Metabolic And Transcriptional Analysis Of Purple Tomato Fruit And Identification Of Anthocyanin Regulators

Purple tomato is a relatively special tomato variety,which appears purple because of the accumulation of anthocyanins in the fruit.Generally,tomato fruits do not accumulate anthocyanin components,but the accumulation of anthocyanins in cultivated tomato fruits was achieved by introgression from a closely related wild tomato.Anthocyanin is a water-soluble pigment,belonging to flavonoids,with strong antioxidant capacity and high health care effect.As people’s requirements for fruit quality are getting higher and higher,purple tomatoes have also attracted much attention.So far,there are three loci that regulate the accumulation of anthocyanins in cultivated tomato fruits:Aft from Solanum chilense,Abg from Solanum lycorpersicoides,and atv from Solanum cheesmaniae.Most of the reports on purple tomato focus on the search for regulatory genes and the study of the regulatory mechanism.The regulation mode of Aft and atv has been thoroughly studied,but the research on the characteristics of purple tomato fruit metabolites and fruit quality has not been reported yet.The purple tomato（No.19861）used in this study is an anthocyanin-containing tomato.After the ovary is enlarged,the epidermis begins to accumulate anthocyanin in the place exposed to sunlight,and the purple color gradually deepens.For the tomato of this special germplasm resource,we have carried out a more comprehensive analysis around the fruit.Firstly,the characteristics of anthocyanin accumulation in purple tomato fruit were investigated,and the content of anthocyanin was determined;genetic population was constructed by hybridization,and the gene regulating the accumulation of anthocyanin in purple tomato fruit was searched by combining BSA technology;The fruits were subjected to metabolome detection to compare the differential metabolites between purple tomato and pink tomato（No.19890）;The transcriptome detection of purple tomato fruits and pink tomato fruits was carried out to enrich the relevant differential genes in the metabolic pathway,and transcription factors associated with anthocyanin accumulation were screened and identified.This study can not only analyze the characteristics of substance accumulation in purple tomato fruits,compare the metabolic differences between purple tomato fruits and pink tomato fruits,and provide a theoretical basis for the fresh eating and product processing of purple tomato fruits.It also provides a theoretical basis for the regulation process of anthocyanins in tomato fruits,provides new insights and has certain reference value and reference significance for the research and utilization of anthocyanin regulatory factors.The main results obtained in this study are as follows:（1）The synthesis of anthocyanins in purple tomato fruits was induced by light,and anthocyanins were mainly accumulated in the fruit epidermis.The mutual grafting of shoots proved that anthocyanins were synthesized in the fruit,rather than being synthesized by other organs and then transported into the fruit.（2）A population with purple tomato 19861 as the female parent and pink tomato 19890 as the male parent was constructed,and the plants in the F₂ population were selected for BSA sequencing analysis,and the genes regulating the accumulation of anthocyanins in purple tomato fruits were identified.It was located in two intervals:one was located on chromosome 7,and the size was about3.8 Mb;the other was located on chromosome 10,and the size was about 6.1 Mb.Through literature search and sequence comparison,it was confirmed that the genes regulating the accumulation of anthocyanins in purple tomato 19861 fruit were Sl AN2-like and Sl MYBATV genes.In addition to fruits,Sl AN2-like and Sl MYBATV genes were also expressed in other organs of 19861 plants where produced the purple phenotype,which might also involve in the synthesis of anthocyanins in other organs.The statistics of the fruits of the F₂ population showed that there were 522 plants that accumulated anthocyanins and 115 plants that did not accumulate anthocyanins,with a ratio of13.62:3.According to the dominant gene of Sl AN2-like and the recessive gene of Sl MYBATV,the theoretical ratio should be 13:3,and the chi-square test resultχ²=0.2029（P>0.05）,indicating that the actual ratio was consistent with the theoretical ratio.（3）A total of 82 flavonoids were detected in the metabolome data,of which 69 were significantly different between the fruits of 19861 and 19890,including 18 flavonoids,25 flavonols,1 flavonoid carbon glycoside,11 anthocyanins,6 flavonoids,6 flavonols and 2 chalcone.Except for a few individual substances,most of the other substances were more abundant in the purple area of the fruit of 19861.Moreover,10 flavonoids did not accumulate in pink tomato fruits,and 7flavonoids were not present in mature pink tomato fruits.In addition to flavonoids,some substances related to the nutritional value of fruits（lignans,coumarin,vitamins）and some substances related to tomato flavor（sugar alcohols,organic acids）were also obvious difference between the fruits of19861 and 19890.（4）The transcriptome results showed that genes related to flavonoid synthesis and metabolism,such as PAL,4CL,CHS,CHI,F3H,DFR,ANS,F3’H,F3’5’H,FLS,etc.,were found much higher transcript level in purple region of 19861 than that of 19890.Fluorescence quantitative assay results were consistent with the trend of transcriptome results.（5）The combined analysis of metabolism and transcriptome showed that differential metabolites and differential genes were significantly enriched in phenylpropane biosynthesis（ko00940）,flavonoid biosynthesis（ko00941）,anthocyanin biosynthesis（ko00942）pathways.In these pathways,the relationship between differential genes and differential metabolite accumulation was determined,and it was the differential expression of these genes that made the accumulation of flavonoids in the purple region of 19861 fruit much stronger than that of 19890.（6）Transcription factors related to the regulation of anthocyanin accumulation in tomato fruit were screened from the transcriptome,and then functional identification was performed by CRISPR technology.The purple anthocyanin spots indicated that b ZIP61-like gene was involved in the regulation of anthocyanin in tomato fruit and could inhibit the accumulation of anthocyanin in fruit.（7）Tomatoes with more anthocyanin accumulation and large fruits were selected from the F₂populations in 19861 and 19890 for breeding.In F_2:3,the fruit surface could produce a purple peel like 19861,and the anthocyanin content determination results showed that it could reach to 56.63mg/100 g FW,and the fruit was about the same size as 19890.