Early selection of novel triploid hybrids of shrub willow with improved biomass yield relative to diploids

BACKGROUND: Genetic improvement of shrub willow (Salix), a perennial energy crop common to temperate climates, has led to the development of new cultivars with improved biomass yield, pest and disease resistance, and biomass composition suitable for bioenergy applications. These improvements have largely been associated with species hybridization, yet little is known about the genetic mechanisms responsible for improved yield and performance of certain willow species hybrids. RESULTS: The top performing genotypes in this study, representing advanced pedigrees compared with those in previous studies, were mostly triploid in nature and outperformed current commercial cultivars. Of the genotypes studied, the diploids had the lowest mean yield of 8.29 oven dry Mg ha(−1) yr(−1), while triploids yielded 12.65 Mg ha(−1) yr(−1), with the top five producing over 16 Mg ha(−1) yr(−1). Triploids had high stem area and height across all three years of growth in addition to greatest specific gravity. The lowest specific gravity was observed among the tetraploid genotypes. Height was the early trait most correlated with and the best predictor of third-year yield. CONCLUSIONS: These results establish a paradigm for future breeding and improvement of Salix bioenergy crops based on the development of triploid species hybrids. Stem height and total stem area are effective traits for early prediction of relative yield performance

Characterization of a large sex determination region in Salix purpurea L. (Salicaceae).

Dioecy has evolved numerous times in plants, but heteromorphic sex chromosomes are apparently rare. Sex determination has been studied in multiple Salix and Populus (Salicaceae) species, and P. trichocarpa has an XY sex determination system on chromosome 19, while S. suchowensis and S. viminalis have a ZW system on chromosome 15. Here we use whole genome sequencing coupled with quantitative trait locus mapping and a genome-wide association study to characterize the genomic composition of the non-recombining portion of the sex determination region. We demonstrate that Salix purpurea also has a ZW system on chromosome 15. The sex determination region has reduced recombination, high structural polymorphism, an abundance of transposable elements, and contains genes that are involved in sex expression in other plants. We also show that chromosome 19 contains sex-associated markers in this S. purpurea assembly, along with other autosomes. This raises the intriguing possibility of a translocation of the sex determination region within the Salicaceae lineage, suggesting a common evolutionary origin of the Populus and Salix sex determination loci

DISSECTION OF GENOTYPIC AND PHENOTYPIC VARIATION IN SHRUB WILLOW (SALIX PURPUREA L.)

Salix spp. and hybrids (shrub willow) are bred as dedicated bioenergy crops around the world, however there is still untapped potential for genetic improvement. Salix is a widely adapted and genetically diverse genus, but few studies have utilized this diversity for trait mapping or development of genomic tools. The studies of this dissertation focused on S. purpurea, a core reference species for breeding shrub willow bioenergy crops in North America, to understand the genetic basis for key traits and identify quantitative trait loci (QTL) that can be utilized for marker-assisted selection (MAS). A genetically diverse germplasm collection of 110 accessions from the Northeastern US was assembled, genotyped using genotyping-by-sequencing (GBS) and extensively phenotyped for key biomass, morphological, phenological, physiological, physical and chemical wood properties, and disease resistance across three years and three replicated experimental sites. The association population was further expanded through the addition of 157 accessions from Europe. Population genetic analysis revealed significant population stratification, subpopulation structuring and differentiation corresponding mainly to geographic regions. Phenotypic analysis of the US population showed wide variation among genotypes and revealed a majority of traits to be sexually dimorphic in favor of male plants including yield, but also a female biased sex-ratio. This suggests that the sex determination locus in Salix may be linked to loci responsible for growth and fitness. The natural phenotypic variation of the US population was evaluated in a genome-wide association study (GWAS), which revealed several candidate genes for high biomass yield and traits significantly correlated to yield, as well as resistance to Melampsora rust. The studies conducted here advance the understanding of traits contributing to increased biomass in woody plants, lay the groundwork for validating the underlying genes, and will contribute to the development of marker-assisted selection and genomic selection to accelerate the efficiency and accuracy of breeding

cDNA Transcriptome of <i>Arabidopsis</i> Reveals Various Defense Priming Induced by a Broad-Spectrum Biocontrol Agent <i>Burkholderia</i> sp. SSG

Burkholderia sp. SSG is a potent biological control agent. Even though its survival on the leaf surface declined rapidly, SSG provided extended, moderate plant protection from a broad spectrum of pathogens. This study used Arabidopsis Col-0 and its mutants, eds16-1, npr1-1, and pad4-1 as model plants and compared treated plants with non-treated controls to elucidate whether SSG triggers plant defense priming. Only eds16-1 leaves with SSG became purplish, suggesting the involvement of salicylic acid (SA) in SSG-induced priming. cDNA sequencing of Col-0 plants and differential gene expression analysis identified 120 and 119 differentially expressed genes (DEGs) at 6- and 24-h post-treatment (hpt) with SSG, respectively. Most of these DEGs encoded responses to biotic and abiotic stimuli or stresses; four DEGs had more than two isoforms. A total of 23 DEGs were shared at 6 and 24 hpt, showing four regulation patterns. Functional categorization of these shared DEGs, and 44 very significantly upregulated DEGs revealed that SSG triggered various defense priming mechanisms, including responses to phosphate or iron deficiency, modulation of defense-linked SA, jasmonic acid, ethylene, and abscisic acid pathways, defense-related gene regulation, and chromatin modification. These data support that SSG is an induced systemic resistance (ISR) trigger conferring plant protection upon pathogen encounter

High-Resolution Melting Analysis Enables Efficient Detection and Differentiation of Two Boxwood Blight Pathogens by qPCR Assays

Boxwood blight is a devastating disease caused by the fungal pathogens Calonectria henricotiae (Che) and C. pseudonaviculata (Cps). Identification and detection of these pathogens from infected plant material could play a significant role in breeding and selection of resistant cultivars and development of disease management strategies in the ornamental nursery industry. We designed a simple, single-tube method for extraction of PCR-amplifiable DNA from boxwood leaves and cultures of the Calonectria pathogens. Previously developed fungal-specific primers based on histone and calmodulin regions were used to detect and distinguish between Che and Cps using real-time PCR and high-resolution melting (HRM) analysis, with discernable melting temperature differences of 0.5°C between amplified products. Here, we describe a single-tube acetone-based DNA extraction method and qPCR-HRM assay targeting single nucleotide polymorphisms within the calmodulin and histone H3 DNA regions as a fast and highly sensitive molecular method to detect and differentiate between Che and Cps species directly from plant tissue. [Graphic: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license

Ploidy Level Affects Important Biomass Traits of Novel Shrub Willow ( Salix ) Hybrids

Polyploidy is a common observation in the genus Salix, including some of the shrub willow species currently being bred as a potential bioenergy feedstock. Breeding of shrub willow has produced new species hybrids, among which a disproportionate number of high-yielding genotypes are triploid, produced from crosses between diploid and tetraploid parents. These novel hybrids display significant variation in biomass compositional quality, including differences according to ploidy. The triploid and tetraploid genotypes possess lower lignin content than diploid genotypes. Biomass composition was also significantly different across the 3-year growth cycle typical of bioenergy plantings. There were differences in syringyl/guaiacyl (S:G) lignin ratios among the 75 genotypes examined, in addition to significant correlations with willow growth traits, yield, and composition. These differences suggest that a long-term strategy of breeding for triploid progeny will generate cultivars with improved growth traits and wood composition for conversion to biofuels

Boxwood phyllosphere fungal and bacterial communities and their differential responses to film-forming anti-desiccants

Abstract Background Anti-desiccant is a class of agrochemicals widely used to protect plants from water stresses, rapid temperature variations, heat and sunburn, frost and freeze damages, transplant shock, and pathogen and pest attack. Although anti-desiccants are generally considered non-toxic to organisms, it is unclear whether they may impact the phyllosphere microbial communities. In this study, three film-forming anti-desiccant products, TransFilm, Vapor Gard, and Wilt-Pruf were applied to the canopy of two boxwood cultivars ‘Vardar Valley’ and ‘Justin Brouwers’ on April 13 and August 26, 2021. Shoot samples were collected from boxwood plants treated with each of the three products, as well as nontreated control on June 16, August 26 (before the second treatment), and October 18. Microbial and plant genomic DNA was isolated together and 16S rRNA gene and the extended internal transcribed spacer regions were amplified with PCR and sequenced on a Nanopore MinION platform for bacterial and fungal identification. Results Bacterial communities were more diverse than fungal communities. At the phylum level, the boxwood phyllosphere was dominated by Proteobacteria and Ascomycota; at the genus level, Methylobacterium and Shiraia were the most abundant bacteria and fungi, respectively. Among the three film-forming anti-desiccants, Vapor Gard and Wilt-Pruf had more impact than TransFilm on the microbial communities. Specifically, broader impacts were observed on fungal than bacterial community composition and structure, with most affected fungi being suppressed while bacteria promoted. Conclusion This study addressed several major knowledge gaps regarding boxwood phyllosphere microbiota and the impact of anti-desiccants on plant microbiome. We identified diverse microbial communities of boxwood, a major evergreen woody crop and an iconic landscape plant. We also found differential effects of three film-forming anti-desiccants on the composition and structure of bacterial and fungal communities. These findings advanced our understanding of the associated microbiome of this landmark plant, enabling growers to fully utilize the potentials of microbiome and three anti-desiccants in improving boxwood health and productivity

Multiple independent recombinations led to hermaphroditism in grapevine

Hermaphroditic (perfect) flowers were a key trait in grapevine domestication, enabling a drastic increase in yields due to the efficiency of self-pollination in the domesticated grapevine (Vitis vinifera L. ssp. vinifera). In contrast, all extant wild Vitis species are dioecious, each plant having only male or female flowers. In this study, we identified the male (M) and female (f) haplotypes of the sex-determining region (SDR) in the wild grapevine species V. cinerea and confirmed the boundaries of the SDR. We also demonstrated that the SDR and its boundaries are precisely conserved across the Vitis genus using shotgun resequencing data of 556 wild and domesticated accessions from North America, East Asia, and Europe. A high linkage disequilibrium was found at the SDR in all wild grape species, while different recombination signatures were observed along the hermaphrodite (H) haplotype of 363 cultivated accessions, revealing two distinct H haplotypes, named H1 and H2. To further examine the H2 haplotype, we sequenced the genome of two grapevine cultivars, 'Riesling' and 'Chardonnay'. By reconstructing the first two H2 haplotypes, we estimated the divergence time between H1 and H2 haplotypes at ∼6 million years ago, which predates the domestication of grapevine (∼8,000 y ago). Our findings emphasize the important role of recombination suppression in maintaining dioecy in wild grape species and lend additional support to the hypothesis that at least two independent recombination events led to the reversion to hermaphroditism in grapevine

Integrative genomics reveals paths to sex dimorphism in Salix purpurea L.

Sex dimorphism and gene expression were studied in developing catkins in 159 F2 individuals from the bioenergy crop Salix purpurea, and potential mechanisms and pathways for regulating sex development were explored. Differential expression, eQTL, bisulfite sequencing, and network analysis were used to characterize sex dimorphism, detect candidate master regulator genes, and identify pathways through which the sex determination region (SDR) may mediate sex dimorphism. Eleven genes are presented as candidates for master regulators of sex, supported by gene expression and network analyses. These include genes putatively involved in hormone signaling, epigenetic modification, and regulation of transcription. eQTL analysis revealed a suite of transcription factors and genes involved in secondary metabolism and floral development that were predicted to be under direct control of the sex determination region. Furthermore, data from bisulfite sequencing and small RNA sequencing revealed strong differences in expression between males and females that would implicate both of these processes in sex dimorphism pathways. These data indicate that the mechanism of sex determination in Salix purpurea is likely different from that observed in the related genus Populus. This further demonstrates the dynamic nature of SDRs in plants, which involves a multitude of mechanisms of sex determination and a high rate of turnover