Ⅰ. HIGHLIGHTS OF THE YEARRIKEN Accel. Prog. Rep. 52 (2019)

Improved and robust method to eﬃciently deplete repetitiveelements from complex plant genomes†
H. Ichida∗1 and T. Abe∗1Wheat is one of the most important staple food crops
around the world, occupying 17% (one-sixth) of the
global crop acreage and serving as a food source forapproximately 40% of the world’s population. Despite
the availability of a high-quality reference genome sequence, whole genome re-sequencing of wheat is stilla major challenge owing to its large and complex
genome. Wheat has an estimated haploid genome sizeof 15.4–15.8 Gb, of which 84.7% is made up of repetitive sequences. To successfully apply genome sequencing to practical agricultural breeding situations, the establishment of an eﬀective strategy to eliminate highly
abundant repetitive elements within the genome is required.
Duplex-speciﬁc nuclease (DSN) provides an enzymatic method to reduce the whole-genome redundancy
and normalization. However, this method has notbeen widely used in the genome analysis of agricultural
crops owing to its sensitivity to reaction conditions anddiﬃculty in DNA recovery after the treatment. We developed an improved design for Illumina-compatible sequencing adapters that avoids duplex formation withina typical annealing temperature range (∼68◦ C) and accidental degradation by the DSN enzyme. The newly
designed adapter includes the 21 bp sequences immediately adjacent to the original TruSeq adapter andforms a hairpin-like structure by placing the necessary
adapter sequences in a complementary orientation at
the 5’ and 3’ ends of a single oligonucleotide at room
temperature during library preparation. Using this design, all necessary sequence elements for Illumina sequencing could then be added using full-length adapter
primers during the recovery PCR, which generates a library with exactly the same structure as the original
TruSeq libraries.
We compared the relative abundance of 18S and
25S rDNA elements in genomic DNA and the DNA
libraries before and after DSN treatment (DSN− andDSN+ , respectively) in rice and diploid, tetraploid,and hexaploid wheat cultivars by quantitative PCR.
In summary, the relative abundances of 18S and25S rDNA were reduced to 1.15% and 3.54% of theDSN− samples in rice. The eﬀectiveness of the DSNtreatment was essentially the same among the threewheat genomes. In particular, the relative abundanceof 18S rDNA was decreased to 7.13%, 4.95%, and4.33% considering the DSN− in diploid, tetraploid, andhexaploid cultivars, respectively. The relative abun†∗1Fig. 1. Comparison of read counts before and after duplexspecific nuclease-based repeat depletion in gene regions.
Reprinted from Plant Science, https://doi.org/10.
1016/j.plantsci.2018.10.021, with permission from
Elsevier.

dance of 25S rDNA was also decreased to 21.73%,8.96%, and 7.54% considering DSN− . These results
indicate that the DSN treatment eﬀectively depleted
repetitive sequences in diﬀerent wheat cultivars, whichhave much larger and more complex genomes compared with rice.
We evaluated the enzymatic depletion of highly
repetitive elements by DSN treatment in an actual Illumina sequencing run using the diploid wheat cultivar KU104-1, which has an estimated genome size of
6.1 Gb, as a model. The sequencing reads mappedon the regions encoding Ty1-Copia and Ty3-Gypsy
were decreased by 61.6% and 33.4%, respectively, compared with the DSN− samples. DNA-type repetitive elements were also successfully depleted by theDSN treatment. In particular, reads mapped to DNAtype transposons and simple sequence repeats (SSRs)
were respectively reduced by 17.1% and 9.0% compared with the untreated control. Therefore, we conclude that this method should be useful in a broad
range of species in which genomic approaches are notcurrently applicable owing to their large and complex
genome structures and the consequent high expenses
of sequencing and analysis.

Condensed from the article in Plant Science,
280, 455-460 (2019)
RIKEN Nishina Center- 26 -