American Journal of Botany: e372–e374. 2011.

AJB Primer Notes & Protocols in the Plant Sciences

CHLOROPLAST MICROSATELLITE PRIMERS FOR CACAO

(THEOBROMA CACAO) AND OTHER MALVACEAE1
Ji Y. Yang2,5, Lambert A. Motilal3, Hannes Dempewolf2, Kamaldeo Maharaj4,
and Q. C. B. Cronk2
2Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia, V6T 1Z4

Canada; 3Cocoa Research Unit, University of the West Indies, St. Augustine, Trinidad, Republic of Trinidad and Tobago, West
Indies; and 4Ministry of Agriculture, Land, and Marine Resources, Central Experiment Station, Centeno, Via Arima P.O.,
Republic of Trinidad and Tobago, West Indies

• Premise of the study: Chloroplast microsatellites were developed in Theobroma cacao to examine the genetic diversity of cacao
cultivars in Trinidad and Tobago.
• Methods and Results: Nine polymorphic microsatellites were designed from the chloroplast genomes of two T. cacao acces-
sions. These microsatellites were tested in 95 hybrid accessions from Trinidad and Tobago. An average of 2.9 alleles per locus
was found.
• Conclusions: These chloroplast microsatellites, particularly the highly polymorphic pentameric repeat, were useful in assessing
genetic variation in T. cacao. In addition, these markers should also prove to be useful for population genetic studies in other
species of Malvaceae.

Key words: chloroplast haplotype; chocolate; Malvaceae; microsatellites; Theobroma cacao; Trinitario.

Theobroma cacao L. (Malvaceae) is one of the most eco- including barley (Provan et al., 1999) and grapevine (Arroyo-
nomically important crops grown in tropical regions of the Garcia et al., 2002). In addition, because of their nonrecombi-
world. There are three major varietal groups of cacao: the nant and uniparentally inherited nature, cpSSRs are the ideal
Forastero, the Criollo, and the Trinitario. The Forastero is the markers for cultivar origin and pedigree studies. Thus, cpSSR
most common; the Criollo is the most prized and expensive, markers should be useful to unravel the history of crosses that
but has a lower yield and is more susceptible to diseases than produced the many Trinitario accessions currently found today.
the Forastero; and the Trinitario is thought to be of hybrid In this study, we designed and characterized nine cpSSR
origin with Criollo and Forastero parents. The Trinitario is markers to estimate the genetic variation of Trinitario cultivars
considered to combine the high flavor quality of Criollo with in Trinidad and Tobago.
the high-yield and disease-resistant properties of Forastero
(http://www.icco.org/about/growing.aspx; Wood and Lass,
1985). As the name implies, Trinitario varieties arose through METHODS AND RESULTS
accidental hybridization in Trinidad and Tobago, and it is the
most common varietal group planted in the country. Further- Using the two complete cpDNA genome sequences from two accessions of
more, there is a large germplasm collection of Trinitarios at T. cacao (Sveinsson et al., 2010; Jansen et al., 2011), we selected for polymor-
phic cpSSR markers. The most common repeat motifs found were mononu-
the International Cocoa Genebank, Trinidad (ICG, T). How- cleotides, and we selected for ones with the longest repeat number. We also
ever, field genebanks of cacao contain many mislabeled and selected one each of polymorphic penta-, hexa-, and octanucleotides because
misidentified plants (Motilal et al., 2009; Irish et al., 2010). their alleles are easier to score. The nine primers were designed using Primer3
There is a need for a standardized, reliable method to geneti- software (Rozen and Skaletsky, 2000) with an expected product size range
cally identify different Trinitario varieties and to estimate their from 150 to 450 bp.
overall genetic diversity. Genomic DNA was extracted from fresh and silica gel–dried leaves using
a QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, California, USA). PCR
Chloroplast simple sequence repeat (cpSSR) markers, or amplifications were performed in a final volume of 15 μL, containing 10 ng
microsatellites, have proved useful as genetic markers for of DNA, 1× reaction PCR buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl), 200
distinguishing among different cultivars in several crop species μM of each dNTP, 1.5 mM MgCl2, 1 U of Taq DNA polymerase (Fermentas
Canada, Burlington, Ontario, Canada), 0.05 μM forward primer, 0.5 μM
1 Manuscript
reverse primer, and 0.5 μM dye-labeled universal primer. Four dyes (VIC,
received 21 June 2011; revision accepted 26 July 2011. FAM, NED, and PET) were used (Applied Biosystems, Carlsbad, California,
The authors thank Dapeng Zhang for sharing his knowledge on USA). PCR reactions were performed using a BIO-RAD thermocycler with a
Theobroma cacao and Nolan Kane and Saimi Sveinsson for their help with touch-down program: 95°C for 3 min; followed by nine cycles of 94°C for 30
bioinformatics. The work was supported by a World Bank Development s, 65°C for 30 s (temperature decreased by 1°C for every cycle), and 72°C for
Marketplace grant awarded to Q. C. B. Cronk and J. M. M. Engels. 45 s; followed by 29 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 45 s; and
5 Author for correspondence: albertoyang@yahoo.com
a final extension at 72°C for 20 min. Each microsatellite marker was amplified
singly and then pooled based on differences in fluorescent labeling and ex-
doi:10.3732/ajb.1100306 pected fragment size. The nine markers were arranged in two multiplex sets

American Journal of Botany: e372–e374, 2011; http://www.amjbot.org/ © 2011 Botanical Society of America
e372
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December 2011] AJB Primer Notes & Protocols—Cacao chloroplast SSRs e373

Table 1. Characterization of nine chloroplast microsatellite primers in Theobroma cacao. For each locus, the primer name, forward and reverse primer
sequence, repeat motif, product size range, number of alleles, unbiased haplotype diversity index, annealing temperature, and GenBank accession
number are shown.

No. of
Primers Primer sequence (5′–3′) Repeat motif Size range (bp) alleles h Ta (°C) GenBank Accession No.

CaCrSSR1 F: CATGGATTCAGCAGCAGTTC (CTTTA)11 359–409 7 0.78 55 JF979116

R: CGAGTCCGCTTATCTCCAAC
CaCrSSR2 F: TTCAACCCAATCGCTCTTTT (A)12 237–239 3 0.63 55 JF979117
R: AATTTGAATGATTACCCGATCT
CaCrSSR3 F: AGAACGAATCCGCTCCTCTT (TAAAAG)2 208–214 2 0.46 55 JF979118
R: GGTCACGGCAACATAACAAC
CaCrSSR4 F: GAACGACGGGAATTGAACC (T)10 180–181 2 0.44 55 JF979119
R: TGATGAATCGTAGAAATGGAAA
CaCrSSR5 F: TCACTTTCACTCCTTTTCCATTT (T)13 199–209 4 0.5 55 JF979120
R: TTTTGACTCCGTTTAGACATAGG
CaCrSSR6 F: CGAATCCCTTTCTTCATACAAA (C)8 175–176 2 0.47 55 JF979121
R: TTTCATGTTTTGATTGCATCG
CaCrSSR7 F: AGGGCTCCGTAAGATCCAGT (T)11 285–286 2 0.46 55 JF979122
R: GTCTTAGGCCTTGCGATTCA
CaCrSSR8 F: TTTCTGATTCACCGGCTCTT (T)11 299–300 2 0.46 55 JF979123
R: TGGTGGAATTCTTTGCATTG
CaCrSSR9 F: TCCACTCAGCCATCTCTCCT (TACTTTAT)11 338–346 2 0.73 55 JF979124
R: GTCCCTTTTGAGCGAAATCA
Note: h = unbiased haploid diversity index; Ta = annealing temperature.

(1) CaCrSSR1 (FAM), CaCrSSR2 (FAM), CaCrSSR3 (VIC), CaCrSSR8 (NED) (Peakall and Smouse, 2006) was from 0.444 to 0.797. The pentameric cpSSR
and (2) CaCrSSR10 (VIC), CaCrSSR4 (PET), CaCrSSR6 (NED), CaCrSSR5 was the most polymorphic with seven alleles and was the most informative in
(FAM), CaCrSSR10 (FAM). An ABI 3730 automated DNA Sequencer (Applied identifying cultivars within the Trinitario varietal group. We detected eight hap-
Biosystems) was used to genotype the multiplex sets. The software GeneMapper lotypes out of the 95 Trinitarios sampled by using all nine cpSSR markers (Table
version 3.2 (Applied Biosystems) was used to call the allele sizes. 2). The eight haplotypes varied in frequency ranging from 0.011 (1 out of 95)
Marker CaCrSSR1, the pentameric repeat, differed by eight repeats in to 0.326 (31 out of 95). These cpSSRs may also prove useful in identifying
the two T. cacao accessions, from which we designed the cpSSR primers. cultivars within and among the 10 population groups said to be present in T.
This marker was the only one to exhibit two alleles. These two alleles cacao (Motamayor et al., 2008).
differed in amplification intensities and level of polymorphism. The lower The high degree of sequence conservation found in chloroplast genomes (Pro-
intensity allele (362 bp) had about a third of the concentration. This allele van et al., 1999) should enable the primers to be used in other species of Malva-
did not create a problem in scoring as it was monomorphic in all 95 samples ceae. We tested the nine cpSSR primers on Gossypium tomentosum Nutt. ex
in the current study with a product size that was easily distinguished Seem. Out of the nine cpSSRs, six amplified successfully (CaCrSSR1, CaCrSSR2,
from the real pentameric product. The higher intensity allele was highly CaCrSSR5, CaCrSSR6, CaCrSSR7, and CaCrSSR8). A BLAST search was con-
polymorphic. We hypothesize that the monomorphic allele may be a cross- ducted on the amplified pentameric cpSSR sequence (CaCrSSR1) from the
amplifying product found in nuclear DNA. Further investigation is, how- National Center for Biotechnology Information website (http://www.ncbi.nlm
ever, necessary to establish definitively the monomorphic nature of this .nih.gov) and obtained nearly perfect hit scores to chloroplasts of three species of
extra product. Gossypium L. The primer sequences matched completely, but the number of
We tested for microsatellite polymorphism in 95 accessions from Trini- pentanucleotide repeats differed. Overall, Gossypium had fewer pentanucleotide
dad and Tobago (see Appendix 1). Sixty-four individuals were collected repeats than T. cacao. GenBank accessions of G. thurberi Tod. and G. barbadense
from 33 different farmers from Trinidad; eight individuals were collected from L. both showed three pentanucleotide repeats while G. hirsutum L. had two. In
six different farmers from Tobago. The remaining 23 came from the ICG, T addition, we also tested the primers on four species of Sidalcea A. Gray
collection (one sample from Grenada Selection [GS], 14 samples from Impe- (S. glaucescens Greene, S. hendersonii S. Watson, S. nelsoniana Piper, and S. oreg-
rial College Selection [ICS], seven samples from Trinidad [TRD], and one ano (Nutt. ex Torr. & A. Gray) A. Gray). The following four primers amplified
sample from Dominica [DOM]). All nine loci were polymorphic, ranging from successfully (CaCrSSR1, CaCrSSR2, CaCrSSR3, and CaCrSSR8). Except for
two to seven alleles with an average of 2.9 alleles per locus (Table 1). The un- primer CaCrSSR3, all these were polymorphic. These cpSSRs may also prove to
biased haploid diversity per locus calculated using GenAlEx 6.1 software be useful in population genetic studies of other species in the Malvaceae.

Table 2. Initial screening of the nine cpSSRs on 95 accessions of Trinitario cultivars. Identified chloroplast haplotypes, haplotype frequencies, and the
alleles pertaining to each haplotype are shown.

Ha Frequency CaCrSSR1 CaCrSSR2 CaCrSSR3 CaCrSSR4 CaCrSSR5 CaCrSSR6 CaCrSSR7 CaCrSSR8 CaCrSSR9

A 0.189 359 239 285 214 338 175 180 300 199
B 0.105 364 237 286 208 338 174 181 299 199
C 0.179 369 237 286 208 338 174 181 300 204
D 0.031 374 238 286 208 346 174 181 299 209
E 0.011 384 239 286 208 346 174 181 299 209
F 0.021 399 239 285 208 338 175 180 300 200
G 0.137 399 239 285 214 338 175 180 300 200
H 0.326 409 239 286 208 346 174 181 299 209
Note: Ha = identified chloroplast haplotypes.
 15372197, 2011, 12, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.3732/ajb.1100306 by Czech Agricultural University, Wiley Online Library on [12/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
e374 American Journal of Botany [Vol. 0

CONCLUSION transfers of rpl22 to the nucleus. Molecular Biology and Evolution

28: 835–847.
The Theobroma cacao cpSSRs revealed a high degree of Motamayor, J. C., P. Lachneaud, J. W. da Silva e Mota, R. Loor, D.
genetic variation within the Trinitario cultivars. Eight haplo- N. Kuhn, J. S. Brown, and R. J. Schnell. 2008. Geographic and
genetic population differentiation of the Amazonian chocolate tree
types were identified out of 95 accessions sampled. (Theobroma cacao L). PLoS ONE 3: e3311.
The pentameric repeat (CaCrSSR1) was the most polymorphic Motilal, L. A., D. Zhang, P. Umaharan, S. Mischke, M. Boccara,
and should prove to be a useful complementary tool for genetic and S. Pinney. 2009. Increasing accuracy and throughput in large-
identification of hybrid cultivars between Forastero and Criollo scale microsatellite fingerprinting of cacao field germplasm collec-
accessions and indeed among all cacao accessions. Furthermore, tions. Tropical Plant Biology 2: 23–37.
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Appendix 1. Accessions used in this study.

Accessions Location

K087, K088, K089, K107 Aripo, Trinidad

K064, K130 Betsy’s Hope, Tobago
K017, K018, K024, K033, K034, K051, K052, K092, K152, K153 Biche, Trinidad
K011, K012, K013, K094, K110, K111, K112, K113, K114, K117, K131 Brasso Seco, Trinidad
K025, K108 Brasso Venado, Trinidad
K050 Carapal, Trinidad
K035, K049, K054 Coromandel, Trinidad
K128, K129 Cumana, Trinidad
K026, K028, K073, K079 Gran Couva, Trinidad
DOM 13, GS 28, ICS 15, ICS 16, ICS 17, ICS 40, ICS 43, ICS 45, ICS 46, ICS 60, ICS 84, ICS 85, ICS 92, ICS 95, ICS 97, ICS ICG, Trinidad
111, TRD 115, TRD 23,TRD 35, TRD 52, TRD 66, TRD 66b, TRD 86
K056, K071, K097, K109, K120 Lopinot, Trinidad
K059, K070, K106, K121, K141, K142, K143, K146, K147, K148, K149, K151 Moruga, Trinidad
K053, K060, K075 Moriah, Tobago
K080 Roxborough, Tobago
K067 Runnemede, Tobago
K133 Tabaquite, Trinidad
K062, K134, K135, K136, K137 Tableland, Trinidad
K055 Tamana, Trinidad
K015, K016, K029 Vega de Oropouche, Trinidad
Note: ICG = International Cocoa Genebank.