Nelzo C. Ereful

The Philippines supplies approximately 86% of the global demand for abaca (Musa textilis Née) fiber. To improve its resistance against pathogens, a ba

The Philippines supplies ∼85% of the global demand for abaca ( Musa textilis Née) fiber. A backcross (BC ₂ ) hybrid, named Bandala, was created by crossing Abuab, an abaca variety with high fiber quality, to Pacol, a wild banana ( Musa balbisiana Colla) variety with resistance against Abaca Bunchy Top Virus (ABTV). To assess regulatory difference and expression heterosis, the parents (Abuab and Pacol) and their BC ₂ were sequenced using RNA-seq. Analysis of expression heterosis showed that a large number of transcripts exhibited non-additive (dominance and transgressive) mode, accounting for ∼83.2% of the total heterotic genes. High-parent Abuab dominant genes in the BC ₂ were identified including genes encoding for cellulose synthase. Results indicated that the combined trans and cis + trans (synergistic interaction) largely explain the combined evolutionary divergence between the parents and repeated backcrossing–selection procedures. Genes exhibiting compensatory interaction are significantly enriched under the transgressive mode of inheritance, contributing largely to the heterotic effect in the backcross under directional selection. Further statistical analysis suggests that regulatory differences strongly influence expression heterosis. We further fitted fixed line equations describing each mode of expression inheritance in a 3D plot. In our concurrent work, the BC ₂ demonstrated overdominance over its parents on two economically important phenotypic traits – fiber length and tensile strength. This study demonstrated that phenotypic and expression heteroses are inherent even in backcrosses owing to the presence of the two alleles in the Bandala genome, providing insights into the genetic mechanisms underlying the heterotic performance of BC ₂ and offering valuable directions for abaca breeding. Key Message: A backcross hybrid (BC ₂ ) derived by crossing Musa textilis var. Abuab and M. balbisiana var. Pacol exhibited expression heteroses which can be explained by the underlying cis and/or trans regulatory factors and their interactions.

Unknown to many, the Philippines is host to a few remaining accessions of the underutilized and understudied cereal foxtail millet (Setaria italica (L.) P. Beauv.). We collected together accessions from different eco-geographical locations within the Philippines, along with a few accessions from Lanyu, Taiwan, to undertake a study of the nutritional value and genetic diversity within accessions of foxtail millet grown in the Philippines. All accessions were field grown in 2022, dry season (DS) at the Institute of Plant Breeding (IPB) Experiment Station, Los Baños, Laguna, Philippines. The accessions were tested for micronutrients, including Zn and Fe, nitrogen as a proxy for protein, β-carotene and a number of phenolic compounds with known nutritional potential. Of the 20 accessions tested, the accessions Bayaras and GB61438 had the highest level of Zn (107.1 mg/kg) and Fe (70.52 mg/kg), respectively, higher than levels found in traditional rice varieties. For β-carotene the highest concentration was found in the accession Balles (∼10µg/g). Twelve phenolic compounds were detected, with catechin, syringic acid, ferulic acid and kaempferol having the highest concentrations and greatest variation between accessions. To assess the genetic diversity of these local foxtail millet accessions, we sequenced a core set of eight accessions to a depth of at least 25-fold. Analysis of the population structure, using genome-wide, high-quality SNPs, showed modest diversity among the accession, with two unadmixed groups. The accessions are monophyletic relative to their earliest common ancestor, with the very light brown accessions emerging earlier than the light brown and reddish-brown varieties. Analysis of Zinc/Iron permease (ZIP) transporters within the foxtail millet reference sequence, var. Yugu1 identified 17 putative ZIP transporters. Variant calling identified SNPs primarily within 3’ and 5’ regions, and introns, indicating variation between foxtail millet accessions within regulatory gene regions rather than in structural proteins. The local foxtail millet accessions found across the Philippines, therefore, represent a potential alternative source of nutrition that would help to address the problems of the double-burden of malnutrition found in the Philippines.

The Musa textilis var. Abuab has high fiber quality (FQ) but is susceptible to abaca bunchy top virus (AbBTV); the Musa balbisiana var. Pacol has low FQ but is resistant against AbBTV. Their backcrosses (BC₂ and BC₃) possess both desirable traits. Analysis using RNA-seq showed that the regulatory divergence of Abuab and Pacol is largely explained by cis differences with 27.4% and 22.3% if we are to assess it using BC₂ and BC₃, respectively. Cis differences between the two genotypes are significantly reduced from BC₂ to BC₃ due to changes in genomic constitution. Trans, on the other hand, is robust to changes in allelic composition. All these are attributed to the loss of heterozygosity in BC₃ relative to BC₂. Further analysis showed that both backcrosses exhibited genome-wide preferential expression of Pacol- over Abuab-specific alleles, despite the wider genetic presence of the latter in the hybrids. The ratio of the two genotype-specific expressed transcripts and the ratio of their corresponding genetic make-up are significantly disproportionate, a phenomenon that we refer to here as “genome–transcriptome incongruence”. We also observed preferential expression switching in which several genes prefer the Abuab- (or Pacol-) specific allele in BC₂ but switched to the Pacol- (or Abuab-) specific allele in the BC₃ genome.

The Musa textilis var. Abuab has high fiber quality (FQ) but has low resistance against abaca bunchy top virus (AbBTV); the Musa balbisiana var. Pacol, has low FQ but high resistance against AbBTV. Their backcrosses (BC2 and BC3) possess both desirable traits. Analysis using RNA-seq showed that the regulatory divergence of Abuab and Pacol is largely explained by cis differences with 27.4% and 22.3% if we are to assess it using BC2 and BC3, respectively. Cis differences between the two genotypes are significantly reduced from BC2 to BC3 due to changes in genomic constitution. Trans, on the other hand, is robust to changes in allelic composition. All these are attributed to the loss of heterozygosity in the BC3 relative to BC2. Further analysis showed that both backcrosses exhibited genome-wide preferential expression of Pacol- over Abuab-specific alleles, despite the wider genetic presence of the latter in the hybrids. The ratio of the two genotype-specific expressed transcripts and the ratio of their corresponding genetic make-up are significantly disproportionate, a phenomenon which we refer here as “genome–transcriptome incongruence”. We also observed preferential expression switching in which several genes prefer Abuab- (or Pacol-) specific allele in the BC2 but switched to Pacol- (or Abuab-) specific allele in the BC3 genome.

Despite the importance of and current demand for abaca (Musa textilis Nee) fiber, there has been limited study that capitalizes on RNA-seq to identify candidate genes associated with high fiber quality and bunchy top virus (AbBTV) resistance. Three varieties (Abuab, Inosa, and Tangongon), one wild banana variety (Musa balbisiana Colla) Pacol, and two developed backcrosses (Abuab × Pacol BC₂ and BC₃) were grown at the Institute of Plant Breeding (IPB), Laguna, Philippines. The pseudostems of 3-month-old suckers of each genotype were sampled for RNA-seq. Datasets were analyzed for differential expression (DE) implementing various model frameworks, including pairwise, genotypic and non-DE models. Results indicate that Abuab and BC₃ induce the highest proportion (70%) of abaca-specific genes. Gene ontology (GO) enrichment analysis showed several genes associated with cellulose synthase activity, callose synthase, ß-glucosidase activity, glucan biosynthetic process, etc. KEGG pathway analysis showed several genes encoding for enzymes involved in the lignin biosynthetic pathway. Analysis using genotypic DE (GDE) between abaca bunchy top virus (AbBTV)-resistant and -susceptible groups revealed genes such as pathogenesis-related protein and NBS-LRR. As the genotypes were not infected with the pathogen, these genes are yet to be confirmed for their roles in disease resistance and are an interesting subject for further investigation.

The indica ecotypes, IR64, an elite drought-susceptible variety adapted to irrigated ecosystem, and Apo (IR55423-01 or NSIC RC9), a moderate drought-tolerant upland genotype together with their hybrid (IR64 × Apo) were exposed to non- and water-stress conditions. By sequencing (RNA-seq) these genotypes, we were able to map genes diverging in cis and/or trans factors. Under non-stress condition, cis dominantly explains (11.2%) regulatory differences, followed by trans (8.9%). Further analysis showed that water-limiting condition largely affects trans and cis + trans factors. On the molecular level, cis and/or trans regulatory divergence explains their genotypic differences and differential drought response. Between the two parental genotypes, Apo appears to exhibit more photosynthetic efficiency even under water-limiting condition and is ascribed to trans. Statistical analyses showed that regulatory divergence is significantly influenced by environmental conditions. Likewise, the mode of parental expression inheritance which drives heterosis (HET) is significantly affected by environmental conditions indicating the malleability of heterosis to external factors. Further analysis revealed that the HET class, dominance, was significantly enriched under water-stress condition. We also identified allelic imbalance switching in which several genes prefer IR64- (or Apo-) specific allele under non-stress condition but switched to Apo- (or IR64-) specific allele when exposed to water-stress condition.

Two indica inbred rice lines, IR64, a drought-sensitive, and Apo, a moderately drought-tolerant genotype, were exposed to non- (control or unstressed) and water-stress treatments. Leaf samples collected at an early flowering stage were sequenced by RNA-seq. Reads generated were analyzed for differential expression (DE) implementing various models in baySeq to capture differences in genome-wide transcriptional response under contrasting water regimes. IR64, the drought-sensitive variety consistently exhibited a broader transcriptional response while Apo showed relatively modest transcriptional changes under water-stress conditions across all models implemented. Gene ontology (GO) and KEGG pathway analyses of genes revealed that IR64 showed enhancement of functions associated with signal transduction, protein binding and receptor activity. Apo uniquely showed significant enrichment of genes associated with an oxygen binding function and peroxisome pathway. In general, IR64 exhibited more extensive molecular re-programming, presumably, a highly energy-demanding route to deal with the abiotic stress. Several of these differentially expressed genes (DEGs) were found to co-localize with QTL marker regions previously identified to be associated with drought-yield response, thus, are the most promising candidate genes for further studies.