Articles
RETROTRANSPOSON BASED MARKERS TO CHARACTERIZE SOMATIC HYBRIDS AND ASSESS VARIATION INDUCED BY PROTOPLAST FUSION OF MONOPLOID POTATO
Article number
619_4
Pages
35 – 43
Language
English
Abstract
IRAP (Inter-Retrotransposon Amplified Polymorphism) is a PCR (polymerase chain reaction) based marker technique that uses the proximity of LTR (long terminal repeat) regions of adjacent retrotransposons to generate markers by use of outward facing primers that anneal to LTR target sequences.
The LTR regions tend to be highly conserved because they contain sequences, essential for expression.
New insertions lead to polymorphism in IRAP banding patterns.
Our primers were designed to target the LTR of potato Tst1 retrotransposons to detect head-to-head orientation.
The potato population under investigation consisted of regenerants following electrofusion of genetically distinct pairs of monoploids (2n=1x=12). Both somaclones from unfused parental protoplasts and somatic hybrids from actual fusions were generated.
The IRAP procedure led to amplification of over 78 bands, of these 2 were unique and clearly distinguished the somatic hybrids from the somaclones among the regenerated plants.
IRAP produced more bands when compared with AFLP. However, polymorphic banding patterns, which could be associated with activation of retrotransposons, were low (4%) suggesting that the Tst1 retrotransposon was inactive during the protoplast-to-plant process.
Fragment analysis revealed high sequence similarities to distantly related Solanaceae implying that this is an ancient retrotransposon.
The LTR regions tend to be highly conserved because they contain sequences, essential for expression.
New insertions lead to polymorphism in IRAP banding patterns.
Our primers were designed to target the LTR of potato Tst1 retrotransposons to detect head-to-head orientation.
The potato population under investigation consisted of regenerants following electrofusion of genetically distinct pairs of monoploids (2n=1x=12). Both somaclones from unfused parental protoplasts and somatic hybrids from actual fusions were generated.
The IRAP procedure led to amplification of over 78 bands, of these 2 were unique and clearly distinguished the somatic hybrids from the somaclones among the regenerated plants.
IRAP produced more bands when compared with AFLP. However, polymorphic banding patterns, which could be associated with activation of retrotransposons, were low (4%) suggesting that the Tst1 retrotransposon was inactive during the protoplast-to-plant process.
Fragment analysis revealed high sequence similarities to distantly related Solanaceae implying that this is an ancient retrotransposon.
Authors
G. Lightbourn, R. Veilleux
Keywords
IRAP, somaclonal variation, AFLP, LTR, Tst1, ALFexpress, fluorophore
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