Exploring DNA barcode for accurate identification of threatened Aconitum L. species from Western Himalaya.

BACKGROUND: Aconitum species, belonging to Ranunculaceae, have high medicinal importance but due to their overexploitation come under IUCN (International Union for Conservation of Nature) red list. The precise identification of the Aconitum species is equally important because they are used in herbal formulations. The present study aimed to develop an efficient DNA barcode system for the authentic identification of Aconitum species. METHODS AND RESULTS: A set of 92 barcode gene sequences (including 12 developed during the present study and 80 retrieved from NCBI) of 5 Aconitum species (A. heterophyllum, A. vialoceum, A. japonicum, A. napellus, and A. stapfianum) were analyzed using three methods (tree-based, distance-based, and similarity-based) for species discrimination. The PWG-distance method was found most effective for species discrimination. The discrimination rate of PWG- distance ranged from 33.3% (rbcL + trnH-psbA) to 100% (ITS, rbcL + ITS, ITS + trnH-psbA and rbcL + ITS + trnH-psbA). Among DNA barcodes and their combinations, the ITS marker had the highest degree of species discrimination (NJ-40%, PWG-100% and BLAST-40%), followed by trnH-psbA (NJ-20%, PWG-60% and BLAST-20%). ITS also had higher barcoding gap as compared to other individual barcodes and their combinations. Further, we also analyzed six Aconitum species (A. balfourii, A. ferox, A. heterophyllum, A. rotundifolium, A. soongaricum and A. violaceum) existing in Western Himalaya. These species were distinguished clearly through tree-based method using the ITS barcode gene with 100% species resolution. CONCLUSION: ITS showed the best species discrimination power and was used to develop species-specific barcodes for Aconitum species. DNA barcodes developed during the present study can be used to identify Aconitum species.

Development of species-specific ISSR-derived SCAR marker for early discrimination between Cinnamomum verum and Cinnamomum cassia.

BACKGROUND: Cinnamomum verum (true cinnamon) and Cinnamomum cassia (cassia cinnamon) are two important species belonging to family Lauraceae. These species are recognized by morphological, chemical composition and essential oil contents. The appropriate identification of species would be considerably improved by a genetic method. The main objective of the present study was to develop molecular markers distinguishing between C. verum and C. cassia. METHODS AND RESULTS: A total 71 ISSR (Inter simple sequence repeat) and four universal barcoding (ITS, rbcL, matK, and psbA-trnH) genes were used to distinguish both the species. No sequence variation was observed between the two species for any DNA barcode gene. However, one ISSR i.e. ISSR-37 showed a clear distinction between the species and produced 570 bp and 746 bp amplicons in C. verum and C. cassia, respectively. The polymorphic bands were converted into species-specific SCAR markers. The SCAR-CV was specific to C. verum and amplified 190 bp band, however there was no amplification seen in the C. cassia samples. CONCLUSION: The SCAR marker generated in this study can be employed as efficient, economical, and reliable molecular tool for the identification of C. verum.

Multi-environment GWAS identifies genomic regions underlying grain nutrient traits in foxtail millet (Setaria italica).

KEY MESSAGE: A total of 104 foxtail millet accessions were evaluated for 11 nutrients in three environments and 67 high-confidence marker-trait associations (MTAs) were identified. Six SNPs showed pleiotropic effect and associated with two or more nutrients, whereas 24 candidate genes were identified for 28 MTAs involving seven traits. Millets are known for their better nutritional profiles compared to major cereals. Foxtail millet (Setaria italica) is rich in nutrients essential to circumvent malnutrition and hidden hunger. However, the genetic determinants underlying this trait remain elusive. In this context, we evaluated 104 diverse foxtail millet accessions in three different environments (E1, E2, and E3) for 11 nutrients and genotyped with 30K SNPs. The genome-wide association study showed 67 high-confidence (Bonferroni-corrected) marker-trait associations (MTAs) for the nutrients except for phosphorus. Six pleiotropic SNPs were also identified, which were associated with two or more nutrients. Around 24 candidate genes (CGs) were identified for 28 MTAs involving seven nutrients. A total of 17 associated SNPs were present within the gene region, and five (5) were mapped in the exon of the CGs. Significant SNPs, desirable alleles and CGs identified in the present study will be useful in breeding programmes for trait improvement.

Cation-Promoted Strain-Release-Driven Access to Functionalized Azetidines from Azabicyclo[1.1.0]butanes.

Access to 1,3-functionalized azetidines through a diversity-oriented approach is highly sought-after for finding new applications in drug-discovery. To this goal, strain-release-driven functionalization of azabicyclo[1.1.0]-butane (ABB) has generated significant interest. Through appropriate N-activation, C3-substituted ABBs are shown to render tandem N/C3-fucntionalization/rearrangement, furnishing azetidines; although, modalities of such N-activation vis-à-vis N-functionalization remain limited to selected electrophiles. This work showcases a versatile cation-driven activation strategy of ABBs. And capitalizes on the use of Csp3 precursors amenable to forming reactive (aza)oxyallyl cations in situ. Herein, N-activation leads to formation of a congested C-N bond, and effective C3 activation. The concept was extended to formal [3+2] annulations involving (aza)oxyallyl cations and ABBs, leading to bridged bicyclic azetidines. Besides the fundamental appeal of this new activation paradigm, operational simplicity and remarkable diversity should engender its prompt use in synthetic and medicinal chemistry.

Integrated omics analysis identified genes and their splice variants involved in fruit development and metabolites production in Capsicum species.

To date, several transcriptomic studies during fruit development have been reported; however, no comprehensive integrated study on expression diversity, alternative splicing, and metabolomic profiling was reported in Capsicum. This study analyzed RNA-seq data and untargeted metabolomic profiling from early green (EG), mature green (MG), and breaker (Br) fruit stages from two Capsicum species, i.e., C. annuum (Cann) and C. frutescens (Cfrut) from Northeast India. A total of 117,416 and 96,802 alternatively spliced events (AltSpli-events) were identified from Cann and Cfrut, respectively. Among AltSpli-events, intron retention (IR; 32.2% Cann and 25.75% Cfrut) followed by alternative acceptor (AA; 15.4% Cann and 18.9% Cfrut) were the most abundant in Capsicum. Around 7600 genes expressed in at least one fruit stage of Cann and Cfrut were AltSpli. The study identified spliced variants of genes including transcription factors (TFs) potentially involved in fruit development/ripening (Aux/IAA 16-like, ETR, SGR1, ARF, CaGLK2, ETR, CaAGL1, MADS-RIN, FUL1, SEPALLATA1), carotenoid (PDS, CA1, CCD4, NCED3, xanthoxin dehydrogenase, CaERF82, CabHLH100, CaMYB3R-1, SGR1, CaWRKY28, CaWRKY48, CaWRKY54), and capsaicinoids or flavonoid biosynthesis (CaMYB48, CaWRKY51), which were significantly differentially spliced (DS) between consecutive Capsicum fruit stages. Also, this study observed that differentially expressed isoforms (DEiso) from 38 genes with differentially spliced events (DSE) were significantly enriched in various metabolic pathways such as starch and sucrose metabolism, amino acid metabolism, cysteine cutin suberin and wax biosynthesis, and carotenoid biosynthesis. Furthermore, the metabolomic profiling revealed that metabolites from aforementioned pathways such as carbohydrates (mainly sugars such as D-fructose, D-galactose, maltose, and sucrose), organic acids (carboxylic acids), and peptide groups significantly altered during fruit development. Taken together, our findings could help in alternative splicing-based targeted studies of candidate genes involved in fruit development and ripening in Capsicum crop.

Leveraging the Domino Skeletal Expansion of Thia-/Selenazolidinones via Nitrogen-Atom Transfer in Hexafluoroisopropanol: Room Temperature Access to Six-Membered S/Se,N-Heterocycles.

Herein, a highly regioselective domino skeletal-expansion process that transforms 2-aminothiazolidinone into six-membered S,N-heterocycle is developed with the aid of TMS-azide in hexafluoroisopropanol (HFIP) at ambient temperature. Functioning of the C2 tertiary amine as latent reactive group on thiazolidinone moiety was the key to this development, which allowed relay substitution with azide and imparted subsequent ring-expansion under metal/acid free-conditions. The reaction also underscored an intermolecular nitrogen-atom transfer process from TMS-azide leading to final products, where any intermediary azidothiazolidinone was absent. The strategy was extendable to analogous synthesis of Se,N-heterocycles, and furthermore, late-stage drug-modification and follow-up transformations were also performed. Density functional theory calculations and control experiments provided important mechanistic insights and highlighted potential roles of HFIP in the transformation.

Pyramiding of genes for grain protein content, grain quality, and rust resistance in eleven Indian bread wheat cultivars: a multi-institutional effort.

Improvement of grain protein content (GPC), loaf volume, and resistance to rusts was achieved in 11 Indian wheat cultivars that are widely grown in four different agro-climatic zones of India. This involved use of marker-assisted backcross breeding (MABB) for introgression and pyramiding of the following genes: (i) the high GPC gene Gpc-B1; (ii) HMW glutenin subunits 5 + 10 at Glu-D1 loci, and (iii) rust resistance genes, Yr36, Yr15, Lr24, and Sr24. GPC increased by 0.8 to 3.3%, although high GPC was generally associated with yield penalty. Further selection among high GPC lines allowed identification of progenies with higher GPC associated with improvement in 1000-grain weight and grain yield in the backgrounds of the following four cultivars: NI5439, UP2338, UP2382, and HUW468. The high GPC progenies (derived from NI5439) were also improved for grain quality using HMW glutenin subunits 5 + 10 at Glu-D1 loci. Similarly, progenies combining high GPC and rust resistance were obtained in the backgrounds of following five cultivars: Lok1, HD2967, PBW550, PBW621, and DBW1. The improved pre-bred lines developed following multi-institutional effort should prove a valuable source for the development of cultivars with improved nutritional quality and rust resistance in the ongoing wheat breeding programmes. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01277-w.

Development of EST-based methylation specific PCR (MSP) markers in Crocus sativus.

BACKGROUND: Saffron (Crocus sativus) is high valued spice crop, but due to its sterile nature, the crop is propagated exclusively through corms. Thus, the genetic base of this crop is very narrow, however, frequency of phenotypic variability is observed; and suggested the potential role of epigenetics in saffron crop growth and development. METHODS AND RESULTS: To facilitate epigenetic studies in saffron, we developed 1525 methylation-specific PCR (MSP) markers using MethPrimer. For this purpose, we used 6767 EST sequences of saffron available on the NCBI database. We also mine CpG islands (2555) and found that 32.7% of EST sequences had CpG islands. Out of 1525 MSP markers developed during the present study, 725 covered the CpG islands and 800 were without CpG islands. PCR amplification was found successful for 82% of MSP markers. A preliminary analysis suggested that 53.7% of genomic sites were methylated and more prominent (60% methylations) in non-CpG island regions, although, more comprehensive studies are required to validate it further. CONCLUSIONS: The epigenetic resource developed during the present study will strengthen the epigenetic studies like epiQTL mapping, epiGWAS to explore the molecular mechanisms and genomic/epigenomic regions associated with phenotype; and further may be utilized for saffron improvement programs through epibreeding.

Development of white-grained PHS-tolerant wheats with high grain protein and leaf rust resistance.

The present study involved incorporation of two major QTLs for pre-harvest sprouting tolerance (PHST) in an Indian wheat cultivar named Lok1, which happens to be PHS susceptible. For transfer of two QTLs, two independent programmes with two different donors (AUS1408, CN19055) were utilized. The recipient cv. Lok1 was crossed with each of the two donors, followed by a number of backcrosses. Each backcross progeny was subjected to foreground and background selections. KASP assay was also used for confirming the presence of PHST QTL. In one case, PHST QTL was later also pyramided with a gene for high grain protein content (Gpc-B1) and a gene for leaf rust resistance (Lr24). The MAS derived lines were screened for PHS using simulated rain chambers leading to selection of 10 PHST lines. Four of these advanced lines carried all the three QTL/genes and exhibited high level of PHST (PHS score 2-3) associated with significant improvement in GPC and resistance against leaf rust. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01234-z.

Single-base cytosine methylation analysis in fruits of three Capsicum species.

Single-base cytosine methylation analysis across fruits of Capsicum annuum, C. chinense and C. frutescens showed global average methylation ranging from 82.8-89.1%, 77.6-83.9%, and 22.4-25% at CG, CHG and CHH contexts, respectively. High gene-body methylation at CG and CHG was observed across Capsicum species. The C. annuum showed the highest proportion (>80%) of mCs at different genomic regions compared to C. chinense and C. frutescens. Cytosine methylation for transposable-elements were lower in C. frutescens compared to C. annuum and C. chinense. A total of 510,165 CG, 583112 CHG and 277,897 CHH DMRs were identified across three Capsicum species. The differentially methylated regions (DMRs) distribution analysis revealed C. frutescens as more hypo-methylated compared to C. annuum and C. chinense, and also the presence of more intergenic DMRs in Capsicum genome. At CG and CHG context, gene expression and promoter methylation showed inverse correlations. Furthermore, the observed correlation between methylation and expression of genes suggested the potential role of methylation in Capsicum fruit development/ripening.

Development and characterization of non-coding RNA based simple sequence repeat markers in Capsicum species.

Plant growth and development are largely regulated by non-coding RNAs (ncRNA); thus ncRNA based markers would be rewarding in molecular breeding. In the present study, for the first time we developed total 623 ncRNA based SSRs including 119 microRNASSRs (miRNASSRs) and 504 long non-coding RNASSRs (lncRNASSRs) distributed across 12 Capsicum chromosomes. Out of 623 ncRNASSRs, 120 (including 60 each miRNASSRs and lncRNASSRs) were used for genotyping of 96 Capsicum accessions belonging to C. annuum, C. chinense and C. frutescens; and 75% SSRs were polymorphic. Model-based and distance-based cluster analyses identified three species specific clusters, i.e. cluster-I (C. annuum), cluster-II (C. frutescens) and cluster-III (C. chinense); therefore, these SSRs may have a potential role to play in interspecific Capsicum breeding. Tissue specific expression of SSR containing ncRNAs and versatile functions of their targets suggested the usefulness of SSRs for mapping of genes/QTLs and breeding of wide range of traits in Capsicum.

Identification of genes involved in fruit development/ripening in Capsicum and development of functional markers.

The molecular mechanism of the underlying genes involved in the process of fruit ripening in Capsicum (family Solanaceae) is not clearly known. In the present study, we identified orthologs of 32 fruit development/ripening genes of tomato in Capsicum, and validated their expression in fruit development stages in C. annuum, C. frutescens, and C. chinense. In silico expression analysis using transcriptome data identified a total of 12 out of 32 genes showing differential expression during different stages of fruit development in Capsicum. Real time expression identified gene LOC107847473 (ortholog of MADS-RIN) had substantially higher expression (>500 folds) in breaker and mature fruits, which suggested the non-climacteric ripening behaviour of Capsicum. However, differential expression of Ehtylene receptor 2-like (LOC107873245) gene during fruit maturity supported the climacteric behaviour of only C. frutescens (hot pepper). Furthermore, development of 49 gene based simple sequence repeat (SSR) markers would help in selection of identified genes in Capsicum breeding.

Mapping QTLs for 15 morpho-metric traits in Arabidopsis thaliana using Col-0 × Don-0 population.

Genome wide quantitative trait loci (QTL) mapping was conducted in Arabidopsis thaliana using F2 mapping population (Col-0 × Don-0) and SNPs markers. A total of five linkage groups were obtained with number of SNPs varying from 45 to 59 per linkage group. The composite interval mapping detected a total of 36 QTLs for 15 traits and the number of QTLs ranged from one (root length, root dry biomass, cauline leaf width, number of internodes and internode distance) to seven (for bolting days). The range of phenotypic variance explained (PVE) and logarithm of the odds ratio of these 36 QTLs was found be 0.19-38.17% and 3.0-6.26 respectively. Further, the epistatic interaction detected one main effect QTL and four epistatic QTLs. Five major QTLs viz. Qbd.nbri.4.3, Qfd.nbri.4.2, Qrdm.nbri.5.1, Qncl.nbri.2.2, Qtd.nbri.4.1 with PVE > 15.0% might be useful for fine mapping to identify genes associated with respective traits, and also for development of specialized population through marker assisted selection. The identification of additive and dominant effect QTLs and desirable alleles of each of above mentioned traits would also be important for future research.

Lewis Acid Catalyzed Nucleophilic Ring Opening and 1,3-Bisfunctionalization of Donor-Acceptor Cyclopropanes with Hydroperoxides: Access to Highly Functionalized Peroxy/(α-Heteroatom Substituted)Peroxy Compounds.

The Lewis acid catalyzed ring opening reaction of Donor-Acceptor (D-A) cyclopropanes with alkyl hydroperoxides is reported to furnish various peroxycarbonyls and 1,3-haloperoxygenated compounds in good to excellent yields. This method adds another instance to scarcely reported noncyclilizing 1,3-bisfunctionalization of D-A cyclopropanes with two different functional groups and relies on the dual role of peroxide as nucleophile and oxidant through an orchestrated reaction sequence. The products obtained, including α-heterosubstituted peroxy compounds, are amenable to useful synthetic elaboration.

QTL mapping for some grain traits in bread wheat (Triticum aestivum L.).

Grain traits are important agronomic attributes with the market value as well as milling yield of bread wheat. In the present study, quantitative trait loci (QTL) regulating grain traits in wheat were identified. Data for grain area size (GAS), grain width (GWid), factor form density (FFD), grain length-width ratio (GLWR), thousand grain weight (TGW), grain perimeter length (GPL) and grain length (GL) were recorded on a recombinant inbred line derived from the cross of NW1014 × HUW468 at Meerut and Varanasi locations. A linkage map of 55 simple sequence repeat markers for 8 wheat chromosomes was used for QTL analysis by Composite interval mapping. Eighteen QTLs distributed on 8 chromosomes were identified for seven grain traits. Of these, five QTLs for GLWR were found on chromosomes 1A, 6A, 2B, and 7B, three QTLs for GPL were located on chromosomes 4A, 5A and 7B and three QTLs for GAS were mapped on 5D and 7D. Two QTLs were identified on chromosomes 4A and 5A for GL and two QTLs for GWid were identified on chromosomes 7D and 6A. Similarly, two QTLs for FFD were found on chromosomes 1A and 5D. A solitary QTL for TGW was identified on chromosome 2B. For several traits, QTLs were also co-localized on chromosomes 2B, 4A, 5A, 6A, 5D, 7B and 7D. The QTLs detected in the present study may be validated for specific crosses and then used for marker-assisted selection to improve grain quality in bread wheat.

Transcription factors involved in drought tolerance and their possible role in developing drought tolerant cultivars with emphasis on wheat (Triticum aestivum L.).

KEY MESSAGE: TFs involved in drought tolerance in plants may be utilized in future for developing drought tolerant cultivars of wheat and some other crops. Plants have developed a fairly complex stress response system to deal with drought and other abiotic stresses. These response systems often make use of transcription factors (TFs); a gene encoding a specific TF together with -its target genes constitute a regulon, and take part in signal transduction to activate/silence genes involved in response to drought. Since, five specific families of TFs (out of >80 known families of TFs) have gained widespread attention on account of their significant role in drought tolerance in plants, TFs and regulons belonging to these five multi-gene families (AP2/EREBP, bZIP, MYB/MYC, NAC and WRKY) have been described and their role in improving drought tolerance discussed in this brief review. These TFs often undergo reversible phosphorylation to perform their function, and are also involved in complex networks. Therefore, some details about reversible phosphorylation of TFs by different protein kinases/phosphatases and the co-regulatory networks, which involve either only TFs or TFs with miRNAs, have also been discussed. Literature on transgenics involving genes encoding TFs and that on QTLs and markers associated with TF genes involved in drought tolerance has also been reviewed. Throughout the review, there is a major emphasis on wheat as an important crop, although examples from the model cereal rice (sometimes maize also), and the model plant Arabidopsis have also been used. This knowledge base may eventually allow the use of TF genes for development of drought tolerant cultivars, particularly in wheat.

Identification and comprehensive analysis of MIPSs in Rosaceae and their expression under abiotic stresses in rose (Rosa chinensis).

The Myo-Inositol-1-phosphate synthase (MIPS) gene family is involved in the myo-inositol synthesis and plays a significant role in signal transduction, membrane biogenesis, oligosaccharides synthesis, auxin storage and transport, programmed cell death and abiotic stress tolerance in plants. This study comprehensively identified the MIPS genes in Rosaceae plant species, and 51 MIPS genes were identified from 26 Rosaceae species. The phylogenetic analysis divided the MIPSs into two clades (clade I; subfamily Amygdaloideae specific, and clade II; subfamily Rosoideae specific). MIPS genes of all 26 Rosaceae species consist of similar gene structure, motif and domain composition, which shows their conserved nature. The cis-regulatory elements (CREs) analysis revealed that most Rosaceae MIPS genes play a role in growth, development, and stress responses. Furthermore, the qRT-PCR analysis also revealed the involvement of RcMIPS gene in plant development and response to abiotic stresses, including drought and heat. The results of the present study contribute to the understanding of the biological function of Rosaceae MIPS genes, and that could be used in further functional validations.

Emerging Roles of SWEET Sugar Transporters in Plant Development and Abiotic Stress Responses.

Sugars are the major source of energy in living organisms and play important roles in osmotic regulation, cell signaling and energy storage. SWEETs (Sugars Will Eventually be Exported Transporters) are the most recent family of sugar transporters that function as uniporters, facilitating the diffusion of sugar molecules across cell membranes. In plants, SWEETs play roles in multiple physiological processes including phloem loading, senescence, pollen nutrition, grain filling, nectar secretion, abiotic (drought, heat, cold, and salinity) and biotic stress regulation. In this review, we summarized the role of SWEET transporters in plant development and abiotic stress. The gene expression dynamics of various SWEET transporters under various abiotic stresses in different plant species are also discussed. Finally, we discuss the utilization of genome editing tools (TALENs and CRISPR/Cas9) to engineer SWEET genes that can facilitate trait improvement. Overall, recent advancements on SWEETs are highlighted, which could be used for crop trait improvement and abiotic stress tolerance.

Integrated analysis of DNA methylation, transcriptome, and global metabolites in interspecific heterotic Capsicum F1 hybrid.

Hybrid breeding is one of the efficacious methods of crop improvement. Here, we report our work towards understanding the molecular basis of F1 hybrid heterosis from Capsicum chinense and C. frutescens cross. Bisulfite sequencing identified a total of 70597 CG, 108797 CHG, and 38418 CHH differentially methylated regions (DMRs) across F1 hybrid and parents, and of these, 4891 DMRs showed higher methylation in F1 compared to the mid-parental methylation values (MPMV). Transcriptome analysis showed higher expression of 46-55% differentially expressed genes (DE-Gs) in the F1 hybrid. The qRT-PCR analysis of 24 DE-Gs with negative promoter methylation revealed 91.66% expression similarity with the transcriptome data. A few metabolites and 65-72% enriched genes in metabolite biosynthetic pathways showed overall increased expression in the F1 hybrid compared to parents. These findings, taken together, provided insights into the integrated role of DNA methylation, and genes and metabolites expression in the manifestation of heterosis in Capsicum.