Identification Of Yellow-flesh Mutant Of 'CN9' Nectarine And Analysis Of Gene Expression Related To Carotenoid Metabolism

Yellow flesh peaches are rich in carotenoids which are beneficial to human beings.Therefore,the exploration of the variation mechanism of white/yellow flesh peach could provide a theoretical basis for fruit breeding with high carotenoids content.In this study,‘CN9' nectarine and its yellow-flesh mutant ‘CN9Y' were used to investigate the mechanism of carotenoid metabolism and the expression of genes related to carotenoid metabolism.The content and composition of total carotenoid was determined by high performance liquid chromatography(HPLC)and spectrophotometry,and genes expression was determined by real-time quantitative polymerase chain reaction(RT-PCR)method,the results showed that the reason of bud sport mutation happening maybe related to carotenoid cleavage dioxygenase 4 gene(CCD4),and carotenoid cleavage dioxygenase gene(CCD4)from the two materials was cloned and characterized,fruits volatile aromatic compounds were determined by gas chromatogram-mass spectrometry(GC-MS),and carotenoid metabolism,temporal and spatial genes expression in other organs were also studied.The results were as follows: 1.In mesocarp of fruits,‘CN9' and ‘CN9Y' were mainly consist of ?-carotene,violaxanthin and lutein during the early development stage,and the total carotenoid content seems similar.At the ripening stage,the total carotenoid content of ‘CN9Y' fruits was approximately 8 folds to ‘CN9',which was mainly composed of violaxanthin,9-cis-violaxanthin,?-carotene and zeaxanthin,while little zeaxanthin and ?-carotene appeared in ‘CN9'.2.The expression of CCD4 gene was obviously up-regualted in mesocarp during fruit development of ‘CN9',which may led to a mass degradation of carotenoid,however,the expression of CCD4 was very low in ‘CN9Y' fruits from young fruit stage to mature stage.Therefore,the difference of CCD4 gene expression levels between ‘CN9' and ‘CN9Y' maybe the reason which led to the huge difference of carotenoid content in mesocarp.3.CCD4 gene in these two cultivars were characterized,and the results showed: The ‘CN9' cultivar harbored the W1 and y2 alleles,while its yellow-flesh mutant ‘CN9Y' was heterozygous for the y1 and y2 alleles.The W1 and y1 alleles differed only in the number of repeats in the microsatellite region.The differences of the repeat number in the microsatellite region of CCD4 might be related to the accumulation of carotenoid in peach fruits.4.The analysis of carotenoid content in other organs showed: In calyx tubes,the total carotenoid content of ‘CN9Y' was approximately 6 folds to ‘CN9',and the ?-carotene content was almost 20 folds to ‘CN9'.In petals,the total carotenoid content was similiar,but there was huge difference between the composition of carotenoids from ‘CN9' and ‘CN9Y'.In leaves,the total carotenoid content of the two materials was similar at young fruit period,after fruit ripening,the total carotenoid content of ‘CN9Y' was significantly higher than that of ‘CN9'.In shoots cortex,the total carotenoid content of ‘CN9' and ‘CN9Y' showed a slowly rising trend,and the carotenoid content of ‘CN9Y' was higher than that of ‘CN9' after fruit ripening.5.The temporal and spatial genes expression indicated that: The expression of carotenoid metabolism relevant genes exhibited obvious organ-specificity.In calyx tubes,the reason of the content of total carotenoid and ?-carotene of ‘CN9Y' were much higher than that of ‘CN9' may mainly came from the different expression of carotenoid biosynthesis genes,but not the carotenoid degradation genes.In leaves,the accumualtion of carotenoids may made a feedback repression to the expression of carotenoid biosynthesis genes,the significant difference of carotenoid content between ‘CN9' and ‘CN9Y' leaves after fruit ripening might be related to the different gene expression levels of CCD4.In petals and shoots cortex,the regulation mechanism of carotenoid metabolism maybe regulated by many factors.6.The analysis of volatile aromatic compounds showed: At early stages of peach fruit development,the volatile aromatic compounds in ‘CN9' and ‘CN9Y' fruit were mainly consisted of(E)-2-Hexenal,Benzaldehyde and Hexanal.At the ripening stage of fruit,the volatile aromatic compounds in ‘CN9' were mainly composed of 2-Hexenal(61.14 %),Hexanal(23.57 %)and(E)-2-Hexenal(6.84 %),however,the volatile aromatic compounds in ‘CN9Y' were mainly composed of(E)-2-Hexenal(70.96 %)and Hexanal(18.83 %).