Transfer of Stress Resilient QTLs and Panicle Traits into the Rice Variety, Reeta through Classical and Marker-Assisted Breeding Approaches.

Reeta is a popular late-maturing high-yielding rice variety recommended for cultivation in the eastern Indian states. The cultivar is highly sensitive to submergence stress. Phosphorus deficiency is an additional constraint for realizing high yield. The quantitative trait loci (QTLs), Sub1, for submergence and Pup1 for low phosphorus stress tolerance along with narrow-grained trait, GW5 were introgressed into the variety from the donor parent, Swarna-Sub1 through marker-assisted breeding. In addition, phenotypic selections for higher panicle weight, grain number, and spikelet fertility were performed in each segregating generation. Foreground selection detected the 3 target QTLs in 9, 8 and 7 progenies in the BC1F1, BC2F1, and BC3F1 generation, respectively. Recurrent parent's genome recovery was analyzed using 168 SSR polymorphic markers. The foreground analysis in 452 BC3F2 progenies showed five pyramided lines in homozygous condition for the target QTLs. No donor fragment drag was noticed in the Sub1 and GW5 QTLs carrier while a segmentwas observed in the Pup1 carrier chromosome. The developed lines were higher yielding, had submergence, and had low phosphorus stress-tolerance alongwith similar to the recipient parent in the studied morpho-quality traits. A promising pyramided line is released in the name of Reeta-Panidhan (CR Dhan 413) for the flood-prone areas of Odisha state.

Mapping the Genomic Regions Controlling Germination Rate and Early Seedling Growth Parameters in Rice.

Seed vigor is the key performance parameter of good quality seed. A panel was prepared by shortlisting genotypes from all the phenotypic groups representing seedling growth parameters from a total of 278 germplasm lines. A wide variation was observed for the traits in the population. The panel was classified into four genetic structure groups. Fixation indices indicated the existence of linkage disequilibrium in the population. A moderate to high level of diversity parameters was assessed using 143 SSR markers. Principal component, coordinate, neighbor-joining tree and cluster analyses showed subpopulations with a fair degree of correspondence with the growth parameters. Marker-trait association analysis detected eight novel QTLs, namely qAGR4.1, qAGR6.1, qAGR6.2 and qAGR8.1 for absolute growth rate (AGR); qRSG6.1, qRSG7.1 and qRSG8.1 for relative shoot growth (RSG); and qRGR11.1 for relative growth rate (RGR), as analyzed by GLM and MLM. The reported QTL for germination rate (GR), qGR4-1, was validated in this population. Additionally, QTLs present on chromosome 6 controlling RSG and AGR at 221 cM and RSG and AGR on chromosome 8 at 27 cM were detected as genetic hotspots for the parameters. The QTLs identified in the study will be useful for improvement of the seed vigor trait in rice.

Molecular Breeding for Incorporation of Submergence Tolerance and Durable Bacterial Blight Resistance into the Popular Rice Variety 'Ranidhan'.

Ranidhan is a popular late-maturing rice variety of Odisha state, India. The farmers of the state suffer heavy loss in years with flash floods as the variety is sensitive to submergence. Bacterial blight (BB) disease is a major yield-limiting factor, and the variety is susceptible to the disease. BB resistance genes Xa21, xa13, and xa5, along with the Sub1 QTL, for submergence stress tolerance were transferred into the variety using marker-assisted backcross breeding approach. Foreground selection using direct and closely linked markers detected the progenies carrying all four target genes in the BC1F1, BC2F1, and BC3F1 generations, and the positive progenies carrying these genes with maximum similarity to the recipient parent, Ranidhan, were backcrossed into each segregating generation. Foreground selection in the BC1F1 generation progenies detected all target genes in 11 progenies. The progeny carrying all target genes and similar to the recipient parent in terms of phenotype was backcrossed, and a total of 321 BC2F1 seeds were produced. Ten progenies carried all target genes/QTL in the BC2F1 generation. Screening of the BC3F1 progenies using markers detected 12 plants carrying the target genes. A total of 1270 BC3F2 seeds were obtained from the best BC3F1 progeny. Foreground selection in the BC3F2 progenies detected four plants carrying the target genes in the homozygous condition. The bioassay of the pyramided lines conferred very high levels of resistance to the predominant isolates of bacterial blight pathogen. These BB pyramided lines were submergence-tolerant and similar to Ranidhan in 13 agro-morphologic and grain quality traits; hence, they are likely to be adopted by farmers.

Ethnobotanical study of endemic and non-endemic medicinal plants used by indigenous people in environs of Gullele botanical garden Addis Ababa, central Ethiopia: A major focus on Asteraceae family.

Gullele Botanical Garden (GBG) in Addis Ababa, Ethiopia is a joint venture of Addis Ababa government and the university. The garden has been built mainly to conserve the endemic plants and to advance the research on the endemic and non-endemic plants collected from different part of Ethiopia. Many traditional healers from the environs of GBG and different subcities of Addis Ababa depend on the garden for their practice of traditional medicine but there is no systematic documentation of the traditional medicinal knowledge of these healers. The main objective of the present study is to comprehensively document the ethnobotanical and ethnomedicinal information from the traditional healers of different ethnic and cultural groups depending on GBG and to create a database of the endemic plants used by these healers. The ethnobotanical and ethnomedicinal data obtained from 60 traditional healers have been analyzed both qualitatively and quantitatively. A total of 81 medicinal plants belonging to 47 families have been identified. Majority of the plants used belonged to Asteraceae (12) family. The most frequently used plant form and plant parts are herbs and leaves. The major method adopted by the healers for preparation and administration of traditional medicine is crushing and topical, respectively. Skin and general diseases are the most important ailments treated by the healers. The three most cited plants used to treat diseases are Echinops kebericho Mesfin (60), Hagenia abyssinica (Bruce) J.F.Gmel (60) and Laggera tomentosa (A.Rich.) Sch.Bip. ex Oliv. & Hiern (58). The present study is the first systematic, qualitative, and quantitative ethnobotanical analysis and documentation done on the use of the medicinal plants from GBG for traditional medicine. In addition, our study reveals that E. kebericho is endemic and endangered plant and is highly used in traditional medicine. Therefore, GBG authorities should take steps for the propagation and restoration of this plant. Further it is suggested that the pharmacological properties of the roots and leaves of E. kebericho should be compared to find the possibility of use of leaves in place of roots for the preparation of traditional medicine which would help in conserving this endemic plant of Ethiopia.

Genomic organization and hypoxia inducible factor responsive regulation of teleost hsp90ß gene during hypoxia stress.

BACKGROUND: The physiological significance of a large family of heat-shock proteins (HSPs), comprised of the cytosolic HSP90A and the endoplasmic reticulum component of HSPB, is evident in prokaryotes and eukaryotes. The HSP90A is believed to play critical roles in diverse physiological functions of cell viability and chromosomal stability including stress management. Heightened abundance of hsp90ß transcript was documented in Channa striatus, a freshwater fish, which is capable of surviving within an extremely hypoxic environment. METHODS AND RESULTS: To better understand the mechanism of hsp90ß gene expression, we investigated its genomic organization. Eleven exons were identified, including a long upstream intron with a remarkable similarity with human, but not with chicken counterpart. Dual-luciferase assays identified promoter activity in a 1366 bp 5'-flanking segment beyond the transcription initiation site. Examination detected a minimal promoter of 754 bp containing a TATA-box, CAAT-enhancer in addition to providing clues regarding other enhancer and repressor elements. The driving capability of this minimal promoter was further validated by its binding ability with TATA-box binding protein and the generation of GFP expressing transgenic zebrafish (F2). Further, deletion of an inverted HIF (hypoxia inducible factor) motif RCGTG (upstream of the TATA-box) dramatically reduced luciferase expression in a hypoxic environment (CoCl2 treated cultivable cells) and was identified as a cis-acting HIF responsive element, necessary for the hypoxia-induced expression. CONCLUSIONS: The results obtained herein provide an insight regarding how hsp90ß gene expression is controlled by HIF responsive element in teleost both during hypoxia stress management and normal physiological functions, and suggested that the hsp90ß gene promoter could be used as a potential candidate for generating ornamental and food-fish transgenics.

Development of Submergence-Tolerant, Bacterial Blight-Resistant, and High-Yielding Near Isogenic Lines of Popular Variety, 'Swarna' Through Marker-Assisted Breeding Approach.

The rice variety 'Swarna' is highly popular in the eastern region of India. The farmers of eastern India cultivate mainly rainfed rice and face the adverse effects of climate change very frequently. Rice production in this region is not stable. Swarna variety is highly susceptible to bacterial blight (BB) disease and flash floods, which cause a heavy reduction in the yield. Transfer of five target genes/QTLs was targeted into the variety, Swarna by adopting marker-assisted backcross breeding approach. Direct markers for Sub1A, OsSPL14, and SCM2 QTLs and closely linked markers for xa5 and Xa21 BB resistance genes were screened in the backcross progenies. Swarna-Sub1, CR Dhan 800, and Swarna-Habataki near-isogenic lines (NILs) were used as donor parents in the breeding program. True multiple F1 plants were identified for backcrossing, and 796 BC1F1 seeds were generated. Foreground selection detected all the five target genes in six progenies in BC1F1 generation. The progeny containing all the target genes and more similar with the recipient parent was backcrossed, and a total of 446 BC2F1 seeds were produced. Foreground screening detected four BC2F1 plants carrying the five target genes. A total of 2,145 BC2F2 seeds were obtained from the best BC2F1 progeny. Screening of the progenies resulted in one plant with all five desirable genes, three plants with four, and another three progenies carrying three genes in homozygous conditions. The pyramided lines showed higher BB resistance and submergence tolerance than the recipient parent, Swarna. Culm strength of the pyramided lines showed higher breaking force than the recipient parent, Swarna. The pyramided line, SSBY-16-68-69 yielded the highest grain yield of 7.52 t/ha followed by the lines SSBY-16-68-511 (7.34 t/ha) and SSBY-16-68-1633 (7.02 t/ha). The best-pyramided line showed a yield advantage of 18% over the recipient parent and 6.8% over the yield component donor parent. Seven pyramided lines showed higher yield than the recipient parent, while five lines were better yielders than the yield component donor parent. The pyramided line SSBY-16-68-69 produced 365 grains/panicle, while the recipient had 152. The main morphologic and grain quality features of the recipient parent were retained in the pyramided lines.

Identification of QTLs for high grain yield and component traits in new plant types of rice.

A panel of 60 genotypes comprising New Plant Types (NPTs) along with indica, tropical and temperate japonica genotypes was phenotypically evaluated for four seasons in irrigated situation for grain yield per se and component traits. Twenty NPT genotypes were found promising with an average grain yield varying from 5.45 to 8.8 t/ha. A total of 85 SSR markers were used in the study to identify QTLs associated with grain yield per se and related traits. Sixty-six (77.65%) markers were found to be polymorphic. The PIC values varied from 0.516 to 0.92 with an average of 0.704. A moderate level of genetic diversity (0.39) was detected among genotypes. Variation to the tune of 8% within genotypes, 68% among the genotypes within the population and 24% among the populations were observed (AMOVA). This information may help in identification of potential parents for development of transgressive segregants with very high yield. The association analysis using GLM and MLM models led to the identification of 30 and 10 SSR markers associated with 70 and 16 QTLs, respectively. Thirty novel QTLs linked with 16 SSRs were identified to be associated with eleven traits, namely tiller number (qTL-6.1, qTL-11.1, qTL-4.1), panicle length (qPL-1.1, qPL-5.1, qPL-7.1, qPL-8.1), flag leaf length (qFLL-8.1, qFLL-9.1), flag leaf width (qFLW-6.2, qFLW-5.1, qFLW-8.1, qFLW-7.1), total no. of grains (qTG-2.2, qTG-a7.1), thousand-grain weight (qTGW-a1.1, qTGW-a9.2, qTGW-5.1, qTGW-8.1), fertile grains (qFG-7.1), seed length-breadth ratio (qSlb-3.1), plant height (qPHT-6.1, qPHT-9.1), days to 50% flowering (qFD-1.1) and grain yield per se (qYLD-5.1, qYLD-6.1a, qYLD-11.1).Some of the SSRs were co-localized with more than two traits. The highest co-localization was identified with RM5709 linked to nine traits, followed by RM297 with five traits. Similarly, RM5575, RM204, RM168, RM112, RM26499 and RM22899 were also recorded to be co-localized with more than one trait and could be rated as important for marker-assisted backcross breeding programs, for pyramiding of these QTLs for important yield traits, to produce new-generation rice for prospective increment in yield potentiality and breaking yield ceiling.

In Silico Analysis of nsSNPs of Carp TLR22 Gene Affecting its Binding Ability with Poly I:C.

Immune response mediated by toll-like receptor 22 (TLR22), only found in teleost/amphibians, is triggered by double-stranded RNA binding to its LRR (leucine-rich repeats) ecto-domain. Accumulated evidences suggested that missense mutations in TLR genes affect its function. However, information on mutation linked pathogen recognition for TLR22 was lacking. The present study was commenced for predicting the effect of non-synonymous single-nucleotide polymorphisms (nsSNPs) on the pathogen recognizable LRR domain of TLR22 of farmed carp, Labeo rohita. The sequence-based algorithms (SIFT, PROVEAN and I-Mutant2.0) indicated that three SNPs (out of 27) such as p.L159F (rs76759876) and p.L529P (rs749355507) of LRR, and p.I836M (rs750758397) of intracellular motifs could potentially disrupt protein function. The 3D structure was generated using MODELLER 9.13 and further validated by SAVEs server. The simulated molecular docking of native TLR22 and mutants with poly I:C ligand indicated that mutations positioned at p.L159F and p.L529P of the LRR region affects the binding affinity significantly. This is the first kind of study of predicting nsSNPs of teleost TLR22 with disturbed ligand binding affinity with its extra-cellular LRR domain and thereby likely hindrance in subsequent signal transduction. This study serves as a guide for in vivo evaluation of impact of mutation on immune response mediated by teleost TLR22 gene.

Identification of Deleterious Mutations in Myostatin Gene of Rohu Carp (Labeo rohita) Using Modeling and Molecular Dynamic Simulation Approaches.

The myostatin (MSTN) is a known negative growth regulator of skeletal muscle. The mutated myostatin showed a double-muscular phenotype having a positive significance for the farmed animals. Consequently, adequate information is not available in the teleosts, including farmed rohu carp, Labeo rohita. In the absence of experimental evidence, computational algorithms were utilized in predicting the impact of point mutation of rohu myostatin, especially its structural and functional relationships. The four mutations were generated at different positions (p.D76A, p.Q204P, p.C312Y, and p.D313A) of MSTN protein of rohu. The impacts of each mutant were analyzed using SIFT, I-Mutant 2.0, PANTHER, and PROVEAN, wherein two substitutions (p.D76A and p.Q204P) were predicted as deleterious. The comparative structural analysis of each mutant protein with the native was explored using 3D modeling as well as molecular-dynamic simulation techniques. The simulation showed altered dynamic behaviors concerning RMSD and RMSF, for either p.D76A or p.Q204P substitution, when compared with the native counterpart. Interestingly, incorporated two mutations imposed a significant negative impact on protein structure and stability. The present study provided the first-hand information in identifying possible amino acids, where mutations could be incorporated into MSTN gene of rohu carp including other carps for undertaking further in vivo studies.

Identification and characterization of differentially expressed transcripts in the gills of freshwater prawn (Macrobrachium rosenbergii) under salt stress.

The giant freshwater prawn, Macrobrachium rosenbergii, is an economically important species. It is a euryhaline shrimp, surviving in wide-range salinity conditions. A change in gene expression has been suggested as an important component for stress management. To better understand the osmoregulatory mechanisms mediated by the gill, a subtractive and suppressive hybridization (SSH) tool was used to identify expressed transcripts linked to adaptations in saline water. A total of 117 transcripts represented potentially expressed under salinity conditions. BLAST analysis identified 22% as known genes, 9% as uncharacterized showing homologous to unannotated ESTs, and 69% as unknown sequences. All the identified known genes representing broad spectrum of biological pathways were particularly linked to stress tolerance including salinity tolerance. Expression analysis of 10 known genes and 7 unknown/uncharacterized genes suggested their upregulation in the gills of prawn exposed to saline water as compared to control indicating that these are likely to be associated with salinity acclimation. Rapid amplification of cDNA ends (RACE) was used for obtaining full-length cDNA of MRSW-40 clone that was highly upregulated during salt exposure. The sequenced ESTs presented here will have potential implications for future understanding about salinity acclimation and/or tolerance of the prawn.

Cloning of cDNA and prediction of peptide structure of Plzf expressed in the spermatogonial cells of Labeo rohita.

The promyelocytic leukemia zinc finger (Plzf) gene containing an evolutionary conserved BTB (bric-a-brac/tramtrack/broad complex) domain plays a key role in self-renewal of mammalian spermatogonial stem cells (SSCs) via recruiting transcriptional co-repressors. Little is known about the function of Plzf in vertebrate, especially in fish species. To gain better understanding of its role in fishes, we have cloned Plzf from the testis of Labeo rohita (rohu), a commercially important freshwater carp. The full-length cDNA contains an open reading frame (ORF) of 2004bp translatable to 667 amino acids (aa) containing a conserved N-terminal BTB domain and C-terminal C(2)H(2)-zinc finger motifs. L. rohita Plzf, which is phylogenetically related to Danio rerio counterpart, abundantly expressed in spermatogonial stem cells (SSCs). A three-dimensional (3D) model of BTB domain of Plzf protein was constructed by homology modeling approach. Molecular docking on this 3D structure established a homo-dimer between two BTB domains creating a charged pocket containing conserved aa residues: L33, C34, D35 and R49. Thus, Plzf of SSC is structurally and possibly functionally conserved. The conserved aa residues in the cleft resulting from Plzf BTB self-association are likely to be the binding platform for interaction with recruited co-repressor peptides. The identified Plzf could be the first step towards exploring its role in rohu SSC behavior.