Barley doubled-haploid production by uniparental chromosome elimination

Uniparental elimination of chromosomes, which occurs in interspecific crosses between Hordeum vulgare (cultivated barley) and H. bulbosum (bulbous barley grass), is a process which can be used to produce doubled-haploid barley plants in breeding programs. We review the procedure of haploid production and the mechanism underlying selective elimination of one of the genomes during the early development of species hybrid embryos.     

Chromosome instability in Hordeum vulgare x H. bulbosum hybrids

Chromosome number in Hordeum vulgare x H. bulbosum hybrids ranged between the haploid and diploid number but with peaks in frequency occurring at the 14 and 7 chromosome level. This was reflected in a gradual change from hybrid morphology to that of haploid H. vulgare. The rate of chromosome elimination differed significantly between hybrids, while within each hybrid, differences in mean chromosome number were recorded between and within individual tillers. An increase in temperature from 25–30° C caused a significant increase in the rate of elimination of H. bulbosum chromosomes.     

A comparison of Hordeum bulbosum-mediated haploid production efficiency in barley using in vitro floret and tiller culture

Summary A high efficiency of Hordeum bulbosum-mediated haploid production in barley has been achieved using a floret culture technique in which florets pollinated with Hordeum bulbosum are cultured on modified N₆ media containing 0.5 mg/l kinetin and 1.2 mg/l2,4-D. Cultures were maintained at 25 °C with a 16 h photoperiod for 9 days before embryo rescue. In a comparison of haploid production efficiency using five F₁ hybrids from winter x winter and winter x spring barley crosses, 41.6 haploid plants/100 florets pollinated were produced using floret culture. Using detached tiller culture, 13.5 haploid plants/100 florets pollinated were produced. Higher efficiencies achieved with floret culture are attributed to the formation of larger, differentiated embryos. Such embryos lead to higher frequencies of plant regeneration. The F₁ from a winter x winter cross was inferior in haploid production compared to F₁s from winter x spring crosses. No genotype x technique interaction was observed.Oregon Agricultural Experiment Station Technical Paper No. 8653     

Anther culture and Hordeum bulbosum-derived barley doubled haploids mutations and methylation

Anther culture and Hordeum bulbosum-derived doubled haploid (DH) lines of barley (Hordeum vulgare L.) were analyzed for RFLP and RAPD polymorphisms. Polymorphisms were not detected in the anther culture-or H. bulbosum-derived DH lines among 273 RFLP and 89 polymerase chain reaction (PCR)-amplified DNA fragments assayed. It was calculated that base substitution or small deletion/insertion mutations had not been induced among 401 640 by screened. Large deletion/insertion mutations were not observed among 33 Mb screened. Polymorphisms were observed when DNA was digested with the methylation-sensitive restriction enzymes HpaII and MspI: these RFLPs originated primarily from the anther culture-derived doubled haploids. The data indicate that heritable DNA methylation changes had occurred during DH production, particularly with the anther culture method.     

Comparative mapping of the barley genome with male and female recombination-derived, doubled haploid populations

Male (anther culture) and female (Hordeum bulbosum) derived, doubled haploid populations were used to map the barley genome and thus determine the different recombination rates occurring during meiosis in the F1 hybrid donor plants. The anther culture-derived (male recombination) population showed an 18% overall increase in recombination rate. This increased recombination rate was observed for every chromosome and most of the chromosome arms. Examination of linkage distances between individual markers revealed eight segments with significantly higher recombination in the anther culture-derived population, and one in the Hordeum bulbosum-derived population. Very strong distortions of single locus segregations were observed in the anther culture-derived population, but map distances were not affected significantly by these distortions. There were 1.047 and 0.912 recombinations per chromosome in the anther culture and Hordeum bulbosum-derived doubled haploid populations, respectively.     

Effect of gibberellic acid treatment,and nutrient supply through detached tillers,upon haploid frequency in barley

Summary Haploids from Hordeum vulgare (2n = 14) X H. Bulbosum (2n = 14) crosses result after fertilization from the subsequent elimination of bulbosum chromosomes during early embryo development. Seed set from the cross is high but embryo culture is necessary to obtain seedlings. Application of gibberellin A₃ (GA₃) to pollinated florets was effective for increasing the frequency of haploid seedlings produced on both nutrient-fed detached tillers and intact plants. GA₃ increased both seed set and embryo yield. The number of cells per embryo during its transition to the haploid state was increased two to three times following GA₃ treatments. Enhanced embryo and endosperm development was attributed to increased mitotic activity. The number of visibly differentiated embryos was doubled to about 35 % of the cultured embryos after GA₃ was applied to detached tillers in nutrient solution. About 70 % of the resulting haploid plants developed from the visibly differentiated embryos. The detached tiller technique offers a convenient method of culturing haploids from field-grown plants.     

An RFLP map of diploid Hordeum bulbosum L. and comparison with maps of barley (H. vulgare L.) and wheat (Triticum aestivum L.)

This paper describes the first extensive genetic map of Hordeum bulbosum, the closest wild relative of cultivated barley. H. bulbosum is valuable for haploid production in barley breeding, and because of desirable agronomic characteristics, it also has potential for trait introgression into barley. A H. bulbosum map will assist introgression and provide a basis for the identification of QTLs for crossability with barley and other potentially useful genes. The present study used a population of 111 individuals from a PB1×PB11 cross to develop a genetic linkage map of diploid H. bulbosum (2n=2x=14) based on barley, wheat and other ”anchor” cereal RFLP markers previously mapped in other species. Because of the cross-pollinating and highly polymorphic nature of H. bulbosum, up to four alleles showed segregation at any one locus, and five different segregation types were found. This enabled maps to be developed for the PB1 and PB11 parents, as well as a combined map. In total, 136 RFLP loci were mapped with a marker coverage of 621 cM. The markers were generally colinear with barley but H. bulbosum had less recombination in the centromeric regions and similar or more in the distal regions. Cytological studies on pollen mother cells at metaphase-I showed marked distal localization of chiasmata and a frequency consistent with the genetic map length. This study showed that H. bulbosum was highly polymorphic, making it suitable for trait analysis and supplementing maps of barley. Received: 20 November 2000 / Accepted: 5 January 2001     

Characterisation of progeny from backcrosses of triploid hybrids between Hordeum vulgare L. (2x) and H. bulbosum L (4x) to H. vulgare

Interspecific hybridisations between Hordeum vulgare L. (cultivated barley) and H. bulbosum L. (bulbous barley grass) have been carried out to transfer desirable traits, such as disease resistance, from the wild species into barley. In this paper we report the results of an extensive backcrossing programme of triploid hybrids (H. vulgare 2x x H. bulbosum 4x) to two cultivars of H. vulgare. Progenies were characterised cytologically and by restriction fragment length polymorphism analysis and comprised (1) haploid and diploid H. vulgare plants, (2) hybrids and aneuploids, (3) single and double monosomic substitutions of H. bulbosum chromosomes into H. vulgare and (4) chromosomal rearrangements and recombinants. Five out of the seven possible single monosomic chromosome substitutions have now been identified amongst backcross progeny and will be valuable for directed gene introgression and genome homoeology studies. The presence amongst progeny of 1 plant with an H. vulgare-H. bulbosum translocated chromosome and one recombinant indicates the value of fertile triploid hybrids for interspecific gene introgression.     

The transfer of a powdery mildew resistance gene from Hordeum bulbosum L to barley (H. vulgare L.) chromosome 2 (2I)

Hordeum bulbosum L. is a source of disease resistance genes that would be worthwhile transferring to barley (H. vulgare L.). To achieve this objective, selfed seed from a tetraploid H. vulgare x H. bulbosum hybrid was irradiated. Subsequently, a powdery mildew-resistant selection of barley phenotype (81882/83) was identified among field-grown progeny. Using molecular analyses, we have established that the H. bulbosum DNA containing the powdery mildew resistance gene had been introgressed into 81882/83 and is located on chromosome 2 (2I). Resistant plants have been backcrossed to barley to remove the adverse effects of a linked factor conditioning triploid seed formation, but there remains an association between powdery mildew resistance and non-pathogenic necrotic leaf blotching. The dominant resistance gene is allelic to a gene transferred from H. bulbosum by co-workers in Germany, but non-allelic to all other known powdery mildew resistance genes in barley. We propose Mlhb as a gene symbol for this resistance.     

Comparative responses of tetraploid wheats pollinated with Zea mays L. and Hordeum bulbosum L.

Ten different tetraploid wheat (Triticum turgidum) genotypes were pollinated with maize (Zea mays). Fertilization was achieved in all ten genotypes and no significant difference in fertilization frequency between the tetraploid wheat genotypes was detected. A mean of 41.1% of pollinated ovaries contained an embryo. All these crosses were characterized by the elimination of the maize chromosomes, and the resulting embryos were haploids. Six of the tetraploid wheat genotypes were also pollinated with Hordeum bulbosum. Fertilization frequencies with H. bulbosum were much lower (mean=13.4%), and significant differences between the tetraploid wheat genotypes were detected. Observation of pollen tube growth revealed that part of the incompatibility reaction between tetraploid wheats and H. bulbosum was due to an effect similar to that of the Kr genes, namely pollen tube growth inhibition. These results indicate that pollinations with maize may have potential as a broad spectrum haploid production system for tetraploid wheats. Present address: Agriculture Canada, Research Branch, Central Experimental Farm, Bldg 50, Ohawa, Ontario, Canada K1A OC6     

Chromosomal instabilities in callus tissue from haploid barley (Hordeum vulgare L.)

Callus culture was derived from haploid barley embryos after crossing withHordeum bulbosum. The callus tissue is cytologically heterogeneous, containing haploid, diploid and polyploid cells. Aneuploidy and karyokinetic irregularities were also observed. Some problems of chromosomal instabilities in plant tissue cultures are discussed.     

Anther culture and Hordeum bulbosum-derived barley doubled haploids mutations and methylation

Anther culture and Hordeum bulbosum-derived doubled haploid (DH) lines of barley (Hordeum vulgare L.) were analyzed for RFLP and RAPD polymorphisms. Polymorphisms were not detected in the anther culture-or H. bulbosum-derived DH lines among 273 RFLP and 89 polymerase chain reaction (PCR)-amplified DNA fragments assayed. It was calculated that base substitution or small deletion/insertion mutations had not been induced among 401 640 by screened. Large deletion/insertion mutations were not observed among 33 Mb screened. Polymorphisms were observed when DNA was digested with the methylation-sensitive restriction enzymes HpaII and MspI: these RFLPs originated primarily from the anther culture-derived doubled haploids. The data indicate that heritable DNA methylation changes had occurred during DH production, particularly with the anther culture method.     

Genetic Control of Chromosome Elimination during Haploid Formation in Barley

Genetic control over chromosome stability in the interspecific hybrid embryos of Hordeum vulgare and H. bulbosum has been hypothesized to reside on specific chromosomes. In this study, crosses between the primary trisomic lines for the seven different H. vulgare chromosomes and tetraploid H. bulbosum revealed that both chromosomes 2 and 3 of H. vulgare were involved in the control of chromosome elimination. Subsequent crosses using the available monotelotrisomics for chromosomes 2 and 3 led to the conclusion that both arms of chromosome 2 and the short arm of chromosome 3 most likely contain major genetic factors.—From the results of this study and the genome balance observed in the interspecific crosses between H. vulgare and H. bulbosum at the diploid and tetraploid cytotypes, it appears that the factors causing the elimination of the bulbosum chromosomes are located on the H. vulgare chromosome. These factors are offset or balanced by factors on the H. bulbosum chromosomes which, when present in sufficient dosage, either neutralize the effects of the vulgare factors or are able to "protect" the bulbosum chromosomes.     

Rph22: mapping of a novel leaf rust resistance gene introgressed from the non-host Hordeum bulbosum L. into cultivated barley (Hordeum vulgare L.)

A resistance gene (Rph22) to barley leaf rust caused by Puccinia hordei was introgressed from the non-host species Hordeum bulbosum into cultivated barley. The H. bulbosum introgression in line ‘182Q20’ was located to chromosome 2HL using genomic in situ hybridisation (GISH). Using molecular markers it was shown to cover approximately 20 % of the genetic length of the chromosome. The introgression confers a very high level of resistance to P. hordei at the seedling stage that is not based on a hypersensitive reaction. The presence of the resistance gene increased the latency period of the leaf rust fungus and strongly reduced the infection frequency relative to the genetic background cultivar ‘Golden Promise’. An F₂ population of 550 individuals was developed and used to create a genetic map of the introgressed region and to determine the map position of the underlying resistance gene(s). The resistance locus, designated Rph22, was located to the distal portion of the introgression, co-segregating with markers H35_26334 and H35_45139. Flanking markers will be used to reduce the linkage drag, including gene(s) responsible for a yield penalty, around the resistance locus and to transfer the gene into elite barley germplasm. This genetic location is also known to harbour a QTL (Rphq2) for non-hypersensitive leaf rust resistance in the barley cultivar ‘Vada’. Comparison of the ‘Vada’ and H. bulbosum resistances at this locus may lead to a better understanding of the possible association between host and non-host resistance mechanisms.     

Unlocking the secondary gene‐pool of barley with next‐generation sequencing

Crop wild relatives (CWR) provide an important source of allelic diversity for any given crop plant species for counteracting the erosion of genetic diversity caused by domestication and elite breeding bottlenecks. Hordeum bulbosum L. is representing the secondary gene pool of the genus Hordeum. It has been used as a source of genetic introgressions for improving elite barley germplasm (Hordeum vulgare L.). However, genetic introgressions from H. bulbosum have yet not been broadly applied, due to a lack of suitable molecular tools for locating, characterizing, and decreasing by recombination and marker‐assisted backcrossing the size of introgressed segments. We applied next‐generation sequencing (NGS) based strategies for unlocking genetic diversity of three diploid introgression lines of cultivated barley containing chromosomal segments of its close relative H. bulbosum. Firstly, exome capture‐based (re)‐sequencing revealed large numbers of single nucleotide polymorphisms (SNPs) enabling the precise allocation of H. bulbosum introgressions. This SNP resource was further exploited by designing a custom multiplex SNP genotyping assay. Secondly, two‐enzyme‐based genotyping‐by‐sequencing (GBS) was employed to allocate the introgressed H. bulbosum segments and to genotype a mapping population. Both methods provided fast and reliable detection and mapping of the introgressed segments and enabled the identification of recombinant plants. Thus, the utilization of H. bulbosum as a resource of natural genetic diversity in barley crop improvement will be greatly facilitated by these tools in the future.     

The influence of temperature on chromosome elimination during embryo development in crosses involving Hordeum spp., wheat (Triticum aestivum L.) and rye (Secale cereale L.)

Summary Several interspecific and intergeneric crosses involving five Hordeum species, Triticum aestivum and Secale cereale were carried out to investigate the influence of two contrasting temperatures on chromosome elimination during embryo development. In four of the interspecific Hordeum crosses, chromosome elimination was significantly increased at the higher of the two temperatures, resulting in greater proportions of haploid plant progenies. However, there was no significant effect of temperature in the other interspecific cross between H. lechleri x H. bulbosum nor in the two intergeneric crosses between H. vulgare x S. cereale and T. aestivum x H. bulbosum whose progeny were exclusively hybrid and haploid, respectively.     

Locating introgressions of Hordeum bulbosum chromatin within the H. vulgare genome

Several disease-resistant recombinants between barley (Hordeum vulgare) and bulbous barley grass (H. bulbosum) have been obtained in recent years, but the process of characterization is often laborious and time-consuming. In order to improve the identification and chromosomal location of introgressed chromatin from H. bulbosum into the barley genome, we employed sequential genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). GISH enabled us to establish that an introgression was present in the disease-resistant recombinant line, and the subsequent use of FISH, with a short oligonucleotide sequence as probe, allowed us to locate the introgression on the long arm of barley chromosome 2H. These data were confirmed using RFLP probes that hybridize to barley chromosome 2HL. Received: 16 December 1998 / Accepted: 12 April 1999     

Haploidy from Hordeum interspecific crosses

Summary Interspecific crosses of Hordeum parodii (2n = 42) with H. bulbosum (2n = 14 or 28) and H. vulgare (2n = 14;, and of H. proaerum (2n = 42) with H. bulboswn, H. vulgare and H. parodii were made. Crosses between parodii and diploid bulbosum resulted in haploids (2n = 21) of parodii, whilst the crosses of parodii by tetraploid bulbosum or diploid vulgare gave hybrid progeny. The procerum by diploid bulbosum cross invariably produced haploids (2n = 21) of procerum, whereas procerum by tetraploid bulbosum or diploid vulgare crosses resulted in both hybrids and haploids of procerum. The cross between procerum and parodii gave hybrid progeny which did not reach maturity.Cytological observations on two-week-old embryos obtained from reciprocal crosses revealed chromosome variability (not less than 21 in any cell) in haploid producing crosses. This shows that chromosome elimination leads to haploid formation irrespective of which species was used as female parent.The results indicate that the ratio of the parental genomes in the zygote determines whether predominantly haploids or hybrids will be produced in any cross combination. Furthermore, procerum appears to be not only more efficient in eliminating bulbosum chromosomes in comparison with parodii, but also capable of eliminating vulgare chromosomes. The possibility of stability factors in overcoming chromosome elimination, a hierarchy of chromosome elimination and the general existence of genome balance for chromosome stability in interspecific crosses, are discussed.     

Haploidy from Hordeum interspecific crosses

Summary Hordeum arizonicum (2n=42) and H. lechleri (2n=42) were crossed with both H. bulbosum (2n=14 or 28) and H. vulgare (2n=14 or 28) and progeny plants were obtained through embryoculture. Crosses of arizonicum with diploid bulbosum invariably resulted in haploids (2n=21) of arizonicum, whereas arizonicum by tetraploid bulbosum or diploid vulgare crosses produced both hybrids and haploids of arizonicum. The lechleri by diploid bulbosum or diploid vulgare crosses resulted in haploids of lechleri, while lechleri by tetraploid bulbosum resulted in well differentiated embryos which failed to germinate.Hybrid embryos derived from the haploid producing crosses exhibit chromosome variability, suggesting that chromosome elimination leads to haploid formation.The results also indicate that the ratio of the parental genomes in the zygote is a critical factor which determines the chromosome elimination or stability in any cross combination. Furthermore, both arizonicum and lechleri appear to be of similar genetic strength in eliminating bulbosum and vulgare chromosomes. The possibility of stability factors in overcoming elimination and manipulation towards elimination are discussed.     

Marker development and characterisation of Hordeum bulbosum introgression lines: a resource for barley improvement

A set of 110 diploid putative introgression lines (ILs) containing chromatin introgressed from the undomesticated species Hordeum bulbosum L. (bulbous barley grass) into cultivated barley (Hordeum vulgare L.) has been identified using a high-copy number retrotransposon-like PCR marker, pSc119.1, derived from rye (Secale cereale L.). To evaluate these lines, 92 EST-derived markers were developed by marker sequencing across four barley cultivars and four H. bulbosum genotypes. Single nucleotide polymorphisms and insertions/deletions conserved between the two species were then used to develop a set of fully informative cleaved amplified polymorphic sequence markers or size polymorphic insertion/deletion markers. Introgressed chromatin from H. bulbosum was confirmed and genetically located in 88 of these lines using 46 of the EST-derived PCR markers. A total of 96 individual introgressions were detected with most of them (94.8%) extending to the most distal marker for each respective chromosome arm. Introgressions were detected on all chromosome arms except chromosome 3HL. Interstitial or sub-distal introgressions also occurred, with two located on chromosome 2HL and one each on 3HS, 5HL and 6HS. Twenty-two putative ILs that were positive for H. bulbosum chromatin using pSc119.1 have not had introgressions detected with these single-locus markers. When all introgressions are combined, more than 36% of the barley genetic map has now been covered with introgressed chromatin from H. bulbosum. These ILs represent a significant germplasm resource for barley improvement that can be mined for diverse traits of interest to barley breeders and researchers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.