Genetic Basis Of Flower Color Variation Among Wild Populations Of Aquilegia Yabeana In North China

The flower color of angiosperms is one of the most significant and diverse innovative traits in nature,and the diversity not only among species,but also within species.The diversity of flower color is mostly considered to be related to pollinators,as a key visual signal to attract pollinators.Due to pollinators are different to color perception and detection ability,thus,flower color diversity of angiosperms is thought to be an adaptation to the vast diversity of animal pollinators.In addition,non-pollinator factors may play an important role of selection on flower color variation.It is great significance of the formation of angiosperm biodiversity to study the formation mechanism of flower color variation and the relationship between the maintenance of variation and natural selection.The genus Aquilegia is widely distributed among the world and has undergone rapid radiation evolution,and there are abundant flower color variations in and within species.In previous work,the different pigment molecules and the transcriptome sequencing of perianth tissue of different color plants were completed of A.yabeana.In this paper,based on the previous research of our group,we analyzed the overall differences of transcriptome between the two flower colors of A.yabeana,and screened the key genes that may participate in flower color regulation.Then,we selected new plant individuals to verify the expression level of candidate genes,and analyzed the genetic diversity of promoters and coding regions of possible upstream transcription factors,carried out to find the key genetic variation sites that may be directly related to flower color variation.1)Based on the clustering and enrichment analysis of differentially expressed genes in the transcriptome of purple flower BJ01 and yellow flower ln01,this study found that the genes specifically up-regulated in the perianth slices of the two flower color plants were involved in different biological processes,molecular functions and metabolic pathways.The highly expressed genes of purple flower plants are significantly enriched in flavonoid metabolism pathway.2)Based on hierarchical clustering and enrichment analysis,21 differentially expressed candidate genes were screened,the results of Realtime RT-PCR in more plant individuals showed that the expression levels of the six genes(CHI DFR,GST,TT12-1,MIXTA-2 and pEARLI1)in purple flowers were significantly higher than those in yellow flowers.By analyzing the promoters of the six genes,it was found that there were no genetic variation sites related to flower color difference,and there were multiple binding sites of MYB and bHLH transcription factors in the promoters of the six genes.3)Furthermore,we screened three transcription factors from transcriptome data,verified the gene expression and analyzed the genetic diversity.The results showed that AyMYB3-1 and AyBHLH-1 were differentially expressed in the two flower color plants,but there was no genetic variation related to flower color in their promoters and coding regions.AyMYB3-2 was highly expressed in the perianth of all individuals with purple flower,and the protein sequence and structure are also conserved.AyMYB3-2 was almost not expressed in LN06,and the promoter region of LN06 has large insertion and deletion compared with the purple flower population.In the yellow flower population with high expression of AyMYB3-2,there were 3 different forms of deletion in the coding region of AyMYB3-2,which made the amino acid sequence lose multiple functional domains of MYB transcription factor.4)We continued to hybridize the plants of two flower colors,and constructed the molecular marker of genetic background identification.We observed the flower color of F1 generation after genetic background detection,it can be found that,the perianth color of F1 plants from different cross combinations generally showed the perianth has purple and yellow colors at the same time.It is suggested that the two colors of perianth may be two independent traits,which are controlled by different genes.In this study,we compared the transcriptome level of two flower color A.yabeana,and found that the function of the genes highly expressed in the perianth of two flower color populations was differentiated.The genetic differentiation of AyMYB3-2 in promoter region and gene coding region was consistent with the difference of flower color,which may lead to the loss of anthocyanin synthesis and accumulation in A.yabeana f.luteola,and this study laid a foundation for the research group to clarify the molecular mechanism of flower color variation among wild populations of A.yabeana.