*4.1. Identification, Chromosomal Location, and Structural Organization of GmXTH Family Members in Glycine Max*

All sequence information for genes and proteins was retrieved by searching Phytozome v10.3 database (available online: http://www.phytozome.net) with a BLASTP algorithm using the *AtXTH31* amino acid sequence. The chromosome location of each *GmXTH* was obtained from Phytozome. The exon/intron organizations of *GmXTH*s were visualized with the Gene Structure Display Server program ([59] GSDS: available online: http://gsds.cbi.pku.edu.cn/).

#### *4.2. Protein Sequence Alignment, Phylogenetic Analysis, and Gene Duplications of* GmXTH *Genes*

Multiple sequence alignments were constructed using ClustalW2 (available online: http://www. ebi.ac.uk/Tools/clustalw2/index.html). Subsequently, a phylogenetic tree was constructed using the neighbor-joining method and implemented using the MEGA7 software tool [60]. The reliability of an inferred tree was confirmed with bootstrap analysis performed with 1000 replications. The evolutionary distances were computed using the Poisson correction method [61] and are in the units of the number of amino acid substitutions per site. A total of 123 coding sequences from *Arabidopsis*, rice, and soybean were collected for phylogenetic analysis. All positions containing gaps and missing data were eliminated. The Ks (synonymous substitutions per synonymous site) and Ka (non-synonymous substitutions per non-synonymous site) values were extracted from the Plant Genome Duplication Database (PGDD: available online: http://chibba.agtec.uga.edu/duplication/), and these were used

for calculating the approximate dates of duplication events. The date of duplication events was subsequently estimated according to the equation T = Ks/2λ, in which the mean synonymous substitution rate (λ) for soybean is 6.1 × <sup>10</sup>−<sup>9</sup> [62].

#### *4.3. Plant Growth, Hormonal/Flooding Treatments, and Tissue Collection*

Soybean cultivar Williams 82 was used for gene expression pattern analysis. For ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) treatment, plants were grown in 4-gallon pots (Greenhouse megastore, USA) containing a 3:1 mixture of turface and sand in a growth chamber under the conditions of 28/20 ◦C day/night temperature, 14/10 h light/dark photoperiod, 800 μmol m−<sup>2</sup> s−<sup>1</sup> light intensity and 60% humidity. Two-week-old plants were sprayed with 50 μM ACC (or a mock solution without ACC) on the leaf, irrigated with 2 l of hormone solution in a plastic case and then incubated for 5 h before root tissue collection.

For flooding stress treatment, two-day-old seedlings cultivated on quartz sand were completely submerged in 700 ml of water for 5 days at 25 ◦C with a light/dark cycle (600 μmol m−<sup>2</sup> s−1, 16 h light/8 h dark). The water level was kept at 2 cm above the quartz sand surface, and control seedlings were grown with a water level below the quartz sand surface. Roots and hypocotyls were collected from soybean seedlings. The collected tissues were frozen immediately in liquid nitrogen and stored at −80 ◦C. All samples were collected in biological triplicate.

For waterlogging treatment, transgenic and control seeds were sown in cones filled with turface and sand in the ratio of 2:1. The cones with plants at the V1 stage were kept inside a tub and flooded above the soil level (>2 cm high) for 14 days. The greenhouse temperatures (24~26 ◦C) were exactly the same during day and night. The heating system turned on when temperatures were below 23.8 ◦C. A passive cooling ridge vent opened when temperatures were above 26.6 ◦C. Active cooling fans turned on when temperatures were above 29.4 ◦C. The shade was set to stay open all the time, and the HID lights were set to be on at all levels between 5 am and 7 pm.

#### *4.4. Promoter Analysis*

The DNA sequences 2000 bp upstream of the translation start site (ATG) were extracted from the soybean genome, and the presence and abundance of the known cis-elements were analyzed with the help of the program SOGO (available online: https://sogo.dna.affrc.go.jp/cgi-bin/sogo.cgi?lang=en& pj=640&action=page&page=newplace).

#### *4.5. Expression Profiling Using RNA-seq Datasets*

The RNA-seq data generated by Libault et al. [32] from nine different soybean tissues (William 82 genotype) including flowers, leaves, nodules, pods, roots, root hairs, seeds, shoot apical meristems, and stems were used to analyze expression patterns of *GmXTH*s members. Chen et al. [34] generated RNA-seq data using soybean (Williams 82 genotype) leaf tissue under flooding stress. Briefly, flooding stress was imposed at the soybean V4 stage (four unfolded trifoliate leaves) by placing the pots into a larger pot filled up to a water level of 4 cm above the soil surface for 7 days.

#### *4.6. RNA Extraction for Expression Pattern Analysis*

The frozen samples were ground to powder in liquid nitrogen with a mortar and pestle. Approximately 100 mg tissue samples were used for RNA extraction using a RNeasy Plant mini kit (Cat# 74904, Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. On-Column DNA digestion performed by following the RNase-Free DNase kit (Cat#79254, Qiagen) manufacturer's protocol. The quality and quantity of RNA were assessed using a Nanodrop®1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

#### *4.7. Quantitative RT-PCR Analysis*

A total of 2 μg RNA from each sample was reverse-transcribed to cDNA in 20 μL reaction volume using RNA to cDNA Ecopry™ Premix (Double primed) cDNA Synthesis Kit (Cat# 639549, Clontech, Foster City, CA, USA) according to the manufacturer's protocol. PCR was performed in a 10 μL reaction volume using the Maxima SYBR Green/ROX qPCR Master Mix (Cat# K0223, Thermo, USA) on the ABI7900HT detection system machine (ABI PRISM® 7900HT, Foster City, CA, USA). The results from three biological replicates and two technical replicates were used for data analysis. The PCR conditions were as follows: 50 ◦C for 2 min, 95 ◦C for 10 min, and 40 cycles of 95 ◦C for 15 s, and 60 ◦C for 1 min. To normalize the gene expression levels, the actin (Glyma.18G290800) gene was used as an internal control. All novel primers were designed using the Primer3 web interface (available online: http://frodo.wi.mit.edu/primer3/ [63,64]. The primer sequences are listed in Supplementary Table S2.

#### *4.8. Construction of the pZY101-AtXTH31 Vector, Agrobacterium-mediated Soybean (Glycine max) Transformation and Progeny Segregation Analysis*

The gene-specific primer pair 5 -CATGCCATGGATGGCTTTGTCTCTTATCTTTC-3 and 5 -CATGCCATGGCTAACATTCTGGTGTTTGGG-3 was designed to isolate the full-length CDS of *AtXTH31* from *Arabidopsis*. The PCR product (902 bp) was cloned into the pCR4-TOPO vector, and the positive plasmid was fully sequenced with M13 sequencing primers. The *AtXTH31* gene sequence was inserted into the pCNSH.131. AtMyb2p-Gus vector, which contained the Myb2 promoter. Finally, the whole cassette contained a promoter, and the gene sequence was moved into the pZY101-Asc binary vector. An improved *Agrobacterium*-mediated transformation of the soybean cotyledonary node system [65] was performed using the elite genotype "Maverick". To determine the segregation of gene of interest and selectable marker gene, at least 30 plants from each T0 event were screened using leaf paint (100 mg/L glufosinate, Sigma, St. Louis, MO, USA) analysis carried out for the T0 generation. T2 progeny from the T1 generation was similarly analyzed to identify homozygous T1 lines for subsequent study.

#### *4.9. DNA Extraction and Quantification and PCR Confirmation of Transgenes*

DNA was extracted from the transgenic plant leaves (mixed leaf tissue) using CTAB methods [66]. DNA concentrations and quality were initially estimated using a Nanodrop spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) and then estimated using a QuantiT dsDNA HS Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The concentrations from the Qubit assays were used to quantify the DNA input in each PCR reaction. Primers were designed to detect the *AtXTH31* gene of interest and the bar gene as the selectable marker gene (Supplementary Table S2). PCR conditions were as follows: 95 ◦C hot start 30 s, followed by 36 cycles of 95 ◦C denaturing 10 s, 55 ◦C annealing 10 s, and 72 ◦C extension 1 min, followed by 72 ◦C final extension 10 min. PCR products were analyzed on an agarose gel, and events were considered transgenic if they displayed an approximately 800 bp band for the gene of interest and a 500 bp band for the bar gene. Four positive transgenic events were obtained and used for further phenotype analysis.

#### *4.10. TaqMan Assays and QuantStudio 3D Digital PCR Analysis for Soybean AtXTH31 Transgenic Copy Number Variation*

The following equipment and chemicals were used from Applied Biosystems (Waltham, MA, USA), Thermo Fisher Scientific, USA: QuantStudio™ 3D Imager (Cat#: PN4489084), QuantStudio™ 3D Loader (Cat#: PN4482592), Dual Flat Block GeneAmpR PCR System 9700 (Cat#: PN4428235), Tilt Base & chip adapters (Cat#: PN4486414 and 4485513), QuantStudio™ 3D Digital PCR 20K Chips (Cat#: PN4485507), and QuantStudio™ 3D Digital PCR Master Mix (Cat#: PN4485718). The probe was designed and synthesized by Life Technology Company. The dPCR reaction volume was 20 μL and contained 10 μL 2× TaqMan 3D mix, 1 μL 20× FAM labeled primers and probe, 1 μL 20× VIC labeled primers and probe, 1 μL DNA samples (40 ng/ μL), and 7 μL nuclease-free water. In total, 14.5 μL

each reaction product was loaded on the chips. Data analysis was conducted using QuantStudio™ 3D Analysis Suite™ Cloud Software as described previously [55]. The designed probes could only amplify transgene *AtXTH31* to ensure that no soybean homologous genes were detected. The ratio of the copy number of *AtXTH31* with lectin in the same soybean material was calculated as follows: (copies/μL of the *AtXTH31* transgene)/(copies/μL of the lectin gene Glyma.02G009600) in the same PCR reaction product. Soybean transgenic plants contained a single insert copy when the ratio value was equal to 0.5 and two insert copies when the ratio value was equal to 1. The ratio value was less than 0.5 when chimeric transgenic plants were found. The primers and probe sequences are shown in Supplementary Table S2.
