Plant cytogentics has been progressed at an extremely rapid pace since fluorescence in situ hybridization (FISH) techniques was developed in plant systems. However, in comparing with animal system, FISH in plant system is still a state-of-the-art technique and requires practices of sophisticated skills to consummate the technique. Recently, we have established a FISH laboratory and have reported our investigations on the organization of Oryza genomes at chromosomal level by FISH.
1. An integrated map of rice chromosome 5:
We have mapped 18 BACs/PACs on pachytene chromosome by FISH and presented a cytogenetic map of rice (Oryza sativa L.) chromosome 5 showing the relationship between genetic recombination and cytological features of rice chromosome 5 at molecular level. Detailed comparisons of the correlative positions of markers on the genetic, cytological, and physical maps of rice chromosome 5 provide insight into the molecular architecture of rice chromosome 5, in relation to its cytological features and recombination events on the genetic map.
Fig 1. The localization of rDNAs (red) on Oryza punctata chromosomes (blue).
2. Genome specific marker in Oryza species:
A genome specific RAPD, Opun210 was isolated from Oryza punctata Kotschy ex Steud (W1593), an African native wild species of rice. The Opun210 sequence is 789 base pairs in length and estimated at 5.3 × 104 copies in O. puntata (W1593) haploid genome. Opun210 is highly specific to B genome. Sequence analysis revealed that, the Opun210 sequence at position 430~480 nucleotides putatively encodes a peptide with 88% identity to a Ty3-gypsy retrotransposon protein or a peptide with 94% identity to a hypothetical protein. The results of Southern hybridization and fluorescent in situ hybridization (FISH) indicated that the repetitive Opun210 sequences dispersed throughout the entire genome of O. punctata.
3. Chromosomal polymorphism of rDNAs in the genus Oryza:
We used the coding region of 45S rRNA gene (rDNA) as probe in fluorescent in situ hybridization to localize rDNAs on chromosomes of 15 accessions representing ten Oryza species. Our results reveal polymorphism in the number of rDNA loci, in the number of rDNA repeats, and in their chromosomal positions among Oryza species. The numbers of rDNA loci are various in one to eight among Oryza species. We suggest that chromosomal inversion and the amplification and transposition of rDNA might occur during Oryza species evolution.
4. Study on chromosomes and embryogenesis of orchids:
Slipper orchids, Paphiopedilum species, are considered deep in commercial capacity. However, the difficulty of seeds germination, low fertility or even sterility of hybrids, and longer generation are mainly restricted factors for the current developing of slipper orchids business. Low germination rate and slow growing into plants are physiological and developmental problems. The results of our morphological and physiological studies indicated that seed maturation is accompanied by enlargement in size, the accumulation of storage materials in embryo and cuticular layer around the embryo, decreasing in water content, and increasing in ABA concentration in seeds. Seed is optimum for in vitro germination when the embryo is fully developed in size and filled with storage materials, the suspensor cells are still functional, but the cuticular layer has not yet fully formed. Low fertility or sterility is genetic problem. For this problem, we execute genomic in situ hybridization (GISH) to distinguish genomes contained in Paphiopedilum intergenic or interspecies hybrids. Our results can help to evaluate the possibility for the extension of intergenomic recombination, which will facilitate the intergenomic genes or chromosomal fragments transfer.
Fig 2. Light micrographs showing the fluorescence outline of the developing P. delenatii embryo after Nile red staining. (Ann Bot 98: 1311–1319, 2006)
(A) A developing embryo at globular stage and Nile red staining fluorescence micrograph of the embryo at the same stage (B). (C) A embryo at 150 DAP (days after pollination) with an elongated suspensor (S) and Nile red staining fluorescence micrograph of the embryo at the same stage (D). (E) A longitudinal section through a mature seed (210 DAP) and Nile red staining fluorescence micrograph of the embryo at the same stage (F).
Chung, M.C., Lee, Y.I., Cheng, Y.Y., Chou, Y.J., Lu, C.F. (2007). Chromosomal polymorphism of ribosomal genes in the genus Oryza. Theor Appl Genet (accepted).
Lee, Y.I., Yeung, E.C., Lee, N., and Chung, M.C. (2007). Embryology of Phalaenopsis amabilis var. formosa: embryo development. Botanical Studies (in press).
Lee, Y.I. and Chung, M.C. (2007) Identification of genome relationships among Paphiopedilum species by genomic and fluorescent in situ hybridization. Acta Hort (in press).
Lee, Y.I., Yeung, E.C., and Chung, M.C. (2007). Embryo Development of Orchids. In: Orchid Biotechnology, W. H. Chen and H. H. Chen eds. (World Scientific Publishing , Singapore), pp. 23-44.
Lee, Y.I., Yeung, E.C., Lee, N., Lu, C.F., and Chung, M.C. (2007) Changes in endogenous abscisic acid levels and asymbiotic seed germination of a terrestrial orchid, Calanthe tricarinata Lindl." J. Am. Soc. Hortic. Sci. 132:246-252.
Lee, Y.I., Yeung, E.C., Lee, N., and Chung, M.C. (2006). Embryo development in the lady's slipper orchid, Paphiopedilum delenatii with emphases on the ultrastructure of the suspensor. Annals of Botany 98:1311-1319.
Kao, F.I., Cheng, Y.Y., Liu, S.M., Cheng, C.H., Chen, H.H., Chow, T.Y., and Chung, M.C. (2006). An integrated Map of Oryza sativa L. Chromosome 5. Theor Appl Genet 112:891-902.
Cheng, C.H., Chung, M.C., Liu, S.M., Chen, S.K., Kao, F.Y., Lin, S.J., Hsiao, S.H., Hsing. Y.I.C., Wu, H. P., Chen, C. S., Shaw, J. F., Wu, J., Sasaki, T., Chen, H. H., and Chow, T. Y. (2005). A fine physical map of the Oryza sativa chromosome 5. Molecular Genetics and Genomics 274:337-345.
Lee, Y.I., Lee, N., Yeung, E.C., and Chung, M.C. (2005). Embryo development of Cypripedium formosanum in relation to seed germination in vitro. J. Am. Soc. Hortic. Sci. 130:747-753.
Yang, J.Y., Chung, M.C., Tu, C.Y., and Leu, W.M. (2002). OSTF1: A HD-GL2 family homeobox gene is developmentally regulated during early embryogenesis in rice. Plant Cell Physiol 43:628-638.
Chung, M.C., Chou, S.J., Kuang, L.Y., Charng, Y.Y., and Yang, S.F. (2002). Subcellular localization of 1-aminocyloprpane-1-carboxylic acid oxidase in apple fruit. Plant Cell Physiol 43:549-554.
Wu, J.Y., Wu, H.K., and Chung, M.C. (2002). Co-dominant RAPD markers closely linked t. with two morphological genes in rice (Oryza sativa L). Bot Bull Acad Sin 43:171-180.
Chung, M.C. and Wu, H.K. (1997). Chromosome instability induced by fragments in trisomic rice (Oryza sativa L.) Cytologia 62:67-79.
Chung, M.C. and Wu, H.K. (1994). Cytological identification of the chromosomes involved in Nishimura’s translocation lines. Theor. and Appl. Genet. 88:956-964.
Chung, M.C., Ning, C.N., and Wu, H.K. (1993). Localization of ribosomal RNA genes on rice chromosomes. Bot. Bull. Acad. Sin. 34:43-55.
Wu, H.K., Chung, M.C., Ning, C.N., Wu, T., and Wu, R. (1991). Localization of specific rice repetitive DNA sequence in individual chromosome. Chromosoma 100:330-338.
