Use of diversity arrays technology markers for integration into a cotton reference map and anchoring to a recombinant inbred line map

A diversity array technology (DArT) marker platform was developed for the cotton genome, to evaluate the use of DArT markers compared with AFLP markers in mapping and transferability across the mapping populations. We used a reference genetic map of tetraploid Gossypium L. that already contained ~5000 loci, which coalesced into 26 chromosomes, to anchor newly developed DArT and AFLP markers with the aim of further improving utility and map resolution. Our results indicated that the percentage of polymorphic DArT markers that could be genetically mapped (78.15%) was much higher than that of AFLP markers (22.28%). Sequence analysis of DArT markers indicated that a majority matched known expressed sequence tag (EST) sequences from tetraploid and diploid Gossypium species. A total of 794 Arabidopsis genes were homologous with various DArT marker sequences. Chromosomes 5(A), 7(A), 19(D), 23(D), and 24(D) had more Arabidopsis syntenic DArT markers than the other chromosomes. Anchoring DArT markers from the reference map to a recombinant inbred line (RIL) map indicated that DArT markers will speed the building of maps in de novo RIL populations.     

First-generation SNP/InDel markers tagging loci for pathogen resistance in the potato genome

A panel of 17 tetraploid and 11 diploid potato genotypes was screened by comparative sequence analysis of polymerase chain reaction (PCR) products for single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (InDels), in regions of the potato genome where genes for qualitative and/or quantitative resistance to different pathogens have been localized. Most SNP and InDel markers were derived from bacterial artificial chromosome (BAC) insertions that contain sequences similar to the family of plant genes for pathogen resistance having nucleotide-binding-site and leucine-rich-repeat domains (NBS-LRR-type genes). Forty-four such NBS-LRR-type genes containing BAC-insertions were mapped to 14 loci, which tag most known resistance quantitative trait loci (QTL) in potato. Resistance QTL not linked to known resistance-gene-like (RGL) sequences were tagged with other markers. In total, 78 genomic DNA fragments with an overall length of 31 kb were comparatively sequenced in the panel of 28 genotypes. 1498 SNPs and 127 InDels were identified, which corresponded, on average, to one SNP every 21 base pairs and one InDel every 243 base pairs. The nucleotide diversity of the tetraploid genotypes (pi = 0.72 x 10(-3)) was lower when compared with diploid genotypes (pi = 2.31 x 10(-3)). RGL sequences showed higher nucleotide diversity when compared with other sequences, suggesting evolution by divergent selection. Information on sequences, sequence similarities, SNPs and InDels is provided in a database that can be queried via the Internet.     

Gene expression in diplosporous and sexual Eragrostis curvula genotypes with differing ploidy levels

The molecular nature of gene expression during the initiation and progress of diplosporous apomixis is still unknown. Moreover, the basis of the close correlation between diplospory and polyploidy is not clarified yet. A comparative expression analysis was performed based on expressed sequence tags (ESTs) sequencing and differential display in an Eragrostis curvula diplosporous tetraploid genotype (T, 4x apo), a sexual diploid derivative obtained from tissue culture (D, 2x sex) and an artificial sexual tetraploid obtained from the diploid seeds after colchicine treatment (C, 4x sex). From a total of 8,884 unigenes sequenced from inflorescence-derived libraries, 112 (1.26%) showed significant differential expression in individuals with different ploidy level and/or variable reproductive mode. Independent comparisons between plants with different reproductive mode (same ploidy) or different ploidy level (same reproductive mode) allowed the identification of genes modulated in response to diplosporous development or polyploidization, respectively. Surprisingly, a group of genes (Group 3) were differentially expressed or silenced only in the 4x sex plant, presenting similar levels of expression in the 4x apo and the 2x sex genotypes. A group of randomly selected differential genes was validated by QR-PCR. Differential display analysis showed that in general the 4x apo and 4x sex expression profiles were more related and different from the 2x sex one, but confirmed the existence of Group 3-type genes, in both inflorescences and leaves. The possible biological significance for the occurrence of this particular group of genes is discussed. In silico mapping onto the rice genome was used to identify candidates mapping to the region syntenic to the diplospory locus. Gerardo D. L. Cervigni, Norma Paniego and Silvina Pessino contributed equally to the work.     

Ploidy doubling by in vitro culture of excised protocorms or protocorm-like bodies in <Emphasis Type="Italic">Phalaenopsis</Emphasis> species

Endopolyploidy was observed in the protocorms of diploid Phalaenopsis aphrodite subsp. formosana with ploidy doubling achieved by in vitro regeneration of excised protocorms, or protocorm-like bodies (PLBs). Thirty-four per cent of the PLBs regenerated from the first cycle of sectioned protocorms were found to be polyploids with ploidy doubled once or twice as determined by flow-cytometry. The frequency of ploidy doubling increased as the sectioning cycles increased and was highest in diploid followed by the triploid and tetraploid. Regeneration of the endopolyploid cells in the tissue of the protocorms or PLBs is proposed as the source of the development of ploidy doubled plantlets. The frequency of ploidy doubling was similar in seven other Phalaenopsis species, although the rate of increase within cycles was genotype specific. In two species, a comparison of five parameters between 5-month-old diploid and tetraploid potted plants showed only the stomata density differed significantly. The flowers of the tetraploid plant were larger and heavier than those of the diploids. This ploidy doubling method is a simple and effective means to produce large number of polyploid Phalaenopsis species plants as well as their hybrids. The method will be beneficial to orchid breeding programs especially for the interspecific hybridization between varieties having different chromosome sizes and ploidy levels.     

The phylogenetic relationship of possible progenitors of the cultivated peanut

The cultivated peanut (Arachis hypogaea L.) is an allotetraploid composed of A and B genomes. The phylogenetic relationship among the cultivated peanut, wild diploid, and tetraploid species in the section Arachis was studied based on sequence comparison of stearoyl-ACP desaturase and oleoyl-PC desaturase. The topology of the trees for both fatty acid desaturases displayed two clusters; one cluster with A genome diploid species and the other with B genome diploid species. The two homeologous genes obtained for each of the two fatty acid desaturases from the tetraploid species A. hypogaea and A. monticola were separated into the A and B genome clusters, respectively. The gene phylogenetic trees showed that A. hypogaea is more closely related to the diploid species A. duranensis and A. ipaensis than to the wild tetraploid species A. monticola, suggesting that A. monticola is not a progenitor of the cultivated peanut. In addition, for the stearoyl-ACP desaturase, the A. duranensis sequence was identical with one of the sequences of A. hypogaea and the A. ipaensis sequence was identical with the other. These results support the hypothesis that A. duranensis and A. ipaensis are the most likely diploid progenitors of the cultivated tetraploid A. hypogaea.     

Analysis of random and specific sequences of nuclear and cytoplasmic DNA in diploid and tetraploid american wild rice species (Oryza spp.).

A sample of American wild rice and other accessions of the genus Oryza were studied at polymorphic regions of nuclear, mitochondrial, and chloroplastic genomes. First, flow cytometry, genome-specific RAPD markers, and chromosome counting were utilized to verify the original ploidy and classification of 230 accessions studied. Based on these methods, 8% of the accessions were considered to be misclassified either taxonomically or as a result of contamination. Second, a fine resolution analysis was conducted at genomic regions sampled at random by RAPD markers and at specific sites of the chloroplast and mitochondrial DNA by cleaved amplified polymorphic sequence (CAPS) analysis. Phylogenetic trees resulting from phenetic and cladistic analyses of RAPD, cpDNA, and mtDNA polymorphisms were obtained. The results indicated that the American diploid species O. glumaepatula should be considered an individual species, distinct from O. rufipogon, and confirmed that the American tetraploid species (O. alta, O. grandiglumis, and O. latifolia) belong to the O. officinalis complex. The data indicate that these species should still be treated as a group rather than as three distinct species and that their closest relative is a CC-genome species. It was estimated that the diploid and tetraploid American species diverged from O. sativa - O. nivara (AA genome) and CC- and BBCC-genome species, respectively, 20 million years ago.     

Frequencies and variation in cytosine methylation patterns in diploid and tetraploid cytotypes of Paspalum notatum

Paspalum notatum Flügge is a grass species organized as an agamic complex. The objective of the current research was to survey the frequencies and variation of cytosine methylation at CCGG sequences in diploid and tetraploid genotypes, and to determine the occurrence of methylation changes associated with tetraploidization by using methylation-sensitive amplification polymorphism (MSAP) markers. No differences were found in the average proportions of methylated CCGG sites between cytotypes, but methylation patterns were significantly more variable in tetraploids. In both groups of plants, epigenetic and non-epigenetic variation correlated significantly when compared by Mantel tests. The evaluation of 159 common MSAP markers showed that 18.86 % of them differed in their methylation status in the different ploidies. Dendrogram analysis, reflecting epigenetic distances, showed that the four diploids and one experimentally-obtained sexually-reproducing tetraploid, grouped together. MSAP analysis performed on a diploid plant and its autotetraploid derivative showed that new epialleles emerged after tetraploidization. Sequencing of several MASP markers showed homologies with low copy genes, non-coding sequences and transposon/retrotransposon elements.     

AFLP variation in 25 Avena species

Current molecular characterization of ex situ plant germplasm has placed more emphasis on cultivated gene pools and less on exotic gene pools representing wild relative species. This study attempted to characterize a selected set of germplasm accessions representing various Avena species with the hope to establish a reference set of exotic oat germplasm for oat breeding and research. The amplified fragment length polymorphism (AFLP) technique was applied to screen 163 accessions of 25 Avena species with diverse geographic origins. For each accession, 413 AFLP polymorphic bands detected by five AFLP primer pairs were scored. The frequencies of polymorphic bands ranged from 0.006 to 0.994 and averaged 0.468. Analysis of molecular variance revealed 59.5% of the total AFLP variation resided among 25 oat species, 45.9% among six assessed sections of the genus, 36.1% among three existing ploidy levels, and 50.8% among eight defined genome types. All the species were clustered together according to their ploidy levels. The C genome diploids appeared to be the most distinct, followed by the Ac genome diploid A. canariensis. The Ac genome seemed to be the oldest in all the A genomes, followed by the As, Al and Ad genomes. The AC genome tetraploids were more related to the ACD genome hexaploids than the AB genome tetraploids. Analysis of AFLP similarity suggested that the AC genome tetraploid A. maroccana was likely derived from the Cp genome diploid A. eriantha and the As genome diploid A. wiestii, and might be the progenitor of the ACD genome hexaploids. These AFLP patterns are significant for our understanding of the evolutionary pathways of Avena species and genomes, for establishing reference sets of exotic oat germplasm, and for exploring new exotic sources of genes for oat improvement.     

Marker utility of miniature inverted-repeat transposable elements for wheat biodiversity and evolution

Transposable elements (TEs) account for up to 80% of the wheat genome and are considered one of the main drivers of wheat genome evolution. However, the contribution of TEs to the divergence and evolution of wheat genomes is not fully understood. In this study, we have developed 55 miniature inverted-repeat transposable element (MITE) markers that are based on the presence/absence of an element, with over 60% of these 55 MITE insertions associated with wheat genes. We then applied these markers to assess genetic diversity among Triticum and Aegilops species, including diploid (AA, BB and DD genomes), tetraploid (BBAA genome) and hexaploid (BBAADD genome) species. While 18.2% of the MITE markers showed similar insertions in all species indicating that those are fossil insertions, 81.8% of the markers showed polymorphic insertions among species, subspecies, and accessions. Furthermore, a phylogenetic analysis based on MITE markers revealed that species were clustered based on genus, genome composition, and ploidy level, while 47.13% genetic divergence was observed between the two main clusters, diploids versus polyploids. In addition, we provide evidence for MITE dynamics in wild emmer populations. The use of MITEs as evolutionary markers might shed more light on the origin of the B-genome of polyploid wheat.     

In vitro regeneration ability of diploid and autotetraploid plants of Cichorium intybus L.

Polyploidy has played a significant role in the evolutionary history of plants and is a valuable tool for obtaining useful characteristics. Because of the novelty of polyploids, comparison of their in vitro culture responses with diploids would be notable. In this study, leaf explants from diploid, autotetraploid and mixoploid plants of Cichorium intybus L. were cultured in vitro on the similar media and under same conditions. The ploidy level of the obtained calluses and regenerants were determined by flow cytometry analysis. The callogenic response of leaf explants cultured on the callus induction medium did not depend on the ploidy level of their parental plants. According to the flow cytometry analysis, the increased ploidy levels (4x) and (8x) were observed in the callus cultures with diploid and tetraploid origin, respectively. A considerable difference was observed between the ploidy level of mixoploid plants and their calluses, indicating the dominance of diploid cells in the callus tissue. The results showed that polyploidy led to the loss of organogenic potential as the tetraploid origin calluses failed to regenerate, while the diploid origin calluses successfully regenerated to whole plants.     

Effect of ploidy increase on transgene expression: example from <Emphasis Type="Italic">Citrus</Emphasis> diploid cybrid and allotetraploid somatic hybrid expressing the EGFP gene

Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from ‘Murcott’ tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic ‘Valencia’ orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy level conversion can affect transgene expression and citrus diploid cybrid and allotetraploid somatic hybrid represents another example of gene regulation coupled to ploidy.     

<Emphasis Type="Italic">Phytophthora colocasiae</Emphasis> from Vietnam,China, Hawaii and Nepal: intra- and inter-genomic variations in ploidy and a long-lived,diploid Hawaiian lineage.

Phytophthora colocasiae is an important pathogen of taro and is widely distributed. Our goal was to develop whole genome sequence and single nucleotide polymorphism (SNP) markers to characterize historical and current populations from Hawaii (2010 and 2016, HA), historical isolates from Vietnam and China (2010, VN and CH) and current isolates from Nepal (2016, NEP). Seven isolates (VN = 2, CH = 1, HA = 1, NEP = 3) were sequenced (NCBI BioProject PRJNA378784) and compared using the reference genome of the closely related vegetable pathogen P. capsici. Genome-wide SNP analysis using 27,537 markers revealed genomes of diploid, triploid, tetraploid and higher ploidy. Ploidy varied within and between populations, with HA being primarily diploid, CH primarily triploid, VN containing diploid and triploid isolates, and NEP having predominantly triploid, tetraploid and higher ploidy. A total of 37 SNP markers were genotyped in 89 samples (grown in culture or directly from infected tissue) using targeted-sequencing. Analyses indicate a single clonal lineage dominated populations in HA from 2010 to 2016 and targeted-sequencing was useful to estimate ploidy. The implications for adaptation and evolution of P. colocasiae are discussed, as well as consequences for selection and breeding of resistant taro cultivars.     

In vitro induction of tetraploids in cassava variety ‘Xinxuan 048’ using colchicine

Polyploids have great breeding value as they could have higher vegetative yields, higher qualities and greater tolerances against biotic and abiotic stresses. This research is focusing on in vitro colchicine-induced tetraploids in cassava. The survival rate of explants decreased with the increase of colchicine concentrations. Based on the survival rate, the treatment with 0.05 g l^?1 colchicine for 2 days was the best protocol for tetraploid induction in the cassava variety ‘Xinxuan 048’. The determination of ploidy levels showed that 28 autotetraploidy and 12 mixoploidy plantlets were obtained from 44 plantlets. Thus, 90.9% of the plants were variants. Significant differences in morphology and anatomy were found between the diploid and tetraploid plants. Tetraploid plants showed better photosynthetic capacities than the original diploid plants. The tetraploid cassava regenerated herein will enrich the germplasm spectrum and open a new arena for breeding novel triploids with elite cultivars by interploid crossing in the future.     

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model ??diploid embryogenic callus protoplast?+?diploid mesophyll-derived protoplast??. Protoplasts were isolated from embryogenic calli of ??Pera?? and ??Westin?? sweet orange cultivars (Citrus sinensis) and from young leaves of ??Fremont??, Nules??, and ??Thomas?? mandarins (C. reticulata), and ??Nova?? tangelo [C. reticulata?×?(C. paradisi?×?C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when ??Pera?? sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of ??Pera?? sweet orange?+???Fremont?? mandarin, 3 ??Pera?? sweet orange?+???Nules?? mandarin, and 2 ??Pera?? sweet orange?+???Nova?? tangelo, and all the diploid regenerated plants showed the ??Pera?? sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96?h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.     

Phenotypic and gene expression analyses of a ploidy series of maize inbred Oh43

Polyploidization has repeatedly occurred during plant evolution. Although autopolyploidy is the best model to characterize the polyploidization effects in a highly controlled manner, there are limited studies on autopolyploids compared to allopolyploids. To improve our understanding of autopolyploidy effects in maize, we developed an inbred Oh43 ploidy series consisting of the diploid (2X), tetraploid (4X) and hexaploid (6X) lines and compared their phenotypes and gene expression in the mature adult leaf tissue. Our phenotypic study showed that plants of higher ploidy exhibit increased cell size but slower growth rate, later flowering, fewer tassel branches, reduced stature and fertility. Two-dimensional difference gel electrophoresis (2D DIGE) and gel electrophoresis followed by liquid chromatography and mass spectrometry (GeLC-MS) assays of the leaf proteomes revealed ~40 and 26% quantitative differentially expressed (DE) proteins, respectively, at the per genome level. A small number of qualitative DE proteins were also identified in the GeLC-MS assay. The majority of the quantitative DE proteins found in the 2D DIGE assay were present in either the 4X versus 6X or the 2X versus 6X comparison but not the 2X versus 4X comparison. Aneuploidy in some 6X plants might contribute to the more extensive changes of gene expression per genome in the 6X. Most changes of the protein expression per genome are less than twofold. Less than 5% of the DE genes exhibit a positive or negative continuous correlation through the ploidy series between their protein expression per genome, and the genome copy number. Hence, in the Oh43 ploidy series, expression for most proteins in a cell increases linearly with ploidy.     

DNA reassociation kinetics in diploid and phylogenetically tetraploid cyprinidae.

Four diploid and three phylogenetically tetraploid Cyprinidae (Ostariophysi) have been characterized as for nuclear DNA content, modal chromosome number and DNA reassociation kinetics (hydroxyapatite chromatography). Among the diploid species nuclear DNA content (10(-12) g DNA/2C) was 1.62 for Tinca tinca, 1.87 for Scardinius erythrophthalmus, 2.53 for Leuciscus cephalus and 2.75 for Alburnus alburnus, while the phylogenetically tetraploid species Carassius auratus, Barbus barbus and Cyprinus carpio attained 3.40, 3.66 and 3.80 respectively. Modal chromosome number was 2n = 48-50 for diploid individuals and 2n = 100-104 for phylogenetically tetraploid ones. In all the species 5--8% of the genome is represented by highly repetitive and foldback DNA. In DNA reassociation kinetics of phylogenetically tetraploid Cyprinidae a distinct plateau separates an intermediate reassociating sequence fraction (about 22% of the genome; with average repetition frequencies between 1,000 and 1,400) from a slow reassociating one (unique DNA; about 72% of the genome). These two genome fractions are not clearly distinguishable from each other in Cot curves of the diploid Cyprinidae, where a similar plateau is not evident. Since simple ploidy changes are not expected to affect DNA reassociation kinetics we suggest a different evolution in the genome organization of the two ploidy groups. Some possible hypotheses are discussed.     

ADAPTATION OF DIPLOID AND TETRAPLOID CHAMERION ANGUSTIFOLIUM TO ELEVATION BUT NOT LOCAL ENVIRONMENT

Polyploid organisms often have different geographic ranges than their diploid relatives. However, it is unclear whether this divergence is maintained by adaptation or results from historical differences in colonization. Here, we conducted a reciprocal transplant experiment with diploid and autotetraploid Chamerion angustifolium to test for adaptation at the ploidy and population level. In the Rocky Mountains, pure diploid populations occur at high elevations and pure autotetraploid populations occur at low elevations with mixed ploidy populations between. We planted 3134 seedlings in 2004 and 3890 juveniles (bolting) in 2005 among nine plots, three in each of the diploid, mixed ploidy, and tetraploid zones, and monitored survival until 2008. For both seedlings and juvenile plants, elevation significantly influenced survival. The juvenile plants also showed a significant ploidy by elevation interaction, indicating that diploids and tetraploids survived best at their native elevations. In contrast, we found no evidence of local adaptation to plot within elevation. This suggests that the current distribution of diploids and tetraploids across elevations is the result of adaptation and that genome duplication may have facilitated the invasion of lower elevation habitats by limiting the movement of maladapted alleles from diploid populations at higher elevations.     

长穗偃麦草中E和E1基因组高分子量麦谷蛋白基因启动子部分序列的进化分析

通过PCR克隆的方法,获得了分别来自二倍体长穗偃麦草的E基因组和四倍体长穗偃麦草的E_1基因组的4个高分子量麦谷蛋白亚基(HMW-GS)基因启动子的部分序列。序列分析表明,它们之间的同源性较高,两个x型亚基启动子序列之间只有1个碱基的差异,而两个y型亚基启动子序列完全相同,x和y型亚基启动子序列之间的长度和部分碱基位点都有差异。推测四倍体长穗偃麦草中的E_1基因组可能起源于二倍体的E基因组。与来自小麦族的A、B、D和G基因组部分亚基基因的启动子序列比较表明,小麦族的这一区域在进化上是相当保守的,不同基因组来源的序列同源性都在90％以上。经过对这些序列的聚类分析,表明长穗偃麦草的y型HMW-GS基因与其他亚基基因的进化关系较远,而x型亚基基因与一个来自小麦1B染色体的亚基基因关系最近。     

Ploidy level and origin of the European invasive weed Senecio inaequidens (Asteraceae)

Native to South-Africa, species of the Senecio inaequidens complex are presently invasive in Europe, Australia and South-America. Previously, different ploidy levels have been found in these different areas, with only tetraploid individuals reported in Europe, and only diploids in South-Africa and Australia. In the present study chromosome counts and flow cytometry were used to survey DNA ploidy levels in a large sample of 66 native and 21 European invasive populations. One Mexican individual was also added to the study. We found only tetraploid individuals occurring in Europe, whereas both ploidy levels, diploid and tetraploid, were found in South-Africa. Moreover, based on genome size, we suggest that two largely allopatric varieties of diploids exist in South-Africa. The Mexican individual was diploid. We suggest that European tetraploid individuals come from South-Africa and hypothesize that a hybridization event between the two DNA types of diploids occurred in the Lesotho area. The taxonomic difficulties surrounding species of theS. inaequidens complex are briefly discussed.     

Molecular evolution of ribosomal intergenic spacers in Odontophrynus americanus 2n and 4n (Amphibia: Anura).

Ribosomal intergenic spacers (IGSs) of Odontophrynus americanus 2n and 4n were cloned, restriction mapped, and partially sequenced. Three distinct regions, namely alpha, beta, and delta, were identified in the IGSs. The alpha and beta regions flanked the 28S and 18S rRNA genes, respectively, conserving an identical restriction pattern at each ploidy level. The delta region, located between alpha and beta, was highly variable in size and restriction pattern, enclosing different BamHI subrepeats (B-SR), 87- to 530-bp-long. Sequence analysis showed that B-SRs were composed mainly of different arrangements of similar blocks of sequences. Another family of repetitive sequences was found in the delta region, clustered inside large BamHI fragments. These subrepeats are 189-bp-long and, although very similar in diploid and tetraploid IGSs, show a pattern of concerted evolution. A hypothetical functional role for the 189-bp repeats is discussed in view of their predicted secondary structure and presence of potential E2 binding sites inside diploid subrepeats. Although the same structural elements were present both in diploid and tetraploid IGSs, the higher level of repeatability of tetraploid IGSs suggests that common ancestor sequences have undergone several rounds of amplification after O. americanus polyploidy.