Applications in Plant Sciences 2013 1(12): 1300047
Applicati Ap tions ons
in Pl Plantt Scien Sciences ces
PRIMER NOTE
DEVELOPMENT AND CHARACTERIZATION OF MICROSATELLITE MARKERS FROM THE TRANSCRIPTOME OF FIRMIANA DANXIAENSIS (MALVACEAE S.L.)1 QIANG FAN2, SUFANG CHEN2, MINGWAN LI2, SHIYANG HE2, RENCHAO ZHOU2, AND WENBO LIAO2,3 2State
Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China
• Premise of the study: Firmiana consists of 12–16 species, many of which are narrow endemics. Expressed sequence tag (EST)– simple sequence repeat (SSR) markers were developed and characterized for size polymorphism in four Firmiana species. • Methods and Results: A total of 102 EST-SSR primer pairs were designed based on the transcriptome sequences of F. danxiaensis; these were then characterized in four Firmiana species—F. danxiaensis, F. kwangsiensis, F. hainanensis, and F. simplex. In these four species, 17 primer pairs were successfully amplified, and 14 were polymorphic in at least one species. The number of alleles ranged from one to 13, and the observed and expected heterozygosities ranged from 0 to 1 and 0 to 0.925, respectively. The lowest level of polymorphism was observed in F. danxiaensis. • Conclusions: These polymorphic EST-SSR markers are valuable for conservation genetics studies in the endangered Firmiana species. Key words: Firmiana danxiaensis; Malvaceae; microsatellites; transcriptome; transferability.
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 highquality 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be divided into two groups, i.e., Firmiana and Erythropsis, based on morphological and molecular data (Fan et al., unpublished data).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 Gotz, 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, Table 1).
1 Manuscript received 29 May 2013; revision accepted 17 July 2013. 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). 3 Author for correspondence:
[email protected]
doi:10.3732/apps.1300047
Applications in Plant Sciences 2013 1(12): 1300047; http://www.bioone.org/loi/apps © 2013 Fan et al. Published by the Botanical Society of America. This work is licensed under a Creative Commons Attribution License (CC-BY-NC-SA). 1 of 4
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GTTCCAGTTACAGCCTCAG CAGCAGGGTGATGATGAATA CCGTTGACATCTGTAATATCTC GCTGGACATGGTGGTTAG TGGCATCTTCAAGGCATTA GCTGTGACTAGGAGGAGA
CGTGGAGAGTCATCTTGG GGTAGTTCTACTTGATCGGATA CAATTACTCTACTCAGCATC GTGTGGAGGTTATGACTAA GAAACTCGTGAAACAGGAAA TGGAAGAACAGTGAAGAAGA CTTGGCTGCTTCAGAGTG TTGGAGTAGTATATGTGGTGAG CTGTCAATCAAATCCCTTCA ATTCCTTCCACTGTGTCTT TACACGATTAGCAGCAGAA AGCAGAGAAGGAGAGGAG CTACTGTTCGGCTACTCAA AGATAGAGAAGAAGGCTGAC GCAGTCAACATGAACTTACT CTTGCCATCTCCATACCAT TCGGAATTGGTTGTTCAGA GAGGTGCAAGCTCAGATT CGGTTACAGGTGTTGAGTT TTACGGCGAGGCAGATAA
F: R: F: R: F: R:
F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: (CT)9
(CA)9
(TG)9
(TC)9
(AG)9
(AG)9
(AG)9
(AG)10
(CT)10
(TC)7…(CA)6
(ACC)6…(CAG)6
(TTC)6…(TC)6
(GCA)8…(ACA)5…(ACA)8
(CAG)5…(CAT)6
(GGA)6…(TC)7
(CCT)5…(TTG)5
(CCA)5…(CAC)5
Repeat motifa
57
50
50
50
50
50
50
50
50
50
55
57
57
50
50
50
50
Ta (°C)
181
250
115
189
216
167
214
214
181
204
149
156
164
197
169
166
248
Expected allele size (bp)
6 (179–195)
3 (238–256)
13 (107–159)
1 (189)
10 (211–239)
5 (159–173)
8 (212–226)
9 (199–223)
11 (172–202)
5 (202–210)
7 (133–166)
8 (145–177)
1 (164)
7 (189–217)
8 (164–184)
1 (166)
9 (216–246)
A (Allele size range [bp])b
KF313465
KF048055
KF313464
KF048053
KF048052
KF048051
KF048050
KF048049
KF048048
KF048047
KF048046
KF048045
KF048044
KF048043
KF048042
KF048041
KF048040
GenBank accession no.
mitochondrial carrier
virion binding
SWIB/MDM2 domaincontaining protein probable lactoylglutathione lyase, chloroplast-like disease resistance protein RPS2 no hit
ubiquitin-conjugating enzyme E2-18 scarecrow-like protein 14-like orange protein
B3 domain-containing transcription factor NGA1-like bZIP transcription factor bZIP28 nudix hydrolase chloroplastic-like probable calciumbinding protein CML48-like exostosin-like protein
flowering time control protein FCA-like no hit
no hit
Putative functionc
Note: F = forward primer; R = reverse primer; Ta = annealing temperature. a Ellipses in repeat motifs signify intervening non-repeat sequence (≤100 bp) between two microsatellite repeats. b Values from F. danxiaensis, F. kwangsiensis, F. hainanensis, and F. simplex. c Annotation of putative homology acquired by searching the National Center for Biotechnology Information (NCBI) nonredundant database with BLASTX with the expected value
