Abstracts and Short Papers from Oral Presentations at the North American Pulse Improvement Association (NAPIA) November 2-4, 2011 Meeting San Juan, Puerto Rico

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Abstracts and Short Papers from Oral Presentations at the

North American Pulse Improvement Association (NAPIA)

November 2-4, 2011 Meeting

San Juan, Puerto Rico

Cool season food legume genome database: translating genomics for crop

improvement

Bodah, E.T.¹*, Cheng, C.¹,

Main, D.\McGee, R.², Coyne, C.³,

Ficklin, S.¹, Jung, S.¹ and Zheng, P.

Washington State University, Pullman, WA USA

²USDA-ARS, GLGPRU, Pullman, WA USA

³USDA-ARS, PGIT, Pullman, WA, USA

^Presenter (eliane.bodah@email.wsu.edu)

Genomics-assisted breeding offers tools to optimize plant breeding efficiency by identify genes and/or

markers related to traits of interest. The Cool Season Food Legume Genome Database

(http://www.coolseasonfoodlegume.org) is being developed to assist in translating genomics into crop

improvement. The main objective is to facilitate cool season food legume breeding and research by

serving as a genomic, genetic and breeding data resource. Although databases exist for the model legumes,

the Cool Season Food Legume Genome Database is specifically designed to collect and centralize data for

pea, chickpea and lentil while using data from the model sequenced legumes for comparison and further

curation. The database is built using Tripal which provides simplified site development by merging the

power of Drupal, a popular web Content Management System (CMS), with that of Chado, a community

derived database schema for storage of genomic, genetic and other biological data. Each page of the web

site has a header bar composed of various categories for easy navigation: 'Home', 'About', 'Community',

'Crops', 'Maps', 'Tools', 'Search', 'Contact', 'Calendar', 'Publications', 'SCRI' and a link to join the 'Mailing

List'. The 'Home', 'About', 'Community', 'Contact', 'Calendar', 'Publications' and 'SCRI' pages are self-

explanatory and straight forward. The 'Crops', 'Maps', 'Tools' and 'Search' pages are described below.

The 'Crops' page has a dropdown table of the currently available cool season food legume crops (pea,

lentil and chickpea). For each crop, a unigene build for the publicly available ESTs along with the

functional annotation data of the unigene set is available for users to browse. The functional annotation

includes homologs in other model species, GO terms and KEGG pathway terms. The 'Crops' pages also

have links to the genetic map data that can be viewed using the comparative map viewer, CMap, as well

as links to NCBI and GRIN. The 'Tools' page provides SSR and BLAST servers, Medigaco GBrowse,

Soybean GBrowse and Lotus GBrowse. The SSR server is an online tool that allows a user to upload

sequences as a batch file, select the type of motifs they are interested in identifying and search the

sequences for microsatellites. The results are returned by email for each SSR containing sequence.

Sequences can be compared to the available datasets, such as the unigenes or the individual ESTs, using

the BLAST server. The Genome Browsers are tools that allow the user to view the features of a genome

such as predicted genes, markers and mapped transcripts. In the Cool Season Food Legume Genome

Database, transcripts from pea, chickpea and lentil are mapped to regions/genes in the module legumes.

This is useful for both function identification and fine mapping. The putative functions of transcribed

genes can be assigned using various functional annotations. Functional annotation data of the EST

unigenes can be browsed in web pages, downloaded in an excel file or queried using various categories in

the EST/unigene 'Search' page. From the EST/unigene detail page, information on ESTs, gene ontology

assignment, KEGG and InterProScan annotations along with homologs in other databases are available.

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Sequences can also be downloaded in Fasta format. GO terms provide keywords of the type of

processes/functions that the gene is involved in, KEGG annotation identifies potential pathways that the

gene product belongs to, and the InterProScan analysis identifies functional domains in the gene product.

These various annotations help build evidence for the function of a specific gene which can then be

verified experimentally. Users can also query for sequences using a simple or advanced form. The

advanced EST 'Search' page offers the option of EST or unigene search. Users can perform combinatorial

search by name, assembly, sequence type, length or putative function. Registered users may acquire the

collaborators status which allows them to view and analyze private research data. COS analysis,

Gbrowse mapping, M. trunculata SSR analysis, and P.sativum SSR analysis are some of the private

options stored under Research. Planned future development includes adding cool season food legume

genome sequences as they become available, addition of more genetic maps and implementation of a

publication search site, a breeder's survey form and an integrated breeder's toolbox such as the one we

have developed for the genome database for Rosaceae (ww.rosaceae.org).

KnowPulse: A breeder-focused web portal that integrates genetics and

genomics of pulse crops with model genomes

Sanderson, L., Krilow, C., Vandenberg, A., University of Saskatchewan, Saskatoon, SK, Canada

Warkentin, T., Tar'an, B. and Bett, K.*

^Presenter

Current sequencing technologies have the ability to deliver vast amounts of genotypic data to

unsuspecting plant breeders. The past few years has seen a huge increase in the amount of genotypic

information now available for pulse crops, including pea, chickpea, lentil and common bean. These data

are of limited use to breeders until associated with phenotypic information and recently several projects

have been initiated to address this both at the CDC and abroad.

The trouble with vast amounts of data is how to store it and how to access it in a manner that is useful to

breeders and geneticists. Traditionally, literature searches or word-of-mouth techniques have been used

to find markers for use in breeding programs. However, as the number of available markers increases, this

becomes less and less feasible. A clear need exists for resources, designed with the breeder's objectives

and viewpoint in mind, that provide centralized access to marker details. The web portal KnowPulse

(http://pulse.usask.ca) is being developed to address these needs. It is designed for pulse crop breeders

and currently includes genotypic information and molecular markers for common bean, chickpea, lentil

and field pea.

Within KnowPulse, each genomic marker has an information page meant to facilitate the integration of

the marker into a breeding program. This includes details like the type and location of the marker and

any known protocols for marker detection. A summary of observed alleles and the genotypes of all

germplasm surveyed to date is also included on the marker page, allowing a researcher to determine the

likelihood of a marker being polymorphic for a given variety or population.

Genotypic data can also be accessed through dynamic tables which allow researchers to select

germplasm entries of interest. Filters are available allowing researchers to select markers that are

polymorphic in specific germplasm, or specific types of markers associated with a given project. Data are

exportable as spreadsheets or marked-up FASTA files. The marked-up FASTA follows the submission

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requirements for design of KBioscience KASP assays and allows bulk design of markers for this

technology platform.

Genomic markers stored in KnowPulse are not required to be associated with phenotypes in the same

species. If a phenotype-associated marker has not yet been developed for a trait of interest, KnowPulse

can be used to find existing genomic markers in regions homologous to a gene thought to be involved in

your trait based on studies in other species. A GMOD GBrowse (1) with a Medicago truncatula backbone

makes it easy to visually inspect a region in Medicago for homologous sequences or genomic markers in

multiple pulse species.

The cross-species GBrowse is also useful to geneticists due to the ease with which homology among

pulse crop species can be visualized. This can help identify potential sequences for gene identification

based on information from other species. KnowPulse hosts its own BLAST server (2, 3) with many pulse

crop-specific and model plant datasets allowing researchers to find homologous sequences based on

sequence similarity. Both of these tools are meant to make it easy to find either sequence or genotypic

data based on information from other legume species.

Currently, KnowPulse contains all sequence data generated as part of 454 sequencing-based projects

carried out at the CDC in conjunction with the NRC Plant Biotechnology Institute (PBI) in Saskatoon,

the Lens culinaris EST collection generated previously at CDC/PBI (already deposited to NCBI), all

legume DFCI Gene Indices (4) and the latest Medicago truncatula assembly (5). All genotypes and

markers generated as part of the CDC-led Implementation of Markers for Pulses (iMAP) project are

being loaded into KnowPulse. We intend to have most genotypic information generated in our lab

publically available through this portal. Any sequence or genotypic information generated by other

research groups is welcome and will be included upon request of the originators.

Future development plans for KnowPulse include the ability to store phenotypes including experimental

details, environmental conditions and the phenotypes observed for any germplasm surveyed. Once this

feature is available, phenotypes can be associated with genotypes, improving the searching capabilities

for finding markers associated with a trait of interest. It is also our intention to promote inter-website

communication and data-sharing between KnowPulse and other legume-specific web portals including

the Legume Information System (6) and the Cool Season Food Legume Genome Database (7).

References

1. Stein L.D et al. The generic genome browser: a building block for model organism system

database. 2002. Genome Research 12: 1599-1610.

2. Morgulis Aet al. Database indexing for production MegaBLAST searches. 2008. Bioinformatics

24(16): 1757-1764.

3. Papanicolaou A.,Heckel D.G. The GMOD Drupal Bioinformatic Server Framework. 2010.

Bioinformatics 26(24):3119-3124.

4. Quackenbush J. et al. The TIGR Gene Indices: analysis of gene transcript sequences in highly

sampled eukaryotic species. 2001. Nucleic Acids Research 29(1): 159-164.

5. Young N.D, et al., The Medicago genome provides insight into the evolution of rhizobial

symbioses. 2011. Nature doi:10.1038/nature10625

6. Gonzales M.D. et al. The Legume Information System (LIS): an integrated information resource

for comparative legume biology. 2005. Nucleic Acids Research 33(1): D660-D665.

7. Cool Season Food Legume Genome Database: http://www.gabcsfl.org/

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Progress report on enhancing faba bean (Vicia faba L.) germplasm for

improved winter-hardiness at Pullman, Washington

Hu, J.^1, ³*, McGee, R.J.^2, ³,

Landry, E.J.³, Mwengi, J.E.

and Coyne, C.J.^1, ³

¹USDA-ARS, PGITU, Pullman, WA USA

²USDA-ARS, GLGRPU,Pullman, WA USA

Washington State University, Pullman, WA USA

^Presenter

Introduction

Numerous reports have documented that grain legumes have positive effects on the succeeding crop yield

(1, 2). Winter-hardy peas and lentils are currently being used in rotation with wheat in the Palouse

region of Washington and Idaho. Murray et al. (3) reported that faba bean (Vicia faba L.) has the same

winter-hardiness as lentil and better than pea. Aiming at developing an alternative rotation crop for

cool-temperate regions, we initiated a research project to enhance faba bean germplasm for improved

winter-hardiness at Pullman, Washington in 2008. This report summarizes the progress made in the

past three years.

Materials and methods

Available faba bean accessions maintained by the USDA-ARS Western Regional Plant Introduction

Station in Pullman, WA were used for this project. For the first year experiment we used 43 accessions

collected from various countries and 12 winter-hardy cultivars/breeding lines from W. Link of Georg-

August University, Germany (4).

Two approaches were taken in screening for winter-hardiness. The first approach is the traditional

replicated field trial of single row plots at two locations (Pullman and Central Ferry, WA). Thirty seeds

from each entry were planted and observation notes were taken through the growing season. A weather

logger was placed near the planting site to collect ambient and soil temperatures through the season. The

second approach is a modified "mass selection". Seeds bulked from 466 accessions were planted at a high

density in both locations in fall of 2010. Plants that survived through the winter at each location were

kept to produce seeds for further testing.

Results and discussion

The first year results were reported earlier (5). We planted 43 accessions in both Pullman and Central

Ferry and the 12 winter-hardy lines from Germany were planted in Central Ferry in October 2008. We

observed a high level of variation in winter-hardiness among the accessions during the first year. The data

loggers recorded minimum ambient temperatures that were at or below -14 °C with at least 5 consecutive

nights of hard frost. The number of consecutive days that temperatures stayed below zero ranged from 7

to 14. The extreme was a period in Pullman that reached -22.2 °C and 11 consecutive nights of hard frost.

All accessions survived in Central Ferry, while 30 of the 43 accessions were completely killed in Pullman.

Accessions that had a higher survival rate in Central Ferry also had a few plants which survived in

Pullman. We also observed that some accessions had the ability to send out shoots from the lower nodes

of the stems from the plants that were damaged or killed by low temperatures. This ability to "regrow"

could be used as one of the criteria to measure winter-hardiness of faba bean.

In October 2009, we planted 75 accessions in both Pullman and Central Ferry. These included 55

accessions that were tested in the first year plus 20 new accessions. Although the winter was not as cold

as the previous year, there was little snow to cover the seedlings during the cold months. All the

accessions in Pullman were completely dead. Approximately 20 accessions in Central Ferry survived and

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the number of plants that survived of each accession varied from one to eleven plants. There was a

significant location by year by genotype interaction. Based on the results of survival rate and seed yield

potential obtained in the past two years, we selected 16 most winter-hardy accessions for further

observation.

In October 2010, the selected accessions were planted in two locations. We also planted nine selected F3

families derived from a cross between a non-winter hardy vegetable-type variety 'Extra Precoce Vioetto'

and a winter-hardy genotype 'Hiverna/2-5EP1' from Germany. All the selected accessions had plants that

survived at both locations. Survival rates ranged from 16 - 100%. The plants that survived produced an

average of 75g (range 33-150 g) of seeds per plant. All nine F3 families survived in Central Ferry while

only two survived in Pullman. Of these two families, one had only one, and the other had two plants

survived. These two families also had a higher survival rate in Central Ferry.

We tried a novel approach of "mass selection" for winter-hardiness. Seeds bulked from 466 accessions

were planted at three locations in the Palouse region in October of 2010. The percentage of winter

survival was estimated at <1% in Pullman, 5% in Central Ferry and 5-10% in Dayton. Seeds from these

plants were harvested for further testing.

In summary, sufficient amount of variation has been captured in the USDA faba bean germplasm

collection and satisfactory progress has been made towards establishing a winter-hardy faba bean

population in the past three years. These genotypes that survived through the harsh winter in Pullman

formed the foundation for developing an alternative fall-sown rotation crop for the Palouse region.

Acknowledgements

Technical assistance from Kristy Ann Ott, Landon Charlo, Wayne Olson, Kurt Tetrick, Sean Vail and

Leslie Elberson is gratefully appreciated. Funding includes a Germplasm Evaluation Grant from the

USDA Cool Season Food Legume Crop Germplasm Committee to WRPIS, Washington State University

and USDA ARS CRIS Project 5348-21 000-026-00D.

References

1. Chalk P.M. 1998. Aust. J. Agric. Res. 4:303-316.

2. Plancquaert P., Desbureaux J. 1985. In: Hebblewaithe et al.(ed.) The Pea Crop. Butterworth,

London. pp. 193-202.

3. Murray G.A., Eser D., Gusta L.V., Eteve G. 1988. In: Summerfield R.J. (ed.) World Crops: Cool

Season Food Legumes. Kluwer, London. pp. 831-843.

4. Arbaoui M., Balko C., Link W. 2008. Field Crops Res. 106:60-67.

5. Hu J., Mwengi J.E., Coyne C.J. and Pan, W.L. 2009. Pisum Genetics. 41:57-58.

Revisiting strategies in lentil breeding: Wild species update

Tullu, A.¹*, Bett, K.¹ Saha, S.¹, University of Saskatchewan, Saskatoon, SK Canada

Vail, S.² and Vandenberg, A¹ ²Agriculture and Agri-Food Canada, Saskatoon, SK, Canada

*Presenter (a. tullu@usask. ca)

Canada is the largest exporter and second largest producer of lentils (L. culinaris Medikus) in the world

and 98% of it is produced in Saskatchewan. When first introduced to Saskatchewan, lentil was grown

with relatively fewer problems, however as lentil acreage increased, the crop is now host for many

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diseases, such as, anthracnose, ascochyta, stemphyllium, Gray mold, white mold, powdery mildew and

root rot complex. A narrow genetic base led to lack of genetic diversity among lentil cultivars. Seven

taxa in four species have been reported in lentil gene pool and only Lens ervoides species has the highest

frequency of resistance followed by L. nigricans and L. lamottei to both anthracnose and ascochyta diseases.

Little or no resistance has been found in the cultivated background for race-Ct0 of anthracnose.

In the breeding program, new lentil cultivars with greater productivity, enhanced quality traits and

improved resistance to multiple diseases are becoming increasingly important as a way of maintaining

genetic gain in yield. The first breeding approach is to establish interspecific genetic populations to

study inheritance pattern and transfer resistance and other key traits to the cultivated background.

Recombinant inbred lines (RILs) have been developed using L. ervoides and L. culinaris, sub sp. culinaris

crosses. Also, a RIL development between L. ervoides accessions is underway. RILs for key traits were

selected and backcrossed to adapted backgrounds to transfer favorable genes. Selections derived from

the backcross entered into preliminary unreplicated yield trials and results so far are promising.

Another strategy to accelerate utilization favorable genes of wild species is the use of a broad array of

genomic and genetic resources. It has been impossible to utilize wild species to improve yield because

the superior traits of interest can not be identified phenotypically alone. Additional methods of

identifying resistance and key quantitative traits (QTLs) are needed that are more rapid, reliable and

useable in earlier generation breeding materials. Significant advances have been made in the

development and use of embryo rescue technology, wide hybridizations, library of introgression lines,

generating interspecific populations, BAC library, 454-SNPs, COS markers and resistance gene

homologues (RGHs).

Developing technology platform for implementing marker assisted selection (MAS) is also another

approach in breeding. The amount of introgressed segments can be assessed with SNP genotyping in

backcross generations and the introduction of undesirable characteristics such as bushiness, small

seededness, etc. during introgression will be minimized using a forground and background breeding.

The goal is to broaden the working germplasm base and increase the genetic gain in yield. As a result, the

number of useful new genes is likely to increase and the use of wild species is likely to continue to grow

in importance for lentil breeders.

References

1. Vail, S., et al. (2012). Field Crops Research. 126: 145-151.

2. Tullu, A., et al. (2010). Plant Genetic Resources: Characterization and Utilization. 9(01):19-29.

3. Alo, F., et al. (2011). Journal of Heredity. 102 (3): 315-329.

Nitrogen fixation and amino acids in faba-developing a screening tool for

improvement

Bueckert, R.A.*, Pritchard, J. University of Saskatchewan, Saskatoon, SK, Canada

and Vandenberg, A.

*Presenter (rosalind.bueckert@usask.ca)

Pulses can supply most of their N requirements through N₂ fixation, a sustainable means of supplying N

to high protein crops. Our goal was to assess 15 genotypes (cultivars and breeding lines of colored flower

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types and white flower / low tannin types) of faba (Viciafaba) in the field. Faba was grown at two

locations in SK in 2009 and 2010, and measured for their ability to take up N and to fix N2. The overall

goal was to develop a screening method for detecting high N2 fixation via free amino acids found in leaf,

stem and reproductive tissue.

Genotypes fixed N₂ (via a commercial inoculant Rhizobium leguminosarum which is marketed to include

faba), and the fixed N2 accounted for 51 to 88% of its requirements depending on environment, the rest

coming from root N uptake from the soil. Faba grew more with increasing moisture, but became more

indeterminate, and despite higher N accumulation values, yield was not necessarily increased. A range of

yield, biomass and N accumulation was seen for Rosthern 2009. Saskatoon 2009 was drier and yields

were slightly lower, along with less biomass and slightly less N accumulated. Data for Rosthern for 2010

came from a record wet year, and so biomass was high and maturity was delayed. The 2010 Saskatoon

site suffered from waterlogged soil, and despite limited sampling throughout the season, data were not

analyzed due to missing plots and unrepresentative plants.

In 2009, Florent, Taboar, Melodie, FB2210 and Divine had the highest total plant N accumulation,

coming from vegetative parts but especially from reproductive growth. The total shoot N content of the

15 genotypes ranged from 32 to 47 g N m^-2. The total shoot nitrogen content can be considered as 8 parts,

with 1 part made up of leaf N, less than 1 part made up of stem N, about 1.5 parts being made up of pod

shell, and the seed making up the remaining 5 parts. The nitrogen budget, as in the amount in stubble left

for succeeding crops, was calculated and was far higher than reported data from Alberta (typical of

western Canada), reflecting wet and excellent growing seasons. Additionally, our data are likely to be

overestimates due to small plot edge effects. Assuming all N from above ground vegetative biomass at

maturity became available to succeeding crops, faba genotypes supplied between 10 and 15 g N m^-2, or 100

to 150 kg N ha^-1. The yield portion removed an additional 20 to 35 g N m^-2, or 200 to 350 kg N ha^-1, due to

faba being a high yielding crop with a high seed protein content (about 30%). For faba to be a good N-

supplying crop to a succeeding crop in the rotation, breeding for less yield or lower seed protein

concentration would enable more N to be retained in stover.

Despite being an indeterminate crop, faba was efficient at partitioning its above ground biomass to yield

with harvest indices ranging from 0.34 to 0.45 in a cool, dry year. For Rosthern in 2009, cultivars had

significantly different yields, ranging from 499 to 617 g m^-2 (4,990 to 6,170 kg ha^-1). At Saskatoon, yield

differences were less obvious among the 15 faba genotypes, ranging from 447 to 650 g m^-2. Snowbird had

the highest yield at both locations in 2009. At Rosthern, Snowbird, NPZ5 7680, Divine and Imposa were

in the high-yielding group (617-590 g m^-2). So far, faba's physiological characteristics show a reasonably

efficient crop with lower variability when compared to chickpea and lentil (from previous research), but

with high amounts of N accumulated in yield and stover. The above ground biomass partitioning of faba

was <1 part leaf, >1 to 1.5 parts stem, 1 part pod shell, and the remaining 2 parts seed. The biomass range

was 850 to 1670 g m^-2 or 8,500 to 16,700 kg ha^-1 in 2009, depending on genotype. Faba is a large biomass

crop for an annual crop, likely at the upper end for the prairies.

Faba is classified as a cool season crop like pea and lentil, having amino acid metabolism where the main

transport forms of N from N₂ fixation are typically the amides glutamine or asparagine, or amino acids

aspartate and glutamate. But faba may have N2 fixation metabolism in the shoot that appears to be

intermediate between cool season pulses and the warm season legumes like common bean (Phaseolus) and

soybean. Warm season legumes use ureides, which are non-structural cyclic amino acids (allantoin and

allantoate), as the major form of N arising from N2 fixation. Ureides are an efficient means of supplying

amino N to the shoot, and may play a role in more stress-tolerant N2 fixation metabolism, making the

crop better suited to dryland production. Ureides may also have a role in stress tolerant N metabolism,

aside from N2 fixation as seen in warm season legumes. Our goal was to measure the ureide

concentrations by a colorimetric assay (glyogylate production) for leaves, stem and pod material at

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flowering, mid pod fill, and close to physiological maturity, and to relate these to plant nitrogen content

and yield. Ureides were found in all plant partitions, and appeared to be associated with stress

metabolism, as was the amino acid proline, due to consistent negative correlations of these amino acids

with N accumulation, fixation and yield. Ureide concentration ranged from 2 to 16 umol g^-1dry tissue,

depending on whether leaf, stem or pods were sampled, the environment, and growth stage. The range of

ureides seen in faba leaf appear to be greater than in non-droughted chickpea and pea leaf , in stem they

are about the same as non-droughted chickpea stem (2 to 5 umol g^-1 stem) and less than non-droughted

pea stem (3 to 15 umol g^-1 stem). The ranges and concentrations of both leaf and stem ureides in faba are

lower than soybean leaf (10 to 30 umol g^-1 leaf) and petioles (15 to 50 umol g^-1).

Free amino acids were also measured by gas chromatography in various plant partitions throughout the

lifecycle and correlated against yield, total plant N accumulated and the N2 fixed. Briefly, faba plants

operate mainly with six amino acids, asparagine the major amide, and alanine, serine, tryptophan,

cysteine and proline. The major N cycling amino acid is asparagine, followed by leaf metabolism using

alanine. Glutamine also plays a role but less is present. Lesser to minimal amounts of aspartic acid and

glutamic acid are used. Ureides and proline indicated more stress. We were expecting to find a single

amino acid concentration or even a ratio using the amides asparagine or glutamine to aspartate and

glutamate to be useful, based on limited studies where amino acid concentrations have been previously

published. We found ratios of amino acids more indicative of plant N status than single amino acids, and

we plan to use the sum of asparagine plus aspartic acid plus glutamic acid plus glutamine, compared to

proline, and a second ratio of alanine to proline, in screening for higher N2 fixation and N accumulation in

faba nurseries. We are currently verifying the amino acid ratios on 2011 plants to test whether this

screening tool does find us elite yielding and high N2 fixing cultivars.

Acknowledgements

Funding from Saskatchewan Pulse Growers Association and the Agriculture Development Fund of

Saskatchewan; ureide measurements were done by Ms. Denise Broersma.

Vicia faba - a potential rootstock for lentil breeding

Yuan, H.Y.¹*, Lulsdorf, M.¹, University of Saskatchewan, Saskatoon, SK Canada

Tullu, A.¹, Gurusamy, V.² ²Canadian Wheat Board, Saskatoon, SK Canada

and Vandenberg, A.¹

* Presenter( haiying.yuan@usask.ca )

Wild lentil species are an increasingly important genetic resource for lentil breeding programs. An

accession of each of the six wild Lens species was used as the scion in grafts to faba bean breeding line

FB50-9 rootstock. Successful grafts were obtained for all species with survival of grafts to seed maturity

between 70.7% and 87.7% except for Lens orientalis PI 72735 with 55.3% survival. Flowering time of

grafted scions compared to ungrafted controls was not affected for four species but scions of L. nigricans PI

72560 and L. orientalis PI 72735 had flowering delays of 9 and 7 days respectively. For all six wild species,

pod length, seed diameter and seed weight were not significantly different between non-grafted controls

and scions grafted onto faba bean rootstocks. This simple approach opens the possibility of using

intergeneric in vivo grafting techniques to rescue interspecific hybrids of lentil. The technique has

potential as a useful tool in lentil breeding, as a means of improving seed multiplication rate of rare

genetic resources of wild lentil and as a way to reduce the costs of germplasm multiplication of wild

lentil species.

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Variety Adaptability and Yield Stability Analysis for a State-wide Variety

Testing Study in Montana

Ito, D., Chen, C.*, Neill, K., Heser, J., Montana State University, Moccasin, MT USA

Eckhoff, J., Kephart, K., Lamb, P.,

Mason, H., McVay, K., Miller, J.,

Miller, P. and Westcott, M.

*Presenter (cchen@montana.edu)

Introduction

Dry pea (PisumsativumL.) and lentil (Lens culinaris Medik) production in Montana has been rapidly

increasing in recent years. Planted acreage of dry pea has been over 200,000 acres since 2006 placing

Montana as second largest producer in the nation. Lentil production reached to 260,000 acres in 2010

doubled from the planted acreage in 2009. Pea and lentil fit well in the wheat-based cropping systems as

rotation crops to replace fallow, providing many agronomic and economic benefits. However, the most

suitable cultivars and agronomic practices have not been well understood in Montana where

considerable variation in geography, soil, and the climate exist. Multi-location testing is important to the

development and sustainability of the pea and lentil industry in Montana. This paper reports a

coordinated statewide dry pea and lentil variety testing from 2008 to 2011. Our objective is to show

adaptable varieties with stable yields using additive main effects and multiplicative interactions (AMMI)

biplot analysis.

Materials and methods

Selected commercial varieties and breeding lines of dry pea and lentil were planted at nine locations

across Montana from 2008 to 2011. Dry pea evaluation consisted of 22 smooth green and yellow cultivars,

of which, nine are commercially available, and four are experimental lines from the USDA-ARS Grain

Legume Genetics and Physiology program at Pullman, Washington. Lentil evaluation consisted of 13

different classes cultivars, of which, ten are commercially available, and three are experimental lines from

the USDA-ARS Grain Legume Genetics and Physiology program. The experiments were randomized

complete block design with three to four replications. The grain yield data were subjected to analysis of

variance within each year and for combined four years respectively to determine interaction between

variety (G) and location (E). Subsequent PCA analysis followed using the same data where each

combination between nine locations and four years generating 31 environments for the dry pea variety

evaluation and 28 environments for lentil variety evaluation. Genotype plus genotype x environment

interaction biplots were generated from the first two principal components (PC1 and PC2). Correlation

among the environment was calculated from the angle between two environment vectors.

Results and discussion

Grain yield of both dry pea and lentil varied greatly among the locations across Montana. Significant

interactions between variety and location were also observed. The interaction effects (genotype over

environment or vice versa) can be described as distance from the plot origin. Thus, the most responsive

varieties can be visualized in the biplot at the corner or vertex. Majoret, Medora, Stirling, PS0010836, and

PS9910140 were identified as extremes. Aragorn, Bridger, Meadow, Patrick and Spider were the most

stable varieties among all environments. Among the test sites, Moccasin in all test years was relatively

clustered and close to the plot origin indicating less interactive, hence, stable and repeatable location for

testing general adaptability in Montana.

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The biplot was analyzed for lentil in the same manner as that of dry peas. Brewer, Crimson, Essex,

Meteor, Vantage, LC01602245P, and LC01602300R were the most responsive lentil cultivars to the

environments while Pennel was the least responsive. Bozeman, Moccasin, and Richland were close to the

plot origin indicating these locations were relatively stable. Bozeman and Moccasin were relatively

clustered and hence could be good repeatable locations.

Biplot analysis allowed us to identify the most or the least responsive varieties in the various

environments. This information is a useful tool to evaluate variety adaptability and yield stability in a

particular environment.

Characterization of mycosphaerella blight resistance, lodging resistance, and

micronutrient concentration in a pea recombinant inbred line population

Liu, Y.¹*, Tar'an, B.¹, Banniza, S.¹, University of Saskatchewan, Saskatoon, SK, Canada

Sharpe, A.² and Warkentin, T.¹ ²NRC-PBI Saskatoon, SK, Canada

*Presenter (yol828@mail.usask.ca)

Abstract

Moderate variation for adult plant resistance to mycosphaerella blight, pre-harvest lodging and selenium

accumulation have been observed in field pea. In order to map the quantitative trait loci (QTLs)

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associated with these traits, a population of 142 F₉ and F₁₀ recombinant inbred lines (RILs) derived from a

cross between Carrera (susceptible to mycosphaerella blight and lodging) and CDC Striker (moderately

resistant to mycosphaerella blight and lodging) were phenotyped in Saskatoon and Rosthern, SK,

Canada in 2010 and 2011. Based on phenotypic data collected from 2010 and 2011, area under the disease

progress curve (AUDPC) of mycosphaerella blight ranged from 131 to 205 and 144 to 235 for Saskatoon

and Rosthern, respectively. At physiological maturity, lodging ratings of the RILs ranged from 3.8 to 8.3

at Saskatoon and 4.5 to 8.5 at Rosthern. Micronutrient (Se, Zn and Fe) concentrations in the seeds of

each RIL were determined using an atomic absorption spectrophotometer for the 2010 samples. A genetic

linkage map was generated using 65 SSR (microsatellite) markers resulting in 13 linkage groups that

cover 290.3cM of the pea genome. A region between AA491 and AA278 on linkage group 13 was identified

as putative QTLs associated with mycosphaerella blight, lodging, Zn and Fe concentrations. All QTLs

were derived from CDC Striker, except the one associated with higher Zn concentration which was

derived from Carrera.

Introduction and objective

Field pea production in western Canada is negatively affected by several fungal diseases and lodging. The

major fungal disease is mycosphaerella blight. Complete resistance to mycosphaerella blight is lacking in

field pea germplasm. Pea cultivars with weak stems show severe lodging after flowering, causing

reductions in forage and seed yield (Stelling, 1994). Lodging resistance enhances harvest and is associated

with reduced severity of mycosphaerella blight (Banniza et al. 2005). Soils in Saskatchewan are rich in

Se. Field pea cultivars grown in Saskatchewan displayed a moderate level of variation in Se accumulation

(Thavarajah et al. 2010), whereas large regions of Asia and Europe have soils deficient in Se (Gawalko et

al. 2009), thus a good opportunity is available to market Canadian peas to these regions for nutritional

benefits. The objective of this study is to determine the genetic control of several traits in field pea

including mycosphaerella blight resistance, lodging resistance and selenium concentration by genotyping

and phenotyping a recombinant inbred line population segregating for these traits.

Methods

A pea population consisting of 142 RILs was developed from a cross between Carrera and CDC Striker.

In 2010 and 2011 field trials, pea seeds were planted in microplots (1 m²) with two replications in each of

two locations (Saskatoon and Rosthern, SK). Fertility and management practices were provided

sufficient for pea production in these regions. From two weeks after flowering until maturity,

assessments were made to record mycosphaerella blight severity and lodging. Mycosphaerella blight was

rated under natural infection on the basis of all plants in a plot using 0-9 scale where 0 = no disease and 9

= whole plant severely blighted. Four assessments of mycosphaerella blight were done and calculated into

area under disease progress curve (AUDPC). Lodging was assessed 4 times during the season and one

time at physiological maturity on a 1-9 scale where 1 = completely upright and 9 = completely lodged.

Regarding genotyping, young fresh leaves were randomly sampled from 5 plants from each RIL grown in

a greenhouse for genomic DNA extraction by hexadecyltrimethyl ammonium bromide (CTAB) method.

A total of 330 Simple Sequence Repeat (SSR) primers derived from the Agrogene consortium (France)

were screened on the parents using polyacrylamide gel electrophoresis (PAGE). Polymorphic markers

between parents were screened among RILs by using an Applied Biosystem 3730 DNA analyzer.

Results and discussion

Significant effects of genotype, location and year were detected for AUDPC (mycosphaerella blight) and

lodging in 2010 and 2011. At Saskatoon, AUDPC ranged from 131 to 205 among RILs, while at Rosthern it

ranged from 144 to 235. At physiological maturity, lodging ratings of the RILs ranged from 3.8 to 8.3 at

Saskatoon and 4.5 to 8.5 at Rosthern. Lodging was significantly correlated with mycosphaerella blight

score (r = 0.35; P < 0.001), which suggests that RILs that are less susceptible to lodging may escape disease

to some extent. A total of 112 out of 330 SSR markers were polymorphic between Carrera and CDC

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Striker and 65 out of 112 SSRs were polymorphic among the RILs. A total of 13 linkage groups were

generated consisting of 56 SSRs with 9 unlinked by using Carthagene 1.2.2 (De Keyser et al. 2010). QTL

analysis was conducted by composite interval mapping (CIM) using the Qgene program to detect

associations between phenotype and genotype. A region between AA491 and AA278 on linkage group 13

was identified as putative QTLs associated with mycosphaerella blight, lodging, Zn and Fe

concentrations. All QTLs were derived from CDC Striker, except the one associated with higher Zn

concentration which was derived from Carrera. Phenotypic variation explained by these QTLs

associated with mycosphaerella blight resistance, lodging resistance, Zn and Fe concentrations were

28.5%, 10.1%, 7.4% and 13.1%, respectively.

Acknowledgments

We would like to thank Ms. Reshma Rizvi, Mr. Brent Barlow, Dr. Aziz Rehman, and Ms. Carmen

Breitkreutz for their great help during this project. The technical support from the field lab crew was also

appreciated. Funding for this research was supplied by Saskatchewan Pulse Growers and Saskatchewan

Ministry of Agriculture.

References

1. Banniza, S., Hashemi, P., Warkentin, T.D., Vandenberg, A. and Davis, A.R. 2005. The

relationships among lodging, stem anatomy, degree of lignification and resistance to

mycosphaerella blight in field pea (Pisumsativum). Can. J. Bot. 83: 954-967.

2. De Keyser, E. D., Shu, Q. Y., Bockstaele, E.V., and Riek, J.D. 2010. Multipoint-likelihood maximization

mapping on 4 segregating populations to achieve an integrated framework map for QTL analysis in pot

azalea (Rhododendron simsiihybrids). BMC MolBiol, 11 (1): 1-20.

3. Gawalko, E., Garrett, R.G., Warkentin, T., Wang, N., and Richter, A. 2009. Trace elements in

Canadian field peas: a grain safety assurance perspective. Food Additives and Contaminants.26

(7): 1002-1012.

4. Joehanes, R., and Nelson, J.C. 2008. Qgene 4.0, an extensible Java QTL-analysis platform.

Bioinformatics Applications Note, 24: 2788-2789.

5. Semagn, K., Bjornstad, A., and Ndjiondjop, M.N. 2006. An overview of molecular marker methods

for plants.African Journal of Biotechnology.5 (25): 2540-2568.

6. Thavarajah, D., Warkentin, T. and Vandenberg, A. 2010. Natural enrichment of selenium in

Saskatchewan field peas (Pisumsativum L.).Can. J. Plant Sci. 90: 38 3-389.

Comparison of transcriptomes between Sclerotinia sclerotiorum and S.

trifoliorum using 454 Titanium RNA sequencing

Qiu, D.¹, Vandemark, G.² and Chen, W.²* Washington State University, Pullman, WA, USA

²USDA-ARS, Pullman, WA, USA

*Presenter

Both Sclerotinia sclerotiorum and S. trifoliorum cause Sclerotinia stem and crown rot of chickpea and white

mold on many economically important crops. The host range of S. trifoliorum is mainly on cool season

forage and grain legumes of about 40 plant species, whereas the host range of S. sclerotiorum encompasses

more than 400 plant species including all the host plant species of S. trifoliorum. Despite of morphological

and ecological differences between the two species, both species are equally pathogenic on chickpea.

Extensive research has been conducted on S. sclerotiorum and its genome sequences are available.

However, relatively very little is known about S. trifoliorum. To take advantages of the genomic

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information of S. sclerotiorum, we compared the transcriptome of S. trifoliorum with that of S. sclerotiorum in

order to gain a better understanding of the biology of both species. Total mRNAs of both species during

vegetative growth were extracted and sequenced using the latest 454 Titanium RNA sequencing

technology. A total of 23325 unique transcripts with average length of 534 nt (12.5 mb genome coverage)

were obtained from S. sclerotiorum, whereas 21214 unique transcripts with average length of 509 nt (10.8

mb genome coverage) were obtained from S. trifoliorum. Comparison of the transcripts between the two

species will be presented and their implications will be discussed.

Insertional mutation at the Cu-Zn-superoxide dismutase gene reduces virulence of

Sclerotinia sclerotiorum on pea (Pisum sativum)

Xu, L.¹ and Chen, W.²* Washington State University, Pullman, WA, USA

²USDA-ARS, Pullman, WA, USA

*Presenter

Sclerotinia sclerotiorum causes white mold disease on pea and on many other economically important pulse,

vegetable and field crops, demonstrating a non-host-specific pathogenic mechanism. Despite extensive

studies on this pathogen, its pathogenic mechanisms are still incompletely understood. In order to gain

insight in understanding its non-specific host-pathogen interactions, Agrobacterium-mediated

transformation (AMT) was used to generate random mutations and to identify potential

virulence/pathogenicity factors in S. sclerotiorum. Among several hundreds of AMT transformants

screened, two stable mutants showed significantly less virulence in comparison with the wild type strain

as measured by colonizing pea leaves in detached leaf assays. Southern hybridization and inverse PCR

analyses showed that the mutation was due to a single T-DNA insertion at the gene Cu-Zn-superoxide

dismutase (SsSOD1, SS1G 00699) of S. sclerotiorum. In addition to reduced virulence, the mutant had

reduced tolerance to heavy metal toxicity and oxidative stress. The SsSOD1 gene was able to functionally

complement SOD in a yeast strain defective of the SOD gene. There was more accumulation of

superoxide in disease lesions caused by the mutant than that caused by the wild type strain. Evidence

showed that the SsSOD1 gene is an important virulence factor of S. sclerotiorum.

Rhizoctonia root rot of lentil caused by Rhizoctonia solaniAG 2-1

Paulitz, T.C.¹, Schroeder, K.L.¹ ¹USDA-ARS-RDBCU, Pullman, WA, USA

and Chen, W²* ²USDA-ARS-GLGPRU, Pullman, WA, USA

*Presenter

Lentil root rot symptoms were observed in commercial fields in the US Pacific Northwest during the

unusually cool and moist spring weather of 2010. Symptoms included sunken lesions on root and stem

with brown discoloration, resembling diseases caused by Rhizoctonia solani. Rhizoctonia solani was isolated

from diseased plants and from surrounding soils and were identified to be AG 2-1 based on ITS

sequences. Pathogenicity tests were conducted at 16C 12 h day and 10 C night temperature conditions

using three isolates of R. solani AG 2-1 from lentil. Isolates were severely pathogenic to lentil cvs. Pardina

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and Merrit, as well as to spring canola cv Sunrise, and the pathogen was reisolated from the inoculated

plants. Rhizoctonia solani AG 2-1 is known to be a pathogen of canola in Australia, Canada and US Pacific

Northwest, but not previously isolated from lentil. This is the first report of Rhizoctonia solani AG 2-1

infecting lentil.

Field evaluation of fungicides for control of ascochyta blight of chickpeas in

North Dakota

Wunsch, M.J^F,Bradbury, G.², ¹Carrington Research Extension Center, Carrington, ND USA

Schaeffer, M. ¹ and Schatz, B.G. ¹ ²Williston Research Extension Center, Williston, ND USA

*Presenter (michael.wunsch@ndsu.edu)

Registered and experimental fungicides, including several products with anticipated registration in 2012

and 2013, were evaluated for their control of ascochyta blight caused by Ascochtya rabiei in Carrington,

Minot, and Williston, ND in 2011. Significant differences in fungicide efficacy were observed among

products, and at least one experimental chemistry gave excellent disease control. Disease efficacy, seed

yield, and seed quality results will be reported.

Field evaluation of fungicides for control of anthracnose, botrytis gray mold,

and sclerotinia stem rot of lentil in North Dakota

Wunsch, M.J,¹* Pederson, J. ², ¹Carrington Research Extension Center, Carrington, ND USA

Schaeffer, M. ¹ and Schatz, B.G. ¹ ²North Central Research Extension Center, Minot, ND USA

*Presenter (michael.wunsch@ndsu.edu)

Registered and experimental fungicides, including several products with anticipated registration in 2012

and 2013, were evaluated for their control of anthracnose, caused by Colletotrichum truncatum, and

sclerotinia stem rot, caused by Sclerotinia sclerotiorum, in Carrington and Williston, ND in 2011. Fungicide

timing was also evaluated in Carrington and Minot, ND for the control of anthracnose and botrytis gray

mold, caused by Botrytis cinerea. Significant differences were observed among treatments; disease efficacy,

seed yield, and seed quality results will be reported.

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Field evaluation of fungicides for control of ascochyta and mycosphaerella

blights of field peas in North Dakota

Wunsch, M.J.*, Schaeffer, M. Carrington Research Extension Center, Carrington, ND USA

and Schatz, B.G.

*Presenter (michael.wunsch@ndsu.edu)

Registered and experimental fungicides, including several products with anticipated registration in 2012

and 2013, were evaluated for their control of Ascochyta and Mycosphaerella blights caused by Ascochtya

pisi, A. pinodes, and/or Phoma pinodella in Carrington and Newburg, ND in 2010 and 2011. Significant

differences in fungicide efficacy were observed among products, and several experimental chemistries

gave excellent disease control. Disease efficacy, seed yield, and seed quality results will be reported.

Natural outcrossing rate of faba bean under Pullman field conditions and its

implication to germplasm management and enhancement

Hu, J.^1, ²*, Landry, E.J.², ¹USDA-ARS, WRPIS, Pullman, WA USA

Mwengi, J.E² and Coyne, C.J.^1, ² ²Washington State University, Pullman, WA USA

*Presenter

Abstract

Knowledge of the natural outcrossing rate of faba bean (Viciafaba L.) under Pullman field conditions will

enable us to refine strategies for both germplasm management and enhancement. We used the white

flower phenotype governed by a recessive gene in the investigation of the natural outcrossing rate of faba

bean. Seeds from white flowered plants grown in Pullman, 2010 were harvested and planted plant-to-

row in spring 2011. During flowering, the number of plants with white or regular colored flowers was

recorded for each row. The percentage of plants with regular colored flowers was used as an estimate of

natural outcrossing rate, which averaged 30.8% with a range from 0 to 82.6 % among 50 rows. This

observed outcrossing rate is within the range of previous reports for faba bean grown in various

locations. The high outcrossing rate is likely the result of abundant bumble bees and honey bees which

visited the faba bean flowers frequently during bloom. Therefore, for germplasm management it is

necessary to regenerate faba bean accessions using insect-proof cages to maintain the genetic integrity of

individual accessions. For germplasm enhancement using phenotypic selection, it is also crucial to

physically isolate the selected plants with insect-proof bags to prevent unwanted cross-pollinations and

produce self-pollinated seeds for subsequent generations.

Introduction

Faba bean (Viciafaba L.) is a partially allogamous crop and the outcrossing rate has been investigated by

many researchers in various locations. These research results reported prior to 1980s were summarized

by Bond and Poulsen (1) and ranged 4 to 84%. More recent reports were within this range (2; 3 and 4). It

has been reported that the outcrossing rate for faba bean is strongly influenced by local environmental

and climate conditions as well as the presence of pollinating insects such as species of bumble-bees,

honey bees, and solitary bees.

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The U.S. National Plant Germplasm System (NPGS) is one of the world's largest national genebank

networks focusing on preserving the genetic resources of crops and wild relative species for the

continuing improvement of agricultural productivity. The USDA faba bean germplasm collection is

maintained by the Western Regional Plant Introduction Station (WRPIS) in Pullman, Washington.

Knowledge of the natural outcrossing rate of this crop under Pullman field conditions will enable us to

refine strategies for both germplasm management and enhancement.

Materials and methods

Plants with pure white flowers were found in 13 among 466 accessions that were planted in 2010 for

evaluation. Two of the 13 accessions consisted of only white flowered plants while the remaining 11

accessions were segregating. Open-pollinated seeds from these white flowered plants were harvested in

fall 2010 and planted plant-to-row in spring 2011. During flowering, plants with white and plants with

regular colored flowers were counted and recorded for each row. The percentage of plants with regular

colored flowers was calculated with the formula of (number of plants with regular colored flower/total

number of plants)*100 and used as an estimate of natural outcrossing rate.

Results and discussion

There were a total of 810 plants scored for flower color in the 50 rows, each of which was derived from

the open-pollinated seeds of a single white-flowered plant grown in Pullman in 2010. The number of

plants per row varied from two to 25. A wide range of outcrossing rate from 0 to 82.6 % was observed

among the 50 rows. The average was 30.8% and the standard error was 2.98 (Figure 1). The high

outcrossing rate is likely the result of abundant bumble bees and honey bees which visited the faba bean

flowers

Figure 1. Wide range of natural outcrossing rates observed among the 50

single-plant-derived rows.

frequently during bloom. Therefore, for germplasm management it is necessary to regenerate faba bean

accessions using insect-proof cages to maintain the genetic integrity of individual accessions. For

germplasm enhancement using phenotypic selection, it is also crucial to physically isolate the selected

plants with insect-proof bags to prevent unwanted cross-pollinations and produce self-pollinated seeds

for subsequent generations.

Acknowledgements

Assistance from Landon Charlo, Wayne Olson, Kurt Tetrick, Sean Vail and Leslie Elberson is gratefully

appreciated. Funding includes a Germplasm Evaluation grant from the USDA Cool Season Food Legume

Crop Germplasm Committee and the USDA-ARS CRIS Project 5348-21 000-026-00D.

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References

1. Bond, D. A., Poulsen, M. H. 1983. In: Hebblethwaite PD, editor. The Faba Bean (Viciafaba L.).

Butterworths, London , pp. 77-101.

2. Cartujo, F., Suzo, M. J., Pierre. J., Moreno, M. T., Le Guen, J. 1998. Proc. International Symposium

on Breeding of Protein and Oil crops. Eucarpia, Pontevedra, Spain. p. 49-50

3. Suso, M. J., Moreno, M. T. 1999. Plant Breeding 118:347-350.

4. Suso, M. J., Pierre J., Moreno, M.T., Esnault, R. and Le Guen, J. 2001. J. Agric. Sci. Camb. 136: 399-

405.

Genome mapping and molecular markers for Ascochyta Blight resistance in

pea (Pisum sativum L.)

Miranda, A.* and McPhee, K. North Dakota State University, Fargo, ND USA

^Presenter (andre.miranda@ndsu.edu)

Ascochyta blight is the most common disease of economic importance in peas (Pisum sativum L.) in North

Dakota. It is caused by three pathogens: Ascochyta pisi Lib., and Mycosphaerellapinodes (Berk & Bloxam)

Vestergr, which causes leaf and pod spot; and Ascochytapinodela, recently designated Phoma medicaginis var.

pinodella (L. K. Jones) Boerema, which causes foot rot. The ultimate goal of this research is to reduce the

economic impact of Ascochyta blight on the pea crop. Objectives of this research are: 1) to implement

marker-assisted selection (MAS) in the breeding program to enhance plant selection for cultivar

development, 2) to develop pea varieties that are resistant to the Ascochyta blight that occurs in ND, and

3) integration of field and laboratory technologies to provide sustainable disease management that is

compatible with overall crop production in the region. An F7-derived recombinant inbred line population

of 394 lines was developed from the cross 'Lifter'/'Radley' and DNA was extracted from each RIL for

PCR-based marker analysis. Phenotypic evaluations were conducted in the greenhouse during the

summer of 2011. Five replicate plants were scored using 0 to 5 scale, where 0 = no disease and 5 = high

incidence of disease or plant death. Disease ratings were taken on 5 different dates, starting 21 days after

planting and every 3 days subsequently. Forty-three lines showed a high level of resistance and RIL-369

and RIL-387 showed the greatest level of resistance. QTL analysis was conducted to identify DNA

markers associated with Ascochyta blight resistance genes in the Lifter/Radley population that can be

applied in the pea breeding program.

Developing a method to scale up production of solanapyrone toxins by

Ascochyta rabiei

Kim, W.¹, Vandemark, G.² ¹Department of Plant Pathology, WSU, Pullman, WA, USA

and Chen, W.²* ²USDA-ARS, Pullman, WA, USA

*Presenter

Ascochyta rabiei, the causal agent of Ascochyta blight of chickpea, produces solanapyrone toxins. The

toxins may play a role in pathogenesis, and a toxin assay using chickpea plant tissues may enable

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screening breeding materials at early generations for resistance to blight. In order to develop toxin assays

for screening chickpea genotypes and to investigate the role of the toxins in developing the disease

Ascochyta blight, sufficient quantity of the toxins is needed for replicated and repeated experiments. A

new method with solid medium using natural substrates is being developed to replace the previously

published method with liquid medium in static culture. The new method significantly improved

production in larger quantity of the solaynapyrone toxins with much reduced requirement of laboratory

space, and will facilitate future research employing and exploiting the solanapyrone toxins.

First report of stemphylium blight of lentil in Montana and North Dakota:

evidence, distribution, and prospects for management.

Wunsch, M.J.* Carrington Research Extension Center, Carrington, ND USA

*Presenter (michael.wunsch@ndsu.edu)

Stemphylium blight, caused by the fungal pathogen Stemphylium botryosum, was observed for the first time

in Montana and North Dakota in 2010 and 2011. The disease occurred in all production regions surveyed,

sometimes at moderate to high severity. Stemphylium blight severity data collected in a fungicide timing

trial and variety performance trials suggest that commonly employed fungicide application strategies

may not effective against the disease but that several lentil varieties adapted to the region may show

some resistance.

Response of chickpea varieties to foliar fungicides

Pederson, S.¹* and McPhee, K². ¹NCREC, North Dakota State University, Minot, ND USA

²North Dakota State University, Fargo, ND USA

*Presenter (shana.pederson@ndsu. edu)

Ascochyta blight (Ascochyta rabiei) can be devastating to chickpea (Cicer arietinum L.) production in North

Dakota. Ascochyta susceptible varieties, as well as resistance in the pathogen to strobilurin fungicides,

have impacted production. In order to determine an effective screening method for the chickpea breeding

program, an evaluation of the response of varieties from different market classes to foliar fungicides was

essential. Eight chickpea varieties were grown at four North Dakota locations. Agronomic performance

and quality of each variety was compared in a fungicide/no fungicide management system. In 2010, seed

yield, canopy height, 1000KWT, disease ratings and seed sizing between fungicide treatment and the

untreated control at two locations were significant. Preliminary results from 2011 indicate a similar trend.

Significant yield differences of each variety in response to foliar fungicide were more evident at higher

yielding locations. Other production constraints may have impacted this response at lower yielding

locations. Based on two years of evaluations, it is clear that fungicide treatment of breeding materials is

necessary in North Dakota environments to ensure seed production and prevent loss of valuable

germplasm. Even in the presence of fungicide treatment, significant disease is evident and sufficient to

allow differential selection among germplasm lines. This research will allow breeding programs to

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effectively manage Ascochyta blight to an advantage and avoid complete loss and setbacks in breeding

and selection of improved varieties.

Pea germplasm with partial resistance to sclerotinia sclerotiorum that extends

the time required by the pathogen to infect host tissue

Porter, L.D* USDA-ARS, VFCRU, Prosser, WA, USA

*Presenter (Lyndon.porter@ars.usda.gov)

White mold, caused by the fungus S. sclerotiorum can be a serious disease on pea. Currently there are no

pea genotypes with complete resistance to this pathogen. Selected wild pea genotypes from the Pisum

Core Collection and cultivars were assessed for the time required by S. sclerotiorum to severely infect these

genotypes at all combinations of five temperatures (15.6, 18.3, 21.1, 23.9, 29.4°C) and four (12, 24, 48 and

72 h) periods of high relative humidity (PHRH). Commercial genotypes did not prevent severe infection

at any temperature/PHRH combination. Three wild pea genotypes were capable of preventing severe

infection for up to 24 hours. PI169603 and PI240515 are recommended to pea breeders as the best

germplasm to extend the time required for serious infection by S. sclerotiorum.

Use of resistant-susceptible cultivar mixture to preserve er-1gene rendering

resistance to powdery mildew of field pea (pisum sativum L.)

Bing, D.J ^1#, Gan, Y. ² ¹AAFC, Lacombe Research Centre, Lacombe, AB Canada

and Warkentin, T. ³ ²AAFC, Semiarid Prairie Agricultural Research Centre, Swift Current, SK, Canada

³CDC, University of Saskatchewan, Saskatoon, SK, Canada

*Presenter (dengjin.bing@agr.gc.ca)

Powdery mildew (caused by Erysiphepisi DC. var pisi.) resistance of field pea (Pisumsativum L.) cultivars is

rendered by a recessive gene er-1. Cultivation of resistant cultivars over large geographic areas creates a

typical gene monoculture, which encourages the pathogen evolution for more virulent races and

breakdown of the resistance. We investigated the use of mixtures made up of resistant and susceptible

cultivars to limit the pathogen evolution and to preserve the resistance gene. Since severe powdery

mildew infection can cause significant yield reduction, it is necessary to define a proper ratio for such

cultivar mixtures. Seed of three powdery mildew resistant cultivars was mixed with 10, 20, or 30% of a

susceptible cultivar, and grown in replicated field trials. The yield reduction in cultivar mixtures

depended on the yield potential and performance of component cultivars and disease severity. When

disease severity was high and the resistant cultivar yielded well, the susceptible cultivar could comprise

10-30% of the mixture without significant yield reduction compared with the resistant cultivars in pure

stand. The results suggest that such cultivar mixtures may be used in field pea production by providing

more substrate to the pathogen so that the breakdown of resistance gene er-1 may be delayed.

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Powdery mildew is a common disease of field pea, and can cause significant yield losses. The majority of

powdery mildew resistant field pea cultivars carry a single recessive gene erJ. Cultivation of such

resistant cultivars over large geographic areas could press the pathogen to mutate for more virulent

race(s), accompanied by a sudden breakdown of the resistance. We propose to use cultivar mixtures

consisting of resistant and susceptible cultivars in an appropriate ratio to provide more substrate to the

pathogen, and therefore to reduce selection pressure on the pathogen, so that the life time of the erJ-

rendered resistance may be extended. The main objective of this study was to define proper ratios of

resistant-susceptible cultivar mixtures with no or little yield reduction compared to the resistant

cultivars in pure stands.

Materials and methods

Three powdery mildew resistant field pea cultivars, Agassiz, Cutlass, and Reward were mixed with the

powdery mildew susceptible cultivar, CDC Striker, in 10, 20 or 30% by number of seeds to make

resistant-susceptible cultivar mixtures. Such mixtures were grown in replicated field trials along with

the four cultivars in pure stands. Also included in the study was CDC Striker sprayed with the fungicide

Kumulus (denoted as CDC Striker + Kumulus), where powdery mildew was controlled by spaying

Kumulus on the plants when the disease symptoms occurred on the plants. The seeding rate was

adjusted to 85 viable seeds m^-2. The yield difference between CDC Striker+Kumulus and CDC Striker

was the measure of yield reduction caused by powdery mildew, and the yield of each cultivar in pure

stand was compared with the yield of each of its mixtures with CDC Striker to determine the yield

reduction of each cultivar mixture. The experimental design was a randomized complete block with four

replications. The field trials were grown

in four locations: Lacombe, AB;

Table 1. Yield difference of cultivars in pure stands and cultivar mixtures

Saskatoon and Swift Current, SK; and

Morden, MB in 2006 and 2007. However,

the disease severity at other three

locations in both years was much less

than the disease severity in Lacombe,

which may lead to biased estimations of

the impact of the disease on the yield.

Therefore, only the results from Lacombe

were reported here.

Results and discussion

Yield reduction caused by powdery mildew.

CDC Striker+Kumulus yielded 3766 kg

ha^-1 in 2006 and 3760 kg ha^-1 in 2007. In

comparison, CDC Striker yield 3033 kg

ha^-1 and 2483 kg ha^-1 in 2006 and 2007,

respectively. Thus, CDS Striker yielded

24% and 32% less than CDC

Striker+Kumulus, This yield reduction

was attributed to the powdery mildew

infection. The higher yield reduction in

2007 than in 2006 was attributed to the

higher disease severity in 2007. This

signifies the importance to preserve the

resistance conferred by gene erJ.

Proper ratio of resistant vs. susceptible cultivar

in a cultivar mixture. No significant

^a Yield difference (kg ha^-1) between resistant cultivar in pure stands and

cultivar mixture. * = p < 0.05._

PISUM GENETICS

2011-VOLUME 43

NAPIA ABSTRACTS

difference was found between any of the three cultivars in pure stands and their mixtures with CDC

Striker in 2006 (Table 1), whereas in 2007, Agassiz had significantly higher yield than Agassiz80+CDC

Striker20, and nearly significant higher yield than Agassiz70+CDC Striker30. Cutlass had significantly

higher yield than Cutlass70 + CDC Striker30 in 2007. There was no significant difference between

Reward and its mixtures with CDC Striker. The difference between 2006 and 2007 was attributed to the

combination of two factors. In general yield and powdery mildew severity were lower in 2006 than in

2007. Thus, the yield potential of the resistant cultivars was not fully expressed in 2006, and the yield

reduction caused by powdery mildew was less in 2006 than in 2007.

Conclusion

The proper ratio of resistant vs. susceptible cultivar in a cultivar mixture depends on yield potential of

component cultivars and powdery mildew severity. When disease severity was high and the resistant

cultivar yielded well, the susceptible cultivar could comprise 10-30% of the mixture without significant

yield reduction compared with the resistant cultivar in pure stand. Such cultivar mixtures may be used

in field pea production by providing more substrate to the pathogen so that the breakdown of resistance

gene erJ may be delayed.

Variability for micronutrient composition of pea and lentil grown in

Montana

McPhee, K.¹*, Thavarajah, D.², ¹North Dakota State University, Dept. 7670,Fargo, ND USA

Thavarajah, P.¹ and Chen, C.³ ²North Dakota State University, Dept. 7640,Fargo, ND USA

³Montana State University, CARC, Moccasin, MT USA

*Presenter (kevin.mcphee@ndsu.edu)

Pulse crop production in North Dakota and the Midwest region including eastern Montana and South

Dakota has increased over the past 15 years to become the primary production region for these crops in

the US. Pea and lentil are well regarded as highly nutritious foods and serve as the primary protein source

in diets for many populations worldwide. In addition to serving as an excellent source of protein, starch

and fiber, pea and lentil seed contain many additional micronutrients that contribute to the daily

nutritional requirements. This research was conducted to explore the environmental effects on

micronutrient accumulation in pea and lentil and to provide a base line for future variety development.

Seed harvested from genotypes comprising the statewide yield trials conducted at eight locations in

Montana were analyzed for micronutrient composition using high performance liquid chromatogrphy

(HPLC) and inductively coupled plasma emission spectroscopy (ICP-Emission) methodology. Iron (Fe)

concentration ranged from 46.3 to 68.0 mg/kg among 21 lentil genotypes and from 50.5 to 69.1 mg/kg

across the eight locations. Fe concentration in pea ranged from 37.3 to 51.2 mg/kg among 27 genotypes

and from 38.2 to 50.5 mg/kg across nine locations. Zinc (Zn) concentration ranged from 31.2 to 43.7

mg/kg among 21 lentil genotypes and from 27.6 to 50.0 mg/kg across eight locations. Zn content among

27 pea genotypes ranged from 31.9 to 129.0 mg/kg and from 29.4 to 124.1 mg/kg across nine locations.

Additional micronutrients and antinutrients including caroteinoids and phytic acids were also analyzed.

Proximity of locations appeared to show a trend in seed micronutrient content. These data indicate that

environmental factors, i.e. soil type, may have a significant role in accumulation of micronutrients in pea

and lentil. This research serves as the basis for future variety development focused on increasing the

micronutrient composition of pea and lentil seed with intent of providing greater nutrition to

undernourished populations worldwide.