Identification, Characterization, and Utility of MITEs in Agrostis.

Miniature Inverted-repeat Transposable Elements (MITEs) are a class of non-autonomous transposable elements characterized by flanking 2-3 bp target site duplications (TSD), 10-15 bp terminal inverted repeats (TIR), short size, and high copy number. This class of transposable elements has not been previously described in turfgrasses. The objective of this study was to identify candidate MITEs from Agrostis and assess their value as a molecular marker tool. 7529 Agrostis capillaris L. and 8535 Agrostis stolonifera L. DNA sequences, were screened using the FindMITE program to identify candidate MITE sequences containing 11 bp TIRs allowing one mismatched nucleotide, with a max size of 900 bp, and the TSDs TA, TTA, TAT, TAA, NNNN, and NNNNNNNN. The computer program mfold was used to predict the secondary structure of the candidate MITEs and these were manually searched for hairpin loop formation, a common characteristic of MITEs. Polymerase chain reaction primers internal to the MITE sequences were manually designed and labeled with the 5' 6-FAM fluorophor. MITE display was used to screen six Agrostis parent plants from three experimental mapping populations. The resulting DNA fragments were processed through an ABI310 or ABI3730 genetic analyzer (Applied Biosystems, Foster City, CA). FindMITE identified 202 MITE-like sequences, or 1.26% of the 16,064 sequences. The MITE display markers had a significantly higher polymorphism rate (0.42) between the mapping population parents than Amplified Fragment Length Polymorphism (AFLP) markers at 0.28. The MITE display protocol would be an effective tool for diversity analyses and mapping in Agrostis.