Chromosome numbers and meiotic behavior of some Paspalum accessions

Chromosome number and meiotic behavior were evaluated in 36 Brazilian accessions of the grass Paspalum (which had never previously been analyzed) to determinate which accessions might be useful in interspecific hybridizations. The analysis showed that one accession of Paspalum coryphaeum was diploid (2n = 2x = 20) and one accession of Paspalum conspersum hexaploid (2n = 6x = 60), the remaining 34 accessions being tetraploid (2n = 4x = 40). The pairing configuration was typical for the ploidy level i.e. in the diploid, chromosomes paired as 10 bivalents, in tetraploids as bi-, triand quadrivalents, and in hexaploid as 30 bivalents. A low frequency of meiotic abnormalities (less than 10%) was observed in the diploid, hexaploid and some tetraploid accessions, although the majority of tetraploid accessions showed a high frequency of meiotic irregularities. The use of accessions with a low frequency of meiotic abnormalities in breeding programs is discussed.


Introduction
The genus Paspalum (L.), one of the most economically important grass genera of the Paniceae tribe, includes approximately 330 species distributed in tropical and subtropical regions of America, with a few taxa growing in the Old World (Denham et al., 2002).A large number of Paspalum species provide excellent grazing in native grasslands of the warm regions of South America (Bonilha and Quarín, 1997) and several members of the genus are also used as turf grass and for ornamental purposes (Burson and Bennett, 1971).Several species occur in the various Brazilian ecosystems, however only a few native species (especially those belonging to the Dilatata, Notata and Plicatula groups) have been cultivated as forage (Quarín et al., 1996).Although great progress has been made in cytological studies of members of the genus, little or no information is available for a large number of members of species and accessions held in herbariums.However, approximately 80% of the Paspalum species which have been cytologically investigated are polyploids, or have polyploid races, with about 50% of these polyploids being tetraploids of which most are apomictic (Quarín, 1992).This type of cytology complicates breeding programs.
During the last three decades, several collection trips have been undertaken by Embrapa Cenargen throughout Brazil, Paraguay, and Argentina to establish a base Paspalum collection.As a result, a germplasm collection comprising more than 1500 accessions of different Paspalum species is available at Embrapa Cenargen (Brasilia, DF), with a small part of this collection being duplicated at the Institute of Botany (IBONE) at Corrientes, Argentina (Quarín et al., 1997).Part of the Cenargen Paspalum germplasm collection was allocated to the Embrapa/Centro de Pesquisa de Pecuária do Sudeste (CPPSE), São Carlos, São Paulo state, Brazil) where the agronomic aspects of 216 Paspalum accessions (known as the CPPSE collection) are currently being evaluated (Batista et al., 1995) with the aim of selecting accessions for intra-and interspecific crosses to be used for obtaining new varieties.
Cytological analyses have been performed on more than 80 accessions from the CPPSE collection (Freitas et al., 1997;Takayama et al., 1998;Adamowski et al., 1998Adamowski et al., , 2000;;Pagliarini et al., 1998Pagliarini et al., , 1999Pagliarini et al., , 2000Pagliarini et al., , 2001) ) and the data presented in the present paper extends this study to a further 36 previously unanalyzed Paspalum accessions, including P. notatum Fluegge, P. compressifolium Swallen, P. usterii Hack., P. plicatulum Michx., P. atratum Swallen, P. coryphaeum Trin., P. lividum, P. rhodopedum L. B. Smith & Wasshausen, P. limbatum Henrard, P. conspersum Schrad and unidentified accessions belonging to the Plicatula group.The Plicatula group is an unofficial taxonomic rank created by Chase (1929) to group species related to P. plicatulum, this group having its geographical center of variation in central and western Brazil, eastern Bolivia and Paraguay (Quarín et al., 1997).Of the informal taxonomic groups into which the genus Paspalum has been subdivided (Chase, 1929), the Plicatula group is of the greatest agronomic interest because it encompasses a wide variation of forms and species, several of which are well accepted by cattle (Quarín et al., 1997).Since knowledge of the cytology and reproductive behavior of a species is es-sential for interspecific hybridization programs the aim of this research was to investigate the chromosome number and meiotic behavior of 36 Embrapa CPPSE germplasm accessions which had not before been analyzed.

Material and Methods
Cytogenetic studies were carried out on 36 accessions from the Embrapa CPPSE Paspalum collection, these accessions having been collected from several different regions of Brazil (Table 1).Of the 36 accessions analyzed, 18 Plicatula group accessions were not identified to species level.
Young inflorescences were collected at Embrapa CPPSE and fixed in 3:1 ethyl alcohol:acetic acid for 24 h at room temperature and then transferred to 70% (v/v) aque-774 Adamowski et al. ous alcohol and stored in a freezer until needed.Microsporocytes (PMCs) were prepared from the inflorescences by squashing and staining with 1% propionic carmine.The chromosome number and the pairing configuration were determined in 20 meiocytes at diakinesis, and the meiotic behavior determined in more than 2000 microsporocytes up to this phase to the end of meiosis with a similar number of cells in each phase.
The pairing configuration at diakinesis varied among accessions with the same ploidy level (Table 2).In the diploid P. coryphaeum accession only bivalents were recorded (Figure 1a) but among the tetraploid accessions the pairing behavior was irregular and diverse, with uni-, bi-, tri-and quadrivalents being observed (Figure 1b).In general, tetraploid accessions showed a predominance of bivalent chromosome associations.In three accessions (BRA-009661, BRA-006378, and BRA-006513), diakinesis was not adequate for analysis of chromosome pairing.For the diploid and tetraploid levels of ploidy, the behavior of chromosome 776 Adamowski et al. association was in agreement with previous reports on the same or other Paspalum species (Burson and Bennett, 1971;Quarín et al., 1996;Takayama et al., 1998;Pagliarini et al., 2001).For the hexaploid accession of P. conspersum, chromosomes paired as bivalents (Figure 1c).Hexaploid accessions reported in literature presented chromosome association including uni-, bi-, tri-and quadrivalents (Burson and Bennett, 1971;Quarín and Burson, 1991;Takayama et al., 1998;Pagliarini et al., 2001).
The tetraploid accessions could have been produced either by chromosome duplication or by interspecific hybrid-ization followed by duplication.The low frequency of quadrivalents in tetraploid accessions reported in several Paspalum species has been interpreted as resulting from segmental allopolyploidy (Burson and Bennett, 1971;Norrmann et al., 1989;Quarín et al., 1996;Takayama et al., 1998;Pagliarini et al., 2001).Although low multivalent frequency is an argument frequently used in advocating segmental allopolyploidy, Sybenga (1996a) pointed out that this character is not necessarily a reliable indication of limited pairing affinity, and thus of homology, because even true autopolyploids may form quadrivalents with frequencies substantially lower than theoretically possible.Quarín (1992) has proposed that at least in apomictic Panicoid grasses, polyploidy is a condition for apomixis and that apomixis is associated with autoploidy rather than alloploidy.Evidence for autoploidy was provided by Quarín et al. (1998) in a tetraploid accession of P. rufum Ness.
It has been hypothesized that hexaploid accessions are produced by hybridization (Katayama and Ikeda, 1975) or the fertilization of unreduced eggs in apomictic tetraploid forms (Quarín and Burson, 1991).Hexaploidy in Paspalum, with chromosomes pairing as 30 bivalents during meiosis, has been reported in the Uruguayan biotype of P. dilatatum Poiret (Burson et al., 1991), this biotype having I and J genomes as well as an unidentified third genome (designated X) giving it the genomic form IIJJXX (Burson, 1991).The meiotic behavior at diakinesis in the hexaploid accession of P. conspersum, with chromosomes pairing only as bivalents, suggest that it resulted from hybridization followed by chromosome duplication to produce an amphidiploid with a diploid-like meiotic process.
Our analysis of meiotic behavior showed that it was compatible with ploidy level and the status of chromosome pairing (Table 3).In the diploid accession of P. coryphaeum, with chromosomes pairing as bivalents (10 II), a low frequency of abnormalities was recorded in all meiotic phases, resulting in a high frequency of normal tetrads (99.6%).In all accessions analyzed the abnormalities recorded were related to irregular chromosome segregation and resulted from precocious chiasmata termination.In the hexaploid accession of P. conspersum, which presented a diploid-like meiotic process, with chromosomes pairing as 30 bivalents, microsporogenesis was initially irregular.Asynchrony in chiasma termination among bivalents in this accession led to precocious chromosome migration to the poles in metaphase I and laggards in anaphase I.However, the majority of such chromosomes were included in the telophase I nuclei and the second meiotic division presented a low frequency of meiotic irregularities.For tetraploid accessions, however, meiotic behavior varied among accessions, with the percentage of abnormal tetrad ranging from 0.6% in the accession BRA-009008 to 88.5% in BRA-01252 and with only three presenting less than 10% of abnormal tetrads.Except for a few other tetraploid accessions, the frequency of abnormalities during meiosis leading to abnormal tetrads was very high.In all accessions analyzed, the meiotic abnormalities were related to irregular chromosome segregation during both meiotic divisions, such as precocious chromosome migration to the poles during the metaphases (Figure 1d) and anaphase laggards (Figure 1e), resulting in micronuclei in the telophases (Figure 1f).Unbalanced gametes generated from these irregularities could compromise pollen fertility.
Some Paspalum species, such as some biotypes of P. dilatatum (Venuto et al., 2003), P. simplex Morong ex Britton (Urbani et al., 2002), P. atratum (Quarín et al., 1997), P. compressifolium (Quarín et al., 1996) and P. notatum (Tischler and Burson, 1995) provide excellent pasture in cattle production systems in several countries, including the Brazil, Thailand and the USA.The Paspalum cultivars used are generally selected based on natural genetic variability, because of which any lack of diversity represents an obvious risk to the ecosystem as well as to cattle production.Therefore rational exploitation of the diversity present in the germplasm, especially for species with good forage value, is of fundamental importance in maintaining diversity in the field.New cultivars are urgently needed and intra-and interspecific hybridizations are desirable as means of introgressing genes but certain difficulties, including differences in ploidy level among accessions and reproduction by apomixis, have delayed the development of new hybrids.
Regardless of an adequate germplasm base, breeding programs demand a knowledge of the mode of reproduction, chromosome number and meiotic behavior to direct the crosses.Apomixis has been widely recognized as a common method of reproduction in the Paspalum.In general, diploids are sexual and most polyploids are apomictic (Quarín and Burson, 1991), however, some polyploids reproduce sexually and many apomictics are facultative (Burson, 1997).Sexual reproduction in tetraploids has invariably been reported to occur in individuals with normal meiosis (Burson and Bennett, 1971;Quarín and Normann, 1987).Despite identifying promising accessions, the Paspalum breeding program in effect at Embrapa CPPSE depends on sexual accessions to act as female genitors in hybridizations.Tetraploid apomictic accessions with nearly regular meiosis, such as were found in some accessions in the present study, could be used as male genitors.Thus, wide cytological screening is a pre-requisite in Paspalum breeding programs.Our investigation revealed that among the 36 accessions evaluated, the diploid P. coryphaeum accession BRA-000841 could be used as the sexual female genitor and four tetraploid accessions (P.notatum BRA-006513, P. limbatum BRA-009008, P. rhodopedum BRA-006602 and Paspalum sp BRA-008630), with less than 10% of abnormal tetrads might have a potential for use in an interspecific hybridization program as male genitors.Our cytogenetic characterization of 36 CPPSE accessions has increased the possibilities of crosses between the Paspalum accessions available at Embrapa CPPSE because in the first analysis of 52 CPPSE accessions only tetra-and hexaploid cytotypes were found (Freitas et al., 1997;Takayama et al., 1998) while in the second analysis of 24 CPPSE accessions (Pagliarini et al., 2001)

Cytological studies in Paspalum 777 Figure 1 -
Figure 1 -Meiotic behavior in different Paspalum cytotypes.a) Diakinesis in the diploid accession of P. coryphaeum showing 10 II.b) Diakinesis in tetraploid accession showing 18 II and 1 IV (arrowhead).c) Diakinesis in the hexaploid accession of P. conspersum with 30 II. d) Metaphase I in tetraploid accession showing precocious chromosome migration to the poles.e) Anaphase I in tetraploid accession showing laggards.f) Early telophase I in tetraploid accession with micronuclei.f) Prophase II with micronuclei.g) Tetrad with micronuclei.h) Microspore with micronuclei.Scale bar = 1 mm.

Table 1 -
Species investigated in the present study with their accession numbers and collection site details.

Table 1 (cont.)
*The two-letter code after the collection site refers to the Brazilian state, DF = Distrito Federal, MS = Mato Grosso do Sul, PR = Paraná, RG = Rio Grande do Sul, SC = Santa Catarina.

Table 2 -
Chromosome numbers and meiotic chromosome associations at diakinesis.

Table 3 -
six were diploids.These results have implications for breeding programs and shed some more light on the genetic structure of this important grass genus.Number of microsporocytes analyzed and the percentage of cells with meiotic abnormalities.