Fine Mapping Of Clorf Colorless Non-ripening Locus In Orange Flesh Of Watermelonn

Watermelon is an important fruit crop of the Cucurbitaceae family and is fascinated by consumers due to its sweet and delicious flavor.China is the largest producer and consumer of watermelon.Carotenoids are an important source of vitamin synthesis precursors in some parts of the human body.In order to ensure human health,the consumtion of carotenoids enriched foods has become the main demand for people’s dietary structure due to the self syntheizing inability of the human body by themselves.The flesh color of watermelon mainly depends on the composition and content of carotenoids in the flesh.Watermelons with different flesh colors can provide people with carotenoids with different nutritional values.Orange-fleshed watermelon is rich in pre-lycopene orβ-Antioxidants such as carotenoids have important effects on maintaining human immunity and delaying aging.Therefore,cultivating orange-fleshed watermelon varieties plays an important role in meeting people’s intake of carotenoids.This experiment is based on previous research in the laboratory;the red flesh watermelon line（W1-1）was used as the female parent,the orange watermelon line（W1-61）was used as the male parent,and a six-generation（P₁,P₂,F₁,F₂,BC₁P₁,and BC₁P₂）population was developed.Genetic inheritance analysis showed that watermelon orange is controlled by a pair of single recessive genes named Clorf（Orange flesh）.The Clorf gene locus was preliminarily located within the physical position（30860537～31546441 bp）of chromosome 10,exhibiting the approximately685.90 kb genetic interval of watermelon genome,using the bulked segregant sequencing analysis（BSA-seq）combined with molecular marker technology.On this basis,this study expanded the F2population and conducted fine mapping and candidate gene analysis on Clorf.The main results are as follows:1.An expanded F₂generation population consisting of 1026 plants was developed,genetic markers on both sides of the preliminary positioning interval were designed,and a total of 26 screened recombinant plants were obtained.A total of 8 pairs of molecular markers were designed with in the primary genetic interval position and genotyped with 26recombinant plants and F_2:3population phenotype.The orange flesh locus（Clorf）of watermelon was located between two molecular markers of Chr10＿30936001 and Chr10＿31240618（30936001 bp to 31240618 bp）,showing 304.62 kb interval.After further analysis,it was found that there are no further molecular markers that can be developed within this interval.Based on gene annotation and base sequence mutation screening of2.Cucurbitaceae dataset,total of 32 candidate genes were predicted in the candidate interval.It is thought that ClCrtiso（carotenoid isomerase,Cla97C10G200950）is a candidate gene regulating the Clorf.3.Further,using two types of watermelon germplasm with different flesh colors（10 red flesh and 1 orange flesh）for base mutation similarity analysis within the mapping populations.It was found that there was a non-identical mutation（SNP＿31017592）with100%similarity in the ClCrtiso coding region of W1-61.The variant flanking sequence was used to develop KASP（kompetitive allele specific PCR）markers for genotyping in the F₂population.It was found that the genotype and phenotype of this marker were completely co-separated,and only W1-61 contained this mutation as compared to 18watermelon natural population.The subsequent sequencing data from the ClCrtiso coding region cloned from the parent cDNA also confirmed this result.4.RNA was extracted from the flesh samples of the parent materials at different days after pollination（10,18,26,34,and 42）,reverse transcription was done to get the cDNA,and qRT-PCR was performed to detect the expression pattern of six genes related to carotenoid metabolism.It was found that the expression of ClCrtiso was significantly higher at the critical period（34-42 days）of the color formation of the orange color watermelon flesh than in the red flesh.So,it was speculated that the low expression of this gene led to the inability of pre-lycopene orange to be isomerized into red lycopene,accumulating a large amount to form the orange flesh.