| Narcissus,also known as daffodil,is a famous bulbous plant in the world for its unique flower shape,brilliant flower color and high ornamental value.However,the flower color of Narcissus is relatively monotonous,usually white,yellow or orange.Various colors have always been an important breeding objective in Narcissus.The widely cultivated Narcissus cultivar ‘Slim Whitman' has white perianth and pale yellow corona,which gradually pale to white during late flowering stages.The cultivar ‘Pinza' possesses yellow perianth and yellow-orange corona.These two cultivars are representative for their distinctive flower color.However,the molecular mechanism of different flower color among cultivars has not been revealed.The understanding of molecular mechanism of flower color formation can provide theoretical basis for artificial regulation and genetic improvement of Narcissus flower color.In this study,a large number of F1 lines were obtained through hybridization for the first time.Among the hybrid lines,hybrid offsprings of ‘Slim Whitman' and ‘Pinza' showed great differences in flower color and were used as the main experimental materials to study the mechanism of difference in flower color of Narcissus.Based on the measurement of the composition and content of carotenoids and transcriptome sequencing analysis in perianth and corona of ‘Slim Whitman' and ‘Pinza',the differentially expressed genes related to the formation of pigments in Narcissus were identified.The structural genes related to carotenoid biosynthesis pathway were identified by bioinformatics and quantitative fluorescence PCR.The key gene NpCCD4 involved in carotenoid degradation was identified and functional studies were carried out to reveal the molecular mechanism of flower color differences between different cultivars.The main results were as follows:1.Hybrid evaluation of NarcissusIn this study,using 7 common cultivars as parents,101 F1 lines with different genotypes were obtained by hybridization for the first time.The growth,reproduction and phenotypic characteristics of the hybrids were studied in detail during 2016 to 2018.Some hybrid lines had high reproductive rate and good disease resistance.After asexual propagation,they had reproduced to a certain amount and become valuable Narcissus germplasm resources.2.Morphological analyses of reciprocal cross hybrids of ‘Slim Whitman' and ‘Pinza'Twenty-seven hybrid lines of ‘Slim Whitman' and ‘Pinza',including 15 Slim Whitman×Pinza offsprings and 12 Pinza×Slim Whitman offsprings,were obtained by cross-breeding.20 phenotypic traits of ‘Slim Whitman' and ‘Pinza' and their hybrids were investigated in detail,and there were large genetic variations in the hybrid lines.In cluster analysis according to the twenty morphological characteristics,27 hybrids and their parents were divided into different groups.Of these hybrids,SP04 got novelty flowers with white perianth and a yellow-orange corona and had the potential to become a new popular Narcissus cultivar.3.Carotenoids composition and content in perianth and corona of ‘Slim Whitman',‘Pinza' and their hybridsThe carotenoids in perianth and corona of ‘Slim Whitman' and ‘Pinza' and their hybrid progenies were analyzed by HPLC-MS/MS.The results showed that 10 kinds of carotenoids,such as all-trans-lutein,9-cis-violaxanthin and all-trans-?-carotene,were detected.The total carotenoid content in perianth and corona of ‘Slim Whitman' rose first and then decreased,reaching the highest value in stage 3.After stage 4,the carotenoid components except all-trans-neoxanthin and 9-cis-neoxanthin,decreased dramatically,which were in consistent with the color fading phenomenon.The total carotenoids contents in perianth and corona of ‘Pinza' were much higher than that in ‘Slim Whitman',and there was no degradation of carotenoids at the late stages of flower development.In the hybrids,the carotenoids content in white perianth lines were lower than that in yellow perianth.The carotenoid contents in perianths were lower than that in corona in both cultivars.The orange rim at the corona of ‘Pinza' and some hybrid lines mainly came from the accumulation of all-trans-?-carotene.4.Transcriptome analysis of perianths and coronas of ‘Slim Whitman' and ‘Pinza'To investigate the mechanism of color formation,RNA-Seq of perianths and coronas of ‘Slim Whitman' and ‘Pinza' was performed.A total of 67.13 GB clean reads were generated,and then assembled into 84,463 unigenes.A total of 30,156 unigenes(36.88%)were annotated based on the public protein databases.730 and 3,317 differentially expressed genes were obtained in SWP vs SWC and PZP vs PZC,and 1,022 and 3,880 differentially expressed genes were obtained in SWP vs PZP and SWC vs PZC.Further differential gene enrichment analysis showed that differentially expressed genes in “carotenoid biosynthesis pathway(ko00906)” between cultivars and tissues were significantly enriched.5.Expression level of genes involved in carotenoid biosynthesis pathway in NarcissusThe accumulation of carotenoids is the main factor determining the flower color of Narcissus.In this study,qRT-PCR technique was used to analyze the expression levels of key genes of carotenoid biosynthesis pathway in different cultivars and tissues using ACT as internal reference genes.The results showed that the expression levels of upstream genes such as PSY in the coronas of ‘Slim Whitman' and ‘Pinza' were higher than those in the perianths,but there was no significant difference between different cultivars.Nevertheless,the expression of CCD gene(c77684.graph_c0)was significantly different between the two cultivars: it was abundantly expressed in perianth and corona of ‘Slim Whitman',but almost not detected in perianth and corona of ‘Pinza'.It was speculated that the expression of PSY and other upstream genes affected the accumulation of carotenoids in perianth and corona of the same cultivar,while the CCD gene(c77684.graph_c0)affected the accumulation of carotenoids in different cultivars.6.Cloning and functional study of NpCCD4 geneIn this study,the carotenoid degrading gene NpCCD4 was cloned in Narcissus by PCR for the first time.The coding region of the gene is 1806 bp and coding for 601 amino acids.Sequence analyses revealed that the NpCCD4 protein contains highly conserved structures of CCD.The gene was mainly expressed in perianth and corona of ‘Slim Whitman' after blooming,but was low in the bulb,leaf and scape,and almost not expressed in ‘Pinza'.Silencing of NpCCD4 resulted in a significant increase in carotenoid accumulation,especially in all-trans-?-carotene and corona of positive plants turned to orange red.Therefore,NpCCD4 had the biological activity of degrading carotenoids.NpCCD4 is a crucial factor that causes the low carotenoid content and color fading phenomenon of ‘Slim Whitman' by mediating carotenoid turnover. |
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