Table 1.
Description of the candidate reference genes, primer sequence and qRT-PCR amplification efficiencies.
Fig 1.
Phenotypic characterization of Taichung65-4x and its corresponding diploid rice.
(A) Morphologies of Taichung65-4x and Taichung65-2x. Bars = 10 cm. (B) and (C) Panicles of Taichung65-4x and Taichung65-2x. Bars = 2 cm. (D) and (E) Grain size of Taichung65-4x and Taichung65-2x. Bars = 1 mm. (F) and (G) Floral organs of Taichung65-4x and Taichung65-2x. Bars = 1 mm. (H) and (I) Anthers of Taichung65-4x and Taichung65-2x were observed by using the WE-CLSM (Whole-mount eosin B-staining confocal laser scanning microscopy). Bars = 100 μm. (J) and (K) Pollens of Taichung65-4x and Taichung65-2x, respectively. Bars = 100 μm.
Fig 2.
Microgametogenesis of Taichung65-4x and its corresponding diploid rice.
(A-D) Microgametogenesis of Taichung65-2x. (A) Late microspore stage. (B) Early bicellular pollen stage. (C) Late bicellular pollen stage. (D) Mature pollen stage. (E-P) Microgametogenesis of Taichung65-4x. (E) Middle microspore stage, vacuolization. (F) Late microspore stage. (G) Late microspore stage. (H) Early bicellular pollen stage. (I) Middle bicellular pollen stage. (J) Late bicellular pollen stage. (K) Middle microspore stage, cell shrinkage. (L) Late bicellular pollen stage, multiple apertures. (M) Late bicellular pollen stage, cell shrinkage and multiple apertures. (N) Late bicellular pollen stage, arrow indicate the cell shrinkage. (O) Middle bicellular pollen stage, arrow indicate the cell shrinkage. (P) Mature pollen stage, arrow indicate the asynchronous pollens. Bars = 40μm.
Fig 3.
Specificity of primers and its amplification size.
(A) Amplification of PCR products for 14 genes in agarose gel (1.5%) electrophoresis. (M: DNA Marker 2000bp; lanes 1–14: OsActin1, UBQ5, OsAOC, β-TUB, GAPDH, CPI, EF-1a, SRP, RIC1, PFP, FLO16, Cytochrome b5, OsATG8a and CPuORF7, respectively. (B to O) Melting curves of 14 candidate reference genes show single peaks.
Table 2.
Stability analysis of candidate reference genes assayed by ΔCt method.
Fig 4.
Stability analysis of candidate reference genes using the geNorm software.
(A) All samples of autotetraploid and diploid rice. (B) All samples of diploid rice. (C) All samples of autotetraploid rice. (D) Samples of pre-meiosis stage in diploid and autotetraploid rice. (E) Samples of meiosis stage in diploid and autotetraploid rice. (F) Samples of single microspore stage in diploid and autotetraploid rice. (G) Samples of bicellular pollen stage in diploid and autotetraploid rice. Total indicate the all samples of diploid and autotetraploid rice. Diploid indicate the all samples of diploid rice; Auto indicate the all samples of autotetraploid rice; PMA indicate the samples of pre-meiosis stage in diploid and autotetraploid rice; MA indicate the samples of meiosis stage in diploid and autotetraploid rice; SCP indicate the samples of single microspore stage in diploid and autotetraploid rice; BCP indicate the samples of bicellular pollen stage in diploid and autotetraploid rice.
Fig 5.
Determination of the optimal number of reference genes for normalization by pairwise variation (V) using geNorm software.
The average pairwise variations (Vn/Vn+1) were analyzed to measure the effect of adding reference gene in the qRT-PCR. Total indicate the all samples of diploid and autotetraploid rice. Diploid indicate the all samples of diploid rice; Auto indicate the all samples of autotetraploid rice; PMA indicate the samples of pre-meiosis stage in diploid and autotetraploid rice; MA indicate the samples of meiosis stage in diploid and autotetraploid rice; SCP indicate the samples of single microspore stage in diploid and autotetraploid rice; BCP indicate the samples of bicellular pollen stage in diploid and autotetraploid rice.
Table 3.
Stability analysis of candidate reference genes assayed by NormFinder software.
Table 4.
Stability analysis of candidate reference genes assayed by BestKeeper software.
Table 5.
Stability analysis of candidate reference genes assayed by RefFinder software.
Fig 6.
Relative expression of pollen development related and stage specific genes using the selected reference genes for normalization.
(A) The expression of EAT1 in different pollen development stages was normalized using the most stable genes and least stable reference genes. (B) The expression of CYP703A3 in different pollen development stages was normalized using the two most stable genes and least stable reference genes. (C) The expression of OsABCG26 in different pollen development stages was normalized using the most stable genes and least stable reference genes. (D) The expression of CRP in different pollen development stages was normalized using the most stable genes and least stable reference genes. (E) The expression of CYP94C4 in different pollen development stages was normalized using the most stable genes and least stable reference genes. (F) The expression of OsGLP8-3 in different pollen development stages was normalized using the most stable genes and least stable reference genes (UBQ5 and OsActin1). PMA indicate the samples of pre-meiosis stage in diploid and autotetraploid rice; MAI indicate the samples of meiosis stage I in diploid and autotetraploid rice; MAII indicate the samples of meiosis stage II in diploid and autotetraploid rice; SCP indicate the samples of single microspore stage in diploid and autotetraploid rice; BCP indicate the samples of bicellular pollen stage in diploid and autotetraploid rice.