B-Chromsomes in Sorghum Stipoideum

Heredity 68 (1992) 457—463 The Genetical Society of Great Britain

B-chromsomes in Sorghum stipoideum

TA-PEN WU Department of Botany, National Taiwan University, Taipei, Taiwan, Republic of China

It is clear from pachytene analysis that the B-chromosome of Sorghum stipoideum is a euchromatic iso-chromosome which has probably originated from centromere misdivision of one of the A-chromosomes. B-bivalents behave in a more stable and regular fashion at meiosis than do B-univalents. These B-univalents frequently lag and divide precociously during male meiosis. Mosaicism of B-chromosomes is found in microsporocytes and tapetal cells, while they are totally eliminated from stems and leaves.

Keywords:B-chromosomes,elimination of B-chromosomes, mosaicism of B-chromosomes, pachytene analysis, Sorghum stipoideum.

obtained from identifiable pachytene chromosomes of Introduction other cells, as individual chromosome landmarks were B-chromosomeshave been reported in numerous plant not visible in every cell. and animal taxa (Jones & Rees, 1982). The origin and cytological behaviour of B-chromosomes in Sorghum Results nitidum and S. purpureosericeum have been described previously (Wu, 1980, 1984). This paper deals with the Pachytene chromosome morphology cytological behaviour of B-chromosomes which have been found in another Sorghum species, S. stipoideum. The synapsis of homologous A-chromosomes at The pachytene karyotype of the plant with B-chromo- pachytene was complete and normal in Sorghum somes was also analysed in order to compare the stipodeum. Typical pachytene A-chromosome bivalents morphology and relationship between A- and had darkly staining heterochromatic regions on both sides of the centromere and light staining distal euchro- B-chromosomes in this species. matic regions. As the appearance and length of the chromosomes varies constantly during pachytene, it Materialsand methods was difficult to identify individual chromosomes based Asingle 2B plant of Sorghum stipoideum was grown only on the chromosome lengths and the arm ratios. from seeds supplied by the Division of Plant Industry, However, each chromosome exhibited a definite pat- CSIRO, Australia. The present study was made using tern involving features such as length and position of 14 progeny produced by the 2B female parent. Young heterochromatic regions, position of deeply staining spikes were fixed in 3:1 ethanol acetic acid for 24 h for knobs, chromomeres and nucleolus organizers which cytological analysis and then stored in 70 per cent made it possible to identify individual chromosomes. ethanol. Meiosis in microsporocytes and somatic mito- Of the five A-bivalents at the pachytene stage, chromo- sis in anther walls and meristematic tissues were some 1 (Figs la and b and 2) is the most asymmetrical. studied by using the propiono-carmine squash tech- Its short arm is entirely heterochromatic. These con- nique. Observations and photomicrographs were made spicuous features make it easily distinguishable from using temporary slides. the rest of the complement. In this species, chromo- At the pachytene stage, measurements of chromosome 4 (Figs la, b, and e and 2) is the main nucleolus- some and heterochromatic lengths were made on nine organizing chromosome. The nucleolus is formed on cells in which all A- and B-chromosomes could be the short arm close to the centromere. Chromosome 3 followed from end-to-end and the positions of all (Figs la, b, e and 2) occasionally participates in organiz- centromeres were discernible. Measurements of lengths ing the nucleolus, and its nucleolus organizer is located and positions of chromosome landmarks were also at a subterminal position on the short arm. 457 458 TA-PENWU

2 s. - 3 "4 (b) Fig. 1 (a) and (b) photomicrograph and interpretative drawing of the pachytene chromosome complement of S. stipoideum. (c) B-univalent showing • inter-arm pairing at pachytene in the F, teSs ;J. lB plant. (d) 2B chromosomes showing 4'. <_ "! I— inter-chromosome pairing at pachytene r. in the 2B plant. (e) Pachytene stage z showing two chromosome bivalents associated with the nucleus: chromo- I,, I some 3 with a subterminal point of %/1 J attachment and chromosome 4 with an ( intercalary point of attachment. Bars ______Cd) Ce) equal 10 um in all Figures. Arrows 1' indicate the centromeres.

Themorphology and behaviour of the B-chromo- ized in Table 1. An ideogram has been constructed somes at pachytene were also studied. When two Bs (Fig. 2) incorporating the distinctive features of each were present they were always paired to form a hi- chromosome. valent, but did not pair with the A-chromosomes (Figs la, b, and d, 4a and b). The average length of the B-bivalent is 26.00 1.17 4um, which is distinctly shorter Thebehaviour of B-chromosomes during male than any of the A-bivalents. The B-bivalent (Figs la, b me/os/s and d and 2) has an approximately median centromere and is euchromatic along its whole length. The B, when Themeiotic behaviour of A-chromosomes was found present singly, exhibited inter-arm pairing with its to be normal in plants with and without B-chromo- centromere terminal (Fig. ic). The single B-chromosomes. However, the behaviour of the Bs depended some did not pair with the standard complement (Figs upon the number present. In the lB plants, the B was ic, 3a and b). The mean value of the B-univalent at univalent during diakinesis (Fig. 3a) and metaphase I pachytene is 13.60 0.60 um which is about half the (Fig. 3b). It divided precociously in the majority of AT length of the B-bivalent. It seems that the B-chromo- cells (67.4 per cent). In some cells, the two sister somes of S. stipoideum are all identical in structure, B-chromatids moved to opposite poles so that (—) and that they are iso-chromosomes. distributionresulted (Fig. 3d), and in some others, The measurements of total length, arm lengths, B-chromatids lagged (Figs 3e and f). In most of the heterochromatic lengths and arm ratio of the individual remaining cells (161 out of 195) the B moved A- and B-bivalents at the pachytene stage are summar- undivided to one pole, giving a chromosome distribu- B-CHROMOSOMES IN SORGHUM STIPO/DEUM 459 tion of (1—0) (Fig. 3c). In only 34 cells did the undi- and one-to-one pole of the two dyads (—0:--0) in 18.6 vided B lag on the equatorial plate. The B-chromo- per cent (Fig. 3h). The former distribution probably somes which did not divide at anaphase I divided at resulted from the (1—0) distribution of B-chromosome anaphase II. at anaphase I, while the latter resulted from precocious At anaphase II, 135 of the 204 dyad groups in the division at anaphase I. Most tetrads of lB plants con- lB plants had lagging B-chromatids, while the remain- tained micronuclei (72.2 per cent) (Fig. 3i). No micro- der had no laggards. Two daughter B-chromatids were nuclei were found in tetrads of normal plants. distributed to the opposite poles of one dyad and none Micronuclei must be formed by the eliminated to the other (—:0—0) in 15.2 per cent of cells (Fig. 3g), B-chromosomes. When two B-chromosomes were present, their 2 3 4 5 B meiotic behaviour was nearly regular. They were usually associated as bivalents at diakinesis (Fig. 4a) and metaphase I (Fig. 4b). The B-bivalent separation at anaphase I was as regular as A-bivalents with 97.3 per cent of Al cells showing normal disjunction. Con- sequently a (1—1) distribution was formed (Fig. 4c). II Rarely (1.7 per cent) the B-bivalent failed to disjoin and moved to one pole, giving a (2—0) distribution (Fig. 4d). Only eight cells (0.6 per cent) showed precocious division of B-chromsomes. Out of 1234 Al cells, only I five had B laggards (Figs 4e and f). B-chromosome division of the 2B plants usually occurred at the second I anaphase, giving distribution patterns of (—:—)in317 tetrads and (+ + :0—0)in only seven. The former pattern (Fig. 4g) has probably arisen from a (1—1) dis- H tribution at first anaphase, and the latter from a (2—0) distribution. No B laggards were seen at All. Examina- tion of 239 tetrads in 2B plants showed only four with Fig. 2 Pachytene ideograms of the five A-bivalents and the micronuclei. The meiotic distributions of the B-chromo- B-bivalent of S. stipoideum. Lengths of chromosome arms somes as described above are summarized in Tables 2 and heterochromatic regions were obtained from the data presented in Table 1. The spaces in the lines show centro- and 3. meres. The thin lines represent euchromatic regions, the solid black portions of lines represent heterochromatic Numericalvariation of the B-chromosomes in the regions and barrel shaped knobs, and the hollow portions of germ line and somatic cells lines represent the faintly dark stained segments. Small dots show chromomeres. Letter N indicates the nucleolus organ- Fourteenoffspring of the 2B female parent were izing regions. Bar equals 10 sum. examined cytologically at microsporogenesis. One is an

Table I Mean values hum) for pachytene chromosome lengths in Sorghum stipoideum Heterochromatic Heterochromatic Arm Chromosome Long Short region on region on short arm ratio pair number Total arm arm long arm 11.04 3.01 1 44.l1±1.17** 33.08±0.87 11.04±0.38 1.36 2 42.50±1.03 25.21±0.65 17.29±0.63 1.42 1.36 1.47 1.90 3* 39.71±1.04 25.96±0.69 13.75±0.48 1.19 1.29 1.37 4* 35.03±1.00 20.25±0.65 14.78±0.41 1.37 1.40 1.14 5 31.44±1.13 16.70±0.62 14.75±0.58 1.42 1.27 lB 13.60±0.60 2B 26.00± 1.17 13.69 12.31 1.12

*Nucleolus..organizing chromosome. **Standard error of the mean. 460 TA-PENWU 4' S C tsr t a 4; a p a, j (a) ___) (b) — (c) •

"3, S S S a - H 4 3. if, IA Fig. 3 (a) Diakinesis with five A-biva- (e) lents and one B-univalent. (b) Meta- phase I with five A-bivalents and one .'b B-univalent. (c) Anaphase I of the lB 2 'I plant. The B-chromosome has moved — g at to one of the poles. (d) Anaphase I of —. 'at.' the lB plant. The B-chromosome has •-'. • a a •! l. precociously divided into two chroma- 4 tids and the two members move to the / opposite poles. (e) Anaphase I of the — (9) lB plant. The B-chromosome divides and lags behind. (1) Anaphase I of the lB plant. The B-chromosome has precociously divided into two chromatids. One member moves to the pole and the ) q. ,, other lags. (g) The lB plant showing ft (—:0—0) distribution of B-chromatids

V at anaphase II. (h) The lB plant show- a ing (—0:—0) distribution of B- chromatids at anaphase IL (1) Tetrad ii) group of the lB plant with micronuclei. Bars equal 10 m in all Figures.

OBplant, while the rest contain B-chromosomes. In containing individuals, but not in normal plants. All nine of these B-containing plants, mosaicism was cells of stem and leaf meristems in plants with and observed for the presence or absence of B-chromo- without B contained only 10 chromosomes (Fig. 4h). somes between spikes. Among these nine plants four B-chromosomes are totally eliminated from these showed between-spikelet variation in B number. The vegetative tissues. remaining four plants with Bs had rather uniform B-numbers in their inflorescences. Two of these had lB Discussion in nearly all cells, only an extremely low percentage of cells possessed no Bs (0.5percent). Both of them are Basedon pachytene analysis (Wu, 1980, 1984), the regarded as lB plants, and in the other two individuals B-chromosomes of Sorghum nitidum and S. purpureo- with B-chromosomes, 2B is the predominant number sericeum are known to be heterochromatic iso- (99 per cent), OB, lB and 3B cells could be found in an chromosomes with the same origin. They probably unusually rare frequency (1 per cent). They may be arose from a nucleolus-organizing chromosome by loss considered as 2B plants. of the long arm after centromere misdivision and sub- In the somatic cells, the Bs (Figs 4i and j)areclearly sequent structural change. This opinion is based on the distinguishable from the A-chromosomes. The number fact that the B-chromosome at pachytene bears a strik- of B-chromosomes in tapetal cells was unstable and ing resemblance to the short arm of the nucleolus- apparently decreasing. During interphase, a micro- organizing chromosome. Centromere misdivision and nucleus (Fig. 4k) was occasionally observed in B- formation of iso-chromosomes is an important origin B-CHROMOSOMES IN SORGHUM STIPOIDEUM 461

"I. I,

La • • C. C. C -, (b) (c)

.7 V Fig. 4 (a) Diakinesis with five A-bivalents and one B-bivalent. (b) Metaphase 5. I with five A-bivalents and one B-biva- (e) lent. (c) The 2B plant showing regular segregation of the 2B chromosomes at anaphase I. (d) The 2B plant showing V. — non-disjunction of the 2B chromo- • • •4 F somes at anaphase I. (e) The two B I. chromosomes laggard behind at ana- a a 1, phase I of the 2B plant. (f) The 2B plant showing the two divided B-chromo- —. — somes laggard behind at anaphase I. (g) The 2B plant showing (——) — (9) _It distribution of B-chromatids at anaphase II. (h) The intercalary men- stematic cell of the lB plant at prophase containing no B-chromosome. (i) The tapetal cell of the lB . BC9 plant at prophase containing a B-chromosome. (j) The tapetal cell of 2 ' the 2B plant at metaphase containing ;r two B-chromosomes. (k) A tapetal cell 2 of the lB plant at interphase containing a micronucleus. Bars equal 10 um in all (i) '¼ N (k) Figures. —

of B-chromosomes (Battaglia, 1964) as proposed for meiotic behaviour by Weimarck (1978). One is repre- Gasteria (Darlington & Kefallinou, 1957). It is clear sented by Hierochloë (Weimarck, 1978), in which the that the B-chromosomes have probably also originated B-chromosomes frequently show aberrations in cen- from centromere misdivision of an A-chromosome and tromeric activity. These B-chromosomes usually lag subsequent structural changes, but the resemblance and divide precociously when univalent. They fre- between B and the A-chromosome complement of quently fail to be included in the daughter nuclei and a S. stipoideum cannot be established. considerable amount of elimination may occur during In the B-containing individuals of S. stipoideum, the male meiosis. The B-chromosomes of S. stipoideum fall number of B-chromosomes was not constant in pollen into this category. The other category is represented by mother cells. Analysis of the mosaicism revealed the B-chromosomes of Anthoxanthum and many other numerical variation not only between spikes of the grasses (Ostergren, 1947; Bosemark, 1957). These same plant but also within spikelets. By contrast, the B-chromosomes rarely divide into chromatids at ana- B-chromosome of Sorghum nitidum was meiotically phase I. They seldom lag or are eliminated during stable (Wii, 1980). B-chromosomes of different organ- meiosis. A stable system of centromeric activity under isms were classified into two categories on the basis of univalent conditions seems to have been acquired by 462 TA-PENWU

Table2Meioticbehaviour of the B-univalent in the lB plants

Distributions of B-univalents at Al Number of cells observed Per cent

1—0 161 26.92 236 39.46 2 0.33 Laggards 0—1—0 34 5.69 0—+—0 142 23.75 23 3.85 Total number of cells 598

Distributions of B-univalents at All Number of dyad groups observed 31 15.20 38 18.63 Laggards 86 42.15 49 24.02 Total number of dyad groups 204

Note: shows B chromatid. 1 represents B chromosome.

Table 3 Meiotic behaviour of the B-bivalent in the 2B plants

Distributions of B-bivalents at A! Number of cells observed Per cent

1—1 1201 97.33 12—0 II 1 21 1.70 2 22 2 . 1—+ 2 0.16 1+— 1 0.08 Laggards 0—2—0 2 0.16 1—1—0 2 0.16 0— +,+—0 1 0.08 Total number of cells 1234

Distributions of B-bivalents at All Number of dyad groups observed 1 1.1 1 22.22 .7 97.84 7 2.16 Laggards 0 0 Total number of dyad groups 324

Note: I shows B chromatid. 1 represents B chromosome. these B-chromosomes during evolution. The meiotic other plants. In Sorghum purpureo-sericeum, Xan- behaviour of B-chromosomes inS. nitidum(Wu, 1980), thisma texanum and Aegilops speltoides (Darlington & place it in the Anthoxanthum class. Thomas, 1941; Berger & Witkus, 1954; Mendelson & The B-chromosome of S. stipoideum is variable Zohary, 1972) the B-chromosomes are eliminated in number in the microsporocytes, but the number from the roots but preserved in shoots and germ cells. typically decreases in the tapetal tissue, and is zero Poa alpina has B-chromosomes in primary roots and in the stem and leaf meristems. Complete loss of the pollen mother cells, but they are excluded from adven- B-chromosomes from some tissues has been found in titious roots and leaves (Muntzing & Nygren, 1955). B-CHROMOSOMES N SORGHUM STIPOIDEUM 463

The mechanisms of B-elimination are not known, but DARLINGTON, C. D. AND KEFALLINOU, M. 1957. Correlated may result from non-disjunction and lagging at mitotic chromosome aberrations at meiosis in Gasteria. Chroino- soma, 8, 364—370. anaphase. DARL!NGTON, C. D. AND THOMAS, P. T. 1941. Morbid mitosis and the activity of inert chromosomes in Sorghum. Proc. Roy. Soc. London B, 130, 127—250. Acknowledgments JONES, R. N, AND REES, H. 1982. B chromosomes. Academic Theauthor sincerely thanks Dr J. S. Parker of Press, London. University of London for valuable suggestions and for MENDELSON, D. AND ZOHARY, D. 1972. Behaviour and trans- critical reading of the manuscript. This work was sup- mission of supernumerary chromosomes in Aegilops spel- ported by a grant NSC78-0211-B002-31 from the toides. Heredity, 29, 329—339. National Science Council, R.O.C. MUNTZING, A. AND NYGREN, A. 1955. A new diploid variety of Poa alpina with two accessory chromosomes at meiosis. Hereditas 41,405—422. References OSTERGREN, G. 1947. Heterochromatic B chromosomes in Ant hoxanthum. Hereditas, 33, 261—296. BATrAGLIA,E. 1964. Cytogenetics of B-chromosomes. Caryo- WEIMARCK, G.1978.Behaviour of B chromosomes in logia, 17, 245—299. Hierochloè' repens (Gramineae) during male meiosis. BERGER, C.A.AND WITKUS, E. R. 1954. The cytology of Xan- flereditas, 88,7—11. thisma texanum DC. I. Differences in the chromosome WU TA-PEN. 1980. Cytogenetic studies of the B chromosomes number of root and shoot. Bull. Torry Bog. Club, 81, in Sorghum nitidum. Proc. Nat. Sci. Counc. ROE. 4, 489-491. 297—306. BOSEMARK, N. o. 1957. Further studies on accessory chromo- WU TA-PEN. 1984. B chromosomes in Sorghum purpureo- somes in grasses. Hereditas, 43, 236—297. sericeum. Proc. Nat. Sci. Counc. B. ROC. 8, 198—209.