Cytogenetics of modern sugar canes

Modern sugar cane varieties are derived from interspecific crosses involving as many as four species. Because a chromosome increase accompanies certain crosses and backcrosses, modern varieties have very high aneuploid chromosome numbers and complicated genetics. Despite this complexity, the chromosome behavior of some modern varieties approaches that of allopolyploids. In achieving homozygosity, therefore, such varieties should respond to inbreeding almost like diploids. The meiotic chromosome behavior of F₁ hybrids and modern varieties indicates little or no genetic exchange between chromosomes ofSaccharum officinarum andS. spontaneum. Irradiation may break linkages between desirable and undesirableS. spontaneum genes not ordinarily broken by crossing-over between the chromosomes of the two species. The quick success of nobilizingS. spontaneum (recurrently back-crossing to “noble canes”) depends on a peculiar increase of the chromosomes ofS. officinarum. Experience with nobilizingS. spontaneum should not make breeders impatient when they turn to interspecific crosses unaccompanied by chromosome increases.     

Noncanonical meiosis in the nematode <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> as a model for studying the molecular bases of the homologous chromosome synapsis,crossing over,and segregation

The nematode C. elegans is a classic study object of developmental biology and genetics, which is particularly suitable for studying the molecular bases of meiosis. Developing meiocytes are located in the threadlike gonads of C. elegans in linear gradient order of the stages of meiosis, which facilitates studying the order of intracellular events during meiosis. C. elegans has polycentric chromosomes. This causes a special order of events during meiosis, and as a consequence, meiosis in C. elegance differs from canonical meiosis of most eukaryotes. In the meiotic prophase I, all chromosomes carry single protein “pairing centers.” They are responsible for joining homologous chromosomes in pairs. This initiates the formation of synaptonemal complexes (SCs). Programmed double-stranded DNA breaks appear after initiation of the SC assembly, and they give rise to meiotic recombination. The initiation of meiotic recombination after the chromosome pairing distinguishes the C. elegans meiotic pattern from those in the absolute majority of eukaryotes studied. C. elegans has strict crossing over interference, which allows for the formation of one chiasma per bivalent. In the late prophase I, the polycentric centromeres are remodeled, one of the chromosome ends acquires a cuplike kinetochore, and during two meiotic divisions, chromosomes behave as monocentric. The study of meiosis in C. elegans allows for separate investigation of synapsis and recombination of homologous chromosomes and provides material for studying the evolution of meiosis.     

The quantitative analysis of chromosome pairing and chiasma formation based on the relative frequencies of M I configurations. III. Telocentric trisomics

The estimation of the crossing-over potentials of the two arms of a specific chromosome that can not be recognized in the diploid, was earlier found to be inefficient with the use of the primary trisomic. With the telocentric trisomics two groups of two different configurations each can be recognized that permit a reasonably exact estimation of the two parameters. Each telocentric trisomic yields estimates for both arms. The trisomic arm is underestimated as a result of partner-exchange and/or interference by the nucleolus in a nucleolus bearing arm. The other arm is estimated more correctly. Thus the two telocentrics together give a complete picture of the chromosome. After a correction for differences in overall chiasma frequencies the ratio of the crossing-over potentials of the two arms of the satellite chromosome ofSecale cereale was found to be approximately 2. This is large for a submedian chromosome in comparison with the ratio for the genome as a whole and it is attributed tentatively to the nucleolus interfering with chiasma formation in the short arm. It is suggested that the three homologous arms, especially the long arms, differ in respect to the tendency to pair and in chiasma frequency.     

Cytogenetic characterization of <Emphasis Type="Italic">Solanum lycopersicoides</Emphasis> Dun. Alien chromosome additions in cultured tomato

Cytogenetic analysis of five Solanum lycopersicoides monosomic alien addition lines of tomato was carried out. Meiotic analysis showed that additional chromosomes caused serious abnormalities. It was demonstrated that different chromosomes of S. lycopersicoides had different effects on chromosome pairing. For instance, associations formed between chromosomes II and IV of S. lycopersicoides and chromosomes of cultured tomato were trivalents, while chromosome XI in all cells was present as a univalent. Pachytene analysis showed that chromosomes of homeologous group II paired at their long arms, and their nucleolus organizer regions were of different sizes. The use of molecular markers provided accelerated identification of the introgression of S. lycopersicoides genetic material.     

The quantitative analysis of chromosome pairing and chiasma formation based on the relative frequencies of MI configurations. V. Interchange trisomics

Information obtained previously and presently on chromosome pairing and chiasma formation in trisomics and in interchange heterozygotes has been applied in newly constructed models for calculating expected MI configuration frequencies in interchange trisomics. Good fit betwen calculated and observed frequencies in some and poor fit in other cases confirmed the expectation of genetic variation in the crossing-over potentials of some or all chromosome regions. If conclusions in respect of chromosome pairing pattern are to be based on relative frequencies of MI configurations, valid values for crossing-over potentials are required. These can only be obtained from genetically comparable material. A few more disturbing factors are recognised. Environmental effects are one of these factors but may have a relatively simple character. Good agreement between expected and observed frequencies of configurations was taken to indicate the validity of the assumption that homologous chromosome end segments have equal probability of being involved in pairing, irrespective of the length of the segment. This conclusion was confirmed by the segregation of chromosomal types in the progenies of interchange trisomics: the excess chromosome was combined as frequently with the interchange set and with the normal set respectively, as expected on basis of the same models, assuming 60–80% viability of trisomes compared to diploids.     

End-to-End Association of <Emphasis Type="Italic">X</Emphasis> and <Emphasis Type="Italic">Y</Emphasis> Chromosomes in Mouse Meiosis

ACCORDING to the hypothesis of Crew and Koller¹ and Koller and Darlington², there are homologous segments in the X and Y chromosomes of the mouse and other mammals. The homologous regions in the mouse were believed to be localized in the extremely short arms proximal to the kinetochores. The end-to-end association at meiosis was thought to be the result of the formation of a chiasma between these homologous regions³. Electron microscopy revealed a short synaptonemal complex in mouse meiotic cells⁴. However, partial sex linkage has never been demonstrated in the mouse⁵ and other authors^6–10 believe that the X and Y chromosomes associate only by connexion between the chromosome ends furthest from the centromeres.     

Intraspecific divergence in wheats of the Emmer group using in situ hybridization with the Spelt-1 family of tandem repeats

Fluorescence in situ hybridization (FISH) was used to study the distribution of Spelt-1 repetitive DNA sequences on chromosomes of 37 accessions representing eight polyploidy wheat species of the Emmer evolutionary lineage: Triticum dicoccoides Körn, T. dicoccum (Schrank) Schuebel, T. durum Desf., T. polonicum L., T. carthlicum Nevski, T. aethiopicum Jakubz., T. aestivum L., and T. spelta L. Substantial polymorphism in the number, distribution, and the sizes of the Spelt-1 loci was revealed. On the chromosomes of the accessions examined, Spelt-1 tandem repeats were found in seven different positions (per haploid chromosome set). These were “potential hybridization sites”, including the subtelomeric regions of either short or long arms of chromosomes 2A and 6B, the short arm of chromosome 1B, and the long arms of chromosomes 2B and 3B. However, in individual genotypes, only from one to three Spelt-1 loci were revealed. Furthermore, no hybridization with Spelt-1 probe was detected on chromosomes from 12 accessions. Thus, the total number of Spelt-1 sites in karyotypes varied from zero to three, with the average number of 1.16. This was substantially lower than in the species of the Timopheevi section and diploid Aegilops speltoides Tausch, a putative donor of the B genome. The decrease of the content of Spelt-1 sequences in the genomes of the Emmer group wheats in comparison with the species of the Timopheevii group and diploid Ae. speltoides was assumed to result from the repetitive sequences reorganization during polyploidization and the repeat elimination during wheat evolution.     

The origin of the North Indian sugarcanes

The “North Indian sugarcanes”, cultivated by Indian peasants during many centuries, have been studied morphologically very exactly byC. A. Barber from 1910 to 1920. They were named “Saccharum Barberi” byJeswiet. Barber distinguished four groups. In 1931 the present author found the following chromosome numbers in these groups: 2n=116 and 2n=82 in the Sunnabile group; 2n=82 in the Mungo group; 2n=124 and 2n=107 in the Nargori group and about 91 in the Saretha group. The first three groups are sterile, the last is fertile. It is shown that the North Indian sugarcanes are hybrids between ancient indigenous sugar canes with a basic number of 17 chromosomes, and forms ofS. spontaneum withn=40,n=48 andn=56 respectively. Differences in the numbers of chromosomes contributed by the mother type may in part have their origin in endo-duplication, as commonly observed inSaccharum hybrids. Details are presented in Table 2. The differences found between different forms of IndianS. spontaneum in respect to chromosome number, sugar content and mosaic resistance may be attributed to intercrossing with canes of the fertile Saretha group.     

Homoeologous recombination in the presence of <Emphasis Type="Italic">Ph1</Emphasis> gene in wheat

A crossover (CO) and its cytological signature, the chiasma, are major features of eukaryotic meiosis. The formation of at least one CO/chiasma between homologous chromosome pairs is essential for accurate chromosome segregation at the first meiotic division and genetic recombination. Polyploid organisms with multiple sets of homoeologous chromosomes have evolved additional mechanisms for the regulation of CO/chiasma. In hexaploid wheat (2n = 6× = 42), this is accomplished by pairing homoeologous (Ph) genes, with Ph1 having the strongest effect on suppressing homoeologous recombination and homoeologous COs. In this study, we observed homoeologous COs between chromosome 5M^g of Aegilops geniculata and 5D of wheat in plants where Ph1 was fully active, indicating that chromosome 5M^g harbors a homoeologous recombination promoter factor(s). Further cytogenetic analysis, with different 5M^g/5D recombinants, showed that the homoeologous recombination promoting factor(s) may be located in proximal regions of 5M^g. In addition, we observed a higher frequency of homoeologous COs in the pericentromeric region between chromosome combination of rec5M^g#2S·5M^g#2L and 5D compared to 5M^g#1/5D, which may be caused by a small terminal region of 5DL homology present in chromosome rec5M^g#2. The genetic stocks reported here will be useful for analyzing the mechanism of Ph1 action and the nature of homoeologous COs.     

Molecular and cytogenetic analyses provide evidence of the introgression of chromosomal segments from the wild <Emphasis Type="Italic">Cucumis hystrix</Emphasis> into the cultivated cucumber through the bridge of a synthetic allotetraploid

Cucumis × hytivus (2n = 4× = 38) is a synthetic allotetraploid obtained from interspecific hybridization between the cucumber (2n = 2× = 14) and its wild relative C. hystrix (2n = 2× = 24). The synthesis of this species built a bridge for cucumber improvement through gene introgression. Allotriploid and introgression lines (ILs) have previously been produced and characterized with respect to morphology, cytology, and molecular markers. However, no clear evidence of how the chromosomal segments of C. hystrix were introgressed and inherited was found owing to the small size of chromosomes. In the present study, cucumber-C. hystrix introgression lines were developed by backcrossing the allotriploid to North China cucumber breeding line “P01” followed by self-pollination. The introgressed segments of C. hystrix in the ILs were revealed by meiotic pachytene chromosome analysis. Fluorescence in situ hybridization (FISH) was performed on pachytene chromosomes using fosmid clones from cucumber, which confirmed that introgression occurred in the long arm of chromosome 7. Molecular analysis using a set of 53 simple sequence repeats (SSRs) indicated that the chromosomal segments of C. hystrix were introduced into 4 cucumber chromosomes, the short arms of chromosomes 2 and 6, and long arms of chromosomes 3 and 7. The inheritance of alien sequences in the long arm of chromosome 7 was investigated with 21 SSRs in self-pollinated progenies. C. hystrix-specific bands of several SSRs were still present in some individuals, indicating that the introgressed segment was partially preserved. The first unambiguous identification of alien chromosome segments in cucumber ILs using combined molecular cytogenetics could facilitate the determination of effects of wild alleles and promote cucumber improvement.     

Induced Bivalent Interlocking and the Course of Meiotic Chromosome Synapsis

WHEN chromosomes pair at meiosis the bivalents so formed do not normally interlock. Heat-treatments can, however, induce bivalent interlocking in the locust Locusta migratoria. Only the longest bivalents interlock and usually only two are found per cell; two “rod” bivalents, with single chiasmata, two “ring” bivalents, each with two or three chiasmata, or one “rod” and one “ring” bivalent (Fig. 1a, b and c). The nature of this interlocking and the metaphase orientational and congressional properties of interlocked bivalents are analysed in detail elsewhere¹.     

Dual-color FISH karyotype analyses using rDNAs in three Cucurbitaceae species

Dual-color fluorescence in situ hybridization (FISH) analysis of three Cucurbitaceae species from different genera was conducted using 5S and 45S rDNA probes. In Benincasa hispida (Thunb.) Cogn. (2n=24), the 45S rDNA probe hybridized on two chromosomes, one in the short arm of a medium-sized metacentric chromosome and another at the satellite of a chromosome. The 5S rDNA hybridized at a site proximal to the centromere of the same short arm of the 45S rRNA gene locus that occupied almost the entire short arm. For Citrullus lanatus (Thunb.) Matsum & Nakai (2n=22), the 45S rDNA probe hybridized at sites in the short arms of two chromosomes and the 5S rDNA probe was co-localized with the 45S rRNA locus at the region proximal to the centromere in one chromosome. The 45S rRNA loci occupied almost all of the short arms in both chromosomes. In Cucurbita moschata Duch. (2n=40), the 45S rDNA probe hybridized in five chromosomes in which the 45S rRNA genes occupied almost two-thirds of the chromosomes in two large chromosomes and the entire short arm of a medium-sized chromosome. Two other loci were present in two medium-sized chromosomes, one in the proximal region in the short arm of a chromosome and another at the tip of the long arm of a chromosome. Chromosomes of B. hispida were relatively larger than those of the other two species. The karyotype of B. hispida is composed of two metacentrics and 10 submetacentrics, while that of C. lanatus is composed of seven metacentrics and four submetacentrics and that of C. moschata is composed of 18 metacentrics and two submetacentrics. Comparative chromosome evolution among the three Cucurbitaceae species was attempted using the karyotypes and the chromosomal distribution patterns of the 5S and 45S rDNAs. The results presented herein will be useful in elucidating the phylogenetic relationships among Cucurbitaceae species, and will provide basic data for their breeding programs.     

Research on meiotic chromosome pairing in <Emphasis Type="Italic">Roegneria sinica</Emphasis> var. <Emphasis Type="Italic">media</Emphasis> evaluated using genomic in situ hybridization

The analysis of chromosome pairing during meiosis is important for understanding the relationships between different genomes. To evaluate the diversity of chromosome pairing behavior in the wild species of Roegneria sinica var. media Keng with St and H genomes in Triticeae (Poaceae), differences and similarities in the meiotic chromosome pairing behaviors of the two genomes in two populations of R. sinica var. media, were analyzed using genomic in situ hybridization. Chromosome pairing at meiotic metaphase I in the two populations of R. sinica var. media mainly formed bivalents, although several univalents, trivalents and quadrivalents also occurred. Chromosome pairings occurred mainly between homologous chromosomes. However, some non-homologous pairings were observed under natural conditions. No significant differences in karyotype were found between the St and H genomes. Chromosome pairing behaviors differed between and within the two populations. Genetic variation occurred mainly within populations (94.04 %), and variation was more abundant in one population than the other. The genomes St and H differed, but there was some relationship between the two genomes. These findings suggest that homoeologous pairing of chromosomes or exchanges occurred between different genomes of the wild species in Triticeae during evolution. The findings also provide conclusive cytological evidence for genetic variation within the wild species, which forms the basis of their genetic diversity.     

Weitere Untersuchungen über Chromosomenverhältnisse und DNS-Gehalt bei Anuren (Amphibia)

The karyotypes of the toad Bufo marinus L. (2n=22) and the frogs Limnodynastes tasmaniensis Gthr. (2n=24), Rana temporaria L., R. esculenta L. (both 2n=26) and R. arvalis Nills. (2n=24) were analysed in colchicine treated leukocyte and spermatogonial metaphases and/or embryonic and larval mitoses. The DNA content of Feulgen stained erythrocyte nuclei was measured microspectrophotometrically. Heteromorphic sex chromosomes are absent in all species. L. tasmaniensis has the lowest DNA content among these species. The south American toad B. marinus shows a karyotype similar to the other known toad species and contains the same amount of DNA as the European species B. calamita with the lowest DNA amount among the European toads. In southern German populations of R. temporaria besides animals with the “standard”-karyotype (2n=26) individuals with 1 or 2, in rare cases with 3 or 4 supernumerary chromosomes have been found. The supernumeraries are heterochromatic and smaller than the smallest chromosome of the “standard”-karyotype. If only 1 or 2 supernumerary chromosomes are present, they seem to show normal mendelian inheritance as a rule. The observation of a few tadpoles with intraindividual different numbers of supernumeraries points to the occurrence of unequal distribution of these chromosomes in individuals containing a higher number of supernumerary chromosomes. The karyotype of R. esculenta is very similar to the “standard”-karyotype of R. temporaria, but the chromosomes of R. esculenta are somewhat longer than those of R. temporaria. R. esculenta contains about 54% more DNA than R. temporaria in the erythrocyte nuclei, so that it must be assumed that all chromosomes of R. esculenta contain more DNA than their homologues in R. temporaria. R. arvalis possesses about 28% more DNA than R. temporaria. It is supposed that these interspecific differences in DNA content of the Rana species — as observed earlier in Bufo species — are not a consequence of differential polyteny but are caused during evolutionary processes by local increase in DNA in the chromosomes of R. esculenta and R. arvalis.     

The Karyotype of <Emphasis Type="Italic">Amoeba borokensis</Emphasis> from a “<Emphasis Type="Italic">proteus</Emphasis>-like” Amoeba Group (Amoebozoa: Euamoebida)

The karyotype of Amoeba borokensis was studied for the first time. At the metaphase of mitosis, this species has a haploid set of chromosomes (n = 27). This is exactly half of the diploid karyotype in Amoeba proteus (strain B). At first glance, it confirms the idea that has recently appeared that one species arises from another via multiple changes in chromosome number. However, comparative cytogenetic analysis revealed that four orthologous chromosomes from haploid sets of these two species were not distinguished by the pattern of DAPI-stained bands. It shows that the genetic distance between A. proteus and A. borokensis is higher than was believed earlier and these two species have diverged from each other. Analysis of data on the life cycles of A. proteus and A. borokensis shows that a kind of so-called “cyclic polyploidy” takes place in “proteus-like” amoebae. “Cyclic polyploidy” has recently been considered as an alternative to the sexual process for genetic recombination in agamic protists from different macrotaxons.     

Similarities and differences in the nuclear genome organization within Pooideae species revealed by comparative genomic in situ hybridization (GISH)

In this paper, we highlight the affinity between the genomes of key representatives of the Pooideae subfamily, revealed at the chromosomal level by genomic in situ hybridization (GISH). The analyses were conducted using labeled probes from each species to hybridize with chromosomes of every species used in this study based on a “round robin” rule. As a result, the whole chromosomes or chromosome regions were distinguished or variable types of signals were visualized to prove the different levels of the relationships between genomes used in this study. We observed the unexpected lack of signals in secondary constrictions of rye (RR) chromosomes probed by triticale (AABBRR) genomic DNA. We have also identified unlabeled chromosome regions, which point to species-specific sequences connected with disparate pathways of chromosome differentiation. Our results revealed a conservative character of coding sequence of 35S rDNA among selected species of the genera Aegilops, Brachypodium, Festuca, Hordeum, Lolium, Secale, and Triticum. In summary, we showed strong relationships in genomic DNA sequences between species which have been previously reported to be phylogenetically distant.     

Genetic Collection of Meiotic Mutants of Rye <Emphasis Type="Italic">Secale cereale</Emphasis> L.

Genetic collection of meiotic mutants of winter rye Secale cereale L. (2n = 14) was created. Mutations were detected in inbred F₂ generations after self-fertilization of the F₁ hybrids, obtained by individual crossing of rye plants (cultivar Vyatka) or weedy rye with plants from autofertile lines. The mutations cause partial or complete plant sterility and are maintained in collection in a heterozygous state. Genetic analysis accompanied by cytogenetic study of meiosis has revealed six mutation types. (1) Nonallelic asynaptic mutations sy1 and sy9 caused the formation of only axial chromosome elements in prophase and anaphase. The synaptonemal complexes (SCs) were absent, the formation of the chromosome “bouquet” was impaired, and all chromosomes were univalent in meiotic metaphase I in 96.8% (sy1) and 67% (sy2) of cells. (2) Weak asynaptic mutation sy3, which hindered complete termination of synapsis in prophase I. Subterminal asynaptic segments were always observed in the SC, and at least one pair of univalents was present in metaphase I, but the number of cells with 14 univalents did not exceed 2%. (3) Mutations sy2, sy6, sy7, sy8, sy10, and sy19, which caused partially nonhomologous synapsis: change in pairing partners and fold-back chromosome synapsis in prophase I. In metaphase I, the number of univalents varied and multivalents were observed. (4) Mutation mei6, which causes the formation of ultrastructural protrusions on the lateral SC elements, gaps and branching of these elements. (5) Allelic mutations mei8 and mei8-10, which caused irregular chromatin condensation along chromosomes in prophase I, sticking and fragmentation of chromosomes in metaphase I. (6) Allelic mutations mei5 and mei10, which caused chromosome hypercondensation, defects of the division spindle formation, and random arrest of cells at different meiotic stages. However, these mutations did not affect the formation of microspore envelopes even around the cells, whose development was blocked at prophase I. Analysis of cytological pictures of meiosis in double rye mutants reveled epistatic interaction in the mutation series sy9 > sy1 > sy3 > sy19, which reflects the order of switching these genes in the course of meiosis. The expression of genes sy2 and sy19 was shown to be controlled by modifier genes. Most meiotic mutations found in rye have analogs in other plant species.     

Chromosome painting in meiosis reveals pairing of specific chromosomes in polyploid <Emphasis Type="Italic">Solanum</Emphasis> species

Analysis of chromosome pairing has been an important tool to assess the genetic similarity of homologous and homoeologous chromosomes in polyploids. However, it is technically challenging to monitor the pairing of specific chromosomes in polyploid species, especially for plant species with a large number of small chromosomes. We developed oligonucleotide-based painting probes for four different potato chromosomes. We demonstrate that these probes are robust enough to monitor a single chromosome throughout the prophase I of meiosis in polyploid Solanum species. Cultivated potato (Solanum tuberosum, 2n?=?4x?=?48) is an autotetraploid. We demonstrate that the four copies of each potato chromosome pair as a quadrivalent in 66–78% of the meiotic cells at the pachytene stage. Solanum demissum (2n?=?6x?=?72) is a hexaploid and has been controversial regarding its nature as an autopolyploid or allopolyploid. Interestingly, no hexavalent pairing was observed in meiosis. Instead, we observed three independent bivalents in 83–98% of the meiotic cells at late diakinesis and early metaphase I for the four chromosomes. These results suggest that S. demissum has evolved into a cytologically stable state with predominantly bivalent pairing in meiosis.     

Suppression of Homoeologous Pairing by <Emphasis Type="Italic">B</Emphasis> Chromosomes in a <Emphasis Type="Italic">Lolium</Emphasis> Species Hybrid

INTERSPECIFIC hybridization together with polyploidy has been an important force in the evolution of many of our graminaceous crop plants. Both wheat (Triticum aestivum) and oats (Avena sativa), for example, are natural allohexaploids derived in each case from the hybridization of three separate but related diploid species. The efforts of plant breeders to synthesize stable and fertile polyploids of this kind have, on the whole, been unsuccessful. The main reason for this is that whereas meiosis in natural allopolyploids such as wheat is extremely regular this is not the case with “synthetic” polyploids. In wheat precise control over pairing at meiosis is achieved by a gene or a cluster of genes on chromosome SB. The gene acts by restricting the pairing to homologous chromosomes with the result that only bivalents are formed, disjunction is regular and inheritance is completely disomic^1,2. In the artificial polyploids at pachytene there is pairing between both homologous chromosomes (from the same species) and “corresponding” homoeologous chromosomes (from different species). The result is an extremely irregular metaphase 1 comprising multivalents and univalents as well as bivalents. Segregation is irregular and a certain degree of infertility is inevitable.     

Controle de validite des caryogrammes application au caryotype de lolium perenne L.

The precise morphological study of the chromosomes of Lolium perenne L. puts forwards the lack of a statistical method allowing to appreciate the distinction between the elements of a chromosomal stock for the establishment of a caryogram. The present method is based upon the comparisons of variances, using, as a check value, a comprehensive estimation of the purely random variation. This estimation must be done by avoiding two sources of error, which were not taken in consideration until now and result from the fluctuation process only: 1. Arbitrary separation in each cell of a set of 4 chromosomes which are not really distinguishable in two pairs, one to which a relatively high value is attributed, and the second to which a lower value is given. Both distributions obtained present very low variances which, when employed in order to test the significance of the difference between both means, wrongly confirm the value of the initial distinction. 2. Confusion of both arms in symmetrical chromosomes when establishing the C/L ratio. This has two consequences: decreasing the value of the variance and under estimation of the symmetry, and this so much the more than the actual symmetry is more complete. — Application of this method leads to consider as identical the f and g chromosomes of Lolium perenne and to separate by means of the total lengths (or the ratio T/Θ) the c and d pairs the morphological identity of which is, in other respects, remarkable. In addition, it is also interesting to note the clear manifestation of a satisfying homogeneity in the variation of the chromosomes and of their constituent arms.