Assembly Of Pan-genome And Analyze Of Agronomic Traits Of Pepper Based On Genomics And Transcriptome

The rapid development of genomics and transcriptomics technology can accelerate the basic research process of crops,and it has been used to analyze the formation mechanism of a number of important agronomic traits in crops such as tomato,corn and rice.The formation mechanism of a group of important agronomic traits.Pepper is one of the most important vegetables in the world,its planting area and economic benefits are among the top of the vegetable crop.In 2014,Ko rea and China completed the assembly of the reference genome of pepper respectively,which laid a foundation for the application of genomics and transcriptomics to analysis of complex agronomic traits of pepper.In this study,the pan-pan genus genome was assembled based on genome-wide resequencing and transcriptome.In addition,4 significant traits of pepper including capsaicin,capsanthin,fasciculate inflorescence and cytoplasmic male sterile were studied.The main research results are as follows:（1）Assembly of pepper pan-genome.In order to better understand the effect of genomic variation on the phenotype of pepper plants,383 pepper germplasm materials were subjected to genome-wide resequencing of approximately 11-fold depth.The resequenced reads were assembled,including data with a total length of956 Mb containing 610292 new contigs,and then the published reference genome Zunla-1 was combined with the data obtained by resequencing to assemble into the pepper pan-genome.A total of 51,757 high quality genes were predicted for pepper pan-genome,28,930 of which were defined as core genes.Based on the presence-absence variations（PAVs）analysis on the predicted genes,the cultivars were clearly clustered into 4 different groups,consistent w ith the classification of four cultivated species based on morphological data.（2）Genome wide association analysis（GWAS）based on PAV.To reveal the relationship between genes and traits in pepper,we built a Pepper Pan（http://47.74.21.101:8012/pepperpan）database and constructed a JBrowse-based web interface to enable users to browse,search and visualize the pan-genome annotation and reads mapping data for any of the 383 pepper cultivars.The relationship between capsaicin and capsanthin content of pepper fruit and gene PAV was analyzed by PAV-GWAS,and the results showed that the PAVs of Ccs gene（pan06g005570,capsanthin/capsorubin synthase）were found to be significantly associated with capsanthin contents.The read coverage analyses showed that 26 cultivars with yellow or orange fruits had two types of deletion around Ccs（pan06g005570）region,we confirmed that 24 of 26 cultivars indeed have a 4.43 kb deletion of the Ccs gene region and that there maining 2 cultivars have a 2.96 kb deletion compared to the red-fruit cultivar NJ03,confirming the GWAS result.We also studied the read coverage for phytoene synthase gene（Psy）and found that four cultivars without the red color in fruits have the same 20 kb deletion that spans the pan04g025380（Psy）region completely.In addition,a 2.5 kb deletion of Pungent1（Pun1,pan02g021380）region in 51 cultivars with low capsaicin contents.（3）Mapping and identifying candidate genes involved in the novel fasciculate inflorescence in pepper based on the genomics and transcriptomics.In our present study,a new mutant with fasciculate inflorescence named CL74 was derived from our germplasm resources,and the genetic analysis of CL74 indicated that its fasciculate inflorescence trait was controlled by a single recessive nuclear gene.A F₂ population was constructed and used for bulked segregant analysis（BSA）and linkage analysis,these findings revealed that the candidate gene was mapped to a segment with a physical distance of approximately 998 kb between the marker Indel-197064077 and Indel-198110136.There were two agamous-like MADS-box protein genes Capanan11g001832 and Capanan11g001834 in the candidate region,there were two single nucleotide polymorphisms（SNPs）existed on the coding region of Capanan11g001832 in the CL74 mutant compared with the single-flower plants.The two SNPs were verified and fully co-segregated with fasciculate inflorescence phenotype in the F₂ individuals.Collectively,Capanan11g001832was predicted a strong candidate gene for the fascicu late inflorescence.Furthermore,transcriptome analysis indicated that three WUSCHEL-like genes（Capana00g000667,Capana06g000476 and Capana11g000671）were down-regulated and CLV1（Capana04g000175）was up-regulated in CL74.Moreover,we speculated that there was a complex relationship among the genes Capanan11g001832,Capanan11g001834,the three WUSCHEL-like genes,and CLV1,and these genes might co-regulate the formation of fasciculate inflorescence in pepper.（4）Expression pattern analysis of coding and long non-codingRNA（LncRNA）in pepper cytoplasmic male sterile line 9704A and maintainer line 9704B during flower bud development based on transcriptomics.A total of 547,976 and 2,416differentially expressed genes were identified in the stage S1,S2 and S3 of 9704A and 9704B based on the transcriptomics technology,respectively,and more than70%of the differentially expressed genes were down-regulated in the sterile line.And a total of 10,655 putative LncRNAs were identified during the developmental process of floral buds in 9704A and 9704B,1137 LncRNAs were unique differentially expressed in the three stages,and their cis and trans target genes were predicted.In addition,Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicate that were highly correlated with pollen development.Our findings lays a foundation for further studies on the function of coding genes and LncRNAs in pollen development and provide valuable insights into the regulatory mechanism leading to cytoplasmic male sterility.