Identification of intergenomic recombinations in unisexual salamanders of the genus Ambystoma by genomic in situ hybridization (GISH)

Unisexual salamanders in the genus Ambystoma (Amphibia, Caudata) are endemic to eastern North America and are mostly all-female polyploids. Two to four of the bisexual species, A. laterale, A. jeffersonianum, A. texanum and A. tigrinum, contribute to the nuclear genome of unisexuals and more than 20 combinations that range from diploid to pentaploid have been identified in this complex. Because the karyotypes of the four bisexual species are similar, homologous and homoeologous chromosomes in the unisexuals can not be distinguished by conventional or banded karyotypes. We chose two widespread unisexual genomic combinations (A.laterale-2 jeffersonianum [or LJJ] and A. 2 laterale-jeffersonianum [or LLJ]) and employed genomic in situ hybridization (GISH) to identify the genomes in these unisexuals. Under optimum conditions, GISH reliably distinguishes the respective chromosomes attributed to both A.laterale and A. jeffersonianum. Of four populations examined, two were found to have independently evolved homoeologous recombinants that persist in both LJJ and LLJ individuals. Our results refute the previous hypothesis of clonal integrity and independent evolution of the genome combinations in these unisexuals. Our data provide evidence for intergenomic interactions between maternal chromosomes during meiosis in unisexuals and help to explain previously observed non-homologous bivalents and/or quadrivalents among lampbrush chromosomes that were possibly initiated by partial homosequential pairing among the homo(eo)logues. To explore the utility of GISH in other members of the complex, probes developed from A. laterale were also applied to unisexuals that contained A. tigrinum and A. texanum genomes. GISH is an effective tool that can be used to identify and to quantify genomic constituents and to investigate intergenomic interactions in unisexual salamanders. GISH also has potential application to examine possible genomic evolution in other unisexuals.     

Two rare aneutriploids in the unisexual Ambystoma (Amphibia, Caudata) identified by GISH indicating two different types of meiotic errors

We report two types of aneutriploids in unisexual salamanders Ambystomalaterale-2jeffersonianum (LJJ) and Ambystoma 2 laterale-jeffersonianum (LLJ). One karyotype has 3n = 42: L27 (L8-); J15 (J8p+), and we suggest that it was induced by homoeologous pairing after premeiotic endomitosis followed by an unequal L8;J8 segregation. The second karyotype has 3n = 43: L14 (L10q); J29 (J12+), which can be explained by meiotic nondisjunction followed by unbalanced segregation. These two rare aneutriploids demonstrate two different types of meiotic errors that might help to explain the high mortality observed in this complex. Case one also indicates that contemporary intergenomic exchanges and homoeologous recombinations may occur after a premeiotic chromosome doubling event. Our study provides additional evidence for the extremely flexible reproduction of unisexual Ambystoma.     

Unisexual salamanders (genus Ambystoma) present a new reproductive mode for eukaryotes.

To persist, unisexual and asexual eukaryotes must have reproductive modes that circumvent normal bisexual reproduction. Parthenogenesis, gynogenesis, and hybridogenesis are the modes that have generally been ascribed to various unisexuals. Unisexual Ambystoma are abundant around the Great Lakes region of North America, and have variously been described as having all 3 reproductive modes. Diploid and polyploid unisexuals have nuclear genomes that combine the haploid genomes of 2 to 4 distinct sexual species, but the mtDNA is unlike any of those 4 species and is similar to another species, Ambystoma barbouri. To obtain better resolution of the reproductive mode used by unisexual Ambystoma and to explore the relationship of A. barbouri to the unisexuals, we sequenced the mitochondrial control and highly variable intergenic spacer region of 48 ambystomatids, which included 28 unisexuals, representatives of the 4 sexual species and A. barbouri. The unisexuals have similar sequences over most of their range, and form a close sister group to A. barbouri, with an estimated time of divergence of 2.4-3.9 million years ago. Individuals from the Lake Erie Islands (Kelleys, Pelee, North Bass) have a haplotype that demonstrates an isolation event. We examined highly variable microsatellite loci, and found that the genetic makeup of the unisexuals is highly variable and that unisexual individuals share microsatellite alleles with sexual individuals within populations. Although many progeny from the same female had the same genotype for 5 microsatellite DNA loci, there was no indication that any particular genome is consistently inherited in a clonal fashion in a population. The reproductive mode used by unisexual Ambystoma appears to be unique; we suggest kleptogenesis as a new unisexual reproductive mode that is used by these salamanders.     

Analysis of karyotypic stability of homoeologous-pairing (ph) mutants in allopolyploid wheats

Karyotypic analysis of wheat lines with different genotypes for the homoeologous-pairing loci Ph1 and Ph2 was carried out by means of a genomic in situ hybridization method that allowed unequivocal identification of the A, B and D genomes. Chromosomal rearrangements mainly affecting the A and D genomes were found in all plants of allohexaploid wheat (AABBDD) lacking Ph1 activity. The frequency of intergenomic exchanges per plant in ph1b mutant and nulli-5B lines was 4.31 and 3.40, respectively. In addition, an unbalanced genomic constitution was found in a few plants, some even showing a euploid chromosomal number. By contrast, rearranged karyotypes were detected neither in the ph1 mutant line (ph1c) of allotetraploid wheat (AABB) nor in the allohexaploid wheat lines lacking Ph2 activity, namely ph2b mutant and nulli-3D lines. These results were compared with the chromosomal pairing behaviour displayed by mutant lines ph1c, ph1b and ph2b at first meiotic metaphase. Despite the finding of standard, nonrearranged karyotypes in the phlc tetraploid mutant, the frequency of A-B homoeologous metaphase I association was similar to that observed in the ph1b hexaploid mutant. The results presented clearly demonstrate that inactivity of the Ph1 locus induces karyotypic instability in wheat. Intergenomic exchanges have probably been accumulating since the original ph1 mutant and aneuploid lines were obtained, which should be taken into account when it is planned to use these lines for basic research on Ph1 function or in applied wheat breeding programmes.     

Identification of intergenomic translocations involving wheat,Hordeum vulgare and Hordeum chilense chromosomes by FISH

Intergenomic translocations between wheat, Hordeum chilense and Hordeum vulgare have been obtained in tritordeum background. Advanced lines from the crosses between three disomic chromosome addition lines for chromosome 2Hv, 3Hv, and 4Hv of barley (Hordeum vulgare) in Triticum aestivum cv. Chinese Spring (CS) and hexaploid tritordeum (2n = 6x = 42, AABBHchHch) were analyzed. Multicolor FISH using both genomic DNA from H. chilense and H. vulgare were used to establish the presence and numbers of H. vulgare introgressions into tritordeum. Interspecific H. vulgare/H. chilense and intergeneric wheat/H. vulgare and wheat/H. chilense translocations were identified. Frequencies of plants containing different kinds of intergenomic translocations between chromosome arms are presented. These lines can be useful for introgressing into tritordeum characters of interest from H. vulgare.     

An unexpected recent ancestor of unisexual Ambystoma

Previous research has shown that members of the unisexual hybrid complex of the genus Ambystoma possess a mitochondrial genome that is unrelated to their nuclear parental species, but the origin of this mitochondrion has remained unclear. We used a 744-bp fragment of the mitochondrial gene cytochrome b within a comparative phylogenetic framework to infer the maternal ancestor of this unisexual lineage. By examining a broader range of species than has previously been compared, we were able to uncover a recent maternal ancestor to this complex. Unexpectedly, Ambystoma barbouri, a species whose nuclear DNA has not been identified in the unisexuals, was found to be the recent maternal ancestor of the individuals examined through the discovery of a shared mtDNA haplotype between the unisexuals and A. barbouri. Based on a combination of sequence data and glacial patterning, we estimate that the unisexual lineage probably originated less than 25 000 years ago. In addition, all unisexuals examined showed extremely similar mtDNA sequences and the resultant phylogeny was consistent with a single origin for this lineage. These results confirm previous suggestions that the unisexual Ambystoma complex was formed from a hybridization event in which the nuclear DNA of the original maternal species was subsequently lost.     

Intergenomic rearrangements after polyploidization of Kengyilia thoroldiana (Poaceae: Triticeae) affected by environmental factors

Polyploidization is a major evolutionary process. Approximately 70–75% species of Triticeae (Poaceae) are polyploids, involving 23 genomes. To investigate intergenomic rearrangements after polyploidization of Triticeae species and to determine the effects of environmental factors on them, nine populations of a typical polyploid Triticeae species, Kengyilia thoroldiana (Keng) J.L.Yang et al. (2n = 6x = 42, StStPPYY), collected from different environments, were studied using genome in situ hybridization (GISH). We found that intergenomic rearrangements occurred between the relatively large P genome and the small genomes, St (8.15%) and Y (22.22%), in polyploid species via various types of translocations compared to their diploid progenitors. However, no translocation was found between the relatively small St and Y chromosomes. Environmental factors may affect rearrangements among the three genomes. Chromosome translocations were significantly more frequent in populations from cold alpine and grassland environments than in populations from valley and lake-basin habitats (P<0.05). The relationship between types of chromosome translocations and altitude was significant (r = 0.809, P<0.01). Intergenomic rearrangements associated with environmental factors and genetic differentiation of a single basic genome should be considered as equally important genetic processes during species'' ecotype evolution.     

Intergenomic single nucleotide polymorphisms as a tool for bacterial artificial chromosome contig building of homoeologous Brassica napus regions

Background

Homoeologous sequences pose a particular challenge if bacterial artificial chromosome (BAC) contigs shall be established for specific regions of an allopolyploid genome. Single nucleotide polymorphisms (SNPs) differentiating between homoeologous genomes (intergenomic SNPs) may represent a suitable screening tool for such purposes, since they do not only identify homoeologous sequences but also differentiate between them.

Results

Sequence alignments between Brassica rapa (AA) and Brassica oleracea (CC) sequences mapping to corresponding regions on chromosomes A1 and C1, respectively were used to identify single nucleotide polymorphisms between the A and C genomes. A large fraction of these polymorphisms was also present in Brassica napus (AACC), an allopolyploid species that originated from hybridisation of A and C genome species. Intergenomic SNPs mapping throughout homoeologous chromosome segments spanning approximately one Mbp each were included in Illumina’s GoldenGate® Genotyping Assay and used to screen multidimensional pools of a Brassica napus bacterial artificial chromosome library with tenfold genome coverage. Based on the results of 50 SNP assays, a BAC contig for the Brassica napus A subgenome was established that spanned the entire region of interest. The C subgenome region was represented in three BAC contigs.

Conclusions

This proof-of-concept study shows that sequence resources of diploid progenitor genomes can be used to deduce intergenomic SNPs suitable for multiplex polymerase chain reaction (PCR)-based screening of multidimensional BAC pools of a polyploid organism. Owing to their high abundance and ease of identification, intergenomic SNPs represent a versatile tool to establish BAC contigs for homoeologous regions of a polyploid genome.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-560) contains supplementary material, which is available to authorized users.     

Fluorescence in situ hybridization mapping of Avena sativa L. cv. SunII and its monosomic lines using cloned repetitive DNA sequences.

Fluorescent in situ hybridization (FISH) employing multiple probes was used with mitotic or meiotic chromosome spreads of Avena sativa L. cv. SunII and its monosomic lines to produce physical chromosome maps. The probes used were Avena strigosa pAs120a (which hybridizes exclusively to A-genome chromosomes), Avena murphyi pAm1 (which hybridizes exclusively to C-genome chromosomes), A. strigosa pAs121 (which hybridizes exclusively to A- and D-genome chromosomes), and the wheat rDNA probes pTa71 and pTa794. Simultaneous and sequential FISH employing two-by-two combinations of these probes allowed the unequivocal identification and genome assignation of all chromosomes. Ten pairs were found carrying intergenomic translocations: (i) between the A and C genomes (chromosome pair 5A); (ii) between the C and D genomes (pairs 1C, 2C, 4C, 10C, and 16C); and (iii) between the D and C genomes (pairs 9D, 11D, 13D, and 14D). The existence of a reciprocal intergenomic translocation (10C-14D) is also proposed. Comparing these results with those of other hexaploids, three intergenomic translocations (10C, 9D, and 14D) were found to be unique to A. sativa cv. SunII, supporting the view that 'SunII' is genetically distinct from other hexaploid Avena species and from cultivars of the A. sativa species. FISH mapping using meiotic and mitotic metaphases facilitated the genomic and chromosomal identification of the aneuploid chromosome in each monosomic line. Of the 18 analyzed, only 11 distinct monosomic lines were actually found, corresponding to 5 lines of the A genome, 2 lines of the C genome, and 4 lines of the D genome. The presence or absence of the 10C-14D interchange was also monitored in these lines.     

Cytogenetic analysis of reciprocal hybrids and their parents between <Emphasis Type="Italic">Larix leptolepis</Emphasis> and <Emphasis Type="Italic">Larix gmelinii</Emphasis>: implications for identifying hybrids

We studied the parental taxa and the interspecific reciprocal hybrids between Larix leptolepis with Larix gmelinii, using classical cytogenetic methods, as well as fluorescence in situ hybridization (FISH) and genomic in situ hybridization. A high frequency (>90%) of complete bivalent formation was observed in reciprocal hybrids. Less than 10% of pollen mother cells exhibited abnormalities. The most frequent abnormalities were bridges. Multivalent chromosome associations were also observed in both reciprocal hybrids, suggesting that some chromosome interchange events did occur, and introgressions from one to the other species were possible. Intergenomic recombination indicates that genes might be readily introgressed into one species from the other in the genus Larix. Interspecific hybridization may be a potential method for genetic improvement in larch. FISH markers documented that the recombinant genomes of reciprocal hybrids were strictly additive and stable, indicating that FISH also might be a useful tool in Larix breeding.     

Intergenomic translocations of polyploid oats (genus Avena) revealed by genomic in situ hybridization

Wild and cultivated hexaploid oats share the same genomes (AACCDD) and display a considerable level of interspecific variation in both plant and chromosome morphology. The GISH was utilized to detect the interspecific genomic compositions in four hexaploid and two tetraploid oats using total genomic DNA of Avena eriantha (a C-genome diploid) as probe. Intergenomic translocations between A/D and C-genome chromosomes were frequently observed in hexaploid and tetraploid species. In the hexaploid, two pairs of A/D genome segments on C-genome chromosome (A/D-C) translocation and four to six pairs of C-genome segments on A/D genome chromosome (C-A/D) translocation were clearly identified whilst the number of A/D-C translocations was constant among species. In the tetraploid A. maroccana (AACC), a pair of A-C and four pairs of C-A translocations were observed. Moreover, the A/D translocation segments on chromosome 5C was detected only in A. byzantina and A. maroccana, whilst A/D-C translocations were observed on the 1C and 7C of A. sativa, A. fatua and A. sterilis. A. byzantina did however also carry the 1C rearrangement. This result shows that A. byzantina has retained a similar genomic constitution to the tetraploid ancestor of hexaploid oats, A. maroccana. Three pairs of A-C translocations were detected only in A. murphyi (AACC), and two pairs of those were the 1C and 7C as well as the three hexaploid species except A. byzantina.     

Molecular cytogenetic analysis of Aegilops cylindrica host.

The genomic constitution of Aegilops cylindrica Host (2n = 4x = 28, DcDcCcCc) was analyzed by C-banding, genomic in situ hybridization (GISH), and fluorescence in situ hybridization (FISH) using the DNA clones pSc119, pAs1, pTa71, and pTA794. The C-banding patterns of the Dc- and Cc-genome chromosomes of Ae. cylindrica are similar to those of D-and C-genome chromosomes of the diploid progenitor species Ae. tauschii Coss. and Ae. caudata L., respectively. These similarities permitted the genome allocation and identification of the homoeologous relationships of the Ae. cylindrica chromosomes. FISH analysis detected one major 18S-5.8S-25S rDNA locus in the short arm of chromosome 1Cc. Minor 18S-5.8S-25S rDNA loci were mapped in the short arms of 5Dc and 5Cc. 5S rDNA loci were identified in the short arm of chromosomes 1Cc, 5Dc, 5Cc, and 1Dc. GISH analysis detected intergenomic translocation in three of the five Ae. cylindrica accessions. The breakpoints in all translocations were non-centromeric with similar-sized segment exchanges.     

Genomic Origin and Organization of the Hybrid Poa jemtlandica(Poaceae) Verified by Genomic In Situ Hybridization and Chloroplast DNA Sequences

Chloroplast DNA sequencing and genomic in situ hybridization(GISH) were used to investigate the genomic origin and organizationof the alpine grass Poa jemtlandica. Using genomic probes ofP. alpina and P. flexuosa, GISH clearly distinguished betweenthese two putative parental genomes and thus confirmed the hybridnature of P. jemtlandica. The chloroplast trn L intron and trnL–trn F intergenic spacer (IGS) sequence genotypes ofP. flexuosa and P. jemtlandica were 100% identical but differedfrom those of P. alpina by a total of ten or 11 nucleotide substitutionsand six indels over 866 aligned positions, identifying P. flexuosaas the maternal parent of the P. jemtlandica population studiedhere and supporting a relatively recent origin of the hybrid.GISH revealed the presence of intergenomic translocations inthe hybrid genome, indicating that the two parental genomeshave undergone some rearrangements following hybridization.It is likely that some of these chromosome changes took placesoon after hybridization in order to overcome the adverse interactionsbetween the nuclear and the cytoplasmic genomes and to facilitatethe successful establishment of the newly formed hybrid. Thepresence of intergenomic chromosome changes may play an importantrole in the evolution of natural hybrids and the establishmentof new evolutionary lineages. Copyright 2000 Annals of BotanyCompany Natural hybridization, genome origin, intergenomic translocations, GISH, chloroplast DNA sequences, Poa jemtlandica     

Cryptic sex? Estimates of genome exchange in unisexual mole salamanders (Ambystoma sp.)

Cryptic sex has been argued to explain the exceptional longevity of certain parthenogenetic vertebrate lineages, yet direct measurements of genetic exchange between sexual and apparently parthenogenetic forms are rare. Female unisexual mole salamanders (Ambystoma sp.) are the oldest known unisexual vertebrate lineage (~5 million years), and one hypothesis for their persistence is that allopolyploid female unisexuals periodically exchange haploid genomes ‘genome exchange’ during gynogenetic reproduction with males from sympatric sexual species. We test this hypothesis by using genome‐specific microsatellite DNA markers to estimate the rates of genome exchange between sexual males and unisexual females in two ponds in NE Ohio. We also test the prediction that levels of gene flow should be higher for ‘sympatric’ (sexual males present) genomes in unisexuals compared to ‘allopatric’ (sexual males absent) unisexual genomes. We used a model testing framework in the coalescent‐based program MIGRATE‐N to compare models where unidirectional gene flow is present and absent between sexual species and unisexuals. As predicted, our results show higher levels of gene flow between sexuals and sympatric unisexual genomes compared to lower (likely artefactual) levels of gene flow between sexuals and allopatric unisexual genomes. Our results provide direct evidence that genome exchange between sexual and unisexual Ambystoma occurs and demonstrate that the magnitude depends on which sexual species are present. The relatively high levels of gene flow suggest that unisexuals must be at a selective advantage over sexual forms so as to avoid extinction due to genetic swamping through genome exchange.     

Translocations of Chromosome End-Segments and Facultative Heterochromatin Promote Meiotic Ring Formation in Evening Primroses

Due to reciprocal chromosomal translocations, many species of Oenothera (evening primrose) form permanent multichromosomal meiotic rings. However, regular bivalent pairing is also observed. Chiasmata are restricted to chromosomal ends, which makes homologous recombination virtually undetectable. Genetic diversity is achieved by changing linkage relations of chromosomes in rings and bivalents via hybridization and reciprocal translocations. Although the structural prerequisite for this system is enigmatic, whole-arm translocations are widely assumed to be the mechanistic driving force. We demonstrate that this prerequisite is genome compartmentation into two epigenetically defined chromatin fractions. The first one facultatively condenses in cycling cells into chromocenters negative both for histone H3 dimethylated at lysine 4 and for C-banding, and forms huge condensed middle chromosome regions on prophase chromosomes. Remarkably, it decondenses in differentiating cells. The second fraction is euchromatin confined to distal chromosome segments, positive for histone H3 lysine 4 dimethylation and for histone H3 lysine 27 trimethylation. The end-segments are deprived of canonical telomeres but capped with constitutive heterochromatin. This genomic organization promotes translocation breakpoints between the two chromatin fractions, thus facilitating exchanges of end-segments. We challenge the whole-arm translocation hypothesis by demonstrating why reciprocal translocations of chromosomal end-segments should strongly promote meiotic rings and evolution toward permanent translocation heterozygosity. Reshuffled end-segments, each possessing a major crossover hot spot, can furthermore explain meiotic compatibility between genomes with different translocation histories.     

Molecular Analysis of the Triticale Lines with Different <Emphasis Type="Italic">Vrn</Emphasis> Gene Systems Using Microsatellite Markers and Hybridization In Situ

Hexaploid triticale (×Triticosecale Wittmack) lines were examined using molecular markers and the hybridization in situ technique. Triticale lines were generated based on wheat varieties differing by the Vrn gene systems and the earing times. Molecular analysis was performed using Xgwm and Xrms microsatellite markers with the known chromosomal localization in the common wheat Triticum aestivum, and rye Secale cereale genomes. Comparative molecular analysis of triticale lines and their parental forms showed that all lines contained A and B genomes of common wheat and also rye homoeologous chromosomes. In the three lines the presence of D genome markers, mapped to the chromosomes 2D and 7D, was demonstrated. This was probably the consequence of the translocations of homoeologous chromosomes from wheat genomes, which took part during the process of triticale formation. The data obtained by use of genomic in situ hybridization supported the data of molecular genetic analysis. In none of the lines wheat-rye translocations or recombinations were observed. These findings suggest that the change of the period between the seedling appearance and earing time in triticale lines compared to the initial wheat lines, resulted from the inhibitory effect of rye genome on wheat vernalization genes.     

Cryptic homoeology analysis in species and hybrids of genus Zea

Cryptic intergenomic pairing of genus Zea was induced by the use of a diluted colchicine solution in order to elucidate the phylogenetic relations and differentiation of the homoeologous genomes. Results indicate that in species and hybrids with 2n = 20, there was chromosome pairing between the homoeologous A and B genomes with a maximum of 5IV, with the exception of Zea diploperennis and their interspecific hybrids where cryptic homoeologous chromosome pairing was not induced. In almost all 2n = 30 hybrids, observed cryptic pairing increased to a maximum of 10III although Z. mays × Z. mays with 2n = 30 did not show significant differences between treated and untreated materials. Pairing was also observed in species and hybrids with 2n = 40, in which a maximum of 10IV was observed, with the exception of Z. mays with 2n = 40 where treated and untreated cells did not differ significantly.     

Identification of C-genome chromosomes involved in intergenomic translocations in Avena sativa L., using cloned repetitive DNA sequences

Four anonymous non-coding sequences were isolated from an Avena strigosa (A genome) genomic library and subsequently characterized. These sequences, designated As14, As121, As93 and As111, were 639, 730, 668, and 619 bp long respectively, and showed different patterns of distribution in diploid and polyploid Avena species. Southern hybridization showed that sequences with homology to sequences As14 and As121 were dispersed throughout the genome of diploid (A genome), tetraploid (AC genomes) and hexaploid (ACD genomes) Avena species but were absent in the C-genome diploid species. In contrast, sequences homologous to sequences As93 and As111 were found in diploid (A and C genomes), tetraploid (AC genomes) and hexaploid (ACD genomes) species. The chromosomal locations of the 4 sequences in hexaploid oat species were determined by fluorescent in situ hybridization and found to be distributed over the length of the 28 chromosomes (except in the telomeric regions) of the A and D genomes. Furthermore, 2 C-genome chromosome pairs with the As14 sequence, and 4 with As121, were discovered to beinvolved in intergenomic translocations. These chromosomes were identified as 1C, 2C, 4C and 16C by combining the As14 or As121 sequences with two ribosomal sequences and a C-genome-specific sequence as probes in fluorescence in situ hybridization. These sequences offer new tools for analyzing possible intergenomic translocations in other hexaploid oat species. Received: 8 April 1999 / Accepted: 30 July 1999     

A molecular screening method for unisexual <Emphasis Type="Italic">Ambystoma</Emphasis> using mitochondrial DNA

The geographical range of unisexual Ambystoma overlaps with four bisexual species that also breed in spring ponds. Several of these species are of conservation concern, and both adults and larvae can be difficult to distinguish morphologically from unisexuals. Here we present a rapid molecular method for screening unisexuals, whose mtDNA is most similar to Ambystoma barbouri. A 258 bp segment of the cytochrome b gene was amplified in six Ambystoma species and exemplar unisexuals by PCR using taxon-specific primers. An internal 113 bp segment was amplified only in unisexuals and A. barbouri using Universal forward and Hybrid reverse primers. Multisequence alignment comparing the nucleotide sequence where Hybrid reverse primer anneals revealed nucleotide diversity in this region among Ambystoma species. This simple method for discriminating between unisexuals and bisexuals, excluding A. barbouri, can be applied prior to further research on these declining species.     

Construction of Brassica B genome synteny groups based on chromosomes extracted from three different sources by phenotypic, isozyme and molecular markers

The three B genomes of Brassica contained in B. nigra, B. carinata and B. juncea were dissected by addition in B. napus. Using phenotypic, isozyme and molecular markers we characterized 8 alien B-genome chromosomes from B. nigra and B. carinata and 7 from B. juncea by constructing synteney groups. The alien chromosomes of the three different sources showed extensive intragenomic recombinations that were detected by the presence of the same loci in more than one synteny group but flanked by different markers. In addition, intergenomic recombinations were observed. These were evident in euploid AACC plants of the rapeseed phenotype derived from the addition lines carrying a few markers from the B genome due to translocations and recombinations between non-homoeologous chromosomes. The high plasticity of the Brassica genomes may have been an powerful factor in directing their evolution by hybridization and amphiploidy.