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Non-Mendelian transmission of mitochondria has been well established across most eukaryotes, however the genetic mechanism that governs this uniparental inheritance remains unclear. Plants in the genus Cucumis , specifically melon and cucumber, exhibit paternal transmission of the mitochondrial (mt) DNA, making them excellent models for exploring the molecular mechanisms underlying mitochondrial transmission. Here, we develop a toolkit to screen for mutants in mitochondrial inheritance ( mti ), and use fine mapping to successfully identify a mitochondrially targeted endonuclease gene ( MTI1 ) controlling mitochondrial transmission. Knockout of MTI1 results in a shift from paternal to bi-parental inheritance of the mtDNA, confirming the crucial role of MTI1 in uniparental inheritance of mitochondria. Moreover, we demonstrate that MTI1 exhibits robust endonuclease activity both in vitro and in vivo, specifically expresses in mitochondria of the fertilized ovule within 24 h of pollination. Collectively, this study reveals that a nuclear-encoded but mitochondria-targeted gene plays a causative role in governing the non-Mendelian mitochondrial inheritance, revolutionizing our knowledge about mitochondrial DNA transmission. Shen et al. established a strategy to screen for mutants showing altered mitochondrial transmission in Cucumis and cloned a nuclear endonuclease gene ( MTI1 ) that controls paternal versus biparental mitochondrial inheritance.

Key message Through a novel phenotyping method, four QTLs were consistently associated with increased parthenocarpic fruit set in North American processing cucumber that accounted for over 75   % of observed phenotypic variation. Parthenocarpy is a desirable trait with potential for increasing yield and quality in processing cucumber production. Although many successful parthenocarpic fresh market cucumber varieties have been developed, the genetic and molecular mechanisms behind parthenocarpic expression in cucumber remain largely unknown. Since parthenocarpy is an important yield component, it is difficult to separate the true parthenocarpic character from other yield related traits. In the present study, we developed a novel phenotypic approach for parthenocarpic fruit set focusing on early fruit development. Two hundred and five F 3 families derived from a cross between the highly parthenocarpic line 2A and low parthenocarpic line Gy8 were phenotypically evaluated in three greenhouse experiments. Seven QTLs associated with parthenocarpic fruit set were detected. Among them, one each on chromosomes 5 and 7 ( parth5.1 and parth7.1 ) and two on chromosome 6 ( parth6.1 and parth6.2 ) were consistently identified in all experiments, but their relative contribution to the total phenotypic variation was dependent on plant growth stages. While each of the four QTLs had almost equal contribution to the expression of the trait at commercial harvest stage, parth7.1 played an important role in early parthenocarpic fruit set. The results suggested that parthenocarpic fruit set can be accurately evaluated with as few as 20 nodes of growth. The QTLs identified in this study for parthenocarpic fruit set are a valuable resource for cucumber breeders interested in developing parthenocarpic cultivars and to researchers interested in the genetic and molecular mechanisms of parthenocarpic fruit set.

Epicuticular waxes on the surface of plant leaves are important for the tolerance to abiotic stresses and plant–parasite interactions. In the onion (Allium cepa L.), the variation for the amounts and types of epicuticular waxes is significantly associated with less feeding damage by the insect Thrips tabaci (thrips). Epicuticular wax profiles are measured using used gas chromatography mass spectrometry (GCMS), which is a labor intensive and relatively expensive approach. Biochemical spectroscopy is a non-destructive tool for measurement and analysis of physiological and chemical features of plants. This study used GCMS and full-range biochemical spectroscopy to characterize epicuticular waxes on seven onion accessions with visually glossy (low wax), semi-glossy (intermediate wax), or waxy (copious wax) foliage, as well as a segregating family from the cross of glossy and waxy onions. In agreement with previous studies, GCMS revealed that the three main waxes on the leaves of a wild type waxy onion were the ketone hentriacontanone-16 (H16) and fatty alcohols octacosanol-1 (Oct) and triacontanol-1 (Tri). The glossy cultivar “Odourless Greenleaf” had a unique phenotype with essentially no H16 and Tri and higher amounts of Oct and the fatty alcohol hexacosanol-1 (Hex). Hyperspectral reflectance profiles were measured on leaves of the onion accessions and segregating family, and partial least-squares regression (PLSR) was utilized to generate a spectral coefficient for every wavelength and prediction models for the amounts of the three major wax components. PLSR predictions were robust with independent validation coefficients of determination at 0.72, 0.70, and 0.42 for H16, Oct, and Tri, respectively. The predicted amounts of H16, Oct, and Tri are the result of an additive effect of multiple spectral features of different intensities. The variation of reflectance for H16, Oct, and Tri revealed unique spectral features at 2259 nm, 645 nm, and 730 nm, respectively. Reflectance spectroscopy successfully revealed a major quantitative trait locus (QTL) for amounts of H16, Oct, and Tri in the segregating family, agreeing with previous genetic studies. This study demonstrates that hyperspectral signatures can be used for non-destructive measurement of major waxes on onion leaves as a basis for rapid plant assessment in support of developing thrips-resistant onions.

Onion thrips (Thrips tabaci Lindeman) is a major pest in organic onion production and effective integrated pest management strategies are lacking. Our objective was to evaluate combinations of semi-glossy (“Rossa di Milano” and B5336AxB5351C) and waxy (“Bradley”) onion cultivars with reflective mulch, with or without biopesticides (spinosad + neem oil tank mix), to manage T. tabaci in organic onion production. Thrips densities were assessed weekly and bulbs graded and weighed at harvest. Onions sprayed with spinosad + neem oil had fewer T. tabaci (adults: 74% (2019); larvae: 40% (2018), 84% (2019) and produced higher yields (13% (2018), 23% (2019)) than onions that were unsprayed, regardless of mulch type or onion cultivar. “Rossa di Milano” had relatively fewer adult and larval thrips populations compared with “Bradley” (21% (2018), 32% (2019)) and B5336AxB5351C. However, “Rossa di Milano” had the lowest marketable yield in both years. Reflective mulch reduced densities on certain dates in both years compared to white mulch, but the largest and most consistent reduction only occurred in 2019. Reflective mulch had no impact on bulb yield. While spinosad + neem oil reduced thrips numbers and increased yield alone, none of the treatment combinations were effective at suppressing populations of thrips. Future T. tabaci management in organic onions will require optimization of the available effective biopesticides.

Cucumber (Cucumis sativus L.) is a warm-season crop that is sensitive to chilling temperatures and a maternally inherited cold tolerance exists in the heirloom cultivar ‘Chipper’ (CH). Because the organelles of cucumber show differential transmission (maternal for chloroplast and paternal for mitochondrion), this cold tolerance is hypothesized to be chloroplast-associated. The goal of this research was to characterize the cold tolerant phenotype from CH and determine its genetic basis. Doubled haploid (DH) lines were produced from CH and cold susceptible cucumbers, reciprocal hybrids with identical nuclear genotypes were produced, and plants were subjected to cold treatments under lights at 4 °C for 5.5 h. Hybrid plants with CH as the maternal parent had significantly higher fresh and dry weights 14 days after cold treatment compared to the reciprocal hybrid, revealing an enhanced cold recovery phenotype maternally conferred by CH. Results from analyses of the nuclear transcriptome and reactive oxygen species (ROS) between reciprocal hybrids were consistent with the cold recovery phenotype. Sequencing of the chloroplast genome and transcriptome of the DH parents and reciprocal hybrids, respectively, revealed one maternally transmitted non-synonymous single nucleotide polymorphism (SNP) in the chloroplast F1FO-ATP synthase (CF1FO-ATPase) beta-subunit gene (atpB) of CH which confers an amino acid change from threonine to arginine. Protein modeling revealed that this change is located at the interface of the alpha- and beta-subunits in the CF1FO-ATPase complex. Polymorphisms in the CF1FO-ATPase complex have been associated with stress tolerances in other plants, and selection for or creation of polymorphic beta-subunit proteins by chloroplast transformation or gene editing could condition improved recovery from cold stress in plants.

Melon is a useful plant species for studying mitochondrial genetics because it contains one of the largest and structurally diverse mitochondrial genomes among all plant species and undergoes paternal transmission of mitochondria. We used droplet digital (dd) PCR in combination with flow cytometric determination of nuclear DNA quantities to determine the absolute per-cell copy numbers of four mitochondrial genes ( nad9 , rps1 , matR , and atp6 ) across four stages of melon leaf development. The copy numbers of these mitochondrial genes not only varied during leaf development but also differed among each other, and there was no correlation between the copy numbers of the mitochondrial genes and their transcript levels. The gene copy numbers varied from approximately 36.8 ± 4.5 ( atp6 copies in the 15th leaf) to approximately 82.9 ± 5.7 ( nad9 copies in the 9th leaf), while the mean number of mitochondria was approximately 416.6 ± 182.7 in the 15th leaf and 459.1 ± 228.2 in the 9th leaf. These observations indicate that the leaf cells of melon do not contain sufficient copies of mitochondrial genes to ensure that every mitochondrion possesses the entire mitochondrial genome. Given this cytological evidence, our results indicate that mtDNA in melon exists as a sub-genomic molecule rather than as a single-master circle and that the copy numbers of individual mitochondrial genes may vary greatly. An improved understanding of the molecular mechanism(s) controlling the relative prevalence and transmission of sub-genomic mtDNA molecules should provide insights into the continuity of the mitochondrial genome across generations.

Fusarium basal rot (FBR) is a serious disease of onion (Allium cepa). We identified sources of FBR resistance, assessed efficacy of selection for increased resistance, and investigated its genetic control. Onion accessions were evaluated for FBR resistance, and percentage survival ranged from 0% to 78%. Survivors were intercrossed, and progenies from one cycle of selection showed increased survival by 18% to 52%. Selections were crossed to male-sterile lines, and hybrids showed specific combining ability for FBR resistance. Segregating families were produced, and quantitative trait loci (QTLs) were identified on chromosomes 2 and 4 conditioning FBR resistance. A second QTL on chromosome 4 was identified that decreased FBR resistance. Plants from families with different genotypes across the 1.5 logarithm of odds (LOD) regions on chromosomes 2 and 4 were self-pollinated, and resulting families were evaluated for FBR survival. Genomic regions on chromosomes 2 and 4 associated with resistance were validated at p = 0.05 and 0.10, respectively. The region on chromosome 4 associated with increased susceptibility was validated at p = 0.05. These results are in agreement with previous studies reporting high heritability and specific combining ability for FBR resistance and should be useful for selection of FBR-resistant onion.

Parent-of-origin effects have long been recognized and exploited in plant breeding and genetic studies. These effects can be conferred by preferential expression of an allele from one parent, organellar effects, or altered organellar-nuclear interaction. The goal of this work was to evaluate parent-of-origin effects on seed, cotyledon, and early growth traits in cucumber using a full eight-by-eight diallel from crossing two doubled haploids (DHs) extracted from each of four cucumber populations. Significant general combining ability (GCA), specific combining ability (SCA), and reciprocal effects were observed for all traits, and direction and magnitude of effects were DH rather than population specific. Transcriptome analyses of reciprocal hybrids with and without significant reciprocal effects for early plant growth revealed that different pathways were associated with the significant reciprocal differences. These findings are consistent with the DH-specific nature of combining abilities and reciprocal effects across cucumber populations. Because reciprocal effects were DH and hybrid-combination specific, cucumber breeders should generate and evaluate both hybrids from reciprocal crossing for improved hybrid development.

Flowers were produced on sterile cucumber (Cucumis sativus L.) plants grown in vitro from seed and micropropagated shoots from stem fragments. The highest numbers of flowers on plants from both sources were produced on Murashige and Skoog (MS) medium without plant growth regulators (PGR), as well as with 6 μM of kinetin (Kin). Plants cultured on MS medium supplemented with 8.9 μM benzyladenine (BA) and 1.1 μM 1-naphthaleneacetic acid (NAA) did not flower. In vitro grown plants produced fewer, smaller flowers compared with greenhouse-grown plants. Male and female flowers developed on plants grown in vitro from seed and were morphologically similar to flowers on greenhouse grown plants. Micropropagated shoots produced male flowers with altered morphology. The highest viability (72.9 ± 4.2%) and germination (69.5 ± 4.1%) of pollen were observed for plants grown from seed on MS medium supplemented with 6 μM Kin. Cytological observations of meiosis in anthers of male flowers from in vitro grown plants revealed abnormalities, such as monads, dyads, triads, polyads, microcytes and degeneration of tetrads, causing reduced viability and germination of pollen. The fewest meiotic irregularities in pollen mother cells were observed in plants grown on MS medium that was PGR-free (12.1 ± 0.9%) or with 6 μM Kin (20.9 ± 1.7%).

KEY MESSAGE : Tyramide FISH was used to locate relatively small genomic amplicons from molecular markers linked to Ms locus onto onion chromosome 2 near the centromere, a region of relatively low recombination. Fluorescence in situ hybridization (FISH) has not been readily exploited for physical mapping of molecular markers in plants due to the technical challenge of visualizing small single-copy probes. Signal amplification using tyramide (tyr) FISH can increase sensitivity up to 100-fold. We used tyr-FISH to physically locate molecular markers tightly linked to the nuclear male-fertility (Ms) restoration locus of onion onto mitotic metaphase, pachytene, and super-stretched pachytene chromosomes. Relatively short genomic amplicons (846–2251 bp) and a cDNA clone (666 bp) were visualized in 9–42 % of observed cells. The markers were assigned to proximal locations close to the centromere on the long arm of chromosome 2, a region of lower recombination, revealing that tightly linked markers may be physically distant from Ms. This result explains why several labs have identified molecular markers tightly linked to the Ms locus after screening relatively few DNA clones or primers and segregating progenies. Although these markers are still useful for marker-aided selection, our results indicate that map-based cloning of Ms will likely be difficult due to reduced recombination near this gene.