Identification of intergenomic translocations involving wheat,Hordeum vulgare and Hordeum chilense chromosomes by FISH

Intergenomic translocations between wheat, Hordeum chilense and Hordeum vulgare have been obtained in tritordeum background. Advanced lines from the crosses between three disomic chromosome addition lines for chromosome 2Hv, 3Hv, and 4Hv of barley (Hordeum vulgare) in Triticum aestivum cv. Chinese Spring (CS) and hexaploid tritordeum (2n = 6x = 42, AABBHchHch) were analyzed. Multicolor FISH using both genomic DNA from H. chilense and H. vulgare were used to establish the presence and numbers of H. vulgare introgressions into tritordeum. Interspecific H. vulgare/H. chilense and intergeneric wheat/H. vulgare and wheat/H. chilense translocations were identified. Frequencies of plants containing different kinds of intergenomic translocations between chromosome arms are presented. These lines can be useful for introgressing into tritordeum characters of interest from H. vulgare.     

Rapid verification of wheat-Hordeum introgressions by direct staining of SCAR, STS, and SSR amplicons.

A range of single tagged site (STS), simple sequence repeat (SSR), and sequence-characterized amplified region (SCAR) markers were screened for their utility in detecting Hordeum vulgare and H. chilense chromosomes in a wheat background. PCR conditions were optimized for specific amplification of the targeted sequences and to avoid cross-species amplification. Two H. vulgare derived STSs, six H. vulgare derived SSRs, and nine H. chilense derived SCARs were usable for the detection of five H. vulgare and three H. chilense chromosomes by direct ethidium bromide staining of the PCR products in test tubes, avoiding the more costly and time-consuming DNA electrophoresis step. The practical application of the method is illustrated by the identification of a monotelosomic substitution of H. vulgare chromosome 6HS in tritordeum and a monosomic addition of H. chilense chromosome 6Hch in durum wheat.     

Molecular cytogenetic analysis of durum wheat x tritordeum hybrids.

Southern and in situ hybridization were used to examine the chromosome constitution, genomic relationships, repetitive DNA sequences, and nuclear architecture in durum wheat x tritordeum hybrids (2n = 5x = 35), where tritordeum is the fertile amphiploid (2n = 6x = 42) between Hordeum chilense and durum wheat. Using in situ hybridization, H. chilense total genomic DNA hybridized strongly to the H. chilense chromosomes and weakly to the wheat chromosomes, which showed some strongly labelled bands. pHcKB6, a cloned repetitive sequence isolated from H. chilense, enabled the unequivocal identification of each H. chilense chromosome at metaphase. Analysis of chromosome disposition in prophase nuclei, using the same probes, showed that the chromosomes of H. chilense origin were in individual domains with only limited intermixing with chromosomes of wheat origin. Six major sites of 18S-26S rDNA genes were detected on the chromosomes of the hybrids. Hybridization to Southern transfers of restriction enzyme digests using genomic DNA showed some variants of tandem repeats, perhaps owing to methylation. Both techniques gave complementary information, extending that available from phenotypic, chromosome morphology, or isozyme analysis, and perhaps are useful for following chromosomes or chromosome segments during further crossing of the lines in plant breeding programs.     

Cloning and molecular characterization of B-hordeins from Hordeum chilense (Roem. et Schult.)

One of the main limitations of cereal breeding is the lack of genetic variability within cultivated crops. Hordeum chilense is a wild relative of Hordeum vulgare, which has been successfully used in the synthesis of amphiploids by crossing with Triticum spp. Among the agronomic traits of these new amphiploids, the allelic variation in the endosperm storage proteins and their influence on breadmaking and malting quality are of special interest. B-hordeins are sulfur rich prolamins, which account for 70–80% of the total hordein fraction in barley. In this work, rapid amplification of cDNA ends by PCR (RACE-PCR) has been used for the cloning of the full-length open reading frame (ORF) of six sequences of B3-hordeins from two lines of H. chilense. Two consensus sequences of 813 and 822 bp for the H1 and H7 lines, respectively, were determined by alignment of all the sequences generated. Between both lines, differences involving single base changes, which could correspond to single nucleotide polymorphisms (SNP), insertions and deletions were observed. Of these differences, only six out of the 13 within the ORF caused a change of amino acid. Two insertions/deletions of 9 and 12 bp were also observed between both lines. The derived amino acid sequences showed a similar structure to the B-hordeins from cultivated barley and other prolamins. The repetitive region is based on the repetition of the motif PQQPFPQQ. The copy number of the B3-hordeins was estimated as a minimum of nine and five copies for the H1 and H7 lines, respectively. The expression profile of the B-hordeins through the developing endosperm is also described in this work. This study of the storage proteins of H. chilense is a useful contribution to the knowledge of the genetic diversity available in wild relatives of cultivated barley. In addition, the origin of the different prolamins can be better understood with an in-depth knowledge of its wild equivalent.     

Genomic differentiation of Hordeum chilense from H. vulgare as revealed by repetitive and EST sequences

Hordeum vulgare, cultivated barley, and its wild relative, H. chilense, have several important traits that might be useful for wheat improvement. Here, in situ hybridization and barley expressed sequence tag (EST) markers were used to characterize and compare the chromosomes of H. chilense with those of H. vulgare. FISH with four repetitive DNA sequences, AG, AAG, 5S rDNA and 45S rDNA, was applied to the mitotic chromosomes of H. vulgare, H. chilense and available wheat-H. chilense addition and substitution lines. FISH with the AAG repeat differentiated the individual chromosomes of H. chilense and H. vulgare. The patterns of FISH signals in the two species differed greatly. The 45S rDNA signals were observed on two pairs of chromosomes in both species, while the 5S rDNA signals were observed on four pairs of chromosomes in H. vulgare and on one pair in H. chilense. The AG repeat showed FISH signals at the centromeric regions of all chromosomes of H. vulgare but none of the chromosomes of H. chilense. These results indicate that the chromosomes of the two species are highly differentiated. To study the homoeology between the two species, 209 EST markers of H. vulgare were allocated to individual chromosomes of H. chilense. One hundred and forty of the EST markers were allocated to respective chromosomes of H. chilense using the wheat-H. chilense addition and substitution lines. Twenty-six EST markers on average were allocated to each chromosome except to the chromosome 2H(ch)S, to which only 10 markers were allocated. Ninety percent of the allocated EST markers in H. chilense were placed on H. vulgare chromosomes of the same homo-eologous group, indicating that the expressed sequences of the two species were highly conserved. These EST markers would be useful for detecting chromatin introgressed from these species into the wheat genome.     

Prospects for exploitation of disease resistance from Hordeum chilense in cultivated cereals

Hordeum chilense is a South American wild barley with high potential for cereal breeding given its high crossability with other members of the Triticeae. In the present paper we consider the resistance of H. chilense to several fungal diseases and the prospects for its transference to cultivated cereals. All H. chilense accessions studied are resistant to the barley, wheat and rye brown rusts, the powdery mildews of wheat, barley, rye and oat, to Septoria leaf blotch, common bunt and to loose smuts, which suggests that H. chilense is a non-host of these diseases. There are also lines resistant to wheat and barley yellow rust, stem rust and to Agropyron leaf rust, as well as lines giving moderate levels of resistance to Septoria glume blotch, tan spot and Fusarium head blight. Some H. chilense lines display pre-appressorial avoidance to brown rust. Lines differ in the degree of haustorium formation by rust and mildew fungi they permit, and in the degree to which a hypersensitive response occurs after haustoria are formed. Unfortunately, resistance of H. chilense to rust fungi is not expressed in tritordeum hybrids, nor in chromosome addition lines in wheat. In tritordeum, H. chilense contributes quantitative resistance to wheat powdery mildew, tan spot and loose smut. The resistance to mildew, expressed as a reduced disease severity, is not associated with macroscopically visible necrosis. Hexaploid tritordeums are immune to Septoria leaf blotch and to common bunt although resistance to both is slightly diluted in octoploid tritordeums. Studies with addition lines in wheat indicate that the resistance of H. chilense to powdery mildew, Septoria leaf blotch and common bunt is of broad genetic basis, conferred by genes present on various chromosomes.     

Introgression of wheat chromosome 2D or 5D into tritordeum leads to free-threshing habit.

Hexaploid tritordeum is the amphiploid derived from the cross between the diploid wild barley Hordeum chilense and durum wheat. The non-free-threshing habit is a constraint to this species becoming a new crop. Three tritordeum lines (HT374, HT376, and HT382) showing the free-threshing habit were selected from crosses between tritordeum and bread wheat. All three lines were euploids, as revealed by mitotic chromosome counting. Genomic in situ hybridization analysis made it possible to distinguish differences among these lines. While the line HT382 carries only 10 chromosomes from H. chilense, the lines HT374 and HT376 have 12. These results suggest that HT382 is a double chromosome substitution line between H. chilense and the wheat D genome, while HT374 and HT376 each have one pair of H. chilense (Hch) chromosomes substituted by wheat D chromosomes. Molecular characterization revealed that HT382 is a 1D/(1Hch), 2D/(2Hch) chromosome substitution line, whereas HT374 and HT376 have 5D/(5Hch) substitutions. On the basis of previous knowledge, it seems that the absence of chromosome 2Hch or 5Hch is more important for producing the free-threshing habit than the presence of chromosome 2D or 5D, while chromosome 1Hch seems to be unrelated to the trait. These free-threshing tritordeum lines constitute an important advance in the tritordeum breeding program.     

D hordeins of <Emphasis Type="Italic">Hordeum chilense</Emphasis>: a novel source of variation for improvement of wheat

The high molecular weight subunits of wheat (Triticum aestivum L.) glutenin (HMW-GS) are important in determining the bread-making quality of flour and dough. There is therefore interest in transferring orthologous HMW-GS present in other grass species into wheat by wide crossing in order to extend the range of end use properties. In this work, we have isolated and characterized two genes encoding D hordeins from Hordeum chilense (Roem. et Schult.) lines H1 and H7, representing two ecotypes. The fragments were 4,305 bp for line H1 and 4,227 for line H7 and contained the promoter, coding and terminator regions. Both sequences differ in the presence of single base changes (SNPs) and insertions/deletions in the open reading frame (ORF). The encoded proteins comprise 870 and 896 amino acids for lines H1 and H7, respectively. The primary structure is similar to those of D hordeins of cultivated barley (H. vulgare L.) and HMW-GS from wheat. However, the D hordeins from H. chilense are significantly larger than those from cultivated barley due to the presence of longer repetitive regions. The H. chilense D hordeins also differ from those of cultivated barley in the distribution of the cysteine residues: whereas the D hordeins of cultivated barley contain ten cysteines with four in the repetitive domain, only nine are present in the H. chilense proteins with two in the repetitive domain. As in the HMW-GS, the central part of the D hordein proteins comprises repeated sequences based on short peptide motifs. The repetitive domain is divided in three regions named as R1 (N-terminal repeats), R2 (central degenerate repeats) and R3 (C-terminal repeats). Hexapeptide motifs are present throughout the repetitive domains of D hordeins with a consensus motif of PFQGQQ in R1 and R2 and PHQGQQ in R3. In addition, the tetrapeptide motif TTVS, which is characteristic of D hordeins of cultivated barley is present in the repetitive domain close to the protein C-terminus.     

Development of wild barley (Hordeum chilense)-derived DArT markers and their use into genetic and physical mapping

Diversity arrays technology (DArT) genomic libraries were developed from H. chilense accessions to support robust genotyping of this species and a novel crop comprising H. chilense genome (e.g., tritordeums). Over 11,000 DArT clones were obtained using two complexity reduction methods. A subset of 2,209 DArT markers was identified on the arrays containing these clones as polymorphic between parents and segregating in a population of 92 recombinant inbred lines (RIL) developed from the cross between H. chilense accessions H1 and H7. Using the segregation data a high-density map of 1,503 cM was constructed with average inter-bin density of 2.33 cM. A subset of DArT markers was also mapped physically using a set of wheat-H. chilense chromosome addition lines. It allowed the unambiguous assignment of linkage groups to chromosomes. Four segregation distortion regions (SDRs) were found on the chromosomes 2H(ch), 3H(ch) and 5H(ch) in agreement with previous findings in barley. The new map improves the genome coverage of previous H. chilense maps. H. chilense-derived DArT markers will enable further genetic studies in ongoing projects on hybrid wheat, seed carotenoid content improvement or tritordeum breeding program. Besides, the genetic map reported here will be very useful as the basis to develop comparative genomics studies with barley and model species.     

Comparative FISH mapping of two highly repetitive DNA sequences in Hordeum chilense (Roem. et Schult.)

Hordeum chilense Roem. et Schult. (2n = 14) is an autogamous wild barley from Chile and Argentina included in the section Anisolepis Nevski. This species shows interesting agronomic traits that can be incorporated into crop plant species. Hordeum chilense has been successfully crossed with species of the genus Aegilops. Among the amphiploids obtained, the hexaploid tritordeum (2n = 6x = 42, AABBHchHch) is outstanding and shows good agronomic characteristics, suggesting its potential either as a new crop or as a bridge species to introgress interesting traits into cultivated cereals. The aim of the present work was to study the hybridization patterns of the two repetitive DNA probes pAs1 and pSc119.2 to evaluate their utility for the identification of H. chilense chromosomes. Fourteen lines of H. chilense were analyzed with fluorescent in situ hybridization using probes pSc119.2 and pAs1. The probe pAs1 was more widely dispersed than pSc119.2 over the H. chilense (Hch) genome. We found 89 different signals for pAs1, distributed evenly over the whole genome, and 10 for pSc119.2, located mainly over the telomeric regions. Five distinct hybridization signals were found for pAs1 and four distinct signals for pSc119.2. These signals allow the identification of different H. chilense lines. For example, centromeric signals for pAs1 on the short arms of chromosomes 1 and 7 identify line H46, and a telomeric signal for pSc119.2 on the short arm of chromosome 2 identifies line H1. A high degree of polymorphism in the hybridization patterns was found, confirming the extensive variability present in H. chilense. This work provides tools for the identification of H. chilense chromosomes in different genetic backgrounds.     

Hordein-gene expression during development of the barley (Hordeum vulgare) endosperm.

A previous study [Rahman, Shewry & Miflin (1982) J. Exp. Bot. 33, 717-728] showed differential accumulation of the major storage proteins (called B and C hordeins) in developing endosperms of barley (Hordeum vulgare). To determine how this accumulation is regulated, we have studied mRNA fractions prepared from similar endosperms. Hordein-related mRNA species were detected some days before the deposition of hordeins in vivo. The translation products in vivo directed by polyribosomes, polysomal RNA and total cellular RNA showed similar changes in the proportions of the hordein products to those observed in the accumulations of the proteins in vivo. There was a relative increase in one of the subfamilies of B hordeins (called B1 hordein) and a decrease in the second subfamily of B hordeins (B3 hordein) and in C hordeins. The populations of RNA species related to these three groups of hordeins were studied by 'dot hybridization', with specific complementary-DNA probes for B1-, B3- and C-hordein-related sequences. This showed a 10-15-fold increase in sequences related to the B1 hordein during endosperm development, but only a 4-fold increase in sequences related to B3 and C hordeins. These results indicate that the rates of synthesis of hordeins are related to the abundance of their respective mRNA species. The different results observed for the two subfamilies of B hordeins are of interest, since they indicate differential expression of two subfamilies of genes present at a single multigenic locus.     

Molecular cloning and analysis of cDNA sequences derived from poly A+ RNA from barley endosperm: identification of B hordein related clones.

A collection of over 130 cDNA clones has been constructed in the bacterial plasmids pPH207 and pBR322 using as template the poly A+ RNA from membrane-bound polysomes of barley endosperm (cv. Sundance). Fifty four B hordein cDNA clones have been identified by cross-hybridization analysis and in vitro translation of plasmid-selected mRNAs. Hybridization of 11 of the B hordein cDNA clones to Northern blots of size-fractionated RNA indicated that the B hordein mRNA is ca. 1300 nucleotides long. One cDNA clone, pHvE-c16, has been partially sequenced and shown by comparison with C-terminal and other peptide sequences to be related to B1 hordein polypeptides. The results obtained from the analysis of the B hordein cDNA clones support the idea that the Hor 2 locus, which specifies the B hordeins, is complex and codes for a family of related mRNA species.     

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.     

Cross-species amplification of the Hordeum chilense genome using barley sequence-tagged-sites (STSs)

A selection of 51 barley Sequence-Tagged Sites (STSs) were studied for their utility in Hordeum chilense. They included four primer sets from wheat origin and six primer sets from oat origin. Forty-four primer pairs amplified H. chilense products consistently. Five primer pairs were suitable for studying the introgression of H. chilense in wheat because they amplified H. chilense products of distinct size. Six of the STSs showed polymorphism between different H. chilense accessions. The results showed that barley STSs could be useful for the genetic characterization of H. chilense, tritordeums and derived introgression lines.     

Molecular analysis of a mutation conferring the high-lysine phenotype on the grain of barley (Hordeum vulgare)

We have analyzed the molecular nature of the Riso 56 mutation that occurs in barley. This mutation results in a depression of hordein accumulation in the grain and consequently in a higher overall lysine content. In particular, the amount of B hordein, which is encoded by the complex locus Hor-2, is decreased by about 75% because of the absence of the major components. The synthesis of certain minor polypeptides, with properties similar to the major B hordeins, remains unaffected. Analysis of endosperm RNA, by in vitro translation and hybridization to various cloned cDNAs derived from hordein mRNA, shows that mRNA for the major B hordeins is not present in the endosperm. Hybridization of a B hordein cDNA clone to gel-fractionated restriction digests of mutant and wild-type DNA indicates that at least 85 kb of DNA has been deleted from the Hor-2 locus in the high-lysine mutant.