Genome Organization and Comparative Genomics - Wheat Science and Trade - page 333

chromosomes per second, and to collect several
hundred thousand chromosomes per day. More-
over, the use of double ditelosomic (dDt) lines
(i.e., lines carrying both arms of a chromosome as
pairs of telosomes) enabled the simultaneous
sorting of the long and short chromosome arms
(Fig. 15.3d). Depending on the size of the arm,
the typical daily yield ranges from 100 to 200 ng
DNA and, within several weeks, suffi cient micro-
grams of DNA are available for BAC library con-
struction. However, it was not clear that the DNA
of fl ow-sorted chromosomes would be intact, a
critical aspect, until Vrána et al. (2000) demon-
strated that DNA of fl ow-sorted wheat chromo-
somes was of high molecular weight. Subsequently,
ˇ imková et al. (2003) developed a protocol for
preparing high-molecular-weight DNA from
fl ow-sorted chromosomes, and this study opened
a way to the construction of high-quality, chro-
mosome (arm)-specifi c BAC libraries.
Construction of the fi rst-ever chromosome-
specifi c BAC library from a eukaryote genome, a
BAC library specifi c for wheat chromosome 3B,
was reported by ˇ afᡠet al. (2004). The library
was constructed from 1.8 million 3B chromo-
somes from Chinese Spring (approximately 4 μg

DNA) that were sorted in 18 working days. In
total, 4,000 seeds were used to prepare 200 samples
of chromosome suspensions for fl ow sorting. The
library was constructed using a pIndigoBAC
vector and the 67,968 BAC clones were ordered
in 177 × 384-well plates. With an average insert
size of about 103 kb, as estimated by pulse fi eld
electrophoresis, the library represents 6.2-fold
coverage of chromosome 3B (995 Mb) ( ˇ afáˇ
et al., 2004).
The reproducibility of BAC library construc-
tion from sorted chromosomes was confi rmed by
Janda et al. (2004), who constructed a library
from chromosomes 1D, 4D, and 6D, that were
sorted as a group from the composite peak I of
the Chinese Spring fl ow karyotype (Table 15.2,
Fig. 15.3a). While these experiments demon-
strated the feasibility of developing genomic
resources from sorted chromosomes, the inability
to sort other chromosomes individually or in
small groups necessitated the construction of
libraries from chromosome arms. The fi rst library
from a wheat chromosome arm (1BS) was
reported by Janda et al. (2006). With only 65,280
BAC clones, the library provided 14.5-fold
coverage of the short arm. Similar libraries have

Table 15.2 BAC libraries that have been constructed from fl ow-sorted chromosomes of hexaploid wheat.

BAC library ( Hin dIII cloning site)

Molecular
Size (Mb)

Genome
Fraction (%)

Number
of Clones

Mean Insert
Size (kb)

Chromosome
Coverage Cultivar Chromosome Reference

Chinese Spring 1D, 4D, 6D 1,964 11.6 87,168 85 3.4x Janda et al. (2004)

ˇ afá ˇ et al.
(pers. comm.)

Chinese Spring 1D, 4D, 6D 1,964 11.6 181,632 102 8.4x

ˇ afá ˇ et al. (2004) Chinese Spring 3B 993 5.9 67,968 103 6.2x

ˇ imková et al.
(pers. comm.)

Hope 3B 993 5.9 92,160 78 6.0x

Pavon 1BS 314 1.9 65,280 82 14.5x Janda et al. (2006)

ˇ imková et al.
(2007)

Chinese Spring 3AS 360 2.1 55,296 80 10.9x

ˇ afá ˇ et al.
(pers. comm.)

Chinese Spring 3AL 468 2.8 55,296 106 10.2x

ˇ afá ˇ et al.
(pers. comm.)

Chinese Spring 3DS 321 1.9 36,864 110 11.0x

ˇ afᡠet al.
(pers. comm.)

Chinese Spring 3DL 449 2.7 64,512 105 12.2x

ˇ imková et al.
(pers. comm.)

Chinese Spring 7DS 381 2.0 49,152 114 12.2x