| Cassava（Manihot esculenta Crantz） is one of the most important energy crop and the staple food source for over 700 million people in the world. It has extremely drought tolerance and survives in tender long dry season,which also has a ability of rapid growth and development after raining.Although there were some studies on the molecular mechanisms of drought resistance in cassava,whether there was relationship between drought tolerance and DNA methylation in cassava or not still is unknow.We use a new tool to mine whole genome methylation which is called Amplified Fragment SNP and Methylation （AFSM） to detect DNA methylation levels in the genome under drought stress.In this experiment, we used cassava cultivars SC 124 and Arg7 as the materials,of which SC124 is tolerance to drought stress, and Arg7 seems sensitive to drought.The whole genome DNA methylation levels were detected by comparison among drought treated 3 days, 12 days and 18 days with control. The main gains as the followings:1.We build the high quality cassava genomic DNA AFSM library. EcoR Ⅰ-Hpa Ⅱ and EcoR Ⅰ-Msp Ⅰ were used to reduce the complexity of the entire genomic DNA by cleaving the DNA. Through the linker, PCR amplification, available for sequencing of the library.The results showed that 36 samples of EcoR Ⅰ, Hpa Ⅱ and Msp Ⅰ were identified in the PCR amplification library by monoclonal detection, bacterial liquid PCR and subsequent bacterial fermentation. Subsequent sequencing data is accurate and reliable, in line with the AFSM database requirements.2. We obtained 30G original data with Illumina HiSeq 2000 paired end sequencing a lane,and map the reads filtered with a perl script according to barcode to the cassava AM560 reference genome. Then, the methylation site information in the data was found by AFSMmethxiaV1.0.pl, a methylation site mining software dedicated to AFSM, and finally a total of 12906 methylation sites were obtained in the all 36 samples of genome.We compared the methylation level between drought treatments and controls with double varieties.It was shown that the DNA methylation increased under drought stresses generally. But there were different response between drought tolerant and sensitive varieties.The methylation level of SC124 in stem increased first and then lower with the increaed drought stress time,while the Arg7 manifested the opposite trend, which was proved that DNA methylation behaved variety specificity in cassava under drought stress.The AFSM analysis showed that the genomic DNA methylation level of leaves in cassava were 8.2%-13%.The DNA methylation level of roots in cassava were 4.4%-27.8%. The study reveals that the pattern of DNA methylation under drought stress is temporal specific and tissue specific.3. The BiNGO analysis of all genes involved in DNA methylation sites in drought stress shown that the biological classification of the genes corresponding to the methylation-changing sites was closely related to the process of drought resistance, such as glycosylation of protein amino acids, transcription factor composition, signal transduction,substance transport, response to external signals and so on. It indicated that DNA methylation plays an important role in regulating drought stress in cassava.4. Combining the RNA-Seq data and the methylation site gene derived from AFSM,we found some genes with up-regulation under drought stress, and clarified the relationship between plant resistance gene and DNA methylation. The research shed a light on the molecular mechanism of DNA methylation in the regulation of drought resistance in cassava. |
