Reference genome-assisted identification of stem rust resistance gene Sr62 encoding a tandem kinase

The wild relatives and progenitors of wheat have been widely used as sources of disease resistance (R) genes. Molecular identification and characterization of these R genes facilitates their manipulation and tracking in breeding programmes. We developed a reference-quality genome assembly of the wild diploid wheat relative Aegilops sharonensis and used positional mapping, mutagenesis, RNA-Seq and transgenesis to identify the stem rust resistance gene Sr62, which was also transferred to common wheat. This gene encodes a tandem kinase, homologues of which exist across multiple taxa in the plant kingdom. Stable Sr62 transgenic wheat lines showed high levels of resistance against diverse isolates of the stem rust pathogen, highlighting the utility of Sr62 for deployment as part of a polygenic stack to maximize the durability of stem rust resistance.

Comparison of Dosimetry Protocols for Electron Beam Radiotherapy Calibrations and Measurement Uncertainties

This paper presents guidelines for the calibration of radiation beams that were issued by the International Atomic Energy Agency (IAEA TRS 398), the American Association of Physicists in Medicine (AAPM TG 51) and the German task group (DIN 6800-2). These protocols are based on the use of an ionization chamber calibrated in terms of absorbed dose to water in a standard laboratory’s reference quality beam, where the previous protocols were based on air kerma standards. This study aims to determine uncertainties in dosimetry for electron beam radiotherapy using internationally established high-energy radiotherapy beam calibration standards. Methods: Dw was determined in 6-, 12- and 18 MeV electron energies under reference conditions using three cylindrical and two plane-parallel ion chambers in concert with the IAEA TRS 398, AAPM TG 51 and DIN 6800-2 absorbed dose protocols. From mean measured Dw values, the ratio TRS 398/TG 51 was found to vary between 0.988 and 1.004, while for the counterpart TRS 398/DIN 6800-2 and TG 51/DIN 6800-2, the variation ranges were 0.991–1.003 and 0.997–1.005, respectively. For the cylindrical chambers, the relative combined uncertainty (k = 1) in absorbed dose measurements was 1.44%, while for the plane-parallel chambers, it ranged from 1.53 to 1.88%. Conclusions: A high degree of consistency was demonstrated among the three protocols. It is suggested that in the use of the presently determined dose conversion factors across the three protocols, dose intercomparisons can be facilitated between radiotherapy centres.

Genome sequence for the blue-flowered Andean shrub Iochroma cyaneum reveals extensive discordance across the berry clade of Solanaceae

The tomato family, Solanaceae, is a model clade for a wide range of applied and basic research questions. Currently, reference-quality genomes are available for over 30 species from seven genera, and these include numerous crops as well as wild species (e.g., Jaltomata sinuosa and Nicotiana attenuata). Here we present the genome of the showy-flowered Andean shrub Iochroma cyaneum, a woody lineage from the tomatillo subfamily Physalideae. The assembled size of the genome (2.7Gb) is more similar in size to chilipepper (2.6Gb) than to other sequenced diploid members of the berry clade of Solanaceae (e.g., potato, tomato, and Jaltomata). Our assembly recovers 92% of the conserved orthologous set, suggesting a nearly complete genome for this species. Most of the genomic content is repetitive (69%), with gyspy elements alone accounting for 52% of the genome. Despite the large amount of repetitive content, most of the 12 Iochroma chromosomes are highly syntenic with tomato. Bayesian concordance analysis provides strong support for the berry clade, including Iochroma, but reveals extensive discordance along the backbone, with placement of pepper and Jaltomata being highly variable across gene trees. The Iochroma genome contributes to a growing wealth of genomic resources in Solanaceae and underscores the need for expanded sampling of diverse berry genomes to dissect major morphological transitions.

A reference-quality, fully annotated genome from a Puerto Rican individual

Until 2019, the human genome was available in only one fully-annotated version, GRCh38, which was the result of 18 years of continuous improvement and revision. Despite dramatic improvements in sequencing technology, no other genome was available as an annotated reference until 2019, when the genome of an Ashkenazi individual, Ash1, was released. In this study, we describe the assembly and annotation of a second individual genome, from a Puerto Rican individual whose DNA was collected as part of the Human Pangenome project. The new genome, called PR1, is the first true reference genome created from an individual of African descent. Due to recent improvements in both sequencing and assembly technology, and particularly to the use of the recently completed CHM13 human genome as a guide to assembly, PR1 is more complete and more contiguous than either GRCh38 or Ash1. Annotation revealed 37,755 genes (of which 19,999 are protein-coding), including 12 additional gene copies that are present in PR1 and missing from CHM13. 57 genes have fewer copies in PR1 than in CHM13, 9 map only partially, and 3 genes (all non-coding) from CHM13 are entirely missing from PR1.

The FORCE Panel: An All-in-One SNP Marker Set for Confirming Investigative Genetic Genealogy Leads and for General Forensic Applications

The FORensic Capture Enrichment (FORCE) panel is an all-in-one SNP panel for forensic applications. This panel of 5422 markers encompasses common, forensically relevant SNPs (identity, ancestry, phenotype, X- and Y-chromosomal SNPs), a novel set of 3931 autosomal SNPs for extended kinship analysis, and no clinically relevant/disease markers. The FORCE panel was developed as a custom hybridization capture assay utilizing ~20,000 baits to target the selected SNPs. Five non-probative, previously identified World War II (WWII) cases were used to assess the kinship panel. Each case included one bone sample and associated family reference DNA samples. Additionally, seven reference quality samples, two 200-year-old bone samples, and four control DNAs were processed for kit performance and concordance assessments. SNP recovery after capture resulted in a mean of ~99% SNPs exceeding 10X coverage for reference and control samples, and 44.4% SNPs for bone samples. The WWII case results showed that the FORCE panel could predict first to fifth degree relationships with strong statistical support (likelihood ratios over 10,000 and posterior probabilities over 99.99%). To conclude, SNPs will be important for further advances in forensic DNA analysis. The FORCE panel shows promising results and demonstrates the utility of a 5000 SNP panel for forensic applications.

The FORCE panel: An all-in-one SNP marker set for confirming investigative genetic genealogy leads and for general forensic applications

The FORensic Capture Enrichment (FORCE) panel is an all-in-one SNP panel for forensic applications. This panel of 5,422 markers encompasses common, forensically relevant SNPs (identity, ancestry, phenotype, X- and Y-chromosomal SNPs), a novel set of 3,931 autosomal SNPs for extended kinship analysis, and no clinically rele-vant/disease markers. The FORCE panel was developed as a custom hybridization capture assay utilizing ~20,000 baits to target the selected SNPs. Five non-probative, previously identified World War II (WWII) cases were used to assess the kinship panel. Each case included one bone sample and associated family reference DNA samples. Additionally, seven reference quality samples, two 200-year-old bone samples, and four control DNAs were processed for kit performance and concordance assessments. SNP recovery after capture resulted in a mean of ~99% SNPs exceeding 10X coverage for reference and control samples, and 44.4% SNPs for bone samples. The WWII case results showed that the FORCE panel could predict 1st to 5th degree relationships with strong statisti-cal support (likelihood ratios over 10,000 and posterior probabilities over 99.99%). To conclude, SNPs will be important for further advances in forensic DNA analysis. The FORCE panel shows promising results and demonstrates the utility of a 5,000 SNP panel for forensic applications.

Efficient iterative Hi-C scaffolder based on N-best neighbors

Background Efficient and effective genome scaffolding tools are still in high demand for generating reference-quality assemblies. While long read data itself is unlikely to create a chromosome-scale assembly for most eukaryotic species, the inexpensive Hi-C sequencing technology, capable of capturing the chromosomal profile of a genome, is now widely used to complete the task. However, the existing Hi-C based scaffolding tools either require a priori chromosome number as input, or lack the ability to build highly continuous scaffolds. Results We design and develop a novel Hi-C based scaffolding tool, pin_hic, which takes advantage of contact information from Hi-C reads to construct a scaffolding graph iteratively based on N-best neighbors of contigs. Subsequent to scaffolding, it identifies potential misjoins and breaks them to keep the scaffolding accuracy. Through our tests on three long read based de novo assemblies from three different species, we demonstrate that pin_hic is more efficient than current standard state-of-art tools, and it can generate much more continuous scaffolds, while achieving a higher or comparable accuracy. Conclusions Pin_hic is an efficient Hi-C based scaffolding tool, which can be useful for building chromosome-scale assemblies. As many sequencing projects have been launched in the recent years, we believe pin_hic has potential to be applied in these projects and makes a meaningful contribution.

The haplotype-resolved chromosome pairs and transcriptome of a heterozygous diploid African cassava cultivar

Background: Cassava (Manihot esculenta) is an important clonally propagated food crop in tropical and sub-tropical regions worldwide. Genetic gain by molecular breeding is limited because cassava has a highly heterozygous, repetitive and difficult to assemble genome. Findings: Here we demonstrate that Pacific Biosciences high-fidelity (HiFi) sequencing reads, in combination with the assembler hifiasm, produced genome assemblies at near complete haplotype resolution with higher continuity and accuracy compared to conventional long sequencing reads. We present two chromosome scale haploid genomes phased with Hi-C technology for the diploid African cassava variety TME204. Genome comparisons revealed extensive chromosome re-arrangements and abundant intra-genomic and inter-genomic divergent sequences despite high gene synteny, with most large structural variations being LTR-retrotransposon related. Allele-specific expression analysis of different tissues based on the haplotype-resolved transcriptome identified both stable and inconsistent alleles with imbalanced expression patterns, while most alleles expressed coordinately. Among tissue-specific differentially expressed transcripts, coordinately and biasedly regulated transcripts were functionally enriched for different biological processes. We use the reference-quality assemblies to build a cassava pan-genome and demonstrate its importance in representing the genetic diversity of cassava for downstream reference-guided omics analysis and breeding. Conclusions: The haplotype-resolved genome allows the first systematic view of the heterozygous diploid genome organization in cassava. The completely phased and annotated chromosome pairs will be a valuable resource for cassava breeding and research. Our study may also provide insights into developing cost-effective and efficient strategies for resolving complex genomes with high resolution, accuracy and continuity.