Arachis hypogaea gene expression atlas for fastigiata subspecies of cultivated groundnut to accelerate functional and translational genomics applications

Abstract

Spatio-temporal and developmental stage-specific transcriptome analysis plays a crucial role in systems biology-based improvement of any species. In this context, we report here the Arachis hypogaea gene expression atlas (AhGEA) for the world's widest cultivated subsp. fastigiata based on RNA-seq data using 20 diverse tissues across five key developmental stages. Approximately 480 million paired-end filtered reads were generated followed by identification of 81 901 transcripts from an early-maturing, high-yielding, drought-tolerant groundnut variety, ICGV 91114. Further, 57 344 genome-wide transcripts were identified with ≥1 FPKM across different tissues and stages. Our in-depth analysis of the global transcriptome sheds light into complex regulatory networks namely gravitropism and photomorphogenesis, seed development, allergens and oil biosynthesis in groundnut. Importantly, interesting insights into molecular basis of seed development and nodulation have immense potential for translational genomics research. We have also identified a set of stable expressing transcripts across the selected tissues, which could be utilized as internal controls in groundnut functional genomics studies. The AhGEA revealed potential transcripts associated with allergens, which upon appropriate validation could be deployed in the coming years to develop consumer-friendly groundnut varieties. Taken together, the AhGEA touches upon various important and key features of cultivated groundnut and provides a reference for further functional, comparative and translational genomics research for various economically important traits.

Keywords: Arachis hypogaea; allergens; gene expression atlas; gravitropism; oil biosynthesis; photomorphogenesis.

Figure 1

Samples used for developing groundnut gene expression atlas (AhGEA). (a) The 20 tissues targeted five growth stages including germinal (cotyledons, emerging radicle, hypocotyl, embryo and pre‐soaked seeds), seedling (root_seedling, and shoot_seedling), vegetative (leaves_veg, root_veg and stem_veg), reproductive (immature bud, flower, peg, seeds_05, seeds_15, seeds_25, pod wall_immature and pod wall_mature) and senescence (nodules and leaves_senescence). (b) Proportion of transcripts expressed at different levels (based on FPKM) in 20 tissues during the five plant growth stages. The bars indicated the number of transcripts expressed in each sample. Transcripts were categorized based on their expression: (i) no expression (0 < FPKM ≤ 1), (ii) low expression (1 < FPKM ≤ 10), (iii) moderate expression (10 < FPKM ≤ 100) and high expression transcripts (100 < FPKM). (c) Venn diagram of the numbers of shared and specifically expressed transcripts in germinal, seedling, vegetative, reproductive and senescence stage tissues.

Figure 2

Grouping of tissues according to the global transcript expression patterns. Heatmap of Pearson's correlation‐based hierarchical clustering (using corrplot and heatmaply function in R) of 20 tissues selected for developing the AhGEA. Genes with a normalized expression level (FPKM ≥ 1) in at least one of the 20 tissues analysed were log2‐transformed and are designated as expressed. The colour scale indicates the degree of correlation. Samples were clustered based on their pairwise correlations.

Figure 3

Seed development in groundnut. (a) A glimpse of the important biological processes involved during various stages of groundnut seed development. (b) Venn diagram of transcripts identified across four seed samples. (c) Venn diagram of transcripts identified in immature and mature pod walls. Venn diagrams were created using http://bioinfogp.cnb.csic.es/tools/venny/.

Figure 4

Expressed transcripts associated with gravitropism and photomorphogenesis. (a) Gene ontology analysis depicting the number of transcripts enriched for gravitropism and photomorphogenesis with their corresponding functional annotation. The colour of the horizontal axis shows the enrichment P‐values, the maximum of 0.05 in yellow and minimum in red. (b) The heatmap represents the expression (log transformed FPKM values) of differentially expressed genes (DEGs) involved in (1) embryonic development (purple bar), (2) gravitropism (orange bar), (3) hormone synthesis and response (blue bar), (4) photomorphogenesis (green bar), and (5) photosynthesis and light signal transduction (red bar). The DEGs have been identified from the nine pairwise comparison, namely seed_5 vs peg, seed_15 vs peg, seed_25 vs peg, seed_5 vs shoot_seedling, seed_15 vs shoot_seedling, seed_25 vs shoot_seedling, seed_5 vs stem_veg, seed_15 vs stem_veg, seed_25 vs stem_veg. First three categories mentioned above (related to gravitropism) have up‐regulated genes, and the other two categories (related to phototropism) have down‐regulated genes collectively. The blue color represents up‐regulation (FPKM ≥ 1) and red color denotes downregulation (FPKM ≤ 1) of the transcripts.

Figure 5

Global expression of allergen encoding transcripts across 20 selected groundnut tissues. (a) Distribution of allergens encoding transcripts across 20 selected groundnut tissues, (b) number of expressed transcripts across the tissues, (c) expression patterns of four major (Ara h1, Ara h2, Ara h3 and Ara h6) and one minor (Ara h8) across the selected 20 tissues.

Figure 6

Oil content and transcript expression pattern in developing groundnut seeds. (a) Phenotyping of oil and four different fatty acids at three different seed developmental stages, (b) Pairwise correlation of oil, protein and four different fatty acids for three seed developmental stages, (c) A conceptual diagram of transcripts encoding for enzymes related to fatty acid biosynthesis and TAG synthesis pathways.