Homoeologous evolution of the allotetraploid genome of Poa annua L.

BACKGROUND: Poa annua (annual bluegrass) is an allotetraploid turfgrass, an agronomically significant weed, and one of the most widely dispersed plant species on earth. Here, we report the chromosome-scale genome assemblies of P. annua's diploid progenitors, P. infirma and P. supina, and use multi-omic analyses spanning all three species to better understand P. annua's evolutionary novelty. RESULTS: We find that the diploids diverged from their common ancestor 5.5 - 6.3 million years ago and hybridized to form P. annua ≤ 50,000 years ago. The diploid genomes are similar in chromosome structure and most notably distinguished by the divergent evolutionary histories of their transposable elements, leading to a 1.7 × difference in genome size. In allotetraploid P. annua, we find biased movement of retrotransposons from the larger (A) subgenome to the smaller (B) subgenome. We show that P. annua's B subgenome is preferentially accumulating genes and that its genes are more highly expressed. Whole-genome resequencing of several additional P. annua accessions revealed large-scale chromosomal rearrangements characterized by extensive TE-downsizing and evidence to support the Genome Balance Hypothesis. CONCLUSIONS: The divergent evolutions of the diploid progenitors played a central role in conferring onto P. annua its remarkable phenotypic plasticity. We find that plant genes (guided by selection and drift) and transposable elements (mostly guided by host immunity) each respond to polyploidy in unique ways and that P. annua uses whole-genome duplication to purge highly parasitized heterochromatic sequences. The findings and genomic resources presented here will enable the development of homoeolog-specific markers for accelerated weed science and turfgrass breeding.

Chromosome-Scale Genome Assembly and Annotation of Allotetraploid Annual Bluegrass (Poa annua L.).

Poa annua L. is a globally distributed grass with economic and horticultural significance as a weed and as a turfgrass. This dual significance, and its phenotypic plasticity and ecological adaptation, have made P. annua an intriguing plant for genetic and evolutionary studies. Because of the lack of genomic resources and its allotetraploid (2n = 4x = 28) nature, a reference genome sequence would be a valuable asset to better understand the significance and polyploid origin of P. annua. Here we report a genome assembly with scaffolds representing the 14 haploid chromosomes that are 1.78 Gb in length with an N50 of 112 Mb and 96.7% of BUSCO orthologs. Seventy percent of the genome was identified as repetitive elements, 91.0% of which were Copia- or Gypsy-like long-terminal repeats. The genome was annotated with 76,420 genes spanning 13.3% of the 14 chromosomes. The two subgenomes originating from Poa infirma (Knuth) and Poa supina (Schrad) were sufficiently divergent to be distinguishable but syntenic in sequence and annotation with repetitive elements contributing to the expansion of the P. infirma subgenome.

Variation Among Orchardgrass (Dactylis glomerata) Germplasm for Choke Prevalence Caused by Epichloë typhina.

Orchardgrass, or cocksfoot (Dactylis glomerata L.), is a cool-season forage grass susceptible to the choke disease caused by Epichloë typhina. Choke has been reported in orchardgrass seed production fields across the temperate regions of the world, but fungicides have not been efficacious in reducing choke incidence or prevalence. To assess the potential for genetic resistance or tolerance of orchardgrass to choke, we evaluated the variation in orchardgrass cultivars and accessions for choke prevalence and characterized infected plants for endophyte secondary metabolite and mating type gene presence. Significant variation was detected across years and locations. Choke prevalence did not always increase with the age of the stand, nor did choke prevalence correlate with flowering time or swathing time of the entries. Both mating types of E. typhina were detected in approximately equal proportions, and no evidence for loline, ergot alkaloid, or indole-diterpene biosynthesis was found. Plants with multiple infected tillers often showed more than one mating type present in the plant, indicating multiple infection events rather than a single infection event that spread to multiple tillers. Both accessions and cultivars with significant choke, and no choke, were detected, which constitute sources of germplasm for further testing and breeding.

Association of candidate genes with heading date in a diverse Dactylis glomerata population.

Flowering occurs in response to cues from both temperature and photoperiod elicitors in cool-season, long-day forage grasses, and genes involved in sensing the elicitors and inducing downstream flowering responses have been associated with heading date and flowering time in perennial forage grasses as well as cereal grasses. In this study we test for association between orchardgrass (Dactylis glomerata L.) heading date and polymorphisms in the CONSTANS (DgCO1), FLOWERING TIME (DgFT1), a VRN1 like MADS-box (DgMADS), and PHOTOPERIOD (DgPPD1-like) containing genes. A diverse population of 150 genotypes was measured for heading date across three years, genotyped, and candidate genes sequenced. Although pairwise population kinship values were generally low, the genotypes fit into a two-group structure model. Linkage disequilibrium decayed rapidly, reaching r2 levels below 0.2 within the 500bp of each gene. SNPs significantly associated with heading date were detected in equal-dose and tetraploid dosage models. The DgCO1 gene had the most significant polymorphisms and those with the largest effects, while DgMADS had several significant polymorphisms in its first intron with smaller effects. These polymorphisms can be used for further validation, selection, and development of breeding lines of orchardgrass.

Phylogenetic relationships among low-ploidy species of Poa using chloroplast sequences.

Species of the genus Poa are taxonomically and genetically difficult to delineate owing to high and variable polyploidy, aneuploidy, and challenging breeding systems. Approximately 5% of the proposed species in Poa are considered to include or comprise diploids, but very few of those diploids are represented in seed collections. Recent phylogenetic studies of Poa have included some diploid species to elucidate Poa genome relationships. In this study, we build upon that foundation of diploid Poa relationships with additional confirmed diploid species and accessions, and with additional chloroplast sequences. We also include samples of P. pratensis and P. arachnifera to hone in on possible ancestral genomes in these two agronomic and highly polyploidy species. Relative to most species of Poa, Poa section Dioicopoa (P. ligularis, P. iridifolia, and P. arachnifera) contained relatively large chromosomes. Phylogenies were constructed using the TLF gene region and five additional chloroplast genes, and the placement of new species and accessions fit within chloroplast lineages previously reported better than by taxonomic subgenera and sections. Low-ploidy species in the P chloroplast lineage, such as P. iberica and P. remota, grouped closest to P. pratensis.

Transcriptome profiling of Kentucky bluegrass (Poa pratensis L.) accessions in response to salt stress.

BACKGROUND: Kentucky bluegrass (Poa pratensis L.) is a prominent turfgrass in the cool-season regions, but it is sensitive to salt stress. Previously, a relatively salt tolerant Kentucky bluegrass accession was identified that maintained green colour under consistent salt applications. In this study, a transcriptome study between the tolerant (PI 372742) accession and a salt susceptible (PI 368233) accession was conducted, under control and salt treatments, and in shoot and root tissues. RESULTS: Sample replicates grouped tightly by tissue and treatment, and fewer differentially expressed transcripts were detected in the tolerant PI 372742 samples compared to the susceptible PI 368233 samples, and in root tissues compared to shoot tissues. A de novo assembly resulted in 388,764 transcripts, with 36,587 detected as differentially expressed. Approximately 75 % of transcripts had homology based annotations, with several differences in GO terms enriched between the PI 368233 and PI 372742 samples. Gene expression profiling identified salt-responsive gene families that were consistently down-regulated in PI 372742 and unlikely to contribute to salt tolerance in Kentucky bluegrass. Gene expression profiling also identified sets of transcripts relating to transcription factors, ion and water transport genes, and oxidation-reduction process genes with likely roles in salt tolerance. CONCLUSIONS: The transcript assembly represents the first such assembly in the highly polyploidy, facultative apomictic Kentucky bluegrass. The transcripts identified provide genetic information on how this plant responds to and tolerates salt stress in both shoot and root tissues, and can be used for further genetic testing and introgression.

Genome evolution of intermediate wheatgrass as revealed by EST-SSR markers developed from its three progenitor diploid species.

Intermediate wheatgrass (Thinopyrum intermedium (Host) Barkworth & D.R. Dewey), a segmental autoallohexaploid (2n = 6x = 42), is not only an important forage crop but also a valuable gene reservoir for wheat (Triticum aestivum L.) improvement. Throughout the scientific literature, there continues to be disagreement as to the origin of the different genomes in intermediate wheatgrass. Genotypic data obtained from newly developed EST-SSR primers derived from the putative progenitor diploid species Pseudoroegneria spicata (Pursh) Á. Löve (St genome), Thinopyrum bessarabicum (Savul. & Rayss) Á. Löve (J = J(b) = E(b)), and Thinopyrum elongatum (Host) D. Dewey (E = J(e) = E(e)) indicate that the V genome of Dasypyrum (Coss. & Durieu) T. Durand is not one of the three genomes in intermediate wheatgrass. Based on all available information in the literature and findings in this study, the genomic designation of intermediate wheatgrass should be changed to J(vs)J(r)St, where J(vs) and J(r) represent ancestral genomes of present-day J(b) of Th. bessarabicum and J(e) of Th. elongatum, with J(vs) being more ancient. Furthermore, the information suggests that the St genome in intermediate wheatgrass is most similar to the present-day St found in diploid species of Pseudoroegneria from Eurasia.

A genetic linkage map of tetraploid orchardgrass (Dactylis glomerata L.) and quantitative trait loci for heading date.

Orchardgrass (Dactylis glomerata L.), or cocksfoot, is indigenous to Eurasia and northern Africa, but has been naturalized on nearly every continent and is one of the top perennial forage grasses grown worldwide. To improve the understanding of genetic architecture of orchardgrass and provide a template for heading date candidate gene search in this species, the goals of the present study were to construct a tetraploid orchardgrass genetic linkage map and identify quantitative trait loci associated with heading date. A combination of SSR markers derived from an orchardgrass EST library and AFLP markers were used to genotype an F1 population of 284 individuals derived from a very late heading Dactylis glomerata subsp. himalayensis parent and an early to mid-heading Dactylis glomerata subsp. aschersoniana parent. Two parental maps were constructed with 28 cosegregation groups and seven consensus linkage groups each, and homologous linkage groups were tied together by 38 bridging markers. Linkage group lengths varied from 98 to 187 cM, with an average distance between markers of 5.5 cM. All but two mapped SSR markers had homologies to physically mapped rice (Oryza sativa L.) genes, and six of the seven orchardgrass linkage groups were assigned based on this putative synteny with rice. Quantitative trait loci were detected for heading date on linkage groups 2, 5, and 6 in both parental maps, explaining between 12% and 24% of the variation.

Orchardgrass (Dactylis glomerata L.) EST and SSR marker development, annotation, and transferability.

Orchardgrass, or cocksfoot [Dactylis glomerata (L.)], has been naturalized on nearly every continent and is a commonly used species for forage and hay production. All major cultivated varieties of orchardgrass are autotetraploid, and few tools or information are available for functional and comparative genetic analyses and improvement of the species. To improve the genetic resources for orchardgrass, we have developed an EST library and SSR markers from salt, drought, and cold stressed tissues. The ESTs were bi-directionally sequenced from clones and combined into 17,373 unigenes. Unigenes were annotated based on putative orthology to genes from rice, Triticeae grasses, other Poaceae, Arabidopsis, and the non-redundant database of the NCBI. Of 1,162 SSR markers developed, approximately 80% showed amplification products across a set of orchardgrass germplasm, and 40% across related Festuca and Lolium species. When orchardgrass subspecies were genotyped using 33 SSR markers their within-accession similarity values ranged from 0.44 to 0.71, with Mediterranean accessions having a higher similarity. The total number of genotyped bands was greater for tetraploid accessions compared to diploid accessions. Clustering analysis indicated grouping of Mediterranean subspecies and central Asian subspecies, while the D. glomerata ssp. aschersoniana was closest related to three cultivated varieties.

Development and annotation of perennial Triticeae ESTs and SSR markers.

Triticeae contains hundreds of species of both annual and perennial types. Although substantial genomic tools are available for annual Triticeae cereals such as wheat and barley, the perennial Triticeae lack sufficient genomic resources for genetic mapping or diversity research. To increase the amount of sequence information available in the perennial Triticeae, three expressed sequence tag (EST) libraries were developed and annotated for Pseudoroegneria spicata, a mixture of both Elymus wawawaiensis and E. lanceolatus, and a Leymus cinereus x L. triticoides interspecific hybrid. The ESTs were combined into unigene sets of 8 780 unigenes for P. spicata, 11 281 unigenes for Leymus, and 7 212 unigenes for Elymus. Unigenes were annotated based on putative orthology to genes from rice, wheat, barley, other Poaceae, Arabidopsis, and the non-redundant database of the NCBI. Simple sequence repeat (SSR) markers were developed, tested for amplification and polymorphism, and aligned to the rice genome. Leymus EST markers homologous to rice chromosome 2 genes were syntenous on Leymus homeologous groups 6a and 6b (previously 1b), demonstrating promise for in silico comparative mapping. All ESTs and SSR markers are available on an EST information management and annotation database (http://titan.biotec.uiuc.edu/triticeae/).

Genetic basis of resistance to fall armyworm (Lepidoptera: Noctuidae) and southwestern corn borer (Lepidoptera: Crambidae) leaf-feeding damage in maize.

Leaf-feeding damage by first generation larvae of fall armyworm, Spodopter frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), and southwestern corn borer, Diatraea grandiosella Dyar (Lepidoptera: Crambidae), cause major economic losses each year in maize, Zea mays L. A previous study identified quantitative trait loci (QTL) contributing to reduced leaf-feeding damage by these insects in the maize line Mp704. This study was initiated to identify QTL and their interactions associated with first generation leaf-feeding damage by fall armyworm and southwestern corn borer. QTL associated with fall armyworm and southwestern corn borer resistance in resistant line Mp708 were identified and compared with Mp704. Multiple trait analysis (MTA) of both data sets was then used to identify the most important genetic regions affecting resistance to fall armyworm and southwestern corn borer leaf-feeding damage. Genetic models containing four and seven QTL explained southwestern corn borer and fall armyworm resistance, respectively, in Mp708. Key genomic regions on chromosomes 1, 5, 7, and 9 were identified by MTA in Mp704 and Mp708 that confer resistance to both fall armyworm and southwestern corn borer. QTL regions on chromosomes 1, 5, 7, and 9 contained resistance to both insects and were present in both resistant lines. These regions correspond with previously identified QTL related to resistance to other lepidopteran insects, suggesting that broad-spectrum resistance to leaf feeding is primarily controlled by only a few genetic regions in this germplasm.

Chemical composition of caneberry (Rubus spp.) seeds and oils and their antioxidant potential.

Caneberries (Rubus spp. L.) are grown primarily throughout the Pacific Northwestern United States and Canada. Processing of caneberry fruit typically removes the seed, and the development of a value-added use of seeds could expand the market for caneberries and the profit margins for growers. An initial step toward the use of the seeds is a characterization of seed and oil. Our investigation has described compositional characteristics for seeds of five commonly grown caneberry species: red raspberry, black raspberry, boysenberry, Marion blackberry, and evergreen blackberry. Seeds from all five species had 6-7% protein and 11-18% oil. The oils contained 53-63% linoleic acid, 15-31% linolenic acid, and 3-8% saturated fatty acids. The two smaller seeded raspberry species had higher percentages of oil, the lowest amounts of saturated fatty acid, and the highest amounts of linolenic acid. Antioxidant capacities were detected both for whole seeds and for cold-pressed oils but did not correlate to total phenolics or tocopherols. Ellagitannins and free ellagic acid were the main phenolics detected in all five caneberry species and were approximately 3-fold more abundant in the blackberries and the boysenberry than in the raspberries.

Acetohydroxyacid synthase mutations conferring resistance to imidazolinone or sulfonylurea herbicides in sunflower.

Wild biotypes of cultivated sunflower ( Helianthus annuus L.) are weeds in corn ( Zea mays L.), soybean ( Glycine max L.), and other crops in North America, and are commonly controlled by applying acetohydroxyacid synthase (AHAS)-inhibiting herbicides. Biotypes resistant to two classes of AHAS-inhibiting herbicides-imidazolinones (IMIs) or sulfonylureas (SUs)-have been discovered in wild sunflower populations (ANN-PUR and ANN-KAN) treated with imazethapyr or chlorsulfuron, respectively. The goals of the present study were to isolate AHAS genes from sunflower, identify mutations in AHAS genes conferring herbicide resistance in ANN-PUR and ANN-KAN, and develop tools for marker-assisted selection (MAS) of herbicide resistance genes in sunflower. Three AHAS genes ( AHAS1, AHAS2, and AHAS3) were identified, cloned, and sequenced from herbicide-resistant (mutant) and -susceptible (wild type) genotypes. We identified 48 single-nucleotide polymorphisms (SNPs) in AHAS1, a single six-base pair insertion-deletion in AHAS2, and a single SNP in AHAS3. No DNA polymorphisms were found in AHAS2 among elite inbred lines. AHAS1 from imazethapyr-resistant inbreds harbored a C-to-T mutation in codon 205 ( Arabidopsis thaliana codon nomenclature), conferring resistance to IMI herbicides, whereas AHAS1 from chlorsulfuron-resistant inbreds harbored a C-to-T mutation in codon 197, conferring resistance to SU herbicides. SNP and single-strand conformational polymorphism markers for AHAS1, AHAS2, and AHAS3 were developed and genetically mapped. AHAS1, AHAS2, and AHAS3 mapped to linkage groups 2 ( AHAS3), 6 ( AHAS2), and 9 ( AHAS1). The C/T SNP in codon 205 of AHAS1 cosegregated with a partially dominant gene for resistance to IMI herbicides in two mutant x wild-type populations. The molecular breeding tools described herein create the basis for rapidly identifying new mutations in AHAS and performing MAS for herbicide resistance genes in sunflower.