Illumina HiSeq 4000 paired end sequencing; Identification of Rf  genes in hexaploid wheat (Triticum aestivum L.) by RNA-seq.
Identification of Rf  genes in hexaploid wheat (Triticum aestivum L.) by RNA-seq.
Sample 8_p
Anthers were harvested from spikes at tetrad and late uninucleate microspore stages after completing of ear emergence (59 Zadox growth scale).  Plants of two non-restorer varieties of hexaploid winter wheat (Astoria, Grana ) and two restorers (Patres and Primépi ), were grown in growth chamber (ForClean, ZalMed Sp. z.o.o) under the following conditions: temperature - 18˚C/15˚C day/night, 14/10 h photoperiod and constant relative humidity of 50%. Plants were cultivated in pots filled with a mixture of peat and perlite (1:1, v/v), three plants per single pot (19 cm diameter). The plants were hand irrigated daily to maintain soil water content close to field capacity. GeneMATRIX Universal RNA Purification kit (EURx, Gdańsk, Poland) was used for total RNA isolation from the anthers of two fertility restoring and non-restoring wheat cultivars according to the manufacturer's protocol. The quantity and purity of each RNA sample was determined by the absorbance (Abs) at 260 and 280 nm by Nanodrop 2000 spectrophotometer and was measured using a Qubit® 2.0 Fluorometer (Invitrogen, Waltham, MA, USA). RNA Integrity Number (RIN) of the total RNA was assessed using the BioAnalyzer 2100, Plant RNA Pico or RNA 6000 Nano Kit (Agilent Technologies,  Santa Clara, CA, USA). Total RNA from harvested anthers was subjected to poly(A) enrichment using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). Twenty four strand-specific cDNA libraries for four cultivars, two anthers' developmental stages, and three biological replications were constructed with NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs).