Parental genome separation in reconstructions of somatic and … · 2005. 8. 22. · Hordeum vulgare cv. Tuleen 346 (barley) X H. bulbosum was shown to have seven chromosomes originating

Journal of Cell Science 101, 13-24 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

13

Parental genome separation in reconstructions of somatic and premeiotic

metaphases of Hordeum vulgare x H. bulbosum

T. SCHWARZACHER, J. S. HESLOP-HARRISON, K. ANAMTHAWAT-J6NSSON

Karyobiology Group, Department of Cell Biology, JJ Centre for Plant Science Research, Norwich NR4 7UJ, UK

R. A. FINCH and M. D. BENNETTJodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK

Summary

A stable interspecific sexual plant hybrid betweenHordeum vulgare cv. Tuleen 346 (barley) X H. bulbosumwas shown to have seven chromosomes originating fromeach parent by genomic in situ hybridization. Electronmicroscope serial thin-section reconstructions of meta-phases and comparison with light micrograph karyo-types enabled chromosomes to be identified from theirmorphology. The three-dimensional positions of then-centromeres were established and analysed in thereconstructions of somatic root tip metaphases and cellsat mitotic metaphase near their entry hi to meiosis.Parental genomes tended to lie in spatially separated

domains in both tissues. Although varying in mor-phology, the two sets of chromosomes had similar meansizes, so size differences did not cause the separationobserved. In the EM, the centromere-associated struc-tures of the chromosomes of the more central genome,originating from H. vulgare, were larger than those ofthe more peripheral genome of H. bulbosum origin.

Key words: nuclear organization, nuclear architecture,three-dimensional reconstruction, meiosis, Hordeum,genome separation.

Introduction

The spatial separation of parental genomes at meta-phase has been reported in many intergeneric andinterspecific hybrids, and species, from both plants(Finch et al. 1981; Bennett, 1982; Gleba et al. 1987;Heslop-Harrison and Bennett, 1984, 1990; Linde-Laursen and Jensen, 1991) and animals (Ordartschenkoand Keneklis, 1973; Zelesco and Marshall Graves,1988; Brandriff et al. 1991). The phenomenon isimportant because of the consequences that spatialorganization of chromosomes in the nucleus might havefor mechanical chromosome behaviour, gene ex-pression (transcription) and DNA replication. Aspectsof genome evolution - including speciation - may also beaffected by nuclear organization. The study of thephysical organization of the chromosomes within thenucleus of man-made and natural hybrids will assist theunderstanding of such phenomena.

Wide hybrids, such as barley {Hordeum vulgare) xH. bulbosum discussed here, are important for plantbreeding. The genetic base of many crops, particularlyinbreeding species like barley is very narrow. Newgenes can be introduced by crossing with wild relatives,such as H. bulbosum, before backcrossing to produceimproved varieties (see Snape et al. 1991). A second

application of wide hybrids relies on uniparentalchromosome elimination. New cereal crops must beuniform, which normally needs many generations ofinbreeding following intercrossing of two varieties. Thecross H. vulgare x H. bulbosum is used in barleybreeding because, in some genotypes, the H, bulbosumchromosomes are eliminated, giving a haploid plant thechromosome number of which can be doubled withdrugs such as colchicine. The doubled haploid ishomozygous ("true breeding") and hence can beselected immediately for agronomic characters withoutvariation. Understanding of stable and eliminatinghybrids, and knowledge of the mechanisms of hybridstability, may enable favourable manipulation in breed-ing programmes.

We have previously described genome separation inthe wide hybrids H. vulgare X Secale africanum (Finchet al. 1981) and H. chilense X S. africanum (Schwarz-acher-Robinson et al. 1987). In these intergenerichybrids, the individual chromosomes of Hordeumorigin were all smaller than those of S. africanumorigin. Reconstructions of mitotic metaphase cellsshowed that centromeres of the two parental haploidsets tended to be spatially separate, and centromeresfrom one particular parent usually tended to be in theperipheral region of the metaphase plate that sur-

14 T. Schwarzacher and others

rounded the central region containing the centromeresfrom the other parent. In the hybrid H. vulgare x S.africanum, the centromeres of the larger chromosomesfrom S. africanum tended to be peripheral, while in H.chilense X S. africanum, the centromeres of the smallerchromosomes from H. chilense tended to be peripheral.As centromeres of the parental set with the largerchromosomes (i.e. Secale) can be either less or moreperipheral than centromeres from the parental genomewith the smaller chromosomes (i.e. Hordeum), atendency for a concentric separation of parentalgenomes is not a packing phenomenon determined bychromosome size per se, but is presumably undergenetic control (Schwarzacher-Robinson et al. 1987).These papers described genome separation in meta-phases from intergeneric hybrids, where genomesdiffered substantially in DNA sequence, and all thechromosomes of one genome were bigger than those ofthe other genome. Here, we aimed to examinecentromere disposition at metaphase in hybrids be-tween related species of the same genus, where the twoparents have very similar mean chromosome sizes.

Chromosomes vary in their behaviour at differentstages of the life cycle. In somatic tissues of variousdiploid cereal species, we have shown that there is noassociation of homologous centromeres at mitoticmetaphase (Heslop-Harrison et al. 1988). At meiosis,chromosomes from the two parental genomes cometogether and pair, which involves substantial physicalmovement of homologous pairs of chromosomes. Thework reviewed by Loidl (1990) suggests that somealignment of chromosomes is present by the first stageof meiotic prophase when chromosome strands can firstbe visualized. It is unknown when the movement fromnon-association to association starts, and few studies ofcentromere disposition at the important premeioticstages before meiotic prophase I (Bennett, 1979,1984a)have been carried out. Therefore, the second aim of thepresent work was to examine centromere disposition inboth root tip (somatic) cells and male premeioticmetaphases.

Materials and methods

Plant materialThe sexual Fl hybrid individuals (code number C245 80/7 forelectron microscopy and C244 80/15 for light microscopy)between Hordeum vulgare L. cv. Tuleen 346 (barley; 2n = 2x= 14; Finch and Bennett, 1982) and H. bulbosum L. clone L6(2n = 2x = 14; Simpson et al. 1980) were obtained on thesame day in 1980 by cross pollination and embryo cultureusing standard techniques. Ramets of the plants used weremaintained in growth cabinets and glasshouses for more than20 months before analysis.

To initiate new root growth, vegetative ramets were splitfrom hybrid plants in soil and grown in hydroponics (Finch etal. 1981) for 4 to 6 days. Root tips were excised and placed inice-water for 24 h before fixation.

In situ hybridizationSpread chromosome preparations were made from ethanol/acetic acid-fixed root tips. In situ hybridization and fluo-

rescence microscopy were modified from Schwarzacher et al.(1989) and Anamthawat-J6nsson et al. (1990). Total genomicDNA from H. bulbosum was labelled with digoxigenin-11-dUTP and used as a probe with 35-fold excess of unlabelledgenomic DNA from H. vulgare to block non-specifichybridization. Sites of hybridization were detected withfluorescein isothiocyanate (FITC) conjugated to sheep anti-digoxigenin and amplified with rabbit anti-sheep secondaryantibody conjugated to FITC. Chromosomes were counter-stained with propidium iodide.

Electron microscopy (EM)Root tips

Root tips were fixed in 5% glutaraldehyde in 0.1 M phosphatebuffer (pH 6.8) for 4 h. They were embedded in Spurr's resin,sectioned (0.1 /an thick) and stained in lead citrate and uranylacetate for electron microscopy using standard techniques(see Schwarzacher-Robinson et al. 1987).

AnthersPlants came into flower under variable conditions withincreasing day length. One of the three synchronouslydeveloping anthers in each floret was removed, measured andstained with aceto-carmine. Archesporial cells were identifiedwithin the anther loculi and their chromosome morphologywas examined in the light microscope. When an antherincluded male archesporial cells at a late or the last mitosisbefore meiosis (a premeiotic mitosis), the other two anthersfrom the same floret were fixed for 19 h and prepared for theEM as described above.

Serial section reconstructionThe techniques for reconstruction were similar to thosedescribed previously (see Heslop-Harrison and Bennett,1983). All sections through metaphases selected only forcompleteness were photographed in the transmission electronmicroscope and printed at a final magnification of 11,800. Ineach metaphase, the volume of each chromosome arm, of thesatellite when present and of the centromere-associatedstructures, was measured by summing the areas (/an2) cut oneach consecutive print and multiplying by 0.1 /an, the sectionthickness.

For each metaphase, a "composite transparency" withprojections of the cell wall and the central centromereposition for each centromere, as seen in the plane of thesections, was made using previously established methods(Heslop-Harrison and Bennett, 1983). Two arbitrary axeswere erected at right angles near the outside of the cell in theplane of the micrographs. The coordinates, X' and Y', of eachcentromere were taken from these axes and entered into amicrocomputer through a digitizing tablet. The coordinate inthe Z' axis was equal to the serial section number where thecentre of the centromere-associated structures occurred.Coordinates were converted to micrometers using the printmagnification (x 11,800) for X' and Y', and the sectionthickness (0.1 /an) for Z'. To visualize the metaphase plate asseen from a spindle pole, the central centromere coordinateswere rotated until the minimum possible variance was presentin the rotated Z axis, and the maximum variance was in the Xaxis (see Heslop-Harrison and Bennett, 1983). Original datafor arm volumes and centromere positions are available fromthe authors.

For each metaphase, the centre of the metaphase plate wasestablished as the mean three-dimensional position of all itscentromeres (the centroid; see Finch et al. 1981). Distances tothe mid-centromere position were calculated in /an for all

Fig. 1. A metaphase chromosome preparation from a roottip of the sexual hybrid plant Hordeum vulgare (barley) xH. bidbosum after genomic in situ hybridization usingdigoxigenin-labelled H. bulbosum DNA as a probe. Sitesof hybridization were detected with yellow-fluorescingfluorescein conjugates. Chromosomes were counterstainedwith orange-red-fluorescing propidium iodide. Sevenstrongly labelled chromosomes of H. bulbosum origin arepresent in the metaphase with seven less-labelledchromosomes of H. vulgare origin. Bar, 10 jon.

Genome separation in reconstructed metaphases 15

centromeres and converted to percentages of the sum of all 14centromere distances in each metaphase. The averagedistance for each genome in each metaphase and in the pooleddata was determined.

Genome separation or a tendency towards genome separ-ation was tested by drawing either a circle or a polygonaround the centromeres of the chromosomes from oneparental genome and assessing the positions of the centro-meres of the other parental genome in relation to the circle orpolygon on the X and Y axes of the rotated plots. Genomeseparation or a tendency towards genome separation wastaken to be present when the results differed significantlyfrom random expectations (Schwarzacher-Robinson et al.1987).

DNA measurementsDNA contents of prophase (4C) nuclei from the parental andhybrid plants were measured by Feulgen microdensitometryof root tip spreads using standard techniques (Bennett andSmith, 1976).

Results

In situ hybridizationIn situ hybridization using total genomic DNA as aprobe permits identification of the parental origin ofchromosomes in plant hybrids (Schwarzacher et al.1989; Anamthawat-J6nsson et al. 1990). Fig. 1 shows afluorescent light micrograph of a metaphase from a roottip chromosome spread of the hybrid H. vulgare X H.bulbosum. Total genomic DNA from H. bulbosum

together with an excess of unlabelled DNA from H.vulgare enabled the parental origin of all chromosomesto be determined. The sites of probe hybridization,detected by yellow fluorescence, were preferentially onthe H. bulbosum-origin chromosomes, while the H.vu/gare-origin chromosomes showed only the orange,propidium iodide, counterstain. All metaphases with 14chromosomes had seven chromosomes originating fromeach parent.

Reconstruction of metaphasesSix root tip metaphases (code numbers 82,83,90, 92,93and 102, all from the same root tip) and four premeioticmetaphases (code numbers 103, 104, 110 (one anther)and 112 (a second anther from the same spike)) werereconstructed from one plant. Fig. 2 shows a typicalsection through an anther loculus. Archesporial cells atinterphase (Fig. 2A) are seen next to metaphase cell 104(Fig. 2A, B). The number of sections per metaphaseranged from 70 to 189. In four metaphases one section,and in two metaphases two sections, were obscured ormissing (an overall loss of 8 in 1188 sections or 0.7%).One section was thicker than normal in each of fivemetaphases. In each metaphase, 14 chromosomes werepresent.

Appearance of centromere-associated structuresCentromere-associated structures were identified bytheir appearance as less electron-dense areas in thedark pieces of chromatin (Fig. 2B) at a corresponding

Fig. 2. Electron micrographs through an anther loculus of the hybrid H. vulgare x H. bulbosum, showing two sections ofthe reconstructed metaphase coded 104. (A) Section number 15 at low magnification, showing the relationship of thepremeiotic, archesporial, cells, to the anther tapetum and wall. Chromatin at both interphase and metaphase is seen aselectron-dense areas. Bar, 10 um. (B) A higher magnification micrograph of section 21, 0.7 um above A. The identitynumbers of the chromosomes are given (in black for H. bulbosum and white for H. vulgare; numbers are the identity ofthe centromere, so a white 1 indicates Tl-5, and a black 3, B3). Some chromosomes are bent and hence seen in two parts.Centromere-associated structures of three chromosomes of H. vulgare origin can be seen as lighter grey areas(arrowheads). Bar, 2 um.


position on several successive sections (illustrated, seeHeslop-Harrison and Bennett, 1983, where it is referredto as a "centromere"). In the hybrid H. vulgare x H.bulbosum, the centromere-associated structures variedin their clarity, although most of the chromosomes hadclear structures. On four of the chromosomes (lateridentified as originating from H. bulbosum) clearcentromere-associated structures were not visible, andthe position of the centromere was deduced from bendsor constrictions in the chromatids.

Chromosome morphologyNo difficulties were encountered in measuring thevolumes of chromosome arms. Compensations weremade for the very few missing, obscured or thicksections. In each cell, the chromosomes were ranked bytheir total volume (using the sum of the volumes of thetwo arms), and arm volume ratios (the size of the largerarm divided by that of the smaller arm) were calculated.Individual chromosome volumes were converted topercentages of the total volume of all chromosomes ineach cell before comparison with chromosomes in othercells and published karyotypes of the two parentalspecies.

In some chromosomes a second lighter area, inaddition to that at the centromere, together with a gapor sharp constriction, was seen at the position of thenucleolar organizer region (NOR). This secondaryconstriction separated a distal portion (the satellite)from the rest of the chromosome arm, and its presencewas used to assist in chromosome identification.

Chromosome identificationResults from in situ hybridization (Fig. 1) and previousstudies of the karyotypes of H. vulgare cv. Tuleen 346(which includes three reciprocal translocations involv-

ing six of the seven chromosomes; Finch and Bennett,1982) and H. bulbosum (Lange and Jochemsen, 1976)indicate that the parental origin of all the chromosomescan be determined in the hybrid by sizes, arm ratios andthe presence of NORs. Examination of the rankedvolumes of the chromosomes in the reconstructionsconfirmed that the chromosomes from H. vulgare couldbe separated from those from H. bulbosum by theirvolume, arm volume ratios and the presence or absenceof secondary constrictions at the NORs. Moreover,most individual chromosomes could be identified. Fig. 3shows the mean volumes of each chromosome typeplotted against the mean arm volume ratios. Themorphology of all chromosomes of H. vulgare origin issignificantly different from that of chromosomes of H.bulbosum origin. Most individual chromosome typescould be identified from volume and arm volume ratio,although in some cells, the identification of chromo-somes 2 and 3 of H. bulbosum was less certain. Fig. 4 isan idiogram of the average morphology of all thechromosomes.

All ten cells had seven chromosomes originating fromeach parent. Three chromosomes from H. vulgare (T2-6, Tl-5, and T3-7; Finch and Bennett, 1982) were largerthan any chromosome from H. bulbosum, and T3-7always had an expressed NOR. T4 was generally largerthan the chromosomes from H. bulbosum, although intwo cases, it was similar in size and arm volume ratio toa chromosome from H. bulbosum. Chromosome T7-3had a high arm volume ratio that always distinguished itfrom any H. bulbosum chromosome. Chromosome T6-2 always had an expressed NOR, while T5-1 was thesmallest chromosome in every metaphase (see Figs 3,4).

The remaining seven chromosomes had sizes andmorphologies that were compatible with their origin

3-

O 2.5-

1 -

T7-3

-5

T2-6

4 5 6 7 8 9

Volume (%)10 11

Fig. 3. A scatter diagram ofarm volume ratio againstvolume showing the means andstandard deviation for allchromosomes in tenreconstructions of the hybridH. vulgare x H. bulbosum.Identities of the chromosomesare indicated next to thepoints. Asterisks indicatechromosomes identified by thepresence of a secondaryconstriction at the nucleolarorganizer region rather thanvolume criteria. Allchromosomes can be assignedto their genome of origin, andmost individual chromosometypes are separated from anyother.


T1-5 T2-6 T3-7 T4 B1 B2 B3 T7-3 B7 B4 B6 B5 T&-2 T6-1

IVol (*,) 10.60 10.33 9.07 6.03 7.19 7.14 7.13 6.73 6.69 6.30 5.88 B.47 6.12 4 43

AVU 1.47 1.28 0.87 1.20 1.06 1.38 1.4S 2.88 2.27 1.40 109 1 61 1 74 1.60

Fig. 4. An idiogram of the hybrid H. vulgare X H.bulbosum from volume measurements of ten reconstructedserially sectioned metaphase cells. For each chromosometype relative mean volumes (Vol, in percentages of the sumof all chromosome volumes per cell) and arm volume ratios(AVR, long arm volume divided by short arm volume) aregiven. Chromosome types are arranged by decreasingvolume. Chromosome types from H. vulgare (black, filled)are numbered after Finch and Bennett (1982) and thosefrom H. bulbosum (gTey, hatched) after Lange andJochemsen (1976, B2 and B3 are reversed). The NORs ofchromosomes T3-7 and T6-2 from H. vulgare wereexpressed and the mean size of the satellite was calculatedand drawn accordingly. The NOR of chromosome B6 fromH. bulbosum was not seen in the hybrid. Its position isindicated by a line (based on two NORs in a reconstructeddiploid H. bulbosum cell).

from the H. bulbosum genome. The standard karyo-type numbering system of Lange and Jochemsen (1976)was used, but chromosomes 2 and 3 were reversed (nowB3 and B2) as described by Heslop-Harrison andBennett (1984).

The three largest chromosomes from H. bulbosum,Bl , B2 and B3, were similar to one another (Figs 3, 4).The arm volume ratio of Bl was lower, while B2 wasseparated from B3 by its lower arm volume ratio andgenerally higher volume. In metaphases 104 and 110,however, identifying B2 from B3 was uncertain. Thetwo similar sized chromosomes, B4 and B7, and the twosmallest chromosomes, B5 and B6, have distinct armvolume ratios (Figs 3,4). The NOR of chromosome B6was not expressed in any cell.

It is important to note that each reconstructed cellcontained a clearly identified complete haploid set ofseven characteristic chromosomes from H. vulgare withclear centromere-associated structures, and seven re-maining chromosomes from H. bulbosum. Thus, therewas no problem in identifying individual chromosomesaccording to their parental origin. The only potentialambiguity could be in distinguishing between particularchromosomes of known H. bulbosum origin in threecells.

DNA measurements and volumes of each genomeFeulgen microdensitometry indicated that the 4C DNAcontent of H. vulgare cv. Tuleen 346 was 22.72 pg, of H.bulbosum L6 was 21.45 pg and of the interspecifichybrid, 22.05 pg. The DNA content of the hybrid is notsignificantly different from the expectation of 22.09 pg,the average of the DNA contents of the two parents.After identification of the parental origin of eachchromosome, the total volume of each genome wascalculated in the ten cells (Table 1). The average ratioof the volumes of the genomes of H. vulgare to H.bulbosum is 1.18:1 (54.2:45.8), which is highly signifi-

Table 1. Total chromosome volumes (pun3) from reconstructed root tip and premeiotic metaphases o / H . vulgarecv. Tuleen 346 X H. bulbosum and the proportion (as percentages) represented by each parent

Metaphaseorigin

Root tip

Premeiotic

Pooled data

s.d.=standard deviation.

Cellnumber

8283909293

102Mean (s.d.)

103104110112

Mean (s.d.)

Mean (s.d.)

Total

(m3)103.9109.090.584.689.387.494.1 (9.0)

82.496.3

114.8116.9102.60 (14.1)

97.5 (12.1)

Chromosome volume

H. vulgare(%)

54.953.554.053.354.853.854.0 (0.6)

55.354.054.154.454.5 (0.6)

54.2 (0.6)

H. bulbosum(%)

45.146.546.046.745.246.246.0 (0.6)

44.746.045.945.645.5 (0.6)

45.8 (0.6)


Table 2. Mean centromere volumes (f-im3) and standard deviation (s.d.) for the chromosomes from H. vulgareand H. bulbosum in ten reconstructed root tip and premeiotic metaphases in the hybrid

Metaphaseorigin

Root tip

Premeiotic

Cellnumber

82

83

90

92t

93

102

Mean (s.d.)

103

104

110

112

Mean (s.d.)

Genome

H. vulgareH. bulbosumH. vulgareH. bulbosumH. vulgareH. bulbosumH. vulgareH. bulbosumH. vulgareH. bulbosumH. vulgareH. bulbosum

H. vulgareH. bulbosumH. vulgareH. bulbosumH. vulgareH. bulbosumH. vulgareH. bulbosum

Mean

0.0800.0390.1610.0730.1090.0540.0960.0610.1250.0660.1140.069

0.1730.0720.1170.0190.1770.0410.2850.080

Centromere volume (;a

(s.d.)

(0.019)(0.021)(0.035)(0.041)(0.015)(0.027)(0.026)(0.026)(0.024)(0.023)(0.022)(0.020)

1:1.90 (0.22)

(0.077)(0.030)(0.018)(0.015)(0.026)(0.024)(0.069)(0.047)

1:4.12 (1.36)

m3)

Ratio

2.05:1

2.21:1

2.02:1

1.57:1

1.89:1

1.65:1

2.40:1

6.15:1

4.32:1

3.56:1

Mest

t

3.627

4.039

4.368

2.269

4.300

3.717

2.999

10.494

9.562

5.670

n**• *

***

•

• •

*

* • *

* * *

• • *

Ratios and results of one-tailed Mests significance (12 degTees of freedom) are given.tThe centromere of chromosome B2 was obscured by a scratch, so the centromere is omitted from the analysis (11 degrees of freedom).%P, probability; • = P<0.05, *• = /><0.01, *** = P<0.001.

cantly different from the expectation of 1.06:1 based onDNA contents.

Volumes of centromere-associated structuresThe volumes of the lighter grey areas of the centro-mere-associated structures of the identified chromo-somes are given in Table 2. The Mests showed that thestructures from H. bulbosum were significantly smallerthan those from H. vulgare. The difference in averagevolume between the two genomes was higher in thepremeiotic cells than in the root tip cells.

Positions of centromeresPolar views of the positions of the centromeres areshown for the reconstructed root tip metaphases in Fig.5, and for premeiotic metaphases in Fig. 6. In all tencells, the centromeres of chromosomes from H. vulgarewere closer to the mean position of all the centromeresthan were the centromeres of chromosomes from H.bulbosum (Table 3). The tendency for the centromeresfrom H. bulbosum to be more peripheral is seen in Figs5 and 6.

Two methods were used to test for parental genomeseparation. In the first test, a cell was taken to showconcentric separation if it has a significantly greatergenome mean distance (GMD) to the mid centromereposition for one genome than for the other genome(Finch et al. 1981). GMDs for the hybrid H. vulgare XH. bulbosum, and the results of one-tailed Mestsbetween the GMDs are given in Table 3. In all tenmetaphases, the GMDs for H. bulbosum centromeres

exceeded those for H. vulgare. Individual differenceswere significant in two of the six root tip metaphasesand three of the four premeiotic metaphases. In pooleddata, the centromeres of the chromosomes from H.bulbosum were very highly significantly farther fromthe mid-centromere position than were the centromeresof the chromosomes from H. vulgare, showing a veryhighly significant tendency towards concentric parentalgenome separation.

In the second test, genome separation was taken tooccur when all centromeres from one parental genomelay within a circle drawn in the plane of the metaphaseplate that excluded all centromeres from the otherparental genome, a distribution expected to occur bychance alone in not more than 1.23% of cells (Bennett,1984b). Such separation occurs in three premeioticmetaphases (Fig. 6).

In Schwarzacher-Robinson et al. (1987), the test forgenome separation was extended to use polygons ratherthan circles to accommodate irregular cell shape. Thepolygons of minimum perimeter that contained all thecentromeres from one parental genome in a metaphasewere drawn. The numbers of centromeres of eitherparent contained in the polygon for the other parentwere counted and compared with random expectation(i.e. 2.1932 per metaphase when 2n=14; Schwarzacher-Robinson et al. 1987). A score of zero for both parentalgenomes indicates side-by-side separation, while ascore of zero for one genome and seven for the othergenome indicates concentric parental genome separ-ation.

In the six hybrid root tip metaphases, the.number of


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Fig. 5. Polar views of positions of H. vulgare ( • ) and H. bulbosum (O) centromeres on metaphase plates in root tip cells82, 83, 90, 92, 93 and 102 of H. vulgare x H. bulbosum reconstructed from serial sections. Identities of the centromeres ofthe chromosomes are given adjacent to the circles (e.g. 1 adjacent to a black circle indicates Tl-5, 1 adjacent to a whitecircle Bl). Polygons of least perimeter including all centromeres of the chromosomes from H. vulgare (broken lines), andthe polygons of the centromeres from H. bulbosum (dotted line, only for cell 92) are shown. The metaphase plates are onaverage 11.6 nm wide (maximum separation between centromeres on the X axis); centromeres that lay between 1.5 and 3jan above or below the plane shown are indicated by + (above) or — (below). No centromere lay more than 3 fun awayfrom the plane.

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Fig. 6. Polar views of positions of H.vulgare (•) and H. bulbosum (O)centromeres on metaphase plates ofpremeiotic metaphase cells 103, 104,110 and 112 of H. vulgare x H.bulbosum reconstructed from serialsections. Identities of the centromeresof the chromosomes are given adjacentto the circles. Identification ofchromosomes B2 and B3 was uncertainin cells 104 and 110; the more likelyidentification used for the analysis inFig. 7 is underlined. A polygon ofleast perimeter (cell 103) and circles(cell 104, 110 and 112) including allcentromeres of the chromosomes fromH. vulgare are shown. The plates areon average 9.8 /wn wide; centromeresthat lay between 1.5 and 3 jan awayfrom the plane shown are indicated by+ (above) or - (below). Onecentromere (indicated ++) lay 5 j/mabove the plane.


Table 3. Genome mean distances (GMD) and standard deviations (s.d.) between centromeres and the cell meancentromere position for the chromosomes from H. vulgare and H. bulbosum in ten reconstructed root tip andpremeiotic metaphases in their hybrid. Ratios and results of one-tailed t-tests of significance of differences in

GMDs between centromeres from H. vulgare and H. bulbosum are given

Metaphase origin

Root tipt

Premeiotict

Summary!Mean root tips (%)

Mean premeiotic (%)

Mean pooled data (%)

O i lV—Cll

number

82

83

90

92

93

102

103

104

110

112

Genome

H. vulgareH. bulbosumH. vulgareH. bulbosumH. vulgareH. bulbosumH. vulgareH. bulbosumH. vulgareH. bulbosumH. vulgareH. bulbosum

H. vulgareH. bulbosumH. vulgareH. bulbosumH. vulgareH. bulbosumH. vulgareH. bulbosum

H. vulgareH. bulbosumH. vulgareH. bulbosumH. vulgareH. bulbosum

Mean

3.004.462.875.483.784.042.593.273.005.873.033.40

2.503.602.484.282.373.902.044.99

5.918.385.169.125.618.68

Genome distance

(s.d.)

(1.48)(1.06)(0.97)(2.60)(1.84)(1.83)(1.09)(1.08)(1.08)(0.58)(1.19)(1.12)

(0.93)(1.10)(0.95)(0.95)(0.85)(0.92)(0.66)(1-20)

(2.68)(3.01)(2.00)(2.37)(2.46)(2.79)

Ratio

1:1.49

1:1.91

1:1.07

1:1.26

1:1.96

1:1.12

1:1.44

1:1.72

1:1.65

1:2.45

1:1.42

1:1.76

1:1.55

Mest

t

1.971

2.301

0.246

1.082

5.738

0.546

1.868

3.259

3.007

5.262

3.923

6.633

6.841

n.s

*

n.s.

n.s.

**•

n.s.

n.s.

*

***

***

•*•

***

tEach GMD value is in ^m (individual cells); 12 degTees of freedom.JEach GMD value is the mean of all 56, 84 or 140 percentage distances of centromeres from their cell mean centromere position (for 4,

6 or 10 cells; 54, 82 or 138 degrees of freedom), where the sum of the 14 distances in a cell = 100%.§P, probability; n.s., not significant; • = P<Q.Q5, ** = P<0.01, • • • = P<0.001.

centromeres of chromosomes from H. bulbosum con-tained in polygons for H. vulgare (Fig. 5) was zero inthree metaphases and one to three in the other threemetaphases (an average of one per metaphase). The sixpolygons for centromeres from H. bulbosum in root tipmetaphases contained an average of 2.83 centromeresfrom H. vulgare per metaphase. These numbers aredifferent from random expectation, indicating a tend-ency towards concentric separation, but not signifi-cantly so. The four premeiotic metaphases, however,showed significant concentric genome separation withzero centromeres from H. bulbosum contained in theH. vulgare polygons and an average of 5.5 centromeresfrom H. vulgare contained in the H. bulbosum polygons(Fig. 6).

In root tip metaphase 92, where the two parentalgenomes lay next to each other, no centromere from theH. bulbosum genome was contained within the polygonof centromeres from H. vulgare, and no centromerefrom H. vulgare within the polygon of centromeresfrom H. bulbosum (Fig. 5). Thus cell 92 showed side-by-side parental genome separation.

Relationship between volumes of centromere-associated structures and centromere position withingenomesA linear regression analysis of the relationship betweenvolumes of centromere-associated structures from H.bulbosum-origin chromosomes, and distances of cen-tromeres from the mid-centromere position are given inTable 4. There was a negative correlation in eight of theten cells. The relationship was significant in fourindividual cells, and highly significant when all ten cellswere pooled. Thus the smaller centromere-associatedstructures from H. bulbosum-origin chromosomestended to be more peripheral in the cell than the largerones. Similar analyses for the centromere-associatedstructures of the chromosomes from H. vulgare in thesame metaphases showed no significant relationship.

Fig. 7 shows regression lines of mean distancesagainst mean volume of centromere-associated struc-tures for individual chromosomes from H. bulbosumfor root tip, premeiotic and pooled data. Centromeresof individual chromosome types occupied differentpositions along the regression line. Chromosome B4


Table 4. Correlation coefficients and their probabilities in linear regression analysis of centromere volume andcentromere distance from the cell mean centromere position in ten reconstructed root tip and premeiotic

metaphases o/H. vulgare x H. bulbosum

Metaphaseorigin

Root tip

Premeiotic

Pooled data

Cellnumber

8283909293

102Sum

103104110112

Sum

Sum

H. vulgare

Correlationcoefficient

0.080-0.181

0.650-0.274-0.543

0.073-0.047

-0.4450.297

-0.4540.179

-0.167

-0.096

Pt

n.s.n.s.n.s.n.s.n.s.n.s.n.s.

n.s.n.s.n.s.n.s.n.s.

n.s.

H. bulbosum

Correlationcoefficient

-0.526-0.770-0.337

0.275-0.289-0.009-0.341

-0.7870.210

-0.722-0.812-0.454

-0.369

PI

n.s*

n.sn.sn.sn.s*

*n.s

***•

**>

, probability; n.s., not significant; • = P<0.05, ** = F<0.01, •*• = P<0.001.

had the largest mean centromere of the chromosomesfrom H. bulbosum and was on average the closest to thecentre while the centromere of chromosome B7 tendedto be more peripheral with the smallest volume.

Discussion

Identification of chromosomes and karyotypeIn the six root tip and four premeiotic metaphasesdescribed here, volume measurements in serial sectionreconstructions from electron micrographs (e.g. Fig. 2)permitted unequivocal identification of all individualchromosomes to their genome of origin (Figs 3, 4; andconfirmed by in situ hybridization results, Fig. 1).Volumes, arm volume ratios and the presence ofsecondary constrictions also enabled 133 chromosomes(of the 140 analysed) to be accurately identifiedindividually (i.e. all 70 chromosomes from H. vulgareand 63 out of the 70 chromosomes from H. bulbosum).The morphology of the chromosomes of H. vulgare cv.Tuleen 346 measured in the present work agrees wellwith arm lengths measured from light microscopespreads of Tuleen 346 (Finch and Bennett, 1982), andthe same numbering scheme is adopted here. Thekaryotype of the H. bulbosum chromosomes is closelysimilar to that described by Lange and Jochemsen(1976), and the numbering system is the same exceptthat H. bulbosum chromosomes 2 and 3 are reversed toindicate the probable homoeologies with H. vulgare(Heslop-Harrison and Bennett, 1984). The locations ofthe centromeres that were harder to see or interpret onchromosomes from H. bulbosum, particularly in pre-meiotic cells, were apparently correct as they invariablyyielded only normally expected idiograms for thesecells. There were no notable differences between thechromosome arm volumes obtained from reconstruc-tions of either root tip or premeiotic metaphases. The

data in Table 1 show that the total volumes ofchromosomes at the two stages are not significantlydifferent (t=0.176).

Spatial distribution of centromeresA strong tendency towards parental genome separation(Table 3) was found in five of the six root tip metaphasereconstructions (Fig. 5) and all the cells at premeioticmitosis (Fig. 6). Previous investigations have demon-strated parental genome separation in intergenerichybrids where all the chromosomes originating fromone parent were larger than those from the other (Finchet al. 1981; Schwarzacher-Robinson et al. 1987). A highfrequency of concentric parental genome separationwas also found in reconstructions of the unstable hybridH. marinum x H. vulgare (Finch and Bennett, 1983),where chromosome disposition was examined in earlyembryos before uniparental genome elimination oc-curred. The total volumes of the two parental sets weremore similar to each other, although still significantlydifferent, in the hybrid investigated in the presentwork, which involved two related species in the samegenus, Hordeum. In the hybrid, several individualchromosomes of H. vulgare cv. Tuleen 346 origin werealways either larger or smaller than those of H.bulbosum origin, yet the centromeres of all chromo-somes from H. vulgare tended to be more centrallylocated. Hence the separation of parental genomescannot be related to chromosome size.

Interestingly, the tendency to genome separation waseven more pronounced in the premeiotic cells than inthe root tip cells. The premeiotic cells studied herewould have entered meiosis soon had they not beenfixed. Evidence for general premeiotic alignment ofhomologous pairs of chromosomes is controversial (seeLoidl, 1990), but Allium species, at least, show somealignment of homologous chromosomes immediately


20-

10 20 30 %

B

30 %

20-

8 iocD

10 20

Centromere Volume

30 %

Fig. 7. Linear regression lines between the volumes ofcentromere-associated structures and distances ofcentromeres from the metaphase centre for thechromosomes from H. bulbosum in the hybrid with H.vulgare in serial-sectioned metaphase cells. Values (inpercentages of the sum of the centromere-associatedstructure volumes, or centromere distances for thechromosomes from H. bulbosum) are shown as means foreach chromosome type in the six root tip cells (A), thefour premeiotic metaphase cells (B) and all ten cells (C),standard deviations for each mean are indicated as bars.

before synaptonemal complex formation (Albini andJones, 1987), and nuclear envelope associations oftelomeres may be responsible for some presynapticalignment (Loidl, 1988). If homologous telomeres anddiscrete intercalary sites are in proximity beforesynapsis (Loidl, 1990), when is the association initiated?The hybrid described here undergoes pairing andchiasma formation between the H. vulgare and H.bulbosum chromosomes. At metaphase I, 68% of thecells had 14 chromosomes; of these, 43% of chromo-somes were involved in bivalents, similar to the reportof Kasha and Sadasivaiah (1971). Hence analysis ofbehaviour in the hybrid studied here, where it ispossible to identify the parental origin of each chromo-some, is at the least an indication of chromosomebehaviour in true species. Previous papers have de-scribed the somatic association of homologous centro-meres at metaphase in both plants (see Avivi andFeldman, 1980) and animals (see Hubert and Bour-geois, 1986); if these analyses reflected the true relativepositions of chromosomes, homologous centromereswould not only be together shortly before meiosis, butin other somatic cells as well. However, our reconstruc-tions of serially sectioned metaphases showed noevidence for the somatic association of homologues invarious diploid grass species (Heslop-Harrison et al.1988), and those data are supported by the evidence forgenome separation, presented in the present work, inboth root tip and premeiotic divisions. Thus centro-meres seem likely to change their relative dispositionshortly before meiotic prophase as argued by Bennett(1984a) from previous observations of reconstructedpremeiotic mitosis in barley. In wheat, the interphasebetween the last premeiotic mitosis and meiotic pro-phase is exceptionally long, lasting some 55 hours(Bennett et al. 1973), compared with 8 hours in root tipcells. Perhaps the extended period is required for, or toprepare for, the major physical reorganization of thenucleus. Corroborative evidence for the importance ofthis period comes from its great sensitivity to tempera-ture shocks and drug treatments, which may inducefailure of meiotic pairing (Dover and Riley, 1977).

In the insects, there are several species in which theparental genomes behave differently at meiosis, show-ing that the parental chromosome sets can remaindistinct throughout the life cycle. In the scale insects,whole genomes may become condensed into hetero-chromatin (Nur, 1990) that clearly maintains a form ofparental genome separation. A most interestingphenomenon has been shown in Sciara coprophilamales: the maternally derived chromosome set movesto the spindle pole, while the paternally derived set islost at meiosis (Kubai, 1982). Reconstructions ofgenome positions in germ-line nuclei demonstratedparental genome separation, with the two chromosomesets lying in consistent positions relative to a cellularaxis, but there were important changes in relativeposition during prophase I of meiosis (Kubai, 1987).Kubai suggested that the change may relate to themitotic-meiotic transition, again demonstrating theimportance of mechanisms for controlling chromosome


disposition at meiosis. Whole parental genomes alsosegregate together at meiosis in hybrid frogs; oneparental genome is excluded during oogenesis (Tunnerand Heppich-Tunner, 1991). These examples demon-strate that parental genome separation may be commonin higher organisms.

Chromosome volumes and positionThe chromosomes of the genome that tended to beperipheral in the present hybrid, H. bulbosum,accounted for a smaller proportion of the total volumethan expected from the ratio of the DNA contents ofthe two parental species (Table 1). In one of the hybridsstudied previously, H. chilense x 5. africanum, the H.chilense centromeres were peripheral, and again had asmaller volume than expected (Table 2 of Schwar-zacher-Robinson et al. 1987). However, in the hybridH. vulgare x S. africanum, the peripheral chromo-somes were larger than expected from the DNAcontents (Table 3 of Finch et al. 1981). Data from theanalysis of chromosome volumes in rye, Secale cereale,also tended to show that more peripheral chromosomeshave slightly larger volumes than expected from theirDNA contents (Fig. 4 of Bennett, 1984b). In anotherHordeum x Secale hybrid, Linde-Laursen and Bothmer(1989) reported allocycly, with the Secale chromosomesbeing longer than expected from their DNA contents.Thus detectable allocycly occurred in many of thenuclei investigated, and is apparently a consistentfeature from nucleus to nucleus within a hybrid.

In all ten reconstructed cells, secondary constrictionsat the NOR were visible on chromosomes T3-7 and T6-2 from the central genome, H. vulgare, but noconstrictions were seen on the NOR chromosome B6from the more peripheral genome, H. bulbosum.Expression of the rRNA genes at a locus duringinterphase has been correlated with the presence of asecondary constriction at the subsequent metaphase.Thus it is likely that the rRNA genes of one parentalgenome are not expressed as reported in other hybridsof both plants (Bennett, 1984b; Linde-Laursen andBothmer, 1989; Pohler, 1989) and animals (Miller et al.1976; Gold et al. 1991). Expression of the rRNA geneshas been shown to be restricted to the more centralgenome in various diploid intergeneric hybrids (Ben-nett, 1984b) as found in the present interspecific hybrid.Thus the correlation between expression of the rRNAgenes and the spatial position of the NOR-bearingchromosomes is again observed.

Morphology and volumes of centromere-associatedstructuresThe chromosomes originating from H. bulbosum hadsmaller centromeric structures (as described above)than those from H. vulgare (Table 2). The hybriddescribed here is relatively stable, and all ten cellsincluded seven chromosomes from each parentalgenome. The same hybrid can be made with othergenotypes of H. bulbosum as the male parent, when allseven of the H. bulbosum chromosomes may be

eliminated in the first few divisions after fertilization(Bennett et al. 1976; Finch and Bennett, 1983). Finch(1983) examined chromosomes from H. vulgare and H.marinum that were tending to be eliminated andshowed that they lay off the flat metaphase plate formedby the chromosomes that were retained, and hadsmaller centric constrictions in light microscope prep-arations. The H. bulbosum chromosomes in thereconstructions from electron micrographs describedhere showed both of these features: their centromerestended to be more peripheral (Table 3, Figs 5-7), andsome were found off the metaphase plate (in particularchromosome Bl in cell 112), and to have smallercentromeric structures (Table 2) than the chromosomesfrom H. vulgare. For chromosomes from H. bulbosum,a significant correlation between the volume of thecentromere-associated structures and the distance ofthe centromeres from the metaphase centre was found(Table 4, Fig. 7), with smaller centromere-associatedstructures being more peripheral.

In mammalian systems, various recent reports haverelated the lagging of some chromosomes duringcongression on to the metaphase plate, and duringanaphase, to the size and hence activity of thekinetochore/centromere structure, to which the micro-tubules responsible for moving chromosomes bind(Ault and Nicklas, 1989; Jabs et al. 1991). In the presenthybrid, chromosomes from H. bulbosum are moreperipheral and have the weaker (or smaller) centro-mere-associated structures. The activity, rate or timingof production of the centromeric structures is likely tobe under genetic control, and might be correlated withthe tendency of chromosomes of particular genomes tobe lost in unstable hybrids.

We thank J.B. Smith for preparation of the seriallysectioned cells described here. We thank BP and VentureResearch International for enabling part of this work to bedone.

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{Received 16 July 1991 - Accepted 7 October 1991)

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Parental genome separation in reconstructions of somatic and … · 2005. 8. 22. · Hordeum vulgare cv. Tuleen 346 (barley) X H. bulbosum was shown to have seven chromosomes originating