Genetic Research And Expression Analysis Of Flower Color Diversity In Nicotiana Alata

Nicotiana alata is a landscape plant with colorful flowers and a long flowering period,which has high ornamental value.However,the heredity laws and gene expression patterns among different flower colors of N.alata were rarely reported.To explore the heredity laws of different flower colors in N.alata,reciprocal cross experiments were conducted on white,purple,red and green flowers of N.alata in this research.Meanwhile,transcriptome sequencing was used to analyze the gene expression profiles in corollas of these four colors at the mature stage to explore the differences of genes expression formed by flower color diversity in N.alata.The results are helpful to preliminarily understand the heredity laws of different flower colors and the differences of genes expression in the process of flower colors formation,which provide the genetic and hereditary bases for further analyzing the formation mechanism of flower color diversity and breeding new varieties of N.alata.The main results of this research are as follows:（1）It was found that the red and green colors of flowers can be superimposed.The F₁generation of reciprocal cross between red and green flowers all showed red flowers,and the segregation ratio of green and non-green flowers was about 3:1.The segregation ratios of red and non-red,green and non-green flowers of the F₁ generation of reciprocal cross between purple and green flowers were the same,approximately 1:1.In the reciprocal cross between white and green flowers,there was no red flowers in the F₁ generation,and the segregation ratio of green and non-green flowers was about 1:1.According to the flower colors distribution of F₁ generation,it was inferred that the genotypes of the red,purple,white,and green flower parents were Cx IIRRGg,Cx Iirrgg,Cxiixxgg,and Cxii Rr Gg,respectively（the x means uncertain genotype）.（2）By transcriptome sequencing of four corollas of different colors of N.alata,333,046unigenes were obtained and 33,445 differentially expressed genes（DEG）were identified after quality control and denovo assembly.Through GO and KEGG enrichment analysis of DEG,it was found that the chlorophyll metabolism pathway and the flavonoid biosynthesis pathway may play important roles in the formation of flower color diversity in N.alata.（3）32 DEG homologous to chlorophyll metabolism genes were identified.The results showed that compared with chlorophyll degradation,the differences in expression of chlorophyll synthesis genes may be the reason for the difference between green and other colors.HEMA is the first key rate-limiting gene of chlorophyll synthesis.The expression of HEMA homologous DEG（Nala236120）was significantly up-regulated in green flowers than that in the other three flowers.（4）41 DEG homologous to anthocyanin biosynthesis genes were identified.The results showed that the competition for substrates between anthocyanin biosynthesis pathway and other branch pathways in the flavonoid biosynthesis pathway may be the reason for the difference between red and other colored flowers.DFR is the key gene of anthocyanin biosynthesis.Compared with white and green flowers,the expression of DFR homologous DEG was significantly up-regulated in red and purple flowers.FLS promotes flavonol biosynthesis pathway to compete with substrates from anthocyanin biosynthesis pathway.Compared with red and purple flowers,the expression of FLS homologous DEG was significantly up-regulated in white flowers.And compared with red flowers,the expression of ANR homologous DEG（Nala2007339）was significantly up-regulated in purple flowers.（5）Through homologous alignment,conservative domain analysis and motif analysis between DEG and transcription factors coding genes related to anthocyanin biosynthesis in other species.And the protein interaction network analysis of proteins which encoded by DEG related to anthocyanin biosynthesis pathway was performed.17 putative transcription factors coding genes were identified,among which HY5 homologous DEG（Nala995950）,AN4 homologous DEG（Nala727362）and AN1 homologous DEG（Nala719468）may play positive roles in the regulation of anthocyanin biosynthesis in N.alata.While,MYB12homologous DEG（Nala1964987）may be a negative regulator of anthocyanin synthesis.