Identification of differentially expressed genes and SNPs linked to harvest body weight of genetically improved rohu carp, Labeo rohita.

In most of the aquaculture selection programs, harvest body weight has been a preferred performance trait for improvement. Molecular interplay of genes linked to higher body weight is not elucidated in major carp species. The genetically improved rohu carp with 18% average genetic gain per generation with respect to harvest body weight is a promising candidate for studying genes' underlying performance traits. In the present study, muscle transcriptome sequencing of two groups of individuals, with significant difference in breeding value, belonging to the tenth generation of rohu carp was performed using the Illumina HiSeq 2000 platform. A total of 178 million paired-end raw reads were generated to give rise to 173 million reads after quality control and trimming. The genome-guided transcriptome assembly and differential gene expression produced 11,86,119 transcripts and 451 upregulated and 181 downregulated differentially expressed genes (DEGs) between high-breeding value and low-breeding value (HB & LB) groups, respectively. Similarly, 39,158 high-quality coding SNPs were identified with the Ts/Tv ratio of 1.23. Out of a total of 17 qPCR-validated transcripts, eight were associated with cellular growth and proliferation and harbored 13 SNPs. The gene expression pattern was observed to be positively correlated with RNA-seq data for genes such as myogenic factor 6, titin isoform X11, IGF-1 like, acetyl-CoA, and thyroid receptor hormone beta. A total of 26 miRNA target interactions were also identified to be associated with significant DETs (p-value < 0.05). Genes such as Myo6, IGF-1-like, and acetyl-CoA linked to higher harvest body weight may serve as candidate genes in marker-assisted breeding and SNP array construction for genome-wide association studies and genomic selection.

SEDDS Basic Design and Recent Formulation Advancement: A Concurrent Review.

In the present scenario, lipid-based novel drug delivery systems are the area of interest for the formulation scientist in order to improve the bioavailability of poorly water-soluble drugs. A selfemulsifying drug delivery system (SEDDS) upon contact with the gastrointestinal fluid, forms an o/w emulsion. SEDDS has gained popularity as a potential platform for improving the bioavailability of the lipophilic drug by overcoming several challenges. The various advantages like improved solubility, bypassing lymphatic transport, and improvement in bioavailability are associated with SMEDDS or SNEDDS. The extent of the formation of stable SEDDS depends on a specific combination of surfactant, co-surfactant, and oil. The present review highlighted the different aspects of formulation design along with optimization and characterization of SEDDS formulation. It also gives a brief description of the various aspects of the excipients used in SEDDS formulation. This review also includes the conflict between types of SEDDS based on droplet size. There is an extensive review of various research regarding different solidification techniques used for SEDDS in the last three years.

De novo assembly, transcriptome characterization and marker discovery in Indian major carp, Labeo rohita through pyrosequencing.

Labeo rohita, one of the Indian major carps, is the most popular culture species in Indian subcontinent due to its consumer preference and delicacy. A selective breeding program for harvest body weight has resulted in an average genetic gain of 17% per generation. Transcriptome resource for this species is scanty. Here, we have characterized the liver and muscle transcriptomes of rohu using Roche 454 GS-FLX next generation sequencing platform. In total, 1.2 million reads were generated, de novo assembly and clustering resulted in 4171 transcripts. Out of these, 4171 had significant blast hit against NCBI nr database, and 2130 transcripts were successfully annotated. In total, 289 SSRs were identified with an identification rate of 5.8%, and dinucleotide repeat motifs were observed to be the most abundant SSRs. Further, 2231 putative SNPs were identified with high confidence. Validation of eight putative SNPs using Sanger sequencing resulted in 100% true SNPs. Significant allelic imbalance of M1, M4 and M5 loci between growth selected and control individual were observed. Furthermore, 13 transcription factors were identified in the present study belonging to six different transcription factor families. The present study demonstrated the utility of RNAseq to develop genomics resources in non-model fish species, and the marker resources developed would support the genetic improvement program of this species.

Molecular characterization, computational analysis and expression profiling of Dmrt1 gene in Indian major carp, Labeo rohita (Hamilton 1822).

Sexual dimorphism of fish morphology, physiology and behavior is diverse and complex in nature. Doublesex and mab-3 related transcription factor (Dmrt) is a large protein family whose function is sexual development and differentiation in vertebrates. Here, we report a full-length cDNA sequence of Labeo rohita (rohu) Dmrt1 of 907 bp length having 798 bp of open reading frame encoding 265 amino acids. The molecular weight of rohu DMRT1 protein was found to be 28.74 KDa and isoelectric point was 7.53. DMRT1 protein contains 23 positively and 24 negatively charged amino acids with a GRAVY score of -0.618. A characteristic DM domain was found in DMRT1 protein, which is a novel DNA-binding domain. Phylogenetic analysis showed maximum similarity with Cyprinus carpio when compared with DMRT1 of other vertebrates. Molecular docking study identified active sites to be targeted for drug designing. Rohu DMRT1 was observed to interact with other proteins such as FOXL2, CYP19a1a, AMH and SOX9a. Differential expression study revealed higher expression in testis tissue implying its role in male sex differentiation and testicular development. The information generated in the present work could facilitate further research to resolve the issues related to gonadal maturation and reproduction of commercially important aquaculture species.

Population structure and genetic diversity of hatchery stocks as revealed by combined mtDNA fragment sequences in Indian major carp, Catla catla.

Catla catla is the second most important Indian major carp due to high growth rate and acceptance to consumers for food value. It is widely cultured in the Indian subcontinent as monoculture or polyculture. In the present study, genetic diversity among hatchery stocks (total 218 samples of catla) collected from different geographical regions of India was examined using mtDNA fragment sequence of Cyt b (306 bp) and D loop (710 bp). High numbers (57) of population specific haplotypes were observed in the present study. The results revealed significant genetic heterogeneity for the sequence data (FST = 0.27546, p < .05). Analysis of molecular variance revealed significant genetic differentiation among different catla populations. The information generated in present study could be useful to develop broad genetic base populations of catla.

Rapid recovery of complete mitogenome of Indian major carp, Catla catla from low depth paired end Illumina sequencing.

Here we report the reconstruction of the catla (Catla catla) complete mitochondrial genome sequence from low depth paired end Illumina sequencing. The genome is of 16,597 bp in size. Similar to other vertebrate mtgenomes, it consists of 13 protein-coding genes, 22 tRNAs, 2 rRNAs and a putative control region. The present mtgenome is 3 bp longer than the earlier reported catla mtgenome from our laboratory. Majority of the mitochondrial genes are encoded by the H-strand. Phylogenetics analysis revealed that Catla catla is closer to Labeo rohita than other labeo species. Present study demonstrated the power of next generation sequencing towards hassle free and rapid sequencing of mitochondrial genomes of non-model organisms.

Genetic relationship and population structure of three Indian local chicken populations as revealed by mtDNA D-loop.

The genetic information obtained from the mitochondrial DNA D-loop region has paramount importance in understanding the evolution of closely related individuals, and designing proper breeding or conservation plans. The present study was conducted using partial D-loop sequences of three local poultry populations from Odisha, India. The partial D-loop sequences were found to be highly polymorphic having 164 polymorphic sites with 89 singletons and 75 parsimony informative sites. Furthermore, 25 insertion and deletion sites were observed. High genetic diversity was observed within three local chicken populations. Highest genetic difference was observed between Gujuri and Kalua population (0.2230) followed by Gujuri and Hansli (0.199) and Kalua with Hansli (0.166). The pairwise mismatch distribution showed that all populations are of constant size over time. Phylogenetic tree analysis indicated that the said three populations were close to the referred population of China, Sri Lanka, Indonesia and Japan than Aseel and Kadaknath (Indian native breeds).

Low-depth shotgun sequencing resolves complete mitochondrial genome sequence of Labeo rohita.

Labeo rohita, popularly known as rohu, is a widely cultured species in whole Indian subcontinent. In the present study, we used in-silico approach to resolve complete mitochondrial genome of rohu. Low-depth shotgun sequencing using Roche 454 GS FLX (Branford, Connecticut, USA) followed by de novo assembly in CLC Genomics Workbench version 7.0.4 (Aarhus, Denmark) revealed the complete mitogenome of L. rohita to be 16 606 bp long (accession No. KR185963). It comprised of 13 protein-coding genes, 22 tRNAs, 2 rRNAs and 1 putative control region. The gene order and organization are similar to most vertebrates. The mitogenome in the present investigation has 99% similarity with that of previously reported mitogenomes of rohu and this is also evident from the phylogenetic study using maximum-likelihood (ML) tree method. This study was done to determine the feasibility, accuracy and reliability of low-depth sequence data obtained from NGS platform as compared to the Sanger sequencing. Thus, NGS technology has proven to be competent and a rapid in-silico alternative to resolve the complete mitochondrial genome sequence, thereby reducing labors and time.

Preliminary genetic linkage map of Indian major carp, Labeo rohita (Hamilton 1822) based on microsatellite markers.

Linkage map with wide marker coverage is an essential resource for genetic improvement study for any species. Sex-averaged genetic linkage map of Labeo rohita, popularly known as 'rohu', widely cultured in the Indian subcontinent, was developed by placing 68 microsatellite markers generated by a simplified method. The parents and their F1 progeny (92 individuals) were used as segregating populations. The genetic linkage map spans a sex-averaged total length of 1462.2 cM, in 25 linkage groups. The genome length of rohu was estimated to be 3087.9 cM. This genetic linkage map may facilitate systematic searches of the genome to identify genes associated with commercially important characters and marker-assisted selection programmes of this species.

Assembly and variation analyses of Clarias batrachus mitogenome retrieved from WGS data and its phylogenetic relationship with other catfishes.

Whole genome sequencing (WGS) using next generation sequencing technologies paves the way to sequence the mitochondrial genomes with greater ease and lesser time. Here, we used the WGS data of Clarias batrachus, generated from Roche 454 and Ion Torrent sequencing platforms, to assemble the complete mitogenome using both de novo and reference based approaches. Both the methods yielded almost similar results and the best assembled mitogenome was of 16,510 bp size (GenBank Acc. No. KM259918). The mitogenome annotation resulted in 13 coding genes, 22 tRNA genes, 2 rRNA genes and one control region, and the gene order was found to be identical with other catfishes. Variation analyses between assembled and the reference (GenBank Acc. No. NC_023923) mitogenome revealed 51 variations. The phylogenetic analysis of coding DNA sequences and tRNA supports the monophyly of catfishes. Two SSRs were identified in C. batrachus mitogenome, out of which one was unique to this species. Based on the relative rate of gene evolution, protein coding mitochondrial genes were found to evolve at a much faster pace than the d-loop, which in turn are followed by the rRNAs; the tRNAs showed wide variability in the rate of sequence evolution, and on average evolve the slowest. Among the coding genes, ND2 evolves most rapidly. The variations present in the coding regions of the mitogenome and their comparative analyses with other catfish species may be useful in species conservation and management programs.

Complete mitochondrial genome sequence of Cirrhinus mrigala (Hamilton, 1822).

The complete mitochondrial genome of Cirrhinus mrigala was determined using the polymerase chain reaction. The mitogenome (16,594 bp) has the typical vertebrate mitochondrial gene arrangement, including 13 protein-coding genes, 22 tRNA genes, two rRNA genes and one control region. The overall base composition on the heavy strand was as follows: A: 32.0%, G: 15.5%, C: 28.0%, T: 24.55% and the A+T content: 56.5%. The control region contains a dinucleotide repeat motif, (TA)14, a termination-associated sequence and three conserved sequence blocks. These mitogenome sequence data would play an important role in population genetics and the molecular taxonomy of cultivable cyprinids in India.

Complete mitochondrial genome sequence of Catla catla and its phylogenetic consideration.

Complete nucleotide sequence of mitochondrial genome (mitogenome) of the Catla catla (Ostariophysi: Cypriniformes: Cyprinidae) was determined in the present study. Its length is 16,594 bp and contains 13 protein coding genes, 22 transfer RNAs, two ribosomal RNAs and one non-coding control region. Most of the genes were encoded on the H-strand, while the ND6 and eight tRNA (Gln, Ala, Asn, Cys, Tyr, Ser (UCN), Glu and Pro) genes were encoded on the L-strand. The reading frames of two pair of genes overlapped: ATPase 8 with 6 and ND4L with ND4 by seven nucleotides each. The main non-coding region was 929 bp, with three conserved sequence blocks (CSB-I, CSB-II, and CSB-III) and an unusual simple sequence repeat, (TA)(7). Phylogenetic analyses based on complete mitochondrial genome sequences were in favor of the traditional taxonomy of family Cyprinidae. In conclusion present mitogenome of Catla catla adds more information to our understanding of diversity and evolution of mitogenome in fishes.

Complete mitochondrial genome of Labeo rohita.

The complete mitochondrial genome of Labeo rohita, an important cultivable fish, was determined for the first time. The genome is 16,611 bp in length and consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and one control region. The gene organisation and its order were similar to other vertebrates. The overall base composition on heavy strand was as follows A: 32.5%, G: 15.2%, C: 27.7%, T: 24.47%, and the A+T content 56.9%. The control region contains a microsatellite, (TA)(12), a putative termination-associated sequence and three conserved sequence blocks. This mitogenome sequence data would play an important role in population genetics and phylogenetics of Indian major carps.

Excess copper induced oxidative stress and response of antioxidants in rice.

To investigate the effects of copper (Cu), rice plant (Oryza sativa. L. var. MSE-9) was treated with different Cu concentrations (0, 10, 50 and 100 µM) for 5 days in hydroponic condition. Gradual decrease in shoot and root growth was observed with the increase of Cu concentration and duration of treatment where maximum inhibition was recorded in root growth. Cu was readily absorbed by the plant though the maximum accumulation was found in root than shoot. Hydrogen peroxide (H(2)O(2)) production and lipid peroxidation were found increased with the elevated Cu concentration indicating excess Cu induced oxidative stress. Antioxidant enzymes superoxide dismutase (SOD), guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) and glutathione reductase (GR) were effectively generated at the elevated concentrations of Cu though catalase (CAT) did not show significant variation with respect to control. Ascorbate (ASH), glutathione (GSH) and proline contents were also increased in all the Cu treated plants compared with the control. SOD isoenzyme was greatly affected by higher concentration of Cu and it was consistent with the changes of the activity assayed in solution. The present study confirmed that excess Cu inhibits growth, induced oxidative stress by inducing ROS formation while the stimulated antioxidative system appears adaptive response of rice plant against Cu induced oxidative stress. Moreover proline accumulation in Cu stress plant seems to provide additional defense against the oxidative stress.

Can the skin smear examination in NLEP be reconsidered?

Slit and skin smear examination is an important laboratory test which was made optional in the NLEP. In a laboratory managed by LEPRA Society, this test was provided to voluntary reporting cases and those referred by the general health service institutions of the area. Total cases tested were 1180, out of which 152 (12.9%) were positive. Of the 152 smear reports, 121 were from Koraput district and constitute the sample for this study. Regarding the spread of site wise highest BI, 56% cases had BI of 3.1+ to 6+. Of the 121 cases, 112 (92.6%) cases completed 12 doses of MDT. Seventy patients, from whom detailed information was collected by personal contact, responded well to MDT resulting in regression of lesions. Out of them, 46 cases had repeat smear showing negative smear in 39% and decline of 81 in the rest of cases. There are 16 contact cases with 2 family clusters. A rough block-wise mapping showed 4 strata with a dense clustering in the urban area where the laboratory facility is available. The probable reasons for uneven distribution of cases and causes for late diagnosis were discussed. The study reflects the leprosy problem in urban pockets which high infection pool. Together these two factors remain unexposed due to winding up of smear labs and pose a potential threat to elimination. The paper also emphasized on the reasons for past failure of the test along with suggestions to revive this facility with minor modifications.

Additional virulence genes in conjunction with efficient selection scheme, and compatible culture regime enhance recovery of stable transgenic plants in cowpea via Agrobacterium tumefaciens-mediated transformation.

A critical step in the development of robust Agrobacterium tumefaciens-mediated transformation system in recalcitrant grain legume, cowpea is the establishment of optimal conditions for efficient T-DNA delivery into target tissue and recovery of transgenic plants. A dramatic increase in efficiency of T-DNA delivery was achieved by constitutive expression of additional vir genes in resident pSB1 vector in Agrobacterium strain LBA4404. A geneticin based selection system permitted rapid and efficient identification of transgenic shoots without interfering with their regeneration, and eliminated the bulk of escapes. Supplementation of 0.5 microM kinetin to medium containing 5.0 microM benzyl aminopurine after 1 week of culture followed by 3 weeks of culture were found critical for optimal multiplication and elongation of transformed shoots from cotyledonary node explants. Combining these three developments, we recovered fertile transgenic plants at a frequency of 1.64%, significantly higher than previous reports. The presence, integration, expression and inheritance of transgenes were confirmed by molecular analysis. The protocol developed for cultivar Pusa Komal will facilitate the transfer of desirable traits into cowpea.