Mol Breeding (2016)36:74

DOI 10.1007/s11032-016-0493-8

Marker-assisted breeding of Musa balbisiana genitors

devoid of infectious endogenous Banana streak virus
sequences
Marie Umber . Jean-Philippe Pichaut . Benot Farinas .
Nathalie Laboureau . Berenger Janzac . Kassa Plaisir-Pineau .
Gersende Pressat . Franc-Christophe Baurens . Matthieu Chabannes .
Pierre-Olivier Duroy . Chantal Guiougou . Jean-Marie Delos .
Christophe Jenny . Marie-Line Iskra-Caruana . Frederic Salmon .
Pierre-Yves Teycheney
Received: 16 September 2015 / Accepted: 18 May 2016
Springer Science+Business Media Dordrecht 2016

Abstract Breeding new interspecific banana hybrid

varieties relies on the use of Musa acuminata and M.
balbisiana parents. Unfortunately, infectious alleles of
endogenous Banana streak virus (eBSV) sequences
are present in the genome of Musa balbisiana genitors.
Upon activation by biotic and abiotic stresses, these
infectious eBSVs lead to spontaneous infections by
several species of Banana streak virus in interspecific
hybrids harboring both Musa acuminata and M.
balbisiana genomes. Here we provide evidence that
seedy M. balbisiana diploids display diverse eBSV
allelic combinations and that some eBSVs differ

structurally from those previously reported. We also

show that segregation of infectious and non-infectious
eBSV alleles can be achieved in seedy M. balbisiana
diploids through self-pollination or chromosome doubling of haploid lines. We report on the successful
breeding of M. balbisiana diploid genitors devoid of
all infectious eBSV alleles following self-pollination
and on the potential of breeding additional M.
balbisiana diploid genitors free of infectious eBSVs
by crossing parents displaying complementary eBSV
patterns. Our work paves the way to the safe use of M.
balbisiana genitors for breeding banana interspecific
hybrid varieties with no risk of activation of infectious
eBSVs.

Electronic supplementary material The online version of

this article (doi:10.1007/s11032-016-0493-8) contains supplementary material, which is available to authorized users.
M. Umber
J.-P. Pichaut
B. Farinas

B. Janzac
K. Plaisir-Pineau
G. Pressat

C. Guiougou
J.-M. Delos
F. Salmon

P.-Y. Teycheney (&)
CIRAD UMR AGAP, 97130 Capesterre-Belle Eau,
Guadeloupe, France
e-mail: teycheney@cirad.fr

N. Laboureau
M. Chabannes
P.-O. Duroy

M.-L. Iskra-Caruana
CIRAD UMR BGPI, 34398 Montpellier, France

Present Address:
M. Umber
INRA UR ASTRO, Domaine Duclos, 97170 Petit-Bourg,
France

F.-C. Baurens
C. Jenny

CIRAD UMR AGAP, 34098 Montpellier, France

P.-O. Duroy
Institut de Biotechnologie UNIL, EPFL-LBTM, CH A1398, Station 6, 1015 Lausanne, Switzerland

J.-P. Pichaut
Vilmorin SA, Route du Manoir, 49250 La Menitre, France

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Page 2 of 11

Keywords Musa
Endogenous viral element

Banana streak virus
Infectious alleles
Markerassisted breeding
Abbreviations
A
Musa acuminata genome
B
Musa balbisiana genome
BSD
Black sigatoka disease
BSV
Banana streak virus
BSGFV Banana streak GF virus
BSIMV Banana streak IM virus
BSOLV Banana streak OL virus
CIRAD Centre International de Cooperation en
Recherche Agronomique pour le
Developpement
CRBBiological Resources Center of Tropical
PT
Plants
eBSV
Endogenous Banana streak virus
EVE
Endogenous viral element
FAO
Food and Agriculture Organization of the
United Nations
ITC
International Transit Center
PKW
Pisang Klutuk Wulung

Introduction
Banana is the fourth largest exported fruit worldwide
(FAO 2014) and an essential asset to the economy of
many developing countries. Hundreds of millions of
people in the tropics and subtropics depend upon
banana for their daily food intake; therefore, it is also
one of the most important staple foods worldwide and
it plays a key role in global food security.
Bananas (Musa spp) are particularly susceptible to
several diseases and pests. These include fungalinvasive species, such as Mycosphaerella fijiensis, the
causing agent of black sigatoka disease (BSD), and
Fusarium oxysporum f. sp. cubense, which cause
severe yield losses and are therefore considered major
threats to banana production (Ploetz 2006; Churchill
2011). Although chemical control is effective against
fungal leaf diseases such as BSD, the use of fungicides
is not sustainable because it has a negative impact on
the environment, it is too expensive for small-scale
farmers in developing countries, and it promotes the
emergence of fungicide-resistant strains (De Lapeyre

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Mol Breeding (2016)36:74

et al. 2010). Furthermore, no chemical control is

available to fight several other major diseases of
bananas, such as those caused by viruses or soil-borne
fungi, whose impact can only be alleviated by cultural
practices that are sometimes difficult to implement in
developing countries. Therefore, sustainable control
of banana diseases relies primarily on breeding
disease-resistant cultivars, which is now facilitated
by access to genomic resources (DHont et al. 2012;
Ortiz and Swennen 2014).
Musa acuminata (A genome) and M. balbisiana (B
genome) are the main progenitors of cultivated
bananas. Following natural hybridization between
sexually compatible types, allopolyploid triploids
AAA, AAB and ABB edible cultivars were domesticated and disseminated worldwide by humans (Perrier
et al. 2011). Within the last century, breeding
programs led to the development of new hybrid
varieties, using M. acuminata and M. balbisiana
parents. Both A and B genomes carry genetic traits
of interest to breeders: A genomes are the main source
of fruit quality and disease resistance, whereas B
genomes are a source of improved vigor and tolerance
to biotic and abiotic stresses (Robinson 1996; Davey
et al. 2013; Ravi et al. 2013). However, efforts to breed
improved interspecific hybrids are currently hampered
by the presence of infectious endogenous Banana
streak virus sequences (eBSVs) in the genome of all
known M. balbisiana progenitors (Geering et al. 2001;
Iskra-Caruana et al. 2010; Duroy et al. 2016). Recent
studies showed that M. balbisiana diploid model
species Pisang Klutuk Wulung (PKW), often used as
a genitor in breeding programs, harbors infectious
eBSVs from three distinct BSV species, namely
Banana streak OL virus (BSOLV), Banana streak
GF virus (BSGFV) and Banana streak IM virus
(BSIMV) (Gayral et al. 2008; Chabannes et al. 2013).
Interestingly, these studies showed that eBSOLV,
eBSGFV and eBSIMV are each present at a single
locus under allelic forms: eBSOLV and eBSGFV have
two distinct alleles and only one is infectious, whereas
eBSIMV has two structurally identical alleles which
are both considered infectious (Chabannes et al.
2013).
Infectious eBSV alleles can spontaneously release
full-length viral genomes upon their activation by
biotic and abiotic stresses, including cell culture and
temperature differences (Dallot et al. 2001; Cote et al.
2010; Iskra-Caruana et al. 2010; Chabannes and Iskra-

Mol Breeding (2016)36:74

Caruana 2013), leading to BSV infections in AB, AAB

and AAAB hybrids. Infected plants can then serve as
reservoirs for further transmission of BSVs by their
natural mealybug vectors (Meyer et al. 2008). Therefore, activation of infectious eBSVs has the potential
to cause BSV outbreaks. As a consequence, infectious
eBSVs are now considered the major constraint for the
breeding and movement of interspecific hybrids
(Bakry et al. 2009; Jain et al. 2009). They have forced
the CIRAD breeding program to give up on the use of
M. balbisiana genitors, further narrowing the genetic
diversity available for breeding (Hippolyte et al.
2012).
In this study, we combined the use of molecular
markers allowing the discrimination of infectious and
non-infectious eBSV alleles (Gayral et al. 2010;
Chabannes et al. 2013) and molecular hybridization
to establish the eBSV pattern of seedy M. balbisiana
diploid genitors. We show that different allelic
combinations exist among these resources and that
some eBSV alleles differ from those previously
reported in model genotype PKW. We also show that
some allelic combinations make it possible to undertake the segregation of infectious and non-infectious
eBSV alleles using self-pollination, chromosome
doubling of haploid lines and crosses between M.
balbisiana accessions with complementary eBSV
allelic patterns. This work led to the successful
breeding of M. balbisiana genitors devoid of eBSV
infectious alleles and paves the way to the breeding of
improved interspecific banana hybrids with no risk of
activation of infectious eBSVs.

Materials and methods

Plant material and genomic DNA isolation
Plants used in this study were seedy Musa balbisiana
diploids Pisang Klutuk Wulung, Pisang Klutuk,
Pisang Batu, Klue Tani, ITC 626, Honduras,
Butuhan, Lal Velchi, Singapuri and Cameroun,
and Musa acuminata tetraploid IDN 110 T. All
originated from the open-field Musa collection of the
Guadeloupe Biological Resources Center of Tropical
Plants (CRB-PT) at Station de Neufchateau, Capesterre Belle-Eau, Guadeloupe, French West Indies.
Plants were submitted to monthly fertilizer applications (100 g/plant of complete fertilizer, 15-4-30

Page 3 of 11

74

NPK) and standard cultivation practices including

desuckering and bunch care. Total genomic DNA was
extracted as described by Risterucci et al. (2000) from
2 g of freshly harvested leaves with the following
modifications. Two rounds of chloroform/isoamylic
alcohol (24:1 v/v) extractions were performed on leaf
extracts, and DNA was suspended in 300 lL of
ultrapure water.
Plant populations
Haploid lines were created from male flower buds of
PKW according to Bakry et al. (2008). Spontaneous
chromosome doubling of these lines was assessed by
flow cytometry as described by Bakry et al. (2008).
Their homozygosity was assessed by the analysis of
simple sequence repeats (SSR) using 36 SSR loci
spread over the eleven M. acuminata chromosomes
(Supplementary Table 1), based on the DH-Pahang
reference genome sequence (Grapin et al. 1998;
Lagoda et al. 1998; Hippolyte et al. 2010; DHont
et al. 2012). Genotyping was carried out using Applied
Biosystems 3500xL Genetic Analyzer and
GeneMapper version 4.1 software (Applied Biosystems; www.appliedbiosystems.com) with a standard
protocol optimized for Musa DNA (Christelova et al.
2011). Automated scoring of the data was corrected by
manual checking.
Selfed progenies of seedy M. balbisiana diploids
Pisang Batu, Klue Tani and Honduras were
created by self-pollination. For this, pollen was
collected from male flower buds and transferred to
female flowers of the same plant, using a paintbrush.
Following pollination, flowers were covered by a
mesh bag to prevent cross-pollination. Seeds were
collected from ripe fruits and subjected to embryo
rescue, as described by Bakry (2008), and ploidy of the
progeny was assessed by flow cytometry.
Interspecific crosses between seedy M. balbisiana
diploids Honduras or Cameroun (female parent)
and M. acuminata tetraploid IDN 110 T (male
parent) and intraspecific cross between seedy M.
balbisiana diploids HND102 (female parent) and
Cameroun (male parent) were performed similarly
to self-pollination except that male and female flowers
originated from separate plants. Following embryo
rescue, progenies were conserved under insect-proof
greenhouse conditions. Progenies from interspecific
crosses
(Honduras 9 IDN
110
T
and

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Page 4 of 11

Cameroun 9 IDN 110 T) were placed under

maximized abiotic stress conditions favoring the
expression of infectious eBSV alleles (several months
in cell culture followed by several months under
insect-proof greenhouse and water stress conditions)
prior to virus indexing.
eBSV genotyping
eBSV allelic genotyping was assessed by PCR-based
screening performed on total genomic DNA using the
conditions described by Gayral et al. (2008) and
Chabannes et al. (2013). eBSOLV, eBSGFV and
eBSIMV patterns were determined using six, ten and
ten specific primer couples, respectively (Supplementary Table 2). In order to discriminate between GF7/
GF9 and GF9/GF9 patterns, PCR products raised by
derived cleaved amplified polymorphic sequences
(dCAPS) difGF markers were digested for 2 h at
37 C with 2 U of restriction enzyme Hpych4III (New
England Biolabs, Evry, France), analyzed by electrophoresis in 2 % agarose gels and compared to
reference patterns of model species PKW, according
to Gayral et al. (2008) and Chabannes et al. (2013).
Southern blot hybridization
Total plant genomic DNA (2025 lg) and DNA from
BAC clones (5 lg) containing the eBSOLV, eBSGFV
or eBSIMV alleles from PKW (Chabannes et al. 2013)
were digested overnight with 50 U of restriction
enzymes. Hind III and DraI (Promega, Charbonnie`res,
France) were used to discriminate infectious and noninfectious eBSOLV and eBSGFV alleles, respectively, whereas BglII was used in Southern blot
experiments targeting eBSIMV sequences. Digested
DNA was purified by dialysis on a cellulose membrane (Millipore, Molsheim, France), electrophoresed
in a 1 % w/v agarose gel for 16 h at 40 V and
transferred to positively charged nylon membrane
Hybond N ? (GE Healthcare, Velizy-Villacoublay,
France) according to the manufacturers instructions.
Membranes were hybridized overnight at 65 C with
radiolabeled probes containing the complete genome
of BSOLV, BSGFV or BSIMV (Chabannes et al.
2013) and then treated according to the manufacturers
instructions. Autoradiography was performed using a
phosphorimager (Typhoon FLA 9000-GE Healthcare)
following a 3- to 24-h exposure.

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Mol Breeding (2016)36:74

Virus indexing
Virus indexing was performed according to Le Provost
et al. (2006) with the following modifications.
Immunocapture was performed for 30 min at 37 C,
and samples were treated with 2 U of RQ1 DNAse
prior to PCR (Promega, Charbonnie`res, France) as
described by Gambley et al. (2008). PCR amplifications were performed separately, with 120, 60 and
200 nM of BSOLV-, BSGFV- and BSIMV-specific
primers, respectively (Geering et al. 2000, 2011).

Results
eBSV allelic patterns of seedy diploid Musa
balbisiana resources
The presence and structural organization of eBSOLV,
eBSGFV and eBSIMV were monitored by PCR-based
genotyping in a set of 10 genitors from CRB-PTs
collection representative of the genetic diversity of M.
balbisiana described by Gayral et al. (2010). Detailed
results of these analyses are provided in Supplementary Table 2. Additional analyses of eBSOLV and
eBSGFV patterns were performed by Southern blot
(Fig. 1). Both PCR-based screening and Southern blot
showed that seedy M. balbisiana diploids Pisang
Klutuk, Pisang Batu and Klue Tani display an
eBSV pattern similar to that of model species PKW
(Table 1). Conversely, several other M. balbisiana
diploids displayed modified eBSV allelic patterns for
all three eBSVs (Lal Velchi and Singapuri), for
eBSOLV and eBSGFV (Cameroun) or for eBSOLV
only (Butuhan). Honduras was the only analyzed
diploid M. balbisiana accession to be devoid of
eBSIMV.
Segregation of eBSOLV and eBSGFV alleles
Segregation of eBSOLV and eBSGFV alleles was
attempted for several seedy M. balbisiana diploids by
chromosome doubling of haploid lines and selfpollination. Progenies were screened using PCR-based
genotyping as described above. eBSOLV and
eBSGFV patterns of selected progenies were confirmed by Southern blot (Fig. 1).
Eight doubled haploid lines of Pisang Klutuk
Wulung (PKW) were obtained from eight distinct

Mol Breeding (2016)36:74

Page 5 of 11

PKW B4/HD1

PKW A2/HD3

PKW B4/HD3

PKW HD-4

PKW HD-2

PKW HD-3

PHD III-6

PKW HD-1

IDN 110 T (AAAA)

PKW (BB)

PKW doubled
haploids

TAF XI-8

TAF VII-7

TAF VIII-8

TAF V-9

TAF VI-3

Klue Tani (BB)

TAF V-7

Klue Tani
selfed progeny

BAF VI-6

BAF V-2

BAF V-5

BAF I-1

Pisang Batu (BB)

Honduras (BB)

Cameroun (BB)

Lal Velchi (BB)

ITC 626 (BB)

MBP_073B22 (OL2)

Butuhan (BB)

MBP_031O07 (OL1)

Ladder

BAF I-4

Pisang Batu
selfed progeny

74

12 kb
10 kb
9 kb
8 kb
7 kb

TAF VI-8

TAF VII-7

Klue Tani (BB)

BAF VIII-8

BAF VI-6

BAF I-4

Pisang Batu (BB)

Honduras (BB)

Pisang Batu
Klue Tani
selfed progeny selfed progeny

PKW B4/HD1

PKW A2/HD3

PKW HD-2

PKW HD-4

PHD III-6

PKW (BB)

IDN 110 T (AAAA)

Pisang Klutuk (BB)

Cameroun (BB)

Lal Velchi (BB)

Sigapuri (BB)

Butuhan (BB)

ITC 626 (BB)

MBP_094I16 (GF9)

MBP_071C19 (GF7)

Ladder

PKW HD-1

PKW doubled
haploids

10 kb
6 kb
4 kb

3 kb
2.5 kb

Fig. 1 eBSOLV (a) and eBSGFV (b) allelic patterns of

selected seedy Musa balbisiana diploids and progenies of
selfed crosses and doubled haploids. Southern blot hybridizations were performed on total DNA digested by restriction
enzymes HindIII (a) and DraI (b), using viral probes covering
the entire genome of viral species BSOLV (a) and BSGFV (b).

BAC clones MBP_031O07 (a) and MBP_071C19 (b) containing

a copy of infectious allele OL1 (a) and GF7 (b), respectively,
and the BAC clones MBP_073B22 (a) and MBP_094I16
(b) containing a copy of non-infectious allele OL2 (a) or GF9
(b) were used as controls. The name and genotype of analyzed
plants are provided

androgenesis events. Their diploid nature was

assessed by flow cytometry and their homozygosity
by SSR genotyping (data not shown). Table 2 summarizes the eBSV patterns established for these lines.
It shows that segregation of eBSOLV and/or eBSGFV
alleles was achieved in all 8 lines and resulted in
various allelic associations, which are illustrated in
Fig. 1. Simultaneous segregation of eBSOLV and
eBSGFV was observed in four lines (PKW HD2, PKW
HD3, PKW A2/HD3 and PKW B4/HD3) that were
free of both OL1 and GF7 infectious alleles, whereas
two lines (PHD III-6 and PKW B4/HD1) still retained
them both. Two remaining lines (PKW HD1 and PKW

HD4) retained only one infectious allele (GF7 or OL1,

respectively).
A total of eight and seventeen progenies were
obtained following self-pollination performed on five
distinct Pisang Batu parents (numbered I, IV, V, VI
and VIII) and nine distinct Klue Tani parents
(numbered I, III, IV, V, VI, VII, VIII, IX, XII),
respectively. Table 2 summarizes their eBSV patterns
and shows that again various allelic combinations
were obtained, as confirmed by Southern blot (Fig. 1).
Progenies BAF I-4, BAF I-8 and TAF IX-8 were
homozygous for both infectious alleles OL1 and GF7.
In the remaining six progenies from the Pisang Batu

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Mol Breeding (2016)36:74

Table 1 eBSOLV, eBSGFV and eBSIMV allelic patterns of a set of 10 seedy M. balbisiana diploids from CRB-PT Musa collection,
based on PCR-based genotyping and molecular hybridization
Accession

eBSOLV

eBSGFV

eBSIMV

Name

ITC code

PKW

ITC1063

OL1/OL2

GF7/GF9

IM/IM

Pisang Klutuk

ITC1077

OL1/OL2

GF7/GF9

IM*

Pisang Batu

ITC1156

OL1/OL2

GF7/GF9

IM*

Klue Tani

ITC1120

OL1/OL2

GF7/GF9

IM*

ITC 626

ITC0626

OL1*

GF7*

IM*

Honduras

ITC0247

OL1/OL2

GF7/GF9

No eBSIMV

Butuhan

ITC1074

Modified OL*

GF7*

IM*

Lal Velchi

ITC1588

Modified OL*

Modified GF*

Modified IM*

Singapuri

ITC0248

Modified OL*

Modified GF*

Modified IM*

Cameroun

ITC0246

Modified OL*

Modified GF*

IM*

eBSV pattern of Pisang Klutuk Wulung (PKW) model species is shown in bold
OL1 and GF7: infectious alleles
OL2 and GF9: non-infectious alleles
* Homozygous and hemizygous patterns cannot be distinguished
Modified OL, GF or IM: allelic pattern different from that of model species PKW
The reference codes under which the accessions are conserved in the International Musa Germplasm Transit Center (ITC) are
provided

self-pollinations and sixteen progenies from the Klue

Tani self-pollinations, incomplete eBSV allelic cosegregations of the two loci (eBSOLV and eBSGFV)
were observed, with at least one heterozygous eBSV.
No progeny homozygous for both non-infectious
alleles was obtained.
Musa balbisiana genitors devoid of infectious
eBSV
PCR-based genotyping and Southern blot indicated
that seedy M. balbisiana diploid Honduras carries
both infectious and non-infectious alleles of eBSOLV
and eBSGFV and is devoid of eBSIMV sequence
(Table 1 and Fig. 1). Taking advantage of this situation, self-pollination of Honduras was undertaken in
order to exploit the heterozygous status of eBSOLV
and eBSGFV loci and breed improved Honduras
genitors that would be free of all infectious eBSVs at
once. A selfed progeny of 67 individuals from two
separate crosses was screened using PCR-based
genotyping as described above. Table 3 shows the
result of the screening. As expected, all 67 individuals
were devoid of eBSIMV. The expected ratios for

123

allelic
combinations
( OL1/OL1 - OL2/
OL2 - OL1/OL2) were observed for eBSOLV
alleles (v2 = 1.119), but not for eBSGFV since the
progeny followed a GF7/GF9 - GF7/GF7 distribution (v2 = 2.632) and none of the 67 individuals
was homozygous for the GF9 allele. Southern blot
hybridization was carried out on a selection of
progenies representing the different allelic combinations and confirmed the eBSOLV and eBSGFV
patterns established by PCR-based genotyping
(Fig. 2), except for the three selected individuals
(HND66, HND85 and HND102) displaying a GF7/
GF9 PCR pattern, which appeared carrying the GF9
allele in Southern blot (Fig. 2, panels B, lanes 4, 5, 6).
In order to clarify the inconsistencies observed
between PCR and Southern blots and the eBSGFV
pattern and allelic distribution of the accession
Honduras, this seedy M. balbisiana diploid was
crossed with the M. acuminata tetraploid (AAAA)
IDN 110 T which is free of eBSV, resulting in an
AAB progeny of 31 individuals. PCR-based genotyping showed that the eBSOLV allelic segregation
observed in this progeny was compatible with the
ratio expected for single-locus segregation, with

Mol Breeding (2016)36:74

Page 7 of 11

Table 2 eBSOLV and eBSGFV allelic patterns of Pisang

Klutuk Wulung doubled haploid lines, Pisang Batu and Klue
Tani selfed progenies based on PCR-based genotyping

PKW parent

Infectious alleles Non-infectious alleles

OL1
G7
OL2
GF9
+
+
+
+

Doubled haploid lines

PHD III-6
PKW HD-1
PKW HD-2
PKW HD-3
PKW HD-4
PKW A2/HD3
PKW B4/HD1
PKW B4/HD3
Pisang Batu
parent

+
+

+
+
+

+
+
+
+

+
+
+
+
+
+

Selfed progenies

BAF I-1
BAF I-4
BAF I-8
BAF IV-1
BAF V-2
BAF V-5
BAF VI-6
BAF VIII-8
Klue Tani
parent

+
+
+
+
+
+

+
+

+
+

+
+
+

+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+

Selfed progenies

TAF I-3
TAF III-3
TAF III-5
TAF III-9
TAF IV-5
TAF V-7
TAF V-9
TAF VI-3
TAF VI-7
TAF VI-8
TAF VII-7
TAF VII-8
TAF VIII-1
TAF VIII-8
TAF IX-6
TAF IX-8
TAF XII-7

+
+
+
+
+
+

+
+
+

+
+
+

+
+

+
+

+
+
+
+
+
+
+
+
+

eBSV patterns of parents are shown in bold

?: presence of the allele; -: absence of the allele
Lines carrying only non-infectious alleles OL2 and GF9 are
highlighted

74

14/31 (45.2 %) individuals carrying the OL2 allele

and 17/31 (54.8 %) individuals carrying the OL1
allele (v2 = 0.29) (data not shown).
For eBSGFV allelic segregation, GF7 allele was
found in 18/31 individuals (58.1 %) and the remaining
13/31 individuals (41.9 %) displayed a complex
pattern with PCR markers indicating both GF7 and
GF9 characteristics (v2 = 0.806). This segregation is
compatible with the ratio expected for singlelocus segregation. This result strongly suggests that
Honduras carries a structurally modified GF9 allele
harboring a complex GF7/GF9 pattern in PCR-based
genotyping and that this modified GF9 allele is
indistinguishable from the GF9 allele in Southern blot
when restriction enzyme DraI is used to cut genomic
DNA.
In order to characterize the infectious or noninfectious nature of Honduras modified GF9 allele,
individual HND102, which arises from the progeny of
the Honduras self-cross described above and is
homozygous for the modified GF9 allele, was crossed
with IDN 110 T, raising 92 progenies with an AAB
genotype. All 92 progenies were subjected to virus
indexing following a 6-month period under tissue
culture conditions known to trigger the expression of
infectious eBSVs (Cote et al. 2010) and then a
6-month culture period in insect-proof greenhouse
under water stress conditions that are also known to
trigger the expression of infectious eBSVs (IskraCaruana et al. 2010). None of the progenies was found
to be infected by BSOLV, BSGFV or BSIMV,
confirming that both OL2 and modified GF9 alleles
of Honduras are non-infectious and that HND102 is
therefore devoid of infectious eBSV.
Characterization of eBSV modified alleles
of accession Cameroun
PCR-based screening and Southern blot analysis of
several seedy M. balbisiana diploids unveiled the
existence of modified eBSV alleles (see Table 1 and
Fig. 1). The zygosity and infectious status of modified
eBSOLV and eBSGFV alleles were investigated in
Cameroun. For this, Cameroun was used as female
parent in a cross with IDN 110 T, resulting in an
AAB progeny of 67 individuals. The eBSV pattern of

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Mol Breeding (2016)36:74

Table 3 Segregation ratios of OL1, OL2, GF7 and GF9 alleles in the progeny of a Honduras 9 Honduras cross

GF7/GF9
GF7/GF7

OL1/OL2
46.3% (25%)
7.5% (12.5%)

OL1/OL1
20.9% (12.5%)
6.0% (6%)

OL2/OL2
16.4% (12.5%)
3.0% (6%)

eBSOLV locus

53.7% (50%)

26.9% (25%)

19.4% (25%)

eBSGFV locus
83.6% (75%)
16.4% (25%)
2 = 2.632
2
= 1.119

Expected ratios are shown in parentheses, and Chi-squared values are indicated

each progeny established by PCR showed that no

segregation of modified eBSOLV and eBSIMV alleles
occurred (data not shown). This suggests that Cameroun is homozygous for modified eBSOLV and
eBSIMV alleles. On the contrary, segregation of
modified eBSGFV allele was observed in this progeny
(v2 = 1.806), with 39/67 (58 %) individuals having
no eBSGFV insertions and 28/67 (42 %) harboring the
same modified eBSGFV allelic pattern than the
Cameroun female parent. Thus, Cameroun is
hemizygous for the modified eBSGFV allele.
In order to characterize the potential infectious
status of Cameroun modified eBSOLV and eBSGFV
alleles, all 67 progenies were subjected to virus
indexing following tissue culture and culture in an
insect-proof greenhouse under similar stress conditions as those described above, in order to trigger the
expression of infectious eBSVs. Two individuals were
found to be infected by BSGFV and none by BSOLV,
suggesting that the modified eBSGFV allele of
Cameroun is infectious, whereas the modified
eBSOLV allele is not.

Discussion
Although endogenous viral elements (EVEs) are
common in plant genomes (Teycheney and Geering
2011; Geering et al. 2014), only a few of them are
infectious and can lead to spontaneous viral infections
following activation by biotic or abiotic stresses. Such
infectious EVEs have been reported so far in petunia
(Richert-Poggeler et al. 2003), Nicotiana edwardsonii
(Lockhart et al. 2000) and banana (Harper et al. 1999;
Ndowora et al. 1999). eBSVs are currently the most
detrimental infectious EVEs in plants since they affect
crops of worldwide economic importance. They are
considered the major constraint for conserving and

123

exchanging banana germplasm and for breeding much

needed improved banana hybrid varieties.
In this work, we described the molecular characterization of the eBSV allelic patterns of 10 seedy M.
balbisiana diploid genitors that are of interest for
breeding interspecific hybrid varieties. These allelic
patterns were first determined by PCR and then by
Southern blot. Available eBSV-specific PCR-based
molecular markers proved accurate for characterizing
allelic patterns that are similar to that of PKW, the
model species from which these markers were designed.
Although they successfully pinpointed structural differences in modified eBSVs whose PCR-based patterns
differed from those of PKW, these markers reached
their limits when confronted with some modified eBSVs
such as Honduras modified GF9 allele. This was
illustrated by the GF7/GF9 molecular pattern displayed
by AAB hybrids resulting from a Honduras 9 IDN
110 T cross and by homozygous BB progenies from a
Honduras 9 Honduras self-cross in which eBSGFV
alleles had segregated.
None of the ten M. balbisiana diploids analyzed in
this work was free of eBSVs. Four of them (Butuhan,
Singapuri, Lal Velchi and Cameroun) displayed
modified eBSVs. Our result suggests that the modified
eBSGFV insertion of Honduras and the modified
eBSOLV insertion of Cameroun are both non-infectious. The mechanism underlying this phenomenon
remains to be investigated. One hypothesis could be that
structural modifications carried by these modified
alleles make them replication incompetent and/or
prevent the recombination-based reconstitution of functional BSV genomes from infectious eBSVs proposed
by Chabannes and Iskra-Caruana (2013). Sequencing
additional M. balbisiana genomes would allow the full
characterization of modified eBSVs and the design of
specific PCR-based markers adapted to these modified
alleles, for marker-assisted breeding purposes.

Mol Breeding (2016)36:74

Page 9 of 11

74

10

11

OL2

GF7

Modified GF9

IM

OL1

Fig. 2 eBSV allelic patterns of selected selfed progenies of

seedy M. balbisiana diploid Honduras. Southern blot
hybridizations were performed on total DNA digested by
restriction enzymes Hind III (a), DraI (b) and BglII (c), using
viral probes covering the entire genome of viral species BSOLV
(a), BSGFV (b) and BSIMV (c). Lane 1 BAC clones
MBP_031O07 (a), MBP_071C19 (b) and MBP_068C24
(c) containing a copy of infectious allele OL1 (a), GF7

(b) and IM (c), respectively; lane 2 BAC clones MBP_073B22

(a), MBP_094I16 (b) containing a copy of non-infectious allele
OL2 (a) or GF9 (b) or empty lane (c); lane 3 M. acuminata
control IDN 110 T; Honduras selfed cross-progenies #66
(lane 4); #85 (lane 5); #102 (lane 6); #64 (lane 7); #95 (lane 8);
#25 (lane 9); #33 (lane 10); lane 11 Honduras parent. Panel D:
eBSV patterns of the progenies. ?: presence of the allele. -:
absence of the allele

We also report on the successful experimental

segregation of eBSV alleles in several seedy M.
balbisiana diploid genitors harboring both infectious
and non-infectious eBSV alleles, following chromosome doubling of haploid lines or self-pollination. Our
results showed that this approach can lead to improved
M. balbisiana genitors devoid of all infectious eBSV
alleles.
Overall, our results pave the way to the safe use of
M. balbisiana genitors for breeding improved interspecific hybrids with no risk of activating infectious
eBSVs. They add to a recently reported complementary approach involving crosses between an interspecific AAAB female parent harboring infectious
alleles OL1 and GF7 and a diploid AA male parent,

which resulted in the segregation of eBSVs in the

progeny (Noumbissie et al. 2016). We further investigated the possibility to create additional M. balbisiana genitors devoid of infectious eBSVs by
crossing diploid M. balbisiana parents with improved
M. balbisiana diploid HND102. For this, a cross
between HND102 (female) and Cameroun (male)
parents was performed, leading to 143 progenies
which displayed identical eBSV patterns when PCR
genotyped, as expected. A selection of these progenies
is now being backcrossed with the HND102 parent in
order to segregate the infectious and/or modified
eBSV alleles. This promising strategy will be
extended to more diploid M. balbisiana parents with
potential for breeding interspecific hybrids.

123

74

Page 10 of 11

Mol Breeding (2016)36:74

Acknowledgments The authors wish to thank Frederic Bakry

and Leonidas Fereol for providing some doubled haploid lines
and Guillaume Fort for technical help. This work was supported
by the European Regional Development Fund. This paper is
dedicated to the memory of Jacky Ganry.
Compliance with ethical standards
Conflict of interest

The authors declare no conflict of interest.

Ethical standards All experiments and data analysis were

performed according to ethical standards.

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