A comparative analysis of the genetic variability and differentiation of rare medicinal ginseng species, Panax vietnamensis Ha et Grushv. and P. ginseng C.A. Meyer, was carried out using inter-simple sequence repeat markers. It was demonstrated that all the genetic diversity parameters of Vietnamese ginseng were high and considerably exceeded those of P. ginseng. On the contrary, the level of genetic differentiation was higher in true ginseng. It is suggested that the differences in the levels of genetic variability and differentiation of the two ginseng species were influenced by the demographic history, peculiarities of the reproductive system, and human activity.

The Panax vietnamensis var. fuscidiscus samples collected at Phong Tho district, Lai Chau Province have morphological characteristics very similar with those of Panax vietnamensis distributed in Ngoc Linh mountain at Quang Nam and Kon Tum provinces that hinders the identification of these taxons. To supply the information to identify two species and know the genetic distance of these species with other Panax species, we sequenced rbcL and rpoB genes that are among DNA barcoding markers recommended for plant species identification. In the rbcL gene of 700 bp in length, we found 9 nucleotide differences between Panax vietnamensis var. fuscidiscus and Panax vietnamensis and the genetic similarity was 98,8%, while in the rpoB gene of 500 bp in length, only 2 nucleotide differences were discovered and the genetic distance was 0,4%. The sequences of rbcL and rpoB of Panax vietnamensis var. fuscidiscus and Panax vietnamensis were submitted to Genbank with the accession numbers KT194325.1, KT194324.1 and KT154685.1, KT154686.1 respectively. Citation: Nguyen Thi Phuong Trang, Nguyen Thi Hong Mai, Zhuravlev Yury N., Reunova Galina D., 2017. rbcL and rpoB gene sequences of Panax vietnamensis var. fuscidiscus and panax vietnamensis, the background for identification and comparison. Tap chi Sinh hoc, 39(1): 80-85. DOI: 10.15625/0866-7160/v39n1.7870 *Corresponding author: ntptrang@yahoo.com. Received 11 March 2016, accepted 20 March 2017

Genetic variability and differentiation of larch populations (genus Larix Mill.) from Sakhalin, Iturup, Kamchatka, Primorsky Kari and Japan were studied by RAPD method. It was shown that Primorsky and Japanese samples had the highest estimations of the genetic variability, while samples from Sakhalin and Iturup had the lowest ones. Larches from Sakhalin and Kamchatka had the most differentiation. All Russian larch populations formed one cluster on the UPGMA dendrogram and one group in the plane of principal coordinates that may be evidence of their common history of forming. At the same time samples of L. kaempferi Sarg. were genetically too distant from the larch populations of Russian Far East. The conclusion that evolution history of Japanese larch differs from evolution history of other Far-East larch species of Russia is discussed.

Two mitochondrial DNA fragments, nad4(3c-4r) and nad5(1-2r), were sequenced in 58 larch accessions from the range of Larix olgensis A. Henry. Combinations of the nad4 polymorphic sites formed four haplotypes, two of which (H3 and H4) were unique and two (H1, H2) were common. Haplotype H1 was found only in pure L. olgensis from the vicinity of Olga Bay and in a number of accessions from the southern part of the range. Haplotype H2 was detected in the other samples from the range of Olga Bay larch, as well as in hybrid forms. Similarly to the nad4(3c-4r) fragment, the mtDNA fragment UBC460 was able to differentiate larch populations from the range of L. olgensis examined.

Panax ginseng C.A. Meyer is an endangered species in Russia. To restore this species, effective protective measures, including the reintroduction into favorable habitats, must be worked out considering the specificity of genetic structure of ginseng populations. One hundred and thirty-nine P. ginseng plants were collected from the forests of nine administrative areas of Primorsky Territory of Russia and transferred to a collection nursery for further investigation. Microsatellite markers were used to study the genetic diversity and the genetic structure of ginseng populations. For populations studied with SSR, the number of observed alleles was ranging from 15 to 25, allelic richness from 1.83 to 3.04, polymorphic loci from 62.5% to 87.7%, observed heterozygosity from 0.410 to 0.512 (an average of 0.453) and expected heterozygosity from 0.304 to 0.479, with an average of 0.393. The values of the inbreeding coefficient within populations (Fis) ranged from -0.447 to 0.056, and their average value was -0.296. Genetic differentiation among populations was significant (Fst = 0.115) but an isolation-by-distance pattern was not detected. UPGMA and MS-tree confirmed the presence of genetic structure within P. ginseng and visualized genetic relationships of populations with similar pattern. STRUCTURE analysis revealed the genetic admixture between different ginseng populations. It was established with SSR markers that P. ginseng still preserves substantial genetic resources although all populations are largely exhausted. Because ginseng populations are significantly differentiated all of them should be restored. Considering the admixture of ginseng populations it would be advisable to apply the individual assignment test to verify the content of indigenous populations and to identify the "true" population plants to serve as stock material for reintroduction.

Секвенированы нуклеотидные последовательности двух фрагментов nad4(3c-4r) и nad5(1-2r) у 58 представителей лиственницы из ареала Larix olgensis A. Henry. Выявлены комбинации полиморфных сайтов nad4, образующие четыре гаплотипа, два из которых уникальные (H3 и H4) и два общие (H1, H2). Отмечено, что гаплотип H1 встречался только у “чистого” вида L. olgensis из окрестностей зал. Ольги и у нескольких образцов из южной части ареала, а гаплотип H2 свойствен остальным выборкам из ареала лиственницы ольгинской, а также гибридным формам лиственниц. Установлено, что фрагмент мтДНК UBC460 дифференцирует исследованные популяции из ареала L. olgensis подобно nad4(3c-4r).

Genetic variability in ten populations of wild-growing ginseng was assessed using AFLP markers with the application of fragment analysis on a genetic analyzer. The variation indices were high in the populations (P = 55.68%, H S = 0.1891) and for the species (P = 99.65%; H S = 0.2857). Considerable and statistically significant population differentiation was demonstrated (θB = 0.363; Bayesian approach, “full model”; F ST = 0.36, AMOVA). The results of AMOVA and Bayesian analysis indicate that 64.46% of variability is found within the populations. Mantel test showed no correlation between the genetic and geographic distances among the populations (r = −0.174; p = 0.817). Hierarchical AMOVA and analysis of genetic relationships based on Euclidean distances (NJ, PCoA, and MST) identified two divergent population groups of ginseng. Low gene flow between these groups (N m = 0.4) suggests their demographic independence. In accordance to the concept of evolutionary significant units (ESU), these population groups, in terms of the strategy and tactics for conservation and management of natural resources, should be treated as management units (MUs). The MS tree topology suggests recolonization of southern Sikhote-Alin by ginseng along two directions, from south and west.

Род Panax L. (Araliaceae) широко известен в мире благодаря тому, что многие его представители обладают важными медицинскими свойствами. В сравнении с другими видами рода Panax ginseng C.A. Meyer пользуется наибольшей известностью, так как препараты из этого растения стимулируют иммунитет и помогают в борьбе с болезнями и стрессами, а настойка из корней женьшеня традиционно применяется как средство, предупреждающее старение. В настоящее время только в Приморском крае России этот вид сохранился в природе, однако его популяции крайне истощены и нуждаются в восстановлении. Исследована способность молекулярных ДНК маркеров выявлять генетическую изменчивость и дифференцировать популяции женьшеня. Генетическая изменчивость женьшеня, обнаруженная RAPD (p = 4%, Hpop = 0.0130) и ISSR маркерами (p = 9.3%, Hpop = 0.0139), была низкой. Метод AFLP*, в котором ампликоны разделяли в полиакриламидном геле и визуализировали серебром, показал несколько большую вариабельность (p = 21.8%, Hpop = 0.0509), но обнаружил низкую способность дифференцировать популяции, так же как RAPD и ISSR. Метод AFLP , включающий анализ фрагментов на генетическом анализаторе, выявил высокую генетическую изменчивость у женьшеня (p = 94.4%, Hpop = 0.3246). Все популяции, исследованные AFLP** маркерами, были значимо дифференцированы по результатам AMOVA. На основе полученных данных AFLP** маркеры признаны перспективными для характеристики генетической структуры и генетических связей популяций женьшеня и рекомендованы для широкомасштабного популяционно-генетического изучения этого вида с целью разработки предложений по его сохранению.

The genus Panax (Araliaceae) is world-famous because many its members have important medicinal properties. Panax ginseng C. A. Meyer is more popular than other species of the genus because remedies prepared from this plant stimulate immunity, help to prevent diseases, and have antistress effects. In addition, the ginseng root extract is traditionally used as a means against aging. At present, this species is found in the wild only in Primorsky krai, Russia, but its populations are extremely exhausted and need to be restored. In this study, effectiveness of molecular DNA markers in detecting genetic variation and differentiation of the ginseng populations was tested. Genetic variation of ginseng, identified using RAPD (P = 4%; H pop = 0.0130) and ISSR (P = 9.3%; H pop = 0.0139) markers was low. The AFLP* approach, according to which amplicons are separated in polyacrylamide gel and visualized by means of silver staining, showed somewhat higher variability (P = 21.8%; H pop = 0.0509), while its effectiveness in population differentiation was as low as that of RAPD and ISSR. The AFLP** technique, which included analysis of the fragments using genetic analyzer, revealed high genetic diversity of ginseng (P = 94.4%; H pop = 0.3246). All populations examined using the AFLP** markers were statistically significantly differentiated based on the AMOVA results. Our result suggest effectiveness of AFLP** markers for characterization of the genetic structure and genetic relationships of the ginseng populations. These markers are recommended for use in large-scale population genetic studies of this species to develop measures of its conservation.

Выполнено кариологическое исследование представителей популяций лиственницы из разных частей ареала Larix olgensis A. Henry в Приморском крае. Подтверждено, что основным числом хромосом для лиственницы ольгинской является n = 12 (2n = 24). Во всех изученных популяциях наблюдалась миксоплоидия. Выявлено отличие индивидумов из ареала лиственницы ольгинской по количеству клеток с разным уровнем плоидности как от популяции из L. olgensis locus classicus, так и от L. sibirica и L. gmelinii, что по-видимому является следствием их гибридной природы.

Karyological study of larch population members from different parts of the Larix olgensis L. Henry areal in the Primorskii krai has been carried out. The main amount of chromosomes for larch as n = 12 (2n = 24) has been confirmed. Mixoploidy was observed in all studied populations. The difference of individuals from the larch areal based on the cell amount with a different level of ploidity has been found in the population of L. olgensis locus classicus and both L. sibirica and L. gmelinii, which is probably a consequence of their hybrid nature.

BACKGROUND:The natural habitat of wild P. ginseng is currently found only in the Russian Primorye and the populations are extremely exhausted and require restoration. Analysis of the genetic diversity and population structure of an endangered species is a prerequisite for conservation. The present study aims to investigate the patterns and levels of genetic polymorphism and population structures of wild P. ginseng with the AFLP method to (1) estimate the level of genetic diversity in the P. ginseng populations in the Russian Primorsky Krai, (2) calculate the distribution of variability within a population and among populations and (3) examine the genetic relationship between the populations.METHODS:Genetic variability and population structure of ten P. ginseng populations were investigated with Amplified Fragment Length Polymorphism (AFLP) markers. The genetic relationships among P. ginseng plants and populations were delineated.RESULTS:The mean genetic variability within populations was high. The mean level of polymorphisms was 55.68% at the population level and 99.65% at the species level. The Shannon's index ranged between 0.1602 and 0.3222 with an average of 0.2626 at the population level and 0.3967 at the species level. The analysis of molecular variances (AMOVA) showed a significant population structure in P. ginseng. The partition of genetic diversity with AMOVA suggested that the majority of the genetic variation (64.5%) was within populations of P. ginseng. The inter-population variability was approximately 36% of the total variability. The genetic relationships among P. ginseng plants and populations were reconstructed by Minimum Spanning tree (MS-tree) on the basis of Euclidean distances with ARLEQUIN and NTSYS, respectively. The MS-trees suggest that the southern Uss, Part and Nad populations may have promoted P. ginseng distribution throughout the Russian Primorye.CONCLUSION:The P. ginseng populations in the Russian Primorye are significant in genetic diversity. The high variability demonstrates that the current genetic resources of P. ginseng populations have not been exposed to depletion.

The level of within-and among-population variation of larch inhabiting the range of Larix olgensis A. Henry in Primorye was estimated on the basis of 440 RAPD loci identified by means of 12 random primers. In ten populations examined, the proportion of polymorphic loci was 35–60%, the average expected heterozygosity varied from 0.1340 to 0.2169, and the average gene flow estimate was 1.38. According to Fisher’s test for heterogeneity, the pairwise differences of the fragment frequencies between the populations were statistically significant. The subdivision index G ST = 0.2663 indicated that the interpopulation variation component accounted for approximately 27% of the total variation. Coefficients of Nei’s genetic distance between the populations varied from 0.0137 to 0.0934. Most of the samples with high support clustered according to the geographic positions relative to one another within the range. These results suggest that the populations examined are characterized by high genetic variation, like the larch populations of Siberia and the Russian Far East studied earlier, but, in contrast to the latter, exhibit higher among-population variability.

Summary To find more morphological characteristics useful for discussion on aralian or non-aralian Panax affinity, pollen morphological diversity was comparatively analysed in P. ginseng, Aralia elata and Oplopanax elatus collected during their pollination periods. In the anthers of both the buds and open flowers, the pollen average diameter varied between some species-specific maximum and minimal measurement. However, the larger pollen grains were typically found in the buds whereas the smaller pollen prevailed in the open flowers, testifying to the pollen size diminution during anther maturation. Based on this finding, the subsequent examination of pollen according to size decrease was put into operation as a method of pollen modification for the study. The structural mechanisms of pollen metamorphosis were identified as not being species specific but rather universal. These mechanisms are suggested to be the shrinkage of the pollen vegetative cytoplasm, the intine enlargement, the deepening of three colporate apertures provided by exine sunken into enlarged intine areas, the aperture accretion as well as the transformation of the exine from thick/sculptured into thin/less sculptured. During ‘size-reducing metamorphosis’, the pollen grains changed dramatically, going through a species-specific set of intermediate morphs to the final species-specific morphotype. In P. ginseng this morphotype is round (diameter is about 16 μm), in A. elata it is round with a single projection (diameter is about 15 μm) and in O. elatus it is ovoid with a single projection (average diameter is about 18 μm). In addition, every species is peculiar in having the unique vegetative cytoplasm inclusions and individual construction of the largest pollen exine. From a phylogenetic perspective, these findings presumably add support to the option of equal remoteness of P. ginseng from A. elata and O. elatus. The characteristics found seem to be suitable for examination of Panax affinity, by the subsequent study of more Araliaceae representatives.

Aim: The mating system of Panax ginseng , genetics and ontogenetic structure of its natural populations of Primorye (Russia) were investigated. Methods: Genetic diversity was assessed using allozyme and the fluorescently based automated amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) markers. Results: Total genetic diversity at species level is low with allozyme assay (0.023), and high with AFLP (0.255) and SSR (0.259) methods. It is observed within populations according to allozyme (>99%), AFLP (>85%), and SSR (>73%) assays. The indices of genetic variability distribution point out the re-colonization of the Sikhote-Alin by ginseng plants from southern refuges during the warming period in the early Holocene. The capability of ginseng plants to cross- and self-pollinate was shown and the assumption that Panax ginseng is a facultative apomictic plant was confirmed. The reproductive system of ginseng possesses high plasticity and stability of the fertilization process that help the species to survive in stress conditions. Disturbances caused by external or internal factors can be reduced due to the morphogenetic potential of ginseng ovule or apomictic embryo development. Analysis of life stages structure of ginseng populations demonstrates that all of them are not full-constituents because some life stages are absent or occur rarely. Conclusion: In all 3 populations, virgin and young generative individuals are predominant. This means that populations studied are viable and the reintroduction of natural ginseng population is possible yet.

Random amplification polymorphic DNA (RAPD) markers were used to assess genetic differentiation between twelve larch populations from various parts of the Larix olgensis A. Henry range and to reveal the genetic relationships between these populations and pure larch species (L. sibirica, L. gmelinii, L. cajanderi, L. kamtschatica and L. kaempferi) and hybrid larch species (L. amurensis, L. komarovii and L. ochotensis) from Siberia and Russian Far East. Analysis of Nei's genetic distance values and molecular variance revealed a significant differentiation between the L. olgensis range populations that was similar in magnitude to that among species. It was found that these populations are as genetically distant from population collected L. olgensis locus classicus as from other pure larch species. At the same time, the similarity of the L. olgensis range populations to hybrid species located in adjacent territories was shown. A possible scenario to explain these results is suggested.