Mark Sweetingham

Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L.

BackgroundIn the last 30 years, a number of DNA fingerprinting methods such as RFLP, RAPD, AFLP, SSR, DArT, have been extensively used in marker development for molecular plant breeding. However, it remains a daunting task to identify highly polymorphic and closely linked molecular markers for a target trait for molecular marker-assisted selection. The next-generation sequencing (NGS) technology is far more powerful than any existing generic DNA fingerprinting methods in generating DNA markers. In this study, we employed a grain legume crop Lupinus angustifolius (lupin) as a test case, and examined the utility of an NGS-based method of RAD (restriction-site associated DNA) sequencing as DNA fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding.ResultsTwenty informative plants from a cross of RxS (disease resistant x susceptible) in lupin were subjected to RAD single-end sequencing by multiplex identifiers. The entire RAD sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform Solexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencing data, were collected. Sequence comparison among the 20 test plants discovered 8207 SNP markers. Filtration of DNA sequencing data with marker identification parameters resulted in the discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective, simple PCR-based markers. Linkage analysis using marker genotyping data and disease resistance phenotyping data on a F8 population consisting of 186 individual plants confirmed that all these five markers were linked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3 and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM), and are now replacing the markers previously developed by a traditional DNA fingerprinting method for marker-assisted selection in the Australian national lupin breeding program.ConclusionsWe demonstrated that more than 30 molecular markers linked to a target gene of agronomic trait of interest can be identified from a small portion (1/8) of one sequencing run on HiSeq2000 by applying NGS based RAD sequencing in marker development. The markers developed by the strategy described in this study are all co-dominant SNP markers, which can readily be converted into high throughput multiplex format or low-cost, simple PCR-based markers desirable for large scale marker implementation in plant breeding programs. The high density and closely linked molecular markers associated with a target trait help to overcome a major bottleneck for implementation of molecular markers on a wide range of germplasm in breeding programs. We conclude that application of NGS based RAD sequencing as DNA fingerprinting is a very rapid and cost-effective strategy for marker development in molecular plant breeding. The strategy does not require any prior genome knowledge or molecular information for the species under investigation, and it is applicable to other plant species.

Screening techniques and sources of resistance to foliar diseases caused by major necrotrophic fungi in grain legumes

SummaryNecrotrophic pathogens of the cool season food legumes (pea, lentil, chickpea, faba bean and lupin) cause wide spread disease and severe crop losses throughout the world. Environmental conditions play an important role in the development and spread of these diseases. Form of inoculum, inoculum concentration and physiological plant growth stage all affect the degree of infection and the amount of crop loss. Measures to control these diseases have relied on identification of resistant germplasm and development of resistant varieties through screening in the field and in controlled environments. Procedures for screening and scoring germplasm and breeding lines for resistance have lacked uniformity among the various programs worldwide. However, this review highlights the most consistent screening and scoring procedures that are simple to use and provide reliable results. Sources of resistance to the major necrotrophic fungi are summarized for each of the cool season food legumes. Marker-assisted selection is underway for Ascochyta blight of pea, lentil and chickpea, and Phomopsis blight of lupin. Other measures such as fungicidal control and cultural control are also reviewed. The emerging genomic information on the model legume, Medicago truncatula, which has various degrees of genetic synteny with the cool season food legumes, has promise for identification of closely linked markers for resistance genes and possibly for eventual map-based cloning of resistance genes. Durable resistance to the necrotrophic pathogens is a common goal of cool season food legume breeders.

Development and implementation of a sequence-specific PCR marker linked to a gene conferring resistance to anthracnose disease in narrow-leafed lupin (Lupinus angustifolius L.)

Anthracnose caused by Colletotrichum gloeosporioides is the most serious disease of lupins (Lupinus spp). A cross was made between cultivars Tanjil (resistant) and Unicrop (susceptible) in narrow-leafed lupin (L. angustifolius). Analysis of disease reaction data on the F2 population and on the resultant F7 recombinant inbred lines suggested that Tanjil contained a single dominant gene (Lanr1) conferring resistance to anthracnose. The parents and the representative F2 plants were used to generate molecular markers liked to the Lanr1 gene using the MFLP technique. A co-dominant MFLP polymorphism linked to the Lanr1 gene was identified as a candidate marker. The bands were isolated, re-amplified by PCR, cloned and sequenced. The MFLP polymorphism was converted into a co-dominant, sequence-specific, simple PCR-based marker. Linkage analysis by the computer program MAPMAKER indicated that the marker was 3.5 centiMorgans (cM) from the gene Lanr1. This marker is currently being implemented for marker assisted selection in the Australian National Lupin Breeding Program.

Draft genome sequence, and a sequence-defined genetic linkage map of the legume crop species Lupinus angustifolius L.

Lupin (Lupinus angustifolius L.) is the most recently domesticated crop in major agricultural cultivation. Its seeds are high in protein and dietary fibre, but low in oil and starch. Medical and dietetic studies have shown that consuming lupin-enriched food has significant health benefits. We report the draft assembly from a whole genome shotgun sequencing dataset for this legume species with 26.9x coverage of the genome, which is predicted to contain 57,807 genes. Analysis of the annotated genes with metabolic pathways provided a partial understanding of some key features of lupin, such as the amino acid profile of storage proteins in seeds. Furthermore, we applied the NGS-based RAD-sequencing technology to obtain 8,244 sequence-defined markers for anchoring the genomic sequences. A total of 4,214 scaffolds from the genome sequence assembly were aligned into the genetic map. The combination of the draft assembly and a sequence-defined genetic map made it possible to locate and study functional genes of agronomic interest. The identification of co-segregating SNP markers, scaffold sequences and gene annotation facilitated the identification of a candidate R gene associated with resistance to the major lupin disease anthracnose. We demonstrated that the combination of medium-depth genome sequencing and a high-density genetic linkage map by application of NGS technology is a cost-effective approach to generating genome sequence data and a large number of molecular markers to study the genomics, genetics and functional genes of lupin, and to apply them to molecular plant breeding. This strategy does not require prior genome knowledge, which potentiates its application to a wide range of non-model species.

Characterization and Pathogenicity of Rhizoctonia Species on Canola

A total of 112 Rhizoctonia isolates were collected from various canola (Brassica napus) growing areas of Western Australia. Pectic enzyme electrophoresis differentiated these isolates into six distinct zymogram groups: R. solani, 54% ZG5 (AG2-1), 8% ZG6 (AG2-1), and 1% ZG9 (AG10); binucleate Rhizoctonia, 12% CZG1 (CAG1), 4% CZG4, and 6% CZG5 (AGK); and the remainder unidentified binucleate groups (15%). Binucleate groups were also confirmed by fluorescent nuclear staining and hyphal morphology. One or more isolates from each of the above zymogram groups (including four unidentified binucleate groups) and an isolate of ZG1-1 (AG8) that causes bare patch in cereals and legumes were tested for their pathogenicity on canola. Isolates of ZG5 and ZG1-1 were highly pathogenic on canola, delayed seedling emergence, and caused severe hypocotyl and root rot, respectively. ZG5 also induced postemergence damping-off. Increasing the depth of sowing from 1 to 3 cm significantly delayed seedling emergence and increased disease severity. Four unidentified binucleate isolates (WAC9316, WAC9297, WAC9307, and WAC9290) were moderately pathogenic to canola, while two isolates (WAC9307 and WAC9316) caused significant preemergence damping-off. Two CZG5 isolates were weakly pathogenic. Isolates of ZG5 and ZG1-1 were also tested for their pathogenicity on other rotational crops (narrow-leafed lupin, subterranean clover, wheat, oats, barley, and mustard) and two weed species (wild radish and annual ryegrass). ZG5 caused a severe hypocotyl rot on mustard and mild symptoms of hypocotyl rot on narrow-leafed lupin and clover, but failed to infect any of the cereal hosts, such as wheat, oats, barley, and ryegrass. In contrast, all crops tested were highly susceptible to ZG1-1 except mustard, which was only moderately susceptible. Results indicate that ZG5 is most pathogenic to crucifers and is a mildly virulent pathogen of the leguminous crops but not of cereal crops tested. ZG1-1, known to cause bare patch in legumes and cereals, also can cause severe root rot in canola. This is the first report of hypocotyl rot and pathogenicity of ZG5 on canola in Australia.

AnthracnoseTracer: A Spatiotemporal Model for Simulating the Spread of Anthracnose in a Lupin Field

ABSTRACT A spatiotemporal model has been developed to simulate the spread of anthracnose, initiated by infected seed, in a lupin field. The model quantifies the loss of healthy growing points of lupin in all 1-m(2) subunits of a field throughout a growing season. The development of growing points is modeled as a function of temperature using a 1-day time step, and disease-induced compensatory growth is accounted for. Dispersal of spores is simulated explicitly using Monte Carlo techniques. Spread of spores occurs during rainfall events on a 1-h time step. The distance traveled by spores is partially dependent on wind speed and is generated by adding the values selected from half-Cauchy distributions. The direction of travel of the spores is influenced by wind direction. The model has been employed to produce a theoretical assessment of damage from disease in two environments at five levels of seed infection. It was calculated that in a susceptible lupin cultivar with a 0.01% initial seed infection, anthracnose would cause approximately 15% loss of healthy growing points in a high rainfall environment in Western Australia. In a low rainfall environment, similar damage would be unlikely even with a much higher (1%) level of seed infection.

Ecogeography of the Old World lupins. 1. Ecotypic variation in yellow lupin (Lupinus luteus L.)

Agricultural crops and their wild progenitors are excellent candidates for ecophysiologal research because germplasm collections are often extensive and well described, and in its dissemination the crop may explore new habitats. The advent of high-resolution climate models has greatly improved our capacity to characterise plant habitats, and study species’ adaptive responses. The yellow lupin (Lupinus luteus) is ideal because it evolved as a Mediterranean winter-annual in relatively high-rainfall coastal regions, but was domesticated as a summer crop in temperate central Europe. Currently the crop is being developed for Mediterranean south-western Australia, raising an interesting ecophysiological problem: is it more appropriate to concentrate on wild material from Mediterranean habitats, which are likely to be more similar to the target environments, or on European germplasm domesticated for temperate summer cropping? Lupinus luteus collection sites across the natural and domesticated distribution range were characterised by calculating site-specific bioclimatic variables and habitat types defined using multivariate analysis. Germplasm was evaluated in 2 field trials measuring a range of characters describing plant growth, phenology, architecture, and productivity. The earliest phenology and highest vigour and productivity were recorded in domesticated material from central Europe, characterised by short but unstressful growing seasons with reliable rainfall, long day-lengths, and rapidly rising vegetative-phase temperatures levelling out after flowering. Mediterranean habitats were classified by altitude, climate, and growing-season length. Early, productive germplasm came from warmer/low elevation sites with inconsistent rainfall and stronger terminal drought. Germplasm from low temperature/high elevation sites with high, relatively frequent rainfall had late phenology and low growth rates, early vigour, seed yield, and harvest index. Distinct habitats within the distribution range of L. luteus have selected for ecotypes with different phenologies and growth rates, which strongly influence plant architecture, fecundity and yield. It is suggested that variable responses to vernalisation and differences in seed size are important in determining these traits. European germplasm has many of the terminal drought-avoiding characteristics required in a productive Mediterranean ideotype, but may lack drought tolerance, which is likely to be under stronger selection pressure in more stressful Mediterranean habitats.

Identification of anthracnose resistance in Lupinus albus L. and its transfer from landraces to modern cultivars

Anthracnose is a major disease of lupins in Western Australia (WA). The disease wiped out the WA albus lupin industry in 1996 and since then, anthracnose resistance has been a major focus for WA lupin breeding. In an endeavour to find a source of resistance to anthracnose, all available germplasm in WA was screened against anthracnose in New Zealand over the summer of 1997 and 1998, and resistance was identified in Ethiopian landraces. The resistance was present in many Ethiopian landraces within a close geographical distribution, suggesting a similar genetic basis of resistance. Crosses were made between the resistant landraces and agronomically superior lines. The progeny were tested for anthracnose resistance, yield, seed quality, and other agronomic characters. The most superior line, Andromeda, was released for commercial production in WA. It was developed from an F3-derived single-plant selection of a cross between an anthracnose-resistant landrace P27175 from Ethiopia and a well adapted but highly susceptible WA breeding line 89B10A-14. Andromeda has a significantly higher level of resistance to anthracnose than the previous cv. Kiev Mutant and is recommended in the medium- to low-rainfall area of the northern wheatbelt of WA. Further breeding effort has resulted in significant improvement in the level of resistance within the WA breeding program, and early generation lines are more resistant than advanced lines. The best resistant lines are, however, in a late flowering background and only an incremental improvement has been achieved in combining early flowering with anthracnose resistance, which seems to be a complex process.

Anthracnose of lupins in Western Australia

In August 1994 a severe anthracnose was found on Lupinus albus in plots at four sites in Western Australia. Colletotrichum gloeosporioides was isolated from lesions on collapsed stems. At one site the disease had spread to adjacent plots of L. albus, L. angustfolius, L. luteus and L. mutabilis. The primary infection was traced to seed lines of L. albus imported from Germany. In September 1994 procedures were put into place to eradicate the disease.

Effect of temperature on growth of Colletotrichum lupini and on anthracnose infection and resistance in lupins

The impact of temperature was assessed on mycelial growth of Colletotrichum lupini on agar and on anthracnose expression in lupin cultivars with varying degrees of resistance. Growth rate of C. lupini was determined at temperatures from 5 to 3°C. Fungal growth was observed at temperatures from 10 to 30°C with maximum growth rate occurring at 25°C. In growth cabinet experiments, the impact of temperatures ranging from 10 to 26°C was observed on anthracnose development in the Lupinus angustifolius cultivars Myallie (susceptible), Merrit (moderately susceptible) and Wonga (resistant) and the L. albus cultivar Kiev Mutant (extremely susceptible). At 10°C, disease incidence was very low (0–12%) in all cultivars, reflecting the slow in vitro growth of the fungus at this temperature. Increasing temperature from 12 to 26°C reduced the latent period by more than 50% and increased disease incidence, sporulation incidence and lesion severity. Latent period response to temperature was independent of cultivar resistance. However, cultivar×temperature interactionswere evident for disease incidence, sporulation incidence and lesion severity responses. Anthracnose resistance in cv.Wonga was temperature sensitive. At 12 and 18°C, disease incidence and severity inWonga were significantly lower than other L. angustifolius cultivars tested but at 26°C disease incidence and severity were similar to other cultivars.