Pisang Klutuk Wulung Research Articles

In silico Comparisons of the Ethylene Response Factor 1 (ERF1) Gene Between Malaysian Wild Banana (Musa acuminata ssp. malaccensis) and Pisang Klutuk Wulung (Musa balbisiana)

Musa balbisiana (B genome) has been observed to have a higher tolerance of biotic and abiotic stresses than Musa acuminata (A genome). Ethylene Response Factor 1 (ERF1) is a gene activator for pathogenesis-related proteins (PR proteins) such as basic chitinases and beta-1,3-glucanase. There are numerous ERF1 gene studies about Oryza sativa, but information about the banana ERF1 gene, especially in the B genome (Musa balbisiana “Pisang Klutuk Wulung”), has still not been explored thoroughly. Using annotated genomic data in an A genome (Musa acuminata ssp. malaccensis) and genomic data in a B genome (Musa balbisiana “Pisang Klutuk Wulung”), research on the ERF1 gene can be conducted at the gene sequences and amino acid sequences levels. The Musa acuminata (A genome) ERF1 gene nucleotide sequence was retrieved from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The Musa balbisiana (B genome) ERF1 gene nucleotide sequence was identified with the nucleotide Basic Local Alignment Search Tool (BLASTn) using an A genome ERF1 gene sequence as a query. Both ERF1 gene nucleotide sequences and amino acid sequences in the A and B genomes were annotated and compared. Seven annotated genome ERF1 gene sequences from the A and B genomes were identified with the probability that these genes were actively transcribed in cell activity. ERF1 gene comparisons between the A and B genomes showed that nucleotide composition, gene structure, gene position in each respective chromosome, ERF clusterization, identified motif, and amino acid composition in each of the identified motifs have similar characteristics.

Embryo and Seed Germination of Pisang Klutuk Wulung (Musa balbisiana Colla) After Storage

Pisang Klutuk Wulung is one of Musa balbisiana accessions which has potential for genetic improvement of cultivated banana so that its conservation is essential. This research aimed to study the post-storage germination of Indonesian seeds of Pisang Klutuk Wulung. Storage methods were carried out using a factorial completely randomized design with three factors: packing methods using vacuum and non-vacuum plastic bags; storage temperature at 25ºC, 4ºC, and -20ºC; and storage duration by 7, 14, 30, and 50 days. The germinations were done in vitro and ex vitro. The results showed that seeds stored at 25ºC in non-vacuum plastic bags were infested by molds, contrasting to the non-vacuum treatment. The sterilization method using 25% sodium hypoclorite, Tween 20, and 80% alcohol resulted in less contamination than 96% alcohol. In vitro germination from the vacuum treatment had a higher germination rate than non-vacuum treatment. However, ex vitro germination was not affected by the storage method. Similar patterns were seen in vitro and ex vitro germination as storage in 4ºC resulted in better seed germination after 30 and 50 days. In contrast, at -20ºC, no embryo germinated in all storage duration treatments. Pisang Klutuk Wulung seeds could not be stored in the long term as they rapidly lost their viability. Our finding showed that airtight condition by vacuum treatment and low temperature at 4ºC were able to maintain seed viability for longer period of storage. Thus, this finding was useful to improve Musa breeding programs and as an essential step for the long-term conservation of Musa genetic resources.

In silico characterization and comparison of the fruit ripening related beta‐ amylase (BAM) gene family in banana genome A and B

Banana is one of the most important commodities for maintaining global food security. Primary metabolic processes during the ripening of banana greatly affect post‐harvest quality, particularly in starch metabolism. The beta‐ amylase (BAM) gene family is known as a group of genes that plays an important role in starch metabolism regulation. In this study, we focused on the characterization and comparative analysis of the BAM gene family in DH Pahang and Pisang Klutuk Wulung (PKW) varieties, these being the AA and BB genomes, respectively. The sequences of BAM gene family were retrieved from the database of Musa acuminata ’DH Pahang’ and Musa balbisiana ’PKW’ genome, then structural and functional characterization was performed, followed by identification of cis‐acting elements in the BAM promoter regions. The results showed that the BAM gene family structure was relatively conserved in both genomes, and a putative BAM11 gene was found, the function of which has not been studied in other plants. Cis‐acting element analysis showed that they were distinct in the copy number and types of elements that were responsive to various phytohormones. This study suggested that the BAM genes involved in ripening are spatiotemporally regulated. However, further functional genomic analysis is required to describe the specific role and regulation of BAM genes during ripening in banana.

In silico Characterization of the Structure of Genes and Proteins related to β-carotene Degradation in Musa acuminata ‘DH-Pahang’ and Musa balbisiana ‘Pisang Klutuk Wulung’

β-carotene is an important nutritious content in banana. However, its lifetime depends on the enzymes controlling its conversion into strigolactone. To understand the involved enzymes’ activity, which are β-carotene isomerase (D27), carotenoid cleavage dioxygenase 7 (CCD7), and CCD8, would be the key to manipulate the rate of β-carotene degradation. In this research, we characterized the structure of genes and proteins of the D27, CCD7, and CCD8 from Musa acuminata ‘DH-Pahang’ and Musa balbisiana ‘Pisang Klutuk Wulung’ (PKW). We aligned the corresponding sequence of genes from both species to determine similarity and intron/exon positions. We also identified domains and motifs in the sequences of putative proteins of D27, CCD7, and CCD8. We found that D27, CCD7, and CCD8 genes in DH-Pahang and PKW comprise of various nucleotide sequence length, putative proteins, and numbers and length of exons and introns. However, the putative proteins possess the same domains: DUF4033 (domain of unknown function) in D27 and RPE65 (retinal pigment epithelium) in CCD7 and CCD8. Phylogenetic trees showed that D27, CCD7, and CCD8 proteins from DH-Pahang and PKW are conserved and clustered in the same clades with the same proteins of monocot plants. Hence, the results could be useful for future research in optimizing β-carotene content in banana.

Characteristics of banana B genome MADS-box family demonstrate their roles in fruit development, ripening, and stress

MADS-box genes are critical regulators of growth and development in flowering plants. Sequencing of the Musa balbisiana (B) genome has provided a platform for the systematic analysis of the MADS-box gene family in the important banana ancestor Musa balbisiana. Seventy-seven MADS-box genes, including 18 type I and 59 type II, were strictly identified from the banana (Pisang Klutuk Wulung, PKW, 2n = 2x = 22) B genome. These genes have been preferentially placed on the banana B genome. Evolutionary analysis suggested that M. balbisiana MCM1-AGAMOUS-DEFICIENS-SRF (MbMADS) might be organized into the MIKCc, MIKC*, Mα, Mβ, and Mγ groups according to the phylogeny. MIKCc was then further categorized into 10 subfamilies according to conserved motif and gene structure analyses. The well-defined MADS-box genes highlight gene birth and death in banana. MbMADSes originated from the same ancestor as MaMADSes. Transcriptome analysis in cultivated banana (ABB) revealed that MbMADSes were conserved and differentially expressed in several organs, in various fruit developing and ripening stages, and in stress treatments, indicating the participation of these genes in fruit development, ripening, and stress responses. Of note, SEP/AGL2 and AG, as well as other several type II MADS-box genes, including the STMADS11 and TM3/SOC1 subfamilies, indicated elevated expression throughout banana fruit development, ripening, and stress treatments, indicating their new parts in controlling fruit development and ripening. According to the co-expression network analysis, MbMADS75 interacted with bZIP and seven other transcription factors to perform its function. This systematic analysis reveals fruit development, ripening, and stress candidate MbMADSes genes for additional functional studies in plants, improving our understanding of the transcriptional regulation of MbMADSes genes and providing a base for genetic modification of MADS-mediated fruit development, ripening, and stress.

The complete chloroplast genome sequence of wild banana, Musa balbisiana variety ’Pisang Klutuk Wulung’ (Musaceae)

Musa balbisiana is a wild-type species of banana, endemic to Southern China, Eastern South Asia, and Northern Southeast Asia. The M. balbisiana variety ’Pisang Klutuk Wulung’ is one of the possible ancestral parents of modern cultivated bananas, but its wild populations are now in decline. In this study, we report the complete chloroplast genome sequence of wild banana, M. balbisiana diploid variety ’Pisang Klutuk Wulung’. The M. balbisiana chloroplast genome is found to be 169,458 bp in length and has a base composition of A (31.44%), G (18.16%), C (18.61%), and T (31.79%). The genome contained two short inverted repeat (IRa and IRb) regions (35,084 bp), which were separated by a large single copy (LSC) region (87,805 bp) and a small single copy (SSC) region (11,485 bp). The genome encodes 113 unique genes, including 79 protein-coding genes, 30 transfer RNA (tRNA) genes, and four ribosomal RNA (rRNA) genes. Further, phylogenetic analysis suggested that M. balbisiana is closely related to the species of M. textilis. This complete chloroplast genome will provide valuable information for the development of DNA markers for future population and conservation studies of M. balbisiana.

How endogenous plant pararetroviruses shed light onMusaevolution

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). We characterize eBSV distribution among a Musa sampling including seedy BB diploids and interspecific hybrids with Musa acuminate exhibiting different levels of ploidy for the B genome (ABB, AAB, AB). We used representative samples of the two areas of sympatry between M. acuminate 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. 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 theM. balbisiana phylogeography story. 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.

"A draft Musa balbisiana genome sequence for molecular genetics in polyploid, inter- and intra-specific Musa hybrids".

BackgroundModern banana cultivars are primarily interspecific triploid hybrids of two species, Musa acuminata and Musa balbisiana, which respectively contribute the A- and B-genomes. The M. balbisiana genome has been associated with improved vigour and tolerance to biotic and abiotic stresses and is thus a target for Musa breeding programs. However, while a reference M. acuminata genome has recently been released (Nature 488:213–217, 2012), little sequence data is available for the corresponding B-genome.To address these problems we carried out Next Generation gDNA sequencing of the wild diploid M. balbisiana variety ‘Pisang Klutuk Wulung’ (PKW). Our strategy was to align PKW gDNA reads against the published A-genome and to extract the mapped consensus sequences for subsequent rounds of evaluation and gene annotation.ResultsThe resulting B-genome is 79% the size of the A-genome, and contains 36,638 predicted functional gene sequences which is nearly identical to the 36,542 of the A-genome. There is substantial sequence divergence from the A-genome at a frequency of 1 homozygous SNP per 23.1 bp, and a high degree of heterozygosity corresponding to one heterozygous SNP per 55.9 bp. Using expressed small RNA data, a similar number of microRNA sequences were predicted in both A- and B-genomes, but additional novel miRNAs were detected, including some that are unique to each genome. The usefulness of this B-genome sequence was evaluated by mapping RNA-seq data from a set of triploid AAA and AAB hybrids simultaneously to both genomes. Results for the plantains demonstrated the expected 2:1 distribution of reads across the A- and B-genomes, but for the AAA genomes, results show they contain regions of significant homology to the B-genome supporting proposals that there has been a history of interspecific recombination between homeologous A and B chromosomes in Musa hybrids.ConclusionsWe have generated and annotated a draft reference Musa B-genome and demonstrate that this can be used for molecular genetic mapping of gene transcripts and small RNA expression data from several allopolyploid banana cultivars. This draft therefore represents a valuable resource to support the study of metabolism in inter- and intraspecific triploid Musa hybrids and to help direct breeding programs.

Mechanisms of haplotype divergence at the RGA08 nucleotide-binding leucine-rich repeat gene locus in wild banana (Musa balbisiana)

BackgroundComparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp.), two diploid wild species Musa acuminata (A genome) and Musa balbisiana (B genome) contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW).ResultsSequence comparison revealed two regions of contrasting features. The first is a highly colinear gene-rich region where the two haplotypes diverge only by single nucleotide polymorphisms and two repetitive element insertions. The second corresponds to a large cluster of RGA08 genes, with 13 and 18 predicted RGA genes and pseudogenes spread over 131 and 152 kb respectively on each haplotype. The RGA08 cluster is enriched in repetitive element insertions, in duplicated non-coding intergenic sequences including low complexity regions and shows structural variations between haplotypes. Although some allelic relationships are retained, a large diversity of RGA08 genes occurs in this single M. balbisiana genotype, with several RGA08 paralogs specific to each haplotype. The RGA08 gene family has evolved by mechanisms of unequal recombination, intragenic sequence exchange and diversifying selection. An unequal recombination event taking place between duplicated non-coding intergenic sequences resulted in a different RGA08 gene content between haplotypes pointing out the role of such duplicated regions in the evolution of RGA clusters. Based on the synonymous substitution rate in coding sequences, we estimated a 1 million year divergence time for these M. balbisiana haplotypes.ConclusionsA large RGA08 gene cluster identified in wild banana corresponds to a highly variable genomic region between haplotypes surrounded by conserved flanking regions. High level of sequence identity (70 to 99%) of the genic and intergenic regions suggests a recent and rapid evolution of this cluster in M. balbisiana.

UNDERSTANDING PLANT RESPONSES TO BIOTIC STRESS: ONGOING RESEARCH IN MUSA

ISHS III International Symposium on Banana: ISHS-ProMusa Symposium on Recent Advances in Banana Crop Protection for Sustainable Production and Improved Livelihoods UNDERSTANDING PLANT RESPONSES TO BIOTIC STRESS: ONGOING RESEARCH IN MUSA

EXPLORING THE BANANA STREAK VIRUSES - MUSA SP. PATHOSYSTEM: HOW DOES IT WORK?

Banana streak viruses (BSVs) are double-stranded DNA pararetroviruses causing banana streak disease. Recently, numerous outbreaks of the disease occurred in many banana-producing areas in interspecific hybrids (#Musa acuminata# × #Musa balbisiana#) originating from virus-free parents. These infections correlated with the presence of endogenous banana streak viruses (eBSVs), viral DNA sequences integrated in the #M. balbisiana# genome only. Although integration is not needed for the viral replication cycle, some viral integrants are infectious under stress conditions by reconstituting a replication-competent genome after possible homologous recombination events. Even though the wild #M. balbisiana# Pisang Klutuk Wulung (PKW) harbours infectious eBSVs, it is resistant to BSVs. We characterised the genetic and genomic endogenous viral organisation of three BSV species in PKW in order to determine the species responsible for the viral expression in the interspecific F1 progeny. (Resume d'auteur)

Analysis of non-TIR NBS-LRR resistance gene analogs in Musa acuminata Colla: isolation, RFLP marker development, and physical mapping.

BackgroundMany commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes). The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence.ResultsA computational strategy was developed for unbiased conserved motif discovery in NBS and LRR domains in R-genes and homologues in monocotyledonous plant species. Degenerate PCR primers targeting conserved motifs were tested on the wild cultivar Musa acuminata subsp. burmannicoides, var. Calcutta 4, which is resistant to a number of fungal pathogens and nematodes. One hundred and seventy four resistance gene analogs (RGAs) were amplified and assembled into 52 contiguous sequences. Motifs present were typical of the non-TIR NBS-LRR RGA subfamily. A phylogenetic analysis of deduced amino-acid sequences for 33 RGAs with contiguous open reading frames (ORFs), together with RGAs from Arabidopsis thaliana and Oryza sativa, grouped most Musa RGAs within monocotyledon-specific clades. RFLP-RGA markers were developed, with 12 displaying distinct polymorphisms in parentals and F1 progeny of a diploid M. acuminata mapping population. Eighty eight BAC clones were identified in M. acuminata Calcutta 4, M. acuminata Grande Naine, and M. balbisiana Pisang Klutuk Wulung BAC libraries when hybridized to two RGA probes. Multiple copy RGAs were common within BAC clones, potentially representing variation reservoirs for evolution of new R-gene specificities.ConclusionThis is the first large scale analysis of NBS-LRR RGAs in M. acuminata Calcutta 4. Contig sequences were deposited in GenBank and assigned numbers ER935972 – ER936023. RGA sequences and isolated BACs are a valuable resource for R-gene discovery, and in future applications will provide insight into the organization and evolution of NBS-LRR R-genes in the Musa A and B genome. The developed RFLP-RGA markers are applicable for genetic map development and marker assisted selection for defined traits such as pest and disease resistance.

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.

Production of haploids from anther culture of banana [Musa balbisiana (BB)

We report here, for the first time, the production of haploid plants of banana Musa balbisiana (BB). Callus was induced from anthers in which the majority of the microspores were at the uninucleate stage. The frequency of callus induction was 77%. Callus proliferation usually preceded embryo formation. About 8% of the anthers developed androgenic embryos. Of the 147 plantlets obtained, 41 were haploids (n=x=11). The frequency of haploid production depended on genotypes used: 18 haploid plants were produced from genotype Pisang klutuk, 12 from Pisang batu, seven from Pisang klutuk wulung and four from Tani. The frequency of regeneration was 1.1%, which was based on the total number of anthers cultured. Diploid plants (2n=2x=22) were also observed in the regenerated plants. The haploid banana plants that were developed will be important material for the improvement of banana through breeding programmes.