Development and characterization of microsatellite markers for Central American Begonia sect. Gireoudia (Begoniaceae).

PREMISE OF THE STUDY: Transcriptome sequence data were used to design microsatellite primers for two widespread Central American Begonia species, B. heracleifolia and B. nelumbiifolia, to investigate population structure and hybridization. • METHODS AND
RESULTS: The transcriptome from vegetative meristem tissue from the related B. plebeja was mined for microsatellite loci, and 31 primer pairs amplified in the target species. Fifteen primer pairs were combined in two multiplex PCR reactions, which amplified an average of four alleles per locus. •
CONCLUSIONS: The markers developed will be a valuable genetic resource for medium-throughput genotyping of Central American species of Begonia sect. Gireoudia. A subset of these markers have perfect sequence matches to Asian B. venusta, and are promising for studies in other Begonia sections.

Begonia L. is a diverse tropical genus with over 1500 species. Evolutionary research has focused on the early-diverging African species (e.g., Hughes and Hollingsworth, 2008) and the more derived Asian species (e.g., Thomas et al., 2011), with the American species largely overlooked. The most recent common ancestor of Central American Begonia is likely to be relatively recent (Miocene; Dewitte et al., 2011), and subsequent speciation has resulted in high species richness (total c. 690 species; Goodall-Copestake et al., 2010). Population studies of Central American Begonia species will shed light on the evolution of species richness in a morphologically diverse group of neotropical herbs; but to date, studies have been limited by the availability of suitable nuclear markers to complement plastid microsatellite markers (Twyford et al., 2013).

In this study, we describe the development of nuclear microsatellite markers to study gene flow within and between Central American Begonia species. This requires markers that amplify over a broad phylogenetic scope, which can then be cross-amplified in divergent species.

METHODS AND RESULTS

Microsatellite markers were designed from the transcriptome sequence of vegetative meristem tissue from B. plebeja Liebm., a related species from Begonia sect. Gireoudia (European Nucleotide Archive Sequence Read Archive accession number: ERP001195; Brennan et al., 2012). The QDD bioinformatic pipeline (Meglécz et al., 2010), which integrates microsatellite detection, a redundancy check to avoid amplifying multiple PCR products, and designs primers, was used according to Lepais and Bacles (2011). A FASTA file of the B. plebeja transcriptome sequence assembly was analyzed in QDD version 1.3 using default parameters: selecting only primers that amplify a PCR product between 90 and 320 bp in length, with a repeat motif of 2–6 bp repeats, and a minimum length of four repeat units. To make microsatellite amplification in other species more likely, primers were excluded if they did not have a perfect BLAST match to the transcriptome of B. conchifolia A. Dietr. (sect. Gireoudia; Brennan et al., 2012). Reads from which the primers were designed were BLAST searched against the Arabidopsis Information Resource (TAIR) database (http://www.arabidopsis.org) to investigate the putative function of each locus.

Thirty-one primer pairs detected in QDD were tested for amplification in B. heracleifolia Cham. & Schltdl. and B. nelumbiifolia Cham. & Schltdl. These species were chosen because they are two of the most widespread Begonia species in a genus of mostly rare endemics (Hughes and Hollingsworth, 2008). The species are known to hybridize (Burt-Utley, 1985), facilitating studies of species boundaries. Primer amplification was tested in seven individuals of the two species (Appendix 1). A subset of polymorphic markers that amplified reliably in both species was then tested for multiplex compatibility by mixing equimolar ratios of each primer. The PCR multiplexes were then tested on a population of each species (20 individuals) to estimate the genetic diversity of the markers. The primer sequences were BLAST searched against the transcriptome sequence of the divergent Asian species B. venusta King (sect. Platycentrum) to test for likely cross-amplification of primers in other Begonia species.

Approximately 15 mg of silica-dried leaf material was extracted using DNeasy 96-sample kit (QIAGEN, Germantown, Maryland, USA). To overcome an unknown PCR inhibitor that coelutes with DNA extractions in Begonia, extractions were diluted 100-fold with Millipore dH2O to a final DNA concentration of ∼0.1–1.5 μg/mL. PCR reactions were performed using the M13-tailed primer method (Schuelke, 2000) in a final reaction volume of 10 μL containing: 0.5 μL of 1 mM M13-tailed forward primer (Invitrogen, Grand Island, New York, USA), 1 μL reverse primer (1 mM), 1 μL of 1 mM M13 fluorescently modified primer (6-FAM,VIC, NED, PET), 0.25 μL bovine serum albumin (BSA, 0.4%), 1 μL of 10× reaction buffer, 1 μL of 2 mM dNTPs, 0.6 μL of 25 mM MgCl2, 0.05 μL BIOTAQ polymerase (Bioline, London, United Kingdom), 1 μL dilute DNA template, and made up to the final volume using dH2O. PCR cycles consisted of an initial denaturation of 1 min at 95°C, followed by 40 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 57°C, and extension for 1 min at 72°C. Five microliters of each PCR product labeled with the four fluorescent dye colors was pooled and diluted 2× in Millipore dH2O, and the GeneScan 500 LIZ internal size standard (Applied Biosystems, Foster City, California, USA) was added prior to fragment analysis on the ABI 3730xl analyzer (Applied Biosystems; analysis was performed at GenePool, University of Edinburgh, Edinburgh, United Kingdom). Fluorescent traces were analyzed automatically with manual editing using GeneMapper version 4.0 (Applied Biosystems).

A total of 136 primer pairs were located in the B. plebeja transcriptome using the QDD bioinformatic pipeline (Appendix 2). All 31 of the subset of primers tested for amplification yielded a PCR product (Table 1). Sixteen loci had a significant (

Appendix 2.

Microsatellite loci in the transcriptome of Begonia plebeja.

Locus	Forward primer sequence (5′–3′)	Reverse primer sequence (5′–3′)	Repeat motif
BC134*	ATCAGCTCACTCCCTATCCTCT	TGCAATCTCCTTCGGTTCTT	(CT)6
BC192	AAGTCAAACCTGTTGACCCG	ATCCTCATCGGATTCGTCAT	(GAT)9
BC232	TGGAAATGCTGTCGTTGAAT	ATTGGAGAAAAGGCAAAGCA	(TCT)8
BC312*	ATTTCCTTCTGCGAACGATG	ATCGGAACTCTGAGCCTGAA	(GA)5
BC332*	GAACCAGAAGTCAAGGGTTCA	AAACATGATTTTCCTCATCCAA	(TCA)5
BC344*	GAGGGAGGGTCCCTTGTTAG	CCGTCTTACGTTGCATCATC	(GCA)5
BC362*	CTTCACCTCGCCTGAACAAC	GAGGCGAAATATTATGCGGA	(ATG)6
BC42*	GAAGGGGTTTCTTGGTCTCA	TTGTCAATTCTCACCAGACACA	(TGG)6
BC402*	TTACTCGAGCTAGAAGCCGC	AGGGCTTGGAGAGCTAGAGG	(AT)5
BC432*	AAACTCCGATGGATTCAGCA	TTGAAATAAACACACAAACAAAGACA	(TG)5
BC532	TCATTCCGCTTCTATGCTCC	CGTCATCGTCAATATCATCCTC	(TGA)6
BC552*	TGTCTGAGATGGAAACTGCG	TAGTCGAAGGGATCCGAATG	(TG)5
BC602	GCAAAGCAGGTAACTTTTAGCC	ACTCACCGAACTTTGGCAAC	(CAG)5
BC632	CATAGCGCTCAGCTTGCTC	GAGATCTTATACGAGCTACTGGATAGT	(TC)9
BC643	GGAGGAGCTCGGTCATTAGA	AACCACCGGTACCCTCATTT	(CT)6
BC652	TTTCGTCCATGAAGAAAGGC	TCCAGGGAACTCCATCACTC	(GAA)5
BC672*	CCTTGATCGAGAAAGAACCG	AAAGCCAGCTCCTTCCTGTA	(GAA)8
BC692	AACATGGCCGTCACTAGTCC	CAGGCAGACAAAGAAGATTCC	(AG)11
BC752	GGCAGATTTTACTGGGACGA	CGCCCATCTATCTGTATCCAA	(TTC)5
BC762	CAACTCTGCAAATGCAAGGA	ACCCATGACAGCATGAACAA	(CT)5
BC932*	GTAGTCCATCAGTCCGCCAT	GAGTGATGAAGGCGAAGAGG	(GA)5
BI0537	CAGATCAACCCTCTTCCTGC	ATCGAAAACCCATTGACTGC	(CCT)6
BI1195	TGCTGCAGAAACTTTAGCCA	CGGTGATTAAAGAAGAGCAAGAA	(GA)11
BI1430	CACAATTCGTGAAAACACGG	TTCTGCATGATGTTGGCTTT	(GA)5
BI1733	GTTCACCACTCCAATGGCTT	CGAGTTTGCCTTCGAATCTC	(GCCACA)5
BI1816	GTTTTGCGGTTGAGTTTGGT	CAAATGAATCTTCTTCATCCAGTG	(GAT)7
BI1937	TCATCATCGCAGCAGAAGAC	CGAAGCTGGGAGTGAGTTTC	(GGA)6
BI1948	CAAAACTGGCTTTGCAGACA	CACGGGCACTTTCAATTTCT	(TA)5
BI2413	GAATGAAGAGCGAATCGACG	CAGAGCTCCGGAATCTCATC	(AGA)5
BI2675	TTCCATTTACTCTCAGCCGC	CGTTCTCCTTCGAGGACTTG	(GA)7
BI2875	CCCAATCTCCCTGTCTATCG	AAGCTGACGAAGCTCTTCCA	(TC)5
BI2935	TGGAAGAAGGTCTCCATATAAGTCA	CATGTTTTCTTCGCCCATTC	(CAC)9
BI2946	ATTTGAAGCCATTGGGTCTG	AAGACGGGAAAGGGTGAGAG	(TC)6
BI2961	TCGCAAAAGAAGAAATCACAAA	TCCTCCGGCACAATAATCTC	(GAA)6
BI2967	GGTGGCTTGTACGGTGAGAT	TCGATTCTCAAATGCCTTCA	(GAA)5
BI2994	GATTTCCGTGGAGGAAACAA	AAACATCACCAGAGCACAACA	(CT)5
BI3043*	CGACATCCAACCAAACCTG	TTGATAGATGGAAGGGTCGC	(TC)5
BI3069*	AACCACAGTAATCATCCGGC	TGTCCGGTAACTGTGGTGAA	(CA)5
BI3131	ACATTGTGTTCAATGGCGAA	GAGCTCATGCAATGCTTCAA	(GAA)6
BI3233	TATGAAGGACGTGGGAGGAG	GGGAATCAGAAGCCAATCAA	(GA)5
BI3234	AAACAAGGAACGCTCAATCC	GCTCGAGTTGGCTTCATTTC	(AG)5
BI3286	CCTATGATGATAGCGTCCGA	AGGCCGACATTCTTTTCCTT	(CT)10
BI3301	GCATGGAGATTGCCAGATTT	CTATTGCTCAGCGGAGAAGG	(GAA)5
BI3348*	ACTTGTTTCTCGTTGGGAGC	CTGCAGCCCAGTGGATTTAC	(CT)6
BI3377*	AACACAATCATCAGCCGGAC	ACGAAGGAGATGATTATGACGAA	(AGG)5
BI3384	ATAATTGGGCTAGGGTTCGG	GCTTTTGGTTGCTTCAGAGG	(TC)5
BI3403	TGTAGGAACAACGGTTAGCG	CGTAGAGACGATTTCCTTAGCC	(GAA)6
BI3519	TTCAGAGCGCTTTTGGTTTT	ACGCACTATGCCGTTCTTCT	(TA)6
BI3553	TCTGAAATAGCACCGCTTCC	TTTCTTCGATGAACGCACTG	(AAG)5
BI3600	CATTATTTCCTGTCGGGACG	TGCTGAAAAGTTGCAGGAAA	(TGTT)5
BI3727	CCTCCACCAGATTTGCTTAAA	AACAGAAACATTTGCCGGTG	(TC)12
BI3741	GCAACACAGCTCCTCTTCGT	GGTCGGAATCGTCGAGTAAA	(CT)7
BI3820*	AGGACCAGTTTTGACGGCTA	GAAGCTTTTGCTCTTCTGTTGA	(CTT)7
BI3865	ACCTCACTCAACCGCCATAG	TTCAGCATCTGTTGCAGGAC	(CT)5
BI3970	TGTGTTCACTCAATTCTGCCA	TCCTTCACCTGAGACGACAA	(TC)5
BI4004*	TCAGGAAATATTCGATTGGGA	GCATTCCTCTGTGTACAATGC	(AT)5
BI4013	AAGCCAAGATACCCCAAAGG	CCGCTTGTCCTTTCTTCTTG	(AG)5
BI4021	TGTGTTGCCCTGCAAGTAGA	GGAAACCTTTCAGAGCTCCA	(AG)5
BI4028	GTCTTCTCCCCATCGTTGAA	GGGCTTTGGAAACATCTCCT	(CT)15
BI4031	TCTTCGCTCTAAAGGCTTGC	AAATTTCGCCAAACATGGAG	(TC)5
BI4088	GGTTTCGAGATATGGCCTCA	TTGGCAATTTATCCCTCCTC	(GGC)5
BI4128	AAGACAACGCCATTCCAAAC	AGGGACGACCGGAAGTAGAG	(CT)5
BI4166	CGGGACAAATGTTAAGCGAT	CAATAAAGAACTTCCGGCGA	(TG)5
BI4175	GGCGATCAAAGGGTGATTTA	CGATTAGCCTCTTCTCGACG	(AG)5
BI4233	ATGCAGACGTAATCGAAGGC	CAAGTTGGTTGGCAAAGACA	(AG)12
BI4279	GGGAGGAAGAGGAAGAAGCA	TCAGATTCAGCGTCATCAGAA	(AGG)6
BI4329*	CAACCAACAATGGCAGCTT	TCATGGAGATAATGGAGCTGG	(GGA)6
BI4360	CCGCAGATCCTCCATTAGAA	TTATGTCCCAAACTCCGCTC	(TGT)5
BI4477*	GGATCTCCTCTGCTTTGCTG	GGCGAGACCAGAAGAAAAGTT	(CT)9
BI4594	CCAGAATCGTGGTCACTTCC	CGTGAATCGAAACTTCTCCC	(TC)9
BI4600	GCTATGGGAAGTTGCTTGGA	AGCTCTTCCTCCCTTTCTGG	(AGA)7
BI4641	GCCACAGTTTTAGCTGTGCTAT	CTGCAACCACGAGGAGTTTA	(CT)5
BI4721	ACTACCCTCCCAAGGCTGTT	GGCCAGAAGTCAAACCTCAA	(TC)8
BI4740	AGGCACCCTCCCAAAGTAAT	GCCTGTATCTGAAATTGGCA	(GA)7
BI4746	GTCGGAGTCAGCGAGGGA	TGATCCTATGCACTCGTGGT	(AG)5
BI4779	CGAAGGAGGAAGAGACGATG	TGGCACTATAATTCCAAGCTCC	(AACG)5
BI4793	CAGTCCCCGCACTAATCTTC	GAAAGACCAGCTTCGTTTGC	(GA)5
BI4804	TCGCTGATGATTTGTTTGGA	AGAATGCCGACGAAATTGAG	(TCT)10
BI4848*	CGACGCCTCTCAAAGAAGAA	GAGCTTTGAATTTCGCTACG	(AG)6
BI4899	CCCATTTGCTTCCAAAACAT	GAGTCGAGGAGCAGCACTCT	(GAA)5
BI4987	AGTGAAAACCTTGGCACCAC	ACCCTTTTCCTATTCCACGG	(GAG)5
BI5091	TGCTTTCCAGGTTCATAGGG	GGCAAGCTTGGAACTTTTGT	(AGA)7
BI5107	CGCGTTTTACATGGCTGAAT	CGATTGAAAACCTTGAAGATGA	(AT)5
BI5115	AGACCGATGACCGAACAATC	TCCGTCGTTTCTAACCGTTC	(TC)5
BI5162	CTCTGAAACTCGCTCATCCC	GCTCTTTCCGTCTCATTTGC	(AGG)5
BI5174*	GTCGCAGGGTTTGTCTAGGA	GGAAATCAGAGTGCTGGCTC	(CTT)5
BI5285	GGTCAAATGGGTAACATGCC	CTGGTTCATCATCGCTGCTA	(GGT)5
BI5317	GCCCTCAAGTTCCTCCATCT	GGGACCGTCGATTATTCTCA	(AT)5
BI5325	TTCCGGACTGAAAGAAATGG	CGTGAGTGGAGTGGTGATTG	(TC)5
BI5347*	TCAGTCCATTTTCTTAATCAGACC	CTCTATCATTTCCAAGCGATTTC	(CTT)6
BI5377	ATCCTCTTCCTATCCACCGC	GGGAGACGGTGAAACTCTGA	(TC)5
BI5414	GCAAAGCAAAGCTGAAAACC	GGCCCAGTCTACCTGCAATA	(AT)5
BI5423	GCTTCCAATGATGCAAACCT	GAGAAGCGCAGGAGAGCTTA	(AG)5
BI5561	GTTGACTCGTCCTCGTCTCC	GTCGTTTCTGCCGATTCTTC	(CTT)5
BI5588	CAGCTGGTTGAGAAACGTGA	AATCATATCGCCGATCAAGG	(TC)5
BI5593	ACTCCAAATTAGGTGCGTGG	AGATAACGAAGCAAAGCGGA	(AG)9
BI5638	GCTTCTTCGTCCTCTTCTTCC	TTACGGCTCCAGATTCTGCT	(TCT)7
BI5668	TATGGGTCCGGATATGGAAA	AGGAAGAGCTCGAAGAAGCC	(GCG)5
BI5710*	GAAAGTTTTGGAGGAAGCCC	TGGAAGAGATCAGAAGGTACA	(GAA)7
BI5800	CGCCTCCCATATCTCGTAAA	GGAAGGTGATGGTTGTTGCT	(TCT)5
BI5813	CGGTAGATTGAATGGGGAGA	AGCATCGCCTCAAGTTGTCT	(AG)5
BI6067	CAGCTTGGAAAATCAGACCC	AGGGGCGTAAGCATAAAGGT	(TA)5
BI6141	GTCGCCATGACGATAAGGTT	TCTGACCCTGAAGATGGACC	(AG)10
BI6227	GACGCGACGAAGATAAGGTA	ATACATCGGAGGGAAGCAAA	(TCT)5
BI6278*	TGTAGTTGTTGTAGTAGCAGAACTTTG	CAGATGGGTCGGAGATTTTG	(TCC)7
BI6294*	TGCTGGTCTGAATCTTTAATCA	TGGGGTCTTGGTACTCTTTCC	(AT)10
BI6299	CATCGCTCTATGAAGCTGCTACT	CCTGAGACCCTGCTATTCCA	(AT)5
BI6399	CTGTCATCATCCCCATCACA	CAGTGAGAAATGCAGGGTCA	(TC)5
BI6422	TTTGATGGAGAAGATTAGTGAGAAGA	AGGCGGAATACCTTGTCCTT	(TTC)5
BI6423	ATATTGGACATGCCAGCACA	CATGAAACAAGAACTCTGGAGAA	(AG)5
BI6469	TCTAGGCGCCAAAAGAAAGA	CTCCCTCATCACTTGCGAAT	(GA)13
BI6534*	CGTTGCTCTGCTCTAACCCT	AGATACAGCCAACCGGATTC	(TC)6
BI6535	AAAGGGGAAAGCAAGGAAAA	GGGATGGATGGCTGATTAAA	(GAA)7
BI6561	CTTCTGAGACTCGTACCGGC	TAGCTCGGTTCAAAACACCC	(GTG)5
BI6581	TTGCTTTTCCTTTCTCATCCA	CCGATTCCAGCTCTATCAGC	(TTC)6
BI6604*	ATTTTTCCACAGAAGAGCCC	GGCAGAACCCGCAGTATATC	(TA)8
BI6605	TCAAAGCTTCGTTCCCATTC	GGAAAGCGTCAGAGTTGAGG	(TTC)5
BI6701*	AGAATCCCCACTCACTGCAC	GAGATGATGAGGGTTCAGGC	(GA)6
BI6717	GATCTCGGGGATTTGGATTT	ACTGCCATAGCCTCCATCAC	(GTG)5
BI6761	TGTTCTTCCGCTCTCCACTT	ACATGCTCTTCCTGGCTTGT	(TC)5
BI6776	CCAAACAGCAAAACTCTTCG	GTTTTGTGGAAGGGTGGCTA	(AG)5
BI6828	TCGTCTCCTTCTTCGTCTCC	GGTCGTCGCTCTGATTCTTC	(CTC)5
BI6849	CCTCAGATCCAGAGGAAGGG	GCGCCTTTTCCTTTAAGTCC	(TA)6
BI6886	TCTTCTCACGGCTCTCCATT	TGGAAATCAAGGAAAGCACC	(CTT)5
BI6901	CGAACTGGAAGAAGACTACAATCA	GCTGCAGCACGGAGTTTTAG	(AG)8
BI6984*	GTATGCAAAGGAGAGCCGAG	TTGTCAATTCTCACCAGACACA	(TC)6
BI7015	TGGTCCAGATTATGATCAGCC	TCTTCTCCGATTCCGATCAC	(GAA)5
BI7023	TTAAGGCGGTGACACAGAGA	CCTTTCGTCTGCAAATGGAT	(GAA)5
BI7036	TTGAGCAGGCTTCCAAACTT	ATTCGAAGGAAGAAGACGGC	(CTT)5
BI7059	CTCCCTCCGACCTCCATAAC	TAGCCTTCTGCGGAGTGTTT	(CT)5
BI7085	ACTCGCGAATATCTCCGAAA	CACCTCTTCAGCTCGTCTCC	(GA)5
BI7112*	ATCCAATGTCAACCTCTCGG	GTGCATTAGAGTCCCGTGGT	(TTC)6
BI7149	CGGAGAATCGAACCTCTGAT	CCCTGAACGATGGAACTCAT	(CT)5
BI7165	AATGAGCACGAACCTGCTTT	GAGGAATTTGGACCGTCTGA	(AG)5
BI7247*	CTCTTATTCCGCGTCAAAGC	AGCGGAGAAGTCGAAAACAG	(AG)6
BI7287	TTGGGGACAACAAATGATGA	CAGTGCTTTCTTTAACAACGCTT	(TGA)5

Indicates markers tested for amplification and polymorphism.

Table 1.

Characterization of nuclear microsatellites for Central American Begonia species.

						A
Locus	Primer sequences (5′–3′)a	Multiplexb	Fluorescent dye	Tm (°C)	Repeat motifc	her	nel	Allele sizes (bp)d	Putative functione	E-value
Multiplexed loci
BI4329	F: M13-CAACCAACAATGGCAGCTT	1	FAM	59	(GGA)6	4	2	89–104	immunoglobulin E-set superfamily protein	2E-13
	R: CATGGAGATAATGGAGCTGG
BI3043	F: M13-CGACATCCAACCAAACCTG	1	FAM	60	(TC)5	1	2	173–179	—	—
	R: TTGATAGATGGAAGGGTCGC
BC432	F: M13-AAACTCCGATGGATTCAGCA	1	FAM	60	(TG)5	1	1	261–263	endotrans glucosylase/hydrolase	5E-18
	R: TGAAATAAACACACAAACAAAGACA
BC344	F: M13-GAGGGAGGGTCCCTTGTTAG	1	VIC	60	(GCA)5	1	1	105–108	chitinase-like protein	3E-07
	R: CCGTCTTACGTTGCATCATC
BI6278	F: M13-TGTAGTTGTTGTAGTAGCAGAACTTTG	1	VIC	59	(TCC)7	1	3	238–253	DOF zinc finger protein	3E-25
	R: CAGATGGGTCGGAGATTTTG
BI5347	F: M13-TCAGTCCATTTTCTTAATCAGACC	1	VIC	59	(CTT)6	2	1	171–183	unknown gene	0.000002
	R: CTCTATCATTTCCAAGCGATTTC
BC552	F: M13-TGTCTGAGATGGAAACTGCG	1	NED	60	(GT)5	2	2	271–273	—	—
	R: TAGTCGAAGGGATCCGAATG
BI3348	F: M13-ACTTGTTTCTCGTTGGGAGC	1	PET	60	(CT)6	3	3	279–283	—	—
	R: CTGCAGCCCAGTGGATTTAC
BI06534	F: M13-CGTTGCTCTGCTCTAACCCT	1	PET	59	(TC)6	6	2	97–107	sterol 4-alpha-methyl-oxidase 2-1	7E-57
	R: AGATACAGCCAACCGGATTC
BI7112	F: M13-ATCCAATGTCAACCTCTCGG	2	FAM	60	(TCC)6	2	2	109–115	—	—
	R: GTGCATTAGAGTCCCGTGGT
BI3820	F: M13-AGGACCAGTTTTGACGGCTA	2	FAM	59	(CTT)7	5	2	158–176	LOB domain-containing protein	2E-39
	R: GAAGCTTTTGCTCTTCTGTTGA
BI134	F: M13-ATCAGCTCACTCCCTATCCTCT	2	VIC	60	(CT)6	4	2	306–314	—	—
	R: TGCAATCTCCTTCGGTTCTT
BI362	F: M13-CTTCACCTCGCCTGAACAAC	2	NED	60	(ATG)6	4	4	147–159	Acyl-CoA N-acyltransferases (NAT) superfamily protein	1.00E-45
	R: GAGGCGAAATATTATGCGGA
BC332	F: M13-GAACCAGAAGTCAAGGGTTCA	2	PET	59	(TCA)5	4	2	188–200	ATPase	1.00E-122
	R: AAACATGATTTTCCTCATCCAA
Additional loci tested
BC672	F: M13-CCTTGATCGAGAAAGAACCG			60	(CTT)8	3	1	152–158	cellulose-synthase-like C12	2E-57
	R: AAAGCCAGCTCCTTCCTGTA
BI4477	F: M13-GGATCTCCTCTGCTTTGCTG			60	(CT)9	4	2	111–119	—	—
	R: GGCGAGACCAGAAGAAAAGTT
BI06604	F: M13-ATTTTTCCACAGAAGAGCCC			59	(AT)8	6	1	111–127	—	—
	R: GGCAGAACCCGCAGTATATC
BI6294	F: M13-TGCTGGTCTGAATCTTTAATCA			59	(AT)10	1M	1M	148	catalytic LigB subunit of aromatic ring-opening dioxygenase family	3E-13
	R: TGGGGTCTTGGTACTCTTTCC
BI6701	F: M13-AGAATCCCCACTCACTGCAC			60	(GA)6	1M	1M	195	—	—
	R: GAGATGATGAGGGTTCAGGC
BI05710	F: M13-GAAAGTTTTGGAGGAAGCCC			60	(GAA)7	3	1	178–184	—	—
	R: TGGAAGAGATCAGAAGGTACA
BI4848	F: M13-CGACGCCTCTCAAAGAAGAA			59	(AG)6	4	2	71–74	arabinogalactan protein	6E-07
	R: GAGCTTTGAATTTCGCTACG
BC402	F: M13-TTACTCGAGCTAGAAGCCGC			60	(AT)5	1M	1M	92	bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin superfamily protein	3E-09
	R: AGGGCTTGGAGAGCTAGAGG
BC932	F: M13-GTAGTCCATCAGTCCGCCAT			60	(GA)5	2	1	660–662†	cysteine proteinase superfamily protein	0.000001
	R: GAGTGATGAAGGCGAAGAGG
BI3069	F: M13-AACCACAGTAATCATCCGGC			60	(CA)5	1	1	184–192	—	—
	R: TGTCCGGTAACTGTGGTGAA
BI3377	F: M13-AACACAATCATCAGCCGGAC			60	(AGG)5	MP	MP		—	—
	R: GAAGGAGATGATTATGACGAA
BI5174	F: M13-GTCGCAGGGTTTGTCTAGGA			60	(CTT)5	1	1	118–121	stromal cell-derived factor 2-like protein precursor	8E-07
	R: GGAAATCAGAGTGCTGGCTC
BC42	F: M13-GCTATGCAGGTTCTGGTGGT			59	(TGG)6	3	2	147–173	—	—
	R: ACTGGTTGTCACTACTGCCG
BI6984	F: M13-GAAGGGGTTTCTTGGTCTCA			59	(TC)6	3	2	148–164	—	—
	R: TTGTCAATTCTCACCAGACACA
BI7247	F: M13-CTCTTATTCCGCGTCAAAGC			60	(AG)6	1M	1M	135	—	—
	R: AGCGGAGAAGTCGAAAACAG
BC312	F: M13-ATTTCCTTCTGCGAACGATG			60	(GA)5	2	1	178–180	—	—
	R: ATCGGAACTCTGAGCCTGAA

Note: A = number of alleles per locus; her = B. heracleifolia; MP = multiple PCR products amplified; nel = B. nelumbiifolia; Tm = primer melting temperature when amplifi ed individually.

M13 sequence is: CACGACGTTGTAAAACGAC.

Multiplex to which the primer was assigned.

Repeat motif in B. plebeja.

The observed range of PCR product sizes excluding the M13 motif.

Putative function in Arabidopsis.

Monomorphic in all individuals tested.

Large product size assumed to be caused by an intron.

Table 2.

Genetic diversity in population samples of Begonia heracleifolia and B. nelumbiifolia.

	B. heracleifolia			B. nelumbiifolia
Locus	A	Ho	He	A	Ho	He	At
BEI4329	3	0.400	0.524	3	0.500	0.537	5
BEI03043	4	0.000	0.444	3	0.500	0.630	4
BEC432	2	0.100	0.097	2	0.000	0.097	3
BEC344	1	—	—	2	0.000	0.097	2
BEI6278	1	—	—	3	0.353	0.668	4
BEI5347	3	0.300	0.449	1	—	—	4
BEC552	1	—	—	3	0.050	0.229	3
BEI3348	4	0.579	0.604	4	0.500	0.665	5
BEI06534	5	0.500	0.750	4	0.105	0.201	7
BEI7112	2	0.400	0.467	3	0.278	0.522	4
BEI3820	5	0.600	0.623	2	0.000	0.108	6
BEC134	4	0.611	0.732	3	0.050	0.145	5
BEI04004	2	0.059	0.059	3	0.188	0.623	4
BIC362	2	0.050	0.050	2	0.000	0.097	2
BEC332	4	0.250	0.483	3	0.154	0.495	5
Mean	3.333	0.321	0.440	2.857	0.191	0.365	4
SD	1.155	0.228	0.246	0.663	0.199	0.243	1.327

Note: A = number of alleles per locus; At = total alleles observed in the two species; He = expected heterozygosity; Ho = observed heterozygosity.

CONCLUSIONS

We have described the development of nuclear microsatellite primers that amplify in two divergent Central American Begonia species. Some of the primers have exact BLAST matches in the transcriptome of the Southeast Asian species B. venusta and, therefore, may be transferable more widely across the genus. The transferability of markers is important for the study of natural hybrids, and the development of a multiplexed assay of 15 loci should enable accurate assignment to hybrid classes (e.g., F1, backcross). Future studies will use these loci to estimate the genetic structure of populations, the frequency of hybrids, and the extent of introgression in hybrid swarms.