[Comparative analysis of active components and transcriptome between autotetraploid and diploid of Dendrobium huoshanense].

In this study, the roots, stems and leaves of diploid and autotetraploid Dendrobium huoshanense were used as materials to compare their contents of polysaccharides and alkaloids, and the transcriptome sequencing analysis was carried out. The results showed that the contents of polysaccharides and alkaloids in the roots, stems and leaves of tetraploid were 7.6%, 34.5%, 17.2%, 0.01%, 0.024% and 0.035% higher than those of diploid D. huoshanense, respectively. The contents of active components in different tissues were significantly different. There were 3 687 differentially expressed genes in diploid and tetraploid D. huoshanense, of which 2 346 genes were up-regulated and 1 341 down regulated. Go functional analysis showed that these genes were mainly involved in growth and development, stress resistance and other related functions. KEGG pathway analysis showed that most of the differential genes were concentrated in the processes of carbon metabolism, signal transduction, carbohydrate metabolism, amino acid metabolism and energy metabolism. The differential expression of key genes involved in the metabolism of polysaccharides, terpenes and polyketones, amino acid metabolism, hormone synthesis and signal transduction in diploid and tetraploid plants may be the main reason for the high energy content, the increase of active components and the growth potential of tetraploid plants.

[Identification and differential expression of miRNA related to seed dormancy of Paris polyphylla var. chinensis].

The purpose of this study was to explore the expression pattern of miRNA in the process of embryo dormancy and provide a reference for the mechanism of regulating seed dormancy and germination by miRNA. We used high-throughput sequencing technology, bioinformatics analysis and real-time fluorescent quantitative PCR(qPCR) technology to sequence, screen and identify miRNAs of dormant and dormant embryos. The results showed that there were 23 811 977, 24 276 695, 20 611 876 and 20 601 811 unique sequences in the four sample libraries during the period of dormancy and dormancy release. MiRNAs are mainly distributed between 21 and 24 nt, among which the length of 24 nt occurred most frequently. A total of 31 known miRNAs were identified, belonging to 13 different families. 93 new miRNAs were predicted by bioinformatics software. Ten miRNAs(mir156 a-5 p, mir160 a-5 p, mir160 h-1, mir169 a-5 p, mir157 d, mir159 a-1, mir395-3, mir156 f-5 p, mir156-2 and mir171 a-3 p) were screened out. In this study, 10 miRNAs related to seed dormancy release were identified. The target genes mainly involved carbohydrate metabolism, plant hormone signal transduction, cell division and growth. The results of qRT-PCR showed that the sequencing results were consistent with the actual results.

[Transcriptome analysis of seed embryo in dormancy and dormancy release state of Thesium chinense].

We used exogenous GA_3 to break the seed dormancy of Thesium chinense. We used high-throughput sequencing technology was used to sequence the transcriptome of dormant seed embryos and dormancy breaking seed embryos of Th. chinense, and the data was analyzed bioinformatically and systematically. The results showed that exogenous GA_3 could effectively break the seed dormancy of Th. chinense; 73 794 up-regulated genes and 42 776 down regulated genes were obtained by transcriptome sequencing; 116 570 diffe-rential genes were annotated by GO function to GO items such as metabolism process, cell process, cell, cell component, binding and catalytic activity. A total of 133 metabolic pathways were found by Pathway analysis of 26 508 differentially expressed genes. In the process of dormancy release, DEGs were mainly enriched in translation, carbohydrate metabolism, folding, classification, degradation and amino acid metabolism. Based on the annotation results in KEGG database, 20 metabolic pathways related to dormancy release were found. Dormancy release of Th. chinense seeds is a complex biological process, including cell morphology construction, secondary metabolite synthesis, sugar metabolism and plant signal transduction, among which plant hormone signal transduction is one of the key factors to regulate dormancy release. The results of qRT-PCR showed that the sequencing results were consistent with the actual results.

[Analysis of differential genes related to dormancy release of Paris polyphylla var. chinensis seeds by transcriptome sequencing].

The study aims at exploring the expression of differential genes and related metabolic pathways in the process of seed dormancy release. The dormant embryo and the dormant released embryo of Paris polyphylla var. chinensis were used as the test materials, a new generation high-throughput sequencing methods to sequence the transcriptome of the samples was used to carry out systematic bioinformatics analysis. We obtained 62 882 650 and 62 263 366 clean reads from the DNA libraries of the samples before and after dormancy breaking. A total of 69 248 differentially expressed genes(DEGs) were obtained, 56 426 up-regulated genes and 12 822 down-regulated genes. There are 138 267 differentially expressed genes in the process of embryo dormancy release, which were annotated by GO function to 58 subclasses of biological processes, molecular functions and cell components. The annotated differentially expressed genes were closely related to metabolic processes, biological regulation, cell component synthesis and enzyme catalytic activity. We found 139 metabolic pathways through pathway analysis of 58 722 differentially expressed genes. Before and after dormancy, DEGs were mainly enriched in carbon metabolism, secondary metabolite biosynthesis and polysaccharide metabolism. Based on the annotation results in KEGG database, we found 16 metabolic pathways related to the dormancy release of P. polyhoylla var. chinensis. A large number of differentially expressed genes were involved in embryo morphogenesis, polysaccharide decomposition and protein synthesis during seed development and dormancy release. It involves the interaction of multiple metabolic pathways and constitutes a complex regulation network for dormancy relief.

[In vitro autotetraploid induction and analysis on DNA methylation diversity of Platycodon grandiflorum].

In order to investigate the epigenetic variations between diploid and autotetraploid of Platycodon grandiflorus. The diploid buds of P. grandiflorus were soaked in the mixture of different concentration colchicines and 0.002 g•mL ⁻¹ dimethyl sulphoxide (DMSO)．The identification of autotetraploid plants were based on morphological characteristics, chromosome number and flow cytometry. And then the level and pattern of DNA methylation explored by using the technology of methylation sensitive amplified polymorphism (MSAP)．The result demonstrated that the buds soaked in 0.2% colchicines and 0.002 g•mL ⁻¹ DMSO solution for 12 h was ideal conditions to induce autotetraploid of P. grandiflorus, with induction rate of 32.0%.The diploid and tetraploid plants existed distinctly differences in morphological indexes.Totally,1 586 bands were amplified by 20 pairs of selective primers, of which 764 and 822 bands were detected in diploid and autotetraploid respectively. The total methylation ratio,full methylation ratio and hemimethylated ratio were 91.25%,61.25% and 30.65% in diploid of P. grandiflorus,respectively.However,the total methylation ratio,full methylation ratio and hemimethylated ratio of autotetraploid of P. grandiflorus were 86.13%,54.38% and 31.75%, respectively. Compared with diploid, the genomic DNA total methylate ratio and full methylation ratio of autotetration plants decreased by 6.02% and 7.14%.But the hemimethylated ratio of autotetraploid was higher than that of diploid, which more than 1.6%. All this results indicated that DNA methylation patterns have adjusted during the polyploidy process．.

[Morphology and AFLP analysis of tetraploid plantlets of Atractylodes macrocephala].

In order to investigate the genetic basis of morphological variation of tetraploid plantlets of Atractylodes macrocephala, diploid plantlets were taken as experimental material, sterile filtration colchicine was used to soak 0.5-1.0 cm long buds. The difference between morphology and stomatal of diploid and tetraploid of A. macrocephala was compared, and genome polymorphism was explored by AFLP. The results showed that the buds dipped in 0.1% colchicine solution for 36 h was optimal conditions to induce tetraploid of A. macrocephala with induction rate of 32.0%. Morphological indexes such as leaf area index, leaf length and width, the density of stomas and the number of chloroplast of tetraploid were distinctly different from diploid. Four hundred and fifty-one bands ranging with 80-500 bp were amplified with 24 pairs of primers, the rate of polymorphism was 32.59%. These amplification sites of diploid were different from tetraploid of A. macrocephala, and the differences in morphology of them were reflected in the DNA polymorphism.

A comparative morphological and transcriptomic study on autotetraploid Stevia rebaudiana (bertoni) and its diploid.

Stevia rebaudiana is an important medical plant for producing steviol glycosides (SGs) or stevioside. Autotetraploids (4x = 44) show an increasing level of morphology, physiology and tolerances comparing to diploids (2x = 22). However, little information regarded on the comparative transcriptome analysis between diploid and autotetraploid S. rebaudiana was found. In this study, synthetic autotetraploid was induced and morphological features were confirmed. A comprehensive transcriptome of stevia leaf, stem and root from the diploids and autotetraploids was constructed based on RNA-seq, yielded 1,000,892,422 raw reads and subsequently assembled into 251,455 transcripts, corresponded to 146,130 genes. Pairwise comparisons of the six leaf libraries between the diploids and autotetraploids revealed 4114 differentially expression genes (DEGs), in which 2105 (51.17%) were up-regulated in autotetraploids and associated with SGs biosynthesis, plant growth and secondary metabolism. Moreover, weighted gene co-expression network analysis showed co-expressed genes of fifteen genes of SG biosynthesis pathway were enriched in photosynthesis, flavonoid and secondary metabolic process, plant growth and morphogenesis. A hundred of DEGs related to plant resistance were identified by interviewing PlantPReS database. This study has highlighted molecular changes related to SGs metabolism of polyploidy, and advanced our understanding in plant resistance responsible for phenotypic change of autotetraploids.

[Application and study of HPLC fingerprint of Lonicera japonica for identification of geo-authenticity].

OBJECTIVE: To establish a chromatographic fingerprint of Lonicera japonica and apply it to identify geo-authentic of L. japonica. METHOD: The HPLC was applied in chromatographic separation and data were analysed by "computed aided similarity evaluation" software. RESULT: There exited distinct difference of chemical components group between and non-authentic samples. CONCLUSION: The established HPLC fingerprint can be used for the identification of geo-authentic of L. japonica.

[Determination of 18 organophosphate pesticide residues in Flos Lonicerae].

OBJECTIVE: To establish an analysical method for the quantitative determination of 18 kinds of organophosphate pesticide residues in Flos Lonicerae. METHOD: The pesticides in samples were extracted with acetone by Ultrasonic wave Assistant Extraction (UAE), purified by Solid-Phase Extraction (SPE). Using PFPD detecter gas chromatography with HP-1701 column. RESULT AND CONCLUSION: All 18 pesticides were separated well in 30 minutes. The detection limits was 4.0-18.0 microg x kg(-1). The range of recovery and RSD were 83.64%-88.65% and 2.8%-6.0%, respectively. And it met the requirements of pesticide multi-residues analysis.