| As one of the most important quality traits in citrus flavor,the citrus aroma has attracted the attention of consumers.The terpenes are the most important compound in the volatiles of citrus,which not only affects the aroma quality of citrus fruit but also plays an important role in the resistance against pests and diseases.The differences of volatile compounds in different citrus fruit have been a hotspot of research in recent years,but most of them are not systematically.There are hundreds of citrus germplasms and the aroma quality is various,which provides rich and diverse material for the study of citrus aroma quality.Therefore,it is of great significance to evaluate citrus germplasm at both metabolic and molecular levels for the exploration of valuable resources and will benefit the development of the citrus industry.The research was carried in three parts: firstly,the volatiles in different citrus germplasms and different tissues were qualitatively and quantitatively determined,then identify the biomarker compounds to distinguish different citrus species based on the contents of volatiles in fruit peel by PLS-DA;secondly,multi-omics method to identify and functional analysis some tissue-specific expression TPS genes;finally,further verified the xenia effect of C.mangshanensis work in HB pummelo and Feicui pummelo based on our previous studies,and identify the Cit LO1 might play an important role in linalool oxides synthase.The main results were as follows: 1.Volatile profiling of various citrus germplasms and different tissues1.1 The volatile profiles of fruit peels and juice sacs from 108 citrus accessions representing seven species were analyzed using GC–MS.162 and 107 compounds were determined in the peels and juice sacs,respectively.In the peels,monoterpene alcohols were accumulated in loose-skin mandarins;clementine tangerines and papedas were rich in sesquiterpene alcohols,sesquiterpenes,monoterpene alcohols,and monoterpene aldehydes.Furthermore,concentrations of 34 distinctive compounds were selected to best represent the volatile profiles of seven species for HCA,and the clustering results were in agreement with classic citrus taxonomy.Comparison of profiles from different growing seasons and production areas indicated that environmental factors play important roles in volatile metabolism.Besides,some citrus germplasms that accumulated certain compounds were determined as promising breeding materials.1.2 Principal component analysis results were indicated that different citrus species could almost completely be separated based on volatile compounds.Thirty volatiles were identified as potential biomarkers in discriminating loose-skin mandarin,sweet orange,pummelo,and lemon,while 17 were identified as effective biomarkers in discriminating clementine mandarins from the other loose-skin mandarins and sweet oranges.Finally,30 citrus germplasms were used to verify the classification based on β-elemene,valencene,nootkatone,and limettin as biomarkers.The accuracy values were 90.0%,96.7%,96.7%,and 100%,respectively.This research may provide a novel and effective alternative approach to identifying citrus genetic resources.The contents of biomarker compounds in different citrus species might affect the aroma trait of citrus fruit.1.3 A total of 198 and 82 volatile compounds were identified in 62 citrus leaves and 25 flowers,respectively.The dominant volatile terpenoids were more diverse in citrus leaves than in peels,such as d-limonene was the most abundant compounds in citrons;trans-β-ocimene was in C.ichangensis;and linalyl acetate was in sour oranges.In wild or semi-wild germplasms,31 of the 34 commonly volatile terpenoids were over-accumulated,consistent with the increased expression level of 5 and 1 key genes in the mevalonate and methylerythritol phosphate biosynthetic pathways and reduced geranylgeranyl diphosphate synthase expression level in the methylerythritol phosphate pathway compared to those in cultivars,these results suggested a negative effect of citrus domestication of volatiles.In fully open flowers,four terpene alcohols were increased and sabinene was decreased compared with balloon flowers,especially in sweet oranges.2.Multi-omics to identify the terpenoids synthase related genes in citrus2.1 Genome-wide analysis of the TPS gene family in sweet orange,pummelo and tangerine were found the gene number was 76-100,classified into TPS-a,TPS-b,TPS-c,TPS-e/f and TPS-g subfamilies.In the pummelo genome,59 TPS gene loci were unevenly distributed across 7 chromosomes,and chromosomes 2 had the largest number of TPS genes and found a gene cluster including 17 genes in 372 kb region on chromosomes 2.2.2 Transcriptome and metabolomic analysis showed that the expression levels of 9 genes were significantly correlated with the content of various terpenes based on different tissues of Cara cara navel orange.Some tissue specificity expression TPS genes were found in the peel,juice sacs,leaf and flower by RNA-Seq analysis.Transgenic the Cs2g07240 and Cs3g07920 into yeast were found the Cs2g07240 has the function of nerolidol;the Cs3g07920 can synthesize(E)-β-farnesene and α-farnesene.STPS gene was contributed to the production of sabinene in vitro.It was found that most of genes in MEP pathway,MVA pathway and TPS genes in peel were expressed with a higher level,followed by leaf and flower,most of them were a low level in juice sacs,maybe the difference in gene expression results in the content of terpenes with a high level in the peel and a low level in the juice sacs.3.Varify the xenia effect by C.mangshanensis pollen and identify the key CYP450 genes related to linalool oxidesThe contents of cis-and trans-linalool oxides were significantly increased in the juice sacs of HB pummelo and Feicui pummelo cultivars artificially pollinated with the pollen of a wild species C.mangshanensis(MS).The xenia effect of MS pollen affects the volatile compounds in pummelo juice sacs was very stable and does not depend on the environment and regions.A novel cytochrome P450 78A7 gene,named as Cit LO1,was significantly upregulated in the juice sacs of huanong red pummelo pollinated with MS pollen in comparison with open pollination.Comparing to wild type tobacco Bright-Yellow2 cells,transgenic cells over-expressing Cit LO1 promoted 3–4 folds more conversion of(–)-linalool to cis-and trans-linalool oxides.Overall,our results suggest that MS pollen has a xenia effect on pummelo fruit aroma quality,and Cit LO1 is a linalool oxide synthase gene that played an important role in the xenia effect.The above results about the metabolic mechanism of volatiles including exploring a variety of specific valuable resources,biomarker compounds might use to distinguish different citrus species,such as β-elemene,valencene,limettin and nootkatone,and then identify and functional analysis four important terpene synthase genes.This research explored a method to increase the aroma of citrus fruit and provide lots of important materials for further research on the formation of citrus flavor. |
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