Identification of stable pollen development related reference genes for accurate qRT-PCR analysis and morphological variations in autotetraploid and diploid rice.

Jinwen Wu,Yifan Hu,Hong Lin,Muhammad Qasim Shahid,Yinzhi Jiao,Xiangdong Liu,Susu Du,Haibin Guo,Hao Fan,Zijun Lu,Allah Bakhsh

doi:10.1371/journal.pone.0253244

Abstract

Autotetraploid rice exhibited hybrid vigor and greater genetic variation compared to diploid rice, but low pollen fertility is a major hindrance for its utilization. Our previous analysis revealed that large number of pollen fertility genes were exhibited down-regulation in autotetraploid rice. Hence, it is of utmost importance to reveal the expression patterns of pollen fertility genes with high accuracy. To find stable reference genes for autotetraploid rice, we compared the pollen development stages between diploid and autotetraploid rice, and 14 candidate genes were selected based on transcriptome analysis to evaluate their expression levels. Autotetraploid rice (i.e. Taichung65-4x) displayed lower seed set (40.40%) and higher percentage of abnormalities during the pollen development process than its diploid counterpart. To detect the candidate reference genes for pollen development of autotetraploid and diploid rice, we used five different algorithms, including NormFinder, BestKeeper, ΔCt method, geNorm and Re-Finder to evaluate their expression patterns stability. Consequently, we identified two genes, Cytochrome b5 and CPI, as the best candidate reference genes for qRT-PCR normalization in autotetraploid and diploid rice during pre-meiosis, meiosis, single microspore and bicellular pollen development stages. However, Cytochrome b5 was found to be the most stably expressed gene during different pollen development stages in autotetraploid rice. The results of our study provide a platform for subsequent gene expression analyses in autotetraploid rice, which could also be used in other polyploid plants.

Highlights

Autotetraploid rice is a new germplasm derived from diploid rice by chromosomes doubling
Using the Cytochrome b5 or CPI alone or Cytochrome b5 combined with CPI as the reference gene, we found that transcript levels of pollen development stage specific genes were consistent with the transcriptome data and much more different from the transcript levels of UBQ5 and OsActin1 (Fig 6D–6F)
Significant phenotypic variations detected in autotetraploid rice compared to diploid rice

Summary

Introduction

Autotetraploid rice is a new germplasm derived from diploid rice by chromosomes doubling. Significant expression differences were detected in pollen development stage between the autotetraploid and its diploid rice, and 42 meiosis meiosis-related or stage-specific genes showed differential expression patterns by transcriptome analysis [7]. We selected 14 candidate reference genes, including 7 commonly used housekeeping genes and 7 stably expressed genes based on the transcriptome data, to assess their expression stability for qRT-PCR analysis during pollen development in polyploid and diploid rice. The results of the present study will be valuable for future research to assess the expression patterns of target genes by using these reference genes during pollen development in autotetraploid rice, and provide the evidence for the selection of reference genes in polyploid plants

Materials and methods

Results

Discussion