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INDUCTION O F CHROMOSOME DOUBLING A T NIEIOSIS BY THE ELONGATE GENE IN MAIZE M. M. RHOADES Department of AND ELLEN DEMPSEY Botany, Indiana University, Bloomington Received March 15, 1966 EIOSIS is a highly integrated system characterized by the orderly procession of events such as pairing, recombination, chiasma formation, and disjunction, which culminates in a reduction o€ chromosome number in the gametes. That this system is under genic control has long been evident from the many mutant genes reported in plants and animals which affect specific stages of the meiotic system. These meiotic genes are of unusual interest in that a thorough study of the physicochemical basis of their action gives promise of leading to a better understanding of the physiological conditions which bring about the transsition from mitosis to meiosis as well as of the cellular environment essential for such critical events as synapsis, recombination, and disjunction. A discussion of the genetic control of meiosis is given in REES(1961) . A new meiotic gene is reported in this paper which will be a useful tool in cytogenetical studies with maize. This mutant was found in the open-pollinated variety Hays Golden. It is a simple recessive and has not yet been placed in a linkage group. This new meiotic gene is called elongate (el) because of the elongated appearance or despiralization of the chromosomes at both meiotic anaphases, but it has a number of other effects. The most significant of these is the production of unreduced eggs which occur in varying proportions with haploid eggs. Evidence will be presented that the diploid eggs originate by chromosome doubling at the second meiotic division. The recovery of two of the four chromatids of a bivalent in the diploid eggs of elongate plants enabled a half-tetrad analysis and data were obtained which bear on the questions of chromatid interference and centromere mapping. EXPERIMENTAL RESULTS Chromosome numbers in female and male gametes: One of the striking manifestations of the action of the el gene is expressed in the female flowers. Ears borne on el plants have plump and shriveled kernels as well as aborted ovules. The relative frequencies of these vary greatly on different ears and are evidently affected by modifying genes but, with the exception of one progeny, no difficulty was encountered in classifying segregating normal and el plants on the basis of ovule abortion and shriveled kernels. One-to-one ratios of EL to el were obtained in testcrosses and 3:l in F, populations. The plump kernels gave rise to diploid plants, all with 20 chromosomes. A considerable fraction of the shriveled kernels Gcrietics 54: 505-522 August 1966. 506 M. M. RHOADES A N D E. DEMPSEY are so poorly developed that they do not germinate, but chromosome counts were made on root tip cells of 825 individuals from shriveled kernels. All were near the triploid level, indicating that diploid or near diploid eggs were formed by el plants. Although 82% were euploids with 30 chromosomes, as shown in the tabulation below, 18% had aneuploid numbers ranging from 25 to 33. Chromosomenumber in plants from shriveled seed 25 26 27 28 29 30 31 32 33 Frequency 1 1 2 17 71 676 46 10 1 A variable amount of pollen abortion is found in el plants but the functional pollen is haploid. When elongate plants were used as male parents in crosses with diploid females, the 961 offspring were all diploid. Crosses of 4n egg parents by 2n males customarily give only shriveled seed. Pollen from elongate stocks on 4n silks gave rise to 1842 shriveled seeds and 18 plump kernels which proved to have triploid embryos. No plump kernels with 4n zygotes were found. These would occur if the pollen of el plants consisted of a mixture of diploid and haploid grains and the diploid grains effected fertilization. However, haploid grains have a competitive advantage over diploid pollen in pollen mixtures, and the failure to find 4n kernels might mean that the el plants had a low proportion of diploid pollen grains which, together with their disadvantageous handicap, could account for the absence of 4n kernels. This is unlikely. Diploid grains are larger than haploid, and the two can readily be distinguishedin pollen samples from tassels chimeric for 4n and Qin tissue. Although there is some abortion, pollen samples from elongate tassels show no indication of diploid grains. Furthermore, in the cytological studies of microsporogenesis all cells observed at metaphase I (MI) and MI1 contained either ten bivalents or ten dyads respectively and none had double that number. It may be concluded that no viable diploid pollen is produced. Among the effects found at microsporogenesis in el plants are the following: ( 1 ) The chromonemata are relatively uncoiled at anaphase and telophase of both meiotic divisions giving elongated or stretched chromosomes (Figure la, d) . (2) Misdivision of the centromere occurs occasionally at MII. (3) Neocentric regions, resembling those produced by abnormal chromosome 10 (RHOADESand VILKOMERSON 1942), occur sporadically at MI1 (Figure l f ) but have not been observed at M I or anaphase I (AI). (4) Immediately after the quartet stage the haploid spores may undergo another division without further chromosomal replication. The ten chromosomes are distributed randomly to one or the other pole (Figure lg). This behavior is similar to that produced by the plymitotic gene described by BEADLE(1931) except that in polymitotic plants the super- ELONGATE MAIZE 507 numerary mitoses take place while the spores are in quartets. Of these four cytological effects of the elongate gene, only the despiralization of the chromonemata is consistently found in all strains of elongate, Also observed in some elongate stocks were duets of cells at interphase between the first and second meiotic divisions that possessed two nucleoli in each sister cell and presumably had an unreduced chromosomal complement (Figure 1b,c) . These putative unreduced cells were never seen to undergo division. They apparently degenerate and may account for some of the pollen abortion observed in el plants, Hypotheses on the action of the el gene: It is evident from the above presentation that el plants produce both haploid eggs with ten chromosomes and unreduced eggs at the diploid level although some have less than 20 chromosomes and some have more. These unreduced eggs could arise in various ways. Among the possibilities are (1 ) somatic doubling in the sporogenous cells to form tetraploid tissue, ( 2 ) doubling in the gametophytic generation, ( 3 ) suppression of the first meiotic division followed by a normal second division (as used in this paper, “suppression of the first meiotic division” means that the meiotic events proceed normally up to AI, but disjunction fails to occur and a restitution nucleus is formed), (4) a normal first division with omission of the second, and ( 5 ) a normal first division with chromosomal replication occurring during interphase to form 20 dyads in each sister cell, followed by the second meiotic division. The logical way to decide among these five alternatives would be by direct cytological examination of megasporogenesis and gametogenesis, but this is technically difficult to do in maize. Some observations have have been made of megasporogenesis in sectioned ovules. They are in accord with the conclusions reached by the genetic analysis but by themselves are not conclusive. However, a cytogenetic study did permit discrimination among the various hypotheses. Observations of megasporogenesis disclosed only ten pairs at diakinesis and MI, but a limited number of cells were found in these stages and, since part of the eggs are diploid and part haploid, it is possible that only normal cells were examined. Nevertheless, the observations indicated that nucellar doubling is not responsible for the diploid eggs. Confirmation of this conclusion came from the ratio of dominant to recessive phenotypes found in the diploid eggs of el plants heterozygous for the wx gene close to the centromere of chromosome 9. The observed ratio of 3Wz:2wz is far from the approximate 5:1 expected if the diploid eggs are derived from duplex tetraploid cells. Cytological evidence of unreduced megaspores is based on observations on the nuclei of embryo sacs. Several ovules were found in which the nuclei contained two nucleoli and were undoubtedly diploid. Diploid nuclei were also observed at the interphase between the first and second meiotic divisions and, if 2n megaspores arise from these cells, doubling in the gametophyte generation (hypothesis 2 ) would be ruled out. Genetic studies have revealed that many of the diploid eggs are heterozygous for one or more marked loci. Obviously such heterozygous eggs could not have arisen by chromosome doubling in the gametophytic generation because, on such a mechanism, both homologues would carry the same alleles. I n order to differentiate between hypothesis 3 and 4, plants homozygous for 508 M. M. RHOADES AND E. DEMPSEY el and heterozygous for Sh Wx/shwx were crossed by pollen from recessive plants. The Wx locus is in the short arm of chromosome 9 and is known to lie within a few crossover units of the centromere (ANDERSON and RANDOLPH1945). Single exchanges may occur between Wx and the centromere, but double exchanges should be rare. The Sh, locus is also in the short arm of 9, approximately 20 crossover units distal to Wx. The frequencies of wx homozygosis in the diploid eggs expected from populations of megasporocytes with different frequencies of single-exchange tetrads is given in the tabulation below on the hypothesis of suppression of the first division and on the hypothesis of omission of the second division. Percentage of diploid eggs homozygous for wx coming from megasporocyte populations with different frequencies of single-exchange tetrads Megasporocyte population Suppression of first division 100% no-exchange Wx-centromere 80% no-exchange,20%single exchanges 100% single exchanges 0 50 5 25 41) Omission of second division 0 If no exchanges occur, 50% of the diploid eggs will be homozygous for the dominant allele of the segregating locus and 50% will be homozygous for the recessive allele on the hypothesis of omission of the second division. If, for example, 20% of the bivalents have a single exchange between the centromere and the tested locus and 80% have no exchange, then 40% of the diploid eggs will be homozygous for the recessive allele. Quite different expectations will come from the hypothesis of first division failure according to the above tabulation. No homozygosity will be found if no recombination occurs between the segregating locus and its centromere and only 5% of the diploid eggs will be homozygous for the recessive allele if single exchanges take place in 120% of the cells. Even with 100% single chiasmata between the locus and the centromere, the frequency of the homozygous recessive would be only 25 %. In a population of 509 diploid eggs, 39.5% were homozygous for the recessive wx allele while among 156 diploid eggs whose genotypes were determined, 19.2% were homozygous for the recessive sh allele. The high percentage of wx cannot be explained on the hypothesis of first division suppression. Moreover, the more distally located sh allele would be expected to give a higher and not a lower frequency of homozygosis if homozygosis was dependent on crossing over between the locus and the centromere. These data clearly indicate that the hypothesis of first division suppression is incorrect; they are wholly in accord with second division doubling as the mechanism by which diploid eggs originate. The validity of this conclusion was tested by an analysis of the genotypes of 2n gametes arising from elongate plants heterozygous for the Zg, and al loci in the long arm of chromosome 3. (Plants with the dominant Lg allele have a ligule at the base of the leaf while Zg plants are liguleless; the dominant A allele produces anthocyanin color in the aleurone and plant, while aa plants have colorless 509 ELONGATE MAIZE aleurone and are green.) The lg locus is situated near the middle of the arm and the A gene lies more distally. The standard recombination value between Lg and A is 36%. Both the plump and shriveled kernels on the testcrossed ears were planted and the progenies scored for the Zg and a phenotypes. The plump kernels gave rise to diploid offspring. The shriveled kernels required special germination techniques and even then many failed to grow. The viable seedlings from shriveled seeds were transplanted to the field and classified for the segregating phenotypes. The chromosome number of each plant was determined; all were at the triploid level with the great majority having 30 chromosomes. The euploid plants with 30 chromosomes were testcrossed both as the male and female parent in order to ascertain the genotypic constitution of the diploid eggs from the phenotypes and their proportions in the testcross progenies. Because of sterility, a great number of triploids failed to produce sufficient seed to allow the elucidation of the genotypic constitution of the diploid eggs from which they arose. However, in the crosses involving chromosome 3, the number of genotypic determinations was sufficient for a genetic analysis. Listed below are the genotypes of diploid eggs coming from diploid elel Lg A/Zg a plants used as the female parent in testcrosses. Genotype Number l g a __ L g A __ L g A -1gA LgA Lga L 1gA --g a L g A lga lga Lga lga 1gA l g a 1 g A 1gA LgA __ 8 5 66 26 37 21 16 22 3 2=204 The frequencies of homozygosis for Zg and for a do not yield useful information in support of either first or second division doubling since these loci in chromosome 3 are too far removed from the centric region. However, if the first division is normal, the frequency of tetrads with different exchanges can be determined from the kinds and numbers of half-tetrads recovered in the diploid eggs, They are as follows: combinations Lg A/Lg A and lg a/lg a come from no-exchange tetrads, Lg A/Zg a from singles in region 1 and from one half of the 3-strand doubles in regions 1 and 2, L g A / L g a and l g A / l g a from single exchanges in region 2, Lg A/lg A and Lg a/lg a from 2- and 4-strand doubles in regions 1 and 2, L g a / l g A from one hali of the 3-strand doubles in regions 1 and 2 and ZgA/lgA from 4-strand doubles in region 2. Region 1 is the segment from the centromere to Lg; region 2 is the Lg-A interval. When allowance is made for undetected combinations arising from doubles in region 2, the tetrads giving rise to the 204 diploid eggs consisted of 4.9% with no exchanges in either regions 1 or 2, 21.6% with a single exchange in region 1. 27.9% with a single exchange in region 2, 39.7% with one exchange in both regions 1 and 2, and 5.9% with two exchanges in region 2. On the assumption that doubling took place by formation of a restitution nucleus at the first division, a n entirely different array of tetrads is obtained. Double exchange tetrads are estimated at 59.3%, no-exchange tetrads are 16.7% and the single exchanges in (1) are 9.8%. Singles in (2) cannot be distinguished from doubles in (2); these would total 2.3%. Triple exchange tetrads with two 510 M. M. RHOADES A N D E. DEMPSEY exchanges in region 2 and one exchange in (1 ) would represent the remaining 12%. The tetrad frequencies calculated on the basis of first or second division doubling may be compared with those derived from G1, LghA,/gl, lgaal heterozygotes (RHOADESand DEMPSEY 1966) where the Gl-Lg interval is comparable to the centromere-Lg segment in the elongate material. Data from plants with knobless chromosomes 3 gave the following frequencies: no exchange tetrads 6.0%, single exchanges in Gl-Lg 20.2%, single exchanges in Lg-A 43.0%, and double exchanges 30.8%. The figures based on hypothesis 4 are in closer agreement with the above distribution than those calculated on hypothesis 3, especially when the doubles in region 2 are combined with the singles in that region. At this stage in the analysis it could be claimed that the diploid eggs originate from the omission of the second meiotic division (hypothesis 4) and that the problem of their origin was solved. However, the same array of half tetrads in the diploid eggs would be expected if the megasporocytes had undergone two normal meiotic divisions with an extra chromosomal replication occurring at interphase after the first division. Hypothesis 5 is the most unorthodox since it invokes a phenomenon that is believed to be unique. An extra replication of univalent chromosomes presumably occurs during meiosis in some triploids and species hybrids (see AVERS1954) since these chromosomes divide equationally at both meiotic divisions. The postulated behavior on hypothesis 5 differs from the previous studies since it presumes a replication of dyads which have come from paired bivalents. The ten dyads found in each cell at the end of TI may give rise to 20 monads which then replicate to produce 20 dyads in each sister cell of the duet. Interphase cells were observed at microsporogenesis and megasporogenesis in which each sister nucleus possessed two nucleoli (Figure Ib,c). They could have arisen either by interphase replication or by the ten dyads separating into 20 monads without replication (hypothesis 4). Although the interphase cells in microsporogenesis were never seen to divide and their diploid constitution was inferred from the number of nucleoli, the occurrrence of interphase replication during megasporogenesis would, if followed by a normal second meiotic division, lead to the formation of a linear set of four diploid megaspores. These have not been found but the number of ovules examined at the crucial stage was not great. On hypothesis 4 there would be two diploid spores at the end of meiosis which would resemble the diploid interphase cells already described. Although neither the cytological nor the genetic data permit an unequivocal choice between hypothesis 4 and 5, they clearly indicate that doubling of chromosome number takes place at the second meiotic division. The postulated interphase replication could be tested by a comparison of the DNA content in the duets of microspores with one nucleolus in each interphase nucleus and in the putative diploid duets with two nucleoli in each nucleus. According to hypothesis 4,the nuclei of the diploid duets would have a 2n level of DNA while on hypothesis 5 the amount of DNA should be at the 4n level following replication. I n addition to production of diploid megaspores, elongate plants also show variable pollen abortion, and considerable ovule abortion is characteristic of el ears. Any explanation of the action of the el gene must account for the inviability ELONGATE MAIZE 511 FIGURE1.-(a) Anaphase I in microsporogenesis of elongate showing stretched or uncoiled chromonemnta. (b) and (c) Duets of cells fr3m elongate microsporocytes at interphase after the first meiotic division. The two nucleoli in each putative diploid nucleus are indicated by arrows. (d) Anaphase I1 in elongate microsporogenesis illustrating the relatively uncoiled condition of the chromosomes. (e) Anaphase I1 in a normal plant showing the contracted chromosomrs characteristic of this stage. Compare with (d). ( f ) Neocentromeres a t MI1 in elongate microsporogmesis. ( g ) Supernumerary mitoses occurring in young microspores from a n elongate plant. No replication of the chromosomes has occurred and the ten chromosomes pass a t random to the two poles. This supernumerary mitosis is the primary cause of the pollen abortion found in elongate plants. 512 M. M. RHOADES A N D E. DEMPSEY of pollen and ovules as well as for the aneuploid chromosome numbers observed in some eggs at the diploid level. Inasmuch as misdivision of the centromeres was occasionally observed at MI1 in microsporogenesis, it was assumed that a similar phenomenon took place in megasporogenesis to form telocentric chromosomes. Plants with 31 chromosomes were, on this supposition, expected to have arisen from eggs with two telocentric chromosomes. Plants with 32 and 33 chromosomes could be accounted for by centromere misdivision of more than one chromosome. These suppositions are reasonable, but cytological examination of 31-chromosome plants revealed that they had nine trivalents and one quadrivalent at pachynema; no telocentrics were present and three intact homologous chromosomes had been contributed to the zygote by the 2n 4-1 eggs from the el parent. When plants with 32 chromosomes were studied, it was found that they possessed eight trivalents and two quadrivalents; in these cases two nonhomologous chromosomes were present in a trisomic condition in the 2n 2 eggs. There is no evidence for nondisjunction of individual chromosomes at the, first division since all plump kernels on el ears had zygotes with 20 chromosomes. Unlike the putative diploid interphase cells observed in microsporogenesis which do not develop further, diploid megaspores continue development to form embryo sacs although it is uncertain whether there is a second meiotic division following interphase replication (hypothesis 5 ) or whether they pass directly into the first of the three mitotic divisions of gametogenesis (hypothesis 4). I n either case, nondisjunction may occur when the 20 chromosomes congress onto a mitotic spindle which normally accommodates ten chromosomes. Failure of some of the 20 chromosomes to attain proper orientation owing to crowding on the spindle plate could lead to nondisjunction and thus give eggs with more and with less than 20 chromosomes. Ovule abortion would come from aneuploid cells having extreme chromosome unbalance. This explanation of the cause of nondisjunction is a mechanical one. It is perhaps more probable that there is a disturbance in the timing of the normally ordered mitotic events which leads to faulty orientation and consequently to nondisjunction. That unusual chromosome behavior does occur in microsporogenesis is evident from the modification of the coiling cycle, sporadic neocentromere formation, and centromere misdivision. Pollen abortion varies widely in different el strains but in those with high abortion a considerable fraction of the haploid microspores were observed to undergo division without chromosome replication having occurred (Figure lg) . Inasmuch as the ten chromosomes were distributed to the two poles in an apparent random manner, a vast majority of the resulting spores would be deficient for one or more chromosomes and hence abort. The inability of the diploid duets to undergo further development would be an additional cause of poor pollen production. A gene causing omission of the second meiotic division was reported in Datura by SATINAand BLAKESLEE(1935). I n their material a low percentage of both mega- and microsporocytes underwent a normal second division but the great majority gave rise to two spores instead of a quartet at the end of meiosis. The abnormal sporocytes had a prolonged telophase and resting stage following the + ELONGATE MAIZE 513 first division, during which time chromosome replication must have occurred in preparation for the first gametophytic mitosis. In the elongate material the chromosomes at telophase I resemble those of an interphase nucleus. The premature loss of condensation and the early despiralization may provide the extended resting condition needed for chromosome replication at the interphase between meiotic divisions postulated to occur by hypothesis 5. The cytological studies of megasporogenesis do not provide critical evidence for or against the hypothesis of omission of the second meiotic division and that of an extra chromosomal replication accompanied by a second division. However, the omission of the second division is the simpler of the two hypotheses and accounts for the cytogenetic data in as satisfactory a manner as does the hypothesis of an extra replication; perhaps it should be the favored mechanism even though there are at present no data which afford an unequivocal decision between the two schemes. Chromatid interference: The genotypic constitutions of 204 diploid eggs from el plants have already been presented. They permit a study of chromatid interference. Plants of el el, Lg A / l g a constitution produce diploid eggs of L g A / l g A and Lg a/lg a genotypes from both 2- and 4-strand doubles in regions 1 and 2. Three-strand doubles in the same regions yield equal numbers of L g a/lg A and L g A / l g a diploid eggs. The latter type cannot be distinguished from diploid eggs originating from single exchanges in region 1 but the origin of the former class is unambiguous. If there is no chromatid interference, the number of combined 2- and 4-strand combinations should be twice as frequent as the recognizable %strand doubles. There were 21 L g A/lg A , 16 Lg a/lg a and 22 Lg a/lg A diploid eggs, a proportion consistent with that expected with no chromatid interference. The crossover regions followed in this study are long enough to permit the occurrence of undetected double exchanges since 30% or more recombination occurs in both regions 1 and 2. That double exchanges do take place in region 2 is evidenced by the diploid eggs of lg A / l g A constitution which arise only from 4-strand doubles in (2) and cannot originate from other types of exchanges. Before accepting the evidence for random chromatid involvement in double exchanges in regions 1 and 2, it is necessary to consider the effect of double exchanges in (1) o r in (2). The diploid eggs from double exchanges in region 1 consist of equal numbers of L g A / L g A and lg a/lg a noncrossover combinations and of Lg A / l g a eggs which are identical to those coming from single exchanges in (1 ) . No combinations classifiable as 2, 4, or the identifiable 3-strand doubles in (1-2) are formed. Likewise double exchanges in region 2 yield no diploid eggs which could be classified as coming from 2-, 3-, or 4-strand doubles in (1-2). However, 4-strand doubles in region 2 will lead to the formation of L g a/Lg a and lg A / l g A compounds; three of the latter were found. These are unique types which come only from 4-strand doubles. No combinations are produced that are similar to the double exchanges in 1 and 2. It follows that doubles in either region 1 or in region 2 will have no effect on the question of chromatid interference in 1-2 doubles. 5 14 M. M. RHOADES AND E. DEMPSEY Triple exchanges with a double in region 1 and a single exchange in region E2 should be exceedingly rare events but if they do occur the ratio of recognizable 2-, 3-, and 4-strand doubles is identical to that coming from double exchanges in regions 1 and 2. Only from triples with a single exchange in region 1 and a double in region 2 is there a deviation from the ratio of 2: Ifor the combined 2- and 4-strand doubles to one class of 3-strand doubles. These infrequent triples produce a ratio of 2: 3 for the above combinations. However, it is improbable that the number of these triples is high enough to have a measureable effect. The conclusion that chromatid interference does not exist in the regions of chromosome 3 investigated appears to be justified although the demonstration suffers from the fact that 2- and 4-strand doubles could not be differentiated from one another and were grouped into one class. Centromere mapping: Centromere mapping in Ascomycetes with ordered spore arrangement is determined from the frequency of second division segregations. Since a single crossover between gene and centromere results in metaphase I1 segregation, the frequency of such segregations may be equated to chiasma frequency or to twice the recombination frequency. With unordered tetrads, centromere distances may be calculated from frequencies of tetratype asci if three 1949; PERKINS 1949; W H I T E independent loci are segregating ( LINDEGREN HOUSE 1950). The transformation of MI1 segregation frequencies into recombination values becomes less accurate as distance from the centromere increases since recombination values approach a maximum of 50% while MI1 frequencies approach 67% in the absence of chiasma interference. SPIEGELMAN’S (1952) equation for conversion of MI1 frequencies into crossover percentages is based on a modification of the formula derived by RIZET and ENGELMANN (1949) and PAPAZIAN (1951) which assumes a Poisson distribution of chiasmata. Centromere-gene distances determined from one half the frequency of MI1 segregation are underestimated whereas those derived from equations assuming no chiasma interference are overestimated. This relationship has been graphically presented by PERKINS (1962), who plotted the observed frequencies of tetratypes in Neurospora and four times the homozygosis values from attached-X Drosophila against map distance. Theoretical expectancies of crossover distances based on complete interference (one half of MI1 segregations) and with no chiasma interference were compared with the observed values. These fell between the two theoretical extremes, indicating a considerable amount of chiasma interference. With the exception of Aspergillus, chiasma interference has been reported in all organisms where tetrad or halftetrad analyses have been made. HOWE’S( 1963) determination of centromere distances in Neurospora tetrasperma, where the asci contain four binucleate ascospores, represents an interesting variant of the method used for those species with &spored asci where the frequency of MI1 segregation is considered to be twice the crossover distance from the centromere. In tetrasperma the overlapping spindles of both the second meiotic division and the third (mitotic) division result in the juxtaposition of pairs of nonsister nuclei which are cut out into the same ascospore. When no 515 ELONGATE MAIZE exchange occurs between a given locus and its centromere, all of the ascospores of an ascus are heterocaryotic (each of the two nuclei carrying a different allele). Following a single exchange, one half of the asci would be identical to those coming from no-exchange tetrads (Type I) while the remaining half would have homocaryotic ascospores (Type 11). Since only one half of the asci have homocaryotic ascospores following an exchange, the frequency of homocaryotic asci equals the recombination value. In species of filamentous fungi in which a sexual stage is unknown, it has been possible to locate centromeres because of the occurrence of haploidization and mitotic crossing over (PONTECORVO and KAFER 1958). Single chromosome arms have been mapped using the relative frequencies of homozygosis of recessive markers following crossing over. All of the genes on one chromosome are inherited as a unit in haploidization and the junction of the linkage maps of the two arms represents the centromere location. In sexually reproducing organisms where the haploid gametes contain a single chromatid of the four comprising each bivalent and there is no mechanism for sequestering the four products of meiosis, the location of the centromere is not so readily accomplished. It has, however, been achieved in a number of ways. In the early days of Drosophila genetics the location of the centromeres on the linkage maps of the two large autosomes was estimated by the absence of chiasma interference in specific short, adjacent regions. Another method of locating centromeres involves the determination of the linkage of mutant loci with a structural aberration which has one breakpoint close to the centric region. It was not possible to measure recombination values between the centromere and a given locus in Drosophila until the discovery of females with attached-X chromosomes (L. V. MORGAN 1922). Since two X chromosomes were regularly recovered in the eggs, half-tetrad analyses were possible and the amount of recombination as well as map distance between the centromere and mutant loci could be calculated from the genotypic constitutions of the exceptional daughters (ANDERSON 1925; EMERSON and BEADLE1933; BEADLEand EMERSON 1935). The opportunity provided by the el gene for half-tetrad analysis is comparable to that furnished by attached-X’s in Drosophila. Only part of the eggs are diploid in el maize, but a sufficient number are produced for half-tetrad studies. The array of diploid eggs from tests involving genes on chromosome 3 has already been presented in another section. Elongate plants heterozygous for sh wx on chromosome 9 were also testcrossed and gave rise to the following diploid eggs: ShWx ~ _ Sh Wx 24 shwx_ _ shwx S_ h W x_ sh wx 28 30 S-h W-x - shwx _ _ShWx sh W x Shwx Sh wx 35 33 4 sh wx shWx 2 x=156 Another group of diploid eggs was obtained from el el, Ws, Lgr Gle/ws, lg, gl, plants, but the triploid progeny was not analyzed for genotypic constitution and only the phenotypic classification is available. The data are given below: 516 M. M. RHOADES AND E. DEMPSEY Ws Lg G1 ws 1g gl ws Lg G1 ws lg G1 371 28 10 12 ws Lg gl Ws Lg gl Ws lg gl 2 30 23 2 = 476 The wss (white sheath), Zgl (liguleless), and gl, (glossy) loci lie in the short arm of chromosome 2 in the order listed with ws near the distal end and gl closest to the centromere. Analyses of these three sets of data have made possible the tentative location of the centromeres on the genetic maps of chromosomes 2 , 3 , and 9. There is a direct relationship between recombination values and the percentage of homozygosis in diploid eggs from el plants. With no recombination between a marked locus and the centromere, 50% of the diploid eggs are homozygous for the recessive allele and 50% are homozygous for the dominant allele. With 10% recombination, 40% of the diploid eggs would be homozygous for the recessive allele, 40% homozygous for the dominant allele, and 20% heterozygous. Thus, for short regions, recombination is equal to 50 minus the percentage of the homozygous recessive class, or one half the frequency of the heterozygous class. With recombination values greater than 1 OX, the percentage of equational segregation ( A a gametes) no longer represents twice the recombination value because multiple exchanges give rise to undetected crossovers. Table l gives the frequencies of homozygosis observed in the 3n progenies of el plants heterozygous for the gene loci listed and the calculated frequencies of equational segregation on the assumption that the homozygous a a class is equal to the A A . The recombination percentage between each locus and its centromere is based on one half the frequency of equational disjunction. The best estimation of centromere position is that determined for chromosome 9 as based on wx homozygosis. The Wx-centromere interval is short and no distortion of the recombination value by double or triple exchanges should occur. It may be concluded that the centromere lies approximately 11 map units from W x . This value is considerably greater than that reported by ANDERSON and RANDOLPH(1945) from translocation studies, but crossing over is reduced in TABLE 1 Recombination values in the gene-centromere segment for seven loci based on observed percentages of homozygosis in diploid eggs Gene ,wx Shl Lg, AI G4 Lg, WS, Percent homozygous recessive Percent equational disjunction 38.5* 18.6* 19.4* 13.0* 17.5 13.3 10.9 23.0 63.6 61.2 74.0 65.0 73.4 78.2 * Average of homozygous dominant and homozygous recessive classes. Percent recombination (Equational disjunction X l/) 11.5 31.8 30.6 37.0 32.5 36.7 39.1 517 ELONGATE MAIZE translocation heterozygotes and their value of 2% is certainly less than the amount occurring in structural homozygotes. Lower percentages of homozygosis (and higher frequencies of equational segregation) were found for the other loci, which are known to be more distally located. In three cases, the frequencies of equational disjunction were greater than 67%, the theoretical limit when chiasma frequencies follow a Poisson distribution. The complete genotypic analysis of the 2n eggs segregating for genes on chromosome 3 and for genes on chromosome 9 made possible a calculation of the distribution of chiasmata in the A-centromere and the Sh-centromere regions. In neither case did the calculated distribution conform with the Poisson distribution expected with the average chiasma frequencies indicated for each set of data. The high frequencies of equational disjunction are therefore due to deviations from a Poisson distribution in which single chiasmata are more frequent than expected and no-exchange tetrads are less frequent. Comparable deviations in chiasma frequencies were reported by PERKINS (1962) for Neurospora and by KNAPP (1960) for the liverwort Sphaerocarpus. Although the four spores in Sphaerocarpus are unordered, the regular disjunction of the X and Y chromosomes to opposite poles at AI permitted identification of the equational or reductional segregation of loci in other chromosomes. Chiasma frequencies were also calculated for the chromosome 2 data from elongate plants. Since only phenotypes were scored, a number of the recombinant types are included in the triple dominant class and their frequencies must be estimated on the basis of detectable crossovers. Although this places undue emphasis on some of the less frequent classes, a reasonable distribution of chiasma frequencies is obtained. These are listed in Table 2, as are the values determined for the chromosome 9 and chromosome 3 data. Double chiasmata in the Wxcentromere, Lg,-centromere, and GL-centromere regions cannot be detected and have been ignored. On the basis of these chiasma frequencies, a corrected recombination value may be obtained. Bivalents with no exchanges give only homozygous diploid eggs and no recombinant strands. Single exchanges lead to 100% A a eggs and 50% recombinant chromatids. Double exchanges give 50% A a eggs and 50% recombination. The corrected recombination exceeds the value obtained from the frequency of equational disjunction by the percentage of 4-strand douTABLE 2 Calculated chiasma frequencies and corrected recombination values in four gene-centromere intervals Calculated chiasma frequencies Region None Single Double Ws,-Centromere Lg,-Centromere Sh,-Centromere A,-Centromere 11.7 68.1 68.5 60.9 49.5 20.2 Corrected percent recombination ~ 21 .4 33.3 4.9 10.1 5.8 45.6 ~~ 44.2 39.3 33.4 47.6 518 M. M. RHOADES AND E. DEMPSEY bles since these are considered to be nonrecombinant on the basis of their reductional segregation but actually contain all recombinant strands. The objection may be raised that the abnormal chromosome behavior in megasporocytes giving rise to diploid eggs could also affect recombination and the values obtained would be unreliable. That this is not the case is indicated by a comparison of recombination in the haploid and diploid eggs from the same ears. The amount of recombination between Lg and A in the 408 chromosomes of the 204 diploid eggs was 36.7%, while 34.1 % was found in a population of 452 plants arising from the haploid eggs. A crossover value of 23.7% was found for the Sh-Wx region in diploid eggs and 99% in the haploid eggs. Moreover, the percentages of recombination in both haploid and diploid eggs of elongate plants agree well with the standard values. It may be concluded that crossing over is normal in megasporocytes which are destined to give rise to diploid eggs and the estimations of centromere distances are considered to be valid. Centromere mapping has been attempted in other higher organisms. LINDSLEY, FANKHAUSER and HUMPHREY (1956) determined the frequency of homozygosis is diploid eggs of the Mexican axolotl produced when the second meiotic division was suppressed as a result of refrigeration. Heterozygous females were mated to recessive males and the triploid offspring arising from the diploid eggs were scored for three loci. The frequency of equational separation was determined from the percentage of homozygous recessive off spring. The value of approximately 67% equational separation found for all three loci was interpreted to mean that the frequency of crossing over in the studied regions was high enough to lead to random assortment. The suggestion was made that all three genes lie near the ends of their respective arms. This need not be the case if the number of chiasmata in different tetrads are not in accord with that expected by the Poisson distribution. For example, the Sh, locus in chromosome 9 is not more than 35 map units from the centromere region, yet in 64% of the diploid eggs from elongate plants there was an equational separation of the Sh:sh alleles. This value is very close to the 67% equational separation which would be realized if the Sh locus was segregating independently of the centric region but this obviously cannot occur for a region 35 map units long. The conclusion reached by LINDSLEY et al. that the three tested loci in the axolotl segregated independently of their centric regions should not be unequivocally accepted. A situation somewhat similar to that in the axolotl has been studied in the honey bee. Diploid progeny are occasionally produced by unmated queens. TUCKER (1958) believes these arise by fusion of two of the four products of meiosis, specifically those arising from different secondary oocytes. The cytological scheme proposed was based on observed percentages of homozygous recessive phenotypes in the offspring of queens heterozygous f o r three unlinked loci. The very low frequency of homozygous ivory was attributed to a combination of (1) its location close to the centromere, (2) union of nuclei derived from different secondary oocytes and (3) reductional centromere division at AI. Interference with oviposition led to increased frequencies of impaternate workers on resumption of egg laying. Since the egg remains at the AI stage until after it is ELONGATE MAIZE 519 laid, it n'as argued that aging of the egg by inhibition of oviposition might cause a failure of spindle reorientation which normally occurs at this stage. This would lead to the union of the two adjacent nuclei resulting from different second division spindles. The observed frequencies of homozygosis could not be explained by the occurrence of meiosis in tetraploid tissue or by fusion of the products of the first cleavage after meiosis. However, TUCKER'S data could be more simply accounted for if there was a failure to complete the first meiotic division and a restitution nucleus was formed. On this hypothesis, a gene close to the centromere would show very low frequencies of homozygosis. Cytological studies are needed to discriminate between the failure of the first division and the spindle reorientation hypothesis. Although the effective suppression of first meiotic division in the unisexual wasp Devorgilla should lead to estimation of valid centromere distances, it is uncertain whether or not unambiguous conclusions can be reached with the I gene studied by SPEICHER,SPEICHER and ROBERTS (1965). Distinction between +/I and females depends on the percentage of abortion in their progenies. This varied from 0 to 23.9%. Females with 0-2% egg lethality were considered to be while those whose egg abortion ranged from 3.77 to 23.9% were scored as heterozygotes. The difference between the two classes is not of great magnitude. In polyploid plants which tolerate a deficiency of one chromosome arm, it is possible to determine gene-centromere distances by the use of telocentric chromosomes. Plants having a heteromorphic pair consisting of a normal chromosome and a telocentric and heterozygous for a gene located in this pair of chromosomes map be testcrossed and the progeny classified for phenotype and for the presence of the telocentric. Centromeres have been located with respect to genes on chromosomes 6B and 3B of wheat by this method (SEARS1962) and a modification allowing use of F, data has resulted in placement of the centromere of chromosome 2A (DRISCOLL 1964). Essentially the same method has been used in maize to locate the centromere of chromosome 3. Plants heterozygous for mutant loci and for a normal chromosome 3 and two chromosome fragments consisting of the short and long arms were testcrossed. The progenies were scored for the segregating alleles and for chromosome constitution (RHOADES, unpublished). I n determining centromere-gene recombination values, the telocentric method has an advantage over the other methods in that the centromeres are distinguishable by virtue of one being terminal and the other interstitial. Recognition of 4-strand doubles is possible with a telocentric chromosome, but comparable doubles in tetrad or half-tetrad analysis produce an apparent noncrossover tetrad. The recombination value for the centromere-A interval determined by the elongate method should be smaller than the correct value by the number of 4-strand doubles. Such doubles comprise 11.4% of the tetrads. The uncorrected centromere-A recombination value from elongate half-tetrads was 37.0%. The sum of 11.4 and 37.0 or 48.4% is a more accurate estimate of the recombination percentage between the centromere and A. Significantly, the centromere-A recombination value determined by the telocentric method indicated that A segregated independently of the centromere. +/+ +/+ 520 M. M. RHOADES AND E. DEMPSEY It is evident from the above discussion that centromere mapping is possible in higher forms by employing unusual cytogenetic mechanisms. However, as LINDSLEY et al. (1956) have emphasized, caution should be exercised before accepting these centromere distances unless it is possible to establish that the amount of recombination has not been modified by the genetic system itself. Polyploid series: The el gene has been used to obtain an interesting series of polyploids. Triploids come irom the cross of el x 2n. Crosses of el by 4n gave some plump kernels which were tetraploids. By self-pollination 4n el stocks were obtained. When these were self-pollinated or crossed by 4n plants, 6n plants came from the union of unreduced 4n eggs with 2n pollen. The 6n plants came from shriveled kernels while the plump kernels on the same ear gave rise to 4n plants. Hexaploid plants homozygous for el have been produced but no seed was obtained when they were self-pollinated. However, plants at the 7n level have arisen from the cross of 4n el x 6n when unreduced 4n eggs were fertilized by 3n pollen. Pentaploids (5n) have come from crosses of 4n el x 6n where reduced 2n eggs were fertilized by 3n pollen. Vigorous plants are obtained up to the 5n level but beyound this level the plants are dwarfed and female sterile. Accurate chromosome counts are difficult to make at high levels of ploidy and the statement that a plant is hexaploid means only that it is at the 6n level. It is of some interest that the chloroplasts were of the same size in haploid leaves as in hexaploid and heptaploid leaves. They are more numerous per cell in the latter but no size difference is apparent. Whether this represents genetic autonomy of the plastids or is an adaptation to optimum size for photosynthesis is uncertain. This manuscript was prepared while the senior author was a guest investigator in the Department of Genetics of the Australian ,National University and in the Division of Plant Industry, C.S.I.R.O.,Canberra, Australia. Appreciation of their hospitality is gratefully acknowledged. SUMMARY Reduced and unreduced eggs are produced by plants homozygous for the recessive elongate ( e l ) gene. When el plants are crossed by pollen from a 2n plant, plump and shriveled kernels as well as aborted ovules are found. The plump kernels give rise to 2n plants and the shriveled kernels to plants at the 3n level. Eighty-two percent of the unreduced eggs had 20 chromosomes while 18% had aneuploid numbers ranging from 15 to 23. The aborted ovules presumably come from megaspores with so extreme a chromosomal unbalance that lethality results. -All functional pollen of el plants is haploid. Duets of cells with an unreduced chromosome number are found in some el microsporocytes at interphase. They do not develop further and are believed to contribute to the poor pollen production. Pollen abortion results from a supernumerary cell division of the microspores at a time before the chromosomes have replicated.-The elongated or stretched appearance of the chromosomes in microsporocytes of el plants at AI and AI1 indicated that the chromonemata are relatively uncoiled. Centromere misdivision and neocentromere formation sporadically occur at MI1.-Half-tetrad ELONGATE MAIZE 521 analysis of the 20-chromosome eggs indicated random chromatid involvement in double exchanges.-The percentage of recombination between a gene and its centromere can be determined from the percentage of diploid eggs homozygous for the recessive or dominant allele. Centromere recombination values were obtained for the wx and sh, loci in chromosome 9, for the lg, and a, loci in chromosome 3, and for the ws3,lg, and gl, loci in chromosome 2.-Unreduced eggs are produced by 4n plants homozygous for el. This made possible a polyploid series up to the heptaploid level-The unreduced eggs originate either by omission of the second meiotic division or from an extra chromosomal replication during interphase between the first and second meiotic divisions. 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