RESUMEN
The isolation of nucleic acids from a biological sample is an important step for many molecular biology applications and medical diagnostic assays. This chapter describes an efficient protocol using established acidic CTAB (with a pH value of 5.0 to 6.8) based extraction method for isolation and/or purification of high molecular weight genomic DNA from a range of fresh and difficult sources from plant, animal, fungi, and soil material. This protocol is suitable for many sequencing and genotyping applications, including large-scale sample screening.
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Fraccionamiento Químico/métodos , ADN/aislamiento & purificación , Animales , ADN/análisis , Alimentos , Biología Molecular/métodos , Plantas/genética , Suelo/química , EspectrofotometríaRESUMEN
Genome walking (GW) refers to the capture and sequencing of unknown regions in a long DNA molecule that are adjacent to a region with a known sequence. A novel PCR-based method, palindromic sequence-targeted PCR (PST-PCR), was developed. PST-PCR is based on a distinctive design of walking primers and special thermal cycling conditions. The walking primers (PST primers) match palindromic sequences (PST sites) that are randomly distributed in natural DNA. The PST primers have palindromic sequences at their 3'-ends. Upstream of the palindromes there is a degenerate sequence (8-12 nucleotides long); defined adapters are present at the 5'-termini. The thermal cycling profile has a linear amplification phase and an exponential amplification phase differing in annealing temperature. Changing the annealing temperature to switch the amplification phases at a defined cycle controls the balance between sensitivity and specificity. In contrast to traditional genome walking methods, PST-PCR is rapid (two to three hours to produce GW fragments) as it uses only one or two PCR rounds. Using PST-PCR, previously unknown regions (the promoter and intron 1) of the VRN1 gene of Timothy-grass (Phleum pratense L.) were captured for sequencing. In our experience, PST-PCR had higher throughput and greater convenience in comparison to other GW methods.
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Genoma de Planta , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuencias Invertidas Repetidas , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN/métodos , Cartilla de ADN/química , Cartilla de ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Intrones , Proteínas de Plantas/genética , Poaceae/genética , Reacción en Cadena de la Polimerasa/normas , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Análisis de Secuencia de ADN/normas , TemperaturaRESUMEN
The purpose of the prospective research was to evaluate the benefit of urine specimen as a collection technique for biological forensic evidence in adult volunteers following consensual intercourse. For detecting Y-chromosomal material Buccal Swab Spin Protocol(®) was used in DNA extraction and purification and samples were analysed with Quantifiler Y Human Male DNA Quantification Kit(®). The time frame for positive Y-DNA was evaluated. Immediate microscopy for detection of spermatozoa was performed. Y-DNA was detected in 173/205 (84.4%) urine samples. Of the 86 first post-coital void urine samples available, Y-DNA was detected in 83 (96.5%) specimens. Of the 119 urine samples from volunteers with post-coital activities Y-DNA was still measurable in 70 (58.8%) urine specimens. The male DNA amount was below 0.023 ng/µl in 28/153 (18.3%) urine samples. Of the 22 urine samples obtained after 24 post-coital hours, 9 (40.9%) were still Y-DNA positive. No associations were found between coital durance, coital frequency during the past two weeks prior to the study intercourse, post-coital activities, and the urine sample Y-DNA positivity. Of the 111 urine samples where the immediate microscopy was performed, in 66 (59.5%) samples spermatozoa were verified and one sample even contained motile spermatozoa. Microscopy detected 66 (67.3%) and failed to detect spermatozoa in 32 (32.7%) of Y-DNA positive samples. In addition to conventional invasive swab techniques, urine samples seem to be an effective biological trace collection method for Y-DNA and spermatozoa within 24 h following penile-vaginal penetration. Furthermore, it may be considered as a non-invasive collection method in suspected acute child sexual abuse cases to diminish time delay in forensic evidence collection and to improve patients' positive attitudes towards evidence collection.
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Cromosomas Humanos Y , Coito , ADN/orina , Espermatozoides/citología , Toma de Muestras de Orina , Adulto , Femenino , Medicina Legal , Humanos , Masculino , Microscopía de Contraste de Fase , Persona de Mediana Edad , Estudios Prospectivos , Factores de Tiempo , Orina/citología , Adulto JovenRESUMEN
Timothy is a perennial forage grass grown commonly in Boreal regions. This study explored the effect of vernalization and photoperiod (PP) on flowering and growth characteristics and how this related to changes in expression of three flowering related genes in accessions from different geographic origin. Large variation was found in accessions in their vernalization and PP responses. In southern accessions vernalization response or requirement was not observed, the heading date remained unchanged, and plants flowered without vernalization. On the contrary, northern types had obligatory requirement for vernalization and long PP, but the tiller elongation did not require vernalization at 16-h PP. Longer vernalization or PP treatments reduced the genotypical differences in flowering. Moreover, the vernalization saturation progressed stepwise from main tiller to lateral tillers, and this process was more synchronized in southern accessions. The expression of PpVRN1 was associated with vernalization while PpVRN3 accumulated at long PP. A crucial role for PpVRN3 in the transition to flowering was supported as in southern accession the transcript accumulated in non-vernalized plants after transfer to 16-h PP, and the apices transformed to generative stage. Differences in vernalization requirements were associated with variation in expression levels of PpVRN1 and PpVRN3, with higher expression levels in southern type. Most divergent transcript accumulation of PpMADS10 was found under different vernalization conditions. These differences between accessions can be translated into agronomic traits, such as the tiller composition of canopy, which affects the forage yield. The southern types, with minimal vernalization response, have fast re-growth ability and rapidly decreasing nutritive value, whereas northern types grow slowly and have better quality. This information can be utilized in breeding for new cultivars for longer growing seasons at high latitudes.
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The purpose of the research was to evaluate gynecological evidence collection techniques; the benefit of cervical canal brush sample compared to vaginal fornix and cervical swab samples and the time frame for detecting Y-chromosomal material QiAmp DNA Mini Kit(®) and Quantifiler Y Human Male DNA Quantification Kit(®) in adult volunteers following consensual intercourse. Eighty-four adult female volunteers following consensual intercourse were recruited for the study. By combining all sample collecting techniques, 81.0% of the volunteers were Y-DNA positive. Up to 60 h the conventional swab sampling techniques detected more Y-DNA positive samples when compared to the brush technique. However, after 60 h, the cervical canal brush sample technique showed its benefit by detecting 27.3% (6/22) of Y-DNA positive samples, which were Y-DNA negative in both conventional swab sampling techniques. By combining swab and brush techniques, 75% of the volunteers were still Y-DNA positive in 72-144 post-coital hours. The rate of measurable Y-DNA decreased approximately 3% per hour. Despite reported consensual intercourse, 6.8% (3/44) of volunteers were Y-DNA negative within 48 h. Y-DNA was not detected after 144 post-coital hours (6 days). In conclusion, the brush as a forensic evidence collection method may provide additional biological trace evidence from the cervical canal, although the best biological trace evidence collection can be obtained by combining all three sampling techniques. The time frame for gynecological forensic evidence sample collection should be considered to be at least a week if sexual violence is suspected.
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Cuello del Útero/química , Cromosomas Humanos Y , Coito , ADN/aislamiento & purificación , Manejo de Especímenes/instrumentación , Vagina/química , Adulto , Femenino , Humanos , Masculino , Microscopía , Persona de Mediana Edad , Factores de Tiempo , Adulto JovenRESUMEN
Lignin amount and subunit composition were analyzed from stems and leaf sheaths of timothy (Phleum pratense L.) clones of different in vitro digestibility. Lignin concentration in stems and leaf sheaths was higher in clones of low digestibility than those of high digestibility. No change in lignin concentration occurred in stems as digestibility decreased. Intriguingly, the lignin concentration was lower and the syringyl/guaiacyl (S/G) ratio was higher in stems compared to leaf sheaths at all developmental stages studied. The developmental-associated decrease in digestibility correlated with the increase in S units in lignin in stems and leaf sheaths and in the amounts of p-coumaric acid and ferulic acid residues in the cell wall of stems. Yields of copper oxidation products increased in stems during maturation indicating qualitative changes in the lignin structure. This correlated strongly with the developmentally linked decrease in digestibility. The information obtained is valuable for breeding and for DNA marker development.
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Alimentación Animal/análisis , Dieta/veterinaria , Fibras de la Dieta/análisis , Digestión , Lignina/análisis , Modelos Biológicos , Phleum/química , Animales , Clonación de Organismos , Fibras de la Dieta/metabolismo , Finlandia , Lignina/biosíntesis , Lignina/química , Estructura Molecular , Phleum/genética , Phleum/crecimiento & desarrollo , Phleum/metabolismo , Hojas de la Planta/química , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Tallos de la Planta/química , Tallos de la Planta/crecimiento & desarrollo , Tallos de la Planta/metabolismo , RumiantesRESUMEN
Timothy (Phleum pratense) is a widely grown perennial forage grass in the Nordic region. The canopy consists of three tiller types, of which the stem forming vegetative elongating (ELONG) tiller and generative (GEN) tillers contribute the most to dry matter yield. In this study, the regulation of tiller formation by vernalization, day length (DL) [12 h, short day length (SD); 16 h, long day length (LD)] and gibberellic acid (GA) was investigated in two timothy cultivars. Vernalization resulted in a shift of ELONG to GEN tillers. No vernalization was required for the development of ELONG tillers but SD strictly arrested stem elongation. Vernalization is an important regulator of tiller development but it seemed to be upstream regulated by DL. LD was essential for floral transition and could not be substituted by GA and/or vernalization treatments. Genotypic variation was found in the development of GEN tillers. The ability to produce GEN tillers was associated with significant upregulation of PpVRN3. PpVRN1 expression peaked at the time of vegetative/generative transition, and PpVRN3 after the transfer to LD, suggesting them to have similar functions with cereal vernalization genes. PpVRN1 alone was not sufficient to activate flowering, and upregulation of PpVRN3 possibly together with PpPpd1 was required. Although vernalization downregulated PpMADS10, this gene did not act as a clear flowering repressor. Our results show that flowering signals alter the tiller composition, so they have important effects on yield formation of timothy.
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Regulación de la Expresión Génica de las Plantas , Giberelinas/metabolismo , Phleum/fisiología , Reguladores del Crecimiento de las Plantas/metabolismo , Transducción de Señal , Biomasa , Frío , Flores/genética , Flores/crecimiento & desarrollo , Flores/fisiología , Flores/efectos de la radiación , Biblioteca de Genes , Genotipo , Anotación de Secuencia Molecular , Phleum/genética , Phleum/crecimiento & desarrollo , Phleum/efectos de la radiación , Fotoperiodo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/fisiología , Hojas de la Planta/efectos de la radiación , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/fisiología , Brotes de la Planta/efectos de la radiación , Tallos de la Planta/genética , Tallos de la Planta/crecimiento & desarrollo , Tallos de la Planta/fisiología , Tallos de la Planta/efectos de la radiación , Estaciones del Año , Análisis de Secuencia de ADNRESUMEN
In spring turnip rape (Brassica rapa L. spp. oleifera), the most promising F1 hybrid system would be the Ogu-INRA CMS/Rf system. A Kosena fertility restorer gene Rfk1, homolog of the Ogura restorer gene Rfo, was successfully transferred from oilseed rape into turnip rape and that restored the fertility in female lines carrying Ogura cms. The trait was, however, unstable in subsequent generations. The physical localization of the radish chromosomal region carrying the Rfk1 gene was investigated using genomic in situ hybridization (GISH) and bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) methods. The metaphase chromosomes were hybridized using radish DNA as the genomic probe and BAC64 probe, which is linked with Rfo gene. Both probes showed a signal in the chromosome spreads of the restorer line 4021-2 Rfk of turnip rape but not in the negative control line 4021B. The GISH analyses clearly showed that the turnip rape restorer plants were either monosomic (2n=2x=20+1R) or disomic (2n=2x=20+2R) addition lines with one or two copies of a single alien chromosome region originating from radish. In the BAC-FISH analysis, double dot signals were detected in subterminal parts of the radish chromosome arms showing that the fertility restorer gene Rfk1 was located in this additional radish chromosome. Detected disomic addition lines were found to be unstable for turnip rape hybrid production. Using the BAC-FISH analysis, weak signals were sometimes visible in two chromosomes of turnip rape and a homologous region of Rfk1 in chromosome 9 of the B. rapa A genome was verified with BLAST analysis. In the future, this homologous area in A genome could be substituted with radish chromosome area carrying the Rfk1 gene.
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Brassica rapa/genética , Mapeo Cromosómico/métodos , Cromosomas de las Plantas/genética , Proteínas de Plantas/genética , Raphanus/genética , Cromosomas Artificiales Bacterianos , Fertilidad/genética , Marcadores Genéticos/genética , Hibridación Fluorescente in Situ , Proteínas de Plantas/metabolismoRESUMEN
BACKGROUND: Timothy is a long-day grass species well adapted for cultivation in northern latitudes. It produces elongating tillers not only in spring growth but also later in summer. As the quantity and quality of harvested biomass is dictated by canopy architecture and the proportion of stem-forming flowering tillers, the regulation of flowering is of great interest in forage grass production. METHODS: Canopy architecture, stem morphology and freezing tolerance of vernalized timothy were investigated in greenhouse and field experiments. The molecular control of development was examined by analysing the relationship between apex development and expression of timothy homologues of the floral inducer VRN1 and repressor VRN2. KEY RESULTS: True stem formation and lignification of the sclerenchyma ring occur in both vernalized and regrowing stems irrespective of the developmental stage of the apex. The stems had, however, divergent morphology. Vernalization enhanced flowering, and the expression of the VRN1 homologue was elevated when the apex had passed into the reproductive stage. High VRN1 homologue expression was not associated with reduction in freezing tolerance and the expression coincided with increased levels of the floral repressor VRN2 homologue. Field experiments supported the observed linkage between the upregulation of the VRN1 homologue and the transition to the reproductive stage in vernalized tillers. The upregulation of putative VRN1 or VRN2 genes was restricted to vernalized tillers in the spring yield and, thus, not detected in non-vernalized tillers of the second yield; so-called regrowth. CONCLUSIONS: The formation of a lignified sclerenchyma ring that efficiently reduces the digestibility of the stem was not related to apex development but rather to a requirement for mechanical support. The observed good freezing tolerance of reproductive timothy tillers could be one important adaptation mechanism ensuring high yields in northern conditions. Both VRN1 and VRN2 homologues required a vernalization signal for expression so the development of yield-forming tillers in regrowth was regulated independently of the studied genes.
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Flores/crecimiento & desarrollo , Flores/metabolismo , Phleum/crecimiento & desarrollo , Phleum/metabolismo , Tallos de la Planta/crecimiento & desarrollo , Tallos de la Planta/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
Effects of Se fertilization on potato processing quality, possible changes in Se concentration and form in tubers during storage, and retransfer of Se from seed tubers were examined. Potato plants were grown at five selenate (SeO4(2-)) concentrations. Tubers were harvested 16 weeks after planting and were stored at 3-4 degrees C prior to analysis. The results showed that the Se concentration did not decrease during storage for 1-12 months. In tubers, 49-65% of total Se was allocated in protein fraction, which is less than found in plant leaves in a previous study. The next-generation tubers produced by the Se-enriched seed tubers had increased Se concentrations, which evidenced the relocation of Se from the seed tubers. At low levels, Se improved the processing quality of potato tubers by diminishing and retarding their raw darkening. The value of Se-enriched potato tubers as a Se source in the human diet was discussed.
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Selenio/metabolismo , Selenio/farmacología , Solanum tuberosum/química , Solanum tuberosum/metabolismo , Fenómenos Bioquímicos , Bioquímica , Oscuridad , Factores de TiempoRESUMEN
Potatoes (Solanum tuberosum L.) supplemented with increasing amounts of sodium selenate were analyzed for glycoalkaloid (GA) content. GAs were extracted with 5% acetic acid from freeze-dried tubers of two potato cultivars, Satu and Sini, harvested 10 weeks after planting as immature. The GAs alpha-solanine and alpha-chaconine were quantified by reverse-phase high-performance liquid chromatography (RP-HPLC) with diode array detection. Two independent experiments were performed. In the first experiment, the total GA concentration +/- standard error of the tubers ranged between 105 +/- 9 and 124 +/- 10 mg kg(-1) fresh weight in Satu and between 194 +/- 26 and 228 +/- 10 mg kg(-1) fresh weight in Sini. The ratio of alpha-solanine to alpha-chaconine was 0.2 in Satu and 0.5-0.6 in Sini. In the second experiment, the total GA concentration +/- standard error was 75 +/- 4 to 96 +/- 11 mg kg(-1) fresh weight, and the ratio of alpha-solanine to alpha-chaconine was 0.3-0.4 in Satu. A high sodium selenate supplementation (0.9 mg of Se kg(-1) quartz sand) slightly decreased the GA content in Satu, but this decrease was not statistically significant. Furthermore, at this addition level the Se concentration increased to a very high level of 20 microg g(-1) dry weight, which cannot be recommended for human consumption. In both experiments, the Se concentration in tubers increased with increasing sodium selenate application levels. Our results show that acceptable application levels of selenate did not have an effect on the GA concentration in immature potato tubers.
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Alcaloides/análisis , Compuestos de Selenio/administración & dosificación , Solanina/análogos & derivados , Solanum tuberosum/química , Cromatografía Líquida de Alta Presión , Tubérculos de la Planta/química , Ácido Selénico , Solanina/análisisRESUMEN
The effect of selenium (Se) treatments on potato growth and Se, soluble sugar, and starch accumulation was investigated. Potato plants were cultivated in quartz sand without or with sodium selenate (0, 0.075, 0.3 mg Se kg(-1) sand). In young potato plants, Se treatment resulted in higher starch concentrations in upper leaves. The tuber yield of Se-treated potato plants was higher and composed of relatively few but large tubers. At harvest, the starch concentration in tubers did not differ significantly between treatments. The higher Se addition (0.3 mg Se kg(-1)) may have delayed the aging of stolons and roots, which was observed as high concentrations of soluble sugar and starch. Together with the earlier results showing elevated starch concentration in Se-treated lettuce, the findings of this research justify the conclusion that Se has positive effects also on potato carbohydrate accumulation and possibly on yield formation.