Anther-derived microspore embryogenesis in pepper hybrids orobelle and Bomby.

BACKGROUND: Traditional breeding methods have long been employed worldwide for the evaluation and development of pepper cultivars. However, these methods necessitate multiple generations of screening, line development, evaluation, recognition, and crossing to obtain highly homozygous lines. In contrast, in vitro anther-derived microspore culture represents a rapid method to generate homozygous lines within a single generation. In the present study, we have optimized a protocol for microspore embryogenesis from anther cultures of pepper hybrids Orobelle and Bomby. RESULTS: We achieved early and successful embryo formation from both genotypes by subjecting the buds to a cold pretreatment at 4 °C for 4 days. Our optimized culture medium, comprised of MS medium supplemented with 4 mg/L NAA, 1 mg/L BAP, 0.25% activated charcoal, 2.6 g/L gelrite, 30 g/L sucrose, and 15 mg/L silver nitrate, exhibited the highest efficiency in embryo formation (1.85% and 1.46%) for Orobelle and Bomby, respectively. Furthermore, successful plant regeneration from the anther derived microspore embryos was accomplished using half-strength MS medium fortified with 2% sucrose and 0.1 mg/L 6-benzylaminopurine (BA), solidified with 2.6 g/L gelrite. The ploidy status of the microspore-derived plantlets was analyzed using flow cytometry technique. Notably, the haploid plants exhibited distinct characteristics such as reduced plant height, leaf length, leaf width, and shorter internode length when compared to their diploid counterparts derived from seeds. CONCLUSION: Our findings highlight the potential of anther culture and microspore embryogenesis as an advanced method for accelerating pepper breeding programs, enabling the rapid production of superior homozygous lines.

Candidate effectors for leaf rust resistance gene Lr28 identified through transcriptome and in-silico analysis.

Puccinia spp. causing rust diseases in wheat and other cereals secrete several specialized effector proteins into host cells. Characterization of these proteins and their interaction with host's R proteins could greatly help to limit crop losses due to diseases. Prediction of effector proteins by combining the transcriptome analysis and multiple in-silico approaches is gaining importance in revealing the pathogenic mechanism. The present study involved identification of 13 Puccinia triticina (Pt) coding sequences (CDSs), through transcriptome analysis, that were differentially expressed during wheat-leaf rust interaction; and prediction of their effector like features using different in-silico tools. NCBI-BLAST and pathogen-host interaction BLAST (PHI-BLAST) tools were used to annotate and classify these sequences based on their most closely matched counterpart in both the databases. Homology between CDSs and the annotated sequences in the NCBI database ranged from 79 to 94% and with putative effectors of other plant pathogens in PHI-BLAST from 24.46 to 54.35%. Nine of the 13 CDSs had effector-like features according to EffectorP 3.0 (≥0.546 probability of these sequences to be effector). The qRT-PCR expression analysis revealed that the relative expression of all CDSs in compatible interaction (HD2329) was maximum at 11 days post inoculation (dpi) and that in incompatible interactions (HD2329 + Lr28) was maximum at 3 dpi in seven and 9 dpi in five CDSs. These results suggest that six CDSs (>0.8 effector probability as per EffectorP 3.0) could be considered as putative Pt effectors. The molecular docking and MD simulation analysis of these six CDSs suggested that candidate Lr28 protein binds more strongly to candidate effector c14094_g1_i1 to form more stable complex than the remaining five. Further functional characterization of these six candidate effectors should prove useful for a better understanding of wheat-leaf rust interaction. In turn, this should facilitate effector-based leaf rust resistance breeding in wheat.

Unravelling the Genetic Basis of Moisture Deficit Stress Tolerance in Wheat for Seedling Vigour-Related Traits and Root Traits Using Genome-Wide Association Study.

A key abiotic stress that negatively affects seed germination, plant development, and crop yield is moisture deficit stress. Achieving higher vigour and uniform germination under stress conditions is essential for crop establishment and productivity and to enhance the yield. Hence, revealing wheat's capacity to withstand moisture deficit stress during seed germination and early growth stages is fundamental in improving its overall performance. However, the genetic regulation of moisture deficit stress tolerance during the seed germination phase remains largely unexplored. In this study, a total of 193 wheat genotypes were subjected to simulated moisture deficit stress using PEG-6000 (-0.4 MPa) during the seed germination stage. The induced moisture deficit stress significantly reduced various seedling-vigour-related traits. The genetic regions linked to these traits were found using a genome-wide association study (GWAS). The analysis identified 235 MTAs with a significance -log10(p) value of >4. After applying the Bonferroni correction, the study identified 47 unique single nucleotide polymorphisms (SNPs) that are linked to candidate genes important for the trait of interest. The current study emphasises the effectiveness of genome-wide association studies (GWAS) in identifying promising candidate genes, improving wheat seedling vigour and root traits, and offering essential information for the development of wheat cultivars tolerant to moisture deficit stress.

Cross species/genera transferability of simple sequence repeat markers, genetic diversity and population structure analysis in gladiolus (Gladiolus × grandiflorus L.) genotypes.

Background: Genetic analysis of gladiolus germplasm using simple sequence repeat (SSR) markers is largely missing due to scarce genomic information. Hence, microsatellites identified for related genera or species may be utilized to understand the genetic diversity and assess genetic relationships among cultivated gladiolus varieties. Methods: In the present investigation, we screened 26 genomic SSRs (Gladiolus palustris, Crocus sativus, Herbertia zebrina, Sysirinchium micranthum), 14 chloroplast SSRs (Gladiolus spp., chloroplast DNA regions) and 25 Iris Expressed Sequence Tags (ESTs) derived SSRs across the 84 gladiolus (Gladiolus × grandiflorus L.) genotypes. Polymorphic markers detected from amplified SSRs were used to calculate genetic diversity estimates, analyze population structure, cluster analysis and principal coordinate analysis (PCoA). Results: A total of 41 SSRs showed reproducible amplification pattern among the selected gladiolus cultivars. Among these, 17 highly polymorphic SSRs revealed a total of 58 polymorphic alleles ranging from two to six with an average of 3.41 alleles per marker. Polymorphic information content (PIC) values ranged from 0.11 to 0.71 with an average value of 0.48. A total of 4 SSRs were selectively neutral based on the Ewens-Watterson test. Hence, 66.66% of Gladiolus palustris, 48% of Iris spp. EST, 71.42% of Crocus sativus SSRs showed cross-transferability among the gladiolus genotypes. Analysis of genetic structure of 84 gladiolus genotypes revealed two subpopulations; 35 genotypes were assigned to subpopulation 1, 37 to subpopulation 2 and the remaining 12 genotypes could not be attributed to either subpopulation. Analysis of molecular variance indicated maximum variance (53.59%) among individuals within subpopulations, whereas 36.55% of variation among individuals within the total population. The least variation (9.86%) was noticed between two subpopulations. Moderate (FST = 0.10) genetic differentiation between two subpopulations was observed. The grouping pattern of population structure was consistent with the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on simple matching dissimilarity coefficient and PCoA. Conclusion: SSR markers from the present study can be utilized for cultivar identification, conservation and sustainable utilization of gladiolus genotypes for crop improvement. Genetic relationships assessed among the genotypes of respective clusters may assist the breeders in selecting desirable parents for crossing.

Marker-assisted selection for transfer of QTLs to a promising line for drought tolerance in wheat (Triticum aestivum L.).

Wheat crop is subjected to various biotic and abiotic stresses, which affect crop productivity and yield. Among various abiotic stresses, drought stress is a major problem considering the current global climate change scenario. A high-yielding wheat variety, HD3086, has been released for commercial cultivation under timely sown irrigated conditions for the North Western Plain Zone (NWPZ) and North Eastern Plain Zone NEPZ of India. Presently, HD3086 is one of the highest breeder seed indented wheat varieties and has a stable yield over the years. However, under moisture deficit conditions, its potential yield cannot be achieved. The present study was undertaken to transfer drought-tolerant QTLs in the background of the variety HD3086 using marker-assisted backcross breeding. QTLs governing Biomass (BIO), Canopy Temperature (CT), Thousand Kernel Weight (TKW), Normalized Difference Vegetation Index (NDVI), and Yield (YLD) were transferred to improve performance under moisture deficit conditions. In BC1F1, BC2F1, and BC2F2 generations, the foreground selection was carried out to identify the plants with positive QTLs conferring drought tolerance and linked to traits NDVI, CT, TKW, and yield. The positive homozygous lines for targeted QTLs were advanced from BC2F2 to BC2F4 via the pedigree-based phenotypic selection method. Background analysis was carried out in BC2F5 and obtained 78-91% recovery of the recurrent parent genome in the improved lines. Furthermore, the advanced lines were evaluated for 2 years under drought stress to assess improvement in MABB-derived lines. Increased GWPS, TKW, and NDVI and reduced CT was observed in improved lines. Seven improved lines were identified with significantly higher yields in comparison to HD3086 under stress conditions.

Neuroimaging findings in 4342 hospitalized COVID-19 subjects: A multicenter report from the United States and Europe.

BACKGROUND AND PURPOSE: To determine the incidence of acute neuroimaging (NI) findings and comorbidities in the coronavirus disease of 2019 (COVID-19)-infected subjects in seven U.S. and four European hospitals. METHODS: This is a retrospective study of COVID-19-positive subjects with the following inclusion criteria: age >18, lab-confirmed COVID-19 infection, and acute NI findings (NI+) attributed to COVID-19 on CT or MRI brain. NI+ and comorbidities in total hospitalized COVID-19-positive (TN) subjects were assessed. RESULTS: A total of 37,950 COVID-19-positive subjects were reviewed and 4342 underwent NI. NI+ incidence in subjects with NI was 10.1% (442/4342) including 7.9% (294/3701) in the United States and 22.8% (148/647) in Europe. NI+ incidence in TN was 1.16% (442/37,950). In NI (4342), incidence of ischemic stroke was 6.4% followed by intracranial hemorrhage (ICH) (3.8%), encephalitis (0.5%), sinus venous thrombosis (0.2%), and acute disseminated encephalomyelitis (ADEM) (0.2%). White matter involvement was seen in 57% of NI+. Hypertension was the most common comorbidity (54%) before cardiac disease (28.8%) and diabetes mellitus (27.7%). Cardiac disease (p < .025), diabetes (p < .014), and chronic kidney disease (p < .012) were more common in the United States. CONCLUSION: This multicenter, multinational study investigated the incidence and spectrum of NI+ in 37,950 hospitalized adult COVID-19 subjects including regional differences in incidences of NI+, associated comorbidities, and other demographics. NI+ incidence in TN was 1.16% including 0.95% in the United States and 2.09% in Europe. ICH, encephalitis, and ADEM were common in Europe, while ischemic strokes were more common in the United States. In this cohort, incidence and distribution of NI+ helped characterize the neurological complications of COVID-19.

Development of bread wheat (Triticum aestivum L) variety HD3411 following marker-assisted backcross breeding for drought tolerance.

Marker-assisted backcross breeding enables selective insertion of targeted traits into the genome to improve yield, quality, and stress resistance in wheat. In the current investigation, we transferred four drought tolerance quantitative trait loci (QTLs) controlling traits, viz canopy temperature, normalized difference vegetative index, chlorophyll content, and grain yield from the drought-tolerant donor line, C306, into a popular high-yielding, drought-sensitive variety, HD2733. Marker-assisted selection coupled with stringent phenotypic screening was used to advance each generation. This study resulted in 23 improved lines carrying combinations of four drought tolerance QTLs with a range of 85.35%-95.79% background recovery. The backcross-derived lines gave a higher yield under moisture-deficit stress conditions compared with the recipient parent. They also showed higher phenotypic mean values for physiological traits and stability characteristics of HD2733. A promising genotype, HD3411, derived from this cross was identified for release after national multi-location coordinating trials under the All India Coordinated Wheat Improvement Project. Our study is a prime example of the advantages of precision breeding using integrating markers and phenotypic selection to develop new cultivars with desirable traits like drought tolerance.

Mapping of the QTLs governing grain micronutrients and thousand kernel weight in wheat (Triticum aestivum L.) using high density SNP markers.

Biofortification is gaining importance globally to improve human nutrition through enhancing the micronutrient content, such as vitamin A, iron, and zinc, in staple food crops. The present study aims to identify the chromosomal regions governing the grain iron concentration (GFeC), grain zinc concentration (GZnC), and thousand kernel weight (TKW) using recombinant inbred lines (RILs) in wheat, developed from a cross between HD3086 and HI1500. The experiment was conducted in four different production conditions at Delhi viz., control, drought, heat, and combined heat and drought stress and at Indore under drought stress. Grain iron and zinc content increased under heat and combined stress conditions, while thousand kernel weight decreased. Medium to high heritability with a moderate correlation between grain iron and zinc was observed. Out of 4,106 polymorphic markers between the parents, 3,407 SNP markers were used for linkage map construction which spanned over a length of 14791.18 cm. QTL analysis identified a total of 32 chromosomal regions governing the traits under study, which includes 9, 11, and 12 QTLs for GFeC, GZnC, and TKW, respectively. A QTL hotspot was identified on chromosome 4B which is associated with grain iron, grain zinc, and thousand kernel weight explaining the phenotypic variance of 29.28, 10.98, and 17.53%, respectively. Similarly, common loci were identified on chromosomes 4B and 4D for grain iron, zinc, and thousand kernel weight. In silico analysis of these chromosomal regions identified putative candidate genes that code for proteins such as Inositol 1,3,4-trisphosphate 5/6-kinase, P-loop containing nucleoside triphosphate hydrolase, Pleckstrin homology (PH) domains, Serine-threonine/tyrosine-protein kinase and F-box-like domain superfamily proteins which play role in many important biochemical or physiological process. The identified markers linked to QTLs can be used in MAS once successfully validated.

Molecular Mapping of Biofortification Traits in Bread Wheat (Triticum aestivum L.) Using a High-Density SNP Based Linkage Map.

A set of 188 recombinant inbred lines (RILs) derived from a cross between a high-yielding Indian bread wheat cultivar HD2932 and a synthetic hexaploid wheat (SHW) Synthetic 46 derived from tetraploid Triticum turgidum (AA, BB 2n = 28) and diploid Triticum tauschii (DD, 2n = 14) was used to identify novel genomic regions associated in the expression of grain iron concentration (GFeC), grain zinc concentration (GZnC), grain protein content (GPC) and thousand kernel weight (TKW). The RIL population was genotyped using SNPs from 35K Axiom® Wheat Breeder's Array and 34 SSRs and phenotyped in two environments. A total of nine QTLs including five for GPC (QGpc.iari_1B, QGpc.iari_4A, QGpc.iari_4B, QGpc.iari_5D, and QGpc.iari_6B), two for GFeC (QGfec.iari_5B and QGfec.iari_6B), and one each for GZnC (QGznc.iari_7A) and TKW (QTkw.iari_4B) were identified. A total of two stable and co-localized QTLs (QGpc.iari_4B and QTkw.iari_4B) were identified on the 4B chromosome between the flanking region of Xgwm149-AX-94559916. In silico analysis revealed that the key putative candidate genes such as P-loop containing nucleoside triphosphatehydrolase, Nodulin-like protein, NAC domain, Purine permease, Zinc-binding ribosomal protein, Cytochrome P450, Protein phosphatase 2A, Zinc finger CCCH-type, and Kinesin motor domain were located within the identified QTL regions and these putative genes are involved in the regulation of iron homeostasis, zinc transportation, Fe, Zn, and protein remobilization to the developing grain, regulation of grain size and shape, and increased nitrogen use efficiency. The identified novel QTLs, particularly stable and co-localized QTLs are useful for subsequent use in marker-assisted selection (MAS).

QTL mapping: insights into genomic regions governing component traits of yield under combined heat and drought stress in wheat.

Drought and heat frequently co-occur during crop growth leading to devastating yield loss. The knowledge of the genetic loci governing component traits of yield under combined drought and heat stress is essential for enhancing the climate resilience. The present study employed a mapping population of 180 recombinant inbred lines (RILs) derived from a cross between GW322 and KAUZ to identify quantitative trait loci (QTLs) governing the component traits of yield under heat and combined stress conditions. Phenotypic evaluation was conducted across two consecutive crop seasons (2021-2022 and 2022-2023) under late sown irrigation (LSIR) and late sown restricted irrigation (LSRI) conditions at the Indian Council of Agricultural Research Institute-Indian Agricultural Research Institute (ICAR-IARI), New Delhi. Various physiological and agronomic traits of importance were measured. Genotyping was carried out with 35K SNP Axiom breeder's genotyping array. The linkage map spanned a length of 6769.45 cM, ranging from 2.28 cM/marker in 1A to 14.21 cM/marker in 5D. A total of 35 QTLs were identified across 14 chromosomes with 6B containing the highest (seven) number of QTLs. Out of 35 QTLs, 16 were major QTLs explaining the phenotypic variance greater than 10%. The study identified eight stable QTLs along with two hotspots on chromosomes 6B and 5B. Five QTLs associated with traits thousand-grain weight (TGW), normalized difference vegetation index (NDVI), and plant height (PH) were successfully validated. Candidate genes encoding antioxidant enzymes, transcription factors, and growth-related proteins were identified in the QTL regions. In silico expression analysis highlighted higher expression of transcripts TraesCS2D02G021000.1, TraesCS2D02G031000, TraesCS6A02G247900, and TraesCS6B02G421700 under stress conditions. These findings contribute to a deeper understanding of the genetic architecture underlying combined heat and drought tolerance in wheat, providing valuable insights for wheat improvement strategies under changing climatic conditions.

Marker-assisted backcross breeding for heat tolerance in bread wheat (Triticum aestivum L.).

Manipulation of flowering time for adaptation through natural or genetic approaches may combat heat-stress damage that occurs at the reproductive stages in production conditions. HD2733, a popular wheat variety of the eastern plains of India, is largely sensitive to heat stress. Therefore, the current study aims to improve heat tolerance of HD2733 by introgression of QTLs associated with early anthesis and high kernel weight linked to markers Xbarc186 and Xgwm190, respectively, through marker-assisted backcross breeding (MABB) from a tolerant donor, WH730. A total of 124 simple sequence repeat (SSR) markers distributed evenly across the genome were used for the background selection. The alleles of Xbarc186 and Xgwm190 were fixed in BC2F1 and BC1F2 generations by selecting individual plants heterozygous for both marker loci and backcrossed with HD2733 and simultaneously selfed to generate BC2F1 and BC1F2 populations, respectively. Furthermore, the selected BC1F2 were selfed to generate the BC1F4 population. By background screening, a total of 39 BC2F3 and 21 BC1F4 families homozygous for the targeted QTLs with 90.9-97.9% and 86.8-88.3% RPG recoveries were selected. The best performing 17 BC2F3 and 10 BC1F4 lines were evaluated for various morpho-physiological traits. Phenotypic evaluation and multi-location trials of the introgressed lines under late sown conditions led to the selection of three promising lines with early anthesis and higher grain yield. The improved lines will serve as an excellent genetic material for functional genomics and expression studies to understand the molecular mechanisms and pathways underlying the stress tolerance.

Leaf rust responsive miRNA and their target genes in wheat.

Small RNA sequencing (sRNA-seq) and degradome analysis were used for the identification of miRNAs and their target host genes in a pair of near-isogenic lines (NILs), which differed for the presence of leaf rust resistance gene Lr28. The study led to identification of (i) 506 known and 346 novel miRNAs; and (ii) 5054 target genes including 4557 in silico predicted and 497 degradome-based genes using 105 differentially expressed (DE) miRNAs. A subset of 128 targets (67 in silico + 61 degradome-based) was differentially expressed in RNA-seq data that was generated by us earlier using the same pair of NILs; among these 128 targets, 58 target genes exhibited an inverse relationship with the DE miRNAs (expression of miRNAs and activation/suppression of target genes). Eight miRNAs which belonged to the conserved miRNA families and were known to be induced in response to fungal diseases in plants included the following: miR156, miR158, miR159, miR168, miR169, miR172, miR319, miR396. The target genes belonged to the following classes of genes known to be involved in downstream disease resistance pathways; peroxidases, sugar transporters, auxin response signaling, oxidation-reduction, etc. It was also noticed that although a majority of miRNAs and target genes followed the above classical inverse relationship, there were also examples, where no such relationship was observed. Among the target genes, there were also 51 genes that were not only regulated by miRNAs, but were also differentially methylated at sequences including the following segments: promotors, introns, TSS, exons. The results of the present study suggest a complex interplay among miRNA genes, target genes, and various epigenetic controls, which regulate the expression of genes involved in downstream pathways for disease resistance.

Marker assisted improvement for leaf rust and moisture deficit stress tolerance in wheat variety HD3086.

There is a significant yield reduction in the wheat crop as a result of different biotic and abiotic stresses, and changing climate, among them moisture deficit stress and leaf rust are the major ones affecting wheat worldwide. HD3086 is a high-yielding wheat variety that has been released for commercial cultivation under timely sown irrigated conditions in the Indo-Gangetic plains of India. Variety HD3086 provides a good, stable yield, and it is the choice of millions of farmers in India. It becomes susceptible to the most prevalent pathotypes 77-5 and 77-9 of Puccinia triticina (causing leaf rust) in the production environment and its potential yield cannot be realized under moisture deficit stress. The present study demonstrates the use of a marker-assisted back cross breeding approach to the successful transfer of leaf rust resistance gene Lr24 and QTLs linked to moisture deficit stress tolerance in the background of HD3086. The genotype HI1500 was used as a donor parent that possesses leaf rust-resistant gene Lr24, which confers resistance against the major pathotypes found in the production environment. It possesses inbuilt tolerance under abiotic stresses with superior quality traits. Foreground selection for gene Lr24 and moisture deficit stress tolerance QTLs linked to Canopy temperature (CT), Normal Differential Vegetation Index (NDVI) and Thousand Kernel Weight (TKW) in different generations of the backcrossing and selection. In BC2F2, foreground selection was carried out to identify homozygous lines based on the linked markers and were advanced following pedigree based phenotypic selection. The selected lines were evaluated against P. triticina pathotypes 77-5 and 77-9 under controlled conditions. Recurrent parent recovery of the selected lines ranged from 78-94%. The identified lines were evaluated for their tolerance to moisture stress under field conditions and their resistance to rust under artificial epiphytotic conditions for two years. In BC2F5 generation, eight positive lines for marker alleles were selected which showed resistance to leaf rust and recorded an improvement in component traits of moisture deficit stress tolerance such as CT, NDVI, TKW and yield compared to the recurrent parent HD3086. The derived line is named HD3471 and is nominated for national trials for testing and further release for commercial cultivation.

Identification of genomic regions of wheat associated with grain Fe and Zn content under drought and heat stress using genome-wide association study.

Wheat is the staple food crop of global importance for its grain nutrient quality. Grain iron and zinc content of the wheat grain is an important quantitatively inherited trait that is influenced by the environmental factors such as drought and heat stress. Phenotypic evaluation of 295 advanced breeding lines from the wheat stress breeding program of IARI was carried out under timely sown irrigated (IR), restricted irrigated, and late-sown conditions at New Delhi during the cropping season of 2020-21, and grain iron (GFeC) and zinc (GZnC) contents were estimated from both control and treatments. A statistically significant increase in GFeC and GZnC was observed under stress conditions compared to that of the control. Genotyping was carried out with the SNPs from the 35K Axiom Breeder's array, and marker-trait association was identified by GWAS analysis. Of the 23 MTAs identified, seven were linked with GFeC and sixteen were linked with GZnC. In silico analysis revealed a few important transcripts involved in various plant metabolism, growth, and development activities such as auxin response factor, root UVB sensitive proteins, potassium transporter, glycosyl transferase, COBRA, and F-box-like domain. The identified MTAs can be used for molecular breeding after validation and also for rapid development of micronutrient-rich varieties of wheat to mitigate hidden hunger.

Genome-wide association studies reveal putative QTLs for physiological traits under contrasting phosphorous conditions in wheat (Triticum aestivum L.).

A Genome-wide association (GWAS) study was conducted for phosphorous (P)-use responsive physiological traits in bread wheat at the seedling stage under contrasting P regimes. A panel of 158 diverse advanced breeding lines and released varieties, and a set of 10,800 filtered single nucleotide polymorphism (SNP) markers were used to study marker-trait associations over the eight shoot traits. Principle component analysis separated the two environments (P regimes) because of the differential response of the traits indicating the essentiality of the separate breeding programmes for each environment. Significant variations for genotypic, environmental, and genotype × environment (GEI) effects were observed for all the traits in the combined analysis of variance with moderately high broad sense heritability traits (0.50-0.73). With the different algorithms of association mapping viz., BLINK, FarmCPU, and MLM, 38 unique QTLs under non-limiting P (NLP) and 45 QTLs for limiting P (LP) conditions for various shoot traits were identified. Some of these QTLs were captured by all three algorithms. Interestingly, a Q.iari.dt.sdw.1 on chromosome 1D was found to explain the significant variations in three important physiological traits under non-limiting phosphorus (NLP) conditions. We identified the putative candidate genes for QTLs namely Q.iari.dt.chl.1, Q.iari.dt.sdw.16, Q.iari.dt.sdw.9 and Q.iari.dt.tpc.1 which are potentially involved in the mechanism regulating phosphorus use efficiency through improved P absorption due to improved root architectural traits and better mobilization such as sulfotransferase involved in postembryonic root development, WALLS ARE THIN1 (WAT1), a plant-specific protein that facilitates auxin export; lectin receptor-like kinase essentially involved in plant development, stress response during germination and lateral root development and F-box component of the SKP-Cullin-F box E3 ubiquitin ligase complex and strigolactone signal perception. Expression profiling of putative genes located in identified genomic regions against the wheat expression atlas revealed their significance based on the expression of these genes for stress response and growth development processes in wheat. Our results thus provide an important insight into understanding the genetic basis for improving PUE under phosphorus stress conditions and can shape the future breeding programme by developing and integrating molecular markers for these difficult-to-score important traits.

Genome-wide association mapping for component traits of drought and heat tolerance in wheat.

Identification of marker trait association is a prerequisite for marker-assisted breeding. To find markers linked with traits under heat and drought stress in bread wheat (Triticum aestivum L.), we performed a genome-wide association study (GWAS). GWAS mapping panel used in this study consists of advanced breeding lines from the IARI stress breeding programme produced by pairwise and complex crosses. Phenotyping was done at multi locations namely New Delhi, Karnal, Indore, Jharkhand and Pune with augmented-RCBD design under different moisture and heat stress regimes, namely timely sown irrigated (IR), timely sown restricted irrigated (RI) and late sown (LS) conditions. Yield and its component traits, viz., Days to Heading (DH), Days to Maturity (DM), Normalized Difference Vegetation Index (NDVI), Chlorophyll Content (SPAD), Canopy temperature (CT), Plant Height (PH), Thousand grain weight (TGW), Grain weight per spike (GWPS), Plot Yield (PLTY) and Biomass (BMS) were phenotyped. Analysis of variance and descriptive statistics revealed significant differences among the studied traits. Genotyping was done using the 35k SNP Wheat Breeder's Genotyping Array. Population structure and diversity analysis using filtered 10,546 markers revealed two subpopulations with sufficient diversity. A large whole genome LD block size of 7.15 MB was obtained at half LD decay value. Genome-wide association search identified 57 unique markers associated with various traits across the locations. Twenty-three markers were identified to be stable, among them nine pleiotropic markers were also identified. In silico search of the identified markers against the IWGSC ref genome revealed the presence of a majority of the SNPs at or near the gene coding region. These SNPs can be used for marker-assisted transfer of genes/QTLs after validation to develop climate-resilient cultivars.

BS-Seq reveals major role of differential CHH methylation during leaf rust resistance in wheat (Triticum aestivum L.).

Epigenetic regulation of the activity of defense genes during onset of diseases or resistance against diseases in plants is an active area of research. In the present study, a pair of wheat NILs for leaf rust resistance gene Lr28 (R) in the background of an Indian cultivar HD2329 (S) was used for a study of DNA methylation mediated regulation of gene expression. Leaf samples were collected at 0 h before (S0 and R0) and 96 h after inoculation (S96 and R96). The DNA samples were subjected to BS-Seq and sequencing data were used for identification of differentially methylated/demethylated regions/genes (DMRs and DMGs). Following four pairs of comparisons were used for this purpose: S0 vs S96; S0 vs R0; R0 vs R96; S96 vs R96. Major role of CHH methylation relative to that of CG and CHG methylation was observed. Some important observations include the following: (i) abundance of CHH methylation among DMRs; (ii) predominance of DMRs in intergenic region, relative to other genomic regions (promoters, exons, introns, TSS and TTS); (iii) abundance of transposable elements (TEs) in DMRs with CHH context; (iv) demethylation mediated high expression of genes during susceptible reaction (S0 vs S96) and methylation mediated low expression of genes during resistant reaction (R0 vs R96 and S96 vs R96); (v) major genes under regulation encode proteins, which differ from those encoded by genes regulated during susceptible reaction and (vi) ~ 500 DMGs carried differential binding sites for H3K4/K27me3 marks suggesting joint involvement of DNA and H3 methylation. Thus, CHH methylation either alone or in combination with histone methylation plays a major role in regulating the expression of genes involved in wheat-leaf rust interaction.

Genetic dissection of marker trait associations for grain micro-nutrients and thousand grain weight under heat and drought stress conditions in wheat.

Introduction: Wheat is grown and consumed worldwide, making it an important staple food crop for both its calorific and nutritional content. In places where wheat is used as a staple food, suboptimal micronutrient content levels, especially of grain iron (Fe) and zinc (Zn), can lead to malnutrition. Grain nutrient content is influenced by abiotic stresses, such as drought and heat stress. The best method for addressing micronutrient deficiencies is the biofortification of food crops. The prerequisites for marker-assisted varietal development are the identification of the genomic region responsible for high grain iron and zinc contents and an understanding of their genetics. Methods: A total of 193 diverse wheat genotypes were evaluated under drought and heat stress conditions across the years at the Indian Agricultural Research Institute (IARI), New Delhi, under timely sown irrigated (IR), restricted irrigated (RI) and late sown (LS) conditions. Grain iron content (GFeC) and grain zinc content (GZnC) were estimated from both the control and treatment groups. Genotyping of all the lines under study was carried out with the single nucleotide polymorphisms (SNPs) from Breeder's 35K Axiom Array. Result and Discussion: Three subgroups were observed in the association panel based on both principal component analysis (PCA) and dendrogram analysis. A large whole-genome linkage disequilibrium (LD) block size of 3.49 Mb was observed. A genome-wide association study identified 16 unique stringent marker trait associations for GFeC, GZnC, and 1000-grain weight (TGW). In silico analysis demonstrated the presence of 28 potential candidate genes in the flanking region of 16 linked SNPs, such as synaptotagmin-like mitochondrial-lipid-binding domain, HAUS augmin-like complex, di-copper center-containing domain, protein kinase, chaperonin Cpn60, zinc finger, NUDIX hydrolase, etc. Expression levels of these genes in vegetative tissues and grain were also found. Utilization of identified markers in marker-assisted breeding may lead to the rapid development of biofortified wheat genotypes to combat malnutrition.

Gene Action Governing the Inheritance of Stomatal Conductance in Four Wheat Crosses Under High Temperature Stress Condition.

The knowledge pertaining to gene action and interactions involved in the inheritance of a character in different generations is crucial for determining the breeding strategies in crop improvement program. In the present study, the gene action of stomatal conductance was determined in four wheat populations under high elevated temperatures coupled with late sowing at experimental farm, ICAR-Indian Agricultural Research Institute, New Delhi, India. Steady-state SC-1 leaf porometer was used to record stomatal conductance on adaxial and abaxial leaf surface at late boot (Z 49-50), early milk (Z 73), and late milk (Z 77) growth stages. Evidence for nuclear genetic control of stomatal conductance was strong, with large and repeatable genetic difference observed for parents and progeny across all the four crosses. Mean stomatal conductance for genotypes, GW 322 and GW 366, was consistently low at late boot, early milk, and late milk under timely sown, late sown, and very late sown condition, whereas the converse was true for the high-conducting parents, KAUZ/AA//KAUZ and RAC 875. Additive and additive x additive epistatic effects were large and reasonably consistent at three stages and in all crosses. Detection of epistasis and evidence of transgressive segregation suggested that variation for stomatal conductance was under oligo or polygenic control. Thus, it is conceivable that independent alleles at two or more loci could be pyramided into a single family for increased or decreased stomatal conductance. Additive-based gene action also facilitates with simple selection at early generation to improve stomatal conductance in expected direction. This is the first report on estimates of gene action for stomatal conductance of flag leaf under heat stress condition during reproductive and grain filling stage.

Quality evaluation of near-isogenic line of the wheat variety HD2733 carrying the Lr24/Sr24 genomic region.

A near-isogenic line (NIL) of the Indian wheat variety HD2733, carrying an introgressed Lr24/Sr24 genomic region was used for studying the effect of this introgression on quality traits. Data on the grain yield and 21 quality traits were recorded in this NIL and its recurrent parent (RP), both of which were grown in a randomized block design for two consecutive years. The statistical analysis revealed that grain yield was on par between the NIL and the RP. The NIL and its RP were both hard grained but the NIL showed a grain hardness index reduced by 9.7%. However, quality traits such as grain weight, protein content, sedimentation value, gluten traits, and solvent retention capacity were significantly higher in the NIL. The NIL also showed an increase in dough stability, a lower degree of softening and a higher farinograph quality number. These results indicated that the NIL could be utilized for hard grain, high protein and strong gluten-based products. An overall improvement in the quality of the NIL over its recurrent parent and without any yield penalty suggests that the Lr24/Sr24 genomic region could be gainfully utilized in wheat breeding for improving the industrial quality of wheat without jeopardising grain yield. The authors suggest that the improved quality of the NIL may be due to the genomic segment carried along with the Lr24/Sr24 genes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02679-x.