Tung tree, Vernicia fordii (Hemsl.) Airy Shaw (Euphorbiaceae), is a native economic tree species in China. It is the most important species used to produce industrial oil (tung oil) and has been cultivated for over a thousand years in China. Today, the remnant plantation areas of V. fordii include Sichuan, Hunan, Hubei, Guizhou, and Chongqing provinces, as well as adjacent regions (Zhang and Peng, 2011). In addition to its irreplaceable role in industry for the manufacture of paints and coatings, tung oil has been reported to be a promising feedstock in biodiesel production (Shang et al., 2010). Vernicia fordii is adaptive to drought and barren mountainous areas. Thus, its development will both meet the energy demands without endangering the food supply chain and provide employment in poor mountainous regions. Molecular markers are efficient in revealing genetic diversity (Peng et al., 2000) and in assisting tree breeding (Li et al., 2008; Zhao et al., 2011). In this study, we developed a set of microsatellite (simple sequence repeat [SSR]) markers based on the specific genomic sequences of V. fordii, and evaluated their efficiency in amplifying the DNA of the related species V. montana Lour.
METHODS AND RESULTS
Leaf tissues collected from adult tung trees were immediately preserved in silica gel for fast drying and then stored in a −70°C freezer. The dried leaf tissues were ground into fine powder in liquid nitrogen just before DNA extraction. Genomic DNA was isolated using a modified cetyltrimethylammonium bromide (CTAB)–based plant DNA extraction method (Doyle and Doyle, 1987; Zhang et al., 2013). The SSR-containing fragments were isolated using a protocol based on the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol (Zane et al., 2002). Total genomic DNA (∼500 ng) was digested by the MseI restriction enzyme (New England Biolabs, Beverly, Massachusetts, USA) at 37°C for 3.5 h and then ligated to an MseI adapter pair (5′-TACTCAGGACTCAT-375′-GACGATGAGTCCTGAG-3′) with T4 DNA ligase (Fermentas International, Burlington, Ontario, Canada) in a 25-µL reaction mixture. The ligation product was diluted (1:10) and amplified by PCR with the adapter-specific primers MseI-N (5′-GATGAGTCCTGAGTAAN-3′) (25 µM). The amplified DNA fragments were enriched for SSR repeats by magnetic bead selection with 5′-biotinylated (AC)13 and (AG)13 probes, respectively. PCR products were purified using an E.Z.N.A. Gel Extraction Kit (Omega Bio-Tek, Guangzhou, China). The purified DNA fragments were ligated into the pMD18-T vector and transformed into DH5α cells (TaKaRa Biotechnology Co., Dalian, Liaoning, China). Positive clones were detected by PCR using the M13-tailed PCR method. All PCR reactions were performed using the following procedure: an initial denaturation of 5 min at 95°C; followed by 30 cycles of 40 s at 94°C, 30 s at 55°C, and 45 s at 72°C; and a final extension at 72°C for 8 min (Pan et al., 2009).
Among 400 colonies, a total of 196 (49%) fragments were found to contain SSR repeats, and the sequences were deposited in GenBank. For the SSR sequences containing adequate flanking regions, PCR primers were designed with Primer3 software (Rozen and Skaletsky, 2000). Eighty-one individuals of V. fordii from a local population (Huangpi, 31°06′15.66″N, 114°11′53.46″E) were used to test the polymorphism of the microsatellite markers. Vouchers were deposited at Wuhan Botanical Garden (Appendix 1). PCR products were separated by 6% denaturing Polyacrylamide gels. Allele sizes were estimated visually using a 10-bp DNA ladder as a size reference. Of the 78 designed primer pairs, only 26 (33.33%) failed to generate PCR amplification products, and 52 (66.67%) could successfully yield PCR products. Forty of the 52 efficient SSR markers were polymorphic and the polymorphism rate reached 76.92% (40/52) (Table 1). Population-level Hardy–Weinberg equilibrium tests were conducted using POPGENE version 1.32 (Yeh et al., 1999). P values indicated there was no significant departure from Hardy–Weinberg equilibrium (P < 0.01). The number of alleles per locus ranged from two to eight with an average of 2.9750, and the average observed heterozygosity, expected heterozygosity, and Shannon information index were 0.4596, 0.3773, and 0.6411, respectively (Table 2). The same set of 78 SSR markers was used to test six accessions of the related species, V. montana. Of these tested SSR markers, 52 (66.67%) also amplified in V. montana, and 25 (48.08%) of the 52 markers that amplified showed polymorphism. The number of alleles per locus ranged from two to three, and the average Shannon information index was 0.5140 (Table 2).
TABLE 2.
Population genetic parameters for the polymorphic SSR markers developed in Vernicia fordii and V. montana.
CONCLUSIONS
This set of highly polymorphic SSR markers could be applied to further studies on genetic diversity in V. fordii. Because the SSR markers were tested in the local tung tree population, we expect that a higher level of genetic diversity will be detected in the equal-size tung tree population consisting of nationwide collections. The SSR-revealed genetic diversity in this species is important for conservation and proper utilization of tung tree germplasm. The newly developed SSR markers are also helpful for marker-assisted breeding in this important biodiesel plant species.
