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Reference gene selection for cross-species and cross-ploidy level comparisons in Chrysanthemum spp.

Wang H, Chen S, Jiang J, Zhang F, Chen F - Sci Rep (2015)

Bottom Line: Here, ten candidate reference genes are compared in the context of gene transcription in the genus Chrysanthemum.EF-1α and PGK (phosphoglycerate kinase) was the best combination for the complete set of four taxa.These results suggest that when making cross-species comparison of transcript abundance involving different ploidy levels, care needs to be taken in the selection of reference gene(s).

View Article: PubMed Central - PubMed

Affiliation: 1] College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China [2] Jiangsu Province Engineering Lab for Modern Facility Agriculture Technology &Equipment, Nanjing 210095, China.

ABSTRACT
The establishment of a (set of) stably expressed reference gene(s) is required to normalize transcription data. Polyploidy is very common in the plant kingdom, but it is not necessarily the case that a reference gene which works well at the diploid level will also work well at the polyploid level. Here, ten candidate reference genes are compared in the context of gene transcription in the genus Chrysanthemum. The robustness of some, but not all, of these was shown to be high across ploidy levels. MTP (metalloprotease) and ACTIN (actin) were the most stable in diploid and tetraploid C. nankingense, while PSAA (photosynthesis-related plastid gene representing photosystem I) and EF-1α (elongation factor-1α) were the most stable in tetraploid and hexaploid C. zawadskii. EF-1α and PGK (phosphoglycerate kinase) was the best combination for the complete set of four taxa. These results suggest that when making cross-species comparison of transcript abundance involving different ploidy levels, care needs to be taken in the selection of reference gene(s).

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Relative abundance of RAD51 transcript, estimated from qPCRs based on either stable or unstable reference genes.(a) The contrast 2xvs 4xC. nankingense, using as reference genes either PSAA or MTP/ACTIN, (b) the contrast 4xvs 6xC. zawadskii, using as reference genes either SKIP16 or PSAA/EF-1α.
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f7: Relative abundance of RAD51 transcript, estimated from qPCRs based on either stable or unstable reference genes.(a) The contrast 2xvs 4xC. nankingense, using as reference genes either PSAA or MTP/ACTIN, (b) the contrast 4xvs 6xC. zawadskii, using as reference genes either SKIP16 or PSAA/EF-1α.

Mentions: To demonstrate the usefulness of the validated candidate reference genes, the transcription levels of RAD51 in both C. nankingense, and C. zawadskii were estimated using qPCR (the isolation and primer details provided in Supplementary information). Current knowledge of the functions of the RAD51 was known to be a key pathway in cells for the homologous recombination and repair of DNA damage26. This gene was chosen because polyploidization events in the genus Chrysanthemum are associated with a certain range of DNA repair disorder27, which can be expected for a high transcript abundance of RAD51 in 4xC. nankingense and 6xC. zawadskii. However, when normalized using a variety of reference genes, there was a substantial degree of divergence in its estimated relative transcript abundance. Based on the unstable reference genes PSAA and SKIP16, the level of transcription of RAD51 was almost the same in 2x and 4xC. nankingense and similarly between 4x and 6xC. zawadskii. In contrast, estimates based on normalization using the most stable reference gene combination (i.e., ACTIN and EF-1α), suggested that there was a more than two fold difference in transcript abundance in the higher ploidy species (Fig. 7).


Reference gene selection for cross-species and cross-ploidy level comparisons in Chrysanthemum spp.

Wang H, Chen S, Jiang J, Zhang F, Chen F - Sci Rep (2015)

Relative abundance of RAD51 transcript, estimated from qPCRs based on either stable or unstable reference genes.(a) The contrast 2xvs 4xC. nankingense, using as reference genes either PSAA or MTP/ACTIN, (b) the contrast 4xvs 6xC. zawadskii, using as reference genes either SKIP16 or PSAA/EF-1α.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4308696&req=5

f7: Relative abundance of RAD51 transcript, estimated from qPCRs based on either stable or unstable reference genes.(a) The contrast 2xvs 4xC. nankingense, using as reference genes either PSAA or MTP/ACTIN, (b) the contrast 4xvs 6xC. zawadskii, using as reference genes either SKIP16 or PSAA/EF-1α.
Mentions: To demonstrate the usefulness of the validated candidate reference genes, the transcription levels of RAD51 in both C. nankingense, and C. zawadskii were estimated using qPCR (the isolation and primer details provided in Supplementary information). Current knowledge of the functions of the RAD51 was known to be a key pathway in cells for the homologous recombination and repair of DNA damage26. This gene was chosen because polyploidization events in the genus Chrysanthemum are associated with a certain range of DNA repair disorder27, which can be expected for a high transcript abundance of RAD51 in 4xC. nankingense and 6xC. zawadskii. However, when normalized using a variety of reference genes, there was a substantial degree of divergence in its estimated relative transcript abundance. Based on the unstable reference genes PSAA and SKIP16, the level of transcription of RAD51 was almost the same in 2x and 4xC. nankingense and similarly between 4x and 6xC. zawadskii. In contrast, estimates based on normalization using the most stable reference gene combination (i.e., ACTIN and EF-1α), suggested that there was a more than two fold difference in transcript abundance in the higher ploidy species (Fig. 7).

Bottom Line: Here, ten candidate reference genes are compared in the context of gene transcription in the genus Chrysanthemum.EF-1α and PGK (phosphoglycerate kinase) was the best combination for the complete set of four taxa.These results suggest that when making cross-species comparison of transcript abundance involving different ploidy levels, care needs to be taken in the selection of reference gene(s).

View Article: PubMed Central - PubMed

Affiliation: 1] College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China [2] Jiangsu Province Engineering Lab for Modern Facility Agriculture Technology &Equipment, Nanjing 210095, China.

ABSTRACT
The establishment of a (set of) stably expressed reference gene(s) is required to normalize transcription data. Polyploidy is very common in the plant kingdom, but it is not necessarily the case that a reference gene which works well at the diploid level will also work well at the polyploid level. Here, ten candidate reference genes are compared in the context of gene transcription in the genus Chrysanthemum. The robustness of some, but not all, of these was shown to be high across ploidy levels. MTP (metalloprotease) and ACTIN (actin) were the most stable in diploid and tetraploid C. nankingense, while PSAA (photosynthesis-related plastid gene representing photosystem I) and EF-1α (elongation factor-1α) were the most stable in tetraploid and hexaploid C. zawadskii. EF-1α and PGK (phosphoglycerate kinase) was the best combination for the complete set of four taxa. These results suggest that when making cross-species comparison of transcript abundance involving different ploidy levels, care needs to be taken in the selection of reference gene(s).

Show MeSH