Characterization of sugarcane mutants developed through gamma irradiations for their lignin content and caffeic acid-O-methyl transferase (COMT) gene mutations.

PURPOSE: Bagasse, the residue left after extracting juice from sugarcane stalks, is rich in lignocellulosic biomass. The lignin present in this plant biomass is the key factor that hinders the efficient extraction of ethanol from the bagasse. In the current study, γ-irradiated sugarcane mutants were evaluated for variation in lignin content and its corresponding caffeic acid-O-methyl transferase (COMT) gene. MATERIALS AND METHODS: The acetyl bromide method was used to estimate lignin content in sugarcane mutants. PCR-based cloning of the COMT gene was performed in low lignin mutants as well as control plants in E. coli (strain DH5α) to understand the mechanism of variation at the molecular level. The Sanger sequencing for cloned gene was performed to check variation in gene sequence. RESULTS: In comparison to the control (21.5%), the mutant plants' lignin content ranged from 13 to 28%. The Sanger sequencing revealed approximately the same length of the gene from mutants as well as a control plant. In comparison to the reference gene, the mutated gene showed SNPs and indels in different regions, which may have an impact on lignin content. CONCLUSIONS: Therefore, γ-irradiated mutagenesis is an acceptable approach to develop novel mutants of sugarcane with low lignin content to enhance bioethanol production from waste material using bioprocess technology.

An online database for einkorn wheat to aid in gene discovery and functional genomics studies.

Diploid A-genome wheat (einkorn wheat) presents a nutrition-rich option as an ancient grain crop and a resource for the improvement of bread wheat against abiotic and biotic stresses. Realizing the importance of this wheat species, reference-level assemblies of two einkorn wheat accessions were generated (wild and domesticated). This work reports an einkorn genome database that provides an interface to the cereals research community to perform comparative genomics, applied genetics and breeding research. It features queries for annotated genes, the use of a recent genome browser release, and the ability to search for sequence alignments using a modern BLAST interface. Other features include a comparison of reference einkorn assemblies with other wheat cultivars through genomic synteny visualization and an alignment visualization tool for BLAST results. Altogether, this resource will help wheat research and breeding. Database URL  https://wheat.pw.usda.gov/GG3/pangenome.

Einkorn genomics sheds light on history of the oldest domesticated wheat.

Einkorn (Triticum monococcum) was the first domesticated wheat species, and was central to the birth of agriculture and the Neolithic Revolution in the Fertile Crescent around 10,000 years ago1,2. Here we generate and analyse 5.2-Gb genome assemblies for wild and domesticated einkorn, including completely assembled centromeres. Einkorn centromeres are highly dynamic, showing evidence of ancient and recent centromere shifts caused by structural rearrangements. Whole-genome sequencing analysis of a diversity panel uncovered the population structure and evolutionary history of einkorn, revealing complex patterns of hybridizations and introgressions after the dispersal of domesticated einkorn from the Fertile Crescent. We also show that around 1% of the modern bread wheat (Triticum aestivum) A subgenome originates from einkorn. These resources and findings highlight the history of einkorn evolution and provide a basis to accelerate the genomics-assisted improvement of einkorn and bread wheat.

Physical mapping of the wheat genes in low-recombination regions: radiation hybrid mapping of the C-locus.

KEY MESSAGE: This work reports the physical mapping of an important gene affecting spike compactness located in a low-recombination region of hexaploid wheat. This work paves the way for the eventual isolation and characterization of the factor involved but also opens up possibilities to use this approach to precisely map other wheat genes located on proximal parts of wheat chromosomes that show highly reduced recombination. Mapping wheat genes, in the centromeric and pericentromeric regions (~ 2/3rd of a given chromosome), poses a formidable challenge due to highly suppressed recombination. Using an example of compact spike locus (C-locus), this study provides an approach to precisely map wheat genes in the pericentromeric and centromeric regions that house ~ 30% of wheat genes. In club-wheat, spike compactness is controlled by the dominant C-locus, but previous efforts have failed to localize it, on a particular arm of chromosome 2D. We integrated radiation hybrid (RH) and high-resolution genetic mapping to locate C-locus on the short arm of chromosome 2D. Flanking markers of the C-locus span a physical distance of 11.0 Mb (231.0-242 Mb interval) and contain only 11 high-confidence annotated genes. This work demonstrates the value of this integrated strategy in mapping dominant genes in the low-recombination regions of the wheat genome. A comparison of the mapping resolutions of the RH and genetic maps using common anchored markers indicated that the RH map provides ~ 9 times better resolution that the genetic map even with much smaller population size. This study provides a broadly applicable approach to fine map wheat genes in regions of suppressed recombination.

Transcriptional dynamics of maize leaves, pollens and ovules to gain insights into heat stress-related responses.

Heat stress (HS) is one of the alarming issues today due to global warming and is the foremost detrimental to crop production. Maize is one of the versatile crops grown over different agro-climatic conditions. However, it is significantly sensitive to heat stress, especially during the reproductive phase. The heat stress tolerance mechanism is yet to be elucidated at the reproductive stage. Thus, the present study focused on identifying transcriptional changes in two inbreds, LM 11 (sensitive to HS) and CML 25 (tolerant to HS), under intense heat stress at 42°C during the reproductive stage from three tissues viz. flag leaf, tassel, and ovule. Samples from each inbred were collected after 5 days of pollinations for RNA isolation. Six cDNA libraries were constructed from three separate tissues of LM 11 and CML 25 and sequenced using an Illumina HiSeq2500 platform. A total of 2,164 (1127 up-regulated and 1037 down-regulated) differentially expressed genes (DEGs) were identified with 1151, 451, and 562 DEGs in comparisons of LM 11 and CML 25, corresponding to a leaf, pollen, and ovule, respectively. Functional annotated DEGs associated with transcription factors (TFs) viz. AP2, MYB, WRKY, PsbP, bZIP, and NAM, heat shock proteins (HSP20, HSP70, and HSP101/ClpB), as well as genes related to photosynthesis (PsaD & PsaN), antioxidation (APX and CAT) and polyamines (Spd and Spm). KEGG pathways analyses showed that the metabolic overview pathway and secondary metabolites biosynthesis pathway, with the involvement of 264 and 146 genes, respectively, were highly enriched in response to heat stress. Notably, the expression changes of the most common HS-responsive genes were typically much more significant in CML 25, which might explain why CML 25 is more heat tolerant. Seven DEGs were common in leaf, pollen, and ovule; and involved in the polyamines biosynthesis pathway. Their exact role in maize heat stress response would warrant further studies. These results enhanced our understanding to heat stress responses in maize.

Whole genome resequencing and comparative genome analysis of three Puccinia striiformis f. sp. tritici pathotypes prevalent in India.

Stripe rust disease of wheat, caused by Puccinia striiformis f. sp. tritici, (Pst) is one of the most serious diseases of wheat worldwide. In India, virulent stripe rust races have been constantly evolving in the North-Western Plains Zone leading to the failure of some of the most widely grown resistant varieties in the region. With the goal of studying the recent evolution of virulent races in this region, we conducted whole-genome re-sequencing of three prevalent Indian Pst pathotypes Pst46S119, Pst78S84 and Pst110S119. We assembled 58.62, 58.33 and 55.78 Mb of Pst110S119, Pst46S119 and Pst78S84 genome, respectively and found that pathotypes were highly heterozygous. Comparative phylogenetic analysis indicated the recent evolution of pathotypes Pst110S119 and Pst78S84 from Pst46S119. Pathogenicity-related genes classes (CAZyme, proteases, effectors, and secretome proteins) were identified and found to be under positive selection. Higher rate of gene families expansion were also observed in the three pathotypes. A strong association between the effector genes and transposable elements may be the source of the rapid evolution of these strains. Phylogenetic analysis differentiated the Indian races in this study from other known United States, European, African, and Asian races. Diagnostic markers developed for the identification of three Pst pathotypes will help tracking of yellow rust at farmers field and strategizing resistance gene deployment.

BSA-seq Identifies a Major Locus on Chromosome 6 for Root-Knot Nematode (Meloidogyne graminicola) Resistance From Oryza glaberrima.

Root-knot nematode (Meloidogyne graminicola) is one of the emerging threats to rice production worldwide that causes substantial yield reductions. There is a progressive shift of the cropping system from traditional transplanting to direct-seeded water-saving rice production that favored the development of M. graminicola. Scouting and deploying new resistance genes is an economical approach to managing the root-knot nematodes. Here, we report that the inheritance of root-knot nematode resistance in Oryza glaberrima acc. IRGC102206 is governed by a single dominant gene. Traditional mapping coupled with BSA-seq is used to map nematode resistance gene(s) using the BC1F1 population derived from a cross of O. sativa cv. PR121 (S) and O. glaberrima acc. IRGC102206 (R). One major novel genomic region spanning a 3.0-Mb interval on chromosome 6 and two minor QTLs on chromosomes 2 and 4 are the potential genomic regions associated with rice root-knot nematode resistance. Within the QTL regions, 19 putative candidate genes contain 81 non-synonymous variants. The detected major candidate region could be fine mapped to accelerate marker-assisted breeding for root-knot nematode resistance in rice.

Virtual screening, molecular dynamics and binding energy-MM-PBSA studies of natural compounds to identify potential EcR inhibitors against Bemisia tabaci Gennadius.

Whitefly (Bemisia tabaci Gennadius) is a hemipteran phyto polyphagous sucking insect pest which is an important pest of cotton that causes economic losses to the crop by reducing its yield and quality. Ecdysteroids such as 20-hydroxy ecdysone (20-E), play a significant role in larval moulting, development, and reproduction in pterygota insects. Receptor of 20-E, that is Ecdysone Receptor (BtEcR) of Bemisia tabaci has been targeted to prevent fundamental developmental processes. To identify potent inhibitors of BtEcr, 98,072 natural compounds were retrieved from ZINC database. A structure-based virtual screening of these compounds was performed for evaluating their binding affinity to BtEcR, and top two compounds (ZINC08952607 and ZINC04264850) selected based on lowest binding energy. Molecular dynamics simulation (MDS) study was performed for analyzing the dynamics and stability of BtEcR and top-scoring ligand-BtEcR complexes at 50 ns. Besides, g_mmpbsa tool was also used to calculate and analyse the binding free energy of BtEcR-ligand complexes. Compounds ZINC08952607 and ZINC04264850 had shown a binding free energy of -170.156 kJ mol-1 and -200.349 kJ mol-1 in complex with BtEcR respectively. Thus, these compounds can be utilized as lead for the development of environmentally safe insecticides against the whitefly.

GAAP: A GUI-based Genome Assembly and Annotation Package.

Background: Next-generation sequencing (NGS) technologies are being continuously used for high-throughput sequencing data generation that requires easy-to-use GUI-based data analysis software. These kinds of software could be used in-parallel with sequencing for the automatic data analysis. At present, very few software are available for use and most of them are commercial, thus creating a gap between data generation and data analysis. Methods: GAAP is developed on the NodeJS platform that uses HTML, JavaScript as the front-end for communication with users. We have implemented FastQC and trimmomatic tool for quality checking and control. Velvet and Prodigal are integrated for genome assembly and gene prediction. The annotation will be done with the help of remote NCBI Blast and IPR-Scan. In the back- end, we have used PERL and JavaScript for the processing of data. To evaluate the performance of GAAP, we have assembled a viral (SRR11621811), bacterial (SRR17153353) and human genome (SRR16845439). Results: We have used GAAP software to assemble, and annotate a COVID-19 genome on a desktop computer that resulted in a single contig of 27994bp with 99.57% reference genome coverage. This assembly predicted 11 genes, of which 10 were annotated using annotation module of GAAP. We have also assembled a bacterial and human genome 138 and 194281 contigs with N50 value 100399 and 610, respectively. Conclusion: In this study, we have developed freely available, platform-independent genome assembly and annotation (GAAP) software (www.deepaklab.com/gaap). The software itself acts as a complete data analysis package with quality check, quality control, de-novo genome assembly, gene prediction and annotation (Blast, PFAM, GO-Term, pathway and enzyme mapping) modules.

Comparative transcriptomics in alternate bearing cultivar Dashehari reveals the genetic model of flowering in mango.

Flowering is a complex developmental process, with physiological and morphological phases influenced by a variety of external and internal factors. Interestingly, many mango cultivars tend to bear fruit biennially because of irregular flowering, and this has a negative impact on mango flowering and the subsequent yield, resulting in significant economic losses. In this article, transcriptome analysis was carried out on four tissues of mango cv. Dashehari (bearing tree leaf, shoot apex, inflorescence, and non-bearing tree leaf). De novo transcriptome assembly of RNA-seq reads of Dashehari using the Trinity pipeline generated 67,915 transcripts, with 25,776 genes identified. 85 flowering genes, represented by 179 transcripts, were differentially expressed in bearing vs. non-bearing leaf tissues. Gene set enrichment analysis of flowering genes identified significant upregulation of flowering related genes in inflorescence tissues compared to bearing leaf tissues. The flowering genes FT, CO, GI, ELF 4, FLD, FCA, AP1, LHY, and SCO1 were upregulated in the bearing leaf tissues. Pathway analysis of DEGs showed significant upregulation of phenylpropanoid and sucrose and starch pathways in non-bearing leaf tissue compared with bearing leaf tissue. The comparative transcriptome analysis performed in this study significantly increases the understanding of the molecular mechanisms driving the flowering process as well as alternative bearing in mango.

Transcriptome based identification and validation of heat stress transcription factors in wheat progenitor species Aegilops speltoides.

Wheat, one of the major cereal crops worldwide, get adversely affected by rising global temperature. We have identified the diploid B genome progenitor of wheat, Aegilops speltoides (SS), as a potential donor for heat stress tolerance. Therefore, the present work was planned to study the total transcriptome profile of heat stress-tolerant Ae. speltoides accession pau3809 (AS3809) and compare with that of tetraploid and hexaploid wheat cultivars PDW274 and PBW725, respectively. The comparative transcriptome was utilized to identify and validate heat stress transcription factors (HSFs), the key genes involved in imparting heat stress tolerance. Transcriptome analysis led to the identification of a total of 74 K, 68 K, and 76 K genes in AS3809, PDW274, and PBW725, respectively. There was a high uniformity of GO profiles under the biological, molecular, and cellular functions across the three wheat transcriptomes, suggesting the conservation of gene function. Twelve HSFs having the highest FPKM value were identified in the AS3809 transcriptome data, while six of these HSFs namely HSFA3, HSFA5, HSFA9, HSFB2a, HSFB2b, and HSFC1b, were validated with qRT PCR. These six HSFs were identified as an important component of thermotolerance in AS3809 as evident from their comparative higher expression under heat stress.

Development of Genome-Wide Functional Markers Using Draft Genome Assembly of Guava (Psidium guajava L.) cv. Allahabad Safeda to Expedite Molecular Breeding.

Guava (Psidium guajava L.), a rich source of nutrients, is an important tropical and subtropical fruit of the Myrtaceae family and exhibits magnificent diversity. Genetic diversity analysis is the first step toward the identification of parents for hybridization, genetic mapping, and molecular breeding in any crop species. A diversity analysis based on whole-genome functional markers increases the chances of identifying genetic associations with agronomically important traits. Therefore, here, we sequenced the genome of guava cv. Allahabad Safeda on an Illumina platform and generated a draft assembly of ~304 MB. The assembly of the Allahabad Safeda genome constituted >37.95% repeat sequences, gene prediction with RNA-seq data as evidence identified 14,115 genes, and BLAST n/r, Interproscan, PfamScan, BLAST2GO, and KEGG annotated 13,957 genes. A comparative protein transcript analysis of tree species revealed the close relatedness of guava with Eucalyptus. Comparative transcriptomics-based SSR/InDel/SNP-PCR ready genome-wide markers in greenish-yellow skinned and white fleshed-Allahabad Safeda to four contrasting cultivars viz apple-color-skinned and white-fleshed-Lalima, greenish-yellow-skinned and pink-fleshed-Punjab Pink, purple-black-skinned and purple-fleshed-Purple Local and widely used rootstock-Lucknow-49 were developed. The molecular markers developed here revealed a high level of individual heterozygosity within genotypes in 22 phenotypically diverse guava cultivars. Principal coordinate, STRUCTURE clustering, and neighbor-joining-based genetic diversity analysis identified distinct clusters associated with fruit skin and flesh color. The genome sequencing of guava, functional annotation, comparative transcriptomics-based genome-wide markers, and genetic diversity analysis will expand the knowledge of genomes of climacteric fruits, facilitating trait-based molecular breeding and diversifying the nutritional basket.

Molecular mapping of quantitative trait loci for ascochyta blight and botrytis grey mould resistance in an inter-specific cross in chickpea (Cicer arietinum L.) using genotyping by sequencing.

Ascochyta blight (AB) and botrytis grey mould (BGM) are the most devastating fungal diseases of chickpea worldwide. The wild relative of chickpea, C. reticulatum acc. ILWC 292 was found resistant to BGM whereas, GPF2 (Cicer arietinum L.) is resistant to AB. A total of 187 F8 Recombinant Inbred Lines (RILs) developed from an inter-specific cross of GPF2 × C. reticulatum acc. ILWC 292 were used to identify quantitative trait loci (QTLs) responsible for resistance to AB and BGM. RILs along with parents were evaluated under artificial epiphytotic field/laboratory conditions for two years. Highly significant differences (P < 0.001) were observed for reaction to both pathogens in both years. Parents and RILs were genotyped-by-sequencing to identify genome wide single nucleotide polymorphism (SNPs). A total of 1365 filtered and parental polymorphic SNPs were used for linkage map construction, of which, 673 SNPs were arranged on eight linkage groups. Composite interval mapping revealed three QTLs for AB and four QTLs for BGM resistance. Out of which, two QTLs for AB and three QTLs for BGM were consistent in both years. These QTLs can be targeted for further fine mapping for deployment of resistance to AB and BGM in elite chickpea cultivars using marker-assisted-selection.

Genome assisted molecular typing and pathotyping of rice blast pathogen, Magnaporthe oryzae, reveals a genetically homogenous population with high virulence diversity.

Genome sequence-driven molecular typing tools have the potential to uncover the population biology and genetic diversity of rapidly evolving plant pathogens like Magnaporthe oryzae. Here, we report a new molecular typing technique -a digitally portable tool for population genetic analysis of M. oryzae to decipher the genetic diversity. Our genotyping tool exploiting allelic variations in housekeeping and virulence genes coupled with pathotyping revealed a prevalence of genetically homogenous populations within a single-field and plant niches such as leaf and panicle. The M. oryzae inciting leaf-blast and panicle-blast were confirmed to be genetically identical with no or minor nucleotide polymorphism in 17 genomic loci analyzed. Genetic loci such as Mlc1, Mpg1, Mps1, Slp1, Cal, Ef-Tu, Pfk, and Pgk were highly polymorphic as indicated by the haplotype-diversity, the number of polymorphic sites, and the number of mutations. The genetically homogenous single field population showed high virulence variability or diversity on monogenic rice differentials. The study indicated that the genetic similarity displayed by the isolates collected from a particular geographical location had no consequence on their virulence pattern on rice differentials carrying single/multiple resistance genes. The data on virulence diversity showed by the identical Sequence Types (STs) is indicative of no congruence between polymorphic virulence genes-based pathotyping and conserved housekeeping genes-based genotyping.

Cloning, expression analysis and In silico characterization of HSP101: a potential player conferring heat stress in Aegilops speltoides (Tausch) Gren.

Heat shock protein (HSP101) function as molecular chaperones and confer thermotolerance to plants. In the present investigation, identification, comprehensive expression analysis, phylogeny and protein modelling of HSP101 gene has been done in Aegilops speltoides accession Pau3583. In the present study, we cloned and in silico characterized a HSP101C gene designated as AsHSP101C-Pau3583. AsHSP101C-Pau3583 is 4180 bp long with seven exons and six introns and encoded a polypeptide of 910 amino acids predicted by FGENESH. We have identified 58 SNPs between the AsHSP101C-Pau3583 and reference gene sequence extracted from Ae. speltoides TGAC assembly. Real-time RT-PCR analysis of expression levels of HSP101 gene in two wheat genotypes under heat stress revealed that gene namely HSP101C was up-regulated in Aegilops speltoides acc. Pau3583 by > fourfold in comparison to Triticum aestivum cv. PBW343 under heat stress signifies that it plays a role in conferring heat tolerance. Sequence comparison and phylogenetic analysis of AsHSP101C-Pau3583 with seven wheat homologs Triticum aestivum, Aegilops speltoides (TGAC), Triticum durum cv Cappelli, Triticum durum cv Strongfield, Triticum monococcum, Aegilops tauschii and Triticum urartu showed significant similarities with highly conserved coding regions and functional domains (AAA, AAA + 2, ClpB domains), suggesting the conserved function of HSP101C in different species. The illustration of the protein models of HSP101C in homologs provided information for the ATP-binding motifs within the nucleotide binding domains (NBD), specific for the chaperone activity. These findings are important and identified SNPs could be used for designing markers for ensuring the transfer of AsHSP101C-Pau3583 gene into hexaploid wheat and its role in heat tolerance. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01005-2.

An approach for high-resolution genetic mapping of distant wild relatives of bread wheat: example of fine mapping of Lr57 and Yr40 genes.

KEY MESSAGE: The article reports a powerful but simple approach for high-resolution mapping and eventual map-based cloning of agronomically important genes from distant relatives of wheat, using the already existing germplasm resources. Wild relatives of wheat are a rich reservoir of genetic diversity for its improvement. The effective utilization of distant wild relatives in isolation of agronomically important genes is hindered by the lack of recombination between the homoeologous chromosomes. In this study, we propose a simple yet powerful approach that can be applied for high-resolution mapping of a targeted gene from wheat's distant gene pool members. A wheat-Aegilops geniculata translocation line TA5602 with a small terminal segment from chromosome 5 Mg of Ae. geniculata translocated to 5D of wheat contains genes Lr57 and Yr40 for leaf rust and stripe rust resistance, respectively. To map these genes, TA5602 was crossed with a susceptible Ae. geniculata 5 Mg addition line. Chromosome pairing between the 5 Mg chromosomes of susceptible and resistant parents resulted in the development of a high-resolution mapping panel for the targeted genes. Next-generation-sequencing data from flow-sorted 5 Mg chromosome of Ae. geniculata allowed us to generate 5 Mg-specific markers. These markers were used to delineate Lr57 and Yr40 genes each to distinct ~ 1.5 Mb physical intervals flanked by gene markers on 5 Mg. The method presented here will allow researchers worldwide to utilize existing germplasm resources in genebanks and seed repositories toward routinely performing map-based cloning of important genes from tertiary gene pools of wheat.

RNA-sequencing based gene expression landscape of guava cv. Allahabad Safeda and comparative analysis to colored cultivars.

BACKGROUND: Guava (Psidium guajava L.) is an important fruit crop of tropical and subtropical areas of the world. Genomics resources in guava are scanty. RNA-Seq based tissue specific expressed genomic information, de novo transcriptome assembly, functional annotation and differential expression among contrasting genotypes has a potential to set the stage for the functional genomics for traits of commerce like colored flesh and apple color peel. RESULTS: Development of fruit from flower involves orchestration of myriad molecular switches. We did comparative transcriptome sequencing on leaf, flower and fruit tissues of cv. Allahabad Safeda to understand important genes and pathways controlling fruit development. Tissue specific RNA sequencing and de novo transcriptome assembly using Trinity pipeline provided us the first reference transcriptome for guava consisting of 84,206 genes comprising 279,792 total transcripts with a N50 of 3603 bp. Blast2GO assigned annotation to 116,629 transcripts and PFam based HMM profile annotated 140,061 transcripts with protein domains. Differential expression with EdgeR identified 3033 genes in Allahabad Safeda tissues. Mapping the differentially expressed transcripts over molecular pathways indicate significant Ethylene and Abscisic acid hormonal changes and secondary metabolites, carbohydrate metabolism and fruit softening related gene transcripts during fruit development, maturation and ripening. Differential expression analysis among colored tissue comparisons in 3 cultivars Allahabad Safeda, Punjab Pink and Apple Color identified 68 candidate genes that might be controlling color development in guava fruit. Comparisons of red vs green peel in Apple Color, white pulp vs red pulp in Punjab Pink and fruit maturation vs ripening in non-colored Allahabad Safeda indicates up-regulation of ethylene biosynthesis accompanied to secondary metabolism like phenylpropanoid and monolignol pathways. CONCLUSIONS: Benchmarking Universal Single-Copy Orthologs analysis of de novo transcriptome of guava with eudicots identified 93.7% complete BUSCO genes. In silico differential gene expression among tissue types of Allahabad Safeda and validation of candidate genes with qRT-PCR in contrasting color genotypes promises the utility of this first guava transcriptome for its potential of tapping the genetic elements from germplasm collections for enhancing fruit traits.

Cloning and Characterization of Limonoid Glucosyltransferase from Kinnow Mandarin (Citrus reticulata Blanco).

Kinnow mandarin (Citrus reticulata Blanco) is a popular citrus crop of northwestern India and it occupies maximum fruit area in Punjab. However, citrus juice processing industry is still suffering from delayed bitterness problem caused mainly by limonoid aglycones such as limonin. In order to study citrus limonoid metabolism, limonoid glucosyltransferase (LGT) gene, which encodes a natural debittering enzyme, was isolated from the fruit tissues of Kinnow mandarin. After confirmation and characterization, its full-length gene sequence (1533 bp) was submitted to National Centre for Biotechnology Information. Citrus reticulata limonoid glucosyltransferase (CrLGT) occupies a position on an independent branch in the largest subgroup and is phylogenetically different from those in other mandarin species like C. unshiu, showing its uniqueness in several features. The transcript expression of CrLGT, evaluated in different tissues such as young leaf, flavedo, albedo, sac covering and seed of Kinnow mandarin during early (90 days after flowering (DAF)), mid (150-210 DAF) and late (240 DAF) fruit developmental stages using semi-quantitative method, showed the highest expression in flavedo. Thus, it was concluded that the isolated LGT gene has an effect on limonoid metabolic engineering in citrus. Overexpression of this gene can reduce the delayed bitterness problem in citrus juice and enhance the accumulation of specific glucosides that have anticancer effects.

Red rot resistant transgenic sugarcane developed through expression of ß-1,3-glucanase gene.

Sugarcane (Saccharum spp.) is a commercially important crop, vulnerable to fungal disease red rot caused by Colletotrichum falcatum Went. The pathogen attacks sucrose accumulating parenchyma cells of cane stalk leading to severe losses in cane yield and sugar recovery. We report development of red rot resistant transgenic sugarcane through expression of ß-1,3-glucanase gene from Trichoderma spp. The transgene integration and its expression were confirmed by quantitative reverse transcription-PCR in first clonal generation raised from T0 plants revealing up to 4.4-fold higher expression, in comparison to non-transgenic sugarcane. Bioassay of transgenic plants with two virulent C. falcatum pathotypes, Cf 08 and Cf 09 causing red rot disease demonstrated that some plants were resistant to Cf 08 and moderately resistant to Cf 09. The electron micrographs of sucrose storing stalk parenchyma cells from these plants displayed characteristic sucrose-filled cells inhibiting Cf 08 hyphae and lysis of Cf 09 hyphae; in contrast, the cells of susceptible plants were sucrose depleted and prone to both the pathotypes. The transgene expression was up-regulated (up to 2.0-fold in leaves and 5.0-fold in roots) after infection, as compared to before infection in resistant plants. The transgene was successfully transmitted to second clonal generation raised from resistant transgenic plants. ß-1,3-glucanase protein structural model revealed that active sites Glutamate 628 and Aspartate 569 of the catalytic domain acted as proton donor and nucleophile having role in cleaving ß-1,3-glycosidic bonds and pathogen hyphal lysis.

Identification of immunotherapeutic epitope of E5 protein of human papillomavirus-16: An in silico approach.

Cervical cancer is one of the most common gynaecological cancer in India and contributes 1/3rd of global burden. High risk-human papillomavirus (HR-HPV) is the major etiological factor for development of cervical cancer. Two available HPV vaccines provide protection against HPV induced cervical malignancy. However, vaccines having therapeutic values are of utmost priority. Till date, most of HPV therapeutic vaccines are focused on two major HPV oncoproteins (E6/E7). HPV-E5 which acts by altering the activity of cellular proteins, mainly growth factor pathways emerges as a new therapeutic target. In present study, we predicted the candidate B-cell and T-cell epitopes of HPV16-E5, which can be used for HPV immunotherapy. We identified that epitope SAFRCFIVYIIFVY as most potent peptide for HLA-A*11:01 having percentile value of 0.5 and immunogenicity score of 0.69558. For MHC-II, epitopes IPLFLIHTHARFLIT for HLA-DRB1*14:01 alleles have the lowest IC50 value (18.13 nM). The identification of structural feature and immunogenic epitopes provides the best information for development of drugs or vaccine. In conclusion, the expression of E5 protein was detected in the early phase of the HPV infection, which gives an opportunity to target HPV-E5 that would help in the prevention and progression of the precancerous lesion to invasive carcinomas.