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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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Amplicon dissociation curves and PAGE analysis.(a) Melting curve analysis of the ten reference genes. (b) PAGE analysis of the amplicons. Marker from top to bottom: 300 bp, 200 bp and 100 bp. The samples in each PAGE separation are ordered from left to right as EF-1α, PP2A, GAPDH, ACTIN, MTP, SKIP16, PGK, TIP41, TUB and PSAA.
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f1: Amplicon dissociation curves and PAGE analysis.(a) Melting curve analysis of the ten reference genes. (b) PAGE analysis of the amplicons. Marker from top to bottom: 300 bp, 200 bp and 100 bp. The samples in each PAGE separation are ordered from left to right as EF-1α, PP2A, GAPDH, ACTIN, MTP, SKIP16, PGK, TIP41, TUB and PSAA.

Mentions: A total of ten genes were selected as candidate reference genes in leaves (the third and fourth true leaves from the shoot apex without any treatment) from diploid (2x) and tetraploid (4x) C. nankingense, and 4x and hexaploid (6x) C. zawadskii. The primer and amplicon characteristics of these ten genes are summarized in Table 1. Melting curve (Fig. 1a) and PAGE (Fig. 1b) analyses showed that each primer pair amplified a single PCR product of the expected size. Analysis using the LinRegPCR program showed that amplification efficiency ranged from 0.8926 to 0.9952, and that the correlation coefficients ranged in value between 0.9922 and 0.9985 (Table 1).


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)

Amplicon dissociation curves and PAGE analysis.(a) Melting curve analysis of the ten reference genes. (b) PAGE analysis of the amplicons. Marker from top to bottom: 300 bp, 200 bp and 100 bp. The samples in each PAGE separation are ordered from left to right as EF-1α, PP2A, GAPDH, ACTIN, MTP, SKIP16, PGK, TIP41, TUB and PSAA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Amplicon dissociation curves and PAGE analysis.(a) Melting curve analysis of the ten reference genes. (b) PAGE analysis of the amplicons. Marker from top to bottom: 300 bp, 200 bp and 100 bp. The samples in each PAGE separation are ordered from left to right as EF-1α, PP2A, GAPDH, ACTIN, MTP, SKIP16, PGK, TIP41, TUB and PSAA.
Mentions: A total of ten genes were selected as candidate reference genes in leaves (the third and fourth true leaves from the shoot apex without any treatment) from diploid (2x) and tetraploid (4x) C. nankingense, and 4x and hexaploid (6x) C. zawadskii. The primer and amplicon characteristics of these ten genes are summarized in Table 1. Melting curve (Fig. 1a) and PAGE (Fig. 1b) analyses showed that each primer pair amplified a single PCR product of the expected size. Analysis using the LinRegPCR program showed that amplification efficiency ranged from 0.8926 to 0.9952, and that the correlation coefficients ranged in value between 0.9922 and 0.9985 (Table 1).

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
Related in: MedlinePlus