Intron polymorphism pattern in AOX1b of wild St John's wort (Hypericum perforatum) allows discrimination between individual plants

Abstract

The present paper deals with the analysis of natural polymorphism in a selected alternative oxidase (AOX) gene of the medicinal plant, St John's wort. Four partial AOX gene sequences were isolated from the genomic DNA of a wild plant of Hypericum perforatum L. Three genes belong to the subfamily AOX1 (HpAOX1a, b and c) and one to the subfamily AOX2 (HpAOX2). The partial sequence of HpAOX1b showed polymerase chain reaction (PCR) fragment size variation as a result of variable lengths in two introns. PCR performed by Exon Primed Intron Crossing (EPIC)-PCR displayed the same two-band pattern in six plants from a collection. Both fragments showed identical sequences for all exons. However, each of the two introns showed an insertion/deletion (InDel) in identical positions for all plants that counted for the difference in the two fragment sizes. The InDel in intron 1 influenced the predictability of a pre-microRNA site. The almost identical PCR fragment pattern was characterized by a high variability in the sequences. The InDels in both introns were linked to repetitive intron single nucleotide polymorphisms (ISNP)s. The polymorphic pattern obtained by InDels and ISNPs from both fragments together was appropriate to discriminate between all individual plants. We suggest that AOX sequence polymorphism in H. perforatum can be used for studies on gene diversity and biodiversity. Further, we conclude that AOX sequence polymorphism of individual plants should be considered in biological studies on AOX activity to exclude the influence of genetic diversity. The identified polymorphic fragments are available to be explored in future experiments as a potential source for functional marker development related to the characterization of origins/accessions and agronomic traits such as plant growth, development and yield stability.

Development of 5123 Intron-Length Polymorphic Markers for Large-Scale Genotyping Applications in Foxtail Millet

“…Overall, multiple sequence alignment has shown that they were homologous to each other and comprised conserved exon regions at two end positions and non-conserved or variable intron region in the middle. Similar kind of observation was also reported in rice, 18 Hypericum perforatum 31 and foxtail millet. 20 Further, higher level of transferability of ILPs compared with previously identified markers reflects the conserved nature of exon positions in gene and variability in the non-coding sequences.…”

Alternative oxidase involvement in Daucus carota somatic embryogenesis

“…Induced single nucleotide polymorphism (SNP) was shown to affect a quantitative trait locus (QTL) for thermo tolerance in Oryza sativa L. (Abe et al 2002). AOX genes demonstrate a rich natural source for polymorphic within‐gene sequences in various species that might be explored in the future for functional marker candidates (Cardoso et al 2009, Ferreira et al 2009, Holtzapffel et al 2003, Macedo et al 2009).…”

The gymnosperm Pinus pinea contains both AOX gene subfamilies, AOX1 and AOX2

“…2). These observations suggest that defined SNPs appear rather non‐casually in pre‐determined positions as it was also found in D. carota (Cardoso et al 2009), O. europaea (Macedo et al 2009) and H. perforatum (Ferreira et al 2009), where repetitive polymorphisms were identified. Three of the six amino acid substitutions observed in the two Pp AOX1bs (positions 194, 195 and 244) also provide uniqueness to Pp AOX1b2 in otherwise conserved sites across species (Fig.…”

“…Recently, the group of Arnholdt‐Schmitt et al (2006) focussed on AOX functional marker development and naturally occurring evidence for across‐species AOX polymorphisms. Several cases of AOX polymorphisms have been highlighted, including intron length polymorphisms (ILPs) in Hypericum perforatum L. (St. John's Wort), Daucus carota L. and Vitis vinifera L. (Cardoso et al 2009, Costa et al 2009b, Ferreira et al 2009), synonymous and non‐synonymous SNPs in H. perforatum , D. carota and Olea europaea L. (Cardoso et al 2009, Ferreira et al 2009, Macedo et al 2009), micro heterogeneity through 3′‐untranslated region (UTR) length variability in O. europaea (Macedo et al 2009) and retrotransposon integration in an intron of AOX2 in V. vinifera (Costa et al 2009b). ILPs and SNPs observed in AOX genes demonstrate repetitive patterns and indicate non‐casual occurrence.…”