Climate-growth relationships of Pinus tabuliformis along an altitudinal gradient on Baiyunshan Mountain,Central China

A set of standard chronologies for tree-ring width(TRW),earlywood width(EWW)and latewood width(LWW)in Pinus tabuliformis Carr.along an altitudi-nal gradient(1450,1400,and 1350 m a.s.l.)on Baiyunshan Mountain,Central China to analyze the effect of varying temperature and precipitation on growth along the gradi-ent.Correlation analyses showed that at all three altitudes and the TRW and EWW chronologies generally had signifi-cant negative correlations with mean and maximum tem-peratures in the current April and May and with minimum temperatures in the prior July and August,but significant positive correlations with precipitation in the current May.Correlations were generally significantly negative between LWW chronologies and all temperatures in the prior July and August,indicating that the prior summer temperature had a strong lag effect on the growth of P.tabuliformis that increased with altitude.The correlation with the standard-ized precipitation evapotranspiration index(SPEI)confirmed that wet conditions in the current May promoted growth of TR and EW at all altitudes.Significant altitudinal differences were also found;at 1400 m,there were significant positive correlations between EWW chronologies and SPEI in the current April and significant negative correlations between LWW chronologies and SPEI in the current September,but these correlations were not significant at 1450 m.At 1350 m,there were also significant negative correlations between the TRW and the EWW chronologies and SPEI in the prior October and the current July and between LWW chronology and SPEI in the current August,but these cor-relations were not significant at 1400 m.Moving correlation results showed a stable response of EWW in relation to the SPEI in the current May at all three altitudes and of LWW to maximum temperature in the prior July-August at 1400 m from 2002 to 2018.The EWW chronology at 1400 m and the LWW chronology at 1450 m were identified as more suitable for climate reconstruction.These results provide a strong scientific basis for forest management decisions and climate reconstructions in Central China.

Effects of Ion Implantation on in Vitro Pollen Germination and Cellular Organization of Pollen Tube in Pinus thunbergii Parl. (Japanese Black Pine)

Low-energy ion implantation, as a new technology to produce mutation in plant breeding, has been widely applied in agriculture in China. But so far there is a little understanding of the underlying mechanisms responsible for its biological effects at the cellular level. Here we report the biological effects of a nitrogen ion beams of 30 keV on the pollen grains of Pinus thunbergii Parl. In general, ion implantation inhibited pollen germination. The dose-response curve presented a particular saddle-like pattern. Ion implantation also changed the dimension of the elongated tubes and significantly induced tip swelling. Confocal microscopy indicated that the pollen tube tips in P. thunbergii contained an enriched network of microtubules. Ion implantation led to the disruption of microtubules especially in swollen tips. Treatment with colchicine demonstrated that tip swelling was caused by the disruption of microtubules in the tip, indicating a unique role for microtubules in maintaining the tip integrality of the pollen tube in conifer. Our results suggest that ion implantation induce the disruption of microtubule organization in pollen and pollen tubes and subsequently cause morphological abnormalities in the pollen tubes. This study may provide a clue for further investigation on the interaction between low-energy ion beams and pollen tube growth.

Metabolites from Resistant and Susceptible <i>Pinus thunbergii</i>after Inoculation with Pine Wood Nematode

Pine wilt disease (PWD), which is caused by pine wood nematodes (PWN), is one of the most serious forest diseases worldwide. To clarify the mechanism of resistance to PWD, we compared metabolites from resistant and susceptible Japanese black pine (Pinus thunbergii) families after inoculation with PWN. After 2 weeks to 1 month post inoculation, the number of PWN dramatically increased in susceptible plants, but not in resistant plants. At this PWN-proliferation phase, ethyl acetate soluble fractions extracted from PWN-inoculated plants were analyzed by gas chromatogramphy-mass spectrometry (GC-MS). Although most compounds were qualitatively and quantitatively similar between resistant and susceptible plants, resistant plants accumulated 2.0-fold more linoleic acid (LA) than susceptible plants. On the other hand, benzoic acid (BA) was barely detected in resistant plants, but it accumulated in susceptible plants as the number of PWN increased. Susceptible plants contained greater levels of the nematicidal compounds pinosylvin and pinosylvin monomethyl ether, compared with resistant plants. These results suggested that LA is involved in the resistance reaction against PWN-proliferation, and that BA could be a good biomarker for PWD.

Optimization of Ultrasonic Extraction Conditions of Polyphenols from the Needles of Black Pine (Pinus thunbergii) Using Response Surface Methodology (RSM)

Ultrasonic extraction conditions of polyphenols from the needles of black pine(Pinus thunbergii) were optimized by single factors analysis and Box-Behnken experiment design. The results showed that both of ethanol concentration and ultrasonic time had a significant effect on polyphenol extraction, and there was significant interaction between any two variables in the three parameters of ethanol concentration, ultrasonic temperature and ultrasonic time. The optimal parameters for polyphenol extraction were the ethanol concentration of 52.67%, ultrasonic temperature at 49.21 ℃and ultrasonic time of 30.76 min. Under these conditions, the actual content of polyphenols was close to the predicted value. Therefore, the process can be used to extract polyphenols from black pine needles.

Performance of Weibull function as a diameter distribution model for Pinus thunbergii stands in the eastern coast of South Korea

This study was carried out to determine the performance of percentile-based Weibull diameter distribution model for Pinus thunbergii stands thriving along the eastern coast of South Korea. The parameter recovery technique was used to estimate the three parameters of the Weibull model. The analysis demonstrated satisfactory results based on the following test statistics for the principal percentile models： fit index （FI） range from 0.501（minimum diameter） to 0.932 （50th diameter percentiles） and root mean square error （RMSE） range from 0.112 （quadratic mean diameter） to 3.572 （minimum diameter）. The developed model was further evaluated by determining the mean bias （E） in trees per ha （TPH） for each diameter class, and the results showed highest over-prediction in the 20 cm, and under-prediction in the x6 cm and 24 cm diameter classes. The goodness of fit tested by Kolmogorov- Smirnov （KS） test showed no significant differences （P〉0.05） between the observed and predicted diameter distributions for almost all plots. Using site index and aboveground biomass （AGB） models developed for P. thunbergii in South Korea, a model to predict the AGB per ha for each diameter class and subsequently the total AGB of the stand was created. An application guide was also created, which will serve as a decision-support tool for forest managers in quantifying the future total AGB in P. thunbergii stands located in the eastern coast of South Korea and, subsequently, the quantification of potential carbon stocks aside from being a vital input in designing efficient management and protection strategies for these stands.

Ion Implantation Hampers Pollen Tube Growth and Disrupts Actin Cytoskeleton Organization in Pollen Tubes of Pinus thunbergii

Pollen grains of Pinus thunbergii Parl. （Japanese black pine） were implanted with 30 keV nitrogen ion beams and the effects of nitrogen ion implantation on pollen tube growth in vitro and the organization of actin cytoskeleton in the pollen tube cell were investigated using a confocal laser scanning microscope after fluorescence labeling. Treatment with ion implantation significantly blocked pollen tube growth. Confocal microscopy showed that ion implantation disrupted actin filament cytoskeleton organization in the pollen tube. It was found that there was a distinct correlation between the inhibition of pollen tube growth and the disruption of actin cytoskeleton organization, indicating that an intact actin cytoskeleton is essential for continuous pollen tube elongation in Pinus thunbergii. Although the detailed mechanism for the ion-implantation-induced bioeffect still remains to be elucidated, the present study assumes that the cytoskeleton system in pollen grains may provide a key target in response to ion beam implantation and is involved in mediating certain subsequent cytological changes.

Allelopathic Effect of Black Pine( Pinus thunbergii) Needles and Litter on Four Cover Plants

Black pine( Pinus thunbergii) is one of the main afforestation species in the sandy coast and the preferred tree species for landscape green in northern China.Allelopathic activity could be one of the reasons of the sparse vegetation under black pine forests.To explore the allelopathic potential of P.thunbergii,the effects of litter and fresh needles were evaluated against the common cover plants seeds and seedlings,i.e.Tateges erecta,Orychophragmus violaceus,Ophiopogon japonicas and Trifolium repens.The results showed that P.thunbergii possessed allelopathic potential to the accompanying plants.Increasing concentrations of aqueous extracts of needles and litter affected target plant development in different levels.Growth and germination inhibition were directly related to the extract concentrations of needles or litters.Fresh needles played a more important role in the inhibition to target plants than litter because of the less germination rate and biomass in the same concentration.In addition,the adaptability of cover plants to the allelopathic effects of P.thunbergii is obviously different.The sensitivities of the four cover plants to the allelopathic effects of P.thunbergii were in order of T.repens > O.japonicas > O.violaceus > T.erecta.Therefore,T.repens is least suitable for concomitant planting with P.thunbergii,and T.erecta is the most suitable,with other two plants in between in landscape configuration.

PCR-Mediated Detection of Endophytic and Phytopathogenic Fungi from Needles of the Japanese Black Pine, <i>Pinus thunbergii</i>

A specific and sensitive polymerase chain reaction (PCR) assay based on the internal transcribed spacer (ITS) region of rDNA sequences was developed to detect endophytic and phytopathogenic fungi from needles of the Japanese black pine, Pinus thunbergii. Sequences of the ITS regions of Lophodermium conigenum, Lecanosticta acicola, Pestalotiopsis neglecta, Rhizosphaera kalkhoffii, and Septorioides pini-thunbergii were compared, and each specific primer pair for these species was designed. First, the designed primer pairs were tested for their specificity to detect each species. A PCR product was amplified only each combination of species and its specific primer pair, confirming the specificity of the designed primer pairs. These primer pairs were also tested on DNA extracted from the needles of P. thunbergii. The PCR products were amplified not only in needles with lesions but also in healthy needles without symptoms. Furthermore, several endophytic and phytopathogenic fungi could be simultaneously detected from the same region in a needle. The PCR-mediated detection method developed in this study will be a valuable tool for the detection of the endophytic and phytopathogenic fungi, not only as a rapid diagnostic tool for early detection but also for monitoring variations in both the quality and quantity of the endophytic and phytopathogenic fungi in needles in Japanese black pines.

Potential reduction in carbon fixation capacity under climate change in a Pinus koraiensis forest

There has been an increasing recognition of the crucial role of forests, responsible for sequestering atmospheric CO_(2), as a moral imperative for mitigating the pace of climate change. The complexity of evaluating climate change impacts on forest carbon and water dynamics lies in the diverse acclimations of forests to changing environments. In this study, we assessed two of the most common acclimation traits, namely leaf area index and the maximum rate of carboxylation(V_(cmax)), to explore the potential acclimation pathways of Pinus koraiensis under climate change. We used a mechanistic and process-based ecohydrological model applied to a P. koraiensis forest in Mt. Taehwa, South Korea. We conducted numerical investigations into the impacts of(i) Shared Socioeconomic Pathways 2–4.5(SSP2-4.5) and 5–8.5(SSP5-8.5),(ii) elevated atmospheric CO_(2) and temperature, and(iii) acclimations of leaf area index and V_(cmax)on the carbon and water dynamics of P. koraiensis. We found that there was a reduction in net primary productivity(NPP) under the SSP2-4.5 scenario, but not under SSP5-8.5, compared to the baseline, due to an imbalance between increases in atmospheric CO_(2) and temperature. A decrease in leaf area index and an increase in V_(cmax)of P. koraiensis were expected if acclimations were made to reduce its leaf temperature. Under such acclimation pathways, it would be expected that the well-known CO_(2) fertilizer effects on NPP would be attenuated.

Microenvironmental effects on growth response of Pinus massoniana to climate at its northern boundary in the Tongbai Mountains,Central China

The Tongbai Mountains is an ecologically sensi-tive region and the northern boundary of Pinus massoniana Lamb.To analyze the effect of different microenvironments on tree growth response to climate factors,we developed standard chronologies for earlywood width(EWW),late-wood width(LWW),and total ring width(TRW)of P.massoniana at two sampling sites on slopes with different orientations,then analyzed characteristics of the chronolo-gies and their correlations with climate variables from five stations in the region and with a regional normalized differ-ence vegetation index(NDVI).Statistical results showed that the TRW/EWW/LWW chronology consistency and charac-teristics(mean sensitivity,signal to noise ratio,expressed population signal)for trees growing on the southeastern slope were much higher than for trees on the northeastern slope.Correlations indicated that temperature in current March and August has a significant positive effect on TRW/EWW/LWW formation,and the effect on the northeastern slope was weaker than on the southeastern slope.Compared to temperature,precipitation has more complicated effects on tree growth,but the effect on the northeastern slope was also generally weaker than on the southeastern slope.Step-wise linear regression analyses showed that temperature in August was the main limiting factor at the two sampling sites.Similarly,the response of tree growth on the southeast-ern slope as determined by the NDVI is better than on the northeastern slope,and the TRW/EWW/LWW chronologies for the southeastern slope explained over 50%of the total NDVI variances in June.Overall,the results indicate that the difference in the climate response of P.massoniana at two sampling sites is clearly caused by differences in the microenvironment,and such differences should be properly considered in future studies of forest dynamics and climate reconstructions.

Old Pinus massoniana forests benefit more from recent rapid warming in humid subtropical areas of central-southern China

Trees progress through various growth stages,each marked by specific responses and adaptation strate-gies to environmental conditions.Despite the importance of age-related growth responses on overall forest health and management policies,limited knowledge exists regarding age-related effects on dendroclimatic relationships in key subtropical tree species.In this study,we employed a den-drochronological method to examine the impact of rapid warming on growth dynamics and climatic sensitivity of young(40–60 years)and old(100–180 years)Pinus mas-soniana forests across six sites in central-southern China.The normalized log basal area increment of trees in both age groups increased significantly following rapid warming in 1984.Trees in young forests further showed a distinct growth decline during a prolonged severe drought(2004–2013),whereas those in old forests maintained growth increases.Tree growth was more strongly influenced by temperature than by moisture,particularly in old forests.Spring tem-peratures strongly and positively impacted the growth of old trees but had a weaker effect on young ones.Old forests had a significantly lower resistance to extreme drought but faster recovery compared to young forests.The“divergence problem”was more pronounced in younger forests due to their heightened sensitivity to warming-induced drought and heat stress.With ongoing warming,young forests also may initially experience a growth decline due to their heightened sensitivity to winter drought.Our findings underscore the importance of considering age-dependent changes in forest/tree growth response to warming in subtropical forest man-agement,particularly in the context of achieving“Carbon Peak&Carbon Neutrality”goals in China.

Stability assessment of tree ring growth of Pinus armandii Franch in response to climate change based on slope directions at the Lubanling in the Funiu Mountains,China

Global warming will affect growth strategies and how trees will adapt.To compare the response of tree radial growth to climate warming in different slope directions,samples of Pinus armandii Franch were collected and treering chronologies developed on northern and western slopes from the Lubanling in the Funiu Mountains.Correlation analyses showed that two chronologies were mainly limited by temperatures in the previous June–August and the combination of temperatures and moisture in the current May–July.The difference of the climate response to slopes was small but not negligible.Radial growth of the LBL01 site on the northern slope was affected by the combined maximum and minimum temperatures,while that of the LBL02 site was affected by maximum temperatures.With regards to moisture,radial growth of the trees on the north slope was influenced by the relative humidity in the current May–July,while on the western slope,it was affected by the relative humidity in the previous June–August,the current May–July and the precipitation in the current May–July.With the change in climate,the effects of the main limiting factors on growth on different slopes were visible to a certain extent,but the differences in response of trees on different slopes gradually decreased,which might be caused by factors such as different slope directions and the change in diurnal temperature range.These results may provide information for forest protection and ecological construction in this region,and a scientific reference for future climate reconstruction.

Stability assessment of tree ring growth of Pinus armandii Franch in response to climate change based on slope directions at the Lubanling in the Funiu Mountains,China

Global warming will affect growth strategies and how trees will adapt.To compare the response of tree radial growth to climate warming in different slope directions,samples of Pinus armandii Franch were collected and tree-ring chronologies developed on northern and western slopes from the Lubanling in the Funiu Mountains.Correlation analyses showed that two chronologies were mainly limited by temperatures in the previous June-August and the com-bination of temperatures and moisture in the current May-July.The difference of the climate response to slopes was small but not negligible.Radial growth of the LBLO1 site on the northern slope was affected by the combined maximum and minimum temperatures,while that of the LBLO2 site was affected by maximum temperatures.With regards to moisture,radial growth of the trees on the north slope was influenced by the relative humidity in the current May-July,while on the western slope,it was affected by the relative humidity in the previous June-August,the current May-July and the precipitation in the current May-July.With the change in climate,the effects of the main limiting factors on growth on different slopes were visible to a certain extent,but the differences in response of trees on different slopes gradually decreased,which might be caused by factors such as different slope directions and the change in diurnal temperature range.These results may provide information for forest protection and ecological construction in this region,and a scientific reference for future climate reconstruction.

Advances in Research of Drought Stress in Major Pinus spp.: A Bibliometric Analysis and Narrative Review

Climate change has caused fluctuations in the frequency and severity of droughts,favoring extended periods of drought associated with anthropic actions and triggering other stressful abiotic effects that threaten terrestrial ecosystems.As climate warming intensifies,drought is a major challenge for forest growth.Pine(Pinus Linn.)is an important genus of forest in the Northern Hemisphere and has a certain tolerance to drought.This article analyzes and reviews the advances in research about drought stress of major Pinus spp.plants in recent years and discusses understanding and future core problems.To adapt to water-deficient environments,pine plants adapt to drought by changing growth traits,closing some stomata on leaves,changing the growth and structure of roots,and adjusting their physiological activities.Moreover,the expression of specific genes is altered,causing changes in the expression of several signaling molecules and metabolites to counteract drought stress.

Effects of drought on non-structural carbohydrates and C,N,and P stoichiometric characteristics of Pinus yunnanensis seedlings

To study non-structural carbohydrate character-istics and nutrient utilization strategies of Pinus yunnanen-sis under continuous drought conditions,2-year-old seed-lings were planted in pots with appropriate water,light and moderate and severe drought treatments[(80±5),(65±5),(50±5),and(35±5)%of field water-holding capacity].Non-structural carbohydrates,carbon(C),nitrogen(N),and phosphorus(P)concentrations were measured in each plant component.The results show that:(1)With increasing drought,non-structural carbohydrates gradually increased in leaves,stems,and coarse roots,while gradually decreased in fine roots;(2)C concentrations of all were relatively stable under different stress levels.Phosphorous utilization of each component increased under light and moderate drought conditions,while N and P utilization efficiency of each plant component decreased under severe drought.Growth was mainly restricted by N,first decreasing and then increasing with increased drought;(3)There was a correlation between the levels of non-structural carbohydrates and C,N,and P in each component.Changes in N concentration affected the interconversion between soluble sugar and starch,which play a regulatory role in the fluctuation of the concentration of non-structural carbohydrates;and,(4)Plasticity analysis showed that P.yunnanensis seedlings responded to drought mainly by altering starch concentration,the ratio of soluble sugar to starch in leaves and stems,and further by alter-ing N and P utilization efficiencies.Overall,these results suggest that the physiological activities of all organs of P.yunnanensis seedlings are restricted under drought and that trade-offs exist between different physiological indicators and organs.Our findings are helpful in understanding non-structural carbohydrate and nutrient adaptation mechanisms under drought in P.yunnanensis seedlings.

The Identification of Phenylalanine Ammonia-Lyase(PAL)Genes from Pinus yunnanensis and an Analysis of Enzyme Activity in vitro

Phenylalanine ammonia lyase(PAL)is the rate-limiting and pivotal enzyme of the general phenylpropanoid path-way,but few reports have been found on PAL genes in Pinus yunnanensis.In the present study,three PAL genes were cloned and identified from P.yunnanensis seedlings for thefirst time,namely,PyPAL-1,PyPAL-2,and PyPAL-3.Our results indicated that the open-reading frames of PyPAL genes were 2184,2157,and 2385 bp.Phylogenetic tree analysis revealed that PyPALs have high homology with other known PAL genes in other plants.In vitro enzymatic analysis showed that all three PyPAL recombinant proteins could catalyze the deamination of L-phenylalanine to form trans-cinnamic acid,but only PAL1 and PAL2 can catalyze the conversion of L-tyrosine toρ-coumaric acid.Three PyPAL genes were expressed in different tissues in 1-year-old P.yunnanensis,and such genes had different expression patterns.This study lays a foundation for further understanding of the biosynthesis of secondary metabolites in P.yunnanensis.

Effects of tree size and organ age on variations in carbon,nitrogen,and phosphorus stoichiometry in Pinus koraiensis

Carbon(C),nitrogen(N),and phosphorus(P)are of fundamental importance for growth and nutrient dynamics within plant organs and deserve more attention at regional to global scales.However,our knowledge of how these nutrients vary with tree size,organ age,or root order at the individual level remains limited.We determined C,N,and P contents and their stoichiometric ratios(i.e.,nutrient traits)in needles,branches,and fine roots at different organ ages(0-3-year-old needles and branches)and root orders(1st-4th order roots)from 64 Pinus koraiensis of varying size(Diameter at breast height ranged from 0.3 to 100 cm)in northeast China.Soil factors were also measured.The results show that nutrient traits were regulated by tree size,organ age,or root order rather than soil factors.At a whole-plant level,nutrient traits decreased in needles and fine roots but increased in branches with tree size.At the organ level,age or root order had a negative effect on C,N,and P and a positive effect on stoichiometric ratios.Our results demonstrate that nutrient variations are closely related to organ-specific functions and ecophysiological processes at an individual level.It is suggested that the nutrient acquisition strategy by younger trees and organ fractions with higher nutrient content is for survival.Conversely,nutrient storage strategy in older trees and organ fractions are mainly for steady growth.Our results clarified the nutrient utilization strategies during tree and organ ontogeny and suggest that tree size and organ age or root order should be simultaneously considered to understand the complexities of nutrient variations.

Study on Antibacterial Effects of Extracts from Pinus massoniana Lamb.Needles

[Objectives]To study the antibacterial effects of extracts from Pinus massoniana Lamb.needles.[Methods]In this experiment,the components from Pinus massoniana Lamb.needles were extracted by systematic solvent extraction method,and the antibacterial activity against four common bacteria,Escherichia coli,Staphylococcus aureus,Bacillus subtilis,Aspergillus flavus and the antibacterial active component were examined for by punch method.[Results]Different solvent extraction rate was different,the rates of petroleum ether,chloroform,ethyl acetate,n-butanol,water extracts were 4.2%,16.7%,17.4%,21.1%,40.6%.All extracts showed inhibitory effect against test bacteria.It was observed that the inhibition of G+was stronger than G-,and the extracts had the best antibacterial activity to Staphylococcus aureus while the weakest to Aspergillus flavus.The antibacterial activity of the components decreased in the order:ethyl acetate extract>n-butanol extract>chloroform extract>petroleum ether extract>aqueous phase.The extracts were stable under ultraviolet radiation(UV)light and long term storage.The antibacterial activity of the extracts was weaker with the increase of pH value when the pH value≤8.[Conclusions]It is inferred that the antibacterial components in the extract of Pinus massoniana needles are widely distributed,and the components with medium polarity or above are the main antibacterial components.

Chitosan Oligosaccharide Induces Plant Resistance Gene Expression in Pinus massoniana

Chitosan oligosaccharides(COSs)are the main degradation products from chitosan or chitin and have been reported to induce resistance to diseases in herbaceous plants like cucumber and Arabidopsis.Concomitantly,pine wilt disease(PWD)is a devastating disease of conifer tree species.Here,we hypothesized that COSs induce plant resistance gene(PRG)expression in the woody plant Masson pine,Pinus massoniana.COSs were inoculated into P.massoniana seedlings and the BGISEQ-500 platform was used to generate transcriptomes from COSs-treated P.massoniana and control seedlings.A total of 501 differentially expressed genes(DEGs)were identified by comparing the treatment and control groups.A total of 251(50.1%)DEGs were up-regulated in the treatment relative to the control seedlings and 250(49.9%)were down-regulated.Inoculation of COSs induced the expression of 31 PRGs in P.massoniana seedlings and the relative expression levels of six of the PRGs were verified by RT-qPCR.This is the first study to demonstrate that COS induces the expression of PRGs in a tree species.These results provide important insights into the function of COSs and further the prospects of developing a COS-based immune inducer for controlling PWD.

Characterization of 14 anonymous nuclear loci in Pinus thunbergii and their cross-species transferability

We characterized 14 anonymous nuclear loci from Pinus thunbergii Parl., an important pine species native to Japan. One hundred and twenty-six single nucleotide polymorphisms （SNPs） were identified from these loci, giving a frequency of 1 SNP per 51 bp. Nucleotide di- versity （0） ranged from 1.06 × 10^-3 to 11.87 × 10^-3, with all average of 4.99 × 10^-3. Only one locus （mK45） deviated significantly from the Hardy-Weinberg equilibrium. Thirteen of 14 loci were applicable in other pine species. These loci will be useful for nucleotide variation studies and will provide material for SNP-based marker development in P. thun- bergii and related species.