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  • Palo, Outi M (Helsingin yliopisto, 2010)
    Bipolar disorder (BP) is a complex psychiatric disorder characterized by episodes of mania and depression. BP affects approximately 1% of the world’s population and shows no difference in lifetime prevalence between males and females. BP arises from complex interactions among genetic, developmental and environmental factors, and it is likely that several predisposing genes are involved in BP. The genetic background of BP is still poorly understood, although intensive and long-lasting research has identified several chromosomal regions and genes involved in susceptibility to BP. This thesis work aims to identify the genetic variants that influence bipolar disorder in the Finnish population by candidate gene and genome-wide linkage analyses in families with many BP cases. In addition to diagnosis-based phenotypes, neuropsychological traits that can be seen as potential endophenotypes or intermediate traits for BP were analyzed. In the first part of the thesis, we examined the role of the allelic variants of the TSNAX/DISC1 gene cluster to psychotic and bipolar spectrum disorders and found association of distinct allelic haplotypes with these two groups of disorders. The haplotype at the 5’ end of the Disrupted-in-Schizophrenia-1 gene (DISC1) was over-transmitted to males with psychotic disorder (p = 0.008; for an extended haplotype p = 0.0007 with both genders), whereas haplotypes at the 3’ end of DISC1 associated with bipolar spectrum disorder (p = 0.0002; for an extended haplotype p = 0.0001). The variants of these haplotypes also showed association with different cognitive traits. The haplotypes at the 5’ end associated with perseverations and auditory attention, while the variants at the 3’ end associated with several cognitive traits including verbal fluency and psychomotor processing speed. Second, in our complete set of BP families with 723 individuals we studied six functional candidate genes from three distinct signalling systems: serotonin-related genes (SLC6A4 and TPH2), BDNF -related genes (BDNF, CREB1 and NTRK2) and one gene related to the inflammation and cytokine system (P2RX7). We replicated association of the functional variant Val66Met of BDNF with BP and better performance in retention. The variants at the 5’ end of SLC6A4 also showed some evidence of association among males (p = 0.004), but the widely studied functional variants did not yield any significant results. A protective four-variant haplotype on P2RX7 showed evidence of association with BP and executive functions: semantic and phonemic fluency (p = 0.006 and p = 0.0003, respectively). Third, we analyzed 23 bipolar families originating from the North-Eastern region of Finland. A genome-wide scan was performed using the 6K single nucleotide polymorphism (SNP) array. We identified susceptibility loci at chromosomes 7q31 with a LOD score of 3.20 and at 9p13.1 with a LOD score of 4.02. We followed up both linkage findings in the complete set of 179 Finnish bipolar families. The finding on chromosome 9p13 was supported (maximum LOD score of 3.02), but the susceptibility gene itself remains unclarified. In the fourth part of the thesis, we wanted to test the role of the allelic variants that have associated with bipolar disorder in recent genome-wide association studies (GWAS). We could confirm findings for the DFNB31, SORCS2, SCL39A3, and DGKH genes. The best signal in this study comes from DFNB31, which remained significant after multiple testing corrections. Two variants of SORCS2 were allelic replications and presented the same signal as the haplotype analysis. However, no association was detected with the PALB2 gene, which was the most significantly associated region in the previous GWAS. Our results indicate that BP is heterogeneous and its genetic background may accordingly vary in different populations. In order to fully understand the allelic heterogeneity that underlies common diseases such as BP, complete genome sequencing for many individuals with and without the disease is required. Identification of the specific risk variants will help us better understand the pathophysiology underlying BP and will lead to the development of treatments with specific biochemical targets. In addition, it will further facilitate the identification of environmental factors that alter risk, which will potentially provide improved occupational, social and psychological advice for individuals with high risk of BP.
  • Saarinen, Silva (Helsingin yliopisto, 2013)
    Lymphomas are classified into Hodgkin and non-Hodgkin lymphomas (HL and NHL), and they are further categorized into tens of different subtypes. This thesis has focused on two rare lymphoma subtypes: nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and primary mediastinal large B-cell lymphoma (PMBCL). Although the majority of lymphomas are sporadic, a familial component has been observed in both HL and NHL. The aim of this thesis was to study the genetic background of familial NLPHL and PMBCL predisposition. HL is classified into NLPHL and classical Hodgkin lymphoma (cHL). Germline mutations of the KLHDC8B gene have been observed in some families with cHL. In this study, the KLHDC8B gene was analyzed in four families with NLPHL, but mutations were not detected. The results suggest that germline mutations of KLHDC8B do not underlie familial NLPHL susceptibility. To clarify the genetic basis of NLPHL predisposition, a Finnish family with NLPHL susceptibility was studied with new genome-wide methods, SNP microarray and exome sequencing. Data from these two efforts were integrated, and a germline deletion of two nucleotides (c.2437-2438delAG) was observed in the NPAT gene. Subsequently, NPAT was screened in a number of familial and sporadic HL cases, and an in-frame deletion of one serine residue was found to be more frequent in the cases versus healthy controls (odds ratio 4.11, p = 0.018). Thus, NPAT was identified as a candidate gene for NLPHL predisposition. The familial risk in NLPHL was studied by using the Finnish Cancer Registry (FCR) and the Finnish Population Registries. Altogether 693 patients with NLPHL were identified, and a family member cohort comprising of the first-degree relatives of 692 patients was collected. Cancer data for the relatives was derived from the FCR. We calculated the standardized incidence ratios (SIRs) for all cancers, NHL, cHL and NLPHL in the family member cohort. In addition, the primary tumor samples from 20 first-degree relatives with HL diagnosis were histopathologically reviewed. In the family member cohort, the SIR for NLPHL was 19. Thus, a high familial risk in NLPHL was observed, suggesting that familial factors contribute to NLPHL susceptibility. An increased relative risk for NHL and cHL was also observed. We studied a family of three siblings with PMBCL and a cousin affected by diffuse large B-cell lymphoma (DLBCL). Re-examination of tumor samples revealed that all four tumors resembled each other histopathologically, proposing a common background. Genome-wide SNP array, linkage analysis and exome sequencing were performed to identify the genetic factor underlying PMBCL/DLBCL in this family. A missense change c.5533C>A (His1845Asn) was detected in the MLL gene and was the only non-database variation residing in linked regions and segregating in lymphoma affected family members. To our knowledge, this is the first description of familial clustering of PMBCL. Our findings suggest that sometimes PMBCL may arise in the context of familial predisposition, and MLL was identified as a candidate gene for PMBCL susceptibility. However, our observations are preliminary, and need to be confirmed in future studies.
  • Renkonen, Elise (Helsingin yliopisto, 2006)
    Hereditary nonpolyposis colorectal cancer (HNPCC) and familial adenomatous polyposis (FAP) are characterized by a high risk and early onset of colorectal cancer (CRC). HNPCC is due to a germline mutation in one of the following MMR genes: MLH1, MSH2, MSH6 and PMS2. A majority of FAP and attenuated FAP (AFAP) cases are due to germline mutations of APC, causing the development of multiple colorectal polyps. To date, over 450 MMR gene mutations and over 800 APC mutations have been identified. Most of these mutations lead to a truncated protein, easily detected by conventional mutation detection methods. However, in about 30% of HNPCC and FAP, and about 90% of AFAP families, mutations remain unknown. We aimed to clarify the genetic basis and genotype-phenotype correlation of mutation negative HNPCC and FAP/AFAP families by advanced mutation detection methods designed to detect large genomic rearrangements, mRNA and protein expression alterations, promoter mutations, phenotype linked haplotypes, and tumoral loss of heterozygosity. We also aimed to estimate the frequency of HNPCC in Uruguayan CRC patients. Our expression based analysis of mutation negative HNPCC divided these families into two categories: 1) 42% of families linked to the MMR genes with a phenotype resembling that of mutation positive, and 2) 58% of families likely to be associated with other susceptibility genes. Unbalanced mRNA expression of MLH1 was observed in two families. Further studies revealed that a MLH1 nonsense mutation, R100X was associated with aberrant splicing of exons not related to the mutation and an MLH1 deletion (AGAA) at nucleotide 210 was associated with multiple exon skipping, without an overall increase in the frequency of splice events. APC mutation negative FAP/AFAP families were divided into four groups according to the genetic basis of their predisposition. Four (14%) families displayed a constitutional deletion of APC with profuse polyposis, early age of onset and frequent extracolonic manifestations. Aberrant mRNA expression of one allele was observed in seven (24%) families with later onset and less frequent extracolonic manifestations. In 15 (52%) families the involvement of APC could neither be confirmed nor excluded. In three (10%) of the families a germline mutation was detected in genes other than APC: AXIN2 in one family, and MYH in two families. The families with undefined genetic basis and especially those with AXIN2 or MYH mutations frequently displayed AFAP or atypical polyposis. Of the Uruguayan CRC patients, 2.6% (12/461) fulfilled the diagnostic criteria for HNPCC and 5.6% (26/461) were associated with increased risk of cancer. Unexpectedly low frequency of molecularly defined HNPCC cases may suggest a different genetic profile in the Uruguayan population and the involvement of novel susceptibility genes. Accurate genetic and clinical characterization of families with hereditary colorectal cancers, and the definition of the genetic basis of "mutation negative" families in particular, facilitate proper clinical management of such families.
  • Georgitsi, Marianthi (Helsingin yliopisto, 2008)
    Much of the global cancer research is focused on the most prevalent tumors; yet, less common tumor types warrant investigation, since A rare disorder is not necessarily an unimportant one . The present work discusses a rare tumor type, the benign adenomas of the pituitary gland, and presents the advances which, during the course of this thesis work, contributed to the elucidation of a fraction of their genetic background. Pituitary adenomas are benign neoplasms of the anterior pituitary lobe, accounting for approximately 15% of all intracranial tumors. Pituitary adenoma cells hypersecrete the hormones normally produced by the anterior pituitary tissue, such as growth hormone (GH) and prolactin (PRL). Despite their non-metastasizing nature, these adenomas can cause significant morbidity and have to be adequately treated; otherwise, they can compromise the patient s quality of life, due to conditions provoked by hormonal hypersecretion, such as acromegaly in the case of GH-secreting adenomas, or due to compressive effects to surrounding tissues. The vast majority of pituitary adenomas arise sporadically, whereas a small subset occur as component of familial endocrine-related tumor syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 is caused by germline mutations in the MEN1 tumor suppressor gene (11q13), whereas the majority of CNC cases carry germline mutations in the PRKAR1A gene (17q24). Pituitary adenomas are also encountered in familial settings outside the context of MEN1 and CNC, but unlike in the latter syndromes, their genetic background until recently remained elusive. Evidence in previous literature supported the notion that a tumor suppressor gene on 11q13, residing very close to but still distinct from MEN1, causes genetic susceptibility to pituitary tumors. The aim of the study was to identify the genetic cause of a low penetrance form of Pituitary Adenoma Predisposition (PAP) in families from Northern Finland. The present work describes the methodological approach that led to the identification of aryl hydrocarbon receptor interacting protein (AIP) as the gene causing PAP. Combining chip-based technologies (SNP and gene expression arrays) with traditional gene mapping methods and genealogy data, we showed that germline AIP mutations cause PAP in familial and sporadic settings. PAP patients were diagnosed with mostly adenomas of the GH/PRL-secreting cell lineage. In Finland, two AIP mutations accounted for 16% of all patients diagnosed with GH-secreting adenomas, and for 40% of patients being younger than 35 years of age at diagnosis. AIP is suggested to act as a tumor suppressor gene, a notion supported by the nature of the identified mutations (most are truncating) and the biallelic inactivation of AIP in the tumors studied. AIP has been best characterized as a cytoplasmic interaction partner of aryl hydrocarbon receptor (AHR), also known as dioxin receptor, but it has other partners as well. The mechanisms that underlie AIP-mediated pituitary tumorigenesis are to date largely unknown and warrant further investigation. Because AIP was identified in the genetically homogeneous Finnish population, it was relevant to examine its contribution to PAP in other, more heterogeneous, populations. Analysis of pituitary adenoma patient series of various ethnic origins and differing clinical settings revealed germline AIP mutations in all cohorts studied, albeit with low frequencies (range 0.8-7.4%). Overall, PAP patients were typically diagnosed at a young age (range 8-41 years), mainly with GH-secreting adenomas, without strong family history of endocrine disease. Because many PAP patients did not display family history of pituitary adenomas, detection of the condition appeared challenging. AIP immunohistochemistry was tested as a molecular pre-screening tool on mutation-positive versus mutation-negative tumors, and proved to be a potentially useful predictor of PAP. Mutation screening of a large cohort of colorectal, breast, and prostate tumors did not reveal somatic AIP mutations. These tumors, apart from being the most prevalent among men and women worldwide, have been associated with acromegaly, particularly colorectal neoplasia. In this material, AIP did not appear to contribute to the pathogenesis of these common tumor types and other genes seem likely to play a role in such tumorigenesis. Finally, the contribution of AIP in pediatric onset pituitary adenomas was examined in a unique population-based cohort of sporadic pituitary adenoma patients from Italy. Germline AIP mutations may account for a subset of pediatric onset GH-secreting adenomas (in this study one of seven GH-secreting adenoma cases or 14.3%), and appear to be enriched among young (≤25 years old) patients. In summary, this work reveals a novel tumor susceptibility gene, namely AIP, which causes genetic predisposition to pituitary adenomas, in particular GH-secreting adenomas. Moreover, it provides molecular tools for identification of individuals predisposed for PAP. Further elaborate studies addressing the functional role of AIP in normal and tumor cells will hopefully expand our knowledge on endocrine neoplasia and reveal novel cellular mechanisms of pituitary tumorigenesis, including potential drug targets.
  • Wan, Xing (2012)
    Class IIa (pediocin-like) bacteriocins are a major group of bacteriocins produced by lactic acid bacteria (LAB) characterised by their antilisterial activity. As a protective LAB strain for meat products, Leuconostoc carnosum 4010 kills Listeria by producing two class IIa bacteriocins, the well characterised leucocin A (LeuA) and the less studied leucocin C (LecC). Although the amino acid sequence of the secreted LecC has been published, the genes required for its production remain unknown. The aims of this study were to characterise the genes needed for LecC production and to express the lecC gene in Lactococcus lactis. The lecC gene was localised by Southern blot in a large plasmid different from the one harbouring LeuA genes in Ln. carnosum 4010 genome. Five genes in two operons were identified mainly by PCR-based methods and sequencing, namely, the structural gene (lecC) with a 72-bp signal sequence, the immunity gene (lecI) encoding a 97-aa immunity protein, two genes lecTS for an ABC transporter and the gene lecX for an accessory protein. The immunity function of LecI was demonstrated by expressing the lecI gene in LecC sensitive Listeria monocytogenes. Compared to the wild type, LecI-producing Listeria was more tolerant to LecC, thus corroborating the immunity function of LecI. For heterologous expression of LecC, the lecC gene was fused to the lactococcal usp45 signal sequence in the nisin-selectable and nisin-inducible food-grade secretion vector pLEB690. Consequently, bioactive LecC was secreted efficiently by the recombinant Lc. lactis. In conclusion, novel genes for the production of LecC in Ln. carnosum 4010 were identified. The findings indicate that LecC is produced by a dedicated system independent of LeuA. The successful production of functional LecC in Lc. lactis offers an attractive approach for the future application of bacteriocins in food production.
  • Metlin, Artem (Helsinki University Printing House, 2008)
    Rabies is a fatal disease that affects the central nervous system of all warmblooded mammals. The rabies virus belongs to the order Mononegavirales, family Rhabdoviridae, genus Lyssavirus. This virus has a negative single-stranded RNA genome and the virions are bullet-shaped. Rabies is reported in many countries throughout the world and has been registered in all continents except Australia, where only the bat Lyssaviruses have been found, and in Antarctica where the main vectors of rabies are absent. Russia and most of the bordering countries are affected by rabies. Finland was a rabies-free country from 1959 to 1988, when a sylvatic rabies epidemic appeared with raccoon dogs as the main host and vector of infection. That epidemic was eradicated by the oral vaccination of wild carnivores and the parenteral immunization of dogs and cats; and Finland has been rabies-free since 1991. However, this status is constantly under threat because rabies is endemic in Russia and Estonia. In June 2003, a horse imported to Finland from Estonia was clinically and laboratory diagnosed as rabies positive. The close relationship of the isolated equine virus strain with the current Estonian strains was verified during subsequent molecular epidemiological studies. Because the case was imported, it did not affect Finland’s rabies-free status. Also in 2007 another 2 imported cases of rabies were recorded: one in a human being from Philippines and the other in a dog from India. Five different antigenic variants of the rabies virus were identified among rabies positive field samples from Russia, Finland, and Estonia by using antinucleocapsid monoclonal antibodies. Two rabies virus field isolates showed a different reaction pattern that was similar to that of the vaccine strains of the SAD group, which might suggest a new antigen variant or reverted vaccine strain. Nevertheless, the sequence analysis showed that the vaccine strains RV-97 and SAD B19 included in the oral anti-rabies vaccine “Sinrab” (Russia) and “Fuchsoral” (Germany), respectively, differ considerably from all the field strains. Field rabies viruses collected in recent years from different regions of the Russian Federation were chosen on the basis of mAb studies and geographical origin for molecular epidemiological studies to characterize their genetic heterogeneity and to study their molecular epidemiology. In addition to the Russian viruses, archival samples from Estonia and Finland and Russian vaccine strains were also included in this study. Among the field viruses studied, two main phylogenetic groups were found, and designated as the Pan-Eurasian and Caucasian based on their geographical origin. The Pan-Eurasian 7 group including some reference viruses from Europe was further divided into four subgroups. All the vaccine strains were clearly different from the field strains. No recombination between the field and vaccine virus strains was observed. The critical roles of geographical isolation, the limitation of the genetic clustering, and the evolution of the rabies virus were shown during this study. The rabies virus vaccine strain RV-97 is widely used in Russia as a component of the oral anti-rabies vaccine “Sinrab”. To characterize the molecular properties of this strain, entire genome sequencing was conducted. A simple technique was developed to obtain this sequence, including the 3’- and 5’- ends. The entire genome sequence and deduced amino-acid sequences of the major viral proteins were compared with the sequences of other known fixed rabies viruses. The strain RV-97 formed a separate phylogenetic branch and seems to be more related to the group of Japanese strains. The field strains from the Caucasian group seem to be phylogenetically the nearest group to the RV-97 strain. The data shown herein makes it possible to develop molecular methods for distinguishing between the field rabies viruses from the vaccine strains for the rapid recognition of the vaccine strains that are unstable or have reverted back to their pathogenic form. The wide genetic heterogeneity verified in this study indicates that it is important to remain on permanent alert for the appearance of rabies.
  • Hytönen, Marjo (Helsingin yliopisto, 2013)
    Since the sequencing of the Canis lupus familiaris genome the dog has become a powerful tool for scientists. Selective breeding has created more than 400 different breeds each representing genetic isolates with breed-specific morphological and behavioral characteristics. Unique population history, available genealogical records, veterinary diagnostics and novel genomic tools greatly facilitate gene mapping studies in dogs. Given that over 600 genetic disorders have been described in dogs and that most of them are similar to human conditions, dogs have emerged as a clinically relevant model for human inherited disorders. This study explores the genetics of three different inherited developmental defects in dogs, caudal dysplasia, ectodermal dysplasia, and mucopolysaccharidosis VII, which all have counterparts in human. In this study, various clinical and pathological techniques have been used to characterize the phenotypes, and genetic methods such as genome-wide association studies and next-generation sequencing to resolve the genetics of the diseases. Moreover, functional studies in mice have been performed to explore the molecular role during embryonic development. The discoveries made here have established the affected breeds as models to further explore disease mechanisms and therapeutic methods, identified new disease pathways, and offered novel approaches for further developmental studies. Furthermore, this work has enabled the development of genetic tests for breeding purposes. Three different phenotypes have been investigated in this study. First, we studied genetics of caudal dysplasia, which in its mildest form is presenting as short-tail phenotype in dogs. A mutation in T (brachyury homolog) was earlier identified to cause this phenotype in Pembroke Welsh Corgis. Homozygous mutations of T in mouse result in severe caudal dysplasia and embryonic lethality suggesting an essential role for the T gene during mammalian development. The presence of the documented T mutation, c.189C>G, was investigated in 23 other breeds demonstrating that short-tailed dogs from 17 breeds were heterozygous for the mutation that associated completely with the phenotype. The homozygous mutation was suggested to be lethal, as no dogs homozygous for the mutation were found and an approximately 30% decrease was seen in the size of Swedish Vallhund litters when both parents were short-tailed. However, short-tailed dogs were found from six breeds that did not carry the known substitution or any other mutations in the T coding regions and therefore other genetic factors are yet to be discovered that affect the development of the posterior mesodermal region. The short-tailed dogs which do not have T mutation will serve as models in future studies to identify possible novel genetic factors for caudal dysplasia and related medical conditions. Second, a new gene was identified for a hairless phenotype and some of its upstream regulators were characterized. Hairless dog breeds show a breed characteristic which is in clinical terms an ectodermal dysplasia. In this study, the causative mutation for canine ectodermal dysplasia (CED) was sought and subsequently the function of this novel gene in the ectodermal organ development was explored. Accordingly, a genome-wide association study was performed and CED was mapped to dog chromosome 17. Haplotype association testing revealed a 160-kb haplotype, which was fine-mapped using three different breeds. The causative genetic mutation for CED was identified as a 7-bp duplication producing a frameshift and premature stop codon in a previously uncharacterized canine gene forkhead box protein I3 (FOXI3). The study provided a novel gene focus to aid research into ectodermal development. Therefore, a detailed expression pattern of murine Foxi3 during the development of the ectodermal organs was constructed and a series of tissue culture experiments and expression analyses with mouse embryos were performed to assess the function of Foxi3 in mammalian embryogenesis. The results suggest that Foxi3 regulates hair follicle and tooth formation as well as the development of mammary and salivary gland, nail, and eye. Ectodysplasin and activin A were identified as upstream regulators of Foxi3. Third, Brazilian Terriers with severe skeletal defects at early puppyhood were identified through information provided by breeders. Subsequently, a major aim of this work was to describe the clinical and pathological features of the syndrome and to identify its genetic cause. Clinicopathological examinations and pedigree analysis demonstrated that the affected puppies had a recessive spondyloepihyseal dysplasia. The disease locus was mapped to chromosome 6 and a mutation leading to pathogenic p.P289L change in a conserved functional domain of β-glucuronidase (GUSB) was identified. Elevated glycosaminoglycans were detected in urine and only a residual β-glucuronidase activity was observed in the serum of the affected dogs, which confirmed the pathogenity of the mutation. GUSB defects result in mucopolysaccharidosis VII (MPS VII) in several species and thus the mutation defined the syndrome as MPS VII in Brazilian Terriers. Overall, this study illustrates how unique morphological diversity and enriched genetic alterations in closed populations can be efficiently harnessed to gain new insights into developmental biology across species. For example, the identification of the CED mutation in FOXI3 revealed a completely novel gene with a previously unknown essential function in ectodermal development. This work has established several novel large animal models to further explore disease mechanisms and to develop therapeutic methods. Moreover, several new DNA tests have been developed for different breeds of dogs to eradicate or, to control better, the conditions through improved breeding plans. This will improve the welfare of our beloved pets.
  • Aragon Obando, Erwin Manuel (2009)
    Genetic composition of Theobroma cacao L., including 60 Nicaraguan farmers accessions, was investigated using nine microsatellite (SSR) markers. Fourteen breeders accessions from Experimental Center “El Recreo”, INTA, Nicaragua, two Criollos accession from CATIE, Costa Rica, and two accessions from Ecuador were included as reference material. The average PIC value (0.78) indicated a high power of discrimination for the nine loci used. A total of 155 alleles were detected at the nine loci. The number of alleles per marker ranged from 10 to 22 with a means of 17.22 alleles per locus. A heterozygosity deficiency (HExp < HObs) was registered for all microsatellite loci. The average expected heterozygosity was=0.68 among Nicaraguan farmers accessions. The Analysis of molecular variance (AMOVA) showed a low level of differentiation among populations. The genetic distances determined for the groups of farmers accessions RAAS and Pacifico Sur are closely similar, while a great genetic distance was observed between RAAN and RAAS groups. The cluster analysis presented a strong genetic relation between the Criollo 13 from CATIE and farmers accession MAT0404. The principal component analysis showed that 7 farmers accession from Nicaragua are genetically related with the accessions Criollo 13 and Yucatan from the international accessions. The present study suggested a good possibility to select farmers accessions to be included in breeding programs, especially those accessions related to know Criollo accessions.
  • Koskela, Elli (2009)
    Strawberry (Fragaria × ananassa) is the most important berry crop cultivated in Finland. Due to the species' economic importance, there is a national breeding programme aimed at extending the cropping season from the current one month to up to three months. This could be achieved by growing cultivars which would initiate flowers throughout the summer months, without the requirement of a period of short days as is the case with currently grown cultivars. The cultivated strawberry is an octoploid and therefore has complex patterns of inheritance. It is desirable to study the genetic mechanisms of flowering in the closely related but diploid species F. vesca (L). In the diploid Fragaria, a mutation in a single locus, namely the SEASONAL FLOWERING LOCUS (Sfl), changes the flowering phenotype from seasonal to perpetual flowering. There is also an array of genetic tools available for F. vesca, which facilitate genetic studies at molecular level. Experiments described here aimed at elucidating the identity of the gene which confers perpetual flowering in F. vesca by exploring the flowering characteristics and genotypes of five F2 populations (crosses between seasonal × perpetual flowering cultivars). The study took advantage of a genetic map for diploid Fragaria, publicly available EST and genomic Fragaria sequences and a recently developed BAC library. Sequence information was used for designing gene–specific primers for a host of flowering–related candidate genes, which were subsequently mapped on the diploid Fragaria genetic map. BAC library was screened with molecular markers supposedly located close to the Sfl, with the aim of positionally cloning the Sfl. Segregation of flowering phenotypes in the five F2 populations showed, that the Sfl indeed controls flowering in all the tested cultivars. A genetic map was constructed of the chromosome with the Sfl, and a positional cloning attempt was initiated with the closest flanking markers. 45 gene–specific primers pairs were designed for 21 flowering–related genes, and eight genes were successfully mapped on the diploid Fragaria map. One of the mapped genes, namely PRR7, located very close to the Sfl, and is a potential candidate for the gene that has evaded identification so far.Mansikka (Fragaria × ananassa) on tärkein Suomessa viljelty marjakasvi. Nykyisin mansikan satokausi kestää vain noin kuukauden. Satokautta pidentämällä viljelijän olisi mahdollista tasata tuotantohuippuja ja saada sesongin ulkopuolisesta tuotteesta parempaa hintaa. Mansikalle onkin perustettu jalostusohjelma, joka tähtää jatkuvasatoisten, Suomen oloihin soveltuvien lajikkeiden jalostamiseen. Jatkuvasatoiset lajikkeet pystyvät muodostamaan kukka–aiheita läpi kesän, toisin kuin nykysin viljellyt lajikkeet, jotka tarvitsevat kukka–aiheiden muodostamiseen lyhyenpäivän oloja. Viljelty mansikka on oktoploidi, jonka periytyminen on monimutkaista. Tämän takia on kannattavaa tutkia kukkimisen geneettisiä mekanismeja lähisukuisessa diploidissa lajissa, ahomansikassa (F. vesca). Ahomansikassa yhden lokuksen, niin kutsutun SEASONAL FLOWERING–lokuksen (Sfl), mutaatio saa aikaan jatkuvan kukkimisen. Ahomansikkaa on tutkittu paljon ja lajille on kehitetty monia geneettisiä työkaluja, jotka mahdollistavat molekyylitason geneettisten tutkimusten teon. Tässä työssä pyrittiin selvittämään Sfl:ssa sijaitsevan geenin identitetti tutkimalla viiden ahomansikan F2–populaation kukintaa ja genotyyppejä. Tutkimus hyödynsi diploidille mansikalle kehitettyä geneettistä karttaa, julkisia EST– ja genomisia mansikan sekvenssejä sekä äskettäin kehitettyä BAC–kirjastoa. Sekvenssitietoja käytettiin tunnetuille kukintageeneille spesifisten alukeparien suunnitteluun. BAC–kirjastoa seulomalla yritettiin löytää klooni(t), jotka sisältäisivät Sfl:n. Kukintafenotyyppien segregaatio viidessä F2–populaatiossa osoitti, että Sfl kontrolloi kukintaa kaikissa kokeessa mukana olleissa lajikkeissa. Kromosomille, jossa Sfl sijaitsee, kehitettiin geneettinen kartta. Sfl:n lähelle sijoittuvien molekyylimerkkien avulla aloitettiin BAC–kirjaston seulonta, jonka tarkoituksena oli Sfl:n kloonaaminen. 21 kukintageenille suunniteltiin yhteensä 45 geenispesifistä alukeparia. Kokeessa onnistuttiin sijoittamaan kahdeksan kukintageeniä geneettiselle kartalle. Yksi kartalle sijoitetuista geeneistä, PRR7, on hyvin lähellä Sfl:a, ja on hyvä kandidaatti ahomansikan keskeiseksi kukintageeniksi.
  • Velmala, Sannakajsa (Helsingin yliopisto, 2014)
    This study was carried out in order to reveal the degree to which host-tree factors influence the interaction between Norway spruce (Picea abies (L.) Karst.) and the endophytic (EN) and ectomycorrhizal fungi (EMF) found in their needles and roots, respectively. We also explored how susceptibility to fungal infection and the composition and functionality of associated fungal communities relates to seedling growth performance. In multiple glasshouse experiments, we challenged Norway spruce seedlings and clonal cuttings with pure culture and natural EMF inoculum. Clonal cuttings from healthy spruce were used to assess the heritability of EMF communities and needle endophytes. The relationship between the susceptibility to fungal infection and EMF community function with host-tree performance was studied using seedlings originating from families known to have different growth rates at later life stages. We also examined resource allocation and root architecture of the fast- and slow-growing Norway spruce seedlings. Hypotheses derived prior to and during this work were tested with molecular tools and appropriate statistical techniques. Host genotype partly controlled the colonization of EMF and EN species but future growth performance was not associated with susceptibility. Norway spruce seedlings originating from differently growing seed orchards were colonized similarly by EMF, and did not show any consistent bias in terms of infection rate or the function of single ectomycorrhizas. However, the short root architecture was found to be moderately heritable and varied consistently between the fast- and slow-growing origins. We observed seedlings of fast-growing origins to have sparse and widespread rootlets that enable a greater allocation of below-ground biomass and higher exoenzyme capacity compared to slow-growing seedlings. Norway spruce does not show a strong genetic signal for within-population selection towards its mutualistic fungi at the species level. Formation of the associated EMF community may be an effect rather than cause of seedling physiological state. The superior growth of fast-growing genotypes seems to be a consequence of resource allocation and optimal root structuring in the juvenile stage rather than the extent of colonization by fungal mutualists. We accept that root physiological factors may subsequently lead to a higher capacity for symbiotic interactions in heterogeneous forest soil and a higher diversity and functionality of associated EMF. An adequate and versatile means of nutrient acquisition is an important factor enabling fast growth, but might also provide the basis for positive feedback via enhanced relationships with mutualistic fungi.
  • Elshibli, Sakina (Helsingin yliopisto, 2009)
    Acquiring sufficient information on the genetic variation, genetic differentiation, and the ecological and genetic relationships among individuals and populations are essential for establishing guidelines on conservation and utilization of the genetic resources of a species, and more particularly when biotic and abiotic stresses are considered. The aim of this study was to assess the extent and pattern of genetic variation in date palm (Phoenix dacttylifera L) cultivars; the genetic diversity and structure in its populations occurring over geographical ranges; the variation in economically and botanically important traits of it and the variation in its drought adaptive traits, in conservation and utilization context. In this study, the genetic diversity and relationships among selected cultivars from Sudan and Morocco were assessed using microsatellite markers. Microsatellite markers were also used to investigate the genetic diversity within and among populations collected from different geographic locations in Sudan. In a separate investigation, fruits of cultivars selected from Sudan, involved morphological and chemical characterization, and morphological and DNA polymorphism of the mother trees were also investigated. Morphological and photosynthetic adjustments to water stress were studied in the five most important date palm cultivars in Sudan, namely, Gondaila, Barakawi, Bitamoda, Khateeb and Laggai; and the mechanism enhancing photosynthetic gas exchange in date palm under water stress was also investigated. Results showed a significant (p < 0.001, t-test) differentiation between Sudan and Morocco groups of cultivars. However, the major feature of all tested cultivars was the complete lack of clustering and the absence of cultivars representing specific clones. The results indicated high genetic as well as compositional and morphological diversity among cultivars; while, compositional and morphological traits were found to be characteristic features that strongly differentiate cultivars as well as phenotypes. High genetic diversity was observed also in different populations. Slight but significant (p < 0.01, AMOVA) divergence was observed for soft and dry types; however, the genetic divergence among populations was relatively weak. The results showed a complex genetic relationships between some of the tested populations especially when isolation by distance was considered. The results of the study also revealed that date palm cultivars and phenotypes possess specific direct or interaction effects due to water availability on a range of morphological and physiological traits. Soft and dry phenotypes responded differently to different levels of water stress, while the dry phenotype was more sensitive and conservative. The results indicated that date palm has high fixation capacity to photosynthetic CO2 supply with interaction effect to water availability, which can be considered as advantageous when coping with stresses that may arise with climate change. In conclusion, although a large amount of diversity exists among date palm germplasm, the findings in this study show that the role of biological nature of the tree, isolation by distance and environmental effects on structuring date palm genome was highly influenced by human impacts. Identity of date palm cultivars as developed and manipulated by date palm growers, in the absence of scientific breeding programmes, may continue to mainly depend on tree morphology and fruit characters. The pattern of genetic differentiation may cover specific morphological and physiological traits that contribute to adaptive mechanisms in each phenotype. These traits can be considered for further studies related to drought adaptation in date palm.
  • Pohjanmies, Tähti (2014)
    Genetic variation within a population is shaped by the life history traits of the species and the properties of the surrounding ecosystem. It is an important factor in the preservation of populations. According to the emerging field of community genetics, genetic variation within a population of one species may also influence the dynamics and diversity of associated species, extending the conservational relevance of intraspecific genetic diversity. Finnish populations of pedunculate oak (Quercus robur) offer an interesting study system for population genetics. Q. robur grows in south-western Finland at the northern limit of its natural range. Here, its distribution has been shaped by long-term climatic and geological changes as well as by human disturbance, and the current populations are small and strongly fragmented. As Q. robur supports a high diversity of associated species, it is considered to have great ecological and conservational importance. In this thesis, I studied the amount and distribution of genetic diversity within and among three Q. robur populations in south-western Finland using population genetic parameters. I also described the spatial and temporal sub-population structure of one population, on the island of Wattkast. The genetic data was based on 15 nuclear microsatellite loci. Additionally, I examined the effect of the genetic diversity and genotypic identity of the oaks within Wattkast on associated herbivore communities. In the analysis, I used observational data from two years. As predicted for widespread, long-lived tree species, the microsatellite loci showed high levels of diversity within the populations, but also significant differentiation among them. This may be due to fragmentation and to the marginality of the populations. Within the population on Wattkast, I observed patterns of spatial and temporal sub-population differentiation. The characteristics of the site, including the ongoing shift to less extensive land use, suggest that the population is in genetic disequilibrium. As both the genetic distance and the community dissimilarity between pairs of trees increased with increasing geographic distance, I could not conclude the genotypic identity of the host trees to have an effect on the herbivore community structure. However, higher heterozygosity was associated with higher richness and abundance of species. This result supports the notion that intraspecific genetic variation may increase associated species richness. Based on the results of my study, both the life history traits of the species and the historic habitat changes may be observed in the genetic structure of Q. robur populations in Finland. The results also suggest that preservation of genetic variation within the remaining stands may be a factor not only in the preservation of these populations, but also in the conservation of associated species diversity.
  • Halinen, Katrianna (Helsingfors universitet, 2008)
    Cyanobacteria (blue-green algae) form blooms in the Baltic Sea during the warmest summer months. According to paleolimnological data, cyanobacteria have long history in the Baltic Sea, going back at least 7000 years. However, the intensity as well as the expanse of cyanobacterial blooms has increased during recent decades. Blooms attract regular attention in the media because of their visibility and the potential health risk they pose to humans and animals. The Gulf of Finland is the most eutrophied area of the Baltic Sea, and cyanobacterial blooms are widely believed to be the result of intense anthropogenic nutrient loading. Cyanobacterial blooms are formed mainly by species of three genera in the Baltic Sea, Nodularia, Anabaena and Aphanizomenon. The focus of present-day research on Baltic Sea cyanobacteria has been on Nodularia and Aphanizomenon, while the genus Anabaena has been neglected. Anabaena is often considered to play a minor role in cyanobacterial blooms. However, Anabaena can form a significant part of the blooms, especially in the northern part of the Baltic Sea. Cyanobacterial blooms in the Baltic Sea are invariably toxic due to the production of hepatotoxic nodularin by Nodularia spumigena. According to systematic studies, Aphanizomenon flos-aquae was not found to produce hepatotoxins in the Baltic Sea. However, it has been speculated that Baltic Sea Anabaena spp. could produce microcystins. The genetic structure of the Anabaena populations in the Baltic Sea has not been systematically explored. The aim of this present study was to increase our understanding of the Anabaena - a component of the Baltic Sea phytoplankton. Altogether, 49 planktonic Anabaena strains were isolated from the Gulf of Finland, five of which were microcystin-producing. This study provided unequivocal evidence that Baltic Sea Anabaena is able to produce microcystins. Each microcystin-producing Anabaena strain produced two to four dominant microcystin variants, including the highly toxic microcystin-LR. In this study, a culture-independent method was designed to detect putative microcystin and nodularin producers. By means of this DGGE method, microcystin-producing Anabaena populations were detected in cyanobacterial bloom samples from the summers of 2003 and 2004. Microcystin-producing Anabaena populations were detected throughout the Gulf of Finland. This excluded the possibility that the presence of microcystin-producing Anabaena was a chance phenomenon. Results suggest that salinity may limit the distribution of the microcystin-producing Anabaena although further studies are needed to confirm the interdependence of salinity and microcystin production. Microcystin-producing Anabaena populations were found to be highly diverse on analyses of the 16S rRNA, rbcL, rpoC1, and mcyE gene sequences. In previous studies, freshwater microcystin-producing Anabaena strains were grouped together in phylogenetic analyses. All microcystin-producing Baltic Sea Anabaena strains belonged to this hepatotoxic cluster, with the exception of a single strain. Both planktonic and benthic Anabaena populations were genetically heterogeneous and closely related to freshwater Anabaena strains. However, genetic diversity in benthic Anabaena strains was higher than in planktonic strains. In phylogenetic analyses, novel Anabaena lineages, possibly specific to the Baltic Sea, were identified. This suggests ecotypic diversification within Anabaena populations. We found two planktonic Anabaena strains which carried the entire mcy gene cluster, but were nonetheless incapable of producing microcystins. Natural genetic inactivation of the mcy gene cluster was identified in Anabaena strain BIR259. This strain carried insertions which most likely caused the inactivation of the mcy genes. The insertions documented here were surprisingly common in the Baltic Sea bloom samples and they were present in samples from both studied summers, 2003 and 2004. However, these insertions were not identified in freshwater strains or in field samples from freshwater lakes. The aim of this study was to establish a strain collection of Baltic Sea Anabaena and to shed light on the phylogeny, microcystin-production, and genetic diversity of the Baltic Sea Anabaena populations. In addition to strain isolation, these research goals were approached by in situ molecular methods. Systematic toxin screening showed that Anabaena is able to produce microcystins, and this should be taken into account in future toxin monitoring programmes.
  • Palo, Jukka (Helsingin yliopisto, 2003)
  • Säisä, Marjatta (Helsingin yliopisto, 2009)
    We described the patterns and extent of microsatellite DNA variation in historical and present-day Atlantic salmon (Salmo salar L.) stocks in the Baltic Sea and neighbouring areas, and in European whitefish (Coregonus lavaretus) ecotypes, populations and run-timing types in Finland. Moreover, the amount and pattern of genetic diversity in historical salmon populations before human impact were described, and the proportion of diversity maintained in the present hatchery stocks evaluated. Salmon populations in the Baltic Sea were, on average, significantly less variable than eastern Atlantic populations, and the diversity of landlocked populations (Lakes Vänern, Saimaa, Onega and Ladoga) was in turn significantly lower than that of anadromous salmon populations in the Baltic Sea populations. Within the Baltic Sea, the anadromous populations of Atlantic salmon formed three clear groups, corresponding to the northern (Gulf of Bothnia), eastern (Gulf of Finland and eastern Baltic Main Basin) and southern (western Baltic Main Basin) regions. Based on microsatellite data, three salmon population groups in the Baltic Sea were considered potentially different colonization lineages. In short- and long-term breeding programmes of Atlantic salmon, the average observed rate of loss of alleles was 4.9% and 2.0% per generation and the average rate of loss of heterozygosity was 1.4% and 1% per generation, respectively. When comparing the genetic parameters of stocks before and after hatchery breeding of several successive generations (Rivers Iijoki and Oulujoki), statistically significant changes in allele frequencies were common, while large wild stock in the Teno River has remained temporally very stable over 56 years. Despite the observed losses of genetic diversity in broodstock breeding, a large proportion of the genetic resources of the extirpated stocks are still conserved in the broodstocks. Genetic differentiation among European whitefish ecotypes was generally low, thus giving support to the hypothesis of one native European whitefish species in Fennoscandia. Among the ecotypes, the northern, large sparsely rakered, bottom-dwelling whitefish was the most unique. The known genetic differences in quantitative traits have thus either developed independently of potential phylogenetic lineages, or the lineages have mixed and the quantitative traits of the ecotypes, like gill-raker number, have later changed according to environment and selection pressures. Overall, genetic distances between the anadromous whitefish populations along the Finnish coast, especially in the Bothnian Bay area, were small. Wild whitefish populations studied had slightly higher allelic diversity than hatchery-reared populations in corresponding rivers.
  • Rehnström, Karola (Helsingin yliopisto, 2009)
    Positional cloning has enabled hypothesis-free, genome-wide scans for genetic factors contributing to disorders or traits. Traditionally linkage analysis has been used to identify regions of interest, followed by meticulous fine mapping and candidate gene screening using association methods and finally sequencing of regions of interest. More recently, genome-wide association analysis has enabled a more direct approach to identify specific genetic variants explaining a part of the variance of the phenotype of interest. Autism spectrum disorders (ASDs) are a group of childhood onset neuropsychiatric disorders with shared core symptoms but varying severity. Although a strong genetic component has been established in ASDs, genetic susceptibility factors have largely eluded characterization. Here, we have utilized modern molecular genetic methods combined with the advantages provided by the special population structure in Finland to identify genetic risk factors for ASDs. The results of this study show that numerous genetic risk factors exist for ASDs even within a population isolate. Stratification based on clinical phenotype resulted in encouraging results, as previously identified linkage to 3p14-p24 was replicated in an independent family set of families with Asperger syndrome, but no other ASDs. Fine-mapping of the previously identified linkage peak for ASDs at 3q25-q27 revealed association between autism and a subunit of the 5-hydroxytryptamine receptor 3C (HTR3C). We also used dense, genome-wide single nucleotide polymorphism (SNP) data to characterize the population structure of Finns. We observed significant population substructure which correlates with the known history of multiple consecutive bottle-necks experienced by the Finnish population. We used this information to ascertain a genetically homogenous subset of autism families to identify possible rare, enriched risk variants using genome-wide SNP data. No rare enriched genetic risk factors were identified in this dataset, although a subset of families could be genealogically linked to form two extended pedigrees. The lack of founder mutations in this isolated population suggests that the majority of genetic risk factors are rare, de novo mutations unique to individual nuclear families. The results of this study are consistent with others in the field. The underlying genetic architecture for this group of disorders appears highly heterogeneous, with common variants accounting for only a subset of genetic risk. The majority of identified risk factors have turned out to be exceedingly rare, and only explain a subset of the genetic risk in the general population in spite of their high penetrance within individual families. The results of this study, together with other results obtained in this field, indicate that family specific linkage, homozygosity mapping and resequencing efforts are needed to identify these rare genetic risk factors.
  • Jiménez Caldera, Oswalt Rafael (Helsingin yliopisto, 2014)
    The common bean (Phaseolus vulgaris L.) is an important component of food security programs aiming to provide better human nutrition in developing countries. However, low yields, climate change affecting production and variable local demands of specific types of cultivars suggest that we should consider the utilization of local bean genetic resources with high market acceptance in local breeding programs. Those efforts would complement regional breeding agendas. In this study, the genetic diversity of a collection of landraces was found to be considerably higher (mean 8.9 alleles per microsatellite locus) than previously reported, with a population structure of three main clusters grouped according to seed weights. Of these landraces, two promising divergent sources of genetic variation, accessions PV0006 and PV0023, were chosen for single crosses. The hybridity of F1 generation was tested using polymorphic microsatellite markers. Computer simulations demonstrated that the selection of F1 individuals possessing the highest degree of allele recombination following the pedigree method, instead of using the whole set of F1 individuals as is usually done, could improve the selection gains for yield. Between 128 and 1024 pure lines could be obtained after a reasonable number of generations. Subsequently, 420 F2 plants originating from 15 marker-selected F1 plants were established in three augmented blocks together with both parents and check cultivar INTA ROJO . Variables PP, SP, SW and YP were measured for each plant. PP and YP were considered the most appropriate traits for selection based on ANOVAs, high heritability values, and genetic gains obtained for both traits. After conducting plant selection using mixed model analyses, 81 and 74 F2 plants (with 61 plants in common for both groups) were selected based on their superior yield potential compared with both parents and check cultivar. Resistance to BCMV, BGYMV, ANT and rust was confirmed in the segregating population, and their higher potential over 40 bean landraces was validated by computer simulations constructed using genotype frequencies. The level of resistance was unexpectedly found to be similar to that of current cultivars in use. However, further experiments to confirm resistance should be conducted. Moreover, resistance genes for BCMV (bc-3 and bc-12) absent in our segregating plants were detected in bean landraces PV0015, PV0016, PV0017, PV0026, PV0031, PA0001, PA0002, and PA0003. It would be beneficial to pyramid broader resistance to this seed-borne virus in our populations by means of new crosses. All these findings emphasize the feasibility of utilizing local landraces for genetic improvement using efficient statistical methods aided by molecular markers. Additionally, molecular markers were efficiently used to test the genetic purity of cultivar INTA ROJO . The detected significant changes in genotype frequencies in four seed categories were probably caused by inadequate roguing procedures during seed production. Similarly, molecular markers were able to discriminate off-type seeds and plants from INTA ROJO , opening the possibility to complement current phenotypic methods employed in the genetic quality testing of seed lots.
  • Onkamo, Päivi (Helsingin yliopisto, 2002)
  • Manninen, Outi (Helsingin yliopisto, 2000)