persea americana (avocado): bringing ancient flowers to fruit in the genomics era
TRANSCRIPT
Persea americana (avocado):bringing ancient flowers tofruit in the genomics eraAndre S. Chanderbali,1,2 Victor A. Albert,3* Vanessa E.T.M. Ashworth,4
Michael T. Clegg,4 Richard E. Litz,5 Douglas E. Soltis,1 and Pamela S. Soltis2
SummaryThe avocado (Persea americana) is a major crop com-modity worldwide. Moreover, avocado, a paleopolyploid,is an evolutionary ‘‘outpost’’ among flowering plants,representing a basal lineage (the magnoliid clade) nearthe origin of the flowering plants themselves. Followingcenturies of selective breeding, avocado germplasm hasbeen characterized at the level of microsatellite and RFLPmarkers. Nonetheless, little is known beyond thesegeneral diversity estimates, and much work remains tobe done to develop avocado as a major subtropical-zonecrop. Among the goals of avocado improvement are todevelop varieties with fruit that will ‘‘store’’ better on thetree, show uniform ripening and have better post-harveststorage. Avocado transcriptome sequencing, genomemapping and partial genomic sequencing will represent amajor step toward the goal of sequencing the entireavocado genome, which is expected to aid in improvingavocado varieties and production, as well as under-standing the evolution of flowers from non-floweringseed plants (gymnosperms). Additionally, continuedevolutionary and other comparative studies of flowerand fruit development in different avocado strains can beaccomplished at the gene expression level, including incomparison with avocado relatives, and these shouldprovide important insights into the genetic regulation offruit development in basal angiosperms. BioEssays30:386–396, 2008. � 2008 Wiley Periodicals, Inc.
Introduction
Our ‘‘favorite plant’’ is the avocado (Persea americana Mill.),
widely celebrated for providing the guacamole of Mexican
cuisine. Our interest in avocado, however, has little to do with
gastronomic delight. We search for the secrets of flowering
plant reproductive genetics and, as one of the rare crop plants
among the basal angiosperms, avocado provides an oppor-
tunity to explore this fundamental botanical process in an
unprecedented context. In this review, we describe the
botanical significance of the avocado, summarize existing
genetic resources and current research avenues, and
introduce opportunities that would pave the way for avocado
into the genomics era.
Avocado: a long history of production
and consumption
Avocado is a major fruit crop; world production was 3,222,069
metric tons (Mt) valued at $606,608,000 in 2004.(1) Leading
producers include Mexico (987,000 Mt), the United States
(247,000 Mt), Indonesia (263,575Mt), Colombia (185,811 Mt),
Brazil (182,000 Mt), Chile (163,000 Mt), the Dominican
Republic (140,000 Mt), Peru (102,000 Mt), China (85,000 Mt),
and South Africa (59,534 Mt). The European Union and North
America are the largest importers of avocados. Although the
avocado is consumed primarily as a fresh fruit, it is also a rich
source of oil that is of rapidly growing use in the cosmetics
industry.(2) The health-related benefits of avocado, including
cancer prevention, are also gaining attention.(3–6)
Although relatively new to international commerce, the
avocado has been used for at least 9000 years in and near its
center of origin in Mexico and Central America.(7,8) There
are eight well-defined varieties or geographical ecotypes of
Persea americana, of which three, referred to as horticultural
races in the literature, comprise the commercial avocado.(9)
The Mexican race, P. americana var. drymifolia (Schltdl. &
Cham.) S.F. Blake, is adapted to the tropical highlands. The
Guatemalan race, P. americana var. guatemalensis (L.O.
Williams) Scora, is adapted to medium elevations in the
tropics, and the West Indian race, P. americana var.
americana, is adapted to the lowland humid tropics.(10)
Commercial avocado production is based on selections within
these three races and of hybrids among them, most commonly
1Department of Botany, University of Florida, Gainesville, FL.2Florida Museum of Natural History, University of Florida, Gainesville,
FL.3Department of Biological Sciences, University at Buffalo (SUNY),
Buffalo, NY.4Ecology and Evolutionary Biology, University of California, Irvine, CA.5Horticultural Sciences, Tropical Research & Education Center,
University of Florida, Hemestead, FL.
Funding agencies: Our research on avocado is supported by California
Avocado Commission to MTC, VETMA, and REL, USDA Tropical and
Subtropical Agricultural Research (TSTAR) grants to REL, and NSF
grants PGR-0115684 (Floral Genome Project) and DBI-0638595
(Ancestral Angiosperm Genome Project) to DES, PSS, VAA and ASC.
*Correspondence to: Victor A. Albert, Department of Biological
Sciences, University of Buffalo (SUNY), 109 Cooke Hall, Buffalo, NY
14260-1300. E-mail: [email protected]
DOI 10.1002/bies.20721
Published online in Wiley InterScience (www.interscience.wiley.com).
386 BioEssays 30.4 BioEssays 30:386–396, � 2008 Wiley Periodicals, Inc.
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Mexican�Guatemalan (e.g. ‘Hass’). Table 1, modified from
Crane et al. 2005,(11) summarizes the major characteristics of
the three races of avocado.
An evolutionary outpost among
basal angiosperms
Avocado is a member of Lauraceae (Laurales; Fig. 1), a large
pantropical family of about 50 genera and approximately
2500–3000 species of trees, rarely shrubs, and one genus of
parasitic vines, Cassytha.(12) Lauraceae have both econom-
ical and cultural importance. The Greeks offered leaves of
Laurus nobilis, the commercial bay laurel, to reward accom-
plishment, a practice echoed by the term ‘‘laureate’’ in modern
vocabulary. Other uses include cinnamon and camphor from
Cinnamomum, bases for perfume oils from Aniba, and durable
timber from Chlorocardium, Eusideroxylon, Persea, and
Mezilaurus. Avocado, however, is undoubtedly the most-
important commodity from Lauraceae. Lauraceae belong to
a clade containing most of the ‘‘primitive angiosperms’’ of
earlier classification schemes,(13,14) the magnoliids, and which
appear to be sister to the eudicot and monocot clades that
accommodate most extant angiosperms.(15,16) Most magno-
liids have large flowers with numerous, spirally arranged
stamens, carpels and tepals (perianth parts that are not
differentiated into petals and sepals). The phylogeny of
magnoliids resolves as four major lineages with Magnoliales
(magnolias and relatives) sister to Laurales (avocado and
relatives) in one clade and Piperales (black pepper relatives)
sister to Canellales (white cinnamon) in the other.(15,16)
Laurales are characterized by flowers in which the ovary is
frequently deeply embedded in a fleshy receptacle.(17) Within
Laurales many prominent trends in floral evolution are evident.
For example, floral phyllotaxy ranges from spiral to whorled,
fusion among floral parts sometimes occurs, and both
reductions and increases in the number of floral organs are
evident in several lineages.(17) Laurales have a rich fossil
record and were especially abundant and widespread in the
Mid-Cretaceous,(18) but the extant species richness (2800–
3500 taxa) and their widespread geographic distribution is
mostly provided by Lauraceae.(19)
In the phylogenetic and biogeographic reconstructions for
Lauraceae,(20) avocado is placed in a large terminal clade
that radiated in Laurasia during the early Eocene. Its closest
relatives are all taxa with fruits lacking the persistent floral
cup typical of most Lauraceae. These constitute the Persea
generic group and have an amphipacific disjunction between
(sub)tropical America (Persea) and Asia (Alseodaphne,
Dehaasia, Machilus, Nothaphoebe, Phoebe) with two species
(Apollonias barbujana, Persea indica) in Macronesia. This
geographic pattern may result from climatic cooling at the end
of the Eocene that restricted essentially continuous Northern
Hemisphere floras to tropical latitudes. Relationships among
these genera and within Persea have yet to be addressed
through molecular phylogenetic approaches.
Avocado and ancient polyploidy
It has been proposed that Lauraceae, as well as other
angiosperm families having high basic chromosome numbers,
represent ancient polyploid lineages.(21) The chromosome
number (2n¼ 24) for avocado is typical of most Lauraceae and
represents the lowest chromosome number in the family.
This high base number (x¼ 12) suggested that all extant
Lauraceae resulted from an ancient polyploidy event (or
events), with the original diploid progenitors now extinct.(21)
Isozyme data testing the hypothesis of ancient polyploidy
for Lauraceae (including avocado), and several other families,
indicate that, in both Lauraceae and Magnoliaceae, 25–29%
of the loci were duplicated and could have arisen via
polyploidy.(22) All species of Lauraceae investigated share
the same gene duplications, suggesting that a single genome
duplication may have predated the diversification of the family.
Likewise, all species of Magnoliaceae share a set of dupli-
cations, indicating genome duplication in the early evolution
of that family as well. Furthermore, because Lauraceae and
Magnoliaceae share similar duplications, a single genome
duplication may have occurred in the common ancestor of
these two sister groups. Further evidence of ancient polyploidy
in Lauraceae and Magnoliaceae(23) was provided by genomic
Table 1. Characteristics of the three horticultural races of commercial avocado
Characteristics West Indian Guatemalan Mexican
Origin Tropical lowlands Tropical highlands Tropical highlands
Foliage No odor No odor Anise-scented
Blooming season February to March March to April January to February
Maturity season May to September September to January June to October
Development period 5 to 8 months 10 to 15 months 6 to 8 months
Fruit size 0.5 to 2 kg 0.2 to 2 kg Not over 0.5 kg
Skin texture Leathery-smooth Woody-rough Papery-smooth
Fruit oil content Low Medium to high Medium to high
Cold hardiness Low Moderate to high High
Fruit ripening No on-tree storage On-tree storage On-tree storage
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BioEssays 30.4 387
data for avocado and Liriodendron (Magnoliaceae),(24) corro-
borating the earlier isozyme analyses. Analyses of paralogous
gene sets suggested polyploidy in a common ancestor of
avocado and Liriodendron at least 100 million years ago,
followed by a second, later episode of ancient polyploidy in the
Lauraceae lineage.(23) If this scenario is correct, the duplicated
isozyme loci observed in both Magnoliaceae and Lauraceae
may have arisen from a shared genome doubling event
that predated the separation of the Magnoliales and Laurales.
This intriguing hypothesis can be assessed by phylogenetic
analyses of gene families containing duplicated lineages in
both Lauraceae and Magnoliaceae.
Figure 1. Strongly supported summary angiosperm phylogeny (modified from Ref. 109) in accordance with recent congruent chloroplast
genome phylogenies.(15,16) Persea americana (Laurales) occupies a phylogenetically pivotal position in the magnoliids near the base of the
topology. Black boxes indicate the position of established genetic models in the Brassicales (Arabidopsis), Lamiales (Antirrhinum),
Solanales (tomato), and Poales (rice, maize). Other non-model fruit based crops with extensive genomic resources include cotton and
cocoa (Malvales), apple, plum, peach and strawberry (Rosales), blueberry (Ericales), grape (Vitales), and orange (Sapindales), all
eudicots.
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388 BioEssays 30.4
Avocado evo-devo: a glimpse of ancient whorls
Research in floral evolution and floral development (often
referred to as part of a new field, the evolution of development,
or ‘‘evo-devo’’) has as its underlying framework what is widely
known as ‘‘the ABC model’’ of floral organ identity. Based on
detailed genetic studies of Arabidopsis thaliana (a member of
the mustard family, or Brassicaceae) and the snapdragon,
Antirrhinum majus (a member of the Plantaginaceae), the
ABC model posits that three functions (A, B and C) act in
a combinatorial manner to specify the identities of the four
primary floral organs: the A function alone specifies sepals,
the A and B functions together specify petals, the B and C
functions together specify stamens, and the C function alone
specifies carpels.(25) The specification of ovule identity via the
D function,(26) and transcriptional activation of the ABC genes
by the E function,(27) have since been added to the original
ABC model. Elegant in its simplicity, the ABC(DE) model has
served as a unifying paradigm for floral developmental genetic
research for over two decades,(28) and many aspects of the
model also seem to be applicable to a wide array of model
eudicots as well as model monocots, specifically, rice and
maize.(29,30)
We have begun to assess the applicability of the ABC(DE)
model to, and/or modification in, avocado.(31) Avocado flowers
are trimerous with two perianth whorls of nearly identical
laminar organs (tepals), three whorls of stamens, and a fourth
inner whorl of staminodes surrounding a single carpel. The
distinction of green leaf-like sepals with protective function
from colorful, often delicately textured petals functioning in
pollinator attraction is usually straightforward in the core
eudicots, but it is not present in avocado. Avocado tepals are
typically densely pubescent with a greenish yellow coloration,
neither distinctly sepaloid nor petaloid (Fig. 2). Except for their
undifferentiated perianth, avocado flowers are structurally
similar to the eudicot models and thus would be predicted to
be in general regulatory accordance with the ABC(DE) model.
Indeed, expression of homologs of the E function gene
SEPALLATA3 (SEP3) has been detected in all floral whorls,
SEP3 and AGAMOUS (AG) homologs (C function) in carpels,
and these together with homologs of APETALA3 (AP3) and
PISTILLATA (PI) (B function) in stamens.(31) Therefore the B,
C and E components appear to be deployed in avocado as they
are in stamens and carpels of other angiosperms. However,
the situation in the perianth is discordant. Remarkably,
homologs of AG, together with B and E function homologs,
have been detected in the perianth. The presence of B and E
function genes might be expected of petals, but these together
with C function homologs would only be expected of stamens.
The C function of AG homologs is considered evolutionarily
conserved in seed plants, specifying reproductive organ
identity in diverse angiosperms and gymnosperms.(32) Their
expression in avocado tepals might therefore represent the
genetic footprints of stamen ancestors, a vestige of a staminal
past. In support of a ‘‘staminodial’’ scenario for the evolution of
avocado perianth, it is perhaps significant that a homolog
of AG is expressed in staminodes of the water lily Nuphar
advena,(33) consistent with the AþBþC genetic equation
envisioned for staminode identity.(34) Tepaloid staminodes
have replaced outer stamen whorls in other Lauraceae,
including Dicypellium, Phyllostemonodaphne, Eusideroxylon
and Endlicheria,(12,35) perhaps indicating a genetic predis-
position that underlies the evolutionary origin of Lauraceae
tepals in general.(31) Avocado is therefore an attractive subject
for further investigations of perianth evolution, especially
vis-a-vis stamen sterilization in an ancient angiosperm
lineage, and perhaps for reproductive development in general.
From avocado flowers to avocado fruits
Fruits are unique to flowering plants and a major component
of human and animal diets. A wide variety of fruit types has
evolved, and this diversity is poorly represented in current
genetic models. Arabidopsis has dry dehiscent capsules,
maize (Zea) and rice (Oryza) are cereal grains, and tomato
(Solanum) produces fleshy berries. Tomato has become the
most tractable economically targeted system for molecular
genetic analysis of fleshy fruit development and ripening.(36)
Recent genetic insights have also come from large EST
sequencing projects with grape (Vitis), also a berry(37) (for
which there is now a genome sequence),(38) strawberry
(Fragaria), an aggregate of achenes (dry single seeded fruits)
Figure 2. Avocado flowers have an undifferentiated perianth
of two whorls of three tepals, three whorls of stamens, and one
whorl of staminodes, surrounding a single central carpel.
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BioEssays 30.4 389
on a fleshy receptacle,(39) and apple (Malus), a pome, in which
the ovary and seeds are encased in a fleshy receptacle.(40,41)
Little, however, is known about the developmental genetics of
the fleshy fruits of the earliest angiosperms. As avocado
produces fleshy fruit of commercial importance, and is also a
basal angiosperm, it provides an opportunity to examine the
developmental genetics of fleshy fruits in an unprecedented
context.
The avocado is a large drupe (a fleshy fruit containing a
single seed; Fig. 3) with the highest oil content of all fruits, with
the possible exception of the olive, and may be the most
nutritious of all fruits.(42) Barlow(43) considers the avocado ‘‘an
overbuilt and extravagant fruit’’ that co-evolved with large
dispersal agents that are now extinct. Fruit development and
ripening in avocado are unique when compared with other
angiosperms, including tomato, the model from which our
understanding of the genetic regulation of fruit development
and ripening has been primarily derived. The development of
most fleshy fruits typically proceeds through an early phase of
rapid cell division followed by cell enlargement.(44) In contrast,
cell division continues in the mesocarp of avocado as long as it
is attached to the tree.(45) Differences in fruit size, therefore,
derive mainly from differences in cell number rather than cell
size.
Regulation of avocado fruit ripening
The avocado is strongly climacteric,(46–48) as are tomato,
apple, banana, and most stone fruits; i.e. they ripen through a
rapid increase in respiration rate and ethylene evolution.(36)
However, while most fleshy fruit will ripen while attached to the
parent plant, fruit of Guatemalan and Mexican avocado will
remain attached to the tree after horticultural maturity, and will
not ripen unless harvested. Healthy fruits remaining on trees
stay firm and continue to grow and to accumulate oil for
several months after maturation. Fruits of cultivar ‘Fuerte’ have
remained on trees for 10 months after attaining horticultural
maturity.(49) This ‘‘on-tree storage’’ leads to extended seasons
for producers, limited only by a gradual decline in fruit pala-
tability, and together with different climatic conditions, has
enabled producers in many areas to concentrate on a single
cultivar, ‘Hass’.(50) West Indian, and West Indian� Guatemalan
hybrids, in contrast, do not exhibit ‘‘on-tree storage’’, but ripen
and drop if not harvested at maturity. To assure year-round
production of West Indian and West Indian�Guatemalan fruit
in the tropics, several cultivars having different maturity dates
must be grown simultaneously.(51)
Avocado ripening is biphasic, with a lag phase or
preclimacteric period during which ethylene levels are low,
followed by the second phase or climacteric peak with elevated
ethylene levels that cause and accompanya respiration climax
attended by ripening phenomena.(48,52–54) Therefore, the
failure of attached fruit to ripen, or ‘‘on-tree storage’’, has
been attributed to (i) presence of an ethylene inhibitor in the
fruit stem,(47,55) (ii) translocation of an ethylene inhibitor into
the fruit from the tree(46,48,56) and (iii) emission of trace
amounts of ethylene from fruits that are attached to the
tree.(52) Ethylene inhibition may be attributable to the action
Figure 3. Avocados are large fleshy fruits with considerable
variety in shape, skin color and pulp texture. Cultivars ‘Russell’
(top) and ‘Hardee’ (bottom) are both members of the West
Indian race but show striking differences in length, approx-
imately 30 and 20 cm, respectively, fruit shape and skin color.
The gourd-like shape of ‘Russell’ and red skin of ‘Hardee’ are
unique. The shiny black skin and smaller size (approximately
10 cm in length) of cultivar ‘Brogdon’ (middle) are typical of
Mexican avocado varieties.
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390 BioEssays 30.4
of senescence-retarding hormones, including auxins,
gibberellins and cytokinins that reduce tissue sensitivity to
ethylene.(57)
The avocado offers unique insights into fruit
developmental genetics
The on-tree storage of some avocado cultivars provides
a unique opportunity to examine the genetic regulation of
delayed ripening. In particular, gene expression profiling of
mature fruit during on-tree storage (pre-climacteric), com-
pared to immature and ripening (climacteric) fruit, will provide
considerable insights into the developmental genetic regu-
lation of these key phases of fruit development. Further,
although a great deal is known regarding specific downstream
ripening processes in a number of fruits, little is known about
the upstream regulation of ethylene-mediated ripening.(36)
Recent evidence of MADS box gene regulation of ripening in
both tomato and strawberry suggests common regulatory
mechanisms operating early in both climacteric and non-
climacteric species.(56) Elucidation of the molecular basis of
early and common events in fruit ripening is an active research
frontier,(36) and the inclusion of an important basal angiosperm
fruit, avocado (Fig. 1), will provide valuable insights into fruit
development close to the evolutionary origin of the angio-
sperms, and therefore of the origin of the fruit itself.
In addition to the unique insights that the avocado deve-
lopmental program can provide, a broad range of phenotypic
diversity in commercial avocado cultivars (Table 2) can be
exploited to assess the genetic regulation of complex fruit
quality traits. The international market standard for avocados
is the Guatemalan�Mexican hybrid black-skinned cultivar
‘Hass’, and to a much lesser extent, the green-skinned
‘Fuerte’. Several cultivars that closely resemble ‘Hass’ have
been released to supplement this selection, particularly in its
off-season, including ‘Gwen’, ‘Jim’, ‘Lamb Hass’ and ‘Reed’.
Optimum fruit size for most markets is about 8–12 g,(59) but
size is highly variable in each genotype and is affected by
stage of maturity, cultural practices and climatic condi-
tions.(60,61) The shapes of pyriform ‘Hass’ and obovate ‘Bacon’
and ‘Gwen’ are desirable. The easily removed peel of ‘Fuerte’
and ‘Hass’ is preferred. The association between phenotypes
and gene expression profiles can potentially provide valuable
insights into the genetic regulation of such commercially
important morphological traits.
Current genetic and genomic
resources for avocado
Four main avenues of molecular research have been
pursued in avocado: (i ) identification of specific genes and
ESTs, (ii ) generation and application of various markers,
(iii ) genetic transformation, and (iv ) floral microarrays.
Gene cloning and ESTs
Many genes have been cloned and sequenced from avocado,
several relevant to fruit ripening, e.g. cellulase,(62–65) cyto-
chrome P-450,(66,67) and polygalacturonase, PG.(68,69) AVOe3
mRNA has been identified as a ripening-related gene,(70) and
23 cDNA clones have been identified that had homologies to
PG, endochitinase, a cysteine proteinase inhibitor and several
stress-related proteins.(71) Nearly 10,000 EST sequences
from early development of avocado flowers have been
collected by the Floral Genome Project (FGP),(24) and
85,000 more will be produced during 2007–2008 as part of
the Ancestral Angiosperm Genome Project (AAGP), funded
by the US National Science Foundation.Among the FGP ESTs
are many of the key genes known to be involved in flower
development in Arabidopsis, including homologs of AG,
APETALA1 (AP1), AP3, PI, and SEP3, plus many important
housekeeping genes (e.g. b-ACTIN and UBIQUITIN). These
sequences have been used in several analyses of floral gene
family evolution: AP3 and PI,(72) APETALA2,(73) SEPALLATA,(74)
and SHAGGY-LIKE KINASEs.(75) To facilitate comparisons
of floral development among divergent lineages of flowering
plants, landmarks in floral development have been identified,(76)
and corresponding expression patterns of floral MADS box
Table 2. Comparison of several commercially important avocado cultivars
Cultivar Parentage PeelsSeedSize
SkinTexture
FlowerType*
FruitShape
Skin ColorRipe
SkinThickness
Average FruitWeight (kg)
‘Bacon’ Mexican Poorly Large Smooth B Obovate Green Thin 0.28 to 0.51
‘Fuerte’ G�M Well Large Medium B Obovate Green Medium 0.25 to 0.45
‘Gwen’ G�M Poorly Medium Pebbly A Obovate Green Medium 0.17 to 0.45
‘Hass’ G�M Well Medium Pebbly A Pyriform Black Medium 0.17 to 0.39
‘Jim’ Mexican Poorly Medium Smooth B Pyriform Green Thin 0.17 to 0.45
‘Lamb Hass’ G�M Well Medium Pebbly A Obovate Black Medium 0.28 to 0.51
‘Reed’ Guatemalan Well Large Medium A Spheroid Green Medium 0.48 to 0.68
‘Zutano’ Mexican Poorly Medium Smooth B Obovate Green Thin 0.31 to 0.39
*A¼ female in the morning *B¼male in the morning. G�M¼Guatemalan�Mexican hybrid. Modified from the UC Avocado Information Home Page http://
ucavo.ucr.edu.
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BioEssays 30.4 391
genes (A, B, C and E function homologs) have been
examined.(31)
Genetic markers and marker-assisted selection
Molecular markers are increasingly being exploited in avocado
breeding and genetic analysis. Isozymes were initially used as
markers,(77) but recent research has focused on a diverse
array of DNA markers, including restriction fragment length
polymorphisms (RFLPs),(78–81) variable numbers of tandem
repeats (VNTRs), including minisatellites,(82) and simple
sequence repeats (SSRs, or microsatellites).(83–87) A prelimi-
nary analysis of nucleotide sequence diversity in the wild
progenitors of cultivated avocado suggests moderate levels of
single nucleotide polymorphism (SNP) diversity (yw¼ 0.0071,
where yw is Watterson’s diversity statistic(88)), indicating that
SNP discovery will be a valuable source of future markers.(89)
This work, based on resequencing four loci in 21 wild acces-
sions, also revealed that intralocus linkage disequilibrium
decays relatively rapidly to half initial values within about 1 kb.
The relationships among avocado cultivars and races have
been explored using microsatellites,(90,91) and more recently
using assignment analysis based on nucleotide sequence
diversity at four loci in 33 cultivated and 21 wild avocado
accessions.(89) These studies indicate that (i) the three races
can be distinguished genetically, (ii) heterozygosity and gene
diversity are moderate, (iii) the characterization of cultivars
based on morphological characters of the tree and fruit can be
unreliable, and (iv) it is possible to quantify the contribution of
various hybrid sources to cultivar genomes.
A preliminary genetic map is available for avocado. This
map, based on 51 microsatellites, 17 RAPD markers and
23 DFP markers, consists of 12 linkage groups having 2–
5 markers in each group, and covers about 357 cM.(92) Markers
associated with harvest duration (length of harvesting
season), skin color, skin thickness, skin surface texture, fruit
weight, seed sizeand peeling ability have been identified.(84,85)
A linkage map is being developed from progeny of a reciprocal
cross between a West Indian variety (‘Simmonds’) and
Guatemalan�West Indian hybrid (‘Tonnage’) using micro-
satellite markers (James Borrone, pers. comm).
Marker-trait associations are also being studied via
quantitative trait locus (QTL) analysis.(93) This research is
centered on a clonally replicated experimental population of
ca. 200 progeny genotypes of ‘Gwen’, each genotype clonally
replicated four times, with two clones growing at each of two
locations in California. The ‘Gwen’ progeny genotypes are
further characterized by pollen parent, with ca. 25% sired by
‘Bacon’, 25% by ‘Fuerte’, and 25% by ‘Zutano’. In parallel with
the collection of allele data for ca. 100 microsatellite markers,
quantitative genetic analyses are being applied to growth- and
yield-related trait measurements. Growth rate (measured in
terms of tree height, trunk girth and canopy diameter) and
flowering are revealing low but adequate levels of heritability.(94)
Trait correlations were calculated among the three measures
of growth rate and between growth rate and fruit set.
Interesting effects of the pollen donor on progeny growth,
flowering and fruit set have emerged as part of this study.
For example, ‘Gwen’ progeny sired by ‘Fuerte’ pollen are
significantly slower growing than ‘Gwen’ progeny sired by
‘Bacon’ or ‘Zutano’, but their mean canopy diameter is
significantly larger than that of the other progeny. Progeny
pollinated by ‘Zutano’ had significantly higher fruit set than
those sired by the other paternal genotypes.(94) QTL analysis
will be used to detect associations between traits and genetic
markers, and the markers will be used to guide future plant
improvement strategies.
Genetic transformation
Regeneration pathways of elite (mature phase) tropical fruit
and forest trees, particularly via somatic embryogenesis,
have proven successful. The avocado has become a model
for genetic engineering studies, including genetic transfor-
mation (Fig. 4) and in vitro mutagenesis, and related
technologies such as cryopreservation,(95) in vitro and ex vitro
micrografting.(96,97)
Genetic transformation studies in avocado have had two
research objectives: (1) engineering enhanced resistance to
Phytophthora root rot in candidate rootstocks using different
pathogenesis-related genes with the CaMV 35S promoter;
and (2) introducing the on-tree fruit storage character to the
tropical West Indian and West Indian�Guatemalan cultivars
using the gene S-adenosyl-L-methionine hydrolase (SAMase)
with a cellulase promoter isolated from avocado fruit.
To circumvent the long juvenile period (it can take up to
seven years for trees to produce fruit), shoots from trans-
formed somatic embryos are micrografted onto seedling
rootstocks, and then regrafted serially onto clonal (grafted)
shoots in the greenhouse (Fig. 3). With this procedure, almost
500 independently transformed avocado plants are now under
assessment. Additional transformants will be soon developed
carrying RNAi silencing cassettes constructed for MADS box
genes, including homologs of AG, AP1, AP3, TM8, PI and
SEP3.
Floral microarrays
Comparisons of expression patterns of genes involved in
particular biological processes have the potential to provide
detailed and specific information about evolutionary change.
Evolution involves biological variations at many levels, and one
of the major challenges is to understand how genes interact to
perform particular biological processes. In this context, the
accumulation of microarray data from evolutionarily distant
species such as avocado and eudicot models provides new
opportunities to discover how genes interact to perform this
specific biological process and to study the evolution of
expression network properties.
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392 BioEssays 30.4
Microarray technology has been used to examine the floral
transcriptional program in several model plants including
Arabidopsis,(98–101) Zea,(102) Lotus(103) and Gerbera.(104)
Custom microarrays for avocado that contain in situ synthe-
sized 60-mer oligonucleotide probes targeting 6068 floral
transcripts have been constructed from the FGP ESTset using
a pipeline that designs oligos with nearly uniform hybridization
rates for each unigene in our EST library while avoiding
stretches of sequence that might be conserved among closely
related gene family members.(105) Initial findings identify
over 1000 transcripts with more than two-fold upregulation
in flowers relative to leaves, including homologs of AG, AP3,
PI and SEP3, known floral regulators in Arabidopsis
(A. Chanderbali, N. Altman, DE Soltis and PS Soltis,
unpublished data) Moreover, more than 50 of the floral
transcripts, including bHLH, MYB, YABBY and WRKY
transcription factors, were actually most highly expressed
in young fruit. Significantly, homologs of the MADS box
transcription factors AG, AP3 and SEP3 were also highly
expressed in young fruit, complementing genetic evidence
from tomato that floral developmental regulators also play a
role in fleshy fruit development.(58)
The future: genome mapping and sequencing
Genomic research on avocado has so far been focused on
ripening-related genes and the floral transcriptome. An
important step towards elucidation of genome structure and
eventual whole-genome sequencing of avocado is the
construction of a physical map using Bacterial Artificial
Chromosome fingerprints (BAC) and BAC End Sequencing
(BES). Physical maps are crucial for genome sequencing,
positional cloning, and understanding the relative organization
of genes and markers. Map-building methods such as high
information content fingerprinting (HICF), which uses multiple
DNA restriction digests and fragment capture via capillary
DNA analysis, are particularly suitable.(106) BAC end sequenc-
ing is used to anchor clones to physical contigs that lack
sufficient matches to be identified by fingerprinting alone.
Additionally, BES is useful in creating successful markers for
genetic anchoring of BACs to maps and for initial character-
ization of gene density and repeat-type frequencies across the
full genome. Following annotation of avocado BES, clones
containing genes of particular interest can be sequenced
completely. A similar project is underway on the basalmost
extant angiosperm, Amborella (undertaken by the Ancestral
Angiosperm Genome Project). Given the relatively basal
phylogenetic position of avocado, its genome sequence could
be expected to be the closest available to that of Amborella in
terms of sequence similarity, thereby concomitantly increasing
the ease of both species’ gene annotation via Arabidopsis and
rice nomenclature.
BES data will also be used to search available genome
sequences for regions of microsynteny. Paired BAC ends from
papaya with low-copy sequences have been mapped to the
Arabidopsis, Populus, and rice genomes.(107) The fraction of
low-copy paired BAC ends mapping in the correct orientation
to the same chromosome was higher for the Populus
comparisons (2.23%) than the Arabidopsis ones (1.67%),
despite the more-recent common ancestry of Arabidopsis and
Figure 4. A, B: Regenerated shoots from transformed
embryogenic cultures of ‘Saurdia’ and ‘Hass’ avocado,
respectively. C: Transgenic ‘Saurdia’ avocado ex vitro contain-
ing the S-adenosylmethionine hydrolase and nptII selection
marker genes. D: ‘Hass’ avocado ex vitro transformed with the
pdf1.2 defensin gene and the BastaTM herbicide selection
marker gene bar; transformed plant (center) and negative
controls (left and right) following application of BastaTM.
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BioEssays 30.4 393
papaya (both are members of the Brassicales). The fraction
was only 0.49% for rice. These results could be explained by a
slower rate of genome evolution in the tree species Populus
and papaya relative to herbaceous Arabidopsis and rice.
Avocado and Amborella, like Populus and papaya, are woody
species with slower rates of nucleotide sequence evolution
than Arabidopsis or rice.(23) As such, it could be expected that
some microsynteny between avocado and Amborella BAC
ends will be visible with the available genome sequences
despite their phylogenetic distance. Finally, given that evi-
dence of paleopolyploidy was detected in avocado yet none in
Amborella,(23) it may be found that avocado BES markers map
more than once to Amborella sequence.
Conclusions
Avocado provides access to an ancient and largely unexplored
branch of flowering plants, the magnoliids. Given its impor-
tance as a fruit crop, substantial genetic resources have
already been assembled. Among these, genetic transforma-
tion procedures,(108) extensive transcriptome sequencing,(24)
and genetic marker assessments on clonally replicated
genotypes(93) promise deep insights into avocado reproduc-
tive genetics. Already, we have shown that MADS box gene
expression in avocado flowers departs from the ABC(DE)
model, and instead indicates a perianth developmental
program that may have evolved from a regulatory network
inherited from stamens.(31) Avocado flowers therefore provide
a rare opportunity to examine the shifting regulatory controls
that necessarily accompany this avenue of perianth evolution.
Fruit development, maturation and ripening in avocado also
depart significantly from that of model plants. Elucidation of the
genetic basis of on-tree-storage in avocado will open
opportunities to transfer this agriculturally desirable trait to
other fruit crops.
Ultimately, any insights obtained for this phylogenetically
important basal angiosperm, when coupled with ongoing
genomics studies for other flowering plants, will help elucidate
floral and fruit developmental genetics across major lineages
of flowering plants. Because avocado can be genetically
transformed, with its rapidly growing transcriptome and
expression databases, it can serve as a much needed model
for functional genetics in basal angiosperms. No other func-
tional models currently exist among these phylogenetically
pivotal flowering plants; thus, avocado offers unique oppor-
tunities to test hypotheses of gene action and evolution in early
angiosperms. Because functional models are difficult to
develop in basal angiosperms, given that most are woody,
the transformability of avocado is of broad importance to
developmental genetics and evolutionary biology. Further
advancement of genetic resources for avocado, including
whole genome mapping and sequencing, will open new
avenues for comparative genetics across the angiosperms.
Acknowledgments
We thank lan Maguire (TREC-UF) and Hong Ma (PSU) for
providing photographs of avocado fruits, and an Arabidopsis
flower, respectively.
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