American Journal of Plant Sciences, 2011, 2, 425-432
doi:10.4236/ajps.2011.23048 Published Online September 2011 (
Copyright © 2011 SciRes. AJPS
Assessment of Genetic Diversity in Contrasting
Sugarcane Varieties Using Inter-Simple
Sequence Repeat (ISSR) Markers*
Maria Lucília M. da Costa1, Lidiane L. Barbosa Amorim2, Alberto V. C. Onofre2, Luiz J. O. Tavares de
Melo3, Maria Betânia M. de Oliveira2, Reginaldo de Carvalho1, Ana M. Benko-Iseppon2#
1Universidade Federal Rural de Pernambuco (UFRPE), Genetics Department, Recife, Brazil; 2Universidade Federal de Pernambuco
(UFPE), Genetics Department, Laboratory of Plant Genetics and Biotechnology, Recife, Brazil; 3Estação Experimental de Cana-de-
Açúcar do Carpina (EECAC/UFRPE), Carpina, Brazil.
Received May 23rd, 2011; revised June 29th, 2011; accepted July 15th, 2011.
Sugarcane is an important tropical crop, responsible for two thirds of the world sugar production, gaining actually
importance as a source of biofuel. Drought tolerance is a very important feature considering the actual climate change
scenario throughout the world. This study aimed to determine the genetic diversity between sugarcane varieties with
contrasting features under drought. For this purpose, twelve ISSR primers were used to characterize nine sugarcane
varieties under cultivation in different countries including selected drought resistant material from Northeast Brazil
and two varieties from India as contrasting genotypes. 317 scorable bands were generated, among which 301 com-
prised polymorphic markers, with an average of 25 polymorphic bands per primer. In the generated dendrogram the
accessions were placed in clusters, where cluster A included two varieties from India (Co331 and Co419), and B com-
prised plants eight Brazilian accessions and a Barb ado variety. Within this clade, drought tolerant and susceptible
varieties were clearly separated. The present evaluation revealed important contrasting parental candidates regarding
their drought response, very promising for future mapping approaches aiming the identification of quantitative trait
loci (QTLs) associated to drought in sugarcane. The selected primers were used for the first time in sugarcane, repre-
senting valuable tools for future evaluations, with emphasis to diversity characterization and genetic mapping.
Keywords: Saccharum, Genetic Variability, ISSR Marker, Drought Tolerance
1. Introduction
Sugarcane is among the most important industrial crops
of tropical and subtropical regions and is cultivated in
more than 90 countries around the globe primarily for its
ability to store high concentrations of sugar and more
recently for the production of ethanol, very demanded as
biofuel. Modern sugarcane are complex hybrids derived
largely from the interspecific crosses involving Saccha-
rum officinarum L. (2n = 80) and the wild species S.
spontaneum L. (2n = 40 - 128) [1].
The choice of the variety is one of the most important
factors in sugarcane breeding and production. Different
varieties have different yield potentials, pest and disease
resistance and are bred for different ecological and eco-
nomic conditions [2-4]. Therefore, the establishment of
the adequate variety to be grown in a given region, char-
acterized by water deficiency originated by irregular dis-
tribution of rainfall, is of paramount importance.
Molecular markers are powerful tools to uncover the
complex genetics of sugarcane and to assist breeders in
genetic improvement of varieties [5-7]. The employment
of new technologies to help in the association of traits
with genetic markers and genetic maps can aid in
achieving further yield increases in breeding programs
Information regarding genetic variability may contrib-
ute to germplasm conservation and use, such as by iden-
tifying duplicates in databanks, in monitoring the genetic
integrity of accessions, and by providing additional in-
formation that may help in the establishment of relation-
ships among genotypes. Traditionally, the choice of pa-
*CNPq Conselho Nacional de Desenvolvimento Científico e Tecnológico,
Assessment of Genetic Diversity in Contrasting Sugarcane Varieties Using Inter-Simple
Sequence Repeat (ISSR) Markers
rental lines in sugarcane breeding programs has been
defined on the basis of agronomic characters and pedi-
gree records, using bi-parental crosses or polycrosses
between elite genotypes. However, the lack of genealogy
data and the inadequate identification of some genotypes
have impaired an accurate estimation of the genetic di-
versity (GD) among sugarcane accessions based on
pedigree data. In addition, the continuous selection for
the same traits such as sucrose content in breeding pro-
grams has caused a reduction in GD, limiting further
advances in sugarcane breeding [11].
Several different molecular markers have been used in
previous studies that have examined diversity among
sugarcane cultivars from different regions worldwide.
These include RFLP [12], RAPD [13,14], AFLP [15,16],
SSR [17] and TRAP [16]. As shown in the different ap-
proaches carried out, the employment of molecular mar-
kers bring significant improvements to assist in the asso-
ciation of traits with genetic markers and genetic maps,
helping in the achievement of significant yield increases
in breeding programs [8].
The objective of this study was to establish genetic
diversity within a collection of selected sugarcane varie-
ties with important contrasting features under drought
conditions, identified in breeding programs in north-
eastern Brazil, a region that includes extensive semi-arid
areas with few agronomic activities. This approach is
important to identify contrasting parental candidates for
future mapping approaches aiming to identity QTLs as-
sociated to drought tolerance/susceptibility and also for
the development of comparative expression essays re-
garding the same traits.
2. Material and Methods
2.1. Plant Material
This study evaluated nine sugarcane varieties already
cultivated in different countries and widely used in breed-
ing stations located in northeast Brazil. These comprise
eight Brazilian sugarcane varieties (SP79-1011, SP70-
1143, SP78-4764, RB98710, RB943365, RB763710,
RB75126 and RB863129) from two breeding programs
(COPERSUCAR: Sugar and Alcohol Production Syndi-
cate of the São Paulo State, and RIDESA: Inter Univer-
sity Network for Development of Sugar and Alcohol Se-
ctor) and one Barbado variety. Plants of SP (COPER-
SUCAR) and RB (RIDESA) varieties have been main-
tained by conventional bud propagation in the experi-
mental area of the Carpina Sugarcane Experimental Sta-
tion (EECAC). All eight SP and RB varieties have im-
portant agronomic traits, representing promising material
for the development of new cultivars. Additionally, two
Indian sugarcane varieties (Co331 and Co419), often
cultivated under a wide range of agroclimatic conditions,
were employed, representing external contrasting mate-
rial still not used in Brazilian breeding programs (Table 1).
2.2. DNA Isolation
DNA was isolated from young leaf tissues using a CTAB
(cetyl-trimethyl-amoniumbromide) protocol [18], with
minor modifications [19]. Contaminating polysaccharides
were selectively precipitated [20] and DNA concentra-
tions were determined comparatively by electrophoresis
Table 1. Identification of 11 sugarcane varieties used in the ISSR-based genetic diversity assessment, including origin, pedi-
gree and response to drought.
Order Variety Origin Progenitor Accessions Response to drought
1 SP79-1011 COPERSUCAR NA56-79 × Co775 Tolerant
2 SP70-1143 COPERSUCAR IAC48-65 × unknown Tolerant
3 RB98710 RIDESA Unknown Tolerant
4 RB763710 RIDESA F147 × unknown Tolerant
5 RB863129 RIDESA RB863411 × unknown Tolerant
6 B8008 Barbados B73348 × B74172 Sensible
7 RB75126 RIDESA C278 × unknown Sensible
8 SP78-4764 COPERSUCAR H66-6254 × unknown Sensible
9 RB943365 RIDESA ROC3 × RB83100 Sensible
10 Co331 SBI, Coimbatore, India Co213 × Co214 Unknown
11 Co419 SBI, Coimbatore, India POJ2828 × Co290 Unknown
Legend for abbreviations: COPERSUCAR = Sugar and Alcohol Production Syndicate of the state of Sao Paulo, Brazil; RIDESA = Inter University Network for
Development of Sugar and Alcohol Sector, Brazil; SBI = Sugarcane Breeding Institute. Genotypes order follow the same arrangement presented in the dendro-
gram (Figure 1).
Copyright © 2011 SciRes. AJPS
Assessment of Genetic Diversity in Contrasting Sugarcane Varieties Using Inter-Simple 427
Sequence Repeat (ISSR) Markers
in agarose gel 1.2% using known amounts of phage λ-
DNA as a reference.
2.3. ISSR Analysis
The ISSR amplification reactions contained 15 ηg of
genomic DNA, 2.0 μL 10 × buffer, 2.5 mM MgCl2, 200
μM of each dNTP (Fermentas), 50 μM primers and 0.5 U
Taq DNA polymerase (Invitrogen), with the final volume
adjusted to 20 μL with H2O bidest. The amplification
reaction was carried out in an Eppendorf Mastercycler
Gradient or Techn TC-412 thermal cycler. The reaction
included an initial denaturation step of 4 min at 94˚C,
followed by 30 cycles, each consisting of a denaturation
step of 30 s at 94˚C, annealing of 1 min at 50.4˚C to
60.5˚C (depending on the primer) and an extension of 2
min at 72˚C. PCR was terminated with a final extension
of 7 min at 72˚C. ISSR reaction products were separated
on 1.8% horizontal agarose gels, in TBE buffer with a
voltage of 5 V/cm and visualized under ultraviolet light
after staining in 0.5 μg/mL ethidium bromide. Digital
photo documentation was taken for each gel. The 100 bp
DNA ladder plus molecular weight marker was used to
compare the molecular weight of amplified products.
Twelve ISSR primers previously selected for Poaceae
species, common cordgrass [21], barley [22] and rice [23]
were chosen and ordered from MWG-Biotech for appli-
cation on sugarcane varieties (Table 2).
2.4. Data Analysis
Polymorphic ISSR markers were scored as binary data:
presence (1) or absence (0). Only clearly resolved bands
were used in the genetic analysis. The genetic similarity
among the varieties was calculated by Jaccard Similarity
Coefficient using NTSYS-pc 2.1 software [24]. A den-
drogram was constructed based on genetic distance using
neighbor-joining method and bootstrap analysis (1000
replicates) with MEGA program (Molecular Evolution-
ary Genetic Analysis), Version 4 for Windows [25].
3. Results
3.1. PCR Analysis
The ISSR analysis, carried out in 11 varieties, produced
317 bands, from which 301 (94.9%) were polymorphic
among the sugarcane varieties, with an average of 25
ISSR polymorphic bands per primer. All primers ampli-
fied fragments, with a number of amplicons varying from
39 (primer 857) to 14 (primers 807 and 815) fragments
per reaction, with sizes varying from ~200 bp to ~2.0 kb
(Table 2). No single band was specific to any individual
variety, nor to a given feature. Some primers produced
polymorphic bands specific to a set of genotypes (Figure 1).
Table 2. List of ISSR primers used, including their nucleotide sequence, annealing temperature, number of total and poly-
morphic bands, as well as percentage of polymor phic bands.
Primer Temp. 5'-3' Sequence Total # bands # Polym. bands % Polym.
UBC 857 54.0 5'ACACACACACACACACYG3' 41 39 90.2
UBC 834 52.8 5'AGAGAGAGAGAGAGAGYT3' 37 35 94.6
UBC 848 54.0 5'CACACACACACACACARG3' 37 35 94.6
UBC 810 50.4 5'GAGAGAGAGAGAGAGAT3' 32 31 96.9
UBC855 52.0 5'ACACACACACACACACYT3' 32 30 93.7
UBC 811 52.8 5'GAGAGAGAGAGAGAGAC3' 27 26 96.3
UBC 812 52.0 5'GAGAGAGAGAGAGAGAA3' 22 21 95.4
K18 54.0 5'DVHCACACACACACACA3 22 21 95.4
UBC 828 52.8 5'TGTGTGTGTGTGTGTGA3' 18 18 100
UBC823 52.0 5'TCTCTCTCTCTCTCTCC3' 18 17 94.4
UBC 807 52.0 5'AGAGAGAGAGAGAGAGT3' 16 14 87.6
UBC 815 54.0 5'CTCTTCTCTCTCTCTCTG3' 15 14 93.3
ISSR primers were obtained from the University of British Colombia (Ayres and Strong 2001, Fernández et al. 2002, Jeung et al. 2005) R = (A,G); Y = (C,T);
D = (A,G,T) (i.e. not C); H = (A,C,T) (i.e. not G), V = (A,C,G) (i.e. not T). Legend for abbreviations: Temp. = Annealing temperature; # = number; Polym. =
Copyright © 2011 SciRes. AJPS
Assessment of Genetic Diversity in Contrasting Sugarcane Varieties Using Inter-Simple
Sequence Repeat (ISSR) Markers
3.2. Clustering Analysis
The neighbor-joining method generated a dendrogram
with two main clusters, where A included two varieties
from India (Co331 and Co419) and B comprised one
Barbado and eight Brazilian varieties. The drought sus-
ceptible accession RB94-3365 occupied a basal position
considering the upper branch. The majority of sugarcane
varieties clearly grouped into two major clusters in the
dendrogram (Figure 2). The first branch included all
tolerant varieties divided into two groups. The first group
Figure 1. Examples of ISSR amplification results in sugar-
cane. (a) Amplicons using primer ISSR 815; (b) amplicons
using the primers ISSR 810 and 812. M: 100 bp ladder
(molecular weight marker). Arrows indicate major polymor-
phic sites. Order of the genotypes: RB763710, RB863129,
SP79-1011, SP70-1143, RB98710, B8008, RB75126, SP78-
4764, RB943365, Co331, Co419.
Figure 2. Consensus tree of the analyzed sugarcane acces-
sions based on ISSR data after the Neighbor Joining analy-
sis (complete deletion and p-distance) using MEGA pro-
gram. Bar indicates genetic distance. Bootstrap values > 80
[1000 replicates] are depicted on the branches. Black circles
represent drought tolerant varieties and triangles represent
susceptible varieties.
comprised two subgroups comprising the varieties SP79-
1011; SP-70-1143; RB98710 and the second one with
RB763710; RB863129 varieties. Within this group the
accessions RB763710 and RB863129 grouped with high
bootstrap value (100). The second branch included all
drought susceptible genotypes plus the variety B8008 in
a basal position.
Genetic similarity estimates varied from 0.14 (between
RB763710 and Co419) to 0.54 (between RB863129 and
RB763710) (Table 3). Low coefficients of similarity
were detected among sugarcane varieties, with an aver-
age similarity value of 0.25. The lowest similarity coeffi-
cient among Brazilian varieties was detected between
RB763710 and SP78-4764 (15.8%), also reflecting their
contrasting performance under drought (tolerance and
sensitivity to water stress, respectively) (Table 1).
4. Discussion
Similar results were reported for other accessions during
the evaluation of the genetic diversity of sugarcane varie-
ties with ISSR markers [1] with 78.48% of the bands
produced being polymorphic. However, the result ob-
tained in the present study bear higher polymorphism
levels than those previously reported by other authors
using RAPD [13,26-28].
The high polymorphism level detected by ISSR mark-
ers was expected due to the use of the external Indian
varieties and also considering the segmental allopoly-
ploid nature of sugarcane, generally attributed to the in-
terspecific hybridization crosses used during breeding
programs that generated the actual breeding accessions.
The nature of ISSR, targeting regions especially rich in
microsatellites may also justify the higher level of poly-
morphism, since those regions are known to accumulate
a larger number of mutations due to DNA polymerase
slippage during replication and unequal crossing-over
[29]. Despite of that, researchers who have compared
RAPD and ISSR methods have found that ISSR markers
exhibit higher level of reproducibility, when compared to
RAPD [30-32].
Reduced coefficient of similarity between RB763710
and SP78-4764 varieties of sugarcane has been observed
by other authors. Specific regions of the sugarcane ge-
nome related to drought tolerance, rather than the entire
genome, were sampled to evaluate the genetic variability
of candidate parents for breeding purposes [11]. In their
study, the lowest similarity value for drought was ob-
tained among the genotypes IACSP95-2078 and SP86-42
(0.44), illustrating that this cross would probably result in
the highest variability for drought among the genotypes
sampled. The SP86-42 variety presents excellent per-
formance in the Brazilian areas of “Cerrado” vegetation
Copyright © 2011 SciRes. AJPS
Assessment of Genetic Diversity in Contrasting Sugarcane Varieties Using Inter-Simple 429
Sequence Repeat (ISSR) Markers
Table 3. Similarity matrix among the sugarcane varieties obtained by Jaccard Similarity Coefficient using NTSYS-pc 2.1
RB793710 RB863129 SP79-1011 SP70-1143 RB98780 B8008
RB863129 0.5346535
SP79-1011 0.2586207 0.3185841
SP70-1143 0.2338710 0.2580645 0.5638298
RB98780 0.2540984 0.2786885 0.3738318 0.4528302
B8008 0.2320000 0.2868852 0.2649573 0.3025210 0.4090909
RB75126 0.2137405 0.2366412 0.2338710 0.2307692 0.2800000 0.4907407
SP78-4764 0.1587302 0.2016129 0.2173913 0.1951220 0.1951220 0.3097345
RB943365 0.1666667 0.1984733 0.2032520 0.1923077 0.2204724 0.2187500
Co331 0.1600000 0.1472868 0.1680672 0.1869919 0.1680000 0.1854839
Co419 0.1451613 0.1600000 0.1826087 0.1532258 0.1532258 0.1520000
RB75126 SP78-4764 RB943365 Co331 Co419
SP78-4764 0.5000000
RB943365 0.2578125 0.3333333
Co331 0.1692308 0.1880342 0.4000000
Co419 0.1640625 0.1826087 0.3090909 0.4062500
(fire adapted savannas characterized by poor and miner-
alized soils), being also grown in drought-prone envi-
Data analysis using the neighbor-joining method gen-
erated a dendrogram with two main clusters that grouped
individuals that share the same gene pool of origin. Cluster
“A” included two varieties whose genomic background is
mainly from plants used in breeding programs from India
(Co331 and Co419), and cluster “B” included eight Bra-
zilian varieties and one Barbado variety. All accessions
included in this last cluster have been used as parental in
crosses carried out in Brazilian breeding programs aim-
ing to generate drought tolerant material.
The accession RB94-3365 is the genetic basis of all
analyzed Brazilian varieties, due to its high productivity,
despite of drought susceptibility, justifying its basal posi-
tion considering the upper branch. The majority of sug-
arcane varieties clearly grouped into two major clusters
in the dendrogram with a bootstrap of 99. Within this
group, the first branch emerged with bootstrap 84, in-
cluding all tolerant varieties divided into two groups.
The first group within the upper cluster included two
subgroups, one comprising three (SP79-1011; SP-70-1143;
RB98710) and the second with two (RB763710; RB-
863129) accessions. Within this branch, RB763710 and
RB863129 grouped with a bootstrap of 100. Besides be-
ing tolerant to drought, both varieties (RB763710 and
SP79-1011) are sources of resistance against rust and
present contrasting maturation time, growth habit and
sugar yield potentials, being very useful candidates in
future breeding programs.
The second branch within the upper cluster presented
bootstrap 80 and included all drought susceptible geno-
types, with the Barbado variety (B8008) in a basal posi-
tion, a material identified as source of resistance against
rust and bearing high sugar production.
Differential responses to abiotic stress were observed
among clusters. The association of drought-tolerance
present in RB varieties (RB863129, RB763710 and
RB98710) and SP varieties (SP70-1143 and SP79-1011)
in the same branch, was supported by high bootstrap
scores (84), indicating that these accessions are also in-
Copyright © 2011 SciRes. AJPS
Assessment of Genetic Diversity in Contrasting Sugarcane Varieties Using Inter-Simple
Sequence Repeat (ISSR) Markers
teresting for breeding of this feature [33,34]. Considering
salinity sensitive varieties, two groups had been formed:
the first containing one variety (RB943365) and the sec-
ond with three varieties (SP78-4764, RB75126 and
B8008). This is promising, especially considering that
other authors have shown that the genetic variations
identified with aid of molecular markers are useful in
evaluating upland accessions for drought-tolerance with
related morphology [35].
The same was observed regarding sugarcane geno-
types with contrasting response to red rot disease [36].
The authors reported the usefulness of ISSR markers to
separate red-rot disease resistant, moderately resistant
and susceptible among sugarcane genotypes. The sugar-
cane accessions Co 8011, Co 86010, Co 85061, Co 62198,
Co 86032, GSBT 9 and Co 8014 that are resistant to
moderately resistant to red rot disease were grouped into
one cluster, while remaining genotypes Co 7804, Co
62175, Co 8371 and Co 671, susceptible to red rot dis-
ease, were grouped into another cluster.
Several authors have reported high genetic similarity
among sugarcane varieties using RAPD [13,14,27]. This
genetic homogeneity, in spite of regional adaptation and
selection history, is probable due to the repeated use of
few sets of nearly related varieties. In addition, consider-
ing the supposed narrow genetic basis of sugarcane va-
rieties based on RAPD markers, one may suppose that
other techniques have to be tested in the search for more
efficient polymorphism identification. For example, us-
ing AFLP to analyze commercial sugarcane varieties
grown under tropical and subtropical regions of India the
level of genetic diversity among the tropical and sub-
tropical cultivars was much higher than most of the pair-
wise diversity measures within each of these two adap-
tive groups. The AFLP-based clustering of the cultivars
also corresponded well with their known pedigree [37].
This method is really more effective than RAPD, but
presents the disadvantage of being more expensive and
time consuming than ISSR.
The results obtained here using ISSR were comparable
to other studies with Poaceae. In a study using AFLP and
ISSR molecular markers to evaluate the genetic diversity
and relationships of 56 waxy rice accessions, a single
ISSR primer BDB(AC)7 produced a total of 88 bands
ranging from 200 to 600 bp, including 77 polymorphic
bands [38]. The average polymorphism of the total ISSR
markers was 92.2%, much higher than that observed for
Similarly, authors reported the usefulness of ISSR in
surveying genetic variation of 46 barley accessions [32].
For this purpose, 18 primers were used, generating a total
of 107 bands with 105 (98.13%) polymorphic bands,
with two to 10 polymorphic bands per primer.
The present results revealed considerable levels of ge-
netic diversity to design crosses for mapping purposes
employing linkage analysis aiming to identify QTLs with
aid of DNA markers considering the analyzed varieties.
Moreover, unambiguous discrimination and identifica-
tion of cultivars using ISSR markers are of significance
in the context of establishing clone fidelity, cultivar cer-
tification and germplasm management.
The here tested ISSR primers were tested for the first
time in sugarcane and presented better performance re-
garding the detection of polymorphisms in this crop, than
previously used ISSR primers. Especially considering the
narrow genetic basis of many existing genetic banks in
sugarcane, they represent additional possibilities for di-
versity screening. In the case of the present evaluation
they helped in the planning of futures crosses using
drought tolerant varieties with the most productive
drought sensible cultivars, aiming the direct breeding of
this important feature, as well as for the identification of
most contrasting drought tolerant and sensible materials
for a cross aiming the construction of a genetic map for
QTL identification.
5. Acknowledgements
To CNPq (Conselho Nacional de Desenvolvimento Cien-
tífico e Tecnológico, Brazil) for fellowships and research
support. For the concession of varieties we thank the
RIDESA (Rede Interuniversitária para o Desenvolvimento
do Setor Sucroalcooleiro, Brazil) through the EECAC
(Estação Experimental de Cana-de-Açúcar do Carpina,
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