The Wheat Microsatellite Consortium

The WMC microsatellites are derived from an international collaboration involving many institutes and companies (see Acknowledgments).  The project was originated and coordinated by Peter Isaac, whilst Scientific Director at Agrogene.

Libraries

The libraries were made by Prof. Keith Edwards whilst at Long Ashton Research Station, now of the University of Bristol.  The methods have been described in detail elsewhere (Edwards et al, Biotechniques 20 758-760 (1996) "Microsatellite libraries enriched for several microsatellite sequences in plants").  The genotype used as a source of the libraries was Chinese Spring.  The two libraries used differed only in the restriction enzyme used to cut the wheat DNA.  The first library used Rsa1 cut DNA (up to microtitre plate number 16), the second used Ssp1.

Modus operandi

Each member of the WMC, upon signing a collaboration agreement and payment of a joining fee, became responsible for the sequencing of 48 clones from the enriched libraries.  Later, they would also be responsible for synthesis of primers from those sequences.  The batch of 48 clones for which each member was responsible became their property,  although the sequences were freely exploitable within the WMC.  The sequences, database information and primer sequences from this project remained confidential within the WMC until the end of December 31 2003,  except that individual members had the right to publish information on their own 48 sequences, and could publish information of sequences belonging to other groups, provided that they had written permission from that group.

The sequences were processed at Agrogene through a semi-automated database system based on Hypercard.  Sequences were processed through the following steps:-

 trimming of vector (where possible).  The cloning technique destroys the original restriction site in the wheat DNA. Where remnants of the restriction site could be identified,  the restriction site was reinstated in the sequence.

tidying up into 5 blocks of 50 per line

duplicate sequences and close relatives were identified

microsatellites were identified and picked out in italic

primers designed using Agrogene's in house software

All of the above steps were annotated in the database.  Following primer design, the primers were tested on four wheat varieties:

            Chinese Spring (library source)

            Amigo (1A1R - North American extraction)

            Brigadier (1B1R UK/European feed wheat)

            Soisson (French industrial breadmaking wheat)

In the early stages,  Amigo, Brigadier and Soisson were tested as a bulk DNA, and Chinese Spring as a single DNA.  All tests used AmpliTaq Gold enzyme,  25ul reaction volumes. Where possible from the predicted Tm of the oligos, an anneal temperature of 61C anneal was used. Failing this,  PCRs were run at 55C or 51C anneal, according to the design of the primers.  No magnesium or other optimisation was carried out. The products of the PCR were resolved on polyacrylamide sequencing gels and silver stained. Following the intial tests

            comments regarding the primers were included in the database

            the current primer lists was output as a single file

            the text of the database was output as a single file

            the database contents were output as an HTML format

            the three output formats were emailed to the members

Results

In the last report to the consortium (number 13,  23 March 2001)  the following details were provided. There were 1992 potential sequences.  Of these 230 (11.5%) did not yield sequence information, 40 (2%) did not contain inserts , 235 were duplicates of other sequences (11.8%), 503 did not contain microsatellites or small microsatellites (25.3%),  300 did not have enough room to design primers (15.1%),  111 were too difficult or complex to design primers (5.6%), primers could be designed to 573 sequences (28.8%, actually yielding 587 microsatellites as some clones contained multiple inserts for which it was possible to design two sets of primers).

Other WMC microsatellites

Another WMC project was begun at the conclusion of the  first project. This second project exploited a different library,  and included more genotypes in the original screen, as well as mapping information.  The information on these microsatellites will remain confidential  for a while yet.

Further, a third project was begun to exploit sequences in the first database, which were originally marked as being too difficult, or with insufficient room to develop primers.  This third project developed a hundred or so additional primers, which again will remain confidential.

Note on the WMC nomenclature

The initials WMC have been registered with Gary Hart for a wheat gene symbol,  and all the WMC sequences where primers have been designed have been given WMC numbers that should appear in all publications. You should note that symbols on the wheat maps must have a numerical basis following a three letter designator (WMC).  The plate reference (eg 1A1) is not an allowable nomenclature.

Other species

The WMC project was the fore-runner of several other projects on other species:-

            Pea

Grape

Tomato

            Lettuce

            Onion

            Melon

            Cucumber

Information on these other projects will also pass into the public domain after a few years.

Acknowledgments

The following is a list of collaborators in the WMC project (not listed in any particular order):-

Pierre Lacaze,  Laurie Bertrand, Patrick Cosson,  Helene Geoffroy, Vincent Wickaert, Isabelle Giraud,(Agrogene), Keith Edwards (LARS and University of Bristol), Daryl Somers (Agriculture Canada)  Michel Debrand (Limagrain/Biogemma), Thierry Ronsin (Nickerson), Pierre Devaux (Florimond Desprez), Jorge Dubcovsky (Argentina and Davis,  Ca), Robbie Waugh (SCRI), Dr. Wang (USDA), Mark Jordan and Greg Penner (Agriculture Canada), Gerard Flore ( Verneuil), Tom Blake (Montana), Robert Henry (Lismore, Australia), Steve Rogers (Monsanto), Michel Bernard (INRA, Clermont), Matthew Morell (Canberra Australia) , Peter Jack (Monsato/PBI, UK), Pierre Benoist (Benoist, France), Prof. Gupta (Meerut), Peter Sharp(Sydney), Monika Messmer  and Beat Keller (Zurich), Maria Jager-Gussen , Joerg Schondelmaier, Carsten Knaak (Saaten Union), Dr. Rouse (Novartis), Hjorstholm (SEJET), M. Khairallah (CIMMYT)),  Dr. Weibull (Svalof Weibull), Stephen Smith (Pioneer), Dr. Mohler (Munich), Dr. Jacquemin (Gembloux), Dr. Westphal (IGS), Dr. Romestant  (RAGT), Mike Gale and Pauline Stephenson (John Innes Centre),  Peter Langridge (CRC), Francois Couvreur (ITCF),  Scott Tingey and Wayne Powell (Dupont),  Jizeng  Jia (Beijing), Dr. G. Darozes (Serasem),  Pat Byrne (Colorado),  Simon Bright (Zeneca/Syngenta ), Michel Madre (Secobra),  Dr. Momont (Momont), Song Weining (Queensland) and Georges Freyssinet (Aventis)

Peter Isaac

IDna Genetics

Norwich

UK

2 June 2004