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Abstract Rice blast disease is the most devastating disease constraining crop productivity. Vertical resistance to blast disease is widely studied despite its instability. Clusters of genes or QTLs conferring blast resistance that offer durable horizontal resistance are important in resistance breeding. In this study, we aimed to refine the reported QTLs and identify stable meta-QTLs (MQTLs) associated with rice blast resistance. A total of 435 QTLs were used to project 71 MQTLs across all the rice chromosomes. As many as 199 putative rice blast resistance genes were identified within 53 MQTL regions. The genes included 48 characterized resistance gene analogs and related proteins, such as NBS–LRR type, LRR receptor-like kinase, NB-ARC domain, pathogenesis-related TF/ERF domain, elicitor-induced defense and proteins involved in defense signaling. MQTL regions with clusters of RGA were also identified. Fifteen highly significant MQTLs included 29 candidate genes and genes characterized for blast resistance, such as Piz, Nbs-Pi9, pi55-1, pi55-2, Pi3/Pi5-1, Pi3/Pi5-2, Pikh, Pi54, Pik/Pikm/Pikp, Pb1 and Pb2. Furthermore, the candidate genes (42) were associated with differential expression (in silico) in compatible and incompatible reactions upon disease infection. Moreover, nearly half of the genes within the MQTL regions were orthologous to those in O. sativa indica, Z. mays and A. thaliana, which confirmed their significance. The peak markers within three significant MQTLs differentiated blast-resistant and susceptible lines and serve as potential surrogates for the selection of blast-resistant lines. These MQTLs are potential candidates for durable and broad-spectrum rice blast resistance and could be utilized in blast resistance breeding.

Introduction: Nodulin-26-like intrinsic proteins (NIPs) are integral membrane proteins belonging to the aquaporin family, that facilitate the transport of neutral solutes across the bilayer. The OsNIP2;1 a member of NIP-III class of aquaporins is permeable to beneficial elements like silicon and hazardous arsenic. However, the atomistic cross-talk of these molecules traversing the OsNIP2;1 channel is not well understood. Objective: Due to the lack of genomic variation but the availability of high confidence crystal structure, this study aims to highlight structural determinants of metalloid permeation through OsNIP2;1. Methods: The molecular simulations, combined with site-directed mutagenesis were used to probe the role of specific residues in the metalloid transport activity of OsNIP2;1. Results: We drew energetic landscape of OsNIP2;1, for silicic and arsenous acid transport. Potential Mean Force (PMF) construction illuminate three prominent energetic barriers for metalloid passage through the pore. One corresponds to the extracellular molecular entry in the channel, the second located on ar/R filter, and the third size constriction in the cytoplasmic half. Comparative PMF for silicic acid and arsenous acid elucidate a higher barrier for silicic acid at the cytoplasmic constrict resulting in longer residence time for silicon. Furthermore, our simulation studies explained the importance of conserved residues in loop-C and loop-D with a direct effect on pore dynamics and metalloid transport. Next we assessed contribution of predicted key residues for arsenic uptake, by functional complementation in yeast. With the aim of reducing arsenic uptake while maintaining beneficial elements uptake, we identified novel OsNIP2;1 mutants with substantial reduction in arsenic uptake in yeast. Conclusion: We provide a comprehensive assessment of pore lining residues of OsNIP2;1 with respect to metalloid uptake. The findings will expand mechanistic understanding of aquaporin’s metalloid selectivity and facilitate variant interpretation to develop novel alleles with preference for beneficial metalloid species and reducing hazardous ones.

Measurement(s) Genome Assembly Sequence Technology Type(s) PacBio Sequel System, Illumina sequencing Factor Type(s) Lathyrus Pusa-24 Sample Characteristic - Organism Lathyrus sativus Sample Characteristic - Environment leaf Sample Characteristic - Location India

von Willebrand factor A (vWA) genes are well characterized in humans except for few BONZAI genes, but the vWA genes are least explored in plants. Considering the novelty and vital role of vWA genes, this study aimed at characterization of vWA superfamily in rice. Rice genome was found to have 40 vWA genes distributed across all the 12 chromosomes, and 20 of the 40 vWA genes were unique while the remaining shared large fragment similarities with each other, indicating gene duplication. In addition to vWA domain, vWA proteins possess other different motifs or domains, such as ubiquitin interacting motif in protein degradation pathway, and RING finger in protein-protein interaction. Expression analysis of vWA genes in available expression data suggested that they probably function in biotic and abiotic stress responses including hormonal response and signaling. The frequency of transposon elements in the entire 3K rice germplasm was negligible except for 9 vWA genes, indicating the importance of these genes in rice. Structural and functional diversities showed that the vWA genes in a blast-resistant rice variety Tetep had huge variations compared to blast-susceptible rice varieties HP2216 and Nipponbare. qRT-PCR analysis of vWA genes in Magnaporthe oryzae infected rice tissues indicated OsvWA9, OsvWA36, OsvWA37 and OsvWA18 as the optimal candidate genes for disease resistance. This is the first attempt to characterize vWA gene family in plant species.

Abstract Galactomannan is a polymer of high economic importance and is extracted from the seed endosperm of clusterbean (C. tetragonoloba). In the present study, we worked to reveal the stage-specific galactomannan biosynthesis and its regulation in clusterbean. Combined electron microscopy and biochemical analysis revealed high protein and gum content in RGC-936, while high oil bodies and low gum content in M-83. A comparative transcriptome study was performed between RGC-936 (high gum) and M-83 (low gum) varieties at three developmental stages viz. 25, 39, and 50 days after flowering (DAF). Total 209,525, 375,595 and 255,401 unigenes were found at 25, 39 and 50 DAF respectively. Differentially expressed genes (DEGs) analysis indicated a total of 5147 shared unigenes between the two genotypes. Overall expression levels of transcripts at 39DAF were higher than 50DAF and 25DAF. Besides, 691 (RGC-936) and 188 (M-83) candidate unigenes that encode for enzymes involved in the biosynthesis of galactomannan were identified and analyzed, and 15 key enzyme genes were experimentally validated by quantitative Real-Time PCR. Transcription factor (TF) WRKY was observed to be co-expressed with key genes of galactomannan biosynthesis at 39DAF. We conclude that WRKY might be a potential biotechnological target (subject to functional validation) for developing high gum content varieties.

Abstract Based upon the morphological characteristics, tea is classified botanically into 2 main types i.e. Assam and China, which are morphologically very distinct. Further, they are so easily pollinated among themselves, that a third category, Cambod type is also described. Although the general consensus of origin of tea is India, Burma and China adjoining area, yet specific origin of China and Assam type tea are not yet clear. Thus, we made an attempt to understand the origin of Indian tea through the comparative analysis of different chloroplast (cp) genomes under the Camellia genus by performing evolutionary study and comparing simple sequence repeats (SSRs) and codon usage distribution patterns among them. The Cp genome based phylogenetic analysis indicated that Indian Tea, TV1 formed a different group from that of China tea, indicating that TV1 might have undergone different domestications and hence owe different origins. The simple sequence repeats (SSRs) analysis and codon usage distribution patterns also supported the clustering order in the cp genome based phylogenetic tree.

Melatonin is an indolic compound derived from tryptophan and has an important role in the general processes of plants including germination, photosynthesis, circadian rhythms, flowering, senescence, and alleviating stress. Integrating melatonin application to agronomical practices reportedly provides numerous benefits. More particularly seed priming with melatonin has been shown to improve seed germination as well as improve resilience under adverse conditions. This review discusses in detail the studies showing elevated drought, salinity, heat, and heavy metal stress resistance as a result of melatonin seed priming. Differences in transcript and protein levels have been observed in plants/seedlings germinated via melatonin primed seeds, providing the foundation for further studies. Many pieces of research have also shown primed seeds possess a greater content of osmoprotectants, upregulation of energy-producing pathways, and an increase in protein content, which may lead to enhancing abiotic stress response in plants. The primary function attributed to melatonin is reactive oxygen species scavenging, which consequently leads to enhancing plant stress resistance against multiple stresses. Here, we highlight all the potential mechanisms involved in melatonin meditated induction of abiotic stress tolerance. We review the reports emphasizing the role played by melatonin under stress conditions in multiple crop species, the modification at genome, ribonucleic acid, and protein level involved, and the underlying mechanisms. Information provided here will be helpful to better understand the melatonin role in plants and its efficient exploration in the cropping system.

Soybean with enriched nutrients has emerged as a prominent source of edible oil and protein. In the present study, a meta-analysis was performed by integrating quantitative trait loci (QTLs) information, region-specific association and transcriptomic analysis. Analysis of about a thousand QTLs previously identified in soybean helped to pinpoint 14 meta-QTLs for oil and 16 meta-QTLs for protein content. Similarly, region-specific association analysis using whole genome re-sequenced data was performed for the most promising meta-QTL on chromosomes 6 and 20. Only 94 out of 468 genes related to fatty acid and protein metabolic pathways identified within the meta-QTL region were found to be expressed in seeds. Allele mining and haplotyping of these selected genes were performed using whole genome resequencing data. Interestingly, a significant haplotypic association of some genes with oil and protein content was observed, for instance, in the case of FAD2-1B gene, an average seed oil content of 20.22% for haplotype 1 compared to 15.52% for haplotype 5 was observed. In addition, the mutation S86F in the FAD2-1B gene produces a destabilizing effect of (ΔΔG Stability) −0.31 kcal/mol. Transcriptomic analysis revealed the tissue-specific expression of candidate genes. Based on their higher expression in seed developmental stages, genes such as sugar transporter, fatty acid desaturase (FAD), lipid transporter, major facilitator protein and amino acid transporter can be targeted for functional validation. The approach and information generated in the present study will be helpful in the map-based cloning of regulatory genes, as well as for marker-assisted breeding in soybean.

Alternaria brassicae is an important necrotrophic pathogen that infects the Brassicaceae family. A. brassicae, like other necrotrophs, also secretes various proteinaceous effectors and metabolites that cause cell death to establish itself in the host. However, there has been no systematic study of A. brassicae effectors and their roles in pathogenesis. The availability of the genome sequence of A. brassicae in public domain has enabled the search for effectors and their functional characterization. Nep1-like proteins (NLPs) are a superfamily of proteins that induce necrosis and ethylene biosynthesis. They have been reported from a variety of microbes including bacteria, fungi, and oomycetes. In this study, we identified two NLPs from A. brassicae viz. AbrNLP1 and AbrNLP2 and functionally characterized them. Although both AbrNLPs were found to be secretory in nature, they localized differentially inside the plant. AbrNLP2 was found to induce necrosis in both host and non-host species, while AbrNLP1 could not induce necrosis in both species. Additionally, AbrNLP2 was shown to induce pathogen-associated molecular pattern (PAMP)-triggered immunity in both host and non-host species. Overall, our study indicates that AbrNLPs are functionally and spatially (subcellular location) distinct and may play different but important roles during the pathogenesis of A. brassicae.

Silicon (Si) is gaining widespread attention due to its prophylactic activity to protect plants under stress conditions. Despite Si’s abundance in the earth’s crust, most soils do not have enough soluble Si for plants to absorb. In the present study, a silicate-solubilizing bacterium, Enterobacter sp. LR6, was isolated from the rhizospheric soil of rice and subsequently characterized through whole-genome sequencing. The size of the LR6 genome is 5.2 Mb with a GC content of 54.9% and 5182 protein-coding genes. In taxogenomic terms, it is similar to E. hormaechei subsp. xiangfangensis based on average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH). LR6 genomic data provided insight into potential genes involved in stress response, secondary metabolite production, and growth promotion. The LR6 genome contains two aquaporins, of which the aquaglyceroporin (GlpF) is responsible for the uptake of metalloids including arsenic (As) and antimony (Sb). The yeast survivability assay confirmed the metalloid transport activity of GlpF. As a biofertilizer, LR6 isolate has a great deal of tolerance to high temperatures (45 °C), salinity (7%), and acidic environments (pH 9). Most importantly, the present study provides an understanding of plant-growth-promoting activity of the silicate-solubilizing bacterium, its adaptation to various stresses, and its uptake of different metalloids including As, Ge, and Si.

Rice is a global food grain crop for more than one-third of the human population and a source for food and nutritional security. Rice production is subjected to various stresses; blast disease caused by Magnaporthe oryzae is one of the major biotic stresses that has the potential to destroy total crop under severe conditions. In the present review, we discuss the importance of rice and blast disease in the present and future global context, genomics and molecular biology of blast pathogen and rice, and the molecular interplay between rice–M. oryzae interaction governed by different gene interaction models. We also elaborated in detail on M. oryzae effector and Avr genes, and the role of noncoding RNAs in disease development. Further, rice blast resistance QTLs; resistance (R) genes; and alleles identified, cloned, and characterized are discussed. We also discuss the utilization of QTLs and R genes for blast resistance through conventional breeding and transgenic approaches. Finally, we review the demonstrated examples and potential applications of the latest genome-editing tools in understanding and managing blast disease in rice.

Nutritional quality improvement of rice is the key to ensure global food security. Consequently, enormous efforts have been made to develop genomics and transcriptomics resources for rice. The available omics resources along with the molecular understanding of trait development can be utilized for efficient exploration of genetic resources for breeding programs. In the present study, 80 genes known to regulate the nutritional and cooking quality of rice were extensively studied to understand the haplotypic variability and gene expression dynamics. The haplotypic variability of selected genes were defined using whole-genome re-sequencing data of ~4700 diverse genotypes. The analytical workflow identified 133 deleterious single-nucleotide polymorphisms, which are predicted to affect the gene function. Furthermore, 788 haplotype groups were defined for 80 genes, and the distribution and evolution of these haplotype groups in rice were described. The nucleotide diversity for the selected genes was significantly reduced in cultivated rice as compared with that in wild rice. The utility of the approach was successfully demonstrated by revealing the haplotypic association of chalk5 gene with the varying degree of grain chalkiness. The gene expression atlas was developed for these genes by analyzing RNA-Seq transcriptome profiling data from 102 independent sequence libraries. Subsequently, weighted gene co-expression meta-analyses of 11,726 publicly available RNAseq libraries identified 19 genes as the hub of interactions. The comprehensive analyses of genetic polymorphisms, allelic distribution, and gene expression profiling of key quality traits will help in exploring the most desired haplotype for grain quality improvement. Similarly, the information provided here will be helpful to understand the molecular mechanism involved in the development of nutritional and cooking quality traits in rice.

Mungbean (Vigna radiata L. Wilczek) is an annual grain legume crop affected by low availability of phosphorus. Phosphorus deficiency mainly affects the growth and development of plants along with changes in root morphology and increase in root-to-shoot ratio. Deciphering the genetic basis of phosphorus use efficiency (PUE) traits can benefit our understanding of mungbean tolerance to low-phosphorus condition. To address this issue, 144 diverse mungbean genotypes were evaluated for 12 PUE traits under hydroponics with optimum- and low-phosphorus levels. The broad sense heritability of traits ranged from 0.63 to 0.92 and 0.58 to 0.92 under optimum- and low-phosphorus conditions, respectively. This study, reports for the first time such a large number of genome wide Single nucleotide polymorphisms (SNPs) (76,160) in mungbean. Further, genome wide association study was conducted using 55,634 SNPs obtained by genotyping-by-sequencing method. The results indicated that total 136 SNPs shared by both GLM and MLM models were associated with tested PUE traits under different phosphorus regimes. We have identified SNPs with highest p value (–log10(p)) for some traits like, TLA and RDW with p value (–log10(p)) of more than 6.0 at LP/OP and OP condition. We have identified nine SNPs (three for TLA and six for RDW trait) which was found to be present in chromosomes 8, 4, and 7. One SNP present in Vradi07g06230 gene contains zinc finger CCCH domain. In total, 71 protein coding genes were identified, of which 13 genes were found to be putative candidate genes controlling PUE by regulating nutrient uptake and root architectural development pathways in mungbean. Moreover, we identified three potential candidate genes VRADI11G08340, VRADI01G05520, and VRADI04G10750 with missense SNPs in coding sequence region, which results in significant variation in protein structure at tertiary level. The identified SNPs and candidate genes provide the essential information for genetic studies and marker-assisted breeding program for improving low-phosphorus tolerance in mungbean.

Microalgae, due to their complex metabolic capacity, are being continuously explored for nutraceuticals, pharmaceuticals, and other industrially important bioactives. However, suboptimal yield and productivity of the bioactive of interest in local and robust wild-type strains are of perennial concerns for their industrial applications. To overcome such limitations, strain improvement through genetic engineering could play a decisive role. Though the advanced tools for genetic engineering have emerged at a greater pace, they still remain underused for microalgae as compared to other microorganisms. Pertaining to this, we reviewed the progress made so far in the development of molecular tools and techniques, and their deployment for microalgae strain improvement through genetic engineering. The recent availability of genome sequences and other omics datasets form diverse microalgae species have remarkable potential to guide strategic momentum in microalgae strain improvement program. This review focuses on the recent and significant improvements in the omics resources, mutant libraries, and high throughput screening methodologies helpful to augment research in the model and non-model microalgae. Authors have also summarized the case studies on genetically engineered microalgae and highlight the opportunities and challenges that are emerging from the current progress in the application of genome-editing to facilitate microalgal strain improvement. Toward the end, the regulatory and biosafety issues in the use of genetically engineered microalgae in commercial applications are described.

The first draft of the pigeonpea (Cajanus cajan (L.) Millsp. cv. Asha) genome with 511 Mbp of assembled sequence information has low genome coverage of about sixty percent. Here we present an improved version of this genome with 648.2 Mbp of assembled sequence of this popular pigeonpea variety, which is liked by the millers and has resistance to fusarium wilt and sterility mosaic diseases. With the addition of 137 Mb of assembled sequence information the present version has the highest available genome coverage of pigeonpea till date. We predicted 56,888 protein-coding genes of which 54,286 (96.7%) were functionally annotated. In the improved genome assembly we identified 158,432 SSR loci, designed flanking primers for 85,296 of these and validated them in-silico by e-PCR. The raw data used for the improvement of genome assembly are available in the SRA database of NCBI with accession numbers SRR5922904, SRR5922905, SRR5922906, SRR5922907. The genome sequence update has been deposited at DDBJ/EMBL/GenBank under the accession AFSP00000000, and the version described in this paper is the second version (AFSP02000000).

Diseases caused by Puccinia graminis are some of the most devastating diseases of wheat. Extensive genomic understanding of the pathogen has proven helpful not only in understanding host- pathogen interaction but also in finding appropriate control measures. In the present study, whole-genome sequencing of four diverse P. graminis pathotypes was performed to understand the genetic variation and evolution. An average of 63.5 Gb of data per pathotype with about 100× average genomic coverage was achieved with 100-base paired-end sequencing performed with Illumina Hiseq 1000. Genome structural annotations collectively predicted 9273 functional proteins including ~583 extracellular secreted proteins. Approximately 7.4% of the genes showed similarity with the PHI database which is suggestive of their significance in pathogenesis. Genome-wide analysis demonstrated pathotype 117-6 as likely distinct and descended through a different lineage. The 3–6% more SNPs in the regulatory regions and 154 genes under positive selection with their orthologs and under negative selection in the other three pathotypes further supported pathotype 117-6 to be highly diverse in nature. The genomic information generated in the present study could serve as an important source for comparative genomic studies across the genus Puccinia and lead to better rust management in wheat.

Abstract Background Genome-wide microarray has enabled development of robust databases for functional genomics studies in rice. However, such databases do not directly cater to the needs of breeders. Here, we have attempted to develop a web interface which combines the information from functional genomic studies across different genetic backgrounds with DNA markers so that they can be readily deployed in crop improvement. In the current version of the database, we have included drought and salinity stress studies since these two are the major abiotic stresses in rice. Results RiceMetaSys, a user-friendly and freely available web interface provides comprehensive information on salt responsive genes (SRGs) and drought responsive genes (DRGs) across genotypes, crop development stages and tissues, identified from multiple microarray datasets. ‘Physical position search’ is an attractive tool for those using QTL based approach for dissecting tolerance to salt and drought stress since it can provide the list of SRGs and DRGs in any physical interval. To identify robust candidate genes for use in crop improvement, the ‘common genes across varieties’ search tool is useful. Graphical visualization of expression profiles across genes and rice genotypes has been enabled to facilitate the user and to make the comparisons more impactful. Simple Sequence Repeat (SSR) search in the SRGs and DRGs is a valuable tool for fine mapping and marker assisted selection since it provides primers for survey of polymorphism. An external link to intron specific markers is also provided for this purpose. Bulk retrieval of data without any limit has been enabled in case of locus and SSR search. Conclusions The aim of this database is to facilitate users with a simple and straight-forward search options for identification of robust candidate genes from among thousands of SRGs and DRGs so as to facilitate linking variation in expression profiles to variation in phenotype. Database URL: http://14.139.229.201

Here, we present the draft genome (377.3 Mbp) of Corchorus olitorious cv. JRO-524 (Navin), which is a leading dark jute variety developed from a cross between African (cv. Sudan Green) and indigenous (cv. JRO-632) types. We predicted from the draft genome a total of 57,087 protein-coding genes with annotated functions. We identified a large number of 1765 disease resistance-like and defense response genes in the jute genome. The annotated genes showed the highest sequence similarities with that of Theobroma cacao followed by Gossypium raimondii. Seven chromosome-scale genetically anchored pseudomolecules were constructed with a total size of 8.53 Mbp and used for synteny analyses with the cocoa and cotton genomes. Like other plant species, gypsy and copia retrotransposons were the most abundant classes of repeat elements in jute. The raw data of our study are available in SRA database of NCBI with accession number SRX1506532. The genome sequence has been deposited at DDBJ/EMBL/GenBank under the accession LLWS00000000, and the version described in this paper will be the first version (LLWS01000000).

Magnaporthe oryzae is one of the fungal pathogens of rice which results in heavy yield losses worldwide. Understanding the genomic structure of M. oryzae is essential for appropriate deployment of the blast resistance in rice crop improvement programs. In this study we sequenced two M. oryzae isolates, RML-29 (avirulent) and RP-2421 (highly virulent) and performed comparative study along with three publically available genomes of 70-15, P131, and Y34. We identified several candidate effectors (>600) and isolate specific sequences from RML-29 and RP-2421, while a core set of 10013 single copy orthologs were found among the isolates. Pan-genome analysis showed extensive presence and absence variations (PAVs). We identified isolate-specific genes across 12 isolates using the pan-genome information. Repeat analysis was separately performed for each of the 15 isolates. This analysis revealed ∼25 times higher copy number of short interspersed nuclear elements (SINE) in virulent than avirulent isolate. We conclude that the extensive PAVs and occurrence of SINE throughout the genome could be one of the major mechanisms by which pathogenic variability is emerging in M. oryzae isolates. The knowledge gained in this comparative genome study can provide understandings about the fungal genome variations in different hosts and environmental conditions, and it will provide resources to effectively manage this important disease of rice.

Metal Tolerance Proteins (MTPs) are the class of membrane proteins involved in the transport of metals, mainly Zn, Mn, Fe, Cd, Co and Ni, and confer metal tolerance in plants. In the present study, a comprehensive molecular analysis of rice MTP genes was performed to understand the evolution, distribution and expression dynamics of MTP genes. Exploration of the whole genome re-sequencing information available for three thousand rice genotypes highlighted the evolution and allelic diversity of MTP genes. Based on the presence of non-synonymous single nucleotide polymorphism (SNP), MTP1, MTP6, MTP8 and MTP9 were found to be the most conserved genes. Furthermore, results showed localization of MTP1, MTP8.1 and MTP9, and MTP11, respectively with QTLs/m-QTLs for Zn and Cd accumulation, making these genes promising candidates to understand the QTL regulation. Expression profiling of the entire set of 10 MTP genes revealed root and shoot specific expressions of MTP9 and MTP8.1, respectively, under all tested vegetative stages. Expression of seed-specific MTPs increased as seed maturation progressed, which revealed their potential role in transporting metals during seed filling. Upon exposure to harmful heavy metals, expression of most MTP genes decreased in root and increased in shoot, suggests that different mechanisms are being employed by MTPs in different tissues. Contrastingly, only a few MTPs were found to be responsive to Fe and/or Zn starvation conditions. The extensive analysis of MTPs presented here will be helpful in identifying candidate MTP genes for crop biofortification and bioremediation purposes.

Rice blast, caused by the ascomycete fungus Magnaporthe oryzae is a destructive disease of rice and responsible for causing extensive damage to the crop. Pi54, a dominant blast resistance gene cloned from rice line Tetep, imparts a broad spectrum resistance against various M. oryzae isolates. Many of its alleles have been explored from wild Oryza species and landraces whose sequences are available in the public domain. Its cognate effector gene AvrPi54 has also been cloned from M. oryzae. Complying with the Flor's gene-for-gene system, Pi54 protein interacts with AvrPi54 protein following fungal invasion leading to the resistance responses in rice cell that prevents the disease development. In the present study Pi54 alleles from 72 rice lines were used to understand the interaction of Pi54 (R) proteins with AvrPi54 (Avr) protein. The physiochemical properties of these proteins varied due to the nucleotide level polymorphism. The ab initio tertiary structures of these R- and Avr- proteins were generated and subjected to the in silico interaction. In this interaction, the residues in the LRR region of R- proteins were shown to interact with the Avr protein. These R proteins were found to have variable strengths of binding due to the differential spatial arrangements of their amino acid residues. Additionally, molecular dynamic simulations were performed for the protein pairs that showed stronger interaction than Pi54tetep (original Pi54 from Tetep) protein. We found these proteins were forming h-bond during simulation which indicated an effective binding. The root mean square deviation values and potential energy values were stable during simulation which validated the docking results. From the interaction studies and the molecular dynamics simulations, we concluded that the AvrPi54 protein interacts directly with the resistant Pi54 proteins through the LRR region of Pi54 proteins. Some of the Pi54 proteins from the landraces namely Casebatta, Tadukan, Varun dhan, Govind, Acharmita, HPR-2083, Budda, Jatto, MTU-4870, Dobeja-1, CN-1789, Indira sona, Kulanji pille and Motebangarkaddi cultivars show stronger binding with the AvrPi54 protein, thus these alleles can be effectively used for the rice blast resistance breeding program in future.

Aquaporins (AQPs) play a pivotal role in the cellular transport of water and many other small solutes, influencing many physiological and developmental processes in plants. In the present study, extensive bioinformatics analysis of AQPs was performed in Aquilegia coerulea L., a model species belonging to basal eudicots, with a particular focus on understanding the AQPs role in the developing petal nectar spur. A total of 29 AQPs were identified in Aquilegia, and their phylogenetic analysis performed with previously reported AQPs from rice, poplar and Arabidopsis depicted five distinct subfamilies of AQPs. Interestingly, comparative analysis revealed the loss of an uncharacterized intrinsic protein II (XIP-II) group in Aquilegia. The absence of the entire XIP subfamily has been reported in several previous studies, however, the loss of a single clade within the XIP family has not been characterized. Furthermore, protein structure analysis of AQPs was performed to understand pore diversity, which is helpful for the prediction of solute specificity. Similarly, an AQP AqcNIP2-1 was identified in Aquilegia, predicted as a silicon influx transporter based on the presence of features such as the G-S-G-R aromatic arginine selectivity filter, the spacing between asparagine-proline-alanine (NPA) motifs and pore morphology. RNA-seq analysis showed a high expression of tonoplast intrinsic proteins (TIPs) and plasma membrane intrinsic proteins (PIPs) in the developing petal spur. The results presented here will be helpful in understanding the AQP evolution in Aquilegia and their expression regulation, particularly during floral development.

Oryza coarctata plant, collected from Sundarban delta of West Bengal, India, has been used in the present study to generate draft genome sequences, employing the hybrid genome assembly with Illumina reads and third generation Oxford Nanopore sequencing technology. We report for the first time the draft genome with the coverage of 85.71 % and deposited the raw data in NCBI SRA, with BioProject ID PRJNA396417.

Valeriana jatamansi, a medicinally important species of the family Valerianaceae, has been cytologically studied in different geographical areas of North-Western Himalayan region of India. The tetraploid cytotype with chromosome numbers 2n=32 is in conformity with the earlier reports of the species from different parts of the world. An octoploid cytotype (2n=64) makes a new addition for the species on a worldwide basis, whereas the diploid cytotype (2n=16) is new to India have been reported for the first time in India. These cytotypes (2n=16, 32, 64) show significant variations with respect to morphology as well as geographical distribution in the Western Indian Himalayas. Further, anomalous populations have been marked with meiotic abnormalities in the form of cytomixis, chromosomal stickiness, unoriented bivalents, formation of laggards and bridges resulting in abnormal microsporogenesis, and production of heterogeneous-sized fertile pollen grains along with reduced pollen fertility.

Oryza coarctata plants, collected from Sundarban delta of West Bengal, India, have been used in the present study to generate draft genome sequences, employing the hybrid genome assembly with Illumina reads and third generation Oxford Nanopore sequencing technology. We report for the first time that more than 85.71 % of the genome coverage and the data have been deposited in NCBI SRA, with BioProject ID PRJNA396417.

Pigeonpea (Cajanus cajan L.), a diploid legume crop, is a member of the tribe Phaseoleae. This tribe is descended from the millettioid (tropical) clade of the subfamily Papilionoideae, which includes many important legume crop species such as soybean (Glycine max), mung bean (Vigna radiata), cowpea (Vigna ungiculata), and common bean (Phaseolus vulgaris). It plays major role in food and nutritional security, being rich source of proteins, minerals and vitamins. We have developed a comprehensive Pigeonpea Transcription Factors Database (PpTFDB) that encompasses information about 1829 putative transcription factors (TFs) and their 55 TF families. PpTFDB provides a comprehensive information about each of the identified TFs that includes chromosomal location, protein physicochemical properties, sequence data, protein functional annotation, simple sequence repeats (SSRs) with primers derived from their motifs, orthology with related legume crops, and gene ontology (GO) assignment to respective TFs. (PpTFDB: http://14.139.229.199/PpTFDB/Home.aspx) is a freely available and user friendly web resource that facilitates users to retrieve the information of individual members of a TF family through a set of query interfaces including TF ID or protein functional annotation. In addition, users can also get the information by browsing interfaces, which include browsing by TF Categories and by, GO Categories. This PpTFDB will serve as a promising central resource for researchers as well as breeders who are working towards crop improvement of legume crops.

Rapid advances in DNA sequencing technologies have resulted in the accumulation of large data sets in the public domain, facilitating comparative studies to provide novel insights into the evolution of life. Phylogenetic studies across the eukaryotic taxa have been reported but on the basis of a limited number of genes. Here we present a genome-wide analysis across different plant, fungal, protist, and animal species, with reference to the 36,002 expressed genes of the rice genome. Our analysis revealed 9831 genes unique to rice and 98 genes conserved across all 49 eukaryotic species analysed. The 98 genes conserved across diverse eukaryotes mostly exhibited binding and catalytic activities and shared common sequence motifs; and hence appeared to have a common origin. The 98 conserved genes belonged to 22 functional gene families including 26S protease, actin, ADP-ribosylation factor, ATP synthase, casein kinase, DEAD-box protein, DnaK, elongation factor 2, glyceraldehyde 3-phosphate, phosphatase 2A, ras-related protein, Ser/Thr protein phosphatase family protein, tubulin, ubiquitin and others. The consensus Bayesian eukaryotic tree of life developed in this study demonstrated widely separated clades of plants, fungi, and animals. Musa acuminata provided an evolutionary link between monocotyledons and dicotyledons, and Salpingoeca rosetta provided an evolutionary link between fungi and animals, which indicating that protozoan species are close relatives of fungi and animals. The divergence times for 1176 species pairs were estimated accurately by integrating fossil information with synonymous substitution rates in the comprehensive set of 98 genes. The present study provides valuable insight into the evolution of eukaryotes.

A precise understanding of the molecular mechanism involved in stress conditions has great importance for crop improvement. Biomolecules, such as WRKY proteins, which are the largest transcription factor family that is widely distributed in higher plants, plays a significant role in plant defense response against various biotic and abiotic stressors. In the present study, an extensive homology-based three-dimensional model construction and subsequent interaction study of WRKY DNA-binding domain (DBD) in CcWRKY1 (Type I), CcWRKY51 (Type II), and CcWRKY70 (Type III) belonging to pigeonpea, a highly tolerant crop species, was performed. Evaluation of the generated protein models was done to check their reliability and accuracy based on the quantitative and qualitative parameters. The final model was subjected to investigate the comparative binding analysis of different types of WRKY−DBD with DNA-W-box (a cis-acting element) by protein−DNA docking and molecular dynamics (MD) simulation. The DNA binding specificity with WRKY variants was scrutinized through protein−DNA interaction using the HADDOCK server. The stability, as well as conformational changes of protein−DNA complex, was investigated through molecular dynamics (MD) simulations for 100 ns using GROMACS. Additionally, the comparative stability and dynamic behavior of each residue of the WRKY−DBD type were analyzed in terms of root mean square deviation (RMSD), root mean square fluctuation (RMSF)values of the backbone atoms for each frame taking the minimized structure as a reference. The details of DNA binding activity of three different types of WRKY−DBD provided here will be helpful to better understand the regulation of WRKY gene family members in plants.

Genome-editing, a recent technological advancement in the field of life sciences, is one of the great examples of techniques used to explore the understanding of the biological phenomenon. Besides having different site-directed nucleases for genome editing over a decade ago, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) based genome editing approach has become a choice of technique due to its simplicity, ease of access, cost, and flexibility. In the present review, several CRISPR/Cas based approaches have been discussed, considering recent advances and challenges to implicate those in the crop improvement programs. Successful examples where CRISPR/Cas approach has been used to improve the biotic and abiotic stress tolerance, and traits related to yield and plant architecture have been discussed. The review highlights the challenges to implement the genome editing in polyploid crop plants like wheat, canola, and sugarcane. Challenges for plants difficult to transform and germline-specific gene expression have been discussed. We have also discussed the notable progress with multi-target editing approaches based on polycistronic tRNA processing, Csy4 endoribonuclease, intron processing, and Drosha ribonuclease. Potential to edit multiple targets simultaneously makes it possible to take up more challenging tasks required to engineer desired crop plants. Similarly, advances like precision gene editing, promoter bashing, and methylome-editing will also be discussed. The present review also provides a catalog of available computational tools and servers facilitating designing of guide-RNA targets, construct designs, and data analysis. The information provided here will be useful for the efficient exploration of technological advances in genome editing field for the crop improvement programs.

Hybrid proline-rich proteins (HyPRPs) belong to the family of 8-cysteine motif (8CM) containing proteins that play important roles in plant development processes, and tolerance to biotic and abiotic stresses. To gain insight into the rice HyPRPs, we performed a systematic genome-wide analysis and identified 45 OsHyPRP genes encoding 46 OsHyPRP proteins. The phylogenetic relationships of OsHyPRP proteins with monocots (maize, sorghum, and Brachypodium) and a dicot (Arabidopsis) showed clustering of the majority of OsHyPRPs along with those from other monocots, which suggests lineage-specific evolution of monocots HyPRPs. Based on our previous RNA-Seq study, we selected differentially expressed OsHyPRPs genes and used quantitative real-time-PCR (qRT-PCR) to measure their transcriptional responses to biotic (Magnaporthe oryzae) and abiotic (heat, cold, and salt) stresses and hormone treatment (Abscisic acid; ABA, Methyl-Jasmonate; MeJA, and Salicylic acid; SA) in rice blast susceptible Pusa Basmati-1 (PB1) and blast-resistant near-isogenic line PB1+Pi9. The induction of OsHyPRP16 expression in response to the majority of stresses and hormonal treatments was highly correlated with the number of cis-regulatory elements present in its promoter region. In silico docking analysis of OsHyPRP16 showed its interaction with sterols of fungal/protozoan origin. The characterization of the OsHyPRP gene family enables us to recognize the plausible role of OsHyPRP16 in stress tolerance.

Insertional mutagenesis is an indispensable tool for engendering a mutant population using exogenous DNA as the mutagen. The advancement in the next-generation sequencing platform has allowed for faster screening and analysis of generated mutated populations. Rice is a major staple crop for more than half of the world’s population; however, the functions of most of the genes in its genome are yet to be analyzed. Various mutant populations represent extremely valuable resources in order to achieve this goal. Here, we have reviewed different insertional mutagenesis approaches that have been used in rice, and have discussed their principles, strengths, and limitations. Comparisons between transfer DNA (T-DNA), transposons, and entrapment tagging approaches have highlighted their utilization in functional genomics studies in rice. We have also summarised different forward and reverse genetics approaches used for screening of insertional mutant populations. Furthermore, we have compiled information from several efforts made using insertional mutagenesis approaches in rice. The information presented here would serve as a database for rice insertional mutagenesis populations. We have also included various examples which illustrate how these populations have been useful for rice functional genomics studies. The information provided here will be very helpful for future functional genomics studies in rice aimed at its genetic improvement.

Pigeonpea (Cajanus cajan L.), a protein-rich legume, is a major food component of the daily diet for residents in semi-arid tropical regions of the word. Pigeonpea is also known for its high level of tolerance against biotic and abiotic stresses. In this regard, understanding the genes involved in stress tolerance has great importance. In the present study, identification, and characterization of WRKY, a large transcription factor gene family involved in numerous biological processes like seed germination, metabolism, plant growth, biotic and abiotic stress responses was performed in pigeonpea. A total of 94 WRKY genes identified in the pigeonpea genome were extensively characterized for gene-structures, localizations, phylogenetic distribution, conserved motif organizations, and functional annotation. Phylogenetic analysis revealed three major groups (I, II, and III) of pigeonpea WRKY genes. Subsequently, expression profiling of 94 CcWRKY genes across different tissues like root, nodule, stem, petiole, petal, sepal, shoot apical meristem (SAM), mature pod, and mature seed retrieved from the available RNAseq data identified tissue-specific WRKY genes with preferential expression in the vegetative and reproductive stages. Gene co-expression networks identified four WRKY genes at the center of maximum interaction which may play a key role in the entire WRKY regulations. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) expression analysis of WRKY genes in root and leaf tissue samples from plants under drought and salinity stress identified differentially expressed WRKY genes. The study will be helpful to understand the evolution, regulation, and distribution of the WRKY gene family, and additional exploration for the development of stress tolerance cultivars in pigeonpea and other legumes crops.

Fast neutron (FN) radiation mediated mutagenesis is a unique approach among the several induced mutagenesis methods being used in plant science in terms of impacted mutations. The FN mutagenesis usually creates deletions from few bases to several million bases (Mb). A library of random deletion generated using FN mutagenesis lines can provide indispensable resources for the reverse genetic approaches. In this review, information from several efforts made using FN mutagenesis has been compiled to understand the type of induced mutations, frequency, and genetic stability. Concerns regarding the utilization of FN mutagenesis technique for a plant with different level of ploidy and genome complexity are discussed. We have highlighted the utility of next-generation sequencing techniques that can be efficiently utilized for the characterization of mutant lines as well as for the mapping of causal mutations. Pros and cons of mapping by mutation (MutMap), mutant chromosome sequencing (MutChromSeq), exon capture, whole genome sequencing, MutRen-Seq, and different tilling approaches that can be used for the detection of FN-induced mutation has also been discussed. Genomic resources developed using the FN mutagenesis have been catalogued wooing to meaningful utilization of the available resources. The information provided here will be helpful for the efficient exploration for the crop improvement programs and for better understanding of genetic regulations.

Rice blast caused by Magnaporthe oryzae is one of the most important diseases of rice. Pi54, a rice gene that imparts resistance to M. oryzae isolates prevalent in India, was already cloned but its avirulent counterpart in the pathogen was not known.. After decoding the whole genome of an avirulent isolate of M. oryzae, we predicted 11440 protein coding genes and then identified four candidate effector proteins which are exclusively expressed in the infectious structure, appresoria. In silico protein modeling followed by interaction analysis between Pi54 protein model and selected four candidate effector proteins models revealed that Mo-01947_9 protein model encoded by a gene located at chromosome 4 of M. oryzae, interacted best at the Leucine Rich Repeat domain of Pi54 protein model. Yeast-two-hybrid analysis showed that Mo-01947_9 protein physically interacts with Pi54 protein. Nicotiana benthamiana leaf infiltration assay confirmed induction of hypersensitive response in the presence of Pi54 gene in a heterologous system. Genetic complementation test also proved that Mo-01947_9 protein induces avirulence response in the pathogen in the presence of Pi54 gene. Here, we report identification and cloning of a new fungal effector gene which interacts with resistance gene Pi54 gene in rice.

Rice sheath blight disease caused by Rhizoctonia solani is one of the most devastating diseases in rice leading to heavy yield losses. Due to the polygenic nature of resistance, no major resistance gene with complete host resistance against R. solani has been reported. In this study, we have performed molecular and functional analysis of the genes associated with the major R. solani-resistance QTL qSBR11-1 in the indica rice line Tetep. Sequence analysis revealed the presence of a set of 11 tandem repeats containing genes with a high degree of homology to class III chitinase defense response genes. Real-time quantitative PCR analysis showed that all the genes are strongly induced 36 hours after R. solani infection. Comparison between the resistant Tetep and the susceptible HP2216 lines shows that the induction of the chitinase genes is much higher in the Tetep line. Recombinant protein produced in vitro for six of the eleven genes showed chitinolytic activity in gel assays but we did not detect any xylanase inhibitory activity. All the six in vitro expressed proteins show antifungal activity with a clear inhibitory effect on the growth of the R. solani mycelium. The characterized chitinase genes can provide an important resource for the genetic improvement of R. solani susceptible rice lines for sheath blight resistance breeding.

Mango (Mangifera indica L.) is called "king of fruits" due to its sweetness, richness of taste, diversity, large production volume and a variety of end usage. Despite its huge economic importance genomic resources in mango are scarce and genetics of useful horticultural traits are poorly understood. Here we generated deep coverage leaf RNA sequence data for mango parental varieties 'Neelam', 'Dashehari' and their hybrid 'Amrapali' using next generation sequencing technologies. De-novo sequence assembly generated 27,528, 20,771 and 35,182 transcripts for the three genotypes, respectively. The transcripts were further assembled into a non-redundant set of 70,057 unigenes that were used for SSR and SNP identification and annotation. Total 5,465 SSR loci were identified in 4,912 unigenes with 288 type I SSR (n ≥ 20 bp). One hundred type I SSR markers were randomly selected of which 43 yielded PCR amplicons of expected size in the first round of validation and were designated as validated genic-SSR markers. Further, 22,306 SNPs were identified by aligning high quality sequence reads of the three mango varieties to the reference unigene set, revealing significantly enhanced SNP heterozygosity in the hybrid Amrapali. The present study on leaf RNA sequencing of mango varieties and their hybrid provides useful genomic resource for genetic improvement of mango.

Black rot caused by Xanthomonas campestris pv. campestris (Pam.) Dowson is the most destructive disease of cauliflower causing huge loss to the farmers throughout the world. Since there are limited sources of resistance to black rot in B. oleracea (C genome Brassica), exploration of A and B genomes of Brassica was planned as these were thought to be potential reservoirs of black rot resistance gene(s). In our search for new gene(s) for black rot resistance, F2 mapping population was developed in Brassica carinata (BBCC) by crossing NPC-17, a susceptible genotype with NPC-9, a resistant genotype. Out of 364 Intron length polymorphic markers and microsatellite primers used in this study, 41 distinguished the parental lines. However, resistant and susceptible bulks could be distinguished by three markers At1g70610, SSR Na14-G02 and At1g71865 which were used for genotyping of F2 mapping population. These markers were placed along the resistance gene, according to order, covering a distance of 36.30 cM. Intron length polymorphic markers At1g70610 and At1g71865 were found to be linked to black rot resistance locus (Xca1bc) at 6.2 and 12.8 cM distance, respectively. This is the first report of identification of markers linked to Xca1bc locus in Brassica carinata on B-7 linkage group. Intron length polymorphic markers provided a novel and attractive option for marker assisted selection due to high cross transferability and cost effectiveness for marker assisted alien gene introgression into cauliflower.

Rice is one of the important crops grown worldwide and is considered as an important crop for global food security. Rice is being affected by various fungal, bacterial and viral diseases resulting in huge yield losses every year. Deployment of resistance genes in various crops is one of the important methods of disease management. However, identification, cloning and characterization of disease resistance genes is a very tedious effort. To increase the life span of resistant cultivars, it is important to understand the molecular basis of plant host–pathogen interaction. With the advancement in rice genetics and genomics, several rice varieties resistant to fungal, bacterial and viral pathogens have been developed. However, resistance response of these varieties break down very frequently because of the emergence of more virulent races of the pathogen in nature. To increase the durability of resistance genes under field conditions, understanding the mechanismof resistance response and its molecular basis should be well understood. Some emerging concepts like interspecies transfer of pattern recognition receptors (PRRs) and transgenerational plant immunitycan be employed to develop sustainable broad spectrum resistant varieties of rice.

The genome of tomato ( L.) is being sequenced by an international consortium of 10 countries (Korea, China, the United Kingdom, India, the Netherlands, France, Japan, Spain, Italy, and the United States) as part of the larger “International Solanaceae Genome Project (SOL): Systems Approach to Diversity and Adaptation” initiative. The tomato genome sequencing project uses an ordered bacterial artificial chromosome (BAC) approach to generate a high-quality tomato euchromatic genome sequence for use as a reference genome for the Solanaceae and euasterids. Sequence is deposited at GenBank and at the SOL Genomics Network (SGN). Currently, there are around 1000 BACs finished or in progress, representing more than a third of the projected euchromatic portion of the genome. An annotation effort is also underway by the International Tomato Annotation Group. The expected number of genes in the euchromatin is ∼40,000, based on an estimate from a preliminary annotation of 11% of finished sequence. Here, we present this first snapshot of the emerging tomato genome and its annotation, a short comparison with potato ( L.) sequence data, and the tools available for the researchers to exploit this new resource are also presented. In the future, whole-genome shotgun techniques will be combined with the BAC-by-BAC approach to cover the entire tomato genome. The high-quality reference euchromatic tomato sequence is expected to be near completion by 2010.

Rice blast resistant gene, Pi54 cloned from rice line, Tetep, is effective against diverse isolates of Magnaporthe oryzae. In this study, we prospected the allelic variants of the dominant blast resistance gene from a set of 92 rice lines to determine the nucleotide diversity, pattern of its molecular evolution, phylogenetic relationships and evolutionary dynamics, and to develop allele specific markers. High quality sequences were generated for homologs of Pi54 gene. Using comparative sequence analysis, InDels of variable sizes in all the alleles were observed. Profiling of the selected sites of SNP (Single Nucleotide Polymorphism) and amino acids (N sites ≥ 10) exhibited constant frequency distribution of mutational and substitutional sites between the resistance and susceptible rice lines, respectively. A total of 50 new haplotypes based on the nucleotide polymorphism was also identified. A unique haplotype (H_3) was found to be linked to all the resistant alleles isolated from indica rice lines. Unique leucine zipper and tyrosine sulfation sites were identified in the predicted Pi54 proteins. Selection signals were observed in entire coding sequence of resistance alleles, as compared to LRR domains for susceptible alleles. This is a maiden report of extensive variability of Pi54 alleles in different landraces and cultivated varieties, possibly, attributing broad-spectrum resistance to Magnaporthe oryzae. The sequence variation in two consensus region: 163 bp and 144 bp were used for the development of allele specific DNA markers. Validated markers can be used for the selection and identification of better allele(s) and their introgression in commercial rice cultivars employing marker assisted selection.

The resistance (R) genes and defense response (DR) genes have become very important resources for the development of disease resistant cultivars. In the present investigation, genome-wide identification, expression, phylogenetic and synteny analysis was done for R and DR-genes across three species of rice viz: Oryza sativa ssp indica cv 93-11, Oryza sativa ssp japonica and wild rice species, Oryza brachyantha. We used the in silico approach to identify and map 786 R -genes and 167 DR-genes, 672 R-genes and 142 DR-genes, 251 R-genes and 86 DR-genes in the japonica, indica and O. brachyanth a genomes, respectively. Our analysis showed that 60.5% and 55.6% of the R-genes are tandemly repeated within clusters and distributed over all the rice chromosomes in indica and japonica genomes, respectively. The phylogenetic analysis along with motif distribution shows high degree of conservation of R- and DR-genes in clusters. In silico expression analysis of R-genes and DR-genes showed more than 85% were expressed genes showing corresponding EST matches in the databases. This study gave special emphasis on mechanisms of gene evolution and duplication for R and DR genes across species. Analysis of paralogs across rice species indicated 17% and 4.38% R-genes, 29% and 11.63% DR-genes duplication in indica and Oryza brachyantha, as compared to 20% and 26% duplication of R-genes and DR-genes in japonica respectively. We found that during the course of duplication only 9.5% of R- and DR-genes changed their function and rest of the genes have maintained their identity. Syntenic relationship across three genomes inferred that more orthology is shared between indica and japonica genomes as compared to brachyantha genome. Genome wide identification of R-genes and DR-genes in the rice genome will help in allele mining and functional validation of these genes, and to understand molecular mechanism of disease resistance and their evolution in rice and related species.

Oryza coarctata plant, collected from Sundarban delta of West Bengal, India, has been used in the present study to generate draft genome sequences, employing the hybrid genome assembly with Illumina reads and third generation Oxford Nanopore sequencing technology. We report for the first time the draft genome with the coverage of 85.71 % and deposited the raw data in NCBI SRA, with BioProject ID PRJNA396417.