Role of transporters in plant disease resistance.

Plants being sessile have evolved numerous mechanisms to meet the changing environmental and growth conditions. Plant pathogens are responsible for devastating disease epidemics in many species. Transporter proteins are an integral part of plant growth and development, and several studies have documented their role in pathogen disease resistance. In this review, we analyze the studies on genome-wide identifications of plant transporters like sugars will eventually be exported transporters (SWEET), multidrug and toxic compound extrusion (MATE) transporters, ATP-binding cassette (ABC) transporters, natural resistance-associated macrophage proteins (NRAMP), and sugar transport proteins (STPs), all having a significant role in plant disease resistance. The mechanism of action of these transporters, their solute specificity, and the potential application of recent molecular biology approaches deploying these transporters for the development of disease-resistant plants are also discussed. The applications of genome editing tools, such as CRIPSR/Cas9, are also presented. Altogether the information included in this article gives a better understanding of the role of transporter proteins during plant-pathogen interaction.

Blast resistance gene Pi54 over-expressed in rice to understand its cellular and sub-cellular localization and response to different pathogens.

Rice blast resistance gene, Pi54 provides broad-spectrum resistance against different strains of Magnaporthe oryzae. Understanding the cellular localization of Pi54 protein is an essential step towards deciphering its place of interaction with the cognate Avr-gene. In this study, we investigated the sub-cellular localization of Pi54 with Green Fluorescent Protein (GFP) as a molecular tag through transient and stable expression in onion epidermal cells (Allium cepa) and susceptible japonica cultivar rice Taipei 309 (TP309), respectively. Confocal microscopy based observations of the onion epidermal cells revealed nucleus and cytoplasm specific GFP signals. In the stable transformed rice plants, GFP signal was recorded in the stomata, upper epidermal cells, mesophyll cells, vascular bundle, and walls of bundle sheath and bulliform cells of leaf tissues. These observations were further confirmed by Immunocytochemical studies. Using GFP specific antibodies, it was found that there was sufficient aggregation of GFP::Pi54protein in the cytoplasm of the leaf mesophyll cells and periphery of the epidermal cells. Interestingly, the transgenic lines developed in this study could show a moderate level of resistance to Xanthomonas oryzae and Rhizoctonia solani, the causal agents of the rice bacterial blight and sheath blight diseases, respectively. This study is a first detailed report, which emphasizes the cellular and subcellular distribution of the broad spectrum blast resistance gene Pi54 in rice and the impact of its constitutive expression towards resistance against other fungal and bacterial pathogens of rice.

Nitric oxide and hydrogen sulfide crosstalk during heavy metal stress in plants.

Gases such as ethylene, hydrogen peroxide (H2 O2 ), nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2 S) have been recognized as vital signaling molecules in plants and animals. Of these gasotransmitters, NO and H2 S have recently gained momentum mainly because of their involvement in numerous cellular processes. It is therefore important to study their various attributes including their biosynthetic and signaling pathways. The present review provides an insight into various routes for the biosynthesis of NO and H2 S as well as their signaling role in plant cells under different conditions, more particularly under heavy metal stress. Their beneficial roles in the plant's protection against abiotic and biotic stresses as well as their adverse effects have been addressed. This review describes how H2 S and NO, being very small-sized molecules, can quickly pass through the cell membranes and trigger a multitude of responses to various factors, notably to various stress conditions such as drought, heat, osmotic, heavy metal and multiple biotic stresses. The versatile interactions between H2 S and NO involved in the different molecular pathways have been discussed. In addition to the signaling role of H2 S and NO, their direct role in posttranslational modifications is also considered. The information provided here will be helpful to better understand the multifaceted roles of H2 S and NO in plants, particularly under stress conditions.

Corrigendum: Cloning and functional validation of early inducible Magnaporthe oryzae responsive CYP76M7 promoter from rice.

[This corrects the article on p. 371 in vol. 6, PMID: 26052337.].

Silencing of HaAce1 gene by host-delivered artificial microRNA disrupts growth and development of Helicoverpa armigera.

The polyphagous insect-pest, Helicoverpa armigera, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of Bt transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement Bt to tackle insect-pests menace. In this study, we report host-delivered silencing of HaAce1 gene, encoding the predominant isoform of H. armigera acetylcholinesterase, by an artificial microRNA, HaAce1-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with HaAce1-amiR1/HaAce1-amiR1* sequences. The recombinant HaAce1-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing HaAce1-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of HaAce1 mRNA was 70-80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of HaAce1 gene for H. armigera management.

Analysis of differential transcript expression in chickpea during compatible and incompatible interactions with Fusarium oxysporum f. sp. ciceris Race 4.

The present study reports the transcriptome analysis of resistance (WR315) and susceptible (JG62) genotypes of chickpea in response to Fusarium oxysporum f. sp. ciceris (Foc) race 4 using the method of suppression subtractive hybridization. Altogether, 162 chickpea-expressed sequence tags (ESTs) were identified from two libraries and analyzed to catalog eight functional categories. These ESTs could be assembled into 18 contigs and 144 singletons with 10 contigs and 68 singletons from compatible and 8 contigs and 70 singletons from incompatible interaction. The largest category consisted of ESTs which encode for proteins related to hypothetical proteins (22.8%), followed by energy and metabolism (20.3%)-related genes, defense and cell rescue-related genes (17.9%) and signal transduction-related genes (16%). Among them, 17.1 and 18.7% were defense-related genes in compatible and incompatible interaction, respectively. These ESTs mainly includes various putative genes related to oxidative burst, pathogenesis and secondary metabolism. Induction of putative superoxide dismutase, metallothionein, 4-coumarate-CoA ligase, heat shock proteins and cysteine proteases indicated oxidative burst after infection. The ESTs belonged to various functional categories which were directly and indirectly associated with defense signaling pathways. Quantitative and semi-quantitative polymerase chain reaction exhibited differential expression of candidate genes and detected higher levels in incompatible interaction compared to compatible interaction. The present study revealed partial molecular mechanism associated with the resistance in chickpea against Foc, which is the key to design a strategy for incorporation of resistance via either biotechnological means or introgression of resistance genes.

Genome-wide discovery of tissue-specific miRNAs in clusterbean (Cyamopsis tetragonoloba) indicates their association with galactomannan biosynthesis.

Owing to the presence of 80% soluble dietary fibre, high protein content and high value gum, clusterbean (Cyamopsis tetragonoloba) has recently emerged as an economically important legume. The developing clusterbean seeds accumulate 90% galactomannans in the endosperm and, therefore, can be used as a model crop to understand galactomannan biosynthesis and its regulation. miRNAs are tiny master regulators of their corresponding target genes, resulting in variations in the amounts of their metabolic end products. To understand the role of these regulators in galactomannan biosynthesis regulation, small RNA libraries were prepared and sequenced from five tissues of clusterbean genotype RGC-936, and miRanalyzer and DSAP programs were used to identify conserved miRNAs and novel small RNAs. A total of 187 known and 171 novel miRNAs were found to be differentially expressed, of which 10 miRNAs were validated. A complicated network topology and 35% sharing of the target mRNAs between known and novel miRNAs suggest random evolution of novel miRNAs. The gene ontology (GO) annotation of potential target genes revealed the genes coding for signalling and carbohydrate metabolism (50.10%), kinases and other enzymes (20.75%), transcription factors (10.20%), transporters (8.35%) and other targets (10.6%). Two novel unigenes were annotated as ManS (mannosyltransferase/mannan synthase) and UGE (UDP- D-glucose 4-epimerase) and validated as targets for three novel miRNAs, that is Ct-miR3130, Ct-miR3135 and Ct-miR3157. Our findings reveal that these novel miRNAs could play an important role in the regulation of the galactomannan pathway in C. tetragonoloba and possibly other galactomannan-producing species.

MiSNPDb: a web-based genomic resources of tropical ecology fruit mango (Mangifera indica L.) for phylogeography and varietal differentiation.

Mango is one of the most important fruits of tropical ecological region of the world, well known for its nutritive value, aroma and taste. Its world production is >45MT worth >200 billion US dollars. Genomic resources are required for improvement in productivity and management of mango germplasm. There is no web-based genomic resources available for mango. Hence rapid and cost-effective high throughput putative marker discovery is required to develop such resources. RAD-based marker discovery can cater this urgent need till whole genome sequence of mango becomes available. Using a panel of 84 mango varieties, a total of 28.6 Gb data was generated by ddRAD-Seq approach on Illumina HiSeq 2000 platform. A total of 1.25 million SNPs were discovered. Phylogenetic tree using 749 common SNPs across these varieties revealed three major lineages which was compared with geographical locations. A web genomic resources MiSNPDb, available at http://webtom.cabgrid.res.in/mangosnps/ is based on 3-tier architecture, developed using PHP, MySQL and Javascript. This web genomic resources can be of immense use in the development of high density linkage map, QTL discovery, varietal differentiation, traceability, genome finishing and SNP chip development for future GWAS in genomic selection program. We report here world's first web-based genomic resources for genetic improvement and germplasm management of mango.

Introgression of Black Rot Resistance from Brassica carinata to Cauliflower (Brassica oleracea botrytis Group) through Embryo Rescue.

Black rot caused by Xanthomonas campestris pv. campestris (Xcc) is a very important disease of cauliflower (Brassica oleracea botrytis group) resulting into 10-50% yield losses every year. Since there is a dearth of availability of resistance to black rot disease in B. oleracea (C genome), therefore exploration of A and B genomes was inevitable as they have been reported to be potential reservoirs of gene(s) for resistance to black rot. To utilize these sources, interspecific hybrid and backcross progeny (B1) were generated between cauliflower "Pusa Sharad" and Ethiopian mustard "NPC-9" employing in vitro embryo rescue technique. Direct ovule culture method was better than siliqua culture under different temperature regime periods. Hybridity testing of F1 inter-specific plants was carried out using co-dominant SSR marker and Brassica B and C genome-specific (DB and DC) primers. Meiosis in the di-genomic (BCC) interspecific hybrid of B. oleracea botrytis group (2n = 18, CC) × B. carinata (2n = 4x = 34, BBCC) was higly disorganized and cytological analysis of pollen mother cells revealed chromosomes 2n = 26 at metaphase-I. Fertile giant pollen grain formation was observed frequently in interspecific F1 hybrid and BC1 plants. The F1 inter-specific plants were found to be resistant to Xcc race 1. Segregation distortion was observed in BC1 generation for black rot resistance and different morphological traits. The At1g70610 marker analysis confirmed successful introgression of black rot resistance in interspecific BC1 population. This effort will go a long way in pyramiding gene(s) for resistance against black rot in Cole crops, especially cauliflower and cabbage for developing durable resistance, thus minimize dependency on bactericides.

A high-density intraspecific SNP linkage map of pigeonpea (Cajanas cajan L. Millsp.).

Pigeonpea (Cajanus cajan (L.) Millsp.) is a major food legume cultivated in semi-arid tropical regions including the Indian subcontinent, Africa, and Southeast Asia. It is an important source of protein, minerals, and vitamins for nearly 20% of the world population. Due to high carbon sequestration and drought tolerance, pigeonpea is an important crop for the development of climate resilient agriculture and nutritional security. However, pigeonpea productivity has remained low for decades because of limited genetic and genomic resources, and sparse utilization of landraces and wild pigeonpea germplasm. Here, we present a dense intraspecific linkage map of pigeonpea comprising 932 markers that span a total adjusted map length of 1,411.83 cM. The consensus map is based on three different linkage maps that incorporate a large number of single nucleotide polymorphism (SNP) markers derived from next generation sequencing data, using Illumina GoldenGate bead arrays, and genotyping with restriction site associated DNA (RAD) sequencing. The genotyping-by-sequencing enhanced the marker density but was met with limited success due to lack of common markers across the genotypes of mapping population. The integrated map has 547 bead-array SNP, 319 RAD-SNP, and 65 simple sequence repeat (SSR) marker loci. We also show here correspondence between our linkage map and published genome pseudomolecules of pigeonpea. The availability of a high-density linkage map will help improve the anchoring of the pigeonpea genome to its chromosomes and the mapping of genes and quantitative trait loci associated with useful agronomic traits.

Novel Chitinase Gene LOC_Os11g47510 from Indica Rice Tetep Provides Enhanced Resistance against Sheath Blight Pathogen Rhizoctonia solani in Rice.

Sheath blight disease (ShB), caused by the fungus Rhizoctonia solani Kühn, is one of the most destructive diseases of rice (Oryza sativa L.), causing substantial yield loss in rice. In the present study, a novel rice chitinase gene, LOC_Os11g47510 was cloned from QTL region of R. solani tolerant rice line Tetep and used for functional validation by genetic transformation of ShB susceptible japonica rice line Taipei 309 (TP309). The transformants were characterized using molecular and functional approaches. Molecular analysis by PCR using a set of primers specific to CaMv 35S promoter, chitinase and HptII genes confirmed the presence of transgene in transgenic plants which was further validated by Southern hybridization. Further, qRT-PCR analysis of transgenic plants showed good correlation between transgene expression and the level of sheath blight resistance among transformants. Functional complementation assays confirmed the effectiveness of the chitinase mediated resistance in all the transgenic TP309 plants with varying levels of enhanced resistance against R. solani. Therefore, the novel chitinase gene cloned and characterized in the present study from the QTL region of rice will be of significant use in molecular plant breeding program for developing sheath blight resistance in rice.

Analysis of genetic structure and interrelationships in lentil species using morphological and SSR markers.

Genetic structure and relationships of 130 lentil accessions belonging to six taxa were analysed. For this purpose, seven morphological traits and 31 polymorphic simple sequence repeat (SSR) primers were used for this purpose. Morphological traits grouped lentil accessions into five main clusters. SSR primers collectively amplified 139 polymorphic alleles in a range of 2-10 with an average of 4.48 alleles. The size of amplified alleles varied from 50 to 650 bp. Polymorphism information content (PIC) ranged from 0.02 to 0.85 with an average of 0.46. Neighbour-joining tree grouped accessions broadly according to their taxonomic ranks, except L. culinaris ssp. odemensis. Analysis of molecular variance (AMOVA) revealed that a major portion (82.0%) of genetic variance resided within species, while only 18% resided among species. Bayesian model-based STRUCTURE analysis assigned all accessions into five clusters and showed some admixture within individuals. Cluster analysis showed that cultivated Lens accessions of Ethiopian origin clustered separately, from other cultivated accessions indicating its distinct lineage. Among the analysed lentil species, L. culinaris ssp. odemensis seemed to have conserved genetic background and needs revision of its taxonomic status. Results of present study provide important information on genetic diversity and relationships among different wild and cultivated taxa of lentil. Thus, these results can be useful in designing breeding strategies for future improvement and taxonomic implications in lentil.

Understanding Host-Pathogen Interactions with Expression Profiling of NILs Carrying Rice-Blast Resistance Pi9 Gene.

Magnaporthe oryzae infection causes rice blast, a destructive disease that is responsible for considerable decrease in rice yield. Development of resistant varieties via introgressing resistance genes with marker-assisted breeding can eliminate pesticide use and minimize crop losses. Here, resistant near-isogenic line (NIL) of Pusa Basmati-1(PB1) carrying broad spectrum rice blast resistance gene Pi9 was used to investigate Pi9-mediated resistance response. Infected and uninfected resistant NIL and susceptible control line were subjected to RNA-Seq. With the exception of one gene (Pi9), transcriptional signatures between the two lines were alike, reflecting basal similarities in their profiles. Resistant and susceptible lines possessed 1043 (727 up-regulated and 316 down-regulated) and 568 (341 up-regulated and 227 down-regulated) unique and significant differentially expressed loci (SDEL), respectively. Pathway analysis revealed higher transcriptional activation of kinases, WRKY, MYB, and ERF transcription factors, JA-ET hormones, chitinases, glycosyl hydrolases, lipid biosynthesis, pathogenesis and secondary metabolism related genes in resistant NIL than susceptible line. Singular enrichment analysis demonstrated that blast resistant NIL is significantly enriched with genes for primary and secondary metabolism, response to biotic stimulus and transcriptional regulation. The co-expression network showed proteins of genes in response to biotic stimulus interacted in a manner unique to resistant NIL upon M. oryzae infection. These data suggest that Pi9 modulates genome-wide transcriptional regulation in resistant NIL but not in susceptible PB1. We successfully used transcriptome profiling to understand the molecular basis of Pi9-mediated resistance mechanisms, identified potential candidate genes involved in early pathogen response and revealed the sophisticated transcriptional reprogramming during rice-M. oryzae interactions.

Dissection of genomic features and variations of three pathotypes of Puccinia striiformis through whole genome sequencing.

Stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici, is one of the important diseases of wheat. We used NGS technologies to generate a draft genome sequence of two highly virulent (46S 119 and 31) and a least virulent (K) pathotypes of P. striiformis from the Indian subcontinent. We generated ~24,000-32,000 sequence contigs (N50;7.4-9.2 kb), which accounted for ~86X-105X sequence depth coverage with an estimated genome size of these pathotypes ranging from 66.2-70.2 Mb. A genome-wide analysis revealed that pathotype 46S 119 might be highly evolved among the three pathotypes in terms of year of detection and prevalence. SNP analysis revealed that ~47% of the gene sets are affected by nonsynonymous mutations. The extracellular secreted (ES) proteins presumably are well conserved among the three pathotypes, and perhaps purifying selection has an important role in differentiating pathotype 46S 119 from pathotypes K and 31. In the present study, we decoded the genomes of three pathotypes, with 81% of the total annotated genes being successfully assigned functional roles. Besides the identification of secretory genes, genes essential for pathogen-host interactions shall prove this study as a huge genomic resource for the management of this disease using host resistance.

Analysis of Magnaporthe oryzae Genome Reveals a Fungal Effector, Which Is Able to Induce Resistance Response in Transgenic Rice Line Containing Resistance Gene, Pi54.

Rice blast caused by Magnaporthe oryzae is one of the most important diseases of rice. Pi54, a rice gene that imparts resistance to M. oryzae isolates prevalent in India, was already cloned but its avirulent counterpart in the pathogen was not known. After decoding the whole genome of an avirulent isolate of M. oryzae, we predicted 11440 protein coding genes and then identified four candidate effector proteins which are exclusively expressed in the infectious structure, appresoria. In silico protein modeling followed by interaction analysis between Pi54 protein model and selected four candidate effector proteins models revealed that Mo-01947_9 protein model encoded by a gene located at chromosome 4 of M. oryzae, interacted best at the Leucine Rich Repeat domain of Pi54 protein model. Yeast-two-hybrid analysis showed that Mo-01947_9 protein physically interacts with Pi54 protein. Nicotiana benthamiana leaf infiltration assay confirmed induction of hypersensitive response in the presence of Pi54 gene in a heterologous system. Genetic complementation test also proved that Mo-01947_9 protein induces avirulence response in the pathogen in presence of Pi54 gene. Here, we report identification and cloning of a new fungal effector gene which interacts with blast resistance gene Pi54 in rice.

Functional Characterization of Novel Chitinase Genes Present in the Sheath Blight Resistance QTL: qSBR11-1 in Rice Line Tetep.

Rice sheath blight disease caused by Rhizoctonia solani is one of the most devastating diseases in rice leading to heavy yield losses. Due to the polygenic nature of resistance, no major resistance gene with complete host resistance against R. solani has been reported. In this study, we have performed molecular and functional analysis of the genes associated with the major R. solani-resistance QTL qSBR11-1 in the indica rice line Tetep. Sequence analysis revealed the presence of a set of 11 tandem repeats containing genes with a high degree of homology to class III chitinase defense response genes. Real-time quantitative PCR analysis showed that all the genes are strongly induced 36 h after R. solani infection. Comparison between the resistant Tetep and the susceptible HP2216 lines shows that the induction of the chitinase genes is much higher in the Tetep line. Recombinant protein produced in vitro for six of the eleven genes showed chitinolytic activity in gel assays but we did not detect any xylanase inhibitory activity. All the six in vitro expressed proteins show antifungal activity with a clear inhibitory effect on the growth of the R. solani mycelium. The characterized chitinase genes can provide an important resource for the genetic improvement of R. solani susceptible rice lines for sheath blight resistance breeding.

Comparative Temporal Transcriptome Profiling of Wheat near Isogenic Line Carrying Lr57 under Compatible and Incompatible Interactions.

Leaf rust caused by Puccinia triticina (Pt) is one of the most important diseases of bread wheat globally. Recent advances in sequencing technologies have provided opportunities to analyse the complete transcriptomes of the host as well as pathogen for studying differential gene expression during infection. Pathogen induced differential gene expression was characterized in a near isogenic line carrying leaf rust resistance gene Lr57 and susceptible recipient genotype WL711. RNA samples were collected at five different time points 0, 12, 24, 48, and 72 h post inoculation (HPI) with Pt 77-5. A total of 3020 transcripts were differentially expressed with 1458 and 2692 transcripts in WL711 and WL711+Lr57, respectively. The highest number of differentially expressed transcripts was detected at 12 HPI. Functional categorization using Blast2GO classified the genes into biological processes, molecular function and cellular components. WL711+Lr57 showed much higher number of differentially expressed nucleotide binding and leucine rich repeat genes and expressed more protein kinases and pathogenesis related proteins such as chitinases, glucanases and other PR proteins as compared to susceptible genotype. Pathway annotation with KEGG categorized genes into 13 major classes with carbohydrate metabolism being the most prominent followed by amino acid, secondary metabolites, and nucleotide metabolism. Gene co-expression network analysis identified four and eight clusters of highly correlated genes in WL711 and WL711+Lr57, respectively. Comparative analysis of the differentially expressed transcripts led to the identification of some transcripts which were specifically expressed only in WL711+Lr57. It was apparent from the whole transcriptome sequencing that the resistance gene Lr57 directed the expression of different genes involved in building the resistance response in the host to combat invading pathogen. The RNAseq data and differentially expressed transcripts identified in present study is a genomic resource which can be used for further studying the host pathogen interaction for Lr57 and wheat transcriptome in general.

A web-based microsatellite database for the Magnaporthe oryzae genome.

Microsatellites have been widely utilized for molecular marker development. Codominant and multiallelic nature of these simple repeats have several advantages over other types of molecular markers. Their broad applicability in the area of molecular biology like gene mapping, genome characterization, genome evolution, and gene regulation has been reported in various crop plants, animals and fungi. Considering these benefits of the SSR markers, a MMDB (Magnaporthe oryzae Microsatellite Database) was developed to help in understanding about the pathogen and its diversity at strains level of a particular geographic region, which can help us to make a proper utilization of blast resistance genes in the region. This microsatellite database is based on whole genome sequence of two M. oryzae isolates, RML-29 (2665 SSRs from 43037792 bp) and RP-2421 (3169 SSRs from 45510614 bp). Although, first M. oryzae genome (70-15) was sequenced in 2005, but this sequenced isolate is not a true field isolate of M. oryzae. Therefore, MMDB has great potential in the study of diversification and characterization of M. oryzae and other related fungi. AVAILABILITY: http://14.139.229.199/home.aspx.

Construction of a Genetic Linkage Map and Identification of QTLs for Seed Weight and Seed Size Traits in Lentil (Lens culinaris Medik.).

Seed weight and seed size both are quantitative traits and have been considered as important components of grain yield, thus identification of quantitative trait loci (QTL) for seed traits in lentil (Lens culinaris) would be beneficial for the improvement of grain yield. Hence the main objective of this study was to identify QTLs for seed traits using an intraspecific mapping population derived from a cross between L. culinaris cv. Precoz (seed weight-5.1g, seed size-5.7mm) and L. culinaris cv. L830 (seed weight-2.2g, seed size-4mm) comprising 126 F8-RILs. For this, two microsatellite genomic libraries enriched for (GA/CT) and (GAA/CTT) motif were constructed which resulted in the development of 501 new genomic SSR markers. Six hundred forty seven SSR markers (including 146 previously published) were screened for parental polymorphism and 219 (33.8%) were found to be polymorphic among the parents. Of these 216 were mapped on seven linkage groups at LOD4.0 spanning 1183.7cM with an average marker density of 5.48cM. Phenotypic data from the RILs was used to identify QTLs for the seed weight and seed size traits by single marker analysis (SMA) followed by composite interval mapping (CIM) which resulted in one QTL each for the 2 traits (qSW and qSS) that were co-localized on LG4 and explained 48.4% and 27.5% of phenotypic variance respectively. The current study would serve as a strong foundation for further validation and fine mapping for utilization in lentil breeding programs.

Extensive sequence variation in rice blast resistance gene Pi54 makes it broad spectrum in nature.

Rice blast resistant gene, Pi54 cloned from rice line, Tetep, is effective against diverse isolates of Magnaporthe oryzae. In this study, we prospected the allelic variants of the dominant blast resistance gene from a set of 92 rice lines to determine the nucleotide diversity, pattern of its molecular evolution, phylogenetic relationships and evolutionary dynamics, and to develop allele specific markers. High quality sequences were generated for homologs of Pi54 gene. Using comparative sequence analysis, InDels of variable sizes in all the alleles were observed. Profiling of the selected sites of SNP (Single Nucleotide Polymorphism) and amino acids (N sites ≥ 10) exhibited constant frequency distribution of mutational and substitutional sites between the resistance and susceptible rice lines, respectively. A total of 50 new haplotypes based on the nucleotide polymorphism was also identified. A unique haplotype (H_3) was found to be linked to all the resistant alleles isolated from indica rice lines. Unique leucine zipper and tyrosine sulfation sites were identified in the predicted Pi54 proteins. Selection signals were observed in entire coding sequence of resistance alleles, as compared to LRR domains for susceptible alleles. This is a maiden report of extensive variability of Pi54 alleles in different landraces and cultivated varieties, possibly, attributing broad-spectrum resistance to Magnaporthe oryzae. The sequence variation in two consensus region: 163 and 144 bp were used for the development of allele specific DNA markers. Validated markers can be used for the selection and identification of better allele(s) and their introgression in commercial rice cultivars employing marker assisted selection.