variegated leaf individuals
Fifty variegated leaf individuals and fifty green leaf individuals of F2 populations were selected to construct two bulks, Var and CK, for BSR-seq analysis. Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen), and were quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agrose gel. The RNA with RIN value above 7 from fifty individual variegated leaves and fifty green leaves, were mixed to construct two libraries, respectively. The next generation sequencing library preparations were constructed according to the manufacturers protocol (NEBNext Ultra RNA Library Prep Kit for Illumina). NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) was used to perform the poly(A) mRNA isolation. The mRNA fragmentation and priming was performed using NEBNext First Strand Synthesis Reaction Buffer and NEBNext Random Primers. First strand cDNA and second-strand cDNA were synthesized using ProtoScript II Reverse Transcriptase and Second Strand Synthesis Enzyme Mix, respectively. Two libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrument according to manufacturers instructions (Illumina, San Diego, CA, USA). The sequences were processed and analyzed by GENEWIZ (Nanjing, Jiangsu, China).