Author Archives: Mike

Colletotrichum genome sequencing

The genome of Colletotrichum graminicola will be sequenced in 2007.  The project is supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, (grant number 2007-35600-17829). The principal investigators of the grant are Lisa Vaillancourt (University of Kentucky), Li-Jun Ma (the Broad Institute of MIT and Harvard), Martin Dickman (Institute for Plant Genomics and Biotechnology, Texas A&M University), Jeffrey Osborn (University of Kentucky), and Michael Thon (Texas A&M University).

The objectives of the project are:

  1. Produce and release to the public sequence reads from 8X shotgun coverage of the genome of C. graminicola strain M1.001.
  2. Construct a high quality draft assembly and annotation, and make this accessible to the public via the Broad Institute’s Fungal Genomics website.
  3. An optical map and an EST resource will be developed to further validate and enhance these data. The map and the EST data will also be publicly released.
  4. Lay the groundwork for a comparative genomics resource at Texas A&M University focused on understanding the evolutionary and functional relationships among hemibiotrophy and other plant-parasitic fungal lifestyles.
  5. Develop an educational unit for Appalachian high school teachers and students that provides a solid understanding of genomics and bioinformatics and how basic genomics research contributes to the advancement of the science of agriculture.

Overview of Sequencing: Genome Seqencing: Our goal is to produce a total of ~615,000 reads representing ~8x coverage in Q20 bases and ~64x in physical coverage. Two plasmid libraries with insert sizes of 4 and 10 kb will be used to provide the majority of sequence coverage. Fosmids, which contain 40kb average inserts from randomly sheared genomic DNA, provide long-range linking information that allows large scaffold sizes to be achieved.The Broad’s assembly program, Arachne, will be used to assemble the Colletotrichum genome sequence. To provide an independent view of the genome and a measure of the quality of the genome assembly, an optical map will be commissioned from OpGen Inc. (Madison, WI, The quality of the Broad assembly will be assessed by comparing the predicted restriction fragment lengths from the assembled genome with the observed fragment lengths in the optical map. The map also provides a means to anchor sequence to the genome, adding order and orientation to the sequence contigs where there previously was none.The Calhoun whole genome analysis system will be used to identify repeat features, call genes using a variety of gene prediction tools, assign functional annotation to genes, predicts protein domains, and provides access to genome annotation data and support various analyses.EST Sequencing: EST sequences developed at the University of Kentucky will be used to train the gene prediction algorithm. We propose to sequence both ends of a total of 15,000 clones. The sequences will be collated into unigene sets with the aid of stackPACK. BLAST and conserved domain and Prosite signature motif searches will provide information about possible functional classes.

Anticipated Timeline:

Spring 2007:

  • Optical Map of the genome
  • Whole Genome Shotgun Sequencing

Fall 2007

  • Genome Assembly
  • EST Resource
  • Design, develop, and implement the educational curriculum

Spring 2008

  • Automated Annotation of the genome
  • Assess High School student participants

Fall 2008

  • Comparative Genomics web site

Colletotrichum Meeting after the Fungal Genetics Conference at Asilomar

We are pleased to announce that the Colletotrichum Workshop will be held in conjunction with the Fungal Genetics Conference at the Asilomar Conference Grounds, Pacific Grove California on Sunday, March 25 2007.

Colletotrichum Meeting Organizers:

  • Lisa Vaillancourt, University of Kentucky, USA (email)
  • Dov Prusky, Agricultural Research Organization, The Volcani Center, Israel

Tentative Schedule: (please follow this link)

Time and Place: The meeting is scheduled for 9:00 AM to 5:00 PM at the Asilomar conference center. Details will be announced during the Fungal Genetics Conference.

Housing: Since the meeting will take the whole day on March 25th, many people have elected to book a room for an extra night at the Asilomar Conference Grounds. Lynn Epstein has coordinated the booking for most people and it may be too late to add additional people. If you need to stay an extra night, please contact Lisa Vaillancourt of Lynn Epstein for more information.

Posters: We would like to compile a list of posters and talks that will be presented during the fungal genetics conference. If you are planning a presenetation involving Colletotrichum during the meeting, please send your title to Lisa Vaillancourt and we can incude it in the list.

Schedule for The Colletotrichum Meeting

Tentative Schedule for the 2007 Colletotrichum Meeting

9:00-9:30 Colletotrichum genome informational meeting: Lijun Ma, Marty Dickman, Lisa Vaillancourt, Mike Thon
9:30-9:55 Model systems: Colletotrichum higginsianum R. O’Connell
9:55-10:20 Model systems: Colletotrichum trifolii B. Dumas
10:40-11:05 Yasuyuki Kubo
11:05-11:30 Yoshitaka Takano
11:30-11:55 Saurabh Kulshrethsa
1:00-1:25 Weihua Tang
1:25-1:50 Stefan Wirsel
1:50-2:15 Yong Hwan Lee
2:15-2:40 Dov Prusky
3:00-3:25 June Simpson
3:25-3:50 Joanne Crouch
3:50-5:00 Discussion of the future of the Colletotrichum group, including taxonomy initiatives.


Protoplast Preparation

Here is a protocol for preparation of protoplasts. I first used it for C. graminicola while I was a postdoc in Lisa Vaillancourt’s lab at the University of Kentucky, but we’ve made a few modifications to it since then.

FCG LAB, 2005

1. Harvest conidia from 2 or more plates that are 3-4 weeks old, center inoculated. Expect about 1×108 conidia per plate.
2. Wash conidia once with sterile water. Resuspend the last time in Fries medium.
3. Count with hemacytometer.
4. Incubate in Fries medium for 48 hours with shaking at 30C. I use a 100 mL volume culture in a 250 mL Erlenmyer flask and shake at 150 to 175 rpm. Apparently if the culture receives too much aeration the culture will be induced to vegetative growth rather than the production of oval conidia.
5. Filter through Nytex membrane.
6. Count with a hemacytometer. Expect about 20X to 25X the number of starting falcate conidia.
7. Pellet in 50 mL centrifuge tubes in a bench-top centrifuge, 3000 rpm, 5 min at room temp. Resuspend in enzyme solution to a conidium concentration of 1.5×108/mL.
8. Incubate at 30C with slow shaking for 4-5 hours.
9. Pellet in a bench-top centrifuge, 3000 rpm, 5 min, 4C. Resuspend in 40 mL STC, ice cold. Keep the protoplasts on ice for the rest of the procedure. Count with a hemacytometer.
10. Pellet as in the previous step. Resuspend in STC medium to a concentration of 108/mL.
11. Divide into aliquots and freeze at -80C. I usually use 500 uL aliquots, which is enough for 5 transformations.

Enzyme solution
Glucanex*, 100 mg/mL dissolved in 0.7M NaCl.
Filter through a 0.2 uM filter before use. The solution will have a high solids content so only 6-7 mL of this solution can be passed through a 0.2 uM syringe filter.

*in the United States:
Novo Nordisk Biochem NA
State Road 1003 Box 576
Franklington NC 27525
Phone 800-879-6686
(note – this is no longer available directly from Novo Nordisk. Lysing Enzyme from Sigma (Catalog # L1412) is reported to be the same product.

STC 50 Ml

1.2 M Sorbitol (10.9 g)
10mM Tris (500 uL of a 1 M stock)
50mM CaCl2 (2.5 mL of a 1 M stock)
Adjust to pH 7.5