# Brachypodium Genomics

Authors: Bahar Sogutmaz Ozdemir; Pilar Hernandez; Ertugrul Filiz; Hikmet Budak
Journal: International Journal of Plant Genomics
 (2008)

---

## Abstract

Brachypodium distachyon (L.) Beauv. is a temperate wild grass species; its morphological and genomic characteristics make it a model system when compared to many other grass species. It has a small genome, short growth cycle, self-fertility, many diploid accessions, and simple growth requirements. In addition, it is phylogenetically close to economically important crops, like wheat and barley, and several potential biofuel grasses. It exhibits agricultural traits similar to those of these target crops. For cereal genomes, it is a better model than Arabidopsis thaliana and Oryza sativa (rice), the former used as a model for all flowering plants and the latter hitherto used as model for genomes of all temperate grass species including major cereals like barley and wheat. Increasing interest in this species has resulted in the development of a series of genomics resources, including nuclear sequences and BAC/EST libraries, together with the collection and characterization of other genetic resources. It is expected that the use of this model will allow rapid advances in generation of genomics information for the improvement of all temperate crops, particularly the cereals.


---

## Body

## 1. Introduction

Brachypodium P. Beauv (from the Greek brachys “short” and podion “a little foot,” referring to its subsessile spikelets, [1]) is a genus representing some
temperate wild grass species. In particular, Brachypodium distachyon (L.) Beauv., also described as “purple false broom,” has recently emerged as a new model plant for the diverse and
economically important group of temperate grasses and herbaceous energy crops [2]. Temperate crops such as
wheat, barley, and forage grasses are the basis for the food and feed supply.
However, the size and complexity of their genomes are major barriers to genomics research and molecular breeding. Similarly, although the herbaceous energy crops (especially grasses)
are becoming novel target sources of renewable energy, very little is known
about the biological basis underlying their bioenergy traits. Therefore, there
is a growing need for a temperate grass model to address questions directly
relevant both for improving grain crops and forage grasses that are
indispensable to our food/feed production systems, and for developing grasses
into superior energy crops. The present status of genomics research conducted
in this model grass species is briefly summarized in this review.


## 2.Brachypodium Genomics as a Model System

### 2.1. Desirable Attributes

B. distachyon has many attributes that
make it a suitable model for conducting functional genomics research not only
among cereal crops like wheat and barley, but also for biofuel crops like Switchgrass
[2]. Due to its small haploid
genome (~355 Mbp) and availability of a polyploid series with a basic
chromosome number of x = 5, (2n = 2x = 10), the diploid race of B. distachyon can be used as a
model for the much larger polyploid genomes of crops such as bread wheat (16979 Mbp, 2n = 6x = 42), durum wheat (12030 Mbp, 2n = 4x = 28), and barley
(5439 Mbp, 2n = 2x = 14) (all C-values from [3]). Besides its small genome
size, other desirable attributes include a small physical stature
(approximately 20 cm), self-fertility, lack of seed-shattering, a short
lifecycle that is normally completed within 11–18 weeks depending on the
vernalization requirement [2] (might be as fast as 8 weeks
under optimized conditions, [4]), and simple growth
requirements with large planting density and easy genetic transformation [4, 5].
This combination of desirable attributes, together with the biological
similarities with its target crops, is responsible for the recent research
interest in this species. A few ecotypes of this taxon collected from diverse
geographic regions of Turkey are shown in Figure 1, indicating a high level of variation
among different accessions.TheBrachypodium distachyon lines grown under greenhouse conditions. (a) Seed heads are covered to prevent crosspollination in case it persists. (b), (c), and (d) seeds were collected from a diverse geographic region of Turkey.
(a)(b)(c)(d)Brachypodium species range from annuals
to strongly rhizomatous perennials that exhibit breeding systems ranging from strictly
inbreeding to highly self-incompatible [6].
Some of the characteristic features of the genus Brachypodium include the following [17]: (i) hairy
terminal ovary appendage, (ii) the single starch grains, (iii) the outermost
thick layer of the nucellus, (iv) the long narrow caryopsis, (v) spicate or
racemose inflorescences, and (vi) hairy nodules [7].


### 2.2. Brachypodium as a Model System: A Comparison with Arabidopsis and Oryza

The
available genome sequences of the model plantsArabidopsis [9] and rice [10] are considered to be the major
resources for plant genomics research. Nevertheless, these model species are
not suitable for the functional genomics studies of temperate grasses. Arabidopsis has all the desirable attributes for a model plant: it is small in size, grows
easily and quickly (reaching maturity in 6 weeks), has a small diploid genome,
is self-compatible and easily transformable. Its utility as a model system has
been proven by the wealth of genomic discoveries, useful for a broad range of
crops (including cereals) it has generated. However, as a dicot species, it
does not share with grass crops most of the biological features related to
agricultural traits and in this sense, rice would provide a better alternative.
The rice plant, however, does not fulfill the requirements of short size, rapid
life cycle, inbreeding reproductive strategy, simple growth requirements, or
easy transformation, thus imposing practical limitations. As a tropical
species, it does not display all agronomic traits that are relevant to
temperate grasses, especially to forage grasses; these agronomic traits include
resistance to specific pathogens, freezing tolerance, vernalization,
perenniality, injury tolerance, meristem dormancy mechanisms, mycorrhizae,
sward ecology, or postharvest biochemistry of silage [2]. Moreover, rice is
phylogenetically distant from the Pooidae subfamily that includes wheat, barley, and temperate grasses [11], whereas Brachypodium diverged from the ancestral Pooidae clade immediately prior to the radiation of the modern
“core pooids” (Triticeae, Bromeae, and Avenae), which
include majority of important temperate cereals and forage grasses [8]. Based upon cytological,
anatomical, and physiological studies, Brachypodium is placed into its
own tribe Brachypodieae of the Poaceae family [12]. In fact, the perennial
outbreeding species, B. sylvaticum (2n = 18) was considered suitable for study of archetypal grass centromere
sequences, which allowed detection of repetitive DNA sequences that are conserved
among wheat, maize, rice, and Brachypodium [13]. Several species of this
genus were studied using combined sequences of chloroplast ndhF gene and
nuclear ITS to reconstruct phylogeny among these selected species within the
genus [8]. Similarly, RFLPs and RAPDs
were used for nuclear genome analysis to establish the evolutionary position of
the genus. The genus Brachypodium constitutes
morphologically more or less closely resembling species that are native to
different ecological regions such that B. distachyon is the
Mediterranean annual, nonrhizomatous B. mexicanum is from the New World, B. pinnatum and B. sylvaticum are Eurasian, and B. rupestre is a European taxon [7]. In view of the above, B. distachyon has been proposed as an
alternative model for functional genomics of temperate grasses [2].


### 2.3.Brachypodium and the Tribe Triticeae

The tribeTriticeae Dumort belongs to the grass family, Poaceae, and
constitutes one of the economically most important plant groups. It includes
three major cereal crops, wheat, barley, and rye (belonging to the genera Triticum, Hordeum, and Secale, resp.), which are
traditionally cultivated in the temperate zone. The tribe has basic chromosome
number of seven, and contains taxa ranging from diploids (2n = 2x = 14) to
duodecaploids (2n = 12x = 84), including all intermediate
ploidy levels [14]. Polyploidy (the most
important cytogenetic process in higher plants, [15]) and more specifically,
allopolyploidy, has played and plays a main role in the tribe’s evolution. With
around 350 species, crossability barriers are poorly understood, and it is
remarkable how its species, even species in different genera, can be made to
hybridize even if they do not hybridize naturally. Therefore, the tribe also
includes man-made crops such as ×Triticosecale (triticale) and ×Tritordeum (an amphiploid of Triticum aestivum ×Hordeum chilense [16, 17] and Triticum aestivum × Leymus
arenarius [18]). It has also been possible to apply
interspecific and intergeneric hybridization to increase the genetic
variability of crops belonging to this tribe (mainly wheat, [19, 20]).As mentioned earlier, the chromosome
numbers withinB. distachyon accessions range from 10 to 30 [21], and the haploid genome size
in diploid Brachypodium (2n = 2x =
10) varies from 172 Mbp to 355 Mbp [2, 22], although the former value
may be an underestimate [22]; the genome size is thus assumed
to be approximately 355 Mbp. Therefore, within Poaceae, B. distachyon carries one of the smallest genomes, which is
intermediate between the genomes of Arabidopsis
thaliana with 157 Mbp, and rice with 490 Mbp (All C-values from [3]). These data are consistent
with previous reports that species of Brachypodium have the smallest 5S rDNA spacer among the grasses, and contain less than 15%
highly repetitive DNA [7]. Additionally, GISH analysis
of somatic chromosomes has shown preponderance of repetitive DNA in the
pericentromeric regions, reflecting the compactness and economy of this genome [12]. This study also revealed
structural uniformity of the diploid accessions ABR1 and ABR5, confirming their
status as model genotypes, from which two BAC libraries have recently been
prepared for functional genomics analysis [23]. Recent analysis of BAC end
sequences (BESs) also corroborates this unusually compact genome [24, 25]. The other accessions having
chromosome numbers in multiples of 10 suggested that this species has evolved as
a polyploid series based upon 2n = 2x = 10, and that ecotypes that deviate
from multiples of 10 evolved due to aneuploid or dysploid changes in chromosome
number [21]. A cytotaxonomic analysis of
the members of the polyploid series has revealed  hybrid origin of several of the polyploid
genotypes, suggesting a complex evolution of this species that is not entirely
based on chromosome doubling [12]. For example, allotetraploid
artificial hybrids between B. distachyon and B. sylvaticum exhibited irregular
meiosis and infertility [6], although allotetraploids
were fertile, one of them (ABR100) showed normal meiosis [12]. This indicates that either the
constituent diploids of this allotetraploid are more compatible in hybrids, or
the hybrids themselves have evolved pairing control mechanisms similar to those
of wheat and other allopolyploids. It has also been shown using GISH that the
genomes of the constituent diploids remain separated in the allotetraploid, and
that there is no recombination between homoeologous chromosomes. These features
make the natural polyploid hybrids within the genus Brachypodium a suitable material for the isolation and characterization
of diplodizing genes.For the reasons stated above,
the whole tribe Triticeae is considered to be an enormous gene pool for crop
improvement, deserving efforts not only for its morphological, physiological,
genetic, and genomic characterization, but also for the establishment of
phylogenetic relationships among different species of the tribe. The large and
complex genomes of some members of the tribe are a main constraint for genomics
research within this tribe, which would be greatly facilitated with the
availability of a suitable model species likeB. distachyon.


## 2.1. Desirable Attributes

B. distachyon has many attributes that
make it a suitable model for conducting functional genomics research not only
among cereal crops like wheat and barley, but also for biofuel crops like Switchgrass
[2]. Due to its small haploid
genome (~355 Mbp) and availability of a polyploid series with a basic
chromosome number of x = 5, (2n = 2x = 10), the diploid race of B. distachyon can be used as a
model for the much larger polyploid genomes of crops such as bread wheat (16979 Mbp, 2n = 6x = 42), durum wheat (12030 Mbp, 2n = 4x = 28), and barley
(5439 Mbp, 2n = 2x = 14) (all C-values from [3]). Besides its small genome
size, other desirable attributes include a small physical stature
(approximately 20 cm), self-fertility, lack of seed-shattering, a short
lifecycle that is normally completed within 11–18 weeks depending on the
vernalization requirement [2] (might be as fast as 8 weeks
under optimized conditions, [4]), and simple growth
requirements with large planting density and easy genetic transformation [4, 5].
This combination of desirable attributes, together with the biological
similarities with its target crops, is responsible for the recent research
interest in this species. A few ecotypes of this taxon collected from diverse
geographic regions of Turkey are shown in Figure 1, indicating a high level of variation
among different accessions.TheBrachypodium distachyon lines grown under greenhouse conditions. (a) Seed heads are covered to prevent crosspollination in case it persists. (b), (c), and (d) seeds were collected from a diverse geographic region of Turkey.
(a)(b)(c)(d)Brachypodium species range from annuals
to strongly rhizomatous perennials that exhibit breeding systems ranging from strictly
inbreeding to highly self-incompatible [6].
Some of the characteristic features of the genus Brachypodium include the following [17]: (i) hairy
terminal ovary appendage, (ii) the single starch grains, (iii) the outermost
thick layer of the nucellus, (iv) the long narrow caryopsis, (v) spicate or
racemose inflorescences, and (vi) hairy nodules [7].


## 2.2. Brachypodium as a Model System: A Comparison with Arabidopsis and Oryza

The
available genome sequences of the model plantsArabidopsis [9] and rice [10] are considered to be the major
resources for plant genomics research. Nevertheless, these model species are
not suitable for the functional genomics studies of temperate grasses. Arabidopsis has all the desirable attributes for a model plant: it is small in size, grows
easily and quickly (reaching maturity in 6 weeks), has a small diploid genome,
is self-compatible and easily transformable. Its utility as a model system has
been proven by the wealth of genomic discoveries, useful for a broad range of
crops (including cereals) it has generated. However, as a dicot species, it
does not share with grass crops most of the biological features related to
agricultural traits and in this sense, rice would provide a better alternative.
The rice plant, however, does not fulfill the requirements of short size, rapid
life cycle, inbreeding reproductive strategy, simple growth requirements, or
easy transformation, thus imposing practical limitations. As a tropical
species, it does not display all agronomic traits that are relevant to
temperate grasses, especially to forage grasses; these agronomic traits include
resistance to specific pathogens, freezing tolerance, vernalization,
perenniality, injury tolerance, meristem dormancy mechanisms, mycorrhizae,
sward ecology, or postharvest biochemistry of silage [2]. Moreover, rice is
phylogenetically distant from the Pooidae subfamily that includes wheat, barley, and temperate grasses [11], whereas Brachypodium diverged from the ancestral Pooidae clade immediately prior to the radiation of the modern
“core pooids” (Triticeae, Bromeae, and Avenae), which
include majority of important temperate cereals and forage grasses [8]. Based upon cytological,
anatomical, and physiological studies, Brachypodium is placed into its
own tribe Brachypodieae of the Poaceae family [12]. In fact, the perennial
outbreeding species, B. sylvaticum (2n = 18) was considered suitable for study of archetypal grass centromere
sequences, which allowed detection of repetitive DNA sequences that are conserved
among wheat, maize, rice, and Brachypodium [13]. Several species of this
genus were studied using combined sequences of chloroplast ndhF gene and
nuclear ITS to reconstruct phylogeny among these selected species within the
genus [8]. Similarly, RFLPs and RAPDs
were used for nuclear genome analysis to establish the evolutionary position of
the genus. The genus Brachypodium constitutes
morphologically more or less closely resembling species that are native to
different ecological regions such that B. distachyon is the
Mediterranean annual, nonrhizomatous B. mexicanum is from the New World, B. pinnatum and B. sylvaticum are Eurasian, and B. rupestre is a European taxon [7]. In view of the above, B. distachyon has been proposed as an
alternative model for functional genomics of temperate grasses [2].


## 2.3.Brachypodium and the Tribe Triticeae

The tribeTriticeae Dumort belongs to the grass family, Poaceae, and
constitutes one of the economically most important plant groups. It includes
three major cereal crops, wheat, barley, and rye (belonging to the genera Triticum, Hordeum, and Secale, resp.), which are
traditionally cultivated in the temperate zone. The tribe has basic chromosome
number of seven, and contains taxa ranging from diploids (2n = 2x = 14) to
duodecaploids (2n = 12x = 84), including all intermediate
ploidy levels [14]. Polyploidy (the most
important cytogenetic process in higher plants, [15]) and more specifically,
allopolyploidy, has played and plays a main role in the tribe’s evolution. With
around 350 species, crossability barriers are poorly understood, and it is
remarkable how its species, even species in different genera, can be made to
hybridize even if they do not hybridize naturally. Therefore, the tribe also
includes man-made crops such as ×Triticosecale (triticale) and ×Tritordeum (an amphiploid of Triticum aestivum ×Hordeum chilense [16, 17] and Triticum aestivum × Leymus
arenarius [18]). It has also been possible to apply
interspecific and intergeneric hybridization to increase the genetic
variability of crops belonging to this tribe (mainly wheat, [19, 20]).As mentioned earlier, the chromosome
numbers withinB. distachyon accessions range from 10 to 30 [21], and the haploid genome size
in diploid Brachypodium (2n = 2x =
10) varies from 172 Mbp to 355 Mbp [2, 22], although the former value
may be an underestimate [22]; the genome size is thus assumed
to be approximately 355 Mbp. Therefore, within Poaceae, B. distachyon carries one of the smallest genomes, which is
intermediate between the genomes of Arabidopsis
thaliana with 157 Mbp, and rice with 490 Mbp (All C-values from [3]). These data are consistent
with previous reports that species of Brachypodium have the smallest 5S rDNA spacer among the grasses, and contain less than 15%
highly repetitive DNA [7]. Additionally, GISH analysis
of somatic chromosomes has shown preponderance of repetitive DNA in the
pericentromeric regions, reflecting the compactness and economy of this genome [12]. This study also revealed
structural uniformity of the diploid accessions ABR1 and ABR5, confirming their
status as model genotypes, from which two BAC libraries have recently been
prepared for functional genomics analysis [23]. Recent analysis of BAC end
sequences (BESs) also corroborates this unusually compact genome [24, 25]. The other accessions having
chromosome numbers in multiples of 10 suggested that this species has evolved as
a polyploid series based upon 2n = 2x = 10, and that ecotypes that deviate
from multiples of 10 evolved due to aneuploid or dysploid changes in chromosome
number [21]. A cytotaxonomic analysis of
the members of the polyploid series has revealed  hybrid origin of several of the polyploid
genotypes, suggesting a complex evolution of this species that is not entirely
based on chromosome doubling [12]. For example, allotetraploid
artificial hybrids between B. distachyon and B. sylvaticum exhibited irregular
meiosis and infertility [6], although allotetraploids
were fertile, one of them (ABR100) showed normal meiosis [12]. This indicates that either the
constituent diploids of this allotetraploid are more compatible in hybrids, or
the hybrids themselves have evolved pairing control mechanisms similar to those
of wheat and other allopolyploids. It has also been shown using GISH that the
genomes of the constituent diploids remain separated in the allotetraploid, and
that there is no recombination between homoeologous chromosomes. These features
make the natural polyploid hybrids within the genus Brachypodium a suitable material for the isolation and characterization
of diplodizing genes.For the reasons stated above,
the whole tribe Triticeae is considered to be an enormous gene pool for crop
improvement, deserving efforts not only for its morphological, physiological,
genetic, and genomic characterization, but also for the establishment of
phylogenetic relationships among different species of the tribe. The large and
complex genomes of some members of the tribe are a main constraint for genomics
research within this tribe, which would be greatly facilitated with the
availability of a suitable model species likeB. distachyon.


## 3. Current Status ofBrachypodium Genomics

### 3.1. Development of Inbred Lines

Inbred lines
make an important resource for genomics research. Keeping this in view, diploid
inbred lines  have been developed inB. distachyon by selfing [4] as well as through selection
from segregating populations derived from crosses among diploid ecotypes [26].


### 3.2. Development of Transformation and  Regeneration Protocols

An efficient
transformation procedure and an optimized plant regeneration protocol have been
developed inB. distachyon. For
instance, in a study reported in 1995, callus induction and plant regeneration
from mature embryos, as well as in vitro clonal propagation of shoots were successfully
achieved in B. distachyon [27]. In our own studies also, efficient
callus formation from mature embryos of B. distachyon was successfully achieved
using MS basal medium supplemented with sucrose and 2,4-dichlorophenoxyacetic (2,4-D)
acid at a concentration ranging from 2.5 to 5 mg∖L (see Figure 2).
The results are suggesting that in Brachypodium species, higher
rates of callus induction can be achieved through the use of
(i) MS basal medium [43] rather than LS basal
medium [44]  (ii) sucrose rather than maltose, and (iii) higher concentrations
of auxin as a plant growth regulator (unpublished
data).Figure 2
Callus induction of matureBrachypodium distachyon embryos supplemented with
5 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid).Agrobacterium-mediated
transformation involving insertions of single genes has also been achieved in
several genotypes of B. distachyon (including diploid and polyploid taxa), giving T1 transgenic plants [4]. These transformation studies
also involved the diploid genotype Bd21, which has also been used for
construction of BAC libraries [28] and for generating 20440 ESTs
[29]. Agrobacterium-mediated transformation was successful in 10 out of
the 19 lines, with efficiencies ranging from 0.4% to 15% [4]. Embryogenic calli derived from
immature embryos were also transformed through biolistic transformation leading
to transgene expression in T1 progeny [2, 5]. In later study, transformation
with an average efficiency of 5.3% was achieved. In this study, testing of T0 as well as T1 generations and seed production in T2 was
achieved within one year due to the short life cycle of B. distachyon [5],
confirming its importance as a model plant species.


### 3.3. BAC Libraries and Expression Sequence Tags (ESTs)

BAC libraries of
two diploid ecotypes ofB. distachyon,
ABR1 and ABR5, have also been constructed and have been used to determine synteny
among rice, Brachypodium, and other species of Poaceae
family. For this purpose, BACs were marker-selected (BAC landing) using primers
designed according to previously mapped rice and Poaceae sequences. Most BACs
hybridized as single loci in known Brachypodium chromosomes, whereas contiguous BACs colocalized on individual chromosomes,
thus confirming conservation of genome synteny [23].


### 3.4. Mutagenesis

Mutagenesis with
sodium azide was also successful inB. distachyon although response to this mutagen differed among different accessions [30]. The results obtained were
comparable with those earlier obtained in barley and rice under higher
concentrations of mutagens. Application of ethylmethane sulphonate (EMS) is
currently on the way in diploid Brachypodium accessions.


## 3.1. Development of Inbred Lines

Inbred lines
make an important resource for genomics research. Keeping this in view, diploid
inbred lines  have been developed inB. distachyon by selfing [4] as well as through selection
from segregating populations derived from crosses among diploid ecotypes [26].


## 3.2. Development of Transformation and  Regeneration Protocols

An efficient
transformation procedure and an optimized plant regeneration protocol have been
developed inB. distachyon. For
instance, in a study reported in 1995, callus induction and plant regeneration
from mature embryos, as well as in vitro clonal propagation of shoots were successfully
achieved in B. distachyon [27]. In our own studies also, efficient
callus formation from mature embryos of B. distachyon was successfully achieved
using MS basal medium supplemented with sucrose and 2,4-dichlorophenoxyacetic (2,4-D)
acid at a concentration ranging from 2.5 to 5 mg∖L (see Figure 2).
The results are suggesting that in Brachypodium species, higher
rates of callus induction can be achieved through the use of
(i) MS basal medium [43] rather than LS basal
medium [44]  (ii) sucrose rather than maltose, and (iii) higher concentrations
of auxin as a plant growth regulator (unpublished
data).Figure 2
Callus induction of matureBrachypodium distachyon embryos supplemented with
5 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid).Agrobacterium-mediated
transformation involving insertions of single genes has also been achieved in
several genotypes of B. distachyon (including diploid and polyploid taxa), giving T1 transgenic plants [4]. These transformation studies
also involved the diploid genotype Bd21, which has also been used for
construction of BAC libraries [28] and for generating 20440 ESTs
[29]. Agrobacterium-mediated transformation was successful in 10 out of
the 19 lines, with efficiencies ranging from 0.4% to 15% [4]. Embryogenic calli derived from
immature embryos were also transformed through biolistic transformation leading
to transgene expression in T1 progeny [2, 5]. In later study, transformation
with an average efficiency of 5.3% was achieved. In this study, testing of T0 as well as T1 generations and seed production in T2 was
achieved within one year due to the short life cycle of B. distachyon [5],
confirming its importance as a model plant species.


## 3.3. BAC Libraries and Expression Sequence Tags (ESTs)

BAC libraries of
two diploid ecotypes ofB. distachyon,
ABR1 and ABR5, have also been constructed and have been used to determine synteny
among rice, Brachypodium, and other species of Poaceae
family. For this purpose, BACs were marker-selected (BAC landing) using primers
designed according to previously mapped rice and Poaceae sequences. Most BACs
hybridized as single loci in known Brachypodium chromosomes, whereas contiguous BACs colocalized on individual chromosomes,
thus confirming conservation of genome synteny [23].


## 3.4. Mutagenesis

Mutagenesis with
sodium azide was also successful inB. distachyon although response to this mutagen differed among different accessions [30]. The results obtained were
comparable with those earlier obtained in barley and rice under higher
concentrations of mutagens. Application of ethylmethane sulphonate (EMS) is
currently on the way in diploid Brachypodium accessions.


## 4.Brachypodium Genomes: Advances on the Way

### 4.1. BAC-Based Physical Maps

A BAC-based
physical map ofB. distachyon is
being developed at the John Innes Centre (Norwich, UK) as an aid to the
international effort to make BAC-based physical maps of the genomes of Chinese
Spring bread wheat [31]. Since establishing a physical
map of the genome of bread wheat, one of the most important crops worldwide, is
a major challenge due to the enormous size of the genome and its hexaploid
constitution, it is expected that the availability of a Brachypodium physical map will greatly facilitate this task. The
close phylogenetic relationship of Brachypodium to wheat leads to high similarity in gene sequences. Unambiguous hybridization
signals are also generated, when Brachypodium probes are used on wheat BAC filters and southern blots. Preliminary experiments
have also shown that it is feasible to anchor Brachypodium BACs to the rice genome by BES to create an outline
physical map. An outline physical map of B.
distachyon genotype, Bd3-1 using BES and fingerprinting, is being established
and will be used to start assembling contigs in wheat chromosome groups [31].AnotherB. distachyon physical map is being
developed at the University of California and US Department of Agriculture (USDA)
[25] by using two BAC libraries
constructed from B. distachyon genotype,
BD-21. These BACs are being fingerprinted using snapshot-based fingerprinting. This
physical map of B. distachyon will
also be integrated with BES, again providing genome-wide Brachypodium resources for sequence assembly, comparative genome
analysis, gene isolation, and functional genomics analysis.


### 4.2. B. distachyon Genome and Retrotransposons

The genome ofB. distachyon is also being examined for
the presence, diversity, and distribution of the major classes of plant
transposable elements, particularly the retrotransposons [32], since retrotransposons
comprise most of the existing DNA between genes in the large cereal genomes.
The compact genome of B. distachyon contains relatively few retrotransposons, which include copia, gypsy, TRIM, and
LARD groups of elements. The availability of retrotransposon sequences will
facilitate the development of retrotransposon-based molecular markers like IRAP,
REMAP, SSAP, and RBIP markers, which have a variety of applications.


### 4.3. Genetic Linkage Maps

A genetic map ofB. distachyon genotype, Bd21 is being
developed by the International Brachypodium Initiative [33]. Genetic maps will provide
anchor points linking the genome of Brachypodium with those of rice, wheat, and some biofuel crops, and will establish
chromosome-scale physical maps of BACs for whole genome sequencing. In order to
develop these genetic maps, mapping populations are being developed, which currently
comprise several hundred F2 lines derived from the cross Bd21 ×
Bd3-1.
These will be advanced to F6 to establish RILs that can serve as a common
mapping resource for the community. Several approaches have been used to
identify polymorphisms between parents of the mapping population. First, conserved
orthologous sequence (COS) markers derived from wheat and millet were used to
identify a set of 80 confirmed polymorphisms between these two parental lines
(Bd21, Bd3-1). Another strategy was the use of ESTs derived from Bd21 in order
to identify additional polymorphisms [29]. The most productive approach
has been to predict introns in Brachypodium genes, based on a comparison of Bd21 ESTs with the annotated rice genome
sequence; nearly all primers designed from predicted introns gave amplified products
in PCR reactions. Most markers developed thus were polymorphic among the 5 diploid
inbred lines used for testing, and thus proved to be useful markers for genetic
mapping.


### 4.4. Whole Genome Sequencing

TheBrachypodium nuclear genome is currently
being sequenced within a project that was funded in early 2006 by the US
Department of Energy (DOE). A draft genome sequence is expected to be completed
by the end of 2007. This project is generating a whole-genome shotgun sequence
of B. distachyon genotype, Bd21 genome,
and is coupled with another project aimed at generating nearly 250.000 ESTs. Data
from both projects will be made publicly available through an online database
(BrachyBase at http://www.brachybase.org) and a community-dedicated
portal (http://www.brachypodium.org). BrachyBase will enable efficient
exploitation of genome and transcriptome sequences to identify genes underlying
traits and will facilitate comparisons with other grass genomes [34].Generation and
analysis of over 60 000 BES from large-insert BAC clones has provided the first
view ofBrachypodium genome
composition, structure, and organization [35]. In this study, ~10% of the
BES show similarity to known repetitive DNA sequences in existing databases,
whereas ~40% matched sequences in the EST database, which suggests that a
considerable portion of the Brachypodium genome is transcribed. Gene-related BESs that were identified for the Brachypodium genome were also aligned in silico to the rice genome
sequences. On the basis of gene colinearity between Brachypodium and rice, conserved and diverged regions were
identified. BES with significant matches to wheat ESTs that have been mapped to
individual chromosome and bin positions were also identified. These BACs
represent regions that are colinear with mapped ESTs and will be useful in
identifying additional markers for specific regions of wheat chromosomes.A 371-kb region inB. sylvaticum has already been sequenced
was also compared with orthologous regions from rice and wheat genomes [36]. In this region, Brachypodium and wheat showed perfect
macrocolinearity, whereas rice contains an approximately 220-kb inversion.
Using conserved genomic and EST sequences, divergence between Brachypodium and wheat was estimated to
be 35–40 million years, which is significantly more recent than the divergence
of rice and wheat, which is estimated to have occurred approximately 50 million
years [37].Chosen target
loci fromBrachypodium genome are
also being sequenced and compared with genomic sequences from a variety of
plant species including the following: (i) wheat species (Triticum and Aegilops) with
different ploidy levels, (ii) rice, and (iii) B. sylvaticum, for which a BAC library is available [38, 39]. This comparison revealed
that there is a better conservation of microcolinearity between wheat and Brachypodium orthologous regions than
between wheat and rice, as was also shown in an earlier study [36]. For instance,
sequence comparison at the grain hardness locus shows that genes responsible
for grain hardness/softness, which is seed quality trait in wheat, are absent
from the rice orthologous region, but present in the B. sylvaticum orthologous region.  The gene density found in B. sylvaticum genome is comparable to that of rice (one gene per 8 kb). These results illustrate that Brachypodium species may represent an intermediate model for wheat genome analysis.To test the
potential ofBrachypodium as a model
for the functional analysis of ryegrass (Lolium
perenne) flowering genes, expression of two Terminal Flower 1 orthologs, namely, LpTFL 1 (from L. perenne) and TFL 1
(from Arabidopsis), was examined in two different B. distachyon accessions [40]. Both these repressors
significantly delayed heading date. The short life cycle of Brachypodium and the rapid
transformation system allowed heading date scoring of T1s within the first year
after transformation, thus demonstrating the potential of Brachypodium as a model for ryegrass (L. perenne) also.Brachypodium is also being explored as a model for the genomics research involving study of cereals-pathogen
interactions. For instance, varying degrees of susceptibility and resistance to Magnaporthe grisea (economically
destructive pathogen and casual agent of Rice Blast disease that can also
infect temperate cereals and forage grasses) have been found in several Brachypodium accessions. Aetiology of
fungal development and disease progression in Brachypodium closely resembled those of rice infections; an overexpression
of genes that were homologous with barley genomic probes was also observed [41]. Recent advances in Brachypodium genomics also involved use of
metabolic profiling using Fourier-transform infrared spectroscopy (FT-IR) for
high-throughput metabolic fingerprinting and electrospray ionization mass
spectrometry (ESI-MS). These metabolomic approaches have shown considerable
differential phospholipids processing of membrane lipids during
M. grisea-B. distachyon accessions ABR1
(susceptible) and ABR5 (resistant) interactions
[42]. Brachypodium distachyon, being
a host for M. grisea and other disease-causing pathogens of Pooid
cereals [42], is a suitable model for conducting
functional genomics research involving study of M. grisea pathology and
plant responses [41].


## 4.1. BAC-Based Physical Maps

A BAC-based
physical map ofB. distachyon is
being developed at the John Innes Centre (Norwich, UK) as an aid to the
international effort to make BAC-based physical maps of the genomes of Chinese
Spring bread wheat [31]. Since establishing a physical
map of the genome of bread wheat, one of the most important crops worldwide, is
a major challenge due to the enormous size of the genome and its hexaploid
constitution, it is expected that the availability of a Brachypodium physical map will greatly facilitate this task. The
close phylogenetic relationship of Brachypodium to wheat leads to high similarity in gene sequences. Unambiguous hybridization
signals are also generated, when Brachypodium probes are used on wheat BAC filters and southern blots. Preliminary experiments
have also shown that it is feasible to anchor Brachypodium BACs to the rice genome by BES to create an outline
physical map. An outline physical map of B.
distachyon genotype, Bd3-1 using BES and fingerprinting, is being established
and will be used to start assembling contigs in wheat chromosome groups [31].AnotherB. distachyon physical map is being
developed at the University of California and US Department of Agriculture (USDA)
[25] by using two BAC libraries
constructed from B. distachyon genotype,
BD-21. These BACs are being fingerprinted using snapshot-based fingerprinting. This
physical map of B. distachyon will
also be integrated with BES, again providing genome-wide Brachypodium resources for sequence assembly, comparative genome
analysis, gene isolation, and functional genomics analysis.


## 4.2. B. distachyon Genome and Retrotransposons

The genome ofB. distachyon is also being examined for
the presence, diversity, and distribution of the major classes of plant
transposable elements, particularly the retrotransposons [32], since retrotransposons
comprise most of the existing DNA between genes in the large cereal genomes.
The compact genome of B. distachyon contains relatively few retrotransposons, which include copia, gypsy, TRIM, and
LARD groups of elements. The availability of retrotransposon sequences will
facilitate the development of retrotransposon-based molecular markers like IRAP,
REMAP, SSAP, and RBIP markers, which have a variety of applications.


## 4.3. Genetic Linkage Maps

A genetic map ofB. distachyon genotype, Bd21 is being
developed by the International Brachypodium Initiative [33]. Genetic maps will provide
anchor points linking the genome of Brachypodium with those of rice, wheat, and some biofuel crops, and will establish
chromosome-scale physical maps of BACs for whole genome sequencing. In order to
develop these genetic maps, mapping populations are being developed, which currently
comprise several hundred F2 lines derived from the cross Bd21 ×
Bd3-1.
These will be advanced to F6 to establish RILs that can serve as a common
mapping resource for the community. Several approaches have been used to
identify polymorphisms between parents of the mapping population. First, conserved
orthologous sequence (COS) markers derived from wheat and millet were used to
identify a set of 80 confirmed polymorphisms between these two parental lines
(Bd21, Bd3-1). Another strategy was the use of ESTs derived from Bd21 in order
to identify additional polymorphisms [29]. The most productive approach
has been to predict introns in Brachypodium genes, based on a comparison of Bd21 ESTs with the annotated rice genome
sequence; nearly all primers designed from predicted introns gave amplified products
in PCR reactions. Most markers developed thus were polymorphic among the 5 diploid
inbred lines used for testing, and thus proved to be useful markers for genetic
mapping.


## 4.4. Whole Genome Sequencing

TheBrachypodium nuclear genome is currently
being sequenced within a project that was funded in early 2006 by the US
Department of Energy (DOE). A draft genome sequence is expected to be completed
by the end of 2007. This project is generating a whole-genome shotgun sequence
of B. distachyon genotype, Bd21 genome,
and is coupled with another project aimed at generating nearly 250.000 ESTs. Data
from both projects will be made publicly available through an online database
(BrachyBase at http://www.brachybase.org) and a community-dedicated
portal (http://www.brachypodium.org). BrachyBase will enable efficient
exploitation of genome and transcriptome sequences to identify genes underlying
traits and will facilitate comparisons with other grass genomes [34].Generation and
analysis of over 60 000 BES from large-insert BAC clones has provided the first
view ofBrachypodium genome
composition, structure, and organization [35]. In this study, ~10% of the
BES show similarity to known repetitive DNA sequences in existing databases,
whereas ~40% matched sequences in the EST database, which suggests that a
considerable portion of the Brachypodium genome is transcribed. Gene-related BESs that were identified for the Brachypodium genome were also aligned in silico to the rice genome
sequences. On the basis of gene colinearity between Brachypodium and rice, conserved and diverged regions were
identified. BES with significant matches to wheat ESTs that have been mapped to
individual chromosome and bin positions were also identified. These BACs
represent regions that are colinear with mapped ESTs and will be useful in
identifying additional markers for specific regions of wheat chromosomes.A 371-kb region inB. sylvaticum has already been sequenced
was also compared with orthologous regions from rice and wheat genomes [36]. In this region, Brachypodium and wheat showed perfect
macrocolinearity, whereas rice contains an approximately 220-kb inversion.
Using conserved genomic and EST sequences, divergence between Brachypodium and wheat was estimated to
be 35–40 million years, which is significantly more recent than the divergence
of rice and wheat, which is estimated to have occurred approximately 50 million
years [37].Chosen target
loci fromBrachypodium genome are
also being sequenced and compared with genomic sequences from a variety of
plant species including the following: (i) wheat species (Triticum and Aegilops) with
different ploidy levels, (ii) rice, and (iii) B. sylvaticum, for which a BAC library is available [38, 39]. This comparison revealed
that there is a better conservation of microcolinearity between wheat and Brachypodium orthologous regions than
between wheat and rice, as was also shown in an earlier study [36]. For instance,
sequence comparison at the grain hardness locus shows that genes responsible
for grain hardness/softness, which is seed quality trait in wheat, are absent
from the rice orthologous region, but present in the B. sylvaticum orthologous region.  The gene density found in B. sylvaticum genome is comparable to that of rice (one gene per 8 kb). These results illustrate that Brachypodium species may represent an intermediate model for wheat genome analysis.To test the
potential ofBrachypodium as a model
for the functional analysis of ryegrass (Lolium
perenne) flowering genes, expression of two Terminal Flower 1 orthologs, namely, LpTFL 1 (from L. perenne) and TFL 1
(from Arabidopsis), was examined in two different B. distachyon accessions [40]. Both these repressors
significantly delayed heading date. The short life cycle of Brachypodium and the rapid
transformation system allowed heading date scoring of T1s within the first year
after transformation, thus demonstrating the potential of Brachypodium as a model for ryegrass (L. perenne) also.Brachypodium is also being explored as a model for the genomics research involving study of cereals-pathogen
interactions. For instance, varying degrees of susceptibility and resistance to Magnaporthe grisea (economically
destructive pathogen and casual agent of Rice Blast disease that can also
infect temperate cereals and forage grasses) have been found in several Brachypodium accessions. Aetiology of
fungal development and disease progression in Brachypodium closely resembled those of rice infections; an overexpression
of genes that were homologous with barley genomic probes was also observed [41]. Recent advances in Brachypodium genomics also involved use of
metabolic profiling using Fourier-transform infrared spectroscopy (FT-IR) for
high-throughput metabolic fingerprinting and electrospray ionization mass
spectrometry (ESI-MS). These metabolomic approaches have shown considerable
differential phospholipids processing of membrane lipids during
M. grisea-B. distachyon accessions ABR1
(susceptible) and ABR5 (resistant) interactions
[42]. Brachypodium distachyon, being
a host for M. grisea and other disease-causing pathogens of Pooid
cereals [42], is a suitable model for conducting
functional genomics research involving study of M. grisea pathology and
plant responses [41].


## 5. Conclusions

With the small
genome size and simple growth requirements,Brachypodium provides us with a genome, which is a model for in-depth understanding of functional
genomics of temperate grass genome. As a model, it overcomes some of the
drawbacks that are inherent in the genomes of Arabidopsis and rice that have
already been sequenced and have been hitherto considered models for the improvement
of crop species like wheat and barley. Therefore, elucidation and an improved understanding of Brachypodium genomics has enormous potential to benefit all phases
of society. It provides improved, efficient, and effective genetics and genomics
program. The knowledge on Brachypodium genome is also useful for an in-depth understanding of evolutionary
relationships among different plant genomes. This will play a pivotal role in
comparative studies in diverse fields such as ecology, molecular evolution, and
comparative genetics.



---

*Source: 536104-2008-02-06.xml*