Identification and characterization of pseudogenes in the rice gene complement

<p>Abstract</p> <p>Background</p> <p>The Osa1 Genome Annotation of rice (<it>Oryza sativa </it>L. ssp. <it>japonica </it>cv. Nipponbare) is the product of a semi-automated pipeline that does not explicitly predict pseudogenes. As such, it is likely to mis-annotate pseudogenes as functional genes. A total of 22,033 gene models within the Osa1 Release 5 were investigated as potential pseudogenes as these genes exhibit at least one feature potentially indicative of pseudogenes: lack of transcript support, short coding region, long untranslated region, or, for genes residing within a segmentally duplicated region, lack of a paralog or significantly shorter corresponding paralog.</p> <p>Results</p> <p>A total of 1,439 pseudogenes, identified among genes with pseudogene features, were characterized by similarity to fully-supported gene models and the presence of frameshifts or premature translational stop codons. Significant difference in the length of duplicated genes within segmentally-duplicated regions was the optimal indicator of pseudogenization. Among the 816 pseudogenes for which a probable origin could be determined, 75% originated from gene duplication events while 25% were the result of retrotransposition events. A total of 12% of the pseudogenes were expressed. Finally, F-box proteins, BTB/POZ proteins, terpene synthases, chalcone synthases and cytochrome P450 protein families were found to harbor large numbers of pseudogenes.</p> <p>Conclusion</p> <p>These pseudogenes still have a detectable open reading frame and are thus distinct from pseudogenes detected within intergenic regions which typically lack definable open reading frames. Families containing the highest number of pseudogenes are fast-evolving families involved in ubiquitination and secondary metabolism.</p

Overcoming Self-Incompatibility in Diploid Potato Using CRISPR-Cas9

Potato breeding can be redirected to a diploid inbred/F1 hybrid variety breeding strategy if self-compatibility can be introduced into diploid germplasm. However, the majority of diploid potato clones (Solanum spp.) possess gametophytic self-incompatibility that is primarily controlled by a single multiallelic locus called the S-locus which is composed of tightly linked genes, S-RNase (S-locus RNase) and multiple SLFs (S-locus F-box proteins), which are expressed in the style and pollen, respectively. Using S-RNase genes known to function in the Solanaceae gametophytic SI mechanism, we identified S-RNase alleles with flower-specific expression in two diploid self-incompatible potato lines using genome resequencing data. Consistent with the location of the S-locus in potato, we genetically mapped the S-RNase gene using a segregating population to a region of low recombination within the pericentromere of chromosome 1. To generate self-compatible diploid potato lines, a dual single-guide RNA (sgRNA) strategy was used to target conserved exonic regions of the S-RNase gene and generate targeted knockouts (KOs) using a Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (Cas9) approach. Self-compatibility was achieved in nine S-RNase KO T0 lines which contained bi-allelic and homozygous deletions/insertions in both genotypes, transmitting self compatibility to T1 progeny. This study demonstrates an efficient approach to achieve stable, consistent self-compatibility through S-RNase KO for use in diploid potato breeding approaches

Genome Editing for Crop Improvement – Applications in Clonally Propagated Polyploids With a Focus on Potato (Solanum tuberosum L.)

Genome-editing has revolutionized biology. When coupled with a recently streamlined regulatory process by the U.S. Department of Agriculture and the potential to generate transgene-free varieties, genome-editing provides a new avenue for crop improvement. For heterozygous, polyploid and vegetatively propagated crops such as cultivated potato, Solanum tuberosum Group Tuberosum L., genome-editing presents tremendous opportunities for trait improvement. In potato, traits such as improved resistance to cold-induced sweetening, processing efficiency, herbicide tolerance, modified starch quality and self-incompatibility have been targeted utilizing CRISPR/Cas9 and TALEN reagents in diploid and tetraploid clones. However, limited progress has been made in other such crops including sweetpotato, strawberry, grapes, citrus, banana etc., In this review we summarize the developments in genome-editing platforms, delivery mechanisms applicable to plants and then discuss the recent developments in regulation of genome-edited crops in the United States and The European Union. Next, we provide insight into the challenges of genome-editing in clonally propagated polyploid crops, their current status for trait improvement with future prospects focused on potato, a global food security crop

Intron gain and loss in segmentally duplicated genes in rice

BACKGROUND: Introns are under less selection pressure than exons, and consequently, intronic sequences have a higher rate of gain and loss than exons. In a number of plant species, a large portion of the genome has been segmentally duplicated, giving rise to a large set of duplicated genes. The recent completion of the rice genome in which segmental duplication has been documented has allowed us to investigate intron evolution within rice, a diploid monocotyledonous species. RESULTS: Analysis of segmental duplication in rice revealed that 159 Mb of the 371 Mb genome and 21,570 of the 43,719 non-transposable element-related genes were contained within a duplicated region. In these duplicated regions, 3,101 collinear paired genes were present. Using this set of segmentally duplicated genes, we investigated intron evolution from full-length cDNA-supported non-transposable element-related gene models of rice. Using gene pairs that have an ortholog in the dicotyledonous model species Arabidopsis thaliana, we identified more intron loss (49 introns within 35 gene pairs) than intron gain (5 introns within 5 gene pairs) following segmental duplication. We were unable to demonstrate preferential intron loss at the 3' end of genes as previously reported in mammalian genomes. However, we did find that the four nucleotides of exons that flank lost introns had less frequently used 4-mers. CONCLUSION: We observed that intron evolution within rice following segmental duplication is largely dominated by intron loss. In two of the five cases of intron gain within segmentally duplicated genes, the gained sequences were similar to transposable elements

Evaluation of Methods to Assess in vivo Activity of Engineered Genome-Editing Nucleases in Protoplasts

Genome-editing is being implemented in increasing number of plant species using engineered sequence specific nucleases (SSNs) such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9), Transcription activator like effector nucleases (TALENs), and more recently CRISPR/Cas12a. As the tissue culture and regeneration procedures to generate gene-edited events are time consuming, large-scale screening methodologies that rapidly facilitate validation of genome-editing reagents are critical. Plant protoplast cells provide a rapid platform to validate genome-editing reagents. Protoplast transfection with plasmids expressing genome-editing reagents represents an efficient and cost-effective method to screen for in vivo activity of genome-editing constructs and resulting targeted mutagenesis. In this study, we compared three existing methods for detection of editing activity, the T7 endonuclease I assay (T7EI), PCR/restriction enzyme (PCR/RE) digestion, and amplicon-sequencing, with an alternative method which involves tagging a double-stranded oligodeoxynucleotide (dsODN) into the SSN-induced double stranded break and detection of on-target activity of gene-editing reagents by PCR and agarose gel electrophoresis. To validate these methods, multiple reagents including TALENs, CRISPR/Cas9 and Cas9 variants, eCas9(1.1) (enhanced specificity) and Cas9-HF1 (high-fidelity1) were engineered for targeted mutagenesis of Acetolactate synthase1 (ALS1), 5-Enolpyruvylshikimate- 3-phosphate synthase1 (EPSPS1) and their paralogs in potato. While all methods detected editing activity, the PCR detection of dsODN integration provided the most straightforward and easiest method to assess on-target activity of the SSN as well as a method for initial qualitative evaluation of the functionality of genome-editing constructs. Quantitative data on mutagenesis frequencies obtained by amplicon-sequencing of ALS1 revealed that the mutagenesis frequency of CRISPR/Cas9 reagents is better than TALENs. Context-based choice of method for evaluation of gene-editing reagents in protoplast systems, along with advantages and limitations associated with each method, are discussed

NUCLEC ACDS ENCODING PSEUDOMONAS HOP PROTEINS AND USE THEREOF

The present invention relates to isolated nucleic acid mol ecules encoding a type III—secreted bacterial protein capable of modifying a cell death pathway in a plant cell. One aspect of the present invention involves an isolated nucleic acid molecule having a nucleotide sequence that encodes the HopPtol)2 protein of Pseudomonas syringae pv. syringae DC 3000. Expression vectors, host cells, and transgenic plants which include the DNA molecules of the present invention are also disclosed. The nucleic acid mol ecules of the present invention can be used to impart disease resistance to a plant and to make a plant hypersusceptible to colonization by nonpathogenic bacteria

NUCLEC ACDS ENCODING PSEUDOMONAS HOP PROTEINS AND USE THEREOF

The present invention relates to isolated nucleic acid mol ecules encoding a type III—secreted bacterial protein capable of modifying a cell death pathway in a plant cell. One aspect of the present invention involves an isolated nucleic acid molecule having a nucleotide sequence that encodes the HopPtol)2 protein of Pseudomonas syringae pv. syringae DC 3000. Expression vectors, host cells, and transgenic plants which include the DNA molecules of the present invention are also disclosed. The nucleic acid mol ecules of the present invention can be used to impart disease resistance to a plant and to make a plant hypersusceptible to colonization by nonpathogenic bacteria

Comparative analyses of six solanaceous transcriptomes reveal a high degree of sequence conservation and species-specific transcripts

BACKGROUND: The Solanaceae is a family of closely related species with diverse phenotypes that have been exploited for agronomic purposes. Previous studies involving a small number of genes suggested sequence conservation across the Solanaceae. The availability of large collections of Expressed Sequence Tags (ESTs) for the Solanaceae now provides the opportunity to assess sequence conservation and divergence on a genomic scale. RESULTS: All available ESTs and Expressed Transcripts (ETs), 449,224 sequences for six Solanaceae species (potato, tomato, pepper, petunia, tobacco and Nicotiana benthamiana), were clustered and assembled into gene indices. Examination of gene ontologies revealed that the transcripts within the gene indices encode a similar suite of biological processes. Although the ESTs and ETs were derived from a variety of tissues, 55–81% of the sequences had significant similarity at the nucleotide level with sequences among the six species. Putative orthologs could be identified for 28–58% of the sequences. This high degree of sequence conservation was supported by expression profiling using heterologous hybridizations to potato cDNA arrays that showed similar expression patterns in mature leaves for all six solanaceous species. 16–19% of the transcripts within the six Solanaceae gene indices did not have matches among Solanaceae, Arabidopsis, rice or 21 other plant gene indices. CONCLUSION: Results from this genome scale analysis confirmed a high level of sequence conservation at the nucleotide level of the coding sequence among Solanaceae. Additionally, the results indicated that part of the Solanaceae transcriptome is likely to be unique for each species

Gene and genome duplications in the evolution of chemodiversity : perspectives from studies of Lamiaceae

Plants are reservoirs of extreme chemical diversity, yet biosynthetic pathways remain underexplored in the majority of taxa. Access to improved, inexpensive genomic and computational technologies has recently enhanced our understanding of plant specialized metabolism at the biochemical and evolutionary levels including the elucidation of pathways leading to key metabolites. Furthermore, these approaches have provided insights into the mechanisms of chemical evolution, including neofunctionalization and subfunctionalization, structural variation, and modulation of gene expression. The broader utilization of genomic tools across the plant tree of life, and an expansion of genomic resources from multiple accessions within species or populations, will improve our overall understanding of chemodiversity. These data and knowledge will also lead to greater insight into the selective pressures contributing to and maintaining this diversity, which in turn will enable the development of more accurate predictive models of specialized metabolism in plants

Analysis of 90 Mb of the potato genome reveals conservation of gene structures and order with tomato but divergence in repetitive sequence composition

<p>Abstract</p> <p>Background</p> <p>The Solanaceae family contains a number of important crop species including potato (<it>Solanum tuberosum</it>) which is grown for its underground storage organ known as a tuber. Albeit the 4^thmost important food crop in the world, other than a collection of ~220,000 Expressed Sequence Tags, limited genomic sequence information is currently available for potato and advances in potato yield and nutrition content would be greatly assisted through access to a complete genome sequence. While morphologically diverse, Solanaceae species such as potato, tomato, pepper, and eggplant share not only genes but also gene order thereby permitting highly informative comparative genomic analyses.</p> <p>Results</p> <p>In this study, we report on analysis 89.9 Mb of potato genomic sequence representing 10.2% of the genome generated through end sequencing of a potato bacterial artificial chromosome (BAC) clone library (87 Mb) and sequencing of 22 potato BAC clones (2.9 Mb). The GC content of potato is very similar to <it>Solanum lycopersicon </it>(tomato) and other dicotyledonous species yet distinct from the monocotyledonous grass species, <it>Oryza sativa</it>. Parallel analyses of repetitive sequences in potato and tomato revealed substantial differences in their abundance, 34.2% in potato versus 46.3% in tomato, which is consistent with the increased genome size per haploid genome of these two <it>Solanum </it>species. Specific classes and types of repetitive sequences were also differentially represented between these two species including a telomeric-related repetitive sequence, ribosomal DNA, and a number of unclassified repetitive sequences. Comparative analyses between tomato and potato at the gene level revealed a high level of conservation of gene content, genic feature, and gene order although discordances in synteny were observed.</p> <p>Conclusion</p> <p>Genomic level analyses of potato and tomato confirm that gene sequence and gene order are conserved between these solanaceous species and that this conservation can be leveraged in genomic applications including cross-species annotation and genome sequencing initiatives. While tomato and potato share genic features, they differ in their repetitive sequence content and composition suggesting that repetitive sequences may have a more significant role in shaping speciation than previously reported.</p