Firmiana Marsili (Malvaceae sensu lato [s.l.]) is distributed in the Asiatic continent, Malaysia, and the Pacific Islands (Kostermans, 1957). This genus consists of 12–16 species, many of which are narrow endemics (Mabberley, 1997; Tang et al., 2007). Some species of Firmiana are cultivated as ornamental plants for their graceful shape and beautiful flowers. The wood of F. simplex W. Wight has been used by the Chinese to make high-quality traditional Asian musical instruments for thousands of years. This genus could be divided into two groups, i.e., Firmiana and Erythropsis, based on morphological and molecular data (Fan et al., unpublished data). There are five Firmiana species endemic to China. Among them, F. danxiaensis H. H. Hsue & H. S. Kiu is distributed only in Mount Danxia, Guangdong Province; F. kwangsiensis H. H. Hsue occurs only in Jingxi County, Guangxi Province; F. hainanensis Kosterm. and F. pulcherrima H. H. Hsue are confined in the central-south mountains of Hainan Province; and F. major (W. W. Sm.) Hand.-Mazz. is restricted to Yunnan Province and southwestern Sichuan Province (Tang et al., 2007). All of these species are listed as threatened in the China Species Red List (Wang and Xie, 2004).
Because of the lack of efficient molecular markers, there have been no studies on genetic diversity analyses and conservation genetics of Firmiana species. The transcriptome of F. danxiaensis was recently sequenced using the Illumina platform, and sequences for tens of thousands of transcripts were obtained by de novo assembly (Chen et al., unpublished data). Based on these sequences, 17 novel polymorphic expressed sequence tag (EST)–simple sequence repeat (SSR) markers were developed and characterized for size polymorphism among four Firmiana species: F. danxiaensis, F. kwangsiensis, F. hainanensis, and F. simplex. Of these species, F. kwangsiensis belongs to the Erythropsis group, while the other species belong to the Firmiana group of the genus Firmiana.
METHODS AND RESULTS
One seedling of F. danxiaensis was collected from Danxia Mountain, Guangdong Province, China, and planted in the greenhouse of Sun Yat-sen University. One year later, fresh leaves of the seedling were collected for RNA extraction, and the subsequent protocols for the transcriptome sequencing followed Chen et al. (2011). A total of 26.95 million 90-nucleotide paired-end reads were obtained and de novo assembled into 33,522 contigs using Trinity (Grabherr et al., 2011) with a minimal length of 300 bp and an average length of 834 bp. Functional annotation for these contigs was performed using the automatic annotation tool Blast2GO (Conesa and Götz, 2008). Using MISA ( http://pgrc.ipk-gatersleben.de/misa), we detected 2069 SSRs in 1897 contigs. Among them, trinucleotide repeats (58.1%) were the most common, followed by dinucleotide (36.1%), tetranucleotide (3.3%), hexanucleotide (1.6%), and pentanucleotide repeats (0.9%). Using Primer3 (Rozen and Skaletsky, 2000; http://frodo.wi.mit.edu/primer3), 102 pairs of primers were designed from contigs containing at least nine repeats for dinucleotide and trinucleotide motifs, or seven repeats for tetranucleotide motifs. Contigs containing these 102 SSR loci were deposited in GenBank ( Appendix S1 (APPSD1300047_AppendixS1.doc), Table 1).
Characteristics of 17 EST-SSR markers developed in Firmiana danxiaensis.
Results of initial primer screening in four Firmiana species.
Polymorphisms of these primer sets were assessed in four Firmania species. Among them, F. danxiaensis was collected from Mt. Danxia, Guangdong Province (25°01′N, 113°44′E); F. hainanensis was collected from Jianfengling and Bawangling, Hainan Province (18°45′N, 108°53′E; 19°07′N, 109°07′E); F. kwangsiensis was collected from Jingxi County, Guangxi Province (23°00′N, 106°40′E); and F. simplex was collected from Taroko National Park, Taiwan (24° 10′N, 121°31′E), Mt. Malan, Shenzhen (22°37′N, 114°19′E), and Daye, Hubei Province (30°04′N, 114°56′E). Voucher specimens for the four species were deposited at the Herbarium of Sun Yat-sen University (Appendix 1). We extracted the genomic DNA for all individuals from silica-dried leaves using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987). For the 102 primer pairs, we first selected three individuals of each species to screen universal primers for all four Firmiana species. PCR amplifications were performed in 20-µL reaction volumes, containing 25 ng of genomic DNA, 2 µL 10× buffer (with Mg2+), 0.25 mM of dNTPs, 0.2 µM of each primer, and 1 U of Easy-Taq DNA polymerase (TransGen Biotech Co. Ltd., Beijing, China). The PCR reactions were conducted with the following conditions: initial denaturing at 94°C for 2 min; followed by 35 cycles of 94°C for 30 s, appropriate annealing temperature (Table 1) for 30 s, and 72°C for 40 s; and a final extension at 72°C for 5 min. The PCR products were first electrophoresed on a 10% Polyacrylamide denaturing gel and visualized by silver nitrate staining. The band size was estimated by comparison with a 25-bp DNA ladder (Fermentas, Vilnius, Lithuania). Of the 102 primer sets, 17 were successfully amplified in the four Firmiana species (Table 1).
The above 17 primer sets were then labeled with fluorescent dye, and PCR amplifications were carried out for the 32 individuals of the four Firmiana species (eight for each species). Their PCR products were analyzed on an Applied Biosystems 3730xL DNA Analyzer (Applied Biosystems, Foster City, California, USA), and the allele size was estimated using Peak Scanner Software version 1.0 (Applied Biosystems). The genetic diversity indices, including the number of alleles (A), observed heterozygosity (Ho), and expected heterozygosity (He), were calculated using the software POPGENE (Yeh and Boyle, 1997). The results showed 14 of these 17 primer sets were polymorphic in at least one Firmiana species. A ranged from one to three in F. danxiaensis, from one to seven in F. kwangsiensis, from one to eight in F. hainanensis, and from one to nine in F. simplex. Ho and He ranged from 0 to 0.500 and 0 to 0.592 in F. danxiaensis, from 0 to 1 and 0 to 0.833 in F. kwangsiensis, from 0 to 0.875 and 0 to 0.883 in F. hainanensis, and from 0 to 0.750 and 0 to 0.925 in F. simplex, respectively (Table 2).
In our study, 17 EST-SSRs were identified from the transcriptome of F. danxiaensis and were amplified in three other Firmiana species. Because the four species of Firmiana belong to different clades, these 17 SSR markers show good transferability in the genus. These polymorphic markers are valuable for conservation genetic studies in these endangered Firmiana species.
 The authors thank the Administrative Committee of Danxiashan National Park for assistance during the field work. This work was supported by the National Natural Science Foundation of China (31100159, 31170202, and 31170213), the Doctoral Foundation of the Ministry of Education of China (20090171120034), and the Fundamental Research Funds for the Central Universities (13lgpy07).