Anther Culture-Derived Regenerants from Hordeum vulgare × H. bulbosum Crosses

Hordeum bulbosum has several desirable attributes, including disease resistance, which would be worthwhile transferring to H. vulgare. Despite homoeologous chromosome pairing in the interspecific hybrids, there have been few reports of successful gene introgression between the two species. A possible explanation for this is that recombinant male gametes are at a competitive disadvantage with normal balanced gametes during post-pollination events. To circumvent this problem, the possibility of obtaining plants directly from immature pollen grains was investigated. Anthers from diploid, triploid and tetraploid H. vulgare × H. bulbosum hybrids were cultured on defined media. Only hybrids with dehiscent anthers in vivo responded in culture, and after transfer of calli and embryoids to regeneration medium, 36 albino and 12 green plants were obtained. Seven of the green regenerants survived, one of which contained 15 H. vulgare chromosomes (including one acrocentric chromosome) and one H. bulbosum chromosome. Another regenerant (Ac166) resembled a diploid H. vulgare × H. bulbosum hybrid but had partial anther dehiscence and a slightly modified chromosome constitution. Mostly normal H. vulgare progeny were obtained from crosses between H. vulgare cv.‘Emir’ and Ac166, but three plants involved chromosome additions and substitutions.     

Association of leaf rust and powdery mildew resistance in a recombinant derived from a Hordeum vulgare × Hordeum bulbosum hybrid

While studying powdery mildew resistance in a recombinant line (code 81882) derived from a Hordeum vulgare (cv. ‘Vada’) ×Hordeum bulbosum hybrid, a low infection type of resistance to leaf rust was observed. To determine the mode of inheritance of the leaf rust resistance and whether there was linkage between the two resistances, F₂ and F₃ progenies from crosses between 81882 and ‘Vada’ were inoculated with the leaf rust and powdery mildew pathogens. Southern blots were prepared using restricted DNA extracted from leaves of 82 F₂ plants and four chromosome 2HS sequences were hybridized with the blots to define the length of the introgression. The leaf rust resistance appears to be inherited as a single dominant gene on chromosome 2HS, which co-segregates with the powdery mildew resistance. There was an almost complete association between the resistances and the respective molecular markers, but it is likely that the strong linkage results from the frequent inheritance of the introgressed H. bulbosum DNA as an intact segment of chromatin with only low levels of recombination within the segment.     

Inheritance and chromosomal location of Septoria passerinii resistance introgressed from Hordeum bulbosum into Hordeum vulgare

Septoria speckled leaf blotch (SSLB), caused by Septoria passerinii, has become one of the most serious diseases of barley in the Upper Midwest region of the USA. The recombinant line 36L5 derived from a backcross of the susceptible barley cultivar ‘Emir’ and a resistant Hordeum bulbosum parent Cb2920/4/Colch was found to be resistant to S. passerinii. Two doubled haploids derived from 36L5 were backcrossed to cv.‘Emir’ to obtain two BCF₂ populations for determining the inheritance of resistance to S. passerinii. BCF₂ progeny and BCF_2:3 families were evaluated at the seedling stage in the greenhouse for reaction to S. passerinii. BCF₂ progeny and BCF_2:3 families from both crosses segregated 3 : 1 (resistant : susceptible), and 1:2:1 (resistant : segregating : susceptible), respectively, indicating that the H. bulbosum‐derived SSLB resistance is conferred by a single dominant gene. The H. bulbosum introgressions were positioned on chromosome 4HL by genomic and fluorescent in situ hybridizations (GISH and FISH, respectively) and by Southern hybridization with the rye repetitive sequence pSc119.2. These findings indicate that SSLB resistance in H. bulbosum has the potential to be transferred and utilized in barley breeding programs.     

Gibberellic Acid + 2,4-D Improves Seed Quality in Hordeum vulgare L. ×H. bulbosum L. Crosses

Doubled haploid production from crosses between barley (Hordeum vulgare L.) and H. bulbosum L. is a well established technique for inducing homozygosity. One problem which occurs using certain barley cultivars is premature seed degeneration. In this paper, a post-pollination spray of gibberellic acid and 2,4-dichlorophenoxyacetic acid that delays the onset of senescence is described. The technique improved the number of high quality seeds to more than 75% of the seed set. This improvement should result in increased survival of embryos for culture and subsequent plant regeneration.     

The transfer of a gene conferring resistance to scald (Rhynchosporium secalis) from Hordeum bulbosum into H. vulgare chromosome 4HS

Scald is a serious foliar disease that infects barley (Hordeum vulgare L.) causing reduced yields and adversely affecting quality. A means to combat the disease is to breed cultivars that possess genetic resistance. However, all known resistance alleles have so far originated from within the primary genepool of barley. This reliance on H. vulgare and H. vulgare ssp. spontaneum as resistance sources may encourage virulent forms of the pathogen to become established. To broaden the genetic base of cultivated barley and provide novel resistances to many diseases we have used a species from the secondary genepool of barley, H. bulbosum, in a resistance‐breeding programme. In this study we describe the development and trialling of a scald‐resistant recombinant line derived from a hybrid between H. vulgare and H. bulbosum. The scald resistance is simply inherited and located on the short arm of barley chromosome 4 (4HS).     

Reduced chromosome association between the short arms of 5H homologues in Hordeum vulgare L. at metaphase I

Rod or ring bivalents at metaphase I (MI) reflect whether crossing-over has occurred on one or both pairs of chromosome arms, respectively, during meiotic prophase (pachytene). In cultivated barley ( Hordeum vulgare ) the highest frequencies of rod bivalents were found for satellite chromosomes (either 5H or 6H). To distinguish between chromosome 5H and 6H associations, a recombinant line derived from an H. vulgare × H. bulbosum hybrid with a hemizygous introgression from H. bulbosum on the short arm of chromosome 6H was probed with 45S rDNA and pSc119.2 using fluorescent in situ hybridization and compared with non-recombinant H. vulgare. The frequencies of rod bivalents, caused by failure of the short arms of 5H chromosomes (5HS) to associate during meiosis in the recombinant line, were similar to the frequencies of rod bivalents involving the satellite chromosomes in H. vulgare. Hence, rod bivalent formation in H. vulgare satellite chromosomes is probably based on reduced association of 5HS, corresponding to a low frequency of genetic recombination. 5HS is, therefore, more likely to show linkage disequilibrium than the other chromosome arms.     

Studies on Low Crossabilities Encountered with the Hordeum bulbosum Method for Haploid Production of Barley, Hordeum vulgare L.

Crosses were made between four spring barley (Hordeum vulgare L.) cultivars and five F₁, hybrids with one genotype of Hordeum bulbosum L. in two locations to investigate further previous low crossabilities which had been found in the barley cultivar ‘Apex’ with H. bulbosum. Data at all the main steps of the H. bulbosum technique were recorded and statistically analyzed. Significant differences between barley genotypes were demonstrated for all characters. It was confirmed that ‘Apex’ has poor crossability with H. bulbosum. Out of the three F₁ hybrids having ‘Apex’ as one parent, two exhibited low crossability similarly to ‘Apex’ but one showed significantly higher seed setting than ‘Apex’. The effect of the location was only significant on seed setting, while genotype X location interactions were significant on seed setting, seed quality and rate of haploid plants in relation 10 florets pollinated. Another problem which has influenced the success rate of the H. bulbosum method was found in the cultivar ‘Havilla’. Although seed setting and seed quality were high for this cultivar, embryo differentiation was low. However, this latter problem was found to influence less the overall success rate than poor crossability. Mahalanobis's distances were calculated and the dendrite of the shortest distances between barley genotypes was plotted.     

Hordeum bulbosum-a new source of disease resistance-transfer of resistance to leaf rust and mosaic viruses from H. bulbosum into winter barley

An outline is given of results for the transfer of new resistances against leaf rust and barley mosaic viruses from Hordeum bulbosum into winter barley. Progenies from backcrosses of barley cultivars with H. bulbosum hybrids were tested both in conventional breeding trials and in additional tests under controlled conditions. Resistance to both pathogens proved to be stable and of good heritability, with differences occurring which depended on the combinations used. Lines with resistance to all leaf rust and mosaic viruses tested, or to either one, were selected. Both resistances segregated independently.     

The resistance to leaf rust and powdery mildew of recombinant lines of barley (Hordeum vulgare L.) derived from H. vulgare × H. bulbosum crosses

A set of 23 recombinant lines (RLs) of barley ( Hordeum vulgare L.) derived from H. vulgare  ×  H. bulbosum L. crosses was inoculated with barley leaf rust ( Puccinia hordei ) and powdery mildew ( Blumeria graminis f.sp. hordei ) at the seedling stage to identify their levels and mechanisms of resistance. Eight RLs were studied further in glasshouse and field tests. All three barley parents ('Emir', 'Golden Promise' and 'Vada') were highly susceptible to powdery mildew and leaf rust isolates. Several RLs showed partial resistance expressed as high relative latency periods and low relative infection frequencies against leaf rust. This high level of partial resistance was due to a very high level of early aborting colonies without host cell necrosis. Several RLs showed hypersensitive resistance to some or all isolates. For powdery mildew, one RL was completely resistant to the CC1 isolate and had a hypersensitive resistance to the CO-02 isolate. Three RLs derived from 'Emir' were completely resistant to both powdery mildew isolates, and three more RLs tested in the field had higher levels of partial resistance than their parents. The results indicate that H. bulbosum contains major and minor gene(s) for resistance to leaf rust and powdery mildew that can be transferred to cultivated barley.     

The resistance of Hordeum bulbosum and its hybrids with H. vulgare to common fungal pathogens

Summary Two tetraploid and two diploid clones of Hordeum bulbosum were screened for resistance to five isolates of powdery mildew which are virulent on cultivated barley. All were resistant and this resistance was also expressed in hybrids with H. vulgare. The tetraploid genotypes were also resistant to isolates of yellow rust and brown rust. These results show that H. bulbosum contains useful genes for resistance to these diseases and that there is a potential to transfer these into cultivated barley.     

A skeletal linkage map of Hordeum bulbosum L. and comparative mapping with barley (H. vulgare L.)

A restriction fragment length polymorphism (RFLP) based linkage map of a cross between two diploid Hordeum bulbosum (2n = 2x = 14) clones, PB1 and PB11, was constructed from 46 recombinant progeny clones. Since both parents are heterozygous, separate and combined parental maps were constructed. All of the RFLP markers screened had previously been mapped in barley (H. vulgare L.) so that comparative maps could be produced. The PB1 linkage map consists of 20 RFLP marker loci assigned to four linkage groups covering 94.3 cM. The PB11 linkage map consists of 27 RFLP marker loci assigned to six linkage groups covering 149.1 cM. Thirteen markers polymorphic in both parents were used as ‘anchors’ to create a combined linkage map consisting of 38 loci assigned to six linkage groups and covering a genetic distance of 198 cM. Marker order was highly conserved in a comparison with the linkage map of H. vulgare (Laurie etal., 1995). However, in contrast, the genetic distances for the same markers were very different being 649 cM and 198 cM respectively, a genetic distance ratio of 1: 3.3. Thus although the map was short, it can be presumed to cover half the genome of H. bulbosum. This study provides further confirmation of the close relationship between the two species and gives a basis for the development of marker mediated introgression through interspecific hybridisation between the two species. This revised version was published online in July 2006 with corrections to the Cover Date.     

The influence of genotype and environment on seed and embryo development in barley (Hordeum vulgare L.) after crossing with Hordeum bulbosum L.

Summary Seed and embryo development was studied in crosses between H. bulbosum and the barley genotypes VK 16032, Vada and Vogelsanger Gold and subsequently the F₁ and F₂ progenies from VK 16032 x Vada and VK 16032 x Vogelsanger Gold. Both seed and embryo development are strongly influenced by the barley genotype. Favourable environmental conditions can promote seed and embryo development in genotypes with existing good characters. Dominant inheritance for good seed development and incomplete dominance for large embryos is evident. Linkage between small embryo size and winter habit exists in Vogelsanger Gold.     

Production of a Trisomic Series in Hordeum vulgare cv. 'Golden Promise' and its Use in Locating a Recessive Chlorina Gene

A trisomic series was produced from a triploid plant of barley (Hordeum vulgare L. cv. ‘Golden Promise’) derived from tissue culture. Its characteristics are briefly described and compared with two trisomic series reported previously. Trisomic number 1 performed poorly under glasshouse conditions. Number 2 failed to set any seed after selfing and must be maintained by pollinating with ‘Golden Promise’. The series was subsequently used to locate a recessive chlorina gene on barley chromosome 3.     

Use of Isozyme-Marked Segments from Wild Barley (Hordeum spontaneum) in Barley Breeding

Fifty-six accessions of Hordeum spontaneum from various Israeli habitats were hybridized with, and their progeny backcrossed three times to H. vulgare cv. ‘Clipper’. Selected chromosome segments marked by one of 24 isozyme loci were transferred to form 84 backcross lines which were nearly isogenic with ‘Clipper’. Each line was made homozygous for a single isozyme-marked segment. These lines were evaluated in preliminary and advanced field trials. The yield, malt extract percentage and grain size of the lines approached or equalled, and sometimes significantly surpassed, that of ‘Clipper’ and more recent commercial cultivars. Three isozyme-marked segments were combined in pairs and the three resultant doubly-marked lines were compared with the reconstituted parental, singly-marked lines and with the ‘Clipper’ recombinant from the same F²-array. The additive effects of single segments on yield or gram size were not significant, whereas half of the six interactions were significant, but in the unfavorable direction. It was concluded that near-isogenic lines can identify segments of wild barley germplasm that are useful for improving yield, but that pairs of these from different habitats may interact unfavourably.     

Genetic analysis of powdery-mildew (Erysiphe graminis f.sp. hordei) resistance derived from wild barley (Hordeum vulgare ssp. spontaneum)

The inheritance of resistance to powdery mildew was investigated in 20 accessions of Hordeum spontaneum and in 20 F₄ lines derived from crosses between the variety ‘Aramir’ and 13 accessions of H. spontaneum. Two resistance genes were detected in 17 accessions, and three resistance genes in one accession. In two accessions, only one resistance gene was present. The 20 breeding lines showed a large variation in infection type and infection level. The genetic relationship between the resistance genes detected was investigated in the seven most resistant F₄ lines. These F₄ lines were divided into three groups which carried different resistance genes. In two lines, the detected resistance gene was shown to be race-specific.     

Cloning, genetic and physical mapping of resistance gene analogs in barley (Hordeum vulgare L.)

The majority of verified plant disease resistance genes isolated to date belong to the NBS‐LRR class, encoding proteins with a predicted nucleotide binding site (NBS) and a leucine‐rich repeat (LRR) region. Using degenerate primers, designed from the conserved motifs of the NBS region in tobacco N and Arabidopsis RPS2 genes, we isolated 190 resistance gene analogs (RGA) clones from barley genomic DNA. A total of 13 single‐ and low‐copy RGAs were genetically mapped onto chromosomes 1H–7H (except 5H) using three barley double haploid (DH) mapping populations: Steptoe × Morex, Harrington × TR306 and LUGC × Bowman. Sequence analysis of the RGAs showed that they are members of a diverse group. As a result of BLAST searches, one RGA proved unique as it did not detect any significant hit. Another RGA is putatively functional, because it detected several barley expressed sequence tag (EST) matches. To physically map the RGAs, 13 sequences were used to screen a 6.3 × cv. ‘Morex’ bacterial artificial chromosome (BAC) library. After fingerprint analysis, eight contigs were constructed incorporating 62 BAC clones. These BAC contigs are of great value for positional cloning of disease resistance genes, because they span the regions where various barley R genes have been genetically mapped.     

Genetic diversity in landrace populations of Hordeum vulgare L. from Sardinia, Italy, as revealed by RAPDs, isozymes and morphophenological traits

Barley is the second most widely cultivated cereal on the island of Sardinia. It is grown for green fodder, grain and straw, all of which are used for animal feet. The cultivation of modem varieties is limited and many farmers grow local populations of the six-row landrace ‘S'orgiu sardu’. Twelve local populations (20 strains per population) were evaluated for 13 random amplified polymorphic DNA (RAPD) markers, sisisozyme loci and five morphophenological traits. Results showed that Sardinian barley is characterized by a lax six-row spike and is. in general, a spring type. Isozyme polymorphism was delected for only two esterase loci (Est and Est 4). The genetic variation for all types of murkers, was found located mostly within population (about 84%, for isozymes aud morphaphenological traits and 89%, for RAPDs). This finding appears to confirm the hypothesis that the populations studied belong to the same land race called ‘S'orgiu sardu’ by farmers. A geographical differential ion between southern and northern populations was foand in relation to distinct environmental characteristics. Taking into account all types of marker 209 (90.5%) different genotypes were identified out of 231 individuals. The overall results confirm the high variability that can be found within landrace populations, underlining the value of landraccs as a source of genetic variation.     

Genetic analysis of β-amylase thermostability to develop a DNA marker for malt fermentability improvement in barley, Hordeum vulgare

β‐Amylase thermostability is one of the major factors affecting fermentability in the brewing process; consequently, it could be used as a selection marker for the trait. In order to clarify what controls its thermostability, the linkage analysis of β‐amylase thermostability and its genotype as restriction fragment length polymorphism patterns was performed in three cross populations. Then, β‐amylase cDNAs cloned from the three varieties which had a different thermostability type were expressed in Escherichia coli. According to the results of the linkage analysis and gene expression test, it was concluded that β‐amylase thermostability resulted from a difference in its structural gene. Furthermore, to construct an STS marker for the gene, the gDNA sequences of β‐amylase were compared among the three varieties, which had different thermostabilities. Although there were many differences in the intron sequence, few nucleotides differed in the exon region. Based on the variation in the intron region, a sequence‐tagged‐site marker was constructed to detect β‐amylase genotypes in breeding material.     

A novel barley scald resistance gene: genetic mapping of the Rrs15 scald resistance gene derived from wild barley, Hordeum vulgare ssp. spontaneum

Most genes for resistance to barley leaf scald map either to the Rrs1 locus on the long arm of chromosome 3H, or the Rrs2 locus on the short arm of chromosome 7H. Other loci containing scald resistance genes have previously been identified using lines derived from wild barley, Hordeum vulgare ssp. spontaneum. A single dominant gene conditioning resistance to scald was identified in a third backcross (BC3F3) line derived from an Israeli accession of wild barley. The resistance gene is linked to three microsatellite markers that map to the long arm of chromosome 7H; the closest of these loci, HVM49, maps 11.5 cM from the resistance gene. As no other scald resistance genes have been mapped to this chromosome arm, it is considered to be a novel scald resistance locus. As the Acp2 isozyme locus is linked to this scald resistance locus, at 17.7 cM, Acp2 is assigned to chromosome 7H. Molecular markers linked to the novel scald resistance gene, designated Rrs15, can be used in breeding for scald resistance.     

The influence of photoperiod on the Vrn-H2 locus (4H) which is a major determinant of plant development and reproductive fitness traits in a facultative × winter barley (Hordeum vulgare L.) mapping population

To determine the effect of Vrn‐H2 locus on plant developmental and agronomic traits, detailed controlled environment tests involving a factorial set of vernalization and photoperiod treatments were carried out using doubled haploid lines developed from a facultative (Vrn‐H2^?) × winter (Vrn‐H2⁺) barley cross. The allele phase at the Vrn‐H2 locus influenced heading date as well as the developmental and agronomic traits. The performance of Vrn‐H2⁺ lines was significantly influenced by vernalization: reproductive fitness traits showed significant decreases without vernalization. However, the effects of alleles at the Vrn‐H2 locus extended beyond simple satisfaction of the vernalization requirement. Vrn‐H2⁺ lines showed increased reproductive fitness compared with Vrn‐H2^? lines when vernalization was followed by a long photoperiod. The responses of the two Vrn‐H2 allele classes to photoperiod duration were quite different in terms of heading date, developmental and agronomic traits. These results suggest that alleles at the Vrn‐H2 locus – and/or tightly linked gene(s) – respond primarily to the exogenous signal of vernalization (temperature), but when the vernalization requirement has been fulfilled, they also respond to photoperiod duration.