Сельскохозяйственная биология, 2012, № 5, с. 100-110.

doi: 10.15389/agrobiology.2012.5.100eng

УДК 635.33:575.822

CLASS II MGE-LIKE SEQUENCES IN GENOMES OF Brassica L. SPECIES

A.M. Artemyeva, A.G. Dubovskaya, A.E. Solov’eva, Yu.V. Chesnokov

Basing on the literature data and own results, an unstability of genomes in species of Brassica genus is considered, and possibility to apply the class II mobile genetic elements-like nucleotide sequences, Ас, MuDR, Far1 and CACTA, for creation of molecular markers which could be used to evaluate genetic diversity is discussed. Using PCR, a distribution and polymorphism of markers based on Ас, MuDR, Far1 and CACTA elements are investigated in samples of Brassicaceae L. from the world collection of the N.I. Vavilov Institute of Plant Industry (VIR), with regard to their phylogeny.

Keywords: species Brassica, mobile genetic elements Ас, MuDR, Far1, CACTA, preserved genetic biodiversity.

Vavilov’s theory of mobilizing and studying biosphere’s genetic resources based on knowledge of botany, geography, history, the theory of evolution, is becoming increasingly important in the view of a global climate change and increased human impact resulting in the reduce of gene pool of both cultivated and wild species. To develop this theory, since the beginning of XX century N.I. Vavilov and his associates performed target botanical-agronomic expeditions for collection of objects for the world’s largest collection of cultivated plant species maintained by VIR (All-Russia Research Institute of Plant Industry; named after N.I. Vavilov in 1968), including economically important crops of the family Brassicaceae L.
The first samples of vegetable and oilseed Brassicaceae were delivered to VIR collection in 1921 from Vavilov’s expeditions to Western Europe, USA and Canada, later - from Afghanistan, Iran and Armenia. Russian breeding varieties and local cultivars were obtained first through the All-Union Agricultural Exhibition, then from expeditions to the North-West of USSR, the Altai and the Far East. Other VIR expeditions visited the countries of ancient agriculture: Mediterranean Europe, Ethiopia, Western China (headed by N.I. Vavilov), Asia Minor (P.M. Zhukovsky), India (V.V. Markovic), etc. Even in Vavilov’s time, VIR collection of cabbage, for example, included the 1500 samples. These activities have developed “... the source potential of species diversity of morphological and physical properties needed for breeding and genetics” (1). Botanical-geographic study of oilseeds and root vegetables of Brassicae was performed in VIR under the direction of E.N. Sinskaya since 1921, cabbage crops – under the direction of T.V. Lizgunova since 1926. Results of these studies were published in monographs describing taxonomy, variability of traits, characteristics of varieties and varieties-groups, and genetic bases of breeding (2-5).
Origin, evolution and phylogenetic relations of six Brassica species known as “triangle of U” (triangle Brassica) - B. nigra (L.) Koch., B. oleracea L., B. rapa L., B. napus L., B. juncea (L.) Czern., and B. carinata A. Braun are generally defined. N. U (6) proposed a diagram showing their phylogenetic relations: allo-tetraploids as sides of a triangle connecting the vertices – constituent source diploids. According to N. U, B. napus (AACC, 2n = 4Ѕ = 38), B. juncea (AABB, 2n = 4Ѕ = 36) and B. carinata (BBCC, 2n = 4Ѕ = 34) originated from natural interspecific hybridization between pairs of diploid species – respectively, B. rapa (AA, 2n = 2Ѕ = 20) Ѕ B. oleracea (CC, 2n = 2Ѕ = 18), B. rapa Ѕ B. nigra (BB, 2n = 2Ѕ = 16) and B. nigra Ѕ B. oleracea.
Cultivated Brassicae are extremely morphologically diverse. However, taxonomic status of some wild species of the genus, as well as intraspecific taxa of cabbage B. oleracea and turnip B. rapa is still discussed (7-9). Thus, the noted modern botanist and taxonomist of Brassicaceae C. Gomez-Campo (7) divided the genus Brassica into two subgenera: Brassica (27 species) and Brassicaria (Godr.) Gomez-Campo (11 species). The subgenus Brassica consists of five sections: Brassica, Rapa (Miller) Salmeen, Micropodium DC, Brassicoides Boiss. and Sinapistrum Willkomm. The section Brassica includes cabbage B. oleracea, Abyssinian cabbage B. carinata (n = 17, BC genome), as well as Mediterranean species related to cabbage (n = 9, C genome). It is assumed that genes of the latter ones introgressed to B. oleracea and, therefore, the formation of different cultivated cabbage varieties was influenced by wild species –  B. cretica Lam., B. incana Ten, B. rupestris Rafin, B. macrocarpa Guss., B. montana Pourret, B. villosa Raimondo and Mazzola, B. insularis Moris., B. hilarionis Post., and B. botteri Vis. At the same time T. Gladis and K. Hammer (9) considered them as subspecies of B. oleracea. It’s also unclear a taxonomic position of close taxa previously described as species, - B. alboglabra Bailey and the only wild species in the former USSR, Crimea endemic B. taurica Tzvel. Most of researchers assume B. alboglabra as subspecies of B. oleracea, and B. taurica - of B. incana (8, 10). The origin of Tournefort cabbage B. tournefortii Gouan. is still unknown (n = 10).
The section Rapa includes turnip B. rapa, rapeseed B. rape and mustard B. juncea. Within the species B. rapa L. there are important oilseeds, vegetables and forage leafy and root crops widespread in the world. The species is represented by such a huge variety of forms developed during evolution and domestication, that many intraspecific taxa were ranked as species in previous classifications (11, 12). G. Olsson (13) proved free crossability according to Bailey (11) and common karyotype, and considered them as a single species B. rapa. Recent classification of the species (8, 9, 14) is still preliminary due to the lack of knowledge about origin, formation and intraspecific relations, which necessitates the improvement based on molecular and genetic research that also can reveal the factors affecting frequency and range of genotypic variation and determining morphobiological diversity of Brassica species.
The whole tribe Brassiceae includes about 240 species among which are those of the genus Brassica. Using various methods of analysis, Brassica species were distinguished in two evolutionary branches: B. nigra (B genome) and B. rapa (A) / B. oleracea (C) genetically isolated about 7,9 million years ago. Genome A has developed from the earlier C genome 3,3-4,0 million years ago. Genomic reduction resulting from deletions was detected in triplicated blocks of B. oleracea, B. rapa and B. napus in comparison with the corresponding genomic regions of Arabidopsis thaliana (L.) Heynh. Approximately one third of Brassica’s genes are homologues to the relevant regions of the genome of Arabidopsis. However, the genome of Brassica contains much more transposon-like sequences and pseudo-genes.
Polyploidy, frequent in the genus Brassica, plays an important role in evolution of plant genomes (15). This fact probably can be explained by features of polyploidy: while a definite increase in the genome above the previous one, it also leads to certain structural and functional modifications, which, undoubtedly, are an important source of new formations (16, 17). A recent study has shown that that the genome of polyploids that are in a process of formation is generally unstable, dynamic and influenced by genetic and epigenetic regulation (18, 19). One of the factors determining such state may be mobile genetic elements, bringing to the host’s genome plasticity so necessary for adaptation to stressors.
Mobile genetic elements (MGE) first have been described 60 years ago in maize. Peculiarities of mobility (the presence or absence of an intermediate RNA during transposition) allow dividing them into two classes: Class I – retrotransposons (transposition is mediated by the formation of an intermediate RNA using reverse transcriptase for its conversion to DNA), Class II – DNA-transposons (translocated directly from DNA to DNA). This research focuses on MGE Class II. The first MGO studied in detail was Activator (Ac) (20, 21). Ac is a simply arranged and relatively small (total 4565 bp) autonomous mobile element containing TIRs (terminal inverted repeats) of 11 bp length whose the most distant nucleotides are non-complementary (22). The size, structural arrangement and sequence of TIR nucleotides of Ac element is significant similar to those of Tam3 in Antirrhinum majus (23), as well as P and hobo elements of Drosophila melanogaster (24, 25). Nucleotide composition of Ac is tendentious. Thus, the content of G + C in the regions of 240 bp in 5'- and 3'-ends are, respectively 45 and 40%. On the contrary, in a long non-translated sequence the proportion of G + C is 68%, and in the coding region of the element - 38% (26). Such nonuniform nucleotide composition of different segments of Ac indicates their different functions. In addition, numerous CpG motifs on the ends of Ac may mean that these sequences are protected by proteins (perhaps constantly), because many of them contain the site of transposase recognition (27, 28).
The next MGE Class II which controls the transposition of MGE family Mutator (Mu) in maize is MuDR. American scientists have identified two major MuDR-homologous transcripts, the presence of which correlates with activity of Mu elements. All active types of Mutator contain MuDR. For example, a typical Mu includes from 5 to 30 such elements (29), although only one MuDR element was found in some of them (30, 31). In single-copy lines, elimination of MuDR leads to inactivation of Mu element (30). When multicopy Mutator spontaneously lose activity, they still continue maintaining MuDR elements in the genomes (32, 33). Despite the presence of MuDR, inactivated lines don’t show the expression of MuDR transcripts (29). Therefore, such transcripts may encode proteins required for transposition of Mu elements (34). That’s why the authors have chosen MuDR to design one of primers’ classes for this research.
Similarity to Mutator transposases was observed in FHY3 (far-red elongated hypocotils 3) and FAR1 (far-red impaired response) – homologous proteins required for the response of controlled phytochrome A to far-red light part of the spectrum (λ = 730 nm) in Arabidopsis thaliana (35, 36). In the genome of arabidopsis there are12 additional associated genes FHY3/FAR1. Polypeptides of this family vary in length from 531 to 851 amino acids, and identical amino acids occupy from 12,0 to 82,4% length of the entire molecule. The study of available databases and phylogenetic relations has revealed the sequences similar to those of FHY3/FAR1 genes in different Angiosperms; they belong to several phylogenetic clusters interspersed with Mutator family encoding transposase and similar sequences (36).
CACTA elements is a group of Class II transposons, whose distinctive feature is the presence in their ends most remote from the center of terminal inverted repeats (TIR) usually of 10-28 bp length with a terminal conservative motif 5'-CACTA-3'. The interior sequences of CACTA are highly variable. In addition, these MGE before the insertion usually create a target duplication site of 3 bp. Sub-terminal repeats, as a rule, serve as binding sites for transposases and jointly with TIR act as cis-elements of transposons (37, 38). For the first time CACTA elements were found in 1953 in maize (39): En (Enhancer)-I (Inhibitor) and Spm (Suppressor-Mutator)-dspm. Today, similar elements are known in snapdragons (38), soybean (40), carrots (41), sorghum (42), petunia (43), peas (44), rice (45) and arabidopsis (46).
It should be noted that, despite bulk databases and many published works on this issue, the genus Brassica wasn’t among the plant species studied for the presence of the abovementioned MGE Class II, except for A. thaliana whose genome was completely sequenced (www.arabidopsis.org) including the elements Far1 and CACTA. However, Arabidopsis and Brassica genera belong to different tribes of the family.
Comparative analysis assisted by bioinformatics has shown that genomes of a model plant A. thaliana L. and cabbage B. oleracea L. (N = 9, genome SS), from which originated B. rapa, include identical MGE although in different ratios, due to many reasons including differences in genome size (47). This result reflects a high degree of genomic conservatism in the two species diverged 15-20 million years ago (48). The genome of arabidopsis contains all known types of MGE, and it is completely sequenced (49), while in Brassica it is still poorly studied at the molecular level in respect to these elements, only a few studies were performed focused on MGE Class I. At the same time, CACTA is the most common type of MGE in the genome of B. oleracea (47). CACTA transposon Bot1 in the genus Brassica passed through several cycles of amplification only in the genome of B. oleracea unlike B. rapa, which was the reason of subsequent divergence of these two genomes (50). These researchers have identified Bot1-segment specific to C genome. In the genomes of B. rapa and B. napus (AACC), they found BOT1-like CACTA elements, determined their size and identified TIR-sequence. During S-SAP analysis (sequence-specific amplification polymorphism) the authors of this work established the amount of Bot1-like CACTA elements in the genome of B. rapa; it was shown that these sequences can be used to design specific markers for revision of intraspecific classification of B. rapa. These experiments have also demonstrated the effect of joint use of two types of molecular markers created upon different groups of repetitive DNA sequences (tandemly repeated microsatellites and CACTA dispersed over the genome) in phylogenetic studies of the species B. rapa (51).
The purpose of this work was identification in the genomes of Brassica species of nucleotide sequences similar to mobile genetic elements Class II - Ac, MuDR, Far1 and CACTA. This knowledge would help better understanding of genetic bases of diversity, phylogenetic relations and evolution of species, which were considered by N.I. Vavilov as major tasks in genetic studies of collections of genetic resources.
Technique. The study was performed on genetically and morphologically diverse representatives of four sections within the subgenus Brassica; totally 45 samples from the world’s collection of Vavilov All-Russia Research Institute of Plant Industry (VIR) were examined, including field mustard Sinapis arvensis L., the carrier of genome S.
PCR was performed in a 25 ul reaction mixture, which contained Tris-HCl (66 mM, pH 8,4), ammonium sulphate (16 mM), magnesium chloride (2 mM), Tween 20 (0,1%), glycerol (7%), bovine serum albumin (100 ug/ml), dNTP (0,2 mM of each), primer (5 pM for each one), and 1,25 units Taq-polymerase. The reaction was conducted under following conditions: denaturation at 95 °C (1 min), elongation at 72 °C (1 min); annealing temperature and the number of amplification cycles were optimized for the experiment (52-54) (thermal cycler C1000, “BioRad”, USA); electrophoretic marker of molecular mass - 100 kb Ladder (“Gibco BRL”, UK; ”Fermentas”, Lithuania). Primers were homologous to fragments of sequences Ac and MuDR in the genome of maize, as well as Far1 and CACTA - in the genome of arabidopsis.
Primers for detection of Ас (respectively, forward and reverse):
E16 (5'-aat ccc gta ccg acc gtt atc-3') and E17 (5'-aga gag gca gag cag cgt tc-3'),
Е15 (5'-CAG GGA TGA AAG TAG GAT GGG A-3') and D3 (5'-GAA ACG GTC GGG AAA CTA GCT C-3'),
Е20 (5'-TGA CAG ATG AGC CTT GGT TGT AAT-3') and Е21 (5'-CGA ACG GGA TAA ATA CGG TAA TCG-3'),
D2 (5'-CCC GTC CGA TTT CGA CTT T-3') and Е22 (5'-TTA ACT TGC GGG ACG GAA AC-3');
primers for MuDR (respectively, forward and reverse):
D12 (5'-GGT TGA AGC AGT TAA GGC CTC A-3') and D13 (5'-ATG CTA TTC AAG AAA TGA GGA GGC-3'),
D14 (5'-TCA TCT ACG GAA GGG TTG TC-3') and D15 (5'-GGT CGT TTA TCT CTT CGA ACC TGT-3'),
Е4 (5'-CGC GGT ATT TGT TGC TGA GA-3') and Е5 (5'-TTG CTG AGA AGG AGG CCA AG-3'),
Е6 (5'-CCT CAT CGA ATG TGG TAT GGA TTA-3') and Е7 (5'-ttt ccc ata gct ctg gat ctt ctg-3');
primers for Far1 (respectively, forward and reverse):
D10 (5'-CAT GGC TTG CTG ATT CGT GAA-3') and D11 (5'-TTG GGC AGA ACT CAA ATG CTC-3'),
Е11 (5'-TCG GCA TGC TTT GAT GAT TC-3') and Е13 (5'-TGG TTG CAA GCT CTG TTG AGA-3');
primers for САСТА:
D5 (5'-CCC TTG GTT GTG CAT GAA GA-3') (forward),
D6 (5'-GCA CCT GAC GCA TCC AGA A-3') and D7 (5'-AGC AGT GCG GCT CTC ATA GG-3') (both reverse).
Results. The objects of study were fairly diverse genomes – representatives of four sections within the subgenus Brassica (of 5 known sections), including polyploids (Table 1).
The primers designed for PCR were homologous to 5'- and 3'-region of Ac sequence in the genome of maize 52), as well as to regions of MudrB area (53) of MuDR MGE in NCBI GenBank database. In addition, there were similarly developed the primers for genetic-molecular analysis of Far1 and CACTA sequences (54).

The study has revealed a significant interspecific polymorphism within the genus Brassica and the close genus Sinapis. It was totally identified 19 polymorphic fragments (Table 2) with the size of amplified DNA varying from 150 to 1000 bp. Among them there were fragments specific for all the natural species with B genome: a 150-bp amplicons obtained with primers E11-E14, and 200 bp – with D5-D6, as well as fragments unique to the only source diploid species (black mustard, B genome) of 270, 330 and 520 bp length obtained with primers E11-E14. At the same time, synthetic hexaploid B.composita (ABC genome) contained the only fragment present in all investigated samples of Brassica and in Sinapis arvensis (S genome) (200 bp, primers D5-D7).

One fragment (550 bp, primers D5-D7) was detected in the vast majority of species with C genome - cabbage, related wild Mediterranean species, and B. tournefortii (genome T), as well as in a half of rapeseed and rutabaga samples with AC genome. At the same time, this fragment can’t be considered specific to C genome, as it wasn’t found in Abyssinian cabbage – the carrier of BC genome. However, this fact confirms the known idea about a closeness of genomes A and C, and a distance from them of B genome. In some samples (for example, kohlrabi from Pakistan) there were detected rare alleles, but it’s yet unclear whether they were specific to the named botanical taxa or not. The presence of a common fragment (370 bp, primers E16-E17) in Mediterranean species B. incana and some samples of cultivated cabbage and rapeseed may indicate the participation of B. incana in their formation, possibly through introgression of the genetic material into ancient forms of B. oleracea.
According to the authors’ research, Tournefort cabbage can be assumed as a separate species that supposedly originated by one of two ways: the introgression of genetic material from the complex B. oleracea / B. rapa genome to black mustard B. nigra, or vice versa. The studied Tournefort cabbage didn’t carry any fragments peculiar to the species with B genome, but it contained an amplicon observed in many C genome species. No marker fragments of A genome were revealed. One detected fragment was specific only to field mustard (280 bp, primers D5-D6). On electrophoretic profiles the amplified fragments were often represented by one, rarely – two bands, and even more rarely – by three or four bands,  i.e. a low polymorphism.
Thus, results of this research indicate the presence in the genomes of all studied Brassica species of sequences similar to MGE Class II (Ac, MuDR, Far1, CACTA).
As already noted, the data about transposon-like sequences and pseudo-genes in Brassica is incomplete, and in some ways contradictory. According to recent phylogenetic studies, six species of the genus Brassica are carriers of clusters Ty1/copia and LINE-like elements, and the third detected cluster was subdivided to Ty3/gypsy, Attila and virus-like branches, which confirms that sub-branches within this group can be seen as gypsy-like elements of plants (55). Dendrograms didn’t show the branches correlated with known genomic relations of Brassica species (probably, because the elements of studied MGE families were present in a common ancestor Brassica unlike other repetitive sequences). Possibly, it was a convergent evolution or horizontal gene transfer. The findings of K. Alix et al. about sequences of retroelements (55) didn’t allow any conclusions on phylogeny of the genus, although Southern-hybridization has confirmed amplification in some retroelements of certain species of the subfamily. Other authors (56) performed identification and characterization of major repeats in centromeric and pericentromeric heterochromatin in the species B. rapa. They identified three copies of the centromere-specific retrotransposon Brassica (CRB), variable-pericentromeric retrotransposons (PCRBr), as well as 24 copies of centromeric repeats (CentBr) of 176 bp. They also revealed a mosaic structure consisting of nine PCRBr and wide block of tandem repeats (TR238). CRB was determined as the major component of all centromeres in diploid and allotetraploid Brassica species. However, centromeric repeats (CentBr) weren’t found in the most distant of studied species – B. nigra. It was shown that PCRBr and TR238 are major components of pericentromeric heterochromatin blocks of four chromosomes in B. rapa. These repetitive elements were assumed as specific to A genome as they weren’t detected in B. oleracea and B. nigra.
However, all the abovementioned MGE do not belong to the object of this study – MGE Class II, which suggests the necessity of exploring this unclear issue. Along with it, the authors findings allow to conclude that amplified sequences of MGE Class II known as diverse and prevalent in the genomes of Brassica species are suitable for design of molecular markers, particularly S-SAP (51); these markers may be used directly for the analysis of genetic diversity in the genus Brassica, genome mapping and phylogenetic analysis. Unfortunately, a low polymorphism of detected PCR amplicons doesn’t allow to develop a phylogenetic tree of botanical and taxonomic relations of cultivated and wild Brassica species. It can be a task for future studies to determine the degree of homology of PCR-amplified sequences with known sequences of MGE Class II, including those used for design of primers applied by the authors.
So, this work shows the results of PCR analysis and estimation of polymorphism of the markers based on Ac, MuDR, Far1, and CACTA elements in samples of different species belonging to the family Brassicaceae L. from the world’s plant collection of VIR, in connection with a discussion about their phylogeny, evolution and polyploidy. The authors have reviewed genetic instability of genomes in the genus Brassica and prospects for the use of primary nucleotide sequences similar to those of mobile genetic elements (MGE) Class II (Ac, MuDR, Far1, and CACTA) in design of molecular markers for analysis of genetic diversity within a model family Brassicaceae L. This approach can be also applied to other families for better understanding of the nature of genetic variation, molecular mechanisms of species’ evolution and their phylogenetic relationship.

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