Allelic Configuration Research Articles

A genetic method to infer ploidy and aberrant inheritance in triploid organisms.

Polyploidy occurs naturally across eukaryotic lineages and has been harnessed in the domestication of many crops and vertebrates. In aquaculture, triploidy can be induced as a biocontainment strategy, as it creates a reproductive barrier preventing farm-to-wild introgression, which is currently a major conservation issue for the industry. However, recent work suggests that triploidisation protocols may, on occasion, produce 'failed triploids' displaying diploidy, aneuploidy and aberrant inheritance. The potentially negative consequences for conservation and animal welfare motivate the need for methods to evaluate the success of ploidy-manipulation protocols early in the production process. We developed a semi-automated version of the MAC-PR (microsatellite DNA allele counting - peak ratios) method to resolve the allelic configuration of large numbers of individuals across a panel of microsatellite markers that can be used to infer ploidy, pedigree and inheritance aberrations. We demonstrate an application of the approach using material from a series of Atlantic salmon (Salmo salar) breeding experiments where ploidy was manipulated using a hydrostatic pressure treatment. We validated the approach to infer ploidy against blood smears, finding a > 99% agreement between these methods, and demonstrate its potential utility to infer ploidy as early as the embryonic stage. Furthermore, we present tools to assign diploid and triploid progeny to families and to detect aberrant inheritance, which may be useful for breeding programmes that utilise ploidy manipulation techniques. The approach adds to the ploidy verification toolbox. The increased precision in detecting ploidy and inheritance aberrations will facilitate the ability of triploidisation programmes to prevent farm-to-wild introgression.

Custom-Designed Probes for the Accurate Determination of Epidermal Growth Factor Receptor Mutations and Their Allelic Configuration.

The identification of single nucleotide polymorphisms (SNPs) is of paramount importance for disease diagnosis and clinical prognostication. In the context of nonsmall cell lung cancer (NSCLC), the emergence of resistance mutations, exemplified by the epidermal growth factor receptor (EGFR) T790 M and C797S, is intricately linked to the therapeutic efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Herein, a highly efficient and specific SNP detection platform for T790 M and C797S mutations has been engineered through the integration of an asymmetric polymerase chain reaction (PCR) and an ingeniously tailored four-way junction (4WJ) probe. Notably, a molecular beacon (MB) probe was judiciously designed to discern the allelic configuration of these mutations. The administration of first- and third-generation EGFR-TKIs demonstrates therapeutic efficacy solely when the mutations are in the trans configuration, characterized by a low fluorescence signal. In contrast, significant fluorescence by the MB probe is indicative of the C797S mutation being in a cis arrangement with T790M, thereby rendering the cells refractory to the therapeutic interventions of both first- and third-generation EGFR-TKIs. The assay is capable of concurrently detecting two point-mutations and ascertaining their allelic positions in a single test within 1.5 h, enhancing both efficiency and simplicity. It also exhibits high accuracy in the identification of clinical samples, offering promising implications for therapeutic guidelines. By enabling tailored treatment plans based on specific genetic profiles, our approach not only advances the precision of NSCLC treatment strategies but also marks a significant contribution to personalized medicine.

Novel genotype–phenotype correlations, differential cerebellar allele-specific methylation, and a common origin of the (ATTTC)n insertion in spinocerebellar ataxia type 37

Spinocerebellar ataxia subtype 37 (SCA37) is a rare disease originally identified in ataxia patients from the Iberian Peninsula with a pure cerebellar syndrome. SCA37 patients carry a pathogenic intronic (ATTTC)n repeat insertion flanked by two polymorphic (ATTTT)n repeats in the Disabled-1 (DAB1) gene leading to cerebellar dysregulation. Herein, we determine the precise configuration of the pathogenic 5ʹ(ATTTT)n–(ATTTC)n–3ʹ(ATTTT)n SCA37 alleles by CRISPR–Cas9 and long-read nanopore sequencing, reveal their epigenomic signatures in SCA37 lymphocytes, fibroblasts, and cerebellar samples, and establish new molecular and clinical correlations. The 5ʹ(ATTTT)n–(ATTTC)n–3ʹ(ATTTT)n pathogenic allele configurations revealed repeat instability and differential methylation signatures. Disease age of onset negatively correlated with the (ATTTC)n, and positively correlated with the 3ʹ(ATTTT)n. Geographic origin and gender significantly correlated with age of onset. Furthermore, significant predictive regression models were obtained by machine learning for age of onset and disease evolution by considering gender, the (ATTTC)n, the 3ʹ(ATTTT)n, and seven CpG positions differentially methylated in SCA37 cerebellum. A common 964-kb genomic region spanning the (ATTTC)n insertion was identified in all SCA37 patients analysed from Portugal and Spain, evidencing a common origin of the SCA37 mutation in the Iberian Peninsula originating 859 years ago (95% CI 647–1378). In conclusion, we demonstrate an accurate determination of the size and configuration of the regulatory 5ʹ(ATTTT)n–(ATTTC)n–3ʹ(ATTTT)n repeat tract, avoiding PCR bias amplification using CRISPR/Cas9-enrichment and nanopore long-read sequencing, resulting relevant for accurate genetic diagnosis of SCA37. Moreover, we determine novel significant genotype–phenotype correlations in SCA37 and identify differential cerebellar allele-specific methylation signatures that may underlie DAB1 pathogenic dysregulation.

Individual bioenergetic capacity as a potential source of resilience to Alzheimer’s disease

AbstractBackgroundBrain glucose hypometabolism is among the earliest pathogenic changes in Alzheimer’s disease (AD). This metabolic dysfunction points to the personal bioenergetic capacity, defined as the ability to maintain energy homeostasis under all circumstances including deregulated glucose uptake, as a potential source of resilience to the disease. Fasting blood acylcarnitine profiles are a central readout for this capacity in the absence of dietary glucose and capture the activity and efficiency of glucose‐independent routes of mitochondrial energy metabolism.MethodWe used fasting serum acylcarnitine profiles of 1,531 participants (465 with normal cognition, 762 with mild cognitive impairment, and 304 with clinical AD) from the AD Neuroimaging Initiative to perform unsupervised subgroup identification using hierarchical clustering. Identified subgroups were investigated for differences in A/T/N biomarker profiles and cognitive status. The contributions of genetic and potentially modifiable factors defining the subgroups were quantified using analysis of explained variance. The influence of the strongest determining factors on longitudinal cognitive trajectories was estimated using linear mixed‐effects models and gene‐by‐environment interaction analysis.ResultWe found several bioenergetically distinct subgroups with significant differences in AD biomarker profiles and cognitive function. The strongest genetic contribution to this bioenergetic endophenotype seems to be specifically linked to succinylcarnitine metabolism and significantly modulates the rate of future cognitive decline. In contrast, potentially modifiable sustainment of beta‐oxidation efficiency seems to decelerate bioenergetic aging, thus creating a bioenergetic reserve that delays progression of cognitive decline. Using gene‐by‐environment interaction analysis, we demonstrate that this molecular framework identifies a subgroup of individuals that is likely to benefit significantly from personalized therapeutic, dietary or lifestyle interventions tailored to increase resilience against bioenergetic disturbances in AD.ConclusionOur study reports on a set of genetic and metabolic markers that define bioenergetically distinct subgroups with significant differences on the AD biomarker and cognitive level. Longitudinal data suggest that targeting the modifiable fraction of this endophenotype might be a promising strategy to slow down disease progression in individuals with specific allelic configurations.

Identification of QTL controlling the ratio of linalool to linalyl acetate in the flowers of Lavandula angustifolia Mill var. Hemus

The content of linalyl acetate and the ratio of linalool to linalyl acetate (L/LA ratio) are one of the important parameters that determine the quality of lavender oil. The characterization of a segregating population derived from a self-pollinated Lavandula angustifolia var. Hemus resulted in the identification of a single quantitative trait locus controlling the L/LA ratio (L/LA-QTL) and located on chromosome 8 of the lavender reference genome. The L/LA data analysis demonstrated that plants homozygous for one of the L/LA-QTL alleles had significantly higher linalool content, lower linalyl acetate content and higher L/LA ratio, than the plants which were either heterozygous or homozygous for the other allele. No significant difference was observed for the sum of linalool and linalyl acetate content among these three groups of plants, suggesting that the identified L/LA-QTL is related to an enzyme conversion of linalool to linalyl acetate. The BLAST search revealed that the L/LA-QTL region included a sequence of the LiAAT4 gene of monoterpene acetyltransferase, considered as a candidate gene for the L/LA-QTL locus. Sequence analysis of the LiAAT4 gene of var. Hemus revealed the presence of two alleles differing in two nucleotides and predicted amino acid substitutions. The comparison of the allele configurations of SSR, SRAP and LiAAT4 loci and the L/LA ratio of plants showing recombination in the L/LA-QTL region provided further support that LiAAT4 is a candidate gene underlying the identified L/LA-QTL and controlling the L/LA ratio. The application of molecular markers for the identified L/LA-QTL is discussed.

Genome sequencing revealed the red-flower trait candidate gene of a peach landrace.

Peach (Prunus persica) is an economically important fruit crop globally and an excellent material for genomic studies. While considerable progress has been made in unveiling trait-associated genes within cultivars and wild relatives, certain novel genes controlling valuable traits in peach landraces, such as the red-flowering gene, remained unclear. In this study, we sequenced and assembled the diploid genome of the red-flower landrace 'Yingzui' (abbreviated as 'RedY'). Multi-omics profiling of red petals of 'RedY' revealed the intensified red coloration associated with anthocyanins accumulation and concurrent decline in flavonols. This phenomenon is likely attributed to a natural variant of Flavonol Synthase (FLS) harboring a 9-bp exonic insertion. Intriguingly, the homozygous allelic configurations of this FLS variant were only observed in red-flowered peaches. Furthermore, the 9-bp sequence variation tightly associated with pink/red petal color in genome-wide association studies (GWAS) of collected peach germplasm resources. Functional analyses of the FLS variant, purified from procaryotic expression system, demonstrated its diminished enzymatic activity in flavonols biosynthesis, impeccably aligning with the cardinal trait of red flowers. Therefore, the natural FLS variant was proposed as the best candidate gene for red-flowering trait in peach. The pioneering unveiling of the red-flowered peach genome, coupled with the identification of the candidate gene, expanded the knowledge boundaries of the genetic basis of peach traits and provided valuable insights for future peach breeding efforts.

Isoniazid-induced liver injury risk level in different variants of N-acetyltransferase 2 (NAT2) polymorphisms: A literature review

Individual NAT2 genotype identity data should be enriched to prevent Isoniazid-induced liver injury (IDILI) and optimize the dose of Isoniazid (INH). Therefore, this study aims to present the level of IDILI risk for specific genotype alleles. The data collection involves literature indexed by Google Scholar, Scopus, and Pubmed databases. The search uses a combination of the following keyword variants “INH” OR “INH”, “liver injury” OR “hepatotoxicity”, “polymorphism” OR “pharmacogenomic”, and “N-acetyltransferase 2” OR “NAT2”. Furthermore, the screening results of library sources were narrowed to 11 original articles that met the inclusion criteria. The IDILI risk assessment analysis due to NAT2 enzyme polymorphism following the odds ratio has a 95% confidence interval. The results showed that the IDILI risk level of the slow acetylator group was 3.11 times higher than other populations. Meanwhile, the rapid and intermediate acetylator groups were not at risk. Three variants related to *6 allele were classified as high risk; *6A/*6A risk 5.76 times, *6A/*7B (5.54 times), and *6/*7 (4 times). The three allele configurations of the *5 and *7 were also classified as a risk; *5B/*7B (5 times), *7B/*7B (3.23 times), and *5/*7 (2,74 times).

Geographic Variation in Genomic Signals of Admixture Between Two Closely Related European Sepsid Fly Species

The extent of interspecific gene flow and its consequences for the initiation, maintenance, and breakdown of species barriers in natural systems remain poorly understood. Interspecific gene flow by hybridization may weaken adaptive divergence, but can be overcome by selection against hybrids, which may ultimately promote reinforcement. An informative step towards understanding the role of gene flow during speciation is to describe patterns of past gene flow among extant species. We investigate signals of admixture between allopatric and sympatric populations of the two closely related European dung fly species Sepsis cynipsea and S. neocynipsea (Diptera: Sepsidae). Based on microsatellite genotypes, we first inferred a baseline demographic history using Approximate Bayesian Computation. We then used genomic data from pooled DNA of natural and laboratory populations to test for past interspecific gene flow based on allelic configurations discordant with the inferred population tree (ABBA–BABA test with D-statistic). Comparing the detected signals of gene flow with the contemporary geographic relationship among interspecific pairs of populations (sympatric vs. allopatric), we made two contrasting observations. At one site in the French Cevennes, we detected an excess of past interspecific gene flow, while at two sites in Switzerland we observed lower signals of past microsatellite genotypes gene flow among populations in sympatry compared to allopatric populations. These results suggest that the species boundaries between these two species depend on the past and/or present eco-geographic context in Europe, which indicates that there is no uniform link between contemporary geographic proximity and past interspecific gene flow in natural populations.

GWAS of adventitious root formation in roses identifies a putative phosphoinositide phosphatase (SAC9) for marker-assisted selection.

Rose propagation by cuttings is limited by substantial genotypic differences in adventitious root formation. To identify possible genetic factors causing these differences and to develop a marker for marker-assisted selection for high rooting ability, we phenotyped 95 cut and 95 garden rose genotypes in a hydroponic rooting system over 6 weeks. Data on rooting percentage after 3 to 6 weeks, root number, and root fresh mass were highly variable among genotypes and used in association mappings performed on genotypic information from the WagRhSNP 68 K Axiom SNP array for roses. GWAS analyses revealed only one significantly associated SNP for rooting percentage after 3 weeks. Nevertheless, prominent genomic regions/peaks were observed and further analysed for rooting percentage after 6 weeks, root number and root fresh mass. Some of the SNPs in these peak regions were associated with large effects on adventitious root formation traits. Very prominent were ten SNPs, which were all located in a putative phosphoinositide phosphatase SAC9 on chromosome 2 and showed very high effects on rooting percentage after 6 weeks of more than 40% difference between nulliplex and quadruplex genotypes. SAC9 was reported to be involved in the regulation of endocytosis and in combination with other members of the SAC gene family to regulate the translocation of auxin-efflux PIN proteins via the dephosphorylation of phosphoinositides. For one SNP within SAC9, a KASP marker was successfully derived and used to select genotypes with a homozygous allele configuration. Phenotyping these homozygous genotypes for adventitious root formation verified the SNP allele dosage effect on rooting. Hence, the presented KASP derived from a SNP located in SAC9 can be used for marker-assisted selection in breeding programs for high rooting ability in the future.

Novel scheme for defining the clinical implications of TP53 mutations in myeloid neoplasia

BackgroundTP53 mutations (TP53MT) occur in diverse genomic configurations. Particularly, biallelic inactivation is associated with poor overall survival in cancer. Lesions affecting only one allele might not be directly leukemogenic, questioning the presence of cryptic biallelic subclones in cases with dismal prognosis.MethodsWe have collected clinical and molecular data of 7400 patients with myeloid neoplasms and applied a novel model by identifying an optimal VAF cutoff using a statistically robust strategy of sampling-based regression on survival data to accurately classify the TP53 allelic configuration and assess prognosis more precisely.ResultsOverall, TP53MT were found in 1010 patients. Following the traditional criteria, 36% of the cases were classified as single hits, while 64% exhibited double hits genomic configuration. Using a newly developed molecular algorithm, we found that 579 (57%) patients had unequivocally biallelic, 239 (24%) likely contained biallelic, and 192 (19%) had most likely monoallelic TP53MT. Interestingly, our method was able to upstage 192 out of 352 (54.5%) traditionally single hit lesions into a probable biallelic category. Such classification was further substantiated by a survival-based model built after re-categorization. Among cases traditionally considered monoallelic, the overall survival of those with probable monoallelic mutations was similar to the one of wild-type patients and was better than that of patients with a biallelic configuration. As a result, patients with certain biallelic hits, regardless of the disease subtype (AML or MDS), had a similar prognosis. Similar results were observed when the model was applied to an external cohort. In addition, single-cell DNA studies unveiled the biallelic nature of previously considered monoallelic cases.ConclusionOur novel approach more accurately resolves TP53 genomic configuration and uncovers genetic mosaicism for the use in the clinical setting to improve prognostic evaluation.

Correspondence of Yolk Sac and Embryonic Genotypes in F0 Mouse CRISPants.

CRISPR-mediated genome editing in vivo can be accompanied by prolonged stability of the Cas9 protein in mouse embryos. Then, genome edited variant alleles will be induced as long as Cas9 protein is active, and unmodified wildtype target loci are available. The corollary is that CRISPR-modified alleles that arise after the first zygotic cell division potentially could be distributed asymmetrically to the cell lineages that are specified early during morula and blastocyst development. This has practical implications for the investigation of F0 generation individuals, as cells in embryonic and extraembryonic tissues, such as the visceral yolk sac, might end up inheriting different genotypes. We here investigated the hypothetically possible scenarios by genotyping individual F0 CRISPants and their associated visceral yolk sacs in parallel. In all cases, we found that embryonic genotype was accurately reflected by yolk sac genotyping, with the two tissues indicating genetic congruence, even when the conceptus was a mosaic of cells with distinct allele configurations. Nevertheless, low abundance of a variant allele may represent a private mutation occurring only in the yolk sac, and in those rare cases, additional genotyping to determine the mutational status of the embryo proper is warranted.

Genetic Heterogeneity and Tissue-specific Patterns of Tumors with Multiple PIK3CA Mutations.

To comprehensively characterize tissue-specific and molecular subclasses of multiple PIK3CA (multi-PIK3CA) mutations and assess their impact on potential therapeutic outcomes. We profiled a pan-cancer cohort comprised of 352,392 samples across 66 tumor types using a targeted hybrid capture-based next-generation sequencing panel covering at least 324 cancer-related genes. Molecularly defined subgroups, allelic configuration, clonality, and mutational signatures were identified and tested for association with PI3K inhibitor therapeutic response. Multi-PIK3CA mutations are found in 11% of all PIK3CA-mutant tumors, including 9% of low tumor mutational burden (TMB) PIK3CA-mutant tumors, and are enriched in breast and gynecologic cancers. Multi-PIK3CA mutations are frequently clonal and in cis on the same allele and occur at characteristic positions across tumor types. These mutations tend to be mutually exclusive of mutations in other driver genes, and of genes in the PI3K pathway. Among PIK3CA-mutant tumors with a high TMB, 18% are multi-PIK3CA mutant and often harbor an apolipoprotein B mRNA-editing enzyme, catalytic polypeptide (APOBEC) mutational signature. Despite large differences in specific allele combinations comprising multi-PIK3CA mutant tumors, especially across cancer types, patients with different classes of multi-PIK3CA mutant estrogen receptor-positive, HER2-negative breast cancers respond similarly to PI3K inhibition. Our pan-tumor study provides biological insights into the genetic heterogeneity and tissue specificities of multi-PIK3CA mutations, with potential clinical utility to guide PI3K inhibition strategies.

A Clinically Practicable Approach to Predict TP53 Allelic Configurations in Myeloid Neoplasia

A Clinically Practicable Approach to Predict TP53 Allelic Configurations in Myeloid Neoplasia

Effect of Arbuscular Mycorrhizal Fungal Seed Coating on Grain Protein and Mineral Composition of Old and Modern Bread Wheat Genotypes

The effect of arbuscular mycorrhizal fungi (AMF) on yield and quality was investigated on a set of seven bread wheat genotypes with varying years of release, including five old genotypes and two modern varieties. A two-year field trial was conducted in central Italy under rainfed conditions. The effect of AM fungal seed coating was proved by assessing the AM fungal root colonization and studied on agronomic and quality traits, and in particular on gluten-forming proteins and grain mineral composition. AMF seed coating led to a general yield improvement in old genotypes (+24%). Concerning the effects on grain quality, while modern genotypes showed an increase in protein content (+16%), in the old ones an improvement of gluten quality was observed, with an increased proportion of HMW-GS from +17% to +92%. The gluten index results were mostly influenced by HMW-GS allelic configuration and amount, showing a significant correlation with gliadin-to-glutenin ratio and HMW-GS to LMW-GS. Concerning mineral uptake, AM fungal treatment determined a general increase in P content, which was more marked in the modern group (+44%). Furthermore, AMF significantly increased mean Fe concentration in Verna (+53%) and Bologna (+45%). Finally, phytate content did not increase with AMF, without affecting mineral bioavailability.

Developmental genomics of limb malformations: Allelic series in association with gene dosage effects contribute to the clinical variability.

SummaryGenetic heterogeneity, reduced penetrance, and variable expressivity, the latter including asymmetric body axis plane presentations, have all been described in families with congenital limb malformations (CLMs). Interfamilial and intrafamilial heterogeneity highlight the complexity of the underlying genetic pathogenesis of these developmental anomalies. Family-based genomics by exome sequencing (ES) and rare variant analyses combined with whole-genome array-based comparative genomic hybridization were implemented to investigate 18 families with limb birth defects. Eleven of 18 (61%) families revealed explanatory variants, including 7 single-nucleotide variant alleles and 3 copy number variants (CNVs), at previously reported “disease trait associated loci”: BHLHA9, GLI3, HOXD cluster, HOXD13, NPR2, and WNT10B. Breakpoint junction analyses for all three CNV alleles revealed mutational signatures consistent with microhomology-mediated break-induced replication, a mechanism facilitated by Alu/Alu-mediated rearrangement. Homozygous duplication of BHLHA9 was observed in one Turkish kindred and represents a novel contributory genetic mechanism to Gollop-Wolfgang Complex (MIM: 228250), where triplication of the locus has been reported in one family from Japan (i.e., 4n = 2n + 2n versus 4n = 3n + 1n allelic configurations). Genes acting on limb patterning are sensitive to a gene dosage effect and are often associated with an allelic series. We extend an allele-specific gene dosage model to potentially assist, in an adjuvant way, interpretations of interconnections among an allelic series, clinical severity, and reduced penetrance of the BHLHA9-related CLM spectrum.

Genome-wide screening for genes involved in the epigenetic basis of fragile X syndrome.

SummaryFragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. The epigenetic factors responsible for FMR1 inactivation are largely unknown. Here, we initially demonstrated the feasibility of FMR1 reactivation by targeting a single epigenetic factor, DNMT1. Next, we established a model system for FMR1 silencing using a construct containing the FXS-related mutation upstream to a reporter gene. This construct was methylated in vitro and introduced into a genome-wide loss-of-function (LOF) library established in haploid human pluripotent stem cells (PSCs), allowing the identification of genes whose functional loss reversed the methylation-induced silencing of the FMR1 reporter. Selected candidate genes were further analyzed in haploid- and FXS-patient-derived PSCs, highlighting the epigenetic and metabolic pathways involved in FMR1 regulation. Our work sheds light on the mechanisms responsible for CGG-expansion-mediated FMR1 inactivation and offers novel targets for therapeutic FMR1 reactivation.

Variation of Chromosome Composition in a Full-Sib Population Derived From 2x × 3x Interploidy Cross of Populus.

Interploidy cross commonly results in complex chromosome number and structural variations. In our previous study, a progeny with segregated ploidy levels was produced by an interploidy cross between diploid female parent Populus tomentosa × Populus bolleana clone TB03 and triploid male parent Populus alba × Populus berolinensis ‘Yinzhong’. However, the chromosome compositions of aneuploid genotypes in the progeny were still unclear. In the present study, a microsatellite DNA allele counting–peak ratios (MAC-PR) method was employed to analyze allelic configurations of each genotype to clarify their chromosome compositions, while 45S rDNA fluorescence in situ hybridization (FISH) analysis was used to reveal the mechanism of chromosome number variation. Based on the MAC-PR analysis of 47 polymorphic simple sequence repeat (SSR) markers distributed across all 19 chromosomes of Populus, both chromosomal number and structural variations were detected for the progeny. In the progeny, 26 hypo-triploids, 1 hyper-triploid, 16 hypo-tetraploids, 10 tetraploids, and 5 hyper-tetraploids were found. A total of 13 putative structural variation events (duplications and/or deletions) were detected in 12 genotypes, involved in chromosomes 3, 6, 7, 14, 15, 16, and 18. The 46.2% (six events) structural variation events occurred on chromosome 6, suggesting that there probably is a chromosome breakpoint near the SSR loci of chromosome 6. Based on calculation of the allelic information, the transmission of paternal heterozygosity in the hypo-triploids, hyper-triploid, hypo-tetraploids, tetraploids, and hyper-tetraploids were 0.748, 0.887, 0.830, 0.833, and 0.836, respectively, indicating that the viable pollen gains of the male parent ‘Yinzhong’ were able to transmit high heterozygosity to progeny. Furthermore, 45S rDNA–FISH analysis showed that specific-chromosome segregation feature during meiosis and chromosome appointment in normal and fused daughter nuclei of telophase II of ‘Yinzhong,’ which explained that the formation of aneuploids and tetraploids in the progeny could be attributed to imbalanced meiotic chromosomal segregation and division restitution of ‘Yinzhong,’ The data of chromosomal composition and structural variation of each aneuploid in the full-sib progeny of TB03 × ‘Yinzhong’ lays a foundation for analyzing mechanisms of trait variation relying on chromosome or gene dosages in Populus.

Evidence for hexasomic inheritance in Chrysanthemum morifolium Ramat. based on analysis of EST-SSR markers.

Chrysanthemums (Chrysanthemum morifolium Ramat.) are ornamental flowers, which are famous worldwide. The mode of inheritance has great implications for the genetic analysis of polyploid species. However, genetic analysis of chrysanthemum has been hampered because of its controversial inheritance mode (disomic or hexasomic). To classify the inheritance mode of chrysanthemums, an analysis of three approaches was carried out in an F1 progeny of 192 offspring using 223 expressed sequence tag-simple sequence repeat (EST-SSR) markers. The analysis included segregation analysis, the ratio of simplex marker alleles linked in coupling to repulsion, as well as the transmission and segregation patterns of EST-SSR marker alleles. After segregation analysis, 204 marker alleles fit hexasomic inheritance and 150 marker alleles fit disomic inheritance, showing that marker alleles were inherited predominantly in a hexasomic manner. Furthermore, the results of the analysis of allele configuration and segregation behavior of five EST-SSR markers also suggested random pairing of chromosomes. Additionally, the ratio of simplex marker alleles linked in coupling to repulsion was 1:0, further supporting hexasomic inheritance. Therefore, it could be inferred that chrysanthemum is a complete or near-complete hexasome.

Genotyping of polyploid plants using quantitative PCR: application in the breeding of white-fleshed triploid loquats (Eriobotrya japonica)

BackgroundPloidy manipulation is effective in seedless loquat breeding, in which flesh color is a key agronomic and economic trait. Few techniques are currently available for detecting the genotypes of polyploids in plants, but this ability is essential for most genetic research and molecular breeding.ResultsWe developed a system for genotyping by quantitative PCR (qPCR) that allowed flesh color genotyping in multiple tetraploid and triploid loquat varieties (lines). The analysis of 13 different ratios of DNA mixtures between two homozygous diploids (AA and aa) showed that the proportion of allele A has a high correlation (R2 = 0.9992) with parameter b [b = a1/(a1 + a2)], which is derived from the two normalized allele signals (a1 and a2) provided by qPCR. Cluster analysis and variance analysis from simulating triploid and tetraploid hybrids provided completely correct allelic configurations. Four genotypes (AAA, AAa, Aaa, aaa) were found in triploid loquats, and four (AAAA, AAAa, AAaa, Aaaa; absence of aaaa homozygotes) were found in tetraploid loquats. DNA markers analysis showed that the segregation of flesh color in all F1 hybrids conformed to Mendel's law. When tetraploid B431 was the female parent, more white-fleshed triploids occurred among the progeny.ConclusionsqPCR can detect the flesh color genotypes of loquat polyploids and provides an alternative method for analyzing polyploid genotype and breeding, dose effects and allele-specific expression.

Integrative single-cell analysis of allele-specific copy number alterations and chromatin accessibility in cancer.

Cancer progression is driven by both somatic copy number aberrations (CNAs) and chromatin remodeling, yet little is known about the interplay between these two classes of events in shaping the clonal diversity of cancers. We present Alleloscope, a method for allele-specific copy number estimation that can be applied to single-cell DNA- and/or transposase-accessible chromatin-sequencing (scDNA-seq, ATAC-seq) data, enabling combined analysis of allele-specific copy number and chromatin accessibility. On scDNA-seq data from gastric, colorectal and breast cancer samples, with validation using matched linked-read sequencing, Alleloscope finds pervasive occurrence of highly complex, multiallelic CNAs, in which cells that carry varying allelic configurations adding to the same total copy number coevolve within a tumor. On scATAC-seq from two basal cell carcinoma samples and a gastric cancer cell line, Alleloscope detected multiallelic copy number events and copy-neutral loss-of-heterozygosity, enabling dissection of the contributions of chromosomal instability and chromatin remodeling to tumor evolution.