Echinocystis lobata mityba azotu priklausomai nuo buveinės

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Molecular markers search for evaluation of genetic diversity of wild cucumber (Echinocystis lobata (Michx.) Torr. & A. Gray)

Dygliavaisis virkštenis (Echinocystis lobata (Michx.) Torr. & A. Gray) Lietuvoje ir daugelyje pasaulio vietų – pripažintas invazine rūšimi ir laikoma viena iš daugelio pavojingiausių Europoje. Gali būti auginamas kaip dekoratyvinis augalas, dėl savo žiedų ir spygliuotų, dekoratyviai atrodančių vaisių. Dygliavaisis virkštenis – žolinis, vienametis augalas, dažniausiai augantis drėgnose upių, ežerų ir kitų vandens telkinių pakrantėse. Darbo tikslas buvo atlikti molekulinių žymenų paiešką Lietuvos paupių dygliavaisio virkštenio (Echinocystis lobata) populiacijų molekulinės įvairovės vertinimui. Populiacijų paieška ir individų rinkimas tyrimams vyko 2015–2016 metais. Iš viso Lietuvoje rastos 35 dygliavaisio virkštenio populiacijos. Iš visų populiacijų tyrimams buvo surinkti 342 individai. Dygliavaisio virkštenio DNR išskyrimui buvo išbandyti trys metodai: CTAB metodas, universalus genominės DNR išskyrimo rinkinys „Genomic DNA purification kit“ K0512, papildomai DNR priemaišas valant sorbitoliu ar kolonėlėmis (OneStep™ PCR Inhibitor Removal Kit). Išbandžius skirtingus DNR išskyrimo metodus buvo parinktas tinkamiausias Echinocystis lobata DNR išskyrimo būdas – naudojant gamyklinį DNR išskyrimo rinkinį „Genomic DNA purification kit“ K0512 (Thermo Scientific, Lietuva). Pagal naudotą skirtingą augalo sausosios masės (s. m.) kiekį, įvertinta išskirtos DNR koncentracija ir jos grynumas. Atrinktu metodu dygliavaisio virkštenio DNR išskirta iš visų 35 tiriamų populiacijų rastų prie įvairių Lietuvos upių. Pirmiausiai buvo bandoma pritaikyti mikrosatelitų (PKS) pradmenis. Tam buvo pasirinktos artimiausių dygliavaisiam virkšteniui Cucurbitaceae šeimos augalų tyrimams naudotos 29 mikrosatelitų pradmenų poros. Pradmenų poros (N1, N6, N12, S9, S13, S15, S26) buvo sukurtos ir panaudotos raukšlėtojo svaidenio (Momordica charantia) tyrimuose, pritaikius reakcijos sąlygas ir pradmenų prikibimo temperatūras iš šių porų tiko 6. Kitos 22 poros buvo paprastojo agurko (Cucumis sativus) ir atlikus šių pradmenų pritaikymo bandymus, teigiamus rezultatus davė 11 pradmenų porų (SSR05723, SSR23220, SSR23370, SSR05012, SSR07543, SSR19998, SSR16068, SSR31399, SSR20218, SSR29620, SSR11340). Remiantis užsienio literatūra apie Cucurbitaceae šeimos augalų tyrimus, pagausintų fragmentų ilgio polimorfizmams tirti buvo išbandytos 8 PFIP pradmenų poros. Visos jos davė teigiamus rezultatus, todėl jų pagalba išanalizuota 6 populiacijų, iš trijų šalių, dygliavaisio virkštenio genetinė įvairovė. Su visomis 8 pradmenų poromis buvo gauti polimorfiniai lokusai. Bendras visų populiacijų polimorfizmas lygus 47,82 %. Didžiausias Šenono informacijos indeksas buvo Lapių (0,363) populiacijoje. Ši populiacija pasižymėjo ir kraštutinėmis Nei genetinės įvairovės vertėmis, atitinkamai 0,253 ir 0,120. Genetiniai atstumai tarp E. lobata populiacijų kito nuo 0,126 iki 0,265. Genetinės įvairovės procentas tarp populiacijų buvo daug mažesnis (9,22 %), nei populiacijų viduje (90,78 %). Įvertinta genetinė įvairovė gana maža, ФPT = 0,092. Bajeso tikimybių analizės duomenimis tirtos dygliavaisio virkštenio populiacijos sudaro tris klasterius. Remiantis užsienyje atliktais sekoskaitos tyrimais buvo išbandytos 15 pradmenų porų taikant skirtingas PGR reakcijos programas, pritaikant pradmens prikibimo temperatūrą kiekvienai pradmenų porai. Pabandžius pritaikyti Lietuvos dygliavaisio virkštenio populiacijoms šias pradmenų poras su 7 poromis gauti teigiami rezultatai, todėl šios pradmenų poros naudojamos tolimesniems sekoskaitos tyrimams.Wild cucumber – herbal, annual plant, usually growing in moist rivers, lakes and other coast. It is native in North America. It can be grown as an ornamental plant for its flowers and spiny, ornamental fruits. Wild cucumber (Echinocystis lobata) in Lithuania and in other parts of the Europe is recognized as invasive species and considered to be one of the most dangerous according to the spread along continent. The objective of present study was to search for molecular markers for evaluation of genetic diversity of wild cucumber (Echinocystis lobata (Michx.) Torr. & A. Gray) in Lithuanian populations. Populations and individuals search and collection for the studies took place in 2015–2016. At total 35 wild cucumber populations were found in Lithuania. Total number of 342 individuals out of all populations was collected. Wild cucumber DNA extraction was performed following three methods: CTAB method, universal genomic DNA extraction kit “Genomic DNA purification kit, K0512”, additional DNA contaminant cleaning with sorbitol or columns (OneStep™ PCR Inhibitor Removal Kit) also was done. Comparison of DNA quantities and quality revealed that among tested different DNA extraction methods, the best Echinocystis lobata DNA extraction method was to apply “Genomic DNA purification kit K0512“ (Thermo Scientific, Lithuania) and follow protocol suggested by company. Extracted DNA concentration and purity was tested with the purpose to select the most favourable dry mass (d. m.) of the plant. DNA extraction was applied for individuals of 35 populations of wild cucumber collected in various riverbank places near various Lithuanian rivers. Firstly, selection and adaptation of the microsatellite (SSR) primers was tested. For this purpose, 29 microsatellite primer pairs were chosen based on the usage for closest plants in Cucurbitaceae family. The primers pairs (N1, N6, N12, S9, S13, S15, S26) were created and used in the studies of Momordica charantia. The best conditions of polymerase chain reaction were determined and 6 pairs showed positive results. The other 22 pairs were selected of Cucumis sativus and after applying best PCR conditions 11 primer pairs (SSR05723, SSR23220, SSR23370, SSR05012, SSR07543, SSR19998, SSR16068, SSR31399, SSR20218, SSR29620, SSR11340) gave positive results. Based on foreign literature on Cucurbitaceae family plant studies, eight AFLP primer pairs were tested. Polymorphic loci have been obtained with all 8 primer pairs. The genetic diversity of 6 populations from three countries was analysed. The mean polymorphism of all populations is 47.8 %. The largest index of the Shannon information was in Lapės (0.363) population. This population was also marked by extreme Nei genetic diversity values of 0.253 and 0.120. Genetic distances between E. lobata populations ranged from 0.126 to 0.265. Percentage of genetic diversity among populations was much smaller (9.22 %) compared to inside of populations (90.78 %). Genetic diversity is relatively low, ФPT = 0,092. According to the Bayesian cluster analysis populations have three clusters. Based on sequencing studies carried out in foreign countries, 15 primer pairs were tested using different PCR reaction programs, adjusting annealing temperature for each primer pair. 7 pairs generated positive results and these primer pairs are used for further sequencing studies

Phenological characteristics of wild cucumber (Echinocystis lobata (Michx.) Torr. et A. Gray.) in Nemunas coastal populations

Wild cucumber – herbaceous, annual plant, usually growing in coastal habitats like banks of the rivers, around the lakes and other over moistured sites also wastelands. This plant is native in North America. It can be cultivated as an ornamental plant for its flowers and spiny, ornamental fruits. Wild cucumber (Echinocystis lobata) in Lithuania and in other parts of Europe is recognized as invasive species, and considered to be one of the most dangerous according to intensity of the spread along continent part with the temperate climate. The objective of present study was to perform comparative phenology of the wild cucumber (Echinocystis lobata) populations and to collect plant material. Population detection and sampling took place in April–September, 2015. For observations of the plant development during vegetation season 4 out of 25 riparian populations of the Nemunas basin were selected – below Kaunas hydroelectric power station (Kaunas, Panemunė district), before tributary Nevėžis flows into the Nemunas (Kaunas, Lampėdžiai district), and sites upper and lower than Jurbarkas city. Morphometric analyses were monthly performed at selected sites. Our analysis revealed that main and lateral shoot length (cm), averaged per plant has expanded from 62 cm in June to 1152 cm in August. The average length of the main stem (cm) in June–August has augment from 62 cm to 471 cm. The avarage number of plant tendrils augment from 5 to 69 during growing season. In the course of the growing season plant stem diameter has increased from 3.9 mm to 6.2 mm. Within vegetation average number of the leaves (longer than 1 cm) per plant increased from 6 to 71 during the growing season. The length of the longest plant leaflet ranged from 5.8 cm to 12.5 cm. The biggest width of the leaflet (mean per plant) ranged from 6.6 cm to 16.7 cm.[...]Biologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

Investigation of mosaic virus in wild cucumber Echinocystis lobata (Michx.) Torr. et A. Gray

Program "Sustainability of agro-, forest and ecosystems" (project no. SIT-2/2015)Biologijos katedraGamtos mokslų fakultetasLietuvos sodininkystės ir daržininkystės institutasVytauto Didžiojo universiteta

Selection of dominant markers for investigation of genetic diversity of Cucurbitaceae family species

In addition to cultivated for food and forage representatives of Cucurbitaceae family, some species belonging to Echinocystis, Bryonia, Ecballium and Sicyos are either cultivated for ornamental purposes or growing wild in Lithuania. The objective of present study was to select dominant Inter Simple Sequence Repeat (ISSR) markers for evaluation of genetic diversity of ornamental species Echinocystis lobata and Sicyos angulata. The total DNA was extracted by DNA Purification Kit (#KO512, Thermo Scientific, Lithuania). Three randomly chosen ISSR markers (UBC 810, UBC 881 and UBC 890) were selected and conditions suitable for DNA amplification were screened. Amplification reactions were carried out in volumes of 20 μl containing of 10 μl PCR Master mix (0.05 U/μl Taq DNA Polymerase; 4 mM MgCl2; 0.4 mM each dNTP), 0.4 μl 20 mg/ml BSA (0.6 μl of BSA was also tested), 0.5 μl 10 μM primer and 80 ng of DNA (40 ng of DNA was also tested). Two different polymerase chain reaction (PCR) programs were tested. The first one consisted of an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s, extension at 72 °C for 45 s and a final extension at 72 °C for 10 min. According to this program, none of the fragments of DNA were amplified. The second type PCR protocol included an initial denaturation at 94 °C for 5 min, followed by 45 cycles of denaturation at 95 °C for 45 s, annealing at 54–56 °C for 45 s, extension at 72 °C for 2 min and a final extension at 72 °C for 5 min, the following program allowed to amplify DNA fragments with all selected markers, although for separate primers annealing temperatures were different. To obtain polymorphic and reproducible fragments of DNA the most favorable annealing temperature for the marker UBC 810 was 54 °C and for the markers UBC 881, UBC 890 temperature was 56 °C. [...]Biologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

Selection of codominant markers for investigation of genetic diversity of Echinocystis lobata (Michx.) Torr. et A. Gray populations

Study was funded by Lithuanian Research Council, Grant number No. SIT-02/2015Wild cucumber (Echinocystis lobata) – climbing annual herbaceous plant, growing in coastal habitats, riverbanks, lakesides, wetlands and wastelands. It is native in North America, but in many European countries is recognized as invasive species. Within last two decades E. lobata is very intensively spreading in Lithuania and is considered to be one of the most aggressive invasive plants. Until now there is a lack of information about genetic diversity of Wild cucumber populations. Simple Sequence Repeats (SSR) markers appeared to be the most often used markers for understanding relations between invasive species and components of natural ecosystems. The objective of present study was to select codominant SSR markers for evaluation of genetic diversity of Echinocystis lobata populations. The DNA was extracted by DNA Purification Kit. Thirty SSR markers (N1, N6, N12, S9, S13, S15, S26, SSR10018, SSR05723, SSR16695, SSR16226, SSR23220, SSR22653, SSR23370, SSR01738, SSR16056, SSR05012, SSR05125, SSR07543, SSR19998, SSR16068, SSR02895, SSR20852, SSR31399, SSR20218, SSR29620, SSR14861, SSR11340, SSR11043) were tested for buffer type, temperature and other conditions of DNA amplification. DNA fragments were evaluated after electrophoresis on 1.5 % agarose gel. In our study seventeen (N6, N12, S9, S13, S15, S26, SSR05723, SSR23220, SSR23370, SSR05012, SSR07543, SSR19998, SSR16068, SSR31399, SSR20218, SSR29620, SSR11340) out of thirty markers showed positive resultsBiologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

Methodical aspects of molecular studies of some riparian plant species

Nemaža dalis į sausumą išmestų teršalų yra išplaunami ir nunešami į ežerus bei upes. Akivaizdu, kad upių ir jų pakrančių augalija patiria nepalankų cheminį poveikį, kurį sustiprina fizinė žmogaus skvarba. Duomenys apie Lietuvos vandens augalų būklę nėra gausūs. Šiuolaikiniuose augalų įvairovės tyrimuose greta morfologija, fiziologija pagrįstų vertinimo būdų plačiai taikomi molekulinės biologijos metodai, naudojant DNR žymenis. Pirmas žingsnis taikant šiuos žymenis, yra kokybiškos DNR išskyrimas. Žemės ūkio auginamų augalų rūšių DNR išskyrimo metodai yra labai ištobulinti ir nuodugniai aprašyti. Natūraliai augančių augalų rūšių populiacijų genetinės įvairovės tyrimai nėra gausūs ir juose aprašomi metodai dažniausiai pateikiami labai apibendrintai, be tikslesnių, išsamesnių aprašų. Tarp augalų DNR išskyrimui naudojamų būdų dažnai minimi metodai, kuriuose panaudojamas cetiltrimetilamonio bromidas (CTAB), natrio dodecilsulfatas, įvairūs paruošti gamykliniai, nežinomos sudėties rinkiniai. Darbo tikslu buvo atrinkti tinkamiausius DNR išskyrimo metodus kai kuriems Lietuvoje augantiems Cucurbitaceae, Lythraceae šeimų bei Poaceae šeimos Aveneae tribos augalams, paplitusiems Nemuno paupiuose. Pasirinktiems, neretai greta augantiems augalams, atrinkti DNR išskyrimo būdai buvo labai skirtingiA large part of the land falling within a range of several pollutants are washed away and finally carried to lakes, streams and rivers. In addition to chemical pollution riparian vegetation is affected by severe human physical penetration. Data concerning health of aquatic plants are not abundant in Lithuania. In addition to traditional assessment based on morphology, physiology, modern plant diversity studies include molecular genetics techniques, where markers of the nucleus, chloroplast or mitochondrial DNA are employed. The first step in the application of these markers is extraction of high quality DNA. DNA extraction methods are perfectly developed and thoroughly described for cultivated plant species, their varieties. Population studies concerning genetic diversity of naturally growing plant species are not numerous. In many cases DNA extraction methods for these plants are presented in very general way, without more detailed methodological descriptions. The most frequently mentioned methods for plant DNA extraction are techniques employing cetyltrimethylammonium bromide, sodium dodecyl sulfate and big variety of ready-use DNA extraction kits of unknown composition. Our study is aimed at selection of DNA extraction methods for some naturally growing riparian plant species of Cucurbitaceae, Lythraceae and Poaceae (Aveneae tribe) spread in Nemunas basin. For several species selected the most efficient DNA extraction methods were different despite close vicinity of habitatsBiologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

Nitrogen nutrition of Echinocystis lobata depends on habitat

Climate change is often associated with global warming. Vegetation is very sensitive to temperature changes. Echinocystis lobata is annual, thermophilic species, invasive in Lithuania. This plant grows along river banks, hydrochory is one of the reasons of successful its spread. Aquatic ecosystems are heavily affected by human activities, fertilisers in special. However, there is still a lack of information on this topic in the Baltic States. Plant nitrogen (N) concentrations could be helpfull tool assessing the impact of pollution on alien species spread. The aim of our study is to estimate the nitrogen concentration of the leaves of Echinocystis lobata populations depending on some parameters of the habitats: river size, condition, origin, land use types and geographical location. The 18 populations of E. lobata were collected in the Nemunas basin. Leaf N concentration expressed as a percentage of the dry mass (% d. m.) ranged from 3.72 % d. m. (Nemunas population) up to 4.91% d. m. (Atmata population). The most contrasting populations differed 1.32 times (p < 0.05). The mean leaf N concentration in the populations was 4.18% d. m. Compared to the most common adjacent riparian species of Lithuania - Lythrum salicaria, Stuckenia pectinata, Phalaris arundinacea, Bidens frondosa, Phragmites australis, Nuphar lutea, E. lobata populations had the highest leaf N concentration. Higher than average N concentrations were found in the populations growing in the center of Kaunas city, were former big enough N pollution from point sources was documented. Large rivers are more vulnerable to plant invasions, as can be seen in the case of E. lobata, which has spread intensively in Lithuania in recent decades. Larger Lithuanian rivers flow through the most populated cities, which are good donors of seeds of alien plant species grown as ornamental plants, thus facilitating multiple introductions.[...]Biologijos katedraGamtos mokslų fakultetasVilniaus Gedimino technikos universitetasVytauto Didžiojo universiteta

Vandens augalų rūšių azoto koncentracijų sąsajos su žemės dangos tipu ir kitais aplinkos veiksniais

Baltijos šalių azoto (N) iškritų duomenys kartu su sausumos vandenų rodikliais rodo, kad azoto apkrova gali lemti augmenijos pokyčius. Nors upių eutrofikacija yra plačiai analizuojama, vis dar trūksta informacijos apie pakrantės augalų rūšių fiziologinius rodiklius. Tyrimo tikslas – palyginti Lietuvos vandens pakrančių augalų rūšių populiacijų lapų azoto koncentracijų duomenis, juos susiejant su tyrimo vietoms pagal gretimų žemių dangos tipu ir naudojimo paskirtimi, taikant aplinkos informacijos koordinuotos sistemą (CORINE), upių būkle bei dydžiu, žemės ūkio intensyvumu 1991–1996 metais ir upių vagų keitimu. Tyrimo medžiagą surinkus per 2015 m. rugpjūčio mėn. pirmąjį dešimtadienį iš pagrindinių Lietuvos upių 241 vietos, buvo įvertintos plačiai paplitusių penkių pakrančių ir dviejų vandens augalų (natūralių ir invazinių) azoto koncentracijos. Analizėms naudoti augalų lapai, kurių azoto koncentracijos buvo nustatytos Kjeldalio metodu. Didžiausi (1,7 karto, p < 0,05) lapų azoto koncentracijos skirtumai tarp populiacijų nustatyti Lythrum salicaria. Pagal populiacijų lapų azoto koncentracijų (N % sausos masės, SM) vidutines vertes, tirtų rūšių augalai išdėstė taip: Lythrum salicaria (3,0) < Stuckenia pectinata (3,1) < Phalaris arundinacea (3,5) < Bidens frondosa (3,8) < Phragmites australis (4,0) < Nuphar lutea (4,1) < Echinocystis lobata (4,2). Iš esmės didesnės (p < 0,05) lapų azoto koncentracijos buvo nustatytos Lythrum salicaria populiacijų, augančių šalia mažų upių (3,4 % N SM) nei augančių šalia didelių upių (2,8 % N SM). Skirtingomis savybėmis pasižyminčiose upių vietose rinkti vandens augalai reikšmingai nesiskyrė pagal lapų azoto koncentracijas. [...]Nitrogen (N) deposition data, together with inland water parameters, provide evidence that N load may affect the vegetation of Baltic States. There is much concern about eutrophication of the rivers, although information about physiological parameters of riparian plant species is still poor. The present study is aimed at comparison of leaf N concentration among populations of aquatic plant species of Lithuania, relating N concentration data to the type of land cover (classification system of COoRdinate Information on the Environment, CORINE) in the neighbouring areas, river state and size, intensity of agriculture in 1991–1996, and natural vice versa regulated fragments of the riverbed. The leaf N concentrations of widely spread native and invasive species (5 riparian and 2 water plants) were estimated for 241 sites (collection time 1st ten-day period of August, 2015) of the main river catchments of Lithuania. Only leaf blades were used for analyses and N concentrations were determined by the Kjeldahl method. The biggest (1.7 times) variation (p < 0.05) in leaf N concentration among populations was documented for Lythrum salicaria. According to the mean values (N % of dry mass, DM) of the leaf N concentration, species could be arranged into following order: Lythrum salicaria (3.0) < Stuckenia pectinata (3.1) < Phalaris arundinacea(3.5) < Bidens frondosa (3.8) < Phragmites australis (4.0) < Nuphar lutea (4.1) < Echinocystis lobata (4.2). Significantly higher (p < 0.05) concentrations of leaf N were found for L. salicaria populations growing near the small rivers (3.4% N DM) compared to the large ones (2.8% N DM). Other selected river and its environment parameters in most cases did not have significant effect on leaf N concentrations of aquatic plants of Lithuania. [...]Aplinkos tyrimų centrasBiologijos katedraGamtos mokslų fakultetasGamtos tyrimų centrasMatematikos ir statistikos katedraVilniaus Gedimino technikos universitetasVilniaus universitetasVytauto Didžiojo universiteta