Analysis Of Cold Tolerance In Chongyi Wild Mandarin Based On Physiological Indexes And Transcriptome Profiling

As one of the most important fruit crops in the world,citrus is widely cultivated in China.However,citrus is often suffered from severe cold damage,which has great negative impact on the citrus industry.One of the ways to solve this problem is to use wild citrus germplasm for cold-tolerant breeding.Chongyi wild mandarin（Citrus reticulata）is a wild citrus variety discovered in the mountainous region of Chongyi County in the south of Jiangxi Province.Matured branches of Chongyi wild mandarin and Poncirus trifoliata were detached and treated under 0 ^oC.In order to compare the cold tolerance between these two materials,low temperature-related physiological and biochemical indexes were determined.Moreover,RNA-Seq was applied to screen the low temperature responsive differentially expressed genes（DEGs）in chongyi wild mandarin.The result of RNA-Seq was partially verified by q RT-PCR.It is of great significance to explore the physiological response of chongyi wild mandarin under low temperature stress and dissect its cold tolerance molecular mechanism as well as to single out candidate genes that could be used in breeding new cultivars with enhance cold tolerance.The main results were as follows:1.Based on the changes of physiological and biochemical indexes of cold-challenged leaves from Chongyi wild mandarin and P.trifoliata,the relative electrolyte leakage（EL）and the content of malondialdehyde（MDA）in the former were significantly lower than in the later,indicating that the membrane system of Chongyi wild mandarin was less damaged by cold than that of P.trifoliata;the content of H₂O₂and the activities of peroxidase（POD）,catalase（CAT）,superoxide dismutase（SOD）and anti-superoxide anion(Anti-O₂^?-)were higher in chongyi wild mandarin,implying that it was more tolerant to oxidative stress caused by cold.The contents of proline and soluble sugars were higher in chongyi wild mandarin throughout the low temperature treatment,indicating that Chongyi wild mandarin had a strong ability to relieve osmotic stress caused by low temperature.Based on the obtained physiological data,membership function（MFV）analysis value was calculated and the comprehensive evaluation index of Chongyi wild mandarin was higher than that of P.trifoliate,suggesting that Chongyi wild mandarin was more cold-tolerant.2.RNA-Seq was performed with the leaves of Chongyi wild mandarin under 0 ^oC low temperature stress for 0 d,1 d and 5 d.Totally,64.94 Gbp clean reads were obtained,comparing with 0 d,there were totally 3921 DEGs（1585 up-regulated and 2036 down-regulated）were identified in 1 d,and 5995 DEGs（3141 up-regulated and 2854 down-regulated）were identified in5 d.3.GO enrichment analysis showed that DEGs were mainly enriched to the metabolic process,oxidation-reduction process and catalytic activity functional groups in 1 d,while in 5 d,DEGs were mostly annotated to the functional groups of cellular protein metabolic process,macromolecule modification and phosphotransferase activity.4.Five hundred and thirty-three transcription factors（TFs）from 67 TF families were identified in RNA-Seq data of Chongyi wild mandarin.Among the identified DEGs,22 genes were related to CBF-dependent/independent transcriptional regulatory pathway,24 genes related to ABA signal pathway,60 genes related to GA signal pathway,42 genes related to carbohydrate synthesis and metabolism,35 genes related to ROS,19 genes related to LEA protein and 5 genes related to proline synthesis and metabolism,which may be the key genes in response to low temperature stress in Chongyi wild mandarin.5.Seventeen candidate genes were selected for q RT-PCR verification,they all showed similar expression trends in q RT-PCR with that in RNA-Seq data,indicating that the sequencing results of RNA-seq were reliable.