Ty1-copia retrotransposon-based S-SAP (sequence-specific amplified polymorphism) for genetic analysis of sweetpotato

Abstract

RNaseH-LTR regions of the Ty1-copia retrotransposon were amplified and cloned from the sweetpotato genome using RNaseH gene-specific degenerate primers and restriction site-specific adaptor primers. Ninety clones out of the 240 sequenced were identified with a variable degree of homology to the Ty1-copia RNaseH gene. Three ( Str6, Str85, Str187) of the 90 had characteristic RNaseH-gene, stop codon, polypurine track and putative 3' LTR sequence elements. Analysis of nine selected genotypes representing Africa, South and Central America, as well as Papua New Guinea, by the established S-SAP technique revealed that the majority of the Ty1-copia transposon insertions were unique (33 to 64%) and only few common bands were detected. Analysis of 177 East African varieties further supported this finding and showed that most of the copia retrotransposon locations were represented only by some genotypes. Considering that sweetpotato has been present in the East African region for only about 500 years, and the number of genotypes introduced was possibly limited, a surprisingly high level of genetic variability of the transposon insertion sites was detected. These findings may indicate the putative activity of the retrotransposon in sweetpotato in the recent past. Comparison of the copia retrotransposon insertion-based S-SAP method to AFLP and RAPD showed that the majority of the markers were more polymorphic (97-99%) in the case of S-SAP in comparison to AFLP (70-90%) and RAPD (88%). Thus demonstrating the transposon-based molecular marker system was very efficient for genotyping.

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

“…S-SAP, a technique of revealing the genetic distribution of retrotransposons in a genome, is a combination of the general principle of amplified fragment length polymorphism (AFLP) and sequence-specific polymerase chain reaction (PCR). Compared with conventional molecular marker systems such as AFLP, random amplified polymorphic DNA (RAPD), intersimple sequence repeat (ISSR), and simple sequence repeat (SSR), the S-SAP method produces a higher density of polymorphisms, a more even distribution across the genome, and a better quality of amplification products Berenyi et al 2002;Syed et al 2005;Tam et al 2005;Lou and Chen 2007). Furthermore, it is worth mentioning that S-SAP is effective for identifying variation among somaclones (Breto et al 2001;Tahara et al 2004;Venturi et al 2006Venturi et al , 2009, which could not be done by conventional molecular markers (Wünsch and Hormaza 2002).…”

Genetic and physical mapping of sequence-specific amplified polymorphic (SSAP) markers on the 1RS chromosome arm of rye in a wheat background

“…Sequence-specific amplification polymorphism has been demonstrated to be a potent marker system in several plant species (Waugh et al 1997;Ellis et al 1998;Gribbon et al 1999;Yu and Wise 2000;Melayah et al 2001;Berenyi et al 2002). The technique described in the present communication differs from those of previous studies in that various kinds of rye repeated sequences were used, instead of retrotransposons, to anchor the sequence-specific primers.…”

Development of retrotransposon primers and their utilization for germplasm identification in Diospyros spp. (Ebenaceae)

“…To our knowledge, it was the first report on the development of retrotransposon LTR and PPT primers through suppression PCR in this genus. The method reported herein has several advantages over previous studies on the development of retrotransposon primers Pearce et al 2000;Berenyi et al 2002;Price et al 2003;Galindo et al 2004;Bousios et al 2007). Our method is relatively cost effective, as it was done independent of specific magnetic separation equipment and biotinylated RNaseH primers.…”

“…Several retrotransposon-based molecular maker systems have been devised, such as interretrotransposon amplified polymorphism (IRAP; Kalendar et al 1999), retrotransposonmicrosatellite amplified polymorphism (REMAP; Kalendar et al 1999), and sequence-specific amplified polymorphism (SSAP; Waugh et al 1997). So far, they have been successfully employed for fingerprinting (Bretó et al 2001;Antonius-Klemola et al 2006;Venturi et al 2006), linkage analysis and mapping of agronomic traits (Manninen et al 2000;Bernet and Asíns 2003), analysis of genome evolution and genetic diversity (Ellis et al 1998;Gribbon et al 1999;Kalendar et al 2000;Pearce et al 2000;Berenyi et al 2002;Soleimani et al 2005;Sanz et al 2007;Petit et al 2007;Bousios et al 2007;Natali et al 2007;Yang et al 2007). However, information on application of retrotransposon-based markers in the genus Diospyros is limited.…”

“…Pearce et al (1999) firstly reported that a novel technique was developed for rapid isolation of plant Ty1-copia retrotransposon terminal repeat sequences for molecular marker analysis, which is a fast and effective method of developing retrotransposon primers by isolating RNaseH-LTR sequences as a prelude to primer design. Recently, similar modified methods have been successfully utilized to obtain retrotransposon primers and then developing retrotransposon molecular markers in a wide range of plant species (Pearce et al 2000;Berenyi et al 2002;Price et al 2003;Galindo et al 2004;Bousios et al 2007), whereas there is no related report on the genus Diospyros.…”