Mapping And Candidate Gene Analysis Of Purple Leaf Gene BnaA.PL1 In Brassica Napus L

Purple leaf, one of the morphological variations in the Brassica napus, is dued to the accumulation of anthocyanins in leaf. The purple leaf can be used as an indicator in the utilization of heterosis, and as secondary metabolites of plants for light protection, resistance to drought or cold, and resistance against diseases and insect pests. Meanwhile, anthocyanins have biological health care functions, such as antioxidation, antitumor, improve cardiovascular. Taken together, the anthocyanins-related traits have become one of research hot spots in recent years.In this study, the purple leaf mutant (6-1029) in B. napus was observated for leaf colour and the distribution of the anthocyanins anatomy at the whole growth period. A segregation population was developed together a nearly isogenic line (NIL) population by crossing purple leaf mutant with wild type (6-1025). The contents of chlorophyll, anthocyanins, and the photosynthesis characteristics of the three kinds of color leaf plants in NIL were investigated. And the genetic model and mapping for purple leaf were performed. The candidate genes were predicted by annonating in the reference genomes of B. rapa and B. napus. The major results are as follows:1 The whole growth period morphological observation of 6-1029 leaf colorThe color of 6-1029 cotyledon was initially green, then turned pale purple at six days after germination. The primary true leaf was green, while the leaf vein appeared pale purple 3 days later. At seedling, the color of leaves of 6-1029 gradually deepened and showed typical purple characters until 35 days after germination. Since the beginning of the bolting stage, the purple leaves of 6-1029 began to fade, and turned completely green at the final flowering time. Therefore, the colour of 6-1029 purple leaf was in association with the growth period.2 Anthocyanins distribution of 6-1029Anthocyanins are distributed on epithelial cells in both of the 6-1029 and F1, not in 6-1025. And the density of anthocyanin in F1 leaves was lower than that of 6-1029. It was in accordance with the phenotypic observation at seedling, i.e. the colour of leaf was purple, pale purple and green in 6-1029, F1 and 6-1025, respectively.3 Comparison of chlorophyll and anthocyanin contentAmong three kinds of leaf in NILs, no significant difference was found for the content of chlorophyll, while significant difference was detected for the ratio of chlorophyll a/b, and the content of anthocyanin, chlorophyll b and total chlorophyll content. The content of chlorophyll b and total chlorophyll was highest in green leaf, while the content of anthocyanin and the ratio of chlorophyll a/b were highest in purple leaf. The pale purple leaf was in the middle of purple and green leaf for those traits.4 The photosynthesis comparison of NILThe A-Q Curve and A-Ci Curve of NIL 3 color leaves plants have been detected by LI-6400 photosynthetic system. Although the trends of A-Q Curve and A-Ci Curve among the 3 color blade plants were basically samilary, the photosynthetic rate of purple leaves plants were higher than that of pale purple leaves plants and green leaves plants under the same light intensity or the same CO2 concentration. Meanwhile, there was no difference of net photosynthesis rate between pale purple leaves plants and green leaves plants under the same light intensity or the same CO2 concentration. The photosynthetic parameters of 3 kinds of leaf were not significantly different.5 Genetic analysis and fine mapping of BnaA. PL1The colour of F1 exhibited pale purple, which was in the middle of two parents. Three kinds of leaf colour in F2 population were found, purple, pale purple and green. And the the segregation pattern, purple:pale purple:green fited 1:2:1. It indicated that purple leaf was controlled by an incompletely dominant gene, referred as BnaA.PLl. By using the method of BSA, BnaA.PL1 was located at end of the A3 in B. napus, corresponding to B. rapa genome Scaffold000096. Followed, the interval of the candidate gene was narrowed in a region with 99kb using a fine mapping population with 2,646 individuals. Unfortunately, all of the linked markers are located on the same side of BnaA.PL1.6 Identification of the candidate geneIn the 99 kb sequence of B. rapa Scaffold000096 and its homologous region in B. napus, there were eleven annotated genes. Expression analysis in NIL plants showed that only BnaA03g59800D was highly expressed, and the expression level of the BnaA03g59800D gene decreased from the leaves of purple plants to pale purple plants to green plants. Three InDel and 18 SNP loci were found among the gene sequences from the two parents. One InDel and seven SNPs were found in the exons resulted in three deletions and one substitution of amino acid.RNA-seq analysis was performed using RNAs from three color leaf plants in NILs at the 5 leaf stage. The results showed that there were 22 high expression genes related to the synthesis of anthocyanins, the differential expression pattern of which was in line with the phenotype (PL>PPL>GL). There was only one gene BnaA03g45610D located on the chromosome A03, but far from our candidate region. In Scaffold000096, only BnaA03g59800D was the high expression gene, consistant with the results of qPCR, and thus the BnaA03g59800D gene was considered as the candidate gene.