Haplotype Sequences Research Articles

Haplotype-resolved genome assembly and resequencing analysis provide insights into genome evolution and allelic imbalance in Pinus densiflora.

Haplotype-level allelic characterization facilitates research on the functional, evolutionary and breeding-related features of extremely large and complex plant genomes. We report a 21.7-Gb chromosome-level haplotype-resolved assembly in Pinus densiflora. We found genome rearrangements involving translocations and inversions between chromosomes 1 and 3 of Pinus species and a proliferation of specific long terminal repeat (LTR) retrotransposons (LTR-RTs) in P. densiflora. Evolutionary analyses illustrated that tandem and LTR-RT-mediated duplications led to an increment of transcription factor (TF) genes in P. densiflora. The haplotype sequence comparison showed allelic imbalances, including presence-absence variations of genes (PAV genes) and their functional contributions to flowering and abiotic stress-related traits in P. densiflora. Allele-aware resequencing analysis revealed PAV gene diversity across P. densiflora accessions. Our study provides insights into key mechanisms underlying the evolution of genome structure, LTR-RTs and TFs within the Pinus lineage as well as allelic imbalances and diversity across P. densiflora.

Cellulose synthase-like OsCSLD4: a key regulator of agronomic traits, disease resistance, and metabolic indices in rice.

Cellulose synthase-like OsCSLD4 plays a pivotal role in regulating diverse agronomic traits, enhancing resistance against bacterial leaf blight, and modulating metabolite indices based on the multi-omics analysis in rice. To delve deeper into this complex network between agronomic traits and metabolites in rice, we have compiled a dataset encompassing genome, phenome, and metabolome, including 524 diverse accessions, 11 agronomic traits, and 841 metabolites, enabling us to pinpoint eight hotspots through GWAS. We later discovered four distinct metabolite categories, encompassing 15 metabolites that are concurrently present on the QTL qC12.1, associated with leaf angle of flag and spikelet length, and finally focused the cellulose synthase-like OsCSLD4, which was pinpointed through a rigorous process encompassing sequence variation, haplotype, ATAC, and differential expression across diverse tissues. Compared to the wild type, csld4 exhibited significant reductions in the plant height, flag leaf length, leaf width, spikelet length, 1000-grain weight, grain width, grain thickness, fertility, yield per plant, and bacterial blight resistance. However, there were significant increase in tiller numbers, degree of leaf rolling, flowering period, growth period, grain length, and empty kernel rate. Furthermore, the content of four polyphenol metabolites, excluding metabolite N-feruloyltyramine (mr1268), notably rose, whereas the levels of the other three polyphenol metabolites, smiglaside C (mr1498), 4-coumaric acid (mr1622), and smiglaside A (mr1925) decreased significantly in mutant csld4. The content of amino acid L-tyramine (mr1446) exhibited a notable increase, whereas the alkaloid trigonelline (mr1188) displayed a substantial decrease among the mutants. This study offered a comprehensive multi-omics perspective to analyze the genetic mechanism of OsCSLD4, and breeders can potentially enhance rice's yield, bacterial leaf blight resistance, and metabolite content, leading to more sustainable and profitable rice production.

Potential approaches to create ultimate genotypes in crops and livestock.

Many thousands and, in some cases, millions of individuals from the major crop and livestock species have been genotyped and phenotyped for the purpose of genomic selection. 'Ultimate genotypes', in which the marker allele haplotypes with the most favorable effects on a target trait or traits in the population are combined together in silico, can be constructed from these datasets. Ultimate genotypes display up to six times the performance of the current best individuals in the population, as demonstrated for net profit in dairy cattle (incorporating a range of economic traits), yield in wheat and 100-seed weight in chickpea. However, current breeding strategies that aim to assemble ultimate genotypes through conventional crossing take many generations. As a hypothetical thought piece, here, we contemplate three future pathways for rapidly achieving ultimate genotypes: accelerated recombination with gene editing, direct editing of whole-genome haplotype sequences and synthetic biology.

GL5.2, a Quantitative Trait Locus for Rice Grain Shape, Encodes a RING-Type E3 Ubiquitin Ligase.

Grain weight and grain shape are important traits that determine rice grain yield and quality. Mining more quantitative trait loci (QTLs) that control grain weight and shape will help to further improve the molecular regulatory network of rice grain development and provide gene resources for high-yield and high-quality rice varieties. In the present study, a QTL for grain length (GL) and grain width (GW), qGL5.2, was firstly fine-mapped into a 21.4 kb region using two sets of near-isogenic lines (NILs) derived from the indica rice cross Teqing (TQ) and IRBB52. In the NIL populations, the GL and ratio of grain length to grain width (RLW) of the IRBB52 homozygous lines increased by 0.16-0.20% and 0.27-0.39% compared with the TQ homozygous lines, but GW decreased by 0.19-0.75%. Then, by analyzing the grain weight and grain shape of the knock-out mutant, it was determined that the annotation gene Os05g0551000 encoded a RING-type E3 ubiquitin ligase, which was the cause gene of qGL5.2. The results show that GL and RLW increased by 2.44-5.48% and 4.19-10.70%, but GW decreased by 1.69-4.70% compared with the recipient. Based on the parental sequence analysis and haplotype analysis, one InDel variation located at -1489 in the promoter region was likely to be the functional site of qGL5.2. In addition, we also found that the Hap 5 (IRBB52-type) increased significantly in grain length and grain weight compared with other haplotypes, indicating that the Hap 5 can potentially be used in rice breeding to improve grain yield and quality.

Relics of interspecific hybridization retained in the genome of a drought-adapted peanut cultivar.

Peanut (Arachis hypogaea L.) is a globally important oil and food crop frequently grown in arid, semi-arid, or dryland environments. Improving drought tolerance is a key goal for peanut crop improvement efforts. Here we present the genome assembly and gene model annotation for 'Line8', a peanut genotype bred from drought tolerant cultivars. Our assembly and annotation are the most contiguous and complete peanut genome resources currently available. The high contiguity of the Line8 assembly allowed us to explore structural variation both between peanut genotypes and subgenomes. We detect several large inversions between Line8 and other peanut genome assemblies, and there is a trend for the inversions between more genetically diverged genotypes to have higher gene content. We also relate patterns of subgenome exchange to structural variation between Line8 homeologous chromosomes. Unexpectedly, we discover that Line8 harbors an introgression from A.cardenasii, a diploid peanut relative and important donor of disease resistance alleles to peanut breeding populations. The fully resolved sequences of both haplotypes in this introgression provide the first in situ characterization of A.cardenasii candidate alleles that can be leveraged for future targeted improvement efforts. The completeness of our genome will support peanut biotechnology and broader research into the evolution of hybridization and polyploidy.

Detecting serologically difficult ABO blood groups using single-molecule real-time sequencing technology.

Recently, third-generation long-read sequencing technology has been increasingly applied to the detection of various blood group systems. Because of its long read length and use of single-molecule sequencing, it is capable of obtaining the sequences of blood group genes in their entirety as well as of distinguishing haplotypes. Therefore, here, we collected ABO blood group samples that were difficult to classify serologically and analysed the sequences of the coding regions of the ABO genes as well as the sequences upstream and downstream of the coding regions. Samples with ABO antigen typing and reverse serum typing discrepancies were screened in a total of 21 patients. All samples were subjected to serological testing and preliminary ABO genotyping (polymerase chain reaction with sequence-specific primers [PCR-SSP]), followed by single-molecule real-time (SMRT) sequencing to obtain complete ABO gene sequences. PCR sequence-based typing (PCR-SBT) was performed to validate the results. Of the 21 samples, 15 had common ABO types, and 6 had rare ABO subtypes. One new allele, ABO*B.NEW (c.861C>T), and one allelic base recombination event was identified. Forty-two haplotype sequences were obtained via SMRT sequencing with intronic single-nucleotide variants (SNVs) specific to the ABO allele, and all of the exon region sequences were consistent with the PCR-SBT results. SMRT sequencing is capable of accurately obtaining complete ABO gene sequences, distinguishing haplotypes and identifying allelic recombination.

Universal Targeted Haplotyping by Droplet Digital PCR Sequencing and Its Applications in Noninvasive Prenatal Testing and Pharmacogenetics Analysis.

The analysis of haplotypes of variants is important for pharmacogenomics analysis and noninvasive prenatal testing for monogenic diseases. However, there is a lack of robust methods for targeted haplotyping. We developed digital PCR haplotype sequencing (dHapSeq) for targeted haplotyping of variants, which is a method that compartmentalizes long DNA molecules into droplets. Within one droplet, 2 target regions are PCR amplified from one template molecule, and their amplicons are fused together. The fused products are then sequenced to determine the phase relationship of the single nucleotide polymorphism (SNP) alleles. The entire haplotype of 10s of SNPs can be deduced after the phase relationship of individual SNPs are determined in a pairwise manner. We applied dHapSeq to noninvasive prenatal testing in 4 families at risk for thalassemia and utilized it to detect NUDT15 diplotypes for predicting drug tolerance in pediatric acute lymphoblastic leukemia (72 cases and 506 controls). For SNPs within 40 kb, phase relation can be determined with 100% accuracy. In 7 trio families, the haplotyping results for 97 SNPs spanning 185 kb determined by dHapSeq were concordant with the results deduced from the genotypes of both parents and the fetus. In 4 thalassemia families, a 19.3-kb Southeast Asian deletion was successfully phased with 97 downstream SNPs, enabling noninvasive determination of fetal inheritance using relative haplotype dosage analysis. In the NUDT15 analysis, the variant status and phase of the variants were successfully determined in all cases and controls. The dHapSeq represents a robust and scalable haplotyping approach with numerous clinical and research applications.

Genome-wide association study identifies candidate genes for bacterial soft rot resistance in cucumber seedlings

Bacterial soft rot (BSR) caused by Pectobacterium carotovorum subsp. brasiliense (Pcb) is a serious bacterial disease which negatively impact yield and quality in cucumber. However, the genetic mechanism of BSR resistance in cucumber has not been reported. Here, we investigated the BSR resistance of 119 cucumber core germplasm worldwide at the seedling stage and identified 26 accessions highly resistant to BSR. A total of 1 642 740 single-nucleotide polymorphisms (SNPs) were used to conduct GWAS, and five loci associated with BSR resistance were detected on four chromosomes: gBSR2.1, gBSR2.2, gBSR3.1, gBSR4.1 and gBSR5.1. Based on haplotype analysis, sequence polymorphisms, functional annotation and qRT-PCR analysis, six candidate genes were identified within the five loci. CsaV3_2G014450, CsaV3_2G014490, CsaV3_2G016000, CsaV3_3G000850, CsaV3_4G033150, and CsaV3_5G000390 each had nonsynonymous SNPs, and were significantly up-regulated in the resistant genotypes after inoculation. And CsaV3_5G000390 in the susceptible genotype was significantly up-regulated after inoculation. The identification of these candidate genes lays a foundation for understanding the genetic mechanism of BSR resistance in cucumber. Generally, our study mined genes associated with BSR resistance in cucumber seedlings and will assist the breeding of BSR-resistant cucumber cultivars.

CD59 gene: 143 haplotypes of 22,718 nucleotides length by computational phasing in 113 individuals from different ethnicities.

CD59 deficiency due to rare germline variants in the CD59 gene causes disabilities, ischemic strokes, neuropathy, and hemolysis. CD59 deficiency due to common somatic variants in the PIG-A gene in hematopoietic stem cells causes paroxysmal nocturnal hemoglobinuria. The ISBT database lists one nonsense and three missense germline variants that are associated with the CD59-null phenotype. To analyze the genetic diversity of the CD59 gene, we determined long-range CD59 haplotypes among individuals from different ethnicities. We determined a 22.7 kb genomic fragment of the CD59 gene in 113 individuals using next-generation sequencing (NGS), which covered the whole NM_203330.2 mRNA transcript of 7796 base pairs. Samples came from an FDA reference repository and our Ethiopia study cohorts. The raw genotype data were computationally phased into individual haplotype sequences. Nucleotide sequencing of the CD59 gene of 226 chromosomes identified 216 positions with single nucleotide variants. Only three haplotypes were observed in homozygous form, which allowed us to assign them unambiguously as experimentally verified CD59 haplotypes. They were also the most frequent haplotypes among both cohorts. An additional 140 haplotypes were imputed computationally. We provided a large set of haplotypes and proposed three verified long-range CD59 reference sequences, based on a population approach, using a generalizable rationale for our choice. Correct long-range haplotypes are useful as template sequences for allele calling in high-throughput NGS and precision medicine approaches, thus enhancing the reliability of clinical diagnostics. Long-range haplotypes can also be used to evaluate the influence of genetic variation on the risk of transfusion reactions or diseases.

Resolution of RHCE Haplotype Ambiguities in Transfusion Settings.

Red blood cell (RBC) transfusion, limited by patient alloimmunization, demands accurate blood group typing. The Rh system requires specific attention due to the limitations of serological phenotyping methods. Although these have been compensated for by molecular biology solutions, some RhCE ambiguities remain unresolved. The RHCE mRNA length is compatible with full-length analysis and haplotype discrimination, but the RHCE mRNA analyses reported so far are based on reticulocyte isolation and molecular biology protocols that are fastidious to implement in a routine context. We aim to present the most efficient reticulocyte isolation method, combined with an RT-PCR sequencing protocol that embraces the phasing of all haplotype configurations and identification of any allele. Two protocols were tested for reticulocyte isolation based either on their size/density properties or on their specific antigenicity. We show that the reticulocyte sorting method by antigen specificity from EDTA blood samples collected up to 48 h before processing is the most efficient and that the combination of an RHCE-specific RT-PCR followed by RHCE allele-specific sequencing enables analysis of cDNA RHCE haplotypes. All samples analyzed show full concordance between RHCE phenotype and haplotype sequencing. Two samples from the immunohematology laboratory with ambiguous results were successfully analyzed and resolved, one of them displaying a novel RHCE allele (RHCE*03 c.340C>T).

Haemonchus contortus, an obligatory haematophagus worm infection in small ruminants: Population genetics and genetic diversity

Haemonchus contortus, a stomach worm, is prevalent in ruminants worldwide. They particularly hamper profitable small ruminant production. Here, we estimate the genetic variation of H. contortus collected from slaughtered goats and sheep from various geographic zones of Bangladesh using multiple genes. To perform this, adult parasites were isolated from the abomasum of slaughtered animals (sheep and goats). Among them, 79 male H. contortus were identified by microscopy. Following the extraction of DNA, ITS-2 and cox1 genes were amplified and subsequently considered for sequencing. After alignment and editing, sequences were analyzed to find out sequence variation, diversity pattern of genes, and population genetics of isolates. Among the sequence data, the analyses identified 19 genotypes of ITS-2 and 77 haplotypes of cox1 genes. The diversity of nucleotides was 0.0103 for ITS-2 and 0.029 for cox1 gene. The dendogram constructed by the genotype and haplotype sequences of H. contortus revealed that two populations were circulating in Bangladesh without any demarcation of host and geographic regions. Analysis of population genetics demonstrated a high flow of genes (89.2 %) within the population of the worm in Bangladesh. The Fst value showed very little amount of genetic difference among the worm populations of Bangladesh but marked genetic variation between different continents. The findings are expected to help explain the risks of anthelmintic resistance and the transmission pattern of the parasite, and also provide a control strategy against H. contortus.

Gene Polymorphisms LEP, LEPR, 5HT2A, GHRL, NPY, and FTO-Obesity Biomarkers in Metabolic Risk Assessment: A Retrospective Pilot Study in Overweight and Obese Population in Romania

Genome-wide association studies (GWAS) have successfully revealed numerous susceptibility loci for obesity. The PREDATORR study (2014) shows that in Romania, 346% of adults aged 20–79 y/o are overweight, and 31.4% are obese with a high risk of cardiometabolic complications, a number that puts almost 67% of Romania’s population in the abnormal weight group. Our study aims to investigate the current status of the genetic foundation in metabolic disease associated with obesity, applied to a pilot group of patients specifically examining the impact of known polymorphisms and their haplotype of six food intake-regulating genes, namely leptin (LEP), leptin receptor (LEP-R), serotonin receptor (5HTR2A), ghrelin (GHRL), neuropeptide Y (NPY), and fat-mass and obesity-associated protein (FTO) with the following polymorphisms: LEP A-2548G, LEPR A-223G, 5HTR2A G-1439A, GHRL G-72T, NPY T-29063C, FTO A-T, and body mass index (BMI). A notable link between the LEP-2548 rs7799039 gene’s AG genotype and the risk of obesity was observed, particularly pronounced in males aged 40–49, with an approximately seven-fold increased likelihood of obesity. The 5HTR2A rs6311 AA genotype was associated with a higher BMI, which was not statistically significant. The FTO rs9939609 gene’s AA genotype emerged as a significant predictor of obesity risk. Besides these significant findings, no substantial associations were observed with the LEPR, 5HTR2A, GHRL, and NPY genes. Haplotype association analysis showed a suggestive indication of GRGMLA (rs7799039, rs1137101, rs6311, rs696217, rs16139, rs9939609 sequence) haplotype with a susceptibility effect towards obesity predisposition. Linkage disequilibrium (LD) analysis showed statistically significant associations between LEP and LEPR gene (p = 0.04), LEP and GHRL gene (p = 0.0047), and GHRL and FTO gene (p = 0.03). Our study, to the best of our knowledge, is one of the very few on the Romanian population, and aims to be a starting point for further research on the targeted interventional strategies to reduce cardiometabolic risks.

Molecular Detection of Anaplasma phagocytophilum in Small Mammals and Infesting Ticks in Laikipia County, Kenya.

Anaplasmosis is a set of disease conditions of various mammals caused by bacteria species of the genus Anaplasma. These are sub-microscopic, Gram-negative, obligate intracellular pathogens that infect both vertebrate and invertebrate hosts. Significant species that infect domestic and wildlife animals include Anaplasma marginale, Anaplasma ovis, Anaplasma mesaeterum, Anaplasma platys, and Anaplasma phagocytophilum. Although A. phagocytophilum has a widespread distribution, there are only a few epidemiological reports from sub-Saharan Africa. This study focused on molecular detection and characterization of A. phagocytophilum in small mammals and their infesting ticks in Laikipia County, Kenya. A total of 385 blood and 84 tick archival samples from small mammals (155 females and 230 males) were analyzed. The blood samples were subjected to a nested PCR-HRM melt analysis using species-specific primers to amplify the 16S ribosomal RNA genes. The ticks were also subjected to nested PCR-HRM involving 16S rRNA gene primers. Anaplasma phagocytophilum DNA was detected in 19 out of 385 samples using species-specific 16S rRNA gene primers giving a prevalence of 4.9% for A. phagocytophilum. Analysis of the tick's samples using 16S rRNA gene species-specific primers also detected A. phagocytophilum in 3 samples from Haemaphysalis leachi ticks (3/84) equivalent to prevalence of 3.6%. Sequencing of 16S rRNA PCR products confirmed A. phagocytophilum in small mammals and ticks' samples. Phylogenetic analysis of the haplotype from this study demonstrated a close ancestral link with strains from Canis lupus familiaris, Alces alces, Apodemus agrarius, and ticks (Haemaphysalis longicornis) reported in Europe, China, and Africa. Comparison was also made with a known pathogenic A. phagocytophilum variant HA and a nonpathogenic variant 1 that were clustered into a distinctive clade different form haplotypes detected in this study. All the haplotype sequences for A. phagocytophilum from this study were submitted and registered in GenBank under the accession numbers OQ308965-OQ308976. Our study shows that small mammals and their associated ticks harbor A. phagocytophilum. The vector competence for H. leachi in A. phagocytophilum transmission should further be investigated.

Mannose-binding lectin gene sequence data in Kelantan population.

The human mannose-binding lectin (MBL) gene encodes a polymorphic protein that plays a crucial role in the innate immune response. Human MBL deficiency is associated with immunodeficiencies, and its variants have been linked to autoimmune and infectious diseases. Despite this significance, gene studies concerning MBL sequencing are uncommon in Malaysia. Therefore, we aimed to preliminary described the human MBL sequencing dataset based on the Kelantan population. Blood samples were collected from 30 unrelated individuals and underwent DNA extraction, genotyping, and sequencing. The sequencing data generated 886 bp, which were deposited in GenBank (ON619541-ON619546). Allelic variants were identified and translated into six MBL haplotypes: HYPA, HYPB, LYPB, LXPB, HXPA, and LXPA. An evolutionary tree was constructed using the haplotype sequences. These findings contribute to the expansion of MBL information within the country, providing a valuable baseline for future research exploring the association between the gene and targeted diseases.

Dark Morph of the Oriental Honey-Buzzard (Pernis ptilorhynchus orientalis) is Attributable to Specific MC1R Haplotypes.

A thorough understanding of the development of complex plumages in birds necessitates the acquisition of genetic data pertaining to the mechanism underlying this phenomenon from various avian species. The oriental honey-buzzard (Pernis ptilorhynchus orientalis), a tropical summer migrant to Northeast Asia, including Japan, exemplifies this aspect owing to the diversity of its ventral coloration and intra-feather barring patterns. However, genetic polymorphism responsible for this diversity has not been identified yet. This study aimed to investigate the link between dark-plumed phenotypes of this subspecies and haplotypes of the melanocortin-1-receptor (MC1R) gene. A draft sequence of MC1R was constructed using next generation sequencing and subsequently amplified using designed polymerase chain reaction (PCR) primers. The genome sequences of 32 honey-buzzard individuals were determined using PCR, and 12 MC1R haplotype sequences were obtained. Among these haplotypes, we found that unique haplotypes with nine non-synonymous substitutions and four or five synonymous substitutions in the coding region had a perfect correlation with the dark-plumed phenotype. The lack of correlation between the genotype of ASIP coding region and plumage phenotype reiterated that the dark morph is attributable to specific MC1R haplotypes. The absence of a correlation between genetic polymorphisms of MC1R and the intra-feather barring patterns, as well as the diversity observed within lighter ground color classes (pale and intermediate), implies the involvement of alternative molecular mechanisms in the manifestation of the aforementioned phenotypes.

Current status of Nosema spp. infection cases in apis mellifera in eurasian countries and Ptp3 gene haplotypes in the Republic of Tatarstan, Russia.

The current status of Nosema spp. infections in A. mellifera throughout Eurasia was characterized using electronic databases. Although N. ceranae was predominantly detected in southwestern and south-central regions and N. apis in northwestern and north-central areas, most studies reported the occurrence of both species in Eurasia. In addition, the occurrence of Nosema spp. and Ptp3 gene haplotypes was investigated in the Republic of Tatarstan, Russia. Most of the examined honey bees were infected with both N. apis and N. ceranae. N. apis and N. ceranae isolates were either heterozygous or belonged to different strains and showed infection with more than one strain. New haplotypes were found for N. apis and N. ceranae in the Republic of Tatarstan, Russia. This study expands the data regarding existing haplotypes of Nosema species: there are currently 9 shared and 56 unique Ptp3 nucleotide sequence haplotypes of N. ceranae, and 2 shared and 7 unique haplotypes of N. apis, respectively.

Clinical and Pathological Features of FTDP-17 with MAPT p.K298_H299insQ Mutation.

MAPT is a causative gene in frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), a hereditary degenerative disease with various clinical manifestations, including progressive supranuclear palsy, corticobasal syndrome, Parkinson's disease, and frontotemporal dementia. To analyze genetically, biochemically, and pathologically multiple members of two families who exhibited various phenotypes of the disease. Genetic analysis included linkage analysis, homozygosity haplotyping, and exome sequencing. We conducted tau protein microtubule polymerization assay, heparin-induced tau aggregation, and western blotting with brain lysate from an autopsy case. We also evaluated abnormal tau aggregation by using anti-tau antibody and PM-PBB3. We identified a variant, c.896_897insACA, p.K298_H299insQ, in the MAPT gene of affected patients. Similar to previous reports, most patients presented with atypical parkinsonism. Biochemical analysis revealed that the mutant tau protein had a reduced ability to polymerize microtubules and formed abnormal fibrous aggregates. Pathological study revealed frontotemporal lobe atrophy, midbrain atrophy, depigmentation of the substantia nigra, and four-repeat tau-positive inclusions in the hippocampus, brainstem, and spinal cord neurons. The inclusion bodies also stained positively with PM-PBB3. This study confirmed that the insACA mutation caused FTDP-17. The affected patients showed symptoms resembling Parkinson's disease initially and symptoms of progressive supranuclear palsy later. Despite the initial clinical diagnosis of frontotemporal dementia in the autopsy case, the spread of lesions could explain the process of progressive supranuclear palsy. The study of more cases in the future will help clarify the common pathogenesis of MAPT mutations or specific pathogeneses of each mutation.

The Identification of Drought Tolerance Candidate Genes in Oryza sativa L. ssp. Japonica Seedlings through Genome-Wide Association Study and Linkage Mapping

Drought stress poses a significant threat to rice production, necessitating the identification of genes associated with drought tolerance. This study employed a combination of genome-wide association study (GWAS) and linkage mapping to pinpoint seedling drought tolerance genes in Japonica rice. Using the leaf rolling scale (LRS) as the phenotypic index, we assessed rice drought tolerance under polyethylene glycol-induced drought during the seedling stage. A lead SNP C8_28933410 by GWAS was identified, which was located within qLRS-8-1 identified by linkage mapping on chromosome 8. Combing the LD block analyses and QTL interval, a 138.6 kb overlap interval was considered as the candidate region. Haplotype analysis, qRT-PCR, sequence analysis, and mutant phenotype verification led to the speculation that LOC_Os08g05520 is a candidate gene associated with drought tolerance. Our findings provide a valuable reference for breeders aiming to enhance rice drought tolerance.

A New ToxA Haplotype in the Wheat Fungal Pathogen Bipolaris sorokiniana.

The necrotrophic effector ToxA is a well-studied virulence factor produced by several fungal necrotrophs. Initially cloned from the wheat tan spot pathogen Pyrenophora tritici-repentis in 1996, ToxA was found almost a decade later in another fungal pathogen, Parastagonospora nodorum, and its sister species, Parastagonospora pseudonodorum. In 2018, ToxA was detected in a third wheat fungal pathogenic species, Bipolaris sorokiniana, which causes spot blotch disease. However, unlike the case with P. tritici-repentis and P. nodorum, the ToxA in B. sorokiniana has only been investigated in recent years. In this report, five Australian B. sorokiniana isolates were assessed for the presence of ToxA. Four isolates were found to contain ToxA. While one isolate harbored the previously reported ToxA haplotype sequence (ToxA19), three isolates contain a different haplotype, designated herein as ToxA25, which has a nonsynonymous mutation resulting in an amino acid change of glycine to arginine at position 168. Both B. sorokiniana ToxA isoforms, when heterologously expressed in Escherichia coli, exhibited the classic ToxA necrosis-inducing activity on ToxA-sensitive Tsn1 cultivars. Preliminary analysis of the B. sorokiniana isolates in Australian wheat cultivars showed that isolates with ToxA19, ToxA25, or ToxA-deficient displayed various degrees of virulence, with the most aggressive isolates observed for those producing ToxA. Differences in spot blotch disease severity between Tsn1 and tsn1 cultivars were observed; however, this was not limited to the ToxA-producing isolates. The overall results suggest that the virulence of the Australian B. sorokiniana isolates is diverse, with the significance of ToxA-Tsn1 interactions depending on individual isolates.

Incipient functional SARS-CoV-2 diversification identified through neural network haplotype maps

Since its introduction in the human population, SARS-CoV-2 has evolved into multiple clades, but the events in its intrahost diversification are not well understood. Here, we compare three-dimensional (3D) self-organized neural haplotype maps (SOMs) of SARS-CoV-2 from thirty individual nasopharyngeal diagnostic samples obtained within a 19-day interval in Madrid (Spain), at the time of transition between clades 19 and 20. SOMs have been trained with the haplotype repertoire present in the mutant spectra of the nsp12- and spike (S)-coding regions. Each SOM consisted of a dominant neuron (displaying the maximum frequency), surrounded by a low-frequency neuron cloud. The sequence of the master (dominant) neuron was either identical to that of the reference Wuhan-Hu-1 genome or differed from it at one nucleotide position. Six different deviant haplotype sequences were identified among the master neurons. Some of the substitutions in the neural clouds affected critical sites of the nsp12-nsp8-nsp7 polymerase complex and resulted in altered kinetics of RNA synthesis in an in vitro primer extension assay. Thus, the analysis has identified mutations that are relevant to modification of viral RNA synthesis, present in the mutant clouds of SARS-CoV-2 quasispecies. These mutations most likely occurred during intrahost diversification in several COVID-19 patients, during an initial stage of the pandemic, and within a brief time period.