Inheritance And Genetic Mapping Of Seed Coat Color In Cucurbita Maxima

Pumpkin is an annual herb of Cucurbita in Cucurbitaceae.Its seeds have high nutritional value and medical health value.Seed coat color is one of the important commercial characters of seed pumpkin due to the preference for white seed coat of pumpkin in the consumer market and the benefit value of actual production.However,the research on seed coat color formation and development regulation of Cucurbita maxima has not been carried out,which limits the aggregation of excellent characters and variety selection of seed type Cucurbita maxima.Map-based cloning and functional analysis of seed coat color regulation genes will help to clarify the molecular regulation mechanism of seed coat color formation and provide basis for seed pumpkin germplasm innovation.In this experiment,two inbred assessions with different seed coat color,Wuminglv（yellow seed coat inbred assessions）and Agol（white seed coat inbred assessions）were used as parents to study the formation process of seed coat color at different grain development stages and construct the six generations population.The In Del molecular markers were developed by genome re-sequencing to map the seed coat color control gene,and an interval closely linked to the target trait was obtained.The recessive recombinant plants were screened by encrypting flanking linkage molecular markers,and the candidate genes were fine mapped.By combining genome annotation,gene sequence alignment,transcriptome sequencing and gene expression analysis,it was predicted that a MYB transcription factor might be a candidate gene controlling seed coat color of Cucurbita maxima.Molecular marker of seed coat color gene was developed to screen seed coat color of Cucurbita maxima.The main results are as follows:1.The important color changing period of seed coat is about 21-28 DPA,and the significant increase period of proanthocyanidins（PAs）content during seed coat development is 21-28 DPA.The structure of seed coat is composed of five layers:epidermis,hypodermis,sclerenchyma,parenchyma and chlorenchyma.The sclerenchyma of Wuminglv differentiated into two layers of cells,while Agol had only one layer.2.Genetic analysis of 6 generations showed that the seed coat color of Cucurbita maxima was a quality trait controlled by a single gene.The yellow seed coat was dominant to the white seed coat,and the gene was named White Seed Coat（WSC）.3.On the basis of the genetic map constructed by predecessors in the laboratory,the In Del markers were further developed by using the parent re-sequencing data.Combined with the seed coat color phenotype of F₂individual plant,the seed coat color gene was located in a 1.1 Mb region between the markers M1503 and M1504 on chromosome 15.Fourty six pairs of In Del markers were designed in the initial mapping interval,and 23 pairs of primers with good polymorphism were selected to screen F₂populations in autumn 2019 and spring 2020.Finally,the WSC gene was located in a 60.4 kb region between the markers SD-4 and s1546.4.According to the annotation information of Cucurbita maxima genome,there are nine genes in the region.Through the sequence alignment of the nine candidate genes in the parents,15 stable mutations in the promoter region of and intron region of Cma Ch15G005270 were found,which were linked with seed coat color.It was speculated as the candidate gene for regulating seed coat color.According to the functional annotation,the gene encodes an R2-MYB transcription factor.5.Fifty-four accessions and 222 F₂segregating population and RILs plants were used to validate the accuracy of the closest flanking In Del marker S1548.The accuracy of S1548 marker was 100%and exhibited validation in the molecular assistance breeding program in edible seed pumpkin in Cucurbita maxima.6.q RT-PCR showed that the candidate gene Cma Ch15G005270 was highly expressed in leaf,flower,pulp and seed coat,with the highest expression level in the seed coat of Wuminglv at 28 DPA,and the expression level in the male parent was significantly higher than that in the female parent.RNA-seq analysis showed that the amount of DEGs was most remarkable at 28 DPA,in accordance with the period of seed coat color formation.KEGG enrichment analysis showed that phenylpropanoid was significantly enriched in the metabolic pathway,suggesting that the difference in seed coat color was closely related to phenylpropanoid biosynthesis pathway.It was verified that the expression of some genes in this pathway increased significantly in the stage of seed coat color changes.