Fatty Acid Profile and Unigene-Derived Simple Sequence Repeat Markers in Tung Tree (Vernicia fordii)

et al. (2014) Fatty Acid Profile and Unigene-Derived Simple Sequence Repeat Markers in Tung Tree (Vernicia fordii). PLoS ONE 9(8): e105298. doi:10.1371/journal.pone.0105298 Fatty Acid Profile and Unigene-Derived Simple Sequence Repeat Markers in Tung Tree (Vernicia fordii ) Lin Zhang 0 1 Baoguang Jia 0 1 Xiaofeng Tan 0 1 Chandra S. Thammina 0 1 Hongxu Long 0 1 Min Liu 0 1 Shanna Wen 0 1 Xianliang Song 0 1 Heping Cao 0 1 Manoj Prasad, National Institute of Plant Genome Research, India 0 Current address: State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University , Taian, Shandong Province , People's Republic of China 1 1 Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees, Ministry of Education, Central South University of Forestry and Technology , Changsha, Hunan Province , People's Republic of China, 2 Department of Plant Science and Landscape Architecture, University of Connecticut, Storrs, Connecticut, United States of America, 3 U.S. Department of Agriculture, Agricultural Research Service, Southern Regional Research Center , New Orleans, Louisiana , United States of America Tung tree (Vernicia fordii) provides the sole source of tung oil widely used in industry. Lack of fatty acid composition and molecular markers hinders biochemical, genetic and breeding research. The objectives of this study were to determine fatty acid profiles and develop unigene-derived simple sequence repeat (SSR) markers in tung tree. Fatty acid profiles of 41 accessions showed that the ratio of a-eleostearic acid was increasing continuously with a parallel trend to the amount of tung oil accumulation while the ratios of other fatty acids were decreasing in different stages of the seeds and that aeleostearic acid (18:3) consisted of 77% of the total fatty acids in tung oil. Transcriptome sequencing identified 81,805 unigenes from tung cDNA library constructed using seed mRNA and discovered 6,366 SSRs in 5,404 unigenes. The di- and tri-nucleotide microsatellites accounted for 92% of the SSRs with AG/CT and AAG/CTT being the most abundant SSR motifs. Fifteen polymorphic genic-SSR markers were developed from 98 unigene loci tested in 41 cultivated tung accessions by agarose gel and capillary electrophoresis. Genbank database search identified 10 of them putatively coding for functional proteins. Quantitative PCR demonstrated that all 15 polymorphic SSR-associated unigenes were expressed in tung seeds and some of them were highly correlated with oil composition in the seeds. Dendrogram revealed that most of the 41 accessions were clustered according to the geographic region. These new polymorphic genic-SSR markers will facilitate future studies on genetic diversity, molecular fingerprinting, comparative genomics and genetic mapping in tung tree. The lipid profiles in the seeds of 41 tung accessions will be valuable for biochemical and breeding studies. - Competing Interests: The authors have declared that no competing interests exist. Tung tree or tung oil tree (Vernicia fordii) is a native woody oil plant in subtropical areas of China. This important economical tree has been grown in China for the production of tung oil or ornamental garden for centuries [1]. Tung tree was introduced to the United States in 1904 [2] and grown mainly in the Southern regions of the United States [2,3]. Tung seeds contain 5060% oil with about 80 mole % a-eleostearic acid (9cis, 11trans, 13trans octadecatrienoic acid) [4]. Tung oil is oxidized easily due to the three conjugated double bonds in eleostearic acid. Dried tung oil possesses excellent characteristics such as insulation, acid and alkali resistance and anticorrosion. Unlike other drying oils, tung oil does not darken with age and it becomes a widely used drying ingredient in paints, varnishes, coatings and finishes [5,6]. Tung oil has also been used as a raw material to produce biodiesel [7], polyurethane and wood flour composites [8], thermosetting polymer [9] and repairing agent for self-healing epoxy coatings [10]. Major efforts have been directed at understanding the genetic control of tung oil biosynthesis. Many tung oil biosynthetic genes have been identified, including those coding for diacylglycerol acyltransferases (DGAT) [11,12], delta-12 oleic acid desaturase (FAD2) and delta-12 fatty acid conjugase (FADX) [13], acyl-CoA binding proteins [14] and oleosins [15,16]. The expression of some tung genes has been studied by northern blotting [1113], quantitative real-time PCR (qPCR) [12,14,1618] and western blotting [12]. A few tung proteins have been expressed in heterologous systems including E. coli [14,19,20], fungi [2022] and Arabidopsis [11,14]. However, selection of target genes for genetic engineering of plant oils is difficult because oil is biosynthesized by at least 10 enzymatic steps and each step is catalyzed by multiple isozymes [11,23,24]. Furthermore, it has been difficult to study tung oil biosynthesis at the protein level because these enzymes are mostly hydrophobic and membranelocalized proteins [19,20]. Understanding fatty acid composition and genetic diversity among tung tree germplasm resources is essential for tung tree Town, County, Province Geographical coordinates breeding and clonal improvement. A series of elite V. fordii clones were released in China in the 1980s for cultivation on the basis of field survey, collection and evaluation data [1]. However, these economically important germplasm resources were severely damaged by human errors and environmental factors over the past 20 years [1]. In recent years, the importance of V. fordii germplasm resources has been more widely recognized. We initiated germplasm collection in 2007. Some superior germplasm were collected from main distribution areas of V. fordii in China and planted at the Central South University of Forestry and Technology Germplasm Repository. Microsatellites, also known as simple sequence repeats (SSRs) or short tandem repeats, are repeating sequences of 26 base pairs of DNA [25]. They are widely used as molecular markers in genetics and used for studies of gene duplication or deletion, marker assisted selection and fingerprinting [2529]. Therefore, SSR markers could be powerful tools for genetic diversity evaluation, molecular fingerprinting identification, comparative genomics analysis and genetic mapping in tung tree. Tung tree SSR markers have been analyzed in two studies. In one study, authors analyzed 2,407 expressed sequence tag (EST) sequences from the database and identified 22 V. fordii-specific EST-SSR markers [30]. In the other study, 40 polymorphic SSR markers were identified from the V. fordii genomic DNA by AFLP of Sequences Containing repeats protocol [31]. Clearly, there is a need for developing more SSR markers for tung tree improvement. Great progress has been developed in high throughput sequencing technology, i.e. Next Generation Sequencing, utilizing the Roche/454 Genome S (...truncated)