Genome relationships among Lotus species based on random amplified polymorphic DNA (RAPD)

The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lotus was evaluated for several geographically dispersed accessions of four diploid Lotus species, L. tennis Waldst. et Kit, L. alpinus Schleich., L. japonicus (Regel) Larsen, and L. uliginosus Schkuhr and for the tetraploid L. corniculatus L., in order to ascertain whether RAPD data could offer additional evidence concerning the origin of the tetraploid L. corniculatus. Clear bands and several polymorphisms were obtained for 20 primers used for each species/accession. The evolutionary pathways among the species/accessions presented in a cladogram were expressed in terms of treelengths giving the most parsimonious reconstructions. Accessions within the same species grouped closely together. It is considered that L. uliginosus which is most distantly related to L. corniculatus, may be excluded as a direct progenitor of L. corniculatus, confirming previous results from isoenzyme studies. Lotus alpinus is grouped with accessions of L. corniculatus, which differs from previous studies. With this exception, these findings are in agreement with previous experimental studies in the L. corniculatus group. The value of the RAPD data to theories on the origin of L. corniculatus is discussed.     

The origin of Lotus corniculatus

Summary Earlier students of the origin of Lotus corniculatus suggested that this tetraploid species arose as an autotetraploid of the closely related diploid species L. tenuis or L. alpinus. More recent studies suggested that L. alpinus and L. japonicus could be ancestral forms. The present study of tannin content, phenolic content, cyanide production, morphology, cytogenetics, Rhizobium specificity and self-incompatibility in the corniculatus group virtually excludes the possibility that L. corniculatus could have arisen through autopolyploidy of L. tenuis or L. alpinus, and suggests that L. corniculatus arose through hybridization of L. alpinus and/or L. tenuis (probably as female parent) with L. uliginosus (probably as male parent), followed by chromosome doubling in the hybrid.     

An isoenzyme study in the genus Lotus (Fabaceae). Experimental protocols and genetic basis of electrophoretic phenotype

Summary An isoenzyme survey of some taxa in the genus Lotus (Fabaceae) was undertaken to increase the number of genetic markers available to breeders and to students of Lotus phylogeny. Twenty-one enzymes were examined using starch gel electrophoresis and nine buffer systems. Clear, consistent banding patterns were obtained for PGI, TPI, MDH, IDH (NADP), PGM, 6-PGDH, and ME. Clear but inconsistent banding patterns were obtained for FDP, G₃PDH (NADP), -EST, LAP, MDH, DIA, and NADHDH. Phenotypes of the seven consistently resolved enzyme systems were obtained for different tissues for each of several genotypes at different stages of development. Variation in enzyme phenotypes of the same individuals under different growth conditions indicated the presence of different isozymic forms of these enzymes. Shoot tissue of plants over 6 weeks of age was found to be suitable material for further genetic studies, since phenotype for this tissue was constant despite changes in growing conditions. A formal genetic analysis of segregation and/or recombination of allozymes for the enzymes PGM, TPI, MDH, IDH, and 6-PGDH was undertaken. Isoenzyme phenotypes were examined for the diploids L. alpinus Schleich., L. burttii Sz. Borsos, L. conimbricensis Brot., L. ornithopodioides L., L. tennis Waldst. et Kit., and L. uliginosus Schkuhr; and for the diploid interspecific hybrids L. alpinus x L. conimbricensis, L. burttii x L. ornithopodioides, and L. japonicus x L. alpinus. Several new loci were identified for Lotus, namely, Idh1, Idh2, Mdh3, Pgi1, Pgi2, Tpi1, Tpi2, and 6-Pdgh1. Duplications of loci of IDH, MDH, PGI, and 6-PGDH were detected in the diploid (2n=12) interspecific hybrid L. japonicus x L. alpinus.     

An isoenzyme study in the genus Lotus (Fabaceae)

Summary Segregation of the cytosolic Pgi2 locus was studied among progeny of the synthetic allotetraploid (L. japonicus × L. alpinus)², the synthetic autotetraploid (L. alpinus)², and the cultivated tetraploid species L. corniculatus L. Evidence of an original diploid duplication found within the interspecific hybrid L. japonicus × L. alpinus was also found within the synthetic allotetraploid (quadruplication of loci). Evidence suggesting quadruplication of loci was also found in the tetraploid L. corniculatus, but not in the synthetic autotetraploid (L. alpinus)². It is suggested that the original duplication resulted from unequal crossing-over between homoeologues and that it provides evidence that L. corniculatus is a segmental allotetraploid. Quadruplication of loci in L. corniculatus could explain previously reported distorted tetrasomic ratios for segregation of qualitative characters in this species.     

Evidence for the existence of 2n gametes in Lotus tenuis Wald. et Kit. (2n=2x=12): their relevance in evolution and breeding of Lotus corniculatus L. (2n=4x=24)

Summary Crosses between male sterile L. corniculatus (2n=4x=24) and L. tenuis (2n=2x=12) plants were performed in order to verify the presence of 2n gametes in L. tenuis. All but one of the plants from these crosses had 2n=4x=24 and the L. corniculatus phenotype; this plant had 2n=2x=12 and the L. tenuis phenotype. The plants also showed good quantity of pollen at tripping, good pollen fertility and good percentage of seed setting in the backcross to L. corniculatus. On the whole, both cytological and morphological observations, showing that all but one of the plants from L. corniculatus x L. tenuis were normal tetraploids, suggest the existence of diploandrous gametes in L. tenuis. On the other hand, haploid parthenogenesis probably gave origin to the dihaploid plant 2n=2x=12.     

Chloroplast-DNA variation in the genus Lotus (Fabaceae) and further evidence regarding the maternal parentage of Lotus corniculatus L.

 To resolve the maternal parentage of the tetraploid Lotus corniculatus, restriction-site variation of chloroplast DNA (cpDNA) was studied in several accessions of that species, in the four putative parental diploid species, L. tenuis, L. alpinus, L. japonicus and L. uliginosus, and in four phylogenetically more distant diploid species, L. hispidus, L. edulis, L. ornithopodoides and Tetragonolobus maritimus var. siliquosus. Evidence of cpDNA maternal inheritance was obtained by using reciprocal controlled crosses between plants of L. corniculatus and natural tetraploid individuals of L. alpinus showing very distinct restriction patterns. Interspecific cpDNA variation in the eight Lotus species and T. siliquosus was analysed by comparing cpDNA fragment patterns produced by five restriction endonucleases and totalling 304 distinct fragments. Genetic differentiation in cpDNA was very high between the L. corniculatus group and L. hispidus on the one hand, and the three other species on the other hand. Sixteen restriction-site mutations and eight length polymorphisms were identified among the five species of the L. corniculatus group and L. hispidus, Lotus uliginosus, L. alpinus and L. japonicus showed at least six DNA changes with regard to the molecule of L. corniculatus. Accordingly, these species should be excluded as maternal progenitors of L. corniculatus. Conversely, the cpDNA of L. tenuis differed from that of L. corniculatus by only two small-length mutations. As also suggested previously from an analysis of several nuclear markers, the results reported here show decisively that L. tenuis may be considered as the most probable maternal ancestor of L. corniculatus. Received: 23 February 1997/Accepted: 28 February 1997     

Effect of oxygen availability on nitrogen fixation by two Lotus species under flooded conditions

The pasture legumes Lotus uliginosus (Schk.) and Lotus corniculatus (L.), known to differ in their tolerance to flooding, were inoculated with Rhizobium loti and flooded for 60 d while subjected to two levels of dissolved pO2: 0.241 and 0.094 mol ml^-1. L. uliginosus showed significantly greater growth (shoot and root) and N2 fixation under both pO2s, compared to L. corniculatus, although growth and N2 fixation by L. corniculatus was not affected by the low pO2. Surprisingly, in L. uliginosus., growth, nodulation and N2 fixation were all increased by low pO2 while nodulation of L. corniculatus where low pO2 plants showed a significant increase over that of the higher pO2 plants while L. uliginosus plants showed a decline. Root porosity of L. uliginosus doubled in the low pO2-treatment from a mean of 14.5% in high pO2 roots to 28.5%, whereas that of L. corniculatus was relatively unaffected by pO2, being 7% and 9% for high and low pO2 plants, respectively. The structure of nodules differed little between species and treatments, although nodules/nodulated roots from the L. uliginosus plants had particularly profuse lenticels and aerenchyma. However, L. corniculatus nodules, especially those grown in the lower pO2 showed signs of early senescence with vacuolation of infected cells and green coloration when cut open. Leghaemoglobin (Lb) concentrations in nodules from both species were unaffected by low pO2, although that of L. corniculatus nodules, regardless of pO2, was significantly greater than L. uliginosus. Concentrations of the intercellular glycoprotein recognized by the monoclonal antibody MAC265 were significantly reduced in nodules from the low pO2 treatment in both species. Immunogold labelling showed that the MAC265 antigen was localized primarily within intercellular spaces within nodule cortices from both Lotus species. A marked decrease in deposition of the MAC265 antigen within the cortices of L. uliginosus nodules grown in the lower pO2, is discussed in terms of the relative abilities of the two Lotus spp. to maintain an O2 supply to the N2-fixing bacteroids within submerged nodules.Keywords: Lotus uliginosus, Lotus corniculatus, N2 fixation, flooding, oxygen.      

Growth and ion relations in response to combined salinity and waterlogging in the perennial forage legumes Lotus corniculatus and Lotus tenuis

Lotus tenuis (Wadst. & Kit.) is a perennial legume widely grown for pasture in the flood-prone and salt affected Pampa region of Argentina. The physiology of salt and waterlogging tolerance in L. tenuis (four cultivars) was evaluated, and compared with Lotus corniculatus (three cultivars); the most widely cultivated Lotus species. Overall, L. tenuis cultivars accumulated less Na⁺ and Cl⁻, and more K⁺ in shoots than L. corniculatus cultivars, when exposed to 200 mM NaCl for 28 days in aerated or in stagnant solutions. Root porosity was higher in L. tenuis cultivars due to greater aerenchyma formation. In a NaCl dose–response experiment (0–400 mM NaCl in aerated solution), L. tenuis (cv. Chaja) accumulated half as much Cl⁻ in its shoots than L. corniculatus (cv. San Gabriel) at all external NaCl concentrations, and about 30% less shoot Na⁺ in treatments above 250 mM NaCl. Ion distributions in shoots were determined for plants at 200 mM NaCl. L. tenuis (cv. Chaja) again accumulated about half as much Cl⁻ in old leaves, young leaves and stems, compared with concentrations in L. corniculatus (cv. San Gabriel). There were not, however, significant differences between the two species for Na⁺ concentrations in the various shoot tissues. The higher root porosity, and maintenance of lower shoot Cl⁻ and Na⁺ concentrations in L. tenuis, compared with L. corniculatus, contributes to the greater tolerance to combined salt and waterlogging stress in L. tenuis. Moreover, significant variation for tolerance to combined salinity and waterlogging stress was identified within both L. tenuis and L. corniculatus.     

Chemical defense production in Lotus corniculatus L. I. The effects of nitrogen source on growth,reproduction and defense

Summary The carbon to nitrogen balance theory was examined for a legume, Lotus corniculatus L., which allocates carbon to nitrogen fixation. N-fixation can influence the ratio of carbon to nitrogen in legumes by providing nitrogen in nutrient-poor habitats, and by consuming carbon for support of symbiotic N-fixation. L. corniculatus clones (genotypes) were grown under two levels of nitrogen fertilization: a treatment which suppressed nodulation with fertilization and a treatment which received no additional fertilization. These plants relied solely on symbiotic N-fixation. Plants which supported symbionts had lower biomass and lower tannin concentrations than fertilized plants; this appears to be a result of the large carbon demand on N-fixation. Plants supporting symbionts often had relatively lower protein concentrations than fertilized plants. Cyanide concentration was influenced by plant genotype but not by nitrogen source. Although symbiotic N-fixing plants were smaller, they had three times the reproductive output of fertilized plants.     

Stability of CaMV 35S-gus gene expression in (bird's foot trefoil) hairy root cultures under different growth conditions

Lotus corniculatus is an agronomically important forage legume. Genetic engineering offers opportunities both to improveL. corniculatus as a crop and to increase basic understanding of plant biochemistry and metabolism. Biosynthesis of secondary products and nitrogen fixation are two areas in which gene expression has been studied using hairy root cultures ofL. corniculatus. The stability of foreign gene expression in these cultures is critically important. TwoL. corniculatus root culture lines containing a reporter gene (CaMV 35S-gus) were used to investigate the stability of expression of a foreign gene under a range of conditions likely to be encountered in experiments. The hairy root culture lines were grown under varying conditions of light, temperature, nutrient supply, and in the presence of the auxin 2,4-D, or the elicitor glutathione. Expression of thegus gene, detected by measuring GUS activity, was found to be relatively stable under all of the conditions investigated.     

Electrophoretic identification of polyploidCamellia japonica (Theaceae) cultivars and evidence for their sexual origin

Electrophoretic examination of allozymes from 189Camellia japonica cultivars revealed some banding patterns not explainable by codominant diploid genetics. At several loci encoding dimeric enzymes, 5 and 6 banded patterns were observed in 7 cultivars. These patterns are interpreted as resulting from triploidy or aneuploidy, where three variant alleles code for products which are electrophoretically distinguishable and associate to form three homodimers and three heterodimers. The presence of allozyme multiplicity in these clones suggests a sexual rather than a somatic mode of triploid origin.     

Inheritance,intracellular localization,and genetic variation of phosphoglucomutase isozymes in maize (Zea mays L.)

Phosphoglucomutase (PGM; EC 2.7.5.1) isozyme variants were studied in a large number of inbred lines, crosses, and races of maize (Zea mays L.). Patterns of Mendelian inheritance demonstrated for PGM isozyme variants indicated that they are encoded by nuclear genes. Two unlinked loci, Pgm1 and Pgm2, located on the long arm of chromosome 1 and the short arm of chromosome 5, respectively, specify the observed electrophoretic variation on starch gels. No intra- or interlocus hybrid bands were found, suggesting that each isozyme band consists of a single polypeptide. PGM isozymes were present in all plant parts studied and the activity specified by both loci appears to reside in the cytoplasm. In studies of 520 racial collections of maize from Latin America, a single allele at each locus predominated in most collections. Likewise, the same alleles predominated in a set of 406 inbred lines of maize from the United States and Canada.This work was supported in part by NIH Research Grant GM 11546.Paper No. 8496 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina.     

Species relationships and genetic variation in the diploid wheats (Triticum,Aegilops) as revealed by starch gel electrophoresis

Twenty enzyme loci were examined in the diploid species ofTriticum andAegilops for allelic variation by starch gel electrophoresis. SectionSitopsis, including the five species,Ae. speltoides, Ae. lingissima, Ae. sharonensis, Ae. bicornis andAe. searsii form a close subgroup withAe. speltoides slightly removed from the others.T. monococcum s. lat., was found to be closest to the species of theSitopsis group.Ae. comosa, Ae. umbellulata andAe. uniaristata form a second subgroup withAe. caudata most closely related to these species.Ae. squarrosa appears almost equally related to all of the species, showing no special affinity for any one species group. Nineteen out of twenty loci examined were polymorphic with a mean of 6.7 alleles per locus. Species could be, for most loci, characterized by the presence of predominant alleles. A conspicious genetic characteristic ofTriticum-Aegilops is the sharing of these predominant alleles between species. Within species variation is characterized by a diffuse distribution of secondary alleles.     

In vitro propagation ofMedicago andLotus species by node culture

Summary Nodes ofMedicago sativa, Lotus corniculatus, Lotus tenuis, andLotus pedunculatus were cultured on MS basal media with different growth regulators. InM. sativa each node produced one shoot and the apical dominance was unaffected by high levels of cytokinins, and subsequent cycles of culture. Shoot development was stimulated by the presence ofN ⁶-isopentenyl-adenine in the culture medium and was dependent on the genotype of the explant. Shoot development was not affected by the original position of the node on the plant nor by the plant age. Shoots rooted in MS medium gelled with starch and containing 2 mg·liter⁻¹ indol-3-acetic acid. In the threeLotus species, node culture was a more effective technique than inM. sativa. The number of shoots per node increased with the time of culture and with the presence of 0.05 mg·liter⁻¹ of 6-benzylaminopurine. The highest number of shoots derived from one node was achieved inL. pedunculatus and inL. tenuis by culturing single nodes, whereas inL. corniculatus stem segments had to be totally covered by the medium for success. Rooting was easily achieved in MS medium with or without auxins.     

Independent origins of tetraploid cryptic species in the fern <Emphasis Type="Italic">Ceratopteris thalictroides</Emphasis>

Ceratopteris thalictroides (L.) Brongn is a tetraploid fern species that contains at least three cryptic species, the south, the north and the third type. In this study we combined data from both chloroplast DNA (cpDNA) and nuclear DNA sequences of three diploid species and three cryptic species of C. thalictroides to unravel the origin of the cryptic species, particularly of the reticulate relationships among the diploid and tetraploid taxa in the genus Ceratopteris. Of the three diploid species examined, C. cornuta had cpDNA identical to that of the tetraploid third type plants, and this diploid species is a possible maternal ancestor of the tetraploid third type. Analysis of the homologue of the Arabidopsis thaliana LEAFY gene (CLFY1) identified ten alleles in the genus Ceratopteris, with six alleles found in C. thalictroides. The unrooted tree of the CLFY1 gene revealed four clusters. Each cryptic species showed fixed heterozygosity at the CLFY1 locus and had two alleles from different clusters of the CLFY1 tree. Consideration of the cpDNA sequences, CLFY1 genotypes of the cryptic species and CLFY1 gene tree in concert suggested that the cryptic species of C. thalictroides had originated through independent allopolyploidization events involving C. cornuta and two unknown hypothetical diploid species.     

Inheritance and mapping of isoenzymes in pea (Pisum sativum L.)

Summary Three isoenzyme systems (amylase, esterase and glutamate oxaloacetate transaminase) were examined in seeds of pea (Pisum sativum L.) and shown to give clear variation in their band patterns on gel electrophoresis between different lines. The inheritance of these isoenzyme systems, and the location of their genes on the pea genome was investigated. Reciprocal crosses were made between lines, F2 seeds were analysed for segregation in the band patterns of the isoenzymes, and F2 plants were investigated to find linkage between the genes for these isoenzymes and genes for selected morphological markers. The results obtained showed that each of the investigated isoenzyme systems is genetically controlled by co-dominant alleles at a single locus. The gene for amylase was found to be on chromosome 2, linked to the loci k and wb (wb ... 9 ... k ... 25 ... Amy). The gene for esterase was found to be linked with the gene Br (chromosome 4) but the exact location is uncertain because of the lack of the morphological markers involved in the cross. The gene for glutamate oxaloacetate transaminase was found to be on chromosome 1 and linked with the loci a and d (a... 24... Got... 41 ... d).     

Allozyme variation in Japanese species ofChionographis (Liliaceae)

Allozyme variation was examined in three diploid taxaChionographis japonica var.japonica, var.kurokamiana, andC. koidzumiana and three tetraploid taxaC. japonica var.kurohimensis, ssp.hisauchiana, and ssp.minoensis. Results show thatC. japonica var.kurokamiana is genetically closer toC. koidzumiana than to var.japonica. In the tetraploid taxa, fixed heterozygosities were found at several loci, and this supports the hypothesis that these taxa are allotetraploids. Furthermore, the tetraploid taxa have many unique alleles not found in the diploid taxa. This suggests that sufficient time has passed since the origin of tetraploids for new mutations to have been fixed.     

Investigations of the host feeding preferences ofSitona weevils found commonly on white clover (Trifolium repens) in the UK

A series of laboratory and field studies were done to evaluate a range of leguminous plant species for their feeding potential by adult weevils of the genusSitona Germar. (Coleoptera: Curculionidae). Three species ofSitona, S. lineatus L.,S. flavescens Marsh. andS. hispidulus F. all of which are found commonly on white clover (Trifolium repens L.) in the UK were offered a range of 11 legume species,T. repens (white clover, cv. Olwen),T. pratense L. (red clover, cv. Marcom),T. fragiferum L. (strawberry clover, cv. Palestine),T. hybridum L. (hybrid clover, cv. Tetra),T. incarnatum L (crimson clover),T. dubium Sibth. (lesser yellow trefoil),Lotus corniculatus L. (birdsfoot-trefoil, cv. Leo),L. uliginosus Schkuhr. (large birdsfoot-trefoil),Melilotus alba Desr. (white melilot),Medicago sativa L. (lucerne, cv. Europe) andM. lupulina L. (black medick) in two laboratory experiments. The weevils were offered a choice of these legumes in one experiment whilst in the other they did not have a choice of food material. These legumes were also sown in the field and a number of measurements of damage, together with counts ofSitona spp., were made. In the laboratoryS. lineatus andS. hispidulus favoured some of the legumes to a greater or lesser extent than white clover.S. flavescens was more restricted in its feeding than the other two weevil species. In the field studyS. lineatus invaded the experimental area quickly and tended to favourMedicago spp. andMelilotus spp. Later in the yearS. flavescens dominated the sitona fauna on the experiment, with the exception of aggregations ofS. lineatus onM. sativa andM. alba. In a separate screen of 5 varieties of white clover (cvs Donna, Menna, Kersey, Olwen and Grasslands Huia), cv. Olwen appeared to be the most susceptible to sitona attack.     

Analysis of the Brassica oleracea genome by the generation of B. campestris-oleracea chromosome addition lines: characterization by isozymes and rDNA genes

Summary This study aimed at generating chromosome addition lines and disclosing genome specific markers in Brassica. These stocks will be used to study genome evolution in Brassica oleracea L., B. campestris L. and the derived amphidiploid species B. napus L. B. campestris-oleracea monosomic and disomic chromosome addition plants were generated by crossing and backcrossing the natural amphidiploid B. napus to the diploid parental species B. campestris. The pollen viability of the derived sesquidiploid and hyperploid ranged from 63% to 88%, while the monosomic and disomic addition plants had an average pollen fertility of 94% and 91%, respectively. The addition lines were genetically characterized by genome specific markers. The isozymes for 6PGD, LAP, PGI and PGM, and rDNA Eco RI restriction fragments were found to possess the desired genome specificity. Duplicated loci for several of these markers were observed in B. campestris and B. oleracea, supporting the hypothesis that these diploid species are actually secondary polyploids. A total of eight monosomic and eight disomic addition plants were identified and characterized on the basis of these markers. Another 51 plants remained uncharacterized due to the lack of additional markers. rDNA genes were found to be distributed in more than one chromosome, differing in its restriction sites. Intergenomic recombination for some of the markers was detected at frequencies between 6% and 20%, revealing the feasibility of intergenomic gene transfer.     

High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae)

The objectives of the present study were to develop microsatellite markers for the wild strawberry, Fragaria virginiana, to evaluate segregation patterns of microsatellite alleles in this octoploid species, and assess genetic variability at microsatellite loci in a wild population. A genomic library was screened for microsatellite repeats and several PCR primers were designed and tested. We also tested the use of heterologous primers and found that F. virginiana primers amplified products in cultivated strawberry, Fragaria × ananassa Duch. and Fragaria chiloensis. Similarly, microsatellite loci developed from cultivated strawberry also successfully amplified F. virginiana loci. We investigated four microsatellite loci in detail, three developed from F. virginiana and one from cultivated strawberry. A survey of 100 individuals from a population of F. virginiana in Pennsylvania demonstrated high heterozygosities (H_e or gene diversity ranged from 0.80 to 0.88 per locus) and allelic diversity (12–17 alleles per locus), but individual plants had no more than two alleles per locus. Segregation patterns in parents and progeny of two controlled crosses at these four loci were consistent with disomic Mendelian inheritance. Together these findings suggest that the genome of F. virginiana is "highly diploidized" and at least a subset of microsatellite loci can be treated as codominant, diploid markers. Significant heterozygote deficiencies were found at three of the four loci for hermaphroditic individuals but for only one locus among females in this gynodioecious species.Communicated by J. Dvorak