Origins of domestication and polyploidy in oca (Oxalis Tuberosa: Oxalidaceae). 2. Chloroplast-expressed glutamine synthetase data

In continuing study of the origins of the octoploid tuber crop oca, Oxalis tuberosa Molina, we used phylogenetic analysis of DNA sequences of the chloroplast-active (nuclear encoded) isozyme of glutamine synthetase (ncpGS) from cultivated oca, its allies in the "Oxalis tuberosa alliance," and other Andean Oxalis. Multiple ncpGS sequences found within individuals of both the cultigen and a yet unnamed wild tuber-bearing taxon of Bolivia were separated by molecular cloning, but some cloned sequences appeared to be artifacts of polymerase chain reaction (PCR) recombination and/or Taq error. Nonetheless, three classes of nonrecombinant sequences each joined a different part of the O. tuberosa alliance clade on the ncpGS gene tree. Octoploid oca shares two sequence classes with the Bolivian tuber-bearing taxon (of unknown ploidy level). Fixed heterozygosity of these two sequence classes in all ocas sampled suggests that they represent homeologous loci and that oca is allopolyploid. A third sequence class, found in eight of nine oca plants sampled, might represent a third homeologous locus, suggesting that oca may be autoallopolyploid, and is shared with another wild tuber-bearing species, tetraploid O. picchensis of southern Peru. Thus, ncpGS data identify these two taxa as the best candidates as progenitors of cultivated oca.     

Suitability of AFLP markers for the study of genomic relationships within theOxalis tuberosa alliance

O. tuberosa is an Andean crop that belongs to the worldwide distributed genusOxalis. On the basis of their chromosome numbers the following species (O. herrerae, O. lotoides, O. medicaginea, O. mollissima, O. oblongiformis, O. peduncularis, O. spiralis, O. subintegra, O. tabaconanensis, O. tuberosa, O. villosula) were placed in an alliance. To analyse five species belonging to theOxalis tuberosa alliance (O. oblongiformis, O. peduncularis, O. tabaconanensis, O. tuberosa andO. villosula) and a distant member of the genus (O. articulata), we examined 253 AFLP markers generated after amplification using four primer combinations. Within the alliance, two main clusters were observed, one containing the diploid species and the other group with the polyploid speciesO. tuberosa. All of the primer combinations assayed showed the same clustering pattern. Grouping of accessions of each species by data analysis corresponded largely with their previous taxonomic classifications. The concordance between the clustering of the individuals belonging to different species obtained in this work show the appropriateness of AFLP markers for this type of study. The results obtained are in good agreement with the cytogenetic hypothesis and showed a clustering behaviour, which is similar to the one previously obtained using ITS rDNA nucleotide sequence comparison.     

Biogeography of theOxalis tuberosa alliance

TheOxalis tuberosa alliance is a group of morphologically similarOxalis species allied to the Andean tuber crop oca,O. tuberosa. Originally described by cytologists as a dozen species sharing a base chromosome number rare inOxalis (x = 8), the alliance as defined here includes additional species for which cytological information is not yet available but which are supported as members on molecular and/or morphological grounds. The alliance includes members found in the Andean region from Venezuela to northern Argentina, with one species at high elevations in Central America. They occur from the high Andean steppes (páramo and puna) to the cloud forests of middle elevations and include both restricted endemics and variable widespread species complexes. Geographical and altitudinal distributions of members of the alliance and selectedOxalis species outside the alliance were compared with a combined phylogenetic analysis of DNA sequence data of ITS and ncpGS (chloroplast-expressed glutamine synthetase). Groups within the alliance (i.e., major clades on the molecular trees) occur across widespread, overlapping regions in the Andes, with only partial ecological separation. The hypothesis that theO. tuberosa alliance may have developed in the Andes of southern Peru and northwestern Bolivia and radiated southward and, especially, northward along the Andean axis is suggested by patterns of distributions of members of the alliance and outgroups. In spite of uncertain species delimitations, it is clear that the alliance includes many endemic species and ecotypes that have very restricted distributions. As relatives of the Andean tuber cropOxalis tuberosa, the genetic diversity represented by this geographical variability should be a high priority for conservation.     

Genetic diversity of the Andean tuber-bearing species, oca (Oxalis tuberosa Mol.), investigated by inter-simple sequence repeats.

The Andean tuber-bearing species, Oxalis tuberosa Mol., is a vegetatively propagated crop cultivated in the uplands of the Andes. Its genetic diversity was investigated in the present study using the inter-simple sequence repeat (ISSR) technique. Thirty-two accessions originating from South America (Argentina, Bolivia, Chile, and Peru) and maintained in vitro were chosen to represent the ecogeographic diversity of its cultivation area. Twenty-two primers were tested and 9 were selected according to fingerprinting quality and reproducibility. Genetic diversity analysis was performed with 90 markers. Jaccard's genetic distance between accessions ranged from 0 to 0.49 with an average of 0.28 +/- 0.08 (mean +/- SD). Dendrogram (UPGMA (unweighted pair-group method with arithmetic averaging)) and factorial correspondence analysis (FCA) showed that the genetic structure was influenced by the collection site. The two most distant clusters contained all of the Peruvian accessions, one from Bolivia, none from Argentina or Chile. Analysis by country revealed that Peru presented the greatest genetic distances from the other countries and possessed the highest intra-country genetic distance (0.30 +/- 0.08). This suggests that the Peruvian oca accessions form a distinct genetic group. The relatively low level of genetic diversity in the oca species may be related to its predominating reproduction strategy, i.e., vegetative propagation. The extent and structure of the genetic diversity of the species detailed here should help the establishment of conservation strategies.     

Origins of domestication and polyploidy in oca (Oxalis tuberosa; Oxalidaceae). 3. AFLP data of oca and four wild, tuber-bearing taxa

Many crops are polyploids, and it can be challenging to untangle the often complicated history of their origins of domestication and origins of polyploidy. To complement other studies of the origins of polyploidy of the octoploid tuber crop oca (Oxalis tuberosa) that used DNA sequence data and phylogenetic methods, we here compared AFLP data for oca with four wild, tuber-bearing Oxalis taxa found in different regions of the central Andes. Results confirmed the divergence of two use-categories of cultivated oca that indigenous farmers use for different purposes, suggesting the possibility that they might have had separate origins of domestication. Despite previous results with nuclear-encoded, chloroplast-expressed glutamine synthetase suggesting that O. picchensis might be a progenitor of oca, AFLP data of this species, as well as different populations of wild, tuber-bearing Oxalis found in Lima Department, Peru, were relatively divergent from O. tuberosa. Results from all analytical methods suggested that the unnamed wild, tuber-bearing Oxalis found in Bolivia and O. chicligastensis in NW Argentina are the best candidates as the genome donors for polyploid O. tuberosa, but the results were somewhat equivocal about which of these two taxa is the more strongly supported as oca's progenitor.     

A Flow Cytometric Analysis of the Nuclear 2C DNA Content in 17 Phaseolus Species (53 Genotypes)

The genus Phaseolus is characterized by a highly stable karyotype of 2n = 22. Despite this constancy, the size of the chromosomes varies, and crossing of species is possible only in a few cases. We determined the 2C nuclear DNA content of a number of Phaseolus species, cultivars and genotypes by flow cytometry, in order to realize the interspecific and intraspecific variation of the 2C value. The data range from 1.03 pg to 2.18 pg without any clear correlation to systematic relationships. The mean DNA values of wild and cultivated forms, as well as those of Andean and Mesoamerican genotypes, do not differ significantly. The variation is interpreted in terms of some nucleotypic adaptations. The data may be useful for molecular biological analyses, as well as for biotechnological and classical breeding programmes.     

Flow cytometric measurements do not reveal different ploidy levels in Minthostachys (Lamiaceae)

Ten of the 17 species of the taxonomically difficult Andean mint genus Minthostachys (Lamiaceae) were submitted to flow cytometric measurements of nuclear DNA content to test the hypothesis of the occurrence of different ploidy levels within the genus. Nuclear DNA content was found to vary from 1.643 to 1.775 pg, i.e by only ca. 8% between individual accessions, thus providing no evidence for polyploidy in Minthostachys. While these results do not preclude the possibility that the genus contains polyploid species nor the occurrence of heteroploidy with nearly identical nuclear DNA contents, they suggest that polyploidy did not play a major role in its diversification.     

Genome size variation in Chenopodium quinoa (Chenopodiaceae)

The extent and significance of intraspecific genome size variation were analysed in quinoa (Chenopodium quinoa Willd.), a pseudocereal important for human consumption in the Andean region of South America. Flow cytometry, with propidium iodide as the DNA stain, was used to estimate the genome size of 20 quinoa accessions from Ecuador, Peru, Bolivia, Argentina, Chile and the USA. Limited genome size variation was found among the analysed accessions. The differences between the accessions were statistically significant but the maximum inter-accession difference between the populations with the largest and the smallest genome reached only 5.9%. The largest genome was found in population C4 from Chile (mean 3.077 pg/2C) and the smallest in the Peruvian population P2 (mean 2.905 pg/2C). The variation was not correlated with collection site; however, the quinoa accessions analysed in this study belonged to three distinct geographical groups: northern highland, southern highland and lowland.     

Collapse of morphological species in the wild potato Solanum brevicaule complex (Solanaceae: sect. Petota)

The major cultivated potato, Solanum tuberosum, and six other related cultivated species, are hypothesized to have arisen from a group of weedy relatives indigenous to the central Andes of central Peru, Bolivia, and northern Argentina. A major problem hindering investigations of the origins of the cultivated species has been a continuing debate over the species boundaries of their putative progenitors. This study investigated the morphological phenetic species boundaries of these putative progenitors and five cultivated taxa, here collectively referred to as the Solanum brevicaule complex. Two hundred fifteen accessions of 30 taxa in the S. brevicaule complex and 42 accessions of six taxa outside of the complex were assessed for 53 morphological traits in replicate plots in a common garden, resulting in a total of over 81;t3000 data points. Phenetic analyses of these data are unable to support 30 taxa, suggesting instead a single variable complex at best only weakly divided into three widely intergrading sets of populations: (1) Peruvian and geographically adjacent Bolivian accessions (including wild species and all the cultigens), (2) Bolivian and Argentinian accessions and S. verrucosum from Mexico (including only wild species), and (3) the Bolivian and Argentinian wild species S. oplocense. These and other data suggest that Hawkes's 1990 treatment (The Potato: Evolution, Biodiversity, and Genetic Resources, Smithsonian Institute Press, Washington, DC.) of 232 morphological species is an overestimate for sect. Petota.     

Origins of domestication and polyploidy in oca (Oxalis tuberosa : Oxalidaceae): nrDNA ITS data

As part of a study aimed at elucidating the origins of the octoploid tuber crop "oca," Oxalis tuberosa, DNA sequences of the internal trancribed spacer of nuclear ribosomal DNA (nrDNA ITS) were determined for oca and several wild Oxalis species, mostly from Bolivia. Phylogenetic analysis of these data supports a group of these species as being close relatives of oca, in agreement with morphology and cytology, but at odds with traditional infrageneric taxonomy. Variation in ITS sequences within this group is quite low (0-7 substitutions in the entire ITS region), contrasting with the highly divergent (unalignable in some cases) sequences within the genus overall. Some groups of morphologically differentiated species were found to have identical sequences, notably a group that includes oca, wild populations of Oxalis that bear small tubers, and several other clearly distinct species. The presence of a second, minor sequence type in at least some oca accessions suggests a possible contribution from a second genome donor, also from within this same species group. ITS data lack sufficient variation to elucidate the origins of oca precisely, but have identified a pool of candidate species and so can be used as a tool to screen yet unsampled species for possible progenitors.     

Quantitative nuclear DNA differences associated with genome evolution in Guizotia (Compositae)

The present communication deals with 2C nuclear genome size variation in a fairly small genus Guizotia. Twenty-four accessions belonging to six species, out of seven known, were analysed in order to elucidate the extent of DNA variation both at an intra—as well as interspecific level. At the intraspecific level none of the species exhibited significant differences in their genome size. Between the species, the 2C DNA amounts ranged from 3.61 pg in G. reptans to 11.37 pg in G. zavattarii; over three-fold DNA variation is evident. Apparently these interspecific DNA differences have been achieved independent of the numerical chromosomal change(s), as all the Guizotias share a common chromosome number 2n=2x=30. The cultivated oilseed crop, G. abyssinica (7.57 pg), has accommodated nearly 78% extra DNA in its chromosome complement during the evolutionary time scale of its origin and domestication from the wild progenitor G. schimperi (4.25 pg). The extent of genomic DNA difference(s) between the species has been discussed in the light of their interrelationships and diversity.     

Nuclear and chloroplast DNA differentiation in Andean potatoes.

Over 3500 accessions of Andean landraces have been known in potato, classified into 7 cultivated species ranging from 2x to 5x (Hawkes 1990). Chloroplast DNA (ctDNA), distinguished into T, W, C, S, and A types, showed extensive overlaps in their frequencies among cultivated species and between cultivated and putative ancestral wild species. In this study, 76 accessions of cultivated and 19 accessions of wild species were evaluated for ctDNA types and examined by ctDNA high-resolution markers (ctDNA microsatellites and H3 marker) and nuclear DNA restriction fragment length polymorphisms (RFLPs). ctDNA high-resolution markers identified 25 different ctDNA haplotypes. The S- and A-type ctDNAs were discriminated as unique haplotypes from 12 haplotypes having C-type ctDNA and T-type ctDNA from 10 haplotypes having W-type ctDNA. Differences among ctDNA types were strongly correlated with those of ctDNA high-resolution markers (r = 0.822). Differentiation between W-type ctDNA and C-, S-, and A-type ctDNAs was supported by nDNA RFLPs in most species except for those of recent or immediate hybrid origin. However, differentiation among C-, S-, and A-type ctDNAs was not clearly supported by nDNA RFLPs, suggesting that frequent genetic exchange occurred among them and (or) they shared the same gene pool owing to common ancestry.     

2C DNA variation and relationships among New World species of the genus Lupinus (Fabaceae)

The 2C DNA values in 38 species and accessions of the genus Lupinus (Fabaceae) from the New World have been analysed using flow cytometry. They are representatives of North and South American species (the Atlantic and the Andean regions). Estimated 2C DNA values ranged from 1.08 pg in L. pusillus to 2.68 pg in L. albicaulis (both from North America), that is a variation of more than 2.5-fold. The variation for North American lupins was much higher than that for South American ones. Statistical analysis of the data resulted in a grouping that showed for North American lupins some correlation with the length of life cycle. Discussion concerns some aspects of the evolution of the genus.     

The effects of nuclear DNA content (C-value) on the quality and utility of AFLP fingerprints

BACKGROUND AND AIMS: Nuclear DNA content (C-value) varies approximately 1000-fold across the angiosperms, and this variation has been reported to have an effect on the quality of AFLP fingerprints. Various methods have been proposed for circumventing the problems associated with small and large genomes. Here we investigate the range of nuclear DNA contents across which the standard AFLP protocol can be used. METHODS: AFLP fingerprinting was conducted on an automated platform using the standard protocol (with 3 + 3 selective bases) in which DNA fragments are visualized as bands. Species with nuclear DNA contents ranging from 1C = 0.2 to 32.35 pg were included, and the total number of bands and the number of polymorphic bands were counted. For the species with the smallest C-value (Bixa orellana) and for one of the species with a large C-value (Damasonium alisma), alternative protocols using 2 + 3 and 3 + 4 selective bases, respectively, were also used. KEY RESULTS: Acceptable AFLP traces were obtained using the standard protocol with 1C-values of 0.30-8.43 pg. Below this range, the quality was improved by using 2 + 3 selective bases. Above this range, the traces were generally characterized by a few strongly amplifying bands and noisy baselines. Damasonium alisma, however, gave more even traces, probably due to it being a tetraploid. CONCLUSIONS: We propose that for known polyploids, genome size is a more useful indicator than the 1C-value in deciding which AFLP protocol to use. Thus, knowledge of ploidy (allowing estimation of genome size) and C-value are both important. For small genomes, the number of interpretable bands can be increased by decreasing the number of selective bases. For larger genomes, increasing the number of bases does not necessarily decrease the number of bands as predicted. The presence of a small number of strongly amplifying bands is likely to be linked to the presence of repetitive DNA sequences in high copy number in taxa with large genomes.     

Flow cytometric analysis reveals different nuclear DNA contents in cultivated Fonio (Digitaria spp.) and some wild relatives from West-Africa

Nuclear DNA amounts of 118 cultivated fonio accessions representing 94 landraces collected from the major growing areas of West-Africa (Benin, Burkina Faso, Guinea, Mali and Togo) and eight accessions of four wild relatives were investigated by Laser flow cytometry. In cultivated species, average 2C-values ranged from 1.848 ± 0.031 pg for Digitaria iburua to 1.956 ± 0.004 pg for D. exilis. In D. exilis landraces the chromosome number was determined at 2n = 36. The closely related wild species D. longiflora and D. ternata showed similar 2C DNA contents of 1.869 ± 0.035 pg and 1.775 ± 0.070 pg, respectively. Distinctly larger genomes were identified for more distant species D. lecardii and D. ciliaris with 2.660 ± 0.070 pg and 2.576 ± 0.030 pg per 2C nucleus, respectively. Intra-specific variations were found to be slight and insignificant, suggesting genome size stability mainly within the cultivated gene pool. These results support the distance of cultivated fonio species D. exilis and D. iburua from D. lecardii and D. ciliaris as well as their close relationships with D. longiflora and D. ternata. Relevance of the results for ploidy level considerations in fonio millets is discussed.     

Genome size variation in Corchorus olitorius (Malvaceae s.l.) and its correlation with elevation and phenotypic traits

In this study, we report genome size variations in Corchorus olitorius L. (Malvaceae s.l.), a crop species known for its morphological plasticity and broad geographical distribution, and Corchorus capsularis L., the second widely cultivated species in the genus. Flow cytometric analyses were conducted with several tissues and nuclei isolation buffers using 69 accessions of C. olitorius and 4 accessions of C. capsularis, representing different habitats and geographical origins. The mean 2C nuclear DNA content (± SD) of C. olitorius was estimated to be 0.918 ± 0.011 pg, with a minimum of 0.882 ± 0.004 pg, and a maximum of 0.942 ± 0.004 pg. All studied plant materials were found to be diploid with 2n = 14. The genome size is negatively correlated with days to flowering (r = -0.29, p < 0.05) and positively with seed surface area (r = 0.38, p < 0.05). Moreover, a statistically significant positive correlation was detected between genome size and growing elevation (r = 0.59, p < 0.001) in wild populations. The mean 2C nuclear DNA content of C. capsularis was estimated to be 0.802 ± 0.008 pg. In comparison to other economically important crop species, the genome sizes of C. olitorius and C. capsularis are much smaller, and therewith closer to that of rice. The relatively small genome sizes will be of general advantage for any efforts into genomics or sequencing approaches of these species.     

Origin of chloroplast DNA diversity in the Andean potatoes

Summary Wide chloroplast DNA (ctDNA) diversity has been reported in the Andean cultivated tetraploid potato, Solanum tuberosum ssp. andigena. Andean diploid potatoes were analyzed in this study to elucidate the origin of the diverse ctDNA variation of the cultivated tetraploids. The ctDNA types of 58 cultivated diploid potatoes (S. stenotomum, S. goniocalyx and S. phureja), 35 accessions of S. sparsipilum, a diploid weed species, and 40 accessions of the wild or weed species, S. chacoense, were determined based on ctDNA restriction fragment patterns of BamHI, HindIII and PvuII. Several different ctDNA types were found in the cultivated potatoes as well as in weed and wild potato species; thus, intraspecific ctDNA variation may be common in both wild and cultivated potato species and perhaps in the higher plant kingdom as a whole. The ctDNA variation range of cultivated diploid potatoes was similar to that of the tetraploid potatoes, suggesting that the ctDNA diversity of the tetraploid potato could have been introduced from cultivated diploid potatoes. This provided further evidence that the Andean cultivated tetraploid potato, ssp. andigena, could have arisen many times from the cultivated diploid populations. The diverse but conserved ctDNA variation noted in the Andean potatoes may have occurred in the early stage of species differentiation of South American tuber-bearing Solanums.     

Flow cytometric analysis of nuclear genome of the Ethiopian cereal tef [Eragrostis tef (Zucc.) Trotter]

Flow cytometric analysis of nuclear DNA content was performed by using nuclei isolated from young leaf tissue of tef (Eragrostis tef). The method was very useful for rapid screening of ploidy levels in cultivars and lines of tef representing the phenotypic variability of this species in Ethiopia. The results of the analysis showed that all cultivars were tetraploid. Flow cytometry was also used to determine nuclear DNA content in absolute units (genome size) in four tef cultivars. Nuclei isolated from tomato (Lycopersicon esculentum, 2C=1.96 pg) were used as an internal reference standard. The 2C DNA content of individual tef cultivars ranged from 1.48 to 1.52 pg (1C genome size: 714 Mbp-733 Mbp), the differences among them being statistically nonsignificant. The fact that the nuclear genome of tef is only about 50% larger than that of rice should make it amenable for analysis and mapping at the molecular level.     

Variation in chromosomal DNA associated with the evolution of Arachis species.

The 2C nuclear DNA amounts were determined for 99 accessions, representing 23 Arachis species from 8 of 9 taxonomic sections, and two synthetic amphidiploids. Mean 2C DNA amounts varied by 15.20%, ranging from 10.26 to 11.82 pg, between accessions of Arachis hypogaea (2n = 4x = 40). Nuclear DNA content variation (5.33-5.91 pg) was also detected among Arachis duranensis (2n = 2x = 20) accessions. The intraspecific variation in the two species may have resulted from indirect selection for favourable genome sizes in particular environmental conditions. The accessions belonging to A. hypogaea ssp. hypogaea (mean value 11.27 pg) with longer life cycle had significantly larger mean DNA content than the accessions of A. hypogaea ssp. fastigiata (mean value 10.97 pg). For 20 diploid (2n = 2x = 20) species of the genus, 2C nuclear DNA amounts ranged from approximately 3 to 7 pg. The diploid perennial species of section Arachis have about 12% more DNA than the annual species. Comparisons of DNA amounts show that evolutionary rating is not a reliable guide to DNA amounts in generic sections of the genus; lower DNA values with evolutionary advancement were found in sections Heteranthae and Triseminatae, but the same was not true for sections Arachis and Caulorrhizae. Similarly, there is evidence of significant differences in DNA content between 4 ancient sections (Procumbentes, Erectoides, Rhizomatosae, and Extranervosae) of the genus. The occurrence of genome size plasticity in both A. duranensis and A. hypogaea provides evidence that A. duranensis could be one of the diploid progenitors of A. hypogaea. The DNA content in the two synthetic amphidiploids corresponded to the sum value estimated for parental species. Key words : Arachis species, genome size, Arachis hypogaea, Arachis duranensis, intraspecific variation.     

Nutritional evaluation of three underexploited andean tubers:Oxalis tuberosa (Oxalidaceae),Ullucus tuberosus (Basellaceae), andTropaeolum tuberosum (Tropaeolaceae)

Three species of edible tubers endemic to and domesticated in the Andes were studied for their nutritional value. Collected samples ofOxalis tuberosa, Ullucus tuberosus, andTropaeolum tuberosum show a high amount of variation in both percent protein and quality of essential amino acids. A protein difference of120% is present amongT. tuberosum cultivars and a protein difference of 300% is present among the three species. The data indicate that previous published Andean tuber crop food values may need revision. The introduction of "improved" crop varieties and less nutritious foodstuffs threatens the base ofcultivar diversity that has been selected by Andean agriculturists over centuries. This rapid erosion of Andean tuber diversity indicates the importance of identifying and conserving Andean tuber cul-tivars throughout the Andes.