Environmental Engineering Reference
In-Depth Information
were used to generate BAC end sequences from 64,694 clones, and the resulting 38.2 Mbp of
sequence covers approximately 11% of the Brachypodium genome (Huo et al. 2007). This sequence
was used to anchor the BAC clones to the rice genome and indicated that the Brachypodium genome
contains 45.9% GC content, approximately 18% repetitive DNA (11% with homology to known
repetitive sequence and 7.3% unique to Brachypodium), and 21.2% coding sequence. In addition,
the Arizona Genomics Institute (www.genome.arizona.edu/) has constructed one library from the
inbred line Bd3-1 and two libraries from Bd21 (M. Bevan, personal communication).
One BAC library exists for the perennial species
B. sylvaticum
. This library contains 30,228
clones with an average insert size of 102 kb (6.6 genome equivalents, on the basis of a genome size
of 470 Mbp) (Foote et al. 2004). From this library, repetitive DNA content was estimated to be
approximately 50%, and analyses demonstrated that synteny was maintained among rice, wheat,
and
B. sylvaticum
BAC contigs over several regions of chromosome 9. The percentage of repetitive
DNA in
B. sylvaticum
is much higher than in
B. distachyon
and largely explains the greater size of
the
B. sylvaticum
genome.
23.4.4 m
apS
and
m
arkErS
Mapping resources for Brachypodium are developing rapidly. A physical map has been constructed
from two of the Bd21 BAC libraries mentioned above
(Gu et al. 2009).
This map contains over
67,000 BAC clones assembled into 671 contigs and can be accessed at http://phymap.ucdavis.
edu:8080/brachypodium/. In addition, a second physical map using two different Bd21 libraries has
been constructed (M. Bevan, personal communication). A high-density linkage map based on 562
single nucleotide polymorphism (SNP) markers has also been constructed (N. Huo, unpublished).
The markers fell into five linkage groups corresponding to the five chromosomes of the haploid
Brachypodium genome. The resulting map was used to assemble the whole-genome shotgun
sequence into chromosome-scale assemblies (International Brachypodium Initiative 2010).
Linking individual BACs contained in physical contigs and ultimately genomic sequences to
specific chromosomes can be accomplished through a technique called “BAC landing.” In this
technique, entire BACs are fluorescently labeled and used for FISH. In this fashion, BACs were
assigned to specific chromosomes, and 32 of 39 BACs hybridized to a single locus, underscoring the
compact nature of the Brachypodium genome (Hasterok et al. 2006). A more extensive application of
the technique will be highly instructive in verifying the whole genome assembly and for comparing
the evolutionary relationships among genomes of various grasses (Wolny and Hasterok 2009).
Genetic markers are essential for many experiments, including positional cloning, mapping
QTLs, association mapping, ECOTILLING, and analysis of genotypic diversity in populations.
Polymerase chain reaction (PCR)-based markers are particularly useful because they are fast, easy
to score, and can be used by any laboratory with routine molecular biology tools. A recent publication
describes the development of 398 SSR markers for Brachypodium (Vogel et al. 2009). SSRs, also
known as microsatellites, are genomic areas with simple, short repeat units. The number of repeats
is highly polymorphic, making SSRs powerful markers. As previously discussed in Section 23.3.1,
the utility of these SSRs was demonstrated by showing that genetic diversity in a large number of
new Brachypodium accessions correlated with significant differences in easily scored phenotypes
such as seed size, vernalization requirements, and the presence of hairs (Vogel et al. 2009). Another
study used 12 SSR markers to examine introduced populations of polyploid Brachypodium and
showed that there have been multiple introductions of Brachypodium into the state of California
(Bakker et al. 2009).
23.4.5 w
holE
-g
EnomE
S
EquEncing
A completely sequenced genome underpins a host of tools, including efficient map-based cloning,
sequence-indexed T-DNA populations, gene chips, and reverse genetic approaches such as TILLING
