Octoploid Species Research Articles

Solitary bees – Potential vectors for gene flow from cultivated to wild strawberries

The genus Fragaria (Rosaceae) contains 24 plant species, including hybrid species such as the octoploid garden strawberry (F.×ananassa). Natural hybridization between Fragaria species has repeatedly been reported, and the potential future cultivation of genetically modified strawberries has made the study of hybridization potential between F.×ananassa and its wild relatives increasingly important. In Europe, F.×ananassa is the only octoploid species present, and the most likely candidate for hybridization is the common diploid woodland strawberry (F. vesca). To date, it is unknown whether pollinator spectra of the two Fragaria species overlap and thus might promote interspecific gene flow. We carried out a survey of flower visitors in northwestern Switzerland to identify major flower visitors of F. vesca and F.×ananassa. This survey indicated that wild bees are the most important shared flower visitors of F.×ananassa and F. vesca. Therefore, we studied flower choice behavior of the common wild bee Osmia bicornis in a greenhouse experiment. Osmia bicornis did not discriminate between F.×ananassa and F. vesca flowers. We conclude that wild bees are important shared flower visitors of both F.×ananassa and F. vesca and are potential vectors for gene flow between cultivated and wild strawberries.

Insights into phylogeny, sex function and age of Fragaria based on whole chloroplast genome sequencing

The cultivated strawberry is one of the youngest domesticated plants, developed in France in the 1700s from chance hybridization between two western hemisphere octoploid species. However, little is known about the evolution of the species that gave rise to this important fruit crop. Phylogenetic analysis of chloroplast genome sequences of 21 Fragaria species and subspecies resolves the western North American diploid F. vesca subsp. bracteata as sister to the clade of octoploid/decaploid species. No extant tetraploids or hexaploids are directly involved in the maternal ancestry of the octoploids.There is strong geographic segregation of chloroplast haplotypes in subsp. bracteata, and the gynodioecious Pacific Coast populations are implicated as both the maternal lineage and the source of male-sterility in the octoploid strawberries. Analysis of sexual system evolution in Fragaria provides evidence that the loss of male and female function can follow polyploidization, but does not seem to be associated with loss of self-incompatibility following genome doubling. Character-state mapping provided insight into sexual system evolution and its association with loss of self-incompatibility and genome doubling/merger. Fragaria attained its circumboreal and amphitropical distribution within the past one to four million years and the rise of the octoploid clade is dated at 0.372–2.05 million years ago.

Karyotype analysis using FISH (fluorescence in situ hybridization) in Fragaria

The karyotypes of Fragaria species, diploid (Fragaria vesca ‘UC-01’, ‘Hawaii-30’, Fragaria nilgerrensis ‘Whiteberry’) and octoploid (Fragaria×ananassa ‘Akihime’), were analyzed using a method based on fluorescence in situ hybridization (FISH), using ribosomal DNA probes (5S rDNA and 45S rDNA). All diploid species of F. vesca and F. nilgerrensis had 2n=14 chromosomes, while F.×ananassa, an octoploid species, were 2n=56. The total length of somatic metaphase chromosome was measured as 12.22, 13.61, and 11.19μm for ‘UC-01’, ‘Hawaii-30’, and ‘Whiteberry’, respectively, and ‘Akihime’ measured 94.23μm. The karyotypes were composed of 6 pairs of metacentric and 1 pair of submetacentric in ‘UC-01’ and ‘Whiteberry’, and 5 pairs of metacentric and 2 pairs of submetacentric in ‘Hawaii-30’. 5S rDNA loci were detected in 1 pair from ‘UC-01’, ‘Hawaii-30’ and ‘Whiteberry’, and 45S rDNA loci were detected in 3 pairs from ‘UC-01’ and ‘Hawaii-30’, whereas 45S rDNA loci were detected in 2 pairs from ‘Whiteberry’. In octoploid strawberry ‘Akihime’, 45S rDNA signals were observed at 12 chromosomes, and 5S rDNA signals were observed at 2 chromosomes.

Comparative Genetic Mapping Points to Different Sex Chromosomes in Sibling Species of Wild Strawberry (Fragaria)

Separate sexes have evolved repeatedly from hermaphroditic ancestors in flowering plants, and thus select taxa can provide unparalleled insight into the evolutionary dynamics of sex chromosomes that are thought to be shared by plants and animals alike. Here we ask whether two octoploid sibling species of wild strawberry--one almost exclusively dioecious (males and females), Fragaria chiloensis, and one subdioecious (males, females, and hermaphrodites), F. virginiana--share the same sex-determining chromosome. We created a genetic map of the sex chromosome and its homeologs in F. chiloensis and assessed macrosynteny between it and published maps of the proto-sex chromosome of F. virginiana and the homeologous autosome of hermaphroditic diploid species. Segregation of male and female function in our F. chiloensis mapping population confirmed that linkage and dominance relations are similar to those in F. virginiana. However, identification of the molecular markers most tightly linked to the sex-determining locus in the two octoploid species shows that, in both, this region maps to homeologues of chromosome 6 in diploid congeners, but is located at opposite ends of their respective chromosomes.

Description of a new octoploid frog species (Anura: Pipidae: Xenopus ) from the Democratic Republic of the Congo, with a discussion of the biogeography of African clawed frogs in the Albertine Rift

We describe a new octoploid species of African clawed frog (Xenopus) from the Lendu Plateau in the northern Albertine Rift of eastern Democratic Republic of the Congo. This species is the sister taxon of Xenopus vestitus (another octoploid), but is distinguished by a unique morphology, vocalization and molecular divergence in mitochondrial and autosomal DNA. Using a comprehensive genetic sample, we provide new information on the species ranges and intra-specific diversity of African clawed frogs from the Albertine Rift, including the details of a small range extension for the critically endangered Xenopus itombwensis and previously uncharacterized variation in Xenopus laevis. We also detail a new method for generating cytogenetic preparations in the field that can be stored at room temperature for up to 3 weeks. While extending our understanding of the extant diversity in the Albertine Rift, this new species highlights components of species diversity in ancestral African clawed frogs that are not represented by known extant descendants.

Generation and analysis of ESTs from strawberry (Fragaria xananassa) fruits and evaluation of their utility in genetic and molecular studies.

BackgroundCultivated strawberry is a hybrid octoploid species (Fragaria xananassa Duchesne ex. Rozier) whose fruit is highly appreciated due to its organoleptic properties and health benefits. Despite recent studies on the control of its growth and ripening processes, information about the role played by different hormones on these processes remains elusive. Further advancement of this knowledge is hampered by the limited sequence information on genes from this species, despite the abundant information available on genes from the wild diploid relative Fragaria vesca. However, the diploid species, or one ancestor, only partially contributes to the genome of the cultivated octoploid. We have produced a collection of expressed sequence tags (ESTs) from different cDNA libraries prepared from different fruit parts and developmental stages. The collection has been analysed and the sequence information used to explore the involvement of different hormones in fruit developmental processes, and for the comparison of transcripts in the receptacle of ripe fruits of diploid and octoploid species. The study is particularly important since the commercial fruit is indeed an enlarged flower receptacle with the true fruits, the achenes, on the surface and connected through a network of vascular vessels to the central pith.ResultsWe have sequenced over 4,500 ESTs from Fragaria xananassa, thus doubling the number of ESTs available in the GenBank of this species. We then assembled this information together with that available from F. xananassa resulting a total of 7,096 unigenes. The identification of SSRs and SNPs in many of the ESTs allowed their conversion into functional molecular markers. The availability of libraries prepared from green growing fruits has allowed the cloning of cDNAs encoding for genes of auxin, ethylene and brassinosteroid signalling processes, followed by expression studies in selected fruit parts and developmental stages. In addition, the sequence information generated in the project, jointly with previous information on sequences from both F. xananassa and F. vesca, has allowed designing an oligo-based microarray that has been used to compare the transcriptome of the ripe receptacle of the diploid and octoploid species. Comparison of the transcriptomes, grouping the genes by biological processes, points to differences being quantitative rather than qualitative.ConclusionsThe present study generates essential knowledge and molecular tools that will be useful in improving investigations at the molecular level in cultivated strawberry (F. xananassa). This knowledge is likely to provide useful resources in the ongoing breeding programs. The sequence information has already allowed the development of molecular markers that have been applied to germplasm characterization and could be eventually used in QTL analysis. Massive transcription analysis can be of utility to target specific genes to be further studied, by their involvement in the different plant developmental processes.

Genetic Variance in Cadmium Tolerance and Accumulation in Wheat Materials Differing in Ploidy and Genome at Seedling Stage

AbstractCadmium (Cd) tolerance and accumulation in wheat varieties differing in ploidy and genome were investigated at seedling stage under controlled environmental conditions. The wheat varieties included six diploid, eight tetraploid, seven hexaploid and three octoploid species together with wheat relatives containing different genomes. Parameters of growth and photosynthesis were measured and the ratio of a given parameter under Cd treatment to that of the control was referred to as the Cd‐tolerant index of this parameter. Cd concentrations in shoots and roots, Cd accumulation in shoots (SCA) and Cd translocation (ratio of SCA to total Cd accumulation in plants) of the wheat varieties differing in ploidies generally decreased from octoploid to diploid, except for material containing the CCUU genome. It was found that Cd tolerance of these 24 wheat varieties varied with different ploidy and genomes at seedling stage. B and D genomes played a positive role, whereas the R genome played a negative role in Cd tolerance. By contrast, there were no significant differences between ploidies in Cd tolerance based on the observed growth and photosynthesis parameters. Using principal components analysis and cluster analysis, Triticum boeoticum Boiss was found to be the most Cd‐tolerant variety exhibiting low Cd translocation rates, whereas T. aestivum cv. Huixianhong and Jinghui 1 were identified as the most Cd‐sensitive varieties showing also higher Cd translocation rates.

MITOCHONDRIAL DNA TRANSMISSION, ANCESTRY, AND SEQUENCES IN FRAGARIA

Of the three plant genomic components (nuclear, plastid, and mitochondrial) the mitochondrial DNA (mtDNA) has received the least research attention. We describe a phylogenetically informative sequence in the matR gene region of the Fragaria (strawberry) mtDNA, and the first mtDNA marker developed for this economically important genus. The sequence was obtained from a fosmid genomic library clone of F. vesca subsp. americana 'Pawtuckaway'. New mtDNA PCR primer sets work on members of the Rosaceae family, and have potential for broad applicability in the angiosperms. An informative mtDNA CAPS (cleaved amplified polymorphic site) marker provided evidence that mtDNA was transmitted maternally in two interspecific crosses, and that diploid F. iinumae is the likely mtDNA donor to the octoploid species F. chiloensis and F. virginiana, but not to decaploid F. iturupensis. We expanded the database of Fragaria mtDNA sequence by assembling a 67 kb mtDNA contig from Illumina 36 bp paired-end reads from 'Pawtuckaway', providing a basis to develop additional mtDNA markers.

Karyotype differentiation in three species of Tripogandra Raf. (Commelinaceae) with different ploidy levels

Most species of the genus Tripogandra (Commelinaceae) are taxonomically poorly circumscribed, in spite of having a relatively stable basic number x = 8. Aiming to estimate the cytological variation among Tripogandra species carrying this base number, several structural karyotypic characters were investigated in the diploid T. glandulosa, the hexaploid T. serrulata, and the octoploid T. diuretica. A careful evaluation of chromosome size and morphology did not reveal clear chromosome homeologies among karyotypes. The mean chromosome size was strongly reduced in the octoploid species, but not in the hexaploid species. They also differed largely in the CMA+ banding pattern and in the number of 5S and 45S rDNA sites per monoploid chromosome complement. All three species showed proximal DAPI + heterochromatin, although in T. serrulata this kind of heterochromatin was only visible after FISH. Further, the meiosis in T. serrulata was highly irregular, suggesting that this species has a hybrid origin. The data indicate that, in spite of the conservation of the base number, these species are karyologically quite different from each other.

STRAWBERRY BIOGEOGRAPHY, GENETICS AND SYSTEMATICS

Based on chromosome numbers, branching of stolons, sex expression, pollen morphology and the geographic distribution, the species of the genus Fragaria and their phylogeny are discussed. Two groups can be distinguished. A Eurasian-American group with originally six diploid species, one tetraploid, one hexaploid and all octoploid species and a Southeast-East Asian group with four diploid species which are ancestral to four tetraploid species. A few remaining species can still not be attached to one of these groups.

MORPHOLOGICAL AND POLYPLOIDY VARIATION OF STRAWBERRY PLANTS OBTAINED BY CROSS POLLINATION OF FRAGARIA VESCA AND F. × ANANASSA

For future production in Japan using genetically modified (GM) octoploid strawberry plants, the possibility exists of genetic pollution of wild diploid strawberry plants through spontaneous hybridization with GM plants. The objective of this study was to clarify the possibility of hybridization between an octoploid cultivar and F. vesca, which is the most closely related species to octoploid species. As male and female parents, F. × ananassa cv. 'Sachinoka' (2n=56) and F. vesca cv. 'Baron Solemacher' (2n=14) were used, respectively. In all, 66 seedlings were obtained from 100 seeds. The petiole length, the leaf blade length and width, and the numbers of runners, vegetative buds and inflorescences were recorded. In addition, polyploidy levels of the hybrid plants were analyzed using flow cytometry and chromosome counting. The hybrids were divided into A type (23 plants) and B type (43 plants) according to their morphological characteristics. Results of flow-cytometry analysis and chromosome counting showed that A type was a diploid of F. vesca and that B type was a hybrid of pentaploid. Consequently, we inferred that hybrids that had the genes of octoploid strawberry cultivars appeared at high frequency.

FRAGARIA ITURUPENSIS: A NEW SOURCE FOR STRAWBERRY IMPROVEMENT?

Fragaria iturupensis, a new strawberry species from the island of Iturup, Kurils, was first described by Staudt in 1973 and was recollected in 2005. The value of this only octoploid species occurring outside of the Americas for breeding cultivated strawberries was investigated by cross combination with Fragaria ×ananassa. F 1 and parent species were examined for fertility, resistance to common diseases and aroma volatiles.

Decaploidy in Fragaria iturupensis (Rosaceae)

The strawberry genus, Fragaria (Rosaceae), has a base chromosome number of x = 7. Cultivated strawberries (F. ×ananassa nothosubsp. ananassa) are octoploid (2n = 8x = 56) and first hybridized from F. chiloensis subsp. chiloensis forma chiloensis × F. virginiana subsp. virginiana. Europe has no known native octoploid species, and only one Asian octoploid species has been reported: F. iturupensis, from Iturup Island. Our objective was to examine the chromosomes of F. iturupensis. Ploidy levels of wild strawberry species, include diploid (2n = 2x = 14), tetraploid (2n = 4x = 28), pentaploid (2n = 5x = 35), hexaploid (2n = 6x = 42), octoploid (2n = 8x = 56), and nonaploid (2n = 9x = 63). Artificial triploid (2n = 3x = 21), tetraploid, pentaploid, octoploid, decaploid (2n = 10x = 70), 16-ploid, and 32-ploid plants have been constructed and cultivated. Surprisingly, chromosome counts and flow cytometry revealed that F. iturupensis includes natural decaploid genotypes with 2n = 10x = 70 chromosomes. This report is the first of a naturally occurring decaploid strawberry species. Further research on F. iturupensis and exploration on northern Pacific islands is warranted to ascertain the phylogeny and development of American octoploid species.

A new species of clawed frog (genus Xenopus) from the Itombwe Massif, Democratic Republic of the Congo: implications for DNA barcodes and biodiversity conservation

Here we describe a new octoploid species of clawed frog from the Itombwe Massif of South Kivu Province, Democratic Republic of the Congo. This new species is the sister taxon of Xenopus wittei, but is substantially diverged in morphology, male vocalization, and mitochondrial and autosomal DNA. Analysis of mitochondrial “DNA barcodes” in polyploid clawed frogs demonstrates that they are variable between most species, but also reveals limitations of this type of information for distinguishing closely related species of differing ploidy level. The discovery of this new species highlights the importance of the Itombwe Massif for conservation of African biodiversity south of the Sahara.

Chromosome numbers, nuclear DNA content, and polyploidy in Consolea (Cactaceae), an endemic cactus of the Caribbean Islands

Polyploidy, an important mechanism of plant evolution, was investigated in Consolea, an endemic Caribbean opuntioid genus represented by nine subdioecious species with very narrow distributions, including species classified as rare or threatened. Standard chromosome counting and flow cytometric analyses were used to determine chromosome numbers and ploidy of each taxon. Compared to the base number (x = 11), the mitotic and meiotic counts indicated that there are seven hexaploid (2n = 66) and two octoploid species (2n = 88); no diploids were found. Histograms of intact nuclei confirmed that all species are polyploid, with C-DNA values ranging from 4.88-9.50 pg. The variation of DNA content was significantly higher for the octoploids than for the hexaploids. Male and female sexual morphs had similar DNA content, suggesting that there are no sex chromosomes. Cytomixis between cells and microsporocytes with no chromatin were observed. This provides a mechanism whereby gametes with variable chromosome numbers are produced, influencing reproduction and promoting speciation. In conclusion, C-DNA content and chromosome number separated Consolea species into two groups, which may correspond to two phylogenetic lineages or indicate that polyploidization occurred independently, with comparable effects on C-DNA content.

Assessment of SSR Marker Transfer from the Cultivated Strawberry to Diploid Strawberry Species: Functionality, Linkage Group Assignment, and Use in Diversity Analysis

The cultivated strawberry, Fragaria ×ananassa Duchesne ex Rozier, originated via hybridization between octoploids F. chiloensis (L.) Mill. and F. virginiana Mill. These three octoploid species are thought to share a putative genome composition of AAA`A'BBB`B'. Diploid F. vesca L., is considered to have donated the A genome. Current attention to the development of a diploid model system for strawberry genomics warrants the assessment of simple sequence repeat (SSR) marker transferability between the octoploid and diploid species in Fragaria L. In the present study, 23 SSR primer pairs derived from F. ×ananassa `Earliglow' by genomic library screening were evaluated for their utility in six diploid Fragaria species, including eight representatives of F. vesca, four of F. viridis Weston, and one each of F. nubicola (Hook. f.) Lindl. ex Lacaita, F. mandshurica Staudt, F. iinumae Makino, and F. nilgerrensis Schltdl. ex J. Gay. SSR primer pair functionality, as measured by amplification success rate (= 100% - failure rate) in each species, was ranked (from highest to lowest) as follows: F. vesca (98.4%) > F. iinumae (93.8%) = F. nubicola (93.8%) > F. mandshurica (87.5%) > F. nilgerrensis (75%) > F. viridis (73.4%). The extent to which these octoploid-derived SSR primer pairs generated markers that could be added to the F. vesca linkage map also was assessed. Of the 13 F. ×ananassa SSR markers that segregated codominantly in the F. vesca mapping population, 11 were assigned to linkage groups based upon close linkages to previously mapped loci. These markers were distributed over six of the seven F. vesca linkage groups, and can serve as anchor loci defining these six groups for purposes of comparative mapping between F. vesca and F. ×ananassa.

Karyotype analysis and chromosome evolution in southernmost South American species of Crotalaria (Leguminosae)

Karyotypes of 18 populations belonging to eight of the southernmost species of South American Crotalaria were examined. All had x = 8, except C. incana which had x = 7. Diploid, tetraploid and octoploid species were found. The octoploid is a new record and is the highest ploidy level detected so far in the genus. Species within the same ploidy level could be distinguished by quantitative karyotype parameters and by the size and position of the satellites. From an evolutionary point of view, our results support a decrease of the basic chromosome number by dysploidy. Polyploidy is one of the more important mechanisms of chromosome change, even though some structural rearrangements may explain variations of the karyotype formula. Gigas effects, due to polyploidy, were evident in the length of the stomatal guard cells and in pollen size, but were less clear in seed weight. Nucleotype effects, caused by the DNA quantity in a nucleus, were also evident for pollen and stomatal guard cell size and seed weight, although only in diploids with x = 8 for the latter character. Chromosome data are further discussed in the light of the infrageneric systematic arrangement.

Collection and Preliminary Evaluation of Turkish Strawberry Germplasm

Fragaria species are in different plodiy levels (from 2n = 2x = 14 to 2n = 8x = 56) and distributed in almost all parts of the arable areas of the world. The flora of Turkey has wild strawberries, some of which are harvested for their small, but very aromatic berries. There are also old cultivars found in Turkey that are known for their aromatic fruits. We made collection trips to the Marmara and Black Sea areas to collect both wild strawberries and old cultivars in Summer 2004. During these trips, we sampled 50 populations from the altitudes of 6 to 2007 m, lat. 35°91'N to 41°76'N latitudes, and long. 26°81'E to 42°65'E. The samples were propagated in a greenhouse and evaluated in a replicated trail for both taxonomic and horticultural traits in an unheated greenhouse. The chromosome numbers of the genotypes were also determined. The results indicate that Turkish strawberry germplasm consists of both diploid and octoploid species. The principal components and cluster analysis separated the genotypes into three groups: octoploid cultivars and/or derivatives (e.g., `Madame Moutot'), and two diploid Fragaria species, vesca and viridis. Further relationships of the genotypes will be discussed.

Iturup and Sakhalin Island Strawberries

A plant-collecting expedition to Iturup and Sakhalin Islands, Russian Federation, occurred between 21 July and 12 Sept. 2003. Actinidia, Rubus, Ribes, and Vaccinium, as well as seven accessions of strawberries, Fragaria L., were collected. Among them, a wild strawberry, Fragaria iturupensis Staudt, was collected on the eastern slope of Atsonupuri Volcano, Iturup Island, at 630 and 650 m elevation. This species was similar in habit, leaf color and overall appearance to the American strawberry species F. virginiana Mill. Fruits were bright red, oblate spherical, about 1.4 cm in diameter, and had exerted achenes. The native distribution of this species was limited to the middle elevation of the slope of this volcano, and only on Iturup Island. Chromosome counts indicated that these plants were octoploid. Initial DNA fingerprinting aligned this sample with other octoploid species. In addition to this species, samples of F. yezoensis H. Hara (syn. F. nipponica Makino) were collected on Kuibyshewskii Bay of Cape Otlivnoy, Iturup, and near Ujno-Sakhalinsk City, Sakhalin Island. F. iinumae Makino was observed in Ogon'ki Village, Sakhalin Island. This report confirmed the existence of F. iturupensis, the only known native Asian octoploid strawberry, and documented its limited range. Seeds of these strawberry species are available for research by request from the U.S. Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository in Corvallis.

Inheritance of Day-neutrality in Octoploid Species of Fragaria

The inheritance of day-neutrality in octoploid Fragaria L. was investigated in crosses between day-neutral (DN) × short day (SD) and DN × DN types using F. ×ananassa Duchesne in Lamarck cultivars and elite selections of F. virginiana Miller and F. chiloensis (L.) Miller. Genotypes were considered as DN if they flowered under both the SDs of spring before 30 May (<14 hours) and the long days of summer after 24 July (>15 hours). Wide ranges in the percentage of DN progeny were found among the families regardless of species background (30% to 87% in DN × SD and 22% to 93% in DN × DN crosses). None of the families fit the segregation ratios expected if DN was regulated by recessive alleles at one locus, and only about half of the families fit the segregation ratios expected if a single dominant allele regulated DN. Several two-gene models fit the segregation data better than the single locus ones, but none of the genetic models tested fit the DN segregation ratios at the ends of the distribution range. The wide range observed in the percentage of DN progeny across all the families is most consistent with a polygenic model. Several other kinds of observations supported the multigenic regulation of DN: 1) Different DN parents crossed to the same SD genotype often produced different percentages of DN progeny, 2) Some of the day-neutrality sources were more powerful than others in producing of DNs, and 3) None of the DN parents produced 100% DN progeny, which would be expected if there were homozygous dominant DN individuals. Specific combining abilities for DN and flowering strength were significant, while general combining abilities for these traits were not. Our results suggest that parental combinations can be selected that will generate very high proportions of DN progeny that bloom for long periods of time.