By Y. Sinoto and D. Sato (With one figure)

Transcription

1 Received March 13, 1940 Basikaryotype and Its Analysis1) By Y. Sinoto and D. Sato (With one figure) Introduction Chromosome studies have been actively carried on in the field of cytogenetics since Rosenberg (1904) observed the chromosome behavior in the meiosis of a hybrid between two Drosercc species, and the multiple relation of the chromosome numbers or polyploidy has been found in allied forms. Generally two sets of chromosomes are found in somatic cells, while only one set is found in the sexual cells or gametes, the former being called diploid (2 n) and the latter haploid (n). The polyploids which have more than two sets of chromosomes are called triploid, tetraploid, pentaploid, etc. according to the number of chromosome sets observed. In these cases the chromosome numbers in the somatic cells are expressed 3 n, 4 n, 5 n etc. when the basic number is symbolized as n. The use of these formulae to represent the gametic numbers of chromosomes is not adequate. The symbol x was suggested by Sansome and Philp (1932) for the basic number instead of n, while on the other hand the senior author (Sinoto 1929) proposed h which is the initial letter of a basis or a basic number of chromosomes. This symbol has been used in our laboratory since then and has been supported by Gates (1935) and his school. Gates emphasized that it was necessary to find a new symbol in place of x in order to avoid confusion, since x would then be em ployed in three ways, namely in the case of X-rays, X-chromosome and as the symbol of the chromosome number. Thus the investigation on the chromosome numbers has lead to the development of the conception of the basic number and recent studies have resulted in the elucidation not only of the chromosome numbers but also of the size and shape of the chromosomes. Delaunay (1922) observed the following fact in some genera; namely that all the species of a certain genus studied has generally a similar chromosome type especially in size classes. He gave a term "Karyotypus" to this particular chromosome type in the genus 1) Contributions from the Divisions of Plant-Morphology and of Genetics, Botanical Institute, Faculty of Science, Tokyo Imperial University, No. 244.

2 530 Y. SINOTO and D. SATO Cytologia 10 and thought that it corresponds with the systematic unit, genus. Levitsky (1924) independently suggested a new conception of "Karyotyp" which was different from that of Delaunay in that no assumption was made concerning its relationship to a systematic unit such as the genus, the meaning he ascribed to it being "the type of chromosomal complex characteristic of any individual or group of allied forms" (Sharp, 1934, p. 126). The karyotypel) has become important in chromosome studies and progress made in the method of karyotype analysis was advanced having stimulated the investigators who came to take an interest in the study of the morphology of chromosomes in a set corresponding to the basic number. This has necessitated suggesting a new conception of karyotype for which we propose the term "basikaryotype" In the following we will explain in detail the conception of the basikaryo type and the method of its analysis. Karyotype analysis In karyotype analysis not only the length and breadth of individual chromosomes but also other morphological characteristics such as primary and secondary constrictions and trabants have to be investigated as carefully as possible. When all of the chromo somes in the set cannot actually be analyzed, certain particular chromosomes such as SAT and nucleolar chromosomes, allosomes, sex-chromosomes and sometimes larger chromosomes need to be analyzed. Even when none of the chromosomes can be distinguished owing to their similar, often small size and shape, that is, when they are not suited for karyotype analysis except as regards their number, then basikaryotype analysis in the sense later specified may be rendered to some extent possible by adopting the conception of secondary association in meiosis (cf. appendix). For the present it is interesting to note that such secondary association of chro mosomes has been reported in a number of plants most of which have small similar chromosomes. By karyotype analysis the basic number of chromosomes and their morphology and further basikaryotypes can be determined. Such basikaryotypes, however, may be distinguished in some cases but not in others only by their morphological differences. Accord 1) The definition of the karyotype seems to differ with different authors. For instance, Bruun (1932) states: "By karyotype is, therefore, here meant the sum total of those nuclei which have most character in common." (p. 196). Variants in a karyotype were called "facies" and a higher rank was called "karyoforms" by Bruun (p ). We use the term karyotype in the broader sense as including the three terms used by Bruun.

3 1940 Basikaryotype and its analysis 531 ingly the question of homology of the basikaryotypes or that of the chromosomes composing the basikaryotypes must be left for further investigation, according to the method of basikaryotype analysis which will be explained later in detail. Even when the differences among basikaryotypes can not be made out by karyotype analysis in the somatic cells, these differences may be detected by observing chromosome alterations such as invertion, translocation etc. shown by the chromosome behavior in meiosis of the plants or hybrids which have the basikaryotypes in question. It is nothing but a basikaryotype analysis to look in such a manner for the homology among individual chromosomes belonging to the different basikaryotypes. Genome analysis As already mentioned above, the conception of the basic number of chromosomes has been established with the progress of the chromosome studies. Winkler (1920) proposed a term "Genom" for the particular chromosome set.1) The basic number simply implies the chromosome number, but the genome suggests a genetical constitution of the chromosome group composing the basic number. A certain plant has AA genomes, and another BB, so their hybrid may have AB genomes. The homology between the genomes is induced by the mode of conjugation of the chromosomes observed in meiosis of the hybrid. Only bivalents are formed in meiosis of the plant with either AA or BB genomes. The A genome is different from or non-homologous with the B, so only univalents are observed in meiosis of the plant having the AB genomes. When the homology between the two genomes is not complete, some of the chromosomes may not conjugate but remain as univalents. In this case these two genomes are said to be "partially homologous." Thus difference or homology of genomes can be traced by using such affinities existing more or less among different genomes. This method, genome analysis, has already done much for the advancement of cytogenetics. In this line the genome analysis in Triticum and its allied genera by Prof. Kihara and his school is renowned. In practice, in genome analysis, the analysators are crossed with an unknown plant to be analysed and the homology of the genomes is judged by the chromosome conjugations in meiosis of these hybrids. Thus a plant with AA genomes is crossed with a plant with unknown genomes XX to form a hybrid with AX genomes. 1) The conception of the genome seems to have wide implication as seen by Kihara's (1939) historical and critical review on this term,

4 532 Y,SlNOTO and D. SATO Cytologia 10 When only bivalents are found in meiosis of the hybrid the X genome is assumed to be homologous with the A and is determined as equivalent with the A genome. On the other hand only univalents may be found when the A genome is non-homologous with the X, which is then presumed as another genome B or etc. If both bivalents and univalents are formed, the A genome is partially homologous with the X which is then determined as an A' genome.1) In recent investigations partially homologous genomes have come to be found rather frequently and moreover the existence of partial homology has been established by observation not only in the genome as mentioned above, but also in individual chromosomes themselves composing the genome. In other words the chromosome alterations such as inversion, translocation, deletion etc. have been detected in certain regions of the chromosomes. Consequently the conception of genome may be modified by taking the karyotype into consideration or it may be related to the conception of further development from the stand-point of the karyotype alteration. The conception of genome however seems to have considerable implica tion in so far as the papers hitherto published go and suggests further development in various lines of genetics and evolution. We shall however attempt here to deal only with the one aspect of the nature of the genome in which the karyotype is chiefly concerned. Basikaryotype analysis By karyotype analysis made in individuals or allied groups (species, section, genus, family etc.) the basic numbers of chromo somes (bases) are determined and consequently various basikaryo types can also be detected in them in different combinations. The resemblane of basikaryotypes is assumed to suggest a phylogentic relationship, but further investigations in regard to the homology or phylogeny of basikaryotypes make it necessary to consider hy bridization between the two forms having the basikaryotypes in question. Accordingly when hybrids cannot be got by crossing them, further analysis of the basikaryotypes is impossible. On the other hand when the two plants can be hybridized, the analysis can be continued, homology between the basikaryotypes being judged from the chromosome behavior in meiosis of the hybrid. In short, the basikaryotype can be determined by the karyotype analysis and the homology, either complete or partial, among in dividual chromosomes composing the basikaryotype can be deduced 1) For detail of genome analysis, see Kihara, Yamamoto and Hosono 1936 "Studies on the Chropnosome Nunmbers in Plants," pp

5 1940 Basikaryotype and its analysis 533 from the state of pairing in meiosis, which is assumed to show homology of chromosomes or parts of the chromosomes. And the latter method of investigation is what we propose to call a basi karyotype analysis. Even the mere basikaryotype may suggest that chromosome changes such as fragmentation, fusion, translocation, inversion or deletion have occurred, and in some cases it may be true, but strictly speaking this is but a presumption and it can hardlly be said that any convincing karyogenetical evidence has been obtained, until the basikaryotype analysis has been made. The basikaryotypes can be determined in the forms in which the karyotypes have been analysed in detail; for instance the karyotype in Paeonia (2n=10) (cf. Sinoto 1938) is generally shown as follows,1) K=2Lt1+2Lt2+2M+2St1+2St2, hence the basikaryotype B is Lt1Lt2MSt1St2. In Tricyrtis formosana var. stolonifera (2n=25) (cf. Sato 1939), K=2L1+2Lt2+M+2St+18S; hence two basikaryotypes with different chromosome numbers are shown as follows, B1=13=L1Lt2St10S and B2=12=L1Lt2MSt8S. In this case the M-chromosome suggests by its three constrictions that it has been derived from the fusion of the two small chromosomes (S). When various karyotypes are found in one and the same species such as Scilla permixta (2n=14, 15, 16) (cf. Sato 1936), the basi karyotypes are also complicated. The I-type is 2n=16, K=2L +2M1+2M2+2M3+2M4+2S1+2S2+2St3, the II-type is 2n=15, K=2L +2M1+2M2+2M3+M4+Mt4+2S1+2S2+St 3 and the III-type is 2n=14, K=2L+2M1+2M2+M3+M3S1+M4+Mt4+2S1+2S2, And then the basikaryotypes are presumed to be as follows, B1=8=LM1M2M3M4S1S2S3, B2=7=LM1M2M3Mt4S1S2 and B3=7 =LM1M2M3S3M4S1S2. Consequently the three karyotypes may be shown as combinations of the three basikaryotypes as follows: the I-type B1B1, the II-type B1B2 and the III-type B2B3. In these cases, the Mt 4 is a M 4-chromosome with a trabant which has possibly been translocated from St3. The M3S3 is also a SAT-chromosome with a secondary constriction which is derived from the translocation or fusion of the M3 and St3 chromosomes. In Scirpus lacustris (2n=38, 40, 42) (cf. Tanaka 1938) three different karyotypes with or without compound chromosomes (C) are observed, that is, S. lacustris var. typicus (2n=38), K=2C+ 36S, S. lacustris var. typicus f. pictus (2n=40), K=C+39 S and S. lacustris var. tab ern.aemontani f. zebrinus (2n=42), K=42 S. 1) K=karyotype, B=basikaryotype, L=large chromosome, M=medium chromosome, S=small chromosome, t=trabant.

6 534 Y. SINOTO and D. SATO Cytologia 10 Consequently two basikaryotypes may be detected, namely, B1=19 =C18S, B2=21=21S. Then the three forms typicus, pictus and zebrinus are represented as B1B1, B1B2 and B2B2 respectively. In these cases one compound chromosome (C) seems to be homologous with three small chromosomes (S). In Scilla japonica (2n=16,18,26,34,35,43) (cf. Sato 1940), various karyotypes were already found, but these polyploid series can be explained by the combinations of the two basikaryotypes, B 1 with 8 chromosomes and B 2 with 9 chromosomes, namely, K=16=B1B1, K=18=B2B2, K=26=B1B2B21), K=34=B1B1B2B21), L=35=B1B2B2B21) and K=43=B1B1B2B2B21). In these cases, V shaped chromosomes belonging to the B1 give the suggestion that they have been formed by translocation or fusion of the two chro mosomes belonging to the B2. As mentioned above various basikaryotypes are found in the same species or genus, and in order to know what genetical.connec tion exists between these basikaryotypes, the necessity is here in sisted upon of observing the chromosome behavior in meiosis of the plants and their hybrids having these basikaryotypes, namely of carrying out the basikaryotype analysis. Basikaryotype analysis in Aloinae In Aloinae there are three genera, namely, Aloe, Gasteria and Haworthia and the analysis, of their karyotypes suggests the similarity or phylogenetic relationship of their basikaryotypes. In general most species are diploids (2n=14) although tetra-, penta and hexaploids are also found on rare occasions. The karyotype is shown in the diploid as follows, K=8L+6 S including generally four SAT-chromosomes. The position of the trabants is at the distal ends of the two pairs of large chromosomes in Gasteria and Haworthia, while in many Aloe plants it is at the distal ends of one pair of large chromosomes and at the proximal ends of one pair of small chromosomes. Four pairs of large chromosomes are distinguished according to the lengths of their short arms, namely, L1, L2, L2 and L4. The SAT-chromosomes in Gasteria and Haworthia are 2Lt1+2 Lt4 and those in Aloe have various combinations such as 2Lt4+2St4, etc. Aloe variegata has a similar karyotype, in cluding 2Lt1+2Lt4 SAT-chromosomes, with that of Gasteria. Considering the basikaryotypes found these plants are not always homozygotic, but some are heterozygotes or hybrids. It is 1) These are also represented as B12B 2 2B2B3, B13B2 and 2B13B1 respec tively

7 1940 Basikaryotype and its analysis 535 necessary, as already stated, for hybridization to occur if one is to pursue the phylogenetic relation among these basikaryotypes. In the interspecific or intergeneric hybrids the chromosome behavior in meiosis must be analyzed to see the state of conjugation of the chromosomes. Three intergeneric hybrids between Aloe and Gasteria were obtained, that is, Aloe variegate ~Gasteria verrucosa var. latifolia, Aloe variegata ~Gasteria gyuzetu and Gasteria gyuzetu ~Aloe variegata, and the basikaryotype analysis has been carried on in these hybrids. In the first hybrid (Aloe variegate 2n=14 ~Gasteria verrucosa var. latifolia 2n=14) seven bivalents are formed in meiosis of more than half of the pollen mother cells (table 1), which show the homology of the two basikaryotypes, but two or four univalents of the large chromosomes are also observed and sug gest that there is partial homology of the chromo somes. As a result of careful observation four kinds of chromatid bridges accompanied by fragments can be found. Such abnormal behavior of the bivalent chromo somes may be due to in version of a chromosome segment which includes no kinetochore. The in versions at the distal arms of the large chromo somes release small frag ments and those of the long arms near the kine tochore release large fragments. In the case of inversion when there Fig. 1. Karyotypes of Aloe variegate Š (A) and Gasteria verrucosa var. latifolia (B) and their hybrid (C). Clear portions of the chromosomes represent inverted regions and dotted portions are homologous with each other. Arrows indicate direction of homologous segments, ~3000. is a kinetochore the abnormal chromosome behavior shows a feature somewhat different from that of chromatid bridges with fragments. In practice such a case was detected by a character istic separation of heteromorphic chromosomes of which one chro Cytologia

8 536 Y. SINOTO and D. SATO Cytologia 10 matid has both the long arms of the two chromosomes. Accordingly, five kinds of inversion were analysed, these being located in four pairs of the large chromosomes. Besides these inversions one translocation was found, that is, the distal end of a large chromo some (L2) was homologous with the proximal end of another large L, chromosome; moreover, this homologous region was also associated with the distal region of one of the small chromosomes. Con sequently a tetrapartite consisting of the two large and one small bivalents is rarely formed. This translocation of the chromosome segments between large and small chromosomes may explain the difference in the typical Aloe and Gasteria as regards the AT-chro mosomes, that is, the Table 1. Chromosome configurations in the first latter in Aloe are meiotic metaphases of pollen mother cells in Aloe 2Lt4+2St1 and in Gas variegata ~Gasteria verrucosa var. latifollia, 2n=14. teria 2Lt1+2Lt4. It is an interesting fact that two heteromor phic large bivalents (Lt1Lt4 and Lt4Lt1) are al ways found in the first meiotic metaphase. In the second hybrids (Aloe variegata 2n=14 ~Gasteria gyu zetu 2n=14 and its reciprocal hybrid) partial homologous chromo somes forming a maximum of six univalents are similarly found and three inversions and one translocation are clearly observed. Besides these cases of abnormal chromosome behavior which were also found in the first hybrid, one tripartite of large chromosomes is often found. It consists of one pair of the most stable bivalents (L3L3) and an Ll chromosome, judging from the chromosome configurations in meiosis (these will be reported in detail elsewhere). Table 2. Chromosome configurations in the first meiotic mataphases of pollen mother cells in Aloe variegataxgasteria gyuzetu, 2n=14. *a univalent of a small chromosome instead of a large one was observed once in each case. As stated above, it is postulated that by the basikaryotype analysis the occurrence of karyotype alterations such as fragmenta tion, fusion, inversion, translocation, deletion etc., which are pre sumed on the basis of the karyotype analysis to have occurred, can

9 1940 Basikaryotype and its analysis 537 be karyologically proved; moreover, it is possible to determine what regions of the individual chromosomes are homologous or non homologous with each other, while the phylogenetic relationship of individuals or groups can to a certain extent be traced. Summary The conception of the basic number of chromosomes has been established by the studies made on the chromosome numbers during the last four decades, and a basic number and morphology of the chromosomes composing it have been investigated by the method of karyotype analysis. We propose applying the term basikaryotype to such a chromosome type which is specific to individuals or groups, corresponding in number to the basic number, and viewed from the stand-point of their morphology. The chromosome behavior in meiosis has to be observed in order to pursue the investigation of the phylogenetic relation of the basikaryotypes. The criterion of the homology of chromosomes is assumed to be the pairing of the chromosomes. By this method the existence of those karyotype alterations which were either expected to have occurred or are not capable of being distinguished by the karyotype analysis may be established on karyogenetical grounds. This method is here called a basikaryotype analysis. Comparing the basikaryotype analysis with the genome analysis, the former originated in the karyotype, while the latter started from hybridization as a premise. With the recent advances in the study of chromosome morphology the conception of the genome has come to take the karyotypes into consideration, and accordingly the new stand-point has been developed to facilitate discussion of the genetical connection of individuals or groups based upon the karyotype. The karyotype analysis is different from the genome analysis in that the former enables one to presume the relationship between the forms which can not be hybridized with each other, while it further enables determination of the basikaryotypes. The basikaryotypes may be detected in the forms in which the karyotype analysis was carried out. Some examples have been given (p. 533). The homology of the basikaryotypes may be traced by the method of basikaryotype analysis here proposed. An example of this method in the case of Aloinae was described (p. 534). Appendix: The basikaryotype analysis can be applied in the case of forms having chromosomes of distinct morphological char acteristics, while its application is difficult in the case of forms having extremely small similar chromosomes, but the conception of 35*

10 538 Y. SINOTO and D. SATO Cytologia 10 partial homology of individual chromosomes which is emphasized here seems to be universally valid in the case of such small chromo somes if one adopts the conception of the secondary association developed by Darlington (1928) and his school, and other investi gators (cf. Matsuura 1939). The expence of the present study was partly defrayed by a grant from the Japan Society for the Advancement of Cytology, to which the writers express their cordial thanks. Division of Genetics, Bet. Inst., Fac. of Sci., Tokyo Imp. Univ. Literature Bruun, H. G Cytological studies in Primula. Symb. bot. Upsal. 1. Upsala. Delaunay, L Vergleichende karyologische Untersuchungen einiger Muscari Mill. and Bellevalia Lapeyr.-Arten. Moniteur du Lard. Bot. de Tiflis, Nr. 11, Ser. 1. (Cited after Lewitsky and Tron 1930). Gates, R. R Symbols for chromosome numbers. Nature 135: 188. Kihara, H Studies on polyploidy, I. The history cf the studies on po'yploidy. (Japanese). Bet. & Zool. (Tokyo) 7: Lewitsky, G. A Materielle Grundlagen der Vererbung (russisch). (Cited after Lewitsky and Tron 1930).- u. Tron, E. J Zur Frage der karyotypischen Evolution der Gattung Muscari Mill. Planta 9 : Sansome, F. W. and Philp, J Recent advances in plant genetics. London. Sato, D Chromosome studies in Scilla, III. SAT-chromosomes and karyo type analysis in Scilla and other genera. Cytologia 7: Karyotype alteration and phylogeny, I. Analysis of karyotypes in Aloinae with special reference to the SAT-chromosome. Cytologia, Fujii Jub. Vol.: Cyto-genetical studies on Tricyrtis, II. Karyotype analysis in Tricyrtis and Brachycyrtis with special reference to SAT and nucleolar chromo some. Cytologia 10: Polyploidy and nucleoli. Bet. Mag. (Tokyo) 54: Sharp, L. W Introduction to cytology. 3. ed. N.Y. Sinoto, Y Chromosome studies in some dioecious plants, with special refer ence to the allosomes. Cytologia 1: Karyotype analysis in Paconia, I. Cytologia 9: Tanaka, N Chromosome studies in Cyperaceae, II. Scirpus lacustris L. Cytologia 8: Winkler, H Verbreitung and Ursache der Parthenogenesis im Pflanzen and Tierreiche. Jena.