Development of molecular markers against endogenous banana
streak virus sequences (eBSV) to secure both banana germplasm
exchange and the use of M. Balbisiana genome in banana and
plantain breeding programs.
Matthieu CHABANNES
1
, Serge GALZI
1
, Nathalie LABOUREAU
1
, Philippe GAYRAL
1,2
, Guy NOUMBISSIE
1
,
Pierre Olivier DUROY
1,3
and Marie Line ISKRA-CARUANA
1
Contact: matthieu.chabannes@cirad.fr
003-08
1UMR BGPI, Univ Montpellier, CIRAD, INRA, Montpellier SupAgro, Montpellier, France
2 Institut de Recherche sur la Biologie de l'Insecte, UMR 7261, CNRS, Université François-Rabelais, 37200 Tours, France
3 Université de Lausanne, Institut de biotechnologie, EPFL FSB – LBTM, CH B1-391 (Bâtiment CH), Station 6, CH-1015 Lausanne, Switzerland
Introduction
Infections of banana and plantain by banana streak viruses (BSV) can occur in the absence of vector-mediated transmission, through the activation of infective endogenous BSV sequences (eBSVs). Such eBSVs are present in the Musa balbisiana (B) genome. Once activated by biotic or abiotic stresses, these viral integrants cause spontaneous infection in both natural and synthetic interspecific hybrids harbouring one copy of the B genome. We realised an in depth characterisation of integrants present within the seedy Musa balbisiana Pisang klutuk wulung (PKW) genome for three BSV species ; Goldfinger (eBSGFV) -Imove (eBSIMV), and Obino l’Ewai (eBSOLV). Based on their sequences and their structures we developed several PCR and Derived Cleaved Amplified Polymorphic Sequences (deCAPS) specific markers for genotyping eBSVs.
Those markers enabled to propose a strategy for the distribution of germplasm containing eBSV alleles present in the B genome, while minimising any risks associated with the distribution of BSV to the recipient country. They were also used to genotype M. balbisiana germplasm, unveiling the presence of modified eBSV alleles in several accessions and finally were used as phylogenetic markers to shed light on Musa evolution. In collaboration with colleagues from CIRAD Guadeloupe, we obtained breeding improved M. balbisiana progenitors devoid of infectious eBSGFV and/or eBSOLV alleles through self-pollination and chromosome doubling of haploid lines. Finally we also produced triploid AAB (3x) plantain progeny without eBSV in collaboration with the Centre Africain de Recherche sur Bananiers et Plantains (CARBAP) in Cameroon by crossing a diploid AA parent (2x) with a tetraploid AAAB parent (4x).
These results pave the way to the safe use of M. balbisiana in breeding programs, and open new perspectives for breeding improved banana and plantain hybrid varieties.
Results
1) Molecular structure of eBSVs discovered in PKW genome
and position of the different molecular markers developed
Chabannes et
Chabannes et
eBSGFV-7 (13.3 kbp) eBSGFV-9 (15.6 kbp) VM 1F/ R) VM 2F/ R) Di fG fF /R Di fG fF /R VV1F/ R) VV2F/ R) VV4F/ R) VV6F/ R) VV3F/ R) VM 1F/ R) VM 2F/ R) Di fG fF /R Di fG fF /R Di fG fF /R VV5F/ R) VV1F/ R) VV2F/ R) VV4F/ R) VV6F/ R) VV3F/ R) eBSOLV-1 (22.9 kbp) Sig 1 Sig 2 M us a jo n 1 M us a jo n 2 M ar 2 OL 1 Dif OL (H ae 3) Dif OL (A hd 1) eBSOLV-2 (23.2 kbp) Sig 1 Sig 2 M us a jo n 1 M us a jo n 2 M ar 2 OL 2 M ar 1 OL 2 Dif OL (A hd 1) Dif OL (H ae 3) eBSIMV (15,8 kbp) M usa /F2 F5/ M usa F1/ F3 F3/ F4 F4/ F5Molecular markers for : - Integrated area/locus - Internal rearranged area - Alleles recognition eB SG FV eB SO LV eB SIM V ORF1 ORF2 ORF3 IG 7,5 kbp Episomal BSV genome (PCR markers) (dCAPS markers)
2) eBSV markers as a tool to shed light on Musa evolution
Chabannes et al., 2013 - J. Virol. 87(15):8624-8637
0 0.2 ABB Auko BB Cam BB 626 ABB Burro BB But OG NNM AAB KM BB LCK AAB Luba AA Maia AAB Moa OG Baj OG Coc AA Pah AAB Kap ABB Bung BB PK AAB Nang AAB Porp ABB Beng ABB Blue AAB Corn ABB Daru AAB Gama AAB Kuna AAB Tig AAB Lady BB EK ABB Peli BB LBA ABB Dole ABB Foug AA Agu AAB Ori AAB Figue ABB Saba AAB Foco AA IDN BB KT AA LoTA AB Safet AAB Prata AB Kunn AAB PRB BB Hond BB PKW BB Lal ABB Khom AAB PR AAB Ceyl BB Batu AAB Mur OG Lat AB Eko BB Chi1 BB Chi2 BB Chi3 OG Man BB Mont BB ButIA BB 545 BB 852 BB 342 BB 211 OG Vel BB I63 OG Orn BB 63-80 AA Bank OG Tex BB 1016 AAB Slen AAB Tay AAB ChuM AAB Yang2 Msat-4 Msat-7 Awak et Silk Pome Bluggoe/ Saba Plantain Mai’a popoulou BB and ABB AAB from Asia AAB from India AA and out-groups eBSV as phylogenetic markers
✖ Relationship between ABB and BB
We established lineage
between BB and ABB/AAB demonstrating the eBSV
capacity to serve as
phylogenetic markers
Duroy et al., 2016 - Ann Bot. 117 (4):625-641
Chabannes et
3) eBSV markers to assist the early selection of banana plants
carrying non-infective eBSVs or eBSV-free banana plants
Chabannes et
Musa
Musa clones
without B genome
BSV not detected BSV detected
DISTRIBUTION
Musa clones with B genome
Infectious eBSV allele
Therapy + indexing + selection*
BSV detected*
Musa clones with B genome
Non-infectious eBSV allele
BSV not detected* Informed consent from the importer to
receive germplasm.
DISTRIBUTION Include disclaimer in the health
statement : BSVs genomes are present in
the B genome of Musa (banana and plantain). Consequently, almost all
accessions containing the B genome may develop BSV infection and may express symptoms during any stage of growth.
INDEXING FOR BSV
*Note: Usual procedures to eliminate episomal BSV infections not originated from eBSV will be applied. However viral particles may still occur even after treatments due to
activation of eBSV allele as indicated in the disclaimer statement.
BEFORE PROPOSITION
INDEXING FOR BSV*
Therapy + indexing + selection
4) Decision tree for distributing germplasm with BSV and/or eBSV
Thomas et al., 2015 MusaNet-Bioversity International (Position paper on a strategy to distribute banana (Musa) germplasm with endogenous Banana streak virus genomes)
BA C OL1 BA C OL2 eB SV fre e pla nt (AA) BSOLV BSGFV BSIMV BAC G F7 BAC G F9 BAC IM Ho ndur as B B Offsprings obtained by self-pollination GF9
Umber et al., 2016 – Mol. Breeding 36:74
Improved BB genitors
Improved plantain banana
Offsprings (184) 24 35 120 5 eBSOLV-1 -+ + eBSGFV-7 -+ + -13% 19% 65% 3% % 155 (84%) 125 (68%)
Noumbissié et al., 2016 – Mol. Breeding 36:38
Pahang AA Parents Hybrids Gametes AA or AB A AA A (50%) & AAB (50%) CRBP39 AAAB Grand parents French Clair AAB
