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19
Citrus Genomics and Breeding: Identification of Candidate Genes by
the Use of Mutants and Microarrays
M. Talon, M. Cercos, D.J. Iglesias, J.M. Colmenero-Flores, V. Ibáñez, J. Brumos,
M.A. Herrero-Ortega, G. Rios, J. Terol and F.R. Tadeo
Centro de Genómica, Instituto Valenciano de Investigaciones Agrarias (IVIA)
Moncada-46113, Valencia
Spain
Keywords: comparative genomic hybridization, fruit quality, transcriptomic
Abstract
The survival of the citrus industry is critically dependent on genetically
superior cultivars. Improvements in these traits through traditional techniques are
unfortunately extremely difficult due to the unusual combination of biological
characteristics of citrus. Genomic science holds promise of improvements in
breeding and the main goal of our group is to develop genomics tools for the
generation of new genotypes. We pursue the identification of candidate genes, alleles
and genotypes improving citrus fruit quality and performance, correlating
phenotypic analyses, metabolic profiling and gene expression. At completion, genes
and alleles with major functions in nutritional quality and stress tolerance could be
selected and genotypes with improved fruit composition searched among existing or
generated collections. This goal is supported by two complementary strategies the
identification of genes of agricultural interest by the use of mutants and microarrays
and the elucidation of the citrus genome sequence. In this communication the efforts
we are developing in this first approach are revised while the other strategy will be
resumed elsewhere.
GOALS OF CITRUS BREEDING
Major goals of variety breeding in citrus are mostly related to fruit quality,
productivity and harvesting period. In a broad sense, citrus fruit quality includes many
physical attributes like fruit size, shape, colour, texture, seedlessness, peelability and
durability. The chemical characteristics of the fruit, such as sugar and acid content,
flavour and aroma compounds (organoleptic properties) are also important. Citrus contain
the largest number of carotenoids in addition to an extensive array of secondary
compounds with pivotal nutritional properties that greatly contribute to the supply of
anticancer agents and other nutraceutical substances essential to prevent cardiovascular
and degenerative diseases, thrombosis, cancer, atherosclerosis and obesity. These traits
are acquired along fruit growth, from pollination to ripening although they can be
strongly modified during post-harvest storage. Extension of the ripening period is also a
key target for the market while abscission behaviour plays a major role in fruit
production.
Citrus Industry basically all over the world is claiming for much more knowledge
on the characteristics related to fruit quality. In addition, development of more modern
know-how and protocols to generate both phenotypic variability to generate new citrus
lines and original procedures for variety authentication are also mayor requirements of the
sector.
BREEDING PROBLEMS IN CITRUS
The reasons for the low impact of traditional breeding of this major fruit crop are
related to the peculiarities of citrus reproductive biology (see Talon and Gmitter, 2008).
Citrus show apomixy, heterozygosis, facultative parthenocarpy, sterility, and
gametophytic self- and cross-incompatibility. In addition to this unusual reproductive
biology, Citrus also possess a rare combination of intriguing biological characteristics
including non-climacteric fruit ripening, juvenility, dormancy, surprising root/shoot
Proc. IIn
d
IS on Citrus Biotechnology
Eds.: A. Gentile and S. La Malfa
Acta Hort. 892, ISHS 2011
20
interactions, and several other specific tree-traits. Overall, variety development is
extremely slow and inherently costly because it requires many years and efforts to
adequately evaluate fruit quality improvement. Furthermore, many of the commercial
citrus types produce polyembryonic seeds through nucellar embryony. These
circumstances and the narrow germplasm bases actually represented in citrus have
precluded breeding as a strategy for cultivar development and improvement.
In Spain mandarin germplasm has been the major source for breeding programs to
develop new types of mandarin hybrids using selections that produce monoembryonic
seeds containing true zygotic embryos. In general, these crosses do not render seedless
lines and therefore are not fully appreciated. This approach, hybridization and selection,
however, may produce interesting commercial rootstock and may result in productive
approaches. Manipulation of ploidy level has been recently introduced with promising
results. In all these cases, however, limited genetic understanding of the inheritance and
control of critical traits remains a substantial issue.
In addition, breeding programs based on induced mutations through irradiation
have also been developed in many countries, including Spain. Below we summarized the
work performed by our team in this connexion.
PROMISES AND HOPES OF GENOMICS
It is a pleasant surprise that in recent years several genetic, genomic, and
proteomic tools and technologies have been quickly adapted by the citrus research
community to address major challenges of this plant system (Talon and Gmitter, 2008).
Critical functional and expression analyses through microarrays with several platforms
have been published and analyses of ESTs in public databases have been initiated.
Moreover, genetic linkage maps have been produced with increasing value and resolution,
following the evolution of new marker systems. Steps for whole genome sequencing of
citrus have also been taken and a collaborative effort has been initiated to sequence a
haploid clementine genome. Strategies based on genome-wide mutagenesis are being
explored since these approaches are non-transgenic and have particular interest for the
industry.
Mutants are invaluable plant materials to help understand the relationship between
genotype and phenotype. As explained below, collections of induced mutants have been
generated through the use of physical and chemical mutagenic agents. These collections
have expanded the natural citrus mutant resources and are enabling the emergence of new
insights into the regulation of citrus fruiting. In these collections a whole lot of
phenotypic characteristics and other deviations from wild type standards can be analyzed
and mutants with altered physiological processes related to fruit shape, size, and quality
have been identified. Thus, current progress in citrus research including the rapid
development of genomic and molecular biology resources may certainly channel new
breeding efforts.
These studies will likely provide new set of tools, including methods to rapidly
identify improved genotypes in already existing collections and manipulate genes that
influence fruit quality.
INDUCED MUTATIONS
Breeding programs based on induced mutations through irradiation have also been
developed in many countries including Spain. Mutation induction has arisen as a useful
technique to increase genetic variability in plant populations, not only in citrus, but also in
other cultivars of commercial relevance. The International Atomic Energy Agency has
registered more than 2000 different mutant cultivars officially released and obtained
through irradiation mutagenic programs. Several studies have shown that irradiation
induced a large number of functional variations in plants, although the efficiency depends
on the dosage, type of mutagen and species. With this strategy, the generation of new
mutants and cultivars through mutagenesis arises as a optimum alternative for citrus
breeding, with a double interest: i) from a commercial point of view, new genotypes can
21
be directly introduced in the markets or been used as parental in breeding programs; ii)
from a scientific perspective, they constitute a unique plant material for functional
analyses.
A collection of new lines of Clementina de Nules was generated through
irradiation of buds with both gamma rays and fast neutrons, to induce a wide range of
genome alterations. Irradiation was applied in the dose range referenced for other wood
species for both gamma rays and fast neutrons. After irradiation, buds were grafted onto
citrange Carrizo rootstocks and to avoid chimeras, selection was performed in the second
bud generation. Buds finally were grafted onto adult commercial trees. Survival after
irradiation reached 50% of the number of irradiated buds (LD50). The lines pre-selected
from this collection that are listed below, showed a regular behaviour through, at least,
three years, with one or more agronomical traits significantly different from the parental
genotypes:
- Late-ripening lines. Those lines showed a delayed colour change respect to the parental
Clementina de Nules. The line 39B3 shows a strongest delay in colour break (a month
later) while other lines show the same pattern, accompanied with an additional
yellowish rind colour when reached maturity. Some lines also exhibited a good size of
fruits and significant delay in colour break (Fig. 1).
- Seedless lines. A high number of lines showed standard characteristics of clementine
fruits with minimal number of seeds in manual pollinated fruits. Some lines presented
no seeds in manual pollinated fruits after two consecutive years of study while others
are also male sterile (Fig. 2).
- Early-ripening lines. All lines of this set reached maturity significantly faster than the
parentals and a few of them showed slight reduction in fruit size.
- Durable traits. There are some lines included in this set that showed good performance
during storage on tree.
- Lines with altered fruit size/shape. These mutations include several genotypes with
altered size, shape, morphology and appearance.
- Lines with altered internal quality. Genotypes included in this subcollection show
standard characteristics except the internal ripening traits. There are lines with high and
low acidity.
Several of these lines are been analyzed via microarrays in order to identify genes
of potential agronomical interest.
COMPARATIVE GENOMIC HYBRIDIZATION AND TRANSCRIPTOMIC
ANALYSES OF MUTANTS
Several selected genotypes from this collection of mutants have been subjected to
array-Comparative Genomic Hybridization (CGH) and to transcriptomic analyses.
Recently we have used CGH for the successful identification of genes included in a
hemizygous deletion resulting in colour ripening delay induced by fast neutron irradiation
on Citrus clementina (Rios et al., 2008). Microarray-based CGH was used to identify
underrepresented genes in a citrus mutant that shows colour break delay. Subsequent
confirmation of gene doses through quantitative PCR and comparison of best hits of
putative deleted citrus genes against annotated genomes from other eudicots, especially
poplar, enabled the prediction that these genes were clustered into a 700 kb fragment. The
availability of Citrus BAC end sequences helped to draw a partial physical map of the
deletion. Furthermore, gene content and order in the deleted segment was established by
PCR location of gene hits on the physical map. Finally, a lower chlorophyll a/b ratio was
found in green tissues from the mutant, an observation that can be related to the
hemizygous deletion of a ClpC-like gene, coding a putative subunit of a multifunctional
protease complex located into the chloroplast. Analysis of gene content and order inside
this deletion led to the conclusion that microsynteny and local gene colinearity with
Populus trichocarpa were higher than with the phylogenetically closer Arabidopsis
thaliana genome. Thus, a combined strategy including genomics tools and induced citrus
mutations has been proved to be a successful approach to identify genes with major roles
22
in citrus fruit development. Basically, the study provided the gene content and order
leading to the identification of candidate genes for this phenotype. These analyses that are
currently extended to other mutations have identified additional regions deleted in
chromosomes of the mutants involved in seedlessness (Fig. 3).
Other citrus mutants affected in fruit colour have been identified and are currently
being studied. For instance, the so called nan (“navel negra”, black navel) mutant is a
spontaneous mutation of Washington Navel that exhibits an abnormal brown colour in the
ripe flavedo (Alós et al., 2007). Analysis of pigment composition in the wild type (WT)
and nan flavedo revealed typical ripening-related chlorophyll (Chl) degradation, but not
carotenoids biosynthesis, was impaired in the mutant, identifying nan as a Type C stay-
green mutant. nan exhibited normal expression of Chl biosynthetic and catabolic genes
and chlorophyllase activity, and a typical distribution of Chl derivatives during ripening,
suggesting that the mutation is not related to a lesion in any of the principal enzymatic
steps in Chl catabolism. However, transcript profiling using a citrus microarray revealed
that a citrus ortholog of a number of SGR (stay green) genes was expressed at
substantially lower levels in nan both prior to, and during, ripening. The lack of
accumulation of Chl catabolites and the reduced expression of SGR suggests that nan is
distinct from previously described stay-green mutants. Moreover, the mutation appears to
be associated with an upstream regulatory step, that rather than directly influencing a
specific component of Chl catabolism. Transcriptomic analysis and comparative
proteomic profiling using 2D-DIGE further indicated that the nan mutation resulted in the
suppressed expression of numerous photosynthesis-related genes and in the induction of
genes that are associated with oxidative stress. These data, in addition to the metabolite
analyses, suggest that nan fruit employ a number of molecular mechanisms to compensate
for the elevated Chl levels and associated photo-oxidative stress.
We have also examined gene expression during development and ripening of self-
incompatible C. clementina (Cercós et al., 2006). As many as 2243 unigenes showed
significant expression changes while functional classification revealed that genes
encoding for regulatory proteins were very abundant (more than 10% of the expression
changes). The most abundant family of up-regulated transcription factors in developing
citrus pulp was the NAC family. These genes were grouped in a very large family of
plant-specific transcription factors characterized by a highly conserved N-terminal
domain. The results of microarray analyses also identified additional transcription factor
families that were apparently deeply involved in fruit development and ripening such as
MYB and MADS genes. Genes encoding proteins with potential functions in signal
transduction mechanisms were also found, such as protein phosphatases, Ras-related
small GTP binding proteins and serine/threonine protein kinases.
We have also used two independent mutations showing delayed colour break to
isolate genes related to peel colour change. In this investigation, pigment analyses
revealed that the pattern of change in both was dissimilar although colour break in both
was equally delayed during one month. The transcriptomic analyses identified around 80
genes that were up- and down-regulated simultaneously in both mutants. These genes
were mostly included in functional categories related to metabolism, photosynthesis and
also to transport, a category specially over-represented with down-regulated mineral
transporters. Interestingly only a MYB-CC type transcriptional factor, coded by CcMYR1
gene, was found to be repressed in both. The study also revealed that the yellowish colour
of fully ripe flavedo of one of the mutants was due to a defective synthesis of β-citraurin.
CcMYR1 is therefore an important component of the regulatory pathway of colour change
in citrus.
Citrate degradation is an important matter and has been the subject of much
research. Once in the cytosol, citrate is apparently metabolized to isocitrate by cytosolic
aconitase and then into 2-oxoglutarate by NADP isocitrate dehydrogenase. Further data
on 2-oxoglutarate utilization (Cercós et al., 2006) propose that citrate is sequentially
metabolized to glutamate that is either used for thiamine biosynthesis or catabolized
through the gamma-aminobutirate (GABA) shunt. This observation is of special relevance
23
since it links an efficient major proton consuming reaction with the occurrence of high
acid levels. It is known that in higher plants GABA is biosynthetized through a proton
consuming reaction catalyzed by glutamate decarboxylase. The suggestion provides a
convincing explanation for the strong reduction of both citrate and cytoplasmic acidity
that takes place in citrus fruit flesh during development and ripening. Crossing microarray
data from several mutants with dissimilar acid levels it has also been possible to identify a
set of genes deeply involved in acidity regulation (Fig. 4). For instance, a strong increase
in the expression of several genes of gluconeogenesis was found to occur in parallel to a
decrease in acidity, linking in this way acid catabolism with an increase in the
carbohydrate content in mature citrus fruits.
CONCLUSIONS AND FUTURE PERSPECTIVES (CONCLUSIONS)
It is a pleasant surprise that in recent years several genetic, genomic, and
proteomic tools and technologies have been quickly adapted by the citrus research
community to address major challenges of this plant system (Talon and Gmitter, 2008).
Critical functional and expression analyses through microarrays with several platforms
have been published and analyses of ESTs in public databases have been initiated (Terol
et al., 2007). Recently, strategies based on genome-wide mutagenesis are being explored
since these approaches are non-transgenic and have particular interest for the industry.
Mutants generated in this way are invaluable plant materials to help understand the
relationship between genotype and phenotype. As explained above, collections of induced
citrus mutants have been generated through the use of physical and chemical mutagenic
agents. These collections have expanded the natural citrus mutant resources and are
enabling the emergence of new insights into the regulation of citrus fruiting. In these
collections a whole lot of phenotypic characteristics and other deviations from wild type
standards can be analyzed and mutants with altered physiological processes related to
fruit shape, size, and quality have been identified. Thus, current progress in citrus
research including the rapid development of genomic, mutants and molecular biology
resources may certainly channel further goals in breeding. Genomics developments can
provide researchers with a new set of tools, including methods to rapidly identify
improved genotypes in already existing collections and manipulate genes that influence
fruit quality. These studies will likely provide knowledge that may allow more rapid
production of new citrus varieties with enhanced nutritional or agricultural values.
In conclusion, the work summarized above corroborates the potential of Citrus
genomic resources to assist mutagenesis-based approaches for functional genetics,
structural studies and comparative genomics, and hence to facilitate citrus variety
improvement.
ACKNOWLEDGMENTS
Work at Centro de Genómica was supported by the Ministerio de Educación y
Ciencia-FEDER grant AGL2007-65437-C04-01/AGR.
Literature Cited
Alós, E., Roca, M., Iglesias, D.J., Mínguez-, M.I., Damasceno, C.M.B., Thannhauser,
T.W., Rose, J.K.C., Talon, M. and Cercos, M. 2008. An evaluation of the basis and
consequences of a stay-green mutation in the navel negra (nan) citrus mutant using
transcriptomic and proteomic profiling and metabolite analysis. Plant Physiol. 147:
1300-1315.
Cercós, M., Soler, G., Iglesias, D.J., Gadea, J., Forment, J. and Talón, M. 2006. Global
analysis of gene expression during development and ripening of citrus fruit flesh. A
proposed mechanism for citric acid utilization. Plant Mol. Biol. 62:513-527.
Ríos, G., Naranjo, M.A., Iglesias, D.J., Ruiz-Rivero, O., Geraud, M., López-García, A.
and Talon, M. 2008. Characterization of hemizygous deletions in Citrus using array-
comparative genomic hybridization and microsynteny comparison with the poplar
genome. BMC Genomics 9:381.
24
Talon, M. and Gmitter, F.G. Jr. 2008. Citrus Genomics. Intl. J. Plant Genomics,
doi:10.1155/2008/528361.
Terol, J., Conesa, A., Colmenero-Flores, J.M., Cercos, M., Tadeo, F.R., Agustí, J., Alós,
E., Andres, F., Soler, G., Brumos, J., Iglesias, D,J., Götz, S., Legaz, F., Argout, X.,
Courtois, B., Ollitrault, P., Dossat, C., Wincker, P., Morillon, R. and Talon, M. 2007.
Analysis of 13000 unique Citrus clusters associated with fruit quality, production and
salinity tolerance. BMC Genomics 8:31.
Figures
Fig. 1. Pictures of clementine mutant 809 B and clementine control fruits taken at the
beginning of March when control fruits are clearly overripe.
Fig. 2. Pollen germination ability of mutant 345C and control clementine.
25
Fig. 3. Schematic representation in poplar chromosomes of putative homolog deleted
regions in citrus.
C
oe
fi
c
i
ente
d
e
C
orre
l
ac
ió
n
-1 -0.5 0 0.5 1 1.5 2
= -0.72
p < 0.01
Fig. 4. Correlation between acidity and expression of CcPFNL, a gene involved in the
regulation of gluconeogenesis. Differential gene expression obtained by
microarrays analyses was studied in 10 different citrus mutants.
Acidity (mutant-control/control)
Differential gene expression
1.0
0.5
0.0
-0.5
-1.0
A
13.5 Mb
19.1 Mb
B
19.1 Mb
1 2 3
4 5
1 2
