Taxonomic evaluations of the genusPinus (Pinaceae) based on electrophoretic data of salt soluble and insoluble seed storage proteins

Electrophoretic studies on seed storage proteins of various gymnosperm species showed that both salt soluble and insoluble protein fractions give taxonomic information. Among species of the genusPinus evident differences are found between the subgeneraHaploxylon (Strobus) andDiploxylon (Pinus). In sect.Strobus the two subsectt.Strobi andCembrae are readily distinguished fromP. bungeana (sect.Parrya) which appears to fall in an intermediate position between haploxyl and diploxyl pines. Among the species of subg.Diploxylon, mountain pines show very similar protein patterns in agreement with their recent speciation (Quaternary).     

Structure and organization of the 5S rRNA genes (5S DNA) inPinus radiata (Pinaceae)

A 5S rRNA gene (5S DNA) from the coniferPinus radiata D. Don has been cloned and characterized at the nucleotide, genomic and chromosomal levels. Sequencing revealed a repeat unit of 524 base pairs which is present in approximately 3000 copies per diploid genome. Two-dimensional gel electrophoresis indicated that these copies are organized in tandem arrays of various length. Using in situ hybridization techniques, the tandem arrays appear to be present on all of the chromosomes. This complexity of chromosomal organization contrasts markedly with the few sites of uniform length found in angiosperm plants such as wheat, pea, and maize.     

Chloroplast DNA-based phylogeny of AsianPinus species (Pinaceae)

The genusPinus includes over 90 species with approximately 24 species native to Asia. We have analyzed the chloroplast (cp) DNA variation of 18Pinus species, including 15 Asian, two Eurasian, and one European species using seven restriction enzymes and ten non-overlapping probes and inferred their phylogenetic relationships. Results of phenetic and cladistic approaches to phylogeny reconstruction were largely in agreement, suggesting two major lineages within the genus and confirmed the ancient character of haploxylon and diploxylon subgenera. Species from sectionParrya appear to have diverged earliest from the hypothesized phylogenetic centre for the haploxylon pines, withP. bungeana andP. gerardiana forming two basal, monotypic lineages. The range of estimated pairwise nucleotide substitutions per site ( ) was higher among haploxylon pines than among diploxylon species. CpDNA divergence was found to be low within the sectionSylvestres, relative to the divergence among haploxylon species, suggesting that the radiation of this group of taxa from its common ancestor occurred after the diversification of other groups. The low cpDNA divergence in this subsection corroborated earlier evidence for its phylogenetic cohesiveness and existence as a monophyletic group.     

Intra- and interspecific genetic differentiation in closely related pines fromPinus subsectionSylvestres (Pinaceae) in the former Soviet Union

Thirty-seven natural populations of four closely related species ofPinus subsect.Sylvestres, P. mugo, P. funebris, P. pallasiana, andP. sylvestris, occurring in the former Soviet Union were investigated by starch-gel electrophoresis. In the populations assayed 127 allelic variants at 25 loci were revealed.Nei's distance coefficient (Dn) was used to estimate the level of genetic differentiation amongP. sylvestris races and among closely related species. A dendrogram constructed using Dn values shows that of the fiveP. sylvestris races analyzed only the geographically isolated var.hamata exhibited sufficient differences at theDia-2 locus (a mean Dn value relative to the other four races is 0.025) to recognize it as a distinct taxon. The remaining races, sylvestris, cretacea, lapponica, and sibirica, have a similar gene pool (Dn values are not greater than 0.010), and they should be regarded as a single taxon,P. sylvestris var.sylvestris. Interspecific comparisons revealed thatP. sylvestris andP. mugo have the closest genetic affinities to each other withNei's genetic distance of 0.108. The dendrogram demonstrates thatP. funebris is closer toP. sylvestris andP. mugo thanP. pallasiana. The available paleontologic data allowed us to conclude thatNei's (1975) time scale estimate for the time of divergence of the taxa was more accurate thanNei's (1971) time scale estimate.     

Population structure,gene diversity,and differentiation in natural populations of Cedar pines (Pinus subsect.Cembrae,Pinaceae) in the USSR

Twelve natural populations of four cedar pine species,Pinus sibirica, P. cembra, P. pumila, andP. koraiensis, occurring in the Soviet Union were investigated by starch-gel electrophoresis. Frequencies of 55 alleles at 19 loci were determined. Interpopulation genetic diversity inP. sibirica andP. pumila was only 2–4 per cent of the total genetic diversity. Nei's distance coefficient (Dn) was used to estimate the level of genetic differentiation among conspecific populations and among species. Dn values among populations ranged from 0.006 to 0.038. A dendrogram constructed using Dn values divided cedar pines species into 2 clusters:sibirica-cembra (Dn = 0.030) andpumila-koraiensis (Dn = 0.143). Nei's distance between these clusters was 0.232. On the basis of the data obtained it was possible to draw the following conclusion:P. sibirica, P. pumila, andP. koraiensis are distinct species, whileP. cembra should apparently be regarded as geographicalP. sibirica race.     

Genetics of seed storage proteins in the love tree Cercis siliquastrum L. (Fabaceae)

The patterns of C. siliquastrum seed storage proteins (cercins) are described using SDS-polyacrylamide gel electrophoresis. The polypeptides detected had very different molecular weights (ranging from 168 to 34 KDa) which, together with their high homogeneity, produced a very good resolution of bands. These proteins could be ascribed to five different loci. The analysis of seed sets of individual trees indicated that the love tree is almost completely autogamous with less than 5% of outcrosses. Although this mode of reproduction seems to produce a decrease in heterozygote frequency among the seeds of the population analysed, the levels of variability detected were very high for an autogamous plant: all of the loci were polymorphic, with a mean heterozygosity of 0.327 and a polymorphic index of 0.412. Protein segregation revealed a strong genetic linkage between three cercin loci (a, c and d) while the other two are independent.     

Immunoelectrophoretic analysis of seed proteins from Pisum sativum L.

Summary Soluble proteins of pea seed were investigated by quantitative immunological methods. Vicilin, legumin, pea seed lectin (PEA), 26 albumins and a globulin (B₁) were detected and observed during seed development, germination and under different extraction and fractionation procedures. Vicilin and legumin were found to be immunologically distinctly different. Legumin was found to be comprised of two similar proteins, Legumin species I and II. Vicilin, but no legumin, was detected in the embryonic axis.Three albumins, B₁ and PEA were found to be synthesized after the onset of legumin synthesis.Among the pea lines investigated, one line exhibited distinct differences with respect to the albumins and PEA.Some observations indicate that PEA might interact with other seed proteins of pea.     

Molecular heterogeneity of Cowpea (Vigna unguiculata Fabaceae) seed storage proteins

81 wild forms and 110 cultivated cowpea,Vigna unguiculata, accessions from 21 countries of Africa were screened for variability in seed storage proteins. Total seed proteins, albumin and globulin fractions were investigated by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) of nonreduced and/or reduced samples in one- and two-dimensional procedures. The globulin fraction is heterogeneous in molecular weight and contains both legumin-like components and three to six nondisulfide-linked subunits. Three globulin subunits, with molecular weights 110, 76, and 41 kD were found to be composed of disulfide-linked polypeptides. In the nondisulfide-linked fraction, both cultivated and wild forms exhibited patterns of four types (A–D). This fraction contains polypeptide subunits of molecular weights 62, 56, and 52 kD for A type, 62, 56, 54, and 52 kD for B type, 62, 56, 52, and 50 kD for C type, and at least 62, 56, 54, 52, 50, and 49 kD for D type. These subunits present similar multiple charge forms but C and D types possess more basic specific 50 and 49 kD nondisulfide linked components. Major albumin fraction contains subunits of 94, 86, 32, and 24kD. No infraspecific variation was observed in albumin or legumin-like fractions. The discussion is focussed on the relations between genetic variability assessed by storage protein coding genes and phenotypic variability.     

Accumulation of seed storage proteins and the taxonomy ofPoaceae

The accumulation of specific seed proteins is a taxonomically valuable feature and can be used to additionally characterize plant taxa. To date, mainly crop proteins have been analysed in thePoaceae. In this investigation seed proteins from 147 species were screened with emphasis on legumin-like proteins and prolamins. The groups resulting from evaluation of the protein profiles correspond with well-known subfamilies and tribes.Panicoideae are clearly separated fromPooideae. WithinPooideae, theBromeae plusTriticeae tribes revealed obvious similarities.Lolium, Festuca andVulpia, generally included in the tribeFestuceae, revealed a protein profile similar to the profile of theBromeae/Triticeae. Legumin-like proteins are accumulated abundantly inBambusoideae andPooideae exceptBromeae/Triticeae, however, only the species included in theAveninae subtribe produce soluble (globulin-type) legumins as already known fromAvena sativa. Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Biochemical genetics of a 7S globulin-like protein from Pinus pinaster seed

The megagametophytes of seeds of Pinus pinaster Ait. contain two types of oligomeric globulins of approximately 175 and 190 kDa that are comprised of 47-kDa and 27- and 22-kDa, monomers, respectively, joined by weak interactions. The 27- and 22-kDa components were purified and their N-terminal sequences determined. Both polypeptides were inherited as if they were coded by a single unit of recombination. The results obtained suggest that these two polypeptides originate from a single protein that undergoes proteolytic processing. The characteristics of this P. pinaster globulin indicate that it is a member of the 7S globulin family of seed storage proteins.     

Phylogenetic relationships amongPinus species (Pinaceae) inferred from different numbers of 6PGDH loci

Electrophoretic examination of variousPinus species from both subgenera revealed that several taxa differ in the number of loci that control the enzyme system 6-phosphogluconate dehydrogenase (6PGDH). Based on inheritance analyses and published data, it was established that all species of subg.Pinus possess only two 6PGDH loci, whereas all stone pines of subg.Strobus exhibit four controlling loci. In order to trace the phylogenetic links at which one or two gene duplications occurred during pine evolution, several species of subsect.Strobi (sectionStrobus) and two species of sect.Parrya were additionally investigated. Based on conclusions about the uniqueness of gene duplications and the different numbers of 6PGDH loci, a phylogenetic tree of the pine taxa was constructed. This tree shows some new features not recognized in earlier studies and supports several novel assignments postulated in very recent pine classifications.     

The 5S RNA genes inPinus radiata and the spacer region as a probe for relationships betweenPinus species

The 5S RNA genes inPinus radiata occur in two size classes of about 850 and 525 bp in length. Representatives from both the long and short size classes were cloned and sequenced. The primary difference in the two size classes was shown to be a 330 bp insertion in the spacer region of the long units. Within an individual breeding clone ofP. radiata there was some sequence heterogeneity between representatives of the short class. The 5S RNA genes in thirty pine species were characterised using either a clone of the short size class or a subclone of the 330 bp insertion characterizing the long size class. Eleven species of subg.Strobus were examined and all lacked the long type of unit of radiata pine. The New World species of subg.Pinus all had both short and long units whereas the Old World species had long units. The implications of these results for the evolution of the 5S DNA sequences and the phylogenetic relationships withinPinus are discussed.     

Phylogenetic analysis of pines using ribosomal DNA restriction fragment length polymorphisms

Phylogenetic relationships among 30 species of the genusPinus were studied using restriction site polymorphism in the large subunit of nuclear rDNA. Of the 58 restriction sites scored, 48 were phylogenetically informative, and the 30 species reduced to ten taxa when species with identical restriction site patterns were combined. These ten taxa corresponded to the currently recognized subsections of the genus, with the sole exception ofP. leiophylla, which was identical in its pattern of restriction sites to all three species included from subsect.Oocarpae despite its being in a different section of subg.Pinus (Pinea instead ofPinus). A measure of the proportion of phylogenetic information contained within the data set (Homoplasy Excess Ratio, or HER) revealed that the character states were significantly non-randomly distributed among the ten taxa (HER = 0.71, p < 0.01). Branchand-bound searches using either Wagner or Dollo parsimony as the optimization criterion were carried out using PAUP in order to estimate phylogenetic relationships among the ten taxa. Three taxa (Picea pungens, Tsuga canadensis, andLarix decidua) were used independently as outgroups for purposes of rooting the trees. Despite the extreme differences in the assumptions underlying the Wagner and Dollo parsimony, the two gave surprisingly similar estimates of phylogeny, with both analyses supporting the monophyly of the two major subgeneraPinus andStrobus and differing in topology only in the placement of subsect.Ponderosae within subg.Pinus. The likelihood for the Wagner tree was only slightly higher than that computed for the Dollo tree.     

The geographical distribution of genetic variation of silver fir (Abies alba,Pinaceae) in relation to its migration history

Silver fir presently occurs in many mountainous regions of eastern, western, southern and Central Europe. In order to elucidate the biogeographic history of fir populations in different European areas, the distribution of area-specific alleles at eight enzyme loci and the allele frequency distributions at seven polymorphic enzyme loci were investigated in seed or bud samples from 48 provenances comprising 98 single stands. Due to great genetic divergence among different populations located near the ancient glacial retreats, it was concluded that silver fir survived in five refugia during the last glaciation but migrated from only three refugia to its present natural range. The occurrence of unique alleles in particular fir areas indicated its migratory routes to the north and the existence of a few introgression zones where silver fir from different refugia has met during its expansion.     

The evolution of angiosperm seed proteins: A methionine-rich legumin subfamily present in lower angiosperm Clades

Analysis of legumin-encoding cDNAs fromDioscorea caucasica Lipsky (Dioscoreaceae) and fromAsarum europaeum L. (Aristolochiaceae) shows that there is an especially methionine-rich legumin subfamily present in the lower angiosperm clades including the Monocotyledoneae. It is characterized by a methionine content of 3–4 mol% which is roughly triple the methionine proportion of most other legumins. These “MetR” legumins, if present, still have to be detected in the higher angiosperms including the important seed crops. Evolutionary analysis suggests that the MetR legumins are the result of a gene duplication allowing the differentiation of legumin genes according to their sulfur content. The duplication event must have taken place before the split into mono- and dicotyledonous plants but probably after the separation of angiosperms and gymnosperms. Correspondence to: H. Fischer     

Genetic control of seed proteins in wheat

Summary Electrophoretic profiles of crude protein extracts from seed of F₁ hybrids and reciprocal crosses among diploid, tetraploid and hexaploid wheats were compared with those of their respective parental species. The electrophoretic patterns within each of three pairs of reciprocal crosses, T.boeoticum X T.urartu, T.monococcun X T. urartu and T.dicoccum X T. araraticum, were different from one another but were identical with those of their respective maternal parents. Protein bands characteristic of the paternal parents were missing in F₁ hybrid seed suggesting that the major seed proteins in wheat were presumably regulated by genotype of the maternal parent rather than by the seed genotype. However, in another three pairs of reciprocal crosses, T.boeoticum X T. durum, T.dicoccum X T.aestivum and T. zhukovskyi x T. aestivum, protein bands attributable to the paternal parents were present in the F₁ hybrid seeds indicating that the seed proteins were not always exclusively regulated by the maternal genotype. The expression of paternal genomes is presumably determined by dosage and genetic affinity of the maternal and paternal genomes in the hybrid endosperm. The maternal regulation of seed protein content is probably accomplished through the maternal control over seed size. The seed protein quality may, however, depend upon the extent of expression of the paternal genome.     

A taxometric analysis of seed proteins in the genusBriza s. l. (Poaceae)

The species ofBriza L. s. l. are disjunctly distributed between Eurasia and South America. A taxometric analysis has been carried out, using data from electrophoresis of seed polypeptides and published morphological characters. Evidence from a cluster analysis of seed polypeptide data strongly supports the recognition of two different natural groups, a S. American group and a Eurasian group, and is consistent with karyotype data, but does not support the generic groupings of South American taxa derived from the morphological data.     

Comparative HPLC analysis of seed albumins fromVicia faba andV. kalakhensis (Fabaceae)

Previously reported electrophoretic seed albumin data have shown an unexpected association ofVicia faba withV. kalakhensis. In the present work, seed albumins ofV. faba (subsp.paucijuga and subsp.faba) were compared with those ofV. kalakhensis using ionexchange (IE) and reversed-phase (RP) high-performance liquid chromatography (HPLC). Two subspecies ofV. faba displayed similar seed albumin profiles. On the other hand, seed albumin profiles ofV. faba andV. kalakhensis showed no major protein peak in common either in IE-HPLC or RP-HPLC chromatograms. The reported differences in seed albumin composition ofV. faba andV. kalakhensis are consistent with other taxonomical data showingV. faba to be genetically distant from the wild relatives.     

Chromosome analysis and DNA homology in threePicea species,P. mariana,P. rubens,andP. glauca (Pinaceae)

A detailed karyotype analysis was made on the somatic complement ofPicea rubens andP. glauca. B-chromosomes were observed in someP. glauca populations. The karyotypes are generally asymmetrical with most of the chromosomes having median to median-submedian centromeres.Picea glauca chromosomes 2, 3, 7, and 8 have secondary constriction on their short arm and chromosome 10 has a secondary constriction on the long arm. Chromosome 3 was the most easily identifiable, as it has two secondary constrictions located on the short arm. InP. rubens, all the chromosomes but chromosomes 8 and 9 have one to four distinctive secondary constrictions. In general, the diagrammatic comparisons show a high degree of similarity amongP. mariana, P. rubens, andP. glauca. GenomicP. mariana probe strongly hybridized to dots of genomic DNA fromP. rubens andP. glauca indicating that there is a high sequence homology among these three species. The synchronizing agent, hydroxyurea was used at different concentrations to enhance the mitotic index of cell suspensions derived from embryogenic cultures. Hydroxyurea at 1.25 mM increased significantly the mitotic index. An increase of hydroxyurea from 1.25 mM to 5 mM and 10 mM resulted in a steady decrease of mitotic index.     

Ovule and seed ontogeny inGnetum gnemon L.

The ovule ofGnetum gnemon has three envelopes around the nucellus. The outer one forms two clear swellings at the lateral sides during the early developmental stages. The middle envelope also shows two swellings in many cases arranged decussately with respect to those of the outer one. All these swellings become obscure or disappear later. The inner envelope arises as an annular primordium and forms several manifest lobes at the stage of pollination. it develops two proliferating structures from its middle portion, viz., a flange and a micropyle-closing tissue. The three envelopes differentiatiate into the fleshy outer, sclerenchymatic middle and compressed inner layers of the seed coat, respectively. The inner one, however, remains restricted to the apical part of the seed owing to endochalazal growth. The outer envelope is derived from both dermal and subdermal cells of the ovule primordium and, therefore, is of dual origin. The middle and inner envelopes are subdermal in origin. The present study has cleared up some conflicting reports in the previous publications.