Phusion master mix

A GBS strategy was used to develop SNP markers as previously described (Zhang et al., 2016 (link)). Briefly, approximately 0.1 to 1 µg of genomic DNA was incubated at 37°C with MseI (New England Biolabs, NEB), T4 DNA ligase (New England Biolabs, NEB), ATP (New England Biolabs, NEB), and an MseI Y adapter N containing barcodes, and the samples were then heat-inactivated at 65°C. Two additional enzymes, HaeIII and EcoRI (New England Biolabs, NEB), were simultaneously added into the MseI digestions to further digest the fragments at 37°C. Then, the digested fragments with ligations were purified with Agencourt AMPure XP beads (Beckman Coulter, Inc.) and subjected to PCR amplification with the Phusion Master Mix (New England Biolabs, NEB) using both universal primers as well as i5 and i7 index primers (Illumina). The PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter, Inc.), pooled, and separated by electrophoresis on a 2% agarose gel. Fragments of 400 to 450 bp (with indexes and adaptors) were excised from the gel and purified using a gel extraction kit (QIAGEN). These purified products were further cleaned using Agencourt AMPure XP beads (Beckman Coulter, Inc.) prior to sequencing. Then, paired-end 150 bp sequencing was performed on the selected tags on the Illumina HiSeq 4000 platform.

GBS sequencing was conducted according to the method of Elshire et al., 2011 [29 (link)], with minor improvements. Briefly, the gDNA from 154 grasspea plants (2 parents and 152 F₂ progeny) was first digested by MseI (5’-T!TAA-3’) (New England Biolabs, ‘NEB’, Ipswich, MA, USA) at 37 °C, then subjected to restriction-ligation by T4 DNA ligase (NEB) and ATP (NEB) with a MseI Y-adapter N-containing barcode at 65 °C, followed by a second digestion with HaeIII (5’-GG!CC-3’) (NEB) at 37 °C. The digested fragments were purified using Agencourt AMPure XP (Beckman Coulter, ‘Beckman’, Brea, CA, USA) and used for PCR amplification, utilizing Phusion Master Mix (NEB) to add universal and index primers, as well as i5 and i7 index sequences, to the digested fragments. The purified PCR fragments (425–490 bp including indexes and adaptors) were screened and extracted using a 2% agarose gel with the Gel Extraction Kit (Qiagen, Valencia, CA, USA). The amplification products were purified using Agencourt AMPure XP (Beckman) and diluted before sequencing. Finally, an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) was used to perform 150-bp paired-end sequencing by Tianjin Novogene Technology Co., Ltd. (Tianjin, China).

Tissue samples were collected from young leaves, freeze-dried, and pulverized using GenoGrinder 2000 (Spex CertiPrep Inc. Metuchen, NJ, USA) at 650 strokes/min for 15 s. Genomic DNA was isolated using 96-well silica filter plates (Epoch Life Science #2020-001). DNA was quantified using PicoGreen (Thermo Fisher Scientific, Waltham, MA) on a Synergy™ HT plate reader (Bio-Tek Instruments, Winooski, VT, USA) and diluted to 50 ng/µl in EB buffer. Genomic libraries were constructed using the two-enzyme GBS approach^{12 (link)} using simultaneous restriction-ligation with HindIII-HF, MseI, and T4 DNA Ligase (New England Biolabs). Restriction-ligation reactions were pooled by 96-well plate, purified using AMPure XP beads, PCR-amplified using Phusion Master Mix (NEB #M0531), and purified again using AMPure XP beads. A DNA7500 chip in an Agilent 2100 Bioanalyzer was used to determine average library size and concentration, and to dilute each library to 10 nM before submission for sequencing on an Illumina Hiseq 2500 (SR100) at the UC Davis Genome Center (Davis, CA).

GBS libraries were constructed in accordance with the modified protocol [45 (link)]. Genomic DNA was incubated at 37℃ with MspI (New England Biolabs, NEB), T4 DNA ligase (NEB), ATP (NEB), and MspI Y adapter N-containing barcode. Restriction-ligation reactions were heat-inactivated at 65℃ and digested with the additional restriction enzyme EcoRI (NEB) at 37℃. The restricted ligation samples were purified using Agencourt AMPure XP (Beckman). The purified samples were PCR amplified with Phusion Master Mix (NEB) universal primer and index primer to add the index. The PCR products were purified, pooled, and electrophoresed on 2% agarose gel. Fragments between 265 and 315 bps were selected and purified. Pair-end sequencing was performed on the selected tags using the Illumina PE150 platform.

The microbial DNA from the frozen fecal samples was extracted with the Magnetic Soil and Stool DNA Kit (TIANGEN BIOTECH) according to the manufacturer's instructions. After evaluating the concentration and purity, the reactions of PCR were conducted with Bio‐Rad T100 Gradient PCR Instrument. The V3‐V4 region of the16S rRNA was amplified with 341F (5′‐barcode‐CCTAYGGGRBGCASCAG‐3′) and 806R (5′‐GGACTACNNGGGTATCTAAT −3′) primers set. All PCRs were carried out with Phusion Master Mix (New England BioLabs Inc). The PCR products were mixed and purified with GeneJET PCR Purification Kit (Thermo Scientific) according to the protocol provided by the manufacturer.²¹