Transcriptome Profile And DNA Methylation Analysis Of Continuously Cropped Andrographis Paniculata

Continuous cropping obstacle, also called "Replant problem", is a major problem in agricultural production, which manifests itself in crops that were planted in a field where had cultivated the same species for a long time. It is well known that continuous cropping can decrease the yield, quality, and disease resistances of crops, especially in medicinal plants. Many medicinal plants have replant problem in varied degrees, such as Panax ginseng, Panax quinquefolius, Rehmannia glutinosa, etc. Continuous cropping obstacle has become an agricultural production problem that causes serious damage, and demands prompt solution.Andrographis herba is the dried aboveground parts of Andrographis paniculata (Burm. f.) Nees belongs to Acanthaceae family. It is widely used in the clinic and is also the raw material of many Chinese patent medicine. There are reports mentioned that A. paniculata has replant problem in herb farming, however, research studies mainly focused on the allelopathy of its phytoma; the effects of continuous cropping on the growth of A. paniculata remains unknown.In order to confirm the effects of continuous cropping on A. paniculata growth, growth indexes, physiological traits, yields and effective components of A. paniculata samples continuously cropped for 0-,1-, and 2 years, were measured and analyzed. Moreover, transcriptome and DNA methylation analysis of continuously cropping A. paniculata were also carried out to elucidate the mechanism of continuous cropping obstacle. The methods and conclusions are summarized below.1 Effects of continuous cropping on growth of A. paniculataResults showed that the plant height, leaf area, chlorophyll content and yield of A. paniculata significantly increased with the length of continuously cropping years (P<0.05). The flowering and primary fruit stages were both brought forward for 5-7 days and the content of effective components reduced after continuous cropping. The content of MDA, POD, SOD, and CAT also increased with the length of continuously cropping years.2 Transcriptomic analysis of continuously cropped A. paniculataThe global transcriptome of continuously cropped A. paniculata was sequenced using Illumina 2000 high-throughput sequencing technology. A total of 231,536,164 clean reads and 2.58 Gb of nucleotides were generated from the cDNA libaray. These clean reads were assembled into 43,683 Unigenes with a mean length of 1,045 bp. After bio informatics annotation,10,584 Unigenes were classified into 24 function categories in COG database, 21,158,22,596 and 12,884 Unigenes were sorted into three main categories respectively, including biobgical process, cellular component and molecular function in GO database. Searching against the KEGG database,8,024 Unigenes were mapped into 122 pathways. Moreover, we also constructed three expression profile of continuously cropped A. paniculata and 8,691 differential expressed genes (DEGs) were found. Using the STEM (Short Times-series Expression Miner) software, we obtained eight expression trends. By analysing pathways related to chemical components biosythesis of A. paniculata, eight key enzyme genes of phenyklanine metabolism pathway,9 key enzyme genes of flavonoid biosynthesis pathway and 6 key enzyme genes of diterpenoid biosynthesis were annotated and their expressed levels were determined in different continuously cropping years using quantitative PCR.3 DN A me thy latin analysis of continuously cropped A. paniculataThe total DNA methylation level of A. paniculata leaf continuously cropped for 0-,1-, and 2 years, were determined using LC-MS/MS and MS AP. Both results indicated that the DNA methylation level gradually decreased with the increase of the continuously cropping years. The DNA methylation level was 10.26%～13.91% determined by LC-MS/MS, and was 8.20%-9.24% detected by MSAP and the demethylation was the main pattern of CCGG sites.In conclusion, this study demonstrated the influence of continuous cropping on A. paniculata. The transcriptome data base of continuously cropped A. paniculata was constructed and series of DEGs were screened out. Furthermore, we also determined the DNA methylation level of continuously cropped A. paniculata. Research combining transcriptome and DNA methylation will be beneficial for understanding of the mechanism of continuous cropping in A. paniculata, it will also provide a theoretical basis for standardized planting, and a novel and considerable research perspective for continuous cropping obstacle of other medicinal plants.