ABSTRACT
italic>Tussilago farfara L. is a perennial herb of Tussilago genus in the Compositae family. Its dried buds and leaves have good biological activities and have a long history of medicinal use in China and Europe. In this paper, we investigated the whole chloroplast genome characteristics, sequence duplication, structural variation and phylogeny of the Tussilago farfara L. After sequencing the Tussilago farfara L. chloroplast genome using Illumination technology, the complete Tussilago farfara L. chloroplast genome was further obtained by assembly and annotation, followed by a series of inverted repeat-large single copy/small single copy region contraction and expansion analysis, genome sequence variation, etc. The sequences of 13 homologous plants downloaded from NCBI were used to construct a neighbor-joining phylogenetic tree. The results showed that the total GC content of the chloroplast genome was 37.4% and the length was 150 300 bp; 125 genes were annotated, including 82 protein-coding genes, 35 tRNAs and 8 rRNAs; 148 (simple sequence repeats, SSR) loci were detected, and the relative synonymous codon usage showed that 31 codons out of 64 codons had a usage of >1. In the phylogenetic analysis, the chloroplast genomes of the seven species of Asteraceae, including the Yulin Tussilago farfara L., were highly conserved, and the sequence variation of the (large single-copy, LSC) and (small single-copy, SSC) regions was higher than that of the (inverted repeat, IR) region. This is in general agreement with the reported phylogeny of Yulin Tussilago farfara L. In this study, we obtained a high quality chloroplast genome and analyzed its genome characteristics, codon preference, SSR characteristics, SC/IR boundary, sequence variation and phylogeny, which can provide a basis for species identification, genetic diversity analysis and resource development of this medicinal plant.
ABSTRACT
Objective: To screen candidate genes involved in the terpenoid biosynthetic pathway of Tussilago farfara. Methods: The transcriptome of buds and leaves of wild T. farfara were respectively sequenced using the Illumina HiSeq 2500 high-throughput sequencing platform. The clean reads were de novo assembled by Trinity software, and the assembled sequences then followed by a series of bioinformatics analysis such as gene function annotation and differential expression gene. According to sequence annotation and differentially expressed genes analysis, the key enzyme genes related to the terpenoid biosynthesis were identified. Results: After high through-put sequencing, a total of 39 912 371 clean reads were obtained (SRA accession: SRR9113366, SRR9113367). The clean reads were then assembled into 91 118 unigenes. A total of 55 830 unigenes were annotated by a similarity search against NR, Swiss-Port, GO, COG, KEGG five public databases. Base on KEGG annotation and differentially expressed genes, totally 129 catalytic enzyme genes referring to the terpenoid biosynthesis were identified, including 91 terpenoid backbone biosynthesis genes, 32 terpene synthases, and 6 cytochrome P450 (CYP450) genes. Among them, 25 genes were differentially expressed. The expression of four enzyme genes in MVA pathway in leaves were higher than that in buds, while the five enzyme genes in MEP pathway were lower in leaves than that in buds. In addition, 10 genes were highly expressed in leaves, and nine genes were highly expressed in buds. According to the high expression of differentially expressed HMGR, TPS, AS, CYP450 genes in buds, it was speculated that these genes may be related to the high content of terpenoids in flower buds. Conclusion: This work obtained candidate key enzyme genes that may be involved in the biosynthesis of terpenoid by transcriptome sequencing. The results laid a foundation for further elucidating the molecular mechanism of terpenoid biosynthetic pathway in T. farfara.
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OBJECTIVE: To compare the chemical compositions of the stems (STL) and leaves (LTL) of Tussilago farfara L. using NMR-based metabolomic approach. METHODS: The STL and LTL were analyzed by NMR, then the differential components were determined by multivariate statistical METHODS:, including PCA, PLS-DA, OPLS-DA, and univariate analysis. RESULTS: Fifty compounds were tentatively identified in the NMR spectra of STL and LTL. Multivariate coupled with univariate analysis revealed that some metabolites, such as valine,leucine,isoleucine,proline,chlorogenic acid,3,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, and tussilagone,were present at higher levels in the LTF, while the STF contained higher levels of α-glucose and β-glucose.For some metabolites, such as malic acid, sucrose and choline, no significant differences were observed between STF and LTF. CONCLUSION: This study reveals the chemical differences between STF and LTF in a holistic way, and lays the scientific foundation for the resource utilization of the stems and leaves of T. farfara L.
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Objective: The SSR loci information in the transcriptome of Tussilago farfara was analyzed and specific primers were designed, so as to provide powerful tools for molecular marker-assisted breeding in this plant. Methods: SSR loci in 18 938 unigenes with length of 1 kb or more obtained by transcriptome sequencing were searched by using MISA. SSR primers were designed by Primer3 and 55 pairs were randomly selected for the polymorphic analysis on 18 samples collected from different habitats. Results: A total of 4 688 SSRs were detected in the transcriptome of T. farfara, distributed in 3 844 unigenes with the distribution frequency of 24.75%. SSR loci occurred every 7 979 bp. Trinucleotide repeats appeared to be the most abundant SSRs with a frequency of 37.12%, followed by mononucleotide (32.36%) and dinucleotide (28.20%). Among all 60 repeat motifs, A/T (31.42%), AG/CT (12.80%), and ATC/ATG (9.62%) were the predominant repeat types. For validating the availability of the SSR primers, 55 pairs of primers were randomly selected for polymorphism analysis. Among them, 42 pairs (76.36%) produced clear and reproductive bands and 14 pairs showed polymorphism. Eighteen plants were divided into three groups by UPGMA. Conclusion: The SSR markers in the transcriptome of T. farfara show high frequency, rich type, and high polymorphism, which will provide the abundant candidate markers for genetic diversity, genetic mapping construction and marker-assisted breeding study for this plant.
ABSTRACT
Objective: To compare the expression of genes in the leaves of Tussilago farfara that involved in biosynthesis of phenylpropanoids in different developmental stages, and infer the accumulation period of biosynthesis of phenylpropanoids and provide a scientific basis for the resource utilization of leaves of T. farfara. Methods: The Illumina HiSeq2500 highthroughput sequencing method was used to analyze the transcriptome of the leaves of T. farfara in different periods. After obtaining transcriptome data, bioinformatics analysis of gene function annotation was performed to compare the expression of genes related to phenylpropanoid biosynthesis in different periods. Results: A total of 46 793 unigenes were obtained by transcriptome sequencing and the average length was 952.144 8 bp. Among them, 4 774 unigenes were annotated in the public databases NR, Swiss-Prot, eggNOG, GO, and KEGG. According to the assignment of KEGG pathway, 144 unigenes were involved in terpenoid biosynthesis, phenylpropanoid biosynthesis and flavonoids, 65 unigenes were involved in terpenoid biosynthesis, 64 unigenes were involved in phenylpropanoid and 15 unigenes were involved in flavonoids biosynthesis. The enzyme genes involved in the phenylpropanoid biosynthesis were also compared in different development stages, and the results indicated that the expression of PAL, 4CL, HCT, and CCoAOMT, which were closely related to biosynthesis of phenylpropanoids, were highest in September, which means that the contents of these compounds might be highest in September. Conclusion: This study lays the foundation for the biosynthetic pathway and regulation analysis of phenylpropanoids, and provides a scientific basis for the development and the resource utilization of leaves of T. farfara.