Øystein Johnsen

Genetic consequences of glacial survival and postglacial colonization in Norway spruce: combined analysis of mitochondrial DNA and fossil pollen

Norway spruce (Picea abies [L.] Karst.) is a broadly distributed European conifer tree whose history has been intensively studied by means of fossil records to infer the location of full‐glacial refugia and the main routes of postglacial colonization. Here we use recently compiled fossil pollen data as a template to examine how past demographic events have influenced the species’ modern genetic diversity. Variation was assessed in the mitochondrial nad1 gene containing two minisatellite regions. Among the 369 populations (4876 trees) assayed, 28 mitochondrial variants were identified. The patterns of population subdivision superimposed on interpolated fossil pollen distributions indicate that survival in separate refugia and postglacial colonization has led to significant structuring of genetic variation in the southern range of the species. The populations in the northern range, on the other hand, showed a shallow genetic structure consistent with the fossil pollen data, suggesting that the vast northern range was colonized from a single refugium. Although the genetic diversity decreased away from the putative refugia, there were large differences between different colonization routes. In the Alps, the diversity decreased over short distances, probably as a result of population bottlenecks caused by the presence of competing tree species. In northern Europe, the diversity was maintained across large areas, corroborating fossil pollen data in suggesting that colonization took place at high population densities. The genetic diversity increased north of the Carpathians, probably as a result of admixture of expanding populations from two separate refugia.

Dehydrins expression related to timing of bud burst in Norway spruce

Cold deacclimation and preparation to flushing likely requires rehydration of meristems. Therefore, water stress related genes, such as dehydrins (DHN), might play an important role in providing protection during winter dormancy, deacclimation and bud burst timing processes. Here we report the sequence analysis of several Norway spruce DHN identified in late and early flushing suppressive subtraction hybridization cDNA libraries and in our Norway spruce EST database. We obtained 15 cDNAs, representing eight genes from three distinct types of DHN, and studied differential expression of these genes before and during bud burst in spring, using qRT-PCR. We found the visible reduction in transcript level of most DHN towards the bud burst, supported by a significant down-regulation of the DHN in needles during experimental induction of bud burst applied at three time points during autumn in Norway spruce grafts. For most of the DHN transcripts, their expression levels in late-flushing spruces were significantly higher than in the early flushing ones at the same calendar dates but were remarkably similar at the same bud developmental stage. From our results we may conclude that the difference between the early and the late families is in timing of the molecular processes leading to bud burst due to differences in their response to the increasing temperature in the spring. They are induced much earlier in the early flushing families.

Effect of frost nights and day and night temperature during dormancy induction on frost hardiness, tolerance to cold storage and bud burst in seedlings of Norway spruce

For trees, the ability to obtain and maintain sufficient levels of frost hardiness in late autumn, winter and spring is crucial. We report that temperatures during dormancy induction influence bud set, frost hardiness, tolerance to cold storage, timing of bud burst and spring frost hardiness in seedlings of Norway spruce (Picea abies (L.) Karst.). Bud set occurred later in 12°C than in 21°C, and later in cool nights (7°C) than in constant temperature. One weekly frost night (−2.5°C) improved frost hardiness. Cool nights reduced frost hardiness early, but improved hardiness later during cold acclimation. Buds and stems were slightly hardier in 21°C than in 12°C, while needles were clearly hardier in 12°C. Cold daytime temperature, cool nights and one weekly frost night improved cold storability (0.7°C). Seedlings receiving high daytime temperatures burst buds later, and were less injured by light frost some days after bud burst.

Does maternal environmental condition during reproductive development induce genotypic selection in Picea abies

In forest trees, environmental conditions during reproduction can greatly influence progeny performance. This phenomenon probably results from adaptive phenotypic plasticity but also may be associated with genotypic selection. In order to determine whether selective effects during the reproduction are environment specific, single pair-crosses of Norway spruce were studied in two contrasted maternal environments (warm and cold conditions). One family expressed large and the other small phenotypic differences between these crossing environments. The inheritance of genetic polymorphism was analysed at the seed stage. Four parental genetic maps covering 66 to 78% of the genome were constructed using 190 to 200 loci. After correcting for multiple testing, there is no evidence of locus under strong and repeatable selection. The maternal environment could thus only induce limited genotypic-selection effects during reproductive steps, and performance of progenies may be mainly affected by a long-lasting epigenetic memory regulated by temperature and photoperiod prevailing during seed production.RésuméChez les arbres forestiers, les conditions environnementales durant la reproduction peuvent influencer les performances des descendants. Ce phénomène reflète probablement la plasticité phénotypique, mais également il pourrait être associé à une sélection génotypique. Afin de déterminer si des effets sélectifs durant la reproduction sont spécifiques d’un environnement donné, deux familles d’épicéa commun non apparentées ont été obtenues par croisements dirigés dans deux environnements maternels contrastés (conditions chaude et froide). La première famille exprimait de larges différences phénotypiques entre les deux environnements tandis que la seconde ne montrait pas de différence significative. La transmission des polymorphismes génétiques a été étudiée au stade de la graine. Quatre cartes génétiques parentales couvrant 66 à 78 % du génome ont été construites. Aucun effet de sélection n’a été mis en évidence aux différents locus étudiés. L’environnement maternel n’induirait donc que des effets de sélection génotypique relativement faibles durant les stades de la reproduction. Les performances des descendants seraient principalement affectées par une mémoire épigénétique durable régulée par la température et la photopériode régnant durant la production des graines.

Effects of early long-night treatment on diameter and height growth, second flush and frost tolerance in two-year-old Picea abies container seedlings

Abstract The object of this study was to obtain Norway spruce seedlings with buds set, ready for summer planting from 1 July. An early long-night treatment prevented flushing of the newly formed terminal buds and ceased height growth, but slightly reduced hardiness in buds and needles. Nevertheless, a sufficient hardiness level in the autumn was acquired at a Norwegian nursery at 59°46′ N, with plants of the local provenance given a long-night treatment (14 h) for 13 days from 25 June. Similar treatment at a nursery at 64°30′ N did not give the same result; all treatments led to a second flush with resumed growth of the local provenance. A trial with seed lots from several provenances was therefore performed at this nursery, and a significant correlation was found between the critical night length of the seed lot and their ability to produce non-flushing buds; the longer the critical night length of the seed lot, the fewer non-flushing buds. Responses at the northern nursery are probably due to the lack of a dark period after termination of the treatment, and too short a treatment period to attain bud dormancy. An early and successful long-night treatment will also produce shorter seedlings with a larger root collar diameter.

Variation in spring and autumn freezing resistance among and within Spanish wild populations of Castanea sativa

Abstract• Genetic variation in freezing resistance was evaluated among and within six populations of Spanish wild chestnut (Castanea sativa Miller). The extent to which frost susceptibility was related to phenology and the relationship between population differentiation and climatic conditions was studied.• Twigs were collected in March and November from saplings (5-year-old trees) of 41 open-pollinated families from the six populations in a provenance-progeny test, and were subjected to artificial freezing. Damage to each twig was assessed as visible browning of bud and of stem tissues.• Population differences as regards frost damage traits were highly significant (p < 0.01) in both spring and autumn. Family differences within populations were low, often non-significant, and in all cases smaller than differences among populations. Population means were closely correlated with the parental drought and frost conditions. Populations originating from dry areas or from regions where frost seldom occurs were the least resistant.• Drought is suggested to be the one of the most important selective agents that shapes population differentiation in Spanish wild chestnut, while frost may be more important in northern Spain. Phenological differences are not always good predictors of the degree of frost damage. Thus, freezing tests should be used to detect frost susceptibility in chestnuts.Résumé• La variation génétique de la résistance au gel a été évaluée entre et dans six populations espagnoles sauvages de châtaignier (Castanea sativa Miller). L’ampleur avec laquelle la sensibilité au gel est liée à la phénologie et aux relations entre la différenciation de la population et les conditions climatiques a été étudiée.• Des rameaux ont été recueillis en mars et novembre à partir de jeunes arbres (âgés de 5 ans) de 41 familles à pollinisation libre issues de six populations en test de descendances/provenances, et ont été soumis à une congélation artificielle. Les dommages à chaque rameau ont été évalués par le brunissement visible des bourgeons et des tissus de la tige.• Les différences entre population pour ce qui concerne les dégâts causés par le gel sont très significatifs (p < 0.01) au printemps et en automne. Les différences entre familles dans les populations sont faibles, souvent non significatives, et dans tous les cas plus petites que les différences parmi les populations. Les moyennes des populations sont étroitement corrélées avec les conditions de sécheresse et de gel auxquelles sont soumis les parents.Les populations originaires des régions sèches ou de régions où le gel se produit rarement sont les moins résistantes.• On suggère que la sécheresse est l’un des plus importants agents sélectifs qui forme la différenciation dans la population espagnole de châtaigniers sauvages, tandis que le gel peut être plus important dans le nord de l’Espagne. Les différences phénologiques ne sont pas toujours de bons indices du degré de dommages par le gel. Ainsi, les essais de gel doivent être utilisés pour détecter la sensibilité au gel des châtaigniers.

Effect of bud burst forcing on transcript expression of selected genes in needles of Norway spruce during autumn

Expression of selected genes in needles of Norway spruce (Picea abies [L.] Karst) was investigated by following their transcription levels during late autumn. Transcription was assessed in mature needles which likely serve as sensor of environmental cues that enable trees in the temperate and boreal regions to change between stages of growth, frost tolerance and bud dormancy. Samples were collected from grafts kept under outdoor conditions and after bud burst forcing in greenhouse at 20 degrees C (12 h darkness) for one week. Transcription was assayed with real-time RT-PCR. During the sampling period, chilling requirement was partially fulfilled, and time to bud burst after forcing was decreased. Of the 27 transcripts studied, expression of 16 was significantly affected either by forcing, sampling time, or interaction between them. PaSAP, PaACP, PaSGS3, PaWRKY, PaDIR9, PaCCCH and dehydrin genes responded drastically to forcing temperatures at all sampling points, showing no correlation with readiness for bud burst. Expression patterns of some vernalization pathway gene homologs PaVIN3, and also of PaMDC, PaLOV1 and PaDAL3 had a clear opposite trends between forcing and outdoor conditions, which could imply their role in chilling accumulation and bud burst regulation/cold acclimation. These genes could constitute putative candidates for further detailed study, whose regulation in needles may be involved in preparation towards bud burst and chilling accumulation sensing.

Secondary buds in Scots pine trees infested with Gremmeniella abietina

In winter 2000–2001, there was a serious outbreak of Gremmeniella abietina Morelet in southeastern Norway. During the outbreak, we noted that injured Scots pine trees (Pinus sylvestris L.) developed secondary buds in response to the fungus attack, and we decided to study the relationship between injury, appearance of secondary buds and recovery of the trees thereafter. For this purpose, 143 trees from 10 to 50 years of age were chosen and grouped into crown density classes. Injury was assessed in detail, and buds were counted before bud burst in the spring of 2002. In addition, a subset of 15 trees was followed through the summer of 2002 to assess recovery. All injured trees developed secondary buds, with a clear overweight of dormant winter buds in proportion to interfoliar buds. Healthy control trees did not develop secondary buds at all. The secondary buds appeared predominantly on the injured parts of the tree; interfoliar buds in particular developed just beneath the damaged tissue. Most of the secondary buds died during the winter of 2001–2002, mainly because the fungus continued to spread after the first outbreak. Many of the remaining buds developed shoots with abnormal growth during the summer. Secondary buds may help trees to recover from Gremmeniella attacks, but this strategy may fail when the fungus continues to grow and injure the newly formed buds and shoots.