Genome-wide transcriptome analysis of Chinese pollination-constant nonastringent persimmon fruit treated with ethanol
© Luo et al.; licensee BioMed Central Ltd. 2014
Received: 8 August 2013
Accepted: 4 February 2014
Published: 8 February 2014
The persimmon Diospyros kaki Thunb. is an important commercial and deciduous fruit tree. The fruits have proanthocyanidin (PA) content of >25% of the dry weight and are astringent. PAs cause astringency that is often undesirable for human consumption; thus, the removal of astringency is an important practice in the persimmon industry. Soluble PAs can be converted to insoluble PAs by enclosing the fruit in a polyethylene bag containing diluted ethanol. The genomic resource development of the persimmon is delayed because of its large and complex genome. Second-generation sequencing is an efficient technique for generating huge sequences that can represent a large number of genes and their expression levels.
We used 454 sequencing for the de novo transcriptome assembly of persimmon fruit treated with 5% ethanol (Tr library) and without treatment as the control (Co library) to investigate the genes and pathways that control PA biosynthesis and other secondary metabolites. We obtained 374.6 Mb in clean nucleotides comprising 624,690 and 626,203 clean sequencing reads from the Tr and Co libraries, respectively. We also identified 83,898 unigenes; 54,719 (~65.2%) unigenes were annotated based on similarity searches with known proteins. Up to 14,954 of the unigenes were assigned to the protein database Clusters of Orthologous Groups (COG), 24,337 were assigned to the term annotation database of Gene Ontology (GO), and 45,506 were assigned to 200 pathways in the database of Kyoto Encyclopedia of Genes and Genomes (KEGG). The two libraries were compared to identify the differentially expressed unigenes. The expression levels of genes involved in PA biosynthesis and tannin coagulation were analysed, and some of them were verified using quantitative real time PCR (qRT-PCR).
This study provides abundant genomic data for persimmon and offers comprehensive sequence resources for persimmon research. The transcriptome dataset will improve our understanding of the molecular mechanisms of tannin coagulation and other biochemical processes in persimmons.
The persimmon Diospyros kaki Thunb. (2n = 6X = 90) originated in China and was principally cultivated in China, Korea and Japan . Persimmon cultivars are classified into four types, including pollination-constant nonastringent (PCNA), pollination-constant astringent (PCA), pollination-variant nonastringent (PVNA), and pollination-variant astringent (PVA); this classification is based on the effect of pollination on flesh colour and the natural loss of astringency at the harvest time on the tree . The PCNA type includes Japanese PCNA (JPCNA) and Chinese PCNA (CPCNA), which differ in their genetic characteristic of PCNA trait . The natural loss of astringency is a trait that is qualitatively inherited and recessive in JPCNA cultivars [3, 4] but dominant in CPCNA cultivars. When the CPCNA cultivar ‘Luotian-tianshi’ is crossed as the maternal parent to a JPCNA or non-PCNA type, the F1 offspring are segregated into a 1:1 ratio for PCNA:non-PCNA types [5, 6]. CPCNA cultivars have attracted attention in the breeding industry because of their natural ability to lose astringency, which is a dominant trait. In addition, CPCNA has the potential to be an important parent in PCNA persimmon breeding in the future.
Persimmon resources are widely distributed in China. However, almost all traditional cultivars native to China are of the PCA type; some of these cultivars include ‘Mopanshi’, ‘Fuping-jianshi’, and ‘Gongcheng-shuishi’ . ‘Luotian-tianshi’ (D. kaki Thunb.; 2n = 6X = 90) is the first PCNA persimmon native to China, and it is only distributed in Dabieshan Mountain around the junction of Hubei, Henan, and Anhui provinces in central China [4, 8].
Most persimmon fruits accumulate proanthocyanidins (PAs) in their flesh during development, causing the sensation of astringency due to the coagulation of oral proteins . PAs or condensed tannins are synthesised via the shikimate and flavonoid biosynthetic pathways [10–12]. To date, many genes encoding the structural proteins and transcription factors involved in PA biosynthesis, transportation, and polymerisation have been isolated by homology-based cloning [13–23]. However, the primary genes involved in PA biosynthesis have not yet been determined.
High-throughput sequencing technologies developed in recent years provide a convenient way of establishing a rapid and efficient molecular research platform. Next-generation sequencing (NGS) is related to the Sanger sequencing method, which is represented by first-generation sequencing technologies. Currently, the three mainstream NGS technologies are Roche/454 pyrosequencing (developed in 2005, http://www.454.com), Illumina/Solexa sequencing (developed in 2006, http://www.illumina.com), and ABI/SOLiD sequencing (developed in 2007, http://www.appliedbiosystems.com). These NGS technologies vary in their input requirements and sequence output with regard to the total bases sequenced, length of each sequence read, and price per megabase of sequence information . Among these technologies, 454 sequencing, which generates a minimum number of sequence reads, produces the longest reads (i.e. from 100 bp to ~800–1000 bp). Long reads are optimal for initial genome and transcriptome characterisation because longer pieces are assembled more efficiently than shorter pieces . Given their rapid processing, high throughput, and cost effectiveness, NGS technologies have been successfully used to study genomes and transcriptomes of species with and without sequenced genomes. Many novel and functional genes can be obtained from massive amounts of data.
Abundant genetic resources for persimmons are currently available. However, genomic information and EST sequences for this fruit tree are lacking. In addition, molecular data on persimmons are insufficient when compared to those of other fruit trees, such as apple, pear, peach, citrus, and grape. Accordingly, we performed large-scale transcriptome sequencing of CPCNA persimmon fruit using Roche/454 technology to create a transcript sequence database of the persimmon and identify candidate genes involved in PA biosynthesis and tannin coagulation. We used IDEG6 to filter the differentially expressed genes in the treatment (Tr) and control (Co) libraries. We also verified the differentially expressed unigenes by quantitative real time PCR (qRT-PCR). The present study provides a platform for studying the genes involved in persimmon tannin coagulation and tannin biosynthesis to analyse the relationship between differentially expressed genes and persimmon fruit deastringency and clarify the mechanism of astringency loss for CPCNA.
Sequencing and assembly
Summary of the sequencing and assembly
No. of HQ reads
Total nucleotides (nt)
Mean length of read (bp)
No. of unigenes of combined data
Total nucleotides (nt) of unigenes
Mean length of unigenes (bp)
No. of contigs above 500 bp
Annotation of non-redundant unigenes
Number of annotated unigenes
Percentage of annotated unigenes (%)
Detection of differentially expressed unigenes in the Tr and Co libraries
Summary of differentially expressed unigenes
Number of unigenes
Differentially expressed in two library
Up (Tr vs Co)
Down (Tr vs Co)
Expressed both in Tr and Co
Expressed only in Tr
Expressed only in Co
Quantitative real-time PCR confirmation
Currently, the most common application of NGS in nonmodel species is transcriptome characterisation [30–34]. Among the currently available NGS technologies, 454 pyrosequencing produces the longest reads; thus, it has emerged as a powerful tool for transcriptome sequencing. In addition, many studies have used de novo assembly of such data to produce genome-level resources for non-model organisms [35–38].
Genetic studies of the persimmon are difficult to implement because of the hexaploid nature of the species and lack of linkage maps and whole genome sequences [i.e. only 14,189 EST sequences deposited in GenBank (accessed on 10/11/2013)]. Previous genetic studies have focused on the diversity and phylogeny of cultivated persimmons and related wild species. The transcriptome characterisation described in the present study will provide the initial information needed for the functional study of persimmons. A total of 624,690 and 626,203 reads were generated from the Tr and Co libraries, with average sequence lengths of 319 and 309 bp, respectively. We obtained 83,898 unigenes from these raw reads (Table 1). The average unigene length for persimmons in this study was comparable to that observed in other species such as Oncidium (493 bp) , Pinus contorta (500 bp) , Fraxinus (649 bp) , and Vicia faba (615 bp) . Further, the unigene was longer than that observed in Ziziphus celata (408 bp)  and Olea europaea (355 bp)  but shorter than that in Castanea mollissima (731 bp)  and Pyrus pyrifolia (853 bp) . The length of unigenes may be related to the sequencing technique and assembly tools used. However, the unigenes assembled in the present study could provide resources for future persimmon genetic and genomic research.
BLAST searches against public databases, such as NR, SwissProt, GO, and KEGG, provided annotation data for the persimmon, with 54,719 (65.2%) unique hits (Table 2). The annotation of persimmon gained more descriptive information than that observed in other species such as Conyza canadensis (51.3%) , Bupleurum chinense (52.6%) , Dendrocalamus latiflorus (54.9%) , Lens culinaris (55.6%) , and Panax quinquefolius (63.6%) . However, it was lower than that observed in Fagopyrum (66.7%) , Taxus cuspidata (68.6%) , Capsicum annuum (72.04%) , Olea europaea (73%) , Pyrus pyrifolia (74.1%) , Dendrocalamus latiflorus (78.9%) , Eichhornia paniculata (87.0%) , and Fraxinus (99%) . However, comparing this information across species studies is difficult because of the different sequencing depths or BLAST parameters utilized in each report .
A total of 31,211 persimmon unigenes were mapped into 200 KEGG pathways (Additional file 2). The genetic information processing pathway (12,838) and cellular processes pathway (2606) were highly represented categories in the metabolic pathways. Most persimmon fruits accumulate PAs in their flesh during development; PAs cause astringency due to coagulation of oral proteins and are synthesised from metabolites via the shikimate and flavonoid pathways [10–12]. Therefore, we focused on the pathways pertaining to phenylpropanoid biosynthesis (ko00940, 123 unigenes) and flavonoid biosynthesis (ko00941, 62 unigenes). Most of the genes related to PA biosynthesis in these two pathways were found in our transcriptome sequencing data. PAs cause astringency that is often undesirable for human consumption; thus, the removal of astringency is an important practice in the persimmon food industry. Soluble PAs can be converted to insoluble PAs by enclosing the fruit in a polyethylene bag containing diluted ethanol . Acetaldehyde formed in situ from ethanol is involved in the direct insolubilisation of soluble PAs, causing a loss of astringency . Pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are two important enzymes in this process, which is involved in the glycolysis/gluconeogenesis pathway (ko00010, 496 unigenes). Moreover, six and 31 unigenes were classified into the PDC and ADH families, respectively.
Summary of genes involved in PA biosynthesis and tannin coagulation
Number of unigenes
Number of reads/Co
Number of reads/Tr
We found that the gene aldehyde dehydrogenase family 2 (ALDH2) was highly expressed (1409 reads) in the Co library and downregulated in the Tr library (545 reads), with a total of 11 unigenes in both libraries (Table 4). ALDH2 has a broad expression pattern and is most notably involved in the second step of ethanol metabolism, (i.e. acetaldehyde oxidation). The decrease in ALDH2 in the Tr library might have inhibited the conversion of acetaldehyde to acetic acid, which, consequently, led to acetaldehyde accumulation. Large amounts of acetaldehyde triggered the coagulation of tannins (insolubilisation of soluble PAs) causing the loss of astringency in the treated persimmon fruits. This result suggests that the ALDH2 gene, together with the ADH and PDC genes, might have important functions in tannin coagulation.
The present study on persimmon transcriptome has several biological implications. First, the plant material persimmon, which accumulates PAs (condensed tannins) in its flesh during development, can be considered a model plant for tannin research. Second, the loss of astringency in CPCNA fruits treated with ethanol is an imitation of the natural loss of astringency, especially for tannin coagulation. This imitation helped us to understand mechanism of astringency loss in CPCNA. Third, the current study, based on the present transcriptome data (even in the absence of complete genome sequences for persimmons), will facilitate the advancement future genetic studies.
This work presents the first de novo transcriptome sequencing analysis of the CPCNA persimmon fruit using the 454 GS FLX Titanium platform. A total of 374.6 Mb of data were generated and assembled into 83,898 unigenes. Persimmon unigenes related to PA biosynthesis were characterised, and differentially expressed unigenes in the two libraries were verified using qRT-PCR. ADH, PDC, and the newly discovered persimmon gene ALDH2 were found to have important functions in tannin coagulation. To the best of our knowledge, this study is the first to employ the 454 sequencing technology to investigate the whole transcriptome of the persimmon fruit. The assembly of the reads was also conducted without a reference genome. The transcriptome characterisation described in the present study will provide the initial information needed for the functional study of persimmons to elucidate the molecular mechanisms of tannin coagulation and other biochemical processes in this fruit tree.
In previous analysis of tannin concentration per fruit, JPCNA and CPCNA varied considerably. Both types accumulate PA in their fruits at an early stage. PA accumulation is halted in JPCNA at 7–9 weeks after bloom (WAB), and a low concentration of PA is observed at 10 WAB. On the other hand, CPCNA continuously accumulates PA until the late stages and maintains a very high PA concentration [22, 23]. Thus, it appears that at 9–10 WAB, JPCNA and CPCNA exhibit different PA accumulation patterns. In the present study, 30 young fruits on a CPCNA-type persimmon tree (D. kaki ‘Luotian-tianshi’, 2n = 6X = 90) grown in the Persimmon Repository of Huazhong Agricultural University, China, were enclosed with polyethylene bags containing 10 mL of 5% ethanol on 12 July 2011 (10 WAB). Control (untreated) fruits were enclosed with polyethylene bags containing 10 mL of water. Three days later, all treated and control fruits were sampled and peeled. The flesh of the fruits was diced into small pieces, frozen in liquid nitrogen, and stored at −80°C until use for RNA isolation.
The concentrations of soluble and insoluble tannins in the control and treated fruit flesh were measured by the Folin–Ciocalteu method after 3 d of treatment . Soluble tannins of the fruit flesh were also examined after 3 d of treatment by the printing method , which is a convenient way of identifying persimmon astringency loss. FeCl2 reacts with the soluble PAs; thus, the darker the resulting product, the more astringent the fruits.
RNA extraction, cDNA library construction, and 454 sequencing
For each sample (5% ethanol treated and control), approximately 10 g of mixed flesh (10 individuals) was used for RNA preparation and tannin concentration measurements. Total RNA was extracted using TRIzol Reagent (Invitrogen, USA) following the manufacturer’s protocol. The quality and quantity of the total RNA was analysed using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and gel electrophoresis.
Approximately 1 μg of RNA was used to generate double-stranded cDNA using the SMARTTM cDNA Library Construction Kit (Clontech, USA). Finally, ~5 mg of cDNA was used to construct a 454 library. Roche GS-FLX 454 pyrosequencing was conducted by the Oebiotech Company in Shanghai, China.
454 de novo transcriptome assembly and analysis
A Perl program was written to remove vector sequences and the PolyA (T) tail from sequences; reads with lengths <100 bp were removed before assembly. Then, high-quality reads were assembled with MIRA  to construct unique consensus sequences. The 454 setting parameters were used by MIRA (−−job = denovo,est,normal, 454; -SK:mnr = yes; -SK:rt = 2; 454_SETTINGS -LR:mxti = no; -CL:qc = no).
The functions of the unigenes were annotated by BLASTX with an E-value threshold of 10−5 to the protein databases, including NCBI-NR, Swiss-Prot, KEGG , and COG . InterPro domains  were annotated by InterProScan  Release 16.0, and functional assignments were mapped onto GO . WEGO  was used for GO classification and GO tree construction.
Differentially expressed unigene detection
A freely available web tool IDEG6  was used to identify unigenes showing statistically significant differences in relative abundance (as reflected by the total count of individual sequence reads) between the Tr and Co libraries. The general Chi-squared method was used because it was the most efficient analytical method . Finally, unigenes with P ≤ 0.01 were deemed significantly different between the two libraries.
RNA extraction, first-strand cDNA synthesis, and qRT-PCR analysis
A total of 50 unigenes generated by 454 sequencing were selected for experimental validation. The total RNA used for qRT-PCR analysis was extracted from the flesh of the Tr and Co fruits. After RNA extraction, first-strand cDNA was synthesised from 1.0 μg of RNA using the PrimeScript® RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer’s protocol. The cDNA was diluted threefold and used as the template for qRT-PCR. qRT-PCR was performed on a LightCycler® 480 II System (Roche Diagnostics) using SYBR® Premix Ex TaqTM II (TaKaRa). The reaction was composed as described in the manual and was performed in quadruplicate. A negative control (no template) was included in each run. The standard amplification protocol consisted of an initial denaturing step of 95°C for 30 s, followed by 45 cycles of 95°C for 5 s, 60°C for 10 s, 72°C for 15 s, and a melting temperature cycle with constant fluorescence data acquisition from 65°C to 95°C. The gene quantification method was based on the relative expression of the target gene versus the reference gene (DkActin), and the ratio was calculated with the LightCycler® 480 software. All primers are listed in Additional file 3.
This research was supported by the Natural Science Foundation of China (31171929) and the Special Scientific Research Fund of Agricultural Public Welfare Profession of China (201203047).
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