Pleiotropic effects on the ml-o mildew resistance gene in barley in different genetical backgrounds

Summary The effects on quantitative characters of the ml-o5 allele has been investigated by means of doubled haploid lines produced by the Hordeum bulbosum method. In one series of experiments the genetic background in each cross was found significantly to modify the negative pleiotropic effects. Doubled haploids were also produced from a F1 cross and tested in the field during two years, one with strong and one with weak mildew infections. In the first year only an insignificant reduction in grain yield was detected in the mlo lines, in the second it was significantly reduced with 5.9%. Thousand grain weights were, however, higher in the resistant lines. In both series of experiments the possibility to extract very high yielding mlo genotypes was demonstrated. The nature of the genetic background effect is discussed.     

The evaluation of superior Hordeum bulbosum L. genotypes for use in a doubled haploid barley breeding programme

Summary Eight Hordeum bulbosum selections were produced from a cross between Cb 2920/4 and Cb 2929/1, two genotypes widely used in doubled haploid breeding programmes. The selections were hybridized with barley to evaluate their ability to produce high proportions of well-differentiated haploid embryos compared with Cb 2929/1 as control. We report here an initial small-scale investigation followed by a larger-scale test in two different environments to assess seed setting, haploid embryo differentiation rates and VB hybrid formation. These VB embryos contain both parental sets of chromosomes and occur more frequently in the glasshouse during the winter. Two of the eight selections were identified as combining the desirable characteristics of both parents, namely high seed setting on cv. Vada which is partially incompatible with H. bulbosum, large numbers of well-differentiated haploid embryos and a low incidence of VB hybrids. The selections are available for release to interested research workers and plant breedersAbbreviations DH doubled haploid - VB a hybrid from H. vulgare x H. bulbosum which contains both parental sets of chromosomes     

Allotetraploid hybrids produced by protoplast fusion for seedless triploid <Emphasis Type="BoldItalic">Citrus</Emphasis> breeding

In connection with crop improvement strategies for marginal regions it has been proposed to increase the outcrossing rate in barley which would presuppose high and persistent pollen viability to ensure successful cross fertilisation. The present study was designed to investigate the viability of mature pollen from extruded and non-extruded anthers of cultivated and wild barley (Hordeum vulgare ssp vulgare and ssp spontaneum, respectively) in comparison with the closely related but obligatory outcrossing species H. bulbosum. Pollen viability (PV) was assessed employing the p-phenylenediamine-peroxidase-test on pollen derived from spikes or anthers immediately after collection and after treatment at 20, 30 and 40 ^∘C for 1, 2, 4, 8 and 16 h. The latter treatment was interposed after 8 h by a 10 h period of darkness at 12 ^∘C and thus called 26 h treatment, consecutively. Initial PV was high with 98% across all genotypes and even at 40 ^∘C did not fall below 80% after 8 h. After the 26 h treatment, PV of two H. vulgare genotypes originating from semi-arid regions and of H. bulbosum fell below 60% while the other genotypes retained a PV of > 80%. Viability of pollen of extruded anthers in H. vulgare ssp. spontaneum was on average slightly but significantly lower than PV of non-extruded anthers but still remained above 90%, even after the 26 h treatment at 40 ^∘C. Pollen viability of the outcrossing species H. bulbosum ranged on a very similar level as PV of H. vulgare. Results indicate that pollen of H. vulgare retains a sufficiently high level of viability to ensure successful cross-fertilisation over a period of at least 26 h even at high temperatures of up to 40 ^∘C.     

Comparison of Anther Culture and Hordeum bulbosum Method for the Production of Doubled Haploid in Winter Barley I. Production of Green Plants

The relative response to two-major techniques of producing haploids in barley – anther culture and the Hordeum bulbosyum method – were compared using seven F₁ hybrids from a winter barley breeding programme. Both techniques were Applied to the same plants with the same number of replications. 358 viable green plants were obtained from 420 spikes ot donor plants used: 147 through anther culture and 211 through the H. bulbosum method. From the data analysis we found that the response to the two techniques was not significantly different, genotype effect and genotype × technique interaction were highly significant. The possibility of combining the two techniques in a doubled haploid production programme is discussed.     

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 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.     

The assessment of variation in two populations of Hordeum bulbosum L. for improving success rates in a doubled haploid barley programme

Summary Improvements in the success of doubled haploid production have been achieved partly by screening new accessions of Hordeum bulbosum. In order to assess levels of variation within stocks of this species, rates of embryo differentiation from the cross between H. vulgare × H. bulbosum were recorded for selections derived from two different stocks of H. bulbosum. There was little difference within stocks for this character despite variation in the morphology and banding patterns of two enzyme systems of one of the stocks. It is proposed that to obtain further increases in success rates a few selections from many accessions of H. bulbosum should be screened rather than many selections from a few stocks.     

Morphological and molecular analysis of androgenetic, selfed and backcrossed plants produced from a Hordeum vulgare x H, bulbosum hybrid

Anther culture (AC) was carried out on a fertile triploid hybrid between Hordeum vulgare L. (cultivated barley) and H. bulbosum L, (bulbous barley grass) to determine whether AC-derived regenerants differed from progeny obtained through selfing and backcrossing. Chromosome counts were carried out on all plants and DNA was extracted from them to prepare Southern blots for molecular analysis. To identify true recombinants, the blots were probed with rye repetitive sequence probes (pSc119.1 and pScl19.2). which hybridize strongly and specifically to H. bulbosum DNA. Twenty probes that detect single- or low-copy sequences were hybridized with Southern blots containing restricted DNA extracted from 25 AC-derived plants, 11 selfed and six backcrossed progeny that showed hybridizations with pScll9. Although restriction fragment length polymorphisms (RFLPs) were only observed using probes that map to four of the possible 14 chromosome arms, an introgression associated with chromosome 6HS was frequently observed among plants derived from AC. selfing and backcrossing. Plants from AC differed from selfed and backcrossed progeny in their chromosome number; unique RFLP bands that were occasionally observed may indicate chromosomal rearrangements.     

Influence of variety, seed position and seed source on screening for coleoptile length in bread wheat (Triticum aestivum L.)

A study was conducted to evaluate the usefulness of somaclonal variation as a means to obtain powdery mildew resistance in the background of an agronomically elite, high-yielding barley (Hordeum vulgare L.) cultivar. A total of 170 R_o-derived lines were regenerated from embryo-induced callus of the barley cultivar Léger. Forty-five lines were selected and evaluated in replicated field plots at two locations in Eastern Canada. In comparison with Léger, one of the 45 lines was higher yielding, one produced a greater test weight, two had a greater seed weight, and one was shorter in plant height. Three lines were found to segregate for resistance to powdery mildew (Erysiphe graminis DC ex Merat f. sp. hordei EM). Many of the single-plant selections from the three resistant lines showed resistance to powdery mildew under field conditions for two years. Three lines were eventually promoted to the official registration tests in Ontario. One of the three lines was subsequently registered as a new cultivar (AC Malone) in Canada. To our knowledge, AC Malone is the world's first barley cultivar selected from somaclonal variation. The results of this study demonstrate that it is possible to obtain high- yielding or disease-resistant lines from callus culture in barley. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison of Anther Culture and the Hordeum bulbosum Method for the Production of Doubled Haploids in Winter Barley

The comparison, between the efficiency of anther culture and the Hordeum bulbosum method in barley was extended to the chromosome number distributions of all planes derived from the two techniques and the proportions of fertile doubled haploid plants which survived until maturity. The frequencies of haploid and spontaneously doubled haploid plants which were useful for practical breeding purposes were found to be around 90 % for both techniques. The remainder consisted of polyploid, mixoploid and aneuploid variants in the case of microspore-derived plants and diploid interspecific hybrids in the progeny of the H. bulbosum method. The ploidy level distributions of the microspore- and H. bulbosum-derived plants appeared to be independent of the genotype of the donor. There were no significant differences between techniques regarding the proportions, of plants which survived a severe winter and the production of fertile doubled haploid plants. Both techniques can therefore complement each other in a breeding programme and their relative merits are discussed. Possible ways of improving doubled haploid production in barley are suggested for increasing its use in breeding schemes.     

Attempts to overcome partial incompatibility between Hordeum vulgare L. and H. bulbosum L.

Summary Partial incompatibility has already been reported between Hordeum vulgare and H. bulbosum during the course of doubled haploid production, and in this paper attempts to overcome the breeding barrier are described. The methods which seem to offer most success are those of environmental adjustments and the adoption of new genotypes of H. bulbosum. Further cultivars of H. vulgare exhibiting this phenomenon are also noted.     

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.     

Segregation of 12 isozyme genes among doubled haploid lines derived from a japonica x indica cross of rice (Oryza sativa L.)

Summary The segregation of 12 heterozygous isozyme markers was analyzed among F₂ plants and 51 anther culture (AC)-derived lines obtained from the japonica × indica cross of rice, IRAT 177 × Apura. All the lines except two were homozygous products of recombination of the two parental phenotypes. Doubled haploid (DH) lines derived from plants regenerated from the same callus were identical, confirming previously obtained results in rice. Surprisingly, some lines derived from different calli were also identical, suggesting a phenomenon of early callus fragmentation. All these observations at the isozyme level were confirmed by field evaluation. Deviations of segregations from the expected 1 : 1 ratio were observed at 4 loci among the DH lines. Among these, two were also noted among the F₂ plants. The two other distortions, both in favor of the japonica allele, were observed specifically in the AC-derived materials.Although this concerns a small proportion of the genes under study, it suggests that the embryogenic microsporal population does not represent a random gametic array. On the other hand, evaluation of recombination between isozyme genes located on chromosome 6 appears consistent with F₂ data and data previously recorded on the other japonica × indica crosses. The potential use of isozymes in breeding doubled haploids derived from remote crosses in rice is discussed.Abbreviations MCPA = 2-methyl-4-chlorophenoxyacetic acid - IAA = indolacetic acid - AC plant or line = anther culture-derived plant or line - DH line = doubled haploid line     

Comparison of Spring Barley Lines Produced by Single Seed Descent, Pedigree Inbreeding and Doubled Haploidy

Samples of inbred lines of Hordeum vulgare produced by pedigree inbreeding, doubled haploidy and single seed descent have been compared for a range of agronomic characters. Differences between the means of the in bred s produced by single seed descent and pedigree breeding were due to differing levels of inbreeding. The present study confirmed chat recombinant inbred families which out-perform the higher scoring parent may be produced by either method. Caution should, however, be exercised when simply inherited characters controlled by major genes are fixed in early generations. Differences between the doubled haploid and single seed descent samples for the character thousand grain weight were attributable to coupling linkages involving epistatic genes. The production and evaluation of random inbred lines should be considered as an alternative to pedigree methods of barley breeding.     

Segregation distortion for agronomic traits in doubled haploid lines of barley

Four barley doubled haploid populations were produced by anther culture from the reciprocal crosses between two six‐row barley cultivars, ‘Plaisant’ and ‘Orria’; the doubled haploid lines (DHLs) derived from each cross were subsequently assigned to weak or vigorous populations according to the weak or vigorous nature of the originating embryos. Well‐formed embryos at day 25 on the induction medium were considered vigorous, whereas embryos maturing later were considered weak. The classification of vigorous and weak was closely associated with the ratio of green to albino plantlets regenerated. A random set of 25 DHLs from each of the four populations were selected for field testing in a replicated trial. Furthermore, a second set consisting of a total of 454 unreplicated DHLs from the four populations were also field assessed for grain yield. Distortion during in vitro culture may impede regeneration of a random array of microspores from a given cross, and may bias genetic estimates of specific trait/marker association in genetic studies. However, no significant differences were detected in this study among the four populations for days to heading, height, grain yield and thousand‐kernel‐weight when measured on the replicated trial of 100 DHLs, nor for grain yield in the second collection of 454 entries. This suggests that the likelihood of producing improved agronomic pure lines is independent of the direction of crossing and, more importantly, independent of the time when embryos matured in the induction media, at least for these particular six‐row cultivars and for the anther culture method used.     

The assessment of in vitro characters and their influence on the success rates of doubled haploid production in barley

Summary A assessment of in vitro characters: embryo size, differentiation stage and germination time of haploid embryos, was carried out with a view to developing a selection procedure to increase the efficiency of doubled haploid (DH) production using the Hordeum bulbosum system. The differentiation stage of haploid embryos at the time of excision was found to be the most promising character for selection. In the two spring crosses examined 55% of excised embryos were undifferentiated and 45% were differentiated, with overall success rates, in terms of the percentage of DHs produced, of 7.91% and 25.16%, respectively. 17% of excised embryos produced callus. These embryos performed poorly in terms of regeneration and subsequent growth. It is suggested that only differentiated non-callusing embryos are maintained in order to maximize the efficiency of DH production. It is also shown that the overall success rate depends not only on initial stages such as seed setting but also on genotypic differences in response to colchicine treatment at later stages.     

The effect of pollination bag type on seed quality and size in Hordeum inter- and intraspecific hybridization

Summary The effects of different spike covering materials have been investigated in crosses between Hordeum vulgare and H. bulbosum and in selfed H. vulgare. It was found that after lemmas and paleas were clipped, improvements in seed quality (H. vulgare × H. bulbosum) and weight (selfed H. vulgare) were obtained by covering heads with small brown paper bags compared with other treatments. Possible reasons for these effects are discussed but so far the mechanism has not been clarified. However, light is suggested as playing a major role.     

The production and behaviour of hexaploid hybrids between Hordeum vulgare and H. Bulbosum

Summary To produce hexaploid (or other polyploid) hybrids, diploid or tetraploid Hordeum vulgare was crossed with hexaploid or octoploid H. bulbosum, and perennial triploid hybrids between the two species were treated with colchicine. The crosses did not yield viable plants: seedset was low, the seed aborted and embryo culture was unsuccessful. The colchicine treatments geve rise to plants in which hexaploid chromosome numbers were observed. At the hexaploid level chromosomal instability occurred, resulting in chromosome elimination.The colchicine-treated triploid hybrids showed in the first years after the treatment better fertility after open flowering than untreated plants, but the level of fertility remained very low. The offspring consisted of haploid, diploid and approximately triploid plants like H. vulgare, tetraploid and approximately tetraploid plants like H. bulbosum, and plants with hybrid morphology and unstable chromosome number, which were highly sterile. Thus the crossing barrier between H. vulgare and H. bulbosum could not be broken down at higher ploidy level.     

Embryo development in two barley genotypes after self pollination and pollination with Hordeum bulbosum L.

Summary Embryo development in vivo was compared in the two barley genotypes VK 16032 and Vogelsanger Gold after self pollination and after pollination with H. bulbosum. Embryo growth in VK 16032 after crossing with H. bulbosum showed a similar growth pattern to that of the self pollinated embryos, although the size increase was smaller. The hybrid embryos continued to grow until day 18 after pollination. The embryos from the cross between Vogelsanger Gold and H. bulbosum ceased to grow 8 days after pollination. Arrested embryo growth with subsequent abortion in Vogelsanger Gold was associated with a very early depletion and break down of the endosperm. Use of barley genotypes less sensitive to the genomic disturbances after crossing with H. bulbosum in combination with genotypes like Vogelsanger Gold is recommended in haploid breeding work.     

Crosses between Hordeum vulgare L. and H. bulbosum L. I. Production,morphology and meiosis of hybrids,haploids and dihaploids

Summary From crosses between diploid and autotetraploid cytotypes of Hordeum vulgare L. (cultivated barley) and H. bulbosum L. (bulbous barley grass) diploid, triploid and tetraploid interspecific hybrids were produced. Both directly and after vegetative segregation crosses in either direction also gave rise to haploids and dihaploids resembling H. vulgare. The use of embryo culture was necessary. Plant morphology of the hybrids was much like that of H. bulbosum, although the hybrid plants were less vigorous. Meiosis in the hybrids was more or less disturbed, and this seemed to be the main cause of the high level of sterility.