Bryon Sosinski

The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution

Rosaceae is the most important fruit-producing clade, and its key commercially relevant genera (Fragaria, Rosa, Rubus and Prunus) show broadly diverse growth habits, fruit types and compact diploid genomes. Peach, a diploid Prunus species, is one of the best genetically characterized deciduous trees. Here we describe the high-quality genome sequence of peach obtained from a completely homozygous genotype. We obtained a complete chromosome-scale assembly using Sanger whole-genome shotgun methods. We predicted 27,852 protein-coding genes, as well as noncoding RNAs. We investigated the path of peach domestication through whole-genome resequencing of 14 Prunus accessions. The analyses suggest major genetic bottlenecks that have substantially shaped peach genome diversity. Furthermore, comparative analyses showed that peach has not undergone recent whole-genome duplication, and even though the ancestral triplicated blocks in peach are fragmentary compared to those in grape, all seven paleosets of paralogs from the putative paleoancestor are detectable.

Sequence and genetic map of Meloidogyne hapla: A compact nematode genome for plant parasitism

We have established Meloidogyne hapla as a tractable model plant-parasitic nematode amenable to forward and reverse genetics, and we present a complete genome sequence. At 54 Mbp, M. hapla represents not only the smallest nematode genome yet completed, but also the smallest metazoan, and defines a platform to elucidate mechanisms of parasitism by what is the largest uncontrolled group of plant pathogens worldwide. The M. hapla genome encodes significantly fewer genes than does the free-living nematode Caenorhabditis elegans (most notably through a reduction of odorant receptors and other gene families), yet it has acquired horizontally from other kingdoms numerous genes suspected to be involved in adaptations to parasitism. In some cases, amplification and tandem duplication have occurred with genes suspected of being acquired horizontally and involved in parasitism of plants. Although M. hapla and C. elegans diverged >500 million years ago, many developmental and biochemical pathways, including those for dauer formation and RNAi, are conserved. Although overall genome organization is not conserved, there are areas of microsynteny that may suggest a primary biological function in nematodes for those genes in these areas. This sequence and map represent a wealth of biological information on both the nature of nematode parasitism of plants and its evolution.

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Abstract Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)n- and (CA)n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L.

Multiple Models for Rosaceae Genomics

The plant family Rosaceae consists of over 100 genera and 3,000 species that include many important fruit, nut, ornamental, and wood crops. Members of this family provide high-value nutritional foods and contribute desirable aesthetic and industrial products. Most rosaceous crops have been enhanced by human intervention through sexual hybridization, asexual propagation, and genetic improvement since ancient times, 4,000 to 5,000 B.C. Modern breeding programs have contributed to the selection and release of numerous cultivars having significant economic impact on the U.S. and world markets. In recent years, the Rosaceae community, both in the United States and internationally, has benefited from newfound organization and collaboration that have hastened progress in developing genetic and genomic resources for representative crops such as apple (Malus spp.), peach (Prunus spp.), and strawberry (Fragaria spp.). These resources, including expressed sequence tags, bacterial artificial chromosome libraries, physical and genetic maps, and molecular markers, combined with genetic transformation protocols and bioinformatics tools, have rendered various rosaceous crops highly amenable to comparative and functional genomics studies. This report serves as a synopsis of the resources and initiatives of the Rosaceae community, recent developments in Rosaceae genomics, and plans to apply newly accumulated knowledge and resources toward breeding and crop improvement.

Epigenomic consequences of immortalized plant cell suspension culture.

Plant cells grown in culture exhibit genetic and epigenetic instability. Using a combination of chromatin immunoprecipitation and DNA methylation profiling on tiling microarrays, we have mapped the location and abundance of histone and DNA modifications in a continuously proliferating, dedifferentiated cell suspension culture of Arabidopsis. We have found that euchromatin becomes hypermethylated in culture and that a small percentage of the hypermethylated genes become associated with heterochromatic marks. In contrast, the heterochromatin undergoes dramatic and very precise DNA hypomethylation with transcriptional activation of specific transposable elements (TEs) in culture. High throughput sequencing of small interfering RNA (siRNA) revealed that TEs activated in culture have increased levels of 21-nucleotide (nt) siRNA, sometimes at the expense of the 24-nt siRNA class. In contrast, TEs that remain silent, which match the predominant 24-nt siRNA class, do not change significantly in their siRNA profiles. These results implicate RNA interference and chromatin modification in epigenetic restructuring of the genome following the activation of TEs in immortalized cell culture.

Arabidopsis thaliana chromosome 4 replicates in two phases that correlate with chromatin state.

DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about replication programs in plants. We used flow cytometry and tiling microarrays to profile DNA replication of Arabidopsis thaliana chromosome 4 (chr4) during early, mid, and late S phase. Replication profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes the remaining euchromatin and essentially all of the heterochromatin. Termination zones were consistent between experiments, allowing us to define 163 putative replicons on chr4 that clustered into larger domains of predominately early or late replication. Early-replicating sequences, especially the initiation zones of early replicons, displayed a pattern of epigenetic modifications specifying an open chromatin conformation. Late replicons, and the termination zones of early replicons, showed an opposite pattern. Histone H3 acetylated on lysine 56 (H3K56ac) was enriched in early replicons, as well as the initiation zones of both early and late replicons. H3K56ac was also associated with expressed genes, but this effect was local whereas replication time correlated with H3K56ac over broad regions. The similarity of the replication profiles for early and mid S phase cells indicates that replication origin activation in euchromatin is stochastic. Replicon organization in Arabidopsis is strongly influenced by epigenetic modifications to histones and DNA. The domain organization of Arabidopsis is more similar to that in Drosophila than that in mammals, which may reflect genome size and complexity. The distinct patterns of association of H3K56ac with gene expression and early replication provide evidence that H3K56ac may be associated with initiation zones and replication origins.

Whole genome comparisons of Fragaria, Prunus and Malus reveal different modes of evolution between Rosaceous subfamilies

BackgroundRosaceae include numerous economically important and morphologically diverse species. Comparative mapping between the member species in Rosaceae have indicated some level of synteny. Recently the whole genome of three crop species, peach, apple and strawberry, which belong to different genera of the Rosaceae family, have been sequenced, allowing in-depth comparison of these genomes.ResultsOur analysis using the whole genome sequences of peach, apple and strawberry identified 1399 orthologous regions between the three genomes, with a mean length of around 100 kb. Each peach chromosome showed major orthology mostly to one strawberry chromosome, but to more than two apple chromosomes, suggesting that the apple genome went through more chromosomal fissions in addition to the whole genome duplication after the divergence of the three genera. However, the distribution of contiguous ancestral regions, identified using the multiple genome rearrangements and ancestors (MGRA) algorithm, suggested that the Fragaria genome went through a greater number of small scale rearrangements compared to the other genomes since they diverged from a common ancestor. Using the contiguous ancestral regions, we reconstructed a hypothetical ancestral genome for the Rosaceae 7 composed of nine chromosomes and propose the evolutionary steps from the ancestral genome to the extant Fragaria, Prunus and Malus genomes.ConclusionOur analysis shows that different modes of evolution may have played major roles in different subfamilies of Rosaceae. The hypothetical ancestral genome of Rosaceae and the evolutionary steps that lead to three different lineages of Rosaceae will facilitate our understanding of plant genome evolution as well as have a practical impact on knowledge transfer among member species of Rosaceae.

Synteny of Prunus and other model plant species

BackgroundFragmentary conservation of synteny has been reported between map-anchored Prunus sequences and Arabidopsis. With the availability of genome sequence for fellow rosid I members Populus and Medicago, we analyzed the synteny between Prunus and the three model genomes. Eight Prunus BAC sequences and map-anchored Prunus sequences were used in the comparison.ResultsWe found a well conserved synteny across the Prunus species – peach, plum, and apricot – and Populus using a set of homologous Prunus BACs. Conversely, we could not detect any synteny with Arabidopsis in this region. Other peach BACs also showed extensive synteny with Populus. The syntenic regions detected were up to 477 kb in Populus. Two syntenic regions between Arabidopsis and these BACs were much shorter, around 10 kb. We also found syntenic regions that are conserved between the Prunus BACs and Medicago. The array of synteny corresponded with the proposed whole genome duplication events in Populus and Medicago. Using map-anchored Prunus sequences, we detected many syntenic blocks with several gene pairs between Prunus and Populus or Arabidopsis. We observed a more complex network of synteny between Prunus-Arabidopsis, indicative of multiple genome duplication and subsequence gene loss in Arabidopsis.ConclusionOur result shows the striking microsynteny between the Prunus BACs and the genome of Populus and Medicago. In macrosynteny analysis, more distinct Prunus regions were syntenic to Populus than to Arabidopsis.

Starch self-processing in transgenic sweet potato roots expressing a hyperthermophilic α-amylase

Sweet potato is a major crop in the southeastern United States, which requires few inputs and grows well on marginal land. It accumulates large quantities of starch in the storage roots and has been shown to give comparable or superior ethanol yields to corn per cultivated acre in the southeast. Starch conversion to fermentable sugars (i.e., for ethanol production) is carried out at high temperatures and requires the action of thermostable and thermoactive amylolytic enzymes. These enzymes are added to the starch mixture impacting overall process economics. To address this shortcoming, the gene encoding a hyperthermophilic α‐amylase from Thermotoga maritima was cloned and expressed in transgenic sweet potato, generated by Agrobacterium tumefaciens‐mediated transformation, to create a plant with the ability to self‐process starch. No significant enzyme activity could be detected below 40°C, but starch in the transgenic sweet potato storage roots was readily hydrolyzed at 80°C. The transgene did not affect normal storage root formation. The results presented here demonstrate that engineering plants with hyperthermophilic glycoside hydrolases can facilitate cost effective starch conversion to fermentable sugars. Furthermore, the use of sweet potato as an alternative near‐term energy crop should be considered.

Rosaceaous Genome Sequencing: Perspectives and Progress

The long-term goal of plant genomics is to identify, isolate and determine the function of plant genes that are associated with both vegetative and reproductive phenotypes. Most phenotypes require the coordinated activity and regulatory control of suites of genes over time and in precise positions within the plant. Until recently, the idea of establishing a comprehensive approach to isolate and characterize all the genes involved in any complex phenotype was a daunting one, and oftentimes it has been necessary to perform whole genome sequencing to obtain all of the gene sequences. The sequence of several plant genomes have been generated including the Arabidopsis, poplar and rice genomes, with the model legume Medicago and sorghum well underway. In addition, large amounts of expressed sequence tag (EST) information are being obtained for many other plants, including rosaceaous plants. The advances in genomics, informatics, and phylogenetics have been developed and refined by these reference projects, to the point that it is now thought that, in many cases, the vast majority of genes of a plant can be identified without the complete genome sequence, however the EST approach to gene identification does not provide valuable information regarding promoters and other non-coding regulatory elements. One of the first eukaryotic genomes to be completely sequenced was that of the small mustard species Arabidopsis thaliana. During the past decade, Arabidopsis has emerged as one of the most widely used model organisms for studying the biology of higher plants. Its genome was chosen for sequencing because it is highly compact, about 125 Mb, with little interspersed repetitive DNA (Arabidopsis Genome Initiative, 2000). However, since Arabidopsis is rather distantly related to the cereal crops that provide the bulk of the world food supply, the genome of rice has also been sequenced. Rice was chosen because, in addition to its importance as a food source for about one quarter of the human population, it has one of