Grain protein variability among species of Triticum and Aegilops: quantitative SDS-PAGE studies

Summary Total proteins were extracted from degermed seeds of various species of Triticum and Aegilops with solutions containing sodium dodecyl sulfate (SDS) and mercaptoethanol. The reduced, dissociated proteins were fractionated according to molecular weight (MW) by high-resolution polyacrylamide gel electrophoresis in buffers containing SDS (SDS-PAGE). Stained SDS-PAGE patterns were measured by densitometric scanning over a suitable range of optical density. The data were normalized to equivalent total areas for each of the densitometric scans by means of a computer program that also permitted the construction of patterns of hypothetical amphiploids by averaging patterns of two or three diploid species. The grain proteins of most species examined had distinctive qualitative and quantitative aspects that were characteristic of the species even though nearly every accession or cultivar of a species exhibited at least minor differences in pattern from other accessions or cultivars. The main protein components (probably prolamins) of Triticum monococcum ssp. monococcum, T. monococcum ssp. boeoticum, T. urartu, and Aegilops squarrosa had MW's in the range 29–36 X 10³ whereas the most important components of Ae. speltoides, Ae. longissima, and Ae. searsii had MW's in the range 37–55 × 10³. Changes in the quantitative expression of particular genes, especially those coding for storage protein components, may have been associated with speciation. The strong predominance of proteins with MW's in the range 29–36 × 10³ in some accessions of AB genome tetraploids, such as T. turgidum ssp. dicoccoides, may indicate contributions to the B genome of these tetraploids by T. monococcum ssp. boeoticum, T. urartu, or Ae. squarrosa.     

Wheat phylogeny determined by RFLP analysis of nuclear DNA. 3. Intra- and interspecific variations of five Aegilops Sitopsis species

The level of intra- and interspecific variations on nuclear DNA in five Aegilops species of the Sitopsis section were investigated using restriction fragment length polymorphism (RFLP) analysis. A total of 18 accessions, i.e. 7 of Ae. speltoides, 3 of Ae. longissima, 2 of Ae. searsii, 3 of Ae. sharonensis and 3 of Ae. bicornis, were used. One accession each of Triticum aestivum, T. durum, T. urartu and Ae. squarrosa was included as reference material. Five enzymes and 20 probes were used. Among the five Sitopsis species studied, Ae. speltoides had the largest intraspecific variation (=0.061), which was as high as the interspecific variation observed among the other four species. The section Sitopsis was divided into two distinct groups: one containing only Ae. speltoides and the other, Ae. longissima, Ae. searsii, Ae. sharonensis and Ae. bicornis. This grouping by RFLP analysis is in agreement with the taxonomical classification of the subsections.     

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.     

CHy-banding patterns and chromatin organization inAegilops andTriticum species (Poaceae)

The distribution of CHy-banded heterochromatin was studied in the chromosomes ofAegilops longissima, Ae. speltoides, Triticum monococcum, andT. turgidum. Interphase nuclei were measured after Feulgen staining at different thresholds of optical density; the curves so obtained indicated the relationship among the species with respect to the different fractions of the genomic DNA. The karyological and cytophotometric analyses indicate differences betweenAe. speltoides andAe. longissima, the latter species being enriched in heterochromatin. Similar results were demonstrated for the genusTriticum, in whichT. turgidum showed more heterochromatin when compared withT. monococcum. The results suggest that the B genome of the cultivated wheats possesses a type of heterochromatin that resembles the type present inAe. longissima.     

Restriction Fragment Length Polymorphism (RFLP) for protein disulfide isomerase (PDI) gene sequences in Triticum and Aegilops species

RFLP variation revealed by protein disulfide isomerase (PDI) coding gene sequences was assessed in 170 accessions belonging to 23 species of Triticum and Aegilops. PDI restriction fragments were highly conserved within each species and confirmed that plant PDI is encoded either by single-copy sequences or by small gene families. The wheat PDI probe hybridized to single EcoRI or HindIII fragments in different diploid species and to one or two fragments per genome in polyploids. Four Aegilops species in the Sitopsis section showed complex patterns and high levels of intraspecific variation, whereas Ae. searsii possessed single monomorphic fragments. T. urartu and Ae. squarrosa showed fragments with the same mobility as those in the A and D genomes of Triticum polyploid species, respectively, whereas differences were observed between the hybridization patterns of T. monococcum and T. boeoticum and that of the A genome. The single fragment detected in Ae. squarrosa was also conserved in most accessions of polyploid Aegilops species carrying the D genome. The five species of the Sitopsis section showed variation for the PDI hybridization fragments and differed from those of the B and G genomes of emmer and timopheevi groups of wheat, although one of the Ae. speltoides EcoRI fragments was similar to those located on the 4B and 4G chromosomes. The similarity between the EcoRI fragment located on the 1B chromosome of common and emmer wheats and one with a lower hybridization intensity in Ae. longissima, Ae. bicornis and Ae. sharonensis support the hypothesis of a polyphyletic origin of the B genome. Received: 25 June 1999 / Accepted: 14 September 1999     

Chromosomal location of genes for Rubisco small subunit and Rubisco-binding protein in common wheat

Summary The genes coding for the Rubisco small subunit (SSU) and for the -subunit of the Rubisco-binding protein were located to chromosome arms of common wheat. HindIII-digested total DNA from the hexaploid cultivar Chinese Spring and from ditelosomic and nullisomic-tetrasomic lines was probed with these two genes, whose chromosomal location was deduced from the disappearance of or from changes in the relative intensity of the relevant band(s). The Rubisco SSU pattern consisted of 14 bands, containing at least 21 different types of DNA fragments, which were allocated to two homoeologous groups: 15 to the short arm of group 2 chromosomes (4 to 2AS, 7 to 2BS, and 4 to 2DS) and 6 to the long arm of group 5 chromosomes (2 on each of arms 5AL, 5BL, and 5DL). The pattern of the Rubisco-binding protein consisted of three bands, each containing one type of fragment. These fragments were located to be on the short arm of group 2 chromosomes. The restriction fragment length polymorphism (RFLP) patterns of several hexaploid and tetraploid lines were highly conserved, whereas the patterns of several of their diploid progenitors were more variable. The variations found in the polyploid species were mainly confined to the B genome. The patterns of the diploids T. monococcum var. urartu and Ae. squarrosa were similar to those of the A and D genome, respectively, in polyploid wheats. The pattern of T. monococcum var. boeoticum was different from the patterns of the A genome, and the patterns of the diploids Ae. speltoides, Ae. longissima, and Ae. Searsii differed from that of the B genome.     

The presence of the enhanced K/Na discrimination trait in diploid Triticum species

Summary A number of accessions of the three species of diploid wheat, Triticum boeoticum, T. monococcum, and T. urartu, were grown in 50 mol m^-3 NaCl+2.5 mol m^-3 CaCl₂. Sodium accumulation in the leaves was low and potassium concentrations remained high. This was not the case in T. durum grown under the same conditions, and indicates the presence in diploid wheats of the enhanced K/Na discrimination character which has previously been found in Aegilops squarrosa and hexaploid wheat. None of the accessions of diploid wheat showed poor K/Na discrimination, which suggests that if the A genome of modern tetraploid wheats was derived from a diploid Triticum species, then the enhanced K/Na discrimination character became altered after the formation of the original allopolyploid. Another possibility is that a diploid wheat that did not have the enhanced K/Na discrimination character was involved in the hybridization event which produced tetraploid wheat, and that this diploid is now extinct or has not yet been discovered.     

High molecular weight glutenin subunit variation in wild and cultivated einkorn wheats (Triticum spp.,Poaceae)

Variation in high molecular weight (HMW) glutenin subunit composition among wild and cultivated einkorn wheats (2n = 2x = 14, AA) was investigated using one- (SDS-PAGE and urea/SDS-PAGE) and two-dimensional (IEF × SDS-PAGE) electrophoretic analyses. The material comprised 150 accessions ofTriticum urartu, 160 accessions ofT. boeoticum, 24 accessions ofT. boeoticum subsp.thaoudar and 74 accessions of primitive domesticatedT. monococcum from many different germplasm collections. The biochemical characteristics of HMW-glutenin subunits ofT. boeoticum andT. monococcum were highly similar to one another but distinctly different from those ofT. urartu. All the species analysed were characterised by large intraspecific variation and only three HMW-glutenin subunit patterns were identical betweenT. boeoticum andT. monococcum. Consistent with the distinct nature ofT. urartu, all its HMW-glutenin patterns were different from those found inT. boeoticum andT. monococcum. The differences detected between these species might reflect their reproductive isolation and are consistent with recent nomenclatural and biosystematic treatments that recogniseT. urartu as separate species fromT. boeoticum andT. monococcum. The presence of three distinct glutenin components in some accessions of the species studied seems to be evidence for the existence of at least three active genes controlling the synthesis of the HMW-glutenin subunits in the A genome of wild and primitive domesticated diploid wheats. Results indicate also that HMW-glutenin subunits could represent useful markers for the evaluation of genetic variability present in different wild diploid wheat collections and subsequently for their conservation and future utilisation.     

Quality Traits, Carbon Isotope Discrimination and Yield Components in Wild Wheats

Seventy-one wild and primitive diploid accessions of the S-,A- and D-genome species of Aegilops and Triticum, one tetraploidwheat, T. turgidum L. var. durum Desf., ‘Mexicali’,and two hexaploid wheats, T. aestivum L., ‘Anza’and ‘Yecora Rojo’ were evaluated and compared forprotein and lysine contents, carbon isotope discrimination,and various agronomic traits in the 1987–88 season underfield conditions. Significant variability was observed amongthe 71 accessions and among the three genomes for all traits.For most characters, the D-genome species exhibited the mostvariation, followed by the A- and S-genome species. Aegilopssquarrosa, T. urartu, and T. boeoticum showed large variationfor harvest index. Large variation for grain yield was exhibitedby Ae. squarrosa, Ae. sharonensis and Ae. longissima, whichcould be exploited in hybridization and breeding programs withmodern cultivars. The mean protein and lysine values of thediploids were significantly higher than those of the moderncultivars. The S- and A-genome accessions had higher proteinand lysine contents than the D genome. Among genomes, the meanvalues for yield and harvest index were significantly greaterin S- and D-genome accessions than in the A-genome accessions. The correlation pattern between yield and quality traits wasdifferent in the three genomes. Superior accessions with regardto both grain yield and quality traits were identified in eachspecies studied, except Ae. longissima and Ae. sharonensis.The advantages and uses of these accessions in wheat breedingprograms are discussed. Aegilops spp, Triticum spp, protein and lysine contents, yield components, carbon isotope discrimination     

Towards a legal framework for systematic conservation: identification and development of allele-specific PCR markers for conspecific varieties of an endangered perennial herb <Emphasis Type="Italic">Primula kisoana</Emphasis> Miquel based on sequence variation of chloroplast DNA

Primula kisoana var. kisoana (Primulaceae) is a narrow endemic found only in extremely restricted areas of central Honshu Island, Japan. Although the species is included as ‘Critically Endangered (CR)’ in the latest Japanese Red List, it is not covered by legislation such as the ‘Law for the Conservation of Endangered Species of Wild Fauna and Flora, Japan’. This poor conservation status is due, largely, to the presence of another conspecific variety, var. shikokiana, which is regarded as less threatened than var. kisoana. In this study, we investigated the genetic dissimilarity between the two varieties by examining sequence variation in three noncoding regions of chloroplast DNA. Ten distinct haplotypes were detected, none of which were distributed across populations and varieties. The genetic differentiation between the two varieties revealed by an AMOVA and a minimum spanning network suggests that var. kisoana and var. shikokiana should be conserved and managed as separate units. Using the allele-specific PCR method, four primer pairs were developed at the point mutations and insertion/deletion loci that exhibited only inter-variety variation. These markers will facilitate reliable identification of var. kisoana, assisting in the implementation of restoration programs including the introduction of seedlings and construction of a legal framework for conservation.     

Assessment of the degree of restriction fragment length polymorphism in Brassica

Summary The feasibility of creating a restriction fragment length polymorphism (RFLP) linkage map in Brassica species was assessed by screening EcoRI-, HindIII-, or EcoRV-digested total genomic DNA from several accessions of B. campestris, B. oleracea, and B. napus using random genomic DNA clones from three Brassica libraries as hybridization probes. Differences in restriction fragment hybridization patterns occurred at frequencies of 95% for comparisons of accessions among species, 79% for comparisons of accessions among subspecies within species, and 70% for comparisons among accessions within subspecies. In addition, species differences in the level of hybridization were noted for some clones. The high degree of polymorphism found even among closely related Brassica accessions indicates that RFLP analysis will be a very useful tool in genetic, taxonomic, and evolutionary studies of the Brassica genus. Development of RFLP linkage maps is now in progress.     

D genome doners for Aegilops cylindrica (CCDD) and Triticum aestivum (AABBDD) deduced from esterase isozyme analysis

Summary Putative D genome donors for Aegilops cylindrica (2n = 28, CCDD) and Triticum aestivum (2n = 42, AABBDD) were studied with the isoelectric focusing patterns of esterase isozymes. 103 strains of Ae. cylindrica were uniform in their isozyme pattern. 30 strains of the putative parent, Ae. caudata, showed no zymogram variation, whereas the other parent, Ae. squarrosa, comprised 3 phenotypes. Natural Ae. cylindrica had an isozyme pattern which corresponded to a mixture of esterases from Ae. caudata and type 3 Ae. squarrosa. Therefore, it is concluded that the D genome donor of Ae. cylindrica is derived from type 3 Ae. squarrosa. These results suggest that Ae. cylindrica originated with a single amphiploidy event, and the C and D genomes have remained remarkably constant regarding esterase isozyme composition.On the other hand, T. aestivum comprised three zymogram phenotypes. These phenotypes contain bands which can be ascribed to the D genome of type 2 Ae. squarrosa. These results suggest that the D genome of Ae. cylindrica differs from that of T. aestivum. Evolution of the AB and D genomes of T. aestivum is indicated by the zymogram polymorphism. The origin of Ae. cylindrica is possibly more recent than that of T. aestivum.Contribution No. 433 of the Laboratory of Genetics, Faculty of Agriculture, Kyoto University     

Electrophoretic survey of seedling esterases in wheats in relation to their phylogeny

Summary Evolutionary and ontogenetic variation of six seedling esterases of independent genetic control is studied in polyploid wheats and their diploid relatives by means of polyacrylamide gel electrophoresis. Four of them are shown to be controlled by homoeoallelic genes in chromosomes of third, sixth and seventh homoeologous groups.The isoesterase electrophoretic data are considered supporting a monophyletic origin of both the primitive tetraploid and the primitive hexaploid wheat from which contemporary taxa of polyploid wheats have emerged polyphyletically and polytopically through recurrent introgressive hybridization and accumulation of mutations. Ancestral diploids belonging or closely related to Triticum boeoticum, T. urartu, Aegilops speltoides and Ae. tauschii ssp. strangulata are genetically the most suitable genome donors of polyploid wheats. Diploids of the Emarginata subsection of the section Sitopsis, Aegilops longissima s.str., Ae. sharonensis, Ae. searsii and Ae. bicornis, are unsuitable for the role of the wheat B genome donors, being all fixed for the esterase B and D electromorphs different from those of tetraploid wheats.     

NADP-dependent aromatic alcohol dehydrogenase in polyploid wheats and their diploid relatives. On the origin and phylogeny of polyploid wheats

Summary The three major isoenzymes of the NADP-dependent aromatic alcohol dehydrogenase (ADH-B), distinguished in polyploid wheats by means of polyacrylamide gel electrophoresis, are shown to be coded by homoeoalleles of the locus Adh-2 on short arms of chromosomes of the fifth homoeologous group. Essentially codominant expression of the Adh-2 homoeolleles of composite genomes was observed in young seedlings of hexaploid wheats (T. aestivum s.l.) and tetraploid wheats of the emmer group (T. turgidum s.l.), whereas only the isoenzyme characteristic of the A genome is present in the seedlings of the timopheevii-group tetraploids (T. timopheevii s.str. and T. araraticum).The slowest-moving B³ isoenzyme of polyploid wheats, coded by the homoeoallele of the B genome, is characteristic of the diploid species Aegilops speltoides S.l., including both its awned and awnless forms, but was not encountered in Ae. bicornis, Ae. sharonensis and Ae. longissima. The last two diploids, as well as Ae. tauschii, Ae. caudata, Triticum monococcum s.str., T. boeoticum s.l. (incl. T. thaoudar) and T. urartu all shared a common isoenzyme coinciding electrophoretically with the band B² controlled by the A and D genome homoeoalleles in polyploid wheats. Ae. bicomis is characterized by the slowest isoenzyme, B⁴, not found in wheats and in the other diploid Aegilops species studied.Two electrophoretic variants of ADH-B, B¹ and B², considered to be alloenzymes of the A genome homoeoallele, were observed in T. dicoccoides, T. dicoccon, T. turgidum. s.str. and T. spelta, whereas B² was characteristic of T. timopheevii s.l. and only B¹ was found in the remaining taxa of polyploid wheats. The isoenzyme B¹, not encountered among diploid species, is considered to be a mutational derivative which arose on the tetraploid level from its more ancestral form B² characteristic of diploid wheats.The implication of the ADH-B isoenzyme data to the problems of wheat phylogeny and gene evolution is discussed.     

Intra- and interspecific phylogenetic relationships among diploid Triticum-Aegilops species (Poaceae) based on base-pair substitutions, indels, and microsatellites in chloroplast noncoding sequences

This study analyzes intra- and interspecific variation in chloroplast DNA (cpDNA) in diploid Triticum-Aegilops species. This analysis focused on DNA sequence variation in noncoding regions of cpDNA, which included base-pair substitutions, insertion/deletions (indels, 50 loci pooled), microsatellites (7 loci pooled), and inversions. Nine of 13 Triticum-Aegilops species were successfully identified and genotyped using these data. Sixty-two haplotypes were detected in 115 accessions of 13 diploid species. Because of the large number of characters examined, novel deep relationships within and among Triticum-Aegilops species could be identified and evaluated. Phylogenetic trees for the genus Triticum-Aegilops were constructed with Hordeum vulgare and Dasypyrum villosum as outgroups, and the results were compared to previous studies. These data support the following inferences: (1) Aegilops species should be included in Triticum; (2) groups D, T, M, N, U, and section Sitopsis (except Ae. speltoides) underwent speciation concurrently, but most diploid species evolved independently; (3) Ae. mutica does not occupy a basal position in Triticum-Aegilops; (4) Ae. speltoides is in a basal position and differs significantly from other Sitopsis species; (5) Ae. caudata is polyphyletic in all trees; (6) the genus Aegilops is paraphyletic with Secale.     

Paternal regulation of seed development in wheat hybrids

Summary Diallel crosses among Triticum boeoticum (4 lines from different geographical areas), T.urartu, Aegilops squarrosa and Ae. speltoides exhibited reciprocal differences in hybrid seed morphology, endosperm development, and embryo viability. T. urartu and Ae. squarrosa as females with T. boeotiaum and Ae. speltoides lead to shrivelled inviable seed. T.boeoticum accessions as female with Ae.speltoides also lead to shrivelled seeds. The reciprocal crosses produced plump seeds which either resembled the maternal parent or showed size differences. By altering the endospermic genome ratios, hybrid seeds with 1 (PF)/1 (PM) showed extreme shrivelling whereas those with 4 (PF)/1 (PM) were medium shrivelled to plump. Genetic experiments involving hybrids of T. boeoticum, T. urartu and T. monococcum showed that a factor is present in pollen or male gametes, which shows dosage effect and which, by interacting with the maternal genome, leads to endosperm abortion.     

Comparative genetic maps reveal extreme crossover localization in the Aegilops speltoides chromosomes

A total of 137 loci were mapped in Aegilops speltoides, the closest extant relative of the wheat B genome, using two F₂ mapping populations and a set of wheat-Ae. speltoides disomic addition (DA) lines. Comparisons of Ae. speltoides genetic maps with those of Triticum monococcum indicated that Ae. speltoides conserved the gross chromosome structure observed across the tribe Triticeae. A putative inversion involving the short arm of chromosome 2 was detected in Ae. speltoides. A translocation between chromosomes 2 and 6, present in the wheat B genome, was absent. The ligustica/aucheri spike dimorphism behaved as allelic variation at a single locus, which was mapped in the centromeric region of chromosome 3. The genetic length of each chromosome arm was about 50 cM, irrespective of its physical length. Compared to T. monococcum genetic maps, recombination was virtually eliminated from the proximal 50–100 cM and was localized in short distal regions, which were often expanded compared to the T. monococcum maps. The wheat B genome and the genome of Ae. longissima, a close relative of Ae. speltoides, do not show the extreme localization of crossovers observed in Ae. speltoides.     

Intergeneric transfer of cytoplasmic male sterility between Raphanus sativus (cms line) and Brassica napus through cytoplast-protoplast fusion

Summary Cytoplasts isolated from hypocotyl protoplasts of Raphanus sativus cv Kosena (cms line) by ultracentrifugation through Percoll/mannitol discontinuous gradient were fused with iodoacetamide(IOA)-treated protoplasts of Brassica napus cv Westar. Seventeen randomly selected regenerated plants were characterized for morphology and chromosome numbers. All of the regenerated plants had morphology identical to B. napus and 10 of them possessed the diploid chromosome number of B. napus. The remaining plants had chimeric or aneuploid chromosome numbers. The mitochondrial genomes in the 10 fusion products possessing the diploid chromosome numbers of B. napus were examined by Southern hybridization analysis. Four of the 10 plants contained mitochondrial DNA showing novel hybridization patterns. Of these 4 plants, 1 was male sterile, and 3 were male fertile. The remaining plants showed mitochondrial DNA patterns identical to B. napus and were male fertile.     

Genetic diversity in wild diploid wheats Triticum monococcum var. boeoticum and T. urartu (Poaceae)

Summary The genetic diversity of two wild diploid wheat species, Triticum monococcum var. boeoticum and T. urartu, was assessed using starch gel electrophoresis. Genetic diversity is uniformly low in both species. Number of alleles per locus was very low with a mean of 1.22 for T. monococcum var. boeoticum and 1.19 in T. urartu. Percentage of polymorphic loci was also low, with a mean of 19.71 for T. monococcum var. boeoticum and a mean of 18.35 for T. urartu. Mean gene diversity was low with a mean of 0.052 in populations of T. monococcum var. boeoticum and a mean of 0.040 in populations of T. urartu. Genetic affinities of the species and of populations were computed using Nei's identity index (NI). Overall genetic affinities of the two species are NI=0.697. The genetic affinities of different populations of a species are uniformly high with NIs ranging from 0.894 to 1.000 in T. monococcum var. boeoticum and from 0.898 to 1.000 in T. urartu.Research supported by the California Agricultural Experiment Station and the International Board of Plant Genetic Resources     

Molecular analysis of the phylogenetic relationships among the diploid <Emphasis Type="Italic">Aegilops</Emphasis> species of the section <Emphasis Type="Italic">Sitopsis</Emphasis>

RAPD analysis was used to study the intraspecific variation and phylogenetic relationships of Sgenome diploid Aegilops species regarded as potential donors of the B genome of cultivated wheat. In total, 21 DNA specimens from six S-genome diploid species were examined. On a dendrogram, Ae. speltoides and Ae. aucheri formed the most isolated cluster. Among the other species, Ae. searsii was the most distant while Ae. longissima and Ae. sharonensis were the closest species. The maximum difference between individual accessions within one species was approximately the same (0.18–0.22) in Ae. bicornis, Ae. longissima, Ae. sharonensis, and Ae. searsii. The difference between the clusters of questionable species Ae. speltoides and Ae. aucheri corresponded to the intraspecific level; the difference between closely related Ae. longissima and Ae. sharonensis corresponded to the interspecific level.