Geneclean spin kit

Total DNA was isolated from dry feces (ca. 2.9–59.2 mg) using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and purified using a Geneclean Spin Kit (MP-Biomedicals, Santa Ana, CA, USA). PCR amplification and rbcL amplicon sequencing by HTS was performed using the DNA metabarcoding primer for rbcL (S1 Table) and the same reaction mixture, conditions and instruments that were used for constructing the local ITS2 database, except that the annealing temperature used for rbcL was 56°C instead of 55°C in the initial PCR. On the other hand, PCR amplification and ITS2 amplicon sequencing by HTS was performed using the same reaction mixture, conditions and instruments that were used to construct the local ITS2 database.

Genomic DNA isolated from callus, regenerated plants or seedlings, was used for polymerase chain reaction (PCR) using primers spanning the target sites (Table 4). The PCR products were resolved on agarose gel and extracted using Geneclean Spin Kit (MP Biomedicals, CA, USA) for sequencing from both ends using forward and reverse primers by the Sanger Sequencing method at Eurofins Genomics USA. The sequences were viewed on Sequence Scanner 2 software (Applied Biosystems Inc.) and aligned with the reference sequences using CLUSTAL-Omega multiple sequence alignment tool. CRISPR-ID tool was used to separate superimposed overlapping spectrum in Sanger sequencing traces, characteristic of heterozygous or chimeric mutations (Dehairs et al. 2016 (link)). The type of indel was identified by cloning PCR amplicon into pCR2.1 vector using TA cloning kit (Thermo-Fisher Scientific, NY) as per manufacturer’s instructions and sequencing individual colonies by Sanger sequencing.

Total community DNA (TC-DNA) was extracted from the microbial pellets using the FastDNA SPIN Kit (MP Biomedicals, Heidelberg, Germany) as described by the manufacturer after a harsh lysis step with the FastPrep-24 Instrument (MP Biomedicals, Heidelberg, Germany). The TC-DNA was purified with GENECLEAN SPIN Kit (MP Biomedicals, Heidelberg, Germany) according to the manufacturer. The purified TC-DNA was diluted 1∶10 with 10 mM Tris HCl before use.
For amplification of 16S rRNA gene fragments, PCR reactions were performed with TC-DNA obtained from rhizosphere samples with the primers F984-GC and R1378 as described by Heuer [41] (link) using Taq DNA polymerase (Stoffel fragment, ABI, Darmstadt, Germany). The PCR products were analyzed by DGGE approach as described by Weinert et al. [42] (link).
Bacterial fingerprints were evaluated with GELCOMPAR II version 6.5 (Applied Maths, Sint-Martens-Latern, Belgium) as described by Schreiter et al. [37] (link). The obtained Pearson similarity matrices were used for construction of a dendrogram by an Unweighted Pair-Group Method with Arithmetic mean (UPGMA) as well as of statistical analysis by the permutation test, calculating the d-value from the average overall correlation coefficients within the groups minus the average overall correlation coefficients between samples from treatments compared as suggested by Kropf et al. [43] .

The roots of the respective plants, which were studied for gene expression, were used for analysis of rhizosphere microbiota (bacteria, archaea, and fungi). At first, roots were washed with sterile tap water (Schreiter et al., 2014b (link)). The rhizosphere fraction was obtained from 5 g of representative root samples, which were collected from complete root systems, by 1 min Stomacher treatment (Seward Ltd., Worthing, United Kingdom) followed by centrifugation (Schreiter et al., 2014a (link)). Rhizosphere pellets were kept at −20°C for total community (TC)-DNA extraction. Root-associated soil was sampled from the soil fraction loosely adhering to the root system collected after vigorous shaking. After collection, the soil samples were immediately frozen at −20°C.
Total community-DNA was extracted from root-associated soil (0.5 g) and rhizosphere pellets using the FastPrep-24 bead-beating system and FastDNA Spin Kit for Soil. DNAs were purified with the GeneClean Spin Kit (both MP Biomedicals, Santa Ana, CA, United States).

Total community DNA from nematodes was extracted using the FastPrep FP120 bead beating system and FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA, United States) as described by the manufacturer. The DNA was purified with GENECLEAN SPIN Kit (MP Biomedicals) according to the manufacturer’s instructions. For the nematode species composition analysis, primers G18S4F and G18S4-22R were used to amplify approximately 345 bp of the 18S rRNA gene (Blaxter et al., 1998 (link)). PCR of 25 μl contained 2.5 μl of 10× GoTaq buffer (Promega, Mannheim, Germany), 3.75 μl 25 mM MgCl_2, 2.5 μl 2 mM dNTP (each), 0.5 μl of each primer (10 μM), 1.25 μl of 2 mg/ml BSA, 2 μl of 50% acetamid, 0.2 μl 5 U/μl GoTaq DNA polymerase (Promega), ca. 2 ng DNA. The following PCR cycler conditions was used: initial denaturation of 5 min at 94°C, 27 cycles of (94°C for 45 s; 54°C for 30 s; 72°C for 1 min) and a final extension of 5 min at 72°C. The resulting PCR product was purified using High Pure PCR Purification kit (Roche Diagnostics GmbH) following the manufacturers instruction. Barcoded amplicon sequencing of the 18S rRNA genes was done by 2 × 250 bp paired-end high-throughput sequencing on an Illumina MiSeq platform (Illumina, San Diego, CA, United States).

The complete ovine PEG11 ORF was amplified by PCR as seven partially overlapping fragments from BAC 229G11 [6 (link), 31 (link), 32 (link)]. These were digested at endogenous restriction enzyme sites (BspeI, BglI, NsiI, AatII, BspHI, MluI) to generate paired cohesive ends which allowed for the reconstitution by ligation of a 4,178 bp fragment spanning from codon two to 175 bp downstream of the TGA stop codon of the ovine PEG11 gene (oPEG11). The corresponding fragment was digested with BglII and ligated in frame with the vector’s ATG initiation codon of the pMlc3F-nlacZ-2E vector [9 (link)]. A 7.5 Kb fragment encompassing Mlc 3F promotor, oPEG11 ORF, bovine GH polyadenylation signal and Mlc 2E enhancer was released from the remainder of the vector by digestion with NotI and SmaI, subjected to agarose gel electrophoresis, and purified with the Geneclean Spin kit (MP Biomedicals, Santa Ana, CA).