Intronic polymorphisms in Daucus carota AOX2b generate ......Intronic polymorphisms in Daucus carota...
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Intronic polymorphisms in Daucus carota AOX2b generate Intronic polymorphisms in Daucus carota AOX2b generate putative genotype specific miRNAs Hélia G. Cardoso, M. Doroteia Campos and Birgit Arnholdt-Schmitt* Hélia G. Cardoso , M. Doroteia Campos and Birgit Arnholdt-Schmitt* 1 EU Marie Curie Chair , ICAAM, University of Évora, Apartado 94, 7002-554 Évora - Portugal *[email protected] ABSTRACT Plant mitochondria play an important role in diverse metabolic pathways and are involved in pathogen stress responses and in the process of programmed cell death. Under diverse biotic as well as abiotic stress conditions, plant mitochondrial can control the reactive oxygen species (ROS) generation by stress conditions, plant mitochondrial can control the reactive oxygen species (ROS) generation by means of energy-dissipating systems (Clifton et al. 2005. Plant Mol Biol 58:193-212). In higher plants, AOX is codified by a small multigene family with at least five genes (Clifton et al. 2006. In higher plants, AOX is codified by a small multigene family with at least five genes (Clifton et al. 2006. Biochim Biophys Acta 1757:730-741) belonging to two subfamilies: AOX1-type and AOX2-type genes. Results achieved in our laboratory has demonstrated that the D. carota AOX multigene family is characterized by two AOX1 (DcAOX1a and DcAOX1b) and two AOX2 (DcAOX2a and DcAOX2b) genes The involvement of introns in the regulation of gene expression can be due to intronic sites for important regulatory elements, such as miRNAs. This knowledge together (Costa et al. 2009. Plant Physiol Biochem 47:753-759). The involvement of introns in the regulation of gene expression can be due to intronic sites for important regulatory elements, such as miRNAs. This knowledge together with the recent identification of a putative pre-miRNA in a polymorphic region of the DcAOX2a (Cardoso et al. 2009. Physiol Plant 137:592-608) made it reasonable to investigate the effect of the variability in intron sequences on the predictability of putative miRNAs also in DcAOX2b in view of the development of functional marker candidates for carrot plant breeding. A study on several individual plant genotypes of D. carota cv . Rotin confirmed a frequent occurrence of intron length polymorphisms candidates for carrot plant breeding. A study on several individual plant genotypes of D. carota cv . Rotin confirmed a frequent occurrence of intron length polymorphisms in DcAOX2b that result in three different sizes of intron 1 (Cardoso et al. 2011. Plant Genet Res: Char Util 9:177-180). Here we will present the result of an in silico analysis performed at the intron 1 sequences of different D. carota genotypes in order to identify putative pre-miRNA sites. MATERIALS AND METHODS Cloning of DcAOX2b Complete DcAOX2b genomic sequences were amplified in 14 Daucus carota L. cv . Rotin Intron 1 sequence analysis For sequence analysis EditSeq and Clustal W algorithm of Megalign (Lasergene, Complete DcAOX2b genomic sequences were amplified in 14 Daucus carota L. cv . Rotin genotypes. Specific primers were designed in the 5’UTR (Fw: 5’- TGCATGCGTCCTTCCTTATTTTTC-3’) and 3’UTR (Rev: 5’- GCTCTGCTGTGATTTTCTGGAC-3’) of DcAOX2b (acc. EU286576). Amplicons were For sequence analysis EditSeq and Clustal W algorithm of Megalign (Lasergene, GATC Biotech, Konstanz) were applied. Putative miRNA precursors (pre-miRNAs) were searched by using the software miR-abela (http://www .mirz.unibas.ch/cgi/pred_miRNA_genes.cgi). For validation of potential GCTCTGCTGTGATTTTCTGGAC-3’) of DcAOX2b (acc. EU286576). Amplicons were generated by using Phusion™ High-Fidelity DNA Polymerase (Finnzymes Oy) and cloned in a pGEM®-T Easy System I vector (Promega). Two plant genotypes representing each band pattern were selected for sequence analysis. (http://www .mirz.unibas.ch/cgi/pred_miRNA_genes.cgi). For validation of potential pre-miRNAs the software MiPred was applied (Jiang et al., 2008. Clin Cancer Res 14:419-427). Prediction of the secondary structure of pre-miRNA was run on the web-based software MFOLD 3.1 (http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi) Two plant genotypes representing each band pattern were selected for sequence analysis. From each those genotypes three bacterial clones were sequenced. web-based software MFOLD 3.1 (http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi) (Zuker, 2003. Nucleic Acids Res 31:3406-3415). RESULTS AND DISCUSSION Five putative pre-miRNAs were predicted: 2 exclusively of fragment a (intron of 1019 bp), Previous analysis of DcAOX2b performed by Exon Primed Intron Crossing (EPIC)-PCR allowed to the identification of differences at the intron 1 size. Five putative pre-miRNAs were predicted: 2 exclusively of fragment a (intron of 1019 bp), [minimal free energy (MFE)= -16.60 and -23.30], and 1 of the fragment b (intron of 822 bp) (MFE= -19.70). intron 1 size. Sequence analysis of the three intron 1 sequences revealed that the intron 1 size can be 1019, 822 or 557 bp (named fragment a, b and c). Those differences are due by Insertions/deletions (InDels) located along An additional pre-miRNA was found in both larger introns (MFE= -16.40 or -18.90, depending on the existence of single nucleotide polymorphisms (SNPs). No pre-miRNA was predicted in the smallest version of intron 1 (fragment c corresponding to Those differences are due by Insertions/deletions (InDels) located along the sequence. No pre-miRNA was predicted in the smallest version of intron 1 (fragment c corresponding to intron size of 557bp). Pre-miRNAs predicted in intron 1 a (1019bp) b (822bp) a and b Sequences of fragment a and b are more similar between each other whereas fragment c shows a higher degree of divergence. whereas fragment c shows a higher degree of divergence. 17 InDels with positions conserved in all analyzed sequences were identified: - 9 within 1 - 10 bp (2 bp was the most frequent); - 9 within 1 - 10 bp (2 bp was the most frequent); - 5 within 10 - 50 bp; - 2 within 50 - 100 bp (61 and 97 bp); - 1 higher than 100 bp (246/248 bp). 15 putative single-nucleotide polymorphisms (SNPs) were identified - 1 higher than 100 bp (246/248 bp). between intron fragments a and b. The comparison of the three intron fragments allowed to identify 74 putative SNPs, 3 exclusively of fragment a (427: T/C, 611: A/C and 852: MFE: -23.30 MFE: -16.60 MFE: -19.70 MFE: -16.40 MFE: -18.90 putative SNPs, 3 exclusively of fragment a (427: T/C, 611: A/C and 852: T/C). CONCLUSIONS ACKNOWLEDGEMENTS ► Polymorphisms at intron 1 sequence influence the pre-miRNA prediction. ► Functional analysis should also consider introns to establish the link between the DNA polymorphisms and the phenotype. This work was supported by the European Commission through the Marie Curie Chair and by Fundação para a Ciência e a Tecnologia (FCT). phenotype. ► Further work will be made in order to confirm the existence of the predicted miRNAs and its function at the plant.
Transcript of Intronic polymorphisms in Daucus carota AOX2b generate ......Intronic polymorphisms in Daucus carota...
Intronic polymorphisms in Daucus carota AOX2b generate Intronic polymorphisms in Daucus carota AOX2b generate putative genotype specific miRNAs
Hélia G. Cardoso, M. Doroteia Campos and Birgit Arnholdt-Schmitt*
putative genotype specific miRNAs
Hélia G. Cardoso, M. Doroteia Campos and Birgit Arnholdt-Schmitt*
1EU Marie Curie Chair, ICAAM, University of Évora, Apartado 94, 7002-554 Évora - PortugalEU Marie Curie Chair, ICAAM, University of Évora, Apartado 94, 7002-554 Évora - Portugal*[email protected]
ABSTRACT
Plant mitochondria play an important role in diverse metabolic pathways and are involved in pathogenstress responses and in the process of programmed cell death. Under diverse biotic as well as abioticstress conditions, plant mitochondrial can control the reactive oxygen species (ROS) generation bystress conditions, plant mitochondrial can control the reactive oxygen species (ROS) generation bymeans of energy-dissipating systems (Clifton et al. 2005. Plant Mol Biol 58:193-212).
In higher plants, AOX is codified by a small multigene family with at least five genes (Clifton et al. 2006.In higher plants, AOX is codified by a small multigene family with at least five genes (Clifton et al. 2006.Biochim Biophys Acta 1757:730-741) belonging to two subfamilies: AOX1-type and AOX2-type genes.Results achieved in our laboratory has demonstrated that the D. carota AOX multigene family ischaracterized by two AOX1 (DcAOX1a and DcAOX1b) and two AOX2 (DcAOX2a and DcAOX2b) genes
The involvement of introns in the regulation of gene expression can be due to intronic sites for important regulatory elements, such as miRNAs. This knowledge together
characterized by two AOX1 (DcAOX1a and DcAOX1b) and two AOX2 (DcAOX2a and DcAOX2b) genes(Costa et al. 2009. Plant Physiol Biochem 47:753-759).
The involvement of introns in the regulation of gene expression can be due to intronic sites for important regulatory elements, such as miRNAs. This knowledge togetherwith the recent identification of a putative pre-miRNA in a polymorphic region of the DcAOX2a (Cardoso et al. 2009. Physiol Plant 137:592-608) made it reasonable toinvestigate the effect of the variability in intron sequences on the predictability of putative miRNAs also in DcAOX2b in view of the development of functional markercandidates for carrot plant breeding. A study on several individual plant genotypes of D. carota cv. Rotin confirmed a frequent occurrence of intron length polymorphismscandidates for carrot plant breeding. A study on several individual plant genotypes of D. carota cv. Rotin confirmed a frequent occurrence of intron length polymorphismsin DcAOX2b that result in three different sizes of intron 1 (Cardoso et al. 2011. Plant Genet Res: Char Util 9:177-180). Here we will present the result of an in silico analysisperformed at the intron 1 sequences of different D. carota genotypes in order to identify putative pre-miRNA sites.
MATERIALS AND METHODS
Cloning of DcAOX2b
Complete DcAOX2b genomic sequences were amplified in 14 Daucus carota L. cv. Rotin
Intron 1 sequence analysis
For sequence analysis EditSeq and Clustal W algorithm of Megalign (Lasergene,Complete DcAOX2b genomic sequences were amplified in 14 Daucus carota L. cv. Rotingenotypes. Specific primers were designed in the 5’UTR (Fw: 5’-TGCATGCGTCCTTCCTTATTTTTC-3’) and 3’UTR (Rev: 5’-GCTCTGCTGTGATTTTCTGGAC-3’) of DcAOX2b (acc. EU286576). Amplicons were
For sequence analysis EditSeq and Clustal W algorithm of Megalign (Lasergene,GATC Biotech, Konstanz) were applied. Putative miRNA precursors (pre-miRNAs)were searched by using the software miR-abela
(http://www.mirz.unibas.ch/cgi/pred_miRNA_genes.cgi). For validation of potentialGCTCTGCTGTGATTTTCTGGAC-3’) of DcAOX2b (acc. EU286576). Amplicons weregenerated by using Phusion™ High-Fidelity DNA Polymerase (Finnzymes Oy) and clonedin a pGEM®-T Easy System I vector (Promega).Two plant genotypes representing each band pattern were selected for sequence analysis.
(http://www.mirz.unibas.ch/cgi/pred_miRNA_genes.cgi). For validation of potentialpre-miRNAs the software MiPred was applied (Jiang et al., 2008. Clin Cancer Res14:419-427). Prediction of the secondary structure of pre-miRNA was run on theweb-based software MFOLD 3.1 (http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi)Two plant genotypes representing each band pattern were selected for sequence analysis.
From each those genotypes three bacterial clones were sequenced.web-based software MFOLD 3.1 (http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi)(Zuker, 2003. Nucleic Acids Res 31:3406-3415).
RESULTS AND DISCUSSION
Five putative pre-miRNAs were predicted: 2 exclusively of fragment a (intron of 1019 bp),Previous analysis of DcAOX2b performed by Exon Primed IntronCrossing (EPIC)-PCR allowed to the identification of differences at theintron 1 size.
Five putative pre-miRNAs were predicted: 2 exclusively of fragment a (intron of 1019 bp),[minimal free energy (MFE)= -16.60 and -23.30], and 1 of the fragment b (intron of 822 bp) (MFE=-19.70).intron 1 size.
Sequence analysis of the three intron 1 sequences revealed that theintron 1 size can be 1019, 822 or 557 bp (named fragment a, b and c).Those differences are due by Insertions/deletions (InDels) located along
An additional pre-miRNA was found in both larger introns (MFE= -16.40 or -18.90, depending onthe existence of single nucleotide polymorphisms (SNPs).
No pre-miRNA was predicted in the smallest version of intron 1 (fragment c corresponding toThose differences are due by Insertions/deletions (InDels) located alongthe sequence.
No pre-miRNA was predicted in the smallest version of intron 1 (fragment c corresponding tointron size of 557bp).
Pre-miRNAs predicted in intron 1
a (1019bp) b (822bp) a and b a (1019bp) b (822bp) a and b
Sequences of fragment a and b are more similar between each otherwhereas fragment c shows a higher degree of divergence.whereas fragment c shows a higher degree of divergence.
17 InDels with positions conserved in all analyzed sequences wereidentified:
- 9 within 1 - 10 bp (2 bp was the most frequent);- 9 within 1 - 10 bp (2 bp was the most frequent);- 5 within 10 - 50 bp;- 2 within 50 - 100 bp (61 and 97 bp);- 1 higher than 100 bp (246/248 bp).
15 putative single-nucleotide polymorphisms (SNPs) were identified
- 1 higher than 100 bp (246/248 bp).
15 putative single-nucleotide polymorphisms (SNPs) were identifiedbetween intron fragments a and b.
The comparison of the three intron fragments allowed to identify 74putative SNPs, 3 exclusively of fragment a (427: T/C, 611: A/C and 852:
MFE: -23.30 MFE: -16.60 MFE: -19.70 MFE: -16.40 MFE: -18.90
putative SNPs, 3 exclusively of fragment a (427: T/C, 611: A/C and 852:T/C).
CONCLUSIONS ACKNOWLEDGEMENTS
► Polymorphisms at intron 1 sequence influence the pre-miRNA prediction.► Functional analysis should also consider introns to establish the link between the DNA polymorphisms and thephenotype.
This work was supported by the European Commissionthrough the Marie Curie Chair and by Fundação para aCiência e a Tecnologia (FCT).
phenotype.► Further work will be made in order to confirm the existence of the predicted miRNAs and its function at the plant.
