Malus Wild Species of Kazakhstan and Their Conservation In Situ

Abstract

Kazakhstan has concentrated unique genetic resources of plant agrobiodiversity of world significance. It has received international recognition for fruit agro-biodiversity and, above all, wild apple, which is highly resistant to many diseases, high frost resistance, and wide ecological plasticity. Kazakhstan is the original genetic center of biological variability of wild apples and, historically, has formed their rich gene pool. Wild apple species in Kazakhstan are genetically kindred to cultivated varieties of the world. Genetic diversity and polymorphism of Malus Sieversii (Ledeb.) from Ile Alatau and Dzhungraian Alatau were studied with ISSR markers.

1. Introduction

The wild apple tree of Kazakhstan is represented by M. Sieversii, M. Kirghisorum, and M. Niedzwetzkyana. There are quite serious disagreements regarding the systematic position of these species. So F.D. Likhonos [1] believes that according to the morphological characteristics of the Nedzvetsky apple tree—M. Niedzwetzkyana is very close to the Sievers apple tree—M. Sieversii and is defined as a variety of M. Sieversii subsp. Sieversii var. Niedzwetzkyana (Dieck) Likh. An undeniable fact is that the Nedzvetsky apple tree is distributed within the area of the Sievers apple tree, but according to V.T. Langenfeld [2], the presence of only red pigmentation of vegetative parts, flowers, and fruits of the plant does not allow considering this apple tree as an independent species. The apple tree of the Kyrgyz is M. Kirghisorum, which does not have large morphological differences from the apple tree of Sievers V.T. Langenfeld, proposes to consider as a subspecies M. Sieversii subsp. Kirghisorum (Theod.et.Fed.) Likh. In modern conditions, these species, according to the peculiarities of addition and distribution, by their valuable role and share of participation in the vegetation cover, perform to various degrees. Thus, M. Sieversii occupies a wider and defined range, while M. Kirghisorum’s role in the formation of the modern landscape looks secondary and topographically limited. M. Niedzwetzkyana, discovered by B.A. Bykov in 1957 in Karatau and Ile Alatau, is insignificant in number and range and does not form large populations anywhere.

So, the natural forests of wild apple of the Kazakhstan in terms of scale, uniqueness, genetic potential, scientific, and practical significance can be classified as one of the most valuable plant communities of the earth [3]. In this regard, their study and preservation in situ and ex situ are important and relevant for the scientific community.

2. Specific features of Malus wild species of Kazakhstan and implication for their conservation

2.1 Specific features of Malus wild species of Kazakhstan

Sievers’ apple tree is 2–10 (14) m tall. The leaves are large from short-elliptic to oblong with a wedge-shaped, less often rounded base, at the apex usually suddenly turning into a short pointed tip, along the edge the leaf plate is town-shaped. The flowers are 3.5–6 cm in diameter, on long felt pedicels. The petals are pale pink. Fruits are 3–4 cm in diameter, spherical, or flattened-spherical. Along mountainous slopes, gorge bottoms, river valleys in the belt of woody-shrubby vegetation (from 900 to 2300–2600 m above sea level) form apple forests.

M. Sieversii is a very polymorphic species. Experimental data from population genetics show that in plants in the mountains, due to the small size of populations and the variety of natural conditions, random combinations of features that are little controlled by selection prevail. This leads to the emergence of new qualities and the rapid pace of their evolution, which was the main reason for the emergence in the mountainous regions of Central Asia of a huge variety of forms and polymorphism of apple trees. In mountain forests, wild individuals are distinguished by an amazing variety of fruits. These apple trees are drought-tolerant, frost-tolerant, and durable (about 150 years) [4].

Wild apple hybrids are characterized by a huge intraspecific polymorphism as a consequence of genetic differences. Within one population, each apple tree is an independent form with reliably distinctive features and properties from the neighboring apple tree. The practical value of these forms and their hereditary properties vary greatly; therefore, in order to preserve the most valuable and well adapted to the specific environmental conditions of the forms, it is necessary to be planted with planting material genetically corresponding to the selected forms. Vegetative propagation meets this condition. Wild apple tree refers to a culture that is easily propagated by root growth, but difficult to form subordinate roots on the stem parts. Non-specialized parts and organs of the plant that serve the purpose of vegetative propagation are not naturally separated from the mother plant. On horizontally located lateral roots of apple trees, adventitious (appendage) and dormant buds develop, from which, under favorable conditions, root offspring form. M. Kirghisorum and M. Sieversii are distinguished by great energy in the formation of root offspring and large natural stands, numbering up to several hundred trunks, turn out to be connected by a common root system. And among them is a viable mother tree. The formation of root offspring occurs throughout the horizontal roots at a depth of 30–40 cm. Numerous offspring arise at a distance of 15–20 m from the tree and grow in rows, sometimes up to 500 pieces in one tree [5], forming apple groves, having a common root system, and genetically being one vegetatively propagated specimen. It has been established that vegetative apple trees do not reduce growth processes and do not show signs of aging trees.

Recently, the wild apple tree of Kazakhstan has been represented by depleted populations in habitats, greatly destroyed by the established practice of nature management. The area of these forests in 2000 was only 7% compared with 1930 [5].

Therefore, along with the problem of conservation, there is a global problem of restoring wild apple in situ and creating ex situ collections. In situ, biodiversity conservation is the conservation of ecosystems and natural habitats, as well as the maintenance and restoration of viable species populations in their natural environment. The ex situ method of preservation and restoration includes methods such as cryopreservation, field gene banks (uteri of root and seed material), propagation, and preservation in vitro.

The general provisions of the theory of conservation of species through their reintroduction have been repeatedly discussed in the literature [6]; however, practical work with a specific species requires the development of a special strategy due to the specific reasons that cause the “rarity” of the species. Most researchers indicate that when reintroducing a species, it is necessary to follow a number of rules to achieve genetic and ecological prosperity of populations mainly in the former habitats of the species [7]. To do this, the mother material must be represented by the most heterozygous plants from the populations with the highest polymorphism indicators. An important step is the identification of parent and daughter plants and their compliance with reintroduction sites [8, 9].

Efficient conservation and use of plant genetic resources require a thorough assessment of the genetic variation they possess.

Genetic variation can be measured at two levels:

• Phenotype—combinations of individual traits determined by the genotype and its interaction with the environment.

• Genotype—specific genetic structure of the organism.

The reintroduction strategy for biodiversity recovery should involve recovery of population-typical allele frequencies. Historically, all patterns of variability and heredity have been studied by analyzing morphological characteristics, which until now have remained the main criterion in plant taxonomy, in the study of the mutation process, in studies of phylogenetic connections. However, morphological features do not always provide complete informatively, since they are strongly influenced by environmental conditions. In this regard, along with morphological features, physiological and biochemical indicators are widely used, for example, polymorphism of isoenzymes and spare proteins; however, isoenzymes and proteins are products of gene expression, the degree of expression of which is influenced by various factors, for example, plant age and tissue specificity [10]. If we consider that in higher eukaryotes only about 1% of the genome are protein-coding sequences, then the main part of the genome escapes the attention of researchers.

Currently, methods based on polymerase valuable reaction (PCR) using molecular markers characterizing regions of variability in nuclear, chloroplast, and ribosomal DNA are widely used worldwide in taxonomy and population genetics.

Various evolutionary events result in different variants in the DNA sequence that describes the polymorphism. Polymorphism manifests itself in genotype differences and is visualized as different band profiles found when using molecular markers in PCR and electrophoresis of PCR products.

The most simple and preferable in experiments where DNA of a large number of representatives at several loci is studied are methods using DNA amplification by single primers with an arbitrary sequence of SSR [11] and ISSR nucleotides [12].

ISSR uses known microsatellite sequences as primers and carries at one end a sequence of two to four arbitrary nucleotides. Such primers allow amplification of DNA fragments that are located between two sufficiently close microsatellite sequences (usually unique DNA). The obtained PCR product patterns are species-specific [13]. ISSR markers refer to markers of dominant inheritance type whose polymorphism is tested by the presence/absence of a band. The method has good reproducibility and, along with AFLP, is used to identify interspecies and intraspecific genetic variation, identify species, populations, lines, and individual genotyping [14].

2.2 Molecular genetic assessment of M. Sieversii in Kazakhstan¹

With ISSR markers, polymorphism and genetic diversity of M. Sieversii in the Ile Alatau and Dzungarian Alatau populations have been studied [15].

Plant material of M. Sieversii was selected from the populations of Ile Alatau and Dzungar Alatau on expeditions in 2009–2011 organized by UN in Kazakhstan. Samples of young leaves without signs of bacterial and fungal infections taken closer to the point of growth of shoots were fixed in silica gel. Genomic DNA from 400 samples M. Sieversii (Ile Alatau and the Dzungarian Ile Alatau) was extracted from dry leaves (20 mg of each sample) using whales of NucleoSpin Plant (Macherey-Negel, Germany) according to the protocol of the producer and was stored at t -25°C.

M. Sieversii inter-simple sequence repeats (ISSRs) were amplified using primers (Table 1) on a Mastercyeler ep gradient amplifier (Eppendorf). The primers used were synthesized at the Russian company Synthol (Table 1). All polymerase chain reactions (PCRs) took place in a total volume of 20 μl.

№ primer	Nucleotide sequence (5′-3′)	№ primer	Nucleotide sequence (5′-3′)
М2	аса сас аса сас аса с(сt)g	М8	gtg gtg gtg gtg gtg
М3	gag aga gag aga gag a(ct)c	М9	gac acg aca cga cac gac ac
М4	aga gag aga gag aga g(ct)c	М11	cac aca cac aca (ag)
М7	cag cag cag cag cag	М12	cac aca cac aca (ag)(ct)

Table 1.

Primer nucleotide sequence.

In the course of work on the selection of ISSR markers, polymorphic between the studied genotypes for all markers, the same PCR conditions were used (primer annealing was set individually), allowing to obtain the maximum amount of reaction product.

PCR parameters used for analysis included: 3 minutes at 95°C—initial denaturation, the following 35 cycles:

30 seconds denaturation at 94°C, annealing primers at the appropriate temperature—30 seconds,

40 seconds elongation at 72°C + addition of 2 seconds per cycle.

The reaction mixture (20 μl) contained 10–20 ng of DNA, 20 pmoles of primer and a finished reaction mix (Biocom) containing Taq-inhibited DNA polymerase, deoxynucleoside triphosphates, and magnesium chloride with final concentrations of 1u, 200 μM, and 2.5 mM, respectively, as well as an optimized buffer system for PCR.

Initially, the annealing temperature of primers was calculated using the formula:

$$\mathrm{Т}=4°\mathrm{C}\mathrm{х}\left(\mathrm{C}+\mathrm{G}\right)+2°\mathrm{C}\mathrm{х}\left(\mathrm{A}+\mathrm{T}\right)-3,$$E1

where C, G, A, and T are the amount of cytosine, guanidine, adenine, and thymine bases, respectively. Subsequently, the optimal annealing temperature of the primers was empirically selected by decreasing or increasing it, depending on the quality of the PCR product obtained. The primer annealing temperature was specifically for primers M11 to 44.8°C, M12 to 49.5°C, M2 to 49.5°C, M9 to 50°C, M8 to 52.7°C, M3 to 52.7°C, M4 to 50.8°C, and M7 to 52.7°C.

For electrophoretic separation of PCR products, 1.7% agarose gel in 1x tris-borate buffer (50 mM Tris, 50 nM boric acid, 1 mM EDTA, pH 8.0) with ethidium bromide (0.5 μg/mL) was used when 100V for 45 minutes, followed by photographing the obtained PCR products and photo-processing in adobe program. Photographs of agarose gels were analyzed in Cross Checker 2.91 [13] with the composition of binary arrays of the presence/absence of fragments of the same length.

The level of heterozygosity of marker data is identified according to the average similarity frequency of alternative alleles. At the same time, 50% of the main alleles versus 50% of alternative alleles (0.5) correspond to a high level of heterozygosity, while 0.9 by 0.1 (90% versus 10%) corresponds to a low level.

Figures 1–5 show electropherograms of amplification of DNA fragments of M. Sieversii samples obtained with three primers M8, M3, and M2. ГД—Golden Delicious cultivar, AP—Aport cultivar.

Figure 1.

Electropherogram of amplification products of apple tree DNA fragments obtained by M8 primer.

Figure 2.

Electropherogram of amplification products of apple tree DNA fragments obtained by M3 primer.

Figure 3.

Electropherogram of apple tree DNA fragment amplification products obtained using primers M8 and M3.

Figure 4.

Electropherogram of apple tree DNA fragment amplification products obtained using primers M8 and M3.

Figure 5.

Electropherogram of apple tree DNA fragment amplification products obtained using primers M2, M8 and M3.

Table 2 shows the genetic diversity of M. Sieversii from the Ile Alatau population, and Table 3 shows the genetic diversity of M. Sieversii from the Dzungar Alatau population, which was calculated as the percentage of alternative amplicon ISSR profiles relative to the overall ISSR profiles of M. domestica amplicons.

Sample no.	Common amplicons with cultural apple, %	Genetic diversity, %	Sample no.	Common amplicons with cultural apple, %	Genetic diversity, %
1-1	40	60	23	40	60
1-2	50	50	29, 32, 33	50	50
1-3	50	50	37	67	33
1-4	33	67	41, 44	50	50
1-5	28,5	71,5	42	50	50
1-6, 1-15	50	50	43	43	57
1-7, 1-13	33	67	45	50	50
1-8, 1-9, 1-14	50	50	47	75	25
1-10	50	50	48, 49, 52	33	67
1-11	40	60	51	60	40
1-12	50	50	53	66	34
1	50	50	54, 55	50	50
4,7	67	33	56	40	60
12, 16, 27, 40	40	60	57	78	22
AP, GD, 13, 36	100	–	58	67	33
18,19	50	50	60	50	50
21	50	50

Table 2.

Genetic diversity of M. Sieversii (Ile Alatau).

Sample no.	Common amplicons with cultural apple, %	Genetic diversity, %	Sample no.	Common amplicons with cultural apple, %	Genetic diversity, %
1	93	7	18	50	50
2	78	22	19	30	70
3	45	55	20	50	50
4	30	70	21	50	50
5	74	26	22	60	40
6	70	30	23	50	50
7	85	15	24	33	67
8	80	20	25	60	40
9	56	44	26	90	10
10	43	57	27	36	64
11	47	53	28	43	57
12	43	57	29	46	54
13	50	50	30	45	55
14	45	55	31	42	58
15	43	57	32	44	56
16	26	74	33	46	54
17	80	20	34	42	58

Table 3.

Genetic diversity of M. Sieversii (Dzungar Alatau).

As can be seen from Tables 4–8, the Golden Delicious and Aport cultivars and wild forms have the same ISSR profiles containing several amplicons, which characterizes their relation to the genus Malus and various amplicons showing polymorphism of the species. Overall, the polymorphism of M. Sieversii and in Ile Alatau and Dzungar Alatau was 73%, with genetic diversity in the Ile Alatau population being higher at 82% and in Dzungar Alatau at 70%. At the same time, in Ile Alatau, 60% of samples were grouped with M. domestica, and in Dzungar Alatau—25%.

Sample no.	Common amplicons with cultural apple, %	Genetic diversity, %	Sample no.	Common amplicons with cultural apple, %	Genetic diversity, %
50, 56, 57, 58, 59, 61, 62, 63, 83, 89, 92: 97	100		79; 81; 82	40	60
52; 53	49	51	86; 87	35	65
79; 81; 82	30	70	98; 99; 100; 101; 103	50	50
64; 65	45	55	51; 55; 60; 68; 69; 70; 71; 73; 74; 76; 83; 84; 88; 90; 93; 94; 95; 102; 104		100
78; 80	43	57

Table 4.

Genetic diversity of M. Sieversii (Dzungar Alatau).

Sample no.	Common amplicons with cultural apple, %	Genetic diversity, %	Sample no.	Common amplicons with cultural apple, %	Genetic diversity, %
ChR-17; ChR-21; ChR-22; ChR-11; ChR-20	100		KR-1; KR-2	45	55
ChR-2; ChR-12 and ChR-23	48	52	KR-18; KR-4; KR-22	40	60
ChR-16; ChR-24	49	51	SR-3-4	100
ChR-9; ChR-10	50	50	SR-3-6; SR-3-11	49	51
ChR-25; ChR-3; ChR-8; ChR-18; ChR-7; ChR-6; ChR-1; ChR-14; ChR-15; ChR-5		100	SR-3-13; SR-3-3	45	55
KR-19; KR-24; KR-14; KR-23; KR-26	100		SR-2; SR-3-11; SR-3-9; SR-3-7; SR-3-8; SR-3-10; SR-3-5		100
KR-5; KR-3; KR-6; KR-21; KR-12; KR-13; KR-10; KR-11; KR-25; KR-27; KR-7; KR-15; KR-28; KR-8; KR-20; KR-18		100

Table 5.

Genetic diversity of Malus Sieversii (Dzungar Alatau).

Sample no.	Common amplicons with cultural apple, %	Genetic diversity, %	Sample no.	Common amplicons with cultural apple, %	Genetic diversity, %
Ssh -2 №7 and Ssh -2 №9	49	51	AP, PS-17; PS-7; PS-12 and PS-18	100
Ssh -2 №4 кв9; Ssh -1-2; Ssh -1-3		100	105, 106 and 107 (Kuznetsova Gorge)	45	55
DA KD –2 №17 and DA KD–2 №18; DA KD–2 №16 and; DA KD–2 №19; DA Ssh 1-5; DA Ssh 1-2 and; DA Ssh 1-1;	40	60	PS-8; PS-9; PS-14; PS-15; PS-11; PS-10; 108 and 109 (Kuznetsova Gorge); DA KD–2 №14; DA KD–2 №15; DA Ssh 1-3 and; DA Ssh 1-4		100

Table 6.

Genetic diversity of M. Sieversii (Dzungar Alatau).

Sample no.	Common amplicons with cultural apple, %	Genetic diversity, %	Sample no.	Common amplicons with cultural apple, %	Genetic diversity, %
KD 1-8; 1-13; 1-14; 1-15; 1-16	35	65	2-№19; 2-№20	35	65
KD 1-2;1-3; 1-4; 1-12; 1-16; 1-6; 1-11; 1-1;1-7; 1-9 and 1-10		100	2-№4; 2- №5; 2- №9		100
2-№11; 2-№12	40	60	AP, DA Ssh 1-6; Ssh 1-7; Ssh 1-8; Ssh 1-9; Ssh 1-10; Ssh 1-15; Ssh 1-16; Ssh 1-17	100
2-№1; 2-№6; 2-№7; 2-№8; 2-№10; 2-№13	33	67	Ssh 1-20; Ssh 1-21	50	50
2-№2; 2-№3	35	65	Ssh 1-12; Ssh 1-14; Ssh 1-18; Ssh 1-19		100
2-№14; 2-№15; 2-№16	45	55

Table 7.

Genetic diversity of M. Sieversii (Dzungar Alatau).

Sample no.	Common amplicons with cultural apple, %	Genetic diversity, %	Sample no.	Common amplicons with cultural apple, %	Genetic diversity, %
KZh-22; KZh-18; KZh-38; KZh-40	100		KZh-43 and KZh-44	45	55
KZh-23; KZh-25 and KZh-34	49	51	D-9; гз-14 and GZ-15	40	60
KZh-20 and KZh-21	45	65	USh-4 and USh -5	35	65
KZh-32; KZh-33 and KZh-46	49	51	DA Ssh -2 №9 and; DA Ssh -2 №11	49	51
KZh-45; KZh-47; KZh-48 and KZh-49	49	51	KZh-19; KZh-24; KZh-26; KZh-27; KZh-28; KZh-29; KZh-30; KZh-31; KZh-35; KZh-36; KZh-37; KZh-39; KZh-41; KZh-42; KZh-50; D-7; D-8; D-10; D-11; D-12; GZ-13; GZ-16; GZ-17; KT-1; KT-2; KT-3; UB-6; DA Ssh -2 №2; DA Ssh - №5		100
51х, 52х and 54х	50	50	53х		100

Table 8.

Genetic diversity of M. Sieversii (Dzungar Alatau).

3. Conclusion

Thus, through molecular genetic analysis of 400 wild apple samples from the Ile Alatau and Dzungarian Alatau populations, ISSR markers have established the presence of loci that can serve as markers for identifying species and populations and identifying intraspecific genetic variation. To create living collections of ex situ and reintroduction of M. Sieversii. 22% of M. Sieversii forms from Ile Alatau and 63% of M. Sieversii forms from Dzungar Alatau, which amounted to, in total, 170 samples of wild apple. The theory of conservation of plant species of reintroduction provides for the admissibility of introducing into a population only those genotypes that are historically present in it. Otherwise, the genetic structure of the population is disturbed, which can lead to irreversible consequences. Therefore, the restoration of degraded plantations should be carried out with seedlings containing the genotypes of these particular plantations, and at the same time free of cultivated fruit genes.

Acknowledgments

Studies were carried out within the framework of the Project of the Government of the Republic of Kazakhstan, GEF/UNDP “Conservation in situ of mountain agro-biodiversity in Kazakhstan.” Project partially funded and published under the scientific program BR10765032 of the Ministry of Agriculture of the Republic of Kazakhstan.