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УДК 634.8 + 631.52 + 581.167
PROGRESS IN GERMPLASM IDENTIFICATION AND GENOTYPING METHODS IN THE STUDY OF THREE TABLE GRAPE VARIETIES1
Troshin Leonid Petrovich Dr.Sci.Biol., professor
Milovanov Alexander Valerievich training master
Mahovickiy Bogdan Alexandrovich postgraduate student
Kuban State Agrarian University, Krasnodar, Russia
In the present article, we have described data of comparative ampelography of biometric evaluation of leaf parameters of the three table grapes: Preobragenie, Victor and Jubiley Novocherkassk, widespread in the amateur and farming areas of Russia and the Ukraine. Showed results of molecular genetic analysis of DNA from these table grapes
Keywords: GRAPES, GRADE, FEATURES, PROPERTIES, SHEETS, COMPARATIVE AMPELOGRAPHY, MORPHOLOGY, BIOMETRIC EVALUATION, PREOBRAGENIE, VICTOR, JUBILEY NOVOCHERKASSK, COMBINATIVE SELECTION, SIBLINGS
1ЮС 634.8 + 631.52 + 581.167
ИДЕНТИФИКАЦИЯ И ГЕНОТИПИРОВАНИЕ ЗАРОДЫШЕВОЙ ПЛАЗМЫ ТРЕХ СТОЛОВЫХ СОРТОВ ВИНОГРАДА
Трошин Леонид Петрович д.б.н., профессор
Милованов Александр Валерьевич учебный мастер
Маховицкий Богдан Александрович аспирант
Кубанский государственный аграрный университет, Краснодар, Россия
В представленной статье освещены данные сравнительной ампелографии о биометрической оценке листовых параметров трех столовых сортов винограда Преображение, Виктор и Юбилей Новочеркасска, широко распространенных на любительских и фермерских участках России и Украины. Приведены результаты молекулярно-генетического анализа ДНК этих столовых сортов
Ключевые слова: ВИНОРРАД, СОРТ, ПРИЗНАКИ, СВОЙСТВА, ЛИСТ, СРАВНИТЕЛЬНАЯ АМПЕЛОРРАФИЯ, МОРФОЛОРИЯ, БИОМЕТРИЧЕСКАЯ ОЦЕНКА, ПРЕОБРАЖЕНИЕ, ВИКТОР, ЮБИЛЕЙ НОВОЧЕРКАССКА, КОМБИНАТИВНАЯ СЕЛЕКЦИЯ, СИБСЫ
INTRODUCTION
Derived by combinative breeding three new high-yielding table grape varieties Preobragenie, Victor and Jubilej Novocherkassk with elevated resistance to Phylloxera and fungal diseases and suitable for own-rooted cultivation became a basis for solutions a two hundred year problem in grape growing [1-12, 16-17, 19-30].
It plays an important role a market demand for spreading of these and similar "optimal" hybrid forms of spectacular appearance as a consumer of breeding material, it is difficult to visually distinguishable by morphometry of leaves and therefore needed to recognize when attracting an interactive ampelography [4, 13-15, 18].
Material and Methods
1 Cost action FA1003:
East-West Collaboration for Grapevine Diversity Exploration and Mobilization of Adaptive Traits for Breeding
Three table grape sibling varieties were taken for studies, bred by amateur wine-grower Victor Nikolaevich Kraynov from city Novocherkassk under supervising of a professional Ivan Aleksandrovich Kostrikin. The breeding scheme was the following:
Talisman [Frumoasa Alba (Guzal kara x Saves Villard 20-473) x Vostorg (Dawn North x Dolores) x Russian early] x Kishmish Luchistyi (Cardinal x Kishmish rozovyi, pink) [2, 6-7, 9, 19, 22-23] (Fig. 1 and 2).
Fig. 1. Cluster of Talisman [25] Fig. 2. Cluster of Kishmish Tuchistyi [25]
While these three (Preobragenie, Victor and Jubilej Novocherkassk) varieties have quite similar phenotypes (Fig. 3-17) its become necessary to conduct comparative morphometric study of leaves [13-15].
Fig. 3-6. Open bud, shoot tip, matured leaf and cluster of Preobrcigenie Preobragenie. The variety has very early ripening period - 110-115 days. The
clusters are large, in range from 0,7 to 1,5 kg, conic, without berry shot. The berries are very
large, 12-18 g, oval-oblong, pink, with harmonious taste. The flesh is fleshy-juicy, the skin
is edible. The productivity is high. It can be cultivated as own-rooted plant having
medium-power growth. The shoot ripening is good, rooting ability of cuttings is easily, the
compatibility with rootstocks is good. The frost resistance is -21°C. Resistance to fungal
diseases is of 3,0-3,5 points. The transportability is good with very high market ability.
Damaging by wasps is medium. This variety has similar morphological characters as Victor,
but vigor of growth is higher respect of it. The variety has ability to give an additional crop on stepson [19].
Fig. 7-10. Open bud, shoot tip, matured leaf and cluster of Victor
Victor. Variety is very early ripening period 110 days. Clusters are of medium loose, from 0,7 to 1 kg, tapered, are not exposed breakage, very interesting. Berries are large, 12-18 g, elongated, dark pink, harmonious taste. Sredneplotnaya pulp, peel eaten. 1-2 seeds per berry pieces. Can be cultivated in own-rooted culture and medium-power growth. Shoots ripen well, cuttings take root easily, compatibility with the stocks is good,
freezeproof to -23 °С. То fungal diseases is relatively stable - 3 points. Portability is good, very high marketability. Wasps damaged in the medium. Morphologically similar to Preobragenie, but has a more moderate force growth. Able to give a second crop on stepson
Fig. 11-14. Open bud, shoot tip, matured leaf and cluster of Jubilee Novocherkassk
Jubilee Novocherkassk. Variety is very early ripening period: 110-115 days. The clusters are large, 0,8 to 1,6 kg, tapered, are not exposed breakage. Berries are very large, 12-18 g, oblong-oval, white and pink, harmonious taste. Flesh fleshy-juicy, the skin is eaten. Can be cultivated in own-rooted culture and medium-power growth. Shoots ripen well, cuttings root easily, compatibility with the stocks is good. Average frost resistance, can withstand temperatures of -23 °C. Resistance to fungal diseases 3 points. Transportability
and high marketability. Wasps damaged in the medium. Morphologically similar to the sort Victor and Preobragenie, but differs from them in a more subtle berry. Currently proliferate growers Russia and Ukraine [19, 25].
The task of research: a comparison of morphological features descriptor leaves of three grape varieties growing in the testing plots of ОАО "Juzhnaja", Temryuk district of the Krasnodar Territory, and to find out their phenotypic identity and DNA- analysis of this siblings.
RESULTS
To determine the morphological features of leaves of compared three varieties of siblings: Preobragenie, Victor and Jubilee Novocherkassk first made their scan and detailed measurement on every sheet of 22 quantitative traits (Fig. 15-30, Table 1).
Fig. 15-16. The leaves of bushes number 4 and number 18 varieties Preobragenie
Рис. 17-18. Double-sided sheet of bush number 9 varieties Preobragenie
Fig. 19-20. Double-sided sheet number 14 of the bush varieties Preobragenie
Fig. 23-24. Double-sided sheet number 5 of the bush varieties Victor
Fig. 27-28. The leaves of the bush number 4 varieties Jubilee Novocherkassk
Fig. 29-30. The leaves of bushes number 23 and 24 varieties Jubilee Novocherkassk
Description scanned 426 raised, that is, adult leaves was conducted using SIAMS Photolab. Information about 01V codes are automatically stored in Excel-table in the following order: 065-1 or 651 in our work, - the length of the leaf blade, 065-2, or 652, -width of the leaf blade, 092 - the length of the petiole, 601 - length of midrib; 602 - length of the upper lateral veins; 603 - length of the lower lateral veins; 604 - length from the intersection of the lower lateral veins to its lower edge 605 - distance to top bay, 606 -distance to lower bay, 607 - alpha angle, 608 - beta angle, 609 - angle zeta, 610 - angle gamma, 611 - the width of the intersection of the lower lateral veins of the lower edge, 612 -the length of the far right tooth, 613 - the width of the far right tooth, 614 - length of the right lower tooth; 615 - the width of the right lower tooth; 616 - the number of teeth the upper side of the blade, 617 - the width of the upper side of the blade, 618 - the distance between the lower lobes; 619 - length from the point of attachment of the petiole to the leaf
blade to the end point of the lower teeth [13-15].
Technical work of measurement leafs was performed by A. Milovanov and B. Mahovitskii (Table 1). Provision of selected natural leaf samples carried out by agronomists of ОАО "Juzhnaja", Temryuk district of the Krasnodar Territory O.B. Belizovskii and L.A. Lashenko, for which we express our sincere gratitude to them.
Table 1. — Data of interactive measurement adult leaves of three grape varieties and 22 original settings
№ ofleaves Ampelographic features (OIV codes)
651 652 092 601 602 603 604 605 606 607 608
Preobragenie
4-5 12,7 12,1 5,9 9,6 8,8 6,1 5,2 3,2 3,4 31,8 43,2
9-1 13,6 12,0 9,0 9,9 8,3 5,5 4,9 4,1 3,7 45,6 45,8
14-1 11,2 11,8 5,4 8,8 8,5 5,8 4,8 3,4 3,5 46,3 44,8
18-1 12,8 13,2 7,1 10,4 8,5 5,9 4,8 5,3 3,8 39,7 54,1
Victor
2-6 13,9 12,6 6,4 10,8 9,1 6,0 5,2 4,8 4,3 38,6 40,4
4 13,1 14,5 7,7 10,9 ЮД 7,5 4,2 4,8 4,2 47,1 39,2
5 14,4 13,7 6,4 11,4 10,1 6,7 3,6 4,8 4,1 38,3 40,7
15 13,7 13,6 8,2 10,8 9,5 7,2 3,1 3,7 4,2 45,1 56,9
17 15,6 13,7 11,1 12,3 10,1 7,0 3,8 4,0 3,3 37,8 36,7
Jubilee Novocherkassk
4-18 12,4 11,7 6,4 9,8 8,9 6,2 2,8 3,2 3,6 40,8 49,6
4-19 13,8 11,7 7,4 10,8 9,5 5,5 2,8 4,4 4,6 46,9 56,5
23b 12,4 11,3 6,6 9,4 8,6 5,6 2,9 4,0 3,4 40,6 46,3
23c 12,0 11,3 7,1 ЮД 8,2 6,4 3,4 4,2 3,1 42,2 51,4
23h ПД 9,8 3,8 8,4 7,9 5,0 2,8 3,0 2,9 43,1 43,3
24b 12,7 ЮД 6,2 9,4 7,8 4,9 3,1 3,0 3,2 48,1 44,5
24h 9,5 9,8 6,5 7,5 6,5 4,7 3,6 4,1 2,8 49,5 43,6
Continuation of Table 1
609 610 611 612 613 614 615 616 617 618 619
Preobragenie
4-5 71,5 81,7 3,2 1,4 1,2 1,1 1,0 4 5,0 2,5 4,3
9-1 68,7 65,5 3,4 1,9 1,7 1,1 1,5 4 5,0 1,1 4,4
14-1 52,5 61,7 2,9 1,4 1,1 0,9 1,4 4 5,3 1,5 4,2
18-1 63,4 58,1 2,8 1,6 1,6 1,1 1,6 5 4,7 2,8 3,8
Victor
2-6 70,4 66,5 3,4 1,8 1,8 1,6 1,2 3 3,7 3,3 4,3
4 70,1 52,2 2,8 1,0 1,2 0,7 0,9 5 4,2 3,4 5,1
5 72,8 66,1 2,9 0,5 0,7 1,0 1,3 6 4,3 3,1 4,2
15 55,1 52,3 2,4 0,7 1,0 0,3 0,8 6 4,6 3,3 4,6
17 75,4 64,5 2,4 1,1 0,9 0,6 1,0 5 4,7 2,7 4,7
Jubilee Novocherkassk
4-18 14,2 66,6 2,0 1,1 1,0 0,4 0,9 5 5,0 1,7 3,4
4-19 66,2 73,9 1,5 0,8 0,9 0,9 1,1 6 4,4 1,7 3,5
23b 84,6 66,5 2,0 0,9 0,8 0,6 0,9 5 3,9 1,1 3,5
23c 76,9 57,5 1,7 0,7 0,7 0,4 0,7 5 3,7 0,8 3,3
23h 64,0 65,4 1,7 0,6 0,8 0,8 0,8 5 3,9 1,7 3,7
24b 79,7 65,7 1,6 0,7 0,6 0,6 0,8 6 3,4 1,6 3,6
24h 65,6 61,4 3,0 1,7 1,5 1,2 0,9 3 3,4 0,8 3,1
For comparative studies, leaves of varieties were also evaluated 18 additional index features [15].
Results of biometric data analysis of interactive three measurements of 426 leaves of grapes are shown in Table 2 and 3.
Table 2. - Variation analysis of the data leaves of the three grape varieties on 22 ampelographic
featured
Ampelographic features (OIV codes)
651 652 092 601 602 603 604 605 606 607 608
Preobragenie, n = 148
X 13,58 13,28 7,51 10,28 9,37 6,66 5,66 4,51 4,12 41,99 49,44
Sx 0,15 0,17 0,15 0,13 0,11 0,10 0,09 0,08 0,07 0,71 0,81
lim 9,5 11,1 9,6 10,9 6,8 5,9 5,5 6,5 5,6 58,9 53,9
min 9,9 8,6 2,6 5,8 6,9 4,2 2,9 2,0 1,5 13,7 25,0
max 19,4 19,7 12,2 16,8 13,7 10,1 8,4 8,5 7,1 72,6 78,9
CV 13,4 15,4 24,4 15,3 14,3 17,8 18,4 20,3 21,6 20,5 19,9
Victor, n = 140
"x 13,61 13,37 7,71 10,35 9,39 6,72 5,72 4,52 4,13 41,94 49,36
Sx 0,16 0,17 0,16 0,13 0,12 0,10 0,09 0,08 0,08 0,74 0,84
lim 9,5 11,1 n,o 9,6 8,8 5,3 5,5 6,5 5,6 58,9 53,9
min 9,9 8,6 2,0 7,1 4,9 4,8 2,9 2,0 1,5 13,7 25,0
max 19,4 19,7 13,0 16,7 13,7 10,1 8,4 8,5 7,1 72,6 78,9
CV 13,5 15,2 24,8 14,9 14,7 17,5 17,9 20,5 22,0 20,9 20,1
Jubilee Novocherkassk, n = [38
"x 12,85 12,22 6,92 9,51 8,72 5,97 5,03 4,42 3,97 40,30 48,05
sx 0,16 0,16 0,14 0,12 0,11 0,09 0,08 0,08 0,06 0,65 0,73
lim 11,3 9,7 8,7 8,9 8,2 5,4 4,4 6,9 3,9 42,1 64,2
min 7,5 7,2 2,9 4,9 4,7 3,4 2,9 2,2 1,6 18,4 22,8
max 18,8 16,9 11,6 13,8 12,9 8,9 7,3 9,1 5,5 60,5 87,0
CV 14,6 15,2 24,0 14,8 15,4 17,8 18,7 22,4 18,1 18,9 17,8
Continuation o f Table 2
609 610 611 612 613 614 615 616 617 618 619
Preobragenie
X 60,02 64,45 3,50 1,82 1,72 1,24 1,37 4,05 5,41 2,24 4,55
Sx 0,85 0,85 0,07 0,04 0,04 0,02 0,03 0,03 0,07 0,08 0,07
lim 66,5 62,3 7,3 2,6 3,7 1,8 2,7 2 5,3 6,8 5,2
min 29,8 25,8 2,0 1,1 1,0 0,6 0,5 3 3,5 0,7 2,5
max 96,3 88,1 9,3 3,7 4,7 2,4 3,2 5 8,8 7,5 7,7
CV 17,3 16,1 23,0 25,6 28,1 24,0 23,9 10,1 15,7 43,5 19,6
Victor
"x 60,10 64,65 3,52 1,83 1,75 1,25 1,39 4,06 5,40 2,20 4,59
sx 0,89 0,89 0,07 0,04 0,05 0,03 0,03 0,04 0,07 0,08 0,07
lim 66,5 62,3 7,3 2,6 3,7 1,8 2,7 2 5,3 4,1 5,2
min 29,8 25,8 2,0 1,1 1,0 0,6 0,5 3 3,5 0,7 2,5
max 96,3 88,1 9,3 3,7 4,7 2,4 3,2 5 8,8 4,8 7,7
CV 17,6 16,3 23,2 25,6 30,6 24,2 24,2 10,8 15,9 40,6 19,2
Jubilee Novocherkassk
"x 60,05 61,11 3,43 1,68 1,54 1,19 1,24 2,51 3,65 2,28 4,01
sx 0,77 0,84 0,06 0,04 0,03 0,02 0,02 0,05 0,06 0,07 0,06
lim 69,9 58,5 5,0 2,3 2,7 1,6 1,3 2 3,9 4,2 4,3
min 20,7 31,5 1,4 0,6 0,9 0,6 0,6 1 1,9 0,4 1,7
max 90,6 90,0 6,4 2,9 3,6 2,2 1,9 3 5,8 4,6 6,0
CV 15,2 16,2 21,5 25,4 26,2 21,8 20,5 22,2 19,4 35,9 17,6
where: _ x - arithmetic mean, sx - error of the mean, lim - the interval of variation, min - minimum value, max - the maximum value, CV - coefficient of variation, %.
According to the data presented in Table 3 and below the Table 4, you can make the following conclusion. The coefficients of variation CV directly measured 22 ampelographic characteristics of leaves, without their biometric transformation from Preobragenie vary within wide ranges: 10,1 - 43,5 under his average of 19,7% in CV Victor almost in the same range: 10,7 - 40,6 at 19,9%, and the cultivar Jubilee Novocherkassk in a smaller amplitude: 14,6-35,9 at 19,4%.
Table 4. - Variation analysis of these calculations 18 index traits of leaves in three grape varieties
Amelographic indexes
651x652 651/652 601/92 603/601 604/601 611/601 613/612 615/614 602/605
Preobragenie
X 183,27 1,03 1,43 0,65 0,55 0,35 0,97 1,13 2,14
Sx 4,32 0,01 0,03 0,01 0,01 0,01 0,02 0,02 0,04
lim 278,75 0,67 2,59 0,58 0,49 0,70 1,64 1,69 2,61
min 98,79 0,71 0,74 0,43 0,36 0,18 0,51 0,54 1,20
max 377,54 1,38 3,33 1,01 0,85 0,88 2,15 2,24 3,81
CV 28,7 9,3 23,0 13,2 14,1 24,4 26,1 23,1 20,7
Victor
"x 185,08 1,02 1,41 0,65 0,56 0,34 0,98 1,14 2,14
Sx 4,47 0,01 0,03 0,01 0,01 0,01 0,02 0,02 0,04
lim 278,75 0,67 3,20 0,58 0,42 0,70 1,72 1,69 2,61
min 98,79 0,71 0,91 0,43 0,33 0,18 0,51 0,54 1,20
max 377,54 1,38 4,12 1,01 0,75 0,88 2,23 2,24 3,81
CV 28,6 9,3 27,7 12,7 13,6 23,8 28,0 23,7 20,0
Jubilee Novocherkassk
"x 159,86 1,06 1,43 0,63 0,53 0,36 0,96 1,06 2,03
Sx 3,85 0,01 0,02 0,01 0,01 0,01 0,03 0,02 0,03
lim 262,87 0,53 1,97 0,43 0,41 0,43 2,37 1,16 2,38
min 53,83 0,83 0,88 0,44 0,37 0,15 0,50 0,57 1,16
max 316,70 1,36 2,85 0,87 0,78 0,58 2,87 1,73 3,54
CV 28,3 8,7 20,5 12,4 13,8 18,3 31,7 19,1 18,8
Continue of table 4
602/601 603/606 614/ 615 612/613 607+608 607+610 607+608 607+608 +609 +610 616*617
Preobragenie
X 0,92 1,69 0,93 1,08 91,42 106,44 151,44 155,87 21,90
Sx 0,01 0,04 0,02 0,02 0,99 1,07 1,37 1,24 0,34
lim 0,78 3,13 1,39 1,50 79,01 69,66 86,68 89,06 23,94
min 0,67 0,88 0,45 0,47 58,03 74,55 109,67 106,22 11,29
max 1,45 4,01 1,84 1,97 137,04 144,20 196,35 195,28 35,23
CV 11,3 29,2 23,6 21,3 13,2 12,2 11,0 9,7 18,8
Victor
"x 0,91 1,71 0,93 1,08 91,30 106,59 151,41 155,95 21,96
Sx 0,01 0,04 0,02 0,02 1,04 1,11 1,43 1,30 0,36
lim 0,65 3,13 1,39 1,52 79,01 69,66 86,68 89,06 23,94
min 0,59 0,88 0,45 0,45 58,03 74,55 109,67 106,22 11,29
max 1,24 4,01 1,84 1,97 137,04 144,20 196,35 195,28 35,23
CV 10,2 30,3 24,9 21,9 13,5 12,4 11,2 9,9 19,5
Jubilee Novocherkassk
"x 0,92 1,53 0,98 1,11 88,35 101,41 148,40 149,46 9,17
Sx 0,01 0,02 0,02 0,02 0,96 1,06 1,22 1,13 0,24
lim 0,53 2,38 1,18 1,63 71,76 68,42 94,17 71,52 15,05
min 0,64 0,95 0,58 0,35 61,26 57,48 102,46 113,22 2,47
max 1,17 3,33 1,76 1,98 133,02 125,91 196,63 184,74 17,52
CV 9,1 18,2 19,9 23,1 12,7 12,2 9,7 8,9 30,2
If we analyze the coefficients of variation of 18 index traits of leaves in the same http://ej.kubagro.ru/2013/06/pdf/49.pdf
three grape varieties (Table 4), they are reflected in the following figures: 9,3 - 29,2 at 18,5%, 9,3 - 30,3 at 18,9% and 8,7 - 31,7 at 17,5%. The overall mean CV of all index attributes accounted for more than 18% - almost 2% less than the original, that is, direct measurement of the 22 signs.
When combined with 40 CV source and index the basis of their average values in the cultivars were at the same level: 19,5, 19,8 and 18,9.
Carried out by the Student's evaluation of differences expressivity 40 ampelographic characteristics in the studied grape varieties has resulted in: between varieties Preobragenie and Victor significant arithmetic differences were found (t < 0,92 for P > 0.05) - the leaves they were twins phenotypic and grade Jubilee of Novocherkassk in comparison with his two above-named siblings metrically different in 21 and 22 cases out of 40 (t > 2,57 at P < 0.01).
For a contemporary analysis of the differences of genotypes was used molecular genetic method. When DNA isolation and PCR reactions were involved in the standard of our research methods and parameters previously highlighted in the literature [30].
DNA extraction was performed upgraded CTAB-method [30]. The paper studied the following protoclones grapes: Victor 29-5 (1), Victor 29-4 (2), Preobragenie 24-6 (3), Preobragenie 24-1 (4), Jubilee Novocherkassk 1-3 (5), Jubilee Novocherkassk 7-1 (6). For a description was used one microsatellite primer: VrZag79.
As a result, imaging and separation of products of the amplification on the acrylamide gel were obtained the following results.
m.w. 1 2 3 4 5 6 m.w.
* m.w. - molecular weight marker
Table 5. - The molecular weight of the identified a leles
Viet. 29-5 Viet. 29-4 Pr. 24-6 Pr. 24-1 J.N. 7-3 J.N. 7-1
VrZag 260 262 262 262 262 262
79 237 242 246 245 247 249
When comparing it can be concluded that, despite the close relationship, the individuals have different alleles on the number of nucleotides.
Conclusions
Recent evidence table grapes Preobragenie, Victor and Jubilee Novocherkassk an indisputable scientific and production value, and so they create a real basis for profitable production. Ampelometric information of leaf varieties of Preobragenie and Victor is very similar, which is phenotypically geminate and varieties Jubilee Novocherkassk - markedly
different from the previous two siblings.
Molecular-genetic specifically PCR-method revealed that varieties Victor, the Preobragenie and the Jubilee Novocherkassk are different genotypes.
Along with these three table grape siblings-"heroes" in OAO "Juzhnaja " and OOO "Fanagoria-Agro" on the Anapa-Tainan zone of Krasnodar region notable are varieties Anthonii Velikii, Anjuta, Bogotyanovskii, Gelios (Arcadia rozovaya), Gurrnan rannii, Dolgozhdannyi, Kishmish Vengerskii, Kubattik, Livia-k, Nizina, Roschfor-k, Super-Extra:
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