| The black-seeded pumpkin(Cucurbita ficifolia)belongs to Cucurbita family,originated from the high altitude area of America,and has a long history of cultivation in Yunnan,China.It is often used as the grafting rootstock of melon,and has high cultivation and utilization value.In recent years,more and more attention has been paid to the exploration of genetic resources and genetic diversity of black-seeded pumpkin.Chloroplast genome structure is conserved,nucleic acid replacement rate is moderate,and has an independent genetic route,which is suitable for plant phylogenetic relationship research and development of molecular identification technology.In this study,the chloroplast genome of black seed pumpkin was sequenced using the second-generation high-throughput sequencing technique.Bioinformatics tools were used to assemble and annotate the chloroplast genome,and the basic characteristics of the chloroplast genome,Codon usage bias and repeat sequence and other basic characteristics.Of Cucurbita and 5species chloroplast genome to conduct a comprehensive analysis,comparative genomics based on pumpkin plant chloroplast genome and 24 other different genus of cucurbitaceae chloroplast genome constructed phylogenetic tree,according to five kinds of pumpkins belong to compare the results of the chloroplast genome,the genus specific DNA barcode research and development of SSR molecular markers,The main results are as follows:(1)The first chloroplast genome sequence of The black-seeded pumpkin was obtained and published.The chloroplast genome of The black-seeded pumpkin was composed of single-loop double-stranded DNA molecules,with a total length of 157,533 bp and GC content of 37%,presenting a conserved tetrad structure.The genome sequence encodes a total of 130 genes,including 44 genes related to photosynthesis,36 t RNA genes and 8r RNA genes.The gene arrangement and frequency of codon use are similar to other published chloroplast genomes of cucurbit family.A total of 31 tandem repeats,48 scattered repeats and 67 simple repeats were detected in the chloroplast genome of The black-seeded pumpkin.(2)The chloroplast genome of The black-seeded pumpkin was applied to comparative genomics.The results showed that the sequence structure and basic characteristics of the chloroplast genome of the five genera of Cucurbita were relatively similar,and the coding region of the genome sequence was more conserved.The genome was colinear and no rearrangement was found.There was little difference in the number of repeat sequences of different types in the chloroplast genome of Cucurbita,and the junction between IR region and SC region was also very close.The five species of Cucurbita in the phylogenetic tree showed close genetic relationship.In general,the chloroplast genome of Cucurbita was conservative in the evolutionary process,and the heritage diversity was narrow.(3)Based on comparative genomics analysis,two molecular identification techniques of pumpkin specific DNA barcodes and SSR molecular markers were studied.The results showed that the combination fragment trn L-ndh J + trn L-ndh K + ycf1-trn N had the strongest species identification ability among the seven specific candidate DNA barcodes of Pumpkin.It can be used as a specific DNA barcode of Cucurbita for subsequent species identification.The results of SSR cluster analysis with polymorphism were similar to that of DNA Barcoding evolutionary tree,Forty-two SSR markers were developed,among which 10 pairs of primers could amplify specific bands.In this study,we published the first black-seeded pumpkin chloroplast genome,conducted comparative genomic analysis of pumpkin chloroplast genome,identified the phylogenetic position of Cucurbita in cucurbit family,and developed suitable Cucurbita specific DNA barcodes and SSR molecular markers.It is of great significance for the study of systematization,population heredity,nucleo-cytoplasmic interaction,breeding of superior varieties and identification of germplasm resources of Cucurbita. |
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