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.

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.

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.

Isolation, cloning and transgenic expression of hepatitis B surface antigen (HBsAg) in Solanum lycopersicum L.

The Hepatitis B virus (HBV) infection is one of the most widespread viral infections of humans. HBV causes acute and chronic hepatitis. Chronic hepatitis leads to hepatocellular carcinoma, which is a significant cause of death. DNA-based immunization programs to control the spread of Hepatitis B in developing countries are costly and require special storage and transportation. The alternative way is to express Hepatitis B surface antigen (HBsAg) in plants to develop oral vaccines. In this study, HBsAg gene was isolated, cloned, and then transformed in tomato plants. The transgenic tomato plants were confirmed through RT-qPCR. HBsAg expression was analysed in mature green and red stages of tomato fruit through quantitative real-time PCR. It was observed that expression of HBsAg was high in matured red tomato as compared to mature green. The present study is the first step to developing Solanum lycopersicum as an edible vaccine production system in this world region.

Role of Wheat Phosphorus Starvation Tolerance 1 Genes in Phosphorus Acquisition and Root Architecture.

The wheat plant requires elevated phosphorus levels for its normal growth and yield, but continuously depleting non-renewable phosphorus reserves in the soil is one of the biggest challenges in agricultural production worldwide. The Phosphorus Starvation Tolerance 1 (PSTOL1) gene has been reported to play a key role in efficient P uptake, deeper rooting, and high yield in rice. However, the function of the PSTOL1 gene in wheat is still unclear. In this study, a total of 22 PSTOL1 orthologs were identified in the wheat genome, and found that wheat PSTOL1 orthologs are unevenly distributed on chromosomes, and these genes were under strong purifying selection. Under different phosphorus regimes, wheat PSTOL1 genes showed differential expression patterns in different tissues. These results strengthen the classification of Pakistan-13 as a P-efficient cultivar and Shafaq-06 as a P-inefficient cultivar. Phenotypic characterization demonstrated that Pakistan-13 wheat cultivar has significantly increased P uptake, root length, root volume, and root surface area compared to Shafaq-06. Some wheat PSTOL1 orthologs are co-localized with phosphorus starvation's related quantitative trait loci (QTLs), suggesting their potential role in phosphorus use efficiency. Altogether, these results highlight the role of the wheat PSTOL1 genes in wheat P uptake, root architecture, and efficient plant growth. This comprehensive study will be helpful for devising sustainable strategies for wheat crop production and adaptation to phosphorus insufficiency.

Impact of Pre-Anthesis Drought Stress on Physiology, Yield-Related Traits, and Drought-Responsive Genes in Green Super Rice.

Optimum soil water availability is vital for maximum yield production in rice which is challenged by increasing spells of drought. The reproductive stage drought is among the main limiting factors leading to the drastic reduction in grain yield. The objective of this study was to investigate the molecular and morphophysiological responses of pre-anthesis stage drought stress in green super rice. The study assessed the performance of 26 rice lines under irrigated and drought conditions. Irrigated treatment was allowed to grow normally, while drought stress was imposed for 30 days at the pre-anthesis stage. Three important physiological traits including pollen fertility percentage (PFP), cell membrane stability (CMS), and normalized difference vegetative index (NDVI) were recorded at anthesis stage during the last week of drought stress. Agronomic traits of economic importance including grain yield were recorded at maturity stage. The analysis of variance demonstrated significant variation among the genotypes for most of the studied traits. Correlation and principal component analyses demonstrated highly significant associations of particular agronomic traits with grain yield, and genetic diversity among genotypes, respectively. Our study demonstrated a higher drought tolerance potential of GSR lines compared with local cultivars, mainly by higher pollen viability, plant biomass, CMS, and harvest index under drought. In addition, the molecular basis of drought tolerance in GSR lines was related to upregulation of certain drought-responsive genes including OsSADRI, OsDSM1, OsDT11, but not the DREB genes. Our study identified novel drought-responsive genes (LOC_Os11g36190, LOC_Os12g04500, LOC_Os12g26290, and LOC_Os02g11960) that could be further characterized using reverse genetics to be utilized in molecular breeding for drought tolerance.

Evaluation of Green Super Rice Lines for Agronomic and Physiological Traits under Salinity Stress.

Rice (Oryza sativa) is an important staple food crop worldwide, especially in east and southeast Asia. About one-third of rice cultivated area is under saline soil, either natural saline soils or irrigation with brackish water. Salinity stress is among the devastating abiotic stresses that not only affect rice growth and crop productivity but also limit its cultivation area globally. Plants adopt multiple tolerance mechanisms at the morphological, physiological, and biochemical levels to tackle salinity stress. To identify these tolerance mechanisms, this study was carried out under both a controlled glass house as well as natural saline field conditions using 22 green super rice (GSR) lines along with two local varieties ("IRRI 6 and Kissan Basmati"). Several morpho-physiological and biochemical parameters along with stress-responsive genes were used as evaluation criteria under normal and salinity stress conditions. Correlation and Principal Component Analysis (PCA) suggested that shoot-related parameters and the salt susceptible index (SSI) can be used for the identification of salt-tolerant genotypes. Based on Agglomerative Hierarchical Cluster (AHC) analysis, two saline-tolerant ("S19 and S20") and saline-susceptible ("S3 and S24") lines were selected for further molecular evaluation. Quantitative RT-PCR was performed, and results showed that expression of 1-5-phosphoribosyl -5-5-phosphoribosyl amino methylidene amino imidazole-4-carboxamide isomerase, DNA repair protein recA, and peptide transporter PTR2 related genes were upregulated in salt-tolerant genotypes, suggesting their potential role in salinity tolerance. However, additional validation using reverse genetics approaches will further confirm their specific role in salt tolerance. Identified saline-tolerant lines in this study will be useful genetic resources for future salinity breeding programs.

Genome-wide and molecular characterization of the DNA replication helicase 2 (DNA2) gene family in rice under drought and salt stress.

Rice plants experience various biotic (such as insect and pest attack) and abiotic (such as drought, salt, heat, and cold etc.) stresses during the growing season, resulting in DNA damage and the subsequent losses in rice production. DNA Replication Helicase/Nuclease2 (DNA2) is known to be involved in DNA replication and repair. In animals and yeast DNA2 are well characterized because it has the abilities of both helicase and nuclease, it plays a crucial role in DNA replication in the nucleus and mitochondrial genomes. However; they are not fully examined in plants due to less focused on plants damage repair. To fill this research gap, the current study focused on the genome-wide identification and characterization of OsDNA2 genes, along with analyses of their transcriptional expression, duplication, and phylogeny in rice. Overall, 17 OsDNA2 members were reported to be found on eight different chromosomes (2, 3, 4, 6, 7, 9, 10, and 11). Among these chromosomes (Chr), Chr4 contained a maximum of six OsDNA2 genes. Based on phylogenetic analysis, the OsDNA2 gene members were clustered into three different groups. Furthermore, the conserved domains, gene structures, and cis-regulatory elements were systematically investigated. Gene duplication analysis revealed that OsDNA2_2 had an evolutionary relationship with OsDNA2_14, OsDNA2_5 with OsDNA2_6, and OsDNA2_1 with OsDNA2_8. Moreover, results showed that the conserved domain (AAA_11 superfamily) were present in the OsDNA2 genes, which belongs to the DEAD-like helicase superfamily. In addition, to understand the post-transcriptional modification of OsDNA2 genes, miRNAs were predicted, where 653 miRNAs were reported to target 17 OsDNA2 genes. The results indicated that at the maximum, OsDNA2_1 and OsDNA2_4 were targeted by 74 miRNAs each, and OsDNA2_9 was less targeted (20 miRNAs). The three-dimensional (3D) structures of 17 OsDNA2 proteins were also predicted. Expression of OsDNA2 members was also carried out under drought and salt stresses, and conclusively their induction indicated the possible involvement of OsDNA2 in DNA repair under stress when compared with the control. Further studies are recommended to confirm where this study will offer valuable basic data on the functioning of DNA2 genes in rice and other crop plants.