Development and characterization of microsatellite loci for Campomanesia xanthocarpa (Myrtaceae) and cross amplification in related species

Campomanesia xanthocarpa is a native tree, of common occurrence in almost all Brazilian Forest formations, which has its fruits and timber with high commercial value. Using an enriched genomic library we isolated and characterized microsatellite loci for C. xanthocarpa (Myrtaceae), in order to estimate genetic diversity parameters for this and related species. Twenty-eight microsatellite loci were identified and ten of them successfully amplified and showed polymorphism in a sample of 96 individuals, from four natural populations. The number of alleles per locus ranged from two to eight, and the observed and expected heterozygosities varied from 0.042 to 1.000 and from 0.294 to 0.855, respectively. These markers were tested and validated in two related species (C. eugenioides and C. guazumifolia). The microsatellite markers will be used in further studies of population genetics of C. xanthocarpa, in order to understand the genetic variability and to define the strategies needed for the conservation of the species.


Introduction
Campomanesia xanthocarpa O. Berg (Myrtaceae) is a tree species commonly known as Gabirobeira or just Gabiroba, which can reach from 10 to 20 m high with an elongated and dense top (Lorenzi, 2002). This species occurs naturally in Brazil in almost all forest formation and it can also be found in Uruguay, Argentina and Paraguay (Biavatti et al., 2004).
Campomanesia xanthocarpa produces a large quantity of seeds that are widely disseminated by the avifauna. The species has an important role in environmental preservation, mainly for the restoration of riparian vegetation and for the recovery of degraded areas. The fruits of C. xanthocarpa have great economic value, either as consumed 'fresh' or in the preparation of candies, sorbets and homemade liqueurs (Lorenzi, 2002). Its wood is used in the production of musical instruments, agriculture, firewood, coal, fence and plank. The species presents medicinal value in the fight against dysentery, fever, scurvy, and urinary tract diseases (Cravo, 1994;Alice, Siqueira, Mentz, Silva, & José, 1995).
Due to the selective logging, C. xanthocarpa is undergoing severe loss of genetic variability that can lead to the extinction of many natural populations, thus, it is necessary to know the genetic structure of the species in order to maintain their genetic variability (Chaves et al., 2015).
The simple sequence repeat (SSR) marker is a powerful technique for characterizing allelic diversity and it is frequently used in population studies due to their high level of polymorphism and codominant inheritance (Ellegren, 2004;Deng, Wang, Zhu, Wen, & Yang, 2015). The purpose of our study was to develop and characterize microsatellite loci for C. xanthocarpa for further application in population genetic studies and to boost strategies for conserving the genetic resources of this species and to assess crossamplification in two related species. PCR amplification and the efficiency of each primer pair were tested in a sample of 96 individuals from four populations of C. xanthocarpa (24 individual per population). Three collection sites were from Paraná state, including Fazenda Doralice (23º16'00.0"S and 51º03'00.0"W; Voucher FUEL 17164), Parque Estadual Mata São Francisco (23º27'00.0"S and 51º15'00.0"W; Voucher FUEL 55710) and RPPN Fazenda Duas Barras (23°0'2"S and 52°54′50"W; Voucher UPCB 18338); and the last one was from São Paulo state, Horto Florestal de Palmital (22º48´00.0"S and 50º16´00.0" W; Voucher FUEL 55282). Cross-amplification was tested in other two species of Campomanesia; C. eugenioides (24° 1'12.23"S and 50°53'48.77"W; Voucher FUEL 42813) and C. guazumifolia (23°19'43.37"S and 51°12'10.86"W; Voucher FUEL 55400) using 10 plants from each species.

Results and discussion
The sequences of the 10 microsatellite loci were submitted to Genbank and registered under the accession numbers cited in Table 1. The genotyping of 96 individuals of C. xanthocarpa identified a total of 61 alleles, with the sizes of the fragments varying from 114 (Cxan2) to 330 bp (Cxan1) ( Table 2). The polymorphic information content varied from 0.492 (Cxan5) to 0.851 (Cxan1). According to Botstein, White, Skolnick, and Davis (1980), the level of polymorphism at a specific loci can be considered high when the PIC > 0.5 (Table 1), therefore, most of the analyzed loci had high polymorphic information content. The number of alleles per locus ranged from two to eight (Table 2)  Pairwise comparisons for multiple tests among the polymorphic loci showed significant linkage disequilibrium between loci (Table 3), while 8 loci showed significant evidence for the presence of null alleles (Cxan2, Cxan5, Cxan6, Cxan10,Cxan13, Cxan16, Cxan27, Cxan28) according to Bonferroni correction (p ≤ 0.05), which can indicate homozygosis excess.
The 10 loci were tested for cross-amplification using annealing temperature gradients in two species of the genus Campomanesia: C. guazumifolia and C. eugenioides (Table 4). The primers Cxan 2, Cxan13 and Cxan27 showed inconsistent amplification for all species tested, while primers Cxan1, Cxan5, Cxan6 and Cxan18 amplified for all species. Loci Cxan10 and Cxan16 showed amplification only for C. guazumifolia and Cxan28 only for C. eugenioides. The patterns of genetic polymorphism observed in C. xanthocarpa for 10 microsatellite loci suggest that these markers can be used as valuable sources of information for the actual level of genetic conservation of C. xanthocarpa in their distribution area. A similar study was carried out by Ruas et al. (2009) and the microsatellite loci developed by those authors were employed by Conson et al. (2013), to verify if the forest fragmentation that began last century, in South and Southeast Brazil affected the genetic variability of the tree species Luehea divaricata. Chaves et al. (2016) used microsatellite loci, developed by Ramos et al. (2011), to demonstrate the apomictic and sexual reproduction, polen and seed dispersion of the tree species Aspidosperma polyneuron.
We also tested the developed markers for cross-species amplification, with a success in the transferability rate of five and six loci for C. eugenioides and C. guazumifolia, respectively (Table 4).
The transferability of polymorphic microsatellite markers in plants is likely to be successful, mainly within genera (success rate close to 60% in eudicots and close to 40% in the monocots). Between genera, transfer rates are approximately 10% for eudicots, and researchers of monocots, such as orchids or grasses, are very unlikely to get away without isolating novel markers from the genomes of new target genera. An exception is the large adaptive radiations with low levels of DNA sequence divergence such as Bromeliaceae, where polymorphic markers are readily transferred between species of the same subfamily and beyond (Barbará et al., 2007).
Comparing our results employing the 10 microsatellite loci developed and validated for C. xanthocarpa with similar work cited in literature we observe the high potential of these markers, to estimate parameters of genetic diversity and population structure in further studies for this species, as well, for other related species tested for cross-amplification. Also, the produced data will be valuable in conservation and management programs aiming the recovering of degraded areas.

Conclusion
In this study, we developed 28 microsatellite markers for the tree species C. xanthocarpa, 10 of which were polymorphic. Tested for cross-amplification, five and six loci successfully amplified in the related species C. eugenioides and C. guazumifolia, respectively. The development of novel microsatellite markers will be an important tool to understand the consequences of selective logging that have affected the genetic diversity of C. xanthocarpa and other related species present in the same area.