High performance liquid chromatography (HPLC) based genetic diversity profiling of chilli germplasm for fruit pungency and phytochemical contents.

Chilli peppers are widely consumed for their pungency, as used in flavoring the food and has many pharmaceutical and medicinal properties. Based on these properties an experiment was held using 83 varieties of chilli (Hot pepper and sweet pepper) were grown in suitable environment using Augment Block design and evaluated for fruit pungency and phytochemical contents using high proficiency liquid chromatography. Analysis of variance (ANOVA) of traits showed highly significant for all traits except for fruit length and capsaicin contents. The value of Least significant increase (LSI)was ranged 0.27-1289.9 for all traits showed high variation among varieties. Highly significant correlation was found among fruit diameter to fruit weight 0.98, while moderate to high correlation was present among all traits. The most pungent genotype 24,634 was 4.8 g in weight, while the least pungent genotypes i.e. PPE-311 (32.8 g), green wonder (40.67) had higher in weight. The genotypes 24,627, 32,344, 32,368 and 1108 marked as higher number of seeds in their placental region. It was observed that chilli genotype 24,621 had maximum length with considerable high amount of pungency act as novel cultivar. Principal component analysis (PCA) showed the high variability of 46.97 for two PCs with the eigen value 2.6 and 1.63 was recorded. Biplot analysis showed a considerable variability for fruit pungency, while huge variability was found for all traits among given varieties. PPE-311, T5 and T3 are found as highly divergent for all traits. The findings of this study are instrumental for selecting parents to improve desirable traits in future chilli pepper breeding programs. It will help plant/vegetable breeders for development of highly nutrient and pungent varieties and attractive for the consumer of food sector.

Association mapping for abiotic stress tolerance using heat- and drought-related syntenic markers in okra.

BACKGROUND: Considerable production losses are caused by heat and drought stress in okra. Germplasm evaluation at genetic level is essential for the selection of promising genotypes. Lack of genomic information of okra limits the use of genetic markers. However, syntenic markers of some related family could be used for molecular characterization of major economic traits. METHODS AND RESULTS: Herein, 56 okra genotypes were evaluated for drought and heat tolerance. Sixty-one expressed sequence tags (ESTs) identified for heat and drought tolerance in cotton were searched from literature surveys and databases. The identified ESTs were BLAST searched into okra unigene database. Primers of selected okra unigenes were synthesized and amplified in all genotypes using standard polymerase chain reaction (PCR) protocol. Marker trait association (MTA) of the syntenic unigenes were identified between genotypic and phenotypic data on the basis of linkage disequilibrium Functional syntenic analysis revealed that out of these 61 cotton ESTs 55 had functional homology with okra unigenes. These 55 unigenes were used as markers for further analysis (amplification). Okra genotypes showed significance variations for all the physo-morphological parameters under heat and drought stress. Genotypes Perbhani Karanti, IQRA-III, Selection Super Green, Anmol and Line Bourd performed better under drought stress whereas genotypes Perbhani Karanti, IQRA-III, Green Gold, OK-1501 and Selection Super Green showed heat tolerance. Fifty markers showed amplification in okra. Fifty-six okra genotypes were clustered into three distinct populations. LD analysis has shown most significant linkage between markers Unigene43786 and Unigene3662. MTAs using MLM and GLM models revealed that 23 markers have significant associations (p < 0.05) with different traits under control and stressed conditions. Relative water content is associated with four markers (Unigene10673, Unigene99547, Unigene152901, and Unigene129684) under drought conditions. Whereas, Electrolyte leakage was associated with 3 markers (Unigene109922, Unigene28667 and Unigene146907) under heat stress. CONCLUSION: These identified unigenes may be helpful in the development of drought and heat tolerant genotypes in okra.

Genetic diversity and marker trait association for phytophthora resistance in chilli.

BACKGROUND: Chilli is an important commercial crop with positive returns tendency. Phytophthora root rot causes drastic damage to chilli plant. Dearth of detecting marker trait associations is a major hinderance in practicing marker assisted selection in chilli breeding. METHODS AND RESULTS: Herein, 110 chilli accessions were assessed for 15 agronomic traits under control and disease infected conditions for two crop seasons (2018-2019). The SSR genotyping revealed high values of major allele frequency (MAF = 0.70), genetic diversity (GD = 0.39) and Polymorphic Information Content (PIC = 0.31). Principal coordinate analysis and population structure analysis showed distribution of diverse genotypes in all groups by dividing 110 genotypes in three populations and nine sub-populations. The UPGMA based Archaeopteryx tree was in concordance with population structure analysis. Linkage disequilibrium analysis evaluated that LD decays within 3-10 bp. Marker trait association (MTA) revealed the associations of 35 SSRs with 14 morphological traits. The significant MTA for marker CAeMS073 with relative leaf damage (RLD, 0.183 R2) under control and treated conditions was consistently observed in both models. The markers, CAMS173 and CAMS194 were found to be strongly associated with RLD and Disease Index (DI), respectively. The absence of MTA was detected for height of first branch. CONCLUSION: The MTAs reported in this study can facilitate marker assisted breeding for developing chilli germplasm resistant against Phytophthora capsici.

Single nucleotide polymorphism based assessment of genetic diversity in local and exotic onion genotypes.

BACKGROUND: Onion is an economically important vegetable cultivated worldwide on a large scale. Liberal exchange of germplasm and frequent selection caused narrow genetic diversity in most crops, including onion. Thus, it is essential to estimate and understand genetic diversity before launching of any breeding program. The current study was conducted to explore genetic diversity among 39 short-day onion genotypes (indigenous and exotic). METHODS AND RESULTS: All the genotypes were evaluated for various phenotypic traits by using single nucleotide polymorphism (SNP) genotyping based on KASPar assays. Principal component analysis (PCA) was performed to determine the variability among genotypes. The four principal components with eigenvalue greater than 1 accounted for 67.5656% variability for quantitative traits, whereas first five principal components with eigenvalue greater than 0.7 accounted for 86.24% variation among the genotypes for qualitative traits. The principal component analysis identified diverse traits including bulb weight, bulb diameter, plant height, number of survived plants and vitamin C. These traits were further analyzed through ANOVA (Analysis of Variance) following augmented block design to describe genotypic variability for selected traits. Onion genotypes showed significant variation for bulb weight, bulb diameter and Vitamin C. Genotypic clustering based on PCA showed that 15 indigenous genotypes were clustered with exotic genotypes (14) while remaining indigenous genotypes (10) were distant. A total of 30 SNPs were used for assessment of genetic diversity out of these, 24 SNPs were detected with polymorphic loci (0.8%, heterozygosity), while only six markers were with monomorphic sites (0.2% heterozygosity). Subsequently, population structure analysis revealed three different populations indicating significant variability. CONCLUSION: Conclusively, a significant similarity between exotic and a group of indigenous genotypes indicates direct adoption of exotic genotypes or their sister lines. A further broadening of the genetic base is required and could be done by crossing distant genotypes.

SNP-Based Genome-Wide Association Mapping of Pollen Viability Under Heat Stress in Tropical Zea mays L. Inbred Lines.

Global environmental changes with more extreme episodes of heat waves are major threats to agricultural productivity. Heat stress in spring affects the reproductive stage of maize, resulting in tassel blast, pollen abortion, poor pollination, reduced seed set, barren ears and ultimately yield loss. As an aneamophelous crop, maize has a propensity for pollen abortion under heat stress conditions. To overcome the existing challenges of heat stress and pollen abortion, this study utilized a broad genetic base of maize germplasm to identify superior alleles to be utilized in breeding programs. A panel of 375 inbred lines was morpho-physiologically screened under normal and heat stress conditions in two locations across two consecutive planting seasons, 2017 and 2018. The exposure of pollen to high temperature showed drastic decline in pollen germination percentage. The average pollen germination percentage (PGP) at 35 and 45°C was 40.3% and 9.7%, respectively, an average decline of 30.6%. A subset of 275 inbred lines were sequenced using tunable genotyping by sequencing, resulting in 170,098 single nucleotide polymorphisms (SNPs) after filtration. Genome wide association of PGP in a subset of 122 inbred lines resulted in ten SNPs associated with PGP35°C (p ≤ 10-5), nine with PGP45°C (p ≤ 10-6-10-8) and ten SNPs associated with PGP ratio (p ≤ 10-5). No SNPs were found to be in common across PGP traits. The number of favorable alleles possessed by each inbred line for PGP35°C, PGP45°C, and the PGP ratio ranged between 4 and 10, 3-13 and 5-13, respectively. In contrast, the number of negative alleles for these traits ranged between 2 and 8, 3-13 and 3-13, respectively. Genetic mapping of yield (adjusted weight per plant, AWP-1) and flowering time (anthesis-silking interval, ASI) in 275 lines revealed five common SNPs: three shared for AWP-1 between normal and heat stress conditions, one for ASI between conditions, and one SNP, CM007648.1-86615409, was associated with both ASI and AWP-1. Variety selection can be performed based on these favorable alleles for various traits. These marker trait associations identified in the diversity panel can be utilized in breeding programs to improve heat stress tolerance in maize.

Nutraceutical profiling of elite onion germplasm and breeding hybrids with improved nutraceutical quality.

Onion (Allium cepa L) is a major reservoir of important nutraceutical ingredients. Herein, nutraceutical profiling of elite germplasm was assessed and hybrids with improved nutraceutical quality were selected. The nutraceutical components were screened through Fourier Transform Infrared Spectroscopy (FTIR) analysis (scan range 4000-400cm-1) followed by spectrophotometric/colorimetric quantification in oven dried bulb samples. Line × Tester (L×T) analysis was used to identify potential hybrids with better nutraceutical quality. Based on common functional groups obtained from FTIR analysis, as well as bulb color, the onion genotypes were categorized into six groups viz., white, yellowish brown, light brown, dark brown, brown and purplish brown. Results indicated that the purplish brown, yellowish brown and dark brown genotypes had maximum concentration of pyruvic acid, total flavonoids and total phenolic content, while vitamin C content showed weak association with color pigmentation. The onion variety 'Onion Swat' contained the highest level of pyruvic acid (17.18 µM) and 'MKS8823GO' had the highest vitamin C content (13.83mg/100mL). The L×T analysis revealed that out of 35 crosses, 'MKS-77127 × Onion Swat' and 'MKS-77127 × MKS777' were the best hybrids with improved nutraceutical quality. Further, observations for specific combining ability, general combining ability, genetic versus environmental variance, heritability and heterosis indicated that the studied parameters were genetically inherited and could be improved significantly by adopting an appropriate breeding strategy.

Genetic Potential and Inheritance Pattern of Phenological Growth and Drought Tolerance in Cotton (Gossypium Hirsutum L.).

Cotton has prime importance in the global economy and governs socio-economic affairs of the world. Water scarcity and high temperature are major constraints that badly affect cotton production, which shows the need for the development of drought-tolerant varieties. Ten cotton genotypes, including three drought-tolerant and seven susceptible, were identified from a panel of diverse cotton genotypes at the seedling stage under two contrasting water regimes. Three lines were crossed with seven testers under line × tester mating design. The 21 F1 cross combinations along with 10 parents were evaluated under 100% non-stress (NS) and 50% drought stress (DS) filed capacity to assess the effects of drought stress and its inheritance in the next generation. All the genotypes were evaluated till the maturity stage for combining ability, heritability, and other genetic factors to understand the drought tolerance mechanisms. The proportional contribution of lines in the total variance evidenced that lines had a significant higher contribution in total variance for days to boll opening (DBO) of 10% and proline contents (PC) of 13% under DS conditions. It indicates that lines contributed more positive alleles for such traits. Under DS condition, DTV-9 × BT-252 and DTV-9 × DTV-10 had maximum negative specific combining ability (SCA) effects for DBO. Simultaneously, DBO also had higher heritability (h2) which indicates its dominant gene action and meanwhile, the importance of these combinations for the early mature and short duration variety development. The results revealed that most of the studied traits, including days taken to maturity, yield traits, and physiological traits, are under significant genetic control, with a strong genetic basis and have a huge potential for improving drought tolerance in cotton. Drought tolerance was found to have a strong association with early maturity and agro-climatic conditions of the cultivated areas. Identified superior parents in this study are suggested to use in the future breeding program to advance the cotton growth and drought tolerance.

Phylogenetic and expression dynamics of tomato ClpB/Hsp100 gene under heat stress.

Heat shock proteins (Hsps) are stress-responsive molecular chaperones, which uphold proper protein folding in response to external and internal stresses. The Hsp100 gene family plays a substantial role in thermos-tolerance of plants. This study investigated evolutionary relationship and expression of ClpB/Hsp100 gene family in tomato under heat stress. Six SlHsp100 genes were identified using bioinformatics tools. In silico sub-cellular localization indicated that of these 6 ClpB/Hsp100 members, 4 are found in chloroplast, 1 in mitochondria and 1 in the cytoplasm. For evolutionary study, 36 SlHsp100 genes were included in the phylogenetic tree showing a hierarchical clustering shared by the members of the kingdoms Plantae, Archaea, Chromista, Fungi and Bacteria. A total 4 pairs of orthologous and 5 pairs of paralogous genes were identified. Functional divergence between different Hsp100 clusters showed considerable functional homology. Thermo-tolerance measured in terms of cell viability, cell membrane stability and pollen viability indicated that it was paralleled by thermal resistance of Hsps. Reverse transcriptase polymerase chain reaction was used to analyze gene expression in leaves of five-week-old tomato seedlings following exposure to heat stress (45°C) and control (25°C). Chloroplastic LeHSP110/ClpB gene was upregulated in all tomato genotypes after exposure to heat stress highlighting the crucial role of this gene family in acquired thermo-tolerance.

Effect of Drought Stress on Capsaicin and Antioxidant Contents in Pepper Genotypes at Reproductive Stage.

Pepper is one of the most important vegetables and spices in the world. Principal pungency is contributed by secondary metabolites called capsaicinoids, mainly synthesized in the placenta of pepper fruit. Various factors, including drought, limit pepper production. Flowering is one of the most sensitive stages affected by drought stress. The current study was conducted to determine the effect of drought on different pepper genotypes at the flowering and pod formation stages. Hot pepper (Pusajuala and Ghotki) and Bell pepper (Green Wonder and PPE-311) genotypes were subjected to drought (35% field capacity) at two different stages (flowering (DF) and pod formation (DP) stage). In comparison, control plants were maintained at 65% field capacity. The data regarding flowering survival rates, antioxidant protein activity, and proline content, were collected. Results indicated that parameters like flower survival percentage, number of fruits per plant, and fruit weight had significant differences among the genotypes in both treatments. A high proline level was observed in Green Wonder at the pod formation stage compared to other genotypes. Capsaicin contents of hot pepper genotypes were affected at the pod formation stage. Antioxidants like GPX were highly active (190 units) in Ghotki at pod formation. Bell pepper genotypes had a high APX activity, highly observed (100 units) in PPE-311 at pod formation, and significantly differ from hot pepper genotypes. In the catalase case, all the genotypes had the highest values in DP compared to control and DF, but Pusajuala (91 units) and Green Wonder (83 units) performed best compared to other genotypes. Overall, the results indicate that drought stress decreased reproductive growth parameters and pungency of pepper fruit as most of the plant energy was consumed in defense molecules (antioxidants). Therefore, water availability at the flowering and pod formation stage is critical to ensure good yield and pepper quality.

Incredible Role of Osmotic Adjustment in Grain Yield Sustainability under Water Scarcity Conditions in Wheat (Triticum aestivum L.).

Interrogations of local germplasm and landraces can offer a foundation and genetic basis for drought tolerance in wheat. Potential of drought tolerance in a panel of 30 wheat genotypes including varieties, local landraces, and wild crosses were explored under drought stress (DS) and well-watered (WW) conditions. Considerable variation for an osmotic adjustment (OA) and yield components, coupled with genotype and environment interaction was observed, which indicates the differential potential of wheat genotypes under both conditions. Reduction in yield per plant (YP), thousand kernel weight (TKW), and induction of OA was detected. Correlation analysis revealed a strong positive association of YP with directly contributing yield components under both environments, indicating the impotence of these traits as a selection-criteria for the screening of drought-tolerant genotypes for drylands worldwide. Subsequently, the association of OA with TKW which contributes directly to YP, indicates that wheat attains OA to extract more water from the soil under low water-potential. Genotypes including WC-4, WC-8 and LLR-29 showed more TKW under both conditions, among them; LLR-29 also has maximum OA and batter yield comparatively. Result provides insight into the role of OA in plant yield sustainability under DS. In this study, we figure out the concept of OA and its incredible role in sustainable plant yield in wheat.

Efficacy assessment of soft and hard acaricides against Varroa destructor mite infesting honey bee (Apis mellifera) colonies, through sugar roll method.

The parasitic mite Varroa destructor is amongst the most serious problems of honey bees, Apis mellifera (Hymenoptera: Apidae) around the world including Pakistan. The present study estimates the mite density through powdered sugar roll method and evaluates the effectiveness of five miticides (fluvalinate, flumethrin, amitraz, formic acid, and oxalic acid) on A. mellifera colonies in German modified beehives. The results indicated that by treating the bees with one strip and two strips of fluvalinate per colony; the mite population remained below the economic threshold level (ETL) for 14 days and 25 days, respectively. Treatment of flumthrin @1 strip and @ 2 strips per colony resulted in mite population suppressed for 14 days and 39 days, respectively below ETL. Application of Amitraz @ 2 mL per 1.5 L water after every three days interval on sealed brood effectively controlled mites below ETL for 21 days. Formic acid @10 mL per colony applied through plastic applicator proved effective (below 3 mites per bee sample) for 24 days and oxalic acid applied through shop towel method resulted in mite population control for fifteen days. Use of powdered sugar roll method for easy sampling of Varroa mites and application of acaricides on precise economic threshold level during different seasons of the year for integrated management of Varroa mite is hereby advocated by current studies.

Quantitative trait loci (QTL) mapping for physiological and biochemical attributes in a Pasban90/Frontana recombinant inbred lines (RILs) population of wheat (Triticum aestivum) under salt stress condition.

Salt stress causes nutritional imbalance and ion toxicity which affects wheat growth and production. A population of recombinant inbred lines (RILs) were developed by crossing Pasban90 (salt tolerant) and Frontana (salt suceptible) for identification of quantitative trait loci (QTLs) for physiological traits including relative water content, membrane stability index, water potential, osmotic potential, total chlorophyll content, chlorophyll a, chlorophyll b and biochemical traits including proline contents, superoxide dismutase, sodium content, potassium content, chloride content and sodium/potassium ratio by tagging 202 polymorphic simple sequence repeats (SSR) markers. Linkage map of RILs comprised of 21 linkage group covering A, B and D genome for tagging and maped a total of 60 QTLs with major and minor effect. B genome contributed to the highest number of QTLs under salt stress condition. Xgwm70 and Xbarc361 mapped on chromosome 6B was linked with Total chlorophyll, water potential and sodium content. The increasing allele for all these QTLs were advanced from parent Pasban90. Current study showed that Genome B and D had more potentially active genes conferring plant tolerance against salinity stress which may be exploited for marker assisted selection to breed salinity tolerant high yielding wheat varieties.

Genome-wide identification, classification, expression profiling and DNA methylation (5mC) analysis of stress-responsive ZFP transcription factors in rice (Oryza sativa L.).

Cytosine DNA methylation (5mC) is an epigenetic mark that regulates gene expression in plant responses to environmental stresses. Zinc-finger protein (ZFP) is the largest family of DNA-binding transcription factors that also plays an essential role in eukaryote. In plant we have already identified and characterized different useful ZFP-genes. While, the main objective of this research was to observe and identify more targeted stress responsive genes of ZFPs epigenetically throughout genome in rice for the first time. A comprehensive correlation analysis was performed through methylated DNA immunoprecipitation (MeDIP)-chip hybridization in rice under salt and osmotic stresses. High salinity and drought are two major abiotic hazards that are destroying the crop world-wide. As a result, Through-out genome 14 unique stress responsive transcription factors of ZFP-genes with varying level of methylation and expression under two conditions (control vs. stress) were isolated. All the identified genes were confirmed from different databases for their specific structure, cis-regulatory elements, phylogenetic analysis, and synteny analysis. Moreover, the tissue-specific expression patterns, and expression under abiotic and phytohormones stresses were also investigated. Phylogenetically all the genes were divided into 6 distinct subgroups with Arabidopsis and orthologous proteins were find-out through synteny analysis. Available RNA-seq data in response to various phytohormones provided hormone inducible gene expression profile. Through Reverse Transcriptase qPCR (RT-qPCR) analysis tissue-specific expression in shoot and root over various time points against salt and osmotic stresses exhibited the diverse expression patterns of identified genes. Overall, the present study providing a foundation for in-depth characterization of identified genes and to further understand the epigenetic role of DNA methylation for genes expression and environmental stresses regulation in higher plant.

Functional analysis of OsHSBP1 and OsHSBP2 revealed their involvement in the heat shock response in rice (Oryza sativa L.).

The heat shock response (HSR) induces the production of heat shock proteins (HSPs) through the activation of heat shock factors (HSF). HSF binding protein (HSBP) is reported to modulate the function of HSF by binding to their trimer and hence to regulate HSR. This report describes the role of OsHSBP1 and OsHSBP2 in the regulation of the HSR and seed development of rice. Both genes expressed ubiquitously in all tissues under normal growth conditions while their expression levels were significantly increased during recovery after heat shock treatment. Subcellular localization revealed the cytosol-nuclear localization of both OsHSBP1 and OsHSBP2 in onion epidermal cells. The yeast two-hybrid assay depicted the self-binding ability of both genes. Both genes were also important for seed development, as their knock-down lines were associated with significant seed abortion. The thermotolerance assay revealed that OsHSBP1 and OsHSBP2 are negative regulators of HSR and involved in acquired thermotolerance but not in basal thermotolerance since their over-expression transgenic lines pre-heated at sublethal temperature, showed significantly decreased seedling survival after heat shock treatment. Furthermore, antioxidant activity and gene expression of catalase and peroxidase was significantly increased in knock-down transgenic seedlings of OsHSBP1 and OsHSBP2 after heat stress compared with the wild type. The expression of heat specific HSPs was also increased significantly in knockdown line of both genes but in a specific manner, suggesting the involvement of HSBP genes in different pathways. Overall, the present study reveals the role of OsHSBP1 and OsHSBP2 in the regulation of the HSR and seed development of rice.