Phenotypic, biochemical and genomic variability in generations of the rapeseed (Brassica napus L.) mutant lines obtained via chemical mutagenesis
Phenotypic, biochemical and genomic variability in generations of the rapeseed (Brassica napus L.) mutant lines obtained via chemical mutagenesis
Alexandra V. AmosovaID 0 2
Svyatoslav A. Zoshchuk 0 2
Valentina T. Volovik 1 2
Anna V. Shirokova 2
Nickolai E. Horuzhiy 2
Galina V. Mozgova 2
Olga Yu. Yurkevich 0 2
Margarita A. ArtyukhovaID 0 2
Valentina A. Lemesh 2
Tatiana E. Samatadze 0 2
Olga V. Muravenko 0 2
0 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences , Moscow, Russian Federation
1 Federal Williams Research Center of Forage Production and Agroecology , Lobnya, Moscow region, Russian Federation , 3 Koltzov Institute of Developmental Biology, Russian Academy of Sciences , Moscow, Russian Federation , 4 Institute of Genetics and Cytology, National Academy of Sciences of Belarus , Minsk , Belarus
2 Editor: Xiu-Qing Li, Agriculture and Agri-Food Canada , CANADA
The phenotypic, biochemical and genetic variability was studied in M2-M5 generations of ethyl methansulfonat (EMS, 0.2%) mutagenized rapeseed lines generated from canola, '00', B. napus cv. Vikros. EMS mutagenesis induced extensive diversity in morphological and agronomic traits among mutant progeny resulted in selection of EMS populations of B. napus- and B. rapa-morphotypes. The seeds of the obtained mutant lines were high-protein, low in oil and stabilized in contents of main fatty acids which make them useful for feed production. Despite the increased level of various meiotic abnormalities revealed in EMS populations, comparative karyotype analysis and FISH-based visualization of 45S and 5S rDNA indicated a high level of karyotypic stability in M2-M5 plants, and therefore, the obtained mutant lines could be useful in further rapeseed improvement. The revealed structural chromosomal reorganizations in karyotypes of several plants of B. rapa-type indicate that rapeseed breeding by chemical mutagenesis can result in cytogenetic instability in the mutant progeny, and therefore, it should include the karyotype examination. Our findings demonstrate that EMS at low concentrations has great potential in rapeseed improvement.
Data Availability Statement: All relevant data are
within the manuscript.
Funding: This work was supported by the Russian
Foundation for Basic Research, No.
17-2908034ofi_m, to OVM, and the Program of
Fundamental Research for State Academies, No.
0120136 3824, to OVM. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Rapeseed (Brassica napus L.) is one of the most economically important crops widely used in
different industries as an important source of edible vegetable oil, animal fodder and biodiesel
]. B. napus is considered to be a natural amphidiploid (genome AACC, 2n = 38) originated
from spontaneous hybridization between the ancestors of B. rapa L. (AA; 2n = 20) and B.
oleracea L. (CC; 2n = 18) followed by diploidization [
]. The polyphyletic origin of B. napus has
also been confirmed by results of organelle and nuclear RFLP analyses . Although both B.
oleracea and B. rapa have a great diversity of morphotypes with various origins, B. napus is
characterized by a relatively narrow genetic diversity [
]. Moreover, breeding selection
resulted in a decrease of genetic basis of current rapeseed cultivars. Therefore, new genetic
sources and approaches are needed to diversify the genetic basis of rapeseed germplasm,
which will make the current breeding programs more effective [
]. Examples of such
approaches may include intraspecific hybridization and a recombinant DNA technology [
], creation of synthetic rapeseed lines via artificial crosses between various Brassica species
containing A and C genomes [
] and also chemical and physical mutagenesis [
Chemical mutagenesis is an effective and simple method for obtaining valuable starting
material that can further be used in crop improvement programs [
]. Chemical mutagens (e.g.,
azide, diethyl sulphate, dimethyl sulphate, ethyl methanesulphonate and N-nitroso
compounds) are known to induce non-lethal point DNA mutations at a high rate and create novel
genetic diversity in various crops [
]. Particularly, this approach is widely used in
rapeseed breeding to produce new cultivars with the desired morpho-agronomic traits and/or
biochemical profile which are difficult to obtain though crossbreeding and selection [
The fatty acid biosynthesis pathway is a primary metabolic pathway in oil-bearing plants
]. Acetyl-CoA is the basic component of the fatty acid chain, involved in the synthesis of
16- or 18-carbon products, which are the major (up to 90%) fatty acids in plants. Various
desaturases located in the plastids and the endoplasmic reticulum are responsible for catalyzing
these fatty acids to become monounsaturated (palmitoleic acid, C16:1, and C18:1) or
polyunsaturated ones (C18:2 and C18:3). The fatty acid composition of the rapeseed oil is the main trait
determined its utilization mode and range [
]. Seeds of the double-low varieties (canola, ?00?,
with very low glucosinolates and erucic acid content) produce oil containing approximately
7% of saturated fatty acids (including palmitic (C16:0) and stearic (C18:0)), 61% of the
monounsaturated oleic acid (C18:1) and polyunsaturated fatty acids (linoleic (C18:2, 20%), linolenic
(C18:3, 10%) and eicosenoic (C20:1, 1%)). This fatty acid composition is considered optimal for
nutritional purposes [
]. However, due to the food- and non-food use of the oil, the demand
for rapeseed oils with other fatty acid compositions exists in the market [
The investigation of mutant rapeseed genomes is mostly related to the allele polymorphism
analysis and mapping of the mutant genes associated to agronomic traits. The content of
erucic acid in B. napus is found to be under additive control of alleles of FAE1.1 and FAE1.2 genes
encoding the enzyme of erucic acid synthesis, 3-ketoacyl-CoA synthase, from the oleoyl-CoA
]. It was shown that loss of functions of FAE1.2 (C subgenome) and one base pair
substitution in FAE1.1 gene (A subgenome) led to formation of canola, ?00?, plants [
content of oleic acid is controlled by the fatty acid desaturase 2 (FAD2) gene that encodes
endoplasmic delta-12 fatty acid desaturase 2 (112-FAD2) which converts the precursors of
oleic acid to the precursors of linoleic acid in the lipid biosynthetic pathway [
homologous FAD2 genes (BnFAD2-1, BnFAD2-2, BnFAD2-3, and BnFAD2-4) located
separately on rapeseed chromosomes of A and C subgenomes, and their possible role in the
rapeseed genome is oleic acid regulation [
]. The linolenic acid content in B. napus is
controlled by two fatty acid desaturase 3 (FAD3) genes (BnaA.FAD3 and BnaC.FAD3),
encoding delta-15 linoleate desaturase which is responsible for dehydration of linoleic acid to
linolenic acid [
]. These genes were detected in the A and C subgenomes of B. napus [
mapped in the N4 (A4) and N14 (C4) linkage groups, correspondingly [
More variability of rapeseed germplasms can be created via mutagenesis [
23, 36, 39, 41?42
At the same time, experimental mutagenesis in allopolyploid B. napus might result in various
genetic, chromosomal and genomic reorganizations promoting genetic instability in the
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progeny. However, in karyotypes of rapeseed mutants, the structure of chromosomes and
possible intra- and intergenomic structural rearrangements and substitutions are poorly
investigated. Due to small rapeseed chromosomes (1.53?3.30 ?m) [
], the detailed chromosomal
analysis is still problematic and needs special approaches, e.g., chromosome elongation with
the use of DNA intercalators, application of chromosomal markers allowing identification of
individual rapeseed chromosomes and their subgenomic affiliation [
study of the genotypic variability in mutant rapeseed lines in combination with the karyotype
structure analysis (chromosomal complements in A and C subgenomes, the presence of
chromosome rearrangements, chromosome substitutions and additions), description of
phenotypic and biochemical variability was not performed. Integration of mutation techniques with
the molecular, cytogenetic and biochemical analyses provides exciting opportunities for
rapeseed breeding. Such approach could be useful in developing reliable tools for improving
selection methods and also for introducing novel traits into rapeseed cultivars.
The objectives of the present study were to analyze phenotypic, biochemical and
cytogenomic variability in M1-M5 generations of the ethyl methanesulfonate (EMS) mutagenized
progeny of the spring canola B. napus cv. Vikros in order to reveal agronomically valuable and
genetically stable rapeseed mutant genotypes. The current approach based on the analysis of
morphological and agronomic traits, the biochemical profile, SNaPshot detection of mutant
and wild-type FAD3 genes, meiosis and FISH localization of 5S and 5S rDNA has been
Materials and methods
This study including plant sample collection and experimental research conducted on these
materials was according to the federal law on environmental protection approved by the
Council of the Russian Federation.
Seeds of the spring canola, ?00?, B. napus cv. Vikros (3480, Russian Federation) were obtained
from the germplasm collections of Federal Williams Research Center of Forage Production and
Agroecology, Lobnya, Moscow, Russian Federation. Before the mutagenesis assays, the progeny
of three succeeding generations (I1-I3) of this B. napus cv. Vikros was tested for hidden effects
of inbreeding (self-pollination), and no deviations from the standard characteristics of the
original cultivar were revealed. To diversify the genetic basis of the rapeseed germplasm, the seeds of
the original cultivar were treated with aqueous solution of ethyl methanesulphonate in
concentrations of 0.2% for 16 h. All studied rapeseed plants were grown with the use of pre-grown
seedlings: seeds were sowing in the greenhouse followed (30?40 days later) the outdoor planting
of at least 50 seedlings. In mutant plants, the leading shoots were isolated at floral initiation
stage to obtain self-pollinated seeds. At maturity, seed siliqua were collected from the leading
shoots. The identification of the plants was performed according to the Manual of Brassica
napus L. [
]. The progeny selection for morphological and agronomic characters was carried
out in 2-5 plants. Statistical data analysis was performed using standard functions of Microsoft
Excel 2013. For each generation, at least 50 plants in every mutant line were analyzed.
The biochemical profile was analysed for 20 plants of each mutant line. The fatty acid
composition and total oil content were determined in milled seeds (2 g from one plant, 15 plants of
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each line) using the gas chromatograph Kristall 2000M (Chromateck, Yoshkar Ola, RF) with
Zebron ZB-FFAP Capillary GC Column 25m x 0.20mm x 0.30?m (Phenomenex, Torrance,
USA) according to the manufacturer?s protocol. The content of protein was estimated
photometrically using a biochemical flowing auto analyzer of chemical composition CIAK-K
(Kinzh-Agro, Moscow, RF) according to the manufacturer?s protocol. Statistical data analysis
was performed using standard functions of Microsoft Excel 2013.
Total genomic DNA was extracted from green leaves and seedlings using the ?Genomic DNA
Purification Kit? (Thermo Fisher Scientific, Vilnius, Lithuania) according to the
manufacturer?s protocol. The DNA concentration and purification degree were determined using the
Implen Nano Photometer NP60 spectrophotometer (Implen, Munich, Germany). Fifteen
individual plants of each mutant line were used to estimate the genetic heterogeneity.
Allelic forms of the B. napus FAD3 genes were identified by PCR amplifications of the gene
fragments comprising wild-type and mutation sites followed the detection of the mutant alleles
by the microsequencing method (SNaPshot) with locus-specific primers.
Target DNA fragments were amplified in two independent reactions with genome-specific
primer pairs FAD3Af/FAD3Ar for the BnaA.FAD3 gene and FAD3Cf/FAD3Cr for the BnaC.
FAD3 gene as it was described earlier [
]. Amplification was carried out on SimpliAmp
Thermal Cycler (Applied Biosystems, Foster City, USA) in following conditions: 4 min at 95?C
followed by 30 steps with 30 s at 95?C, 30 s at 55?C and 30 s at 72?C, and with the final elongation
step for 30 min at 72?C. Reaction mix included 100 ng of genomic DNA template, 1 ?M
dNTP, 1.5 mM MgCl2, 10x PCR-buffer (650 mM Tris-HCl, 166 mM (NH4)2SO4, 0,2% tween
20, pH 8.8), 0.25 ?M each primer, and 1 U Taq polymerase (Primetech, Minsk, Belarus) in a
total volume of 25 ?l. PCR products were separated by electrophoresis in 1.5% agarose gel with
an addition of ethidium bromide solution to a final concentration of 0.5 ?g/ml at a voltage of
100 V with the use of 100 bp Plus DNA-ladder (Thermo Scientific, Vilnius, Lithuania). After
amplification, post-PCR purification was performed as follows: 5 ?l of the PCR product was
incubated with 1 U of FAST alkaline phosphatase and 2 U of exoI (Thermo Fisher Scientific,
Vilnius, Lithuania) for 1 h at 37?C, followed by 15 min at 80?C for enzyme inactivation.
The amplified on the first step fragments were used for the detection of FAD3 mutant and
wild-type alleles by SNaPshot technique. In the SNaPshot analysis, previously described
primers mutA-1f and mutC-45F [
] modified with a poly-A tail, were used. To discriminate these
fragments, the s550 high density size standard for fragment analysis (Synthol, Moscow, RF)
was used. Primer extension reactions were carried out independently for FAD3A and FAD3C
in a final volume of 10 ?l containing 2 ?l exoI/FAST treated PCR product (5?50 ng DNA) as a
template, 2 ?l of the SNaPshot Ready Reaction Mix (Applied Biosystems, Foster City, USA)
and 0.2 ?M primer. The following amplification protocol was applied: 35 cycles of 10 s at
95?C, 5 s at 50?C and 30 s at 60?C. After the extension reaction, PCR products were treated
with FAST alkaline phosphatase (1 unit per sample) for 1 h at 37?C. For electrophoresis, 0.5 ?l
of the purified primer extension reaction products were combined and mixed with 9 ?l of
HiDi (highly deionized) formamide and 0.5 ?l of s550 size standard (Synthol, Moscow, Russia),
denatured for 5 min at 95?C and separated by capillary electrophoresis on an ABI Prism 310
Genetic Analyser (Applied Biosystems, Foster City, USA) using POP6 polymer. Alleles of the
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FAD3A and FAD3C were scored using Gene Mapper 4.1 software (Applied Biosystems, Foster
City, USA). The presence of polymorphic alleles was visualized by colour depending on the
included in the SNaPshot-PCR product ddNTP which carried the corresponding fluorescent
? A?dR6G label?green
? C?dTAMRA label?black
? G?dR 110 label?blue
? T(U)?dROX label?red
? FAD3A (wild-type allele)?black
? fad3A (mutant allele)?red
? FAD3C (wild-type allele)?blue
? fad3C (mutant allele)?green
Considering that the alleles of FAD3 genes differed from each other by one nucleotide
(FAD3A ?C; fad3A ?T; FAD3C ?G; fad3C ?A), the following coloured peaks were visualized
as a result of SnaPshot-PCR with a single locus-specific forward primer:
For chromosome spread preparation, rapeseed root tips (1?0.5 cm) were incubated (16?24 h)
in ice-cold water with 1 ?g/mL of 9-AMA (Sigma, St. Louis, USA) to inhibit chromosome
condensation process and accumulate prometaphase chromosomes [
]. Then, the roots were
treated in ethanol: glacial acetic acid fixative (3:1) for 48 h at room temperature and after that
stored at ?20?C until use. Chromosome spreads were prepared according to the technique
described previously [
For meiotic chromosome preparation, young floral buds (prefoliation) were fixed in
ethanol:acetic acid (3:1) fixative for 30 min at 4?C and then chromosome spreads were prepared as
previously described [
]. After freezing in liquid nitrogen, the cover glasses were removed,
and the slides were stored in 96% ethanol at ?20?C until use.
DNA probe preparation and FISH
Following probes were used for FISH:
1. pTa71 containing a 9 kb long repeated DNA sequence of common wheat including
18S5.8S-26S rDNA [
2. pTa794 containing a 420 bp long repeated DNA sequence of wheat including 5S rDNA
DNA probes were labelled directly with SpectrumRed or SpectrumAqua fluorochromes
(Abbott Molecular, Wiesbaden, Germany) by nick translation according to manufacturer?s
protocol. FISH procedure was performed as described previously [
]. After hybridization
(16?20 h), the slides were washed twice with 0.1xSSC at 44 C for 10 min, twice with 2xSSC at
44 C for 5 min followed by a 5-min wash in 2xSSC and three washes in PBS for 3 min each at
room temperature. Then the slides were dehydrated through a graded ethanol series and air
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After the FISH procedure, chromosome slides were stained with 0.1 ?g/mL DAPI
(40,6-diamidino-2-phenylindole) (Serva, Heidelberg, Germany) in Vectashield mounting medium
(Vector laboratories, Peterborough, UK). DAPI-banding analysis was used as an additional
parameter for the identification of individual chromosomes [
The slides were examined using Olympus BX61 epifluorescence microscope (Olympus,
Tokyo, Japan) combined with a monochrome CCD camera (Cool Snap, Roper Scientific Inc.,
Tucson, USA). The captured images were processed with Adobe Photoshop 10.0 software
(Adobe Systems Inc., Birmingham, USA). At least 30 plants of each line and 15 metaphase
plates of each plant were analyzed. In karyotypes, the cytological numerical designation of the
chromosomes of A and C subgenomes was according to Levan?s criterion [
the identification of chromosomes and genome affiliation were performed based on the
chromosome morphology, revealed chromosome markers as well as earlier described data [
]. The meiotic chromosome preparations were analyzed as described previously .
Analysis of pollen
The examination of pollen grains was performed with the use of a scanning electron
microscope (SEM) JEOL JSM? 6380LA (accelerating voltage 20 kV, SEI mode) (Jeol, Tokyo, Japan).
In each line, pollen grains were collected from six plants (three flowers from the main
inflorescence). Fresh pollen was mounted on carbon adhesive tape. The analysis of pollen grains was
performed with the use of SEM Control User Interface, Version 7.11 (Jeol, Tokyo, Japan). For
each line, ten ocular views (250 x) of pollen grains were analysed. Statistical data analysis was
performed using standard functions of Microsoft Excel 2013.
Within the M2-M3 progeny, a segregation of morphological traits was found, and plants of B.
napus-like and B. rapa-like morphotypes displaying distinct morphological differences were
revealed. In M4-M5 generations, the progeny of B. napus-type plants presented constant
morphotypes. Within the progeny of the B. rapa-type line, further segregation of morphological
traits was observed, and both B. rapa- and B. napus- (up to 12%) morphotypes were detected.
At the stage of the third pair of true leaves, these morphological differences became more
evident. Plants of the B. rapa-like morphotype had tender, thin, round and puberulent leaf
blades and bright green (non-glaucous) leaves, stems and siliqua (Fig 1). The pubescence
disappeared at the stage of the fifth pair of true leaves. Rapeseed-like plants had more coriaceous
and smooth leaf blades and glaucous leaves, stems and siliqua (Fig 1).
In most mutant plants, we observed moderate decrease in the mean value of plant height
compared to the original cultivar. However, this character was highly variable (Table 1). The
plants of B. rapa-type had longer hypocotyl (Fig 1) and were more liable to lodging at the stage
of early flower bud formation if compared with the original cultivar and rapeseed-type plants.
Then, the leading shoot checked in growth, and the plant height in such plants became
contingent on first-order shoot development. Basal first-order shoots in B. rapa-type plants, were
well-developed and grew subopposite from the hypocotyl (vs. in B. napus-type plants, they
were also well-developed but grew from the root neck). In plants of B. rapa-type, shoots III
were also observed though side shoots II were less developed compared to B. napus-type
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Fig 1. Plants of original B. napus cv. Vikros and its mutant progeny. Plants of B. napus cv. Vikros (a1), mutant
plants of B. rapa-type (b1) and B. napus-type (c1) at the rosette vegetative growth stage; position and shape of siliqua in
B. napus cv. Vikros (a2), in plants of B. rapa-type (b2-1) and B. napus-type (c2); a plant of B. rapa-type with a long
(Table 1). Siliqua in B. rapa-like plants were thinner and grew more vertical (vs. in B.
napustype plants, the angle was about 45?) (Fig 1).
All studied plants had yellow racemose inflorescences. In mutant plants, inflorescences
were shorter and few-flowered. In plants of B. rapa-type, flowers were a little smaller and the
flower colour was lighter than in rapeseed-like plants (Fig 2). In all studied plants, the pollen
grains were tricolpate (typical for Brassicaceae) (Fig 2). However, more imperfect and/or
deformed pollen grains were revealed in plants of B. rapa-type compared to the original
cultivar and rapeseed-like plants (Fig 2, Table 1). Also, in plants of B. rapa-type, the number of
seeds per silique was more variable; seeds were red-brown, irregular-shaped and smaller in
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size; and seed productivity was less if compared with the plants of rapeseed-type (Fig 2,
Biochemical profile and fatty acid composition
Biochemical analysis of seeds showed that the oil content in the mutant lines was rather low
(39?42%) especially, in B. rapa-type plants (28?39%), and the protein content was high (about
30%, in plants B. rapa- and segregated B. napus-type it reached 35%). Biochemical analysis
also revealed a high level of variability in seed crude fiber contents among the mutant plants of
different morphotypes (Fig 3).
The analysis of fatty acid (FA) compositions in seeds showed that in mutant plants, the
palmitic (C16:0) fatty acid was synthesized more extensively compared to the original B. napus cv.
Vikros. The contents of the other main fatty acids were roughly comparable with the original
cultivar. Besides, small amount ( 2%) of 7?8 other fatty acids which were not typical for
rapeseed (?8:0, ?14:0, ?16:2, ?16:3, ?17:0, ?17:1, ?24:0, ?24:1) were detected in the studied rapeseed
plants (Table 2).
The SNaPshot analysis was performed for 20 M4-M5 plants of different types including plants
with abnormal karyotypes and also the original cultivar. This analysis detected only wild-type
alleles of FAD3 genes in both A and C subgenomes in all studied samples (Table 3).
Chromosomal structural variations in the EMS populations
In most studied maternal pollen cells of B. napus cv. Vikros, regular meiotic chromosome
behavior with normal chromosome disjunction and nineteen bivalents (19II) was observed
(Fig 4A). Besides, in the reduction division, few common meiotic abnormalities were detected.
As an example, the occurrence of some chromosomes outside the metaphase spread is shown
in Fig 4B. However, the cumulative percentage of these irregularities in maternal pollen cells
was nonessential (~1.5%).
In both constant and segregated populations of B. napus-type plants, common meiotic
abnormalities were detected in 0.15?5.1% of the maternal pollen cells. For instance,
chromosome lagging and chromosome bridges at anaphase I are shown in Fig 4C and Fig 4D,
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Fig 2. Vegetative parameters in B. napus cv. Vikros and mutant plants. Inflorescences of B. napus cv. Vikros (a1),
plants of B. rapa-type (b1) and B. napus-type (c1); SEM images of pollen grains in B. napus cv. Vikros (a2), in plants of
B. rapa-type (b2) and B. napus-type (c2); seeds of B. napus cv. Vikros (a3), plants of B. rapa-type (b3) and B.
napustype (c3). Scale bar? 50 ?m.
Fig 3. Biochemical composition of seeds in B. napus cv. Vikros and M5 plants. Contents of crude fiber (blue), oil
(red) and crude protein (green) (the vertical axis, %) in the original cultivar and EMS populations (the horizontal axis).
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Each meaning represents the mean value ? standard deviation
The values are significantly different at P 0.05
correspondingly. Besides, at anaphase II, the spindle function related abnormalities
(asynchronous division and lagging) were also detected in maternal pollen cells.
In the B. rapa-type plants, the cumulative percentage of common meiotic abnormalities in
maternal pollen cells was ranged from 0.15% to 11.8%. For example, univalents and
quadrivalents in the reduction devision (M-1) as well as chaotic disjunction and chromosome lagging
at A-I are presented in Fig 4E and Fig 4F, correspondingly.
In one M5 plant of B. rapa-type, multiple meiotic abnormalities (elimination of
chromosome groups at anaphase-telophase I, micronuclei in dyads, chromosome elongation and
chaotic chromosome distribution at metaphase II, chromatin agglutination, three-polar
configurations and asynchronous division within one meiocyte) were revealed in 0.17%?
35.7% of the studied maternal pollen cells. For example, a three-polar configuration with
chromatin agglutination and asynchronous division within one meiocyte are shown in Fig 4 g and
Fig 4H, correspondingly.
In the original cultivar and most studied M2-M5 plants, rapeseed karyotypes with 2n = 38
chromosomes were observed. The exception was one M5 plant with 2n = 40 chromosomes
(Figs 5 and 6).
In karyotypes of the original cultivar, FISH analysis revealed separate 45S rDNA sites in the
secondary constriction regions (subtelomere positions of the short arms) of two large
chromosome pairs 7 and 8 (C subgenome) and also in the pericentromeric region of one middle-sized
chromosome pair 2 (A subgenome). Separate 5S rDNA sites were detected in the
pericentromeric and interstitial positions (the long arm) of one large chromosome pair 4 (C subgenome)
and in the subtelomere region of the short arm of the smallest chromosome pair 10 (A
subgenome). Co-localized 45S and 5S rDNA sites were found in the pericentromeric region of
middle-sized chromosome pairs 1, 3 and 4 (A subgenome) and also in the secondary constriction
region (subtelomere positions of the short arms) of the pair of a middle-sized chromosome
pair 5 (A subgenome) (Figs 5 and 6).
In karyotypes of most studied mutant plants, patterns chromosomal distribution of 45S and
5S rDNA were similar to those observed in the original cultivar with the exception of one M3
plant of B. rapa-type having only separate 45S rDNA sites on chromosome pairs 4 (A
subgenome); one M5 plant of B. rapa-type with double trisomy (2n = 40) and also one M5 plant of
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B. rapa-type with a homeologous substitution of one chromosome pair (4) between A and C
subgenomes (Figs 5 and 6).
EMS is included among the so-called ?supermutagens? which can be used to generate the
important recessive and dominant genomic mutations at a high rate and thereby create a basis
Fig 4. Meiosis in maternal pollen cells in B. napus cv. Vikros and mutant plants. (a) A-I, 19II; (b) several
chromosomes are localized outside the metaphase plate; (c) A-I, chromosome lagging; (d) A-I, chromosomal bridges;
(e) M-I, 14II+2IV(short arrows)+2I (long arrows); (f) A-I, chaotic disjunction and chromosome lagging; (g) three-polar
configuration with chromatin agglutination; (h) asynchronous division within one meiocyte.
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Fig 5. FISH-based localization of 45S and 5S rDNA on chromosomes of B. napus cv. Vikros and M2-M5 mutant
plants. Metaphase plates of B. napus cv. Vikros (A), M2 plant of B. napus-type (B), M3 plant of B. rapa-type (C), M4-4
plant of B. rapa-type (D), M-17 plant of B. napus-type (segregated) (E), M5-3 plant of B. rapa-type (F), M5-2 plant of
B. rapa-type (G). The correspondent probes and their pseudo-colours are specified in the upper right-hand corner.
DAPI-banding (blue). Scale bar? 5 ?m.
for useful genetic variations required for plant breeding programs [
mutagenesis is an effective approach to create mutations in genes of the polyploid species such as B.
napus. These mutagens was found to induce non-lethal point DNA mutations which could be
retained in the genome due to its capacity for self-pollination [
]. These induced genetic
variations correlate to variability in agronomic and phenotypic traits in rapeseed mutant
]. In the present study, EMS mutagenesis induced extensive morphological
diversity among mutant progeny of canola B. napus cv. Vikros. As a result, we could
successfully select EMS populations of B. napus- and B. rapa-morphotypes displaying distinct
differences in morphological and agronomic traits. Within the progeny of the B. rapa-type line,
further segregation of morphological traits was observed indicating that EMS had induced the
heterozygous mutations in genomes of B. rapa-type plants, and both B. rapa- and B.
napus(up to 12%) morphotypes were revealed. As it was quite possible that the mutagenesis could
result in genotypic differences between constant and segregated populations of B. napus-type,
we performed comparative analysis among the EMS populations of different types. Currently,
producing short-stem lines is a high-priority task, and due to breeding efforts for growth
limitation of lateral meristems in joints of a plant stem, plant height is considered to be an
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Fig 6. Karyotypes of B. napus cv. Vikros and M2-M5 mutant plants. Karyograms of the metaphase plates shown in
Fig 5 after DAPI-banding (blue) and FISH with 45S (green) and 5S rDNA (red). Scale bar? 5 ?m.
important agronomic trait. The decrease in plant height was described earlier in several EMS
mutagenized populations of crops including Brassica species [
]. In the studied EMS
populations of different types, we observed moderate decrease in plant height and also high variability
of this feature. These findings indicate that plant height could probably be reduced by a further
selection process in the EMS progeny.
Also, plants of B. rapa-type and segregated B. napus-type plants were found to have more
extensively developed shoots I and shoots II. Probably, due to high density of plant tillers, poor
flowering, lower number of siliqua and lower level of seed productivity were observed in those
plants compared to plants of the original cultivar and constant rapeseed-type.
Biochemical analysis showed that seeds of the studied mutant plants were high-protein and
low in oil which makes them useful for feed production. Also, seeds of B. rapa-type plants had
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the highest crude fiber content, but this character was more variable compared to the protein
contents in the seeds.
Different fatty acid components of rapeseed oil make it best suited to particular uses.
Canola ?00? (low erucic acid and low glucosinolate) produces seeds that are used to generate
excellent edible oil that is lower in saturated fat and higher in omega-3 fatty acids than most
other commercially available oils [
]. These attributes have been shown to have a significant
positive impact on human health, reducing diseases such as cancer, heart disease and some
neurological disorders [
]. EMS mutagenesis can induce genetic changes in plants and
modify the levels of fatty acids in seed oil . In this study, however, the treatment of canola
seeds with EMS at low concentration did not influence the contents of main fatty acids in
canola seeds with the exception of a palmitic (C16:0) acid which level was higher compared to
the original cultivar. One of the rapeseed breeding goals is to obtain genotypes producing
naturally stable oil. Particularly, a low content ( 10%) of the linolenic acid prevents oxidation
and rancidification of seed oil which is important for healthy food production [
high stability of the oil with low linolenic acid content makes it an important source of raw
material for biofuel production. Genetic analyses revealed that the fatty acid composition of
rapeseed varied depending on the allelic composition of FAD3 genes as well as the ratio of
mutant fad3a, fad3c alleles and FAD3, FAD3A, FAD3C wild-type alleles [
single-nucleotide mutations detected in mutant rapeseed lines resulted in a decrease in the
content of linolenic acid in rapeseed oil [
]. SNaPshot analysis using SNP markers is an
effective approach for detecting mutant alleles of the FAD3 genes in B. napus [
]. In the
present study, the performed SNaPshot analysis did not detect any single-nucleotide
polymorphisms in FAD3 genes in both A and C subgenomes indicating the homozygous state of these
genes in the studied lines. Considering also that the original canola cultivar and the plants of
B. rapa- and B. napus-morphotypes had related meanings of linolenic (C18:3) fatty acid
contents (8?10%), our results showed that mutagenesis did not influence the stability of this
essential fatty acid in the obtained mutant lines.
Chemical mutagens can influence the plant genome and cause the meiotic disorders
manifested themselves as typical anaphase aberrations (chromosome fragments, bridges, lagging,
etc.) as well as fragmentation, nondisjunction, chromosome stickiness and other abnormalities
]. In most studied here maternal pollen cells of the original rapeseed cultivar and mutant
lines, normal chromosome disjunction (19:19) was observed. However, typical meiotic
abnormalities including chromosome fragments, chaotic chromosome disjunction and lagging at
anaphase I; occurrence of some chromosomes outside the metaphase spread and bridges were
also revealed. Chromosome nondisjunction, occurred at anaphase I, is considered to be a
serious meiotic abnormality which resulted in chromosome loss as well as unequal distribution of
genetic material. These disorders could appear due to the paracentric inversions as previously
described in tomatoes and Nigella sativa [
Besides, deviations from the normal bivalent conjugation could be displayed as univalent
and multivalent formation at metaphase I stage [
]. In this study, univalents at diakinesis
were also detected in maternal pollen cells of the studied mutant plants. The mutagen-induced
univalent formation was supposed to be a result of chromosome structure changes followed by
the reduction of chiasma frequency due to restriction of pairing to homologs [
In the original B. napus plants, the cumulative percentage of meiotic irregularities in
maternal pollen cells was nonessential (~1.5%). However, the percentage of cells with meiotic
disorders was higher in the studied plants rapeseed-type (up to 5.1%) and B. rapa-type (up to
11.8%) compared to the original cultivar. In one M5 plant of B. rapa-type, multiple meiotic
abnormalities including elimination of chromosome groups at anaphase-telophase I,
micronuclei in dyads, chromosome elongation and chaotic chromosome distribution at metaphase II,
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chromatin agglutination, three-polar configurations and asynchronous division within one
meiocyte were revealed. The analysis of meiotic chromosome behaviour indicated that in
plants of the obtained EMS populations, various chromosome rearrangements could occur.
Probably, the observed high level of phenotypic variability could also be related to these
chromosomal variations. However, zygotes with chromosome abnormalities (appeared due to
disorders during meiosis) were shown not always to produce viable seeds, and most meiotic
abnormalities were eliminated before the tetrade stage and therefore, do not influence the
pollen quality [
]. However, several meiotic irregularities, such as chromatin agglutination, as
well as high level of abberrations (35.7%) revealed in this sample could reduce the quality and
fertility of pollen and subsequently, result in reductions in seed yield.
The amphidiploid genome of B. napus consists of closely related A and C subgenomes [
] which display numerous deviations from parental Brassica species additivity [
Consequently, B. napus is considered to be an important model species to study the processes
of genomic reorganizations in recently formed polyploids [
]. The examples of such
processes could be different chromosomal rearrangements and intragenomic substitutions
observed in natural and resynthesized rapeseed lines which could probably be related to the
maintenance of genomic stability [
]. Also, it was previously shown that an enhanced
genome instability in resynthesized rapeseed lines developed under the pressure of selection
resulted in chromosome rearrangements or/and deletions and even elimination of the whole
parental genome in hybrids in the succeeding generations [
]. Besides, intraspecific
polymorphism in pattern of chromosomal distribution of 45S and 5S rDNA was previously described
for B. napus [
]. In this study, the molecular cytogenetic analysis of the original B. napus
cultivar and obtained mutant lines of B. rapa- and B. napus-morphotypes indicated a high degree
of karyotypic stability despite the fact that the cumulative percentage of microsporocytes with
meiotic disorders was higher in mutant plants compared to the original cultivar. FISH analysis
showed that all the studied karyotypes in B. napus-type plants and most karyotypes in B.
rapatype plants did not differ in chromosome number, morphology and pattern of 45S and 5S
rDNA chromosomal distribution from the original cultivar. However, among M3-M5 progeny
of B. rapa-type, chromosomal reorganizations including variations in number of 45S and 5S
rDNA, trisomy and substitutions between homeological chromosomes were also revealed. It
should be noted that the observed chromosomal reorganizations correlated to the higher levels
of different meiotic abnormalities, differences in plant morphology and also low seed
productivity detected in B. rapa-type progeny, and this could be related to the EMS induced
mutations. Different cytogenetical abnormalities induced by EMS mutagenesis were observed
earlier in tomatoes and Nigella sativa [
]. Our findings demonstrate that rapeseed
breeding via chemical mutagenesis could result in cytogenomic instability in the obtained mutant
progeny, and therefore, should include karyotype examination.
Thus, molecular cytogenetic analysis of the original B. napus cv. Vikros and its EMS
mutagenized progeny indicated that the processes of mutagenesis and also selection for
morphological and agronomic traits did not induce changes in chromosomal structure of both constant
and segregated mutant lines of B. napus-type, and these mutant lines could be a basis for
further rapeseed improvement. The revealed structural chromosomal reorganizations in
karyotypes of the mutant plants of B. rapa-type showed that it can be useful for the development of
rapeseed forms with trisomy and also chromosome addition/substitution lines. Such
aneuploidy lines are important for rapeseed breeding as they provide the opportunity to produce
introgression lines and also offer the way to check heterologous gene expression and
interaction between recipient genome and donor chromosomes in plants [
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In the present study, EMS mutagenesis induced extensive diversity in morphological and
agronomic traits among mutant progeny of canola B. napus cv. Vikros resulted in selection of EMS
populations of B. napus- and B. rapa-morphotypes. The obtained unique data on phenotypic,
biochemical and cytogenomic variability within these populations showed distinct differences
among them. The mutant plants with abnormal karyotypes revealed within the EMS
populations indicate that rapeseed breeding by chemical mutagenesis can induce chromosome
instability in the mutant progeny, and therefore, it should include karyotype examination. Our
findings demonstrate that EMS at low concentrations has great potential in rapeseed
The authors acknowledge Dr. N.N. Kozlov and Dr. V.L. Korovina (Laboratory of Genetic
Resources of Fodder Plants, FWRC of Forage Production and Agroecology, Lobnya, Moscow
region, RF) for providing us valuable plant material and Dr. A.G. Bogdanov (Laboratory of
Electron Microscopy, Faculty of Biology, Lomonosov Moscow State University, Moscow, RF)
for SEM technical assistance.
Conceptualization: Alexandra V. Amosova, Svyatoslav A. Zoshchuk, Valentina A. Lemesh,
Olga V. Muravenko.
Formal analysis: Alexandra V. Amosova, Svyatoslav A. Zoshchuk, Valentina T. Volovik,
Anna V. Shirokova, Nickolai E. Horuzhiy, Galina V. Mozgova, Olga Yu. Yurkevich,
Margarita A. Artyukhova, Tatiana E. Samatadze.
Investigation: Alexandra V. Amosova, Svyatoslav A. Zoshchuk, Valentina T. Volovik, Anna
V. Shirokova, Nickolai E. Horuzhiy, Galina V. Mozgova, Olga Yu. Yurkevich, Margarita A.
Artyukhova, Tatiana E. Samatadze, Olga V. Muravenko.
Methodology: Alexandra V. Amosova, Svyatoslav A. Zoshchuk, Olga V. Muravenko.
Resources: Valentina T. Volovik, Anna V. Shirokova.
Supervision: Olga V. Muravenko.
Validation: Valentina A. Lemesh, Olga V. Muravenko.
Visualization: Alexandra V. Amosova, Svyatoslav A. Zoshchuk, Valentina T. Volovik, Anna
V. Shirokova, Nickolai E. Horuzhiy, Galina V. Mozgova, Olga Yu. Yurkevich, Margarita A.
Artyukhova, Tatiana E. Samatadze.
Writing ? original draft: Alexandra V. Amosova, Svyatoslav A. Zoshchuk, Valentina T.
Volovik, Anna V. Shirokova, Nickolai E. Horuzhiy, Galina V. Mozgova, Olga Yu. Yurkevich,
Valentina A. Lemesh, Tatiana E. Samatadze, Olga V. Muravenko.
Writing ? review & editing: Alexandra V. Amosova, Svyatoslav A. Zoshchuk, Valentina A.
Lemesh, Tatiana E. Samatadze, Olga V. Muravenko.
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