Research On Transcriptional Regulation Mechanism Of Fruit Color Changes In Wild Fragaria Pentaphylla

Fragaria pentaphylla Lozinsk is a specific plant of China,which belongs to the strawberry genus of Rosaceae.It distributes mainly in Sichuan,Shaanxi and Gansu provinces,which often been found in the hillside or grassland with the altitude between of 1,000 meters to 2,300 meters.The main characteristics of F.pentaphylla include five pinnate leaflets,cyme and fleshy receptacle formed fruit.So far,the main research directions of strawberry are focused on cultivation,preservation and quality control of the fruit,while analysis at genomic and transcriptomic level are rare.Searching in the field,researchers found two typical wild varieties of F.pentaphylla.When maturation,the fruit color of one species becomes white but the other species becomes red.In this study,we sequenced the transcriptome of F.pentaphylla from seven groups(21 samples in total)through next generation sequencing(NGS)technology,which produced 9.8 GB data per sample on average.We studied the mechanisms of fruit color change of F.pentaphylla at RNA-Seq level,LncRNA level and co-expression network level and obtained following conclusions:1.Identification of the expression pattern of genes associated with fruit color change:a)rRNA was removed before the full transcriptome library was constructed,and 150 bp pair-end sequencing was performed using illumina HiSeq 4000.In total,206GB Clean Data were obtained,which represents 9.8 GB per sample on average.b)GO and KEGG Pathway enrichment analyses were performed on the DE genes.The results showed that DE genes were mainly enriched in pathways of secondary metabolic,plant-pathogen interaction,phenylpropanoid metabolic and flavonoid metabolic.Interestingly,DE genes that are enriched in the phenylpropanoid metabolic pathway are mainly related to peroxidase,anthocyanin 3-0-glucosyltransferase,cytochrome p450,β-glucosidase,peroxidase,cation Oxidase,4-coumaric acid-ligase,peroxidase,mannitol dehydrogenase,lysosomal β-glucosidase and catechol 7-O-methyltransferase.It indicates that phenylpropanoid metabolic pathway may play an important role in the process of anthocyanin synthesis.2.The function of IncRNA in the fruit development and fruit color formation of strawberry:1)30,393 lncRNA were strictly identified by following processes:(a)transcripts should with the length equal or longer than 200 nt.(b)When the transcripts were translated into proteins,the number of amino acids should lower than 100.(c)Public databases such as NCBI NR,Pfam,InterProscan and etc.,were used as reference to compare.2)Based on statistical analysis,we found the candidate IncRN As have large expression variances among different materials,which range from 0 to 22,666.18 fpkm.Most IncRNA have low expression and only expressed in one sample,only 2,526 lncRNAs were found to express in all samples.3)During the third developmental stage,only 64 IncRNAs were differentially expressed between white strawberry and white red strawberries.The fruit color is dramatically different between at this stage W and R had significant differences in fruit color.These specifically expressed IncRNAs may be involved in the color formation of strawberries.4)Based on the prediction of the target genes of 612 DE IncRNAs,7,996 trans-target genes were identified,resulted in 86,754 high associated IncRNA-gene pairs.Interestingly,some IncRNAs can multiple genes while some target genes were also regulated by multiple IncRNAs.Based on the location on the genome,we successfully identified the targets of 2,228 IncRNAs which may play role in cis-acting,most of them(-73%)located in intergenic region and only a small portion of IncRNAs were located within a gene.5)Based on the annotation of the target genes of these DE IncRNAs,we found 30 genes were involved in the synthesis of phenylpropionic acid,including 4-coumarate:CoA ligase(4CL),B-glucosidase(BGLU),cinnamyl-alcohol dehydrogenase(CAD),peroxidase(POD),caffeic acid-3-aminotransferase(caffeic acid 3 O-methyltransferase,COMT),coniferyl-aldehyde dehydrogenase(CALDH).3.The construction of co-expression network of genes related to fruit development and fruit color formation:1)PR10 is one important member of disease-related protein family,which not only involved in both biotic and abiotic stress responses but also plays important role in plant development and co-existence with rhizobium.Genes in the gene module correlated with fruit color are significantly enriched in the biological process of plant-pathogen interaction and stress responses,which also contain multiple PR proteins.Further analysis focused on the PR10 protein family has help to identify 29 PR10 protein family members,which all contain the conserved domain Bet vl-like(cd07816).In addition,except TRINITY_DN49653_c0_g2,MSTRG.5876 and TRINITY_DN118165_c0_g1,the other members of PR10 family all contain the highly conserved glycine-rich loop sequence(EG(D/N)GG(V/P)G(T/S)).Among them,TRINITY_DN111701_c0_g3 and MSTRG.13759 are the orthologous of Fra a 1E,which is known to involve in flavonoid synthesis.Both of them showed significantly different expression between white(W)and red(R)fruit,and the expression are higher in R.The expression fold changes range from 1.45 to 5.12 among the three developmental stages.MSTRG.13759,which is identified in this study is a new member of PR 10 family and probably have similar function as Fra a 1E.Further analysis is needed to confirm whether its N’ extended sequence affect the intermediary metabolism formation of flavonoid synthesis.2)Gene annotation of DE genes between W and R among the three developmental stages revealed that they are enriched in plant-pathogen interaction and fatty acid metabolism.Genes in the MEpink module are mainly involved in fatty acid metabolism and wax coat biosynthesis(9 genes).We also identified 7 DE genes related to elongation of fatty acids and 7 DE genes related to cuticle and waxiness biosynthesis.These above mentioned genes are continuously displayed expression variance between the two materials during different development stages,which provide a candidate list for future analysis.3)In this study,we constructed a complex strawberry fruit development and fruit color formation related molecular regulation network by analyzing co-expression network,protein-protein interaction and target prediction of miRNA-mRNA,miRNA-lncRNA and IncRNA-mRNA.A protein interaction network containing 20,770 genes was constructed.Within this network,there were "one to one","one to many" and "many to many" interactions,and a total of 2.8 M protein interaction pairs were obtained.The results showed that lncRNA could be involved in the development of strawberry fruit and fruit color formation process by a variety of methods.Meanwhile,we also identified some unknown function genes that involved in the above process.Further studies focus on the functions of these genes would greatly help to study the molecular regulation mechanisms of fruit development and color formation of strawberry.