Marker-assisted breeding of Musa balbisiana genitors devoid of infectious endogenous Banana streak virus sequences

The main edible and cultivated banana varieties are intra- and interspecific hybrids of the two main Musa species, Musa acuminata and Musa balbisiana, having diploid genomes denoted A and B, respectively. The B genome naturally hosts sequences of banana streak virus (BSV) named endogenous BSV (eBSV). Upon stress, eBSVs are identified as the origin of BSV infection for at least three BSV species, causing banana streak disease. For each of the three species, BSV and eBSV share >99.9 % sequence identity, complicating PCR-based diagnosis of viral infection in the B genome-containing bananas. Here, we designed a quantitative PCR-based method to only quantify episomal BSV particles produced, overcoming the limitation of eBSV also being detected by qPCR by using it as a ‘calibrator’. However, our results revealed unexpected variation of eBSV amplification in calibrator plants composed of a clonal population of 53 replicating virus-free banana hybrids with the same AAB genotype. Our in-depth molecular analyses suggest that this calibrator variation is due to the variable abundance of non-encapsidated extrachromosomal viral DNA, likely produced via the transcription of eBSVs, followed by occasional reverse transcription. We also present evidence that accumulation of viral transcripts in AAB plants is downregulated both at post-transcriptional and transcriptional levels by an RNA interference mechanism that keeps the plants free of virus infection. Finally, we recommend that such eBSV amplification variation be taken into account to establish a quantitative viral diagnostic for banana plants with the B genome.

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Creation of a BAC resource to study the structure and evolution of the banana (Musa balbisiana) genome

Genome ◽

10.1139/g04-062 ◽

2004 ◽

Vol 47 (6) ◽

pp. 1182-1191 ◽

Cited By ~ 34

Author(s):

Jan Šafář ◽

Juan Carlos Noa-Carrazana ◽

Jan Vrána ◽

Jan Bartoš ◽

Olena Alkhimova ◽

...

Keyword(s):

Flow Cytometry ◽

Bacterial Artificial Chromosome ◽

Bac Library ◽

Artificial Chromosome ◽

Streak Virus ◽

Insert Size ◽

Banana Streak Virus ◽

Musa Balbisiana ◽

Bac Clones ◽

Nuclei Isolation

The first bacterial artificial chromosome (BAC) library of the banana species Musa balbisiana 'Pisang Klutuk Wulung' (PKW BAC library) was constructed and characterized. One improved and one novel protocol for nuclei isolation were employed to overcome problems caused by high levels of polyphenols and polysaccharides present in leaf tissues. The use of flow cytometry to purify cell nuclei eliminated contamination with secondary metabolites and plastid DNA. Furthermore, the usefulness of the inducible pCC1BAC vector to obtain a higher amount of BAC DNA was demonstrated. The PKW BAC library represents nine haploid genome equivalents of M. balbisiana and its mean insert size is 135 kb. It consists of two sublibraries, of which the first one (SN sublibrary with 24 960 clones) was prepared according to an improved standard nuclei isolation protocol, whereas the second (FN sublibrary with 11 904 clones) was obtained from flow-sorted nuclei. Screening with 12 RFLP probes, which were genetically anchored to 8 genetic linkage groups of the banana species Musa acuminata, revealed an average of 11 BAC clones per probe, thus confirming the genome coverage estimated based on the insert size, as well as a high level of conservation between the two species of Musa. Localization of selected BAC clones to mitotic chromosomes using FISH indicated that the BAC library represented a useful resource for cytogenetic mapping. As the first step in map-based cloning of a genetic factor that is involved in the activation of integrated pararetroviral sequences of Banana streak virus (BSV), the BSV expressed locus (BEL) was physically delimited. The PKW BAC library represents a publicly available tool, and is currently used to reveal the integration and activation mechanisms of BSV sequences and to study banana genome structure and evolution.Key words: bacterial artificial chromosome library, banana, BAC-FISH, flow cytometry, Musa balbisiana, Banana streak virus, BSV.

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Rolling Circle Amplification Is More Sensitive than PCR and Serology-Based Methods in Detection of <i>Banana streak virus</i> in <i>Musa</i> Germplasm

American Journal of Plant Sciences ◽

10.4236/ajps.2012.311191 ◽

2012 ◽

Vol 03 (11) ◽

pp. 1581-1587 ◽

Cited By ~ 5

Author(s):

Moses C. Wambulwa ◽

Francis N. Wachira ◽

Laura S. Karanja ◽

Samuel M. Muturi

Keyword(s):

Rolling Circle Amplification ◽

Streak Virus ◽

Banana Streak Virus ◽

Rolling Circle

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PCR multiplex para a detecção de BSV e CMV em bananeiras micropropagadas

Summa Phytopathologica ◽

10.1590/s0100-54052007000300003 ◽

2007 ◽

Vol 33 (3) ◽

pp. 229-232 ◽

Cited By ~ 2

Author(s):

Daniel Vasquez Figueiredo ◽

Paulo Sergio Torres Brioso

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Streak Virus ◽

Banana Streak Virus ◽

Pcr Multiplex

Um protocolo de PCR multiplex foi estabelecido para a detecção do Banana streak virus (BSV) e do Cucumber mosaic virus (CMV) em bananeiras micropropagadas. Estes vírus são responsáveis por perdas na produção de bananas em todo o mundo. Alguns trabalhos descrevem a integração do BSV no genoma B da bananeira. Contudo, a existência de bananeiras híbridas livres do BSV tem sido demonstrada. Ademais, determinadas estirpes do CMV não são transmitidas mecanicamente sob condições de laboratório, nem tampouco detectadas por testes sorológicos. Como conseqüência, a indexação de matrizes para cultura de tecido algumas vezes se mostra ineficiente. A metodologia apresentada neste trabalho sobrepõe esta dificuldade, pois se baseia na detecção do ácido nucléico viral presente em amostras foliares de bananeira. Na reação, foram usados os oligonucleotídeos BADNA 1A e BADNA 4, para a detecção do BSV, e "CMV senso" e "CMV antisenso" para a detecção do CMV. Após a eletroforese foi verificada a presença de dois fragmentos de DNA amplificados simultaneamente, um dos quais com 597 pb correspondente ao BSV e o outro, com 488 pb, correspondente ao CMV. Este resultado indica que o PCR multiplex pode ser utilizado como uma ferramenta adicional na indexação do BSV e do CMV em bananeiras propagadas por cultura de tecido.

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Vector transmission of Banana streak virus in the screenhouse in Uganda

Annals of Applied Biology ◽

10.1111/j.1744-7348.2001.tb00128.x ◽

2001 ◽

Vol 139 (1) ◽

pp. 37-43 ◽

Cited By ~ 31

Author(s):

J KUBIRIBA ◽

J P LEGG ◽

W TUSHEMEREIRWE ◽

E ADIPALA

Keyword(s):

Streak Virus ◽

Banana Streak Virus ◽

Vector Transmission

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A Single Banana Streak Virus Integration Event in the Banana Genome as the Origin of Infectious Endogenous Pararetrovirus

Journal of Virology ◽

10.1128/jvi.00212-08 ◽

2008 ◽

Vol 82 (13) ◽

pp. 6697-6710 ◽

Cited By ~ 77

Author(s):

Philippe Gayral ◽

Juan-Carlos Noa-Carrazana ◽

Magali Lescot ◽

Fabrice Lheureux ◽

Benham E. L. Lockhart ◽

...

Keyword(s):

Artificial Chromosome ◽

Streak Virus ◽

Banana Streak Virus ◽

Genetic Cross ◽

In The Wild ◽

Virus Integration ◽

High Degree ◽

First Time ◽

Viral Sequences ◽

Integration Event

ABSTRACTSequencing of plant nuclear genomes reveals the widespread presence of integrated viral sequences known as endogenous pararetroviruses (EPRVs). Banana is one of the three plant species known to harbor infectious EPRVs.Musa balbisianacarries integrated copies ofBanana streak virus(BSV), which are infectious by releasing virions in interspecific hybrids. Here, we analyze the organization of the EPRV of BSV Goldfinger (BSGfV) present in the wild diploidM. balbisianacv. Pisang Klutuk Wulung (PKW) revealed by the study ofMusabacterial artificial chromosome resources and interspecific genetic cross. cv. PKW contains two similar EPRVs of BSGfV. Genotyping of these integrants and studies of their segregation pattern show an allelic insertion. Despite the fact that integrated BSGfV has undergone extensive rearrangement, both EPRVs contain the full-length viral genome. The high degree of sequence conservation between the integrated and episomal form of the virus indicates a recent integration event; however, only one allele is infectious. Analysis of BSGfV EPRV segregation among an F1 population from an interspecific genetic cross revealed that these EPRV sequences correspond to two alleles originating from a single integration event. We describe here for the first time the full genomic and genetic organization of the two EPRVs of BSGfV present in cv. PKW in response to the challenge facing both scientists and breeders to identify and generate genetic resources free from BSV. We discuss the consequences of this unique host-pathogen interaction in terms of genetic and genomic plant defenses versus strategies of infectious BSGfV EPRVs.

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