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Turczaninowia 2013, 16 (2) : 134-137
биотехнология и генетика растений biotechnology and plant genetics
УДК 575.1:582.734 (571.1)
M.G. Kutsev1 М.Г. Куцев
T.A. Sinitsyna1 Т.А. Синицына
K. Kondo2 К. Кондо
GENETIC DIvERSITY BETwEEN THREE SPECIES OF SANGUISORBA L.
from west Siberia based on randomly amplified dna fingerprints
генетические различия между ТРЕМЯ видами SANGUISORBA L. из западной сибири на основе метода случайно АМпЛИфИцИРОвАННыХ фрагментов дНК (RAF)
Summary. Genetic differences between Sanguisorba officinalis L., S. alpina Bunge and S. azovtsevii Krasnob. et Pschen. were studied. Randomly amplified DNA fingerprints (RAF) technique demonstrated a high degree of genetic identity between S. alpina and S. azovtsevii. Placement of S. officinalis and S. azovtsevii into the same species is shown to be unjustified. Allopolyploid origin of S. azovtsevii on the basis of S. alpina genome with a small contribution of S. officinalis is confirmed.
Key words: Sanguisorba, genetic diversity, randomly amplified DNA fingerprints, species differentiation.
Аннотация. Изучены генетические различия между Sanguisorba officinalis L., S. alpina Bunge и S. azovtsevii Krasnob. et Pschen. Метод случайно амплифицированных фрагментов (RAF) показал высокую степень генетической идентичности S. alpina и S. azovtsevii. Объединение видов S. officinalis и S. azovtsevii под одним названием также неоправданно. Подтверждено аллополиплоидное происхождение S. azovtsevii на основе генома S. alpina с небольшим вкладом генома S. officinalis.
Ключевые слова: Sanguisorba, генетические различия, метод случайно амплифицированных фрагментов ДНК, видовая дифференциация.
Introduction. The genus Sanguisorba L. cm wide. Chromosome numbers of S. officinalis in
(Rosaceae) includes about 20 species. For our inves- Siberia: 2n=28 (Seminskiy Pass), 56 (Azovtsev et
tigation, we chose three species: S. officinalis L., S. al- Zaitseva, 1971).
pina Bunge and S. azovtsevii Krasnob. et Pschen. Sanguisorba alpina is common in the Sibe-Within the territory of Russia, the most rian mountains, and differs clearly from S. officina-important species is S. officinalis. It is a medicinal lis by its long inflorescence. Sanguisorba alpina is plant, which is widely used. Sanguisorba officinalis not used as a medicinal plant although it is a pois broadly distributed from Europe to Far East and tentially valuable source of biologically active sub-North America. In traditional Russian medicine, S. stances, triterpenoids (Jia et al., 1993). Inflorescence officinalis roots were used to treat intestinal prob- nodding, spicate, cylindric, rarely ellipsoid, 1.5-7 cm lems (bloody dysentery, stomatitis and others). The long, 1-1.5 cm wide, pendent. Chromosome number methanol extract of S. officinalis demonstrated anti- of S. alpina in Siberia: 2n=28 (Mishima et al., 2002). cancer and antithrombin activity (Goun et al., 2002). Sanguisorba azovtsevii (Pschenichnaja et Inflorescences erect, spicate, 1-2 cm long, 0.5-1 Krasnoborov, 1986) was described as an interspeci-
1Altai State University, Lenina str., 61; 656049, Barnaul, Russia; e-mail: m_kucev@mail.ru
2Faculty of Agriculture, Tokyo University of Agriculture, Atsugi Campus, 1737, Hunako, Atsugi-shi, Kanagawa Prefecture, 243-0034, Japan; e-mail: k3kondo@nodai.ac.jp
1Алтайский государственный университет, пр-т Ленина, 61; 656049, Барнаул, Россия;
2Сельскохозяйственный факультет, Токийский аграрный университет, кампус Ацуги, 1737, Хунако, Ацуги-ши, префектура Канагава, 243-0034, Япония
Поступило в редакцию 20.08.2012 г. Submitted 20.08.2012
Принято к публикации 28.05.2013 г. Accepted 28.05.2013
Kutsev M.G., Sinitsyna T.A., Kondo K. Genetic diversity between three species of Sanguisorba L. from West Siberia based on randomly amplified DNA fingerprints
Fig. 1. Inflorescence shape of three studied species
of Sanguisorba.
fic hybrid between S. officinalis and S. alpina. This species is allied to S. officinalis, but differs from it by having longest stamens and inflorescences. Inflorescence cylindrical, 2-5 cm long, 1-1.5 cm wide, erect, sometimes pendent. However, S. azovtse-vii differs from S. alpina by different biochemical content of the stamens. Chromosome number of S. azovtsevii: 2n=42 (Azovtsev et Zaitseva, 1971).
The species status of S. azovtsevii has been doubted; it was suggested that this species is one of the polyploid forms of S. officinalis since morphological differences between these two species are insignificant (Cherepanov, 1995) (Fig. 1). In order to establish taxonomic status of S. azovtsevii, we conducted population analysis of all three species with the goal to investigate their molecular-genetic differences.
In our work, we used RAF (Randomly amplified DNA fingerprints) technique, which is a modified DAF protocol (Waldron et al., 2002). Recently RAF was used effectively for evaluation of genetic diversity and interpopulational relationships both in animals and plants (Chan et al., 2008; Cunningham et al., 2002; Nand et al., 2005; Ramage et al., 2004).
Materials and methods. Plant material was collected in August 2010 within Russian Federation: 9 individuals of S. officinalis from Tuva Republic, at the bank of Uyuk River (55° 04' 10'' N, 94° 11' 01'' E); 10 individuals of the same species from Altai Republic, at Chibit Village (50° 21' 29'' N, E 87° 22' 47'' E); and 9 individuals of each S. azovtsevii and S. alpina from Altai Republic, at Seminskiy Pass (51° 05' 50'' N, E 85° 36' 51'' E). Fresh leaf material was dried using silica gel.
DNA was isolated using Diamond DNA kit (ABT Llc., Russia) according to the manufacturer's instructions. Experimental amplifications were done using primers of series A - 01-10 and B - 01-11 (Carl Roth, Germany). For further work, we selected primers A04 (5' - AATCGGGCTG - 3') and B06 (5' - TGCTCTGCCC - 3').
PCRs were carried out in 25 ^L reaction mix included 5 ng DNA, 2.5 ^L 10x Buffer and 25 mM MgCl2 (Sibenzyme Llc., Russia), 1 ^L 5mM of mix dNTPs (Medigen Llc., Russia), 1 ^L of each 10mM primer and 1 unit Taq DNA polymerase (Sibenzyme Llc., Russia) in the MyCycler thermal cycler (BioRad, USA) using RAF protocol: 94.0°C for 5 min. [94.0°C for 30 sec., 57.0°C for 1 min., 56.0°C for 1 min., 55.0°C for 1 min., 54.0°C for 1 min., 53.0°C for 1 min.]x35, 72.0°C for 10 min., 4.0°C until the end of the process.
S. alpina s azovtsevii 1 S. officinalis (Chibit) S. officinalis (Tuva)
L 1 23456789 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 smiiüi L
1500 H H 030.0 B m = mi= ibii-bs-bsi. m
700 .0 M H 500.0 4UU.U m i il ■m ■ i i ¡i m m M m un ■ m [imán m 111 m = - Mil III Mill ■ III ni hh uni m i «m il m nui c h mu i m nu H H 11 mi 1 1 illllIII 11 i il i il m 1 II ill du D III III un 111 Nil d III II lililí IHIII 1 I II II III II III 1 II m min il ■■min ii i « i i h ai i i i i i il 1111 m h 1 iii ii» i n il m i il m i -
= |i|!|=|" m hiii m i Il n i m i i i i m uni m m m il i mil -
500.0 _ i-IEiiii | =ÍI~"==™== i ni m i i i h 1 i i i i i il i i ii —
151111 — 100 0 es i m i m 11 i mil K 1 i in i mini i s 5-=S= iii m 11 i iiiiiii ii il m ii un h i m in 0 lili
_—— —_—_
i ■ ■ ■ i h " iii i mi ■ i ni ■ i ■■■■{■»--■-«»I llll
Fig. 2. The virtual gel photography of Sanguisorba individuals' RAF-polymorphisms.
Kutsev M.G., Sinitsyna T.A., Kondo K. Genetic diversity between three species of Sanguisorba L.
from West Siberia based on randomly amplified DNA fingerprints
Table 1
Nei's unbiased measures of genetic identity and genetic distance between different population of Sanguisorba
Population S. alpina S. azovtsevii S. officinalis (Chibit) S. officinalis (Tuva)
S. alpina *** 0.9682 0.5887 0.5823
S. azovtsevii 0.0323 *** 0.6726 0.6499
S. officinalis (Chibit) 0.5298 0.3966 *** 0.9340
S. officinalis (Tuva) 0.5407 0.4310 0.0682 ***
Note: Nei's (1978) genetic identity (above diagonal)
DNA fragments were separated by micro-fluidic electrophoresis in Automated Electroforesis Station Experion (Bio-Rad, USA) using Experion DNA 1K Analysis Kit (Bio-Rad, USA). Resulting image (virtual gel) of fragments separation is presented on Fig. 2.
In the analysis we formed a matrix on the basis of presence (1) or absence (0) fragments of the equal length. There were 52 fragments for 37 individuals (4 populations) for further analysis: in populations of S. alpina, S. azovtsevii and S. officinalis from Tuva we used only 9 individuals. This dataset was used to calculate genetic diversity estimates, GST (Nei, 1973) and genetic distances (Nei, 1978) among populations and individual samples using the Popgene (v. 1.31), a software package of Yeh and Boyle (1997).
Multidimensional scaling (MDS) (Kruskal, 1964) was carried out using program for phenetic analysis NTSYS-pc, Numerical Taxonomy System, version 2.1 (Rohlf, 1992). Pairwise genetic distances were calculated using the coefficient of Lynch (1990).
Results and discussion. As a result of this study, we have revealed genetic diversity within populations of all three species of Sanguisorba. The highest genetic diversity was in S. azovtsevii (GST= 0.2830), which indicates a high intrapopulational polymorphism of this species. Sanguisorba alpina had GST=0.2137; and S. officinalis, GST=0.2569 (Chibit) and GST= 0.2289 (Tuva). Total genetic diversity for all populations GST=0.4053, therefore one cannot speak about free gene exchange among S. alpina, S. azovtsevii and S. officinalis. We interpret high genetic diversity in S. azovtsevii as a result of its hybrid origin from S. alpina and S. officinalis. At the same time, combined genetic diversity for S. alpina and S. azovtsevii was much lower (0.2678) than combined genetic diversity for S. azovtsevii and S. officinalis (GST=0.3913). This attests to high similarity between genomes of S. alpina and S. azovtsevii. The same is demonstrated by the analysis of genetic identity and genetic distance between different populations of Sanguisorba (Table 1).
The lowest genetic distance is observed between S. alpina and S. azovtsevii, and is very
and genetic distance (below diagonal).
close to the genetic distance between populations of S. officinalis. It must be noted that in angiosperm species Nei's unbiased measure of genetic distance between populations based on RAF analysis do not exceed 0.3 even when populations are geographically distant (Gao et al., 2000; Huang et al., 2000; Hu et al., 2010; Yamskikh et al., 2011). However, distance-matrix methods (measures of gene diversity, genetic identity and genetic distance) do not always provide a correct estimate of genetic similarity since different characters in RAF analysis have different weight, which is not taken into account here. One of the methods that allows to minimize errors of statistical analysis in case of unequal weights is Multidimensional Scaling (MDS) (Weising, Nybom, 2005). A result of MDS analysis of RAF data is a three-dimensional graph that reflects the degree of similarity between the specimens (Fig. 3).
Studied specimens of Sanguisorba are differentiated into two highly separate groups. One group is formed by S. officinalis while the second includes S. alpina and S. azovtsevii; this is comparable to the data of genetic identity and genetic diversity analysis. Therefore, we established
Q -S. alpina Q - S. azovtsevii
Fig. 3. Differentiation of Sanguisorba species based on multidimensional scaling analysis.
Turczaninowia 2013, 16 (2) : 134-137
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a very high degree of relatedness of S. alpina and S. azovtsevii. Based on the results of RAF DNA analysis, we confirm cytogenetic data on allopolyploid origin of S. azovtsevii on the basis of S. alpina genome, but not that of S. officinalis. Placement of S. officinalis and S. azovtsevii into the same species, suggested by Cherepanov (1995), is therefore unjustified. However, in our opinion, placement of S. alpina and S. azovtsevii into the same species is also not justified due to the existence
of numerous cytogenetic, molecular-genetic and morphological differences that have been observed over more than 30 years.
Acknowledgements. The study was funded by the Ministry of Education and Science of the Russian Federation (Federal Targeted Program "Scientific and Scientific-Pedagogical Personnel of the Innovative Russia in 2009-2013"), Contract no. P483; project14. B37.21.0110; and Scientific School - 250.2012.4.
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