c Indian Academy of Sciences

RESEARCH ARTICLE

Isolation and characterization of microsatellite markers in Garcinia

gummi-gutta by next-generation sequencing and cross-species
amplication

K. V. RAVISHANKAR1 , R. VASUDEVA2 , BYATROY HEMANTH1 , B. S. SANDYA1 , B. R. STHAPIT3 ,

V. A. PARTHASARATHY1 and V. RAMANATHA RAO4

1
ICAR-Indian Institute of Horticultural Research, Bengaluru 560 089, India
2
Department of Forest Biology and Tree Improvement, College of Forestry Sirsi, University of Agricultural Sciences,
Dharwad 581 401, India
3
Biodiversity International, 93.4, Dharhara Marg, Fulbari, Ward no.11, Pokhara, Nepal
4
Bioversity International, University of Horticultural Sciences, GKVK, Bengaluru 560 065, India

Abstract
Garcinia gummi-gutta (L.) Roxb. (Clusiaceae) is an endemic, semidomesticated, fruit-yielding tree species distributed in the
Western Ghats of India and Sri Lanka. Various bioactive phytochemicals, such as garcinol, benzophenones and xanthones
are isolated from G. gummi-gutta and have shown antibacterial, antiviral and antioxidant activities. We sequenced the total
genomic DNA using Illumina Hiseq 2000 platform and examined 241,141,804 bp high quality data, assembled into 773,889
contigs. In these contigs, 27,313 simple-sequence repeats (SSRs) were identied, among which mononucleotide repeats were
predominant (44.98%) followed by dinucleotide and trinucleotide repeats. Primers were designed for 9964 microsatellites
among which 32 randomly selected SSR primer pairs were standardized for amplication. Polymerase chain reaction (PCR)
amplication of genomic DNA in 30 G. gummi-gutta genotypes revealed polymorphic information content (PIC) across all
32 loci ranging from 0.867 to 0.951, with a mean value of 0.917. The observed and expected heterozygosity ranged from 0.00
to 0.63 and 0.896 to 0.974, respectively. Alleles per locus ranged from 12 to 27. This is the rst report on the development of
genomic SSR markers in G. gummi-gutta using next-generation sequencing technology. The genomic SSR markers developed
in this study will be useful in identication, mapping, diversity and breeding studies.

[Ravishankar K. V., Vasudeva R., Hemanth B., Sandya B. S., Sthapit B. R., Parthasarathy V. A. and Rao V. R. 2017 Isolation and charac-
terization of microsatellite markers in Garcinia gummi-gutta by next-generation sequencing and cross-species amplication. J. Genet. 96,
xxxx]

Introduction rind of the uppage fruit has been traditionally used in India
and Sri Lanka as a culinary additive and sh preserva-
Garcinia gummi-gutta (L.) Roxb., popularly known as Mal-
tive (Samarajeewa and Shanmugapirabu 1983; Bhagyavanth
abar Gamboge (in English) uppage (in Kannada) belongs
et al. 2010). Many studies showed that hydroxycitric acid
to the family Clusiaceae. It is a moderate-sized dioecious
(HCA), a secondary compound present in the rind of uppage
tree with round canopy, drooping branches and smooth dark
fruit is effective in weight loss (Jena et al. 2002). The
barks. It is common in lower Shola forests of the West-
ern Ghats, India, up to an altitude of 1800 mean sea level. extract obtained from this fruits has exhibited the property of
G. gummi-gutta is recognized in Ayurveda, the traditional antiobesity. Recently, a number of studies have shown that
Indian system of medicine, for better digestion and is pre- G. gummi-gutta fruit extracts are rich in HCA and are effec-
scribed against abdominal disorders and heart diseases. The tive in reducing body weight (Shara et al. 2004; Saito et al.
fruit rind is ground and used as a sour avouring spice in 2005).
the preparation of curries and to garnish sh preparations. The Germplasm utilization and conservation requires precise
information on the genetics, genetic relationships and diver-
For correspondence. E-mail: kv_ravishankar@yahoo.co.in; sity. Earlier, a few studies attempted to examine diversity
kvravi@iihr.res.in. using random amplied polymorphic DNA (RAPD) and

Keywords. microsatellite markers; cross-species amplication; next-generation sequencing; Garcinia gummi-gutta.

Journal of Genetics, DOI 10.1007/s12041-017-0756-0

 K. V. Ravishankar et al.

inter-simple sequence repeat (ISSR) markers (Mohan et al. PCR and genotyping
2012; Parthasarathy et al. 2013). However, development of
We selected 50 SSR primer sets randomly and synthe-
microsatellite markers have helped in ngerprinting unique
sized with M13 tail. These M13 tailed primers were
trees, assessing degree of diversity in the populations, and
rst screened for amplication using pooled total genomic
identifying marker tightly linked to the important agronomic
DNA from ve randomly selected genotypes. We used
traits like active ingredients, and resistance to biotic and
uorescence-based M13 tailing PCR method following
abiotic stresses. Therefore, the development of markers has
Schuelke (2000) to amplify the microsatellites in a quick,
become a prerequisite for genetic studies (Bohra et al. 2011;
accurate and efcient manner. The forward primer tailed
Dutta et al. 2011). Keeping this in view, here, we report the
with 5 -GTAAAACGACGGCCAGT-3 and reverse primer
development and standardization of microsatellite markers
tailed with 5 -GTTTCTT-3 . PCR was carried out in 20 L
of G. gummi-gutta using next-generation sequencing (NGS)
reaction volume containing 2 L of 10 reaction buffer,
and their cross species transferability.
2.0 L of 1 mM dNTPs, 0.9 L (5 pmol) of forward, 0.9 L
reverse primers (5 pmol), labelled M13 probes (HEX, NED,
Materials and methods VIC, TET) 1.2 L (5 pmol), 5.0 L (5075 ng) of tem-
plate genomic DNA, 0.8 L (2 U) of Taq DNA polymerase
Plant materials
and 7.2 L of nuclease free water. The PCR cycling prole
The total genomic DNA from G. gummi-gutta was used for was: initial denaturation at 94 C for 2 min, followed by 35
genome sequencing. We have also included Garcinia indica cycles of 94 C for 30 s, 55 C for 30 s, 72 C for 1 min and a
and Garcinia morella to examine cross species transferabil- nal extension at 72 C for 5 min. PCR reaction was carried
ity of isolated microsatellite markers. The leaf material was out using thermocycler (Eppendrof Master Cycler Gradient,
obtained from the germplasm collection of the College of Germany). Amplied products were initially separated on
Forestry, Sirsi (University of Agricultural Sciences, Dhar- 3% agarose gel to conrm the amplication. Finally, 32 SSR
wad), India (see details in table 1 in electronic supplementary primers were selected based on amplication of clear PCR
material at http://www.ias.ac.in/jgenet/). The leaf samples products. These primers were employed for amplication of
were obtained from Jaddi Gadde collection (14 44 13.2 N 30 genotypes of G. gummi-gutta and one genotype each of
latitude and 74 43 03.2 E longitude with altitude of 498 m). G. indica and G. morella. The PCR products were separated
The authenticated herbarium specimens were deposited to using automated DNA Sequencer (Applied Biosystems, ABI
the herbarium of College of Forestry, Department of Forest 3730 DNA Analyzer) through capillary electrophoresis, at
Biology and Tree improvement. M/S Eurons facility, Bengaluru.

Genome sequencing and assembly

Genetic analysis of SSR markers
High-quality genomic DNA was isolated from the leaves of
30 G. gummi-gutta genotypes, one genotype from each of G. The raw data generated was analysed and compiled using
indica and G. morella (table 1 in electronic supplementary Peak Scanner ver. 1.0 software (Applied Biosystems, Foster
material) following modied CTAB method (Ravishankar Table 1. Details of sequenced genomic data.
et al. 2000). Total genomic DNA was sequenced using NGS
Illumina HiSeq2000 platform at M/s Genotypic Bengaluru Sequencing details
facility following manufactures instructions. High-quality
sequence reads (Q > 20; >70% bases in a read) were used Total number of contigs 773889
Total number of examined sequences (bp) 241141804
for de novo assembly into contigs using SOAPdenovo2 soft-
Total number of identied SSRs 27313
ware (Luo et al. 2012). Assembly with Kmer-63 was selected Number of SSR containing contigs 26663
as it has the optimal readings for N50. Number of SSR containing more than one SSR 631

Survey, identication and design primers for genomic

Table 2. Simple-sequence repeat types in the G. gummi-gutta
microsatellite markers contigs sequences.
The Perl Script software program MISA (http://pgrc.ipk-
gatersleben.de/misa/) (Thiel et al. 2003; Ravishankar et al. 2015) Motif length Number of SSR Frequency (%)
was used for identication of SSRs from assembled con-
Mononucleotide 12286 44.98
tigs (Feng et al. 2009; Ravishankar et al. 2015). MISA Dinucleotide 9638 35.29
les were transferred to Microsoft Excel where SSRs were Trinucleotide 3003 10.99
classied into mononucleotide, dinucleotide, trinucleotide, Tetranucleotide 552 2.02
tetranucleotide, pentanucleotide and hexanucleotide repeats Pentanucleotide 249 0.91
and compound repeats. Primer pairs anking the repeats Hexanucleotide 58 0.21
Complex/compound 1527 5.59
were designed using Primer3 software (Untergrasser et al. Total 27313
2012) (table 2 in electronic supplementary material).

Journal of Genetics
 Table 3. Genetic analysis of microsatellite markers developed for G. gummi-gutta.

Number Observed Observed Expected Polymorphic Probability Cross species

Repeat of allele size range heterozygosity heterozygosity information of identity amplication
Locus name Forward sequence 5 3 Reverse sequence 5 3 type (k) (bp) (Ho ) (He ) content (PI) GI GM

GG_KVRf282 TTTTGCACAAGCACACG TGTAGTCCTCCTTCAGGT (AG)6 24 107163 0.296 0.957 0.936 0.0065 NA NA

CAGGT TCGACG
GG_KVRf283 TTTTCTCAATCTCCATGCA AAAGGCCAAGAGCCTAA (AC)6 24 101255 0.375 0.925 0.900 0.0169 NA NA
TTCCCCAA TATGATGAGC
GG_KVRf293 TTTGCCAGGGGAAAAC TGGGTTTTGGTTTATGCC (AG)7 19 100191 0.05 0.965 0.938 0.0252 NA NA
ACAAATCCA CCAACTCG
GG_KVRf294 TTTCTGCAGCATGGCC TTGAACTAGGGAGGGCA (TG)7 19 100186 0.000 0.969 0.942 0.0243 NA NA
CTTGGC CCCGT
GG_KVRf553 TGTGAACATGCATGCCTT TCGGGTTGTTCCTCACAT (TG)8 24 330460 0.208 0.969 0.946 0.0089 A A
TGGATGTA CCACCT
GG_KVRf619 TGTATATTTTGTGTGGCAG TGGACTAGGGTGAGCC (AC)12 25 132210 0.222 0.914 0.893 0.0133 NA NA
CAAGCCAT ACTACAGA
GG_KVRf659 TGGTTGGCTTGCATATGT CATTCACATTCATGTTGGCCA (AT)11 23 199284 0.087 0.974 0.951 0.0105 NA NA
GGCATGT TGCTC
GG_KVRf716 TGGTATGGATGGCATATGG GGGGGTGAGCAAACAAC (AT)6 27 126278 0.36 0.965 0.943 0.0075 NA NA
TTTGGGT AGGCT
GG_KVRf717 TGGTATGGATGGCATATG GGGGTATCACTATAGC (AT)8 23 160264 0.292 0.951 0.928 0.0120 NA A
GCTTGGT CCCCTN
GG_KVRf796 TGGGCTATACATGGTGACA GCCATGTGGATCCGCTTAG (AT)6 22 192286 0.208 0.964 0.941 0.0098 NA NA
GATGCC AACCA
GG_KVRg194 TGCACCAGACAGTCGATT TGGGGGTTTGATCCATGG (CA)7 20 143241 0.364 0.964 0.939 0.0157 NA A
CCACC AATGGG
GG_KVRg313 TGAGCATGCCATCTATTTGT GGACGGTCACTGGAAAG (AT)6 17 302451 0.143 0.954 0.927 0.0224 NA NA

Journal of Genetics
GGGGA CACCT
GG_KVRj151 AACAACGAGGGCGTCGT CATATCACCATCACCACC (TG)6 18 119215 0.308 0.918 0.893 0.0151 NA A
GGAAC AACACGA
GG_KVRj152 AAATGGTGGCAAGACACAC TGCCACCTTCCAGGCAC (AC)6 15 146243 0.091 0.943 0.916 0.0204 A NA
ATACCTT CATGT
GG_KVRi741 ACCACTGTTCCAACCAT AGAAGGTGGTGGAGGTGTA (CT)6 20 391498 0.115 0.949 0.927 0.0089 NA NA
GGGCA GGTGT
Microsatellite markers in Garcinia gummi-gutta

GG_KVRi010 ACCACTCTGTCACGAC TGGTTGTGTAGGGATGTG (TG)6 15 349394 0.2 0.936 0.906 0.0324 NA A

CCGACT GTAGCC
GG_KVRi011 AGGGGGAGGGGGTTTT GTGTGTGCGTGTGTGG (CA)6 17 241351 0.37 0.943 0.921 0.0093 NA NA
GCTCAT TCTTGC
GG_KVRg565 TCCTATTACCCGCCCC GGAGGGCGGGATATATGTTG (AT)10 18 201247 0.519 0.937 0.914 0.0103 NA A
ACCACt GAGGG
GG_KVRg470 TCGGGTGTCAGAACCGT GGTGTGGTATGCAGCTCTGT (TA)6 17 106188 0.37 0.945 0.923 0.0089 A NA
AGCGA GATCT
GG_KVRg670 TCCACTCACAAGCCAAC ACCCAAAAGAACCGGT (TA)7 12 185285 0.000 0.913 0.88 0.0384 A A
AAGCCA GTGCGG
GG_KVRg756 TCACGAAGAGAACCACTT TCCTCGCCTCGTAACCTC (GA)11 16 145232 0.4 0.942 0.918 0.0120 A A
ACCCCA CCAA
GG_KVRj174 TTTGTCTGAGGCCTGTG GCAATGGAGGCAATGCT (TTG)6 19 143305 0.321 0.939 0.917 0.0097 NA A
AGGCA AGCCCA
GG_KVRj198 TTGCCCCCAACCCAGA TGTTGCACTTGTGCCTGTT (CAA)9 18 102158 0.077 0.937 0.913 0.0116 A A
AATGGC GTTGC
 K. V. Ravishankar et al.

Cross species
City, USA) for determining the exact allele size. Allele sizes
amplication
GM

NA
NA
NA
NA

NA

NA
NA
for each SSR loci were used for genetic analysis using

A
A
Cervus 3.0 software (Kalinowski et al. 2007). We have esti-

NA
NA
NA
NA
mated the number of alleles, observed heterozygosity (Ho ),
GI

A
A

A
A
expected heterozygosity (He ) and PIC. Probability of iden-
Expected Polymorphic Probability
of allele size range heterozygosity heterozygosity information of identity

tity (PI) was analysed for each SSR loci using Identity 1.0

0.0070
0.0163

0.0622
0.0144
0.0093

0.0165

0.0245
0.0089
0.0114
(PI)

software (Wagner and Sefc 1999).

Results and discussion

content

0.932
0.897

0.888
0.916
0.867
0.923

Off the wide range of DNA markers in use, microsatellites or

0.92

0.89
0.91

SSR markers are extensively employed in plant studies. SSR

markers are highly reproducible, multiallelic, PCR based,
highly polymorphic, easy to use and amenable to automa-
tion. Thus, SSRs markers are widely used for mapping, crop

0.953
0.919

0.923
0.941
0.943

0.916
0.932

0.896
0.945
(He )

breeding programmes and population genetics (Varshney et al.

2005). However, the use of microsatellite markers for study-
ing nonmodel species like G. gummi-gutta has been impeded
by lack of available genomic resources. A few years ago,
Number Observed Observed

0.233

0.208
0.593

0.179

0.208
0.259

0.118
0.115

identication of genomic SSRs and subsequent conversion to

(Ho )

0.63

markers were expensive and time-consuming, involving con-

struction and screening of microsatellite-enriched genomic
DNA libraries (Glenn and Schable 2005). Compared to this
237324
128197

175268
103192

105178

227302
226337

309411
110211

hybrid capture method using probes, the present NGS based

(bp)

method is fast, simple, and overcomes a number of technical

difculties. The advent of NGS technologies, such as pyrose-
quencing, has made this process less complicated and easy
(k)

24
17

13
18
18

12
18

14
20

(Zalapa et al. 2012). As a result, a large number of SSR mark-

ers can be developed in a short span of time and at a lower
CCATGAGCCCAAGTGC (TTTTC)5
GGGGAGGGTGTGCGTTG (AATG)5
(AATA)6

cost. This approach is especially useful for many tree crops

(ACC)5
GTGGGGGTGGCAAATGAG (TGG)6
ACTGGTCGCCCTGAGGTG (CCA)5

(TCA)5
(TCA)5
(TTG)6
Repeat
type

where there is no sequence information available.

We used high-throughput IlliminaHiSeq 2000 platform
to develop genomic SSR markers in G. gummi-gutta. The
TGGGGAGAGTGGCATTG

TGGACCTAGCCATGCCC

TGGTGCCATCCAGTTAG
TTGCCACCTAAGCATGC

AGGGTGAGTTTTTGGC

number of assembly of reads of the long sequences was

GI, Garcinia indica; GM, Garcinia morella; A, amplied; NA, not amplied.
Reverse sequence 5 3

773,889 contigs of total length 241 Mb (table 1). An SSR

survey of genomic sequences using MISA software (http://
CAGCCC
CATGAA

GGGTTT
AGCCT

GGCAT
ATCCT

CTTTT

pgrc.ipk-gatersleban.de/misa) revealed that the 773,889 con-

GAAA
GCAA

tigs contained 27,313 SSRs. Mononucleotide repeats are

predominant (44.98%) followed by dinucleotide (35.29%)
and trinucleotide (14.9%) repeats (table 2). Mononucleotide
repeats are present in high number in some monocots
(rice, sorghum and Brachypodium) and also in some dicots
(Arabidopsis, Medicago and Populus) (Sonah et al. 2011).
TGTATTTCGGTCCATTAGC
TCTCATGCGAAAGCTCAT
TGAGGCATAAAATGCAC
TGGGGCATGCTCTGATC

TCCCCATCACCTTCCAC
TGCTGAGATGGCAGCG

GCAACATCCCAAGTGT

ATGCCGCACACCATGC
Forward sequence 5 3

In the present study, apart from mononucleotide repeats, din-

TGTTGGATCCGTTGTT

ucleotide repeats were the most prevalent accounting for

35.29% of all SSRs identied, followed by trinucleotide
GAGGGGT

AAACGA
GGGGGT
GTGCCT

TGCTCA
ATGCTT

GCTATT

GGCCA
CACCA

repeats (10.29%; table 2). While the mononucleotide, dinu-

cleotide and trinucleotide repeats contribute to the major pro-
portion of SSRs (90.56%) and the rest was contributed by
tetranucleotide, pentanucleotide and hexanucleotide repeats
(table 2). The molecular mechanism of the origin and evolu-
tion of microsatellite markers are not clearly understood. The
Table 3 (contd)

GG_KVRk715
GG_KVRk769

GG_KVRk831

relative dominant occurrence of repeat motif of a particular

GG_KVRj225

GG_KVRj345
GG_KVRj447

GG_KVRj541

GG_KVRj603

GG_KVRj938
Locus name

sequence types and its length in plant genome might be the

outcome of selection pressure applied on that specic motif
during evolution. The most common mutation mechanisms
assumed to be operating are replication slippage, and unequal

Journal of Genetics
 Microsatellite markers in Garcinia gummi-gutta

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Received 10 December 2015, in nal revised form 8 July 2016; accepted 11 July 2016
Unedited version published online: 13 July 2016
Final version published online: 17 April 2017

Corresponding editor: RAJIVA RAMAN

Journal of Genetics