Meiotic analysis of four cross-pollinated generations in a synthetic autotetraploid population of husk tomato (<i>Physalis ixocarpa</i>)

The cultivated husk tomato (Physalis ixocarpa) (2n = 2x = 24) is native from Mexico and Central America and shows a wide genetic variation. Presently, it is the fourth horticultural crop in cultivation surface in Mexico. The working team of this research previously developed an autotetraploid population by using colchicine. The objectives of the present work were to analyze the ploidy level and meiotic behavior of the subsequent generations (C3, C4, C5, C6) from the original (C2) composed only by plants with the duplicated genome from the Rendidora cultivar, and to determine pollen viability. As a diploid control the cultivar Rendidora of P. ixocarpa was used. Ploidy level was determined by flow citometry and meiotic analysis. For the meiotic study, the microsporocytes were prepared by the squash method, stained with carmin and analyzed in diakinesis. Pollen viability was evaluated through 0.01% Buffalo Black staining. The tetraploid condition prevailed through four cross-pollinating generations, maintaining a constant chromosome number 2n = 4x = 48. In diakinesis, the chromosomes of the diploid cultivar were associated into bivalents, whereas in tetraploid plants the chromosomes associated into univalents, bivalents and trivalents. Highly significant differences in bivalent pairing were detected between autotetraploid plants and between generations. Pollen viability did not show significant differences between generations and allowed reproduction. These results indicate that it is possible to develop an autotetraploid cultivar, because the polyploid state is naturally maintained and the plants are fertile. Furthermore, given the differences in bivalent pairing between plants and generations, a response to selection toward meiotic stability is expected.     

Stabilization of tetraploid triticale with chromosomes from Triticum aestivum (ABD)(ABD)RR (2n = 28)

Summary F₁ hybrids with the genome constitution ABDERR (2n = 6x = 42) or ABDE(AB)RR (2n = 7x = 49), selected from crosses between either an octoploid Triticum aestivum/Thinopyrum elongatun amphiploid and tetraploid Secale cereale (AABBDDEE x RRRR) or autoallohexaploid triticale [AABBDDEE x (AB)(AB)RRRR], were backcrossed to tetraploid triticale (AB)(AB)RR and selfed for six generations. Thirty-three different tetraploid F₆ progenies were karyotyped using C-banding. The aneuploidy frequency was 6.6% with 4.0% hypoploids and 2.6% hyperploids. Among 71 plants with 28 chromosomes, 53.5% had a stabilized karyotype while 46.5% were unstabilized with at least one homoeologous group segregating for A-, B-, or D-genome chromosomes. The stabilized plants represent 19 different tetraploid karyotypes with six of them not containing any detectable D-genome chromosomes from T. aestivum or E-genome chromosome from Th. elongatum. Thirteen lines were (ABD)(ABD)RR tetraploids with one-to-three disomic substitutions of D-genome chromosomes for A or B-genome chromosomes. No disomic substitution of E-genome chromosomes was identified. On average 0.58 D substitutions per line were determined. Of the seven D-genome chromosomes only four, 1D, 2D, 5D, and 7D, were present in their disomic state. In unstabilized karyotypes, chromosomes 3D, 4D, and 6D were present in their monosomic state. Among all 30 viable plants (42.3%), the order of decreasing frequency of Dgenome chromosomes was 5D (25.0%), 1D (20.0%), 2D (10.0%), 6D (5.0%), and 3D (1.7%). Plants with 4D and 7D chromosomes were not viable. An increase in the number of D-genome chromosomes in the (ABD) genome is associated with a decrease in viability and fertility. Minor differences in the C-banding of chromosomes in homoeologous groups 1, 5, and 6 indicate the possibility of translocations between A-, B-, D-, and E-genome chromosomes. Evolutionary and breeding aspects of tetraploid triticale with mixed genomes are discussed.     

Karyotypes and nuclear DNA amounts in Polypodium L. (Polypodiaceae)

Karyotype studies in several species of Polypodium show that telocentric chromosomes are the most common with acrocentrics forming the remainder of the complement. The relative numbers of these chromosome types can be used as indicators of species relationships although direct comparisons are difficult to make due to the large number of similar-sized chromosomes. The karyotype data support the theory that P. interjectum is a polyploid derived from the hybridization of, P. australe and P. vulgare. Measurements of nuclear DNA content show that the four diploid species P. australe, P. scouleri, P. virginianum and P. glycyrrhiza all have very similar amounts of DNA. The tetraploid P. vulgare has one-and-a-half times the DNA content of the diploids and the hexaploid P. interjectum has two times the DNA content of the diploids. The chromosomes of the tetraploid and hexaploid are smaller than those of the diploids and evolution in Polypodium appears to have been accompanied by either a loss or gain of nuclear DNA; the direction of the change cannot be ascertained by the present study.     

Alteration and preservation of cellular characteristics in long-term culture of tetraploid H-1 (ES) cells

To examine the alteration in cellular characteristics of polyploid ES cells during long-term culturing, tetraploid H-1 (ES) cells were continuously cultured for 180 days. Cellular DNA content of the tetraploid cells decreased and reached a plateau of 3.3 C, where C represents the complement of haploid chromosomes. The chromosome number also decreased, indicating that the DNA loss was induced by chromosome loss. Cell volume was maintained, suggesting that the DNA loss did not involve cytoplasmic loss. The cell cycle parameters were almost the same during the DNA decay process, indicating that cell cycle progression was independent of the quantity of homologous chromosomes. Hypotetraploid cells showed alkaline phosphatase activity and formed teratocarcinomas in mouse abdomens, suggesting that the pluripotent potential was maintained. Cellular morphology was also retained, suggesting that the gene expression specifying morphological characteristics was conserved. We conclude that these initial cellular characteristics of tetraploid H1 (ES) cells were preserved in long-term culture, irrespective of chromosome loss.     

Retention of D genome chromosomes in pentaploid wheat crosses

The transfer of genes between Triticum aestivum (hexaploid bread wheat) and T. turgidum (tetraploid durum wheat) holds considerable potential for genetic improvement of both these closely related species. Five different T. aestivum/T. turgidum ssp. durum crosses were investigated using Diversity Arrays Technology (DArT) markers to determine the inheritance of parental A, B and D genome material in subsequent generations derived from these crosses. The proportions of A, B and D chromosomal segments inherited from the hexaploid parent were found to vary significantly among individual crosses. F(2) populations retained widely varying quantities of D genome material, ranging from 99% to none. The relative inheritance of bread wheat and durum alleles in the A and B genomes of derived lines also varied among the crosses. Within any one cross, progeny without D chromosomes in general had significantly more A and B genome durum alleles than lines retaining D chromosomes. The ability to select for and manipulate this non-random segregation in bread wheat/durum crosses will assist in efficient backcrossing of selected characters into the recurrent durum or hexaploid genotype of choice. This study illustrates the utility of DArT markers in the study of inter-specific crosses to commercial crop species.     

Formation of bipolar spindles with two centrosomes in tetraploid cells established from normal human fibroblasts

Tetraploid cells with unstable chromosomes frequently arise as an early step in tumorigenesis and lead to the formation of aneuploid cells. The mechanisms responsible for the chromosome instability of polyploid cells are not fully understood, although the supernumerary centrosomes in polyploid cells have been considered the major cause of chromosomal instability. The aim of this study was to examine the integrity of mitotic spindles and centrosomes in proliferative polyploid cells established from normal human fibroblasts. TIG-1 human fibroblasts were treated with demecolcine (DC) for 4?days to induce polyploidy, and the change in DNA content was monitored. Localization of centrosomes and mitotic spindles in polyploid mitotic cells was examined by immunohistochemistry and laser scanning cytometry. TIG-1 cells treated with DC became almost completely tetraploid at 2?weeks after treatment and grew at the same rate as untreated diploid cells. Most mitotic cells with 8C DNA content had only two centrosomes with bipolar spindles in established tetraploid cells, although they had four or more centrosomes with multipolar spindles at 3?days after DC treatment. The frequency of aneuploid cells increased as established tetraploid cells were propagated. These results indicate that tetraploid cells that form bipolar spindles with two centrosomes in mitosis can proliferate as diploid cells. These cells may serve as a useful model for studying the chromosome instability of polyploid cells.     

Genome analysis of Elytrigia caespitosa, Lophopyrum nodosum, Pseudoroegneria geniculata ssp. scythica, and Thinopyrum intermedium (Triticeae: Gramineae).

To elucidate the genome constitutions of the tetraploid (2n = 4x = 28) species Elytrigia caespitosa, Lophopyrum nodosum, and Pseudoroegneria geniculata ssp. scythica and the hexaploid (2n = 6x = 42) Thinopyrum intermedium, meiotic pairing was studied in these species as well as 10 hybrids. Karyotype analysis with aceto-orcein stained root-tip cells was performed for the four species and the hybrids of T. bessarabicum with E. caespitosa, P. geniculata ssp. scythica, and T. intermedium. Karyotype analysis by Giemsa C-banding was carried out with the three tetraploid species and the two triploid hybrids involving T. bessarabicum. The species behaved as strict allopolyploids. All hybrids were male sterile with few stainable pollen grains. It is concluded from the results that the three tetraploid species have the genome formula JeJeSS and T. intermedium has the formula JeJeJeJeSS. The chromosomes of the Je and S genomes in these species had C-banding patterns differing from each other and from those of the extant diploid species. Based on these findings, the four species investigated should be placed in the same genus or the same section of a genus. However, new combinations are not proposed at this time pending future taxonomic investigation of the genome constitution of Elytrigia repens (L.) Nevski.     

In situ hybridization identifies the diploid progenitor species of Coffea arabica (Rubiaceae)

 The most important commercial coffee species, Coffea arabica, which is cultivated in about 70% of the plantations world-wide, is the only tetraploid (2n=4x=44) species known in the genus. Genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) were used to study the genome organization and evolution of this species. Labelled total genomic DNA from diploid species (C. eugenioides, C. congensis, C. canephora, C. liberica) closely related to C. arabica was separately used as a probe in combination with or without blocking DNA to the chromosome spreads of C. arabica. GISH discriminated between chromosomes of C. arabica only in the presence of an excess of unlabelled block DNA from the species not used as a probe. Among the range of different species combinations used, DNA from C. eugenioides strongly and preferentially labelled 22 chromosomes of the tetraploid C. arabica, while the remaining 22 chromosomes were labelled with C. congensis DNA. The similarity of observations between C. arabica and the two diploid species using two ribosomal genes with FISH with respect to metaphase chromosomes provided additional support to the GISH results. These results confirm the allopolyploid nature of C. arabica and show that C. congensis and C. eugenioides are the diploid progenitors of C. arabica. Received: 2 February 1998 / Accepted: 12 May 1998     

PARTHENOGENESIS IN TETRAPLOID LILIUM LONGIFLORUM

Emsweller , S. L., and Joseph Uhring . (U.S.D.A., Beltsville, Md.) Parthenogenesis in tetraploid Lilium longiflorum. Amer. Jour. Bot. 49(9): 978–984. Illus. 1962.—Nine maternal polyhaploids from 1 capsule and 1 tetraploid from another were produced following pollination of 2 tetraploid Lilium longiflorum plants with pollen from diploids of the same species. One of the 9 plants had 25 chromosomes; the extra chromosome was identified as a modified D. Two other plants had 2 new chromosomes each and the remaining 6 had 24 unmodified chromosomes. Translocations in meiosis of the tetraploid produced the new chromosomes. One plant obtained from a second capsule had 48 chromosomes. The 9 plants were smaller than diploids and the 48-chromosome plant was considered a diploid until mitosis was observed. The 9 plants originated from unfertilized eggs of the tetraploid, and the 48-chromosome plant presumably from chromosome doubling of an egg cell.     

Rapid response to artificial selection on flower size in Phlox

Quantitative characters are often said to evolve rather slowly, taking many generations to exhibit appreciable differences among populations. We tested this notion experimentally by performing bi-directional selection on corolla diameter of plants from a wild population of Phlox drummondii for three generations. By monitoring flower size, tube length and stigma-anther proximity of flowers, we obtained the direct and indirect responses to selection, and calculated genetic correlations, realized and narrow sense heritabilities using offspring-mother regression. Realized heritability of flower size was high (0.83), whereas genetic correlations among traits were weak or not significant. The per-generation average of the response in corolla diameter was about 5%. We found that P. drummondii has a great capacity to respond rapidly to selection, and this capacity may be in part responsible for the observed high degree of differentiation within the species. We also concluded that rapid evolution of morphological floral traits is possible.     

Chromosome characterization and variability in some Iridaceae from Northeastern Brazil

The chromosomes of 15 species of Iridaceae of the genera Alophia, Cipura, Eleutherine, Neomarica and Trimezia (subfamily Iridoideae) were examined after conventional Giemsa staining. The karyotypes of Alophia drummondii (2n = 14+1B, 28, 42 and 56), Cipura paludosa (2n = 14), C. xanthomelas (2n = 28) and Eleutherine bulbosa (2n = 12) were asymmetric; Neomarica candida, N. caerulea, N. humilis, N. glauca, N. gracilis, N. northiana and Neomarica sp. (2n = 18); N. cf. paradoxa (2n = 28), Trimezia fosteriana (2n = 52), T. martinicensis (2n = 54) and T. connata (2n = 82) were all generally symmetric. New diploid numbers of 2n = 56 for Alophia drummondii, 2n = 18 for N. candida, N. humilis, N. glauca, and N. gracilis, 2n = 28 for N. cf. paradoxa, and 2n = 82 for T. connata are reported. The karyotypic evolution of the studied species is discussed.     

Use of fluorescence in situ hybridization for gross mapping of transgenes and screening for homozygous plants in transgenic barley ( Hordeum vulgare L.)

Introduced transgenes, uidA, sgfp (S65T) and/or bar, were localized using fluorescence in situ hybridization (FISH) on metaphase chromosomes of transgenic barley produced by microparticle bombardment of immature embryos. Of the 19 independent transgenic lines (eight diploid and 11 tetraploid), nine had uidA and ten had s gfp (S65T). All lines tested had three or more copies of the transgenes and 18 out of 19 lines had visibly different integration sites. At a gross level, it appeared that no preferential integration sites of foreign DNA among chromosomes were present in the lines tested; however, a distal preference for transgene integration was observed within the chromosome. In diploid T0 plants that gave a 3:1 segregation ratio of transgene expression in the T1, only single integration sites were detected on one of the homologous chromosomes. Homozygous diploid plants had doublet signals on a pair of homologous chromosomes. All tetraploid T0 plants that gave a 3:1 segregation ratio in the T1 generation had only a single integration site on one of the homologous chromosomes. In contrast, the single tetraploid T0 plant with a 35:1 segregation ratio in the T1 generation had doublet signals on a pair of homologous chromosomes. In the one tetraploid T0 line, which had a homozygote-like segregation ratio (45:0), there were doublet signals at two loci on separate chromosomes. We conclude that the application of FISH for analysis of transgenic plants is useful for the gross localization of transgene(s) and for early screening of homozygous plants.     

苜蓿核糖体基因物理定位及染色体荧光分带

利用核糖体基因为探针对,二倍体和四倍体苜蓿(Medicago sativa)进行原位杂交,结果表明,45s在四倍体、二倍体种中总是以单位点位于核仁组织区,5s则有2～3个位点;以二倍体种的基因组DNA为探针的原位杂交表明,蓝花苜蓿(M.coerulea)和黄花苜蓿(M.falcata)均能与四倍体染色体进行杂交,仅杂交信号强弱的染色体数目有差别;荧光染料DAPI使苜蓿的染色体显示带纹,蓝花苜蓿的DAPI带与C-带基本一致.文章对四倍体苜蓿的可能来源进行了讨论.     

Haploid unit-ploidy transition of tetraploid and octaploid H1 (ES) cells in long-term culturing

Haploid unit-ploidy transition in tetraploid and octaploid mouse H1 (ES) cells (4H1 and 8H1 cells, respectively) during long-term culturing was observed using flow cytometry. The DNA content of 4H1 cells was elevated from 3.5C to 4.5C, and that of 8H1 cells was degraded from 6.5C to 5.5C, in addition to gradual DNA loss (C: complement). The timing of the transition was not predetermined. Cell cycle parameters, doubling time and phase durations, were essentially the same before and after the transition, suggesting that most cells in a cell population were induced to undergo the ploidy transition at the same time. Cellular morphology was altered before and after the transition, suggesting that the ploidy shift changed cellular characteristics; however, pluripotency was maintained irrespective of DNA content. Cell volume correlated with DNA content during the final stage of culturing. Diploid and hexaploid H1 (ES) cells--2H1 and 6H1 cells, respectively--were used as control cells in which the ploidy was maintained for about 300 days of culturing. The haploid unit-ploidy transition was explained using a hypothesis concerning the DNA structure of polyploid cells: closing homologous chromosomes causes inhomogeneous cell division accompanying a haploid DNA set, suggesting the existence of a coupling apparatus connecting DNA fibers with a single haploid DNA set.     

Regain of sex determination system and sexual reproduction ability in a synthetic octoploid male fish

Polyploids in vertebrates are generally associated with unisexual reproduction, but the direct consequences of polyploidy on sex determination system and reproduction mode remain unknown. Here, we synthesized a group of artificial octoploids between unisexual gynogenetic hexaploid Carassius gibelio and sexual tetraploid Carassius auratus. The synthetic octoploids were revealed to have more than 200 chromosomes, in which 50 chromosomes including the X/Y sex determination system were identified to transfer from sexual tetraploid C. auratus into the unisexual gynogenetic hexaploid C. gibelio. Significantly, a few synthetic octoploid males were found to be fertile, and one octoploid male was confirmed to regain sexual reproduction ability,which exhibits characteristics that are the same to sexual reproduction tetraploid males, such as 1:1 sex ratio occurrence, meiosis completion and euploid sperm formation in spermatogenesis, as well as normal embryo development and gene expression pattern during embryogenesis. Therefore, the current finding provides a unique case to explore the effect of sex determination system incorporation on reproduction mode transition from unisexual gynogenesis to sexual reproduction along with genome synthesis of recurrent polyploidy in vertebrates.     

Arrangement of spindle apparatus in mitoses of different ploidy

In non-hypotonically treated mitoses from tissue cultures of Microtus agrestis, both the constitutive heterochromatin of the sex chromosomes and the spindle apparatus were stained by the Giemsa C-banding technique. By means of counting the heterochromatic chromosomes, we determined the cell ploidy and studied the number of centrioles and the spindle arrangement of diploid, triploid, tetraploid and octoploid mitoses. Diploid and triploid prophases contained 2 centrioles in most cases, tetraploid prophases 4, binucleate cells with 2 diploid nuclei likewise 4 and binucleate cells with 2 tetraploid nuclei 8 centrioles. Nearly 99% of diploid and triploid metaphases were bipolar. Of the tetraploid metaphases only 45% were bipolar, 29.5% tripolar, 7.5% quadripolar and 18% formed as a parallel mitosis. In all examined binucleate cells that had had an asynchronous DNA synthesis, a multipolar mitosis was found.     

Mouse IgD half molecules with shortened IgD heavy chain result from alterations within C delta locus

An unusually small (51 KD) IgD myeloma protein was isolated from secretions of TEPC 1017 generation (gen) 24. The delta-chain mRNA and the delta-chain gene in this tumor were compared with those of TEPC 1017 of earlier generations. The gen 24 protein contained one normal-sized kappa-type light chain (21 KD) and one unusually short delta-heavy chain (30 KD). The delta-heavy chain was 15 KD shorter than that of TEPC 1017 of earlier generations, owing to a delta-mRNA (1.15 kb) which was 600 bp shorter than that of TEPC 1017 of earlier generations. TEPC 1017 is a tetraploid tumor, and the gen 24 appears to contain at least two different deletions on different chromosomes. The short mRNA was produced from one of these altered delta-chain genes which had a productive VDJ rearrangement but which had lost the C delta 3 domain and perhaps the C delta H domain as well. Despite these genetic insults, RNA splicing produced delta-mRNA with secreted termini and mRNA with membrane-binding termini. It is suggested that the mouse C delta gene has an unusual predilection for deletions because it normally lacks any vestige of C delta 2 and, during i.p. passage, it suffered further deletions or alterations.     

Long-term stability of transgene expression driven by barley endosperm-specific hordein promoters in transgenic barley

In order to evaluate the long-term stability of transgene expression driven by the B(1)- and D-hordein promoters in transgenic barley ( Hordeum vulgare L., 2 n=2 x=14), we analyzed plants from 15 independent transgenic barley lines [6 for uidA and 9 for sgfp(S65T)] produced via microprojectile bombardment of immature embryos; 4 were diploid and 11 were tetraploid. The expression and inheritance of transgenes were determined by analysis of functional transgene expression, polymerase chain reaction and fluorescence in situ hybridization (FISH). Ability to express transgenes driven by either B(1)- or D-hordein promoter was inherited in T(4) and later generations: T(4) (2 lines), T(5) (8 lines), T(6) (3 lines), T(8) (1 line) and T(9) (1 line). Homozygous transgenic plants were obtained from 12 lines [5 for uidA and 7 for sgfp(S65T)]; the remaining lines are currently being analyzed. The application of the FISH technique for physical mapping of chromosomes was useful for early screening of homozygous plants by examining for presence of the transgene. For example, one line expressing uidA, and shown to have doublet fluorescence signals on a pair of homologous chromosomes was confirmed as a homozygous line by its segregation ratio; additionally this line showed stable inheritance of the transgene to T(9) progeny. The expression of transgenes in most lines (14 out of 15 lines) driven by hordein promoters was stably transmitted to T(4) or later generations, although there was a skewed segregation pattern (1:1) from the T(1) generation onward in the remaining line. In contrast, transgene silencing or transgene loss under the control of the maize ubiquitin promoter was observed in progeny of only 6 out of 15 lines.     

Die Verteilung der Chromosomen auf die Tochterzellkerne multipolarer Mitosen in euploiden Gewebekulturen von Microtus agrestis

In kidney epithelial cultures from female Microtus agrestis, 3,55% of all mitoses are multipolar, 94% of them tripolar. Feulgen photometric measurements of 21 tripolar mitoses reveal a total DNA amount corresponding to the mitotic diploid value (4c) in 5 cases, and to the tetraploid value (8c) in 16 cases, Diploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei each with a haploid DNA value (1c). Most tetraploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei with a triploid DNA value (3c). Also the sex chromosomes are distributed to the daughter nuclei in the relation of 2∶3∶3. This can be seen in anaphase figures as well as in interphase nuclei presumably derived from tripolar mitoses, showing chromocenters according to the number of X-chromosomes. In two cases of tripolar tetraploid mitoses the resulting nuclei have a haploid, a triploid and a tetraploid DNA value. The DNA replication pattern is always identical in the daughter nuclei of diploid and tetraploid tripolar mitoses. — Our observations suggest segregation and distribution of haploid chromosome sets or multiples of haploid sets to the daughter nuclei of multipolar mitoses. They also show a possible way of formation of haploid and triploid cells in a basically diploid tissue. Presumably triploid nuclei (with 3 chromocenters) are capable of DNA synthesis.     

The use of double fluorescence in situ hybridization to physically map the positions of 5S rDNA genes in relation to the chromosomal location of 18S-5.8S-26S rDNA and a C genome specific DNA sequence in the genus Avena.

A physical map of the locations of the 5S rDNA genes and their relative positions with respect to 18S-5.8S-26S rDNA genes and a C genome specific repetitive DNA sequence was produced for the chromosomes of diploid, tetraploid, and hexaploid oat species using in situ hybridization. The A genome diploid species showed two pairs of rDNA loci and two pairs of 5S loci located on both arms of one pair of satellited chromosomes. The C genome diploid species showed two major pairs and one minor pair of rDNA loci. One pair of subtelocentric chromosomes carried rDNA and 5S loci physically separated on the long arm. The tetraploid species (AACC genomes) arising from these diploid ancestors showed two pairs of rDNA loci and three pairs of 5S loci. Two pairs of rDNA loci and 2 pairs of 5S loci were arranged as in the A genome diploid species. The third pair of 5S loci was located on one pair of A-C translocated chromosomes using simultaneous in situ hybridization with 5S rDNA genes and a C genome specific repetitive DNA sequence. The hexaploid species (AACCDD genomes) showed three pairs of rDNA loci and six pairs of 5S loci. One pair of 5S loci was located on each of two pairs of C-A/D translocated chromosomes. Comparative studies of the physical arrangement of rDNA and 5S loci in polyploid oats and the putative A and C genome progenitor species suggests that A genome diploid species could be the donor of both A and D genomes of polyploid oats. Key words : oats, 5S rDNA genes, 18S-5.8S-26S rDNA genes, C genome specific repetitive DNA sequence, in situ hybridization, genome evolution.