Qubit dna assay kit in qubit 2.0 flurometer

Genomic DNA and RNA were extracted with CTAB method [52 (link)]. Genomic DNA and RNA degradation and contamination were monitored on 1% agarose gels. DNA and RNA purity were checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). DNA concentration was measured using Qubit DNA Assay Kit in Qubit 2.0 Flurometer (Life Technologies, CA, USA).

A segregating population of 199 F₂ plants derived from an intra-specific cross between Zea mays L. SG5 and Zea mays L. SG7 was grown in November 2014 at the Panxian Maize Breeding Station in Guizhou, China. Young healthy leaves from the two parents and each of the 199 F₂ individuals were collected and frozen in liquid nitrogen, and then transferred to a −80°C freezer. Genomic DNA from the F₂ population and parents were extracted following the manufacturer's protocols with the Plant Genomic DNA Kit (TIANGEN, Beijing, China). DNA degradation and contamination were monitored on 1% agarose gels. DNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). DNA concentration was measured using Qubit® DNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA).

Genomic DNA was extracted using the CTAB-based method. The purity and concentration of the extracted DNA were checked using a Nanophotometer® spectrophotometer (IMPLEN, CA, USA) and the Qubit® DNA Assay Kit in Qubit 2.0 Flurometer (Life Technologies, CA, USA), respectively. To prepare the DNA for sequencing, it was fragmented into 200–300-bp fragments and subjected to end repair with adenylation. Subsequently, bisulfite treatment was performed twice using the EZ DNA Methylation-GoldTM Kit (Zymo Research, Irvine, CA, USA). The resulting single-strand DNA fragments were then amplified using the KAPA HiFi HotStart Uracil + ReadyMix (2X; Zymo Research) and sequenced on an Illumina Hiseq 4000 (Illumina, USA) in 150 bp paired-end reads. Following the removal of low-quality reads, the remaining reads were mapped to the pear reference genome v1.0 (http://peargenome.njau.edu.cn/) using Bismark software v0.16.3 [72 ], with a requirement of no mismatch allowed. Methylation sites were identified, and their levels were calculated according to a previously established methodology [73 (link)]. DMR was identified within the promoter of each gene using the DSS software [74 (link)].

Leaf tissues were collected from all genotypes and DNAs were extracted using the Qiagen DNeasy 96 Plant kit (Qiagen, CA, United States). DNA degradation and contamination were monitored on 1% agarose gels. DNA purity and concentration were checked using the NanoPhotometer^® spectrophotometer (IMPLEN, CA, United States) and Qubit^® DNA Assay Kit in Qubit^® 2.0 Flurometer (Life Technologies, CA, United States), respectively. DNA was digested by MseI [New England Biolabs (NEB)] restriction enzyme. The reduced representation libraries were constructed for individual genotypes according to published GBS protocol (Elshire et al., 2011 (link)) and sequenced using Illumina HiSeq2000 platform. Raw data were submitted to the NCBI Sequence Read Archive with a reference number of SRP081825. The Tassel 3.0 Universal Network Enabled Analysis Kit (UNEAK) pipeline (Lu et al., 2013 (link)) was used for de novo SNP discovery and genotype calling following Li et al. (2014) (link).

The putative homokaryotic (CGMCC 5.545 SS20), heterokaryotic strains (5.78 SS40, 5.78 SS46, and 5.78 SS84), and parent strain 5.78 were fermented in potato broth for DNA extraction. The mycelia were transferred to 50 mL sterile centrifuge tubes, washed twice with sterile water, and stored at -80°C until DNA extraction. Total DNA of W. cocos strain was extracted using blood and cell culture DNA midi kit (Q13343, QIAGEN, Dusseldorf, Germany). DNA degradation and contamination were monitored on 1% agarose gels. DNA purity was checked using the NanoPhotometer^®spectrophotometer (IMPLEN, CA, United States) and concentration was measured using Qubit^®DNA Assay Kit in Qubit^®2.0 Flurometer (Life Technologies, CA, United States). The qualified samples were sequenced using the Illumina NovaSeq platform in Nextomics Biosciences (Wuhan, China). Clean reads were obtained by eliminating unmatched reads and low-quality reads, connector contamination, and duplication reads by fastp³ (Chen et al., 2018 (link)). Quality control was performed using FastQC⁴. Clean data (accession number of NCBI: PRJNA644235) were used to estimate the heterozygous ratio based on analysis of k-mer depth distribution using Jellyfish⁵ (Marcais and Kingsford, 2011 (link)) and GenomeScope⁶ (Liu et al., 2013 ; Vurture et al., 2017 (link)).

At the vegetative stage (V2: one fully expanded trifoliolate leaf presence), leaves were sampled from young, healthy plants of accessions that exhibited extreme contrasting differences for tocopherol composition and used for genomic DNA isolation according to the standard protocol of the Novel Plant Genomic DNA Extraction Kit (DP320, Tiangen Biotech, Beijing, China). The RNase A was used to remove RNA contamination. The concentration and quality of each genomic DNA were measured by the NanoPhotometer^® spectrophotometer (Implen, Westlake Village, CA, USA) and 1% agarose gel electrophoresis, respectively. DNA concentration was measured using Qubit^® DNA Assay Kit in Qubit^® 2.0 Flurometer (Life Technologies, Carlsbad, CA, USA). Only genomic DNA samples with an OD260/280 value ranging from 1.8 to 2.2 were considered good quality DNA for further analyses.