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Brazilian Journal of Botany ISSN 0100-8404 Braz. J. BotDOI 10.1007/s40415-013-0012-7

Transferability of 10 nuclear microsatelliteprimers to Vriesea minarum(Bromeliaceae), a narrowly endemic andthreatened species from Brazil

P. Lavor, C. van den Berg &L. M. Versieux

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SHORT COMMUNICATION

Transferability of 10 nuclear microsatellite primersto Vriesea minarum (Bromeliaceae), a narrowly endemicand threatened species from Brazil

P. Lavor • C. van den Berg • L. M. Versieux

Received: 12 November 2012 / Accepted: 18 March 2013

� Botanical Society of Sao Paulo 2013

Abstract Vriesea minarum is an endemic rupiculous

bromeliad species, with naturally fragmented populations,

restricted to the Iron Quadrangle, in Minas Gerais, Brazil.

It is a threatened species, which is suffering from habitat

loss due to the growth of cities and mining activities. Thus,

it is extremely important to know its genetic structure to set

strategies for its in situ and ex situ conservation. Here, we

tested 14 nuclear microsatellite primers (SSRs) for one

population of V. minarum to search for polymorphisms and

evaluated the transferability of previously developed

primers. We succeeded in the amplification of 10 loci in

which we also found polymorphisms. The expected and

observed heterozygosity found here is similar to other

Bromeliaceae population genetics studies. Our results show

the great potential in working with these co-dominant

markers for V. minarum, which may help in developing

conservation actions for the species in the future.

Keywords Conservation genetics � Cross-amplification �Iron Quadrangle � Metallophytes � SSR

Introduction

Vriesea minarum L.B.Sm. (Bromeliaceae), is an endemic

species of bromeliad, restricted to the Iron Quadrangle

region, Minas Gerais, Brazil (Versieux and Wendt 2007;

Jacobi et al. 2007) and its populations are naturally frag-

mented. This metallophyte species has suffered from

increasing mining activities and urban growth, which have

caused the decline in its area of occupancy and quality of

habitat (Versieux 2011). Thus, some studies have consid-

ered this species as endangered (Versieux and Wendt 2007;

Versieux 2011). Currently, V. minarum is classified as

threatened (EN: Endangered category of the IUCN) by the

Brazilian Official Plant Red List (CNCFlora 2013)

(Figs. 1–3).

Therefore, it is necessary to know the genetic structure

of V. minarum populations to have a conservation plan for

the species. Studies of conservation genetics in Bromelia-

ceae have provided a rich source of primers for microsat-

ellite (SSR) loci. The SSR primers may be transferred to

different plant species depending on the extent to which the

primer sites flanking SSRs are conserved among related

taxa, and the stability of the SSR throughout evolution

(Powell et al. 1996). This study aims to optimize a set of

previously published microsatellite loci primers for V.

minarum.

Materials and methods

Leaf samples from 20 individuals were collected in Pedra

Rachada, in Sabara municipality. The genomic DNA was

extracted following Doyle and Doyle (1990). Fourteen SSR

loci previously described were tested: Vriesea gigantea

Gaud. (Palma-Silva et al. 2007—loci: VgB10, VgC01,

P. Lavor (&) � L. M. Versieux

Programa de Pos-Graduacao em Sistematica e Evolucao,

Laboratorio Botanica Evolutiva, Departamento de Botanica,

Ecologia e Zoologia, Universidade Federal do Rio Grande do

Norte, Lagoa Nova, Natal, RN 59072-970, Brazil

e-mail: pamelalavor@hotmail.com

C. van den Berg

Laboratorio de Biologia Molecular de Plantas, Universidade

Estadual de Feira de Santana, BR-116, Km 03, Feira de Santana,

BA 44031-460, Brazil

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DOI 10.1007/s40415-013-0012-7

Author's personal copy

VgF01, VgF02, VgG02, VgG03, and VgG5), Tillandsia

fasciculata Sw. and Guzmania monostachya Rusby (Boneh

et al. 2003—loci: e6, p2p19, e19, and CT5), Pitcairnia

albiflos Herb. (Paggi et al. 2008—locus: PaZ01) and Fos-

terella rusbyi (Mez) L.B.Sm. (Wohrmann et al. 2012—

loci: ngFos_6 and ngFos_22).

Three primers were used for each SSR locus: a forward

SSR-specific primer with the M13 tail at its 50 end, a

reverse locus-specific primer, and a universal M13 (50

CACGACGTTGTAAAACGAC 30) primer labeled with

four fluorescent dyes (Applied Biosystems matrix DS-33,

with 6-FAM, VIC, NED, and PET). The amplifications

were done using the GeneAmp PCR system 9700 (Applied

Biosystems) thermal cyclers and Swift.maxpro (Esco).

For seven loci (VgB10, VgC01, VgF02, VgG03, VgG5,

and ngFos_6 ngFos_22), the following ‘‘touchdown’’ pro-

gram was used: 95 �C for 3 min, then 10 cycles of 94 �C

for 30 s, 58 �C decreasing to 48 �C at 1 �C per cycle for

30 s, 72 �C for 30 s followed by 35 cycles of 94 �C for

30 s, 48 �C for 30 s, 72 �C for 30 s, followed by a final

extension of 30 min at 72 �C before cooling down to 4 �C.

For five SSR loci (PaZ01, e6, p2p19, e19 and CT5) the

program used was: 94 �C for 3 min, 40 cycles of 94 �C for

20 s, 51 �C for 40 s, 72 �C for 20 s, followed by a final

extension of 30 min at 72 �C before cooling down to 4 �C.

And for the two remaining SSR loci (VgF01 and VgG02),

the program was: 94 �C for 3 min, 40 cycles of 94 �C for

20 s, 54 �C for 40 s, 72 �C for 20 s, followed by a final

extension of 30 min at 72 �C before cooling down to 4 �C.

For all the programs, we added 8 cycles of 94 �C for 1 min,

53 �C for 1 min, 72 �C for 1 min before the final extension

to incorporate the M13 tail.

Microsatellite alleles were resolved on an ABI 3130XL

Genetic Analyzer with a 50 cm capillary array (Applied

Biosystems) and were precisely sized using GeneMapper

4.0 (Applied Biosystems). GenAlex 6.5 software was used

to calculate the estimates of allelic diversity and test for

departure from Hardy–Weinberg equilibrium (HWE). The

inbreeding coefficient (Fis) was estimated by Fstat version

2.9.3 software (Goudet 1995). The Microchecker software

was used for detection and correction of null alleles

(Brookfield 1 estimator). The polymorphic information

content (PIC) was calculated using PICcalc (http://w3.

georgikon.hu/pic/english/default.aspx).

Results and discussion

Of the 14 primers tested in the present study, 10 showed

amplifications. All of these 10 loci were polymorphic,

displaying an average allele number of 6.4 (2–13). The

average observed heterozygosity (Ho) was of 0.432, and

the average expected heterozygosity (He) was of 0.579. Six

loci showed null alleles. The mean of the inbreeding

coefficient (Fis) was 0.225. Deviations from HWE were

found in five loci, where levels of observed heterozygosity

were lower than expected (Table 1).

The values of observed and expected heterozygosity are

within the values found in other studies of Bromeliaceae,

such as: Palma-Silva et al. (2009) for V. gigantea

(Ho = 0.424 and He = 0.714); Barbara et al. (2007a) for

Alcantarea imperialis (Carriere) Harms (Ho = 0.362 and

He = 0.615) and A. geniculata (Wawra) J.R. Grant

(Ho = 0.357 and He = 0.429); Zanella et al. (2011) for

Bromelia antiacantha Bertol. (Ho = 0.369 and He =

0.746). All of these studies indicate an observed hetero-

zygosity smaller than the expected, due to a deficit of

heterozygotes. The same pattern is observed in this study

with V. minarum. The inbreeding coefficient was high

probably because V. minarum is partially self-compatible

(P Lavor, unpublished data), it also has a developed clonal

growth (Versieux 2011) and the inbreeding coefficient

value found here is also similar to those found in other

Figs. 1–3 Specimens of Vriesea minarum in habitat. 1. population; 2.

Blooming individual; 3. Detail of the inflorescence (Voucher: Lavor

13939, UFRN herbarium)

P. Lavor et al.

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Bromeliaceae (e.g., Palma-Silva et al. 2011; Zanella et al.

2011).

We consider the level of transferability obtained here for

V. minarum as high, since 10 (*70 %) out of 14 primer pairs

provided positive results. Apparently, the ability to transfer

primers seen in this study is not related to the phylogenetic

position of the taxon in which the original microsatellite was

isolated. Vriesea minarum is placed within the Tillandsioi-

deae subfamily, and we transferred primers developed for the

Pitcairnioideae subfamily (Fosterella and Pitcairnia) that

worked well. In contrast, four primers that were isolated from

other species of Tillandsioideae did not amplify.

This successful cross-amplification in Bromeliaceae

could perhaps be one indication of the great adaptive

radiation within the family, leading to rapid speciation and

to low levels of divergence in DNA sequences, allowing

the SSRs to be transferred among species of the same or

different subfamilies (Barbara et al. 2007b).

Thus, given the high number of bromeliad species

threatened with extinction in Brazil the transferring of

previously developed markers may save time and eliminate

part of the costs, allowing more studies in population

genetics to be done. This will certainly help in drawing up

concrete targets for conservation in the future.

Acknowledgments We thank CAPES for the first author’s M.Sc.

fellowship and FAPESB for financial support (PNX0014/2009).

CVDB thanks CNPq for his fellowship (PQ-1D). We thank two

anonymous reviewers for their comments, F. F. Carmo for field

assistance, and M. C. Lopez-Roberts for training in molecular labo-

ratory techniques.

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Table 1 Characteristics of the 10 primers transferred for one population of Vriesea minarum

Organism Locus Reference Size PB Ta (�C) A He Ho Fisa

Tillandsia fasciculata and

Guzmania monostachya

e6 Boneh et al. (2003) 134–148 51 3 0.471 0.500 -0.035

T. fasciculata and G. monostachya e19 Boneh et al. (2003) 129–137 51 2 0.054 0.056 0.000

T. fasciculata and G. monostachya p2p19 Boneh et al. (2003) 204–222 51 8 0.798 0.643 0.230

Fosterella rusbyi ngFos_6 Wohrmann et al. (2012) 140–188 58–48 2 0.142 0.154 -0.043

F. rusbyi ngFos_22 Wohrmann et al. (2012) 170–232 58–48 6 0.695 0.368 0.357**

Vriesea gigantea VgB10 Palma-Silva et al. (2007) 132–174 58–48 12 0.864 0.684 0.206

V. gigantea VgC01 Palma-Silva et al. (2007) 230–270 58–48 13 0.843 0.444 0.379***

V. gigantea VgF02 Palma-Silva et al. (2007) 130–174 58–48 8 0.776 0.200 0.625***

V. gigantea VgG03 Palma-Silva et al. (2007) 219–233 58–48 3 0.397 0.389 0.048***

V. gigantea VgG05 Palma-Silva et al. (2007) 168–216 58–48 7 0.754 0.882 -0.140***

Ta annealing temperature, A number of alleles, Ho observed heterozygosity, He expected heterozygosity, Fis inbreeding coefficienta Departures from Hardy–Weinberg equilibrium are indicated by asterisks: ** P \ 0.01, *** P \ 0.001

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