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Introduction

Assimilation, Cryptic variability (Lauter 2002), Dolores’s previous paper (Piperno 2014).

Methods

Growth Chamber Experiment

RNA sequencing

For the RNAseq, the total RNA was isolated from the leaf tissue of the plants with RNeasy mini Kit (Qiagen) following the manufacturer’s protocol. Under 400ppm and 265ppm of CO2, respectively 12 and 11 biological replicates were included. RNA quality and concentration was verified using a Bioanalyzer (Agilent RNA Nano). Sequencing library preparation was performed as previously describe (Zhong et al., 2011). Briefly, the mRNA was extracted with Dynabeads oligo(dt)25 (Ambion). After chemical shearing with divalent cations, the First strand synthesis was performed with Random Hexamers Primers (Invitrogen) and SuperScript III (Invitrogen). The second strand cDNA was synthetized with DNApol1 (Thermo Scientific) and RNaseH (New England Biolabs). The cDNA fragments were prepared for Illumina sequencing. First, the cDNA fragments were repaired with the End-Repair enzyme mix (New England Biolab). An deoxyadenosine triphosphate was added at each 3’ ends with the Klenow fragment (New England Biolab). Illumina Trueseq adapters (Affymetrix) were added with the Quick ligase kit (New England Biolab). Between each enzymatic step the cDNA was washed with AMpure beads (Beckman Coulter). Finally the The 23 samples were multiplexed and sequenced in one lane of Hiseq 2500 (UCDavis genome center sequencing facility) for 50 bases single-end reads with an insert size of approximately 300 bases. After demultiplexing, 3.8-8.8 million reads were generated for each sample (Table \ref{tab:readmoms}).

\label{tab:readmoms}

Reads
Sample Reads Maize-like mom
265_4B.1.txt 3814875
400_1A.1.txt 4070011 X
265_3C.1.txt 4139946 X
265_3A.1.txt 4399187
265_4C.1.txt 4746629 X
400_3C.1.txt 5029499 X
265_1B.1.txt 5031069
265_2B.1.txt 5063618 X
400_3B.1.txt 5433424
265_3B.1.txt 5564170
400_2B.1.txt 5812095 X
400_4A.1.txt 5857687
400_1A_2.1.txt 5989185
265_2B_2.1.txt 6467938
265_2A.1.txt 6732943
265_4A.1.txt 7427625
400_2B_2.1.txt 7455893
400_1B.1.txt 7570271
265_1A_2.1.txt 7648528
400_2A.1.txt 7882249
400_3A.1.txt 8630267
265_1A.1.txt 8643790 X
400_4C.1.tx 8836575 X

Data Analysis

Low qaulity (base quality <33) bases were trimmed using fastx toolkit v. 0.0.13 ((http://hannonlab.cshl.edu/fastx_toolkit/)), and adapters were subsequently removed using scythe ((https://github.com/vsbuffalo/scythe)). Trimmed reads were mapped to the AGPv3.21 of the maize genome using STAR version 2.3.0 (Dobin 2013) with default parameters. Read counting was performed with HTseq (Anders 2014) using a modified version of the ENSEMBL Zea_mays.AGPv3.21.gff3 annotation file . Only reads with mapping quality 30 or higher were included in subsequent analyses.

Expression analysis was performed using the EdgeR package (Robinson2013).