Comparative RNA-seq analysis of resistant and susceptible banana genotypes reveals molecular mechanisms in response to banana bunchy top virus (BBTV) infection.

Bananas hold significant economic importance as an agricultural commodity, serving as a primary livelihood source, a favorite fruit, and a staple crop in various regions across the world. However, Banana bunchy top disease (BBTD), which is caused by banana bunchy top virus (BBTV), poses a considerable threat to banana cultivation. To understand the resistance mechanism and the interplay of host suitability factors in the presence of BBTV, we conducted RNA-seq-based comparative transcriptomics analysis on mock-inoculated and BBTV-inoculated samples from resistant (wild Musa balbisiana) and susceptible (Musa acuminata 'Lakatan') genotypes. We observed common patterns of expression for 62 differentially expressed genes (DEGs) in both genotypes, which represent the typical defense response of bananas to BBTV. Furthermore, we identified 99 DEGs exclusive to the 'Lakatan' banana cultivar, offering insights into the host factors and susceptibility mechanisms that facilitate successful BBTV infection. In parallel, we identified 151 DEGs unique to the wild M. balbisiana, shedding light on the multifaceted mechanisms of BBTV resistance, involving processes such as secondary metabolite biosynthesis, cell wall modification, and pathogen perception. Notably, our validation efforts via RT-qPCR confirmed the up-regulation of the glucuronoxylan 4-O-methyltransferase gene (14.28 fold-change increase), implicated in xylan modification and degradation. Furthermore, our experiments highlighted the potential recruitment of host's substrate adaptor ADO (30.31 fold-change increase) by BBTV, which may play a role in enhancing banana susceptibility to the viral pathogen. The DEGs identified in this work can be used as basis in designing associated gene markers for the precise integration of resistance genes in marker-assisted breeding programs. Furthermore, the findings can be applied to develop genome-edited banana cultivars targeting the resistance and susceptibility genes, thus developing novel cultivars that are resilient to important diseases.

A novel SNP panel developed for targeted genotyping-by-sequencing (GBS) reveals genetic diversity and population structure of Musa spp. germplasm collection.

The Philippines is situated in the geographic region regarded as the center of diversity of banana and its wild relatives (Musa spp.). It holds the most extensive collection of B-genome germplasm in the world along with A-genome groups and several natural hybrids with A- and B-genome combinations. Management of this germplasm resource has relied immensely on identification using local names and morphological characters, and the extent of genetic diversity of the collection has not been achieved with molecular markers. A high-throughput and reliable genotyping method for banana and its relatives will facilitate germplasm management and support breeding initiatives toward a marker-based approach. Here, we developed a 1 K SNP genotyping panel based on filtering of high-quality genome-wide SNPs from the Musa Germplasm Information System and used it to assess the genetic diversity and population structure of 183 accessions from a Musa spp. germplasm collection containing Philippine and foreign accessions. Targeted GBS using SeqSNP™ technology generated 70,376,284 next-generation sequencing (NGS) reads with an average effective target SNP coverage of 340 × . Bioinformatics pipeline revealed 971 polymorphic SNPs containing 76.9% homozygous calls, 23.1% heterozygous calls and 4% with missing data. A final set of 952 SNPs detected 2,092 alleles. Pairwise genetic distance varied from 0.0021 to 0.3325 with most pairs of accessions distinguished with 250 to 300 loci. The SNP panel was able to detect seven (k = 7) genetically differentiated groups and its composition through Principal Component Analysis (PCA) with k-means clustering algorithm and Discriminant Analysis of Principal Components (DAPC). Accession-specific SNPs were also identified. The 1 K SNP panel effectively distinguishes between genomic groups and provides relatively good resolution of genome-wide nucleotide diversity of Musa spp. This panel is recommended for low-density genotyping for application in marker-assisted breeding and germplasm management, and could be further enhanced to increase marker density for other applications like genetic association and genomic selection in bananas.

Reference-aided full-length transcript assembly, cDNA cloning, and molecular characterization of coronatine-insensitive 1b (COI1b) gene in coconut (Cocos nucifera L.).

BACKGROUND: In the Philippines, 26% of the total agricultural land is devoted to coconut production making coconut one of the most valuable industrial crop in the country. However, the country's multimillion-dollar coconut industry is threatened by the outbreak of coconut scale insect (CSI) and other re-emerging insect pests promoting national research institutes to work jointly on developing new tolerant coconut varieties. Here, we report the cloning and characterization of coronatine-insensitive 1 (COI1) gene, one of the candidate insect defense genes, using 'Catigan Green Dwarf' (CATD) genome sequence assembly as reference. METHODS AND RESULTS: Two (2) splicing variants were identified and annotated-CnCOI1b-1 and CnCOI1b-2. The full-length cDNA of CnCOI1b-1 was 7919 bp with an ORF of 1176 bp encoding for a deduced protein of 391 amino acids while CnCOI1b-2 has 2360 bp full-length cDNA with an ORF of 1743 bp encoding a deduced protein of 580 amino acids. The 3D structural model for the two (2) isoforms were generated through homology modelling. Functional analysis revealed that both isoforms are involved in various physiological and developmental plant processes including defense response of plants to insects and pathogens. Phylogenetic analysis confirms high degree of COI1 protein conservation during evolution, especially among monocot species. Differential gene expression via qRT-PCR analysis revealed a seven-fold increase of COI1 gene expression in coconut post introduction of CSI relative to base levels. CONCLUSION: This study provided the groundwork for further research on the actual role of COI1 in coconut in response to insect damage. The findings of this study are also vital to facilitate the development of improved insect-resistant coconut varieties for vibrant coconut industry.

Mining and validation of novel simple sequence repeat (SSR) markers derived from coconut (Cocos nucifera L.) genome assembly.

BACKGROUND: In the past, simple sequence repeat (SSR) marker development in coconut is achieved through microsatellite probing in bacterial artificial chromosome (BAC) clones or using previously developed SSR markers from closely related genomes. These coconut SSRs are publicly available in published literatures and online databases; however, the number is quite limited. Here, we used a locally established, coconut genome-wide SSR prediction bioinformatics pipeline to generate a vast amount of coconut SSR markers. RESULTS: A total of 7139 novel SSR markers were derived from the genome assembly of coconut 'Catigan Green Dwarf' (CATD). A subset of the markers, amounting to 131, were selected for synthesis based on motif filtering, contig distribution, product size exclusion, and success of in silico PCR in the CATD genome assembly. The OligoAnalyzer tool was also employed using the following desired parameters: %GC, 40-60%; minimum ΔG value for hairpin loop, -0.3 kcal/mol; minimum ΔG value for self-dimer, -0.9 kcal/mol; and minimum ΔG value for heterodimer, -0.9 kcal/mol. We have successfully synthesized, optimized, and amplified 131 novel SSR markers in coconut using 'Catigan Green Dwarf' (CATD), 'Laguna Tall' (LAGT), 'West African Tall' (WAT), and SYNVAR (LAGT × WAT) genotypes. Of the 131 SSR markers, 113 were polymorphic among the analyzed coconut genotypes. CONCLUSION: The development of novel SSR markers for coconut will serve as a valuable resource for mapping of quantitative trait loci (QTLs), assessment of genetic diversity and population structure, hybridity testing, and other marker-assisted plant breeding applications.

Draft Genomes of Six Philippine Erwinia mallotivora Isolates: Comparative Genomics and Genome-Wide Analysis of Candidate Secreted Proteins.

Erwinia mallotivora is one of the most important bacterial pathogens of papaya and causes bacterial crown rot disease in the Philippines. In this paper, we present the draft genome sequences of six Philippine E. mallotivora isolates to provide insights into the genes involved in host-pathogen interactions and compare their genomes to other Erwinia species. The genomes were sequenced using Illumina Miseq platform. The draft whole-genome assemblies of the E. mallotivora isolates are composed of 36-64 contigs with N50 value ranging from 285 to 332 kbp and cover 96.2-100% of the estimated genome size. Structural genome annotation of these assemblies has predicted 4489-4749 protein-coding genes. Comparative genomic analysis using orthologous gene sets led to the identification of conserved genes within the genus and species-specific gene orthologous groups, which collectively provide a baseline for functional genomic studies to determine genes affecting virulence and host specificity. Secreted proteins of E. mallotivora were also predicted and characterized to unravel putative genes involved in plant-pathogen interactions. This study provides the first draft whole-genome sequences of Philippine isolates of E. mallotivora, thus expanding the genomic knowledge for this species in comparison with other members of the genus Erwinia.

Genome-wide SNP and InDel analysis of three Philippine mango species inferred from whole-genome sequencing.

BACKGROUND: The Philippines is among the top 10 major exporters of mango worldwide. However, genomic studies of Philippine mangoes remain largely unexplored and lacking. Here, we sequenced the whole genome of the three Philippine mango species, namely, Mangifera odorata (Huani), Mangifera altissima (Paho), and Mangifera indica "Carabao" variety using Illumina HiSeq 2500, to identify and analyze their genome-wide variants (SNPs and InDels). RESULTS: The high confidence variants were identified by successfully mapping 93-95% of the quality-filtered reads to the Alphonso and Tommy Atkins mango reference genomes. Using these two currently available mango genomes, most variants were observed in M. odorata (4,353,063 and 4,277,287), followed by M. altissima (3,392,763 and 3,449,917), and lastly, M. indica Carabao (2,755,267 and 2,852,480). Approximately 50, 46, and 38% of the variants were unique in the three Philippine mango genomes. The analysis of variant effects and functional annotation across the three mango species revealed 56,982 variants with high-impact effects mapped onto 37,746 genes, of which 25% were found to be novel. The affected mango genes include those with potential economic importance such as 6945 genes for defense/resistance/immune response, 323 genes for fruit development, and 338 genes for anthocyanin production. CONCLUSIONS: To date, this is the first sequencing effort to comprehensively analyze genome-wide variants essential for the development of genome-wide markers specific to these mango species native to the Philippines. This study provides an important genomic resource that can be used for the genetic improvement of mangoes.

Identification and characterization of genome-wide resistance gene analogs (RGAs) of durian (Durio zibethinus L.).

BACKGROUND: Durian (Durio zibethinus L.) is a tropical fruit crop which is popular in Southeast Asia but recently gaining popularity in other parts of the world. In this study, we analyzed the resistance gene analogs (RGAs) of durian through mining of the currently available reference genome of its 'Musang King' cultivar (PRJNA400310). RESULTS: A total of 2586 RGAs were identified in the durian genome consisting of 47 nucleotide binding site proteins (NBS), 158 NBS-leucine rich repeat proteins (NL), 400 coiled-coil NBS-LRR (CNL), 72 toll/interleukin-1 receptor NBS-LRR (TNL), 54 coiled-coil NBS (CN), 10 toll/interleukin-1 receptor NBS (TN), 19 toll/interleukin-1 receptor with unknown domain (TX), 246 receptor-like proteins (RLP), 1,377 receptor-like kinases (RLK), 185 TM-CC, and 18 other NBS-containing proteins with other domains. These RGAs were functionally annotated and characterized via gene ontology (GO) analysis. Among the RGAs with the highest copies in durian genome include the putative disease resistance RPP13-like protein 1, disease resistance protein At4g27190, disease resistance protein RPS6, Probable disease resistance protein At4g27220, and putative disease resistance protein RGA3, while 35 RGAs were found to be novel. Phylogenetic analyses revealed that the genome-wide RGAs were broadly clustered into four major clades based on their domain classification. CONCLUSION: To our knowledge, this is the most comprehensive analysis of durian RGAs which provides a valuable resource for genetic, agronomic, and other biological research of this important tropical fruit crop.

De Novo Genome Sequence Assembly of Dwarf Coconut (Cocos nucifera L. 'Catigan Green Dwarf') Provides Insights into Genomic Variation Between Coconut Types and Related Palm Species.

We report the first whole genome sequence (WGS) assembly and annotation of a dwarf coconut variety, 'Catigan Green Dwarf' (CATD). The genome sequence was generated using the PacBio SMRT sequencing platform at 15X coverage of the expected genome size of 2.15 Gbp, which was corrected with assembled 50X Illumina paired-end MiSeq reads of the same genome. The draft genome was improved through Chicago sequencing to generate a scaffold assembly that results in a total genome size of 2.1 Gbp consisting of 7,998 scaffolds with N50 of 570,487 bp. The final assembly covers around 97.6% of the estimated genome size of coconut 'CATD' based on homozygous k-mer peak analysis. A total of 34,958 high-confidence gene models were predicted and functionally associated to various economically important traits, such as pest/disease resistance, drought tolerance, coconut oil biosynthesis, and putative transcription factors. The assembled genome was used to infer the evolutionary relationship within the palm family based on genomic variations and synteny of coding gene sequences. Data show that at least three (3) rounds of whole genome duplication occurred and are commonly shared by these members of the Arecaceae family. A total of 7,139 unique SSR markers were designed to be used as a resource in marker-based breeding. In addition, we discovered 58,503 variants in coconut by aligning the Hainan Tall (HAT) WGS reads to the non-repetitive regions of the assembled CATD genome. The gene markers and genome-wide SSR markers established here will facilitate the development of varieties with resilience to climate change, resistance to pests and diseases, and improved oil yield and quality.