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Recent Progress in Model Grass Brachypodium distachyon (Poaceae)

禾草模式植物二穗短柄草的研究进展



全 文 :禾草模式植物二穗短柄草的研究进展
莫新春1ꎬ2
(1 中国科学院昆明植物研究所中国西南野生生物种质资源库ꎬ 云南 昆明  650201ꎻ
2 中国科学院大学ꎬ 北京  100049)
摘要: 二穗短柄草 (Brachypodium distachyon) 是近来开发的一种温带禾草模式植物ꎬ 它具有与粮食作物相
同的许多生物学特性ꎬ 可作为研究粮食作物生物学特性的模式实验植物ꎮ 不同采集地的二穗短柄草具有高
度的表观变异性ꎬ 可帮助研究人员对这些生物学特性从表观到遗传的深入研究ꎮ 二穗短柄草与其他重要经
济作物如小麦、 大麦及其他潜在能源植物一样同属于早熟禾亚科ꎬ 使其成为研究这些重要经济作物无可非
议的模式植物ꎮ 近来ꎬ 由于二穗短柄草基因组序列及其相关注释的完成ꎬ 功能基因组学和其他实验技术手
段的不断进步ꎬ 二穗短柄草可为其他禾草类植物提供序列分析、 基因表达和功能研究等诸多便利ꎮ 本文综
述了利用二穗短柄草作为模式植物来进行比较基因组学、 生物学研究、 转化和 T ̄DNA 突变等方面的最新
研究进展ꎮ
关键词: 二穗短柄草ꎻ 模式系统ꎻ 比较基因组学ꎻ 生物特性研究ꎻ 转化ꎻ T ̄DNA突变
中图分类号: Q 75            文献标识码: A                文章编号: 2095-0845(2014)02-197-11
Recent Progress in Model Grass Brachypodium distachyon (Poaceae)
MO Xin ̄Chun1ꎬ2
(1 Germplasm Bank of Wild Species in Southwest Chinaꎬ Kunming Institute of Botanyꎬ Chinese Academy of Sciencesꎬ
Kunming 650201ꎬ Chinaꎻ 2 University of Chinese Academy of Sciencesꎬ Beijing 100049ꎬ China)
Abstract: Brachypodium distachyonꎬ recently developed model system for temperate grassesꎬ exhibited many traits
with cereal crops and proposed to be an experimental system to access the biological approach. These traits have
shown a surprised degree of phenotypic variation in many collected accessions. Like some important economical cere ̄
alsꎬ B􀆰 distachyon also belongs to subfamily Pooideaeꎬ which make it become an unquestionable model system to re ̄
search the economically important cropsꎬ such as wheatꎬ barley and several potential biofuel plants. Recentlyꎬ ge ̄
nome sequence and annotation of B􀆰 distachyon has been finished. Associated with the development of the functional ge ̄
nomics and other experimental resources establishmentꎬ B􀆰 distachyon will provide a key resource for improving cereal
crops and facilitate the approach of sequence analyze gene expression and functional resources available for a variety of
species. In this article we review and assess the current progress of B􀆰 distachyon as a model system and then focus spe ̄
cifically on recent studies of comparative genomicsꎬ biological improvementꎬ transformation and T ̄DNA mutations.
Key words: Brachypodium distachyonꎻ Model systemꎻ Comparative genomicsꎻ Biological improvementꎻ Transforma ̄
tionꎻ T ̄DNA mutations
  Grasses consist of over 10 000 species distribu ̄
ted widely across the earthꎬ and belong to the fourth
largest plant family (Poaceae) in the world (Watson
and Dallwitzꎬ 1992). They are centrally important
for human existence by directly ( cereals) or indi ̄
rectly (animal feed) serving as the primary source of
human nutrition. Howeverꎬ the size and complexity
of many grass crop genomes become the major barri ̄
植 物 分 类 与 资 源 学 报  2014ꎬ 36 (2): 197~207
Plant Diversity and Resources                                    DOI: 10.7677 / ynzwyj201413108
Received date: 2013-05-08ꎬ Accepted date: 2013-07-12
作者简介: 莫新春 (1980-) 男ꎬ 博士研究生ꎬ 主要从事植物基因组学研究ꎮ E ̄mail: gxumxc@gmail􀆰 com
ers of systematic genetic analysis of traits at the mo ̄
lecular level (Vainꎬ 2011). Moreoverꎬ many grass
species are relatively expensive to work withꎬ com ̄
pared to model plantsꎬ such as Arabidopsis thaliana.
These features lead such research only restrict to
some wealthy and specialized academic institutions
or commercial enterprises.
In generalꎬ model plants were proposed as a
useful tool to understanding biological processes at
geneticꎬ molecular and systematic levels. A􀆰 thalianaꎬ
the first higher plant with complete finished genome
sequence releaseꎬ is arguably the best model species
for higher plants. It is clear that an alternative model
to Arabidopsis was needed for the study of grassesꎬ
for which monocots and dicots had diverged from each
other for over 150 million years (Hands and Dreaꎬ
2012). Prior to the availability of the B􀆰 distachyon
genomeꎬ rice has provided the genomic resources as
the main monocotyledonous model system while
maize formed an extensive resource of developmental
genetics. Howeverꎬ rice is a semi ̄aquatic tropical
species. Its specialized cultivation requirements and
lacking many important temperate cereal traits make
it not an ideal model for the temperate grasses (Vo ̄
gel and Braggꎬ 2009).
A smallꎬ fast ̄growing wild grass species within
the Pooideae familyꎬ B􀆰 distachyon (Hereafter Brachy ̄
podium)ꎬ has been recently developed as a new
model system for the temperate cereals. It is native to
the Middle East and Southern Europe but widely dis ̄
tributed in the temperate areas of Australasiaꎬ Ameri ̄
ca and Asia (Draper et al.ꎬ 2001). Brachypodium
possesses many of the biological traits required for a
tractable model systemꎬ such as small statureꎬ small
genome sizeꎬ rapid life cycleꎬ undemanding growing
environmentꎬ self ̄compatible and easy to transforma ̄
tion ( Bevan et al.ꎬ 2010ꎻ Garvin et al.ꎬ 2008ꎻ
Opanowicz et al.ꎬ 2008). Since Brachypodium was
first proposed as a new model for grass research
(Draper et al.ꎬ 2001)ꎬ a genome sequencing project
of the diploid inbred line Bd21 lead by the Interna ̄
tional Brachypodium Initiative was already finishedꎬ
with a draft (4X) sequence released in 2007 and
the completed genome sequence deposited in 2010
(International Brachypodium Initiativeꎬ 2010).
Studies on Brachypodium resources for molecu ̄
lar genetics and genomics research growing exponen ̄
tially after the full genome sequence released. Con ̄
sequentlyꎬ the significant increases in our knowledge
of grass biology include systems ̄level approaches to
understanding the mechanisms on cereal crops
breeding. Although the track record of Brachypodium
as a model system is currently limitedꎬ it is easy to
achieve the genetic variation through classical or
transgenic mutagenesisꎬ and there is already a tre ̄
mendous resource in terms of natural variation.
When the genomic tools are taken into accountꎬ it
will be possible to unlock these sources of variation
and to address a wide range of important issues in
grass biology. In this contextꎬ we review the recent
progress of Brachypodium as an emerging model sys ̄
tem for the temperate grasses in the area of compara ̄
tive genomicsꎬ biological improvement and transgenic
approach.
1  Comparative genomics of Brachypodium
and cereals
The grass family Poaceaeꎬ contains important
grain crops such as wheatꎬ riceꎬ maize and sor ̄
ghumꎬ become the main resource of human and do ̄
mestic animal nutrition (Bevan et al.ꎬ 2010). Al ̄
though grass genomes greatly vary in size (Wicker
and Kellerꎬ 2007)ꎬ there is an underlying conserved
gene order (Moore et al.ꎬ 1995) existence to reflect
their common ancestry and rapid diversification (Da ̄
vies et al.ꎬ 2004). In 2004ꎬ Foote et al. (2004)
firstly constructed a bacterial artificial chromosome
(BAC) library of B􀆰 sylvaticum with 30 228 clonesꎬ
representing more than six genome equivalents.
Chromosome mapping between B􀆰 sylvaticum BAC
contigs and the major temperate cereals ( wheatꎬ
barleyꎬ maize and oat)ꎬ revealed that the synteny
between the four cereal crops and Brachypodium was
largely maintained over several syntenous regions.
891                                  植 物 分 类 与 资 源 学 报                            第 36卷
Another BAC library of 9 100 clones from B􀆰 distachyon
was subsequently constructed to validate the high
synteny between Brachypodium and Poaceae species
(Hasterok et al.ꎬ 2006)ꎬ with most of the defined
BACs hybridizing as single loci on known chromo ̄
somes. Furthermoreꎬ more studies focus on the pro ̄
tocols of BAC library construction and facilitate the
investigation of comparative genomics between Brachy ̄
podium and cereals ( Farrar and Donnisonꎬ 2007ꎻ
Huo et al.ꎬ 2006ꎻ Jenkins and Hasterokꎬ 2007).
BAC libraries with three times coverage have been
employed to construct physical maps for the Brachy ̄
podium genome (Huo et al.ꎬ 2008). These works
prove that Brachypodium will be a useful tool in the
elucidation of gene content in agronomically impor ̄
tant cereal cropsꎬ complementing rice as a “ grass
genome model” .
To date the complete genome sequences of
B􀆰 distachyonꎬ the US Department of Energy (DOE)
in conjunction with the Joint Genome Institute (JGI)
embarked upon a project to sequence the genome of
the diploid ecotypeꎬ Bd21 (2n= 10). Moreoverꎬ the
expressed ̄sequence tag (EST) libraries of B􀆰 distachyon
have been sequenced (Vogel et al.ꎬ 2006)ꎬ and an
additional 180 000 ESTs are expected as part of the
genome sequencing project. In 2010ꎬ the complete
and annotated genome was released and a sophisti ̄
cated and growing collection of tools and facilities
are already availableꎬ with more becoming available
in the near future (International Brachypodium Initi ̄
ativeꎬ 2010 ). The diploid inbred line Bd21 of
B􀆰 distachyon was sequenced using whole genome
shot ̄gun sequencing and assembled to ten largest
scaffolds spanned 272 Mb covering 99􀆰 6% of se ̄
quenced nucleotides. The genome contains 21􀆰 4%
retrotransposon sequences and annotated about 25 532
protein ̄coding gene loci similar to those predicted in
rice and sorghum (International Brachypodium Initi ̄
ativeꎬ 2010). As DNA transposons evolve rapidly in
plant genomeꎬ Buchmann et al. (2012) investigate
the influence of the intergenic sequences on genome
turnover by compared 1 Mbp of orthologous genomic
sequences from B􀆰 distachyon and B􀆰 sylvaticum. In
total of 219 analyzed genesꎬ only 24 transposable ele ̄
ments of a total of 451 were orthologous. Further a ̄
nalysis proved that the DNA transposon excision
through two ways of double ̄strand break (DSB) re ̄
pair and become a major factor for the rapid turnover
and erosion of intergenic sequences.
A complete genome sequence will favour Brachy ̄
podium in transcriptomics research ( Huan et al.ꎬ
2013ꎻ Walters et al.ꎬ 2013) and association map ̄
pingꎬ which enable us to define both induced and
natural variation. Association mapping might be a
useful tool for identifying alleles and loci responsible
for natural variation in Brachypodium (Cui et al.ꎬ
2012ꎻ Sablok et al.ꎬ 2011ꎻ Zhang et al.ꎬ 2013).
Associated by the published plant genomesꎬ the
epitope databases and the protein sequences of
B􀆰 distachyon are also released to enable researchers
to identify the food allergies and sensitivities related
proteins (Juhasz et al.ꎬ 2012). Wang et al. (2012)
also identified the low molecular weight glutenin
subunit ( LMW ̄GS) genes in B􀆰 distachyonꎬ which
similar to those in wheat. Detailed proteomics and
molecular genetics study revealed that Brachypodium
possessed a highly conserved Glu ̄3 locus closely re ̄
lated to Triticum and related speciesꎬ which sever
B􀆰 distachyon as a model system to study of the
wheat quality attributes. Recentlyꎬ Walters et al.
(2013) profiled a genome ̄wide landscape of alter ̄
native splicing (AS) events in B􀆰 distachyon by u ̄
sing assembled expressed transcript sequences and
subsequent mapping to the corresponding genome.
Approximatelyꎬ 6􀆰 3% of expressed genes are showed
alternatively spliced in B􀆰 distachyon and the majori ̄
ty of the identified AS events were exhibited in the
retained introns ( 55􀆰 5%). Comparative AS tran ̄
script analysis revealed there are 163 and 39 homol ̄
ogous pairs between B􀆰 distachyon and O􀆰 sativa and
between B. distachyon and A􀆰 thalianaꎬ respectively.
In allꎬ 16 AS transcripts are also identified to be
conserved in all three species.
Comparative genomics has revealed the evidence
9912期            MO Xin ̄Chun: Recent Progress in Model Grass Brachypodium distachyon (Poaceae)              
of synteny between Brachypodium and Poaceae spe ̄
cies at the whole genome level ( Hasterok et al.ꎬ
2006ꎻ Huo et al.ꎬ 2009ꎻ Ma et al.ꎬ 2010). Howev ̄
erꎬ many genomic rearrangements and duplications
occurring in the wheat / barley lineage after the diver ̄
gence of the Brachypoideae challenge the signifi ̄
cance of the syntenic relationship between Brachypo ̄
dium and the Triticeaeꎬ appear to alignment frag ̄
mentary at the macro level (Bossolini et al.ꎬ 2007ꎻ
Mur et al.ꎬ 2011ꎻ Wicker et al.ꎬ 2011). Although
gene order appears to be highly conserved within rice
and Brachypodium at nucleotide levelꎬ sequence
similarity between Brachypodium and the Pooids is
generally much higher than in rice (Bossolini et al.ꎬ
2007ꎻ Mur et al.ꎬ 2011ꎻ Vogel et al.ꎬ 2006). This
similarity will facilitate Brachypodium in experimen ̄
tally functional genomics and create higher con ̄
served markers in Brachypodium to temperate spe ̄
cies ( Mur et al.ꎬ 2011). Neverthelessꎬ the gene
family sequence similarity across the Poaceae have
been shown to be comparatively highꎬ as rapid evo ̄
lutionary divergence may occur under domesticationꎬ
it may be addressed an attention to ensure Brachypo ̄
dium will offer the best source of genomic informa ̄
tion for any particular trait under investigationꎬ and
to utilize other grass genomes in comparative ap ̄
proach (Hands and Dreaꎬ 2012ꎻ Mur et al.ꎬ 2011).
Huan et al. (2013) employed a transcriptome analysis
on vernalization ̄memory ̄related genes of B􀆰 distachyon
compare to those of Barley demonstrated that the oxi ̄
dative ̄stress response was the most conserved path ̄
way between these two plant species. Correlation a ̄
nalysis of those vernalization ̄related genes with bar ̄
ley revealed that the vernalization mechanism was
conserved between these two plant species although
several species ̄specific features exist. The Yr26 geneꎬ
an important resistance gene to wheat stripe rustꎬ
was cross ̄species mapped to wheatꎬ B􀆰 distachyon
and rice chromosome for the collinearity of these
three species. Thirty ̄one markers were developed to
saturate the chromosomal region containing the Yr26
locus and six of them cosegregated with the resis ̄
tance gene. These markers had been prove that they
could provide a potential target site for further map ̄
based cloning of Yr26 and should be useful in mark ̄
er assisted selection for pyramiding the gene with
other resistance genes (Zhang et al.ꎬ 2013).
2  Biological improvement
Brachypodium has been demonstrated that it is
a valuable and successful functional genomic model
for temperate grass research ( Opanowicz et al.ꎬ
2008). The original biological features of Brachypo ̄
dium and their similarities to cropsꎬ have a great po ̄
tential for gene discovery (Bevan et al.ꎬ 2010ꎻ Watt
et al.ꎬ 2009). Although some technologies are avail ̄
able in a number of cropsꎬ Brachypodium plants
make it possible to undertake economically feasible
large ̄scale research projects in specialized laborato ̄
ries such as high ̄throughput phenomicsꎬ plant ̄
pathogen interactions or de novo engineering of path ̄
waysꎬ and attribute to dramatically reduce the costs
than in cereal crops (Philippeꎬ 2011). Brachypodi ̄
um was initially proposed to study the plant ̄pathogen
interactions with cereal fungal rust pathogens (Bar ̄
bieri et al.ꎬ 2012ꎻ Draper et al.ꎬ 2001) following
successful interactions with rice blast (Routledge et
al.ꎬ 2004). It also shown resistance to Fusarium head
blight (FHB)ꎬ a major wheat disease causing by the
Fusarium species (F􀆰 graminearum and F􀆰 culmorum)ꎬ
enabling scientists to undertake the studies of Fusari ̄
um head blight and other Fusarium diseases of wheat
in a model system (Peraldi et al.ꎬ 2011). In gener ̄
alꎬ the pathogens will co ̄evolve to exploit the new
plant populations during cereal domesticationꎬ thusꎬ
combating plant disease is become a essential re ̄
quirement in many cereal breeding programs
(Kanyuka et al.ꎬ 2004) and the agrochemical in ̄
dustry (Deacon and Berryꎬ 1993). The prejudicious
influences of disease on crops yield and the desire to
reduce chemical inputs would greatly promote the
plant breeders to wage a never ̄ending battle to breed
new resistant varieties to the current prevalent strains
of pathogens (Opanowicz et al.ꎬ 2008).
002                                  植 物 分 类 与 资 源 学 报                            第 36卷
Brachypodium has been proposed as energy
plant by the U􀆰 S. Department of Energyꎬ which en ̄
courage researchers to learn the genetic mechanisms
of biological traits such as cell wall compositionꎬ bi ̄
omass yieldꎬ stress toleranceꎬ and other phenotypes
relevant to biomass crop developmentꎻ accelerate the
domestication of wild grasses ( e􀆰 g. switchgrass and
Miscanthus) that are promising biomass crops (Of ̄
fice of the Biomass Programꎬ 2006). Of interest in
the context of biomass cropsꎬ Brachypodium has a
typical grass cell wall structure ( Type II)ꎬ differ
from the Type I cell wall in the type of hemicellulose
(primarily xyloglucans in dicots and glucuronoarabi ̄
noxylans in grasses)ꎬ pectin and proteins contentsꎬ
cross ̄linking phenolic compounds and mixed linkage
glucans (Christensen et al.ꎬ 2010). Thusꎬ studies
of the biosynthesis / composition of Type II cell walls
will improve the capture of fermentable products
from biomass grasses (Bevan et al.ꎬ 2010ꎻ Vogelꎬ
2008). Brachypodium also shown the similar cell
wall compositions with Miscanthus ( Gomez et al.ꎬ
2008) and had the similar members of cell wall bio ̄
synthetic enzyme families with rice and sorghum
(International Brachypodium Initiativeꎬ 2010). Fur ̄
ther comparative analysis of four diverse grass ge ̄
nomes confirming the conservation of gene families
involved in cell wall biosynthesisꎬ supporting the use
of Brachypodium as such a general model for diverse
grasses (International Brachypodium Initiativeꎬ 2010).
Cell walls are a major component and have
greatly impacts upon the nutritional quality of cereal
grains. Its distinctive profile in grains is unique with ̄
in the genusꎬ species and even cultivar (Barrero et
al.ꎬ 2012ꎻ Molinari et al.ꎬ 2013ꎻ Rancour et al.ꎬ
2012ꎻ Toole et al.ꎬ 2011). Although Brachypodium
shares the same types of cell wall polysaccharides as
in other cerealsꎬ the relative amounts are quite
difference (Guillon et al.ꎬ 2011ꎻ Opanowicz et al.ꎬ
2011). In most of the domesticated cerealsꎬ the cell
walls of grains typically account for around 3%-8%
of the total grain weight. Howeverꎬ the cell wall pol ̄
ysaccharide content of dehulled Brachypodium grains
is reported to have 60% of the dry weightꎬ which is
three ̄fold of those come from wheatꎬ barley and oat
(Barron et al.ꎬ 2007ꎻ Guillon et al.ꎬ 2011ꎻ Man ̄
they et al.ꎬ 1999ꎻ Oscarsson et al.ꎬ 1996). It seem
that the very thick cell walls of the endosperm con ̄
tains much of the carbohydrate material and relative ̄
ly little starch in the grainꎬ differ from the grains of
barleyꎬ wheatꎬ maize and rice characterized in detail
(Guillon et al.ꎬ 2011). Similar to cell wallsꎬ storage
proteins are other important features of cereal grainsꎻ
also contribute to the nutritional quality. Their occur ̄
rence and relative amounts are distinctive within dif ̄
ferent species. One of the storage proteins ̄globulinsꎬ
widely distributed amongst the flowering plants in
both monocots and dicotsꎬ are typically embryo stor ̄
age proteins in cereal grains. They are normally di ̄
vided into two major classesꎬ the 7S and 11 - 12S
globulins (Shewry and Halfordꎬ 2002). In Brachyp ̄
odiumꎬ 7S globulins are identified to encoding by
the BdGLO1 gene (Bradi1g13040) ( Larre et al.ꎬ
2010ꎻ Laudencia ̄Chingcuanco and Venselꎬ 2008)ꎬ
the orthologue of barley embryo globulin 1 (BEG1)
and wheat globulin 3 ̄A (TaGLO3A) (Heck et al.ꎬ
1993ꎻ Loit et al.ꎬ 2009)ꎬ take account for around a
third of the globulins. The remainder mainly compri ̄
sing 11S proteinsꎬ encoding by the BdGLO2 gene
(Bradi2g38060)ꎬ have similar properties and solu ̄
bility to the globulins predominate in oat and rice
with 70%-80% account to total proteins ( Larre et
al.ꎬ 2010ꎻ Laudencia ̄Chingcuanco and Venselꎬ
2008ꎻ Shewry and Halfordꎬ 2002). Another promi ̄
nent example is a study of globulin gene duplication
event occurring in the Triticeae. The HMW glutenin
geneꎬ encoded a critical protein unique to bread ̄
making properties of wheatꎬ resulted in this duplica ̄
tion and can be identified in Brachypodium whereas
cannot be seen in any of the tropical grass genomes
(Gu et al.ꎬ 2010). The details of storage protein
deposition and sub ̄cellular localization are not yet
well drawn in Brachypodium and this area will bene ̄
fit from further investigation.
Recentlyꎬ the natural diversity of Brachypodium
1022期            MO Xin ̄Chun: Recent Progress in Model Grass Brachypodium distachyon (Poaceae)              
accessions have been reported to response the
drought stress and proposed to provide a means to i ̄
dentify genes and alleles important for the complex
trait of drought tolerance (Luo et al.ꎬ 2011). In or ̄
der to improve the tolerance of temperate cereals to
droughtꎬ a reproducible in vivo drought assay was
developed in B􀆰 distachyon. The result shows that the
cell expansion of leaf was greatly decreased under
the drought stress whereas the cell division remained
largely unaffected. Further transcriptome profiles ap ̄
proach indicated an up ̄regulation of sterol synthesis
associated with increased energy availability in the
proliferation zones might influence the membrane
fluidity in leaf development (Verelst et al.ꎬ 2013).
In additionꎬ the availability of diploidꎬ polyploid
and hybrid Brachypodium accessions also allows us
to learn the mechanisms relevant to polyploid species
in a model systemꎬ such as chromosome pairing and
recombination ( Garvin et al.ꎬ 2008ꎻ Hasterok et
al.ꎬ 2004ꎻ Idziak and Hasterokꎬ 2008). The diver ̄
sity of Brachypodium species ranging from rhizoma ̄
tous perennial outbreed to annual inbred plants could
also provide information on the genetic determinants
of traits such as perenniality and self ̄incompatibility
(Wolny et al.ꎬ 2011). Converselyꎬ investigation on the
terms of grain development / composition (Charles et
al.ꎬ 2009ꎻ Gu et al.ꎬ 2010ꎻ Larre et al.ꎬ 2010ꎻ
Opanowicz et al.ꎬ 2011) or flowering / verbalization
(Higgins et al.ꎬ 2010) within Brachypodium could
bring a new insight into the mechanisms and path ̄
ways underpinning grass development.
3  Transformation and T ̄DNA mutants
With numerous genes were accelerated disco ̄
very in grassesꎬ high efficient transformation tools of
Brachypodium need to be developed to assess the
gene functionꎬ mechanisms understanding and path ̄
ways modeling. It is feasible to achieve the gene
function in Brachypodium by efficient transformation
systems (Pacurar et al.ꎬ 2008ꎻ Vain et al.ꎬ 2008ꎻ
Vogel and Hillꎬ 2008) for increasing (Olsen et al.ꎬ
2006) or suppressing gene expression under RNAi
strategies (Demircan and Akkayaꎬ 2010ꎻ Pacak et
al.ꎬ 2010). Prior to Agrobacterium tumefaciens ̄me ̄
diated transformations establishedꎬ a fast and effi ̄
cient microprojectile bombardment ̄mediated trans ̄
formation protocol of Brachypodium was developed
using embryogenic calli with an average transforma ̄
tion efficiencies (defined as the percentage of callus
pieces reproduce a transgenic plant) of 5􀆰 3%ꎬ and
up to 14% for single bombardments transformation
(Christiansen et al.ꎬ 2005). When the highly effi ̄
cient A􀆰 tumefaciens ̄mediated transformation methods
established (Pacurar et al.ꎬ 2008ꎻ Vain et al.ꎬ 2008ꎻ
Vogel and Hillꎬ 2008)ꎬ the transformation efficien ̄
cies of this method now reached 50% in a transgenic
plants production setting where hundreds of T ̄DNA
insertion lines were generated every week ( for fur ̄
ther protocols updating informationꎬ please visit ht ̄
tp: / / brachypodium􀆰pw􀆰usda􀆰gov / and http: / / www􀆰
brachytag􀆰 org / ) . Most investigators can easy to ac ̄
complish the Brachypodium transformation to assess
the gene function in simply tissue culture trainingꎬ
and searched in two Brachypodium transformation
services which available to the public (http: / / www􀆰
brachytag􀆰 org / and http: / / www􀆰 agron􀆰 iastate􀆰 edu /
ptf / index􀆰 aspx) (Brkljacic et al.ꎬ 2011).
Benefit from the efficient Brachypodium trans ̄
formation methods establishmentꎬ large collections of
sequence ̄indexed T ̄DNA mutants were easy to cre ̄
ate. Two large ̄scale projects were initiated to create
T ̄DNA mutant collections by the John Innes Centre
and U􀆰 S. Department of Agriculture ( USDA )
Brachypodium Genome Resourcesꎬ respectively. The
BrachyTAG project at the John Innes Centre current ̄
ly lists 5 000 T ̄DNA lines (genotype Bd21) and has
distributed mutants since 2008 (http: / / www􀆰 brachy ̄
tag􀆰 org / ) . The collection mainly consists of T ̄DNA
insertion lines (BrachyTAG)ꎬ but also contains se ̄
veral hundred promoter trap lines (BrachyTRAP)
using the reporter gene green fluorescent protein
(GFP). Analysis of these fertile tagged lines from
this collection may help to profiling of T ̄DNA inser ̄
tions in the nuclear genome of Brachypodium (Thole
202                                  植 物 分 类 与 资 源 学 报                            第 36卷
et al.ꎬ 2010). The second Brachypodium T ̄DNA
collection is established by the USDA Brachypodium
Genome Resourcesꎬ which contains 8 700 lines and
aims to create additional 30 000 lines using the geno ̄
type Bd21 ̄3 ( http: / / brachypodium􀆰 pw􀆰 usda􀆰 gov /
TDNA / ). This collection mostly contains lines for
the activation tagging (via a transcriptional enhancer
present in the T ̄DNA) and promoter trapping vec ̄
tors by using ß ̄glucuronidase (GUS) and / or GFP
as reporter genes ( Thole et al.ꎬ 2012). Approxi ̄
mately 50 000 T ̄DNA lines will be made by the In ̄
ternational Brachypodium Tagging Consortium ( IT ̄
BC) within this yearꎬ which produced by eight labo ̄
ratories from five countries (United Statesꎬ United
Kingdomꎬ Chinaꎬ Koreaꎬ and Canada) ( http: / /
www􀆰 brachytag􀆰 org / users􀆰 htm). These mutants will
be integrated into online databases and enable re ̄
searchers to identify and order mutants of interest
from the different collections. This will significantly
accelerate establishing an in silico resource for re ̄
verse genetics in Brachypodium and play a key role
on investigate the contribution of mutant resources in
temperate grass species.
In additionalꎬ high ̄efficiency transformation al ̄
so can be used to the characterization of gene func ̄
tion through over ̄expression or gene silencing in
Brachypodium (Demircan and Akkayaꎬ 2010ꎻ Moli ̄
nari et al.ꎬ 2013ꎻ Olsen et al.ꎬ 2006ꎻ Pacak et al.ꎬ
2010ꎻ Schweiger et al.ꎬ 2013ꎻ Tripathi et al.ꎬ
2012ꎻ Valdivia et al.ꎬ 2013ꎻ Wang et al.ꎬ 2012ꎻ
Zhang et al.ꎬ 2013). Recentlyꎬ a T ̄DNA mutation
harboring eukaryotic initiation factor 4A ( eIF4A)
genes present in Brachypodium nuclear genome has
been identified. The eIF4A homozygous mutant ex ̄
hibited reduced final plant stature due to a decrease
in both cell number and cell sizeꎬ consistent with
roles for eIF4A in both cell division and cell growth.
The gene function of eIF4A was found to be con ̄
served between monocotyledonous and dicotyledon ̄
ous species and complemented with an Arabidopsis
ortholog. This demonstrates Brachypodium as a
bridging model for grass and cereals research (Vain
et al.ꎬ 2011). Furthermoreꎬ biotechnological ap ̄
proaches for crop improvement can be achieved by
introducing and expressing heterologous genes (e􀆰 g.
pectin specific fungal acetylesterases) in Brachypo ̄
dium (Pogorelko et al.ꎬ 2013). Recent in ̄depth re ̄
views critically assess the current status of the
Brachypodium T ̄DNA mutagenesis and A􀆰 tumefaciens ̄
mediated transformation (Thole et al.ꎬ 2012ꎻ Thole
and Vainꎬ 2012).
4  Conclusion and future prospects
With the full annotated genome releasedꎬ Brachy ̄
podium was emerged as an attractive model system
for grass biologyꎬ which played a key foundation to
facilitate the dissection of many traits of agronomic
and ecological importance. A rapidly growing num ̄
ber of researchers have adopted Brachypodium as a
model for their research programs on the elucidation
of grass biology as Brachypodium exhibits extensive
biodiversity and hold great potential for genetic ana ̄
lyses and engineering. As Arabidopsis successful to
serve as an experimental system for the plant biolo ̄
gyꎬ series of common experimental toolkits have
been established to enable large and small laborato ̄
ries around the world to identify important functional
genesꎬ which provided a basic paradigm to the in ̄
vestigation on grass biology by using Brachypodium
as a tractable model system.
Previouslyꎬ many traits of plant biology were
best analyzed as quantitative characteristics though
they were probably multigenic complexꎬ shown by
reports covering a wide range of topics such as ver ̄
nalization and flowering time (Faricelli et al.ꎬ 2010ꎻ
Higgins et al.ꎬ 2010ꎻ Olsen et al.ꎬ 2006ꎻ Schwartz
et al.ꎬ 2010)ꎬ seed storage proteins (Charles et al.ꎬ
2009ꎻ Gu et al.ꎬ 2010ꎻ Larre et al.ꎬ 2010ꎻ Lauden ̄
cia ̄Chingcuanco and Venselꎬ 2008ꎻ Loit et al.ꎬ
2009)ꎬ fatty acid turnover ( Yang and Ohlroggeꎬ
2009)ꎬ plant - pathogen interactions ( Drader and
Kleinhofsꎬ 2010ꎻ Figueroa et al.ꎬ 2013ꎻ Mandadi
and Scholthofꎬ 2012ꎻ Parker et al.ꎬ 2008ꎻ Pogorel ̄
ko et al.ꎬ 2013)ꎬ and wounding / insect responses
3022期            MO Xin ̄Chun: Recent Progress in Model Grass Brachypodium distachyon (Poaceae)              
(Azhaguvel et al.ꎬ 2009ꎻ Mur et al.ꎬ 2004). As
Brachypodium is proposed to serve as a renewable
biomass plantꎬ several reports have focused on the
Brachypodium root development ( Ingram et al.ꎬ
2012ꎻ Kim and Dolanꎬ 2011ꎻ Pacak et al.ꎬ 2010ꎻ
Pacheco ̄Villalobos and Hardtkeꎬ 2012). Similar to
wheatꎬ Brachypodium can readily forms mycorrhizal
associationsꎬ which important for uptake of P in
many cropsꎬ and is especially important for low ̄in ̄
put agriculture as envisioned for the future produc ̄
tion of biomass crops (Chochois et al.ꎬ 2012ꎻ Watt
et al.ꎬ 2009).
Overallꎬ a truly tractable and generic model
system-Brachypodium has been establishedꎬ which
would accelerate the plant biology and crop research
to uncover the link between genotype and phenotype
to exploit and create new genetic variation. With
many investigations undertaken directly in this at ̄
tractive modelꎬ Brachypodium would greatly facili ̄
tate and promote grass research. Thusꎬ Brachypodi ̄
um can play a key contribution for the elucidation of
grass biology as an attractive model system.
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7022期            MO Xin ̄Chun: Recent Progress in Model Grass Brachypodium distachyon (Poaceae)