Genetic Signature Controlling Root System Architecture in Diverse Spring Wheat Germplasm.

Roots are the main sensing organ, initiating multiple signaling pathways in response to abiotic factors, including nutrients, drought, and salt stress. A focus on improving the root system architecture is a key strategy to mitigate these stresses in wheat crop. In the present study, a diversity panel comprising indigenous landraces and historical cultivars from Pakistan was characterized for the root system architecture (RSA) and important loci were identified using a genome-wide association study (GWAS). RSA of the diversity panel was characterized 30 days after sowing in brunch tubes, and root images were taken. A high-throughput root imaging analysis using Rhizovision software was performed by setting the scale to extract the eight RSA traits and four plant biomass-related traits. GWAS identified 323 association signals for 12 root and biomass traits present on all wheat chromosomes, while the most important and reliable genetic loci (based on pleotropic loci and candidate genes) were identified on chromosomes 2A, 2B, 5A, 5D, 6A, 7B, and 7D for RSA. SNP annotation and transcriptome profiling identified nine candidate genes regulating the RSA and plant biomass traits, including ROOTLESS WITH UNDETECTABLE MERISTEM1, MYB TRANSCRIPTION FACTOR4, BRASSINOSTEROID INSENSITIVE1, SLENDER RICE1, AUXIN-RESPONSIVE FACTOR25, SCARECROW, NARROW LEAF2, PIN-FORMED1 AND PHOSPHATE TRANSCRIPTION FACTOR1. This study provided pre-breeding information for deep-rooting genotypes and associated markers that will accelerate the incorporation of such traits in breeding.

Mentha piperita silver nanoparticle-loaded hydrocolloid film for enhanced diabetic wound healing in rats.

OBJECTIVE: To investigate the role of Mentha piperita silver nanoparticle-loaded carbopol gel for enhanced wound healing in a diabetic rat model. This research further aims to explore bioactive compounds derived from Mentha piperita obtained from high altitude. METHOD: Methanolic extracts of Mentha piperita (MP), Mentha spicata (MS) and Mentha longifolia (ML) were used to synthesise silver nanoparticles (AgNP). AgNP synthesis was confirmed by ultraviolet-visible (UV-Vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The antioxidant activity was assessed by 2, 2-diphenyl-1-picrylhydrazyl (DDPH) assay. Antiglycation potential was determined by measuring the fluorescent advanced glycation end products. The bioactive compound identified in the Mentha piperita methanolic (MPM) fraction through electrospray ionisation tandem mass spectrometric analysis (ESI-MS) was responsible for the highest antiglycation. The effects of MPM and MPM.AgNP-loaded Carbopol (Sanare Lab, India) on wound healing were compared in male, alloxan-induced, diabetic albino rats (200-250g), divided into control and treated groups. Effects on wound healing were assessed via histopathology. RESULTS: UV-Vis and FTIR confirmed NP synthesis with peaks for flavonoids and polyphenols. SEM and XRD explored the cubical, 30-63nm crystalline NP. The maximum antioxidant and antiglycation potential was observed in order of; MP.AgNP>MS.AgNP>ML.AgNP. The highest antioxidant activity was observed by methanolic and aqueous MP.AgNPs (88.55% and 83.63%, respectively) at 2mg.ml-1, and (75.16% and 69.73%, respectively) at 1mg.ml-1, compared to ascorbic acid (acting as a positive control, 90.01%). MPM.AgNPs demonstrated the best antiglycation potential of 75.2% and 83.3% at 1mg.ml-1 and 2mg.ml-1, respectively, comparable to positive control (rutin: 88.1%) at 14 days post-incubation. A similar trend was observed for antimicrobial activity against Bacillus subtilis, Micrococcus luteus and Escherichia coli with an inhibition zone of 21mm, 21.6mm and 24.6mm. Rosmarinic acid was the active compound present in Mentha piperita, as identified by ESI-MS. MPM.AgNP-loaded Carbopol resulted in 100% wound closure compared with control at 20 days post-wounding. In the treatment group, re-epithelialisation was achieved by day 18, compared with 25 days for the positive control group. CONCLUSION: MPM.AgNP-loaded Carbopol demonstrated safer and more effective biological properties, hence accelerating the diabetic excision wound healing process in alloxan-induced diabetic rats.

CRISPR/Cas9 editing of wheat Ppd-1 gene homoeologs alters spike architecture and grain morphometric traits.

Mutations in Photoperiod-1 (Ppd-1) gene are known to modify flowering time and yield in wheat. We cloned TaPpd-1 from wheat and found high similarity among the three homoeologs of TaPpd-1. To clarify the characteristics of TaPpd-1 homoeologs in different photoperiod conditions for inflorescence architecture and yield, we used CRISPR/Cas9 system to generate Tappd-1 mutant plants by simultaneous modification of the three homoeologs of wheat Ppd-1. Tappd-1 mutant plants showed no off-target mutations. Four T0-edited lines under short-day length and three lines under long-day length conditions with the mutation frequency of 25% and 21%, respectively. These putative transgenic plants of all the lines were self-fertilized and generated T1 and T2 progenies and were evaluated by phenotypic and expression analysis. Results demonstrated that simultaneously edited TaPpd-1- A1, B1, and D1 homoeologs gene copies in T2_SDL-8-4, T2_SDL-4-5, T2_SDL-3-9, and T2_LDL-10-9 showed similar spike inflorescence, flowering time, and significantly increase in 1000-grain weight, grain area, grain width, grain length, plant height, and spikelets per spike due to mutation in both alleles of Ppd-B1 and Ppd-D1 homoeologs but only spike length was decreased in T2_SDL-8-4, T2_SDL-4-5, and T2_LDL-13-3 mutant lines due to mutation in both alleles of Ppd-A1 homoeolog under both conditions. Our results indicate that all TaPpd1 gene homoeologs influence wheat spike development by affecting both late flowering and earlier flowering but single mutant TaPpd-A1 homoeolog affect lowest as compared to the combination with double mutants of TaPpd-B1 and TaPpd-D1, TaPpd-A1 and TaPpd-B1, and TaPpd-A1 and TaPpd-D1 homoeologs for yield enhancement. Our findings further raised the idea that the relative expression of the various genomic copies of TaPpd-1 homoeologs may have an impact on the spike inflorescence architecture and grain morphometric features in wheat cultivars.

Comprehensive insights into the regulatory mechanisms of lncRNA in alkaline-salt stress tolerance in rice.

BACKGROUND: Alkaline-salt is one of the abiotic stresses that slows plant growth and developmental processes and threatens crop yield. Long non-coding RNAs (lncRNAs) are endogenous RNA found in plants that engage in a variety of cellular functions and stress responses. METHOD: lncRNAs act as competing endogenous RNAs (ceRNA) and constitute a new set of gene control. The precise regulatory mechanism by which lncRNAs function as ceRNAs in response to alkaline-salt stress remains unclear. We identified alkaline-salt responsive lncRNAs using transcriptome-wide analysis of two varieties including alkaline-salt tolerant [WD20342 (WD)] and alkaline-salt sensitive [Caidao (CD)] rice cultivar under control and alkaline-salt stress treated [WD20342 (WDT, and Caidao (CDT)] conditions. RESULTS: Investigating the competitive relationships between mRNAs and lncRNAs, we next built a ceRNA network involving lncRNAs based on the ceRNA hypothesis. Expression profiles revealed that a total of 65, 34, and 1549 differentially expressed (DE) lncRNAs, miRNAs, and mRNAs were identified in alkaline-salt tolerant WD (Control) vs. WDT (Treated). Similarly, 75 DE-lncRNAs, 34 DE-miRNAs, and 1725 DE-mRNAs (including up-regulated and down-regulated) were identified in alkaline-salt sensitive CD (Control) vs. CDT (Treated), respectively. An alkaline-salt stress ceRNA network discovered 321 lncRNA-miRNA-mRNA triplets in CD and CDT, with 32 lncRNAs, 121 miRNAs, and 111 mRNAs. Likewise, 217 lncRNA-miRNA-mRNA triplets in WD and WDT revealed the NONOSAT000455-osa_miR5809b-LOC_Os11g01210 triplet with the highest degree as a hub node with the most significant positive correlation in alkaline-salt stress response. CONCLUSION: The results of our investigation indicate that osa-miR5809b is dysregulated and plays a part in regulating the defense response of rice against alkaline-salt stress. Our study highlights the regulatory functions of lncRNAs acting as ceRNAs in the mechanisms underlying alkaline-salt resistance in rice.

The comparative transcriptome analysis of two green super rice genotypes with varying tolerance to salt stress.

BACKGROUND: Salinity is one of the main abiotic factors that restrict plant growth, physiology, and crop productivity is salt stress. About 33% of the total irrigated land suffers from severe salinity because of intensive underground water extraction and irrigation with brackish water. Thus, it is important to understand the genetic mechanism and identify the novel genes involved in salt tolerance for the development of climate-resilient rice cultivars. METHODS AND RESULTS: In this study, two rice genotypes with varying tolerance to salt stress were used to investigate the differential expressed genes and molecular pathways to adapt under saline soil by comparative RNA sequencing at 42 days of the seedling stage. Salt-susceptible (S3) and -tolerant (S13) genotypes revealed 3982 and 3463 differentially expressed genes in S3 and S13 genotypes. The up-regulated genes in both genotypes were substantially enriched in different metabolic processes and binding activities. Biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and plant signal transduction mechanisms were highly enriched. Salt-susceptible and -tolerant genotypes shared the same salt adaptability mechanism with no significant quantitative differences at the transcriptome level. Moreover, bHLH, ERF, NAC, WRKY, and MYB transcription factors were substantially up-regulated under salt stress. 391 out of 1806 identified novel genes involved in signal transduction mechanisms. Expression profiling of six novel genes further validated the findings from RNA-seq data. CONCLUSION: These findings suggest that the differentially expressed genes and molecular mechanisms involved in salt stress adaptation are conserved in both salt-susceptible and salt-tolerant rice genotypes. Further molecular characterization of novel genes will help to understand the genetic mechanism underlying salt tolerance in rice.

Role of the type-B authentic response regulator gene family in fragrant rice under alkaline salt stress.

Globally, rice is being consumed as a main staple food and faces different kinds of biotic and abiotic stresses such drought, salinity, and pest attacks. Through the cytokinin signaling, Type-B authentic response regulators (ARR-Bs) respond positively towards the environmental stimuli. ARR-Bs are involved in abiotic stress tolerance and plant development but their molecular mechanisms in fragrant rice are still not fully explored. The current study showed the genome-wide characterization of OsARR-B genes under alkaline salt stress. Results showed that in total, 24 OsARR-B genes were found and divided into four subgroups on the basis of a phylogenetic analysis. These genes were located on all rice chromosomes except 8 and 10. Analysis of gene duplications, gene structure, cis-elements, protein-protein interactions, and miRNA were performed. Gene ontology analysis showed that OsARR-B genes are involved in plant development through the regulation of molecular functions, biological processes, and cellular components. Furthermore, 117 and 192 RNA editing sites were detected in chloroplast and mitochondrial genes, respectively, encoding proteins of OsARR-B. In chloroplast and mitochondrial genes, six and nine types of amino acid changes, respectively, were caused by RNA editing, showing that RNA editing has a role in the alkaline salt stress tolerance in fragrant rice. We also used a comparative transcriptome approach to study the gene expression changes in alkaline tolerant and susceptible genotypes. Under alkaline salt stress, OsARR-B5, OsARR-B7, OsARR-B9, OsARR-B10, OsARR-B16, OsARR-B22, and OsARR-B23 showed higher transcript levels in alkaline salt tolerant genotypes as compared to susceptible ones. Quantitative RT-PCR showed upregulation of gene expression in the alkaline tolerant genotypes under alkaline stress. Our study explored the gene expression profiling and RESs of two rice contrasting genotypes, which will help to understand the molecular mechanisms of alkaline salt tolerance in fragrant rice.

Comparative genomic analysis of human GLI2 locus using slowly evolving fish revealed the ancestral gnathostome set of early developmental enhancers.

BACKGROUND: The zinc finger-containing transcription factor Gli2, is a key mediator of Hedgehog (Hh) signaling and participates in embryonic patterning of various organs including the central nervous system (CNS) and limbs. Abnormal expression of Gli2 can impede the transcription of Hh target genes through disruption of proper balance between Gli2 and Gli3 functions. Therefore, delineation of enhancers that are required for complementary roles of Glis would allow the interrogation of those pathogenic variants that cause gene dysregulation, and a corresponding abnormal phenotype. Previously, we reported tissue-specific enhancers for Gli family including Gli2 through direct tetrapod-teleost comparisons. RESULTS: Here, we employed the sequence alignments of slowly evolving spotted gar and elephant shark and have identified six novel conserved noncoding elements in human GLI2 containing locus. Zebrafish-based transgenic assays revealed that combined action of these autonomous CNEs reflects many aspects of Gli2 specific endogenous transcriptional activity, including CNS and pectoral fins. CONCLUSION: Taken together with our previous findings, this study suggests that Hh-signaling controlled deployment of Gli2 activity in embryonic patterning arose in the common ancestor of gnathostomes. These GLI2 specific cis-regulatory modules will help to identify DNA variants that probably reside outside of coding intervals and are associated with congenital anomalies.

Genome-wide characterization and expression analysis of pseudo-response regulator gene family in wheat.

Pseudo-response regulator (PRR) gene family members play a significant role in plant circadian clocks, flowering time inflorescence architecture development during transition from vegetative growth phase to reproductive phase. In current study, we analyzed the expression profiling, phylogenetic relationship, and molecular characterization of PRR gene family members of common wheat by using IWGSC Ref seq v1.1 wheat genome database with a coverage rate of 90%. By using bioinformatic approach total 20 candidate gene sequences were identified and divided into six groups and four clades. It was found that mostly genes have same number of exons and introns showed similar features because they originated through duplication events during evolution processes. Although all the proteins have conserved PRR domains, but some are distinct in their sequences suggesting functional divergence. By comparative synteny analysis it was revealed that Group 1, 2, 3 and 11-D of group 4 have duplication events while group 5 and TaPRR9-B,10-D showed conservation with previously identified PRR members from rice. While expression variation of six groups from each analysis matches with each other. Five groups highly expressed in leaf, spike, and roots in pattern like leaf > spike > root at all three stages booting, heading and anthesis of spike development. This suggests that TaPRR genes play important roles in different photoperiod signaling pathways in different organs at different stages of spike development and flowering via unknown pathway. These findings will also provide comprehensive knowledge about future investigations on wheat PRR family members involved in complex network of circadian system for plant development.

Cis-regulatory control of human GLI2 expression in the developing neural tube and limb bud.

BACKGROUND: GLI2, a zinc finger transcription factor, mediates Sonic hedgehog signaling, a critical pathway in vertebrate embryogenesis. GLI2 has been implicated in diverse set of embryonic developmental processes, including patterning of central nervous system and limbs. In humans, mutations in GLI2 are associated with several developmental defects, including holoprosencephaly and polydactyly. RESULTS: Here, we demonstrate in transient transgenic zebrafish assays, the potential of a subset of tetrapod-teleost conserved non-coding elements (CNEs) residing within human GLI2 intronic intervals to induce reporter gene expression at known regions of endogenous GLI2 transcription. The regulatory activities of these elements are observed in several embryonic domains, including neural tube and pectoral fin. Moreover, our data reveal an overlapping expression profile of duplicated copies of an enhancer during zebrafish evolution. CONCLUSIONS: Our data suggest that during vertebrate history GLI2 acquired a high level of complexity in the genetic mechanisms regulating its expression during spatiotemporal patterning of the central nervous system (CNS) and limbs.

WsMAGO2, a duplicated MAGO NASHI protein with fertility attributes interacts with MPF2-like MADS-box proteins.

MAIN CONCLUSION: WsMAGO2 a duplicated protein in Withania through interactions with MPF2-like proteins affects male fertility by producing fewer flowers and aborted non-viable pollens/seeds regulated by anther-specific GAATTTGTGA motif. The MAGO NASHIs are highly conserved genes that encode proteins known to be involved in RNA physiology and many other developmental processes including germ cell differentiation in animals. However, their structural and functional implications in plants as fertility function proteins remained fragmented. MAGO (shorter name of MAGO NASHI) proteins form heterodimers with MPF2-like MADS-box proteins which are recruited in calyx identity and male fertility in Solanaceous plants. Four MAGO genes namely WsMAGO1 and WsMAGO2 and TaMAGO1 and TaMAGO2 were isolated from Withania somnifera and Tubocapsicum anomalum, respectively. These genes have duplicated probably due to whole genome duplication event. Dysfunction of WsMAGO2 through double-stranded RNAi in Withania revealed suppression of RNA transcripts, non-viable pollens, fewer flowers and aborted non-viable seeds in the developing berry suggesting a role of this protein in many traits particularly male fertility. WsMAGO2 flaunted stronger yeast 2-hybrid interactions with MPF2-like proteins WSA206, WSB206 and TAB201 than other MAGO counterparts. The native transcripts of WsMAGO2 culminated in stamens and seed-bearing berries though other MAGO orthologs also exhibited expression albeit at lower level. Coding sequences of the two orthologs are highly conserved, but they differ substantially in their upstream promoter regions. Remarkably, WsMAGO2 promoter is enriched with many anther-specific cis-motifs common in fertility function genes promoters. Among them, disruption of GAATTTGTGA abolished YFP/GUS gene expression in anthers alluding towards its involvement in regulating expression of MAGO in anther. Our findings support a possible recruitment of WsMAGO2 in fertility trait in Withania. These genes have practical application in hybrid production through cytoplasmic male sterility maintenance for enhancement in crops yield.

Evaluation of functional kompetitive allele-specific PCR (KASP) markers for selection of drought-tolerant wheat (Triticum aestivum) genotypes.

Wheat (Triticum aestivum ) is a major crop around the globe and different techniques are being used for its productivity enhancement. Germplasm evaluation to improve crop productivity mainly depends on accurate phenotyping and selection of genotypes with a high frequency of superior alleles related to the trait of interest. Therefore, applying functional kompetitive allele-specific PCR (KASP) markers for drought-related genes is essential to characterise the genotypes for developing future climate-resilient wheat crop. In this study, eight functional KASP markers and nine morphological traits were employed to evaluate the 40 wheat genotypes for drought tolerance. Morphological traits showed significant variation (P ≤0.05) among the genotypes, except tiller count (TC), fresh root weight (FRW) and dry root weight (DRW). PCA biplot showed that 63.3% phenotypic variation was explained by the first two PCs under control treatment, while 70.8% variation was explained under drought treatment. It also indicated that root length (RL) and primary root (PR) have considerable variations among the genotypes under both treatments and are positively associated with each other. Hence, the findings of this study suggested that both these traits could be used as a selection criterion to classify the drought-tolerant wheat genotypes. KASP genotyping accompanied by morphological data revealed that genotypes Markaz, Bhakar Star, China 2, Aas and Chakwal-50 performed better under drought stress. These outperforming genotypes could be used as parents in developing drought-tolerant wheat genotypes. Hence, KASP genotyping assay for functional genes or significant haplotypes and phenotypic evaluation are prerequisites for a modern breeding program.

Functional evolution of cis-regulatory modules of STMADS11 superclade MADS-box genes.

Evolution of phenotypic morphologies is closely associated with modular organization of cis-regulatory elements underlying expression divergence. The MADS-box gene family is the subject of extensive studies that try to unscramble the structural complexity of flowering plants. This study is envisaged to explore the potential of CRMs in highly constrained non-coding elements of STMADS11superclade MADS-box genes in expression divergence. Phylogenetic reconstruction differentiated the STMADS11 genes into SVP-like, ZMM19-like, MPF1-like and MPF2-like clades. Differential gene expression in vegetative and floral organs was evident within the clades as well as at inter-clade level. The genomic DNA search for clusters of short motifs and sequence conservation of the -2 kb promoter region of particularly, MPF2-like clade permitted to establish three well defined CRMs where transcription factors bind, being CRM1 the activator, CRM2 the repressor, and CRM3 the enhancer element. Similar clusters were also mapped in the large 1st introns in the coding region. Within these CRMs many transcription factor-binding sites, particularly the hotspots for MADS-domain TF binding elements--CArG-boxes, directing sepal specific expression in Arabidopsis--were accrued in the CRM1 of MPF2-like promoters. Site-directed mutagenesis and motif swapping through reporter assays allude towards their implication as functionally active elements. In terms of directional evolution of MPF2-like promoters, CRMs are significantly more conserved than flanking regions, hence, bearing the signatures for purifying selection. Thus, CRMs are the pervasive feature of STMADS11 genes and mutations and/or appearance of new transcription factor binding sites and position of the CRMs are responsible for the divergence in expression patterns in this clade. These results have implications in understanding functional evolution of cis-regulatory modules in plants.

Production of Sitobion avenae-resistant Triticum aestivum cvs using laccase as RNAi target and its systemic movement in wheat post dsRNA spray.

Among the wheat biotic stresses, Sitobion avenae is one of the main factors devastating the wheat yield per hectare. The study's objective was to find out the laccase (lac) efficacy; as a potential RNAi target against grain aphids. The Sitobion avenae lac (Salac) was confirmed by Reverse Transcriptase-PCR. Gene was sequenced and accession number "ON703252" was allotted by GenBank. ERNAi tool was used to design 143 siRNA and one dsRNA target. 69% mortality and 61% reduction in lac expression were observed 8D-post lac DsRNA feeding. Phylogenetic analysis displayed the homology of grain aphid lac gene with peach potato, pea, and Russian wheat aphids. While Salac protein was found similar to the Russian grain, soybean, pea, and cedar bark aphid lac protein multi-copper oxidase. The dsRNAlac spray-induced silencing shows systematic translocation from leaf to root; with maximum lac expression found in the root, followed by stem and leaf 9-13D post-spray; comparison to control. RNAi-GG provides the Golden Gate cloning strategy with a single restriction ligation reaction used to achieve lac silencing. Agrobacterium tumefaciens mediated in planta and in-vitro transformation was used in the study. In vitro transformation, Galaxy 2012 yielded a maximum transformation efficiency (1.5%), followed by Anaj 2017 (0.8%), and Punjab (0.2%). In planta transformation provides better transformation efficiencies with a maximum in Galaxy 2012 (16%), and a minimum for Punjab (5%). Maximum transformation efficiency was achieved for all cultivars with 250 µM acetosyringone and 3h co-cultivation. Galaxy 2012 exhibited maximum transformation efficiency, and aphid mortality post-feeding transgenic wheat.

Evaluation of sugarcane promising clones based on the morphophysiological traits developed from fuzz.

Sugarcane is one of the critical commercial crops and principal sources of ethanol and sugar worldwide. Unfavorable conditions and poor seed setting rates hinder variety development in sugarcane. Countries like Pakistan directly import fuzz (true seed) and other propagation material from the USA, China, Brazil, etc. In this study, we imported fuzz from China, developed 29 genotypes germinating in the glasshouse, and evaluated at field conditions along with two local checks (CPF-251 and HSF-240). Morphophysiological data were recorded, including plant height (PH), cane length (CL), internodal length (IL), tiller number (TN), brix percentage (B), cane diameter (CD), chlorophyll a (Chl. a), chlorophyll b (Chl. b), and total chlorophyll (T. Chl). Results showed highly significant (p < 0.001) differences among the sugarcane accessions for all the studied traits. High broad-sense heritability (81.89% to 99.91%) was recorded for all the studied parameters. Genetic Advance (GA) ranges from 4.6% to 65.32%. The highest GA was observed for PH (65.32%), followed by CL (63.28%). Chlorophyll leaching assay was also performed at different time points (0, 50, 100, 150, and 200 min). All the genotypes showed the same leaching trend at all times, and better performing genotypes showed less leaching compared to poor performing, indicating the high amount of cutin and wax on the leaf surface. Correlation analysis showed that PH, CL, IL, and TN had significant associations. Principal components analysis (PCA) further confirms these results. Based on PCA and correlation results, PH, CL, IL, and TN can be utilized as a selection criterion for sugarcane improvement. Genotypes such as NS-4a, NS-5, NS-6, NS-8, NS-9, and NS-15 are recommended for future breeding programs related to sugarcane variety development.

RNAseq of diverse spring wheat cultivars released during last 110 years.

Here, we performed RNA-seq based expression analysis of root and leaf tissues of a set of 24 historical spring wheat cultivars representing 110 years of temporal genetic variations. This huge 130 tissues RNAseq dataset was initially used to study expression pattern of 97 genes regulating root growth and development in wheat. Root system architecture (RSA) is an important target for breeding stress-resilient and high-yielding wheat cultivars under climatic fluctuations. However, root transcriptome analysis is usually obscured due to challenges in root research due to their below ground presence. We also validated the dataset by performing correlation analysis between expression of RSA related genes in roots and leaves with 25 root traits analyzed under varying moisture conditions and 10 yield-related traits. The Pearson's correlation coefficients between root phenotypes and expression of root-specific genes varied from -0.72 to 0.78, and strong correlations with genes such as DRO1, TaMOR, ARF4, PIN1 was observed. The presented datasets have multiple uses such as a) studying the change in expression pattern of genes during time, b) differential expression of genes in two very important tissues of wheat i.e., leaf and roots, and c) studying customized expression of genes associated with important phenotypes in diverse wheat cultivars. The initial findings presented here provided key insights into understanding the transcriptomic basis of phenotypic variability of RSA in wheat cultivars.

Divergences of MPF2-like MADS-domain proteins have an association with the evolution of the inflated calyx syndrome within Solanaceae.

The inflated calyx syndrome (ICS) is a post-floral novelty within Solanaceae. Previous work has shown that MPF2-like MADS-box genes have been recruited for the development and evolution of ICS through heterotopic expression from vegetative to floral organs. ICS seems to be a plesiomorphic trait in Physaleae, but it has been secondarily lost in some lineages during evolution. We hypothesized that molecular and functional divergences of MPF2-like proteins might play a role in the loss of ICS. In this study we analyzed the phylogeny, selection and various functions of MPF2-like proteins with respect to the evolution of ICS. Directional selection of MPF2-like orthologs toward evolution of ICS was detected. While auto-activation capacity between proteins varies in yeast, MPF2-like interaction with floral MADS-domain proteins is robustly detected, hence substantiating their integration into the floral developmental programs. Dimerization with A- (MPF3) and E-function (PFSEP1/3) proteins seems to be essential for ICS development within Solanaceae. Moreover, the occurrence of the enlarged sepals, reminiscent of ICS, and MPF2-like interactions with these specific partners were observed in transgenic Arabidopsis. The interaction spectrum relevant to ICS seems to be plesiomorphic, reinforcing the plesiomorphy of this trait. The inability of some MPF2-like to interact with either the A-function or any of the E-function partners characterized is correlated with the loss of ICS in the lineages that showed a MPF2-like expression in the calyx. Our findings suggest that, after recruitment of MPF2-like genes for floral development, diversification in their coding region due to directional selection leads to a modification of the MADS-domain protein interacting spectrum, which might serve as a constraint for the evolution of ICS within Solanaceae.

Decoding RNA Editing Sites Through Transcriptome Analysis in Rice Under Alkaline Stress.

Ribonucleic acid editing (RE) is a post-transcriptional process that altered the genetics of RNA which provide the extra level of gene expression through insertion, deletions, and substitutions. In animals, it converts nucleotide residues C-U. Similarly in plants, the role of RNA editing sites (RES) in rice under alkaline stress is not fully studied. Rice is a staple food for most of the world population. Alkaline stress cause reduction in yield. Here, we explored the effect of alkaline stress on RES in the whole mRNA from rice chloroplast and mitochondria. Ribonucleic acid editing sites in both genomes (3336 RESs) including chloroplast (345 RESs) and mitochondria (2991 RESs) with average RES efficiency ∼55% were predicted. Our findings showed that majority of editing events found in non-synonymous codon changes and change trend in amino acids was hydrophobic. Four types of RNA editing A-G (A-I), C-T (C-U), G-A, and T-C were identified in treated and untreated samples. Overall, RNA editing efficiency was increased in the treated samples. Analysis of Gene Ontology revealed that mapped genes were engaged in many biological functions and molecular processes. We also checked the expression of pentatricopeptide repeat (PPR), organelle zinc-finger (OZI), and multiple organellar RNA editing factors/RNA editing factor interacting proteins genes in control and treatment, results revealed upregulation of PPR and OZ1 genes in treated samples. This induction showed the role of these genes in RNA editing. The current findings report that RNA editing increased under alkaline stress which may contribute in adaptation for rice by changing amino acids in edited genes (88 genes). These findings will provide basis for identification of RES in other crops and also will be useful in alkaline tolerance development in rice.

Unveiling forensically relevant biogeographic, phenotype and Y-chromosome SNP variation in Pakistani ethnic groups using a customized hybridisation enrichment forensic intelligence panel.

Massively parallel sequencing following hybridisation enrichment provides new opportunities to obtain genetic data for various types of forensic testing and has proven successful on modern as well as degraded and ancient DNA. A customisable forensic intelligence panel that targeted 124 SNP markers (67 ancestry informative markers, 23 phenotype markers from the HIrisplex panel, and 35 Y-chromosome SNPs) was used to examine biogeographic ancestry, phenotype and sex and Y-lineage in samples from different ethnic populations of Pakistan including Pothwari, Gilgit, Baloach, Pathan, Kashmiri and Siraiki. Targeted sequencing and computational data analysis pipeline allowed filtering of variants across the targeted loci. Study samples showed an admixture between East Asian and European ancestry. Eye colour was predicted accurately based on the highest p-value giving overall prediction accuracy of 92.8%. Predictions were consistent with reported hair colour for all samples, using the combined highest p-value approach and step-wise model incorporating probability thresholds for light or dark shade. Y-SNPs were successfully recovered only from male samples which indicates the ability of this method to identify biological sex and allow inference of Y-haplogroup. Our results demonstrate practicality of using hybridisation enrichment and MPS to aid in human intelligence gathering and will open many insights into forensic research in South Asia.

Genome-wide investigation and functional analysis of RNA editing sites in wheat.

Wheat is an important cereal and half of the world population consumed it. Wheat faces environmental stresses and different techniques (CRISPR, gene silencing, GWAS, etc.) were used to enhance its production but RNA editing (RESs) is not fully explored in wheat. RNA editing has a special role in controlling environmental stresses. The genome-wide identification and functional characterization of RESs in different types of wheat genotypes was done. We employed six wheat genotypes by RNA-seq analyses to achieve RESs. The findings revealed that RNA editing events occurred on all chromosomes equally. RNA editing sites were distributed randomly and 10-12 types of RESs were detected in wheat genotypes. Higher number of RESs were detected in drought-tolerant genotypes. A-to-I RNA editing (2952, 2977, 1916, 2576, 3422, and 3459) sites were also identified in six wheat genotypes. Most of the genes were found to be engaged in molecular processes after a Gene Ontology analysis. PPR (pentatricopeptide repeat), OZ1 (organelle zinc-finger), and MORF/RIP gene expression levels in wheat were also examined. Normal growth conditions diverge gene expression of these three different gene families, implying that normal growth conditions for various genotypes can modify RNA editing events and have an impact on gene expression levels. While the expression of PPR genes was not change. We used Variant Effect Predictor (VEP) to annotate RNA editing sites, and Local White had the highest RESs in the CDS region of the protein. These findings will be useful for prediction of RESs in other crops and will be helpful in drought tolerance development in wheat.

CRISPR/Cas9 mediated disruption of Inositol Pentakisphosphate 2-Kinase 1 (TaIPK1) reduces phytic acid and improves iron and zinc accumulation in wheat grains.

Introduction: Phytic acid (PA) is an important antinutrient agent present in cereal grains which reduces the bioavailability of iron and zinc in human body, causing malnutrition. Inositol pentakisphosphate 2- kinase 1 (IPK1) gene has been reported to be an important gene for PA biosynthesis. Objective: A recent genome editing tool CRISPR/Cas9 has been successfully applied to develop biofortified rice by disrupting IPK1 gene, however, it remained a challenge in wheat. The aim of this study was to biofortify wheat using CRISPR/Cas9. Methods: In this study, we isolated 3 TaIPK1 homeologs in wheat designated as TaIPK1.A, TaIPK1.B and TaIPK1.D and found that the expression abundance of TaIPK1.A was stronger in early stages of grain filling. Using CRISPR/Cas9, we have disrupted TaIPK1.A gene in cv. Borlaug-2016 with two guide RNAs targeting the 1st and 2nd exons. Results: We got several genome-edited lines in the T0 generation at frequencies of 12.7% and 10.8%. Sequencing analysis revealed deletion of 1-23 nucleotides and even an addition of 1 nucleotide in various lines. Analysis of the genome-edited lines revealed a significant decrease in the PA content and an increase in iron and zinc accumulation in grains compared with control plants. Conclusion: Our study demonstrates the potential application of CRISPR/Cas9 technique for the rapid generation of biofortified wheat cultivars.