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Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2010, № 5, p. 42-48.
UDK 633.12:575.174.015.3:577.2.08
ANALYSIS OF GENOMIC DIVERSITY OF SAMPLES AND CULTIVARS IN COMMON BUCKWHEAT (Fagopyrum esculentum Moench) BY THE ISSR-METHOD
G.D. Kadyrova1, F.Z. Kadyrova1, E. V. Martirosyan2, N.N. Ryzhova2
1 Tatarstan Research and Development Institute of Agriculture, Russian Academy of Agricultural Sciences, Kazan 420059, Russia,
e-mail: [email protected]
2 Center ".Bioengineering ", Russian Academy of Sciences, Moscow 117312, Russia, e-mail: [email protected]
Received May 18, 2010 S u m m a r y
For an assessment of a genetic polymorphism of common buckwheat (F. esculentum, 27 samples of different ecologo-geographic origin, including 14 domestic cultivars) the authors determined the ISSR-primers of revealing intraspecific polymorphism and obtained the genomspecific polymorphic ISSR-spectra of DNA fragments. Genomic variability and genomic linkage groups cultivars of common buckwheat were determined for the first time. Obtained data can be used in buckwheat breeding program and further molecular characterization of investigated cultivars.
Key words: buckwheat, ISSR-method, inter-microsatellite sequences, genetic variability of cultivars.
Simple tandemly repeating DNA sequences - so-called mini- and microsatellites - are basic components of all eukaryotic genomes. In general, a total number of microsatellite repeats varies from 5*103 to 3*105 per genome, in which they are distributed fairly evenly (1). As a rule, many mini- and microsatellites are highly variable even in closely related organisms (2). The study of microsatellite polymorphism and intra-microsatellite sequences using ISSR (intersimple sequence repeats) technique (3) is based on amplification of genomic DNA with primers. The primers consist of 15-20 nucleotides homologous to a microsatellite repeat and often including a short (2-4 nucleotides) random “anchor” sequence at the 3'- or 5'-end; the anchor increases selectivity of the primer and reproducibility of ISSR-spectra obtained by DNA amplification between two closely spaced and oppositely oriented microsatellites. ISSR- and RAPD-spectra (random amplified polymorphic DNA) are similar, but ISSR-spectra usually contain a larger set of polymorphic bands (up to 97) which allows to detect a higher level of genomic polymorphism (4, 5).
At present time, ISSR technique is widely used for detection of intraspecific polymorphism, especially in closely related genotypes of cultivated plants. This work is quite actual in connection with contemporary breeding programs of obtaining the varieties of essentially new quality level (4, 6-8), which requires the creation of a wide gene pool of source material carrying multiple economically valuable parameters. This task implies the expansion of present gene pool and a preliminary assessment of natural variability of species.
The species Fagopyrum esculentum Moench the family Polygonaceae (Buckwheat fam.) is one of most valuable cereal crops becoming increasingly important as an essential dietary product; some buckwheat forms are used as a raw material for production of rutin - an important pharmaceutical preparation of flavonoid group. To create a wide genetic pool, local populations of F. esculentum ssp. esculentum were involved into the selection of domestic buckwheat varieties (9, 10). Upon this base, more than 50 local varieties have been locally adapted by the end of 1950ies (11) still being involved in breeding work. Another buckwheat species - F. tataricum ssp. tataricum - is cultivated mainly in highlands of China, Japan, India, Nepal, as well as in several European countries. In breeding work on buckwheat, this species is used as a component of crosses providing improved cold resistance and viability of plants (12).
Extensive use of local natural populations of F. esculentum and samples of the cultivated species F. tataricum significantly increased productivity of domestic buckwheat varieties owing to more intense growth processes and resistance to environmental conditions. However, it’s still unknown how much the genetic pool of modern domestic buckwheat varieties corresponds to intraspecific variability of F. esculentum, and how fully the potential of natural populations is used in breeding work today. The few studies of intraspecific diversity of local wild forms and varieties of F. tataricum and F. esculentum (mostly of Japanese breeding origin) using molecular techniques (AFLP - amplified fragment length polymorphism, RAPD - random amplified polymorphic DNA, SSR - simple sequence repeats) were performed in Japan (13-15). For Russian buckwheat, the only data of RAPD-analysis have been obtained earlier by the authors (16).
The purpose of this work was to perform ISSR-analysis of intraspecific genetic polymorphism of Fagopyrum esculentum and to evaluate the genetic pool of domestic buckwheat cultivars and samples (including F. tataricum) most frequently used in breeding work.
Technique. The molecular analysis of genomes of cultivated species was performed upon 27 buckwheat samples from the gene bank collection of the All-Russia Research and Development Institute of Plant Growing (VIR, St. Petersburg) and the Tatarstan Research and Development Institute of Agriculture (TatNIISKh, Kazan): F. esculentum - 14 varieties and 8 samples, F. tataricum - 5 samples from natural populations of different ecology-geographic origin.
A plant DNA was isolated from 8-10-day-old seedlings along a standard method (17) with additional deproteinization with the mixture of phenol and chloroform. In this work, 13 oligonucleotide sequences homologous to microsatellite repeats were used.
Polymerase chain reaction (PCR), separation and visualization of PCR products were performed by standard techniques (18). The reaction mixture (volume - 15 ul) contained 100 ng of total plant DNA, 0,5 uM primer, 0,2 mM of each dNTP, 2,5 mM MgCl2, 0,3 units Taq-polymerase (“Dialat LTD”, Moscow) in the corresponding 1* buffer. The amplification with preliminary denaturation (94 °C, 5 min) was performed in the thermocycler GeneAmp PCR System2700 (“Applied Biosystems”, USA) under the regime: denaturation - 94 °C, 30 s, annealing of primer - 30 s: (temperature was selected individually for each primer), elongation - 72 °C, 1 min (40 cycles). Melting temperature for each primer was calculated by the formula T = 69,3 + 0,41 (GC,%) - 650/L, where L - a number of nucleotides in the primer; GC,% - a proportion of GC-nucleotides in the primer. Amplification products were separated by electrophoresis in 1,7% agarose gel (High resolution, “Sigma”, MetaPhor, “Cambrex”, USA) in 1 * TBE-buffer with a consequent staining with ethidium bromide.
For statistical processing of data, the binary matrixes (1/0) on each primer were composed to calculate the coefficients of genetic variation (the program STATISTICA 6.0) using groupling methods (tree clustering) and the unweighted pair group method with arithmetic mean UPGMA (http://www.statsoft.com).
Results. Characteristics of the studied samples are shown in Table 1; the samples of F. tataricum selected upon the results of RAPD-analysis (16).
1. The origin of studied buckwheat samples (Fagopyrum)
Species | № the cultivar from VIR catalogue | Geographic origin
F. tataricum k-73 Mongolia
F. tataricum k-65 Republic of Tatarstan, Russia
F. tataricum k-38 Brest province (Brestskaya Oblast’), Russia
F. tataricum k-25 Ternopil’ province (Ternopil’s’ka Oblast’), Ukraine
F. tataricum k-57 Poland
F. esculentum k-4131 Canada
F. esculentum k-4233 Japan
F. esculentum k-549 Japan
F. esculentum k-4170 India
F. esculentum k-4117 China
F. esculentum k-140 China
F. esculentum k-143 China
F. esculentum k-4350 Nepal
F. esculentum Kama TatNIISKh (Kazan, republic of Tatarstan, Russia)
F. esculentum Chatyr Tau TatNIISKh (Kazan, republic of Tatarstan, Russia)
F. esculentum Skorospelaya 86 VNIIZBK (Orel, Russia)
F. esculentum Krasnostreletskaya TatNIISKh (Kazan, republic of Tatarstan, Russia), VNIIZBK (Orel, Russia)
F. esculentum Kazanskaya krupnozernaya TatNIISKh (Kazan, republic of Tatarstan, Russia)
F. esculentum Karakityanka TatNIISKh (Kazan, republic of Tatarstan, Russia)
F. esculentum Saulyk TatNIISKh (Kazan, republic of Tatarstan, Russia)
F. esculentum Cheremshanka TatNIISKh (Kazan, republic of Tatarstan, Russia)
F. esculentum Bogatyr’ Shatilovo experimental station (Orel province (Orlovskaya Oblast’), Russia )
F. esculentum Shatilovskaya 5 VNIIZBK (Orel, Russia)
F. esculentum Ballada VNIIZBK (Orel, Russia)
F. esculentum Molva VNIIZBK (Orel, Russia)
F. esculentum Batyr TatNIISKh (Kazan, republic of Tatarstan, Russia)
F. esculentum Demetra VNIIZBK (Orel, Russia)
Note: TatNIISKh - - Tatarstan Research and Development Institute of Agriculture (Tatarstanskii Nauchno-Issledovatel’skii In-
stitut Sel’skogo Khozyaistva), VNIIZBK - All-Russia Research and Development Institute of Grain Legumes and Cereal Crops (Vserossiiskii Nauchno-Issledovatel’skii Institut Zernobobovykh Kul’tur), VIR - All-Russia Research and Development Institute of Plant Growing (Vserossiiskii Naucho-Issledovatel’skiiInstitutRastenievodstva').
The applied primers (Table 2) mainly consisted of dinucleotide repeats with 1-3 additional selective anchor nucleotides in the 3'-end, as well as tri-, tetra- and pentanucleotide repeats. The primers were selected upon the data on their ability to detect genomic polymorphism (6, 19, 20).
2. Characteristics of the used ISSR-primers and results of amplification
ISSR- primer Nucleotide sequence of the primer Amplification results (number of detected ISSR-fragments of DNA)
M1 (AC)SCG Differential spectra (35 polymorphic fragments)
M2 (AC)„(C/T)G Differential spectra (29 polymorphic fragments)
М3 (GA)„(C/T)C The absence of PCR-product
М4 (AG)„(C/T)C Low-polymorphic spectra
М5 (AT)8 Smear
М6 (CAC)5 Low-polymorphic spectra
M7 (CAG)5 Differential spectra (25 polymorphic fragments)
M8 (GTG)5 Low-polymorphic spectra
M9 (GACAC)4 The absence of PCR-product
M10 (CA)6(A/G)G The absence of PCR-product
M11 (CA)6(A/G) Differential spectra (24 polymorphic fragments)
M12 (CA)6(A/G)(C/T) Low-polymorphic spectra
M13 (AGC)4(C/T) Monomorphic spectra
Testing of ISSR-primers on the limited set of samples (three for each species of buckwheat) has shown that not all of them provided clear multilocus polymorphic spectra of amplified genomic fragments. The primers M4, M12 and M13 (see Table 2) revealed an interspecific polymorphism, but the samples of one species revealed low-polymorphic (M4, M12) or monomorphic spectra (M13). Four primers showed the negative result: an absence of amplification (M3, M9, M10) or smear (M5) were obtained. Smear-detection of amplification products (the primer M5) occurs quite frequently, and this is possibly connected with either the structure of primer sequence, or with frequency of a corresponding microsatellite in the genome. The absence of amplification products when using the primers M3, M9 and M10 can be explained by low frequency of the microsatellite repeats (GA)8(C/T)C, (CA)6(A/G)G and (GACAC)n in buckwheat genome. A number of studies assume the partial self-complementarity of such primers, which results in formation of double-chained primer-primer structures as a possible reason for the detected absence of amplification (8, 21).
Three most informative primers (M1, M2 and M7) providing reproducible amplification and clear polymorphic spectra consisting of no less than 20 fragments were selected for a consequent use. In general, 89 polymorphic ISSR-fragments (the length varied from 2700 bp to 320 bp) for 27 buckwheat genotypes were obtained. All the detected genome fragments were polymorphic, i.e., the degree of polymorphism revealed by the three selected ISSR-fragments was 100%, while at the RAPD-analysis - 88% (16). This fact is consistent with data on higher discriminatory ability of the first technique compared with the second one (4).
Five ISSR-fragments specific for all the studied samples of F. esculentum were marked, as well as three fragments - for F. tataricum and the number of fragments typical for certain samples and varieties. In particular, the unique fragments were found in the sample of Chinese cultivar F. esculentum (k-143) - the three fragments of 780 bp, 990 bp (primer M1) and 1500 bp (primer M7) length, in the varieties Bogatyr’ (1000M2, 1600M2), Skorospelaya 86 (780M7, 970M7), Demetra (850Mi, 1700Mi) Karakityanka (1000Mi,
650M2) and Batyr (910M2). These fragments can be used to design gene-specific DNA markers for buckwheat cultivars, samples and species.
3. Coefficients of intra- and interspecific genetic variation in the studied buckwheat samples (Fagopyrum)
Analyzed group Coefficient of genetic variation (average) Standard error Standard deviation Min Max
F. esculentum (cultivars) 0,169 0,005 0,051 0,056 0,303
F. esculentum (samples) 0,191 0,009 0,048 0,101 0,281
F. esculentum F. esculentum (cultivars)-F. esculentum 0,182 0,003 0,051 0,045 0,315
(samples) 0,190 0,005 0,050 0,045 0,315
F. tataricum 0,099 0,014 0,044 0,011 0,169
F. esculentum (samples)-F. tataricum 0,304 0,012 0,073 0,112 0,438
F. esculentum (cultivars)-F. tataricum 0,292 0,008 0,063 0,135 0,427
F. esculentum-F. tataricum 0,300 0,010 0,070 0,110 0,440
Note: Coefficients of genetic variation were calculated in the program STATISTICA 6.0 using groupling methods (tree clustering) and unweighted pair group method with arithmetic mean UPGMA (http://www.statsoft.com).
The obtained ISSR-spectra and data on polymorphism of DNA fragments were used to calculate pairwise coefficients of genetic variation in the genus Fagopyrum, which were found to range from 0,002 to 0,440. The narrowest limits of interspecific diversity (0,135-0,427) have been established in the pair F. esculentum - F. tataricum (Table 3).
Intraspecific genomic variability in F. esculentum (0,045-0,315) almost twice exceeded that in F. tataricum (0,010-0,169) (see Table. 3), which fact was earlier shown by RAPD-analysis (16).
At the same time, intervarietal differences in F. esculentum were very high (coefficients of pairwise identity - 0,056-0,303) and corresponded to genomic variability of wild samples of F. esculentum (0,101-0,281) from Japan, China, Nepal, thereby indicating a wide genetic basis of domestic buckwheat varieties. Previously, the results of RAPD test suggested a similar conclusion (16).
0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1
4-----1-----1-----1-----1----1-----1-----1-----1—
F.
tatataricum, k-38
tatataricum, k-57
tatataricum, k-25
tatataricum, k-73
tatataricum, k-65
esculentum, k-4170
esculentum, k-4350
esculentum, cv Cheremshanka
esculentum, cv Molva
esculentum, k-4131
esculentum, k-140
esculentum, k-549
esculentum, cv Chatyr Tau
esculentum, k-4233
esculentum, k-143
esculentum, cv Krasnostreletskaya
esculentum, cv Kazanskaya krupjiozemaya
esculentum, cv Bogatyr’
esculentum, k-411?
esculentum, cv Saulyk
esculentum, cv Demetra
esculentum, cv Shatilovskaya 5
esculentum, cv Ballada
esculentum, cv Karakityanka
esculentum, cv Kama
esculentum, cv Skorospelaya 86
esculentum, cv Batyr
Dendrogram showing the degree of genetic identity of studied buckwheat species, cultivars and samples (Fagopyrum) (built using the UPGMA hierarhical culuster analysis based on ISSR-labeling).
The dendrogram built upon data of cluster analysis revealed a clear interspecific differentiation of F. tataricum and F. esculentum: the studied samples were divided into two main groups with reliable bootstrap values (Fig.). The representatives of F. tataricum formed a relatively low-polymorphic group, while the cluster of F. esculentum included three main groups of samples with different similarity levels. The first clade (basal and most polymorphic) was represented by local wild populations of F. esculentum from Nepal and India, as well as two varieties - Cheremshanka and Molva. The second clade was formed by the samples originated from China and Japan, as well as the domestic variety Chatyr Tau. The third clade was fully represented by the studied cultivars. Despite the fact that intervarietal groupings of this clade were supported by low bootstrap values, there was a certain correlation between their degree of identity and morphobiological properties of varieties from these groups. Thus, the group Karakityanka - Kama - Skorospelaya 86 -Batyr was represented by early-ripening and drought-resistant varieties with limited ramification of plants. In this group, the variety Batyr has a longer growing season, but its pedigree includes an ultrafast-ripening cultivar Kazanskaya 309. The group Shatilovskaya 5 - Ballada have close origin, economical and biological properties. The pedigree of cultivar Kazanskaya krupnozernaya showed the presence of material selected from the cultivar Krasnostreletskaya. The variety Demetra formed a separate branch, which may be associated with its significant morphobiological features.
Thus, for the first time ISSR-method was used for genome analysis of buckwheat the genus Fagopyrum. The ISSR-primers capable to detect intraspecific polymorphism have been established. For each sample of F. esculentum and F. tataricum, the unique ISSR-spectra were obtained and the sample- and variety-specific fragments were found, which can be used to develop gene-specific DNA markers for buckwheat. Upon the basis of microsatellite variability and inter-microsatellite sequences, the authors have determined the degree of genetic differences between the samples. Domestic cultivars of F. esculentum have shown high level of intervarietal polymorphism comparable to genetic variability of wild forms. The results of this study can be used for identification of varieties and hybrids in breeding programs.
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