Browsing by department "Avdelningen för lantbruksvetenskaper"

Stress response in plants is influenced by several external and internal factors and is executed in a modular way. Environmental stimuli or stress is sensed by cellular receptors and the signal is transduced inside cell via the phospho-activation of highly conserved intracellular signaling cascades like mitogen activated protein kinase (MAPK) cascades. The signal then activates biosynthesis pathways of major stress response hormones like Salicylic acid (SA). In Arabidopsis about 90% SA is synthesized via isochorismate pathway and Isochorismate synthase 1 (ICS1) is a rate limiting enzyme in this pathway. In this study, goal was to select transgenic ICS1 (homozygous) candidate lines from parent ICS1-CFP by selective regeneration. Then, by molecular and physiological characterization of transgenic ICS1-CFP plants, the function of ICS1 phosphorylation, more specifically, impact of different photoperiods (Long day; LD and Short day; SD) and stress conditions on ICS1 activity would have resolved. However, there were no homozygous candidate line from any parent ICS1-CFP plants after several screening. Nevertheless, ozone treated stress sensitivity test was performed with heterozygous ICS1-CFP candidate plants (T2 generation). Ozone treated stress depends on stomata factor because ozone enters into plants through stomata. Therefore, stomata index analysis was performed with sid2 and WT (Col-0) phenotypes and grown in LD and SD conditions. Since, stomata number was different between LD and SD plants of both sid2 and WT phenotypes, a different method named Xanthine-Xanthine oxidase (X/XO) treatment was applied that induce oxidative stress regardless of stomata. Although, WT and sid2 had shown sensitivity to the treatment, the overall cell death percentage was very low. Lastly, our aim was to observe the impact of different photoperiods on the activation of two particular MAPKs i.e MPK3 and MPK6 under stress conditions. The phosphorylated (P-MPK3 and P-MPK6) are found abundantly in ozone treated plants as an early response. In this experiment, plants were grown in both LD and SD, stressed with both ozone and X/XO treatments, the activation of P-MPK3 and P-MPK6 was observed by protein level analysis (western blotting) in detailed time course. Although, the activation was visualized in both LD and SD plants, qualitatively the pattern was similar between day type samples and activation signal was very weak in both stress methods. In addition, anti-ICS1 antibody provided by Agrsera TM was tested for its efficiency to detect endogenous ICS1 protein in plants in two experimental set-up. Although the antibody could detect overexpressed ICS1-CFP protein in samples, it was not that efficient to detect endogenous ICS1 in any of the experiments.

The world population is growing and is expected to reach over 9 billion in about 30 years. Climate change is also widely expected to worsen famines in certain regions of the world. This will drastically increase global food demand. Food security efforts should be therefore be geared towards promoting food crops that can thrive in these regions and can withstand the condition likely to be brought about by changing climate. Cassava is a typical example of such a crop. This study investigated the use of digital images to estimate growth parameters of young cassava plants. Cassava was cultivated in pots at the University of Helsinki greenhouse at Viikki. The plants were given different water level (100%, 60% and 30% saturation) and potassium (0.1, 1.0, 4.0, 16.0 and 32.0mM) treatments. Digital red-green-blue (RGB) and multispectral images were taken every other week for 5 consecutive times. The images were processed to obtain leaf area, Normalized Difference Vegetation Index (NDVI), and Crop Senescence Index (CSI) and correlated with directly measured growth parameters of the young cassava crops. It was observed that leaf area that was computed from images, and NDVI which was computed from the multispectral images have significant positive correlations with the growth parameters, ie, actual leaf area, chlorophyll content, and plant biomass. CSI however showed weak a correlation between the growth parameters of the young cassava plants. Images leaf area and NDVI were then used to identify the changes in the effects of the water and potassium treatments.

The research of this thesis was focused on anaerobic digestion of cow manure mixed with different types of biowaste, especially those material that are available in Finland. The research was conducted by search, collection, and analysis of different data in literature. Topic of the thesis was predetermined by the Co-Creation Lab project of Helsinki Institute of Sustainability Science (HELSUS), which was seeking solution to achieve carbon neutral cow milk production. The solution was co-approached by three participants conducting thesis from technological, economical, and legislative points of view, in which I was responsible for writing mainly the technological part, and general findings in economic efficiency and legislative terms by the other two co-creators were also included in this thesis. The research was mainly related with the whole cycle of biogas production, including basics about anaerobic digestion (AD), applications of the biogas and digestate as a product and by-product of AD. Substrates for AD were researched with a focus to find the best combination of cow manure (CM) and biowaste in regard with methane yield outcome, especially a mixture of CM and silage waste that suits the cow farming situation in Finland. Methane yields for mono-digestion of various types of biowaste and co-digestion of CM with different biomass were collected and analyzed. Premises for biogas plant establishment were researched briefly, including facility composition, and consideration of feasibility and raw material availability. CM with grass containing 75% timothy and 25% meadow fescue grass at 70%:30% mixing ratio could be the best combination of CM: grass co-digestion, followed by 0.5:0.5 mixed CM and perennial ryegrass. Furthermore, CM mixed with food waste at 52:48% ratio could be the best combination among co-digestion of CM with biowaste other than grass, followed by CM and food waste mixed with 68%:32% ratio, and CM with oat straw mixed at 1:2 ratio could be a considerable combination of CM and crop waste.

Selective degeneration and dysregulation of specific neuronal populations is a common hallmark shared by neurodegenerative diseases affecting the aging population. Parkinson’s disease (PD) is one of the most prevalent neurodegenerative diseases with debilitating clinical manifestations that follow a chronic and progressive course. Pathological hallmarks of PD involve gradual and specific loss of DA (DA) neurons and widespread presence of Lewy body (LB) inclusions that consist of aggregated presynaptic protein, α-Synuclein (αSyn). Treatment of PD remains to be at symptomatic management as the underlying mechanisms that trigger neurodegeneration are still not fully elucidated. Over the past two decades, microRNAs (miRNAs) have become a major area of interest within biomedical fields and gained increasing momentum in the context of neurodegenerative diseases. In recent developments, changes in mature miRNA profiles have been reported in aging tissue and many age-related diseases, including PD. More recently, a number of studies have found that the most essential enzyme in the miRNA biogenesis pathway, Dicer, exhibits reduced expression with aging. To these ends, a genetic mouse model based on heterozygous knockout of Dicer (DicerHET) was introduced to simulate Dicer downregulation. Initial investigations identified the DicerHET model as a promising tool for studying the relationship between disrupted miRNA biogenesis and neurodegeneration associated with PD. To facilitate future investigations and speed up screening of potential therapeutic compounds using this genetic model, in the current work, we aimed to produce a DicerHET in vitro model with a practical and convenient genetic engineering approach. The main focus of this work was to validate the model and establish a standardized reproducible approach suitable for research that addresses the role of miRNA biogenesis in PD. The desired DicerHET genotype was generated in vitro by employing traditional Cre/loxP system in conjunction with a virally mediated Cre expression. More specifically, primary cortical cultures, derived from Dicer flox/+ mice embryos, were transduced with Cre expressing lentiviral vectors (lenti-hSYN-T2A-Cre) to delete the “floxed” Dicer allele. To establish optimal parameters for the procedure, we analysed recombination efficiency under different transduction conditions. The most efficient recombination was achieved after 5 days of induction in cultures. However, we observed that DicerHET genotype did not attenuate survival of the cells, as assessed by immunohistochemistry. Further, as a proof of concept, we exposed the DicerHET cultures to pre-formed fibrils (PFFs) - a PD related stressor that causes αSyn aggregation. pSer129-αSyn-positive LB-like aggregates were detected in all the PFF-treated cultures, however, with a greater accumulation in the DicerHET cultures. Interestingly, increased aggregation was not accompanied by increased cell death, suggesting that DicerHET genotype does not increase vulnerability of cortical neurons to pSer129-αSyn aggregation. Based on our earlier studies we presume that DA neurons may bear a specific vulnerability towards the age-related Dicer depletion. More conclusive evidence on this intriguing relationship could be provided in future research using the DicerHET model that can be readily applied to primary DA cultures.

Tämän tutkielman tavoitteena oli selvittää suomalaisen emolehmätuotannon tuottavuutta kuvaavia tunnuslukuja. Emolehmätuotantoa tarvitaan, koska lypsylehmien määrä pienenee ja näin ollen myös lihantuotantoon käytettävissä olevien maitorotuisten vasikoiden määrä. Emolehmien määrä on noussut Suomessa lähes 20 % kymmenen viime vuoden aikana. Emolehmän tärkein tuotos on elävä vasikka, joka saadaan vieroitettua menestyksekkäästi ja riittävän isossa vieroituspainossa suhteessa emon painoon. Naudanlihantuotannon hiilijalanjälkeen on viime vuosina kiinnitetty yhä enemmän huomiota ja emolehmiin perustuvan lihantuotannon tuottavuutta parantamalla voidaan pienentää myös kasvihuonekaasupäästöjä. Emolehmätuotannon tuottavuutta kuvaavia tunnuslukuja ovat hiehojen poikimaikä, poikimaväli, poikimaprosentti, vasikkakuolleisuus, elinikäistuotos ja poistoikä. Nämä analysoitiin Ruokaviraston nautarekisterin tietojärjestelmistä saatavasta datasta vuosilta 1995-2014. Aineiston tilastollinen analysointi toteutettiin SAS-ohjelmalla. Keskimääräisten tunnuslukujen lisäksi analysoitiin rotukohtaiset erot sekä emolehmätilojen parhaimman ja heikoimman neljänneksen väliset erot. Emolehmähiehot poikivat keskimäärin 27,5 kuukauden iässä. Poikimaväli oli keskimäärin 378 päivää. Angus-rodun hiehojen poikimaikä oli matalin ja lehmien poikimaväli lyhyin lähes koko analysointijakson ajan. Limousinen poikimaikä oli korkein ja poikimaväli pisin. Parhaan neljänneksen tiloilla hiehot poikivat keskimäärin 22 kuukauden iässä ja heikoimman neljänneksen tiloilla yli 42 kuukauden iässä. Poikimavälin keskiarvo parhaan neljänneksen tiloilla oli 364 päivää ja heikoimman neljänneksen tiloilla 400 päivää. Poikimaprosentti oli keskimäärin lähes 86 prosenttia. Anguksen poikimaprosentti oli korkein koko analysointijakson ajan. Parhaan neljänneksen karjoissa poikimaprosentti oli keskimäärin 96 % ja heikoimman neljänneksen karjoissa 73 %. Vasikkakuolleisuus oli keskimäärin 6,2 %. Sonnivasikoiden kuolleisuus oli lähes 2 %-yksikköä suurempi kuin lehmävasikoiden. Ensikoilla vasikkakuolleisuus oli suurempi kuin vanhemmilla lehmillä. Vasikkakuolleisuus oli suurin charolais-rodulla. Emolehmän elinikäistuotos oli keskimäärin 4,4 tuotettua vasikkaa. Elinikäistuotos oli paras anguksella ja limousinella ja heikoin simmentalilla. Poistoiän keskiarvo oli 6,8 vuotta. Korkein poistoikä oli limousinella ja matalin simmentalilla. Tilakohtaiset vaihtelut tuotantotuloksissa olivat suuria. Tuotannon tulisi olla riittävän tehokasta, jotta se olisi ympäristöllisesti kestävää ja emolehmätuotannon hiilijalanjälkeä saataisiin entisestään pienennettyä.

Patellar luxation is a typical orthopaedic disorder in small sized dogs. Patella can luxate either medially or laterally, medial luxation being by far more common than the lateral luxation. PL is considered hereditary since certain breeds have great susceptibility to get the condition, and the symptoms may occur at young age. PL is diagnosed by following the so-called Putnam’s scale where the stifle joint is palpated and manipulated. PL is graded from 0=normal…4=permanent luxation. The aim of this study was to estimate the variance components and the heritability of PL, to visualize PL’s genetic trend and to calculate the genetic correlation between left and right stifle and between PL and hip dysplasia (HD) in Japanese Spitz. The PL, HD and pedigree data were provided by the Finnish Kennel Club. Data were modified with both R-program and Microsoft Office Excel. The pedigree check was performed with RelaX2 program and variance component analyses were done with DMU program using the restricted maximum likelihood method. Heritabilities (h2) were from very low to low depending on the model and breed. When the dependent variable was the mean of left and right patellae of an individual the lowest heritability was in the Pomeranian h2=0.03 and highest in the Chihuahua h2=0.18. The genetic correlation of left and right patellae was 1 in all breeds which suggests that they are genetically the same trait. In the Japanese Spitz the genetic correlation between PL and HD was -0.05. The genetic trend of PL was favorable in the Chihuahua and the Japanese Spitz. In the Pomeranian the trend was negative and neutral in the Finnish Spitz. Based on the results, the selection against PL has not been efficient. In future, patellae health should be controlled by using breeding indexes because the heritability of PL is low. Also, improvements in phenotyping could lead to more accurate selection.

Asteraceae comprises of approximately 10% of all angiosperm plant species. These species are well known for their highly compressed inflorescences known as capitula which consists of morphologically different types of flowers: ray, trans and disc flowers. This immense morphological difference excels Gerbera as an ideal plant to study flower type differentiations. Even though this complex process is governed by several genes, the ray flower identity is believed to be greatly influenced by GhCYC3 promoter mediated gene regulations. In previous studies two TCP transcription factors (TF): GhCIN1and GhCIN2, and two MADS TFs: GAGA1 and RCD5 were identified as the potential upstream regulators of GhCYC3. So, the aim of this study is to test whether these potential upstream regulators physically bind to GhCYC3 promoter in in vitro conditions. In order to achieve the goal, these transcription factor proteins from Gerbera hybrida were successfully expressed in E. coli and purified as fusion proteins to maltose-binding protein (MBP). Physical binding of the purified fusion proteins to the putative target DNA sites in the promoter region of GhCYC3 gene was tested by electrophoretic mobility shift assay (EMSA). The results showed that none of the gerbera transcription factors (GhCIN1, GhCIN2, GAGA1 and RCD5) bind to their putative target sites under the condition tested in this study. However, it might not be justifiable to deduce that these TFs do not interact with GhCYC3 promoter. The absence of in vitro interaction between the tested TFs and GhCYC3 promoter might be caused by either lack of proper folding and activity of the TFs or absence of co-factors which are available in vivo.

The sequencing methods used to study the genome of organisms have become cheaper, resulting in a significant increase in the amount of genomic data available. Knowing the nucleic acid sequence of the DNA does not tell much about an organism. Not without first annotating the genome, which means searching for the locations of genes and defining their products. The programs used for annotation make mistakes and their results must be evaluated in various ways. The vast amount of genomic data encourages fast production of new annotations and this can increase human made errors. Some annotation programs use gene databases, so the number of wrongly annotated genes they contain may increase in the future if the quality control of annotations is not improved. This study examines correlation between selected quality measures and the quality of annotations. The quality metrics used can be divided into two basic types, the first one is based on the basic structures of genes and the second one on comparing the protein product of a gene against a protein database. The study assumes that comparison to a reference is a reliable way to assess the quality of annotations. The comparison is made at genome, exon and nucleotide levels. A single value describing the comparison is calculated at each level. For each gene aligned with a reference gene, sensitivity and specificity are calculated and used to make f-score at the nucleotide level. Four different versions of the wild strawberry (Fragaria vesca) genome and their six annotations were used as data. They were downloaded from the Genome Database for Rosacaea, which is a genome database specializing in rose plants. The correlation coefficients calculated from quality metrics and f-scores were in several cases small but reliable because the p-value was minimal. Correlation coefficients were higher when quality metrics based on protein homology were examined. The correlation coefficient calculated from the mean of the structure-based quality metrics and the f-score received lower values if the studied annotation had a high f-score value. These results detailed in this paper support the view that the selected structure-based quality metrics are not suitable for evaluation of high-grade annotations. They might possibly be used in automated detection of poor-quality annotations. Quality metrics based on protein homology appeared to be promising subjects for further research.

Five wild sea buckthorn populations in Finland were studied using 8 SSR markers to reveal genetic diversity within and among populations. Population samples were collected from four different locations on mainland and one from the Åland Islands. Allelic data obtained from SSR markers were subjected to statistical analysis and AMOVA, and rarefaction was used to correct effects of uneven population sample sizes on some population diversity indices. Results showed moderate to high genetic diversity levels, as mean HE of all populations was 0.55 and HO 0.50. Most populations showed larger observed heterozygosity levels than expected, one showed equal amounts and one population showed lower heterozygosity levels than expected. Most genetic variation of populations was found within individuals across populations (84%) and only some among populations (16%). A weighted neighbor-joining phylogenetic tree showed only some clustering based on populations. The results of this study show, that there is mentionable diversity in Finnish wild sea buckthorn populations, and that populations are only moderately differentiated. This information can be beneficial particularly in sea buckthorn breeding efforts, but also from a possible genetic resources’ and conservation standpoint.

Kasvit tuottavat ensisijaisen aineenvaihdunnan eli primaarimetabolian avulla tarvitsemansa amino-, nukleiini- ja rasvahapot. Näitä molekyylejä kasvit käyttävät kasvuun ja kehitykseen sekä elintoimintojensa turvaamiseen. Suuri osa primaarimetabolian geeneistä on hyvin konservoituneita eli ne ovat säilyneet lähes muuttumattomana kasvilajista toiseen miljoonien vuosien ajan. Sekundaarimetaboliitteja kasvit tuottavat vuorovaikuttaakseen ympäristönsä kanssa. Sekundaarimetaboliittien biosynteesin voi laukaista esimerkiksi kasvin kokema stressi, joka aiheutuu kasvinsyöjistä, taudinaiheuttajista, toisista kasveista tai jos kasvi altistuu liikaa ultraviolettisäteilylle. Ympäristötekijöillä, kuten pH:lla, veden saatavuudella, lämpötilalla ja maaperän suolapitoisuudella on myös suuri vaikutus sekundaarimetaboliittien biosynteesiin sekä vaihteluun saman kasvilajin sisällä. Suurin osa kasvien sekundaarimetaboliiteista syntetisoidaan sikimaatti-, isoprenoidi- ja polyketidireittiä pitkin. Polyketidit toimivat muun muassa pigmentteinä ja osana kasvin puolustusjärjestelmää. Gerberassa esiintyy harvinaista polyketidijohdannaista, 4-hydroksi-5-metyylikumariinia (HMC), jonka biosynteesistä 2-pyronisyntaasi G2PS2:n on päätelty vastaavan. Tässä tutkielmassa selvitettiin g2ps2:n osallisuutta HMC:n biosynteesiin käyttämällä Virus induced gene silencing (VIGS)- menetelmää sekä proteiinin ylituottoa gerberan kallussolukossa. VIGS- menetelmä ei toiminut tässä kokeessa g2ps2- geenin aikaisen ekspression takia. Kokeet gerberan Regina- lajikkeen kallussolukossa sen sijaan vaikuttivat lupaavilta ja kallus osoittautui hyödylliseksi menetelmäksi gerberan polyketidisyntaasien tutkimiseen.