Core Ideas
An R package that performs Next Generation Sequencing Bulk Segregant Analysis was developed.
Two methods for analysis are provided: QTL‐seq and G’.
The QTLseqr package is quick and ...produces publication quality figures and tables.
Next‐Generation Sequencing Bulk Segregant Analysis (NGS‐BSA) is efficient in detecting quantitative trait loci (QTL). Despite the popularity of NGS‐BSA and the R statistical platform, no R packages are currently available for NGS‐BSA. We present QTLseqr, an R package for NGS‐BSA that identifies QTL using two statistical approaches: QTL‐seq and G’. These approaches use a simulation method and a tricube smoothed G statistic, respectively, to identify and assess statistical significance of QTL. QTLseqr can import and filter SNP data, calculate SNP distributions, relative allele frequencies, G’ values, and log10 (p‐values), enabling identification and plotting of QTL. The source code is available at https://github.com/bmansfeld/QTLseqr.
Summary
Cassava (Manihot esculenta Crantz, 2n = 36) is a global food security crop. It has a highly heterozygous genome, high genetic load, and genotype‐dependent asynchronous flowering. It is ...typically propagated by stem cuttings and any genetic variation between haplotypes, including large structural variations, is preserved by such clonal propagation. Traditional genome assembly approaches generate a collapsed haplotype representation of the genome. In highly heterozygous plants, this results in artifacts and an oversimplification of heterozygous regions. We used a combination of Pacific Biosciences (PacBio), Illumina, and Hi‐C to resolve each haplotype of the genome of a farmer‐preferred cassava line, TME7 (Oko‐iyawo). PacBio reads were assembled using the FALCON suite. Phase switch errors were corrected using FALCON‐Phase and Hi‐C read data. The ultralong‐range information from Hi‐C sequencing was also used for scaffolding. Comparison of the two phases revealed >5000 large haplotype‐specific structural variants affecting over 8 Mb, including insertions and deletions spanning thousands of base pairs. The potential of these variants to affect allele‐specific expression was further explored. RNA‐sequencing data from 11 different tissue types were mapped against the scaffolded haploid assembly and gene expression data are incorporated into our existing easy‐to‐use web‐based interface to facilitate use by the broader plant science community. These two assemblies provide an excellent means to study the effects of heterozygosity, haplotype‐specific structural variation, gene hemizygosity, and allele‐specific gene expression contributing to important agricultural traits and further our understanding of the genetics and domestication of cassava.
Significance Statement
The cassava varieties grown by subsistence farmers in Africa largely differ from the partially inbred reference genome due to their highly heterozygous nature. We used multiple sequencing technologies to assemble and resolve both haplotypes in TME7, a farmer‐preferred cassava line, enabling us to study the considerable haplotypic structural variation in this line.
Age-related resistance (ARR) is a developmentally regulated phenomenon conferring resistance to pathogens or pests. Although ARR has been observed in several host-pathogen systems, the underlying ...mechanisms are largely uncharacterized. In cucumber, rapidly growing fruit are highly susceptible to Phytophthora capsici but become resistant as they complete exponential growth. We previously demonstrated that ARR is associated with the fruit peel and identified gene expression and metabolomic changes potentially functioning as preformed defenses.
Here, we compare the response to infection in fruit at resistant and susceptible ages using microscopy, quantitative bioassays, and weighted gene co-expression analyses. We observed strong transcriptional changes unique to resistant aged fruit 2-4 h post inoculation (hpi). Microscopy and bioassays confirmed this early response, with evidence of pathogen death and infection failure as early as 4 hpi and cessation of pathogen growth by 8-10 hpi. Expression analyses identified candidate genes involved in conferring the rapid response including those encoding transcription factors, hormone signaling pathways, and defenses such as reactive oxygen species metabolism and phenylpropanoid biosynthesis.
The early pathogen death and rapid defense response in resistant-aged fruit provide insight into potential mechanisms for ARR, implicating both pre-formed biochemical defenses and developmentally regulated capacity for pathogen recognition as key factors shaping age-related resistance.
SUMMARY
The Pacific crabapple (
Malus fusca
) is a wild relative of the commercial apple (
Malus
×
domestica
). With a range extending from Alaska to Northern California,
M. fusca
is extremely hardy ...and disease resistant. The species represents an untapped genetic resource for the development of new apple cultivars with enhanced stress resistance. However, gene discovery and utilization of
M. fusca
have been hampered by the lack of genomic resources. Here, we present a high‐quality, haplotype‐resolved, chromosome‐scale genome assembly and annotation for
M. fusca
. The genome was assembled using high‐fidelity long‐reads and scaffolded using genetic maps and high‐throughput chromatin conformation capture sequencing, resulting in one of the most contiguous apple genomes to date. We annotated the genome using public transcriptomic data from the same species taken from diverse plant structures and developmental stages. Using this assembly, we explored haplotypic structural variation within the genome of
M. fusca
, identifying thousands of large variants. We further showed high sequence co‐linearity with other domesticated and wild
Malus
species. Finally, we resolve a known quantitative trait locus associated with resistance to fire blight (
Erwinia amylovora
). Insights gained from the assembly of a reference‐quality genome of this hardy wild apple relative will be invaluable as a tool to facilitate DNA‐informed introgression breeding.
Significance Statement
We present a high‐quality, haplotype‐resolved, chromosome‐scale genome assembly and annotation for
Malus fusca
, a wild relative of the commercial apple. This genomic resource is an important contribution to the field of plant genomics and has significant implications for the development of new apple cultivars with enhanced disease and stress resistance.
Very young cucumber (Cucumis sativus) fruit are highly susceptible to infection by the oomycete pathogen, Phytophthora capsici. As the fruit complete exponential growth, at approximately 10-12 days ...post pollination (dpp), they transition to resistance. The development of age-related resistance (ARR) is increasingly recognized as an important defense against pathogens, however, underlying mechanisms are largely unknown. Peel sections from cucumber fruit harvested at 8 dpp (susceptible) and 16 dpp (resistant) showed equivalent responses to inoculation as did whole fruit, indicating that the fruit surface plays an important role in defense against P. capsici. Exocarp from 16 dpp fruit had thicker cuticles, and methanolic extracts of peel tissue inhibited growth of P. capsici in vitro, suggesting physical or chemical components to the ARR. Transcripts specifically expressed in the peel vs. pericarp showed functional differentiation. Transcripts predominantly expressed in the peel were consistent with fruit surface associated functions including photosynthesis, cuticle production, response to the environment, and defense. Peel-specific transcripts that exhibited increased expression in 16 dpp fruit relative to 8 dpp fruit, were highly enriched (P<0.0001) for response to stress, signal transduction, and extracellular and transport functions. Specific transcripts included genes associated with potential physical barriers (i.e., cuticle), chemical defenses (flavonoid biosynthesis), oxidative stress, penetration defense, and molecular pattern (MAMP)-triggered or effector-triggered (R-gene mediated) pathways. The developmentally regulated changes in gene expression between peels from susceptible- and resistant- age fruits suggest programming for increased defense as the organ reaches full size.
Cassava mosaic disease (CMD) suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to cassava mosaic geminiviruses. It has been reported that CMD2-type landraces ...lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for this loss of resistance, whole genome sequencing and genetic variant analysis was performed and the CMD2 locus was fine-mapped to a 190 kilobase interval. Collectively, these data indicate that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase δ subunit 1 (MePOLD1) located within this region. Virus-induced gene silencing of MePOLD1 in a CMD-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional candidate resistance alleles within MePOLD1. Genetic variation of MePOLD1, therefore, could represent an important genetic resource for resistance breeding and/or genome editing, and elucidating mechanisms of resistance to geminiviruses.
Phytophthora fruit rot (PFR) caused by the soilborne oomycete pathogen,
Phytophthora capsici
, can cause severe yield loss in cucumber. With no resistant variety available, genetic resources are ...needed to develop resistant varieties. The goal of this work was to identify quantitative trait loci (QTL) associated with resistance to PFR using multiple genomic approaches and populations. Two types of resistances have been identified: age-related resistance (ARR) and young fruit resistance. ARR occurs at 12-16 days post pollination (dpp), coinciding with the end of exponential fruit growth. A major QTL for ARR was discovered on chromosome 3 and a candidate gene identified based on comparative transcriptomic analysis. Young fruit resistance, which is observed during the state of rapid fruit growth prior to commercial harvest, is a quantitative trait for which multiple QTL were identified. The largest effect QTL,
qPFR5.1
, located on chromosome 5 was fine mapped to a 1-Mb region. Genome-wide association studies (GWAS) and extreme-phenotype genome-wide association study (XP-GWAS) for young fruit resistance were also performed on a cucumber core collection representing > 96% of the genetic diversity of the USDA cucumber germplasm. Several SNPs overlapped with the QTL identified from QTL-seq analysis on biparental populations. In addition, novel SNPs associated with the resistance were identified from the germplasm. The resistant alleles were found mostly in accessions from India and South Asia, the center of diversity for cucumber. The results from this work can be applied to future disease resistance studies and marker-assisted selection in breeding programs.
Effective assessment of pathogen growth can facilitate screening for disease resistance, mapping of resistance loci, testing efficacy of control measures, or elucidation of fundamental host-pathogen ...interactions. Current methods are often limited by subjective assessments, inability to detect pathogen growth prior to appearance of symptoms, destructive sampling, or limited capacity for replication and quantitative analysis. In this work we sought to develop a real-time,
, high-throughput assay that would allow for quantification of pathogen growth. To establish such a system, we worked with the broad host-range, highly destructive, soil-borne oomycete pathogen,
. We used an isolate expressing red fluorescence protein (RFP) to establish a microtiter plate, real-time assay to quantify pathogen growth in live tissue. The system was successfully used to monitor
growth
on cucumber (
) fruit and pepper (
) leaf samples in relation to different levels of host susceptibility. These results demonstrate usefulness of the method in different species and tissue types, allowing for highly replicated, quantitative time-course measurements of pathogen growth
. Analyses of pathogen growth during initial stages of infection preceding symptom development show the importance of very early stages of infection in determining disease outcome, and provide insight into points of inhibition of pathogen growth in different resistance systems.
Tomato (Solanum lycopersicum) diageotropica (dgt) mutants, containing a single mutation in the Cyclophilin1 (SlCyp1) gene, are auxin-insensitive, exhibiting a pleiotropic phenotype including lack of ...geotropism, abnormal xylem structure, lack of lateral roots (LRs), and elevated shoot-to-root ratio. SlCyp1 is a putative peptidyl-prolyl isomerase that can traffic from shoot to root, where it induces changes in auxin response, LR formation, and xylem development, suggesting it has a role as a long-distance signaling molecule. Here, we explored the mechanism underlying SlCyp1 function in the phloem. Expression of SlCyp1 under a phloem-specific (AtSuc2) promoter in dgt plants partially restored the wild-type phenotype, including lateral root development, root branching, and xylem morphology. The observed developmental changes were associated with physiological alternations at the whole-plant level, including a reduction in shoot-to-root ratio, enhanced transpiration, and elevated photosynthetic rates. Conversely, phloem-specific expression of SlCyp1 active-site mutants did not restore the wild-type phenotype. Local inhibition of cyclophilin functioning in the target tissue reduced auxin sensitivity, suggesting that its enzymatic activity in the distant organ is required for its action as a long-distance signalling agent. The data presented suggest that SlCyp1 is a signal molecule trafficking from shoot to root where its activity is required for auxin-mediated lateral root development.
Key message
We demystify recent advances in genome assemblies for the heterozygous staple crop cassava (
Manihot esculenta
), and highlight key cassava genomic resources.
Cassava,
Manihot esculenta
...Crantz, is a crop of societal and agricultural importance in tropical regions around the world. Genomics provides a platform for accelerated improvement of cassava’s nutritional and agronomic traits, as well as for illuminating aspects of cassava’s history including its path towards domestication. The highly heterozygous nature of the cassava genome is widely recognized. However, the full extent and context of this heterozygosity has been difficult to reveal because of technological limitations within genome sequencing. Only recently, with several new long-read sequencing technologies coming online, has the genomics community been able to tackle some similarly difficult genomes. In light of these recent advances, we provide this review to document the current status of the cassava genome and genomic resources and provide a perspective on what to look forward to in the coming years.
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