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A case for Molecular Breeding in Musa

A Case for Molecular Breeding
in Musa
Michael Pillay,1,* Kaliyaperumal Ashokkumar,2,a
Arun Siva Kumar Shunmugam2,b and Sivalingam Elayabalan3
Conventional breeding of Musa that is based mainly on mass phenotypic
recurrent selection is handicapped by a number of factors. The rapid
development of molecular biology techniques and their application to
plant breeding has resulted in signifi cant genetic gains in agricultural
crops. Marker assisted breeding will be very useful for a crop like banana
that has a relatively long life cycle. DNA markers are being sought for
several characters of importance in Musa including resistance to pests
and diseases. Achievements and prospects of molecular breeding for
black Sigatoka, Fusarium, Banana bunchy top virus (BBTV), nematodes
and Xanthomonas wilt resistance are discussed. In addition gains made
in nutritional enhancement of banana are described. The development
of modern plant molecular and quantitative genetics in the last two
decades has the potential to revolutionize what has mostly been
experienced-based empirical plant breeding. This chapter outlines the
value of modern molecular tools for molecular breeding of banana.
Keywords: breeding challenges, molecular markers, molecular breeding
1Vaal University of Technology, Private Bag X021, Vanderbijlpark 1900, Gauteng, South
2Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon,
SK, Canada, S7N 5A8.
3Crop Tech Ltd, P.O. Box 1367 Arusha, Tanzania; e-mail:
*Corresponding author
List of abbreviations after the text.
© 2012 by Taylor & Francis Group, LLC
282 Genetics, Genomics and Breeding of Bananas
15.1 Introduction
Currently the production of improved banana and plantain cultivars that
are nutritionally acceptable to consumers, with resistance or tolerance to
biotic and abiotic stresses, and reduced post-harvest losses, has been met
largely through conventional breeding that has made steady progress over
the years producing a large number of hybrids (Rowe 1984; Vuylsteke et al.
1995). The pressure of an increasing population and consequent increase in
demand for food on the one hand and the depletion of arable land on the
other have placed new emphases on conventional plant breeding (Pillay
et al. 2011). However, conventional breeding of Musa is handicapped by
sterility and a number of other factors that are discussed in Pillay et al.
(2002), and Pillay and Tripathi (2006, 2007).
The rapid development of molecular techniques and their application
to plant breeding has resulted in signifi cant genetic gains in agricultural
crops, some of which have already entered the market (Newell-McGloughlin
2008). Molecular and biotechnological tools such as marker-assisted
breeding, tissue culture, in vitro mutagenesis and genetic transformation can
contribute to solving or reducing some of the constraints of conventional
banana breeding. This chapter examines some aspects of molecular breeding
in Musa.
15.1.1 Breeding Challenges in Musa
Musa is a polyploid crop with ploidy ranging from diploid (2n = 2x = 22) to
tetraploids (2n = 4x = 44). Most cultivated bananas are triploids (2n = 3x =
33) and sterile harboring various combinations of either one, two or three A,
B, S, or T genomes. New banana cultivars are exceptionally cumbersome to
develop. Selection for desirable characters is time consuming and it may take
up to 12 years to develop a new cultivar. Musa breeding is based mainly on
phenotypic mass recurrent selection. The high levels of heterozygosity make
identifi cation of ideal parental material diffi cult and very large populations
are required for selection of individual clones with good agronomic traits.
This is virtually impossible to attain due to the low seed set in crosses.
Generally, few seeds are obtained (an average of 1 to 1.5) and acquiring
large numbers of seeds is a labor intensive and tedious process (Ortiz and
Vuylsteke 1995; Ssebuliba et al. 2006a, b, 2009). The genes for resistance to
diseases and pests are introgressed from wild diploid species. Wild species
also carry many undesirable traits, e.g., low yield and non-parthenocary. The
process of eliminating the unwanted traits requires several backcrosses that
lengthen the breeding process. The multigenic nature and low heritability
© 2012 by Taylor & Francis Group, LLC
A Case for Molecular Breeding in Musa 283
of some traits also slow down the breeding process. Musa breeding is also
problematic due to the narrow genetic diversity of the germplasm (Pillay
et al. 2001; Nyine and Pillay 2011) and the lack of information about wild
species that carry useful agronomic traits. Only a few wild diploids have
been used so far and mostly as male parents by majority of the breeding
programs. As cultivated banana is propagated asexually, its genetic base
is narrow with diversity dependent on somatic mutation. Limited genetic
variation has resulted in a crop lacking resistance to fungal, bacterial
and viral pathogens and numerous pests (Miller et al. 2009). Very little
knowledge exists on the genetics of important agronomic traits in Musa
and precise genetic control is known for relatively few traits (see Chapters
6, 7).
15.1.2 Production Constraints
The production constraints of Musa have been well documented (Pillay
et al. 2002; Pillay and Tripathi 2006, 2007; Tenkouano et al. 2011). Briefl y
the production of bananas worldwide is threatened by a complex of foliar
diseases, nematodes, viruses and pests. The use of resistant varieties is
considered to be the most effective, economical and environmentally
friendly approach to controlling diseases and pests. Two of the most
important fungal diseases include black Sigatoka (Mycosphaerella fi jiensis
Morelet) and fusarium wilt (Fusarium oxysporum Schlect. f.sp. cubense (E.F.
Smith). The main pests include a complex of nematodes (Radopholus similis,
Pratylenchus spp. Helicotylenchus) and the banana weevil (Cosmopolites
sordidus Germar). New diseases such as banana Xanthomonas wilt (BXW)
have been recently identifi ed in East Africa.
15.1.3 Breeding Objectives in Musa
The most important objectives of Musa breeding include:
• increased bunch size and yield
host plant resistance against the major pathogens including those
causing Sigatoka, Fusarium and Xanthomonas wilts, and viruses
• host plant resistance against nematodes and insect pests
• fruit quality traits, e.g., increased vitamin A, iron and zinc levels
• better adaptation to abiotic stresses such as drought, heat and other
stresses that may be enforced by predictions in climate change.
Breeding for yield is a major target followed by breeding for host plant
resistance to pathogens and pests that impact on yield.
© 2012 by Taylor & Francis Group, LLC
284 Genetics, Genomics and Breeding of Bananas
15.2 Molecular Breeding
Molecular breeding (MB) is the generic term used to describe several
modern breeding methods including, (i) marker-assisted selection (MAS)
—the selection of specifi c alleles for traits conditioned by a few loci, (ii)
marker-assisted backcrossing (MABC)—the transfer of a limited number
of loci from one genetic background to another, including transgenes, (iii)
marker-assisted recurrent selection (MARS)—the identifi cation and selection
of several genomic regions involved in the expression of complex traits to
“assemble” the best-performing genotype within a single, or across related,
populations, and (iv) genome wide selection (GWS)—selection based on
markers without signifi cant testing and without identifying a priori a subset
of markers associated with the trait (Ribaut et al. 2010). MABC is one of
the most anticipated and frequently cited benefi ts of molecular markers as
indirect selection tools in breeding programs (Semagn et al. 2006).
Routine use of MAS in ongoing plant breeding programs has not been
achieved as yet. The implementation of MAS has been slow due to the high
relative cost compared to conventional phenotypic selection. To be useful
to plant breeders, gains made from MAS must be more cost-effective than
gains through traditional breeding or MABC must generate signifi cant time
savings to justify the additional cost involved (Semagn et al. 2006).
Since a large number of traits in plants are polygenic, MABC of traits
controlled by single genes is the most effective way of using DNA markers
effectively. The improvement of quantitative trait loci (QTL) through
MABC has produced variable results ranging from limited success and/
or even a failure to a few highly successful stories (Semagn et al. 2006).
Marker-assisted breeding will be very useful for a crop like banana that has
a relatively long life cycle. The use of molecular markers for the indirect
selection of improved cultivars speeds up the selection process by alleviating
time-consuming approaches of direct screening under greenhouse or fi eld
conditions. Some of the most important characters to the Musa breeder have
been reported to have an oligogenic epistatic basis (Ortiz 1995).
15.2.1 Molecular Markers in Musa
It is now generally accepted that molecular markers represent the most
signifi cant advance in breeding technology in the last few decades and
are currently the most important application of molecular biology to plant
breeding. There appears to be no resistance to the use of molecular marker
technology in breeding as there is for genetically-modifi ed organisms
(Pillay et al. 2011). DNA markers are being sought for several characters
of importance in Musa including resistance to pests and diseases. Fruit
quality (color, texture, ripening) are other candidate traits for selecting with
© 2012 by Taylor & Francis Group, LLC
A Case for Molecular Breeding in Musa 285
DNA markers. Most of these traits are expressed only late in the life cycle
of the plants or are diffi cult to screen. Identifi cation of markers linked to
loci governing important traits will facilitate gene introgression and other
MAS applications. Accessing genes from various genomes, including the S
(M. schizocarpa) and T (M. textilis) genomes will increasingly become
important for Musa breeding. To date very few markers have been linked
to traits of interest in Musa and are limited to markers for disease resistance
and the main genomes.
Methylation-sensitive amplifi cation polymorphism (MSAP) markers
were used to identify molecular markers associated with resistance to
Mycosphaerella fi jiensis toxins (black Sigatoka) with a set of reference cultivars
and somaclonal variants (Gimenez et al. 2006). The study identifi ed four
MSAP markers that were associated with resistance to M. fi jiensis toxins.
The MSAP markers showed a high degree of sequence similarity with
resistance gene analogs and with retrotransposon sequences. These markers
were cited as being useful as molecular indicators of tolerance to M. fi jiensis
toxins and resistance to black Sigatoka.
A reliable molecular method to detect Fusarium oxysporum f.sp. cubense
(Foc) race 4 isolates in Taiwan was developed by (Lin et al. 2010). By PCR
amplifi cation, the primer set Foc-1/Foc-2 derived from the sequence of
a random primer OP-A02 amplifi ed fragment produced a 242 bp size
DNA fragment, which was specifi c to Foc race 4. With the optimized PCR
parameters, the molecular method was sensitive and could detect small
quantities of Foc DNA as low as 10 pg in 50 to 2,000 ng host genomic DNA
with high effi ciency.
A putative RAPD marker for Sigatoka resistance has been identifi ed
at the National Research Center for Banana (NRCB), India. The marker
has been cloned, sequenced and converted into a sequence characterized
amplifi ed region (SCAR) marker and is being validated using contrasting
parents for expression of Sigatoka (M. musicola) disease resistance and their
progenies. Parallel studies have led to the identifi cation of a putative random
amplifi ed polymorphic DNA (RAPD) marker for nematode resistance (S.
Uma, pers. comm.) An RAPD marker has been identifi ed for salt tolerance
among clones of cv. “Dwarf Cavendish” that were obtained through induced
mutagenesis (Miri et al. 2009).
A banana somatic embryogenesis receptor-like kinase (SERK) gene,
designated as MaSERK1, isolated from Musa acuminata cv. “Mas” (AA) was
associated with somatic embryogenic competence and disease resistance
response in Musa (Xia et al.. 2010). The gene encoded a protein of 628 amino
acids with identities of above 82% to SERK genes in coconut, rice, maize,
Arabidopsis, carrot, and Medicago truncatula. MaSERK1 was expressed weakly
in male fl ower clusters, but not in male fl ower-derived non-embryogenic
calli. It was highly expressed in male fl ower-derived embryogenic calli
© 2012 by Taylor & Francis Group, LLC
286 Genetics, Genomics and Breeding of Bananas
and embryogenic cell suspensions (ECS). The frequency of somatic
embryogenesis of ECS positively correlated with MaSERK1 transcript levels.
MaSERK1 expression in leaves of cultivar “Dongguan Dajiao” (ABB), known
to be resistant to FOC race 4, was induced by exogenous salicylic acid (SA)
or inoculation with FOC race 4. However, MaSERK1 expression levels in
leaves of “Pisang awak” (ABB), known to be susceptible to FOC race 4, did
not change following either treatment (Xia et al. 2010). It was suggested
that MaSERK1 gene expression not only could serve as a molecular marker
for banana somatic embryogenesis, but could also play a role in host plant
resistance response to banana pathogens.
15.2.2 Marker-Assisted Introgression
Marker-assisted breeding takes advantage of the association between
agronomic traits and allelic variants of genetic markers, mostly molecular
markers (Stam 2003). Generally these associations are the result of
genetic linkages between markers and gene loci underlying the trait(s)
of interest. These associations are also known as linkage disequilibrium.
Linkage disequilibrium arise in experimental populations used for linkage
mapping, e.g., backcross generations (BC), F2 segregating populations,
recombinant inbred lines (RILs) or doubled haploids (DHs) (Stam 2003). In
cross-fertilizing plant species such as Musa a mapping population usually
consists of a large full-sib family resulting from a cross between single
plants of divergent genotypes. Before a plant breeder can utilize linkage-
based associations between traits and markers, the associations have to be
assessed with a certain degree of accuracy, such that it can be safely relied
on, and thus marker genotypes can be used as indicators or predictors of
trait genotypes and phenotypes (Stam 2003). For monogenic traits with
a clear qualitative contrast between genotypes, such as a single gene-
based host plant resistance to pathogens, the assessment of association is
straightforward: mapping a monogenic trait goes along with the mapping of
markers. For quantitative, multigenic traits, however, a reliable assessment
of trait-marker association requires large-scale fi eld experiments as well
as statistical techniques, known as QTL mapping (Stam 2003). Progress in
the breeding of plantain and banana has been restricted by the complex
genetic structure and behavior of cultivated polyploid Musa. Mapping in
Musa has been hampered by the low levels of male and female fertility and
seed viability and the absence of large segregating populations. The key
to successfully integrating marker-aided breeding into breeding programs
will lie in identifying applications in which markers offer real advantages
over conventional breeding methods or complement them in novel ways
(Semagn et al. 2006). Marker-aided breeding offers signifi cant advantages
in the following cases.
© 2012 by Taylor & Francis Group, LLC
A Case for Molecular Breeding in Musa 287
1) When phenotypic screening is expensive, diffi cult or impossible.
2) When the trait is of low heritability (incorporating genes that are highly
affected by environment).
3) When the selected trait is expressed late in plant development, like
fruit and fl ower features or adult characters in species with a juvenile
4) For incorporating genes for host plant resistance to pathogens or pests
that cannot be easily screened for due to special requirement for the
gene to be expressed.
5) When the expression of the target gene is recessive.
6) To accumulate multiple genes for one or more traits within the same
cultivar, a process used is called gene pyramiding (Sharma et al. 2004;
Barone et al. 2005; Yang et al. 2005).
Highly precise MAS approaches require the development of high density
linkage maps. Improved molecular markers systems are required to enhance
the adoption of MAS. Several factors are important when considering MAS
including ease of use, robustness, cost and linkage to trait of interest (de
Koeyer et al. 2010). The ideal marker systems for polyploid crops should be
dosage sensitive and have the ability to distinguish heterozygous genotypes
with multiple haplotypes within the target genomic region by the marker
(de Koeyer et al. 2010).
Currently, several molecular marker methods have been used in Musa
and these differ from each other in their technical requirements, sensitivity
and reliability (see Chapter 4). New markers systems are available and have
not been exploited in Musa as yet.
15.2.3 Gene Pyramiding
Gene pyramiding is the accumulation of multiple genes for one or more
traits within the same cultivar (Barone et al. 2005; XiangYan et al. 2005).
Genetic stocks produced from gene pyramiding can be used in breeding
programs. Gene pyramiding is a very useful approach for the introgression
of genes controlling different agronomic traits into one cultivar to ensure
that the cultivar has acquired several traits simultaneously (Semagn et al.
2006). For example, genes leading to host plant resistance to different races
or biotypes of a pathogen or an insect pest can be pyramided together to
make a line with multi-race or multi-biotype resistances, which could be
more durable than any single-race or single-biotype resistance (Jiang et
al. 2004). The joint expression of pyramided genes was found to provide
numerical increases or a broader spectrum of host plant resistance over
that conferred by single genes through gene interaction and quantitative
complementation (Yoshimura et al. 1995; Singh et al. 2001). Gene pyramiding
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288 Genetics, Genomics and Breeding of Bananas
has been successfully applied in several crop breeding programs, and many
cultivars and lines possessing multiple attributes have been produced
(Porter et al. 2000; Wang et al. 2001; Samis et al. 2002; Jiang et al. 2004).
Traits which are traditionally regarded as quantitative and not targeted
by gene pyramiding programs can be improved using gene pyramiding
if major genes affecting the trait are identifi ed (Ashikari and Matsuoka
2006). Gene pyramiding is, however, diffi cult using conventional breeding
methods due to the dominance and epistasis effects of genes governing
disease resistance (the stronger resistance genes will always mask the less
strong, which cannot be revealed without screening using a virulent strain
on the former—in itself undesirable) (Semagn et al. 2006). Moreover, genes
with similar reactions to two or more races—so called race-non specifi c or
partial resistance—are diffi cult to identify and transfer through conventional
approaches (Singh et al. 2001), and virtually impossible if stronger race-
specifi c genes are present.
Gene pyramiding programs are also thought to be highly cost intensive.
While the breeder’s work will be made easier by using a single donor,
phenotyping is still required to select the desired segregants in fi eld
experiments. It is also highly likely that whatever genes are being stacked
into one cultivar might lose their usefulness by the time they are pyramided
and subsequently by the time they are used by the breeder.
15.2.4 Marker Systems and Germplasm Characterization
The most frequent use of molecular marker methods in Musa has been
limited to germplasm characterization and diversity analysis. There are
about 1,500 to 3,000 Musa accessions with a wide range of morphological
variation and genome constitutions (Heslop-Harrison and Schwarzacher
2007) within the germplasm. About 1,000 Musa cultivars and 180 wild
species, are maintained in tissue culture at the International Transit Centre
(ITC) in the Catholic University of Leuven (KULeuven) in Belgium, and
these provide a valuable reference collection that is mostly in the public
domain and freely accessible for research and breeding. Numerous banana
researchers in Asia have developed fi eld-based germplasm collections and
well-curated internet databases are now disseminating information about
these collections (Pollefeys et al. 2004). Although diversity can be assessed
by morphology and fl ow cytometry, these analyses have limitations and
there remain questions about the presence of multiple genotypes with a
single name or a single genotype with multiple names (Heslop-Harrison
and Schwarzacher 2007). Therefore, DNA-based molecular diversity studies
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A Case for Molecular Breeding in Musa 289
will help to direct plant breeders towards appropriate germplasm to test and
select, and to focus germplasm collections towards representing the full range
of diversity present in the genus at all ploidy levels (Heslop-Harrison and
Schwarzacher 2007). The various techniques used to assess Musa diversity
and phylogenetic relationships have been addressed in Chapter 3.
15.2.5 Transgenic Breeding
Conventional breeding of bananas is hindered by a number of factors
including the long-generation time, triploidy and sterility of most edible
cultivars (Pillay et al. 2002). Sources of resistance to many of the major pests
and diseases have been identifi ed in a few wild diploid species. However,
most landraces are often sterile and cannot be used in breeding, while
crosses involving wild species result in the transfer of many unwanted
traits together with the desired resistance genes. Furthermore, there are
certain diseases such as banana bunchy top virus (BBTV) for which sources
of resistance are not known (Sagi et al. 1998).
Although conventional breeding of Musa is faced with diffi culties,
currently available transformation methods may not solve all these
diffi culties. Increased understanding of responses in Musa to biotic and
abiotic stresses may provide new opportunities for genetic improvement.
Genetic transformation provides an opportunity for single genes or
gene combinations, such as those associated with host plant resistance
to pathogens, to be extracted from the genome of the source organism
and transferred directly into the desired cultivar, which allows to retain
all the original characteristics of the cultivar and adding the desired trait.
Furthermore, since most banana cultivars do not produce seeds under
natural conditions, crosses with other cultivars or species will seldom
occur. In these cases, the introduced gene remains confi ned to the cultivar
in which it has been introduced (Sagi et al. 1998).
Relative success in genetic engineering of bananas and plantains has
been achieved enabling the transfer of foreign genes into some cultivars
(Sagi et al. 2007). But the scarcity of useful genes, factors that affect transgene
expression such as RNA interference, interactions between transgenes and
those already present in the plant and the quantitative nature of some traits
are still problems that must be considered before accepting that genetic
transformation is the only choice for Musa improvement. Protocols for the
introduction of genes, including the effi cient regeneration of shoots in tissue
cultures, and transformation methods still remain as major bottlenecks in
genetic engineering (Sharma et al. 2005).
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290 Genetics, Genomics and Breeding of Bananas
15.3 Achievements and Prospects of Transgenic Breeding in
15.3.1 Resistance to Black Sigatoka and Fusarium Wilt Disease
Transformation of banana and plantains for fungal diseases started in
the 1990s (Sagi et al. 1995). Various transformation techniques have been
used to produce transgenic bananas for the cultivars “Williams” (AAA)
export banana, “Gros Michel” (AAA) fruit banana, “Bluggoe” (ABB)
cooking banana and “Three Hand Planty” (AAB) plantain with antifungal
peptides which are highly active in vitro against major pathogenic fungi
such as black Sigatoka and Fusarium wilt of bananas (Remy et al. 2000).
The transgenics showed resistance to black Sigatoka under laboratory
conditions. Antimicrobial proteins (AMPs) which are stable, cysteine-rich
small peptides isolated from seeds of diverse plant species were also used
in developing transgenics for fungal diseases (Sagi et al. 1998).
Improved resistance to Sigatoka was obtained when banana was
transformed with the endochitinase gene ThEn-42 from Trichoderma
harzianum and the grape stilbene synthase (StSy) gene (Vishnevetsky et
al. 2010). The superoxide dismutase gene Cu,Zn-SOD from tomato, under
control of the ubiquitin promoter, was also added to this cassette to improve
scavenging of free radicals generated during fungal attack. A 4-year fi eld
trial demonstrated several transgenic banana lines with improved tolerance
to Sigatoka. Since the genes conferring Sigatoka tolerance may have a
wide range of antifungal activities the regenerated banana plants were
also inoculated with the fungus Botrytis cinerea. The best transgenic lines
exhibiting Sigatoka tolerance were also found to have tolerance to B. cinerea
in laboratory assays (Vishnevetsky et al. 2010). Gene discovery via analysis
of EST data from cDNA libraries produced from Mycosphaerella fi jiensis-
infected leaf material from M. acuminata ssp. burmannicoides “Calcutta 4”
(resistant) and “Grande Naine” (AAA genome, susceptible) is ongoing in
Brazil (Miller et al. 2009)
15.3.2 Resistance to Banana Bunchy Top Virus (BBTV)
Transgenic research to develop resistance to BBTV has been in progress in
Australia and Hawaii. The replication initiation protein (Rep) of nanoviruses
is the only viral protein essential for viral replication and represents an
ideal target for pathogen derived resistance. In Australia, a Rep-encoded
protein (DNA-S1) was identifi ed that suppressed the replication of BBTV.
Different constructs of the Rep gene were shown to signifi cantly suppress
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A Case for Molecular Breeding in Musa 291
replication of BBTV in banana embryogenic cell suspensions (Tsao and
Tsun-Hui 2008). Using such constructs, transgenic bananas with resistance
to BBTV have been developed in Australia and Hawaii. The resistant lines
have been fi eld tested in Hawaii.
15.3.3 Resistance to Nematodes
The approach adopted for nematode resistance in Musa relies on introducing
an additional plant gene coding for a protein called cystatin that prevents
the digestion in parasitic nematodes. The cystatin suppresses the nematode’s
ability to grow, lay eggs and build to population levels that damage crops.
The advantage of using cystatins is that they are part of the human diet
(e.g., present in cereal seeds or eggs) and have no effect on our digestion or
health. This approach has been already been used in developing a transgenic
Cavendish bananas (AAA) that showed resistance to Radopholous similis one
of the major nematodes of Musa (Atkinson et al. 2004).
15.3.4 Resistance to Banana Xanthomonas Wilt
Banana Xanthomonas or bacterial wilt disease caused by infection with
Xanthomonas campestris pv. musacearum (BXW) has reached epidemic
proportions in the Great Lakes region of East and Central Africa (Biruma et
al. 2007). The lack of banana germplasm exhibiting resistance to the disease
makes it an ideal target for transformation. Transgenic technologies may
hold the key for developing bananas that are resistant to the BXW pandemic.
The ferredoxin-like amphipathic protein (pfl p) and hypersensitive response
assisting protein (hrap), isolated from sweet pepper (Capsicum annuum) are
novel proteins that can intensify the harpinPSS-mediated hypersensitive
response (Chen et al. 2000). Transgenic rice carrying the pfl p gene showed
enhanced resistance to Xanthomonas oryzae pv. oryzae (Tang et al. 2001).
The pfl p has also been shown to enhance resistance in transgenic orchids
against E. carotovora (Liau et al. 2003). The elicitor-induced resistance
is not specifi c against particular pathogens, so it could be a very useful
strategy for developing broad spectrum resistance. This strategy has been
used for developing transgenic banana with resistance to Xanthomonas
wilt. Transgenic lines with pfl p or hrap genes have been developed using a
protocol based on the Agrobacterium tumefaciens technology (Tripathi et al.
2009). These transformed lines of various cultivars have been validated via
PCR assay and Southern blot analysis. They have been tested for disease
resistance under laboratory conditions and transgenic bananas showed
complete resistance. The transgenic lines are now under fi eld trials in
Uganda (Tripathi et al. 2010).
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292 Genetics, Genomics and Breeding of Bananas
15.3.5 Nutritional Enhancement
Banana is rich in natural antioxidants such as vitamin C and vitamin E
(Someya et al. 2002; Amorim et al. 2009a, b). High levels of vitamin A
defi ciency leads to serious health problems, especially in children in low-
income regions of the world, such as parts of Asia, Africa and Latin America
(Bloem et al. 2005). Micronutrient defi ciencies of iron and zinc also results in
serious health problems such as mental and physical retardation, reduced
resistance to infections and hypogonadism (Whittaker 1998). The genetic
enhancement of micronutrient content (i.e., biofortifi cation) of banana by
conventional breeding combined with the use of biotechnological tools has
the potential to increase the concentrations of micronutrients (Fe, Zn) and
vitamin A in new cultivars (Amorim et al. 2011). Improving the nutritional
content of Musa would have a signifi cant impact on vitamin and nutrient
intake for millions of people who depend on the crop for food.
Researchers in Australia are transforming bananas for increased vitamin
A, vitamin E or iron. A large suite of both fruit-specifi c and constitutive
promoters that drive pro-vitamin A, vitamin E, or iron accumulation genes,
have been cloned into vectors. Four cultivars “Nakinyika”, “Mpologoma”,
“Nakasabira”, and “Sukalindizi” have been selected for this study (Dale
and Tushemeirewe 2008). A study by Fungo and Pillay (2011) showed that
cultivars “Nakitembe”, “Entukura” and “Nakhaki” had the highest levels
of vitamin A among 10 East African Highland bananas in Uganda and these
cultivars may be suited for transformation studies for micronutrients.
15.4 Limitations and Prospects of MAS
One of the major limitations in the use of MAS is the high costs associated
with the identifi cation and verifi cation of genetic markers, development
of genetic maps, etc. Economics is the key determinant for the application
of molecular markers in genetic improvement programs (Dekkers and
Hospital 2002). Other factors that infl uence the cost of utilizing marker-aided
breeding include inheritance of the trait, method of phenotypic evaluation,
and high costs. The main factors that slow down using molecular breeding
technologies in most developing countries include poor infrastructure;
inadequate capacity and operational support; and lack of an enabling policy,
statutory and regulatory framework at country level, which in turn affects
research institutions. Despite these diffi culties some developing countries
are making progress in using biotechnology for Musa improvement.
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A Case for Molecular Breeding in Musa 293
15.5 Conclusion
Although conventional breeding programs have their limitations, they have
shown over time that they can be highly successful. Genome manipulations
and interspecifi c crosses have allowed considerable genetic progress in
Musa breeding but much remains to be done in the identifi cation of parental
combinations that are likely to produce progenies with both high mean and
genetic variability (Tenkouano 2001). The development of modern plant
molecular and quantitative genetics in the last two decades has the potential
to revolutionize what has mostly been experience-based empirical plant
breeding (Ye and Smith 2008). Molecular breeding is expected to improve
the effi ciency of crop breeding by selecting and stacking favorable alleles
at target loci (Ribaut et al. 2010). New developments and improvements
in marker technology, the integration of functional genomics with QTL
mapping, and the availability of more high-density maps are the other
factors that will greatly affect the effi ciency and effectiveness of QTL
mapping and marker-aided breeding in the future (Collard et al. 2005).
The development of high-density maps that incorporate new marker types,
such as single nucleotide polymorphisms (SNPs) and expressed sequence
tags (EST) will provide researchers with a greater arsenal of tools for QTL
mapping and marker-aided breeding (Semagn et al. 2010). The number
of EST and genomic sequences available in databases is growing rapidly
(especially from genome sequencing projects), and the accumulation
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potential genetic and economic benefi ts of using molecular breeding need
to be critically compared to those achieved or expected from any existing
conventional breeding programs.
AMPs : antimicrobial proteins
BBTV : banana bunchy top virus
GWS : genome wide selection
MABC : marker-assisted backcrossing
MARS : marker assisted recurrent selection
MAS : marker-assisted selection
MB : molecular breeding
MSAP : methylation-sensitive amplifi cation
© 2012 by Taylor & Francis Group, LLC
294 Genetics, Genomics and Breeding of Bananas
PCR : polymerase chain reaction
pfl p : ferredoxin-like amphipathic protein
QTL : quantitative trait loci
RAPD : random amplifi ed polymorphic DNA
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Full-text available
Bananas are cultivated in more than 107 countries in an area of 4.1 million hectares with a production of 95 million tons and considered the second most produced fruit worldwide. Bananas are attacked by viruses (CMV and BSV), fungi (Yellow and Black Sigatoka and Fusarium), bacteria (Moko), nematodes and insects (Weevils). However, through genetic breeding, it is possible to achieve resistance to most pests and diseases. The center of origin of most of Musa spp. germplasm is the Asian Continent, where diploid, triploid, Tetraploid with genome Musa acuminata and Musa balbisiana bananas are found. In banana breeding, especially for disease resistance, the following methods are employed: introduction and selection of clones; hybridization (crosses between diploids and diploids, triploids and diploids and diploids with tetraploids); chromosome duplication; mutation and transgenic. Methods involving hybridization, however, the method mostly used, is limited by pathernocarpy, sterility, variable number of ploidy levels and low seed production. All varieties produced by the program are then evaluated in banana producing regions in Brazil. Recently new breeding techniques based on genetic information on Musa ssp. are being incremented.
Full-text available
Genetic diversity amongst 21 induced mutant clones tolerant to salinity along with one non-irradiated sensitive clone of banana (Musa acuminata cv. Dwarf Cavendish (AAA)) were studied using morphological and random amplified polymorphic DNA (RAPD) markers. Out of the 30 phenotypic indices screened, 23 were polymorph and two traits, leaf habit and blotches color, were differentiated by non-irradiated clone. RAPDs established 106 major amplified products using 14 primers. Out of 106 markers, eight were monomorph, and the remaining (98) were polymorph. The extent of polymorphism indicated the existence of considerable variation DNA level within induced mutant clones. Primer OPA-02 revealed banding patterns specific to salinity resistant clones. Both morphological and RAPD analyses successfully detected genetic variation within induced mutant clones, RAPD also detected variation between the irradiated and non-irradiated clones, which were morphologically indistinguishable. Results were indicative that induced mutations bear a great potential in improving banana for salinity resistant.
Full-text available
Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice in several countries. Three BB resistance genes, xa5, xa13 and Xa21, were pyramided into cv. PR106, which is widely grown in Punjab, India, using marker-assisted selection. Lines of PR106 with pyramided genes were evaluated after inoculation with 17 isolates of the pathogen from the Punjab and six races of Xoo from the Philippines. Genes in combinations were found to provide high levels of resistance to the predominant Xoo isolates from the Punjab and six races from the Philippines. Lines of PR106 with two and three BB resistance genes were also evaluated under natural conditions at 31 sites in commercial fields. The combination of genes provided a wider spectrum of resistance to the pathogen population prevalent in the region; Xa21 was the most effective, followed by xa5. Resistance gene xa13 was the least effective against Xoo. Only 1 of the BB isolates, PX04, was virulent on the line carrying Xa21 but avirulent on the lines having xa5 and xa13 genes in combination with Xa21.
Full-text available
Health related problems due to vitamin A deficiency affect a large part of the world’s population. Enhancing the levels of vitamin A in staple foods is regarded as a sustainable food-based approach that can have a significant long term beneficial impact on optimising vitamin A intake. Banana and plantain (Musa spp.) is the staple food crop for millions of people in Africa and other parts of the world. Increasing the nutritional qualities of Musa spp. will translate into improved diets. This study used high-performance liquid chromatography (HPLC) to determine the β-carotene content of 47 banana genotypes from the International Institute of Tropical Agriculture (IITA) germplam collection in Uganda and used a color meter to assess the correlation between pulp color intensity and β-carotene levels. There was wide variability in β-carotene levels within and among the different groups of banana studied. Banana genotypes from Papua New Guinea (PNG) had the highest levels of β-carotene with values as high as 2594.0 μg/100 g edible pulp. A positive correlation existed between pulp color intensity and β-carotene concentration. Accessions with relatively high levels of β-carotene, especially the PNG genotypes, could be deployed to regions with high vitamin A deficiency and/or be used as parents for development of vitamin dense varieties. The PNG genotypes could be useful in genetic studies related to vitamin A in banana.
The use of molecular markers linked to resistance genes can highly improve the breeding schemes aimed to both deploying and pyramiding resistance genes. The aim of the present work was to confirm that molecular marker technology could be easily integrated in breeding programs in order to transfer and combine resistances to various pathogens into superior tomato lines. In order to pyramid several resistance genes in the same variety, two near-isogenic lines, each possessing at least three resistant genes, were intercrossed and then selfed for many generations. Selection for resistance was performed using molecular markers linked to the genes in order to fix them at the homozygous level in the same genotype. These were mainly CAPS markers, which are easy to be detected by simple laboratory analyses. After the F4 generation, it was possible to obtain various genotypes, which carried all resistance genes at homozygous condition. This objective was achieved through the early molecular screening for resistance, a very efficient tool in reducing time and space necessary for selection.
Commercial banana cultivars developed from wild species of M. acuminata Colla (A genome) and M. balbisiana Colla (B genome). Whilst wild species are generally seminiferous and fertile, many commercial cultivars are sterile with fruit development resulting from parthenocarpy. As cultivated banana is propagated asexually, its genetic base is narrow with diversity dependent on somatic mutation. Limited genetic variation has resulted in a crop lacking resistance to fungal, bacterial and viral pathogens and numerous pests. Commercial production is often heavily reliant upon agrochemical input. Increased understanding of responses in Musa to biotic stresses will provide new opportunities for genetic improvement. Research is ongoing in characterisation of candidate genes involved in responses to biotic stresses, with groups characterising diverse resistance gene analogs (RGAs) across cultivars and utilising EST approaches for candidate gene discovery. Within our labs, gene discovery is ongoing via analysis of EST data from cDNA libraries produced from Mycosphaerella fijiensis-infected leaf material from M. acuminata ssp. burmannicoides 'Calcutta 4' (resistant) and 'Grande Naine' (AAA genome, susceptible). A large-scale analysis of NBS-LRR RGAs was also conducted in 'Calcutta 4', with polymorphic genetic markers included on a reference map under development at CIRAD. Co-localisation of any RGA markers with QTLs for disease resistance will enable application in marker-assisted selection, facilitating phenotyping for defined traits, such as pest and disease resistance. RGA probes were applied to 'Calcutta 4', 'Grande Naine' and M. balbisiana 'Pisang Klutuk Wulung' BAC libraries revealing numerous putative resistance loci. Shotgun sequencing will provide insight into organisation and evolution of NBS-LRR class R genes in Musa.
For identification of random amplified polymorphic DNA (RAPD) markers tightly linked to Xa-1, which is one of the bacterial blight resistance genes in rice, a near-isogenic line (NlL) for Xa-1 and a recurrent cultivar were screened by RAPD primers. Two out of 280 primers produced DNA fragments tightly linked to Xa-1, namely U087780and Y03700. No recombinant was detected between Xa-1 and Y03700, indicating that this RAPD fragment was closely linked to Xa-1. Y03700 existed in IR-BB I and Kogyoku as a single copy sequence, but did not exist in IR 24. U08750 was linked to Xa I at a drstance of 1 4 ±1.4 cM. These RAPD markers will facilitate selection of Xa-1 for breeding and will be starting points of gene cloning based on chromosome walking in near future.