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Integration of Brassica A genome genetic linkage map between Brassica napus and B. rapa Keita Suwabe, Colin Morgan, and Ian Bancroft
Abstract: An integrated linkage map between B. napus and B. rapa was constructed based on a total of 44 common markers comprising 41 SSR (33 BRMS, 6 Saskatoon, and 2 BBSRC) and 3 SNP/indel markers. Between 3 and 7 common markers were mapped onto each of the linkage groups A1 to A10. The position and order of most common markers revealed a high level of colinearity between species, although two small regions on A4, A5, and A10 revealed apparent local inversions between them. These results indicate that the A genome of Brassica has retained a high degree of colinearity between species, despite each species having evolved independently after the integration of the A and C genomes in the amphidiploid state. Our results provide a genetic integration of the Brassica A genome between B. napus and B. rapa. As the analysis employed sequence-based molecular markers, the information will accelerate the exploitation of the B. rapa genome sequence for the improvement of oilseed rape. Key words: linkage map, Brassica napus, Brassica rapa, microsatellite, SSRs. Re´sume´ : Une carte ge´ne´tique inte´gre´e combinant celles du B. napus et du B. rapa a e´te´ produite a` l’aide de 44 marqueurs communs comprenant 41 SSR (33 BRMS, 6 Saskatoon et 2 BBSRC) et 3 indels. Entre 3 et 7 marqueurs communs ont e´te´ assigne´s a` chacun des groupes de liaison A1 a` A10. La position et l’ordre de la plupart des marqueurs communs ont re´ve´le´ un fort degre´ de coline´arite´ entre les espe`ces, bien que deux petites re´gions situe´es sur A4, A5 et A10 aient affiche´ une apparente inversion locale. Ces re´sultats indiquent que le ge´nome A du genre Brassica a conserve´ une grande coline´arite´ entre les espe`ces, malgre´ le fait que chacune des espe`ces ait e´volue´ inde´pendamment suite a` l’inte´gration des ge´nomes A et C dans l’e´tat amphidiploı¨de. Les re´sultats de ces travaux contribuent une inte´gration ge´ne´tique du ge´nome A entre le B. napus et le B. rapa. Comme l’analyse faisait appel a` des marqueurs mole´culaires de se´quence connue, cette information va permettre d’acce´le´rer l’exploitation du ge´nome du B. rapa pour des fins d’ame´lioration ge´ne´tique du colza. Mots-cle´s : carte ge´ne´tique, Brassica napus, Brassica rapa, microsatellite, SSR. [Traduit par la Re´daction]
Introduction Among the Brassicaceae, the genus Brassica is the most widely cultivated in the world. It includes various important agronomical crops such as oilseed rape, cabbage, broccoli, Chinese cabbage, black mustard, and other leafy vegetables. The extensive morphological variation in traits such as leaf, head, and root size and shape has allowed further selection, which has resulted in a wide range of forms available for cultivation for human consumption. Furthermore, the genetic relationships of Brassica have been well studied and were first described in U’s triangle (U 1935). The genomes of 3 diploid species, B. rapa, B. nigra, and B. oleracea, have been designated as A, B, and C, respectively, while those of the amphidiploids B. juncea, B. napus, and B. carinata have been designated as AB, AC, and BC, respectively. Although these species have complicated genomes, the relationship of their genomes includes a high degree of colinearity between Received 25 May 2007. Accepted 17 November 2007. Published on the NRC Research Press Web site at genome.nrc.ca on 6 February 2008. Corresponding Editor: T. Schwarzacher. K. Suwabe, C. Morgan,1 and I. Bancroft. Department of Crop Genetics, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK. 1Corresponding
author (e-mail:
[email protected]).
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species. Because such genetic and morphological variation has occurred as the result of a natural hybridization of species, the remarkable diversity of Brassica is an excellent example for understanding how genetic and morphological variation have developed during the evolution of plant genomes. The international research community has launched the Multinational Brassica Genome Project, one aim of which is to sequence the genome of one Brassica species. The genome chosen, on the basis that it is the smallest Brassica genome, is that of Chinese cabbage, B. rapa. The cultivated Brassica species represent the group of crops most closely related to Arabidopsis thaliana, the model plant for dicots. Recent studies of this species have yielded new insights into plant genetics and genomics. A pattern of segmental chromosomal colinearity has been identified between Brassica and Arabidopsis (O’Neill and Bancroft 2000; Ryder et al. 2001). Physically and functionally conserved gene(s), known as orthologues and paralogues, have also been identified. The level of genomic synteny between these genera provides a good opportunity to study how genetic and morphological variation have developed during the evolution of the plant genome, including the endurance of certain genetic structures in Arabidopsis and related Brassica species. Such investigation may lead to a better understanding of plant genetics, including molecular biological and physiological aspects that have emerged via evolution.
doi:10.1139/G07-113
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Comparative genome analysis has been carried out in the Brassicaceae at two levels: first, at the whole-genome level, by the genetic mapping of a small number of molecular markers across multiple genomes (Kowalski et al. 1994; Lagercrantz and Lydiate 1996; Parkin et al. 2003); and second, at the microstructure level, in small segments of the genome (O’Neill and Bancroft 2000; Rana et al. 2004). Partial BAC-based physical maps have also been constructed for B. oleracea and B. rapa, and these have been linked to the Arabidopsis genome sequence (http://brassica.bbsrc.ac.uk/ IGF/). However, the number of comparatively mapped genetic markers that are positioned on communal maps is too low to enable detailed map integration between species, only a tiny fraction of each Brassica genome has been analysed at the level of microstructure, and the physical maps have not been integrated with genetic maps. Thus considerable integration is required to take full advantage of the opportunities to take comparative genetic and genomic approaches to solving biological problems in the Brassicaceae. In this paper, we aim to integrate genetic and genomic resources across the Brassicaceae. We focus on the integration of the Brassica A genome in the most economically important Brassica crop species, oilseed rape (B. napus), and the species adopted for genome sequencing, B. rapa. This will contribute to an understanding of the genetic and chromosomal relationship between them and open new avenues for research into the improvement of Brassica crops. It will also yield novel insights into varied research topics such as the evolution of the plant genome including polyploidy, genomic rearrangement, and synteny.
Materials and methods Plant materials A doubled haploid population, TNDH (Qiu et al. 2006), and an F2 population, AG (Suwabe et al. 2006), were used for map construction on B. napus (L.) and B. rapa (L.), respectively. The TNDH population, consisting of 188 lines, was developed from a cross between a European cultivar, Tapidor, and a Chinese cultivar, Ningyou 7. The AG population, consisting of 94 lines, was developed from a cross between the Chinese cabbage parental lines Nou 7 (A9709) and G004. Nomenclature of linkage groups During their meeting on 14 January 2007, the Steering Committee for the Multinational Brassica Genome Project recommended a change to the nomenclature for Brassica linkage groups in order to deal with problems with incorporating the B genome species into the previous convention. Consequently, we refer to linkage groups A1 to A10, which correspond to R1 to R10, respectively, in B. rapa and N1 to N10, respectively, in B. napus in the previous system. In the new nomenclature, O1 to O9 in B. oleracea and N11 to N19 in B. napus become C1 to C9, respectively, and the B genome linkage groups, originating from B. nigra, are named B1 to B8. In our report, linkage groups derived by mapping in
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different populations are identified by suffixes derived from the initial letters of the names of the parental lines. For example, linkage group A1 constructed using the population derived by crossing B. napus cultivars Tapidor and Ningyou 7 is named A1TN. Molecular markers New SSR markers were obtained from three sources: John Innes Centre, UK (designated Na. . . or Ra. . .; Lowe et al. 2002; http://www.brassica.bbsrc.ac.uk/BrassicaDB/); National Institute of Vegetable and Tea Science, Japan (designated BRMS. . .; http://vegetea.naro.affrc.go.jp); and Agriculture and Agri-Food Canada (designated sN. . ., sR. . ., or sS. . .; http://brassica.agr.gc.ca/index_e.shtml). Additionally, because there was only one SSR marker, BRMS-247, that mapped to A9 in both TNDH and AG (see Results), 7 SNP/indel markers (designated by the letters IGF; Qiu et al. 2006; http://www.brassica.bbsrc.ac.uk/ IMSORB/) that had been previously mapped to TNDH A9 were screened for polymorphisms in B. rapa, and 3 of these were used for constructing the map of A9AG. Parental allele sizes and genotyping data for the new markers are given as supplementary data (Tables S1 and S2, respectively2). Genotyping Genotyping of the SSR markers in the populations was carried out using PCR with fluorescent dyes, performed according to Schuelke (2000) with some modifications. The M13(–21) universal sequence (18 bp) was fused to the 5’ end of the original forward primer, and the M13(–21) universal primer was labeled with one of the following fluorescent dyes: 6-FAM, VIC, NED, or PET (Applied Biosystems, California, USA). PCRs were performed in a 6.25 mL reaction volume containing 10 ng of template DNA, 4.7 mmol/L of fluorescently labeled M13(–21) universal primer and reverse primer, 0.3 mmol/L of forward primer, and 3.125 mL of HotStarTaq Plus Master Mix (QIAGEN, California, USA). Conditions for the PCR were as follows: initial denaturing and activation of Taq polymerase were carried out at 95 8C for 15 min, followed by 35 cycles at 95 8C for 1 min, 50 8C (slope of 0.5 8C/s) for 1 min, 72 8C (slope of 0.5 8C/s) for 1 min, and a final extension at 72 8C for 10 min. Subsequently, 1 mL of 100 times diluted PCR product was added to 8.9 mL of Hi-DiTM Formamide and 0.1 mL of GeneScanTM 500 LIZTM Size Standard (Applied Biosystems) and applied to an ABI 3730 DNA Analyzer (Applied Biosystems). Data were analysed using ABI GeneMapper1 software. The method for SNP/indel analysis for IGF markers is described by Qiu et al. (2006). Sequence analysis was used to genotype these markers except one SNP/indel marker, IGF5706e, which showed dominant genetic inheritance in the AG population; this marker amplified only in G004 and was genotyped on an agarose gel. Linkage map construction and integration Construction of the linkage maps was carried out using
2 Supplementary
data for this article are available on the journal Web site (http://genome.nrc.ca) or may be purchased from the Depository of Unpublished Data, Document Delivery, CISTI, National Research Council Canada, Building M-55, 1200 Montreal Road, Ottawa, ON K1A 0R6, Canada. DUD 3707. For more information on obtaining material refer to http://cisti-icist.nrc-cnrc.gc.ca/irm/unpub_e.shtml. #
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JoinMap 3.0 (Van Ooijen and Voorrips 2001). The threshold for goodness of fit was set to £5.0, a recombination frequency of
