Illumina MiSeq paired end sequencing; Identification of Rf  genes in hexaploid wheat (Triticum aestivum L.) by next generation sequencing of products of Representational Difference Analysis (RDA-seq).
Identification of Rf  genes in hexaploid wheat (Triticum aestivum L.) by next generation sequencing of products of Representational Difference Analysis (RDA-seq).
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). The first and the second strand of cDNA smART were obtained usin First Strand cDNA Synthesis Kit (EURx, Gdańsk, Poland) and Second Strand cDNA Synthesis Kit (abm, Richmond, BC, Canada), respectively. Representational difference analysis was performed on high quality cDNA according to Hubank and Schatz (1994) procedure with small modifications. Anthers at the microspore stage were used as testers while anthers excised at the stage of pollen tetrads were selected as driver probes. The analysis was taken in three replicates for each variety (Primépi, Patres, Grana, and Astoria). The tester:driver ratio in three subsequent rounds of subtractive hybridization increased from 1:50, and 1:400 to 1:200000. Differential products obtained in the third subtraction of the size range from 200 to 800 bp were sequenced. Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA) was used for library construction according to the manufacturer instructions.