Research On Light Response And Inheritance Of Purple Trait In Pepper Fruit

Flavonoids are important secondary metabolites during plant growth and are widely distributed in nature.Anthocyanins and other flavonoids play an important role in plant antioxidation,drought tolerance,resistance to UV-B damage and pathogens.China’s pepper cultivation area is 2 million hectares,ranking first in vegetables.It is an important vegetable crop in China.Pepper has high nutritional value.In addition to rich vitamin C,capsaicin and carotenoids,the immature fruit of purple pepper also contains a large number of secondary metabolites such as anthocyanins.Purple pepper breeding has become a new direction of pepper breeding.At present,the formation of anthocyanins in purple pepper mostly belongs to light dependent type,which is only synthesized in the epidermis,and generally degrades gradually with the maturity of pepper fruit.Therefore,the study on the synthesis and regulation of anthocyanins in pepper is important for understanding the regulation of pepper anthocyanins and breeding purple pepper.In this study,the synthesis of anthocyanins in purple pepper at different stages was analyzed by p H difference method and liquid chromatography tandem mass spectrometry（LC-MS/MS）.The common transcriptome sequencing technology was used to analyze the light response of purple pepper.Eighteen common transcriptome libraries were constructed.The modules closely related to anthocyanin synthesis were found through weighted gene coexpression network analysis（WGCNA）,and some important structural genes and transcription factors affecting anthocyanin synthesis and transport were identified.Some metabolic pathways related to anthocyanin synthesis were found.Full transcriptome analysis was carried out during the critical period of anthocyanin synthesis in pepper fruit.Six lnc RNA and circ RNA sequencing libraries and six small RNA sequencing libraries were constructed.Three lnc RNA-mi RNA-m RNA gene interaction networks related to light response were constructed.Three hybrid F₂generation populations of purple fruit and green fruit pepper were also constructed.The main results are as follows:1.A total of 38 flavonoid metabolites were identified in HNUCA21 purple pepper germplasm by liquid chromatography tandem mass spectrometry（LC-MS/MS）,of which30 belong to anthocyanins.The content of three anthocyanins was the highest,and reached the maximum in the later stage of light treatment.They are delphinidin-3-o-glucoside（del-3-o-glu）17.13μg/g,delphinidin-3-o-rutin（del-3-o-rut）10.23μg/g and petunia-3-o-p-coumaryl glucoside（pet-3-o-（coumaryl）-Glu）4.77μg/g.2.The peel samples of pepper bagged fruits at the early（48 h）,middle（72 h）and late（168 h）stages after removing the bags,as well as the shading pepper peel samples at these three stages were selected,and the transcriptome sequencing of these materials was carried out.Through the analysis of weighted gene coexpression network,the module related to anthocyanin synthesis was identified.The structural genes of flavonoid synthesis included CHS（107871256 and 107864266）,CHI（107871144 and 107852750）,DFR（107860031）,F3’5’H（107848667）,F3M（107862334）,LDOX（107866341）and TCM（107875406 and10785407）.GST（107861273）,UFGT（107861697 and 107843659）and MATE（10786323410784661）are related to anthocyanin transport function.Transcription factors EGL1（107865400）,b HLH104（107864591）,WRKY44（107843538 and 107843524）belong to this module and may play an important regulatory role in anthocyanin synthesis.Further analysis revealed a coexpression regulatory network with unknown functional gene（107839364）as the central gene.The network includes important genes CHS,DFR,CHI and EGL1 involved in anthocyanin synthesis,as well as two MATE and two WRKY genes.Through regulatory network analysis,genes involved in early,middle and late light response were screened,which provided a reference for further analysis of the regulatory mechanism of anthocyanin biosynthesis in pepper.3.The purple pepper materials under light for 48 hours were analyzed by liquid chromatography tandem mass spectrometry（LC-MS/MS）.It was found that the main anthocyanin was delphinidin 3-o-glucoside.Further,strand specific transcriptome and small RNA analysis were performed.It was found that the response genes in the early stage of flavonoid biosynthesis,including structural genes（SHT,AT-like,CCo AOMT,CHI,CHS1B,CHS2,CYP98A2-like,DFR,F3’5’H,F3H,F3’M and FLS）,related transcription factors and targeted lnc RNAs,were significantly differentially expressed after 48 hours of light,and the differential expression of some genes was confirmed by q RT-PCR.The differential expression of transcription factors MYB4 like（xm＿016725242.1）,MYB113 like（xm＿016689220.1）,MYB308 like（xm＿016696983.1,xm＿01672244.1）and EGL1（xm＿016711673.1）may be involved in the regulation of flavonoid metabolism.Three lnc RNA-mi RNA-m RNA interaction networks with sly-mir5303,stu-mir5303g,stu-mir7997a and stu-mir7997c as the core point were constructed.These results provide important data for understanding the light response of anthocyanin synthesis in pepper.4.We use the main gene and multigene hybrid genetic model to clarify the genetic model of young purple pepper fruit color traits with three F₂populations.It was determined that the L and C values of young purple pepper were controlled by two additive-dominant-dominant polygene models,and the heritability of the main gene was no less than 85.37%.BSA（bulked segregant analysis）method,Illumina sequencing technology and♂HNUCC000293（fruit green）×♀The F₂fruit color segregation population of HNUC000239（fruit purple）were used to map the purple trait of pepper fruit.We obtained about 280G sequencing data,and detected strong signals in specific regions of chromosomes 2,4,5 and 9,with the strongest signal in chromosome 2（27,550,001-28,604,000bp）.