4.3. Bulked Segregant RNA-Seq Analysis

For bulked segregant RNA-Seq (BSR-Seq) analysis, four cDNA pools were constructed with two parent bulks (T01 and T02) and two F₂ segregation bulks (T03 and T04); total RNA was isolated from fresh leaves according to the standard protocol of Illumina. To ensure that a qualified sample was used for the construction of the sequencing library, the RNA concentration was detected using Qubit^® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), while the RNA integrity was measured on Agilent 2100 System. The mRNA was enriched and fragmented as described by Gu et al. (2017) [8]. The first strand cDNAs were synthesized by using the shorted mRNA as PCR templates, and the second cDNA strand was synthesized after adding buffer, dNTPs, RNase H and DNA polymerase I; the cDNA was purified by using AMPure XP beads. Then purified double-stranded cDNA was subjected to end-repair and sequenced on the Illumina HiSeq™ 2500 platform (Illumina, San Diego, CA, USA).

The clean reads were obtained by removing the lower reads and adaptors of raw reads. Filtered reads were mapped to the reference Chinese cabbage genome (http://brassicadb.org/brad/) using HISAT2 (v2.0.5). The gene expression level was assessed by using FPKM (Cuffdiff, Fragments Per Kilobase of transcript per Million fragments mapped) method. The differentially expressed genes (DEGs) between the two set pairs of BSR-Seq bulks (T01 vs. T02, T03 vs. T04) were analyzed by using the edgeR software [55], the dispersion was set as 0.1. The false discovery rate (FDR) was obtained through adjusting the hypothesis significance p-values with the Benjamini and Hochberg method (1995) [56].

The fold change indicated the ratio of expression levels of the same gene between the two groups. Standards for screening differentially-expressed genes: |log₂ (Fold Change)| ≥ 1, FDR < 0.05. The SNPs and small indels were identified by using the GATK toolkit, sliding-window analysis was performed for Δ(SNP-index) association analysis like BSA-Seq analysis, and all DEGs and genes involved in candidate regions were annotated to GO, COG, KOG, KEGG public databases.