Hiseq pe150

Total DNA was extracted from the frozen ruminal samples using a TIANamp Stool DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s protocols. The purity and the quality of the extracted DNA were assessed by electrophoresis and determined spectrophotometrically by measuring the A260/280 (Beckman DU/800; Beckman Coulter, Inc., Fullerton, CA, USA). Qualified DNA samples were randomly broken into approximately 350 bp fragments using Covaris M220 (Gene Company Limited, Woburn, MA, USA) for paired-end library construction and the whole library was prepared using a TruSeq^TM DNA Sample Prep Kit (Illumina Inc., San Diego, CA, USA). Adapters were ligated to the blunt-end fragments, which contained the full complement of sequencing primer hybridization sites. Then, the resulting PCR products were purified and quantified on an Agilent Bioanalyzer 2100 system. Sample labeling was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS. Finally, paired-end sequencing was performed on an Illumina HiSeq PE150 (Illumina Inc., San Diego, CA, USA) platform according to the manufacturer’s instructions.

A modified MutMap method was used to identify the candidate gene for qiu. A total of 20 lines with cylindrical fruits and 20 lines with spherical fruits were selected from the F₂ population. DNA was extracted from each plant and mixed in equal amounts to construct the cylindrical fruit bulk (C-pool) and the spherical fruit mutant bulk (S-pool). DNA from the two parental plants (20 individuals) was extracted to construct the wild-type pool (P₁, 32X) and the mutant pool (P₂, qiu), respectively. DNA from the two parental plants, C-pool and S-pool, were re-sequenced with Illumina HiSeq TMPE150. The raw reads were filtered using the NGSQC toolkit software [26 (link)]. BWA was used to align the clean reads to the reference genome [27 (link)], and single-nucleotide polymorphisms (SNPs) were detected using the Genome Analysis Toolkit (GATK) [28 (link)]. ANNOVAR software was used for functional annotation for SNP detection results [29 (link)].

The 152 gDNA samples were randomly sheared into 350 bp fragments in the Covaris breaker (Covaris, Woburn, MA, USA). Libraries were built based on Illumina’s TruSeq Library Construction Kit (Illumina, San Diego, CA, USA). Briefly, the gDNA fragments were processed by end repair, modified by poly-A tail and sequencing adapter addition, purified, and amplified via PCR to construct the library. Paired-end sequencing libraries were sequenced with a read length of 350 bp using an Illumina HiSeqTM PE150 (Illumina, San Diego, CA, USA).
The parental genotypes were sequenced separately at a sequencing depth of 29.71× and 28.77×. Individual F₂ plants were sequenced at 9.97× coverage. The raw data were filtered to determine the sequencing read quantities, sequencing error rates, Q20, Q30, GC content, and data output. The filtered reads were compared with the reference genome assemblies of sorghum bicolor and used for SNP identification and genotyping.

At the seedling stage, young leaf tissues of the parents and 150 F₂ individuals were harvested to isolate DNA using the CTAB procedure [36 ]. DNA quality was detected using gel electrophoresis and a Nanodrop instrument. High-quality DNA was sequenced on the Illumina HiSeq^TM PE 150 platform by Novogene Bioinformatics Technology Co., Ltd., Beijing, China, using the TruSeq Library Construction Kit.

A modified MutMap method was used to identify the candidate gene for the mutant ebm3. DNA was extracted from 15 F₂ individuals with the early-bolting phenotype and the parental lines using a DNAsecure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. Equal amounts of each DNA from the 15 F₂ individuals were mixed to construct an offspring pool. Sequencing libraries of the mutant ebm3 (ebm3), wild-type line ‘FT’ (‘FT’), and offspring pool (F₂_ebm3) were generated using a TruSeq Nano DNA HT Sample preparation Kit (Illumina, San Diego, CA, USA). The libraries were sequenced using Illumina HiSeq^TMPE150 (Novogene Co., Ltd., Beijing, China). After quality control and filtration, the clean reads of each sample were aligned to the B. rapa reference genome (http://brassicadb.org/brad/, v3.0) using Burrows-Wheeler Alignment tool (BWA) [86 (link)]. Alignment files were converted to BAM files using the SAMtools software [87 (link)]. SNP calling was performed using GATK [88 (link)] and annotated using ANNOVAR [89 (link)]. The screened SNPs between the M and W library were used to calculate the SNP index in offspring-pool library. The sliding window method was used to determine the SNP index of the whole genome in offspring pool library.