# Development in Rice Genome Research Based on

Authors: Takashi Matsumoto; Jianzhong Wu; Baltazar A. Antonio; Takuji Sasaki
Journal: International Journal of Plant Genomics
 (2008)

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## Abstract

Rice is one of the most important crops in the world. Although genetic improvement is a key technology for the acceleration of rice breeding, a lack of genome information had restricted efforts in molecular-based breeding until the completion of the high-quality rice genome sequence, which opened new opportunities for research in various areas of genomics. The syntenic relationship of the rice genome to other cereal genomes makes the rice genome invaluable for understanding how cereal genomes function. Producing an accurate genome sequence is not an easy task, and it is becoming more important as sequence deviations among, and even within, species highlight functional or evolutionary implications for comparative genomics.


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## Body

## 1. Introduction

Food security is a major issue as we aspire toward sustainable development. In spite
of continuous increases in agricultural production due to the introduction of
improved crop cultivars and the wide use of affordable technologies, more than
800 million people still do not have access to sufficient food to meet their
dietary needs [1]. Cereal
crops are basic source of food for humankind, with 85% of total crop
production represented by maize, wheat, and rice. These three crops provide
more than half of the protein and energy required for daily life. However,
increase of world agricultural production in 2006 was less than 1%, which was
due to decrease in cereal production [2]. On the other hand, the world’s
population is expected to reach 9 billion by 2050 [3]. It is therefore
necessary to provide food security to this growing population in the midst of
global environmental problems that deprive us of much arable land and
biodiversity.Worldwide transformation of agriculture
was first achieved with the Green Revolution, which led to significant
increases in agricultural production. It began in the 1940s with the
cultivation of a high-yielding dwarf wheat cultivar with resistance to pests
and diseases. The Green Revolution for rice in the 1960s, based on the cultivar
IR8, also dramatically increased rice production and helped food production to
keep pace with population growth.Now, the second Green
Revolution, which will be based on genomics, is expected to pave the way for
the leap in crop production. The availability of the rice genome sequence
allowed the development of innovative approaches to increasing production. In the last 10 years, the basic syntenic relationships in
gene content and gene order within the grass family have been established
[4–6]. Therefore,
the rice genome could be used as a reference genome for understanding the
evolution of cereal crops and could provide a basis for their improvement [7, 8].Among plants, only the Arabidopsis [9] and rice [10] genome sequences have been
completed so far. A positionally confirmed, quality-validated genome sequence
is obligatory required as a reference for the efficient use of sequence
information, particularly in comparative analysis. Hence, the genome sequence
derived from Oryza sativa ssp. japonica cv. Nipponbare has been recognized as a gold
standard for understanding the genetics and biology of rice at the molecular
level and in the breeding and genetic manipulation of cereal crops.This
chapter presents a past history of the rice genome sequencing efforts and a
present endeavor for analysis of the genome sequence to clarify its structure
and function. Approach to the “difficult” regions whose functions are the
maintenance and regulation of chromosomes—notably the
centromeres and telomeres—is described.
Application of the new sequencing technology toward comparative studies among
genusOryza is also described in the
context of the rice genome as a reference.


## 2. Genome Sequencing through International Collaboration

The
International Rice Genome Sequencing Project (IRGSP) was established in 1997.
The 10 member countries agreed to sequence a standard rice cultivar
(Nipponbare), to use common resources, and to share sequencing of the 12 rice
chromosomes by using a map-based clone-by-clone strategy
(http://rgp.dna.affrc.go.jp/E/IRGSP/index.html). For construction of
sequence-ready physical maps, two complementary approaches were used. The Rice
Genome Research Program (RGP) in Japan anchored the genomic clones
using expressed sequence tags/sequence-tagged sites (EST/STS) and genetic
markers from the genetic and transcript maps of rice [11, 12]. The Clemson
University Genomics Institute, the Arizona Genomics Institute, and the Arizona
Genomics Computational Laboratory used a high-throughput bacterial artificial
chromosome (BAC) fingerprint and automatic BAC contig assembly system using FPC
software [13], and anchored the assembled contigs on the rice genome by hybridization-based screening [14]. The sequence-ready physical maps generated from use of these two strategies covered more than 95% of the rice genome, and 92% to 100% of each chromosome. A total of 3453 PAC/BAC clones forming the
minimum tiling path were selected for sequencing. DNA from a BAC/PAC clone was
purified and fragmented by sonication. The ends of 2000 subclones of each clone
were sequenced with capillary sequencers and assembled using the
phred/phrap
assembler [15]. The genome sequences of each PAC/BAC clone at the
high-throughput
genomic (HTG) phase 2 category were submitted to the DNA Data Bank
of Japan (DDBJ). By December 2002, almost all the clone sequences corresponding
to the minimum tiling path were sequenced to at least HTG phase 2. As a result,
a high-quality draft sequence representing 366 Mb of the rice genome was released
in the public database [16]. Thereafter, the IRGSP continued with the arduous
task of finishing: gap-filling, improving base read quality, and resolving
misassemblies (Figure 1).Figure 1
Four steps used for the finishing process to sequence completion.In
December 2004, the high-quality map-based sequence of the rice genome at HTG
phase 3 category was completed and released in the public domain [10]. The
sequence, ca. 370 Mb in total, covered nearly 95% of the total estimated size
of the genome and about 99% of the euchromatic regions. The sequence also
included three centromeres, parts of the rDNA regions, and regions for various
transposable elements (corresponding up to 35% in the total genome).
This comprehensive, relatively accurate sequence of the rice genome, is
currently considered the gold standard.In
contrast to the hierarchical clone-by-clone strategy used by the IRGSP, a
whole-genome shotgun (WGS) sequencing strategy is widely used in many
sequencing projects [17]. In this strategy, a high-throughput computer program
to reproduce the entire genome sequence assembles millions of shotgun sequences
from the total genome. This method was used in sequencing the 2.9-Gb human
genome [18]. Two independent groups used the WGS strategy to sequence the rice
genome. The Beijing Genome Institute assembled shotgun sequences of the indica line 93-11 with 4× [19] and later 6× [20] genome coverage. A private company, Syngenta (Basel, Switzerland),  also used the WGS strategy to sequence
the Nipponbare [21]. This WGS sequence of Nipponbare was further improved by
reassembling the shotgun sequences and combining the japonica and indica (line
99-11) sequences, resulting in 433 Mb of sequence composed of 50 233
contigs of Nipponbare [20]. Nearly 99% of the rice full-length cDNAs [22] have
been localized in these latest assemblies [20] of the japonica and indica genome.The
effectiveness of the WGS sequencing strategy was compared with that of the
hierarchal clone-by-clone sequencing approach [23, 24]. Although WGS assembly
could readily provide an overview of the genome structure with a practical
level of accuracy, misassembly could result in nonhomologous, misaligned, or
duplicated coverage and some mismatches even in the genic regions. Moreover,
repeat sequences could not be properly assigned to their original positions in
the genome. In the case of rice, which has a lot of repeat sequences, WGS
sequencing is therefore not a highly reliable strategy as it creates
misassembly, particularly in duplicated regions. It is therefore important to
have a highly accurate map-based sequence, which can be obtained by the
hierarchical clone-by-clone strategy. Today projects aiming at obtaining entire
genome sequences of gramineae plants are progressing [25–29]. All the projects,
either using WGS strategy or clone-by-clone strategy, regard the completed rice
genome sequence as sequence reference in reconstruction of chromosome
sequences, emphasizing the importance of “gold standard.”


## 3. Deciphering the Genome through Annotation

Detecting
the gene-coding regions within the genome sequence is one of the most efficient
ways
to characterize the structure and function of the genome.
RGP constructed an annotation system that facilitates gene detection of the
genome sequence in a timely manner. The Rice Genome Automated Annotation System, or RiceGAAS
(http://ricegaas.dna.affrc.go.jp [30]), was designed as a fully automated
system for annotating rice genome sequences. It retrieves rice sequences from
GenBank and analyzes them with gene prediction programs such as Genscan [31]
and FgeneSH (http://www.softberry.com/berry.phtml) and with BLAST [32] for
similarity to proteins, rice ESTs, and rice full-length cDNAs to generate the
most accurate gene models on the basis of available information (Figure 2). A
similar automatic annotation pipeline was established by TIGR (http://rice.tigr.org/tdb/e2k1/osa1/data_download.shtml),
and gene models are improved with rice ESTs and transcripts [33]. Both sets of
gene models are published on the Web to accelerate gene analysis. With
increasing data on nucleic acids and proteins in the public databases, regular
re-evaluation and update of these gene models is necessary. In this respect,
one of the advantages of these full-computational approaches is that whole gene
sets can be relatively easily revised.Figure 2
RiceGAAS annotation view, showing results from application of gene prediction
software and similarity searches. Upper box: a DNA strand from left (5′) to right (3′). Lower box: from right (5′) to left (3′).RGP
has also developed a manual annotation system to facilitate curation of the
gene models by human annotators
(http://rgp.dna.affrc.go.jp/genomicdata/AnnSystem.html). This pipeline directly
takes the output generated from RiceGAAS for in-depth analysis with in-house editing tools. Each
gene model is manually edited to improve the prediction accuracy. The gene
models for each BAC or PAC clone are released to the public domain through the
DDBJ/EMBL/GenBank database. All data can be accessed through the central
database whole genome annotation (WhoGA) on our website at
http://rgp.dna.affrc.go.jp. Initially, only the six chromosomes (1, 2,
6, 7, 8, and 9) assigned to RGP were manually curated. Recently, curation of
the rest was completed, so the manual annotation of the entire genome is now
available. After removal of clone overlaps, a total of 57 724 genes were
predicted, including many hypothetical genes predicted by a single prediction
program. Among them, 24056 gene models are supported by full-length cDNAs. All
the gene models are ordered and organized in a genome browser.Apart
from these individual activities, the IRGSP conceived the establishment of the
Rice Annotation Project (RAP), a community standard annotation project, in
2004. Genes were annotated at regular jamboree-style annotation meetings to facilitate the manual
curation of all gene models in rice. The National Institute of Agrobiological
Sciences has been leading this project, collaborating with IRGSP members and
many international and Japanese laboratories. So far, three RAP meetings have
been held, at which gene models, chiefly constructed by mapping full-length
cDNAs on the latest rice genome assemblies, have been manually curated. This
collaboration confirmed 32000 curated genes, most of which have some degree of
evidence [34]. The RAP-database (RAP-DB, http://rapdb.lab.nig.ac.jp) will be
further improved with the integration of other annotation and functional
genomics data.


## 4. Uncovered Territory—Exploration of the Missing Regions

At
the time of completion of the genome in 2004, IRGSP published nearly 371 Mb of
high-quality DNA sequences, leaving about 5% of its estimated 389 Mb to be
sequenced [10]. These unsequenced genomic regions existed as 62 gaps, including
the telomeres and centromeres in all but two out of 12 chromosomes. One of the
main reasons for the presence of these gaps was that no more clones with
sequence extension into gap regions could be selected from any Nipponbare
genomic resources, including BAC and PAC libraries (both based on partial
digestion of DNA fragments) and fosmid libraries (based on physically sheared
DNA fragments), containing a total of 630 000 clones. For unknown reasons,
specific genomic regions could not be cloned or maintained by using the above
vectors in bacteria. In addition, a number of regions in the genome contain
highly repeated sequences, making it difficult to construct a correct and
complete physical map. However, analysis of sequences from these complicated
genomic regions is not futile. Researchers have reported the importance of
heterochromatic regions in silencing gene expression [35]. Cytological analysis
has been used to define the distribution of such heterochromatin along each
rice chromosome [36]. Through the IRGSP efforts, 2 of the 12 centromeres and 14
of the 24 telomeres have been completely or partly sequenced
(http://rgp.dna.affrc.go.jp/E/IRGSP/index.html). Here, we focus on both regions
because they play essential roles in chromosome maintenance or segregation.


### 4.1. Composition and Structure of Rice Centromeres

Because
of the relatively small amount of centromeric satellite DNA in rice,
significant progress has been made in genomic and molecular studies of the
structures, functions, and evolution of rice centromeres. Two centromeres,
derived from chromosomes 4 and 8, have been completely sequenced, revealing the
complicated composition and structure of the first centromeres to have been
sequenced among eukaryotes [37–39]. Repetitive
sequences occupy ~60% of the whole region (~2 Mb) of the centromere of
chromosome 8 (Cen8). The majority of
copies of the 155-bp centromeric satellite repeat CentO, totaling 68.5-kb, occur in three large clusters in the
center, separated by centromere-specific retrotransposon of rice (CRR) sequences. Numerous sequences of
other transposable elements were also found in its surrounding region. Cen8 contains an ~750-kb core domain
that binds rice CENH3, the centromere-specific H3 histone [37]. It is
surprising to find transcriptionally active genes even within the core domain
of Cen8. A similar result was found
in Cen3, where a much bigger region
(~1881 kb) has been found to have associations with CENH3 [40]. As a
chromosomal site for
kinetochore assembly that plays an
important role in the faithful segregation of sister chromatids during cell
division, the centromere has functions that are well conserved among all higher
eukaryotes. Inter- and extrachromosomal analysis of the centromeres has,
however, revealed the divergence of DNA components and organization patterns
even among closely related species. The amount of CentO satellite DNA in the centromere of individual chromosomes
varies from 60 kb to 1.9 Mb in O. sativa [41]. The number and organization of CentO clusters within the core region differ markedly between Cen4 and Cen8 in the
Nipponbare genome. Cen8 has only
three CentO tracts (clusters) with
442 copies of the 155-bp tandem repeat distributed within a 75-kb region,
whereas Cen4 has up to 18 tracts but
only 379 copies of the repeat within a 124-kb region [38, 39] (Figure 3). CentO repeats, on the other hand, are
absent from several wild rice species, such as Oryza brachyantha [42]. It would be interesting to sequence and
compare the compositional and structural changes in centromeres between
different Oryza species in the
future, since in-depth analysis of the Cen8 and Cen4 sequences has demonstrated
segmental duplication and inversion of centromeric DNA [43]. First glimpse of
this analysis was performed in sequencing the centromere region of chromosome.8  from O. brachyantha, revealing positional
shift of centromere [44].Figure 3
Structural comparisons ofCentO domains between Nipponbare chromosomes 4 and 8. Yellow ovals and red arrows
indicate the position of CentO arrays
and the direction of the 155-bp tandem repeats within each array, respectively.
Length of arrays ranges from 477 to 8571 bp in chromosome 4 and 7616 to
34589 bp in chromosome 8.Rice
is now becoming a model for centromere and heterochromatin research [38, 45, 46].
Further research will lead to insights into the evolutionary dynamics,
processes, and molecular mechanisms of plant centromeres.


### 4.2. Composition and Structure of Rice Telomeres

Like
those of centromeres, the composition and structure of telomere regions in rice
have also been analyzed. Telomeres form the ends of linear eukaryotic
chromosomes, serving as protective caps that prevent end-to-end fusion,
recombination, and degradation of chromosomal ends [47]. The telomeres of most
eukaryotes consist of an array of repeats that contain similar sequences but
vary in length. For example, telomere DNA has a conserved sequence of 5′-TTAGGG-3′ in humans and 5′-TTGGGG-3′ in Tetrahymena (a ciliate protozo) [48, 49]. The first
plant telomere DNA was isolated from Arabidopsis
thaliana and shows tandemly repeated arrays of 5′-TTTAGGG-3′ [50]. Rice telomeres consist of the same
repeat [51]. Sequencing and extensive analysis of seven rice chromosomal ends
revealed several basic features that could provide a platform for analyzing and
understanding the telomere structures and functions. All seven rice telomeres
revealed contain highly conserved TTTAGGG sequences in tandem repeats, although
deletions, insertions, and substitutions of single nucleotides or inverted
copies were found within the arrayed repeats, particularly in the region of the
junction between the telomere and subtelomere. Fluorescent in situ hybridization and
terminal
restriction fragment analyses suggest that the rice telomeres are a
bit longer than those of Arabidopsis but much shorter than those of Nicotiana
tabacum, ranging in a length from 5 to 20 kb, thus hinting at the genetic
control of telomere length in plants [52, 53]. Interestingly, variation in
telomere length is observed not only among different chromosomes, but also
between different species within Oryza;
this variation should provide useful information for future studies of telomere
evolution. Gene annotation in the 7 rice subtelomere regions (each within 500 kb) demonstrated that the genomic region adjacent to the chromosome terminus is
gene-rich (1 gene per 5.9 kb on average). Since nearly half of these annotated
genes match rice full-length cDNAs, these rice subtelomeres could be considered
to have high transcriptional activity. Recently, seven
new rice telomeres were partly sequenced, and their sequences have been
submitted to DDBJ (Table 1;
http://rgp.dna.affrc.go.jp/E/publicdata/telomere2007/index.html). Among the
above 14 chromosomal ends, the telomere and subtelomere regions on the short
arm of chromosome 9 show some specific compositional and structural features.
Sequencing and analysis of the fosmid clone OSJNOa063K24 revealed that the
telomere repeats are colocalized with the ribosomal RNA gene (rDNA) cluster
[54]. Besides the telomere-specific repeat and the long rDNA array (sized in
megabases), the content of repetitive sequences such as retrotransposons within
the 500-kb region proximal to the centromere is relatively high, suggesting
that much of the short arm of rice chromosome 9
is heterochromatic. Rice telomere reverse
transcriptase has also been isolated [55]. It will be interesting to conduct
future studies using rice as a model of telomere research, as has been done for
centromeres, especially to reveal how telomere length (shortening or
elongation) is regulated and whether the telomere repeats and structure affect
the expression of genes in the subtelomere region. The sequence resources obtained
from the telomere and centromere regions of rice chromosomes should thus
provide an unprecedented opportunity for future study, particularly to
construct an artificial chromosome for use in both molecular and applied
biology in plant science.Table 1
Mapped and sequenced rice telomeres.
Clone nameAccession no.Copies*ChrOSJNOa264G09AP008219171SOSJNOa183H18AP006851522SOSJNOa246I10AP008220692LOSJNOa070P15AP009053273SOSJNOa083A04AP009055753LOSJNOa076I12AP0090561294SOSJNOa281H13AP009057684LOSJNOa070B13AP009052535SOSJNOa230J22AP006854376LOSJNOa219C16AP008222177SOSJNOa136M23AP0082231277LOSJNOa162K02AP008224558SOSJNOa063K24AP0090511629SOSJNOa073B23AP0090546210S*Copies of telomere-specific repeats detected from the sequenced clones.


## 4.1. Composition and Structure of Rice Centromeres

Because
of the relatively small amount of centromeric satellite DNA in rice,
significant progress has been made in genomic and molecular studies of the
structures, functions, and evolution of rice centromeres. Two centromeres,
derived from chromosomes 4 and 8, have been completely sequenced, revealing the
complicated composition and structure of the first centromeres to have been
sequenced among eukaryotes [37–39]. Repetitive
sequences occupy ~60% of the whole region (~2 Mb) of the centromere of
chromosome 8 (Cen8). The majority of
copies of the 155-bp centromeric satellite repeat CentO, totaling 68.5-kb, occur in three large clusters in the
center, separated by centromere-specific retrotransposon of rice (CRR) sequences. Numerous sequences of
other transposable elements were also found in its surrounding region. Cen8 contains an ~750-kb core domain
that binds rice CENH3, the centromere-specific H3 histone [37]. It is
surprising to find transcriptionally active genes even within the core domain
of Cen8. A similar result was found
in Cen3, where a much bigger region
(~1881 kb) has been found to have associations with CENH3 [40]. As a
chromosomal site for
kinetochore assembly that plays an
important role in the faithful segregation of sister chromatids during cell
division, the centromere has functions that are well conserved among all higher
eukaryotes. Inter- and extrachromosomal analysis of the centromeres has,
however, revealed the divergence of DNA components and organization patterns
even among closely related species. The amount of CentO satellite DNA in the centromere of individual chromosomes
varies from 60 kb to 1.9 Mb in O. sativa [41]. The number and organization of CentO clusters within the core region differ markedly between Cen4 and Cen8 in the
Nipponbare genome. Cen8 has only
three CentO tracts (clusters) with
442 copies of the 155-bp tandem repeat distributed within a 75-kb region,
whereas Cen4 has up to 18 tracts but
only 379 copies of the repeat within a 124-kb region [38, 39] (Figure 3). CentO repeats, on the other hand, are
absent from several wild rice species, such as Oryza brachyantha [42]. It would be interesting to sequence and
compare the compositional and structural changes in centromeres between
different Oryza species in the
future, since in-depth analysis of the Cen8 and Cen4 sequences has demonstrated
segmental duplication and inversion of centromeric DNA [43]. First glimpse of
this analysis was performed in sequencing the centromere region of chromosome.8  from O. brachyantha, revealing positional
shift of centromere [44].Figure 3
Structural comparisons ofCentO domains between Nipponbare chromosomes 4 and 8. Yellow ovals and red arrows
indicate the position of CentO arrays
and the direction of the 155-bp tandem repeats within each array, respectively.
Length of arrays ranges from 477 to 8571 bp in chromosome 4 and 7616 to
34589 bp in chromosome 8.Rice
is now becoming a model for centromere and heterochromatin research [38, 45, 46].
Further research will lead to insights into the evolutionary dynamics,
processes, and molecular mechanisms of plant centromeres.


## 4.2. Composition and Structure of Rice Telomeres

Like
those of centromeres, the composition and structure of telomere regions in rice
have also been analyzed. Telomeres form the ends of linear eukaryotic
chromosomes, serving as protective caps that prevent end-to-end fusion,
recombination, and degradation of chromosomal ends [47]. The telomeres of most
eukaryotes consist of an array of repeats that contain similar sequences but
vary in length. For example, telomere DNA has a conserved sequence of 5′-TTAGGG-3′ in humans and 5′-TTGGGG-3′ in Tetrahymena (a ciliate protozo) [48, 49]. The first
plant telomere DNA was isolated from Arabidopsis
thaliana and shows tandemly repeated arrays of 5′-TTTAGGG-3′ [50]. Rice telomeres consist of the same
repeat [51]. Sequencing and extensive analysis of seven rice chromosomal ends
revealed several basic features that could provide a platform for analyzing and
understanding the telomere structures and functions. All seven rice telomeres
revealed contain highly conserved TTTAGGG sequences in tandem repeats, although
deletions, insertions, and substitutions of single nucleotides or inverted
copies were found within the arrayed repeats, particularly in the region of the
junction between the telomere and subtelomere. Fluorescent in situ hybridization and
terminal
restriction fragment analyses suggest that the rice telomeres are a
bit longer than those of Arabidopsis but much shorter than those of Nicotiana
tabacum, ranging in a length from 5 to 20 kb, thus hinting at the genetic
control of telomere length in plants [52, 53]. Interestingly, variation in
telomere length is observed not only among different chromosomes, but also
between different species within Oryza;
this variation should provide useful information for future studies of telomere
evolution. Gene annotation in the 7 rice subtelomere regions (each within 500 kb) demonstrated that the genomic region adjacent to the chromosome terminus is
gene-rich (1 gene per 5.9 kb on average). Since nearly half of these annotated
genes match rice full-length cDNAs, these rice subtelomeres could be considered
to have high transcriptional activity. Recently, seven
new rice telomeres were partly sequenced, and their sequences have been
submitted to DDBJ (Table 1;
http://rgp.dna.affrc.go.jp/E/publicdata/telomere2007/index.html). Among the
above 14 chromosomal ends, the telomere and subtelomere regions on the short
arm of chromosome 9 show some specific compositional and structural features.
Sequencing and analysis of the fosmid clone OSJNOa063K24 revealed that the
telomere repeats are colocalized with the ribosomal RNA gene (rDNA) cluster
[54]. Besides the telomere-specific repeat and the long rDNA array (sized in
megabases), the content of repetitive sequences such as retrotransposons within
the 500-kb region proximal to the centromere is relatively high, suggesting
that much of the short arm of rice chromosome 9
is heterochromatic. Rice telomere reverse
transcriptase has also been isolated [55]. It will be interesting to conduct
future studies using rice as a model of telomere research, as has been done for
centromeres, especially to reveal how telomere length (shortening or
elongation) is regulated and whether the telomere repeats and structure affect
the expression of genes in the subtelomere region. The sequence resources obtained
from the telomere and centromere regions of rice chromosomes should thus
provide an unprecedented opportunity for future study, particularly to
construct an artificial chromosome for use in both molecular and applied
biology in plant science.Table 1
Mapped and sequenced rice telomeres.
Clone nameAccession no.Copies*ChrOSJNOa264G09AP008219171SOSJNOa183H18AP006851522SOSJNOa246I10AP008220692LOSJNOa070P15AP009053273SOSJNOa083A04AP009055753LOSJNOa076I12AP0090561294SOSJNOa281H13AP009057684LOSJNOa070B13AP009052535SOSJNOa230J22AP006854376LOSJNOa219C16AP008222177SOSJNOa136M23AP0082231277LOSJNOa162K02AP008224558SOSJNOa063K24AP0090511629SOSJNOa073B23AP0090546210S*Copies of telomere-specific repeats detected from the sequenced clones.


## 5. Genome Sequence for Evolutionary Genomics in Rice

Rice
is believed to have been domesticated from a wild relative 0.2 Mya [56] or 0.44 Mya [57]. Asian cultivated rice (O.
sativa L.) has two subspecies, indica and japonica. Both are important as
modern crops, and there are many phenotypic variations among them, conferring
adaptation to many different environmental and cultural conditions. Crossing of
these subspecies has produced new cultivars of agricultural importance. Knowing
the differences at the molecular level would widen the capacity for rice
breeding. RGP constructed a BAC library of Kasalath, an indica cultivar, generating 78427 high-quality BAC end sequences
from 47194 BAC clones, and mapped these end sequences on Nipponbare chromosome
sequences [58]. Mapping of 12170 clones allowed the construction of 450
Kasalath BAC contigs covering 308.5 Mb. Single-nucleotide polymorphism (SNP)
frequency in the BAC end sequences and corresponding Nipponbare sequences was
0.71% on average. Sequencing of part of the Kasalath genome is in progress and
could in future elucidate the precise gene dynamics in evolution and
domestication. Results of Kasalath BAC physical maps are shown on RGP
homepage (http://rgp.dna.affrc.go.jp/E/publicdata/kasalathendmap/index.html).
Figure 4 is an example of a computer-generated Kasalath BAC physical map. BLAST
searches for Kasalath BAC-end sequence screening could be performed through
website (http://rgp.dna.affrc.go.jp/blast/runblast.html). Other approaches [59, 60] could identify positions of SNPs for high-density SNP markers.Figure 4
In silico physical map of Kasalath
chromosome 8, based on the Nipponbare sequence. Green vertical bars indicate
BAC clones (with K numbers) mapped against Nipponbare genome sequence (shown at
left with landmarks).It
had long been a mystery how Asian rice originated from its wild progenitor,Oryza rufipogon. Recently, the origin has been clarified by comparison of
retrotransposon [56], retroposon [61], chloroplast [62], and gene [63] sequences along the evolutionary lineages. These studies show evidence of
multiple independent domestications of the two major subspecies. Further
molecular studies of domestication will show how the crop and humans coevolved.The
genusOryza has 23 species [64], but
only two species (O. sativa in Asia
and O. glaberrima in Africa) are domesticated and cultivated. This fact is
remarkable given that rice grows under a wide variety of natural conditions.
Consequently, many genetic resources might be waiting to be developed. Study of
the wild relatives might
reveal new genes for hybridization, improved yield, and sustainable production.
The Oryza Map Alignment Project
(OMAP, OMAP, http://www.omap.org/index.html) of the USA and China
aims at the establishment of an experimental platform to unravel and understand
the evolution, physiology, and biochemistry of the genus. The Arizona Genomics
Institute has constructed 12
BAC libraries from the AA (the same as sativa
species) to HHKK (remote species from sativa) species genomes.
Computer-based mapping and filter hybridization screening provided high-density
cross-species physical maps [65, 66].


### 6. Impact of New Sequencing Technologies

The
genome sequences ofO. sativa and its
progenitors are expected to show extensive base substitutions and
rearrangements. Therefore, it would be difficult to reconstruct the genome
sequences of wild rice relatives from cultivars. As resequencing with the
conventional Sanger methodology can take much time and effort, a new
pyrosequencing technology was developed. Massively parallel short reads from
pyrosequencing analysis [67] could sequence more than 20 million bases with
much less cost and less time than with Sanger analysis. In collaboration with
454 Life Sciences and Roche Diagnostics, we compared pyrosequencer and Sanger
sequence data. Eight BAC clones which include OR_CBa0076I05, OR_CBa0091G05, OR_CBa0094N06,
OR_CBa0004O24, OR_CBa0063M01, OR_CBa0075G04, OR_CBa0034E23, and OR_CBa0010H05 from O. rufipogon IRGC105491 (AA species) were
chosen from a
fingerprint contig of the OMAP BAC
library (OR_CBa-FPC contig 51). This contig corresponds to an 800-kb region of
the short arm of Nipponbare chromosome 6 and is expected to contain two genes
for rice flowering
(Hd3a and RFT1). DNA of each BAC clone was purified individually and then
mixed for pyrosequencing on a GS20 genome analyzer (Roche). The output from
this analysis (ca. 20× coverage) contained 286639 reads. Of these,
169130 reads were mapped and 16123462 bases were aligned to the corresponding
Nipponbare sequences, forming 1422 mapped contigs that cover 57.5% of the
entire genomic region. The average depth was 23.39 showing deep coverage.To
compare these sequences with those from Sanger sequencing, we shotgun sequenced
a BAC clone OR_CBa0004O24 and assembled it with phred/phrap software to form
contigs. Each contig sequence from pyrosequencing was aligned to its
corresponding Sanger sequence by BLAST alignment. Statistical results from this
comparison are shown in Table2.Table 2
Sequence
comparison of BAC clone OR_CBa0004O24, Sanger versus Pyrosequencing.
Low-quality thresholdScore 30Score 40Number of alignments checked3434Total length of alignments132229132229Total HQ bases131759130639Total LQ bases4701590“In/del” type discrepancy2020– Sanger insertion, total1515– Pyro insertion, total55LQ insertion, total56– Sanger LQ33– Pyro LQ23HQ insertion, total1514– Sanger HQ insertion1212– Pyro HQ insertion32“SNP” type discrepancy6060– both HQ5447– LQ for Sanger512– LQ for Pyro11– both LQ00Discrepancy rate (%)0.04090.0359Accuracy rate (%)99.959199.9641Comparing only high quality (HQ sequence quality score >
either 30 or 40) nucleotides gave an overall error rate of 0.0409% or 0.0359%.
This means that the high-coverage reads from pyrosequencing show more than
99.95% accuracy. Researchers have pointed out that pyrosequencing is more
problematic in repeats and homopolymers than Sanger technology [68, 69], but we
did not observe this type of discrepancy. We also compared nucleotide sequences
of Hd3a (one of the rice heading date
QTL, corresponding to FT gene of Arabidopsis) between O. sativa cv.
Nipponbare (by Sanger method) and O. rufipogon (by pyrosequencing). Only
3 SNPs and no in/del
were found in exons (540 coding nt), whereas many deviations (20 SNPs, 6
indels) were found in introns; this was evolutionally reasonable. This sequence
conservation might indicate that Hd3a is functionally important and under purifying selection.These
results show that emerging new resequencing technologies (not only
pyrosequencing but also other methods [70]), when properly used in combination
with current methods, will revolutionize the cost and performance of rice
genome resequencing and will help elucidate the evolution of the Oryza genomes.


## 6. Impact of New Sequencing Technologies

The
genome sequences ofO. sativa and its
progenitors are expected to show extensive base substitutions and
rearrangements. Therefore, it would be difficult to reconstruct the genome
sequences of wild rice relatives from cultivars. As resequencing with the
conventional Sanger methodology can take much time and effort, a new
pyrosequencing technology was developed. Massively parallel short reads from
pyrosequencing analysis [67] could sequence more than 20 million bases with
much less cost and less time than with Sanger analysis. In collaboration with
454 Life Sciences and Roche Diagnostics, we compared pyrosequencer and Sanger
sequence data. Eight BAC clones which include OR_CBa0076I05, OR_CBa0091G05, OR_CBa0094N06,
OR_CBa0004O24, OR_CBa0063M01, OR_CBa0075G04, OR_CBa0034E23, and OR_CBa0010H05 from O. rufipogon IRGC105491 (AA species) were
chosen from a
fingerprint contig of the OMAP BAC
library (OR_CBa-FPC contig 51). This contig corresponds to an 800-kb region of
the short arm of Nipponbare chromosome 6 and is expected to contain two genes
for rice flowering
(Hd3a and RFT1). DNA of each BAC clone was purified individually and then
mixed for pyrosequencing on a GS20 genome analyzer (Roche). The output from
this analysis (ca. 20× coverage) contained 286639 reads. Of these,
169130 reads were mapped and 16123462 bases were aligned to the corresponding
Nipponbare sequences, forming 1422 mapped contigs that cover 57.5% of the
entire genomic region. The average depth was 23.39 showing deep coverage.To
compare these sequences with those from Sanger sequencing, we shotgun sequenced
a BAC clone OR_CBa0004O24 and assembled it with phred/phrap software to form
contigs. Each contig sequence from pyrosequencing was aligned to its
corresponding Sanger sequence by BLAST alignment. Statistical results from this
comparison are shown in Table2.Table 2
Sequence
comparison of BAC clone OR_CBa0004O24, Sanger versus Pyrosequencing.
Low-quality thresholdScore 30Score 40Number of alignments checked3434Total length of alignments132229132229Total HQ bases131759130639Total LQ bases4701590“In/del” type discrepancy2020– Sanger insertion, total1515– Pyro insertion, total55LQ insertion, total56– Sanger LQ33– Pyro LQ23HQ insertion, total1514– Sanger HQ insertion1212– Pyro HQ insertion32“SNP” type discrepancy6060– both HQ5447– LQ for Sanger512– LQ for Pyro11– both LQ00Discrepancy rate (%)0.04090.0359Accuracy rate (%)99.959199.9641Comparing only high quality (HQ sequence quality score >
either 30 or 40) nucleotides gave an overall error rate of 0.0409% or 0.0359%.
This means that the high-coverage reads from pyrosequencing show more than
99.95% accuracy. Researchers have pointed out that pyrosequencing is more
problematic in repeats and homopolymers than Sanger technology [68, 69], but we
did not observe this type of discrepancy. We also compared nucleotide sequences
of Hd3a (one of the rice heading date
QTL, corresponding to FT gene of Arabidopsis) between O. sativa cv.
Nipponbare (by Sanger method) and O. rufipogon (by pyrosequencing). Only
3 SNPs and no in/del
were found in exons (540 coding nt), whereas many deviations (20 SNPs, 6
indels) were found in introns; this was evolutionally reasonable. This sequence
conservation might indicate that Hd3a is functionally important and under purifying selection.These
results show that emerging new resequencing technologies (not only
pyrosequencing but also other methods [70]), when properly used in combination
with current methods, will revolutionize the cost and performance of rice
genome resequencing and will help elucidate the evolution of the Oryza genomes.


## 7. Conclusion

The
rice genome sequence has become available as a reference genome, providing a
basis for understanding the wide range of diversity among cultivated and wild
relatives of rice. The continuous efforts in generating a high-quality sequence
have paved the way for clarifying the structures of genomic regions that are
difficult to analyze, including centromeres and telomeres. Comparative genomics
within the genusOryza has also
become a feasible strategy for understanding the evolutionary events that led
to the development of cultivated rice. The syntenic relationships among cereal
crops must be thoroughly exploited from now on. The rice genome sequence will
be the most important tool in explaining the structure and function of other
cereal genomes, and its use may open new opportunities for researchers to look
deeper into the synteny between rice and other cereal crops, which has been
maintained for some 60 million years of evolution [6]. From a more practical aspect,
the rice genome sequence could be the key for developing rice-genomics-based research in
order to improve crop production and food security for humankind.



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*Source: 348621-2008-06-18.xml*