RNA extraction, cDNA library construction, and library sequencing on Illumina HiSeq2500 platform of 12 samples were strictly implemented by Biomarker Technologies (Beijing, China) in accordance with standard procedures (Kong et al. 2020 ). Raw data were filtered by fastp (Chen et al. 2018 (link)) and mapped to the Nipponbare genome (MSU v7.0) using hisat2 (Kim et al. 2015 (link)) with default parameters. The mapped reads were counted by featureCounts (Liao et al. 2014 (link)) and differentially expressed genes (DEGs) in QTLs were identified by DEseq2 with |log2fold change|≥ 1 and a False Discovery Rate (FDR) < 0.01 (Kong et al. 2020 ). The heatmap of DEGs was also conducted by TBtools (Chen et al. 2020 (link)).
Finally, three randomly selected candidate DEGs in QTLs were verified by RT-PCR according to the previously described method (Kong et al. 2019 ). All primers of RT-PCR were designed by Primer 5.0 software (Additional file4 : Table S3). The qRT-PCR reaction (10 μL) was formulated using the 2 X SYBR Green qPCR Master Mix (US Everbright®Inc., Suzhou, China). All qRT-PCRs were carried out on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The gene expression fold change was calculated by the 2−ΔΔCT method from three biological replicates.
Finally, three randomly selected candidate DEGs in QTLs were verified by RT-PCR according to the previously described method (Kong et al. 2019 ). All primers of RT-PCR were designed by Primer 5.0 software (Additional file
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