Introduction

Subfamily Bambusoideae (bamboo) is a major Poaceae group that comprises 115 genera and around 1640 species (Kelchner 2013). Natural bamboo populations are distributed all over the world, except for Europe and Antarctica (Clark et al. 2015). Asia has high diversity of bamboo species, particularly in southern China, where they have economic, ecological and social importance (Yuming et al. 2004). Brazil also has high number of bamboo species, including 18 native and five endemic species in the genus Guadua Kunth, which are used in construction, furniture, and handicrafts (Greco et al. 2015; Brazil Flora 2020).

Phylogenetic studies have shown that subfamily Bambusoideae is divided into three main monophyletic lineages that correspond to three tribes: temperate woody (Arundinarieae), tropical woody (Bambuseae) and herbaceous (Olyreae) bamboos (Kelchner 2013, Wysocki et al. 2015). The temperate woody bamboo species diverged from others at the beginning of the evolutionary history of the subfamily (Zang et al. 2011) and are now a distinct group. Among the described species, woody bamboos can be distinguished from others by their infrequent sexual reproduction, with long flowering intervals that range from 7 to 120 years (Janzen 1976). Guadua is a representative woody bamboo genus with many native South America species (Clark 2001), including G. chacoensis (2n = 2x = 46) (Andrada et al. 2007; Rincón and Castillo 2012). This species is ecologically important in its natural habitat and little is known about its reproductive mechanism (Areta et al. 2009). Further, the genetic structure and diversity of G. chacoensis native populations are almost unknown. Reproductive cycle monitoring studies describe that G. chacoensis flowers at intervals of approximately 31 years (Guerreiro 2014).

The advancement of scientific and technological knowledge about molecular genetics, such as SSR markers, facilitates the characterization of genetic diversity and structure, and also assists in the selection of descriptors for neglected species, such as G. chacoensis. Thus, SSR markers overcome the limitations of previous studies, employing dominant molecular markers, and are expected to yield valuable genetic information (Marulanda et al. 2007; Rugeles-Silva et al. 2012). SSR is one of the most informative molecular markers and its uniqueness and value are intrinsic to its multiallelic nature, co-dominant inheritance, relative abundance, broad coverage of the genome, and simple detection by PCR using oligonucleotide primer pairs (forward and reverse) flanking the SSR locus (Powell et al. 1996; Vieira et al. 2016).

Thus, SSRs can potentially be used in many genetic studies (Varshney et al. 2005). However, they can only be used in two ways, either transferred between species that are usually closely related, like species in the same taxon or genus, or developed (identified and validated) for specific species. Specific SSR development requires sequencing the genome, assembling the sequenced fragments, identifying SSR loci, designing primers that flank the SSR region, validation (Powell et al. 1996), and using the DNA of a certain number of individuals. Accordingly, this study aimed to identify, validate and characterize SSR markers for genetic studies about G. chacoensis and examine their transferability among other bamboo species.

Materials and methods

Plant material

Guadua chacoensis leaf samples were collected from natural populations. Collection was authorized (ICMbio-SISBIO nº 45390 and 48802-1) by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio). The collected leaf samples were dehydrated and stored in silica gel for subsequent genomic DNA extraction. The DNA from a fresh leaf of an individual (voucher FLOR_0058621) grown in Florianópolis, Brazil (27° 35′ 49″ S 48° 32′ 56″ W), was used for sequencing and SSR loci identification. Leaf samples of other bamboo species were also collected from the germplasm maintained at the Federal University of Santa Catarina, namely: Bambusa oldhamii, Chusquea tenella, Dendrocalamus latiflorus, Dendrocalamus yunnanensis, Farguesia gaolinensis, Farguesia yunnanensis, Guadua angustifolia, Guadua paniculata, Guadua paraguaiyana, Merostachys scandens, Merostachys speciosa, Merostachys glauca, Oxytenanthera abyssinica, Phyllostachys aurea, Phyllostachys edulis, Phyllostachys pubescens, Pseudosasa mirabilis and Shibatea kumasasa. Eight plants of G. chacoensis were sampled from six natural populations (1.3 km–12 km apart) in Parque Nacional do Iguaçu, Foz do Iguaçu, Brazil, and one cultivated population from Rancho Queimado, Brazil, which is 570.43 km from the natural populations and was used to validate the SSR markers.

DNA isolation

Genomic DNA was extracted from 100 mg of dried leaves, previously ground using a Precellys® homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France), with NucleoSpin Plant II kit (Macherey–Nagel) according to the manufacturer’s instructions. The 100 µl of extracted DNA was immediately frozen at − 20 °C until analysis. DNA quality and quantity were determined with NanoDrop ND-1000 Spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) and 0.8% agarose gel electrophoresis performed at 6 V cm−1 for 60 min and stained with GelRed™ (Biotium Inc., Hayward, CA, USA). For comparison and quantification, Lambda DNA was loaded into gel at concentrations of 12.5 ng µl−1, 25 ng µl−1, 50 ng µl−1, and 100 ng µl−1. For the NanoDrop analysis, 1 µL of DNA was used at the wavelengths 230, 260, and 280 nm, and both the 260/280 and 230/280 ratios were examined for every sample.

DNA sequencing and SSR identification

A total of 1 ng of genomic DNA was used to prepare sequencing libraries with a Nextera XT DNA Sample Prep Kit (Illumina Inc., San Diego, California, USA) according to the manufacturer’s instructions. The libraries were sequenced using a MiSeq Reagent Kit v3 (600 cycles) and an Illumina MiSeq Sequencer (Illumina Inc., San Diego, California, USA). The obtained paired-end reads (2 × 300 bp) were used for de novo assembly with CLC Genomics Workbench 8.0v. Paired-end sequence reads were trimmed of low-quality data with a quality score limit of 0.01 using CLC Genomics Workbench 8.0v and reads of less than 50 bp in length were discarded. Organellar reads were excluded by mapping against two bamboo mitochondrion genomes (gb|JN120789.1, gb|EU365401.1) and the G. chacoensis chloroplast genome (gb|KT373814.1; Vieira et al. 2015) using the Basic Local Alignment Search Tool (BLAST, Altschul et al. 1990). Trimmed read sequences were de novo assembled. The obtained nuclear contigs with depth coverage (higher than 5X) were selected and analyzed with SSRLocator (Maia et al., 2008) for SSR identification, with a threshold of twelve repeat units for mononucleotide SSR, six repeat units for dinucleotide SSR, four repeat units for trinucleotide SSRs, three repeat units for tetra- and pentanucleotide SSR, and two repeat units for hexanucleotide SSR. Primers were designed with SSR Locator using the PRIMER3 algorithm by setting product size ranges from 100 to 350 bp, primer size from 18 to 22 bp, GC content from 40 to 60%, 1 °C as the maximum difference between the melting temperatures of the left and right primers, and a melting temperature (TM) of 57 °C (minimum) and 63 °C (maximum). Primers were analyzed with the software Gene Runner® to ensure the absence of secondary structure formation.

SSR validation by gel electrophoresis

Among the identified SSRs for primer design, 35 with a higher potential for use in population genetic studies were selected. PCR amplifications were carried out to test annealing temperatures (52–62 °C) and reagent concentrations for each of the selected primers. The following thermal cycle conditions were initially used for PCR reactions: 0.2 µM of each primer, 1 unit of Taq DNA polymerase (Invitrogen), 0.2 mM of each dNTP, 2.0 mM MgCl2, 15 ng of template DNA, and 1 × PCR buffer (Invitrogen); the final volume was adjusted to 10 µl. The PCR profile was the following: 95 °C for 3 min; 35 cycles of denaturation at 95 °C for 30 s, annealing temperature (52–62 °C) for 30 s, and 72 °C for 1 min; followed by a final extension at 72 °C for 30 min in a Veriti® 96-Well Thermal Cycler (Applied Biosystems, California, USA). PCR products were submitted to 3% agarose gel electrophoresis at 6 V cm−1 for 120 min and stained with GelRed™. Product sizes were determined by comparison with a 1-kb ladder (Invitrogen). The PCR reactions with dubious results were submitted to new PCR conditions for optimization. After optimization, polymorphism was evaluated in 4% denaturing polyacrylamide gels stained with silver nitrate. Gels were run with 1 × TBE buffer on a vertical electrophoresis apparatus for 90 min at 75 V. Product sizes were determined by comparison with a 100-bp DNA ladder (Invitrogen) (Figure S1).

SSR characterization by multiplex-ready PCR for fluorescence-based genotyping

SSR characterization was made using plant DNA from six natural populations (24 plants of each population) from Paraná State, Brazil, and 12 plants from a population grown in Santa Catarina State, Brazil. The loci with higher polymorphism potential, validated by gel electrophoresis, were selected and primers were fluorescent dye labeled. Genotyping was performed on the ABI Genetic Analyzer 3500xL platform (Applied Biosystems, Forster City, CA). The different fluorescent dye-labeled primers, with different spectra, allowed for multiplex reactions (3–4 primers per reaction). The polymorphism level of each locus was estimated by the polymorphism information content (PIC). Where \( \begin{aligned} {\text{PIC = 1}} - \sum {p_{i}^{2} } \hfill \\ \hfill \\ \end{aligned} \) and pi is the frequency of the i-th allele (Maroof et al. 1994; Anderson et al. 1993).

SSR transferability to other species

SSR transferability was tested using DNA from 18 different bamboo species. Regarding the polymorphic loci, the primers were considered transferred when the amplification resulted in one or two alleles near the expected size. The PCR condition and electrophoresis protocols were the same used for G. chacoensis.

Results

Sequencing the G. chacoensis genomic DNA resulted in 296,699 high-quality paired-end reads. After trimming (0.01 quality threshold) and excluding organellar reads, we obtained 239,621 paired-end reads (average length = 238.79). These reads were submitted to de novo assembly and 6013 contigs were obtained (N50 = 685).

We analyzed the occurrence, type, and distribution of SSRs in G. chacoensis contigs. In total, 92 SSRs were identified. Among them, trinucleotide repeats were the most common with 40 occurrences; whereas, di- (17), tetra- (18), penta- (8), and hexanucleotide repeats (9) occurred with lower frequency (Fig. 1). The most frequent motifs were AT (7.6%), TA (6.5%), TTC (5.4%) and TTA (4.3%). However, only 70 loci were in possible regions to design primers free of secondary structures (Table S1). Among them, 35 loci were selected for validation (Table 1).

Fig. 1
figure 1

Number of distinct types of genomic simple sequence repeats (SSRs) identified in low-depth sequences of the bamboo Guadua chacoensis, grown in Florianópolis, SC (Brazil), using the Illumina MiSeq Sequencer platform (Illumina Inc., San Diego, California, USA). Di-, tri-, tetra-, penta- and hexanucleotides represent 2, 3, 4, 5 and 6 nucleotides that are the length of an SSR repeat

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Table 1 Features of 35 simple sequence repeat (SSR) markers of Guadua chacoensis selected for validation
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PCR reactions with Gcha17, Gcha19, Gcha22, Gcha23, and Gcha24 revealed no amplification products for any of the tested temperatures (52–62 °C) or plants. Different results were obtained for PCR reactions with primers designed for the Gcha25, Gcha30 and Gcha35 loci, since they showed an abundance of nonspecific bands. These characteristics were used as criteria for selecting the markers to be characterized, as well as the amplicons sharpness and quality revealed after gel electrophoresis. The 12 markers selected for characterization and fluorescent labeling are listed in Table 2.

Table 2 Multiplex sets of loci used for characterization 12 SSR markers of Guadua chacoensis
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The SSR characterization showed that the Gcha33 and Gcha18 loci had nonspecific amplification products for all plants, possibly due to instability during amplification reactions or annealing inconsistencies and hence, these loci were discarded. The Gcha01, Gcha02, Gcha04, Gcha05, Gcha07, Gcha10, Gcha11, Gcha12, Gcha13 and Gcha21 loci were polymorphic, of which the Gcha02 locus had the highest allele number (4) and the highest PIC value (0.507). The Gcha04 locus, with only 2 alleles, of which one is rare (frequency < 5%), had the lowest PIC value (0.039) (Table 3).

Table 3 Allele frequency and polymorphism information content (PIC) for 10 of the characterized SSR markers of Guadua chacoensis
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To further characterize the new SSR markers, five multiplexes were formed. Multiplex “A” was composed of the Gcha04 and Gcha18 loci (Figure S2), multiplex “B” of the Gcha08, Gcha10, Gcha21 and Gcha33 loci (Figure S3), multiplex “C” of the Gcha02, Gcha05 and Gcha06 loci, multiplex “D” of Gcha01 and Gcha07 loci (Figure S4), and multiplex “E” of the Gcha11, Gcha12 and Gcha13 loci (Figure S5). All of these multiplexes showed good and unambiguous amplification results.

The transferability to other bamboo species was successfully achieved for all tested SSR loci in 15 (i.e., all but Phyllostachys pubescens, Phyllostachys edulis and M. glauca) of the 15 species (Table 4, Figs. 6S and 7S).

Table 4 Transferability of SSR loci from Guadua chacoensis to other bamboo species
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Discussion

AT and GC content of the genome has been used in evolutionary ecology studies about monocots (Smarda et al. 2014). The higher AT content than GC content in the G. chacoensis genome, observed in the present work, is in accordance with previous studies about other bamboos (Liu et al. 2012), as well as other plant species, such as Zea mays, Oryza sativa, Beta vulgaris, and Arabidopsis thaliana. On the contrary, in animal genomes, GC content is higher (Beven et al. 1998; Kubo et al. 2000; Tóth et al. 2000; Barow and Meister 2002; McCouch et al. 2002; Jaillon et al. 2004; Yu et al. 2012).

High di- and trinucleotide repeat frequency was already described for the bamboos D. latiflorus (Bhandawat et al. 2015) and Phyllostachys violascens (Cai et al. 2019), as well as for conifer species, such as Pinus taeda and Picea glauca (Bérubé et al. 2007). These SSR motifs are reported as more informative, due to their greater stability compared to mononucleotides, and higher polymorphism compared to tetra-, penta- and hexanucleotide repeats (Samadi et al. 1998; Bérubé et al. 2007).

The annealing temperature is important to develop protocols with accurate results in molecular genetic analyses. An incorrect annealing temperature can cause unspecific amplifications or a lack of amplifications, resulting in a false positive or false negative, respectively, making it impossible to use (Ishii and Fukui 2001; Sipos et al. 2007). During the annealing temperature test, which ranged from 52 to 62 °C, we were able to discard the primers designed for the Gcha17, Gcha19, Gcha22, Gcha23 and Gcha24 loci, due to the absence of amplification at all tested temperatures. We also discarded the Gcha25, Gcha30 and Gcha35 loci, due to the presence of many unspecific amplicons at all tested temperatures. Further studies are necessary to optimize the protocol of these loci and to develop additional primers without these problems. For all other primer loci, the annealing temperature was 58 °C, except for Gcha11, Gcha12, and Gcha13, where 55 °C resulted in the best amplification (Figure S1). Thus, all the multiplex mixes were standardized with this temperature.

Among the polymorphic loci characterized in this study, only the Gcha05 (dinucleotide + tetranucleotide) and Gcha21 (tetranucleotide) loci were not trinucleotide repeats, which demonstrated that trinucleotide repeats were also informative for G. chacoensis. The allele number and PIC values showed reduced diversity for the characterized loci compared to other species (Hammami et al. 2014; Cubry et al. 2014). However, this seems to be an intrinsic feature of bamboo populations, since similar results were found for Dendrocalamus giganteus (Tian et al. 2012). It is worth mentioning that these estimators and values may be evaluated again for further characterization with additional and distant populations to confirm this feature of G. chacoensis.

The phenological cycle and reproductive biology of G. chacoensis are possible causes of the reduced polymorphism found in this study. Flowering and, consequently, allelic recombination in bamboos are governed by poorly understood environmental factors (Campanello et al. 2007). In G. chacoensis species, flowering occurs at intervals of approximately 31 years (Areta et al. 2009), pollen is predominantly dispersed by wind, seeds are predominantly dispersed by associated fauna, and reproductive behavior is similar to other woody bamboo species (Areta and Bodrati 2008; Montti et al., 2011a). These features do not favor the quick establishment or spread of new alleles (Eriksson 1997) or allelic transgressive combinations, yet little is known about the effects of this reproductive behavior on the ecology and genetic structure of bamboo populations (Budke et al. 2010; Montti et al. 2011b).

This is the first report that characterizes SSR loci for G. chacoensis genetic studies. The informative value of each characterized locus obtained in the present study may vary from the analysis of other populations. Therefore, genetic studies with geographically more distant populations can be based on the molecular markers developed here. With additional data, more accurate estimates of the polymorphic potential and allele number of each locus could be made. The polymorphic loci described here represent an advance in phylogenetic and population genetic studies in G. chacoensis and closely related species, in which primer transferability may be possible.

Conclusion

The shotgun genome sequencing of G. chacoensis with the Illumina platform allowed the identification, validation and characterization of 12 SSR markers for this species. Among them, 10 were polymorphic and can be used in G. chacoensis population genetic studies. These markers could be used in analyses about G. chacoensis genetic diversity, relationships between natural populations and phylogenetics, as well as populations of the 12 bamboo species that were found to be transferable.