The presence of synaptic and chromosome disjunction mutants in Cenchrus ciliaris ( Poaceae : Paniceae )

Synaptic mutants are present in Cenchrus ciliaris L This species, due to the presence o f linear bivalents and occasion ­ al trivalents and quadrivalents, is an intermediate desynaptic species. In addition, geographical distribution and environ­ mental factors, such as high temperatures and low humidity, could also have had an influence on the desynapsis observed. The disjunction o f chromosomes during anaphase I was mostly abnormal in this desynaptic species. Precocious disjunction o f chrom osom es into chromatids occurred during anaphase I Due to the high incidence o f this chromosome abnormality, a mutant gene, 'p c \ responsible for the disjunction o f chromosomes, must be present. The absence o f cytokinesis in one spec­ imen indicates a recessive mutant gene, 'v a \ to be active in this species.


INTRODUCTION
Meiosis is a complex process and includes cytogenet ic features such as chromosome pairing, synaptonemal complex formation, recombination, chromosome segre gation and the creation of gametic meiotic products.The precise sequence of meiosis is under the control of vari ous genes (Golubovskaya 1979).These include premeiotic genes, 'as' genes (controlling leptotene and zygotene), 'des' genes (controlling the various stages from pachytene to metaphase I) and chromosomal disjunction or spindle genes (controlling meiotic stages from ana phase I through to the formation of tetrads) (Golu bovskaya 1979).Mutations present in these genes drasti cally change the normal behaviour of chromosomes with in a specimen or species.Reports of synaptic and malesterility mutants predominate, whereas premeiotic and disjunction mutants are relatively rare (Singh 1993).
Synaptic mutants are common in the plant kingdom and were originally discovered in maize (2n = 20) (Beadle & McClintock 1928) and were observed in about 20 high er plant families, consisting of 50 genera and approxi mately 70 species (Koduru & Rao 1981).The majority of these taxa belong to the family Poaceae (Singh 1993).
Meiotic mutants have been mainly identified with the aid of cytogenetic studies, genetic evidence and pollen or ovule abortion.These mutants, which arise mostly spon taneously.may result from interspecific hybridisation or may be induced by mutagenesis (Singh 1993).The aim of this study was to determine whether meiotic mutants are present in Cenchrus ciliaris L.

MATERIALS A N D M ETHODS
The specimens used are listed in Table 1.Voucher her barium specimens are housed in the George Potts Herbarium, Department of Botany and Genetics, Uni versity of the Orange Free State, Bloemfontein (BLFU).Slides, suitable for meiotic studies, were prepared accord ing to the methods described by Visser & Spies (1994).A minimum of 20 cells of each of the following stages were studied: metaphase I, anaphase I, and telophases I and II.The following were recorded when observed: chromosome configurations and the number of univalents (MI), laggards (Al) and micronuclei (TI and Til).
Cells in the diakinesis stage were seldom observed.Well-defined meiotic configurations during diakinesis were observed in one specimen only, Spies 5655 (Figure 1A).The chromosomes were small and configurations consisting of more than one chromosome, were identi fied based on relative size (Figure 1A-D).The chromo somes were often not paired and were distributed as uni valents in the cell (Figure 1A-D).
Meiotic behaviour of the various chromosome config urations observed, varied during metaphase I (Figure 2A-E).Bivalents and occasional trivalents or quadriva lents moved to the equatorial plate, whereas the univa lents were mostly distributed in the cytoplasm (Figure 2A-E).The number of univalents observed during metaphase I, varied within the different microsporocytes in the same specimen.In Spies 5230, zero to 18 univa lents were observed during metaphase I (Table 1).This was the highest number of univalents observed within a single cell of this species (Table 1).More than 95% of the univalents observed were two or multiples of two per cell (Visser et al. 1998a), indicating that they originate from incomplete pairing of a chromosome pair, rather than representing the odd non-pairing chromosome in multivalent formation.
a i a j r j n rsi n a j a i rvi rJ r j r^j rsi r-J rJ r j r J N N r 4 N fS CS rS N  ------------------------------------------------------------------------------------* ---------( , 1  Chromosome pairing during prophase plays a critical role in the sequence of meiotic events that follow.The success of chromosome pairing during the early stages of the first meiotic division, will affect the viability ot the meiotic gametes formed.Incomplete chromosome pair ing will lead to various meiotic irregularities, such as the formation of univalents, the presence of chromosome laggards and micronuclei. The partial or complete loss of chromosome pairing observed during prophase and metaphase I can be attrib uted to one of two processes, namely asynapsis or desy napsis.Asynapsis (Randolph 1928) is the absence of chromosome pairing during the first meiotic division, whereas desynapsis (Li etal. 1945) is the failure to main tain association after first synapsis in prophase.The paired chromosomes, therefore, dissociate during diplotene.The action of asynaptic genes is recognisable when most, or all of the chromosomes, remain as univa lents at diakinesis and metaphase I.These genes also induce polyploid meiocytes, elongated and curved spin dles, and the misdivision of univalents (Miller 1963).Beadle (1930) assigned the gene symbol 'as' to these types of mutants.Prakken (1943) classified desynaptic mutants, depending upon their expressivity, into three categories, namely weakly desynaptic (several univalents), interme diate desynaptic (many univalents) and completely desy naptic (exclusively univalents and rarely any bivalents).The cytogenetic results of this study indicate the pres ence of desynaptic genes.Univalents observed during diplotene and metaphase I were accompanied by biva lents and occasionally quadrivalents (Figure 2C).Not all of the chromosomes present were univalents (Figure 2B, C. E).Cenchrus ciliaris is thus an intermediate desynap tic species.
The variation in the number of univalents present in the cells, excludes the presence of asynaptic genes, since asynapsis is associated with complete lack of chromo some pairing.The variation in the number of univalents also varied among the cells of a particular specimen, and among specimens belonging to the same polyploid level.This suggests that within the chromosome complement of a species, there may be differences among the differ ent chromosomes concerning their requirements for the initiation of pairing (Rees 1958;Swaminathan & Murty 1959;Koduru & Rao 1981).Since chiasmata in desy naptic mutant plants are mostly terminal and rarely inter stitial at metaphase I (Li et al. 1945).the number of rod and ring bivalents were noted particularly during metaphase I.Most of the bivalents observed within the specimens of all three polyploid levels, were linear (Figure 2B, C, E), indicating terminal chiasmata.This observation confirmed the presence of synaptic mutants in this species.
Previous studies indicated that C. ciliaris forms an agamic complex and all ploidy levels represent speci mens which are alloploid or segmental alloploid tending towards alloploidy (References listed in Visser et al. 1998a.b, c).These specimens are consequently sup posed to behave meiotically almost as diploids.Although the very low frequency of multivalents formed in a few specimens could contribute to the formation of univa lents, we regard that contribution as insignificantly low and consider the majority of univalents in this study as the result of desynaptic genes.
Spontaneous synaptic mutants exhibit monogenic recessive inheritance mostly (Koduru & Rao 1981) and have been isolated from natural populations.The major ity of synaptic mutants reported in the higher plants have been identified in diploid species, such as Hordeum vul gare L. (Ramage 1985).Fifteen desynaptic genes have been identified in barley, of which 13 were of sponta neous origin and two were induced (Hernandez-Soriano et al. 1973;Hernandez-Soriano & Ramage 1974. 1975).
Chromosome behaviour during anaphase I differed among the various specimens studied.Univalents lying away from the spindle equator during metaphase I. were randomly distributed as laggards to the poles during anaphase I. Univalents distributed on the metaphase plate orientated themselves axially and divided into chromatids, which in turn lagged during segregation to opposite poles during anaphase I (Figure 3A-I).This precocious disjunction of chromosomes into chromatids during anaphase I. was observed in 24 of the specimens studied (Table 1).These specimens represented all three polyploid levels (Figure 3A-I) (Table 1).The number of chromosomes participating in the precocious disjunction varied from one to a maximum of 15 (Table 1).
The premature segregation of some univalents into chromatids resulted, in the case of a tetraploid specimen (n = 2x = 18) with two univalents, in a 20/20 distribution of chromosomes and chromatids during late anaphase I.In a normal cell, an 18/18-chromosome distribution is expected.The segregating chromatids were, due to their smaller size, distinguished from the normal chromo somes (Figure 3A-E).The segregating chromosomes were organised into dyad nuclei, whereas the chromo some and/or chromatid laggards were included in micronuclei.
Temperature, humidity and chemicals (Prakken 1943;Ahloowalia 1969;Koduru & Rao 1981) may influence the degree of chromosome pairing in synaptic mutants.It also varies from plant to plant, day to day, year to year and between specimens collected at different times dur ing the same day (Prakken 1943;Soost 1951).The degree of expression of each synaptic gene is also vari able.Goodspeed & Avery (1939) reported, with regard to an asynaptic mutant of Nicotiana sylvestris L., that high temperature and low humidity greatly increased asynap sis, whereas high temperatures and high humidity decreased asynapsis.Ahloowalia (1969) recorded, in a desynaptic mutant of Hordeum vulgare (2n = 14).that at lower temperatures (11C C). the mean number of bivalents/cell was 7.71.but that at 28°C. the mean number of bivalents dropped to 5.39 bivalents/cell due to desynap sis.In contrast.Li et al. (1945) observed a greater degree of pairing at higher temperatures and decreased pairing at lower temperatures in desynaptic mutants of Triticum aestivum L.
The Cenchrus ciliaris specimens studied, were col lected in areas with a very low average annual rainfall.These areas represent some of the hottest and least humid geographical regions in South Africa.Therefore, the geo graphical distribution and environmental factors could also have had an influence on the desynapsis observed within specimens belonging to this species.
The disjunction of the chromosomes during anaphase I in C. ciliaris was not normal according to the descrip tion of meiotic behaviour for desynaptic mutants previ ously mentioned.Very few chromosome laggards observed during anaphase I, did not undergo disjunction into chromatids (Figure 3A-E).This chromosome abnor mality was representative of all three polyploid levels and was observed in 47.7% of all the specimens studied (Table 1).The high percentage of precocious disjunction suggests the presence of a mutated gene responsible for the disjunction of chromosomes.
A meiotic mutant that shows precocious centromere division, 'p c \ in Lycopersicon esculentum Mill, was described by Clayberg (1959).Chromosome pairing was normal until metaphase I.The precocity first appeared at anaphase I in some bivalents that often lagged and underwent premature centromere division.The cen tromeres of those chromosomes not lagging during the first division, divided mostly during prophase II.All of these chromosomes were regularly oriented on the metaphase II plate.The precociously divided chromo somes moved at random to the poles without further divi sion.Many chromosomes lagged in the second division and frequently formed restitution nuclei.The mutation segregates as a single recessive gene, tp c \ Although in Cenchrus ciliaris the situation differs to some extent, chromosome pairing was mostly normal until metaphase I.The precocity first appeared at anaphase I in some chromosomes that lagged and under went premature centromere division.The presence of a gene responsible for precocious centromere division, could, therefore, also be functional in this species.
One specimen, Spies 5512 (n = 2x = 18), exhibited nor mal chromosome behaviour until telophase I, but cytoki nesis did not occur in some of the meiocytes studied (Figure 4A-F).Two spindles formed in some of the cells, resulting in four anaphase I poles (Figure 4A-D).This resulted in cells containing a total of approximately 62 to 69 chromosomes, being distributed amongst the four poles during anaphase I.The lack of cytokinesis resulted in gametes with unequal chromosome constitutions.Chro mosomes not segregating to the nearest pole were exclud ed from the main nuclei, and included in additional micronuclei (Figure 4E, F).Another chromosome disjunc tion mutant, namely the recessive mutant 4va\ may be active in this specimen.Beadle (1932) first identified this mutant in maize.He based his hypothesis on the fact that a homozygous plant (vaJva) exhibited a normal prophase I, but cytokinesis was absent during telophase I.This resulted in gametes with diploid and tetraploid chromo some constitutions (Singh 1993).Beadle (1932) stated that failure of cytokinesis might either occur at the first or the second meiotic division.Due to the lack of cytokinesis, an increase in chromosome numbers can occur.This mutant gene could, therefore, be the reason for higher polyploid levels within this species.
Due to the lack of cytokinesis in some of the cells belonging to this specimen, the disjunction gene, 'va\ may be present.Since this specimen was collected near the only hexaploid (n = 3x = 27) specimens studied (Spies 5514, 5515 & 55/7), this mutated gene could have been responsible for the high polyploid levels in this area.

CONCLUSIONS
The cytogenetic results indicate the presence of synaptic mutants in C. ciliaris.This statement is based on incomplete chromosome pairing, which led to the ori gin of various chromosome irregularities observed during meiosis.This species was characterised as an intermediate desynaptic species.Geographical distribution and en vironmental factors, such as high temperatures and low humidity, could also have had an additional influence on the desynapsis observed in this species.
The disjunction of chromosomes during anaphase I was mostly abnormal in this desynaptic species.Precocious disjunction of chromosomes during anaphase I led to the formation of chromatids.Due to the high inci dence of this chromosome abnormality, a mutant gene, 'p c \ responsible for the disjunction of chromosomes, may be present.The lack of cytokinesis in one specimen indicates the possible presence of a recessive mutant gene, 'va' in this species.
It is, therefore, concluded that three meiotic mutant genes could be active in this species, namely the desy naptic mutant 4a s \ active during prophase, and the two chromosome disjunction mutant genes 4 p c ' and 'va .