Fig 1.
Differential pigment accumulation in AJ and GZ spikes.
(A) Developmental stages of zoysiagrass spikes according to the size and the degree of pigmentation. ‘Anyang-Jungji’ (AJ) and ‘Greenzoa’ (GZ) spikes are categorized into six developmental stages (S1–S6) by size, color, and flower development. (B) Aglycone contents of acid-hydrolyzed anthocyanins in zoysiagrass spikes over developmental stages. The left y-axis indicates the amount of total cyanidin chloride and the right y-axis indicates the amount of petunidin chloride as determined by HPLC analysis. Fw, Fresh weight.
Table 1.
Characteristics of AJ and GZ spikes during spike development.
Table 2.
Summary of de novo transcriptome assemblies of AJ and GZ.
Fig 2.
Phylogenetic relationship of de novo assembled zoysiagrass transcriptome to other plant species.
(A) Transcript homology of two zoysiagrass cultivars and Setaria italica. Venn diagram shows the number of orthologous transcripts (E-value < 1.0e-15). The percentage in the brackets indicates the degree of transcriptome homology relative to AJ. (B) Relative similarity of zoysiagrass transcriptome with other plants. The zoysiagrass unigenes annotated with the NR database were matched by TBLASTX hit with E-value < 1.0E-15. (C) Phylogenetic tree based upon the β-ACTIN sequences from various plant species. The tree was constructed by using the maximum-likelihood method with MEGA 5.2 based on the ClustalX-generated multiple sequence alignment. The topological stability of the tree was evaluated by 1,000 bootstrap replications, and the bootstrapping percentage values are indicated by the numbers at the tree nodes. The GenBank accessions of β-ACTIN sequences used for phylogenetic analysis are listed in S4 Table.
Fig 3.
Differentially expressed genes between AJ and GZ spike tissues.
(A) MA plot of differentially expressed transcripts identified in AJ and GZ. The X axis represents the mean expression level, and the Y axis represents the log2 fold-change of GZ transcripts over AJ. Red and blue dots represent differentially expressed genes (DEGs) that are significantly abundant in GZ and AJ, respectively, at p-value < 0.05. Horizontal lines and the values to the right represent the average M-values of corresponding groups of DEGs and non-DEGs. (B) Gene ontology (GO) classification of the DEGs. GO terms of Z. japonica unigenes are based on significant hits against the NR database. The right y-axis indicates the number of upregulated genes in AJ (black) and GZ (grey), respectively. The left y-axis indicates the percentage of each group from the total.
Fig 4.
Zoysiagrass transcripts involved in general anthocyanin biosynthesis pathway.
(A) A schematic view of the core anthocyanin biosynthesis pathway. ANS, anthocyanidin synthase; CHS, chalcone synthase; DFR, dihydroflavonol reductase; F3H, flavanone 3-hydroxylase; F3’H, flavonol 3’-hydroxylase; F3’5’H, flavonol 3’,5’-hydroxylase; FLS, flavonol synthase; PAL, phenylalanine ammonia lyase; UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase. (B) Heatmap of digital expression of candidate transcripts related to anthocyanin biosynthesis pathway in zoysiagrass. The log2-transformed FPKM values and relative expression (AJ vs. GZ) values are represented by the color map. Asterisks indicate statistical DEGs.
Fig 5.
Expression levels of DFR and ANS genes in AJ and GZ spikes at different developmental stages.
The relative expression level of each transcript was determined by quantitative RT-PCR, in which all values are normalized relative to the mean abundance of β-ACTIN. The expression levels are presented relative to the AJ gene abundance at stage S1. Bars represent means ± SD from triplicate biological repeats.
Fig 6.
Analysis of ZjDFR1 and ZjANS1 expression among zoysiagrass cultivars.
(A) Representative spikes of nine zoysiagrass cultivars at developmental stage S6. The scale bar represents 10 mm. Expression levels of ZjDFR1 (B) and ZjANS1(C) genes in spike tissues determined by quantitative RT-PCR. All values are normalized relative to the mean abundance of β-ACTIN. Bars represent means ± SD from triplicate biological repeats.