LIBRO-CONGRESO-CITRUS

In citrus, it would be difficult to recover the elite cultivar (such as a sweet orange) following a cross, so an
alternative method to introduce these ZFN genes will be needed. Such genes could be carried on a virus
that infects the initial cultivar and is later removed by thermotherapy or shoot-tip grafting before the new
mutated cultivar is released. These approaches will allow breeders to reduce or eliminate function of one
copy of a gene. To have a phenotypic effect, the mutation would have to have some level of dominance. In
annual crops new mutations are commonly made homozygous by selfing or sib-mating, methods that are
either not possible in citrus or which yield progeny very unlike the parental cultivar type. Possibly targeted
gene modification can be efficient enough to produce mutations in both alleles. It is also important to
recognize the limits of these methods – they produce mostly loss-of-function mutations, and not all desired
phenotypes can be created by such mutations.
High quality reference genome sequences for citrus have been developed by the International Citrus
Genomics Consortium (haploid from clementine), the University of Florida (diploid sweet orange) and
Huazhong University (haploid of sweet orange). With these reference sequences available, advances in
genome sequencing now make it simple and inexpensive to determine most of the genome sequence of
interesting mutants and cultivars. Comparison of sequences from a few varieties indicates the ancestral
species that contributed each chromosome segment to hybrids and may even indicate the precise
genotype of each parent. Comparative genome sequence information will allow identification of candidate
genes that may cause phenotypic differences among cultivars derived by mutation (navel oranges with
different maturity dates, grapefruit differing in flesh color etc.). Once genes are known, they can be more
easily transferred to other cultivar groups by hybridization-selection, transgenics, or targeted mutation
approaches. Breeding by hybridization-selection methods is also increasing in efficiency through use of
genome sequence information and methods. Several groups have now developed SNP arrays that can be
used to rapidly determine the overall genotype of each individual progeny. Projects that required years of
work using “one at a time” markers such as SSRs can now be completed in a few weeks. Genotyping-bynextgen
sequencing methods, in which a defined fraction of genome fragments is sequenced in multiplex,
is another approach to this end. Development of these methods, particularly when combined with gene
expression studies, will greatly facilitate identification of genes that confer disease resistance, fruit quality
and other important traits. Efficient, high-density genotyping should also facilitate identification of progeny
plants that resemble specific genotypes such as sweet orange or clementine and thereby allow breeders
to develop hybrids that closely resemble these cultivars except for chromosome segments carrying desired
genes. These technologies will reduce the “random” element in hybridization-selection breeding, but
exploiting them will not be inexpensive because achieving these objectives will require molecular analysis
of large populations.
New methods also promise to revolutionize development of citrus cultivars using transgenic approaches. In
the past, nearly all transgenes were introduced using methods such as Agrobacterium or biolistics that result
in integration at more or less random locations in the genome. The genomic context in which a transgene
integrates can affect its expression and this contributes to the need to screen a large number of transgenics
to find those with appropriate expression levels. Random integration also complicates regulatory review
of transgenics. Targeted integration of transgenes is now possible by engineering a “founder line” having a
construct with good gene expression and an integration site developed with a recombinase-mediated cassette
exchange strategy. Such lines can then be transformed with additional genes that will be integrated at this
site. Another significant opportunity is development of cis-genic varieties in which the added DNA originates
from citrus rather than from an unrelated species. It is possible to envision systems in which all genes
introduced, including selectable markers, originate from citrus sequences. Such varieties may encounter less
public opposition than those that contain genes from a non-citrus species. Despite the potential power and
promise of these new tools, real progress will also depend on accurate plant phenotyping and subsequent
field trials to identify superior cultivars. Success will also be facilitated by sharing information widely in the
citrus community because there are many challenges to be addressed and few citrus breeding groups to
address them.
XII INTERNATIONAL CITRUS CONGRESS 2012 - 9