Characterization of microsatellite markers developed from Prosopis rubriflora and Prosopis ruscifolia (Leguminosae - Mimosoideae), legume species that are used as models for genetic diversity studies in Chaquenian areas under anthropization in South America

Background Prosopis rubriflora and Prosopis ruscifolia are important species in the Chaquenian regions of Brazil. Because of the restriction and frequency of their physiognomy, they are excellent models for conservation genetics studies. The use of microsatellite markers (Simple Sequence Repeats, SSRs) has become increasingly important in recent years and has proven to be a powerful tool for both ecological and molecular studies. Findings In this study, we present the development and characterization of 10 new markers for P. rubriflora and 13 new markers for P. ruscifolia. The genotyping was performed using 40 P. rubriflora samples and 48 P. ruscifolia samples from the Chaquenian remnants in Brazil. The polymorphism information content (PIC) of the P. rubriflora markers ranged from 0.073 to 0.791, and no null alleles or deviation from Hardy-Weinberg equilibrium (HW) were detected. The PIC values for the P. ruscifolia markers ranged from 0.289 to 0.883, but a departure from HW and null alleles were detected for certain loci; however, this departure may have resulted from anthropic activities, such as the presence of livestock, which is very common in the remnant areas. Conclusions In this study, we describe novel SSR polymorphic markers that may be helpful in future genetic studies of P. rubriflora and P. ruscifolia.


Background
The genus Prosopis L. belongs to the Leguminosae botanical family, which contains 44 species. Prosopis L. is predominantly restricted to the neotropics [1]. Prosopis rubriflora [2] and Prosopis ruscifolia [3] are tree species known locally as "espinheiro" and "algarroba," respectively. These species are important both economically and ecologically. For example, the fruits and seeds of P. ruscifolia are reported to be good sources of nutrition for humans and animals [4], and the flowers of P. rubriflora, which are present throughout the year, provide important food resources, such as pollen and nectar, for the local fauna [5]. P. rubriflora has a narrow distribution range and is limited to Paraguay and Brazil, but P. ruscifolia is also found in Argentina and Bolivia [6,7].
In Brazil, P. rubriflora and P. ruscifolia are associated with Chaquenian areas [8] and are limited to the southern portion of the Pantanal [9,10]. Both species are excellent indicators of Chaquenian areas in Brazil; P. rubriflora is usually associated with arboreal physiognomy, and P. ruscifolia is frequently associated with forest physiognomy. Both species can be used as models for genetic studies of diversity in these areas. While estimating genetic diversity, the use of molecular markers has been helpful in defining alleles and studying genetic flow, population structure, paternity, inheritability, genetic maps and conservation genetics [11]. Simple sequence repeat markers (SSRs), commonly referred to as microsatellite markers, are desirable tools because they are co-dominant in nature, multiallelic and widely distributed in the genome; they are also currently cheap, reproducible and relatively easy to analyze [12]. This work reports the development, characterization and transferability of microsatellite markers for P. rubriflora and P. ruscifolia.

Construction of a microsatellite-enriched library
DNA was extracted from P. rubriflora and P. ruscifolia using the DNeasy® Plant Mini Kit (Qiagen, Hilden, DE) according to the manufacturer's instructions. Microsatelliteenriched libraries for P. rubriflora and P. ruscifolia were constructed as described by Billote et al. [13]. The genomic DNA was digested with AfaI after enrichment with streptavidin-coated magnetic beads (Streptavidin MagneSphere Paramagnetic Particles, Promega, Madison, WI); biotinylated (CT) 8 and (GT) 8 microsatellite probes were added for the dinucleotide-enriched library. The fragments were amplified by PCR and cloned into the pGEM-T vector (Promega, Madison, WI). XL1-Blue (Escherichia coli) competent cells were transformed with the recombinant plasmids and then cultivated on agar medium containing ampicillin (100 mg/ml), X-galactosidase 2% (100 μg/ml) and IPTG (100 mM). The selected clones were added to a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and sequenced using an ABI 377 sequencer (Applied Biosystems, Foster City, CA). The sequences were aligned and edited using SeqMan Software (DNAStar, Madison, WI), and the adapters and restriction sites were removed using Microsat Software (A. M. Risterucci, CIRAD, personal communication). To identify microsatellite-enriched regions, we used the Simple Sequence Repeat Identification Tool (SSRIT) [14] and defined the following numbers of repeats/motifs: five/dinucleotides, four/trinucleotides and three/ tetra-or pentanucleotides. After these steps, primers were designed using the PrimerSelect software (DNAStar, Madison, WI).    [18] was used to estimate adherence to Hardy-Weinberg (HW) equilibrium and possible linkage disequilibrium (LD), and the frequency of null alleles was estimated using FreeNA [19].

Results and discussion
We designed 32 primer pairs: 13 for P. rubriflora and 19 for P. ruscifolia. However, only 10 of the P. rubriflora primer pairs and 13 of the P. ruscifolia primer pairs amplified properly. The nine remaining pairs of primers were discarded because amplification errors were observed in the preliminary tests. Polymorphisms were detected in 9 of the native P. rubriflora markers and 12 of the native P. ruscifolia markers; only one marker from each species had a monomorphic pattern based on the populations analyzed. Eight markers from P. rubriflora successfully crossamplified and were polymorphic for the tested samples, and 2 markers failed during cross-amplification. Eleven P. ruscifolia markers were successfully cross-amplified; 7 were polymorphic, and 2 failed this analysis ( Table 1). The number of P. rubriflora alleles in the sampled remnants ranged from 3 to 12; the polymorphism information content (PIC) values of these markers ranged from 0.073 to 0.791, the observed heterozygosity (H o ) ranged from 0.000 to 0.850, and the expected heterozygosity (H e ) ranged from 0.000 to 0.835. No evidence of null alleles was observed, and no departure from Hardy-Weinberg equilibrium was observed (Table 2). No significant linkage disequilibrium (LD) was observed for any of the markers of this species after Bonferroni correction (P-value for 5% = 0.001389). The number of P. ruscifolia Higher values of H o were observed for the Prb1, Prb2, Prb4, Prb6, Prb7, Prsc1, Prsc3 and Prsc5 markers in this study; these higher values may indicate that an insufficient number of samples was collected or may be related to the reproductive patterns of these populations. The ECD populations are highly disturbed, and the FRC population is currently recovering from a relatively recent suppression (within the last 15 years); these factors may underlie the observed departure from HW and the presence of null alleles. A study with new and conserved populations may produce better results for these markers.
These markers are the first microsatellite markers developed for Prosopis rubriflora and Prosopis ruscifolia, and together with the set of P. ruscifolia markers amplified by Bessega et al. [20], they are expected to be useful tools for studies of the conservation genetics, reproductive biology, phylogeography and taxonomy of these species.

Availability of supporting data
The original sequences of the developed markers were submitted to the GenBank database (http://ncbi.nlm.nih. gov), and the registered codes are available in Table 1.
The testimony samples were deposited at Herbarium Universidade Estadual de Campinas (UEC -Campinas, SP, BR) and registered according to the following: P. rubri