Michael Materne

Transcriptome sequencing of lentil based on second-generation technology permits large-scale unigene assembly and SSR marker discovery

BackgroundLentil (Lens culinaris Medik.) is a cool-season grain legume which provides a rich source of protein for human consumption. In terms of genomic resources, lentil is relatively underdeveloped, in comparison to other Fabaceae species, with limited available data. There is hence a significant need to enhance such resources in order to identify novel genes and alleles for molecular breeding to increase crop productivity and quality.ResultsTissue-specific cDNA samples from six distinct lentil genotypes were sequenced using Roche 454 GS-FLX Titanium technology, generating c. 1.38 × 106 expressed sequence tags (ESTs). De novo assembly generated a total of 15,354 contigs and 68,715 singletons. The complete unigene set was sequence-analysed against genome drafts of the model legume species Medicago truncatula and Arabidopsis thaliana to identify 12,639, and 7,476 unique matches, respectively. When compared to the genome of Glycine max, a total of 20,419 unique hits were observed corresponding to c. 31% of the known gene space. A total of 25,592 lentil unigenes were subsequently annoated from GenBank. Simple sequence repeat (SSR)-containing ESTs were identified from consensus sequences and a total of 2,393 primer pairs were designed. A subset of 192 EST-SSR markers was screened for validation across a panel 12 cultivated lentil genotypes and one wild relative species. A total of 166 primer pairs obtained successful amplification, of which 47.5% detected genetic polymorphism.ConclusionsA substantial collection of ESTs has been developed from sequence analysis of lentil genotypes using second-generation technology, permitting unigene definition across a broad range of functional categories. As well as providing resources for functional genomics studies, the unigene set has permitted significant enhancement of the number of publicly-available molecular genetic markers as tools for improvement of this species.

SNP marker discovery, linkage map construction and identification of QTLs for enhanced salinity tolerance in field pea (Pisum sativum L.)

BackgroundField pea (Pisum sativum L.) is a self-pollinating, diploid, cool-season food legume. Crop production is constrained by multiple biotic and abiotic stress factors, including salinity, that cause reduced growth and yield. Recent advances in genomics have permitted the development of low-cost high-throughput genotyping systems, allowing the construction of saturated genetic linkage maps for identification of quantitative trait loci (QTLs) associated with traits of interest. Genetic markers in close linkage with the relevant genomic regions may then be implemented in varietal improvement programs.ResultsIn this study, single nucleotide polymorphism (SNP) markers associated with expressed sequence tags (ESTs) were developed and used to generate comprehensive linkage maps for field pea. From a set of 36,188 variant nucleotide positions detected through in silico analysis, 768 were selected for genotyping of a recombinant inbred line (RIL) population. A total of 705 SNPs (91.7%) successfully detected segregating polymorphisms. In addition to SNPs, genomic and EST-derived simple sequence repeats (SSRs) were assigned to the genetic map in order to obtain an evenly distributed genome-wide coverage. Sequences associated with the mapped molecular markers were used for comparative genomic analysis with other legume species. Higher levels of conserved synteny were observed with the genomes of Medicago truncatula Gaertn. and chickpea (Cicer arietinum L.) than with soybean (Glycine max [L.] Merr.), Lotus japonicus L. and pigeon pea (Cajanus cajan [L.] Millsp.). Parents and RIL progeny were screened at the seedling growth stage for responses to salinity stress, imposed by addition of NaCl in the watering solution at a concentration of 18 dS m-1. Salinity-induced symptoms showed normal distribution, and the severity of the symptoms increased over time. QTLs for salinity tolerance were identified on linkage groups Ps III and VII, with flanking SNP markers suitable for selection of resistant cultivars. Comparison of sequences underpinning these SNP markers to the M. truncatula genome defined genomic regions containing candidate genes associated with saline stress tolerance.ConclusionThe SNP assays and associated genetic linkage maps developed in this study permitted identification of salinity tolerance QTLs and candidate genes. This constitutes an important set of tools for marker-assisted selection (MAS) programs aimed at performance enhancement of field pea cultivars.

SNP discovery and high-density genetic mapping in faba bean (Vicia faba L.) permits identification of QTLs for ascochyta blight resistance

Ascochyta blight, caused by the fungus Ascochyta fabae Speg., is a common and destructive disease of faba bean (Vicia faba L.) on a global basis. Yield losses vary from typical values of 35-40% to 90% under specific environmental conditions. Several sources of resistance have been identified and used in breeding programs. However, introgression of the resistance gene determinants into commercial cultivars as a gene pyramiding approach is reliant on selection of closely linked genetic markers. A total of 14,552 base variants were identified from a faba bean expressed sequence tag (EST) database, and were further quality assessed to obtain a set of 822 high-quality single nucleotide polymorphisms (SNPs). Sub-sets of 336 EST-derived simple sequence repeats (SSRs) and 768 SNPs were further used for high-density genetic mapping of a biparental faba bean mapping population (Icarus×Ascot) that segregates for resistance to ascochyta blight. The linkage map spanned a total length of 1216.8 cM with 12 linkage groups (LGs) and an average marker interval distance of 2.3 cM. Comparison of map structure to the genomes of closely related legume species revealed a high degree of conserved macrosynteny, as well as some rearrangements. Based on glasshouse evaluation of ascochyta blight resistance performed over two years, four genomic regions controlling resistance were identified on Chr-II, Chr-VI and two regions on Chr-I.A. Of these, one (QTL-3) may be identical with quantitative trait loci (QTLs) identified in prior studies, while the others (QTL-1, QTL-2 and QTL-4) may be novel. Markers in close linkage to ascochyta blight resistance genes identified in this study can be further validated and effectively implemented in faba bean breeding programs.

An AFLP-based survey of genetic diversity among accessions of sea oats (Uniola paniculata, Poaceae) from the southeastern Atlantic and Gulf coast states of the United States

Uniola paniculata, commonly known as sea oats, is a C4 perennial grass capable of stabilizing sand dunes. It is most abundant along the Gulf of Mexico and southeastern Atlantic coastal regions of the United States. The species exhibits low seed set and low rates of germination and seedling emergence, and so extensive clonal reproduction is achieved through production of rhizomes, which may contribute to a decline in genetic diversity. To date, there has been no systematic assessment of genetic variability and population structure in naturally occurring stands in the USA. This study was conducted to assess the genetic relationship and diversity among nineteen U. paniculata accessions representing eight states: Texas, Louisiana, Mississippi, Alabama, Florida, South Carolina, North Carolina, and Virginia, using amplified fragment length polymorphism (AFLP). Twelve AFLP EcoRI+MseI primer combinations generated a wide range of polymorphisms (42–81%) with a mean of 59%. Overall, the sea oats plants exhibited a low range of genetic similarity. Florida accessions, FL-33 and FL-39, were most genetically diverse and the accessions from both Carolinas and Virginia (NC-1, NC-11, SC-15, and VA-53) harbored less genetic variability. Cluster analysis using the UPGMA approach separated U. paniculata plants into four major clusters which were also confirmed by principal coordinate analysis (PCO). Further examination of the different components of genetic variation by analysis of molecular variance (AMOVA) indicated the largest proportion of variability at the state level (47.8%) followed by the variation due to the differences among the genotypes within an accession (34.4%), and the differences among the accessions within a state (17.8%). The relationship between genetic diversity and geographic source of sea oats populations of the United States as revealed through this comprehensive study will be helpful to resource managers and commercial nurseries in identifying suitable plant materials for restoration of new areas without compromising the adaptation and genetic diversity.

Response of lentil ( Lens culinaris ) germplasm to high concentrations of soil boron

For lentil production to expand further in Australia, adaptation to the less favourable soils of the low to medium rainfall zones is required. To improve adaptation to these regions, varieties are required with increased tolerance to soil constraints such as high concentrations of boron (B), salinity and sodicity. To evaluate the range of B tolerance in lentil germplasm, 310 lines were screened in soil with a high concentration of B and tolerance was assessed at the seedling stage. A wide range in response to high concentrations of soil B was observed in the germplasm tested. Current Australian varieties were generally very intolerant to high concentrations of soil B. High levels of B tolerance was identified in germplasm originating from Afghanistan and Ethiopia. A subsequent experiment comparing lentils with different levels of B tolerance found that tolerant accessions (ILL213A and ILL2024) produced greater above and below ground biomass than intolerant accessions. The tolerant accessions had no significant yield loss under a high B treatment (extractable B = 18.20 mg/kg) compared to the control treatment (extractable B = 1.55 mg/kg). The large improvement in B tolerance, at soil concentrations typical of those found in the target regions, suggests there is potential to improve the tolerance level of adapted varieties and expand lentil production areas to regions with higher concentrations of soil B.

Assessment of genetic variation within a global collection of lentil (Lens culinaris Medik.) cultivars and landraces using SNP markers

BackgroundLentil is a self-pollinated annual diploid (2n = 2× = 14) crop with a restricted history of genetic improvement through breeding, particularly when compared to cereal crops. This limited breeding has probably contributed to the narrow genetic base of local cultivars, and a corresponding potential to continue yield increases and stability. Therefore, knowledge of genetic variation and relationships between populations is important for understanding of available genetic variability and its potential for use in breeding programs. Single nucleotide polymorphism (SNP) markers provide a method for rapid automated genotyping and subsequent data analysis over large numbers of samples, allowing assessment of genetic relationships between genotypes.ResultsIn order to investigate levels of genetic diversity within lentil germplasm, 505 cultivars and landraces were genotyped with 384 genome-wide distributed SNP markers, of which 266 (69.2%) obtained successful amplification and detected polymorphisms. Gene diversity and PIC values varied between 0.108-0.5 and 0.102-0.375, with averages of 0.419 and 0.328, respectively. On the basis of clarity and interest to lentil breeders, the genetic structure of the germplasm collection was analysed separately for cultivars and landraces. A neighbour-joining (NJ) dendrogram was constructed for commercial cultivars, in which lentil cultivars were sorted into three major groups (G-I, G-II and G-III). These results were further supported by principal coordinate analysis (PCoA) and STRUCTURE, from which three clear clusters were defined based on differences in geographical location. In the case of landraces, a weak correlation between geographical origin and genetic relationships was observed. The landraces from the Mediterranean region, predominantly Greece and Turkey, revealed very high levels of genetic diversity.ConclusionsLentil cultivars revealed clear clustering based on geographical origin, but much more limited correlation between geographic origin and genetic diversity was observed for landraces. These results suggest that selection of divergent parental genotypes for breeding should be made actively on the basis of systematic assessment of genetic distance between genotypes, rather than passively based on geographical distance.

Cool-season grain legume improvement in Australia—use of genetic resources

Abstract. The cool-season grain legume industry in Australia, comprising field pea (Pisum sativum L.), chickpea (Cicer arietinum L.), faba bean (Vicia faba L.), lentil (Lens culinaris ssp. culinaris Medik.), and narrow-leaf lupin (Lupinus angustifolius L.), has emerged in the last 40 years to occupy a significant place in cropping systems. The development of all major grain legume crops—including field pea, which has been grown for over 100 years—has been possible through large amounts of genetic resources acquired and utilised in breeding. Initially, several varieties were released directly from these imports, but the past 25 years of grain legume breeding has recombined traits for adaptation and yield for various growing regions. Many fungal disease threats have been addressed through resistant germplasm, with varying successes. Some threats, e.g. black spot in field pea caused by Mycosphaerella pinodes (Berk. and Blox.) Vestergr., require continued exploration of germplasm and new technology. The arrival of ascochyta blight in chickpea in Australia threatened to destroy the chickpea industry of southern Australia, but thanks to resistant germplasm, it is now on its way to recovery. Many abiotic stresses including drought, heat, salinity, and soil nutritional toxicities continue to challenge the expansion of the grain legume area, but recent research shows that genetic variation in the germplasm may offer new solutions. Just as the availability of genetic resources has been key to successfully addressing many challenges in the past two decades, so it will assist in the future, including adapting to climate change. The acquisition of grain legume germplasm from overseas is a direct result of several Australians who fostered collaborations leading to new collection missions enriching the germplasm base for posterity.

Breeding for biotic stress resistance in chickpea: progress and prospects

Abstract Chickpea (Cicer arietinum L.) is the third most economically important food legume in the world. Its yield potential is often limited by various biotic stresses, including fungal and viral diseases, insects, nematodes and parasitic weeds. Incorporating genetic resistance into cultivars is the most effective and economical way of controlling biotic stresses and this is a major objective in many breeding programs. Extensive searches for resistances have been conducted by screening commercial varieties, landraces and closely related species. Resistances to disease such as Ascochyta blight and Fusarium wilt have been identified and molecular tools are being used to increase the efficiency of gene transfer from wild species into chickpea elite genotypes. Quantitative trait loci for resistance genes have been located on linkage maps and molecular markers associated with these loci can potentially be used for efficient pyramiding of the traits. Significant chickpea genomic resources have been developed in order to investigate resistance genes. Such resources include an integrated genetic map, expressed sequence tag libraries, bacterial artificial chromosome libraries, microarrays and draft genome sequences. Although these resources have yet to be used to improve chickpea cultivars in the field, this is likely to change in the near future. These genomic resources, as well as high-resolution phenotyping tools and cutting-edge technologies such as next-generation sequencing, promise to increase efficiency as work to identify valuable candidate genes continues.

Genotyping elite genotypes within the Australian lentil breeding program with lentil-specific sequenced tagged microsatellite site (STMS) markers

Lentil (Lens culinaris ssp. culinaris) is consumed in many countries as a rich source of protein in largely vegetarian diets. Australia grows lentil as a cash crop in rotation with cereal and produces predominantly red lentils that are exported throughout the world, particularly to countries in South Asia and the Middle East. Differentiation of varieties is important when exporting products to such markets, maintaining variety purity during seed production and in the collection of end-point royalties. Lentil-specific and fluorescent sequenced tagged microsatellite markers (STMS) markers were used to construct a DNA fingerprint database for 10 Lens culinaris ssp. culinaris genotypes (Northfield, Digger, ILL7537, Nugget, Indianhead, ILL2024, ILL6788, Palouse, Nipper and Boomer) that represent major new cultivars and key breeding lines within the Australian breeding program. All 10 lentil genotypes were distinguished using the assessed STMS loci. Unique alleles were observed for several lines, including Boomer and Nipper, varieties recently released in Australia. This database will play an important role in seed typing for commercial export certification and the commercial management of cultivars.

Breeding for Improvement of Cool Season Food Legumes

Michael Matterne, Antonio Leonforte, Kristy Hobson, Jeffrey Paull and Annathurai Gnanasambandam