| With the development of society and the gradual improvement of people’s living standards,consumers are increasingly demanding the quality of special agricultural products.Xiangyu pumpkin is deeply loved by consumers because of its rich fragrance characteristics.Therefore,it is of great significance to carry out research on the molecular mechanism of fragrance traits of Xiangyu pumpkin for regulating fragrance of Xiangyu pumpkin and cultivating new varieties of Xiangyu pumpkin.In this paper,the leaves of Xiangyu pumpkin(No.312)and non-Xiangyu pumpkin(No.18292)are used as materials.Based on gas chromatography and mass spectrometry(GC-MS)technology,volatile compounds are obtained.Principal component analysis(PCA)and Partial Least Squares Regression Analysis(PLS-DA)analysis of differential compounds.The genetic background was analyzed by simple repeated sequence markers(SSR),and transcriptome sequencing analysis was performed to obtain differentially expressed genes,and the relevant candidate genes for the formation of fragrance traits were selected.At the same time,the fruits of one type of Xiangyu pumpkin(No.44)and four types of non-Xiangyu pumpkin(No.45,301,326,335)were studied through electronic nose,GC-MS,and gas sniffing technology(GC-O)In-depth analysis of volatile compounds of Xiangyu pumpkin fruit and other key substances.The content of 2-acetyl-1-pyrroline(2-AP)in Xiangyu pumpkin leaves at different harvesting times and sprayed with foreign substances was detected.Finally,the Xiangyu pumpkin(No.44,female parent)and non-Xiangyu pumpkin(No.45,male parent)were used as parents to construct the F2 population,and 25 F2 single plants were used to construct the two mixed pools,using DNA-BSA sequencing technology.The taro flavor gene localization segment was obtained,and through the whole genome re-sequencing of the parents,polymorphic molecular markers were developed in the flavor gene localization segment,and molecular markers closely linked to the taro flavor were screened.The main results of this study are as follows:1.Use GC-MS to collect and analyze the volatile components of Xiangyu pumpkin leaves.A total of 59 types of aroma substances were identified,including 18 types of aldehydes,13 types of alcohols,10 types of ketones,8 types of alkenes,5 types of alkanes,2 types of esters,2 types of heterocyclic compounds,and 1 type of phenols Species.The component with the highest content of a single compound is 2-hexenal.Using PCA and PLS-DA analysis,it was found that the aroma components of the two types of materials were significantly different.The contribution rates of the first principal component(PC1)and the second principal component(PC2)were 68.1% and 17.2%,respectively.By analyzing the importance projection(VIP)values of the different compounds,the top five compounds with the highest contribution are caryophyllene,1-nonanol,α-terpineol,2-ethyl-1-hexanol and 2-AP.2.Using polymorphic SSR marker detection,Xiangyu pumpkin(No.312)and non-Xiangyu pumpkin(No.18292)have similar genetic backgrounds and are suitable materials for molecular mechanism research.A transcriptome analysis of pumpkin leaves revealed a total of 543 differential genes,including 302 up-regulated genes and 214down-regulated genes.The differential genes were annotated into 82 metabolic pathways,and the five most abundant pathways were: photosynthesis-antenna proteins,phenylpropanoid biosynthesis,fatty acid degradation,ascorbate and aldarate metabolism,fatty acid metabolism.3.Electronic nose technology can distinguish Xiangyu pumpkin and non-Xiangyu pumpkin resources from the overall fragrance level.GC-MS technology was used to analyze the aroma compounds of Xiangyu pumpkin fruits.PCA analysis was performed on the data obtained.The contribution rates of PC1 and PC2 were 27.87% and 21.02%,respectively,indicating the difference in volatile substances between different resources.A total of 25 different compounds that could be characterized were identified among different pumpkin resources.Among them,there are two different compounds between Xiangyu pumpkin and four non-Xiangyu pumpkins,namely 1-hexanol and 2-AP.4.Using GC-O technology to study the aroma compounds of pumpkin fruits,four flavor compounds with the highest dilution factor were identified.Among them,the volatile substance that had a significant correlation with the change of pumpkin taro aroma index was 2-AP.5.Among the pumpkin fruits,the 2-AP content on the 5th day was the highest,but the content of this substance decreased significantly from the 35 th day on.Spraying foreign substances on the leaves of Xiangyu pumpkin: zinc chloride treatment(three concentrations:50,100,150 mg/kg)and mixed treatment: α-ketoglutarate 5 mmol/L+r-aminobutyric acid 4mmol /L+6-BA 15 mg/L+ethrin 5 mmol/L.After 7 days of treatment,the content of 2-AP in the high-concentration zinc chloride treatment was significantly lower than that in the control,and the content of 2-AP in the mixed treatment on 14 d was significantly higher than that of the control.There was no significant change in each treatment on 21 d.6.The F2 generation single plant material obtained by crossing the pumpkin materials44 and 45 was used to construct a mixed pool and subjected to DNA-BSA sequencing.A total of 22.39 Gbp data volume was obtained.Based on the Euclidean Distance(ED)correlation algorithm,a candidate region related to taro flavor traits was obtained and located on the 10 th chromosome of Chinese pumpkin,with a total length of 2.35 Mb.A total of 508 genes were annotated,including 115 non-synonymous mutant genes and 20 frameshift mutant genes among the parents.7.The primers were designed based on the insertion-deletion polymorphism(In Del)marker sites obtained by whole-genome resequencing of the two parents,and one of the primers with clear and stable bands was selected for the identification of taro flavor resources.The results showed that the aroma phenotypes of different types of Chinese pumpkin resources were consistent with primer PCR amplification results.PCR amplification of improved materials for taro flavor trait breeding showed that the improved materials were divided into homozygous plants with taro flavor traits and heterozygous plants without taro flavor.It can be seen from the phenotypes of the various improved materials that the In Del marker can efficiently and accurately screen out individual plants with taro flavor traits,improving the efficiency of improving taro flavor traits. |
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