Introduction
Until the 1970s, eukaryotic introns were considered to be genetically inert sequences with no function in the genome and not subject to evolutionary selection. They were known as dubbed selfish or junk DNA (Orgel and Crick, Reference Orgel and Crick1980). However, recent evidences show that plant introns play an active role in the control of gene expression (Gianì et al., Reference Gianì, Morello, Bardini and Breviario2003; Fiume et al., Reference Fiume, Christou, Gianì and Breviario2004) and evolution. Insertions/deletions (InDels) and single-nucleotide polymorphisms (SNPs) are the two main types of DNA polymorphism (Nasu et al., Reference Nasu, Suzuki, Ohta, Hasegawa, Yui, Kitazawa, Monna and Minobe2002) found in intron sequences (Wang et al., Reference Wang, Zhao, Zhu and Wu2005). These intronic polymorphisms are becoming important genetic markers for genetic research and breeding in major crop species (Bi et al., Reference Bi, Sanchez-Villeda, Schroedor, Gardiner, Polacco, Soderlund, Wing, Fang and Coe2006; Braglia et al., Reference Braglia, Manca, Mastromauro and Breviario2010).
The recent identification of intron variation in carrot AOX2a (Cardoso et al., Reference Cardoso, Campos, Costa, Campos, Nothnagel and Arnholdt-Schmitt2009) suggested the investigation of intron variation in other carrot AOX genes (Campos et al., Reference Campos, Cardoso, Linke, Costa, Fernandes de Melo, Justo, Frederico and Arnholdt-Schmitt2009; Costa et al., Reference Costa, Cardoso, Campos, Zavattieri, Frederico, Fernandes de Melo and Arnholdt-Schmitt2009a). Alternative oxidase (AOX) is an inner mitochondrial membrane protein involved in stress acclimation and adaptation of diverse nature (e.g. McDonald and Vanlerberghe, Reference McDonald and Vanlerberghe2006; Plaxton and Podestá, Reference Plaxton and Podestá2006). We describe here the intron variation in carrot AOX2b (DcAOX2b) among genotypes, with a view to potential applications in carrot breeding (Arnholdt-Schmitt et al., Reference Arnholdt-Schmitt, Costa and Fernandes de Melo2006; Arnholdt-Schmitt, Reference Arnholdt-Schmitt2009; Polidoros et al., Reference Polidoros, Mylona and Arnholdt-Schmitt2009).
Materials and methods
In a search for intron length polymorphisms (ILPs) in the three introns of DcAOX2b (see gene structure in Campos et al., Reference Campos, Cardoso, Linke, Costa, Fernandes de Melo, Justo, Frederico and Arnholdt-Schmitt2009), exon-primed intron crossing-PCR (EPIC-PCR) was performed using 10 ng DNA of 40 Daucus carota L. genotypes (14 of cv. Rotin and 26 of wild carrot). Specific primers were designed in the exons flanking each intron (Table 1). To avoid the occurrence of heteroduplex fragments, electrophoresis was performed in denaturing conditions in 3-(N-morpholino) propanesulphonic acid buffer (BDH, Haasrode, Belgium) supplied with 2% formaldehyde (Sigma-Aldrich, Steinheim, Germany).
Oligonucleotides applied in the present work
Fw, forward; Rev, reverse; T a, annealing temperature; T e, extension time at 72oC for 35 cycles.
For analysis of intron variation in the DcAOX2b sequence, the gene was isolated from six genotypes of cv. Rotin using a forward primer located in the 5′-untranslated region (UTR) and a reverse in the 3′UTR (Table 1). For sequence analysis, EditSeq and the ClustalW algorithm of Megalign (Lasergene 7; GATC Biotech AG, Konstanz, Germany) were applied. Putative miRNA precursors (pre-miRNAs) located in intron sequences were searched and validated as described in Cardoso et al. (Reference Cardoso, Campos, Costa, Campos, Nothnagel and Arnholdt-Schmitt2009).
Results and discussion
EPIC-PCR fragments from intron 2 and intron 3 show lengths consistently around 430 or 400 bp in all carrot genotypes analyzed. Sequence analysis yielded a size for intron 2 of 91 bp and for intron 3 of 85 bp. All intron 2 sequences were identical. In intron 3 sequences, four putative SNPs were identified.
ILP was found in intron 1 in the genome of individual plants and between plant genotypes. Six polymorphic patterns were identified: three single-band patterns (homozygous) named fragments a, b and c, and three double-band patterns (heterozygous) named ab, ac and bc (Fig. 1). In cv. Rotin and in wild carrot, the PCR-fragment pattern related to the single 1 kb (b) band was found more frequently than other patterns (Fig. 1). No more than two fragments were observed in the same individual plant, consistent with biallelic nature in a diploid species. Breviario et al. (Reference Breviario, Baird, Sangoi, Hilu, Blumetti and Gianì2007), using ILPs as molecular markers, described a link between the number of amplified bands and ploidy level of the taxon. ILPs were also reported in the other member of the AOX2-subfamily in D. carota (Cardoso et al., Reference Cardoso, Campos, Costa, Campos, Nothnagel and Arnholdt-Schmitt2009) and in the AOX1-subfamily of other plant species (Naydenov et al., Reference Naydenov, Takumi, Sugie, Ogihara, Atanassov and Nakamura2005; Costa et al., Reference Costa, Fernandes de Melo, Gouveia, Cardoso, Peixe and Arnholdt-Schmitt2009b; Ferreira et al., Reference Ferreira, Cardoso, Macedo, Breviario and Arnholdt-Schmitt2009).
Agarose gel showing the six-band patterns identified by EPIC-PCR for intron 1 of DcAOX2b. From the homozygote genotypes, 40% demonstrated fragment b (seven genotypes of cv. Rotin and nine of wild genotypes), fragment a was detected once in Rotin and also in wild genotypes, fragment c was not detected in cv. Rotin, but was present in four genotypes of wild carrot. From the heterozygous genotypes, the combination of fragments bc was more frequent than other combinations (22%: two genotypes in cv. Rotin and seven in the wild genotypes), the combination ab amplified in three genotypes of cv. Rotin and wild carrots and ac appeared once in cv. Rotin and twice in wild carrot plants. MM: molecular marker (O'Range Ruler 100bp+500bp Ladder; Fermentas, Ontario, Canada).
The length of inton 1 of DcAOX2b was reported as 822 bp (Cardoso et al., Reference Cardoso, Campos, Costa, Campos, Nothnagel and Arnholdt-Schmitt2009). The present extended analysis reveals that intron 1 has at least three length variants of 1019 (a), 822 (b) and 557 bp (c).
The existence of SNPs and InDels was confirmed by sequencing. Both types of polymorphisms are known as two of the major driving forces in genome evolution (e.g. Gregory, Reference Gregory2004). Twelve putative SNPs were identified between the intron 1 sequences of fragment a and b (the most similar fragments). When fragment c was included in the alignment, 64 additional putative SNPs were identified. Four of them were specific to fragment a (427:T/C, 611:A/C, 852:T/C and 1061:T/C).
Three InDels were identified by alignment of fragments a and b. Integrating the 557 bp intron sequence into the alignment allowed the identification of nine new InDels. In total, 12 InDels were identified, three larger than 50 bp, six with a size between 5 and 50 bp and three shorter than 5 bp. This observation is not in full agreement with Wang et al. (Reference Wang, Zhao, Zhu and Wu2005), who reported that the most frequent InDels (72.6%) had a size of < 5 bp, followed (23.5%) by 5–50 bp and very few (3.9%) longer than 50 bp.
The involvement of introns in the regulation of gene expression can be due to the coding of regulatory elements such as miRNAs, recently associated with plant stress responses (Chiou et al., Reference Chiou, Aung, Lin, Wu, Chiang and Su2006) and development (Wang et al., Reference Wang, Reyes, Chua and Gaasterland2004). In DcAOX2b intron 1 sequences, five pre-miRNAs were predicted: two exclusively in fragment a, one in fragment b and two common to both. No pre-miRNA could be predicted for the short fragment c.
The data obtained for the orthologous gene AOX2b in Vigna unguiculata point to its function in modulating abiotic stress response (Costa et al., Reference Costa, Jolivet, Hasenfratz-Sauder, Orellano, Lima, Dizengremel and Fernandes de Melo2007). McCabe et al. (Reference McCabe, Finnegan, Millar, Day and Whelan1998) reported an enhancement of AOX2b expression during the ageing of soybean cotyledons concomitant to a reduction of AOX2a transcripts and a low level of AOX1 expression.
The polymorphisms identified by us in DcAOX2b are of special interest due to their location in the first intron. Introns that are most proximate to the 5′ end of a gene were observed to exert a more pronounced effect on gene expression (Rose et al., Reference Rose, Elfersi, Parra and Korf2008). In future experiments, genotypes with homozygous and heterozygous polymorphic intron 1 sequences will be explored for an association to diverse abiotic stress phenotypes and differential expression of miRNA.
Acknowledgements
This work was supported by the European Commission through a Marie Curie Chair for Birgit Arnholdt-Schmitt and by the Foundation for Science and Technology (FCT) through the COMPETE programme and for scholarship to M. Doroteia Campos (SFRH/BI/15991/2006). The authors are grateful to James C. Nelson from the Kansas University in USA for comments on the manuscript and language revision.
