Analysis of plant diversity with retrotransposon-based molecular markers

Abstract

Retrotransposons are both major generators of genetic diversity and tools for detecting the genomic changes associated with their activity because they create large and stable insertions in the genome. After the demonstration that retrotransposons are ubiquitous, active and abundant in plant genomes, various marker systems were developed to exploit polymorphisms in retrotransposon insertion patterns. These have found applications ranging from the mapping of genes responsible for particular traits and the management of backcrossing programs to analysis of population structure and diversity of wild species. This review provides an insight into the spectrum of retrotransposon-based marker systems developed for plant species and evaluates the contributions of retrotransposon markers to the analysis of population diversity in plants.

Figure 1

Organization of the LTR retrotransposon genome. The order of coding domains differs between the Copia and Gypsy superfamilies as shown. Retrotransposons are bounded by long terminal repeats (LTRs), which contain the transcriptional promoter and terminator (indicated diagrammatically by a bent arrow and stop sign, respectively). The resultant transcript is indicated as a hatched box between the Gypsy and Copia diagrams. The LTRs contain short inverted repeats at either end (shown as filled triangles). Reverse transcription is primed at the PBS and PPT domains, respectively, for the (−) and (+) strands of the complementary DNA (cDNA). The internal region of the retrotransposon codes for the proteins necessary for the retrotransposon life cycle and is generally divided into two open reading frames: GAG, for the capsid protein, which packages the transcript into a virus-like particle, and POL, for the other proteins. The POL contains: aspartic proteinase (AP), which cleaves the polyprotein; integrase (IN), which inserts the cDNA copy into the genome; reverse transcriptase (RT) and RNaseH (RH), which together copy the transcript into cDNA. An additional open reading frame for the envelope protein (ENV), found in some groups of Gypsy elements, is indicated. The LTRs are generally well conserved within families, and can serve for the design of primers to generate DNA footprints (Figure 2). Direct repeats in the flanking genomic DNA are generated upon retrotransposon integration: these are depicted as short, hatched arrows. The flanking genomic DNA is shown as a wavy line. The apposition of a long element bearing conserved sequences within genomic DNA of random sequence is the basis for retrotransposon marker methods.

Figure 2

Retrotransposon-based molecular marker methods. (a–c) Alternative priming sites in the genome paired with a priming site in a retrotransposon. (a) The SSAP method. Amplification is carried out from genomic DNA cut with two restriction enzymes (R1 and R2), containing a retrotransposon and ligated to an adapter (shown only for R2). Primers are indicated as arrows; the LTR generally serves as the retrotransposon priming site. (b) The IRAP method. The second priming site is also a retrotransposon. (c) The REMAP method. Amplification takes place between a microsatellite domain (labeled simple sequence repeat (SSR)) and a retrotransposon, using a primer anchored to the proximal side of the microsatellite and a retrotransposon primer. (d, e) RBIP. (d) Full sites are scored by amplification between a primer in the flanking genomic DNA (shown as a blue wavy line) and a retrotransposon primer. The single product is shown as a red bar beneath the diagram. The alternative reaction between the primers for the left and right flanks (light blue bar beneath the diagram) is inhibited in the occupied site by the length of the retrotransposon. (e) The flanking RBIP primers are able to amplify the empty site, depicted as a deep blue bar beneath the diagram, but amplification from the retrotransposon primer does not occur (missing product shown as a light red bar) because the TE insert is missing.

Figure 3

TAM fingerprinting of two RBIP markers. A total of 3263 Pisum lines were scored for the RBIP markers (a) Birte-B1 and (b) 1794-2 (Jing et al., 2005) by the TAM approach (Flavell et al., 2003; Jing et al., 2007, 2010). Each spot represents a single sample (sample locations in the array are conserved between slides) and in these two cases a red spot indicates an occupied (retrotransposon insertion present) locus and the green spot an unoccupied locus. Yellow spots indicate an individual heterozygous for the retrotransposon insertion.

Figure 4

IRAP fingerprints for Triticeae species. A barley BARE1 LTR primer (5′-GCCTCTAGGGCATAATTCCAACAC-3′) served in the reaction. (a) Hordeum vulgare cultivars and landraces: 1, Rolfi; 2, CI 9819; 3, Pallidum107; 4, Odesskij 31; 5, Odesskij 17; 6, Sonja; 7, Sultan; 8, Ingri; 9, Beka; 10, Djau Kabutak; 11, W1991; 12, 408; 13, 688; and 14, 1354. (b) Hordeum spontaneum lines: 15, T1 (Turkey); 16, T11(Turkey); 17, J31(Jordan); 18, IN68 (Iran); 19, IN80 (Iran); 20, IS112 (Israel); and 21, IS147 (Israel). (c) Other Hordeum species: 22, H. murinum ssp. glaucum; 23, H. brachyantherum ssp. californicum; 24, H. erectifolium; and 25, H. marinum ssp. gussoneanum. (d) Other Triticeae: 26, Aegilops peregrina; 27, Triticum diccocoides; 28, Triticum aestivum (cv. Bogdarka); 29, Psathyrostachys fragilis ssp. fragilis; 30, Phleum pratense; 31, Avena sativa; and 32, Secale strictum. Marker sizes in bp are indicated on the left axis.