Ilex paraguariensis A. St. Hil, popularly known as erva mate or mate tea, is a tree species belonging to the Aquifoliaceae family and occurring in southern Brazil, Argentina, and Paraguay. This species is dioecious and can reach 30 m in height, but is kept below seven meters in height when managed for leaf extraction (Mazuchowski, 1989). Ilex paraguariensis has been used particularly in the production of beverages, but has also been used in medicines and cosmetics (Alikaridis, 1987; Maccari Junior and Mazuchowski, 2000). Although the cultivation of I. paraguariensis has been implemented with the aim of addressing a growing market demand, extraction of erva mate still largely occurs from natural populations. Knowledge of the genetic structure of a population may be applied to the conservation and management of a species, and can be acquired through the use of microsatellite, or simple sequence repeat (SSR), molecular markers. Microsatellites represent a powerful tool in determining genetic parameters, such as genetic diversity, paternity analysis, gene flow, and genetic drift (Goldstein and Schlotterer, 2001).
METHODS AND RESULTS
A genomic library was constructed for I. paraguariensis by random fragmentation of DNA (shotgun) and by sample sequencing for the detection of microsatellite loci. Fragmentation of 30 µg genomic DNA was performed in 500 µL of shearing buffer in a nebulizer (TOPO Shotgun Subcloning Kit; Invitrogen, Carlsbad, California, USA) at 0.7 bar for 40 s, generating fragments between 500 and 4000 bp. The QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany) was used to separately recover the largest fragments (between 2000 and 4000 bp) and smaller fragments (between 500 and 2000 bp), which were cloned into the vector PCR4Blunt-TOP (Invitrogen) and transformed into E. coli competent cells. Following blue/white colony selection, the plasmids were then extracted using the alkaline lysis miniprep method (Birnboim and Doly, 1979). Positive clones were sequenced in both directions in a reaction volume of 10 µL, containing 100 ng purified plasmid DNA, 3.2 µM M13 forward or M13 reverse primers, and 1 µL BigDye Terminator version 3.1 mix (Applied Biosystems, Foster City, California, USA). Cycle sequencing of clones used a program consisting of an initial denaturing step of 94°C for 2 min, followed by 35 cycles of denaturation at 96°C for 10 s, annealing at 50°C for 5 s, and elongation at 60°C for 4 min. Sequences of approximately 3000 random clones were resolved with an ABI PRISM 3700 sequencer (Applied Biosystems). SSRs were identified using TROLL (Castelo et al., 2002) and the Staden package (Staden, 1996), and primers were designed using Primer3 software (Rozen and Skaletsky, 2000). A total of 1434 (48%) sequences were of high quality and were subjected to reverse-strand sequencing. A total of 98 sequences contained microsatellites, which means that 3.3% of the total clones contained SSR sequences, revealing a high number of microsatellite regions in the I. paraguariensis genome. Of those, 46 contained dinucleotide repeats (47%), 18 tri-, 13 tetra-, 13 penta-, six hexa-, and one a heptanucleotide repeat. Forty-one SSR clones possessed adequate flanking regions for primer design. The pairs of primers were tested in PCR to verify amplification efficiency, and the annealing temperature was optimized using 12 adult individuals from two natural populations from the southern region of Brazil (Putinga, Rio Grande do Sul State, and Jaguariaíva, Paraná State; Appendix 1). The PCRs contained 3 ng DNA, 1× reaction buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl), forward and reverse primers (0.28 µM), 1.5 mM MgCl2, 0.25 mg/mL bovine serum albumin (BSA), 0.25 mM each dNTP, and 1.3 units of Taq DNA polymerase (Invitrogen).
Thermal cycling conditions were: denaturation at 94°C for 5 min, then 30 cycles of denaturation at 94°C for 1 min, annealing temperature for 1 min (Table 1), extension at 72°C for 1 min, and a final elongation at 72°C for 15 min. Reaction products were separated on 4% denaturing Polyacrylamide gels in 1×TBE buffer and visualized by silver staining. The size of the amplified alleles was estimated in comparison with marker fragments of known size (10-bp ladder; Invitrogen). Of the 41 pairs of primers analyzed, 20 SSR loci showed polymorphism and five were monomorphic. The remaining primers did not amplify or showed nonspecific bands. Table 1 shows the forward and reverse primer sequence, repeat motif, observed amplified fragment size, annealing temperature (Ta) in °C, and GenBank accession number of the clone sequence. The forward primers of the polymorphic loci were fluorescently labeled and used to analyze 24 adult trees from two natural populations, collected from the Putinga (24 trees) and Jaguariaíva (24 trees) sites. The PCR products were analyzed by electrophoresis in an ABI PRISM 3700 sequencer (Applied Biosystems). The ROX-labeled fluorescent internal size standard used was developed by Brondani and Grattapaglia (2001).
TABLE 1.
Characteristics of 25 microsatellite markers in Ilex paraguariensis.
The number of alleles per locus (A), mean observed heterozygosity (H o), and mean expected heterozygosity (H e) were calculated for the total number of individuals using Genetic Data Analysis (Lewis and Zaykin, 2001). All loci were individually tested for significant deviations from Hardy—Weinberg equilibrium (HWE). Significant values (P < 0.05) of deviation from HWE were detected in 12 and nine loci in population 1 and 2, respectively (Table 2). This result is very common for tree species. Levels of variability detected in the 20 loci were high, with the number of alleles ranging from two to 10. The average expected heterozygosity (H o = 0.60) was higher than the observed (H e= 0.54) for both populations, showing an increase of homozygous genotypes in relation to that expected under HWE (Table 2), and indicating deviations from random mating. Because self-fertilization is not possible in this dioecious species, occurrence of inbreeding is suggested as the cause of the reduction in heterozygosity, generated by crosses between relatives. This has probably occurred due to the reduction in native forests, combined with their ruthless exploitation, which can cause erosion and genetic drift leading to biparental inbreeding.
TABLE 2.
Genetic characterization of polymorphic microsatellite markers in Ilex paraguariensis.
CONCLUSIONS
The shotgun cloning and sequencing technique proved to be efficient in the detection of microsatellite sequences in I. paraguariensis. The markers developed in this study represent a powerful tool for the generation of population genetic data, allowing rapid and accurate analysis of the current state of the distribution of genetic variability in the fragments of native and planted populations. This forms essential information for the conservation and sustainable management of this species.
LITERATURE CITED
Notes
[1] This publication is a result of the EMBRAPA/NATURA/FUNARBE partnership. The authors thank Natura Inovação e Tecnologia de Produtos Ltda. for financial support. We also thank the Embrapa Florestas group for collecting samples and Mr. Eduardo, from Ervateira Putinguense, and his whole family for allowing us to collect samples inside his property and for help during the collection.
