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ARTICLE Exploring the origin of the D genome of oat by fluorescence in situ hybridization
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Xiaomei Luo, Haiqin Zhang, Houyang Kang, Xing Fan, Yi Wang, Lina Sha, and Yonghong Zhou
Abstract: Further understanding of the origin of cultivated oat would accelerate its genetic improvement. In particular, it would be useful to clarify which diploid progenitor contributed the D genome of this allohexaploid species. In this study, we demonstrate that the landmarks produced by fluorescence in situ hybridization (FISH) of species of Avena using probes derived from Avena sativa can be used to explore the origin of the D genome. Selected sets of probes were hybridized in several sequential experiments performed on exactly the same chromosome spreads, with multiple probes of cytological preparations. Probes pITS and A3-19 showed there might be a similar distribution of pITS between the Ac and D genomes. These results indicated that the Ac genome is closely related to the D genome, and that Avena canariensis (AcAc) could be the D-genome donor of cultivated oat. Key words: D genome, origin, hexaploid oat, Avena canariensis, FISH. Résumé : Une connaissance plus approfondie de l’origine de l’avoine cultivée permettrait d’accélérer les travaux en amélioration génétique. En particulier, il serait utile de déterminer laquelle des espèces diploïdes ancestrales a contribué le génome D chez cette espèce allohexaploïde. Dans ce travail, les auteurs montrent que les bandes obtenues en hybridation in situ en fluorescence (FISH) chez des espèces d’Avena, obtenues en employant des sondes provenant de l’Avena sativa, permettent d’explorer les origines du génome D. Des séries de sondes choisies ont été hybridées de manière séquentielle sur les mêmes étalements chromosomiques et plusieurs préparations cytologiques ayant été examinées. Les sondes pITS et A3-19 ont montré qu’il y aurait une distribution similaire de pITS chez les génomes Ac et D. Ces résultats indiquent que le génome Ac serait très apparenté au génome D et que l’Avena canariensis (AcAc) pourrait s’avérer la source du génome D chez l’avoine cultivée. [Traduit par la Rédaction] Mots-clés : génome D, origine, avoine hexaploïde, Avena canariensis, FISH.
Introduction Current information suggests that Avena sativa L. (2n = 6x = AACCDD) originated from a hybridization between an unknown diploid with D genome and a tetraploid with AC genomes (Rajhathy and Morrison 1959; Thomas 1992; Loskutov 2008). Rajhathy (1966) proposed that Avena ventricosa Bal. ex Coss. was the possible diploid progenitor of the C genome of the hexaploid species. On the basis of morphology, karyotypes, and geographical distribution, Baum et al. (1973) tentatively proposed that the diploid species Avena canariensis Baum, Raj. et Samp. was the probable progenitor of the A genome of the hexaploid species. There is no documented identification of the origin of the third D genome of the hexaploids, and some authors consider that this progenitor may no longer exist (Linares et al. 1998; Loskutov 2008). However, the possible diploid progenitors with A and C genomes did not take part in the origin of the tetraploid species whose genomes are labeled as AC, and nor in the origin of the hexaploid oat (Ladizinsky 2012). Based on the degree of chromosome pairing in F1 hybrids, the A genome can be classified into subgroups and these are denoted with a subscript: As, Ap, Al, Ac, Ad (Rajhathy 1961; Thomas 1992). Differences among the A genome of diploid species have been further characterized using cytological studies (Rajhathy and Thomas 1974; Sánchez and Fominaya 1989; Thomas 1992). The A genome has also been described in relation to genomic affinities
with the D genome in hexaploid oat. Molecular investigations suggest that both the A and D genome of A. sativa are highly homologous to Avena strigosa Schreb. (Chen and Armstrong 1994; Jellen et al. 1994). Attempts to differentiate between the A and D genomes by genomic in situ hybridization were previously not successful (Leggett and Markhand 1995). These reports, together with the absence of D-genome diploid species, formerly suggested that an A-genome diploid species could be the donor of both the A and D genome of the hexaploid oat. More recently, Sanz et al. (2010) were able to distinguish A- versus D-genome chromosomes by subtracting those with affinity to specific A-genome probes. However, to date, the D genome has still not been identified by direct hybridization to D-genome probes. Hence, the donor of the D genome is still unknown and controversial. In the present study, probes A3-19, pITS, pAs120a, and pAm1 were used to explore the origin of the D genome by fluorescence in situ hybridization (FISH).
Materials and methods Five species of Avena (Table 1) were used to analyze mitotic metaphases. Seeds were germinated on sterile wet filter paper. When 1.5–2.0 cm long, root tips were harvested to make cytological preparations for FISH. Using common procedures (e.g., Shuai 2003), root tips were pretreated in ice water for 24–36 h, fixed in Carnoy’s Solution I (3:1 mix of 95% ethanol – glacial acetic acid),
Received 20 March 2014. Accepted 10 October 2014. Corresponding Editor: G. Jenkins. X. Luo. Triticeae Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China; College of Forestry, Sichuan Agricultural University, Yaan 625014, Sichuan, China. H. Zhang, H. Kang, X. Fan, Y. Wang, L. Sha, and Y. Zhou. Triticeae Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China. Corresponding author: Yonghong Zhou (e-mail: [email protected]). Genome 57: 469–472 (2014) dx.doi.org/10.1139/gen-2014-0048
Published at www.nrcresearchpress.com/gen on 14 October 2014.
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Genome Vol. 57, 2014
Table 1. Seeds materials of Avena used in this study. Species
Genome
Accession
Origin or source
Avena canariensis Baum, Raj. et Samp. Avena longiglumis Dur. Avena maroccana Gdgr. Avena vaviloviana (Malz.) Mordv. Avena sativa L.
AcAc AlAl AACC AABB AACCDD
CN 26195 Ciav 9087 Ciav 8330 PI 412766 CN 64226
Canary Islands, Spain Oran, Algeria Morocco Shewa, Ethiopia Rio Grande do Sul, Brazil
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Fig. 1. FISH of mitotic metaphase plates of (a) Avena canariensis and (b) Avena sativa. (a) FISH of digoxigenin-labelled pITS (green) probe. (b) FISH with digoxigenin-labelled pITS (green) and biotin-labelled pAm1 (red) probes. pITS signals are indicated by arrows in both images. All chromosomes were counterstained with DAPI. Scale bar = 5 m.
then squashed in a drop of 45% acetic acid. After freezing with liquid nitrogen, the slides with the squashes were uncovered, air dried, and stored at −20 °C until use. FISH with multiple probes was performed as described by Hao et al. (2011). Four DNA probes were used in this study: (i) pAm1 (Solano et al. 1992; Fominaya et al. 1995) was used to identify the C genome of oat; (ii) pAs120a (Linares et al. 1998) was used to identify the A genome of oat; (iii) pITS (Sanz et al. 2010) was used to locate to the telomeres of 19A, 20D, and 21D of oat; (iv) A3-19 was tested as a novel probe, which was located to the telomere or centromere of oat (Tinker et al. 2009). Clones containing these fragments in pGEM-4Z were grown in LB liquid medium and the plasmids isolated using the High-Speed Plasmid Mini Kit protocol from IBI Scientific (Peosta, Iowa, USA, Cat. No.IB47102). The probes were labeled with digoxigenin-11-dUTP (Roche Diagnostics Gmbh, Mannheim, Germany, REF 11745816910) or biotin-16-dUTP (Roche Diagnostics Gmbh, Mannheim, Germany REF 11745824910) according to the manufacturer’s instructions. Streptavidin-Cy3 (SIGMA ALDRICH CHEMIE Gmbh, Steinheim, Germany, Pcod 1001238444) was used to detect the biotinylated probe and anti-digoxigenin-fluorescein (Roche Diagnostics Gmbh, Mannheim, Germany, REF 11207741910) was used to detect the digoxigenin probe. The preparations were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc., Burlingame, Calif., USA). Slides were examined using an Olympus BX-51 microscope coupled to a Photometric SenSys Olympus DP70 CCD camera (Olympus, Japan). Raw images were processed using Photoshop ver. 7.1 (Adobe Systems Inc., San Jose, Calif., USA).
Results FISH of mitotic metaphase plates of A. canariensis and A. sativa are illustrated in Figs. 1a and 1b, respectively. The probe of pITS was visible in Fig. 1a (green), while the probe of pAm1 was visible in Fig. 1b (red). The results were as follows: (i) C-genome chromosomes from A. sativa were labelled by probe pAm1. (ii) pITS signals were located on interstitial regions of the short arms of the chromosomes. (iii) Six signals in AD-genome chromosomes from A. sativa were labelled by probe pITS, while four signals in A. canariensis were labelled by probe pITS. Together with previous results showing two signals in the A genome and four in the D genome from A. sativa
labelled by probe pITS, this could indicate a similar distribution of pITS between the Ac and D genomes. FISH of mitotic metaphase plates of five species of Avena are shown in Fig. 2. The probe from A3-19 is visible in Figs. 2a–2g (arrows). The probe from pAm1 is visible in Fig. 2f (green), while the probe from pAs120a is visible in Figs. 2e (red) and 2g (green). The A3-19 signal is located on interstitial regions of the short arms of the chromosomes (arrows). Four signals from probe A3-19 were observed in the Al genome (Fig. 2a, A. longiglumis), as well as in the A/C genome (Fig. 2b, A. maroccana). Eight signals in the A/B genome were labelled by probe A3-19 (Fig. 2c, A. vaviloviana), whereas two signals in the Ac genome were labelled by probe A3-19 (Fig. 2d, A. canariensis). Four signals in the A genome and two signals in the C/D genome were labelled by probe A3-19 (Figs. 2e–2g, A. sativa), while six signals in the A/D genome were labelled by probe A3-19 (Fig. 2f, A. sativa). A summary of these results is as follows: (i) Four signals in the A genome and in the B genome were both labelled by probe A3-19. (ii) Two signals in the Ac genome were labelled by probe A3-19. (iii) Two signals in the D genome were also labelled by probe A3-19. These results suggest that the distribution of A3-19 on the Ac genome is different from that in the Al genome, but similar to the D genome. In addition, it suggests that the A genome is closely related to the B genome. Taken together, the hybridization pattern for probes from pITS, A3-19, pAm1, and pAs120a showed a similar distribution between the Ac and D genomes. These results indicate that the Ac genome is closely related to the D genome, and suggest that A. canariensis (AcAc) could be the D genome donor of cultivated oat.
Discussion These results demonstrate that the novel probe A3-19 originating from A. sativa and other probes (pAm1, pAs120a, and pITS) reveal a similar pattern of affinity to both the Ac and D genomes. These results provide a better basis for exploration of the D genome origin in hexaploid oat. Further discussion here will focus on the following points: (i) the relationship between the Ac and D genomes, and (ii) the D-genome diploid progenitor of cultivated oat. Published by NRC Research Press
Luo et al.
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Fig. 2. FISH of mitotic metaphase plates of five species of Avena: (a) Avena longiglumis, (b) Avena maroccana, (c) Avena vaviloviana, (d) Avena canariensis (CN 26195), (e–g) Avena sativa. (a–d) FISH of biotin-labelled A3-19 (red) probe. (e) FISH with digoxigenin-labelled A3-19 (green) and biotin-labelled pAs120a (red) probes. (f) FISH with digoxigenin-labelled pAm1 (green) and biotin-labelled A3-19 (red) probes. (g) FISH with digoxigenin-labelled pAs120a (green) and biotin-labelled A3-19 (red) probes. A3-19 signals are indicated by arrows in all images. All chromosomes were counterstained with DAPI. Scale bar = 5 m.
Relationship between the Ac and D genomes The evolution of nuclear genomes in oat has been an intricate process, involving divergence at the diploid level from a common diploid ancestor, followed by convergence, followed by divergence again at the hexaploid level (Thomas 1992). Known examples of the A genome (As, Ap, Al, Ac, Ad) demonstrate this diploid divergence. Among these genomes, the As genome is considered to be the more primitive genome, while the others are considered to be derivatives (Thomas 1992). The Ap genome is closest to the Al genome (Thomas and Leggett 1974), while the Ac genome is closest to the Ad genome (Leggett 1984). The D genome has remained a mystery as it has yet to be observed in a diploid species. However, these results suggest that the D genome may also be derived from the A genome, and that it is most closely related to the Ac genome. D-genome diploid progenitor of cultivated oat Avena canariensis is the only diploid oat species with denticulate lemma tips, which is a morphological feature in common with hexaploid oat (Baum et al. 1973). This species has remarkable morphological variations, distinctive ecotypes, and polymorphic protein and isozyme electrophoretic patterns (Morikawa and Leggett 1996). As such, it has previously been identified as a diploid progenitor of hexaploid oat. This study further suggests that A. canariensis could be the D-genome donor of cultivated oat. However, the only available cytological data (Thomas and Leggett 1974; Thomas 1992) suggests that the Ac genome from A. canariensis is no closer to the hexaploid than the other modified A genomes. Thus, the evidence provided here should be taken as putative and exploratory. The disparity between these sets of results could be due to the differ-
ent research methods. The cytological data was used to reject A. canariensis as a D-genome progenitor because of low chromosome pairing between A. canariensis and A. sativa. However, no single species with a modified A genome has shown complete pairing with the A genome of the hexaploids (Thomas 1992; Ladizinsky 2012). Similarly, pairing with the presumed C-genome progenitor also shows incomplete homology (Thomas 1992). We speculate that this lack of complete diploid homology is a result of chromosomal rearrangement and structural plasticity within the hexaploid genome; a possibility that is supported by a relative lack of diploid–hexaploid colinearity in existing oat linkage maps (Tinker et al. 2009). Thus, lack of cytological evidence to support Ac genome as the D-genome progenitor is not conclusive. Thus the putative evidence presented here does support the hypothesis that A. canariensis contributed the D genome of hexaploid oat.
Acknowledgement This work was financially supported by the Science and Technology Bureau of Sichuan Province, China.
References Baum, B.R., Rajhathy, T., and Sampson, D.R. 1973. An important new diploid Avena species discovered on the Canary Islands. Can. J. Bot. 51(4): 759–762. doi:10.1139/b73-095. Chen, Q., and Armstrong, K. 1994. Genomic in situ hybridization in Avena sativa. Genome, 37(4): 607–612. doi:10.1139/g94-086. PMID:18470104. Fominaya, A., Hueros, G., Loarce, Y., and Ferrer, E. 1995. Chromosomal distribution of a repeated DNA sequence from C-genome heterochromatin and the identification of a new ribosomal DNA locus in the Avena genus. Genome, 38(3): 548–557. doi:10.1139/g95-071. PMID:7557363. Published by NRC Research Press
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Hao, M., Luo, J., Yang, M., Zhang, L., Yan, Z., Yuan, Z., et al. 2011. Comparison of homoeologous chromosome pairing between hybrids of wheat genotypes Chinese Spring ph1b and Kaixian-luohanmai with rye. Genome, 54(12): 959– 964. doi:10.1139/g11-062. PMID:22070394. Jellen, E.N., Gill, B.S., and Cox, T.S. 1994. Genomic in situ hybridization differentiates between A/D- and C-genome chromatin and detects intergenomic translocations in polyploid oat species (Genus Avena). Genome, 37(4): 613– 618. doi:10.1139/g94-087. PMID:18470105. Ladizinsky, G. 2012. Studies in oat evolution. Springer Heidelberg, New York, Dordrecht, London. Leggett, J.M. 1984. Morphology and metaphase chromosome pairing in three Avena hybrids. Can. J. Genet. Cytol. 26(6): 641–645. doi:10.1139/g84-101. Leggett, J.M., and Markhand, G.S. 1995. The genomic structure of Avena revealed by GISH. In Kew Chromosome Conference IV. Edited by P.E. Brandham and M.D. Bennett. Royal Botanic Gardens, Kew, UK. pp. 133–139. Available from http://www.researchgate.net/publication/251761889 [accessed 20 March 2014]. Linares, C., Ferrer, E., and Fominaya, A. 1998. Discrimination of the closely related A and D genomes of the hexaploid oat Avena sativa L. Proc. Natl. Acad. Sci. U.S.A. 95: 12450–12455. doi:10.1073/pnas.95.21.12450. PMID:9770506. Loskutov, I.G. 2008. On evolutionary pathways of Avena species. Genet. Resour. Crop Evol. 55: 211–220. doi:10.1007/s10722-007-9229-2. Morikawa, T., and Leggett, J.M. 1996. Cytological and morphological variations in wild populations of Avena canarensis from the Canary Islands. Genes Genet. Syst. 71: 15–21. doi:10.1266/ggs.71.15. Rajhathy, T. 1961. Chromosome differentiation and speciation in diploid Avena. Can. J. Genet. Cytol. 3(4): 372–377. doi:10.1139/g63-026.
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Rajhathy, T. 1966. Evidence and a hypothesis for the origin of the C genome of hexaploid Avena. Can. J. Genet. Cytol. 8(4): 774–779. doi:10.1139/g66-092. Rajhathy, T., and Morrison, J.W. 1959. Chromosome morphology in the genus Avena. Can. J. Bot. 37(3): 331–337. doi:10.1139/b59-024. Rajhathy, T., and Thomas, H. 1974. Cytogenetics of oats (Avena L.). Miscellaneous Publications of Genetics Society of Ottawa, Canada, No. 2, pp. 1–90. Sánchez de la Hoz, P., and Fominaya, A. 1989. Studies of isozymes in oat species. Theor. Appl. Genet. 77(5): 735–741. doi:10.1007/BF00261252. Sanz, M., Jellen, E., Loarce, Y., Irigoyen, M.L., and Fominaya, A. 2010. A new chromosome nomenclature system for oat (Avena sativa L. and A. byzantina C. Koch) based on FISH analysis of monosomic lines. Theor. Appl. Genet. 121(8): 1541–1552. doi:10.1007/s00122-010-1409-3. PMID:20658121. Shuai, S.R. 2003. Common genetics experiment tutorials. Sichuan Science and Technology Press, China. pp. 10–12. [In Chinese.] Solano, R., Hueros, G., Fominaya, A., and Ferrer, E. 1992. Organization of repeated sequences in species of the genus Avena. Theor. Appl. Genet. 83: 602–607. doi:10.1007/BF00226904. Thomas, H. 1992. Cytogenetics of Avena. In Oat science and technology. Edited by H.G. Marshall and M.E. Sorrells. American Society of Agronomy, Inc. and Crop Science Society of America, Inc., Madison, Wisc. pp. 473–507. Thomas, H., and Leggett, J.M. 1974. Chromosome relationships between Avena sativa and the two diploid species A. canariensis and A. prostrata. Can. J. Genet. Cytol. 16(4): 889–894. doi:10.1139/g74-096. Tinker, N.A., Kilian, A., Wight, C.P., Heller-Uszynska, K., Wenzl, P., Rines, H.W., et al. 2009. New DArT markers for oat provide enhanced map coverage and global germplasm characterization. BMC Genet. 10: 39–61. doi:10.1186/14712164-10-39.
Published by NRC Research Press
ARTICLE Exploring the origin of the D genome of oat by fluorescence in situ hybridization
Genome Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 06/12/15 For personal use only.
Xiaomei Luo, Haiqin Zhang, Houyang Kang, Xing Fan, Yi Wang, Lina Sha, and Yonghong Zhou
Abstract: Further understanding of the origin of cultivated oat would accelerate its genetic improvement. In particular, it would be useful to clarify which diploid progenitor contributed the D genome of this allohexaploid species. In this study, we demonstrate that the landmarks produced by fluorescence in situ hybridization (FISH) of species of Avena using probes derived from Avena sativa can be used to explore the origin of the D genome. Selected sets of probes were hybridized in several sequential experiments performed on exactly the same chromosome spreads, with multiple probes of cytological preparations. Probes pITS and A3-19 showed there might be a similar distribution of pITS between the Ac and D genomes. These results indicated that the Ac genome is closely related to the D genome, and that Avena canariensis (AcAc) could be the D-genome donor of cultivated oat. Key words: D genome, origin, hexaploid oat, Avena canariensis, FISH. Résumé : Une connaissance plus approfondie de l’origine de l’avoine cultivée permettrait d’accélérer les travaux en amélioration génétique. En particulier, il serait utile de déterminer laquelle des espèces diploïdes ancestrales a contribué le génome D chez cette espèce allohexaploïde. Dans ce travail, les auteurs montrent que les bandes obtenues en hybridation in situ en fluorescence (FISH) chez des espèces d’Avena, obtenues en employant des sondes provenant de l’Avena sativa, permettent d’explorer les origines du génome D. Des séries de sondes choisies ont été hybridées de manière séquentielle sur les mêmes étalements chromosomiques et plusieurs préparations cytologiques ayant été examinées. Les sondes pITS et A3-19 ont montré qu’il y aurait une distribution similaire de pITS chez les génomes Ac et D. Ces résultats indiquent que le génome Ac serait très apparenté au génome D et que l’Avena canariensis (AcAc) pourrait s’avérer la source du génome D chez l’avoine cultivée. [Traduit par la Rédaction] Mots-clés : génome D, origine, avoine hexaploïde, Avena canariensis, FISH.
Introduction Current information suggests that Avena sativa L. (2n = 6x = AACCDD) originated from a hybridization between an unknown diploid with D genome and a tetraploid with AC genomes (Rajhathy and Morrison 1959; Thomas 1992; Loskutov 2008). Rajhathy (1966) proposed that Avena ventricosa Bal. ex Coss. was the possible diploid progenitor of the C genome of the hexaploid species. On the basis of morphology, karyotypes, and geographical distribution, Baum et al. (1973) tentatively proposed that the diploid species Avena canariensis Baum, Raj. et Samp. was the probable progenitor of the A genome of the hexaploid species. There is no documented identification of the origin of the third D genome of the hexaploids, and some authors consider that this progenitor may no longer exist (Linares et al. 1998; Loskutov 2008). However, the possible diploid progenitors with A and C genomes did not take part in the origin of the tetraploid species whose genomes are labeled as AC, and nor in the origin of the hexaploid oat (Ladizinsky 2012). Based on the degree of chromosome pairing in F1 hybrids, the A genome can be classified into subgroups and these are denoted with a subscript: As, Ap, Al, Ac, Ad (Rajhathy 1961; Thomas 1992). Differences among the A genome of diploid species have been further characterized using cytological studies (Rajhathy and Thomas 1974; Sánchez and Fominaya 1989; Thomas 1992). The A genome has also been described in relation to genomic affinities
with the D genome in hexaploid oat. Molecular investigations suggest that both the A and D genome of A. sativa are highly homologous to Avena strigosa Schreb. (Chen and Armstrong 1994; Jellen et al. 1994). Attempts to differentiate between the A and D genomes by genomic in situ hybridization were previously not successful (Leggett and Markhand 1995). These reports, together with the absence of D-genome diploid species, formerly suggested that an A-genome diploid species could be the donor of both the A and D genome of the hexaploid oat. More recently, Sanz et al. (2010) were able to distinguish A- versus D-genome chromosomes by subtracting those with affinity to specific A-genome probes. However, to date, the D genome has still not been identified by direct hybridization to D-genome probes. Hence, the donor of the D genome is still unknown and controversial. In the present study, probes A3-19, pITS, pAs120a, and pAm1 were used to explore the origin of the D genome by fluorescence in situ hybridization (FISH).
Materials and methods Five species of Avena (Table 1) were used to analyze mitotic metaphases. Seeds were germinated on sterile wet filter paper. When 1.5–2.0 cm long, root tips were harvested to make cytological preparations for FISH. Using common procedures (e.g., Shuai 2003), root tips were pretreated in ice water for 24–36 h, fixed in Carnoy’s Solution I (3:1 mix of 95% ethanol – glacial acetic acid),
Received 20 March 2014. Accepted 10 October 2014. Corresponding Editor: G. Jenkins. X. Luo. Triticeae Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China; College of Forestry, Sichuan Agricultural University, Yaan 625014, Sichuan, China. H. Zhang, H. Kang, X. Fan, Y. Wang, L. Sha, and Y. Zhou. Triticeae Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China. Corresponding author: Yonghong Zhou (e-mail: [email protected]). Genome 57: 469–472 (2014) dx.doi.org/10.1139/gen-2014-0048
Published at www.nrcresearchpress.com/gen on 14 October 2014.
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Genome Vol. 57, 2014
Table 1. Seeds materials of Avena used in this study. Species
Genome
Accession
Origin or source
Avena canariensis Baum, Raj. et Samp. Avena longiglumis Dur. Avena maroccana Gdgr. Avena vaviloviana (Malz.) Mordv. Avena sativa L.
AcAc AlAl AACC AABB AACCDD
CN 26195 Ciav 9087 Ciav 8330 PI 412766 CN 64226
Canary Islands, Spain Oran, Algeria Morocco Shewa, Ethiopia Rio Grande do Sul, Brazil
Genome Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 06/12/15 For personal use only.
Fig. 1. FISH of mitotic metaphase plates of (a) Avena canariensis and (b) Avena sativa. (a) FISH of digoxigenin-labelled pITS (green) probe. (b) FISH with digoxigenin-labelled pITS (green) and biotin-labelled pAm1 (red) probes. pITS signals are indicated by arrows in both images. All chromosomes were counterstained with DAPI. Scale bar = 5 m.
then squashed in a drop of 45% acetic acid. After freezing with liquid nitrogen, the slides with the squashes were uncovered, air dried, and stored at −20 °C until use. FISH with multiple probes was performed as described by Hao et al. (2011). Four DNA probes were used in this study: (i) pAm1 (Solano et al. 1992; Fominaya et al. 1995) was used to identify the C genome of oat; (ii) pAs120a (Linares et al. 1998) was used to identify the A genome of oat; (iii) pITS (Sanz et al. 2010) was used to locate to the telomeres of 19A, 20D, and 21D of oat; (iv) A3-19 was tested as a novel probe, which was located to the telomere or centromere of oat (Tinker et al. 2009). Clones containing these fragments in pGEM-4Z were grown in LB liquid medium and the plasmids isolated using the High-Speed Plasmid Mini Kit protocol from IBI Scientific (Peosta, Iowa, USA, Cat. No.IB47102). The probes were labeled with digoxigenin-11-dUTP (Roche Diagnostics Gmbh, Mannheim, Germany, REF 11745816910) or biotin-16-dUTP (Roche Diagnostics Gmbh, Mannheim, Germany REF 11745824910) according to the manufacturer’s instructions. Streptavidin-Cy3 (SIGMA ALDRICH CHEMIE Gmbh, Steinheim, Germany, Pcod 1001238444) was used to detect the biotinylated probe and anti-digoxigenin-fluorescein (Roche Diagnostics Gmbh, Mannheim, Germany, REF 11207741910) was used to detect the digoxigenin probe. The preparations were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc., Burlingame, Calif., USA). Slides were examined using an Olympus BX-51 microscope coupled to a Photometric SenSys Olympus DP70 CCD camera (Olympus, Japan). Raw images were processed using Photoshop ver. 7.1 (Adobe Systems Inc., San Jose, Calif., USA).
Results FISH of mitotic metaphase plates of A. canariensis and A. sativa are illustrated in Figs. 1a and 1b, respectively. The probe of pITS was visible in Fig. 1a (green), while the probe of pAm1 was visible in Fig. 1b (red). The results were as follows: (i) C-genome chromosomes from A. sativa were labelled by probe pAm1. (ii) pITS signals were located on interstitial regions of the short arms of the chromosomes. (iii) Six signals in AD-genome chromosomes from A. sativa were labelled by probe pITS, while four signals in A. canariensis were labelled by probe pITS. Together with previous results showing two signals in the A genome and four in the D genome from A. sativa
labelled by probe pITS, this could indicate a similar distribution of pITS between the Ac and D genomes. FISH of mitotic metaphase plates of five species of Avena are shown in Fig. 2. The probe from A3-19 is visible in Figs. 2a–2g (arrows). The probe from pAm1 is visible in Fig. 2f (green), while the probe from pAs120a is visible in Figs. 2e (red) and 2g (green). The A3-19 signal is located on interstitial regions of the short arms of the chromosomes (arrows). Four signals from probe A3-19 were observed in the Al genome (Fig. 2a, A. longiglumis), as well as in the A/C genome (Fig. 2b, A. maroccana). Eight signals in the A/B genome were labelled by probe A3-19 (Fig. 2c, A. vaviloviana), whereas two signals in the Ac genome were labelled by probe A3-19 (Fig. 2d, A. canariensis). Four signals in the A genome and two signals in the C/D genome were labelled by probe A3-19 (Figs. 2e–2g, A. sativa), while six signals in the A/D genome were labelled by probe A3-19 (Fig. 2f, A. sativa). A summary of these results is as follows: (i) Four signals in the A genome and in the B genome were both labelled by probe A3-19. (ii) Two signals in the Ac genome were labelled by probe A3-19. (iii) Two signals in the D genome were also labelled by probe A3-19. These results suggest that the distribution of A3-19 on the Ac genome is different from that in the Al genome, but similar to the D genome. In addition, it suggests that the A genome is closely related to the B genome. Taken together, the hybridization pattern for probes from pITS, A3-19, pAm1, and pAs120a showed a similar distribution between the Ac and D genomes. These results indicate that the Ac genome is closely related to the D genome, and suggest that A. canariensis (AcAc) could be the D genome donor of cultivated oat.
Discussion These results demonstrate that the novel probe A3-19 originating from A. sativa and other probes (pAm1, pAs120a, and pITS) reveal a similar pattern of affinity to both the Ac and D genomes. These results provide a better basis for exploration of the D genome origin in hexaploid oat. Further discussion here will focus on the following points: (i) the relationship between the Ac and D genomes, and (ii) the D-genome diploid progenitor of cultivated oat. Published by NRC Research Press
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Fig. 2. FISH of mitotic metaphase plates of five species of Avena: (a) Avena longiglumis, (b) Avena maroccana, (c) Avena vaviloviana, (d) Avena canariensis (CN 26195), (e–g) Avena sativa. (a–d) FISH of biotin-labelled A3-19 (red) probe. (e) FISH with digoxigenin-labelled A3-19 (green) and biotin-labelled pAs120a (red) probes. (f) FISH with digoxigenin-labelled pAm1 (green) and biotin-labelled A3-19 (red) probes. (g) FISH with digoxigenin-labelled pAs120a (green) and biotin-labelled A3-19 (red) probes. A3-19 signals are indicated by arrows in all images. All chromosomes were counterstained with DAPI. Scale bar = 5 m.
Relationship between the Ac and D genomes The evolution of nuclear genomes in oat has been an intricate process, involving divergence at the diploid level from a common diploid ancestor, followed by convergence, followed by divergence again at the hexaploid level (Thomas 1992). Known examples of the A genome (As, Ap, Al, Ac, Ad) demonstrate this diploid divergence. Among these genomes, the As genome is considered to be the more primitive genome, while the others are considered to be derivatives (Thomas 1992). The Ap genome is closest to the Al genome (Thomas and Leggett 1974), while the Ac genome is closest to the Ad genome (Leggett 1984). The D genome has remained a mystery as it has yet to be observed in a diploid species. However, these results suggest that the D genome may also be derived from the A genome, and that it is most closely related to the Ac genome. D-genome diploid progenitor of cultivated oat Avena canariensis is the only diploid oat species with denticulate lemma tips, which is a morphological feature in common with hexaploid oat (Baum et al. 1973). This species has remarkable morphological variations, distinctive ecotypes, and polymorphic protein and isozyme electrophoretic patterns (Morikawa and Leggett 1996). As such, it has previously been identified as a diploid progenitor of hexaploid oat. This study further suggests that A. canariensis could be the D-genome donor of cultivated oat. However, the only available cytological data (Thomas and Leggett 1974; Thomas 1992) suggests that the Ac genome from A. canariensis is no closer to the hexaploid than the other modified A genomes. Thus, the evidence provided here should be taken as putative and exploratory. The disparity between these sets of results could be due to the differ-
ent research methods. The cytological data was used to reject A. canariensis as a D-genome progenitor because of low chromosome pairing between A. canariensis and A. sativa. However, no single species with a modified A genome has shown complete pairing with the A genome of the hexaploids (Thomas 1992; Ladizinsky 2012). Similarly, pairing with the presumed C-genome progenitor also shows incomplete homology (Thomas 1992). We speculate that this lack of complete diploid homology is a result of chromosomal rearrangement and structural plasticity within the hexaploid genome; a possibility that is supported by a relative lack of diploid–hexaploid colinearity in existing oat linkage maps (Tinker et al. 2009). Thus, lack of cytological evidence to support Ac genome as the D-genome progenitor is not conclusive. Thus the putative evidence presented here does support the hypothesis that A. canariensis contributed the D genome of hexaploid oat.
Acknowledgement This work was financially supported by the Science and Technology Bureau of Sichuan Province, China.
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