A microsatellite sequence from the rice blast fungus (Magnaporthe grisea) distinguishes between the centromeres of Hordeum vulgare and H. bulbosum in hybrid plants.

A TC/AG-repeat microsatellite sequence derived from the rice blast fungus (Magnaporthe grisea) hybridized to all of the centromeres of Hordeum vulgare chromosomes, but hybridized faintly or not at all to the chromosomes of Hordeum bulbosum. Using this H. vulgare centromere-specific probe, the chromosomes of four F1 hybrids between H. vulgare and H. bulbosum were analyzed. The chromosome constitution in the root tips of the hybrids was mosaic, i.e., 7 (7v, H. vulgare) and 14 (7v + 7b H. bulbosum), or 14 (7v + 7b) and 27 (14v + 13b), or 7 (7v), 14 (7v + 7b), and 27 (14v + 13b). The 27-chromosome tetraploid hybrid cells were revealed to have the NOR (nucleolus organizer region) bearing chromosome of H. bulbosum in a hemizygous state, which might indicate some role for this chromosome in the chromosome instability of the hybrid condition. The chromosomal distribution showed that the chromosomes of H. vulgare were concentric and chromosomes of H. bulbosum were peripheral in the mitotic squash. This non-random chromosome distribution and the centromere-specific repeated DNA differences in the two species were discussed in relation to H. bulbosum chromosome elimination. Meiotic chromosome analyses revealed a high frequency of homoeologous chromosome pairing in early prophase. However, this chromosome pairing did not persist until later meiotic stages and many univalents and chromosome fragments resulted. These were revealed to be H. bulbosum by fluorescence in situ hybridization (FISH) analysis with the H. vulgare centromere-specific probe. Because the chromosome segregation of H. vulgare and H. bulbosum chromosomes at anaphase I of meiosis was random, the possibility for obtaining chromosome substitution lines in diploid barley from the diploid hybrid was discussed.     

Characterization by RFLP analysis and genomic in situ hybridization of a recombinant and a monosomic substitution plant derived from Hordeum vulgare L. x Hordeum bulbosum L. crosses.

Interspecific crosses in Hordeum have been made with the aim of transferring desirable traits, such as disease resistance, from a wild species, Hordeum bulbosum, into cultivated barley (Hordeum vulgare). Interspecific recombinants have previously been identified using several methods, but there are limitations with all the techniques. We improved our ability to characterize progeny from H. vulgare x H. bulbosum crosses by using genomic in situ hybridization (GISH). The plant material comprised a recombinant and a monosomic alien substitution plant derived from H. vulgare x H. bulbosum crosses. The recombinant possesses a pubescent leaf sheath conferred by a gene transferred from H. bulbosum into barley cultivar Golden Promise. The use of GISH on a plant homozygous for the pubescence gene confirmed the presence of H. bulbosum DNA located distally on two barley chromosomes and we mapped the introgression to barley chromosome 4HL using RFLP analysis. Furthermore, by means of an allelism test we found that the transferred gene for pubescence is allelic or closely linked to a gene for pubescence (Hs) located on barley chromosome 4HL. The presence of a single H. bulbosum chromosome in the monosomic substitution plant was confirmed by GISH. A distal introgression of H. bulbosum DNA was also observed on one barley chromosome, which was located on chromosome 3HL by RFLP analysis.     

Recent progress in barley improvement using wild species of Hordeum

In this review we describe recent progress in barley (Hordeum vulgare) improvement through hybridisation with its wild relatives. We have focused on one species in the secondary genepool of cultivated barley, namely H. bulbosum. This wild species has desirable traits, such as disease resistance, that are worthwhile transferring to its cultivated relative. Progress has been achieved through developing partially fertile interspecific hybrids that have been selfed or backcrossed to barley. We present the results of cytogenetic and molecular analyses that have enabled us to characterise and produce agronomically useful recombinant lines obtained from the hybrids.     

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.     

Elimination of chromosomes in Hordeum vulgare x H. bulbosum crosses at mitosis and interphase involves micronucleus formation and progressive heterochromatinization

Uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanism underlying selective elimination of the paternal chromosomes during the development of Hordeum vulgare x H. bulbosum hybrid embryos that is restricted to an early stage of development. In almost all embryos most of the H. bulbosum chromatin undergoes a fast rate of elimination within nine days after pollination. There are differences in the mitotic behaviour between the parental chromosomes, with H. bulbosum chromatids segregating asymmetrically at anaphase. We provide evidence for a chromosome elimination pathway that involves the formation of nuclear extrusions during interphase in addition to postmitotically formed micronuclei. The chromatin structure of nuclei and micronuclei differs and heterochromatinization and disintegration of the nuclear envelope of micronuclei are the final steps of chromosome elimination.     

Characterization of Hordeum vulgare - Hordeum bulbosum chromosome substitution lines by restriction fragment length polymorphism analysis.

Plants obtained from crosses between Hordeum vulgare and H. bulbosum were previously analyzed cytologically and for isozyme composition. They were identified as possessing substitutions of one or more H. vulgare chromosomes by their H. bulbosum homoeologues. To confirm their constitution and assess the merits of molecular techniques, chromosome-specific probes developed for the Triticeae were hybridized to Southern blots of DNA extracted from these plants and their parents. The hybridization patterns in the substitution plants confirmed that particular chromosomes of H. vulgare were replaced by their H. bulbosum homoeologues. For most probes, heterozygosity between pairs of H. bulbosum chromosomes was recorded. A possible duplication involving H. bulbosum homoeologues of barley chromosomes 4 and 7 was observed. Although molecular and cytological methods for analyzing chromosomally engineered plants are complementary, molecular probes may uncover differences not discernible using light microscopy or isozyme analysis.     

The Crossability of Triticum aestivum with Tetraploid Hordeum bulbosum

In order to study the crossability of wheat with H. bulbosum a series of wheat varieties from various sources and their F1 hybrids as well as tetraploid H. bulbosum from different countries were used as parents in this experiment. The main results of the experiment are showed as follows: 1. Twenty-one wheat varieties from Europe, West Asia, America, China, Australia etc. 11 countries and regions respectively as famele parents were crossed with four tetraploid H. bulbosum from Hungary, USSR, Canada and Germany. The seed set percentages in the intergeneric cross combinations ranged from 0.00% to 49.93%. Statistical analysis revealed that there were significant differences between the seed set percentages of wheat varieties. The crossability, with H. bulbosum might differed in different varieties of wheat from same country. 2. Various F1 hybrids resulting from 13 and 26 intervarietal cross combinations of wheat and their parents were crossed with H. bulbosum from Hungary and USSR respectively in different years. The mean seed set percentages of F1 hybrids, their male and female parents were 26.53%, 15.38%, 20.30% and 39.1%, 34.8%, 26.7% respectively. The results indicated that when some wheat varieties having poor-crossability with H. bulbosum were hybridized with other varieties especially with those varieties having high- crossability, the crossability of their F, hybrids probably had higher crossability than their parents having poor-crossability. Six F1 hybrids of wheat obtained from six com- binations of reciprocal crosses, in which the completely non-crossable varieties Hope, Xiao- Bai-Mang and high-crossable varieties Chinese Spring, Fortunate were used as male or female parent alternately, were crossed as female parents with H. bulbosum. All of 6 F1 hybrids were crossable and gave the percentage of seed set from 7.00% to 42.57%, although they ought to carry the dominant Kr genes responsible for non-crossability, which were passed on to F1 hybrids by non-crossable varieties, the parent Hope or Xiao-Bai- Mang. Clearly that is due to the gene interaction between female and male parents. Be- sides Kr loci in wheat, probably other genes can influence the crossability. 3. There were significant differences between clones of H. bulbosum in the crossa- bilities with wheat. In conclusion F1 hybrids of wheat have higher crossability with H. bulbosum than their parents having poor-crossability. This could be advantageous for exploitation of this technique. By selecting even more efficient clones of H. bulbosum and improving embryo culture techniques, the H. bulbosum technique will be used in wheat breeding program- mes probably.     

Genetic and geographic variation of bulbous barley (Hordeum bulbosum L.) assessed by rapd markers

Estimated the genetic relationships among 21 barley accessions from 17 bulbous barley (H. bulbosum L.), 4 cultivated barley (H. vulgare L.) collected from different part of Turkey were investigated using Random Amplified Polymorphic DNA (RAPD). Eleven informative primers amplified 111 markers of which 98 (89.8%) were polymorphic. A dendogram was constructed using the UPGMA method based on the RAPD markers. The range of genetic similarity was from 0.111 to 0.815. The accessions were grouped into two main clusters based on the molecular data. The H. vulgare and H. bulbosum separated into two groups in the Principle Component Analysis.     

The distribution of early recombination nodules on zygotene bivalents from plants.

Early recombination nodules (ENs) are protein complexes approximately 100 nm in diameter that are associated with forming synaptonemal complexes (SCs) during leptotene and zygotene of meiosis. Although their functions are not yet clear, ENs may have roles in synapsis and recombination. Here we report on the frequency and distribution of ENs in zygotene SC spreads from six plant species that include one lower vascular plant, two dicots, and three monocots. For each species, the number of ENs per unit length is higher for SC segments than for (asynapsed) axial elements (AEs). In addition, EN number is strongly correlated with SC segment length. There are statistically significant differences in EN frequencies on SCs between species, but these differences are not related to genome size, number of chromosomes, or phylogenetic class. There is no difference in the frequency of ENs per unit length of SC from early to late zygotene. The distribution of distances between adjacent ENs on SC segments is random for all six species, but ENs are found at synaptic forks more often than expected for a random distribution of ENs on SCs. From these observations, we conclude that in plants: (1) some ENs bind to AEs prior to synapsis, (2) most ENs bind to forming SCs at synaptic forks, and (3) ENs do not bind to already formed SCs.     

RFLP mapping of a Hordeum bulbosum gene highly expressed in pistils and its relationship to homoeologous loci in other Gramineae species

A cDNA sequence (Hbc8-2) isolated from pistils of the self-incompatible species Hordeum bulbosum was analysed for expression pattern and genetic map location. Hbc8-2 was expressed just prior to anthesis in mature pistils, and expression was maintained at a high level throughout anthesis. The same expression pattern was found in self-incompatible rye ( Secale cereale), but no expression was detected in the self-compatible cereals wheat ( Triticum aestivum) or barley ( Hordeum vulgare) at comparable stages of development. However, three wheat expressed sequence tags from a pre-anthesis library had high homology to Hbc8-2. Southern blot analyses using Hbc8-2 as a probe detected hybridising bands in the genomes of various Gramineae species including rye, barley, bread wheat, wild wheat relatives ( Aegilops tauschii and Ae. speltoides), oats ( Avena fatua and A. strigosa), rice ( Oryza sativa) and maize ( Zea mays). This suggests that Hbc8-2-like sequences are present in many species but that high levels of expression may be associated with self-incompatibility. Hbc8-2 was mapped on the long arms of chromosome 2H(b) of H. bulbosum, 2R of rye, and 2B and 2D of wheat and was assigned to chromosome 2H of barley using wheat/barley addition lines. On a H. bulbosum genetic map, Xhbc8-2 was located between Xbcd266 and Xpsr87, while in rye and wheat it was located in a 13.2-cM interval between Xpsr331 and Xpsr932, consistent with previous comparative mapping studies of these species. Mapping in rye suggested that Hbc8-2 is probably proximal to the Z self-incompatibility locus which was previously shown to be tightly linked to Xbcd266.     

Genomic organization, evolution, and structural peculiarities of highly repetitive DNA of Hordeum vulgare

A fraction of highly repeated DNA sequences of Hordeum vulgare has been investigated by cloning 19 separate highly repetitive sequences in the plasmid pBR327. Characteristics studied included genus specificity of isolated sequences, their prevalence, and genome organization. Sequences (pHv7161, pHv7191, pHv7179) have been identified that are the most widespread in the H. vulgare genome and have a complicated arrangement. A tandemly arranged sequence, pHv7141, was also identified. The primary structure of a 999 bp long, BamHI fragment of one of the most widespread sequences, pHv7161, as well as the adjacent pHv7302 and pHv7245 sequences was determined. The fragment abounds in inverted repeats, of which two are flanked by direct repeats, and contains short subrepeats, A, B, and C, and a great variety of potential protein-binding sites. A comparison is drawn between the content and genome organization of highly repeated DNA sequences of H. vulgare and those of the wild barley species Hordeum bulbosum, Hordeum jubatum, Hordeum geniculatum, Hordeum brevisubulatum, Hordeum turkestanicum, and Hordeum murinum. According to the above characters (close copy number and genome organization similarity of highly repetitive sequences) the species under discussion have been classified into four groups. This division is in good agreement with other data on interspecific crossing in Hordeum and on chromosome pairing in hybrid meiosis.     

Specific Isozyme Marker of a Virus Resistant Barley Derived from Interspecies Cross Between Hordeum vulgare and H. bulbosum

A diploid barley cultivar "Supi 1" was crossed with a tetraploid Hordeum bulbosum “GBC141” to transfer the disease resistant traits. Eleven viable triploid F1 plants were produced by means of embryo rescue technique. The resulting triploid hybrids were backcrossed to diploid barley, and seven BC1 plants were obtained. One of the BC1 plants exhibited barley yellow mosaic virus (BaYMV) resistance when grown in the diseased nursery. Isozyme analysis of H. vulgate, H. bulbosum and their backcross hybrids were made via slab polyacrylamide gel electrophoresis technique. The primary results showed that zymogram variation could be obviously found between diploid barley "Supi 1' and tetraploid H. bulbosurn "GBC141”. A peroxidase isozyme (Rf=0.47) from H. bulbosum was detected in the peroxidase isozyme zymogram of young roots of backcross hybrid BC1-2. This peroxidase isozyme was related to the BaYMV resistance but the linkage relation will be determined by the genetic analysis of the F2 population in the future. The BaYMV resistant line of the backcross with isozyme marker is the important resource of barley disease-resistant breeding.     

Male mouse recombination maps for each autosome identified by chromosome painting

Linkage maps constructed from genetic analysis of gene order and crossover frequency provide few clues to the basis of genomewide distribution of meiotic recombination, such as chromosome structure, that influences meiotic recombination. To bridge this gap, we have generated the first cytological recombination map that identifies individual autosomes in the male mouse. We prepared meiotic chromosome (synaptonemal complex [SC]) spreads from 110 mouse spermatocytes, identified each autosome by multicolor fluorescence in situ hybridization of chromosome-specific DNA libraries, and mapped >2,000 sites of recombination along individual autosomes, using immunolocalization of MLH1, a mismatch repair protein that marks crossover sites. We show that SC length is strongly correlated with crossover frequency and distribution. Although the length of most SCs corresponds to that predicted from their mitotic chromosome length rank, several SCs are longer or shorter than expected, with corresponding increases and decreases in MLH1 frequency. Although all bivalents share certain general recombination features, such as few crossovers near the centromeres and a high rate of distal recombination, individual bivalents have unique patterns of crossover distribution along their length. In addition to SC length, other, as-yet-unidentified, factors influence crossover distribution leading to hot regions on individual chromosomes, with recombination frequencies as much as six times higher than average, as well as cold spots with no recombination. By reprobing the SC spreads with genetically mapped BACs, we demonstrate a robust strategy for integrating genetic linkage and physical contig maps with mitotic and meiotic chromosome structure.     

Interspecific crosses inHordeum (Poaceae)

A crossing programme including 30 species and 40 cytotypes within the genusHordeum was undertaken. Viable hybrids were obtained in 302 combinations, 15 of which were intraspecific. Differences in seed set and in germination were observed in crosses between different groups of species. Obtaining crosses between different taxonomic groups was generally more difficult when diploid material was used. Some species, e.g.,H. lechleri, H. jubatum, andH. brachyantherum showed a higher crossability than others. The chromosome numbers of the hybrids were usually those expected from the parental numbers but aneuploid series around the expected numbers were rather frequent. Three cases of unreduced gametes were found. Selective chromosome elimination was restricted to combinations including eitherH. vulgare orH. bulbosum.—Despite a very diverse morphology, all South American diploid species together with the two North American diploidsH. intercedens andH. pusillum appear to be closely related. The hexaploid American speciesH. procerum, H. lechleri, andH. arizonicum are also related. The two North American tetraploid speciesH. jubatum andH. brachyantherum sometimes form semifertile hybrids. The Asiatic speciesH. roshevitzii appears to be related to both North and South American taxa.     

The mildew resistance of Hordeum bulbosum and its transference into H. vulgare genotypes

Mildew from susceptible genotypes (SI and S2) of Hordeum bulbosum was found to be avirulent on all H. vulgare genotypes tested, including such cultivars as Proctor with no known genes for resistance to mildew. The H. bulbosum genotype SI (2n =14) proved resistant to all isolates of mildew from H. vulgare. The mildew isolates used for these tests possessed most of the common virulence factors which attack the current ‘vulgare’ cultivars in Western Europe. Some H. bulbosum genotypes were resistant to the ‘bulbosum’ mildew. Attempts at combining resistance from both species are presented and discussed.     

Linkage Relationships Reflecting Ancestral Tetraploidy in Salmonid Fish

Fifteen classical linkage groups were identified in two salmonid species (Salmo trutta and Salmo gairdneri) and three fertile, interspecific hybrids (S. gairdneri X Salmo clarki, Salvelinus fontinalis X Salvelinus namaycush and S. fontinalis X Salvelinus alpinus) by backcrossing multiply heterozygous individuals. These linkage relationships of electrophoretically detected, protein coding loci were highly conserved among species. The loci encoding the enzymes appeared to be randomly distributed among the salmonid chromosomes. Recombination frequencies were generally greater in females than in males. In males, certain linkage groups were pseudolinked with other linkage groups, presumably because of facultative multivalent pairing and directed disjunction of chromosomes. Five such pseudolinkage groups were identified and they also appeared to be common among species and hybrids. Duplicate loci were never classically linked with each other, although some exhibited pseudolinkage and some showed evidence of exchanging alleles. Gene-centromere recombination frequencies estimated from genotypic distributions of gynogenetic offspring were consistent with map locations inferred from female intergenic recombination frequencies. These linkage relationships support the contention that all extant salmonids arose from a common tetraploid progenitor and that this progenitor may have been a segmental allotetraploid.     

High-resolution crossover maps for each bivalent of Zea mays using recombination nodules

Recombination nodules (RNs) are closely correlated with crossing over, and, because they are observed by electron microscopy of synaptonemal complexes (SCs) in extended pachytene chromosomes, RNs provide the highest-resolution cytological marker currently available for defining the frequency and distribution of crossovers along the length of chromosomes. Using the maize inbred line KYS, we prepared an SC karyotype in which each SC was identified by relative length and arm ratio and related to the proper linkage group using inversion heterozygotes. We mapped 4267 RNs on 2080 identified SCs to produce high-resolution maps of RN frequency and distribution on each bivalent. RN frequencies are closely correlated with both chiasma frequencies and SC length. The total length of the RN recombination map is about twofold shorter than that of most maize linkage maps, but there is good correspondence between the relative lengths of the different maps when individual bivalents are considered. Each bivalent has a unique distribution of crossing over, but all bivalents share a high frequency of distal RNs and a severe reduction of RNs at and near kinetochores. The frequency of RNs at knobs is either similar to or higher than the average frequency of RNs along the SCs. These RN maps represent an independent measure of crossing over along maize bivalents.     

Recombination nodule mapping and chiasma distribution in spermatocytes of the pigeon, Columba livia.

Pigeon spermatocytes were processed with a drying-down technique and their synaptonemal complex (SC) complements were analyzed by electron microscopy. The synaptonemal complex karyotype of the macrobivalents shows an excellent correspondence with the mitotic karyotype. The number and distribution of recombination nodules (RNs) were scored in complete nuclei stained with phosphotungstic acid. The average number of RNs per nucleus is 64.7. The number of nodules per bivalent shows a clear linear relationship with SC length in the 10 longest synaptonemal complexes, while the microbivalents usually bear a single RN. The location of RNs has a non-random distribution along the largest synaptonemal complexes, with lower frequencies near kinetochores and higher frequencies toward the telomeres. The ZZ bivalent is the fourth in size and shows free recombination, having on average 3.8 RNs. The mean number of nodules per cell and the mean number of nodules in the largest bivalents show very good agreement with the corresponding number of chiasmata scored in metaphase-I spermatocytes. It is concluded that the recombination nodules provide a good check for reciprocal exchanges in this and other species of birds. Additionally, a new morphology for the recombination nodules is presented, consisting of groups of electron-dense particles measuring 43 nm in diameter.     

Effect of gamma irradiated Hordeum bulbosum pollen upon haploid frequency in barley following interspecific hybridization

Haploids (monoploids) can be produced in cultivated barley (Hordeum vulgare) by pollination with Hordeum bulbosum and the subsequent elimination of H. bulbosum chromosomes [13]. Pollen of H. bulbosum was gamma-irradiated at doses of 1 to 8 kR to determine if it would lead to a more rapid chromosome elimination and subsequently a higher frequency of haploid barley seedlings. Early embryo development was slower following low dosages of irradiation than at higher dosages and there was no significant improvement in haploid production. At higher dosages, the frequencies of seed set, embryos and haploid seedlings declined. Double fertilization is an apparent pre-requisite of haploid formation and the “intrinsic vigour” of the bulbosum nucleus after fertilization appears to be an important factor in this system of haploid formation.