2. Isolate the plasmids containing ITS and 16S rDNA PCR products with the aid of QIAprep Spin Miniprep Kit (Qiagen) for
sequencing.
3. Sequence the plasmids from both ends by the universal primers
T7 and SP6 with an Applied Biosystems 3730 DNA Analyzer.
4. Sequence the purified gyrB gene PCR products from both ends
with the help of the oligonucleotides with the same sequences
as underlined in primers gyrB-F and gyrB-R [179].
5. Use the individual DNA sequences to acquire the anticipated
endonuclease restriction site map of the ITS1 region.
6. Analyze individual restriction enzyme gel images with the aid of
the Gel Manager software package from Bio Systematica.
7. Use the high-quality sequences to generate alignments of ITS
or gyrB with known nematodes or bacteria for comparative
purposes.
6.2 Phylogenetic
Analysis
1. Inspect manually every aligned sequence and delete the
unaligned sequence ends and unreliable sequences.
2. Treat the gaps in the aligned domains as either missing data or a
fifth base.
3. Use Caenorhabditis elegans and Xenorhabdus nematophila as
outgroup taxa and to root the trees for Heterorhabditis and
Photorhabdus.
4. Align the outgroup sequences separately with MALIGN [180]
to test the presence or absence of different output taxa had an
effect on the alignment and resulting phylogenetic trees.
5. Conduct Parsimony analyses on phylogenetically informative
characters.
6. Apply indel coding to account the multiple gaps that may arise
due to single insertion or deletion events (indels), including all
gaps as independent characters.
7. Treat apparent single indels as a single character.
8. Append the matrix for the indels to the end of the data set.
6.3 Phylogeny
Construction
1. Consider all variable character states of the outgroup taxa as
plesiomorphic.
2. Consider inferred homologous character states of the ingroup
as apomorphic.
3. Eliminate multi-state characters from the analysis for the difficulty in establishing their polarity.
4. Remove autapomorphic and monomorphic characters also
from phylogenetic analysis as they provide no hierarchical
information.
94
Laboratory Techniques for Entomopathogenic Nematodes
sequencing.
3. Sequence the plasmids from both ends by the universal primers
T7 and SP6 with an Applied Biosystems 3730 DNA Analyzer.
4. Sequence the purified gyrB gene PCR products from both ends
with the help of the oligonucleotides with the same sequences
as underlined in primers gyrB-F and gyrB-R [179].
5. Use the individual DNA sequences to acquire the anticipated
endonuclease restriction site map of the ITS1 region.
6. Analyze individual restriction enzyme gel images with the aid of
the Gel Manager software package from Bio Systematica.
7. Use the high-quality sequences to generate alignments of ITS
or gyrB with known nematodes or bacteria for comparative
purposes.
6.2 Phylogenetic
Analysis
1. Inspect manually every aligned sequence and delete the
unaligned sequence ends and unreliable sequences.
2. Treat the gaps in the aligned domains as either missing data or a
fifth base.
3. Use Caenorhabditis elegans and Xenorhabdus nematophila as
outgroup taxa and to root the trees for Heterorhabditis and
Photorhabdus.
4. Align the outgroup sequences separately with MALIGN [180]
to test the presence or absence of different output taxa had an
effect on the alignment and resulting phylogenetic trees.
5. Conduct Parsimony analyses on phylogenetically informative
characters.
6. Apply indel coding to account the multiple gaps that may arise
due to single insertion or deletion events (indels), including all
gaps as independent characters.
7. Treat apparent single indels as a single character.
8. Append the matrix for the indels to the end of the data set.
6.3 Phylogeny
Construction
1. Consider all variable character states of the outgroup taxa as
plesiomorphic.
2. Consider inferred homologous character states of the ingroup
as apomorphic.
3. Eliminate multi-state characters from the analysis for the difficulty in establishing their polarity.
4. Remove autapomorphic and monomorphic characters also
from phylogenetic analysis as they provide no hierarchical
information.
94
Laboratory Techniques for Entomopathogenic Nematodes