Reprogramming in Candida albicans Gene Expression Network under Butanol Stress Abrogates Hyphal Development.

Candida albicans is the causative agent of invasive fungal infections. Its hyphae-forming ability is regarded as one of the important virulence factors. To unravel the impact of butanol on Candida albicans, it was placed in O+ve complete human serum with butanol (1% v/v). The Candida transcriptome under butanol stress was then identified by mRNA sequencing. Studies including electron microscopy demonstrated the inhibition of hyphae formation in Candida under the influence of butanol, without any significant alteration in growth rate. The numbers of genes upregulated in the butanol in comparison to the serum alone were 1061 (20 min), 804 (45 min), and 537 (120 min). Candida cells exhibited the downregulation of six hypha-specific transcription factors and the induction of four repressor/regulator genes. Many of the hypha-specific genes exhibited repression in the medium with butanol. The genes related to adhesion also exhibited repression, whereas, among the heat-shock genes, three showed inductions in the presence of butanol. The fungal-specific genes exhibited induction as well as repression in the butanol-treated Candida cells. Furthermore, ten upregulated genes formed the core stress gene set in the presence of butanol. In the gene ontology analysis, enrichment of the processes related to non-coding RNA, ribosome biosynthesis, and metabolism was observed in the induced gene set. On the other side, a few GO biological process terms, including biofilm formation and filamentous growth, were enriched in the repressed gene set. Taken together, under butanol stress, Candida albicans is unable to extend hyphae and shows growth by budding. Many of the genes with perturbed expression may have fitness or virulence attributes and may provide prospective sites of antifungal targets against C. albicans.

Optimized protocol for assay for transposase-accessible chromatin by sequencing (ATAC-seq) from Drosophila melanogaster brain tissue.

Transposase-accessible chromatin by sequencing (ATAC-seq) has emerged as an advantageous technique to assess chromatin accessibility owing to the robustness of "tagmentation" process and a relatively faster library preparation. A comprehensive ATAC-seq protocol from Drosophila brain tissue is currently unavailable. Here, we have provided a detailed protocol of ATAC-seq assay from Drosophila brain tissue. Starting from dissection and transposition to amplification of libraries has been elaborated. Furthermore, a robust ATAC-seq analysis pipeline has been presented. The protocol can be easily adapted for other soft tissues.

High-quality single amplicon sequencing method for illumina MiSeq platform using pool of 'N' (0-10) spacer-linked target specific primers without PhiX spike-in.

BACKGROUND: Illumina sequencing platform requires base diversity in the initial 11 cycles for efficient cluster identification and colour matrix estimation. This limitation yields low-quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but reduces the overall number of sequencing reads for data analysis. To overcome such low diversity issues during amplicon sequencing on illumina platforms, we developed a high throughput single amplicon sequencing method by introducing 'N' (0-10) spacers in target gene amplification primers that are pooled for simple handling. RESULT: We evaluated the efficiency of 'N' (0-10) spacer-linked primers by targeting bacterial 16S V3-V4 region, demonstrating heterogeneous base library construction. The addition of 'N' (0-10) spacers causes sequencing frameshift at every base that leads to base diversity and produces heterogeneous high quality reads within a single amplicon library. We have written a python based command-line software,"MetReTrim", to trim the 'N' (0-10) spacers from the raw reads ( https://github.com/Mohak91/MetReTrim ). We further demonstrated the accuracy of this method by comparative mock community analysis with standard illumina V3-V4 primer method. The ZymoBIOMICS™ microbial community DNA standard was used as a control for this study. We performed data analysisusing the DADA2 pipeline where taxonomy was assigned using SILVA database as reference. We observed no difference between the communities represented by our method and standard illumina V3-V4 primer method. CONCLUSION: This method eliminates the need for PhiX spike-in for single amplicon sequencing on illumina MiSeq platform. This allows for sequencing of more number of samples in a run and a reduction in the overall cost. Given that Illumina sequencing works on SBS chemistry irrespective of the platform (such as HiSeq, MiSeq, NextSeq, NovaSeq, etc.) we propose that this strategy of using 'N' (0-10) spacer-linked primer design can be adopted for generating high-quality single locus amplicon sequencing in a high throughput manner across the illumina platform subject to further validation.

Active enhancers strengthen insulation by RNA-mediated CTCF binding at chromatin domain boundaries.

Vertebrate genomes are partitioned into chromatin domains or topologically associating domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at domain boundaries correlates with better insulation; however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF and MYC TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer-promoter interactions, and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters, we show that active TAD enhancers, but not promoters, induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs.

Degradome comparison between wild and cultivated rice identifies differential targeting by miRNAs.

BACKGROUND: Small non-coding (s)RNAs are involved in the negative regulation of gene expression, playing critical roles in genome integrity, development and metabolic pathways. Targeting of RNAs by ribonucleoprotein complexes of sRNAs bound to Argonaute (AGO) proteins results in cleaved RNAs having precise and predictable 5` ends. While tools to study sliced bits of RNAs to confirm the efficiency of sRNA-mediated regulation are available, they are sub-optimal. In this study, we provide an improvised version of a tool with better efficiency to accurately validate sRNA targets. RESULTS: Here, we improvised the CleaveLand tool to identify additional micro (mi)RNA targets that belong to the same family and also other targets within a specified free energy cut-off. These additional targets were otherwise excluded during the default run. We employed these tools to understand the sRNA targeting efficiency in wild and cultivated rice, sequenced degradome from two rice lines, O. nivara and O. sativa indica Pusa Basmati-1 and analyzed variations in sRNA targeting. Our results indicate the existence of multiple miRNA-mediated targeting differences between domesticated and wild species. For example, Os5NG4 was targeted only in wild rice that might be responsible for the poor secondary wall formation when compared to cultivated rice. We also identified differential mRNA targets of secondary sRNAs that were generated after miRNA-mediated cleavage of primary targets. CONCLUSIONS: We identified many differentially targeted mRNAs between wild and domesticated rice lines. In addition to providing a step-wise guide to generate and analyze degradome datasets, we showed how domestication altered sRNA-mediated cascade silencing during the evolution of indica rice.

Immune profile and responses of a novel dengue DNA vaccine encoding an EDIII-NS1 consensus design based on Indo-African sequences.

The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens, and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1-4 and a dengue virus (DENV)-2 non-structural protein 1 (NS1) protein-coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on the whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. The vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying intracellular interferon (IFN)-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios, and immune gene profiling suggests a strong Th1-dominant immune response. Finally, the passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse-adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design by offering a novel vaccine candidate with a possible broad-spectrum protection and a successful clinical translation either as a stand alone or in a mix and match strategy.

CDX2 inducible microRNAs sustain colon cancer by targeting multiple DNA damage response pathway factors.

Meta-analysis of transcripts in colon adenocarcinoma patient tissues led to the identification of a DNA damage responsive miR signature called DNA damage sensitive miRs (DDSMs). DDSMs were experimentally validated in the cancerous colon tissues obtained from an independent cohort of colon cancer patients and in multiple cellular systems with high levels of endogenous DNA damage. All the tested DDSMs were transcriptionally upregulated by a common intestine-specific transcription factor, CDX2. Reciprocally, DDSMs were repressed via the recruitment of HDAC1/2-containing complexes onto the CDX2 promoter. These miRs downregulated multiple key targets in the DNA damage response (DDR) pathway, namely BRCA1, ATM, Chk1 (also known as CHEK1) and RNF8. CDX2 directly regulated the DDSMs, which led to increased tumor volume and metastasis in multiple preclinical models. In colon cancer patient tissues, the DDSMs negatively correlated with BRCA1 levels, were associated with decreased probability of survival and thereby could be used as a prognostic biomarker. This article has an associated First Person interview with the first author of the paper.

Antimicrobial Resistance Profiling and Phylogenetic Analysis of Neisseria gonorrhoeae Clinical Isolates From Kenya in a Resource-Limited Setting.

BACKGROUND: Africa has one of the highest incidences of gonorrhea. Neisseria gonorrhoeae is gaining resistance to most of the available antibiotics, compromising treatment across the world. Whole-genome sequencing (WGS) is an efficient way of predicting AMR determinants and their spread in the population. Recent advances in next-generation sequencing technologies like Oxford Nanopore Technology (ONT) have helped in the generation of longer reads of DNA in a shorter duration with lower cost. Increasing accuracy of base-calling algorithms, high throughput, error-correction strategies, and ease of using the mobile sequencer MinION in remote areas lead to its adoption for routine microbial genome sequencing. To investigate whether MinION-only sequencing is sufficient for WGS and downstream analysis in resource-limited settings, we sequenced the genomes of 14 suspected N. gonorrhoeae isolates from Nairobi, Kenya. METHODS: Using WGS, the isolates were confirmed to be cases of N. gonorrhoeae (n = 9), and there were three co-occurrences of N. gonorrhoeae with Moraxella osloensis and N. meningitidis (n = 2). N. meningitidis has been implicated in sexually transmitted infections in recent years. The near-complete N. gonorrhoeae genomes (n = 10) were analyzed further for mutations/factors causing AMR using an in-house database of mutations curated from the literature. RESULTS: We observe that ciprofloxacin resistance is associated with multiple mutations in both gyrA and parC. Mutations conferring tetracycline (rpsJ) and sulfonamide (folP) resistance and plasmids encoding beta-lactamase were seen in all the strains, and tet(M)-containing plasmids were identified in nine strains. Phylogenetic analysis clustered the 10 isolates into clades containing previously sequenced genomes from Kenya and countries across the world. Based on homology modeling of AMR targets, we see that the mutations in GyrA and ParC disrupt the hydrogen bonding with quinolone drugs and mutations in FolP may affect interaction with the antibiotic. CONCLUSION: Here, we demonstrate the utility of mobile DNA sequencing technology in producing a consensus genome for sequence typing and detection of genetic determinants of AMR. The workflow followed in the study, including AMR mutation dataset creation and the genome identification, assembly, and analysis, can be used for any clinical isolate. Further studies are required to determine the utility of real-time sequencing in outbreak investigations, diagnosis, and management of infections, especially in resource-limited settings.

Redefining genetic essentiality in Mycobacterium tuberculosis.

Sequencing transposon mutant libraries have been pivotal in annotating essential and non-essential genes in bacteria. This is particularly very helpful in the case of Mycobacterium tuberculosis with a large part of its genome without known function. It is not known whether there are any variations in the essentiality states as a function of optimal growth in the absence of any selection pressure. We here grow a high-density mutant library of M. tuberculosis through serial cultures and monitor the temporal fluctuations in insertion frequencies across all TA dinucleotides in the genome. Genes that cause morphological and physiological heterogeneity or enable metabolic bypass were found to gradually lose insertions, while genes comprising the toxin-antitoxin systems were found to get enriched with insertions during growth in nutrient replete conditions. High levels of fluctuations were observed in genes involved in cell wall and cell processes, intermediary metabolism, and genes involved in virulence, suggesting new modes of adaptation undertaken by the mutants. We also report the essentiality status of several newly annotated genetic features.

A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signaling.

Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signaling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signaling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. Here, we describe a set of CRISPR-based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.

miR828 and miR858 regulate VvMYB114 to promote anthocyanin and flavonol accumulation in grapes.

MicroRNAs are a class of non-coding small RNAs involved in the negative regulation of gene expression, which play critical roles in developmental and metabolic pathways. Studies in several plants have identified a few microRNAs and other small RNAs that target regulators of the phenylpropanoid metabolic pathway called the MYB transcription factors. However, it is not well understood how sRNA-mediated regulation of MYBs influences the accumulation of specific secondary metabolites. Using sRNA sequencing, degradome analysis, mRNA sequencing, and proteomic analysis, we establish that grape lines with high anthocyanin content express two MYB-targeting microRNAs abundantly, resulting in the differential expression of specific MYB proteins. miR828 and miR858 target coding sequences of specific helix motifs in the mRNA sequences of MYB proteins. Targeting by miR828 caused MYB RNA decay and the production of a cascade of secondary siRNAs that depend on RNA-dependent RNA polymerase 6. MYB suppression and cascade silencing was more robust in grape lines with high anthocyanin content than in a flavonol-rich grape line. We establish that microRNA-mediated silencing targeted the repressor class of MYBs to promote anthocyanin biosynthesis in grape lines with high anthocyanins. We propose that this process regulates the expression of appropriate MYBs in grape lines to produce specific secondary metabolites.

Isolation and molecular characterization of dengue virus clinical isolates from pediatric patients in New Delhi.

OBJECTIVE: To characterize the in vitro replication fitness, viral diversity, and phylogeny of dengue viruses (DENV) isolated from Indian patients. METHODS: DENV was isolated from whole blood collected from patients by passaging in cell culture. Passage 3 viruses were used for growth kinetics in C6/36 mosquito cells. Parallel efforts also focused on the isolation of DENV RNA from plasma samples of the same patients, which were processed for next-generation sequencing. RESULTS: It was possible to isolate 64 clinical isolates of DENV, mostly DENV-2. Twenty-five of these were further used for growth curve analysis in vitro, which showed a wide range of replication kinetics. The highest viral titers were associated with isolates from patients with dengue with warning signs and severe dengue cases. Full genome sequences of 21 DENV isolates were obtained. Genome analysis mapped the circulating DENV-2 strains to the Cosmopolitan genotype. CONCLUSIONS: The replication kinetics of isolates from patients with mild or severe infection did not differ significantly, but the viral titers varied by two orders of magnitude between the isolates, suggesting differences in replication fitness among the circulating DENV-2.

Single nucleotide polymorphism in sugar pathway and disease resistance genes in sugarcane.

KEY MESSAGE: Single nucleotide polymorphism in sugar pathway and disease resistance genes showing genetic association with sugar content and red rot resistance would be useful in marker-assisted genetic improvement of sugarcane. Validation and genotyping of potential sequence variants in candidate genes are necessary to understand their functional significance and trait association potential. We discovered, characterized, validated and genotyped SNPs and InDels in sugar pathway and disease resistance genes of Saccharum complex and sugarcane varieties using amplicon sequencing and CAPS assays. The SNPs were abundant in the non-coding 3'UTRs than 5'UTRs and coding sequences depicting a strong bias toward C to T transition substitutions than transversions. Sequencing of cloned amplicons validated 61.6 and 45.2 % SNPs detected in silico in 21 sugar pathway and 16 disease resistance genes, respectively. Sixteen SNPs in four sugar pathway genes and 10 SNPs in nine disease resistance genes were validated through cost-effective CAPS assay. Functional and adaptive significance of SNP and protein haplotypes identified in sugar pathway and disease resistance genes was assessed by correlating their allelic variation with missense amino acid substitutions in the functional domains, alteration in protein structure models and possible modulation of catalytic enzyme activity in contrasting high and low sugar and moderately red rot resistant and highly susceptible sugarcane genotypes. A strong genetic association of five SNPs in the sugar pathway and disease resistance genes, and an InDel marker in the promoter sequence of sucrose synthase-2 gene, with sugar content and red rot resistance, was evident. The functionally relevant SNPs and InDels, detected and validated in sugar pathway and disease resistance genes, and genic CAPS markers designed, would be of immense use in marker-assisted genetic improvement of sugarcane for sugar content and disease resistance.

Rifampicin reduces advanced glycation end products and activates DAF-16 to increase lifespan in Caenorhabditis elegans.

Advanced glycation end products (AGEs) are formed when glucose reacts nonenzymatically with proteins; these modifications are implicated in aging and pathogenesis of many age-related diseases including type II diabetes, atherosclerosis, and neurodegenerative disorders. Thus, pharmaceutical interventions that can reduce AGEs may delay age-onset diseases and extend lifespan. Using LC-MS(E), we show that rifampicin (RIF) reduces glycation of important cellular proteins in vivo and consequently increases lifespan in Caenorhabditis elegans by up to 60%. RIF analog rifamycin SV (RSV) possesses similar properties, while rifaximin (RMN) lacks antiglycation activity and therefore fails to affect lifespan positively. The efficacy of RIF and RSV as potent antiglycating agents may be attributed to the presence of a p-dihydroxyl moiety that can potentially undergo spontaneous oxidation to yield highly reactive p-quinone structures, a feature absent in RMN. We also show that supplementing rifampicin late in adulthood is sufficient to increase lifespan. For its effect on longevity, rifampicin requires DAF-18 (nematode PTEN) as well as JNK-1 and activates DAF-16, the FOXO homolog. Interestingly, the drug treatment modulates transcription of a different subset of DAF-16 target genes, those not controlled by the conserved Insulin-IGF-1-like signaling pathway. RIF failed to increase the lifespan of daf-16 null mutant despite reducing glycation, showing thereby that DAF-16 may not directly affect AGE formation. Together, our data suggest that the dual ability to reduce glycation in vivo and activate prolongevity processes through DAF-16 makes RIF and RSV effective lifespan-extending interventions.

PHA-4/FOXA-regulated microRNA feed forward loops during Caenorhabditis elegans dietary restriction.

Dietary restriction (DR) increases life span and delays the onset of age-related diseases across species. However, the molecular mechanisms have remained relatively unexplored in terms of gene regulation. InC. elegans, a popular model for aging studies, the FOXA transcription factor PHA-4 is a robust genetic regulator of DR, although little is known about how it regulates gene expression. We profiled the transcriptome and miRNAome of an eat-2 mutant, a genetic surrogate of DR, by Next Generation sequencing and find that most of the miRNAs are upregulated in the young-adult worms, none significantly downregulated. Interestingly, PHA-4 can potentially regulate the expression of most of these miRNA genes. Remarkably, many of the PHA-4-regulated genes that are induced during DR are also targets of the PHA-4-upregulated miRNAs, forming a large feed-forward gene regulatory network. The genes targeted by the feed-forward loops (FFLs) are enriched for functions related to ubiquitin-mediated decay, lysosomal autophagy, cellular signalling, protein folding etc., processes that play critical roles in DR and longevity. Together our data provides a framework for understanding the complex and unique regulatory network employed during DR, suggesting that PHA-4 employs such FFLs to fine-tune gene expression and instil robustness in the system during energy crisis.

The first draft of the pigeonpea genome sequence.

Pigeonpea (Cajanus cajan) is an important grain legume of the Indian subcontinent, South-East Asia and East Africa. More than eighty five percent of the world pigeonpea is produced and consumed in India where it is a key crop for food and nutritional security of the people. Here we present the first draft of the genome sequence of a popular pigeonpea variety 'Asha'. The genome was assembled using long sequence reads of 454 GS-FLX sequencing chemistry with mean read lengths of >550 bp and >10-fold genome coverage, resulting in 510,809,477 bp of high quality sequence. Total 47,004 protein coding genes and 12,511 transposable elements related genes were predicted. We identified 1,213 disease resistance/defense response genes and 152 abiotic stress tolerance genes in the pigeonpea genome that make it a hardy crop. In comparison to soybean, pigeonpea has relatively fewer number of genes for lipid biosynthesis and larger number of genes for cellulose synthesis. The sequence contigs were arranged in to 59,681 scaffolds, which were anchored to eleven chromosomes of pigeonpea with 347 genic-SNP markers of an intra-species reference genetic map. Eleven pigeonpea chromosomes showed low but significant synteny with the twenty chromosomes of soybean. The genome sequence was used to identify large number of hypervariable 'Arhar' simple sequence repeat (HASSR) markers, 437 of which were experimentally validated for PCR amplification and high rate of polymorphism among pigeonpea varieties. These markers will be useful for fingerprinting and diversity analysis of pigeonpea germplasm and molecular breeding applications. This is the first plant genome sequence completed entirely through a network of Indian institutions led by the Indian Council of Agricultural Research and provides a valuable resource for the pigeonpea variety improvement.

Functionally relevant microsatellites in sugarcane unigenes.

BACKGROUND: Unigene sequences constitute a rich source of functionally relevant microsatellites. The present study was undertaken to mine the microsatellites in the available unigene sequences of sugarcane for understanding their constitution in the expressed genic component of its complex polyploid/aneuploid genome, assessing their functional significance in silico, determining the extent of allelic diversity at the microsatellite loci and for evaluating their utility in large-scale genotyping applications in sugarcane. RESULTS: The average frequency of perfect microsatellite was 1/10.9 kb, while it was 1/44.3 kb for the long and hypervariable class I repeats. GC-rich trinucleotides coding for alanine and the GA-rich dinucleotides were the most abundant microsatellite classes. Out of 15,594 unigenes mined in the study, 767 contained microsatellite repeats and for 672 of these putative functions were determined in silico. The microsatellite repeats were found in the functional domains of proteins encoded by 364 unigenes. Its significance was assessed by establishing the structure-function relationship for the beta-amylase and protein kinase encoding unigenes having repeats in the catalytic domains. A total of 726 allelic variants (7.42 alleles per locus) with different repeat lengths were captured precisely for a set of 47 fluorescent dye labeled primers in 36 sugarcane genotypes and five cereal species using the automated fragment analysis system, which suggested the utility of designed primers for rapid, large-scale and high-throughput genotyping applications in sugarcane. Pair-wise similarity ranging from 0.33 to 0.84 with an average of 0.40 revealed a broad genetic base of the Indian varieties in respect of functionally relevant regions of the large and complex sugarcane genome. CONCLUSION: Microsatellite repeats were present in 4.92% of sugarcane unigenes, for most (87.6%) of which functions were determined in silico. High level of allelic diversity in repeats including those present in the functional domains of proteins encoded by the unigenes demonstrated their use in assay of useful variation in the genic component of complex polyploid sugarcane genome.

Combining QTL mapping and transcriptome profiling of bulked RILs for identification of functional polymorphism for salt tolerance genes in rice (Oryza sativa L.).

Identification of genes for quantitative traits is difficult using any single approach due to complex inheritance of the traits and limited resolving power of the individual techniques. Here a combination of genetic mapping and bulked transcriptome profiling was used to narrow down the number of differentially expressed salt-responsive genes in rice in order to identify functional polymorphism of genes underlying the quantitative trait loci (QTL). A population of recombinant inbred lines (RILs) derived from cross between salt-tolerant variety CSR 27 and salt-sensitive variety MI 48 was used to map QTL for salt ion concentrations in different tissues and salt stress susceptibility index (SSI) for spikelet fertility, grain weight, and grain yield. Eight significant QTL intervals were mapped on chromosomes 1, 8, and 12 for the salt ion concentrations and a QTL controlling SSI for spikelet fertility was co-located in one of these intervals on chromosome 8. However, there were total 2,681 genes in these QTL intervals, making it difficult to pinpoint the genes responsible for the functional differences for the traits. Similarly, transcriptome profiling of the seedlings of tolerant and sensitive parents grown under control and salt-stress conditions showed 798 and 2,407 differentially expressed gene probes, respectively. By analyzing pools of RNA extracted from ten each of extremely tolerant and extremely sensitive RILs to normalize the background noise, the number of differentially expressed genes under salt stress was drastically reduced to 30 only. Two of these genes, an integral transmembrane protein DUF6 and a cation chloride cotransporter, were not only co-located in the QTL intervals but also showed the expected distortion of allele frequencies in the extreme tolerant and sensitive RILs, and therefore are suitable for future validation studies and development of functional markers for salt tolerance in rice to facilitate marker-assisted breeding.

Informative genomic microsatellite markers for efficient genotyping applications in sugarcane.

Genomic microsatellite markers are capable of revealing high degree of polymorphism. Sugarcane (Saccharum sp.), having a complex polyploid genome requires more number of such informative markers for various applications in genetics and breeding. With the objective of generating a large set of microsatellite markers designated as Sugarcane Enriched Genomic MicroSatellite (SEGMS), 6,318 clones from genomic libraries of two hybrid sugarcane cultivars enriched with 18 different microsatellite repeat-motifs were sequenced to generate 4.16 Mb high-quality sequences. Microsatellites were identified in 1,261 of the 5,742 non-redundant clones that accounted for 22% enrichment of the libraries. Retro-transposon association was observed for 23.1% of the identified microsatellites. The utility of the microsatellite containing genomic sequences were demonstrated by higher primer designing potential (90%) and PCR amplification efficiency (87.4%). A total of 1,315 markers including 567 class I microsatellite markers were designed and placed in the public domain for unrestricted use. The level of polymorphism detected by these markers among sugarcane species, genera, and varieties was 88.6%, while cross-transferability rate was 93.2% within Saccharum complex and 25% to cereals. Cloning and sequencing of size variant amplicons revealed that the variation in the number of repeat-units was the main source of SEGMS fragment length polymorphism. High level of polymorphism and wide range of genetic diversity (0.16-0.82 with an average of 0.44) assayed with the SEGMS markers suggested their usefulness in various genotyping applications in sugarcane.

Single-copy genes define a conserved order between rice and wheat for understanding differences caused by duplication, deletion, and transposition of genes.

The high-quality rice genome sequence is serving as a reference for comparative genome analysis in crop plants, especially cereals. However, early comparisons with bread wheat showed complex patterns of conserved synteny (gene content) and colinearity (gene order). Here, we show the presence of ancient duplicated segments in the progenitor of wheat, which were first identified in the rice genome. We also show that single-copy (SC) rice genes, those representing unique matches with wheat expressed sequence tag (EST) unigene contigs in the whole rice genome, show more than twice the proportion of genes mapping to syntenic wheat chromosome as compared to the multicopy (MC) or duplicated rice genes. While 58.7% of the 1,244 mapped SC rice genes were located in single syntenic wheat chromosome groups, the remaining 41.3% were distributed randomly to the other six non-syntenic wheat groups. This could only be explained by a background dispersal of genes in the genome through transposition or other unknown mechanism. The breakdown of rice-wheat synteny due to such transpositions was much greater near the wheat centromeres. Furthermore, the SC rice genes revealed a conserved primordial gene order that gives clues to the origin of rice and wheat chromosomes from a common ancestor through polyploidy, aneuploidy, centromeric fusions, and translocations. Apart from the bin-mapped wheat EST contigs, we also compared 56,298 predicted rice genes with 39,813 wheat EST contigs assembled from 409,765 EST sequences and identified 7,241 SC rice gene homologs of wheat. Based on the conserved colinearity of 1,063 mapped SC rice genes across the bins of individual wheat chromosomes, we predicted the wheat bin location of 6,178 unmapped SC rice gene homologs and validated the location of 213 of these in the telomeric bins of 21 wheat chromosomes with 35.4% initial success. This opens up the possibility of directed mapping of a large number of conserved SC rice gene homologs in wheat. Overall, only 46.4% of these SC genes code for proteins with known functional domains; the remaining 53.6% have unknown function, and hence, represent an important, but yet, under explored category of genes.