Genotyping by sequencing for SNP marker development in onion.

Onion (Allium cepa) is not highly tractable for development of molecular markers due to its large (16 gigabases per 1C) nuclear genome. Single nucleotide polymorphisms (SNPs) are useful for genetic characterization and marker-aided selection of onion because of codominance and common occurrence in elite germplasm. We completed genotyping by sequencing (GBS) to identify SNPs in onion using 46 F2 plants, parents of the F2 plants (Ailsa Craig 43 and Brigham Yellow Globe 15-23), two doubled haploid (DH) lines (DH2107 and DH2110), and plants from 94 accessions in the USDA National Plant Germplasm System (NPGS). SNPs were called using the TASSEL 3.0 Universal Network Enabled Analysis (UNEAK) bioinformatics pipeline. Sequences from the F2 and DH plants were used to construct a pseudo-reference genome against which genotypes from all accessions were scored. Quality filters were used to identify a set of 284 high quality SNPs, which were placed onto an existing genetic map for the F2 family. Accessions showed a moderate level of diversity (mean He = 0.341) and evidence of inbreeding (mean F = 0.592). GBS is promising for SNP discovery in onion, although lack of a reference genome required extensive custom scripts for bioinformatics analyses to identify high quality markers.

Reflectance Spectroscopy for Non-Destructive Measurement and Genetic Analysis of Amounts and Types of Epicuticular Waxes on Onion Leaves.

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.

Rare maternal and biparental transmission of the cucumber mitochondrial DNA reveals sorting of polymorphisms among progenies.

KEY MESSAGE: We used a mitochondrial (mt) mutant of cucumber to document rare maternal transmission of mt polymorphisms and demonstrate that polymorphisms can become more prevalent and sort to progenies to increase mt DNA diversity. The mitochondrial (mt) DNAs of most angiosperms show maternal inheritance, although relatively rare biparental or paternal transmission has been documented. The mt DNAs of plants in the genus Cucumis (family Cucurbitaceae) are paternally transmitted in intra- and interspecific crosses. MSC16 is an inbred line of cucumber (Cucumis sativus) with a mitochondrially associated mosaic (MSC) phenotype. MSC16 was crossed as the male parent to wild-type cultivar Calypso, and hybrid progenies were evaluated for the wild-type phenotype in order to screen for rare maternal or biparental transmission of the mt DNA. We then used standard and droplet digital (dd) PCR to study the transmission of polymorphic mt markers across three generations. We observed evidence for occasional maternal and biparental transmission of the mt DNA in cucumber. The transmission of specific regions of the maternal mt DNA could be as high as 17.8%, although the amounts of these maternal regions were often much lower relative to paternally transmitted regions. Different combinations of maternal and paternal mt polymorphisms were detected in progenies across generations, indicating that relatively rare maternal regions can be transmitted to progenies and become predominant to increase mt DNA diversity over generations.

Assembly and characterisation of a unique onion diversity set identifies resistance to Fusarium basal rot and improved seedling vigour.

KEY MESSAGE: A unique, global onion diversity set was assembled, genotyped and phenotyped for beneficial traits. Accessions with strong basal rot resistance and increased seedling vigour were identified along with associated markers. Conserving biodiversity is critical for safeguarding future crop production. Onion (Allium cepa L.) is a globally important crop with a very large (16 Gb per 1C) genome which has not been sequenced. While onions are self-fertile, they suffer from severe inbreeding depression and as such are highly heterozygous as a result of out-crossing. Bulb formation is driven by daylength, and accessions are adapted to the local photoperiod. Onion seed is often directly sown in the field, and hence seedling establishment is a critical trait for production. Furthermore, onion yield losses regularly occur worldwide due to Fusarium basal rot caused by Fusarium oxysporum f. sp. cepae. A globally relevant onion diversity set, consisting of 10 half-sib families for each of 95 accessions, was assembled and genotyping carried out using 892 SNP markers. A moderate level of heterozygosity (30-35%) was observed, reflecting the outbreeding nature of the crop. Using inferred phylogenies, population structure and principal component analyses, most accessions grouped according to local daylength. A high level of intra-accession diversity was observed, but this was less than inter-accession diversity. Accessions with strong basal rot resistance and increased seedling vigour were identified along with associated markers, confirming the utility of the diversity set for discovering beneficial traits. The onion diversity set and associated trait data therefore provide a valuable resource for future germplasm selection and onion breeding.

Spontaneous polyploidization in cucumber.

KEY MESSAGE: This is the first quantitative estimation of spontaneous polyploidy in cucumber and we detected 2.2% polyploids in a greenhouse study. We provide evidence that polyploidization is consistent with endoreduplication and is an on-going process during plant growth. Cucumber occasionally produces polyploid plants, which are problematic for growers because these plants produce misshaped fruits with non-viable seeds. In this study, we undertook the first quantitative study to estimate the relative frequency of spontaneous polyploids in cucumber. Seeds of recombinant inbred lines were produced in different environments, plants were grown in the field and greenhouse, and flow cytometry was used to establish ploidies. From 1422 greenhouse-grown plants, the overall relative frequency of spontaneous polyploidy was 2.2%. Plants possessed nuclei of different ploidies in the same leaves (mosaic) and on different parts of the same plant (chimeric). Our results provide evidence of endoreduplication and polysomaty in cucumber, and that it is an on-going and dynamic process. There was a significant effect (p = 0.018) of seed production environment on the occurrence of polyploid plants. Seed and seedling traits were not accurate predictors of eventual polyploids, and we recommend that cucumber producers rogue plants based on stature and leaf serration to remove potential polyploids.

High-resolution tyramide-FISH mapping of markers tightly linked to the male-fertility restoration (Ms) locus of onion.

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.

QTL mapping of parthenocarpic fruit set in North American processing cucumber.

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 F3 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.

Tyramide-FISH mapping of single genes for development of an integrated recombination and cytogenetic map of chromosome 5 of Allium cepa.

Chromosome 5 of onion carries major quantitative trait loci (QTL) that control dry-matter content, pungency and storability of bulbs, amounts and types of epicuticular waxes, and resistances to abiotic factors, all of which are of interest to breeders. SNPs, SSRs, and RFLPs in expressed regions of the onion genome have been genetically mapped, and we used these clones and sequences from the NCBI database to develop DNA probes for in situ hybridization to integrate the genetic and physical maps of onion chromosome 5. We produced genomic amplicons from expressed regions of the onion genome that carried both exons and introns in order to increase the hybridization specificity of the probes and to enlarge the target DNA sizes. Tyramide-FISH technique was used to increase the detection sensitivity of relatively short target DNA regions, which range from 950 to 2100 bp. Through the integration of genetic and chromosomal maps, we were able to estimate the distribution of recombination events along onion chromosome 5. We demonstrated the efficiency of chromosomal in situ mapping of exon-intron genomic clones for the extremely large genome of onion.

Cucumber Possesses a Single Terminal Alternative Oxidase Gene That is Upregulated by Cold Stress and in the Mosaic (MSC) Mitochondrial Mutants.

Alternative oxidase (AOX) is a mitochondrial terminal oxidase which is responsible for an alternative route of electron transport in the respiratory chain. This nuclear-encoded enzyme is involved in a major path of survival under adverse conditions by transfer of electrons from ubiquinol instead of the main cytochrome pathway. AOX protects against unexpected inhibition of the cytochrome c oxidase pathway and plays an important role in stress tolerance. Two AOX subfamilies (AOX1 and AOX2) exist in higher plants and are usually encoded by small gene families. In this study, genome-wide searches and cloning were completed to identify and characterize AOX genes in cucumber (Cucumis sativus L.). Our results revealed that cucumber possesses no AOX1 gene(s) and only a single AOX2 gene located on chromosome 4. Expression studies showed that AOX2 in wild-type cucumber is constitutively expressed at low levels and is upregulated by cold stress. AOX2 transcripts and protein were detected in leaves and flowers of wild-type plants, with higher levels in the three independently derived mosaic (MSC) mitochondrial mutants. Because cucumber possesses a single AOX gene and its expression increases under cold stress and in the MSC mutants, this plant is a unique and intriguing model to study AOX expression and regulation particularly in the context of mitochondria-to-nucleus retrograde signaling.

Analysis of wild-species introgressions in tomato inbreds uncovers ancestral origins.

BACKGROUND: Decades of intensive tomato breeding using wild-species germplasm have resulted in the genomes of domesticated germplasm (Solanum lycopersicum) being intertwined with introgressions from their wild relatives. Comparative analysis of genomes among cultivated tomatoes and wild species that have contributed genetic variation can help identify desirable genes, such as those conferring disease resistance. The ability to identify introgression position, borders, and contents can reveal ancestral origins and facilitate harnessing of wild variation in crop breeding. RESULTS: Here we present the whole-genome sequences of two tomato inbreds, Gh13 and BTI-87, both carrying the begomovirus resistance locus Ty-3 introgressed from wild tomato species. Introgressions of different sizes on chromosome 6 of Gh13 and BTI-87, both corresponding to the Ty-3 region, were identified as from a source close to the wild species S. chilense. Other introgressions were identified throughout the genomes of the inbreds and showed major differences in the breeding pedigrees of the two lines. Interestingly, additional large introgressions from the close tomato relative S. pimpinellifolium were identified in both lines. Some of the polymorphic regions were attributed to introgressions in the reference Heinz 1706 genome, indicating wild genome sequences in the reference tomato genome. CONCLUSIONS: The methods developed in this work can be used to delineate genome introgressions, and subsequently contribute to development of molecular markers to aid phenotypic selection, fine mapping and discovery of candidate genes for important phenotypes, and for identification of novel variation for tomato improvement. These universal methods can easily be applied to other crop plants.

Chromosome rearrangements during domestication of cucumber as revealed by high-density genetic mapping and draft genome assembly.

Cucumber, Cucumis sativus L. is the only taxon with 2n = 2x = 14 chromosomes in the genus Cucumis. It consists of two cross-compatible botanical varieties: the cultivated C. sativus var. sativus and the wild C. sativus var. hardwickii. There is no consensus on the evolutionary relationship between the two taxa. Whole-genome sequencing of the cucumber genome provides a new opportunity to advance our understanding of chromosome evolution and the domestication history of cucumber. In this study, a high-density genetic map for cultivated cucumber was developed that contained 735 marker loci in seven linkage groups spanning 707.8 cM. Integration of genetic and physical maps resulted in a chromosome-level draft genome assembly comprising 193 Mbp, or 53% of the 367 Mbp cucumber genome. Strategically selected markers from the genetic map and draft genome assembly were employed to screen for fosmid clones for use as probes in comparative fluorescence in situ hybridization analysis of pachytene chromosomes to investigate genetic differentiation between wild and cultivated cucumbers. Significant differences in the amount and distribution of heterochromatins, as well as chromosomal rearrangements, were uncovered between the two taxa. In particular, six inversions, five paracentric and one pericentric, were revealed in chromosomes 4, 5 and 7. Comparison of the order of fosmid loci on chromosome 7 of cultivated and wild cucumbers, and the syntenic melon chromosome I suggested that the paracentric inversion in this chromosome occurred during domestication of cucumber. The results support the sub-species status of these two cucumber taxa, and suggest that C. sativus var. hardwickii is the progenitor of cultivated cucumber.

Sequencing and annotation of the chloroplast DNAs and identification of polymorphisms distinguishing normal male-fertile and male-sterile cytoplasms of onion.

Male-sterile (S) cytoplasm of onion is an alien cytoplasm introgressed into onion in antiquity and is widely used for hybrid seed production. Owing to the biennial generation time of onion, classical crossing takes at least 4 years to classify cytoplasms as S or normal (N) male-fertile. Molecular markers in the organellar DNAs that distinguish N and S cytoplasms are useful to reduce the time required to classify onion cytoplasms. In this research, we completed next-generation sequencing of the chloroplast DNAs of N- and S-cytoplasmic onions; we assembled and annotated the genomes in addition to identifying polymorphisms that distinguish these cytoplasms. The sizes (153 538 and 153 355 base pairs) and GC contents (36.8%) were very similar for the chloroplast DNAs of N and S cytoplasms, respectively, as expected given their close phylogenetic relationship. The size difference was primarily due to small indels in intergenic regions and a deletion in the accD gene of N-cytoplasmic onion. The structures of the onion chloroplast DNAs were similar to those of most land plants with large and small single copy regions separated by inverted repeats. Twenty-eight single nucleotide polymorphisms, two polymorphic restriction-enzyme sites, and one indel distributed across 20 chloroplast genes in the large and small single copy regions were selected and validated using diverse onion populations previously classified as N or S cytoplasmic using restriction fragment length polymorphisms. Although cytoplasmic male sterility is likely associated with the mitochondrial DNA, maternal transmission of the mitochondrial and chloroplast DNAs allows for polymorphisms in either genome to be useful for classifying onion cytoplasms to aid the development of hybrid onion cultivars.

Genetic mapping of paternal sorting of mitochondria in cucumber.

Mitochondria are organelles that have their own DNA; serve as the powerhouses of eukaryotic cells; play important roles in stress responses, programmed cell death, and ageing; and in the vast majority of eukaryotes, are maternally transmitted. Strict maternal transmission of mitochondria makes it difficult to select for better-performing mitochondria, or against deleterious mutations in the mitochondrial DNA. Cucumber is a useful plant for organellar genetics because its mitochondria are paternally transmitted and it possesses one of the largest mitochondrial genomes among all eukaryotes. Recombination among repetitive motifs in the cucumber mitochondrial DNA produces rearrangements associated with strongly mosaic (MSC) phenotypes. We previously reported nuclear control of sorting among paternally transmitted mitochondrial DNAs. The goal of this project was to map paternal sorting of mitochondria as a step towards its eventual cloning. We crossed single plants from plant introduction (PI) 401734 and Cucumis sativus var. hardwickii and produced an F(2) family. A total of 425 F(2) plants were genotyped for molecular markers and testcrossed as the female with MSC16. Testcross families were scored for frequencies of wild-type versus MSC progenies. Discrete segregations for percent wild-type progenies were not observed and paternal sorting of mitochondria was therefore analyzed as a quantitative trait. A major quantitative trait locus (QTL; LOD >23) was mapped between two simple sequence repeats encompassing a 459-kb region on chromosome 3. Nuclear genes previously shown to affect the prevalence of mitochondrial DNAs (MSH1, OSB1, and RECA homologs) were not located near this major QTL on chromosome 3. Sequencing of this region from PI 401734, together with improved annotation of the cucumber genome, should result in the eventual cloning of paternal sorting of mitochondria and provide insights about nuclear control of organellar-DNA sorting.

Polymorphism in the Chloroplast ATP Synthase Beta-Subunit Is Associated with a Maternally Inherited Enhanced Cold Recovery in Cucumber.

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.

Management of Onion Thrips (Thrips tabaci) in Organic Onion Production Using Multiple IPM Tactics.

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.

Non-synonymous single nucleotide polymorphisms in the watermelon eIF4E gene are closely associated with resistance to zucchini yellow mosaic virus.

Zucchini yellow mosaic virus (ZYMV) is one of the most economically important potyviruses infecting cucurbit crops worldwide. Using a candidate gene approach, we cloned and sequenced eIF4E and eIF(iso)4E gene segments in watermelon. Analysis of the nucleotide sequences between the ZYMV-resistant watermelon plant introduction PI 595203 (Citrullus lanatus var. lanatus) and the ZYMV-susceptible watermelon cultivar 'New Hampshire Midget' ('NHM') showed the presence of single nucleotide polymorphisms (SNPs). Initial analysis of the identified SNPs in association studies indicated that SNPs in the eIF4E, but not eIF(iso)4E, were closely associated to the phenotype of ZYMV-resistance in 70 F(2) and 114 BC(1R) progenies. Subsequently, we focused our efforts in obtaining the entire genomic sequence of watermelon eIF4E. Three SNPs were identified between PI 595203 and NHM. One of the SNPs (A241C) was in exon 1 and the other two SNPs (C309A and T554G) were in the first intron of the gene. SNP241 which resulted in an amino acid substitution (proline to threonine) was shown to be located in the critical cap recognition and binding area, similar to that of several plant species resistance to potyviruses. Analysis of a cleaved amplified polymorphism sequence (CAPS) marker derived from this SNP in F(2) and BC(1R) populations demonstrated a cosegregation between the CAPS-2 marker and their ZYMV resistance or susceptibility phenotype. When we investigated whether such SNP mutation in the eIF4E was also conserved in several other PIs of C. lanatus var. citroides, we identified a different SNP (A171G) resulting in another amino acid substitution (D71G) from four ZYMV-resistant C. lanatus var. citroides (PI 244018, PI 482261, PI 482299, and PI 482322). Additional CAPS markers were also identified. Availability of all these CAPS markers will enable marker-aided breeding of watermelon for ZYMV resistance.

Copy numbers of mitochondrial genes change during melon leaf development and are lower than the numbers of mitochondria.

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.

Cytological Evaluations of Advanced Generations of Interspecific Hybrids Between Allium cepa and Allium fistulosum Showing Resistance to Stemphylium vesicarium.

Interspecific crossing is a promising approach for introgression of valuable traits to develop cultivars with improved characteristics. Allium fistulosum L. possesses numerous pest resistances that are lacking in the bulb onion (Allium cepa L.), including resistance to Stemphylium leaf blight (SLB). Advanced generations were produced by selfing and backcrossing to bulb onions of interspecific hybrids between A. cepa and A. fistulosum that showed resistance to SLB. Molecular classification of the cytoplasm established that all generations possessed normal (N) male-fertile cytoplasm of bulb onions. Genomic in situ hybridization (GISH) was used to study the chromosomal composition of the advanced generations and showed that most plants were allotetraploids possessing the complete diploid sets of both parental species. Because artificial doubling of chromosomes of the interspecific hybrids was not used, spontaneous polyploidization likely resulted from restitution gametes or somatic doubling. Recombinant chromosomes between A. cepa and A. fistulosum were identified, revealing that introgression of disease resistances to bulb onion should be possible.

Transcriptome Analyses of Mosaic (MSC) Mitochondrial Mutants of Cucumber in a Highly Inbred Nuclear Background.

Cucumber (Cucumis sativus L.) has a large, paternally transmitted mitochondrial genome. Cucumber plants regenerated from cell cultures occasionally show paternally transmitted mosaic (MSC) phenotypes, characterized by slower growth, chlorotic patterns on the leaves and fruit, lower fertility, and rearrangements in their mitochondrial DNAs (mtDNAs). MSC lines 3, 12, and 16 originated from different cell cultures all established using the highly inbred, wild-type line B. These MSC lines possess different rearrangements and under-represented regions in their mtDNAs. We completed RNA-seq on normalized and non-normalized cDNA libraries from MSC3, MSC12, and MSC16 to study their nuclear gene-expression profiles relative to inbred B. Results from both libraries indicated that gene expression in MSC12 and MSC16 were more similar to each other than MSC3. Forty-one differentially expressed genes (DEGs) were upregulated and one downregulated in the MSC lines relative to B. Gene functional classifications revealed that more than half of these DEGs are associated with stress-response pathways. Consistent with this observation, we detected elevated levels of hydrogen peroxide throughout leaf tissue in all MSC lines compared to wild-type line B. These results demonstrate that independently produced MSC lines with different mitochondrial polymorphisms show unique and shared nuclear responses. This study revealed genes associated with stress response that could become selection targets to develop cucumber cultivars with increased stress tolerance, and further support of cucumber as a model plant to study nuclear-mitochondrial interactions.

The selection of mosaic (MSC) phenotype after passage of cucumber (Cucumis sativus L.) through cell culture - a method to obtain plant mitochondrial mutants.

Mosaic (MSC) mutants of cucumber (Cucumis sativus L.) appear after passage through cell cultures. The MSC phenotype shows paternal transmission and is associated with mitochondrial DNA rearrangements. This review describes the origins and phenotypes of independently produced MSC mutants of cucumber, including current knowledge on their mitochondrial DNA rearrangements, and similarities of MSC with other plant mitochondrial mutants. Finally we propose that passage of cucumber through cell culture can be used as a unique and efficient method to generate mitochondrial mutants of a higher plant in a highly homozygous nuclear background.