Analysis of DNA sequence polymorphism at the cMWG699 locus reveals phylogenetic relationships and allopolyploidy within Hordeum murinum subspecies.

Hordeum murinum L. is one of the most widely distributed species in the genus Hordeum. This species is composed of three subspecies with three ploidy levels, namely subsp. glaucum (2x=14), subsp. murinum (4x=28) and subsp. leporinum (4x=28, 6x=42). These three subspecies are morphologically similar and are frequently referred to as the 'murinum complex'. Although many cytological studies suggest that the murinum complex is allopolyploid, one inter-specific hybridization study suggested that it is autopolyploid. The goals of the present study are to identify nucleotide variation in the cMWG699 locus in the polyploid genomes of the murinum complex, to conduct molecular phylogenetic analysis of this locus, and to clarify the allo- versus auto-polyploidy status of the murinum complex. For this purpose, PCR-RFLP analysis was conducted with HhaI and SspI restriction enzymes on 80 H. murinum accessions. Single enzyme digestion data revealed polymorphism between diploid and polyploids, and double-digestion revealed polymorphism between tetra- and hexaploids. The nucleotide sequences of clones clearly show that polyploid murinum species are allopolyploid. In addition, DNA sequence analysis indicated that one donor of the tetraploid was subsp. glaucum (2x), as has been suggested previously by cytological studies. The other diploid donors were not identified, but at least one group of sequences common to 4x and 6x genomes (namely clonetype B) was highly diverged from 2x subsp. glaucum. The two tetraploid subspecies, 4x subsp. murinum and 4x subsp. leporinum, had identical DNA sequences, suggesting that these two subspecies are not differentiated at the cMWG699 locus.

Structural and distributional variation of mitochondrial rps2 genes in the tribe Triticeae (Poaceae).

The mitochondrial rps2 gene from barley, like that of rice, wheat, and maize, has an extended open reading frame (ORF) at the 3'-region when compared to that from lower plants. However, the extended portions are variable among these cereals. Since barley and wheat belong to the same tribe (Triticeae), it would be interesting to know when and where the two types of rps2 were generated during evolution. To determine this, we utilized the mitochondrial (mt) DNA sequence to examine variations of the rps2 genes in the tribe Triticeae. By means of the variable 3'-region, the distribution of barley (B)-type and wheat (W)-type rps2 sequences was studied in 19 genera of the tribe. The B-type sequence was identified in 10 of the 19 genera, whereas the W-type sequence was present in all 19 genera. Thus, ten of the examined genera have both types of rps2 sequences due to the presence of two copies of the gene. The W-type sequence was also present in the tribe Bromeae and the B-type sequence was also found in Aveneae and Poeae. Phylogenetic trees based on the B-type and W-type sequences were different from those based on other molecular data. This suggests that the mitochondrial genome in Triticeae has a unique evolutionary history.

Physical locations of 5S and 18S-25S rDNA in Asian and American diploid Hordeum species with the I genome.

The physical locations of 5S and 18S-25S rDNA sequences in 15 diploid Hordeum species with the I genome were examined by double-target in situ hybridization with pTa71 (18S-25S rDNA) and pTa794 (5S rDNA) clones as probes. All the three Asian species had a species-specific rDNA pattern. In 12 American species studied, eight different rDNA types were found. The type reported previously in H. chilense (the 'chilense' type) was observed in eight American species. The chilense type had double 5S rDNA sites - two sites on one chromosome arm separated by a short distance - and two pairs of major 18S-25S rDNA sites on two pairs of satellite chromosomes. The other seven types found in American species were similar to the chilense type and could be derived from the chilense type through deletion, reduction or addition of a rDNA site. Intraspecific polymorphisms were observed in three American species. The overall similarity in rDNA patterns among American species indicates the close relationships between North and South American species and their derivation from a single ancestral source. The differences in the distribution patterns of 5S and 18S-25S rDNA between Asian and American species suggest differentiation between the I genomes of Asian and American species. The 5S and 18S-25S rDNA sites are useful chromosome markers for delimiting Asian species, but have limited value as a taxonomic character in American species. On the basis of rDNA patterns, karyotype evolution and phylogeny of the I-genome diploid species are discussed.

Genetic diversity of allozymes in turnip (Brassica rapa L. var. rapa) from the Nordic area.

Genetic diversity and relationships based on isozymes were studied in 31 accessions of turnip (Brassica rapa L. var. rapa). The material included varieties, elite stocks, landraces and older turnip of slash-and-burn type from the Nordic area. A total of 9 isozyme loci and 26 alleles were studied. The isozyme systems were ACO, DIA, GPI, GOT, PGM, PGD and SKD. The level of heterozygosity was reduced in the landraces, but it was high for the variety group 'Ostersundom'. Turnip has a higher genetic variation than other crops within B. rapa and than in other species with the same breeding system. The genetic diversity showed that 18.7% of the genetic variation was within the accessions, and the total H tau value was 0.358. Gpi-I and Pgd-I showed the lowest variation compared with the other loci. The cluster analysis revealed five clusters, with one main cluster including 25 of the 31 accessions. The dendrogram indicated that the variety group 'Ostersundom' clustered together whereas the variety group 'Bortfelder' was associated with country of origin. The landraces were spread in different clusters. The 'slash-and-burn' type of turnip belonged to two groups.

Trigenomic origin of the hexaploid Psammopyrum athericum (Triticeae: Poaceae) revealed by in-situ hybridization.

The genomic constitution of the hexaploid Psammopyrum athericum was studied with in-situ DNA hybridization using both genomic DNA and isolated cloned sequences as probes. A genomic probe from Thinopyrum bessarabicum (E genome) hybridized successfully to 14 chromosomes of Ps. athericum and a probe from Festucopsis serpentinii (L genome) hybridized to another 14 chromosomes. The remaining chromosomes did not hybridize, apart from in the centromeric region, to any of the genomic probes used. It is thus proposed that Ps. athericum contains the genomes E, L and X where X stands for a so-far unknown genome. Psammopyrum athericum has three pairs of pTa71 sites and approximately 30 pSc119:2 sites. The origin of the third genome will be a matter for further research using genomic and genome-specific probes.

Extent and patterns of RAPD variation in landraces and cultivars of rye (Secale cereale L.) from northern Europe.

Little is known about the extent and patterns of distribution of RAPD diversity in outcrossing species. This study is the first step in using RAPD markers to quantify the amount and distribution of genetic variation within and between accessions of 9 landraces and 3 cultivars of cultivated rye from Northern Europe. A high level of RAPD variation was detected, demonstrating the utility of RAPDs for genetic characterisation in rye. The results show that: (1) landraces and improved cultivars maintain roughly the same high levels of RAPD variation, (2) landraces from Norway, Germany and Finland showed the lowest level of variation, probably because of a small amount of seeds from the original samples, (3) most of the RAPD variation was found within rather than between the accessions, which is consistent with the pattern expected for a cross pollinated crop. Both the cluster and the principal coordinates analyses displayed the same pattern of genetic relationship among the accessions studied.

Assessing the allozyme variation in cultivars and Swedish landraces of rye (Secale cereale L.).

Genetic interpretation and diversity of 9 isozyme loci have been estimated in 7 improved varieties and 19 landraces from Sweden by means of starch gel electrophoresis. The isozyme systems were ACO, DIA, GPI, MDH, PGD and PGM. For the statistic analysis we used the following measures: average number of alleles per locus, percentage of polymorphic loci, average heterozygosity direct count and average heterozygosity Hardy-Weinberg expected unbiased estimate. The measures were made on species and population levels. The distribution of the total genetic diversity among populations was also calculated. To illustrate the genetic relationships among populations, genetic distances were measured and principal component analysis performed. As expected in a cross-pollinated crop we found high genetic diversity and a larger variation within than among the populations. Somewhat unexpectedly, however, we found that the currently used varieties have the same high level of heterozygosity as the landraces but in the dendrogram the two groups are separated. The dendrogram showed three main clusters. The large cluster included 21 populations and the two small clusters were clearly distinguishable from the rest. The landrace spring-type could not be separated from the landraces winter-type, but we did detect a difference between different spring types. A few populations had unique alleles for certain loci.

Patterns and levels of genetic differentiation in North American populations of the Alaskan wheatgrass complex.

Levels and distribution of genetic variation were assessed using six allozymes in 27 populations of Alaskan wheatgrass (Elymus alaskanus) from different locations in Canada, USA, Greenland and Russia to obtain information on the genetic structure of these populations. The enzyme systems were ACO, DIA, GPI, MDH, PGM and SKD. Allozyme variation at the species level was high, with 64.3% (Ps) of the loci being polymorphic, an average number of alleles per locus of 1.9 (As), and an average genetic diversity of 0.17 (Hes). Differentiation was found in the populations studied, with the following findings: (1) statistically significant differences were found in allele frequencies among populations for every polymorphic locus (P < 0.001); (2) 63% of the total allozyme variation at polymorphic loci was partitioned among populations (GST = 0.63); (3) relatively low mean genetic distances between the populations were obtained (mean D = 0.029); (4) the genetic structure of Russian populations are clearly distinct from the other populations, the cluster and principal component analyses revealed the same genetic patterns of relationships among populations. This study also indicates that E. alaskanus contains different levels of allozyme variation in its populations. Furthermore, some banding patterns at the loci Aco-1, Aco-2, Gpi-2, Mdh-1, Skd-1, Skd-2 can be used as markers to identify individual populations.

Phylogeny in the genus Hordeum based on nucleotide sequences closely linked to the vrs1 locus (row number of spikelets).

The phylogenetic relationship between four basic genomes designated H, I, Xa, and Xu in the genus Hordeum was studied using a nuclear DNA sequence. The sequence, cMWG699, is single copy in the H. vulgare genome, and tightly linked to the vrs1 locus which controls two- and six-rowed spikes. DNA fragments homologous to cMWG699 were amplified from diploid Hordeum species and the nucleotide sequences were determined. A phylogeny based on both base substitutions and an insertion-deletion event showed that the H- and Xa-genome groups are positioned in one monophyletic group indicating that the Xa-genome taxa should be included in the H-genome group. The large H-genome group is highly homogeneous. The I and Xu genomes are distinctly separated from H and Xa, and form sister groups. Another phylogeny pattern based on data excluding the insertion-deletion gave a result that the Xa genome forms a sister group to the H-genome group. The difference between the H and Xa genomes was affected only by a single base insertion-deletion event, thus the H and Xa genomes are likely to be closely related. The I and Xu genomes were again distinctly separated from the H and Xa genomes.

Genetic diversity in Elymus caninus as revealed by isozyme, RAPD, and microsatellite markers.

Genetic diversity of 33 Elymus caninus accessions was investigated using isozyme, RAPD, and microsatellite markers. The three assays differed in the amount of polymorphism detected. Microsatellites detected the highest polymorphism. Six microsatellite primer pairs generated a total of 74 polymorphic bands (alleles), with an average of 15.7 bands per primer pair. Three genetic similarity matrices were estimated based on band presence or absence. Genetic diversity trees (dendrograms) were derived from each marker technique, and compared using Mantel's test. The correlation coefficients were 0.204, 0.267, and 0.164 between isozyme and RAPD distance matrices, RAPD and microsatellite distance matrices, and between isozyme and microsatellite distance matrices, respectively. The three methodologies gave differing views of the amount of variation present but all showed a high level of genetic variation in E. caninus. The following points may be drawn from this study whether based on RAPD, microsatellite, or isozyme data: (i) The Icelandic populations are consistently revealed by the three dendrograms. The congruence of the discrimination of this accession group by RAPD, microsatellite, and isozyme markers suggests that geographic isolation strongly influenced the evolution of the populations; (ii) The degree of genetic variation within accessions was notably great; and (iii) The DNA-based markers will be the more useful ones in detecting genetic diversity in closely related accessions. In addition, a dendrogram, which took into account all fragments produced by isozymes, RAPDs, and microsatellites, reflected better the relationships than did dendrograms based on only one type of marker.

Characterization of microsatellite loci from Elymus alaskanus and length polymorphism in several Elymus species (Triticeae: Poaceae).

A size-selected genomic library from Elymus alaskanus was screened for the presence of (GA)n, (GT)n, (CAC)n, and (TCT)n microsatellite sequences. A total of 28 positive clones were found for the two dinucleotide repeats, whereas no positive clones were found for the trinucleotide repeats. Positive clones were sequenced to validate the presence of microsatellites and to generate polymerase chain reaction (PCR) primers, based on the sequences flanking the microsatellite. Primer pairs were designed and evaluated for 18 selected microsatellites. The resulting loci were analysed by PCR for their usefulness as molecular markers in an array of 18 accessions representing E. alaskanus and 10 other Elymus species. PCR amplification revealed alleles for the 18 loci, which varied in having 1-10 alleles in these accessions. In the 18 accessions tested, 7 of the 18 loci were polymorphic, with gene diversity values ranging from 0.54 to 0.80 among all species. Within E. alaskanus, gene diversity values ranged from 0.20 to 0.72, with a mean of 0.48. Polymorphism was also detected within accessions. No clear relationship between total repeat length and the degree of polymorphism was observed in this study. Primer pairs designed to amplify microsatellites in E. alaskanus can be used to generate polymorphism products in other species within the genus. Hence, microsatellites are useful markers for studying both inter- and intra-specific genetic variability within Elymus.

Effect of rye A and B chromosomes on meiotic association of Hordeum marinum ssp. gussoneanum (4x) chromosomes in intergeneric hybrids.

Chromosome association at metaphase I was studied in PMCs of eight H. marinum ssp. gussoneanum (4x) x rye hybrids. Differences in the levels of association separated six hybrids with 2n = 23 including 14 Hordeum, 7 rye A and 2 rye B chromosomes into two groups of three plants each, a "low association" group with means of 0.03III + 4.43II (1.55 rings + 2.88 rods) + 5.10I and 6.03 chiasmata/cell, and a "high association" group with means of 0.01IV + 0.03III + 6.40II (3.55 rings + 2.85 rods) + 1.13I and 10.04 chiasmata/cell. The low number of plants studied prevents a safe estimate of the number of genes involved, but the significant difference between groups suggests the presence in the rye genome of two major genes, or two genotypes, for control of meiotic chromosome association. In two additional hybrids with 2n = 25, one of each above-mentioned group, the presence of two extra rye B chromosomes raised chiasma frequencies by ca 1.5, indicating a promoting effect on chromosome association. The level of Hordeum chromosome association in the "high association" group and the observation of up to 7 Hordeum ring bivalents in some cells agree with an autoploid origin of H. marinum ssp. gussoneanum (4x). Hordeum and rye chromosomes formed a few heterogenomic bi- and trivalents. Most rye A chromosomes formed univalents, but 2.7% were included in associations. Rye B chromosomes generally formed rod bivalents. The use of genome analysis in its traditional sense is discussed.

Characterization of a family of tandemly repeated DNA sequences in Triticeae.

The recombinant plasmid dpTa1 has an insert of relic wheat DNA that represents a family of tandemly organized DNA sequences with a monomeric length of approximately 340 bp. This insert was used to investigate the structural organization of this element in the genomes of 58 species within the tribe Triticeae and in 7 species representing other tribes of the Poaceae. The main characteristic of the genomic organization of dpTa1 is a classical ladder-type pattern which is typical for tandemly organized sequences. The dpTa1 sequence is present in all of the genomes of the Triticeae species examined and in 1 species from a closely related tribe (Bromus inermis, Bromeae). DNA from Hordelymus europaeus (Triticeae) did not hybridize under the standard conditions used in this study. Prolonged exposure was necessary to obtain a weak signal. Our data suggest that the dpTa1 family is quite old in evolutionary terms, probably more ancient than the tribe Triticeae. The dpTa1 sequence is more abundant in the D-genome of wheat than in other genomes in Triticeae. DNA from several species also have bands in addition to the tandem repeats. The dpTa1 sequence contains short direct and inverted subrepeats and is homologous to a tandemly repeated DNA sequence from Hordeum chilense.

Phylogenetic analysis of the genus Hordeum using repetitive DNA sequences.

A set of six cloned barley (Hordeum vulgare) repetitive DNA sequences was used for the analysis of phylogenetic relationships among 31 species (46 taxa) of the genus Hordeum, using molecular hybridization techniques. in situ hybridization experiments showed dispersed organization of the sequences over all chromosomes of H. vulgare and the wild barley species H. bulbosum, H. marinum and H. murinum. Southern blot hybridization revealed different levels of polymorphism among barley species and the RFLP data were used to generate a phylogenetic tree for the genus Hordeum. Our data are in a good agreement with the classification system which suggests the division of the genus into four major groups, containing the genomes I, X, Y, and H. However, our investigation also supports previous molecular studies of barley species where the unique position of H. bulbosum has been pointed out. In our experiments, H. bulbosum generally had hybridization patterns different from those of H. vulgare, although both carry the I genome. Based on our results we present a hypothesis concerning the possible origin and phylogeny of the polyploid barley species H. secalinum, H. depressum and the H. brachyantherum complex.

Hordein variation in the genus Hordeum as recognized by monoclonal antibodies.

The composition of the major storage protein, hordein, in wild barley species has been studied by using gel electrophoresis, Coomassie staining, and immunoblot assays. We have shown earlier that it is possible to obtain cross-reaction outside the cultivated barley, with monoclonal antibodies raised against hordeins from the barley cultivar Bomi. These antibodies have now been used to investigate the hordein composition in all species of the Hordeum genus. The results showed that polypeptides similar to the two major hordein groups of cultivated barley, the B- and C-hordeins, are produced in all wild Hordeum species, and that there are both similarities and differences between the two hordein groups. The similarities indicate a common evolutionary origin, while the distinction between B- and C-hordeins in the entire genus clearly shows that the divergence of their coding genes preceded the divergence of the Hordeum species. The presence of the same antigenic site in two different species indicates that they are evolutionarily related. Among the wild species, two rarely occurring sites were exclusively found in H. vulgare ssp. spontaneum and H. bulbosum, which confirms that they are the cultivated barley's closest relatives. Some of the antibodies also gave an extensive reaction pattern with H. murinum, which suggests a fairly close relationship to H. vulgare, though not as close as between H. vulgare and H. bulbosum.

Cytogenetic studies of the intergeneric hybrids between Secale cereale and Elymus caninus, E. brevipes, and E. tsukushiensis (Triticeae: Poaceae).

Integeneric hybridizations were carried out between Secale cereale L. (2n = 14, RR) and three Elymus species, namely, E. caninus (L.) L. (2n = 28, SSHH), E. brevipes (Keng) Löve (2n = 28, SSYY) and E. tsukushiensis Honda (2n = 42, SSHHYY). Chromosome pairing was studied at metaphase I in the parental species and the hybrids. Meiotic configurations of the hybrids were 20.74 1+0.14 II for E. caninus x S. cereale (SHR), 16.35 I+2.17 II+0.09 III for E. brevipes x S. cereale (SYR) and 25.84 I+1.10 II+0.02 III for E. tsukushiensis x S. cereale (SHYR), in addition to some secondary associations in the different hybrids. It is concluded from the study that (1) a certain, different homoeologous relationship exists among "S", "H" and "Y" genomes in the investigated Elymus species; (2) low homoeology is present between genomes of Elymus (S or H or Y) and rye (R); (3) the Secale genome affects homoeologous chromosome pairing between different genomes in E. brevipes and E. tsukushiensis.

Triple hybridization with cultivated barley (Hordeum vulgare L.).

A crossing programme for trispecific hybridization including cultivated barley (Hordeum vulgare L.) as the third parent was carried out. The primary hybrids comprised 11 interspecific combinations, each of which had either H. jubatum or H. lechleri as one of the parents. The second parent represented species closely or distantly related to H. jubatum and H. lechleri. In trispecific crosses with diploid barley, the seed set was 5.7%. Crosses with tetraploid barley were highly unsuccessful (0.2% seed set). Three lines of diploid barley were used in the crosses, i.e. 'Gull', 'Golden Promise' and 'Vada'. Generally, cv 'Gull' had high crossability in crosses with related species in the primary hybrid. It is suggested that 'Gull' has a genetic factor for crossability not present in cv 'Vada' and cv 'Golden Promise'. One accession of H. brachyantherum used in the primary hybrid had a very high crossability (seed set 54.7%) in combination with cv 'Vada' but no viable offspring was produced. In all, two trispecific hybrids were raised, viz. (H. lechleri x H. brevisubulatum) x 'Gull' (2n=7-30) and (H. jubatum x H. lechleri) x 'Gull' (2n=20-22). The first combination invariably had a full complement of seven barley chromosomes plus an additional chromosome no. 7, but a varying number of chromosomes (19-22) of the wild-species hybrid. The second combination had a full set of barley chromosomes. The meiotic pairing was low in both combinations.

Complex interspecific hybridization in barley (Hordeum vulgare L.) and the possible occurrence of apomixis.

Several complex hybrids were produced from the combination [(Hordeum lechleri, 6x xH. procerum, 6 x) × H. vulgare, 2 x]. Crosses with six diploid barley lines resulted in triple hybrids, most of which had a full complement of barley chromosomes (no. 1-7), but were mixoploid with respect to alien chromosomes (19-22). In one combination, chromosome no. 7 was duplicated. Meiosis in triple hybrids showed low, but variable pairing (1.3-5.5 chiasmata per cell). The syndesis probably did not include the barley chromosomes. Direct back-crosses to di- and tetraploid barley lines were unsuccessful. Chromosome doubling of the triple hybrid based on cv 'Pallas' resulted in a plant with 2n = 53-56, which had an increased fertility. Backcrosses to one di- and one tetraploid barley line resulted in offspring. The cross made with the tetraploid line ('Haisa II'), produced a 28-chromosomic plant in which the male parental genome was absent. We suspect that this plant may have arisen through parthenogenetic development of a reduced female gamete. The other cross with a diploid line ('9208/9') resulted in plant with 2n = 51-53. The most likely explanation for this second plant is that an unreduced gamete from the amphiploid was fertilized by a normal gamete from the backcross parent, and during early embryo development, some chromosomes were eliminated.

Elimination and duplication of particular Hordeum vulgare chromosomes in aneuploid interspecific Hordeum hybrids.

Seeds formed in crosses Hordeum lechleri (6x) x H. vulgare (2x and 4x), H. arizonicum (6x) x H. v. (2x), H. parodii (6x) x H. v. (2x), and H. tetraploidum (4x) x H. v. (2x) produced plants at high or rather high frequencies through embryo rescue. Giemsa C-banding patterns were used to analyze chromosomal constitutions and chromosomal locations on the methaphase plate. Among 100 plants obtained from H. vulgare (2x) crosses, 32 plants were aneuploid with 2n=29 (1), 28 (3), 27 (13), 26 (5), 25 (4), 24 (4), or 22 (2); 50 were euploid (12 analyzed), and 18 were polyhaploid (5 analyzed). Four plants had two sectors differing in chromosome number. Two of four hybrids with H. vulgare (4x) were euploid and two were aneuploid. Parental genomes were concentrically arranged with that of H. vulgare always found closest to the metaphase centre. Many plants showed a certain level of intraplant variation in chromosome numbers. Except for one H. vulgare (4x) hybrids, this variation was restricted to peripherally located non-H. vulgare genomes. This may reflect a less firm attachment of the chromosomes from these genomes to the spindle. Interplant variation in chromosome numbers was due to the permanent elimination or, far less common, duplication of the centrally located H. vulgare chromosomes in all 34 aneuploids, and in a few also to loss/gain of non-H, vulgare chromosomes. This selective elimination of chromosomes of the centrally located genome contrasts conditions found in diploid interspecific hybrids, which eliminate the peripherally located genome. The difference is attributed to changed "genomic ratios'. Derivatives of various H. vulgare lines were differently distributed among euploid hybrids, aneuploids, and polyhaploids. Chromosomal constitutions of hypoploid hybrids revealed a preferential elimination of H. vulgare chromosomes 1, 5, 6, and 7, but did not support the idea that H. vulgare chromosomes should be lost in a specific order. H. vulgare SAT-chromosomes 6 and 7 showed nucleolar dominance. Aneuploidy is ascribed to the same chromosome elimination mechanism that produces haploids in cross-combinations with H. vulgare (2x). The findings have implications for the utilization of interspecific Hordeum hybrids.