Gelstrain

When the plants were at a height of approximately 20 cm, 1 mL L^-1 10% Basta solution (8.0 mg l^-1) was used for preliminary screening. The plants were sprayed three times every 6 days. Genomic DNA was extracted from the alfalfa leaves using a Plant Genomic DNA extraction kit (Tiangen, Beijing). The PCR was conducted with a genomic DNA template in PCR premix using two convergent primers that were complementary to the CsALDH gene and another pair of primers that were complementary to the bar gene. DNA amplification was performed at 94°C for 3 min; 35 cycles of 94°C for 30 s, 50°C for 45 s, and 72°C for 1 min 30 s, and then a final extension at 72°C for 10 min. Total RNA was extracted using the UNIQ-10 column total RNA extraction kit (Sangon Biotech, Shanghai). The RT-PCR conditions were identical for both CsALDH and MsActin, as described in the genomic PCR analysis. The PCR products were separated on a 1.2% agarose gel, stained with GelStrain (TransGene Biotech, Beijing), and visualized under UV. Sixteen transgenic plants showing highly expressed CsALDH were selected for further stress tolerance and phenotype analysis. All subsequent experiments were conducted on cloned plants from the T₀ generation of transgenic plants.

All the DNA preparations were analyzed for the presence of E. bieneusi by nested PCR amplification of a 410 bp fragment including the ITS region (243 bp) of the rRNA gene, as previously described [24 ]. Each specimen was subjected to at least two PCR reactions. A positive control (DNA of a human-derived genotype Type IV) and a negative control (nuclease-free water) were included in each PCR test. The secondary PCR products were analyzed by 1.5% agarose gel and visualized under UV by staining the gel with GelStrain (TransGen Biotech., Beijing, China).

Except for the ITS region of the rRNA gene, all the 305 E. bieneusi DNA specimens were also analyzed at the four MLST loci, including three microsatellites (MS1, MS3, and MS7) and one minisatellite (MS4). The primers and the cycle parameters for nested PCR amplifications used in the present study were designed previously by Feng et al. (2011) (link) and the approximately expected fragment lengths of the secondary PCR products were 676 for MS1, 537 for MS3, 885 for MS4 and 471 for MS7 (Feng et al., 2011 (link)). TaKaRa Taq DNA polymerase (TaKaRa Bio Inc., Tokyo, Japan) was used in all the PCR reactions. All secondary PCR products were separated by electrophoresis in a 1.5% agarose gel and visualized on GelDoc^TM EZ Imager (Bio-Rad, United States) by staining the gel with GelStrain (TransGen Biotech., Beijing, China) before sequencing.