Transcriptome Analysis Of Diploid And Tetraploid Watermelon And Ploidy Identification

Watermelon(Citrullus lanatus)is an annual vine herb.Its peel is green and fresh,its flesh is bright red and delicious.It is rich in nutrition,and also has the function of relieving summer heat and thirst.It is one of the most favorite fruits people eat in summer.Polyploidy,as an important marker of chromosome evolution in higher plants,is helpful for plants to adapt to the changes of environment and the formation of new species.Polyploid watermelon has many advantages.For example the fruit of polyploid watermelon is larger,which can improve the yield of watermelon.Using polyploid watermelon can improve the quality and nutrition of watermelon,And it also improves watermelon resistance through polyploidy.There are two key points in watermelon polyploidy breeding,one is chromosome doubling technology,the other is ploidy detection technology.At present,chromosome doubling technology is mature,but ploidy detection cannot be simple and low cost to deployed.There are few reports about the transcriptome of tetraploid watermelon.In this study,we used the herbicide Oryzalin to test the chromosome doubling of watermelon,sequenced the transcriptome of diploid and tetraploid watermelon,analyzed the differentially expressed genes between them,sorted out six genes that may be related to the ploidy of watermelon,and established the q RT-PCR method to detect the ploidy of watermelon.The main results are as follows:1.The tetraploid watermelon plants were successfully induced with20μmol/L Oryzalin.The induction rate was 25%.Because of its high induction efficiency,low concentration and harmelss,amhuangling could be used as a new and efficient tetraploid mutagen for human body and plants.2.RNA-Seq technique was used to sequence the high-throughput transcriptome of 1 diploid watermelon material 515(WMA)and 3 tetraploid watermelon materials 516(WMB),517(WMC)and 518(WMD).After quality control,a total of 18.48 Gb of Clean Data was obtained from 4 samples.The Clean Data of each sample was over 4.41 Gb,and the Q30 was over94.52%.Comparing the Clean Data of each sample with the reference genome,the comparison rate is over 95.27%.Based on the comparison results,the gene expression level of the samples was analyzed quantitatively.2782 differential expression genes(DEGs)were sorted out from three groups of wma and WMB,wma and WMC,wma and WMD.3.According to GO analysis,it was found that the most significant term ofdifferential expression genes between diploid watermelon and tetraploid watermelon in Biological Process was the biosynthesis process of nitrogen compounds,carboxylic acid metabolism process,oxy acid metabolism process,organic acid metabolism process,amine metabolism process,cell ketone metabolism process,etc.In Cellular Component,the most significant and differential gene enriched terms are photosystem,photosystem I reaction center,thylakoid,etc.In Molecular Function,the most significant and differential gene enriched terms are oxidoreductase activity.According to KEGG analysis,it was found that the differentially expressed genes between diploid watermelon and tetraploid watermelon were mainly active in photosynthesis,photosynthesis antenna protein,chlorophyll and porphyrin metabolism,glyoxylate and dicarboxylic acid metabolism.In addition,most of the high level difference genes in diploid and tetraploid watermelon were up-regulated.Oxidative stress protein,abscisic acid receptor,ethylene responsive transcription factor and other genes are up-regulated,which may be the key factors for the formation of watermelon polyploidy.4.Several genes related to ploidy,Cla97C10G205730,Cla97C10G187010,Cla97C01G019450,Cla97C02G026280,Cla97C01G004920 and Cla97C07G140200 were sorted out.According to GO function annotation,Cla97C10G187010,Cla97C02G026280,Cla97C01G004920 and Cla97C07G140200 are genes regulating nuclear components and controlling cell genetics and metabolism of polyploid watermelon.Cla97C10G205730 andCla97C01G019450 are genes regulating cell wall,vacuole membrane,endoplasmic reticulum,Golgi body and other structures of watermelon.On this basis,q RT-PCR was designed to identify the ploidy of watermelon.The results of Cla97C01G019450 and Cla97C02G026280 were good in the materials used in this experiment,but this method can only be used as a supplement to the conventional identification method,and the universality and accuracy need further test verification.