Genetic Map Construction And Carotenoid Metabolism Regulation In Citrus

Carotenoids are important secondary metabolites in the plant, it can not only provided diversity color for plants, but also involved in important biological processes such as photosynthesis and hormone synthesis. As a major carotenoid, β-carotene is an important vitamin A source for human. Moreover, it is also essential health-protecting compounds to reduce the incidence of certain cancers and cardiovascular problems. To exert the effective usage, it is essential to understand the regulation mechanism of β-carotene metabolism. Because of the large diverse carotenoid patterns, citrus is an important model species for the study of plant carotenoids metabolism. In this study, we constructed high-density genetic map derived from two parents with significant differences content of p-carotene. We also used two type materials to research the regulation mechanism of β-carotene metabolism, including CsLCYb transgenic tomato fruits and abnomal β-carotene accumulation mutant from’guanxi’ pummelo. The main results are as follows:1. Creation of hybrid population and construction of high-density genetic linkage map in pummelo1) An F1 population of 550 progeny were derived a cross from the’Guanxi’ pummelo and’Pingshan’ pummelo. The’Pingshan’ pummelo (low β-carotene content) was employed as the male parent. The’Guanxi’ pummelo (high P-carotene content) was employed as the female parent.2) The RAD sequencing library was constructed from DNA of the two parents and 124 F1 individuals. Three conditions were used for filtering mapping SNP markers including sequencing depth, Mendelian segregation by X2 test, and genotype missing. In total, we identified 1,543 reliable SNP loci which can be used for the genetic map construction.3) To facilitate the integration and comparative between different genetic maps, we screened 20 SSR primers from another pummelo map developed previously. These polymorphic primers were amplification in the two parents and 124 F1 individuals. Then we recorded these SSR genotyping of 124 F1 individuals.4) According to pseudo-test-cross strategy, the genetic map was constructed by linkage analysis using Joinmap 4.0 software. The 1,563 markers were assigned to 9 linkage groups with a total genetic length of 976.58 cM and an average of 0.62 cM between adjacent loci. Most of the markers distributed randomly in 9 linkage groups. All 9 linkage groups consisted of 81-285 loci ranging in length from 75.42 to 139.26 cM5) The collinear analysis was used to evaluate the genetic position of SNP markers on the LGs against its physical position in the reference genome. According to our results, most of the markers showed good linear agreement between the physical and genetic maps. These results indicated that this genetic map is reliable. It also confirmed the high similarity between pummelo genome and sweet orange genome.6) Through comparison of shared markers in the map constructed by us and 2 maps constructed by previously researchers, we found that these markers in this study could show better identity with other maps. Moreover, these markers also showed a similar genetic distance. These results indicated that this genetic map is reliable. These common SSR markers allowed us to integrate these genetic maps in the future, which making the map constructed in this study more useful and valueable.2. Molecular mechanisms associated with orange-pericarp mutation in pummelo1) The carotenoid composition and content of pericarp were analysed by HPLC in MT versus WT. The distinctive orange colour in pericarp has clearly been shown to be due to the massive accumulation of β-carotene. The p-carotene content of MT was about 10.5-fold higher than that of the WT. Moreover, the total carotenoid concentration of MT was also7.9-fold higher than that of WT.2) The effect of the mutation on carotenogenic gene expression was examined by qRT-PCR. Results showed that the expression levels of upstream carotenogenic genes (PSY, PDS and CCS) were much higher in WT than in MT. The expression of downstream carotenogenic genes (CCD1, BCH and NCED2) was significantly reduced in MT than in WT.3) The RNA-seq data showed that 303 genes were significantly differentially expressed (padj<0.05) in MT pericarp compared to the WT. Of these genes,135 were up-regulated and 168 were down-regulated in MT. Analysis of the RNA-seq data using the KEGG database revealed that the carbon metabolism, starch and sucrose metabolism, and biosynthesis of amino acids were significantly changed in the MT.4) The GC-MS analysis showed that most of the soluble sugar and amino acids contents in MT were affected in the MT. Most of the soluble sugar contents were significantly reduced in MT pericarp compared to the WT. Interestingly, we detected amount of asparagine in MT but trace in WT. These data suggest that enhanced metabolic flux from glycolysis and down-regulation of downstream genes for P-carotene degradation are critical for the formation of β-carotene accumulation trait in the MT. The down-regulation of upstream carotenoid synthesis genes may caused by the feedback inhibition of endproducts.5) In order to identify potential important or novel genes involved in the β-carotene regulation, we also used RNA-Seq to analysis another β-carotene accumulation fruit mutation. The RNA-seq data showed that 200 genes were significantly differentially expressed (padj<0.05) in MT fruit compared to the WT. Of these genes,92 were up-regulated and 108 were down-regulated in MT. After a comprehensive analysis of two sets RNA-Seq data, we found 10 common differential expressed genes, these genes mainly involved in fatty acid metabolism and resistance response.3. Effect of the citrus lycopene β-cyclase transgene on carotenoid metabolism in transgenic tomato fruits1) By using a PCR-based progeny test, single copy carrier pure line (T217-1) selected from the carotenoid transgenic material engineered previously was used for the further research. The T2 generation line we selected was still showing steady yellow phenotype similar to the To generation, indicating that the transgenic event is very stable.2) The CsLCYb and its wild type fruits at mature green, breaker, and ripe stages were collected for the further research. HPLC analysis revealed that the content of (3-carotene and total carotenoid both significantly increased in CsLCYb overexpressed fruits at all three stages, but the a-branch carotenoids were declined. The a-carotene content was significantly increased in the ripe stage. Moreover, the CsLCYb fruits also accumulated substantial quantities of violaxanthin at the ripe stage, a compound which was not detected in WT fruit. There was not significant difference in plastid development and structure between CsLCYb and wild type at all three developmental stages.3) The expression levels in both CsLCYb and wild type of the carotenogenic genes were assessed by qRT-PCR at all three developmental stages. At the mature green stage, except for PDS and LCYe, the transcript abundance of all of these genes was lower in the transgenic fruit. At the breaker stages, all of the upstream genes were unregulated by the constitutive expression of CsLCYb, while transcript abundance for the three downstream genes (LCYe, BCH and ZEP) was all lower in CsLCYb than in WT. At the ripe stage, all but ZEP were up-regulated in the transgenic fruit. Thus, ZEP appeared to be down-regulated by the constitutive expression of CsLCYb throughout fruit development.4) The microarray data showed 93 genes were significantly differentially expressed (FDR≤0.05 and fold change≥2) in CsLCYb overexpression fruits compared to the wild type. Of these genes,30 were up-regulated and 63 were down-regulated in CsLCYb fruits. GO analysis revealed that the most highly represented categories were concerned with binding, catalytic activity, hydrolase activity and protein binding. Analysis of the microarray data using the KEGG database revealed that the synthesis or degradation of secondary metabolites, fatty acids biosynthesis, starch and sucrose metabolism, and photorespiration were significantly changed in the CsLCYb overexpression fruits.5) According to the expression level of ZEP, we used the LC-MS to determine the ABA concentration. The resulted showed that the ABA content in CsLCYb fruit was lower than in the WT fruit at all three developmental stages sampled. It was consistent with the expression levels of ZEP. According to the microarray data, we used the GC to determine the soluble sugar contents during three developmental stages in CsLCYb and wild type fruits. Results showed that the sucrose content was unaffected by the presence of the transgene at all three development stages, but the concentration of both fructose and glucose was lower in the CsLCYb than in the WT fruit at all stages, especially at the ripe stage.