INTRODUCTION

The apple (Malus × domestica Borkh.) is the most important commercial fruit crop in temperate latitudes. According to various estimates, there are from 7500 to 20 000 apple cultivars in the world; of them, no more than 20 cultivars are widely cultivated in the horticulture industry [1, 2]. Such distribution of a small set of industrial cultivars leads to a displacement of local and old cultivars (and sometimes to their loss); accordingly, individual genes and gene combinations typical, for example, of the cultivars adapted to local conditions are lost, leading to a loss of genetic diversity [3].

Genetic resources of cultivated plants and their wild relatives are one of the most important components of plant biological diversity, since they have a potential value for food production and sustainable development of environmentally friendly agriculture. At present, realizing the strategic importance of plant genetic resources, most countries of the world have developed and are implementing national programs on their conservation and use [4]. National gene banks occupy a key position in solving the problems of plant genetic resources conservation strategy; the conservation of genetic resources ex situ is one of their main functions. In our country, the Vavilov All-Russian Institute of Plant Genetic Resources (VIR) is such an organization; at present, it has become not only a national but also a recognized world center for the conservation and study of plant diversity [5].

VIR holds the largest collection of apple genetic resources in Russia, which includes more than 3800 cultivar and species accessions stored in the field gene banks of six branches of the institute that are located in different ecological and geographical zones of Russia. More than 500 apple accessions are preserved and studied in the conditions of the Northwest region at the research and production base Pushkin and Pavlovsk Laboratories of VIR in the collection gardens; of them, there are more than 100 accessions of Russian and European landraces: old cultivars widely cultivated in Russia and in the world and local cultivars locally grown by gardeners only in certain regions of the country. The apple landraces represented in the collection combine durability, high adaptability, and plasticity to the conditions of the northwest of Russia, as well as resistance to diseases and pests [6].

Domestic and foreign researchers and breeders developed descriptors for the study, estimation, pomological characteristics, and identification of the apple cultivars [7–9]. But the work with them is sometimes difficult because of the polygenic nature of many phenotypic traits and their dependence on environmental conditions, as well as because of a significant amount of time and labor costs required when studying large collections. Molecular markers (DNA markers) are widely used today to study the diversity of gene bank collections and to identify and certify accessions. One of the effective methods used to solve the problems of molecular characterization of species and varietal polymorphism is the multilocus AFLP (Amplified Fragment Length Polymorphism) method. This method was previously used for the identification of apple cultivars [10], creation of genetic linkage maps of two apple cultivars [11], and also, in combination with other methods, it was used for mapping apple resistance genes to aphids and scab [12–14]. AFLP analysis was successfully used to study the genetic diversity of wild species of the genus Malus Mill. from VIR collection [15].

At present, morphological and physiological charasteristics, biochemical composition, and field resistance to main diseases and pests are well studied in apple landraces from VIR collection; however, no molecular genetic studies of these landraces were previously carried out. Therefore, the aim of this work was to study the genetic diversity of old and local apple cultivars from the collection of the research and production base Pushkin and Pavlovsk Laboratories of VIR using AFLP analysis.

MATERIALS AND METHODS

To conduct the study, 106 apple accessions from the collection of the research and production base Pushkin and Pavlovsk Laboratories of VIR were selected: 100 landraces and six Soviet cultivars (Table 1, nos. 1–106), as well as 17 modern commercial Russian and foreign cultivars (Table 1, nos. 107–123) from the collection of VIR and the I.V. Michurin Federal Scientific Center.

Table 1. Apple accessions used in the study
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DNA was isolated from fresh young leaves using the ZR Plant/Seed DNA Mini Prep (Zymo Research) kit according to the manufacturer’s instructions.

AFLP analysis was conducted according to a standard method [16]. EcoRI and MseI endonucleases (NEB) were used for restriction of genomic DNA. A combination of EcoRI-A and MseІ-C primers was used for the first round of amplification; two combinations of primers (EcoRI-ATG/MseI-CAT and EcoRI-ATG/MseI-CTA) were used for the second round. Polymerase chain reaction was conducted using reagent kits manufactured by Dialat-LTD in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems). The products of amplification were separated by electrophoresis in 6% polyacrylamide gel in 1× TBE buffer in a Sequi-Gen® GT Sequencing Cell (BioRad) followed by silver nitrate staining according to the method of Н. Benbouza et al. [17].

The presence or absence of amplification products was registered visually, only clearly reproduced fragments were taken into account, and the results were recorded into a binary matrix (1/0) in Microsoft Excel. The portion of polymorphic fragments was determined as the ratio of the number of polymorphic fragments to their total number, expressed as a percentage. The indicators of expected heterozygosity (Не) and Shannon’s index (I) were calculated using the GenAlEx6.41 program [18]. The calculation of the Dice genetic similarity coefficient between the samples and classical multidimensional scaling (principal coordinates analysis, PCO) were performed using the PAST 3.16 program [19]. The analysis of the genetic structure of the collection was carried out using STRUCTURE 2.3 program [20] with the following parameters: admixture model and allele frequency correlations (since all analyzed samples belong to the same species); the number of tested clusters (K) from 1 to 15, each with five repetitions; burn-in of 300 000 steps; the total number of Markov chain Monte Carlo (MCMC) steps of 1 000 000. Selection of the optimal number of clusters into which the studied samples were divided was performed using the Structure Harvester service (http://taylor0.biology.ucla.edu/structureHarvester/) by the deltaK method.

RESULTS

AFLP analysis of 123 apple accessions made it possible to detect 355 fragments, 319 (89.86%) of which were polymorphic. The expected heterozygosity (He) was 0.243 ± 0.010, while the Shannon’s index (I) was 0.373 ± 0.13. The apple landraces were characterized by a higher genetic diversity level (the portion of polymorphic fragments was 89.20%, Не = 0.238 ± 0.010, I = 0.366 ± 0.13) as compared with the studied modern commercial Russian and foreign apple cultivars (the portion of polymorphic fragments was 74.11%, Не = 0.202 ± 0.011, I = 0.302 ± 0.15). Each of analyzed cultivars was characterized by a unique AFLP spectrum, and sample-specific fragments were identified in the Yalkarnan Kesa and Antonovka Obyknovennaya k-711.

The Dice genetic similarity coefficients between the pairs of samples were established using the PAST 3.16 program. The maximum value of this index was 0.99 between Antonovka Obyknovennaya k-74 and k‑21190 accessions. The high level of similarity (0.95) was also observed between Antonovka Monastyrskaya and Antonovka Obyknovennaya k-74. Among other accessions, Kal’vil’ Belyj Letnij and Medovoe ot Verevkina (0.91), as well as Ananas Berzhanitskogo and Malinovka, Arkad Zimnij and Pudovka, and Pestrushka and Terent’evka (0.90), were the most genetically similar. The minimum level of similarity was observed between Pestrushka and Zimnee ot Berdashkevicha and Vinnoe and Antonovka Obyknovennaya k-711 (0.63) accessions, as well as Litovskoe Sakharnoe and Antonovka Obyknovennaya k-711 (0.64). The average value of the Dice similarity coefficient for the studied sample set was 0.77. The level of genetic differences in modern commercial cultivars was lower (the similarity coefficient varied from 0.72 between Golden Delicious and Bylina to 0.91 between Bylina and Blagovest, as well as Stroevskoe and Kurnakovskoe, with an average value 0.79).

On the basis of the Dice genetic similarity coefficients, principal coordinates analysis (PCO) was conducted (Fig. 1). All studied modern commercial apple cultivars are located on the right side of the graph. Soviet cultivars, as well as some local and old cultivars, are located near the group of modern cultivars: Baltic (Grushovka Gerbnitskogo, Mestnoe Lezhkoe, Seyanets Trebu, Suislepskoe, Tellisaare, Yalkarnan Kesa), Western European cultivar Shtreifling Krasnyi, local cultivar of Grodno region Seyanets Rebristogo, and some Russian landraces (Abrikosovoe Mazyuka, Severnyi Velikan, Sladkoe Zimnee, Slivochnoe, Titovka, Titovka klon, Tyushkinskoe Krasnoe, Fedorovskoe). Other studied landraces (both Russian and foreign) are mainly located in the left and central parts of the graph, and no clear differentiation of the landraces by their origin is observed. Antonovka accessions: Antonovka Monastyrskaya, Antonovka Rzhavaya, and Antonovka Obyknovennaya (k-74 and k-21190) occupy a somewhat isolated position (Fig. 1).

Fig. 1.
figure 1

Location of studied apple accessions in principal coordinates based on AFLP analysis data: (⚫) modern and commercial cultivars; (◻) landraces of Northwest and Central Russia; (▲) landraces of Western Europe and Baltics, (*) Soviet cultivars. Sample numbers on the graph correspond to serial numbers in Table 1.

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Subsequently, the genetic structure of the studied collection was determined using the STRUCTURE 2.3 program (Fig. 2a). Data analysis using the deltaK method demonstrated that the studied accessions most likely can be divided into two clusters (Fig. 2b). The first cluster includes most of the studied apple landraces, and no differentiation of Russian and Baltic landraces was detected. The second cluster includes modern and commercial apple cultivars, Soviet cultivars, and some landraces. In addition, samples that include the components of both groups according to AFLP data were detected (Fig. 2a). Such separation was also observed on the PCO graph, where these samples occupied an intermediate position between modern commercial cultivars and landraces (Fig. 1).

Fig. 2.
figure 2

(a) The probability of ascribing the studied apple accessions to one of the clusters according to the results of the analysis in the STRUCTURE 2.3 program with the number of clusters K = 2. Vertical axis, a posteriori probability of ascribing the sample to one of the clusters; horizontal axis, analyzed samples (() cluster 1; (◼) cluster 2); (b) Estimation of optimal number of clusters using the deltaK method. The sample numbers correspond to serial numbers in Table 1.

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DISCUSSION

The conducted AFLP analysis detected a fairly high level of genetic diversity of the studied collection (89.86%, He = 0.243, I = 0.373); at the same time, the level of genetic diversity of modern Russian and foreign commercial cultivars was lower (74.11%, Не = 0.202, I = 0.302) as compared with landraces (89.2%, Не = 0.238, I = 0.366). The analysis of data obtained demonstrated that most of the studied apple accessions can be attributed with a high probability to one of two groups. The first group includes most of the analyzed landraces, while the second group includes modern Russian and foreign commercial apple cultivars. Within the first group, no clear division of the studied landraces by geographic origin was observed, which may be due to the exchange of genetic material that existed between neighboring territories of northwestern regions of Russia and Baltic countries, where most of the samples originate (Table 1). Previously, such mixing was registered when studying the genetic diversity of the collection of local Finnish and Latvian apple cultivars using microsatellite markers [21, 22].

Besides modern commercial apple cultivars, the second group included Soviet cultivars (Grushovka Zimnyaya and Osennee Aloe) created with the involvement of foreign cultivars and Estonian cultivar Talve Nauding. Zimnee Sikory and Letnee Sladkoe accessions obtained in the collection of VIR from Belorussian breeder I.P. Sikora in the 1960s are also included in this group. The parental forms of these cultivars are unknown, but Sikora often used foreign cultivars in the breeding process [1]. The second group also includes several Baltic (mainly Estonian) landraces (Grushovka Grebnitskogo, Mestnoe Lezhkoe, Seyanets Trebu, Suislepskoe, Tellisaare, Yalkarnan Kesa), Western European cultivar Shtreifling Krasnyi, local Belorussian cultivar Seyanets Rebristogo, and several Russian landraces: Abrikosovoe Mazyuka, Severnyj Velikan (Leningrad region); Sladkoe Zimnee, Tyushkinskoe Krasnoe (Pskov region); Fedorovskoe (Tver region); Slivochnoe, Titovka, Titovka clone (old, once widespread cultivars), which may be associated with the use of landraces in breeding.

Modern Russian cultivars are not significantly differentiated from traditional foreign commercial cultivars; this is apparently a consequence of an increasing involvement of foreign material in domestic breeding programs and can lead to further narrowing of the genetic base of the apple cultivars in our country. However, the problem of apple genetic diversity reduction is global. At present, a relatively small number of apple cultivars are used in world production and breeding programs. Thus, 15 cultivars accounted for 90% of apple production in United States by 2008, out of which just Red Delicious cultivar took 24% [2], while only ten cultivars in Europe in 2012 accounted for 70% of the production. Thus, the Golden Delicious cultivar occupied 35% of the production area in France [23]. Such a small number of cultivars on large areas increases their vulnerability to diseases, pests, and other unfavorable environmental factors.

Wild species of the genus Malus are traditional sources of economically valuable traits, such as resistance to biotic and abiotic stresses [2, 24]. The study of genetic diversity of wild Malus species from VIR collection by AFLP analysis demonstrated high level of their polymorphism (90.2% of polymorphic loci) [15]. But in addition to valuable traits, wild species transmit undesirable traits to the offspring; in particular, the quality of fruit worsens and their size decreases. To solve this problem, re-hybridization with large-fruited best seedlings obtained from the crossing of cultivars with wild species is recommended, but this complicates and slows down the breeding work. Landraces can be a good alternative to wild species in the breeding programs, since they have passed a long selection and have fewer negative traits that are manifested during the crosses. It is necessary to note that some landraces (such as Antonovka Obyknovennaya, Borovinka, and Osennee Polosatoe) were quite widely used and continue to be used by Russian and foreign breeders as sources of early maturity, productivity, and high environmental stability. However, most of the local and old apple cultivars from the collection of Pushkin and Pavlovsk Laboratories of VIR have almost never been used in breeding. When studying these landraces in modern conditions of unstable changing climate, the genotypes with a wide norm of reaction that can be recommended for breeding as sources of valuable traits are distinguished.

Thus, the genetic diversity of the apple landraces from the collection of the research and production base Pushkin and Pavlovsk Laboratories of VIR was for the first time estimated using AFLP analysis. A high polymorphism level of the apple landraces, as well as differentiation between landraces and modern cultivars, was demonstrated. The data obtained once again confirm the importance of local and old apple cultivars preservation as a valuable source of genetic diversity.