4
Genome Sequencing
Shusei Sato and Stig U. Andersen
Abstract
The currentLotus japonicusreference genome sequence is based on a
hybrid assembly of Sanger TAC/BAC, Sanger shotgun and Illumina
shotgun sequencing data generated from the Miyakojima-MG20 acces-
sion. It covers nearly all expressedL. japonicus genes and has been
annotated mainly based on transcriptional evidence. Analysis of repetitive
sequences suggests that they are underrepresented in the reference
assembly, reflecting an enrichment of gene-rich regions in the current
assembly. Characterization of Lotus natural variation by resequencing of
L. japonicusaccessions and diploid Lotus species is currently ongoing,
facilitated by the MG20 reference sequence.4.1 Sequencing and Assembly of
Gene-rich Regions
Large scale sequencing of theLotus japonicus
genome was launched in 2001 at the Kazusa DNA
Research Institute. Miayakojima-MG20 was
chosen as the reference accession because of its
favorable features, including very earlyflowering
and robust growth under both growth chamber and
greenhouse conditions. In addition, MG20 is
polymorphic with respect to the widely used
experimental accession Gifu B-129, and offspring
from MG20 x Gifu crosses can be used to generate
genetic linkage maps, which are essential for
contig ordering and assignment to linkage groups
(chromosomes). Byflow cytometry analysis, the
genome size of MG20 was estimated to be 472 Mb
(Ito et al. 2000 ), and the preliminary genomic
sequencing revealed the presence of large amounts
of various types of repetitive sequences in theL.
japonicusgenome, estimated to make up at least
20 % of the total genome length. Aiming at a cost-
efficient characterization of gene-rich regions, two
independent approaches for focused sequencing of
gene-rich regions were implemented: Clone-by-
clone sequencing from seed points and shotgun
sequencing of selected genomic regions.
For the clone-by-clone approach, genome
libraries were constructed using mainly transfor-
mation-competent artificial chromosome (TAC)S. Sato (&)
Graduate School of Life Sciences, Tohoku
University, Katahira 2-1-1, Aoba-Ku, Sendai
980-8577, Japan
e-mail: [email protected]
S.U. Andersen
Centre for Carbohydrate Recognition and Signalling,
Department of Molecular Biology and Genetics,
Aarhus University, Gustav Wieds Vej 10,
8000 Aarhus C, Denmark
S. Tabata and J. Stougaard (eds.),The Lotus japonicus Genome, Compendium of Plant Genomes,
DOI 10.1007/978-3-662-44270-8_4,©Springer-Verlag Berlin Heidelberg 2014
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