Genetic structure and diversity analysis of tall fescue populations by EST-SSR and ISSR markers.

Tall fescue is a perennial cool-season grass with economic importance especially in temperate regions of the northern hemisphere. This study was done to assess the genetic diversity and population structure of 90 tall fescue populations and cultivars using ISSR and EST-SSR markers in order to categorize valuable populations for breeding programs and to construct the core collection of tall fescue collection in Iran. The 10 EST-SSR primer pairs amplified 92 alleles. The allele numbers varied from 4 to 13 alleles per locus with an average of 9.2 alleles, of which 84 (90.6%) were polymorphic with an average of 8.4 polymorphic bands per primer. The 39 ISSR primers totally produced 387 scorable bands, of which 335 (86.6%) were polymorphic with an average of 8.6 polymorphic bands per primer. The amplified markers by ISSR primers varied from 6 to 14 markers per primer with an average of 9.92 markers per primer. The 90 tall fescue populations using both EST-SSR and ISSR data were classified into two clusters by UPGMA method that was coincide with PCA and structure analysis results. The turf-type and forage-type populations were clearly separated. Based on the results, the Iranian populations provide a valuable and novel germplasm to employ in tall fescue varietal improvement programs for both forage and turf-type applications. This progression is an important step to introduce this collection for development of a core collection of tall fescue germplasm in Iran.

Five species, many genotypes, broad phenotypic diversity: When agronomy meets functional ecology.

PREMISE OF THE STUDY: Current ecological theory can provide insight into the causes and impacts of plant domestication. However, just how domestication has impacted intraspecific genetic variability (ITV) is unknown. We used 50 ecotypes and 35 cultivars from five grassland species to explore how selection drives functional trait coordination and genetic differentiation. METHODS: We quantified the extent of genetic diversity among different sets of functional traits and determined how much genetic diversity has been generated within populations of natural ecotypes and selected cultivars. KEY RESULTS: In general, the cultivars were larger (e.g., greater height, faster growth rates) and had larger and thinner leaves (greater SLA). We found large (average 63%) and trait-dependent (ranging from 14% for LNC to 95.8% for growth rate) genetic variability. The relative extent of genetic variability was greater for whole-plant than for organ-level traits. This pattern was consistent within ecotypes and within cultivars. However, ecotypes presented greater ITV variability. CONCLUSIONS: The results indicated that genetic diversity is large in domesticated species with contrasting levels of heritability among functional traits and that selection for high yield has led to indirect selection of some associated leaf traits. These findings open the way to define which target traits should be the focus in selection programs, especially in the context of community-level selection.

Past climate changes facilitated homoploid speciation in three mountain spiny fescues (Festuca, Poaceae).

Apart from the overwhelming cases of allopolyploidization, the impact of speciation through homoploid hybridization is becoming more relevant than previously thought. Much less is known, however, about the impact of climate changes as a driven factor of speciation. To investigate these issues, we selected Festuca picoeuropeana, an hypothetical natural hybrid between the diploid species F. eskia and F. gautieri that occurs in two different mountain ranges (Cantabrian Mountains and Pyrenees) separated by more than 400 km. To unravel the outcomes of this mode of speciation and the impact of climate during speciation we used a multidisciplinary approach combining genome size and chromosome counts, data from an extensive nuclear genotypic analysis, plastid sequences and ecological niche models (ENM). Our results show that the same homoploid hybrid was originated independently in the two mountain ranges, being currently isolated from both parents and producing viable seeds. Parental species had the opportunity to contact as early as 21000 years ago although niche divergence occurs nowadays as result of a climate-driven shift. A high degree of niche divergence was observed between the hybrid and its parents and no recent introgression or backcrossed hybrids were detected, supporting the current presence of reproductive isolation barriers between these species.

Insight into the genetic variability analysis and cultivar identification of tall fescue by using SSR markers.

BACKGROUND: Genetic diversity of 19 forage-type and 2 turf-type cultivars of tall fescue (Festuca arundinacea Schreb.) was revealed using SSR markers in an attempt to explore the genetic relationships among them, and examine potential use of SSR markers to identify cultivars by bulked samples. RESULTS: A total of 227 clear band was scored with 14 SSR primers and out of which 201 (88.6 %) were found polymorphic. The percentage of polymorphic bands (PPB) per primer pair varied from 62.5 to 100 % with an average of 86.9 %. The polymorphism information content (PIC) value ranged from 0.116 to 0.347 with an average of 0.257 and the highest PIC value (0.347) was noticed for primer NFA040 followed by NFA113 (0.346) whereas the highest discriminating power (D) of 1 was shown in NFA037 and LMgSSR02-01C. A Neighbor-joining dendrogram and the principal component analysis identified six major clusters and grouped the cultivars in agreement with their breeding histories. STRUCTURE analysis divided these cultivars into 3 sub-clades which correspond to distance based groupings. CONCLUSION: These findings indicates that SSR markers by bulking strategy are a useful tool to measure genetic diversity among tall fescue cultivars and could be used to supplement morphological data for plant variety protection.

Phylogenomics of fescue grass-derived fungal endophytes based on selected nuclear genes and the mitochondrial gene complement.

BACKGROUND: Tall fescue and meadow fescue are important as temperate pasture grasses, forming mutualistic associations with asexual Neotyphodium endophytes. The most frequently identified endophyte of Continental allohexaploid tall fescue is Neotyphodium coenophialum, while representatives of two other taxa (FaTG-2 and FaTG-3) have been described as colonising decaploid and Mediterranean hexaploid tall fescue, respectively. In addition, a recent study identified two other putatively novel endophyte taxa from Mediterranean hexaploid and decaploid tall fescue accessions, which were designated as uncharacterised Neotyphodium species (UNS) and FaTG-3-like respectively. In contrast, diploid meadow fescue mainly forms associations with the endophyte taxon Neotyphodium uncinatum, although a second endophyte taxon, termed N. siegelii, has also been described. RESULTS: Multiple copies of the translation elongation factor 1-a (tefA) and ß-tubulin (tub2) 'house-keeping' genes, as well as the endophyte-specific perA gene, were identified for each fescue-derived endophyte taxon from whole genome sequence data. The assembled gene sequences were used to reconstruct evolutionary relationships between the heteroploid fescue-derived endophytes and putative ancestral sub-genomes derived from known sexual Epichloë species. In addition to the nuclear genome-derived genes, the complete mitochondrial genome (mt genome) sequence was obtained for each of the sequenced endophyte, and phylogenetic relationships between the mt genome protein coding gene complements were also reconstructed. CONCLUSIONS: Complex and highly reticulated evolutionary relationships between Epichloë-Neotyphodium endophytes have been predicted on the basis of multiple nuclear genes and entire mitochondrial protein-coding gene complements, derived from independent assembly of whole genome sequence reads. The results are consistent with previous studies while also providing novel phylogenetic insights, particularly through inclusion of data from the endophyte lineage-specific gene, as well as affording evidence for the origin of cytoplasmic genomes. In particular, the results obtained from the present study imply the possible occurrence of at least two distinct E. typhina progenitors for heteropoid taxa, as well the ancestral contribution of an endophyte species distinct from (although related to) contemporary E. baconii to the extant hybrid species. Furthermore, the present study confirmed the distinct taxonomic status of the newly identified fescue endophyte taxa, FaTG-3-like and UNS, which are consequently proposed to be renamed FaTG4 and FaTG5, respectively.

Plant response to climate change varies with topography, interactions with neighbors, and ecotype.

Predicting the future of any given species represents an unprecedented challenge in light of the many environmental and biological factors that affect organismal performance and that also interact with drivers of global change. In a three-year experiment set in the Mongolian steppe, we examined the response of the common grass Festuca lenensis to manipulated temperature and water while controlling for topographic variation, plant-plant interactions, and ecotypic differentiation. Plant survival and growth responses to a warmer, drier climate varied within the landscape. Response to simulated increased precipitation occurred only in the absence of neighbors, demonstrating that plant-plant interactions can supersede the effects of climate change. F. lenensis also showed evidence of local adaptation in populations that were only 300 m apart. Individuals from the steep and dry upper slope showed a higher stress/drought tolerance, whereas those from the more productive lower slope showed a higher biomass production and a greater ability to cope with competition. Moreover, the response of this species to increased precipitation was ecotype specific, with water addition benefiting only the least stress-tolerant ecotype from the lower slope origin. This multifaceted approach illustrates the importance of placing climate change experiments within a realistic ecological and evolutionary framework. Existing sources of variation impacting plant performance may buffer or obscure climate change effects.

Divergence and biogeography of the recently evolved Macaronesian red Festuca (Gramineae) species inferred from coalescence-based analyses.

Studying the biogeography and the phylogeography of the endemic Macaronesian red Festuca species (Loliinae, Poaceae) is of prime interest in understanding the speciation and colonization patterns of recently evolved groups in oceanic archipelagos. Coalescence-based analyses of plastid trnLF sequences were employed to estimate evolutionary parameters and to test different species-history scenarios that model the pattern of species divergence. Bayesian IM estimates of species divergence times suggested that ancestral lineages of diploid Macaronesian and Iberian red fescues could have diverged between 1.2 and 1.57 Ma. When empirical data were compared to coalescence-based simulated distributions of discordance and p-distance statistics, two species-history models were chosen in which the first branching lineage derived in Canarian Festuca agustinii. Its sister lineage could have involved a recent polytomy leading to the Madeiran Festuca jubata, the Azorean Festuca francoi + Festuca petraea and the continental Festuca rivularis lineages (Canarian model) or the sequential branching of lineages leading to F. jubata and finally to the sister clades of F. rivularis and F. francoi + F. petraea (Sequential model). Nested clade phylogeographic analysis (NCPA) and a first adapted host-parasite co-evolutionary ParaFit method were used to detect the phylogeographic signal. NCPA inferred long-distance colonizations for the entire diploid red Festuca complex, but allopatric-fragmentation and isolation-by-distance (IBD) patterns were inferred within archipelagos. In addition, the ParaFit method suggested a generalized pattern of a stepping-stone model at all hierarchical levels. Maximum-likelihood-based dispersal-extinction-cladogenesis (DEC) models were superimposed on the Sequential model species tree. The three-independent-colonization (3IC) model was the best supported biogeographic scenario, concurring with previous analysis based on multilocus AFLP data.

Evolutionary history of tall fescue morphotypes inferred from molecular phylogenetics of the Lolium-Festuca species complex.

BACKGROUND: The agriculturally important pasture grass tall fescue (Festuca arundinacea Schreb. syn. Lolium arundinaceum (Schreb.) Darbysh.) is an outbreeding allohexaploid, that may be more accurately described as a species complex consisting of three major (Continental, Mediterranean and rhizomatous) morphotypes. Observation of hybrid infertility in some crossing combinations between morphotypes suggests the possibility of independent origins from different diploid progenitors. This study aims to clarify the evolutionary relationships between each tall fescue morphotype through phylogenetic analysis using two low-copy nuclear genes (encoding plastid acetyl-CoA carboxylase [Acc1] and centroradialis [CEN]), the nuclear ribosomal DNA internal transcribed spacer (rDNA ITS) and the chloroplast DNA (cpDNA) genome-located matK gene. Other taxa within the closely related Lolium-Festuca species complex were also included in the study, to increase understanding of evolutionary processes in a taxonomic group characterised by multiple inter-specific hybridisation events. RESULTS: Putative homoeologous sequences from both nuclear genes were obtained from each polyploid species and compared to counterparts from 15 diploid taxa. Phylogenetic reconstruction confirmed F. pratensis and F. arundinacea var. glaucescens as probable progenitors to Continental tall fescue, and these species are also likely to be ancestral to the rhizomatous morphotype. However, these two morphotypes are sufficiently distinct to be located in separate clades based on the ITS-derived data set. All four of the generated data sets suggest independent evolution of the Mediterranean and Continental morphotypes, with minimal affinity between cognate sequence haplotypes. No obvious candidate progenitor species for Mediterranean tall fescues were identified, and only two putative sub-genome-specific haplotypes were identified for this morphotype. CONCLUSIONS: This study describes the first phylogenetic analysis of the Festuca genus to include representatives of each tall fescue morphotype, and to use low copy nuclear gene-derived sequences to identify putative progenitors of the polyploid species. The demonstration of distinct tall fescue lineages has implications for both taxonomy and molecular breeding strategies, and may facilitate the generation of morphotype and/or sub-genome-specific molecular markers.

Variable effects of endophytic fungus on seedling establishment of fine fescues.

Seedborne systemic endophytic fungi of grasses are thought to be plant mutualists, because they have been shown to improve their host's resistance against biotic and abiotic stresses. The interactions in plant-endophyte associations vary from mutualistic to parasitic with environmental conditions and the genotypes of interacting species. The possible pros and cons of endophytic fungi are expected to be most evident during the seedling establishment, where host fitness is most directly affected. If this holds true, endophytes may play a focal role in local adaptation of hosts to different environments. We examined if endophyte-infected and uninfected seeds and seedlings of two native grass species, Festuca rubra and F. ovina, differ in seed germination and seedling growth rates under greenhouse conditions. The germination of F. rubra seeds was also studied in the field. This is the first time that the effects of Epichloë endophyte on seedling establishment of fine fescues from natural populations have been experimentally evaluated. Mother plant (seed family) had a marked effect on many response variables in both grass species. Length and mean biomass of tillers of endophyte-infected (E+) F. ovina seedlings were lower, but root:shoot ratios were higher than in endophyte-free (E-) seedlings. In F. rubra, the effects of the endophyte were dependent on the habitat where the seeds were collected. The E+ seeds from river banks germinated faster than E+ seeds from meadows, and E+ seedlings from the river banks produced fewer but taller and heavier tillers than the other seedlings. Our data suggest that the effects of the endophyte infection on the seedling stage of fine fescues are dependent the species of grass, host genetic background and mother plant habitat. The germination strategy and growth form of E+ red fescue seedlings from river banks may be beneficial to surviving in the harsh conditions of that habitat.

Origin and timing of the horizontal transfer of a PgiC gene from Poa to Festuca ovina.

A segregating second locus, PgiC2, for the enzyme phosphoglucose isomerase (PGIC) is found in the grass sheep's fescue, Festuca ovina. We have earlier reported that a phylogenetic analysis indicates that PgiC2 has been horizontally transferred from the reproductively separated grass genus Poa. Here we extend our analysis to include intron and exon information on 27 PgiC sequences from 18 species representing five genera, and confirm our earlier finding. The origin of PgiC2 can be traced to a group of closely interrelated, polyploid and partially asexual Poa species. The sequence most similar to PgiC2 is found in Poa palustris with a divergence, based on synonymous substitutions, of only 0.67%. This value suggests that the transfer took place less than 600,000 years ago (late Pleistocene), at a time when most extant Poa and Festuca species already existed.

Identifying, cloning and structural analysis of differentially expressed genes upon Puccinia infection of Festuca rubra var. rubra.

Differentially expressed genes in response to rust infection (Puccinia sp.) in creeping red fescue (Festuca rubra var. rubra) were identified and quantified using the mRNA differential display technique. The differentially induced genes were identified as homologs of mitogen-activated protein kinase (MAPK) 3 of Arabidopsis thaliana, stem rust resistance protein Rpg1 of barley and Hsp70 of Spinacia oleracea. The change in the steady state expression levels of these genes in response to rust infection was tested by Northern blot analysis and further quantified by real-time PCR. A steady accumulation of transcripts in the course of rust infection was observed. Full-length transcript of a fescue MPK-3 was obtained by RACE PCR. Its corresponding cDNA encodes a protein with a predicted MW of 42.5 kDa which was mapped onto the structural model of homologs MAPK to illustrate the corresponding MAPK signature motifs. This study, for the first time, presents evidence on the rust infection dependent metabolic pathways in creeping red fescue.

Genetic relationships within and among Iberian fescues (Festuca L.) based on PCR-amplified markers.

The genus Festuca comprises approximately 450 species and is widely distributed around the world. The Iberian Penninsula, with more than 100 taxa colonizing very diverse habitats, is one of its main centers of diversification. This study was conducted to assess molecular genetic variation and genetic relatedness among 91 populations of 31 taxa of Iberian fescues, based on several molecular markers (random amplified polymorphic DNA, amplified fragment length polymorphisms, and trnL sequences). The analyses showed the paraphyletic origin of the broad-leaved (subgenus Festuca, sections Scariosae and Subbulbosae, and subgenus Schedonorus) and the fine-leaved fescues (subgenus Festuca, sections Aulaxyper, Eskia, and Festuca). Schedonorus showed a weak relationship with Lolium rigidum and appeared to be the most recent of the broad-leaved clade. Section Eskia was the most ancient and Festuca the most recent of the fine-leaved clade. Festuca and Aulaxyper were the most related sections, in concordance with their taxonomic affinities. All taxa grouped into their sections, except F. ampla and F. capillifolia (section Festuca), which appeared to be more closely related to Aulaxyper and to a new independent section, respectively. Most populations clustered at the species level, but some subspecies and varieties mixed their populations. This study demonstrated the value in combining different molecular markers to uncover hidden genetic relationships between populations of Festuca.

AFLP-based differentiation of tropical African Festuca species compared to the European Festuca complex.

For the first time amplified fragment length polymorphism (AFLP) fingerprinting is applied to classify tropical African Festuca species. Five afro-alpine narrow- and two afro-montane broad-leaved species from Uganda and Ethiopia are compared to ten European grass species. A principal coordinate analysis (PCoA) accounts for 62.5% with its first three coordinates. The PCoA and the neighbor-joining (NJ) distinguish the five narrow-leaved African Festuca species from all other species. The broad-leaved African Festuca africana and Festuca simensis are linked to the broad-leaved European species through Festuca altissima and Festuca gigantea, respectively. The narrow- and broad-leaved European species are separated as expected in the NJ. One narrow-leaved African alpine species recently described appears merged (i.e. Festuca richardii with Festuca abyssinica). We provide chromosome numbers for all seven Ugandan species and compare taxonomy and AFLP classification. Our most striking result is that the narrow-leaved African Festuca species are unique and not clustering with the narrow-leaved European species.

Low variability of internal transcribed spacer rDNA and trnL (UAA) intron sequences of several taxa in the Festuca ovina aggregate (POACEAE).

Identification and classification of numerous Festuca species is still a difficult problem due to the close morphological resemblance. The most difficult fine fescues to identify belong to the Festuca ovina aggregate, which is the largest group in the genus Festuca. Many taxons are considered to be separate species based on quantitative taxonomic characters, differences in ploidy level or the structure of sclerenchyma cells. In order to evaluate the taxonomic value of DNA-based markers, sequence analysis of the internal transcribed spacer (ITS1-5.8S-ITS2) region and the chloroplast trnL (UAA) intron was performed in the ten most problematic fine fescues belonging to the Festuca ovina aggregate. Intraspecific ITS variants were found in a single case while in other cases only intragenomic ITS polymorphisms were detected with 1-2 ambiguous positions. Among the sequences of the trnL (UAA) intron even intragenomic polymorphisms were not detected in any of the Festuca species studied. Thus, the results do not support the species status of these ten taxa.

Ploidy level variability in South American fescues (festuca L., poaceae): use of flow cytometry in up to 5 1/2-year-old caryopses and herbarium specimens.

Ploidy levels and chromosome numbers for 24 species of Festuca L. from 29 sites in Bolivia, Colombia, Ecuador and Venezuela are given. The ploidy level of 22 species is reported for the first time. A higher proportion of tetraploids in northern South America and the high frequency of polyploids in the whole continent are documented. In combination with chromosome counts, ploidy level was determined using flow cytometry in 4- to 5 1/2 -year-old herbarium specimens and mature caryopses. Flow cytometric determination from seeds was more reliable than determination from herbarium specimens. In herbarium specimens, the youngest, fresh green leaves, still hidden in sheaths, seem to be most suitable for cytometric determination. In old, brownish leaves, or poorly preserved herbarium specimens, the degradation of DNA signal in flow histograms was documented. DNA content measured in seeds was always higher than that measured in herbarium specimens, which may be caused by the presence of different cytosolic compounds. Differences of about 15% in relative DNA content of F. sodiroana and F. vaginalis was found in simultaneous measurements in seeds.

Intake, digestion, and N metabolism in steers fed endophyte-free, ergot alkaloid-producing endophyte-infected, or nonergot alkaloid-producing endophyte-infected fescue hay.

A digestion and N balance trial was conducted to compare effects of traditional endophyte-infected (E+), endophyte-free (E-), and nontoxic endophyte infected (NE; MaxQ; Pennington Seed, Inc., Madison, GA) Jesup tall fescue (Festuca arundinacea Schreb.) hay on digestion and N retention in steers. Hay composition (DM basis) was as follows: E+ (10.8% CP, 59.9% NDF, and 29.4% ADF), E- (11.8% CP, 58.5% NDF, and 28.4% ADF), and NE (11.6% CP, 58.6% NDF, and 28.3% ADF). Eight Polled Hereford steers (initial BW 240 +/- 9 kg) were used in a replicated, 3 x 3 Latin square design, with an extra steer allotted to each square. Steers were fed ad libitum for 14 d, followed by a 9-d adaptation to restricted intake (based on the animal with the lowest ad libitum intake for the square) and a 5-d fecal and urine collection. Water intake (20.2 L/d) and urine output (7.40 L/d) did not differ (P > 0.10) during the collection period. Plasma prolactin concentration was less (P < 0.05) for steers on the E+ hay (8.83 ng/mL) than for those on the E- hay (18.03 ng/mL) and intermediate for steers on the NE hay (12.65 ng/mL). Endophyte-infected hay differed (P < 0.05) from E- and NE in ad libitum DMI (5.02 vs. 5.62 and 5.61 kg/d, respectively) and ad libitum DMI as a percentage of BW (1.86 vs. 2.06 and 2.06%, respectively). Restricted DMI during the fecal and urine collection was lower (P < 0.05) for E+ hay than for E- (5.04 vs. 5.24 kg/d), and NE was intermediate (5.19 kg/d). Dry matter digestibility was lower (P < 0.05) for E+ compared with E- and NE (62.3 vs. 67.0 and 65.9%, respectively). Digestibility of ADF was lower (P < 0.05) for E+ than for E-, and was intermediate for NE (61.5, 66.0, and 63.9%, respectively). There were no differences for NDF, cellulose, or hemicellulose digestibilities among hay types. Crude protein digestibility was higher (P < 0.05) for E- and NE than for E+ (54.3 and 52.5 vs. 48.1%, respectively). Nitrogen retention was lower (P < 0.01) for E+ than for E- or NE (15.6 vs. 22.7 or 23.0 g/d, respectively). Hay type did not influence plasma urea N, urine urea N output, or urine urea N as a percentage of urinary N. Results from this study indicate that E+ tall fescue hay was lower in ad libitum DMI, DM digestibility, and N retention than NE or E- hays with similar chemical composition. Hay from NE and E- fescue had nearly identical composition, and did not differ for any variable measured.