Strategies of preserving genetic diversity while maximizing genetic response from implementing genomic selection in pulse breeding programs.

KEY MESSAGE: Genomic selection maximizes genetic gain by recycling parents to germplasm pool earlier and preserves genetic diversity by restricting the number of fixed alleles and the relationship in pulse breeding programs. Using a stochastic computer simulation, we investigated the benefit of optimization strategies in the context of genomic selection (GS) for pulse breeding programs. We simulated GS for moderately complex to highly complex traits such as disease resistance, grain weight and grain yield in multiple environments with a high level of genotype-by-environment interaction for grain yield. GS led to higher genetic gain per unit of time and higher genetic diversity loss than phenotypic selection by shortening the breeding cycle time. The genetic gain obtained from selecting the segregating parents early in the breeding cycle (at F1 or F2 stages) was substantially higher than selecting at later stages even though prediction accuracy was moderate. Increasing the number of F1 intercross (F1i) families and keeping the total number of progeny of F1i families constant, we observed a decrease in genetic gain and increase in genetic diversity, whereas increasing the number of progeny per F1i family while keeping a constant number of F1i families increased the rate of genetic gain and had higher genetic diversity loss per unit of time. Adding 50 F2 family phenotypes to the training population increased the accuracy of genomic breeding values (GEBVs) and genetic gain per year and decreased the rate of genetic diversity loss. Genetic diversity could be preserved by applying a strategy that restricted both the percentage of alleles fixed and the average relationship of the group of selected parents to preserve long-term genetic improvement in the pulse breeding program.

Genome-wide association analysis of stripe rust resistance in modern Chinese wheat.

BACKGROUND: Stripe rust (yellow rust) is a significant disease for bread wheat (Triticum aestivum L.) worldwide. A genome-wide association study was conducted on 240 Chinese wheat cultivars and elite lines genotyped with the wheat 90 K single nucleotide polymorphism (SNP) arrays to decipher the genetic architecture of stripe rust resistance in Chinese germplasm. RESULTS: Stripe rust resistance was evaluated at the adult plant stage in Pixian and Xindu in Sichuan province in the 2015-2016 cropping season, and in Wuhan in Hubei province in the 2013-2014, 2016-2017 and 2018-2019 cropping seasons. Twelve stable loci for stripe rust resistance were identified by GWAS using TASSEL and GAPIT software. These loci were distributed on chromosomes 1B, 1D, 2A, 2B, 3A, 3B, 4B (3), 4D, 6D, and 7B and explained 3.6 to 10.3% of the phenotypic variation. Six of the loci corresponded with previously reported genes/QTLs, including Sr2/Yr30/Lr27, while the other six (QYr.hbaas-1BS, QYr.hbaas-2BL, QYr.hbaas-3AL, QYr.hbaas-4BL.3, QYr.hbaas-4DL, and QYr.hbaas-6DS) are probably novel. The results suggest high genetic diversity for stripe rust resistance in this population. The resistance alleles of QYr.hbaas-2AS, QYr.hbaas-3BS, QYr.hbaas-4DL, and QYr.hbaas-7BL were rare in the present panel, indicating their potential use in breeding for stripe rust resistance in China. Eleven penta-primer amplification refractory mutation system (PARMS) markers were developed from SNPs significantly associated with seven mapped QTLs. Twenty-seven genes were predicted for mapped QTLs. Six of them were considered as candidates for their high relative expression levels post-inoculation. CONCLUSION: The resistant germplasm, mapped QTLs, and PARMS markers developed in this study are resources for enhancing stripe rust resistance in wheat breeding.

Characterization of Stripe Rust Resistance Genes in the Wheat Cultivar Chuanmai45.

The objective of this research was to characterize the high level of resistance to stripe that has been observed in the released wheat cultivar, Chuanmai45. A combination of classic genetic analysis, molecular and cytogenetic methods were used to characterize resistance in an F2 population derived from Chuanmai45 and the susceptible Chuanmai42. Inheritance of resistance was shown to be conferred by two genes in Chuanmai45. Fluorescence in situ hybridization (FISH) was used along with segregation studies to show that one gene was located on a 1RS.1BL translocation. Molecular markers were employed to show that the other locus was located on chromosome 4B. The defeated gene, Yr24/26, on chromosome 1BL was present in the susceptible parent and lines that recombined this gene with the 1RS.1BL translocation were identified. The germplasm, loci, and associated markers identified in this study will be useful for application in breeding programs utilizing marker-assisted selection.

Genomic selection for target traits in the Australian lentil breeding program.

Genomic selection (GS) uses associations between markers and phenotypes to predict the breeding values of individuals. It can be applied early in the breeding cycle to reduce the cross-to-cross generation interval and thereby increase genetic gain per unit of time. The development of cost-effective, high-throughput genotyping platforms has revolutionized plant breeding programs by enabling the implementation of GS at the scale required to achieve impact. As a result, GS is becoming routine in plant breeding, even in minor crops such as pulses. Here we examined 2,081 breeding lines from Agriculture Victoria's national lentil breeding program for a range of target traits including grain yield, ascochyta blight resistance, botrytis grey mould resistance, salinity and boron stress tolerance, 100-grain weight, seed size index and protein content. A broad range of narrow-sense heritabilities was observed across these traits (0.24-0.66). Genomic prediction models were developed based on 64,781 genome-wide SNPs using Bayesian methodology and genomic estimated breeding values (GEBVs) were calculated. Forward cross-validation was applied to examine the prediction accuracy of GS for these targeted traits. The accuracy of GEBVs was consistently higher (0.34-0.83) than BLUP estimated breeding values (EBVs) (0.22-0.54), indicating a higher expected rate of genetic gain with GS. GS-led parental selection using early generation breeding materials also resulted in higher genetic gain compared to BLUP-based selection performed using later generation breeding lines. Our results show that implementing GS in lentil breeding will fast track the development of high-yielding cultivars with increased resistance to biotic and abiotic stresses, as well as improved seed quality traits.

Lr68: a new gene conferring slow rusting resistance to leaf rust in wheat.

The common wheat cultivar Parula possesses a high level of slow rusting, adult plant resistance (APR) to all three rust diseases of wheat. Previous mapping studies using an Avocet-YrA/Parula recombinant inbred line (RIL) population showed that APR to leaf rust (Puccinia triticina) in Parula is governed by at least three independent slow rusting resistance genes: Lr34 on 7DS, Lr46 on 1BL, and a previously unknown gene on 7BL. The use of field rust reaction and flanking markers identified two F(6) RILs, Arula1 and Arula2, from the above population that lacked Lr34 and Lr46 but carried the leaf rust resistance gene in 7BL, hereby designated Lr68. Arula1 and Arula2 were crossed with Apav, a highly susceptible line from the cross Avocet-YrA/Pavon 76, and 396 F(4)-derived F(5) RILs were developed for mapping Lr68. The RILs were phenotyped for leaf rust resistance for over 2 years in Ciudad Obregon, Mexico, with a mixture of P. triticina races MBJ/SP and MCJ/SP. Close genetic linkages with several DNA markers on 7BL were established using 367 RILs; Psy1-1 and gwm146 flanked Lr68 and were estimated at 0.5 and 0.6 cM, respectively. The relationship between Lr68 and the race-specific seedling resistance gene Lr14b, located in the same region and present in Parula, Arula1 and Arula2, was investigated by evaluating the RILs with Lr14b-avirulent P. triticina race TCT/QB in the greenhouse. Although Lr14b and Lr68 homozygous recombinants in repulsion were not identified in RILs, γ-irradiation-induced deletion stocks that lacked Lr68 but possessed Lr14b showed that Lr68 and Lr14b are different loci. Flanking DNA markers that are tightly linked to Lr68 in a wide array of genotypes can be utilized for selection of APR to leaf rust.

Characterization of field pea (Pisum sativum) resistance against Peyronellaea pinodes and Didymella pinodella that cause ascochyta blight.

Ascochyta blight is one of the most destructive diseases in field pea and is caused by either individual or combined infections by the necrotrophic pathogens Peyronellaea pinodes, Didymella pinodella, Ascochyta pisi and Ascochyta koolunga. Knowledge of disease epidemiology will help in understanding the resistance mechanisms, which, in turn, is beneficial in breeding for disease resistance. A pool of breeding lines and cultivars were inoculated with P. pinodes and D. pinodella to study the resistance responses and to characterize the underlying resistance reactions. In general, phenotypic analysis of controlled environment disease assays showed clear differential responses among genotypes against the two pathogens. The released variety PBA Wharton and the breeding line 11HP302-12HO-1 showed high levels of resistance against both pathogens whereas PBA Twilight and 10HP249-11HO-7 showed differential responses between the two pathogens, showing higher resistance against D. pinodella as compared to P. pinodes. OZP1604 had high infection levels against both pathogens. Histochemical analysis of leaves using diamino benzidine (DAB) showed the more resistant genotypes had lower accumulation of hydrogen peroxide compared to susceptible genotypes. The digital images of DAB staining were analyzed using ImageJ, an image analysis software. The image analysis results showed that quantification of leaf disease infection through image analysis is a useful tool in estimating the level of cell death in biotic stress studies. The qRT-PCR analysis of defense related genes showed that partially resistant genotypes had significantly higher expression of PsOXII and Pshmm6 in the P. pinodes treated plants, whereas expression of PsOXII, PsAPX1, PsCHS3 and PsOPR1 increased in partially resistant plants inoculated with D. pinodella. The differential timing and intensity of expression of a range of genes between resistant lines challenged with the same pathogen, or challenged with different pathogens, suggests that there are multiple pathways that restrict infection in this complex pathogen-host interaction. The combination of phenotypic, histochemical and molecular approaches provide a comprehensive picture of the infection process and resistance mechanism of pea plants against these pathogens.

Combining NDVI and Bacterial Blight Score to Predict Grain Yield in Field Pea.

Field pea is the most commonly grown temperate pulse crop, with close to 15 million tons produced globally in 2020. Varieties improved through breeding are important to ensure ongoing improvements in yield and disease resistance. Genomic selection (GS) is a modern breeding approach that could substantially improve the rate of genetic gain for grain yield, and its deployment depends on the prediction accuracy (PA) that can be achieved. In our study, four yield trials representing breeding lines' advancement stages of the breeding program (S0, S1, S2, and S3) were assessed with grain yield, aerial high-throughput phenotyping (normalized difference vegetation index, NDVI), and bacterial blight disease scores (BBSC). Low-to-moderate broad-sense heritability (0.31-0.71) and narrow-sense heritability (0.13-0.71) were observed, as the estimated additive and non-additive genetic components for the three traits varied with the different models fitted. The genetic correlations among the three traits were high, particularly in the S0-S2 stages. NDVI and BBSC were combined to investigate the PA for grain yield by univariate and multivariate GS models, and multivariate models showed higher PA than univariate models in both cross-validation and forward prediction methods. A 6-50% improvement in PA was achieved when multivariate models were deployed. The highest PA was indicated in the forward prediction scenario when the training population consisted of early generation breeding stages with the multivariate models. Both NDVI and BBSC are commonly used traits that could be measured in the early growth stage; however, our study suggested that NDVI is a more useful trait to predict grain yield with high accuracy in the field pea breeding program, especially in diseased trials, through its incorporation into multivariate models.

Advances in lentil production through heterosis: Evaluating generations and breeding systems.

Heterosis is defined as increased performance of the F1 hybrid relative to its parents. In the current study, a cohort of populations and parents were created to evaluate and understand heterosis across generations (i.e., F1 to F3) in lentil, a self-pollinated annual diploid (2n = 2× = 14) crop species. Lentil plants were evaluated for heterotic traits in terms of plant height, biomass fresh weight, seed number, yield per plant and 100 grain weight. A total of 47 selected lentil genotypes were cross hybridized to generate 72 F1 hybrids. The F1 hybrids from the top five crosses exhibited between 31%-62% heterosis for seed number with reference to the better parent. The five best performing heterotic crosses were selected with a negative control for evaluation at the subsequent F2 generation and only the tails of the distribution taken forward to be assessed in the F3 generation as a sub selection. Overall, heterosis decreases across the subsequent generations for all traits studied. However, some individual genotypes were identified at the F2 and sub-selected F3 generations with higher levels of heterosis than the best F1 mean value (hybrid mimics). The phenotypic data for the selected F2 and sub selected F3 hybrids were analysed, and the study suggested that 100 grain weight was the biggest driver of yield followed by seed number. A genetic diversity analysis of all the F1 parents failed to correlate genetic distance and divergence among parents with heterotic F1's. Therefore, genetic distance was not a key factor to determine heterosis in lentil. The study highlights the challenges associated with different breeding systems for heterosis (i.e., F1 hybrid-based breeding systems and/or via hybrid mimics) but demonstrates the potential significant gains that could be achieved in lentil productivity.

Identification of agro-physiological traits of lentil that reduce risks of drought.

Ideotype breeding is an essential approach for selection of desired combination of plant traits for testing in crop growth model for potential yield gain in specific environments and management practices. Here we parameterized plant traits for untested lentil cultivars for the APSIM-lentil model in phenology, biomass, and seed yield. We then tested these against independent data and applied the model in an extrapolated analysis (i) to assess the impact of drought on productivity across different rainfall environments; (ii) to identify impactful plant traits and (iii) to design new lentil ideotypes with a combination of desirable traits that mitigate the impact of drought, in the context of various agronomic practices across a wide range of production environments. Desirable phenological and physiological traits related to yield were identified with RUE having the greatest effect on yield followed by HI rate. Leaf size significantly affected seed yield (p< 0.05) more than phenological phases. The physiological traits were integrated into four ideotype designs applied to two baseline cultivars (PBA Hallmark XT and PBA Jumbo2) providing eight ideotypes. We identified a combination of genetic traits that promises a yield advantage of around 10% against our current cultivars PBA Hallmark XT and PBA Jumbo2. Under drought conditions, our ideotypes achieved 5 to 25% yield advantages without stubble and 20 to 40% yield advantages with stubble residues. This shows the importance of genetic screening under realistic production conditions (e.g., stubble retention in particular environments). Such screening is aided by the employment of biophysical models that incorporate both genetic and agronomic variables that focus on successful traits in combination, to reduce the impact of drought in the development of new cultivars for various environments. Stubble retention was found to be a major agronomic contributor to high yield in water-limiting environments and this contribution declined with increasing growing season rainfall. In mid- and high-rainfall environments, the key drivers of yield were time of sowing, physiological traits and soil type. Overall, the agronomic practices, namely, early sowing, residue retention and narrow row spacing deceased the impact of drought when combined with improved physiological traits of the ideotypes based on long term climate data.

Breeding has selected for architectural and photosynthetic traits in lentils.

Genetic progress in seed yield in lentils (Lens culinaris Medik) has increased by 1.1% per year in Australia over the past 27 years. Knowing which plant traits have changed through breeding during this time can give important insights as to how lentil yield has increased. This study aims to identify morphological and physiological traits that were directly or indirectly selected between 1993 and 2020 in the Australian lentil breeding program using 2 years of experimental data. Major changes occurred in plant architecture during this period. Divergent selection has seen the release of varieties that have sprawling to very upright types of canopies. Despite this genetic diversity in recently released varieties, there is an overall tendency of recently released varieties having increased plant height and leaf size with reduced number of branches. Increased light interception was positively correlated with year of release (YOR) and yield, and likely results from indirect selection of yield and taller plant types. There is an indication that recently released varieties have lower CO2 assimilation rate, stomatal conductance and canopy temperature depression (CTD) at high ambient temperatures (~30°C). Understanding lentil physiology will assist in identifying traits to increase yield in a changing climate with extreme weather events.

Australian Lentil Breeding Between 1988 and 2019 Has Delivered Greater Yield Gain Under Stress Than Under High-Yield Conditions.

The contemporary lentil (Lens culinaris ssp. culinaris) industry in Australia started in the late 1980s. Yield in farmers' fields averages 1.2 t ha-1 nationally and has not increased over three decades. Lack of yield progress can be related to a number of non-mutually exclusive reasons: expansion of lentil to low-yielding environments, lack of genetic gain in yield, lack of progress in agronomic practices, and lack of adoption of superior technologies. The aims of this study were to (i) quantify the genetic gain in lentil yield since 1988, (ii) explore the variation in the expression of genetic gain with the environment, and (iii) identify shifts in crop phenotype associated with selection for yield and agronomic adaptation. We grew a historic collection of 19 varieties released between 1988 and 2019 in eight environments resulting from the factorial combination of two sowing dates, two water regimes, and two seasons. Across environments, yield varied 11-fold from 0.2 to 2.2 t ha-1. The rate of genetic gain averaged 20 kg ha-1 year-1 or 1.23% year-1 across environments and was higher in low-yield environments. The yield increase was associated with substantial shifts in phenology. Newer varieties had a shorter time to flowering and pod emergence, and the rate of change in these traits was more pronounced in slow-developing environments (e.g., earlier sowing). Thermal time from sowing to end of flowering and maturity were shorter in newer varieties, and thermal time from pod emergence to maturity was longer in newer varieties; the rate of change in these traits was unrelated to developmental drivers and correlated with environmental mean yield. Genetic gain in yield was associated with increased grain number and increased harvest index. Despite their shorter time to maturity, newer varieties had similar or higher biomass than their older counterparts because crop growth rate during the critical period increased with the year of release. Genotype-dependent yield increased over three decades in low-yield environments, whereas actual farm yield has been stagnant; this suggests an increasing yield gap requiring agronomic solutions. Genetic improvement in high-yield environments requires improved coupling of growth and reproduction.

Pulse Root Ideotype for Water Stress in Temperate Cropping System.

Pulses are a key component of crop production systems in Southern Australia due to their rotational benefits and potential profit margins. However, cultivation in temperate cropping systems such as that of Southern Australia is limited by low soil water availability and subsoil constraints. This limitation of soil water is compounded by the irregular rainfall, resulting in the absence of plant available water at depth. An increase in the productivity of key pulses and expansion into environments and soil types traditionally considered marginal for their growth will require improved use of the limited soil water and adaptation to sub soil constrains. Roots serve as the interface between soil constraints and the whole plant. Changes in root system architecture (RSA) can be utilised as an adaptive strategy in achieving yield potential under limited rainfall, heterogenous distribution of resources and other soil-based constraints. The existing literature has identified a "'Steep, Deep and Cheap" root ideotype as a preferred RSA. However, this idiotype is not efficient in a temperate system where plant available water is limited at depth. In addition, this root ideotype and other root architectural studies have focused on cereal crops, which have different structures and growth patterns to pulses due to their monocotyledonous nature and determinant growth habit. The paucity of pulse-specific root architectural studies warrants further investigations into pulse RSA, which should be combined with an examination of the existing variability of known genetic traits so as to develop strategies to alleviate production constraints through either tolerance or avoidance mechanisms. This review proposes a new model of root system architecture of "Wide, Shallow and Fine" roots based on pulse roots in temperate cropping systems. The proposed ideotype has, in addition to other root traits, a root density concentrated in the upper soil layers to capture in-season rainfall before it is lost due to evaporation. The review highlights the potential to achieve this in key pulse crops including chickpea, lentil, faba bean, field pea and lupin. Where possible, comparisons to determinate crops such as cereals have also been made. The review identifies the key root traits that have shown a degree of adaptation via tolerance or avoidance to water stress and documents the current known variability that exists in and amongst pulse crops setting priorities for future research.

Automated phenotyping for early vigour of field pea seedlings in controlled environment by colour imaging technology.

Early vigour of seedlings is a beneficial trait of field pea (Pisum sativum L.) that contributes to weed control, water use efficiency and is likely to contribute to yield under certain environments. Although breeding is considered the most effective approach to improve early vigour of field pea, the absence of a robust and high-throughput phenotyping tool to dissect this complex trait is currently a major obstacle of genetic improvement programs to address this issue. To develop this tool, separate trials on 44 genetically diverse field pea genotypes were conducted in the automated plant phenotyping platform of Plant Phenomics Victoria, Horsham and in the field, respectively. High correlation between estimated plant parameters derived from the automated phenotyping platform and important early vigour traits such as shoot biomass, leaf area and plant height indicated that the derived plant parameters can be used to predict vigour traits in field pea seedlings. Plant growth analysis demonstrated that the "broken-stick" model fitted well with the growth pattern of all field pea genotypes and can be used to determine the linear growth phase. Further analysis suggested that the estimated plant parameters collected at the linear growth phase can effectively differentiate early vigour across field pea genotypes. High correlation between normalised difference vegetation indices captured from the field trial and estimated shoot biomass and top-view area confirmed the consistent performance of early vigour field pea genotypes under controlled and field environments. Overall, our results demonstrated that this robust screening tool is highly applicable and will enable breeding programs to rapidly identify early vigour traits and utilise germplasm to contribute to the genetic improvement of field peas.

Identification and evaluation of resistance to powdery mildew and yellow rust in a wheat mapping population.

Deployment of cultivars with genetic resistance is an effective approach to control the diseases of powdery mildew (PM) and yellow rust (YR). Chinese wheat cultivar XK0106 exhibits high levels of resistance to both diseases, while cultivar E07901 has partial, adult plant resistance (APR). The aim of this study was to map resistance loci derived from the two cultivars and analyze their effects against PM and YR in a range of environments. A doubled haploid population (388 lines) was used to develop a framework map consisting of 117 SSR markers, while a much higher density map using the 90K Illumina iSelect SNP array was produced with a subset of 80 randomly selected lines. Seedling resistance was characterized against a range of PM and YR isolates, while field scores in multiple environments were used to characterize APR. Composite interval mapping (CIM) of seedling PM scores identified two QTLs (QPm.haas-6A and QPm.haas-2A), the former being located at the Pm21 locus. These QTLs were also significant in field scores, as were Qpm.haas-3A and QPm.haas-5A. QYr.haas-1B-1 and QYr.haas-2A were identified in field scores of YR and were located at the Yr24/26 and Yr17 chromosomal regions respectively. A second 1B QTL, QYr.haas-1B-2 was also identified. QPm.haas-2A and QYr.haas-1B-2 are likely to be new QTLs that have not been previously identified. Effects of the QTLs were further investigated in multiple environments through the testing of selected lines predicted to contain various QTL combinations. Significant additive interactions between the PM QTLs highlighted the ability to pyramid these loci to provide higher level of resistance. Interactions between the YR QTLs gave insights into the pathogen populations in the different locations as well as showing genetic interactions between these loci.

Validation of molecular markers associated with boron tolerance, powdery mildew resistance and salinity tolerance in field peas.

Field pea (Pisum sativum L.) is an important grain legume consumed both as human food and animal feed. However, productivity in low rainfall regions can be significantly reduced by inferior soils containing high levels of boron and/or salinity. Furthermore, powdery mildew (PM) (Erysiphe pisi) disease also causes significant yield loss in warmer regions. Breeding for tolerance to these abiotic and biotic stresses are major aims for pea breeding programs and the application of molecular markers for these traits could greatly assist in developing improved germplasm at a faster rate. The current study reports the evaluation of a near diagnostic marker, PsMlo, associated with PM resistance and boron (B) tolerance as well as linked markers associated with salinity tolerance across a diverse set of pea germplasm. The PsMlo1 marker predicted the PM and B phenotypic responses with high levels of accuracy (>80%) across a wide range of field pea genotypes, hence offers the potential to be widely adapted in pea breeding programs. In contrast, linked markers for salinity tolerance were population specific; therefore, application of these markers would be suitable to relevant crosses within the program. Our results also suggest that there are possible new sources of salt tolerance present in field pea germplasm that could be further exploited.

Transcriptomic analysis of wheat near-isogenic lines identifies PM19-A1 and A2 as candidates for a major dormancy QTL.

BACKGROUND: Next-generation sequencing technologies provide new opportunities to identify the genetic components responsible for trait variation. However, in species with large polyploid genomes, such as bread wheat, the ability to rapidly identify genes underlying quantitative trait loci (QTL) remains non-trivial. To overcome this, we introduce a novel pipeline that analyses, by RNA-sequencing, multiple near-isogenic lines segregating for a targeted QTL. RESULTS: We use this approach to characterize a major and widely utilized seed dormancy QTL located on chromosome 4AL. It exploits the power and mapping resolution afforded by large multi-parent mapping populations, whilst reducing complexity by using multi-allelic contrasts at the targeted QTL region. Our approach identifies two adjacent candidate genes within the QTL region belonging to the ABA-induced Wheat Plasma Membrane 19 family. One of them, PM19-A1, is highly expressed during grain maturation in dormant genotypes. The second, PM19-A2, shows changes in sequence causing several amino acid alterations between dormant and non-dormant genotypes. We confirm that PM19 genes are positive regulators of seed dormancy. CONCLUSIONS: The efficient identification of these strong candidates demonstrates the utility of our transcriptomic pipeline for rapid QTL to gene mapping. By using this approach we are able to provide a comprehensive genetic analysis of the major source of grain dormancy in wheat. Further analysis across a diverse panel of bread and durum wheats indicates that this important dormancy QTL predates hexaploid wheat. The use of these genes by wheat breeders could assist in the elimination of pre-harvest sprouting in wheat.

A Lycopersicon esculentum phosphate transporter (LePT1) involved in phosphorus uptake from a vesicular-arbuscular mycorrhizal fungus.

In vesicular-arbuscular mycorrhizal symbioses, specialized fungal structures (the arbuscules) are formed which are in intimate contact with plant root cortical cells. It is assumed that these arbuscules are the major sites of solute transfer between the plant and fungus, but there have been no studies that definitively show the extent or types of transfer processes that occur in this structure. Phosphate is one of the major nutrients that is acquired by mycorrhizal fungi and transferred to plants. In this study a single Lycopersicon esculentum cDNA was cloned and shown to be identical to LePT1, a previously cloned inorganic-phosphate transporter. Expression studies revealed that LePT1 transcript levels remained constant in mycorrhizal plants, but increased in phosphate-starved, non-mycorrhizal plants. Localization of the LePT1 transcript by in situ hybridization showed that this gene is highly expressed in arbuscule-containing cortical cells in mycorrhizal plants. In non-mycorrhizal plants LePT1 expression was localized to the stele and cortex. The expression studies suggest that this transporter is involved in phosphate nutrition of L. esculentum and its localization in cells that contain arbuscules indicate that it may be the mechanism used by the plant to take up phosphate that is effluxed across the fungal plasma membrane of the arbuscule. Based on our findings and those of others, an integrated model of inorganic phosphate uptake and transfer in mycorrhizal and non-mycorrhizal plants is presented.

Localization of proton-ATPase genes expressed in arbuscular mycorrhizal tomato plants.

In arbuscular mycorrhizal symbioses, solutes such as phosphate are transferred to the plant in return for photoassimilates. The uptake mechanism is probably facilitated by a proton gradient generated by proton H(+)-ATPases. We investigated expression of Lycopersicon esculentum Mill. H(+)-ATPases in mycorrhizal and non-mycorrhizal plants to determine if any are specifically regulated in response to colonization. Tissue expression and cellular localization of H(+)-ATPases were determined by RNA gel blot analysis and in situ hybridization of mycorrhizal and non-mycorrhizal roots. LHA1, LHA2, and LHA4 had high levels of expression in roots and were expressed predominantly in epidermal cells. LHA1 and LHA4 were also expressed in cortical cells containing arbuscules. The presence of arbuscules in root sections was correlated with lower levels of expression of these two isoforms in the epidermis. These results suggest that LHA1 and LHA4 expression is decreased in epidermal cells located in regions of the root that contain arbuscules. This provides evidence of differential regulation between molecular mechanisms involved in proton-coupled nutrient transfer either from the soil or fungus to the plant.

Differential expression of Glomus intraradices genes in external mycelium and mycorrhizal roots of tomato and barley.

Relative quantitative RT-PCR and western blotting were used to investigate the expression of three genes with potentially regulatory functions from the arbuscular mycorrhizal fungus Glomus intraradices in symbiosis with tomato and barley. Standardisation of total RNA per sample and determination of different ratios of plant and fungal RNA in roots as colonisation proceeded were achieved by relative quantitative RT-PCR using universal (NS1/NS21) and organism-specific rRNA primers. In addition, generic primers were designed for amplification of plant or fungal beta-tubulin genes and for plant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes as these have been suggested as useful controls in symbiotic systems. The fungal genes Ginmyc1 and Ginhb1 were expressed only in the external mycelium and not in colonised roots at both mRNA and protein levels, with the proteins detected almost exclusively in the insoluble fractions. In contrast, mRNA of Ginmyc2 was identified in both external and intraradical mycelium. In mycorrhizal roots, Ginmyc2 and fungal beta-tubulin mRNAs increased in proportion to fungal rRNA as colonisation proceeded, suggesting that accumulation reflected intraradical fungal growth. Fungal alpha-tubulin protein and beta-tubulin mRNA both appeared to be more abundantly accumulated in AM hyphae within heavily colonised roots than in external hyphae, relative to fungal rRNA. Tomato GAPDH mRNA accumulation was proportional to tomato rRNA, but accumulation of tomato beta-tubulin mRNA was reduced in colonised roots compared to non-mycorrhizal roots. These results provide novel evidence of differential spatial and temporal regulation of AM fungal genes, indicate that the expression of tubulin genes of both plant and fungus may be regulated during colonisation and validate the use of multiple 'control' genes in analysis of mycorrhizal gene expression.