Genomic mechanisms of climate adaptation in polyploid bioenergy switchgrass.

Long-term climate change and periodic environmental extremes threaten food and fuel security1 and global crop productivity2-4. Although molecular and adaptive breeding strategies can buffer the effects of climatic stress and improve crop resilience5, these approaches require sufficient knowledge of the genes that underlie productivity and adaptation6-knowledge that has been limited to a small number of well-studied model systems. Here we present the assembly and annotation of the large and complex genome of the polyploid bioenergy crop switchgrass (Panicum virgatum). Analysis of biomass and survival among 732 resequenced genotypes, which were grown across 10 common gardens that span 1,800 km of latitude, jointly revealed extensive genomic evidence of climate adaptation. Climate-gene-biomass associations were abundant but varied considerably among deeply diverged gene pools. Furthermore, we found that gene flow accelerated climate adaptation during the postglacial colonization of northern habitats through introgression of alleles from a pre-adapted northern gene pool. The polyploid nature of switchgrass also enhanced adaptive potential through the fractionation of gene function, as there was an increased level of heritable genetic diversity on the nondominant subgenome. In addition to investigating patterns of climate adaptation, the genome resources and gene-trait associations developed here provide breeders with the necessary tools to increase switchgrass yield for the sustainable production of bioenergy.

Candidate Genes and Molecular Markers Correlated to Physiological Traits for Heat Tolerance in Fine Fescue Cultivars.

Heat stress is one of the major abiotic factors limiting the growth of cool-season grass species during summer season. The objectives of this study were to assess genetic variations in the transcript levels of selected genes in fine fescue cultivars differing in heat tolerance, and to identify single nucleotide polymorphism (SNP) markers associated with candidate genes related to heat tolerance. Plants of 26 cultivars of five fine fescue species (Festuca spp.) were subjected to heat stress (38/33 °C, day/night temperature) in controlled environmental growth chambers. Physiological analysis including leaf chlorophyll content, photochemical efficiency, and electrolyte leakage demonstrated significant genetic variations in heat tolerance among fine fescue cultivars. The transcript levels of selected genes involved in photosynthesis (RuBisCO activase, Photosystem II CP47 reaction center protein), carbohydrate metabolism (Sucrose synthase), energy production (ATP synthase), growth regulation (Actin), oxidative response (Catalase), and stress protection (Heat shock protein 90) were positively correlated with the physiological traits for heat tolerance. SNP markers for those candidate genes exhibited heterozygosity, which could also separate heat-sensitive and heat-tolerant cultivars into clusters. The development of SNP markers for candidate genes in heat tolerance may allow marker-assisted breeding for the development of new heat-tolerant cultivars in fine fescue and other cool-season grass species.

Impact of Growing Environment on Anthracnose Severity of Switchgrass Cultivars and Clones.

Anthracnose (caused by Colletotrichum navitas) has the potential to significantly reduce biomass yield of switchgrass (Panicum virgatum L.); however, limited information is available on the impact of growing environment on tolerance of switchgrass to anthracnose. Therefore, the major objectives of this study were to (i) examine genotype-environment (G × E) effects on anthracnose severity in populations of switchgrass cultivars and individual genotypes and (ii) determine clonal repeatability estimates and stability analysis of anthracnose tolerance on individual switchgrass genotypes. Two experiments were conducted at one prime and two marginal soil locations in New Jersey. In all, 14 switchgrass cultivars were established from seed in 2008 for experiment 1 and 50 replicated switchgrass clones were planted in 2009 for experiment 2 at all three locations. Anthracnose was rated visually in 2010 for experiment 1 and in 2010 and 2011 for experiment 2. Significant G × E interactions were detected for both experiments (P ≤ 0.05) and anthracnose severity varied by location and cultivar. Clonal repeatability estimates for disease tolerance among clones was 0.78 on a clonal basis and 0.32 on a single-plant basis. Lowland ecotypes exhibited less disease overall than upland ecotypes. Results from this study indicate that selection for improved tolerance to anthracnose should be conducted after evaluation across several environments over multiple years.

Characterization of polymorphic microsatellites for the invasive grass Microstegium vimineum (Poaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the invasive plant Microstegium vimineum (Poaceae) to assess its population structure and to facilitate tracking of invasion expansion. METHODS AND RESULTS: Using 454 sequencing, 11 polymorphic and six monomorphic microsatellite primer sets were developed for M. vimineum. The primer sets were tested on individuals sampled from six populations in the United States and China. The polymorphic primers amplified di-, tri-, and tetranucleotide repeats with three to 10 alleles per locus. CONCLUSIONS: These markers will be useful for a variety of applications including tracking of invasion dynamics and population genetics studies.

Anthracnose disease of switchgrass caused by the novel fungal species Colletotrichum navitas.

In recent years perennial grasses such as the native tallgrass prairie plant Panicum virgatum (switchgrass) have taken on a new role in the North American landscape as a plant-based source of renewable energy. Because switchgrass is a native plant, it has been suggested that disease problems will be minimal, but little research in this area has been conducted. Recently, outbreaks of switchgrass anthracnose disease have been reported from the northeastern United States. Incidences of switchgrass anthracnose are known in North America since 1886 through herbarium specimens and disease reports, but the causal agent of this disease has never been experimentally determined or taxonomically evaluated. In the present work, we evaluate the causal agent of switchgrass anthracnose, a new species we describe as Colletotrichum navitas (navitas=Latin for energy). Multilocus molecular phylogenetics and morphological characters show C. navitas is a novel species in the falcate-spored graminicolous group of the genus Colletotrichum; it is most closely related to the corn anthracnose pathogen Colletotrichum graminicola. We present a formal description and illustrations for C. navitas and provide experimental confirmation that this organism is responsible for switchgrass anthracnose disease.

Breeding for disease resistance in the major cool-season turfgrasses.

Over the past several decades, breeding cool-season turfgrasses for improved disease resistance has been the focus of many turfgrass breeding programs. This review article discusses the dramatic improvements made in breeding Kentucky bluegrass (Poa pratensis) for resistance to leaf spot (caused by Drechslera poae), stem rust (caused by Puccinia graminis), and stripe smut (caused by Ustilago striiformis); perennial ryegrass (Lolium perenne) for resistance to gray leaf spot (caused by Pyricularia grisea), stem rust and crown rust (caused by Puccinia coronata); tall fescue (Festuca arundinacea) for resistance to brown patch (Rhizoctonia solani) and stem rust; creeping bentgrass (Agrostis stolonifera) for resistance to dollar spot (caused by Sclerotinia homoeocarpa); and fine fescues (Festuca spp.) for improved disease resistance. Historically, the dramatic improvements in disease resistance of the cool-season grasses have been attributed to traditional/conventional breeding techniques; however, it is likely that functional genomics and molecular techniques will play a more significant role in the development of cultivated turfgrasses as the specific genes and mechanisms for disease resistance are identified in the future.

A first linkage map and downy mildew resistance QTL discovery for sweet basil (Ocimum basilicum) facilitated by double digestion restriction site associated DNA sequencing (ddRADseq).

Limited understanding of sweet basil (Ocimum basilicum L.) genetics and genome structure has reduced efficiency of breeding strategies. This is evidenced by the rapid, worldwide dissemination of basil downy mildew (Peronospora belbahrii) in the absence of resistant cultivars. In an effort to improve available genetic resources, expressed sequence tag simple sequence repeat (EST-SSR) and single nucleotide polymorphism (SNP) markers were developed and used to genotype the MRI x SB22 F2 mapping population, which segregates for response to downy mildew. SNP markers were generated from genomic sequences derived from double digestion restriction site associated DNA sequencing (ddRADseq). Disomic segregation was observed in both SNP and EST-SSR markers providing evidence of an O. basilicum allotetraploid genome structure and allowing for subsequent analysis of the mapping population as a diploid intercross. A dense linkage map was constructed using 42 EST-SSR and 1,847 SNP markers spanning 3,030.9 cM. Multiple quantitative trait loci (QTL) model (MQM) analysis identified three QTL that explained 37-55% of phenotypic variance associated with downy mildew response across three environments. A single major QTL, dm11.1 explained 21-28% of phenotypic variance and demonstrated dominant gene action. Two minor QTL dm9.1 and dm14.1 explained 5-16% and 4-18% of phenotypic variance, respectively. Evidence is provided for an additive effect between the two minor QTL and the major QTL dm11.1 increasing downy mildew susceptibility. Results indicate that ddRADseq-facilitated SNP and SSR marker genotyping is an effective approach for mapping the sweet basil genome.

Magnaporthiopsis meyeri-festucae, sp. nov., associated with a summer patch-like disease of fine fescue turfgrasses.

Summer patch is a common and destructive root disease of turfgrasses. In this study, a new Magnaporthiopsis species, M. meyeri-festucae, was identified from the roots of fine fescue (Festuca spp.) turfgrasses with summer patch-like symptoms. It is described and illustrated on the basis of phenotypic characteristics and partial sequences of rDNA 18S, internal transcribed spacer (ITS), and 28S regions, and of MCM7, RPB1, and TEF1 genes. A key for all seven described species in the genus Magnaporthiopsis is provided. Distinctions between the new species and related species are discussed. Fulfillment of Koch's postulates confirmed Magnaporthiopsis meyeri-festucae as a pathogen causing summer patch-like symptoms of fine fescue turfgrasses. This work is the basis for future studies on biogeography, host range, and impact of summer patch pathogens on a broader scale.

Heritability of dollar spot resistance in creeping bentgrass.

ABSTRACT The dollar spot disease incited by Sclerotinia homoeocarpa is an important disease of creeping bentgrass (Agrostis stolonifera). Genetic resistance is an important control strategy and could reduce fungicide use. Despite recent research, the genetic mechanism of dollar spot resistance in turfgrasses is still not fully understood. The objectives of this study were to (i) determine narrow-sense heritability and predicted gain from selection for dollar spot resistance in creeping bentgrass and (ii) evaluate inheritance characteristics of dollar spot disease resistance. Inheritance characteristics such as the detection of major genes, heterosis, maternal effects, and combining ability were determined by evaluating the disease severity of progeny from crosses between resistant and susceptible bent-grass clones. Parental clones and progenies from crosses were established in a field trial in a randomized complete block design and inoculated with one isolate of S. homoeocarpa applied at a rate of 0.25 g m(-2) of prepared inoculum. Differences in progeny means between crosses were observed over both years. Progeny from resistant x resistant crosses had significantly less disease severity than resistant x susceptible and susceptible x susceptible crosses. High narrow-sense heritability estimates (0.79 [2002], 0.79 [2003]) and large mean squares for general combining ability support the idea that additive gene action plays a significant role in disease resistance and support previous research that dollar spot resistance is most likely quantitatively inherited.

Sclerotinia homoeocarpa overwinters in turfgrass and is present in commercial seed.

Dollar spot is the most economically important disease of amenity turfgrasses in the United States, yet little is known about the source of primary inoculum for this disease. With the exception of a few isolates from the United Kingdom, Sclerotinia homoeocarpa, the causal agent of dollar spot, does not produce spores. Consequently, it was assumed that overwintering of this organism in soil, thatch, and plant debris provides primary inoculum for dollar spot epidemics. Overwintering of S. homoeocarpa in roots and shoots of symptomatic and asymptomatic creeping bentgrass turfgrass was quantified over the course of a three-year field experiment. Roots did not consistently harbor S. homoeocarpa, whereas S. homoeocarpa was isolated from 30% of symptomatic shoots and 10% of asymptomatic shoots in the spring of two out of three years. The presence of stroma-like pathogen material on leaf blades was associated with an increase in S. homoeocarpa isolation and colony diameter at 48 hpi. Commercial seed has also been hypothesized to be a potential source of initial inoculum for S. homoeocarpa. Two or more commercial seed lots of six creeping bentgrass cultivars were tested for contamination with S. homoeocarpa using culture-based and molecular detection methods. A viable, pathogenic isolate of S. homoeocarpa was isolated from one commercial seed lot and contamination of this lot was confirmed with nested PCR using S. homoeocarpa specific primers. A sensitive nested PCR assay detected S. homoeocarpa contamination in eight of twelve (75%) commercial seed lots. Seed source, but not cultivar or resistance to dollar spot, influenced contamination by S. homoeocarpa. Overall, this research suggests that seeds are a potential source of initial inoculum for dollar spot epidemics and presents the need for further research in this area.

Characterization of 215 simple sequence repeat markers in creeping bentgrass (Agrostis stolonifera L.).

Creeping bentgrass (Agrostis stolonifera L.) is a versatile, cross-pollinated, temperate and perennial turfgrass species. It occurs naturally in a wide variety of habitats and is also cultivated on golf courses, bowling greens and tennis courts worldwide. Isozymes and amplified fragment length polymorphisms (AFLPs) have been used to determine genetic diversity, and restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNA (RAPDs) were used to construct a genetic linkage map of this species. In the current report, we developed and characterized 215 unique genomic simple sequence repeat (SSR) markers in creeping bentgrass. The SSRs reported here are the first available markers in creeping bentgrass to date. Eight hundred and eighteen alleles were amplified by 215 SSR loci, an average of 3.72 alleles per locus. Fifty-nine per cent of those alleles segregated in a 1:1 Mendelian fashion (P > 0.05). Twenty-two per cent had a distorted segregation ratio (P ≤ 0.05). These SSR markers will be useful for assessing genetic diversity in creeping bentgrass and will be important for the development of genetic linkage maps and identifying quantitative trait loci. These markers could enhance breeding programmes by improving the efficiency of selection techniques.

Analysis of EST sequences suggests recent origin of allotetraploid colonial and creeping bentgrasses.

Advances in plant genomics have permitted the analysis of several members of the grass family, including the major domesticated species, and provided new insights into the evolution of the major crops on earth. Two members, colonial bentgrass (Agrostis capillaris L.) and creeping bentgrass (A. stolonifera L.) have only recently been domesticated and provide an interesting case of polyploidy and comparison to crops that have undergone human selection for thousands of years. As an initial step of characterizing these genomes, we have sampled roughly 10% of their gene content, thereby also serving as a starting point for the construction of their physical and genetic maps. Sampling mRNA from plants subjected to environmental stress showed a remarkable increase in transcription of transposable elements. Both colonial and creeping bentgrass are allotetraploids and are considered to have one genome in common, designated the A2 genome. Analysis of conserved genes present among the ESTs suggests the colonial and creeping bentgrass A2 genomes diverged from a common ancestor approximately 2.2 million years ago (MYA), thereby providing an enhanced evolutionary zoom in respect to the origin of maize, which formed 4.8 MYA, and tetraploid wheat, which formed only 0.5 MYA and is the progenitor of domesticated hexaploid wheat.

Identification of a gene in the process of being lost from the genus Agrostis.

Lineage-specific gene loss is considered one of the processes contributing to speciation and genome diversity. Such gene loss has been inferred from interspecies comparisons of orthologous DNA segments. Examples of intraspecific gene loss are rare. Here we report identification of a gene, designated Crs-1 (creeping specific-1), that appears to be in the process of being lost from heterozygous populations of the species creeping bentgrass (Agrostis stolonifera). The Crs-1 gene encodes a protein with an N-terminal dirigent protein domain and a C-terminal lectin domain and is similar to the maize (Zea mays) beta-glucosidase aggregating factor. Most individual creeping bentgrass plants examined are lacking Crs-1. Some individuals are hemizygous for the Crs-1 locus, indicating major haplotype noncolinearity at that locus. Crs-1 was not detected in several other Agrostis species, indicating it is being lost from the genus. The Crs-1 locus in creeping bentgrass provides a rare example of the evolutionary process of gene loss occurring within a plant species.

Ploidy Determination in Agrostis Using Flow Cytometry and Morphological Traits.

The taxonomic classification of the genus Agrostis is one of the most complicated of the grass genera. Classification based upon morphological and anatomical characters is difficult and complicated by the presence of intermediate forms and the misapplication of names. Determining ploidy levels of new germplasm can assist in species determination and is necessary before initiating breeding or genetics studies. The objectives of this study were to (i) evaluate the use of laser flow cytometry as a quick, reliable tool to determine ploidy level and aid in Agrostis species determination, and (ii) identify morphological characters associated with DNA content or ploidy level. The six Agrostis species evaluated were A. canina L. subsp. canina, A. canina L. subsp. montana (Hartm.) Hartm., A. palustris Huds. [= A. stolonifera var. palustris (Huds.) Farw.], A. tenuis Sibth. (= A. capillaris L.), A. castellana Boiss. & Reut., and A. alba L. Ploidy level was determined by flow cytometry and root tip chromosome counts. Plant height, panicle height, flag leaf length, flag leaf width, and highest internode length of mature field-grown spaced plants were measured. Significant differences in 2C DNA content were found between species (P < 0.01) differing in ploidy level. Flow cytometry was effective in differentiating between diploid, tetraploid, and hexaploid species. Chromosome numbers previously reported and those observed in this study were positively correlated with 2C nuclear DNA content (r = 0.98, P < 0.01). Flag leaf length was the only morphological measurement taken that was significantly positively correlated to DNA content (r = 0.98, P < 0.001). The results of this study indicate that laser flow cytometry is a quick, reliable tool to determine ploidy levels and infer certain species of AGROSTIS: This technique will aid breeders to quickly and accurately determine ploidy levels of new germplasm collections.