Сельское хозяйство/5.Растениеводство,селекция и семеноводство

C.b.s., Dolgikh S.G. , C.agr.s. Nurtazin M.T.

Kazakh Research Institute of Fruits and Viticulture, Kazakhstan

Study of genetic resources of apricot by means of molecular - genetic markers

The study of apricot in the Kazakstan is explained by its valuable biological and economic peculiarities. Apricot belongs to the precocious and under favorable conditions to annually high-productive fruits. Apricot trees begin to bear fruits in three-four years after planting. In the period of full fruit bearing fruit yield may reach 50 tons per hectare. The apricot trees under favorable conditions are very long-lived-they bear fruits till 100 years, and sometimes live to 200 years. Apricot fruits besides for fresh consumption are the raw-material for processing and dried fruits. At present in the collection of the Pomological Garden apricot is presented by 80 introduced varieties from different parts of world.

However, the regions for commercial culture of apricot are limited and it is explained by very short period of winter dormancy and in the time of early and long thaws fruit buds open from dormancy and are damaged even by slight negative temperatures. Climatic conditions of the south-east of Kazakhstan are distinguished by frequent recurrent spring frosts. Therefore apricot varieties winter hardiness and the resistance to spring frosts are the main indexes for apricot determining the possibility of its growing under conditions of the south-east of Kazakhstan. Proceeding from this selection work on apricot in the Pomological Garden is conducted for enhance of its winter hardiness and frost-resistance – the selection of late-flowering varieties and resistance wild forms.

For the selection of the initial apricot forms great positive part played the suggested by K.F.Kostina [1] the systematic-geographical scheme of the division of the whole apricot variety diversity in four groups: Middle-Asian, Iranian-Caucasian, European and East-Asian. The Middle-Asian group is the most ancient according to it origin and includes all local varieties of Middle Asia, West China, Afghanistan and West India. These varieties have comparatively long period of deep dormancy of fruit buds and later flowering, relatively high frost-resistance, high heat-resistance, high fruit sugar content. However they are distinguished by weak resistance to fungus diseases, later then in other groups the period of fruit bearing, self-sterility, the greatest fruit size, lower acidity and weak aroma.

The European group is the youngest according to the origin and includes a little number of the varieties, introduced in West Europe from Iran, Armenia and North Africa. The varieties of this group are distinguished by relatively best resistance to fungus diseases, precocity, and self-bearing, large size of fruits with typical apricot aroma. But they are less resistant to recurrent frosts and spring frosts.

The Iranian-Caucasian group includes a considerable number of local varieties distributed in Armenia, Georgia, Dagestan and Iran. According to the most their peculiarities the varieties of this group are in the intermediate position between the varieties of European and Middle-Asian groups.

East-Asian or Siberian-Manchurian group includes all varieties in breeding of which east-Asian species participated: Apricot Siberian (Armeniaca sibirica), Apricot Manchurian (A.manshurica) and Apricot Ansu (A.Ansu). The dignity of the varieties of this group is their high frost-resistance; however they have a short period of winter dormancy, small fruits and satisfactory taste. For the breeding of late-flowering and frost-resistant apricot varieties in the Pomological Garden the seedling from open pollination of middle-Asian and European varieties and also wild apricot forms are grown.

Kazakhstan possesses the great gene fund of wild apricot. In the selection programmers germplasm of wild apricot forms are very important as they initially had resistant genes both to the frosts [2].

The majority wild apricot mass of the world is referred to one and the same botanical species- Prunus Armeniaca vulgaris Lam. In the limits of the species 4 main ecology-geographical groups were distinguished: Middle-Asian, Iran-Caucasus, European and Dzhungar-Zailijskei. Dzhungar-Zailiyskei group occupies the very northern part of the Middle-Asian area of apricot stands, entering more northern of 44^o north latitude. This is the youngest sub-group of the Middle-Asian apricot group.

Wild growing forms of Zailijskei and Dzhungarskei Alatau contain a considerable part of the natural apricot gene fund. It is an endemic species of northern and western Tien-Shan, a relatively rare species with strongly decreasing natural habitat. In the northern macro slope of Zailiyskei Alatau, it usually grows in gorges of the rivers Bolshaya and Malaya Almatinka, Talgar, Aksai, Issyk and it is plentiful in Kotur-Bulak and Karakunuz. In Dzhungarskei Alatau, the northern border of its natural habitat, it is found along the banks of the Khorgos, Usek, and Koktal rivers. The total area in the Republic occupied by this species is 1675 ha. The wild apricot of Kazakhstan is also notable for its excellent winter-hardiness, withstanding air temperatures of -37 to 40⁰ C [3]. Kostina K.F. [1], Shitt P.G [4] and other unanimously recognize the mountain origin of cultivated apricot. Academician Komarov V.P. [5] pointed out that Tien Shan wild apricot showed be considered as the ancestor of cultivated apricot. Especially there is a lot of wild apricot in Zailijskei Alatau.

Under conditions of Almaty region a considerable part of natural gene-fund of common apricot is concentrated. However, because of economic man activity natural apricot area reduces, many habitats are irretrievably disturbed and even are lost as a result of tree felling. Wild apricot is registered in Red Book of Kazakhstan. The genetic diversity of the natural, wild apricot populations in the Tien-Shan mountain range in Kazakhstan offers an exceptionally important gene fund for breeding programs aimed at raise frost tolerance, and resistance to pathogens. Therefore, it is of great importance to maintain this valuable genetic material in a germ plasma collection.

Lately time Wild Apricot of Kazakhstan has been presented by shrunken population in locality, strong corrupt by the getting practice on of nature use. In here there is a problem no only of conservation, but recovery of this genus in situ and creation of collections ex situ.

Practice shows that by the moment of keen need in reintroduction genetic and ecologic characteristics of species are un-full and discrepant, that demands additional research which is impossible without application of molecular markers. Using molecular markers, one can carry out directly and precise measurements: variety oh genes and plenty of alleles; subdivision of population; heterozygosis of effective sizes of populations and frequency of alleles; likeness and differences (genetic differences) in specials and populations; genetic maps for using in selection of germplasm in breeding programs.

Now the molecular markers are widespread in taxonomy and population genetics and in particular of DNA, based on PCR.

Development of science is defined by using high technologies allowing automating an analysis in the same time a large size of specimens, what is possible when using ISSR-analyses.

In ISSR as primers it is used known sequence of micro-satellites and bearing in one of ends a sequence of the two-four random nucleotides. Such primers allow amplification fragments of DNA, which are between two enough near located micro-satellite sequences (as a rule it is a unique DNA). The obtained patterns of PCR-products are species-specific. ISSR-markers among the markers of dominate type of inheritance, which polymorphism is tested on a presence/absence of stripe. The method has got a good reproducibility and along with AFLP is used to determine inter-specific variability, identification of species, populations, lines and individual gene-typing.

The aim of the research is: to study gene fund of apricot in Pomological Garden on resistance to frost and polymorphism wild apricot by ISSR-markers.

Materials and Methods

For exploration it was taken 29 varieties of apricot trees from the collection of the Pomological Garden and 16 forms of select wild apricot growing in Pomological Garden.

The samples of young leaves, taken from the dots of sprouts, are fixed in silica gel. Genome DNA was separated from above dried samples by using a set of NucleoSpin Plant (firm “Biokom”, Russia) according to the Producer’s Protocol and had been kept at temperature – 25⁰C. PCR parameters, using for the analysis, included: 3 min at the temperature of 95⁰C – the first denatured, the following 35 cycles: 30 second of denatured at 94⁰C; annealing of primers in corresponding temperature – 30 sec., 40 seconds of elongation at 72⁰C + addition of 2 seconds for each cycle.

Reaction mixture (20µl) contained 10-20 ng DNA, 20 pmols of primers and ready reaction mix (firm “Biokom”, Russia), containing of inhibiting for “hot start” Taq DNA polymerase, dNTP and magnesium chloride with final concentration, correspondently 1 u, 200 mkM and 2,5 mM and also optimized buffer system for the conduction of PCR reaction.

Table. 1 Primer sequences

Primer N	Primer sequences (5’-3’)	Primer N	Primer sequences (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	М12	cac aca cac aca (ag)(ct)

The temperature of the annealing of primers was the following for the primers: M12-49,5⁰C, M2-49,5⁰C, M9-50⁰C, M8-52,7⁰C, М3-52,7⁰C and М4-50,8⁰C.

For the electrophoreses division of the PCR-products it was used 1,7% of agarose gels in 1x TBE buffer (50 mM Tris, 50 nM boric acid, 1 mM EDTA, pH 8,0) with bromine ethide (0,5 mkg/ml) on 100 V during 45 min with the following photograph of the produced PCR-products. Figure photographs of the agarose gels were analyzed in the programmer Cross Checker 2, 91 [6] with the compiling of binary matrixes in present/absent of the fragments of identical length.

Results

At present in the collection of the Pomological Garden apricot is presented by 80 introduced varieties. The characteristic of some apricot varieties, used in the breeding investigations is presented on the Table 2.

Table 2. Characteristics of selected apricot cultivars at the Pomological Garden

№	Cultivar	Color	Fruit wt (g)	Yield capacity	Ripening time	Separate of seed	Taste of seed	Origin
1	Nikitskiy Krasnoshchekiy	Orange with ruddiness	56	high	1 decade of August	good	sweet	Ukraine
2	Arzami	Yellow-cream with ruddiness	58-70	high	1 decade of July	half-separate	sweet	Uzbekistan
3	Alexander	Yellow	33	good	3 decade of June	good	bitter	USA
4	Kolkhozniy	Orange	36	moderate	3 decade of August	good	bitter	Russia
5	Tlor Ziran	Blue	28	good	1decade of August	not-separate	sweet	W. Europe
6	No16 Katchenko	Light orange	25	moderate	3 decade of July	good	bitter	Ukraine
7	Spitak kremoviy	Cream with ruddiness	50	high	3decade of August	good	sweet	Uzbekistan
8	Makhtobi Samarkandskey	White-cream	41	moderate	2 decade of July	good	sweet	Uzbekistan
9	Korolevskiy	Pink-orange	40-50	good	2 decade of July	good	sweet	France
10	Kursadyk	Orange	30	high	2 decade of July	good	sweet	Uzbekistan
11	Melitopolskiy ranniy	Bright orange with ruddiness	37	moderate	3 decade of June	half-separate	sweet	Ukraine
12	Triumf severniy	Orange with ruddiness	45	good	1 decade of August	good	sweet	Russia
13	Badami	Yellow with ruddiness	20	good	3 decade of June	good	sweet	Uzbekistan
14	Vir	Green with red ruddiness	45	good	2 decade of July	good	sweet	Uzbekistan
15	z.Suanpsana	Orange	36	high	3 decade of July	good	sweet	Uzbekistan
16	Michurinez	Light -orange	36	good	3 decade of July	good	sweet	Russia
17	Zarya Vostoka	Cream-yellow with bright ruddiness	50	high	3 decade of July	good	sweet	Uzbekistan
18	Zard	Cream-yellow	30	high	2 decade of July	good	sweet	Uzbekistan
19	Sovetskiy	Bright yellow with ruddiness	45	good	1 decade of August	good	sweet	Uzbekistan
20	Nikitskey	Green with dark red ruddiness	60	high	3 decade of July	good	sweet	Ukraine
21	Darunok Malaxovoy	Bright yellow with ruddiness	40	high	2decade of July	good	bitter	Ukraine
22	Krasnotchekiy	Bright orange	50	high	3 decade of August	good	sweet	Ukraine
23	Zavetniy	Orange-yellow	30	good	3 decade of August	good	sweet	Ukraine
24	Yubileiniy	Orange with ruddiness	45	good	2decade of August	good	sweet	Ukraine
25	Burevestnik	Light - Cream	35		3 decade of July	good	sweet	Ukraine
26	Obilniy	Orange	45	good	1 decade of August	not-separate	sweet	Ukraine
27	D-11	Orange	25	good	1 decade of July	good	sweet	Ukraine
28	Mary Cenad	Orange with ruddiness	40-45	moderate	2 decade of August	good	sweet	Rumania
29	Manitoba	Bright yellow with ruddiness	40	high	2 decade of July	good	sweet	Canada

Winter hardy forms of wild apricot with resistance of blossom -5⁰C were selected from the most northern areas of the mountain range of Tien-Shan: H-1/7, H-1/14 and H-1/23.

Among the apricot varieties the variety of the Ukraine selection hybrid D-11 appeared to be the most resistant to frost and also the varieties Korolevskiy, Manitoba, Yubileiniy, Triumf Severniy. The varieties Zard, Izumrud, Obilniy were selected as resistant to late frosts. The varieties Darunok Malakhovoy, Botsadovskiy, Burevestnik, Mary Cenad by the most average-perennial productivity (74 centre/ha) distinguished. These varieties are used in the breeding programmers and are recommended to the State and Production variety-testing.

The level of heterozygosis marker data is identified according to the mean frequency of the similarity of alternative alleles. For all this 50% of the main alleles against 50% of alternative alleles (0, 5) corresponds to the high level of heterozygosis, whereas 0, 9 on 0, 1 (90% against 10%) corresponds to the low level. For the determination of polymorphism of DNA of investigated samples of wild apricot the markers with arbitrary sequence of nucleotides of DNA were used.

Genetic diversity of A.Vulgaris (wild apricot) was calculated as percent content of alternative profiles of 6ISSR amplicones relatively to the content of common profiles of ISSR amplicones of cultivated apricot - Nikitskei Krasnoshechekei (table.3).

Table 3. – Genetic diversity of A. Vulgaris from Pomological garden

№ sample	Common amplicons with the cultured apricot,%	Genetic diversity, %
1,2	75	25
3, 4	66	34
8	28,5	71,5
5, 7	14	86
9	50	50
10,13,14	25	75
12	50	50
15	37,5	62,5
11,16	43	57

The variety Nikitskei Krasnoshechekei and wild forms have both the identical ISSR profiles, containing several amplicones that characterizes them as concerning to the P. Armeniaca, and the different amplicones, showing polymorphism of the species. Such loci - the ISSR profiles with the content of unique amplicones may as markers for interspecies identification. As a whole the polymorphism of wild apricot from Pomological garden was 57% where 44% of samples contained unique loci. The samples №№ 5, 8, 10, 11 and 15 distinguished the most genetic diversity. They belong to the same genetic group of A.Vulgaris and can use for further breeding work.

Conclusion

Winter hardy forms of wild apricot with resistance of blossom -5⁰C were selected from the most northern areas of the mountain range of Tien-Shan: H-1/7, H-1/14 and H-1/23.

Among the apricot varieties the variety of the Ukraine selection hybrid D-11 appeared to be the most resistant to frost and also the varieties Korolevskiy, Manitoba, Yubileiniy, Triumf Severniy. The varieties Zard, Izumrud, Obilniy were selected as resistant to late frosts. The varieties Darunok Malakhovoy, Botsadovskiy, Burevestnik, Mary Cenad by the most average-perennial productivity (74 centre/ha) distinguished. These varieties are used in the breeding programmers and are recommended to the State and Production variety-testing. The polymorphism of wild apricot from Pomological garden was 57% where 44% of samples contained unique loci. The samples №№ 5, 8, 10, 11 and 15 distinguished the most genetic diversity. They belong to the same genetic group of A.Vulgaris and can use for further breeding work.

Literature:

1. Kostina K.F. Apricot, VASKHNIL,Leningrad, 1936.

2. Nurtazin M.T. Winter hardiness and productivity of apricot in Alma-Ata region. Scientific publication of Kazakh SRI of Fruits and Viticulture, Almaty, 2001, Vol.16, p.16-18.

3. Dzhangaliev A.D., Salova T.N., and Turekhanova P.M. The wild fruit and nut plants of Kazakhstan/ Horticultural Reviews, Volume 29, Edited by Jules Janick ISBN 0-471-21968-1, 2003, p.325-326.

4. Shitt P.G Apricot, Selkhozizdat, 1950.

5. Komarov V.P. The origin of cultivated plants /Agricultural publishing house, 1938.

6.Buntjer J.B. 2000. Cross Checker: computer assisted scoring of genetic AFLP data//Plant & Animal Genome V111 Conference. San.Diego, CA, January 9-12.