De novo Sequencing and Transcriptome Analysis of Pinellia ternata Identify the Candidate Genes Involved in the Biosynthesis of Benzoic Acid and Ephedrine.

BACKGROUND: The medicinal herb, Pinellia ternata, is purported to be an anti-emetic with analgesic and sedative effects. Alkaloids are the main biologically active compounds in P. ternata, especially ephedrine that is a phenylpropylamino alkaloid specifically produced by Ephedra and Catha edulis. However, how ephedrine is synthesized in plants is uncertain. Only the phenylalanine ammonia lyase (PAL) and relevant genes in this pathway have been characterized. Genomic information of P. ternata is also unavailable. RESULTS: We analyzed the transcriptome of the tuber of P. ternata with the Illumina HiSeq™ 2000 sequencing platform. 66,813,052 high-quality reads were generated, and these reads were assembled de novo into 89,068 unigenes. Most known genes involved in benzoic acid biosynthesis were identified in the unigene dataset of P. ternata, and the expression patterns of some ephedrine biosynthesis-related genes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Also, 14,468 simple sequence repeats (SSRs) were identified from 12,000 unigenes. Twenty primer pairs for SSRs were randomly selected for the validation of their amplification effect. CONCLUSION: RNA-seq data was used for the first time to provide a comprehensive gene information on P. ternata at the transcriptional level. These data will advance molecular genetics in this valuable medicinal plant.

Transcriptomic comparison of the self-pollinated and cross-pollinated flowers of Erigeron breviscapus to analyze candidate self-incompatibility-associated genes.

BACKGROUND: Self-incompatibility (SI) is a widespread and important mating system that promotes outcrossing in plants. Erigeron breviscapus, a medicinal herb used widely in traditional Chinese medicine, is a self-incompatible species of Asteraceae. However, the genetic characteristics of SI responses in E. breviscapus remain largely unknown. To understand the possible mechanisms of E. breviscapus in response to SI, we performed a comparative transcriptomic analysis with capitulum of E. breviscapus after self- and cross-pollination, which may provide valuable information for analyzing the candidate SI-associated genes of E. breviscapus. METHODS: Using a high-throughput next-generation sequencing (Illumina) approach, the transcriptionexpression profiling of the different genes of E. breviscapus were obtained, some results were verified by quantitative real time PCR (qRT-PCR). RESULTS: After assembly, 63,485 gene models were obtained (average gene size 882 bp; N50 = 1485 bp), among which 38,540 unigenes (60.70% of total gene models) were annotated by comparisons with four public databases (Nr, Swiss-Prot, KEGG and COG): 38,338 unigenes (60.38% of total gene models) showed high homology with sequences in the Nr database. Differentially expressed genes were identified among the three cDNA libraries (non-, self- and cross-pollinated capitulum of E. breviscapus), and approximately 230 genes might be associated with SI responses. Several these genes were upregulated in self-pollinated capitulum but downregulated in cross-pollinated capitulum, such as SRLK (SRK-like) and its downstream signal factor, MLPK. qRT-PCR confirmed that the expression patterns of EbSRLK1 and EbSRLK3 genes were not closely related to SI of E. breviscapus. CONCLUSIONS: This work represents the first large-scale analysis of gene expression in the self-pollinated and cross-pollinated flowers of E. breviscapus. A larger number of notable genes potentially involved in SI responses showed differential expression, including genes playing crucial roles in cell-cell communication, signal transduction and the pollination process. We thus hypothesized that those genes showing differential expression and encoding critical regulators of SI responses, such as MLPK, ARC1, CaM, Exo70A1, MAP, SF21 and Nod, might affect SI responses in E. breviscapus. Taken together, our study provides a pool of SI-related genes in E. breviscapus and offers a valuable resource for elucidating the mechanisms of SI in Asteraceae.

Transcriptome analysis of Panax vietnamensis var. fuscidicus discovers putative ocotillol-type ginsenosides biosynthesis genes and genetic markers.

BACKGROUND: P. vietnamensis var. fuscidiscus, called "Yesanqi" in Chinese, is a new variety of P. vietnamensis, which was first found in Jinping County, the southern part of Yunnan Province, China. Compared with other Panax plants, this species contains higher content of ocotillol-type saponin, majonoside R2. Despite the pharmacological importance of ocotillol-type saponins, little is known about their biosynthesis in plants. Hence, P. vietnamensis var. fuscidiscus is a suitable medicinal herbal plant species to study biosynthesis of ocotillol-type saponins. In addition, the available genomic information of this important herbal plant is lacking. RESULTS: To investigate the P. vietnamensis var. fuscidiscus transcriptome, Illumina HiSeq™ 2000 sequencing platform was employed. We produced 114,703,210 clean reads, assembled into 126,758 unigenes, with an average length of 1,304 bp and N50 of 2,108 bp. Among these 126,758 unigenes, 85,214 unigenes (67.23%) were annotated based on the information available from the public databases. The transcripts encoding the known enzymes involved in triterpenoid saponins biosynthesis were identified in our Illumina dataset. A full-length cDNA of three Squalene epoxidase (SE) genes were obtained using reverse transcription PCR (RT-PCR) and the expression patterns of ten unigenes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Furthermore, 15 candidate cytochrome P450 genes and 17 candidate UDP-glycosyltransferase genes most likely to involve in triterpenoid saponins biosynthesis pathway were discovered from transcriptome sequencing of P. vietnamensis var. fuscidiscus. We further analyzed the data and found 21,320 simple sequence repeats (SSRs), 30 primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism in 13 P. vietnamensis var. fuscidiscus accessions. Meanwhile, five major triterpene saponins in roots of P. vietnamensis var. fuscidicus were determined using high performance liquid chromatography (HPLC) and evaporative light scattering detector (ELSD). CONCLUSIONS: The genomic resources generated from P. vietnamensis var. fuscidiscus provide new insights into the identification of putative genes involved in triterpenoid saponins biosynthesis pathway. This will facilitate our understanding of the biosynthesis of triterpenoid saponins at molecular level. The SSR markers identified and developed in this study show genetic diversity for this important crop and will contribute to marker-assisted breeding for P. vietnamensis var. fuscidiscus.

[SSR information in Erigeron breviscapus transcriptome and polymorphism analysis].

OBJECTIVE: The SSR information in the transcriptome of Erigeron breviscapus was analyzed in this study, in order to further develop new functional genes SSR markers laid a solid foundation. METHOD: SSR loci were searched in all of 52,060 unigenes by using est_timmer. Perl program and SSR primers were designed by Primer3. Furthermore, 36 pairs of primers were randomly selected for the polymorphism analysis on 13 Erigeron breviscapus plants collected from different places. RESULT: A total of 3639 SSRs were found in the transcriptome of Erigeron breviscapus, distributed in 3260 unigenes with the distribution frequency of 6.99%. Di-nucleotide repeat was the main type, account for as much as 34.41% of all SSRs, followed by mono-nucleotide (31.41%) and tri-nucleotide repeat motif (28.08%). The di-nucleotide repeat motifs of AT/AT and AC/GT were the predominant repeat types (28.71%). The tri-nucleotide repeat motifs of AAT/AT was the predominant repeat types (7.94%). For validation the availability of those SSR primers, we randomly selected 36 pairs of primers for PCR amplification. Among them, 34 pair primers (94.44%) produced clear and reproductive bands, 19 pair primers showed polymorphism (52.78%), and 13 Erigeron breviscapus plants were divided into 2 groups. CONCLUSION: There are numerous SSRs in Erigeron breviscapus transcriptome with high frequency and various types, this will provide abundant candidate molecular markers for genetic diversity study and genetic map in this plant.

Analysis of the transcriptome of Erigeron breviscapus uncovers putative scutellarin and chlorogenic acids biosynthetic genes and genetic markers.

BACKGROUND: Erigeron breviscapus (Vant.) Hand-Mazz. is a famous medicinal plant. Scutellarin and chlorogenic acids are the primary active components in this herb. However, the mechanisms of biosynthesis and regulation for scutellarin and chlorogenic acids in E. breviscapus are considerably unknown. In addition, genomic information of this herb is also unavailable. PRINCIPAL FINDINGS: Using Illumina sequencing on GAIIx platform, a total of 64,605,972 raw sequencing reads were generated and assembled into 73,092 non-redundant unigenes. Among them, 44,855 unigenes (61.37%) were annotated in the public databases Nr, Swiss-Prot, KEGG, and COG. The transcripts encoding the known enzymes involved in flavonoids and in chlorogenic acids biosynthesis were discovered in the Illumina dataset. Three candidate cytochrome P450 genes were discovered which might encode flavone 6-hydroase converting apigenin to scutellarein. Furthermore, 4 unigenes encoding the homologues of maize P1 (R2R3-MYB transcription factors) were defined, which might regulate the biosynthesis of scutellarin. Additionally, a total of 11,077 simple sequence repeat (SSR) were identified from 9,255 unigenes. Of SSRs, tri-nucleotide motifs were the most abundant motif. Thirty-six primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism. The result revealed that 34 (94.40%) primer pairs were successfully amplified and 19 (52.78%) primer pairs exhibited polymorphisms. CONCLUSION: Using next generation sequencing (NGS) technology, this study firstly provides abundant genomic data for E. breviscapus. The candidate genes involved in the biosynthesis and transcriptional regulation of scutellarin and chlorogenic acids were obtained in this study. Additionally, a plenty of genetic makers were generated by identification of SSRs, which is a powerful tool for molecular breeding and genetics applications in this herb.

[SSR information in transcriptome of Pinellia ternata].

OBJECTIVE: In order to establish new molecular markers and to design SSR primers,the SSR information was analyzed in the transcriptome of Pinellia ternata. METHODS: The unigenes in transcriptome were filtered and then redundant sequences were removed by the tool of EST-trimmer and cross-match, respectively. Meanwhile, the non-redundant unigenes were examined by the tool of MISA. RESULTS: 14,468 SSRs were found, and these SSRs were distributed in 12 000 unigenes in the examined sequences. Frequency of occurrences for SSRs was 16.24%, and density of distribution was on average 1/4.33 kb in the present study. Di-nucleotide and tri-nucleotide were the major repeated types,accounting for 51.15% and 41.50%, respectively. AG/CT and CCG/CGG were most frequent motifs in di-nucleotide and tri-nucleotide, accounting for 41.54% and 11. 84%, respectively. 87.14% motifs in the length ranged from 12 bp to 20 bp. CONCLUSION: The SSR site in the transcriptome of Pinellia ternata have the characteristics of high frequency of occurrences, type richness, high density and high potential of polymorphism, and these characteristics might be applied in the study on genetic diversity, marker-assisted breeding,and genetic map of Pinellia ternata.