IC4R016-RNA-Seq-2013-24215296

Project Title

Transcriptomes of isolated Oryza sativa gametes characterized by deep sequencing: evidence for distinct sex-dependent chromatin and epigenetic states before fertilization

The formation of a zygote by the fusion of egg and sperm involves the two gametic transcriptomes. In flowering plants, the embryo sac embedded within the ovule contains the egg cell, whereas the pollen grain contains two sperm cells inside a supporting vegetative cell. The difficulties of collecting isolated gametes and consequent low recovery of RNA have restricted in-depth analysis of gametic transcriptomes in flowering plants. The researchers isolated living egg cells, sperm cells and pollen vegetative cells from Oryza sativa (rice), and identified transcripts for approximately 36 000 genes by deep sequencing.

Egg cells were isolated using flowers harvested at anthesis from rice plants (O. sativa L. ssp. japonica, cultivar ‘Kitaake’) grown in glasshouses under ambient light during the summer.
Sperm cells and Ve were isolated from near anthesis anthers of vigorous, visibly disease-free, field grown rice at Dale Bumpers National Rice Research Center in Stuttgart, AR, USA. A differential centrifugation isolation method was used to separate Sp and Ve

from whole anthers.

RNA from the Sp and Ve was extracted using the QIAGEN RNeasy Plant Mini Kit (Qiagen, http://www.qiagen.com) following the manufacturer’s instructions, including the optional on-column DNAse treatment.
The barcoded FASTQ files of the reads were mapped to rice RGAP 7.0 genomic reference sequences using TOPHAT 2.0.5 with default settings, except that the ‘microexon’ search option was switched on, and minimum and maximum intron sizes were set at 20 and 15 000

bases, respectively.

Illumina sequencing by the RNA-seq protocol was performed on RNA samples from three biological replicates for each of the three cell types.Samples were multiplexed and run with six samples per lane on the Illumina HiSeq 2000 DNA sequencer (Illumina, http://www.illumina.com).

Department of Plant Biology,University of California, Davis, CA 95616, USA,
Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK 73019, USA,
School of Life Sciences, Lanzhou University, Lanzhou 730000, China, and
Department of Plant Sciences, University of California, Davis, CA 95616, USA

Sarah N. Anderson:srussell@ou.edu & Venkatesan Sundaresan:sundar@ucdavis.edu