Mitogenome and Nuclear-encoded Fungicide-target Genes of Thecaphora frezii - Causal Agent of Peanut Smut

Background: Thecaphora frezii Carranza and Lindquist causes smut disease in peanut (Arachis hypogaea L.) resulting in up to 35% yield losses. Fungicides have shown ineffective in controlling the disease; whereas research on the molecular basis of that fungicide resistance has been hindered because of the lack of genetic information about T. frezii. The goal of this work was to provide molecular information about fungicide-target loci in T. frezii, including its mitochondrial genome (mitogenome) and critical nuclear-encoded genes. Results: Here we report the complete annotated mitogenome of T. frezii, a 123,773 bp molecule containing the standard 14 genes that form part of mitochondrial complexes I, III, IV and V, 22 transfer RNAs, small and large subunits of ribosomal RNA, DNA polymerase, ribonuclease P, GII-reverse transcriptase/maturase, nine hypothetical open-reading frames and homing endonucleases (LAGLIDADG, GIY-YIG, HEG). In addition, we report the full-length cDNA sequence of T. frezii cytochrome b (cob) and cytochrome oxidase 1 (cox1) genes; as well as partial sequences of T. frezii succinate dehydrogenase (sdhb), ergosterol biosynthesis (Erg4), cytochrome P450 (cyp51), and beta tubulin (β-tubulin) genes, which are respective targets of strobilurins, quinone oxidation inhibitors, triazoles and beta-tubulin inhibitor fungicides commonly used in the peanut crop. Translation of cob and sdhb genes in this particular T. frezii isolate suggests potential resistance to strobilurin and carboxamide fungicides. Conclusion: The mitogenome and nuclear-encoded gene sequences presented here provide the molecular tools to research T. frezii fungicide-target loci

Representing true plant genomes: haplotype-resolved hybrid pepper genome with trio-binning

As sequencing costs decrease and availability of high fidelity long-read sequencing increases, generating experiment specific de novo genome assemblies becomes feasible. In many crop species, obtaining the genome of a hybrid or heterozygous individual is necessary for systems that do not tolerate inbreeding or for investigating important biological questions, such as hybrid vigor. However, most genome assembly methods that have been used in plants result in a merged single sequence representation that is not a true biologically accurate representation of either haplotype within a diploid individual. The resulting genome assembly is often fragmented and exhibits a mosaic of the two haplotypes, referred to as haplotype-switching. Important haplotype level information, such as causal mutations and structural variation is therefore lost causing difficulties in interpreting downstream analyses. To overcome this challenge, we have applied a method developed for animal genome assembly called trio-binning to an intra-specific hybrid of chili pepper (Capsicum annuum L. cv. HDA149 x Capsicum annuum L. cv. HDA330). We tested all currently available softwares for performing trio-binning, combined with multiple scaffolding technologies including Bionano to determine the optimal method of producing the best haplotype-resolved assembly. Ultimately, we produced highly contiguous biologically true haplotype-resolved genome assemblies for each parent, with scaffold N50s of 266.0 Mb and 281.3 Mb, with 99.6% and 99.8% positioned into chromosomes respectively. The assemblies captured 3.10 Gb and 3.12 Gb of the estimated 3.5 Gb chili pepper genome size. These assemblies represent the complete genome structure of the intraspecific hybrid, as well as the two parental genomes, and show measurable improvements over the currently available reference genomes. Our manuscript provides a valuable guide on how to apply trio-binning to other plant genomes

Gene disruption by structural mutations drives selection in US rice breeding over the last century.

The genetic basis of general plant vigor is of major interest to food producers, yet the trait is recalcitrant to genetic mapping because of the number of loci involved, their small effects, and linkage. Observations of heterosis in many crops suggests that recessive, malfunctioning versions of genes are a major cause of poor performance, yet we have little information on the mutational spectrum underlying these disruptions. To address this question, we generated a long-read assembly of a tropical japonica rice (Oryza sativa) variety, Carolina Gold, which allowed us to identify structural mutations (>50 bp) and orient them with respect to their ancestral state using the outgroup, Oryza glaberrima. Supporting prior work, we find substantial genome expansion in the sativa branch. While transposable elements (TEs) account for the largest share of size variation, the majority of events are not directly TE-mediated. Tandem duplications are the most common source of insertions and are highly enriched among 50-200bp mutations. To explore the relative impact of various mutational classes on crop fitness, we then track these structural events over the last century of US rice improvement using 101 resequenced varieties. Within this material, a pattern of temporary hybridization between medium and long-grain varieties was followed by recent divergence. During this long-term selection, structural mutations that impact gene exons have been removed at a greater rate than intronic indels and single-nucleotide mutations. These results support the use of ab initio estimates of mutational burden, based on structural data, as an orthogonal predictor in genomic selection

The Liverpool alcohol-related liver disease algorithm identifies twice as many emergency admissions compared to standard methods when applied to Hospital Episode Statistics for England

BackgroundEmergency admissions in England for alcohol-related liver disease (ArLD) have increased steadily for decades. Statistics based on administrative data typically focus on the ArLD-specific code as the primary diagnosis and are therefore at risk of excluding ArLD admissions defined by other coding combinations.AimTo deploy the Liverpool ArLD Algorithm (LAA), which accounts for alternative coding patterns (e.g., ArLD secondary diagnosis with alcohol/liver-related primary diagnosis), to national and local datasets in the context of studying trends in ArLD admissions before and during the COVID-19 pandemic.MethodsWe applied the standard approach and LAA to Hospital Episode Statistics for England (2013-21). The algorithm was also deployed at 28 hospitals to discharge coding for emergency admissions during a common 7-day period in 2019 and 2020, in which eligible patient records were reviewed manually to verify the diagnosis and extract data.ResultsNationally, LAA identified approximately 100% more monthly emergency admissions from 2013 to 2021 than the standard method. The annual number of ArLD-specific admissions increased by 30.4%. Of 39,667 admissions in 2020/21, only 19,949 were identified with standard approach, an estimated admission cost of £70 million in under-recorded cases. Within 28 local hospital datasets, 233 admissions were identified using the standard approach and a further 250 locally verified cases using the LAA (107% uplift). There was an 18% absolute increase in ArLD admissions in the seven-day evaluation period in 2020 versus 2019. There were no differences in disease severity or mortality, or in the proportion of admissions with decompensation of cirrhosis or alcoholic hepatitis.ConclusionsThe LAA can be applied successfully to local and national datasets. It consistently identifies approximately 100% more cases than the standard coding approach. The algorithm has revealed the true extent of ArLD admissions. The pandemic has compounded a long-term rise in ArLD admissions and mortality

Development of nuclear microsatellite markers to facilitate germplasm conservation and population genetics studies of five groups of tropical perennial plants with edible fruits and shoots: rambutan (Nephelium lappaceum L.), sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and Garcinia cochinchinensis (Lour.) Choisy) and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth)

Simple sequence repeat (SSR) enriched libraries for five groups of tropical perennial plants with edible fruits and shoots were prepared and sequenced in a GS-FLX Roche 454: sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and G. cochinchinensis (Lour.) Choisy), rambutan (Nephelium lappaceum L.), and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth). For SSR development, these species were organized by their common names in five groups. A total of 3870 SSR primer sets were designed, using capillary electrophoresis 1872 nuclear SSRs were tested on 4 to 10 DNA samples within each plant group, that is 384 loci for each of the four groups of fruit trees and 336 loci for the bamboo group. Only 7.9% of the primers tested did not result in amplification. All 1872 SSRs are provided, we highlight 178 SSRs (between 26 and 47 per group) considered topquality polymorphic SSRs that amplified all the samples, had strong fluorescence signal, presented no stutters and showed minimum non-specific amplification or background fluorescence. A total of 66,057 contig sequences were submitted to GenBank Database. Markers presented here will be useful not only for conservation efforts in banks of germplasm, but also for in-depth analysis of population genetics which usually requires evaluation of large number of loci

Development of nuclear microsatellite markers to facilitate germplasm conservation and population genetics studies of five groups of tropical perennial plants with edible fruits and shoots: rambutan (Nephelium lappaceum L.), sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and Garcinia cochinchinensis (Lour.) Choisy) and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth)

Simple sequence repeat (SSR) enriched libraries for five groups of tropical perennial plants with edible fruits and shoots were prepared and sequenced in a GS-FLX Roche 454: sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and G. cochinchinensis (Lour.) Choisy), rambutan (Nephelium lappaceum L.), and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth). For SSR development, these species were organized by their common names in five groups. A total of 3870 SSR primer sets were designed, using capillary electrophoresis 1872 nuclear SSRs were tested on 4 to 10 DNA samples within each plant group, that is 384 loci for each of the four groups of fruit trees and 336 loci for the bamboo group. Only 7.9% of the primers tested did not result in amplifi- cation. All 1872 SSRs are provided, we highlight 178 SSRs (between 26 and 47 per group) considered top-quality polymorphic SSRs that amplified all the samples, had strong fluorescence signal, presented no stutters and showed minimum non-specific amplification or background fluorescence. A total of 66,057 contig sequences were submitted to GenBank Database. Markers presented here will be useful not only for conservation efforts in banks of germplasm, but also for in-depth analysis of population genetics which usually requires evaluation of large number of loci

Molecular Dissection of the 5S Ribosomal RNA-Intergenic Transcribed Spacers in <i>Saccharum</i> spp. and <i>Tripidium</i> spp.

Due to complex polyploid, sugarcane whole genome sequencing and characterization lag far behind other crops. PCR-based DNA markers are a viable low-cost option to evaluate genetic diversity and verify genotypes. In this study, the 5S ribosomal RNA-intergenic spacer (ITS) of 171 accessions of Saccharum spp. and Tripidium spp. was dissected, including 30 accessions of S. officinarum, 71 of S. spontaneum, 17 of S. robustum, 25 of S. barberi, 13 of S. sinense, 2 of S. edule, 5 sugarcane cultivars (Saccharum spp. hybrids), 6 of Tripidium spp. (formally Erianthus spp.), and 2 of unknown species. The ITS spacers were amplified from 10 ng of the leaf DNA of each accession with the universal PCR primers PI and PII. The PCR-amplified spacers (amplicons) were analyzed by both agarose gel and capillary electrophoresis (CE). While agarose gel electrophoresis revealed five banding patterns, a total of 42 polymorphic amplicons, ranging from 60 to 506 bp, were detected by CE. Three amplicons, 234-, 235-, and 236-bp in size, were amplified from all accessions of six Saccharum species, except for three S. robustum accessions (Molokai 5573, NG 57-054, and NG 77-235) that lacked the 236-bp amplicon. The 234-, 235-, 236-bp banding pattern found in S. spontaneum was less consistent than other Saccharum species, sometimes missing a few but not all the bands in this region. An amplicon of 61-bp was amplified only from the sugarcane hybrid varieties. The PI/PII patterns indicated diversity and subpopulations within Saccharum, which could potentially be used in Breeding. Moreover, all Saccharum-specific amplicons were mostly absent in Tripidium spp. accessions, which produced 405-bp and 406-bp amplicons, and any pattern of the exceptions indicated misidentification. The T. bengalense accession Kalimpong had a unique CE-banding pattern that was different from all other accessions. Although the clustering pattern of the 42 amplicons only discriminated at the genus level, these amplicons helped identify nine misclassified accessions. This study further demonstrates that these PI/PII amplicons could be particularly useful markers for breeders at sugarcane field stations to quickly confirm and discriminate among the accessions of germplasm collections

Development of nuclear microsatellite markers to facilitate germplasm conservation and population genetics studies of five groups of tropical perennial plants with edible fruits and shoots: rambutan (Nephelium lappaceum L.), sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and Garcinia cochinchinensis (Lour.) Choisy) and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth)

Simple sequence repeat (SSR) enriched libraries for five groups of tropical perennial plants with edible fruits and shoots were prepared and sequenced in a GS-FLX Roche 454: sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and G. cochinchinensis (Lour.) Choisy), rambutan (Nephelium lappaceum L.), and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth). For SSR development, these species were organized by their common names in five groups. A total of 3870 SSR primer sets were designed, using capillary electrophoresis 1872 nuclear SSRs were tested on 4 to 10 DNA samples within each plant group, that is 384 loci for each of the four groups of fruit trees and 336 loci for the bamboo group. Only 7.9% of the primers tested did not result in amplification. All 1872 SSRs are provided, we highlight 178 SSRs (between 26 and 47 per group) considered topquality polymorphic SSRs that amplified all the samples, had strong fluorescence signal, presented no stutters and showed minimum non-specific amplification or background fluorescence. A total of 66,057 contig sequences were submitted to GenBank Database. Markers presented here will be useful not only for conservation efforts in banks of germplasm, but also for in-depth analysis of population genetics which usually requires evaluation of large number of loci

Development of nuclear microsatellite markers to facilitate germplasm conservation and population genetics studies of five groups of tropical perennial plants with edible fruits and shoots: rambutan (Nephelium lappaceum L.), sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and Garcinia cochinchinensis (Lour.) Choisy) and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth)

Simple sequence repeat (SSR) enriched libraries for five groups of tropical perennial plants with edible fruits and shoots were prepared and sequenced in a GS-FLX Roche 454: sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and G. cochinchinensis (Lour.) Choisy), rambutan (Nephelium lappaceum L.), and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth). For SSR development, these species were organized by their common names in five groups. A total of 3870 SSR primer sets were designed, using capillary electrophoresis 1872 nuclear SSRs were tested on 4 to 10 DNA samples within each plant group, that is 384 loci for each of the four groups of fruit trees and 336 loci for the bamboo group. Only 7.9% of the primers tested did not result in amplifi- cation. All 1872 SSRs are provided, we highlight 178 SSRs (between 26 and 47 per group) considered top-quality polymorphic SSRs that amplified all the samples, had strong fluorescence signal, presented no stutters and showed minimum non-specific amplification or background fluorescence. A total of 66,057 contig sequences were submitted to GenBank Database. Markers presented here will be useful not only for conservation efforts in banks of germplasm, but also for in-depth analysis of population genetics which usually requires evaluation of large number of loci

Development of a Large Set of Microsatellite Markers in Zapote Mamey (Pouteria sapota (Jacq.) H.E. Moore &amp; Stearn) and Their Potential Use in the Study of the Species

Pouteria sapota is known for its edible fruits that contain unique carotenoids, as well as for its fungitoxic, anti-inflammatory and anti-oxidant activity. However, its genetics is mostly unknown, including aspects about its genetic diversity and domestication process. We did high-throughput sequencing of microsatellite-enriched libraries of P. sapota, generated 5223 contig DNA sequences, 1.8 Mbp, developed 368 microsatellites markers and tested them on 29 individuals from 10 populations (seven wild, three cultivated) from Mexico, its putative domestication center. Gene ontology BLAST analysis of the DNA sequences containing microsatellites showed potential association to physiological functions. Genetic diversity was slightly higher in cultivated than in the wild gene pool (HE = 0.41 and HE = 0.35, respectively), although modified Garza–Williamson Index and Bottleneck software showed evidence for a reduction in genetic diversity for the cultivated one. Neighbor Joining, 3D Principal Coordinates Analysis and assignment tests grouped most individuals according to their geographic origin but no clear separation was observed between wild or cultivated gene pools due to, perhaps, the existence of several admixed populations. The developed microsatellites have a great potential in genetic population and domestication studies of P. sapota but additional sampling will be necessary to better understand how the domestication process has impacted the genetic diversity of this fruit crop