> **Barley Genetics Newsletter (2007) 37: 44-46**

**CAPS markers targeting barley *Rpr1* region**

Ling Zhang\* and Andris Kleinhofs

Department of Crop and Soil Sciences

Washington State University, Pullman, WA, 99164-6420

\*E-mail: <lzhang1@wsu.edu>

Abstract

Eight cleaved amplified polymorphic sequences (CAPS) markers were
developed around *Rpr1* genetic region. Eight markers were co-dominant
between barley cultivars Morex and Steptoe after digestion with
restriction enzymes.

Introduction

In barley, resistance to *Puccinia graminis* f. sp*. tritici* pathotype
MCC requires the presence of at least of two host genes, *Rpg1*
(Brueggeman et al., 2002) and *Rpr1* (Zhang et al., 2006). Mutational
analysis and transcript-based cloning were used to isolate 3 candidate
*Rpr1* genes. These 3 candidate *Rpr1* genes, HU03D17U_s_at,
Contig4901_s_at and Contig7061_s_at were mapped to chromosome 4 bin 5.
Screening recombinants between the three candidate genes will identify
the real *Rpr1* gene. Therefore, molecular markers in this region are
needed.

Cleaved amplified polymorphic sequences (CAPS) markers are PCR-based
markers, requires a small amount of genomic DNA, which will facilitate
the screening of large numbers of genotypes at the seedling stage. 141
probesets representing a major *Rpr1* eQTL served as a starting point to
develop CAPS markers. Here we report the development and mapping of CAPS
markers in the *Rpr1* region.

Materials and Methods

CAPS markers development

Plant genomic DNA extraction was modified from Edwards et al. (1991);
the modification added an extra-step of chloroform-isoamyl alcohol
(24:1) extraction. Barley EST unigene sequences (HarvEST assembly#21;
http://harvest.ucr.edu/) were used as templates for primer design. RFLP
clone MWG058 was sequenced using primers T3 and T7 with the BigDye
terminator system on ABI Prizm 377 DNA sequencer (Applied Biosystems) at
the Bioanalytical Center, Washington State University, Pullman. A pair
of primers was designed from the MWG058 sequence. All the primer pairs
listed in Table 1 were used to amplify genomic DNA from the parent
cultivars Morex and Steptoe. All PCRs of 20μl contained 20-50ng of
genomic DNA, 0.1mMdNTP mix, 12.5 pmol of each primer, 1μl of RedTaq DNA
polymerase (Sigma), and 2 μl of 10xRedTaq reaction buffer. Amplification
was performed in a PTC-100 programmable thermal controller (MJ Research,
Cambridge, MA) at 95°C for 4 min, followed by 35 cycles of 95°C for 1
min, 60°C for 1 min, and 72°C for 1 min; this was followed by 7 min at
72°C. PCR products were purified using the Gel Extraction Kit (Qiagen,
Valencia, CA) and sequenced. Steptoe sequence was compared to the Morex
sequence in order to identify single nucleotide polymorphisms (SNPs)
that could be utilized for CAPS marker development. Sequence analysis
was done by VectorNTI software (Invitrogen). SNPs were identified and
restriction enzymes (New England BioLabs) were selected (Table 1). All
the PCR products were digested directly using restriction enzymes
correspondingly. Cleaved PCR products were then separated on 1% agarose
gel.

## Genetic mapping

The Steptoe x Morex \"minimapper\" population consisting of 35 selected
doubled-haploid lines (DHL), was used to map the molecular markers to
the barley Bin map (Kleinhofs and Graner 2002). CAPS marker genetic
order and the distance between snp_3139 and LZ2502 was estimated based
on segregation data from Steptoe x *Rpr1* F2 population.

#### Results and Discussion

Details of developed CAPS markers are listed in Tables 1 and Fig. 1.
Molecular mapping in Steptoe x Morex population with CAPS markers showed
that LZ6641, LZ13393 and LZ10152 co-segregated with LZMWG058, ABG484 and
BCD453B, respectively. Markers snp_3139 (Druka, personal communication)
and LZ2502 are the closest to *Rpr1* delimiting the 3 markers LZ17u,
LZ4901 and LZ7061 that co-segregate with *Rpr1*. LZ2502 and sn3139 are
about 1cM apart and can be used to screen recombinants in Steptoe x
*rpr1* F2 population.

![](media/image1.png){width="6.497222222222222in"
height="4.959027777777778in"} **Figure 1.** Chromosome locations of
eight CAPS marker in barley chr. 4 (4H).

+--------+------+---------------------------+------+-----------------+
| Ta     |      |                           |      |                 |
| ble 1. |      |                           |      |                 |
| S      |      |                           |      |                 |
| ummary |      |                           |      |                 |
| of     |      |                           |      |                 |
| dev    |      |                           |      |                 |
| eloped |      |                           |      |                 |
| CAPS   |      |                           |      |                 |
| ma     |      |                           |      |                 |
| rkers, |      |                           |      |                 |
| pr     |      |                           |      |                 |
| imers, |      |                           |      |                 |
| restr  |      |                           |      |                 |
| iction |      |                           |      |                 |
| e      |      |                           |      |                 |
| nzymes |      |                           |      |                 |
| and    |      |                           |      |                 |
| annot  |      |                           |      |                 |
| ation. |      |                           |      |                 |
| All    |      |                           |      |                 |
| the    |      |                           |      |                 |
| CAPS   |      |                           |      |                 |
| m      |      |                           |      |                 |
| arkers |      |                           |      |                 |
| were   |      |                           |      |                 |
| mapped |      |                           |      |                 |
| to Chr |      |                           |      |                 |
| 4      |      |                           |      |                 |
| (4H),  |      |                           |      |                 |
| except |      |                           |      |                 |
| L      |      |                           |      |                 |
| Z15227 |      |                           |      |                 |
| was    |      |                           |      |                 |
| mapped |      |                           |      |                 |
| to Chr |      |                           |      |                 |
| 7      |      |                           |      |                 |
| (5H).  |      |                           |      |                 |
+--------+------+---------------------------+------+-----------------+
| M      | Af   | Forward Primer            | En   | Annotation      |
| arkers | fyme |                           | zyme |                 |
|        | trix | Reverse Primer            |      |                 |
|        | P    |                           |      |                 |
|        | robe |                           |      |                 |
|        | sets |                           |      |                 |
+--------+------+---------------------------+------+-----------------+
| LZ2502 | Co   | 2502F:                    | Hpy  | putative IAA1   |
|        | ntig | AGCTTCAGCTTCAGGTCGAT      | CH4V | protein         |
|        |      |                           |      |                 |
|        | 250  | 2502R:                    |      |                 |
|        | 2_at | GAAACTTAGAACCTGAACC       |      |                 |
+--------+------+---------------------------+------+-----------------+
| LZ6641 | Co   | 6641F:                    | A    | putative        |
|        | ntig | TGATTGATCCTTTGCTGTCT      | vaII | expressed SLT1  |
|        |      |                           |      | protein         |
|        | 664  | 6641R:                    |      |                 |
|        | 1_at | CTGGAAAGCGTTCAAATGCT      |      |                 |
+--------+------+---------------------------+------+-----------------+
| LZ6435 | Co   | 6                         | DdeI | phos            |
|        | ntig | 435F:ACACCAGGAAGATCATCGAC |      | phoenolpyruvate |
|        |      |                           |      | carboxy-kinase  |
|        | 643  | 6                         |      | (ATP) -like     |
|        | 5_at | 435R:ACAATGGAGAACACATGGTT |      | protein         |
+--------+------+---------------------------+------+-----------------+
| L      | Co   | 13                        | A    | hypothetical    |
| Z13393 | ntig | 393F:AAGTGGACCGCGAAGCACGT | vaII | protein         |
|        |      |                           |      |                 |
|        | 1339 | 13                        |      |                 |
|        | 3_at | 393R:GCAGCATGTCAGGTTATACA |      |                 |
+--------+------+---------------------------+------+-----------------+
| L      | Co   | 15                        | AseI | microtubule     |
| Z15227 | ntig | 227F:ATGGACTAATGACCCCAACA |      | associated      |
|        |      |                           |      | protein         |
|        | 15   | 15                        |      |                 |
|        | 227_ | 227R:TGCAACACACAAAGCCAGTC |      |                 |
|        | s_at |                           |      |                 |
+--------+------+---------------------------+------+-----------------+
| LZ1321 | Co   | 1321F:                    | S    | calmodulin      |
|        | ntig | CACTATCGACTTCCCGGAAT      | au3A |                 |
|        |      |                           | or   |                 |
|        | 132  | 1321R:                    | MboI |                 |
|        | 1_at | ACTGCAATCAGGGTTCATCA      |      |                 |
+--------+------+---------------------------+------+-----------------+
| L      | Co   | 10152F:                   | S    | putative        |
| Z10152 | ntig | AGATCTCCGGCTACGTGCTG      | au3A | membrane        |
|        |      |                           | or   | protein         |
|        | 1015 | 10152R:                   | MboI |                 |
|        | 2_at | CGTACATCAGCTCGAAGAAA      |      |                 |
+--------+------+---------------------------+------+-----------------+
| LZMWG  |      | MWG058F:                  | BtsI | unknown         |
| 058^a^ |      | ATTCATGCATCTACCCATCTCA    |      |                 |
|        |      | MWG058R:                  |      |                 |
|        |      | TTGGATTGGCTAGAATCCTGGA    |      |                 |
+--------+------+---------------------------+------+-----------------+
| snp_3  |      | 3139F:                    | DdeI | unknown         |
| 139^b^ |      | AACCACGCAGCAAGCCTAT       |      |                 |
|        |      |                           |      |                 |
|        |      | 3139R:                    |      |                 |
|        |      | CTCGCTTCCTCCGTCATCAT      |      |                 |
+--------+------+---------------------------+------+-----------------+
| ^a^LZ  |      |                           |      |                 |
| MWG058 |      |                           |      |                 |
| was    |      |                           |      |                 |
| dev    |      |                           |      |                 |
| eloped |      |                           |      |                 |
| from   |      |                           |      |                 |
| RFLP   |      |                           |      |                 |
| clone  |      |                           |      |                 |
| M      |      |                           |      |                 |
| WG058. |      |                           |      |                 |
|        |      |                           |      |                 |
| ^b^sn  |      |                           |      |                 |
| p_3139 |      |                           |      |                 |
| se     |      |                           |      |                 |
| quence |      |                           |      |                 |
| pr     |      |                           |      |                 |
| ovided |      |                           |      |                 |
| by     |      |                           |      |                 |
| Druka  |      |                           |      |                 |
| A,     |      |                           |      |                 |
| Sc     |      |                           |      |                 |
| ottish |      |                           |      |                 |
| Crop   |      |                           |      |                 |
| Re     |      |                           |      |                 |
| search |      |                           |      |                 |
| Ins    |      |                           |      |                 |
| titute |      |                           |      |                 |
| (      |      |                           |      |                 |
| SCRI), |      |                           |      |                 |
| Inverg |      |                           |      |                 |
| owrie, |      |                           |      |                 |
| Dundee |      |                           |      |                 |
| DD2    |      |                           |      |                 |
| 5DA,   |      |                           |      |                 |
| UK     |      |                           |      |                 |
+--------+------+---------------------------+------+-----------------+

References

Brueggeman R., Rostoks N., Kudrna D., Kilian A., Han F., Chen J., Druka
A., Steffenson B., Kleinhofs A. 2002. The barley stem rust-resistance
gene *Rpg1* is a novel disease-resistance gene with homology to receptor
kinases. Proc. Natl. Acad. Sci. USA 99: 9328-9333.

Edwards K., Johnstone C., Thompson C. 1991. A simple and rapid method
for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids
Res. 19 (6):1349.

Kleinhofs A., Graner A. 2002. An integrated map of the barley genome.
In: R. L. Phillips and I. Vasil, eds., *DNA-Based Markers in Plants*,
2nd Edition. Kluwer Academic Publishers, Boston. pp. 187-199.

Zhang L., Fetch T., Nirmala J., Schmierer D., Brueggeman R., Steffenson
B., Kleinhofs A. 2006. *Rpr1*, a gene required for *Rpg1*-dependent
resistance to stem rust in barley. Theor Appl Genet 113: 847-855