Heredity 57 (1986) 247-254 The Genetical Society of Great Britain Received 16 December 1985 Continuous variation in Y-chromosome structure of Rumex acetosa A. S. Wilby and School of Biological Sciences, J. S. Parker Queen Mary College, Mile End Road, London El 4NS. The dioecious angiosperm Rumex acetosa has an XXIXY1Y2sex-chromosomesystem. Each V-chromosome is heterochromatic except for a minute terminal euchromatic pairing segment. The Vs are constant in size but have a variable centromere position. The centromeres can be located anywhere within the central 40 per cent of the chromosome but are excluded from the two distal 30 per cent regions. In a sample of 270 males from 18 different populations 68 distinct variants have been identified on the basis of V-morphology. All populations are highly polymorphic with a minimum of four variants in a sample of ten males. The origin and significance of this massive variability is considered in this paper. Increased mutation rate of the Ys may be implicated in maintenance of this variation. I NTRO DUCTI ON these "inert" Ys has been described (Vana, 1972) variation in their structure has been overlooked. Sex-determinationin animals is usually genic and Extensive heterochroinatic content is a charac- frequently associated with visibly-differentiated teristic of many Y- and W-chromosomes. Indeed, sex-chromosomes. Sex expression in plants, some have argued that the process of hetero- however, is usually more plastic, and is subject to chromatinisation itself was implicated in the initial environmental influences such as temperature and phase of sex-chromosome differentiation (Jones, photoperiod (Heslop-Harrison, 1957). Addition- 1984). Highly-heterochromatic sex-chromosomes ally, even strictly dioecious plants seldom have exhibit hypervariability in many groups of organ- visibly-differentiated sex-chromosomes and con- isms and this is normally quantitative in character siderable controversy exists concerning the partic- (Hamerton, 1971; Mengden 1981; Vorontsov eta!., ular chromosomes involved in sex-determination 1980). In the human Y, for example, the long arm in species as diverse as the gymnosperm Ginkgo varies more or less continuously with respect to its biloba and the angiosperm Spinacia oleracea C-band content while a single population of the (Lewis and John, 1968). rodent Nesokia indica contained three distinct Y- Highly-differentiated sex-chromosomes have morphs in equilibrium proportions (Rao et a!., been found in a few plant species such as Melan- 1983). drium rubrum and Rumex acetosa. In M. rubrum Structural variation in sex-chromosomes a pair of X-chromosomes is found in pistillate independent of C-band content is unusual, but (female) plants, a single X and a larger Y in a remarkable case has been described in the W- the staminate (male) plants. Detailed studies of chromosomes of the gekkonid lizard Gehyra pur- this active-Y system have been carried out by purascens. Six W-morphs which differ by para- and Westergaard (1958). The subgenus to which Rumex pericentric inversions have been identified in a acetosa belongs is characterised by an aneuploid sample of only 30 females from 13 populations sex-chromosome system, XX in females and (Moritz, 1984). In Rumex acetosa we have found XY1Y2 in males (Kihara and Ono, 1925). Several that the two Y-chromosomes, which are almost studies have shown this to be a dosage compensa- completely heterochromatic, show massive struc- tion system analogous to that in Drosophila (Ono, tural but not quantitative variability. Centromere 1935). Although the heterochromatic nature of location in each Y varies more or less continuously 248 A. S. WILBY AND J. S. PARKER such that in 270 males 68 recognisably-distinct 1 and 6 sometimes carry heterochromatic segments complements have been found. The nature of this terminal on their short arms. distinctive sex-chromosome system is reported in The X-chromosome is metacentric. It is the this paper. largest member of the complement (7 pm), nearly twice the length of chromosome 1. During mitotic prophase the X-chromosomes and autosomes coil MATERIALSAND METHODS regularly, displaying no conspicuous blocks of heterochromatin. The two X-chromosomes in Theplants used in this study were grown from females have identical coiling patterns. seed collected in 18 natural populations of The Y1- and Y2-chromosomes, by contrast, are R. acetosa.Inthree populations—Navestock highly heterochromatic. They are distinguishable Heath, Essex, Torver, Cumbria and Hendra, Corn- from the rest of the complement on the basis of wall—seeds were collected from 40 females. In the coiling and staining at all stages of the mitotic other 15 populations, scattered widely through cycle except metaphase and anaphase. In inter- Britain, 10 seed samples were obtained. From each phase nuclei, one large or two smaller female parent a single male offspring was chromocentres are present. These may either examined giving a total of 270 males. The precise appear as dense masses or as extended chro- locations of these populations will be given in a mosome-like structures. Plants can be reliably later paper. sexed by observations of interphase nuclei. During For chromosome studies roots were pretreated mitotic prophase the Ys are uniformly dark- with 0.05 per cent colchicine for 2 hours, fixed in staining with no evidence of intercalary euchro- 1:3 acetic alcohol and stained by the Feulgen matin. Euchromatin is present, however, and is method. Meiosis was examined in PMCs fixed in limited to a very short terminal region on each Carnoy and stained in 2 per cent acetocarmine. Y-chromosome (fig. 3). These segments on Y1 and Y2 are probably the pairing segments, homologous with the distal regions of the X arms. RESULTS The Y-chromosomes are intermediate in size between the autosomes and the X-chromosome. The Karyotype The Y1 is on average 83 per cent of the X-chro- mosome length at metaphase, while the Y2 is 74 Pistillate (female) plants of Rumex acetosa have per cent of the X. Most frequently both Y-chro- 2n =14chromosomes comprising six pairs of auto- mosomes are metacentric. The position of the somes and a pair of X-chromosomes. The centromeres, however, is remarkably variable and staminate (male) plants carry a single X-chromo- the pattern of this variation is described below. some and two distinguishable Y-chromosomes, designated Y1 and Y2, giving a somatic number of orientation 2n =15. Sex-trivalent The six pairs of autosomes are individually Maintenanceof a 1: 1 sex ratio in R. acetosa recognisable on the basis of length, arm-ratio and depends on the segregation of both Y-chro- secondary constrictions (figs. 1-2). Chromosomes mosomes to the same pole at anaphase-I. This is fr '1 'sf1, Figure la and b' Chromosome complements of Rumexacetosa (a) 9, 2n = 14+1B;X-chromosomesarrowed. (b) d, 2n= 15+2Bs; X,Y1 and Y2 arrowed 'lIar in all figures represents 10 p.m. V-CHROMOSOME STRUCTURE OF RUMEX ACETOSA 249 (b) Y-chromosomes In conventionally-stained preparations chro- mosome morphology is defined by centromere position alone. In the Y-chromosomes of R. acetosa,however,the euchromatic pairing segment defines one telomere so centromere position in acrocentrics can be given a polarity. In about 40 per cent of the males (115/270) both Y1 and Y2 were metacentric while in the rest at least one Y was acrocentric. Four major classes can be defined on the basis of centromere position: both Ys meta- I centric (mm), Y1 or Y2 acrocentric (am and ma respectively), both Y1 and Y2 acrocentric (aa). In addition Y-polarity enables two classes to be dis- tinguished in am and ma and four classes in aa. We designate centric location nearer the pairing segment "+"andaway "—"(seefig. 3). All nine classes have been found in this sample of 270 males. Figure 2 Karyotype of Rumexacetosa, showingthe extreme Y1 and Y2 variants. The Ys are drawn with the pairing segments uppermost C, ensured by regular production of a linear sex- trivalent by two terminal chiasmata and its sub- •0 sequent orientation in a convergent manner (fig. 4). The system is extremely accurate. In 238 meta- .4,,. phase-I cells six trivalents in linear orientation (fig. 5)andthree bivalent+ univalent configurations were found (38 per cent abnormal PMCs) and at Figure 3 Mitotic prophase in male Rumexacetosa. Notethe anaphase-I only five PMCs out of 371 (1.3 per heterochromatic nature of the Ys. The euchromatic pairing cent) were XY: Y. Failure of regular segregation segments are arrowed should give numerical sex-chromosome variants in the progeny and in 1095seedgrown plants two Changes in the apparent position of the centro- XXY individuals were detected. meres could result from addition or deletion of chromosomal material. This can be excluded by comparison of chromosome length in meta- and Sex-chromosomestructural variants acrocentric Y-chromosomes. For Y1 the meta- (a)X-chromosome centrics average 822 per cent of X and acrocentrics The X-chromosome is very stable and few variants 839 per cent, for Y2 735 per cent and 747 per have been detected. Two females carrying single cent respectively. Rather than addition/deletion, acrocentric Xs and a male with an acrocentric X, rearrangements such as pericentric inversions are all of standard length, have been found, presum- implicated in centric transposition. ably resulting from pericentric inversion. In addi- Centromere position is defined by long-arm tion two different X/autosome interchanges have length as a proportion of total length. Thus a been detected: one female was heterozygous for metacentric has an arm-ratio of 05 and a telo- an X/3 interchange while two females and a male centric 1.0. The smallest deviation from meta- from the same population carried an X/2 centricity which can be reliably assigned a polarity exchange. with respect to the euchromatic segment gives an 250 A.