Cabomba Aubl. is a small genus in the family Cabombaceae comprising strictly aquatic plants restricted to Neotropic and adjoining warmer temperate zones (Ørgaard, 1991). The genus contains six (Fassett, 1953) or five (Ørgaard, 1991) species. The last and more complete taxonomic study was made by Ørgaard in 1991, in which five species were recognized, as she synonymizes C. schwartzii Rataj under C. aquatica Aublet. Cabomba aquatica s.l. is distributed along northern, northeastern, and southeastern regions of Brazil and northern South American countries, occurring in habitats such as floodplains, floodplain lakes, creeks, ponds, and swampy areas where sufficient light is available. The color of the flower, morphology of the floating leaves, phyllotaxy of the submerged leaves, and seed size and shape are the main characters used for recognizing species (Ørgaard, 1991). Despite the great contribution of the study by Ørgaard (1991), identification of Cabomba species is still problematic due to vegetative similarity among the taxa, the presence of few morphological characters that are useful in delimiting species, and the necessity of both flower and seed to differentiate some species. Grown for its ornamental value, Cabomba species were one of the most important plants to be commercialized for a long period in the history of aquarism (Francisco and Barreto, 2007). However, the rapid development of Cabomba plants in water reservoirs can negatively affect the water flow in hydroelectric turbines and irrigation channels and can reduce the navigability of watercourses. Every year, countries like Australia and the United States spend millions of dollars on its control to minimize the damage (Francisco and Barreto, 2007). On the other hand, species of Cabomba are important elements of water plant vegetation with a high primary production rate. The plants are ecologically important as a food source and as hiding places for several vertebrate and invertebrate species. They also produce considerable biomass and act as a nutrient reservoir (Esteves, 1998; Silva and Leite, 2011). For these reasons, a set of rapid molecular markers is needed in C. aquatica s.l. In this study, we report the development of 13 microsatellite loci for the species to subsidize further taxonomic and population studies within Cabomba.
METHODS AND RESULTS
The total genomic DNA was extracted from floating leaf tissue dried in silica gel using a cetyltrimethylammonium bromide (CTAB) method, based on Doyle and Doyle (1987), and then digested with AfaI restriction enzyme. Microsatellite DNA loci were isolated from one individual from Parque Nacional do Viruá, Roraima, Brazil (Barbosa, T. D. M. 1230 & Costa, S. M. [UEC 154811]) (Appendix 1), as described in Billotte et al. (1999). Enrichment was performed using a hybridization-based capture with (CT)8 and (GT)8 biotin-linked probes and streptavidin-coated magnetic beads (MagneSphere Magnetic Separation Products; Promega Corporation, Madison, Wisconsin, USA). The enriched fragments were amplified by PCR, and the amplification products were cloned into pGEM-T Easy Vector (Promega Corporation). Competent XL1-Blue Escherichia coli (Stratagene, Agilent Technologies, Santa Clara, California, USA) were transformed with the recombinant plasmids and cultivated on agar medium containing ampicillin and 100 µg/mL of X-galactosidase. Ninety-six recombinant colonies were selected using blue/white screening and sequenced in an automated ABI 3500xL Genetic Analyzer (Perkin Elmer-Applied Biosystems, Foster City, California, USA) using T7 and SP6 primers and the BigDye Terminator version 3.1 Cycle Sequencing Kit (Perkin Elmer-Applied Biosystems). Approximately 86 sequenced clones presented microsatellite motifs, from which 30 primer pairs were designed, using Primer3Plus (Untergasser et al., 2007). As a criterion for the selection of simple sequence repeats (SSRs), sequences that showed at least five dinucleotide repeats, four trinucleotide repeats, or three tetra-, penta-, and hexanucleotide repeats were selected, giving preference to motifs with more repetitions. From the 30 primer pairs developed, 13 did not successfully amplify in PCR and four did not show conclusive results. Thirteen primer pairs amplified PCR products, from which one pair was monomorphic (CS11). The characteristics of the primer pairs and the optimal annealing temperature are given in Table 1. These revealed a two-banded pattern, which is typical for diploid organisms. Preliminary cytogenetic studies reported 2n = 26, 52, or 104 chromosomes for C. aquatica s.l. (Ørgaard, 1991), being diploid, tetraploid, and octoploid, respectively.
Characteristics of 13 successfully amplified SSR loci developed for Cabomba aquatica s.l.
Polymorphism was tested with total DNA of 49 individuals from two populations: (1) a lake on the campus of the Instituto Federal do Amazonas (IFAM), municipality of Manaus, Amazonas State, Brazil (3°06′07″S, 60°01′30″W; n = 29), and (2) Parque Nacional do Viruá, municipality of Caracaraí, Roraima State, Brazil (1°24′44″N, 60°13′00″W; n = 20). The microsatellite fragments were amplified by PCR containing 20 ng of template DNA, 2.5 µL of 1× PCR buffer (20 mM Tris HCl [pH 8.4] and 50 mM KCl), 2.0 µL (10.0 mM) of forward and reverse primer, 1.5 µL of dNTP mix (2.5 mM of each base), 1.5 µL of MgCl2 (1.5 mM), and 1 unit of Taq DNA polymerase (Fermentas, Thermo Fisher Scientific, Pittsburgh, Pennsylvania, USA). The PCR program for all loci amplification consisted of an initial denaturation at 95°C for 3 min, followed by 39 cycles of denaturation at 95°C for 40 s, annealing at specific temperature for 40 s (Table 1), extension at 72°C for 50 s, and a final extension at 72°C for 8 min. PCR products were separated by electrophoresis in denaturing acrylamide gels and silver stained (Creste et al., 2001). The molecular size of the fragments was estimated using a 10-bp ladder (Invitrogen, Carlsbad, California, USA).
For each population, we calculated the number of alleles per locus (A), expected heterozygosity (He), observed heterozygosity (Ho), and fixation index (F) using the R package HIERFSTAT (Goudet, 2005). Adherence to Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) among loci was tested using the Markov chain method in the software GENEPOP version 4.2 (Raymond and Rousset, 1995). Deviations from HWE in the two populations could derive from asexual reproduction, which is reported to be high for Cabomba (Ørgaard, 1991), and from environmental anthropization. No LD was detected between pairs of loci after Bonferroni correction for multiple tests. The number of alleles per locus in the 12 polymorphic loci ranged from two to four across the 49 C. aquatica s.l. individuals. Ho, He, and F vary from 0.0 to 1.0, 0.0 to 0.5, and −1.0 to −0.0667, respectively, in the Manaus population, and from 0.0 to 1.0, 0.0 to 0.6, and −1.0 to 0.4643 in the Viruá population (Table 2).
Genetic diversity values for 49 individuals of Cabomba aquatica s.l. across 12 polymorphic SSR loci.
These are the first SSR markers developed for the Cabomba genus. These loci will allow us to investigate the genetic structure of C. aquatica s.l. populations alongside morpho-anatomical studies to reconsider whether C. schwartzii should be recognized as a distinct species. They will also provide support for the adequate management of this ecologically important species and may be instrumental for further ecological research.
 The authors thank S. M. Costa for help during fieldwork, the staff of the Parque Nacional do Viruá and Instituto Nacional de Pesquisas da Amazônia (INPA) for field support, and A. P. Souza for technical support for the laboratory infrastructure. T.D.M.B. is grateful to the Post-Graduate program in Plant Biology of the Instituto de Biologia (UNICAMP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the scholarship; M.C.E.A. is a Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) research fellow. This work was financed by CAPES (PNADB 457/2010).