| | Presented at the 18th North American Barley Researchers Workshop, July 17-20, 2005, Red Deer, Alberta, Canada
A TaqMan® fluorescent reporter probe replaces gel electrophoresis for the Rpg1 SCAR marker in molecular marker-assisted selection
The markers for the barley stem rust resistance gene Rpg1 (Eckstein et al, 2003) are based on DNA sequence of the isolated gene (Brueggeman et al, 2002) and as such are “perfect” markers, being able to distinguish between resistant and susceptible alleles without recombination. While linkage based markers cannot reliably be used to predict the phenotype of plant lines that have not previously been disease tested, the Rpg1 marker can be used in this fashion. The marker is routinely used in our breeding program and has replaced the need for our local rust nursery. The disease reaction of candidate varieties is confirmed only at the Co-op testing level. In addition, the marker is routinely used to determine the presence or absence of rust resistance in previously uncharacterized materials. As such, the marker is highly useful in the breeding program. The present limiting step in our MMAS program remains the analysis of PCR products by gel electrophoresis. We are investigating the use of TaqMan® fluorescent reporter probes for allelic discrimination in quantitative real-time PCR (qRT-PCR) to replace gel electrophoresis. The Rpg1 markers are ideal as a model since the markers are highly robust and the primers are highly selective due to the 3nt insertion/deletion event in the gene that determines gene functionality and which form the discriminatory basis for the PCR primers. A TaqMan® probe has been designed with the aid of Beacon Designer 4.0 such that the PCR primers are discriminatory and the fluorescent probe itself is universal to both the resistant and the susceptible PCR test. Amplification of the locus, and the amplification mediated increase in fluorescence, is determined by the selective sense primers (specific for either the resistant allele or the susceptible allele). The probe sequence (Rpg1TMP, see below) lies 19 nucleotides downstream of the non-selective primer on the anti-sense strand. The non-selective primer (Rpg1TMR, see below) has been redesigned to amplify a short 120bp fragment for more efficient qRT-PCR assays. PCR amplification conditions are currently being optimized. Cost of the TaqMan® probe is $0.07 per test, which is less than the cost of gel electrophoresis (based on 96 samples per gel). In addition, with fewer steps in the screening process there is less potential for error, and the fluorescent detection of PCR products eliminates one of the steps of the MAS process that is not amenable to automation.
Rpg1TMP...5’...FAM-TTTGGTATAGCTCTCCTTTCCTGCC-Black Hole Quencher1...3’
Rpg1TMR...5’...TACACGCTCAGTAAACTCTT...3’
References:
Brueggeman, R., Rostoks, N., Kudrna, D., Kilian, A., Han, J., Druka, A., Steffenson, B., and A. Kleinhofs. 2002. Proc. Natl. Acad. Sci. USA 99:9328-9333.
Eckstein, P., Rossnagel, B., and G. Scoles. 2003. Barley Genetics Newsletter 33:7-11.
Peter Eckstein, Donna Hay, Brian Rossnagel, and Graham Scoles
Department of Plant Sciences/Crop Development Centre, University of Saskatchewan, Saskatoon, SK, CANADA S7N 5A8
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Genetic analysis of preharvest sprouting in barley
Preharvest sprouting (PHS) can be a problem in barley production, especially of malting barley. Rain or even very high humidity from near physiological maturity onward can cause sprouting in spikes. This has very serious consequences for malting grain, since rapid and complete germination is critical. Much information has been gained by studying the genetic control of dormancy (measured as percent germination) in barley. The objective of this study is to determine if the germplasm developed and QTLs discovered in previous research of dormancy can be applied or related to the genetic control of PHS. PHS was measured in this study as ‘PHS score’ based on visual sprouting in mist chamber-treated spikes at 0 and 14 d after physiological maturity and as ‘alpha-amylase activity’ in kernels taken from mist chamber-treated spikes that showed little or no visible sprouting at physiological maturity (0 d). Germination percentage was also measured at 0 and 14 days after physiological maturity. Many QTLs for dormancy have been previously mapped, most of which are minor and inconsistently expressed across environments. Consistently expressed major and minor dormancy QTLs were previously mapped to barley chromosomes 1 (7H), 4 (4H), and 7 (5H) in the U.S. Barley Genome Project Steptoe (dormant) / Morex (non-dormant) doubled haploid mapping population. Evaluation of this population grown in two environments (greenhouse and field) for PHS score revealed QTL regions on all chromosomes, except chromosome 6 (6H) and for alpha-amylase activity on all seven chromosomes from one or both environments. However, many of the QTLs identified were minor in effect. QTL effects for all traits analyzed ranged from 4 to 36%. The two major dormancy QTLs previously identified on chromosome 7, as well as, the minor ones on chromosomes 1 and 4 were confirmed in this study. Some of the PHS score and alpha-amylase QTLs coincide with known dormancy QTLs, but there appear to be QTLs unique to PHS, as well. Some PHS alpha-amylase activity QTLs coincide with known malt-derived alpha-amylase activity QTLs, but some do not. The major chromosome 7 dormancy QTLs detected from this cross are expressed during PHS, but several previously identified minor dormancy QTLs appear to be more important during preharvest sprouting than during after-ripening or after-harvest dormancy conditions. Whereas, the literature frequently equates dormancy and preharvest sprouting, it appears there is some difference in genetic control of these two somewhat opposite traits. Both traits are complexly inherited, but with some overlap and some uniqueness in gene expression. In addition, several relatively major genes seem to stand out in expression with many minor genes presumably interacting or adding to the expression of the two traits. This study continues with two other mapping populations previously analyzed for dormancy. Ultimately, key QTLs will be identified, which should benefit the breeding efforts of both six-row and two-row barley for a suitable balance between the tendencies for preharvest sprouting and dormancy.
S.E. Ullrich*, J.A. Clancy, H. Lee, F. Han, K. Matsui, I.A. del Blanco
*Corresponding author: ullrich@wsu.edu
Dept. of Crop & Soil Sciences, Washington State University, Pullman, WA 99164-6420, USA
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Effects of ethylene in barley (Hordeum vulgare L.) tissue culture regeneration
Ethylene is a gaseous plant hormone that regulates numerous cellular processes from germination to flowering and senescence. It is produced under stress conditions such as tissue culture and can be physiologically significant in-vitro due to enclosed conditions. This study was conducted to determine genotype-dependent ethylene production and its role in regeneration of barley (Hordeum vulgare L.) callus. Six barley cultivars were examined and found to produce different amounts of ethylene during culture. The highest regeneration was observed in cultivars generating the most ethylene. Ethylene production was correlated with regeneration rates (r2=0.90625). There were no significant genotype by stage interactions for either ethylene production or green plant regeneration. The media was modified by adding the ethylene precursor, ACC (1-amino-cyclopropane-1-carboxylic acid) or the ethylene antagonist silver nitrate (AgNO3) to the media at different stages of callus culture to determine the effects of ethylene during plant regeneration. Highest regeneration in Morex was observed when AgNO3 was added to maintenance stages (M-1, M-2) and lowest regeneration when AgNO3 was applied to the regeneration stage compared to the control. In Golden Promise, AgNO3 added throughout the second maintenance and regeneration stages showed the highest regeneration compared to control. Regeneration was significantly affected with addition of ACC in Morex and highest when ACC was added at the second maintenance stage. Golden Promise did not show improved regeneration when ACC was added at any time. Regeneration was highest for the control. Further manipulation of ethylene synthesis and/or action will be used to identify critical timing and duration for ethylene to effects on plant regeneration from recalcitrant genotypes. Ethylene exposure for briefer time periods will help pinpoint the specific stages when ethylene should be manipulated.
Jha, Ajay K. (1), Lynn S. Dahleen* (2) and Jeff C. Suttle (2)
*Corresponding author: dahleenl@fargo.ars.usda.gov
(1) Department of Plant Sciences, North Dakota State University, Fargo, ND 58105
(2) USDA-ARS, Fargo, ND 58105
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Validation of select diastatic power QTL in elite Western U. S. six-rowed spring barley germplasms
Barley is a major commodity in the western US., especially when utilized for malt production. Spring six-rowed barley germplasm adapted to the western US. is often low in diastatic power (DP), an important malting trait. DP quantitative trait loci (QTL) and linked markers have been identified in a spring six-rowed mapping population grown in western environments, but these QTL have not been validated in elite western six-rowed backgrounds and the QTL are based on restriction fragment length polymorphism (RFLP) markers. The objectives of this study were to validate DP QTL in populations containing elite western germplasm and to identify polymerase chain reaction (PCR) -based markers linked to select DP QTL.
Low DP spring six-rowed cultivars or elite lines were crossed to ‘Morex’, a high DP 6-rowed barley, or to SM#42, a high DP line of the ‘Steptoe’/Morex mapping population. F1 plants were backcrossed to the adapted, low DP lines two or three times, and segregating lines were developed from the backcrossed populations. The low DP parents and backcross-derived segregating lines were genotyped with PCR-based markers closely aligned to five DP QTL with large effects on chromosomes 1H, 4H, and 7H. Lines homozygous for the PCR-based markers and parental checks were planted in 4-m rows at Aberdeen, ID. Quality analysis to determine DP levels was performed by the Cereal Crops Research Unit, Madison, WI. Nearly all the low DP parental lines had marker alleles that resembled either Steptoe or Morex with regard to DNA fragment size. Two lines from Utah State University had DP-negative marker alleles for all loci except the one on the short arm of chromosome 4H. Some had more DP-positive alleles with a few DP-negative alleles, while others had similar numbers of DP-positive and DP-negative alleles. Also, some heterogeneity within lines was detected. We plan to determine if relationships between marker genotype and DP can be identified in the advanced populations and if other traits are affected by DP marker selection. This study should provide useful information for the development of six-rowed malting barleys in the western U.S.
D. Hoffman, A. Hang, and D. Obert
USDA-ARS Small Grains and Potato Germplasm Facility, 1691 South 2700 West Aberdeen, Idaho 83210
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The barley stem rust resistance gene product RPG1 is specifically degraded upon infection with the stem rust fungus Puccinia graminis f. sp. tritici pathotype MCC
Disease resistance in plants, mediated by the gene-for-gene mechanism, involves the direct or indirect recognition by the R-gene product of specific effector molecules produced by the pathogen. This recognition triggers a series of signals regulating an elaborate series of defense mechanisms by the plant. In order to understand the role of the recently cloned barley stem rust resistance gene product RPG1 (Brueggeman et al. 2002) in resistance to the stem rust fungus Puccinia graminis f. sp. tritici, we investigated the fate of the RPG1 protein in response to infection with the P. graminis f. sp. tritici avirulent pathotype MCC. Different barley lines with varying levels of resistance were challenged with the avirulent pathotype MCC and sampled at 0, 12, 16, 20and, 24 and 36 24 h post-infection. The extracts were immuno-precipitated with an RPG1-specific antibody. The precipitate was subjected to SDS-PAGE, and RPG1 was visualized by western blot analysis. Though the endogenous transcript levels of Rpg1 remained unchanged upon infection with the avirulent pathotype MCC (Rostoks et al. 2004), the RPG1 protein disappeared to undetectable levels 20-24h post-infection. The degradationisappearance of the RPG1 protein was localized to the infected tissue and did not spread to the adjoining leaves. The RPG1 protein was shown to be stable in cyclohexamide translation inhibited leaves for up to 48 hrs. These results suggest that the localized disappearance of RPG1 protein is due to proteolysis, probably by the avr-gene product. Since the mere absence of the RPG1 protein does not result in resistance, the degradation products or the process of degradation may trigger the signaling resulting in resistance to the stem rust infection.
J. Nirmala (1), B. Steffenson (2) and A. Kleinhofs* (1,3)
*Corresponding author: andyk@wsu.edu
(1) Department of Crop and Soil Sciences Washington State University, Pullman, WA-99164-USA
(2) Department of Plant Pathology, University of Minnesota, St Paul, MN 55108, USA
(3) School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA
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Saturation mapping of barley chromosome 2H Fusarium head blight resistance QTL
Outbreaks of fusarium head blight (FHB), caused by Fusarium graminearum, within the past two decades have caused large economic losses to farmers in North Dakota and northwestern Minnesota due to the reduced quality of harvested barley by blighted kernels and significant levels of deoxynivalenol (DON), a mycotoxin produced by the fungus. Field practices and chemical control have had limited success, putting greater importance on a genetic approach for control. Two quantitative trait loci (QTL) each for lower FHB severity and plant height, and one major QTL each for DON accumulation and days to heading were mapped with recombinant inbred lines obtained from a cross between CIho 4196, a two-rowed resistant cultivar, and Foster, a six-rowed susceptible cultivar (Horsley et al., submitted). These loci reside in the barley chr. 2H region flanked by the markers ABG306 and MWG882A (bins 8-10). In an attempt to saturate this region with markers, 29 rice chr. 4 BAC clones with synteny to this region were blasted against the barley expressed sequence tag (EST) database. To date, 41 of the ESTs picked by this method have been mapped to this region. Nine other genes, identified based on microarray analysis of the wheat-barley 2HL addition line and comparison to Betzes and Chinese Spring controls (courtesy of Gary Muehlbauer), were also mapped to this region on the Foster x CIho 4196 RFLP map. To date, there are a total of 26 unique loci and 67 markers in this major FHB QTL region on chr. 2. Eighteen markers have been hybridized to the 6x cv. Morex barley BAC library, identifying 131 BAC clones as part of the physical map of the region. Three cleaved amplified polymorphic sequences (CAPS) markers were designed for the major FHB resistance QTL in this region, two flanking and one in the middle, to aid in development of isolines containing fragments of this region from CIho 4196 in a Morex cultivar background.
Christina Maier* (1), Deric Schmierer (1), Thomas Drader (1), Richard Horsley (2), and Andris Kleinhofs (1,3)
*Corresponding author: cmaier@wsu.edu
(1) Dept. of Crop and Soil Sciences, Washington State University, Pullman, WA 99164-6420, USA
(2) Dept. of Plant Sciences, North Dakota State University, Fargo, ND 58105-5051, USA
(3) School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660, USA
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Map-based cloning efforts of the barley spot blotch resistance gene Rcs5
The Rcs5 gene confers seedling resistance to barley spot blotch, caused by the fungus Cochliobolus sativus. Spot blotch is a common and economically important foliar disease of barley in the Midwestern United States. Genetic mapping localized the resistance gene between the markers MWG622 and KAJ154 on the short arm of chromosome 1(7H). A BAC clone physical contig was constructed consisting of 4 clones, 053N3, 612G14, 452P9, and 808M17. Subclone shotgun libraries of BAC clones 612G14 and 808M17 were constructed and sequenced by the Arizona Genomics Institute. Sequence assembly resulted in 25 contigs ranging from 1kb to 28.5kb for 808M17 and 7 contigs ranging from 0.6kb to 63.7kb for 612G14. Analysis of the contigs' sequence by a gene prediction program (FGENESH) with limits within monocot genomic DNA yielded 40 putative genes, 22 with protein homology. Of the 22 protein hits, 19 had equivalent Triticeae EST's. The BAC contig overlap between 612G14 and 452P9 as well as the overlap between 452P9 and 808M17 left an unsequenced gap of ~15kb. The BAC 452P9 38kb NotI subclone, TBD001, covers this region and contains markers KAJ108B.2 and KAJ154, which flank a high recombination region with 11 crossovers and potentially the Rcs5 gene. The subclone TBD001 sequencing is in progress. Physical and genetic mapping of predicted genes will delimit the region of interest. Putative candidate genes will be sequenced from resistant and susceptible cultivars.
Thomas B Drader* (1), Kara A Johnson (2), Robert S Brueggeman (1), Hye Ran Kim (3), Dave Kudrna (3), Rod Wing (3), Brian Steffeson (4), and Andris Kleinhofs (1, 5)
*Corresponding author: tdrader@wsu.edu
(1) Crop and Soil Science, Washington State University, Pullman, WA 99164, USA
(2) USDA, Washington State University, Pullman, WA 99164, USA
(3) Plant Sciences Department University of Arizona, , Tucson, AZ 85721-0036, USA
(4) Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108-6030, USA
(5) Molecular Biosciences, Washington State University, Pullman, WA 99164, USA
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A gene tagging system for Hordeum vulgare
An efficient system for production of knockout plants for functional analysis and gene tagging in cereals would be a significant complement to current cereal genome analyses. Tos17, a rice retrotransposon activated by tissue culture, has been successfully used for generation of knockout plants in rice. More recently, it was reported that Tos17 insertions were predominately located within coding sequences and thus provide a unique system to develop knockout plants at a relatively high frequency. Retrotransposons have the advantage over other transposon systems of yielding stable mutations and low copy numbers which facilitate the identification of genes. Using a novel approach that enables the cultivar-independent regeneration of fertile monocots, transgenic barley T0 was efficiently obtained, and confirmed by southern blot. Results suggest that Tos17 is a very efficient system to generate knockout plants in cereals. Under vegetative growth, the integrated rice retrotransposon Tos 17 is inactive based on the absence of retrotransposase activity in barley leaves and stems. Under tissue culture conditions, the retrotransposase activity was stimulated and readily detected via a reverse transcription-PCR assay. A modification in the number of Tos17 copies in barley genomic DNA (from callus tissues) was detected. We also regenerated fertile plants C0, from 4-5 months callus culture, with novel Tos17 insertion sites. Using real time PCR, we report an increased copy number in C0 barley genome, and new phenotype in offspring C1. We will discuss the advantages of this new system for development of knockout plants in cereals and possible impact for genomic studies in barley.
François Eudes (1)*, André Laroche (1), Michele Frick (1), Jennifer Geddes (1) and Laurian Robert (2)
*Corresponding author: eudesf@em.agr.ca
Agriculture and Agri-Food Canada,
(1) Lethbridge Research Centre, Lethbridge, Alberta T1J 4B1, Canada and
(2) Eastern Cereal and Oilseed Research Centre, Ottawa, Ontario K1A 0C6, Canada
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Unraveling the mysteries of germination using SAGE (Serial Analysis of Gene Expression)
The processes involved in malting are still somewhat a mystery on a genetic level. SAGE (Serial Analysis of Gene Expression) is a technique that allows rapid, detailed analysis of thousands of transcripts in a cell. The process of SAGE relies on two principles. Firstly, a small sequence of nucleotides from the transcript, called a “tag” can effectively identify the original transcript from whence it came. Secondly, linking these tags allows rapid sequencing analysis of multiple transcripts. By examining the transcripts expressed at any time in the cell it is possible to determine which genes and their related proteins are being expressed at that moment in time. In this study the gene expression profile of germinating (malting) barley is being examined at seven intervals over a time course of 120 hours post steeping. This will be compared to a baseline of dry ungerminated seed. The identification of genes for improved malting quality can be identified and examined using SAGE and ultimately used for commercial improvement.
Toni Pacey-Miller*, Jessica White, Allison Crawford, Peter Bundock, Giovanni Cordeiro, Daniel Barbary and Robert Henry
Corresponding author: tpacey@scu.edu.au
Grain Foods CRC, Ltd., Centre for Plant Conservation Genetics, Southern Cross University, Lismore, Australia 2480
The above posters were presented at the 18th North American Barley Researchers Workshop, July 17-20, 2005, Red Deer, Alberta |
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