Using wild strawberry of the Sakha (Yakutia) Republic in breeding garden strawberry (Fragaria × ananassa Duch.)

In Central Yakutia, cultivating of Fragaria × ananassa varieties involves high risk of frost-killing. To c reate winter-resistant cultivars of garden strawberry there was performed open pollination under the conditions of redundant pollen background of lo ca wild strawberry F. mandshurica Staudt. Most seedlings grown from the achenes thus developed retain specific characters of F. × ananassa. The stock of selected seedlings was established. Creation of the vari ties adapted to the local growing conditions proves the selection techn ique to be successful. Fragaria L. genus includes the species following the polypl oidy series: 2n = 2x, 4x, 5x, 6x, 8x, and 10x, where x = 7 [1, 2]. The taxonomy of Fragaria L. genus is considered to comprise 20 wild species, three spontaneous intersp ecific hybrids species and two cultivated hybrid species [3-5]. Among those, the s ynthetic octoploid garden species (Fragaria × ananassa Duch., 2n = 8x = 56) is the most widely-cultivated berry in the w orld [5]. In Russia, the garden strawberry is grown thro ughout the country being very popular with the public. This species appeared in the Royal Botanical garden in France due to occasional crossing of two octoploid species delive red from America, namely Fragaria chiloensis (L.) Mill. and F. virginiana Duchesne [6, 7]. In Russia, garden strawberry varieties brought from Western Europe have been gro wn since the late XVIII century; first, mainly in the gardens of monasteries and landlords and since the XX century they have become mass-cultivated [8]. That means in Russia, F. × ananassa is actually an introduced species with all the attendant problems of low adap tation to frost, especially in Siberian regions. Practically, during the XX century till no wadays, there have been numerous attempts to both enrich the taste and increase the wint r-hardiness of the garden strawberry by introgression of the genetic material of Norther n Eurasia wild strawberries such as F. vesca L. [9-12], F. viridis Duch. [11-14], F. orientalis Los. [15-18], F. moschata Weston [14, 17-19]. Despite the evident progress in creati ng winter-resistant varieties mostly by intraspecific hybridisation F. × ananassa, the varieties with high adaptability to stressors are still needed. That is particularly important fo r cultivation in Eastern Siberia, including the Sakha (Yakutia) Republic, where winter temperat ures are extremely low. The climate in * Corresponding author: SO_baturin@mail.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). BIO Web of Conferences 38, 00012 (2021) Northern Asia Plant Diversity 2021 https://doi.org/10.1051/bioconf/20213800012

Central Yakutia is extreme continental with long severe winters with low snowfall and dry hot summers. The sum of active temperatures above 10°C is 1400-1600°C. The absolute minimum is -64°C, the absolute maximum is +38°C, the range of variability for temperature to demonstrate the climate continentality level comprising 102°C [20]. In such conditions, growing F. × ananassa varieties is extremely difficult and needs special protective measures in winter. In that regard, since 1990 th in M.G. Safronov Yakut Scientific Research Institute of Agriculture there were initiated investigations on strawberry introduction and breeding with selection of winter-resistant samples. Essential attention was paid to inter-specific hybridisation of F. × ananassa varieties with selected samples of local wild strawberry. According to the literature, the Fragaria genus in Yakutia is represented by two species: F. viridis (insignificantly) and F. orientalis (predominantly) [21]. It should be mentioned, the specific belonging interpretation differs in Russian and international botanical nomenclatures referring to the Fragaria genus and F. orientalis particularly for strawberry plants growing in Yakutia, Buryatia and Zabaykalsry Krai. In all the literature but Russian, wild strawberry samples from the listed regions are considered as the species Fragaria mandshurica Staudt. This conclusion results from diploid chromosome level (2n = 2x = 14) and complete flowers of plants from Yakutia, Buryatia and Zabaykalsky Krai, on the contrary to F. orientalis with its tetraploid chromosome number (2n = 4x = 28) and dioecy in populations. More detailed arguments for the necessity to distinguish these species are presented by a prominent taxonomist of Fragaria genus Günter Staudt [22]. In this connection, in the paper, we will follow his recommendation to refer wild strawberry samples from the Central Yakutia to F. mandshurica.
In M.G. Safronov Yakut Scientific Research Institute of Agriculture, for more than 25years have been working on selection of seedlings, which were taken from local F. mandshurica populations growing in 8 uluses of Central Yakutia, by winter-hardiness and productivity; that resulted in creating the varieties Pokrovskaya and Bogdalena [23,24]. Open pollination of selected by winter-resistance varieties of F. × ananassa and selected local F. mandshurica samples led to obtaining Sadovo-Spasskaya, Bersenevskaya, Vladyka Zosima cultivars [24,25]. Conditions of open pollination of alpine forest small-fruited remontant strawberry of Alexandria variety (F. vesca L., 2n = 2x = 14) by pollen of selected forms from Yakut cenopopulations F. mandshurica allowed to breed an adaptive and productive small-fruited variety Alexandra [26]. Experiments on crossing F. × ananassa × F. viridis Duch. were not further enhanced due to low efficiency of hybridisation and sterility of seedlings.
The aim of the paper is to analyse the chromosome number of seed progeny of the remontant variety Moskovsky Delicates selected seedlings with pistillate flowers crossed by F. mandshurica selected samples under the conditions of open pollination with redundant pollen background of F. mandshurica.

Materials and methods
All the wild strawberry populations from the collection of M.G. Safronov Yakut Scientific Research Institute of Agriculture under the study are diploid and by their morphology match the taxonomy keys typical for F. mandshurica [22]. There were two ways of pollination in the experiment: directed pollination F. × ananassa (variety Moskovsky Delicates) × F. mandshurica and open pollination of Moskovsky Delicates with redundant pollen background of F. mandshurica (Fig. 1). For the study, from the seedlings of Moskovsky Delicates (F. × ananassa, 2n = 8x = 56) there were taken the plants with pistillate flowers which do not need castration. In directed pollination F. × ananassa × F. Mandshurica the inflorescences were isolated by technical wrapping cellophane bags tightly fixed by twine with labels. In the isolating bags where pollination was not performed, fruit and seed development was not observed and this proves good isolation and autonomous apomixis absence. As the mother form there were involved seven F. × ananassa plants pollinated by F. mandshurica with the help of a soft brush. The pollen was collected randomly from the nearest plants and dried for 24 hours at +20-22°C. Pollination was performed during the mass strawberry blossom in the second decade of June.
For the second type of pollination, eight Mokovsky Delicates individuals with pistillate flowers were surrounded only by flowering F. mandshurica plants of different selections (total number more than 2000, distance of 1 to 5 m). Thus the achenes in the Moskovsky Delicates seedlings could develop only if the pollen of F. mandshurica was delivered to the carpel stigma by insect pollinators. After three months of stratification, the obtained achenes were germinated in Petri dishes on wet filter paper, seedlings then placed to pricking boxes with fertile soil. When 6-8 leaves developed, the seedlings were planted to the experimental open ground; the total numbers of germinated seeds were 230 in the first and 1090 in the second type of the study. The chromosome number in seedlings were counted at root tips by staining the chromosomes with lacto-propionic orcein [27].

Results and discussion
During the mass blossom of F. mandshurica, F. × ananassa plants successfully develop fruits and achenes (Fig. 2). Achene germination in the two parts of the experiment differed insignificantly, standing at 81.2 ± 3.2% for directed pollination of F. × ananassa, and 85.8 ± 2.4% for open pollination. The results generally characterize good germination ability of received achenes for both cases. The seeds were planted and there were obtained 91 (39.6%) and 610 (56.0%) seedlings in the two parts of the experiment, consequently. Morphological analysis of characters along with blossom and fruitage revealed two groups of seedlings in every experimental version: mother-type (F. × ananassa-type) and hybridtype plants. In both cases, F. × ananassa-type seedlings formed developed achenes and fruits after blossom, although most hybrid-type plants were sterile or produced misshaped fruits with sporadic achenes. Selective count of chromosome number showed 2n = 56 in mother-type plants, while in hybrid-type seedlings, the chromosome number matched the expected result of the heteroploid crossing (8x × 2x), that was 2n = 5x = 35. It is noteworthy, among the off-springs in the first version of the experiment or F. × ananassa × F. mandshurica, there were as many as 23.9% mother-type seedlings. Heteroploid crossings in Fragaria often result in occurrence of matromorphic-type seedlings (mother parent chromosome number) alongside the expected hybrid ones. When the pollinated flowers are isolated, there appear 8x (2n = 56) seedlings showing no any character of a father parent due to realisation of the additional precautionary mechanism of sexual process -agamospermy in a form of pseudogamous diplospory. We have shown this mechanism to be realised in F. × ananassa cultivars only under the conditions when normal megasporogenesis and fertilisation are hindered [28]; thus variability in agamospermic progenies allows to perform selective breeding [29]. Undoubtedly, in Central Yakutia, preparation of F. × ananassa plants to blossom and the blossom process are negatively influenced by low temperatures during their vegetation. Hence a rate of seedlings of agamospermous origin is high. Under the climatic conditions of Novosibirsk, we performed controlled crossings F. × ananassa (8х) × F. orientalis (4х) and F. × ananassa (8х) × F. moschata (6х). In the first crossing, there was 1 agamospermous seedling of 104, and in the second -2 of 129 ones, that comprises 0.95% and 1.55% of all fruit-bearing seedlings, consequently. We suppose occurrence of 1.5% agamospermous off-springs to be a typical frequency rate for the pollination and fertilization norm under the climatic conditions of Novosibirsk Oblast [28]. When blossom of F. × ananassa plants takes place under severe climatic conditions of Central Yakutia, the rate of agamospermous progenies increases manifold.
By open pollination of plants with pistillate flowers, 79.1% seedlings were F. × ananassa-type, while the rate of hybrid-type ones was 20.9%. Moreover, F. × ananassatype off-springs could occur in two cases: either when the egg-cell with unreduced chromosome number was stimulated by F. mandshurica pollen to parthenogenetic development (pseudogamous diplospory), or when F. × ananassa pollen was brought to the carpel stigma by an insect pollinator. In such an experiment, it is not possible to determine exactly due to which process each F. × ananassa-type plant appeared. Nevertheless, the most productive off-springs were selected and thus first cultivars of Yakut breeding were created [24,25]. These varieties are characterised by good taste of fruit and high winterhardiness.
A pollen mixture of a wild species and of garden strawberry varieties to produce productive seedlings from F. × ananassa varieties with pistillate flowers has repeatedly been used by Russian breeders. Thus, selected hybrids (candidates for varieties) were created due to pollination the plants of Komsomolka and Pozdnyaya from Leopoldsghall varieties with pistillate flowers by pollen mixture comprising F. moschata and several F. × ananassa varieties [30,31]. These crossings partly simulate our experiment in Yakutia with open pollination of F. × ananassa cultivars with pistillate flowers under the conditions of redundant pollen background of a wild species F. mandshurica. The rates of F. × ananassatype plants obtained in the experiment with F. moschata are 70.7% and 77.3%, that is also similar to our results equal to 79.1%. As a result, in Central Yakutia, germination of F. × ananassa achenes obtained by open pollination with redundant pollen background of F. mandshurica allows to produce the stock of F. × ananassa seedlings which enables to conduct selection and breed new varieties adapted to cultivation under climatic conditions of Central Yakutia.