Abstract: Fibrinolytic enzyme has potential role as therapeutic agents for the treatment of some blood clotting diseases. It is a novel oral-administered fibrinolytic agent used for the prevention and treatment of cardiac and cerebrovascular diseases. As a drug, it has been widely studied and used for clinical treatment. In the present study, we have cloned and sequenced partial Eisenia fetida fibrinolytic protease (Efp-0) gene from three earthworm strains collected from diverse habitats.

ABSTRACT Introduction: The enzyme cyclooxygenase (COX) has been implicated to be a key enzyme involved in recruiting inflammations. Developing COX inhibitors has remained one of the important aspects of developing novel and safe antiinflammatory agents. An attempt has made to find the medicinal plants as alternatives to presently available NSAIDs (Non steroidal anti-inflammatory drugs).

Plant genomes are complex and contain large amounts of repetitive DNA including microsatellites that are distributed across entire genomes. Whole genome sequences of several monocot and dicot plants that are available in the public domain provide an opportunity to study the origen, distribution and evolution of microsatellites, and also facilitate the development of new molecular markers.

Understanding the distribution of genetic diversity among individuals, populations and gene pools is crucial for the efficient management of germplasm collections and breeding programs. Diversity analysis is routinely carried out using sequencing of selected gene(s) or molecular marker technologies. Here we report on the development of Diversity Arrays Technology (DArT) for pigeonpea (Cajanus cajan) and its wild relatives. DArT tests thousands of genomic loci for polymorphism and provides the binary scores for hundreds of markers in a single hybridization-based assay. We tested eight complexity reduction methods using various combinations of restriction enzymes and selected PstI/HaeIII genomic representation with the largest frequency of polymorphic clones (19.8%) to produce genotyping arrays. The performance of the PstI/HaeIII array was evaluated by typing 96 accessions representing nearly 20 species of Cajanus. A total of nearly 700 markers were identified with the average call rate of 96.0% and the scoring reproducibility of 99.7%. DArT markers revealed genetic relationships among the accessions consistent with the available information and systematic classification. Most of the diversity was among the wild relatives of pigeonpea or between the wild species and the cultivated C. cajan. Only 64 markers were polymorphic among the cultivated accessions. Such narrow genetic base is likely to represent a serious impediment to breeding progress in pigeonpea. Our study shows that DArT can be effectively applied in molecular systematics and biodiversity studies

Background: Pigeonpea [Cajanus cajan (L.) Millspaugh], one of the most important food
legumes of semi-arid tropical and subtropical regions, has limited genomic resources,
particularly expressed sequence based (genic) markers. We report a comprehensive set of
validated genic simple sequence repeat (SSR) markers using deep transcriptome sequencing,
and its application in genetic diversity analysis and mapping.

Assessment of genetic diversity is essential for germplasm characterization, utilization and conservation. Genetic diversity in a set of 132 Indian wild rice accessions belonging to Oryza nivara and Oryza rufipogon and eight cultivated rice varieties was evaluated with 25 highly variable simple sequence repeat (HvSSR) markers using agarose gels and 36 genome-wide SNP markers using Sequenom MassARRAY system. A total of 106 SSR alleles were amplified with an average of 4.24 allele per locus, but only 72 alleles with SNP markers because of its bi-allelic nature. PIC values for HvSSR markers ranged from 0.27 (HvSSR 11-24) to 0.71(HvSSR 05-39) with an average of 0.52 and for SNP it ranged from 0.01(11-3935) to 0.39 (09-209) with an average of 0.19, suggesting more allelic diversity for SSRs over SNP markers. Principal coordinate analysis with SSR and SNP markers revealed that the accessions were uniformly distributed across the two axes in both the plots with 66.53% and 77.65% of cumulative variation, respectively. The analysis of the population structure with SNP markers revealed three sub populations. The comprehensive molecular characterization of the selected set of rice germplasm contributes to the knowledge about levels and distribution of genetic diversity in the Indian wild rice.

Key message Three coding SNPs and one haplotype identified in the OsDREB1F gene have potential to be associated with drought tolerance in rice. Abstract Drought is a serious constraint to rice production worldwide, that can be addressed by deployment of drought tolerant genes. OsDREB1F, one of the most potent drought tolerance transcription activator genes, was re-sequenced for allele mining and association study in a set of 136 wild rice accessions and four cultivated rice. This analysis led to identify 22 SNPs with eight haplotypes based on allelic variations in the accessions used. The nucleotide variation-based neutrality tests suggested that the OsDREB1F gene has been subjected to purifying selection in the studied set of rice germplasm. Six different OsDREB1F protein variants were identified on the basis of translated amino acid residues amongst the orthologues. Five protein variants were truncated due to deletions in coding region and found susceptible to drought stress. Association study revealed that three coding SNPs of this gene were significantly associated with drought tolerance.

Magnaporthe oryzae causes rice blast that is one of the most devastating diseases of rice worldwide. Highly variable nature of this fungus has evolved itself against major resistance genes in newly released rice varieties. Understanding the population structure of this fungus is essential for proper utilization of the rice blast resistance genes in rice crop plants. In the present study, we analyzed 133 isolates of M. oryzae from ten countries to find the allelic variation of Avr-Pita gene that is triggering Pita-mediated resistance in rice plant. The diversity analysis of these alleles showed higher level of nucleotide variation in the coding regions than the noncoding regions. Evolutionary analysis of these alleles indicates that Avr-Pita gene is under purifying selection to favor its major alleles in 133 isolates analyzed in this study. We hypothesize that the selection of favorable Avr-Pita allele in these isolates may occur through a genetic mechanism known as recurrent selective sweeps. A total of 22 functional Avr-Pita protein variants were identified in this study. Insertion of Pot3 transposable element into the promoter of Avr-Pita gene was identified in virulent isolates and was Electronic supplementary material The online version of this article (suggested that mobility of repeat elements in avirulence genes of M. oryzae seems to help in emergence of new virulent types of the pathogen. Allele-specific markers developed in this study will be helpful to identify a particular type of Avr-Pita allele from M. oryzae population which can form the basis for the deployment of Pita gene in different epidemiological regions.

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.

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.

Blast disease caused by Magnaporthe oryzae is
a serious constraint in rice production at global level. Pi54
gene imparts resistance against M. oryzae. Three different
transgenic lines containing Pi54 and its orthologue Pi54rh
were shown to be resistant to different races of M. oryzae.
To determine the chromosomal location of Pi54 gene in
transgenic lines, inverse PCR was performed. Our analysis
showed that in two transgenic lines, Pi54 gene was integrated on chromosomes 6 and 10 at 12.94 and 22.30 Mb,
respectively. Similarly, Pi54rh allele was integrated on
chromosome 1 at 16.25 Mb. The Pi54 gene present on
chromosome 6 was located in a non-coding region whereas
in the other TP-Pi54 line, the gene was introgressed on
chromosome 10 in between the coding region of SAP
domain gene. The Pi54rh was also located in the non
coding region flanked by the retrotransposon genes. These rice lines were evaluated for eight different traits related to
seed and plant morphology and agronomic features for two
consecutive years. The transgenic lines containing Pi54
gene have higher tiller number, grain weight, epicotyl
length, and yield compared to the non-transgenic control.
Multivariate correlation analysis shows that blast resistance
was positively correlated with the number of tillers; thousand grain weight and epicotyl length. These results will
facilitate precise utilization of Pi54 gene and its orthologue
in breeding programs for the development of rice cultivars
with broad spectrum and durable resistance to M. oryzae.