Kimmo Rumpunen

Phylogenetic relationships and differentiation among and within populations of Chaenomeles Lindl. (Rosaceae) estimated with RAPDs and isozymes

Abstract RAPD and isozyme analyses based on numerous markers have been used for the first time to investigate patterns of phenetic and genetic differentiation among and within nine wild populations of the genus Chaenomeles represented by the species C. japonica, C. speciosa, C. cathayensis and C. thibetica. Highly significant correlations were found between the two different marker systems for both phenetic distances and gene diversity estimates. In agreement with previous studies on cultivated Chaenomeles material, C. japonica was clearly differentiated from C. speciosa and C. cathayensis. The recently recognised species C. thibetica appeared to be rather closely related to C. cathayensis. Populations of C. japonica and C. speciosa were considerably more diverse than populations of C. cathayensis and C. thibetica. Correspondingly, most of the total variability could be attributed to the within-population differentiation in the case of C. japonica and C. speciosa, and to the between-population differentiation in the case of C. cathayensis. Differences in mating systems among the species can be suggested as a possible explanation of the results. A discordant pattern was found between RAPDs and isozymes in the analyses of population structure within C. japonica. This may be explained by a higher proportion of non-neutral markers for isozymes than for RAPDs. This finding also shows the importance of using multiple molecular marker systems in studies of population structure.

Carotenoids in sea buckthorn (Hippophae rhamnoides L.) berries during ripening and use of pheophytin a as a maturity marker.

Four cultivars of sea buckthorn berries were analyzed for their carotenoid and chlorophyll contents during ripening in three consecutive years. The different carotenoids generally increased in concentration during ripening and comprised from 120 to 1425 microg/g of DW of total carotenoids (1.5-18.5 mg/100 g of FW) depending on cultivar, harvest time, and year. GLM analyses revealed the effect of cultivar to be considerably larger than that of year and harvest time. The content of pheophytin a, a chlorophyll a derivate, steadily decreased during berry ripening. Pheophytin a therefore acted as a marker of the degree of ripening of sea buckthorn berries and was used here to convert harvest date into an estimated ripening time.

Effects of Plantago major L. leaf extracts on oral epithelial cells in a scratch assay

ETHNOPHARMACOLOGICAL RELEVANCE The present study was undertaken to evaluate the effects from different leaf extracts of the traditional medicinal herb Plantago major L. (plantain) on cell proliferation and migration in vitro, as a test for potential wound healing properties. MATERIALS AND METHODS Water and ethanol-based extracts were prepared from Plantago major fresh and dried leaves, and tested in vitro in a scratch assay with oral epithelial cells. RESULTS The scratch assay produced reliable results after 18 h. Most of the tested extracts increased the proliferation/migration of the oral epithelial cells compared to the negative control. A concentration of 1.0 mg/mL (on dry weight basis) appears to be optimal regardless of type of extract, and among the alternatives, 0.1 mg/mL was always better than 10 mg/mL. Ethanol-based extracts with a concentration of 10 mg/mL had very detrimental effects on cell proliferation/migration. At the other two concentrations, ethanol-based extracts had the most beneficial effect, followed by water extracts of fresh leaves, ethanol plus water extracts of dried leaves and, finally, water extracts of dried leaves. CONCLUSIONS This study suggests that both the water extracts and the more polyphenol-rich ethanol-based extracts of Plantago major leaves have medicinal properties. Further research is, however, needed to determine what compounds are responsible for the wound healing effects.

An Optimized Method for Analysis of Phenolic Compounds in Buds, Leaves, and Fruits of Black Currant (Ribes nigrum L.)

Although the fruits are the economic driver for the black currant industry, the buds and leaves are excellent sources of beneficial phenolic compounds that may contribute to the future value of the crop. In this study, extraction of phenolic compounds for different parts of the black currant plant was optimized, and an efficient method for their separation by HPLC was developed. This allowed the simultaneous quantification of a range of hydroxycinnamic acids, flavan-3-ols, flavonols, and anthocyanins by DAD following their identification by HPLC-ESI-MS(n). A total of 23 compounds were detected in the buds, 22 of which were found in fruit and leaves. To the best of our knowledge, this is the first report of flavonol glycosides of quercetin, myricetin, isorhamnetin, and kaempferol along with hydroxycinnamic acids such as neo-chlorogenic acid and chlorogenic acid in the buds. Additionally, we provide the first evidence of kaempferol-3-O-rutinoside in black currant leaves. This approach offers avenues for superior combined compositional identification and cultivar selection targeted at the generation of polyphenol-rich products derived from the whole crop and not just the fruit.

Tocopherols and Tocotrienols in Sea Buckthorn (Hippophae rhamnoides L.) Berries during Ripening

Sea buckthorn berries ( Hippophae rhamnoides L.) are used in foods, cosmetics, and pharmaceutical products. They are of particular interest for their high content of healthy phytochemicals, including vitamin E-related compounds (tocopherols and tocotrienols). This study investigated the content of tocopherols and tocotrienols during ripening in berries from four cultivars of sea buckthorn over a three-year period. The results showed large variations in tocopherols and tocotrienols depending on harvest date, cultivar, and year. Levels of alpha-tocopherol were higher early in the ripening period, while at later dates, delta-tocopherol levels increased. Great differences in amounts and composition of tocopherols and tocotrienols were observed between cultivars. Tocopherol levels were positively correlated with daily temperature, but this trend varied between years. Variations in tocopherols and tocotrienol levels in sea buckthorn berries due to cultivar, year, and ripening stage should therefore be considered in the production of nutritional products.

RAPD-based analysis of genetic diversity and selection of lingonberry (Vaccinium vitis-idaea L.) material for ex situ conservation

Random amplified polymorphic DNA markers were used to assess relatedness and genetic diversity for 15 lingonberry (Vaccinium vitis-idaea) populations. Seven primers yielding 59 polymorphic bands were used to analyse 13 populations, representing ssp. vitis-idaea from Sweden, Finland, Norway, Estonia and Russia, and two populations, representing ssp. minus from Japan and Canada. A cluster analysis and a multidimensional scaling analysis (MDS) showed similar phenetic patterns among populations, with a pronounced geographic grouping in most cases. Significant correlations were obtained between geographic and genetic distances for the entire set of populations as well as for the 13 ssp. vitis-idaea populations. Mean within-population diversity was 0.206 when estimated with Lynch and Milligans index, and 0.431 when estimated with Shannons index, which is in agreement with the mixed mating system reported for lingonberry. Within-population variability accounted for 68.6% of the total variance when all populations were included, and for 78.8% when only populations of ssp. vitis-idaea were analysed. Two different approaches were applied to the selection of plant material for a potential gene bank: (1) a hierarchical sampling strategy based on a cluster analysis and (2) the Maximum genetic diversity program, developed for the establishment of core collections. Random sampling was undertaken for comparisons with the selected data sets. The most diverse and representative set of lingonberry specimens was obtained when samples were selected with the Maximum diversity program.

Genetic diversity inChaenomeles (Rosaceae) revealed by RAPD analysis

Genetic relatedness inChaenomeles was studied by RAPD analysis in 42 plants representing accessions of three wild species and one hybrid taxon. Amplification with 17 primers yielded a total of 156 polymorphic RAPD bands. Estimates of genetic relatedness suggest thatC. cathayensis andC. japonica are the most distantly related species, and that the former is comparatively homogeneous.Chaenomeles speciosa, which may have arisen through hybridization betweenC. cathayensis andC. japonica, takes an intermediate position between these two species. Analysis of diagnostic bands demonstrate that neitherC. speciosa norC. ×superba has any bands that do not occur in at least one ofC. cathayensis orC. japonica. Moreover,C. speciosa and the partly overlapping taxonC. ×superba are comparatively heterogeneous, which is also in accordance with a hybrid origin. Intraspecific variation was studied mainly inC. japonica; plants obtained from different sources of material formed well separated groups in the cluster analysis.

Phenolic compounds in blackcurrant (Ribes nigrum L.) leaves relative to leaf position and harvest date

Blackcurrant leaves are an essential source of phenolic compounds and this study investigated their variation relative to leaf positions and harvest date. The phenolic content varied between harvest dates, although leaf position on the shoot and interactions also played an important role. The contents of quercetin-malonyl-glucoside, kaempferol-malonyl-glucoside isomer and kaempferol-malonyl-glucoside were higher than that of the other identified phenolic compounds, whereas epigallocatechin was the lowest for all investigated leaf positions and harvest dates. The content of several of the compounds was highest in June, while quercetin-glucoside, kaempferol-glucoside and total phenols, increased towards the end of the season. Leaf position influenced the content of myricetin-malonyl-glucoside, myricetin-malonyl-glucoside isomer, quercetin-malonyl-glucoside and kaempferol-glucoside at the end of the season. Knowledge relating to the influence of ontogenetic and harvest time on the content of specific phenolic compounds might contribute in tailoring functional foods or pharmaceutical products using blackcurrant leaves as natural ingredients.

Pollination, pollen tube growth and fertilization in Chaenomeles japonica (Japanese quince)

The fertilization system in Japanese quince, Chaenomeles japonica (Thunb.) Lindl. (Maloideae, Rosaceae) was studied by fluorescence microscopy. In a compatible combination, pollen grains germinated fast and pollen tubes grew rapidly, through the stylar tissue. Most of pollen tubes reached the base of the ovary within 2‐6 days. In the ovary, pollen tube growth was considerably retarded, and fertilization of ovules started at the earliest 3 days after pollination. In an incompatible combination only few pollen grains germinated, and pollen tubes grew slowly and were often completely arrested in the style. The pollen tubes reached the ovary in 6‐7 days. Fertilization did not take place until 7‐9 days after pollination, and the percentage of fertilized ovules was very low. The same results were obtained when selfing self-incompatible genotypes. However, a small percentage of pollen tubes reached the ovary within 3‐4 days in some genotypes including self-pollination (as in a compatible cross-pollination), but fertilization was not observed or considerably delayed compared to crosspollination. C. japonica thus shows the characteristics of a gametophytic self-incompatibility system. The viable period of the embryo sac was 6‐7 days in most of the investigated genotypes, and the effective period of pollination was estimated to 5‐7 days. Bagging was not necessary to avoid open pollination, provided that the flowers had been emasculated previously. Emasculation or bagging did not decrease fruit set and could, therefore, be used in breeding. # 2002 Elsevier Science B.V. All rights reserved.

Comparison of differentiation estimates based on morphometric and molecular data, exemplified by various leaf shape descriptors and RAPDs in the genus Chaenomeles (Rosaceae)

The efficiency of classical metric descriptors (MDs), normalised elliptic Fourier coefficients (EFCs), and the power series of normalised elliptic Fourier coefficients (PEFCs) in revealing variation in leaf shape was evaluated and compared for species in the genus Chaenomeles, using canonical variates analyses (CVAs) and reclassification tests. For EFCs 30 harmonics (and for PEFCs 40 harmonics) were needed to achieve 100% correct reassignment of plants. By contrast, MDs were considerably less efficient and only 87%, 77% and 66% of the plants were correctly reassigned to species, populations, and maternal families, respectively. Furthermore, when compared with data based on molecular random amplified polymorphic DNA markers (RAPDs), PEFCs produced the most concordant data sets as revealed by cluster analyses. To obtain comparable estimates of variability and differentiation, the AMOVA approach was used both for molecular data and, as a novelty, for quantitative morphometric data. The variation was partitioned by hierarchical extraction of variance components from matrices of standardised squared Euclidean distances, and differentiation estimates were obtained as Φ-statistics. All descriptor sets partitioned the variance in a similar way. Surprisingly high correlations were found between RAPDs and elliptic Fourier transforms for the among-family estimates of variance components (RAPDs vs. PEFCs: 0.89, P = 0.003; RAPDs vs. EFCs: 0.86, P = 0.006). In contrast, only a moderate correlation was found between RAPDs and MDs (0.71, P = 0.049). This may indicate that shape per se (the size component excluded) is a less biased estimator of genetic variation than metric leaf descriptors in the genus Chaenomeles. Estimates of population differentiation based on EFCs and PEFCs were always lower than differentiation estimates based on RAPDs, whereas the differentiation estimates based on MDs were in general higher than estimates based on RAPDs, except for one species including hybrid populations.