关键词: 柑橘, CcCCD基因家族, CcCCD4a, 果肉颜色, 基因表达, 单倍型

Abstract:

【Objective】To reveal the distribution, structure and evolution of carotenoid cleavage dioxygenas gene family in the citrus genome (CcCCD), this study were performed to develope marker-assisted selection of flesh color in citrus breeding program, bioinformatics predication, expression analysis and genotype of CcCCD4a in flesh color development and different germplasm accessions. 【Method】The CCD gene family of Citrus clementina genome were identified by homologous search according to previously reported CCD in other plant species. Phylogenetic analysis, subcellular localization prediction, relative molecular weight, theoretical isoelectric point (PI), conserved motif prediction, and scaffold location were studied by bioinformatics methods. Real-time fluorescence quantitative PCR (qRT-PCR) was used to study the expression of CcCCD4a in 10 citrus accessions during the flesh color development period. Haplotype analysis was performed by Tassel software after sequencing of CcCCD4a in 30 citrus varieties. 【Result】Fourteen CCD family genes were found in the Citrus clementina genome, and these genes could be divided into five subfamily, namely, CcCCD1, CcCCD4, CcCCD7, CcCCD8 and CcNCED. Its theoretical isoelectric point were 6.05 to 8.53 and these CCD family genes encoded 412-611 amino acids. The subcellular localization prediction indicated that CcCCD genes mainly were located in chloroplast and cytoplasm. Phylogenetic analysis showed that CCD genes in citrus were also found in other plant species. Obviously, CCD8 subfamily had farther genetic distance with other CCD. Scaffold localization analysis showed that 14 CcCCD members were unevenly distributed in all scaffolds except scaffold 5. The phenotyping of flesh color in 10 citrus varieties demonstrated that the hue angle of flesh color was decreased along with fruit maturing. The relative expression of CcCCD4a in different citrus varieties was significantly different. The expression of CcCCD4a in the flesh color of orange red was significantly lower than that in the flesh color of orange or light orange yellow (P<0.05). There was a significant positive correlation between the relative expression of CcCCD4a and the hue angle during fruit ripening. Genotyping of CcCCD4a in 30 citrus varieties revealed that hap-1, hap-4 and hap-5 were dominant haplotype at orange red flesh varieties. 【Conclusion】The whole genome of Citrus clementina contained 14 members of CCD gene family. All these CCD gene family members contained RPE65 conserved domain, but they were located in different cell components and unevenly distributed at different scaffolds. CcCCD4a was involved in the development of citrus flesh color, and there was a significant positive correlation between its relative expression and the hue angle. Therefore, CcCCD4a could be used as potential marker for citrus fruit color breeding. Especially, hap-1, hap-4 and hap-5 had a high correlation with the phenotypes of orange red flesh, which might be helpful for selecting candidate hybrids in early stage of citrus breeding program.

Key words: citrus, CCD gene family, CcCCD4a, flesh color, gene expression, haplotype