Cloning and characterization of a norbelladine 4'-O-methyltransferase involved in the biosynthesis of the Alzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus.

Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer's disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4'-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4'-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.

Next generation sequencing in predicting gene function in podophyllotoxin biosynthesis.

Podophyllum species are sources of (-)-podophyllotoxin, an aryltetralin lignan used for semi-synthesis of various powerful and extensively employed cancer-treating drugs. Its biosynthetic pathway, however, remains largely unknown, with the last unequivocally demonstrated intermediate being (-)-matairesinol. Herein, massively parallel sequencing of Podophyllum hexandrum and Podophyllum peltatum transcriptomes and subsequent bioinformatics analyses of the corresponding assemblies were carried out. Validation of the assembly process was first achieved through confirmation of assembled sequences with those of various genes previously established as involved in podophyllotoxin biosynthesis as well as other candidate biosynthetic pathway genes. This contribution describes characterization of two of the latter, namely the cytochrome P450s, CYP719A23 from P. hexandrum and CYP719A24 from P. peltatum. Both enzymes were capable of converting (-)-matairesinol into (-)-pluviatolide by catalyzing methylenedioxy bridge formation and did not act on other possible substrates tested. Interestingly, the enzymes described herein were highly similar to methylenedioxy bridge-forming enzymes from alkaloid biosynthesis, whereas candidates more similar to lignan biosynthetic enzymes were catalytically inactive with the substrates employed. This overall strategy has thus enabled facile further identification of enzymes putatively involved in (-)-podophyllotoxin biosynthesis and underscores the deductive power of next generation sequencing and bioinformatics to probe and deduce medicinal plant biosynthetic pathways.

Prevalence of single nucleotide polymorphism among 27 diverse alfalfa genotypes as assessed by transcriptome sequencing.

BACKGROUND: Alfalfa, a perennial, outcrossing species, is a widely planted forage legume producing highly nutritious biomass. Currently, improvement of cultivated alfalfa mainly relies on recurrent phenotypic selection. Marker assisted breeding strategies can enhance alfalfa improvement efforts, particularly if many genome-wide markers are available. Transcriptome sequencing enables efficient high-throughput discovery of single nucleotide polymorphism (SNP) markers for a complex polyploid species. RESULT: The transcriptomes of 27 alfalfa genotypes, including elite breeding genotypes, parents of mapping populations, and unimproved wild genotypes, were sequenced using an Illumina Genome Analyzer IIx. De novo assembly of quality-filtered 72-bp reads generated 25,183 contigs with a total length of 26.8 Mbp and an average length of 1,065 bp, with an average read depth of 55.9-fold for each genotype. Overall, 21,954 (87.2%) of the 25,183 contigs represented 14,878 unique protein accessions. Gene ontology (GO) analysis suggested that a broad diversity of genes was represented in the resulting sequences. The realignment of individual reads to the contigs enabled the detection of 872,384 SNPs and 31,760 InDels. High resolution melting (HRM) analysis was used to validate 91% of 192 putative SNPs identified by sequencing. Both allelic variants at about 95% of SNP sites identified among five wild, unimproved genotypes are still present in cultivated alfalfa, and all four US breeding programs also contain a high proportion of these SNPs. Thus, little evidence exists among this dataset for loss of significant DNA sequence diversity from either domestication or breeding of alfalfa. Structure analysis indicated that individuals from the subspecies falcata, the diploid subspecies caerulea, and the tetraploid subspecies sativa (cultivated tetraploid alfalfa) were clearly separated. CONCLUSION: We used transcriptome sequencing to discover large numbers of SNPs segregating in elite breeding populations of alfalfa. Little loss of SNP diversity was evident between unimproved and elite alfalfa germplasm. The EST and SNP markers generated from this study are publicly available at the Legume Information System ( http://medsa.comparative-legumes.org/) and can contribute to future alfalfa research and breeding applications.

A comparative transcriptomic study of an allotetraploid and its diploid progenitors illustrates the unique advantages and challenges of RNA-seq in plant species.

PREMISE OF THE STUDY: RNA-seq analysis of plant transcriptomes poses unique challenges due to the highly duplicated nature of plant genomes. We address these challenges in the context of recently formed polyploid species and detail an RNA-seq experiment comparing the leaf transcriptome profile of an allopolyploid relative of soybean with the diploid species that contributed its homoeologous genomes. METHODS: RNA-seq reads were obtained from the three species and were aligned against the genome sequence of Glycine max. Transcript levels were estimated for each gene, relative contributions of polyploidy-duplicated loci (homoeologues) in the tetraploid were identified, and comparisons of transcript profiles and individual genes were used to analyze the regulation of transcript levels. KEY RESULTS: We present a novel metric developed to address issues arising from high degrees of gene space duplication and a method for dissecting a gene's measured transcript level in a polyploid species into the relative contribution of its homoeologues. We identify the gene family likely contributing to differences in photosynthetic rate between the allotetraploid and its progenitors and show that the tetraploid appears to be using the "redundant" gene copies in novel ways. CONCLUSIONS: Given the prevalence of polyploidy events in plants, we believe many of the approaches developed here to be applicable, and often necessary, in most plant RNA-seq experiments. The deep sampling provided by RNA-seq allows us to dissect the genetic underpinnings of specific phenotypes as well as examine complex interactions within polyploid genomes.

Identification of candidate genes underlying an iron efficiency quantitative trait locus in soybean.

Prevalent on calcareous soils in the United States and abroad, iron deficiency is among the most common and severe nutritional stresses in plants. In soybean (Glycine max) commercial plantings, the identification and use of iron-efficient genotypes has proven to be the best form of managing this soil-related plant stress. Previous studies conducted in soybean identified a significant iron efficiency quantitative trait locus (QTL) explaining more than 70% of the phenotypic variation for the trait. In this research, we identified candidate genes underlying this QTL through molecular breeding, mapping, and transcriptome sequencing. Introgression mapping was performed using two related near-isogenic lines in which a region located on soybean chromosome 3 required for iron efficiency was identified. The region corresponds to the previously reported iron efficiency QTL. The location was further confirmed through QTL mapping conducted in this study. Transcriptome sequencing and quantitative real-time-polymerase chain reaction identified two genes encoding transcription factors within the region that were significantly induced in soybean roots under iron stress. The two induced transcription factors were identified as homologs of the subgroup lb basic helix-loop-helix (bHLH) genes that are known to regulate the strategy I response in Arabidopsis (Arabidopsis thaliana). Resequencing of these differentially expressed genes unveiled a significant deletion within a predicted dimerization domain. We hypothesize that this deletion disrupts the Fe-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT)/bHLH heterodimer that has been shown to induce known iron acquisition genes.

Whole-genome nucleotide diversity, recombination, and linkage disequilibrium in the model legume Medicago truncatula.

Medicago truncatula is a model for investigating legume genetics, including the genetics and evolution of legume-rhizobia symbiosis. We used whole-genome sequence data to identify and characterize sequence polymorphisms and linkage disequilibrium (LD) in a diverse collection of 26 M. truncatula accessions. Our analyses reveal that M. truncatula harbors both higher diversity and less LD than soybean (Glycine max) and exhibits patterns of LD and recombination similar to Arabidopsis thaliana. The population-scaled recombination rate is approximately one-third of the mutation rate, consistent with expectations for a species with a high selfing rate. Linkage disequilibrium, however, is not extensive, and therefore, the low recombination rate is likely not a major constraint to adaptation. Nucleotide diversity in 100-kb windows was negatively correlated with gene density, which is expected if diversity is shaped by selection acting against slightly deleterious mutations. Among putative coding regions, members of four gene families harbor significantly higher diversity than the genome-wide average. Three of these families are involved in resistance against pathogens; one of these families, the nodule-specific, cysteine-rich gene family, is specific to the galegoid legumes and is involved in control of rhizobial differentiation. The more than 3 million SNPs that we detected, approximately one-half of which are present in more than one accession, are a valuable resource for genome-wide association mapping of genes responsible for phenotypic diversity in legumes, especially traits associated with symbiosis and nodulation.

Polyploidy did not predate the evolution of nodulation in all legumes.

BACKGROUND: Several lines of evidence indicate that polyploidy occurred by around 54 million years ago, early in the history of legume evolution, but it has not been known whether this event was confined to the papilionoid subfamily (Papilionoideae; e.g. beans, medics, lupins) or occurred earlier. Determining the timing of the polyploidy event is important for understanding whether polyploidy might have contributed to rapid diversification and radiation of the legumes near the origin of the family; and whether polyploidy might have provided genetic material that enabled the evolution of a novel organ, the nitrogen-fixing nodule. Although symbioses with nitrogen-fixing partners have evolved in several lineages in the rosid I clade, nodules are widespread only in legume taxa, being nearly universal in the papilionoids and in the mimosoid subfamily (e.g., mimosas, acacias)--which diverged from the papilionoid legumes around 58 million years ago, soon after the origin of the legumes. METHODOLOGY/PRINCIPAL FINDINGS: Using transcriptome sequence data from Chamaecrista fasciculata, a nodulating member of the mimosoid clade, we tested whether this species underwent polyploidy within the timeframe of legume diversification. Analysis of gene family branching orders and synonymous-site divergence data from C. fasciculata, Glycine max (soybean), Medicago truncatula, and Vitis vinifera (grape; an outgroup to the rosid taxa) establish that the polyploidy event known from soybean and Medicago occurred after the separation of the mimosoid and papilionoid clades, and at or shortly before the Papilionoideae radiation. CONCLUSIONS: The ancestral legume genome was not fundamentally polyploid. Moreover, because there has not been an independent instance of polyploidy in the Chamaecrista lineage there is no necessary connection between polyploidy and nodulation in legumes. Chamaecrista may serve as a useful model in the legumes that lacks a paleopolyploid history, at least relative to the widely studied papilionoid models.

Genome, epigenome and RNA sequences of monozygotic twins discordant for multiple sclerosis.

Monozygotic or 'identical' twins have been widely studied to dissect the relative contributions of genetics and environment in human diseases. In multiple sclerosis (MS), an autoimmune demyelinating disease and common cause of neurodegeneration and disability in young adults, disease discordance in monozygotic twins has been interpreted to indicate environmental importance in its pathogenesis. However, genetic and epigenetic differences between monozygotic twins have been described, challenging the accepted experimental model in disambiguating the effects of nature and nurture. Here we report the genome sequences of one MS-discordant monozygotic twin pair, and messenger RNA transcriptome and epigenome sequences of CD4(+) lymphocytes from three MS-discordant, monozygotic twin pairs. No reproducible differences were detected between co-twins among approximately 3.6 million single nucleotide polymorphisms (SNPs) or approximately 0.2 million insertion-deletion polymorphisms. Nor were any reproducible differences observed between siblings of the three twin pairs in HLA haplotypes, confirmed MS-susceptibility SNPs, copy number variations, mRNA and genomic SNP and insertion-deletion genotypes, or the expression of approximately 19,000 genes in CD4(+) T cells. Only 2 to 176 differences in the methylation of approximately 2 million CpG dinucleotides were detected between siblings of the three twin pairs, in contrast to approximately 800 methylation differences between T cells of unrelated individuals and several thousand differences between tissues or between normal and cancerous tissues. In the first systematic effort to estimate sequence variation among monozygotic co-twins, we did not find evidence for genetic, epigenetic or transcriptome differences that explained disease discordance. These are the first, to our knowledge, female, twin and autoimmune disease individual genome sequences reported.

Characterization of duplicate gene evolution in the recent natural allopolyploid Tragopogon miscellus by next-generation sequencing and Sequenom iPLEX MassARRAY genotyping.

Tragopogon miscellus (Asteraceae) is an evolutionary model for the study of natural allopolyploidy, but until now has been under-resourced as a genetic model. Using 454 and Illumina expressed sequence tag sequencing of the parental diploid species of T. miscellus, we identified 7782 single nucleotide polymorphisms that differ between the two progenitor genomes present in this allotetraploid. Validation of a sample of 98 of these SNPs in genomic DNA using Sequenom MassARRAY iPlex genotyping confirmed 92 SNP markers at the genomic level that were diagnostic for the two parental genomes. In a transcriptome profile of 2989 SNPs in a single T. miscellus leaf, using Illumina sequencing, 69% of SNPs showed approximately equal expression of both homeologs (duplicate homologous genes derived from different parents), 22% showed apparent differential expression and 8.5% showed apparent silencing of one homeolog in T. miscellus. The majority of cases of homeolog silencing involved the T. dubius SNP homeolog (164/254; 65%) rather than the T. pratensis homeolog (90/254). Sequenom analysis of genomic DNA showed that in a sample of 27 of the homeologs showing apparent silencing, 23 (85%) were because of genomic homeolog loss. These methods could be applied to any organism, allowing efficient and cost-effective generation of genetic markers.

Genome sequence of the palaeopolyploid soybean.

Soybean (Glycine max) is one of the most important crop plants for seed protein and oil content, and for its capacity to fix atmospheric nitrogen through symbioses with soil-borne microorganisms. We sequenced the 1.1-gigabase genome by a whole-genome shotgun approach and integrated it with physical and high-density genetic maps to create a chromosome-scale draft sequence assembly. We predict 46,430 protein-coding genes, 70% more than Arabidopsis and similar to the poplar genome which, like soybean, is an ancient polyploid (palaeopolyploid). About 78% of the predicted genes occur in chromosome ends, which comprise less than one-half of the genome but account for nearly all of the genetic recombination. Genome duplications occurred at approximately 59 and 13 million years ago, resulting in a highly duplicated genome with nearly 75% of the genes present in multiple copies. The two duplication events were followed by gene diversification and loss, and numerous chromosome rearrangements. An accurate soybean genome sequence will facilitate the identification of the genetic basis of many soybean traits, and accelerate the creation of improved soybean varieties.