Superior haplotypes of key drought-responsive genes reveal opportunities for the development of climate-resilient rice varieties.

Haplotype-based breeding is an emerging and innovative concept that enables the development of designer crop varieties by exploiting and exploring superior alleles/haplotypes among target genes to create new traits in breeding programs. In this regard, whole-genome re-sequencing of 399 genotypes (landraces and breeding lines) from the 3000 rice genomes panel (3K-RG) is mined to identify the superior haplotypes for 95 drought-responsive candidate genes. Candidate gene-based association analysis reveals 69 marker-trait associations (MTAs) in 16 genes for single plant yield (SPY) under drought stress. Haplo-pheno analysis of these 16 genes identifies superior haplotypes for seven genes associated with the higher SPY under drought stress. Our study reveals that the performance of lines possessing superior haplotypes is significantly higher (p ≤ 0.05) as measured by single plant yield (SPY), for the OsGSK1-H4, OsDSR2-H3, OsDIL1-H22, OsDREB1C-H3, ASR3-H88, DSM3-H4 and ZFP182-H4 genes as compared to lines without the superior haplotypes. The validation results indicate that a superior haplotype for the DREB transcription factor (OsDREB1C) is present in all the drought-tolerant rice varieties, while it was notably absent in all susceptible varieties. These lines carrying the superior haplotypes can be used as potential donors in haplotype-based breeding to develop high-yielding drought-tolerant rice varieties.

Extreme trait GWAS (Et-GWAS): Unraveling rare variants in the 3,000 rice genome.

Identifying high-impact, rare genetic variants associated with specific traits is crucial for crop improvement. The 3,010 rice genome (3K RG) dataset offers a valuable resource for discovering genomic regions with potential applications in crop breeding. We used Extreme Trait GWAS (Et-GWAS), employing bulk pooling and allele frequency measurement to efficiently extract rare variants from the 3K RG. This innovative approach facilitates the detection of associations between genetic variants and target traits, concentrating and quantifying rare alleles. In our study, on grain yield under drought stress, Et-GWAS successfully identified five key genes (OsPP2C11, OsK5.2, OsIRO2, OsPEX1, and OsPWA1) known for enhancing yield under drought. In addition, we examined the overlap of our results with previously reported qDTY-QTLs and observed that OsUCH1 and OsUCH2 genes were located within qDTY2.2 We compared Et-GWAS with conventional GWAS, finding it effectively capturing most candidate genes associated with the target trait. Validation with resistant starch showed similar results. To enhance user-friendliness, we developed a GUI for Et-GWAS; https://et-gwas.shinyapps.io/Et-GWAS/.

Codon Usage Provide Insights into the Adaptation of Rice Genes under Stress Condition.

Plants experience different stresses, i.e., abiotic, or biotic, and to combat them, plants re-program the expression of growth-, metabolism-, and resistance-related genes. These genes differ in their synonymous codon usage frequency and show codon usage bias. Here, we investigated the correlation among codon usage bias, gene expression, and underlying mechanisms in rice under abiotic and biotic stress conditions. The results indicated that genes with higher expression (up- or downregulated) levels had high GC content (≥60%), a low effective number of codon usage (≤40), and exhibited strong biases towards the codons with C/G at the third nucleotide position, irrespective of stress received. TTC, ATC, and CTC were the most preferred codons, while TAC, CAC, AAC, GAC, and TGC were moderately preferred under any stress (abiotic or biotic) condition. Additionally, downregulated genes are under mutational pressure (R2 ≥ 0.5) while upregulated genes are under natural selection pressure (R2 ≤ 0.5). Based on these results, we also identified the possible target codons that can be used to design an optimized set of genes with specific codons to develop climate-resilient varieties. Conclusively, under stress, rice has a bias towards codon usage which is correlated with GC content, gene expression level, and gene length.

Whole-Genome-Based Web Genomic Resource for Water Buffalo (Bubalus bubalis).

Water buffalo (Bubalus bubalis), belonging to the Bovidae family, is an economically important animal as it is the major source of milk, meat, and drought in numerous countries. It is mainly distributed in tropical and subtropical regions with a global population of approximately 202 million. The advent of low cost and rapid sequencing technologies has opened a new vista for global buffalo researchers. In this study, we utilized the genomic data of five commercially important buffalo breeds, distributed globally, namely, Mediterranean, Egyptian, Bangladesh, Jaffrarabadi, and Murrah. Since there is no whole-genome sequence analysis of these five distinct buffalo breeds, which represent a highly diverse ecosystem, we made an attempt for the same. We report the first comprehensive, holistic, and user-friendly web genomic resource of buffalo (BuffGR) accessible at http://backlin.cabgrid.res.in/buffgr/, that catalogues 6028881 SNPs and 613403 InDels extracted from a set of 31 buffalo tissues. We found a total of 7727122 SNPs and 634124 InDels distributed in four breeds of buffalo (Murrah, Bangladesh, Jaffarabadi, and Egyptian) with reference to the Mediterranean breed. It also houses 4504691 SSR markers from all the breeds along with 1458 unique circRNAs, 37712 lncRNAs, and 938 miRNAs. This comprehensive web resource can be widely used by buffalo researchers across the globe for use of markers in marker trait association, genetic diversity among the different breeds of buffalo, use of ncRNAs as regulatory molecules, post-transcriptional regulations, and role in various diseases/stresses. These SNPs and InDelscan also be used as biomarkers to address adulteration and traceability. This resource can also be useful in buffalo improvement programs and disease/breed management.

Genome Wide Prediction, Mapping and Development of Genomic Resources of Mastitis Associated Genes in Water Buffalo.

Water buffalo (Bubalus bubalis) are an important animal resource that contributes milk, meat, leather, dairy products, and power for plowing and transport. However, mastitis, a bacterial disease affecting milk production and reproduction efficiency, is most prevalent in populations having intensive selection for higher milk yield, especially where the inbreeding level is also high. Climate change and poor hygiene management practices further complicate the issue. The management of this disease faces major challenges, like antibiotic resistance, maximum residue level, horizontal gene transfer, and limited success in resistance breeding. Bovine mastitis genome wide association studies have had limited success due to breed differences, sample sizes, and minor allele frequency, lowering the power to detect the diseases associated with SNPs. In this work, we focused on the application of targeted gene panels (TGPs) in screening for candidate gene association analysis, and how this approach overcomes the limitation of genome wide association studies. This work will facilitate the targeted sequencing of buffalo genomic regions with high depth coverage required to mine the extremely rare variants potentially associated with buffalo mastitis. Although the whole genome assembly of water buffalo is available, neither mastitis genes are predicted nor TGP in the form of web-genomic resources are available for future variant mining and association studies. Out of the 129 mastitis associated genes of cattle, 101 were completely mapped on the buffalo genome to make TGP. This further helped in identifying rare variants in water buffalo. Eighty-five genes were validated in the buffalo gene expression atlas, with the RNA-Seq data of 50 tissues. The functions of 97 genes were predicted, revealing 225 pathways. The mastitis proteins were used for protein-protein interaction network analysis to obtain additional cross-talking proteins. A total of 1,306 SNPs and 152 indels were identified from 101 genes. Water Buffalo-MSTdb was developed with 3-tier architecture to retrieve mastitis associated genes having genomic coordinates with chromosomal details for TGP sequencing for mining of minor alleles for further association studies. Lastly, a web-genomic resource was made available to mine variants of targeted gene panels in buffalo for mastitis resistance breeding in an endeavor to ensure improved productivity and the reproductive efficiency of water buffalo.

Computational insights into RNAi-based therapeutics for foot and mouth disease of Bos taurus.

Foot-and-mouth disease (FMD) endangers a large number of livestock populations across the globe being a highly contagious viral infection in wild and domestic cloven-hoofed animals. It adversely affects the socioeconomic status of millions of households. Vaccination has been used to protect animals against FMD virus (FMDV) to some extent but the effectiveness of available vaccines has been decreased due to high genetic variability in the FMDV genome. Another key aspect that the current vaccines are not favored is they do not provide the ability to differentiate between infected and vaccinated animals. Thus, RNA interference (RNAi) being a potential strategy to control virus replication, has opened up a new avenue for controlling the viral transmission. Hence, an attempt has been made here to establish the role of RNAi in therapeutic developments for FMD by computationally identifying (i) microRNA (miRNA) targets in FMDV using target prediction algorithms, (ii) targetable genomic regions in FMDV based on their dissimilarity with the host genome and, (iii) plausible anti-FMDV miRNA-like simulated nucleotide sequences (SNSs). The results revealed 12 mature host miRNAs that have 284 targets in 98 distinct FMDV genomic sequences. Wet-lab validation for anti-FMDV properties of 8 host miRNAs was carried out and all were observed to confer variable magnitude of antiviral effect. In addition, 14 miRBase miRNAs were found with better target accessibility in FMDV than that of Bos taurus. Further, 8 putative targetable regions having sense strand properties of siRNAs were identified on FMDV genes that are highly dissimilar with the host genome. A total of 16 SNSs having > 90% identity with mature miRNAs were also identified that have targets in FMDV genes. The information generated from this study is populated at http://bioinformatics.iasri.res.in/fmdisc/ to cater the needs of biologists, veterinarians and animal scientists working on FMD.

Transcriptional changes during ovule development in two genotypes of litchi (Litchi chinensis Sonn.) with contrast in seed size.

Litchi chinensis is a subtropical fruit crop, popular for its nutritional value and taste. Fruits with small seed size and thick aril are desirable in litchi. To gain molecular insight into gene expression that leads to the reduction in the size of seed in Litchi chinensis, transcriptomes of two genetically closely related genotypes, with contrasting seed size were compared in developing ovules. The cDNA library constructed from early developmental stages of ovules (0, 6, and 14 days after anthesis) of bold- and small-seeded litchi genotypes yielded 303,778,968 high quality paired-end reads. These were de-novo assembled into 1,19,939 transcripts with an average length of 865 bp. A total of 10,186 transcripts with contrast in expression were identified in developing ovules between the small- and large- seeded genotypes. A majority of these differences were present in ovules before anthesis, thus suggesting the role of maternal factors in seed development. A number of transcripts indicative of metabolic stress, expressed at higher level in the small seeded genotype. Several differentially expressed transcripts identified in such ovules showed homology with Arabidopsis genes associated with different stages of ovule development and embryogenesis.

Neuroprotective Role of L-NG-Nitroarginine Methyl Ester (L-NAME) against Chronic Hypobaric Hypoxia with Crowding Stress (CHC) Induced Depression-Like Behaviour.

Improper neuroimmune responses following chronic stress exposure have been reported to cause neuronal dysfunctions leading to memory impairment, anxiety and depression like behaviours. Though several factors affecting microglial activation and consequent alteration in neuro-inflammatory responses have been well studied, role of NO and its association with microglia in stress induced depression model is yet to be explored. In the present study, we validated combination of chronic hypobaric hypoxia and crowding (CHC) as a stress model for depression and investigated the role of chronic stress induced elevated nitric oxide (NO) level in microglia activation and its effect on neuro-inflammatory responses in brain. Further, we evaluated the ameliorative effect of L-NG-Nitroarginine Methyl Ester (L-NAME) to reverse the stress induced depressive mood state. Four groups of male Sprague Dawley rat were taken and divided into control and CHC stress exposed group with and without treatment of L-NAME. Depression like behaviour and anhedonia in rats were assessed by Forced Swim Test (FST) and Sucrose Preference Test (SPT). Microglial activation was evaluated using Iba-1 immunohistochemistry and proinflammatory cytokines were assessed in the hippocampal region. Our result showed that exposure to CHC stress increased the number of active microglia with corresponding increase in inflammatory cytokines and altered behavioural responses. The inhibition of NO synthesis by L-NAME during CHC exposure decreased the number of active microglia in hippocampus as evident from decreased Iba-1 positive cells. Further, L-NAME administration decreased pro-inflammatory cytokines in hippocampus and improved behaviour of rats. Our study demonstrate that stress induced elevation of NO plays pivotal role in altered microglial activation and consequent neurodegenerative processes leading to depression like behaviour in rat.

PmiRExAt: plant miRNA expression atlas database and web applications.

High-throughput small RNA (sRNA) sequencing technology enables an entirely new perspective for plant microRNA (miRNA) research and has immense potential to unravel regulatory networks. Novel insights gained through data mining in publically available rich resource of sRNA data will help in designing biotechnology-based approaches for crop improvement to enhance plant yield and nutritional value. Bioinformatics resources enabling meta-analysis of miRNA expression across multiple plant species are still evolving. Here, we report PmiRExAt, a new online database resource that caters plant miRNA expression atlas. The web-based repository comprises of miRNA expression profile and query tool for 1859 wheat, 2330 rice and 283 maize miRNA. The database interface offers open and easy access to miRNA expression profile and helps in identifying tissue preferential, differential and constitutively expressing miRNAs. A feature enabling expression study of conserved miRNA across multiple species is also implemented. Custom expression analysis feature enables expression analysis of novel miRNA in total 117 datasets. New sRNA dataset can also be uploaded for analysing miRNA expression profiles for 73 plant species. PmiRExAt application program interface, a simple object access protocol web service allows other programmers to remotely invoke the methods written for doing programmatic search operations on PmiRExAt database.Database URL:http://pmirexat.nabi.res.in.

EEG Abnormalities in Children with Speech and Language Impairment.

INTRODUCTION: Epilepsy, a chronic condition of recurrent seizures, affects language, but the extent and nature of the language disturbance varies widely according to the type, severity, and cause of the epilepsy. There is paucity of literature on the electroencephalographic abnormalities in children with speech and language impairment. The present study was therefore planned to find the association of epileptiform EEG abnormalities in children with speech and language impairment and if present, their localization and lateralization to the language areas of the brain that are present predominantly in the left hemisphere. MATERIALS AND METHODS: The study was conducted on Paediatric patients having speech and language impairment (n=94, age-2 to 8 years) selected on the basis of detailed history and neurologic examination. Video Electroencephalography (EEG) was performed as per American Clinical Neurophysiology Society guidelines using 16 channel RMS computerized EEG machine for a minimum of 40 minutes to capture both wakefulness and sleep along with activation procedures like hyperventilation (if feasible) and photic stimulation. EEG was reviewed for any abnormal EEG background, benign variants, interictal epileptiform discharges and ictal discharges. RESULTS: In our cohort, 19.7% boys and 22.2% girls presented with seizures in their infancy and this gender difference was found to be statistically significant (p<0.05). EEG was abnormal in 47.9% children (45 out of 94) with no significant gender difference. Epileptiform EEG was seen in 73.6% of children with history of seizures and 41.3% of children without history of seizures (p<0.05). The EEG abnormities included: abnormal background (64.5%), presence of generalized interictal epileptiform discharges (57.8%), focal epileptiform discharges (20%) exclusively from left hemisphere and multifocal interictal epileptiform discharges (33.3%), each occurring in isolation or associated with other abnormities. CONCLUSION: In the current study, it is definite that presence of generalized abnormalities in EEG are seen in higher frequency and focal interictal epileptiform discharges are solely seen in left hemisphere in children with speech and language impairment. Although, there is no distinct pattern of EEG abnormalities in such patients, we recommend a routine EEG in them and also brain imaging to complement the EEG findings.

ABER Assessment in Pre-school Children with Developmental Speech and Language Impairment.

INTRODUCTION: Developmental speech and language disorders (DLD) constitute a group of disorders when children with normal intelligence and hearing fail to develop language in an age-appropriate manner. There is no definite or surrogate neurophysiologic laboratory marker to quantitate the extent of speech and language impairment. The current study was designed to evaluate the abnormalities in Auditory Brainstem Evoked Responses (ABER) in children with speech and language impairment who do not have a hearing deficit or autism. MATERIALS AND METHODS: ABER recording was done in a cohort of 94 children (age 2-8 y) with DLD without overt hearing deficit or autism. The mean latencies for waves I, II, III, IV and V along with inter peak latencies for I-III, I-V, III-V and amplitude ratio of wave V/I was measured after click stimulus with intensities 110 db until 40 db and compared to age appropriate normograms. RESULTS: The peak latencies for waves I, III & V, inter-peak latencies I-III & I-V, III-V and wave amplitude ratio V/I was found within normal limits in both ears of all the children when compared to age appropriate normograms. CONCLUSION: The current study therefore emphasizes the fact that ABER may not be used/recommended as diagnostic or prognostic tool in children with speech and language impairment without autism or hearing deficit. The results and the recommendations of this study will definitely reduce the burden on electrophysiologist, laboratories and also save time and financial resources.