Indel Events Research Articles

Dynamic Evolution of \u03b1-Gliadin Prolamin Gene Family in Homeologous Genomes of Hexaploid Wheat

Wheat Gli-2 loci encode complex groups of α-gliadin prolamins that are important for breadmaking, but also major triggers of celiac disease (CD). Elucidation of α-gliadin evolution provides knowledge to produce wheat with better end-use properties and reduced immunogenic potential. The Gli-2 loci contain a large number of tandemly duplicated genes and highly repetitive DNA, making sequence assembly of their genomic regions challenging. Here, we constructed high-quality sequences spanning the three wheat homeologous α-gliadin loci by aligning PacBio-based sequence contigs with BioNano genome maps. A total of 47 α-gliadin genes were identified with only 26 encoding intact full-length protein products. Analyses of α-gliadin loci and phylogenetic tree reconstruction indicate significant duplications of α-gliadin genes in the last ~2.5 million years after the divergence of the A, B and D genomes, supporting its rapid lineage-independent expansion in different Triticeae genomes. We showed that dramatic divergence in expression of α-gliadin genes could not be attributed to sequence variations in the promoter regions. The study also provided insights into the evolution of CD epitopes and identified a single indel event in the hexaploid wheat D genome that likely resulted in the generation of the highly toxic 33-mer CD epitope.

An Unbiased Genome-Wide View of the Mutation Rate and Spectrum of the Endosymbiotic Bacterium Teredinibacter turnerae.

Mutations contribute to genetic variation in all living systems. Thus, precise estimates of mutation rates and spectra across a diversity of organisms are required for a full comprehension of evolution. Here, a mutation-accumulation (MA) assay was carried out on the endosymbiotic bacterium Teredinibacter turnerae. After ∼3,025 generations, base-pair substitutions (BPSs) and insertion–deletion (indel) events were characterized by whole-genome sequencing analysis of 47 independent MA lines, yielding a BPS rate of 1.14 × 10−9 per site per generation and indel rate of 1.55 × 10−10 events per site per generation, which are among the highest within free-living and facultative intracellular bacteria. As in other endosymbionts, a significant bias of BPSs toward A/T and an excess of deletion mutations over insertion mutations are observed for these MA lines. However, even with a deletion bias, the genome remains relatively large (∼5.2 Mb) for an endosymbiotic bacterium. The estimate of the effective population size (Ne) in T. turnerae is quite high and comparable to free-living bacteria (∼4.5 × 107), suggesting that the heavy bottlenecking associated with many endosymbiotic relationships is not prevalent during the life of this endosymbiont. The efficiency of selection scales with increasing Ne and such strong selection may have been operating against the deletion bias, preventing genome erosion. The observed mutation rate in this endosymbiont is of the same order of magnitude of those with similar Ne, consistent with the idea that population size is a primary determinant of mutation-rate evolution within endosymbionts, and that not all endosymbionts have low Ne.

Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses

Recent advances using CRISPR-Cas9 approaches have dramatically enhanced the ease for genetic manipulation in rodents. Notwithstanding, the methods to deliver nucleic acids into pre-implantation embryos have hardly changed since the original description of mouse transgenesis more than 30 years ago. Here we report a novel strategy to generate genetically modified mice by transduction of CRISPR-Cas9 components into pre-implantation mouse embryos via recombinant adeno-associated viruses (rAAVs). Using this approach, we efficiently generated a variety of targeted mutations in explanted embryos, including indel events produced by non-homologous end joining and tailored mutations using homology-directed repair. We also achieved gene modification in vivo by direct delivery of rAAV particles into the oviduct of pregnant females. Our approach greatly simplifies the generation of genetically modified mice and, more importantly, opens the door for streamlined gene editing in other mammalian species.

Caracterización de la variabilidad genética de cepas de campo de Brucella canis aisladas en Antioquia

Brucella canis is a facultative intracellular pathogen responsible for canine brucellosis, a zoonotic disease that affects canines, causing abortions and reproductive failure; and the production of non-specific symptoms in humans. In 2005 the presence of B. canis in Antioquia was demonstrated and the strains were identified as type 2. The sequencing of the genome of a field strain denoted Brucella canis str. Oliveri, showed species-specific indel events, which led us to investigate the genomic characteristics of the B. canis strain isolated and to establish the phylogenetic relationships and the divergence time of B. canis str. Oliveri. Conventional PCR sequencing was performed in 30 field strains identifying 5 indel events recognized in B. canis str. Oliveri. ADN from Brucella suis, Brucella melitensis and vaccine strains from Brucella abortus were used as control, and it was determined that all of the studied field strains shared 4 out of the 5 indels of the sequenced Oliveri strain, indicating the presence of more than one strain circulating in the region. Phylogenetic analysis was performed with 24 strains of Brucella using concatenated sequences of genetic markers for species differentiation. The molecular clock hypothesis and Tajima's relative rate test were tested, showing that the Oliveri strain, similarly to other canis species, diverged from B. suis. The molecular clock hypothesis between Brucella species was rejected and an evolution rate and a similar genetic distance between the B. canis were demonstrated.

Abstract LB-280: The landscape of somatic microsatellite indels across cancer: detection and identification of driver events

Abstract Microsatellites (MSs) are tracts of variable-length repeats of short DNA motifs that are abundant in the human genome and exhibit high rates of mutations in the form of insertions or deletions of the repeated motif (MS indels). Despite their prevalence, the contribution of somatic MS indels to cancer is largely unexplored due to difficulties in detecting them and assessing their significance. Here, we present a comprehensive analysis of MS indels across 20 tumor types. We characterize the overall MS indel landscape and detect genes with candidate driver MS indel events. We present two novel tools: MSMuTect for accurate detection of somatic MS indels and MSMutSig for identifying candidate cancer genes containing events at higher frequency than expected by chance. We observe high variability of the frequency of MS indels across tumors and demonstrate that the number and pattern of MS indels can accurately distinguish microsatellite stable (MSS) tumors from tumors with microsatellite instability (MSI). Applying MSMutSig across 6,788 tumors from 20 different tumor types identified 7 genes with significant MS indel hotspots: ACVR2A, RNF43, DOCK3, MSH3, ESRP1, PRDM2 and JAK1. In the four genes that have been previously implicated in cancer (ACVR2A, RNF43, JAK1 and MSH3), we identified previously unreported MS indels events. Three of the genes with significant loci - DOCK3, PRDM2 and ESRP1- had not been previously listed as cancer genes. MS indels in DOCK3, a negative regulator of the WNT pathway, were mutually exclusive with mutations in CTNNB1. MS indels in ESRP1, an RNA processing gene, correlated with alternative splicing of FGFR2, an event associated with the epithelial-to-mesenchymal transition. Overall, our comprehensive analysis of somatic MS indels across cancer highlights their importance, particularly in MSI tumors, significantly contributes to the ongoing global efforts to detect cancer genes, and may improve classification of patients into clinically-relevant subgroups. Citation Format: Yosef E. Maruvka, Kent W. Mouw, Rosa Karlic, Rasanna Parasuraman, Atanas Kamburov, Paz Polak, Nicholas J. Haradhvala, Julian M. Hess, Esther Rheinbay, Yehuda Brody, Lior Z. Braunstein, Alan D’Andrea, Michael S. Lawrence, Adam Bass, Andre Bernards, Franziska Michor, Gad Getz. The landscape of somatic microsatellite indels across cancer: detection and identification of driver events [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-280. doi:10.1158/1538-7445.AM2017-LB-280

Structural variations generated by simian foamy virus-like (SFV) in Crocodylus siamensis

Endogenous retrovirus (ERV) integrates into the germline of its host and could remain in the genome as a molecular fossil. ERV is one of sources that cause INDEL and recombination events in the vertebrate genomes, leading to various genomic and genetic changes in their hosts. There have been many studies conducted on ERVs in the vertebrate genomes to elucidate their evolutionary history. However, ERVs have not been studied well in Crocodylus siamensis. Here, we report structural variations among SFV1 elements (simian foamy virus-like), ERVs in C. siamensis. We initially identified 26 SFV1 candidates in the genome and experimentally verified 9 SFV1_1 and 5 SFV1_10 elements using PCR display. Their structural analyses showed that most of them are solitary-LTRs but two SFV1_1 elements are full-length. Through further analyses, we found that the two full-length elements retain intact ORFs. We examined transcription factor binding sites within their LTR sequences to predict promoter/enhancer activities. In sum, we identified 14 crocodile-specific SFV1 elements and the results of their structural analyses suggest that they could contribute to genomic or phenotypic variations in C. siamensis population.

Validation of an expanded neoantigen identification platform for therapeutic and diagnostic use in immuno-oncology.

11589 Background: Neoantigen identification is increasingly critical for clinical immuno-oncology applications including predicting immunotherapy response and neoantigen-based personalized cancer vaccines. Although standard research pipelines have been developed to aid neoantigen identification, building a robust, validated neoantigen identification platform suitable for clinical applications has been challenging due to the complex processes involved. Methods: To improve neoantigen identification, we extended standard sequencing and informatics methods. We developed an augmented and content enhanced (ACE) exome sequenced at 200X to increase sensitivity to SNPs and indels used for neoantigen identification as well as HLA performance. To accurately identify fusions and variants from RNA, we optimized our ACE transcriptome for FFPE tissue. To improve neoantigen pipelines based on MHC binding algorithms, we developed peptide phasing, high accuracy HLA typing, TCR interaction predictors, and transcript isoform estimation tools to detect neoantigens from indel and fusion events. We performed comprehensive analytical validation of the platform including the ACE Exome, somatic SNV/indel calls, RNA based variant and fusion calls, and HLA typing. This was followed by an overall in silico validation of neoantigen identification using 23 experimentally validated immunogenic neoepitopes spiked into exome data. Results: Analytical validation of our ACE exome platform showed > 97% sensitivity for small variants with a specificity of > 98% at minor allele frequency > 10%. From the ACE transcriptome we achieved a fusion sensitivity of > 99% and RNA based variant calls sensitivity of > 97%. Our ACE exome based HLA typing was 98% and 95% concordant with Class I and II HLA results (respectively) from clinical testing. Our in silico validation of neoantigen predictions resulted in identification of 22 out of 23 immunogenic neoepitopes. Conclusions: We developed sequencing and informatics improvements to standard approaches that can enhance neoantigen identification and demonstrated a comprehensive validation approach that may support neoantigen use in future clinical settings.

Computational prediction of the tolerance to amino-acid deletion in green-fluorescent protein.

Proteins evolve through two primary mechanisms: substitution, where mutations alter a protein’s amino-acid sequence, and insertions and deletions (indels), where amino acids are either added to or removed from the sequence. Protein structure has been shown to influence the rate at which substitutions accumulate across sites in proteins, but whether structure similarly constrains the occurrence of indels has not been rigorously studied. Here, we investigate the extent to which structural properties known to covary with protein evolutionary rates might also predict protein tolerance to indels. Specifically, we analyze a publicly available dataset of single—amino-acid deletion mutations in enhanced green fluorescent protein (eGFP) to assess how well the functional effect of deletions can be predicted from protein structure. We find that weighted contact number (WCN), which measures how densely packed a residue is within the protein’s three-dimensional structure, provides the best single predictor for whether eGFP will tolerate a given deletion. We additionally find that using protein design to explicitly model deletions results in improved predictions of functional status when combined with other structural predictors. Our work suggests that structure plays fundamental role in constraining deletions at sites in proteins, and further that similar biophysical constraints influence both substitutions and deletions. This study therefore provides a solid foundation for future work to examine how protein structure influences tolerance of more complex indel events, such as insertions or large deletions.

BayesCAT: Bayesian co-estimation of alignment and tree.

Traditionally, phylogeny and sequence alignment are estimated separately: first estimate a multiple sequence alignment and then infer a phylogeny based on the sequence alignment estimated in the previous step. However, uncertainty in the alignment is ignored, resulting, possibly, in overstated certainty in phylogeny estimates. We develop a joint model for co-estimating phylogeny and sequence alignment which improves estimates from the traditional approach by accounting for uncertainty in the alignment in phylogenetic inferences. Our insertion and deletion (indel) model allows arbitrary-length overlapping indel events and a general distribution for indel fragment size. We employ a Bayesian approach using MCMC to estimate the joint posterior distribution of a phylogenetic tree and a multiple sequence alignment. Our approach has a tree and a complete history of indel events mapped onto the tree as the state space of the Markov Chain while alternative previous approaches have a tree and an alignment. A large state space containing a complete history of indel events makes our MCMC approach more challenging, but it enables us to infer more information about the indel process. The performances of this joint method and traditional sequential methods are compared using simulated data as well as real data. Software named BayesCAT (Bayesian Co-estimation of Alignment and Tree) is available at https://github.com/heejungshim/BayesCAT.

Efficient Screening of CRISPR/Cas9-Induced Events in Drosophila Using a Co-CRISPR Strategy.

Genome editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated nuclease (Cas9) enables specific genetic modifications, including deletions, insertions, and substitutions in numerous organisms, such as the fruit fly Drosophila melanogaster. One challenge of the CRISPR/Cas9 system can be the laborious and time-consuming screening required to find CRISPR-induced modifications due to a lack of an obvious phenotype and low frequency after editing. Here we apply the successful co-CRISPR technique in Drosophila to simultaneously target a gene of interest and a marker gene, ebony, which is a recessive gene that produces dark body color and has the further advantage of not being a commonly used transgenic marker. We found that Drosophila broods containing higher numbers of CRISPR-induced ebony mutations (“jackpot” lines) are significantly enriched for indel events in a separate gene of interest, while broods with few or no ebony offspring showed few mutations in the gene of interest. Using two different PAM sites in our gene of interest, we report that ∼61% (52–70%) of flies from the ebony-enriched broods had an indel in DNA near either PAM site. Furthermore, this marker mutation system may be useful in detecting the less frequent homology-directed repair events, all of which occurred in the ebony-enriched broods. By focusing on the broods with a significant number of ebony flies, successful identification of CRISPR-induced events is much faster and more efficient. The co-CRISPR technique we present significantly improves the screening efficiency in identification of genome-editing events in Drosophila.

Whole Transcriptome Sequencing (RNAseq) As a Comprehensive, Cost-Efficient Diagnostic Tool for Acute Myeloid Leukemia

Abstract Background Acute myeloid leukemia (AML) is caused by cooperating oncogenic driver mutations that induce uncontrolled proliferation in combination with maturation arrest in myeloid precursor cells. The majority of oncogenic drivers are somatically acquired. Recurrent genetic lesions are used for molecular classification and prognosis of AML. In addition, expression levels of selected genes contribute to AML subclassification (CD34, KIT) and prognostication (e.g. EVI1). Actionable aberrations and pathways are molecular targets for personalized pharmacotherapy. Structures, incidences, and mutual associations of recurrent genetic aberrations have been elucidated by large whole genome/exome sequencing efforts. However, 10-20% of AML appear to be caused by hitherto unrecognized or individual driver lesions. Current AML diagnostics are poorly standardized and comprise a combination of morphology, flow cytometry, cytogenetics, targeted gene amplification with electrophoresis, sequence analysis, and qRT-PCR. We here test the hypothesis that whole transcriptome sequencing (RNAseq) without parallel germ-line sequencing could potentially be used as a single and cost-efficient platform for AML diagnosis and prognostication. Methods Poly(A)+ RNA was isolated from 100 cryopreserved AML samples with a blast count of 10-99% (median: 75%). These samples were obtained from 97 patients (2 diagnosis-relapse pairs, 1 de novo AML with subsequent tAML). RNA was sequenced at a depth of 59x106 paired-end reads per sample with median read length of 126 bp through a ISO17025-accredited Illumina HiSeq 2500 pipeline. After alignment against the GRCh38 reference genome, single nucleotide variants (SNV) and small indels were called by VarScan with a threshold of 20% aberrant reads. Variants were filtered for known polymorphisms occurring at >5% in defined ethnic subpopulations of the 1000 Genomes and Genome of the Netherlands Projects. Internal tandem duplications (ITD) were identified by frequency and distribution of soft clipped reads. Detection of fusion genes was performed on raw reads using STAR-Fusion and FusionCatcher. Geneexpression levels were measured relative to HBMS as a housekeeping gene by counting the number of reads aligned to gene exons normalized to the sum of exon lengths. HAMLET was developed and implemented as an integrated RNAseq pipeline to measure relevant recurrent AML-associated aberrations, i.e. SNV and small indels in recurrently mutated genes, the FLT3-ITD, AML-associated gene fusions, and EVI1 overexpression. Results A total of 221 SNV/indels (15 homozygous) were detected at expected or higher frequencies in NPM1 (33%), FLT3 (ITD+TKD: 37%), DNMT3A (30%), TET2 (27%), IDH1 (14%), IDH2 (14%), RUNX1 (23%), CEBPA (4%), KIT (7%), WT1 (8%), ASXL1 (11%), and TP53 (3%). Sensitivities to detect SNV/small indels and FLT3-ITD were 96% and 94%, respectively, with no apparent relation to the blast count. Fusion genes relevant to the WHO AML classification were found with a sensitivity of 100% when compared to metaphase cytogenetics and FISH. Specifically, 10 cases of CBFB-MYH11, 3 RUNX1-RUNX1T1, 2 PML-RARA, 1 FUS-ERG, 1 KMT2A-MLLT3, and 1 DEK-NUP214 fusion events were correctly detected. In addition, 3 variant KMT2A translocations with MLLT1, MLLT4, and MLLT6 as fusion partners were correctly called. The specificity of HAMLET for recurrent SNV, indels, and fusion genes was 100%. Finally, RNAseq quantitatively detected variability in gene expression as exemplified for EVI1 overexpression and confirmed by qRT-PCR (r=0.85). Conclusions In conclusion, RNAseq analysis of AML samples without concomitant germ-line sequencing detects molecular information with relevance for classification, prognosis, and targeted therapy with 96% overall sensitivity and 100% specificity. HAMLET is currently optimized to further improve the sensitivity to detect SNV/small indel and ITD events. With a cost price of €980,- per case and full accreditation for diagnostic application of the RNAseq raw data, RNAseq facilitates comprehensive and cost-effective AML diagnostics in a single assay. Additional predicted benefits of this RNAseq approach to be explored include identification of non-recurrent individual drivers, neoantigens, and minor histocompatibility antigens, a potential to replace conventional HLA typing, and as a potential alternative for flow cytometry. Disclosures Janssen: GenomeScan BV: Employment.

Comparative genomics of Tunisian Leishmania major isolates causing human cutaneous leishmaniasis with contrasting clinical severity

Zoonotic cutaneous leishmaniasis caused by Leishmania (L.) major parasites affects urban and suburban areas in the center and south of Tunisia where the disease is endemo-epidemic. Several cases were reported in human patients for which infection due to L. major induced lesions with a broad range of severity. However, very little is known about the mechanisms underlying this diversity. Our hypothesis is that parasite genomic variability could, in addition to the host immunological background, contribute to the intra-species clinical variability observed in patients and explain the lesion size differences observed in the experimental model. Based on several epidemiological, in vivo and in vitro experiments, we focused on two clinical isolates showing contrasted severity in patients and BALB/c experimental mice model. We used DNA-seq as a high-throughput technology to facilitate the identification of genetic variants with discriminating potential between both isolates. Our results demonstrate that various levels of heterogeneity could be found between both L. major isolates in terms of chromosome or gene copy number variation (CNV), and that the intra-species divergence could surprisingly be related to single nucleotide polymorphisms (SNPs) and Insertion/Deletion (InDels) events. Interestingly, we particularly focused here on genes affected by both types of variants and correlated them with the observed gene CNV. Whether these differences are sufficient to explain the severity in patients is obviously still open to debate, but we do believe that additional layers of -omic information is needed to complement the genomic screen in order to draw a more complete map of severity determinants.

Carrot plastid terminal oxidase gene (DcPTOX) responds early to chilling and harbors intronic pre-miRNAs related to plant disease defense

The nuclear-encoded plastid terminal oxidase gene (PTOX) is present in photosynthetic species and functions in the oxidation of the plastoquinone pool. It plays an important role on chlororespiration, chromorespiration and carotenoid biosynthesis. Here, we show short-term early response of carrot PTOX (DcPTOX) in leaves of two carrot inbred lines upon chilling. Analysis of the complete gene confirmed DcPTOX as a single gene and revealed an exceptionally large genomic sequence (9422bp) in comparison to other species, comprising nine exons interrupted by eight introns. In silico analysis based on data from whole genome sequencing projects discovered that some plant species present two PTOX genes. A search for sequence variability at genomic level was performed in the heterogeneous, ancient carrot cultivar ‘Rotin’. DcPTOX revealed intron length polymorphisms (ILPs) in intron 2, due to the occurrence of two insertion/deletions (InDels) events. Prediction of pre-miRNA sequences in intronic regions of DcPTOX showed two putative locations coding for putative miRNAs, one located at intron 2 and other located at intron 6. Both putative miRNAs revealed high homology with miRNAs described in Glycine max (gma-miR1520p) and Oryza sativa (osa-miR5494), respectively. Plant disease resistance genes were identified as miRNAs target genes.

135. Optimization of Dual-gRNA Lentiviral Vectors for Targeted Genomic Deletions

CRISPR-Cas9 technology is a powerful tool for genome editing based therapeutic approaches. Despite its potential, the delivery of Cas9 components into primary cells, such as hematopoietic stem progenitor cells (HSPC), is still a major challenge. In addition, several applications - e.g. the use of Cas9-nickase and the generation of genomic deletions/inversions - require the delivery and expression of two different gRNA. Lentiviral vectors (LV) can efficiently transduce HSPC, but the presence and the orientation of direct repeated elements in the gRNA expression cassettes can potentially trigger recombination events that affect vector stability. To address this issue, we designed different LV encoding for two gRNA pairs, which generate deletions of different size (3 and 13 kb) in the beta-globin gene cluster. gRNA expression was driven by murine and human U6 promoters, having little sequence similarity, to avoid potential recombination events of the LV genome. To further reduce LV rearrangements and optimize expression levels, gRNA cassettes were positioned in different orientations with respect to each other (LV Inward, Tandem and Outward). We compared LV from different vector batches in HCT116 cells and we observed that Tandem configuration resulted in higher viral titers and infectivity, although this difference was not statistically significant. Analysis of proviral integrity on genomic DNA of transduced cells showed the intact dual-gRNA cassette and no sign of recombination for all the LV configurations. To understand if different orientations could affect gRNA expression (measured by RT-qPCR) and consequently deletion/inversion efficiency, we co-transduced erythroleukemic K562 cells with LV-Cas9-Blast and the three different LV. Surprisingly, LV Inward showed poor gRNA expression, negligible deletion and inversion frequency and low efficiency of InDel formation at each gRNA target site (2.4 % and 6.8%). Conversely, LV Tandem and Outward configurations allowed efficient cell transduction and significant and comparable levels of gRNA expression. As a results, we observed a good deletion frequency (11.0% ±1.8 of total alleles for LV Tandem and 12.3 % ± 2.5 for LV Outward), a lower proportion of inversions events (5.4% ±1.3 for LV Tandem and 3.8 % ± 0.4 for LV Outward) and up to 58% of InDels events at each gRNA target sites. Comparable results were also obtained in adult HSPC-derived erythroid cell line (HUDEP-2). Overall, our study indicates LV Tandem and LV Outward as promising tools for genome editing of primary cells; both vectors are now being evaluated in primary HSPC.

Molecular systematics of southern African monkey orange Strychnos L. (Loganiaceae)

Strychnos is the largest genus of the Loganiaceae with about 200 species distributed across Africa (including Madagascar), the Americas, Australia and Asia. Recent molecular phylogenetic effort at elucidating relationships globally provided a useful overview for the genus, based on the internal transcribed spacer (ITS) data. However, an understanding of evolutionary and ecological patterns at regional levels is better served by fine scale phylogenetic analysis to resolve species complexes for conservation and allied reasons. In this study, we use plastid (trnL-trnF, trnS-trnG) and nuclear ribosomal ITS sequence data to infer phylogenetic patterns among members of southern Africa Strychnos. We also evaluate sectional validity of the current classification for African members with ITS sequence data. Our findings support the monophyly of Strychnos, although several of the sections are not monophyletic, thus raising the need for sectional reappraisal of the genus. S. xantha is sister to our expanded representation of southern African taxa, while S. aculeata is sister to all African taxa. The uncertain relationships among S. innocua, S. madagascariensis and S. gerrardii were partly resolved in the phylogenetic analysis of combined datasets. S. innocua is sister to the other two species in a well-supported clade. S. gerrardii and S. madagascariensis are also sister taxa that are not yet reciprocally monophyletic, but possess other features to distinguish them. The trnS-trnG marker revealed a relatively large indel event of potential taxonomic value in section Densiflorae. There is also a clear ecological signal in both the plastid and nuclear datasets, as both consistently placed forest species at the base of the phylogeny, and savanna species in more derived positions. We addressed the adaptive significance of such signals as they relate to fruit and growth forms, and submit proposals towards a comprehensive sectional revision of African Strychnos.

Mining of Blast Resistance Gene, Pi2 and Its Novel Allelic Variant from Landraces of Rice from Karnataka

Rice blast is one of the important fungal diseases of rice, caused by Magnaporthe oryzae. Rice blast can be effectively managed by the deployment of R-genes. Pi2 (Piz-5) is one of the major blast resistant genes effective against pathogen populations in various parts of India. In the present study, Pi2 gene was mined from 83 native landraces of rice from Karnataka state, India which revealed the presence of Pi2 in 60 landraces with the frequency of 72.28 %. Further, by using sequencing based allele mining approach, allelic variants of Pi2 from 16 landraces were analysed. High nucleotide variations (InDels) in the middle region of the conserved domain sequence of Pi2 in all variants were obtained. 469 polymorphic sites were observed with a total 570 mutations and 191 InDels among 16 Pi2 variants. Among Pi2 resistant and susceptible variants, 206 mutations with 29 InDel events were observed. Comparative modeling and Ramachandran plot of Pi2 protein variants from resistant and susceptible landraces were studied which revealed the significant differences among them. One potential novel Pi2 allelic variant from landrace Vanasurya was identified for the first time. This novel effective allelic variant can be used in rice breeding programmes to enhance blast resistance.

Genome Sequence Analysis of New Isolates of the Winona Strain of Plum pox virus and the First Definitive Evidence of Intrastrain Recombination Events.

Plum pox virus (PPV) is genetically diverse with nine different strains identified. Mutations, indel events, and interstrain recombination events are known to contribute to the genetic diversity of PPV. This is the first report of intrastrain recombination events that contribute to PPV's genetic diversity. Fourteen isolates of the PPV strain Winona (W) were analyzed including nine new strain W isolates sequenced completely in this study. Isolates of other strains of PPV with more than one isolate with the complete genome sequence available in GenBank were included also in this study for comparison and analysis. Five intrastrain recombination events were detected among the PPV W isolates, one among PPV C strain isolates, and one among PPV M strain isolates. Four (29%) of the PPV W isolates analyzed are recombinants; one of which (P2-1) is a mosaic, with three recombination events identified. A new interstrain recombinant event was identified between a strain M isolate and a strain Rec isolate, a known recombinant. In silico recombination studies and pairwise distance analyses of PPV strain D isolates indicate that a threshold of genetic diversity exists for the detectability of recombination events, in the range of approximately 0.78×10(-2) to 1.33×10(-2) mean pairwise distance. RDP4 analyses indicate that in the case of PPV Rec isolates there may be a recombinant breakpoint distinct from the obvious transition point of strain sequences. Evidence was obtained that indicates that the frequency of PPV recombination is underestimated, which may be true for other RNA viruses where low genetic diversity exists.

Mode and tempo of sequence and floral evolution within the Anserineae

The Chenopodiaceae Tribe Anserineae Dumort was proposed to include the genus Spinacia and the genus Blitum. In addition to the recent domestication of Spinacia, the tribe demonstrates extensive evolution within its floral development. We test whether the development of dioecy, monoecy, and protogyny is reflected differentially among floral developmental versus non-floral developmental genes, and whether recent domestication leaves traces in the phylogenetic relationship within the genus Spinacia. The phylogenetic predictions consistently support the sister relationship of Spinacia sp. to a Blitum clade consisting of Blitum bonus-henricus, Blitum virgatum, and Blitum nuttallianum. Relative rates tests indicate a generally faster rate of nucleotide substitutions within Spinacia. Tests of selection indicate that there is generally purifying selection acting on the sequences. In addition, insertion/deletion (indel) events occur more prominently within the Spinacia clade and occur in both coding and intron regions. The phylogenetic relationships within this tribe calls into question the hypothesis that dioecy in Spinacia evolved from a monoecious grade. The evidence for purifying selection in Spinacia suggests that the increased nucleotide substitution rates are not driving protein evolution, in contrast to evidence of protein sequence and structure evolution driven by indels. There is no footprint of domestication on sequence evolution, and we cannot detect phylogenetic signals that would support separation of the Spinacia accessions into three distinct taxa.

Single Nucleotide Polymorphisms and Indel Markers from the Transcriptome of Garlic

Garlic (Allium sativum) is cultivated worldwide and appreciated for its culinary uses. In spite of primarily being asexually propagated, garlic shows great morphological variation and adaptability to diverse production environments. Molecular markers and phenotypic characteristics have been used to assess the genetic diversity among garlics. In this study, we undertook transcriptome sequencing from a single garlic plant to identify molecular markers in expressed regions of the garlic genome. Garlic sequences were assembled and selected if they were similar to monomorphic sequences from a doubled haploid (DH) of onion (Allium cepa). Single nucleotide polymorphisms (SNPs) and insertion–deletion (indel) events were identified in 4355 independent garlic assemblies. A sample of the indels was verified using the original complementary DNA (cDNA) library and genomics DNAs from diverse garlics, and segregations confirmed by sexual progenies of garlic. These molecular markers from the garlic transcriptome should be useful for estimates of genetic diversity, identification and removal of duplicate accessions from germplasm collections, and the development of a detailed genetic map of this important vegetable crop.

Complete genome sequence of a strain of Actinidia virus X detected in Ribes nigrum cv. Baldwin showing unusual symptoms.

A Ribes-infecting strain of the potexvirus Actinidia virus X (AVX-RV3124) was isolated from black currant plants (Ribes nigrum cv. Baldwin, accession 3124-03D1) showing symptoms of leaf chlorosis and deformity. This is the first description of the complete genome sequence of an isolate of this virus and the first detection of a potexvirus in Ribes. The genome of AVX-RV3124 consists of 6,888 nucleotides (nt) excluding the poly(A) tail at the 3' terminus. When AVX-RV3124 was compared to the available sequence of the AVX isolate in GenBank (accession no. KC568202), two large indel events (72 nt and 33 nt) were identified in the replicase coding region of RV3124. Evidence of recombination was detected upstream of the 3' terminus of the replicase gene of both virus isolates, providing further evidence of a common origin.