Genome and transcriptome exploration reveals receptor-like kinases as potential resistance gene analogs against bacterial blight in pomegranate.

BACKGROUND: Pomegranate (Punica granatum L.) is a tropical fruit crop of pharma-nutritional importance. However, it faces farming challenges due to pests and diseases, particularly bacterial blight and wilt. Developing resistant cultivars is crucial for sustainable pomegranate cultivation, and understanding resistance's genetic basis is essential. METHODS AND RESULTS: We used an extensive resistance gene analogues (RGA) prediction tool to identify 958 RGAs, classified into Nucleotide Binding Site-leucine-rich repeat (NBS-LRR) proteins, receptor-like kinases (RLKs), receptor-like proteins (RLPs), Transmembrane coiled-coil (TM-CC), and nine non-canonical RGAs. RGAs were distributed across all eight chromosomes, with chromosome 02 containing the most RGAs (161), and chromosome 08 having the highest density (4.42 RGA/Mb). NBS-LRR genes were predominantly present on chromosomes 08 and 02, whereas RLKs and RLPs were primarily located on chromosomes 04 and 07. Gene ontology analysis revealed that 475 RGAs were associated with defence against various biotic stresses. Using RNAseq, we identified 120 differentially expressed RGAs, with RLKs (74) being prominent among the differentially expressed genes. CONCLUSION: The discovery of these RGAs is a significant step towards breeding pomegranates for pest and disease resistance. The differentially expressed RLKs hold promise for developing resistant cultivars against bacterial blight, thereby contributing to the sustainability of pomegranate cultivation.

Comprehensive analysis of physiological and metabolomic responses to drought reveals specific modulation of acquired tolerance mechanisms in rice.

Mild stresses induce "acquired tolerance traits" (ATTs) that provide tolerance when stress becomes severe. Here, we identified the genetic variability in ATTs among a panel of rice germplasm accessions and demonstrated their relevance in protecting growth and productivity under water-limited conditions. Diverse approaches, including physiological screens, association mapping and metabolomics, were adopted and revealed 43 significant marker-trait associations. Nontargeted metabolomic profiling of contrasting genotypes revealed 26 "tolerance-related-induced" primary and secondary metabolites in the tolerant genotypes (AC-39000 and AC-39020) compared to the susceptible one (BPT-5204) under water-limited condition. Metabolites that help maintain cellular functions, especially Calvin cycle processes, significantly accumulated more in tolerant genotypes, which resulted in superior photosynthetic capacity and hence water use efficiency. Upregulation of the glutathione cycle intermediates explains the ROS homeostasis among the tolerant genotypes, maintaining spikelet fertility, and grain yield under stress. Bioinformatic dissection of a major effect quantitative trait locus on chromosome 8 revealed genes controlling metabolic pathways leading to the production of osmolites and antioxidants, such as GABA and raffinose. The study also led to the identification of specific trait donor genotypes that can be effectively used in translational crop improvement activities.

Identification of suitable reference genes for expression studies in pomegranate under different biotic and abiotic stress conditions.

Pomegranate (Punica granatum L.) is an important economic fruit crop, facing many biotic and abiotic challenges during cultivation. Several research programs are in progress to understand both biotic and abiotic stress factors and mitigate these challenges using gene expression studies based on the qPCR approach. However, research publications are not available yet to select the standard reference gene for normalizing target gene expression values in pomegranate. The most suitable candidate reference gene is required to ensure precise and reliable results for qPCR analysis. Eight candidate reference genes' stability was evaluated under different stress conditions using different algorithms such as ∆Ct, geNorm, BestKeeper, NormFinder, and RefFinder. The various algorithms revealed that EFA1 and 18S rRNA were common and most stable reference genes (RGs) under abiotic and wilt stress. Whereas comprehensive ranking by RefFinder showed GAPDH and CYPF were the most stable RGs under combined biotic (pooled samples of all biotic stress) and bacterial blight samples. For normalizing target gene expression under wilt, nematode, bacterial blight, and abiotic stress conditions both GAPDH and CYPFreference genes are adequate for qPCR. The above data provide comprehensive details for the selection of a candidate reference gene in various stresses in pomegranate.

Differential gene responses in different varieties of pomegranate during the pathogenesis of Xanthomonas axonopodis pv. punicae.

Bacterial blight (BB) caused by Xanthomonas axonopodis pv. punicae (Xap) is the major scourge in pomegranate cultivation leading to an extensive yield loss up to 60-80%. Hence, identifying a novel resistance source for BB is very necessary for developing a suitable management strategy. Host range analysis and cross-inoculation studies revealed that Xap is specific to pomegranate and there are no alternative hosts to the pathogen. Screening of 149 accessions recorded the varied disease resistance levels with mean disease severity of 30.67%. Accession lines IC318735, IC318724, and IC318762 exhibited maximum disease tolerance by exhibiting the lowest disease severity of 4.91, 5.66, and 6.82%, respectively. Comparative expression analysis of defence genes in IC318724 and IC318735 recorded significant upregulation of phenylalanine ammonia-lyase (PAL), callose synthase-3 (CS3), chitinase, pathogenesis-related protein-1 (PR1), and pathogenesis-related protein-10 (PR10), indicating these genes might be actively involved in conferring disease tolerance. Abiotic elicitors were tested to induce systemic resistance in agronomically superior and widely adapted variety Bhagwa for managing BB of pomegranate. Among the various elicitors tested; proline (600 ppm), gamma-aminobutyric acid (600 ppm), chitosan (600 ppm), ß-aminobutyric acid (200 ppm), laminarin (600 ppm), and eugenol (200 ppm) recorded maximum disease protection in prophylactic treatment with disease protection of 89.59, 88.59, 87.15, 86.08, 81.05, and 78.72%, respectively. Similar observations were recorded when these were applied as curative treatment. The present study will broaden our understanding of host-pathogen interactions during BB infection in pomegranate, also aid in developing ideal approach for developing effective disease management. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02721-y.

Reliable and early diagnosis of bacterial blight in pomegranate caused by Xanthomonas axonopodis pv. punicae using sensitive PCR techniques.

Bacterial blight caused by Xanthomonas axonopodis pv. punicae is a major disease of pomegranate. Bacterial blight drastically reduces the yield and quality of fruits, which are critical for pomegranate production. Precise and early diagnosis of bacterial blight is crucial for active surveillance and effective management of the disease. Symptoms based disease diagnostic methods are labor-intensive, time-consuming and may not detect disease on asymptomatic plants. DNA-based disease diagnostics using polymerase chain reaction (PCR) are reliable, precise, accurate and quick. PCR coupled with agarose gel electrophoresis (PCR-AGE), PCR coupled with capillary electrophoresis (PCR-CE) and real-time PCR (qPCR) were applied for the early and accurate diagnosis of bacterial blight in pomegranate. PCR-CE and qPCR were capable of diagnosing bacterial blight 6 to 10 days before symptom appearance, with detection limits of 100 fg and 10 fg of bacterial DNA respectively. However, conventional PCR-AGE detected pathogen at the onset of disease symptoms with a detection limit of 10 pg of bacterial DNA. qPCR detected bacterial blight in orchards that did not show any disease symptoms. Our data demonstrate that qPCR is more sensitive than other PCR methods along with being reliable for early diagnosis.

Introgression of Root and Water Use Efficiency Traits Enhances Water Productivity: An Evidence for Physiological Breeding in Rice (Oryza sativa L.).

BACKGROUND: Semi-irrigated aerobic cultivation of rice has been suggested as a potential water saving agronomy. However, suitable cultivars are needed in order to sustain yield levels. An introgression of water mining and water use efficiency (WUE) traits is the most appropriate strategy for a comprehensive genetic enhancement to develop such rice cultivars. RESULTS: We report a novel strategy of phenotyping and marker-assisted backcross breeding to introgress water mining (root) and water use efficiency (WUE) traits into a popular high yielding cultivar, IR-64. Trait donor genotypes for root (AC-39020) and WUE (IET-16348) were crossed separately and the resultant F1s were inter-mated to generate double cross F1s (DCF1). Progenies of three generations of backcross followed by selfing were charatcerised for target phenotype and genome integration. A set of 260 trait introgressed lines were identified. Root weight and root length of TILs were 53% and 23.5% higher, while Δ13C was 2.85‰ lower indicating a significant increase in WUE over IR-64. Five best TILs selected from BC3F3 generation showed 52% and 63% increase in yield over IR-64 under 100% and 60% FC, respectively. The trait introgressed lines resembled IR64 with more than 97% of genome recovered with a significant yield advantage under semi-irrigated aerobic conditions The study validated markers identified earlier by association mapping. CONCLUSION: Introgression of root and WUE into IR64, resulted in an excellent yield advantage even when cultivated under semi-irrigated aerobic condition. The study provided a proof-of-concept that maintaining leaf turgor and carbon metabolism results in improved adaptation to water limited conditions and sustains productivity. A marker based multi-parent backcross breeding is an appropriate approach for trait introgression. The trait introgressed lines developed can be effectively used in future crop improvement programs as donor lines for both root and WUE.