A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits

Background: Molecular marker technologies are undergoing a transition from largely serial assays measuring DNA fragment sizes to hybridization-based technologies with high multiplexing levels. Diversity Arrays Technology (DArT) is a hybridization-based technology that is increasingly being adopted by barley researchers. There is a need to integrate the information generated by DArT with previous data produced with gel-based marker technologies. The goal of this study was to build a high-density consensus linkage map from the combined datasets of ten populations, most of which were simultaneously typed with DArT and Simple Sequence Repeat (SSR), Restriction Enzyme Fragment Polymorphism (RFLP) and/or Sequence Tagged Site (STS) markers. Results: The consensus map, built using a combination of JoinMap 3.0 software and several purpose-built perl scripts, comprised 2,935 loci (2,085 DArT, 850 other loci) and spanned 1,161 cM. It contained a total of 1,629 'bins' (unique loci), with an average inter-bin distance of 0.7 ± 1.0 cM (median = 0.3 cM). More than 98% of the map could be covered with a single DArT assay. The arrangement of loci was very similar to, and almost as optimal as, the arrangement of loci in component maps built for individual populations. The locus order of a synthetic map derived from merging the component maps without considering the segregation data was only slightly inferior. The distribution of loci along chromosomes indicated centromeric suppression of recombination in all chromosomes except 5H. DArT markers appeared to have a moderate tendency toward hypomethylated, gene-rich regions in distal chromosome areas. On the average, 14 ± 9 DArT loci were identified within 5 cM on either side of SSR, RFLP or STS loci previously identified as linked to agricultural traits. Conclusion: Our barley consensus map provides a framework for transferring genetic information between different marker systems and for deploying DArT markers in molecular breeding schemes. The study also highlights the need for improved software for building consensus maps from high-density segregation data of multiple populations.

High subsoil chloride concentrations reduce soil water extraction and crop yield on Vertosols in north-eastern Australia

Salinity, sodicity, acidity, and phytotoxic levels of chloride (Cl) in subsoils are major constraints to crop production in many soils of north-eastern Australia because they reduce the ability of crop roots to extract water and nutrients from the soil. The complex interactions and correlations among soil properties result in multi-colinearity between soil properties and crop yield that makes it difficult to determine which constraint is the major limitation. We used ridge-regression analysis to overcome colinearity to evaluate the contribution of soil factors and water supply to the variation in the yields of 5 winter crops on soils with various levels and combinations of subsoil constraints in the region. Subsoil constraints measured were soil Cl, electrical conductivity of the saturation extract (ECse), and exchangeable sodium percentage (ESP). The ridge regression procedure selected several of the variables used in a descriptive model, which included in-crop rainfall, plant-available soil water at sowing in the 0.90-1.10 m soil layer, and soil Cl in the 0.90-1.10 m soil layer, and accounted for 77-85% of the variation in the grain yields of the 5 winter crops. Inclusion of ESP of the top soil (0.0-0.10 m soil layer) marginally increased the descriptive capability of the models for bread wheat, barley and durum wheat. Subsoil Cl concentration was found to be an effective substitute for subsoil water extraction. The estimates of the critical levels of subsoil Cl for a 10% reduction in the grain yield were 492 mg cl/kg for chickpea, 662 mg Cl/kg for durum wheat, 854 mg Cl/kg for bread wheat, 980 mg Cl/kg for canola, and 1012 mg Cl/kg for barley, thus suggesting that chickpea and durum wheat were more sensitive to subsoil Cl than bread wheat, barley, and canola.

Developmental and physiological traits associated with high yield and stay-green phenotype in wheat

Water availability is a key limiting factor in wheat production in the northern grain belt of Australia. Varieties with improved adaptation to such conditions are actively sought. The CIMMYT wheat line SeriM82 has shown a significant yield advantage in multi-environment screening trials in this region. The objective of this study was to identify the physiological basis of the adaptive traits underpinning this advantage. Six detailed experiments were conducted to compare the growth, development, and yield of SeriM82 with that of the adapted cultivar, Hartog. The experiments were undertaken in field environments that represented the range of moisture availability conditions commonly encountered by winter crops grown on the deep Vertosol soils of this region. The yield of SeriM82 was 6-28% greater than that of Hartog, and SeriM82 exhibited a stay-green phenotype by maintaining green leaf area longer during the grain-filling period in all environments where yield was significantly greater than Hartog. However, where the availability of deep soil moisture was limited, SeriM82 failed to exhibit significantly greater yield or to express the stay-green phenotype. Thus, the stay-green phenotype was closely associated with the yield advantage of SeriM82. SeriM82 also exhibited higher mean grain mass than Hartog in all environments. It is suggested that small differences in water use before anthesis, or greater water extraction from depth after anthesis, could underlie the stay-green phenotype. The inability of SeriM82 to exhibit stay-green and higher yield where deep soil moisture was depleted indicates that extraction of deep soil moisture is important.

Analysis of high yielding maize production - A study based on a commercial crop

This paper reports on the use of APSIM - Maize for retrospective analysis of performance of a high input, high yielding maize crop and analysis of predicted performance of maize grown with high inputs over the long-term (>100 years) for specified scenarios of environmental conditions (temperature and radiation) and agronomic inputs (sowing date, plant population, nitrogen fertiliser and irrigation) at Boort, Victoria, Australia. It uses a high yielding (17 400 kg/ha dry grain, 20 500 kg/ha at 15% water) commercial crop grown in 2004-05 as the basis of the study. Yield for the agronomic and environmental conditions of 2004-05 was predicted accurately, giving confidence that the model could be used for the detailed analyses undertaken. The analysis showed that the yield achieved was close to that possible with the conditions and agronomic inputs of 2004-05. Sowing dates during 21 September to 26 October had little effect on predicted yield, except when combined with reduced temperature. Single year and long-term analyses concluded that a higher plant population (11 plants/m2) is needed to optimise yield, but that slightly lower N and irrigation inputs are appropriate for the plant population used commercially (8.4 plants/m2). Also, compared with changes in agronomic inputs increases in temperature and/or radiation had relatively minor effects, except that reduced temperature reduces predicted yield substantially. This study provides an approach for the use of models for both retrospective analysis of crop performance and assessment of long-term variability of crop yield under a wide range of agronomic and environmental conditions.

High resolution genetic and physical mapping of the I-3 region of tomato chromosome 7 reveals almost continuous microsynteny with grape chromosome 12 but interspersed microsynteny with duplications on Arabidopsis chromosomes 1, 2 and 3

The tomato I-3 gene introgressed from the Lycopersicon pennellii accession LA716 confers resistance to race 3 of the fusarium wilt pathogen Fusarium oxysporum f. sp. lycopersici. We have improved the high-resolution map of the I-3 region of tomato chromosome 7 with the development and mapping of 31 new PCR-based markers. Recombinants recovered from L. esculentum cv. M82 × IL7-2 F2 and (IL7-2 × IL7-4) × M82 TC1F2 mapping populations, together with recombinants recovered from a previous M82 × IL7-3 F2 mapping population, were used to position these markers. A significantly higher recombination frequency was observed in the (IL7-2 × IL7-4) × M82 TC1F2 mapping population based on a reconstituted L. pennellii chromosome 7 compared to the other two mapping populations based on smaller segments of L. pennellii chromosome 7. A BAC contig consisting of L. esculentum cv. Heinz 1706 BACs covering the I-3 region has also been established. The new high-resolution map places the I-3 gene within a 0.38 cM interval between the molecular markers RGA332 and bP23/gPT with an estimated physical size of 50-60 kb. The I-3 region was found to display almost continuous microsynteny with grape chromosome 12 but interspersed microsynteny with Arabidopsis thaliana chromosomes 1, 2 and 3. An S-receptor-like kinase gene family present in the I-3 region of tomato chromosome 7 was found to be present in the microsyntenous region of grape chromosome 12 but was absent altogether from the A. thaliana genome.

The use of high resolution melting (HRM) to map single nucleotide polymorphism markers linked to a covered smut resistance gene in barley

Using an established genetic map, a single gene conditioning covered smut resistance, Ruh.7H, was mapped to the telomere region of chromosome 7HS in an Alexis/Sloop doubled haploid barley population. The closest marker to Ruh.7H, abg704 was 7.5 cM away. Thirteen loci on the distal end of 7HS with potential to contain single nucleotide polymorphisms (SNPs) were identified by applying a comparative genomics approach using rice sequence data. Of these, one locus produced polymorphic co-dominant bands of different size while two further loci contained SNPs that were identified using the recently developed high resolution melting (HRM) technique. Two of these markers flanked Ruh.7H with the proximal marker located 3.8 cM and the distal marker 2.7 cM away. This is the first report on the application of the HRM technique to SNP detection and to rapid scoring of known cleaved amplified polymorphic sequence (CAPS) markers in plants. This simple, precise post-PCR technique should find widespread use in the fine-mapping of genetic regions of interest in complex cereal and other plant genomes.

Laboratory information management software for genotyping workflows: Applications in high throughput crop genotyping

Background: With the advances in DNA sequencer-based technologies, it has become possible to automate several steps of the genotyping process leading to increased throughput. To efficiently handle the large amounts of genotypic data generated and help with quality control, there is a strong need for a software system that can help with the tracking of samples and capture and management of data at different steps of the process. Such systems, while serving to manage the workflow precisely, also encourage good laboratory practice by standardizing protocols, recording and annotating data from every step of the workflow Results: A laboratory information management system (LIMS) has been designed and implemented at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) that meets the requirements of a moderately high throughput molecular genotyping facility. The application is designed as modules and is simple to learn and use. The application leads the user through each step of the process from starting an experiment to the storing of output data from the genotype detection step with auto-binning of alleles; thus ensuring that every DNA sample is handled in an identical manner and all the necessary data are captured. The application keeps track of DNA samples and generated data. Data entry into the system is through the use of forms for file uploads. The LIMS provides functions to trace back to the electrophoresis gel files or sample source for any genotypic data and for repeating experiments. The LIMS is being presently used for the capture of high throughput SSR (simple-sequence repeat) genotyping data from the legume (chickpea, groundnut and pigeonpea) and cereal (sorghum and millets) crops of importance in the semi-arid tropics. Conclusions: A laboratory information management system is available that has been found useful in the management of microsatellite genotype data in a moderately high throughput genotyping laboratory. The application with source code is freely available for academic users and can be downloaded from http://www.icrisat.org/bt-software-d-lims.htm

A systems evaluation of high-input management using fortified molasses for beef production in Australia's dry tropics

The potential of beef producers to profitably produce 500-kg steers at 2.5 years of age in northern Australia's dry tropics to meet specifications of high-value markets, using a high-input management (HIM) system was examined. HIM included targeted high levels of fortified molasses supplementation, short seasonal mating and the use of growth promotants. Using herds of 300-400 females plus steer progeny at three sites, HIM was compared at a business level to prevailing best-practice, strategic low-input management (SLIM) in which there is a relatively low usage of energy concentrates to supplement pasture intake. The data presented for each breeding-age cohort within management system at each site includes: annual pregnancy rates (range: 14-99%), time of conception, mortalities (range: 0-10%), progeny losses between confirmed pregnancy and weaning (range: 0-29%), and weaning rates (range: 14-92%) over the 2-year observation. Annual changes in weight and relative net worth were calculated for all breeding and non-breeding cohorts. Reasons for outcomes are discussed. Compared with SLIM herds, both weaning weights and annual growth were >= 30 kg higher, enabling 86-100% of HIM steers to exceed 500 kg at 2.5 years of age. Very few contemporary SLIM steers reached this target. HIM was most profitably applied to steers. Where HIM was able to achieve high pregnancy rates in yearlings, its application was recommended in females. Well managed, appropriate HIM systems increased profits by around $15/adult equivalent at prevailing beef and supplement prices. However, a 20% supplement price rise without a commensurate increase in values for young slaughter steers would generally eliminate this advantage. This study demonstrated the complexity of pro. table application of research outcomes to commercial business, even when component research suggests that specific strategies may increase growth and reproductive efficiency and/or be more pro. table. Because of the higher level of management required, higher costs and returns, and higher susceptibility to market changes and disease, HIM systems should only be applied after SLIM systems are well developed. To increase profitability, any strategy must ultimately either increase steer growth and sale values and/or enable a shift to high pregnancy rates in yearling heifers.

The effects of high stocking rates on milk production from dryland and irrigated Mediterranean pastures

An experiment using herds of similar to 20 cows (farmlets) assessed the effects of high stocking rates on production and profitability of feeding systems based on dryland and irrigated perennial ryegrass-based pastures in a Mediterranean environment in South Australia over 4 years. A target level of milk production of 7000 L/cow.year was set, based on predicted intakes of 2.7 t DM/cow.year as concentrates, pasture intakes from 1.5 to 2.7 t/cow.year and purchased fodder. In years 1 and 2, up to 1.5 t DM/cow.year of purchased fodder was used and in years 3 and 4 the amounts were increased if necessary to enable levels of milk production per cow to be maintained at target levels. Cows in dryland farmlets calved in March to May inclusive and were stocked at 2.5, 2.9, 3.3, 3.6 and 4.1 cows/ha, while those in irrigated farmlets calved in August to October inclusive and were stocked at 4.1, 5.2, 6.3 and 7.4 cows/ha. In the first 2 years, when inputs of purchased fodder were limited, milk production per cow was reduced with higher stocking rates (P < 0.01), but in years 3 and 4 there were no differences. Mean production was 7149 kg/cow.year in years 1 and 2, and 8162 kg/cow.year in years 3 and 4. Production per hectare was very closely related to stocking rate in all years (P < 0.01), increasing from 18 to 34 t milk/ha.year for dryland farmlets (1300 to 2200 kg milk solids/ha) and from 30 to 60 t milk/ha.year for irrigated farmlets (2200 to 4100 kg milk solids/ha). Almost all of these increases were attributed to the increases in grain and purchased fodder inputs associated with the increases in stocking rate. Net pasture accumulation rates and pasture harvest were generally not altered with stocking rate, though as stocking rate increased there was a change to more of the pasture being grazed and less conserved in both dryland and irrigated farmlets. Total pasture harvest averaged similar to 8 and 14 t DM/ha.year for dryland and irrigated pastures, respectively. An exception was at the highest stocking rate under irrigation, where pugging during winter was associated with a 14% reduction in annual pasture growth. There were several indications that these high stocking rates may not be sustainable without substantial changes in management practice. There were large and positive nutrient balances and associated increases in soil mineral content (P < 0.01), especially for phosphorus and nitrate nitrogen, with both stocking rate and succeeding years. Levels under irrigation were considerably higher (up to 90 and 240 mg/kg of soil for nitrate nitrogen and phosphorus, respectively) than under dryland pastures (60 and 140 mg/kg, respectively). Soil organic carbon levels did not change with stocking rate, indicating a high level of utilisation of forage grown. Weed ingress was also high (to 22% DM) in all treatments and especially in heavily stocked irrigated pastures during winter. It was concluded the higher stocking rates used exceeded those that are feasible for Mediterranean pastures in this environment and upper levels of stocking are suggested to be 2.5 cows/ha for dryland pastures and 5.2 cows/ha for irrigated pastures. To sustain these suggested stocking rates will require further development of management practices to avoid large increases in soil minerals and weed invasion of pastures.

A consensus genetic map of sorghum that integrates multiple component maps and high-throughput Diversity Array Technology (DArT) markers

Background: Sorghum genome mapping based on DNA markers began in the early 1990s and numerous genetic linkage maps of sorghum have been published in the last decade, based initially on RFLP markers with more recent maps including AFLPs and SSRs and very recently, Diversity Array Technology (DArT) markers. It is essential to integrate the rapidly growing body of genetic linkage data produced through DArT with the multiple genetic linkage maps for sorghum generated through other marker technologies. Here, we report on the colinearity of six independent sorghum component maps and on the integration of these component maps into a single reference resource that contains commonly utilized SSRs, AFLPs, and high-throughput DArT markers. Results: The six component maps were constructed using the MultiPoint software. The lengths of the resulting maps varied between 910 and 1528 cM. The order of the 498 markers that segregated in more than one population was highly consistent between the six individual mapping data sets. The framework consensus map was constructed using a "Neighbours" approach and contained 251 integrated bridge markers on the 10 sorghum chromosomes spanning 1355.4 cM with an average density of one marker every 5.4 cM, and were used for the projection of the remaining markers. In total, the sorghum consensus map consisted of a total of 1997 markers mapped to 2029 unique loci ( 1190 DArT loci and 839 other loci) spanning 1603.5 cM and with an average marker density of 1 marker/0.79 cM. In addition, 35 multicopy markers were identified. On average, each chromosome on the consensus map contained 203 markers of which 58.6% were DArT markers. Non-random patterns of DNA marker distribution were observed, with some clear marker-dense regions and some marker-rare regions. Conclusion: The final consensus map has allowed us to map a larger number of markers than possible in any individual map, to obtain a more complete coverage of the sorghum genome and to fill a number of gaps on individual maps. In addition to overall general consistency of marker order across individual component maps, good agreement in overall distances between common marker pairs across the component maps used in this study was determined, using a difference ratio calculation. The obtained consensus map can be used as a reference resource for genetic studies in different genetic backgrounds, in addition to providing a framework for transferring genetic information between different marker technologies and for integrating DArT markers with other genomic resources. DArT markers represent an affordable, high throughput marker system with great utility in molecular breeding programs, especially in crops such as sorghum where SNP arrays are not publicly available.

High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira.

A new test for pathogenic Leptospira isolates, based on RAPD-PCR and high-resolution melt (HRM) analysis (which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used to define serovars or detect genotypes. Ten serovars, of leptospires from the species Leptospira interrogans (serovars Australis, Robinsoni, Hardjo, Pomona, Zanoni, Copenhageni and Szwajizak), L. borgpetersenii (serovar Arborea), L. kirschneri (serovar Cynopteri) and L. weilii (serovar Celledoni), were typed against 13 previously published RAPD primers, using a real-time cycler (the Corbett Life Science RotorGene 6000) and the optimised reagents from a commercial kit (Quantace SensiMix). RAPD-HRM at specific temperatures generated defining amplicon melt profiles for each of the tested serovars. These profiles were evaluated as difference-curve graphs generated using the RotorGene software package, with a cut-off of at least 8 'U' (plus or minus). The results demonstrated that RAPD-HRM can be used to measure serovar diversity and establish identity, with a high degree of stability. The characterisation of Leptospira serotypes using a DNA-based methodology is now possible. As an objective and relatively inexpensive and rapid method of serovar identification, at least for cultured isolates, RAPD-HRM assays show convincing potentia.

High resolution satellite imagery and GPS collars can help assist in the assessment of patch selection by grazing cattle in semi-arid savannas.

The selection of different patch types for grazing by cattle in tropical savannas is well documented. Advances in high resolution satellite imagery and computing power now allow us to identify patch types over an entire paddock, combined with GPS collars as a non instrusive method of capturing positional data, an accurate and comprehensive picture of landscape use by cattle can be quantified.

Campylobacter jejuni and Campylobacter coli Genotyping by High-Resolution Melting Analysis of a flaA Fragment.

The highly variable flagellin-encoding flaA gene has long been used for genotyping Campylobacter jejuni and Campylobacter coli. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The objective of this study was to apply HRM analysis to flaA-based genotyping. The initial aim was to identify a suitable flaA fragment. It was found that the PCR primers commonly used to amplify the flaA short variable repeat (SVR) yielded a mixed PCR product unsuitable for HRM analysis. However, a PCR primer set composed of the upstream primer used to amplify the fragment used for flaA restriction fragment length polymorphism (RFLP) analysis and the downstream primer used for flaA SVR amplification generated a very pure PCR product, and this primer set was used for the remainder of the study. Eighty-seven C. jejuni and 15 C. coli isolates were analyzed by flaA HRM and also partial flaA sequencing. There were 47 flaA sequence variants, and all were resolved by HRM analysis. The isolates used had previously also been genotyped using single-nucleotide polymorphisms (SNPs), binary markers, CRISPR HRM, and flaA RFLP.flaA HRM analysis provided resolving power multiplicative to the SNPs, binary markers, and CRISPR HRM and largely concordant with the flaA RFLP. It was concluded that HRM analysis is a promising approach to genotyping based on highly variable genes.

Nitrogen use for high productivity and sustainability in cashew.

Interest in cashew production in Australia has been stimulated by domestic and export market opportunities and suitability of large areas of tropical Australia. Economic models indicate that cashew production is profitable at 2.8 t ha-1 nut-in-shell (NIS). Balanced plant nutrition is essential to achieve economic yields in Australia, with nitrogen (N) of particular importance because of its capacity to modify growth, affect nut yield and cause environmental degradation through soil acidification and off-site contamination. The study on a commercial cashew plantation at Dimbulah, Australia, investigated the effect of N rate and timing on cashew growth, nut production, N leaching and soil chemical properties over five growth cycles (1995-1999). Nitrogen was applied during the main periods of vegetative (December-April) and reproductive (June-October) growth. Commercial NIS yields (up to 4.4 t ha-1 from individual trees) that exceeded the economic threshold of 2.8 t ha-1 were achieved. The yield response was mainly determined by canopy size as mean nut weight, panicle density and nuts per panicle were largely unaffected by N treatments. Nitrogen application confined to the main period of vegetative growth (December-April) produced a seasonal growth pattern that corresponded most consistently with highest NIS yield. This N timing also reduced late season flowering and undesirable post-November nut drop. Higher yields were not produced at N rates greater than 17 g m-2 of canopy surface area (equating to 210 kg N ha-1 for mature size trees). High yields were attained when N concentrations in Mveg leaves in May-June were about 2%, but this assessment occurs at a time when it is not feasible to correct N deficiency. The Mflor leaf of the preceding November, used in conjunction with the Mveg leaf, was proposed as a diagnostic tool to guide N rate decisions. Leaching of nitrate-N and acidification of the soil profile was recorded to 0.9 m. This is an environmental and sustainability hazard, and demonstrates that improved methods of N management are required.

Resilience of a high-conservation-value, semi-arid grassland on fertile clay soils to burning, mowing and ploughing.

In grassland reserves, managed disturbance is often necessary to maintain plant species diversity. We carried out experiments to determine the impact of fire, kangaroo grazing, mowing and disc ploughing on grassland species richness and composition in a nature reserve in semi-arid eastern Australia. Vegetation response was influenced by winter-spring drought after establishment of the experiments, but moderate rainfall followed in late summer-autumn. Species composition varied greatly between sampling times, and the variability due to rainfall differences between seasons and years was greater than the effects of fire, kangaroo grazing, mowing or disc ploughing. In the fire experiment, species richness and composition recovered more rapidly after spring than autumn burning. Species richness and composition were similar to control sites within 12 months of burning and mowing, suggesting that removal of the dominant grass canopy is unnecessary to enhance plant diversity. Two fires (separated by 3 years) and post-fire kangaroo grazing had only minor influence on species richness and composition. Even disc ploughing caused only a small reduction in native richness. The minor impact of ploughing was explained by the small areas that were ploughed, the once-off nature of the treatment, and the high degree of natural movement and cracking in these shrink-swell soils. Recovery of the composition and richness of these grasslands was rapid because of the high proportion of perennial species that resprout vegetatively after fire and mowing. There appears to be little conservation benefit from fire, mowing or ploughing ungrazed areas, as we could identify no native plant species dependent on frequent disturbance for persistence in this grassland community. However, the ability of the Astrebla- and Dichanthium-dominated grasslands to recover quickly after disturbance, given favourable seasonal conditions, suggests that they are well adapted to natural disturbances (e.g. droughts, fire, flooding and native grazing).