Acta Botanica 1-2017 - za web.indd


ACTA BOT. CROAT. 76 (1), 2017 103

Acta Bot. Croat. 76 (1), 103–106, 2017   CODEN: ABCRA 25
DOI: 10.1515/botcro-2016-0050 ISSN 0365-0588
 eISSN 1847-8476

Short communication

Microsatellite allele length variations 
in inter-specifi c hybrids of Eucalyptus
Murugan Sumathi, Yasodha Ramasamy*

Institute of Forest Genetics and Tree Breeding Coimbatore 641 002 Tamilnadu, India

Abstract – The genus Eucalyptus encompasses several species of industrial importance. Many of these spe-
cies have been subjected to genetic characterization using different kinds of DNA markers. More than 1000 
microsatellites have been identifi ed from the genome of eucalypts and they are highly amenable for cross spe-
cies transferability. During cross amplifi cation of microsatellites, homoplasy is reported in many species in 
which although the allele size might be the same, the sequences are not. Thus, it is essential to ascertain the 
DNA sequence homology with source and target microsatellite repeats. Accordingly, fifty five alleles from 
six microsatellite loci (ECc1, ECc2, Eg61, Embra100, Embra1468 and Embra2002) were amplifi ed in two 
inter-specifi c hybrid populations (Eucalyptus tereticornis × E. grandis and E. tereticornis × E. camaldulensis) 
and sequenced. The results showed that all the microsatellite loci were amplifying the target repeat types ex-
cept for the loci Eg61 and Embra2002. The locus Eg61 has target repeat of (CAA)(GAT) but the sampled al-
leles had either (CAA)(GAT) or (GAT) alone. Similarly, the Embra2002 locus was targeting interrupting re-
peats of (CCA)..(CCA), but the sequenced alleles had repeats of (CCA) with or without interruption. 
Nevertheless, the allele size estimated in electrophoresis for hybrids was in conformity with that of the parent 
alleles. This study suggests the need for validation of the repeat characteristics of microsatellites by sequenc-
ing of the alleles particularly in cross species amplifi cation.

Keywords: allele sequences, Eucalyptus, inter-specifi c hybrids, microsatellites, repeat size

* Corresponding author, e-mail: yasodharaja@yahoo.com

Introduction
The species of the genus Eucalyptus are widely planted 

in tropical and temperate regions of the world and are an 
important commercial tree species for paper pulp, bioener-
gy and timber production. Several members of Eucalyptus 
have been genetically characterized because of the ease of 
generating inter-specifi c crosses and vegetative propaga-
tion, its unique biological properties, fast growing nature 
and comparatively small genome size. Various DNA mark-
ers have been applied for the genetic characterization of the 
eucalyptus genome. Microsatellites or simple sequence re-
peats (SSRs) are the DNA marker system of choice, for it is 
highly conserved and easily transferrable across species. 
Many of these SSRs were shown to be potentially involved 
in growth, wood quality and stress responsive pathways 
(Gion et al. 2015). SSR based genetic linkage map has a 
signifi cant place in eucalyptus genetic improvement be-
cause the high cross species transferability led to the inte-
gration of linkage maps across different genetic back-
grounds. Genetic maps in eucalypts were used in mapping 
quantitative trait loci (QTL), synteny and collinearity anal-

ysis and genome sequence assembly (Hudson et al. 2012, 
Bartholome et al. 2015).

Recently, Grattapagalia et al. (2014) showed that 84% 
of the mapped microsatellites had colinearity between link-
age map and physical position in the assembled E. grandis 
genome. Although to date about 1200 SSRs have been 
characterized in different species of eucalypts, only about 
535 SSRs have been utilized for genetic mapping studies. 
Hence, to generate high density genetic map in new combi-
nations of inter-specifi c hybrids requires the addition of a 
higher number of SSR loci. So far, the majority of the SSRs 
mapped (both genomic and expressed sequence tag-derived 
SSRs) were developed from E. grandis and E. urophylla 
and have been transferred to other species. During cross 
transfer, it is essential to verify the allele variations by se-
quencing the PCR fragments, wherein the structural vari-
ants in the form type of repeats, insertion or deletion gener-
ated by the SSRs are accurately detected. In eucalypts, 240 
EST-SSRs were confi rmed for their sequence homology 
with the original EST sequences, however, no information 
on repeat size variations were reported (Zhou et al. 2014). 
Recently, it was emphasized that the sequence verifi cation 



SUMATHI M., RAMASAMY Y.

104 ACTA BOT. CROAT. 76 (1), 2017

of SSR alleles is critical for interpretation of the SSR poly-
morphism for genetic analysis (Barthe et al. 2012). Further, 
size homoplasy is commonly reported among the SSR al-
leles (Curtu et al. 2004, Javed et al. 2014), hence, informa-
tion on DNA sequence is essential before the utilization of 
SSR loci variations. Thus, in the present study, attempts 
were made to sequence the SSR-PCR amplicons generated 
in the inter-specifi c hybrid populations, E. tereticornis × E. 
camaldulensis and E. tereticornis × E. grandis.

Materials and methods
Plant material and DNA isolation

The two inter-specifi c hybrid populations used in this 
study were E. tereticornis (Et86) × E. grandis (Eg9) and E. 
tereticornis (Et217) × E. camaldulensis (Ec17). These spe-
cies belong to the subgenus (Symphyomyrtus) and the sec-
tion Exsertaria (E. tereticornis and E. camaldulensis) and 
Lato angulatae (E. grandis). The number indicated for each 
species represents the parent clone identity. The hybrid 
populations were generated by controlled pollination be-
tween selected parents. F1 seeds generated from controlled 
pollination were established in a fi eld trial at Panampally, 
Kerala. Total genomic DNA was extracted using DNA iso-
lation kit (Qiagen Ltd, UK) from the freshly collected 
leaves of two individual hybrid plants in each population. 
The DNA of parents was isolated from leaf samples of trees 
employed in controlled pollination. DNA quality and quan-
tity was assessed by electrophoresis on 0.8% agarose gel 
with Lambda DNA (Bangalore Genei, Bangalore, India) as 
the standard and spectrophotometry (NanoDrop 8000; Ther-
mo Scientifi c, Wilmington, DE, USA).

SSR amplifi cation

Three hundred and twenty SSRs (Sumathi et al. 2014) 
were tested between the parents of two hybrid populations 
for the selection of polymorphic loci. For sequence analysis 
and repeat motif identifi cation of the parents and hybrids, 6 
loci (4 genomic SSRs – ECc1, ECc2, Eg61, EMBRA100, 
and 2 EST-SSRs -EMBRA1468, EMBRA2002) were ran-
domly selected to represent different repeat motif types and 
source species such as E. camaldulensis (da Silva et al. 2009) 
and E. globulus (Thamarus et al. 2002) and E. grandis (Bron-
dani et al. 2006, Faria et al. 2010). Details on allele size 
range, annealing temperature and repeat motif and NCBI 
accession number of each SSR loci are presented in Tab. 1.

SSR amplifi cation was carried on 40 μl of reaction con-
taining 4 μl of 10× buffer (100 mM Tris–HCl (pH 8.3), 500 
mM KCl and 15 mM MgCl2), 125 μM of dNTPs, 0.4 μM of 
each forward and reverse primer, 4 U of Taq DNA poly-
merase, and 20 ng of template DNA of 2 parents and 2 F1 
individuals of the hybrid populations. The PCR amplifi ca-
tion was carried out for initial denaturation 5 min at 94 °C 
for 1 cycle and 30 cycles of 1 min at 94 °C, 30 s at the pri-
mer specifi c annealing temperature (Tab. 1), 1 min at 72 °C, 
fi nal extension for 15 min at 72 °C. PCR products were size 
separated using 5% denaturing polyacrylamide (PAGE) gels 
of size 21×50 cm (Sequi-Gen GT System, Bio-Rad, USA) 
containing 7 M urea and 1× Tris-borate-EDTA buffer. A DNA 
ladder of 50 bp (MBI, Fermentas, USA) was included dur-
ing electrophoresis of samples in the fi rst and last well of 
the electrophoresis system. The gels were run at 61 W con-
stant power for 3 h and visualized by silver staining.

Sequencing of PCR fragments and sequence analysis

To isolate the specifi c bands of SSRs for sequencing, the 
PCR amplifi ed products were separated on a 3.5% Metaphor 
agarose gel (Cambrex Bio Science, Rockland, ME, USA) in 
1× TBE buffer and visualized with ethidium bromide stain-
ing. Only the best amplifi ed bands for each SSR primer 
combination was removed from the gel and purifi ed using 
QIAquick gel extraction kit (Qiagen, Crawley, UK). These 
DNA fragments were sequenced at Eurofi ns Biotechnolo-
gies Pvt Ltd, Bangalore, India using standard procedures on 
an ABI3700 DNA analyser (Applied Biosystems, USA) by 
employing the specifi c forward or reverse SSR primer.

The sequences obtained were checked for their homol-
ogy with the SSR source sequence in NCBI and the genome 
sequence of Eucalyptus grandis in Phytozome (https://phy-
tozome.jgi.doe.gov/pz/portal.html). All the DNA sequences 
of SSR locus along with the source SSR sequence and E. 
grandis genomic region harbouring SSR were aligned using  
Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) 
to identify the structural variations in microsatellites.

Results and discussion
Microsatellites are well known to generate high poly-

morphism across species due to their mutational character-
istics (Bhargava and Fuentes 2010). Cross transferability of 
these markers across genera and species enabled the use of 
microsatellites, where the DNA sequence is not available. 

Tab. 1. Informat ion on the six microsatellite loci used in this study.

Sa
m

pl
e

SSR code Repeat motif
Annealing temperature (ºC)

Allele
size

Linkage
group

Reference
NCBI

Accession
No.Original 

Optimized 
Et86 × Eg9

Optimized
Et217 × Ec17

1 ECc1 (TC)18 60–45 56 58 157–192 1 da Silva et al. 2009 GQ302860.1 
2 ECc2 (GA)9TATA(GA)6 60–46 57 57 238–255 5 da Silva et al. 2009 GQ302861.1 
3 Eg61 (GAA)9(GAT)6 64 56 61 302–316 2//7 Thamarus et al. 2002 EU699745.1 
4 EMBRA100 (CT)26 56 58 56 195 1 Brondani et al. 2006 BV682837.2
6 EMBRA1468 (TC)12 60 57 59 181 8 Faria et al. 2010 GF101903.1
5 EMBRA2002 (CCA)8…(CCA)8 60 56 64 263 1 Faria et al. 2010 GF101907.1



MICROSATELLITE ALLELE LENGTH VARIATIONS IN EUCALYPTUS

ACTA BOT. CROAT. 76 (1), 2017 105

Hence, it is essential to confi rm the repeat characteristics of 
SSRs to utilize them as markers for DNA fi ngerprinting, 
genetic linkage map generation and quantitative trait loci 
identifi cation. In order to evaluate the variation in repeat re-
gion of SSR alleles, six SSR loci were sequenced in two 
different inter-specifi c hybrid populations of Eucalyptus. 
The lengths of the sequenced fragments varied between 
206–338 bp.

Out of 87 bands (alleles), fi fty fi ve bands showing high 
intensity in the agarose gel were selected for sequencing and 
the DNA sequences were analyzed to estimate the degree of 
sequence divergence among equally sized alleles, repeat 
number variations and repeat composition. The information 
on allele size, repeat motif for the sequenced alleles is pre-
sented in Tab. 2. The SSR locus, ECc1 (GQ302860.1) was 
developed from E. camaldulensis targeting (TC) repeats 
which had amplifi ed same type of repeats in all the sam-
ples. The parents Et86 and Eg9 generated four different al-
leles (247 and 297 bp; and 263 and 275 bp, respectively) 
for the ECc1 locus while their hybrids generated allelic 
combination of 275/297 bp (H1) and 247/263 bp (H2). Se-
quencing of all the four alleles showed repeat number vari-
ations from 16 to 25 (Tab. 2) and no direct relationship be-
tween repeat number and allele size could be established, 
because the allele size variation could be due to the varia-
tions in the fl anking regions also. The parent Et217 ampli-
fi ed two different alleles of size 263 and 297 bp and the 
parent Ec17 amplifi ed a homozygous allele of size 263 bp. 
Sequencing was done only for the 263 bp allele from all the 
four individuals and all the sequences showed same repeat 
number. The 263 bp allele sequence of parent Et217 showed 
19 (TC) repeats followed by an insertion of one (GT) repeat 
and 6 (TC) repeats. Such insertions or interruptions within 
the motif sequence were documented in Vitis (Masi et al. 
2004), Citrus (Barkley et al. 2009) and rice (Dong et al. 
2013). Sequence data on microsatellite variation in birds in-
dicated that size homoplasy exists with no sequence simi-
larity (Anmarkrud et al. 2008). In tree species, Barthe et al. 
(2014) showed the high amount of variations in fl anking re-
gions. The similarity of repeat regions across the genome 
sequence of E. grandis in Phytozome, the SSR source se-
quence and the allele sequences for all the loci analyzed are 
given in On-line Suppl. Material.

The locus ECc2 produced heterozygous alleles in par-
ents Et86 (241/247 bp) and Et217 (235/250 bp) and homo-
zygous alleles in parents Eg9 and Ec17 (238 bp). In the 
original E. camaldulensis sequence of ECc2 (GQ302861.1), 
GA repeat was interrupted with TATA and was not observed 
in any of the sequences analyzed including the genome se-
quence of E. grandis. Instead, all the sequences had (GA) 
repeats in uninterrupted form and the number varied from 
16–19. Similarity across the sequences was also very low 
except in the repeat region. The Eg61 SSR locus generated 
heterozygous alleles in both the parents (ET86 and Eg9). 
Original microsatellite sequence of Eg61 (EU699745.1) 
and E. grandis genome sequence showed compound SSR 
(GAA) (GAT) repeats. The parents Et86 and Eg9 showed 
only (GAT) repeats and their hybrid individuals showed 
(TAT) and (AAT) repeats and this could be attributed to-
wards the error during PCR amplifi cation or sequencing. Ta

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SUMATHI M., RAMASAMY Y.

106 ACTA BOT. CROAT. 76 (1), 2017

The Et217 parent had (GAA and (GAT) motifs and Ec17 
and the hybrids showed only (GAT) motifs.

The SSR loci Embra100 (BV682837.2) was present in 
the E. grandis gene sequence Protein Transparent Testa12 
(Eucgr.A02047), which had 18 repeat units of the target 
motif (CT). This locus was heterozygous in both the crosses 
analyzed. Alleles from the individuals sequenced showed 
(CT) repeats varying from 18 to 25. The EST-SSR locus 
Embra1468 (GF101903.1) showed similarity with the gene 
sequence Acetyl-CoA C-acetyltransferase / Acetoacetyl-
CoA thiolase (Eucgr.H00849). It had heterozygous alleles 
in Et86, Eg 9 and Ec17 whereas the Et217 showed homozy-
gous alleles. The original sequence consisted of 12 repeat 
units of TC and all the alleles analyzed had 13–22 repeat 
units. The SSR locus Embra2002 (GF101907.1) sequence 
showed similarity with Auxin responsive protein (Auxin_in-
ducible) of E. grandis (Eucgr.A01420). Except Ec17 all the 
parents had heterozygous alleles and the target repeat of 
(CCA) was present in varying repeat numbers in interrupt-
ed and uninterrupted form (Tab. 2).

This study demonstrated the successful cross species 
amplifi cation of several eucalypt SSR loci within the genus. 
Microsatellite allele sequencing results of parents and their 

hybrids show almost same repeat type, corroborating the 
earlier results in eucalypt species (Ochieng et al. 2007, He 
et al. 2012). Variations in fl anking regions and repeat num-
bers refl ect the occurrence of mutational processes in both 
the repeat and fl anking regions. Similar observations were 
made in Vitis (Masi et al. 2004) and Shorea (Javed et al. 
2014) and insisting the requirement of information on un-
derlying DNA sequence. However, with the availability of 
genome sequence of E. grandis, the primer sequence simi-
larity search can be applied to fi nd the primer binding re-
gions. Loci with more than one similar site may be avoided 
to limit any discrepancy during genotyping. Although mic-
rosatellites are proven to be highly informative markers and 
continue to be used along with NGS based markers, though 
they throw up lot of challenges during genotyping and al-
lele calling in the form of homoplasy, which demands 
enough care while employing the microsatellites for link-
age mapping and QTL studies.

Acknowledgements
This research was supported by a research grant from 

the Department of Biotechnology (DBT), New Delhi, India.

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