Computer-simulated virtual RFLP patterns generated from in silico digestions of phytoplasma 16S rRNA genes from strains detected in this study belonging to groups: (A) 16SrI using HhaI (MH428961: 16SrI-P *); (B) 16SrIII using BstUI (MH428959: 16SrIII-F *); (C) 16SrIX using RsaI (MH428962:16SrIX-A[infinito]); (D) 16SrXIII using MseI
(MH428958: 16SrXIII-H *); (E, F) 16SrXV using HaeIII and HpalI (MH428963: 16SrXV-A[infinito]), obtained using iPhyClassifier tool.
The PCR reaction for the pre-amplification contained, 35 ng of PstI and MseI
primers, 0.5 uL of dNTP mixture (10 mM), 0.5 uL of MgCl2 stock solution (25 mM), 0.9 uL of 10 x PCR buffer (non-Mg2+), 5 units of Taq DNA Polymerase in a total volume of 25 uL.
The result of sequencing showed that there was a recognition site of MseI
in GG genotype, not in AA genotype.
An identification key was constructed for these species using as two characters of the PCR product its size and the restriction patterns generation using the three restriction enzymes EcoRI, MseI
and MaeI (Table 3).
For each individual, 15 ng of genomic DNA was digested by EcoRI and MseI
, with simultaneous ligation of corresponding adapters to the fragments.
Approximately 10--50 ng of gDNA per sample was dispensed into a single well on a 96-well plate already containing Droplet PCR Supermix (Bio-Rad Laboratories), 1.25 U of the restriction enzyme MseI
(New England Biolabs), 900 nmol/L primers, and 250 nmol/L probe in a final volume of 25 [micro]L.
(2012) and included: 1) restriction of the DNA with Msel and EcoRI restriction enzymes; 2) adapter ligation with Msel and EcoRI adapters; 3) pre-amplification; and 4) selective amplification with 4 combinations of MseI
and EcoRI primers (Table 2).
and let MSEi
denote the mean-square error for the estimation of [x.sub.k,i], that is, (1/M) [[summation].sup.M.sub.j=1] [([x.sup.(j).sub.k,i] - [[??].sup.(j).sub.k|k,i]).sup.2], where i = 1, 2, 3 and M is the number of simulation test.
For this purpose, XbaI (Fermentas Life Sciences, #ER0681), MSEI
or Tru1I (Fermentas Life Sciences, ER# 09825), and TaqI (Fermentas Life Sciences, ER# 0671) enzymes were used.
--Number of markers-- Primers EcoRI (E) --Primers MseI
(M)-- CAG CAT CTA CTC ACC 12 (a) 18 15 0 AAG 15 18 14 16 ACA 0 0 11 10 ACT 14 9 11 7 AAC 5 17 19 14 ACG 0 0 0 0 AGG 3 2 0 0 --Number of polymorphic markers-- E / M CAT CTA CTC Total ACC 8 (b) 4 - 12 AAG 5 8 5 18 AAC - 9 1 10 Total 13 21 6 40 Primer combinations are denoted by the EcoRI--AXX and Msel--CXX selective nucleotides, respectively; (a) Number of markers scored per primer combination; Underlined--AFLP primer combinations selected to investigate the Mendelian inheritance pattern in the full-sibs; (b) Total number of polymorphic markers scored.
The DNA fragments were linked to EcoRI and MseI
adapters (EcoRI adapter, 5'-CTCGTAGACTGCGTACC 3'/3'CTGACGCATGGTTAA-5'; and MseI
adapter, 5'-GACGATGAGTCCTGAG-3'/3' TACTCAGGACTCAT-5') at 20[degrees]C for 2 h.