CHROMOSOME-SPECIFIC DESYNAPSIS IN THE N 2

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1 American Journal of Botany 89(): CHROMOSOME-SPECIFIC DESYNAPSIS IN THE N 2 RACE OF HAPLOPAPPUS GRACILIS (COMPOSITAE) 1 R. C. JACKSON, 2 NGAN NGO, AND HAO NGO Department of Biological Sciences, Texas Tech University, Lubbock, Texas USA During cytological screening for pollen sterility in a population of Haplopappus gracilis (n 2), several partially sterile plants were found that had good pachytene pairing but varying numbers of univalents. Some plants had chromosome A bivalents or A univalents, while in the same cells chromosome B had only bivalents. In other plants the reverse condition occurred; the B chromosome had B bivalents or B univalents and only A bivalents. This demonstrates a chromosome-specific effect for the desynapsis genes. Hybridization between the two homozygous mutant genotypes produced only normal bivalents; this indicates the two mutants are not alleles and each is recessive. An F2 generation showed independent assortment of the desynaptic mutations. The chromosome A bivalent is the larger of the two and normally has one or two chiasmata; the B bivalent normally has a single chiasma. Chiasmata distribution was tested in the desynaptic mutant A bivalents and showed an acceptable fit to a binomial distribution. This occurs also in heterozygous, asynaptic pairing control gene mutations. Analysis of the NOR bivalent in two hologenomic desynaptic mutations in tomato also showed a good fit to a binomial distribution of chiasmata. This indicates the same methods are applicable to diverse species. Key words: bivalents; chromosome-specific; Compositae; desynapsis; genetics; Haplopappus; random chiasmata. Desynapsis was defined by Li, Pao, and Li (194) as failure of homologous pachytene chromosomes to form chiasmata and consequently to become univalents at later prophase and metaphase I stages of meiosis. Research on desynaptic mutations continues in many economically important plants but primarily as a source of trisomics for genetic analysis. Asynapsis refers to chromosomes in the first meiotic division in which synapsis either fails completely or is incomplete (Beadle, 193; Rieger, Michaelis, and Green, 1991). In both kinds of synaptic mutations all potential bivalents are not affected the same way in every cell. Reviews of asynapsis and desynapsis have been published periodically (Baker et al., 1976; Zickler and Kleckner, 1999), but most studies of mutant synaptic genes have not identified chromosome-specific effects or tested fits to various chiasmata or crossover distribution models. The total number of genes essential for meiotic crossing over is unknown although a series of marker mutants have been identified in genetically well-known organisms (Zickler and Kleckner, 1999). Some of the cytogenetically more instructive studies of desynapsis have involved analyses of species with very small chromosome numbers in which individual chromosomes can be identified at various mitotic and meiotic stages. Two carefully analyzed examples with low chromosome numbers are in Hypochoeris radicata (n 4; Parker, 197) and Crepis capillaris (n 3; Tease and Jones, 1976), both in the family Compositae. Haplopappus gracilis (n 2) is the third species with such a desirable genome, and it is better suited than other taxa because of its lower chromosome number and unambiguous identification of each chromosome during meiosis (Jack- 1 Manuscript received 1 November 21; revision accepted 13 December 21. This research was supported in part by a Howard Hughes Medical Institute grant through the Undergraduate Biological Sciences Education Program to Texas Tech University. The authors thank Ann Porter for photograph reproductions. 2 Author for reprint requests (Tel: , FAX: ; e- mail: ray.c.jackson@ttu.edu). 777 son, 199). We have found a chromosome-specific desynaptic mutation in each linkage group of H. gracilis. Our goals in this study were (1) to determine the inheritance pattern of the desynapsis genes for each linkage group, (2) to test chiasmata distribution for fits to models and equations developed for asynaptic mutations, and (3) to analyze the univalent patterns of movement to the two poles. MATERIALS AND METHODS Plant sources and culture Mutant plants used in the analysis were from a n 2 population (8) of Haplopappus gracilis in Sandoval County, New Mexico, USA, about 8 km ( miles) east of the entrance to Bandelier National Monument. A source (82) for normal or type genes was from Kingman, Mojave County, Arizona. After drying in the laboratory for 1 mo, seeds were stored in a refrigerator at C until used. Seeds were germinated in distilled water at room temperature. Seedlings about 1 cm long were transplanted to hot-water-expanded Jiffy-7 peat pots (Jiffy Products, Forestry Suppliers, Jackson, Mississippi, USA) in white plastic dishpans, covered by clear plastic with aeration under light for 3 d, and then grown with small amounts of water without the cover under artificial light until roots were visible. The peat pots were then transferred to 2.4-cm (8-inch) soil-filled pots and the plants grown to maturity in a greenhouse. Cytological methods Seedlings 2 d old were used for karyotype analysis. They were germinated as above, pretreated for about 2 h in.8 mol/l 8- hydroxyquinoline, and then fixed 24 h in a solution of 3 : 1 9% ethanol : propionic acid. Root tips were then excised and placed in 1% HCl for 1 17 min, rinsed with fixative, and placed on a glass slide in a drop of FLPorcein stain. A cover glass was added and tapped gently to separate the cells for examination before squashing. Meiotic material of microsporocytes was fixed as described. Anthers were placed in a drop of stain under a cover glass, and microsporocytes were separated gently by slight tapping with a pencil eraser to maintain the fixed chromosome positions. Those preparations of homozygous mutants used for pachytene stage analysis were squashed as flat as possible. Other cytological analyses of chiasma and univalent frequencies were at very late diakinesis or metaphase I. Chromosome distribution to the poles was scored at late anaphase I or early telophase I. Pollen grains were stained with Buffalo Black B in 7% propionic acid, and those with a uniformly stained cytoplasm were counted as fertile. The A chromosome linkage group is the larger of the two with a near

2 778 AMERICAN JOURNAL OF BOTANY [Vol. 89 median centromere, and the nucleolar organizing region (NOR) chromosome B is smaller with a subterminal centromere (Fig. 1). We designate dsa and dsb, respectively, as the chromosome A and B desynaptic gene symbols. A superscript plus sign ( ) refers to a normal (-type) allele. Testing models and equations Tests of data from numerous sources show that natural selection requires a minimum of one chiasma per bivalent and any excess is randomly distributed among all bivalents (Jackson, 1984, 1991). If univalents occur in normal genetic systems, they are so rare and unpredictable that they need not be considered. Homozygous desynaptic mutants with a maximum of two chiasmata have bivalents with one (II 1 ) or two (II 2 ) chiasmata or two univalents (2 Is). A method for testing chiasma types for a fit to a mutant, random chiasmata distribution model is by use of the binomial (P c Q) 2. The probability (P c ) of a chiasma in a bivalent is obtained by dividing the observed number of chiasmata by the theoretical maximum of two per bivalent. 1 P c Q is lack of a chiasma, and N is cell sample size. So P c2 N (II 2 ), 2(P c Q) N (II 1 ), and Q 2 N (2 Is). Methods for bivalents with more than two chiasmata are given elsewhere (Jackson, 1991). Chiasma frequency data for linkage group A with a maximum of two chiasmata per bivalent were used to test whether crossovers were distributed randomly among the desynaptic meiocytes. The B chromosome bivalents normally have a single chiasma in the long arm, and univalents do not occur. Thus the II number is the same as sample size for the normal genetic system. Univalents and II 1 in the desynaptic genotype have no degrees of freedom for a chi-square test. The movement of four univalents to the two poles, P c and Q, at anaphase I was tested for a fit to a theoretical standard binomial as (P c Q) 4 with equal probability of a univalent moving to either pole. For convenience, the univalents are designated as A, a, B, b, with homologues identified by the same letters. Hybridizations Heads used in crosses were covered with Kimwipes (Kimberly-Clark Corp., Neenah, Wisconsin, USA) laboratory paper prior to anthesis and then kept covered after pollination until stigmas withered. The species has a sporophytic self-incompatible mating system (Jackson, Skvarla, and Chissoe, 2), so sample sizes were large enough for a high probability of successful crosses. The desynaptic mutants were found segregating in a greenhouse population. Plants homozygous for desynaptic genes on chromosomes A and B were initially crossed inter se to preserve the mutant genotypes. They were then crossed to pollen compatible -type 82 individuals to produce an F1 generation. F1s were then intercrossed to produce an F2 generation in which the proposed null hypothesis was an expected F2 ratio of 9 :3:3:1. RESULTS Genetics Analysis of 36 F1 progeny from six different crosses produced only plants with two bivalents ( type) so both desynaptic genes are recessive (Table 1). In addition, separate complementation tests of homozygotes (dsa, dsb dsa, dsb ) always yielded -type F1s and normal bivalents, indicating the two mutants are not alleles. A single F1 plant in the double desynaptic cross to a homozygous -type had one meiocyte with two chromosome B univalents in a total of 127 meiocytes sampled (Table 1). This undoubtedly represents an unpredictable failure of chiasma formation (.83%) in one plant and is not considered significant. This also represents only one failure in 3739 (.27%) F1 meiocytes examined at metaphase I. A small F2 population gave an acceptable fit to a dihybrid ratio for the two desynaptic gene markers (Table 2). Meiotic analysis In a plant desynaptic for both linkage groups, 19 of the 21 pachytene stages examined had normal synapsis. Two meiocytes had very small, unpaired regions in the short arm of the B chromosome bivalents that normally do not have chiasmata (Fig. 2). This was observed in one genotype during another study and was attributed to a duplication of part of this arm on chromosome A (Jackson, 1963). Univalents were clearly identifiable and well spaced at a stage corresponding to diakinesis (Fig. 3). Four univalents, when present, usually were aligned at the metaphase I plane in unsquashed cells as were cells with two univalents (Figs. 4, ). Univalent chromosome B behavior was not always synchronized with A bivalents at early anaphase I. In some cells the univalent Bs were precocious in moving to opposite or to the same poles (Figs. 6, 7). Univalent Bs had precocious chromatid separation in several cells while the A dyads were at opposite poles at pro-metaphase II with chromatids held together in the centromere region (Fig. 8). Thus, chromosome Bs were at anaphase II and not synchronized with the A chromosomes as they normally are. Univalents that were not successful in their polar movements typically formed micronuclei at interkinesis and ultimately produced very small and empty microspores. The different classes of polar distribution of the four univalents were all observed (Figs. 9 11), and the fit to a random distribution was acceptable (P.317; Table 3). Pollen fertility varied among homozygous mutant individuals. For example, dsa-dsb had 1 32% fertility with a mean of 24% in four plants. The complementation hybrids of these two genes had fertilities of 84 98% and a mean of 88% in 12 plants. These counts included very small spores formed by single chromosomes, unbalanced and unreduced types, and those of normal size. Analysis of 167 meiocytes in a chromosome A desynaptic genotype gave an acceptable fit to a binomial distribution of chiasmata (P.3; Table 4). Chiasmata distributions were analyzed in 746 additional meiocytes from nine plants with either normal or desynaptic A and B bivalents and one plant desynaptic for both A and B (Table ). In eight desynaptic B plants analyzed, there was no significant correlation for an increase of chiasmata in A bivalents when there were univalents of chromosome B (R 2.4). Because there were no degrees of freedom for tests of chromosome B desynaptics (Table ), only examples of the A linkage group were tested by chi-square, and all had acceptable fits to a random chiasmata distribution model (P.1). DISCUSSION The extensive literature on desynaptic mutations consists primarily of studies of whole genomes and not of chromosome-specific examples. Some studies have concentrated only on diakinesis or metaphase I and analysis of the pachytene stage was ignored. Therefore, some early examples could and did confuse desynapsis with asynapsis. Also, the term pairing is used incorrectly in some instances so that statements such as pairing at diakinesis and metaphase I are encountered. However, chromosome pairing is initiated at zygotene and is complete at the pachytene stage insofar as a particular genetic system dictates because some organisms normally do not have complete synapsis. Most often, chiasmata data are not adequately quantified for a binomial distribution test. Asynapsis can be differentiated from desynapsis by the fact that in the former chromosome pairing may vary among the chromosomes of genomes such that the same chromosomes

3 May 22] JACKSON ET AL. CHROMOSOME-SPECIFIC DESYNAPSIS IN HAPLOPAPPUS 779 Figs Mitotic and meiotic cells. 1. Late mitotic prophase; two A chromosomes with near medium centromeres and two acrocentric nucleolar organizing B chromosomes with secondary constrictions. 2. Pachytene with a small unpaired region at the arrow. Some chromosome breakage occurred as a result of high squashing power. 3. Diakinesis equivalent with four univalents. 4. Metaphase I equivalent with four univalents aligned at the metaphase plane. Figs. 7. Metaphase I stages with an A bivalent and two B univalents.. B univalents are aligned at the metaphase I plane. 6. The two B univalents have moved precociously to opposite poles. 7. The two B univalents have moved precociously to the same pole.

4 78 AMERICAN JOURNAL OF BOTANY [Vol. 89 Figs Chromosome distribution to the poles. 8. Precocious separation of the B chromosome dyads at anaphase II while the A dyads are at prometaphase II. Figs Types of univalent A and B distribution to the poles at anaphase I-telophase I. 9. Polar distribution in this cell has homologues going to the same pole; this is expected one-third of the time. 1. Three univalents have gone to one pole and one to the other. 11. All four univalents have gone to the same pole. TABLE 1. Microsporocytes (N 3739) from F 1 hybrids of desynaptic A(dsa) andb(dsb) genotypes crossed to -type (normal) plants from population 82 from Kingman, Arizona, USA. Genotype No. plants No. cells Phenotype dsa-7 King.-4 dsa- King.-4 dsa-9 King.-9 dsa-2 King.-4 dsb-1 King.-4 dsa, dsb-4 King (126; 1dsb) may have complete pairing in some meiocytes and partial or no pairing in others. The term for this kind of chromosome behavior is homoeologous pairing (Huskins, 1932) in contrast to normal homologous pairing in desynaptic genotypes. So far as known, asynaptic behavior affects the search for homologues during zygotene and pachytene, but some authors claim the search may commence as early as leptotene (Zickler and Kleckner, 1999). One kind of asynapsis occurs in F1 hybrids heterozygous for pairing control (PC) mutations and is found most often in interspecific or intergeneric hybrids (Jackson, 1991). The end results are variable chiasma and univalent frequencies such as occur in desynaptic mutants. The same binomial equations can be used to accurately predict chiasmata distribution classes in both kinds of mutations. These methods

5 May 22] JACKSON ET AL. CHROMOSOME-SPECIFIC DESYNAPSIS IN HAPLOPAPPUS 781 TABLE 2. and expected F2 numbers of normal and desynaptic phenotypes tested for a fit to a dihybrid ratio ( 2 (3) 4.162, P.162). N 44. Phenotypes dsa dsb dsa dsb dsa dsb dsa dsb TABLE 4. and expected numbers of one (II 1 ) or two chiasmata (II 2 ) bivalents and two univalents (2 Is) in a desynaptic chromosome A mutant with chiasmata distribution fitted to a binomial distribution. 2 (1).83, P.3. II 2 II 1 (P c ) 2 2(P c Q) 2 Is (Q) 2 No. cells can be used also to detect deviations from random univalent movement to the poles at anaphase I, which is of interest genetically. For example, univalent distribution at anaphase I in triploid Allium triquetrum was not random in a high frequency collection as opposed to random distribution in low frequency classes (Balog, 1979). A classic example of polarized, nonrandom univalent segregation at anaphase I was found in the sexually reproducing triploid Leucopogan juniperinus (Smith- White, 1948). We stated earlier that most desynaptic mutants described in the literature affect entire genomes (hologenomic) and are not chromosome-specific. However, if individual bivalents from whole-genome desynapsis can be identified, they can be tested for fits to binomial equations. An example of this is in the NOR chromosome 2 of tomato that was analyzed in a classic study by Soost (191) and later by Moens (1969). Soost (191) gave meiotic configurations and chiasmata frequencies for two of four known desynaptic genes on chromosome 2. We have calculated the expected numbers of diakinesis configurations for these two desynaptic mutants, and there is an acceptable fit to Soost s observed numbers (P.7 and P.; Table 6). Both Haplopappus and tomato show that the same binomial equations can be used to accurately predict diakinesis or metaphase I configurations at the diploid level in desynaptic mutations representing diverse taxonomic groups. Our chiasmata counts were accurate, and random distribution of crossovers was demonstrated. However, we cannot rule out error in chiasmata counts owing to failure of chiasmata maintenance, which has been demonstrated for the desynaptic mutant (dy) in maize (Maguire, 1978, 199; Maguire, Paredes, and Riess, 1991). Pachytene in this mutant was normal and crossing over occurred, but there was a lack of visible chiasmata due to loss of the cohesion (cohesin) matrix that holds sister chromatids together until diakinesis. Cohesin genes have been reported for yeasts, Drosophila, Xenopus (Cohen-Fix, 2), and Arabidopsis (Dong and Makaroff, 21). Loss of cohesin in an arm with a chiasma in a maize bivalent may result in separation of sister chromatids to produce two univalents at diakinesis or a reduction in observable chiasmata. Chiasma counts at diakinesis or metaphase I could not detect TABLE 3. Distribution of observed and expected classes of four univalent chromosomes (A, a, B, b) to the two poles (P c, Q) indesynaptic genotypes of the A and B linkage groups fitted to a binomial distribution of (P c Q) 4. 2 (3) 3.28, P (P c3 Q 1 ) (P c ) 4 (Q) 4 4(P 1 Q 3 ) a 6(P c2 Q 2 ) No. cells a combinations under 6(P c2 Q 2 are {Aa Bb} 1/3): {AB ab} {Ab ab} 2/ this effect. Detection was possible in maize univalents because conspicuous knobs in a heterozygote for this trait showed equational separation, such that one of the two dyads of a univalent had a knob and the other was knobless. This maize mutation may have as its primary effect a defective synaptonemal complex (Maguire, Paredes, and Riess, 1991), but whether this syndrome is common to other desynaptic mutations is yet to be determined. Parker (197) and Tease and Jones (1976) reported other bivalents of complements with low frequencies of univalents in addition to chromosome-specific desynapsis in one bivalent. However, bivalent IV in Hypochoeris clearly shows chromosome-specific desynapsis due to a single recessive gene, and univalent numbers from this bivalent are correlated with a possible increase of chiasmata in the others so there may be a compensation mechanism. F2 data for bivalent A in Crepis showed that a single recessive gene is responsible for desynaptic behavior. However, neither mutant was tested for random chiasmata distribution among cells nor did the data sets include the different crossover classes for individual bivalents. We have examined the data of Richardson (193) for a desynaptic strain-x of Crepis capillaris in which she gave mean chiasma frequencies for bivalents A, C, and D. This strain was completely homozygous, barring a new mutation, because it came from seeds off a diploid branch of a haploid. She also did not give data for bivalent chiasmata classes. However, from her data we calculated the expected 2 I frequencies in a sample of 66 nuclei at late diakinesis in plant from her Tables 1 and. Her observed 2 I numbers are followed by our expected values in parentheses: A 14 (14.8), C 27 (34.69), D 2 (22.7). Although data for the bivalent chiasmata classes were not given, the expected 2 I numbers suggest that this particular plant had random chiasmata distribution among its bivalents. In analyses involving bivalents with different chiasma frequencies, each bivalent should be ana- TABLE. Chiasmata distribution in 746 meiocytes from nine plants with normal or desynaptic homozygotes for linkage groups A or B and one homozygote for both mutants. Plant no. A-2 A-3 A-13 A-7 B-1 B-2 B-3 B-1 Desynaptic A II 2 II 1 2Is Normal A Normal B II 2 II 1 2Is Desynaptic B Desynaptic B Desynaptic A H

6 782 AMERICAN JOURNAL OF BOTANY [Vol. 89 TABLE 6. and expected meiotic configurations of two desynaptic alleles of tomato (Lycopersicum esculentum Solanum lycopersicum) linked to NOR chromosome 2. data are from Soost (191). Allele as 1 Allele as 4 II 2 II 1 2Is 2 (1) P , ,. lyzed separately because cell frequencies alone cannot accurately describe the variation inherent among the different linkage groups. This information is necessary for determining whether chiasmata are distributed randomly. The desynaptic genes in tomato are examples of hologenomic mutations that affect all bivalents (Jackson, 198). A chromosome-specific gene affects a single bivalent as in Haplopappus, Hypochoeris, and Crepis. But both genetic systems likely occur in all eukaryotes. It seems logical also that the chromosome-specific system arose earlier in evolution because the primitive eukaryotes likely had a single linkage system. A master gene then evolved to synchronize chiasma distribution behavior among multiple linkage groups. LITERATURE CITED BAKER, B. S., A. T. C. CARPENTER, M.S.ESPOSITO, R.E.ESPOSITO, AND L. SANDLER The genetic control of meiosis. Annual Review of Genetics 1: BALOG, C Studies on triploid Allium triquetrum. II. Metaphase I univalents and their influence on anaphase I distribution. Chromosoma (Berlin) 73: BEADLE, G. W Genetical and cytological studies of Mendelian asynapsis in Zea mays. Cornell University Agriculture Station Memoirs 129: COHEN-FIX, O. 2. Sister chromatid separation: falling apart at the seams. Current Biology 1: DONG, F., AND C. A. MAKAROFF. 21. Cloning and characterization of two Arabidopsis genes that belong to the Rad21/REC8 family of chromosome cohesin proteins. Gene 21: HUSKINS, C. L A cytological study of Vilmoren s unfixable dwarf wheat. Genetics 2: JACKSON, R. C A study of meiosis in Haplopappus gracilis. American Journal of Botany 46: 4. JACKSON, R. C Variation in the short arm of chromosome B of Haplopappus gracilis. Canadian Journal of Genetics and Cytology : JACKSON, R. C Chromosome pairing in species and hybrids. In W. F. Grant [ed.], Plant biosystematics, Academic Press, Toronto, Ontario, Canada. JACKSON, R. C Genomic differentiation and its effect on gene flow. Systematic Botany 1: JACKSON, R. C Cytogenetics of polyploids and their diploid progenitors. In T. Tsuchiya and P. K. Gupta [eds.], Chromosome engineering in plants, Elsevier, Amsterdam, The Netherlands. JACKSON, R. C., J. J. SKVARLA, AND W. F. CHISSOE. 2. A unique pollen wall mutation in the family Compositae: ultrastructure and genetics. American Journal of Botany 87: LI, H. W., W. K. PAO, AND C. H. LI Desynapsis in the common wheat. American Journal of Botany 32: MAGUIRE, M. P Evidence for separate genetic control of crossing over and chiasma maintenance in maize. Chromosoma (Berlin) 6: MAGUIRE, M. P Sister chromatid cohesioness: vital function, obscure mechanism. Biochemical Cell Biology 68: MAGUIRE, M. P., A. M. PAREDES, AND R. W. RIESS The desynaptic mutant of maize as a combined defect of synaptonemal and chiasma maintenance. Genome 34: MOENS, P. B Genetic and cytological effects of three desynaptic genes in the tomato. Canadian Journal of Genetics and Cytology 11: PARKER, J. S Chromosome-specific control of chiasma formation. Chromosoma (Berlin) 49: RICHARDSON, M II. Failure of pairing in Crepis capillaris (L.) Wallr. Journal of Genetics 31: RIEGER, R., A. MICHAELIS, AND M. M. GREEN Glossary of genetics. Springer-Verlag, Berlin, Germany. SMITH-WHITE, S Polarized segregation in the pollen mother cells of a stable triploid. Heredity 2: SOOST, R. K Comparative cytology and genetics of asynaptic mutants in Lycopersicon esculentum Mill. Genetics 36: TEASE, C., AND G. H. JONES Chromosome-specific control of chiasma formation in Crepis capillaris. Chromosoma (Berlin.) 7: ZICKLER, D., AND N. KLECKNER Meiotic chromosomes: integrating structure and function. Annual Review of Genetics 33: