Origin and diversity of the wild cottons (Gossypium hirsutum) of Mound Key, Florida.

Elucidating genetic diversity within wild forms of modern crops is essential for understanding domestication and the possibilities of wild germplasm utilization. Gossypium hirsutum is a predominant source of natural plant fibers and the most widely cultivated cotton species. Wild forms of G. hirsutum are challenging to distinguish from feral derivatives, and truly wild populations are uncommon. Here we characterize a population from Mound Key Archaeological State Park, Florida using genome-wide SNPs extracted from 25 individuals over three sites. Our results reveal that this population is genetically dissimilar from other known wild, landrace, and domesticated cottons, and likely represents a pocket of previously unrecognized wild genetic diversity. The unexpected level of divergence between the Mound Key population and other wild cotton populations suggests that the species may harbor other remnant and genetically distinct populations that are geographically scattered in suitable habitats throughout the Caribbean. Our work thus has broader conservation genetic implications and suggests that further exploration of natural diversity in this species is warranted.

Innovations in double digest restriction-site associated DNA sequencing (ddRAD-Seq) method for more efficient SNP identification.

We present an improved ddRAD-Seq protocol for identifying single nucleotide polymorphisms (SNPs). It utilizes selected restriction enzyme digestion fragments, quick acting ligases that are neutral with the restriction enzyme buffer eliminating buffer exchange steps, and adapters designed to be compatible with Illumina index primers. Library amplification and barcoding are completed in one PCR step, and magnetic beads are used to purify the genomic fragments from the ligation and library generation steps. Our protocol increases the efficiency and decreases the time to complete a ddRAD-Seq experiment. To demonstrate its utility, we compared SNPs from our protocol with those from whole genome resequencing data from Gossypium herbaceum and Gossypium arboreum. Principal component analysis demonstrated that the variability of the combined data was explained by the genotype (PC1) and methodology applied (PC2). Phylogenetic analysis showed that the SNPs from our method clustered with SNPs from the resequencing data of the corresponding genotype. Sequence alignments illustrated that for homozygous loci, more than 90% of the SNPs from the resequencing data were discovered by our method. Our analyses suggest that our ddRAD-Seq method is reliable in identifying SNPs suitable for phylogenetic and association genetic studies while reducing cost and time over known methods.

Transcriptome analysis of the 2,4-dichlorophenoxyacetic acid (2,4-D)-tolerant cotton chromosome substitution line CS-B15sh and its susceptible parental lines G. hirsutum L. cv. Texas Marker-1 and G. barbadense L. cv. Pima 379.

The cotton chromosome substitution line, CS-B15sh, exhibits 41% lower injury from 2,4-D when applied at the field recommended rate of 1.12 kg ae ha-1 (1×) than does Texas Marker-1 (TM-1). CS-B15sh was developed in the genetic background of Gossypium hirsutum L. cv TM-1 and has chromosome introgression on the short arm of chromosome 15 from Gossypium barbadense L. cv. Pima 379. In a previous experiment, we observed reduced translocation of [14C]2,4-D outside the treated leaf tissue in CS-B15sh, which contrasted with an increased translocation of the herbicide in the tissues above and below the treated leaf in TM-1. Our results indicate a potential 2,4-D tolerance mechanism in CS-B15sh involving altered movement of 2,4-D. Here, we used RNA sequencing (RNA-seq) to determine the differential expression of genes between 2,4-D-challenged and control plants of the tolerant (CS-B15sh) and susceptible lines (TM-1 and Pima 379). Several components of the 2,4-D/auxin-response pathway-including ubiquitin E3 ligase, PB1|AUX/IAA, ARF transcription factors, and F-box proteins of the SCFTIR1/AFB complex-were upregulated with at least threefold higher expression in TM-1 compared with CS-B15sh, while both Pima 379 and TM-1 showed the same fold change expression for PB1|AUX/IAA mRNA. Some genes associated with herbicide metabolism, including flavin monooxygenase (Gohir.A01G174100) and FAD-linked oxidase (Gohir.D06G002600), exhibited at least a twofold increase in CS-B15sh than in TM-1 (the gene was not expressed in Pima 379), suggesting a potential relationship between the gene's expression and 2,4-D tolerance. It is interesting to note that glutathione S-transferase was differentially expressed in both CS-B15sh and Pima 379 but not in TM-1, while cytochrome P450 and other genes involved in the oxidation-reduction process were significantly expressed only in CS-B15sh in response to 2,4-D. Gene set enrichment analysis on the union DEGs of the three cotton genotypes revealed the depletion of transcripts involved in photosynthesis and enrichment of transcripts involved in ABA response and signaling.

Exploring the loblolly pine (Pinus taeda L.) genome by BAC sequencing and Cot analysis.

Loblolly pine (LP; Pinus taeda L.) is an economically and ecologically important tree in the southeastern U.S. To advance understanding of the loblolly pine (LP; Pinus taeda L.) genome, we sequenced and analyzed 100 BAC clones and performed a Cot analysis. The Cot analysis indicates that the genome is composed of 57, 24, and 10% highly-repetitive, moderately-repetitive, and single/low-copy sequences, respectively (the remaining 9% of the genome is a combination of fold back and damaged DNA). Although single/low-copy DNA only accounts for 10% of the LP genome, the amount of single/low-copy DNA in LP is still 14 times the size of the Arabidopsis genome. Since gene numbers in LP are similar to those in Arabidopsis, much of the single/low-copy DNA of LP would appear to be composed of DNA that is both gene- and repeat-poor. Macroarrays prepared from a LP bacterial artificial chromosome (BAC) library were hybridized with probes designed from cell wall synthesis/wood development cDNAs, and 50 of the "targeted" clones were selected for further analysis. An additional 25 clones were selected because they contained few repeats, while 25 more clones were selected at random. The 100 BAC clones were Sanger sequenced and assembled. Of the targeted BACs, 80% contained all or part of the cDNA used to target them. One targeted BAC was found to contain fungal DNA and was eliminated from further analysis. Combinations of similarity-based and ab initio gene prediction approaches were utilized to identify and characterize potential coding regions in the 99 BACs containing LP DNA. From this analysis, we identified 154 gene models (GMs) representing both putative protein-coding genes and likely pseudogenes. Ten of the GMs (all of which were specifically targeted) had enough support to be classified as intact genes. Interestingly, the 154 GMs had statistically indistinguishable (α = 0.05) distributions in the targeted and random BAC clones (15.18 and 12.61 GM/Mb, respectively), whereas the low-repeat BACs contained significantly fewer GMs (7.08 GM/Mb). However, when GM length was considered, the targeted BACs had a significantly greater percentage of their length in GMs (3.26%) when compared to random (1.63%) and low-repeat (0.62%) BACs. The results of our study provide insight into LP evolution and inform ongoing efforts to produce a reference genome sequence for LP, while characterization of genes involved in cell wall production highlights carbon metabolism pathways that can be leveraged for increasing wood production.

Bacterial Diversity Patterns Differ in Soils Developing in Sub-tropical and Cool-Temperate Ecosystems.

Microbial diversity patterns have been surveyed in many different soils and ecosystems, but we are unaware of studies comparing similar soils developing from similar parent materials in contrasting climates. In 2008, developmental chronosequences with ages ranging from 105 to 500,000 years across Georgia (GA) and Michigan (MI) were studied to investigate how bacterial community composition and diversity change as a result of local environmental gradients that develop during pedogenesis. Geographic factors were studied between and within locations spanning two scales: (1) regionally between 0.1 and 50 and (2) ∼1700 km apart. The diversity was surveyed using high-throughput pyrosequencing, and variance partitioning was used to describe the effects of spatial, environmental, and spatio-environmental factors on bacterial community composition. At the local scale, variation in bacterial communities was most closely related to environmental factors (rM = 0.59, p = 0.0001). There were differences in bacterial communities between the two locations, indicating spatial biogeography. Estimates of bacterial diversity were much greater in MI (numbers of OTU, ACE, and Chao1) and remained 2-3× greater in MI than GA after removing the effect of soil properties. The large differences in diversity between geographically separated bacterial communities in different climates need further investigation. It is not known if the rare members of the community, which contributed to greater bacterial diversity in GA relative to MI, play an important role in ecosystem function but has been hypothesized to play a role in ecosystem resiliency, resistance, and stability. Further research on the link between bacterial diversity and spatial variability related to climate needs further investigation.

Transcriptomic dissection of the rice-Burkholderia glumae interaction.

BACKGROUND: Bacterial panicle blight caused by the bacterium Burkholderia glumae is an emerging disease of rice in the United States. Not much is known about this disease, the disease cycle or any source of disease resistance. To understand the interaction between rice and Burkholderia glumae, we used transcriptomics via next-generation sequencing (RNA-Seq) and bioinformatics to identify differentially expressed transcripts between resistant and susceptible interactions and formulate a model for rice resistance to the disease. RESULTS: Using inoculated young seedlings as sample tissues, we identified unique transcripts involved with resistance to bacterial panicle blight, including a PIF-like ORF1 and verified differential expression of some selected genes using qRT-PCR. These transcripts, which include resistance genes of the NBS-LRR type, kinases, transcription factors, transporters and expressed proteins with functions that are not known, have not been reported in other pathosystems including rice blast or bacterial blight. Further, functional annotation analysis reveals enrichment of defense response and programmed cell death (biological processes); ATP and protein binding (molecular functions); and mitochondrion-related (cell component) transcripts in the resistant interaction. CONCLUSION: Taken together, we formulated a model for rice resistance to bacterial panicle blight that involves an activation of previously unknown resistance genes and their activation partners upon challenge with B. glumae. Other interesting findings are that 1) though these resistance transcripts were up-regulated upon inoculation in the resistant interaction, some of them were already expressed in the water-inoculated control from the resistant genotype, but not in the water- and bacterium-inoculated samples from the susceptible genotype; 2) rice may have co-opted an ORF that was previously a part of a transposable element to aid in the resistance mechanism; and 3) resistance may have existed immediately prior to rice domestication.

Adventures in the enormous: a 1.8 million clone BAC library for the 21.7 Gb genome of loblolly pine.

Loblolly pine (LP; Pinus taeda L.) is the most economically important tree in the U.S. and a cornerstone species in southeastern forests. However, genomics research on LP and other conifers has lagged behind studies on flowering plants due, in part, to the large size of conifer genomes. As a means to accelerate conifer genome research, we constructed a BAC library for the LP genotype 7-56. The LP BAC library consists of 1,824,768 individually-archived clones making it the largest single BAC library constructed to date, has a mean insert size of 96 kb, and affords 7.6X coverage of the 21.7 Gb LP genome. To demonstrate the efficacy of the library in gene isolation, we screened macroarrays with overgos designed from a pine EST anchored on LP chromosome 10. A positive BAC was sequenced and found to contain the expected full-length target gene, several gene-like regions, and both known and novel repeats. Macroarray analysis using the retrotransposon IFG-7 (the most abundant repeat in the sequenced BAC) as a probe indicates that IFG-7 is found in roughly 210,557 copies and constitutes about 5.8% or 1.26 Gb of LP nuclear DNA; this DNA quantity is eight times the Arabidopsis genome. In addition to its use in genome characterization and gene isolation as demonstrated herein, the BAC library should hasten whole genome sequencing of LP via next-generation sequencing strategies/technologies and facilitate improvement of trees through molecular breeding and genetic engineering. The library and associated products are distributed by the Clemson University Genomics Institute (www.genome.clemson.edu).

Empirical comparison of ab initio repeat finding programs.

Identification of dispersed repetitive elements can be difficult, especially when elements share little or no homology with previously described repeats. Consequently, a growing number of computational tools have been designed to identify repetitive elements in an ab initio manner, i.e. without using prior sequence data. Here we present the results of side-by-side evaluations of six of the most widely used ab initio repeat finding programs. Using sequence from rice chromosome 12, tools were compared with regard to time requirements, ability to find known repeats, utility in identifying potential novel repeats, number and types of repeat elements recognized and compactness of family descriptions. The study reveals profound differences in the utility of the tools with some identifying virtually their entire substrate as repetitive, others making reasonable estimates of repetition, and some missing almost all repeats. Of note, even when tools recognized similar numbers of repeats they often showed marked differences in the nature and number of repeat families identified. Within the context of this comparative study, ReAS and RepeatScout showed the most promise in analysis of sequence reads and assembled genomic regions, respectively. Our results should help biologists identify the program(s), if any, that is best suited for their needs.

Is catalase activity one of the factors associated with maize resistance to Aspergillus flavus?

Plant responses to biotic and abiotic stresses are usually accompanied by the release of reactive oxygen species including hydrogen peroxide. Hydrogen peroxide plays a direct role in defense and is involved in many signal transduction pathways that lead to the proliferation of other defenses. Because catalase helps to maintain reactive oxygen homeostasis during biotic and abiotic stress, its activity was measured in various cob tissues during maize ear development. Catalase activity was determined in immature and mature embryos, pericarp, and rachis tissues of maize lines that are resistant and susceptible to Aspergillus flavus infection. The effect of fungal inoculation on catalase activity was also measured. Over two years of field experimentation, a correlation was observed between resistance and the level of catalase-specific activity in immature embryos, which was significantly higher in resistant lines (P < 0.0001). Furthermore, catalase activity in the resistant lines was significantly higher in immature embryos from inoculated ears (P = 0.0199). No correlation was observed between resistance and catalase activity in other ear tissues. Levels of hydrogen peroxide, the catalase substrate, and salicylic acid in the embryo were also determined. The resistant lines showed lower levels of H2O2 (P < 0.0001) and higher levels of salicylic acid (P < 0.0001) as compared with the susceptible lines. Catalase 3 was sequenced from the aflatoxin-resistant (Mp313E) and susceptible (SC212m) inbreds. The predicted amino acid sequence indicated that there was a 20-aa deletion in the resistant inbred that might affect enzymatic activity. Unlike many plant-pathogen interactions, it appears that lowering H2O2 levels helps to prevent A. flavus infection and subsequent aflatoxin accumulation.