LMTdb: A comprehensive transcriptome database for climate-resilient, nutritionally rich little millet (Panicum sumatrense).

Little millet (Panicum sumatrense) a native of Chhattisgarh, belongs to the minor millet group and is primarily known as a climate-resilient and nutritionally rich crop. However, due to the lack of enough Omic studies on the crop, the scientific community has largely remained unaware of the potential of this crop, resulting in less scope for its utilization in crop improvement programs. Looking at global warming, erratic climate change, nutritional security, and limited genetic information available, the Little Millet Transcriptome Database (LMTdb) (https://igkv.ac.in/xenom/index.aspx) was conceptualized upon completion of the transcriptome sequencing of little millet with the aim of deciphering the genetic signatures of this largely unknown crop. The database was developed with the view of providing information about the most comprehensive part of the genome, the 'Transcriptome'. The database includes transcriptome sequence information, functional annotation, microsatellite markers, DEGs, and pathway information. The database is a freely available resource that provides breeders and scientists a portal to search, browse, and query data to facilitate functional and applied Omic studies in millet crops.

Meta-QTL and haplo-pheno analysis reveal superior haplotype combinations associated with low grain chalkiness under high temperature in rice.

Chalk, an undesirable grain quality trait in rice, is primarily formed due to high temperatures during the grain-filling process. Owing to the disordered starch granule structure, air spaces and low amylose content, chalky grains are easily breakable during milling thereby lowering head rice recovery and its market price. Availability of multiple QTLs associated with grain chalkiness and associated attributes, provided us an opportunity to perform a meta-analysis and identify candidate genes and their alleles contributing to enhanced grain quality. From the 403 previously reported QTLs, 64 Meta-QTLs encompassing 5262 non-redundant genes were identified. MQTL analysis reduced the genetic and physical intervals and nearly 73% meta-QTLs were narrower than 5cM and 2Mb, revealing the hotspot genomic regions. By investigating expression profiles of 5262 genes in previously published datasets, 49 candidate genes were shortlisted on the basis of their differential regulation in at least two of the datasets. We identified non-synonymous allelic variations and haplotypes in 39 candidate genes across the 3K rice genome panel. Further, we phenotyped a subset panel of 60 rice accessions by exposing them to high temperature stress under natural field conditions over two Rabi cropping seasons. Haplo-pheno analysis uncovered haplotype combinations of two starch synthesis genes, GBSSI and SSIIa, significantly contributing towards the formation of grain chalk in rice. We, therefore, report not only markers and pre-breeding material, but also propose superior haplotype combinations which can be introduced using either marker-assisted breeding or CRISPR-Cas based prime editing to generate elite rice varieties with low grain chalkiness and high HRY traits.

Identification and characterization of a grain micronutrient-related OsFRO2 rice gene ortholog from micronutrient-rich little millet (Panicum sumatrense).

Minor millets are considered as nutrient-rich cereals having significant effect in improving human health. In this study, a rice ortholog of Ferric Chelate Reductase (FRO2) gene involved in plant metal uptake has been identified in iron-rich Little millet (LM) using PCR and next generation sequencing-based strategy. FRO2 gene-specific primers designed from rice genome amplified 2.7 Kb fragment in LM genotype RLM-37. Computational genomics analyses of the sequenced amplicon showed high level sequence similarity with rice OsFRO2 gene. The predicted gene structure showed the presence of 6 exons and 5 introns and its protein sequence was found to contain ferric reductase and NOX_Duox_Like_FAD_NADP domains. Further, 3D structure analysis of FCR-LM model protein (494 amino acids) shows that it has 18 helices, 10 beta sheets, 10 strands, 41 beta turn and 5 gamma turn with slight deviation from the FCR-Os structure. Besides, the structures of FCR-LM and FCR-Os were modelled followed by molecular dynamics simulations. The overall study revealed both sequence and structural similarity between the identified gene and OsFRO2. Thus, a putative ferric chelate reductase gene has been identified in LM paving the way for using this approach for identification of orthologs of other metal genes from millets. This also facilitates mining of effective alleles of known genes for improvement of staple crops like rice.

Genome Wide Identification of Orthologous ZIP Genes Associated with Zinc and Iron Translocation in Setaria italica.

Genes in the ZIP family encode transcripts to store and transport bivalent metal micronutrient, particularly iron (Fe) and or zinc (Zn). These transcripts are important for a variety of functions involved in the developmental and physiological processes in many plant species, including most, if not all, Poaceae plant species and the model species Arabidopsis. Here, we present the report of a genome wide investigation of orthologous ZIP genes in Setaria italica and the identification of 7 single copy genes. RT-PCR shows 4 of them could be used to increase the bio-availability of zinc and iron content in grains. Of 36 ZIP members, 25 genes have traces of signal peptide based sub-cellular localization, as compared to those of plant species studied previously, yet translocation of ions remains unclear. In silico analysis of gene structure and protein nature suggests that these two were preeminent in shaping the functional diversity of the ZIP gene family in S. italica. NAC, bZIP and bHLH are the predominant Fe and Zn responsive transcription factors present in SiZIP genes. Together, our results provide new insights into the signal peptide based/independent iron and zinc translocation in the plant system and allowed identification of ZIP genes that may be involved in the zinc and iron absorption from the soil, and thus transporting it to the cereal grain underlying high micronutrient accumulation.

Analysis of drought-responsive signalling network in two contrasting rice cultivars using transcriptome-based approach.

Traditional cultivars of rice in India exhibit tolerance to drought stress due to their inherent genetic variations. Here we present comparative physiological and transcriptome analyses of two contrasting cultivars, drought tolerant Dhagaddeshi (DD) and susceptible IR20. Microarray analysis revealed several differentially expressed genes (DEGs) exclusively in DD as compared to IR20 seedlings exposed to 3 h drought stress. Physiologically, DD seedlings showed higher cell membrane stability and differential ABA accumulation in response to dehydration, coupled with rapid changes in gene expression. Detailed analyses of metabolic pathways enriched in expression data suggest interplay of ABA dependent along with secondary and redox metabolic networks that activate osmotic and detoxification signalling in DD. By co-localization of DEGs with QTLs from databases or published literature for physiological traits of DD and IR20, candidate genes were identified including those underlying major QTL qDTY1.1 in DD. Further, we identified previously uncharacterized genes from both DD and IR20 under drought conditions including OsWRKY51, OsVP1 and confirmed their expression by qPCR in multiple rice cultivars. OsFBK1 was also functionally validated in susceptible PB1 rice cultivar and Arabidopsis for providing drought tolerance. Some of the DEGs mapped to the known QTLs could thus, be of potential significance for marker-assisted breeding.

Diversity and functional significance of cellulolytic microbes living in termite, pill-bug and stem-borer guts.

Arthropods living on plants are able to digest plant biomass with the help of microbial flora in their guts. This study considered three arthropods from different niches - termites, pill-bugs and yellow stem-borers - and screened their guts for cellulase producing microbes. Among 42 unique cellulase-producing strains, 50% belonged to Bacillaceae, 26% belonged to Enterobacteriaceae, 17% belonged to Microbacteriaceae, 5% belonged to Paenibacillaceae and 2% belonged to Promicromonosporaceae. The distribution of microbial families in the three arthropod guts reflected differences in their food consumption habits. Most of the carboxymethylcellulase positive strains also hydrolysed other amorphous substrates such as xylan, locust bean gum and ß-D-glucan. Two strains, A11 and A21, demonstrated significant activity towards Avicel and p-nitrophenyl-ß-D-cellobiose, indicating that they express cellobiohydrolase. These results provide insight into the co-existence of symbionts in the guts of arthropods and their possible exploitation for the production of fuels and chemicals derived from plant biomass.

Detection of genomic changes in transgenic Bt rice populations through genetic fingerprinting using amplified fragment length polymorphism (AFLP).

A highly precise molecular marker technique, Amplified Fragment Length Polymorphism (AFLP) was used to characterize the changes in genomic structure occurred due to introduction of foreign gene and/ or due to the induced tissue culture stress during the development of various transgenic Bt rice populations. The transgenic rice plants having complex to simple Bt gene and selectable marker gene organizations were selected from the six insect resistant Bt rice lines. Our results clearly demonstrate that integration pattern, extent of rearrangement of foreign DNA and method of transformation may influence the genomic changes in transgenic rice populations. Eleven of fourteen AFLP primer combinations tested, generated distinct scorable banding pattern, which were further used in this study. Three primer combinations E-TA / M-CTT (IRRI-NPT), E-AG / M-CAC (Tulasi) and E-AA / M-CAG (IR 68899B) produced only monomorphic bands in all the transgenic and control rice plants .A percentage of 0.61% of 430 million bases of haploid rice genome were examined by the use of 11 AFLP primer combinations employed in this study. The DNA fingerprints generated by AFLP analysis, of each Bt rice population was compared with their respective non-transgenic control and was found to be > 97% similar. This indicated that the introduction of Bt gene into the genome of six rice varieties showed few genomic changes. The comparison shows that fewer changes were observed among the transgenic plants developed by Agrobacterium infection than that of developed through particle bombardment. Transgene integration pattern and their copy number were associated with the extent of genomic changes observed in the transgenic Bt rice varieties. AFLP fingerprints of the six transgenic rice varieties evidenced few, but consistent polymorphic bands between the transgenic individuals with different PI values. Relationships among the transgenic populations with their control rice plants were expressed in the form of dendrograms.