# Presentation: 144507

## Development of a Microsatellite Library for Puccinia coronata

- Development of a Microsatellite Library for *Puccinia coronata*
- Hattie R. Dambroski and Martin Carson, USDA-ARS-Cereal Disease Laboratory, St. Paul, MN

- Introduction
*Puccinia coronata* or oat crown rust is a devastating disease in wild oats (*Avena fatua* and *A. sterilis*) and cultivated oats (*A. sativa*) in the United States and other parts of the world. *P**. coronata* is highly polymorphic for virulence and avirulence corresponding to resistance genes found originally in *A. sterilis* and now introgressed into *A. sativa* cultivars (Leonard, K.J. et al 2004). This polymorphism is found in the Northern Great Plains of the United States where the *P. coronata *populations are presumably mostly sexual due to the widespread occurrence of the alternate host, buckthorn (*Rhamnus cathartica*), as well as in the Southern Plains of the United States where the populations are mostly asexual (Leonard, K.J., 2003). One of the most accepted hypothesis is that of the *Puccinia* pathway where cereal rust spores (including *P. coronata*) from the southern plains are blown north later in the season, serving as a significant source of inoculum infecting oats in the northern plains and potentially increasing polymorphism in the north (Simons, M.D. 1970).  Our goal was to form a microsatellite library to test the population genetics of this species. *P. coronata* has no microsatellite (simple sequence repeats, SSR) markers available for use in population studies and microsatellite markers from *P. graminis*, wheat stem rust, and *P. triticina*, wheat leaf rust were tried in *P. coronata*, with no success.

- Materials and Methods
- Germinated urediniospores of *P. coronata* (isolate CDL 03MN 097) was ground by shaking samples with 1 mm glass beads (Lysing Matrix C, Q Bio Gene, Irvine, CA) and 25 mg of diatomaceous earth for 20 s.  DNA was extracted from this ground mixture using the Omni-Prep DNA extraction kit (Genotech, St. Louis, MO).  The microsatellite library was made using the protocol from the Molecular Markers: Tools for Developing Enriched Microsatellite Libraries Workshop given by Ginger Clark and Daniel Brazeau at the Interdisciplinary Center for Biotechnology Research, University of Florida, May 2005.

- Results
- The first CA library had a success rate of only two polymorphic markers out of 192 sequenced clones.  Three more libraries were constructed with enrichment of repeats AAT, AAC and CA.  An additional selection of hybridization with biotin-labeled probes was used on these libraries to reduce the number of false positives.  No markers were found in the AAT (0/56) or AAC (0/65) libraries, but three markers were found from 45 sequenced clones in the second CA library.  These three markers were not polymorphic.  The two markers that did work had repeats of (GC)4 and (CA)10 (CT)6.

- Discussion
- These libraries did not provide enough markers in order to run any sort of population analysis.  We have since contracted with ATG Genetics in Vancouver, BC, Canada to make a library for us and we are currently waiting for that to be completed.  The protocol worked for a number of plant and animal species at the workshop, so why these *P. coronata* libraries did not work is uncertain. It could be that more tinkering needs to be done with the protocol itself in order for it to work on *P. coronata*.

- Literature Cited
- Leonard, K. J. 2003. Regional frequencies of virulence in oat crown rust in the United States from 1990 through 2000.
- Plant Dis. 87: 1301-1310.
- Leonard, K. J., Anikster, Y., and Manisterski, J. 2004. Patterns of virulence in natural populations of *Puccinia *
*coronata* on wild oat in Israel and in agricultural populations on cultivated oat in the United States.
- Phytopathology 94: 505-514.
- Simons, M. D. 1970. Crown rust of oats. Am. Phytopathol. Soc. Monogr No. 5. Heffernan Press, Worcester, MA.

| 1X PCR buffer |
| --- |
| 1.5 mM MgCl2 |
| 2 mM each dNTP |
| 0.5 μM Sau 3A I primer |
| 2.5 U of Taq polymerase |
| 15 μl of linker ligated DNA (about 150 ng) |

-    DNA was digested with the restriction enzyme, *Sau *3A I.
-    Fragments were selected for those size 400 bp and bigger      using a Chroma Spin + TE 400 column (Clontech Laboratories, Inc, Palo Alto, CA) and checked on an agarose gel.
*   **Sau* 3A I linkers were ligated to the selected fragments overnight and the excess linkers were removed using the Chroma Spin column.
- Linker ligated DNA was amplified in 100 μl reactions with a final concentration of:

- Reactions were run in a thermal cycler (MJ Research Model PTC-100) using the program:

| Linker Connection | 72oC | 5 min |
| --- | --- | --- |
| Initial Denaturation | 94oC | 3 min |
|  | 94oC | 1 min |
| 25 Cycles | 68oC | 1 min |
|  | 72oC | 2 min |
| Final Amplification | 72oC | 10 min |

- The fragments were checked on an agarose gel.

-    Amplified fragments were enriched using Dynabeads® M-280 Streptavidin (Dynal Biotech, Lake Success, NY)
-    15 μl of amplified fragments were denatured for 10 mins at 98°C and then chilled on ice for two min
-    The probe mixture (4 μl of biotinylated (CA)15 oligonucleotide [0.125 μg/μl] and 19 μl of 2X hybridization buffer (1M sodium phosphate, pH 7.4, 1% SDS, 1% BSA)) was added to the fragments after the denaturation and allowed to hybridize overnight.
-    At room temperature (RT) Dynabeads were washed in 1X TBST buffer (0.1 M Tris Base, pH 7.5, 150 mM NaCl, 0.1% Tween 20) three times before being used.
-    Then the hybridized probe solution was added to the beads and remained at RT for 15 mins, for biotin to bind to streptavidin.
-    The beads were again washed three times with 1X TBST and then sterile water was added and the beads were incubated at 98oC for 10 mins to cleave the biotin from the streptavidin.
- Using the supernatant from above, a 50 μl PCR reaction was set up with the final concentration

| 1X PCR buffer |
| --- |
| 1.5 mM MgCl2 |
| 100 μl each dNTP |
| 0.5 μl Sau 3A I primer |
| 2.5 U / 50 μl Taq polymerase |
| 10 μl eluted DNA fragments |

- Reactions were run in a thermal cycler (MJ Research Model PTC-100) using the program:

| Initial Denaturation | 94oC | 3 min |
| --- | --- | --- |
|  | 94°C | 1 min |
| 25 cycles | 68°C | 1 min |
|  | 72°C | 2 min |
| Final Amplification | 72°C | 10 min |

-    Reactions were checked on an agarose gel.
-    Cloning was done using Invitrogen’s TOPO TA Cloning Kit.
-    Sequencing was done at the University of Washington’s High-Throughput Genomics Unit, Seattle, WA.

- For the final three libraries an additional selection step of a colony lift hybridization was added after cloning.
-    A nylon membrane was placed on the surface of each plate and allowed to sit at RT for 5 mins and then the membrane spent 5 mins on each of the solution absorbed 3 mm filter paper in the following order:
-   (1) 10% SDS
-   (2) and (3) Denaturing solution (0.5 M NaOH, 1.5 NaCl)
-   (4) Neutralizing solution (0.5 Tris, pH 7.4, 1.5 M NaCl)
-   (5) 2X SSC (20X SSC = 3 M NaCl, 0.3 M Na citrate, pH 7.0).

- The DNA was cross-linked by placing DNA side of membrane down on a UV box for 5 mins.
- The membrane was then treated with a prehybridization solution (6X SSC, 5X Denhardts, 0.5% SDS for a total of 10 ml) for 30 mins at 55°C.
- The prehybridization solution was poured off and the membrane was treated with a hybridization solution (6X SSC, 0.5% SDS and 50 ng biotin labeled repeat probe for a total of 10 ml) for one hour at 55°C.
- The membrane was washed two times at RT for 5 mins with 1X SSC, 0.1% SDS for a total of 25 ml.
- Next it was washed two times at 55°C for 5 mins with 0.1X SSC, 0.1% SDS for a total of 25 ml.
- Finally it was washed briefly in 10 ml of 2X SSC.
- Hybridization for chemiluminescent detection of the repeats was done using the Phototope® - Star Chemiluminescent Detection Kit (New England Biolads, Inc, Ipswich, MA).