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

<div><p>Tung tree (<i>Vernicia fordii</i>) 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 α-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 α-eleostearic 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.</p></div

Correlation between expression levels of polymorphic SSR-associated unigenes and oil and fatty acid composition in tung seeds.

<p>Gray correlation analysis was performed to generate correlation coefficient between gene expression levels and oil content and fatty acid composition. The higher correlation coefficient between the mRNA levels and oil content/fatty acid composition means the more positive effect of the gene product on oil content/fatty acid composition.</p

Tung oil and fatty acid accumulation in developing tung tree seeds.

<p>DAF: Days after flowering. The means and standard deviations of three determinations are presented.</p><p>Tung oil and fatty acid accumulation in developing tung tree seeds.</p

Polymorphism of genic-SSRs revealed by agarose gel and capillary electrophoresis.

<p>PCR primers for VfUg25262, VfUg3139 and VfUg77143 loci (unigenes) were used to amplify DNA fragments from genomic DNA of three tung tree accessions (HUN42, GZ11 and HEN176). The PCR products were separated by 2% agarose gel electrophoresis (left panels) and capillary electrophoresis (right panels). Vf and Ug in the locus name represent the abbreviation of tung tree (<i>Vernicia fordii</i>) and unigene. M represents the DNA size standards (DL600 DNA ladder: 100, 200, 300, 400, 500 and 600 bp). (A) VfUg25262 locus, (B) VfUg3139 locus, (C) VfUg77143 locus.</p

Voucher numbers, collection locations and geographical coordinates of tung tree (<i>Vernicia fordii</i>).

<p>GZ, HB, HEN, HUN and SX under “voucher” column refer to Guizhou, Hubei, Henan, Hunan and Shanxi Provinces. The bolded “HUN42” (accession No. 30) was used for cDNA library construction.</p><p>Voucher numbers, collection locations and geographical coordinates of tung tree (<i>Vernicia fordii</i>).</p

PCR primers for polymorphic SSR-associated unigenes in tung tree (<i>Vernicia fordii</i>).

<p>PCR primers for polymorphic SSR-associated unigenes in tung tree (<i>Vernicia fordii</i>).</p

PCR primers and test results for detecting monomorphism and polymorphism in tung tree (<i>Vernicia fordii</i>).

<p>Vf and Ug under “unigene ID” column represent the abbreviation of tung tree (<i>Vernicia fordii</i>) and unigene followed by the unigene number.</p><p>PCR primers and test results for detecting monomorphism and polymorphism in tung tree (<i>Vernicia fordii</i>).</p

Types and frequencies of SSRs identified from the unigenes from tung seed cDNA library.

<p>The search parameters were set for detection of perfect di-, tri-, tetra-, penta- and hexa-nucleotide SSR motifs with a minimum of six, five, five, four and four repeats, respectively. (A) Distribution of SSR unit type, (B) Frequency of classified SSR motifs.</p

UPGMA dendrogram of the genetic relationships among 41 <i>V. fordii</i> accessions.

<p>The dendrogram was generated using the Jaccard’s similarity coefficient based on 32 polymorphic SSR-associated genes including 15 new genes identified in this study and 17 genes confirmed based on a previous publication <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105298#pone.0105298-Pan1" target="_blank">[31]</a>. The boxed “HUN42” was used for cDNA library construction.</p

Identification of the number and size of polymorphic genic-SSRs by capillary electrophoresis.

<p>The results from VfUg78868 (polymorphic gene) are shown as an example. PCR primers for VfUg78868 locus were used to amplify DNA fragments from genomic DNA of 41 tung tree accessions. The results from four accessions representing the complete set of 4 alleles are presented. The PCR products were separated by capillary electrophoresis. The length of the PCR product on the figure is 18 bp longer than the actual size in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105298#pone-0105298-t003" target="_blank">Table 3</a> because an 18 bp fluorescent primer M13 (–21) (5′-TGTAAAACGACGGCCAGT-3′) was used for labeling PCR products. (A) accession GZ131, (B) accession HEN176, (C) accession HUN42, (D) accession HB60.</p