Using Genomic Data to Develop Chloroplast DNA SSRs for the Neotropical Liana<i>Stizophyllum riparium</i>(Bignonieae, Bignoniaceae)

Keywords: Bignoniaceae, chloroplast microsatellites, cross-amplification, Neotropical flora, Stizophyllum inaequilaterum, Stizophyllum perforatum

Premise of the study: We developed chloroplast microsatellite markers (cpSSRs) to be used to study the patterns of genetic structure and genetic diversity of populations of Stizophyllum riparium (Bignonieae, Bignoniaceae). 
• Methods and Results: We used genomic data obtained through an Illumina HiSeq sequencing platform to develop a set of cpSSRs for S. riparium. A total of 36 primer pairs were developed, of which 28 displayed polymorphisms across 59 individuals from three populations. Two to 12 alleles were recorded, and the unbiased haploid diversity per locus ranged from 0.037 to 0.905. All 28 cpSSRs presented transferability to two closely related species, S. inaequilaterum and S. perforatum. • Conclusions: We report a set of 28 cpSSRs for S. riparium. All markers were shown to be variable in S. riparium, indicating that these markers will be valuable for population genetic studies across S. riparium and congeneric species. 

Applications in Plant Sciences

Stizophyllum Miers (Bignoniaceae) is a small genus of Bignonieae, the largest tribe in the Bignoniaceae (Lohmann and Taylor, 2014). The genus is clearly monophyletic (Lohmann, 2006) and includes three species, i.e., S. inaequilaterum Bureau & K. Schum., S. perforatum (Cham.) Miers, and S. riparium (Kunth) Sandwith (Lohmann and Taylor, 2014). All species of Stizophyllum are lianas with trumpet-shaped flowers that are pollinated by medium-sized bees (Gentry, 1974). Their winged seeds are dispersed by wind (Gentry, 1974). Members of Stizophyllum are easily recognized by their hollow stems, pellucidpunctate leaflets, urceolate calyces, and linear fruits (Lohmann and Taylor, 2014). The genus as a whole is broadly distributed, occurring from southern Mexico to the Atlantic Forest in southern Brazil (Lohmann and Taylor, 2014). Although the generic circumscription is clear, the three species of Stizophyllum currently recognized are morphologically similar and can occur sympatrically in Amazonia. 
Nuclear DNA polymorphisms based on microsatellites (simple sequence repeats [SSRs]) are powerful sources for population genetic studies (Kalia et al., 2011). These molecular markers, present also in the genomes of organelles (e.g., chloroplast), allow us to access genetic information that facilitates genotype identification, mainly due to their multiallelic nature (Masi et al., 2003). The chloroplast microsatellite and nuclear microsatellite markers (cpSSR and nSSR, respectively) have been widely used to study phylogenetic and genetic diversity in plants (Kalia et al., 2011). Unlike nSSRs, which are highly polymorphic, codominant, and biparentally inherited, cpSSRs are a nonrecombinant molecule and uniparentally inherited, allowing us to trace the history of populations through their haplotype diversity (Ebert and Peakall, 2009). Despite these differences, the two markers are complements for understanding the genetic structure in natural populations (Ebert and Peakall, 2009;Kalia et al., 2011). 
Furthermore, the advent of high-throughput sequencing technologies (Metzker, 2010) has allowed the development of SSR markers for multiple taxonomic groups (Zalapa et al., 2012;Francisco et al., 2016). Here, we used chloroplast genome sequence data obtained by de novo and reference-guided assembly to develop and characterize chloroplast microsatellite markers for S. riparium. Cross-amplification of the cpSSR markers developed for S. riparium was tested in all congeneric species to evaluate the utility of those markers for population genetic studies in Stizophyllum as a whole. 

METHODS AND RESULTS

We first obtained the chloroplast genome sequence of S. riparium using an Illumina platform. For that, we extracted the genomic DNA from fresh leaf material dried in silica gel from a single individual of S. riparium (voucher: Nogueira 170; Appendix 1) collected in Manaus (Amazonas State, Brazil) using the Invisorb Spin Plant Mini Kit (Invitek, Berlin, Germany) and following the manufacturer's instructions. Approximately 5 μg of total DNA was fragmented using a Covaris S-Series Focused-ultrasonicator (Covaris, Woburn, Massachusetts, USA) and a short-insert (300 bp) library was constructed with NEBNext DNA Library Prep Master Mix Set and NEBNext Multiplex oligos for Illumina (New England Bio-Labs, Ipswich, Massachusetts, USA), following the manufacturer's protocol. The library concentration was diluted to 10 mM and sequenced (single end) on an Ilumina HiSeq 2000 system (Illumina, San Diego, California, USA) at the University of São Paulo (Escola Superior de Agricultura Luiz de Queiroz da Universidade de São Paulo [ESALq]) in Piracicaba, Brazil. A Perl script was used to filter for quality using a Phred score of 20 or more for the cleaned reads, with the exclusion of reads with more than three uncalled bases, or shorter than 40 bp. We used a combination of reference-guided and de novo assembly to construct the chloroplast genome of S. riparium following Nazareno et al. (2015). The chloroplast genome for S. riparium was annotated using DOGMA (Dual Organellar The Imperfect Microsatellite Extractor (IMEx) interface (Mudunuri and Nagarajaram, 2007) was used to detect perfect microsatellites, with a threshold of 10 repeat units for mononucleotide and six repeats for di-, tri-, tetra-, penta-, and hexanucleotides. The annotated chloroplast genome was used to select markers located in noncoding regions exclusively. Primer pairs were designed from microsatellite sequence sites using Primer3web 4.0 (Rozen and Skaletsky, 1999), with the following parameters: primer size of 18-23 bp, temperature of 50-62°C with maximum difference between forward and reverse primers of 1°C, and GC content of 40-80%. 
In total, 36 primer pairs were designed. The PCR amplifications were performed in a final volume of 10 μL and contained 15 ng of genomic DNA, 0.5 μL (10 mM) of each primer with forward primers labeled with 6-FAM or JOE fluorescent dyes (Macrogen, Seoul, South Korea; Table 1), 0.6 μL (25 mM) MgCl 2 (Promega Corporation, Madison, Wisconsin, USA), and 5 μL 1× of KAPA2G Fast ReadyMix (Kapa Biosytems, Wilmington, Massachusetts, USA). The cycling conditions were as follows: an initial denaturation step of 3 min at 94°C; followed by 30 cycles of 30 s at 94°C for denaturation, 30 s at the specific annealing temperature for each primer pair (Table 1), and 72°C for 60 s; and a final extension of 5 min at 72°C. To test the utility of the individual primers, PCR products were detected using a 1.0% agarose gel electrophoresis with a 100-bp range DNA ladder (Promega Corporation). 
Of the 36 primer pairs tested, 28 successfully amplified (Table 1) and were checked for polymorphism in 59 individuals from three S. riparium populations (ranging from 18 to 22 individuals; Appendix 1). All samples tested were also extracted using fresh leaf material dried in silica gel or from herbarium specimens using the Invisorb Spin Plant Mini Kit, following the manufacturer's instructions. The amplicons with fluorescent labels were resolved to genotype on an ABI 3500 XL automated DNA sequencer with GeneScan 500 ROX Size Standard (Applied Biosystems, Foster City, California, USA). The microsatellite marker profiles were analyzed using GeneMarker (Holland and Parson, 2011). For each cpSSR, the number of alleles (A) and unbiased haploid diversity index (h) were obtained using GenAlEx 6.41 (Peakall and Smouse, 2006). In addition, we calculated a Successful amplification (+) evidenced by the occurrence of a distinct single band on the sequencing gel. chloroplast haplotype variation within populations by estimating the total number of haplotypes and the unbiased haplotype diversity as H e = [n/(n -1)](1 -∑pi 2 ), where n is the number of individuals analyzed in each population and p is the frequency of the i th haplotype in the respective population (Nei, 1978). Crossamplification of polymorphic cpSSRs was tested in six individuals of S. inaequilaterum and S. perforatum using the same PCR conditions described above. 
A partial chloroplast genome (128,239 bp) was obtained and used to develop a set of 28 polymorphic cpSSRs (Table 2). The number of alleles ranged from two to 12, and unbiased haplotype diversity varied from 0.037 (Stiz40) to 0.905 (Stiz45) (Table 2). When alleles at each of the 28 loci were jointly analyzed, we observed 18, 19, and 22 haplotypes for EAM (i.e., east of Amazonia), CAM (i.e., Central America), and SAM (i.e., south of Amazonia) populations, respectively. Furthermore, the maximum unbiased haplotype diversity (H e = 1.00) was observed for all S. riparium populations. All polymorphic cpSSR markers successfully amplified for S. inaequilaterum and S. perforatum (Table 3). 

CONCLUSIONS

We developed and characterized 28 chloroplast microsatellite markers for S. riparium. Due to the high rate of crossamplification (100%), these chloroplast microsatellite markers will be useful for genetic studies involving Stizophyllum as a whole. 

LITERATURE CITED


taBle 1 .

 1 Characteristics of 28 intergenic chloroplast microsatellite markers developed for Stizophyllum riparium. 


								GenBank
	Locus	Primer sequences (5′-3′)	Repeat motif	Allele size range (bp) T a (°C) Fluorescent dye a	Position	accession no.
	Stiz2 Stiz3 Stiz4 Stiz5 Stiz6 Stiz7 Stiz10 Stiz11 Stiz12 Stiz13 Stiz14 Stiz15 Stiz18 Stiz20 Stiz21 Stiz22 Stiz23 Stiz24 Stiz25 Stiz26 Stiz27 Stiz28 Stiz33 Stiz34 Stiz36 Stiz39 Stiz40 Stiz45	F: CCTTGTTGGTTAGTTGAGTTCC R: GGGGTAGCGACTTGTATATAACT F: AGGAAGTTTTCTCCTCGTAC R: F: ATCTAATGATCCGGGGCG R: TTCCCTTTCCGTTGATGACT F: CCCTTCCCGAACCAAACATG R: TTGTCAGAAGAGTCCTCCAAGT F: GCCACACATACATTGCTTTGC R: TCCCTGTCATGTTCCTTGGA F: TCCTAGTATGCTGGCCAACA R: AGAAGAGAAGGTTCCATCGGA F: CCAACCTAAAATTCTTCCGA R: CTTATAGAAAGTGCGGTGC F: CAGATTGCCTTTCCACTTCGA R: TCGAAGAAATCCCCAACCCT F: GGATTTCTTCGATGGGCCCT R: GCCAGTCCCGACAGATCC F: CCTGTTCTTTCCATGACCCC R: AAAGAAAGGGGAATGGCTCG F: GCATAGCTAGCAATCCATTCT R: GGAACAATTGGAATAGAATGCC F: CATTCCTCGGCTTTCATTCGT R: TCTTCCTGCCATTTCTCCCA F: CATTCGTTCTTCAGCCTCA R: TTGAGTTGCATGGATTTGGA F: TTCTAGGAGGCTTGTCTTCC R: TGTCCACTACTTTACTGTACGT F: TACTGCGTCAATTGCCAATT R: TCACCACGCCTGTATTGTAA F: ACCCACCTATACAGTAACGGT R: TCTCCATTGGTAGCAAATGGT F: GATTCTCTATCTGCTGTGTG R: TCTTGAATGGAAAGTAAGGG F: AGTCGAGTATCTGAAACACGA R: AGAACCATTGGATCATCACGT F: TGGAGGAGAAACGATATTAG R: CTTCTCGAGGTATAATGACA F: TCTAGCCATATCAGCATTTCGT R: GTGTCTCACGGCATATACCT F: TCTTCTCATCCAGCTCCTCG R: CGAATAAGCGCTACGACTGA F: GTCCTTTATCCAAGTTTACC R: TCTTACTGATTCACTAGTCG F: TGGATCTCTCAGTCTAAGCAGG R: CCACAAGCCGAGGAGATCTT F: GACACGCAGGATAATTCATA R: TCCCAGAATGAATACAGAAC F: ACATCCCTATTTCCTCCATTG R: TCTTAGCGAGTAGTTCCGA F: CTCTAACCTCTGAGCTAAGC R: CGAAATTCTATATCGCTGCA F: GATCCAAGAAATTACAGGAC R: TGAGCTATCCTGACCATT F: GAAATCCCATATGACCCA R: CCCCAAAGAAAGAGAAAG	(A) 6 (G) 10 (T) 10 (A) 10 (A) 11 (A) 12 (A) 10 (T) 10 (A) 10 (A) 11 (T) 10 (A) 13 (A) 10 (T) 10 (T) 12 (T) 10 (T) 10 (A) 10 (A) 11 (T) 11 (T) 10 (T) 10 (A) 10 (T) 13 (T) 10 (TA) 6 (ATA) 4 (TAA) 4 (TCTCTT) 7	211-216 170-176 160-169 176-181 143-150 164-171 165-173 190-199 167-180 201-210 136-149 90-96 179-184 183-191 186-197 161-169 153-162 179-183 168-175 168-170 243-248 168-169 230-238 182-206 168-170 175-176 130-132 156-171	49.9 54.0 55.8 57.4 57.4 57.4 57.4 54.0 56.9 54.0 54.0 51.7 54.7 56.9 48.6 56.9 56.9 61.4 61.4 61.4 53.2 55.8 49.9 56.9 56.9 58.0 53.2 53.2	JOE JOE 6-FAM JOE JOE JOE 6-FAM JOE 6-FAM JOE JOE 6-FAM JOE 6-FAM 6-FAM JOE 6-FAM 6-FAM JOE 6-FAM JOE JOE 6-FAM JOE 6-FAM JOE JOE JOE	trnK-UUU/rps16 rps16/rps16 psbI/trns-GCU atpF/atpF atpH/atpI rps2/rpsC2 petN/psbM psbM/trnD-GUC psbM/trnD-GUC psbC/trnS-UGA psaA/ycf3 trnT-UGU/trnL-UAA ycf4/cemA petA/psbJ petL/petG rpl20/rpl1s ClpP/psi_psbT psbH/petB infA/rps8 rps8/rpl14 rpl16/rps3 rpl16/rps3 ycf1/ycf1 ycf1/ycf1 trnP-UGG/psaJ trnT-UGU/trnL-UAA trnL-UAA/trnF-GAA ClpP/psi_psbT	KP863512 KP863513 KP863514 KP863515 KP863516 KP863517 KP863519 KP863520 KP863521 KP863522 KP863523 KP863525 KP863527 KP863528 KP863529 KP863531 KP863532 KP863534 KP863535 KP863536 KP863537 KP863538 KP863539 KP863540 KP863530 KP863524 KP863526 KP863533
	Note: T a = annealing temperature.						
	a Fluorescent label used for the forward primer sequence.					

taBle 2 .

 2 Characteristics of 28 polymorphic chloroplast microsatellite markers in three populations of Stizophyllum riparium. a 


										taBle 3. Transferability of 28 chloroplast microsatellite markers
										developed for Stizophyllum riparium across two related Stizophyllum
										species. a			
		CAM (n = 19) SAM (n = 22)	EAM (n = 18)	All (n = 59)				
	Locus	A	h	A	h	A	h	A	h	Locus	Repeat motif	S. perforatum	S. inaequilaterum
	Stiz2	3	0.368	4	0.614	4	0.654	6	0.660	Stiz2	(A) 6 (G) 10	+	+
	Stiz3	4	0.551	2	0.524	4	0.575	6	0.671	Stiz3	(T) 10	+	+
	Stiz4	5	0.719	3	0.626	3	0.451	9	0.773	Stiz4	(A) 10	+	+
	Stiz5	3	0.550	2	0.095	2	0.125	4	0.533	Stiz5	(A) 11	+	+
	Stiz6	3	0.433	4	0.726	2	0.209	6	0.668	Stiz6	(A) 12	+	+
	Stiz7	4	0.618	3	0.511	2	0.529	6	0.719	Stiz7	(A) 10	+	+
	Stiz10	5	0.693	2	0.505	2	0.118	6	0.633	Stiz10	(T) 10	+	+
	Stiz11	2	0.529	3	0.426	4	0.802	5	0.653	Stiz11	(A) 10	+	+
	Stiz12	5	0.683	4	0.671	4	0.712	6	0.760	Stiz12	(A) 11	+	+
	Stiz13	4	0.419	6	0.766	3	0.385	7	0.584	Stiz13	(T) 10	+	+
	Stiz14	7	0.850	8	0.884	4	0.600	10	0.893	Stiz14	(A) 13	+	+
	Stiz15	4	0.575	2	0.467	4	0.582	6	0.627	Stiz15	(A) 10	+	+
	Stiz18	3	0.608	4	0.633	3	0.228	6	0.588	Stiz18	(T) 10	+	+
	Stiz20	3	0.342	8	0.833	2	0.294	9	0.780	Stiz20	(T) 12	+	+
	Stiz21	7	0.596	5	0.745	3	0.601	8	0.717	Stiz21	(T) 10	+	+
	Stiz22	1	0.000	2	0.416	4	0.467	5	0.703	Stiz22	(T) 10	+	+
	Stiz23	4	0.642	4	0.676	3	0.582	6	0.698	Stiz23	(A) 10	+	+
	Stiz24	2	0.441	3	0.706	2	0.111	4	0.665	Stiz24	(A) 11	+	+
	Stiz25	2	0.525	3	0.552	2	0.523	4	0.732	Stiz25	(T) 11	+	+
	Stiz26	3	0.632	2	0.395	1	0.000	3	0.420	Stiz26	(T) 10	+	+
	Stiz27	4	0.608	3	0.386	3	0.600	4	0.625	Stiz28	(T) 10	+	+
	Stiz28	2	0.125	2	0.233	1	0.000	2	0.458	Stiz27	(A) 10	+	+
	Stiz33	4	0.419	5	0.737	2	0.309	9	0.796	Stiz33	(T) 13	+	+
	Stiz34	2	0.485	3	0.338	2	0.476	5	0.725	Stiz34	(T) 10	+	+
	Stiz36	2	0.264	3	0.451	2	0.111	3	0.516	Stiz36	(TA) 6	+	+
	Stiz39	3	0.542	2	0.521	1	0.000	3	0.493	Stiz39	(ATA) 4	+	+
	Stiz40	1	0.000	2	0.100	1	0.000	2	0.037	Stiz40	(TAA) 4	+	+
	Stiz45	5	0.788	9	0.900	3	0.621	12	0.905	Stiz45	(TCTCTT) 7	+	+
	Mean	3.4	0.500	3.6	0.551	2.6	0.381	5.7	0.644				
	Note: A = number of alleles; h = unbiased haploid diversity.					
	a Voucher and locality information are provided in Appendix 1.					



  eBert, d., aNd r. Peakall. 2009. Chloroplast simple sequence repeats (cpSSRs): Technical resources and recommendations for expanding cpSSR discovery and applications to a wide array of plant species. Molecular Ecology Resources 9: 673-690. FraNcisco, J. N. c., a. G. NazareNo, aNd l. G. lohmaNN. 2016. A genomic approach for isolating chloroplast microsatellite markers for Pachyptera kerere (Bignoniaceae). Applications in Plant Sciences 4: 1600055. 



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The authors thank Comissão de Aperfeiçoamento de Pessoal do Nível Superior (CAPES) for a scholarship to M.B., Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for a Pq-1C grant to L.G.L. (307781/2013-5), and Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) for a scholarship to A.G.N. (2013/12633-8) and a regular research grant (2011/508559-2) and a collaborative FAPESP-NSF-NASA Dimensions of Biodiversity Grant (2012/50260-6) to L.G.L. 


Species

Population Note: CAM = Central America; CAZ = Central Amazonia; EAM = east of Amazonia; SAM = south of Amazonia. a Most specimens are deposited at the Herbarium of the University of São Paulo (SPF), São Paulo, Brazil, except for 20 samples ( 1 ) that are deposited at the Missouri Botanical Garden Herbarium (MO), St. Louis, Missouri, USA; one sample ( 2 ) that is deposited at the Herbarium of the Muséum National d'Histoire Naturelle (P), Paris, France; one sample ( 3 ) that is deposited at the New York Botanical Garden Herbarium (NY), Bronx, New York, USA; and one sample ( 4 ) that is deposited at the Herbarium of the Federal University of Ceará (EAC), Fortaleza, Ceará, Brazil. 
* Sample used for DNA extraction. 
aPPeNdix 1. Continued.
Maila Beyer, Alison G Nazareno, Lúcia G Lohmann
Using Genomic Data to Develop Chloroplast DNA SSRs for the Neotropical Liana<i>Stizophyllum riparium</i>(Bignonieae, Bignoniaceae)