Limits...
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).

Show MeSH
Venn diagram to identify stably transcribed reference genes in Chrysanthemum spp., and their stability values calculated using NormFinder.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4308696&req=5

f4: Venn diagram to identify stably transcribed reference genes in Chrysanthemum spp., and their stability values calculated using NormFinder.

Mentions: A geNorm based analysis, carried out to determine which of the reference genes would be most suitable in each of the four taxa, produced the ranking based on M values, as depicted in Fig. 3. On the basis that an M value of <0.5 indicates stability, MTP, ACTIN, PGK, TUB, GAPDH and EF-1α were considered to be stable in the contrast 2xvs 4xC. nankingense, while PSAA, EF-1α, PP2A, ACTIN, PGK and GAPDH were all stable for 4xvs 6xC. zawadskii. The least stable gene in the comparison 2xvs 4xC. nankingense was PSAA, even though this gene was stable in the contrast 4xvs 6xC. zawadskii. The most stable genes across all four taxa were EF-1α and PGK, while PSAA, SKIP16 and TIP41 all performed poorly (Fig. 3). The pairwise variation parameter V calculated by geNorm predicts the optimal number of reference genes required. A Vn/Vn + 1 ratio <0.15 is taken to indicate that no additional reference gene is necessary2223. Here, the optimal number of genes was two in every case (Fig. 3). NormFinder analysis generated a very similar ranking to the GeNorm output. The combinations MTP/ACTIN, EF-1α/PSAA and EF-1α/PGK were predicted to deliver the most reliable level of normalization (Fig. 4).


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)

Venn diagram to identify stably transcribed reference genes in Chrysanthemum spp., and their stability values calculated using NormFinder.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Venn diagram to identify stably transcribed reference genes in Chrysanthemum spp., and their stability values calculated using NormFinder.
Mentions: A geNorm based analysis, carried out to determine which of the reference genes would be most suitable in each of the four taxa, produced the ranking based on M values, as depicted in Fig. 3. On the basis that an M value of <0.5 indicates stability, MTP, ACTIN, PGK, TUB, GAPDH and EF-1α were considered to be stable in the contrast 2xvs 4xC. nankingense, while PSAA, EF-1α, PP2A, ACTIN, PGK and GAPDH were all stable for 4xvs 6xC. zawadskii. The least stable gene in the comparison 2xvs 4xC. nankingense was PSAA, even though this gene was stable in the contrast 4xvs 6xC. zawadskii. The most stable genes across all four taxa were EF-1α and PGK, while PSAA, SKIP16 and TIP41 all performed poorly (Fig. 3). The pairwise variation parameter V calculated by geNorm predicts the optimal number of reference genes required. A Vn/Vn + 1 ratio <0.15 is taken to indicate that no additional reference gene is necessary2223. Here, the optimal number of genes was two in every case (Fig. 3). NormFinder analysis generated a very similar ranking to the GeNorm output. The combinations MTP/ACTIN, EF-1α/PSAA and EF-1α/PGK were predicted to deliver the most reliable level of normalization (Fig. 4).

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