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____________________
C.B.M. Cerqueira-Silva ● E.S.L. dos Santos
Laboratório de Genética Molecular Aplicada, Departamento de Ciências Exatas e Naturais,
Universidade Estadual do Sudoeste da Bahia (UESB), Itapetinga, Bahia, Brazil
e-mail: csilva@uesb.edu.br; cerqueirasilva1@yahoo.com.br; elisalisboa@yahoo.com.br
F.G. Faleiro
Centro de Pesquisa Agropecuária do Cerrado, Empresa Brasileira de Pesquisa Agropecuária
(Embrapa Cerrados), Brasília, Distrito Federal, Brazil
e-mail: fabio.faleiro@embrapa.br
O.N. de Jesus
Centro Nacional de Pesquisa em Mandioca e Fruticultura, Empresa Brasileira de Pesquisa
Agropecuária (Embrapa Mandioca e Fruticultura), Cruz das Almas, Bahia, Brazil
e-mail: onildo.nunes@embrapa.br
A.P. de Souza ()
Centro de Biologia Molecular e Engenharia Genética, Departamento de Biologia Vegetal,
Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, São Paulo,
Brazil
e-mail: anete@unicamp.br
J.M. Al-Khayri et al. (eds.) Advances in Plant Breeding Strategies: Fruits
1
Chapter 30
Passion Fruit (Passiflora spp.) Breeding
Carlos Bernard Moreno Cerqueira-Silva, Fábio Gelape Faleiro,
Onildo Nunes de Jesus, Elisa Susilene Lisboa dos Santos, and Anete
Pereira de Souza
Abstract. The genus Passiflora, commonly known as passion fruit, is
prominent in the family Passifloraceae due to its numerous species
(approximately 520) and economic importance. The biodiversity of this genus
is widely represented in the Americas, where Colombia and Brazil harbor
approximately 170 and 150 species of Passiflora, respectively. The economic
interest in passion fruit species emerged due to the beauty of their flowers,
their active medicinal properties, their essential oils that can be extracted for
the cosmetics industry, and their production of fruit for consumption or for
obtaining derivatives. Brazil is considered the largest producer of passion fruit,
although its national productivity is low (an average of 14 mt/ha-1 year-1)
compared with the potential for passion fruit cultivation (50 mt/ha -1 year-1).
This low productivity is partly caused by a lack of cultivars adapted to
different production regions and their susceptibility to major diseases.
Although the number of passion fruit breeding programs has increased, the
results obtained thus far have been modest compared with the existing
demands. Such programs therefore represent a burgeoning field of research
and financial investment. Among the obstacles faced by breeders, the low
representation of Passiflora in germplasm banks (considering its species
2 C.B.M. Cerqueira-Silva et al.
richness and wide geographical distribution) and the scarcity of biological and
agronomic information for most accessions are the most salient. Despite the
difficulties encountered in Passiflora research over the past two decades, there
has been a notable increase in the use of molecular tools for the
characterization of this genus and in the number of cultivars registered and
effectively available for the large-scale production of passion fruit. Thus, in
this chapter, we present an overview of innovations and modern technologies,
including advances in breeding programs and molecular tools, related to the
availability of genetic resources for Passiflora. These technologies can be used
as strategies to improve every stage of breeding programs, from pre- to post-
breeding. Finally, we discuss future perspectives for studies leading to the
genetic breeding of passion fruit (Passiflora spp.).
Keywords Biotechnology Molecular markers Molecular tools Passion
flower Passifloraceae Plant breeding Plant improvement
30.1 Introduction
30.1.1 Distribution and Biodiversity
The family Passifloraceae Juss. ex DC includes diverse species, numbering
approximately 520 to 700, with highly variable leaves and flowers (Bernacci
2003; Feuillet 2004; Feuillet and MacDougal 2004), making this an exotic,
complex group. These species are mainly categorized as lianas or climbing
plants with tendrils, although some are trees or shrubs without tendrils (Cervi
1997). The family Passifloraceae exhibits great ecological importance not only
for its numerous species but also for their ecological and evolutionary
relationships with their pollinators, such as bees, wasps, hummingbirds, and
bats (Semir and Brown Jr. 1975; Sazima et al. 1999; Varassin et al. 2001) as
well as their predators, such as butterflies (Heliconidae) (Benson et al. 1978;
Plotze et al. 2005). These ecological relationships highlight the complex nature
of interactions between plants and animals.
While many taxonomical uncertainties exist within the family
Passifloraceae, the genus Passiflora is noted for its diversity, including
approximately 520 species (MacDougal and Feuillet 2004), which is a
consequence of the greater diversity (approximately 75%) that exists within
the family (Bernacci 2015; Cerqueira-Silva et al. 2014a; Feuillet 2004; Feuillet
and MacDougal 2004). Passiflora species are commonly known as passion
fruits and/or passion flowers. These species are allogamous and exhibit self-
incompatibility, although some species may be self-compatible (Varassin and
Silva 1999; Bruckner et al. 2005).
The Passifloraceae family is widely distributed in the tropics and warm
30 Passion fruit 3
temperate regions (mainly in America and Africa). Specifically, the vast
majority of species of the genus Passiflora are estimated to be distributed in
tropical and subtropical regions, from the United States to Chile and
Argentina, with the greatest occurrence and diversity being observed in South
America (Martin and Nakasone 1970, Lorenzi et al. 2006; Cerqueira-Silva et
al. 2016). There are also records of Passiflora spp. in India, China, Southeast
Asia, Australia and the Pacific islands and neighboring regions, although they
represent less than 5% of passion fruit species (Cerqueira-Silva et al. 2014a;
Cerqueira-Silva et al. 2016).
The importance of the genus Passiflora is not restricted to the ecological
aspects inherent to the diversity and dispersion of its species, as many
Passiflora species are recognized as natural resources due to their horticultural
value. The beauty and diversity of the foliage and flowers of passion fruit,
including over 400 ornamental hybrids, elicit significant economic interest
(Peixoto 2005; Abreu et al. 2009). The economic interest in passion fruit
species is also due to their medicinal and pharmacological traits (Dhawan et al.
Website information. 2004; Costa and Tupinamba 2005), which represent an
active field of research. Valuable oils and extracts obtained from passion fruit
are utilized in the cosmetics industry for manufacturing creams, soaps, and
shampoos (Zeraik et al. 2010; Faleiro et al. 2011).
In addition to ornamental appeal, the economic value of the genus
Passiflora can be attributed to production and commercialization of the fruits.
At least 70 species of edible Passiflora fruits are recognized (Souza and
Meletti 1997; Coppens d'Eeckenbrugge et al. 2001); however, the commercial
production of passion fruit is based on the cultivation of only a few species,
such as Passiflora edulis Sims, P. alata Curtis, P. setacea DC, P. ligularis A.
Juss, P. nitida Kunth, P. cincinnata Mast, P. tripartita (Juss.) Poir, P.
maliformis L., P. quadrangularis L. (Fig. 1). Among these species, only P.
edulis and P. alata are effectively grown on a commercial scale in Brazil, with
the cultivation of P. edulis accounting for at least 90% of the area designated
for Brazilian pasiculture, including the production of both yellow and purple
fruits (Meletti et al. 2005; Faleiro et al. 2011). The other species are major
components of the production chain of passion fruit in Colombia, which
presents a greater diversification of pasiculture, despite its production being
lower than that of Brazil.
4 C.B.M. Cerqueira-Silva et al.
Fig. 1. Examples of the main species of passion fruit cultivated commercially: P.
edulis Sims (a); P. alata Curtis (b); P. setacea D.C. (c); P. ligularis A. Juss. (d); P.
nitida Kunth (e); P. cincinnata Mast. (f); P. tripartita (Juss.) Poir. (g); P. maliformis
L. (h); P. edulis Sims f. edulis (i); P. quadrangularis L. (j); Ornamental hybrid P.
setacea x P. coccinea cv. BRS Estrela do Cerrado (k); Ornamental hybrid P. edulis x
P. incarnata cv. BRS Céu do Cerrado-BRS CC (l). Photos: Fábio Faleiro, Ana Maria
Costa, Embrapa Cerrados.
Although incipient, in a context of national and international production,
the cultivation and consumption of fruits and the derivatives of wild species
such as P. cincinnata, P. nitida and P. setacea have been described in previous
studies, although this work is still in the early stages (Souza and Meletti 1997;
30 Passion fruit 5
Meletti et al. 2005). The biodiversity inherent to wild passion fruit trees and
their resistance to biotic and abiotic stress factors are important components of
passion fruit breeding programs (Junqueira et al. 2005; Faleiro et al. 2011).
Thus, passion fruit biodiversity is potentially useful for the maintenance
and expansion of passion fruit breeding programs, but additional studies are
required to facilitate the exploitation of this characteristic as a natural resource.
The lack of biological, agronomic and molecular characterization for most
wild species of Passiflora means that many species are omitted from
germplasm banks and collections. An important advance for the morphological
characterization of Passiflora was the elaboration of the list of descriptors
illustrated by Embrapa and partner institutions. This list aims to facilitate and
standardize the morphological and agronomic characterization and
identification of sources of resistance to diseases in a germplasm bank (Jesus
et al., 2017) Based on compilations performed by Ferreira et al. (2005), it is
estimated that only 15% of Passiflora species are represented at least once in
one of the existing germplasm banks worldwide (Cerqueira-Silva et al. 2014a;
Cerqueira-Silva et al. 2016).
Some passion fruit species are adapted to certain biomes; therefore, the
preservation of the species in germplasm banks present in these biomes is
interesting for aiding their preservation. The Embrapa Units have three
Passiflora Germplasm Banks (PGB) located in three distinct biomes (Fig. 2A).
Each bank has its own peculiarities with regard to type, genetic breeding use
and frequency with which a certain species is represented. Embrapa has
approximately 571 accessions, whereas 322, 177 and 72 are preserved in
Embrapa Mandioca e Fruticultura, Embrapa Cerrados and Embrapa
Seminárido, respectively (Fig. 2A-B). Most of the accessions (163) at the PGB
are from the P. edulis Sims species (yellow and purple passion fruits) and the
other 110 from P. cincinnata, also known as passion fruit from the savannas or
bushes (Fig. 1C). In regard to the number of different species in the PGB,
Embrapa Cerrados has 54, followed by Embrapa Mandioca e Fruticultura with
37 and Embrapa Seminarido with 9 (Fig. 2C).
Although there is a scarcity of available information, there has been
enormous growth in the number of studies dedicated to the genetic
characterization of passion fruit species in past decades (Fig. 3). Moreover,
there has been both quantitative and organizational growth of research groups
and national and international partnerships established to enhance our
knowledge and utilization of this natural resource. The first World Congress
on Passion Fruit, conducted in March 2017 in Colombia (www.cepass.org),
was a result of diligent research and genetic improvement programs for
passion fruit.
____________________
C.B.M. Cerqueira-Silva ● E.S.L. dos Santos
Laboratório de Genética Molecular Aplicada, Departamento de Ciências Exatas e Naturais, Universidade Estadual do Sudoeste da Bahia (UESB),
Itapetinga, Bahia, Brazil
e-mail: csilva@uesb.edu.br; cerqueirasilva1@yahoo.com.br; elisalisboa@yahoo.com.br
F.G. Faleiro
Centro de Pesquisa Agropecuária do Cerrado, Empresa Brasileira de Pesquisa Agropecuária (Embrapa Cerrados), Brasília, Distrito Federal, Brazil
e-mail: fabio.faleiro@embrapa.br
O.N. de Jesus
Centro Nacional de Pesquisa em Mandioca e Fruticultura, Empresa Brasileira de Pesquisa Agropecuária (Embrapa Mandioca e Fruticultura), Cruz das
Almas, Bahia, Brazil
e-mail: onildo.nunes@embrapa.br
A.P. de Souza ()
Centro de Biologia Molecular e Engenharia Genética, Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas
(UNICAMP), Campinas, São Paulo, Brazil
e-mail: anete@unicamp.br
J.M. Al-Khayri et al. (eds.) Advances in Plant Breeding Strategies: Fruits 6
Fig. 2. Location of Embrapa Units with accessions preserved ex situ in germplasm
banks (A). Number of passion fruit accessions preserved in germplasm banks in
Embrapa Units (B) and species and distribution of the number of accessions per
Embrapa Unit (C). The colors in Fig. 1B are related to those in Fig. 1C. Source of map:
IBGE maps.
The successful examples of research activities associated with the genetic
improvement of passion fruit include the project entitled "Characterization and
use of germplasm and genetic improvement of passion fruit (Passiflora spp.)
with the aid of molecular markers" (coordinated by EMBRAPA) as well as
actions aimed at strengthening research networks (such as the "Passion fruit:
Germplasm and Improvement" and "Molecular Genetics of Passion Fruit"
networks), which receive support from researchers at different public
institutions [Universidade Estadual do Sudoeste da Bahia (UESB),
Universidade Estadual de Santa Cruz (UESC), Universidade Estadual de
Campinas (UNICAMP), Empresa Brasileira de Pesquisa Agropecuaria
(EMBRAPA), Universidade Estadual da Bahia (UNEB), Universidade de
Brasilia (UNB) and others, and financial support from development agencies
such as the Fundação de Amparo a Pesquisa do Estado da Bahia (FAPESB)
and Conselho Nacional de Pesquisa Científica (CNPQ).
Fig. 3. Trend graph of the number of published articles reporting results related to the
genetic characterization of passion fruit (Passiflora spp.) species. The values were
calculated based on the SCOPUS database (www.scopus.com), adopting the search
terms "Passiflora" and "genetic" and/or "molecular marker" as well as the
corresponding Portuguese words.
In general terms, the actions proposed in these projects strengthen and
contribute to the scientific and technological advancement of the passion fruit
crop as well as accelerate the production of new cultivars for genetic resistance
to diseases. The activities being developed are expected to obtain and use
microsatellite markers to ensure the estimation of the genetic diversity
maintained in the main germplasm banks of passion fruit, the proposition of a
nuclear collection and the identification and protection of cultivars. New
generation technologies are also being used for the construction and analysis
of transcriptome from contrasting passion fruit in reaction to Passion fruit
woodiness virus, allowing the identification of single nucleotide
polymorphisms (SNPs) and differentially expressed genes. In addition, the
reaction of the passion fruit plants kept in the main germplasm banks is being
evaluated for the virus (CABMV).
30.1.2 Objectives and Challenges of Breeding Programs
Considering the richness of the species available in the Passiflora genus and
their potential genetic variability, together with the factors limiting their
expansion and the maintenance of their cultivation, it is possible to highlight
the major challenges faced by the breeding programs in the development of
varieties and hybrids of passion fruit. These challenges include (i) the
production of fresh fruits for market or industrial purposes (with the physical-
chemical quality of fruits and multiple resistance to diseases) and ensuring
suitability for (ii) ornamental purposes, (iii) use in medicinal applications, and
(iv) the functional foods market. In view of these challenges, there is a need
for morpho-agronomic and molecular characterization of both
economically/commercially important species and wild passion fruit species to
support the advancement of breeding programs. Wild species may also
contribute to activities that are complementary to breeding, such as the use of
rootstocks (Lima et al., 2017). Therefore, activities recognized as pre-
improvement actions (prospecting and characterization of germplasm), in
addition to domestication and the development of new commercial passion
fruit, are current critical demands of research associated with the use of this
natural resource.
The number of accessions available for breeding programs is lower than
the total number of accessions registered in active germplasm banks (AGBs),
mainly due to the technical and financial difficulties in the maintenance and
characterization of AGB accessions. The lack of information related to the
accessions maintained in collections and AGBs is the main challenge for their
use in programs focused on genetic variability in breeding (Duvick 1984; Nass
2011).
The biology, agronomic characteristics, and genetics of wild passion fruit
species as well as domestication and breeding activities can contribute to
increasing the number of passion fruit species marketed in Brazil and other
countries, thus increasing fruit production. The potential for diversification in
the production of passion fruit is illustrated by the discrepancy in the number
of species cultivated on a commercial scale in Colombia in relation to the
small number of species cultivated and effectively marketed in Brazil.
Moreover, the prominence of countries such as Brazil in fruit production does
not always stem from efficiency of orchard productivity but from the area of
the land devoted to passion fruit cultivation. The productivity of the Brazilian
crop can reach 40 to 50 T/ha-1 year-1 (Melo et al. 2001; Meletti et al. 2005;
Neves et al. 2013) but has become much lower (approximately 14 T/ha-1 year-
1) in recent years.
Over 400 ornamental passion fruit hybrids are registered worldwide.
However, regarding the effective commercialization of passion fruit species as
ornamental plants in the Brazilian market, improved techniques for launching
cultivars adapted to shaded environments, potting and large-scale flower
production are lacking.
Theoretically, the limiting factors for the expansion and maintenance of
passion fruit cultivation can be classified as biotic stress factors (pests and
pathogens) and abiotic stress factors (low water availability, which is a
characteristic of certain Brazilian regions). The losses resulting from pathogen
attacks cannot be ameliorated by adopting better cultivation techniques for the
use of pesticides; thus, special attention must be paid to these stress factors by
researchers.
Among the common diseases that affect passion fruit, the following can
be highlighted due to their widespread occurrence in the national territory or
the difficulty of combat and control activities: anthracnose (Glomerella
cingulata, anamorfo: Colletotrichum gloeosporioides), verrugose
(Cladosporium cladosporioides and C. herbarum), fusariosis (Fusarium
oxysporum f. sp. passiflorae), bacteriosis (Xanthomonas axonopodis pv.
passiflorae) and passion fruit woodiness disease (Cowpea aphid-borne mosaic
virus) (Junqueira et al. 2005; Meletti et al. 2005; Fischer and Resende 2008).
Although some studies on these diseases have focused on their pathosystems,
the great majority are limited to the identification and epidemiological aspects
of pathogens and do not involve research programs allowing the continuous
evaluation of plant diseases. Furthermore, Fischer and Resende (2008) noted
that passion fruit production is generally associated with small producers, who
sometimes have other growing priorities.
Finally, the scarcity of commercial cultivars exploiting the potential of
wild passion fruit is the primary cause of the underutilization of this
biodiversity. Faleiro et al. (2015) mainly attribute the lack of cultivars
available to producers to the lack of consistency in the productive chain of
passion fruit, leading to high market prices (Faleiro et al. 2015). Thus,
increasing the number and quality of available cultivars will allow producers
to at least cope with crop restrictions imposed by pathogens and water
scarcity, increasing consistency in the production chain and commercial
stability for crop diversification.
30.2 State of the Art in Genetic Improvement
Passion fruit has been commercially cultivated in Brazil for over 40 years, and
regional varieties, such as Maguary, Golden Star, Amafrutas and the
composite IAC-27, have been available since the 1990s. However, the first
cultivars of passion fruit are more recent, having been introduced
approximately 15 years ago (Meletti 2011; Faleiro et al. 2011), and are
associated with Passiflora edulis Sims. The first cultivar related to a wild
species was introduced as recently as 2013, from P. setacea, and a second
cultivar related to a wild species (P. cincinnata) was released in 2016.
Currently, 34 entries are cataloged in the national cultivar registration
system (Ministério da Agricultura, Pecuária e Abastecimento – MAPA).
These entries are associated with the term passion fruit (consultation held in
May 2017,
http://extranet.agricultura.gov.br/php/snpc/cultivarweb/cultivares_registradas.p
hp). Approximately half of these records are related to cultivars that are
available to producers or have been evaluated by the scientific community. In
Table 1, nineteen cultivars are presented, for which dissemination and
commercialization information is available. Data from scientific evaluations of
the fruit production, ornamental use and disease reactions of these cultivars
have also been recorded.
The advances that have been made in the genetic improvement of passion
fruit are due to the establishment of groups and research networks dedicated to
characterization of the genus Passiflora, in public institutions such as
EMBRAPA, UESB, UESC, UNICAMP, Universidade de São Paulo (USP),
and Universidade Estadual do Norte Fluminense (UENF) in Brazil. In these
institutions, steps have been taken to support pre-breeding and breeding
programs that effectively contribute to the release of cultivars and hybrids in
the market.
Among the many possibilities for using biotechnology in genetic
improvement programs for passion fruit, molecular markers are mainly
employed to (i) characterize genetic diversity among accessions of wild
species (Cerqueira-Silva et al. 2012; Pérez-Almeida et al. 2010; Cerqueira-
Silva et al. 2010ab; Junqueira et al. 2007) and commercial varieties (Bellon et
al. 2007; Bellon et al. 2009; Fonseca-Trujillo et al. 2009; Cerqueira-Silva et al.
2010c; Ortiz et al. 2012) present in collections and germplasm banks; (ii)
monitor the variability and recovery of the parent genome over recurrent
selection cycles (Fonseca et al. 2009; Reis et al. 2011; Reis et al. 2012); (iii)
map genes and genomic regions associated with characteristics of interest
(Carneiro et al. 2002; Lopes et al. 2006; Oliveira et al. 2008; Penha et al.
2013); (iv) confirm hybrids (Junqueira et al. 2008; Conceição et al. 2011); and
(v) monitor introduced/marketed varieties, as discussed by Ferreira and Rangel
(2011) and in the next subsection of this chapter.
Practical examples of the use of molecular markers in breeding programs
include reduction of the time and resources required to retrieve the genomes of
parents of interest in backcrossing cycles (Faleiro et al. 2011). Thus, molecular
markers have contributed to the selection of intra- and interspecific passion
fruit hybrids with desirable characteristics (including disease resistance) and a
decreased genetic distance from the recurrent parent for various passion fruit
species (e.g., P. edulis, P. setacea, P. caerulea, and P. coccinea) (Faleiro et al.
2007; Fonseca et al. 2009; Faleiro et al. 2011).
Estimates of diversity based on microsatellite markers, performed in
conjunction with assessments of resistance to the major diseases that affect
passion fruit crops, are also being used to assist in the targeting of breeding
programs (Cerqueira-Silva et al. 2014b,c). For example, Cerqueira-Silva et al.
(2014c) used 23 microsatellite loci and determined the scale based on specific
symptoms for each of the diseases assessed (woodiness virus, scab and
anthracnose), which aided in the establishment of a nuclear collection
including the accessions of Passiflora edulis showing the highest levels of
resistance to diseases and exhibiting greater representativeness of the
microsatellite alleles identified in the germplasm. In addition to the
identification of accessions with a higher level of resistance to each of the
diseases, we also identified accessions with a reasonable resistance profile for
the three diseases considered in the study.
Table 1. Main Brazilian cultivars of passion fruit (varieties and hybrids of Passiflora
spp.) available for cultivation
Identification
Year of cultivar
release
Website information Responsible company
Monte Alegre (IAC-273)
1999
http://www.iac.sp.gov.br/publicacoes/agronomico/
maracuja_amarelo.php Instituto Agronômico de Campinas
http://www.iac.sp.gov.br/
Maravilha (IAC-275)
Joia (IAC-277)
IAC Paulista 2005
http://www.iac.sp.gov.br/publicacoes/agronomico/
pdf/v58_Maracuja_Roxo.pdf
FB 200 - Yellow Master
2008
http://www.viveiroflorabrasil.com.br/site/produtos-
2/
Viveiro Flora Brasil
http://www.viveiroflorabrasil.com.br
FB 300 - Araguari
CPATU Casca Fina 2002
http://dx.doi.org/10.1590/S0100-
29452003000100052
Embrapa
http://www.embrapa.br
BRS Sol do Cerrado (BRS SC1)
2008
http://www.cpac.embrapa.br/lancamentoazedo/
BRS Gigante Amarelo (BRS GA1)
BRS Ouro Vermelho (BRS OV1)
BRS Rubi do Cerrado (BRS RC)
2012
http://www.cpac.embrapa.br/lancamentobrsrubidoc
errado/
BRS Perola do Cerrado (BRS PC)
2013
http://www.cpac.embrapa.br/lancamentoperola/
BRS Sertão Forte (BRS SF)
2016
http://www.cpac.embrapa.br/lancamentosertaoforte
/
BRS Estrela do Cerrado
2007
http://www.cpac.embrapa.br/lancamentoornamenta
l/
BRS Rubiflora
BRS Roseflora
BRS Céu do Cerrado (BRS CC)
2017
http://www.cpac.embrapa.br/lancamentoornamenta
l2016/
BRS Rosea Púrpura (BRS RP)
BRS Mel do Cerrado (BRS MC)
2017
http://www.cpac.embrapa.br/lancamentomeldocerr
ado/
Sul Brasil
2010
AFRUVEC / Fundo Passiflora
SCS437 Catarina 2016
http://www.epagri.sc.gov.br/?p=13866
EPAGRI
(http://www.epagri.sc.gov.br/)
30.3 Genetic Diversity Based on Molecular Markers and
Characteristics of Agronomic Traits
Estimates of genetic diversity are based on molecular data and agronomic
characterization and are essential for the effective targeting and use of genetic
resources. Such assessments are standard in pre-breeding stages, and when
effectively conducted, they facilitate the use of available accessions in
germplasm banks for breeding programs (Lopes et al. 2011). Among the
advances in passion fruit cultivation achieved in recent decades, we highlight
the increasing use of molecular markers to obtain basic knowledge of the
genetic diversity of the genus (Fig. 4). Additionally, we note the effort of
different research groups to associate molecular and agronomic data for more
robust and useful characterization, to prioritize accessions in breeding
programs.
In this context, we have (as practical examples and directly associated
with the genetic improvement) the identification of potential nuclear
collections for some species of passion fruit (Passiflora edulis, P. cincinnata
and P. setacea), which provides information what can aid in their genetic
improvement (Cerqueira-Silva et al. 2014b,c). For these species, the authors
report the possibility of representing 100% of the allelic richness identified
with microsatellite markers through the maintenance growth of approximately
27% (for P. edulis), 75% (for P. cincinnata) and 47% (for P. setacea) of the
accessions maintained in the germplasm bank of Embrapa cassava and fruit.
Although only a few studies have been conducted with the aim of
proposing nuclear collections (or working collections), various authors have
presented genetic estimates based on molecular markers and Passiflora spp.
accessions maintained in germplasm banks (Bellon et al. 2009; Cerqueira et al.
2010abc; Santos et al. 2011; Ortiz et al. 2012; Cerqueira-Silva et al. 2012;
Cerqueira-Silva et al. 2014b,c). In summary, despite the specific objectives of
each study, the estimates of genetic diversity contribute to the understanding
of the variability available in the germplasm banks and the direction of
convergent and divergent crosses that drive the initial stages of breeding.
Fig. 4. Histogram of the percentages of reports associated with genetic-molecular
studies of Passiflora species publishes from 1999 to 2016. The percentages were
calculated based on the SCOPUS database (www.scopus.com), adopting the search
terms "Passiflora" and “molecular marker” as well as the corresponding Portuguese
words
30.4 Genetic Mapping
The first maps were published by Carneiro et al. (2002) and were based on the
characterization of a segregant population of P. edulis through random
amplification of polymorphic DNA (RAPD) markers. The population used to
construct the first map helped identify the first quantitative resistance locus
(QRL) linked to the genus Passiflora, based on amplified fragment length
polymorphism (AFLP) markers (Lopes et al. 2006), which explained
approximately 16% of the phenotypic variation related to the symptoms of
bacterial spot disease caused by Xanthomonas axonopodis pv. passiflorae.
Oliveira et al. (2008) employed map integration strategies to construct a
single map, which was a landmark in genetic mapping studies and the
identification of QTLs in passion fruit. An important advantage of the
integrated construction of a single representative map for a segregant
population is an increase in map saturation and length. Recently, this research
was extended to construct maps for passion fruit plants of another species of
this genus (P. alata) (Vieira et al. 2005). The use of variable AFLP and simple
sequence repeat (SSR) analyses as well as the first characterization of SNPs in
passion fruit plants were included in this map.
The first research dedicated to the construction of physical maps was
based on putative genes identified from a passion fruit genomic library
inserted into bacterial artificial chromosomes (BACs) (Penha et al. 2012;
Santos 2013). These studies are a new development in genetic and genomic
research in passion fruit, allowing the characterization of genomes and the
identification of evolutionary relationships among species and aiding in the
development of new molecular markers, including SSR markers, expressed
sequence tags (ESTs), and SNPs (Cerqueira-Silva et al. 2014a).
Advances in the genetic mapping of passion fruit associated with the
reduction of next-generation sequencing (NGS) costs should enable genetic
characterization based on genotyping-by-sequencing (GBS), at least for
commercial species. Thus, alternative methodologies that decrease costs and
enable GBS, such as the use of restriction enzymes to reduce genome
complexity (Elshire et al. 2011), should be considered in future genetic studies
of passion fruit. These methods can contribute to both population studies and
genome-wide selection in collections and germplasm banks (Cerqueira-Silva
et al. 2014a).
30.5 Genetic Engineering
Genetic engineering in passion fruit has been employed as a strategy for the
development of resistance to diseases such as passion fruit woodiness (PWD)
(caused by cowpea aphid-borne mosaic virus in Brazil) and bacterial spot
disease (caused by Xanthomonas axonopodis pv. passiflorae), against which
chemical control measures are not effective, and resistant cultivars are not
available (Alfenas et al. 2005; Trevisan et al. 2006; Freitas et al. 2007;
Monteiro-Hara et al. 2011). These studies were initiated in the late 1990s and
early 2000s, and their first results were published in 2005 and 2006; in these
studies, P. edulis plants were transformed with fragments of genes obtained
from viral isolates known to cause PWD (Alfenas et al. 2005; Trevisan et al.
2006).
The first transgenic P. alata plants were later generated by Pinto (2010),
who demonstrated the possibility of obtaining transgenic P. alata plants using
methods similar to those employed in studies conducted in P. edulis (Alfenas
et al. 2005; Trevisan et al. 2006). More recently, Correa et al. (2015)
demonstrated that to the possibility of selecting Passiflora alata plants with
gain of resistance to CABMV through the incorporation of CABMV-gene
fragments into the genome of P. alata lines.
Although the available studies dedicated to the production of transgenic
passion fruits are not conclusive, they indicate that, at least for PWD (in
Passiflora edulis and P. alata), it is possible to obtain resistant plants for
cultivation and/or for use in breeding programs dedicated to the production of
resistant cultivars with higher productivity. However, the release of cultivars
related to plants of transgenic passion fruits was not observed.
30.6 Biotechnology Tools in Pre- and Post-Breeding Programs
The various stages of pre-breeding, especially the characterization of material
from natural populations and germplasm banks, are essential for extending the
genetic basis of breeding programs Ferreira and Rangel 2011; Pereira, Pereira
and Viana 2005. Moreover, these steps aid in the identification of genetic
variability, which further facilitates the study of resistance to biotic and abiotic
stress factors (Ferreira and Rangel 2011). Thus, biotechnology and molecular
tools (especially molecular markers) effectively contribute to estimation of the
available variability in germplasm banks, to identify duplicate accessions,
monitor the percentage of the genome recovered in backcrossing cycles, and
confirm inter- or intra-specific hybrids (Cerqueira-Silva et al 2016; Cerqueira-
Silva et al 2014a; Ferreira and Rangel 2011).
The real success of genetic improvement is associated with the use of the
developed products and their acceptance and consumption. Thus, post-
breeding activities essentially highlight (i) the preparation of marketing plans;
(ii) the definition of strategies and logistics for upscaling the production and
commercialization of seeds; (iii) the registration and protection of cultivars by
legal and competent bodies; (iv) the performance of market promotion and
insertion actions; and (v) the strengthening of partnerships with the private
sector, aiming at the validation and application of technologies. In this context,
especially regarding items “ii,” “iii” and “v,” molecular markers have the
potential to accelerate and provide security in the genetic identification and
consequent protection of these products.
Thus, biotechnology and molecular tools can contribute to the
effectiveness of genetic improvement programs and significantly reduce the
amount of time necessary to complete the stages inherent to breeding
programs, thereby bestowing confidence in the monitoring of the marketed
technological product (Cerqueira-Silva et al 2016; Ferreira and Rangel 2011;
Faleiro, Junqueira and Braga 2006; Pereira, Pereira and Viana 2005).
30.7 Conclusions and Prospects
Although population studies of Passiflora species are highly relevant to
conservation and breeding activities, this research is in its nascent stage.
Population studies, associated with increasing numbers of microsatellite
markers and recent advances in the development and use of SNP markers, are
important strategies for achieving sequencing and genotyping on a large scale.
The development and application of large-scale genotyping should allow the
use of strategies for genome-wide selection (or simply genomic selection),
enhancing the association of genetic diversity data with characteristics of
agronomic interest. Moreover, the use of GBS strategies reduces the costs and
time required for germplasm characterization and the selection of passion fruit
genotypes.
In tandem with the population estimates that must be performed for the
genus Passiflora using molecular tools, strategies for the exploration and
conservation of germplasm collections are also necessary to enable morpho-
agronomic characterization and to improve the understanding of diversity.
Thus, interactions involving the accessions present in germplasm banks and
collections and the elite materials used in breeding programs will be enhanced.
Finally, the molecular characterization and evaluation of agronomic
characteristics, particularly those related to disease resistance, will promote
better utilization of Passiflora biodiversity in breeding programs. In this
context, resistance characteristics must be introduced not only to species such
as P. edulis and P. alata but also, more importantly, to cultivars related to new
wild species that can potentiate the diversification of passion fruit cultivation
should be made available.
Acknowledgments The authors thank Empresa Brasileira de Pesquisa
Agropecuaria (Embrapa), Universidade Estadual do Sudoeste da Bahia
(UESB, campus Juvino Oliveira) and Universidade Estadual de Campinas
(UNICAMP) for their support of this chapter; the authors also acknowledge
Fundação de Amparo a Pesquisa de São Paulo (FAPESP 2008/52197-4),
Fundação de Amparo a Pesquisa do Estado da Bahia (FAPESB
DTE0001/2016) and Conselho Nacional para o Desenvolvimento Científico e
Tecnológico (CNPq) for their financial support and fellowships.
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Appendix I: Research institutes and online resources
List of major institutes engaged in research on Passion fruit (Passiflora spp.)
1. Centro Internacional de Agricultura Tropical, 6713, CIAT/Cali, Colombia. e-
mail: jaocampop@unal.edu.co
2. Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina, Florianópolis,
88034-901, Santa Catarina, Brazil. http://www.epagri.sc.gov.br/
3. Empresa Brasileira de Pesquisa Agropecuária, Cruz das Almas, 44380000, Bahia, Brazil.
e-mail: onildo.nunes@embrapa.br
4. Empresa Brasileira de Pesquisa Agropecuária, Brasília, 70770901, Distrito Federal,
Brazil. e-mail: fabio.faleiro@embrapa.br
5. Instituto Agronômico de Campinas, Campinas, 13001970, São Paulo, Brazil. e-mail:
lmmm@iac.sp.gov.br
6. Universidade Estadual de Santa Cruz, Ilhéus, 45662900, Bahia, Brazil. e-mail:
ronanxc@uesc.br
7. Universidade Estadual do Sudoeste da Bahia, Itapetinga, 45700000, Bahia, Brazil. e-mail:
csilva@uesb.edu.br
8. Universidade Estadual do Sudoeste da Bahia, Vitória da Conquista, 45100000, Bahia,
Brazil. e-mail: abelsj@edu.uesb.br
9. Universidade Estadual de Campinas, Campinas, 13083860, São Paulo, Brazil. e-mail:
anete@unicamp.br
10. Universidade de São Paulo, Piracicaba, 13418900, São Paulo, Brazil. e-mail:
mlcvieir@usp.br
11. Universidade Federal de Viçosa, Viçosa, 36571000, Minas Gerais, Brazil. e-
mail: bruckner@ufv.br
12. Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, 28013602, Rio de
Janeiro, Brazil. e-mail: pirapora@uenf.br
13. Universidade Federal do Rio Grande do Sul, Porto Alegre, 91501970, Rio Grande do Sul,
Brazil. e-mail: loreta.freitas@ufrgs.br
14. Universidad Jorge Tadeo Lozano, 22-61, Bogotá, Colombia. e-
mail: javier.hernandez@utadeo.edu.co
15. Viveiro Flora Brasil, Araguari, Minas Gerais, Brazil. e-mail:
florabrasil@viveiroflorabrasil.com.br
Index keywords
Biodiversity
Biotechnology
Genetic conservation
DNA
Fruit
Euphytica
Germplasm conservation
Genetic mapping
Molecular markers
Molecular tools
Passion flower
Passion fruit
Passifloraceae
Plant breeding
Plant improvement
Resistance
Transgenic
