Skip to main content

Development and characterization of nuclear microsatellite markers for Eremanthus erythropappus and their transferability across related species

Abstract

Background

We developed simple sequence repeats (SSR) for Eremanthus erythropappus (DC.) MacLeish, an endangered tree species endemic to the Brazilian Savanna and Atlantic Forest biomes, and tested their transferability to two closely related Eremanthus species.

Results

Using a genomic library enriched with tandem repeat motifs, we identified 16 primer pairs, and characterized them in two populations. Nine primers amplified the expected size fragments and seven SSRs were polymorphic, providing a total of 38 alleles and an average of 4.22 alleles per marker. The polymorphic information content (PIC) ranged from 0.44 to 0.94 with an average of 0.65. The average observed heterozygosity across all loci varied from 0.61 to 1.00. The observed (HO) and expected (HE) heterozygosity within the two populations varied from 0.65 to 1.00 and from 0.31 to 1.00, respectively.

Conclusions

These newly developed SSR markers are a powerful tool for population genetic analyses and may be useful in studies on species ecology, evolution, and taxonomy.

Background

Eremanthus erythropappus (DC.) MacLeish is an endemic tree species native to the high-elevation mountains of the Brazilian Savanna and Atlantic Forest biomes [12, 13]. Regenerants of the Eremanthus genus are light-demanding, as is common with pioneer species [3, 21], and E. erythropappus normally occurs in shallow, rocky soils with low fertility [17]. Historically in Brazil, the species has been over harvested for use as fence posts in rural areas due to the durability of the wood against weathering [22, 23], and for the extraction of a sesquiterpene alcohol, α-bisabolol, which is used in many pharmaceutical products because of its anti-inflammatory, antibacterial, skin-smoothing, and wound healing properties [10, 24].

Microsatellite markers, also known as simple sequence repeats (SSR), are often used to study the genetic diversity of plants and animals. SSR sequences are distributed throughout the euchromatin and eukaryote genome [11, 19] and exhibit high polymorphism and repeatability across laboratories [28]. Microsatellite loci present the highest information content among all classes of molecular markers [27]. These markers can be analyzed using polymerase chain reaction (PCR), allowing for a highly informative evaluation of a large number of loci, as well as assessing effects related to species population genetics, breeding programs, and germplasm conservation [18]. The objective of this study was to develop a set of microsatellite markers to be used to evaluate the genetic diversity and structure of E. erythropappus and other species of the Eremanthus genus.

Results

From the initial 16 primer pairs, nine amplified the expected size fragment while the remaining seven either did not amplify or generated multiple bands indicating non-specific amplification. The optimum annealing temperature was 53 °C for all nine primers. Of the nine amplified primers, seven were polymorphic (Ere02, Ere03, Ere07, Ere08, Ere10, Ere13, and Ere14) and two were monomorphic (Ere04 and Ere09). The observed and expected heterozygosities ranged from 0.31 to 1.00 and from 0.38 to 0.91, respectively. The total number of alleles was 38 and the number of alleles per polymorphic marker ranged from 2 to 18 (Table 1). The mean within-population inbreeding coefficient (FIS) was − 0.23. The polymorphic information content (PIC) ranged from 0.44 to 0.94, with an average of 0.65. The combined exclusion probability is sufficient to perform a paternity/maternity exclusion analysis among breeding populations using the seven polymorphic loci. Thus, for the first parent, the exclusion probability reached 0.98 and for the second parent it reached 0.99. None of the tested loci showed significant deviation from Hardy–Weinberg equilibrium, nor did they exhibit a high frequency of null alleles (\( \overline{r}_{d } \) < 0.5; P > 0.05). Six SSRs (Ere03, Ere07, Ere08, Ere09, Ere13, and Ere14) cross amplified in E. incanus and E. glomerulatus.

Table 1 Genetic characterization of the nine developed microsatellites for Eremanthus erythropappus and its transferability to Eremanthus incanus and Eremanthus glomerulatus

Discussion

The development of SSR markers for E. erythropappus enables the application of new genetic research into this endemic and overexploited tree species. Whereas E. erythropappus presents relevant economic interest and remarkable ecological importance, new studies will enable us to analyze the genetic diversity, gene flow and also possible processes of inbreeding and clonality. Overall, the genetic diversity estimates found herein were considerably higher than those found in previous studies on the species using inter-simple sequence repeat (ISSR) markers [6, 7, 15].

Although SSR markers are sometimes identified as monomorphic through agarose electrophoresis, they could be polymorphic considering the resolution of capillary electrophoresis (CE). While MetaPhorTM Agarose (4 bp) offers a high resolution, CE presents a better resolution and greater separation efficiency (2 bp) [25]. Thus, the two monomorphic primers developed herein may also be valuable for studying genetic parameters [14]. The evidence of previous researches suggests that the high information content found in SSR markers may enable their use as primers.

Conclusions

We developed nine microsatellite markers, of which seven are polymorphic and two are monomorphic. These markers can inform new research on population genetics, genetic diversity, spatial genetic distribution, as well as the sustainability of forest management practices employed for this species. Additionally, these primer pairs may be an important tool to assist breeding programs of this species. The analysis of cross-amplification confirmed the possibility of applying these markers in genetic studies of E. incanus and E. glomerulatus.

Methods

We used a (GA)n and (CA)n microsatellite-enriched library based on Billotte et al. [1]. As such, leaf tissue samples from E. erythropappus adult trees were collected and preserved in silica gel. Total genomic DNA was extracted using the CTAB method according to Doyle and Doyle [4]. Thirty ng of genomic DNA was digested using the RsaI restriction enzyme (Promega, Madison, Wisconsin, USA), and fragments were ligated to adapters (Rsa21 5′-CTCTTGCTTACGCGTGGACTA-3′ and Rsa25 5′-TAGTCCACGCGTAAGCAAGAGCACA-3′). For the enrichment of SSRs sequences, we used (CT)8, (GT)8, and (TTC)8 repeats using biotinylated microsatellite probes, and the target fragments were captured by the use of streptavidin-coated magnetic beads (Promega Corporation, Madison, Wisconsin, USA). The Rsa21 and Rsa25 adapter sequences were used as primer templates for the amplification of fragments. The microsatellite fragments were ligated to a pGEM-T Easy Vector System (Promega Corporation, Madison, Wisconsin, USA). The plasmids were introduced into Escherichia coli XL1-Blue strains, and transformed cells were plated on Petri dishes with Luria-Bertani (LB) agar medium containing ampicillin (100 μg ml−1) and X-galactosidase (5-bromo-4-chloro-indolyl-β-d-galactoside) (50 μg ml−1). The recombinant colonies were sequenced using an ABI 377 automated sequencer and the Big Dye Terminator Kit (Applied Biosystems, Vienna, Austria). We found 16 positive clones that contained microsatellite sequences with at least five tandem repeats. Primer pairs were designed using the software Primer 3 [20] with a product size ranging from 100 to 300 base pairs (bp), primer size from 18 to 22 bp, GC % from 40 to 60, and annealing temperature from 57 to 60 °C.

Polymerase chain reaction (PCR) was performed by screening each primer pair through 10 annealing temperatures (between 46 and 55 °C) for 21 individuals from two different E. erythropappus populations. The final volume of each reaction was 15 μl using 30 ng of template DNA added to 12 μl reaction mixture containing 3.33 mM IB Phoneutria buffer (consisting of 100 mM Tris–HCl pH 8.4; 500 mM KCl; 1% Triton X-100; 15 mM MgCl2), 1.5 mM MgCl2, 0.28 mM of each dNTP, 1 U Taq polymerase, and 0.22 mM of each primer (forward and reverse). The temperature regime was assessed separately for each primer pair; as such, we tested a total of 17 temperatures (from 46 to 62 °C) for six individuals from two populations using MJ Mini™ Thermal Cycler (Bio-Rad, Singapore). The optimal PCR profile used for the amplification of each microsatellite consisted of an initial 3 min at 94 °C, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing temperature (Table 2) for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 7 min. Amplifications were performed using a MJ Mini™ Thermal Cycler.

Table 2 Characteristics of nine microsatellite loci developed for Eremanthus erythropappus from Minas Gerais State, Brazil

Additionally, we sampled 42 individuals from population 1 (Itamonte: 22°16′45.00″S 44°46′25.60″W) and population 2 (Lavras: 21°19′51.85″S 44°57′54.76″W) to evaluate the SSR primer pairs. Voucher specimens were deposited in the ESAL herbarium of the Federal University of Lavras (UFLA), Brazil. Amplification was conducted using the thermocycler GeneAmp PCR System 9700. We applied the same reaction components and PCR thermal cycle used in the validation process. We separated the PCR products using a 3% high-resolution MetaPhor™ agarose (Lonza, Rockland, Maine, USA) stained with GelRed. Allele sizes were estimated by comparison to a 10-bp DNA Ladder standard (Invitrogen, Carlsbad, California, USA). Individuals that failed to amplify at a minimum of three primer pairs were excluded.

To analyze genotyping errors due to the presence of null alleles, stuttering, and allele dropout, we applied the Brookfield 1 method [2] using 1000 permutations and the Micro-Checker 2.2.3 software [26]. We estimated allele richness using the MSA software [5]. The number of alleles per locus (NA), observed heterozygosity (HO), and expected heterozygosity (HE) for each population and locus according to the Hardy–Weinberg equilibrium were calculated using GenAlEx 6.4 [16]. The within-population inbreeding coefficients (FIS) were determined using FSTAT 2.9.3.2 [8], applying a Bonferroni correction for multiple comparisons. The probability of non-exclusion for each locus, the combined probability of paternity exclusion, and the PIC were calculated using CERVUS 3.0 [9].

We also tested for cross-amplification into two other species, E. incanus and E. glomerulatus. We amplified the DNA of 10 individuals for each species from one population located in the Beautiful River Falls Ecological Park, Lavras, Minas Gerais State, Brazil (21°19′44.98″S; 44°58′24.58″W). PCR reactions and electrophoresis were performed following the same protocol described above. Subsequently, we compared amplification bands to identify the primers that showed amplification patterns for the two species.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

PCR:

Polymerase chain reaction

CTAB:

Cetyltrimethylammonium bromide

SSR:

Simple sequences repeats

PIC:

Polymorphic information content

DNA:

deoxyribonucleic acid

HO :

Observed heterozygosity

HE :

Expected heterozygosity

FIS :

Inbreeding coefficient

References

  1. 1.

    Billotte N, Lagoda PJL, Risterucci AM, Baurens FC. Microsatellite-enriched libraries: applied methodology for the development of SSR markers in tropical crops. Fruits. 1999;54:277–88.

    CAS  Google Scholar 

  2. 2.

    Brookfield J. A simple new method for estimating null allele frequency from heterozygote deficiency. Mol Ecol. 1996;5:453–5.

    CAS  Article  Google Scholar 

  3. 3.

    Carvalho PER. Espécies florestais brasileiras: recomendações silviculturais, potencialidade e uso da madeira. Brasília: EMBRAPA; 1994.

    Google Scholar 

  4. 4.

    Doyle JJ, Doyle JL. Isolation of plant DNA from fresh tissue. Geog Genet. 1987;12:13–5.

    Google Scholar 

  5. 5.

    Dieringer D, Schlötterer C. Microsatellite analyser (MSA): a platform independent analysis tool for large microsatellite data sets. Mol Ecol Notes. 2003;19:167–9.

    Article  Google Scholar 

  6. 6.

    Estopa RA, Souza AD, Moura MCO, Botrel MCG, Mendonça EG, Carvalho D. Diversidade genética em populações naturais de candeia (Eremanthus erythropappus (DC.) MacLeish). Sci Forestalis. 2006;70:97–106.

    Google Scholar 

  7. 7.

    Freitas VLO, Lemos-Filho JP, Lovato MB. Contrasting genetic diversity and differentiation of populations of two successional stages in a neotropical pioneer tree (Eremanthus erythropappus, Asteraceae). Genet Mol Res. 2008;7:388–98.

    CAS  Article  Google Scholar 

  8. 8.

    Goudet J. FSTAT (version 1.2): a computer program to calculate F-statistics. J Hered. 1995;86:485–6.

    Article  Google Scholar 

  9. 9.

    Kalinowski ST, Taper ML, Marshall TC. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Mol Ecol. 2007;16:1099–106.

    Article  Google Scholar 

  10. 10.

    Kamatou GP, Viljoen AM. A review of the application and pharmacological properties of α-Bisabolol and α-Bisabolol-rich oils. J Am Oil Chem Soc. 2010;87:1–7.

    CAS  Article  Google Scholar 

  11. 11.

    Katti MV, Ranjekar PK, Gupta VS. Differential distribution of simple sequence repeats in eukaryotic genome sequences. Mol Biol Evol. 2001;18:1161–7.

    CAS  Article  Google Scholar 

  12. 12.

    Loeuille B, Lopes JC, Pirani JR. Taxonomic novelties in Eremanthus (Compositae: Vernonieae) from Brazil. Kew Bull. 2012;67:1–9.

    Article  Google Scholar 

  13. 13.

    MacLeish NF. Revision of Eremanthus (Compositae: Vernonieae). Ann Missouri Bot Gard. 1987;74:265–90.

    Article  Google Scholar 

  14. 14.

    Nazareno AG, Reis MS. The same but different: monomorphic microsatellite markers as a new tool for genetic analysis. Am J Bot. 2011;98(10):265–7.

    Article  Google Scholar 

  15. 15.

    Pádua JAR, Brandão MM, Carvalho D. Spatial genetic structure in natural populations of the overexploited tree Eremanthus erythropappus (DC.) MacLeish (Asteraceae). Biochem Syst Ecol. 2016;66:307–11.

    Article  Google Scholar 

  16. 16.

    Peakall RO, Smouse PE. GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes. 2006;6:288–95.

    Article  Google Scholar 

  17. 17.

    Pérez FMJ, Scolforo SJR, Mello JM, Borges RLF, Camolesi JF. Sistema de manejo para a candeia-Eremanthus erythropappus (DC.) MacLeish a opção do sistema de corte seletivo. Cerne. 2004;10:257–73.

    Google Scholar 

  18. 18.

    Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S, Rafalski A. The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol Breed. 1996;2:225–38.

    CAS  Article  Google Scholar 

  19. 19.

    Phumichai C, Phumichai T, Wongkaew A. Novel chloroplast microsatellite (cpSSR) markers for genetic diversity assessment of cultivated and wild Hevea rubber. Plant Mol Biol. 2015;33:1486–98.

    CAS  Article  Google Scholar 

  20. 20.

    Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Bio Methods Prot. 2000;132:365–86.

    CAS  Google Scholar 

  21. 21.

    Scolforo JRS, Loeuille BFP, Altoé TF. Caracterização da candeia. In: Scolforo JRS, Oliveira AD, Davide AC, editors. O manejo sustentável da candeia: o caminhar de uma nova experiência florestal em Minas Gerais. 1st ed. Lavras: Editora UFLA; 2012.

    Google Scholar 

  22. 22.

    Scolforo JRS, Oliveira AD, Acerbi Junior FW. Inventário Florestal de Minas Gerais: equações de volume, peso de matéria seca e carbono para diferentes Fitofisionomias da Flora Nativa. 1st ed. Lavras: Editora UFLA; 2008.

    Google Scholar 

  23. 23.

    Silva MA, Melo JM, Scolforo JR, Czanck Júnior L, Andrade IS, Oliveira AD. Análise da distribuição espacial da candeia (Eremanthus erythropappus (DC.) MacLeish) sujeita ao sistema de manejo porta-sementes. Cerne. 2008;14:311–6.

    Google Scholar 

  24. 24.

    Sousa OV, et al. Antinociceptive and anti-inflammatory effects of the essential oil from Eremanthus erythropappus leaves. J Pharm Pharmacol. 2008;60:771–7.

    CAS  Article  Google Scholar 

  25. 25.

    Swerdlow H, Gesteland R. Capillary gel electrophoresis for rapid, high resolution DNA sequencing. Nucleic Acids Res. 1989;18(6):1415–9.

    Article  Google Scholar 

  26. 26.

    Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P. MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes. 2004;4:535–8.

    Article  Google Scholar 

  27. 27.

    Varshney RK, Graner A, Sorrells ME. Genic microsatellite markers in plants: features and applications. Trends Biotechnol. 2005;23:48–55.

    CAS  Article  Google Scholar 

  28. 28.

    Vieira ML, Santini L, Diniz AL, Munhoz CD. Microsatellite markers: what they mean and why they are so useful. Genet Mol Biol. 2016;39:312–28.

    Article  Google Scholar 

Download references

Acknowledgements

We are thankful to Eve R Nimmo for English review.

Funding

We are grateful to FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais) for financial support (CRA APQ-02641-14). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Author information

Affiliations

Authors

Contributions

LFR, DC and NRB performed the laboratory work; DC, LFR and AGN analyzed the raw data and contributed with the statistical and genetical analysis. DC also administered the funds of this research. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Lucas Fernandes Rocha.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Rocha, L.F., Paula, N.R., Nazareno, A.G. et al. Development and characterization of nuclear microsatellite markers for Eremanthus erythropappus and their transferability across related species. Biol Res 53, 30 (2020). https://doi.org/10.1186/s40659-020-00298-z

Download citation

Keywords

  • Eremanthus erythropappus
  • Simple sequence repeats
  • Genetic diversity
  • Eremanthus incanus
  • Eremanthus glomerulatus