TBX3 is essential for establishment of the posterior boundary of anterior genes and upregulation of posterior genes together with HAND2 during the onset of limb bud development.

During limb bud formation, axis polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This is a hallmark of the establishment of antero-posterior limb axis polarity, together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we show that TBX3 is required for establishment of the posterior expression boundary of anterior genes in mouse limb buds. ChIP-seq and differential gene expression analysis of wild-type and mutant limb buds identifies TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole-mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to upregulate shared posterior identity target genes in early limb buds.

A draft genome sequence of the pulse crop chickpea (Cicer arietinum L.).

Cicer arietinum L. (chickpea) is the third most important food legume crop. We have generated the draft sequence of a desi-type chickpea genome using next-generation sequencing platforms, bacterial artificial chromosome end sequences and a genetic map. The 520-Mb assembly covers 70% of the predicted 740-Mb genome length, and more than 80% of the gene space. Genome analysis predicts the presence of 27,571 genes and 210 Mb as repeat elements. The gene expression analysis performed using 274 million RNA-Seq reads identified several tissue-specific and stress-responsive genes. Although segmental duplicated blocks are observed, the chickpea genome does not exhibit any indication of recent whole-genome duplication. Nucleotide diversity analysis provides an assessment of a narrow genetic base within the chickpea cultivars. We have developed a resource for genetic markers by comparing the genome sequences of one wild and three cultivated chickpea genotypes. The draft genome sequence is expected to facilitate genetic enhancement and breeding to develop improved chickpea varieties.

Transcriptome sequencing of wild chickpea as a rich resource for marker development.

The transcriptome of cultivated chickpea (Cicer arietinum L.), an important crop legume, has recently been sequenced. Here, we report sequencing of the transcriptome of wild chickpea, C. reticulatum (PI489777), the progenitor of cultivated chickpea, by GS-FLX 454 technology. The optimized assembly of C. reticulatum transcriptome generated 37 265 transcripts in total with an average length of 946 bp. A total of 4072 simple sequence repeats (SSRs) could be identified in these transcript sequences, of which at least 561 SSRs were polymorphic between C. arietinum and C. reticulatum. In addition, a total of 36 446 single-nucleotide polymorphisms (SNPs) were identified after optimization of probability score, quality score, read depth and consensus base ratio. Several of these SSRs and SNPs could be associated with tissue-specific and transcription factor encoding transcripts. A high proportion (92-94%) of polymorphic SSRs and SNPs identified between the two chickpea species were validated successfully. Further, the estimation of synonymous substitution rates of orthologous transcript pairs suggested that the speciation event for divergence of C. arietinum and C. reticulatum may have happened approximately 0.53 million years ago. The results of our study provide a rich resource for exploiting genetic variations in chickpea for breeding programmes.

Gene discovery and tissue-specific transcriptome analysis in chickpea with massively parallel pyrosequencing and web resource development.

Chickpea (Cicer arietinum) is an important food legume crop but lags in the availability of genomic resources. In this study, we have generated about 2 million high-quality sequences of average length of 372 bp using pyrosequencing technology. The optimization of de novo assembly clearly indicated that hybrid assembly of long-read and short-read primary assemblies gave better results. The hybrid assembly generated a set of 34,760 transcripts with an average length of 1,020 bp representing about 4.8% (35.5 Mb) of the total chickpea genome. We identified more than 4,000 simple sequence repeats, which can be developed as functional molecular markers in chickpea. Putative function and Gene Ontology terms were assigned to at least 73.2% and 71.0% of chickpea transcripts, respectively. We have also identified several chickpea transcripts that showed tissue-specific expression and validated the results using real-time polymerase chain reaction analysis. Based on sequence comparison with other species within the plant kingdom, we identified two sets of lineage-specific genes, including those conserved in the Fabaceae family (legume specific) and those lacking significant similarity with any non chickpea species (chickpea specific). Finally, we have developed a Web resource, Chickpea Transcriptome Database, which provides public access to the data and results reported in this study. The strategy for optimization of de novo assembly presented here may further facilitate the transcriptome sequencing and characterization in other organisms. Most importantly, the data and results reported in this study will help to accelerate research in various areas of genomics and implementing breeding programs in chickpea.

Conserved and species-specific chromatin remodeling and regulatory dynamics during mouse and chicken limb bud development.

Chromatin remodeling and genomic alterations impact spatio-temporal regulation of gene expression, which is central to embryonic development. The analysis of mouse and chicken limb development provides important insights into the morphoregulatory mechanisms, however little is known about the regulatory differences underlying their morphological divergence. Here, we identify the underlying shared and species-specific epigenomic and genomic variations. In mouse forelimb buds, we observe striking synchrony between the temporal dynamics of chromatin accessibility and gene expression, while their divergence in chicken wing buds uncovers species-specific regulatory heterochrony. In silico mapping of transcription factor binding sites and computational footprinting establishes the developmental time-restricted transcription factor-DNA interactions. Finally, the construction of target gene networks for HAND2 and GLI3 transcriptional regulators reveals both conserved and species-specific interactions. Our analysis reveals the impact of genome evolution on the regulatory interactions orchestrating vertebrate limb bud morphogenesis and provides a molecular framework for comparative Evo-Devo studies.

Spatial regulation by multiple Gremlin1 enhancers provides digit development with cis-regulatory robustness and evolutionary plasticity.

Precise cis-regulatory control of gene expression is essential for normal embryogenesis and tissue development. The BMP antagonist Gremlin1 (Grem1) is a key node in the signalling system that coordinately controls limb bud development. Here, we use mouse reverse genetics to identify the enhancers in the Grem1 genomic landscape and the underlying cis-regulatory logics that orchestrate the spatio-temporal Grem1 expression dynamics during limb bud development. We establish that transcript levels are controlled in an additive manner while spatial regulation requires synergistic interactions among multiple enhancers. Disrupting these interactions shows that altered spatial regulation rather than reduced Grem1 transcript levels prefigures digit fusions and loss. Two of the enhancers are evolutionary ancient and highly conserved from basal fishes to mammals. Analysing these enhancers from different species reveal the substantial spatial plasticity in Grem1 regulation in tetrapods and basal fishes, which provides insights into the fin-to-limb transition and evolutionary diversification of pentadactyl limbs.

Environmental Enrichment Induces Epigenomic and Genome Organization Changes Relevant for Cognition.

In early development, the environment triggers mnemonic epigenomic programs resulting in memory and learning experiences to confer cognitive phenotypes into adulthood. To uncover how environmental stimulation impacts the epigenome and genome organization, we used the paradigm of environmental enrichment (EE) in young mice constantly receiving novel stimulation. We profiled epigenome and chromatin architecture in whole cortex and sorted neurons by deep-sequencing techniques. Specifically, we studied chromatin accessibility, gene and protein regulation, and 3D genome conformation, combined with predicted enhancer and chromatin interactions. We identified increased chromatin accessibility, transcription factor binding including CTCF-mediated insulation, differential occupancy of H3K36me3 and H3K79me2, and changes in transcriptional programs required for neuronal development. EE stimuli led to local genome re-organization by inducing increased contacts between chromosomes 7 and 17 (inter-chromosomal). Our findings support the notion that EE-induced learning and memory processes are directly associated with the epigenome and genome organization.

5-Methylcytosine and 5-Hydroxymethylcytosine Signatures Underlying Pediatric Cancers.

In addition to the genetic variations, recent evidence has shown that DNA methylation of both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) underlies the pathogenesis of pediatric cancer. Given the high mortality rate, there is an urgent need to study the mechanisms contributing to the pathogenicity of pediatric cancer. Over the past decades, next-generation sequencing (NGS) has enabled us to perform genome-wide screening to study the complex regulatory mechanisms of 5mC and 5hmC underlying pediatric tumorigenesis. To shed light on recent developments on pediatric cancer predisposition and tumor progression, here we discuss the role of both genome-wide and locus-specific dysregulation of 5mC and 5hmC in hematopoiesis malignancy and neuroblastoma, the most common types of pediatric cancer, together with their therapeutic potential.

Comparative analysis of kabuli chickpea transcriptome with desi and wild chickpea provides a rich resource for development of functional markers.

Chickpea (Cicer arietinum L.) is an important crop legume plant with high nutritional value. The transcriptomes of desi and wild chickpea have already been sequenced. In this study, we sequenced the transcriptome of kabuli chickpea, C. arietinum (genotype ICCV2), having higher commercial value, using GS-FLX Roche 454 and Illumina technologies. The assemblies of both Roche 454 and Illumina datasets were optimized using various assembly programs and parameters. The final optimized hybrid assembly generated 43,389 transcripts with an average length of 1065 bp and N50 length of 1653 bp representing 46.2 Mb of kabuli chickpea transcriptome. We identified a total of 5409 simple sequence repeats (SSRs) in these transcript sequences. Among these, at least 130 and 493 SSRs were polymorphic with desi (ICC4958) and wild (PI489777) chickpea, respectively. In addition, a total of 1986 and 37,954 single nucleotide polymorphisms (SNPs) were predicted in kabuli/desi and kabuli/wild genotypes, respectively. The SNP frequency was 0.043 SNP per kb for kabuli/desi and 0.821 SNP per kb for kabuli/wild, reflecting very low genetic diversity in chickpea. Further, SSRs and SNPs present in tissue-specific and transcription factor encoding transcripts have been identified. The experimental validation of a selected set of polymorphic SSRs and SNPs exhibited high intra-specific polymorphism potential between desi and kabuli chickpea, suggesting their utility in large-scale genotyping applications. The kabuli chickpea gene index assembled, and SSRs and SNPs identified in this study will serve as useful genomic resource for genetic improvement of chickpea.

Genome-wide survey and expression analysis suggest diverse roles of glutaredoxin gene family members during development and response to various stimuli in rice.

Glutaredoxins (GRXs) are glutathione-dependent oxidoreductase enzymes involved in a variety of cellular processes. In this study, our analysis revealed the presence of 48 genes encoding GRX proteins in the rice genome. GRX proteins could be classified into four classes, namely CC-, CGFS-, CPYC- and GRL-type, based on phylogenetic analysis. The classification was supported with organization of predicted conserved putative motifs in GRX proteins. We found that expansion of this gene family has occurred largely via whole genome duplication events in a species-specific manner. We explored rice oligonucleotide array data to gain insights into the function of GRX gene family members during various stages of development and in response to environmental stimuli. The comprehensive expression analysis suggested diverse roles of GRX genes during growth and development in rice. Some of the GRX genes were expressed in specific organs/developmental stages only. The expression of many of rice GRX genes was influenced by various phytohormones, abiotic and biotic stress conditions, suggesting an important role of GRX proteins in response to these stimuli. The identification of GRX genes showing differential expression in specific tissues or in response to environmental stimuli provide a new avenue for in-depth characterization of selected genes of importance.

Isolation, characterization and structural studies of amorpha - 4, 11-diene synthase (ADS(3963)) from Artemisia annua L.

With the escalating prevalence of malaria in recent years, artemisinin demand has placed considerable stress on its production worldwide. At present, the relative low-yield of artemisinin (0.01-1.1 %) in the source plant (Artemisia annua L. plant) has imposed a serious limitation in commercializing the drug. Amorpha-4, 11-diene synthase (ADS) has been reported a key enzyme in enhancing the artemisinin level in Artemisia annua L. An understanding of the structural and functional correlations of Amorpha-4, 11-diene synthase (ADS) may therefore, help in the molecular up-regulation of the enzyme. In this context, an in silico approach was used to study the ADS3963 (3963 bp) gene cloned by us, from high artemisinin (0.7-0.9% dry wt basis) yielding strain of A. annua L. The full-length putative gene of ADS3963 was found to encode a protein consisting of 533 amino acid residues with conserved aspartate rich domain. The isoelectric point (pI) and molecular weight of the protein were 5.25 and 62.2 kDa, respectively. The phylogenetic analysis of ADS genes from various species revealed evolutionary conservation. Homology modeling method was used for prediction of the 3D structure of ADS3963 protein and Autodock 4.0 version was used to study the ligand binding. The predicted 3D model and docking studies may further be used in characterizing the protein in wet laboratory.