Fine Mapping And Linkage Marker Development Of The Dark Green Stem Gene Cp Dsc-1 In Pumpkin (Cucurbita Pepo L.)

The pumpkin（Cucurbita pepo）is one of the most important cultivars of the pumpkin genus Cucurbitaceae and is very rich in colour.Currently,research on the colour traits of Cucurbita pepo crops has focused on the rind and leaves.The dark green trait in pumpkin crops is abundantly variable and independently inherited in leaves,fruits and stems.Stem colour is also an important agronomic trait.On the one hand,it is one of the most desirable seedling marker traits,which can eliminate hybrids,reduce labour intensity at later stages and is of great value in plant breeding;on the other hand,dark green stems are rich in chloroplasts,which are able to carry out some photosynthesis and promote plant growth and development.Therefore,the genetic analysis of dark green stem traits and the study of their regulatory genes are of great importance.In this study,we completed the localization of the dark green trait in pumpkin stems by genetic analysis,analysis of chlorophyll content and chloroplast structure,BSA sequencing,validation of F₂ isolates,bioinformatics analysis and q RT-PCR validation,and finally identified the key candidate gene Cp4.1LG15g03420.1.Detailed results are as follows:1.An F₂ population was created using the pumpkin inbred lines 274 and 296 as parents and genetic analysis of stem colour traits in progeny isolates showed that pumpkin stem colour（dark green/light green）is controlled by a single dominant gene（CpDsc-1）for quality traits.Transmission electron microscopy results showed that chloroplast defects were the main cause of the light green colour of 274 stems.2.The CpDsc-1 gene was initially localized within a 2.09 Mb interval on chromosome 15 by BSA sequencing;based on the parental resequencing results,highly polymorphic Indel markers were developed and screened within the candidate interval,and the interval was narrowed by searching for recombinant single strains in the expanded F₂ population（1259 strains）,and the target gene CpDsc-1 was finally localized within the marker between SNP3 and SNP1 at a physical distance of approximately 65.2 kb,an interval that included seven candidate genes.3.By sequence alignment and differential expression analysis of seven genes within the locus interval,only two genes,Cp4.1LG15g03360.1 and Cp4.1LG15g03420.1,were found to have sequence differences between the two parents.Based on the results of biparental genome resequencing,they were compared in IGV（version,8）and the results showed that: the 296 genome sequence is relatively complete and there is no variation in gene structure;in the 274 material,the Cp4.1LG15g03360.1 gene has a 2.1kb deletion at the 3’ end and the Cp4.1 LG15g03420.1 gene has a 2kb deletion at the 5’ end.Differential expression analysis of the seven genes showed that274 and 296 differed significantly for the dark/light green trait of the stem only on the Cp4.1LG15g03420.1 gene,thus inferring that the Cp4.1LG15g03420.1 gene is the key gene controlling the dark/light green trait of the stem.4.Based on the results of gene annotation and protein sequence alignment in Cucurbitaceae,the Cp4.1LG15g03420.1 gene was found to encode a PRR2two-component response regulatory protein related to chlorophyll synthesis with high homology to Arabidopsis thaliana,cucumber,watermelon and melon.Subcellular localization results showed that the Cp4.1LG15g03420.1 gene was finally localized to the nucleus.5.Spatiotemporal expression analysis showed that the expression level of Cp4.1LG15g03420.1 gene was significantly higher in the stems of 296 plants.Meanwhile,the expression of Cp4.1LG15g03420.1 gene was significantly increased at 30 d after germination,and the chlorophyll content was also significantly increased at the same period.This further verified that Cp4.1LG15g03420.1 is likely to be involved in chlorophyll synthesis.6.Analysis of the candidate gene promoter elements revealed that candidate genes are involved in important responses in both the light response element and the hormone response element.Biparental treatment with hormones and stresses revealed significant inhibition of expression under the hormones salicylic acid（SA）,abscisic acid（ABA）,growth hormone（IBA）and dark stress,consistent with the results of the promoter analysis.7.To clarify the diversity of the Cp4.1LG15g03420.1 gene,the closely linked markers G4563 and Fra3 were developed and validated in a natural population of pumpkin,showing that the phenotypic and genotypic concordance of the marker G4563 was around 93%;The validation of the chromosomal fragment deletion marker Fra3 showed that the deletion of the Cp4.1LG15g03420.1 gene on chromosome 15 was prevalent in pumpkin material with light green stems,and that there were differences in the deletion fragments.Also,validation results in Chinese pumpkin and Indian pumpkin indicated that this locus is a unique trait in pumpkin.In conclusion,this study conducted a genetic analysis of dark and light green traits in pumpkin stems and located its key regulatory gene,CpDsc-1,within a 65.2kb interval on chromosome 15,and found the Cp4.1LG15g03420.1 gene to be the only candidate gene within this interval.In addition,markers were developed and natural populations were analysed.This study provides an ideal seedling marker trait for seedling selection in pumpkin,as well as new genes and markers for molecular marker-assisted selection breeding in American pumpkin,and lays the foundation for further in-depth analysis of the molecular regulatory mechanisms of dark and light green stems in American pumpkin.