Transferability of SSR markers from Eugenia uniflora L. to Myrtaceae species of the Atlantic Forest

ABSTRACT

The Myrtaceae family is home to several species of economic and ecological importance, but many are in areas of constant environmental degradation, demanding studies aiming at the conservation status evaluation or other characteristics of interest. One of the widely used tools for these purposes is SSR markers. This study sought to evaluate the transferability of 11 new SSR markers characterized in Eugenia uniflora to 14 different species of the family Myrtaceae. Out of the 11 markers tested, nine were amplified in at least one of the examined species. For seven species, this is the first report of cross-species transfer of SSR markers. These markers may contribute to the characterization of the genetic diversity of the species and planning policies for the conservation and breeding of these genetic resources.

Keywords:
genetic diversity; microsatellite markers; Myrtoideae; molecular diversity; molecular markers

Introduction

The Myrtaceae family is widely represented among botanical families, dispersed in various regions of the world (Govaerts et al. 2008). In Brazil, it is present in several ecosystems, mainly in the Atlantic Forest (Guilherme et al. 2004), a biome suffering a historical decrease in its natural area. Despite the degradation and fragmentation, it is estimated that the Atlantic Forest holds about 20,000 species, including several endemic and endangered ones, being recognized as a region of high priority for the conservation of global biodiversity (Myers et al. 2000).

In general, Brazilian Myrtaceae are not used as a source of timber, like species of Eucalyptus, but are used to supply firewood, manufacture small objects (Costella et al. 2013), in popular medicine and provide a variety of eatable fruits. Despite the recognized economic, cultural, and ecological importance of several Brazilian Myrtaceae (e.g., Brazilian cherry, guava, jaboticaba, and feijoa), scientific data for the conservation of less prominent species is still incipient. In turn, molecular markers based on the direct analysis of the DNA are useful tools for generating sound information about the genetic diversity of such species.

Nuclear SSR markers are one of the predominant genetic markers for molecular ecology studies. However, researchers usually demand universal markers that can be used in different species. Such markers facilitate comparisons among closely related taxa for addressing the mechanisms involved in population divergence and speciation, as well as comparisons among multiple co-occurring species for studying the interaction of diversity patterns at the genetic and community levels (Barbará et al. 2007). Given the high polymorphism and codominant nature of nuclear SSRs, the potential of cross-species transfer of these markers has been gaining increased importance. Particularly for forest species with incipient commercial importance, the transferability of SSR markers may be the main tool for researchers accessing genetic diversity and the structure of natural populations. Aiming to provide a new set of molecular markers for genetic studies of Myrtaceae species from the Atlantic Forest, this study investigated the transferability of 11 newly characterized SSR markers from E. uniflora to 14 species of this family.

The SSR markers were prospected through the partial genome sequencing of E. uniflora (Sarzi et al. 2019; Stefenon et al. 2019), while the 14 species investigated correspond to six key genera of Myrtaceae native to the Atlantic Forest. Leaf samples were collected from adult individuals in forest fragments in the Central region of the Rio Grande do Sul State, Atlantic Forest domain. Species were identified based on morphological characteristics described in the specialized literature (Landrum & Kawasaki 1997; Souza & Lorenzi 2005; Lorenzi et al. 2006; Sobral et al. 2015). Vouchers of each species (Figure 1) were deposited in the Bruno Edgar Irgang Herbarium, Federal University of the Pampa, São Gabriel, Brazil. Total DNA was isolated from the leaves of two distinct trees of each species using the CTAB method (Doyle & Doyle 1987). The concentration, purity, and integrity of the DNA were verified by electrophoresis in 0.8% agarose gel and through spectrophotometry in a NanoVue™ spectrophotometer.

Figure 1
Vouchers of each species used in this study, deposited in the Bruno Edgar Irgang Herbarium, Federal University of the Pampa, São Gabriel, Brazil, and their corresponding herbarium ID. (A) Acca sellowiana (O. Berg) Burret - HBEI 1626. (B) Campomanesia guazumifolia (Cambess.) O. Berg - HBEI 1625. (C) Campomanesia xanthocarpa Mart. ex O. Berg - HBEI 1632. (D) Eugenia involucrata DC. - HBEI 1627. (E) Eugenia pyriformis Cambess. - HBEI 1628. (F) Eugenia ramboi D. Legrand. - HBEI 1629. (G) Eugenia rostrifolia D. Legrand. - HBEI 1630. (H) Eugenia uruguayensis Cambess. - HBEI 1621. (I) Eugenia verticillata (Vell.) Angely - HBEI 1631. (J) Myrcianthes pungens (O. Berg) D. Legrand - HBEI 1633. (K) Plinia rivularis (Cambess.) Rotman - HBEI 1566. (L) Plinia peruviana (O.Berg) Kausel - HBEI 1634. (M) Psidium cattleyanum Sabine - HBEI 1635. (N) Psidium guajava L. - HBEI 1636. The red bar above each voucher corresponds to the number of amplified SSR markers in the species.

PCR amplifications were performed in 12.5 μL reaction mix containing 100 ng of DNA, 0.25 μM of buffer, 0.5 μM MgCl₂, 1 U of Taq DNA-Polymerase, 0.05 μM each dNTP, 0.125 μM of each primer, and 0.01% BSA. Amplification reactions were performed on a BIO-RAD C1000 Touch™ Thermal Cycler with an initial step of 94 °C for 3 minutes, followed by 39 cycles with a step of denaturation at 94 °C for 45 seconds, the specific annealing temperature of each primer (Beise et al. 2022) for 30 seconds, and extension at 72 °C for 1 minute, with a final extension of 72 °C for 10 minutes.

The products of the amplifications were resolved using electrophoresis on 3% agarose gel, stained with GelRed^®, and visualized using UV light (Figure 2). A 100 bp ladder was used as a reference. The sizing of the alleles was performed using the software TotalLab TL 120 1D v. 2009 (Nonlinear Dynamics, UK).

Figure 2
Example of the electrophoresis gel (3% agarose) visualized using UV light. (A) Transferability test of the SSR marker Pit38. (B) Transferability test of the SSR marker Pit123. Two different samples from each species were employed.

The cross-species transferability was effective in 11 species (Table 1), with successful PCR amplification within the expected size, based on the genome of E. uniflora. No marker was amplified in E. verticillata, M. pungens, and P. rivularis. From two (in C. xanthocarpa and P. guajava) to seven (in E. involucrata and P. cattleyanum) markers were transferred across the species included in this study. The PCR amplification of markers Pit57 and Pit115 failed for all species. The number of amplified alleles ranged from one to 11 (Table 1). Markers Pit26 and Pit138 are linked to genes FRI (involved in the regulation of flowering time) and LECRKS7 (involved in resistance response to the pathogenic oomycetes) respectively. Gene-linked SSR markers are expected to show a high degree of transferability since they are found linked to conserved expressed regions (Petry et al. 2019). However, marker Pit26 cross-amplified only in P. peruviana and P. cattleyanum, while Pit138 amplified in nine species (Table 1).

Table 1
SSR markers prospected in E. uniflora and tested for cross-species transferability to species of Myrtaceae. +: successfully transferred; -: failed cross-species transferability; Alleles: number of alleles amplified overall species.

Species	Marker
	Pit26	Pit38	Pit53	Pit57	Pit71	Pit98	Pit115	Pit119	Pit123	Pit138	Pit140
E. uniflora	+	+	+	+	+	+	+	+	+	+	+
E. involucrata	-	+	+	-	+	-	-	+	+	+	+
E. pyriformis	-	+	+	-	+	-	-	-	+	+	+
E. ramboi	-	+	-	-	-	+	-	-	+	+	-
E. rostrifolia	-	+	+	-	-	+	-	-	+	+	+
E. uruguayensis	-	+	-	-	-	-	-	-	+	+	+
E. verticillata	-	-	-	-	-	-	-	-	-	-	-
A. sellowiana	-	-	+	-	-	-	-	-	+	+	-
C. guazumifolia	-	+	-	-	-	-	-	-	+	+	-
C. xanthocarpa	-	+	-	-	-	-	-	-	-	+	-
M. pungens	-	-	-	-	-	-	-	-	-	-	-
P. rivularis	-	-	-	-	-	-	-	-	-	-	-
P. peruviana	+	+	+	-	-	-	-	+	+	-	-
P. cattleyanum	+	+	+	-	-	-	-	+	+	+	+
P. guajava	-	-	+	-	-	-	-	-	-	-	+
Alleles	4	11	5	1	4	4	1	4	9	10	7

It has been shown that SSR markers transferability is more likely to occur among taxonomically close species (65% between species within a genus, 43% between genera within a family, and 28% between families within an order; Barbará et al. 2007). Our results were comparatively higher with successful transferability reaching 73% of the markers to Eugenia species (ranging from 0% to 83.3% of the markers) and 63.6% of the markers to other genera within the family Myrtaceae (ranging from 0% to 75% of the markers).

The SSR markers transferability among species of Myrtaceae was reported by Santos et al. (2007), Ferreira-Ramos et al. (2014), Nogueira et al. (2016), and Petry et al. (2019). In this study, we add to the scientific literature, a set of nine SSR markers able to be used in genetic studies of nine Myrtaceae species of the Atlantic Forest. To the best of our knowledge, this is the first report of marker transfer to E. ramboi, E. rostrifolia, E. uruguayensis, C. guazumifolia, C. xanthocarpa, M. pungens, and P. peruviana, even though SSR markers were recently developed for C. xanthocarpa (Petry et al. 2019). These transferred markers may contribute to the characterization of the genetic diversity of the species and planning conservation policies and breeding of these genetic resources.

Acknowledgments

The authors thank CNPq (Process 302501/2017-7) for a research grant to VMS and CAPES (Process 001) for financial support.

References

• Barbará T, Palma-Silva C, Paggi GM, Bered F, Fay MF, Lexer C. 2007. Cross-species transfer of nuclear microsatellite markers: Potential and limitations. Molecular Ecology 16: 3759-3767.
• Beise DC, de Oliveira LO, dos Santos DD, Stefenon VM. 2022. Assessing genetic structure of Eugenia uniflora L. populations along an environmental gradient using a novel set of SSR markers. South African Journal of Botany 149: 530-536.
• Costella E, Garcia LSC, Corneleo NS, Schünemann AL, Stefenon VM. 2013. Anthropogenic use of gallery forests in the Brazilian Pampa. Acta Scientiarum: Biological Sciences 35: 211-217.
• Doyle JD, Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11-15.
• Ferreira-Ramos R, Accoroni KAG, Rossi A et al 2014. Genetic diversity assessment for Eugenia uniflora L., E. pyriformis Cambess., E. brasiliensis Lam. and E. francavilleana O. Berg neotropical tree species (Myrtaceae) with heterologous SSR markers. Genetic Resources and Crop Evolution 6: 267-272.
• Govaerts R, Sobral M, Ashton P, Barie F. 2008. World checklist of Myrtaceae. Kew Publishing, Royal Botanic Gardens.
• Guilherme FAG, Morellato LPC, Assis MA. 2004. Horizontal and vertical tree community structure in a lowland Atlantic rain forest, Southeastern Brazil. Revista Brasileira de Botânica 27: 725-737.
• Landrum LR, Kawasaki ML. 1997. The genera of Myrtaceae in Brazil: An illustrated synoptic treatment and identification keys. Brittonia 49:508-536.
• Lorenzi H, Bacher L, Lacerda M, Sartori S. 2006. Frutas brasileiras e exóticas cultivadas. Nova Odessa: Instituto Plantarum.
• Myers N, Mittermeier RA, Mittermeier CG, Fonseca GAB, Kent J. 2000. Biodiversity hotspots for conservation priorities. Nature 403: 853-858.
• Nogueira AM, Ferreira A, da Silva Ferreira MF. 2016. Transferability of Microsatellites from Psidium guajava to Eugenia, Myrciaria, Campomanesia, and Syzygium Species (Myrtaceae). Plant Molecular Biology Reporter 34: 249-256.
• Petry VS, Stefenon VM, Machado LO, Klabunde GHF, Pedrosa FO, Nodari RO. 2019. Repetitive genomic elements inCampomanesia xanthocarpa:Prospection, characterization and cross amplification of molecular markers. 3 Biotech 9: 423. doi: 10.1007/s13205-019-1953-8
  » https://doi.org/10.1007/s13205-019-1953-8
• Santos KL, Welter LJ, Dantas ACM, Guerra MP, Ducroquet JPHJ, Nodari RO. 2007. Transference of microsatellite markers from Eucalyptus spp to Acca sellowiana and the successful use of this technique in genetic characterization. Genetics and Molecular Biology 30: 73-79.
• Sarzi DS, Justolin B, Da Silva C, Lemos RMP, Stefenon VM. 2019. Discovery and characterization of SSR markers in Eugenia uniflora L. (Myrtaceae) using low coverage genome sequencing. Annals of the Brazilian Academy of Sciences 91: e20180420
• Sobral M, Proença C, Souza M, Mazine F, Lucas E. 2015. Myrtaceae in Lista de Espécies da Flora do Brasil. Rio de Janeiro: Jardim Botânico do Rio de Janeiro.
• Souza VC, Lorenzi H. 2005. Botânica Sistemática: Guia ilustrado para identificação de Angiospermas da flora brasileira, baseado em APG II. Nova Odessa: Instituto Plantarum .
• Stefenon VM, Sarzi DS, Roesch LFW. 2019. High-throughput sequencing analysis of Eugenia uniflora: Insights into repetitive DNA, gene content and potential biotechnological applications. 3 Biotech 9: 200.

Publication Dates

• Publication in this collection
  25 Aug 2023
• Date of issue
  2023

History

• Received
  07 Mar 2023
• Accepted
  11 July 2023