Insight into morphological and molecular variations across Iranian spinach landraces.

Spinach is salt-tolerant leafy vegetable stemmed from central Asia. It has been well adapted to different climates of Iran. We aimed to study genetic diversity between several landraces as a prerequisite for crop improvement programs using molecular markers including microsatellites (SSRs) and morphological traits. Genetic diversity was studied among 22 spinach landraces using morphological and molecular tools. We developed 17 genic and genomic SSR markers based on the information acquired from NCBI resources. Morphological evaluation indicated high variability for economic traits including leaf color, leaf thickness, leaf wrinkle and seed type, across Iranian landraces. The molecular results exhibited that 12 out of 17 primer pairs successfully amplified genomic DNA with explicit bands. The results verified that genic markers were superior to genomic markers to detect polymorphism and genetic diversity. In this regard, PIC for genomic and genic SSRs was in the range of 0.4616-0.6621 and 0.5188-0.7394, respectively. Polymorphic genic SSRs were identified to be directly and indirectly involved in biotic/abiotic stresses. High degree of polymorphism, which was detected across the landraces by genic SSRs, could assist us to select 11 landraces for the second experiment. The second experiment was designed to evaluate the response of selected landraces to salinity stress. The results confirmed genetic variability among the landraces in terms of salinity tolerance. A highly diverse germplasm of Iranian spinach based on molecular and morphological characteristics along with the tolerance to oxidative stress provides an ample opportunity for plant breeders to select superior genotypes.

Genetic diversity and association analysis of leafminer (Liriomyza langei) resistance in spinach (Spinacia oleracea).

Leafminer (Liriomyza langei) is a major insect pest of many important agricultural crops, including spinach (Spinacia oleracea). Use of genetic resistance is an efficient, economic, and environment-friendly method to control this pest. The objective of this research was to conduct association analysis and identify single nucleotide polymorphism (SNP) markers associated with leafminer resistance in spinach germplasm. A total of 300 USDA spinach germplasm accessions were used for the association analysis of leafminer resistance. Genotyping by sequencing (GBS) was used for genotyping and 783 SNPs from GBS were used for association analysis. The leafminer resistance showed a near normal distribution with a wide range from 1.1 to 11.7 stings per square centimeter leaf area, suggesting that the leafminer resistance in spinach is a complex trait controlled by multiple genes with minor effect in this spinach panel. Association analysis indicated that five SNP markers, AYZV02040968_7171, AYZV02076752_412, AYZV02098618_4615, AYZV02147304_383, and AYZV02271373_398, were associated with the leafminer resistance with LOD 2.5 or higher. The SNP markers may be useful for breeders to select plants and lines for leafminer resistance in spinach breeding programs through marker-assisted selection.

Effect of spinach cultivar and bacterial adherence factors on survival of Escherichia coli O157:H7 on spinach leaves.

Similar to phytopathogens, human bacterial pathogens have been shown to colonize the plant phylloplane. In addition to environmental factors, such as temperature, UV, relative humidity, etc., the plant cultivar and, specifically, the leaf blade morphological characteristics may affect the persistence of enteropathogens on leafy greens. This study was conducted to evaluate the effect of cultivar-dependent leaf topography and the role of strain phenotypic characteristics on Escherichia coli O157:H7 persistence on organic spinach. Spinach cultivars Emilia, Lazio, Space, and Waitiki were experimentally inoculated with the foodborne E. coli O157:H7 isolate EDL933 and its isogenic mutants deficient in cellulose, curli, or both curli and cellulose production. Leaves of 6-week-old plants were inoculated with 6.5 log CFU per leaf in a biosafety level 2 growth chamber. At 0, 1, 7, and 14 days, E. coli O157:H7 populations were determined by plating on selective medium and verified by laser scanning confocal microscopy. Leaf morphology (blade roughness and stoma density) was evaluated by low-temperature and variable-pressure scanning electron microscopy. E. coli O157:H7 persistence on spinach was significantly affected by cultivar and strain phenotypic characteristics, specifically, the expression of curli. Leaf blade roughness and stoma density influenced the persistence of E. coli O157:H7 on spinach. Cultivar Waitiki, which had the greatest leaf roughness, supported significantly higher E. coli O157:H7 populations than the other cultivars. These two morphological characteristics of spinach cultivars should be taken into consideration in developing intervention strategies to enhance the microbial safety of leafy greens.

Effect of cultivar on chemical composition of some Iranian spinach.

This research was carried out to determine fatty acids, phenolic compounds, oxalic acid, protein, fat, total fiber and mineral elements of Iranian spinach cultivars (Varamin 1, Varamin 2, Varamin 3, Zabol, Kashan, Qom and Zanjan). Results showed that cultivar Zabol had the highest protein (2.59%). The highest fat percentage, phenolic compounds, P, linoleic acid (18:2) and the lowest oxalic acid were found in cultivar Kashan. Cultivar Varamin 2 had the highest total fiber (2.22%). Dry matter, K, Ca, Cu and Zn contents of cultivar Zanjan were higher than the other cultivars. Cultivar Qom had the highest amounts of Mg and Fe. Cultivar Varamin 3 had the highest Na (93.8 mg/100 g fw). Cultivars Qom and Varamin l had the highest percentage of linolenic acid (18:3). Present results for chemical composition of studied cultivars, were similar to values reported by other researchers.

Characterization of SpAPETALA3 and SpPISTILLATA, B class floral identity genes in Spinacia oleracea, and their relationship to sexual dimorphism.

Floral organ identity B class genes are generally recognized as being required for development of petals and stamens in angiosperm flowers. Spinach flowers are distinguished in their complete absence of petals in both sexes, and the absence of a developed stamen whorl in female flowers. As such, we hypothesized that differential expression of B class floral identity genes is integral to the sexual dimorphism in spinach flowers. We isolated two spinach orthologs of Arabidopsis B class genes by 3' and 5' RACE. Homology assignments were tested by comparisons of percent amino acid identities, searches for diagnostic consensus amino acid residues, conserved motifs, and phylogenetic groupings. In situ hybridization studies demonstrate that both spinach B class genes are expressed throughout the male floral meristem in early stages, and continue to be expressed in sepal primordia in reduced amounts at later stages of development. They are also highly expressed in the third whorl primordia when they arise and continue to be expressed in these tissues through the development of mature anthers. In contrast, neither gene can be detected in any stage in female flowers by in situ analyses, although northern blot experiments indicate low levels of SpAP3 within the inflorescence. The early, strong expressions of both B class floral identity genes in male floral primordia and their absence in female flowers demonstrate that B class gene expression precedes the origination of third whorl primordia (stamen) in males and is associated with the establishment of sexual floral dimorphism as it initiates in the first (sepal) whorl. These observations suggest that regulation of B class floral identity genes has a role in the development of sexual dimorphism and dioecy in spinach rather than being a secondary result of organ abortion.