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ISSN 10227954, Russian Journal of Genetics, 2015, Vol. 51, No. 7, pp. 647–652. © Pleiades Publishing, Inc., 2015. Original Russian Text © M.E. Omasheva, S.V. Chekalin, N.N. Galiakparov, 2015, published in Genetika, 2015, Vol. 51, No. 7, pp. 759–765.

PLANT GENETICS

Evaluation of Molecular Genetic Diversity of Wild Apple Malus sieversii Populations from Zailiysky Alatau by Microsatellite Markers M. E. Omashevaa, S. V. Chekalinb, and N. N. Galiakparova a

Institute of Plant Biology and Biotechnology, Almaty, 05040 Republic of Kazakhstan email: [email protected] b Institute of Botany and Phytointroduction, Almaty, 05040, Republic of Kazakhstan email: [email protected] Received July 24, 2014; in final form, September 25, 2014

Abstract—The territory of Kazakhstan is part of the distribution range of Malus sieversii, which is one of the ancestors of cultivated apple tree varieties. The collected samples of Sievers apple leaves from five populations growing in the Zailiysky Alatau region served as a source not only for the creation of a bank of genomic DNA but also for determination of the wild apple genetic polymorphism. The seven microsatellite markers used in this study revealed 86 alleles with different frequencies, as well as the characteristic pools of rare alleles for each of the populations. Molecular genetic analysis showed a high level of genetic diversity (Ho = 0.704, PIC = 0.752, I = 1.617). Moreover, interpopulation variability accounted only for 7.5% of total variability, confirming the genetic closeness of the populations examined. Based on phylogenetic analysis, it was dem onstrated that the Bel’bulak and Almaty Reserve populations were closest to each other, while the most dis tant were the Ketmen and Great Almaty gorge populations, which suggests the dependence of genetic dis tance on the geographical. DOI: 10.1134/S1022795415070108

INTRODUCTION According to N.I. Vavilov [1], the territory of Kaza khstan is one of the centers of origin of the wild apple. In Flora of Kazakhstan, several species of the genus Malus were described [2]. One of the most important species is Malus sieversii, which is recognized as the ancestor and stock for the Malus domestica Borkh cul tivars [3]. Sievers apple, along with M. neidzwetzkyana apple, is listed in the Red Book of Threatened Species of Kazakhstan. At the same time, the points of view of the taxonomists on the species assignment of wild apples of Kazakhstan still differ. A.D. Dzhangaliev [4] recog nizes three species, including Malus sieversii, Malus kirghisorum, and Malus niedzwetzkyana. S.A. Abdulina [5] believes that there are actually only two species, Malus sieversii and Malus niedzwetzkyana. In addition, molecular genetic studies show that Malus niedzwetz kyana is a relative but is not identical to Malus sieversii and that it is genetically more distant from domestic apple varieties [6]. The suggestion that the natural apple population of Central Asia could contain wild species, feral cul tivars, and hybrids between them was expressed by M.G. Popov [7] and E.P. Korovin [8]. Obviously, there is a need for molecular genetic characterization of the wild apple species composition for Kazakh stan. This need is based on fundamental and practical

aspects, since recognition of the importance and use fulness of the wild apple gene pool conservation requires the definition and validation of the subject of conservation. The importance of studying wild species and the need to study the phylogenetics of cultivated plants and their relationships with wild relatives with the use of modern genetic knowledge was stressed in the works of N.I. Vavilov [1]. In the process of increasing pro ductivity, cultivated plants lose some important fea tures, such as resistance to environmental stress fac tors. Genetically homogeneous cultivars replace genetically more diverse ones, leading to the loss of genes of wild relatives, especially those responsible for resistance to pests and pathogens and adaptability to climate change. It is obvious that populations of wild apple trees on the territory of Kazakhstan are indis pensable donors of the stress resistance genes that were lost by cultivated varieties [9]. The maintenance of genetic diversity is of great importance for the ecological plasticity of the popula tions. The presence in a population of a number of alleles at certain loci provides for population adapta tion to varying environmental conditions [10]. Anthropogenic pressure mediates a dramatic reduc tion in the distribution range of wild apple trees in the mountain regions of Central Asia. The establishment of apple orchards near the forests of wild apples leads

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Kapchagay

Shelek Shonzhy

Bayserke 2 3 1

4 5 Esik

Talgar Almaty

Kegen 48 km Fig. 1. Map of the distribution of the examined wild apple populations in Zailiysky Alatau (1, Great Almaty gorge; 2, Bel’bulak; 3, Almaty Reserve (Right Talgar); 4, Tau Turgen (Kuznetsova gapping); 5, Ketmen).

to a genetic erosion of natural populations and thereby disturbs the biodiversity. Molecular genetic systemat ics and the organization of natural gene pool conser vation will ensure the preservation of the wild apple germplasm and the development of breeding programs to improve the gene pool of cultivated apple. MATERIALS AND METHODS The plant material used in the study was repre sented by five populations of wild apple from Zailiysky Alatau. Leaves were collected from 180 accessions, of which 40 accessions were from the Tau Turgen popu lation (Kuznetsova gapping); 48 accessions were from the Almaty Reserve population (Right Talgar); 36 accessions were from the Bel’bulak gorge popula tion; 48 accessions were from the Great Almaty gorge population; and eight accessions were from the Ket men population (Fig. 1). For each of the five wild apple populations of Zailiysky Alatau, the GPS coor dinates were obtained for each tree from which leaves were taken. Leaves were collected from trees of differ ent ages, ranging from 10 to more than 140 years old. The collected leaves were stored at –80° until iso lation of genomic DNA. DNA isolation. Genomic DNA was isolated accord ing to a previoulsy procedure described [11]. Analysis of the quality and quantity of the extracted DNA was car ried out in 1% agarose gel by spectrophotometry. The purity of the DNA samples was determined by the ratio

of absorbance at 260 and 280 nm (A260/280). DNA sam ples with a ratio of 1.7 to 1.9 were used for further anal ysis. SSR analysis. To characterize the genetic diversity, seven microsatellite markers previously used in studies on apple genotyping were used [12]. Three nonspecific oligonucleotides labeled with different fluorescent dyes were also used. The nucleotide sequences of these oligonucleotides were added to the 5' end of one of the primers of each marker. This modification made it possible to reduce the cost of SSR analysis [13]. Polymerase chain reaction (PCR) was conducted in two steps. The first PCR was carried out in a total vol ume of 20 µL containing 2 µL 10× Taq buffer (750 mM TrisHCl, pH 8.8; 200 mM (NH4)2SO4; 0.1% Tween 20); 2.5 mM MgCl2; 0.2 mM of each deoxynucleotide triphosphate (dNTP); 1 unit Taq polymerase; and 40 ng DNA. The reaction conditions consisted of initial denaturation at 94°C for 2 min followed by seven cycles of 1 min at 94°C; 2 min at 56°C; and 2 min at 72°C; and then 20 cycles of 1 min at 94°C; 2 min at 53 to 51°C; 2 min at 72°C; and final extension at 72°C for 10 min. After PCR, 10 µL of each reaction was tested by elec trophoresis in 1% agarose gel. The rest of the reaction was diluted 10 times with water and 1 µL was used for the second PCR. The reaction conditions for second step PCR were identical to the first step except for for ward oligonucleotides, instead of which fluorescent labeled tails were used. Furthermore, the duration of final extension was increased from 10 to 30 min.

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Table 1. Values of genetic diversity indices in five populations of Zailiysky Alatau at microsatellite loci Locus

Na

Ne

Ho

He

PIC

PI

I

GD12

11

5.030

0.795

0.800

0.804

0.052

1.755

GD96

15

4.327

0.698

0.762

0.784

0.058

1.718

GD142

15

6.230

0.872

0.832

0.845

0.033

2.034

GD147

13

4.189

0.595

0.755

0.791

0.059

1.603

GD162

18

4.190

0.651

0.714

0.760

0.065

1.713

GD100

6

2.911

0.425

0.644

0.631

0.156

1.132

GD103

8

3.140

0.887

0.679

0.650

0.135

1.368

12.285

4.288

0.704

0.741

0.752



1.617

0.486

0.244

0.030

0.014





0.058

Mean value SE

Na, number of alleles per locus; Ne, effective number of alleles per locus; Ho, observed heterozygosity; He, expected heterozygosity; PIC, polymorphic information content; PI, probability of identity; I, Shannon’s information index; SE, standard error of the mean.

PCR products generated with seven markers from one template were pooled with the addition of forma mide and a size standard labeled with LIZ dye (Size standard 500 LIZ, Applied Biosystems). The total vol ume of the mixture constituted 10 µL. In the mixture, PCR products processed with VIClabeled oligonule otide primers were diluted 540 times, those processed with NEDlabeled primers were diluted 120 times, and those processed with 6FAMlabeled primers were diluted 360 times to align the signal intensity. The fragments were analyzed with the help of cap illary electrophoresis on the ABI Prism 310 analyzer (Applied Biosystems). The obtained data were pro cessed with the GeneMapper 4.0 software program. Statistical treatment of the data. The allele sizes determined by the GeneMapper 4.0 program were converted and listed in the MS Excel 2007 software program. The Golden Delicious cultivar was chosen as reference. They were used to correct the exact allele with the TANDEM v. 1.09 software program [14]. The allele frequencies, allele number, average num ber of alleles per locus (Na), effective number of alleles (Ne), number and frequency of rare alleles for each pop ulation, Shannon’s index, and Mantel test were calcu lated in the GenAlex 6.5 software program [15]. The expected and observed heterozygosity, as well as the measure of the informativeness of a marker (PIC), were calculated in the Excel Microsatellite Tool Kit. The dendrogram was constructed with the UPGMA approach and based on Nei’s genetic distance as imple mented in the Tree Figure Drawing Tool v. 1.4 software program [16]. RESULTS AND DISCUSSION Seven primer pairs used in the genetic analysis of wild apple populations showed 100% polymorphism. RUSSIAN JOURNAL OF GENETICS

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The number of alleles for all loci ranged from 18 for GD162 to six for GD100, with the average number of alleles per locus constituting 12.285 (Table 1). The most frequent alleles of all seven loci were common to all five populations. In almost all loci and populations, the effective number of alleles was lower than the actual per locus, with an average of 4.288 ± 0.244 at an average level of heterozygosity of Ho = 0.704, which means that alleles are distributed in the populations with different frequencies. The level of polymorphism of the loci examined was assessed by the PIC (polymorphic information content) index. These values were quite high, ranging from 0.845 for the GD142 locus to the lowest, 0.631, for the GD100 locus, with an average value of 0.752. The PIC value increases proportionally to the value of heterozygosity, and the frequency of rare alleles has less influence on this process than that of common alleles [12]. The average level of observed heterozygosity (Ho) for all seven loci was 0.704 ± 0.030, which was enough to be considered a high level of intraspecific polymor phism. The Ho values at individual loci varied from 0.887 to 0.425, which was lower than the values of expected heterozygosity (He) at all loci except for GD103 (Table 1). According to the Hardy–Weinberg principle, under certain conditions, the frequencies of different alleles of a single gene remain constant from one generation to the next, i. e., the genetic equilib rium is preserved while the level of expected heterozy gosity (He) is close to the value of observed heterozy gosity (Ho). One reason for the difference between the two measures (Ho; He) is the presence of null alleles. It is suggested that the majority of the observed homozy gotes can be heterozygotes, in which one allele is visi ble and the other is not. These types of null alleles can be formed when mutations make it impossible for the primer to bind to the target region. However, their 2015

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Table 2. Frequencies of rare alleles in the populations Population Tau Turgen

Almaty Reserve

Bel’bulak

Locus

Allele

Frequency

GD12

146

0.013

GD96

154

GD142

Allele

Frequency

184

0.017

0.051

Great Almaty GD12 gorge GD147

129

0.017

148

0.026

GD147

153

0.017

GD96

190

0.015

GD162

246

0.017

GD162

222

0.013

GD162

252

0.017

GD162

226

0.013

GD100

247

0.019

GD162

228

0.025

GD103

84

0.017

GD142

128

0.017

GD96

178

0.083

GD147

137

0.036

GD147

123

0.214

GD162

242

0.017

GD162

212

0.063

presence is difficult to prove experimentally, because it is first necessary to examine the previous generations and the segregation for this character [17]. Shannon’s index was calculated to describe the polymorphism of five populations. It is often used to assess the diversity of both molecular genetic and mor phological characters. In five populations of wild apple, the maximum value of Shannon’s diversity index (I), 2.034, was observed for the GD142 locus. The I value for the GD100 locus was the lowest (1.132). However, the average level of polymorphism over all loci was 1.617 ± 0.058, which was rather high in light of its range from 0 to 3.5, depending on the material under study [18]. In addition, the probability of identity index (PI), which is an estimate of the mean probability of detec tion of the same multilocus genotype in two unrelated individuals selected from the same population, was also calculated. The PI value is inversely proportional to the PIC value; the smaller the PI is, the more infor mative the marker is. The lowest PI value was observed for the GD142 locus (PI = 0.033), confirming that this locus was the most polymorphic relative to the others. The highest index value was observed for the GD100 locus, at which PI = 0.156. The frequencies of rare alleles typical for each pop ulation were determined (Table 2). This provided not only the diagnostic character but also important data for the construction of the core collection. In the study of population differentiation, an important point is the determination of the degree of inbreeding that increases the level of homozygosity, thus leading to the loss of genetic diversity. Thus, the inbreeding coefficient (Fis, Wright’s fixation index) was calculated to quantify this process [19]. This sta

Population

Ketmen

Locus

tistic is defined as the probability that the developing zygote of the next generation in one of its loci will con tain alleles identical by descent, such that the appear ing individual will be autozygous for the locus. In this case, identity by descent means that these alleles are copies of the same allele present in the genotype of one of the ancestors of given individual. Calculation of the Wright’s fixation index (Table 3), which reflects the degree of excess or deficit of het erozygotes in the populations of wild apple, showed that all five populations revealed a small deficit of heterozy gotes (in view of the fact that all index values were posi tive). The largest positive mean Fis values were found for the Tau Turgen (+0.115) and Almaty Reserve (+0.061) populations, pointing to the possibility of homozygoti zation in these populations. This was also seen in terms of the Ho and He indices, which almost coincided, i.e., were in a state of genetic equilibrium. While an excess of heterozygotes was recorded for some loci (Tab. 4), the mean index value for all populations and loci remained positive and constituted Fis = +0.053. In this case, the deficit of heterozygotes for the apple is not a measure of inbreeding, because the apple is not capable of selfpol lination as a result of its high autosterility. The reasons for the lack of heterozygotes can be the isolation of indi vidual panmictic groups, which leads to effects similar to inbreeding in a large, unseparated population. The proportion of homozygotes at the same time increases with increasing interpopulation variance in gene fre quencies because of the reduction in the proportion of heterozygotes. Another possible explanation is associ ated with negative selection of heterozygous individu als. It is associated with chromosomal mutations (trans locations) and the lower adaptation of heterozygotes to environmental conditions as compared to homozygotes

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Table 3. Genetic variability parameters in five populations of Zailiysky Alatau Population

Ns

Ne

I

Ho

He

Fis

Mean

7.286

3.763

1.515

0.631

0.716

0.115

SE

0.969

0.387

0.107

0.071

0.031

0.094

Mean

9.143

4.562

1.707

0.707

0.753

0.061

SE

1.388

0.734

0.146

0.076

0.030

0.100

Mean

8.000

5.173

1.755

0.771

0.787

0.019

SE

1.069

0.598

0.135

0.069

0.029

0.090

Mean

8.714

4.202

1.685

0.704

0.747

0.054

SE

0.969

0.417

0.119

0.053

0.026

0.079

Mean

5.429

3.741

1.426

0.705

0.701

0.007

SE

0.571

0.481

0.130

0.075

0.043

0.065

Tau Turgen

Almaty Reserve

Bel’bulak

Great Almaty gorge

Ketmen Ns, average number of alleles per locus; Ne, effective number of alleles per locus; Ho, observed heterozygosity; He, expected heterozygosity; I, Shannon’s information index; SE, standard error of the mean; Fis, Wright’s fixation index.

[20]. Significant differences in the genotypic propor tions associated with the lack of heterozygous genotypes were identified at different levels of statistical signifi cance in the four loci. The highest heterozygote defi ciency was observed at the GD100 locus (+0.34), while the GD142 and GD103 loci demonstrated an excess of heterozygotes (Table 4). The most significant deficit of heterozygotes was observed for the Tau Turgen popula tion, with the Fis = 0.115, which means that homozygo tization of an individual relative to the population was 11.5%. The value Fit index, which reflects the homozy gotization of an individual relative to the entire metap opulation as a whole, was slightly higher, with an aver age of 0.121. The subdivision index (Fst) for all multiple alleles was calculated as a weighted average over all of the populations studied. At polymorphic loci, its value ranged from 0.04 (GD103) to 0.136 (GD162) with an average value of 0.075, suggesting that interpopulation variability accounted for only 7.5% of total variability, while the remaining variability was found within the five populations of M. sieversii. Based on the calculation of Nei’s genetic distance (1972), a UPGMA dendrogram was constructed (Fig. 2). As can be seen from Fig. 2, the most genetically distant population was Ketmen. This can be explained by its large geographical distance from other populations (Fig. 1) or by its small population size. The Bel’bulak and Almaty Reserve populations were found to be close to each other, with a Nei’s dis tance between them of 0.088. At the same time, the distance between the Ketmen and Great Almaty gorge populations was 0.827. To confirm the nonnull hypothesis that geographical distance and genetic dis RUSSIAN JOURNAL OF GENETICS

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tance were interrelated, the Mantel test was per formed. The correlation coefficient between the two distance matrices was R2 = 0.908 (P < 0.0001) with 9999 permutations. These figures confirm phyloge netic relationships between the populations that are based on geographical distance. Table 4. Values of Wright’s F statistics for the seven loci in the examined populations Locus

Fis

Fit

Fst

GD12

0.005

0.058

0.053

GD96

0.084

0.163

0.086

GD142

–0.048

–0.019

0.028

GD147

0.213

0.293

0.102

GD162

0.088

0.212

0.136

GD100

0.340

0.393

0.079

GD103

–0.308

–0.256

0.040

Mean

0.053

0.121

0.075

SE

0.078

0.081

0.014

Fis, inbreeding coefficient of individuals relative to subpopula tion; Fit, inbreeding coefficient of individuals relative to metap opulation; Fst, the effect of subpopulation relative to metapop ulation. 2015

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Tau Turgen Great Almaty gorge Almaty Reserve Bel’bulak 0.04 Fig. 2. Dendrogram of the phylogenetic relationships between five populations of Zailiysky Alatau.

CONCLUSIONS Malus sieversii (or the Sievers apple), a wild fruit tree species from the foothills of Kazakhstan, is the recognized ancestor of cultivated apple tree varieties. Evaluation of the genetic diversity of Sievers apple populations from Zailiysky Alatau by the modern molecular genetic approaches described in this study will surely serve as the basis for proper gene pool con servation and management. One of the most impor tant issues in this case is the future creation of a collec tion of the most valuable genotypes, which are charac terized by a sufficient level of genetic diversity for further use in breeding programs to improve the gene pool of cultivated apple. ACKNOWLEDGMENTS This study was supported by the Grant on the subp riority “Fundamental Studies in the Area of Natural Sciences,” Budget Program 055 of the Scientific Committee of the Ministry of Education and Science of the Republic of Kazakhstan (grant no. 0043/GF). REFERENCES 1. Vavilov, N.I., The role of Central Asia in the origin of cul tivated plants, in Proiskhozhdenie i geografiya kul’turnykh rastenii (Origin and Geography of Cultivated Plants), Leningrad: Nauka, 1987, pp. 132–134. 2. Flora Kazakhstana (Flora of Kazakhstan), AlmaAta, 1961, vol. 4. 3. Forte, A.V., Ignatov, A.N., Ponomarenko, V.V., et al., Phylogeny of the Malus (apple tree) species, inferred from its morphological traits and molecular DNA anal ysis, Russ. J. Genet., 2002, vol. 38, no. 10, pp. 1150– 1160. 4. Dzhangaliev, A.D., Dikaya yablonya Kazakhstana (Wild Apple Tree of Kazakhstan), AlmaAta, 1977, pp. 5–34.

5. Abdulina, S.A., Spisok sosudistykh rastenii Kazakhstana (Vascular Plants List of Kazakhstan), Almaty, 1999. 6. Harris, S.A., Robinson, J.P., and Juniper, B.E., Genetic clues to the origin of the apples, Trends Genet., 2002, vol. 18, pp. 426–430. 7. Popov, M.G., Wild apple and plum in the mountains of Chimgan, in Izbrannye Proizvedeniya (Selected Works), Ashkhabad, 1958, pp. 121–125. 8. Korovin, E.P., Rastitel’nost’ Srednei Azii i Yuzhnogo Kazakhstana (Vegetation of Central Asia and Southern Kazakhstan), Tashkent, 1962, book 2. 9. Forsline, P.L. and Aldwinckle, H.S., Evaluation of Malus sieversii seedling populations for disease resis tance and horticultural traits, Acta Hort., 2004, no. 663, pp. 529–534. 10. Altukhov, Yu.P., Geneticheskie protsessy v populyatsi yakh (Genetic Processes in Populations), Moscow: Akademkniga, 2003, 3rd ed. 11. Aubakirova, K., Omasheva, M., Ryabushkina, N., et al., Evaluation of five protocols for DNA extraction from leaves of Malus sieversii, Vitis vinifera, and Arme niaca vulgaris, Genet. Mol. Res., 2014, vol. 13, no. 1, pp. 1278–1287. 12. Hokanson, S.C., SzewcMcFadden, A.K., Lam boy, W.F., and McFerson, G.R., Microsatellite (SSR) markers reveal genetic identities, genetic diversity and relationships in a Malus domestica Borkh. core subset collection, Theor. Appl. Genet., 1998, no. 94, pp. 671– 683. 13. Missiaggia, A. and Grattapaglia, D., Plant microsatel lite genotyping with 4color fluorescent detection using multipletailed primers, Genet. Mol. Res., 2006, vol. 5, pp. 72–78. 14. Matschiner, M. and Salzburger, W., TANDEM: inte grating automated allele binning into genetics and genomics workflows, Bioinformatics, 2009, vol. 25, no. 15, pp. 1982–1983. 15. Peakall, R. and Smouse, P.E., GenAlEx 6.5: genetic analysis in Excel: population genetic software for teaching and research update, Bioinformatics, 2012, no. 19, pp. 2537–2539. 16. Nei, M., Genetic distance between populations, Am. Nat., 1972, vol. 106, pp. 283–292. 17. Hoffman, J.I. and Amos, W., Microsatellite genotyping errors: detection approaches, common sources and consequences for paternal exclusion, Mol. Ecol., 2005, vol. 14, pp. 599–612. 18. Shannon, C.E., A mathematical theory of communica tion, Bell Syst. Tech. J., 1948, vol. 27, pp. 379–423, 623–656. 19. Wright, S., Evolution and the genetics of populations, in Variability within and among Natural Populations, Chicago: University of Chicago Press, 1978, vol. 4, pp. 242–322. 20. Lebedeva, N.V. and Krivolutskii, D.A., Biologicheskoe raznoobrazie i metody ego otsenki (Biological Diversity and Methods of Its Evaluation), division 1: Geografiya i monitoring bioraznoobraziya (Geography and Monitor ing of Biodiversity), Moscow: Nauchnyi i Uchebno Metodicheskiy Tsentr, 2002.

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Translated by N. Maleeva Vol. 51

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