Comparative genome analysis reveals extensive conservation of genome organisation for Arabidopsis thaliana and Capsella rubella

Genome colinearity has been studied for two closely related diploid species of the Brassicaceae family, Arabidopsis thaliana and Capsella rubella. Markers mapping to chromosome 4 of A. thaliana were found on two linkage groups in Capsella and colinear segments spanning more than 10 cM were revealed. Detailed analysis of a 60 kbp region in A. thaliana and its counterpart in C. rubella showed virtually complete conservation of gene repertoire, order and orientation. The comparison of orthologous genes revealed very similar exon-intron structures and sequence identities of 90% or more were found for exon sequences. This extensive genome colinearity at the genetic and molecular level allows the efficient transfer of data from the well-studied A. thaliana genome to other species in the Brassicaceae family, substantially facilitating genome analysis studies for species of this family.     

Comparative genetic mapping in Boechera stricta, a close relative of Arabidopsis

The angiosperm family Brassicaceae contains both the research model Arabidopsis (Arabidopsis thaliana) and the agricultural genus Brassica. Comparative genomics in the Brassicaceae has largely focused on direct comparisons between Arabidopsis and the species of interest. However, the reduced genome size and chromosome number (n = 5) of Arabidopsis complicates comparisons. Arabidopsis shows extensive genome and chromosome reshuffling compared to its close relatives Arabidopsis lyrata and Capsella rubella, both with n = 8. To facilitate comparative genomics across the Brassicaceae we recently outlined a system of 24 conserved chromosomal blocks based on their positions in an ancestral karyotype of n = 8, rather than by their position in Arabidopsis. In this report we use this system as a tool to understand genome structure and evolution in Boechera stricta (n = 7). B. stricta is a diploid, sexual, and highly self-fertilizing species occurring in mostly montane regions of western North America. We have created an F(2) genetic map of B. stricta based on 192 individuals scored at 196 microsatellite and candidate gene loci. Single-nucleotide polymorphism genotyping of 94 of the loci was done simultaneously using an Illumina bead array. The total map length is 725.8 cM, with an average marker spacing of 3.9 cM. There are no gaps greater than 19.3 cM. The chromosomal reduction from n = 8 to n = 7 and other genomic changes in B. stricta likely involved a pericentric inversion, a chromosomal fusion, and two reciprocal translocations that are easily visualized using the genomic blocks. Our genetic map will facilitate the analysis of ecologically relevant quantitative variation in Boechera. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers DU 667459 to DU 708532.     

Geographic patterns of microsatellite variation in Boechera stricta, a close relative of Arabidopsis

The genus Boechera is a widespread North American group with great potential for studies of ecology and evolution: Boechera is closely related to Arabidopsis and exhibits different ecological and reproductive strategies. Boechera stricta (previously Arabis drummondii) is a morphologically and genetically well-defined, perennial crucifer species. Fifteen natural populations of diploid individuals from the Rocky Mountains were analysed using 21 microsatellite loci. In accordance with our expectation for this predominately inbreeding species, a high F IS value (0.89) was observed. Furthermore, populations of B. stricta were highly differentiated, as indicated by F ST = 0.56. Three clusters were identified using structure- the majority of populations belonged to either the Northern or Southern cluster. Together, the north-south partitioning and evenness of genetic variation across the two clusters suggested multiple refugia for this perennial herb in the Rocky Mountains. Pleistocene glaciation, together with the topographically and climatologically heterogeneous cordillera, has profoundly influenced the genetic architecture of B. stricta. Genetic population structure was also influenced by relatively recent genome admixture at two levels: within species (involving individuals from the Northern and Southern clusters) and between species (with the hybridization of B. stricta and Boechera holboellii). This complexity of population structure at presumably neutral microsatellite loci located throughout the genome in B. stricta provides a baseline against which to test whether functional genetic variation is undergoing local adaptive evolution throughout the natural species range.     

Local coexpression domains in the genome of rice show no microsynteny with Arabidopsis domains

Plant responses to abiotic stress are determined both by the severity of the stress as well as the metabolic status of the plant. Abscisic acid (ABA) is a key component in integrating these various signals and controlling downstream stress responses. By screening for plants with decreased RD29A:LUC expression, we isolated two alleles, glutamate:glyoxylate transferase1-1 (ggt1-1) and ggt1-2, of a mutant with altered ABA sensitivity. In addition to reduced ABA induction of RD29A, ggt1-1 was altered in ABA and stress regulation of Δ ¹ -pyrroline-5-carboxylate synthase, proline dehydrogenase and 9-cis-epoxycarotenoid dioxygenase 3, which encode enzymes involved in Pro and ABA metabolsim, respectively. ggt1-1 also had altered ABA and Pro contents after stress or ABA treatments while root growth and leaf water loss were relatively unaffected. A light-dependent increase in H₂O₂ accumulation was observed in ggt1-1 consistent with the previously characterized role of GGT1 in photorespiration. Treatment with exogenous H₂O₂, as well as analysis of a mutant in nucleoside diphosphate kinase 2 which also had increased H₂O₂ content but is not involved in photorespiration or amino acid metabolism, demonstrated that the greater ABA stimulation of Pro accumulation in these mutants was caused by altered H₂O₂ content as opposed to other metabolic changes. The results suggest that metabolic changes that alter H₂O₂ levels can affect both ABA accumulation and ABA sensitivity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Life-history QTLS and natural selection on flowering time in Boechera stricta, a perennial relative of Arabidopsis

Plants must precisely time flowering to capitalize on favorable conditions. Although we know a great deal about the genetic basis of flowering phenology in model species under controlled conditions, the genetic architecture of this ecologically important trait is poorly understood in nonmodel organisms. Here, we evaluated the transition from vegetative growth to flowering in Boechera stricta, a perennial relative of Arabidopsis thaliana. We examined flowering time QTLs using 7920 recombinant inbred individuals, across seven laboratory and field environments differing in vernalization, temperature, and photoperiod. Genetic and environmental factors strongly influenced the transition to reproduction. We found directional selection for earlier flowering in the field. In the growth chamber experiment, longer winters accelerated flowering, whereas elevated ambient temperatures delayed flowering. Our analyses identified one large effect QTL (nFT), which influenced flowering time in the laboratory and the probability of flowering in the field. In Montana, homozygotes for the native allele at nFT showed a selective advantage of 6.6%. Nevertheless, we found relatively low correlations between flowering times in the field and the growth chambers. Additionally, we detected flowering-related QTLs in the field that were absent across the full range of laboratory conditions, thus emphasizing the need to conduct experiments in natural environments.     

Characterizing the citrus cultivar Carrizo genome through 454 shotgun sequencing

The citrus cultivar Carrizo is the single most important rootstock to the US citrus industry and has resistance or tolerance to a number of major citrus diseases, including citrus tristeza virus, foot rot, and Huanglongbing (HLB, citrus greening). A Carrizo genomic sequence database providing approximately 3.5×genome coverage (haploid genome size approximately 367 Mb) was populated through 454 GS FLX shotgun sequencing. Analysis of the repetitive DNA fraction indicated a total interspersed repeat fraction of 36.5%. Assembly and characterization of abundant citrus Ty3/gypsy elements revealed a novel type of element containing open reading frames encoding a viral RNA-silencing suppressor protein (RNA binding protein, rbp) and a plant cytokinin riboside 5′-monophosphate phosphoribohydrolase-related protein (LONELY GUY, log). Similar gypsy elements were identified in the Populus trichocarpa genome. Gene-coding region analysis indicated that 24.4% of the nonrepetitive reads contained genic regions. The depth of genome coverage was sufficient to allow accurate assembly of constituent genes, including a putative phloem-expressed gene. The development of the Carrizo database (http://citrus.pw.usda.gov/) will contribute to characterization of agronomically significant loci and provide a publicly available genomic resource to the citrus research community.     

Whole genome shotgun sequencing of one Colombian clinical isolate of Mycobacterium tuberculosis reveals DosR regulon gene deletions

Several genomes of different Mycobacterium tuberculosis isolates have been completely sequenced around the world. The genomic information obtained have shown higher diversity than originally thought and specific adaptations to different human populations. Within this work, we sequenced the genome of one Colombian M.?tuberculosis virulent isolate. Genomic comparison against the reference genome of H37Rv and other strains showed multiple deletion and insertions that ranged between a few bases to thousands. Excluding PPE and PG-PGRS genes, 430 proteins present changes in at least 1 amino acid. Also, novel positions of the IS6110 mobile element were identified. This isolate is also characterized by a large genomic deletion of 3.6?kb, leading to the loss and modification of the dosR regulon genes, Rv1996 and Rv1997. To our knowledge, this is the first report of the genome sequence of a Latin American M.?tuberculosis clinical isolate.     

Multilocus Patterns of Nucleotide Diversity, Population Structure and Linkage Disequilibrium in Boechera stricta, a Wild Relative of Arabidopsis

Information about polymorphism, population structure, and linkage disequilibrium (LD) is crucial for association studies of complex trait variation. However, most genomewide studies have focused on model systems, with very few analyses of undisturbed natural populations. Here, we sequenced 86 mapped nuclear loci for a sample of 46 genotypes of Boechera stricta and two individuals of B. holboellii, both wild relatives of Arabidopsis. Isolation by distance was significant across the species range of B. stricta, and three geographic groups were identified by structure analysis, principal coordinates analysis, and distance-based phylogeny analyses. The allele frequency spectrum indicated a genomewide deviation from an equilibrium neutral model, with silent nucleotide diversity averaging 0.004. LD decayed rapidly, declining to background levels in ~10 kb or less. For tightly linked SNPs separated by <1 kb, LD was dependent on the reference population. LD was lower in the specieswide sample than within populations, suggesting that low levels of LD found in inbreeding species such as B. stricta, Arabidopsis thaliana, and barley may result from broad geographic sampling that spans heterogeneous genetic groups. Finally, analyses also showed that inbreeding B. stricta and A. thaliana have ~45% higher recombination per kilobase than outcrossing A. lyrata.     

Fine mapping in tomato using microsynteny with the Arabidopsis genome: the Diageotropica (Dgt) locus

Background  

The Arabidopsis thaliana genome sequence provides a catalog of reference genes applicable to comparative microsynteny analysis of other species, facilitating map-based cloning in economically important crops. We have applied such an analysis to the tomato expressed sequence tag (EST) database to expedite high-resolution mapping of the Diageotropica (Dgt) gene within the distal end of chromosome 1 in tomato (Lycopersicon esculentum).     

Systematic inference of copy-number genotypes from personal genome sequencing data reveals extensive olfactory receptor gene content diversity

Copy-number variations (CNVs) are widespread in the human genome, but comprehensive assignments of integer locus copy-numbers (i.e., copy-number genotypes) that, for example, enable discrimination of homozygous from heterozygous CNVs, have remained challenging. Here we present CopySeq, a novel computational approach with an underlying statistical framework that analyzes the depth-of-coverage of high-throughput DNA sequencing reads, and can incorporate paired-end and breakpoint junction analysis based CNV-analysis approaches, to infer locus copy-number genotypes. We benchmarked CopySeq by genotyping 500 chromosome 1 CNV regions in 150 personal genomes sequenced at low-coverage. The assessed copy-number genotypes were highly concordant with our performed qPCR experiments (Pearson correlation coefficient 0.94), and with the published results of two microarray platforms (95-99% concordance). We further demonstrated the utility of CopySeq for analyzing gene regions enriched for segmental duplications by comprehensively inferring copy-number genotypes in the CNV-enriched >800 olfactory receptor (OR) human gene and pseudogene loci. CopySeq revealed that OR loci display an extensive range of locus copy-numbers across individuals, with zero to two copies in some OR loci, and two to nine copies in others. Among genetic variants affecting OR loci we identified deleterious variants including CNVs and SNPs affecting ~15% and ~20% of the human OR gene repertoire, respectively, implying that genetic variants with a possible impact on smell perception are widespread. Finally, we found that for several OR loci the reference genome appears to represent a minor-frequency variant, implying a necessary revision of the OR repertoire for future functional studies. CopySeq can ascertain genomic structural variation in specific gene families as well as at a genome-wide scale, where it may enable the quantitative evaluation of CNVs in genome-wide association studies involving high-throughput sequencing.     

Progress in Arabidopsis genome sequencing and functional genomics

Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.     

Modeling the feasibility of whole genome shotgun sequencing using a pairwise end strategy

In pairwise end sequencing, sequences are determined from both ends of random subclones derived from a DNA target. Sufficiently similar overlapping end sequences are identified and grouped into contigs. When a clone's paired end sequences fall in different contigs, the contigs are connected together to form scaffolds. Increasingly, the goals of pairwise strategies are large and highly repetitive genomic targets. Here, we consider large-scale pairwise strategies that employ mixtures of subclone sizes. We explore the properties of scaffold formation within a hybrid theory/simulation mathematical model of a genomic target that contains many repeat families. Using this model, we evaluate problems that may arise, such as falsely linked end sequences (due either to random matches or to homologous repeats) and scaffolds that terminate without extending the full length of the target. We illustrate our model with an exploration of a strategy for sequencing the human genome. Our results show that, for a strategy that generates 10-fold sequence coverage derived from the ends of clones ranging in length from 2 to 150 kb, using an appropriate rule for detecting overlaps, we expect few false links while obtaining a single scaffold extending the length of each chromosome.     

Comparison of C. elegans and C. briggsae genome sequences reveals extensive conservation of chromosome organization and synteny

To determine whether the distinctive features of Caenorhabditis elegans chromosomal organization are shared with the C. briggsae genome, we constructed a single nucleotide polymorphism–based genetic map to order and orient the whole genome shotgun assembly along the six C. briggsae chromosomes. Although these species are of the same genus, their most recent common ancestor existed 80–110 million years ago, and thus they are more evolutionarily distant than, for example, human and mouse. We found that, like C. elegans chromosomes, C. briggsae chromosomes exhibit high levels of recombination on the arms along with higher repeat density, a higher fraction of intronic sequence, and a lower fraction of exonic sequence compared with chromosome centers. Despite extensive intrachromosomal rearrangements, 1:1 orthologs tend to remain in the same region of the chromosome, and colinear blocks of orthologs tend to be longer in chromosome centers compared with arms. More strikingly, the two species show an almost complete conservation of synteny, with 1:1 orthologs present on a single chromosome in one species also found on a single chromosome in the other. The conservation of both chromosomal organization and synteny between these two distantly related species suggests roles for chromosome organization in the fitness of an organism that are only poorly understood presently.     

Synteny between Arabidopsis thaliana and rice at the genome level: a tool to identify conservation in the ongoing rice genome sequencing project

BLASTX alignment between 189.5 Mb of rice genomic sequence and translated Arabidopsis thaliana annotated coding sequences (CDS) identified 60 syntenic regions involving 4–22 rice orthologs covering ≤3.2 cM (centiMorgan). Most regions are <3 cM in length. A detailed and updated version of a table representing these regions is available on our web site. Thirty-five rice loci match two distinct A.thaliana loci, as expected from the duplicated nature of the A.thaliana genome. One A.thaliana locus matches two distinct rice regions, suggesting that rice chromosomal sequence duplications exist. A high level of rearrangement characterizing the 60 syntenic regions illustrates the ancient nature of the speciation between A.thaliana and rice. The apparent reduced level of microcollinearity implies the dispersion to new genomic locations, via transposon activity, of single or small clusters of genes in the rice genome, which represents a significant additional effector of plant genome evolution.     

Plasticity of plant defense and its evolutionary implications in wild populations of Boechera stricta

Phenotypic plasticity is thought to impact evolutionary trajectories by shifting trait values in a direction that is either favored by natural selection (“adaptive” plasticity) or disfavored (“nonadaptive” plasticity). However, it is unclear how commonly each of these types of plasticity occurs in natural populations. To answer this question, we measured glucosinolate defensive chemistry and reproductive fitness in over 1500 individuals of the wild perennial mustard Boechera stricta, planted in four common gardens across central Idaho, United States. Glucosinolate profiles—including total glucosinolate concentration as well as the relative abundances and overall diversity of different compounds—were strongly plastic both among habitats and within habitats. Patterns of glucosinolate plasticity varied greatly among genotypes. Plasticity among sites was predicted to affect fitness in 27.1% of cases; more often than expected by chance, glucosinolate plasticity increased rather than decreased relative fitness. In contrast, we found no evidence for within‐habitat selection on glucosinolate reaction norm slopes (i.e., plasticity along a continuous environmental gradient). Together, our results indicate that glucosinolate plasticity may improve the ability of B. stricta populations to persist after migration to new habitats.     

Comparative physical mapping reveals features of microsynteny between Glycine max, Medicago truncatula, and Arabidopsis thaliana.

To gain insight into genomic relationships between soybean (Glycine max) and Medicago truncatula, eight groups of bacterial artificial chromosome (BAC) contigs, together spanning 2.60 million base pairs (Mb) in G. max and 1.56 Mb in M. truncatula, were compared through high-resolution physical mapping combined with sequence and hybridization analysis of low-copy BAC ends. Cross-hybridization among G. max and M. truncatula contigs uncovered microsynteny in six of the contig groups and extensive microsynteny in three. Between G. max homoeologous (within genome duplicate) contigs, 85% of coding and 75% of noncoding sequences were conserved at the level of cross-hybridization. By contrast, only 29% of sequences were conserved between G. max and M. truncatula, and some kilobase-scale rearrangements were also observed. Detailed restriction maps were constructed for 11 contigs from the three highly microsyntenic groups, and these maps suggested that sequence order was highly conserved between G. max duplicates and generally conserved between G. max and M. truncatula. One instance of homoeologous BAC contigs in M. truncatula was also observed and examined in detail. A sequence similarity search against the Arabidopsis thaliana genome sequence identified up to three microsyntenic regions in A. thaliana for each of two of the legume BAC contig groups. Together, these results confirm previous predictions of one recent genome-wide duplication in G. max and suggest that M. truncatula also experienced ancient large-scale genome duplications.