Genomic and Transcriptomic Comparisons of the Twig Blight Pathogen, Passalora sequoiae, with Mycosphaerellaceae Foliar and Conifer Pathogens.

Passalora sequoiae is a foliar pathogen to conifer tree species. In this study, we conducted whole-genome and transcriptome analyses on isolates of P. sequoiae collected from symptomatic Leyland cypress leaves from a Christmas tree farm in Mississippi. The objectives for this research were to elucidate the pathogenicity mechanisms of P. sequoiae by characterizing the genome and transcriptome and possibly identify unique and shared predicted genes in comparison with non-conifer/canker and foliar pathogens in the family Mycosphaerellaceae. P. sequoiae was found to be similar to other foliar Mycosphaerellaceae pathogens and likely represents a hemibiotrophic lifestyle based on comparisons across pathogens. The genome and in planta transcriptome highlighted some unique features of P. sequoiae: the significant presence of chitin synthases and fructose-degrading carbohydrate-degrading enzymes, trans-AT PKS genes, and antibiotic gene clusters that were unique to P. sequoiae compared with the other Mycosphaerellaceae species genomes. Several transcripts that were highly expressed in planta were identified as effectors, yet the functions were not characterized. These targets provide ample resources to continue to characterize pathogen-conifer host interactions in conifer foliar pathogens. Furthermore, this research helps build genomic resources for an important plant pathogen on Leyland cypress that will further our ability to develop novel management practices that could begin with breeding for resistance.

Long-read, chromosome-scale assembly of Vitis rotundifolia cv. Carlos and its unique resistance to Xylella fastidiosa subsp. fastidiosa.

BACKGROUND: Muscadine grape (Vitis rotundifolia) is resistant to many of the pathogens that negatively impact the production of common grape (V. vinifera), including the bacterial pathogen Xylella fastidiosa subsp. fastidiosa (Xfsf), which causes Pierce's Disease (PD). Previous studies in common grape have indicated Xfsf delays host immune response with a complex O-chain antigen produced by the wzy gene. Muscadine cultivars range from tolerant to completely resistant to Xfsf, but the mechanism is unknown. RESULTS: We assembled and annotated a new, long-read genome assembly for 'Carlos', a cultivar of muscadine that exhibits tolerance, to build upon the existing genetic resources available for muscadine. We used these resources to construct an initial pan-genome for three cultivars of muscadine and one cultivar of common grape. This pan-genome contains a total of 34,970 synteny-constrained entries containing genes of similar structure. Comparison of resistance gene content between the 'Carlos' and common grape genomes indicates an expansion of resistance (R) genes in 'Carlos.' We further identified genes involved in Xfsf response by transcriptome sequencing 'Carlos' plants inoculated with Xfsf. We observed 234 differentially expressed genes with functions related to lipid catabolism, oxidation-reduction signaling, and abscisic acid (ABA) signaling as well as seven R genes. Leveraging public data from previous experiments of common grape inoculated with Xfsf, we determined that most differentially expressed genes in the muscadine response were not found in common grape, and three of the R genes identified as differentially expressed in muscadine do not have an ortholog in the common grape genome. CONCLUSIONS: Our results support the utility of a pan-genome approach to identify candidate genes for traits of interest, particularly disease resistance to Xfsf, within and between muscadine and common grape.

Genomic insight into the developmental history of southern highbush blueberry populations.

Interspecific hybridization is a common breeding approach for introducing novel traits and genetic diversity to breeding populations. Southern highbush blueberry (SHB) is a blueberry cultivar group that has been intensively bred over the last 60 years. Specifically, it was developed by multiple interspecific crosses between northern highbush blueberry [NHB, Vaccinium corymbosum L. (2n = 4x = 48)] and low-chill Vaccinium species to expand the geographic limits of highbush blueberry production. In this study, we genotyped polyploid blueberries, including 105 SHB, 17 NHB, and 10 rabbiteye blueberry (RE) (Vaccinium virgatum Aiton), from the accessions planted at Poplarville, Mississippi, and accessions distributed in Japan, based on the double-digest restriction site-associated DNA sequencing. The genome-wide SNP data clearly indicated that RE cultivars were genetically distinct from SHB and NHB cultivars, whereas NHB and SHB were genetically indistinguishable. The population structure results appeared to reflect the differences in the allele selection strategies that breeders used for developing germplasm adapted to local climates. The genotype data implied that there are no or very few genomic segments that were commonly introgressed from low-chill Vaccinium species to the SHB genome. Principal component analysis-based outlier detection analysis found a few loci associated with a variable that could partially differentiate NHB and SHB. These SNP loci were detected in Mb-scale haplotype blocks and may be close to the functional genes related to SHB development. Collectively, the data generated in this study suggest a polygenic adaptation of SHB to the southern climate, and may be relevant for future population-scale genome-wide analyses of blueberry.

Identification of Winter Habit Bread Wheat Landraces in the National Small Grains Collection with Resistance to Emerging Stem Rust Pathogen Variants.

Wheat stem rust caused by Puccinia graminis f. sp. tritici is a widespread and recurring threat to wheat production. Emerging P. graminis f. sp. tritici variants are rapidly overcoming major gene resistance deployed in wheat cultivars and new sources of race-nonspecific resistance are urgently needed. The National Small Grains Collection (NSGC) contains thousands of wheat landrace accessions that may harbor unique and broadly effective sources of resistance to emerging P. graminis f. sp. tritici variants. All NSGC available facultative and winter-habit bread wheat landraces were tested in a field nursery in St. Paul, Minnesota, against a bulk collection of six common U.S. P. graminis f. sp. tritici races. Infection response and severity data were collected on 9,192 landrace accessions at the soft-dough stage and resistant accessions were derived from single spikes. Derived accessions were tested in St. Paul a second time to confirm resistance and in a field nursery in Njoro, Kenya against emerging races of P. graminis f. sp. tritici with virulence to many known resistance genes including Sr24, Sr31, Sr38, and SrTmp. Accessions resistant in the St. Paul field were also tested at the seedling stage with up to 13 P. graminis f. sp. tritici races, including TTKSK and TKTTF, and with 19 molecular markers linked with known stem rust resistance genes or genes associated with modern breeding practices. Forty-five accessions were resistant in both U.S. and Kenya field nurseries and lacked alleles linked with known stem rust resistance genes. Accessions with either moderate or strong resistance in the U.S. and Kenya field nurseries and with novel seedling resistance will be prioritized for further study.

Mapping of the stem rust resistance gene Pg13 in cultivated oat.

KEY MESSAGE: The widely deployed, oat stem rust resistance gene Pg13 was mapped by linkage analysis and association mapping, and KASP markers were developed for marker-assisted selection in breeding programs. Pg13 is one of the most extensively deployed stem rust resistance genes in North American oat cultivars. Identification of markers tightly linked to this gene will be useful for routine marker-assisted selection, identification of gene pyramids, and retention of the gene in backcrosses and three-way crosses. To this end, high-density linkage maps were constructed in four bi-parental mapping populations using SNP markers identified from 6K oat Infinium iSelect and genotyping-by-sequencing platforms. Additionally, genome-wide associations were identified using two sets of association panels consisting of diverse elite oat lines in one set and landrace accessions in the other. The results showed that Pg13 was located at approximately 67.7 cM on linkage group Mrg18 of the consensus genetic map. The gene co-segregated with the 7C-17A translocation breakpoint and with crown rust resistance gene Pc91. Co-segregating markers with the best prediction accuracy were identified at 67.7-68.5 cM on Mrg18. KASP assays were developed for linked SNP loci for use in oat breeding.

Comparison of Whole Plant and Detached Leaf Screening Techniques for Identifying Anthracnose Resistance in Strawberry Plants.

Anthracnose is a destructive disease of strawberry caused by several Colletotrichum species including C. acutatum, C. fragariae, and C. gloeosporioides. Identification of anthracnose resistant strawberry germplasm has commonly relied on inoculation of whole plants with isolates of these pathogens. In this study, whole plants and detached leaves from 81 germplasm lines were inoculated with a conidial suspension of isolates of C. acutatum, C. fragariae, and C. gloeosporioides, incubated in the dark at 30°C, 100% relative humidity, for 48 h, and assessed for disease severity based on symptoms on inoculated petioles and leaves. The correlation between the disease severity ratings of the whole plants rated 30 days after inoculation and detached leaves rated 5 days after inoculation was determined. Based on leaf symptoms and petiole lesions, the association between the whole plant leaf disease severity rating (DSR) and detached leaf DSR was positive (rp = 0.70), and the association between the whole plant DSR and the detached leaf DSR was also positive (rp = 0.66). Whole plant and detached leaf DSRs were used to assign each germplasm line to a resistance category, and a posthoc Tukey's test showed that the whole plant DSR means and the detached leaf DSR means for each resistance category differed significantly at p < 0.05. This research was used to develop a strawberry detached leaf assay which can reliably and quickly determine the degree of resistance of strawberry germplasm to anthracnose.

Markers Linked to Wheat Stem Rust Resistance Gene Sr11 Effective to Puccinia graminis f. sp. tritici Race TKTTF.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici, can cause severe yield losses on susceptible wheat varieties and cultivars. Although stem rust can be controlled by the use of genetic resistance, population dynamics of P. graminis f. sp. tritici can frequently lead to defeat of wheat stem rust resistance genes. P. graminis f. sp. tritici race TKTTF caused a severe epidemic in Ethiopia on Ug99-resistant 'Digalu' in 2013 and 2014. The gene Sr11 confers resistance to race TKTTF and is present in 'Gabo 56'. We identified seven single-nucleotide polymorphism (SNP) markers linked to Sr11 from a cross between Gabo 56 and 'Chinese Spring' exploiting a 90K Infinium iSelect Custom beadchip. Five SNP markers were validated on a 'Berkut'/'Scalavatis' population that segregated for Sr11, using KBioscience competitive allele-specific polymerase chain reaction (KASP) assays. Two of the SNP markers, KASP_6BL_IWB10724 and KASP_6BL_IWB72471, were predictive of Sr11 among wheat genetic stocks, cultivars, and breeding lines from North America, Ethiopia, and Pakistan. These markers can be utilized to select for Sr11 in wheat breeding and to detect the presence of Sr11 in uncharacterized germplasm.

Quantitative Trait Loci from Two Genotypes of Oat (Avena sativa) Conditioning Resistance to Puccinia coronata.

Developing oat cultivars with partial resistance to crown rust would be beneficial and cost-effective for disease management. Two recombinant inbred-line populations were generated by crossing the susceptible cultivar Provena with two partially resistant sources, CDC Boyer and breeding line 94197A1-9-2-2-2-5. A third mapping population was generated by crossing the partially resistant sources to validate the quantitative trait locus (QTL) results. The three populations were evaluated for crown rust severity in the field at Louisiana State University (LSU) in 2009 and 2010 and at the Cereal Disease Laboratory (CDL) in St. Paul, MN, in 2009, 2010, and 2011. An iSelect platform assay containing 5,744 oat single nucleotide polymorphisms was used to genotype the populations. From the 2009 CDL test, linkage analyses revealed two QTLs for partial resistance in the Provena/CDC Boyer population on chromosome 19A. One of the 19A QTLs was also detected in the 2009 LSU test. Another QTL was detected on chromosome 12D in the CDL 2009 test. In the Provena/94197A1-9-2-2-2-5 population, only one QTL was detected, on chromosome 13A, in the CDL 2011 test. The 13A QTL from the Provena/94197A1-9-2-2-2-5 population was validated in the CDC Boyer/94197A1-9-2-2-2-5 population in the CDL 2010 and 2011 tests. Comparative analysis of the significant marker sequences with the rice genome database revealed 15 candidate genes for disease resistance on chromosomes 4 and 6 of rice. These genes could be potential targets for cloning from the two resistant parents.

Evaluating global Vaccinium germplasm for resistance against invasive Drosophila suzukii (Diptera: Drosophilidae).

Control of spotted-wing Drosophila, Drosophila suzukii, in small fruits emphasizes biological, cultural, and chemical approaches, whereas studies of host plant resistance as a form of genetic control are just getting underway. The identification of resistance patterns among genotypes of host plants whose fruit, leaves, roots, stems, or seeds are specifically targeted by an invasive pest is the first step in the development of an effective genetic control. Therefore, a detached fruit bioassay was developed to screen for D. suzukii oviposition and larval infestation within berries from 25 representative species and hybrids of wild and cultivated Vaccinium. Ten Vaccinium species showed strong resistance; among them, two wild diploids originating from within the fly's native range: V. myrtoides and V. bracteatum. Other resistant species came from the sections Pyxothamnus and Conchophyllum. They included New World V. consanguineum and V. floribundum. Large-cluster blueberry, V. amoenum, and three Floridian genotypes of related rabbiteye blueberry, V. virgatum, were the only hexaploids expressing strong resistance against D. suzukii. Most screened blueberry genotypes from managed lowbush and cultivated highbush types were susceptible to the flies' attacks (i.e., oviposition). Tetraploid blueberries tended to host the most eggs, whereas diploids and hexaploids harbored 50%-60% fewer eggs, on average. D. suzukii cannot lay eggs or complete development in the smallest, sweetest, and firmest diploid fruits. Likewise, certain genotypes of large-fruited tetraploid and hexaploid blueberry strongly curbed D. suzukii egg-laying and larval growth, indicating the possibility of heritable resistance operating against this invasive fly species.

There and back again; historical perspective and future directions for Vaccinium breeding and research studies.

The genus Vaccinium L. (Ericaceae) contains a wide diversity of culturally and economically important berry crop species. Consumer demand and scientific research in blueberry (Vaccinium spp.) and cranberry (Vaccinium macrocarpon) have increased worldwide over the crops' relatively short domestication history (~100 years). Other species, including bilberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), and ohelo berry (Vaccinium reticulatum) are largely still harvested from the wild but with crop improvement efforts underway. Here, we present a review article on these Vaccinium berry crops on topics that span taxonomy to genetics and genomics to breeding. We highlight the accomplishments made thus far for each of these crops, along their journey from the wild, and propose research areas and questions that will require investments by the community over the coming decades to guide future crop improvement efforts. New tools and resources are needed to underpin the development of superior cultivars that are not only more resilient to various environmental stresses and higher yielding, but also produce fruit that continue to meet a variety of consumer preferences, including fruit quality and health related traits.

High-quality reference genome and annotation aids understanding of berry development for evergreen blueberry (Vaccinium darrowii).

Vaccinium darrowii Camp (2n = 2x = 24) is a native North American blueberry species and an important source of traits such as low chill requirement in commercial southern highbush blueberry breeding (Vaccinium corymbosum, 2n = 4x = 48). We present a chromosomal-scale genome of V. darrowii generated by the combination of PacBio sequencing and high throughput chromatin conformation capture (Hi-C) scaffolding technologies, yielding a total length of 1.06 Gigabases (Gb). Over 97.8% of the genome sequences are scaffolded into 24 chromosomes representing the two haplotypes. The primary haplotype assembly of V. darrowii contains 34,809 protein-coding genes. Comparison to a V. corymbosum haplotype assembly reveals high collinearity between the two genomes with small intrachromosomal rearrangements in eight chromosome pairs. With small RNA sequencing, the annotation was further expanded to include more than 200,000 small RNA loci and 638 microRNAs expressed in berry tissues. Transcriptome analysis across fruit development stages indicates that genes involved in photosynthesis are downregulated, while genes involved in flavonoid and anthocyanin biosynthesis are significantly increased at the late stage of berry ripening. A high-quality reference genome and accompanying annotation of V. darrowii is a significant new resource for assessing the evergreen blueberry contribution to the breeding of southern highbush blueberries.

Genome-Wide Identification of Loci Associated With Phenology-Related Traits and Their Adaptive Variations in a Highbush Blueberry Collection.

Genetic variation in phenological traits is the key in expanding production areas of crops. Southern highbush blueberry (SHB) is a blueberry cultivar group adapted to warmer climates and has been developed by multiple interspecific hybridizations between elite northern highbush blueberry (NHB) (Vaccinium corymbosum L.) and low-chill Vaccinium species native to the southern United States. In this study, we employed a collection of diverse SHB accessions and performed a genome-wide association study (GWAS) for five phenology-related traits [chilling requirement (CR), flowering date, ripening date, fruit development period, and continuous flowering] using polyploid GWAS models. Phenology-related traits showed higher heritability and larger correlation coefficients between year replications, which resulted in the detection of robust phenotype-genotype association peaks. Notably, a single association peak for the CR was detected on Chromosome 4. Comparison of genotypes at the GWAS peaks between NHB and SHB revealed the putative introgression of low-chill and late-flowering alleles into the highbush genetic pool. Our results provide basic insights into the diversity of phenological traits in blueberry and the genetic establishment of current highbush cultivar groups.

Draft genome assembly of Passalora sequoiae a needle blight pathogen on Leyland cypress.

OBJECTIVE: Passalora sequoiae (family Mycosphaerellaceae) causes a twig blight on Leyland cypress that requires numerous fungicide applications annually to minimize economic losses for ornamental plant nursery and Christmas tree producers. The objective was to generate a high-quality draft assembly of the genome of P. sequoiae as a resource for primer development to investigate genotype diversity. DATA DESCRIPTION: We report here the genome sequence of P. sequoiae 9LC2 that was isolated from Leyland cypress 'Leighton Green' in 2017 in southern Mississippi, USA. The draft genome was obtained using Pacific Biosciences (PacBio) SMRT and Illumina HiSeq 2500 sequencing. Illumina reads were mapped to PacBio assembled contigs to determine base call consistency. Based on a total of 44 contigs with 722 kilobase (kb) average length (range 9.4 kb to 3.4 Mb), the whole genome size was estimated at 31,768,716 bp. Mapping of Illumina reads to PacBio contigs resulted in a 1000 × coverage and were used to confirm accuracy of the consensus sequences.

Comparative Analysis of Rhizosphere Microbiomes of Southern Highbush Blueberry (Vaccinium corymbosum L.), Darrow's Blueberry (V. darrowii Camp), and Rabbiteye Blueberry (V. virgatum Aiton).

Plants are inhabited by millions of parasitic, commensal, and mutualistic microorganisms that coexist in complex ecological communities, and profoundly affect the plant's productivity, health, and capacity to cope with environmental stress. Therefore, a better understanding of the rhizosphere microbiome may open a yet untapped avenue for the rational exploitation of beneficial plant-microbe interactions in modern agriculture. Blueberries encompass several wild and cultivated species of shrubs of the genus Vaccinium that are native to North America. They are grown commercially for the production of fruits, which are considered a health food due to the rich content of minerals, trace elements, and phenolic compounds with antioxidant, antitumor, and anti-inflammatory properties. Despite a long history of breeding and extensive commercial use, remarkably little is known about the composition and function of the blueberry root microbiome. To address this gap, we employed molecular approaches to characterize and compare microbial communities inhabiting the roots of rabbiteye blueberry (Vaccinium virgatum), Darrow's blueberry (Vaccinium darrowii), and southern highbush blueberry (SHB; an interspecific hybrid of Vaccinium corymbosum and V. darrowii). Our results revealed that these plant species share a common core rhizobiome, but at the same time differ significantly in the diversity, relative abundance, richness, and evenness of multiple groups of prokaryotic and eukaryotic microorganisms. Although the host signature effects were especially pronounced at the plant species level, we also observed genotype-level variations in the distribution of specific microbial taxa, which suggests that the assembly of the blueberry microbiome is shaped by the plant genotype and modifications associated with the domestication and breeding of members of the Vaccinium genus. We also demonstrated that the studied Vaccinium species differ in the abundance of beneficial rhizobacteria and ericoid mycorrhizal fungi, which play a vital role in their adaptation to soils with low pH and slow turnover of organic matter.

Draft Genome Sequences of Xylella fastidiosa subsp. fastidiosa Strains OK3, VB11, and NOB1, Isolated from Bunch and Muscadine Grapes Grown in Southern Mississippi.

We report here high-quality draft whole-genome assemblies of Xylella fastidiosa subsp. fastidiosa strains OK3, VB11, and NOB1, which were isolated from symptomatic bunch and muscadine grape plants grown in southern Mississippi.

Genome-Wide Association Mapping of Crown Rust Resistance in Oat Elite Germplasm.

Oat crown rust, caused by f. sp. , is a major constraint to oat ( L.) production in many parts of the world. In this first comprehensive multienvironment genome-wide association map of oat crown rust, we used 2972 single-nucleotide polymorphisms (SNPs) genotyped on 631 oat lines for association mapping of quantitative trait loci (QTL). Seedling reaction to crown rust in these lines was assessed as infection type (IT) with each of 10 crown rust isolates. Adult plant reaction was assessed in the field in a total of 10 location-years as percentage severity (SV) and as infection reaction (IR) in a 0-to-1 scale. Overall, 29 SNPs on 12 linkage groups were predictive of crown rust reaction in at least one experiment at a genome-wide level of statistical significance. The QTL identified here include those in regions previously shown to be linked with seedling resistance genes , , , , , and and also with adult-plant resistance and adaptation-related QTL. In addition, QTL on linkage groups Mrg03, Mrg08, and Mrg23 were identified in regions not previously associated with crown rust resistance. Evaluation of marker genotypes in a set of crown rust differential lines supported as the identity of . The SNPs with rare alleles associated with lower disease scores may be suitable for use in marker-assisted selection of oat lines for crown rust resistance.

A Consensus Map in Cultivated Hexaploid Oat Reveals Conserved Grass Synteny with Substantial Subgenome Rearrangement.

Hexaploid oat ( L., 2 = 6 = 42) is a member of the Poaceae family and has a large genome (∼12.5 Gb) containing 21 chromosome pairs from three ancestral genomes. Physical rearrangements among parental genomes have hindered the development of linkage maps in this species. The objective of this work was to develop a single high-density consensus linkage map that is representative of the majority of commonly grown oat varieties. Data from a cDNA-derived single-nucleotide polymorphism (SNP) array and genotyping-by-sequencing (GBS) were collected from the progeny of 12 biparental recombinant inbred line populations derived from 19 parents representing oat germplasm cultivated primarily in North America. Linkage groups from all mapping populations were compared to identify 21 clusters of conserved collinearity. Linkage groups within each cluster were then merged into 21 consensus chromosomes, generating a framework consensus map of 7202 markers spanning 2843 cM. An additional 9678 markers were placed on this map with a lower degree of certainty. Assignment to physical chromosomes with high confidence was made for nine chromosomes. Comparison of homeologous regions among oat chromosomes and matches to orthologous regions of rice ( L.) reveal that the hexaploid oat genome has been highly rearranged relative to its ancestral diploid genomes as a result of frequent translocations among chromosomes. Heterogeneous chromosome rearrangements among populations were also evident, probably accounting for the failure of some linkage groups to match the consensus. This work contributes to a further understanding of the organization and evolution of hexaploid grass genomes.

Population Genomics Related to Adaptation in Elite Oat Germplasm.

Six hundred thirty five oat ( L.) lines and 4561 single-nucleotide polymorphism (SNP) loci were used to evaluate population structure, linkage disequilibrium (LD), and genotype-phenotype association with heading date. The first five principal components (PCs) accounted for 25.3% of genetic variation. Neither the eigenvalues of the first 25 PCs nor the cross-validation errors from = 1 to 20 model-based analyses suggested a structured population. However, the PC and = 2 model-based analyses supported clustering of lines on spring oat vs. southern United States origin, accounting for 16% of genetic variation ( < 0.0001). Single-locus -statistic () in the highest 1% of the distribution suggested linkage groups that may be differentiated between the two population subgroups. Population structure and kinship-corrected LD of = 0.10 was observed at an average pairwise distance of 0.44 cM (0.71 and 2.64 cM within spring and southern oat, respectively). On most linkage groups LD decay was slower within southern lines than within the spring lines. A notable exception was found on linkage group Mrg28, where LD decay was substantially slower in the spring subpopulation. It is speculated that this may be caused by a heterogeneous translocation event on this chromosome. Association with heading date was most consistent across location-years on linkage groups Mrg02, Mrg12, Mrg13, and Mrg24.