Barley CAP Participants Guide (updated 4-2-2008)

(Click here for pdf format)

I.  GERMPLASM and SEED

Barley CAP germplasm – refers to 96 breeding lines that will be selected in each of the four years of the project by each of the 10 participating breeding programs to be included in the CAP.  The Barley CAP germplasm must meet the following requirements: 
1) must be lines generated in the breeding program and included in the breeding programs standard yield trials; 
2) must attempt to represent the phenotypic diversity of the current breeding germplasm for traits that are the subject of the CAP; 
3) must be inbred lines at the F4 generation or later; 
4) breeders must be able to provide ~50 grams of seed to be distributed by April 1 for DNA extraction, field disease screening (spring) and the Ft. Collins / Crookston Seed Multiplication;
5) must have pedigree information for each line that can be uploaded to THT database.

To clarify the source of the seed produced for the CAP lines, we will use the following definitions. 
Breeders Source Seed – refers to the source of seed that the breeders use to plant their normal breeding trials with the 96 CAP lines they submit each year.  This will likely be from a winter nursery or from the breeding programs previous years yield trials.  These lines meet the above criteria for barley CAP germplasm.  In addition, this is the source of seed for:
1) growing plants for tissue to extract DNA and DNA source seed.
2) the spring planted CAP Seed Multiplication at Ft Collins, CO. (Cooper) and Crookston, MN. (Smith)
3) the spring planted field disease trials spot blotch at MN (Steffenson) spring lines only and FHB at MN and ND (Smith, Dill-Macky, Horsley, Neate, Steffenson) Midwest lines only.
4)  Vernalization requirement screen for winter lines only in Oregon (Hayes).

DNA Source Seed   A single plant will be grown in the greenhouse at Minnesota for each of the CAP lines using the Breeders Source Seed.  The tissue from each plant will be harvested and sent to Chao for DNA isolation.  The seed will be harvest from each single plant in December.  Five g of seed will be stored at Minnesota and the remaining seed sent to the National Small Grains Collection (NSGC). 
Crookston/Ft. Collins CAP Seed Multiplication – refers to the production of seed for the spring habit breeding lines at two seed production trials in Ft. Collins, CO (Cooper) and Crookston, MN (Smith).  Each of the spring breeding programs will send 25 g of breeders source seed to Smith at the University of Minnesota for seed increase, DNA growout, and Steffenson’s spot blotch nursery.  Each of the spring breeding programs will also send 10 g of seed to Blake Cooper at Ft. Collins for a backup growout.  The two winter programs will be responsible for their own seed multiplication.  The source for this seed increase will be Breeders Source Seed.  The seed produced from these sites will be used for:

• Grain for food quality trait analyses (Baik, and Wise)
• Grain for LOX and Beta-glucanase analyses (Schwarz)
• Seed for fall disease screening (Dill-Macky, Steffenson, Jackson, Griffey),
• Seed for fall Pre-harvest sprouting screening (Horsley)
• Seed for harvest index and drought adaptation yield trials in the subsequent year (Hole, Blake)
• Seed for common root rot trial in the subsequent year (Neate)

Summary of Seed Distribution For Phenotyping

Breeders Source Seed goes to:                                                                  Date
         10 g to Hayes WINTER LINES (Vernalization)                                    Feb 15
         25 g to Smith SPRING LINES  (seed increase & spot blotch)              April 1
         1 g to Smith ALL LINES (DNA Source)                                              April 1
         10 g to Cooper  SPRING LINES (Ft. Collins seed production trial)      April 1
         16 g to Smith FHB (Midwest lines only)                                                April 1
         12 g to Neate FHB (Midwest lines only)                                               April 1
         8 g to Horsley FHB (Midwest lines only)                                               April 1

Crookston/Ft. Collins CAP Seed Multiplication Seed goes to:               
            250 g to Baik (food quality)                                                               Oct 1
            40 g to Neate (common root rot)                                                       Oct 1
30 g to Schwarz (LOX and beta-glucanase)                                       Oct 1
10 g to Wise (beta-glucan)                                                                 Oct 1
12 g to Jackson (net blotch, scald, BYDV, stripe rust)                        Oct 1
40 g to Blake (drought adaptation)                                                     Oct 1
15 g to Hole (harvest index)                                                               Oct 1
5 g to Horsley spring lines only (pre-harvest sprout)                            Oct 1
12 g to Griffey for Leaf Rust screening                                               Oct 1
1 g to Dill-Macky for greenhouse Net blotch screening                       Oct 1   
1 g to Steffenson for greenhouse Septoria screening                            Oct 1

416 g total required     

II.  Barley CAP Genotype and Pedigree Formats

CAP Codes for Genotypes

Each of the 96 Barley CAP genotypes submitted by collaborators each year will be assigned a unique identifier code. The code will start with the year of harvest, followed by a standard two-letter abbreviation for the breeding program, then a dash, and then the numbers 1-96.

The abbreviations for the breeding programs are:

MN      U of Minn
N2       NDSU 2 -row
N6       NDSU 6-row
BA       Busch Ag
OR       Oregon State
MT      Montana
AB       Aberdeen
VT       Virginia Tech
WA      Washington State
UT       Utah State

For example, entries from Minnesota in 2006 would be designated 06MN-1 through 06MN-96.

Pedigree Formats

1)   Breeders can submit pedigrees in text format:
        Mentor/Minerva//Vada mutant/4/Carlsberg/Union//Opavsky/Salle/3/Ricardo/5/Oriol/6153P40
      We are using standard Purdy notation (Purdy et al., Crop Sci 8: 405-406).
Pedigrees in this format will be stored in a text field for reference by breeders.

2)   Breeders should indicate if the parent in a cross was in the F1, F2, or F3 generation; if no information is provided it will be assumed that the parent was fully inbred (F4s and F5s are close enough).

3)   Where possible, provide names of intermediate parents in the pedigrees. If a parent is already in the database, then it is not necessary to expand the pedigree to include earlier generations.

• For data input, use the following naming conventions:
• Use all capital letters for line names:                                     BARONESSE
• Keep dashes within names:                                                 6B97-2245
• Remove nonessential spaces between letters and numbers:   BCD47  (not BCD 47)
• Use an underscore for essential spaces within a name:          AC_METCALFE

 III.  TRAITS
We will try to use traits and trait measurement methods that have been defined by GRAMENE trait ontology which can be found at:  http://www.gramene.org/db/ontology
I have included the trait ontology description (in italics) and TO number if one exists for the traits we are measuring for the BarleyCAP.

Breeders Traits – refers to the standard set of traits that are measured in each program in the yield trials conducted by that program.  It is expected that the Barley CAP germplasm will be evaluated in at least two replicated field trials.  Additional data for lines that are normally evaluated in subsequent years by the breeding program may be included in the CAP data set as well.

Traits measured for all programs.  Eight agronomic traits will be evaluated for all of the lines in standard breeding program trials.  Common checks for the spring germplasm trials will be Robust, Harrington, and Baronesse.  Common checks for the winter germplasm trials will be Strider and 88Ab536.
Yield –    GRAMENE trait ontology grain yield (TO:0000396).   The grain yield, measured in kilograms per hectare at 14 percent moisture.  Each breeder will describe the plot size and harvested area as part of the annotation to the data set.
Plant Height – GRAMENE trait ontology plant height (TO:0000207) #1389 actual measurement of plant height from soil surface to the highest point in plant as identified in the study.  This will be the mean of 2 measurements per plot of the distance from the soil surface to tip of spike excluding awns and reported in cM.
Stem Length - GRAMENE trait ontology stem length (TO:0000576) and culm length (TO:0000309) measure from soil surface to highest point on stem.  This measurement will be the mean of 2 measurements per plot of the distance from the soil surface to the base of the spike and can be taken at the same time as plant height and reported in cM.
Heading Date - GRAMENE trait ontology days to heading (TO:0000137)  Number of days required to 50 percent panicles/tassel exsertion from the flag leaf in a field or study.  Number of days is calculated from either the day of sowing of seeds or from the day of transplanting the seedlings depending on type of study.  We will report days to heading from the planting date.  Breeders will provide planting date as part of the annotation to the data set.
Lodging – GRAMENE trait ontology lodging incidence (TO:0000068) Measure of percentage of plants that lodged.  This will be measured on a 0 – 10 rating scale which should correspond to 0 – 100% lodged plants in a plot.  The data will be reported as a percent.
Plump Grain Percentage -  GRAMENE trait ontology seed width (TO:0000149)  Determined by the actual measurement of width in millimeters as the distance across the seed (with hull).  We will measure this using the current methods for each program and report the data in the units of percent kernels (by weight) that stay on a 6/64” sieve.  Each breeder will describe the method used to measure plump grain percentage as part of the annotation to their data set.
Test Weight -  GRAMENE trait ontology  seed density (TO:0000612)  Test weight is a measure of the density or weight per unit of volume of a grain at a standardized moisture level. This will be measured by each program in their standard method and should be reported in grams per liter.  Each breeder will describe the method used to measure test weight as part of the annotation to their data set.
Grain Protein Concentration - GRAMENE trait ontology  protein content (TO:0000598)  The total protein content measured in a plant or a plant part.  This will be measured by each program in their standard method and should be reported in percent units.  Each breeder will describe the method used to measure GPC as part of the annotation to their data set.
Malting Quality data will be generated for lines that are normally evaluated for malting quality by the individual breeding programs. For each program, this will include those lines that are bred for malting quality.  However, breeders are encouraged to submit lines that are not malting quality (feed) that are part of the CAP 96 lines to provide additional data and phenotypic variation for mapping.  Breeders will also include the standard yield trial checks in the malting analysis to facilitated comparisons across environments.  The USDA Cereal Crops Research Unit (M. Schmitt and Al Budde) in Madison, Wisconsin will conduct analysis of nine malting quality traits (kernel weight, barley color, malt extract, wort color, wort protein, soluble protein/total protein, diastatic power, alpha amylase, and malt beta-glucan).  Samples submitted to the CCRU for malting quality must fit within existing breeders quotas.  The Barley CAP does not pay for samples outside normal breeder quotas.  The BARI (B. Cooper) breeding program will generate similar data from their own analysis lab.
Winter-hardiness will be evaluated by winter programs on their CAP breeding lines.
Additional Traits – refers to traits that will be measured in trials and experiments that are in addition to the normal yield trials conducted by the breeding programs.  All of the traits listed below will be evaluated on all of the 960 breeding lines each year unless otherwise noted.

Various morphological traits (Kevin Smith) will be measured (kernels per spike, tiller per unit area, awn length, spike density, leaf width, leaf length, peduncle length, and hull adherence) in the CAP Seed Multiplication trial planted by Smith in Crookston.    
Harvest index  (David Hole) will examine traits related to harvest index (above-ground dry matter, grain dry matter, number of fertile tillers, sink size per fertile tiller, 1000 kernel weight, number of seeds per spike) in a field trial in Utah.  Harvest index traits will be measured in 2007 and 2008 with the CAP06 and CAP07 breeding lines.   Seed source will be Crookston/Ft. Collins CAP Seed Multiplication.
Drought Adaptation – (Tom Blake) will measure yield, height, heading date, lodging, plump grain percentage, test weight at two field trials in Montana for the spring breeding lines.  One trial will be in a drought stressed environment (Huntley) and the other in an unstressed environment (Bozeman).  Seed source will be Crookston/Ft. Collins CAP Seed Multiplication.
Vernalization requirement (Pat Hayes)  Only winter breeding lines.  Two plantings the first in April and the second in Fall at Corvallis.  Breeders source seed will be used. 
Pre- harvest sprouting (Rich Horsley) Greenhouse screen of spring lines only.  Seed source will be Crookston/Ft. Collins CAP Seed Multiplication.

Disease Traits
Net blotch (Lee Jackson)  field screen for net blotch, stripe rust, BYDV and scald (see below).  Seed source will be Crookston/Ft. Collins CAP Seed Multiplication.
Stripe rust field screen (Lee Jackson) – see net blotch above
Barley yellow dwarf field screen (Lee Jackson) – see net blotch above
Scald field screen (Lee Jackson) – see net blotch above
Net Blotch greenhouse seedling screen (Ruth Dill-Macky)  Seed source will be Crookston/Ft. Collins CAP Seed Multiplication.
Septoria speckled leaf blotch seedling greenhouse screen (Brian Steffenson).  Seed source will be Crookston/Ft. Collins CAP Seed Multiplication.
Spot blotch field screen in St. Paul (Brian Steffenson)  Breeders Seed Source will be used. 
Leaf rust seedling greenhouse screen (Carl Griffey).  Reaction to three races of leaf rust will be evaluated in the greenhouse in the Fall of each year. Seed source will be Crookston/Ft. Collins CAP Seed Multiplication. 
Common root rot field screen  (Stephen Neate)  Field evaluation in large plots (8 row, 8 replicates) in ND.  Seed source will be Crookston/Ft. Collins CAP Seed Multiplication from previous year. 
Fusarium head blight field screen  (Kevin Smith, Ruth Dill-Macky, Brian Steffenson, Rich Horsley, Stephen Neate, Linnea Skoglund, and B. Cooper) will be screened on a subset (384 lines) from the four Midwest breeding programs (MN, NDSU six and two rowed, and BARI) in five summer field nurseries (St. Paul, MN, Crookston, MN, Fargo, ND, Langdon, ND, Osnabrock, ND) and a winter nursery in Hangzhou, China.  Breeders Seed Source will be distributed to MN and NDSU by April 1.

 Quality Traits

Lipoxygenase (LOX) activity in malt  (Paul Schwarz)  Seed source will be Crookston/Ft. Collins CAP Seed Multiplication.
Beta-glucanase activity in malt  (Paul Schwarz)  Beta-glucanase will be determined on green malt (and perhaps kilned malts as well...as this may shed some light on thermal stability).  I will need to malt and freeze-dry samples. Seed source will be Crookston/Ft. Collins CAP Seed Multiplication.
Grain hardness (Byung-Kee Baik) will be assessed along with the other food quality traits using 250 g. from Crookston/Ft. Collins CAP Seed Multiplication.
Starch amylose content (Byung-Kee Baik) (see above).
Phenolic compound content (Byung-Kee Baik) (see above).
Polyphenol oxidase activity (Byung-Kee Baik) (see above).
Barley beta-glucan content (Mitch Wise)  will be assessed using 10 g. sample of grain harvested from Crookston/Ft. Collins CAP Seed Multiplication.

IV.  PHENOTYPIC DATA

THT Phenoypic Data – refers to the actual data that will be uploaded to THT from each of the experiments for breeding or additional traits.  For breeder data two files will be sent to the trait coordinator (Jenifer Kling).  The first file will contain the raw values (individual reps) for the 8 standard traits from the breeding yield trails.  This will include all of the data from the trial from which the Barley CAP lines were grown.  In addition, one sheet will contain the plot plan for the trial showing the range and row positions for each of the plots in a trial.  In many cases, the Barley CAP lines will be evaluated across more than one experiment.  The rational for uploading all the data is to store the entire data set for other kinds of analyses not necessarily outlined in this proposal for association mapping. 
The second file will be a file with the 96 entries and checks and the mean values for each of the standard traits at each location the CAP lines were evaluated.  The mean value should be the mean that the breeder normally calculates (arithmetic, least squares, etc..) the column next to the mean should be the standard error for the experiment containing the entries.  This value can be used to normalize means in THT.  So for example, you might evaluate your 96 CAP lines across three different experiments, each at three locations.  Then you would have a column that designates location, another for experiment within location, another for mean value, and another for standard error of the experiment.  In the SE column, the number would be the same for each entry that was evaluated in a particular experiment.   If you have questions about this contact the trait coordinator (Kling).
For the additional traits, a standard excel spreadsheet will be designed by the person conducting the phenotyping and the trait coordinator and used to store and upload data to THT. 
For all trait data include information describing the methods, experimental design (replication, plot size, sample and or subsample size etc…) along with ANOVA results for the experiment.

DATA SUBMISSION DEADLINES  (shown for 2006 CAP lines)

December 1, 2006
      Breeder trials:  yield, heading date, height, lodging, test weight.  (Tom Blake Mar 1)
Spot blotch  (Steffenson)
FHB Severity data (Smith, Horlsey, Neate)
Winter-hardiness (Griffey, Hayes)
Vernalization requirement (Hayes)

June 1, 2007
Breeders trials: plump grain percentage, grain protein concentration, malting quality (if available)
Pre-Harvest Sprouting (Horsley)
Net Blotch, Stripe Rust, Scald, BYD (Jackson)
Net Blotch (Dill-Macky)
Septoria (Steffenson)
Leaf Rust (Griffey)
DON Midwest Lines (Smith, Horlsey, Neate, Schwarz)
Barley Beta Glucan (Wise)

December 1, 2007
Food Quality (Baik)

V.  GENOTYPING

Genotyping – refers to the evaluation of the CAP breeding germplasm with 3,072 SNP markers developed and mapped by the Barley CAP.  Shiaoman Chao will conduct the Genotyping using the Illumina BeadStation at the USDA Genotyping Lab in Fargo, ND.  For spring programs, the seed for DNA growout will be from the 25 g that you send by April 1 each year.  Winter programs will need to send ~ 20 seeds from each of their 96 lines to Smith in April of each year.  Smith will sow the seeds in the greenhouse and thin to 1 plant per line.  Leaf tissue will be sampled from each plant and sent to Chao. This tissue will be the source for DNA extractions that will be used for the SNP genotyping carried out using the Illumina technology in the Chao lab.  The single plant for each line will be grown to maturity, the seed harvested, and bulked to form the DNA source seed.  

VI.  Contact Information and Shipping Addresses

Byung-Kee Baik