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Japanese Team Applies Sequencing to Transposon-based DNA Fingerprinting Method

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NEW YORK (GenomeWeb) – Researchers from Okayama University have come up with a sequencing-based DNA fingerprinting method that uses active retrotransposon insertion patterns to identify specific plant cultivars — an approach that can be applied to species that have not yet had their full genomes sequenced.

"[T]he low cost and time-saving DNA fingerprinting methods developed in this study could facilitate the precise identification of cultivars" and can be used to assess genetic diversity and conduct linkage analyses, senior author Makoto Tahara, an environmental and life science researcher at Okayama University, and colleagues wrote in a study published online earlier this month in DNA Research.

There, the researchers demonstrated that cultivar-specific retrotransposon insertion sites can serve as markers in DNA fingerprinting experiments. These "sequence-characterized, amplified region" (SCAR) markers appear to be present in a range of plant species and can be derived using insertion sites created by transposons from several different families.

Through targeted Illumina MiSeq sequencing on dozens of sweet potato cultivars, for example, the team uncovered almost 400 SCAR markers — polymorphisms produced when active retrotransposons from the Rtsp-1 or LIb families inserted themselves into the sweet potato genome during cultivar development.

Consistent with their cultivar-specific nature, these markers provided phylogenetic information that roughly matched known relationships between the cultivars at hand.

The ability to narrow in on cultivar-specific markers has a number of potential applications in non-model plants such as sweet potato, a hexaploid plant with a highly heterozygous genome, according to the study's authors.

While such plants await the type of genome sequencing and population resequencing studies needed to ascertain genome-wide SNP patterns, the investigators argued that SCAR marker analyses could provide a peek at breeding histories for various cultivars.

Moreover, the team noted that the approach may serve as a fast, accurate, and cost-effective way of fingerprinting plants from other reference genome-less species to narrow in on informative targets for PCR-based identity testing.

The retrotransposon-based DNA fingerprinting project, funded by a grant from Japan's Ministry of Agriculture, Forestry, and Fisheries, was motivated by an interest in finding cultivar-specific DNA fingerprinting methods that can be applied to key crop plant that have not been fully genetically characterized by other means.

In particular, the researchers hope to develop schemes for identifying DNA markers that can be used for everything from reliable food labeling and inspection of plant imports to the identification and protection of cultivars produced by plant breeders in Japan.

"The breeder's right extends to not only seed or seedling but also to the products from the cultivar," Tahara and first author Yuki Monden, also with Okayama University, told In Sequence in an email. They noted that "[m]any food products are prepared by mixing or blending different cultivars, such as wheat flour, fruit or vegetable juice, jam, tea and coffee."

For these and other applications, identifying DNA markers needs to be easy, but also cultivar-specific and sensitive enough to pick up on the presence of a given cultivar in mixtures of plant material.

"The simple and efficient test procedure is a basic requirement for on-site inspection: the customs inspection to prevent the protected cultivar products illegally produced outside of Japan from import," Monden and Tahara said. "This is likewise true for an inspection of ingredient labeling on processed food products in the marketplace."

For the most part, researchers have focused on simple sequence repeats (SSR) or short tandem repeat (STR) sequences as a means of genetically fingerprinting plants and animals, respectively.

While those multiple locus-based approaches have proven useful in a variety of settings, including human forensics, the authors of the new study noted that it's tricky to discern fingerprints for materials in complicated mixtures or contaminated samples using the SSR- and STR-based methods.

In an effort to overcome such potential limitations, they decided to focus on polymorphisms produced when retrotransposons skip across genome sequences, inserting themselves more or less at random.

Retrotransposons from most families lost their transposition abilities long ago, they explained. But some retrotransposon families remain active, making them more apt to introduce unique, new insertions during the process of plant cultivar development, Monden and Tahara explained. "In general, active retrotransposons are useful for DNA fingerprinting because this type of family shows high insertion polymorphism among modern cultivars."

In the case of sweet potato, for example, the researchers saw signs of active transposition by Rtsp-1 and LIb retrotransposons when they scrutinized the plant's RNA transcripts for previous studies.

For the new analysis, the team took a closer look at the insertion sites, sequencing Rtsp-1 and LIb insertions in leaf samples from 38 different sweet potato cultivars simultaneously on one lane of the Illumina's MiSeq.

Prior to sequencing, the team amplified each insertion site using a nested PCR method and primers designed to recognize retrotransposon and adaptor sequences, ultimately producing amplicons in the 300 to 500 base pair range.

Within the more than 4.4 million MiSeq reads they generated for the 38 sweet potato cultivars, the researchers identified almost 1,500 Rtsp-1 insertion sites and 527 LIb insertion sites (nearly 258 Rtsp-1 and 93 LIb insertions per plant, on average).

Their subsequent analyses suggested that 1,327 of the Rtsp-1 insertions and 523 of the LIb insertions were polymorphic in the plants tested. Of those, 376 Rtsp-1 or LIb insertions appeared to have shared patterns within just one of the 38 sweet potato cultivars tested.

As it turned out, a phylogenetic analysis of the plants that relied on those cultivar-specific insertions produced a tree that matched the previously documented pedigree patterns for the plants.

The researchers have already designed primers matching five of the cultivar-specific Rtsp-1 insertion sites, which are expected to prove useful for future SCAR marker studies of the sweet potato cultivars.

While the current study centered on the previously unsequenced sweet potato species, the team noted that the availability of whole-genome sequence information may complement the approach by making it possible to see how retrotransposon insertion sites relate to other polymorphisms and/or protein-coding sequences in the reference genome.

The same general strategy is expected to be applicable in a range of crop plant species, so long as active retrotransposons are detectable. In a study published online last week in the journal Genome, for example, members of the same research team used Illumina's HiSeq 2000 to catalogue active retrotransposon insertion sites across the strawberry plant genome, with an eye toward finding insertion sites that may help fingerprint key strawberry cultivars from Japan.

Once cultivar-specific insertion sites are identified for strawberry, Tahara and Monden noted, it should be possible to specifically assay for just a small number of the most informative insertion sites.

In a Journal of Biotechnology study published in mid-June, for instance, the researchers demonstrated the utility of a PCR-based approach called single tag hybridization chromatic printed array strip, or "STH chromatographic PAS," for genotyping strawberry plants at eight polymorphic FaRE1 retrotransposon insertion sites.

Tahara noted that he and his colleagues hope to commercialize a cultivar identification kit based on the STH chromatographic PAS method following additional research and validation.

In addition to their use in identifying specific plant cultivars, the researchers noted that SCAR markers could also find favor amongst those assembling complicated plant genome sequences. Their preliminary experiments suggest insertion sites identified through rapid sequencing on a platform such as the Illumina MiSeq can help cobble together genetic linkage maps for plants with complicated, polyploid genomes.

In the interest of further improving the SCAR method, the researchers are considering ways to optimize the sequencing and library construction steps. As it stands now, library construction steps can take up to three or four weeks, Tahara and Monden said, followed by up to two weeks of sequencing time using a sequencing service and a few days for data analysis.

The sequencing side of the current sweet potato analysis cost roughly $55 to $65 per sample, though the study authors noted that that price varies depending on whether researchers have a sequencing instrument available in house and the level of multiplexing used. For their part, Tahara and Monden noted that they have since tested as many as 100 SCAR samples simultaneously on a single MiSeq lane.

The approach is expected to be compatible with a range of available high-throughput sequencing platforms, according to the study's authors, though the analytical method used to pinpoint precise insertion sites may need to be modified slightly if long-read platforms are used.

Along with improving the methodology itself, the team plans to develop a database outlining the collections of retrotransposon insertion sites in plant species and cultivars as they are detected.

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