Subcategoría didáctica (estudiantes)

The standard PCR reaction contained 5 to 10 ng genomic DNA, 2.5 l 10× PCR buffer (-Mg, Solis Biodyne, Tartu, Estonia), 1.5 l MgCl2 (25 mM), 2 l dNTP

mixture (1.25 mM), 0.2 l FIREPol® DNA Polymerase (5 U/μl, Solis Biodyne) and 0.5 l primer mix (10 μM each) in a reaction volume of 25 l. The cycling conditions used were 4 min at 94℃, 35 cycles of 10 s at 95℃, 30 s at 50 to 60℃ and 72℃ for 1 min per Kb, and 10 min at 72℃.

2.4.2 High fidelity PCR

PCR products required to be sequenced were amplified with Platinum® Taq DNA Polymerase, High Fidelity (Invitrogen). The PCR components included 5 to 10 ng genomic DNA, 2.5 l 10× High Fidelity PCR buffer (-Mg), 1 l MgSO4 (50 mM), 0.2

l dNTP mixture (25 mM), 0.1 l Platinum® Taq High Fidelity (5 U/μl, Invitrogen) and 1 l primer mix (10 μM each) in a reaction volume of 25 l. The cycling conditions were the same as above.

2.4.3 Real time PCR

2.4.3.1 Primer and probe design

Beacon Designer™ software (version 7.0, Premier Biosoft, Palo Alto, California) was used in primer and probe design. Probes (Table 2) were custom synthesized by Biosearch Technologies (Novato, CA, USA) with fluorophores chosen according to the dye selection chart provided by the manufacturer (Biosearch-Technologies 2013).

2.4.3.2 Cycling conditions

Real time PCR was performed on a LightCycler® 480 (Roche, Germany). A LightCycler® 480 Probes Master mix kit (catalog number 04707494001, Roche) was used to perform the real time PCR. The simplex (PCR with only one probe/primer combination) reaction mix contained 5 L of probe master mix, and a final concentration of 200 nM of each primer and probe. The duplex (PCR with two probe/primer combinations) reaction mix contained 7.5 L of probe master mix, 0.05 mM additional dNTPs and a final concentration of 200 nM of each of ltmG and TB1

primers and probes. In each case 1 L (with a concentration in the range of 1 to 10 ng/L) DNA template was used in a final volume of 10 L. All real time PCR assays had the same cycling conditions: 5 min at 95℃, 45 cycles of 10 s at 95℃, 20 s at 60℃ and 10s at 72℃, and 10s at 40℃.

2.4.3.3 Standard curves

For generating standard curves, a mix of ryegrass and NZCT DNA was used, with the highest concentrations of 100 ng/L ryegrass and 1 ng/L endophyte DNA. Ten-fold dilutions over six orders of magnitude of the template concentrations, each with three replicates, were used to generate all standard curves. The average crossing points (the point at which the fluorescence of a sample rises above background fluorescence) were calculated and plotted as a function of the Log10 of the template

input amount in nanograms.

2.4.3.4 Colour compensation

As fluorophores have overlapping emission spectra, which can result in over- or under-estimation of data, colour compensation was applied to compare the emission spectra when the reporter dyes are used in separate reactions. LightCycler® 480 software uses those spectra to make adjustments when the reporter dyes are used together, and thus to eliminate errors associated with this spectrum overlap. To perform colour compensation, six replicates of simplex reactions containing the same amount of master mix and primer/probe as used in multiplex PCR, and 10 ng WT E+ genomic DNA template, were used for each reporter dye. Another six replicate reactions containing the same amount of master mix and template but no primer/probe were used as blank. The colour compensation was performed after 5 min pre incubation at 95℃ and 45 amplification cycles (10 s at 95℃, 20 s at 60℃ and 10s at 72℃) when reactions reached the plateau phase. The thermocycling profile consisted of 95℃for 1sec at 4.4℃/s, 40℃ for 30s at 2.5℃/s, and 80℃ at 0.1℃/s with continuous acquisition mode.

2.4.3.5 Calculation

Standard curves of each primer/probe were used to calculate the DNA biomass of endophyte or grass by using the advanced relative quantification method according to the LightCycler® 480 system manual, taking the colour compensation correction into account. The concentration was expressed as a ratio of copy number of endophyte gene (ltmG, ltmJ or perA) to ryegrass gene (TB1). One NZCT-infected ryegrass DNA sample was used as calibrator in all quantification experiments so that adjustments

could be made to compensate for between-experiment variation. For instance, the formula for calculating endophyte concentration using the ltmG gene is:

2.4.3.6 Primer/probe specificity, efficiency and reliability evaluation

Primer/probe specificity was evaluated in a real time PCR assay using genomic DNA individually extracted from cultures of NZCT, AR1 and AR37 as well as from ryegrass plants that were endophyte-free or infected with NZCT. Clover DNA was used as a negative control. In addition to this, the specificity of the fungal primers was confirmed by lack of amplification of 16 fungal species from a range of classes and families (listed in Table 2.2).

To evaluate the reliability of the assays with other ryegrass and hybrid cultivars, DNA was extracted from 16 glasshouse-grown cultivars. The presence of endophyte in all endophyte-infected grasses was confirmed by immunoblotting (see section 2.6). Subsequently, real time PCR was done with DNA from the same samples and using all primer/probe combinations.

2.4.3.7 Evaluation of a dual ltmG/ TB1 assay on quantification of

endophyte concentration

To evaluate the correlation between endophyte nucleotide concentration and the ratio of endophyte biovolume/ryegrass biovolume, grass pseudostems (leaf sheaths and un-emerged leaf lamina) were collected from the base of the tiller upwards (approximately 1 cm long sections). The top and bottom parts (1-2 mm slices) of these pseudostem sections, including all leaf sheaths and emerging lamina, were cut and prepared for determination of the ratio of endophyte biovolume/ryegrass biovolume (see details in 2.8), whilst the central section was used for real time PCR analysis. Duplex real time PCR using ltmG and TB1 primer/probe combinations was performed with DNA extracted from the central part of the grass pseudostem to quantify endophyte concentration.

ltmG crossing point value

TB1 crossing point value Sample

ltmG crossing point value

TB1 crossing point value Calibrator

=

Table 2.2 Fungi used in endophyte primer specificity evaluation.

Fungi

Division

Class

Family

Trichoderma atroviride Ascomycota Sordariomycetes Hypocreaceae

Trichoderma viride Ascomycota Sordariomycetes Hypocreaceae

Pestalotiopsis sp. Ascomycota Sordariomycetes Amphisphaeriaceae

Glomerella cingulata Ascomycota Sordariomycetes Glomerellaceae

Xylariaceae sp. Ascomycota Sordariomycetes Xylariaceae

Eutypa lata Ascomycota Sordariomycetes Diatrypaceae

Lophodermium conigenum Ascomycota Leotiomycetes Rhytismataceae

Cyclaneusma minus Ascomycota Leotiomycetes Rhytismataceae

Lophodermium pinastri Ascomycota Leotiomycetes Rhytismataceae

Sclerotinia sp. Ascomycota Leotiomycetes Sclerotiniaceae

Alternaria alternata Ascomycota Dothideomycetes Pleosporaceae

Phoma glomerata Ascomycota Dothideomycetes Didymellaceae

Passalora arachidicola Ascomycota Dothideomycetes Mycosphaerellaceae

Aspergillus nidulans Ascomycota Eurotiomycetes Trichocomaceae

Strasseria geniculata Ascomycota Not assigned Not assigned

2.4.3.8 Evaluation of

ltmJ and perA

primer/probes for detecting

contaminating endophytes in seed

AR37-infected ryegrass seeds (‘Grasslands Samson’) were mixed with either NZCT-infected ‘Grasslands Samson’ or AR1-infected ‘Grasslands Samson’ at proportions by weight of 3%, 5%, 10%, 15%, 20% and 25% for the purpose of measuring each in the mixtures. Mixed seeds were ground into fine powder in liquid nitrogen, and 30 mg of the powder used for DNA extraction using a plant genomic DNA mini kit (Geneaid, Taipei, Taiwan) following the manufacturer’s instructions. Three replicates were extracted separately for each mixed seed sample. Real time PCR analyses using ltmJ and perA primer/probe combinations were conducted to detect NZCT and AR1 contamination in AR37-infected ryegrass seed.

2.5 RNA isolation and manipulation