3. Identification of extra chromosomes of six IR36 Triplo lines

We have been engaged in studies of chromosomes of IR36 Triplo lines since late 1986, under the auspice of the RGC. We have examined all the Triplo lines with respect to both pachytene and somatic prometaphase stages. In this note, however, our presentation is confined to the pachytene of six lines remaining unsettled after the joing work by Drs. N. Kurata and R.J. Singh. The numbering system of chromosomes employed in this note is that given by Fukui et al. (1988; this issue) as was proposed in the preceding note (Oka and Wu). It differs from that used by Wu and coworkers in that chromosomes 8 and 9 are replaced with each other and also from the system used by Kurata and others in that chromsomes 2 and 3 are replaced with each other.

Materials used were the plants raised from the seeds kindly made available by Dr. G.S. Khush in 1982 and 1986. The technique used was that described by Wu (1967). Chromosome length was measured on photographs with the aid of a digitizer. A difficulty we met with in this work was the occurrence of structural changes involving extra chromosomes in certain lines, which produced tertiary trisomics as will be reported elsewhere. We then made efforts to select nuclei that appeared to be primary trisomics as the objects of observation. In Triplo 12, the extra chromosome was broken into two telocentric fragments. We used R77-24-2 which was a sib line as the substitute of Triplo 12.

The method of identification of an extra chromosome will have to be mentioned first. In a primary trisomic, triplex chromosomes form either a trivalent or a bivalent and a univalent. In a good preparation, the trivalent can be identified by comparing its size and shape with those of bivalents of the control and by identifying all other vibalent chromosomes. But univalents are by no means suited for identification. When trivalents are identified in a number of cells consistently, their measurements of relative length and arm ratio are compared statistically with those of the corresponding bivalents of diploid plants that are used as the control, so as to confirm the result of identification.

A trivalent chromosome gives three measurements each standing for one of its components. The mean of the three values was used for determining the relative length and arm ratio of a trivalent. It may be questioned whether or not the data fro trivalents are comparable to those for bivalents. To answer this question, the trivalent data were subjected to analysis of varance in each Triplo line, and the variance of single components of trivalents, that of their mean values, and that for corresponding bivalents, were compared with one another (Table 1). The results showed that in both relative length and arm ratio, the variance of trivalent means was comparable to that of bivalents, although the variance for three single components of trivalents was often larger significantly indicating the fluctuation of measurements. These comparison of variances may serve as an evidence justifying the comparison of mean measurements of trivalents with those of corresponding bivalents.

The following three controls were used for identifying trivalent chromosomes. CK-1 is the data for IR36 presented by Wu et al. (1985, also by Chung and Wu 1987). CK-2 is the mean for data presented by different authors given in the preceding note (Oka and Wu, relative length in Table 1; arm ratio in Table 5). CK-3 is the general mean for six Triplo lines and IR36 given in Table 4 of this note.

A condition imposed on our identification work was that the chromosomes to be assigned to the six triplo lines in question (Triplos 3,4,6,8,11 and 12) were limited to the six not adopted by Drs. N. Kurata and R.J. Singh in their agreement. In addition, the extra chromosomes of Triplos 4 and 8 were suggested to be 2 (originally 3) and 8 in the 1987 RGC meeting at the IRRI (RGN 4, p. 1- 2), so that the scope of choice was narrowed to only four chromosomes, 4,6,11 and 12. However, this limitation has not led us to a maze; no unjustifiable treatment of the data was made in our data analysis.

The relative lengths of respective pachytene chromosomes obtained in the six Triplo lines are given in Table 2, and the relative lengths and arm ratios of trivalent chromosomes are in Table 3. The table of arm ratios of respective chromosomes obtained in the six Triplo lines is omitted from this note for brevity. The general means for relative length and arm ratio of the six lines and IR36 are given in Table 4. The trivalent chromosomes in respective Triplo lines were identified as follows:

Triplo 3: The relative length and arm ratio of trivalents showed no significant differences from thos of chromosome 6 of the controls except for the case of arm ratio in CK-3, and they showed significant differences from those of chromosome 5 as well as of 7. Therefore, the extra chromosome was judged to be chromosome 6 (Table 3).

Triplo 4: In the 1987 RGC meeting, the extra chromosome of this line was designated as chromosome 3, which corresponds to chromosome 2 according to the numbering system adopted in this note. This designation was endorsed in our data, as the measurements of trivalents agreed well with those of chromosome 2 and disagreed with those of chromosomes 1 and 3 (Table 3).

Triplo 6: The measurements of trivalents of this line agreed with those of chromosome 12 of the controls. Their relative length differed from those of chromosome 11 of CK-1 and CK-3, although their arm ratio showed no significant difference from those of chromosome 11. As the trivalent was not attached to the nucleolus, it could not be chromosome 10 (Table 3).

Triplo 8: The extra chromosome of this line was designated as chromosome 8 in the 1987 RGC meeting. This was confirmed in our data. The arm ratio of the trivalents was close to that of chromosome 8 of the controls and differed significantly from that of chromosome 7, although their relative length differed from that of chromosome 7 only when CK-3 was used for comparison (Table 3). As the trivalents were not attached to the nucleolus, they could not be chromosome 9 or 10.

Triplo 11: The measurements of trivalents showed better agreement with those of chromosome 11 of the controls than with those of chromosome 12 (Table 3). As the trivalents were not nucleolar, they could not be chromosome 9 or 10. Accordingly, the extra chromosome of this line was considered to be chromosome 11.

Triplo 12: The extra chromosome was represented by two fragments which appeared to correspond to the long arm of chromosome 4. Its sib line, R77-24-2, was then used as substitute. The trivalent chromosome was characterized by a subterminal feature, and its measurements were closest to those of chromosome 4 of the control (Table 3).

In conclusion, the extra chromosomes of the four Triplo lines remaining undecided are designated as follows: Triplo 3-chr. 6, Triplo 6-chr. 12, Triplo 11-chr. 11, and Triplo 12-chr. 4. This conclusion holds true when plants of primary trisomics are selected for observation. The numbering system of chromosomes adopted in this note, those used by Kurata, Wu and coworkers in their respective papers, and the Triplo lines carrying these chromosomes in extra, are compared collectively in Table 4. We are indebted to Dr. H.I. Oka for his careful checking up of statistical analysis of our data and critical discussion.

Table 1. Variations in measurements of trivalents as compared with those of corresponding bivalents (showing variance at single-determination level) RL-Relative length in %; L/S-Arm ratio

Table 2. Relative lengths of pachytene chromosomes of Triplo lines (mean+=standard deviation, in %; under lines show extra chromosome)

Table 3. Measurements of trivalent chromosomes of 6 Triplo lines and their identification by comparison with control data (t-test)

Table 4. Mean measurement of respective chromosomes and the extra chromosomes of Triplo lines corresponding to them

Chung, M.C. and H.K. Wu, 1987. Karyotypes analysis of IR36 and two trisomic lines of rice, Bot. Bull. Acad. Sinica 28: 289-304. (Chinese/English)

Fukui, K., K. Iijima and K. Kakeda, 1988. Analysis and utility of chromosome information, 21. Karyotypes of rice chromosomes. Jpn. J. Breed. 38, Suppl. 1:474-475. (in Japanese)

Wu, H.K., 1967. Note on preparing of pachytene chromosomes by double mordants. Scientific Agriculture (Taipei) 15: 40-44. (Chinese/English)

Wu, H.K., M.C. Chung and M.H. Chen, 1985. Karyotype analysis of cultivar IR36. RGN2:54-57.