Genetic Variation Study Of Tieguanyin Based On Whole Genome Resequencing And Transcriptome Analysis

Camellia Sinensis ‘Tieguanyin’ originates from Anxi County,Fujian Province.It is an elite tea cultivar in China.But there are species variations in the process of asexual reproduction for many years.There is a saying that Tieguanyin is good to drink but not easy to cultivate.In this study,the mother tree of Tieguanyin,its 3 consecutive asexual reproductions and 3Tieguanyin trees from multi-generation that used for actual production were used as experimental materials.High-resolution genome resequencing was carried out to analyze the Tieguanyin somatic mutation,screening of key genes that may cause species variation.Transcriptome sequencing technology was used to analyze Tieguanyin,Fengyuanchun(the natural hybrid offspring of Tieguanyin),and the local varieties Baiguanyin,Jinmianguanyin,Zaoguanyin,Hongying,Qigaixian,Xianghua No.1 in Anxi county.Pearson distance,GO,KEGG enrichment and transcription factor analysis were used to explore the differences between different varieties.This study preliminarily reveals the genetic variation mechanism of Tieguanyin asexual reproduction,and provides a reference for the research on the promation and rejuvenation of clonal tea tree varieties.The main results were as follows:1.Study on genetic variation of Tieguanyin tea trees asexual reproduction based on high-depth genome resequencingThe genome of Tieguanyin mother tree and its 3 consecutive asexual reproductions and 3 Tieguanyin trees from multi-generation that used for actual production were resequenced with over 50 X in this study.Through SNP and In Del detection and annotation,it was found that most of the variants were located in intergenic regions,only 1.09%～1.13% of SNPs and 0.51%～0.61% of In Dels were located in gene coding regions.In the whole genome and coding regions,the total number of mutation sites of the 3 consecutive asexual reproductions had little differences,and the number of genes involved in the mutation was basically the same in each sample.It was speculated that the varietal characteristics of Tieguanyin had been generally stable during the long-trem of asexual reproduction.The KEGG enrichment analysis of differential genes with the mother tree showed that purine and carbohydrate metabolic pathways were significantly enriched in 3 Tieguanyin trees used for actual production,and the caffeine metabolic pathway was extremely significant enriched.It was speculated that some of the quality and flavor-related metabolic pathway genes were varied during the long-term asexual reproduction process in Tieguanyin.Compared with the 3 consecutive asexual reproductions,the sequence of three genes such as FAR1,WAT1,and MYB that related to growth and development and five genes such as PP2 A,BTB/POZ,BAT2,RPP13,and Lr10 that related to stress response had been mutated in 3Tieguanyin trees used for actual production.The above results can provide reference for the study of genetic variation of tea plants asexual reproduction.2.Structural Variation analysis of Tieguanyin tea tree in multi generation asexual reproductionStructural Variation is another important mutation in the genome apart from SNP and In Del,which plays an important role in plant adaptive evolution and phenotypic diversity.Structural variation analysis was carried out on Tieguanyin mother tree and its 3 consecutive asexual reproductions and 3 Tieguanyin trees from multi-generation that used for actual production,and four types of structural variation were mainly detected: insertion(INS),deletion(DEL),inversion(INV)and intrachromosomal translocation(ITX).The average ratio of DEL was more than 70%,and more than 40% of the structural variations were less than1 kb in length.The number of SVs in the 3 samples of Tieguanyin used for actual production were more than the mother tree of Tieguanyin and its 3consecutive asexual reproductions,among which the number of Xiping Tieguanyin(AT)and Cannei Tieguanyin(BT)increased more.The common differential genes associated with SVs in the 3 samples of Tieguanyin used for actual production were enriched in sugar and amino acid metabolism pathways,suggesting that Tieguanyin tea trees may have been artificially domesticated during years of asexual reproduction.3.Genetic analysis of Tieguanyin series cultivars based on transcriptome sequencingThe shoots of Tieguanyin(TGY)and Anxi local varieties Baiguanyin(BGY),Jinmian Guanyin(JMGY),Zaoguanyin(ZGY),Hongying(HY),Qigaixian(QGX),and Xianghua No.1(XH1H),and Tieguanyin’s natural hybrid offspring Fengyuanchun(FYC)were used as materials for transcriptome sequencing analysis.Through principal component analysis and Pearson correlation distance analysis,the similarity between the tested varieties and Tieguanyin from high to low is: Fengyuanchun > Hongying >Jinmianguanyin > Zaoguanyin > Qigaixian > Xianghua No.1 > Baiguanyin.Using the KEGG database,the metabolic pathway enrichment analysis of differentially expressed genes was carried out,and it was found that the cultivars had significant differences in zeatin and brassinosteroid metabolism pathways related to growth and development,fatty acid and terpenoid metabolism pathways related to aroma formation,and carbohydrate-related metabolic pathways compared with Tieguanyin.The analysis of transcriptional factors of differentially expressed genes showed that the expression levels of GRF transcription factors related to growth and development in Tieguanyin,Fengyuanchun,Zaoguanyin,and Hong Ying were significantly lower than those in Jinmianguanyin,Baiguanyin,Qigaixian,and Xianghua No.1.The WGCNA analysis results indicated that Csb HLH and Cs MYB may affect the accumulation of catechins,gallic catechins,and epigallocatechins in tea plants by regulating Cs PAL and Cs ANS genes that related to flavonoid biosynthesis.4.Identification and genetic analysis of Tieguanyin GRF transcription factorsGrowth regulating factor(GRF)plays an important role in the growth and development of plants.Previous transcriptome analysis showed that the expression of GRF transcription factor in Tieguanyin was low.Bioinformatics analysis were used to analyze the GRF transcription factors in Tieguanyin and 15 Cs GRF transcription factors were identified.Based on phylogenetic relationships,the Cs GRF genes of Tieguanin were divided into 6 groups and each group shared the similar gene structure and conserved protein motifs.A large number of cis-acting elements related to light response,hormone response and stress response were revealed from upstream promoter region of the Cs GRF transcription factors.The results of tissue-specific expression indicate that Cs GRF genes were strongly expressed in tender shoots,while the expression levels in mature leaves were low or not.Genetic analysis of the expression level of Cs GRF in Tieguanyin and its offsprings showed that the expression levels of Cs GRF genes in Tieguanyin were low.It was speculated that the low expression of Cs GRF genes in Tieguanyin tea tree was related to its low growth vigor.And the expression levels of Cs GRF genes varied among different artificial F1 hybrid progenies.The heterosis expression of some Cs GRF in hybrid offspring may be related to the formation of heterosis of tea plant growth vigor.