Transient gene expression assays in rose tissues using a Bio-Rad Helios® hand-held gene gun

Rose tissues of different varieties were transformed using a Bio-Rad Helios® hand-held biolistic gun. Parameters for optimum transient expression were optimized and included rose variety, flower age, tissue, gold particle size, DNA loading ratio. Smooth flowers without thick waxy layers and young unopened actively growing flowers were found to be better suited for the transient expression assays. The DNA amounts, gold particle amounts and size etc were not found to influence the efficiency of the transient transformation in these tissues. These studies indicate that biolistic transformation using hand-held guns can be used for successful transient expression assays in rose flower tissues. This is especially useful for a quick and easy analysis of genes and their expression before attempting stable transformation.http://www.elsevier.com/locate/saj

Expression of Aprotinin in anther causes male sterility in tobacco var Petit havana

Expression of many proteinases has been documented during anther development. Although their roles are not completely understood, their inhibition could possibly result in impairment of anther development leading to male sterility. We proposed that such an impairment of anther development can be engineered in plants resulting in male sterile plants that can be used for hybrid seed production. Here, we report that antherspecific expression of Aprotinin gene (serine proteinase inhibitor) in tobacco has resulted in male sterility. Southern analysis and zymogram analysis confirmed the integration and expression of Aprotinin gene in the anthers of the transgenic plants. Transverse sections of anthers of transgenic male sterile plants showed damaged tapetum. The pollen germination in the transgenic plants ranged between 2% and 65% that confirmed the impairment in pollen production leading to male sterility and low seed yield. Thus, inhibition of serine proteinases that are expressed during anther development has resulted in impaired pollen production and male sterility, though the exact role of these proteinases in anther development still has to be elucidated.Council for Scientific and Industrial Research.http://www.springerlink.com/content/0735-9640/nf201

Transient gene expression assays in rose tissues using a Bio-Rad Helios® hand-held gene gun

Rose tissues of different varieties were transformed using a Bio-Rad Helios® hand-held biolistic gun. Parameters for optimum transient expression were optimized and included rose variety, flower age, tissue, gold particle size, DNA loading ratio. Smooth flowers without thick waxy layers and young unopened actively growing flowers were found to be better suited for the transient expression assays. The DNA amounts, gold particle amounts and size etc were not found to influence the efficiency of the transient transformation in these tissues. These studies indicate that biolistic transformation using hand-held guns can be used for successful transient expression assays in rose flower tissues. This is especially useful for a quick and easy analysis of genes and their expression before attempting stable transformation.http://www.elsevier.com/locate/saj

Random amplification of genomic ends (RAGE) as an efficient method for isolation and cloning of promoters and uncloned genomic regions

Isolation of complete coding sequences and regulatory regions is critical for the complete characterization of a gene. Efficient methods to obtain complete genomic or regulatory is important in the process of isolation. The utility of the available genome walking methods are influenced by factors like the size of the genome and the length of the desired sequence. This study utilizes a genome walking method -random amplification of genomic ends (RAGE) efficiently to obtain the 5' -regulatory sequence of a rice stress inducible gene OsAsr1 and to obtain the full length sequence and promoter of the HetR gene of Cylindrospermum stagnale (Cylindrospermum sp. A1345). We demonstrate that this technique can be used for cloning of full length gene and promoters in organisms where whole genome data is unavailable utilising very little sequence information. Our studies show that RAGE can be a strong tool in functional genomics especially in the study of promoter

Whole Genome Sequencing and Comparative Genomic Analysis Reveal Allelic Variations Unique to a Purple Colored Rice Landrace (Oryza sativa ssp. indica cv. Purpleputtu)

Purpleputtu (Oryza sativa ssp. indica cv. Purpleputtu) is a unique rice landrace from southern India that exhibits predominantly purple color. This study reports the underlying genetic complexity of the trait, associated domestication and de-domestication processes during its coevolution with present day cultivars. Along-with genome level allelic variations in the entire gene repertoire associated with the purple, red coloration of grain and other plant parts. Comparative genomic analysis using ‘a panel of 108 rice lines’ revealed a total of 3,200,951 variants including 67,774 unique variations in Purpleputtu (PP) genome. Multiple sequence alignment uncovered a 14 bp deletion in Rc (Red colored, a transcription factor of bHLH class) locus of PP, a key regulatory gene of anthocyanin biosynthetic pathway. Interestingly, this deletion in Rc gene is a characteristic feature of the present-day white pericarped rice cultivars. Phylogenetic analysis of Rc locus revealed a distinct clade showing proximity to the progenitor species Oryza rufipogon and O. nivara. In addition, PP genome exhibits a well conserved 4.5 Mbp region on chromosome 5 that harbors several loci associated with domestication of rice. Further, PP showed 1,387 unique when SNPs compared to 3,023 lines of rice (SNP-Seek database). The results indicate that PP genome is rich in allelic diversity and can serve as an excellent resource for rice breeding for a variety of agronomically important traits such as disease resistance, enhanced nutritional values, stress tolerance, and protection from harmful UV-B rays

Draft genome sequence of Sclerospora graminicola, the pearl millet downy mildew pathogen:Genome sequence of pearl millet downy mildew pathogen

Sclerospora graminicola pathogen is one of the most important biotic production constraints of pearl millet worldwide. We report a de novo whole genome assembly and analysis of pathotype 1. The draft genome assembly contained 299,901,251 bp with 65,404 genes. Pearl millet [Pennisetum glaucum (L.) R. Br.], is an important crop of the semi-arid and arid regions of the world. It is capable of growing in harsh and marginal environments with highest degree of tolerance to drought and heat among cereals (1). Downy mildew is the most devastating disease of pearl millet caused by Sclerospora graminicola (sacc. Schroet), particularly on genetically uniform hybrids. Estimated annual grain yield loss due to downy mildew is approximately 10?80 % (2-7). Pathotype 1 has been reported to be the highly virulent pathotype of Sclerospora graminicola in India (8). We report a de novo whole genome assembly and analysis of Sclerospora graminicola pathotype 1 from India. A susceptible pearl millet genotype Tift 23D2B1P1-P5 was used for obtaining single-zoospore isolates from the original oosporic sample. The library for whole genome sequencing was prepared according to the instructions by NEB ultra DNA library kit for Illumina (New England Biolabs, USA). The libraries were normalised, pooled and sequenced on Illumina HiSeq 2500 (Illumina Inc., San Diego, CA, USA) platform at 2 x100 bp length. Mate pair (MP) libraries were prepared using the Nextera mate pair library preparation kit (Illumina Inc., USA). 1 ?g of Genomic DNA was subject to tagmentation and was followed by strand displacement. Size selection tagmented/strand displaced DNA was carried out using AmpureXP beads. The libraries were validated using an Agilent Bioanalyser using DNA HS chip. The libraries were normalised, pooled and sequenced on Illumina MiSeq (Illumina Inc., USA) platform at 2 x300 bp length. The whole genome sequencing was performed by sequencing of 7.38 Gb with 73,889,924 paired end reads from paired end library, and 1.15 Gb with 3,851,788 reads from mate pair library generated from Illumina HiSeq2500 and Illumina MiSeq, respectively. The sequences were assembled using various assemblers like ABySS, MaSuRCA, Velvet, SOAPdenovo2, and ALLPATHS-LG. The assembly generated by MaSuRCA (9) algorithm was observed superior over other algorithms and hence used for scaffolding using SSPACE. Assembled draft genome sequence of S. graminicola pathotype 1 was 299,901,251 bp long, with a 47.2 % GC content consisting of 26,786 scaffolds with N50 of 17,909 bp with longest scaffold size of 238,843 bp. The overall coverage was 40X. The draft genome sequence was used for gene prediction using AUGUSTUS. The completeness of the assembly was investigated using CEGMA and revealed 92.74% proteins completely present and 95.56% proteins partially present, while BUSCO fungal dataset indicated 64.9% complete, 12.4% fragmented, 22.7% missing out of 290 BUSCO groups. A total of 52,285 predicted genes were annotated using BLASTX and 38,120 genes were observed with significant BLASTX match. Repetitive element analysis in the assembly revealed 8,196 simple repeats, 1,058 low complexity repeats and 5,562 dinucleotide to hexanucleotide microsatellite repeats.publishersversionPeer reviewe

H yposidra talaca NPV (HytaNPV): a potential baculovirus for efficient control of the black inch worm, Hyposidra talaca Walker (Lepidoptera: Geometridae), a major pest of tea Camellia sinensis (Ericales: Theaceae (L.) O. Kuntze)

Abstract Background The black inch worm (BIW), Hyposidra talaca Walker (Lepidoptera: Geometridae), is a pest that defoliates tea leaves in India, posing a significant threat to the tea industry. Nucleopolyhedrovirus (NPV) is capable of infecting larvae of this species, which has raised the possibility of its use as a biocontrol agent. Results Rearing larvae in a semi-synthetic artificial diet produced healthy adults, which is sufficient for mass culture of H. talaca to support one of the IPM components using baculovirus. In artificial diets, the NPV was evaluated for its insecticidal activity against H. talaca. The bioassay findings of inoculated H. talaca nucleopolyhedrovirus virus (HytaNPV) at various concentrations showed that it was effective in killing the BIW. Purified polyhedral inclusion bodies (PIBs) were estimated to a concentration of 1 × 1010 PIBs per ml by mixing with water, and various concentrations of 0.25, 0.5, 1, 2, 5, 7.5, 10 ml/l were evaluated against BIW. Both laboratory and field studies revealed that HytaNPV is an eco-friendly and ecologically safe agent for controlling BIW. Besides no residue was estimated in made tea after the seventh day of exposure, and it is nontoxic to non-target species. Conclusion It was found that NPV is environmentally beneficial for the control of pests on tea plants and in production of pesticide-free tea. Tea ecosystems can reduce their reliance on conventional insecticides by using HytaNPV as an alternative bio-insecticide

Random amplification of genomic ends (RAGE) as an efficient method for isolation and cloning of promoters and uncloned genomic regions

Isolation of complete coding sequences and regulatory regions is critical for the complete characterization of a gene. Efficient methods to obtain complete genomic or regulatory is important in the process of isolation. The utility of the available genome walking methods are influenced by factors like the size of the genome and the length of the desired sequence. This study utilizes a genome walking method - random amplification of genomic ends (RAGE) efficiently to obtain the 5' – regulatory sequence of a rice stress inducible gene OsAsr1 and to obtain the full length sequence and promoter of the HetR gene of Cylindrospermum stagnale (Cylindrospermum sp. A1345). We demonstrate that this technique can be used for cloning of full length gene and promoters in organisms where whole genome data is unavailable utilising very little sequence information. Our studies show that RAGE can be a strong tool in functional genomics especially in the study of promoters

De Novo Sequencing and Hybrid Assembly of the Biofuel Crop <i>Jatropha curcas</i> L.: Identification of Quantitative Trait Loci for Geminivirus Resistance

Jatropha curcas is an important perennial, drought tolerant plant that has been identified as a potential biodiesel crop. We report here the hybrid de novo genome assembly of J. curcas generated using Illumina and PacBio sequencing technologies, and identification of quantitative loci for Jatropha Mosaic Virus (JMV) resistance. In this study, we generated scaffolds of 265.7 Mbp in length, which correspond to 84.8% of the gene space, using Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis. Additionally, 96.4% of predicted protein-coding genes were captured in RNA sequencing data, which reconfirms the accuracy of the assembled genome. The genome was utilized to identify 12,103 dinucleotide simple sequence repeat (SSR) markers, which were exploited in genetic diversity analysis to identify genetically distinct lines. A total of 207 polymorphic SSR markers were employed to construct a genetic linkage map for JMV resistance, using an interspecific F2 mapping population involving susceptible J. curcas and resistant Jatropha integerrima as parents. Quantitative trait locus (QTL) analysis led to the identification of three minor QTLs for JMV resistance, and the same has been validated in an alternate F2 mapping population. These validated QTLs were utilized in marker-assisted breeding for JMV resistance. Comparative genomics of oil-producing genes across selected oil producing species revealed 27 conserved genes and 2986 orthologous protein clusters in Jatropha. This reference genome assembly gives an insight into the understanding of the complex genetic structure of Jatropha, and serves as source for the development of agronomically improved virus-resistant and oil-producing lines