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Cross-amplification of EST-derived markers among 16 grass species
Authors:M. Zeid  J.K. Yu  I. Goldowitz  M.E. Denton  Denise E. Costich  C.T. Jayasuriya  M. Saha  R. Elshire  D. Benscher  F. Breseghello  J. Munkvold  R.K. Varshney  G. Belay  M.E. Sorrells
Affiliation:1. Dept. of Plant Breeding and Genetics, Cornell University, Ithaca, NY 14853, USA;2. Syngenta Seeds Inc., 317 330th Street, Stanton, MN 55018, USA;3. Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA;4. Institute for Genomic Diversity, Cornell University, Ithaca, NY 14853, USA;5. USDA-ARS, Robert W. Holley Center for Agriculture and Health, Ithaca, NY, USA;6. Embrapa Rice and Beans, P.O. Box 179, Santo Antônio de Goiás, GO 75375-000, Brazil;g Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853, USA;h International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, India;i Ethiopian Institute of Agricultural Research, Debre Zeit Agricultural Research Center, P.O. Box 32, Debre Zeit, Ethiopia
Abstract:The availability of a large number of expressed sequence tags (ESTs) has facilitated the development of molecular markers in members of the grass family. As these markers are derived from coding sequences, cross-species amplification and transferability is higher than for markers designed from genomic DNA sequences. In this study, 919 EST-based primers developed from seven grass species were assessed for their amplification across a diverse panel of 16 grass species including cereal, turf and forage crops. Out of the 919 primers tested, 89 successfully amplified DNA from one or more species and 340 primers generated PCR amplicons from at least half of the species in the panel. Only 5.2% of the primers tested produced clear amplicons in all 16 species. The majority of the primers (66.9%) were developed from tall fescue and rice and these two species showed amplification rate of 41.6% and 19.0% across the panel, respectively. The highest amplification rate was found for conserved-intron scanning primers (CISP) developed from pearl millet (91%) and sorghum (75%) EST sequences that aligned to rice sequences. The primers with successful amplification identified in this study showed promise in other grass species as demonstrated in differentiating a set of 13 clones of reed canary grass, a species for which very little genomic research has been done. Sequences from the amplified PCR fragments indicated the potential for the transferable CISP markers for comparative mapping purposes. These primer sets can be immediately used for within and across species mapping and will be especially useful for minor grass species with few or no available molecular markers.
Keywords:Poaceae   Expressed sequence tag (EST)   EST-SSR   CISP   Comparative mapping   Homology   Sequence conservation
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