The genus *Melastoma* L. (Melastomataceae) is distributed in tropical and subtropical Asia and northern Australia ([@bib4]).  Previous studies have identified around 50--100 species within this genus ([@bib1]; [@bib6]); however, only 22 species were recognized in the latest taxonomic revision by [@bib4].  Natural hybridization between two species of this genus has been reported, with more cases of interspecific hybridization being expected, because many species in this genus have overlapping geographic distributions and flowering periods ([@bib2]).  According to the Flora of China, there are nine *Melastoma* species in China, which are distributed south of the Yangtze River ([@bib1]). * Melastoma dodecandrum* Lour.  is a small shrub that is distributed primarily in southern China, extending to northern Vietnam ([@bib1]).  Unlike other *Melastoma* species, which are restricted to the area south of the Nanling Mountains, *M. dodecandrum* extends north to Zhejiang Province, exhibiting higher levels of cold tolerance ([@bib1]).  Population genetic studies hold promise toward inferring the dispersal routes of this species and studying molecular adaptation to colder climates.  Microsatellite markers are codominant and often highly polymorphic, and hence are increasingly used in population genetic studies.  However, no microsatellite markers have been available in *Melastoma* to date to investigate the genetic diversity of this species, or hybridization and introgression in this genus.  In this study, we developed and characterized 14 microsatellite markers for *M. dodecandrum* and tested their transferability to two other congeneric species, *M. candidum* D. Don and *M. sanguineum* Sims. 

METHODS AND RESULTS
===================

Two populations of *M. dodecandrum* were sampled from Dafu Mountain (22°56′18″N, 113°19′08″E) and Maofeng Mountain (23°17′07″N, 113°20′18″E) in Guangzhou, Guangdong, China.  In addition, three individuals each of *M. candidum* and *M. sanguineum* were collected from Dafu Mountain.  Voucher specimens (Dafu Mountain population: RZ20111015; Maofeng Mountain population: RZ20111006; *M. sanguineum*: RZ20111016; *M. candidum*: RZ20111017) were deposited in the Herbarium of Sun Yat-sen University (SYS).  Genomic DNA was extracted from silica-dried leaves with the cetyltrimethylammonium bromide (CTAB) method ([@bib3]).  The genomic DNA of one individual of *M. dodecandrum* from Dafu Mountain was used for the development of microsatellites.  Approximately 300 ng of genomic DNA was digested with *Mse*I restriction enzyme (New England Biolabs, Ipswich, Massachusetts, USA), and then ligated to an *Mse*I adapter (5′-TACTCAGGACTCAT-3′/5′-GACGATGAGTCCTGAG-3′) using T4 DNA ligase (New England Biolabs).  The ligation product was diluted and amplified with the *Mse*I-N primer (5′-GATGAGTCCTGAGTAAN-3′) under a PCR program with initial denaturation of 3 min at 95°C; followed by 30 cycles of 30 s at 94°C, 60 s at 53°C, and 60 s at 72°C; and a final extension of 10 min at 72°C.  The PCR products were denatured at 95°C for 5 min and hybridized with each of the two 5′-biotinylated probes, (AG)~15~ and (AC)~15~, in 300 μL of hybridization solution (20× saline sodium citrate \[SSC\], 10% sodium dodecyl sulfate \[SDS\], 100 μM probe) at 48°C for 2 h to construct two independent microsatellite libraries.  Streptavidin magnesphere paramagnetic particles (Promega Corporation, Madison, Wisconsin, USA) were used to separate the DNA fragments hybridized to the probes.  The fragments were washed three times in TEN~100~ for 8 min and three times in TEN~1000~ for 8 min.  The recovered DNA fragments were amplified with adapter-specific primers by following the same procedure as mentioned above.  The PCR products were purified with the EZgene Gel/PCR Extraction Kit (Biomiga, San Diego, California, USA), ligated into pMD18-T vector (TaKaRa Biotechnology Co., Dalian, Liaoning, China), and then transformed into *E. coli* strain DH5α (TaKaRa Biotechnology Co.).  Transformants were selected on Luria--Bertani (LB) agar medium containing 100 mg/mL ampicillinum natricum.  Two hundred forty positive clones were selected and tested by PCR using (AG)~10~, (AC)~10~, and M13 universal primers, of which 197 clones contained potential microsatellite motifs.  These positive clones were sequenced on an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, California, USA).  Sequences containing more than five tandem repeats were considered as microsatellites. 

In total, 64 clones were found to contain microsatellites, of which 43 with five or more repeats were selected for primer design using Primer3 ([@bib5]).  To test these microsatellites, PCR amplifications were conducted using three individuals for each of the three species in a final volume of 20 μL, containing 25 ng of genomic DNA, 10× PCR buffer (with Mg^2+^), 2.5 mM of each dNTP, 10 μM of each primer set, and 1 U r*Taq* DNA polymerase (TaKaRa Biotechnology Co.).  The PCR reactions were carried out under standard conditions for all primers with the following cycling conditions: 3 min of denaturation at 95°C; followed by 30 cycles of 30 s at 94°C, 30 s at 55°C, and 40 s at 72°C; with a final extension of 10 min at 72°C.  Amplification products were first electrophoresed through 1.5% agarose gels to assess whether amplification was successful and the expected sizes were obtained.  Our results showed that PCR products of expected sizes were successfully amplified for 14 primer pairs in *M. dodecandrum* ([Table 1](#tbl1){ref-type="table"}), and that these primer pairs exhibited successful amplification in the two congeneric species, *M. candidum* and *M. sanguineum*.  We then labeled the forward primers of the 14 primer pairs with the fluorescent dye FAM (Invitrogen, Carlsbad, California, USA) and conducted the PCR amplifications for both populations of *M. dodecandrum* using the conditions mentioned above.  Using ROX 500 as an internal size standard, the fragment sizes of these PCR products were determined on an ABI PRISM 3100 DNA Analyzer with Genotyper 4.0 (Applied Biosystems).  Population genetics parameters for *M. dodecandrum* were calculated using POPGENE version 1.31 ([@bib7]). 

###### 

Characteristics of 14 microsatellite loci of *Melastoma dodecandrum*. 

  Locus   Primer sequences (5′--3′)       Repeat motif        Size (bp)   *T*~a~ (°C)   GenBank accession no. 
  ------- ------------------------------- ------------------- ----------- ------------- -----------------------
  C33     F: GGGCTGAGACTTGAGAAAGAGA       (AC)~10~            208         55            JX126109
          R: GTTTTCCCGTGGAACAAAAA                                                       
  C38     F: TTCACCCTCCTCTAATGACCTC       (AC)~5~             201         55            JX126110
          R: CTATGTCGATGGGTGGATGATT                                                     
  C54     F: TGTGTTATTGGCAAGACAAAGG       (GT)~7~             214         55            JX126111
          R: AGAGATCTCGATTATGCCCAAG                                                     
  C67     F: CGCCTCGAGTCTTTCTTTCTT        (AC)~8~             201         55            JX126112
          R: CATACAAATCCCAGCAAATGTT                                                     
  C71     F: GTTACAGCCCACCAGAAAGAAA       (AG)~10~(TAC)~10~   250         55            JX126113
          R: CTTAGCCCTTGTCCGTAAGAAT                                                     
  C73     F: CGGCCGACATTTGAAGTTAGTA       (AC)~6~             199         55            JX126114
          R: GGGTCGTTTTATCCATCTTCTTC                                                    
  C91     F: GTTTTCCCGTGGAACAAAAA         (AC)~10~            204         55            JX126115
          R: ACCGATGCATATTTTGGAATTT                                                     
  C96     F: TTCTGGTCCCCAGTACTTCAAT       (CA)~8~             217         55            JX126116
          R: TCGTTGGGGAATCTTTACAGTC                                                     
  C102    F: GAGAAGAAAATTTTTACATGTCCCTA   (AC)~10~            190         55            JX126117
          R: ATGGCAAATACCAGCAGATGAT                                                     
  G80     F: GAATGACAACACCGGCAAG          (CT)~11~            204         55            JX126118
          R: CCTCCACGTTCTCTTGAATGTC                                                     
  G90     F: TAAGCAACAGGCAAGAATGAGA       (AG)~9~             201         55            JX126119
          R: GTGACCCTTGCCTAGTTTTGAC                                                     
  G92     F: GGGCTGAGACTTGAGAAAGAGA       (GA)~10~            224         55            JX126120
          R: TTAGGAGAGGATCGTCTGATGC                                                     
  G107    F: CCAATCAGTCAGCCACAAAGT        (CT)~6~             184         55            JX126121
          R: GGAGACCTAACTTCATGTCAATG                                                    
  G119    F: GAAAATAAAAACAGCCGGATTG       (CT)~6~             200         55            JX126122
          R: GACTTCATTGGATCAAGACACG                                                     

*Note*: *T*~a~ = annealing temperature. 

All but one of the microsatellite loci exhibited polymorphisms, with the number of alleles per locus ranging from two to 15 ([Table 2](#tbl2){ref-type="table"}).  The observed and expected heterozygosity from the polymorphic loci ranged from 0.235 to 0.941 and 0.219 to 0.922, respectively.  Four loci (C67 and C73 in the Dafu Mountain population, and C33 and C91 in the Maofeng Mountain population) deviated significantly from Hardy--Weinberg equilibrium (*P* \< 0.05).  There was no significant linkage disequilibrium between locus pairs; therefore, all of the loci should be considered as being independent across the genome. 

###### 

Genetic diversity in two populations of *Melastoma dodecandrum*. 

         Dafu Mountain (*n* = 17)   Maofeng Mountain (*n* = 18)                        
  ------ -------------------------- ----------------------------- ------- ---- ------- -------
  C33    7                          0.824                         0.839   6    0.389   0.737
  C38    2                          0.353                         0.499   3    0.389   0.465
  C54    6                          0.765                         0.752   6    0.778   0.754
  C67    5                          0.412                         0.726   4    0.500   0.671
  C71    11                         0.941                         0.881   12   0.889   0.854
  C73    3                          0.529                         0.604   4    0.778   0.657
  C91    7                          0.824                         0.840   7    0.389   0.737
  C96    3                          0.235                         0.219   3    0.333   0.294
  C102   3                          0.706                         0.570   3    0.556   0.513
  G80    15                         0.941                         0.922   9    0.722   0.779
  G90    7                          0.647                         0.629   3    0.611   0.541
  G92    11                         0.647                         0.620   6    0.778   0.605
  G107   1                          0.000                         0.000   1    0.000   0.000
  G119   2                          0.471                         0.428   2    0.389   0.322

*Note*: *A* = number of alleles; *H*~e~ = expected heterozygosity; *H*~o~ = observed heterozygosity; *n* = sample size for each population. 

CONCLUSIONS
===========

This is the first study to report microsatellite markers for species of *Melastoma*.  All of the 14 markers for *M. dodecandrum* show good transferability in two congeneric species.  The primers developed here are suitable for investigating the genetic diversity and population structure of *M. dodecandrum* and other congeneric species, and for detecting hybridization and introgression within this genus. 

[^1]: This work was supported by the National Natural Science Foundation of China (31170213), the Fundamental Research Funds for the Central Universities (10lgpy20), and Chang Hungta Science Foundation of Sun Yat-sen University.