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AbstractFruit of Lonicera caerulea L. is a rich source of phenolic compounds such as phenolic acids as well as anthocyanins, proanthocyanidins and other flavonoids, which display potential healthpromoting effects. However, its genetics and genomic information is limited. Transcriptome and digital gene expression (DGE) profiles of fruit at four different development stages were compared. Related genes with anthocyanin biosynthesis were compared using realtime quantitative PCR (qRTPCR). RNASeq generated 6.26Gb clean reads, which was then de novo assembled into 45,656 UniGenes with a mean length 837 bp. A total of 7 928 unigenes were annotated into 32 known metabolic or signaling pathways in which a few primary, intermediate, and secondary metabolic pathways are directly related to fruit quality. The result of DGE and qRTPCR indicates that UFGT (comp20132_c0) may play a key role in honeysuckle coloration. This provides a reference for the study of complicated metabolism in nonmodel perennial species.
Key wordsLonicera caerulea L.; RNASeq; DGE; Transcriptome
Received: May 3, 2018Accepted: August 16, 2018
Supported by Agricultural Science and Technology Innovation Project of China.
Qingtian ZHANG (1981-), male, P. R. China, assistant researcher, devoted to research about genetic breeding of fruit trees.
*Corresponding author. Email: aijun1005@163.com.
Lonicera caerulea L. (also known as haskap, blue honeyberry/honeysuckle) belongs to Caprifoliaceae family and comprises more than 200 species［1］. Blue honeysuckle berries are rich source of phenolic compounds such as phenolic acids as well as anthocyanins, proanthocyanidins and other flavonoids, which display potential healthpromoting effects. Chemopreventive, antimicrobial, antiadherence and antioxidant effects, among others, have been described for these compounds［2］. The anthocyanin content in honeysuckle is up to 657.10 mg/100 g［3］, and its anthocyanin composition was analyzed［4］. However, a comprehensive description of genes that expressed in L. caerulea fruit during the fruit development and maturation period remains unavailable. Based on genomewide expression profiles by sequencing, DGE is a powerful tool to identify and quantify gene expression on the whole genome level［5］.
This study has generated a large set of blue honeysuckle transcript sequences using the Illumina RNASeq method, which can be used to discover tissuespecific functions and the mechanisms of secondary metabolism. The dataset will also make it possible to construct high density microarrays for further characterization of gene expression profiles during these processes［6］. RNAseq and DGE Sequencing
Total RNA was extracted using modified CTAB method［7-8］, and RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using Qubit RNA Assay Kit in Qubit 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
The eight DGE libraries prepared from the samples at four different development stages (5, 10, 15, and 20 d after flowering) were constructed using Sample Preparation Kit. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500 platform and pairedend reads were generated by Novogene company.
qRTPCR Analysis of Selected Genes
Sixteen unigenes were chosen for validation using qRTPCR. The qRTPCR was performed with the ABI 7500 Fast RealTime Detection System (Applied Biosystems) with the Ultra SYBR Mix (with ROX) (CWBIO, Beijing, China). The thermal profile for SYBR Green I RTPCR was 95 ℃ for 10 min, followed by 40 cycles of 95 ℃ for 15 s and 55 ℃ for 1 min. Each plate was repeated three times in independent runs for all reference and selected genes.
The reference gene (βACTIN) was used for normalization. The comparative CT method (2ΔΔCT method) was used to analyze the expression levels of the different genes.
Results and Discussion
In total, there were about 64.5 million raw reads generated. All the 45656 unigenes were annotated. Digital gene expression (DGE) method generates direct gene expression measurements, which avoids the inherent limitation of microarray analysis. Eight DGE libraries with one biological repeat corresponding to four developmental stages of blue honeysuckle were sequenced with 6.93 to 8.58 million raw tags per library (Table 1). The number of clean tags per library ranged from 6.90 to 8.52 million after filtering out the low quality reads. Of all clean tags, 94.03% to 95.03% were mapped to unigenes.
The predicted biosynthesis pathways for the unigenes differed between the libraries, and most of the unigenes involved in these pathways were differentially expressed. In particular, we found that the unigenes predicted to be involved in anthocyanin biosynthesis significantly increased or decreased in different DGE libraries. The correlation between fruit anthocyanin content and the expression of anthocyanin biosynthetic genes has been studied in many crops, such as sweet cherry, grape and apple［9-11］. In this study, 15 key enzymeencoding candidate unigenes associated with anthocyanin biosynthesis in fruit were identified from the DEG data including PAL (3 unigenes), 4CL (1 unigene), CHS (2 unigene), CHI (1 unigene), F3H (2 unigene), DFR (3 unigenes), ANS (1 unigene) and UFGT (2 unigenes). These unigenes showed basically upregulated expression during the ripening process, particularly when the fruit turned red (stage 3 and stage 4) (Table 2). The result is consistent with our qRTPCR (Fig. 1) and previously reported results［9］. Most anthocyanin synthesis related genes in stage 4 exhibited higher expression than those in stage 3. This result indicates that the anthocyanin biosynthesis takes place rapidly during fruit coloring and ripening. Previous studies have shown that UFGT might play a regulatory role in anthocyanin biosynthesis at the transcriptional level and is a key enzyme gene in regulating anthocyanin synthesis in many plants, such as grape, malus and lychee［10-12］. This result indicates that UFGT (comp20132_c0) may play a key role in honeysuckle coloration.
Key wordsLonicera caerulea L.; RNASeq; DGE; Transcriptome
Received: May 3, 2018Accepted: August 16, 2018
Supported by Agricultural Science and Technology Innovation Project of China.
Qingtian ZHANG (1981-), male, P. R. China, assistant researcher, devoted to research about genetic breeding of fruit trees.
*Corresponding author. Email: aijun1005@163.com.
Lonicera caerulea L. (also known as haskap, blue honeyberry/honeysuckle) belongs to Caprifoliaceae family and comprises more than 200 species［1］. Blue honeysuckle berries are rich source of phenolic compounds such as phenolic acids as well as anthocyanins, proanthocyanidins and other flavonoids, which display potential healthpromoting effects. Chemopreventive, antimicrobial, antiadherence and antioxidant effects, among others, have been described for these compounds［2］. The anthocyanin content in honeysuckle is up to 657.10 mg/100 g［3］, and its anthocyanin composition was analyzed［4］. However, a comprehensive description of genes that expressed in L. caerulea fruit during the fruit development and maturation period remains unavailable. Based on genomewide expression profiles by sequencing, DGE is a powerful tool to identify and quantify gene expression on the whole genome level［5］.
This study has generated a large set of blue honeysuckle transcript sequences using the Illumina RNASeq method, which can be used to discover tissuespecific functions and the mechanisms of secondary metabolism. The dataset will also make it possible to construct high density microarrays for further characterization of gene expression profiles during these processes［6］. RNAseq and DGE Sequencing
Total RNA was extracted using modified CTAB method［7-8］, and RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using Qubit RNA Assay Kit in Qubit 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
The eight DGE libraries prepared from the samples at four different development stages (5, 10, 15, and 20 d after flowering) were constructed using Sample Preparation Kit. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500 platform and pairedend reads were generated by Novogene company.
qRTPCR Analysis of Selected Genes
Sixteen unigenes were chosen for validation using qRTPCR. The qRTPCR was performed with the ABI 7500 Fast RealTime Detection System (Applied Biosystems) with the Ultra SYBR Mix (with ROX) (CWBIO, Beijing, China). The thermal profile for SYBR Green I RTPCR was 95 ℃ for 10 min, followed by 40 cycles of 95 ℃ for 15 s and 55 ℃ for 1 min. Each plate was repeated three times in independent runs for all reference and selected genes.
The reference gene (βACTIN) was used for normalization. The comparative CT method (2ΔΔCT method) was used to analyze the expression levels of the different genes.
Results and Discussion
In total, there were about 64.5 million raw reads generated. All the 45656 unigenes were annotated. Digital gene expression (DGE) method generates direct gene expression measurements, which avoids the inherent limitation of microarray analysis. Eight DGE libraries with one biological repeat corresponding to four developmental stages of blue honeysuckle were sequenced with 6.93 to 8.58 million raw tags per library (Table 1). The number of clean tags per library ranged from 6.90 to 8.52 million after filtering out the low quality reads. Of all clean tags, 94.03% to 95.03% were mapped to unigenes.
The predicted biosynthesis pathways for the unigenes differed between the libraries, and most of the unigenes involved in these pathways were differentially expressed. In particular, we found that the unigenes predicted to be involved in anthocyanin biosynthesis significantly increased or decreased in different DGE libraries. The correlation between fruit anthocyanin content and the expression of anthocyanin biosynthetic genes has been studied in many crops, such as sweet cherry, grape and apple［9-11］. In this study, 15 key enzymeencoding candidate unigenes associated with anthocyanin biosynthesis in fruit were identified from the DEG data including PAL (3 unigenes), 4CL (1 unigene), CHS (2 unigene), CHI (1 unigene), F3H (2 unigene), DFR (3 unigenes), ANS (1 unigene) and UFGT (2 unigenes). These unigenes showed basically upregulated expression during the ripening process, particularly when the fruit turned red (stage 3 and stage 4) (Table 2). The result is consistent with our qRTPCR (Fig. 1) and previously reported results［9］. Most anthocyanin synthesis related genes in stage 4 exhibited higher expression than those in stage 3. This result indicates that the anthocyanin biosynthesis takes place rapidly during fruit coloring and ripening. Previous studies have shown that UFGT might play a regulatory role in anthocyanin biosynthesis at the transcriptional level and is a key enzyme gene in regulating anthocyanin synthesis in many plants, such as grape, malus and lychee［10-12］. This result indicates that UFGT (comp20132_c0) may play a key role in honeysuckle coloration.