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Discovery and comparative profiling of microRNAs in a sweet orange red-flesh mutant and its wild type - PubMed

  • ️Fri Jan 01 2010

Discovery and comparative profiling of microRNAs in a sweet orange red-flesh mutant and its wild type

Qiang Xu et al. BMC Genomics. 2010.

Abstract

Background: Red-flesh fruit is absent from common sweet orange varieties, but is more preferred by consumers due to its visual attraction and nutritional properties. Our previous researches on a spontaneous red-flesh mutant revealed that the trait is caused by lycopene accumulation and is regulated by both transcriptional and post-transcriptional mechanisms. However, the knowledge on post-transcriptional regulation of lycopene accumulation in fruits is rather limited so far.

Results: We used Illumina sequencing method to identify and quantitatively profile small RNAs on the red-flesh sweet orange mutant and its wild type. We identified 85 known miRNAs belonging to 48 families from sweet orange. Comparative profiling revealed that 51 known miRNAs exhibited significant expression differences between mutant (MT) and wild type (WT). We also identified 12 novel miRNAs by the presence of mature miRNAs and corresponding miRNA*s in the sRNA libraries. Comparative analysis showed that 9 novel miRNAs are differentially expressed between WT and MT. Target predictions of the 60 differential miRNAs resulted 418 target genes in sweet orange. GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in transcription regulation, protein modification and photosynthesis. The expression profiles of target genes involved in carotenogenesis and photosynthesis were further confirmed to be complementary to the profiles of corresponding miRNAs in WT and MT.

Conclusion: This study comparatively characterized the miRNAomes between the red-flesh mutant and the wild type, the results lay a foundation for unraveling the miRNA-mediated molecular processes that regulate lycopene accumulation in the sweet orange red-flesh mutant.

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Figures

Figure 1
Figure 1

Fruit development of the red-flesh mutant 'Hong anliu' sweet orange (MT) and its wild type (WT). The schematic presentation shows the strategy of transcriptional analysis by the Massive Parallel Signature Sequencing (previously) and post-transcriptional analysis by sRNA sequencing (this study) on the MT and WT. DAF stands for Days After Flowering.

Figure 2
Figure 2

Length distribution of sweet orange small RNA.

Figure 3
Figure 3

Known miRNAs from sweet orange (Citrus sinensis), designated as csi on the first column, and their homologs in other plant species. The picture is modified from Axtell et al. [45]. Scale 0, miRNA only predicted; scale 1, miRNA sequenced; scale 2, miRNA/miRNA* accumulation detected; scale 3, miRNA detected by small RNA blot or qRT-PCR; scale 4, miRNAs with validated targets. The general information is based on miRBase 13.0 statistics. Abbreviations: ath, Arabidopsis thaliana; bna, Brassica napus; csi, Citrus sinensis; eca, Eschscholzia californica; mtr, Medicago truncatula; osa, Oryza sativa; pta, Pinus taeda; ptc, Populus trichocarpa; ppt, Physcomitrella patens; sly; Solanum lycopersicum; vvi, Vitis vinifera; zma, Zea mays. Some of the data is renewed with information from publications including Arabidopsis [24,62], California poppy [46], grape [60], rice [44], and tomato [23,55].

Figure 4
Figure 4

Expression confirmation of citrus miRNAs derived from high throughput sequencing. (A). novel miRNAs expression detected by stem-loop qRT-PCR; (B) differentially expressed known miRNAss expression detected by stem-loop qRT-PCR; (C) electrophoresis of the stem-loop PCR products. Each primer has been used for the PCR amplifications on two samples, mutant and wild type.

Figure 5
Figure 5

Gene ontology categories of the predicted target genes of the 60 differential miRNAs. Categorization of miRNA-target genes was performed according to the cellular component (A), molecular function (B), and biological process (C).

Figure 6
Figure 6

The model for the biological processes possibly regulated by miRNAs in the red-flesh mutant of sweet orange. The pathway was modified based on Xu et al. (2009) [13]. The two biological processes, the carotenogenesis and photosynthesis, were high ranked processes that differential miRNA target genes involved in by gene ontology analysis. The data of photosynthetic rate were extracted from our previous in situ photosynthesis analysis [13]. The expression profiles of miRNA319 and miR1857 are complementary to the profiles of the corresponding miRNA-target genes (phosphoenolpyruvate carboxylase [TC11997]) and lycopene β-cyclase [LYCb]), implied the possible roles of these miRNAs in regulation the biological processes on posttranscriptional way.

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