| Ascorbic acid (AsA) also known as vitamin C is a multifunctional metabolites and could directly or indirectly to clean up the reactive oxygen species (ROS) in plant. Since the human body can not synthesize AsA, it can only be obtained from food. AsA content is an important index of the quality of vegetables. In addition, flowering is the most important transition from vegetative to reproductive growth and flowering period affect on the vegetable yields. Both AsA content and flowering are regulated by complex networks in plant. Although AsA and flowering related genes have been identified in model plant Arabidopsis, extending the study of these genes from Arabidopsis to crops could shed light on the evolution of AsA and flower related genes in plants and inform crop breeding.Brassica rapa as important crop in Brassica genus, mainly contains Chinese Cabbage (B.rapa ssp. pekinensis) and non-heading Chinese Cabbage (B. rapa ssp. chinensis). With the completion of B. rapa whole-genome sequencing, comparative genomics as a strategy for agricultural applications. Through comparison of important genes between Arabidopsis and Brassicas genome to reveal the evolution patterns and duplication divergence of these genes and also to obtain the key genes. Meanwhile, in order to obtain the key genes about excellent traits, the QTL mapping analysis were performed in non-heading Chinese cabbage. The main results are as follows:1. Patterns of evolutionary conservation of ascorbic acid-related genes following whole-genome triplication in Brassica rapaWe identifried 102 AsA-related genes in B. rapa and they mainly diverged 12-18 million years ago accompanied by the Brassica-specific genome triplication event. During their evolution, these AsA-related genes were preferentially retained, consistent with the gene dosage hypothesis. The putative proteins were highly conserved, but their expression patterns varied. Although the number of AsA-related genes is higher in B. rapa than in A.thaliana, the AsA contents and the numbers of expressed genes in leaves of both species are similar, the genes that are not generally expressed may serve as substitutes during emergencies.2. Conservation and adaptability of ascorbic acid L-galactose pathway genes in Brassica rapaThe L-galactose pathway is the major plant AsA biosynthetic pathway, thus, genes of this pathway are conservation during evolution. In this study,we further explored the conservation of these genes in Brassicas and the functional divergence in B. rapa. Similar retention and a high degree of gene sequence similarity were identified in B. rapa (A),Brassica oleracea (C) and Brassica napus (AC). However, among these genes, the number of genes which encode the same enzymes were various. Except GME has nine genes, there were one to four genes encode other enzymes, respectively. Moreover, we found functional divergence exist widely among them: i) VTC2 and VTC5 were paralogous genes, but only VTC5 might be influenced by FLC; ii) under light treatment, PMI1 co-regulated the AsA pool size with other D-Man/L-Gal pathway genes, while PMI2 was regulated only by darkness; iii) under NaCl, Cu2+, MeJA and wounding stresses, most of paralogs exhibited different expression patterns. Additionally, GME and GPP were the key regulatory enzymes that limit AsA biosynthesis under these treatments.3. Molecular evolutionary analysis of AKR genes in plant kingdom and cloned them in pak-choi (Brassica rapa ssp. chinensis)The aldo-keto reductase (AKR) superfamily are involved in a wide range of plant metabolic pathways. GalUR, one of ascorbic acid biosynthetic enzymes, belong to this family. Through investigation of the evolutionary patterns,footprints and conservation of AKRs genes in plants, we identified that they are divided into two distinct groups (groups A and B) and group A genes were considered as GalUR candidate genes. By nucleotides distance,selective pressure and protein structure analyses,we concluded that group A AKRs were more conservative than group B AKRs. We found that the retention of AKRs was conservative in Brassica rapa after the specific Brassica-Arabidopsis thaliana split.Furthermore, tandemly gene duplication was the reasons for their expansion. Next, we cloned and sequenced 12 group A AKRs from cDNA libraries of stress-induced Pak-choi,and verified the relationship between their expression and AsA content under multiple stresses. They exhibit diverged expression patterns, BrcAKR22 had correlation with AsA content, BrcAKR23 had a central role in response under the stresses.4. Comprehensive analysis of the polygalacturonase and pectin methylesterase genes in Brassica rapa shed light on their different evolutionary patternsPolygalacturonases (PGs) and pectin methylesterases (PMEs), major components of the pectin remodeling and disassembly network, are involved in AsA Galacturonate pathway and in cell separation processes during many stages of plant development,especially in pollen development. A comprehensive study of these genes in plants could shed light on the evolution patterns of their structural development. In this study, we conducted whole-genome annotation,molecular evolution and gene expression analyses of PGs and PMEs in Brassica rapa and 8 other plant species. A total of 100 PGs and 110 PMEs were identified in B. rapa; they primarily diverged from 12-18 MYA and PMEs were retained more than PGs. Along with another 305 PGs and 348 PMEs in the 8 species, two different expansion or evolution types were discovered: a new branch of class A PGs appeared after the split of gymnosperms and angiosperms, which led to the rapid expansion of PGs; the pro domain was obtained or lost in the proPMEs through comprehensiveanalyses among PME genes. In addition, the PGs and PMEs exhibit diverged expression patterns.5. Comprehensive analysis of the flowering genes in Chinese cabbage and examination of evolutionary pattern of Co-like genes in plant kingdomIn this study, we identified 798 floweing related genes in Brassica rapa and then analysed the evolutionary pattern, duplication and expression level of these genes.According to characterization of flowering genes in monocots and eudicots, including the identification of family-specific, orthologous and collinear genes, we obtained that flora meristem and photoperiod pathway were important in B. rapa. Then we identified 25 CONSTANS-like genes in B. rapa and explored the evolution of COL genes in plant kingdom and constructed the evolutionary pattern of COL genes. Through comparative analysis of CO genes between Brassicaceae and Grass, we identified that several family-specific amino acids, and revealed that CO protein structures were similar in B. rapa and Arabidopsis but different in rice. Finally, we explored the expression patterns of photoperiod pathway-related genes in B. rapa under different photoperiod treatments by qPCR. This analysis will provide resources for understanding the flowering mechanisms and evolutionary pattern of COL genes. In addition, this genome-wide comparative study of COL genes may also provide clues for evolution of other flowering genes.6. Genome-wide analysis of the wads-box gene family in Brassica rapa (Chinese cabbage)The MADS-box gene family functions in almost every developmental process in plants, especially in flower development. In this study, 160 MADS-box transcription factors were identified from the entire Chinese cabbage genome. Phylogenetic analysis of the BrMADS genes, together with their Arabidopsis and rice counterparts, showed that the BrMADS genes were categorised into type ? (M?,M?,M?) and type ? (MIKCc,MIKC*)groups,and the MIKCc proteins were further divided into 13 subfamilies. The Chinese cabbage type ? group has 95 members, which is twice as much as the Arabidopsis type ?group, indicating that the Chinese cabbage type ? genes have been retained more frequently than the type ? genes. Finally, RNA-seq transcriptome data and quantitative real-time PCR analysis revealed that BrMADS genes are expressed in a tissue-specific manner similar to Arabidopsis. Interestingly, a number of BrMIKC genes showed responses to different abiotic stress treatments,suggesting a function for some of the genes in these processes as well.7. High-density genetic linkage map construction and identification of main QTLs in non-heading Chinese cabbage using whole genome resequencingAsA content and flowering time directly affect the quality and production of B. rapa.Thus, revealing the location of trait control genes on linkage groups is of significant value.A high-density linkage map is a valuable tool for fine mapping quantitative trait loci (QTL)and map-based gene cloning for molecular breeding. In this study, we constructed a high-density linkage map of NHCC using 5440 SNPs, developed by the whole genome resequencing 100 recombinant inbred lines. The length of the map was 1915.07 cM with an average marker interval of 0.35 cM. A high level of genomic synteny between this linkage map and B. rapa genome physical maps was detected that anchoring SNPs were able to anchor ten linkage groups to their corresponding chromosomes. Based on this high-density integrated linkage map and leaf phenotyping, a total of 6 potential QTLs for AsA content and flowering time identified and positioned on the genetic map using composite interval mapping (CIM) model. It is significant in molecular breeding and comparative genome analysis, especially in Brassicas crops. |
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