Optical Genome Mapping for Applications in Repeat Expansion Disorders.

Short tandem repeat (STR) expansions are associated with more than 60 genetic disorders. The size and stability of these expansions correlate with the severity and age of onset of the disease. Therefore, being able to accurately detect the absolute length of STRs is important. Current diagnostic assays include laborious lab experiments, including repeat-primed PCR and Southern blotting, that still cannot precisely determine the exact length of very long repeat expansions. Optical genome mapping (OGM) is a cost-effective and easy-to-use alternative to traditional cytogenetic techniques and allows the comprehensive detection of chromosomal aberrations and structural variants >500 bp in length, including insertions, deletions, duplications, inversions, translocations, and copy number variants. Here, we provide methodological guidance for preparing samples and performing OGM as well as running the analysis pipelines and using the specific repeat expansion workflows to determine the exact repeat length of repeat expansions expanded beyond 500 bp. Together these protocols provide all details needed to analyze the length and stability of any repeat expansion with an expected repeat size difference from the expected wild-type allele of >500 bp. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genomic ultra-high-molecular-weight DNA isolation, labeling, and staining Basic Protocol 2: Data generation and genome mapping using the Bionano Saphyr® System Basic Protocol 3: Manual De Novo Assembly workflow Basic Protocol 4: Local guided assembly workflow Basic Protocol 5: EnFocus Fragile X workflow Basic Protocol 6: Molecule distance script workflow.

GWAS elucidated grain yield genetics in Indian spring wheat under diverse water conditions.

KEY MESSAGE: Underpinned natural variations and key genes associated with yield under different water regimes, and identified genomic signatures of genetic gain in the Indian wheat breeding program. A novel KASP marker for TKW under water stress was developed and validated. A comprehensive genome-wide association study was conducted on 300 spring wheat genotypes to elucidate the natural variations associated with grain yield and its eleven contributing traits under fully irrigated, restricted water, and simulated no water conditions. Utilizing the 35K Wheat Breeders' Array, we identified 1155 quantitative trait nucleotides (QTNs), with 207 QTNs exhibiting stability across diverse conditions. These QTNs were further delimited into 539 genomic regions using a genome-wide LD value of 3.0 Mbp, revealing pleiotropic control across traits and conditions. Sub-genome A was significantly associated with traits under irrigated conditions, while sub-genome B showed more QTNs under water stressed conditions. Favourable alleles with significantly associated QTNs were delineated, with a notable pyramiding effect for enhancing trait performance. Additionally, allele of only 921 QTNs significantly affected the population mean. Allele profiling highlighted C-306 as a most potential source of drought tolerance. Moreover, 762 genes overlapping significant QTNs were identified, narrowing down to 27 putative candidate genes overlapping 29 novel and functional SNPs expressing (≥ 0.5 tpm) relevance across various growth conditions. A new KASP assay was developed, targeting a gene TraesCS2A03G1123700 regulating thousand kernel weight under severe drought condition. Genomic selection models (GBLUP, BayesB, MxE, and R-Norm) demonstrated an average prediction accuracy of 0.06-0.58 across environments, indicating potential for trait selection. Retrospective analysis of the Indian wheat breeding program supported a genetic gain in GY at the rate of ca. 0.56% per breeding cycle, since 1960, supporting the identification of genomic signatures driving trait selection and genetic gain. These findings offer insight into improving the rate of genetic gain in wheat breeding programs globally.

Genome wide association study of seedling and adult plant leaf rust resistance in two subsets of barley genetic resources.

Leaf rust (LR) caused by Puccinia hordei is a serious disease of barley worldwide, causing significant yield losses and reduced grain quality. Discovery and incorporation of new sources of resistance from gene bank accessions into barley breeding programs is essential for the development of leaf rust resistant varieties. To identify Quantitative Trait Loci (QTL) conferring LR resistance in the two barley subsets, the Generation Challenge Program (GCP) reference set of 142 accessions and the leaf rust subset constructed using the Focused Identification of Germplasm Strategy (FIGS) of 76 barley accessions, were genotyped to conduct a genome-wide association study (GWAS). The results revealed a total of 59 QTL in the 218 accessions phenotyped against barley leaf rust at the seedling stage using two P. hordei isolates (ISO-SAT and ISO-MRC), and at the adult plant stage in four environments in Morocco. Out of these 59 QTL, 10 QTL were associated with the seedling resistance (SR) and 49 QTL were associated with the adult plant resistance (APR). Four QTL showed stable effects in at least two environments for APR, whereas two common QTL associated with SR and APR were detected on chromosomes 2H and 7H. Furthermore, 39 QTL identified in this study were potentially novel. Interestingly, the sequences of 27 SNP markers encoded the candidate genes (CGs) with predicted protein functions in plant disease resistance. These results will provide new perspectives on the diversity of leaf rust resistance loci for fine mapping, isolation of resistance genes, and for marker-assisted selection for the LR resistance in barley breeding programs worldwide.

Cold tolerance SNPs and candidate gene mining in the soybean germination stage based on genome-wide association analysis.

KEY MESSAGE: Three QTLs associated with low-temperature tolerance were identified by genome-wide association analysis, and 15 candidate genes were identified by haplotype analysis and gene expression analyses. Low temperature is a critical factor affecting the geographical distribution, growth, development, and yield of soybeans, with cold stress during seed germination leading to substantial productivity loss. In this study, an association panel comprising 260 soybean accessions was evaluated for four germination traits and four cold tolerance index traits, revealing extensive variation in cold tolerance. Genome-wide association study (GWAS) identified 10 quantitative trait nucleotides (QTNs) associated with cold tolerance, utilizing 30,799 single nucleotide polymorphisms (SNPs) and four GWAS models. Linkage disequilibrium (LD) analysis positioned these QTNs within three cold-tolerance quantitative trait loci (QTL) and, with QTL19-1, was positioned by three multi-locus models, underscoring its importance as a key QTL. Integrative haplotype analysis, supplemented by transcriptome analysis, uncovered 15 candidate genes. The haplotypes within the genes Glyma.18G044200, Glyma.18G044300, Glyma.18G044900, Glyma.18G045100, Glyma.19G222500, and Glyma.19G222600 exhibited significant phenotypic variations, with differential expression in materials with varying cold tolerance. The QTNs and candidate genes identified in this study offer substantial potential for marker-assisted selection and gene editing in breeding cold-tolerant soybeans, providing valuable insights into the genetic mechanisms underlying cold tolerance during soybean germination.

High resolution mapping of a novel non-transgressive hybrid susceptibility locus in barley exploited by P. teres f. maculata.

Hybrid genotypes can provide significant yield gains over conventional inbred varieties due to heterosis or hybrid vigor. However, hybrids can also display unintended negative attributes or phenotypes such as extreme pathogen susceptibility. The necrotrophic pathogen Pyrenophora teres f. maculata (Ptm) causes spot form net blotch, which has caused significant yield losses to barley worldwide. Here, we report on a non-transgressive hybrid susceptibility locus in barley identified between the three parental lines CI5791, Tifang and Golden Promise that are resistant to Ptm isolate 13IM.3. However, F2 progeny from CI5791 × Tifang and CI5791 × Golden Promise crosses exhibited extreme susceptibility. The susceptible phenotype segregated in a ratio of 1 resistant:1 susceptible representing a genetic segregation ratio of 1 parental (res):2 heterozygous (sus):1 parental (res) suggesting a single hybrid susceptibility locus. Genetic mapping using a total of 715 CI5791 × Tifang F2 individuals (1430 recombinant gametes) and 149 targeted SNPs delimited the hybrid susceptibility locus designated Susceptibility to Pyrenophora teres 2 (Spt2) to an ~ 198 kb region on chromosome 5H of the Morex V3 reference assembly. This single locus was independently mapped with 83 CI5791 × Golden Promise F2 individuals (166 recombinant gametes) and 180 genome wide SNPs that colocalized to the same Spt2 locus. The CI5791 genome was sequenced using PacBio Continuous Long Read technology and comparative analysis between CI5791 and the publicly available Golden Promise genome assembly determined that the delimited region contained a single high confidence Spt2 candidate gene predicted to encode a pentatricopeptide repeat-containing protein.

Quantitative trait locus analysis of gray leaf spot resistance in the maize IBM Syn10 DH population.

KEY MESSAGE: The exploration and dissection of a set of QTLs and candidate genes for gray leaf spot disease resistance using two fully assembled parental genomes may help expedite maize resistance breeding. The fungal disease of maize known as gray leaf spot (GLS), caused by Cercospora zeae-maydis and Cercospora zeina, is a significant concern in China, Southern Africa, and the USA. Resistance to GLS is governed by multiple genes with an additive effect and is influenced by both genotype and environment. The most effective way to reduce the cost of production is to develop resistant hybrids. In this study, we utilized the IBM Syn 10 Doubled Haploid (IBM Syn10 DH) population to identify quantitative trait loci (QTLs) associated with resistance to gray leaf spot (GLS) in multiple locations. Analysis of seven distinct environments revealed a total of 58 QTLs, 49 of which formed 12 discrete clusters distributed across chromosomes 1, 2, 3, 4, 8 and 10. By comparing these findings with published research, we identified colocalized QTLs or GWAS loci within eleven clustering intervals. By integrating transcriptome data with genomic structural variations between parental individuals, we identified a total of 110 genes that exhibit both robust disparities in gene expression and structural alterations. Further analysis revealed 19 potential candidate genes encoding conserved resistance gene domains, including putative leucine-rich repeat receptors, NLP transcription factors, fucosyltransferases, and putative xyloglucan galactosyltransferases. Our results provide a valuable resource and linked loci for GLS marker resistance selection breeding in maize.

Detection of closely linked QTLs and candidate genes controlling germination indices in response to drought and salinity stresses in barley.

The aim of current study was to identify closely linked QTLs and candidate genes related to germination indices under control, salinity and drought conditions in barley. A total of nine (a major), 28 (eight major) and 34 (five major) closely linked QTLs were mapped on the seven chromosomes in response to control, drought and salinity conditions using genome-wide composite interval mapping, respectively. The major QTLs can be used in marker-assisted selection (MAS) projects to increase tolerance to drought and salinity stresses during the germination. Overall, 422 unique candidate genes were associated with most major QTLs. Moreover, gene ontology analysis showed that candidate genes mostly involved in biological process related to signal transduction and response to stimulus in the pathway of resistance to drought and salinity stresses. Also, the protein-protein interaction network was identified 10 genes. Furthermore, 10 genes were associated with receptor-like kinase family. In addition, 16 transcription factors were detected. Three transcription factors including B3, bHLH, and FAR1 had the most encoding genes. Totally, 60 microRNAs were traced to regulate the target genes. Finally, the key genes are a suitable and reliable source for future studies to improve resistance to abiotic stress during the germination of barley.

Enhanced recombination empowers the detection and mapping of Quantitative Trait Loci.

Modern plant breeding, such as genomic selection and gene editing, is based on the knowledge of the genetic architecture of desired traits. Quantitative trait loci (QTL) analysis, which combines high throughput phenotyping and genotyping of segregating populations, is a powerful tool to identify these genetic determinants and to decipher the underlying mechanisms. However, meiotic recombination, which shuffles genetic information between generations, is limited: Typically only one to two exchange points, called crossovers, occur between a pair of homologous chromosomes. Here we test the effect on QTL analysis of boosting recombination, by mutating the anti-crossover factors RECQ4 and FIGL1 in Arabidopsis thaliana full hybrids and lines in which a single chromosome is hybrid. We show that increasing recombination ~6-fold empowers the detection and resolution of QTLs, reaching the gene scale with only a few hundred plants. Further, enhanced recombination unmasks some secondary QTLs undetected under normal recombination. These results show the benefits of enhanced recombination to decipher the genetic bases of traits.

Identification, validation and candidate gene analysis of major QTL for Supernumerary spikelets in wheat.

BACKGROUND: The number of spikelets per spike is a key trait that affects the yield of bread wheat (Triticum aestivum L.). Identification of the QTL for spikelets per spike and its genetic effects that could be used in molecular assistant breeding in the future. RESULTS: In this study, four recombinant inbred line (RIL) populations were generated and used, having YuPi branching wheat (YP), with Supernumerary Spikelets (SS) phenotype, as a common parent. QTL (QSS.sicau-2 A and QSS.sicau-2D) related to SS trait were mapped on chromosomes 2 A and 2D through bulked segregant exome sequencing (BSE-Seq). Fourteen molecular markers were further developed within the localization interval, and QSS.sicau-2 A was narrowed to 3.0 cM covering 7.6 Mb physical region of the reference genome, explaining 13.7 - 15.9% the phenotypic variance. Similarly, the QSS.sicau-2D was narrowed to 1.8 cM covering 2.4 Mb physical region of the reference genome, and it explained 27.4 - 32.9% the phenotypic variance. These two QTL were validated in three different genetic backgrounds using the linked markers. QSS.sicau-2 A was identified as WFZP-A, and QSS.sicau-2D was identified a novel locus, different to the previously identified WFZP-D. Based on the gene expression patterns, gene annotation and sequence analysis, TraesCS2D03G0260700 was predicted to be a potential candidate gene for QSS.sicau-2D. CONCLUSION: Two significant QTL for SS, namely QSS.sicau-2 A and QSS.sicau-2D were identified in multiple environments were identified and their effect in diverse genetic populations was assessed. QSS.sicau-2D is a novel QTL associated with the SS trait, with TraesCS2D03G0260700 predicted as its candidate gene.

Identification of candidate genes controlling cold tolerance at the early seedling stage from Dongxiang wild rice by QTL mapping, BSA-Seq and RNA-Seq.

BACKGROUND: The cold tolerance of rice is closely related to its production and geographic distribution. The identification of cold tolerance-related genes is of important significance for developing cold-tolerant rice. Dongxiang wild rice (Oryza rufipogon Griff.) (DXWR) is well-adapted to the cold climate of northernmost-latitude habitats ever found in the world, and is one of the most valuable rice germplasms for cold tolerance improvement. RESULTS: Transcriptome analysis revealed genes differentially expressed between Xieqingzao B (XB; a cold sensitive variety) and 19H19 (derived from an interspecific cross between DXWR and XB) in the room temperature (RT), low temperature (LT), and recovery treatments. The results demonstrated that chloroplast genes might be involved in the regulation of cold tolerance in rice. A high-resolution SNP genetic map was constructed using 120 BC5F2 lines derived from a cross between 19H19 and XB based on the genotyping-by-sequencing (GBS) technique. Two quantitative trait loci (QTLs) for cold tolerance at the early seedling stage (CTS), qCTS12 and qCTS8, were detected. Moreover, a total of 112 candidate genes associated with cold tolerance were identified based on bulked segregant analysis sequencing (BSA-seq). These candidate genes were divided into eight functional categories, and the expression trend of candidate genes related to 'oxidation-reduction process' and 'response to stress' differed between XB and 19H19 in the RT, LT and recovery treatments. Among these candidate genes, the expression level of LOC_Os12g18729 in 19H19 (related to 'response to stress') decreased in the LT treatment but restored and enhanced during the recovery treatment whereas the expression level of LOC_Os12g18729 in XB declined during recovery treatment. Additionally, XB contained a 42-bp deletion in the third exon of LOC_Os12g18729, and the genotype of BC5F2 individuals with a survival percentage (SP) lower than 15% was consistent with that of XB. Weighted gene coexpression network analysis (WGCNA) and modular regulatory network learning with per gene information (MERLIN) algorithm revealed a gene interaction/coexpression network regulating cold tolerance in rice. In the network, differentially expressed genes (DEGs) related to 'oxidation-reduction process', 'response to stress' and 'protein phosphorylation' interacted with LOC_Os12g18729. Moreover, the knockout mutant of LOC_Os12g18729 decreased cold tolerance in early rice seedling stage signifcantly compared with that of wild type. CONCLUSIONS: In general, study of the genetic basis of cold tolerance of rice is important for the development of cold-tolerant rice varieties. In the present study, QTL mapping, BSA-seq and RNA-seq were integrated to identify two CTS QTLs qCTS8 and qCTS12. Furthermore, qRT-PCR, genotype sequencing and knockout analysis indicated that LOC_Os12g18729 could be the candidate gene of qCTS12. These results are expected to further exploration of the genetic mechanism of CTS in rice and improve cold tolerance of cultivated rice by introducing the cold tolerant genes from DXWR through marker-assisted selection.

Genome-wide atlas of rust resistance loci in wheat.

Rust diseases, including leaf rust, stripe/yellow rust, and stem rust, significantly impact wheat (Triticum aestivum L.) yields, causing substantial economic losses every year. Breeding and deployment of cultivars with genetic resistance is the most effective and sustainable approach to control these diseases. The genetic toolkit for wheat breeders to select for rust resistance has rapidly expanded with a multitude of genetic loci identified using the latest advances in genomics, mapping and cloning strategies. The goal of this review was to establish a wheat genome atlas that provides a comprehensive summary of reported loci associated with rust resistance. Our atlas provides a summary of mapped quantitative trait loci (QTL) and characterised genes for the three rusts from 170 publications over the past two decades. A total of 920 QTL or resistance genes were positioned across the 21 chromosomes of wheat based on the latest wheat reference genome (IWGSC RefSeq v2.1). Interestingly, 26 genomic regions contained multiple rust loci suggesting they could have pleiotropic effects on two or more rust diseases. We discuss a range of strategies to exploit this wealth of genetic information to efficiently utilise sources of resistance, including genomic information to stack desirable and multiple QTL to develop wheat cultivars with enhanced resistance to rust disease.

A catalogue of recombination coldspots in interspecific tomato hybrids.

Increasing natural resistance and resilience in plants is key for ensuring food security within a changing climate. Breeders improve these traits by crossing cultivars with their wild relatives and introgressing specific alleles through meiotic recombination. However, some genomic regions are devoid of recombination especially in crosses between divergent genomes, limiting the combinations of desirable alleles. Here, we used pooled-pollen sequencing to build a map of recombinant and non-recombinant regions between tomato and five wild relatives commonly used for introgressive tomato breeding. We detected hybrid-specific recombination coldspots that underscore the role of structural variations in modifying recombination patterns and maintaining genetic linkage in interspecific crosses. Crossover regions and coldspots show strong association with specific TE superfamilies exhibiting differentially accessible chromatin between somatic and meiotic cells. About two-thirds of the genome are conserved coldspots, located mostly in the pericentromeres and enriched with retrotransposons. The coldspots also harbor genes associated with agronomic traits and stress resistance, revealing undesired consequences of linkage drag and possible barriers to breeding. We presented examples of linkage drag that can potentially be resolved by pairing tomato with other wild species. Overall, this catalogue will help breeders better understand crossover localization and make informed decisions on generating new tomato varieties.

Genome-wide association study reveals genetic loci for ten trace elements in foxtail millet (Setaria italica).

KEY MESSAGE: One hundred and fifty-five QTL for trace element concentrations in foxtail millet were identified using a genome-wide association study, and a candidate gene associated with Ni-Co-Cr concentrations was detected. Foxtail millet (Setaria italica) is an important regional crop known for its rich mineral nutrient content, which has beneficial effects on human health. We assessed the concentrations of ten trace elements (Ba, Co, Cr, Cu, Fe, Mn, Ni, Pb, Sr, and Zn) in the grain of 408 foxtail millet accessions. Significant differences in the concentrations of five elements (Ba, Co, Ni, Sr, and Zn) were observed between two subpopulations of spring- and summer-sown foxtail millet varieties. Moreover, 84.4% of the element pairs exhibited significant correlations. To identify the genetic factors influencing trace element accumulation, a comprehensive genome-wide association study was conducted, identifying 155 quantitative trait locus (QTL) for the ten trace elements across three different environments. Among them, ten QTL were consistently detected in multiple environments, including qZn2.1, qZn4.4, qCr4.1, qFe6.3, qFe6.5, qCo6.1, qPb7.3, qPb7.5, qBa9.1, and qNi9.1. Thirteen QTL clusters were detected for multiple elements, which partially explained the correlations between elements. Additionally, the different concentrations of five elements between foxtail millet subpopulations were caused by the different frequencies of high-concentration alleles associated with important marker-trait associations. Haplotype analysis identified a candidate gene SETIT_036676mg associated with Ni accumulation, with the GG haplotype significantly increasing Ni-Co-Cr concentrations in foxtail millet. A cleaved amplified polymorphic sequence marker (cNi6676) based on the two haplotypes of SETIT_036676mg was developed and validated. Results of this study provide valuable reference information for the genetic research and improvement of trace element content in foxtail millet.

Mapping of Aegilops speltoides derived leaf rust and stripe rust resistance genes using 35K SNP array.

Wheat is an essential food commodity cultivated throughout the world. However, this crop faces continuous threats from fungal pathogens, leaf rust (LR) and stripe rust (YR). To continue feeding the growing population, these major destructors of wheat must be effectively countered by enhancing the genetic diversity of cultivated germplasm. In this study, an introgression line with hexaploid background (ILsp3603) carrying resistance against Pt pathotypes 77-5 (121R63-1), 77-9 (121R60-1) and Pst pathotypes 46S119 (46E159), 110S119 (110E159), 238S119 (238E159) was developed from donor wheat wild progenitor, Aegilops speltoides acc pau 3603. To understand the genetic basis of resistance and map these genes (named Lrsp3603 and Yrsp3603), inheritance studies were carried out in F6 and F7 mapping population, developed by crossing ILsp3603 with LR and YR susceptible cultivar WL711, which revealed a monogenic (single gene) inheritance pattern for each of these traits. Bulk segregant analysis combined with 35 K Axiom SNP array genotyping mapped both genes as separate entities on the short arm of chromosome 6B. A genetic linkage map, comprising five markers, 1 SNP, 1 PLUG and three gene based SSRs, covered a genetic distance of 12.65 cM. Lrsp3603 was flanked by markers Tag-SSR14 (located proximally at 2.42 cM) and SNP AX-94542331 (at 3.28 cM) while Yrsp3603 was mapped at one end closest to AX-94542331 at 6.62 cM distance. Functional annotation of Lrsp3603 target region (∼ 1 Mbp) revealed 10 gene IDs associated with disease resistance mechanisms including three encoding typical R gene domains.

Phenotypic characterization and gene mapping of hybrid necrosis in Triticum durum-Haynaldia villosa amphiploids.

KEY MESSAGE: Phenotypical, physiological and genetic characterization was carried out on the hybrid necrosis gene from Haynaldia villosa, and the related gene Ne-V was mapped to chromosome arm 2VL. Introducing genetic variation from wild relatives into common wheat through wide crosses is a vital strategy for enriching genetic diversity and promoting wheat breeding. However, hybrid necrosis, a genetic autoimmunity syndrome, often occurs in the offspring of interspecific or intraspecific crosses, restricting both the selection of hybrid parents and the pyramiding of beneficial genes. To utilize the germplasms of Haynaldia villosa (2n = 2x = 14, VV), we conducted wide hybridization between durum wheat (2n = 4x = 28, AABB) and multiple H. villosa accessions to synthesize the amphiploids (2n = 6x = 42, AABBVV). This study revealed that 61.5% of amphiploids derived from the above crosses exhibited hybrid necrosis, with some amphiploids even dying before reaching maturity. However, the initiation time and severity of necrosis varied dramatically among the progenies, suggesting that there were multiple genetic loci or multiple alleles in the same genetic locus conferring to hybrid necrosis in H. villosa accessions. Genetic analysis was performed on the F2 and derived F2:3 populations, which were constructed between amphiploid STH59-1 with normal leaves and amphiploid STH59-2 with necrotic leaves. A semidominant hybrid necrosis-related gene, Ne-V, was mapped to an 11.8-cM genetic interval on the long arm of chromosome 2V, representing a novel genetic locus identified in Triticum-related species. In addition, the hybrid necrosis was correlated with enhanced H2O2 accumulation and cell death, and it was influenced by the temperature and light. Our findings provide a foundation for cloning the Ne-V gene and exploring its molecular mechanism.

High-Throughput Mapping of Chromosomal Conformations in E. coli Under Physiological Conditions Using Massively Multiplexed Mu Transposition.

Many approaches for measuring three-dimensional chromosomal conformations rely upon formaldehyde crosslinking followed by subsequent proximity ligation, a family of methods exemplified by 3C, Hi-C, etc. Here we provide an alternative crosslinking-free procedure for high-throughput identification of long-range contacts in the chromosomes of enterobacteria, making use of contact-dependent transposition of phage Mu to identify distant loci in close contact. The procedure described here will suffice to provide a comprehensive map of transposition frequencies between tens of thousands of loci in a bacterial genome, with the resolution limited by the diversity of the insertion site library used and the sequencing depth applied.

In Vivo Genomic Supercoiling Mapping Using Psora-seq.

Supercoiling is a fundamental property of DNA that governs all strand opening reactions, including DNA replication, transcription, and homologous recombination. However, traditional genomic supercoiling assays are difficult and lack sensitivity. Building on prior assays using the DNA intercalator psoralen, we developed a supercoil mapping assay that is robust and sensitive to a wide range of supercoiling while requiring only commercially available reagents and common laboratory equipment. This method, psoralen affinity purification with genomic sequencing (Psora-seq), utilizes biotinylated psoralen and streptavidin-conjugated magnetic beads to facilitate efficient pull-down of psoralen-bound DNA, followed by deep sequencing to identify and quantify supercoiling at 1 kb resolution. Psora-seq overcomes two major bottlenecks associated with existing psoralen pull-down assays, inefficient photo-binding of psoralen-bound molecules, and poor recovery of cross-linked DNA.

Refinement of rice blast disease resistance QTLs and gene networks through meta-QTL analysis.

Rice blast disease is the most devastating disease constraining crop productivity. Vertical resistance to blast disease is widely studied despite its instability. Clusters of genes or QTLs conferring blast resistance that offer durable horizontal resistance are important in resistance breeding. In this study, we aimed to refine the reported QTLs and identify stable meta-QTLs (MQTLs) associated with rice blast resistance. A total of 435 QTLs were used to project 71 MQTLs across all the rice chromosomes. As many as 199 putative rice blast resistance genes were identified within 53 MQTL regions. The genes included 48 characterized resistance gene analogs and related proteins, such as NBS-LRR type, LRR receptor-like kinase, NB-ARC domain, pathogenesis-related TF/ERF domain, elicitor-induced defense and proteins involved in defense signaling. MQTL regions with clusters of RGA were also identified. Fifteen highly significant MQTLs included 29 candidate genes and genes characterized for blast resistance, such as Piz, Nbs-Pi9, pi55-1, pi55-2, Pi3/Pi5-1, Pi3/Pi5-2, Pikh, Pi54, Pik/Pikm/Pikp, Pb1 and Pb2. Furthermore, the candidate genes (42) were associated with differential expression (in silico) in compatible and incompatible reactions upon disease infection. Moreover, nearly half of the genes within the MQTL regions were orthologous to those in O. sativa indica, Z. mays and A. thaliana, which confirmed their significance. The peak markers within three significant MQTLs differentiated blast-resistant and susceptible lines and serve as potential surrogates for the selection of blast-resistant lines. These MQTLs are potential candidates for durable and broad-spectrum rice blast resistance and could be utilized in blast resistance breeding.

Nitrogen deficiency tolerance conferred by introgression of a QTL derived from wild emmer into bread wheat.

KEY MESSAGE: Genetic dissection of a QTL from wild emmer wheat, QGpc.huj.uh-5B.2, introgressed into bread wheat, identified candidate genes associated with tolerance to nitrogen deficiency, and potentially useful for improving nitrogen-use efficiency. Nitrogen (N) is an important macronutrient critical to wheat growth and development; its deficiency is one of the main factors causing reductions in grain yield and quality. N availability is significantly affected by drought or flooding, that are dependent on additional factors including soil type or duration and severity of stress. In a previous study, we identified a high grain protein content QTL (QGpc.huj.uh-5B.2) derived from the 5B chromosome of wild emmer wheat, that showed a higher proportion of explained variation under water-stress conditions. We hypothesized that this QTL is associated with tolerance to N deficiency as a possible mechanism underlying the higher effect under stress. To validate this hypothesis, we introgressed the QTL into the elite bread wheat var. Ruta, and showed that under N-deficient field conditions the introgression IL99 had a 33% increase in GPC (p < 0.05) compared to the recipient parent. Furthermore, evaluation of IL99 response to severe N deficiency (10% N) for 14 days, applied using a semi-hydroponic system under controlled conditions, confirmed its tolerance to N deficiency. Fine-mapping of the QTL resulted in 26 homozygous near-isogenic lines (BC4F5) segregating to N-deficiency tolerance. The QTL was delimited from - 28.28 to - 1.29 Mb and included 13 candidate genes, most associated with N-stress response, N transport, and abiotic stress responses. These genes may improve N-use efficiency under severely N-deficient environments. Our study demonstrates the importance of WEW as a source of novel candidate genes for sustainable improvement in tolerance to N deficiency in wheat.

Haplotype-based association mapping of genomic regions associated with Zymoseptoria tritici resistance using 217 diverse wheat genotypes.

BACKGROUND: Septoria tritici blotch (STB) is considered to be one of the most destructive foliar wheat diseases and is caused by Zymoseptoria tritici. The yield losses are severe and in Northwestern Europe can reach up to 50%. The efficacy of fungicides is diminishing due to changes in the genetic structure of the pathogen. Therefore, resistance breeding is the most effective strategy of disease management. Recently, genome-wide association studies (GWAS) have become more popular due to their robustness in dissecting complex traits, including STB resistance in wheat. This was made possible by the use of large mapping populations and new sequencing technologies. High-resolution mapping benefits from historical recombination and greater allele numbers in GWAS. RESULTS: In our study, 217 wheat genotypes of diverse origin were phenotyped against five Z. tritici isolates (IPO323, IPO88004, IPO92004, IPO86036 and St1-03) and genotyped on the DArTseq platform. In polytunnel tests two disease parameters were evaluated: the percentage of leaf area covered by necrotic lesions (NEC) and the percentage of leaf area covered by lesions bearing pycnidia (PYC). The disease escape parameters heading date (Hd) and plant height (Ht) were also measured. Pearson's correlation showed a positive effect between disease parameters, providing additional information. The Structure analysis indicated four subpopulations which included from 28 (subpopulation 2) to 79 genotypes (subpopulation 3). All of the subpopulations showed a relatively high degree of admixture, which ranged from 60% of genotypes with less than 80% of proportions of the genome attributed to assigned subpopulation for group 2 to 85% for group 4. Haplotype-based GWAS analysis allowed us to identify 27 haploblocks (HBs) significantly associated with analysed traits with a p-value above the genome-wide significance threshold (5%, which was -log10(p) > 3.64) and spread across the wheat genome. The explained phenotypic variation of identified significant HBs ranged from 0.2% to 21.5%. The results of the analysis showed that four haplotypes (HTs) associated with disease parameters cause a reduction in the level of leaf coverage by necrosis and pycnidia, namely: Chr3A_HB98_HT2, Chr5B_HB47_HT1, Chr7B_HB36_HT1 and Chr5D_HB10_HT3. CONCLUSIONS: GWAS analysis enabled us to identify four significant chromosomal regions associated with a reduction in STB disease parameters. The list of valuable HBs and wheat varieties possessing them provides promising material for further molecular analysis of resistance loci and development of breeding programmes.