Stizophyllum Miers (Bignoniaceae) is a small genus of Bignonieae, the largest tribe in the Bignoniaceae (Lohmann and Taylor, 2014). The genus is clearly monophyletic (Lohmann, 2006) and includes three species, i.e., S. inaequilaterum Bureau & K. Schum., S. perforatum (Cham.) Miers, and S. riparium (Kunth) Sandwith (Lohmann and Taylor, 2014). All species of Stizophyllum are lianas with trumpet-shaped flowers that are pollinated by medium-sized bees (Gentry, 1974). Their winged seeds are dispersed by wind (Gentry, 1974). Members of Stizophyllum are easily recognized by their hollow stems, pellucid-punctate leaflets, urceolate calyces, and linear fruits (Lohmann and Taylor, 2014). The genus as a whole is broadly distributed, occurring from southern Mexico to the Atlantic Forest in southern Brazil (Lohmann and Taylor, 2014). Although the generic circumscription is clear, the three species of Stizophyllum currently recognized are morphologically similar and can occur sympatrically in Amazonia.
Nuclear DNA polymorphisms based on microsatellites (simple sequence repeats [SSRs]) are powerful sources for population genetic studies (Kalia et al., 2011). These molecular markers, present also in the genomes of organelles (e.g., chloroplast), allow us to access genetic information that facilitates genotype identification, mainly due to their multiallelic nature (Masi et al., 2003). The chloroplast microsatellite and nuclear microsatellite markers (cpSSR and nSSR, respectively) have been widely used to study phylogenetic and genetic diversity in plants (Kalia et al., 2011). Unlike nSSRs, which are highly polymorphic, codominant, and biparentally inherited, cpSSRs are a nonrecombinant molecule and uniparentally inherited, allowing us to trace the history of populations through their haplotype diversity (Ebert and Peakall, 2009). Despite these differences, the two markers are complements for understanding the genetic structure in natural populations (Ebert and Peakall, 2009; Kalia et al., 2011).
Furthermore, the advent of high-throughput sequencing technologies (Metzker, 2010) has allowed the development of SSR markers for multiple taxonomic groups (Zalapa et al., 2012; Francisco et al., 2016). Here, we used chloroplast genome sequence data obtained by de novo and reference-guided assembly to develop and characterize chloroplast microsatellite markers for S. riparium. Cross-amplification of the cpSSR markers developed for S. riparium was tested in all congeneric species to evaluate the utility of those markers for population genetic studies in Stizophyllum as a whole.
METHODS AND RESULTS
We first obtained the chloroplast genome sequence of S. riparium using an Illumina platform. For that, we extracted the genomic DNA from fresh leaf material dried in silica gel from a single individual of S. riparium (voucher: Nogueira 170; Appendix 1) collected in Manaus (Amazonas State, Brazil) using the Invisorb Spin Plant Mini Kit (Invitek, Berlin, Germany) and following the manufacturer's instructions. Approximately 5 µg of total DNA was fragmented using a Covaris S-Series Focused-ultrasonicator (Covaris, Woburn, Massachusetts, USA) and a short-insert (300 bp) library was constructed with NEBNext DNA Library Prep Master Mix Set and NEBNext Multiplex oligos for Illumina (New England Bio-Labs, Ipswich, Massachusetts, USA), following the manufacturer's protocol. The library concentration was diluted to 10 mM and sequenced (single end) on an Ilumina HiSeq 2000 system (Illumina, San Diego, California, USA) at the University of São Paulo (Escola Superior de Agricultura Luiz de Queiroz da Universidade de São Paulo [ESALq]) in Piracicaba, Brazil. A Perl script was used to filter for quality using a Phred score of 20 or more for the cleaned reads, with the exclusion of reads with more than three uncalled bases, or shorter than 40 bp. We used a combination of reference-guided and de novo assembly to construct the chloroplast genome of S. riparium following Nazareno et al. (2015). The chloroplast genome for S. riparium was annotated using DOGMA (Dual Organellar GenoMe Annotator; Wyman et al., 2004; http://dogma.ccbb.utexas.edu/). Start and stop codons were inspected and adjusted manually.
Characteristics of 28 intergenic chloroplast microsatellite markers developed for Stizophyllum riparium.
Characteristics of 28 polymorphic chloroplast microsatellite markers in three populations of Stizophyllum riparium.a
The Imperfect Microsatellite Extractor (IMEx) interface (Mudunuri and Nagarajaram, 2007) was used to detect perfect microsatellites, with a threshold of 10 repeat units for mononucleotide and six repeats for di-, tri-, tetra-, penta-, and hexanucleotides. The annotated chloroplast genome was used to select markers located in noncoding regions exclusively. Primer pairs were designed from microsatellite sequence sites using Primer3web 4.0 (Rozen and Skaletsky, 1999), with the following parameters: primer size of 18–23 bp, temperature of 50–62°C with maximum difference between forward and reverse primers of 1°C, and GC content of 40–80%.
In total, 36 primer pairs were designed. The PCR amplifications were performed in a final volume of 10 µL and contained 15 ng of genomic DNA, 0.5 µL (10 mM) of each primer with forward primers labeled with 6-FAM or JOE fluorescent dyes (Macrogen, Seoul, South Korea; Table 1), 0.6 µL (25 mM) MgCl2 (Promega Corporation, Madison, Wisconsin, USA), and 5 µL 1× of KAPA2G Fast ReadyMix (Kapa Biosytems, Wilmington, Massachusetts, USA). The cycling conditions were as follows: an initial denaturation step of 3 min at 94°C; followed by 30 cycles of 30 s at 94°C for denaturation, 30 s at the specific annealing temperature for each primer pair (Table 1), and 72°C for 60 s; and a final extension of 5 min at 72°C. To test the utility of the individual primers, PCR products were detected using a 1.0% agarose gel electrophoresis with a 100-bp range DNA ladder (Promega Corporation).
Of the 36 primer pairs tested, 28 successfully amplified (Table 1) and were checked for polymorphism in 59 individuals from three S. riparium populations (ranging from 18 to 22 individuals; Appendix 1). All samples tested were also extracted using fresh leaf material dried in silica gel or from herbarium specimens using the Invisorb Spin Plant Mini Kit, following the manufacturer's instructions. The amplicons with fluorescent labels were resolved to genotype on an ABI 3500 XL automated DNA sequencer with GeneScan 500 ROX Size Standard (Applied Biosystems, Foster City, California, USA). The microsatellite marker profiles were analyzed using GeneMarker (Holland and Parson, 2011). For each cpSSR, the number of alleles (A) and unbiased haploid diversity index (h) were obtained using GenAlEx 6.41 (Peakall and Smouse, 2006). In addition, we calculated chloroplast haplotype variation within populations by estimating the total number of haplotypes and the unbiased haplotype diversity as He = [n/(n – 1)](1 – Σpi2), where n is the number of individuals analyzed in each population and p is the frequency of the ith haplotype in the respective population (Nei, 1978). Cross-amplification of polymorphic cpSSRs was tested in six individuals of S. inaequilaterum and S. perforatum using the same PCR conditions described above.
Transferability of 28 chloroplast microsatellite markers developed for Stizophyllum riparium across two related Stizophyllum species.a
A partial chloroplast genome (128,239 bp) was obtained and used to develop a set of 28 polymorphic cpSSRs (Table 2). The number of alleles ranged from two to 12, and unbiased haplotype diversity varied from 0.037 (Stiz40) to 0.905 (Stiz45) (Table 2). When alleles at each of the 28 loci were jointly analyzed, we observed 18, 19, and 22 haplotypes for EAM (i.e., east of Amazonia), CAM (i.e., Central America), and SAM (i.e., south of Amazonia) populations, respectively. Furthermore, the maximum unbiased haplotype diversity (He = 1.00) was observed for all S. riparium populations. All polymorphic cpSSR markers successfully amplified for S. inaequilaterum and S. perforatum (Table 3).
The authors thank Comissão de Aperfeiçoamento de Pessoal do Nível Superior (CAPES) for a scholarship to M.B., Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for a Pq-1C grant to L.G.L. (307781/2013-5), and Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) for a scholarship to A.G.N. (2013/12633-8) and a regular research grant (2011/508559-2) and a collaborative FAPESP-NSFNASA Dimensions of Biodiversity Grant (2012/50260-6) to L.G.L.