Klaus Oldach

Sodium exclusion QTL associated with improved seedling growth in bread wheat under salinity stress

Worldwide, dryland salinity is a major limitation to crop production. Breeding for salinity tolerance could be an effective way of improving yield and yield stability on saline-sodic soils of dryland agriculture. However, this requires a good understanding of inheritance of this quantitative trait. In the present study, a doubled-haploid bread wheat population (Berkut/Krichauff) was grown in supported hydroponics to identify quantitative trait loci (QTL) associated with salinity tolerance traits commonly reported in the literature (leaf symptoms, tiller number, seedling biomass, chlorophyll content, and shoot Na+ and K+ concentrations), understand the relationships amongst these traits, and determine their genetic value for marker-assisted selection. There was considerable segregation within the population for all traits measured. With a genetic map of 527 SSR-, DArT- and gene-based markers, a total of 40 QTL were detected for all seven traits. For the first time in a cereal species, a QTL interval for Na+ exclusion (wPt-3114-wmc170) was associated with an increase (10%) in seedling biomass. Of the five QTL identified for Na+ exclusion, two were co-located with seedling biomass (2A and 6A). The 2A QTL appears to coincide with the previously reported Na+ exclusion locus in durum wheat that hosts one active HKT1;4 (Nax1) and one inactive HKT1;4 gene. Using these sequences as template for primer design enabled mapping of at least three HKT1;4 genes onto chromosome 2AL in bread wheat, suggesting that bread wheat carries more HKT1;4 gene family members than durum wheat. However, the combined effects of all Na+ exclusion loci only accounted for 18% of the variation in seedling biomass under salinity stress indicating that there were other mechanisms of salinity tolerance operative at the seedling stage in this population. Na+ and K+ accumulation appear under separate genetic control. The molecular markers wmc170 (2A) and cfd080 (6A) are expected to facilitate breeding for salinity tolerance in bread wheat, the latter being associated with seedling vigour.

Genetic analysis of tolerance to Boron toxicity in the legume Medicago truncatula

BackgroundMedicago 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.ResultsGenetic 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.ConclusionThe 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.

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.

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.

Quantitative trait loci for agronomic and physiological traits for a bread wheat population grown in environments with a range of salinity levels

Worldwide, salinity is a major environmental stress affecting agricultural production. Sodium (Na+) exclusion has long been recognised as a mechanism of salinity tolerance (ST) in cereals and several molecular markers have been suggested for breeding. However, there have been no empirical studies to show that selection for Na+ exclusion markers could improve grain yield in bread wheat under dryland salinity. In six field trials, a bread wheat mapping population was grown to validate Na+ exclusion quantitative trait loci (QTL) identified earlier in hydroponics, to determine the impact of Na+ exclusion on grain yield, and to identify QTL for yield-related traits. The traits included grain yield, grain number per m2, 1,000-grain weight, maturity, plant height, and leaf Na+ and K+ concentrations. The presence of numerous QTL with minor effects for most traits indicated the genetic complexity of these traits, and thus limited prospects for pyramiding at present. Considerable QTL-by-environment interactions were observed, with the stable QTL generally being co-located with maturity or early vigour/height genes, which demonstrates the importance of measuring major agronomic traits in order to discover genuine QTL for ST. Several QTL for seedling biomass and Na+ exclusion identified earlier in hydroponics were also detected in field trials but with marginal impact on grain yield. These results suggest that selection for Na+ exclusion and the use of hydroponics-based seedling assays may not necessarily result in improved ST. However, as this is the first report of its kind, there is an urgent need for testing other mapping populations in realistic environments to discover novel ST-QTL for breeding programs. In the meantime, grain yield QTL independent of maturity and height may offer potential to improve ST.

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.

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

BackgroundThe 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.ResultsA 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.ConclusionThe 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.