Investigation On Population Traits Of Spinach Anthocyanin F 2 And Screening Of Key Genes

Spinach?Spinacia oleracea L.?is one of the important leaf vegetables in China.The annual gross production of spinach was approximately 21.94 million tons in 2014.Spinach have a great variety of cultivars,but the purple red cultivars is rare.The purpleor red vegetables,riched with high anthocyanin accumulations are beneficial to human health,and have certain ornamental features,leading them to attract more attention from consumers.In this study,the green and purple spinach materials are used to generate F₂ group,explore the color-formationand identify the key genes invovled in anthocyanin biosynthesis in purple spinach,according to the spinach transcriptome and genome databases.The results above enhanced our understanding about the molecular mechanisms of anthocyanin biosynthesis in purple spinach,and will accelerate the agricultural improvement of spinach breeding.This study dentified 22 biosynthesis genes and 25 regulatory genesin spinach,using bioinformatics methods,and predicted the functions of the relevant transcription factor;Using 322 F₂ segregation population,botanical traits are investigated,including appearance characters,nutrient quality traits and pigment contents;The genetic diversity of botanical traits of segregation populationis evaluated by multivariate statistical analysis;The anthocyanin related gene expression level and the relationship between each other are analyzed using qRT-PCR in spinach;The F₂ segregation population constructedin the study is helpful to construction of high density genetic map and development of anthocyanin related molecular markers,and also to provide materials for red spinach breeding.The research results of this study are as follows:1.Through bioinformatics analysis,forty-five anthocyanin related genesare screened in spinach genome,including twenty-two structural genes and twenty-five regulating genes.There are two 4CL,FLS,ANS genes,four CHS genes,five UGT genes,and the rest of structural genes are only one copy.There are six MYB proteins,fifteen bHLH proteins and four WD40 proteins,and the functions of related transcription factors are predicted using phylogenetic analysis,which can be divided into four categories: light regulation,hormone regulation,stress regulation,the unknown function,among them,light regulation genes have a large proportionin spinach.2.The survey of color of leaves and petioles of F₂ population found that 265 plants showred characters,inclucding leaves and petioles,and 87 plants show green characters,the ratio of the number of the red plants and the green plants is about 3:1,accordance with Mendelian law,which is conformed by chi-square test?P<0.01?,suggesting that purpleor red trait is controlled by a single dominant gene in spinach.3.Evaluating the genetic diversity of F₂ populationby multivariate statistical analysis shown that 322 spinach plants can be assessed by eight factors,including anthocyanin factor,represent for leaf color,petiole color and anthocyanins content of leaf and petiole;plant size factor,leaf shape factor and leaf smooth factor mainly represent forthe agronomic traits,soluble sugar factor,nitric nitrogen factor,oxalate factor and VC factor mainly represent fornutrition quality.The F₂ population are divided into eight groupsusing the Ward method and the anthocyanin content of group ? are the highest,which can be used in the study of anthocyanin,the agronomic traits of group ? are excellent,with lower oxalic acid and nitric nitrogen content,hight VC and soluble sugar content,and medium of anthocyanin content,which can be used for spinach breeding.4.The pigments contents analysis of F₂ population shows that accumulation of anthocyanins are the mainly pigment responsible for red coloration in spinach,and plants with red traits display higher anthocyanin,flavonoid,total phenol content and anthocyanin/chlorophyll ratio than that of plants with green traits,except for reddish group,and there are no significant differences between the total chlorophyll,chlorophyll a,chlorophyll b and carotenoid content in red and green spinach.5.Analyzing anthocyanin metabolism related gene expression of leaf and petiolein green plants NO.38 and NO.208,red plants NO.42 and NO.270 in F₂ segregation population,and obtaining the following results: the key enzyme genes mainly have high expression level in the leaf of NO.42 and NO.270 with red traits,it significantly higher than the leaf and petiole of green materials and the petiole of red materials,including early anthocyanin synthesis gene SoCHS1 has the highest expression level,its relative expression is 205 times and 172 times than the green materials in the leaf of two red materials,moreover the expression of late anthocyanin synthesis genes are also similar to early genes,SoUGT78D2 has the highest expression level,its relative expression is 112 times and 305 times than the green materials in the leaf of two red materials,therefore two structural genes SoCHS1 and SoUGT78D2 may play a key role in spinach anthocyanin synthesis.The expression patterns of Spinach regulator genesare similar to structural genes,also show the tissue specificity,mainly have high expression level in the leaf of NO.42 and NO.270 with red traits,including the R2R3-MYB class regulator genes SoMYB1 and SoMYB5;WD40 class regulator genes SoWD1,SoWD2 and SoWD4;bHLH class regulator genes SobHLH1,SobHLH2 and SobHLH9.