Cloning And Expression Analysis Of S Locus Related Gene Based On RNA-Seq In Ping'ou Hybrid Hazelnut(Corylus Heterophylla Fisch.× Corylus Avellana L.)

Ping'ou hybrid hazelnut,belonging to economic forest,is the main cultivated species of Chinese Corylus,and is mostly characteristic of self-incompatibility.In production,it is necessary to arrange suitable pollinated cultivars for the main cultivars.And cross breeding is now a main method to obtain new cultivars of Ping'ou hybrid hazelnut.So the compatibility study of Ping'ou hybrid hazelnut plays a very important role in high-yielding cultivation and germplasm innovation.According to classical genetics,hazelnut belongs to sporophytic self-incompatibility.However,the molecular biology study on S locus and its related genes are still at the initial stage in hazelnut.In this study,the main cultivars 'Dawei' of Ping'ou hybrid hazelnut were used as materials,and RNA-seq was performed to explore the transcriptome profile of pistils under non-pollination,compatible pollination and incompatible pollination in Ping'ou hybrid hazelnut.Based on the RNA-seq dataset,the stable reference gene suitable for real-time quantitative PCR was screened and verified.Finally,eight S locus related genes were cloned by RACE technology.All the researches in this study aims to reveal the molecular mechanism of self-incompatibility in Corylus.The main results are as follows: 1.The RNA-Seq of pistils in Ping'ou hybrid hazelnutApproximately 42,046,035,660 nt data from Ping'ou hybrid hazelnut was generated by Illumina HiSeq 2000 RNA-seq platform,with 56,177 annotated Unigene.There was totally obtained 53,054 matched and predicted coding protein box,including 26 COG classifications,55 GO classifications and 128 metabolic pathways.The comparison between IC4 treatment and other treatment samples showed that the 4th hour after self-incompatibility pollination treatment was a key stage to study self-incompatibility of hazelnut.In addition,the Blast and SMART comparison results,based on the RNA-seq data,confirmed that the hazelnut plants did belong to the sporophyte self-incompatible.2.Identification and validation of reference genes for RT-qPCR analysis in Ping'ou hybrid hazelnutTwelve candidate reference genes were screened based on RNA-seq data and previous studies.The agarose gel electrophoresis of reverse transcription PCR product and the melting curves of real-time quantitative PCR analysis showed that the primer specificity of the candidate reference genes was good.The results of primers evaluation showed that the E-value of primers amplification efficiency was between 86.3-121.6% and R2 value was more than 0.98,and the linear regression equation was reliable.The expression stability of the 12 candidate reference genes in 24 samples were evaluated by four different programs geNorm,NormFinder,BestKeeper and Delta C.The 24 samples included different tissues or organs,the pistils at different time after self-pollination and the pistils at 4 hours after compatible or incompatible pollination.ChaActin and ChaEF1-? were ranked as the most stable reference gene across all samples by the four evaluated programs together.Finally,the expression of published gene Cav Prx was analyzed to prove their practicability.The results showed that the selected reference gene were useful for detecting the gene expression in Corylus,and can be used for the expression analysis of genes related to self-incompatibility in hazelnut.3.Cloning and analysis of S locus related genes in Ping'ou Hybrid HazelnutBased on RACE cloning technology and the screened unigenes,the full length cDNA of eight S locus related genes were cloned.These genes included ChaTHL1/ChaTHL2,ChaMLPK,ChaExo70A1,ChaARC1,ChaMOD1/ChaMOD2 and ChaKAPP.The bioinformatics analysis,including the analysis of the physical and chemical properties of protein,the domain and functional prediction of amino acid sequence and evolutionary analysis,showed that: 1)Cha THL1 and Cha THL2 was a stable protein,had one thioredoxin domain,with protein disulfide oxidoreductase activity.In evolution analysis,ChaTHL1/Cha THL2 belonged to THL-like protein;2)ChaMLPK was a basic hydrophilic stable protein,with rich phosphorylation sites and one STKs domain.Phylogenetic analysis displayed that ChaMLPK was closest to the protein kinase APK 1A sequence of Juglans regia;3)Cha ARC1 was acid stable hydrophilic protein,had one U-box and two ARM domains,with ubiquitin transferase activity Phylogenetic analysis indicated ChaARC1 was cluster to U-box domain-containing protein 14 of Juglans regia;4)Cha Exo70A1 was a hydrophilic protein,which had one Exo domain.It belonged to the extracellular complex,and had the closest relationship with Exo70A1 of Juglans regia;5)Both Cha MOD1 and Cha MOD2 were basic hydrophobic protein,belonged to the PIP family.They were part of the membrane and could participate in the process of substance transport;6)Cha KAPP was an acid hydrophilic protein,with phosphoprotein phosphatase activity.This protein harbored one FHA and one PP2 C domain,showed a close genetic relationship with PP2 C 70 of Juglans regia.In the study,the stable genes(ChaActin and ChaEF1-a)were screened as double internal reference genes.The expressions of the above genes were analyzed by real-time quantitative PCR,ChaActin and ChaEF1-a was used as double reference genes.The expression analysis indicated that: 1)ChaMLPK and ChaMOD1/ChaMOD2 all had a certain amount of expression in the tissues or organs,except in pollen.However,the other 5 genes were constitutively expressed in all various tissues or organs;2)within 24 hours after self-incompatible pollination,ChaTHL1/ChaTHL2,ChaARC1 and ChaKAPP had the highest expression at 2 h,while ChaMLPK,ChaMOD1 and ChaExo70A1 reached the highest expression at 24 h,and the highest expression of ChaMOD2 was at 1 h.