SLAF-seq of tobacco: F2 individual aa
A F2 population derived from a cross between 'Yunyan 85' and 'Dabaijin 599'. Specific-locus amplified fragment sequencing (SLAF-seq) was used to genotype F2 individuals and the two parents. Genomic DNA was first incubated at 37°C with MseI [New England Biolabs (NEB), Ipswich, MA, USA], T4 DNA ligase (NEB), ATP (NEB), and MseI adapter. Restriction/ligation reactions were heat-inactivated at 65°C and digested with HaeIII and BfaI restriction enzymes at 37°C. Then, polymerase chain reactions (PCR) were carried out in the reaction solutions containing the diluted restriction/ ligation samples, dNTP, Taq DNA polymerase (NEB), and MseI-primer containing barcode 1. PCR products were purified using an E.Z.N.A.® Cycle Pure Kit (Omega BioTek, Norcross, GA, USA) and pooled. Pooled samples were incubated at 37°C with MseI, T4 DNA ligase, ATP, and Solexa adapter, purified using a Quick Spin column (Qiagen, Hilden, Germany), and run on a 2% agarose gel. Fragments of 400–450 bp (with indexes and adaptors) were isolated using a Gel Extraction Kit (Qiagen) and subjected to PCR amplification with Phusion Master Mix (NEB) and Solexa Amplification primer mix (Illumina, Inc., San Diego, CA, USA) to add barcode 2 according to the Illumina sample preparation guide. After samples were gel purified, DNA fragments (SLAFs) of 330–380 bp were excised and diluted for pair-end sequencing on an Illumina high-throughput sequencing platform.