Is CRISPR/Cas9-based multi-trait enhancement of wheat forthcoming?

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies have been implemented in recent years in the genome editing of eukaryotes, including plants. The original system of knocking out a single gene by causing a double-strand break (DSB), followed by non-homologous end joining (NHEJ) or Homology-directed repair (HDR) has undergone many adaptations. These adaptations include employing CRISPR/Cas9 to upregulate gene expression or to cause specific small changes to the DNA sequence of the gene-of-interest. In plants, multiplexing, i.e., inducing multiple changes by CRISPR/Cas9, is extremely relevant due to the redundancy of many plant genes, and the time- and labor-consuming generation of stable transgenic plant lines via crossing. Here we discuss relevant examples of various traits, such as yield, biofortification, gluten content, abiotic stress tolerance, and biotic stress resistance, which have been successfully manipulated using CRISPR/Cas9 in plants. While existing studies have primarily focused on proving the impact of CRISPR/Cas9 on a single trait, there is a growing interest among researchers in creating a multi-stress tolerant wheat cultivar 'super wheat', to commercially and sustainably enhance wheat yields under climate change. Due to the complexity of the technical difficulties in generating multi-target CRISPR/Cas9 lines and of the interactions between stress responses, we propose enhancing already commercial local landraces with higher yield traits along with stress tolerances specific to the respective localities, instead of generating a general 'super wheat'. We hope this will serve as the sustainable solution to commercially enhancing crop yields under both stable and challenging environmental conditions.

Flexible, humidity- and contamination-resistant superhydrophobic MXene-based electrospun triboelectric nanogenerators for distributed energy harvesting applications.

The low surface-charge density, poor stability and irreparable surface of triboelectric materials under harsh environments are still some obstacles for developing high-performance triboelectric nanogenerators (TENGs). In particular, a two-dimensional MXene material's surface is likely to be corroded by water molecules under high humidity conditions owing to its hydrophilic nature, limiting the output performance and stability of TENGs. Herein, an approach for fabricating a humidity- and contamination-resistant MXene-based TENG is established using the electrospinning technique. First, nanofibrous layers of MXene/MoS2 composites blended in a cellulosic polymer matrix were prepared, benefitting the high surface roughness and controlled air-trapping pores. Furthermore, the prepared nanofibrous layers were chemically modified with stearic acid (SA), which enhances the hydrophobicity and electronegativity of MXene/MoS2 composites. In a typical synthesis, four different compositions of MXene/MoS2/cellulose acetate nanofibers were prepared, which illustrates that an increasing concentration of MoS2 could effectively tune the surface oxidation, hydrophilic nature, and surface roughness of MXene as well as induce a piezoelectricity-enhanced triboelectric potential. On the other side, the SA modification ultimately generated a superhydrophobic surface with low surface energy and a high water contact angle of ∼154°. The integrated TENG displayed an enhanced output voltage of ∼140 V and an instantaneous power density of ∼2975 mW cm-2 with long-term stability under high humidity conditions. Additionally, the self-cleaning properties were demonstrated, ensuring the sustainability and reusability of the TENG in a contaminated environment. Moreover, the fabricated MXene-based superhydrophobic layer can harvest the energy on dripping water droplets based on the liquid-solid contact-electrification TENG mode. Overall, this work paves the way for the design and development of humidity- and contamination-resistant triboelectric materials and guides the study of harvesting of distributed environmental energy efficiently.

Plasma membrane aquaporins regulate root hydraulic conductivity in the model plant Setaria viridis.

The high rate of productivity observed in panicoid crops is in part due to their extensive root system. Recently, green foxtail (Setaria viridis) has emerged as a genetic model system for panicoid grasses. Natural accessions of S. viridis originating from different parts of the world, with differential leaf physiological behavior, have been identified. This work focused on understanding the physiological and molecular mechanisms controlling root hydraulic conductivity and root-to-shoot gas exchange signaling in S. viridis. We identified 2 accessions, SHA and ZHA, with contrasting behavior at the leaf, root, and whole-plant levels. Our results indicated a role for root aquaporin (AQP) plasma membrane (PM) intrinsic proteins in the differential behavior of SHA and ZHA. Moreover, a different root hydraulic response to low levels of abscisic acid between SHA and ZHA was observed, which was associated with root AQPs. Using cell imaging, biochemical, and reverse genetic approaches, we identified PM intrinsic protein 1;6 (PIP1;6) as a possible PIP1 candidate that regulates radial root hydraulics and root-to-shoot signaling of gas exchange in S. viridis. In heterologous systems, PIP1;6 localized in the endoplasmic reticulum, and upon interaction with PIP2s, relocalization to the PM was observed. PIP1;6 was predominantly expressed at the root endodermis. Generation of knockout PIP1;6 plants (KO-PIP1;6) in S. viridis showed altered root hydraulic conductivity, altered gas exchange, and alteration of root transcriptional patterns. Our results indicate that PIPs are essential in regulating whole-plant water homeostasis in S. viridis. We conclude that root hydraulic conductivity and gas exchange are positively associated and are regulated by AQPs.

The Alteration of Tomato Chloroplast Vesiculation Positively Affects Whole-Plant Source-Sink Relations and Fruit Metabolism under Stress Conditions.

Changes in climate conditions can negatively affect the productivity of crop plants. They can induce chloroplast degradation (senescence), which leads to decreased source capacity, as well as decreased whole-plant carbon/nitrogen assimilation and allocation. The importance, contribution and mechanisms of action regulating source-tissue capacity under stress conditions in tomato (Solanum lycopersicum) are not well understood. We hypothesized that delaying chloroplast degradation by altering the activity of the tomato chloroplast vesiculation (CV) under stress would lead to more efficient use of carbon and nitrogen and to higher yields. Tomato CV is upregulated under stress conditions. Specific induction of CV in leaves at the fruit development stage resulted in stress-induced senescence and negatively affected fruit yield, without any positive effects on fruit quality. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/CAS9) knockout CV plants, generated using a near-isogenic tomato line with enhanced sink capacity, exhibited stress tolerance at both the vegetative and the reproductive stages, leading to enhanced fruit quantity, quality and harvest index. Detailed metabolic and transcriptomic network analysis of sink tissue revealed that the l-glutamine and l-arginine biosynthesis pathways are associated with stress-response conditions and also identified putative novel genes involved in tomato fruit quality under stress. Our results are the first to demonstrate the feasibility of delayed stress-induced senescence as a stress-tolerance trait in a fleshy fruit crop, to highlight the involvement of the CV pathway in the regulation of source strength under stress and to identify genes and metabolic pathways involved in increased tomato sink capacity under stress conditions.

Pollen viability as a potential trait for screening heat-tolerant wheat (Triticum aestivum L.).

High temperature during reproductive stage of winter crops causes sterility of pollen grains and reduced yield. It is essential to find the genotypes with higher pollen viability, as it is most sensitive to temperature extremes. A field study was conducted with wheat (Triticum aestivum L.) genotypes to understand the effect of high temperature on pollen viability and grain yield for 2years under timely (TS) and late sown (LS) conditions. A strong correlation was observed between higher pollen viability and higher grain yield under heat stress condition. Genotypes like K7903, HD2932, WH730 and RAJ3765 showed higher pollen viability, whereas DBW17, HUW468, RAJ4014 and UP2425 had lower pollen viability under LS condition. Further, the quantification of antioxidant enzymes activity mainly, Super oxide dismutase (SOD), Catalase (CAT), Peroxidase (POD) and Glutathione peroxidase (GPX) has showed significant variation among study genotypes. Thus, the identified high pollen viability genotypes can serve as a potential source for trait based breeding under heat stress in wheat. The present study is a first of its kind to assess more number of wheat genotypes for pollen viability and antioxidants activity under field condition. It also confirms that pollen viability can be used as a potential trait to screen genotypes for heat stress tolerance in wheat.

Meprin A1 subunit beta gene polymorphism is associated with the length of post-partum anestrus interval in Murrah buffaloes.

Postpartum anestrus interval (PPAI) is the interval between parturition and the first postpartum estrus exhibition in animals. Appearance of both normal and PPA buffaloes under the same farm conditions indicates the role of possible genetic predisposition to PPA. To identify the genetic and non-genetic factors associated with PPA in buffaloes, we collected data on PPAI and other 38 non-genetic variables from 575 Murrah buffaloes in the field conditions and identified the PPA associated non-genetic factors in our previous study. To explore the genetic factors associated with the unexplained variation in PPAI residuals, the present study identified 41 single nucleotide polymorphisms (SNPs) in 13 candidate genes using Sanger sequencing. Exploration of their association with the PPAI residuals of 50 extreme PPA and 50 normal buffaloes identified the significant (P < 0.01) association of the SNP (g.37219977A>G) in the 3'-UTR region of the Meprin A 1 subunit beta (Mep1b) gene with PPAI, which was further validated (P = 0.058) in a large population sample (n = 417). Bioinformatics analysis of the 3'-UTR region has identified three miRNA, bta-miR-2420, bta-miR-2325b and bta-miR-453 that could regulate Igf-1 in the plasma of animals with different genotypes (GG, AG and AA). The higher Igf-1 levels in the GG genotypes than that of AA and AG genotypes of this SNP (g.37219977A>G) further suggest the association of Mep1b gene with PPA condition in Murrah buffaloes. As a result of this study, we propose that buffaloes with protective alleles at this SNP be selected to improve the herd's reproductive efficiency.

Automatic detection of COVID-19 disease using U-Net architecture based fully convolutional network.

The severe acute respiratory syndrome coronavirus 2, called a SARS-CoV-2 virus, emerged from China at the end of 2019, has caused a disease named COVID-19, which has now evolved as a pandemic. Amongst the detected Covid-19 cases, several cases are also found asymptomatic. The presently available Reverse Transcription - Polymerase Chain Reaction (RT-PCR) system for detecting COVID-19 lacks due to limited availability of test kits and relatively low positive symptoms in the early stages of the disease, urging the need for alternative solutions. The tool based on Artificial Intelligence might help the world to develop an additional COVID-19 disease mitigation policy. In this paper, an automated Covid-19 detection system has been proposed, which uses indications from Computer Tomography (CT) images to train the new powered deep learning model- U-Net architecture. The performance of the proposed system has been evaluated using 1000 Chest CT images. The images were obtained from three different sources - Two different GitHub repository sources and the Italian Society of Medical and Interventional Radiology's excellent collection. Out of 1000 images, 552 images were of normal persons, and 448 images were obtained from COVID-19 affected people. The proposed algorithm has achieved a sensitivity and specificity of 94.86% and 93.47% respectively, with an overall accuracy of 94.10%. The U-Net architecture used for Chest CT image analysis has been found effective. The proposed method can be used for primary screening of COVID-19 affected persons as an additional tool available to clinicians.

Body condition score, parity, shelter cleanliness and male proximity: Highly associated non-genetic factors with post-partum anestrus in Murrah buffalo in field conditions.

Postpartum anestrus (PPA) is a major reproductive problem in buffalo. Although both genetic and non-genetic factors influence the incidence of PPA, identifying associated non-genetic factors would be helpful for effective management of this problem. No systematic study, however, has been conducted to identify the association of non-genetic factors with PPA in buffalo, especially in field conditions. Data for 39 variables affecting postpartum anestrus interval (PPAI), health, nutrition, management and environmental status of the animals were collected on 575 buffalo from 15 villages. Data were analyzed using a general linear model considering PPAI as a dependent variable and the remaining 38 non-genetic parameters as independent variables. The results from the analysis indicated body condition score (BCS) variables, such as pelvic girdle score (P <  0.0001) and girth (P <  0.05), parity (P < 0.0001), shelter cleanliness (P <  0.001), male proximity (P <  0.005), amount and frequency of concentrate feeding during the early postpartum period (P <  0.05), general feeding frequency in a day (P <  0.05) and the treatments with oxytocin at the time of milking to facilitate milk release from the udder (P <  0.05) were associated with PPAI in Murrah buffalo. Plotting the PPAI residuals indicated the possibility of other unexplained factors, including genetics, that could also affect the differences in values for this variables in Murrah buffalo. In conclusion, a greater understanding of associated non-genetic factors with PPAI would help farmers in conducting proper managemental practices for early induction of reproductive cycles following parturition in Murrah buffalo.

Buffalo liver transcriptome analysis suggests immune tolerance as its key adaptive mechanism during early postpartum negative energy balance.

Females undergo negative energy balance (NEB) during the early postpartum period to meet the lactation demands. The liver, being the key metabolic organ, plays a major role in handling NEB. Dairy animals handling high lactation demands are better models to understand the liver adaptive mechanisms during this phase. Therefore, we analyzed the liver transcriptome of dairy buffaloes during early postpartum. Liver biopsies were performed on three lactating buffaloes on the 15th and the 30th days of early postpartum and three heifers (controls) at the diestrous stage. Paired-end Next Generation Sequencing (NGS) identified 509 significantly differentially expressed genes (SDE) in the liver among the three groups. The SDE with log2 fold change > 3 and the unique SDE revealed the promotion of immune suppression (e.g., TCR), apoptosis (e.g., CCDC103), PGF2α synthesis, fat accumulation (e.g., BGLAP) and liver regeneration (e.g., FGF10) pathways, and the downregulation of antigen presentation (e.g., BOLA-DQA) on the 15th day of lactation. Consistently upregulated genes on the 15th and 30th days of early postpartum indicated the promotion of immune tolerance (e.g., IFITM3), medium and long-chain fatty acids' oxidation (e.g., ACSM3), and lipid accumulation (e.g., INSIG1). However, consistently downregulated genes during early postpartum showed immunosuppression, the downregulation of gluconeogenesis from amino acids (e.g., DDO), and the biosynthesis of taurine (e.g., CSAD) and unsaturated fatty acids (e.g., SCD). Functional annotation and network analyses also indicated the promotion of immune tolerance, fat accumulation and decreased gluconeogenesis from amino acids, and estrogen metabolism on the 15th day of lactation. Overall, the liver showed immune tolerance as an adaptive mechanism during early postpartum of buffaloes.

Optimization of Agrobacterium-mediated transformation in spring bread wheat using mature and immature embryos.

Wheat is the most widely grown staple food crop in the world and accounts for dietary needs of more than 35% of the human population. Current status of transgenic wheat development is slow all over the world due to the lack of a suitable transformation system. In the present study, an efficient and reproducible Agrobacterium-mediated transformation system in bread wheat (Triticum aestivum L.) is established. The mature and immature embryos of six recently released high yielding spring bread wheat genotypes were used to standardize various parameters using Agrobacterium tumefaciens strain EHA105 harbouring binary vector pCAMBIA3301 having gus and bar as marker genes. The optimum duration for embryo pre-culture, inoculation time and co-cultivation were 2 days, 30 min and 48 h, respectively. The bacterial inoculum concentration of OD of 1 at 600 nm showed 67.25% transient GUS expression in the histochemical GUS assay. The filter paper based co-cultivation limits the Agrobacterium overgrowth and had 82.3% explants survival rate whereas medium based strategy had 22.7% explants survival only. The medium having picloram 4 mg/l along with antibiotics (cefotaxime 500 mg/l and timentin 300 mg/l) was found best suitable for initial week callus induction. The standardized procedure gave overall 14.9% transformation efficiency in immature embryos and 9.8% in mature embryos and confirmed by gene-specific and promoter-specific PCR and southern analysis. These results indicate that the developed Agrobacterium-mediated transformation system is suitable for diverse wheat genotypes. The major obstacle for the implication of the CRISPR-based genome editing techniques is the non-availability of a suitable transformation system. Thus, the present system can be exploited to deliver the T-DNA into the wheat genome for CRISPR-based target modifications and transgene insertions.

An efficient method for extracting next-generation sequencing quality RNA from liver tissue of recalcitrant animal species.

The next-generation RNA sequencing technologies expedite the discovery of a large number of novel transcripts and genes associated with various pathophysiological conditions. These technologies involve poly(A) enrichment, which in turn requires micrograms of high-quality total RNA. Unfortunately, the available RNA isolation approaches produce poor quality total RNA from difficult-to-isolate animal tissues, such as the liver with high glycogen content. Moreover, the extraction efficiencies of these approaches vary significantly depending on the animal species. To address this challenge, we optimized a three-step protocol for the extraction of high-yield and high-quality total RNA from the liver tissue (LT). The procedure effectively resolved the problem of glycogen coprecipitation by its stepwise removal. No signs of RNA degradation on gel electrophoresis analysis and RNA integrity number values ≥8.5 indicated that the extracted RNA is suitable for downstream processing, such as poly(A) enrichment and transcriptome profiling. To demonstrate the robustness of the novel protocol, a comparison was made with other currently available RNA extraction approaches from diverse resources. Whereas other protocols yielded partially degraded bands with either decreased or reversed ribosomal RNA (rRNA) ratio, our protocol yielded intact rRNA with a ratio of 2.0 ± 0.1. This optimized protocol was also successfully followed for other animal tissues, such as the bone and muscles. In conclusion, the study has described a highly efficient method for the next-generation sequencing quality RNA isolation from LT across a broad range of animal species, with extended applicability to other difficult-to-isolate tissues.

A highly efficient method for extracting next-generation sequencing quality RNA from adipose tissue of recalcitrant animal species.

The next-generation sequencing (NGS) based RNA sequencing (RNA-Seq) and transcriptome profiling offers an opportunity to unveil complex biological processes. Successful RNA-Seq and transcriptome profiling requires a large amount of high-quality RNA. However, NGS-quality RNA isolation is extremely difficult from recalcitrant adipose tissue (AT) with high lipid content and low cell numbers. Further, the amount and biochemical composition of AT lipid varies depending upon the animal species which can pose different degree of resistance to RNA extraction. Currently available approaches may work effectively in one species but can be almost unproductive in another species. Herein, we report a two step protocol for the extraction of NGS quality RNA from AT across a broad range of animal species.

Mutations in the plastidic ACCase gene endowing resistance to ACCase-inhibiting herbicide in Phalaris minor populations from India.

Littleseed canarygrass (Phalaris minor Retz.) is one of the most common and troublesome weeds infesting wheat crop in India. Repeated use during the last two decades of the ACCase-inhibiting herbicide (clodinafop) to control this weed has resulted in the occurrence of resistance. Fifty-three P. minor populations were collected from wheat fields in Haryana and Punjab states of India. The dose-response assays indicated that 29 populations were resistant, 23 populations were susceptible and one population was moderately resistant to clodinafop. Sequence analysis of the CT domain of ACCase gene among resistant and susceptible populations revealed two non-synonymous mutations, Trp2027 to Cys and Ile2041 to Asn in the resistant populations. Allele-specific PCR markers were developed to differentiate between wild-type and resistant codons at positions 2027 and 2041 of ACCase in P. minor which enables molecular assays for rapid detection and resistance diagnosis for efficient weed management in wheat. This is the first report from India of a target site mutation corresponding to resistance to clodinafop in P. minor.

Mapping of stripe rust resistance QTL in Cappelle-Desprez × PBW343 RIL population effective in northern wheat belt of India.

Stripe rust caused by Puccinia striiformis f. sp. tritici is most important and devastating disease of wheat worldwide, which affects the grain yields, quality and nutrition. To elucidate, the genetic basis of resistance, a mapping population of recombinant inbred lines was developed from a cross between resistant Cappelle-Desprez and susceptible cultivar PBW343 using single-seed descent. Variety PBW343 had been one of the most popular cultivars of North Western Plains Zone, for more than a decade, before succumbing to the stripe rust. Cappelle-Desprez, a source of durable adult plant resistance, has maintained its resistance against stripe rust for a long time in Europe. Map construction and QTL analysis were completed with 1012 polymorphic (DArT and SSR) markers. Screenings for stripe rust disease were carried out in field condition for two consecutive crop seasons (2012-2013 and 2013-2014). Susceptible parent (PBW343) achieved a significant level of disease i.e., 100 % in both the years. In present investigations, resistance in Cappelle-Desprez was found stable and response to the rust ranged from 0 to 1.5 % over the years. The estimated broad-sense heritability (h 2) of stripe rust rAUDPC in the mapping population was 0.82. The relative area under the disease progress curve data showed continuous distributions, indicating that trait was controlled multigenically. Genomic region identified on chromosome 2D, was located within the short arm, with flanking markers (Xgwm484-Xcfd73), explained phenotypic variation (PVE) ranged from 13.9 to 31.8 %. The genomic region identified on chromosome 5B was found with the effect of maximum contribution with flanking DArT markers (1376633|F|0-1207571|F|0), PVE ranged from 24 to 27.0 %. This can, therefore, be utilized for marker assisted selection in developing much needed stripe rust resistant lines for the northern wheat belt of India.

Molecular genotyping of herbicide resistance in P. minor: ACCase resistance.

Little seed canary grass (Phalaris minor Retz.) populations resistant to herbicides that inhibit acetyl-CoA carboxylase (ACCase) represent an increasingly important weed control problem in northern India. The objective of this study was to develop DNA-based markers to differentiate herbicide-resistant and herbicide-susceptible population of P. minor. Primers were designed to amplify the conserved region carrying two reported mutations Trp2027 to Cys and Ile2041 to Asn conferring ACCase inhibitor resistance in several grass weeds and subjected to single-strand conformational polymorphism (SSCP) to detect the mutations. Five distinctive electrophoretic patterns on non-denaturing PAGE were observed, and four patterns were found to be associated with ACCase herbicide resistance in P. minor. The PCR-SSCP test developed in this study confirmed 17 resistant populations to contain mutations in CT domain of ACCase gene. This is the first report of rapid and easy molecular diagnosis of ACCase herbicide-resistant and herbicide-sensitive population of P. minor through PCR-SSCP analysis.