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There is an increasing awareness that as a result of structural variation, a reference sequence representing a genome of a single individual is unable to capture all of the gene repertoire found in the species. A large number of genes affected by presence/absence and copy number variation suggest that it may contribute to phenotypic and agronomic trait diversity. Here we show by analysis of the Brassica oleracea pangenome that nearly 20% of genes are affected by presence/absence variation. Several genes displaying presence/absence variation are annotated with functions related to major agronomic traits, including disease resistance, flowering time, glucosinolate metabolism and vitamin biosynthesis. Brassica oleracea is a single species that includes diverse crops such as cabbage, broccoli and Brussels sprouts. Here, the authors identify genes not captured in existing B. oleracea reference genomes by the assembly of a pangenome and show variations in gene content that may be related to important agronomic traits

Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in children globally, with nearly all children experiencing at least one infection by the age of two. Partial sequencing of the attachment glycoprotein gene is conducted routinely for genotyping, but relatively few whole genome sequences are available for RSV. The goal of our study was to sequence the genomes of RSV strains collected from multiple countries to further understand the global diversity of RSV at a whole-genome level. We collected RSV samples and isolates from Mexico, Argentina, Belgium, Italy, Germany, Australia, South Africa, and the USA from the years 1998-2010. Both Sanger and next-generation sequencing with the Illumina and 454 platforms were used to sequence the whole genomes of RSV A and B. Phylogenetic analyses were performed using the Bayesian and maximum likelihood methods of phylogenetic inference. We sequenced the genomes of 34 RSVA and 23 RSVB viruses. Phylogenetic analysis showed that the RSVA genome evolves at an estimated rate of 6.72 × 10(-4) substitutions/site/year (95% HPD 5.61 × 10(-4) to 7.6 × 10(-4)) and for RSVB the evolutionary rate was 7.69 × 10(-4) substitutions/site/year (95% HPD 6.81 × 10(-4) to 8.62 × 10(-4)). We found multiple clades co-circulating globally for both RSV A and B. The predominant clades were GA2 and GA5 for RSVA and BA for RSVB. Our analyses showed that RSV circulates on a global scale with the same predominant clades of viruses being found in countries around the world. However, the distribution of clades can change rapidly as new strains emerge. We did not observe a strong spatial structure in our trees, with the same three main clades of RSV co-circulating globally, suggesting that the evolution of RSV is not strongly regionalized.

Chickpea (Cicer arietinum L.), an important grain legume crop of the world is seriously challenged by terminal drought and salinity stresses. However, very limited number of molecular markers and candidate genes are available for undertaking molecular breeding in chickpea to tackle these stresses. This study reports generation and analysis of comprehensive resource of drought- and salinity-responsive expressed sequence tags (ESTs) and gene-based markers...

Human Parainfluenza viruses (HPIV) type 1 and 3 are important causes of respiratory tract infections in young children globally. HPIV infections do not confer complete protective immunity so reinfections occur throughout life. Since no effective vaccine is available for the two virus subtypes, comprehensive understanding of HPIV-1 and HPIV-3 genetic and epidemic features is important for diagnosis, prevention, and treatment of HPIV-1 and HPIV-3 infections. Relatively few whole genome sequences are available for both HPIV-1 and HPIV-3 viruses, so our study sought to provide whole genome sequences from multiple countries to further the understanding of the global diversity of HPIV at a whole-genome level. We collected HPIV-1 and HPIV-3 samples and isolates from Argentina, Australia, France, Mexico, South Africa, Switzerland, and USA from the years 2003-2011 and sequenced the genomes of 40 HPIV-1 and 75 HPIV-3 viruses with Sanger and next-generation sequencing with the Ion Torrent, Illumina, and 454 platforms. Phylogenetic analysis showed that the HPIV-1 genome is evolving at an estimated rate of 4.97 × 10-4 mutations/site/year (95% highest posterior density 4.55 × 10-4 to 5.38 × 10-4) and the HPIV-3 genome is evolving at a similar rate (3.59 × 10-4 mutations/site/year, 95% highest posterior density 3.26 × 10-4 to 3.94 × 10-4). There were multiple genetically distinct lineages of both HPIV-1 and 3 circulating on a global scale. Further surveillance and whole-genome sequencing are greatly needed to better understand the spatial dynamics of these important respiratory viruses in humans.

Phosphorus (P) is an essential element for plant growth. Crop production of common bean (Phaseolus vulgaris), the most important legume for human consumption, is often limited by low P in the soil. Functional genomics were used to investigate global gene expression and metabolic responses of bean plants grown under P-deficient and P-sufficient conditions. P-deficient plants showed enhanced root to shoot ratio accompanied by reduced leaf area and net photosynthesis rates. Transcript profiling was performed through hybridization of nylon filter arrays spotted with cDNAs of 2,212 unigenes from a P deficiency root cDNA library. A total of 126 genes, representing different functional categories, showed significant differential expression in response to P: 62% of these were induced in P-deficient roots. A set of 372 bean transcription factor (TF) genes, coding for proteins with Inter-Pro domains characteristic or diagnostic for TF, were identified from The Institute of Genomic Research/Dana Farber Cancer Institute Common Bean Gene Index. Using real-time reverse transcription-polymerase chain reaction analysis, 17 TF genes were differentially expressed in P-deficient roots; four TF genes, including MYB TFs, were induced. Nonbiased metabolite profiling was used to assess the degree to which changes in gene expression in P-deficient roots affect overall metabolism. Stress-related metabolites such as polyols accumulated in P-deficient roots as well as sugars, which are known to be essential for P stress gene induction. Candidate genes have been identified that may contribute to root adaptation to P deficiency and be useful for improvement of common bean.

Background: Pigeonpea [Cajanus cajan (L.) Millsp.] is an important legume crop of rainfed agriculture. Despite of concerted research efforts directed to pigeonpea improvement, stagnated productivity of pigeonpea during last several decades may be accounted to prevalence of various biotic and abiotic constraints and the situation is exacerbated by availability of inadequate genomic resources to undertake any molecular breeding programme for accelerated crop improvement. With the objective of enhancing genomic resources for pigeonpea, this study reports for the first time, large scale development of SSR markers from BAC-end sequences and their subsequent use for genetic mapping and hybridity testing in pigeonpea. Results: A set of 88,860 BAC (bacterial artificial chromosomes)-end sequences (BESs) were generated after constructing two BAC libraries by using HindIII (34,560 clones) and BamHI (34,560 clones) restriction enzymes. Clustering based on sequence identity of BESs yielded a set of >52 K non-redundant sequences, comprising 35 Mbp or >4% of the pigeonpea genome. These sequences were analyzed to develop annotation lists and subdivide the BESs into genome fractions (e.g., genes, retroelements, transpons and non-annotated sequences). Parallel analysis of BESs for microsatellites or simple sequence repeats (SSRs) identified 18,149 SSRs, from which a set of 6,212 SSRs were selected for further analysis. A total of 3,072 novel SSR primer pairs were synthesized and tested for length polymorphism on a set of 22 parental genotypes of 13 mapping populations segregating for traits of interest. In total, we identified 842 polymorphic SSR markers that will have utility in pigeonpea improvement. Based on these markers, the first SSR-based genetic map comprising of 239 loci was developed for this previously uncharacterized genome. Utility of developed SSR markers was also demonstrated by identifying a set of 42 markers each for two hybrids (ICPH 2671 and ICPH 2438) for genetic purity assessment in commercial hybrid breeding programme. Conclusion: In summary, while BAC libraries and BESs should be useful for genomics studies, BES-SSR markers, and the genetic map should be very useful for linking the genetic map with a future physical map as well as for molecular breeding in pigeonpea

Recent RNA-seq studies reveal that the transcriptomes in animals and plants are more complex than previously thought, leading to the inclusion of many more splice isoforms in annotated genomes. However, it is possible that a significant proportion of the transcripts are spurious isoforms that do not contribute to functional proteins. One of the current hypotheses is that commonly used mRNA extraction methods isolate both pre-mature (nuclear) mRNA and mature (cytoplasmic) mRNA, and these incompletely spliced pre-mature mRNAs may contribute to a large proportion of these spurious transcripts. To investigate this, we compared a traditional RNA-seq dataset (total RNA-seq) and a ribosome-bound RNA-seq dataset (polyribosomal RNA-seq) from Arabidopsis thaliana. An integrative framework that combined de novo assembly and genome-guided assembly was applied to reconstruct transcriptomes for the two datasets. Up to 44.8% of the de novo assembled transcripts in total RNA-seq sample were of low abundance, whereas only 0.09% in polyribosomal RNA-seq de novo assembly were of low abundance. The final round of assembly using PASA (Program to Assemble Spliced Alignments) resulted in more transcript assemblies in the total RNA-seq than those in polyribosomal sample. Comparison of alternative splicing (AS) patterns between total and polyribosomal RNA-seq showed a significant difference (G-test, p-value<0.01) in intron retention events: 46.4% of AS events in the total sample were intron retention, whereas only 23.5% showed evidence of intron retention in the polyribosomal sample. It is likely that a large proportion of retained introns in total RNA-seq result from incompletely spliced pre-mature mRNA. Overall, this study demonstrated that polyribosomal RNA-seq technology decreased the complexity and diversity of the coding transcriptome by eliminating pre-mature mRNAs, especially those of low abundance.

Optical mapping has been widely used to improve de novo plant genome assemblies, including rice, maize, Medicago, Amborella, tomato and wheat, with more genomes in the pipeline. Optical mapping provides long-range information of the genome and can more easily identify large structural variations. The ability of optical mapping to assay long single DNA molecules nicely complements short-read sequencing which is more suitable for the identification of small and short-range variants. Direct use of optical mapping to study population-level genetic diversity is currently limited to microbial strain typing and human diversity studies. Nonetheless, optical mapping shows great promise in the study of plant trait development, domestication and polyploid evolution. Here we review the current applications and future prospects of optical mapping in the field of plant comparative genomics.

Root induction by auxins is still not well understood at the molecular level. In this study a system has been devised which distinguishes between the two active auxins indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA). IBA, but not IAA, efficiently induced adventitious rooting in Arabidopsis stem segments at a concentration of 10 μM. In wild-type plants, roots formed exclusively out of calli at the basal end of the segments. Root formation was inhibited by 10 μM 3,4,5-triiodobenzoic acid (TIBA), an inhibitor of polar auxin transport. At intermediate IBA concentrations (3–10 μM), root induction was less efficient in trp1, a tryptophan auxotroph of Arabidopsis with a bushy phenotype but no demonstrable reduction in IAA levels. By contrast, two mutants of Arabidopsis with measurably higher levels of IAA (trp2, amt1) show root induction characteristics very similar to the wild type. Using differential display, transcripts specific to the rooting process were identified by devising a protocol that distinguished between callus production only and callus production followed by root initiation. One fragment was identical to the sequence of a putative regulatory subunit B of protein phosphatase 2A. It is suggested that adventitious rooting in Arabidopsis stem segments is due to an interaction between endogenous IAA and exogenous IBA. In stem explants, residual endogenous IAA is transported to the basal end of each segment, thereby inducing root formation. In stem segments in which the polar auxin transport is inhibited by TIBA, root formation does not occur.