By assessing diversity variations of bacterial communities under different rhizocompartment types (i.e., roots, rhizosphere soil, root zone soil, and inter-shrub bulk soil), we explore the structural variability of bacterial communities in different root microenvironments under desert leguminous plant shrubs. 16S rRNA high-throughput genome sequencing was used to characterise the bacterial diversity in the rhizocompartments of three xeric leguminous plants (Caragana microphylla, Hedysarum scoparium, and Hedysarum mongolicum) in the Mu Us Desert.

16S rRNA genome sequencing and bioinformatics analysis

E.Z.N.A. soil DNA kits (OMEGA, the USA) were used with 0.5 g of fresh sample to extract DNA samples, as per the manufacturer’s instructions. All samples were stored at -80 °C. After thawing on ice, extracted DNA samples were separately centrifuged and fully mixed; sample quality was determined using a NanoDrop instrument, and 30 ng DNA was used for PCR amplification. PCR amplification was performed in 25 μL reaction volumes containing 10×PCR buffer, 0.5 μL dNTPs, 1 μL of each primer, 3 μL bovine serum albumin (2 ng/μL), 12.5 μL KAPA 2G Robust Hot Start Ready Mix, ultrapure H₂O, and 30 ng template DNA. The PCR amplification program included initial denaturation at 95 °C for 5 min, followed by 25 cycles of denaturing at 95 °C for 45 s, annealing at 55 °C for 50 s, and extension at 72 °C for 45 s. Finally, the PCR amplification program was completed at 4 °C. Forward primer F799 (5′-AACMGGATTAGATACCCKG-3′) and reverse primer R1193 (5′-ACGTCATCCCCACCTTCC-3′) were used to target the V5-V7 regions of 16S rRNA. Both primers contained Illumina adapters, and the forward primer contained an 8 bp barcode sequence unique to each sample. An Agarose Gel DNA purification kit (Axygen Biosciences, Union City, CA, the USA) was used for the purification and combination of PCR amplicons. After purification, PCR amplicons were mixed at an equal molar concentration, followed by pair-end sequencing using the Illumina MiSeq sequencing system (Illumina, the USA) according to a standardized process.

MiseqPE300 (Illumina, the USA) sequencing results were recorded in the Fastq format. Quantitative insights into microbial ecology software (QIIME; Version 1.8 http://qiime.org/) was used to analyze original Fastq files and to undertake quality control according to the following criteria : (i) base sequences with a quality score <20 at read tails were removed, and the window was set at 50 bp. When the mean quality score in the window was <20, posterior-end base sequences were discarded from the window and reads shorter than 50 bp were removed after quality control; (ii) paired reads were assembled into one sequence according to the overlapping relationship between reads (minimum overlapping length: 10 bp); (iii) the maximum allowable mismatch ratio of the overlapping areas of assembled sequences was set to 0.1, and sequences failing to meet this criterion in pairs were removed; (iv) samples were distinguished according to barcodes and primers at the head and tail ends of sequences, and the sequence directions were adjusted based on the number of mismatches allowed by barcodes (0); and (v) different reference databases were selected according to the type of sequencing data. Chimeras were removed using the Usearch program V8.1861 (http://www.drive5.com/usearch/), and clean tags of high-quality sequences were acquired after smaller-length tags were discarded using mothur software. Sequences were clustered into Operational Taxonomic Units (OTUs) using UPARSE V7.1 (http://drive5.com/uparse/) based on a 97% sequence similarity cutoff (excluding single sequences). Representative sequences and an OTU table were obtained.