Kevin McPhee

Screening techniques and sources of resistance to root diseases in cool season food legumes

SummarySoil-borne fungal diseases are among the most important factors, limiting the yield of grain legumes in many countries worldwide. Root rot, caused by Aphanomyces euteiches, Rhizoctonia solani, Fusarium solani and wilt, caused by several formae speciales of Fusariumoxysporum are the most destructive soil-borne diseases of pea, chickpea, lentil, fababean and lupin. The most effective control of these diseases is achieved through the use of resistant varieties. In this paper, recent advances in conventional and innovative screening methods for disease resistance are presented. Many grain legume accessions, which are maintained in national and international germplasm collections, have been evaluated for disease resistance and numerous resistant varieties have been released following incorporation of identified resistance genes from these sources. Recent identification of molecular markers tightly linked to resistance genes has greatly enhanced breeding programs by making marker assisted selection (MAS) possible and allowing the development of varieties with multiple disease resistance. Progress in the understanding of the biology of soil-borne fungal pathogens of grain legumes is also reviewed with particular reference to the genetic structure of their populations, diagnosis and host–pathogen interaction.

Genetic diversity, population structure and genome-wide marker-trait association analysis emphasizing seed nutrients of the USDA pea (Pisum sativum L.) core collection

Genetic diversity, population structure and genome-wide marker-trait association analysis was conducted for the USDA pea (Pisum sativum L.) core collection. The core collection contained 285 accessions with diverse phenotypes and geographic origins. The 137 DNA markers included 102 polymorphic fragments amplified by 15 microsatellite primer pairs, 36 RAPD loci and one SCAR (sequence characterized amplified region) marker. The 49 phenotypic traits fall into the categories of seed macro- and micro-nutrients, disease resistance, agronomic traits and seed characteristics. Genetic diversity, population structure and marker-trait association were analyzed with the software packages PowerMarker, STUCTURE and TASSEL, respectively. A great amount of variation was revealed by the DNA markers at the molecular level. Identified were three sub-populations that constituted 56.1%, 13.0% and 30.9%, respectively, of the USDA Pisum core collection. The first sub-population is comprised of all cultivated pea varieties and landraces; the second of wild P. sativum ssp. elatius and abyssinicum and the accessions from the Asian highland (Afghanistan, India, Pakistan, China and Nepal); while the third is an admixture containing alleles from the first and second sub-populations. This structure was achieved using a stringent cutoff point of 15% admixture (q-value 85%) of the collection. Significant marker-trait associations were identified among certain markers with eight mineral nutrient concentrations in seed and other important phenotypic traits. Fifteen pairs of associations were at the significant levels of P ≤ 0.01 when tested using the three statistical models. These markers will be useful in marker-assisted selection to breed pea cultivars with desirable agronomic traits and end-user qualities.

Marker assisted selection (MAS) for developing powdery mildew resistant pea cultivars

In this contribution we review the state of the art for genetic resistance to powdery mildew, caused by Erysiphe pisi, in pea (Pisum sativum L.) and potential use of marker-assisted selection (MAS) for developing disease resistant cultivars. Powdery mildew is important in many production regions worldwide and reduces yield and crop quality when present in epidemic proportions. Although genetic resistance to powdery mildew is available (er1 and er2) and has been durable since its characterization in 1969, recently a new dominant gene (Er3) has been reported in Pisum fulvum, a wild relative of pea that is different from previously reported er1 and er2. The efficacy of these genes may be at risk from the point of view of new pathotypes and pathogens. Erysiphe trifolii has been reported that was not previously known as a pathogen of pea powdery mildew. A continued search for new and diverse resistant sources remains a priority in pea breeding and special emphasis should be paid to selection of resistance that will prolong durability of existing resistance genes. Marker assisted selection is a new emerging approach for target breeding that has been intensively employed especially in cereals and has recently got popularity among legume breeders. With the advancement of genomic research, especially related to quantitative traits loci, the MAS is potentially anticipated future technique for routine plant breeding that is scarce in legumes at present. In pea, various DNA markers have been reported linked to er1, er2 and Er3 at varying distances in different mapping populations that are currently being used in breeding programs. Currently MAS of single gene is the most powerful approach and successes have been witnessed. If single marker is not close enough to the gene of interest then two flanking markers are considerably utilized to improve the correct identification that is being successfully employed in MAS for powdery mildew resistance in pea.

Rapid transcriptome characterization and parsing of sequences in a non-model host-pathogen interaction; pea- Sclerotinia sclerotiorum

BackgroundWhite mold, caused by Sclerotinia sclerotiorum, is one of the most important diseases of pea (Pisum sativum L.), however, little is known about the genetics and biochemistry of this interaction. Identification of genes underlying resistance in the host or pathogenicity and virulence factors in the pathogen will increase our knowledge of the pea-S. sclerotiorum interaction and facilitate the introgression of new resistance genes into commercial pea varieties. Although the S. sclerotiorum genome sequence is available, no pea genome is available, due in part to its large genome size (~3500 Mb) and extensive repeated motifs. Here we present an EST data set specific to the interaction between S. sclerotiorum and pea, and a method to distinguish pathogen and host sequences without a species-specific reference genome.Results10,158 contigs were obtained by de novo assembly of 128,720 high-quality reads generated by 454 pyrosequencing of the pea-S. sclerotiorum interactome. A method based on the tBLASTx program was modified to distinguish pea and S. sclerotiorum ESTs. To test this strategy, a mixture of known ESTs (18,490 pea and 17,198 S. sclerotiorum ESTs) from public databases were pooled and parsed; the tBLASTx method successfully separated 90.1% of the artificial EST mix with 99.9% accuracy. The tBLASTx method successfully parsed 89.4% of the 454-derived EST contigs, as validated by PCR, into pea (6,299 contigs) and S. sclerotiorum (2,780 contigs) categories. Two thousand eight hundred and forty pea ESTs and 996 S. sclerotiorum ESTs were predicted to be expressed specifically during the pea-S. sclerotiorum interaction as determined by homology search against 81,449 pea ESTs (from flowers, leaves, cotyledons, epi- and hypocotyl, and etiolated and light treated etiolated seedlings) and 57,751 S. sclerotiorum ESTs (from mycelia at neutral pH, developing apothecia and developing sclerotia). Among those ESTs specifically expressed, 277 (9.8%) pea ESTs were predicted to be involved in plant defense and response to biotic or abiotic stress, and 93 (9.3%) S. sclerotiorum ESTs were predicted to be involved in pathogenicity/virulence. Additionally, 142 S. sclerotiorum ESTs were identified as secretory/signal peptides of which only 21 were previously reported.ConclusionsWe present and characterize an EST resource specific to the pea-S. sclerotiorum interaction. Additionally, the tBLASTx method used to parse S. sclerotiorum and pea ESTs was demonstrated to be a reliable and accurate method to distinguish ESTs without a reference genome.

Analysis of the Accumulation of Pea enation mosaic virus Genomes in Seed Tissues and Lack of Evidence for Seed Transmission in Pea (Pisum sativum)

Pea enation mosaic virus (PEMV) is an important virus disease of pea. International movement of commercial pea cultivars and germplasm can be problematic due to uncertainty about seed transmission of the viruses responsible for the disease. Whether PEMV is seedborne was assessed by collecting developing seed from infected plants and determining the relative concentrations of the PEMV-1 and PEMV-2 viral genomes using quantitative real-time reverse-transcription polymerase chain reaction. The relative accumulation of PEMV-1 and PEMV-2 was approximately 1,240 and 13,000 times higher, respectively, in leaf than in embryo tissues. Accumulation of PEMV-1 and PEMV-2 RNA was also significantly higher in pod walls and seed coats than in cotyledons or embryo axes. No evidence was obtained for seed transmission of PEMV in pea. Although PEMV-1 and PEMV-2 genomic RNAs were found in developing seed, no PEMV symptoms were observed in the field on more than 50,000 plants from seed derived from PEMV-infected source plants. These data demonstrate that PEMV is seedborne in pea but do not support a previous report that PEMV is seed transmitted. Absence of seed transmission may result from the low abundance of PEMV viral genomes in embryo tissue.

Development and Characterization of 37 Novel EST-SSR Markers in Pisum sativum (Fabaceae)

Premise of the study: Simple sequence repeat markers were developed based on expressed sequence tags (EST-SSR) and screened for polymorphism among 23 Pisum sativum individuals to assist development and refinement of pea linkage maps. In particular, the SSR markers were developed to assist in mapping of white mold disease resistance quantitative trait loci. Methods and Results: Primer pairs were designed for 46 SSRs identified in EST contiguous sequences assembled from a 454 pyrosequenced transcriptome of the pea cultivar, ‘LIFTER’. Thirty-seven SSR markers amplified PCR products, of which 11 (30%) SSR markers produced polymorphism in 23 individuals, including parents of recombinant inbred lines, with two to four alleles. The observed and expected heterozygosities ranged from 0 to 0.43 and from 0.31 to 0.83, respectively. Conclusions: These EST-SSR markers for pea will be useful for refinement of pea linkage maps, and will likely be useful for comparative mapping of pea and as tools for marker-based pea breeding.

Isolation and Characterization of Novel EST-Derived Genic Markers in Pisum sativum (Fabaceae)

Premise of the study: Novel markers were developed for pea (Pisum sativum) from pea expressed sequence tags (ESTs) having significant homology to Medicago truncatula gene sequences to investigate genetic diversity, linkage mapping, and cross-species transferability. Methods and Results: Seventy-seven EST-derived genic markers were developed through comparative mapping between M. truncatula and P. sativum in which 75 markers produced PCR products and 33 were polymorphic among 16 pea genotypes. Conclusions: The novel markers described here will be useful for future genetic studies of P. sativum; their amplification in lentil (Lens culinaris) demonstrates their potential for use in closely related species.

Development of Molecular Markers for Iron Metabolism Related Genes in Lentil and Their Expression Analysis under Excess Iron Stress

Multiple genes and transcription factors are involved in the uptake and translocation of iron in plants from soil. The sequence information about iron uptake and translocation related genes is largely unknown in lentil (Lens culinaris Medik.). This study was designed to develop iron metabolism related molecular markers for Ferritin-1, BHLH-1 (Basic helix loop helix), or FER-like transcription factor protein and IRT-1 (Iron related transporter) genes using genome synteny with barrel medic (Medicago truncatula). The second objective of this study was to analyze differential gene expression under excess iron over time (2 h, 8 h, 24 h). Specific molecular markers were developed for iron metabolism related genes (Ferritin-1, BHLH-1, IRT-1) and validated in lentil. Gene specific markers for Ferritin-1 and IRT-1 were used for quantitative PCR (qPCR) studies based on their amplification efficiency. Significant differential expression of Ferritin-1 and IRT-1 was observed under excess iron conditions through qPCR based gene expression analysis. Regulation of iron uptake and translocation in lentil needs further characterization. Greater emphasis should be given to development of conditions simulating field conditions under external iron supply and considering adult plant physiology.

Chapter 11 – Virus Resistance Breeding in Cool Season Food Legumes: Integrating Traditional and Molecular Approaches

In cool season food legumes, a large number of viral diseases cause severe yield losses. The description of disease symptoms is crucial for distinguishing various viruses; however, different virus species cannot be distinguished reliably by symptoms in many of the legumes, and infection by mixtures of viruses is common. That is why identification of viruses by serological and nucleic-acid-based techniques is important for making early and informed decisions on disease management strategies. Generally, breeders develop improved cultivars for yield and quality which may not possess resistance to various diseases. Protection from virus infection can be obtained by deploying either resistance genes present in the existing germplasm, or from non-host resistant sources. Screening of germplasm collections for virus resistance has provided breeders with genetic variation not found in available cultivars or enhanced germplasm. Inheritance of virus resistance can be monogenic or multigenic and may be either dominant or recessive. Availability of high-density molecular genetic maps has allowed identification of closely linked markers for a trait of interest and has therefore allowed marker-assisted selection (MAS) for improving concerned traits in breeding programs. However, little known information about closely linked markers for selection of virus-resistant genotypes has become a major limitation for exercising MAS in cool season food legume breeding programs. Development of markers based on genomic information from model species has great potential to facilitate efficient selection of virus resistance genes but may not be accomplished easily because of the relatively larger genome sizes of cool season food legumes. Here, the present state of the art concerning molecular breeding for resistance to these viruses is reviewed, and future perspectives for gene isolation and breeding at allelic level are briefly discussed.

Molecular and phenotypic characterization of variation related to pea enation mosaic virus resistance in lentil (Lens culinaris Medik.)

Jain, S., Porter, L. D., Kumar, A., Mir, R. R., Eigenbrode, S. D. and McPhee, K. E. 2014. Molecular and phenotypic characterization of variation related to pea enation mosaic virus resistance in lentil (Lens culinaris Medik.). Can. J. Plant Sci. 94: 1333-1344. Identification of genetically diverse lentil germplasm with resistance to pea enation mosaic virus (PEMV) through the combined approach of molecular marker analysis and phenotyping could prove useful in breeding programs. A total of 44 lentil (Lens culinaris Medik.) accessions, were screened for resistance to PEMV. Two accessions (PI 431663 and PI 432028) were identified with resistance to PEMV in field tests while several accessions were found resistant in greenhouse screenings. Thirty-six polymorphic simple sequence repeat (SSR) markers which produced 43 loci with 2 to 12 alleles per locus were used for genetic diversity analysis. The polymorphic information content (PIC) values for these markers ranged from 0.22-0.85 with a mean of 0.55 per marker. Using allelic data of 36 SSR primer pairs, dissimilarity ranging from 0.12 to 0.74 was calculated. Cluster analysis performed using the unweighted pair group method with arithmetic mean (UPGMA) determined that most of PEMV-resistant accessions were grouped in one cluster along with other accessions from Iran, Chile, Ethiopia, India, Pakistan, Turkey, Afghanistan and Lebanon. All the adapted cultivars originating from North and South America were grouped in another cluster along with some European accessions. The 44 accessions were classified into 4 subpopulations using Structure 2.2 software complimenting the results of UPGMA analysis and indicated the effect of geographical origin on the grouping of accessions. The results of this study can be used to select genetically diverse PEMV-resistant accessions for lentil improvement programs.