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UDC 577.21+575.22+633.16 http://dx.doi.org/10.15407/biotech9.03.061
SSR ANALYSIS IN THE STUDY OF GENETIC DIVERSITY AND SIMILARITY OF BARLEY CULTIVARS
O. R. Lakhneko1 institute of Cell Biology and Genetic Engineering
B. V. Morgun1 of the National Academy of Sciences of Ukraine, Kyiv
R. M. Kalendar2 2Republican State Enterprise "National Center for Biotechnology",
A. I. Stepanenko1 Astana, Kazakhstan
A. V. Troianovska3 3Plant Breeding and Genetics Institute — National Center
O. I. Rybalka3 of Seed & Cultivar Investigation of the National Academy
of Agrarian Sciences of Ukraine, Odesa
E-mail: molgen@icbge.org.ua
Received 17.03.2016
The aim of research was to develop an evaluation system of the genetic polymorphism for barley cultivars of Ukrainian and foreign origin based on the analysis of simple sequence repeats and valuable agricultural trait loci as well as to compose the molecular genetic passports for those cultivars. PCRs with the following separation of amplification products by agarose and polyacrylamide electrophoresis were performed to find out genetic polymorphism. Unweighted Pair Group Method with Arithmetic Mean was used for phylogenetic relationship detection. The dandrogram of phylogenetic relationships of 55 barley cultivars was constructed and molecular genetic passports were developed. Molecular genetic passports can be involved in verification for the compliance with standards cultivars, stability and seed purity.
Key words: Hordeum vulgare, SSR markers, PCR analysis, molecular genetic passports.
Barley (Hordeum vulgare L.) is an important crop, which is used as fodder, raw material for malt production, and as human food [1, 2]. Barley is second only after wheat as the most important nutritional grain crop grown in low rainfall environments [2]. In addition, the cost of barley production is significantly lower than for other crops. Approximately 3.4 million hectares of spring and 400-500 thousand hectares of winter barley are sown in Ukraine annually [3, 4].
The study of genetic diversity of barley cultivars gives resource of potential genes donors in order to create and maintain modern crop cultivars and for direct farmer use [2, 5].
Molecular markers are of great value for characterization and evaluation of barley genetic diversity. Different types of molecular markers, used for characterizing germplasm, are based on different methods that differ fundamentally in application, type and number of detected polymorphism, cost and need for time [6].
Simple sequence repeats (SSR) markers are widely used in the study of barley genome. SSRs, also called microsatellite, are tandem repeats of 1-5 base pairs (bp), and are usually presented in the eukaryotes genomes [7]. The main benefit of all SSR loci is codominance, so they are useful for different breeding programs [8], e. g. genetic mapping [9] and the genetic diversity assessment [10]. SSRs are extremely unstable and this feature allows to distinguish similar plant cultivars [11]. Polymorphism of SSRs is easily analyzed with PCR. Finally, SSR markers are technically efficient, cost-effective and widely spread in barley genome [7, 9, 12-15]. These features make them effective molecular marker system for many types of genetic analyzes.
Besides the study of genetic diversity, results of SSR analysis should be used for composing molecular genetic passports of cultivars. Such a passport is a document that reflects the particular structure of cultivar's, line's, or hybrid's DNA and enables their
identification. Genotype passportization is performed by writing a formula that reflects characteristic of variable loci. DNA typing isn't a prerequisite for a cultivar registration in Ukraine yet. However, representation of a cultivar in the form of molecular genetic formula gives an idea of the cultivar structure, its compliance with the homogeneity and stability [16].
The process of the passport composing can take a month and a half instead of 2-3 years of field trials with UPOV DUS test [16, 17]. DUS test (test for distinctness, uniformity and stability [18]) does not provide registration of hybrids. Genetic formula allows us to determine varietal purity, carry out differentiation and identification of cultivars [16]. These passports can be complemented with data on valuable agricultural trait loci.
The objective of the work was to develop a system to evaluate genetic polymorphism of Ukrainian and foreign barley cultivars based on analysis of both SSR loci and valuable agricultural trait loci as well as to compose molecular genetic passports for barley cultivars.
Materials and Methods
The subject of the research were 55 barley cultivars of Ukrainian and foreign origing.
Total DNA was isolated with the modified CTAB method [19].
The reaction mixture of 20 pl included 0.5 pM of forward and reverse primers, 1 x DreamTaq™ Green Buffer (Thermo Fisher Scientific), 0.2 mM of each deoxy-ribonucleotide-3-phosphate (Thermo Fisher Scientific), 1 unit of polymerase DreamTaq™ DNA Polymerase (Thermo Fisher Scientific), 30 ng of total plant DNA.
Primers for loci EBmac0715, EBmac0874, MGB391, MGB402tt1, Bmag13, GMS1 and MGB318 were used in the study [9, 20].
The PCR products were evaluated for polymorphisms on agarose (3%) and polyacrylamide (12%) gels using sodium borate and Tris-borate-EDTA buffers respectively [21-23]. Visualization of results was performed in UV-light with the photosystem Canon EOS 600D. Detection of the cultivars phylogenetic relationships was performed by the unweighed pair-group method using arithmetic averages cluster analysis (UPGMA) with the software DARwin 6.0.010 [24].
Results and Discussion
The subject of the study were 7 microsatellite loci (Table 1) to determine the level of genetic diversity and relationship of 55 barley cultivars of Ukrainian and foreign origin (Table 2). These selected microsatellite loci showed varying degree of polymorphism. To measure the informativeness of each SSR marker, the polymorphism information content (PIC) was calculated [25]. Using primers EBmac0874 three fragments were amplified, among them, two were polymorphic and the alleles of these loci are found in various combinations. This fact enhances the value of the marker EBmac0874 for the study of cultivars genotypes. The loci MGB402tt1 and Bmag13 also showed a high degree of polymorphism, and the loci MGB391 and MGB318 had a relatively low polymorphism (Fig. 1). The results demonstrated the effectiveness of using microsatellite markers in the genetic diversity study of barley cultivars, and detection the heterozygous forms.
Molecular genetic passports
Applying the results and the data on valuable agricultural trait loci (Bmy1 determines the thermostability of P-amylase; ITR1 — the synthesis of SE-protein, which causes the beer clouding; Wax — the synthesis of amylose) [26-28], molecular genetic passports were developed for 55 Ukrainian and foreign barley cultivars (Table 3).
The genotype passportization was carried out by writing a formula, which reflects the characteristic of variable loci. A locus was encoded with a letter of the Latin alphabet, and allele's molecular weight was pointed at subscript. The molecular weight of an allele was indicated in the case of the homozygous state of a locus (gene), and the molecular weight of several alleles — for heterozygous forms. Thus, SSR-loci were designated as A — Ebmac0715, B — Ebmac0874, C — MGB391, D — MGB402tt1, E — Bmag13, F — MGB318, G — GMS1; the valuable agricultural traits loci: H — Bmy1, I — ITR1, J — Wax.
Genetic diversity and similarity of barley cultivars
The results obtained during SSR analysis and the data on valuable agricultural trait loci (Bmy1, ITR1 and Wax) were used in the research of the genetic diversity among 55 cultivars of Ukrainian and foreign origin [26-28]. The phylogenetic tree (Fig. 2) was
1 2 3 4 5 6 7 K M
-ÎOObp
155 bp
1 2 3 4 5 6 M
228 bp-l
160 bp-
-300 bp
-100 bp
1 2 3 4 5 6 K M
226 bp-2U6 bp
-300 bp -200 bp
M 1 2 3 4 5 6 7 8
200 bp -
100 bp-
13Sbp 123 bp
Fig. 1. Agarose gels showing the allelic segregation of the SSR markers:
A — EBmac0715; B — MGB391; C — MGB318; D — GMS1; Lane 1-8, samples; K — negative control without DNA; M — marker of molecular weight GeneRuler™ DNA Ladder Mix
Table 1. The characteristic of barley SSR markers
Marker Chromosome location Fragment size, bp Polymorphic amplified fragments PIC
EBmac0715 2H (2) 150-155 2 0.401
EBmac0874 6H (6) 83-89 167-216 3 5 0.526 0.489
MGB391 2H (2) 206-226 2 0.370
MGB402tt1 5H (1) 215-260 7 0.792
Bmag13 3H (3) 146-156 5 0.651
MGB318 5H (7) 160-228 4 0.346
GMS1 5H (7) 123-131 3 0.410
performed by means of the method UPGMA using DARwin software.
The dendrogram consists of two main clusters. The first cluster group includes 54 cultivars, and only one Ukrainian cultivar Medikum 46 belongs to another cluster group. The first group divided into two subgroups. Two Canadian cultivars CDC Alamo and CDC Candle belong to one of them, the other Ukrainian and foreign cultivars — to another subgroup.
The dendrogram shows that cultivars of different breeding have significant genetic similarity. In addition, the similarity among a number of Ukrainian and foreign barley cultivars (e.g. Slavutych and Shakira, Ukrainian and German cultivars) is obvious. This fact can be an evidence of the possibility
of the use of foreign elite barley to improve the genetic pool of national culture.
The various samples of a cultivar (e.g. Jennifer and Akhiles), that originated from different collections, are included in the various cluster groups, that is to say, they have different genotypes. The fact can indicate that barley cultivars from different growing areas don't comply with the standard. This confirms the necessity to control cultivars purity and cultivars to standards using DNA markers.
The detection of genetic diversity between a pair of Ukrainian cultivars Odeskyi 14 and Stepovyk failed using seven SSR loci and three valuable agricultural trait loci. Some additional loci should be included in further research to solve the problem.
b
a
d
c
Table 2. Barley cultivars and their origin
Cultivar Country Cultivar Country Cultivar Country Cultivar Country
Akhiles Ukraine Ebson Czech Republic Kovzan Ukraine Odeskyi 69 Ukraine
Annabell Germany Enei Ukraine Kozatskyi Ukraine Odeskyi 70 Ukraine
Barke Germany Gladys Netherlands Xanadu Palidum 32 Ukraine
Beatris Germany Golden promise Ukraine Luka Ukraine Quench United Kingdom
Beatrix Germany Halychyn Ukraine Malz Czech Republic Rosalina Denmark
Bojos Czech Republic Helios Ukraine Marthe Germany Scarlett Czech Republic
CDC Alamo Canada Henrike Germany Medicum 46 Ukraine Shakira Germany
CDC Candle Canada JB Maltasia Germany Modern Ukraine Slavutych Ukraine
CDC Gainer Canada Jennifer Germany Nutans 106 Ukraine Stepovyk Ukraine
Chorno-morets Ukraine Jersey Netherlands Nutans 244 Ukraine Sviatohor Ukraine
Claire Germany KBC Aliciana Germany Odeskyi 9 Ukraine Yuzhnyi Ukraine
Cristalia United Kingdom KBC Bambina Germany Odeskyi14 Ukraine
Danuta Germany Khadar Ukraine Odeskyi18 Ukraine
Datcha France Komandor Ukraine Odeskyi 36 Ukraine
Û
4=i
-cE
Sla\utyc(i
Sriaklra
Akhiles 1
Odeskiy 35
Gladys
Datcha
Beatri se
Jennifer 1
Modem
Quench
Marths
Cristal!»
CDC Gainer
JB Vlallasia
KBC Aiiciana
Henrike
Jersey
Kovcan
KBC Bambina Jennifer 2 Barke Beatrix Rusalma Odeskyi 15 Nutans .106
Odeskyi 9 Mutans 244 oaeskyi 70 Odeskyi 39 Golden promise Khadar Odeskyi 14 Stepowk KozatSKyi Ch entornareis Luka
Akhiles 2
Ksanadu
BQJOS
Halychyn
Scalen
Ebson
Claire
Malz
Helios
<omandor
Sviatohor
Palidum 32
□anuta Annabel I CDC Candle CDC Alamo Medic um 46
Fig. 2. The dendrogram showing similarity and clustering of 55 barley genotypes
Table 3. Molecular genetic passports for barley cultivars
Cultivar Formula
Akhiles 1 A155 B89,205 C226 D246 E149 F167 G123 H516 ^455 J802
Akhiles 2 A155 B89,167 C206,226 D225,235 E149 F172 G123 H643 ^55 J802
Annabell A155 B83,170 C206 D215 E150 F167 G123 H516 ^0 J1010
Barke A155 B83,170 C226 D225 E150 F167 G123 H516 ^ J802
Beatris A155 B83,170 C226 D215 E149 F172 G123 H516 ^455 J802
Beatrix A155 B89,170 C226 D225 E150 F167 G123 H516 ^ J802
Bojos A155 B89,170 C206 D240 E149 F167 G123 H643 ^455 J802
CDC Alamo A150 B89,170,205 C206,226 D230 E149,150 F167 G131,138 H643 ^55 J592,802
CDC Candle A150 B89,205 C226 D230 E150 F167 G138 H643 ^455 J592
CDC Gainer A155 B89,170 C226 D225 E149 F172 G123 H516 ^0 J802
Chornomorets A155 B89,170,205 C226 D230 E146,150 F167,228 G123,131 H516,643 ^0,455 J802,1010
Claire A155 B89,170 C206 D215 E149 F167 G123 H516 ^455 J802
Cristalia A155 B89,170 C226 D215 E150 F167 G123 H516 ^455 J802
Danuta A155 B83,170 C206 D215 E150 F167 G123 H516 ^0 J1010
Datcha A155 B89,170 C226 D246 E150 F167 G123 H516 ^55 J1010
Ebson A155 B93,170 C206 D230 E149 F167 G123 H516 ^0 J802
Enei A155 B83,170 C206 D225 E150 F172 G123 H643 ^ J1010
Gladys A155 B89,170 C226 D246 E149 F167 G131 H516 ^455 J802
Golden promise A150 B83,170 C226 D230 E150 F172 G123 H643 ^55 J802
Halychyn A155 B89,170 C206 D230 E149 F167 G131 H643 ^455 J1010
Helios A155 B93,205 C226 D225 E150 F160 G131 H516 ^55 J802
Henrike A150 B83,170 C226 D235 E149 F167 G123 H516 ^0 J802
JB Maltasia A155 B83,170 C226 D230 E149 F167 G123 H516 ^ J802
Jennifer 1 A155 B83,170 C226 D240 E148 F172 G123 H516 ^455 J802
Jennifer 2 A150 B89,170 C226 D235 E150 F167 G123 H516 ^ J802
Jersey A155 B89,205 C226 D230 E148 F167 G123 H516 ^0 J802
KBC Aliciana A155 B83,170 C226 D230 E148 F167 G123 H516 ^0 J802
KBC Bambina A150 B89,170 C226 D230 E150 F167 G123 H516 ^0 J802
Khadar A150 B89,216 C226 D215,225 E150 F167 G123 H643 ^455 J802
Komandor A155 B83,170 C206 D246 E148 F167 G131 H643 ^ J1010
Kovzan A155 B89,205 C226 D230 E150 F167 G123 H516 ^0 J802
Kozatskyi A155 B83,89,167,173 C226 D235 E148 F167 G123 H516,643 ^55 J802
Ksanadu A155 B89,170 C206 D215 E149 F167 G131 H643 ^455 J802
Luka A155 B89,216 C206 D225 E149 F172 G131 H516 ^55 J802
Malz A155 B89,170 C206 D230 E150 F167 G123 H516 ^455 J802
Marthe A155 B83,170 C226 D215 E150 F167 G123 H516 ^55 J802
Medicum 46 A155 B83,89,167,173 C226 D230,266 E146 F167,228 G131,138 H643 ^0 J802
Modern A155 B89,170 C226 D215 E148 F167 G123 H643 ^55 J802
Nutans 106 A150 B89,216 C226 D230 E150 F167 G123 H516,643 ^455 J802
Nutans 244 A150 B83,170 C226 D225 E150 F167 G123 H516 ^55 J802
Table 3. End
Cultivar Formula
Odeskyi 9 A150 B83,216 C226 D230 E150 F167 G123 H516 I455 J1010
Odeskyi14 A150 B89,170 C226 D225 E150 F167 G123 H643 I455 J802
Odeskyi18 A150 B83,89,170,216 C226 D230 E150 F167 G123 H516 I455 J802
Odeskyi 36 A155 B89,170 C226 D246 E149 F167 G123 H643 I455 J802
Odeskyi 69 A150 B83,170 C226 D225 E150 F167,172 G123 H643 I0,455 J802
Odeskyi 70 A150,155 B83,89,170,205 C226 D225,235 E146,150 F167 G123 H516 I455 J802
Palidum 32 A155 B83,170 C206 D246 E156 F172 G138 H643 I0 J1010
Quench A155 B83,170 C226 D215,230 E148 F167 G123 H516 I455 J802
Rosalina A155 B89,170 C226 D215 E150 F167 G123 H516 I0 J802
Scarlett A155 B93,170 C206 D215 E149 F167 G131 H516 I455 J802
Shakira A155 B89,170 C226 D246 E149 F167 G123 H516 I455 J802
Slavutych A155 B89,170 C226 D225,246 E149 F167 G123 H516 I455 J802
Stepovyk A150 B89,170 C226 D225 E150 F167 G123 H643 I455 J802
Sviatohor A155 B83,170 C226 D246 E149 F167 G131 H643 I0 J802
Yuzhnyi A150 B89,170,216 C226 D215,230 E146,150 F167 G123 H516 I455 J802,1010
The system of genetic diversity evaluation of barley cultivars with the SSR markers was developed. The results of SSR analysis and the data on valuable agricultural trait loci determined the genetic relationship or similarity between Ukrainian and foreign
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SSR-АНАЛШ У ДОСЛ1ДЖЕНН1 ГЕНЕТИЧНОГО Р1ЗНОМАН1ТТЯ ТА СПОР1ДНЕНОСТ1 СОРТ1В ЯЧМЕНЮ
О. Р. Лахнеко1 Б. В. Моргун1 Р. M. Календар2 А. I. Степаненко1 А. В. Трояновська3 О. I. Рибалка3
Институт кл^инно! бмлоги та генетично!
шженери НАН Укра!ни, Ки!в 2Республiканське державне тдприемство "Нащональний центр бмтехнологи", Астана, Казахстан 3Селекцшно-генетичний шститут — Нацiональний центр насшнезнавства та сортовивчення Нащонально! академп аграрних наук Укра!ни, Одеса
E-mail: molgen@icbge.org.ua
Метою роботи було провести ощнювання ге-нетичного полiморфiзму сорив ячменю укра!н-сько! та зарубiжноl селекцй на основi SSR-ана-лiзу та локусiв цiнних сiльськогосподарських ознак, а також скласти молекулярно-генетичш паспорти сорив. Для встановлення генетичного полiморфiзму здшснювали ПЛР з наступним роздшенням продуктiв амплiфiкацü методом електрофорезу в агарозних та полiакриламiд-них гелях. Для встановлення фшогенетичних зв'язкiв було застосовано метод незваженого попарного середнього — UPGMA. Побудовано дендрограму фшогенетичних зв'язшв 55 сорив ячменю та складено !хш молекулярно-генетич-нi паспорти, як можуть бути використанi для перевiрки сортiв на вiдповiднiсть еталонам, стабшьшсть та чистоту.
Ключовi слова: Hordeum vulgare, SSR-марке-ри, ПЛР-аналiз, молекулярно-генетичнi пас-порти.
SSR-АНАЛИЗ В ИССЛЕДОВАНИИ ГЕНЕТИЧЕСКОГО РАЗНООБРАЗИЯ И СРОДСТВА СОРТОВ ЯЧМЕНЯ
О. Р. Лахнеко1 Б. В. Моргун1 Р. Н. Календарь2 А. И. Степаненко1 А. В. Трояновская3 О. И. Рыбалка3
1Институт клеточной биологии и генетической инженерии НАН Украины, Киев 2Республиканское государственное предприятие "Национальный центр биотехнологии", Астана, Казахстан 3Селекционно-генетический институт —
Национальный центр семеноведения и сортоизучения Национальной академии аграрных наук Украины, Одесса
E-mail: molgen@icbge.org.ua
Целью работы было провести оценку генетического полиморфизма сортов ячменя украинской и зарубежной селекции на основе SSR-анализа и локусов ценных сельскохозяйственных признаков, а также составить моле-кулярно-генетические паспорта сортов. Для установления генетического полиморфизма осуществляли ПЦР с последующим разделением продуктов амплификации методом электрофореза в агарозных и полиакриламидных гелях. Для установления филогенетических связей был использован метод невзвешенного попарного среднего — UPGMA. Построена ден-дрограмма филогенетических связей 55 сортов ячменя и составлены их молекулярно-генети-ческие паспорта, которые могут быть использованы для проверки сортов на соответствие стандартам, стабильность и чистоту.
Ключевые слова: Hordeum vulgare, SSR-маркеры, ПЦР-анализ, молекулярно-генети-ческие паспорта.
