Summary of the workshop


Following is the summary of the workshop held at KSU.

Summary of the EST mapping workshop of NSF project

The first workshop on the NSF EST mapping project was held at the Wheat
Genetics Resource Center, Kansas State University from Feb. 11 to 16,  2001.
Workers from all the ten laboratories participating in the mapping effort
attended. We tried to resolve some problems that most of mapping labs are
faced with. The participants spent half a day on  C-banding and FISH
analyses. The workshop program focused on the following topics:

1. Analysis of deletion lines:

Detailed introduction of deletion lines with special emphases on checking
some deletion lines before use (see attached file ³Information for checking
deletions²) was provided.

PCR products of the probes used for checking all deletion lines at KSU were
prepared , and DNA samples were provided to each  mapping lab.

2. DNA isolation:

Every participant  isoated DNA from one tissue sample of Chinese Spring.
Everyone was able to obtain DNA in large quantity and of high quality (see
attached file ³DNA test²).

3. Comparison of DNA samples from different Labs:

Twenty micrograms DNA of twenty-eight  samples, three from each lab, were
digested with EcoRI, and run in the gel of 0.8% agarose. Most of DNA samples
were okay for quality and quantity (see attached file ³DNA digestion²).

4. Southern blotting:

The procedure included DNA digestion, loading DNA samples, heating lambda
DNA of size marker, gel running, and southern blotting. The DNA samples in
the each lane of the gel should have similar amount (see attached file ³KSU
DNA samples²). In order to avoid missing data, it is better to have  all
deletion and ditelo lines selected in the gel.

5. Southern hybridization:

Eight hybridization, separating two groups, was set up with  two EST probes.
Each southern had four filters from a mapping lab and one control filter of
fifth gel from KSU. All of control filters worked very well (see attached
files ³image 1-8²). Most of the  filters from different labs also gave good
results.

A common problem is missing DNA samples in the blots (see attached files
³image 1-8²). In the blots of UC-Davis and Cornell, problem line DNA was not
loaded but it should be stressed that even problem deletion DNA is
informative as some of them have secondary deletions. There are many missing
samples in the blots of other labs partly caused by DNA degradation. Blots
of UNL-Nebraska used HindIII; we all must use EcoR1. As mentioned before, in
order to get more information, all deletion, NT, and Dt lines selected for
EST mapping should be in the blots. We recommend making new blots for the
labs with missing lanes or lanes with degraded DNA.

We are keeping all of autorads in our lab, and are sending images and
stripped filters back to each lab.

6. Scoring data from autorads:

      Nine participants were divided into three groups and each group scored
sixteen sets of autorads as time permitted. These autorads involved in seven
homoeologous groups with southern patterns from three to eleven bands. On
the last day , we compared the data and discussed potential problems and
fine points of scoring data.


7. Submission of image and scoring data

Based on detail discussion, submission of image and scoring data was
standardized. Gerry Lazo will create the templates for image and scoring
data, and Shiaoman Chao will give  guidelines for data entry in the Excel
file.

PS: Please go over the information carefully and compare your results of
autorads with KSU 5th gel standard and with others and make improvements
accordingly.

B.S. Gill and Lili Qi