The transcriptome landscape of early maize meiosis

Dukowic-Schulze, Stefanie; Sundararajan, Anitha; Mudge, Joann; Ramaraj, Thiruvarangan; Farmer, Andrew; Wang, Minghui; Sun, Qi; Pillardy, Jarosław; Kianian, Shahryar F.; Retzel, Ernest F.; Pawlowski, Wojciech P.; Chen, Changbin

doi:10.1186/1471-2229-14-118

Cited by 44 publications

(54 citation statements)

References 78 publications

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“…In fact, for some wheat genotypes as those carrying the Ph1 deletion, meiosis is not synchronized 43 , making the identification of each meiosis stage even more complicated. In addition, and as far as we know, although there are some massive approaches to study meiosis using transcriptomics and proteomics in cereals like maize [44][45][46] , rice 47,48 and other plants 49 , the only massive transcriptomic study covering the whole meiosis process in wheat anthers was performed by Crismani and colleagues 23 , using the Affymetrix GeneChip Wheat Genome Array. Recent studies have examined gene expression in wheat meiotic anthers using RNA-seq 31,38 .…”

Section: Discussionmentioning

confidence: 99%

Identification and validation of reference genes for RT-qPCR normalization in wheat meiosis

Garrido

Aguilar

Prieto

2020

Sci Rep

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Meiosis is a specialized type of cell division occurring in sexually reproducing organisms to generate haploid cells known as gametes. In flowering plants, male gametes are produced in anthers, being encased in pollen grains. Understanding the genetic regulation of meiosis key events such as chromosome recognition and pairing, synapsis and recombination, is needed to manipulate chromosome associations for breeding purposes, particularly in important cereal crops like wheat. Reverse transcription-quantitative PCR (RT-qPCR) is widely used to analyse gene expression and to validate the results obtained by other transcriptomic analyses, like RNA-seq. Selection and validation of appropriate reference genes for RT-qPCR normalization is essential to obtain reproducible and accurate expression data. In this work, twelve candidate reference genes were evaluated using the mainstream algorithms geNorm, Normfinder, BestKeeper and ΔCt, then ranked from most to least suitable for normalization with RefFinder. Different sets of reference genes were recommended to normalize gene expression data in anther meiosis of bread and durum wheat, their corresponding genotypes in the absence of the Ph1 locus and for comparative studies among wheat genotypes. Comparisons between meiotic (anthers) and somatic (leaves and roots) wheat tissues were also carried out. To the best of our knowledge, our study provides the first comprehensive list of reference genes for robust RT-qPCR normalization to study differentially expressed genes during male meiosis in wheat in a breeding framework. The study of biological processes usually involves gene expression analyses and quantification. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most widely used technique nowadays to analyse gene expression due to factors like cost-effectiveness, specificity and sensitivity 1. However, to achieve accurate and reliable results, sample-to-sample variation and experimental error need to be controlled by making use of normalization strategies 2,3. The most common and effective method for RT-qPCR normalization is the use of reference genes (RGs), often referred as control genes or housekeeping genes, as internal controls. RGs need to be validated for a given experimental setup, since there are no universal RGs suitable for every tissue or experimental condition as the vast scientific literature on this topic proves. Validation is a critical step, since the use of random, putative or unvalidated RGs introduces significant biases in results 1,4. The main premise for RGs is that their expression remains unchanged or relatively invariant in the experimental context under study, which is not always the case in practice 1. Thus, the available validation methods perform a selection based on the expression stability of the candidate RGs. That is to say, the least variable genes are the most stably expressed and the most suitable for normalization. Among the available RG methods, the most frequently used are geNorm 3 , or its updated version, qB...

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Section: Discussionmentioning

confidence: 99%

Identification and validation of reference genes for RT-qPCR normalization in wheat meiosis

Garrido

Aguilar

Prieto

2020

Sci Rep

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“…Transcriptome analysis has been extensively used to help address fundamental questions in maize anther development. In addition to analyzing whole anthers at various developmental stages (Ma et al, 2007(Ma et al, , 2008Skibbe et al, 2009;Wang et al, 2010;Nan et al, 2011;Sekhon et al, 2011;Zhang and Yang, 2014;, cell type-specific transcriptomes have also been reported for AR cells undergoing the first phase of mitotic proliferation in 0.30-to 0.35-mm and 0.7-mm anthers (Kelliher and Walbot, 2014; and prophase I meiocytes in anthers about 2 mm long (Dukowic-Schulze et al, 2014a). Rapid progress has been made in understanding the mechanisms of AR cell fate specification and meiotic progression.…”

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confidence: 99%

Stage-Specific Gene Profiling of Germinal Cells Helps Delineate the Mitosis/Meiosis Transition

Yuan

Huang

et al. 2017

Plant Physiol.

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In flowering plants, germ lines are induced from somatic meristems within reproductive organs. Within anthers, germinal cell initials first undergo several rounds of mitotic proliferation before synchronously entering meiosis. Our understanding of the progression and the molecular basis of this mitosis to meiosis transition is still limited. Taking advantage of the correlation between anther length and premeiotic germinal cell development in maize (), we studied the transcriptome dynamics of germinal cells at three sequential stages, mitotic archesporial cells, enlarging pollen mother cells at the premeiosis interphase, and pollen mother cells at the early prophase of meiosis, using laser microdissection-based expression profiling. Our analysis showed that cells undergoing the mitosis-meiosis switch exhibit robust transcriptional changes. The three stages are distinguished by the expression of genes encoding transcription factor subsets, meiotic chromosome recombination proteins, and distinct E3 ubiquitin ligases, respectively. The transcription level of genes encoding protein turnover machinery was significantly higher in these three stages of germinal cells than in mature pollen, parenchyma cells, or seedlings. Our experimental results further indicate that many meiotic genes are not only transcribed, but also translated prior to meiosis. We suggest that the enlarging pollen mother cells stage represents a crucial turning point from mitosis to meiosis for developing germinal cells.

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“…Several methods have been previously described or proposed for the isolation of male meiocytes [6], such as micromanipulation ( Plumbago [10, 11], Nicotiana [12], Brassica [13, 14], Arabidopsis [15–17] and sunflower [18]), capillary collection of meiocytes (CCM) ( Arabidopsis [19, 20] and maize [21, 22]), laser capture microdissection (LCM) in rice [23–26], Percoll gradient separation ( Arabidopsis [27, 28], rice [29] and Brassica [30]) and isolation of nuclei tagged in specific cell types (INTACT) in Arabidopsis [31]. However, these methods have drawbacks that affect their use in the simultaneous isolation of large numbers of meiocytes from prophase I sub-stages for omics studies.…”

Section: Introductionmentioning

confidence: 99%

MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat

et al. 2018

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BackgroundMolecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific genes and associated regulatory networks.ResultsWe describe an optimized method termed MeioCapture for simultaneous isolation of uncontaminated male meiocytes from wheat (Triticum spp.), specifically from the pre-meiotic G2 and the five sub-stages of meiotic prophase I. The MeioCapture protocol builds on the traditional anther squash technique and the capillary collection method, and involves extrusion of intact sporogenous archesporial columns (SACs) containing meiocytes. This improved method exploits the natural meiotic synchrony between anthers of the same floret, the correlation between the length of anthers and meiotic stage, and the occurrence of meiocytes in intact SACs largely free of somatic cells. The main advantage of MeioCapture, compared to previous methods, is that it allows simultaneous collection of meiocytes from different sub-stages of prophase I at a very high level of purity, through correlation of stages with anther sizes. A detailed description is provided for all steps, including the collection of tissue, isolation and size sorting of anthers, extrusion of intact SACs, and staging of meiocytes. Precautions for individual steps throughout the procedure are also provided to facilitate efficient isolation of pure meiocytes. The proof-of-concept was successfully established in wheat, and a light microscopic atlas of meiosis, encompassing all stages from pre-meiosis to telophase II, was developed.ConclusionThe MeioCapture method provides an essential technique to study the molecular basis of chromosome pairing and exchange of genetic information in wheat, leading to strategies for manipulating meiotic recombination frequencies. The method also provides a foundation for similar studies in other crop species.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1514-z) contains supplementary material, which is available to authorized users.

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The transcriptome landscape of early maize meiosis

Cited by 44 publications

References 78 publications

Identification and validation of reference genes for RT-qPCR normalization in wheat meiosis

Identification and validation of reference genes for RT-qPCR normalization in wheat meiosis

Stage-Specific Gene Profiling of Germinal Cells Helps Delineate the Mitosis/Meiosis Transition

MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat

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