persea americana (avocado): bringing ancient flowers to fruit in the genomics era

11
Persea americana (avocado): bringing ancient flowers to fruit in the genomics era Andre ´ 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. Soltis 2 Summary The 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) near the origin of the flowering plants themselves. Following centuries of selective breeding, avocado germplasm has been characterized at the level of microsatellite and RFLP markers. Nonetheless, little is known beyond these general diversity estimates, and much work remains to be done to develop avocado as a major subtropical-zone crop. Among the goals of avocado improvement are to develop varieties with fruit that will ‘‘store’’ better on the tree, show uniform ripening and have better post-harvest storage. Avocado transcriptome sequencing, genome mapping and partial genomic sequencing will represent a major step toward the goal of sequencing the entire avocado genome, which is expected to aid in improving avocado varieties and production, as well as under- standing the evolution of flowers from non-flowering seed plants (gymnosperms). Additionally, continued evolutionary and other comparative studies of flower and fruit development in different avocado strains can be accomplished at the gene expression level, including in comparison with avocado relatives, and these should provide important insights into the genetic regulation of fruit development in basal angiosperms. BioEssays 30:386–396, 2008. ß 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 1 Department of Botany, University of Florida, Gainesville, FL. 2 Florida Museum of Natural History, University of Florida, Gainesville, FL. 3 Department of Biological Sciences, University at Buffalo (SUNY), Buffalo, NY. 4 Ecology and Evolutionary Biology, University of California, Irvine, CA. 5 Horticultural 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, USDATropical 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, ß 2008 Wiley Periodicals, Inc. My favorite plant

Upload: andre-s-chanderbali

Post on 06-Jun-2016

215 views

Category:

Documents


2 download

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.

My favorite plant

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

My favorite plant

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.

My favorite plant

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.

My favorite plant

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.

My favorite plant

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.

My favorite plant

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.

My favorite plant

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.

My favorite plant

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.

References1. FAOSTAT. 2006 http://apps.fao.org

2. Swisher HE. 1988. Avocado oil: from food use to skin care. J Amer Oil

Chemists’ Soc 65:1704–1706.

3. Birkbeck J. 2002. Health benefits of avocado oil. Food NZ 40–42.

4. Lu Q-Y, Arteaga JR, Qifeng Z, Huerta S, Go VLW, et al. 2005.

Inhibition of prostate cancer cell growth by an avocado extract: Role

of lipid-soluble bioactive substances. J Nutr Biochem 16:23–30.

5. Ashton OBO, Wong M, McGhie TK, Vather R, Wang Y, et al. 2006.

Pigments in avocado tissue and oil. J Agric Food Chem 54:10151–

10158.

6. Ding H, Chin YW, Kinghorn AD, D’Ambrosio SM. 2007. Chemo-

preventive characteristics of avocado fruit, Seminars in Cancer Biology.

DOI:10.1016/j.semcancer.2007.04.003

7. Smith CE Jr. 1966. Archaeological evidence for selection of avocados.

Econ Bot 20:169–175.

8. Smith CE Jr. 1969. Additional notes on pre-conquest avocados in

Mexico. Econ Bot 23:135–140.

9. Bergh BO, Ellstrand NC. 1986. Taxonomy of the avocado. Yrbk Calif

Avocado Soc 70:135–145.

10. Popenoe W. 1941. The avocado- a horticultural problem. Trop Agric

18:3–7.

11. Crane JH, Balerdi CF, Maguire I. 2005. The avocado Circular 1034.

Florida Cooperative Extension Service, Institute of Food and Agricultural

Sciences, University of Florida, Gainesville.

12. Rohwer JG. 1993. Lauraceae. In: Kubitzki K, Rohwer J, Bittrich V,

editors. The families and genera of flowering plants. Berlin: Springer-

Verlag. pp 426–437.

13. Cronquist A. 1988. The Evolution and Classification of Flowering Plants.

second edition. New York: The New York Botanical Garden.

14. Takhtajan A. 1997. Diversity and Classification of Flowering Plants. New

York: Columbia University Press.

15. Moore MJ, Bell CD, Soltis PS, Soltis DE. 2007. Using plastid genome-

scale data to resolve enigmatic relationships among basal angio-

sperms. PNAS 104:19363–19368.

16. Jansen RK, Cai Z, Raubeson LA, Daniell H, dePamphilis CW, et al.

2007. Analysis of 81 genes from 64 plastid genomes resolves

relationships in angiosperms and identifies genome-scale evolutionary

patterns. PNAS 104:19369–19374.

17. Renner S. 1999. Circumscription and phylogeny of the Laurales:

evidence from molecular and morphological data. Amer J Bot 86:

1301–1315.

18. Friis EM, Eklund H, Pedersen KR, Crane PR. 1994. Virgininanthus

calycanthoides gen. et sp. nov.—A calycanthaceous flower from the

Potomac group (Early Cretaceous) of eastern North America. Int J Plant

Sci 155:772–785.

19. Renner SS. 2004. Variation in diversity among Laurales, Early

Cretaceous to Present. Biol Skr 55:441–458.

20. Chanderbali AS, Renner SS, van der Werff H. 2001 Phylogeny and

historical biogeography of Lauraceae: Evidence from the chloroplast

and nuclear genomes. Ann Missouri Bot Gard 88:104–134.

21. Stebbins GL. 1971. Chromosomal Variation in Higher Plants. London:

Arnold.

22. Soltis DE, Soltis PS. 1990. Isozyme evidence for ancient polyploidy in

primitive angiosperms. Sys Bot 15:328–337.

23. Cui L, Wall PK, Leebens-Mack J, Lindsay BG, Soltis DE, et al. 2006.

Widespread genome duplications throughout the history of flowering

plants. Genome Res 16:738–739.

24. Albert VA, Soltis DE, Carlson JE, et al. 2005. Floral gene resources from

basal angiosperms for comparative genomics research. BMC Plant Biol

5:1–15.

25. Coen ES, Meyerowitz EM. 1991. The war of the whorls: genetic

interactions controlling flower development. Nature 353:31–37.

My favorite plant

394 BioEssays 30.4

26. Colombo L, Franken J, Koetje E, Vanwent J, Dons HJM, et al. 1995. The

petunia MADS box gene FBP11 determines ovule identity. Plant Cell 7:

1859–1868.

27. Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF. 2000. B and C

floral organ identity functions require SEPALLATA MADS-box genes.

Nature 405:200–203.

28. Soltis PS, Soltis DE, Kim S, Chanderbali AS, Buzgo M. 2006. Expression

of Floral Regulators in Basal Angiosperms and the Origin and Evolution

of ABC-function. Adv Bot Res 44:485–506.

29. Kyozuka J, Shimamoto K. 2002. Ectopic expression of OsMADS3, a

rice ortholog of AGAMOUS, caused a homeotic transformation of

lodicules to stamens in transgenic rice plants. Plant Cell Phys 43:130–

135.

30. Ambrose BA, Lerner DR, Ciceri P, Padilla CM, Yanofsky MF, et al. 2000.

Molecular and genetic analyses of the silky1 gene reveal conservation

in floral organ specification between eudicots and monocots. Mol Cell

5:569–579.

31. Chanderbali AS, Kim S, Buzgo M, Zheng Z, Oppenheimer DG, et al.

2006. Genetic footprints of stamen ancestors guide perianth evolution in

Persea (Lauraceae). Int J Plant Sci 167:1075–1089.

32. Kramer EM, Hall JC. 2005. Evolutionary dynamics of genes controlling

floral development. Curr Opin Plant Biol 8:13–18.

33. Kim S, Koh J, Yoo M-J, Kong H, Hu Y, et al. 2005 Expression of floral

MADS-box genes in basal angiosperms: implications for the evolution of

floral regulators. Plant J 43:724–744.

34. Albert VA, Gustafsson MHG, Di Laurenzio L. 1998. Ontogenetic

systematics, molecular developmental genetics, and the angio-

sperm petal. In: Soltis PS, Soltis DE, Doyle JJ, editors. Molecular

Systematics of Plants II. Boston: Kluwer Academic Publishers. pp 349–

374.

35. Chanderbali AS. 2004. Endlicheria (Lauraceae) Fl Neotrop Monogr 91:

1–141.

36. Giovannoni J. 2004. Genetic Regulation of Fruit Development and

Ripening. Plant Cell 16:S170–S180.

37. da Silva FG, Iandolino A, Al-Kayal F, Bohlmann MC, Cushman MA, et al.

2005. Characterizing the grape transcriptome. Analysis of expressed

sequence tags from multiple Vitis species and development of a

compendium of gene expression during berry development. Plant

Physiol 139:574–597.

38. Jaillon O, et al. 2007. The grapevine genome sequence suggests

ancestral hexaploidization in major angiosperm phyla. Nature 449:463–

467.

39. Aharoni A, O’Connell A. 2002. Gene expression analysis of strawberry

achene and receptacle maturation using DNA microarrays. J Exp Bot

53:2073–2087.

40. Newcomb RD, Crowhurst RN, Gleave AP, Rikkerink EHA, Allan AC,

et al. 2006. Analyses of Expressed Sequence Tags from Apple. Plant

Phys 141:147–166.

41. Park S, Sugimoto N, Larson MD, Beaudry R, van Nocker S. 2006.

Identification of Genes with Potential Roles in Apple Fruit Development

and Biochemistry through Large-Scale Statistical Analysis of Expressed

Sequence Tags. Plant Phys 141:811–824.

42. Purseglove JW. 1968. Persea americana Mill. In Tropical Crops:

Docotyledons/1. London Longmans pp 11–15.

43. Barlow C. 2000. The Ghosts of Evolution. Basic Books. New York.

44. Nitsch JP. 1950. Physiology of flower and fruit development. Encycl

Plant Physiol 15:1537–1647.

45. Schroeder CA. 1953. Growth and development of the Fuerte avocado

fruit. Proc. Amer Soc Hort Sci 61:103–109.

46. Adato I, Gazit S. 1977. Post harvest response of avocado fruits of differ-

ent maturity to delayed ethylene treatments. Plant Physiol 53:899–902.

47. Morton JF. 1987. Fruits of warm climates. Winterville NC: Creative

Resource Systems, Inc.

48. Kays SJ. 1997. Postharvest physiology of perishable plant products.

Exon Press Athens.

49. Blumenfeld A, Gazit S. 1974. Development of Seeded and Seedless

Avocado Fruits. J Amer Soc Hort Sci 99:442–448.

50. Griswold HB. 1945. The ‘Hass’ avocado. Yrbk Calif Avocado Soc 30:

27–31.

51. Crane JH, Balerdi CF, Campbell CW. 1996. The avocado Circular 1034.

Gainesville: Florida Cooperative Extension Service, Institute of Food.

and. Agricultural Sciences, University of Florida.

52. Sitrit Y, Riov J, Blumenfeld A. 1986. Regulation of ethylene biosynthesis

in avocado fruit during ripening. Plant Physiol 81:130–135.

53. Starret AA, Laties GG. 1991. The effect of ethylene and propylene

pulses on respiration, ripening advancement, ethylene-forming enzyme,

and 1-aminocyclopropane-1-carboxylate synthase activity in avocado

fruit. Plant Physiol 95:921–927.

54. Starret AA, Laties GG. 1993. Ethylene and wound-induced gene

expression in the preclimateric phase of ripening avocado fruit and

mesocarp disc. Plant Physiol 103:227–234.

55. Tingwa PO, Young RE. 1975. The effect of Indole-3-acetic acid and the

other growth regulators on the ripening of avocado fruit. Plant Physiol

55:937–940.

56. Whiley AW. 1992. Persea americana Mill. In: Verheij EWM, Coronel RE,

editors. Plant Resources of South-East Asia No. 2 Edible fruits and nuts.

Wageningen: Pudoc-DLO. pp 249–254.

57. Frenkel C, Dyck R. 1973. Auxin Inhibition of Ripening in Bartlett Pears.

Plant Physiol 51:6–9.

58. Vrebalov J, Ruezinsky D, Padmanabhan V, White R, Medrano D, et al.

2002. A MADS-Box Gene Necessary for Fruit Ripening at the Tomato

Ripening-Inhibitor (Rin) Locus. Science 296:343–346.

59. Lahav E, Lavi U. 2002. Genetics and breeding. In: Whiley AW, Schaffer

B, Wolstenholme BN, editors. Avocado Botany Production and Uses.

Wallingford, Oxon: CAB International. pp 39–69.

60. Lahav E, Kalmar D. 1977. Water requirement of avocado in Israel. II:

influence on yield, fruit growth and oil content. Aust J Agric Res 28:869–

877.

61. Whiley AW, Schaffer B. 1994. Avocado. In: Schaffer B, Andersen PC,

editors. Handbook of Environmental Physiology of Fruit Crops, Volume

2, Subtropical and Tropical Crops. Boca Raton: CRC Press Inc.

pp 165–197.

62. Christoffersen RE, TuckerML, Laties GG. 1984. Cellulase gene

expression in ripening avocado fruit: the accumulation of cellulase

mRNA and protein as demonstrated by cDNA hybridization and

immunodetection. Plant Mol Biol 3:385–391.

63. Tucker ML, Durbin ML, Clegg MT, Lewis LN. 1987. Avocado cellulase:

nucleotide sequence of a putative full-length cDNA clone and evidence

for a small gene family. Plant Mol Biol 9:197–203.

64. Cass LG, Kirven KA, Christoffersen RE. 1990. Isolation and character-

ization of a cellulase gene family member expressed during avocado

fruit ripening. Mol Gen Genet 223:76–86.

65. Tonutti P, Cass LG, Christoferssen RE. 1995. The expression of

cellulase gene family members during induced avocado fruit abscission

and ripening. Plant Cell Environ 18:709–713.

66. Bozak KR, Yu H, Sirevag R, Christoffersen RE. 1990. Cloning and

sequence analysis of ripening-related cytochrome P-450 cDNAs from

avocado fruit. Proc Nat Acad Sci 87:3904–3908.

67. O’Keefe DP, Bozak KR, Christoffersen RE, Tepperman JA, Dean C,

et al. 1992. Endogenous and engineered cytochrome P-450 mono-

oxygenase in plants. Biochem Soc Trans 20:357–361.

68. Kanellis AK, Solomos T, Roubelakis-Angelakis KA. 1991. Suppression

of cellulase and polygalacturonase and induction of alcohol

dehyrogenase isoenzymes in avocado fruit mesocarp subjected to

oxygen stress. Plant Physiol 96:269–274.

69. Kutsunai S, Lin AC, Percival FW, Laties GG, Christoffersen RE. 1993.

Ripening related polygalacturonase cDNA from avocado. Plant Physiol

103:289–290.

70. McGarvey DG, Sirevag R, Christoffersen RE. 1990. Ripening related

gene from avocado fruit. Plant Physiol 98:554–559.

71. Dopico B, Lowe AL, Wilson ID, Merodio C, Grierson D. 1993. Cloning

and characterization of avocado fruit mRNAs and their expression

during ripening and low temperature storage. Plant Mol Biol 21:437–

449.

72. Kim S, Yoo M-J, Albert VA, Farris JS, Soltis PS, et al. 2004. Phylogeny

and diversification of B-function MADS-box genes in angiosperms:

evolutionary and functional implications of a 260-million-year-old

duplication. Am J Bot 91:2102–2118.

My favorite plant

BioEssays 30.4 395

73. Kim S, Soltis PS, Wall K, Soltis DE. 2005a. Phylogeny and Domain

Evolution in the APETALA2-Like Gene Family. Mol Biol Evol 23:107–120.

74. Zahn LM, Kong H, Leebens-Mack JH, Kim S, Soltis PS, et al. 2005.

The Evolution of the SEPALLATA Subfamily of MADS-Box Genes: A

Preangiosperm Origin With Multiple Duplications Throughout Angio-

sperm History. Genetics 169:2209–2223.

75. Yoo M-J, Albert VA, Soltis PS, Soltis DE. 2006. Phylogenetic

diversification of glycogen synthase kinase 3/SHAGGY-like kinase

genes in plants. BMC Plant Biol 6:3.

76. Buzgo M, Chanderbali AS, Kim S, Zheng Z, Oppenheimer D, et al. 2007.

Floral developmental morphology of Persea americana (avocado,

Lauraceae): the oddities of male organ identity. In l Plant Sci 168:

261–284.

77. Torres AM, Bergh BO. 1980. Fruit and leaf isozymes as genetic markers

in avocado. J Amer Soc Hort Sci 105:614–619.

78. Furnier GR, Cummings MP, Clegg MT. 1990. Evolution of the

avocados as revealed by DNA restriction site variation. J Hered 81:

183–188.

79. Bufler G, Ben Ya’acov A. 1992. A study of the avocado germplasm

resources 1988–1990.Ribosomal DNA repeat unit polymorphism in

avocado. In: Lovatt C, Holthe PA, Arpaia ML, editors. Proceedings of

the Second World Avocado Congress. Vol 2. Riverside: University of

California. pp 545–550.

80. Davis J, Henderson D, Kobayashi M, Clegg MT. 1998. Genealogical

relationships among cultivated avocado as revealed through RFLP

analyses. J Hered 89:319–323.

81. Kobayashi M, Lin J-Z, Davis J, Francis L, Clegg MT. 2000. Quantitative

analysis of avocado outcrossing and yield in California using RAPD

markers. Scientia Horticulturae 86:135–149.

82. Lavi U, Hillel J, Vainstein A, Lahav E, Sharon D. 1991. Application of

DNA fingerprints for identification and genetic analysis of avocado.

J Amer Soc Hort Sci 116:1078–1081.

83. Lavi U, Akkaya M, Bhagwat A, Cregan PB. 1994. Methodology

of generation and characterization of simple sequence repeat DNA

markers in avocado (Persea americana Mill.). Eupytica 80:171–177.

84. Mhameed S, Hillel J, Lahav E, Sharon D, Lavi U. 1995. Genetic

association between DNA fingerprint fragment and loci controlling

agriculturally important traits in avocado (Persea americana Mill.).

Euphytica 81:81–87.

85. Sharon D, Hillel J, Mhameed S, Cregan TB, Lahav E, et al. 1998.

Association between DNA markers and loci controlling avocado traits.

J Amer Soc Hort Sci 123:1016–1022.

86. Ashworth VETM, Kobayashi MC, De La Cruz M, Clegg MT. 2004.

Microsatellite markers in avocado (Persea americana Mill.): develop-

ment of dinucleotide and trinucleotide markers. Scient Hort 101:255–

267.

87. Borrone JW, Schnell RJ, Violi HA, Ploetz R. 2007. Seventy microsatellite

markers from Persea americana Miller (avocado) expressed sequence

tags. Molecular Ecology 7:439–444.

88. Watterson GA. 1975. On the number of segregating sites in genetical

models without recombination. Theor Popul Biol 7:256–276.

89. Chen H, Morrell PL, de la Cruz M, Clegg MT. (In press) Nucleotide

diversity and linkage disequilibrium in wild avocado (Persea americana

Mill.). Journal of Heredity.

90. Ashworth VETM, Clegg MT. 2003. Microsatellite markers in avocado

(Persea americana Mill.): genealogical relationships among cultivated

avocado genotypes. J Hered 94:407–415.

91. Schnell RJ, Brown JS, Olano CT, Power EJ, Krol CA. 2003. Evaluation of

Avocado Germplasm Using Microsatellite Markers. J Amer Soc Hort Sci

128:881–889.

92. Sharon D, Cregan PB, Mhameed S, Hillel J, Lahav E, et al. 1997. An

integrated genetic linkage map of avocado. Theor Appl Gen 95:911–921.

93. Ashworth VETM, Chen H, Clegg MT. 2006. Chapter 17: Avocado. In:

Kole C, editor. Genome mapping and molecular breeding in plants.

Fruits and nuts 4. Berlin: Springer Verlag. pp. 325–329.

94. Chen H, Ashworth VETM, Xu S, Clegg MT. 2007. Quantitative genetic

analysis of growth rate in avocado. J Amer Soc Hort Sci 132:691–696.

95. Efendi D. 2003. Transformation and cryopreservation of embryogenic

avocado (Persea americana Mill.) cultures. Ph.D. dissertation,

University of Florida, Gainesville.

96. Suarez IE, Schnell RA, Kuhn DA, Litz RE. 2004. Micrografting of ASBVd-

infected avocado (Persea americana Mill.) plants. Plant Cell Tiss Org

Cult 80:179–185.

97. Raharjo SH-T, Litz RE. 2005. Micrografting and ex vitro grafting for

somatic embryo rescue and plant recovery in avocado (Persea

americana). Plant Cell Tiss Org Cult 82:1–9.

98. Zik M, Irish VF. 2003. Global Identification of Target Genes Regulated

by APETALA3 and PISTILLATA Floral Homeotic Gene Action. Plant Cell

15:207–222.

99. Wellmer F, Riechmann JL, Alves-Ferreira M, Meyerowitz EM. 2004.

Genome-wide analysis of spatial gene expression in Arabidopsis

flowers. Plant Cell 16:1314–1326.

100. Wellmer F, Alves-Ferreira M, Dubois A, Luis J, Meyerowitz EM. 2006.

Genome-wide analysis of gene expression during early Arabidopsis

flower development. Plos Gen 2:1012–1024.

101. Gomez-Mena C, de Folter S, Costa MMR, Angenent GC, Sablowski R.

2005. Transcriptional program controlled by the floral homeotic gene

AGAMOUS during early organogenesis. Development 132:429–438.

102. Cho Y, Fernandes J, Kim S-H, Walbot V. 2002. Gene-expression profile

comparisons distinguish seven organs of maize. Gen Biol 3: research

0045.1-0045.16

103. Endo M, Matsubara H, Kokubun T, Masuko H, Takahata Y, et al. 2002.

The advantages of cDNA microarray as an effective tool for

identification of reproductive organ-specific genes in a model legume,

Lotus japonicus. FEBS Lett 514:229–237.

104. Laitinen RAE, Immanen J, Auvinen P, Rudd S, Alatalo E, et al. 2005.

Analysis of the floral transcriptome uncovers new regulators of organ

determination and gene families related to flower organ differentiation

in Gerbera hybrida (Asteraceae). Genome Res 15:475–486.

105. Altman N, Leebens-Mack J, Zahn L, Chanderbali AS, Tian D, et al. 2006.

Behind the Scenes: Planning a Multispecies Microarray Experiment.

Chance 19:28–39.

106. Nelson WM, Bharti AK, Butler E, Wei F, Fuks G, et al. 2005. Whole-

genome validation of high-information-content fingerprinting. Plant Phys

139:27–38.

107. Lai CWJ, Yu Q, Hou S, Skelton RL, Jones MR, et al. 2006. Analysis of

papaya BAC end sequences reveals first insights into the organization

of a fruit tree genome. Mol Genet Genomics 276:1–12.

108. Litz RE, Witjakso, Raharjo S, Efendi D, Pliego Alfaro F, et al. 2005.

Persea americana Avocado. In: Litz RE, editor. Biotechnology of Fruit

and Nut Crops. Cambridge: CABI Publishing. pp 325–348.

109. Soltis PS, Soltis DE, Chase MW. 1999. Angiosperm phylogeny inferred

from multiple genes as a tool for comparative biology. Nature 402:

402–404.

My favorite plant

396 BioEssays 30.4