Just after three extra weeks, plants were transplanted into one gallon pots and moved to a greenhouse bench. The hybrid identity of putative F1 plants was con firmed via examination of external phenotypes and mo lecular markers. Visible phenotypic markers integrated pigmentation on the base of your stem, leaf form, plant branching, and production of foliar glandular trichomes. Molecular marker phenotypes had been observed by ex traction of genomic DNA and amplification of two loci previously established to vary in dimension involving cmsHA89 and Pet2152. PCR primers, amplification situations, and re presentative gel photos are provided in Further file one, Supplemental Procedures 1. mRNA extraction and sequencing At 45 days post germination, leaf tissue was collected from 8 F1 plants and 2 plants from every parent ac cession.
The youngest fully expanded leaf was minimize from each plant, positioned right into a 50 ml conical tube, and imme diately frozen in liquid nitrogen. Complete RNA was extracted from approximately 50 mg of ground tissue as described. Preparation of non normalized cDNA libraries and entire transcriptome shotgun sequencing via Illumina HiSeq 2000 had been carried out with the Michael Smith Genome Sciences Centre in Vancouver, knowing it British Columbia, Canada. Samples had been multiplexed with 3 samples per lane. Sequence data processing and evaluation Paired end, 100bp RNA Seq reads were aligned to a H. annuus derived transcriptome reference. This reference, assembled from 93428 EST sequen ces, consists of 16312 exceptional contigs with a total length of 17. 062 million bases. Fasta formatted sequence for the transcript reference is obtainable at datadryad.
org. The median insert size involving paired finish reads ranged from 131 to NVPAUY922 151 bases per sample. Approxi mately 52% of reference contigs were assigned to genetic map positions inside of the H. annuus genome by way of identity to sequenced markers appearing on a map of H. annuus derived from recombinant inbred lines from the population RHA280 ? RHA801. Genetic map positions assigned for the transcript reference are available as Extra file 2, Table S3. Alignments had been performed implementing the Burrows Wheeler Aligner resources aln and sampe utilizing a highest insert dimension of one thousand and a top quality filter of thirty to trim reads. Aligned BAM files had been sorted and PCR duplicates eliminated utilizing SAMtools utilities sort and rmdup. Reads per contig have been counted for every sam ple making use of coverageBed. Read through counts have been analyzed in R using the DESeq pac kage to review counts of reads aligned to a offered refe rence contig. The DESeq package deal makes use of a modified Fishers precise check with data fit to a unfavorable binomial distribution to check for pair wise differences in count information amongst sample courses, permitting inside of transcript comparisons across a broad dynamic variety.