KARYOTYPE ANALYSIS OF THE PLANT- PARASITIC NEMATODE HETERODERA GLYCINES BY ELECTRON MICROSCOPY

Size: px

Start display at page:

Download "KARYOTYPE ANALYSIS OF THE PLANT- PARASITIC NEMATODE HETERODERA GLYCINES BY ELECTRON MICROSCOPY"

Kerry Harrison
5 years ago
Views:

1 J. Cell Sci. 40, (1979) 171 Printed 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. Nine normal synaptonemal complexes (SC) were detected following 3-dimensional reconstruction of pachytene nuclei from electron microscopy of serial sections. Regions of unique 'modified synaptonemal complexes' (MSC) were observed along 2 SCs. These consist of a heterochromatic knob within which the SC appears either disorganized or stacked in layers of lateral elements. Its function is not known. Recombination nodules and 'cylindrical granular complexes', were not observed in H. glycines. INTRODUCTION Analyses of the meiotic system of diverse organisms have revealed the presence of synaptonemal complexes (SCs) during the pachytene stage of prophase I (for review see Westergaard & von Wettstein, 1972; Gillies, 1975). The SC appears to be highly conserved in structure and function from primitive to advanced eukaryotes. Abnormalities along the SC do exist in some cases as in autopolyploids that exhibit switching of pairing partners (Moens, 1968) and during the second phase of pairing of nonhomologous regions of chromosomes (Moses, 1977; Rasmussen, 1977). Several peculiarities in SC structure have been observed also in the plant-parasitic nematode Meloidogyne hapla whose SC consists of 2 lateral elements but, apparently, lacks a central element (Goldstein & Triantaphyllou, 1978 a). Other modifications of the SC in M. hapla have been described as 'double SCs' and 'decondensed chromatin regions' of unknown function (Goldstein & Triantaphyllou, 19786). We have assumed that the modification of the SC of M. hapla is associated with the parthenogenetic reproduction of this nematode and may represent a step toward the evolution of ameiotic type of maturation of oocytes. Such an assumption could be clarified if the SC of M. liapla is compared to that of other amphimictic and meiotic parthenogenetic relatives. We have chosen for study Heterodera glycines, a close relative of M. hapla, which reproduces by cross-fertilization and undergoes regular meiosis 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-

2 172 P. Goldstein and A. C. Triantaphyllou some numbers (n = 12-15). An analysis of the SCs of all 3 forms is of much interest as it may elucidate the chromosome pairing pattern in different states of ploidy. This study has revealed peculiarities in SC structures not previously reported in other organisms. 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 plant parasite. An analysis of the tetraploid and hybrid forms will be presented in a separate paper. 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 in Eastern North Carolina and has been propagated on soybean seedlings in the greenhouse during the past 2 years. Species and race identification have been based on morphological characteristics and host specificity. A cytogenetic study revealed that it is a diploid with n = 9 chromosomes and reproduces by cross-fertilization. Treatment of young, egg-laying females for electron microscopy was carried out in a similar manner to that described for Meloidogyne hapla (Goldstein & Triantaphyllou, 1978a). One nucleus 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 Computer with 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 the nucleus in each serial section. The total number of boxes was then multiplied by the area of each 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 arranged peripherally around a central rachis, as in Meloidogyne hapla (Goldstein & Triantaphyllou, 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 /tm 3 ) and the mitochondria of the oocytes are normal in appearance (Fig. 1). The cytoplasmic organelles are displaced to one side of the cell at pachytene, whereas they are evenly distributed in the cytoplasm at earlier and later stages 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 'modified SC region' (msc) is present and is surrounded by condensed chromatin (cli). The nuclear morphology 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 along the 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.

3 Karyotype analysis of Heterodera CEL 40

4 174 P- Goldstein and A. C. Triantaphyllou There are 9 SCs present in the pachytene nucleus that vary in length from 9-0 to 50-2 fim (Table 1) (Fig. 5). Each SC has only one end attached to the inner membrane of the nuclear envelope (ne) (Fig. 3) and the distribution of the ends appears to be random (Fig. 4). This lack of a bouquet arrangement is similar to other nematodes analysed, 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 females of Heterodera glycines SCno Total karyotype, fim Nuclear vol., fim 3 No. MSC t Nucleus no. 1 Length, fim * * t A Relative length, % 3-5 4' i Nucleus no. 2 Length, fim IO-I * 2I-I 247* i-5 47it A Relative Length, length, % /"n 38 4' i = Modified synaptonemal complex region = Nucleolar organizer region (NOR). 2 (MSC) Average Relative length, % 365 4' '55! were not observed. Pairing of the homologues is regular and complete. The average karyotype length is 260 fim and the relative length of each SC is similar in the 2 nuclei studied (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 in each of the nuclei reconstructed. These 'modified SC regions' (Figs. 7-12) (Tables 1, 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 normal upon entering and exiting this structure (Figs. 8, 9, 11). The 2 MSCs are located on different SCs (Table 1) and at different relative positions (Table 2). For example, MSC no. 1 is located on a small SC at 59% from the end that is attached to the nuclear envelope while MSC no. 2 is located on a mid-size SC at % from the attached end. The nucleolar organizer region (NOR) is located on a long SC (Figs. 4, 5) about halfway from each end (Tables 1, 2). In both nuclei, the MSCs and the NOR are located on different SCs.

5 Karyotype analysis of Heterodera _17.B ' r^: Fig. 4. Diagrammatic reconstruction of the 9 SCs of an oocyte nucleus of Heterodera glycines from 80 serial sections. Each SC is attached by one end to the nuclear envelope while the other end is free in the nucleoplasm (open circle). The SCs are numbered according 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 lengths are in fim. a = modified SC region, b = nucleolar organizer region. SCs from nucleus no. 1 are drawn in continuous lines and from nucleus no. 2 in dashed lines. Fig. 6. Reconstruction of modified synaptonemal complex region (MSC) from four consecutive sections (as in Figs. 7-10). The SC appears normal upon entering and exiting 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 formation and structure.

6 176 P. Goldstein and A. C. Triantaphyllon DISCUSSION In this study, the diploid H. glycines appears to be normal with regard to SC formation and structure. Pairing probably occurs during the first phase of SC formation, i.e. via attraction of homologous chromosomes, as opposed to the non-homologous pairing 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 able to recognize the attachment sites on the nuclear envelope but actual attachment may occur randomly or through a selective mechanism. The formation of the SC occurs without prior axial core formation. The lateral elements are apparently organized from a pool of precursors simultaneously with the SC (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 present prior 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 has demonstrated the presence of regular bivalent chromosomes at metaphase I (Triantaphyllou & Hirschmann, 1962). Crossing over has not been demonstrated genetically in Heterodera, but it is very likely that it occurs, in spite of the apparent absence of recombination 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 of the parthenogenetic M. hapla. This observation is consistent with our earlier assumption that the modified SC of M. hapla may represent an evolutionary step toward ameiotic type of maturation of oocytes and a step toward mitotic parthenogenesis. The 2 modified SC regions (MSC) are located on different SCs within the same nucleus. MSC no. 2 is located on SC no. 5 in both nuclei (Tables 1, 2), but MSC no. 1 is located on SC no. 2 in nucleus no. 1 and on SC no. 3 in nucleus no. 2. The location of the NOR also appears to be variable (Tables 1, 2). This variability is most probably due to differential condensation of the various SCs during pachytene which makes identification of the SCs according to their length unreliable in this case. It is likely that the presence of MSC and NOR are more precise SC markers than absolute length of the SCs. Figs The modified SC region (msc) is a unique structure since within the heterochromatic knob (lik) the SC appears disorganized. The SC appears normal upon entering and exiting this structure. Figs are 4 consecutive sections through an MSC. Fig. 11 is a magnification of Fig. 8 to show detail of the entrance of the SC into the heterochromatic knob. Fig. 12 illustrates the organization of the SC within the heterochromatic knob, ce, central element; le, lateral element. In Figs. 7-10, scale bar is 02 /Jtn; in Figs. 11, 12, 01 fim.

7 Karyotype analysis of Heterodera / W : " 11

8 178 P. Goldstein and A. C. Triantaphyllou Modified SC regions Modified SC regions are unique in that a normal SC becomes disorganized as it enters the heterochromatic knob and assumes a normal form as it leaves. In other cases where the SC is associated with heterochromatin, i.e. centromeric regions, the SC is not differentiated (Counce & Meyer, 1973; Gillies, 1973). The disorganized appearance of the SC within the heterochromatic knob of the MSC suggests that it may be a precursor pool or it may represent a longitudinal multiplication of that SC region. The presence of such a thick layer of heterochromatin completely covering the MSC suggests that this region may have an important function since heterochromatin has been designated as having the function of protecting vital areas from external 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 Distance to nuclear envelope MSC no. 1, fan Relative position, MSC no. 2, /tm Relative position, NOR, /tm Relative position, /o 0/ /o /o Nucleus no. 1 I-I 5' o Nucleus no. 2 2' S5-o ' Individual sex chromosomes often appear as univalents during pachytene, e.g. the heterochromatic masses in the nematode Ascaris (Goldstein, 1978). Sex chromosomes have not been identified in H. glycines by light-microscopic analysis and this is confirmed by the absence of univalents at pachytene. Thus, it is expected that those regions of chromatin that influence the sex-differentiation system must be located on the autosomes. Often, specific modifications along the SC suggest specific function, e.g. the centromere and nucleolar organizer. Differences along the SC in M. hapla, described as ' decondensed chromatin regions' were suggested to be the site of sex-determining chromatin (Goldstein & Triantaphyllou, 1978 b). It may be that the MSCs in H. glycines represent the location of the sex-determining chromatin. We thank Mr Eugene McCabe for valuable technical assistance. Part of this work was done in the laboratory of Dr M. J. Moses, Department of Anatomy, Duke University, and we thank him for his co-operation and discussions. Financial assistance was provided by the National Science Foundation Grant DEB A02 to A. C. Triantaphyllou and by the International Meloidogyne Project, contract no. AID/ta8-C-i234. Paper no of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, N.C.

9 Karyotype analysis of Heterodera 179 REFERENCES BOCDANOV, Yu. F. (1977). Formation of cytoplasmic synaptonemal-like polycomplexes at leptotene and normal synaptonemal complexes at zygotene in Ascaris suttm male meiosis. Chromosoma 61, COUNCE, S. & MEYER, G. F. (1973). Differentiation of the synaptonemal complex and the kinetochore in Locusta spermatocytes studied by whole mount electron microscopy. Chromosoma 44, CRAIG-CAMERON, T. A., SOUTHERN, O. I. & PELL, P. E. (1973). Chiasmata and the synaptinemal complex in male meiosis of Glossina. Cytobios 8, FIIL, A. (1978). Meiotic chromosome pairing and synaptonemal complex transformation in Culex pipiens oocytes. Chromosoma 69, FIIL, A., GOLDSTEIN, P. & MOENS, P. B. (1977). Precocious formation of synaptonemal-like polycomplexes and their subsequent fate in female Ascaris lumbricoides var. suum. Chromosoma 65, GILLIES, C. B. (1973). Ultrastructural analysis of maize pachytene karyotypes by three-dimensional reconstruction of the synaptonemal complexes. Chromosoma 43, GILLIES, C. B. (1975). Synaptonemal complex and chromosome structure. A. Rev. Genet. 9, GOLDSTEIN, P. (1978). Ultrastructural analysis of sex determination in Ascaris suum. Chromosoma 66, GOLDSTEIN, P. & MOENS, P. B. (1976). Karyotype analysis of Ascaris lumbricoides var. suum male and female pachytene nuclei by 3-D reconstruction from electron microscopy of serial section. Chromosoma 58, ioi-m. GOLDSTEIN, P. & TRIANTAPHYLLOU, A. C. (1978a). Occurrence of synaptonemal complexes and recombination nodules in a meiotic race of Meloidogyne hapla and their absence in a mitotic race. Chromosoma 68, GOLDSTEIN, P. & TRIANTAPHYLLOU, A. C. (1978b). Karyotype analysis of Meloidogyne hapla by 3-D reconstruction of synaptonemal complexes from electron microscopy of serial sections. Chromosoma 70, MOENS, P. B. (1968). Synaptonemal complexes of Lilum trigrinum (tiploid) sporocytes. Can. J. Genet. Cytol. 10, MOSES, M. J. (1977). Microspreading and the synaptonemal complex in cytogenetic studies. Chromosomes Today 6, RASMUSSEN, W. E. (1974). Studies on the development of the synaptonemal complex in Drosophila melartogaster. C. r. Trav. Lab. Carlsberg 39, 443, 468. RASMUSSEN, S. W. (1976). The meiotic prophase in Bombyx mori females analysed by 3-D reconstructions of synaptonemal complexes. Chromosoma 54, RASMUSSEN, S. W. (1977). Chromosome pairing in triploid females of Bombyx mori analysed by 3-D reconstructions of synaptonemal complexes. Carhberg Res. Commun. 42, TRIANTAPHYLLOU, A. C. (1971). Genetics and cytology. In Plant Parasitic Nematodes, vol. 2 (ed. B. Zuckerman, W. Mai & R. Rhode), pp New York: Academic Press. TRIANTAPHYLLOU, A. C. & HIRSCHMANN, H. (1962). Oogenesis and mode of reproduction in the soybean cyst nematode, Heterodera glycines. Nematologica 7, TRIANTAPHYLLOU, A. C. & Rices, R. (1979). Polyploidy in an amphimictic population of Heterodera glycines. J. Nematol. (in Press). WESTERGAARD, M. & VON WETTSTEIN, D. (1972). The synaptonemal complex. A. Rev. Genet. 6, YUNIS, J. J. & YASMINEH, W. G. (1971). Heterochromatin, satellite DNA, and cell function. Science, N.Y. 174, {Received 8 May 1979)

The Gametic Cell Cycle

The Gametic Cell Cycle Diploid Parents MEIOSIS Haploid Gametes FERTILIZATION The Somatic Cell Cycle Diploid Zygote MITOSIS Diploid organism Activities of meiosis that differ from mitosis? Life cycle of