Capitalizing genebank core collections for rare and novel disease resistance loci to enhance barley resilience.

In the realm of agricultural sustainability, the utilization of plant genetic resources (PGRs) for enhanced disease resistance is paramount. Preservation efforts in genebanks are justified by their potential contributions to future crop improvement. To capitalize on the potential of PGRs, we focused on a barley core collection from the German ex situ genebank, and contrasted it with a European elite collection. The phenotypic assessment included 812 PGRs and 298 elites with a particular emphasis on four disease traits (Puccinia hordei, Blumeria graminis hordei, Ramularia collo-cygni, and Rhynchosporium commune). An integrated genome-wide association study, employing both Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway (BLINK) and a linear mixed model, was performed to unravel the genetic underpinnings of disease resistance. A total of 932 marker-trait associations were identified and assigned to 49 quantitative trait loci. The accumulation of novel and rare resistance alleles significantly bolstered the overall resistance level in PGRs. Three PGR donors with high counts of novel/rare alleles and exhibited exceptional resistance to leaf rust and powdery mildew were identified, offering promise for targeted pre-breeding goals and enhanced resilience in forthcoming varieties. Our findings underscore the critical contribution of PGRs to strengthening crop resilience and advancing sustainable agricultural practices.

High-resolution mapping of Ryd4Hb, a major resistance gene to Barley yellow dwarf virus from Hordeum bulbosum.

KEY MESSAGE: We mapped Ryd4Hb in a 66.5 kbp interval in barley and dissociated it from a sublethality factor. These results will enable a targeted selection of the resistance in barley breeding. Virus diseases are causing high yield losses in crops worldwide. The Barley yellow dwarf virus (BYDV) complex is responsible for one of the most widespread and economically important viral diseases of cereals. While no gene conferring complete resistance (immunity) has been uncovered in the primary gene pool of barley, sources of resistance were searched and identified in the wild relative Hordeum bulbosum, representing the secondary gene pool of barley. One such locus, Ryd4Hb, has been previously introgressed into barley, and was allocated to chromosome 3H, but is tightly linked to a sublethality factor that prevents the incorporation and utilization of Ryd4Hb in barley varieties. To solve this problem, we fine-mapped Ryd4Hb and separated it from this negative factor. We narrowed the Ryd4Hb locus to a corresponding 66.5 kbp physical interval in the barley 'Morex' reference genome. The region comprises a gene from the nucleotide-binding and leucine-rich repeat immune receptor family, typical of dominant virus resistance genes. The closest homolog to this Ryd4Hb candidate gene is the wheat Sr35 stem rust resistance gene. In addition to the fine mapping, we reduced the interval bearing the sublethality factor to 600 kbp in barley. Aphid feeding experiments demonstrated that Ryd4Hb provides a resistance to BYDV rather than to its vector. The presented results, including the high-throughput molecular markers, will permit a more targeted selection of the resistance in breeding, enabling the use of Ryd4Hb in barley varieties.

Fine mapping of root lesion nematode (Pratylenchus thornei) resistance loci on chromosomes 6D and 2B of wheat.

KEY MESSAGE: Resistance QTL to root lesion nematode (Pratylenchus thornei) in wheat (Triticum aestivum), QRlnt.sk-6D and QRlnt.sk-2B, were mapped to intervals of 3.5 cM/1.77 Mbp on chromosome 6D and 1.4 cM/2.19 Mbp on chromosome 2B, respectively. Candidate resistance genes were identified in the QTL regions and molecular markers developed for marker-assisted breeding. Two previously known resistance QTL for root lesion nematode (Pratylenchus thornei) in bread wheat (Triticum aestivum), QRlnt.sk-6D and QRlnt.sk-2B, were fine-mapped using a Sokoll (moderately resistant) by Krichauff (susceptible) doubled haploid (DH) population and six newly developed recombinant inbred line populations. Bulked segregation analysis with the 90K wheat SNP array identified linked SNPs which were subsequently converted to KASP assays for mapping in the DH and RIL populations. On chromosome 6D, 60 KASP and five SSR markers spanned a total genetic distance of 23.7 cM. QRlnt.sk-6D was delimited to a 3.5 cM interval, representing 1.77 Mbp in the bread wheat cv. Chinese Spring reference genome sequence and 2.29 Mbp in the Aegilops tauschii genome sequence. These intervals contained 42 and 43 gene models in the respective annotated genome sequences. On chromosome 2B, 41 KASP and 5 SSR markers produced a map spanning 19.9 cM. QRlnt.sk-2B was delimited to 1.4 cM, corresponding 3.14 Mbp in the durum wheat cv. Svevo reference sequence and 2.19 Mbp in Chinese Spring. The interval in Chinese Spring contained 56 high-confidence gene models. Intervals for both QTL contained genes with similarity to those previously reported to be involved in disease resistance, namely genes for phenylpropanoid biosynthetic pathway-related enzymes, NBS-LRR proteins and protein kinases. The potential roles of these candidate genes in P. thornei resistance are discussed. The KASP markers reported in this study could potentially be used for marker-assisted breeding of P. thornei-resistant wheat cultivars.

A comparative gene analysis with rice identified orthologous group II HKT genes and their association with Na(+) concentration in bread wheat.

BACKGROUND: Although the HKT transporter genes ascertain some of the key determinants of crop salt tolerance mechanisms, the diversity and functional role of group II HKT genes are not clearly understood in bread wheat. The advanced knowledge on rice HKT and whole genome sequence was, therefore, used in comparative gene analysis to identify orthologous wheat group II HKT genes and their role in trait variation under different saline environments. RESULTS: The four group II HKTs in rice identified two orthologous gene families from bread wheat, including the known TaHKT2;1 gene family and a new distinctly different gene family designated as TaHKT2;2. A single copy of TaHKT2;2 was found on each homeologous chromosome arm 7AL, 7BL and 7DL and each gene was expressed in leaf blade, sheath and root tissues under non-stressed and at 200 mM salt stressed conditions. The proteins encoded by genes of the TaHKT2;2 family revealed more than 93% amino acid sequence identity but ≤52% amino acid identity compared to the proteins encoded by TaHKT2;1 family. Specifically, variations in known critical domains predicted functional differences between the two protein families. Similar to orthologous rice genes on chromosome 6L, TaHKT2;1 and TaHKT2;2 genes were located approximately 3 kb apart on wheat chromosomes 7AL, 7BL and 7DL, forming a static syntenic block in the two species. The chromosomal region on 7AL containing TaHKT2;1 7AL-1 co-located with QTL for shoot Na(+) concentration and yield in some saline environments. CONCLUSION: The differences in copy number, genes sequences and encoded proteins between TaHKT2;2 homeologous genes and other group II HKT gene families within and across species likely reflect functional diversity for ion selectivity and transport in plants. Evidence indicated that neither TaHKT2;2 nor TaHKT2;1 were associated with primary root Na(+) uptake but TaHKT2;1 may be associated with trait variation for Na(+) exclusion and yield in some but not all saline environments.

Uncoupling of sodium and chloride to assist breeding for salinity tolerance in crops.

The separation of toxic effects of sodium (Na(+)) and chloride (Cl(-)) by the current methods of mixed salts and subsequent determination of their relevance to breeding has been problematic. We report a novel method (Na(+) humate) to study the ionic effects of Na(+) toxicity without interference from Cl(-), and ionic and osmotic effects when combined with salinity (NaCl). Three cereal species (Hordeum vulgare, Triticum aestivum and Triticum turgidum ssp. durum with and without the Na(+) exclusion gene Nax2) differing in Na(+) exclusion were grown in a potting mix under sodicity (Na(+) humate) and salinity (NaCl), and water use, leaf nutrient profiles and yield were determined. Under sodicity, Na(+)-excluding bread wheat and durum wheat with the Nax2 gene had higher yield than Na(+)-accumulating barley and durum wheat without the Nax2 gene. However, under salinity, despite a 100-fold difference in leaf Na(+), all species yielded similarly, indicating that osmotic stress negated the benefits of Na(+) exclusion. In conclusion, Na(+) exclusion can be an effective mechanism for sodicity tolerance, while osmoregulation and tissue tolerance to Na(+) and/or Cl(-) should be the main foci for further improvement of salinity tolerance in cereals. This represents a paradigm shift for breeding cereals with salinity tolerance.

Genetic analysis of tolerance to the root lesion nematode Pratylenchus neglectus in the legume Medicago littoralis.

BACKGROUND: The nematode Pratylenchus neglectus has a wide host range and is able to feed on the root systems of cereals, oilseeds, grain and pasture legumes. Under the Mediterranean low rainfall environments of Australia, annual Medicago pasture legumes are used in rotation with cereals to fix atmospheric nitrogen and improve soil parameters. Considerable efforts are being made in breeding programs to improve resistance and tolerance to Pratylenchus neglectus in the major crops wheat and barley, which makes it vital to develop appropriate selection tools in medics. RESULTS: A strong source of tolerance to root damage by the root lesion nematode (RLN) Pratylenchus neglectus had previously been identified in line RH-1 (strand medic, M. littoralis). Using RH-1, we have developed a single seed descent (SSD) population of 138 lines by crossing it to the intolerant cultivar Herald. After inoculation, RLN-associated root damage clearly segregated in the population. Genetic analysis was performed by constructing a genetic map using simple sequence repeat (SSR) and gene-based SNP markers. A highly significant quantitative trait locus (QTL), QPnTolMl.1, was identified explaining 49% of the phenotypic variation in the SSD population. All SSRs and gene-based markers in the QTL region were derived from chromosome 1 of the sequenced genome of the closely related species M. truncatula. Gene-based markers were validated in advanced breeding lines derived from the RH-1 parent and also a second RLN tolerance source, RH-2 (M. truncatula ssp. tricycla). Comparative analysis to sequenced legume genomes showed that the physical QTL interval exists as a synteny block in Lotus japonicus, common bean, soybean and chickpea. Furthermore, using the sequenced genome information of M. truncatula, the QTL interval contains 55 genes out of which five are discussed as potential candidate genes responsible for the mapped tolerance. CONCLUSION: The closely linked set of SNP-based PCR markers is directly applicable to select for two different sources of RLN tolerance in breeding programs. Moreover, genome sequence information has allowed proposing candidate genes for further functional analysis and nominates QPnTolMl.1 as a target locus for RLN tolerance in economically important grain legumes, e.g. chickpea.

QTL for resistance to root lesion nematode (Pratylenchus thornei) from a synthetic hexaploid wheat source.

KEY MESSAGE: A whole genome average interval mapping approach identified eight QTL associated with P. thornei resistance in a DH population from a cross between the synthetic-derived wheat Sokoll and cultivar Krichauff. Pratylenchus thornei are migratory nematodes that feed and reproduce within the wheat root cortex, causing cell death (lesions) resulting in severe yield reductions globally. Genotypic selection using molecular markers closely linked to Pratylenchus resistance genes will accelerate the development of new resistant cultivars by reducing the need for laborious and expensive resistance phenotyping. A doubled haploid wheat population (150 lines) from a cross between the synthetic-derived cultivar Sokoll (P. thornei resistant) and cultivar Krichauff (P. thornei moderately susceptible) was used to identify quantitative trait loci (QTL) associated with P. thornei resistance. The resistance identified in the glasshouse was validated in a field trial. A genetic map was constructed using Diversity Array Technology and the QTL regions identified were further targeted with simple sequence repeat (SSR) and single-nucleotide polymorphism (SNP) markers. Six significant and two suggestive P. thornei resistance QTL were detected using a whole genome average interval mapping approach. Three QTL were identified on chromosome 2B, two on chromosome 6D, and a single QTL on each of chromosomes 2A, 2D and 5D. The QTL on chromosomes 2BS and 6DS mapped to locations previously identified to be associated with Pratylenchus resistance. Together, the QTL on 2B (QRlnt.sk-2B.1-2B.3) and 6D (QRlnt.sk-6D.1 and 6D.2) explained 30 and 48 % of the genotypic variation, respectively. Flanking PCR-based markers based on SSRs and SNPs were developed for the major QTL on 2B and 6D and provide a cost-effective high-throughput tool for marker-assisted breeding of wheat with improved P. thornei resistance.

Characterization of resistance to Pratylenchus thornei (Nematoda) in wheat (Triticum aestivum): attraction, penetration, motility, and reproduction.

Lines from a cross between two wheat (Triticum aestivum) cultivars with contrasting resistance phenotypes to Pratylenchus thornei (Nematoda) were investigated to determine the stage at which resistance occurs. Host resistance was examined at nematode attraction to and penetration of roots and nematode motility, maturation, and reproduction within roots. There was no significant difference in the rate at which P. thornei was attracted toward or penetrated resistant or susceptible roots. However, suppression of migration, juvenile maturation, and reproduction in and near resistant roots was evident, suggesting that resistance acts post penetration. No preferential root penetration zone was observed in contrast to other studies. The inhibitory compounds from resistant wheat plants appeared to be constitutively expressed and water soluble because nematode migration was suppressed in roots and root exudates of unchallenged seedlings. The effects of these compounds were reversible and affected P. thornei but not P. neglectus. Apart from migration, nematode multiplication was greatly inhibited by resistance because only a few juveniles (10%) developed past stage three in roots of resistant compared with susceptible plants. Earlier in the life cycle, egg deposition and hatch of P. thornei were also significantly reduced in resistant roots and root exudates, suggesting the presence of hatching inhibitors.

Genetic analysis of tolerance to boron toxicity in the legume Medicago truncatula.

BACKGROUND: Medicago truncatula Gaertn. (barrel medic) is cultivated as a pasture legume for its high protein content and ability to improve soils through nitrogen fixation. Toxic concentrations of the micronutrient Boron (B) in agricultural soils hamper the production of cereal and leguminous crops. In cereals, the genetic analysis of B tolerance has led to the development of molecular selection tools to introgress and maintain the B tolerance trait in breeding lines. There is a comparable need for selection tools in legumes that grow on these toxic soils, often in rotation with cereals. RESULTS: Genetic variation for B tolerance in Medicago truncatula was utilised to generate two F2 populations from crosses between tolerant and intolerant parents. Phenotyping under B stress revealed a close correlation between B tolerance and biomass production and a segregation ratio explained by a single dominant locus. M. truncatula homologues of the Arabidopsis major intrinsic protein (MIP) gene AtNIP5;1 and the efflux-type transporter gene AtBOR1, both known for B transport, were identified and nearby molecular markers screened across F2 lines to verify linkage with the B-tolerant phenotype. Most (95%) of the phenotypic variation could be explained by the SSR markers h2_6e22a and h2_21b19a, which flank a cluster of five predicted MIP genes on chromosome 4. Three CAPS markers (MtBtol-1,-2,-3) were developed to dissect the region further. Expression analysis of the five predicted MIPs indicated that only MtNIP3 was expressed when leaf tissue and roots were assessed. MtNIP3 showed low and equal expression in the roots of tolerant and intolerant lines but a 4-fold higher expression level in the leaves of B-tolerant cultivars. The expression profile correlates closely with the B concentration measured in the leaves and roots of tolerant and intolerant plants. Whereas no significant difference in B concentration exists between roots of tolerant and intolerant plants, the B concentration in the leaves of tolerant plants is less than half that of intolerant plants, which further supports MtNIP3 as the best candidate for the tolerance trait-defining gene in Medicago truncatula. CONCLUSION: The close linkage of the MtNIP3 locus to B toxicity tolerance provides a source of molecular selection tools to pasture breeding programs. The economical importance of the locus warrants further investigation of the individual members of the MIP gene cluster in other pasture and in grain legumes.

The Potential of Genome-Wide Prediction to Support Parental Selection, Evaluated with Data from a Commercial Barley Breeding Program.

Parental selection is at the beginning and contributes significantly to the success of any breeding work. The value of a cross is reflected in the potential of its progeny population. Breeders invest substantial resources in evaluating progeny to select the best performing genotypes as candidates for variety development. Several proposals have been made to use genomics to support parental selection. These have mostly been evaluated using theoretical considerations or simulation studies. However, evaluations using experimental data have rarely been conducted. In this study, we tested the potential of genomic prediction for predicting the progeny mean, variance, and usefulness criterion using data from an applied breeding population for winter barley. For three traits with genetic architectures at varying levels of complexity, ear emergence, plant height, and grain yield, progeny mean, variance, and usefulness criterion were predicted and validated in scenarios resembling situations in which the described tools shall be used in plant breeding. While the population mean could be predicted with moderate to high prediction abilities amounting to 0.64, 0.21, and 0.39 in ear emergence, plant height, and grain yield, respectively, the prediction of family variance appeared difficult, as reflected in low prediction abilities of 0.41, 0.11, and 0.14, for ear emergence, plant height, and grain yield, respectively. We have shown that identifying superior crosses remains a challenging task and suggest that the success of predicting the usefulness criterion depends strongly on the complexity of the underlying trait.

New insights into the infection process of Rhynchosporium secalis in barley using GFP.

Through the use of a Rhynchosporium secalis isolate transformed with the green fluorescent protein gene and LASER scanning confocal microscopy (LSCM), fungal development during the R. secalis/barley interaction was analysed. Each infection stage was investigated from 0.5h to 14 days post-inoculation (p.i.) with extensive sampling within the first 48 h p.i. Early germination events were observed that had not been previously described. A specific time of germination was noted, with germ tube formation appearing as early as 1h p.i. Conidia were observed within anticlinal grooves of epidermal cells and the formation of bubbles within these pectin-rich regions was observed within 24h p.i. The study reports R. secalis pectinase production and suggests degradation of these pectin-rich regions. Reactive oxygen species were present during early penetration, 3h p.i. and co-localised with fungal development. LSCM allowed the visualisation of fungal growth deep within tissues at the later stage of the infection.

Genes mapping to boron tolerance QTL in barley identified by suppression subtractive hybridization.

Boron tolerance is a quantitative trait controlled by multiple genes. Suppression subtractive hybridization was carried out on root cDNA from bulked boron tolerant and intolerant doubled haploid barley lines grown under moderate boron stress to identify genes associated with boron tolerance. One hundred and eleven clones representing known proteins were found to be up-regulated in the tolerant bulk upon boron stress. Nine clones were genetically mapped to previously reported boron tolerance QTL. These include a clone identical to the boron transporter gene Bot1 and a clone coding for a bromo-adjacent homology domain-containing protein, mapping to the 6H boron tolerance locus and co-segregating with reduced boron intake in a Clipper x Sahara-3771 mapping population. A third clone mapping to the 2H QTL region encoding an S-adenosylmethionine decarboxylase precursor was found to provide tolerance to high boron by heterologous expression. Yeast cells expressing Sahara SAMDC were able to grow on 15 mm boron solid media and maintained cellular boron concentrations at 13% lower than control cells expressing empty vector. The data suggest that an antioxidative response mechanism involving polyamines and the ascorbate-glutathione pathway in Sahara barley may provide an advantage in tolerating high soil concentrations of boron.

Bread Wheat With High Salinity and Sodicity Tolerance.

Soil salinity and sodicity are major constraints to global cereal production, but breeding for tolerance has been slow. Narrow gene pools, over-emphasis on the sodium (Na+) exclusion mechanism, little attention to osmotic stress/tissue tolerance mechanism(s) in which accumulation of inorganic ions such as Na+ is implicated, and lack of a suitable screening method have impaired progress. The aims of this study were to discover novel genes for Na+ accumulation using genome-wide association studies, compare growth responses to salinity and sodicity in low-Na+ bread Westonia with Nax1 and Nax2 genes and high-Na+ bread wheat Baart-46, and evaluate growth responses to salinity and sodicity in bread wheats with varying leaf Na+ concentrations. The novel high-Na+ bread wheat germplasm, MW#293, had higher grain yield under salinity and sodicity, in absolute and relative terms, than the other bread wheat entries tested. Genes associated with high Na+ accumulation in bread wheat were identified, which may be involved in tissue tolerance/osmotic adjustment. As most modern bread wheats are efficient at excluding Na+, further reduction in plant Na+ is unlikely to provide agronomic benefit. The salinity and sodicity tolerant germplasm MW#293 provides an opportunity for the development of future salinity/sodicity tolerant bread wheat.

Phenotypic and molecular characterisation of novel Vicia faba germplasm with tolerance to acetohydroxyacid synthase-inhibiting herbicides (AHAS) developed through mutagenesis techniques.

BACKGROUND: Faba bean (Vicia faba L.) is an important crop in Australian farming systems, however, weed control is a major constraint due to a lack of in-crop broadleaf herbicide options. To address this, we developed acetohydroxyacid synthase (AHAS) inhibitor herbicide tolerance in faba bean using mutagenesis techniques. Dose-response experiments, agronomic field evaluation and DNA sequencing of the AHAS gene were used to quantify and validate tolerance traits. RESULTS: Four M2 faba bean single-plant biotypes (IMI-1, IMI-2, IMI-3 and IMI-4) at a frequency of 3.63 × 10-6 were successfully recovered. Molecular characterisation of the AHAS gene identified two known target site mutations (resulting in protein substitutions Ala205Val and Ser653Asn) conferring tolerance. Phenotypic characterisation found that both mutations conferred high levels of tolerance to the imidazolinone herbicide imazapyr. However, although the Ala205Val substitution showed improved levels of cross-tolerance to a range of sulfonylurea chemistries, the Ser653Asn substitution did not. In the field, IMI-3 showed the highest level of agronomic tolerance across a range of imidazolinone herbicides. CONCLUSIONS: Mutagenesis techniques were successful in the development of tolerance to AHAS inhibitor herbicides in faba bean, and could facilitate the first safe in-crop broadleaf herbicide control option in Australian faba bean production. © 2019 Society of Chemical Industry.

Identification of a chemically induced point mutation mediating herbicide tolerance in annual medics (Medicago spp.).

BACKGROUND AND AIMS: Sulfonylurea (SU) herbicides are used extensively in cereal-livestock farming zones as effective and cheap herbicides with useful levels of residual activity. These residues can persist beyond the cropping year, severely affecting legumes in general, and annual medics in particular, resulting in reduced dry matter production, lower seed yields and decreased nitrogen fixation. A strand medic cultivar, Medicago littoralis 'Angel', has been developed via chemical mutagenesis with tolerance to SU soil residues. Identifying the molecular basis of the observed tolerance was the aim of this study. METHODS: Two F(2) populations were generated from crosses between 'Angel' and varieties of intolerant M. truncatula, the male-sterile mutant tap and the cultivar 'Caliph'. Genetic mapping with SSR (single sequence repeat) and gene-based markers allowed identification of the trait-defining gene. Quantitative gene expression studies showed the activity of the respective alleles. KEY RESULTS: Segregation ratios indicated the control of SU-herbicide tolerance by a single dominant gene. SU herbicides inhibit the biosynthesis of the branched-chain amino acids by targeting the acetolactate synthase enzyme, allowing the choice of a mapping approach using acetolactate synthase (ALS) gene homologues as candidates. SSR-marker analysis suggested the ALS-gene homologue on chromosome 3 in M. truncatula. The ALS-gene sequences from 'Angel' and intolerant genotypes were sequenced. In 'Angel', a single point mutation from C to T translating into an amino acid change from proline to leucine was identified. The polymorphism was used to develop a diagnostic marker for the tolerance trait. Expression of the mutant ALS allele was confirmed by quantitative RT-PCR and showed no differences at various seedling stages and treatments to the corresponding wild-type allele. CONCLUSIONS: The identification of the trait-defining gene and the development of a diagnostic marker enable efficient introgression of this economically important trait in annual medic improvement programs.

Expression of transgenic stilbene synthases in wheat causes the accumulation of unknown stilbene derivatives with antifungal activity.

The expression of foreign phytoalexins in a new host is thought to increase fungal resistance, since host-specific pathogens have not experienced selection for detoxifying or metabolising the novel antifungal compounds. Two resveratrol synthase genes vst1 and vst2 from grapevine (Vitis vinifera L.) and the pinosylvin synthase gene pss from pine (Pinus sylvestris L.) were stably transformed into bread wheat. The expression of the target genes is regulated by stress-inducible grapevine promoters. The vst1 and vst2 promoters were functional in wheat and retained their expression profiles described for grapevine. ALL vst and pss transgenic lines accumulated stilbene derivatives upon induction by UV light. The detected stilbenes showed a remarkable similarity to resveratrol and pinosylvin, however were found to be more hydrophilic than resveratrol and pinosylvin. Upon inoculation with the biotrophic pathogen Puccinia recondita f.sp. tritici several vst expressing wheat lines showed a significant reduction of disease symptoms (19 +/- 9% to 27 +/- 8%) compared to wild-type plants. The reduction of disease symptoms was even more obvious after inoculation with the facultative biotrophic pathogen Septoria nodorum Berk. and ranged from 42 +/- 13% to 71 +/- 4%. None of the four tested pss expressing lines showed a reduction in disease incidence.

A major locus for chloride accumulation on chromosome 5A in bread wheat.

Chloride (Cl-) is an essential micronutrient for plant growth, but can be toxic at high concentrations resulting in reduced growth and yield. Although saline soils are generally dominated by both sodium (Na+) and Cl- ions, compared to Na+ toxicity, very little is known about physiological and genetic control mechanisms of tolerance to Cl- toxicity. In hydroponics and field studies, a bread wheat mapping population was tested to examine the relationships between physiological traits [Na+, potassium (K+) and Cl- concentration] involved in salinity tolerance (ST) and seedling growth or grain yield, and to elucidate the genetic control mechanism of plant Cl- accumulation using a quantitative trait loci (QTL) analysis approach. Plant Na+ or Cl- concentration were moderately correlated (genetically) with seedling biomass in hydroponics, but showed no correlations with grain yield in the field, indicating little value in selecting for ion concentration to improve ST. In accordance with phenotypic responses, QTL controlling Cl- accumulation differed entirely between hydroponics and field locations, and few were detected in two or more environments, demonstrating substantial QTL-by-environment interactions. The presence of several QTL for Cl- concentration indicated that uptake and accumulation was a polygenic trait. A major Cl- concentration QTL (5A; barc56/gwm186) was identified in three field environments, and accounted for 27-32% of the total genetic variance. Alignment between the 5A QTL interval and its corresponding physical genome regions in wheat and other grasses has enabled the search for candidate genes involved in Cl- transport, which is discussed.

Analysis of high pI α-Amy-1 gene family members expressed in late maturity α-amylase in wheat (Triticum aestivum L.).

Late maturity α-amylase (LMA) is a genetic defect involving the synthesis of high pI isozymes of α-amylase encoded by α-Amy-1 genes during the later stages of grain development. The aims of this investigation were to determine both the number of expressed α-Amy-1 genes and their relative transcript abundance. Sub-cloning and sequencing of expressed high pI α-amylase genes in developing wheat seeds revealed three insertion/deletion patterns in the 3' untranslated region and numerous single nucleotide polymorphisms at the 3' end of α-Amy-1. The genetic variations defined 36 α-Amy-1 gene sequences that were expressed on the onset of LMA in doubled haploid progenies (SpM25, SpM52 and SpM127) derived from the cross Spica (LMA)/Maringa (non-LMA). Five isoelectric point groups were predicted based on the translated partial coding sequences. The potential application of quantitative real-time RT-PCR in screening wheat genotypes for LMA is discussed.

Genetic structure of South Australian Pyrenophora teres populations as revealed by microsatellite analyses.

The aim of this study was to determine the genetic structure of South Australian field populations of the barley net blotch pathogens, Pyrenophora teres f. sp. teres (PTT) and P. teres f. sp. maculata (PTM), using microsatellite DNA markers. Three PTT populations (76 isolates total) and two PTM populations (43 isolates total) were sampled from separate fields during a single growing season. The results showed that of the 20 microsatellite loci examined, 17 (85%) were polymorphic within the PTT and PTM populations. In total, 120 distinct alleles were identified of which only 11 (9%) were shared between the two population types. Nei's measure of gene diversity across the PTT and PTM populations was similar at 0.38 and 0.40, respectively, and also much higher than previously reported from studies in which other types of molecular markers were used. The coefficient of genetic differentiation among both populations was the same (G(ST)=0.03) and the low and insignificant estimates of F(ST), as indicated by θ, between populations of the same type (PTT: θ<0.008, PTM: θ=0.014) indicated that isolates sampled from different areas within the same field were genetically similar. In contrast, high and significant genetic differentiation was observed among and between populations of different type (G(ST)=0.42, θ>0.567). The high number of unique multilocus haplotypes observed within the PTT (84%) and PTM (100%) populations, combined with a 1:1 distribution of both mating types, suggested that sexual reproduction was predominant among these populations. However, tests for multilocus associations showed that both PTT and PTM populations were in significant linkage disequilibrium. Although the levels of disequilibrium were low, an asexual reproductive component could not be excluded.

Rust and downy mildew resistance in pearl millet (Pennisetum glaucum) mediated by heterologous expression of the afp gene from Aspergillus giganteus.

The cDNA encoding the antifungal protein AFP from the mould Aspergillus giganteus was introduced into two pearl millet (Pennisetum glaucum) genotypes by particle bombardment. Stable integration and expression of the afp gene was confirmed in two independent transgenic T0 plants and their progeny using Southern blot and RT-PCR analysis. In vitro infection of detached leaves and in vivo inoculation of whole plants with the basidomycete Puccinia substriata, the causal agent of rust disease, and the oomycete Sclerospora graminicola, causal agent of downy mildew, resulted in a significant reduction of disease symptoms in comparison to wild type control plants. The disease resistance of pearl millet was increased by up to 90% when infected with two diverse, economically important pathogens. This is the first report of genetic enhancement of Pennisetum glaucum against fungal infections.