Advances in the identification of novel factors required in soybean nodulation, a process critical to sustainable agriculture and food security

Nodulation is a process of organogenesis that results from a symbiotic relationship between legume plants and soil-dwelling, nitrogen-fixing bacteria, called rhizobia. The rhizobia are housed in newly formed structures on the host roots, called nodules. Within nodules, the rhizobia fix atmospheric N2 into useable forms of nitrogen for the plant. This process is highly important to agriculture, as nitrogen is critical for plant growth and development and is typically the main component of fertilizers. Although fertilizers are effective, they are expensive and often pollute, making biological alternatives, such as legume nodulation, attractive for use in agriculture. Nodulation is regulated by the auto regulation of nodulation (AON) pathway, which enables the host plant to balance its needs between nitrogen acquisition and energy expenditure. Current research is elucidating the nodule development and AON signalling networks. Recent technological advances, such as RNA-sequencing, are revolutionizing the discovery of genes that are critical to nodulation. The discovery of such genes not only enhances our knowledge of the nodulation signalling network, but may help to underpin future work to isolate superior legume crops via modern breeding and engineering practices. Here, recent advances using the cutting-edge technique of RNA sequencing to identify new nodulation genes in soybean are discussed

Molecular analysis of lipoxygenases associated with nodule development in soybean

We utilized transcriptional profiling to identify genes associated with nodule development in soybean. Many of the candidate genes were predicted to be involved in processes such as defense, metabolism, transcriptional regulation, oxidation, or iron storage. Here, we describe the detailed characterization of one specific class of genes that encode the enzyme lipoxygenase (LOX). The LOX9 and LOX10 genes identified by microarray analysis represent novel soybean LOXs expressed in developing nodules. LOX expression during nodulation was relatively complex, with at least eight different LOX genes expressed in soybean nodules. Histochemical analyses utilizing LOX9 promoter::beta-glucuronidase (GUS) fusion constructs in transgenic soybean hairy roots suggest that this gene is involved in the growth and development of specific cells within the root and nodules. In soybean roots, LOX9 was expressed specifically in the developing phloem. In nodules, the expression of LOX9 was correlated with the development of cells in the vasculature and lenticels. The use of RNAi in transgenic hairy roots reduced LOX expression by approximately 95%. Despite this significant reduction in LOX expression, there was no detectable effect on the development of roots or nodules. Our findings are discussed with respect to the potential function of LOXs in nodulation

Are the current gRNA ranking prediction algorithms useful for genome editing in plants?

Introducing a new trait into a crop through conventional breeding commonly takes decades, but recently developed genome sequence modification technology has the potential to accelerate this process. One of these new breeding technologies relies on an RNA-directed DNA nuclease (CRISPR/Cas9) to cut the genomic DNA, in vivo, to facilitate the deletion or insertion of sequences. This sequence specific targeting is determined by guide RNAs (gRNAs). However, choosing an optimum gRNA sequence has its challenges. Almost all current gRNA design tools for use in plants are based on data from experiments in animals, although many allow the use of plant genomes to identify potential off-target sites. Here, we examine the predictive uniformity and performance of eight different online gRNA-site tools. Unfortunately, there was little consensus among the rankings by the different algorithms, nor a statistically significant correlation between rankings and in vivo effectiveness. This suggests that important factors affecting gRNA performance and/or target site accessibility, in plants, are yet to be elucidated and incorporated into gRNA-site prediction tools

BioNano genome mapping of individual chromosomes supports physical mapping and sequence assembly in complex plant genomes

The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC-by-BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high-resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high-resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome-scale analysis of repetitive sequences and revealed a ~800-kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone-by-clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC-contig physical map and validate sequence assembly on a chromosome-arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome-by-chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules

Mutation Analysis of 2009 Pandemic Influenza A(H1N1) Viruses Collected in Japan during the Peak Phase of the Pandemic

BACKGROUND: Pandemic influenza A(H1N1) virus infection quickly circulated worldwide in 2009. In Japan, the first case was reported in May 2009, one month after its outbreak in Mexico. Thereafter, A(H1N1) infection spread widely throughout the country. It is of great importance to profile and understand the situation regarding viral mutations and their circulation in Japan to accumulate a knowledge base and to prepare clinical response platforms before a second pandemic (pdm) wave emerges. METHODOLOGY: A total of 253 swab samples were collected from patients with influenza-like illness in the Osaka, Tokyo, and Chiba areas both in May 2009 and between October 2009 and January 2010. We analyzed partial sequences of the hemagglutinin (HA) and neuraminidase (NA) genes of the 2009 pdm influenza virus in the collected clinical samples. By phylogenetic analysis, we identified major variants of the 2009 pdm influenza virus and critical mutations associated with severe cases, including drug-resistance mutations. RESULTS AND CONCLUSIONS: Our sequence analysis has revealed that both HA-S220T and NA-N248D are major non-synonymous mutations that clearly discriminate the 2009 pdm influenza viruses identified in the very early phase (May 2009) from those found in the peak phase (October 2009 to January 2010) in Japan. By phylogenetic analysis, we found 14 micro-clades within the viruses collected during the peak phase. Among them, 12 were new micro-clades, while two were previously reported. Oseltamivir resistance-related mutations, i.e., NA-H275Y and NA-N295S, were also detected in sporadic cases in Osaka and Tokyo

Integrated physical map of bread wheat chromosome arm 7DS to facilitate gene cloning and comparative studies

Bread wheat (Triticum aestivum L.) is a staple food for a significant part of the world’s population. The growing demand on its production can be satisfied by improving yield and resistance to biotic and abiotic stress. Knowledge of the genome sequence would aid in discovering genes and QTLs underlying these traits and provide a basis for genomics-assisted breeding. Physical maps and BAC clones associated with them have been valuable resources from which to generate a reference genome of bread wheat and to assist map-based gene cloning. As a part of a joint effort coordinated by the International Wheat Genome Sequencing Consortium, we have con- structed a BAC-based physical map of bread wheat chromosome arm 7DS consisting of 895 contigs and covering 94% of its estimated length. By anchoring BAC contigs to one radiation hybrid map and three high resolution genetic maps, we assigned 73% of the assembly to a distinct genomic position. This map integration, inter- connecting a total of 1713 markers with ordered and sequenced BAC clones from a minimal tiling path, provides a tool to speed up gene cloning in wheat. The process of physical map assembly included the integration of the 7DS physical map with a whole-genome physical map of Aegilops tauschii and a 7DS Bionano genome map, which together enabled efficient scaffolding of physical-map contigs, even in the non-recombining region of the genetic centromere. Moreover, this approach facilitated a comparison of bread wheat and its ancestor at BAC-contig level and revealed a reconstructed region in the 7DS pericentromere