Study On Genetic And Molecular Localization Of Coloration Trait In Yunnan Red Radish

Anthocyanins,which soluble in water,are a kind of natural pigments widely found in plants.It provide plants with bright colors and attracts pollinators and seed-spreading animals.Anthocyanins are also effective free radical scavengers and protect plants from biotic and abiotic stresses.In recent years,anthocyanins have become a research hotspot due to their biological activities,For example,anthocyanins have the properties of inhibiting tumor production and development,decreasing blood pressure,antibacterial,anti-inflammatory,antioxidant and so on.Vegetables and fruits rich in anthocyanins and industrial products containing natural anthocyanins are becoming increasingly popular.Yunnan red radish（Raphanus sativus L.）is a kind of special germplasm resources rich in anthocyanins represented by“Tonghai wuluobo”and“Lanping ziluobo”.Furthermore,it has diverse industrial uses（e.g.,production of food products,medicines,and cosmetics）because of its rose color and highly stable red pigment.With suitable climatic conditions,since the 1990s,radish red pigment from Yunnan has been exported to more than 10 countries and regions at home and abroad for its bright red color and good stability,accounting for more than 60%of internatianl market shares.However,because of years of non-standardized cultivation,the mixed germplasm of the radish varieties currently used for commercial production has affected the quality of the red pigment in radish.To generate highly pure hybrids,the flesh color instability of the commercially cultivated varieties needs to be addressed.This requires a more thorough understanding of the genetic mechanism underlying anthocyanin synthesis in Yunnan red radish.To achieve the objective above,firstly,10 cross combinations of high generation inbred lines of red radish were used to analyze the effects of main agronomic traits on the yield of pigment in fleshy roots.Secondly,the high generation of inbred lines"YAAS-RR1"（bell shape with red root-skin and red root-flesh）was crossed with"YAAS-WR1"（long cylindrical shape with white root-skin and white root-flesh）to generate BC₁P₁,BC₁P₂,and F₂populations.Genetic analysis of these populations showed that two genes controlled the coloration trait of radish plants.The high density genetic map of Yunnan red radish was constructed via SLAF-seq,and Linked markers were developed to predict and structurally analyze the candidate genes potentially applicable for the marker-assisted selection-based breeding of new radish varieties with improved pigment production.The primary results in this study are as follows:Correlation analysis,partial correlation analysis,path analysis and principal component analysis were used to analyze 10 yunnan carrot cross combinations.In the harvest period of radish,root length,plant height,leaf length,development degree,root diameter,leaf weight,exposed length,single root weight,leaf width,and pigment yield of fleshy root were investigated.And then,the partial correlation,the correlation,the path analysis and the principal component analysis were analyzed using DPS between pigment yield and main agronomic characters of Yunnan red radish.The results showed that the variation coefficient of the 11 agronomic traits ranged from 0.09%to 10.42%.,and the variation coefficient of developmental degree was the largest.Among all traits,root weight had the greatest effect on pigment yield,followed by root width.In breeding aimed at high anthocyanin content,combinations with moderate development degree,leaf size and exposure length,and large root width,root length and single root weight should be selected.The result will clarify the effects of main agronomic characters on pigment yield of radish fleshy roots.Genetic analysis was carried out using the established population.The results revealed that the pigmented/non-pigmented（Pi N）and purple/red（PR）traits were controlled by two genetic loci.Combined with the character segregation ratio of BC₁P₁and BC₁P₂populations,it could be inferred that the Pi N-related gene,which was designated Rs Pi（pigmented）,is dominant homozygous in YAAS-RR1.The PP/PR-related gene,which was designated Rs PP（purple pigment）,is dominant homozygous in YAAS-WR1.The frequency distribution of anthocyanin content in root flesh and root skin in F₂population showed that the genetic mechanism underlying pigment accumulation differs between these two tissues.However,in the F₂population,all individuals with pigmented root flesh also had pigmented root skin,suggesting that the gene controlling the pigment accumulation in the root skin was also involved in regulating the accumulation of pigment in the root flesh.Therefore,we mainly focused on identifying the dominant gene Rs Pi based on the root skin pigment content of F₂individuals.Base on F₂population,a high density genetic map with a total map spacing of 1230.16 c M was constructed usingthe specific-locus amplified fragment sequencing（SLAF-seq）technique.In the map,4032 markers constituted 9 linkage groups,with a mean distance between markers of 0.31c M.Further QTL mapping results indicated that two QTL loci were found to be correlated with coloration trait in radish,and these two sites are named q AC1 and q AC2,respectively.In additioan,one locus for root length,,one locus for root width and one locus for root weight were identified respectively..A bioinformatics analysis of the QAC1 region identified 58 predicted protein-coding genes.Among these genes,Rs F3’H,which associated with anthocyanin biosynthesis,may be a candidate gene related to PR trait.The results was further verified by analyzing the structure and the expression of alleles of Rs F3’H of two parents.Regarding QAC2,through function prediction and expression analysis,Rs MYB1.3 was identified as an important candidate gene for Pi N.trait,with a 4-bp insertion in the first exon that introduced a premature termination codon in the YAAS-WR1 sequence.The results of yeast one hybridization,yeast two hybridization and double luciferase report experiments showed that Rs MYB1.3 can interacte with Rs TT8 and then activates Rs TT8 and Rs UFGT expression.These findings may help to elucidate the complex regulatory mechanisms of anthocyanin synthesis in Yunnan red radish.Furthermore,this study’s results may be relevant for the molecular breeding of radish to improve the anthocyanin content and appearance of the taproots.