A genotype × environment experiment reveals contrasting response strategies to drought between populations of a keystone species (Artemisia tridentata; Asteraceae).

Western North America has been experiencing persistent drought exacerbated by climate change for over two decades. This extreme climate event is a clear threat to native plant communities. Artemisia tridentata is a keystone shrub species in western North America and is threatened by climate change, urbanization, and wildfire. A drought Genotype × Environment (G × E) experiment was conducted to assess phenotypic plasticity and differential gene expression in A. tridentata. The G × E experiment was performed on diploid A. tridentata seedlings from two populations (one from Idaho, USA and one from Utah, USA), which experience differing levels of drought stress during the summer months. Photosynthetic data, leaf temperature, and gene expression levels were compared between treatments and populations. The Utah population maintained higher photosynthetic rates and photosynthetic efficiency than the Idaho population under drought stress. The Utah population also exhibited far greater transcriptional plasticity than the Idaho population and expressed genes of response pathways distinct from those of the Idaho population. Populations of A. tridentata differ greatly in their drought response pathways, likely due to differences in response pathways that have evolved under distinct climatic regimes. Epigenetic processes likely contribute to the observed differences between the populations.

Four Invasive Plant Species in Southwest Saudi Arabia Have Variable Effects on Soil Dynamics.

Predicting the direction and magnitude of change in soil dynamics caused by invasive plant species has proven to be difficult because these changes are often reported to be species- and habitat-specific. This study was conducted to determine changes in three soil properties, eight soil ions, and seven soil microelements under established stands of four invasive plants, Prosopis juliflora, Ipomoea carnea, Leucaena leucocephala, and Opuntia ficus-indica. Soil properties, ions, and microelements were measured in sites invaded by these four species in southwest Saudi Arabia, and these values were compared to the results for the same 18 parameters from adjacent sites with native vegetation. Because this study was conducted in an arid ecosystem, we predict that these four invasive plants will significantly alter the soil properties, ions, and microelements in the areas they invaded. While the soils of sites with the four invasive plant species generally had higher values for soil properties and ions compared to sites with native vegetation, in most instances these differences were not statistically significant. However, the soils within sites invaded by I. carnea, L. leucocephala, and P. juliflora had statistically significant differences for some soil parameters. For sites invaded by O. puntia ficus-indica, no soil properties, ions, or microelements were significantly different compared to adjacent sites with native vegetation. Sites invaded by the four plant species generally exhibited differences in the 11 soil properties, but in no instance were these differences statistically significant. All three soil properties and one soil ion (Ca) were significantly different across the four stands of native vegetation. For the seven soil microelements, significantly different values were detected for Co and Ni, but only among stands of the four invasive plant species. These results indicate that the four invasive plant species altered soil properties, ions, and microelements, but for most of the parameters we assessed, not significantly. Our results do not support our initial prediction, but are in general agreement with previous published findings, which indicate that the effects of invasive plants on soil dynamics vary idiosyncratically among invasive species and among invaded habitats.

Suppression of Seedling Survival and Recruitment of the Invasive Tree Prosopis juliflora in Saudi Arabia through Its Own Leaf Litter: Greenhouse and Field Assessments.

Many studies have focused on how leaf litter depth affects seed germination and seedling growth because the seedling stage is the most vulnerable portion of a plant's life cycle. Invasive plants with the most severe ecological consequences are those that modify ecosystems, and this can occur through the formation of thick litter layers which can suppress the emergence, survival, and recruitment of native plant seedlings; in addition, in some cases, these litter layers can suppress invasive plant seedling recruitment. Prosopis juliflora is a thorny shrub that is native to arid and semi-arid portions of North America, parts of South America, and the Caribbean. It has invaded millions of hectares around the world, including Saudi Arabia. The objective of this study is to evaluate whether P. juliflora leaf litter reduces the recruitment of its own seedlings under greenhouse and field conditions in Saudi Arabia. In both the greenhouse and the field, the number of days to first emergence increased and germination percentage decreased with increasing litter depth. With the 1, 2, and 4 cm litter depth treatments, the number of viable seeds generally decreased, with no emergence, germination, or viable seeds detected for the 8 cm litter depth treatment. Results of this study reveal that increasing the depth of P. juliflora leaf litter suppresses the survival and recruitment of its own seedlings. Future search should assess the actual mechanisms through which P. juliflora seeds are suppressed, the role of allelopathic compounds in this process, and whether viable seeds are dormant and will persist in the soil seed bank.

Meta-Analysis Reveals Challenges and Gaps for Genome-to-Phenome Research Underpinning Plant Drought Response.

Severe drought conditions and extreme weather events are increasing worldwide with climate change, threatening the persistence of native plant communities and ecosystems. Many studies have investigated the genomic basis of plant responses to drought. However, the extent of this research throughout the plant kingdom is unclear, particularly among species critical for the sustainability of natural ecosystems. This study aimed to broaden our understanding of genome-to-phenome (G2P) connections in drought-stressed plants and identify focal taxa for future research. Bioinformatics pipelines were developed to mine and link information from databases and abstracts from 7730 publications. This approach identified 1634 genes involved in drought responses among 497 plant taxa. Most (83.30%) of these species have been classified for human use, and most G2P interactions have been described within model organisms or crop species. Our analysis identifies several gaps in G2P research literature and database connectivity, with 21% of abstracts being linked to gene and taxonomy data in NCBI. Abstract text mining was more successful at identifying potential G2P pathways, with 34% of abstracts containing gene, taxa, and phenotype information. Expanding G2P studies to include non-model plants, especially those that are adapted to drought stress, will help advance our understanding of drought responsive G2P pathways.

A haploid pseudo-chromosome genome assembly for a keystone sagebrush species of western North American rangelands.

Increased ecological disturbances, species invasions, and climate change are creating severe conservation problems for several plant species that are widespread and foundational. Understanding the genetic diversity of these species and how it relates to adaptation to these stressors are necessary for guiding conservation and restoration efforts. This need is particularly acute for big sagebrush (Artemisia tridentata; Asteraceae), which was once the dominant shrub over 1,000,000 km2 in western North America but has since retracted by half and thus has become the target of one of the largest restoration seeding efforts globally. Here, we present the first reference-quality genome assembly for an ecologically important subspecies of big sagebrush (A. tridentata subsp. tridentata) based on short and long reads, as well as chromatin proximity ligation data analyzed using the HiRise pipeline. The final 4.2-Gb assembly consists of 5,492 scaffolds, with nine pseudo-chromosomal scaffolds (nine scaffolds comprising at least 90% of the assembled genome; n = 9). The assembly contains an estimated 43,377 genes based on ab initio gene discovery and transcriptional data analyzed using the MAKER pipeline, with 91.37% of BUSCOs being completely assembled. The final assembly was highly repetitive, with repeat elements comprising 77.99% of the genome, making the Artemisia tridentata subsp. tridentata genome one of the most highly repetitive plant genomes to be sequenced and assembled. This genome assembly advances studies on plant adaptation to drought and heat stress and provides a valuable tool for future genomic research.

Establishing a Health Information Technology for the Vaccination of National Institutes of Health Staff.

Introduction: Healthcare organizations faced unique operational challenges during the COVID-19 pandemic. Assuring the safety of both patients and healthcare workers in hospitals has been the primary focus during the COVID-19 pandemic. Methods: The NIH Vaccine Program (VP) with the Vaccine Management System (VMS) was created based on the commitment of NIH leadership, program leadership, the development team, and the program team; defining Key Performance Indicators (KPIs) of the VP and the VMS; and the NIH Clinical Center's (NIH CC) interdisciplinary approach to deploying the VMS. Results: This article discusses the NIH business requirements of the VP and VMS, the target KPIs of the VP and the VMS, and the NIH CC interdisciplinary approach to deploying an organizational VMS for vaccinating the NIH workforce. The use of the DCRI Spiral-Agile Software Development Life Cycle enabled the development of a system with stakeholder involvement that could quickly adapt to changing requirements meeting the defined KPIs for the program and system. The assessment of the defined KPIs through a survey and comments from the survey support that the VP and VMS were successful. Conclusion: A comprehensive program to maintain a healthy workforce includes asymptomatic COVID testing, symptomatic COVID testing, contact tracing, vaccinations, and policy-driven education. The need to develop systems during the pandemic resulted in changes to build software quickly with the input of many more users and stakeholders then typical in a decreased amount of time.

A draft genome provides hypotheses on drought tolerance in a keystone plant species in Western North America threatened by climate change.

Climate change presents distinct ecological and physiological challenges to plants as extreme climate events become more common. Understanding how species have adapted to drought, especially ecologically important nonmodel organisms, will be crucial to elucidate potential biological pathways for drought adaptation and inform conservation strategies. To aid in genome-to-phenome research, a draft genome was assembled for a diploid individual of Artemisia tridentata subsp. tridentata, a threatened keystone shrub in western North America. While this taxon has few genetic resources available and genetic/genomics work has proven difficult due to genetic heterozygosity in the past, a draft genome was successfully assembled. Aquaporin (AQP) genes and their promoter sequences were mined from the draft genome to predict mechanisms regulating gene expression and generate hypotheses on key genes underpinning drought response. Fifty-one AQP genes were fully assembled within the draft genome. Promoter and phylogenetic analyses revealed putative duplicates of A. tridentata subsp. tridentata AQPs which have experienced differentiation in promoter elements, potentially supporting novel biological pathways. Comparison with nondrought-tolerant congener supports enrichments of AQP genes in this taxon during adaptation to drought stress. Differentiation of promoter elements revealed that paralogues of some genes have evolved to function in different pathways, highlighting these genes as potential candidates for future research and providing critical hypotheses for future genome-to-phenome work.

Inheritance of heat tolerance in perennial ryegrass (Lolium perenne, Poaceae): evidence from progeny array analysis.

BACKGROUND: Heat stress is considered one of the most important environmental factors influencing plant physiology, growth, development, and reproductive output. The occurrence and damage caused by heat stress will likely increase with global climate change. Thus, there is an urgent need to better understand the genetic basis of heat tolerance, especially in cool season plants. MATERIALS AND METHODS: In this study, we assessed the inheritance of heat tolerance in perennial ryegrass (Lolium perenne L. subspecies perenne) , a cool season grass, through a comparison of two parental cultivars with their offspring. We crossed plants of a heat tolerant cultivar (Kangaroo Valley) with plants of a heat sensitive cultivar (Norlea), to generate 72 F1 hybrid progeny arrays. Both parents and their progeny were then exposed to heat stress for 40 days, and their photosynthetic performance (Fv/Fm values) and leaf H2O2 content were measured. RESULTS: As expected, Kangaroo Valley had significantly higher Fv/Fm values and significantly lower H2O2 concentrations than Norlea. For the F1 progeny arrays, values of Fv/Fm decreased gradually with increasing exposure to heat stress, while the content of H2O 2 increased. The progeny had a wide distribution of Fv/Fm and H 2O2 values at 40 days of heat stress. Approximately 95% of the 72 F1 progeny arrays had Fv/Fm values that were equal to or intermediate to the values of the two parental cultivars and 68% of the progeny arrays had H2O2 concentrations equal to or intermediate to their two parents. CONCLUSION: Results of this study indicate considerable additive genetic variation for heat tolerance among the 72 progeny arrays generated from these crosses, and such diversity can be used to improve heat tolerance in perennial ryegrass cultivars. Our findings point to the benefits of combining physiological measurements within a genetic framework to assess the inheritance of heat tolerance, a complex plant response.

Abundance, distribution, and growth characteristics of three keystone Vachellia trees in Gebel Elba National Park, south-eastern Egypt.

This study was conducted to evaluate the abundance and distribution pattern of three keystone Vachellia taxa in wadi Khoda and wadi Rahaba, Gebel Elba National Park, a protected area in south-eastern Egypt. These taxa included Vachellia tortilis subsp. tortilis, Vachellia tortilis subsp. raddiana, and Vachellia ehrenbergiana. In wadi Khoda, only two of these taxa were detected (V. tortilis subsp. raddiana and V. tortilis subsp. tortilis), while all three taxa were encountered in wadi Rahaba. The density of trees in wadi Khoda was 34.3 plant ha-1 compared to 26.3 plant ha-1 in wadi Rahaba. Vachellia tortilis subsp. raddiana was the most frequently observed tree, with an average of 47.93% and 68.25% in wadi Rahaba and wadi Khoda, respectively. Crown depth and tree height were mainly associated with elevation, indicating that elevation plays a key role in the growth of these Vachellia taxa. Our results suggest that human activities have had limited effect on the distribution of these three keystone taxa. Thus, management in Gebel Elba National Park should continue to limit the impact of human activities on these three keystone species.

Genetic analysis of Bromus tectorum (Poaceae) in the Mediterranean region: biogeographical pattern of native populations.

Genetic diversity within and among 42 native populations of Bromus tectorum (cheatgrass) was characterized within two regions, the eastern Mediterranean and the western Mediterranean. Two hypotheses were tested for the genetic diversity of these populations: (1) populations from the eastern Mediterranean are more genetically diverse compared with populations to the west, a potential consequence of the species' westward dispersal with the spread of agriculture, and (2) populations across the Mediterranean contain comparable genetic diversity but display high genetic differentiation, a potential consequence of both regions having served as refugia during glacial advances in the late Quaternary Period. Populations in the eastern Mediterranean possess 16 polymorphic loci and 37 multilocus genotypes. In contrast, populations from the western Mediterranean include a subset of these polymorphic loci (9) and fewer multilocus genotypes (19), consistent with the dispersal of B. tectorum with the east-west Holocene spread of agriculture. Among the 19 multilocus genotypes identified in populations from the western Mediterranean, 13 are undetected among eastern Mediterranean populations. Average genetic diversity within populations from the eastern Mediterranean is nonetheless comparable to the genetic diversity in populations from the Iberian Peninsula, whereas diversity is the lowest in the populations from southern France. Our results suggest a prominent role for agriculture in the grass's western spread, although glacial history and environmental heterogeneity also could have influenced the grass's genetic diversity. The exceptionally high level of self-pollination (>99%) in B. tectorum has contributed to preserving the genetic signature associated with the species' biogeographical history across the Mediterranean region.

RNAi targeting of rootworm Troponin I transcripts confers root protection in maize.

Western corn rootworm, Diabrotica virgifera virgifera, is the major agronomically important pest of maize in the US Corn Belt. To augment the repertoire of the available dsRNA-based traits that control rootworm, we explored a potentially haplolethal gene target, wings up A (wupA), which encodes Troponin I. Troponin I, a component of the Troponin-Tropomyosin complex, is an inhibitory protein involved in muscle contraction. In situ hybridization showed that feeding on wupA-targeted dsRNAs caused systemic transcript knockdown in D. v. virgifera larvae. The knockdown of wupA transcript, and by extension Troponin I protein, led to deterioration of the striated banding pattern in larval body muscle and decreased muscle integrity. Additionally, the loss of function of the circular muscles surrounding the alimentary system led to significant accumulation of food material in the hind gut, which is consistent with a loss of peristaltic motion of the alimentary canal. In this study, we demonstrate that wupA dsRNA is lethal in D. v. virgifera larvae when fed via artificial diet, with growth inhibition of up to 50% within two days of application. Further, wupA hairpins can be stably expressed and detected in maize. Maize expressing wupA hairpins exhibit robust root protection in greenhouse bioassays, with several maize transgene integration events showing root protection equivalent to commercial insecticidal protein-expressing maize.

Plant Biotechnology Applications of Zinc Finger Technology.

With ever-increasing genomic information combined with modern tools for genome modification, we are entering a new era of plant biotechnology. One major tool used for genome modification is the zinc finger nuclease (ZFN). Here, we discuss how ZFNs have proven useful in many genome modification applications. In order to remove the function of a gene or genes, targeted mutagenesis using ZFNs has been readily demonstrated creating numerous gene knockouts, and gene deletion has been demonstrated with removal of gene segments both native and transgenic up to 9 Mb. Applications for gain of function have also been demonstrated. Precision gene editing using ZFNs has resulted in the development of herbicide tolerance, and numerous forms of targeted gene addition have been exhibited. In addition to genome modification, this chapter also describes the use of zinc finger protein transcription factors (ZFP-TFs) for gene regulation in order to provide useful modification of gene expression resulting in altered phenotypes.

RNase If -treated quantitative PCR for dsRNA quantitation of RNAi trait in genetically modified crops.

BACKGROUND: RNA interference (RNAi) technology has been widely used to knockdown target genes via post-transcriptional silencing. In plants, RNAi is used as an effective tool with diverse applications being developed such as resistance against insects, fungi, viruses, and metabolism manipulation. To develop genetically modified (GM) RNAi traits for insect control, a transgene is created and composed of an inversely-repeated sequence of the target gene with a spacer region inserted between the repeats. The transgene design is subject to form a self-complementary hairpin RNA (hpRNA) and the active molecules are > 60 bp doubled-stranded RNA (dsRNA) derived from the hpRNA. However, in some cases, an undesirable intermediate such as single-stranded RNA (ssRNA) may be formed, which is not an active molecule. The aforementioned characteristics of RNAi traits lead to increase the challenges for RNAi-derived dsRNA quantitation. RESULTS: To quantify the dsRNA and distinguish it from the ssRNA in transgenic maize, an analytical tool is required to be able to effectively quantify dsRNA which contains a strong secondary structure. Herein, we develop a modified qRT-PCR method (abbreviated as RNase If -qPCR) coupled with a ssRNA preferred endonuclease (i.e., RNase If). This method enables the precise measurement of the active molecules (i.e., dsRNA) derived from RNAi traits of GM crops and separately quantifies the dsRNA from ssRNA. Notably, we also demonstrate that the RNase If -qPCR is comparable to a hybridization-based method (Quantigene Plex 2.0). CONCLUSIONS: To our best knowledge, this is the first report of a method combining RNase If with modified qRT-PCR protocol. The method represents a reliable analytical tool to quantify dsRNA for GM RNAi crops. It provides a cost-effective and feasible analytical tool for general molecular laboratory without using additional equipment for other methods. The RNase If -qPCR method demonstrates high sensitivity (to 0.001 pg/ µL of dsRNA), precision and accuracy. In this report, we demonstrated the deployment of this method to characterize the RNAi events carrying v-ATPase C in maize during trait development process. The method can be utilized in any application which requires the dsRNA quantification such as double-stranded RNA virus or sprayable dsRNA as herbicide.

Resolving relationships at the animal-fungal divergence: A molecular phylogenetic study of the protist trichomycetes (Ichthyosporea, Eccrinida).

Trichomycetes is a group of microorganisms that was considered a class of fungi comprising four orders of commensal, gut-dwelling endosymbionts obligately associated with arthropods. Since molecular phylogenies revealed two of those orders (Amoebidiales and Eccrinales="protist trichos") to be closely related to members of the protist class Ichthyosporea (=Mesomycetozoea), trichomycetes have been considered an ecological association of both early-diverging fungi and protists. Understanding of the taxonomy, evolution, and diversity of the protist trichos is lacking largely due to the difficulties inherent in species collection that have contributed to undersampling and understudy. The most recent classification divides the protist trichos between two families, Amoebidiidae and Eccrinidae (suborder Trichomycina, order Eccrinida). However, there is no comprehensive molecular phylogeny available for this group and major questions about the systematics of protist trichos remain unanswered. Therefore, we generated 18S and 28S rDNA sequences for 106 protist tricho samples and combined them with publicly available Eccrinida sequences for phylogenetic analyses. We also sequenced a conserved protein-coding gene (heat-shock 70 protein) to obtain a multigene data set. We conducted ancestral state reconstruction (ASR) and Bayesian tip-association significance test (BaTS) analyses by mapping six morphological and ecological characters onto the resulting phylogenetic trees. Our results demonstrate: (1) several ecological and morphological character states (habitat, host type, host stage at time of infestation, location within host, spore production, and growth form) are significantly correlated with the phylogeny, and (2) two additional protist tricho families should be incorporated into the taxonomy to reflect phylogenetic relationships. Our data suggest that an integrated strategy that combines morphological, ecological, and molecular characters is needed to further resolve and clarify the systematics of the Eccrinida.

Trait stacking in modern agriculture: application of genome editing tools.

Advances in plant transgenic technology in the 20th century overcame the major hurdle for transfer of genetic material between species. This not only enabled fundamental insights into plant biology, but also revolutionized commercial agriculture. Adoption of transgenic plants in industrial agriculture has reduced pesticide application, while bringing significant increase in crop yields and farmers' profits. The progress made in transgenic technology over the last three decades paved the way mainly for simple single-gene insect and herbicide tolerance (HT) trait products. Modern agriculture demands stacking and pyramiding of complex traits that provide broad-spectrum insect and HT with other agronomic traits. In addition, more recent developments in genome editing provide unique opportunities to create precise on-demand genome modifications to enhance crop productivity. The major challenge for the plant biotech industry therefore remains to combine multiple forms of traits needed to create commercially viable stacked product. This review provides a historical perspective of conventional breeding stacks, current status of molecular stacks and future developments needed to enable genome-editing technology for trait stacking.

A trait stacking system via intra-genomic homologous recombination.

MAIN CONCLUSION: A gene targeting method has been developed, which allows the conversion of 'breeding stacks', containing unlinked transgenes into a 'molecular stack' and thereby circumventing the breeding challenges associated with transgene segregation. A gene targeting method has been developed for converting two unlinked trait loci into a single locus transgene stack. The method utilizes intra-genomic homologous recombination (IGHR) between stably integrated target and donor loci which share sequence homology and nuclease cleavage sites whereby the donor contains a promoterless herbicide resistance transgene. Upon crossing with a zinc finger nuclease (ZFN)-expressing plant, double-strand breaks (DSB) are created in both the stably integrated target and donor loci. DSBs flanking the donor locus result in intra-genomic mobilization of a promoterless selectable marker-containing donor sequence, which can be utilized as a template for homology-directed repair of a concomitant DSB at the target locus resulting in a functional selectable marker via nuclease-mediated cassette exchange (NMCE). The method was successfully demonstrated in maize using a glyphosate tolerance gene as a donor whereby up to 3.3 % of the resulting progeny embryos cultured on selection medium regenerated plants with the donor sequence integrated into the target locus. The process could be extended to multiple cycles of trait stacking by virtue of a unique intron sequence homology for NMCE between the target and the donor loci. This is the first report that describes NMCE via IGHR, thereby enabling trait stacking using conventional crossing.

Consequences of inoculation with native arbuscular mycorrhizal fungi for root colonization and survival of Artemisia tridentata ssp. wyomingensis seedlings after transplanting.

In arid environments, the propagule density of arbuscular mycorrhizal fungi (AMF) may limit the extent of the plant-AMF symbiosis. Inoculation of seedlings with AMF could alleviate this problem, but the success of this practice largely depends on the ability of the inoculum to multiply and colonize the growing root system after transplanting. These phenomena were investigated in Artemisia tridentata ssp. wyomingensis (Wyoming big sagebrush) seedlings inoculated with native AMF. Seedlings were first grown in a greenhouse in soil without AMF (non-inoculated seedlings) or with AMF (inoculated seedlings). In spring and fall, 3-month-old seedlings were transplanted outdoors to 24-L pots containing soil from a sagebrush habitat (spring and fall mesocosm experiments) or to a recently burned sagebrush habitat (spring and fall field experiments). Five or 8 months after transplanting, colonization was about twofold higher in inoculated than non-inoculated seedlings, except for the spring field experiment. In the mesocosm experiments, inoculation increased survival during the summer by 24 % (p = 0.011). In the field experiments, increased AMF colonization was associated with increases in survival during cold and dry periods; 1 year after transplanting, survival of inoculated seedlings was 27 % higher than that of non-inoculated ones (p < 0.001). To investigate possible mechanisms by which AMF increased survival, we analyzed water use efficiency (WUE) based on foliar (13)C/(12)C isotope ratios (δ (13)C). A positive correlation between AMF colonization and δ (13)C values was observed in the spring mesocosm experiment. In contrast, inoculation did not affect the δ (13)C values of fall transplanted seedlings that were collected the subsequent spring. The effectiveness of AMF inoculation on enhancing colonization and reducing seedling mortality varied among the different experiments, but average effects were estimated by meta-analyses. Several months after transplanting, average AMF colonization was in proportion 84 % higher in inoculated than non-inoculated seedlings (p = 0.0042), while the average risk of seedling mortality was 42 % lower in inoculated than non-inoculated seedlings (p = 0.047). These results indicate that inoculation can increase AMF colonization over the background levels occurring in the soil, leading to higher rates of survival.

A modular gene targeting system for sequential transgene stacking in plants.

A modular, selection-based method was developed for site-specific integration of transgenes into a genomic locus to create multigene stacks. High-frequency gene targeting was obtained using zinc finger nuclease (ZFN)-mediated double-strand break (DSB) formation at a pre-defined target genomic location using a unique intron directly downstream of a promoter driving a selectable marker gene to facilitate homology between target and donor sequences. In this system, only insertion into the target locus leads to a functional selectable marker, and regeneration from random insertions of the promoterless donor construct are reduced on selection media. A new stack of transgenes can potentially be loaded with each successive cycle of gene targeting by exchanging the selectable marker gene using the intron homology. This system was tested in maize using the pat selectable marker gene, whereby up to 30% of the plants regenerated on Bialaphos-containing medium were observed to have the donor construct integrated into the target locus. Unlike previous gene targeting methods that utilize defective or partial genes for selecting targeted events, the present method exchanges fully functional genes with every cycle of targeting, thereby allowing the recycling of selectable marker genes, hypothetically for multiple generations of gene targeting.

Speciation within Columnea section Angustiflora (Gesneriaceae): islands, pollinators and climate.

Despite many advances in evolutionary biology, understanding the proximate mechanisms that lead to speciation for many taxonomic groups remains elusive. Phylogenetic analyses provide a means to generate well-supported estimates of species relationships. Understanding how genetic isolation (restricted gene flow) occurred in the past requires not only a well-supported molecular phylogenetic analysis, but also an understanding of when character states that define species may have changed. In this study, phylogenetic trees resolve species level relationships for fourteen of the fifteen species within Columnea section Angustiflorae (Gesneriaceae). The distributions of sister species pairs are compared and ancestral character states are reconstructed using Bayesian stochastic mapping. Climate variables were also assessed and shifts in ancestral climate conditions were mapped using SEEVA. The relationships between morphological character states and climate variables were assessed with correlation analyses. These results indicate that species in section Angustiflorae have likely diverged as a result of allopatric, parapatric, and sympatric speciation, with both biotic and abiotic forces driving morphological and phenological divergence.

An eight-gene molecular phylogeny of the Kickxellomycotina, including the first phylogenetic placement of Asellariales.

Kickxellomycotina is a recently described subphylum encompassing four zygomycete orders (Asellariales, Dimargaritales, Harpellales, Kickxellales). These fungi are united by the formation of disciform septal pores containing lenticular plugs. Morphological diversification and life history evolution has made the relationships within and among the four orders difficult to resolve on those grounds alone. Here we infer the phylogeny of the Kickxellomycotina based on an eight-gene supermatrix including both ribosomal rDNA (18S, 28S, 5.8S) and protein sequences (MCM7, TSR1, RPB1, RPB2, ß-tubulin). The results of this study demonstrate that Kickxellomycotina is monophyletic and related to members of the Zoopagomycotina. Eight unique clades are distinguished in the Kickxellomycotina, including the four defined orders (Asellariales, Dimargaritales, Harpellales, Kickxellales) as well as four genera previously placed within two of these orders (Barbatospora, Orphella, Ramicandelaber, Spiromyces). Dimargaritales and Ramicandelaber are the earliest diverging members of the subphylum, although the relationship between these taxa remains uncertain. The remaining six clades form a monophyletic group, with Barbatospora diverging first. The next split divides the remaining members of the subphylum into two subclades: (i) Asellariales and Harpellales and (ii) Kickxellales, Orphella and Spiromyces. Estimation of ancestral states for four potentially informative morphological and ecological characters reveals that arthropod endosymbiosis might have been an important factor in the early evolution of the Kickxellomycotina.