Structural characterization of Brachypodium genome and its syntenic relationship with rice and wheat.

Abstract

Brachypodium distachyon (Brachypodium) has been recently recognized as an emerging model system for both comparative and functional genomics in grass species. In this study, 55,221 repeat masked Brachypodium BAC end sequences (BES) were used for comparative analysis against the 12 rice pseudomolecules. The analysis revealed that ~26.4% of BES have significant matches with the rice genome and 82.4% of the matches were homologous to known genes. Further analysis of paired-end BES and ~1.0 Mb sequences from nine selected BACs proved to be useful in revealing conserved regions and regions that have undergone considerable genomic changes. Differential gene amplification, insertions/deletions and inversions appeared to be the common evolutionary events that caused variations of microcolinearity at different orthologous genomic regions. It was found that ~17% of genes in the two genomes are not colinear in the orthologous regions. Analysis of BAC sequences also revealed higher gene density (~9 kb/gene) and lower repeat DNA content (~13.1%) in Brachypodium when compared to the orthologous rice regions, consistent with the smaller size of the Brachypodium genome. The 119 annotated Brachypodium genes were BLASTN compared against the wheat EST database and deletion bin mapped wheat ESTs. About 77% of the genes retrieved significant matches in the EST database, while 9.2% matched to the bin mapped ESTs. In some cases, genes in single Brachypodium BACs matched to multiple ESTs that were mapped to the same deletion bins, suggesting that the Brachypodium genome will be useful for ordering wheat ESTs within the deletion bins and developing specific markers at targeted regions in the wheat genome.

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Title:
Structural characterization of Brachypodium genome and its syntenic relationship with rice and
wheat
Author:
Huo, Naxin; Vogel, John P.; Lazo, Gerard R.; You, Frank M.; Ma, Yaqin; McMahon, Stephanie;
et al.
Publication Date:
2009
Publication Info:
Postprints, Multi-Campus
Permalink:
http://escholarship.org/uc/item/7ss61705
DOI:
10.1007/s11103-009-9456-3
Abstract:
Brachypodium distachyon (Brachypodium) has been recently recognized as an emerging model
system for both comparative and functional genomics in grass species. In this study, 55,221 repeat
masked Brachypodium BAC end sequences (BES) were used for comparative analysis against
the 12 rice pseudomolecules. The analysis revealed that ~26.4% of BES have significant matches
with the rice genome and 82.4% of the matches were homologous to known genes. Further
analysis of paired-end BES and ~1.0 Mb sequences from nine selected BACs proved to be useful
in revealing conserved regions and regions that have undergone considerable genomic changes.
Differential gene amplification, insertions/deletions and inversions appeared to be the common
evolutionary events that caused variations of microcolinearity at different orthologous genomic
regions. It was found that ~17% of genes in the two genomes are not colinear in the orthologous
regions. Analysis of BAC sequences also revealed higher gene density (~9 kb/gene) and lower
repeat DNA content (~13.1%) in Brachypodium when compared to the orthologous rice regions,
consistent with the smaller size of the Brachypodium genome. The 119 annotated Brachypodium
genes were BLASTN compared against the wheat EST database and deletion bin mapped wheat
ESTs. About 77% of the genes retrieved significant matches in the EST database, while 9.2%
matched to the bin mapped ESTs. In some cases, genes in single Brachypodium BACs matched
to multiple ESTs that were mapped to the same deletion bins, suggesting that the Brachypodium
genome will be useful for ordering wheat ESTs within the deletion bins and developing specific
markers at targeted regions in the wheat genome.

Page 2

Structural characterization of Brachypodium genome
and its syntenic relationship with rice and wheat
Naxin Huo Æ Æ John P. Vogel Æ Æ Gerard R. Lazo Æ Æ Frank M. You Æ Æ
Yaqin Ma Æ Æ Stephanie McMahon Æ Æ Jan Dvorak Æ Æ Olin D. Anderson Æ Æ
Ming-Cheng Luo Æ Æ Yong Q. Gu
Received: 1 July 2008/Accepted: 7 January 2009/Published online: 29 January 2009
? The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract
been recently recognized as an emerging model system for
both comparative and functional genomics in grass species.
In this study, 55,221 repeat masked Brachypodium BAC
end sequences (BES) were used for comparative analysis
against the 12 rice pseudomolecules. The analysis revealed
that *26.4% of BES have significant matches with the rice
genome and 82.4% of the matches were homologous to
known genes. Further analysis of paired-end BES and
*1.0 Mb sequences from nine selected BACs proved to be
useful in revealing conserved regions and regions that have
undergone considerable genomic changes. Differential
gene amplification, insertions/deletions and inversions
appeared to be the common evolutionary events that caused
variations of microcolinearity at different orthologous
genomic regions. It was found that *17% of genes in the
two genomes are not colinear in the orthologous regions.
Analysis of BAC sequences also revealed higher gene
Brachypodium distachyon (Brachypodium) has
density (*9 kb/gene) and lower repeat DNA content
(*13.1%) in Brachypodium when compared to the
orthologous rice regions, consistent with the smaller size of
the Brachypodium genome. The 119 annotated Brachypo-
dium genes were BLASTN compared against the wheat
EST database and deletion bin mapped wheat ESTs. About
77% of the genes retrieved significant matches in the EST
database, while 9.2% matched to the bin mapped ESTs. In
some cases, genes in single Brachypodium BACs matched
to multiple ESTs that were mapped to the same deletion
bins, suggesting that the Brachypodium genome will be
useful for ordering wheat ESTs within the deletion bins and
developing specific markers at targeted regions in the
wheat genome.
Keywords
Comparative genomics ? Gene density ? Colinearity ?
Repetitive DNA elements ? Genome evolution
Brachypodium distachyon ?
Introduction
The large grass family (Poaceae), which includes major
important cereals such as rice, wheat, and maize, encom-
passesover 10,000 species
comparative genetic mapping based on RFLP markers has
revealed considerable synteny between grass species,
despite the great variation in genome size and evolutionary
divergence times up to 60 million years (Moore et al. 1995;
Gale and Devos 1998; Keller and Feuillet 2000). Because
of the syntenic relationship among grass species, it is
expected that the knowledge gained from an ideal model
grass species will greatly facilitate the study of other
important cereal crops. Rice was logically chosen as one
model system for cereal crop genomics owing to its small
(Kellogg 2001).Early
Electronic supplementary material
article (doi:10.1007/s11103-009-9456-3) contains supplementary
material, which is available to authorized users.
The online version of this
N. Huo ? J. P. Vogel ? G. R. Lazo ? F. M. You ? S. McMahon ?
O. D. Anderson ? Y. Q. Gu
Genomics and Gene Discovery Research Unit, USDA-ARS,
Western Regional Research Center, 800 Buchanan Street,
Albany, CA 94710, USA
N. Huo ? F. M. You ? Y. Ma ? J. Dvorak ? M.-C. Luo
Department of Plant Sciences, University of California, Davis,
CA 95616, USA
Y. Q. Gu (&)
U.S. Department of Agriculture, Agriculture Research Service,
Western Regional Research Center, 800 Buchanan Street,
Albany, CA 94710, U.S.A
e-mail: Yong.Gu@ars.usda.gov
123
Plant Mol Biol (2009) 70:47–61
DOI 10.1007/s11103-009-9456-3

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genome size and importance as a staple crop (Goff 1999;
Shimamoto and Kyozuka 2002; IRGSP 2005). The com-
pletion of the rice genome sequence has fueled its use in
comparative genomics to understand the evolution of grass
genomes as well as in map-based cloning of important
genes in other cereal crops such as wheat (Song et al. 2002;
Ling et al. 2003; Lai et al. 2004; Yan et al. 2004; Yan et al.
2006; Bruggmann et al. 2006; Wei et al. 2007).
Despite their close relationship, grass genomes are
evolutionarily labile for many characteristics including
chromosome number, ploidy, and genome size. In addition,
common sequence changes such as insertion, deletion,
duplication, and translocation can further complicate the
use of many regions of the rice genome for cross-species
comparison with other grasses (Sorrells et al. 2003;
Bruggmann et al. 2006). Therefore, comparative analysis
using more than two grass genomes could allow for elu-
cidation of the nature of sequence changes that occurred in
specific lineages during the evolutionary history of grass
species (Song et al. 2002; Lai et al. 2004; Wei et al. 2007;
Salse et al. 2008). For genome studies in Triticeae species
including wheat and barley, a more closely related model
grass genome will serve as a better intermediate for com-
parative and functional analysis.
The genus Brachypodium belongs to the Brachypodieae
tribe, which is sister group to the four major cool season
grass tribes of great economic importance—Triticeae,
Aveneae, Poeae, and Bromeae (Draper et al. 2001; Kellogg
2001; Vogel et al. 2006). Hence, the Brachypodium gen-
ome is expected to show greater gene colinearity to the
genomes of major cool season cereal grain and forage
grasses and will be more useful in gene discovery in large
Triticeae genomes such as wheat and barley (Garvin et al.
2008; Opanowicz et al. 2008; Ozdemir et al. 2008). In
addition, the annual species in the genus Brachypodium
possess a range of desirable biological features that make it
well suited for functional genomics. For example, diploid
Brachypodium has one of the smallest genomes in grasses
(*300 Mb). The plant also has small physical stature,
short life cycle, and undemanding growth requirements—
all of which make it amenable to high-throughput genetic
screening. Therefore, Brachypodium could serve as a util-
ity model system for various types of plant research
(Beckmann et al. 2008; Li et al. 2008; Idziak and Hasterok
2008; Parker et al. 2008; Spielmeyer et al. 2008).
Despite the growing interest in utilization of Brachypo-
diumasamodelgrass,littleisstillknownaboutthestructure
andorganizationoftheBrachypodiumgenome.Theutilityof
Brachypodium for grass crop genomics remains to be thor-
oughly tested. So far, comparative analysis with rice and
wheat have been only conducted in a few genetic regions
from a perennial, outbreeding species B. sylvativum (Foote
et al. 2004; Griffiths et al. 2006; Bossolini et al. 2007; Faris
et al. 2008; Spielmeyer et al. 2008), which has a genome
reported to be *470 Mb (Foote et al. 2004) with a size
similar to that of rice, but larger than that of B. distachyon.
B. sylvaticum has proved useful in mapping the wheat Ph1
gene(Griffithsetal.2006).Itwasshownthattheorthologous
sequences from B. sylvaticum are more colinear to wheat as
compared to those of rice. B. sylvaticum and wheat shared
such high sequence identity that probes derived from
B. sylvaticum sequences can be directly used for wheat
mapping (Griffiths et al. 2006). Comparative studies on the
wheat Lr34 region also indicated that B. sylvaticum and
wheat showed perfect macro-colinearity of genetic markers,
whereas rice contained a *200-kb inversion in the orthol-
ogous region (Bossolini et al. 2007). It was estimated that
B. sylvaticum and wheat diverged 35–40 Mya, significantly
more recently than the divergence of rice and wheat
(Bossolini et al. 2007). As far as we know, comparative
analysis using B. distachyon genome has not been reported.
A genome study on B. distachyon will provide important
insightsintothegene distributionandevolutionofrepetitive
DNA in a compact grass genome.
Previously, we sequenced *65,000 BAC ends from
Brachypodium BAC libraries to generate *38 Mb of
random short genomic sequence, representing 10% of the
Brachypodium genome (Huo et al. 2007). Analysis of BES
revealed low repetitive DNA content and close phyloge-
netic relationship with the Triticeae species. In this study,
we compared the BES against the rice genome to assess
sequence conservation between these two compact grass
genomes. To further analyze the colinearity of the orthol-
ogous regions, we sequenced nine Brachypodium BAC
clones selected on basis of BAC-end matches to the rice
genome. Our study provides the first comparison of the
Brachypodium and rice genomes at multiple genetic loci.
To evaluate the utility of Brachypodium for cereal crops
with large genomes, the annotated Brachypodium genes
were BLASTN searched against the wheat EST database
and deletion-bin mapped wheat ESTs. Comparative anal-
ysis using the Brachypodium genome could offer a
potentially useful strategy for the development of wheat
genetic and linkage maps.
Materials and methods
Blast search of Brachypodium BES against the rice
genome
To anchor Brachypodium BACs onto the rice genome, the
55,221 repeat masked BES were compared to the rice
genome sequence using BLASTN. An expectation value
(E) of e-10was used as the significant threshold (www.
tigr.org). The BES were assigned to individual rice
48Plant Mol Biol (2009) 70:47–61
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chromosomes based on their best match to the rice genome.
BES matching the rice genome were also analyzed by
BLASTX against NCBI non-redundant protein database
(http://www.ncbi.nlm.nih.gov/BLAST).
Sequencing of Brachypodium BAC clones
and sequence assembling
BAC DNA was isolated with the Qiagen Large Construct
Kit (Qiagen, Valencia, CA) and shotgun libraries were
constructed as described previously (Gu et al. 2003). In
brief, the purified DNA was sheared and size selected by
agarose gel electrophoresis. Fragments with sizes between
3 and 5 kb were ligated into the pCR4TOPO vector using a
TOPO cloning kit (Invitrogen, Carlsbad, CA). The ligation
mixture was transformed into electrocompetent TOP10
cells. Plasmid DNA was isolated using the PerfectPrep
Direct Bind Kit (Eppendorf, Boulder, CO). For each BAC,
768 subclones were sequenced from both ends using T7
and T3 primers and BigDye terminator chemistry (Applied
Biosystems, Foster City, CA) on an ABI3730XL auto-
mated sequencer (Applied Biosystems). Gaps were filled
by sequencing PCR products amplified directly from the
BAC clones.
The BAC sequences were assembled using the Laser-
gene SeqMan Module (DNAStar, www.DNAStar.com) as
described previously (Gu et al. 2003). In this module, we
set the stringency for base calling and quality assessment to
‘‘high’’ to generate the most accurate consensus sequence
possible. The sequence assembling was performed using a
40-bp window size and a 95% match requirement.
Annotation of Brachypodium repetitive elements
To define the Brachypodium repetitive element, a survey of
the composition and contents of the Brachypodium repeat
element sequences in the sequenced BACs was conducted
usingthe RepeatMasker
masker.org). The BAC sequences were also searched
using BLASTN against the Triticeae Repeat Sequence
(TREP) database (http://wheat.pw.usda.gov/ITMI/Repeats)
and the local BLAST database containing unique Brac-
hypodium repeat element sequences (Huo et al. 2007).
program(http://www.repeat
Gene annotation
To annotate coding sequences, a combination of BLASTN
and BLASTX against non-redundant nucleotide and pro-
tein databases were utilized to identify all putative gene
sequences. In addition, coding regions in the BAC
sequences were also predicted using FGENESH (http://
www.softberry.com) set for a monocot model. Predicted
genes were then compared to the nonredundant and dbEST
databases of NCBI (March 2008) using BLASTN and
BLASTX. If a hypothetical protein was predicted, the
sequence was searched against UniProt (Ver. 9.7) of
European Bioinformatics Institute database (March 2008).
Only matches with E values smaller than e-10were
accepted. The complete annotation of each sequenced BAC
clone has been submitted to GenBank (Accession numbers
EU730894-EU730902).
Brachypodium sequence comparison with rice
and wheat
Orthologous rice sequences and annotations in VISTA
format were downloaded from Gramene (http://www.
gramene.org) and TIGR Rice Genome Browser (http://
www.tigr.org). The orthologous rice sequences were
re-annotated with the same criteria used to annotate the
Brachypodium BACs. For comparative analysis between
rice and Brachypodium, the rice CDS annotated in
orthologous regions were aligned with the Brachypodium
sequences to identify genes that were colinear. Sequence
alignment analysis was performed using the VISTA pro-
gram (Mayor et al. 2000). To compare Brachypodium and
wheat, the annotated Brachypodium genes were compared
tothe deletionbin mapped
(http://wheat.pw.usda.gov/wEST/) using BLASTN. Brac-
hypodium genes were also compared to NCBI wheat and
rice EST databases using BLASTN.
wheat ESTdatabase
Results
Anchoring Brachypodium BES onto the rice genome
Analysis of *65,000 Brachypodium BES revealed its
relatively simple genome with low repetitive DNA content
and high gene density (Huo et al. 2007). To provide an
initial genome-wide comparison of rice and Brachypodium,
both with compact genomes, we attempted to anchor
Brachypodium BES onto the rice genome. Since the coding
sequences of transposable elements (TE) often identify
multiple noncolinear matches between the two grass spe-
cies, it was critical to use repeat-masked BES in the
analysis. Our BLASTN analysis showed that 14,547 out of
55,221 repeat-masked BES (26.3%) had matches to the rice
genome sequence (E\e-10) at the nucleotide level. Fur-
ther analysis using BLASTX showed that 11,982 (82.4%)
out of these 14,547 BES matched to known protein-coding
genes at E\e-10. The nature of the remaining 17.4% of
the matches is unclear, however, some of these sequences
could represent conserved noncoding sequences as previ-
ously suggested (Bossolini et al. 2007).
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The matched BES were plotted along the 12 rice chro-
mosomes to view the distribution pattern of these
conserved sequences. The number of BES anchored to rice
Chromosome 1 (chr1), chr2, and chr3 are higher than those
aligning to the rest of the chromosomes. There were 2,018
(13.8%), 1,923 (13.2%) and 1,915 (13.2%) BES matched to
the sequences on rice chromosome 1, 2 and 3, respectively,
with a total of 40.2% of matched BES (Fig. 1a). More
matches to these chromosomes are expected since they
contain more genes than other chromosomes (Wu et al.
2002; IRGSP 2005). The number of matches to rice chr4 to
chr12 ranged from 626 (4.3%) to 1247 (8.6%) (Fig. 1a).
Among the BES, there were 1,734 BAC clones for
which the paired ends both showed significant matches to
the rice genome. Since the approximate distance between
these matches is known, these BAC clones are more
informative in comparative studies to identify putative
orthologous regions (Zhao et al. 2001, 2004). According to
the positions of the two paired-end matches in the rice
genome, these BACs can be placed into six different
classes (Fig. 1b). For Classes I to V, both ends of the BAC
clone matched rice sequences on the same chromosome.
The distance separating the two ends ranged from less than
50 kb to over 1 Mb. For Class VI, the best matches of the
two BAC ends to the rice sequences are on different
chromosomes. The result showed that the number of BAC
clones in each category was 116 (6.7%), 734 (42.3%), 58
(3.3%), 20 (1.2%), 153 (8.8%) and 653 (37.7%), respec-
tively (Fig. 1b). We randomly selected 10 BAC clones
from each category for determination of BAC insert size by
CHEF gel electrophoresis. All 10 BAC clones in Class I
contained an insert smaller than 50 kb, suggesting that the
small distances between two matches in the rice genome
reflected the small insert size in Brachypodium BAC
clones. The BAC clones in other classes all had an insert
with sizes ranging from 80 to 170 kb (data not shown),
suggesting that most of the large size difference between a
Brachypodium BAC and the orthologous rice region was
the result of genomic changes that have occurred since the
divergence of the two grass genomes.
BAC selection for sequencing
Considering the small insert size of Class I BACs, it is
likely that most of the BACs in this category identified a
colinear rice region with no major sequence rearrange-
ments. This might be also true for many BACs in the Class
II category, although some of the size difference between
the Class II BAC insert and the corresponding rice region
could be explained by the lower amount of repetitive DNA
content in the smaller genome of Brachypodium (Huo et al.
2007). However, the large size differences observed in
Class III, IV and V BACs and the scenario in Class VI
BACs are likely due to substantial sequence difference
between the two genomes. We examined the possibility
that the two BAC ends in Class VI might match to the
duplicated regions on two different rice chromosomes.
23.3% of these BACs showed that the two ends matched to
two different regions originated from ancient genome
duplication or chromosomal duplications in rice (Yu et al.
2005).
Dynamic sequence changes such as insertion, deletion,
inversion, and translocation, coupled with rapid but dif-
ferential repetitive DNA amplification and removal, are
major evolutionary forces constantly reshaping individual
plant genomes (SanMiguel et al. 1996; Vicient et al. 1999;
Fu et al. 2002; Jiang et al. 2004; Kazazian 2004; Ma et al.
2004). In order to gain a more detailed view of the
sequence changes in the regions represented by these
paired BAC clones, we sequenced one or two BAC clones
Rice chromosome
No. of BES matches
0
Chr1
500
1000
1500
2000
2500
(A)
(B)
Chr2
Chr3
Chr4
Chr5
Chr6
Chr7
Chr8
Chr9
Chr10
Chr11
Chr12
Match to rice genome
Match to known gene
6.7%6.7%
6.7%
3.3%3.3%
1.2%1.2%
1.2%
8.8%8.8%
37.7%37.7%
42.3%42.3%
42.3%
5.0%
10.0%
15.0%
20.0%
25.0%
30.0%
35.0%
40.0%
45.0%
Percentge of BACs
Class
0.0%
I IIIII VIV IV
3.3%
8.8%
37.7%
<50kb50-300
Kb
300-600
Kb
600-1000
Kb
>1000
Kb
Different
Chr.
Fig. 1 Alignment of Brachypodium BES onto the rice genome. a
Distribution of Brachypodium BES matched on 12 rice chromosomes.
Repeat-masked Brachypodium BES was compared against the rice
genome using BLASTN and BLASTX. The top match of each BES in
BLASTN was used to determine its assignment to the rice chromo-
some. The number of total matches to each rice chromosome was
counted and plotted. b Percentage of paired BES in each category.
1,734 BAC clones with paired ends both matching to the rice genome
were categorized based on the distance of two matches on the same
chromosome as indicated. BAC clones with two matches on different
chromosomes are classified into category VI
50Plant Mol Biol (2009) 70:47–61
123