Isolation of <italic>Ty1-copia-like</italic> Retrotransposon Sequences from the Apple Genome by Chromosome Walking Based on Modified SiteFinding-polymerase Chain Reaction

Abstract

Long terminal repeat (LTR) retrotransposons are powerful tools for studying genetic biodiversity, genome evolution, gene mutation, gene cloning and gene expression. The scarcity of retrotransposon sequence information restricts the development of these studies in higher plants. In the present study, 31 reverse transcriptase (RT) genes of Ty1-copia-like retrotransposons were identified from the apple genome by amplifying the RT coding region using degenerate primers. Nineteen RT genes showed extreme heterogeneity in terms of fragment size, base pair composition and open reading frame integrality. Originating from one 266 bp cloned RT gene, a 1966 bp Ty1-copia-like retrotransposon (named Tcrm1), including RT-ribonuclease H-LTR domain sequences, was achieved by chromosome walking based on modified SiteFinding-polymerase chain reaction. The comparison between Tcrm1 and other LTR retrotransposons in gene structure and sequence homology shows that Tcrm1 is the first Ty1-copia-like retrotransposon including an LTR domain in the apple genome. Dot blot analysis revealed that Tcrm1 copy number in the apple was approximately 1 x 10(3) copies per haploid genome.

Isolation of two novel complete Ty1-copia retrotransposons from apple and demonstration of use of derived S-SAP markers for distinguishing bud sports of Malus domestica cv. Fuji

“…Although previous studies have isolated partial sequences of Ty1-copia retrotransposons in the apple genome and show that they were highly heterogeneous and in high copy number (Tignon et al 2001;Zhao et al 2007;Sun et al 2008), almost no information about complete Ty1-copia retrotransposons is available in the apple literature. One objective of this study was to isolate and characterize complete Ty1-copia retrotransposons in apple.…”

“…Previously, we used improved chromosome walking based on SiteFinding-PCR to isolate 1,966 bp of RT-RNaseH-LTR domain sequence of the Ty1-copia retrotransposon Tcrm1 (GenBank accession no. DQ898280), proving that this method without enzyme restriction or adaptor ligation is simple, fast, and inexpensive (Zhao et al 2007). However, a key point pertaining to SiteFinding-PCR is the number and even distribution of 4, 5, or 6 nt sequences in the genome homologous to the 3′ end of the SiteFinder oligonucleotides (Tan et al 2005).…”

“…One sequence of 1,966 bp containing an RT-RNaseH-LTR fragment (named Tcrm1, GenBank accession no. DQ898280) was obtained previously by the modified SiteFinding-PCR method (Zhao et al 2007). …”

“…Fuji and 19 bud sports are listed in Table 1. DNA was isolated from leaf tissues using the cetyltrimethylammonium bromide method according to procedures described previously (Zhao et al 2007). The S-SAP analysis was performed with one leaf sample for each bud sport.…”

“…Sixty-four RT fragments (Sun et al 2008) and one RT-RNaseH-LTR fragment (named Tcrm1; Zhao et al 2007) were isolated, and they were present in high copy numbers. We report here the isolation and characterization of complete Ty1-copia retrotransposons in apple using a genome walking strategy based on adaptermediated nested PCR, and we demonstrate the feasibility of using high-resolution S-SAP markers based on novel retrotransposons to distinguish bud sports of the Fuji apple cultivar.…”