Quantitative trait loci and candidate genes associated with freezing tolerance of winter triticale (× Triticosecale Wittmack).

Freezing tolerance of triticale is a major trait contributing to its winter hardiness. The identification of genomic regions - quantitative trait loci (QTL) and molecular markers associated with freezing tolerance in winter hexaploid triticale - was the aim of this study. For that purpose, a new genetic linkage map was developed for the population of 92 doubled haploid lines derived from 'Hewo' × 'Magnat' F1 hybrid. Those lines, together with parents were subjected to freezing tolerance test three times during two winter seasons. Plants were grown and cold-hardened under natural fall/winter conditions and then subjected to freezing in controlled conditions. Freezing tolerance was assessed as the plants recovery (REC), the electrolyte leakage (EL) from leaves and chlorophyll fluorescence parameters (JIP) after freezing. Three consistent QTL for several fluorescence parameters, electrolyte leakage, and the percentage of the survived plants were identified with composite interval mapping (CIM) and single marker analysis (SMA). The first locus Qfr.hm-7A.1 explained 9% of variation of both electrolyte leakage and plants recovery after freezing. Two QTL explaining up to 12% of variation in plants recovery and shared by selected chlorophyll fluorescence parameters were found on 4R and 5R chromosomes. Finally, main locus Qchl.hm-5A.1 was detected for chlorophyll fluorescence parameters that explained up to 19.6% of phenotypic variation. The co-located QTL on chromosomes 7A.1, 4R and 5R, clearly indicated physiological and genetic relationship of the plant survival after freezing with the ability to maintain optimal photochemical activity of the photosystem II and preservation of the cell membranes integrity. The genes located in silico within the identified QTL include those encoding BTR1-like protein, transmembrane helix proteins like potassium channel, and phosphoric ester hydrolase involved in response to osmotic stress as well as proteins involved in the regulation of the gene expression, chloroplast RNA processing, and pyrimidine salvage pathway. Additionally, our results confirm that the JIP test is a valuable tool to evaluate freezing tolerance of triticale under unstable winter environments.

Mechanisms involved in the regulation of photosynthetic efficiency and carbohydrate partitioning in response to low- and high-temperature flooding triggered in winter rye (Secale cereale) lines with distinct pink snow mold resistances.

In terms of climate changes and global warming, winter hardiness could be determined by unfavorable environmental conditions other than frost. These could include flooding from melting snow and/or rain, coincident with fungal diseases. Therefore, we designed an experiment to identify potential common mechanisms of flooding tolerance and snow mold resistance, involving the regulation of photosynthetic efficiency and carbohydrate metabolism at low temperatures. Snow mold-resistant and susceptible winter rye (Secale cereale) plants were characterized by considerably different patterns of response to flooding. These differences were clearer at low temperature, thus confirming a possible role of the observed changes in snow mold tolerance. The resistant plants were characterized by lower PSII quantum yields at low temperature, combined with much higher energy flux for energy dissipation from the PSII reaction center. During flooding, the level of soluble carbohydrates increased in the resistant plants and decreased in the susceptible ones. Thus increase in resistant line was connected with a decrease in the energy dissipation rate in PSII/increased photosynthetic activity (energy flux for electron transport), a lower rate of starch degradation and higher rates of sucrose metabolism in leaves. The resistant lines accumulated larger amounts of total soluble carbohydrates in the crowns than in the leaves. Irrespective of flooding treatment, the resistant lines allocated more sugars for cell wall composition, both in the leaves and crowns. Our results clearly indicated that studies on carbohydrate changes at low temperatures or during anoxia should investigate not only the alterations in water-soluble and storage carbohydrates, but also cell wall carbohydrates. The patterns of changes observed after low and high-temperature flooding were different, indicating separate control mechanisms of these responses. These included changes in the photosynthetic apparatus, starch accumulation and cell wall carbohydrate accumulation.

Influence of short-term drought conditions and subsequent re-watering on the physiology and proteome of Lolium multiflorum/Festuca arundinacea introgression forms, with contrasting levels of tolerance to long-term drought.

Festuca arundinacea is a drought tolerant species. Lolium multiflorum has better forage quality but lower tolerance to abiotic stresses. Their hybrids offer an opportunity to perform research on the molecular basis of tolerance to drought. The aim of this work was to recognise the mechanisms of response to short-term drought (11 days) in a glasshouse in two L. multiflorum/F. arundinacea introgression forms with distinct levels of tolerance to long-term drought (14 weeks) in the field. Measurements of physiological parameters, analyses of protein accumulation profiles using two-dimensional gel electrophoresis, and mass spectrometry identification of proteins, which were accumulated differentially between the selected genotypes during short-term drought, were performed. Genotype 7/6, with lower yield potential during 14 weeks of drought, and lower ability to re-grow after watering, had a higher capacity for photosynthesis during 11 days of drought. Genotype 4/10, more tolerant to long-term drought, was able to repair damaged cell membranes after watering and was also characterised by lower transpiration during short-term drought. A total of 455 proteins were analysed, and the 17 that were differentially accumulated between the two genotypes were identified. The results of physiological and proteomic research led to a hypothesis that the higher photosynthetic capacity of genotype 7/6 could be due to a more efficient Calvin cycle, supported by higher accumulation of crucial proteins involving chloroplast aldolase.

Introgression mapping of genes for winter hardiness and frost tolerance transferred from Festuca arundinacea into Lolium multiflorum.

Genes for winter hardiness and frost tolerance were introgressed from Festuca arundinacea into winter-sensitive Lolium multiflorum. Two partly fertile, pentaploid (2n = 5x = 35) F(1) hybrids F. arundinacea (2n = 6x = 42) x L. multiflorum (2n = 4x = 28) were generated and backcrossed twice onto L. multiflorum (2x). The backcross 1 (BC(1)) and backcross 2 (BC(2)) plants were preselected for high vigor and good fertility, and subsequently, a total of 83 BC(2) plants were selected for winter hardiness after 2 Polish winters and by simulated freezing tests. Genomic in situ hybridization (GISH) was performed on 6 winter-hardy plants selected after the first winter and shown to be significantly (P < 0.05) more frost tolerant than the L. multiflorum control. Among the analyzed BC(2) winter survivors, only diploid (2n = 2x = 14) plants were found. Five plants carried 13 intact L. multiflorum chromosomes and 1 L. multiflorum chromosome with a single introgressed F. arundinacea terminal chromosome segment. The sixth BC(2) winter survivor appeared to be Lolium without any Festuca introgression capable of detection by GISH. A combined GISH and fluorescence in situ hybridization analysis with rDNA probes of the most winter-hardy (after 2 winters) and frost-tolerant BC(2) plant revealed the location of an F. arundinacea introgression on the nonsatellite arm of L. multiflorum chromosome 2, the same chromosome location reported previously as a site for frost tolerance genes in the diploid and winter-hardy species Festuca pratensis.

GISH/FISH mapping of genes for freezing tolerance transferred from Festuca pratensis to Lolium multiflorum.

The first backcross breeding programme for the transfer of freezing-tolerance genes from winter hardy Festuca pratensis to winter-sensitive Lolium multiflorum is described. A partly fertile, triploid F(1) hybrid F. pratensis (2n=2x=14) x L. multiflorum (2n=4x=28) was employed initially, and after two backcrosses to L. multiflorum (2x) a total of 242 backcross two (BC(2)) plants were generated. Genomic in situ hybridisation (GISH) was performed on 61 BC(2) plants selected for their good growth and winter survival characters in the spring following one Polish winter (2000-2001). Among the winter survivors, diploid chromosome numbers were present in 80% of plants. An appropriate single Festuca introgression in an otherwise undisturbed Lolium genome could provide increased freezing tolerance without compromise to the good growth and plant vigour found in Lolium. Among all the diploids, a total of 20 individuals were identified, each with a single F. pratensis chromosome segment. Another diploid plant contained 13 Lolium chromosomes and a large metacentric F. pratensis chromosome, identified as chromosome 4, with two large distal Lolium introgressions on each chromosome arm. Three of the diploid BC(2), including the genotype with Festuca chromosome 4 DNA sequences, were found to have freezing tolerance in excess of that of L. multiflorum, and in one case in excess of the F. pratensis used as control. A detailed cytological analysis combining GISH and fluorescence in situ hybridisation analyses with rDNA probes revealed that the other two freezing-tolerant genotypes carried a Festuca chromosome segment at the same terminal location on the non-satellite arm of Lolium chromosome 2.

Cold-deacclimation of oilseed rape (Brassica napus var. oleifera) in response to fluctuating temperatures and photoperiod.

The aim of this work was to establish the role of factors that may trigger elongation growth in the dehardening response, namely temperature during daylight, photoperiod and vernalization. Fully cold-acclimated seedlings of winter (with incomplete vernalization) and spring oilseed rape were subjected to deacclimation under temperatures of 2/12, 12/2, 12/12, 12/20, 20/12 and 20/20 degrees C (day/night) and a 12 h photoperiod. Plants were also deacclimated under photoperiods of 8 and 16 h at constant temperatures of 12 and 20 degrees C. After deacclimation, plants were subjected to reacclimation. Results suggest that the level of growth activity induced during deacclimation affects both the deacclimation rate and the capacity for reacclimation. Deacclimation is fully reversible if it is not accompanied by induction of elongation growth. In such cases the rate of the decrease in freezing tolerance depends on the mean temperature of deacclimation. Deacclimation becomes partially or completely irreversible when it is connected with promotion of elongation growth. The stimuli triggering elongation growth during deacclimation may be the growth-promoting temperature (20 degrees C) during the day and the lack of vernalization blockage of elongation growth. When elongation growth was stimulated by other factors such as long-day treatments, rehardening was also disturbed.

The initiation of elongation growth during long-term low-temperature stay of spring-type oilseed rape may trigger loss of frost resistance and changes in photosynthetic apparatus.

The aim of the present investigation was to determine if the loss of frost resistance observed in spring-type oilseed rape during winter may be the effect of the tendency to start elongation growth during the prolonged low-temperature stay. Interactions between elongation growth rate, properties of photosynthetic apparatus and frost resistance were studied under these conditions in spring and winter cultivars of oilseed rape. Both spring and winter cultivars of oilseed rape reached the maximal frost resistance after 6 weeks at +5 degrees C. Photosynthetic apparatus of both cultivars acclimated to functioning in cold. The resistance of winter type plants remained unchanged at the end of the experiment (10 weeks) whereas spring-type plants lost the maximal resistance in subsequent weeks. It was preceded in the 7th week of low-temperature stay by acceleration of elongation growth without an increase in dry matter accumulation. A gradual loss of photosynthetic activity was also observed during this period. It was manifested as a decrease in antenna trapping efficiency, photochemical and non-photochemical fluorescence quenching and actual quantum yield of PSII without affecting apparent quantum yield of PSII. At the 70th day of the experiment, a decrease in CO(2) exchange and dry matter accumulation were even observed. The possible relationships between growth rate and functioning of photosynthetic apparatus are discussed.