DNA markers able to distinguish species or genera with high specificity are valuable in the identification of introgressed regions in interspecific or intergeneric hybrids. Intergeneric hybridization between the genera of Lolium and Festuca, leading to the reciprocal introgression of chromosomal segments, can produce novel forage grasses with unique combinations of characteristics. To characterize Lolium/Festuca introgressions, novel PCR-based expression sequence tag (EST) markers were developed. These markers were designed around intronic regions which show higher polymorphism than exonic regions. Intronic regions of the grass genes were predicted from the sequenced rice genome. Two hundred and nine primer sets were designed from Lolium/Festuca ESTs that showed high similarity to unique rice genes dispersed uniformly throughout the rice genome. We selected 61 of these primer sets as insertion-deletion (indel)-type markers and 82 primer sets as cleaved amplified polymorphic sequence (CAPS) markers to distinguish between Lolium perenne and Festuca pratensis. Specificity of these markers to each species was evaluated by the genotyping of four cultivars and accessions (32 individuals) of L. perenne and F. pratensis, respectively. Evaluation using specificity indices proposed in this study suggested that many indel-type markers had high species specificity to L. perenne and F. pratensis, including 15 markers completely specific to both species. Forty-nine of the CAPS markers completely distinguish between the two species at bulk level. Chromosome mapping of these markers using a Lolium/Festuca substitution line revealed syntenic relationships between Lolium/Festuca and rice largely consistent with previous reports. This intron-based marker system that shows a high level of polymorphisms between species in combination with high species specificity will consequently be a valuable tool in Festulolium breeding.
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We gratefully acknowledge Dr. M.W. Humphreys for having provided of F. pratensis ‘S215’ genomic DNA, and Ms. Mayumi Hata and Ms. Azusa Kameyama for their technical assistance. This work was supported, in part, by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Green Technology Project DM-1406) and by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.
Communicated by E. Guiderdoni.
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Tamura, K., Yonemaru, J., Hisano, H. et al. Development of intron-flanking EST markers for the Lolium/Festuca complex using rice genomic information. Theor Appl Genet 118, 1549–1560 (2009). https://doi.org/10.1007/s00122-009-1003-8
- Simple Sequence Repeat Marker
- Cleave Amplify Polymorphic Sequence
- Polymerase Chain Reaction Fragment
- Indel Marker
- Intergeneric Hybrid