MicroRNA Identification, Target Prediction, and Validation for Crop Improvement.

Micro-RNAs (mi-RNAs) are regulatory elements that play a vital role in the growth, development, and metabolic regulation of plants. In current research, the isolation of miRNAs is a tedious and difficult task using in vitro methods. However, recent exploration into the remarkably highly conserved nature of nucleotide sequences of miRNAs assists in the identification of miRNAs in plant species through homologous approaches. Here, we describe the in silico-based method for identification of miRNAs from the EST database which is emerging as a faster and more reliable approach along with the development of miRNA-SSR markers. This approach has the potential to accelerate research into the regulation of gene expression in various plant species such as tea, potato, tomato, tobacco, and orphan crops like cluster bean.

In silico Identification of miRNAs and Their Targets in Cluster Bean for Their Role in Development and Physiological Responses.

Cluster bean popularly known as "guar" is a drought-tolerant, annual legume that has recently emerged as an economically important crop, owing to its high protein and gum content. The guar gum has wide range of applications in food, pharma, and mining industries. India is the leading exporter of various cluster bean-based products all across the globe. Non-coding RNAs (miRNAs) are involved in regulating the expression of the target genes leading to variations in the associated pathways or final protein concentrations. The understanding of miRNAs and their associated targets in cluster bean is yet to be used to its full potential. In the present study, cluster bean EST (Expressed Sequence Tags) database was exploited to identify the miRNA and their predicted targets associated with metabolic and biological processes especially response to diverse biotic and abiotic stimuli using in silico approach. Computational analysis based on cluster bean ESTs led to the identification of 57 miRNAs along with their targets. To the best of our knowledge, this is the first report on identification of miRNAs and their targets using ESTs in cluster bean. The miRNA related to gum metabolism was also identified. Most abundant miRNA families predicted in our study were miR156, miR172, and miR2606. The length of most of the mature miRNAs was found to be 21nt long and the range of minimal folding energy (MFE) was 5.8-177.3 (-kcal/mol) with an average value of 25.4 (-kcal/mol). The identification of cluster bean miRNAs and their targets is predicted to hasten the miRNA discovery, resulting in better knowledge of the role of miRNAs in cluster bean development, physiology, and stress responses.

Promising versions of a commercial pearl millet hybrid for terminal drought tolerance identified through MAS.

Extreme climatic conditions like drought are a major threat to global food production. Terminal drought stress causes severe yield losses in pearl millet. Development of climate-resilient varieties/hybrids can minimize the yield losses to the farmers caused due to climatic extremes. In the present study, marker-assisted selection (MAS) was employed with an aim to develop improved version of HHB 226 by introgression of QTLs for terminal drought stress tolerance into the male parent of the hybrid (HBL 11). HBL 11 (recurrent parent) was crossed with PRLT 2 (donor) to develop F1 and backcrossed four times to raise BC4F1 and further selfed twice to raise BC4F3. Four polymorphic SSR markers were used to track the QTL introgressed lines in each subsequent generation until BC4F2. The recurrent parent genome recovery was assessed using 25 polymorphic SSRs. Morpho-physiological analysis of BC4F3 generation at field-level under terminal drought stress conditions showed that the QTL introgressed lines showed higher, grain yield, 1000-seed weight, relative water content (%), and lower electrolyte leakage (%) than the recurrent parent. Line number 63 performed best with all the four foreground markers, 97.20% recurrent parent genome recovery, 7.27 g 1000-seed weight, 73.27% relative water content, 65.06% electrolyte leakage, 0.58 (fv/fm) chlorophyll fluorescence, and 53.25 g grain yield per plant. Finally, the Improved version of HHB 226 was developed by using the Improved HBL 11 developed through MAS. Besides this, HBL 11 is the male parent of other commercial hybrids like HHB 223 and HHB 197 as well making Improved HBL 11 an asset to improve these pearl millet hybrids.

High-Throughput Phenotyping: A Platform to Accelerate Crop Improvement.

Development of high-throughput phenotyping technologies has progressed considerably in the last 10 years. These technologies provide precise measurements of desired traits among thousands of field-grown plants under diversified environments; this is a critical step towards selection of better performing lines as to yield, disease resistance, and stress tolerance to accelerate crop improvement programs. High-throughput phenotyping techniques and platforms help unraveling the genetic basis of complex traits associated with plant growth and development and targeted traits. This review focuses on the advancements in technologies involved in high-throughput, field-based, aerial, and unmanned platforms. Development of user-friendly data management tools and softwares to better understand phenotyping will increase the use of field-based high-throughput techniques, which have potential to revolutionize breeding strategies and meet the future needs of stakeholders.

Evaluating stripe rust resistance in Indian wheat genotypes and breeding lines using molecular markers.

Stripe rust (yellow rust), caused by Puccinia striiformis f. sp. tritici (Pst), is a serious disease of wheat worldwide, including India. Growing resistant cultivars is the most cost-effective and eco-friendly approach to manage the disease. In this study, 70 publically available molecular markers were used to identify the distribution of 35 Yr genes in 68 wheat genotypes. Out of 35 Yr genes, 25 genes amplified the loci associated with Yr genes. Of the 35, 18 were all-stage resistance ASR (All-stage resistance) genes and 7 (Yr16, Yr18, Yr29, Yr30, Yr36, Yr46 &Yr59) were APR (Adult-plant resistance) genes. In the field tests, evaluation for stripe rust was carried out under artificial inoculation of Pst. Fifty-three wheat genotypes were found resistant to yellow rust (ITs 0), accounting for 77.94% of total entries. Coefficients of infection ranged from 0 to 60 among all wheat genotypes. Two genotypes (VL 1099 & VL 3002) were identified with maximum 15 Yr genes followed by 14 genes in VL 3010 and HI8759, respectively. Maximum number of all-stage resistance genes were identified in RKD 292 (11) followed by ten genes in DBW 216, WH 1184 and VL 3002. Maximum number of adult-plant resistance gene was identified in VL 3009 (6), HI 8759 (5) and Lassik (4) respectively. Genes Yr26 (69.2%), Yr2 (69.1%), Yr64 (61.7%), Yr24 (58.9%), Yr7 (52.9%), Yr10 (50%) and Yr 48 (48.5%) showed high frequency among selected wheat genotypes, while Yr9 (2.94%), Yr36 (2.94%), Yr60 (1.47%) and Yr32 (8.8%) were least frequent in wheat genotypes. In future breeding programs, race specific genes and non-race specific genes should be utilised to pyramid with other effective genes to develop improved wheat cultivars with high-level and durable resistance to stripe rust. Proper deployment of Yr genes and utilizing the positive interactions will be helpful for resistance breeding in wheat.

SRAPs and EST-SSRs provide useful molecular diversity for targeting drought and salinity tolerance in Indian mustard.

Abiotic stress tolerance is one of the target trait in crop breeding under climate change scenario. Selection of suitable gene pools among available germplasm is first requisite for any crop improvement programme. Drought and salinity traits, being polygenic, are most difficult to target. The present investigation aimed at exploring and assessment of the genetic variability in Indian mustard at molecular level. A total of twenty-five genotypes and five related species were used. Sixty-three molecular markers including sequence related amplified polymorphism (SRAP) markers along with twenty-three expressed sequence tag-simple sequence repeats (EST-SSRs) were used for diversity analysis. Thirty-seven SRAPs and 18 EST-SSRs showed amplification producing a total of 423 alleles of which 422 were polymorphic. These markers gave an overall polymorphism of 99.78%, with 99.67% polymorphism in SRAPs and 100% polymorphism in EST-SSRs. The study revealed the genetic relationships among different genotypes of B. juncea and related species which could be used for Indian mustard improvement for targeting drought and salinity tolerance in future. Four SRAP and two EST-SSRs identified unique bands which may be related to abiotic stress tolerance. EST sequence BRMS-040 (IM7) was similar to Brassica and radish sequences related to PR-5 (pathogenesis-related) protein.

Molecular breeding of ameliorating commercial pearl millet hybrid for downy mildew resistance.

Downy mildew (DM) caused by Sclerospora graminicola is the most calamitous disease of pearl millet. Therefore, for introgression of DM resistance (DMR) in HHB 197 (MH-1302), an elite pearl millet hybrid, a marker-assisted breeding was undertaken by targeting three DMR loci on linkage groups (LGs) 1, 2 and 4. Breeding programme was initiated by crossing HBL 11 (DM susceptible), male parent of HHB 197 hybrid with ICMP 451 (DM-resistant) to produce true F1 plants. By conducting three rounds of backcrossing and selection, BC3F1 lines were generated. Foreground selection was employed using six polymorphic simple sequence repeat (SSR) markers of the 18 total selected markers. Four of these markers were linked to LG 1, five to LG 2 and nine to LG 4. Background selection was performed in BC3F1 generation using 33 polymorphic SSR markers of a total of 56 evenly spread SSR markers in the pearl millet genome to check recovery of recurrent parent genome. On the basis of genotypic selection (foreground as well as background) using selected SSR markers, agronomic performance in field and DM screening in greenhouse; 10 improved HBL 11 lines were selected and crossed with ICMA 97111 to produce DM-resistant HHB 197 hybrid versions. Six putatively improved HHB 197 hybrids were successfully tested in first year trials at Hisar and Bawal locations of Haryana and two selected versions with higher yield and zero DM incidence will be further tested in multilocation trials.

Nanotechnology based approaches for detection and delivery of microRNA in healthcare and crop protection.

Nanobiotechnology has the potential to revolutionize diverse sectors including medicine, agriculture, food, textile and pharmaceuticals. Disease diagnostics, therapeutics and crop protection strategies are fast emerging using nanomaterials preferably nanobiomaterials. It has potential for development of novel nanobiomolecules which offer several advantages over conventional treatment methods. RNA nanoparticles with many unique features are promising candidates in disease treatment. The miRNAs are involved in many biochemical and developmental pathways and their regulation in plants and animals. These appear to be a powerful tool for controlling various pathological diseases in human, plants and animals, however there are challenges associated with miRNA based nanotechnology. Several advancements made in the field of miRNA therapeutics make it an attractive approach, but a lot more has to be explored in nanotechnology assisted miRNA therapy. The miRNA based technologies can be employed for detection and combating crop diseases as well. Despite these potential advantages, nanobiotechnology applications in the agricultural sector are still in its infancy and have not yet made its mark in comparison with healthcare sector. The review provides a platform to discuss nature, role and use of miRNAs in nanobiotechnology applications.

High Quality Unigenes and Microsatellite Markers from Tissue Specific Transcriptome and Development of a Database in Clusterbean (Cyamopsis tetragonoloba, L. Taub).

Clusterbean (Cyamopsis tetragonoloba L. Taub), is an important industrial, vegetable and forage crop. This crop owes its commercial importance to the presence of guar gum (galactomannans) in its endosperm which is used as a lubricant in a range of industries. Despite its relevance to agriculture and industry, genomic resources available in this crop are limited. Therefore, the present study was undertaken to generate RNA-Seq based transcriptome from leaf, shoot, and flower tissues. A total of 145 million high quality Illumina reads were assembled using Trinity into 127,706 transcripts and 48,007 non-redundant high quality (HQ) unigenes. We annotated 79% unigenes against Plant Genes from the National Center for Biotechnology Information (NCBI), Swiss-Prot, Pfam, gene ontology (GO) and KEGG databases. Among the annotated unigenes, 30,020 were assigned with 116,964 GO terms, 9984 with EC and 6111 with 137 KEGG pathways. At different fragments per kilobase of transcript per millions fragments sequenced (FPKM) levels, genes were found expressed higher in flower tissue followed by shoot and leaf. Additionally, we identified 8687 potential simple sequence repeats (SSRs) with an average frequency of one SSR per 8.75 kb. A total of 28 amplified SSRs in 21 clusterbean genotypes resulted in polymorphism in 13 markers with average polymorphic information content (PIC) of 0.21. We also constructed a database named 'ClustergeneDB' for easy retrieval of unigenes and the microsatellite markers. The tissue specific genes identified and the molecular marker resources developed in this study is expected to aid in genetic improvement of clusterbean for its end use.

Hsp transcript induction is correlated with physiological changes under drought stress in Indian mustard.

Brassica juncea is an important oilseed crop and drought stress is major abiotic stress that limits its growth and productivity. RH0116 (drought tolerant) and RH8812 (drought sensitive) genotypes were undertaken to study some of the physiological parameters and hsp gene expression related to stress tolerance under drought stress conditions. Differential response in terms of seed germination, electrolyte leakage, RWC, osmotic potential was observed in the selected genotypes. In vitro seed germination studies using PEG stress treatments indicated reduced seed germination with increasing levels of stress treatment. Electrolyte leakage increased, whereas, relative water content and osmotic potential decreased in stressed seedlings. Expression of hsp gene was found to be upregulated during drought stress as the transcripts were present only in the stressed plants and disappeared upon rehydration. The drought tolerant variety showed higher transcript accumulation as compared to the sensitive variety. The study showed that drought induced changes in gene expression in two contrasting genotypes were consistent with the physiological response.

MYB transcription factor genes as regulators for plant responses: an overview.

Regulation of gene expression at the level of transcription controls many crucial biological processes. Transcription factors (TFs) play a great role in controlling cellular processes and MYB TF family is large and involved in controlling various processes like responses to biotic and abiotic stresses, development, differentiation, metabolism, defense etc. Here, we review MYB TFs with particular emphasis on their role in controlling different biological processes. This will provide valuable insights in understanding regulatory networks and associated functions to develop strategies for crop improvement.

Sequence related amplified polymorphism (SRAP) analysis for genetic diversity and micronutrient content among gene pools in mungbean [Vigna radiata (L.) Wilczek].

Vigna radiata (L.) Wilczek, commonly called mungbean is an important pulse crop. Commercial cultivars contain low levels of iron and zinc and it is important to assess genetic variability in the available germplasm for improving micronutrient content in commercial cultivars. The present study was undertaken to study molecular diversity using Sequence-related amplified polymorphism (SRAP) among 21 Vigna radiata genotypes. Twenty nine SRAP primer combinations produced a total of 121 amplified bands which were polymorphic with an average of 4.65 bands per primer. The size of amplified bands ranged from 70 bp to 3,000 bp and 6 out of 29 SRAP primers were most useful in fingerprinting Vigna radiata genotypes under study. The similarity coefficients between different genotypes ranged from 0.45 to 0.96 with an average similarity value of 0.71. At an arbitrary cut-off at 60 % similarity level on a dendrogram, the Vigna radiata accessions were categorized into two major clusters. ML1108 and 2KM115 were found to be genetically similar. SMH99-1A and ML776 showed high iron and zinc content while Satya was poor in iron as well as zinc content. Mapping population involving ML776 and Satya could be used for tagging gene(s) for micronutrient content. The results indicated that SRAP markers were efficient for identification of Vigna radiata genotypes and assessment of the genetic relationships among them.

Efficient protocol for in vitro direct plant regeneration in chickpea Cicer arietinum L.

An efficient plant regeneration system was developed for two important Indian chickpea cultivars, C-235 and HC-1. Immature cotyledons (7-8 mm) directly formed shoots without an intervening callus phase on MS medium containing B5 vitamins, BAP (2.0 mg/l), IBA (0.125 mg/l), AgNO3 (1.69 mg/l) and phytagel (2.5 g/l). The regenerated shoots had normal morphology and were successfully rooted in half strength MS medium under partial dark conditions. Regenerated plants were transferred to potted soil. However, the survival rate of pot house transferred plants was 17.6 per cent.