Phenotypic Segregation Research Articles

Pediatric onset neuronal ceroid lipofuscinoses: Unraveling clinical and genetic specifications.

To unravel the clinical and genetic specifications of Neuronal ceroid lipofuscinosis (NCL). This is a retrospective cross-sectional study conducted in the Department of Pediatric Neurology Children Hospital and University of Child Health Sciences, Lahore, Pakistan from March 2017 to March 2022. The primary outcome was to measure genotype-phenotype correlation by segregation of phenotypes according to genotype. The secondary outcomes included a correlation between genotype and distribution of age(s) of onset. One hundred fifty three patients clinically diagnosed with NCL underwent genetic testing and pathologic mutation was identified in 32.7% of patients. About 59.6% were male and 37.2% had an affected sibling. The median age was 5.46±1.95 years at the onset of the first symptom i.e., myoclonic seizures in 68%, and motor difficulty in 24%. Other features found were global developmental delay (56%), hypotonia (23%), visual impairment (80%), ataxia (22%), and disc pallor (56%). The most common type was CLN6 (Ceroid lipofuscinosis neuronal) (42%), CLN2 (16%) followed by CLN7 (12%). When 50 patients with recognized mutations were compared with 103 patients with no mutation, family history (p=0.049), early visual loss (p=0.016), hypotonia (p=0.001), white matter signals (p=0.026) and pan-atrophy(p=0.047) was statistically significant in the genetically confirmed NCL. Multiple pairwise comparisons indicated that the estimated age of onset for the CLN1 and CLN2 mutation group was significantly lower than other genotypes including CLN6 (p 0.012), CLN10 (p 0.007) and CLN12 (p 0.007). Following a detailed review of NCL symptomatology, a clinically-oriented approach should be used for a rapid diagnosis with confirmation by targeted molecular testing for future genetic counseling.

CAVaLRi: An Algorithm for Rapid Identification of Diagnostic Germline Variation

Clinical exome and genome sequencing (ES/GS) have become indispensable diagnostic tools for rare genetic diseases (RGD). However, the interpretation of ES/GS presents a substantial operational challenge in clinical settings. Test interpretation requires the review of hundreds of genetic variants, a task that has become increasingly challenging given the rising use of ES/GS. In response, we present Clinical Assessment of Variants by Likelihood Ratios (CAVaLRi), which employ a modified likelihood ratio (LR) framework to assign diagnostic probabilities to candidate germline disease genes. CAVaLRi models aspects of the clinical variant assessment process, taking into consideration the predicted impact of the variant, the proband and parental genotypes, and the proband’s clinical characteristics. It also factors in computational phenotype noise and weighs the relative significance of genotype, phenotype, and variant segregation information. We trained and tested CAVaLRi on variant and phenotype data from an internal cohort of 655 clinical ES cases. For validation, CAVaLRi’s performance was benchmarked against four leading gene prioritization algorithms (Exomiser’s hiPHIVE and PhenIX prioritizers, LIRICAL, and XRare) using a distinct cohort of 12,832 ES cases. Our findings reveal that CAVaLRi significantly outperforms its counterparts when clinician-curated phenotype sets are used, as evidenced by its superior precision-recall curve (PR AUC: 0.701) and average diagnostic gene rank (1.59). Notably, even when substituting highly focused clinician-curated phenotype sets with large and potentially nonspecific computationally derived phenotypes, CAVaLRi retains its precision (PR AUC: 0.658; diagnostic gene average rank: 1.68) and markedly outperforms other tools. In a large, heterogeneous validation cohort, CAVaLRi stood out as the most precise prioritization algorithm (PR AUC: 0.335; average diagnostic rank: 1.91). In conclusion, CAVaLRi presents a robust solution for prioritizing diagnostic genes, surpassing current methods. It demonstrates resilience to noisy, computationally-derived phenotypes, providing a scalable strategy to help labs focus on the most diagnostically relevant variants, thus addressing the growing demand for ES/GS interpretation.

Phenotypic characterization and candidate gene analysis of a short kernel and brassinosteroid insensitive mutant from hexaploid oat (Avena sativa).

In an ethyl methanesulfonate oat (Avena sativa) mutant population we have found a mutant with striking differences to the wild-type (WT) cv. Belinda. We phenotyped the mutant and compared it to the WT. The mutant was crossed to the WT and mapping-by-sequencing was performed on a pool of F2 individuals sharing the mutant phenotype, and variants were called. The impacts of the variants on genes present in the reference genome annotation were estimated. The mutant allele frequency distribution was combined with expression data to identify which among the affected genes was likely to cause the observed phenotype. A brassinosteroid sensitivity assay was performed to validate one of the identified candidates. A literature search was performed to identify homologs of genes known to be involved in seed shape from other species. The mutant had short kernels, compact spikelets, altered plant architecture, and was found to be insensitive to brassinosteroids when compared to the WT. The segregation of WT and mutant phenotypes in the F2 population was indicative of a recessive mutation of a single locus. The causal mutation was found to be one of 123 single-nucleotide polymorphisms (SNPs) spanning the entire chromosome 3A, with further filtering narrowing this down to six candidate genes. In-depth analysis of these candidate genes and the brassinosteroid sensitivity assay suggest that a Pro303Leu substitution in AVESA.00010b.r2.3AG0419820.1 could be the causal mutation of the short kernel mutant phenotype. We identified 298 oat proteins belonging to orthogroups of previously published seed shape genes, with AVESA.00010b.r2.3AG0419820.1 being the only of these affected by a SNP in the mutant. The AVESA.00010b.r2.3AG0419820.1 candidate is functionally annotated as a GSK3/SHAGGY-like kinase with homologs in Arabidopsis, wheat, barley, rice, and maize, with several of these proteins having known mutants giving rise to brassinosteroid insensitivity and shorter seeds. The substitution in AVESA.00010b.r2.3AG0419820.1 affects a residue with a known gain-of function substitution in Arabidopsis BRASSINOSTEROID-INSENSITIVE2. We propose a gain-of-function mutation in AVESA.00010b.r2.3AG0419820.1 as the most likely cause of the observed phenotype, and name the gene AsGSK2.1. The findings presented here provide potential targets for oat breeders, and a step on the way towards understanding brassinosteroid signaling, seed shape and nutrition in oats.

OcBSA: An NGS-based bulk segregant analysis tool for outcross populations

Constructing inbred lines for self-incompatible species and species with long generation times is challenging, making the use of F1 outcross/segregating populations the main strategy for genetic studies of such species. However, there is a lack of dedicated algorithms/tools for rapid quantitative trait locus (QTL) mapping using the F1 populations. To this end, we have designed and developed an algorithm/tool called OcBSA specifically for QTL mapping of F1 populations. OcBSA transforms the four-haplotype inheritance problem from the two heterozygous diploid parents of the F1 population into the two-haplotype inheritance problem common in current genetic studies by removing the two haplotypes from the heterozygous parent that do not contribute to phenotype segregation in the F1 population. Testing of OcBSA on 1800 simulated F1 populations demonstrated its advantages over other currently available tools in terms of sensitivity and accuracy. In addition, the broad applicability of OcBSA was validated by QTL mapping using seven reported F1 populations of apple, pear, peach, citrus, grape, tea, and rice. We also used OcBSA to map the QTL for flower color in a newly constructed F1 population of potato generated in this study. The OcBSA mapping result was verified by the insertion or deletion markers to be consistent with a previously reported locus harboring the ANTHOCYANIN 2 gene, which regulates potato flower color. Taken together, these results highlight the power and broad utility of OcBSA for QTL mapping using F1 populations and thus a great potential for functional gene mining in outcrossing species. For ease of use, we have developed both Windows and Linux versions of OcBSA, which are freely available at: https://gitee.com/Bioinformaticslab/OcBSA.

ВАЛИДАЦИЯ ДНК-МАРКЕРА УСТОЙЧИВОСТИ ПОДСОЛНЕЧНИКА К РАСЕ G ЗАРАЗИХИ НА МАЛЫХ ВЫБОРКАХ

The genotypic and phenotypic segregation of 57 plants of a progeny of two heterozygous F1 sunflower hybrids was analyzed (by DNA-marker) by a trait of resistance to a broomrape race G. The observed segregation by a genotype corresponded reliably to the theoretically expected ones in monohybrid crosses due to models 3:1 and 1:2:1. The segregation by a phenotype corresponded completely to the segregation by a marker of a model 3:1. A developed marker allows selecting by a trait of sunflower resistance to the broomrape race G.

Genetics of begomovirus resistance in Cucurbita moschata Duchesne: A novel resistant source PVR-1343

Whitefly transmitted begomoviruses are the most destructive virus(es) that causes quantitative and qualitative losses to pumpkin (Cucurbita moschata) crop worldwide. In present study we have molecularly characterized three types of viral infections, which were caused by squash leaf curl China virus (SLCCNV), tomato leaf curl New Delhi virus (ToLCNDV), and their mixed infection (MI-Sq/To). Screening of diverse germplasm through whitefly (Bemisia tabaci mitotype Asia II) mediated artificial inoculation and PCR confirmed the 'PVR-1343' genotype as a resistance source against identified begomoviruses. To understand the genetic control of begomovirus resistant gene(s), segregating populations derived from crosses of 'PVR-1343' and four susceptible genotypes (MVSR-6711, Punjab Samrat, MBN-6242 and P-278) inoculated with SLCCNV, ToLCNDV and MI-Sq/To by whitefly in closed environment for three consecutive years (2018–2020). Disease scoring at 75 days after sowing rendered all F1 progenies as susceptible indicating recessive nature of resistance with no maternal or cytoplasmic effects. Chi-square analysis indicated a good fit of 9(S):3(MS):3(MR):1(R) ratio in F2 progenies of all crosses. Backcross of the F1’s to the resistant parent segregated into 1:1:1:1 ratio, while all progeny were susceptible in backcross of the F1’s with susceptible parent. Phenotypic segregation ratio of F2:3 confirmed the F2 genotypic ratio of 1:2:2:4:1:2:1:2:1. This substantiated that multiple begomovirus resistance in 'PVR-1343' is controlled by digenic recessive genes. Moreover, F2:3 progenies resistant to SLCCNV were also resistant to ToLCNDV and MI-Sq/To and vice versa, suggesting that common or tightly linked gene(s) might be involved in resistance to both viruses.

A Frameshift Mutation in the Mg-Chelatase I Subunit Gene OsCHLI Is Associated with a Lethal Chlorophyll-Deficient, Yellow Seedling Phenotype in Rice.

Chlorophyll biosynthesis is a crucial biological process in plants, and chlorophyll content is one of the most important traits in rice breeding programs. In this study, we identified a lethal, chlorophyll-deficient, yellow seedling (YS) phenotype segregating in progeny of CR5055-21, an F2 plant derived from a backcross between Korean japonica variety 'Hwaseong' (Oryza sativa) and CR5029, which is mostly Hwaseong with a small amount of Oryza grandiglumis chromosome segments. The segregation of the mutant phenotype was consistent with a single gene recessive mutation. Light microscopy of YS leaf cross-sections revealed loosely arranged mesophyll cells and sparse parenchyma in contrast to wildtype. In addition, transmission electron microscopy showed that chloroplasts did not develop in the mesophyll cells of the YS mutant. Quantitative trait loci (QTL)-seq analysis did not detect any significant QTL, however, examination of the individual delta-SNP index identified a 2-bp deletion (AG) in the OsCHLI gene, a magnesium (Mg)-chelatase subunit. A dCAPs marker was designed and genotyping of a segregating population (n = 275) showed that the mutant phenotype co-segregated with the marker. The 2-bp deletion was predicted to result in a frameshift mutation generating a premature termination. The truncated protein likely affects formation and function of Mg-chelatase, which consists of three different subunits that together catalyze the first committed step of chlorophyll biosynthesis. Transcriptome analysis showed that photosynthesis and carbohydrate metabolism pathways were significantly altered although expression of OsCHLI was not. Chlorophyll- and carotenoid-related genes were also differentially expressed in the YS mutant. Our findings demonstrated that OsCHLI plays an important role in leaf pigment biosynthesis and leaf structure development in rice.

Floral trait variation in a putative hybrid zone between specialist pollination systems: how could it impact pollinator attraction?

Abstract Floral traits are used as signals to attract pollinators and play an important role in species identification and isolation. Nicotiana is a genus with a diverse range of flower morphologies, colours, and pollination systems related to a natural history of hybridization, a driver of speciation in this genus. Nicotiana alata and Nicotiana forgetiana are pollinated by hawkmoths and hummingbirds, respectively, and a putative hybrid population was recently found. This population presents flowers with intermediate phenotypes providing an opportunity to investigate the impact of hybridization on floral trait variation. Here we investigated the floral shape, floral pigments, and nectar traits in a putative hybrid population, hereafter termed atypical populations (AP) compared to phenotypes of allopatric populations of both species. We found a high variation in floral pigmentation in AP plants and observed phenotype segregation in some genotypes. Nicotiana forgetiana and AP plants showed the same flavanol peaks as Nicotiana alata, suggesting the same UV-absorbent phenotype attractive to hawkmoths. The geometric morphometric results showed that the intermediate-coloured flowers had similar floral shape and size to N. alata. Our results suggest that the putative hybrid population would be able to attract the same parental pollinator—hawkmoths and hummingbirds—making backcrossing events possible and attracting other pollinators, such as bees.

The R2R3-MYB transcription factor GaPC controls petal coloration in cotton

Although a few cases of genetic epistasis in plants have been reported, the combined analysis of genetically phenotypic segregation and the related molecular mechanism remains rarely studied. Here, we have identified a gene (named GaPC) controlling petal coloration in Gossypium arboreum and following a heritable recessive epistatic genetic model. Petal coloration is controlled by a single dominant gene, GaPC. A loss-of-function mutation of GaPC leads to a recessive gene Gapc that masks the phenotype of other color genes and shows recessive epistatic interactions. Map-based cloning showed that GaPC encodes an R2R3-MYB transcription factor. A 4814-bp long terminal repeat retrotransposon insertion at the second exon led to GaPC loss of function and disabled petal coloration. GaPC controlled petal coloration by regulating the anthocyanin and flavone biosynthesis pathways. Expression of core genes in the phenylpropanoid and anthocyanin pathways was higher in colored than in white petals. Petal color was conferred by flavonoids and anthocyanins, with red and yellow petals rich in anthocyanin and flavonol glycosides, respectively. This study provides new insight on molecular mechanism of recessive epistasis, also has potential breeding value by engineering GaPC to develop colored petals or fibers for multi-functional utilization of cotton.

Transgressive segregation in mating traits drives hybrid speciation.

Hybridization can rapidly generate novel genetic variation, which can promote ecological speciation by creating novel adaptive phenotypes. However, it remains unclear how hybridization, creating novel mating phenotypes (e.g., mating season, genitalia shapes, sexual displays, mate preferences), affects speciation especially when the phenotypes do not confer adaptive advantages. Here, based on individual-based evolutionary simulations, we propose that transgressive segregation of mating traits can drive incipient hybrid speciation. Simulations demonstrated that incipient hybrid speciation occurred most frequently when the hybrid population received moderate continued immigration from parental lineages causing recurrent episodes of hybridization. Recurrent hybridization constantly generated genetic variation, which promoted the rapid stochastic evolution of mating phenotypes in a hybrid population. The stochastic evolution continued until a novel mating phenotype came to dominate the hybrid population, which reproductively isolates the hybrid population from parental lineages. However, too frequent hybridization rather hindered the evolution of reproductive isolation by inflating the variation of mating phenotypes to produce phenotypes allowing mating with parental lineages. Simulations also revealed conditions for the long-term persistence of hybrid species after their incipient emergence. Our results suggest that recurrent transgressive segregation of mating phenotypes can offer a plausible explanation for hybrid speciation and radiations that involved little adaptive ecological divergence.

Spatiotemporal segregation by migratory phenotype indicates potential for assortative mating in lake sturgeon

Migratory diversity can promote population differentiation if sympatric phenotypes become temporally, spatially, or behaviorally segregated during breeding. In this study, the potential for spatiotemporal segregation was tested among three migratory phenotypes of lake sturgeon (Acipenser fulvescens) that spawn in the St. Clair River of North America's Laurentian Great Lakes but differ in how often they migrate into the river and in which direction they move after spawning. Acoustic telemetry over 9 years monitored use of two major spawning sites by lake sturgeon that moved north to overwinter in Lake Huron or south to overwinter in Lake St. Clair. Lake St. Clair migrants were further distinguished by whether they migrated into the St. Clair River each year (annual migrants) or intermittently (intermittent migrants). Social network analyses indicated lake sturgeon generally co-occurred with individuals of the same migratory phenotype more often than with different migratory phenotypes. A direct test for differences in space use revealed one site was almost exclusively visited by Lake St. Clair migrants whereas the other site was visited by Lake Huron migrants, intermittent Lake St. Clair migrants, and, to a lesser extent, annual Lake St. Clair migrants. Analysis of arrival and departure dates indicated opportunity for co-occurrence at the site visited by all phenotypes but showed Lake Huron migrants arrived approximately 2 weeks before Lake St. Clair migrants. Taken together, our results indicated partial spatiotemporal segregation of migratory phenotypes that may generate assortative mating and promote population differentiation.

Genetic characteristics and linkage of virulence genes of the Puccinia striiformis f. sp. tritici TSA-6 isolate to Yr5 host resistance.

To understand inheritance of the TSA-6 Puccinia striiformis f. sp. tritici isolate, virulent to Yr5, that was recently detected in China, we analyzed avirulence and virulence of 120 selfed progeny lines from Berberis shensiana. The results showed that the TSA-6 isolate is virulent against the Yr5 resistance gene, and overall progeny lines were categorized into 73 virulence phenotypes (VPs), and of these, 72 VPs differed from the isolate TSA-6, and only 1 VP including 3 progeny was identical to the parental isolate. The analyses indicated that the TSA-6 isolate is homozygous for avirulence at Yr10, Yr15 and Yr26 resistance loci, and virulence at YrA resistance locus. The TSA-6 isolate is heterozygous for avirulence at the Yr2, Yr3, Yr5, Yr7 and Yr8 resistance loci, which are controlled by a dominant/recessive relationship. The Yr1, Yr6, Yr9, Yr17, Yr27, Yr25, Yr28, Yr29, Yr32, YrTr1 and YrSP resistance loci are governed by two complementary dominant/recessive genes. Avirulence against heterozygous Yr4, Yr43，Yr44, Yr76 and YrExp2 resistance loci is regulated by a dominant and recessive, or a dominant and suppressor gene pairs. Totally, 117 multi-locus genotypes (MLGs) were detected at 24 KASP-SNP marker loci among the 120 progeny. Using these marker loci, we constructed a linkage map with a genetic distance interval spanning 624.5 cM. Quantitative trait loci (QTL) corresponding to phenotypic segregation for virulence at 20 Yr resistance loci in addition to the Yr1 resistance locus were identified. These results facilitate our understanding of Pst virulence evolution, and simplify breeding of wheat cultivars with effective resistance to wheat stripe rust.

The Human Early Maternal–Embryonic Interactome

Background: Single cell transcriptomics offers an avenue for predicting, with improved accuracy, the gene networks that are involved in the establishment of the first direct cell–cell interactions between the blastocyst and the maternal luminal epithelium. We hypothesised that in silico modelling of the maternal–embryonic interface may provide a causal model of these interactions, leading to the identification of genes associated with a successful initiation of implantation. Methods: Bulk and single cell RNA-sequencing of endometrial epithelium and scRNAseq of day 6 and 7 trophectoderm (TE) were used to model the initial encounter between the blastocyst and the maternal uterine lining epithelium in silico. In silico modelling of the maternal–embryonic interface was performed using hypernetwork (HN) analysis of genes mediating endometrial–TE interactions and the wider endometrial epithelial transcriptome. A hypernetwork analysis identifies genes that co-ordinate the expression of many other genes to derive a higher order interaction likely to be causally linked to the function. Potential interactions of TE with non-ciliated luminal cells, ciliated cells, and glandular cells were examined. Results: Prominent epithelial activities include secretion, endocytosis, ion transport, adhesion, and immune modulation. Three highly correlated clusters of 25, 22 and 26 TE-interacting epithelial surface genes were identified, each with distinct properties. Genes in both ciliated and non-ciliated luminal epithelial cells and glandular cells exhibit significant functional associations. Ciliated cells are predicted to bind to TE via galectin–glycan interaction. Day 6 and day 7 embryonic–epithelial interactomes are largely similar. The removal of aneuploid TE-derived mRNA invoked only subtle differences. No direct interaction with the maternal gland epithelial cell surface is predicted. These functional differences validate the in silico segregation of phenotypes. Single cell analysis of the epithelium revealed significant change with the cycle phase, but differences in the cell phenotype between individual donors were also present. Conclusions: A hypernetwork analysis can identify epithelial gene clusters that show correlated change during the menstrual cycle and can be interfaced with TE genes to predict pathways and processes occurring during the initiation of embryo–epithelial interaction in the mid-secretory phase. The data are on a scale that is realistic for functional dissection using current ex vivo human implantation models. A focus on luminal epithelial cells may allow a resolution to the current bottleneck of endometrial receptivity testing based on tissue lysates, which is confounded by noise from multiple diverse cell populations.

Integrative analysis reveals the divergence and speciation between sister Sooty Copper butterflies Lycaena bleusei and L. tityrus

The comparison of closely related taxa is cornerstone in biology, as understanding mechanisms leading up to differentiation in relation to extant shared characters are powerful tools in interpreting the evolutionary process. Hotspots of biodiversity such as the west-Mediterranean, where many lineages meet are ideal grounds to study these processes. We set to explore the interesting example of Sooty Copper butterflies: widespread Eurasian Lycaena tityrus (Poda, 1761) comes into contact in Iberia with closely related and local endemic, L. bleusei (Oberthür, 1884), which hasn’t always been considered a distinct species. An integrative analysis was designed, combining the use of extensive molecular data (five genes), geometric morphometrics analyses, verified and up-to-date distribution data, and environmental niche modelling, aimed at deciphering their true relationship, their placement within European Lycaena and trace their evolutionary history. We revealed several levels of differentiation: L. bleusei and L. tityrus appear to be reciprocally monophyletic independent gene-pools, distinct in all genes analysed, having mutually diverged 4.8 Ma ago. L. tityrus but not L. bleusei, further displays a genetic structure compatible with several glacial refugia, where populations assignable to infraspecific taxa surface. Conversely, L. bleusei shows a loss in mtDNA diversity in relation to nuDNA. Morphological analyses differentiate both species according to size and shape but also discriminate strong seasonal and sexual traits and a geographical phenotype segregation in L. tityrus. Finally, updated distribution and its modelling for current and glacial timeframes reveal both species respond differently to environmental variables, defining a mostly parapatric distribution and an overlapping belt where sympatry was recovered. During the last glacial maximum, a wider expansion in L. bleusei distribution explains current isolated populations. Our study highlights the importance of gathering several lines of evidence when deciphering the relationships between closely related populations in the fringe of cryptic species realm.

Mapping a mutation causing pale yellow petals in Brassica rapa.

Petal color is an important trait for both ornamental purposes and also for attracting pollinators. Here, we report a mutation of Brassica rapa R-o-18 with pale yellow petals that we retrieved from an EMS population and named whiter shade of pale ( wsp ). Phenotypic segregation ratio of an F2 mapping population indicates the phenotype is controlled by a single recessive gene. Mapping data from the whole genome sequencing coupled with allele frequency analysis suggests the mutation is located in a ~2 Mbp interval on chromosome 2. The interval contains a putative esterase/lipase/thioesterase protein previously demonstrated to account for floral color in B. rapa . We demonstrate that wsp carries a G to A missense mutation causing an aspartate to asparagine substitution within the putative lysophospholipid acyltransferase domain.

Challenges for the implementation of next generation sequencing-based expanded carrier screening: Lessons learned from the ciliopathies

Next generation sequencing (NGS) can detect carrier status for rare recessive disorders, informing couples about their reproductive risk. The recent ACMG recommendations support offering NGS-based carrier screening (NGS-CS) in an ethnic and population-neutral manner for all genes that have a carrier frequency >1/200 (based on GnomAD). To evaluate current challenges for NGS-CS, we focused on the ciliopathies, a well-studied group of rare recessive disorders. We analyzed 118 ciliopathy genes by whole exome sequencing in ~400 healthy local individuals and ~1000 individuals from the UK1958-birth cohort. We found 20% of healthy individuals (1% of couples) to be carriers of reportable variants in a ciliopathy gene, while 50% (4% of couples) carry variants of uncertain significance (VUS). This large proportion of VUS is partly explained by the limited utility of the ACMG/AMP variant-interpretation criteria in healthy individuals, where phenotypic match or segregation criteria cannot be used. Most missense variants are thus classified as VUS and not reported, which reduces the negative predictive value of the screening test. We show how gene-specific variation patterns and structural protein information can help prioritize variants most likely to be disease-causing, for (future) functional assays. Even when considering only strictly pathogenic variants, the observed carrier frequency is substantially higher than expected based on estimated disease prevalence, challenging the 1/200 carrier frequency cut-off proposed for choice of genes to screen. Given the challenges linked to variant interpretation in healthy individuals and the uncertainties about true carrier frequencies, genetic counseling must clearly disclose these limitations of NGS-CS.

Reporting Two Novel Mutations in Two Iranian Families with Cystic Fibrosis, Molecular and Bioinformatic Analysis

Cystic fibrosis is the most common heredity disease among the Caucasian population. More than 350 known pathogenic variations in the CFTR gene (NM_000492.4) cause CF. Herein, we report the outcome of our investigation in two unrelated Iranian families with CF patients. We conducted phenotypic examination, segregation, linkage analysis, and CFTR gene sequencing to define causative mutations. We found two novel mutations in the present study. The first one was a deletion causing frameshift, c.299delT p.(Leu100Profs*7), and the second one was a missense mutation, c.1857G>T, at nucleotide binding domain 1 of the CFTR protein. Haplotype segregation data supported our new mutation findings. Findings of this study expand the spectrum of CFTR pathogenic variations and can improve prenatal diagnosis and genetic counseling for CF.

Map-based cloning and transcriptome analysis of the more-tiller and small-grain mutant in rice.

A G to T nucleotide substitution of OsTSG2 led to more tillers and smaller grains in rice by participating in phytohormone signal transduction and starch and sucrose metabolism. Rice is one of the most important food crops worldwide. Grain size and tiller number are the most important factors determining rice yield. The more-tiller and small-grain 2 (tsg2) mutant in rice, developed by ethyl methanesulfonate (EMS) mutagenesis, has smaller grains, more tillers, and a higher yield per plant relative to the wild-type (WT). Based on the genetic analysis, the tsg2 traits were conferred by a single recessive nuclear gene located on the long arm of chromosome 2. After fine-mapping the OsTSG2 locus, a G to T nucleotide substitution was identified, which resulted in an A to S mutation in a highly conserved domain of the growth-regulation factor protein. The single-strand conformation polymorphism (SSCP) marker was developed based on the SNP associated with the phenotypic segregation of traits. The functional complementation of OsTSG2 from the tsg2 mutant to the WT led to an increase in grain size and weight. The differentially expressed genes (DEGs) identified by RNA sequencing were involved in phytohormone signal transduction and starch and sucrose metabolism. Enzyme-linked immunosorbent assay (ELISA) analysis detected variation in the indole acetic acid (IAA) and jasmonic acid (JA) content in the tsg2 inflorescence, while the cellular organization, degree of chalkiness, gel consistency, amylose content, and alkaline spreading value were affected in the tsg2 grains. The findings elucidated the regulatory mechanisms of the tsg2 traits. This mutant could be used in marker-assisted breeding for high-yield and good-quality rice.

Molecular Characterizations of the er1 Alleles Conferring Resistance to Erysiphe pisi in Three Chinese Pea (Pisum sativum L.) Landraces.

Powdery mildew caused by Erysiphe pisi DC. is a major disease affecting pea worldwide. This study aimed to confirm the resistance genes contained in three powdery mildew-resistant Chinese pea landraces (Suoshadabaiwan, Dabaiwandou, and Guiwan 1) and to develop the functional markers of the novel resistance genes. The resistance genes were identified by genetic mapping and PsMLO1 gene sequence identification. To confirm the inheritance of powdery mildew resistance in the three Landraces, the susceptible cultivars Bawan 6, Longwan 1, and Chengwan 8 were crossed with Suoshadabaiwan, Dabaiwandou, and Guiwan 1 to produce F1, F2, and F2:3 populations, respectively. All F1 plants were susceptible to E. pisi, and phenotypic segregation patterns in all the F2 and F2:3 populations fit the 3:1 (susceptible: resistant) and 1:2:1 (susceptible homozygotes: heterozygotes: resistant homozygotes) ratios, respectively, indicating powdery mildew resistance in the three Landraces were controlled by a single recessive gene, respectively. The analysis of er1-linked markers and genetic mapping in the F2 populations suggested that the recessive resistance genes in three landraces could be er1 alleles. The cDNA sequences of 10 homologous PsMLO1 cDNA clones from the contrasting parents were obtained. A known er1 allele, er1-4, was identified in Suoshadabaiwan. Two novel er1 alleles were identified in Dabaiwandou and Guiwan 1, which were designated as er1-13 and er1-14, respectively. Both novel alleles were characterized with a 1-bp deletion (T) in positions 32 (exon 1) and 277 (exon 3), respectively, which caused a frame-shift mutation to result in premature termination of translation of PsMLO1 protein. The co-dominant functional markers specific for er1-13 and er1-14, KASPar-er1-13, and KASPar-er1-14 were developed and effectively validated in populations and pea germplasms. Here, two novel er1 alleles were characterized and their functional markers were validated. These results provide powerful tools for marker-assisted selection in pea breeding.

A single QTL harboring multiple genetic variations leads to complicated phenotypic segregation in apple flesh firmness and crispness.

Within a QTL, the genetic recombination and interactions among five and two functional variations at MdbHLH25 and MdWDR5A caused much complicated phenotype segregation in apple FFR and FCR. The storability of climacteric fruit like apple is a quantitative trait. We previously identified 62 quantitative trait loci (QTLs) associating flesh firmness retainability (FFR) and flesh crispness retainability (FCR), but only a few functional genetic variations were identified and validated. The genetic variation network controlling fruit storability is far to be understood and diagnostic markers are needed for molecular breeding. We previously identified overlapped QTLs F16.1/H16.2 for FFR and FCR using an F1 population derived from 'Zisai Pearl' × 'Red Fuji'. In this study, five and two single-nucleotide polymorphisms (SNPs) were identified on the candidate genes MdbHLH25 and MdWDR5A within the QTL region. The SNP1 A allele at MdbHLH25 promoter reduced the expression and SNP2 T allele and/or SNP4/5 GT alleles at the exons attenuated the function of MdbHLH25 by downregulating the expression of the target genes MdACS1, which in turn led to a reduction in ethylene production and maintenance of higher flesh crispness. The SNPs did not alter the protein-protein interaction between MdbHLH25 and MdWDR5A. The joint effect of SNP genotype combinations by the SNPs on MdbHLH25 (SNP1, SNP2, and SNP4) and MdWDR5A (SNPi and SNPii) led to a much broad spectrum of phenotypic segregation in FFR and FCR. Together, the dissection of these genetic variations contributes to understanding the complicated effects of a QTL and provides good potential for marker development in molecular breeding.