Pachytene Analysis and Observations of Chromosome Association in Haploid Rice

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1 Pachytene Analysis and Observations of Chromosome Association in Haploid Rice Yaw-En Chu National Institute of Genetics, Misima, Japan Received March 30, 1966 The chromosome morphology of Oryza sativa has been studied by many researchers; Rau (1929), Nandi (1936) and Pathak (1940) investigated somatic chromosomes of diploid rice, Yasui (1941) and Hu (1959, 1960) those of haploids, and Shastry (1960) studied pachytene chromosomes of diploids. The karyotypes given by those workers are not in complete agree ment, due to differences in materials and methods. On the other hand, after Kuwada's (1910) first observation of secondary association of meiotic chromosomes in Oryza sativa, Sakai (1935), Nandi (1936) and Parthasarathy (1938) assumed that the basic chromosome number of rice is five. It has generally been taken for granted that in rice, the secondary association of bivalents is due the same kind of inter chromosomal attraction which causes the association of univalents in haploid plant. After a statistical analysis of the frequency distribution of chromosomes of haploid rice, Hu (1958, 1962) concluded that the association pattern does not fit the Poisson distribution and, consequently, it cannot be a random phenomenon. A number of cytologists have dealt with this phenomenon in different plant groups, namely, Pyrus (Darlington 1930), Brassica (Catchside 1937), Oryzeae and Zinzanieae (Hirayoshi 1937), Ricinus (Kurita 1936 and Jacob 1957), Luzula (Brown 1950) and Triticum (Riley 1960 and 1963). Their opinions are divided, some considering secondary association to be the expression of residual chromosomes homology, while others emphasize randomness. The present author investigated chromosomes of rice haploids regarding the patterns of their association at different meiotic stages. This report is mainly concerned with the karyotype and association patterns found at pachytene as well as their frequency distribution at diakinesis and metaphase-i. Materials and methods The materials used are haploid plants of spontaneous origin obtained from six rice varieties, Norin 8, Norin 22, Norin 25, Shin-ai, Senbon-asahi and Saitama-mochi (all belonging to the japonica type). As no varietal differences could be noticed, pooled data are presented in this paper. For observing P. M. C.'s, young panicles collected in the forenoon between ten and eleven were fixed in Farmer's fluid for 24 hours, and were stored in 75% alcohol. Preparations were made by aceto-carmine or propion-carmine smear method. Results I. Karyological studies in the pachytene stage The pachytene chromosomes of haploid rice plants appeared to be more slender than those of the diploids at the same stages. The determination of the centromere position was not always possible. The karyotypes arranged in the order of length are diagrammatically shown in Figs. I and 2. At the proper

2 88 Y. E. Chu pachytene stage, the longest chromosomes become shortened as the stage proceeds. Chromosome 1 was 2.8 times as long as chromosome 12. The chromosomes could be divided into three groups by their arm ratio: 1) nos. 3, 4 and 7 were of median type (arm ratio ranging from 1.00 to 0.75), 2) no. 11 was subterminal (0.50 to 0.01) and 3) the remaining eight were submedian (0.75 to 0.50). In the majority of cells, chromosomes 9 (or 10) and 12 were found to be attached to the nucleolus, the latter not as often as the former. Darkly stained knobs were constantly found in the short arm of chromosome 11. Fig. 1. Pachytene chromosomes in a haploid plant of Oryza sativa. Fig. 2. Diagram of the pachytene chromosomes of haploid O. sativa. II. Association of chromosomes The pattern of association was observed in pachytene, diakinesis and metaphase-i. Two chromosomes attached to each other by a stainable thread were considered to be associated. At diakinesis and metaphase-i, 30 different patterns were distingui shed, the number of associations ranging from 0 to 8. The average number of associations per cell at pachytene, diakinesis and metaphase- I was 3.23, 2.90 and 2.48, respectively (Table 1). Most of the pre vious workers considered the maximum associa tion pattern to be 2 (3)+ 3 (2). But such associa tion as 3 (3) and 1 (4), which show a higher number of associations than the previously assumed maximum, were found as shown in Figs The frequency of such patterns was 36/680, while that of 2 (3)+3 (2) was only 4/680, being lower than the frequencies of other types with the same number of association. It therefore seems that the pattern 2 (3)+3 (2) does not represent the maximum association which was the basis of the hypothesis of the basic number 5. If the association of haploid chromosomes and the secondary association in diploids were similarly due to specific affinities between partially homologous segments, they should occur between definite chromosomes at definite points.

3 1967 Pachytene Analysis and Observations of Chromosome Association in Haploid Rice 89 In an effort to distinguish the individual chromsomes on the basis of length and arm ratio the place of association was recorded for each chromosome. The results are given in Table 2. Identification of the individual chromosomes was not quite reliable, particularly for nos. 2 and 3, and nos. 5 and 6, but no. 1 could always be distinguished from the others. No. 1 was found to be associated with nos. 2 Table 1. Frequency distribution of association types at diakinesis and metaphase-i or 3, 5 or 6 and 11 or 12. Further, the places of association in this chromosome were as shown in Fig. 6. In 11 cells in which the position of the centromere could be determined, at least three different places of association were found, namely one at the distal part of the long arm, and two at the distal and the proximal part of the short arm. Thus, chromosome 1 can associate with three other chromosomes simultaneously, forming a group of four as the author's observation at diakinesis and metaphase-i proves.

4 90 Y. t. Chu Cytologia 92 Also in the other chromosomes, the distal part showed more association sites than the proximal part (Fig. 7). In some cases the configuration appeared to involve a chiasma (Fig. 8). Figs Association of univalent chromosomes in diakinesis and metaphase-i. 3, 1(4)+ 2(2)+2(1). 4, 2(4)+4(1). 5, 1(4)+1(2)+6(1). Table 2. Frequency of associations between chromosomes in pachytene No. of cells observed: 30 Aver. no, of associations: Karyotype Discussion As to the chromosome morphology of Oryza sativa, Nandi (1936) first reported that its 12 chromosomes could be divided into five groups, i.e., three, each consisting of two chromosomes of similar size, and two, each consisting of three median chromosomes. Later, Morinaga (1939), Yasui (1941) and Pathak (1940)

1967 Pachytene Analysis and Observations of Chromosome Association in Haploid Rice 91 reported that three to four chromosomes are of median type, the rest being sub median or sub-terminal.

5 1967 Pachytene Analysis and Observations of Chromosome Association in Haploid Rice 91 reported that three to four chromosomes are of median type, the rest being sub median or sub-terminal. Recently, Hu (1964) as well as Shastry (1960) obtained similar results, using haploid somatic chromosomes and diploid pachytene chromosomes, respectively. In Table 3 the results of those workers are compared with findings of the present author. Assuming that Fig. 6. Positions of associa tion in chromosome 1 at pachytene. the relative lengths of chromosomes are not different in haploid and diploid cells, and also the same in mitotic and meiotic cells, the results obtained by the various workers seem to be in agreement in that chromosomes 1, 2, 5, 8, 9 and 12 are sub-median, chromosome 3 is median, and chromosome 6 is sub-terminal. As for chromosome 4, Hu (1964) as well as the present author consider it to be median (arm ratio being 0.80 and 0.92, res pectively), while Shastry (1960) treats it as sub median (arm ratio being 0.48). As the chromosomes have been numbered in the order of length, such a discrepancy may arise from a bias in measuring Figs Association between nonhomologous chromosomes in pachytene. Arrows show the position of association. 8. Chiasma-like structure between nonhomologous chromosomes in pachytene. the length. Shastry and the present author consider chromosome 7 to be median (arm ratio being 0.96 and 1.00), but according to Hu it is sub-median (arm ratio

6 92 Y. E. Chu Cytologia 32 being 0.50). Further, Hu and the present author consider chromosome 10 to be sub-median (arm ratio being 0.82 and 0.72), but Shastry considers it as sub terminal (arm ratio being 0.17); both Hu and the present author failed to find such a low arm ratio. It is possible that Shastry has mistaken the position of centromere in this chromosome. Chromosome 11 is according to Shastry and the present author sub-median (arm ratio being 0.64 and 0.70 respectively), but according to Hu it is sub-terminal (arm ratio being 0.35). Further, according to Hu, this chromosome has a secondary constriction, but the author has found that chromo some 12, not 11, is attached to the nucleolus. Hu's chromosome 11 seems to be comparable with the author's no. 12. Its arm ratio is then given as 0.35 (Hu), 0.33 (Shastry) and 0.50 (present author). Table 3. Comparison of karyotype data published by three workers Hu (1964): haploid somatic chromosome Shastry (1960): diploid pachytene chromosome The present author: haploid pachytene chromosome Regarding the relative length, the longest chromosome is 2.8 times as long as the shortest. Hu as well as Shastry obtained similar results, namely figures 2.4 and 3.7, respectively. Nandi (1937) concluded that the majority of the chromosomes were of similar size and shapes, but other workers failed to confirm his conclusion. The chromosomes of rice are too small for a clear determination of the distribution of the achromatic regions and chromomeres. Li et al. (1963) noticed the presence of heterochromatic regions in the pachytene chromosomes of Oryza australiensis, but did not describe their distribution. It may be concluded from the above comparisons that most of the chromosomes are identifiable though some discrepancy still remains among the workers. 2. Association The occurrence of secondary association in Oryza sativa was pointed out first by Sakai (1935), and later by many other workers. Most of them have assumed the maximum association to be 3(2)+2(3), leading to the hypothesis that the basic

7 1967 Pachytene Analysis and Observations of Chromosome Association in Haploid Rice 93 chromosome number of rice is five. Hu (1962) showed that the frequency distribu tion of cells with different numbers of associations in haploid meiosis did not fit the Poisson distribution, concluding non-randomness of the association. Oka (1964) suggested the secondary polyploid nature of rice on the basis of duplication of various sterility genes (Gametic development genes, complementary semi-lethals, and duplicate fertility genes). Two kinds of associations can be observed in haploic meiosis. Primary associa tion, resulting in true bivalent formation with chiasmata, might be due to pachytene pairing along the entire chromosome length, while in secondary association the chromosomes might be segmentally associated. According to Hu's (1960) observa tion, the frequency of secondary association was more than ten times as frequent as that of primary pairing. According to Kihara (1933), Tuchiya (1962) as well as Heneen (1965), at meiosis of haploid wheat, barley and rye some association of two chromosomes lying side by side had similar patterns of heteropycnotic differentia tion, while end-to-end thread like contacts showed no similarity in sturcture. In the present study, association of non-homologous chromosomes in haploid meiosis was clearly recognized, but higher associations than 2 (3)+3 (2) were ascertained. Both in diakinesis and metaphase-i, such associations as 3 (3) and 1 (4) were found with similar frequencies as that of 2 (3)+3 (2). The occurrence of these high associations must be taken into consideration in making speculations as to the basic chromosome number of rice. If those associations were only chance events, the frequency of such high associations 3 (3) and 1 (4) should be lower than the previously assumed maximum. Katayama (1965) also pointed out the occurrence of association types disproving the previous hypothesis of 5 as basic number. Therefore, in the light of the new finding the basic number of five appears doubtful. The mechanism of secondary association is an unsettled issue. Gustafsson (1946) considered that secondary association could be attributed to terminal affinity or to fusion of chromosome pellicles. Heilborn (1936) ascribed it to a differential operation of repulsion forces between bivalents of different sizes. On the other hand, Thomas and Revell (1946), from an analysis of induced autopoly ploids in Cicer, concluded that secondary association occurs mainly between homologous chromosomes, and is actually due to a fusion of heterochromatic portions of chromosomes at pachytene. Two bivalents in secondary association are then potentially capable of forming a quadrivalent. Recently, a critical analysis of the relative position of bivalents in common wheat was made by Riley (1960, 1963 and 1964). He could identify certain bivalents morphologically and concluded that homologous chromosomes tended to be located near each other. This strongly suggests that secondary association represents genetic interrelationships between the chromosomes. A similar situation might be the case of secondary association in rice. Jacob (1957) found in the pachytene of Ricinus that three large macro chromomeres were similarly located in the long arm of chromosomes E and in the short arm of G and that secondary association took place between them at a

8 94 Y. E. Chu Cytologia 32 high frequency. Thus, secondary association seems to represent some residual homology between the chromosomes. The various association types found in the present study might reflect relative affinities between the chromosomes, but they might also be influenced by the relative position of chromosomes in a cell which is determined by chance. The distribution of association types may be considered to be a function of relative affinity and relative position. In this connection, Kudo and Katayama (1965) considered that free chromosomes could have more chance to associate with each other than with those already associated. These considerations lead the author to conclude that secondary association may be essentially due to a certain homology between chromosomes, but the patterns of association actually observed are not only due to homology relation ships but also to chance events. It is possible that rice is a secondary polyploid, but it seems difficult to postulate the basic number from the patterns of secondary association. Summary The karyotype of haploid plants of cultivated rice (Oryza sativa) was inves tigated in a pachytene analysis. At diakinesis and metaphase-i, 30 different association types were found. Though the maximum association of haploid chromosomes has been regarded as 2(3)+3(2), such types as 3 (3) and 1 (4) were also found with high frequency. There fore, 2(3)+(3)2 can not be the maximum association. At pachytene, chromosome I showed associations with three different chromo somes. This chromosome could simultaneously associate with three others form ing a group of four. The distribution of association types may be considered to be a function of relative affinity and relative position. Taking it for granted that the association in a haploid cell indicates the presence of homologous chromosome segments, the situation disproves the hypothesis that the basic number of rice could be five. Acknowlegment The writer wishes to express his sincere thanks to the late Dr. Y. Takenaka and Dr. H. I. Oka of the National Institute of Genetics for their valuable suggestions and review of the manuscript. Literature cited Brown, S. W Supurious secondary association and asymmetric spindles in Luzula. Cytologia 15: Catcheside, D. G Secondary pairing in Brassica olerracea. Cytologia Fujii. Jub. Vol.: Darlington, C. D. and A. A. Moffett Primary and secondary chromosome balance in Tyrus. Jour. Genet. 22: Gustafsson, A Primary and secondary association in Taraxacum. Hereditas 20: Heilborn, O The mechanics of so-called secondary association between chromosomes. Hereditas 22:

9 1967 Pachytene Analysis Observatiaon of Chromosome Association in Haploid Rice 95 Heneen, W. K On the meiosis of haploid rye. Hereditas 52: Hirayoshi, I The chromosomal relationships in Oryzeae and Zinzanieae. Proc. Inter. Genet. Symposia, Tokyo Hu, C. H Karyological studies in haploid rice. II. Analysis of karyotype and somatic pairings. Jap. Jour. Genet. 33: Studies of meiosis in Oryza species with special reference to secondary association. Cytologia 27: Further studies on the chromosome morphology in Oryza saliva. L. Rice Genetics and Cytogenetics. (Elservier, Amsterdam): Jacob, K. M Secondary association in the Castor oil plant. Cytologia 22: Katayama, T On so-called secondary association in rice plants. I. Cytological observa tions. Jap. Jour. Genet. 40: Kempanna, C. and R. Riley Secondary association between genetically equivalent bivalents. Heredity 19: Kudo, A. and T. Katayama On so-called secondary association in the rice plants. II. Statistical analysis. Jap. Jour. Genet. 40: Kurita, M Secondary association of chromosomes in the castor oil plant. Jap. Jour. Genet, 21: 63. Kuwada, Y A cytological study of Oryza saliva L. Bot. Mag. Tokyo 24: Li, H. W., C. C. Chen, H. K. Wu and K. C. L. Lu Cytogenetical studies of Oryza saliva L. and its related species. 5. Differential condensation and chromosome pairing in the hybrid O, saliva ~O. australiensis. Cytologia 28: Nandi, H. K The chromosome morphology, secondary association and origin of cultivated rice. Jour. Genetics 33: Oka, H. I Considerations on the genetic basis of intervarietal sterility in Oryza saliva. Rice Genetics and Cytogenetics (Elsevier, Amsterdam): Pathasarathy, N Cytological studies in Oryza and Phalarideas. II. Farther studies in Oryza. Cytologia 9: Pathak, G. N Studies in the cytology of cereals. Jour. Genet. 39: Riley, R. 1960a. The secondary pairing of bivalents with genetically similar chromosomes. Nature 185: Sakai, K Chromosome study of Oryza saliva L. I. The secondary association of the meiotic chromosomes. Jap. Jour. Genet. 11: Shastry, S. V. S., D. R. Ranga and R. N. Misra Pachytene analysis in Oryza. I. Chromosome morphology in Oryza saliva. Ind. Jour. Genet. and Plant. Breed. 20: Thomas, P. T. and Revell, S. H Secondary association and heterochromatic attraction. Ann. Bot. 10: Tsuchiya, T Haploid plants in barley. Chromosome Information Service No. 3: Yasui, K Diploid-bud formation in a haploid Oryza saliva with same remarks on the behaviour of nucleolus in mitosis. Cytologia 11:

PLANT CYTOGENETICS. Ram J. Singh Department of Agronomy University of Illinois Urbana, Illinois. CRC Press Boca Raton Ann Arbor London Tokyo

PLANT CYTOGENETICS. Ram J. Singh Department of Agronomy University of Illinois Urbana, Illinois. CRC Press Boca Raton Ann Arbor London Tokyo f{ PLANT CYTOGENETICS Ram J. Singh Department of Agronomy University of Illinois Urbana, Illinois CRC Press Boca Raton Ann Arbor London Tokyo TABLE OF CONTENTS Chapter 1. INTRODUCTION 1 Chapter 2. THE