Salix Purpurea Research Articles

Indigo carmine biodegradation by endophytic Microbacterium zeae K5: Enzymatic insights, degradation mechanism, and ecotoxicity analysis

The enormous amount of colored effluent release from dying industries into fresh and marine reservoirs, causing ecotoxicity and serious health problems. Pertaining to this, the current research emphasis on the decolorization and degradation treatment of Indigo carmine (IC). The decolorization of IC with endophytic Microbacteium zeae K5, isolated from the root of the Salix purpurea plant was studied. In a minimal salt medium with shaking, full dye decolorization (400 mg/L) was achieved within a 24 h incubation period. During dye degradation, the activities of enzymes such as laccase (12.02 U/g), manganese peroxidase (4.23 U/g), and quinone dehydrogenase (0.09 U/g) were also observed. The biodegradation of IC into isatin sulfonic acid and isatin was confirmed by UV–VIS spectrophotometry and GC-MS analysis of dye samples extracted with ethyl acetate. Finally, phytotoxicity studies revealed that the IC degraded metabolites toxicity was lower than that of the parent dye compound. The current study demonstrated isolate M. zeae K5 has ability to efficiently break down IC, indicating its potential for future bioremediation uses.

Genetically-clustered antifungal phytocytokines and receptor protein family members cooperate to trigger plant immune signaling.

Phytocytokines regulate plant immunity by cooperating with cell-surface proteins. Populus trichocarpa RUST INDUCED SECRETED PEPTIDE 1 (PtRISP1) exhibits an elicitor activity in poplar, as well as a direct antimicrobial activity against rust fungi. PtRISP1 gene directly clusters with a gene encoding a leucine-rich repeat receptor protein (LRR-RP), that we termed RISP-ASSOCIATED LRR-RP (PtRALR). In this study, we used phylogenomics to characterize the RISP and RALR gene families, and molecular physiology assays to functionally characterize RISP/RALR pairs. Both RISP and RALR gene families specifically evolved in Salicaceae species (poplar and willow), and systematically cluster in the genomes. Despite a low sequence identity, Salix purpurea RISP1 (SpRISP1) shows properties and activities similar to PtRISP1. Both PtRISP1 and SpRISP1 induced a reactive oxygen species (ROS) burst and mitogen-activated protein kinases (MAPKs) phosphorylation in Nicotiana benthamiana leaves expressing the respective clustered RALR. PtRISP1 also triggers a rapid stomatal closure in poplar. Altogether, these results suggest that plants evolved phytocytokines with direct antimicrobial activities, and that the genes coding these phytocytokines co-evolved and physically cluster with genes coding LRR-RPs required to initiate immune signaling.

Impacts of in-vitro zebularine treatment on genome-wide DNA methylation and transcriptomic profiles in Salix purpurea L.

Renewable energy resources such as biomass are crucial for a sustainable global society. Trees are a major source of lignocellulosic biomass, which can vary in response to different environmental factors owing to epigenetic regulation, such as DNA C-methylation. To investigate the effects of DNA methylation on plant development and wood formation, and its impacts on gene expression, with a focus on secondary cell wall (SCW)-associated genes, Salix purpurea plantlets were cloned from buds derived from a single hybrid tree for both treatment and control conditions. For the treatment condition, buds were exposed to 50 μM zebularine in vitro and a combined strategy of whole-genome bisulfite sequencing (WGBS) and RNA-seq was employed to examine the methylome and transcriptome profiles of different tissues collected at various time points under both conditions. Transcriptomic and methylome data revealed that most of the promoter and gene body demethylation had no marked effects on the expression profiles of genes. Nevertheless, gene expression tended to decrease with the increased methylation levels of genes with highly methylated promoters. Results indicated that demethylation is less evident in centromeric regions and sex chromosomes. Promoters of secondary cell wall-associated genes, such as 4-coumarate-CoA ligase-like and Rac-like GTP-binding protein RHO, were differentially methylated in the secondary xylem samples collected from two-month potted treated plants compared to control samples. Our results provide novel insights into DNA methylation and gene expression landscapes and a basis for investigating the epigenetic regulation of wood formation in S. purpurea as a model plant for bioenergy species.

Soil mite communities (Acari: Gamasina) from different ecosystem types from Romania

The study aimed to characterize the distribution of soil mite populations (Acari: Gamasina) from several terrestrial ecosystems, some of them little studied in Romania as well as in Europe. We studied the Gamasina communities in seven ecosystem types from the Doftana Valley, Prahova County: Luzulo-Fagetum beech forest; Medio-European limestone beech forest of the Cephalanthero-Fagion; Pannonic woods with Quercus petraea and Carpinus betulus; Galio-carpinetum oak – hornbeam forests; Alpine rivers and their ligneous vegetation with Myricaria germanica; alluvial shrub, characteristic for a hilly-mountain area, with Salix purpurea; adjacent area to an inland cliff ecosystem. A total of 240 soil samples, 63 species and 475 individuals were analysed. The samples were taken from May 2006 till September 2010. The highest numerical abundance and species diversity was found in the area located nearby to an inland cliff, which is an early succession stage ecosystem. Considering the dominance and constancy indices quantified for mites from all investigated areas, the highest percent was recorded by the recedent-subrecedent and accessory-accidental species. Based on similarity index two different characteristic groups of gamasid populations were delimited: one for deciduous forests and one for shrubs. DCA ordination successfully handled the variation in soil mite species communities taking account of the soil type.

Diversity and distribution of Phytophthora species across different types of riparian vegetation in Italy with the description of Phytophthora heteromorpha sp. nov.

Riparian formations encompass a diverse suite of transitional zones between terrestrial and aquatic ecosystems. During the last decades, these formations have been impacted by several emerging diseases. The first outbreaks were detected on alder formations, but have progressively also been observed on other plant species such as Betula pubescens, Nerium oleander, Populus alba, Salix alpina, Salix purpurea and Tamarix gallica. Declining plants showed a plethora of symptoms (leaf spot, shoot blight, bleeding cankers and root rot) indicative of Phytophthora infections. Since there is little information about the aetiology of these pathosystems, from November 2019 to March 2023, an in-depth study was conducted in 46 riparian ecosystems spanning from the Mediterranean to Alpine regions. Overall, 744 symptomatic samples (stem bleeding cankers and root with rhizosphere) from 27 host species were collected for Phytophthora isolation. Based on morphology and DNA sequence data, 20 known Phytophthora species belonging to seven phylogenetic clades have been identified: P. plurivora (202 isolates), P. gonapodyides (156), P. pseudosyringae (84), P. lacustris (57), P. acerina (31), P. idaei (30), P. alpina (20), P. pseudocryptogea (19), P. cambivora (13), P. pseudotsugae (13), P. cactorum (9), P. honggalleglyana (6), P. pseudogregata (6), P. debattistii (4), P. multivora (4), P. cinnamomi (3), P. bilorbang (2) P. crassamura (2), P. ilicis (2) and P. inundata (2). In addition, 26 isolates of a new putative species obtained from Alnus incana and Pinus sylvestris are described here as Phytophthora heteromorpha sp. nov. The new species proved to be pathogenic on grey alder causing symptoms congruent with field observations. This study represents the most comprehensive investigation on the Phytophthora species associated with declining riparian vegetation in Italy and highlights that the polyphagous pathogen P. plurivora represents a growing threat to Mediterranean, temperate and alpine ecosystems.

Revealing the transitory and local effect of zebularine on development and on proteome dynamics of Salix purpurea.

DNA methylation plays major roles in the epigenetic regulation of gene expression, transposon and transcriptional silencing, and DNA repair, with implications in developmental processes and phenotypic plasticity. Relevantly for woody species, DNA methylation constitutes a regulative layer in cell wall dynamics associated with xylogenesis. The use of methyltransferase and/or demethylase inhibitors has been proven informative to shed light on the methylome dynamics behind the regulation of these processes. The present work employs the cytidine analog zebularine to inhibit DNA methyltransferases and induce DNA hypomethylation in Salix purpurea plantlets grown in vitro and in soil. An integrative approach was adopted to highlight the effects of zebularine on proteomic dynamics, revealing age-specific (3 weeks of in vitro culture and 1 month of growth in soil) and tissue-specific (stem and root) effects. After 3 weeks of recovery from zebularine treatment, a decrease of 5-mC levels was observed in different genomic contexts in the roots of explants that were exposed to zebularine, whereas a functionally heterogeneous subset of protein entries was differentially accumulated in stem samples, including entries related to cell wall biosynthesis, tissue morphogenesis, and hormonal regulation. Significant proteomic remodeling was revealed in the development from in vitro to in-soil culture, but no significant changes in 5-mC levels were observed. The identification of tissue-specific proteomic hallmarks in combination with hypomethylating agents provides new insights into the role of DNA methylation and proteome in early plant development in willow species. Proteomic data are available via ProteomeXchange with identifier PXD045653. WGBS data are available under BioProject accession PRJNA889596.

Draft Genome of Nocardia canadensis sp. nov. Isolated from Petroleum-Hydrocarbon-Contaminated Soil.

The bacterial strain WB46 was isolated from the rhizosphere of willow plants (Salix purpurea L.) growing in soil contaminated with petroleum hydrocarbons. The strain was subjected to whole-genome shotgun sequencing using Illumina HiSeq. Its draft genome is 7.15 Mb, with a 69.55% GC content, containing 6387 protein-coding genes and 51 tRNA and 15 rRNA sequences. The quality and reliability of the genome were assessed using CheckM, attaining an estimated genome completeness of 98.75% and an estimated contamination of 1.68%. These results indicate a high-quality genome (>95%) and low contamination (<5%). Many of these genes are responsible for petroleum hydrocarbon degradation, such as alkane 1-monooxygenase (alkB) and naphthalene dioxygenase (ndo). 16S rRNA gene analysis, including in silico DNA-DNA hybridization (DDH) and average nucleotide identity (ANI), showed that strain WB46 belongs to the genus Nocardia, and the most closely related species is Nocardia asteroides. The strain WB46 showed a distance of 63.4% and sequence identity of 88.63%, respectively. These values fall below the threshold levels of 70% and 95%, respectively, suggesting that the strain WB46 is a new species. We propose the name of Nocardia canadensis sp. nov. for this new species. Interestingly, the sequence divergence of the 16S rRNA gene showed that the divergence only occurred in the V2 region. Therefore, the conventional V3-V4, V5-V7, or V8-V9 targeting metabarcoding, among others, would not be able to assess the diversity related to this new species.

Functional analysis of Salix purpurea genes support roles for ARR17 and GATA15 as master regulators of sex determination.

The Salicaceae family is of growing interest in the study of dioecy in plants because the sex determination region (SDR) has been shown to be highly dynamic, with differing locations and heterogametic systems between species. Without the ability to transform and regenerate Salix in tissue culture, previous studies investigating the mechanisms regulating sex in the genus Salix have been limited to genome resequencing and differential gene expression, which are mostly descriptive in nature, and functional validation of candidate sex determination genes has not yet been conducted. Here, we used Arabidopsis to functionally characterize a suite of previously identified candidate genes involved in sex determination and sex dimorphism in the bioenergy shrub willow Salix purpurea. Six candidate master regulator genes for sex determination were heterologously expressed in Arabidopsis, followed by floral proteome analysis. In addition, 11 transcription factors with predicted roles in mediating sex dimorphism downstream of the SDR were tested using DAP-Seq in both male and female S. purpurea DNA. The results of this study provide further evidence to support models for the roles of ARR17 and GATA15 as master regulator genes of sex determination in S. purpurea, contributing to a regulatory system that is notably different from that of its sister genus Populus. Evidence was also obtained for the roles of two transcription factors, an AP2/ERF family gene and a homeodomain-like transcription factor, in downstream regulation of sex dimorphism.

Genome-Wide Identification of Pleiotropic Drug Resistance (PDR) Transporters in Salix purpurea and Expression Analysis in Response to Various Heavy Metal Stresses

Pleiotropic drug resistance (PDR) transporters, which are part of the ABCG subfamily of ATP-binding cassette (ABC) transporters, have been found to be involved in heavy metal tolerance. Salix species (willow) is widely regarded as a perfect candidate for phytoremediation of heavy metals because of its substantial biomass, strong tolerance, and remarkable capacity to accumulate heavy metals. However, the phylogeny and mechanisms underlying the response to heavy metals within the PDR family in willow have yet to be determined. In this study, we discovered and valuated a total of 21 PDR genes in the genome of Salix purpurea. The phylogenetic relationships of these genes were used to classify them into five major clades. The SpPDRs exhibited variations in exon-intron distribution patterns and gene lengths across different branches. Cis-acting elements linked to stress response, drought induction, low temperature, and defense response were discovered in the promoters of PDRs. Significant variations in the transcription levels of various PDR genes were observed across different tissues under heavy metal stress, with distinct heavy metals regulating different PDR members. In roots, PDR4 and PDR21 exhibited high expression levels. Meanwhile, PDR7 and PDR17 showed similar transcription patterns across all analyzed tissues. Furthermore, there was a significant and positive correlation between PDR5 and PDR16, whereas a significant and negative correlation was detected between PDR3 and PDR9, suggesting that the response of PDR members to heavy metals is complex and multifaceted. These findings will establish a vital basis for comprehending the biological functionalities of PDR genes, specifically their involvement in the regulation of willow’s tolerance to heavy metals.

Экологическая оценка пространственного варьирования и сезонной динамики потоков N2O и CH4 из почвы в условиях города

The article considers the ecological assessment of the seasonality of greenhouse gas emissions such as methane and nitrogen oxide (I), their spatial variation depending on the microrelief, which affects the conditions of humidification, in urban conditions in the planting of Purple Willow (Salix purpurea) at the Ecological Station of the Russian State Agrarian University-Moscow State Agricultural Academy named after K.A. Timiryazev. The assessment took into account indicators such as soil temperature and humidity, which are the determining characteristics in the intensity of emission fluxes of methane and nitrogen oxide(I) from soils. The dependence of seasonality on the emission of N2O and CH4 fluxes in various microrelief variants in the plantings of the Purple Willow (Salix purpurea) in the city has been established. Keywords: GREENHOUSE GAS FLOWS, EMISSION OF GASES INTO THE ATMOSPHERE, URBAN SOILS, NITROUS OXIDE, METHANE, CLIMATE CHANGE, GREENHOUSE GAS EMISSIONS INVENTORY

Inter- and intraspecific diversity of Salix bark phenolic profiles – A resource for the pharmaceutical industry

Due to their content of phenolic compounds, willow bark preparations are used as an herbal remedy. The large diversity of phenolic secondary metabolites in Salix still provides a resource for the identification of bioactive compounds in particular species, including species not yet in focus from a phytopharmaceutical perspective. The present study describes the bark phenolic profile of 13 Salix species analyzed by HPLC-MS: Salix alba, Salix babylonica, Salix daphnoides, Salix fragilis, Salix hastata, Salix myrsinifolia, Salix pentandra, Salix purpurea, Salix repens (including subspecies S. repens ssp. arenaria and S. repens ssp. repens), Salix rosmarinifolia, Salix sachalinensis, Salix triandra and Salix viminalis. The analyzed profiles comprised the chemical groups of salicylates, flavonoids, procyanidins, phenolic acid derivatives, and some unclassified phenolics. Particular compounds were detected in species where they have not been previously reported. Apart from interspecific diversity, qualitative variability within species was observed as certain components were detected only in some of the analyzed genotypes. The knowledge on specific phenolic profiles of species and genotypes is the basis for the selection of suitable willow bark material with certain desired bioactive properties. Furthermore, the high inter- and intraspecific variability points out the necessity for product standardization of willow bark raw material.

Structural variation of a sex-linked region confers monoecy and implicates GATA15 as a master regulator of sex in Salix purpurea.

The Salicaceae, including Populus and Salix, are dioecious perennials that utilize different sex determination systems. This family provides a useful system to better understand the evolution of dioecy and sex chromosomes. Here, a rare monoecious genotype of Salix purpurea, 94003, was self- and cross-pollinated and progeny sex ratios were used to test hypotheses on possible mechanisms of sex determination. To delimit genomic regions associated with monoecious expression, the 94003 genome sequence was assembled and DNA- and RNA-Seq of progeny inflorescences was performed. Based on alignments of progeny shotgun DNA sequences to the haplotype-resolved monoecious 94003 genome assembly and reference male and female genomes, a 1.15 Mb sex-linked region on Chr15W was confirmed to be absent in monecious plants. Inheritance of this structural variation is responsible for the loss of a male-suppressing function in what would otherwise be genetic females (ZW), resulting in monoecy (ZWH or WWH ), or lethality, if homozygous (WH WH ). We present a refined, two-gene sex determination model for Salix purpurea, mediated by ARR17 and GATA15 that is different from the single-gene ARR17-mediated system in the related genus Populus.

SALICIN CONTENT FROM SALIX ALBA L. AND SALIX PURPUREA L. EXTRACTS AND ITS ANTIBACTERIAL EFFECTS

The species Salix alba L. and Salix purpurea L. are medicinal herbs, frequently used in the pharmaceutical industry. This study performs phytochemical and antibacterial analysis of ethanolic extracts from the bark of these two species. The qualitative and quantitative phytochemical analyses were performed by thin-layer chromatography identifying the salicin and salicylic acid; the salicin content was assessed by high performance liquid chromatography. The bark extracts yielded 4.300 μg/ml-1 salicin for Salix alba and 1.167 μg/ml-1 salicin for Salix purpurea. The plant extracts were tested on Gram negative and positive bacteria (Escherichia coli and Staphylococcus aureus). Both tested strains showed a mild resistance, but S. aureus showed slightly higher inhibition by all extract samples.

Protoplast-Based Transient Expression and Gene Editing in Shrub Willow (Salix purpurea L.).

Shrub willows (Salix section Vetrix) are grown as a bioenergy crop in multiple countries and as ornamentals across the northern hemisphere. To facilitate the breeding and genetic advancement of shrub willow, there is a strong interest in the characterization and functional validation of genes involved in plant growth and biomass production. While protocols for shoot regeneration in tissue culture and production of stably transformed lines have greatly advanced this research in the closely related genus Populus, a lack of efficient methods for regeneration and transformation has stymied similar advancements in willow functional genomics. Moreover, transient expression assays in willow have been limited to callus tissue and hairy root systems. Here we report an efficient method for protoplast isolation from S. purpurea leaf tissue, along with transient overexpression and CRISPR-Cas9 mediated mutations. This is the first such report of transient gene expression in Salix protoplasts as well as the first application of CRISPR technology in this genus. These new capabilities pave the way for future functional genomics studies in this important bioenergy and ornamental crop.

Nantucket's Neglected Herbivores II: Diptera

Records of Diptera and their hymenopteran parasitoids are reported from a ten-year study of herbivorous insects of Nantucket Island, Massachusetts, USA, with a focus on searching for galls, leaf mines, and other characteristic feeding evidence on host plants. Our field observations and reared specimens are supplemented with herbarium records and Johnson's (1930) list of the insect fauna of Nantucket. Compared with Johnson's list of nine species of Agromyzidae, we identified 53 along with 15 others determined only to genus or higher taxa (a few of the latter could conceivably be conspecific with the former or with one another). We found 37 named species of Cecidomyiidae (including eight of the nine on Johnson's list), as well as 26 others that are undescribed or are currently unidentifiable for other reasons (a few of which could conceivably be conspecific with one of the identified species, or represent galls not actually caused by midges). A few rearings and collections of Anthomyiidae, Chloropidae, Ephydridae, Phoridae, Sphaeroceridae, and Tephritidae are reported, adding another seven species and two genera to Johnson's list. Cerodontha (Dizygomyza) edithae Eiseman and Lonsdale (Agromyzidae), an as yet undescribed species of Ophiomyia Braschnikov (Agromyzidae), Megaselia nantucketensis Eiseman and Hartop (Phoridae), and the cecidomyiid parasitoids Platygaster tephrosiae Buhl and Eiseman and P. vitisiellae Buhl and Eiseman (Hymenoptera: Platygastridae) are known only from specimens reared as part of this study, which also produced paratypes of Liriomyza pistilla Lonsdale (Agromyzidae). Noteworthy cecidomyiid records include the first known specimens of an undescribed Asphondylia Loew on Solidago sempervirens L. (Asteraceae), galls of an undetermined lasiopterid species on Tephrosia virginiana (L.) Pers. (Fabaceae) that have only been found on Nantucket, and previously unreported galls on Gaylussacia baccata (Wangenh.) K. Koch (Ericaceae), Ionactis linariifolia (L.) Greene (Asteraceae) (Dasineura Rondani sp.), Quercus prinoides Willd. (Fagaceae), Salix purpurea L. (Salicaceae), and Solidago latissimifolia Mill. (Asteraceae) (Asphondylia sp., Rhopalomyia Rübsaamen sp.).

Evidence of Asexual Overwintering of Melampsora paradoxa and Mapping of Stem Rust Host Resistance in Salix.

Melampsora rust is a devastating disease of shrub willow in North America. Previous work has identified Melampsora paradoxa as one of two identified rust species in New York State that infect Salix purpurea and other important Salix host species, however little is known about the population of this rust species in this region. Genotyping-by-sequencing was used to identify single nucleotide polymorphisms (SNPs) and assess population diversity of M. paradoxa isolates collected from three Salix breeding populations in Geneva, NY between 2015 and 2020. Statistical analyses of SNP revealed that all isolates collected were clonally derived even though they were collected across years. In 2020, isolates were collected from stem infections where uredospore pustules were observed, and these isolates were also identical to M. paradoxa collected in previous seasons. These data suggest that M. paradoxa sampled across multiple years overwintered and reproduced asexually and that stem infection is a possible mechanism for overwintering, both of which are novel findings for this rust species. Additionally, field disease ratings were conducted on a S. purpurea × S. suchowensis F1 breeding population with high disease severity, enabling the discovery of QTL for resistance on chromosomes 1 and 19. Lastly, Colletotrichum salicis was frequently associated with stem rust and may play a role in M. paradoxa stem infection. Together, this work is the first substantial exploration into M. paradoxa population biology, stem infection, and host resistance in Salix.

Analysis of the NAC Gene Family in Salix and the Identification of SpsNAC005 Gene Contributing to Salt and Drought Tolerance

The NAC gene family is of great value for plant stress resistance and development. In this study, five NAC genes with a typical NAM domain were isolated from Salix psammophila, which is a stress-resistant willow endemic to western China. Two hundred sixty-two NAC genes from Salix psammophila, Salix purpurea, and Arabidopsis were used to construct the phylogenetic tree to examine the phylogenetic relationship. Five NAC genes in Salix psammophila were the focus of bioinformatics analysis and conserved structural domain analysis. The SpsNAC005 gene was overexpressed in Populus hopeiensis, and the transgenic lines were subjected to salt and simulated drought stress to analyze their phenotype changes and tolerance to stress. The results showed that transgenic poplar height and leaf area increased by 29.73% and 76.36%, respectively, compared with those of wild-type plants. Under stress treatment, the height growth rates and ground diameter growth rates of the transgenic lines were significantly higher than those of the wild-type, whereas their fresh weight and dry weight were decreased compared to those of the wild-type. The SOD activities, POD activities, and Pro contents of the transgenic plants were significantly increased, and the accumulation of MDA was significantly lower than that in the wild-type, and the transgenic lines showed clear tolerance to salt and drought. The expressions of the SOS1, MPK6, HKT1, and P5CS1 genes were downregulated in the transgenic lines. The expression of the PRODH1 gene was downregulated in the transgenic lines. These results indicate that overexpression of the SpsNAC005 gene in transgenic plants can promote plant growth and development and improve tolerance to salt and drought.

Genetic mapping of sexually dimorphic volatile and non-volatile floral secondary chemistry of a dioecious willow.

Secondary chemistry often differs between sexes in dioecious plant species, a pattern attributed to its possible role in the evolution and/or maintenance of dioecy. We used GC-MS to measure floral volatiles emitted from, and LC-MS to quantitate non-volatile secondary compounds contained in, female and male Salix purpurea willow catkins from an F2 family. Using the abundance of these chemicals, we then performed quantitative trait locus (QTL) mapping to locate them on the genome, identified biosynthetic candidate genes in the QTL intervals, and examined expression patterns of candidate genes using RNA-seq. Male flowers emitted more total terpenoids than females, but females produced more benzenoids. Male tissue contained greater amounts of phenolic glycosides, but females had more chalcones and flavonoids. A flavonoid pigment and a spermidine derivative were found only in males. Male catkins were almost twice the mass of females. Forty-two QTL were mapped for 25 chemical traits and catkin mass across 16 of the 19 S. purpurea chromosomes. Several candidate genes were identified, including a chalcone isomerase associated with seven compounds. A better understanding of the genetic basis of the sexually dimorphic chemistry of a dioecious species may shed light on how chemically mediated ecological interactions may have helped in the evolution and maintenance of dioecy.

Mapping the sex determination region in the Salix F1 hybrid common parent population confirms a ZW system in six diverse species.

Within the genus Salix, there are approximately 350 species native primarily to the northern hemisphere and adapted to a wide range of habitats. This diversity can be exploited to mine novel alleles conferring variation important for production as a bioenergy crop, but also to identify evolutionarily important genes, such as those involved in sex determination. To leverage this diversity, we created a mapping population by crossing 6 Salix species (Salix viminalis, Salix suchowensis, Salix integra, Salix koriyanagi, Salix udensis, and Salix alberti) to common male and female Salix purpurea parents. Each family was genotyped via genotyping-by-sequencing and assessed for kinship and population structure as well as the construction of 16 backcross linkage maps to be used as a genetic resource for breeding and selection. Analyses of population structure resolved both the parents and F1 progeny to their respective phylogenetic section and indicated that the S. alberti parent was misidentified and was most likely S.suchowensis. Sex determining regions were identified on Salix chromosome 15 in the female-informative maps for seven of the eight families indicating that these species share a common female heterogametic ZW sex system. The eighth family, S. integra × S. purpurea, was entirely female and had a truncated chromosome 15. Beyond sex determination, the Salix F1 hybrid common parent population (Salix F1 HCP) introduced here will be useful in characterizing genetic factors underlying complex traits, aid in marker-assisted selection, and support genome assemblies for this promising bioenergy crop.

The Melampsora americana Population on Salix purpurea in the Great Lakes Region Is Highly Diverse with a Contributory Influence of Clonality.

Shrub willows (Salix spp.) are emerging as a viable lignocellulosic, second-generation bioenergy crop with many growth characteristics favorable for marginal lands in New York State and surrounding areas. Willow rust, caused by members of the genus Melampsora, is the most limiting disease of shrub willow in this region and remains extremely understudied. In this study, genetic diversity, genetic structure, and pathogen clonality were examined in Melampsora americana over two growing seasons via genotyping-by-sequencing to identify single-nucleotide polymorphism markers. In conjunction with this project, a reference genome of rust isolate R15-033-03 was generated to aid in variant discovery. Sampling between years allowed regional and site-specific investigation into population dynamics, in the context of both wild and cultivated hosts within high-density plantings. This work revealed that this pathogen is largely panmictic over the sampled areas, with few sites showing moderate genetic differentiation. These data support the hypothesis of sexual recombination between growing seasons because no genotype persisted across the two years of sampling. Additionally, clonality was determined as a driver of pathogen populations within cultivated fields and single shrubs; however, there is also evidence of high genetic diversity of rust isolates in all settings. This work provides a framework for M. americana population structure in the Great Lakes region, providing crucial information that can aid in future resistance breeding efforts.