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Background and Aims: The genetic basis of increased rooting below the plough layer, post-anthesis in the field, of an elite wheat line (Triticum aestivum 'Shamrock') with recent introgression from wild emmer (T. dicoccoides), is investigated. Shamrock has a non-glaucous canopy phenotype mapped to the short arm of chromosome 2B (2BS), derived from the wild emmer. A secondary aim was to determine whether genetic effects found in the field could have been predicted by other assessment methods. Methods: Roots of doubled haploid (DH) lines from a winter wheat ('Shamrock' × 'Shango') population were assessed using a seedling screen in moist paper rolls, in rhizotrons to the end of tillering, and in the field post-anthesis. A linkage map was produced using single nucleotide polymorphism markers to identify quantitative trait loci (QTLs) for rooting traits. Key Results: Shamrock had greater root length density (RLD) at depth than Shango, in the field and within the rhizotrons. The DH population exhibited diversity for rooting traits within the three environments studied. QTLs were identified on chromosomes 5D, 6B and 7B, explaining variation in RLD post-anthesis in the field. Effects associated with the non-glaucous trait on RLD interacted significantly with depth in the field, and some of this interaction mapped to 2BS. The effect of genotype was strongly influenced by the method of root assessment, e.g. glaucousness expressed in the field was negatively associated with root length in the rhizotrons, but positively associated with length in the seedling screen. Conclusions: To our knowledge, this is the first study to identify QTLs for rooting at depth in field-grown wheat at mature growth stages. Within the population studied here, our results are consistent with the hypothesis that some of the variation in rooting is associated with recent introgression from wild emmer. The expression of genetic effects differed between the methods of root assessment.
The dynamics of a long-term cold acclimation (CA) was studied in spring barley cultivar Atlas 68, winter barley cultivar Igri and a set of doubled haploid (DH) lines derived from an Atlas 68xIgri cross. The aim was to evaluate the effect of plant development on the ability to induce frost tolerance (FT) and to accumulate dehydrin 5 (DHN5) during CA. The plant developmental stage was evaluated by phenological development of the shoot apex and by determination of days to heading after a certain period of CA. FT was determined by direct frost tests. Plant winter survival was also determined. DHN5 was evaluated by densitometric analysis of protein gel blots. Cold led to the induction of increased FT and to the accumulation of DHN5 in both spring and winter lines. However, with the progression of CA, differences between the growth habits occurred as the winter lines were able to maintain increased FT and DHN5 levels for a significantly longer period of time than the spring lines. After vegetative/reproductive transition, a significant decrease in DHN5 accumulation was found in all lines; however, a discrepancy between the acquired FT level and DHN5 accumulation in vernalized winter barley plants was found. A correlation between DHN5 accumulation and plant winter survival was found when the studied lines were differentiated according to their developmental stage and DHN5 level. Possible explanations for these phenomena are provided.
- MeSH
- aklimatizace genetika fyziologie MeSH
- chov MeSH
- haploidie MeSH
- ječmen (rod) genetika fyziologie MeSH
- listy rostlin metabolismus fyziologie MeSH
- nízká teplota MeSH
- rostlinné proteiny biosyntéza genetika metabolismus MeSH
- výhonky rostlin fyziologie MeSH
- zmrazování MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The production of doubled haploid (DH) barley plants through anther culture is a very useful yet simple in vitro technique. DH plants derive from divisions of haploid microspores that have undergone a developmental switch under the appropriate conditions. The successive divisions lead to the formation of an embryo or callus rather than the formation of mature pollen grains. Plants that regenerate from these embryos are often either haploid, in which case their chromosome set can be doubled by treatment with colchicine, or spontaneous double haploids. The efficiency of DH plant production is highly variable depending on the genotype of the source material. Despite this limitation, DH plants have been widely used in breeding and research programs. Compared to conventional approaches, breeding strategies that makes use of DH plants achieve a homozygous state, allowing transgene or mutation stabilization in the genome, within a considerably shorter time, thus accelerating workflow or reducing work volume.
- MeSH
- barvení a značení MeSH
- DNA rostlinná genetika MeSH
- haploidie MeSH
- ječmen (rod) růst a vývoj MeSH
- kultivační média MeSH
- květy růst a vývoj MeSH
- pyl růst a vývoj MeSH
- regenerace MeSH
- somatická embryogeneze rostlin MeSH
- sterilizace MeSH
- techniky tkáňových kultur metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The detection of meiotic crossovers in crop plants currently relies on scoring DNA markers in a segregating population or cytological visualization. We investigated the feasibility of using flow-sorted haploid nuclei, Phi29 DNA polymerase-based whole-genome-amplification (WGA) and multi-locus KASP-genotyping to measure meiotic crossovers in individual barley pollen grains. To demonstrate the proof of concept, we used 24 gene-based physically mapped single nucleotide polymorphisms to genotype the WGA products of 50 single pollen nuclei. The number of crossovers per chromosome, recombination frequencies along chromosome 3H and segregation distortion were analysed and compared to a doubled haploid (DH) population of the same genotype. The number of crossovers and chromosome wide recombination frequencies show that this approach is able to produce results that resemble those obtained from other methods in a biologically meaningful way. Only the segregation distortion was found to be lower in the pollen population than in DH plants.
- MeSH
- chromozomy rostlin MeSH
- crossing over (genetika) * MeSH
- DNA rostlinná MeSH
- genom rostlinný MeSH
- haploidie MeSH
- ječmen (rod) genetika MeSH
- lokus kvantitativního znaku MeSH
- meióza genetika MeSH
- multilokusová sekvenční typizace * MeSH
- pyl genetika MeSH
- rekombinace genetická MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The moss Physcomitrella patens is unique for the high frequency of homologous recombination, haploid state, and filamentous growth during early stages of the vegetative growth, which makes it an excellent model plant to study DNA damage responses. We used single cell gel electrophoresis (comet) assay to determine kinetics of response to Bleomycin induced DNA oxidative damage and single and double strand breaks in wild type and mutant lig4 Physcomitrella lines. Moreover, APT gene when inactivated by induced mutations was used as selectable marker to ascertain mutational background at nucleotide level by sequencing of the APT locus. We show that extensive repair of DSBs occurs also in the absence of the functional LIG4, whereas repair of SSBs is seriously compromised. From analysis of induced mutations we conclude that their accumulation rather than remaining lesions in DNA and blocking progression through cell cycle is incompatible with normal plant growth and development and leads to sensitive phenotype.
- MeSH
- analýza jednotlivých buněk MeSH
- bleomycin farmakologie MeSH
- buněčný cyklus genetika MeSH
- haploidie * MeSH
- homologní rekombinace genetika MeSH
- mechy genetika růst a vývoj MeSH
- mutace MeSH
- mutageneze genetika MeSH
- mutageny farmakologie MeSH
- oprava DNA genetika MeSH
- oxidační stres účinky léků MeSH
- poškození DNA účinky léků MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné proteiny biosyntéza genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In male germ cells the repair of DNA double strand breaks (DSBs) differs from that described for somatic cell lines. Irradiation induced immunofluorescent foci (IRIF's) signifying a double strand DNA breaks, were followed in spermatogenic cells up to 16 h after the insult. Foci were characterised for Mdc1, 53BP1 and Rad51 that always were expressed in conjecture with gamma-H2AX. Subsequent spermatogenic cell types were found to have different repair proteins. In early germ cells up to the start of meiotic prophase, i.e. in spermatogonia and preleptotene spermatocytes, 53BP1 and Rad51 are available but no Mdc1 is expressed in these cells before and after irradiation. The latter might explain the radiosensitivity of spermatogonia. Spermatocytes from shortly after premeiotic S-phase till pachytene in epithelial stage IV/V express Mdc1 and Rad51 but no 53BP1 which has no role in recombination involved repair during the early meiotic prophase. Mdc1 is required during this period as in Mdc1 deficient mice all spermatocytes enter apoptosis in epithelial stage IV when they should start mid-pachytene phase of the meiotic prophase. From stage IV mid pachytene spermatocytes to round spermatids, Mdc1 and 53BP1 are expressed while Rad51 is no longer expressed in the haploid round spermatids. Quantifying foci numbers of gamma-H2AX, Mdc1 and 53BP1 at various time points after irradiation revealed a 70% reduction after 16 h in pachytene and diplotene spermatocytes and round spermatids. Although the DSB repair efficiency is higher then in spermatogonia where only a 40% reduction was found, it still does not compare to somatic cell lines where a 70% reduction occurs in 2 h. Taken together, DNA DSBs repair proteins differ for the various types of spermatogenic cells, no germ cell type possessing the complete set. This likely leads to a compromised efficiency relative to somatic cell lines. From the evolutionary point of view it may be an advantage when germ cells die from DNA damage rather than risk the acquisition of transmittable errors made during the repair process.
- MeSH
- chromozomální proteiny, nehistonové MeSH
- DNA vazebné proteiny MeSH
- DNA * účinky záření MeSH
- dvouřetězcové zlomy DNA * MeSH
- fluorescenční protilátková technika MeSH
- fosfoproteiny * metabolismus MeSH
- histony metabolismus MeSH
- inbrední kmeny myší MeSH
- intracelulární signální peptidy a proteiny fyziologie genetika metabolismus MeSH
- meióza fyziologie MeSH
- myši knockoutované MeSH
- myši MeSH
- oprava DNA * MeSH
- rekombinasa Rad51 metabolismus MeSH
- rentgenové záření MeSH
- spermatocyty * účinky záření MeSH
- spermatogonie metabolismus MeSH
- vztah dávky záření a odpovědi MeSH
- zárodečné buňky metabolismus účinky záření MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
During meiotic prophase I, tightly regulated processes take place, from pairing and synapsis of homologous chromosomes to recombination, which are essential for the generation of genetically variable haploid gametes. These processes have canonical meiotic features conserved across different phylogenetic groups. However, the dynamics of meiotic prophase I in non-mammalian vertebrates are poorly known. Here, we compare four species from Sauropsida to understand the regulation of meiotic prophase I in reptiles: the Australian central bearded dragon (Pogona vitticeps), two geckos (Paroedura picta and Coleonyx variegatus) and the painted turtle (Chrysemys picta). We first performed a histological characterization of the spermatogenesis process in both the bearded dragon and the painted turtle. We then analyzed prophase I dynamics, including chromosome pairing, synapsis and the formation of double strand breaks (DSBs). We show that meiosis progression is highly conserved in reptiles with telomeres clustering forming the bouquet, which we propose promotes homologous pairing and synapsis, along with facilitating the early pairing of micro-chromosomes during prophase I (i.e., early zygotene). Moreover, we detected low levels of meiotic DSB formation in all taxa. Our results provide new insights into reptile meiosis.
- Publikační typ
- časopisecké články MeSH
Flowering time variation was identified within a mapping population of doubled haploid lines developed from a cross between the introgressive line 8.1 and spring bread wheat cv. Tähti. The line 8.1 carried introgressions from tetraploid Triticum militinae in the cv. Tähti genetic background on chromosomes 1A, 2A, 4A, 5A, 7A, 1B and 5B. The most significant QTL for the flowering time variation was identified within the introgressed region on chromosome 5A and its largest effect was associated with the VRN-A1 locus, accounting for up to 70% of phenotypic variance. The allele of T. militinae origin was designated as VRN-A1f-like. The effect of the VRN-A1f-like allele was verified in two other mapping populations. QTL analysis identified that in cv. Tähti and cv. Mooni genetic background, VRN-A1f-like allele incurred a delay of 1.9-18.6 days in flowering time, depending on growing conditions. Sequence comparison of the VRN-A1f-like and VRN-A1a alleles from the parental lines of the mapping populations revealed major mutations in the promoter region as well as in the first intron, including insertion of a MITE element and a large deletion. The sequence variation allowed construction of specific diagnostic PCR markers for VRN-A1f-like allele determination. Identification and quantification of the effect of the VRN-A1f-like allele offers a useful tool for wheat breeding and for studying fine-scale regulation of flowering pathways in wheat.
Chromera velia is an alveolate alga which represents the closest known phototrophic relative to apicomplexan parasites. Although the nuclear, mitochondrial, and plastid genomes of this alga have been sequenced, the number of chromosomes and ploidy of C. velia are unknown. We explored ploidy in the vegetative cell, the predominant stage in cultures of Chromera, using the tyramide signal amplification-fluorescence in situ hybridization (TSA-FISH) in isolated nuclei of C. velia. Probes were derived from three single copy genes coding for 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP) synthase and Topoisomerase II. Our results indicate that the vegetative cell of C. velia is haploid, as each probe produced a single fluorescent signal, although the possibility of diploidy with somatic pairing of homologous chromosomes cannot be completely excluded. Restriction analysis and hybridization with the telomere probe produced eight bands suggesting the presence of four chromosomes in haploid vegetative cells of C. velia. However, when the chromerid-specific telomere probe (TTTAGGG)4 was used for TSA-FISH, we consistently obtained a double signal. This may indicate that the four chromosomes are organized in clusters in interphase nuclei of C. velia, which is a chromosome organization similar to that of their apicomplexan relatives.
Telomeres represent the repetitive sequences that cap chromosome ends and are essential for their protection. Telomere length is known to be highly heritable and is derived from a homeostatic balance between telomeric lengthening and shortening activities. Specific loci that form the genetic framework underlying telomere length homeostasis, however, are not well understood. To investigate the extent of natural variation of telomere length in Arabidopsis thaliana, we examined 229 worldwide accessions by terminal restriction fragment analysis. The results showed a wide range of telomere lengths that are specific to individual accessions. To identify loci that are responsible for this variation, we adopted a quantitative trait loci (QTL) mapping approach with multiple recombinant inbred line (RIL) populations. A doubled haploid RIL population was first produced using centromere-mediated genome elimination between accessions with long (Pro-0) and intermediate (Col-0) telomere lengths. Composite interval mapping analysis of this population along with two established RIL populations (Ler-2/Cvi-0 and Est-1/Col-0) revealed a number of shared and unique QTL. QTL detected in the Ler-2/Cvi-0 population were examined using near isogenic lines that confirmed causative regions on chromosomes 1 and 2. In conclusion, this work describes the extent of natural variation of telomere length in A. thaliana, identifies a network of QTL that influence telomere length homeostasis, examines telomere length dynamics in plants with hybrid backgrounds, and shows the effects of two identified regions on telomere length regulation.
- MeSH
- Arabidopsis genetika MeSH
- genetická variace * MeSH
- jednonukleotidový polymorfismus MeSH
- lokus kvantitativního znaku MeSH
- mapování chromozomů MeSH
- molekulární evoluce MeSH
- populační genetika MeSH
- selekce (genetika) * MeSH
- telomery * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
