Pollen epigenetics

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Published on: October 11, 2012

Biology learned in school and as a first year undergraduate is easily forgotten if it is not relevant to your current research. Today’s highlighted article required me to refresh my memory of plant germ line development, so I included my basic research here.

Highlighted article: Joseph P. Calarco, Filipe Borges, Mark T.A. Donoghue, Frédéric Van Ex, Pauline E. Jullien, Telma Lopes, Rui Gardner, Frédéric Berger, José A. Feijó, Jörg D. Becker and Robert A. Martienssen (2012) Reprogramming of DNA Methylation in Pollen Guides Epigenetic Inheritance via Small RNA. Cell 151:194-205.

Germline biosynthesis: A pollen mother cell undergoes meiosis to make haploid microspores, which unevenly split into a larger vegetative cell and a small generative cell. The generative cell splits symmetrically into two – these are the plant ‘sperm’ cells. Each pollen grain contains two sperm cells, which are surrounded by a vegetative cell. The vegetative nucleus contains completely decondensed heterochromatin, but DNA in generative nuclei is tightly condensed.

The female gametophyte develops from a megaspore mother cell. Both the megaspore mother cell and pollen mother cell are specified from somatic cells in developing flowers.

GFP staining in the two sperm nuclei and vegetative nucleus in the vegetative cell.

Bisulphite sequencing is a DNA sequencing method which determines methylation pattern by treating DNA with sodium bisulphite before sequencing it using a conventional DNA sequencing method. Bisulphite induces the conversion of unmethylated cytosines to uracil, but this is not a perfect technique so unmethylated DNA may be recorded as methylated. Additionally, bisulphite treatment can cause DNA degradation. Sequencing the DNA of interest multiple times, in the case of Calarco et al., anywhere from 7 to 17 times, improves reliability of the method. There is a brief overview of DNA methylation in this post. (more…)

Traditional varieties are key to modern rice farming

Analysing root growth and yield of rice plants.

Highlighted article: Rico Gamuyao, Joong Hyoun Chin, Juan Pariasca-Tanaka, Paolo Pesaresi, Sheryl Catausan, Cheryl Dalid, Inez Slamet-Loedin, Evelyn Mae Tecson-Mendoza, Matthias Wissuwa & Sigrid Heuer (2012). The protein kinase Pstol1 from traditional rice confers tolerance of phosphorus deficiency. Nature 488, 535–539 doi:10.1038/nature11346

Over centuries, many local rice varieties have been bred into a few modern varieties which are extensively farmed throughout much of Asia. In regions where soil is poor such as western India and Thailand, rice crops are dependent on rainfall, frequently suffering from floods and draughts, and importantly also require phosphorus fertilizer. Phosphorus is an essential plant nutrient, and as phosphorus fertilizer is made from a finite store of phosphorus rock the current situation in the parts of Asia with poor soil is not sustainable.

A solution to this problem was found in a traditional rice variety, Kasalath. Another traditional rice variety has already supplied modern rice breeders with submergence tolerant gene SUB1, which enables rice plants to survive up to two weeks of flooding. A decade ago, a major quantitative trait locus was identified in Kasalath that conferred tolerance to phosphorus deficient soil. This locus was labelled Pup1, and last year the Heuer group at the International Rice Research Institute defined a core set of Pup1 markers and used them to backcross Pup1 into modern rice varieties, which were grown in their natural environments and all produced significantly more rice in P-deficient conditions than their wildtype counterpart. These Pup1 introgression lines also showed improved root growth under stress. (more…)

Method: New and improved multiplex PCR

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Published on: August 30, 2012

Highlighted article: Daxing Wen and Chuqing Zhang (2012) Universal Multiplex PCR: a novel method of simultaneous amplification of multiple DNA fragments. Plant Methods 8:32 (Online preview) doi: 10.1186/1746-4811-8-32

Background

Multiplex PCR allows amplification of multiple targets in a single PCR experiment. It is possible to amplify several sections of a single template, or to amplify different templates using a number of primer sets. If there are multiple primers in a reaction, it can be difficult optimise the PCR reaction to maximise the efficiency of every primer, and it is likely that some cross-hybridisation and mis-priming will occur.

Figure 3B from Wen and Zhange (2012). A comparison of multiplex PCR (Lanes 1-4) and universal multiplex PCR (lanes 5-8), using the same primers with universal adaptors. The band intensity from traditional PCR is very variable, but it is consistently strong when the universal adaptors are used. 

Image credit: BioMed Central

 

The Method

Wen and Zhang from Shandong Agricultural University have devised a way around the inconveniences of multiplex PCR to develop a universal multiplex PCR method. ‘Universal adaptors’ are linked to specific primers, making the annealing temperature of the adaptor-primer structures 70°C. (more…)

From bench to bountiful harvest … MASC roadmap summarised in current Plant Cell Paper

Highlighted article: Lavagi I., Estelle M., Weckwerth W., Beynon J., and Bastow R. (2012) From Bench to Bountiful Harvests: A Road Map for the Next Decade of Arabidopsis Research. Plant Cell Advance Online Publication.

The Multinational Arabidopsis Steering Committee (MASC), as you might expect from their title, is an international group of Arabidopsis researchers who steer research in a productive direction. MASC reduces redundancy in research and encourages collaborations.  Over the last 20 years, MASC has neatly guided the Arabidopsis community to achievements in genome sequencing, understanding of plant hormones, development of open access bioinformatics resources and much more. Now MASC has planned a roadmap for the next ten years of Arabidopsis research entitled From Bench to Bountiful Harvest.

The roadmap consists of five broad objectives: (more…)

New TAIR10-compatible CDF files and review of RNA labelling methods

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Published on: July 17, 2012

A current paper in Plant Methods assessed the pros and cons of two RNA labeling methods for AGRONOMICS1 tiling arrays, concluding that random priming is more suitable for organelle transcriptome analysis as it can label non-polyadenylated transcripts effectively. They also generated new TAIR-10 based CDF files, which can be used to re-analyse existing AGRANOMICS1 CEL files. The new CDFs can be accessed here.

First of all, the authors gave an overview of the AGRONOMICS1 tiling array. It contains all the probes from the traditionally used Affymetrix ATH1 array, but has additional probes which mean the AGRONOMICS1 array yields expression data for over 7000 more genes, around a third of the genome. 90% of annotated genes on the TAIR9 database are on the array. Mitochondrial and chloroplast genomes are completely represented, and sRNA, tRNA and miRNA can also be detected. The AGRONOMICS1 array has probes that represent both strands of the entire Arabidopsis genome, allowing epigenetic profiling. The quality is comparable to that of the ATH1 array.

Müller et al. compared the GeneChip© IVT express kit, an oligo-dT based RNA labeling technique, with the GeneChip© whole transcript (WT) Sense Target Labeling Assay which uses random hexamers tagged with T7 promotor sequences. Both kits are from Affymetrix, Santa Carla, CA. (more…)

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