Pachyptera kerere (Aubl.) Sandwith (Bignoniaceae) is a Neotropical liana that is widely distributed from Belize to central Amazon in Brazil (Lohmann and Taylor, 2014). This species occurs in humid and often flooded forest vegetation almost entirely along stream banks and rivers, where it is found in low densities. The flowers of P. kerere are white and infundibuliform and bloom throughout the year, providing a constant nectar source for different species of Euglossa, which are the most likely pollinators (Gentry, 1974, 1976). This species falls within the Anemopaegma flower type and steady-state phenology proposed by Gentry (1974). Specialized secretory glands are concentrated near the calyx margin and on the upper portion of the corolla tube. In addition, glands are also present at the interpetiolar region and the petiole apex, and play an important role in ant–plant interactions (Lohmann and Taylor, 2014). The seeds of P. kerere are corky and most likely water dispersed (Gentry, 1979). The broad distribution of P. kerere, combined with its habitat specificity and morphology, make it an interesting model to study the biological processes that determine the patterns of intra- and interpopulation variation of plant species in the Amazon.
Microsatellites (simple sequence repeats [SSRs]) constitute an important genomic resource for botanical studies (Ellegren, 2004) and have been widely used to study the ecological and evolutionary processes that shape plant populations (Ebert and Peakall, 2009). Next-generation sequencing (NGS) technologies now allow us to easily isolate and develop SSR markers from nuclear and plastid genomes (Egan et al., 2012). In this study, we reconstructed the chloroplast genome of P. kerere and used this genome to develop a set of chloroplast microsatellite markers (cpSSRs) for population genetic studies of P. kerere. We also tested the transferability of these markers to P. kerere var. incarnata (Aubl.) A. H. Gentry and the three other recognized species of Pachyptera DC. ex Meisn. (Lohmann and Taylor, 2014): P. aromatica (Barb. Rodr.) L. G. Lohmann, P. erythraea (Dugand) A. H. Gentry, and P. ventricosa (A. H. Gentry) L. G. Lohmann.
METHODS AND RESULTS
Whole genomic DNA was extracted from silica-dried leaf tissue of one individual of P. kerere (collection A. Nogueira 162) using a mini-scale cetyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle, 1987). An aliquot of 5 µg of total DNA was fragmented using a Covaris S-series sonicator (Covaris, Woburn, Massachusetts, USA) and used to construct short-insert libraries (300 bp) using the NEBNext DNA Library Prep Master Mix Set and the NEBNext Multiplex Oligos for Illumina (New England BioLabs, Ipswich, Massachusetts, USA) following the manufacturer's instructions. The P. kerere library was diluted to a concentration of 10 mM, indexed by tags, and sequenced on an Illumina HiSeq 2000 system (Illumina, San Diego, California, USA) at the Universidade de São Paulo (Escola Superior de Agricultura Luiz de Queiroz [ESALQ], Piracicaba, Brazil). Clean reads (100-bp single-end) were filtered for quality using a Perl script that trimmed reads from the ends until there were three consecutive bases with a Phred quality score of 20 or more. Reads with more than three uncalled bases or fewer than 40 bp in length were removed from the data set. The chloroplast genome of P. kerere was reconstructed using a combination of de novo and reference-guided assembly following Nazareno et al. (2015). The chloroplast genome for P. kerere was annotated using the software Geneious version 4.7.5 (Biomatters Ltd., Auckland, New Zealand). Start and stop codons were inspected and adjusted manually.
We used the Imperfect Microsatellite Extractor (IMEx) interface (Mudunuri and Nagarajaram, 2007) to detect perfect and imperfect microsatellites, with minimum thresholds of four repeat units for tri-, tetra-, penta-, and hexa-; six for di-; and 10 for mononucleotide repeats, respectively. Chloroplast microsatellite– flanking primers for cpSSRs found only on intergenic regions were designed using the software Primer3 (Rozen and Skaletsky, 1999) and the following settings: (i) length ranging from 20 to 23 nucleotides, (ii) annealing temperature from 50°C to 62°C, and (iii) minimum GC content of 50%.
In total, 24 primer pairs were designed. To validate those primer pairs, PCR amplifications were performed in 8.5-µL reactions containing 10 ng of template DNA, 0.5 µL 10 mM of each primer with forward primers labeled with 6-FAM or JOE fluorescent dyes (Macrogen, Seoul, South Korea), 5 µL 1× of Kapa2G Fast Ready Mix (Kapa Biosystems, Wilmington, Massachusetts, USA), and 0.6 µL 25 mM MgCl2 (Promega Corporation, Madison, Wisconsin, USA). PCR conditions were as follows: 94°C for 3 min; 20 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, 72°C for 1 min; and a final elongation step at 72°C for 5 min. Initial screens were performed with three P. kerere individuals, and their amplicons were visualized on an agarose gel (0.8%) with a 100-bp ladder (Promega Corporation).
Characteristics of 21 intergenic chloroplast microsatellite primers developed for Pachyptera kerere.a
Twenty-one of the 24 primer pairs produced a single band with strong amplification and were selected for polymorphism assessment in 65 P. kerere samples. These samples were grouped in three populations (11–39 individuals per population; Appendix 1). For these samples, genomic DNA was extracted from silica-dried leaves using an Invisorb Plant Mini Kit (Invitek, Berlin, Germany) following the manufacturer's protocol. Fluorescently labeled amplicons were resolved to genotype on an automated sequencer (ABI 3730XL) with GeneScan 500 ROX Size Standard (Applied Biosystems, Foster City, California, USA). Chloroplast microsatellite profiles were analyzed with GeneMarker (Holland and Parson, 2011). Each cpSSR was considered a locus at a specific site and the length variants were considered alleles. For each polymorphic locus, we obtained the number of alleles (A) and unbiased haploid diversity index (h) using the program GenAlEx version 6.41 (Peakall and Smouse, 2006). Transferability of polymorphic cpSSRs was tested in five individuals of each of the following taxa: P. aromatica, P. erythraea, P. ventricosa, and P. kerere var. incarnata. The PCR amplification profile followed the same conditions described above.
We obtained a partial chloroplast genome (149,076 bp) and used it to develop a set of 21 polymorphic chloroplast microsatellite markers (Table 1). Considering all samples (n = 65), A ranged from three to nine and h ranged from 0.207 (Pac28) to 0.817 (Pac04) (Table 2). Most of the polymorphic primers (96%) successfully amplified for P. kerere var. incarnata and for all species of Pachyptera (Table 3).
Characteristics of 21 polymorphic chloroplast microsatellite loci in three populations of Pachyptera kerere.a
Transferability of 21 microsatellite markers developed for Pachyptera kerere across four different taxa of Pachyptera.
We developed and amplified a set of polymorphic chloroplast microsatellite markers for P. kerere. These markers will be useful for evolutionary and phylogeographic studies. The applicability of these microsatellite loci in Pachyptera congeneric species was confirmed by successful transferability. We plan to use these markers to assess patterns of genetic structure of Pachyptera species in the Amazon rainforest.
The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for a scholarship to J.N.C.F. and for a Pq-1C grant to L.G.L. We also thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for a scholarship to A.G.N. (2013/12633-8), a regular research grant to L.G.L. (2011/50859-2), and a collaborative Dimensions of Biodiversity Grant supported by FAPESP (2012/50260-6), the U.S. National Science Foundation, and the National Aeronautics and Space Administration.