Probe-based loop-mediated isothermal amplification assay for rapid detection of two Clarireedia spp., the causal agent of dollar spot of turfgrass.

Dollar spot is a major fungal disease affecting turfgrass worldwide and can quickly destroy turfgrass swards. An assimilating probe-based loop-mediated amplification (LAMP) assay was developed to detect Clarireedia monteithiana and C. jacksonii, the causal agents of dollar spot within the continental US. Five LAMP primers were designed to target the calmodulin gene with the addition of a 6-carboxyl-fluorescein florescent assimilating probe and the temperature amplification was optimized for C. jacksonii and C. monteithiana identification. The minimum amount purified DNA needed for detection was 0.05 ng µL-1. Specificity assays against host DNA and other turfgrass pathogens were negative. Successful LAMP amplification was also observed for dollar spot infected turfgrass field samples. Further, a DNA extraction technique via rapid heat-chill cycles and visualization of LAMP results via a florescent flashlight was developed and adapted for fast, simple and reliable detection in 1.25 hours. This assimilating probe-based LAMP assay has proved successful as a rapid, sensitive, and specific detection of C. monteithiana and C. jacksonii in pure cultures and from symptomatic turfgrass leaves blades. The assay represents a promising technology to be used in the field for on-site, point-of-care pathogen detection.

Dose-dependent physiological effects of UV-C radiation on seashore paspalum.

Positive effects of ultraviolet-C (UV-C) radiation on plants have been documented in previous literature with a focus on extending shelf life and reducing disease development. However, its effect on plant growth habits has been scarcely explored, especially in turfgrass where a compact shoot growth is a desirable trait. Seashore paspalum (Paspalum vaginatum) is a warm-season perennial turfgrass requiring low fertilizer and pesticide inputs. This project aimed to test the effects of different doses of UV-C radiation on growth and performance of seashore paspalum cv. Seastar. Here, we provide evidence of dose-dependent effects. Lower UV-C doses (6 s and 1 min daily) improved the performance of seashore paspalum, as manifested by higher tiller density, reduced clipping yields, increased chlorophyll level on selected dates as well as enhanced photosynthetic efficiency compared to control. Contrastingly, higher doses (6 min and 30 min daily) resulted in severe damage with 30-min treatment being lethal to seashore paspalum, causing marked declines in all measured parameters. This is the first time that UV-C-induced growth response was reported in turf. Conclusions drawn from this study would shed light into the effects of UV-C radiation on the growth and performance of seashore paspalum and offer exciting potential for the utilization of UV-C at non-lethal dosage in turfgrass management.

Genetic Diversity, Mycotoxin Profiles, and Population Structure of Fusarium spp. Associated with Fusarium Head Blight in Georgia, U.S.A.

Fusarium head blight (FHB) has become a limiting factor in soft red winter wheat production in the southeast US. Recent epidemics have occurred in Georgia, however genetic information on the Fusarium species responsible for FHB is unknown. This study aimed to assess pathogen population structure and genetic diversity, trichothecene profiles, and representative pathogenicity of 196 Fusarium isolates collected from 44 wheat (n = 85) and 53 corn (n = 111) fields in Georgia. Phylogenetic analysis using the translation elongation factor 1-alpha (635 bp) and RNA polymerase second largest subunit (930 bp) sequence data resolved isolates into 185 haplotypes, representing 12 Fusarium species grouped under five species complexes. F. graminearum with 15-acetyl-deoxynivalenol (15ADON) chemotype (75.6%) and F. incarnatum (57.7%) predominated in wheat and corn, respectively, with a surprisingly higher frequency of NIV F. graminearum (21.8%). Using nine variable number of tandem repeat markers, 82 multilocus genotypes out of 86 F. graminearum isolates were identified and grouped into two genetic clusters, pop1fg (n = 29) and pop2fg (n = 32), as part of the North American populations (NA1 and NA2), but with no chemotype differentiation. F. graminearum populations in Georgia are mostly clonal and might have evolved through at least two introductions from the northeast US and Canada and local adaptation to maintain high genetic diversity. Pathogenicity of F. graminearum isolates from wheat and corn had high FHB severity (>60%) in wheat, depicting the risk they can pose towards future FHB outbreaks. Overall, this baseline study provided important information on Fusarium species diversity including F. graminearum associated with FHB in Georgia that will be useful to formulate integrated disease management incorporating improved host resistance and fungicide spray program.

Quantitative Trait Locus Analysis of Hessian Fly Resistance in Soft Red Winter Wheat.

The Hessian fly (HF) is an invasive insect that has caused millions of dollars in yield losses to southeastern US wheat farms. Genetic resistance is the most sustainable solution to control HF. However, emerging biotypes are quickly overcoming resistance genes in the southeast; therefore, identifying novel sources of resistance is critical. The resistant line "UGA 111729" and susceptible variety "AGS 2038" were crossbred to generate a population of 225 recombinant inbred lines. This population was phenotyped in the growth chamber (GC) during 2019 and 2021 and in field (F) trials in Georgia during the 2021-2022 growing seasons. Visual scoring was utilized in GC studies. The percentage of infested tillers and number of pupae/larvae per tiller, and infested tiller per sample were measured in studies from 2021 to 2022. Averaging across all traits, a major QTL on chromosome 3D explained 42.27% (GC) and 10.43% (F) phenotypic variance within 9.86 centimorgans (cM). SNP marker IWB65911 was associated with the quantitative trait locus (QTL) peak with logarithm of odds (LOD) values of 14.98 (F) and 62.22 (GC). IWB65911 colocalized with resistance gene H32. KASP marker validation verified that UGA 111729 and KS89WGRC06 express H32. IWB65911 may be used for marker-assisted selection.

Unlocking the story of resistance to Zymoseptoria tritici in Tunisian old durum wheat germplasm based on population structure analysis.

BACKGROUND: Septoria tritici blotch (STB) remains a significant obstacle to durum wheat cultivation on a global scale. This disease remains a challenge for farmers, researchers, and breeders, who are collectively dedicated to reduce its damage and improve wheat resistance. Tunisian durum wheat landraces have been recognized as valuable genetic ressources that exhibit resistance to biotic and abiotic stresses and therefore play a crucial role in breeding program aimed at creating new wheat varieties resistant to fungal diseases as STB, as well as adapted to climate change constraints. RESULTS: A total of 366 local durum wheat accessions were assessed for resistance to two virulent Tunisian isolates of Zymoseptoria tritici Tun06 and TM220 under field conditions. Population structure analysis of the durum wheat accessions, performed with 286 polymorphic SNPs (PIC > 0.3) covering the entire genome, identified three genetic subpopulations (GS1, GS2 and GS3) with 22% of admixed genotypes. Interestingly, all of the resistant genotypes were among GS2 or admixed with GS2. CONCLUSIONS: This study revealed the population structure and the genetic distribution of the resistance to Z. tritici in the Tunisian durum wheat landraces. Accessions grouping pattern reflected the geographical origins of the landraces. We suggested that GS2 accessions were mostly derived from eastern Mediterranean populations, unlike GS1 and GS3 that originated from the west. Resistant GS2 accessions belonged to landraces Taganrog, Sbei glabre, Richi, Mekki, Badri, Jneh Khotifa and Azizi. Furthermore, we suggested that admixture contributed to transmit STB resistance from GS2 resistant landraces to initially susceptible landraces such as Mahmoudi (GS1), but also resulted in the loss of resistance in the case of GS2 susceptible Azizi and Jneh Khotifa accessions.

Genome analyses reveal population structure and a purple stigma color gene candidate in finger millet.

Finger millet is a key food security crop widely grown in eastern Africa, India and Nepal. Long considered a 'poor man's crop', finger millet has regained attention over the past decade for its climate resilience and the nutritional qualities of its grain. To bring finger millet breeding into the 21st century, here we present the assembly and annotation of a chromosome-scale reference genome. We show that this ~1.3 million years old allotetraploid has a high level of homoeologous gene retention and lacks subgenome dominance. Population structure is mainly driven by the differential presence of large wild segments in the pericentromeric regions of several chromosomes. Trait mapping, followed by variant analysis of gene candidates, reveals that loss of purple coloration of anthers and stigma is associated with loss-of-function mutations in the finger millet orthologs of the maize R1/B1 and Arabidopsis GL3/EGL3 anthocyanin regulatory genes. Proanthocyanidin production in seed is not affected by these gene knockouts.

Sensitivity of Clarireedia spp. to benzimidazoles and dimethyl inhibitors fungicides and efficacy of biofungicides on dollar spot of warm season turfgrass.

Dollar spot caused by Clarireedia spp. (formerly Sclerotinia homoeocarpa) is an economically destructive fungal disease of turfgrass that can significantly compromise turf quality, playability, and aesthetic value. Fungicides are frequently used to manage the disease but are costly and potentially unfavorable to the environment. Repeated use of some active ingredients has resulted in reduced efficacy on C. jacksonii causing dollar spot in cool-season turfgrasses in the US. Experiments were conducted to study fungicide sensitivity of Clarireedia spp. as well as to develop alternatives to fungicides against dollar spot on warm-season turfgrass in Georgia. First, 79 isolates of Clarireedia spp. collected across the state were tested on fungicide-amended agar plates for their sensitivity to thiophanate-methyl (benzimidazole) and propiconazole (dimethyl inhibitor). Seventy-seven isolates (97.5%) were sensitive (0.001 to 0.654 µg/mL) and two isolates (2.5%) were found resistant (>1000 µg/mL) to thiophanate-methyl. However, in the case of propiconazole, 27 isolates (34.2%) were sensitive (0.005 to 0.098 µg/mL) while 52 isolates (65.8%) were resistant (0.101 to 3.820 µg/mL). Next, the efficacy of three bio- and six synthetic fungicides and ten different combinations were tested in vitro against C. monteithiana. Seven bio- and synthetic fungicide spray programs comprising Bacillus subtilis QST713 and propiconazole were further tested, either alone or in a tank mix in a reduced rate, on dollar spot infected bermudagrass 'TifTuf' in growth chamber and field environments. These fungicides were selected as they were found to significantly reduce pathogen growth up to 100% on in vitro assays. The most effective spray program in growth chamber assays was 100% B. subtilis QST713 in rotation with 75% B. subtilis QST713 + 25% propiconazole tank mix applied every 14 days. However, the stand-alone application of the biofungicide B. subtilis QST713 every seven days was an effective alternative and equally efficacious as propiconazole, suppressing dollar spot severity and AUDPC up to 75%, while resulting in acceptable turf quality (>7.0) in field experiments. Our study suggests that increased resistance of Clarireedia spp. to benzimidazoles and dimethyl inhibitors warrants continuous surveillance and that biofungicides hold promise to complement synthetic fungicides in an efficacious and environmentally friendly disease management program.

Deciphering resistance to Zymoseptoria tritici in the Tunisian durum wheat landrace accession 'Agili39'.

BACKGROUND: Septoria tritici blotch (STB), caused by Zymoseptoria tritici (Z. tritici), is an important biotic threat to durum wheat in the entire Mediterranean Basin. Although most durum wheat cultivars are susceptible to Z. tritici, research in STB resistance in durum wheat has been limited. RESULTS: In our study, we have identified resistance to a wide array of Z. tritici isolates in the Tunisian durum wheat landrace accession 'Agili39'. Subsequently, a recombinant inbred population was developed and tested under greenhouse conditions at the seedling stage with eight Z. tritici isolates and for five years under field conditions with three Z. tritici isolates. Mapping of quantitative trait loci (QTL) resulted in the identification of two major QTL on chromosome 2B designated as Qstb2B_1 and Qstb2B_2. The Qstb2B_1 QTL was mapped at the seedling and the adult plant stage (highest LOD 33.9, explained variance 61.6%), conferring an effective resistance against five Z. tritici isolates. The Qstb2B_2 conferred adult plant resistance (highest LOD 32.9, explained variance 42%) and has been effective at the field trials against two Z. tritici isolates. The physical positions of the flanking markers linked to Qstb2B_1 and Qstb2B_2 indicate that these two QTL are 5 Mb apart. In addition, we identified two minor QTL on chromosomes 1A (Qstb1A) and chromosome 7A (Qstb7A) (highest LODs 4.6 and 4.0, and explained variances of 16% and 9%, respectively) that were specific to three and one Z. tritici isolates, respectively. All identified QTL were derived from the landrace accession Agili39 that represents a valuable source for STB resistance in durum wheat. CONCLUSION: This study demonstrates that Z. tritici resistance in the 'Agili39' landrace accession is controlled by two minor and two major QTL acting in an additive mode. We also provide evidence that the broad efficacy of the resistance to STB in 'Agili 39' is due to a natural pyramiding of these QTL. A sustainable use of this Z. tritici resistance source and a positive selection of the linked markers to the identified QTL will greatly support effective breeding for Z. tritici resistance in durum wheat.

Detection of Maternal and Cytoplasmic Effects on Resistance to Zymoseptoria tritici in Durum Wheat.

Septoria tritici blotch (STB) is a major disease problem of wheat worldwide. To optimize the introgression of resistance genes in elite genotypes throughout traditional or molecular breeding programs, a full understanding of the quantitative inheritance of resistance to Zymoseptoria tritici, plant height (PH), and thousand kernel weight (TKW) is needed. In this study, maternal and cytoplasmic effects of resistance to STB were investigated using P1 (susceptible, high-yielding line) and P2 (resistant, low-yielding line) durum wheat lines and their F1, RF1, F2, RF2, BC1, RBC1, BC2, and RBC2 progeny, assessed for resistance to STB during three growing seasons. Duncan mean's analysis revealed significant differences between generation means for STB, PH, and TKW. The two parents had an extreme pattern. The F1 and RF1 segregated close to their respective parents, suggesting the presence of cytoplasmic and maternal genetic effects for Z. tritici resistance, PH, and TKW. Separate generation mean's analysis confirmed the results of the Duncan test. A three-parameter model was found to be not adequate for all traits in all three growing years; while a digenic epistatic model with cytoplasmic or/and maternal effect was adequate for all cases. Narrow-sense heritability was in the range of 50-60%, 30-69%, and 28-31% for STB, PH, and TKW, respectively. For STB, high heritability and the presence of fixable epistatic effect is encouraging and could lead to creating varieties with the right female parent to exploit cytoplasmic and maternal effects in order to improve resistance to Z. tritici in durum wheat.

Whole genome sequencing of Clarireedia aff. paspali reveals potential pathogenesis factors in Clarireedia species, causal agents of dollar spot in turfgrass.

Dollar spot is one of the most damaging diseases in turfgrass, reducing its quality and playability. Two species, Clarireedia monteithiana and C. jacksonii (formerly Sclerotinia homoeocarpa) have been reported so far in the United States To study the Clarireedia genome, two isolates H2 and H3, sampled from seashore paspalum in Hawaii in 2019 were sequenced via Illumina paired-end sequencing by synthesis technology and PacBio SMRT sequencing. Both isolates were identified as C. aff. paspali, a novel species in the United States Using short and long reads, C. aff. paspali H3 contained 193 contigs with 48.6 Mbp and presented the most completed assembly and annotation among Clarireedia species. Out of the 13,428 protein models from AUGUSTUS, 349 cytoplasmic effectors and 13 apoplastic effectors were identified by EffectorP. To further decipher Clarireedia pathogenicity, C. aff. paspali genomes (H2 and H3), as well as available C. jacksonii (LWC-10 and HRI11), C. monteithiana (DRR09 and RB-19) genomes were screened for fifty-four pathogenesis determinants, previously identified in S. sclerotiorum. Seventeen orthologs of pathogenicity genes have been identified in Clarireedia species involved in oxalic acid production (pac1, nox1), mitogen-activated protein kinase cascade (pka1, smk3, ste12), appressorium formation (caf1, pks13, ams2, rgb1, rhs1) and glycolytic pathway (gpd). Within these genes, 366 species-specific SNPs were recorded between Clarireedia species; twenty-eight were non-synonymous and non-conservative. The predicted protein structure of six of these genes showed superimposition of the models among Clarireedia spp. The genomic variations revealed here could potentially lead to differences in pathogenesis and other physiological functions among Clarireedia species.

New Approaches to an Old Problem: Dollar Spot of Turfgrass.

Dollar spot, caused by fungal pathogens Clarireedia spp. (formerly Sclerotinia homoeocarpa), is the most common and widely distributed disease of turfgrass worldwide. It can drastically reduce the quality of turfgrass species and affect their aesthetic value and playability. Management of dollar spot typically includes a costly program of multiple application of fungicides within a growing season. Consequently, there have been reported cases of fungicide resistance in populations of Clarireedia spp. Host resistance could be an important component of dollar spot management; however, this approach has been hampered by the lack of sources of resistance because nearly all known warm- and cool-season turfgrass species are susceptible. With the recent advancement in genome sequencing technologies, studies on pathogen genomics and host-pathogen interactions are emerging with the hope of revealing candidate resistance genes in turfgrass and genes for virulence and pathogenicity in Clarireedia spp. Large-scale screening of turfgrass germplasm and quantitative trait locus (QTL) analysis for dollar spot resistance are important for resistance breeding, but only a handful of such studies have been conducted to date. This review summarizes currently available information on the dollar spot pathosystem, taxonomy, pathogen genomics, host-pathogen interaction, genetics of resistance, and QTL mapping and also provides some thoughts for future research prospects to better manage this disease.

First Report of Gray Leaf Spot Caused by Pyricularia oryzae (synonym: Magnaporthe oryzae) in Oat (Avena sativa) in Georgia, USA.

In southeastern U.S., oat (Avena sativa L.) is predominantly grown as a grain or forage crop due to its exceptional palatability (Buntin et al. 2009). In November 2020, leaf spot symptoms were observed in an oat field (cv. Horizon 720) in Screven County, Georgia (GPS: 32°38'57.6"N 81°31'32.178"W). Lesions were oblong, whitish to gray in color, and surrounded by dark brown borders. Symptomatic oat leaves were sampled from the field and cut into 1 cm2 sections that were surface sterilized, plated onto Potato Dextrose Agar (PDA) media and incubated in the dark at 23°C. To obtain pure cultures, fungal hyphal tips were transferred onto fresh PDA plates 3 times. The pathogen was identified as Pyricularia (Magnaporthe) based on typical conidial morphology (Ellis 1971). Conidia were hyaline, pyriform, 2-septate, and displayed a basal hilum. Conidia measured 5.32 to 10.64 µm (average 8.24 µm) wide by 15.96 to 29.26 µm (average 25.40 µm) long. The identification of Pyricularia was further confirmed genetically via PCR amplification followed by sequencing. Genomic DNA was extracted from a 14-day old pure culture using a CTAB method (Doyle and Doyle 1987). The internal transcribed spacer (ITS) region of ribosomal DNA, calmodulin (CaM) gene, and -tubulin (TUB) gene were amplified using ITS5-ITS4 (White et al. 1990), CMD5-CMD6 (Hong et al. 2005), and Bt2a- Bt2b (Glass and Donaldson 1995) primer sets, respectively. Amplicons were Sanger sequenced and blasted against the NCBI database. Results exhibited 100% (ITS), 100% (CaM), and 99.61% (TUB) homology with Pyricularia oryzae Cavara (GenBank accession no. LC554423.1, CP050920.1, and CP050924.1, respectively). The ITS, CaM, and TUB sequences of the isolate were deposited in GenBank as MZ295207, MZ342893, and MZ342894, respectively. In a greenhouse (23°C, 80% RH), Koch's postulates were carried out by using oat seedlings cv. Horizon 270 grown in Kord sheet pots filled with Sun Gro professional growing mix, and a P. oryzae spore suspension containing 104 conidia ml-1. The spore suspension (10 ml) was sprayed with an air sprayer onto 7 pots of oat seedlings at the two-leaf stage. Seven supplementary pots of oat seedlings of the same cultivar were sprayed with sterile water to act as controls. After inoculation, plants were covered with black plastic bags that had been sprayed with sterile water to maintain high humidity and incubated overnight in the greenhouse. The bags were removed the next day, and plants were evaluated for symptoms in the following days. Seven days after inoculation, plants displayed symptoms similar to those found in the original field sample. Control plants showed no symptoms. Pyricularia oryzae was consistently re-isolated from inoculated symptomatic oat tissues. To our knowledge, this is the first report of gray leaf spot caused by P. oryzae on oat in the state of Georgia and in the continental United States. Pyricularia oryzae can infect several graminaceous plants, including agronomically important crops such as rice (Oryza sativa) and wheat (Triticum spp.) (Chung et al. 2020). Phylogenetic analysis on the ITS region using 6 different host lineages was performed and revealed that this oat isolate was most closely related to the Lolium lineage. This outbreak could have economic implications in oat production.

First report of stem rust, caused by Puccinia graminis, and crown rust, caused by Puccinia coronata var. avenae f. sp. avenae, on tall fescue (Festuca arundinacea) in Georgia, USA.

Stem rust, caused by Puccinia graminis, and crown rust, caused by P. coronata, are common rust diseases on cool-season grasses (Karakkat et al. 2018), for which long-distance spore dispersal was recorded in northern US (Harder and Haber 1992). During the summers of 2019 and 2020, severe infection of stem rust and crown rust was observed on > 60% of tall fescue (Festuca arundinacea) germplasm plants in a breeding nursery located at the University of Georgia, Griffin GA. Rust-infected leaves first presented uredinia pustules, then black telia towards the end of the season. The uredinia pustules of stem rust and crown rust were brick-red and, yellow and arranged along the host veins, respectively. The urediniospores were one-celled, round to ovoid and measured from 20.75±2.44 µm (crown rust) to 27±3.60 µm long (stem rust). The teliospores were two-celled and measured from 45.75±10.14 µm (stem rust) to 51.60±4.0 µm long (crown rust) (Leonard et al. 2005; Cummins 1971). Urediniospores of both rusts were collected from infected plants in the field in April of 2020 using a Piston vacuum pump (Welch by Gardner Denver Ltd.) and stored at -80 °C in 1.5 ml Eppendorf tubes. Genomic DNA was extracted by grinding the urediniospores in liquid nitrogen using mortar and pestle, followed by the cetyltrimethylammonium bromide method (Doyle and Doyle 1987). The internal transcribed spacer (ITS) region of the ribosomal DNA was amplified using the ITS5-ITS4 primers (White et al. 1990). BLASTn and phylogenetic analyses revealed that the sequence of stem rust (GenBank acc. no. MW430963) and crown rust (GenBank acc. no. MW431324) pathogens had >99% similarity with P. graminis (GenBank acc. no. HQ317538) and P. coronata var. avenae f. sp. avenae (clade V; Liu and Hambleton 2013) (GenBank acc. no. EU014044), respectively. Pathogenicity tests were conducted on the tall fescue cultivar 'Bandit'. For each rust, 12 pots (10 cm × 10 cm) were planted, each containing 13 seeds in a Sungro professional growing mix soil (Sun Gro Horticulture Distribution Inc.). The plant materials were kept in the greenhouse at 20°C/ 25°C (night/day),15-hrs of light, and watered twice a week for 4-weeks. Urediniospores were recovered from -80°C and allowed to acclimate at room temperature for 1 h. For each rust, 20 ml of suspension containing 1×105 urediniospores ml-1 and 5 µl of Tween-twenty (Agdia Inc. Elkhart, IN) were used to inoculate 6 pots; while 6 control pots were sprayed with sterile water. After inoculation, plants were allowed to dry for 1 h and then transferred to a dark chamber at 20°C and 90% of humidity for 12-15 h. At 10-days post inoculation, all inoculated plants developed rust symptoms identical to those observed in the field, whereas control plants had no symptoms. Stem and crown rust pathogens were re-isolated from the artificially inoculated tall fescue plants. Based on form, size, color and numbers of cells forming the spores, a 1947 Festuca elatior specimen from Georgia mentioning Puccinia coronata (Hanlin 1966), held at the Julian H. Miller Mycological Herbarium (Catalog No. GAM00013162), was discarded as an earlier record of P. coronata var. avenae and could have been misdiagnosed. Due to the fragile integrity of the original infected plant sample as well as the incipient infection, DNA identification was unsuccessful. To our knowledge, this is the first morphological, genetic and taxonomic report of P. graminis and P. coronata var. avenae f. sp. avenae on tall fescue in Georgia, USA.

Quantitative trait locus mapping combined with variant and transcriptome analyses identifies a cluster of gene candidates underlying the variation in leaf wax between upland and lowland switchgrass ecotypes.

KEY MESSAGE: Mapping combined with expression and variant analyses in switchgrass, a crop with complex genetics, identified a cluster of candidate genes for leaf wax in a fast-evolving region of chromosome 7K. Switchgrass (Panicum virgatum L.) is a promising warm-season candidate energy crop. It occurs in two ecotypes, upland and lowland, which vary in a number of phenotypic traits, including leaf glaucousness. To initiate trait mapping, two F2 mapping populations were developed by crossing two different F1 sibs derived from a cross between the tetraploid lowland genotype AP13 and the tetraploid upland genotype VS16, and high-density linkage maps were generated. Quantitative trait locus (QTL) analyses of visually scored leaf glaucousness and of hydrophobicity of the abaxial leaf surface measured using a drop shape analyzer identified highly significant colocalizing QTL on chromosome 7K (Chr07K). Using a multipronged approach, we identified a cluster of genes including Pavir.7KG077009, which encodes a Type III polyketide synthase-like protein, and Pavir.7KG013754 and Pavir.7KG030500, two highly similar genes that encode putative acyl-acyl carrier protein (ACP) thioesterases, as strong candidates underlying the QTL. The lack of homoeologs for any of the three genes on Chr07N, the relatively low level of identity with other switchgrass KCS proteins and thioesterases, as well as the organization of the surrounding region suggest that Pavir.7KG077009 and Pavir.7KG013754/Pavir.7KG030500 were duplicated into a fast-evolving chromosome region, which led to their neofunctionalization. Furthermore, sequence analyses showed all three genes to be absent in the two upland compared to the two lowland accessions analyzed. This study provides an example of and practical guide for trait mapping and candidate gene identification in a complex genetic system by combining QTL mapping, transcriptomics and variant analysis.

Morphological characterization and genetic diversity analysis of Tunisian durum wheat (Triticum turgidum var. durum) accessions.

BACKGROUND: Tunisia is considered a secondary center of diversification of durum wheat and has a large number of abandoned old local landraces. An accurate investigation and characterization of the morphological and genetic features of these landraces would allow their rehabilitation and utilization in wheat breeding programs. Here, we investigated a diverse collection of 304 local accessions of durum wheat collected from five regions and three climate stages of central and southern Tunisia. RESULTS: Durum wheat accessions were morphologically characterized using 12 spike- and grain-related traits. A mean Shannon-Weaver index (H') of 0.80 was obtained, indicating high level of polymorphism among accessions. Based on these traits, 11 local landraces including Mahmoudi, Azizi, Jneh Khotifa, Mekki, Biskri, Taganrog, Biada, Badri, Richi, Roussia and Souri were identified. Spike length (H' = 0.98), spike shape (H' = 0.86), grain size (H' = 0.94), grain shape (H' = 0.87) and grain color (H' = 0.86) were the most polymorphic morphological traits. The genetic diversity of these accessions was assessed using 10 simple sequence repeat (SSR) markers, with a polymorphic information content (PIC) of 0.69. Levels of genetic diversity were generally high (I = 0.62; He = 0.35). In addition, population structure analysis revealed 11 genetic groups, which were significantly correlated with the morphological characterization. Analysis of molecular variance (AMOVA) showed high genetic variation within regions (81%) and within genetic groups (41%), reflecting a considerable amount of admixture between landraces. The moderate (19%) and high (59%) levels of genetic variation detected among regions and among genetic groups, respectively, highlighted the selection practices of farmers. Furthermore, Mahmoudi accessions showed significant variation in spike density between central Tunisia (compact spikes) and southern Tunisia (loose spikes with open glume), may indicate an adaptation to high temperature in the south. CONCLUSION: Overall, this study demonstrates the genetic richness of local durum wheat germplasm for better in situ and ex situ conservation and for the subsequent use of these accessions in wheat breeding programs.

Fusarium Head Blight and Rust Diseases in Soft Red Winter Wheat in the Southeast United States: State of the Art, Challenges and Future Perspective for Breeding.

Among the biotic constraints to wheat (Triticum aestivum L.) production, fusarium head blight (FHB), caused by Fusarium graminearum, leaf rust (LR), caused by Puccinia triticina, and stripe rust (SR) caused by Puccinia striiformis are problematic fungal diseases worldwide. Each can significantly reduce grain yield while FHB causes additional food and feed safety concerns due to mycotoxin contamination of grain. Genetic resistance is the most effective and sustainable approach for managing wheat diseases. In the past 20 years, over 500 quantitative trait loci (QTLs) conferring small to moderate effects for the different FHB resistance types have been reported in wheat. Similarly, 79 Lr-genes and more than 200 QTLs and 82 Yr-genes and 140 QTLs have been reported for seedling and adult plant LR and SR resistance, respectively. Most QTLs conferring rust resistance are race-specific generally conforming to a classical gene-for-gene interaction while resistance to FHB exhibits complex polygenic inheritance with several genetic loci contributing to one resistance type. Identification and deployment of additional genes/QTLs associated with FHB and rust resistance can expedite wheat breeding through marker-assisted and/or genomic selection to combine small-effect QTL in the gene pool. LR disease has been present in the southeast United States for decades while SR and FHB have become increasingly problematic in the past 20 years, with FHB arguably due to increased corn acreage in the region. Currently, QTLs on chromosome 1B from Jamestown, 1A, 1B, 2A, 2B, 2D, 4A, 5A, and 6A from W14, Ning7840, Ernie, Bess, Massey, NC-Neuse, and Truman, and 3B (Fhb1) from Sumai 3 for FHB resistance, Lr9, Lr10, Lr18, Lr24, Lr37, LrA2K, and Lr2K38 genes for LR resistance, and Yr17 and YrR61 for SR resistance have been extensively deployed in southeast wheat breeding programs. This review aims to disclose the current status of FHB, LR, and SR diseases, summarize the genetics of resistance and breeding efforts for the deployment of FHB and rust resistance QTL on soft red winter wheat cultivars, and present breeding strategies to achieve sustainable management of these diseases in the southeast US.

Intra- and interspecific diversity analyses in the genus Eremurus in Iran using genotyping-by-sequencing reveal geographic population structure.

Eremurus species, better known as 'Foxtail Lily' or 'Desert Candle', are important worldwide in landscaping and the cut-flower industry. One of the centers of highest diversity of the genus Eremurus is Iran, which has seven species. However, little is known about the genetic diversity within the genus Eremurus. With the advent of genotyping-by-sequencing (GBS), it is possible to develop and employ single nucleotide polymorphism (SNP) markers in a cost-efficient manner in any species, regardless of its ploidy level, genome size or availability of a reference genome. Population structure and phylogeographic analyses of the genus Eremurus in Iran using a minimum of 3002 SNP markers identified either at the genus level or at the species level from GBS data showed longitudinal geographic structuring at the country scale for the genus and for the species E. spectabilis and E. luteus, and at the regional scale for E. olgae. Our analyses furthermore showed a close genetic relatedness between E. olgae and E. stenophyllus to the extent that they should be considered subspecies within an E. olgae/stenophyllus species complex. Their close genetic relatedness may explain why crosses between these two (sub)species have been found in the wild and are exploited extensively as ornamentals. Last, current species identification, while robust, relies on flower morphology. A subset of seven SNPs with species-specific (private) alleles were selected that differentiate the seven Eremurus species. The markers will be especially useful for cultivar protection and in hybrid production, where true hybrids could be identified at the seedling stage.

Phoenix phylogeny, and analysis of genetic variation in a diverse collection of date palm (Phoenix dactylifera) and related species.

Date palm (Phoenix dactylifera), one of the most ancient crops, is grown commercially in >30 countries. Using whole plastome assemblies, phylogenetic analyses revealed that cultivated date palm accessions share the same clade with P hoenix sylvestris, P hoenix pusilla and P hoenix acaulis, which are native to the Indian subcontinent, and Phoenix caespitosa that is native to the Arabian Peninsula and the deserts of Somalia. Analysis of genetic diversity and genetic relationships among date palm accessions from 13 producing countries involved 195 date palm accessions that were genotyped at 19 microsatellite loci. Extensive genetic diversity was observed, with many accessions heterozygous for most markers in this clonally propagated crop. The average number of alleles per locus (42.1), expected heterozygosity (0.8), observed heterozygosity (0.47) and fixation indices (FST = 0.42) demonstrated substantial genetic diversity and population structure. Iraqi accessions were found to have the richest allelic diversity, and the most private alleles. The model-based Bayesian method indicated that these accessions could be broadly divided into two structure groups, one group with predominantly African accessions and another predominantly Asian. Some germplasm, especially from Tunisia and Iraq, deviated from this generalization. Many accessions in the STRUCTURE-derived groups were found to be genetic admixtures, with gene flow between Asian and African groups. Indian and Pakistani date palms were found to be most closely related to North African germplasm.

QTL Mapping Combined With Comparative Analyses Identified Candidate Genes for Reduced Shattering in Setaria italica.

Setaria (L.) P. Beauv is a genus of grasses that belongs to the Poaceae (grass) family, subfamily Panicoideae. Two members of the Setaria genus, Setaria italica (foxtail millet) and S. viridis (green foxtail), have been studied extensively over the past few years as model species for C4-photosynthesis and to facilitate genome studies in complex Panicoid bioenergy grasses. We exploited the available genetic and genomic resources for S. italica and its wild progenitor, S. viridis, to study the genetic basis of seed shattering. Reduced shattering is a key trait that underwent positive selection during domestication. Phenotyping of F2:3 and recombinant inbred line (RIL) populations generated from a cross between S. italica accession B100 and S. viridis accession A10 identified the presence of additive main effect quantitative trait loci (QTL) on chromosomes V and IX. As expected, enhanced seed shattering was contributed by the wild S. viridis. Comparative analyses pinpointed Sh1 and qSH1, two shattering genes previously identified in sorghum and rice, as potentially underlying the QTL on Setaria chromosomes IX and V, respectively. The Sh1 allele in S. italica was shown to carry a PIF/Harbinger MITE in exon 2, which gave rise to an alternatively spliced transcript that lacked exon 2. This MITE was universally present in S. italica accessions around the world and absent from the S. viridis germplasm tested, strongly suggesting a single origin of foxtail millet domestication. The qSH1 gene carried two MITEs in the 5'UTR. Presence of one or both MITEs was strongly associated with cultivated germplasm. If the MITE insertion(s) in qSH1 played a role in reducing shattering in S. italica accessions, selection for the variants likely occurred after the domestication of foxtail millet.

Natural variation in genes potentially involved in plant architecture and adaptation in switchgrass (Panicum virgatum L.).

BACKGROUND: Advances in genomic technologies have expanded our ability to accurately and exhaustively detect natural genomic variants that can be applied in crop improvement and to increase our knowledge of plant evolution and adaptation. Switchgrass (Panicum virgatum L.), an allotetraploid (2n = 4× = 36) perennial C4 grass (Poaceae family) native to North America and a feedstock crop for cellulosic biofuel production, has a large potential for genetic improvement due to its high genotypic and phenotypic variation. In this study, we analyzed single nucleotide polymorphism (SNP) variation in 372 switchgrass genotypes belonging to 36 accessions for 12 genes putatively involved in biomass production to investigate signatures of selection that could have led to ecotype differentiation and to population adaptation to geographic zones. RESULTS: A total of 11,682 SNPs were mined from ~ 15 Gb of sequence data, out of which 251 SNPs were retained after filtering. Population structure analysis largely grouped upland accessions into one subpopulation and lowland accessions into two additional subpopulations. The most frequent SNPs were in homozygous state within accessions. Sixty percent of the exonic SNPs were non-synonymous and, of these, 45% led to non-conservative amino acid changes. The non-conservative SNPs were largely in linkage disequilibrium with one haplotype being predominantly present in upland accessions while the other haplotype was commonly present in lowland accessions. Tajima's test of neutrality indicated that PHYB, a gene involved in photoperiod response, was under positive selection in the switchgrass population. PHYB carried a SNP leading to a non-conservative amino acid change in the PAS domain, a region that acts as a sensor for light and oxygen in signal transduction. CONCLUSIONS: Several non-conservative SNPs in genes potentially involved in plant architecture and adaptation have been identified and led to population structure and genetic differentiation of ecotypes in switchgrass. We suggest here that PHYB is a key gene involved in switchgrass natural selection. Further analyses are needed to determine whether any of the non-conservative SNPs identified play a role in the differential adaptation of upland and lowland switchgrass.