Fine Mapping And Color Formation Mechanism Of Skin Color Trait In Cucurbita Maxima

Cucurbita maxima is an important horticultural and economic crop worldwide.The skin color is an important agronomic trait of the C.maxima.It is the key factor for the fruit appearance and quality.The skin color formation of squash is related to the accumulation,varieties and ratio of chlorophyll and carotenoids.Research on the genetics and molecular mechanism of squash skin color traits will improve the quality of fruit appearance and provide a theoretical basis for breeding.In this study,inbred lines of C.maxima‘2013-12’with red skin and‘9-6’with grey skin was used as materials to create generation population for the identification of the phenotypic and genetic relues on skin color traits;the polymorphisms molecular markers were developed to map the genes that control the skin color trait;The regulation mechanism of carotenoid synthesis was studied based on transcriptome and carotenoid metabolome analysis of skin samples at 0,15 and 30 DAP.The main conclusions were as follows:1.The pigments content in ripen skin of the two parental lines was determined.The results showed that the main pigments in the skin of‘2013-12’is carotenoid.The main pigment accumulated in the skin of‘9-6’was chlorophyll.Chlorophyll a was the the main component of chlorophyll in‘9-6’.The microstructure of ripen skin showed that the skin color of two parental lines was basically the same as the color of the main pigment layer.In this study,generation population was constructed by the parental inbred lines of‘2013-12’and‘9-6’.The results showed that the red skin trait was regulated by a dominant single gene,which named as CmaRd.2.Polymorphic molecular markers were developed to scan the 2027 F₂ populations.The CmaRd was located in 12.21 Kb candidate region between markers M＿13871494 and M＿13883706,which contained 3 candidate genes.Through the analysis of qRT-PCR,gene cloning and gene function prediction,CmaCh14G019730 is supposed to be the candidate gene of CmaRd.There is 3non-synonymous SNP mutation sites between two parental lines,which exist in the PLN00113 super family and LRRNT＿2 domains,respectively.Phylogenetic tree analysis indicated that CmaCh14G019730 clustered to the Ⅳ subgroup of LRR-RLK family in Arabidopsis.It was found that3 SNP in CmaCh14G019730 gene were 86.36%associated with the ripen skin color in the germplasm resources of C.maxima.3.In this study,transcriptomes data of parental skin samples at 0,15 and 30 DAP were obtained.All genes were annotated according to the comparison results with GO,KEGG and other databases.DEGs were mainly enriched in GO annotation of‘photosynthesis’,‘photosynthetic system’,‘tetrapyrrole binding’,‘heme binding’,et al;DEGs were mainly enriched in‘secondary metabolite biosynthesis’,‘photosynthesis-antenna protein’and‘plant hormone signal transduction’in KEGG pathway.It was found that in the chlorophyll and carotenoid metabolism pathways,the number of differential genes between 0 DAP vs 15 and 30 DAP was more than 15 vs 30 DAP.According to the functional annotation,it was found that there were 32 differentially expressed genes related to chlorophyll synthesis,26 related to chlorophyll metabolism,and 51 related to carotenoid synthesis.4.Carotenoid metabolome were analyzed in skin of C.maxima.68 carotenoids,including 6carotenoids,47 carotenoid esters and 15 xanthophylls,were detected at 0,15,and 30 DAP in‘2013-12’and‘9-6’.KEGG enrichment analysis showed that differential accumulated metabolites were mainly enriched in‘metabolic pathways’,‘carotenoid biosynthesis’and‘secondary metabolite biosynthesis’.With fruit development,carotenoids showed increase trend in the skin of both parental lines.The main carotenoid component accumulated in the skin of‘2013-12’was lutein>zeaxanthin>β-carotene>β-cryptoxanthin.The main carotenoids accumulated in‘9-6’were lutein>β-carotene.The lutein content was the highest in both parental lines.The zeaxanthin was most differential pigment accumulated between the two parental lines at 15 and 30 DAP.5.The skin color formation of‘2013-12’was related to the content and proportion of carotenoids.With the development of the fruit,the highest accumulation carotenoid components in skin is lutein.Zeaxanthin is a key metabolite responsible for skin color change from yellow to red.The CmaPSY1gene was significantly positively correlated with the contents of lutein,zeaxanthin andβ-cryptoxanthin in skin of‘2013-12’.The CmaBCH2 was the key gene regulating the content of zeaxanthin andβ-cryptoxanthin.The deepening of skin color of‘9-6’mainly related to the pigments accumulation in stead of proportion.The up-regulated expression of CmaChl H gene and the down-regulated expression of chlorophyll degradation pathway-related genes regulated high chlorophyll accumulation in‘9-6’.The synthesis of chlorophyll in skin of‘2013-12’was mainly hindered between coproporphyrinogen Ⅲ and protoporphyrin Ⅸ.The down-regulated expression of CmaChl H gene and the up-regulated expression of genes related to chlorophyll metabolic pathway were contributed to the low chlorophyll accumulation in the skin of‘2013-12’.