Biology Reference
In-Depth Information
would presumably be active (our unpublished
data). Yet, BR28 is manifestly sensitive, raising
the possibility that the BR28 genetic background
may have additional loci suppressing the effects
of Saltol , at least on whole-plant performance.
More data are needed to explore the SKC1 alle-
les in other recipient varieties and further inves-
tigate the usefulness of the Pokkali Saltol QTL
for breeding applications.
Thus, cloning of QTLs is of significant value
in plant breeding, both in terms of develop-
ing perfect markers for MABC and in accu-
rate identification of potential recipient lines.
Yet, the cloning of QTLs still relies on one of
two techniques, both costly and labor-intensive.
Either the QTL must be fine-mapped in a
large population to narrow down the limits, or
researchers must sort through many candidate
genes, testing such parameters as expression
changes under treatment, sequence polymor-
phisms (cloning), and eventually transgenic vali-
dation. Both approaches have been used success-
fully in salinity research (fine-mapping of SKC1 ,
Ren et al. 2005; the candidate gene approach for
wheat Nax1 and Nax2 , Byrt et al. 2007; Huang
et al. 2006), but neither is simple or “high-
throughput” in nature. Yet, the large number -
and relatively small effect - of many salinity-
tolerance QTLs means that, to achieve high
tolerance, multiple QTLs will need to be intro-
gressed into any potential recipient germplasm.
This will multiply the number of genes that
must be identified, while at the same time mak-
ing it more and more likely that the mutations
responsible for tolerance alleles will be sub-
tle in nature and possibly difficult to identify.
Thus, it remains challenging to identify the genes
and causal mutations responsible for salinity-
tolerance QTLs. In some respects, approaches
that do not rely on QTL isolation, such as marker-
assisted recurrent selection and genomic selec-
tion, may prove useful as alternative molecular
breeding strategies in the future (Bernardo 2008).
However, the benefits of isolating the genes
underlying QTLs makes the challenge worth-
while, since it enables more efficient allele min-
ing, development of perfect markers, and char-
acterization of the molecular mechanisms under-
lying salinity tolerance.
Next-generation Sequencing:
Advances and Limitations
The significant advances made in genomics
techniques, from microarrays to very-high-
throughput sequencing and SNP detection,
provide enormous opportunities to sidestep and
overcome these limitations in QTL mapping,
candidate gene identification, and marker-
assisted breeding. Much of the utility of
next-generation genomics techniques revolves
around the revolution in sequencing technology
that started with MPSS (massively-parallel
signature sequencing; Brenner et al. 2000) and
was brought to its first widely useful appli-
cations in the 454 pyrosequencing (454 Life
Sciences, www.my454.com; now part of Roche
Diagnostics) and Illumina (formerly Solexa,
www.illumina.com) sequencing platforms.
Other platforms include sequencing-by-ligation
techniques such as those used by the Applied
Biosystems SOLiD platform and the Complete
Genomics system, the IonTorrent system,
Pacific Biosciences' single-molecule real-time
(SMRT) sequencing, and methods still under
development,
such
as
Oxford's
Nanopore
sequencing technique.
Many of these technologies are based around
principles similar to the dideoxy terminator tech-
nique used in traditional Sanger sequencing.
However, they differ in a few key details. The
Sanger process involves a single, static reac-
tion using a single, defined primer on a single,
pure template, and produces a complex mixture
of products that are subsequently resolved via
a high-resolution electrophoresis system. This
allows the Sanger process to produce long reads
of high-quality contiguous sequence, but it lim-
its the process to sequencing pure, defined tem-
plates (e.g., PCR product or cloned DNA) at
defined locations (determined by the sequencing
primer). This makes it difficult, laborious, and
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