGACAT  (CGT)4. .E− Hypothetical protein
SORBIDRAFT g
SYMS R GAGGTCTCATCGGAGCTTA
SYMS F AGGTCGTTTTAATTCCTTCC  (GTTTT)3. .E− Preprotein translocase Sec
SYMS R CGTAAATATGAACGAGGTCAG
SYMS F AGGTCGTTTTAATTCCTTCC  (TTTA)6. .E− TPA: hypothetical protein
SYMS R CGTAAATATGAACGAGGTCAG
SYMS F GGACTGTACAAGGACGACAG —(GCT)
4—.E−
Zinc nger A and AN
domains-containing
protein
SYMS R TCTGCTTTCTTGGATATGGTA
SYMS F TCCAAGGATTTAGCTATGGAT —(TGT)
10 — .E− TPA: seed maturation
protein
SYMS R TTCAACTACACCCTTCTGTTG
SYMS F GCGTCAGAGTGTTAAAACAAG —(GCT)
4—.E− Hypothetical protein
SYMS R ATTGTCACTTGCTATCCATTT
SYMS F CACCTTCTTTCCTTCTCCTC —(CGC)
4—.E− V-type proton ATPase
 kDa proteolipid subunit
SYMS R GTAGATACCGAGCACACCAG
SYMS F TCAGTTCAGGGATGACAATAG  (CCGTGG)3. .E−
Homeodomain-like
transcription factor
superfamily protein
SYMS R GGATAGACTGAAATCTGCTCA
SYMS F CAACTCGACTCTTTTCTCTCA — (CTC)5— .E− Protein transport protein
SEC
SYMS R GGAGGTGGAACTTCCTGA
SYMS F GGACTGTACAAGGACGACAG —(GCT)
4—.E−
Protein transport protein
Sec subunit beta-like
isoform
SYMS R TCTGCTTTCTTGGATATGGTA
SYMS F GGACTGTACAAGGACGACAG —(GCT)
4— .E−
Protein transport protein
Sec subunit beta-like
isoform
SYMS R TCTGCTTTCTTGGATATGGTA
SYMS F AAACGATCAGATACCGTTGTA —(CG)
6—.E− Caltractin
SYMS R ATCAAAGAGATCAAAGGCTTC
SYMS F CATTTCGAAGCTCCTCCT  (CCTCCG)6. .E−
Zinc nger A and AN
domains-containing
protein
SYMS R TAGGCTGCACAACAATAGTCT
SYMS F CTCCCCCATTTCTCTTCC  (GCAGCC)6. .E− Predicted: reticulon-like
protein B
SYMS R CAAGTACTCCAGCAGAGATGT
SYMS F CTTTTCCCTCTTCCTCTCTC — (CCG)5—.E− Predicted:uncharacterized
tRNA-binding protein
SYMS R TGTCACTAACACGAATCACAA
SYMS F CCCTCTCCCTGCTCTTTC  (TCC)5. .E− Actin-depolymerizing
factor 

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SYMS R CAGTCACAAAGTCGAAATCAT
SYMS F ACAACTCTTCAGTCTTCACGA  (CAAC)3. .E− Truncated alcohol
dehydrogenase
SYMS R CCAATCTTGACATCCTTGAC
SYMS F GCACGGTGAAGTTCTAGTTC  (TCGAT)4. .E− Hypothetical protein
SORBIDRAFT g
SYMS R CAGCTTCACTCATGAATTTTT
SYMS F GGACTGTACAAGGACGACAG —(GCT)
4—.E−
Protein transport protein
Sec subunit beta-like
isoform
SYMS R TCTGCTTTCTTGGATATGGTA
SYMS F AACACAAGCAAGAAATAAACG  (GGT)4. .E− Dirigent-like protein
SYMS R AACACTATGGTCAAGGTGGTA
SYMS F GCGTCAGAGTGTTAAAACAAG  (GCT)4. .E−
Protein transport protein
Sec subunit beta-like
isoform
SYMS R GAAATCGCTCTATAAGGTTCC
SYMS F TCTCTCTGAAGATGATGCTTT  (AAG)5. .E− Hypothetical protein
SORBIDRAFT g
SYMS R GTTAAGAGGCTTCCAAAGAAC
SYMS F CAGCTCGTCGTCTTCTTTT —(GTC)
5.E−
Putative
ubiquitin-conjugating
enzyme family
SYMS R GTGGCTTGTTTGGATATTCTT
SYMS F GGACTGTACAAGGACGACAG  (GCT)4. .E−
Protein transport protein
Sec subunit beta-like
isoform
SYMS R CGTCAGACGTACTGAAATGTT
SYMS F AACACAAGCAAGAAATAAACG  (GGT)4. .E− Putative dirigent protein
SYMS R AACACTATGGTCAAGGTGGTA
SYMS F GGACTGTACAAGGACGACAG —(GCT)
4—.E−
Protein transport protein
Sec subunit beta-like
isoform
SYMS R TCTGCTTTCTTGGATATGGTA
SYMS F CCCTCTCCCTGCTCTTTC  (TCC)4. .E− Actin-depolymerizing
factor 
SYMS R CAGTCACAAAGTCGAAATCAT
SYMS F GGACTGTACAAGGACGACAG  (GCT)4. .E−
Protein transport protein
Sec subunit beta-like
isoform
SYMS R TCTGCTTTCTTGGATATGGTA
SYMS F GGACTGTACAAGGACGACAG  (GCT)4. .E− Preprotein translocase Sec
SYMS R TCTGCTTTCTTGGATATGGTA
SYMS F GCACCCCCAATTCGAACG  (ACG)3. .E− TPA: general regulatory
factor 
SYMS R CGGTAGTCCTTGATGAGTGT
SYMS F GGACTGTACAAGGACGACAG  (GCT)4. .E−
Protein transport protein
Sec subunit beta-like
isoform
SYMS R TCTGCTTTCTTGGATATGGTA
SYMS F CACGCAACGCAAGCACAG  (CCAT)3. .E− Hypothetical protein
SORBIDRAFT g

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SYMS R AAGTTGATTCACCCTCATTCT
SYMS F CACGCAACGCAAGCACAG  (CGATC)3. .E−
Translocon-associated
protein alpha subunit
precursor
SYMS R AAGTTGATTCACCCTCATTCT
SYMS F GGACTGTACAAGGACGACAG  (GCT)4. .E−
Protein transport protein
Sec subunit beta-like
isoform
SYMS R TCTGCTTTCTTGGATATGGTA
SYMS F CTTGATCCTTGACAAAAGAGA  (AG)6. .E−
Predicted:
ubiquitin-conjugating
enzyme E
SYMS R ATTGCTGTTGATATTTGGATG
SYMS F GCGTCAGAGTGTTAAAACAAG  (GCT)4. .E−
Protein transport protein
Sec subunit beta-like
isoform
SYMS R GTGTAGAACTGGAGCATTGAG
SYMS F TATCAACAAGCCTTCCATTC  (GTG)4. .E− Glycine-rich RNA-binding
protein 
SYMS R GGCTATAGTCACCACGGTAG
SYMS F CGACAGGGAGAAGAGTACAG  (GCT)4. .E−
Protein transport protein
Sec subunit beta-like
isoform
SYMS R GACTCTGCTTTCTTGGATATG
SYMS F GCGTCAGAGTGTTAAAACAAG  (GCT)4. .E−
Protein transport protein
Sec subunit beta-like
isoform
SYMS R AATCGCTCTATAAGGTTCCTC
SYMS F CTCTTCTTCACCAATTCCTCT — (CCG)6—.E−
Protein transport protein
Sec subunit beta-like
isoform
SYMS R CAAACCTCATAAAGAGTGCAG
SYMS F GGGCAAGCAAGAAACCAC  (TCC)4. .E− TPA: translation initiation
factor 
SYMS R CGTACATGAACGTAGTCCTTT
SYMS F GCGTCAGAGTGTTAAAACAAG —(GCT)
4—.E− Protein transport protein
Sec beta subunit
SYMS R AATCGCTCTATAAGGTTCCTC
SYMS F TTATAAGGAAATCCCCCACT —(GCC)
4—.E− Hypothetical protein
SORBIDRAFT g
SYMS R CACCAAGTACTCATCCATCAT
SYMS F CATCTCCTGCTAACAATTCAC  (TGC)4. .E− Predicted:NAC
domain-containing protein
SYMS R ATTTATAGGTTGGCACCAGAG
SYMS F GCGTCAGAGTGTTAAAACAAG  (GCT)4. .E−
Protein transport protein
Sec subunit beta-like
isoform
SYMS R GTGTAGAACTGGAGCATTGAG

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0
20
40
60
80
100
120
140
AC/GT
AG/CT
AT/AT
CG/CG
AAC/GTT
AAG/CTT
AAT/ATT
ACC/GGT
ACG/CGT
ACT/AGT
AGC/CTG
AGG/CCT
ATC/ATG
CCG/CGG
AAAG/CTTT
AAAT/ATTT
AAGG/CCTT
AATG/ATTC
AATT/AATT
ACGC/CGTG
ACTC/AGTG
AGCC/CTGG
AGCG/CGCT
AGGC/CCTG
AGGG/CCCT
CCGG/CCGG
AAAAG/CTTTT
AATAT /ATAT T
ACAGG/CCTGT
ACCTC/AGGTG
AGCGG/CCGCT
AACACC/GGTGTT
AGCAGG/CCTGCT
Types of motifs
Frequency
F : Details of  dierent types of nucleotide repeat motifs belonging to di-, tri-, tetra-, penta-, and hexanucleotide repeat motifs with
sequence complementarity.
0
10
20
30
40
50
60
70
Ala
Arg
Asn
Asp
Cys
Gln
Glu
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Val
Types of amino acids
Frequency
Ser
Stop
Tyr
r
F : Details of dierent types of predicted amino acids encoded by trinucleotide repeat motifs.
transcripts. Selected EST-SSRs were associated with various
pathways of metabolic process, namely, GO: DNA
repair, GO: postreplication repair, GO:
RNA metabolic process, GO: RNA metabolic
process, GO: regulation of translational initiation,
GO: ATP hydrolysis coupled proton transport,
GO: lipid metabolic process, GO: protein
transport, GO: transcription factor complex,
GO: microtubule organizing centre, GO:
translation initiation factor activity, GO: 󸀠-
tyrosyl-DNA phosphodiesterase activity, GO: actin
lament depolymerization, and GO: hydrogen ion
transmembrane transporter activity, and so forth (see the
complete details of the most promising hits of gene ontology
of EST-SSRs in the supplementary table available online at
http://dx.doi.org/.//).
3.3. Assessment of EST-SSR Marker in Selected Plants. Aset
of  EST-SSR primers were evaluated for PCR optimization,
polymorphism, and cross amplication in twenty genotypes
belonging to cereals plants and Saccharum related genera and
Saccharum species and their commercial cultivars, of which
 EST-SSR primers produced successful amplications with
both expected and unexpected sizes (Figure ). Among 
EST-SSRs, twenty-eight belonged to trinucleotide repeats
with then seven of tetra-, three of penta-, three of hexa-, and
one of dinucleotide repeats. Meanwhile, PCR amplications
produced  alleles (expected size) at  loci with an average
of.allelesperlocus.isresultiscomparablewithearlier
studies that reported on various plant species, namely, .
alleles/locus in rice varieties [], . to . alleles per locus
in maize [], and . alleles/locus in rye []. However,
our result of alleles per locus is lower compared to previous
studies that reported on sugarcane, that is, . alleles/locus
[], . alleles/locus [], and . alleles/locus []. e
polymorphic information content (PIC) was extended from
. to . with an average of .. It could be encompassed
that low and high range of allelic amplications with EST-
SSRs correspond to marker polymorphism and low level
of polymorphism from EST-SSRs might be due to possible
selection against alterations in the conserved sequences of
EST-SSRs [, ].
3.4. Cross Transferability. e potentials of EST-SSR primers
were examined for cross transferability among  plant
species belonging to cereals and Saccharum related genera
and Saccharum species and their cultivars under the same
PCR conditions. However,  EST-SSRs showed successful
amplications among all the selected plants. e cross trans-
ferability was estimated to be .% in wheat, .% in
maize, .% in barely, .% in rice, .% in pearl millet,
.% in oat, .% in Sorghum, .% in Narenga, .%
in Sclerostachya, .% in Erianthus, .% in Miscanthus,
.% in Bandjermasin Hitam,.%inGunjera,.%in

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Primary metabolic process
Cellular metabolic process
Response to stress
Macromolecule metabolic process
Cellular response to stimulus
Tra nsp o rt
Response to other organisms
Response to biotic stimulus
Establishment of protein localization
Response to abiotic stimulus
Immune response
Response to chemical stimulus
Regulation of biological process
Cellular component organization
Cellular component organization or
biogenesis at cellular level
Nitrogen compound metabolic process
Small molecule metabolic process
Catabolic process
Oxidation-reduction process
Establishment of localization in cell
Biosynthetic process
Anatomical structure morphogenesis
Actin lament-based process
Cell cycle process
Vesicle-mediated transport
Post-embryonic development
Interspecies interaction between organisms
Developmental process involved in reproduction
Cellular developmental process
Activation of immune response
Cell growth
Regulation of biological quality
Immune eector process
Modication of morphology or
physiology of other organism
Secondary metabolic process
Embryo development
Cellular localization
Seed germination
Multicellular organismal reproductive process
Anatomical structure formation
involved in morphogenesis
Transmembrane transport
Dormancy process
Cellular component movement
Cell division
Response to endogenous stimulus
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Biological_process of GO Iv3
(a)
F : Continued.

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0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Cellular_component of GO Iv3
0.90.80.7 1.0
0.0 0.60.40.30.20.1 0.5
External encapsulating structure
Envelope
Vesicle
Cell-cell junction
Membrane part
Ribonucleoprotein complex
Protein complex
Organelle membrane
Non-membrane-bounded organelle
Intracellular organelle part
Membrane
Membrane-bounded organelle
Intracellular organelle
Intracellular part
(b)
0.80.7 0.9 1.00.5 0.60.30.20.1 0.4
0.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Molecular_function of GO Iv3
transcription factor activity
Sequence-specic DNA binding
Transmembrane transporter activity
Tetrapyrrole binding
Oxidoreductase activity
Ligase activity
Nucleotide binding
Hydrolase activity
Ion binding
Protein binding
Nucleic acid binding
Substrate-specic transporter activity
(c)
F : Most promising results of gene ontology (GO) as horizontal bar graphs. ese graphs represent the distribution of GO terms
categorized as a biological process (a), cellular component (b), and molecular function (c).
NG, .% in N, .% in CoS , .% in CoS
, .% in UP , .% in CoS , and .%
in CoS . Meanwhile, the frequency distributions of cross
transferability of EST-SSRs ranged from .% for Sorghum
to .% for Sclerostachya,withanaverageof.%
(Table ). Saccharum related genera (.%) and Saccharum
species (.%) showed high rate of cross transferability
compared to other groups. is is in agreement with previous
studies reported on Saccharum species and Saccharum related
genera [, , ]. Several earlier studies related to cross
transferabilityhavebeenreportedondistinctplantgroups
from dierent families using EST-SSRs markers [, , ].
is suggests that transferring ability of genic markers makes
it compatible to determine genetic studies across the taxa for
utilization in mapping of genes from related species along
with genera and identication of suspended hybridization.
is can also aid vigilance of the introgression of genetic
entity from wild relatives to cultivated, comparative mapping
and establishing evolutionary relationship between them.
us, microsatellites derived from expressed region of the
genome are expected to be more conserved and more trans-
ferable across taxa.
3.5. Genetic Diversity Analysis by EST-SSRs. In order to
evaluate the potential of EST-SSRs, the genetic analysis was
done among  genotypes belonging to  cereals (wheat,
maize, barley, rice, pearl millet, oat, and Sorghum),  Sac-
charum related genera (Erianthus,Miscanthus,Narenga,and
Sclerostachya),  Saccharum species (NG (S. robustum),
N (S. spontaneum), and two of S. ocinarum clones
(Gunjera and Bandjermasin Hitam)), and  sugarcane com-
mercial cultivars (CoS , CoS , CoS , UP ,
and CoS ). e generated allelic data were used for
genetic relationships analysis by making dendrogram based
on Jaccard’s and neighbour-joining algorithm using FreeTree
and TreeView soware. e dendrogram fell into three major
clusters with several edges, cluster I with eight genotypes
comprising most of Saccharum species and their commercial
cultivars, cluster II encompassing six genotypes of most of
cereals species, and cluster III with six species comprising

Genetics Research International 
T : Details of cross transferability of  EST-SSR markers in twenty genotypes belonging to cereals and Saccharum related genera and Saccharum speciesandtheircultivars.Lanesto
 represent number of bands produced in wheat, maize, barley, rice, pearl millet, oat, Sorghum,Narenga,Sclerostachya,Erianthus,Miscanthus,Bandjermasin Hitam,Gunjera, NG, N,
CoS , CoS , UP , CoS , and CoS , respectively.
S. number/lane          Primer
polymorphism (%)
SY   
SY                  
SY   
SY    
SY                
SY   
SY              
SY                    
SY              
SY   
SY               
SY                    
SY   
SY   
SY                    
SY                     
SY    
SY                    
SY                    
SY   
SY                    
SY               
SY                    
SY                 
SY                  
SY                    
SY                  
SY                     
SY                    
SY                  
SY                    
SY                  
SY           
SY                     
SY                  
SY                     
SY                   
SY               
SY                     

 Genetics Research International
T  : C ontinu e d .
S. number/lane  Primer
polymorphism (%)
SY   
SY          
SY        
Ave rage of
transferability . . . . . . . . . . . . . . . . . . . .

Genetics Research International 
20
191817
16151413121110987654321M
50 bp
100 bp
200 bp
300 bp
400 bp
600 bp
1050 bp
1350 bp
F : e gel represents PCR amplication prole with SYMS
primer among twenty dierent plant species. Lanes:  wheat,maize,
barley,rice,pearl millet,oat,Sorghum,Narenga,
Schlerostachya,Erianthus,Miscanthus,Bandjermasin Hitam,
 Gunjera,51NG56,N58,CoS 92423,CoS 88230,UP
9530,CoS 91230,andCoS 8436.
CoS 92423
51NG56
Bandjermasin Hitam
Gunjera
CoS 88230
UP 9530
CoS 8436
CoS 91230
Barley
Rice
Maize
Wheat
Pearl millet
Sorghum Erianthus
Narenga
Miscanthus
N58
Oat
Sclerostachya
I
II
III
F : Dend rogram is constructed b ased on allelic data produ ced
from  EST-SSR markers using FreeTree and TreeView soware.
most of the Saccharum related genera along with some
interventions (Figure ). is relationship is in agreement
with previous studies reported by other authors [, , , ].
Our EST-SSRs markers showed close syntenic relationship
and their evolutionary nature among the  genotypes
into three major clusters with some genotypes divergence.
ese relationships have resulted from the expansion and
contraction of SSRs in conserved EST sequences within the
same group of plant species along with some variation having
resulted from higher evolutionary divergence among them.
Several earlier studies also reported on genetic diversity
analysis within and across the plant taxa using molecular
marker[,,,,,–].us,microsatellitemarkers
distinguished all the genotypes to certain extent and also
provided the realistic estimate of genetic diversity among
them.
3.6. Bulk Segregation Analysis (BSA) in Sugarcane. All the
EST-SSRmarkerswereevaluatedinpooledDNAbulks
SYMS69SYMS66SYMS53SYMS45
SYMS30
SYMS89SYMS83SYMS82SYMS81SYMS72
F : e gel represents polymorphism and discrimination
between bulks of pooled DNA with contrasting high and low plant
diameter through bulk segregation analysis.
of contrasting trait of sugarcane cultivars (CoS  (CoS
 ×Co ) cross with CoS  (MS / ×Co ))
for the identication of reporter EST-SSR markers based
on their allelic dierences between them. Interestingly, 
markers showed polymorphic nature and apparently dis-
criminating potential between bulks through bulk segrega-
tion analysis (Figure ). Among these, markers SYMS,
SYMS, SYMS, and SYMS showed a better response to
discriminating the bulks. BSA is the strategy that involves
the identication of genetic markers associated with char-
acter or trait which are based on their allelic dierences
between bulks []. Earlier studies have been established
in sugarcane for the most prominent molecular markers
detection linked to desirable traits through BSA. For example,
molecular markers apparently linked to high ber content in
Saccharum species [–] and molecular markers used for
QTL analysis and utilized for generating genetic maps around
resistance genes in sugarcane against diseases and pests
through BSA [, , ]. Several other studies also reported
on selection of dierent agronomic traits in sugarcane for
breeding programme with the development of molecular
markers through BSA [, –]. Alternatively, BSA approach
has been recently used for various purposes against the
identication of dierential expressed gene associated with
both qualitative and quantitative using of the cDNA-AFLP
approach [–]. us, BSA approach provides the easiest
way in the direction of trait linked marker identication and
also makes it possible to select informative markers beside
evaluationsofeachmarkerinthewholeprogeny.

 Genetics Research International
4. Conclusion
e present study was intended for identication and char-
acterization of SSR in Saccharum spp. expressed sequence
tag which is retrieved from public database (NCBI). Further,
functional annotation was feasible to identify the most emi-
nent EST-SSR markers selection. erefore, this is the bypass
way for EST-SSR markers development which reduces cost
and time and provides an ecient way to analyze the tran-
scribed portion of genome besides expense of own libraries
development. A total of  EST-SSR markers were developed
and experimentally validated for cross transferability along
with their genetic relationships and also used for dieren-
tiation between pooled DNA bulks of Saccharum cultivars.
ese markers showed successful transferability rate among
the twenty genotypes and established genetic diversity among
cereals, Saccharum species/cultivars, and Saccharum related
genera with some inconsistency. Further, some prominent
marker also distinguished pooled DNA bulks of sugarcane
cultivars based on stem diameter. Consequently, these EST-
SSR markers were found to be more convenient which made
it easy for us to use them as informative markers in further
genetic studies in sugarcane breeding programme.
Competing Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-
ogy, UP Council of Sugarcane Research, for providing an
opportunity and facilities for research works. Authors are
also grateful to Director, UP Council of Sugarcane Research,
Shahjahanpur, UP, India for their moral support. Authors
also acknowledge University of Rajasthan for providing DBT-
IPLS and DBT-BIF facilities.
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