Abstract
Chinese chestnut (Castanea mollissima Blume) seed kernels (CCSK) with high quality and quantity of starch has emerged as a potential raw material for food industry, but the molecular regulatory mechanism of starch accumulation in developing CCSK is still unclear. In this study, we firstly analyzed the fruit development, starch accumulation, and microscopic observation of dynamic accumulation of starch granules of developing CCSK from 10 days after flowering (DAF) to 100 DAF, of which six representative CCSK samples (50–100 DAF) were selected for transcriptome sequencing analysis. Approximately 40 million valid reads were obtained, with an average length of 124.95 bp, which were searched against a reference genome, returning 38,146 unigenes (mean size = 1164.19 bp). Using the DESeq method, 1968, 1573, 1187, 1274, and 1494 differentially expressed unigenes were identified at 60:50, 70:60, 80:70, 90:80 and 100:90 DAF, respectively. The relationship between the unigene transcriptional profiles and starch dynamic patterns in developing CCSK was comparatively analyzed, and the specific unigenes encoding for metabolic enzymes (SUSY2, PGM, PGI, GPT, NTT, AGP3, AGP2, GBSS1, SS1, SBE1, SBE2.1, SBE2.2, ISA1, ISA2, ISA3, and PHO) were characterized to be involved potentially in the biosynthesis of G-1-P, ADPG, and starch. Finally, the temporal transcript profiles of genes encoding key enzymes (susy2, pgi2, gpt1, agp2, agp3, gbss1, ss1, sbe1, sbe2.1, sbe2.2, isa1, isa2, isa3, and pho) were validated by quantitative real-time PCR (qRT-PCR). Our findings could help to reveal the molecular regulatory mechanism of starch accumulation in developing CCSK and may also provide potential candidate genes for increasing starch content in Chinese chestnut or other starchy crops.
Highlights
Abbreviations CCSK Chinese chestnut seed kernels days after flowering (DAF) Days after flowering RNA-Seq RNA sequencing quantitative real-time PCR (qRT-PCR) Quantitative real-time PCR differentially expressed genes (DEGs) Differentially expressed genes sucrose synthase (SUS) Sucrose synthase INV Invertase FK Fructose kinase UGP Uridyl diphosphate glucose pyrophosphorylase HK Hexokinase GPT Glucose-6-phosphate/phosphate transporter GLT Glucose transporter TPT Triose phosphate/phosphate translocator nucleotide translocator (NTT) ATP/ADP-transporter
The fruit shape index showed a significant decrease from 10 DAF (0.96 ± 0.01) to 60 DAF (0.76 ± 0.01), and increased to 0.86 ± 0.01 at 100 DAF, revealing that fruit shape changed from the initial oval to hemispheric during development (Fig. 1b, c), which corresponded to the previous study on C. sativa[9]
We found that transcripts of AGP2/3, GBSS1, SS1/3, SBE2.1/2.2, ISA3, and PHO all were significantly upregulated at 50–90 DAF, whereas those of SBE1, GBSS2, and ISA1/2 had high transcript abundance at early developmental stages (50–70 DAF), and low transcript levels were observed for AGP1 and SS4 throughout development (Additional file 9: Table S5 and Fig. 6)
Summary
Abbreviations CCSK Chinese chestnut seed kernels DAF Days after flowering RNA-Seq RNA sequencing qRT-PCR Quantitative real-time PCR DEGs Differentially expressed genes SUS Sucrose synthase INV Invertase FK Fructose kinase UGP Uridyl diphosphate glucose pyrophosphorylase HK Hexokinase GPT Glucose-6-phosphate/phosphate transporter GLT Glucose transporter TPT Triose phosphate/phosphate translocator NTT ATP/ADP-transporter. Chestnut is a major arboreal nut crop and its chemical composition reveals that starch is the most abundant component, comprising approximately 47–80% of the dry kernel weight[9,10], differing significantly from other temperate seeds, which are o ily[11]. This starchy property makes Chinese chestnut a potential substrate for fermentation, which could be developed for biological production in addition to utilization in the food industry. RNA-Seq analysis of developing CCSK could support a more comprehensive and reliable genetic information database, which could be used for research into chestnut molecular breeding to promote the development of chestnut resources for food, forest biomass energy or industrial material
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