1. Current status in rice chromosome research 

Hokuriku National Agricultural Experiment Station 

Joetsu 943-01, Japan

This review summarizes the current status of rice chromosome research with special reference to technological aspects. Whole view of the recent advances in rice chromosome research has recently been reported (Fukui in press). Main new technologies which appeared in these years are such as, imaging methods, molecular cytology, chromosome painting and laser dissection methods.

I. Imaging methods in rice chromosome research

The most important advance in imaging fields is that the new image parameter, CP was defined and applied to identification of plant chromosomes and development of a

mitotic rice chromosome map (Pukui and Mukai 1988; Fukui and Iijima 1991). The CP is a profile of density distribution along a mid-rib of chromatid of rice chromosomes, which represents the condensation pattern of rice chromosome at the mitotic pro-metaphase stage. Fig. 1 shows the condensation pattern of rice chromosome 11 and definition of a quantitative chromosome map by thresholding the CP at the two gray levels. Fig. 2 shows the quantitative chromosome map for rice mitotic chromosomes rearranged in the order of the new rice chromosome number (Fukui and lijima 1991). All the rice chromosomes were also characterized by their morphology and the detailed
 
 

3D Rice Genetics Newsletter Vol. 13
 

	same (P=83.3%). When a	B. Characteristics of the condensed regions	C. Characteristics of the dispersed regions
Chromosome 6	Metacentrics. relative length 7.9	The condensation degree of the	There is a FUSC in the central
(Phantom or	± 0.6, arm ratio 1.68 ± 0.21.	pCR is greater than that of the	part of the pDR (P=40.0%, e.g.,
Terumodoki)	This chromosome shows vari-	qCR (P=83.3%).	chromosomes 6b and 6f). There
	able patterns. The typical type		are several FUSCs in the qDR (
	exhibits a very small qCR com-		P=96.7%, e.g., chromosomes 6a
	pared with the pCR (e.g., chro-		and 6c).
	mosomes 6a and 6c) and the
	distinct dispersed regions on
	both arms, whereas the pDR is
	smaller than the qDR (e.g., chro-
	mosomes 6a and 6b). A great-
	er part of the short arm is some-
	times condensed with simulta-
	neous occurrence of bifurcation
	at the end of the chromatids to
	the outside, with tapering off to
	a point (e.g., chromosome
	6d). The end of the chromatids
	of the long arm converged (e.g.,
	chromosomes 6a and 6f).
Chromosome 7	Metacentrics, relative length 7.6	The condensation degree of the	The long arm often has a FUSC
(Disturber)	± 0.5, arm ratio 1.61 ± 0.31. The	pCR is the same or slightly	at the qDR (P=66.7%, e.g.,
	chromosome shows variable	greater than that of the qCR (P	chromsome 7d).
	patterns. In the typical pattern,	=86.6%). The short arm has a
	the lengths of the pCR and the	telomeric FUSC (P=76.7%,
	qCR are identical or the qCR is	e.g., chromosomes 7a and
	slightly longer. The qDR is	7f). The whole region of the
	distinct, whereas the pDR is	short arm is often condensed (P
	very small. A typical short	=70.0%, e.g., chromosomes 7d
	arm appears as the junction of	and 7e).
	the pCR and the telomeric con-
	densed region (e.g., chromoso-
	mes 7b and 7f).
Chromosome 8	Metacentrics, relative length 6.6	The degree of condensation of	The ends of the chromatids at
(Rocket)	± 0.4, arm ratio 1.24 ± 0.13. The	the pCR is the same or slightly	the pter are not separated (P=
	whole part of the short arm is	lower than that of the qCR (P	53.3%, e.g., chromosomes 8c
	condensed. The visual pCR is	=76.7%). The pCR frequently	and 8d), although they are sepa-
	frequently longer than the qCR	appears longer than the qCR	rated at the qter (P=80.0%, e.g.,
	and the long arm has a relatively	from visual inspection of the	chromosomes 8d and 8f).
	long qDR. As an alternative	photographs (e.g., chromo-
	pattern, the pCR is divided into	somes 8b and 8c), although the
	two segments and the pter is	average length determined by
	relatively darker than usual.	the CP analysis is almost the
	Consequently three darkly stain-	same (P=83.3%). When a
	ed blocks appear, two at the	gap-like structure appears in the
	short arm and one at the long	pCR, the CP line of the pCR
	arm. The qter of the chromo-	becomes variable (P= 16.7%,
	some is sometimes bifurcate	e.g., chromosomes 8a).
	(e.g., chromosomes 8d and 80.
Cromosome 9	Metacentrics, relative length 6.6	One and two condensed regions	There are no FUSCs at the short
(Trimodal or	± 0.5, arm ratio 1.26 ± 0.11. The	frequently appear at the respec-	arm (P=86.7%, e.g., chromo-
Mitsuyama)	greater part of the chromosome	tive short and long arms	somes 9b and 9c). The chro-
	is condensed. Three condens-	(P=96.7%, e.g., chromosome	matids are not separated at the
	ed blocks appear or, alternative-	9a).	telomeric ends of the long arm
	ly a slit can be observed in the		(P=80%,, e.g., chromosomes
	center of the qCR (e.g., chromo-		9d and 9f).
	somes 9a and 9c).

 

descriptions were given to all the rice chromosomes with statistical data as shown in Table 1 (Fukui and Iijima 1992).

An automatic identification method of rice chromosomes based on CP has also been established (Kamisugi et al. 1992). The three statistical methods such as, a discrimination chart method, a linear discrimination method, and a minimum distance classifier method to identify the chromosome number automatically were tested. As a result, 92.2% of the rice chromosomes are correctly identified by the minimum distance classifier method. The discrimination chart and linear discrimination methods gave respective 91.1 and 84.4% of correct identification. These results are good proof that the CP contains sufficient information to determine the chromosome number and is the stable parameter representing morphological information of rice chromosomes.

Fig. 3. Fluorescence in situ hybridization by 45SrDNA in 0. officinalis. Six FITC fluorescent signals were clearly observed on PI counter-stained rice chromosomes. An improved FISH method especially for rice chromosomes and imaging methods were employed (Fukui et al. 1994).

Development of a fluorescence in situ hybridization (FISH) method improved the detection efficiency and sensitivity. Fukui et al. (1994) detected one rDNA locus in 0. sativa (japonica), 0. rufipogon, and 0. brachyantha. Two 45S rDNA loci were found in 0. sativa (indica and javanica), 0. rufipogon, and 0. australiensis. Three loci were revealed in 0. officinalis (Fig. 3). It seems that there is no species dependent tendency but geographical trend in the number of 45S rDNA loci. It is worth noticing that the variation of one and two 45S rDNA loci detected in japonica and indica also exists in their ancestral species of 0. rufipogon.

5S rDNA has also physically been mapped by ISH on chromosome 9 of indica rice (Song and Gustafson 1993) and on chromosome II of japonica rice (Kamisugi et al. 1994). The 5S rDNA locus is localized at the position 4% away from centromere toward the distal end on the short arm in japonica rice. Further research on localization of 5S rDNA using several rice species by FISH revealed that rice has only one locus and they are located on chromosome 11 without exceptions (Shishido et al. 1994).

Ohmido and Fukui (1995) developed a multicolor FISH (McFISH) method using 45S and 5S rDNA probes simultaneously in the African cultivated rice, 0. glaberrima and detected identical localization of both the rDNAs on the chromosomes as in 0. sativa ssp. japonica.

Several repeated sequences of rice such as TrsA, TrsB, TrsC, and RIREI (Ohtsubo et al. 1991; Noma et al. 1996) have been successfully mapped on the rice chromosomes

Jiang et al. (1995) reported mapping BAG clones on the chromosomes by FISH. The successful mapping in YAC, BAC, cosmid clones and even RFLP markers on rice chromosomes have also been obtained (Ohmido and Fukui in preparation). Therefore it is anticipated that the reproducible mapping of single copy genes with less than 1 kb nucleotide sequences will be a common technique in rice chromosomes.

III. Chromosome painting

Chromosome painting is a variation of ISH using total genomic DNAs as the probe. The method is also called as genomic in situ hybridization, GISH. GISH has been used for phylogenetic studies and identification of alien chromosome(s) from different genome(s) in the genus Oryza. As the probe is the mixture of unique and repetitive sequences, the signals uniformly cover all over the chromosome(s).

Chromosome painting using tetraploid rice species with different genomes by GISH was tried (Fukui et al. in preparation). They extracted genomic DNA from 0. officinalis, a diploid C genome species and use the genomic DNA as the probe to paint the chromosomes originated from C genome diploid species in the two amphidiploid species of 0. minuta, BBCC and 0. latifolia, CCDD.

Twenty four chromosomes derived from an ancestral diploid C genome species were clearly painted both in 0. minuta and 0. latifolia. The chromosomes originated from a D genome species have been identified for the first time under the microscope. The fact that the differences in the intensity of the fluorescent signals between the B and C genome chromosomes were clearer than that between the C and D genome indicates that C and D genomes are closely related to each other than the B and C genomes.

IV. Laser dissection of rice chromosomes

Recent advances in laser optics makes it possible to dissect plant chromosomes (laser knife, Fukui et al. 1995). Laser microdissection seems to be a useful technology in chromosome research. Fig. 4 shows that the representative steps of laser dissection of rice chromosomes (Fukui et al. 1992). Fig. 4a shows the rice chromosomal spread prepared by the enzymatic maceration/air drying (EMA) method (Fukui and Iijima 1992; Fukui 1996). Two chromosomal regions of chromosome 4 were dissected in this case. Scattered cytoplasmic debris was eliminated at first and unnecessary chromosomes at the peripheral region were laser ablated (Fig. 4b). Then all the rice chromosomes within the spread except for chromosome 4 were removed by laser ablation (Fig. 4c). A fine laser beam was then applied to eliminate the condensed region to obtain the dispersed tail region of the chromosome 4. For the other chromosome 4, the

60. Detection of 5S rDNA locus in Oryza spp. by multicolor FISH. Japan. J. Breed. 44(Suppl. 1 ): 47 Song, Y.C. and J.P. Gustafson, 1993. Physical mapping of the 5S rDNA gene complex in rice (Oryza sativa).