Phylogeny of <Emphasis Type="Italic">Banana Streak Virus</Emphasis> Reveals Recent and Repetitive Endogenization in the Genome of Its Banana Host (<Emphasis Type="Italic">Musa</Emphasis> sp.)

Banana streak virus (BSV) is a plant dsDNA pararetrovirus (family Caulimoviridae, genus badnavirus). Although integration is not an essential step in the BSV replication cycle, the nuclear genome of banana (Musa sp.) contains BSV endogenous pararetrovirus sequences (BSV EPRVs). Some BSV EPRVs are infectious by reconstituting a functional viral genome. Recent studies revealed a large molecular diversity of episomal BSV viruses (i.e., nonintegrated) while others focused on BSV EPRV sequences only. In this study, the evolutionary history of badnavirus integration in banana was inferred from phylogenetic relationships between BSV and BSV EPRVs. The relative evolution rates and selective pressures (d_N/d_S ratio) were also compared between endogenous and episomal viral sequences. At least 27 recent independent integration events occurred after the divergence of three banana species, indicating that viral integration is a recent and frequent phenomenon. Relaxation of selective pressure on badnaviral sequences that experienced neutral evolution after integration in the plant genome was recorded. Additionally, a significant decrease (35%) in the EPRV evolution rate was observed compared to BSV, reflecting the difference in the evolution rate between episomal dsDNA viruses and plant genome. The comparison of our results with the evolution rate of the Musa genome and other reverse-transcribing viruses suggests that EPRVs play an active role in episomal BSV diversity and evolution.     

Identification of genetic markers linked to banana streak disease expression in inter-specific Musa hybrids

Recently-introduced inter-specific Musa hybrids, bred for improved yield and resistance to diseases, have been found to be widely infected with banana streak virus (BSV), the causal agent of banana streak disease (BSD). One hypothesis suggests: (1) that BSD occurrence in these inter-specific hybrids results from activation of BSV-Ol endogenous pararetrovirus sequences (EPRV) integrated into the Musa genome rather than from external sources of infection, and (2) that the process of genetic hybridisation may be one factor involved in triggering episomal expression of the BSV integrants. In order to test this hypothesis we carried out a genetic analysis of BSD incidence in a F1 triploid ( Musa AAB) population produced by inter-specific hybridisation between virus and disease-free diploid Musa balbisiana (BB) and tetraploid Musa acuminata (AAAA) parents. Half of the F1 progeny of this cross expressed BSV particles. Using PCR amplification to determine the presence or absence of BSV-Ol EPRVs, it was determined that this endogenous sequence was specific to the M. babisiana genome and occurred in a homozygous state. Using bulk segregant analysis, ten AFLP markers co-segregating with the absence and/or presence of BSV infection were identified in the M. balbisiana genome, but were absent from the M. acuminata genome. Seven of these markers segregated with the presence of a BSV particle and three with the absence of BSV particles. Analysis of the segregation of these markers using a test-cross configuration allowed the construction of a genetic map of the linkage group containing the locus associated with BSV infection in the F1 hybrid population. These data indicate that a genetic mechanism is involved in BSV appearance, and suggest that a monogenic allelic system confers the role of carrier to the M. balbisiana parent.     

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

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.     

Three Infectious Viral Species Lying in Wait in the Banana Genome

Plant pararetroviruses integrate serendipitously into their host genomes. The banana genome harbors integrated copies of banana streak virus (BSV) named endogenous BSV (eBSV) that are able to release infectious pararetrovirus. In this investigation, we characterized integrants of three BSV species—Goldfinger (eBSGFV), Imove (eBSImV), and Obino l''Ewai (eBSOLV)—in the seedy Musa balbisiana Pisang klutuk wulung (PKW) by studying their molecular structure, genomic organization, genomic landscape, and infectious capacity. All eBSVs exhibit extensive viral genome duplications and rearrangements. eBSV segregation analysis on an F1 population of PKW combined with fluorescent in situ hybridization analysis showed that eBSImV, eBSOLV, and eBSGFV are each present at a single locus. eBSOLV and eBSGFV contain two distinct alleles, whereas eBSImV has two structurally identical alleles. Genotyping of both eBSV and viral particles expressed in the progeny demonstrated that only one allele for each species is infectious. The infectious allele of eBSImV could not be identified since the two alleles are identical. Finally, we demonstrate that eBSGFV and eBSOLV are located on chromosome 1 and eBSImV is located on chromosome 2 of the reference Musa genome published recently. The structure and evolution of eBSVs suggest sequential integration into the plant genome, and haplotype divergence analysis confirms that the three loci display differential evolution. Based on our data, we propose a model for BSV integration and eBSV evolution in the Musa balbisiana genome. The mutual benefits of this unique host-pathogen association are also discussed.     

A four-partner plant–virus interaction: enemies can also come from within

Plant viruses are disseminated by either vertical (vegetative multiplication or sexual reproduction) or horizontal (vector-mediated) propagation. Plant pararetroviruses—members of the Caulimoviridae family—have developed an alternative strategy for vertical propagation via integration within the host plant genome, although integration is not required for viral replication. Integrated endogenous pararetrovirus (EPRV) sequences have undergone extensive viral genome rearrangements and contain more than one copy of the viral genome. Furthermore, EPRV can become infectious upon spontaneous escape of active virus following stresses such as wounding, tissue culture, or interspecific crosses. Such infectious EPRV are of great importance, not only in terms of their ability to precipitate epidemic outbreaks but also because of their effect on breeding of numerous plant genomes in temperate and tropical crops. This is especially true for banana, a crop susceptible to banana streak viruses, the causative agents of banana streak disease. Thus, the classical three-component banana–Banana streak virus (BSV)–mealybug pathosystem can be expanded to include endogenous BSV as an alternative source of active virions. The BSV-banana pathosystem is one of only three pathosystems known to date to harbor this remarkable feature, and the present review focuses exclusively on it to illustrate this four-partner interaction.     

Micropropagation by tissue culture triggers differential expression of infectious endogenous Banana streak virus sequences (eBSV) present in the B genome of natural and synthetic interspecific banana plantains

The genome of Musa balbisiana spp. contains several infectious endogenous sequences of Banana streak virus (eBSV). We have shown previously that in vitro micropropagation triggers the activation of infectious eBSOLV (endogenous sequences of Banana streak Obino l'Ewai virus ) in the synthetic tetraploid interspecific hybrid FHIA21 (AAAB). In this work, we show that another synthetic tetraploid (AAAB) hybrid and two natural triploid (AAB) plantains are equally prone to the activation of infectious eBSOLV during tissue culture. These results are a strong indication that such activation is a general phenomenon in interspecific Musa cultivars, whether synthetic or natural. We also report the first in-depth study of the correlation between the duration of tissue culture and the level of activation of infectious eBSOLV, and show that specific and common activation patterns exist in these banana plants. We hypothesize that these patterns result from the concomitant activation of infectious eBSOLV and a decrease in the virus titre in neoformed plantlets, resulting from cell multiplication outcompeting virus replication. We provide experimental data supporting this hypothesis. No activation of infectious eBSGFV (endogenous sequences of Banana streak Goldfinger virus) by tissue culture was observed in the two natural AAB plantain cultivars studied here, whereas such activation occurred in the AAAB synthetic hybrid studied. We demonstrate that this differential activation does not result from differences in the structure of eBSGFV, as all banana genomes harbour eaBSGFV-7.     

Endogenous pararetroviruses: two-faced travelers in the plant genome

Endogenous plant pararetroviruses (EPRVs) were identified as integrated counterparts of most members of the plant virus family Caulimoviridae and represent repetitive elements that are ubiquitous in the plant kingdom. They are often located in pericentromeric regions of plant chromosomes in the vicinity of retrotransposon sequences. Depending on their structure and sequence integrity, some EPRVs are able to replicate and to initiate viral infection. However, conservation of integrated sequences in plant genomes might indicate benefits for the host during evolution. Understanding EPRV activation and control by the host could have important implications for plant breeding strategies to prevent viral disease caused by EPRVs in newly generated cultivars.     

Analysis of the distribution and structure of integrated Banana streak virus DNA in a range of Musa cultivars

Banana streak virus strain OL (BSV-OL) commonly infects new Musa hybrids, and this infection is thought to arise de novo from integrated virus sequences present in the nuclear genome of the plant. Integrated DNA (Musa6+8 sequence) containing the whole genome of the virus has previously been cloned from cv. Obino l’Ewai (Musa AAB group), a parent of many of the hybrids. Using a Southern blot hybridization assay, we have examined the distribution and structure of integrated BSV-OL sequences in a range of Musa cultivars. For cv. Obino l’Ewai, almost every restriction fragment hybridizing to BSV-OL was predicted from the Musa6+8 sequence, suggesting that this is the predominant type of BSV-OL integrant in the genome. Furthermore, since only two junction fragments of Musa/BSV sequence were detected, and the Musa6+8 sequence is believed to be integrated as multiple copies in a tandem array, then the internal Musa spacer sequences must be highly conserved. Similarly sized restriction fragments were detected in four BB group cultivars, but not in six AA or AAA group cultivars, suggesting that the BSV-OL sequences are linked to the B-genome of Musa. We also provide evidence that cv. Williams (Musa AAA group) contains a distinct badnavirus integrant that is closely related to the ‘dead’ virus integrant previously characterized from Calcutta 4 (Musa acuminata ssp. burmannicoides). Our results suggest that the virus integrant from cv. Williams is linked to the A-genome, and the complexity of the hybridization patterns suggest multiple sites of integration and/or variation in sequence and structure of the integrants.     

How endogenous plant pararetroviruses shed light on Musa evolution

Background and Aims Banana genomes harbour numerous copies of viral sequences derived from banana streak viruses (BSVs) – dsDNA viruses belonging to the family Caulimoviridae. These viral integrants (eBSVs) are mostly defective, probably as a result of ‘pseudogenization’ driven by host genome evolution. However, some can give rise to infection by releasing a functional viral genome following abiotic stresses. These distinct infective eBSVs correspond to the three main widespread BSV species (BSOLV, BSGFV and BSIMV), fully described within the Musa balbisiana B genomes of the seedy diploid ‘Pisang Klutuk Wulung’ (PKW).Methods We characterize eBSV distribution among a Musa sampling including seedy BB diploids and interspecific hybrids with Musa acuminata exhibiting different levels of ploidy for the B genome (ABB, AAB, AB). We used representative samples of the two areas of sympatry between M. acuminata and M. balbisiana species representing the native area of the most widely cultivated AAB cultivars (in India and in East Asia, ranging from the Philippines to New Guinea). Seventy-seven accessions were characterized using eBSV-related PCR markers and Southern hybridization approaches. We coded both sets of results to create a common dissimilarity matrix with which to interpret eBSV distribution.Key Results We propose a Musa phylogeny driven by the M. balbisiana genome based on a dendrogram resulting from a joint neighbour-joining analysis of the three BSV species, showing for the first time lineages between BB and ABB/AAB hybrids. eBSVs appear to be relevant phylogenetic markers that can illustrate the M. balbisiana phylogeography story.Conclusion The theoretical implications of this study for further elucidation of the historical and geographical process of Musa domestication are numerous. Discovery of banana plants with B genome non-infective for eBSV opens the way to the introduction of new genitors in programmes of genetic banana improvement.     

Chromosome segregation in an allotetraploid banana hybrid (AAAB) suggests a translocation between the A and B genomes and results in eBSV-free offsprings

Many banana cultivars (including the Plantain type) are triploid interspecific hybrids between Musa acuminata (A genome) and Musa balbisiana (B genome). M. balbisiana contains endogeneous Banana streak virus sequences (eBSVs) that can, in interspecific genome context, spontaneously release infectious viral genomes. We analyzed, a triploid progeny of 184 individuals from a cross between a tetraploid AAAB breeding accession (CRBP39) and the diploid AA accession (Pahang) with 38 SSR and eBSV-specific PCR markers. The results showed that (1) most of the alleles are found/transmitted in the expected frequency to the progeny with only 10 % biased; (2) 70 % of the loci displayed a tetrasomic allele segregation and (3) interspecific intrachromosomal recombinations occurred for all the chromosome segments surveyed. However, half of the offspring obtained resulted from maternal unbalanced gametes transmission. Analysis of gamete composition and marker association suggested the presence of a large translocation between A and B genome involving chromosome 1 and 3. The two infectious eBSVs present in the maternal parent CRBP39 are located on chromosome 1B and appeared in a higher proportion than expected in the progeny. Interestingly, we showed that both eBSVs were absent from 24 offspring that represent promising material for breeding.     

Endogenous pararetroviruses of allotetraploid Nicotiana tabacum and its diploid progenitors, N. sylvestris and N. tomentosiformis

Endogenous pararetroviruses (EPRVs) represent a new class of dispersed repetitive DNA in plants. The genomes of many Nicotiana species and other solanaceous plants are rich in EPRVs. Distinct EPRV families are present in N. sylvestris ( Ns ) and in N. tomentosiformis ( Nto ), the two diploid progenitors of allotetraploid N. tabacum . Nicotiana EPRVs represent an interesting type of repetitive sequence to analyse in polyploids because of their potential impact on plant fitness and the epigenetic architecture of plant genomes. The Ns EPRV family appears identical in N. sylvestris and N. tabacum , indicating little change has occurred in either species since polyploid formation. By contrast, the Nto EPRV family is larger in N. tomentosiformis than in N. tabacum , suggesting either preferential elimination from the polyploid genome or specific accumulation in the diploid genome following polyploidization. The lability of Nto EPRVs might be enhanced by a frequent association with gypsy retrotransposons. Although some EPRVs are probably benign, others are potentially pathogenic or, conversely, determinants of virus resistance. Normally quiescent EPRVs can be reactivated and cause symptoms of infection in hybrids of species that differ in their EPRV content. EPRVs that furnish immunity to the free virus exemplify the selective value of so-called 'junk' DNA. Variation in the abundance and distribution of EPRVs among related species can be useful in taxonomic and evolutionary studies.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 627–638.     

The genome of African yam (Dioscorea cayenensis‐rotundata complex) hosts endogenous sequences from four distinct badnavirus species

Several endogenous viral elements (EVEs) have been identified in plant genomes, including endogenous pararetroviruses (EPRVs). Here, we report the first characterization of EPRV sequences in the genome of African yam of the Dioscorea cayenensis‐rotundata complex. We propose that these sequences should be termed ‘endogenous Dioscorea bacilliform viruses' (eDBVs). Molecular characterization of eDBVs shows that they constitute sequences originating from various parts of badnavirus genomes, resulting in a mosaic structure that is typical of most EPRVs characterized to date. Using complementary molecular approaches, we show that eDBVs belong to at least four distinct Badnavirus species, indicating multiple, independent, endogenization events. Phylogenetic analyses of eDBVs support and enrich the current taxonomy of yam badnaviruses and lead to the characterization of a new Badnavirus species in yam. The impact of eDBVs on diagnosis, yam germplasm conservation and movement, and breeding is discussed.     

Marker-assisted breeding of <Emphasis Type="Italic">Musa balbisiana</Emphasis> genitors devoid of infectious endogenous Banana streak virus sequences

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.     

Molecular characterization of <Emphasis Type="Italic">Banana streak virus</Emphasis> isolate from <Emphasis Type="Italic">Musa Acuminata</Emphasis> in China

Banana streak virus (BSV), a member of genus Badnavirus, is a causal agent of banana streak disease throughout the world. The genetic diversity of BSVs from different regions of banana plantations has previously been investigated, but there are relatively few reports of the genetic characteristic of episomal (non-integrated) BSV genomes isolated from China. Here, the complete genome, a total of 7722bp (GenBank accession number DQ092436), of an isolate of Banana streak virus (BSV) on cultivar Cavendish (BSAcYNV) in Yunnan, China was determined. The genome organises in the typical manner of badnaviruses. The intergenic region of genomic DNA contains a large stem-loop, which may contribute to the ribosome shift into the following open reading frames (ORFs). The coding region of BSAcYNV consists of three overlapping ORFs, ORF1 with a non-AUG start codon and ORF2 encoding two small proteins are individually involved in viral movement and ORF3 encodes a polyprotein. Besides the complete genome, a defective genome lacking the whole RNA leader region and a majority of ORF1 and which encompasses 6525bp was also isolated and sequenced from this BSV DNA reservoir in infected banana plants. Sequence analyses showed that BSAcYNV has closest similarity in terms of genome organization and the coding assignments with an BSV isolate from Vietnam (BSAcVNV). The corresponding coding regions shared identities of 88% and ∼95% at nucleotide and amino acid levels, respectively. Phylogenetic analysis also indicated BSAcYNV shared the closest geographical evolutionary relationship to BSAcVNV among sequenced banana streak badnaviruses.     

A distinct endogenous pararetrovirus family in Nicotiana tomentosiformis, a diploid progenitor of polyploid tobacco

A distinct endogenous pararetrovirus (EPRV) family corresponding to a previously unknown virus has been identified in the genome of Nicotiana tomentosiformis, a diploid ancestor of allotetraploid tobacco (Nicotiana tabacum). The putative virus giving rise to N. tomentosiformis EPRVs (NtoEPRVs) is most similar to tobacco vein clearing virus, an episomal form of a normally silent EPRV family in Nicotiana glutinosa; it is also related to a putative virus giving rise to the NsEPRV family in Nicotiana sylvestris (the second diploid progenitor of tobacco) and in the N. sylvestris fraction of the tobacco genome. The copy number of NtoEPRVs is significantly higher in N. tomentosiformis than in tobacco. This suggests that after the polyploidization event, many copies were lost from the polyploid genome or were accumulated specifically in the diploid genome. By contrast, the copy number of NsEPRVs has remained constant in N. sylvestris and tobacco, indicating that changes have occurred preferentially in the NtoEPRV family during evolution of the three Nicotiana species. NtoEPRVs are often flanked by Gypsy retrotransposon-containing plant DNA. Although the mechanisms of NtoEPRV integration, accumulation, and/or elimination are unknown, these processes are possibly linked to retrotransposon activity.     

Long-range restriction site mapping of a syntenic segment conserved between human chromosome 1 and mouse chromosome 3

A linkage map determined from segregation analysis of 338 meiotic events in an interspecific mouse cross was utilized to help investigate genomic organization of a linkage group conserved between human chromosome 1p and mouse chromosome 3. Using pulsed-field gel electrophoresis, the genes encoding the lymphocyte adhesion molecule human CD2/murine Ly-37, the alpha 1-subunit of Na, K-ATPase, the beta-subunit of thyrotropin, the beta-subunit of nerve growth factor, and muscle adenylate deaminase were similarly positioned on long-range restriction maps in both species. These studies indicate that the development of detailed genetic maps using interspecific Mus crosses facilitates rapid analysis of murine genomic organization and may enable physical mapping of syntenic regions within the human genome. Moreover, the data suggest profound conservation of genomic organization during mammalian evolution.     

Identification of RAPD markers linked to A and B genome sequences in Musa L.

Plantains and bananas (Musa spp. sect. eumusa) originated from intra- and interspecific hybridization between two wild diploid species, M. acuminata Colla. and M. balbisiana Colla., which contributed the A and B genomes, respectively. Polyploidy and hybridization have given rise to a number of diploid, triploid, and tetraploid clones with different permutations of the A and B genomes. Thus, dessert and highland bananas are classified mainly as AAA, plantains are AAB, and cooking bananas are ABB. Classification of Musa into genomic groups has been based on morphological characteristics. This study aimed to identify RAPD (random amplified polymorphic DNA) markers for the A and B genomes. Eighty 10-mer Operon primers were used to amplify DNA from M. acuminata subsp. burmannicoides clone 'Calcutta 4' (AA genomes) and M. balbisiana clone 'Honduras' (BB genomes). Three primers (A17, A18, and D10) that produced unique genome-specific fragments in the two species were identified. These primers were tested in a sample of 40 genotypes representing various genome combinations. The RAPD markers were able to elucidate the genome composition of all the genotypes. The results showed that RAPD analysis can provide a quick and reliable system for genome identification in Musa that could facilitate genome characterization and manipulations in breeding lines.