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.     

A single Banana streak virus integration event in the banana genome as the origin of infectious endogenous pararetrovirus

Sequencing 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 balbisiana carries integrated copies of Banana 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 diploid M. balbisiana cv. Pisang Klutuk Wulung (PKW) revealed by the study of Musa bacterial 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.     

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.     

Ascertaining maternal and paternal lineage within Musa by chloroplast and mitochondrial DNA RFLP analyses.

In banana, the maternal transmission of chloroplast DNA and paternal transmission of the mitochondrial DNA provides an exceptional opportunity for studying the maternal and paternal lineage of clones. In the present study, RFLP combined with hybridization of heterologous mitochondrial and chloroplastic probes have been used to characterize 71 wild accessions and 131 diploid and 103 triploid cultivated clones. In additon to Musa acuminata and Musa balbisiana, other species from the four Musa sections were studied to investigate their contribution to the origin of cultivated bananas. These molecular analyses enable the classification of the Musa complex to be discussed. Results ascertain relationships among and between the wild accessions and the mono- and interspecific diploid and triploid bananas, particularly for the acuminata genome. Parthenocarpic varieties are shown to be linked to M. acuminata banksii and M. acuminata errans, thus suggesting that the first center of domestication was in the Philippines - New Guinea area.     

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.     

Molecular Characterization of Banana streak virus Isolate from Musa Acuminata 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 (BSAc...     

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.     

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.     

Genomes, diversity and resistance gene analogues in Musa species

Resistance genes (R genes) in plants are abundant and may represent more than 1% of all the genes. Their diversity is critical to the recognition and response to attack from diverse pathogens. Like many other crops, banana and plantain face attacks from potentially devastating fungal and bacterial diseases, increased by a combination of worldwide spread of pathogens, exploitation of a small number of varieties, new pathogen mutations, and the lack of effective, benign and cheap chemical control. The challenge for plant breeders is to identify and exploit genetic resistances to diseases, which is particularly difficult in banana and plantain where the valuable cultivars are sterile, parthenocarpic and mostly triploid so conventional genetic analysis and breeding is impossible. In this paper, we review the nature of R genes and the key motifs, particularly in the Nucleotide Binding Sites (NBS), Leucine Rich Repeat (LRR) gene class. We present data about identity, nature and evolutionary diversity of the NBS domains of Musa R genes in diploid wild species with the Musa acuminata (A), M. balbisiana (B), M. schizocarpa (S), M. textilis (T), M. velutina and M. ornata genomes, and from various cultivated hybrid and triploid accessions, using PCR primers to isolate the domains from genomic DNA. Of 135 new sequences, 75% of the sequenced clones had uninterrupted open reading frames (ORFs), and phylogenetic UPGMA tree construction showed four clusters, one from Musa ornata, one largely from the B and T genomes, one from A and M. velutina, and the largest with A, B, T and S genomes. Only genes of the coiled-coil (non-TIR) class were found, typical of the grasses and presumably monocotyledons. The analysis of R genes in cultivated banana and plantain, and their wild relatives, has implications for identification and selection of resistance genes within the genus which may be useful for plant selection and breeding and also for defining relationships and genome evolution patterns within the genus using the multi-copy and variable resistance genes.     

Variable number of tandem repeat markers in the genome sequence of Mycosphaerella fijiensis, the causal agent of black leaf streak disease of banana (Musa spp)

We searched the genome of Mycosphaerella fijiensis for molecular markers that would allow population genetics analysis of this plant pathogen. M. fijiensis, the causal agent of banana leaf streak disease, also known as black Sigatoka, is the most devastating pathogen attacking bananas (Musa spp). Recently, the entire genome sequence of M. fijiensis became available. We screened this database for VNTR markers. Forty-two primer pairs were selected for validation, based on repeat type and length and the number of repeat units. Five VNTR markers showing multiple alleles were validated with a reference set of isolates from different parts of the world and a population from a banana plantation in Costa Rica. Polymorphism information content values varied from 0.6414 to 0.7544 for the reference set and from 0.0400 and 0.7373 for the population set. Eighty percent of the polymorphism information content values were above 0.60, indicating that the markers are highly informative. These markers allowed robust scoring of agarose gels and proved to be useful for variability and population genetics studies. In conclusion, the strategy we developed to identify and validate VNTR markers is an efficient means to incorporate markers that can be used for fungicide resistance management and to develop breeding strategies to control banana black leaf streak disease. This is the first report of VNTR-minisatellites from the M. fijiensis genome sequence.     

Isolation and characterization of the highly repeated fraction of the banana genome

Although the nuclear genome of banana (Musa spp.) is relatively small (1C approximately 610 Mbp for M. acuminata), the results obtained from other sequenced genomes suggest that more than half of the banana genome may be composed of repetitive and non-coding DNA sequences. Knowledge of repetitive DNA can facilitate mapping of important traits, phylogenetic studies, BAC-based physical mapping, and genome sequencing/annotation. However, only a few repetitive DNA sequences have been characterized in banana. In this work, we used DNA reassociation kinetics to isolate the highly repeated fraction of the banana genome (M. acuminata 'Calcutta 4'). Two libraries, one prepared from Cot 相似文献   

Physical mapping of the 18S-25S and 5S ribosomal RNA genes in diploid bananas

Fluorescent in situ hybridisation (FISH) was used to determine the number and distribution of the 18S-25S and 5S rDNA sites on mitotic chromosomes of 6 wild and 2 edible diploid (2n=22) accessions belonging to the two banana species, Musa acuminata and M. balbisiana. FISH with the 18S-25S probe resulted in signals on one pair of chromosomes, the position of signals corresponded to the secondary constriction at the end of a short arm. The intensity of labelling was different between the homologues and the larger site corresponded to a larger secondary constriction. This labelling pattern was observed consistently in all genotypes. On the other hand, differences in the number of 5S sites were observed between the accessions. While in some of the wild seeded species, the 5S rDNA was localised on two pairs of chromosomes, hybridisation signals appeared on three pairs of chromosomes in other wild accessions. Quite unexpectedly, only five sites of 5S rDNA were reproducibly observed in the two vegetatively propagated diploid edible cultivars, Pisang Mas and Niyarma Yik, evidence for structural heterozygosity. A dual colour FISH showed that in all accessions, the satellite chromosomes carrying the 18S-25S loci did not carry the 5S loci. The results demonstrate that molecular cytogenetics can be applied to Musa and that physical cytogenetic maps can be generated. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Exploring wild genetic resources of Musa acuminata Colla distributed in the humid forests of southern Western Ghats of peninsular India using ISSR markers

Musa acuminata ssp. burmannica, one of the wild progenitors contributing 'A genome' to the present-day dessert bananas, has a long evolutionary history intervened by human activities. In this study, ISSR markers were used to analyze the pattern of genetic variation and differentiation in 32 individuals along with two reference samples (viz., Musa acuminata ssp. burmannicoides, var. Calcutta 4 and Musa balbisiana) of wild Musa, which corresponded to three populations across the biodiversity-rich hot spot of southern Western Ghats of India. High levels of genetic diversity were revealed both at the species and population levels, using Nei's diversity indices. The hierarchical analysis of molecular variance showed pronounced genetic differentiation, as 96?% of the total variance was fixed within population and only 4?% among populations. Nei's genetic differentiation coefficient (G (ST)?=?0.1823) and low gene flow (Nm?=?1.18) further confirmed this. The positive correlation (Mantel test) between geographic distance and genetic distance (r?=?0.338 P?相似文献   

Nuclear genome size and genomic distribution of ribosomal DNA in Musa and Ensete (Musaceae): taxonomic implications

Nuclear DNA content and genomic distributions of 5S and 45S rDNA were examined in nineteen diploid accessions of the genus Musa representing its four sections Eumusa, Rhodochlamys, Callimusa and Australimusa, and in Ensete gilletii, which was the outgroup in this study. In the Eumusa (x = 11), 2C DNA content ranged from 1.130 to 1.377 pg, M. balbisiana having the lowest DNA content of all sections. M. beccarii (x = 9), a representative of Callimusa, had the highest 2C nuclear DNA content (1.561 pg). Species belonging to Rhodochlamys (x = 11) and Australimusa (x = 10) had 2C DNA contents ranging from 1.191 to 1.299 pg and from 1.435 to 1.547 pg, respectively. E. gilletii (x = 9) had 2C DNA content of 1.210 pg. The number of 5S rDNA loci in Musa varied from 4 to 8 per diploid cell. While different numbers of 5S rDNA loci were observed within Eumusa and Rhodochlamys, four 5S rDNA loci were observed in all accessions of Australimusa. M. beccarii (Callimusa) and E. gilletii contained 5S rRNA gene clusters on five and six chromosomes, respectively. The number of 45S rDNA loci was conserved within individual sections. Hierarchical cluster analysis of genome size, number of chromosomes and 45S rDNA sites suggested a close relationship between Rhodochlamys and Eumusa; Australimusa was clearly separated as were M. beccarii and E. gilletii. Within the Eumusa-Rhodochlamys group, M. balbisiana, M. schizocarpa and M. ornata formed distinct subgroups, clearly separated from the accessions of M. acuminata, M. mannii, M. laterita and M. velutina, which formed a tight subgroup. The results expand the knowledge of genome size and genomic distribution of ribosomal DNA in Musa and Ensete. They aid in clarification of the taxonomical classification of Musa and show a need to supplement the analyses on the DNA sequence level with cytogenetic studies.     

New microsatellite markers for bananas (Musa spp)

Thirty-four microsatellite markers (SSRs) were identified in EST and BAC clones from Musa acuminata burmannicoides var. Calcutta 4 and validated in 22 Musa genotypes from the Banana Germplasm Bank of Embrapa-CNPMF, which includes wild and improved diploids. The number of alleles per locus ranged from 2 to 14. The markers were considered highly informative based on their polymorphism information content values; more than 50% were above 0.5. These SSRs will be useful for banana breeding programs, for studies of genetic diversity, germplasm characterization and selection, development of saturated genetic linkage maps, and marker assisted selection.     

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.     

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.     

Utility of selected non-coding chloroplast DNA sequences for lineage assessment of Musa interspecific hybrids

Single-copy chloroplast loci are used widely to infer phylogenetic relationship at different taxonomic levels among various groups of plants. To test the utility of chloroplast loci and to provide additional data applicable to hybrid evolution in Musa, we sequenced two introns, rpl16 and ndhA, and two intergenic spacers, psaA-ycf3 and petA-psbJ-psbL-psbF and combined these data. Using these four regions, Musa acuminata Colla (A)- and M. balbisiana Colla (B)-containing genomes were clearly distinguished. Some triploid interspecific hybrids contain A-type chloroplasts (the AAB/ABB) while others contain B-type chloroplasts (the BBA/BBB). The chloroplasts of all cultivars in 'Namwa' (BBA) group came from the same wild maternal origin, but the specific parents are still unrevealed. Though, average sequence divergences in each region were little (less than 2%), we propose that petA-psbJ intergenic spacer could be developed for diversity assessment within each genome. This segment contains three single nucleotide polymorphisms (SNPs) and two indels which could distinguish diversity within A genome whereas this same region also contains one SNP and an indel which could categorize B genome. However, an inverted repeat region which could form hairpin structure was detected in this spacer and thus was omitted from the analyses due to their incongruence to other regions. Until thoroughly identified in other members of Musaceae and Zingiberales clade, utility of this inverted repeat as phylogenetic marker in these taxa are cautioned.