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– Add family information about rice and maize Type II cell walls to compare to A. thaliana Type I cell walls
– Add links to outside information on rice genes like we have for A. thaliana
– Include annotated composite trees of A. thaliana, rice and maize gene families
– Add links to sites used to generate the data
– Add source protein sequence used for our family trees so other researchers can make their own adding their genes of interest
– Generate a tutorial on how researchers can make use of the bioinformatics data on our site
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Too few genes, Blast other sites
N
Genes from
A. thaliana
Blast
TIGR
Homologous rice genes
Choose genes
A thaliana
& rice genes
Make tree
Good tree?
Y
Too many genes, tighten criteria
N
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Publish to website
Draw rice dendrogram

A. thaliana genes
Rice genes
Maize genes
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Draw tree with all family members
Annotate Publish to web

• To be considerate of the bioinformatics community with the number of
BLASTs to be performed and to speed the process, we downloaded the text or “flat file” of the TIGR rice protein sequences (available at: http://www.tigr.org/tdb/e2k1/osa1/data_download.shtml
) and performed local blasts using blastall from NCBI (available at: http://www.ncbi.nlm.nih.gov/BLAST/download.shtml
)
• Direction for use of these tools is available at the above sites and is beyond the scope of this tutorial
• For a small number of BLASTs, you can use web-based methods and common programs such as Word and Excel plus any of a number of downloadable tree drawing programs to make these kinds of trees on your own if you are not familiar with programming languages such as
Perl to automate the process. Although web searches can be more time consuming, they work just as well for a few sequences
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• For smaller numbers of BLASTs to the rice genome, TIGR provides an excellent Web BLAST at: http://tigrblast.tigr.org/eukblast/index.cgi?project=osa1
• You can also use the new BLAST tool at Gramene: http://www.gramene.org/multi/blas tview for most cereal sequences
• Note: gene model versions sometimes differ between Gramene and TIGR as one site may update to the latest model before the other

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• Downloading the protein sequence for
Arabidopsis SUD1
(At3g46440) from
TIGR, you can
BLAST it against the
TIGR Rice
Pseudomolecules –
Protein database using BLASTp

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• You get a series of “hits” to the gene of interest
• A higher score and smaller probability is a better match to the original gene
• This procedure is followed for all of the genes in a family to gather the best possible hits, sort the hits to remove duplicates and choose the best rice matches to the
Arabidopsis families
• You can use NCBI’s
blastall tool for multiple simultaneous blasts as we do for this step

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• This is a word document generated by BLASTing
SUD1 and SUD2 of
Arabidopsis against the
TIGR Rice Protein database
• The hits were copied into word and set to the font
Courier New, 9 pt and saved as a text only document (to remove the HTML code)
• The file was reloaded in
Word and converted to a table (table menu) using other and the character |
(shift \) to separate the columns

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• The Word file is copied into
Excel and the Data – Sort menu is used to sort by the first column
• This brings all of the same named genes together (the two highlighted lines for example)
• Duplicate genes are removed from the spreadsheet and the far right column only
(LOC_Osxxgxxxxxx) tags can be copied back to word

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• You can use the table menu to convert table to text (Paragraph Marks) to generate a list of genes
• These genes can be searched through a downloaded database using the NCBI
fastacmd (included in the BLAST download tools) or you can search them one at a time using a web-based database such as the locus search name on TIGR:
( http://www.tigr.org/td b/e2k1/osa1/LocusName
Search.shtml
)

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• Once you have found all of your sequences, check that each sequence name has a < in front of it
(denoting a new sequence name) and the sequence starts on a new line
• Copy and paste all of your sequences into an alignment program like
ClustalW (we use: http://align.genome.jp/ from the Kyoto
University Bioinformatics center, but any ClustalW program will work)

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• For our trees we use:
Slow/Accurate pair-wise comparisons and Gonnet for our Weight Matrix (two spots on the website)
• Click execute alignment to get your sequence alignment
• At the end of the alignment page will be the information needed for tree drawing programs
• You can click on clustal.dnd for a quick tree or take the information after it – A
Newick format tree and copy it into a new Word file, saving it as a text file
(include all parenthesis)

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• We use the program TreeDyn to generate our trees (available at: http://www.treedyn.org/ )
• This is an example of the
Arabidopsis and rice 1.1 family
• The tree text file was loaded into
TreeDyn and the frame enlarged
• The red text for Arabidopsis sequences was done by changing the font color to red and using the find panel to find all At* sequences (which turn red)
• The scale at the bottom was added by right clicking on that space and choosing the tree name, annotation, and scale sub-menus
• This square tree is useful to see associations of genes for different species

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• This is part of the family
1.1 square dendrogram of
Arabidopsis, rice and maize from our website
• The red names are
Arabidopsis sequence , the
black names are rice, and the green names are maize
• Regions alternate between grey shaded and white backgrounds (added with
Photoshop) to indicate clades of similar sequence genes which may relate function (such as
AUD/SUD or GME, etc)

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• TreeDyn can also draw radial dendrograms such as the one shown for rice family 1.1
• This can be done by right clicking on the tree area to bring up the grey box in TreeDyn, choosing your tree, then Conformation-
Radial
• Treedyn allows you to resize, rotate, and flip clades around (see http://www.treedyn.org/ for detailed tutorials on these processes)
• For our site, we export the radial trees as jpeg images

The TreeDyn tree jpeg is finished as a FLASH file where the ovals and family names are added (Rice family
1.1 shown)
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Each individual clade of a family tree is also prepared in TreeDyn and link buttons added later in FLASH
( AUD/SUD-like shown)

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• We provide protein sequence information you can download and add in your own sequence of interest for comparison to these three species
• Under each tree (family 1.1 shown) is the link “View the protein sequence file”
• Right click and choose Save
Target as… to download the sequence with a filename and location you will remember
• You can do this for each
Arabidopsis, rice, and maize family

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• You may have a sequence you think is related to a particular family such as nucleotide interconversion pathway (family 1.1)
• For example, the wheat
EST CV523101 from
Genebank: http://www.ncbi.nlm.nih.go
v/entrez/viewer.fcgi?db=n ucleotide&val=CV523101 might be related to the
TIGR rice gene:
Os05g29990 in the
AUD/SUD clade of family
1.1 according to information from Gramene

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• You can take the nucleotide sequence and covert it to protein sequence using a program such as Genemark:
( http://opal.biology.gatech.e
du/GeneMark/eukhmm.cgi
)
• Protein sequence returned:
>CV523101_wheat
IARIFNTYGPRMCIDDGRVVSNFVAQALR
KEPLTVYGDGKQTRSFQYVSDLVEGLMRL
MEGDHIGPFNLGNPGEFTMLELAKVVQDT
IDPNARIEFRENTQDDPHKRKPDITKAKE
QLGWEPKIALRDGLPLMVTDFRKRIFGDQ
DSAATATEG

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• Paste all of the sequences for family 1.1 (Arabidopsis, rice, and maize) plus the
Wheat EST,
CV523101_wheat, converted to protein into a
ClustalW program such as: http://align.genome.jp/ from the Kyoto University
Bioinformatics center
• Perform the multiple alignment, copy the Newick tree data generated into a new word file, and save a text file as previously shown

The AUD/SUD clade of the family 1.1 tree for
Arabidopsis (red) , Rice (black) , Maize (green) , and a wheat EST (blue) added to demonstrate how you can visualize relatedness of your own genes using our protein sequences
• Taking the Newick tree from clustalW into
TreeDyn as previously shown will allow you to visualize the tree
• The AUD/SUD clade of the tree generated by
TreeDyn shows that the wheat EST (in blue) is most closely related to the rice gene Os05g29990 in the AUD clade
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– Multiple alignment for trees, ClustalW ( http://align.genome.jp/ )
– Making trees, TreeDyn ( http://www.treedyn.org/ )
– BLASTing NCBI ( http://www.ncbi.nlm.nih.gov/BLAST/ )
– Proteins translated by GeneMark
( http://opal.biology.gatech.edu/GeneMark/eukhmm.cgi
)
– Sequence BLAST using TIGR ( http://www.tigr.org/tdb/e2k1/osa1/ )
– Downloading rice protein sequences from TIGR
( http://www.tigr.org/tdb/e2k1/osa1/LocusNameSearch.shtml
)
– Sequence BLAST using TIGR ZmGI ( http://www.tigr.org/tigrscripts/tgi/T_index.cgi?species=maize )
– Sequence BLAST using Gramene
( http://www.gramene.org/multi/blastview )
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Plant Genome Research and REU Programs