Identification and characterization of SSR markers of Guadua chacoensis (Rojas) Londoño & P.M. Peterson and transferability to other bamboo species

Abstract

The aim of this study was to develop simple sequence repeat (SSR) markers for genetic studies on G. chacoensis, as well as to evaluate their transferability to other bamboo species. Genomic DNA was isolated from G. chacoensis and its partial sequencing was used to find SSR loci. The obtained sequencing data were de novo assembled using the software CLC Genomics Workbench^® 8.0v. The SSR loci primers were identified and designed with the software SSRLocator. The selected markers were validated using 56 plants sampled in seven populations from southern Brazil. The markers with potential polymorphism were selected and fluorescently labeled for characterization by capillary electrophoresis. In total, 92 SSR loci were found in G. chacoensis contigs. Suitable primers were designed for 70 SSR loci, and the remaining 22 SSR loci did not have sequences for primer development. Out of 35 selected SSR markers, after PCR optimization, 10 with high polymorphism potential were characterized. These loci can be used in genetic analyses of G. chacoensis and all of them were successfully transferred to other bamboo species. Non-polymorphic loci require further tests with additional plants, from different populations, to identify possibilities of their use.

Electronic supplementary material

The online version of this article (10.1007/s13205-020-02268-4) contains supplementary material, which is available to authorized users.

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 $\begin{array}{c}\hfill \text{PIC = 1}-\sum p_i^2\\ \hfill \\ \hfill \end{array}$ and p_i 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.

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

Table 1.

Features of 35 simple sequence repeat (SSR) markers of Guadua chacoensis selected for validation

Locus	GenBank accession		Primer sequence (5′–3′)	TM (°C)	Product size (bp)	(Motif) repeat number
Gcha01	MN644912	F	CCAGTTGATATGCAGAACCT	55	238	(GCC)4
		R	TTCTCTCTATCGTCCTCAGC
Gcha02	MN644913	F	TAGCACGTACCATCAAACAA	55	249	(GCG)4
		R	CCTACCGTAAGCTCTCTGAA
Gcha03	MN644914	F	TTCACCTAGACTTCTGGCAT	55	289	(TTC)4
		R	TGAGGAGAGAAAGGTTGAGA
Gcha04	MN644915	F	TTGTGCATGACTTGTTGAGT	55	239	(AGA)4
		R	TCACCTTACAATAATTCGCC
Gcha05	MN644916	F	TAGTCGGGTGATCATGAAAT	55	291	(AT)6-(AATT)3
		R	GTCGATATATTCTTGGCTGC
Gcha06	MN644917	F	AGGGGAATACAGATATGCAA	55	312	(GGA)4
		R	CGTTCTCATGATACTCCCAT
Gcha07	MN644918	F	AACTTCTCCCTCGAAACCT	55	250	(GGA)4-(GGA)5
		R	GTGTCACAGATGACGCAATA
Gcha08	MN644919	F	AGCTTCACAGATGACGAACT	55	285	(AATA)3
		R	AAGATGACAACGACGATGAT
Gcha09	MN644920	F	GCGGGTTGTAGAATTAACAG	55	265	(TGC)4
		R	GAGAATGTTGCACTGGATTT
Gcha10	MN644921	F	CTGTGGACATGAACAATCTG	55	244	(AAC)4
		R	ATGTGCAGCTTTCTCTCAAT
Gcha11	MN644922	F	GATTGAGGAATGAGATGGAA	55	214	(CAAG)3
		R	GAACTTGGCTTGAAAGATTG
Gcha12	MN644923	F	CAATCACATCACTGTTCAGC	55	166	(TA)6
		R	CTGCACCTGGAGAGTTTTAC
Gcha13	MN644924	F	ATGTTGAGGATGAGATCGAC	55	177	(AGC)4
		R	CGAAGAAAATCTGGAACAAG
Gcha14	MN644925	F	GAGTACTTCGCCTTGCTAGA	55	243	(GAA)4
		R	GTGCTGGTGTTTCTTCTTCT
Gcha15	MN644926	F	CTCTTCGTTCACACTTCTCC	55	159	(TCT)4
		R	AAGGAAATACCGAAGAGGAC
Gcha16	MN644927	F	GGTCTGTTCTTCTTCCTTCC	55	246	(TTC)4
		R	CTCCAAAATTGAAGATGAGG
Gcha17	MN644928	F	GGACCTAAACGCAATTTCTA	55	226	(TA)18
		R	CACCAAAGTTGGAGATGTTT
Gcha18	MN644929	F	GAACTACGGCAAGAACAAAG	55	305	(AAG)4
		R	AACACACATGAACGTTAGCA
Gcha19	MN644930	F	GTATTGGGCCGAGTACTGT	55	254	(CCG)5
		R	GGCATAAAAGGTAGCAAATG
Gcha20	MN644931	F	AATGTCTTTGTTCTGGTTGG	55	194	(AT)16
		R	AGACAGGTTTGCAATAGAGC
Gcha21	MN644932	F	AACTAGGGAATTGGAAGCTC	55	310	(TAGC)3
		R	TATGAAGTTGACACCCCTTT
Gcha22	MN644933	F	CGAAGAAAGGAGATCAACTG	55	210	(CGG)4
		R	GGGACGTTACAGCAATAGAG
Gcha23	MN644934	F	TCCGCCTATATTGTTGAGTT	55	244	(TTTA)3
		R	CTAGTGCTTCTTCCTCATGG
Gcha24	MN644935	F	GGTGAAGAAGGATGTATGGA	55	304	(AC)6
		R	TATTGCGTGATGTAGTTTGC
Gcha25	MN644936	F	GAGCCCATATGTCATTGTCT	55	328	(TTCT)3
		R	GATCTTCAATCTCTGCTTGC
Gcha26	MN644937	F	GAGGGCTGTAAGCAACTAAA	55	309	(TTC)4
		R	ACATGAAGAACAGGGATGAG
Gcha27	MN644938	F	AACGTATTTTCGACCGC	55	315	(TCA)4
		R	GTAGATGGATCGAAGATGGA
Gcha28	MN644939	F	TCTATTGTCCTTAGCCAGTCA	55	280	(GGC)4
		R	CTGGAAACATCAATGAGGAC
Gcha29	MN644940	F	GAGCACAAAAACCTCAAAAC	55	297	(ACTC)3
		R	AAGGAATGGATGAGATGCTA
Gcha30	MN644941	F	GGGACTACGAGGTAGGACTT	55	318	(CAAT)3
		R	GAGCTTGGGTTAAATGAGTG
Gcha31	MN644942	F	AGTCCAGTCGCACTCTTCTA	55	167	(CTTC)3
		R	TGTGTAATATAACCCGGAGG
Gcha32	MN644943	F	ATACCGCCTGGAGAAGTT	55	284	(GAC)5
		R	GACAATCATCCTTGGAGAAA
Gcha33	MN644944	F	GATCTCAGAAGATGGATTCG	55	208	(CAAC)3
		R	ATACATAATGAATGGGTGGC
Gcha34	MN644945	F	TTGAGTACAAGGGATGCTCT	55	283	(GT)7
		R	GGATCTAGGTCGAACATTCA
Gcha35	MN644946	F	AACTCAAGACCCTGGACC	55	187	(GAG)4
		R	GATTTCCAACTACGAAGTGC

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

Locus	Motif	Sequence (5′–3′)	ATa	Product size	Fluorescent dye
Multiplex A
Gcha04	(AGA)4	F: TTGTGCATGACTTGTTGAGT	58	235–238	NED
		R: TCACCTTACAATAATTCGCC
Gcha18	(AAG)4	F: GAACTACGGCAAGAACAAAG		–	PET
		R: AACACACATGAACGTTAGCA
Multiplex B
Gcha10	(AAC)4	F: CTGTGGACATGAACAATCTG	58	241–247	NED
		R: ATGTGCAGCTTTCTCTCAAT
Gcha21	(TAGC)3	F: AACTAGGGAATTGGAAGCTC		300–308	VIC
		R: TATGAAGTTGACACCCCTTT
Gcha33	(CAAC)3	F: GATCTCAGAAGATGGATTCG		–	PET
		R: ATACATAATGAATGGGTGGC
Multiplex C
Gcha02	(GCG)4	F: TAGCACGTACCATCAAACAA	58	225–249	FAM
		R: CCTACCGTAAGCTCTCTGAA
Gcha05	(AT)6-(AATT)3	F: TAGTCGGGTGATCATGAAAT		287–291	NED
		R: GTCGATATATTCTTGGCTGC
Multiplex D
Gcha01	(GCC)4	F: CCAGTTGATATGCAGAACCT	58	235–238	FAM
		R: TTCTCTCTATCGTCCTCAGC
Gcha07	(GGA)4-(GGA)5	F: AACTTCTCCCTCGAAACCT		249–252	NED
		R: GTGTCACAGATGACGCAATA
Multiplex E
Gcha11	(CAAG)3	F: GATTGAGGAATGAGATGGAA	55	202–214	FAM
		R: GAACTTGGCTTGAAAGATTG
Gcha12	(TA)6	F: CAATCACATCACTGTTCAGC		153–165	VIC
		R: CTGCACCTGGAGAGTTTTAC
Gcha13	(AGC)4	F: ATGTTGAGGATGAGATCGAC		174–180	NED
		R: CGAAGAAAATCTGGAACAAG

^aAnnealing temperature (°C)

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

Locus	GenBank access	Allele frequency (%)				PIC
		Allele A	Allele B	Allele C	Allele D
Gcha01	MN644912	0.50	0.50			0.500
Gcha02	MN644913	0.57	0.41	0.01	0.01	0.507
Gcha04	MN644915	0.98	0.02			0.039
Gcha05	MN644916	0.51	0.49			0.500
Gcha07	MN644918	0.50	0.50			0.500
Gcha10	MN644921	0.51	0.47	0.02		0.519
Gcha11	MN644922	0.50	0.43	0.07		0.560
Gcha12	MN644923	0.50	0.50			0.500
Gcha13	MN644924	0.85	0.15			0.255
Gcha21	MN644932	0.51	0.49			0.500

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

Species	SSR loci transferable	Species	SSR loci transferable
Bambusa oldhamii	Yesa	Merostachys glauca	Notb
Chusquea tenella	Yes	Merostachys scandens	Yes
Dendrocalamus latiflorus	Yes	Merostachys speciosa	Yes
Dendrocalamus yunnanensis	Yes	Oxytenanthera abyssinica	Yes
Farguesia gaolinensis	Yes	Phyllostachys aurea	Yes
Farguesia yunnanensis	Yes	Phyllostachys edulis	Not
Guadua angustifolia	Yes	Phyllostachys pubescens	Not
Guadua paniculata	Yes	Pseudosasa mirabilis	Yes
Guadua paraguaiyana	Yes	Shibatea kumasasa	Yes

^aYes—all tested loci were successfully transferred, ²Not—none SSR loci amplified in these bamboo species

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.

Electronic supplementary material

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Author contributions

MDR, MPG, RP and RON conceived the research. MDR and RON designed the experiments. MDR, TCT, GHFK and RFS conducted the lab and statistical analyses. MDR and RON wrote the manuscript. RP, RFS, GHFK, TCT, and LNV revised the draft of the manuscript. MPG coordinated and supported the research and revised the manuscript. All authors read and approved the final manuscript version.

Compliance with ethical standards

Conflict of interest

All authors hereby declare that there is no conflict of interest.

Ethical approval

This article does not include any studies with human participants or animals performed by any of the authors.

Informed consent

This article does not involve any informed consent.