64º Congresso Nacional de Botânica
Belo Horizonte, 10-15 de Novembro de 2013
SELECTION OF PRIMERS FOR A STUDY OF THE GENETIC VARIABILITY
OF Pilocarpus microphyllus (RUTACEAE) USING ISSR MARKERS.
1
1
3
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Jefferson A. Rocha , Ivanilza M. de Andrade , Santelmo Vasconcelos , Ana M. Benko-Iseppon , Fabrícia M.
2
1
1
1
M. da Silva , Anne J. Melo , Maria F. S. Silva , João A. L. de Miranda .
1
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Universidade Federal do Piauí; Anidro do Brasil Extrações S.A.; Universidade Federal de Pernambuco;
* [email protected]
Introduction
Pilocarpus microphyllus Stapf ex Wardlew. is native to the
North and Northeast regions of Brazil [1]. Intensive
exploitation for the extraction of pilocarpine, a valuable
alkaloid for the pharmaceutical industry, has killed off or
brought about a loss of vigor of naturally occurring plant
populations [2]. As a result the species is officially listed
as one of the endangered species of the flora of Brazil [3].
This study presents a selection of primers suitable for
ISSR analysis of genetic structure of natural populations
and cultivated collections of P. microphyllus.
Methods
Eight individuals of P. microphyllus were used, two from
each sampled population in Piauí (Parnaíba and
Luzilandia), Maranhão (Mata Roma) and Pará
(Parauapebas). Young leaves were collected from each
individual and immediately stored in CTAB 2% buffer. 48
primers were tested and posteriorly applied statistical
parameters to obtain the total number of polymorphic
fragments, polymorphism information content (PIC),
resolving power (RP) and the marker index (MI) [4].
Results e Discussion
Of the forty-eight primers tested, 34 were informative,
generating 336 markers, of which 226 (67%) were present
in 90-100% of the accessions, and can therefore be
considered polymorphic.
The PIC values vary between 0.045 (UBC-855) and 0.428
(UBC-886), with an average of 0.270. The average MI was
2.06, varying between 0.04 (UBC-855) and 4.23 (UBC886). A strong positive correlation was observed between
the PIC values and the MI for the ISSR tested primers (r =
0.979; p 0.0001). The average RP was 4.09 and values
varied between 1 (UBC-855) and 9.42 (UBC-824). There
was also a positive correlation between RP and PIC (r =
0.724; p
0.0001), similar to that observed between MI
and RP (r = 0.722; p 0.0001).
The primer UBC-873 was one of the best, giving a total of
12 bands, 91% of which were polymorphic [5]. Indeed the
number of fragments by primer may vary considerably
depending on the species. The total numbers of bands
less than 10 (about five) with the primers UBC-807, UBC810, UBC-812, UBC-813, and UBC-856 being relatively
uninformative in the analyses cited [6]. However, reported
an abundant generation of bands for these same primers,
showing on average 20 generated fragments [5]. This
discussion shows the importance of knowing the profile of
bands of a certain primer before beginning a study of the
genetic variability of a species.
Figure. Pilocarpus microphyllus native.
Conclusion
According to the indices analyzed, such as PIC, MI, and
RP, the 34 primers are informative for genetic diversity
analysis in the studied species, and can be used in future
studies aimed at the conservation of natural populations.
Acknowledgements
The authors thank Anidro do Brasil Extrações S.A. and
the Laboratório de Genética e Biotecnologia Vegetal
(LGBV) of the Universidade Federal de Pernambuco
(UFPE), the Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES).
References
[1] Pirani, J.R., Groppo, M. 2012. Rutaceae in Lista de
Espécies
da
Flora
do
Brasil.
Disponível
em:
<http://floradobrasil.jbrj.gov.br/2012/FB000212> acess in 15 jul.
2013.
[2] Pinheiro, C. U. B. 2002 Extrativismo, cultivo e privatização do
jaborandi (Pilocarpus microphyllus Stapf ex Holm.; Rutaceae) no
Maranhão, Brasil. Acta Botânica Brasilica 16: 141 – 150.
[3] Ibama. 2008. Instituto Brasileiro do Meio Ambiente e dos
Recursos Naturais Renováveis. Lista oficial de espécies
Brasileiras ameaçadas de extinção. Instrução normativa MMA
nº 6
[4] Vasconcelos Jr, S. S. 2011. Análise da diversidade
genética entre genótipos de Mamona (Ricinus communis –
Euphorbiaceae) Através de Fingerprinting de DNA com
marcadores AFLP e ISSR. Dissertação (Mestrado em
Genética). Coordenadoria de Pós-graduação, Universidade
Federal de Pernambuco, Recife.
[5] Pharmawati M, Yan G, Finnegan PM. 2005. Molecular
Variation and Fingerprinting of Leucadendron Cultivars
(Proteaceae) by ISSR Markers. Annals of Botany 95: 11631170.
[6] Kareem VK, Rajasekharan PE, Ravish BS, Mini S, Sane A,
Kumar TV. 2012. Analysis of genetic diversity in Acorus calamus
populations in South and North East India using ISSR markers.
Biochemical Systematics and Ecology 40: 156-161.