Bread Wheat Biofortification for Grain Carotenoid Content by Inter-Specific Breeding.

Bread wheat has traditionally been selected for whitish derived flours. As a consequence, the current varieties carry carotenogenic alleles associated with low grain carotenoid. In contrast, high grain yellow pigment content (YPC) has been a major target in durum wheat programs since yellow colour is an important aesthetic factor for pasta production. Phytoene synthase 1 (Psy1) genes have an important role in the determination of the carotenoid content in wheat. In this work, we have transferred the genes Psy1-A1 and Psy1-B1 from durum to bread wheat by inter-specific hybridization in order to evaluate the combined effect of these genes for the improvement of grain carotenoid content, as well as the development of carotenoid-enriched bread wheat lines. Inter-specific breeding coupled with a MAS approach based on Psy1-A1 and Psy1-B1 alleles has allowed the development of bread wheat pre-breeding lines with enhanced grain carotenoid content (16-23% mean). These biofortified lines have the potential to become new varieties or to be used as recurrent parents in bread wheat breeding programs.

Marker-Trait Associations for Total Carotenoid Content and Individual Carotenoids in Durum Wheat Identified by Genome-Wide Association Analysis.

Yellow pigment content is one of the main traits considered for grain quality in durum wheat (Triticum turgidum L.). The yellow color is mostly determined by carotenoid pigments, lutein being the most abundant in wheat endosperm, although zeaxanthin, α-carotene and ß-carotene are present in minor quantities. Due to the importance of carotenoids in human health and grain quality, modifying the carotenoid content and profile has been a classic target. Landraces are then a potential source for the variability needed for wheat breeding. In this work, 158 accessions of the Spanish durum wheat collection were characterized for carotenoid content and profile and genotyped using the DArTSeq platform for association analysis. A total of 28 marker-trait associations were identified and their co-location with previously described QTLs and candidate genes was studied. The results obtained confirm the importance of the widely described QTL in 7B and validate the QTL regions recently identified by haplotype analysis for the semolina pigment. Additionally, copies of the Zds and Psy genes on chromosomes 7B and 5B, respectively, may have a putative role in determining zeaxanthin content. Finally, genes for the methylerythritol 4-phosphate (MEP) and isopentenyl diphosphate (IPPI) carotenoid precursor pathways were revealed as additional sources of untapped variation for carotenoid improvement.

The breeder's tool-box for enhancing the content of esterified carotenoids in wheat: From extraction and profiling of carotenoids to marker-assisted selection of candidate genes.

Carotenoid esterification is a new target for cereal biofortification since esterification increases both accumulation and stability of carotenoids. A xanthophyll acyl transferase is responsible for carotenoid esterification in the endosperm of wheat and related cereals. In this chapter we describe the procedures for transferring the carotenoid esterification attribute into wheat using the wild barley Hordeum chilense as donor of the esterification trait, the outline of the breeding program and the protocols for marker assisted selection and the analysis of carotenoids in grain. Biofortified cereals with increased lutein ester content will help to reduce the risk of developing age-related macular degeneration in human populations with limited access to other dietary sources.

Genome-Wide Association Analysis for Stem Cross Section Properties, Height and Heading Date in a Collection of Spanish Durum Wheat Landraces.

Durum wheat landraces have a high potential for breeding but they remain underexploited due to several factors, including the insufficient evaluation of these plant materials and the lack of efficient selection tools for transferring target traits into elite backgrounds. In this work, we characterized 150 accessions of the Spanish durum wheat collection for stem cross section, height and heading date. Continuous variation and high heritabilities were recorded for the stem area, pith area, pith diameter, culm wall thickness, height and heading date. The accessions were genotyped with DArTSeq markers, which were aligned to the durum wheat 'Svevo' genome. The markers corresponding to genes, with a minor allele frequency above 5% and less than 10% of missing data, were used for genome-wide association scan analysis. Twenty-nine marker-trait associations (MTAs) were identified and compared with the positions of previously known QTLs. MTAs for height and heading date co-localized with the QTLs for these traits. In addition, all the MTAs for stem traits in chromosome 2B were located in the corresponding synteny regions of the markers associated with lodging in bread wheat. Finally, several MTAs for stem traits co-located with the QTL for wheat stem sawfly (WSS) resistance. The results presented herein reveal the same genomic regions in chromosome 2B are involved in the genetic control of stem traits and lodging tolerance in both durum and bread wheat. In addition, these results suggest the importance of stem traits for WSS resistance and the potential of these landraces as donors for lodging tolerance and WSS resistance enhancement. In this context, the MTAs for stem-related traits identified in this work can serve as a reference for further development of markers for the introgression of target traits into elite material.

Tritordeum: Creating a New Crop Species-The Successful Use of Plant Genetic Resources.

Hexaploid tritordeum is the amphiploid derived from the cross between the wild barley Hordeum chilense and durum wheat. This paper reviews the main advances and achievements in the last two decades that led to the successful development of tritordeum as a new crop. In particular, we summarize the progress in breeding for agronomic performance, including the potential of tritordeum as a genetic bridge for wheat breeding; the impact of molecular markers in genetic studies and breeding; and the progress in quality and development of innovative food products. The success of tritordeum as a crop shows the importance of the effective utilization of plant genetic resources for the development of new innovative products for agriculture and industry. Considering that wild plant genetic resources have made possible the development of this new crop, the huge potential of more accessible resources, such as landraces conserved in gene banks, goes beyond being sources of resistance to biotic and abiotic stresses. In addition, the positive result of tritordeum also shows the importance of adequate commercialization strategies and demonstrative experiences aimed to integrate the whole food chain, from producers to end-point sellers, in order to develop new products for consumers.

Durum Wheat (Triticum durum L.) Landraces Reveal Potential for the Improvement of Grain Carotenoid Esterification in Breeding Programs.

Carotenoids are essential in the human diet for their important functions in health. Besides, they are responsible for the yellow pigments desirable for industrial quality in durum wheat. The remarkable carotenoid content of durum wheat endosperm is mostly due to lutein. However, lutein esters have not been previously detected in durum wheat as in other cereals such as common wheat, tritordeum or Hordeum chilense. Esterification increases carotenoid stability and allows greater retention and accumulation through the food chain. Therefore, carotenoid esterification is revealed as a new key target in breeding. We characterized the carotenoid profile of 156 accessions of the Spanish durum wheat collection, searching for landraces with esterification ability. Interestingly, four accessions produced lutein monoesters and diesters. Also, traces of lutein monoesters were detected in eleven accessions. The identification of the first durum wheat accessions with esterification ability reported herein is a remarkable advance for carotenoid biofortification. Furthermore, variation for the relative content of zeaxanthin, α-carotene and ß-carotene was also observed. This diversity for the ß,ε and ß,ß branches of the carotenogenic pathway also represents a new opportunity for breeding for specific carotenoids in biofortification programs.

Mediation of a GDSL Esterase/Lipase in Carotenoid Esterification in Tritordeum Suggests a Common Mechanism of Carotenoid Esterification in Triticeae Species.

Carotenoids are essential in human diet, so that the development of programs toward carotenoid enhancement has been promoted in several crops. The cereal tritordeum, the amphiploid derived from the cross between Hordeum chilense Roem. et Schulz. and durum wheat has a remarkable carotenoid content in the endosperm. Besides, a high proportion of these carotenoids are esterified with fatty acids. The identification of the gene(s) responsible for xanthophyll esterification would be useful for breeding as esterified carotenoids show an increased ability to accumulate within plant cells and have a higher stability during post-harvest storage. In this work, we analyzed five genes identified as candidates for coding the xanthophyll acyltransferase (XAT) enzyme responsible for lutein esterification in H. chilense genome. All these genes were expressed during grain development in tritordeum, but only HORCH7HG021460 was highly upregulated. Sequence analysis of HORCH7HG021460 revealed a G-to-T transversion, causing a Glycine to Cysteine substitution in the protein of H290 (the only accession not producing quantifiable amounts of lutein esters, hereinafter referred as zero-ester) of H. chilense compared to the esterifying genotypes. An allele-specific marker was designed for the SNP detection in the H. chilense diversity panel. From the 93 accessions, only H290 showed the T allele and the zero-ester phenotype. Furthermore, HORCH7HG021460 is the orthologue of XAT-7D, which encodes a XAT enzyme responsible for carotenoid esterification in wheat. Thus, HORCH7HG021460 (XAT-7Hch) is a strong candidate for lutein esterification in H. chilense and tritordeum, suggesting a common mechanism of carotenoid esterification in Triticeae species. The transference of XAT-7Hch to wheat may be useful for the enhancement of lutein esters in biofortification programs.

Transcriptomics, chromosome engineering and mapping identify a restorer-of-fertility region in the CMS wheat system msH1.

KEY MESSAGE: An original RNA-seq mapping strategy, validated with chromosome engineering and physical mapping, identifies candidate genes for fertility restoration in the 6HchS chromosome of Hordeum chilense in the wheat msH1 system. Cytoplasmic male sterility (CMS) is a valuable trait for hybrid seed production. The msH1 CMS system in common wheat results from the incompatibility between the nuclear genome of wheat and the cytoplasm of the wild barley Hordeum chilense. This work aims to identify H. chilense candidate genes for fertility restoration in the msH1 system with a multidisciplinary strategy based on chromosome engineering, differential expression analysis and genome mapping. Alloplasmic isogenic wheat lines differing for fertility, associated with the presence of an acrocentric chromosome Hchac resulting from the rearrangement of the short arms of H. chilense chromosomes 1Hch and 6Hch, were used for transcriptome sequencing. Two novel RNA-seq mapping approaches were designed and compared to identify differentially expressed genes of H. chilense associated with male fertility restoration. Minichromosomes (Hchmi), new smaller reorganizations of the Hchac also restoring fertility, were obtained and used to validate the candidate genes. This strategy was successful identifying a putative restorer-of-fertility region on 6HchS, with six candidate genes, including the ortholog of the barley restorer gene Rfm1. Additionally, transcriptomics gave preliminary insights on sterility and restoration networks showing the importance of energy supply, stress, protein metabolism and RNA processing.

A glycosyl transferase family 43 protein involved in xylan biosynthesis is associated with straw digestibility in Brachypodium distachyon.

The recalcitrance of secondary plant cell walls to digestion constrains biomass use for the production of sustainable bioproducts and for animal feed. We screened a population of Brachypodium recombinant inbred lines (RILs) for cell wall digestibility using commercial cellulases and detected a quantitative trait locus (QTL) associated with this trait. Examination of the chromosomal region associated with this QTL revealed a candidate gene that encodes a putative glycosyl transferase family (GT) 43 protein, orthologue of IRX14 in Arabidopsis, and hence predicted to be involved in the biosynthesis of xylan. Arabinoxylans form the major matrix polysaccharides in cell walls of grasses, such as Brachypodium. The parental lines of the RIL population carry alternative nonsynonymous polymorphisms in the BdGT43A gene, which were inherited in the RIL progeny in a manner compatible with a causative role in the variation in straw digestibility. In order to validate the implied role of our candidate gene in affecting straw digestibility, we used RNA interference to lower the expression levels of the BdGT43A gene in Brachypodium. The biomass of the silenced lines showed higher digestibility supporting a causative role of the BdGT43A gene, suggesting that it might form a good target for improving straw digestibility in crops.

Physiological, biochemical and molecular characterization of an induced mutation conferring imidazolinone resistance in wheat.

The Clearfield(®) wheat cultivars possessing imidazolinone (IMI)-resistant traits provide an efficient option for controlling weeds. The imazamox-resistant cultivar Pantera (Clearfield(®) ) was compared with a susceptible cultivar (Gazul). Target and non-target mechanisms of resistance were studied to characterize the resistance of Pantera and to identify the importance of each mechanism involved in this resistance. Pantera is resistant to imazamox as was determined in previous experiments. The molecular study confirmed that it carries a mutation Ser-Asn627 conferring resistance to imazamox in two out of three acetolactate synthase (ALS) genes (imi1 and imi2), located in wheat on chromosomes 6B and 6D, respectively. However, the last gene (imi3) located on chromosome 6A does not carry any mutation conferring resistance. As a result, photosynthetic activity and chlorophyll content were reduced after imazamox treatment. Detoxification was higher in the resistant biotype as shown by metabolomic study while imazamox translocation was higher in the susceptible cultivar. Interestingly, imazamox metabolism was higher at higher doses of herbicide, which suggests that the detoxification process is an inducible mechanism in which the upregulation of key gene coding for detoxification enzymes could play an important role. Thus, the identification of cultivars with a higher detoxification potential would allow the development of more resistant varieties.

Fertility of CMS wheat is restored by two Rf loci located on a recombined acrocentric chromosome.

Cytoplasmic male sterility (CMS) results from incompatibility between nuclear and cytoplasmic genomes, and is characterized by the inability to produce viable pollen. The restoration of male fertility generally involves the introgression of nuclear genes, termed restorers of fertility (Rf). CMS has been widely used for hybrid seed production in many crops but not in wheat, partly owing to the complex genetics of fertility restoration. In this study, an acrocentric chromosome that restores pollen fertility of CMS wheat in Hordeum chilense cytoplasm (msH1 system) is studied. The results show that this chromosome, of H. chilense origin and named H(ch)ac, originated from a complex reorganization of the short arm of chromosomes 1H(ch) (1H(ch)S) and 6H(ch) (6H(ch)S). Diversity arrays technology (DArT) markers and cytological analysis indicate that H(ch)ac is a kind of `zebra-like' chromosome composed of chromosome 1H(ch)S and alternate fragments of interstitial and distal regions of chromosome 6H(ch)S. PCR-based markers together with FISH, GISH, and meiotic pairing analysis support this result. A restorer of fertility gene, named Rf6H(ch)S, has been identified on the short arm of chromosome 6H(ch)S. Moreover, restoration by the addition of chromosome 1H(ch)S has been observed at a very low frequency and under certain environmental conditions. Therefore, the results indicate the presence of two Rf genes on the acrocentric chromosome: Rf6H(ch)S and Rf1H(ch)S, the restoration potential of Rf6H(ch)S being greater. The stable and high restoration of pollen fertility in the msH1 system is therefore the result of the interaction between these two restorer genes.

Increase in transcript accumulation of Psy1 and e-Lcy genes in grain development is associated with differences in seed carotenoid content between durum wheat and tritordeum.

Carotenoid rich diets have been associated with lower risk of certain diseases. The great importance of cereals in human diet has directed breeding programs towards carotenoid enhancement to alleviate these deficiencies in developing countries and to offer new functional foods in the developed ones. The new cereal tritordeum (×Tritordeum Ascherson et Graebener) derived from durum wheat (Triticum turgidum ssp. durum) and the wild barley Hordeum chilense, naturally presents carotenoid levels 5-8 times higher than those of durum wheat. The improvement of tritordeum properties as a new functional food requires the elucidation of biosynthetic steps for carotenoid accumulation in seeds that differ from durum wheat. In this work expression patterns of nine genes from the isoprenoid and carotenoid biosynthetic pathways were monitored during grain development in durum wheat and tritordeum. Additionally, a fine identification and quantification of pigments (chlorophylls and carotenoids) during grain development and in mature seeds has been addressed. Transcript levels of Psy1, Psy2, Zds, e-Lcy and b-Lcy were found to correlate to carotenoid content in mature grains. The specific activation of the homeologous genes Psy1, e-Lcy from H. chilense and the high lutein esterification found in tritordeum may serve to explain the differences with durum wheat in carotenoid accumulation.

Cytoplasmic genome substitution in wheat affects the nuclear-cytoplasmic cross-talk leading to transcript and metabolite alterations.

BACKGROUND: Alloplasmic lines provide a unique tool to study nuclear-cytoplasmic interactions. Three alloplasmic lines, with nuclear genomes from Triticum aestivum and harboring cytoplasm from Aegilops uniaristata, Aegilops tauschii and Hordeum chilense, were investigated by transcript and metabolite profiling to identify the effects of cytoplasmic substitution on nuclear-cytoplasmic signaling mechanisms. RESULTS: In combining the wheat nuclear genome with a cytoplasm of H. chilense, 540 genes were significantly altered, whereas 11 and 28 genes were significantly changed in the alloplasmic lines carrying the cytoplasm of Ae. uniaristata or Ae. tauschii, respectively. We identified the RNA maturation-related process as one of the most sensitive to a perturbation of the nuclear-cytoplasmic interaction. Several key components of the ROS chloroplast retrograde signaling, together with the up-regulation of the ROS scavenging system, showed that changes in the chloroplast genome have a direct impact on nuclear-cytoplasmic cross-talk. Remarkably, the H. chilense alloplasmic line down-regulated some genes involved in the determination of cytoplasmic male sterility without expressing the male sterility phenotype. Metabolic profiling showed a comparable response of the central metabolism of the alloplasmic and euplasmic lines to light, while exposing larger metabolite alterations in the H. chilense alloplasmic line as compared with the Aegilops lines, in agreement with the transcriptomic data. Several stress-related metabolites, remarkably raffinose, were altered in content in the H. chilense alloplasmic line when exposed to high light, while amino acids, as well as organic acids were significantly decreased. Alterations in the levels of transcript, related to raffinose, and the photorespiration-related metabolisms were associated with changes in the level of related metabolites. CONCLUSION: The replacement of a wheat cytoplasm with the cytoplasm of a related species affects the nuclear-cytoplasmic cross-talk leading to transcript and metabolite alterations. The extent of these modifications was limited in the alloplasmic lines with Aegilops cytoplasm, and more evident in the alloplasmic line with H. chilense cytoplasm. We consider that, this finding might be linked to the phylogenetic distance of the genomes.

High-throughput genotyping of wheat-barley amphiploids utilising diversity array technology (DArT).

BACKGROUND: Hordeum chilense, a native South American diploid wild barley, is one of the species of the genus Hordeum with a high potential for cereal breeding purposes, given its high crossability with other members of the Triticeae tribe. Hexaploid tritordeum (×Tritordeum Ascherson et Graebner, 2n=6×=42, AABBH(ch)H(ch)) is the fertile amphiploid obtained after chromosome doubling of hybrids between Hordeum chilense and durum wheat. Approaches used in the improvement of this crop have included crosses with hexaploid wheat to promote D/H(ch) chromosome substitutions. While this approach has been successful as was the case with triticale, it has also complicated the genetic composition of the breeding materials. Until now tritordeum lines were analyzed based on molecular cytogenetic techniques and screening with a small set of DNA markers. However, the recent development of DArT markers in H. chilense offers new possibilities to screen large number of accessions more efficiently. RESULTS: Here, we have applied DArT markers to genotype composition in forty-six accessions of hexaploid tritordeum originating from different stages of tritordeum breeding program and to H. chilense-wheat chromosome addition lines to allow their physical mapping. Diversity analyses were conducted including dendrogram construction, principal component analysis and structure inference. Euploid and substituted tritordeums were clearly discriminated independently of the method used. However, dendrogram and Structure analyses allowed the clearest discrimination among substituted tritordeums. The physically mapped markers allowed identifying these groups as substituted tritordeums carrying the following disomic substitutions (DS): DS1D (1H(ch)), DS2D (2H(ch)), DS5D (5H(ch)), DS6D (6H(ch)) and the double substitution DS2D (2H(ch)), DS5D (5H(ch)). These results were validated using chromosome specific EST and SSR markers and GISH analysis. CONCLUSION: In conclusion, DArT markers have proved to be very useful to detect chromosome substitutions in the tritordeum breeding program and thus they are expected to be equally useful to detect translocations both in the tritordeum breeding program and in the transference of H. chilense genetic material in wheat breeding programs.

Hordeum chilense genome, a useful tool to investigate the endosperm yellow pigment content in the Triticeae.

BACKGROUND: The wild barley Hordeum chilense fulfills some requirements for being a useful tool to investigate the endosperm yellow pigment content (YPC) in the Triticeae including its diploid constitution, the availability of genetic resources (addition and deletion stocks and a high density genetic map) and, especially, its high seed YPC not silenced in tritordeums (amphiploids derived from H. chilense and wheat). Thus, the aim of this work was to test the utility of the H. chilense genome for investigating the YPC in the Triticeae. RESULTS: Twelve genes related to endosperm carotenoid content and/or YPC in grasses (Dxr, Hdr [synonym ispH], Ggpps1, Psy2, Psy3, Pds, Zds, e-Lcy, b-Lcy, Hyd3, Ccd1 and Ppo1) were identified, and mapped in H. chilense using rice genes to identify orthologs from barley, wheat, sorghum and maize. Macrocolinearity studies revealed that gene positions were in agreement in H. vulgare and H. chilense. Additionally, three main regions associated with YPC were identified in chromosomes 2Hch, 3Hch and 7Hch in H. chilense, the former being the most significant one. CONCLUSIONS: The results obtained are consistent with previous findings in wheat and suggest that Ggpps1, Zds and Hyd3 on chromosome 2Hch may be considered candidate genes in wheat for further studies in YPC improvement. Considering the syntenic location of carotenoid genes in H. chilense, we have concluded that the Hch genome may constitute a valuable tool for YPC studies in the Triticeae.

Allelic variation, alternative splicing and expression analysis of Psy1 gene in Hordeum chilense Roem. et Schult.

BACKGROUND: The wild barley Hordeum chilense Roem. et Schult. is a valuable source of genes for increasing carotenoid content in wheat. Tritordeums, the amphiploids derived from durum or common wheat and H. chilense, systematically show higher values of yellow pigment colour and carotenoid content than durum wheat. Phytoene synthase 1 gene (Psy1) is considered a key step limiting the carotenoid biosynthesis, and the correlation of Psy1 transcripts accumulation and endosperm carotenoid content has been demonstrated in the main grass species. METHODOLOGY/PRINCIPAL FINDINGS: We analyze the variability of Psy1 alleles in three lines of H. chilense (H1, H7 and H16) representing the three ecotypes described in this species. Moreover, we analyze Psy1 expression in leaves and in two seed developing stages of H1 and H7, showing mRNA accumulation patterns similar to those of wheat. Finally, we identify thirty-six different transcripts forms originated by alternative splicing of the 5' UTR and/or exons 1 to 5 of Psy1 gene. Transcripts function is tested in a heterologous complementation assay, revealing that from the sixteen different predicted proteins only four types (those of 432, 370, 364 and 271 amino acids), are functional in the bacterial system. CONCLUSIONS/SIGNIFICANCE: The large number of transcripts originated by alternative splicing of Psy1, and the coexistence of functional and non functional forms, suggest a fine regulation of PSY activity in H. chilense. This work is the first analysis of H. chilense Psy1 gene and the results reported here are the bases for its potential use in carotenoid enhancement in durum wheat.

Identification of suitable reference genes for normalization of qPCR data in comparative transcriptomics analyses in the Triticeae.

Comparative transcriptomics are useful to determine the role of orthologous genes among Triticeae species. Thus they constitute an interesting tool to improve the use of wild relatives for crop breeding. Reverse transcription quantitative real-time PCR (qPCR) is the most accurate measure of gene expression but efficient normalization is required. The choice and optimal number of reference genes must be experimentally determined and the primers optimized for cross-species amplification. Our goal was to test the utility of wheat-reference genes for qPCR normalization when species carrying the following genomes (A, B, D, R, H ( v ) and H ( ch )) are compared either simultaneously or in smaller subsets of samples. Wheat/barley/rye consensus primers outperformed wheat-specific ones which indicate that consensus primers should be considered for data normalization in comparative transcriptomics. All genes tested were stable but their ranking in terms of stability differed among subsets of samples. CDC (cell division control protein, AAA-superfamily of ATPases, Ta54227) and RLI (68 kDa protein HP68 similar to Arabidopsis thaliana RNase L inhibitor protein, Ta2776) were always among the three most stable genes. The optimal number of reference genes varied between 2 and 3 depending on the subset of samples and the method used (geNorm vs. coefficient of determination between sequential normalization factors). In any case a maximum number of three reference genes would provide adequate normalization independent of the subset of samples considered. This work constitutes a substantial advance towards comparative transcriptomics using qPCR since it provides useful primers/reference genes.

Genetic variability of carotenoid concentration and degree of esterification among tritordeum (xTritordeum Ascherson et Graebner) and durum wheat accessions.

The higher carotenoid content (commonly referred as "yellow pigment content") of tritordeum seeds as compared to wheat and the potential of this species as a donor of useful traits to wheat led us to investigate the detailed carotenoid composition of 53 accessions of hexaploid tritordeums originating from different stages of the tritordeum breeding program developed at IAS-CSIC. In addition, seven durum wheat accessions were also studied for comparison. Lutein was the unique carotenoid detected, either free or esterified with fatty acids. On average, tritordeum had 5.2 times more carotenoids than durum wheat, which suggests a high potential of this species to become a functional food. In addition, the most outstanding result of this work is the high esterification degree of lutein found in tritordeums as compared to durum wheat. This difference may indicate the differential esterification ability between tritordeum and durum wheat species. The implications of this high level of lutein esterification on both carotenoid accumulation and stability are discussed.

Accumulation of genes for susceptibility to rust fungi for which barley is nearly a nonhost results in two barley lines with extreme multiple susceptibility.

Nonhost resistance is the most common type of resistance in plants. Understanding the factors that make plants susceptible or resistant may help to achieve durably effective resistance in crop plants. Screening of 109 barley (Hordeum vulgare L.) accessions in the seedling stage indicated that barley is a complete nonhost to most of the heterologous rust fungi studied, while it showed an intermediate status with respect to Puccinia triticina, P. hordei-murini, P. hordei-secalini, P. graminis f. sp. lolii and P. coronata ff. spp. avenae and holci. Accessions that were susceptible to a heterologous rust in the seedling stage were much more or completely resistant at adult plant stage. Differential interaction between barley accessions and heterologous rust fungi was found, suggesting the existence of rust-species-specific resistance. In particular, many landrace accessions from Ethiopia and Asia, and naked-seeded accessions, tended to be susceptible to several heterologous rusts, suggesting that some resistance genes in barley are effective against more than one heterologous rust fungal species. Some barley accessions had race-specific resistance against P. hordei-murini. We accumulated genes for susceptibility to P. triticina and P. hordei-murini in two genotypes called SusPtrit and SusPmur, respectively. In the seedling stage, these accessions were as susceptible as the host species to the target rusts. They also showed unusual susceptibility to other heterologous rusts. These two lines are a valuable asset to further experimental work on the genetics of resistance to heterologous rust fungi.