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     

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.     

Microsatellite retrieval in lettuce (Lactuca sativa L.).

By using enriched genomic libraries, microsatellite-containing sequences were isolated from lettuce (Lactuca sativa) with high efficiency. With this approach, a sizeable fraction (up to 55%) of the clones contained a microsatellite. In about half of these clones, primers could be designed for PCR amplification of the microsatellite. This yielded 28 primer sets amplifying unambiguously scorable products, of which 26 showed polymorphisms in a test set of six lettuce varieties. Practically all microsatellite-amplifying primer sets yielded products in lettuce's nearest relative, L. serriola, but only half of the primer sets yielded products in the more distant species L. saligna and L. virosa. An average polymorphism information content (PIC) value of 0.55 and an average number of 3.5 alleles per locus were in the normal range for a self-fertilizing species like lettuce. In addition, the incidental cloning of a microsatellite-containing repeat family, apparently specific for Lactuca, is reported and the implications for the efficient retrieval of single-locus microsatellite sequences are discussed. The microsatellite loci isolated will be useful for distinguishing lettuce cultivars and for screening diversity of genetic resources.     

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.     

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.     

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.     

纳米Fe3O4对生菜生长及土壤细菌群落结构的影响

纳米Fe₃O₄的广泛应用增加了其暴露到农田环境的可能性,因此亟待研究纳米Fe₃O₄对农业生态环境的影响.本研究采用盆栽试验方式,研究不同浓度纳米Fe₃O₄颗粒(1、10、100 mg·kg^-1)对生菜生长及土壤细菌群落的影响,并与相应浓度的普通Fe₃O₄处理进行对比.通过测定植物光合速率常数、植株Fe含量来评价植物生长;采用高通量测序技术研究土壤细菌群落结构及组成.结果表明: 不同浓度纳米Fe₃O₄的影响不同.低浓度纳米Fe₃O₄能提高植物生物量,增强植物叶片光合速率,增加土壤中黄单胞菌目的相对丰度,降低蓝细菌、鞘脂杆菌纲的相对丰度,但对群落多样性指数影响不显著.高浓度纳米Fe₃O₄抑制作物生长,提高植株中Fe积累及土壤电导率,降低细菌群落系统发育多样性,降低黄单胞菌目、鞘脂杆菌纲相对丰度,增加蓝细菌相对丰度.此外,一些土壤功能微生物对纳米Fe₃O₄及普通Fe₃O₄处理的响应也存在差异,说明不同粒径及浓度的Fe₃O₄均会对土壤微生物群落产生影响,并可能影响地上部分植物性状.因此,在评估纳米颗粒的生物学效应时需较多关注土壤微生物.     

Association analysis of bacterial leaf spot resistance and SNP markers derived from expressed sequence tags (ESTs) in lettuce (Lactuca sativa L.)

Bacterial leaf spot of lettuce, caused by Xanthomonas campestris pv. vitians, is a devastating disease of lettuce worldwide. Since there are no chemicals available for effective control of the disease, host-plant resistance is highly desirable to protect lettuce production. A total of 179 lettuce genotypes consisting of 29 leaf, 15 crisphead, one stem, 21 romaine, and 113 butterhead types were evaluated for response to X. c. vitians. One source of high resistance and five sources of moderate resistance were identified with four being butterhead lettuce and two leaf lettuce. The population genetic structure based on 350 expressed-sequence-tag-derived single nucleotide polymorphism generated two clades: Clade I and Clade II. The butterhead type was genetically distinct from romaine and crisphead types, while the leaf type was found to frequently exchange genes with other types through the history of breeding. Association mapping identified one single nucleotide polymorphism (QGB19C20.yg-1-OP5) associated with disease severity in Q general linear model and Q + K mixed linear model. Two SNP markers (Contig15389-1-OP1 and Contig6039-19-OP1) were associated with the resistance in the leaf lettuce PI 358000-1 which had no disease symptoms. The marker QGB19C20.yg-1-OP5 is in linkage group 2, while both of Contig15389-1-OP1 and Contig6039-19-OP1 are in linkage group 4. The resistant lines and the associated SNPs should be useful to develop resistant cultivars to battle against the devastating disease in lettuce.     

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; )     

Development of EST-SSR markers for genetic diversity analysis in coconut (Cocos nucifera L.)

Molecular Biology Reports - Genetic improvement in coconut relies on exploiting the vast existing diversity among coconut accessions. Robust molecular markers are a pre-requisite for efficient...     

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.     

Randomization of cortical microtubules in root epidermal cells induces root hair initiation in lettuce (Lactuca sativa L.) seedlings

Root hair formation is induced when lettuce seedlings are transferred from liquid medium at pH 6.0 to fresh medium at pH 4.0. If seedlings are transferred to pH 6.0, no root hairs are formed. We investigated the role of microtubules in this low pH-induced root hair initiation in lettuce. At the hair-forming zone in root epidermal cells, microtubules were perpendicular to the longitudinal axis of the cell just after pre-culture. This arrangement became disordered as early as 5 min after transfer to pH 4.0, and became random by 30 min later. At pH 4.0, the randomization extended to the entire hair-forming zone of seedlings; at pH 6.0, however, randomization did not occur and transverse microtubules were maintained. When seedlings at pH 6.0 were treated with microtubule-depolymerizing drugs, root hairs were formed. In contrast, when a microtubule-stabilizing drug, taxol, was added to the medium, no root hairs formed, even at pH 4.0. These results suggest that the transverse cortical microtubules inhibit root hair formation, and that their destruction is necessary for initiation. Furthermore, the microfilament-disrupting drugs cytochalasin B and latrunculin B inhibited root hair initiation, suggesting that actin filaments are necessary for root hair initiation.     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加。同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少。伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后。小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡。体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大液泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

Cell-Wall Autohydrolysis in Isolated Endosperms of Lettuce (Lactuca sativa L.)

Cell walls prepared from the endosperm tissue of hydrated lettuce (Lactuca sativa L.) seeds undergo autohydrolysis. Release of carbohydrates is most rapid (0.4-0.6 [mu]g per endosperm) within the 1st h of incubation in buffer, but substantial autolysis is sustained for at least 10 h. Autolysis is temperature sensitive, and the optimum rate occurs at pH 5. The rate of autolysis increases markedly in the period just prior to radicle emergence. The cell-wall polysaccharide composition in micropylar and lateral endosperm regions differs significantly; the micropylar walls are rich in arabinose and glucose with substantially lower amounts of mannose. Although walls prepared from both micropylar and lateral regions undergo autolysis, micropylar walls release carbohydrates at a higher rate than lateral walls. Autolysis products elute as large polymers when subjected to size-exclusion chromatography, suggesting that endo-enzyme activity is responsible for release of fragments containing arabinose, galactose, mannose, and uronic acids. Arabinose, galactose, mannose, and glucose are also released as monomers. As a function of time, the ratio of polymers to monomers decreases, indicating that exo-enzyme activity is also present. Thermoinhibition or treatment with abscisic acid suppresses germination and reduces the rates of autolysis of walls isolated from the endosperm by about 25%. Treatments that alleviate thermoinhibition (kinetin and gibberellic acid) increase the rates of autolysis by 20 to 30% when compared to thermoinhibited controls.     

Development of Stomata in Lactuca sativa L.

The structure of the epidermis on various parts of the plantof Lactuca sativa Linn. is described. Development of stomatais studied in young leaves and stems. Tognini (1897) has previouslymentioned that the stomata on stems of L. virosa develop froma meristemoid with four cutting faces but the present studypoints out that the meristemoid cuts off only two mesogene neighbouringcells and the two others are perigene.