J. Cell Sci. 40, 171-179 (1979) 171Printed in Great Britain © Company of Biologists Limited 1979

KARYOTYPE ANALYSIS OF THE PLANT-

PARASITIC NEMATODE HETERODERA

GLYCINES BY ELECTRON MICROSCOPY

I. THE DIPLOID

PAUL GOLDSTEIN AND A. C. TRIANTAPHYLLOU

Departments of Plant Pathology and Genetics,North Carolina State University, Raleigh, North Carolina 27650, U.S.A.

SUMMARY

Hcterodera glycines is a diploid amphimictic nematode with n = 9 chromosomes. Ninenormal synaptonemal complexes (SC) were detected following 3-dimensional reconstruction ofpachytene nuclei from electron microscopy of serial sections. Regions of unique 'modifiedsynaptonemal complexes' (MSC) were observed along 2 SCs. These consist of a hetero-chromatic knob within which the SC appears either disorganized or stacked in layers of lateralelements. Its function is not known. Recombination nodules and 'cylindrical granular com-plexes', were not observed in H. glycines.

INTRODUCTION

Analyses of the meiotic system of diverse organisms have revealed the presence ofsynaptonemal complexes (SCs) during the pachytene stage of prophase I (for reviewsee Westergaard & von Wettstein, 1972; Gillies, 1975). The SC appears to be highlyconserved in structure and function from primitive to advanced eukaryotes. Abnor-malities along the SC do exist in some cases as in autopolyploids that exhibit switchingof pairing partners (Moens, 1968) and during the second phase of pairing of non-homologous regions of chromosomes (Moses, 1977; Rasmussen, 1977).

Several peculiarities in SC structure have been observed also in the plant-parasiticnematode Meloidogyne hapla whose SC consists of 2 lateral elements but, apparently,lacks a central element (Goldstein & Triantaphyllou, 1978 a). Other modifications ofthe SC in M. hapla have been described as 'double SCs' and 'decondensed chromatinregions' of unknown function (Goldstein & Triantaphyllou, 19786).

We have assumed that the modification of the SC of M. hapla is associated with theparthenogenetic reproduction of this nematode and may represent a step toward theevolution of ameiotic type of maturation of oocytes. Such an assumption could beclarified if the SC of M. liapla is compared to that of other amphimictic and meioticparthenogenetic relatives. We have chosen for study Heterodera glycines, a closerelative of M. hapla, which reproduces by cross-fertilization and undergoes regularmeiosis during maturation of the oocytes. In addition, this nematode exists in 2 forms,a diploid, with n = 9 and a tetraploid, with n = 18 chromosomes (Triantaphyllou &Riggs, 1979). Hybrids between the 2 forms are viable and have intermediate chromo-

172 P. Goldstein and A. C. Triantaphyllou

some numbers (n = 12-15). An analysis of the SCs of all 3 forms is of much interestas it may elucidate the chromosome pairing pattern in different states of ploidy. Thisstudy has revealed peculiarities in SC structures not previously reported in otherorganisms.

The present report deals with the analysis of the SCs of the diploid form of H.glycines, which is the prevalent and biologically the most stable form of this plantparasite. An analysis of the tetraploid and hybrid forms will be presented in a separatepaper. The presence of a normal SC in the diploid form is reported and unique'modified SC regions' are described. Their possible function is discussed.

MATERIALS AND METHODS

The nematode population used in this study was originally obtained from a soybean field inEastern North Carolina and has been propagated on soybean seedlings in the greenhouseduring the past 2 years. Species and race identification have been based on morphologicalcharacteristics and host specificity. A cytogenetic study revealed that it is a diploid with n = 9chromosomes and reproduces by cross-fertilization.

Treatment of young, egg-laying females for electron microscopy was carried out in a similarmanner to that described for Meloidogyne hapla (Goldstein & Triantaphyllou, 1978a). Onenucleus was reconstructed from each of 2 different ovaries that were processed at different times.Nucleus no. 1 was reconstructed from 80 serial sections and nucleus no. 2 from 76 serial sections.

Reconstruction and lengths of SCs were calculated as previously described (Goldstein & Moens,1976). The computer program was adapted for use on a Tektronix 405 Graphics Computerwith digitizer by Mr J. Bragg of the Department of Statistics, North Carolina State University.

Nuclear volumes were determined by recording the number of grids needed to cover thenucleus in each serial section. The total number of boxes was then multiplied by the area ofeach box and the average section thickness.

RESULTS

The general morphology of the ovary has been described (Triantaphyllou &Hirschmann, 1962; Triantaphyllou, 1971). The oocytes at pachytene are arrangedperipherally around a central rachis, as in Meloidogyne hapla (Goldstein & Trianta-phyllou, 1978a). Synaptonemal complexes (SCs) are observed in pachytene nuclei,however, no axial cores were observed prior to this stage. The pachytene nuclei(average volume 256 /tm3) and the mitochondria of the oocytes are normal in appear-ance (Fig. 1). The cytoplasmic organelles are displaced to one side of the cell atpachytene, whereas they are evenly distributed in the cytoplasm at earlier and laterstages of prophase I. This behaviour is similar to that reported in Bombyx (Rasmussen,1976).

Fig. 1. Pachytene nucleus from a diploid female of Heterodera glycines. A 'modifiedSC region' (msc) is present and is surrounded by condensed chromatin (cli). The nuclearmorphology is regular. The mitochondria (m) are normal, we, nuclear envelope; nu,nucleolus; sc, synaptonemal complex. Scale bar, 0-5 /tm.Fig. 2. The synaptonemal complex is a tripartite structure and the chromatin alongthe SC is not highly condensed. The central element (ce) is of the scalariform type.le, lateral element. Scale bar, o-i fim.Fig. 3. Attachment of the synaptonemal complex (sc) to the nuclear envelope (ne).Scale bar, o-i fim.

Karyotype analysis of Heterodera

CEL 40

174 P- Goldstein and A. C. Triantaphyllou

There are 9 SCs present in the pachytene nucleus that vary in length from 9-0 to50-2 fim (Table 1) (Fig. 5). Each SC has only one end attached to the inner membraneof the nuclear envelope (ne) (Fig. 3) and the distribution of the ends appears to berandom (Fig. 4). This lack of a bouquet arrangement is similar to other nematodesanalysed, e.g. Ascaris (Goldstein & Moens, 1976) and Meloidogyne (Goldstein &Triantaphyllou, 19786). Univalents, recognizable as chromatic masses without SCs,

Table 1. Synaptonemal complexes from two pachytene nuclei of diploid femalesof Heterodera glycines

SCno.

1

2

3456789

Total karyotype, fim

Nuclear vol., fim3

No. MSC•

t

Nucleus no. 1A

Length,fim

9 0

11-8*1 7 920-625-3*36-542-3t48-55 0 2

262-12 6 0

2

Relativelength, %

3-54'56 97 99 6

1 3 91 6 1

i8-51 9 1

—

—

—

Nucleus no. 2A

Length,fim

IO-I

1 1 7

187*2 I - I

247*35-24i-547it49-2

25932 5 2

2

Average

Relative Length,length,

3 84'57 28-i

9 51 3 616-018-31 9 0

—

—

—

= Modified synaptonemal complex region= Nucleolar organizer region (NOR).

% /"n

9-551175183020-8525-0035-8541-9047-804970

260-7

256

2

(MSC).

Relativelength, %

3654'5©7 0 58 0 0

9'55!37516-0518-401905

were not observed. Pairing of the homologues is regular and complete. The averagekaryotype length is 260 fim and the relative length of each SC is similar in the 2 nucleistudied (Table 1). The dimensions of the SC in cross-sections are: Lateral elements(LE), 9 nm; central element (CE), 20 nm; and the central region (CR), 53 nm (Fig. 2).

There is a structure along 2 of the SCs that is of unusual appearance and occurs ineach of the nuclei reconstructed. These 'modified SC regions' (Figs. 7-12) (Tables1, 2) consist of a heterochromatic knob within which the SC appears to be disorganized(Figs. 6, 8, 10) or may appear as stacks of linear elements (Fig. 12). The SC is normalupon entering and exiting this structure (Figs. 8, 9, 11). The 2 MSCs are located ondifferent SCs (Table 1) and at different relative positions (Table 2). For example,MSC no. 1 is located on a small SC at 5—9% from the end that is attached to thenuclear envelope while MSC no. 2 is located on a mid-size SC at 53-55 % from theattached end.

The nucleolar organizer region (NOR) is located on a long SC (Figs. 4, 5) abouthalfway from each end (Tables 1, 2). In both nuclei, the MSCs and the NOR arelocated on different SCs.

Karyotype analysis of Heterodera

_17.B

•' 18.7

206

- . 1

3 0 5

7 r ^ :

Fig. 4. Diagrammatic reconstruction of the 9 SCs of an oocyte nucleus of Heteroderaglycines from 80 serial sections. Each SC is attached by one end to the nuclear envelopewhile the other end is free in the nucleoplasm (open circle). The SCs are numberedaccording to length (as in Fig. 5). *, Nucleolar organizer region. Scale bar, 10/ im,

Fig. 5. Reconstructed karyotype of oocyte pachytene nucleus as in Fig. 4. The lengthsare in fim. a = modified SC region, b = nucleolar organizer region. SCs from nucleusno. 1 are drawn in continuous lines and from nucleus no. 2 in dashed lines.

Fig. 6. Reconstruction of modified synaptonemal complex region (MSC) from fourconsecutive sections (as in Figs. 7-10). The SC appears normal upon entering andexiting from the heterochromatic knob (hk). Scale bar, 025 /tm.

176 P. Goldstein and A. C. Triantaphyllon

DISCUSSION

In this study, the diploid H. glycines appears to be normal with regard to SC forma-tion and structure. Pairing probably occurs during the first phase of SC formation,i.e. via attraction of homologous chromosomes, as opposed to the non-homologouspairing during the second stage of SC formation (Moses, 1977; Rasmussen, 1977).Although only one end of each SC is associated with the nuclear envelope at pachytene,the ends appear to be structurally very similar. It is possible that both ends are ableto recognize the attachment sites on the nuclear envelope but actual attachment mayoccur randomly or through a selective mechanism.

The formation of the SC occurs without prior axial core formation. The lateralelements are apparently organized from a pool of precursors simultaneously with theSC (Fiil, Goldstein & Moens, 1977). This is similar to the situation in Glossina (Craig-Cameron, Southern & Pell, 1973); Culex (Fiil, 1979); Ascaris (Goldstein & Moens,1976); Meloidogyne (Goldstein & Triantaphyllou, 19786); Drosophila (Rasmussen,1974) and Bombyx (Rasmussen, 1976). Axial cores have been reported to be presentprior to pachytene (Bogdanov, 1977) in what appears to be a different variety of Ascaris.

Recombination nodules (RN) and cylindrical granular complexes, reported in M.hapla (Goldstein & Triantaphyllou, 1978 a) were not observed at any stage in H.glycines. The apparent absence of RNs does not imply the lack of crossing over.Analysis by light microscopy has indicated regular formation of chiasmata and hasdemonstrated the presence of regular bivalent chromosomes at metaphase I (Trianta-phyllou & Hirschmann, 1962). Crossing over has not been demonstrated geneticallyin Heterodera, but it is very likely that it occurs, in spite of the apparent absence ofrecombination nodules.

The SC of the diploid amphimictic H. glycines is normal in structure (tripartite),similar to that of many other amphimictic organisms. It is different from the SC ofthe parthenogenetic M. hapla. This observation is consistent with our earlier assump-tion that the modified SC of M. hapla may represent an evolutionary step towardameiotic type of maturation of oocytes and a step toward mitotic parthenogenesis.

The 2 modified SC regions (MSC) are located on different SCs within the samenucleus. MSC no. 2 is located on SC no. 5 in both nuclei (Tables 1, 2), but MSCno. 1 is located on SC no. 2 in nucleus no. 1 and on SC no. 3 in nucleus no. 2. Thelocation of the NOR also appears to be variable (Tables 1, 2). This variability is mostprobably due to differential condensation of the various SCs during pachytene whichmakes identification of the SCs according to their length unreliable in this case. It islikely that the presence of MSC and NOR are more precise SC markers than absolutelength of the SCs.

Figs. 7-12. The modified SC region (msc) is a unique structure since within theheterochromatic knob (lik) the SC appears disorganized. The SC appears normal uponentering and exiting this structure. Figs. 7-10 are 4 consecutive sections through anMSC. Fig. 11 is a magnification of Fig. 8 to show detail of the entrance of the SC intothe heterochromatic knob. Fig. 12 illustrates the organization of the SC within theheterochromatic knob, ce, central element; le, lateral element. In Figs. 7-10, scalebar is 0 2 /Jtn; in Figs. 11, 12, 0 1 fim.

Karyotype analysis of Heterodera

• /W:"

11

178 P. Goldstein and A. C. Triantaphyllou

Modified SC regions

Modified SC regions are unique in that a normal SC becomes disorganized as itenters the heterochromatic knob and assumes a normal form as it leaves. In othercases where the SC is associated with heterochromatin, i.e. centromeric regions, theSC is not differentiated (Counce & Meyer, 1973; Gillies, 1973). The disorganizedappearance of the SC within the heterochromatic knob of the MSC suggests that itmay be a precursor pool or it may represent a longitudinal multiplication of that SCregion. The presence of such a thick layer of heterochromatin completely coveringthe MSC suggests that this region may have an important function since hetero-chromatin has been designated as having the function of protecting vital areas fromexternal disruptive forces and evolutionary change (Yunis & Yasmineh, 1971).

Table 2. Relative positions of' modified synaptonemal complex regions' (MSC)and nuckolar organizer region (NOR) on synaptonemal complex

MSC no. 1, fanRelative position,MSC no. 2, /tmRelative position,NOR, /tmRelative position,

/o

0//o

/o

Distance to

Nucleusno. 1

I - I

5'06 2

53-o2 0 85 0 0

nuclear envelope

Nucleusno. 2

2'49 0

1 0 3

S5-o27-1

57'°

Individual sex chromosomes often appear as univalents during pachytene, e.g. theheterochromatic masses in the nematode Ascaris (Goldstein, 1978). Sex chromosomeshave not been identified in H. glycines by light-microscopic analysis and this is con-firmed by the absence of univalents at pachytene. Thus, it is expected that those regionsof chromatin that influence the sex-differentiation system must be located on the auto-somes. Often, specific modifications along the SC suggest specific function, e.g. thecentromere and nucleolar organizer. Differences along the SC in M. hapla, describedas ' decondensed chromatin regions' were suggested to be the site of sex-determiningchromatin (Goldstein & Triantaphyllou, 1978 b). It may be that the MSCs in H. glycinesrepresent the location of the sex-determining chromatin.

We thank Mr Eugene McCabe for valuable technical assistance. Part of this work was donein the laboratory of Dr M. J. Moses, Department of Anatomy, Duke University, and we thankhim for his co-operation and discussions. Financial assistance was provided by the NationalScience Foundation Grant DEB 76-20968 A02 to A. C. Triantaphyllou and by the Inter-national Meloidogyne Project, contract no. AID/ta8-C-i234.

Paper no. 5995 of the Journal Series of the North Carolina Agricultural Research Service,Raleigh, N.C.

Karyotype analysis of Heterodera 179

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{Received 8 May 1979)