Homozygous Single Nucleotide Polymorphisms Research Articles

Characterization of a New Citrus Mutant Induced by Gamma Irradiation with a Unique Fruit Shape, Gwonje-Early, and Determination of Specific Selection Markers Using Allele-Specific PCR

Gamma-ray irradiation is one of the most widely used mutagens worldwide. We previously conducted mutation breeding using gamma irradiation to develop new Citrus unshiu varieties. Among these mutants, Gwonje-early had an ovate shape, a protrusion of the upper part of the fruit, and a large fruit size compared with wild-type (WT) fruits. We investigated the external/internal morphological characteristics and fruit sugar/acid content of Gwonje-early. Additionally, we investigated genome-wide single-nucleotide polymorphisms (SNPs) and insertion/deletion (InDel) variants in Gwonje-early using whole-genome re-sequencing. Functional annotation by Gene Ontology analysis confirmed that InDels were more commonly annotated than SNPs. To identify specific molecular markers for Gwonje-early, allele-specific PCR was performed using homozygous SNPs detected via Gwonje-early genome re-sequencing. The GJ-SNP1 and GJ-SNP4 primer sets were effectively able to distinguish Gwonje-early from the WT and other commercial citrus varieties, demonstrating their use as specific molecular markers for Gwonje-early. These findings also have important implications in terms of intellectual property rights and the variety protection of Gwonje-early. Our results may provide insights into the understanding of morphological traits and the molecular breeding mechanisms of citrus species.

Identification of Late Ripening Citrus Mutant, Ara-unshiu (Citrus unshiu), and Its Selectable Marker.

'Miyagawa-wase' mandarin (Citrus unshiu Marc. cv. Miyagawa-wase early) is one of the most widely cultivated citrus varieties on Jeju Island in Korea. Mutation breeding is a useful tool for the induction of genetic diversity for the rapid creation of new plant variants. We previously reported the use of gamma irradiation for the development of new citrus varieties. Here, we report a new mutant, Ara-unshiu, with a unique late fruit ripening phenotype. We investigated the fruit morphological characteristics including weight, vertical/transverse diameter, peel thickness, hardness, and color difference, as well as sugar and acid contents of the Ara-unshiu compared to wild-type controls. We then used whole genome re-sequencing and functional annotation by gene ontology to identify and characterize single nucleotide polymorphism (SNP) and insertion/deletion (InDel) variants in the Ara-unshiu, finding a greater abundance of annotated genes containing InDels compared to SNPs. Finally, we used allele-specific PCR to identify molecular markers among the homozygous SNPs detected from the Ara-unshiu genome sequencing. We report a primer set that effectively distinguishes the Ara-unshiu from the wild-type control and other citrus varieties. Our findings provide insights into the mechanisms controlling the timing of fruit ripening and tools for the molecular breeding of citrus varieties.

Genetic Control of GCF Exudation: Innate Immunity Genes and Periodontitis Susceptibility.

Chronic periodontitis is a bacterial infection associated with dentally adherent biofilm (plaque) accumulation and age-related comorbidities. The disease begins as an inflammatory exudate from gingival margins, gingival crevicular fluid (GCF) in response to biofilm lysine. After a week of experimental gingivitis (no oral hygiene), biofilm lysine concentration was linearly related to biofilm accumulation (plaque index) but to GCF as an arch-shaped double curve which separated 9 strong from 6 weak GCF responders (hosts). Host DNA was examined for single nucleotide polymorphisms (SNPs) of alleles reported in 7 periodontitis-associated genes. Across all 15 hosts, an adenine SNP (A) at IL1B-511 (rs16944), was significant for strong GCF (Fisher's exact test, p < 0.05), and a thymidine SNP (T) at IL1B+3954 (rs1143634) for weak GCF provided 2 hosts possessing IL6-1363(T), rs2069827, were included. The phenotype of IL1B+3954(T) was converted from weak to strong in one host, and of the non-T allele from strong to weak in the other (specific epistasis, Fisher's exact test, p < 0.01). Together with homozygous alternate or reference SNPs at IL10-1082 or CD14-260 in 4 hosts, all hosts were identified as strong or weak GCF responders. The GCF response is therefore a strong or weak genetic trait that indicates strong or weak innate immunity in EG and controllable or uncontrollable periodontal disease, dental implant survival and late-life comorbidities.

Abstract PO-032: Association of a polymorphic variant in JMJD1C with tumor recurrence after adjuvant chemoradiation therapy in head and neck squamous cell carcinoma

Abstract Background: Adjuvant chemoradiation therapy (CRT) is the standard treatment for locally advanced head and neck squamous cell carcinomas (HNSCCs). Over 50% of HNSCC cases are diagnosed at the locally advanced stage, with in-field persistence and recurrence major causes of morbidity and poor survival outcome. Apart from human papillomavirus (HPV) status, there are no predictive biomarkers for CRT outcomes. CRT lethality arises from unrepaired double strand breaks (DSBs) in DNA. Recent work has suggested that dynamic changes in lysine modification of chromatin regulate DSB repair. This study investigated the association of germline single nucleotide polymorphisms (SNPs) in lysine-modifying genes with DSB repair and CRT outcomes in HNSCC. Methods: Blood specimens and associated retrospective clinical data from HNSCC patients (n=90; age range=41-89, median age 64; males=69, females=21) were obtained under IRB# 21-9921. The patients had HPV-negative tumors in the larynx or oral cavity, and had received radiation, with some receiving concurrent chemotherapy. Patients receiving concurrent chemotherapy were: 40% in disease-free group, 21% in recurrence disease group, and 38% in never disease-free group. The patients were segregated into 3 groups: disease-free (n=30), recurrent disease (n=28), and never-disease free (n=32). DNA from blood was genotyped for specific germline SNPs in lysine-modifying genes. Isogenic HNSCC lines (Cal-27, SCC9) carrying either the wild type (WT) or homozygous SNP were generated using CRISPR/Cas9 technology, and bulk RNA sequencing and immunofluorescence (IF)-based studies were performed to assess cellular pathways affected. Results: A homozygous variant SNP in the coding region of JMJD1C was significantly enriched in the disease-free group (n=21/30) versus the recurrent disease (n=3/28) and never disease-free (n=2/32) groups (p=0.0003, OR=0, Fisher’s Exact test). Analysis of SNP frequency in large population datasets found that while it was abundant in European Americans (EAs), it is relatively rare in African Americans (AAs) (EA= 0.3139 vs. AA=0.072; gnomAD population). Cal-27 JMJD1C SNP cells were significantly enriched for altered mRNA levels of genes associated with DNA repair, replication, cell cycle, oxidative phosphorylation, and histone kinase activity versus CAL-27 with WT JMJD1C (q-value&amp;lt;0.05). The SCC9 JMJD1C SNP cells were significantly enriched in extracellular matrix organization, cytokine-cytokine receptor interaction, and humoral immune response versus SCC9 with WT JMJD1C (q-value&amp;lt;0.05). On irradiation, the JMJD1C SNP cells had robust activation of the DSB repair pathway (BRCA1- mediated repair foci) versus the WT cell lines. Conclusion and future work: This work provides novel insight into a germline SNP in a lysine-modifying gene effecting DSB repair and associating with CRT outcomes in HNSCC. It may serve as a predictive biomarker for recurrence. Future research will explore racial disparities in tumor recurrence and confirm results in larger HNSCC datasets. Citation Format: Adria Hasan, Elena V. Demidova, Shreya M. Shah, Philip Czyzewicz, Karthik Devarajan, Thomas Galloway, Barbara Burtness, Erica A. Golemis, Joshua E. Meyer, Sanjeevani Arwesd. Association of a polymorphic variant in JMJD1C with tumor recurrence after adjuvant chemoradiation therapy in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-032.

Homozygosity mapping identified loci and candidate genes responsible for freezing tolerance in Camelina sativa.

Homozygosity mapping is an effective tool for detecting genomic regions responsible for a given trait when the phenotype is controlled by a limited number of dominant or co-dominant loci. Freezing tolerance is a major attribute in agricultural crops such as camelina. Previous studies indicated that freezing tolerance differences between a tolerant (Joelle) and susceptible (CO46) variety of camelina were controlled by a small number of dominant or co-dominant genes. We performed whole genome homozygosity mapping to identify markers and candidate genes responsible for freezing tolerance difference between these two genotypes. A total of 28 F3 RILs were sequenced to ∼30× coverage, and parental lines were sequenced to >30-40× coverage with Pacific Biosciences high fidelity technology and 60× coverage using Illumina whole genome sequencing. Overall, about 126k homozygous single nucleotide polymorphism markers were identified that differentiate both parents. Moreover, 617 markers were also homozygous in F3 families fixed for freezing tolerance/susceptibility. All these markers mapped to two contigs forming a contiguous stretch of chromosome 11. The homozygosity mapping detected 9 homozygous blocks among the selected markers and 22 candidate genes with strong similarity to regions in or near the homozygous blocks. Two such genes were differentially expressed during cold acclimation in camelina. The largest block contained a cold-regulated plant thionin and a putative rotamase cyclophilin 2 gene previously associated with freezing resistance in arabidopsis (Arabidopsis thaliana). The second largest block contains several cysteine-rich RLK genes and a cold-regulated receptor serine/threonine kinase gene. We hypothesize that one or more of these genes may be primarily responsible for freezing tolerance differences in camelina varieties.

46P Long-term residual chemotherapy-induced peripheral neuropathy (CIPN) and relationship with single nucleotide polymorphisms (SNPs) in ovarian cancer patients free of disease in the GINECO Vivrovaire study

Paclitaxel and carboplatin remains a backbone treatment of ovarian cancer (OC) inducing peripheral neuropathy in a significant proportion of patients. Meanwhile, long term survival of OC has dramatically increased with the recent therapeutic progress. Predictive factors for long-term CIPN are needed considering the impact on quality of life (Qol) of patients. We investigated the link between the incidence of CIPN and selected genetic polymorphisms in a cohort of ovarian cancer survivors. Vivrovaire is a French multi-center long-standing cohort of ovarian cancer patients free of disease 3 years after the end of the primary treatment. Long-term analysis of Qol in linked with neuropathy was performed including peripheral neuropathy assessed by the FACT/GOG-Ntx4 self-questionnaire. CIPN scores were correlated with SNPs in the selected CYP2C8, CYP3A4, ERCC1 and XPC genes. 130 patients were included with a median time from the end of chemotherapy of 63 months [35-180]. Median CIPN score was 37 [18-44] with 35 patients (27 %) having a severe score (< 33). SNPs (homozygotous/heterozygotous) were identified as follows: CYP2C8 [n=32 (24.6%)/ n=99 (76%)]; CYP3A4 [n=0 (0%)/ n=8 (6.1%)], ERCC1 [n=21 (16.1%)/ n=57 (43.8%)] and XPC [n=45 (34.6%)/ n=66 (50.8%)]. In univariate analysis, homozygous SNP (ho SNP) in any of the selected genes was associated with CIPN score as a continuous variable (p=0.045) but not with severe score (< 33) (OR: 1.66; 95% CI [0.74-3.88], p=0.22). Patients with CYP2C8_rs1934951 SNP tend to have CIPN score <33, (OR: 2.22; 95% CI [0.98-5.02], p=0.057). In multivariate analyses including age, interval from the end of chemotherapy, type and number of chemotherapy courses, identification of homozygotous or heterozygotous CYP2C8_rs1934951 SNP was associated with a severe CIPN score (OR: 2.41; 95% CI [1.02-5.70], p=0.043). Our study shows that residual CIPN is a frequent concern for ovarian cancer survivors. CYP2C8_rs1934951 SNP may be associated with severe residual CIPN in long-term survivors of ovarian cancer.

Whole genome resequencing identifies candidate genes and allelic diagnostic markers for resistance to Ralstonia solanacearum infection in cultivated peanut (Arachis hypogaea L.).

Bacterial wilt disease (BWD), caused by Ralstonia solanacearum is a major challenge for peanut production in China and significantly affects global peanut field productivity. It is imperative to identify genetic loci and putative genes controlling resistance to R. solanacearum (RRS). Therefore, a sequencing-based trait mapping approach termed "QTL-seq" was applied to a recombination inbred line population of 581 individuals from the cross of Yueyou 92 (resistant) and Xinhuixiaoli (susceptible). A total of 381,642 homozygous single nucleotide polymorphisms (SNPs) and 98,918 InDels were identified through whole genome resequencing of resistant and susceptible parents for RRS. Using QTL-seq analysis, a candidate genomic region comprising of 7.2 Mb (1.8-9.0 Mb) was identified on chromosome 12 which was found to be significantly associated with RRS based on combined Euclidean Distance (ED) and SNP-index methods. This candidate genomic region had 180 nonsynonymous SNPs and 14 InDels that affected 75 and 11 putative candidate genes, respectively. Finally, eight nucleotide binding site leucine rich repeat (NBS-LRR) putative resistant genes were identified as the important candidate genes with high confidence. Two diagnostic SNP markers were validated and revealed high phenotypic variation in the different resistant and susceptible RIL lines. These findings advocate the expediency of the QTL-seq approach for precise and rapid identification of candidate genomic regions, and the development of diagnostic markers that are applicable in breeding disease-resistant peanut varieties.

Self-Hybridization in Leishmania major.

Genetic exchange between different Leishmania strains in the sand fly vector has been experimentally demonstrated and is supported by population genetic studies. In nature, opportunities for Leishmania interstrain mating are restricted to flies biting multiply infected hosts or through multiple bites of different hosts. In contrast, self-mating could occur in any infected sand fly. By crossing two recombinant lines derived from the same Leishmania major strain, each expressing a different drug-resistance marker, self-hybridization in L. major was confirmed in a natural sand fly vector, Phlebotomus duboscqi, and in frequencies comparable to interstrain crosses. We provide the first high resolution, whole-genome sequencing analysis of large numbers of selfing progeny, their parents, and parental subclones. Genetic exchange consistent with classical meiosis is supported by the biallelic inheritance of the rare homozygous single nucleotide polymorphisms (SNPs) that arose by mutation during the generation of the parental clones. In contrast, heterozygous SNPs largely failed to be transmitted in Mendelian ratios for reasons not understood. SNPs that were heterozygous in both parents, however, recombined to produce homozygous alleles in some hybrids. For trisomic chromosomes present in both parents, transmittal to the progeny was only altered by self-hybridization, involving a gain or loss of somy in frequencies predicted by a meiotic process. Whole-genome polyploidization was also observed in the selfing progeny. Thus, self-hybridization in Leishmania, with its potential to occur in any infected sand fly, may be an important source of karyotype variation, loss of heterozygosity, and functional diversity. IMPORTANCE Leishmania are parasitic protozoa that cause a wide spectrum of diseases collectively known as the leishmaniases. Sexual reproduction in Leishmania has been proposed as an important source of genetic diversity and has been formally demonstrated to occur inside the sand fly vector midgut. Nevertheless, in the wild, opportunities for genetic exchange between different Leishmania species or strains are restricted by the capacity of different Leishmania strains to colonize the same sand fly. In this work, we report the first high resolution, whole-genome sequence analysis of intraclonal genetic exchange as a type of self-mating in Leishmania. Our data reveal that self-hybridization can occur with comparable frequency as interstrain mating under experimental lab conditions, leading to important genomic alterations that can potentially take place within every naturally infected sand fly.

Abstract 2266: Host genetic susceptibility to COVID-19 and pandemic -associated stressors

Abstract Background: Single nucleotide polymorphisms (SNPs) in IL-6, IL-1β, and IL-10 genes have been reported to impact infectious disease outcomes and are associated with altered emotional states. High levels of these cytokines, which have a role in regulating inflammation, have been detected among individuals with severe COVID-19 disease. The objective of this study was to examine if pro-inflammatory SNPs in the promoter region of IL-6, IL-1β, and IL-10 genes are associated with more severe COVID-19 disease and/or increased anxiety/stress levels in response to pandemic-associated stressors in a vulnerable Hispanic population that is part of the Puerto Rico Colorectal Cancer Registry. Methods: TaqMan® SNP Genotyping Assays (ThermoFisher Scientific) for IL-1β (rs1143627), IL-6 (rs1800795), and IL-10 (rs1800871) were performed according to the manufacturer’s recommendations in individuals between the ages of 21 to 75 (n=136). We assessed their anxiety and stress levels using the Generalized Anxiety Disorder Questionnaire (GAD-7) and the Perceived Stress Scale (PSS). Of these individuals, we evaluated those that had been previously infected with COVID-19 (n=38) for their symptom severity through a questionnaire. Chi-Square tests and Multivariate Logistic Regressions were used to calculate associations and ORs. Results: Preliminary analysis showed an association between having the homozygous IL-1β pro-inflammatory SNP and reporting fever as a symptom (OR=2.50, p-value=0.05). Individuals with the homozygous IL-10 pro-inflammatory SNP had higher odds of reporting difficulty breathing (OR= 4.51, p-value = 0.02), shortness of breath (OR=7.82, p-value=0.006), and fatigue as symptoms (OR=4.43, p-value=0.020. Further analysis also showed an association between having the homozygous IL-10 pro-inflammatory SNP and reporting at least 1 severe COVID-19 symptom (OR=3.38, p-value=0.046). Those with the homozygous or heterozygous IL-6 pro-inflammatory allele were less likely to report higher anxiety levels than those with the wild-type allele (OR=0.1447, p-value=0.04). We also observed that individuals between the ages of 21 to 49 were more likely to report changes in diet (OR=1.65, p-value=0.059) and constantly disinfecting as a pandemic-related stressor (OR=1.87, p-value=0.04) compared to individuals between the ages of 50 to 79. Conclusions: Pro-inflammatory IL-1β and IL-10 SNPs were associated with reporting fever and more severe symptoms of COVID-19, respectively. Data analysis using a larger sample size is warranted and is currently underway. Studies focused on examining that factors that contribute to more severe COVID-19 symptoms and how pandemic-associated stressors promote higher inflammation levels as a result of increased stress/anxiety are needed to fully understand the long-term effects the pandemic on public health, including possible increases in cancer incidence due to chronic inflammation. Citation Format: Maria Gonzalez-Pons, Valeria Valentin, Ingrid Montes-Rodriguez, Maria J. Sanchez, Fabiola M. Morales, Ibis R. Vera-Urbina, Valerie Maldonado-Pino, Estefania I. Ayala, Ediel Rodriguez, Hilmaris Centeno-Girona, Belisa Suarez, Leslie Casiano, Ana Sala, Marcia Cruz-Correa. Host genetic susceptibility to COVID-19 and pandemic -associated stressors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2266.

Performance characterization of PCR-free whole genome sequencing for clinical diagnosis.

To evaluate the performance of polymerase chain reaction (PCR)-free whole genome sequencing (WGS) for clinical diagnosis, and thereby revealing how experimental parameters affect variant detection.Five NA12878 samples were sequenced using MGISEQ-2000. NA12878 samples underwent WGS with differing deoxyribonucleic acid (DNA) input and library preparation protocol (PCR-based vs PCR-free protocols for library preparation). The depth of coverage and genotype quality of each sample were compared. The performance of each sample was measured for sensitivity, coverage of depth and breadth of coverage of disease-related genes, and copy number variants. We also developed a systematic WGS pipeline (PCR-free) for the analysis of 11 clinical cases.In general, NA12878-2 (PCR-free WGS) showed better depth of coverage and genotype quality distribution than NA12878-1 (PCR-based WGS). With a mean depth of ∼40×, the sensitivity of homozygous and heterozygous single nucleotide polymorphisms (SNPs) of NA12878-2 showed higher sensitivity (>99.77% and >99.82%) than NA12878-1, and positive predictive value exceeded 99.98% and 99.07%. The sensitivity and positive predictive value of homozygous and heterozygous indels for NA12878-2 (PCR-free WGS) showed great improvement than NA128878-1. The breadths of coverage for disease-related genes and copy number variants are slightly better for samples with PCR-free library preparation protocol than the sample with PCR-based library preparation protocol. DNA input also influences the performance of variant detection in samples with PCR-free WGS. All the 19 previously confirmed variants in 11 clinical cases were successfully detected by our WGS pipeline (PCR free).Different experimental parameters may affect variant detection for clinical WGS. Clinical scientists should know the range of sensitivity of variants for different methods of WGS, which would be useful when interpreting and delivering clinical reports.

Genetic variations of adenosine kinase as predictable biomarkers of efficacy of vagus nerve stimulation in patients with pharmacoresistant epilepsy.

Vagus nerve stimulation (VNS) is an alternative treatment option for individuals with refractory epilepsy, with nearly 40% of patients showing no benefit after VNS and only 6%-8% achieving seizure freedom. It is presently unclear why some patients respond to treatment and others do not. Therefore, identification of biomarkers to predict efficacy of VNS is of utmost importance. The objective of this study was to explore whether genetic variations in genes involved in adenosine kinase (ADK), ecto-5'-nucleotidase (NT5E), and adenosine A1 receptor (A1R) are linked to outcome of VNS in patients with refractory epilepsy. Thirty single-nucleotide polymorphisms (SNPs), including 9 in genes encoding ADK, 3 in genes encoding NT5E, and 18 in genes encoding A1R, were genotyped in 194 refractory epilepsy patients who underwent VNS. The chi-square test and binary logistic regression were used to determine associations between genetic differences and VNS efficacy. A significant association between ADK SNPs rs11001109, rs7899674, and rs946185 and seizure reduction with VNS was found. Regardless of sex, age, seizure frequency and type, antiseizure drug use, etiology, and prior surgical history, all patients (10/10 patients [100%]) with minor allele homozygosity at rs11001109 (genotype AA) or rs946185 (AA) achieved > 50% seizure reduction and 4 patients (4/10 [40%]) achieved seizure freedom. VNS therapy demonstrated higher efficacy among carriers of minor allele rs7899674 (CG + GG) (68.3% vs 48.8% for patients with major allele homozygosity). Homozygous ADK SNPs rs11001109 (AA) and rs946185 (AA), as well as minor allele rs7899674 (CG + GG), may serve as useful biomarkers for prediction of VNS therapy outcome.

Heterotic prediction of hybrid performance based on genome-wide SNP markers and the phenotype of parental inbred lines in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Chinese cabbage (Brassica rapa L. ssp. Pekinensis) is the most widely consumed leafy vegetable in Asia. Most commercial cultivars are F1 hybrids with high yield and good quality. To increase the breeding efficiency, heterotic prediction of hybrid performance was studied. Fourteen parental lines of Chinese cabbage were studied in a half-diallel cross to produce 91 hybrids. The average mid-parent heterosis (MPH) for 14 agronomic traits ranged from −5.07% to 106.76%, while the average high-parent heterosis (HPH) ranged from −109.90% to 80.56%. Significant HPH was observed in the growth period (−109.90%), biomass (80.56%), and plant weight (15.20%), which exhibited comprehensive advantages in breeding. Two methods were used to calculate the genetic distance (GD) between the parents based on 2,444,676 single nucleotide polymorphisms (SNPs): total SNPs (GDtotal) and homozygous differential SNPs (GDhomo). Parental clustering based on total SNPs divided the parents into three major types, namely “flat-topped”, “ovate”, and “cylindrical”. These types were more suitable for their traditional pedigree relationships. The correlation between the GD, mean parental phenotype, and heterosis indicated that the phenotype of the parental inbred lines may be useful for predicting heterosis because it is rapid and simple, whereas GD calculated using SNP markers led to a moderate improvement in heterotic prediction. GDhomo was a better predictor of heterosis and F1 performance than GDtotal. These results confirmed that whole-genome SNP markers are an effective tool for evaluating GD in Chinese cabbage and are useful for parent selection in breeding.

Genome-Wide and Exome-Capturing Sequencing of a Gamma-Ray-Induced Mutant Reveals Biased Variations in Common Wheat.

Induced mutagenesis is a powerful approach for the creation of novel germplasm and the improvement of agronomic traits. The evaluation of mutagenic effects and functional variations in crops is needed for breeding mutant strains. To investigate the mutagenic effects of gamma-ray irradiation in wheat, this study characterized genomic variations of wheat early heading mutant (eh1) as compared to wild-type (WT) Zhongyuan 9 (ZY9). Whole-genome resequencing of eh1 and ZY9 produced 737.7 Gb sequencing data and identified a total of 23,537,117 homozygous single nucleotide polymorphism (SNP) and 1,608,468 Indel. Analysis of SNP distribution across the chromosome suggests that mutation hotspots existed in certain chromosomal regions. Among the three subgenomes, the variation frequency in subgenome D was significantly lower than in subgenomes A and B. A total of 27.8 Gb data were obtained by exome-capturing sequencing, while 217,948 SNP and 13,554 Indel were identified. Variation annotation in the gene-coding sequences demonstrated that 5.0% of the SNP and 5.3% of the Indel were functionally important. Characterization of exomic variations in 12 additional gamma-ray-induced mutant lines further provided additional insights into the mutagenic effects of this approach. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) analysis suggested that genes with functional variations were enriched in several metabolic pathways, including plant–pathogen interactions and ADP binding. Kompetitive allele-specific PCR (KASP) genotyping with selected SNP within functional genes indicated that 85.7% of the SNPs were polymorphic between the eh1 and wild type. This study provides a basic understanding of the mechanism behind gamma-ray irradiation in hexaploid wheat.

Single-nucleotide polymorphisms that uniquely identify cultivars of avocado (Persea americana).

PremiseProgeny of avocado (Persea americana) are highly variable due to high levels of heterozygosity. Breeding programs need molecular resources to allow the assessment of genetic differences and the selection of genotypes. Polymorphisms that uniquely identify different avocado cultivars provide a valuable tool to accelerate avocado research and development, including, for example, genotype selection.MethodsA double‐digest restriction site–associated DNA sequencing (ddRADseq) approach was used to screen 10 avocado cultivars for single‐nucleotide polymorphisms (SNPs). The fragments were size selected with Blue Pippin and PCR using universal Illumina primers, and catalog tags were then created with de novo alignment using Stacks software. Catalog tags were tabulated and filtered to identify alleles unique to each cultivar.ResultsA total of 104 million sequences were collected, and 52 homozygous SNPs were identified that uniquely distinguished nine avocado cultivars. The cultivars Carmen Hass and Hass have a strong genetic similarity and no homozygous SNPs distinguishing these cultivars could be identified; therefore, both cultivars were grouped together.DiscussionThe resource described here for cultivars of P. americana presents a new and significant molecular resource that can enable targeted genotype selection, paternity analysis, germplasm genotyping, pollination dynamics investigation, and crop improvement.

Comparative transcriptomic analysis reveals the regulatory mechanism of the gibberellic acid pathway of Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.) dwarf mutants

BackgroundTartary buckwheat is an important minor crop species with high nutritional and medicinal value and is widely planted worldwide. Cultivated Tartary buckwheat plants are tall and have hollow stems that lodge easily, which severely affects their yield and hinders the development of the Tartary buckwheat industry.MethodsHeifeng No. 1 seeds were treated with ethylmethanesulfonate (EMS) to generate a mutant library. The dwarf mutant ftdm was selected from the mutagenized population, and the agronomic characteristics giving rise to the dwarf phenotype were evaluated. Ultra-fast liquid chromatography-electrospray ionization tandem mass spectrometry (UFLC-ESI–MS/MS) was performed to determine the factors underlying the different phenotypes between the wild-type (WT) and ftdm plants. In addition, RNA sequencing (RNA-seq) was performed via the HiSeq 2000 platform, and the resulting transcriptomic data were analysed to identify differentially expressed genes (DEGs). Single-nucleotide polymorphism (SNP) variant analysis revealed possible sites associated with dwarfism. The expression levels of the potential DEGs between the WT and ftdm mutant were then measured via qRT-PCR and fragments per kilobase of transcript per million mapped reads (FPKM).ResultThe plant height (PH) of the ftdm mutant decreased to 42% of that of the WT, and compared with the WT, the mutant and had a higher breaking force (BF) and lower lodging index (LI). Lower GA4 and GA7 contents and higher contents of jasmonic acid (JA), salicylic acid (SA) and brassinolactone (BR) were detected in the stems of the ftdm mutant compared with the WT. Exogenous application of GAs could not revert the dwarfism of the ftdm mutant. On the basis of the transcriptomic analysis, 146 homozygous SNP loci were identified. In total, 12 DEGs with nonsynonymous mutations were ultimately identified, which were considered potential candidate genes related to the dwarf trait. When the sequences of eight genes whose expression was downregulated and four genes whose expression was upregulated were compared, SKIP14, an F-box protein whose sequence is 85% homologous to that of SLY1 in Arabidopsis, presented an amino acid change (from Ser to Asn) and was expressed at a lower level in the stems of the ftdm mutant compared with the WT. Hence, we speculated that this amino acid change in SKIP14 resulted in a disruption in GA signal transduction, indirectly decreasing the GA content and downregulating the expression of genes involved in GA biosynthesis or the GA response. Further studies are needed to determine the molecular basis underlying the dwarf phenotype of the ftdm mutant.ConclusionWe report a Tartary buckwheat EMS dwarf mutant, ftdm, suitable for high-density planting and commercial farming. A significant decrease in GA4 and GA7 levels was detected in the ftdm mutant, and 12 DEGs expressed in the stems of the ftdm mutant were selected as candidates of the dwarfing gene. One nonsynonymous mutation was detected in the SKIP14 gene in the ftdm mutant, and this gene had a lower transcript level compared with that in the WT.

The effect of polymorphisms in FSHR and FSHB genes on ovarian response: a prospective multicenter multinational study in Europe and Asia.

Does the presence of single nucleotide polymorphisms (SNPs) in the FSH receptor gene (FSHR) and/or FSH beta subunit-encoding gene (FSHB) influence ovarian response in predicted normal responders treated with rFSH? The presence of FSHR SNPs (rs6165, rs6166, rs1394205) has a statistically significant impact in ovarian response, although this effect is of minimal clinical relevance in predicted normal responders treated with a fixed dose of 150 IU rFSH. Ovarian reserve markers have been a breakthrough in response prediction following ovarian stimulation. However, a significant percentage of patients show a disproportionate lower ovarian response, as compared with their actual ovarian reserve. Studies on pharmacogenetics have demonstrated a relationship between FSHR or FSHB genotyping and drug response, suggesting a potential effect of individual genetic variability on ovarian stimulation. However, evidence from these studies is inconsistent, due to the inclusion of patients with variable ovarian reserve, use of different starting gonadotropin doses, and allowance for dose adjustments during treatment. This highlights the necessity of a well-controlled prospective study in a homogenous population treated with the same fixed protocol. We conducted a multicenter multinational prospective study, including 368 patients from Vietnam, Belgium, and Spain (168 from Europe and 200 from Asia), from November 2016 until June 2019. All patients underwent ovarian stimulation followed by oocyte retrieval in an antagonist protocol with a fixed daily dose of 150 IU rFSH until triggering. Blood sampling and DNA extraction was performed prior to oocyte retrieval, followed by genotyping of four SNPs from FSHR (rs6165, rs6166, rs1394205) and FSHB (rs10835638). Eligible were predicted normal responder women <38 years old undergoing their first or second ovarian stimulation cycle. Laboratory staff and clinicians were blinded to the clinical results and genotyping, respectively. The prevalence of hypo-responders, the number of oocytes retrieved, the follicular output rate (FORT), and the follicle to oocyte index (FOI) were compared between different FSHR and FSHB SNPs genotypes. The prevalence of derived allele homozygous SNPs in the FSHR was rs6166 (genotype G/G) 15.8%, rs6165 (genotype G/G) 34.8%, and rs1394205 (genotype A/A) 14.1%, with significant differences between Caucasian and Asian women (P < 0.001). FSHB variant rs10835638 (c.-211 G>T) was very rare (0.5%). Genetic model analysis revealed that the presence of the G allele in FSHR variant rs6166 resulted in less oocytes retrieved when compared to the AA genotype (13.54 ± 0.46 vs 14.81 ± 0.61, estimated mean difference (EMD) -1.47 (95% CI -2.82 to -0.11)). In FSHR variant rs1394205, a significantly lower number of oocytes was retrieved in patients with an A allele when compared to G/G (13.33 ± 0.41 vs 15.06 ± 0.68, EMD -1.69 (95% CI -3.06 to -0.31)). A significantly higher prevalence of hypo-responders was found in patients with the genotype A/G for FSHR variant rs6166 (55.9%, n = 57) when compared to A/A (28.4%, n = 29), ORadj 1.87 (95% CI 1.08-3.24). No significant differences were found regarding the FORT across the genotypes for FSHR variants rs6166, rs6165, or rs1394205. Regarding the FOI, the presence of the G allele for FSHR variant rs6166 resulted in a lower FOI when compared to the A/A genotype, EMD -13.47 (95% CI -22.69 to -4.24). Regarding FSHR variant rs6165, a lower FOI was reported for genotype A/G (79.75 ± 3.35) when compared to genotype A/A (92.08 ± 6.23), EMD -13.81 (95% CI -25.41 to -2.21). The study was performed in relatively young women with normal ovarian reserve to eliminate biases related to age-related fertility decline; thus, caution is needed when extrapolating results to older populations. In addition, no analysis was performed for FSHB variant rs10835638 due to the very low prevalence of the genotype T/T (n = 2). Based on our results, genotyping FSHR SNPs rs6165, rs6166, rs1394205, and FSHB SNP rs10835638 prior to initiating an ovarian stimulation with rFSH in predicted normal responders should not be recommended, taking into account the minimal clinical impact of such information in this population. Future research may focus on other populations and other genes related to folliculogenesis or steroidogenesis. This study was supported by an unrestricted grant by Merck Sharp & Dohme (MSD). N.P.P. reports grants and/or personal fees from MSD, Merck Serono, Roche Diagnostics, Ferring International, Besins Healthcare, Gedeon Richter, Theramex, and Institut Biochimique SA (IBSA). N.L.V. and M.T.H. report consultancy and conference fees from Merck, Ferring, and MSD, outside the submitted work. P.D. has received honoraria for lecturing and/or research grants from MSD, Ferring International, and Merck. D.S. reports grants and/or personal fees from MSD, Ferring International, Merck Serono, Cook, and Gedeon Richter. A.R.N., B.A.M., C.S., J.M., L.H.L., P.Q.M.M., H.T., and S.G. report no conflict of interests. NCT03007043.

GPR39 localization in the aging human brain and correlation of expression and polymorphism with vascular cognitive impairment.

IntroductionThe pathogenesis of vascular cognitive impairment (VCI) is not fully understood. GPR39, an orphan G‐protein coupled receptor, is implicated in neurological disorders but its role in VCI is unknown.MethodsWe performed GPR39 immunohistochemical analysis in post mortem brain samples from mild cognitive impairment (MCI) and control subjects. DNA was analyzed for GPR39 single nucleotide polymorphisms (SNPs), and correlated with white matter hyperintensity (WMH) burden on pre mortem magnetic resonance imaging.ResultsGPR39 is expressed in aged human dorsolateral prefrontal cortex, localized to microglia and peri‐capillary cells resembling pericytes. GPR39–capillary colocalization, and density of GPR39‐expressing microglia was increased in aged brains compared to young. SNP distribution was equivalent between groups; however, homozygous SNP carriers were present only in the MCI group, and had higher WMH volume than wild‐type or heterozygous SNP carriers.DiscussionGPR39 may play a role in aging‐related VCI, and may serve as a therapeutic target and biomarker for the risk of developing VCI.

Genetic diversity and genomic inbreeding in Japanese Black cows in the islands of Okinawa Prefecture evaluated using single-nucleotide polymorphism array.

Maintaining genetic diversity and inbreeding control are important in Japanese Black cattle production, especially in remote areas such as the islands of Okinawa Prefecture. Using a single-nucleotide polymorphism (SNP) array, we evaluated the genetic diversity and genomic inbreeding in Japanese Black cows from the islands of Okinawa Prefecture and compared them to those from other locations across Japan. Linkage disequilibrium decay was slower in cows in the islands of Okinawa Prefecture. The estimated effective population size declined over time in both populations. The genomic inbreeding coefficient (FROH ) was estimated using long stretches of consecutive homozygous SNPs (runs of homozygosity; ROH). FROH was higher in the cows on the islands of Okinawa Prefecture than on other locations. In total, 818 ROH fragments, including those containing NCAPG and PLAG1, which are major quantitative trait loci for carcass weight in Japanese Black cattle, were present at significantly higher frequencies in cows in the islands of Okinawa Prefecture. This suggests that the ROH fragments are under strong selection and that cows in the islands of Okinawa Prefecture have low genetic diversity and high genomic inbreeding relative to those at other locations. SNP arrays are useful tools for evaluating genetic diversity and genomic inbreeding in cattle.

Identification of Allelic Variation in Drought Responsive Dehydrin Gene Based on Sequence Similarity in Chickpea (Cicer arietinum L.).

Chickpea (Cicer arietinum L.) is an economically important food legume grown in arid and semi-arid regions of the world. Chickpea is cultivated mainly in the rainfed, residual moisture, and restricted irrigation condition. The crop is always prone to drought stress which is resulting in flower drop, unfilled pods, and is a major yield reducer in many parts of the world. The present study elucidates the association between candidate gene and morpho-physiological traits for the screening of drought tolerance in chickpea. Abiotic stress-responsive gene Dehydrin (DHN) was identified in some of the chickpea genotypes based on the sequence similarity approach to play a major role in drought tolerance. Analysis of variance revealed a significant effect of drought on relative water content, membrane stability index, plant height, and yield traits. The genotypes Pusa1103, Pusa362, and ICC4958 were found most promising genotypes for drought tolerance as they maintained the higher value of osmotic regulations and yield characters. The results were further supported by a sequence similarity approach for the dehydrin gene when analyzed for the presence of single nucleotide polymorphisms (SNPs) and indels. Homozygous indels and single nucleotide polymorphisms were found after the sequencing in some of the selected genotypes.

Development of Kompetitive Allele Specific PCR markers for identification of persimmon varieties using genotyping-by-sequencing

Persimmon ( Diospyros kaki Thunb.) is the most widely cultivated species of the genus Diospyros . In this study, genetic diversity and variations in persimmon genotypes were investigated using single nucleotide polymorphism (SNP) markers identified by genotyping-by-sequencing (GBS) analysis. Ninety-five persimmon accessions grown in the Pear Research Institute, National Institute Horticultural and Herbal Science, were sequenced using the Illumina Hiseq2500 platform and polymorphic SNPs were detected to develop molecular markers. These reliable SNPs were analyzed using the Kompetitive Allele Specific PCR (KASP) assay to discriminate among persimmon genotypes. GBS generated a total of 447,495,724 trimmed reads, of which 89.7% were raw reads. After demultiplexing and sequence quality trimming, 108,876,644 clean reads were mapped to the reference transcriptome. An average of 1,146,070 genotype reads were mapped. Filtering of raw SNPs in each sample led to selection of a total of 1,725,401 high-quality SNPs. The number of homozygous and heterozygous SNPs ranged from 1,933 to 6,834 and from 846 to 5,927, respectively. Of the 49 SNPs selected for development of an identification system for persimmons, 15 SNPs were used in the KASP assay to analyze 32 persimmon accessions. These KASP markers discriminated among all accessions. How to cite: Ma KB, Yang SJ, Jo YS, et al. Development of Kompetitive Allele Specific PCR markers for identification of persimmon varieties using genotyping-by-sequencing. Electron J Biotechnol 2021;49. https://doi.org/10.1016/j.ejbt.2020.11.003