Identification Of Functional Genes And MicroRNAs Relative To Taproot Thickening In Radish (Raphanus Sativus L.)

Radish(Raphanus sativus L.)is an important annual or biennial root vegetable crop belonging to the Brassicaceae family.The fleshy taproot compries the main edible portion of the plant with high nutrition and medical value,and is rich in carbohydrate,folic acid,ascorbic acid and sulforaphane.The size of the fleshy taproot directly determines the yield and quality of radish.Thus.it’s of vital significance to explore the growth process and molecular regulatory networks of taproot thickening in radish.The fleshy taproot thickening of radish is a complex biological process involving morphogenesis and dry matter accumulation.Previous studies mainly focused on the morphological and physio-biochemical levels.Recently,several studies have been invested into investigating the molecular mechanisms underlying taproot development in radish.However,the molecular mechanism underlying radish taproot thickening is still unclear.In this study,to isolate differentially regulated genes as well as miRNAs and their target genes involved in taproot thickening,RNA sequencing(RNA-seq)technology was employed to characterize the de novo transcriptome of radish taproots using an advanced inbred line ’NAU-YH’ as the plant material,and then the gene expression profile and small RNA expression profile were performed during radish taproot thickening.The main achievements obtained were as follows.(1)To develop a comprehensive overview of the radish root transcriptome,a cDNA library,prepared from three equally mixed RNA of taproots at different developmental stages including pre-cortex splitting stage,cortex splitting stage and expanding stages was sequenced using high-throughput Illumina RNA sequencing.From approximately 51 million clean reads,a total of 70,168 unigene with a total length of 50.28 Mb,an average length of 717 bp and a N50 of 994 bp were obtained.In total,63,991(about 91.20%of the assembled unigenes)unigenes were successfully annotated to five public databases including NR,GO,COG.KEGG and Nt.GO term analysis revealed that the majority of these unigenes were predominately involved in basic physiological and metabolic processes,catalytic,binding and cellular process.In addition,a total of 103 unigenes encoding eight enzymes in the sucrose metabolism related pathways were also identified,from which,two genes were validated by T-A cloning and sequencing while six were validated by RT-qPCR analysis.In addition,the full cDNA and genomic DNA sequences of RsCLE41 and RsSAUR genes were isolated.The open reading frame(ORF)sequences of RsCLE41 and RsSAUR genes were 300 bp and 327 bp,encoding 99 and 108 amino acids,respectively.Furthermore,the RT-qPCR analysis showed that RsCLE41 and RsSAUR genes exhibited different expression and regulation during radish taproot thickening.The RsCLE41 gene was highly expressed in the root and stem,especially in root organ of pre-cortex splitting stage.RsSAUR had higher expression profiles in root organ during the different taproot thickening stages,except for pre-cortex splitting stage.Moreover,the highest expression level of RsSAUR was observed at cortex splitting stage.These results would be served as an important public reference platform to identify the related key genes during taproot thickening and facilitate the understanding of molecular mechanisms underlying taproot thickening in radish.(2)To identify key miRNAs involved in the taproot thickening in radish,three small RNA libraries from radish taproots collected at pre-cortex splitting stage(Stagel),cortex splitting stage(Stage2)and expanding stage(Stage3)were constructed by Solexa high-throughput sequencing.In total 175 known and 107 potential novel miRNAs were isolated,from which 85 known and 13 novel miRNAs were found to be significantly differentially expressed during taproot thickening.Furthermore,totally 191 target genes were identified for the differentially expressed miRNAs.These target genes were annotated as transcription factors and other functional proteins,which were involved in various biological functions including plant growth and development,metabolism,cell organization and biogenesis,signal sensing and transduction,and plant defense response.Notably,some target transcripts such as NF-YA2,ILR1,bHLHW4,XTH16,CEL41 and EXPA9 were involved in radish taproot thickening.The expression patterns of five miRNAs and their corresponding target genes were validated by RT-qPCR,and the results were approximately accordant with the Solexa analysis.These results could provide new insights into the regulatory roles of miRNAs during the taproot thickening and facilitate genetic improvement of taproot in radish.(3)To isolate differentially regulated genes involved in radish taproot thickening process,three RNA libraries from radish taproot collected at pre-cortex splitting stage(L1),cortex splitting stage(L2)and expanding stage(L3)were constructed and sequenced by RNA-Seq technology.In all,7,219,043,7,047,977 and 7,161,612 clean reads were obtained from L1,L2 and L3 librariy,respectively;from which 4,717,617(65.35%),4,809,588(68.24%)and 4,973,745(69.45%)reads were matched to the radish reference genes.A total of 85,939 transcripts were generated from three libraries,from which 10,450(LI Vs.L2),12,325(L1 Vs.L3)and 7,392(L2 Vs.L3)were significantly differentially-regulated.GO analysis showed that these differentially-regulated transcripts could involve in cell events,metabolic process,single-organism process and stimulant response.The enrichment analysis of pathway function indicated that these transcripts were assigned to 127(L1 vs.L2),126(L1 vs.L3)and 122(L2 vs.L3)pathways,respectively.Plant hormone signal transduction(ko04075)and starch and sucrose metabolism(ko00500)were the most abundant categories in pair comparison of each library.Additionally,114 miRNA-mRNA pairs(43 unique miRNAs and 92 genes)were co-expressed in three taproot thickening stages by the correlation analysis between DGE and sRNA sequencing.Notably,some genes,including EXPA9,Cyclin,katanin,Syntaxin,MADS-box,SAUR and CalS could play significant roles during radish taproot thickening.The expression patterns of 16 selected genes were validated by RT-qPCR,and the results were approximately accordant with the RNA-seq analysis.These results would provide new insights into the complex molecular mechanism underlying taproot thickening in radish.