Transferability of Cucurbita SSR markers for genetic diversity assessment of Turkish bottle gourd (Lagenaria siceraria) genetic resources

The genetic diversity present in crop landraces represents a valuable genetic resource for breeding and genetic studies. Bottle gourd (Lagenaria siceraria) landraces in Turkey are highly genetically diverse. However, the limited genomic resources available for this crop hinder the molecular characterization of Turkish bottle gourd germplasm for its adequate conservation and management. Therefore, we evaluated the efficacy of 40 SSR markers from major cucurbit crops (Cucurbita pepo L. and Cucurbita moschata L.) in 30 bottle gourd landraces, together with 16 SRAP primer combinations. In addition, we compared the genetic relationship between bottle gourd and 31 other cucurbit accessions (11 Cucurbita maxima, 3 C. moschata, 5 C. pepo subsp. ovifera, 10 C. pepo and 2 Luffa cylindrica). Twenty-seven Cucurbita SSR markers showed transferability to bottle gourd. SSR markers amplified 59 alleles, in bottle gourd genome with an average of 1.64 alleles per locus. Together, SSR and SRAP markers amplified 453 fragments across the 61 accessions, and clearly discriminated L. siceraria and L. cylindrica from the other cucurbit species. Genetic diversity analysis separated edible cucurbit from ornamentals, while population structure analysis classified L. siceraria in two subpopulations defined by fruit shape, rather than geographical origin. The results indicated that the genomic resources available for Cucurbita species are valuable to study and preserve the genetic diversity of bottle gourd in Turkey.     

Resistance to Tomato leaf curl New Delhi virus in Cucurbita spp.

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (family Geminiviridae) first reported in India and its neighbouring countries. ToLCNDV severely affects zucchini crop (Cucurbita pepo) in the main production areas of Southern Spain since 2012. This emerging begomovirus is a serious threat to this and other cucurbit crops. Breeding resistant cultivars is the most promising method for disease control, but requires the identification of sources of resistance in the Cucurbita genus. In this work, we screened for ToLCNDV resistance a large collection of Cucurbita spp. accessions, including landraces and commercial cultivars of the main cultivated species, C. pepo, Cucurbita moschata and Cucurbita maxima and wild species. The screening was performed using mechanical and whitefly inoculation. The level of resistance was assessed by scoring symptom severity and by measuring the virus content with quantitative polymerase chain reaction in selected genotypes. Diversity in the response was observed within and among species. Severe symptoms and high viral amounts were found at 30 days after mechanical and whitefly inoculation in C. pepo, in all accessions belonging to the Zucchini morphotype and to other morphotypes of both subspecies, pepo and ovifera, and even in the wild relative Cucurbita fraterna. C. maxima was also highly susceptible. This species showed characteristic symptoms of leaf decay and intense yellowing, different from those of mosaic, curling and internode shortening found in C. pepo. The only species showing resistance was C. moschata. Four accessions were symptomless or had some plants with only mild symptoms after three independent rounds of mechanical inoculation with different inoculum sources. Two of them also remained symptomless after virus inoculation with viruliferous whiteflies. ToLCNDV was detected in these asymptomatic accessions at 15 and 30 days post inoculation, but viral amounts were much lower than those found in susceptible genotypes, suggesting a high level of resistance. The symptoms in the susceptible accessions of this species were also different, with a characteristic leaf mottling, evolving to a severe mosaic. The newly identified C. moschata resistant accessions are good candidates for breeding programmes to avoid the damage caused by ToLCNDV.     

Inhibition of geranylgeranyl diphosphate synthesis in in vitro systems

The incorporation of [¹⁴C]mevalonate and [¹⁴C]isopentenyl diphosphate into geranylgeranyl diphosphate was investigated in in vitro systems from Cucurbita pepo (pumpkin) endosperm and from Avena sativa etioplasts. Mevalonate incorporation was effectively inhibited in the pumpkin system by geranylgeranyl diphosphate and geranylgeranyl monophosphate but less effectively by phytyl diphosphate or inorganic diphosphate. Membrane lipids, geranyllinalool, or lecithin enhanced mevalonate incorporation in the Cucurbita system. Incorporation of isopentenyl diphosphate was also enhanced by lecithin and inhibited by geranylgeranyl diphosphate in the Cucurbita system. No lipid enhancement was found in the Avena system; inhibition by GGPP required a much higher GGPP concentration than in the Cucurbita system.     

Cadmium induced changes in subcellular glutathione contents within glandular trichomes of Cucurbita pepo L.

Plants cope with cadmium (Cd) stress by complexation with phytochelatins (Pc), metallothioneins and glutathione and sequestration within vacuoles. Especially glutathione was found to play a major role in Cd detoxification as Cd shows a high binding affinity towards thiols and as glutathione is a precursor for Pc synthesis. In the present study, we have used an immunohistochemical approach combined with computer-supported transmission electron microscopy in order to measure changes in the subcellular distribution of glutathione during Cd-stress in mesophyll cells and cells of different glandular trichomes (long and short stalked) of Cucurbita pepo L. subsp. pepo var. styriaca Greb. Even though no ultrastructural alterations were observed in leaf and glandular trichome cells after the treatment of plants with 50 µM cadmium chloride (CdCl₂) for 48 h, all cells showed a large decrease in glutathione contents. The strongest decrease was found in nuclei and the cytosol (up to 76%) in glandular trichomes which are considered as a major side of Cd accumulation in leaves. The ratio of glutathione between the cytosol and nuclei and the other cell compartments was strongly decreased only in glandular trichomes (more than 50%) indicating that glutathione in these two cell compartments is especially important for the detoxification of Cd in glandular trichomes. Additionally, these data indicate that large amounts of Cd are withdrawn from nuclei during Cd exposure. The present study gives a detailed insight into the compartment-specific importance of glutathione during Cd exposure in mesophyll cells and glandular trichomes of C. pepo L. plants.     

Inheritance analysis and identification of SNP markers associated with ZYMV resistance in <Emphasis Type="Italic">Cucurbita pepo</Emphasis>

Cucurbit crops are economically important worldwide. One of the most serious threats to cucurbit production is Zucchini yellow mosaic virus (ZYMV). Several resistant accessions were identified in Cucurbita moschata and their resistance was introgressed into Cucurbita pepo. However, the mode of inheritance of ZYMV resistance in C. pepo presents a great challenge to attempts at introgressing resistance into elite germplasm. The main goal of this work was to analyze the inheritance of ZYMV resistance and to identify markers associated with genes conferring resistance. An Illumina GoldenGate assay allowed us to assess polymorphism among nine squash genotypes and to discover six polymorphic single-nucleotide polymorphisms (SNPs) between two near-isogenic lines, “True French” (susceptible to ZYMV) and Accession 381e (resistant to ZYMV). Two F₂ and three BC₁ populations obtained from crossing the ZYMV-resistant Accession 381e with two susceptible ones, the zucchini True French and the cocozelle “San Pasquale,” were assayed for ZYMV resistance. Molecular analysis revealed an approximately 90% association between SNP1 and resistance, which was confirmed using High Resolution Melt (HRM) and a CAPS marker. Co-segregation up to 72% in populations segregating for resistance was observed for two other SNP markers that could be potentially linked to genes involved in resistance expression. A functional prediction of proteins involved in the resistance response was performed on genome scaffolds containing the three SNPs of interest. Indeed, 16 full-length pathogen recognition genes (PRGs) were identified around the three SNP markers. In particular, we discovered that two nucleotide-binding site leucine-rich repeat (NBS-LRR) protein-encoding genes were located near the SNP1 marker. The investigation of ZYMV resistance in squash populations and the genomic analysis performed in this work could be useful for better directing the introgression of disease resistance into elite C. pepo germplasm.     

Parallel Evolution Under Domestication and Phenotypic Differentiation of the Cultivated Subspecies of <Emphasis Type="Italic">Cucurbita pepo</Emphasis> (Cucurbitaceae)

Parallel Evolution under Domestication and Phenotypic Differentiation of the Cultivated Subspecies of Cucurbita pepo (Cucurbitaceae). Cucurbita pepo (pumpkin, squash, gourd, Cucurbitaceae) is an ancient North American domesticate of considerable economic importance. Based on molecular genetic polymorphisms, two cultivated lineages of this species, each consisting of very many edible–fruited cultigens, have been recognized, C. pepo subsp. pepo and C. pepo subsp. texana. However, the phenotypic commonalities and differences between these two subspecies have not as yet been systematically collected and organized. Among the evolutionary developments common to the two subspecies are the increased size of the plant parts, less plant branching, and premature loss of chlorophyll in the exocarp of the fruits. In both subspecies, bush growth habit, conferred by allele Bu, is common to the cultigens grown for consumption of the immature fruits, as is the deviation from the 1:1 ratio of fruit length to fruit width. A major characteristic differentiating between the edible–fruited cultigens of the respective subspecies are the longitudinal protrusions, in subsp. pepo, versus depressions, in subsp. texana, of the fruit surface corresponding with the subsurface primary carpellary vein tracts. Subsp. pepo also has larger fruits and larger and longer seeds. In addition, some alleles affecting stem color, leaf mottling, multiple flower bud production, and fruit characteristics are frequently occurring to nearly fixed in one subspecies but are rare to less common in the other.