^^^^^^^^^^^^ ЭКСПЕРИМЕНТАЛЬНЫЕ
СТАТЬИ
581.1
Molecular Characterization of the Flowering Time Gene FRIGIDA in Brassica Genomes A and C1 © 2013 O. A. Fadina, A. A. Pankin, E. E. Khavkin
Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, Moscow
Received June 04, 2012
An important determinant of flowering time variation in Arabidopsis, the FRIGIDA (FRI) gene has not been until recently investigated in economically important Brassica species. In diploid Brassica species, this gene exists as two paralogous loci on chromosomes A3 and A4 (B. rapa; A genome), and C3 and C9 (B. oleracea; C genome). Each locus is represented by several genome-specific alleles, which are discerned primarily by polymorphisms in C- and especially N-terminal regions. Locus- and genome-specific sequences of two FRI paralogues are conserved almost completely in the subgenomes A and C of tetraploid B. napus. The phyloge-netic analysis of available FRI sequences presumes that the duplication of FRI loci preceded speciation in the genus Brassica.
Keywords: Arabidopsis - Brassica napus - B. oleracea - B. rapa - FRIGIDA - vernalization - gene divergence -allelic polymorphism
УДК
DOI: 10.7868/S0015330313020073
INTRODUCTION
Several environmental cues, including ambient temperature, regulate floral transition in higher plants. Genes involved in cold induction of flowering (vernalization) are best investigated in Arabidopsis and temperate cereals (for recent reviews, see [1-4]). In Arabidopsis, transition to flowering is repressed early in development by the FLOWERING LOCUS C (FLC) complex, and FRIGIDA (FRI) upregulates this floral repression through a co-transcriptional mechanism [5, 6]. Later, following exposure to cold, the epigenetic changes in FLC chromatin structure reduce FLC expression releasing floral transition [2].
Greenhouse studies with diverse seed-bank lines and field-collected ecotypes of Arabidopsis thaliana revealed extensive allelic variation at FRI, including numerous loss-of-function mutations related to the changes in time to flower across environments. These studies established that flowering time, especially in early-flowering ecotypes, depended on the interplay of strong and weak alleles of FLC and FRI [7-11]. In particular, two FRI alleles, friCol and friLer, originally defined in the common laboratory lines Columbia and Landsberg erecta, were shown to account for a large
1 This text was submitted by the authors in English.
Corresponding author. Khavkin E.E. Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences. Timiryazevskaya ul., 42, Moscow, 127550 Russia. Fax. +7 (499) 977-09-47; e-mail. emil@iab.ac.ru
proportion of polymorphisms found in Europe. Similar to the natural variation in FRI reportedly mediating latitudinal and altitudinal clines in flowering time in A. thaliana [8, 10, 12-14]; the FRI variation was also reported in A. lyrata [15]. These authors explored FRI allelic polymorphism in a South-North gradient across nine populations of A. lyrata from Europe and North America and reported two FRI alleles, which conferred a 15-day difference in flowering time as confirmed by transformation of A. thaliana. On the other hand, several populations of A. lyrata harbored simultaneously both late- and early-flowering FRI alleles, and the intermediate frequencies presumably maintained by balancing selection could not explain the observed variation in flowering time [15]. Several authors reported substantial variation in flowering time independent of FRI and FLC in A. thaliana accessions with and without functional alleles of these genes [16, 17]. Field-plot experiments also demonstrated that in addition to FRI polymorphisms and FLC-FRI allelic interactions, the combined effects of seasonally varying factors (e.g., photoperiod) and other, often pleiotropic, loci were responsible for the variation in flowering time [18-21].
In the hybrids between A. thaliana lines carrying various combinations of strong and weak FRI and FLC alleles, these genes significantly contributed to heterosis for several flowering-time traits [22]. The interaction of FRI and FLC alleles was shown to mediate
flowering time variations in synthetic Arabidopsis allopolyploids [23].
The cultivated species of Brassica (diploid B. rapa, genome A; diploid B. oleracea, genome C; allotetrap-loid B. napus, genome AC) include summer annual, winter annual, and biennial life forms highly polymorphic as regards timing of flowering and requirements for vernalization. Therefore, one can presume that Brassica species contain homologues of the Arabidop-sis FRI gene, and these homologues may similarly participate in the vernalization pathway of flowering control and adaptation to diverse climate conditions.
We identified and structurally characterized several FRI homologues in the A and C Brassica genomes by mining the genetic databases and by cloning from ge-nomic DNA. Our data presumed that, in contrast to
A. thaliana [7], Brassica species comprised two FRIlo-ci, which probably evolved long before speciation in Brassica [24]. Four FRI homologues were described in
B. napus [25]; these authors demonstrated, by linkage analysis and association mapping, that at least one of these genes corresponded to a major cluster of quantitative trait loci (QTL) for flowering time and significantly affected flowering time variation. Recently Irwin et al. [26] reported two FRI paralogues mapped and characterized in B. oleracea and screened a wide range of B. oleracea cultivars for two functional alleles, which complemented FRI alleles in A. thaliana mutants.
On the whole, present comparison of FRI homologues from three Brassica crops supports the existence in diploid B. rapa and B. oleracea of two expressible FRI loci represented by several alleles characteristic of the A and C genomes. Locus- and genome-specific FRI paralogues are retained in the subge-nomes A and C of tetraploid B. napus. These results will help reveal global patterns of FRI variation and assist in future studies on early evolution of FRI loci in Brassica ancestors and the functional significance of two FRI paralogues in the cultivated Brassica species.
MATERIALS AND METHODS
Seeds of Brassica species were obtained from the Centre for Genetic Resources, the Netherlands (CGN) and the Warwick HRI Genetic Resources Unit, United Kingdom (GK). Seeds were germinated on moist filter paper for two days, and the seedlings were transferred into a standard soil mix. Plants were grown for two-three weeks at room temperature and humidity under constant illumination from OSRAM Circolux EL (24W) lamps.
Genomic DNA was isolated from young leaves using the AxyPrep™ Multisource Genomic DNA Mini-prep Kit ("Axygen Biosciences", Union City, United
States). DNA samples were quantified at 260 nm with a NanoPhotometer P 300 ("IMPLEN", Germany). The PCR mix contained 10x PCR buffer, 2 mM MgCl2, 100 ng of genomic DNA, 0.2 mM dNTP, 1 mM forward and 1 mM reverse primers, and 1 U of Taq DNA polymerase ("Fermentas", Germany) or 2.5 U Pfu polymerase ("Fermentas"). PCR was run using the following program: one cycle of 30 s at 94°C; four cycles of 30 s at 94°C, 30 s at 65°C per cycle, 3 min 30 s at 72°C; 30 cycles of 30 s at 94°C, 30 s at 61°C, 3 min 30 s at 72°C; one cycle of15 min at 72°C. PCR products were separated by electrophoresis in 0.8% (w/v) agarose in 1x TAE buffer for 90 min at 6 V/cm and visualized under UV after staining with ethidium bromide. Following electrophoretic separation, PCR-amplified DNA fragments were purified using QIAquick Gel Extraction Kit ("Qiagen", Germany) as recommended by the manufacturer. The sticky-end fragments were ligated into the pTZ57R/T vector; blunt-ended fragments, in the pJET1.2/blunt vector by standard protocols using the InsTAcloneTM and CloneJET™ PCR Cloning Kits ("Fermentas"). Inserts were multiplied in Escherichia coli strain GM 109 ("Promega", United States) and sequenced three to five clones per genotype using a 3130xl Genetic analyzer ("Applied Biosystems", United States). The chromato-grams were visually inspected using Chromas Lite 2.0 (www.technelysium.com.au/chromas_lite.html), and hand-edited sequences were submitted to GenBank (http://www.ncbi.nlm.nih.gov) as accession nos. JN015481, JN015482, JN882592-JN882595, and JN989363.
Homologous sequences were extracted from NCBI GenBank release 161 (nuccore, EST, GSS, and SRA databases), BRAD database (http://brassicadb. org/brad; B. rapa BGI scaffolds v. 1.0), and INRA Brassica.FR (http://www.brassica.fr; 454-reads database) using BLASTN implemented at the corresponding resources. The latest database search was run on May 22, 2012. For multiple sequence alignment, we used ClustalW2 (http://www.ebi.ac.uk/Tools/ msa/clustalw2). Sequences of FRI homeologues were assembled using a combination of the Martinez and Needleman-Wunsch algorithms (SeqMan, Lasergene 7.0; http://www.dnastar.com). The genome-specific insertions/deletions and nucleotide substitutions thus disclosed were further employed for designing genome- and locus-specific primers. Specific PCR primers were selected manually and optimized using the Oligonucleotide Properties Calculator (http:// www.basic.northwestern.edu/biotools) for the following parameters: annealing temperature, GC content, potential hairpins, and self-complementarity. Optimized primers were verified for possible non-specific annealing using BLAST. All primers were synthesized by "Syntol", Moscow (www.syntol.ru).
MOLECULAR CHARACTERIZATION OF THE FLOWERING TIME GENE FRIGIDA F R
(a)
907
ATG
(b)
5'UTR
^BraA.
FRI.b
907
ATG
1203 1375 _ 1468
Donor splice site "GT"
1203 1296__1391
Acceptor splice site "GC"
2178
2293 3'UTR
TAG R
2084 2069 3'UTR
TAA
(c)
E
ATG
■BraA.FRI.b_putative CDS 907 1203
1321 ■TAG
1296 * 1321
F
Fig. 1. Structures of the loci FRI.a and FRI.b in B. rapa.
Arrows F and R indicate the positions of for
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