Cross-amplification of EST-derived markers among 16 grass species

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

A strain of soybean mosaic virus infecting Passiflora spp. in Colombia

Viruses causing severe mosaic, epinasty, defoliation, and premature death of Passiflora spp. were isolated from west central Colombia. Western blot analyses indicated the presence of potyviruses. Two Colombian isolates (COL-22 and -GR) were compared to known potyviruses as to host range, symptomatology, serological activity, amino acid and nucleotide sequence similarity in the coat protein and 3' noncoding region (3'NCR), and reactivity in dot-blot hybridization. Host range and symptomatology of COL-22 and -GR indicated they are similar to soybean mosaic virus (SMV). Also, both isolates reacted strongly with SMV antisera in immunodiffusion plates. Aphid transmission by Aphis gossypii and Toxoptera citricida was confirmed, but seed transmission was not demonstrated. In addition to COL-22 and -GR, five other Colombian isolates were cloned and sequenced. The deduced coat protein amino acid sequences for all of the Colombian isolates were virtually identical and shared a 98% similarity with SMV. Dot-blot hybridization experiments, using DNA complementary to the 3'NCR of the virus as a probe, further confirmed that the potyvirus infecting Passiflora spp. in Colombia is a strain of SMV. To our knowledge, infection of Passiflora spp. by a strain of SMV has not been previously reported

Cross-amplification of EST-derived markers among 16 grass species

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