Transcript (1), NV Punina (1)

Дендрограмма, построенная методом ближнего соседа (NJ) для 25 штаммов Bt, выделенных на территории Украины по суммарным результатам ERIC-, BOX-ПЦР и saAFLP для 36 полиморфных признаков. Значения бутстрепа расчитывались в 1000 повторениях.
Genetic diversity of Rhizobium sp. in Russian Federation and Ukraine
V.S. Zotov (1), N.V. Punina (1), S.A. Khapchaeva (1), S.V. Didovych (2),
T.N. Melnichuk (2), A.F. Topunov (1)
(1) A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia, е-mail: [email protected];
(2) Institute of Agriculture of Crimea of National Academy of Agriculture Sciences of Ukraine, Department of microbiology, Simferopol, Ukraine
ABSTRACT
The new taxonomic marker (hin-region) has been proposed which gives possibility for Rhizobium bacteria study on “species - group of strains” level. Using this marker the groups of Rhizobium strains were
determined which could not be distinguished with other methods, and these results correlated with evolutionary similarity of bacteria. The developed approach for creating of marker systems affords to carry
out effective inventory of inter- and intra-species genetic diversity of nodulating bacteria and to evaluate perspectives of their use in agriculture. The proposed marker system was used for description of
Rhizobium bacteria samples isolated from different ecological-geographical regions of Ukraine and Russia.
INTRODUCTION
MATERIALS AND METHODS
Legume family (Fabaceae) includes over 19,000 described species of
plants that are usually able to enter into a symbiotic relationships with
nitrogen-fixing bacteria forming the nodules on the roots. Bacteria of the
genus Rhizobium is one of the representative symbiont of valuable crops,
such as Pisum, Lens, Vicia faba, Trifolium, Phaseolus, Galega. Study of
the taxonomic structure and biodiversity of this genus is very important
due to the fact that knowledge of their genetic structure and polymorphism
varietal plant lines reveals the evolutionary dynamics of the variability, and
thus predicts the couple of micro-macrosymbiont most adapted to each
other. This coordinated selection will allow creating the combinations of
symbiont bacteria-plant with a more efficient process of nitrogen fixation
for full development and the formation of high yield. At present, as a rule,
taxonomy and evolutionary relationships of the order Rhizobiales are
studied by comparison of the nucleotide sequences of 16S rRNA gene
and MLSA analysis. However, this assessment does not always
correspond to the dates obtained by other loci. Using universal primers
systems, especially when we are working with a mixture of different
microorganisms in one sample, makes MLSA analysis more timeconsuming and limited due to the high divergence of specific markers. The
adequately rhizobia’s isolates classification and taxonomy can’t always be
realizing due to frequent interspecies recombination between different
alleles of taxonomically important genes (ribosomal rRNA operon), and
the horizontal transfer (sym-genes), that’s why we need to obtain
additional genetic data. The genus Rhizobium is particular interest as a
model object of this Order, that has a complex taxonomic structure, which
is difficult to study by current methods.
Previously, in details we’ve studied the genetic diversity of
phytopathogenic bacteria of the genus Xanthomonas, affecting Brassica
napus in the Moscow region and the North Caucasus region of Russia
using hin-region PCR and saAFLP (XbaI) techniques (Zotov et al., 2010).
In this study we used these techniques to research the genetic diversity of
rhizobia from different eco-geographical zones of Ukraine.
Schematic representation of the genomic DNA from rhizobia including reference (blue) and
proposed by us (sand color) target markers: ribosomal gene cluster, restriction sites of
endonuclease with an average cutting frequency, protein-coding genes, symbiotic genes and
intergenic regions.
R.leguminosarum bv. viciae 248b
R.leguminosarum bv. viciae B-8 (its-L)
R.leguminosarum bv. viciae К-29
R.leguminosarum bv. viciae 31 (its-L)
R.leguminosarum bv. viciae 96-st
R.leguminosarum bv. phaseoli FN-6 (its-L)
100
R.leguminosarum bv. phaseoli FК-0
72
R.leguminosarum bv. phaseoli FN (its-L)
R.leguminosarum bv. viciae 245а-st
R.leguminosarum bv. trifolii КК (its-L)
R.leguminosarum bv. trifolii КP012 (its-L)
R.leguminosarum bv. trifolii КL91 (its-L)
R.leguminosarum bv. viciae Y-1 (its-S)
86
Rhizobium leguminosarum LMG 14904 T (AF345271)
R.leguminosarum bv. phaseoli 682
R.leguminosarum bv. trifolii 1326-st
R.leguminosarum bv. viciae 261b-st
R.leguminosarum bv. viciae B-9 (its-L)
R.leguminosarum bv. phaseoli 105
R.leguminosarum bv. viciae B-8 (its-S)
R.leguminosarum bv. trifolii КК (its-S)
R.leguminosarum bv. viciae 31 (its-S)
R.leguminosarum bv. viciae 97-st
R. leguminosarum bv. viciae Y-7
R.leguminosarum bv. trifolii КL91 (its-S)
R.leguminosarum bv. trifolii КP012 (its-S)
R.leguminosarum bv. viciae B-9 (its-S)
91 Rhizobium gallicum R 602 T clone 1 (AF345267)
96 Rhizobium gallicum R 602 T clone 2 (AF345266)
Rhizobium mongolense USDA 1844 T (AF345273)
R.leguminosarum bv. phaseoli FN-6 (its-S)
89 R.leguminosarum bv. phaseoli FА-4
R.leguminosarum bv. phaseoli FN (its-S)
Rhizobium etli LMG 17827 T (AF541974)
97 Rhizobium etli CIAT 656 (NC 010994)
Rhizobium hainanense USDA 3588 T (AF345269)
Rhizobium tropici LMG 9503 T (AF345278)
Rhizobium radiobacter LMG 140 T (AF541973)
Rhizobium rhizogenes DSM 30148 T (AF345275)
Rhizobium lusitanum P1-7 T (AY943641)
Rhizobium undicola LMG 11875 T (AF345280)
Rhizobium vitis LMG 8750 T (AF345279)
86
R.giardinii FS
Rhizobium giardinii H152 T (AF345268)
Rhizobium rubi DSM 6772 T clone 1 (AF345276)
100 Rhizobium rubi DSM 6772 T clone 2 (AF345277)
Rhizobium huautlense LMG 18254 T (AF345270)
R.galegae 926st
100 Rhizobium galegae LMG 6124 T (AF345265)
R. leguminosarum (55)
leguminosarum bv. trifolii 1326st
leguminosarum bv. viceae 31
leguminosarum bv. phaseoli 108*
leguminosarum bv. phaseoli FК-6
leguminosarum bv. phaseoli FN
leguminosarum bv. phaseoli 108**
leguminosarum bv. phaseoli FК-4
leguminosarum bv. phaseoli FК-0
leguminosarum bv. phaseoli 105
R. leguminosarum bv. phaseoli FN-6
Rhizobium radiobacter K84 (CP000628)
84
89 Rhizobium rhizogenes IFO 13257 T (D14501)
97
Rhizobium lusitanum P1-7 T (NR_043150)
Rhizobium tropici CIAT 899 T (EU488752)
86
98
99 Rhizobium hainanense I66 T (U71078)
Rhizobium etli CIAT 652 (NC 010994)
99 R. etli 700-st
Rhizobium sullae IS 123 T (Y10170)
Rhizobium indigoferae CCBAU 71042 T (AF364068)
97
Rhizobium yanglingense SH22623 T (AF003375)
Rhizobium gallicum R602sp T (U86343)
99
Rhizobium mongolense USDA 1844 T (U89817)
Rhizobium loessense CCBAU 7190B T (AF364069)
Rhizobium galegae ATCC 43677 T (D11343)
R. galegae К-3
R. galegae 926-st
98
R. galegae 916-st
R. galegae 913-st
99
R. galegae К-18
R. galegae 912-st
71
Rhizobium huautlense SO2 T (AF025852)
93
Rhizobium undicola LMG 11875 T (Y17047)
Rhizobium vitis NCPPB3554 T (D01258)
Rhizobium rubi IFO 13261 T (D01259)
99
Rhizobium tumefaciens NCPPB2437 T (D01256)
95
Rhizobium giardinii H152 T (U86344)
99 R. giardinii FS
Mesorhizobium plurifarium LMG 11892 T (Y14158)
72 Mesorhizobium amorphae ACCC 19665 T (AF041442)
Mesorhizobium huakuii IFO 15243 T (D13431)
Mesorhizobium mediterraneum UPM-Ca36 T (L38825)
Mesorhizobium tianshanense USDA 3592 T (AF041447)
70
99
Mesorhizobium chacoense PR5 T (AJ278249)
Mesorhizobium ciceri UPM-Ca7 T (U07934)
99 Mesorhizobium loti LMG 6125 T (X67229)
96 Sinorhizobium meliloti LMG 6133 T (X67222)
Sinorhizobium kummerowiae CCBAU 71714 T (AF364067)
98
Sinorhizobium medicae A 321 T (L39882)
Sinorhizobium arboris HAMBI 1552 T (Z78204)
Sinorhizobium kostiense HAMBI 1489 T (Z78203)
97
Sinorhizobium terangae LMG 6463 T (X68387)
Sinorhizobium saheli LMG 7837 T (X68390)
97
Sinorhizobium fredii ATCC 35423 (D01272)
98 Sinorhizobium xinjiangense IAM 14142 T (D12796)
Bradyrhizobium japonicum DSM 30131 T (X87272)
99
R.
R.
R.
R.
R.
R.
R.
R.
R.
Bacterial strains. 73 rhizobial isolates was obtained from
root nodules of legume plants (Pisum satіvum, Trifolium repens,
T. pratense, Vicia faba, Phaseolus vulgaris, Galega orientalis)
growing in different regions of Russia and Ukraine. 12 reference
strains of Rhizobium sp. was obtained from cultures collections
of Department of microbiology Institute of Agriculture of Crimea
of NAAS and All-Russia Research Institute for Agricultural
Microbiology RAAS, was investigated.
DNA isolation. Total genomic DNAs of all Rhizobium strains
were obtained after lysozyme-sodium dodecyl sulfate lysis
followed by phenol-chloroform extraction and ethanol
precipitation.
saAFLP. The AFLP procedure was performed as described
by Valsangiacomo (1995), with some modifications. 80 ng of
DNA was digested with XmaJI (AvrII) restriction enzyme and
ligated with single adapter specific for restriction half-site. The
restriction-ligation reaction was performed at 37°C for 2 h in a
total volume of 20 μl. Primer (Pr.CTAG2: GGCCGTGGTTTTAGctag) and PCR conditions were as described previously (Zotov et
al., 2012).
PCR amplification 16S rRNA gene and 16S-23S rRNA
(ITS). The primers fD1 and rD1 were used to amplify 16S rRNA
genes (Weisburg et al., 1991). For ITS amplification, the primers
FGPS1490-72 (Normand et al., 1992) and ITS_R (Zotov et al.,
2012) were used. PCR products was amplified, purified and
directly sequenced.
Hin-region PCR. Hin-region was amplified by PCR with
primers systems described earlier Zotov et al. (2012). PCR
fragments were amplified, purified, directly sequenced and
aligned using the Clustal W v. 1.75 (Thompson et al., 1994).
Phylogenetic trees were constructed with Mega v. 3.1 (Kumar &
Nei, 2006), using the neighbor-joining algorithms with bootstrap
and genetic distances computed by Kimura’s 2-parameter model
(Kimura, 1980).
R. leguminosarum (65)
R. etli (1)
R. galegae (6)
R. giardinii (1)
Mesorhizobium
Alternative phylogenetic
marker - gyrB gene
Sinorhizobium
Group of strains "R. leguminosarum LMG14904T"
87
89
Group of strains
100
R. leguminosarum (20) - isolates from nodules T.repens, T.pratense and Vicia faba
R. leguminosarum bv. viceae Y-7
78
78 R. leguminosarum bv. viceae 248b
R. leguminosarum bv. viceae Y-9
R. leguminosarum bv. viceae 245а-st
71
Rhizobium leguminosarum bv. trifolii WSM1325 (NC_012850)
98
Rhizobium etli 700-st
Rhizobium etli CIAT652 (NC 010994)
Rhizobium leguminosarum bv. trifolii WSM2304 (NC_011369)
R. leguminosarum bv. phaseoli 108**
100
R. leguminosarum bv. phaseoli 105
99
R. leguminosarum bv. phaseoli FN
R. leguminosarum bv. phaseoli FК-0
- I/b
R. leguminosarum bv. phaseoli 108*
- I/b
R. leguminosarum bv. phaseoli FК-6
- I/b
100 R. leguminosarum bv. phaseoli FК-4
- I/b
Rhizobium leguminosarum bv. viceae 3841 (NC_008380)
R. leguminosarum bv. viceae 96-st
R. leguminosarum bv. viceae 97-st
R. leguminosarum bv. viceae 261b-st
99 R. leguminosarum bv. viceae 31
R. leguminosarum bv. viceae К-29
R. leguminosarum bv. trifolii 1326-st
94
99
Group of strains "R. giardinii H152T"
Group of strains "R. galegae LMG6124T"
R. leguminosarum (26) - isolates from nodules Pisum and Phaseolus
R. leguminosarum bv. phaseoli FN-6
R. giardinii bv. phaseoli FS
R. galegae 913-st
R. galegae 926-st
100
R. galegae K-18
100 R. galegae 916-st
R. galegae 912-st
R. galegae K-3
I/B
V/F
II/C
I/B
I/A
III/D
IV/E
VI/G
0.05
Bradyrhizobium
0.05
0.01
16S rRNA
The nucleotide sequences of 16S rRNA gene analysis showed that isolates
belonging to three species: the majority of isolates belonged to R.leguminosarum,
one isolate to R.giardinii and 6 isolates did to R.galegae. However, the
intraspecific polymorphism in this marker wasn’t identified.
ITS
The study of nucleotide sequences intergenic region 16S-23S rRNA (ITS)
resulted to a significant intraspecific polymorphism. However, due to frequent
recombination of this locus and its several genomic copies using of this region
for taxonomic purposes is limited.
REFERENCES
CONCLUSION
According to the results the collection of the nodule genus
Rhizobium can be represented as follows:
 analysis of nucleotide sequences of 16S rRNA gene reveals of
genera and species belonging isolates;
 test inoculation of host plants determines the specificity of the
strains (i.e. biovars);
 nucleotide polymorphism intergenic region 16S-23S rRNA reflects
the degree of intra- and inter-specific recombination;
 using technique saAFLP we can determine relationships between
groups of strains within the proposed genotypes.
 efficiency of the symbiotic system can be evaluated with hin-region
marker;
The advantage of hin-region PCR analysis include: the ease and
speed of execution, universality of marker system to study intra-generic
relationships, high informativity and reproducibility, the ability to work
with complex objects (such us plant tissue and soil). Hin-region PCR
analysis can be used as a basis for fast and accurate screening of
natural populations of the nodule genus Rhizobium by this features.
Hin-region
Eight genotypes, 4 of which are the form of R.leguminosarum were identified on the basis of
differences in the structure of the hin-regions nucleotide sequences. The level of genetic diversity
within the identified genotypes was 96.2 ÷ 99.9%, and between them - 25.5 ÷ 89.9%.
94
R. leguminosarum bv. viceae 32-2
R. leguminosarum bv. viceae 28
R. leguminosarum bv. viceae 24
R. leguminosarum bv. viceae Y-4
R. leguminosarum bv. viceae 32
R. leguminosarum bv. viceae Y-3
R. leguminosarum bv. viceae 65
100 R. leguminosarum bv. viceae Y-1
R. leguminosarum bv. viceae Ch-14
R. leguminosarum bv. viceae Y-5
R. leguminosarum bv. viceae G-8
R. leguminosarum bv. viceae Y-2
R. leguminosarum bv. viceae Y-11
R. leguminosarum bv. viceae Y-12
R. leguminosarum bv. viceae P-2
R. leguminosarum bv. viceae 34
R. leguminosarum bv. viceae 31
100
R. leguminosarum bv. viceae K-29
R. leguminosarum bv. viceae 261b-st
100 R. leguminosarum bv. viceae 96-st
R. leguminosarum bv. viceae 97-st
99 R. leguminosarum bv. viceae 248b
73
R. leguminosarum bv. viceae Y-9
100
R. leguminosarum bv. viceae 245a-st
R. leguminosarum bv. viceae Y-7
67
R. leguminosarum bv. viceae B-6
R. leguminosarum bv. viceae B-16
99
R. leguminosarum bv. viceae B-9
R. leguminosarum bv. viceae B-8
89 R. leguminosarum bv. viceae B-25
R. leguminosarum bv. viceae B-17
R. leguminosarum bv. viceae B-18
R. leguminosarum bv. viceae B-15
I/A
I/A
Isolates from nodules Pisum sativum
I/A+I/B
I/B
Most of isolates from nodules Vicia faba
I/B
0.01
Previously identified genotypes of rhizobia biovar viciae had variety special specificity, that
was established by studying separately all nodules per plant.
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