Microsatellite fingerprinting in lettuce (Lactuca sativa L.) and wild relatives

Southern hybridisation with a single microsatellite probe, (TCT)₁₀, sufficed to discriminate between a representative set of cultivars/accessions of lettuce, Lactuca sativa L., and its wild relatives L. serriola, L. saligna and L. virosa. Variability within cultivars was tested in a relatively modern cultivar (Hector), where no variation was found, and in an older and morphologically more variable cultivar (Madrilene), where heterogeneity was observed in the TCT fingerprint. (TCT)₁₀ fingerprinting should be useful for variety identification and homogeneity testing in lettuce. Received: 25 July 1997 / Revision received: 5 August 1997 / Accepted: 30 August 1997     

Interaction of [3H]gibberellin A1 with a sub-cellular fraction from lettuce (Lactuca sativa L.) hypocotyls

The relationship between protein synthesis and the incorporation of [³H]gibberellin A₁ ([³H]GA₁) into a 2,000xg pelletable (2KP) fraction from lettuce (Lactuca sativa L.) hypocotyl sections has been investigated. Concentrations of D-2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide (MDMP) between 10^-7 M and 10^-4 M caused increasing inhibition of growth, 2KP labelling and incorporation of [¹⁴C]leucine into soluble protein. Growth and 2KP radioactivity were highly correlated (r=0.996). Transfer to MDMP early or late in the course of GA response caused reductions in both growth and incorporation into the 2KP fraction. Exposure to the inhibitor had more effect at 4 h than at 20 h. The proportions of alkali-soluble and insoluble radioactivity in the 2KP fraction were also altered by this treatment. The implications of these findings are discussed.Abbreviations GA₁ gibberellin A₁ - MDMP D-2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide - 2KP a2,000xg pelletable fraction     

Interaction of [3H]gibberellin A1 with a sub-cellular fraction from lettuce (Lactuca sativa L.) hypocotyls

The relationship between elongation growth and the incorporation of [³H]gibberellin A₁ ([³H]GA₁) into a 2,000g pelletable (2KP) fraction from lettuce (Lactuca sativa L., cv. Arctic) hypocotyl sections has been examined. Sections were loaded with incremental amounts of GA₁ under conditions where growth was arrested (5° C) or permitted (30° C) and, after 16 h, all were transferred to a GA-free medium at 30° C. Growth and 2KP radioactivity were measured at this point and after a further 24 h in the chase medium. Uptake was reduced by 80% at 5° C, as compared to 30° C, but 2KP labelling and protein synthesis were only reduced by half. The growth rate of the 5° C pretreated sections during the chase period was comparable to that observed during the pulse in the 30° C material but the dose/response relationship was flatter. Low temperature sections incorporated a much higher percentage of GA₁ uptake into the 2KP fraction (27% at maximum) but the absolute levels of labelling at this temperature were lower than those measured at 30° C. The data are interpreted as showing that 2KP labelling is not a consequence of growth. It must either precede response or be an unconnected concurrent process.     

Production of hairy root cultures of lettuce (Lactuca sativa L.)

Hairy root cultures of lettuce (Lactuca sativa L.) were obtained by inoculation of cotyledonary leaves of in vitro lettuce seedlings (cvs. Nansen and Ljubljanska ledenka) with Agrobacterium rhizogenes A4M70GUS. Approximately in 96.7% cvs. Nansen and in 91.2% Ljubljanska ledenka inoculated explants produced hairy root when they were incubated on Murashige and Skoog (MS) half-strength medium without plant growth regulators. A total of 54% of all hairy root cultures expressed GUS activity. Every hairy root represented an independent transformation event. Line Ljubljanska ledenka 18 showed the highest biomass (5.5 times the biomass of control root). A PCR analysis of the genomic DNA confirmed the presence of marker and target genes in 15 hairy roots examined.     

Cloning and expression of sesquiterpene synthase genes from lettuce (Lactuca sativa L.)

Sesquiterpenoid lactones (SLs) from lettuce (Lactuca sativa L.) include constitutive components of latex such as lactucin and the induced phytoalexin, lettucenin A. A redundant primer strategy was used to recover two full length cDNA clones (LTC1 and LTC2) encoding sesquiterpene synthases from a cDNA library derived from seedlings with the red spot disorder, which accumulate phytoalexins. Recombinant enzymes produced from LTC1 and LTC2 in Escherichia coli catalysed the cyclisation of farnesyl diphosphate to germacrene A, potentially an early step in the biosynthesis of SLs. RT-PCR analysis showed LTC1 and LTC2 were expressed constitutively in roots, hypocotyls and true leaves but not in cotyledons. Expression in cotyledons was induced by challenge with the downy mildew pathogen Bremia lactucae in the disease resistant cultivar Diana. Southern hybridisation experiments showed that LTC1 and LTC2 were not part of a multigene family. The germacrene A synthases provide targets for modified expression to generate beneficial modifications to the SL profile in lettuce.     

Tipburn in salt-affected lettuce (Lactuca sativa L.) plants results from local oxidative stress

Tipburn in lettuce is a physiological disorder expressed as a necrosis in the margins of young developing leaves and is commonly observed under saline conditions. Tipburn is usually attributed to Ca²⁺ deficiencies, and there has very limited research on other mechanisms that may contribute to tipburn development. This work examines whether symptoms are mediated by increased reactive oxygen species (ROS) production.Two butter lettuce (Lactuca sativa L.) varieties, Sunstar (Su) and Pontina (Po), with contrasting tipburn susceptibility were grown in hydroponics with low Ca²⁺ (0.5 mM), and with or without 50 mM NaCl. Tipburn symptoms were observed only in Su, and only in the saline treatment. Tipburn incidence in response to topical treatments with Ca²⁺ scavengers, Ca²⁺ transport inhibitors, and antioxidants was assessed. All treatments were applied before symptom expression, and evaluated later, when symptoms were expected to occur. Superoxide presence in tissues was determined with nitro blue tetrazolium (NBT) and oxidative damage as malondialdehyde (MDA) content. Superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activities were assayed.Under control and saline conditions, tipburn could be induced in both varieties by topical treatments with a Ca²⁺ scavenger (EGTA) and Ca²⁺ transport inhibitors (verapamil, LaCl₃) and reduced by supplying Ca²⁺ along with a ionophore (A 23187). Tipburn symptoms were associated with locally produced ROS. O₂⁻ and oxidative damage significantly increased in leaf margins before symptom expression, while topical antioxidant applications (Tiron, DPI) reduced symptoms in treated leaves, but not in the rest of the plant. Antioxidant enzyme activity was higher in Po, and increased more in response to EGTA treatments, and may contribute to mitigating oxidative damage and tipburn expression in this variety.     

Development of EST-SSR markers for the study of population structure in lettuce (Lactuca sativa L.)

A set of 61 simple sequence repeat (SSR) markers was developed from the 19,523 Lactuca sativa and Lactuca serriola unigenes. Approximately 4.5% of the unigenes contained a perfect SSR at least 20 bp long, corresponding to roughly 1 perfect SSR per 14.7 kb. Marker polymorphism was tested on a set comprising 96 accessions representing all major horticultural types and 3 wild species (L. serriola, Lactuca saligna, and Lactuca virosa). Both the average marker heterozygosity (UHe = 0.32) and the number of different alleles per locus (Na = 3.56) were significantly reduced in expressed sequence tag (EST)-SSRs as compared with anonymous SSRs (UHe = 0.59, Na = 5.53). Marker transfer rate to the wild species corresponded to the decreasing sexual compatibility with L. sativa and was higher for EST-SSRs (100% L. serriola, 87% L. saligna, and 75% L. virosa) than for anonymous SSRs (93%, 66%, and 42%, respectively). Assessment of population structure among 90 L. sativa cultivars with SSRs was in good agreement with classification into the horticultural types. The average marker heterozygosity was smallest in iceberg (0.097), Latin (0.140), and romaine-type (0.151) cultivars while highest in leaf (green leaf 0.208 and red leaf 0.240) lettuces. The level of marker heterozygosity is in accord with morphological variability observed in different horticultural types.     

A note on the measurement of genetic diversity within genebank accessions of lettuce (Lactuca sativa L.) using AFLP markers

This paper discusses a statistical approach for measuring genetic diversity within genebank accessions of a self-fertilising species. This approach is applied to lettuce (Lactuca sativa L.), using AFLP marker data on a set of 1,390 accessions, representing six different lettuce types. Knowledge of the within-accession genetic diversity is important for decisions about the way accessions have to be maintained by genebanks. It is argued that if the within-accession diversity is small, as can be expected for a self-fertilising species like L. sativa, the best approach is to sample as many accessions as possible with only two plants per accession and estimate the within-accession diversity by the proportion of accessions of which the individuals are different.     

Interactions of Cd with Zn, Cu, Mn and Fe for lettuce (Lactuca sativa L.) in hydroponic culture

Abstract. The effect and accumulation of cadmium (Cd) in lettuce grown by means of the hydroponic technique was investigated by multivariate analysis, and was found to be affected by the concentration of other trace elements. Particularly iron acted in a strongly antagonistic way against Cd. Consequently, no absolute toxicity limits for Cd can be drawn without considering other trace elements.     

Expression analysis of polyphenol oxidase isozymes by active staining method and tissue browning of head lettuce (Lactuca sativa L.)

Browning of plant tissue is generally considered attributable to enzymatic oxidation by polyphenol oxidase (PPO). Electrophoresis followed by activity staining has been used as an effective procedure to visually detect and isolate isozymes; however, it has not been applied for examination of various PPO isozymes in lettuce. Our study demonstrated that different lettuce PPO isozymes could be detected at different pH in active staining, and multiple isozymes were detected only under alkaline conditions. As a result, we concluded that activity staining with approximately pH 8 enabled to detect various PPO isozymes in lettuce. By expression analysis of the PPO isozymes after wounding, PPO isozymes that correlated with time-course of tissue browning were detected. The wound-induced PPO may play a key role in enzymatic browning.     

A high-density, integrated genetic linkage map of lettuce (Lactuca spp.)

An integrated map for lettuce comprising of 2,744 markers was developed from seven intra- and inter-specific mapping populations. A total of 560 markers that segregated in two or more populations were used to align the individual maps. 2,073 AFLP, 152 RFLP, 130 SSR, and 360 RAPD as well as 29 other markers were assigned to nine chromosomal linkage groups that spanned a total of 1,505 cM and ranged from 136 to 238 cM. The maximum interval between markers in the integrated map is 43 cM and the mean interval is 0.7 cM. The majority of markers segregated close to Mendelian expectations in the intra-specific crosses. In the two L. saligna × L. sativa inter-specific crosses, a total of 155 and 116 markers in 13 regions exhibited significant segregation distortion. Data visualization tools were developed to curate, display and query the data. The integrated map provides a framework for mapping ESTs in one core mapping population relative to phenotypes that segregate in other populations. It also provides large numbers of markers for marker assisted selection, candidate gene identification, and studies of genome evolution in the Compositae. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Respiration of Grand Rapids lettuce (Lactuca sativa L.) seeds in relation to chemical and photocontrol of germination

A 10-min irradiation with red light (R) of ‘Grand Rapids’lettuce (Lactuca sativa L.) seeds increased respiratory rates(QO₂ and QCO₂) over those of far red (FR)-irradiated seeds within2 to 3 hr. Differences in respiration between R- and FR-irradiatedseeds became more pronounced with time. The respiration, indarkness at 25?C, of seeds air dried after germination promotingimpregnation treatments with 1.0 mM GA₃+0.5 mM kinetin in acetonewas stimulated slightly by the 6th hour after planting and markedlyby the 12th and 24th hour. Results with seeds in GA₃+kinetindissolved in water were similar. ABA, which inhibits germinationand seedling growth, prevented stimulation in respiration oflight-induced seeds. ABA also repressed respiration when usedwith GA₃+kinetin. The germination and growth retardant, isopropylN-(3-chlorophenyl) carbamate (CIPC), did not affect the respirationof either R- or FR-treated seeds. A comparison of the effectsof chemical and light treatments on respiration indicate thatchemical and photocontrol mechanisms are not identical. (Received May 17, 1976; )     

lyc1, a transgenic mutant of Lactuca sativa (lettuce) tightly linked to chloroplast biogenesis.

A T-DNA tagging mutant of lettuce was studied. The transgenic lettuce mutant, designated light yellowish cotyledon1 (lyc1), exhibited a chloroplast mutant phenotype in which the chloroplast development was arrested at the cotyledon stage and the cotyledon became a light yellowish due to the reduced level of chlorophyll content. A microscopic observation of the mutant showed that the chloroplasts failed to position along the cell wall, but were scattered in the cytoplasm. The number of chloroplasts in the mutant was reduced to 66% compared to that in the wild-type plant. These results suggest that the lyc1 mutation is due to a defect in the biogenesis of chloroplasts. Also, the homozygous lyc1 mutant became lethal in a T2 and F1 progeny analysis, indicating that the mutation is recessive.     

Stage-specific crosstalk between light, auxin, and ethylene during low-pH-induced root hair formation in lettuce (Lactuca sativa L.) seedlings

In the light, transfer of lettuce seedlings precultured on liquid medium at pH 6.0 to fresh medium at pH 4.0 induces root hair formation. However, no root hairs form in the dark. Here, we investigated how light induces root hair formation. Randomization of the transverse cortical microtubule (CMT) arrays which occurs in root epidermal cells in the light prior to root hair initiation was not observed in the dark. However, addition of indole-3-acetic acid (IAA) or 1-aminocyclopropane-1-carboxylic acid (ACC) induced CMT randomization and root hair formation. In these cases, CMT randomization occurred in almost the same time-dependent manner as under light. However, root hair initiation was delayed for several hours in the dark. These results suggest that light promotes CMT randomization and root hair initiation via auxin and ethylene signaling but light additionally influences root hair initiation independently of these signaling mechanisms. Furthermore, addition of a microtubule-depolymerizing drug in the dark disrupted the transverse CMT arrays and initiated root hair formation; however, root hair elongation was still suppressed. Root hairs elongated when IAA or ACC was applied with the drug. These results suggest that light also promotes root hair elongation via auxin and ethylene signaling.     

A controlled-environment chamber for measurement of canopy photosynthesis by small stands of lettuce (Lactuca sativa L.)

Abstract. A controlled-environment chamber constructed from a standard chest freezer was used to grow and measure the CO₂ exchange of small stands of lettuce ( Lactuca sativa L. ev. Ambassador). The chamber, with horizontal air flow, provided good control of air temperature ( c. 6 to 16°C), irradiance (0–300 μmol PAR m ²S¹ and CO₂ (350–1000 μmol mol⁻¹). The photosynthetic response to changes in these variables was measured using an inexpensive CO₂ dosing system which recorded the input rate required to maintain a constant concentration of CO₂ (to ± 2.5%). Characteristis of the growth environment and the changes in response to temperature and irradiance are described.