Hiseq pe150 platform

SNP identification and genotyping were performed by resequencing of the genome of L. polyactis individuals. Sequencing libraries were constructed following Nunes et al.^{39 (link)} with slight modifications. Genomic DNA of each individual was fragmented into about 350 bp fragments by a Covaris crusher. Sequencing libraries were generated using a TruSeq Library Construction Kit following the manufacturer's instructions. Digested DNA fragments were examined by electrophoresis and were then ligated with barcode adaptors. Ligation products were pooled to select for 350–600 bp fragments using Pippin Prep (Sage Science, USA) after clean up with a QIAquick PCR Purification Kit (Qiagen, Germany). Libraries were enriched by PCR using Phusion® High-Fidelity DNA Polymerase (New England Biolabs, USA). Finally, libraries were cleaned using the QIAquick PCR Purification Kit (Qiagen, Germany) and quantified using the KAPA Library Quantification Kit (Kapa Biosystems, USA) for paired-end sequencing on an Illumina HiSeq™ PE150 platform (Illumina, USA), which produced single-end raw reads of 150 bp.

Leaf samples were collected from the 275 RILs in the F₇ generation. Leaf tissues were used to extract total genomic DNA using the CTAB method. The quantity and quality of genomic DNA was determined by NanoDrop ND-1000 Spectrophotometer and 1% agarose gel electrophoresis, respectively (Thermo Scientific, Wilmington, USA).
For 275 RILs, the genomic DNA was incubated with ATP (NEB), T4 DNA ligase (NEB), MseI (New England Biolabs, NEB), and MseI Y adapter N containing barcode at 37°C. Temperature at 65°C was used to inactivate restriction ligation reactions and digested at 37°C by the additional restriction enzyme (NlaIII). Agencourt AMPure XP (Beckman) was utilized to purify samples of the restriction ligation. Finally PCR was done with these purified samples, universal primers of Phusion Master Mix (NEB), index primers to add index, as well as entire i5 and i7 sequences. Purification of products of PCR was carried out by Agencourt AMPure XP (Beckman) then pooled and run on an agarose gel (2%). Gel extraction kit (Qiagen) was utilized to separate fragments of 375-400 bp (with indexes and adaptors) in size. The fragment products were subsequently purified by using Agencourt AMPure XP (Beckman) and diluted to further sequencing. After that paired-end sequencing was done on the selected tags with Illumina HiSeq PE150 platform (Novogene Bioinformatics Technology Co., Ltd, P.R. China).